ISSN 1580-4003 THE SCIENTIFIC JOURNAL OF THE VETERINARY FACULTY UNIVERSITY OF LJUBLJANA SLOVENIAN VETERINARY RESEARCH SLOVENSKI VETERINARSKI ZBORNIK Supplement 22 5th International Scientific Conference of Faculty of Veterinary Medicine Kafrelsheikh University, Egypt Veteinary Medicine Research and Application Hurghada, Egypt 5 - 9 March 2019 Volume Supply ^ 56 22 Slov Vet Res • Ljubljana • 2019 • Vol 56 • Supplement 22 • 1-796 ISSN 1580-4003 THE SCIENTIFIC JOURNAL OF THE VETERINARY FACULTY UNIVERSITY OF LJUBLJANA SLOVENIAN VETERINARY RESEARCH SLOVENSKI VETERINARSKI ZBORNIK Supplement 22 5th International Scientific Conference of Faculty of Veterinary Medicine Kafrelsheikh University, Egypt Veteinary Medicine Research and Application Hurghada, Egypt 5 - 9 March 2019 Volume Suppl^^ 56 22 Slov Vet Res • Ljubljana • 2019 • Vol 56 • Supplement 22 • 1-796 The Scientific Journal of the Veterinary Faculty University of Ljubljana SLOVENIAN VETERINARY RESEARCH SLOVENSKI VETERINARSKI ZBORNIK Supplement 22 The fifth International Scientific Conference of Faculty of Veterinary Medicine, Kafrelsheikh University, Egypt Veterinary Medicine: Research and Application Hurghada, Egypt 5-9 March 2019 Supplement was edited by guest editor Dr. Mohammed Abu El-Magd who has sole responsibility for the quality and originality of the published articles in this issue The articles are reviewed / Članki so recenzirani Editor in Chief / Glavni in odgovorni urednik: Gregor Majdič Co-Editor / Sourednik: Modest Vengušt Technical Editor / Tehnični urednik: Matjaž Uršič Assistants to Editor / Pomočnici urednika: Valentina Kubale Dvojmoč, Klementina Fon Tacer Guest Editor / Gostujoči urednik: Mohammed Abu El-Magd Editorial Board / Uredniški odbor: Vesna Cerkvenik, Robert Frangež, Polona Juntes,Tina Kotnik, Matjaž Ocepek, Joško Račnik, Ivan Toplak, Milka Vrecl, Veterinary Faculty University of Ljubljana / Veterinarska fakulteta Univerze v Ljubljani Editorial Advisers / Svetovalca uredniškega odbora: Gita Grecs-Smole for Bibliography (bibliotekarka), Leon Ščuka for Statistics (za statistiko) Address: Veterinary Faculty, Gerbičeva 60, 1000 Ljubljana, Slovenia Naslov: Veterinarska fakulteta, Gerbičeva 60, 1000 Ljubljana, Slovenija Tel.: +386 (0)1 47 79 100, 47 79 129, Fax: +386 (0)1 28 32 243 E-mail: slovetres@vf.uni-lj.si Sponsored by the Slovenian Research Agency Sofinancira: Javna agencija za raziskovalno dejavnost Republike Slovenije ISSN 1580-4003 Printed by / Tisk: DZS, d.d., Ljubljana Indexed in / Indeksirano v: Agris, Biomedicina Slovenica, CAB Abstracts, IVSI Urlich's International Periodicals Directory, Science Citation Index Expanded, Journal Citation Reports/Science Edition http://www.slovetres.si/ SLOVENIAN VETERINARY RESEARCH SLOVENSKI VETERINARSKI ZBORNIK Supplement 22 Slov Vet Res 2019; 56 (Suppl 22) Original Research Articles Atiba A S, Ghazy A, Farrag F A, Abu El-Magd M, Almadaly E A. Ultrasound-guided brachial plexus nerve block in donkeys............................................................................................................................................7 Gad El-Karim D RS, GA El-Amrawi. Cyclophosphamide hepatotoxicity: the role of 4-hydroxynonenal and cytochrome C oxidase and the possible protective effect of Ganoderma lucidum extract...........................15 Ismail T, Nassef ED, Hegazi ES, Bakr AN, Moustafa E M, Abdo W, Elbialy Z I. The modulatory effect of dietary betaine on intesti-nal absorptive capacity, lipogenesis and expression of lipid metabolism- and growth-related genes in Nile tilapia fed on soybean meal-based diet.....................................................................25 Abou-ElnagaA F, Rehan I F, Thompson R RJ, Abou-Ismail U A, Mahmoud M E, Mohamed R A, Mohammed H H, Sabek A A, Elhussiny M Z. The effects of group versus single housing on home cage behaviour in two strains of laboratory mice............................................................................................................................39 El-Sheshtawy S, El-Gobary G, Omar N, Shawky N. Ameliorating the toxic effects of cypermethrin by sesame oil in male rabbits........................................................................................................................................51 Abdel-Razek M A, Abozeid A M, Eltholth M M, Abouelenien F A, El-Midany S A, Moustafa N Y, Mohamed R A. Bioremediation of a pesticide and selected heavy metals in wastewater from various sources using a consortium of microalgae and cyanobacteria..............................................................................................61 Elmadawya M A, El-Kassasb S, Abdoc S E, Nagaid A, Bunaid Y. Genetic diversity among two common populations of Canis lupus familiaris in Egypt by using mitochondrial DNA HVR1 sequence.....................75 Magouz F, Salem M, Moustafa E, Elkhamy S. Impact of Biomos and Agrimos dietary supplementation on growth performance, feed utilization and immunological parameters of Nile tilapia (Oreochromis niloticus) fingerlings.....................................................................................................................................87 Dawood M A, Shukry M, Zayed M M, Omar A A, Zaineldin A I, El Basuini M F. Digestive enzymes, immunity and oxidative status of Nile tilapia (Oreochromis niloticus) reared in intensive conditions..........................99 Ali M, Eweedah N, Gaafar H, Abd El-Salam M. Utilization of sugar beet by products in feeding growing rahmani lambs ...........................................................................................................................................111 Elkomy H E, Taha A E, Basha H A, Abo-Samaha M I, Sharaf M M. Growth and reproduction performance of japanese quails (Coturnix coturnix japonica) under various environ-ments of light colors.........................119 Shewita R S, El-Naggar K, Abd El Naby W SH. Influence of dietary vitamin C supplementation on growth performance, blood biochemical parameters and transcript levels of heat shock proteins in high stocking density reared broiler chickens..................................................................................................................129 Mohammed H H, Rehan I F, Abou-Elnaga A F, Mohamed R A. Effects of feeder shape on behavioral patterns, performance and egg quality traits of Japanese quail...............................................................................139 Mohammed H H, Abdelaty A I, Saleem A Y, Youssef M I, Abdel-Hamid S EL. Effect of bedding materials on duck's welfare and growth performance....................................................................................................149 Meligy A MA, Al-Taher A Y, Ismail M Al-Naeem, A A, El-Bahr S M, El-Ghareeb W R. Pesticides and toxic metals residues in muscle and liver tissues of sheep, cattle and dromedary camel in Saudi Arabia.........157 Salah HM, Moussa EA, SM El. Bakr, Atta MS, Farrag FA, Hafez AS. Green tea mitigates streptozotocin- induced diabetic mice through antifibrotic activity and modulation of proinflammatory cytokines..............167 Donia MA, Gomaa NA, Abdelmegeid M, Nassif MN. Biomarkers versus duplex ultrasonography for early detection of acute kidney injury in dogs: an experimental study................................................................179 Kamal M A, Khairy M H, ELSadek N A, Hussein M MA. Therapeutic efficacy of zinc oxide nanoparticles in diabetic rats...............................................................................................................................................187 El-Shawadfi A, El-Khyat F, El-Gohary A. Identification of virulent Newcastle disease viruses in broilers in kafrelsheikh governorate through the year 2017.......................................................................................195 Mansour A, Mahfouz NB, Husien MM, El-Magd MA. Molecular identification of Aeromonas hydrophila strains recovered from kafrelsheikh fish farms......................................................................................................201 El-Sharawy M E, Mashaly I M, Atta M S, Kotb M, El-Shamaa I S. Influence of body condition score on blood metabolites and oxidative stress in pre- and post-calving of Friesian dairy cows in Egypt........................209 Abdelfattah-Hassan A, El-Ghazali H M. Effects of diet's acidifying additives on the intestinal histomorphology in ducks.....................................................................................................................................................219 Magouz F I, Khalafalla M M, Moustafa E M, Sadek A S. The impact of different stocking denesities and dietary protein levels on the performance of African catfish..................................................................................229 Ramoun A, E Almadaly, Hattab H, Darwish S, EL- Kon I. Transrectal ultrasonography and rectal palpation for judging uterine and cervical involutions in buffalo: a comparative study..............................................239 El-Ghareeb W R, Almathen, Fayez M M, Alsultan R A. Methicillin resistant Staphylococcus aureus (MRSA) in camel meat: prevalence F S and antibiotic susceptibility.......................................................................249 Alattar R, Abdel Alim A, Abdelmetal S, Abdel Aziz S. Corticosteroid-induced osteoporosis and osteonecrosis: role of oxidative stress.......................................................................................................257 Al-Sultan S I, Abdel-Raheem S M, Abd-Allah S M S, Edris A M. Alleviation of chronic heat stress in broilers by dietary supplementation of novel feed additive combinations...............................................................269 Elkhenany H, Nabil S, Abu-Ahmed H, Mahmoud H, Korritum A, Khalifa H. Treatment and outcome of horses with cutaneous pythiosis, and meta-analysis of similar reports.................................................................281 Khalafalla M, Mohamed A, Zayed M, Metwally A. Using of animal by products as an alternative protein source in Nile tilapia (Oreochromis niloticus) diets................................................................................................293 Kholife M, Moawad A, Diab A, El-keredy M. Mycological examination of fish feed stuff with special reference to mycotoxin production............................................................................................................................303 Saleh E A, Al-Hawary I, Elnajar M M. Antibacterial and anti-oxidant activities of laurel oil against Staphylococcus aureus and Pseudomonas fluorescence in Oreochromis niloticus fillets.........................313 Mahfouz N, Moustafa E, Kassab M, Marzouk W. Seasonal Screening of the Mycotic Infections of Cultured Freshwater Fishes in Kafr El-Sheikh Governorate....................................................................................321 Abdelhakam O, Elsebaie E, Ghazi A, Gouda M. Quality characteristics of beef hamburger enriched with red grape pomace powder during freezing storage.........................................................................................333 El-Katcha M, Soltan M, El-Naggar K, El-Shobokshy S, El-Erian M. Laying performance, fat digestibility and liver condition of laying hens supplemented with vitamin B12 or biotin and/ or bile acids in diet................341 Abdel-Raheem S, Mahmoud G, Senosy W, El-Sherry T. Influence of vitamin E and selenium supplementation on the performance, reproductive indices and metabolic status of Ossimi ewes.......................................353 Moselhy A, Mahdy E. Comparative three dimensional computed tomography (CT) scans and anatomical investigation of rabbit (Oryctolagus cuniculus) and cat (Felis domestica) skull.........................................365 Nassef E, Hegazi E, Sherif A, Elshamy M, Bakr A, Abouzed T. Impact of altering dietary omega 6 to omega 3 fatty acids ratio on growth performance, carcass composition, hemato-biochemical parameters and absorptive capacity of the intestine of Nile tilapia......................................................................................381 Al-Mallah K, Hussien M. Monitoring of pathological lesions in non-gravid ovine uterus.......................................391 El-Nagar S, Helal M, Mahmoud S, Dillard L. Comparative histopathological changes of liver, kidney and appendix of rabbits treated with inorganic nano chromium to ameliorate heat stress effect......................399 Dawood M, Mohsen M, El-dakar A, Abdelraouf E, Moustafa E, Ahmed H. Effectiveness of exogenous digestive enzymes supplementation on the performance of rabbitfish (Siganus rivulatus)........................409 Abdelaal A, Elgioushy M, Gouda S, El-Adl M, Hashish E, Elgaml S, Ali M, El-Gedawy A. Hemato- biochemical and molecular markers (IS900) of cattle infected with Johne's disease in Egypt..................421 Omar A, Gharib H, Said E. Effect of feeding different concentrate roughage ratio on growth, reproductive performance and behavior of sheep..........................................................................................................433 Abd-Elfatah E, El-Mekkawi M, Aboul-Soud E, Fawzi E, El-Soally S. Immunological response of a new trivalent Capripoxvirus vaccine in pregnant ewes and does......................................................................445 Hafez Y M, Moustafa N Y, Magouz A F, Al-Maria N F. Isolation of white spot syndrome virus (WSSV) in Egyptian shrimp using conventional PCR and real time PCR (QPCR) techniques...................................457 El-shafaey E, Sayed-Ahmed M, Shoeib S, El-khodery S. Occurrence and risk factors of digital dermatitis in Egyptian dairies.....................................................................................................................................465 Gaffer W, Gwida M, Abo Samra R, Al- Ashmawy M. Occurrence and molecular characterization of extended spectrum beta-lactamase producing Enterobacteriaceae in milk and some dairy products......................475 Mokh A., Abdelhady D., Ghazy E., Aboumosalem H., Goda W. Sesame oil mitigates initiation stage of diethynitrosamine hepatocarcinogenesis in rats........................................................................................487 Salama M, Morsy W, Mohamed R, El-Midany S. Effect of some feed-additives on the growth performance, physiological response and histopathological response and histopathological changes of rabbits subjected to ochratoxin-A feed contamination...........................................................................................499 El Zeny T, Essa R Y, Bisar B A, Metwalli S M. Effect of using chicory roots powder as a fat replacer on beef burger quality................................................................................................................................509 Mostafa N, Kirrella G, Aideia H, Abo Shaisha J. Assesstment of mould contamination of Tilapia nilotica and Mugil cephalus fish and trials to reduce using natamycin..........................................................................515 Salem A, El-Awady H, Tag EL-Dein M, Eisa D. Effect of aromatic plants oils on immunity, udder health and milk production of Friesian cows........................................................................................................523 Elsheikh H, El-Mekkawi M, Abou-zaid A, Abd El Raof A. Diagnosis and phylogenetic analysis of the circulating peste des petits ruminants virus in Al-Sharqia governate..........................................................................531 Almadaly E, Tawfik F, El-Kon I, Heleil B, Fattouh El. Effect of cryoprotectants on in vitro semen quality and in vivo fertility of buffalo (Bubalus bubalis) bull semen..............................................................................541 Ghazy E., Mokhbatly A., Keniber S., Shoghy K. Synergistic ameliorative effect of Lactobacillus and Spirulina platensis against expermintal colitis in albinorats: antioxidant, histopathological and molecular studies...............................................................................................................................553 Ebissy E, El-Sayed A, Mohamed R. Hematological and biochemical profile in female camels (Camelus dromedarius) during the transition period..................................................................................................571 Ramou A, EL- Kon I, Almadaly E, Aly A, Abo EL- Ghate H. Assessment the synchrony between uterine status and hormonal profiles in modified Ovsync protocols in relation to fertility in buffaloes........579 Shoeib S, Sayed-Ahmed M, El-khodery S. Hypomagnesemic tetany in camel calves (Camelus dromedarius): clinical consequences and treatment outcomes........................................................................................589 Elblehi S S, Gad El-Karim D R. Potential amelorative effect of Panax ginseng on hepatorenal damage ocuured in hydroxurea-treated rats...........................................................................................................595 Abdel-Hay A M, Hamed S A. Zayd M M, Abouelenien F A, Rehan I F, El-Kassas S, Deyab A M, Mohamed R A. Night feeding regime improves water quality, growth performance and feeding behaviour of Eropean eels, Anguilla anguilla.................................................................................................................607 Sherif A A, Nada H S, Moawad A A, El- desouky I E, Helmy S M. Using real-time polymerase chain reaction to study the effect of salicylic acid and sodium chloride on the pyocyanin production of Pseudomonas aeruginosa.................................................................................................................................................615 ALomaisi S A, El-Ghazali H, Nosseur H, Ahmed S, Konsowa M. Prenatal development of the vomeronasal organ in rabbit...........................................................................................................................................623 Elbaz S, Nassef E, Bakr A, Hegazi E, EL-keredy A. Impact of dietary eicosapentaenoic and docosahexaenoic fatty acids supplementation on inflammatory response of post calving cows during transition to lactation.....................................................................................................................................................633 Moawad A, Noor El Deen A, Mahfouz N, Helmy S, Hashem Y Phenotypic and molecular identification of yeasts isolated from cultured tilapia (Oreochromis niloticus) in Kafrelsheikh province, Egypt...................647 Hafez Y, Sobeih A, Mansour N. Pivotal role of Lactobacillus strains in improvement of soft cheese quality and inhibiting the growth of harmful and dangerous bacterial pathogens..................................................657 Kamal T, Dorghamm D, Kahilo K, Elkattawy A, Nassef E, El-sawy H. Impact of L-carnitine supplementation on growth of broiler chicken through determination of changes in the expression of CAT2, MYOD and MYF5 genes..............................................................................................................................................665 Ragab A, Nasr N, Kahilo K. Protective effect of glycyrrhizc acid against carbon tetrachloride-induced liver fibrosis in rats: role of integrin subunit p LIKE 1 (ITG B L1)...............................................................673 Shireen S, Nasr E, Khaled A, Hossam E. Hemotoxic and genotoxic effects of lead acetate and chlorpyrifose on freshwater cat fish (Calarias gariepinus)..............................................................................................681 Allam S, Mostafa N, Kirrella G, Eleiwa N, El-Magd M. Molecular detection of inva and hila virulent genes in salmonella serovars isolated from fresh water fish....................................................................................693 Omar M A, Abdel-Hamid T M, Esam S, Omar A E. Growth and economic performance of using dried tomato pomace for Mallard ducks.........................................................................................................................699 Abdel-hamid T M, Omar M A. Growth and economic performance, some hematological, biochemical and oxidative stress parameters, and carcass traits of summer stressed Californian and crossbred rabbits as affected by dietary supplementation of formic acid...............................................................................707 Aziz M, Zahra A, Kheder Z, Fikry H. The role of L. methionine, L. carnitine, choline and/or silymarin in hepatoprotection against paracetamol intoxication and oxidative stress in broilers...................................717 Shabana S, Helmy S, Hegazy A E. Characterization of class 1 integrons and some anti-microbial resistance genes in Salmonella species isolated from poultry in Egypt......................................................................725 Ghazy E, Mokh A, Abdelhady D, Goda W, Hashem E. The role of thymoquinone in ameliorating the hepatoxic effect of diazinon in male rats....................................................................................................................735 Elmonir W, Essa H, El-Tras W. Ecology of Staphylococcus aureus and its antibiotic resistance genes in dairy farms: contributing factors and public health implications.........................................................................747 Elmonir W, Aglan H, Elmahallawy E, El-Tras W. Antimicrobial phenotypes of geographically matched Staphylococcus aureus isolated from buffalo's milk and clinical human cases in Egypt: potential zoonotic risks..............................................................................................................................755 Abu El-Hamd M, Metwally A, Hegazy M, Ghallab Z, Elateeqy O. Effect of supplementation of omega-3 fatty acids on blood parameters and semen quality of Friesian bulls.........................................765 Omara N A, Elsebaie E, Kassab H, Salama A. Production of chitosan from shrimp shells by microwave technique and its use in minced beef preservation....................................................................................773 Abdelmoez N S, Shawky M M, Abdelhady H A, Lebdah M A, Salama S S. Isolation and identification of some possible causative agents of Swollen Head Syndrome (SHS) in broiler chickens in Egypt.......................781 Salama S, Abdelhady H, Atia L. Field application for experimental inactivated multivalent P. multocida and avian influenza (H9N2) vaccine in poultry.................................................................................................789 Slov Vet Res 2019; 56 (Suppl 22): 7-14 DOI 10.26873/SVR-739-2019 Original Research Article ULTRASOUND-GUIDED BRACHIAL PLEXUS NERVE BLOCK IN DONKEYS Ayman S. Atiba1*, Alaa Ghazy1, Foad A. Farrag2, Mohammed A. El-Magd2, Essam A. Almadaly3 1Departments of Surgery, Anesthesiology and Radiology, Faculty of Veterinary Medicine, Kafrelsheikh University, 33516, Egypt, 2Department of Anatomy and Embryology, Faculty of Veterinary Medicine, Kafrelsheikh University, 33516, Egypt, 3Department of Theriogenology, Faculty of Veterinary Medicine, Kafrelsheikh University, Egypt *Corresponding author, E-mail: atiba_2003@yahoo.co.uk Abstract: This study was conducted to describe and evaluate the ultrasound-guided technique and the anatomical relation for brachial plexus block in donkeys. In the first study, three donkey's cadavers were used to determine the neuroanatomical description of the brachial plexus. In the second study, four donkeys were used to evaluate the ultrasound structure of brachial plexus and the distribution of methylene blue solution that was injected around the nerves during subsequent postmortem anatomical dissection. In the third study, six donkeys were used to evaluate the efficient of the ultrasound-guided brachial plexus block after injection of lidocaine. Ultrasound was used to identify brachial plexus and blood vessels. Anatomical scrutiny assured the relationship between the nerves and the blood vessels. The nerves were adequately stained with methylene blue solution in all donkeys. A volume of 25 mL of lidocaine per injection site resulted in effective sensitive and motor block of forelimbs nerves after brachial plexus block. Based on the obtained data we could conclude that brachial plexus nerve block guided by ultrasound can be used in surgery of donkey distal fore limb. Key words: ultrasound; brachial plexus; nerve block; donkey Introduction The donkey (Equus asinus) is the most important working animal in many parts of the world (1). Donkeys provide one of the most cost effective sources of transport in the rural agricultural sector. There are an estimated 59 millions of donkeys and mules in the world (2). Although greater populations of donkeys live in developing countries, very little scientific information specific for this species exists in the literature. Peripheral nerve block techniques are becoming increasingly popular for performing certain surgeries in both animals and humans (3-5). These techniques do not only decrease general anesthesia drawbacks but they also lead to effective analgesia (6-8). In animals many surgical operations especially in the distal parts of the limbs can be performed under peripheral nerve block such as sciatic and femoral nerves block of the pelvic limbs in cattle, sheep and dogs (9-11) and brachial plexus nerve block in the forelimbs in cattle, dogs and cats (3, 5, 7, 9). Received: January 2019 Accepted for publication: February 2019 8 AS Atiba, A Ghazy, FA Farrag, M Abu El-Magd, EA Almadaly In humans, brachial plexus block is very universal and safe, and allows anesthesia for the upper limb. Therefore, various operations, principally orthopedic, could be carried out under this block (12, 13). Several techniques exist to produce local anesthetic blockade of the brachial plexus include the use of anatomic landmarks (blind technique), a nerve locator technique, and ultrasound guidance technique. Many studies comparing these techniques and all stated that ultrasound-guided one is the most effective technique to facilitate the peripheral nerve block (8, 14, 15). Previous literatures lack detailed description for neuroanatomy and block technique of brachial plexus in donkeys. Thus, this study was designed to describe and evaluate the ultrasound-guided technique and the anatomical relation for brachial plexus block in donkeys. Materials and methods The experimental protocol was approved by the Animal Care and Use Committee of Faculty of Veterinary Medicine, Kafrelsheikh University. Study 1: Anatomical study of the brachial plexus For the anatomical dissection of the brachial plexus, cadavers of three donkeys (23 ± 2 months old, weighing 167 ± 10 kg) were used. The forelimb of the three donkeys were separated carefully from the trunk by cutting the skin, the pectoralis superficialis and pectoralis profundus muscles at their origin on the sternum, brachiocephalicus and trapezius muscles at the cranial border of scapula, serratus ventra-lis, rhomboideus muscles at their insertion near the dorsal border of scapula and latissmus dorsi muscle at the caudal border of scapula. The forelimb was abducted to expose the axillary artery and brachial plexus. The nerves and main blood vessels were dissected and identified. Finally, the dissected region was photographed. Study 2: Ultrasound-guided brachial plexus blockade in cadavers The ultrasound images of the brachial plexus blockade of both forelimbs from four donkeys (18 ± 4 months old, weighing 181 ± 18 kg) were taken using a high frequency 5-10 MHz linear transducer (Mindray Diagnostic Ultrasound System, Z5, Germany). The transducer was placed in front of the cranial border of scapula and the first rib and dorsal to the shoulder joint (Fig. 1C). Color Doppler mode was utilized to aid vessels identification. The location of brachial plexus nerves were injected with 25 mL methylene blue (Sigma-Aldrich, USA) using a 20 gauge, 90 mm spinal needle. The needle was inserted just ventral to the ultrasound transducer and vertical to the nerves then methylene blue was injected when the needle tip reached a depth of 1 mm. After injection, donkeys were euthanized and injection site was dissected to determine staining distributions around nerves. Study 3: Ultrasound-guided brachial plexus blockade in live donkeys For the ultrasound-guided brachial plexus blockade, six donkeys (aged 19 ± 7 months and weighing 188 ± 41 kg) were used. The brachial plexus nerves were blocked unilaterally. Each donkey was used for three times with 1 week interval. The donkeys were mild sedated with 0.3 mg/kg IV xylazine (Xyla-Ject, ADWIA Pharmaceuticals Co. Cairo, Egypt). In the standing position, the skin of the craniolateral aspect of the first rib was clipped. The brachial plexus were blocked with 25 ml of a lidocaine (Debocaine 2% Al-Debeiky Pharmaceutical Industries Co., Egypt). To evaluate analgesia, a pin-prick test was applied with a 21 gauge needle inserted through the skin. The sensitive response was scored using a three-point numerical rating scale (Table 1). The areas of sensitive responses were evaluated according to the sensory branches of brachial plexus (Table 2). The motor effect was evaluated as previously described (3) and was scored using a three-point numerical rating scale (Table 1). The sensitive and motor responses of the fore-limb were evaluated and compared before and after lidocaine injection. Twelve injections were given with 10 min intervals. Moreover, each injected forelimb was compared with the other contralateral non-injected forelimb. Ultrasound-guided brachial plexus nerve block in donkeys 9 Statistical analysis Descriptive statistical data are expressed as medians and ranges. The effects of local anesthetic on brachial plexus in the various anatomical regions were analyzed by the non-parametric Friedman test with pairwise comparisons using SPSS for Windows Version 16 (SPSS Inc., IL, USA). A value of P < 0.05 was considered significant. Results Study 1: Anatomical study and dissection of the brachial plexus nerves The gross dissection of donkeys brachial plexus revealed that it was composed of the anastomosis of the ventral branches of the last three cervical (6th, 7th and 8th) and the first two thoracic spinal nerves. The brachial plexus was located cranial to the first rib and dorsal to sca-lenius medius muscle. It was covered laterally by subclavius and supraspinatus muscles slightly above the shoulder joint (Figs. 1A and B). The brachial plexus gave many branches which innervated the skin and muscles of fore-limb in addition to parts of the lateral wall of thorax and abdomen as well as parts of the shoulder girdle (Fig 1B). The branches of brachial plexus either motor or sensory were summarized in table 2. Study 2 and 3: Ultrasound-guided nerve blockade The brachial plexus was identified as a hy-perechoic band with several intermittent hy-poechoic strips separated by hypoechoic lines (Figs. 2A-C). These hyperechoic structures were confirmed to be brachial plexus after utilizing color Doppler mode, which showed the axillary blood vessels ventral to these hy-perechoic structures (Fig. 2B). The ultrasound transducer was placed cranially near the first rib. In this position the needle was advanced above the brachial plexus and axillary blood vessels. After assuring that blood was not withdrawn, methylene blue was injected. The extent of dye was monitored as an anechoic area proximal to the brachial plexus. Staining of brachial plexus was observed in all donkeys. The dye was spread more than 2 cm in 3 donkeys (Fig. 1D). During this procedure, only one animal was intravascularly injected in axillary vessels. Figs. 2D and E show the scores of the sensitive and motor effects after ultrasound-guided brachial plexus block in 16 forelimbs. One animal was died after 1 week with a reason not related to the current study. The sensory score for the blocked area showed a significant increase at 30, 40 and 50 min in musclocutaneous nerve area, at 30 and 40 min for the area of median nerve, and at 30 min for the area of ulnar nerve (p < 0.05). The motor scores increased significantly at time points 20, 30, 40 and 50 min. A motor effect was extended more than the sensory effect in all examined animals. Discussion To the best of our knowledge, this may be the first study to investigate the ultrasound-guided brachial plexus blocking in donkeys. With this study we were able to demonstrate the feasibility to block the brachial plexus in donkeys with a highly successful rate. Table 1: The score of sensitive and motor response (3) Score_Sensitive response_Motor response_ 0 Normal response Normal gait while walking and no abnormal clinical sign while standing. 1 Diminished response Donkey can walk while bearing mild to moder- ate weight and shows no abnormal sign while standing. 2 No response Donkey can walk but is barely able to bear weight while walking, and shows abnormal _signs while standing._ 10 AS Atiba, A Ghazy, FA Farrag, M Abu El-Magd, EA Almadaly Table 2: Summary of branches of brachial plexus and its motor and sensory innervation Branches of brachial plexus Innervation Motor Sensory Long thoracic nerve Thoracic part of serratus ventra-lis muscle. Dorsal thoracic nerve Latissmus dorsi muscle. Lateral thoracic nerve Cutaneous trunci muscle Skin of lateral thoracic wall Cranial pectoral nerve Superficial pectoral and subclavian muscles Caudal pectoral nerve Deep pectoral muscles Suprascapular nerve Supra spinatus and infra spina-tus muscles Axillary nerve Deltoid, teres major, teres minor, capsularis humeri and cleidobrachialis muscles Skin of craniolateral aspect of brachium and cranial aspect of antebrachium Subscapular nerve Subscapularis muscle Musculocutaneous nerve Coracobrachial, brachialis and biceps brachii muscles Skin of medial part of antebrachium Radial nerve Triceps brachi, anconeus, tensor fascia antebrachii, extensor carpi radialis, common digital extensor, lateral digital extensor, extensor carpi ulnaris and extensor carpi obliqus Skin of the lateral aspect of brachium and antebrachium Ulnar nerve Flexor Carpi Ulnaris, superficial digital flexor and ulnar head of Flexor Digitorum Profundus, interosseous muscles Skin of the caudal aspect of antebrachium and dorsolat-eral aspect of metacarpus and digit. Median nerve Flexor carpi radialis, humoral and radial head of deep digital flexor muscles Skin of the palmar aspect of metacarpus and digit. The detailed description of Equine forelimb nerve supply has been found in several standard anatomical textbooks (16-19). Briefly, this nerve supply comes from the brachial plexus which is a complex network of nerves formed by the anastomoses of the ventral roots of some spinal nerves extending from the 6th cervical nerve to the 2nd thoracic nerve. It passes between the two parts of the scalenius medius muscle and the medial aspect of shoulder in the axillary region. The major nerves emanated from the brachial plexus are the suprascapular, subscapular, musculocutaneous, axillary, radial, median, and ulnar nerves. Brachial plexus supplies sensory and motor innervation of the forelimb (20). The previous description of the nerve supply of forelimb in horses coincides with the findings of the current study regarding the brachial plexus position and nerves emanating from it in donkeys. The higher degree of similarity between donkeys and horses might be attributed to close relation between the two species which belong to the same family (Equi-dae) and genus (Equus). Ultrasound-guided brachial plexus nerve block in donkeys 11 Figure 1: Position, branches of brachial plexus and site of ultrasound transducer. A. Position of brachial plexus (black arrowhead), scalenius medius muscle (white arrowhead) and long thoracic nerve (black arrow). B. Axillary artery (A) surrounded by branches of brachial plexus (B); pectoral nerves (1), musculocutaneous nerve (2), median nerve (3), dorsal thoracic nerve (4), subscapular nerve (5), lateral thoracic nerve (6), radial nerve (7), ulnar nerve (8) and the musculocutaneous with median nerves (9). C. Position of ultrasound transducer in front of cranial border of scapula and first rib and dorsal to the shoulder joint. D. Site of injection of methylene blue dye (white arrow) in the region of brachial plexus 12 AS Atiba, A Ghazy, FA Farrag, M Abu El-Magd, EA Almadaly wivmiunu f: Figure 2: Ultrasound images of the area around the brachial plexus, the sensory and motor score after nerve block. A. Ultrasound image of the brachial plexus (the area surrounded by the arrowheads) and the first rib (R1). B. Color Doppler ultrasound image of the brachial plexus (the area surrounded by the arrowheads) and the axillary blood vessels (arrow). C. Ultrasound image of the brachial plexus after injection of local anesthetic drug (L). D. Sensitive and motor (E) scores after ultrasound-guided brachial plexus block in donkeys. Data are expressed as the median. Symbols indicate significant differences from baseline value within a defined anatomical subarea (P < 0.05) Currently, the use of the ultrasonography has been reported to provide an accurate guide for performance of peripheral nerve block when compared with the use of conventional blind or electrolocation techniques (8, 15). This technique provided the ability to administer a calculated amount of anesthetic drug at the target site under real-time observation (7, 14). Additionally, it minimized complications like maldistribution of local anesthetic or intravascular needle placement (21). In the present study, among all injected cases, only one case exhibited intra-vascular insertion of the needle. Peripheral nerves may look hypoechoic or hyperechoic on ultrasound image (22). In the present study, the brachial plexus appeared hy-perechoic structures dorsal to the axillary vessels. Similar findings were reported for ultrasound-guided nerve blockade of the brachial plexus in humans, calves, dogs and cats (3, 7, 9, 13, 15). The brachial plexus provides the sensory and motor supply of the thoracic limb. Therefore, its successful block would provide complete de-sensitization of the whole forelimb (7, 20). In the present study, the regions dominated by Ultrasound-guided brachial plexus nerve block in donkeys 13 musculocutaneous, median and ulnar nerves showed a significant sensory scores (p < 0.05). While the other regions dominated by radial and axillary nerves showed insignificant increased score as compared to the basal line time. Motor score was increased significantly and extended more than the sensory effect. Our obtained sensory and motor scores were more effective than that obtained after brachial plexus block in calves (3). This may be attributed to the volume of local anesthetic drug used. In calves, the volume was 10 ml 2% lido-caine while we used 25 ml 2% lidocaine. Increasing the dose of local anesthetic associated with efficacy of peripheral nerve block (23). Conclusion To the best of our knowledge, this may be the first description to the anatomical and ultrasound-guided brachial plexus block in donkeys. The ultrasound-guided technique was found to be a feasible and safe method to provide adequate analgesia of the forelimb in donkeys but further trials would be needed to assess the usefulness and the applicability of this technique in the clinical cases. Conflict of interest The authors declare that they have no conflict of interest. References 1 .Lizarraga I, Sumano H, and Brumbaugh G. Pharmacological and pharmacokinetic differences between donkeys and horses. Equine Veterinary Education 2004; 16 (2): 102-12. 2. Al-Salihi K, Farhat S. The Society for the Protection and Welfare of Donkeys and Mules in Egypt "SPWDME": An overview. MRSVA 2014; 3 (3), 1-5. 3. Iwamoto J, Yamagishi N, Sasaki K, Kim D, Devkota B, Furuhama K. A novel technique of ultrasound-guided brachial plexus block in calves. Research in veterinary science 2012; 93(3):1467-71. 4. Gallacher K, Santos L, Campoy L, Bezuiden-hout A, Gilbert R. Development of a peripheral nerve stimulator-guided technique for equine pu-dendal nerve blockade. The Veterinary Journal 2016; 217:72-7. 5. Portela DA, Verdier N, Otero PE. Regional anesthetic techniques for the thoracic limb and thorax in small animals: A review of the literature and technique description. The Veterinary Journal 2018 ; 241: 8-19 6. Edmondson MA. Local and regional anesthesia in cattle. Veterinary Clinics of North America: Food Animal Practice 2008; 24(2): 211-26. 7. Anson A, Laredo FG, Gil F, Soler M, Belda E, Agut A. Evaluation of an ultrasound-guided technique for axillary brachial plexus blockade in cats. Journal of feline medicine and surgery 2017;19 (2): 146-52. 8. Hagag U and Tawfiek MG. Blind versus ultrasound-guided maxillary nerve block in donkeys. Veterinary anaesthesia and analgesia, 2018; 45(1): 103-10. 9. Campoy L, Bezuidenhout AJ, Gleed RD, Martin-Flores M, Raw RM, Santare CL. Ultrasound-guided approach for axillary brachial plexus, femoral nerve, and sciatic nerve blocks in dogs. Veterinary anaesthesia and analgesia 2010; 37(2): 144-53. 10. Re M, Blanco-Murcia J, Villaescusa Fernandez A, De Gaspar Simon I, Gomez de Segura IA. Ultrasound-guided anaesthetic blockade of the pelvic limb in calves. Veterinary Journal 2014; 200 (3):434-9. 11. Waag S, Stoffel MH, Spadavecchia C, Eich-enberger U, Rohrbach H. Ultrasound-guided block of sciatic and femoral nerves: an anatomical study. Laboratory animals 2014; 48(2):97-104. 12. Enneking FK, Chan V, Greger J, Hadzic A, Lang SA, Horlocker TT. Lower-extremity peripheral nerve blockade: essentials of our current understanding. Regional anesthesia and pain medicine 2005; 30(1):4-35. 13. Nowakowski P. Ultrasound guided axillary brachial plexus block. Part 1 Basic sonoanatomy. Anaesthesiol Intensive Ther 2015; 47 (4), 409-416. 14. Singh S, Goyal R, Upadhyay KK, Sethi N, Sharma RM, Sharma A. An evaluation of brachial plexus block using a nerve stimulator versus ultrasound guidance: A randomized controlled trial. Journal of anaesthesiology, clinical pharmacology 2015; 31(3):370. 15. Akasaka, M and Shimizu M. Comparison of ultrasound-and electrostimulation-guided nerve blocks of brachial plexus in dogs. Veterinary anaesthesia and analgesia 2017; 44(3): 625-35. 16. Dams R, Lapras M, Florio R, Barone R. On malformations following the injection of delta-hy-drocortisone acetate into the embryonal chicken egg and their transmission by a sole survivor. Comptes 14 AS Atiba, A Ghazy, FA Farrag, M Abu El-Magd, EA Almadaly rendus des seances de la Societe de biologie et de ses filiales 1964; 158:740-2. 17. Getty R, Sisson S, Grossman J. The anatomy of the domestic animals 1975; 1:739-1211. 18. Nickel R, Schummer A, Seiferle E. The anatomy of the domestic animals. The locomotor system of the domestic mammals 1986; 1:150: 289. 19. Dyce KM, Sack WO and Wensing CJG. Textbook of Veterinary Anatomy-E-Book. Elsevier Health Sciences 2009; 4(3): 830-4. 20. Magilton J, Getty R and Ghoshal N. A comparative morphological study of the brachial plexus of domestic animals (goat, sheep, ox, pig and horse). Iowa State J. Sci, 1968; 42(3): 245-79. 21. Koscielniak-Nielsen Z. Ultrasound-guided peripheral nerve blocks: what are the benefits? Acta Anaesthesiologica Scandinavica 2008; 52(6): 727-37. 22. Marhofer P, Greher M, and Kapral S. Ultrasound guidance in regional anaesthesia. British journal of anaesthesia 2004; 94(1): 7-17. 23. Skarda RT. Local and regional anesthesia in ruminants and swine. Veterinary Clinics: Food Animal Practice. 1996; 12 (3):579-26. Slov Vet Res 2019; 56 (Suppl 22): 15-23 DOI 10.26873/SVR-740-2019 Original Research Article CYCLOPHOSPHAMIDE HEPATOTOXICITY: THE ROLE OF 4-HYDROXYNONENAL AND CYTOCHROME C OXIDASE AND THE POSSIBLE PROTECTIVE EFFECT OF Ganoderma lucidum EXTRACT Dina R. S. Gad El-Karim1*, Gamal El-Amrawi2 *1Department of Pathology, Clinical Pathology branch, Faculty of Veterinary Medicine, Alexandria University, 2Department of Theriogenology, Faculty of Veterinary, Medicine, Alexandria University, 11511, Egypt Corresponding author, E-mail: Gaddidi@yahoo.com Abstract: Cyclophosphamide (CPA) is a widely used anti-neoplastic drug, but its usage is associated with several side effects including hepatotoxicity. This study was designed to evaluate the ameliorative effect of Ganoderma lucidium (GL) extract as a natural antioxidant against CPA hepatotoxicity in male Wistar albino rats. Four groups of rats (n = 7) were used in this study, Group I: served as a control group, group II: was administrated CPA (5 mg/kg), group III: received GL extract (50 mg/kg), and group IV: received CPA and GL extract at the same doses of groups II and III. All the treatments were orally administrated via gastric intubation for 21 consecutive days. Serum activities of the hepatic enzymes (AST, ALT and GGT) in addition to serum proteinogram were evaluated. Moreover, hepatic oxidant-antioxidant markers (4- hydroxynonenal (4-HNE), GSH, CAT and SOD) besides activity of cytochrome C oxidase enzyme were detected. Also, histopatho-logical examination of the hepatic sections (H&E stained) was performed. Serum activity of liver enzymes was elevated, proteinogram was disturbed in CPA-treated animals which accompanied by an increase in hepatic content of 4-HNE, depletion of hepatic antioxidant (GSH, CAT, SOD) and inhibition of hepatic cytochrome C oxidase activity, also hepatic histopathological disruptions were noticed in this group. All these hepatic disturbances were partially ameliorated upon co-administration of GL extract with CPA. In conclusion, production of 4- HNE and inhibition of cytochrome C oxidase enzyme may play a role in occurrence of CPA hepatotoxicity and GL extract may have an ameliorative role against this toxicity. Key words: cyclophosphamide; hepatotoxicity; Ganoderma lucidium; 4-hydroxynonen-alon; cytochrome C oxidase Introduction Cyclophosphamide (CPA) is a drug with a wide spectrum of clinical uses especially in the treatment of cancer (lymphoma, acute and chronic leukemia, multiple myeloma) and autoimmune diseases such as rheumatoid arthritis and vasculitis (1). As a chemotherapy, CPA is an alkylating agent depending on formation of phosphoramide mustard (major chemically reactive CPA metabolite) which brings about interstrand cross-links between opposite DNA strands and hampers their replication and tran- Received: January 2019 Accepted for publication: February 2019 16 D. R. Gad El-Karim, G. A. El-Amrawi scription process and this characterizes the clinical activity of CPA (2). Bioconversion of CPA into its metabolites (acrolein and phospho-ramide mustard) leads to the formation of high level of reactive oxygen species (ROS), which disturb antioxidant activity inducing CPA-related tissue injuries (3-5). Oxidative stress (disruption of redox balance) which generates biochemical and physiological disturbances, mediates the injury of normal tissues (as hepatic cells damage and mutagencity) and this is considered the major limitation of CPA usage as an anti-neoplastic drug (6,7). Several studies suggest that antioxidant supplementation can influence the response to chemotherapeutic drugs and attenuate the development of their adverse side effects (8). Ganoderma lucidum (GL) or Reishi is an oriental fungus which has been recognized as a medicinal mushroom for over 2000 years due to its multiple powerful effects (9). It presents in many commercial forms including dietary supplement, tea or powder which are produced from different mushroom parts as fruits, myce-lia or spores (10). GL polysaccharides (GLPS) is one of the major active bio-components of GL which have multiple pharmacological effects, such as antitumor (11), and antioxidant activity (12) in a dose dependent manner (13) through direct free radicals scavenging and ability to increase antioxidant enzymes activity (14), immunomodulation (15). It also has a well proved hepato-protective effect against chemical or immune-related hepatic damage (16-19). This study was an attempt to prove our hypothesis about the inhibitory effect of 4-hy-droxynonenalon and cytochrome C oxidase enzyme in relation to the occurrence of cyclo-phosphamide hepato-toxicity and possible attenuating role of Ganoderma lucidium against this toxicity in Wistar albino rats. Material and methods Experimental animal Twenty eight adult male Wistar albino rats weighting about 180-200 g were obtained from Alexandria University Research Institute, Egypt and kept under 12 hr-12 hr light/dark cy- cles. They were allowed to standard commercial pelleted food with water ad- libitum. The animals were left without any treatment for 10 days for acclimatization. All the experimental procedures of this study were performed according to the instructions of "Care and Use of Laboratory Animals" prepared by Faculty of Veterinary Medicine, Alexandria University, Egypt. The protocol of this study was approved by the Research Ethics Committee of the University. Drugs Commercially available CPA tablets (En-doxan® 50mg, Baxter Oncology GmbH) and GL whole fruiting body extract (Standardized Reishi, 376 mg capsules standardized to con-tain10% polysaccharides, Nature's Way®, USA) were used to accomplish this study. The accurate doses of the drugs were dissolved in saline solution daily and shortly before administration. Experimental protocol The acclimated animals were randomly divided into four equal groups (n =7) and treated as follow: group I (control group): rats received 1 ml saline; group II (GL-treated group): rats received GL extract at a dose of 50 mg/kg body weight, bw (20); group III (CPA-treated group): rats received CPA at a dose of 5 mg /kg bw (21); and group IV (CPA+GL-treated group): rats received CPA and GL extract at the same doses of groups II and III. All treatment were orally administrated by gastric intubation for 21 consecutive days. Twenty four hours after the last administration and under the effect of light ether anesthesia, blood aliquots were collected from retro-orbital venous plexus and then, the animals were dispatched by cervical dislocation to harvest the liver tissue samples. Serum samples were separated by centrifugation of coagulated blood aliquots for 10 min at 1000 xg and kept frozen at -4 °C for later analysis. Serum biochemical analysis Determination of serum activity of hepatic damage biomarkers enzymes including AST, ALT (22) and GGT (23), in addition to serum Cyclophosphamide hepatotoxicity: the role of cytochrome C oxidase and the possible protective effect 17 level of total protein (TP) (24), albumin (25) and globulins (26) were evaluated using commercially available kits (Biosystems, Spain). Liver homogenate preparation and biochemical assays The liver of each animal was rinsed with 0.9% NaCl and deionized water. The tissues were perfused with phosphate buffer saline (PBS), pH 7.4 containing 0.1 mMethylenedia-minetetraacetate (EDTA) to discard any red blood cell or clots. Small piece of each liver (about 1 g) was homogenized in 10 ml ice-cold PBS using Glas-Col® tissue homogenizer and centrifuged at 10,000 x g for 30 min. Supernatant was separated, filtrated and stored at -80°C in Eppendorfs tubes for detection of catalase enzyme (CAT) activity depending on rate of hydrolysis of hydrogen peroxide (27), superoxide dismutase enzyme (SOD) activity which determined as the rate of inhibition of reaction between superoxide with a WST-1 dye to form a colored product (28) and reduced glutathione (GSH) content which based on reduction of 2-nitrobenzoic acid (DTNB) with glutathione (29) using locally available kits (Biodiagnostic, Egypt and Abnova, Taiwan). Protein content of tissue samples was detected spectrophotome-terically using Bradford reagent (Sigma-Al-drich, USA) (30). Detection of hepatic level of 4-HNE and activity of cytochrome C oxidase According to manufacturer instruction, about 100 mg of hepatic tissues was rinsed with PBS, homogenized in 1 ml of ice-cold PBS and stored overnight at -20°C. Two freeze-thaw cycles were done to break the cell membranes; the homogenates were centrifuged for 5 min at 5000 x g. The supernatant was removed and kept at 4° C for assessment of 4-hydroxynon-enallevel using specific ELISA kits (Mybio-source, USA) in addition to activity of cyto-chrome C oxidase enzyme using specific assay kits (Sigma-Aldrich, USA). Histopathological examination Fixed liver tissues (in 10 % formalin) were sliced into 5 pm thick sections after paraffin embedding and stained with hematoxylin and eosin (H&E) (31) using light microscope. Semiquantitative grading system for hepatic histopathological alterations Briefly, five random fields from each animal liver histopathological sections were examined (x100), the grade of the detected lesion severity was assessed depending on the percentage of affected area/entire section and recorded as follow: (-): absence of lesion, (+): for mild degree of lesions (5-25%), (++): for moderate lesions degree (26-50%) and (+++): for severe degree of lesions (>50%). Statistical analysis The statistical analysis was performed using One-way analysis of variance (ANOVA) to study the effect of different treatment on different studied parameters using analysis SAS system software (32). Results Serum biochemical results As shown in Table (1), serum activities of the hepatic enzymes (AST, ALT and GGT) were significantly elevated in CPA-treated animals (P < 0.05) when compared to the control group. Concerning serum proteinogram, none of the treated groups showed any significant changes from control group in serum total protein level (P < 0.05), but serum albumin level recorded a significant decrease (P < 0.05) in CPA and CPA+GL-treated group when compared to the control group. While, serum globulins level showed a significant increase (P < 0.05) in CPA-treated group which was accompanied by a significant decrease in A/ G ratio when compared to the control group. In comparison with CPA-treated group, co-administration of GL with CPA significantly decreased the evoked serum hepatic enzymes activity, decreased serum globulins level and significantly reversed A/G ratio toward normal control level, but, it did not affect the decreased serum albumin level significantly. Administration of GL extract only to the rats significantly decreased serum activity of different hepatic enzymes (AST, ALT and GGT), (P < 0.05) but, 18 D. R. Gad El-Karim, G. A. El-Amrawi it did not have any significant effect on serum proteinogram of these rats as compared to the control group. Hepatic antioxidant biomarkers In comparison with the control group, the liver content of GSH along with hepatic activities of CAT and SOD enzymes recorded a significant decrement (P < 0.05) in the CPA-treated group. Fortunately, these assessed hepatic antioxidant biomarkers were significantly increased in CPA+GL-treated animals as compared to CPA-treated rats. As compared to the control group, GL extract successfully and significantly increased (P < 0.05) these tested antioxidant biomarkers in hepatic tissues of rats when administrated alone (Table 2). Hepatic level of 4-hydroxynonenal and activity of cytochrome C oxidas enzyme In CPA-treated group, the hepatic level of 4-hydroxynonenal was increased significantly which was coupled with a significant decrease in hepatic activity of cytochrome C oxidase enzyme (P < 0.05) when compared to the control group. These changes were partially and significantly alleviated when GL extract was administrated with CPA. Compared to the control group, administration of GL extract alone to the rats significantly decreased (P < 0.05) liver content of 4-hydroxynonenal and enhanced the activity of cytochrome C oxidase enzyme (Table 2). Histopathological results Light microscopy examination of representative histological liver sections of various treated animals groups showed normal and well detailed hepatocytes which were arranged in regular cords around portal vein in area in both of control and GL extract administrated groups (Fig. 1). However, CPA-treated group exhibited severe hemorrhage (Fig. 2) with broadening of portal area with mononuclear inflammatory cell and fibroblast besides vacuolar degeneration of the majority of hepatocytes (Fig. 3), but CPA+GL extract-treated group showed small focus of necrotic hepatocytes with inflammatory cells infiltration (Fig. 4) with minimal infiltration of these inflammatory cells in portal area (Fig. 5). Table (3) illustrated the results of histo-pathological evaluation of the hepatic lesions of different groups using simple semiquantitative scoring system which affirmed the alleviating role of GL extract on the detected hepatic lesions. Discussion Despite of CPA effectiveness in treatment of a wide variety of neoplastic and autoimmune diseases (1), damage or injury of healthy tissues may be the major factor of limited usage of CPA as an anti-neoplastic treatment (33). CPA hepatotoxicity was reported in several studies Table 1: The effect of different treatments on serum hepatic biomarkers enzymes (AST, ALT and GGT) and serum proteinogram Control GL CPA CPA+GL AST (U/L) 35.20±2.91c 31.80±3.43d 79.80±3.85a 61.40±3.61b ALT(U/L) 25.80±3.12c 22.80±2.63d 69.80±3.81a 40.60±3.16b GGT(U/L) 22.00±2.35c 19.80±1.32d 51.60±2.66a 34.20±2.52b TP (g/dl) 6.52±0.19ab 6.50±0.19ab 6.88±0.13a 6.02±0.13b Albumin(g/dl) 4.18±0.14a 4.22±0.09a 2.98±0.11b 3.56±0.05b Globulins(g/dl) 2.34±0.14b 2.28±0.11b 3.90±0.14a 2.37±0.08b A/G ratio 1.82±0.12a 1.87±0.06a 0.76±0.05c 1.51±0.07b All the values are expressed as mean ±SD. Means within the same raw of different litters are significantly different at (P < 0.05). GL: Ganoderma lucidium extract, CPA: Cyclophosphamide, CPA+GL: Cyclophosphamide+ Ganoderma lucidium extract. Cyclophosphamide hepatotoxicity: the role of cytochrome C oxidase and the possible protective effect 19 Table 2: The effect of different treatments on hepatic antioxidants content (GSH, CAT and SOD), 4-HNE level and cytochrome C oxidase activity Control GL CPA CPA+GL GSH (mmol/mg protein) 79.00±3.18b 110.60±4.45a 37.20±2.80d 55.80±2.84c CAT (U/mg protein) 10.10±0.87b 17.00±0.79a 3.58±0.39d 7.40±0.43c SOD (U/mg protein) 9.72±0.82b 16.04±0.62a 4.02±0.18d 6.76±0.40c 4-HNE (pg/ mg protein) 13.90±1.49c 8.10±0.64d 48.40±3.08a 29.00±2.93b Cytochrome C oxidase (U/mg protein) 51.20±3.34b 66.40±3.04a 25.80±2.22d 35.40±2.80c All the values are expressed as mean ±SD. Means within the same raw of different litters are significantly different at (P < 0.05). Table 3: The score of detected hepatic lesions in male Wistar albino rats of different experimental groups Incidence1 and Severity2 of histopathological lesions CPA-treated rats CPA+ GL treated rats Absent Mild Moderate Severe Absent Mild Moderate Severe Scored liver lesions (-) (+) (++) (+++) (-) (+) (++) (+++) 1-Congestion of blood vessels 2 3 2 0 4 3 0 2-Hepatocytes vacuolar degeneration 0 0 4 3 2 2 2 1 3-Portal Inflammatory cells infiltration 0 1 4 2 1 3 3 0 4-Hepatocellular necrosis 0 2 3 2 2 2 3 0 5-Hyperplasia of biliary epithelium 0 3 2 2 0 5 2 0 6-Hemorrhage 0 4 2 1 7 0 0 0 1Number of rats with lesions per total examined (7 rats). 2Severity of lesions was graded by estimating the percentage area affected in the entire section. (34-37) as the liver is the primary site for microsomal activation of this drug (38). The prime cause may be reactive oxygen species (ROS) generation including hydrogen peroxide and superoxide anion during CPA metabolism which in role exhaust anti-oxidant mechanisms of liver (3, 39). This action may be exaggerated by CPA reactive metabolite (Acrolin) which can react with hepatic GSH causing its depletion (40) which collectively increase susceptibility of lipid peroxidation and hepatic tissues injury (41). The previous mechanisms of CPA-induced hepatotoxicity may explain the significant increase in serum hepatic enzymes activity (AST, ALT and GGT) of CPA-treated animals which in a concomitant with decreased activity of CAT and SOD enzymes in addition to depletion of GSH in hepatic tissues of the same group may represent a sufficient indicators for the oc- currence of oxidative injury of hepatocytes consistently with several previously recorded findings (20, 36, 42). Several studies have reported that GL extract is a good free radical scavenger (13, 43, 44), so treatment with GL rendered some protection against CPA-hepatotoxicity which was evidenced by a significant decrease in serum activity of AST, ALT and GGT which accompanied by a significant increase in hepatic tissues content of GSH and activity of antioxidant enzymes (CAT and SOD) in CPA+GL-treated group when compared to the CPA-treated group. Hepatoprotective effect of GL against oxidative damage was previously detected and proved (18). Albumin is one of plasma proteins which is synthesized by liver so, determination of its levels is considered one of best indicators to evaluate hepatic function efficacy (45) and this may demonstrate its decrease in both of CPA and CPA+GL-treated groups which may be attributed to hepatotoxic 20 D. R. Gad El-Karim, G. A. El-Amrawi Figure 1: (1) Control group, a section in the liver of a rat showing normal hepatocytes (H&E, X200). (2) CPA-treated group, a section in the liver of a rat showing focal hemorrhage (stars, H&E, X200). (3) CPA-treated group, a section in the liver of a rat showing thickening of portal area with mononuclear cells infiltrate and fibrosis (arrow) and hyperplasia of biliary epithelium (arrow heads) besides hydropic degeneration or vacuolation of most of hepatocytes (H&E, X200). (4) CPA+GL-treated group, a section in the liver of a rat showing congested blood vessel (A) and necrosis hepatocytes with inflammatory cells infiltrate (arrow, H&E, X400), (5) CPA+GL-treated group, a section in the liver of a rat showing mononuclear inflammatory cells in the portal area (arrow, H&E, X200) Cyclophosphamide hepatotoxicity: the role of cytochrome C oxidase and the possible protective effect 21 impact of CPA, while hyperglobulinemia of hepatocellular disorders, appearing as an inflammatory response to liver injury due to the probable increase in production alpha and beta globulins by liver and/or immune- globulins by plasma cells (45,46). The previous explanation may clarify the reasons for hypoalbuminemia and hyperglobulinemia which significantly decreased A/G ratio of CPA-treated animals which could be attributed to CPA hepatotoxic effects. The treatment with GL significantly ameliorated these changes in proteinogram and clearly lessens the decrease in A/G ratio caused by CPA treatment, thereby GL may exhibit a considerable suppressive effect on hepatic inflammatory reactions caused by CPA, this ameliorative effect of GL on hepatic inflammatory state was concluded formerly (18). 4-hydroxynonenal is the major lipid perox-idation product which is produced in the tissues as a result of cellular exposure to oxidative stress (47). It has a suppressive effect on cytochrome C oxidase enzyme activity (48,49) which is the key enzyme of aerobic cell respiration that contributes to the formation of ATP (essential energy molecules for cell livability and viability), so deficiency of this enzyme represent a risk for the living cells (50). The proved oxidative stress caused by CPA administration may demonstrate the increment in hepatic tissues level of 4-hydroxynonenalwhich in turn may be the cause for the decrement in these tissues activity of cytochrome C oxidase enzyme. Another explanation for inhibition of hepatic cytochrome C oxidase enzyme in CPA-treated rats may be the great susceptibility of its mRNA as well as protein to oxidative damage (51). The later changes were alleviated by the dosage of powerful antioxidant GL extract to the rats of the CPA+GL group. Hence, the antioxidant activity of GL was reflected on the effects of its supplementation as a sole treatment to the rats of GL-treated group, as it successfully boosted the hepatic antioxidant bi-omarkers (GSH, CAT, SOD), which in role, decreased hepatocytes destruction and leakage of hepatic enzymes to the blood and finally decreased hepatic level of 4-HNE and enhanced the activity of cytochrome C oxidase enzyme as compared to control group. In this study, histopathological examination of liver, affirmed that CPA can cause damage to the liver which was evidenced by presence of several different hepatic lesions, which might be caused by cellular damaging potential of CPA metabolites in relation to generation of ROS in agreement with the current studies on CPA-induced hepatotoxicity (38,42,52). These histopathological changes correlated strongly with the altered serum enzymes activity and hepatic antioxidants depletion of CPA-treated group. Semiquantiative evaluation of hepatic lesion confirmed the biochemical results which illuminated that the treatment with GL extract effectively ameliorated the abnormal hepatic histopathological lesions caused by CPA, and this may be attributed to its powerful antioxidant ability in prevention of ROS membrane damage (12,16,18). Conclusion Our results reported the role of 4-hy-droxynonenal production and inhibition of cy-tochrome C oxidase enzyme in CPA-induced hepatic damage. In addition, it clarified and supported that the treatment with GL extract can reduce oxidative hepato-cyto damage caused by treatment with cyclophosphamide due to its powerful antioxidant effect, so it can be used during treatment with this anti-cancer drug to minimize its deleterious effects on the liver. Conflict of interest The authors declare that she has no conflict of interest. References 1. Dollery C. Cyclophosphamide. In: Therapeutic Drugs. Edinburgh: Churchill Livingstone1999; 349-53. 2. Paolo AD, Danesi R, Tacca MD. Pharmacogenetics of neoplastic disease: New Trends. Pharmacol Res 2004; 49: 331-42. 3. Stankiewicz A, Skrzydlewska E , Makiela M. Effects of amifostine on liver oxidative stress caused by cyclophosphamide administration to rats. Drug Metabol Drug Interact 2002; 19: 67-82. 22 D. R. Gad El-Karim, G. A. El-Amrawi 4. Haque R, Bin-Hafeez B, Parvez S, Pandey S. Aqueous extract of walnut (Juglans regia L.) protects mice against cyclophosphmide induced biochemical toxicity. Human Exp Toxicol 2003; 22:473-80. 5. Bhatia K, Ahmad F, Rashid H, Raisuddin S. Protective effect of S-allylcysteine against cyclo-phosphamide-induced bladder hemorrhagic cystitis in mice. Food Chem Toxicol 2008; 46(11):3368-74. 6. Chakraborty P, Hossain U, Murmu N, Das JK, Pal S, Bhattacharya S. Modulation of cyclo-phosphamide-induced cellular toxicity by diphe-nylmethyl selenocyanate in vivo, an enzymatic study. J Cancer Mol 2009; 4(6): 183-9. 7. Mousa NK, Muhsin EA, Sabbar SS, Ishrak A. Ahmed IA. Liver histopathological of purification cinnamic acid activity against endoxan in mice. J Gen Enviro Reso Cons 2014; 2(1): 22-9. 8. Weijl NI, Cleton FJ, Osanto S. Free radicals and antioxidants in chemotherapy-induced toxicity. Cancer Treat Rev 1997; 23 (4): 209-40. 9. Wasser SP. Reishi or Lingzhi (Ganoderma lu-cidium). Encyclopedia of dietary supplements 2005; 1: 603-22. 10. Watchel-Galore S, Yuen J, Buswell JA. Ganoderma Lucidium (Lingzhi or Reishi): a Medicinal Mushroom. In: Benzie IFF, Watchel-Galore S, editors. Herbal Medicine: Bimolecular and Clinical Aspects. 2nd edition. Boca Raton (FL): CRC Press/Taylor &Francis; 2011. Chapter 9. 11. Lin ZB, Zhang H. Anti-tumor and immuno-regulatory activities of Ganoderma lucidum and its possible mechanisms. Acta Pharmacol Sin; 2004, 25 (11): 1387-95. 12. Lee JM, Kwon H, Jeong H, Lee JW, Lee SY, Baek SJ, Surh YJ. Inhibition of Lipid Peroxidation and Oxidative DNA Damage by Ganoderma lu-cidum. Phytother Res 2001; 15:245-9. 13. Zhu MQ, Chang LK, Wong FS, Li RC. Triterpene antioxidants from Ganoderma lucidum. Phytotherapy Res1999; 13(6): 529-31. 14. Kao CH, Jesuthasan AC, Bishop KS, Glu-cina MP, Ferguson LR. Anti-cancer activities of Ganoderma lucidum: active ingredients and pathways. Fun Foods Health Dis 2013; 3(2):48-65. 15. Lin Z. Cellular and Molecular Mechanisms of Immuno-modulation by Ganoderma lucidum. J Pharmacol Sci 2005; 99: 144-53. 16. Kim KC, Kim IG. Ganoderma lucidum extract protects DNA from strand breakage caused by hydroxyl radical and UV irradiation. Int J Mol Med 1999; 4(3): 273-7. 17. Zhang GL, Wang YH, Ni W, Teng HL, Lin ZB, Lin ZB. Hepatoprotective role of Ganoderma Lucidium polysaccharide against BCG induced immune liver injury in mice. World J Gastroenterol 2002; 8 (4): 728-33. 18. Lin WC, Lin WL. Ameliorative effect of Ganoderma lucidum on carbon tetrachloride-in-duced liver fibrosis in rats. World J Gastroenterol 2006; 12(2):265-70. 19. Wang X, Zhao X, Li D, Lou YQ, Lin ZB, Zhang GL. Effects of Ganoderma lucidum Polysaccharide on CYP2E1, CYP1A2 and CYP3A Activities in BCG-Immune Hepatic Injury in Rats. Biol. Pharm. Bull 2007; 30 (9): 1702-6. 20. Shieh YH, Liu CF, Huang YK, Yang JY, Wu L, Lin CH , Lin SC. Evaluation of the Hepatic and Renal-protective Effects of Ganoderma lu-cidum in Mice. Am Chin Med 2001; 29(3): 501-7. 21. Ghosh S, Ghosh D, Chattopadhyay S, Dep-nath J. Effect of Ascorbic Acid Supplementation on Liver and Kidney Toxicity in Cyclophosphamide Treated Female Albino Rats. The J toxicol Sci 1999; 24(3): 141-4. 22. Reitman S, Frankel S. A coloremetric method for determination of oxaloacetic transaminase and serum glutamic pyruvic transaminase. Am. J. Clin Pathol 1957; 28: 56-63. 23. Szasz ZG. A kinetic photometric method for serum gamma glutamyltranspeptidase. Clin. Chem 1969; 15:124-36. 24. Doumas BT, Bayso DD, Caster RJ, Leters T, Schaffer R. Determination of serum total protein. Clin. Chem 1981; 27: 1642. 25. Drupt F. Dosage del albumin serque part vert de bromocresol. Pharma. Biol 1974; 111: 77. 26. Watson D. Albumin and "total globulin" fraction of blood. In: Advances in Clinical Chemistry. Academic Press Inc. 1965; 8: 238-49. 27. Aebi H. Catalase in vitro. Methods Enzymol 1984;105:121-6. 28. Ukeda H, Maeda S, Ishii T, Sawamura M. Spectrophotometry assay for superoxide dismutase based on tetrazolium salt 3'{1-[(phenylamino)-car-bonyl]-3,4- tetrazolium}-bis(4-methoxy-6-nitro) benzenesulfonic acid hydrate reduction by xan-thine-xanthine oxidase. Anal Biochem1997; 251: 206-9. 29. Beutler E, Duron O, Kelly MB. Reduced glutathione determination. J Lab Clin Med 1963; 61:882. 30. Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding, Anal Biochem1976; 72 (1-2): 248-54. Cyclophosphamide hepatotoxicity: the role of cytochrome C oxidase and the possible protective effect 23 31. Bancroft JD, Stevens A. Theory and Practice of Histological Technique. 4th ed. Churchil liv-ingestone; 1996. 32. SAS. Statistical analysis system. SAS User's Guide, SAS incorp Inst 2004. 33. Patel JM. Stimulation of cyclophosphamide-induced pulmonary microsomal lipid peroxidation by oxygen. Toxicology 1987; 45: 79-91. 34. McDonald GB, Slattery GH, Bouvier ME, Ren S, Batchelder AL et al. Cyclophosphamide metabolism, liver toxicity, and mortality following hematopoietic stem cell transplantation. Blood J 2003;101: 2043-8. 35. Sheeja K, Kuttan. Ameliorating Effects of Andrographis Paniculata Extract Against Cyclo-phosphamide-Induced Toxicity in Mice. Asia Pac Can Prev 2006; 7: 609-14. 36. Alenzi FQ, El-Bolkiny YE, Salem ML. Protective effects of Nigella sativa oil and thymoqui-none against toxicity induced by the anticancer drug cyclophosphamide. BRIT J BIOMED SCI 2010; 67 (1):20-8. 37. Jain R, Jain SK. Effect of Buchanania lanzan Spreng bark extract on cyclophosphamide induced genotoxicity and oxidative stress in mice. Asia Paci J Trop Med 2012; 187-91. 38. Shokrzadeh M, Ahmadi A, Naghshvar F, Chabra A, Jafarinejhad M. Prophylactic Efficacy of Melatonin on Cyclophosphamide-Induced Liver Toxicity in Mice. BioMed Res Int 2014; Article ID 470425. 39. Bhattacharya A , Lawence RA, Krishnan A, Zaman K, Sun D, Fernandes G. Effect of dietary n-3 and n-6 oils with and without food restriction on activity of antioxidant enzymes and lipid peroxida-tion in livers of cyclophosphamide treated autoimmune-prone NZB/W female mice . J Am Coll Nutr 2003; 22: 388-99. 40. Kehrer JP, Biswal SS. The molecular effects of acrolein. Toxicol Sci 2000; 57:6-15. 41. Werts ED, Gould MN. Relationships between cellular superoxide dismutase and susceptibility to chemically induced cancer in the rat mammary gland. Carcinogen1986; 7:1197-201. 42. Shokrzadeh M, Chabra A, Ahmadi A , Naghshvar F, Habibi E , Salehi F , Assadpour S. Hepatoprotective Eff ects of Zataria Multifl ora Ethanolic Extract on Liver Toxicity Induced by Cyclophosphamide in Mice. Drug Res 2014; 64: 1-7. 43. Kim DH, Shim SB, Kim NJ, Jang LS. Beta Glucoronidase Inhibitory Activity and Hepatoprotective Effect of Ganoderma Lucidium. Biol Pharm Bull 1999; 22(2): 162-4. 44. Zhong D, Wang H, Liu M, Li X, Huang M, Zhou H et al. Ganoderma lucidum polysaccharide peptide prevents renal ischemia reperfusion injury via counteracting oxidative stress. Sci Rep 2015; 5:16910. 45. Latimer KS, Mahaffey EA, Prasse KW. Plasma Protein. In: Veterinary Laboratory Medicine, Clinical Pathology. 4th Ed., Iwoa State Press; 2003.p.162-71. 46. Vandenberghe J. Hepatotoxicology: Mechanisms of liver toxicity and methodological aspects. In: Niesink RJM, De Vries J, Hollinger MA. Toxicology: Principle and Applications. New York: CRC Press, 1996; 703-23. 47. Chen JJ, Schenker S, Henderson GI. 4-Hy-droxynonenal Levels Are Enhanced in Fetal Liver Mitochondria by In Utero Ethanol Exposure. HEPATOL 1997; 25(1):142-7. 48. Chen J, Robinson NC, Schenker S, Frosto T, George I. Henderson GI. Formation of 4-Hy-droxynonenal Adducts With Cytochrome c Oxidase in Rats Following Short-Term Ethanol Intake. HEPATOL 1999; 29 (6): 1792-8. 49. Srinivasan S, Avadhani NG. Cytochrome c Oxidase dysfunction in oxidative stress. Free Rad Biol Med 2012; 53(6): 1252-63. 50. Wikstrom M. Cytochrome C Oxidase. eLS. 2001. https://doi.org/10.1002/9780470 015902.a00 00649.pub2. 51. Sohal RS, Toroser D, Bre'ge're C, Mockett RJ and Orr WC. Age-related decrease in expression of mitochondrial DNA encoded subunits of cyto-chrome c oxidase in Drosophila melanogaster. Mech Ageing Dev 2008; 129: 558-61. 52. Senthilkumar S ,Devaki T, Manohar BM, Babu MS. Effect of squalene on cyclophospha-mide-induced toxicity . Clin Chim Acta 2006; 364: 335-42. Slov Vet Res 2019; 56 (Suppl 22): 25-38 DOI 10.26873/SVR-741-2019 Original Research Article THE MODULATORY EFFECT OF DIETARY BETAINE ON INTESTINAL ABSORPTIVE CAPACITY, LIPOGENESIS AND EXPRESSION OF LIPID METABOLISM- AND GROWTH-RELATED GENES IN NILE TILAPIA FED ON SOYBEAN MEAL-BASED DIET Taha Ismail1, El-Desoky Nassef1, El-Sayed Hegazi1, Abdel-Nasser Bakr1, Eman M. Moustafa2*, Walied Abdo3, Zizy I. Elbialy4 department of Nutrition and Clinical Nutrition, Faculty of Veterinary Medicine, Kafrelsheikh University, Egypt. 2Department of Fish Diseases and Management, Faculty of Veterinary Medicine, Kafrelsheikh University. 3Department of Pathology, Faculty of Veterinary Medicine, Kafrelsheikh University. 4Department of Fish Processing and Biotechnology, Faculty of Aquatic and Fisheries Sciences, Kafrelsheikh University Corresponding author, E-mail: emantarek2002@yahoo.com Abstract: The current experiment was randomly designed in a 2^2 factorial design. Two dietary protein sources were utilized; fish meal and soybean meal; with betaine incorporation to both of them. This study aimed to determine the impact of incorporation of betaine into soybean meal-based (SBM) diets through its effects on growth performance, intestinal healthiness and expression of some lipid metabolism- and growth-related genes. Fish (19.84±0.20 g) were stocked in 12 aquaria and allotted into triplicate four groups (10 fish per aquarium). Four test diets were formulated to contain fish meal (FM) as a positive control, FM with betaine (FM + Betaine), SBM diet and SBM with betaine (SBM + Betaine), respectively. After 60 days, dietary betaine improved the growth performance of fish fed FM or SBM as revealed by higher final body weight, body weight gain and average daily gain and lower feed conversion ratio. Monitoring the whole-body composition revealed that addition of betaine to diet relatively augmented flesh protein content and reduced its fat content. In addition, betaine incorporation in diets significantly (P<0.05) increased the intestinal villi length especially in the jujenal portion as well as the numbers goblet cells. Furthermore, betaine had a downregulating effect on expression of lipid metabolism-related genes, fatty acid synthetase (Fas) and lipoprotein lipase (Lpl) and up-regulating effect on insulin growth like factor-1 (Igf-1) gene in liver. It could be concluded that dietary supplementation of betaine incorporation to soybean-based diets enable nutritionists to substitute FM in fish diet. Also, betaine could improve growth performance, carcass quality (through increasing protein and decreasing lipid in fish) and enhance intestinal functions capability. Key words: Betaine; growth performance; insulin like growth hormone factor; lipid metabolism related genes; Nile tilapia; soybean meal Introduction Fish serve as a vital source of human dietary protein worldwide, particularly for African Received: January 2019 Accepted for publication: February 2019 26 T Ismail, D Nassef, ES Hegazi, AN Bakr, EM Moustafa, W Abdo, Z Elbialy people (1-3). Natural fisheries are progressively exhausted and the aquaculture end up basic means to compensate the need in this fundamental wellspring of dietary protein (4). Along these lines, to repay the insufficiency in animal protein resulted from the expanded interest for fish as human sustenance, fish farming is quickly extending everywhere throughout the world (5). Aquaculture contributes the greater part of the overall fish production all over the world. In Egypt, the aquaculture industry gives about 77% of the all national fish production providing arround one million ton annually (68). Progressive enhancement of this industry must be correlated with corresponding improvement of fish feed quality representing 70 % of the total culturing costs (7, 9). Nile tilapia (Oreochromis niloticus) represents the most prevalent freshwater fishes in Egypt, widely cultured because of its high growth rate, cheap price, palatability, ability to withstand stress and diseases, ability to spawn easily and the minimal requirements in regard to management and energy inputs (10). Heightening of aquaculture production frameworks exposes fish to various ecological stressors like; poor water quality, over-crowdness, improper handling and transport which may contrarily influence their growth and health, as well as limiting the outcome of aquaculture systems (11). Nutrition assumes as a basic job in the growth, advancement and prosperity upkeep of fish (12). Years ago, fish meal was used as the basic protein source in tilapia diets (13). Due to the extending cost and unreliable supply of this ingredient, numerous attempts have been admitted to upgrade the growth performance and decrease the production costs of cultured tilapia through replacement of fish meal with another protein sources either from terrestrial animals or plants. This was done by utilization of probi-otics or prebiotics as safe dietary supplements which have neither residue in the cultured fish nor harmful impacts for fish consumers (1415). However, increasing amount of the fish meal alternatives in the diet may result in decreasing the diet palatability to fish; decreasing feed intake and subsequently decreasing fish growth rate (16). Betaine (trimethylglycine) is synthetized from choline oxidation (17), present in most organisms and is fundamentally extracted from sugar beet processing (18). Interestingly, betaine was recorded to improve growth performance, fish health status, feed digestibility as well as flesh quality and immune status of fish species (19, 20). It was used as a feed attractant and appetizer through stimulating the olfactory bulb, increasing feed intake and minimizing the feed wastage and water pollution (21). In addition, betaine can play a role in the osmoregulation through increasing both water retention of perturbed cells and cytoplasmic osmotic pressure via raising the temperature and ionic tolerance of critical enzymes and cellular membranes that results in protection of intracellular enzymes against osmotically or temperature induced inactivation (22, 23). Moreover, betaine acts as a methyl donor so it plays a great vital role in energy metabolism and synthesis of me-thionine, carnitine, phosphatidyl choline, creatine and protein (24, 25). Betaine plays a fundamental role in lipid metabolism suggesting that it is a lipotropic factor having a hepatoprotec-tive function (26-28). Dietary betaine additionally has the ability to reduce body lipid deposition (29, 30). To the best of our knowledge, there is little published data on the supplemental effects of betaine on growth performance, intestinal absorptive capacity, intestinal morphometry, lipid metabolism and growth-related genes in Nile tilapia (O. niloticus). Thus, the current study was conducted to role out the effect of incorporation of betaine to soybean-based diets and to assess its effects on growth performance, intestinal absorptive capacity, and expression of lipid metabolism- and growth-related genes in Nile ti-lapia. Material and methods The feeding trials were carried out at the Nutrition and Clinical Nutrition Department, Faculty of Veterinary Medicine, Kafrelsheikh University, Egypt, for 10 weeks during 2018. Animal ethics committee, Faculty of Veterinary Betaine improves growth performance in Nile tilapia fed on soybean meal-based diets 27 Medicine, Kafrelsheikh University, Egypt; affirmed and approved the protocol and conducting of the study. Preparation of the experimental diets Two basal diets were formulated to be isoni-trogenous and isocaloric in terms of crude protein (30%) and digestible energy (3.05 kcal g-1). Each diet was based on either fish meal (FM) or soybean meal (SBM) as the main source of dietary protein. Then each diet was supplemented with 2 g betaine per kg diet (FM, FM+betaine, SBM and SBM+betaine) (Table 1) as suggested by Luo et al., (31). Betaine was purchased from Biochem Ltd. Company, Egypt and incorporated to the diets after firstly mixed with dical-cium phosphate, limestone, salts and premix until the mixture became homogenous and finally added to the other finely ground concentrate with stirring. Proximate chemical analysis of diets was performed according to Association of Official Analytical Chemists methods as shown in (Table 2). Experimental design The experiment was randomly designed in a 2^2 factorial design. Two dietary protein sources were used with betaine incorporation to both of them. Thus, a total of four treatments run in triplicate were allotted into12 glass aquaria (60x40x30 cm) provided with a constant aeration. One hundred and twenty healthy Nile tilapia (O. niloticus) (initial body weight, 19.84 ± 0.20 g) were obtained from a private farm in Kafrelsheikh governorate, Egypt. Prior to the experiment, fish were acclimatized to basal diet for 2 weeks. At the beginning of the experiment, fish (n= 10) were stocked into each glass aquaria, and each diet was assigned to triplicate aquaria. Fish were fed till apparent satiation twice daily (at 9 a.m. and 5 p.m.) for 60 days and weighted every ten days in order to determine the growth and healthy status. Water parameters were monitored twice a week using water analysis device (Lamotte device, USA). Temperature ranged from (24-26°C), 6 mg L-1 dissolved oxygen, 6.5-7 pH, ammonia adjusted to the normal permissible limits (< 0.1 mg total ammonia) and 14 h of light and 10 h of dark photoperiod with 40 % daily water change. Proximate chemical analysis At the end of the feeding trial, fish were starved for 24 h, harvested and weighed individually then euthanized through over anesthetization using MS222 as a local anesthetic drug. 4 fish from each group were stored at (-20 °C) for proximate chemical analysis of fish tissue. Moisture content was carried out by drying the samples in a hot air oven at 60 °C for 48 h. Ash content was determined by samples incineration at 550 °C for 6 hrs. Crude protein (Nx6.25) was assessed using the Kjeldahl method after acid digestion. Fat and fiber were determined using ANKOM Technology Method. Morphometrical examination Snippets of the different intestinal portions were sampled from another four fish and immediately fixed in a Bouin solution for histological observations. Fixed samples were subjected to dehydration through ascending grades of alcohol solution series (70 to 100%). After dehydration process, tissues were deparafinized in xylene, embedded in paraffin, sectioned (5 pm) and stained with hematoxylin and eosin (H&E). The heights of intestinal villi in all parts were measured using ImageJ version 1.36 (National Institutes of Health). Mean villus height for each section was based on an average of 10 villus heights/section. Goblet cells No. was determined according to the equation: goblet cell number = goblet cell number in 8 fields of x 200 magnification power / total area examined per each fish, and was expressed as (No. of goblet cells/mm2) Quantitative real-time PCR (qRT-PCR) Liver samples were collected, quickly frozen in liquid nitrogen then kept at -80°C for gene expression analysis, then total RNA was extracted from 50 mg of liver tissue (n=3/group) using TRIzol reagent (easy-RED™, iNtRON Biotechnology). RNA quality was verified using formol gel electrophoresis examination. The cDNA was synthesized from each RNA sample (2 |g) using SensiFAST™ cDNA syn- 28 T Ismail, D Nassef, ES Hegazi, AN Bakr, EM Moustafa, W Abdo, Z Elbialy thesis kit (Bioline, United Kingdom). As presented in Table (3), gene specific primers of fatty acid synthase (Fas), lipoprotein lipase (Lpl), and insulin like growth factor-1 (Igf-1), were used to determine the expression in liver tissue and P actin was selected as a reference gene for normalization of gene expression. Real time PCR (qPCR) was carried out using SYBR® Premix and Stratagene MX3000 qPCR system. The reaction mix consisted of a 2 pl cDNA sample, 12.5 pl SYBR® Premix Ex TaqTM, 0.25 pl PCR forward/reverse primers (10 pM), and 10 pl nuclease-free water. The thermo-cycling conditions for the reaction were as follows: 95 °C for 5 min, followed by 40 cycles consisting of 95 °C for 10 s, annealing for 15 s at primer specific annealing temperature. Dissociation curve analyses were performed beginning at 65°C and ending at 95°C, with incremental increases of 0.5°C every 5s to validate the specificity of the PCR products. All samples were run in duplicate. Relative gene expression levels were evaluated using 2-AAct as previously described (32). Calculations and statistical analysis The following variables were calculated: Total body weight gain (TWG)(g/fish) =fi-nal body weight (W2) - initial body weight (W1). Average daily gain (ADG) = (W2 - W1) / t (experiment duration = 60 days) Feed intake (FI, g fish-1 60 days-1) = (offered feed - feed refusal recovered and dried)/number of fish Feed conversion ratio (FCR) = feed intake (g)/weight gain (g). The results were presented as mean ± standard error (SE) of three replicates. All results were analyzed by one-way analysis of variance (ANOVA). When ANOVA identified differences between groups, multiple comparisons among means were made with Duncan's new multiple range test. Differences were considered significant at (P<0.05). All statistical analyses were performed using the SPSS 24. Results Growth performance As shown in table (4), addition of betaine to diets based on soybean meal protein significantly (P<0.05) increased the feed intake. Fish fed on FM+betaine showed the highest FBW and BWG followed by that fed on SBM+beta-ine. Also, supplementation of betaine significantly (P<0.05) increased FBW and BWG in fish fed SBM+ betaine as compared to fish fed SBM only. FCR was markedly decreased in fish fed on FM+ betaine or SBM+ betaine when compared to the other groups. Also, FI significantly differed according to the source of dietary protein either from FM or SBM-based diet. Whole body chemical composition Analysis of whole-body composition presented in table (5) showed that crude protein content was significantly (P<0.05) increased by addition of betaine either with FM or SBM diets. FM+ betaine showed relatively higher CP content than other experimental treatments. Fat content was significantly (P<0.05) decreased by addition of betaine to fish diets. There were no significant differences in moisture, ash, soluble carbohydrate and ADF content among all dietary treatments. Assessment of intestinal absorptive capacity Duodenal, jujenal and ileal villi morphometry were showed in (Table 6) and illustrated in (Fig. 1). Betaine significantly increased the intestinal absorptive capacity of fish supplemented with FM diet (P<0.05), while, SBM markedly decreased intestinal villi parameters in comparison with fish supplemented with FM diet. Interestingly, supplementation of betaine improved the jujenal villi length in FM and SBM groups. Also, the number of goblet cells per area along the intestinal mucosal lining was increased in fish fed FM diets than fish fed with SBM as a protein source. Incorporation of betaine with SBM demonstrated marked restore of goblet cells like FM diet. Betaine improves growth performance in Nile tilapia fed on soybean meal-based diets 29 Expression of Fas, Lpl and Igf-I genes in liver tissue Relative mRNA expression of Fas, Lpl, Igf-1 genes in liver of Nile tilapia fed on different protein sources with and without addition of betaine was shown in Figure 3. Hepatic Fas and Lpl mRNA levels showed upregulation in fish fed on the diets deprived from betaine (P<0.05). Interestingly, addition of dietary betaine to either fish meal or soybean-based diets markedly decreased Fas and Lpl mRNA levels (P<0.05). Fish fed on FM+ betaine had significantly (P<0.05) higher hepatic Fas mRNA level as compared to fish fed SBM+ betaine. Igf-I mRNA level in the liver of Nile tilapia fed on dietary betaine with SBM was significantly (P<0.05) upregulated as compared to fish fed SBM only. Fish fed diet with FM+ betaine showed significantly (P<0.05) higher hepatic Igf-1 expression than other groups. Table 1 : Diets formulation and experimental design Item (%) FM FM+ betaine SBM SBM+ betaine FM 35.5 35.5 — — SBM — — 56.5 56.5 Wheat bran 3.8 3.7 6.7 6.6 Rice polishing 16 16 — — Corn grains 25 25 25 25 Wheat middling 18.9 18.8 4.96 4.86 Soya oil — — 1.2 1.2 Fish oil — — 1.8 1.8 Nacl — — 0.33 0.33 Minerals1 0.05 0.05 0.05 0.05 Vitamins2 0.05 0.05 0.05 0.05 Vitamin C 0.1 0.1 0.1 0.1 Dicalcium phosphate — — 2.4 2.4 Limestone — — — — Lysine hydrochloride — — — — DL methionine — — 0.31 0.31 Antimycotoxin 0.1 0.1 0.1 0.1 CMC 0.5 0.5 0.5 0.5 Betaine — 0.2 — 0.2 1Minerals (Egypt pharma company) each 5 kgs of this product provide the following: 5000 mg Copper; 5 mg..Cobalt; 5000 mg..Iodine; 100 mg..Selenium; 30000 mg Iron; 40000 mg magnesium; 10000 mg manganese; 150000 mg Zinc; Calcium carbonate ad to 1000 gm. 2Vitamins (Egypt pharma company) each 5 kilograms of this product provide the following: 5000000 IU Vit.A; 1000000 IU Vit. D3; 50000 mg Vit. E; 10000 mg K3; 20000 mg B1; 20000 mg B2; 20000 mg B6; 20 mg B12; 100000 mg Niacin; 5000 mg Folic; 100 mg Biotin; 50000 mg Pantothenic Acid; Calcium carbonate ad to 1000 gm. 30 T Ismail, D Nassef, ES Hegazi, AN Bakr, EM Moustafa, W Abdo, Z Elbialy Table 2: Proximate chemical analysis of the basal diets FM SBM DE (Kcal/kg) 3045 3059 CP % 29.9 29.92 Fat % 6.25 3.6 ADF % 6.57 7.17 Ca % 1.9 0.7 Total P % 1.35 1.02 Available p % 0.71 0.46 Na % 0.15 0.15 Lysine % Methionine % 1.98 0.75 1.67 0.73 Threonine % 1.18 0.99 Tryptophane % 0.31 0.24 ADF: Acid detergent fiber DE: digestible energy SBM: Soybean meal CMC: Carboxy-methyl cellulose FM: Fish meal. Table 3: Primer sequences used for qPCR analysis Gene Primer sequence (5'-3') Annealing References Temperature Fas F: TGAAACTGAAGCCTTGTGTGCC 60°c Tian et al., (33) R: TCCCTGTGAGCGGAGGTGATTA Lpl F: TGCTAATGTGATTGTGGTGGAC 59°c Tian et al., (33) R: GCTGATTTTGTGGTTGGTAAGG Igf-I F: TCCTGTAGCCACACCCTCTC 60°c Costa et al., (34) R: ACAGCTTTGGAAGCAGCACT ß-actin F: CCACACAGTGCCCATCTACGA 63°c Qiang et al., (35) R: CCACGCTCTGTCAGGATCTTCA Fas: fatty acid synethase Lpl: lipoprotein lipase Igf-1: insulin like growth factor-1. Betaine improves growth performance in Nile tilapia fed on soybean meal-based diets 31 Table 4: Effect of dietary betaine on growth parameters of Nile tilapia fed on FM and soybean. IW (g) FBW (g) BWG (g) ADG (g) FI (g) FCR FM 19.95±0.21 50.07± 0.42b 30.11±0.27b 0.50±0.00b 48.68±0.52a 1.62± 0.03b FM+ betaine 19.62±0.13 54.61± 0.33a 34.99± 0.20a 0.58±0.00a 50.24±0.25a 1.44± 0.00c SBM 19.90±0.19 43.17± 0.54c 23.27±0.73c 0.39±0.01c 42.67±0.77b 1.84± 0.03a SBM+ betaine 20.00±0.08 51.16±0.30b 31.16±0.34b 0.52±0.01b 49.29±0.55a 1.59± 0.01b IW: Initial weight FBW: Final body weight BWG: Body weight gain ADG: Average daily gain FI: Feed Intake FCR: Feed Conversion Ratio. **Values are means ± standard error (n = 10). Means with different small letter in the same column differ significantly (P< 0.05). Table 5: Effect of dietary betaine on whole-body composition of Nile tilapia fed on FM and soybean_ Soluble carbo- Moisture% CP% Fat% Ash% ADF% hydrates% FM 57.83±0.30 18.78±0.17cb 15.80±0.15a 2.76±0.17 3.49±0.54 1.35 ± 0.17 FM+ betaine 57.92±0.29 19.85±0.15a 14.82±0.36ab 2.79±0.3 3.37±0.41 1.25 ± 0.10 SBM 58.2±0.31 18.52±0.4b 15.33±0.21b 2.79±0.31 3.61±1.07 1.48 ± 0.10 SBM+ betaine 57.62±0.28 19.35±0.34ab 14.59±0.18b 2.87±0.43 4.53±0.71 1.04 ± 0.08 CP: crude protein ADF: acid detergent fiber. **Values are means ± standard error (n=4). Means with different small letter in the same column differ significantly (P< 0.05). Table 6: Effect of dietary betaine on of intestinal villi length of Nile tilapia fed on plant and animal protein sources Treatments Duodenum Villi Length (pm) Jujenum Ileum FM 290.69±18.16 530.35±30.93bc 355.98±30.05a FM+ betaine 351.20±32.03 684.92±26.96a 414.96±37.16a SBM 278.73±23.06 465.48±24.04c 220.44±19.76b SBM+ betaine 290.18±31.43 605.62±43.47ab 255.62±30.66b Values are means±standard error (n = 4). Means with different small letter in the same column differ significantly (P< 0.05). 32 T Ismail, D Nassef, ES Hegazi, AN Bakr, EM Moustafa, W Abdo, Z Elbialy Duodenum Jujenum Ilium O" n> on 03 CD cr fD Figure 1: Haematoxylin & eosin stained photomicrograph, assessed the effect of dietary betaine on the duodenal, jujenal and ileal villi length (pm) of Nile tilapia fed on soybean meal and fish meal for 60 days Figure 2: Effect of dietary betaine on intestinal goblet cells number of Nile tilapia fed on soybean meal and fish meal. Values are means±standard error (n = 4). Means with different small letters on columns for each organ differ significantly (P< 0.05) Betaine improves growth performance in Nile tilapia fed on soybean meal-based diets 33 Figure 3: Effect of dietary betaine and different protein sources on mRNA level in liver (a) Fas: fatty acid synthetase; (b) Lpl: lipoprotein lipase; (c) Igf-1: insulin like growth factor-1 in Nile tilapia fed for 60 days. Values are expressed as the mean ± SE (n = 3). Data with different letters on columns statistically differ (P< 0.05) 34 T Ismail, D Nassef, ES Hegazi, AN Bakr, EM Moustafa, W Abdo, Z Elbialy Discussion Growth is the fundamental key of energy loss and/or gains in the aquatic environment; which can be measured mainly by determination of the weight gain. Because of the expanding cost and untrustworthy supply of fish meal, various trials have been applied to redesign the growth performance and decline the production expenses of refined tilapia through substitution of fish meal with another plant protein sources. This was performed by usage of probiotics or prebiotics as protected dietary enhancements which have neither buildup in the refined fish nor unsafe effects for the fish customers (14, 15). In the current study, fish fed FM showed higher BWG and FBW than SBM-based diets. The result is coincided with Fontainhas-Fer-nandes et al., (36) who reported that dietary FM was the main protein source showing the highest FBW and Khan et al. (37) who reported that inclusion of dietary FM up to 35% showed marked increase in the growth rate. This may be attributed to high protein quality of FM and its palatability. Betaine incorporation to SBM based diet improved feed intake relatively similar to FM. Similarly, betaine was used as a feeding stimulant in red sea bream (38), dover sole (39), European eel (40), Tilapia (31), juvenile grouper (41), and Chinese soft-shelled Turtle (42). This may indicate that betaine improved palatability of the diets which might be through stimulation of cephalic index induced by betaine smell and taste. Conversely, Hughes (43) suggested that betaine not affect the feed intake in Chinook Salmon Fry. In addition, betaine incorporation in SBM-based diets not only increased FI but also growth performance compared to FM diet levels. Similar results were also reported in Indian major carp (25), rainbow trout (44), pike perch (45), and in tilapia fed 0.5% betaine (31). Kasper et al. (46) found that dietary betaine can spare the entire choline requirement in Nile tilapia in contrast with Vieira et al. (47). Regarding to the feed conversion ratio (FCR) which is considered as one of the most economic indicators, addition of betaine to FM or SBM-based diets decreased FCR. Diet based on SBM+ betaine achieved the same effect FM on feed efficiency. This result is similar to that reported by (25) who used 0.25% of betaine in carp fish diet, (48) who used 0.5% betaine in juvenile fresh water prawn fish and (26) who got the best FCR in channel cat fish (26). Decreased FCR might be attributed to the improvement of growth performance (31). Recently, Zou et al. (49) reported that feeding Nile tilapia with 0.6% betaine in diet containing high level of plant protein significantly lowered FCR. The beneficial effect of betaine on growth performance may be attributed to its feeding stimulant effect together with increased intestinal amylase and protease activities (49, 50). The positive effect of betaine on growth performance might be also due to alternation in the metabolism which needs further examination. However, the improvement of FCR in this study was due to the rapid growth rate which saves maintenance requirements. Interestingly, betaine supplementation to SBM-based diets increased the body protein and decreased the fat content. This may be attributed to betaine role as a methyl donner, sparing methionine amino acid needed for protein synthesis. In addition, betaine plays a key role in synthesis of phosphatidyl choline, car-nitine and creatine. Moreover, it was suggested that betaine role as a methyl donner enhances lipid metabolism, stimulating the hepa-toprotective function resulting in prevention of the fatty liver (26). Furthermore, the incorporation of betaine at 0.4% in Allogynogenetic gibel carp diet resulted in lowering in the hepatopancreas, muscle and the whole body lipid content (51). Additionally, Wang et al. (27) reported that supplementation of dietary betaine decreased lipogenesis and increased fatty acids oxidation. The above evidence suggests that betaine incorporation into Nile ti-lapia diets provides the priority for protein synthesis which consumes smaller energy amounts than fat synthesis. Assessment of intestinal healthiness including absorptive capacity and digestive Betaine improves growth performance in Nile tilapia fed on soybean meal-based diets 35 functions through the height of intestinal villi, thickness of muscular layer and the number of goblet cells is an important morphometry indicator in case of evaluating different feed strategies in aquatic animals (52, 53). In the current study, betaine incorporation at 0.2% increased the intestinal villi length and number of goblet cells. Improving the intestinal absorptive and protective functions might be due to increased surface area available for absorption and/or due to secreted mucous from numerous goblet cells which has bactericidal effect through covering receptors of pathogens and keeping the integrity of intestinal epithelium (54, 55). Lipid metabolism variations induced by betaine incorporation was monitored by changing in mRNA level of Fas which affects the synthesis of body fat and thereby affects the body fat accumulation (56). Fas and Lpl are considered key factors in lipid metabolism and fat deposition in the body (57). Current results revealed decreased Fas mRNA level associated with betaine incorporation in diet. Similar results were reported by Kim et al. (58) who revealed that protection of liver against steato-sis could be occurred through supplementation of dietary betaine which decreased Fas and ACC (acetyl-CoA carboxylase) through affecting Forkhead Box O1 (FoxOl) binding peroxisome proliferator activated receptor gamma (PPARy). Current results are similar to that reported in Allogynogenetic gibel carp (51). Additionally, hepatic Lpl mRNA level markedly decreased with supplementation of dietary betaine. Oku et al. (59) reported that the expression of Lpl was tissue specific therefore, the effect of dietary betaine on Lpl expression needs further examination for Nile ti-lapia different organs other than liver. Igf-1 considers the major anabolic factor needed for growth (60). Igf-1 mRNA expression in the current study showed upregulation with addition of dietary betaine and down regulation when fish fed SBM-based diets without betaine which could subsequently reduce growth performance. Similar results were reported by Men et al. (61) who found that when fish fed on plant protein based diets at high level (60%) showed lower hepatic Igf-1 than that fed 30% in Japanese seabass. Conclusion From the obtained results, it could be concluded that betaine incorporation with 0.2% level to SBM-based diet improve palatability, growth performance, intestinal absorptive capacity, body protein on expense of lipid body accumulation, hepatic Igf-1 gene expression and reduce the lipogenesis through markedly decrease Fas and Lpl mRNA level in liver. Acknowledgement The authors are thankful to Dr. Mohsen Kamal, the technical director for Biochem company for his support in practical work in this manuscript and Dr. Safaa El-Sayed Abdo, lecturer of genetic and genetic engineering, Faculty of Veterinary Medicine, Kafrelsheikh University, Egypt, for her valuable cooperation. Conflict of interest The authors declare that they have no conflict of interest. References 1. Hussain SM, Javed M, Javid A, Javid T, Hussain N. Growth responses of Catla catla, Labeo rohita and Cirrhina mrigala during chronic exposure of iron. Pak J Agric Sci. 2011; (1); 48:22530. 2. Kumolu-Johnson CA, Ndimele PE. A review on post-harvest losses in Artisanal fisheries of some African countries. J. Fish. Aquat. Sci. 2011; 6: 365-78. 3. Rubbani B, Afzal M, Mubarik MS, Salim M, Hussain SM. Estimation of apparent digestibility coefficients of soybean meal based diets with different protein levels for Labeo rohita. Pak J Agric Sci. 2011; 1(48):231-5. 4. Fletcher GL, Shears MA, Goddard SV. Transgenic Fish for Sustainable Aquaculture. In: Sustainable Aquaculture: Food for the Future, Svennig, N., H. Reinertsen and M. New (Eds.). Balkema , Rotterdam, 1999; pp: 193-201 5. Mohamed HMA, Walaa FAE, Braeuning A, Hammad S. Detection of aflatoxin -producing fungi isolated from tilapia and fish feed. Excil J. 2017; 16:1308-18. 36 T Ismail, D Nassef, ES Hegazi, AN Bakr, EM Moustafa, W Abdo, Z Elbialy 6. GAFRD. General authority for fish resources development. In: Fish Statistics Year Book. Cairo, Egypt: Ministry of Agriculture and Land Reclamation (2016). 7. El-Sayed AFM, Dickson MW, El-Naggar GO. Value chain analysis of the aquaculture feed sector in Egypt. Aquac 2015; 437: 92-101. 8. FAO. Fishery and Aquaculture Statistics Yearbook 2014. Rome, Italy: Food and Agriculture organization of the United Nations. ISSN 20706057 (2016). 9. El-Tholth M, Fornace K, Grace D, Rush-ton J, Hàsler B. Characterisation of production, marketing and consumption patterns of farmed ti-lapia in the Nile Delta of Egypt. Food Policy 2015; 51: 131-43. 10. Nandlal S, Pickering T. Tilapia fish farming in Pacific Island countries. Vol. 1: Tilapia hatchery operation. Noumea, New Caledonia: Secretariat of the Pacific Community, 2004. 11. Sakai M. Current research status of fish immunostimulants. Aquac. 1999; 172: 63-92. 12. Merrifield DL, Dimitroglou A, Foey A, Davies SJ, Baker RTM. The current status and future focus of probiotic and prebiotic applications for salmonids. Aquac 2010; 302: 1-18. 13. Tacon AG, Metian M. Global overview on the use of fish meal and fish oil in industrially compounded aquafeeds: Trends and future prospects. Aquac, 2008; 285(1-4): 146-58. 14. Hai NV. Research findings from the use of probiotics in tilapia aquaculture: a review. Fish Shellfish Immunol 2015; 45: 592-7. 15. Ibrahem MD. Evolution of probiotics in aquatic world: Potential effects, the current status in Egypt and recent prospectives. J Adv Res 2015; 6: 765-91. 16. Leong-Seng L, Yong ASK, Shapawi R. Terrestrial animal-and plant-based ingredients as alternative protein and lipid sources in the diets for juvenile groupers: current status and future perspectives. Ann Res Rev Biol 2014;4(20): 3071. 17. Muñoz-Clares R, Valenzuela-Soto E. Betaine aldehyde dehydrogenases : evolution, physiological functions, mechanism, kinetics, regulation, structure, and stability. Adv. Protein Phys. Chem., Kerala, India, Res. Sign Post 2008; 279-302 . 18. Eklund M, Bauer E, Wamatu J, Mosenthin R. Potential nutritional and physiological functions of betaine in livestock. Nut Res Rev 2005;18(1): 31-48 . 19. Xue M, Cui Y. Effect of several feeding stimulants on diet preference by juvenile gibel carp (Carassius auratus gibelio), fed diets with or without partial replacement of fish meal by meat and bone meal. Aquac 2001; 198(3-4):281-92. 20. Fekrandish H, Abedian A, Matin Far A, Monfard N, Dehghani A. Influence of betaine and methionine in the diet for stimulating food intake of indian white shrimp (Fenneropenaeus indicus). Pajouhesh and Sazandegi. 2007. 21. Papatryphon E, Soares Jr JH. Optimizing the levels of feeding stimulants for use in high-fish meal and plant feedstuff-based diets for striped bass, Morone saxatilis. Aquac 2001; 202(3-4): 279--88. 22. Hanson AD, Rathinasabapathi B, Rivoal J, Burnet M, Dillon MO, Gage DA. Osmoprotective compounds in the Plumbaginaceae: a natural experiment in metabolic engineering of stress tolerance. Proceed Nat Acad Sci 1994; 91(1):306-10 . 23. De Vooys C, Geenevasen J. Biosynthesis and role in osmoregulation of glycine-betaine in the Mediterranean mussel Mytilus galloprovin-cialis LMK. Comparative Biochemistry andPhys-iology Part B: Biochemistry and Molecular Biology, 2002; 132(2): 409-14 . 24. Sheard N, Zeisel S. Choline: an essential dietary nutrient? Nutrition (Burbank, Los Angeles County, Calif.), 1989; 5(1): 1-5. 25. Shankar R, Murthy S, Pavadi P, Thanuja K. Effect of betaine as a feed attractant on growth , survival, and feed utilization in fingerlings of the Indian major carp, Labeo rohita. Isr. J. Aquac -Bamidgeh 2008; 60(2): 95-9. 26. Wu G, Davis DA. Inter-relationship among methionine, choline, and betaine in channel catfish (Ictalurus punctutus). J World Aquac Soc 2005; 36(3): 337-45. 27. Wang L, Chen L, Tan Y, Wei J, Chang Y, Jin T, Zhu H. Betaine supplement alleviates hepatic triglyceride accumulation of apolipoprotein E deficient mice via reducing methylation of pe-roxisomal proliferator-activated receptor alpha promoter. Lipids in health and disease 2013; 12(1): 34 . 28. Zhang W, Wang LW, Wang LK, Li X, Zhang H, Luo LP,. . . Gong ZJ. Betaine protects against high-fat-diet-induced liver injury by inhibition of high-mobility group box 1 and Toll-like receptor 4 expression in rats. Dig Dis & sci 2013; 58(11): 3198-206 . 29. He S, Zhao S, Dai S, Liu D, Bokhari SG.. Effects of dietary betaine on growth performance, fat deposition and serum lipids in broilers subjected to chronic heat stress. Animal Science J 2015; 86(10): 897-903 . Betaine improves growth performance in Nile tilapia fed on soybean meal-based diets 37 30. Leng Z, Fu Q, Yang X, Ding L, Wen C, Zhou Y. Increased fatty acid P-oxidation as a possible mechanism for fat-reducing effect of betaine in broilers. Animal Science J 2016; 87(8): 100510. 31. Luo Z, Tan XY, Liu XJ, Wen H. Effect of dietary betaine levels on growth performance and hepatic intermediary metabolism of GIFT strain of Nile tilapia Oreochromis niloticus reared in freshwater. Aquac Nut 2011; 17(4):361-7 . 32. Livak KJ, Schmittgen TD. Analysis of Relative Gene Expression Data Using Real-Time Quantitative PCR and the 2-AACT Method. Methods 2001; 25: 402-8. 33. Tian J, Wu F, Yang CG, Jiang M, Liu W, Wen H. Dietary lipid level impact lipoprotein li-pase, hormone-sensitive lipase and fatty acid syn-thetase gene expression in three tissue of adult GIFT strain of Nile Tilapia, Oreochromis niloticus. Fish Physiol Biochem 2015; 41:1-18. 34. Costa LS, Rosa PV, Fortes-Silva R, Sánchez-Vázquez FJ, López-Olmeda JF. Daily rhythms of the expression of genes from the soma-totropic axis: the influence on tilapia (Oreo-chromis niloticus) of feeding and growth hormone administration at different times. Comparative Biochemistry and Physiology Part C: Toxicology & Pharmacology 2016; 181: 27-34. 35. Qiang J, He J, Yang H, Wang H, Kpundeh M, Xu P, Zhu Z. Temperature modulates hepatic carbohydrate metabolic enzyme activity and gene expression in juvenile GIFT tilapia (Oreochromis niloticus) fed a carbohydrate-enriched diet. J Therm Biol 2014; 40: 25-31. 36. Fontainhas-Fernandes A, Gomes E, Reis-Henriques MA, Coimbra J. Replacement of fish meal by plant proteins in the diet of Nile tilapia: digestibility and growth performance. Aquacult Int 1999; 7(1): 57-67. 37. Khan M, Siddique M, Zamal H. Replacement of fish meal by plant protein sources in Nile tilapia (Oreochromis niloticus) diet: growth performance and utilization. Iran J Fish Sci 2013;12(4): 864-72. 38. Goh Y, Tamura T. Effect of amino acids on the feeding behaviour in red sea bream. Comparative Biochemistry and Physiology Part C: Comparative Pharmacology 1980; 66(2):225-9 . 39. Mackie A, Mitchell A. Further studies on the chemical control of feeding behaviour in the Dover sole, Solea solea. Comp Biochem Physiol Part A: Physiology 1982; 73(1): 89-93 . 40. Mackie A, Mitchell A. Studies on the chemical nature of feeding stimulants for the juvenile European eel, Anguilla anguilla (L.). J Fish Biol 1983; 22(4): 425-30 . 41. Lim LS, Chor WK, Tuzan AD, Shapawi R, Kawamura G. Betaine is a feed enhancer for juvenile grouper (Epinephelus fuscoguttatus) as determined behaviourally. J Appl Ani Res 2016; 44(1): 415-8. 42. Sun CX, Xu WN, Zhang DD, Li XF, Li PF, Jiang GZ, Liu WB. Different preference is modulated by the feeding stimulants supplementation in different Chinese soft-shelled turtle (Pelo-discus sinensis) basic diets. Aquac Nut 2018; 24(1): 195-203. 43. Hughes SG. Single feeding response of chinook salmon fry to potential feed intake modifiers. Prog Fish-Cult 1993; 55(1): 40-2 . 44. Tiril SU, Alagil F, Yagci FB, Aral O. Effects of betaine supplementation in plant protein based diets on feed intake and growth performance in rainbow trout (Oncorhynchus mykiss). Isr. J Aq-uace - Bamidgeh 2008; 60(1): 57-64. 45. Zakipour Rahimabadi E, Akbari M, Ar-shadi A, Effatpanah E. Effect of different levels of dietary Betaine on growth performance, food efficiency and survival rate of pike perch (Sander lu-cioperca) fingerlings. Iran J Fish Sci. 2012; 11: 902-10. 46. Kasper CS, White M, Brown P. Betaine can replace choline in diets for juvenile Nile Tilapia, Oreochromis niloticus. Aquac 2002; 205: 119-26. 47. Vieira I, Cyrino JEP, Pezzato LE. Colina e betaina em ra^öes purificadas na nutri^ao da tilapia do Nilo (Oreochromis niloticus). Sci Agric 2001; 675-80. 48. Felix N, Sudharsan M. Effect of glycine betaine, a feed attractant affecting growth and feed conversion of juvenile freshwater prawn Macro-brachium rosenbergii. Aquac Nut 2004; 10:193-. 49. Zou Q, Huang Y, Cao J, Zhao H, Wang G, Li Y, Pan Q. Effects of four feeding stimulants in high plant-based diets on feed intake, growth performance, serum biochemical parameters, digestive enzyme activities and appetite-related genes expression of juvenile GIFT tilapia (Oreochromis sp.). Aquac Nut 2017; 23(5): 1076-85. 50. Srinivas D. Effect of G-probiotic on growth, body composition and survival of giant freshwater prawn, Macrobrachium rosenbergii (de Man) and Indian Major Carp, Labeo rohita (Ham), MF Sc. Thesis submitted to University of Agricultural Sciences 2000; 113. 38 T Ismail, D Nassef, ES Hegazi, AN Bakr, EM Moustafa, W Abdo, Z Elbialy 51. Dong X, Xue W, Hua J, Hang Y, Sun L, Miao S, Wei W, Wu X, Du X. Effects of dietary betaine in Allogynogenetic gibel carp (Carassius auratus gibelio): Enhanced growth, reduced lipid deposition and depressed lipogenic gene expression. Aquac Res 2018; 49: 1967-72. 52. Pirarat N, Pinpimai K, Endo M, Katagiri T, Ponpornpisit A, Chansue N, Maita M. Modulation of intestinal morphology and immunity in nile tilapia (Oreochromis niloticus) by Lactobacillus rhamnosus GG. Res Vet Sci 2011; 91(3): 92-7. 53. Khojasteh SMB. The morphology of the post-gastric alimentary canal in teleost fishes: a brief review. Int J Aquat Sci 2012; 3(2): 71-88. 54. Ring0 E, Olsen RE, Mayhew TM, Mykle-bust R. Electron microscopy of the intestinal microflora of fish. Aquac 2003; 227(1-4): 395-415. 55. Smirnov A, Perez R, Amit-Romach E, Sklan D, Uni Z. Mucin dynamics and microbial populations in chicken small intestine are changed by dietary probiotic and antibiotic growth promoter supplementation. J Nut 2005; 135(2): 18792. 56. Leng X, Wu X, Tian J, Li X, Guan L, Weng D. Molecular cloning of fatty acid synthase from grass carp (Ctenopharyngodon idella) and the regulation of its expression by dietary fat level. Aquac Nut 2012; 18: 551-8. 57. Albalat A, Saera-Vila A, Capilla E, Gutiérrez J, Pérez-Sánchez J, Navarro I. Insulinregulation of lipoprotein lipase (Lpl) activity and expression in gilthead sea bream (Sparus au-rata). Comparative Biochemistry and Physiology PartB: Biochem Mol Biol 2007; 148(2): 151-159. 58. Kim DH, Lee B, Kim MJ, Park MH, An HJ, Lee EK, Chung KW, Park JW, Yu BP, Choi JS. Molecular Mechanism of Betaine on Hepatic Lipid Metabolism: Inhibition of Forkhead Box O1 (FoxO1) Binding to Peroxisome Proliferator-Acti-vated Receptor Gamma (PPARy). J Agr food chem 2016; 64: 6819-6825. 59. Oku H, Koizumi N, Okumura T, Koba-yashi T, Umino T. Molecular characterization of lipoprotein lipase, hepatic lipase and pancreatic li-pase genes: effects of fasting and refeeding on their gene expression in red sea bream Pagrus major. Comp Biochem Physiol B: Biochem Mol Biol 2006; 145:168-178. 60. Thissen JP, Underwood LE, Ketelslegers JM. Regulation of Insulin like Growth Factor-I in Starvation and Injury. Nut Rev 1999; 57(6): 167176. 61. Men K, Ai Q, Mai K, Xu W, Zhang Y, Zhou H. Effects of dietary corn gluten meal on growth, digestion and protein metabolism in relation to Igf-I gene expression of Japanese seabass, Lateolabrax japonicus. Aquac 2014; 428: 303-9. Slov Vet Res 2019; 56 (Suppl 22): 39-49 DOI 10.26873/SVR-742-2019 Original Research Article THE EFFECTS OF GROUP VERSUS SINGLE HOUSING ON HOME CAGE BEHAVIOUR IN TWO STRAINS OF LABORATORY MICE Ahmed F. Abou-Elnaga1, Ibrahim F. Rehan2*, Ralph RJ. Thompson3, Usama A. Abou-Ismail1, Motamed E. Mahmoud4, Radi A. Mohamed5, Hesham H. Mohammed6, Ahmed A. Sabek7, Mohamed Z. Elhussiny8. department of Husbandry and Development of Animal Wealth, Faculty of Veterinary Medicine, Mansoura University, P.O. Box 35516, Egypt, 2Department of Animal Behaviour and Husbandry (management, genetics and breeding), Faculty of Veterinary Medicine, South Valley University, 83523, Egypt, 3University of Edinburgh, UK, 4Department of Animal Behaviour and Husbandry (management, genetics and breeding), Faculty of Veterinary Medicine, Sohag University, 82524, Egypt, 5Department of Aquaculture (Animal and Fish Behaviour and Welfare), Faculty of Aquatic Sciences and Fisheries, University of Kafrelsheikh, P.O. Box 33511, Egypt, 6Department of Veterinary Public Health, Faculty of Veterinary Medicine, Zagazig University, 44511, Egypt, department of Veterinary Hygiene and Management, Faculty of Veterinary Medicine, Benha University, 13736, Egypt, 8Department of Animal & Poultry Behaviour and Management, Faculty of Veterinary Medicine, Aswan University, 81528, Egypt Corresponding author, E-mail: ibrahim.rehan@vet.svu.edu.eg Abstract: Studying the behavioural patterns of animals in their house may help to understand their needs, but there has been a little investigation of home-cage behaviours in commonly used inbred strains of mice such as C57BL/6 and DBA/2. Therefore, understanding behavioural patterns in these mice is important for neuroscience research. For the first time, this experiment was carried out to investigate the long-term effects of housing conditions (single vs. group) on home cage behaviour of C57BL/6 and DBA/2 mice, in order to reveal differences between C57BL/6 and DBA/2 in home-cage behaviours and in response to single-housing. Sixty-four mice (32 mice/strain) were housed either singly (SH) (n= 32) or in four groups, each group contained 8 animals. Home cage behavioural patterns were recorded weekly using ethogram-based instantaneous sampling for 5 consecutive weeks. Regardless of strain, single housed (SH) mice displayed higher levels of grooming and bedding directed-behaviours and were more frequently seen in-the-crawl ball, and had lower levels of feeding behaviour compared to their group housed (GH) conspecifics. There were significant strain differences in anxiety-related behaviours with the DBA/2 strain demonstrating higher levels of sleep, feeding and grooming behaviour and frequent presence in-the-crawl ball, and lower levels of exploration, locomotion and bedding-directed behaviour compared to the C57BL/6 strain. The results therefore suggest that different housing systems influence home cage behaviours of laboratory mice with the mice of the DBA/2 strain appearing more anxious. These findings may also have great implications for researchers to decide the most appropriate phenotype to use in measuring neural response-relevant behaviours in novel animal/human models. Key words: anxiety; behaviour; C57BL/6; DBA/2; ethogram; home-cage Received: January 2019 Accepted for publication: February 2019 40 A Abou-Elnaga, I Rehan, R Thompson, U Abou-Ismail, M Mahmoud, et al. Introduction The housing conditions of laboratory rodents have been demonstrated to induce changes in their behaviour, physiology and pathology (1) which in turn could impact the reproducibility and validity of experimental results. An important aspect of the housing conditions is the social environment (e.g. single versus group housing). Improving the housing conditions of laboratory rodents may enhance welfare in these animals by promoting their ability to cope with the environment, and may also improve the accuracy of experimental data by providing a more valid animal model for research, and may therefore reduce the number of animals used. The hypothesis that alterations in home-cage behaviour can upset neural circuit function has attracted much consideration of the recent years both in the context of disease detection and more commonly to quantify food consumption and activity parameters (2). Moreover, great effort was directed towards the 'mouse phenome' relating characterization of common inbred strains to various behavioural tests (3). Significant differences have been found between inbred mice strains in spontaneously emitted activity, particularly in the open-field test (4). In addition, large genetic differences in movement as a response to stimulant administration (5) could provide evidence of genetic associates with many behaviours, such as sleep (6). Studies carried out on inbred strains of mice have revealed consistent differences in behaviour due to the biological functions of their genotype (7). In addition, many different inbred genetic lines showed differences in behaviour which pose challenges when seeking functional explanations for specific neural mechanisms (8). Therefore, there is interest in assigning particular behavioural phenotypes as characteristic features to easily differentiate each mouse strain and further to develop easy criteria to assess their behavioural competences (9). C57BL/6 and DBA/2 are two of the commonly used inbred strains of laboratory mice. Numerous behavioural differences have been reported between them (10); furthermore, envi- ronmental manipulations have induced remarkable differences in epigenetic effects between strains (11). Studying home-cage behavioural differences between strains can yield several advantages for behavioural phenotypic purposes e.g. reliable measurement of stable behavioural circadian rhythms that are highly responsive to environmental signals, such as light and human interference (12). Individual housing is generally not recommended for social laboratory rodents such as mice as it compromises their welfare, but there remains some confusion surrounding the impact of individual housing on mice. Many authors illustrated that single housing, compared to group housing of mice does not amplify urinary corticosterone level (13-16), whereas others agreed that individual housing for social animals like mice is a stressful condition (17). Measures of physiological stress; however, do not necessarily correlate with subjective welfare of the animal. Together with the conflicting research on stress this highlights the need for a further investigation onto the impact of individual housing on mice, and how this might differ between strains, especially on the consequences for their behaviour. In the present study, the home-cage behaviour of single and group housed C57BL/6 and DBA/2 mice was investigated using ethogram-based behavioural observation. Material and methods General animal housing Sixty-four adult male mice (32 C57BL/6J and 32 DBA/2), purchased from the Animal House, Theodor Bilharz Research Institute, Giza, Egypt, were used in this experiment. The mice were 10 weeks of age on arrival and had an average body weight about 28 g. After transportation, all animals were given 2 weeks to habituate to their new environment and to adjust to the new lighting regime and were therefore 12 weeks of age at the start of the experiment. All cages were supplied with sawdust (as bedding material) and a crawl-ball (115 mm, with 3 x 58 mm holes, polycarbonate ball, Lillico, UK) as an enrichment object. Cages were inspected daily and were cleaned once a week, Effect of housing system on C57BL/6 and DBA/2 mice behaviour 41 during which mice were removed and rehoused in clean cages with new bedding material. Mice had ad-libitum access to pelleted food (Mice chow®, Oil and Soap Manufacturing Company, El-Gharbia, Egypt) and tap water, were maintained under conditions of controlled temperature (22±2 °C) and humidity (60%) and were checked daily. The room was maintained under a 12:12 h light/dark cycle. Lights were turned off at 19:00 h and on at 07:00 h with a continuous dim red light allowing observation in the dark phase. The group housed mice were marked on their tails by using a permanent marker pen to allow individual identification and these marks were refreshed every week throughout the experimental period. Likewise, the single housed mice were handled every week to avoid the behavioural changes between the two groups. All experimental procedures were performed in accordance with the agreement for the Animal Care and Use and Approval of Ethics Committee for Animal Experimentation of Mansoura University, Egypt and care was taken to comply with the 3R concept. Experimental treatments A total of 64 mice representing two strains (32 mice of DBA/2J strain and 32 mice of C57BL/6J strain) were arbitrarily assigned to one of the following two experimental treatments: 1) "Single housing" (SH): 16 mice of DBA/2J strain and 16 mice of C57BL/6J strain (n= 32) were housed singly in Plexiglas cages (27.5 cm length x 16.5 cm width x 21.5 cm height). 2) "Group housing" (GH): 16 mice of DBA/2J strain and 16 mice of C57BL/6J strain (n= 8) were housed in groups of four in Plexiglas cages (27.5 cm length x 16.5 cm width x 21.5 cm height). All animals were introduced to their particular experimental treatments at 12 weeks of age and were naive to the housing conditions. They were kept under the same housing condition until they were 17 weeks old; the age at which data collection was stopped. Behavioural assessment In order to allow mice to habituate to the presence of the observer, the observer entered the experimental room 10 min before the observation started (18). Behaviour of the mice in each cage was recorded in the usual housing location during the dark phase when mice are most active. Observations were made in real time using an instantaneous scan sampling method with 10s intervals. Each sample interval was prompted by an audio cue via headphones, and the behaviour was recorded into a check sheet. Observation sessions, were carried out consecutively on the same day, and lasted 640 s (100s per mouse, yielding 10 scans per mouse for each of 64 mice). Observation was carried out for each cage once per week for five consecutive weeks. This meant a total of 50 scans were recorded per mouse over the entire experimental period. A complete ethogram of mice's behaviours is shown in Table 1. Data analyses Data was organized and summarized and tested for normality, linearity and homogeneity of variance. Then the average percentage of scans spent performing each behavioural pattern for each individual mouse was calculated by dividing the number of scans for each behavioural variable by the total number of scans and the resultant value was multiplied by 100. Two-way ANOVA was used to test for differences in behavioural patterns between the two strains of mice (DBA/2 and C57BL/6J) within the two experimental treatments (group and single housing) and result were expressed as Means ± standard error (M ± SEM) as independent variables followed by Bonferroni-PostHoc tests to compare each strain to its housing type using SPSS V.20. In cases of the significant interactions (interaction between two variables), e.g. housing system*strain, general linear model was used to compare means of behavioural patterns with different housing system as a factor and strain as a dependent variable. 42 A Abou-Elnaga, I Rehan, R Thompson, U Abou-Ismail, M Mahmoud, et al. Results Main effect of experimental treatment The output of the two-way ANOVA-test showed that there was an effect of housing conditions on the home cage behaviour of mice with the mice in the SH treatment displaying higher levels of enrichment-directed behaviours (F3,60= 27.59, P= 0.001) (Fig. 1) than those in the GH, and those of C57BL/6J displayed higher levels of bedding-directed behaviours (F3,60= 5.647, P= 0.035) (Fig. 3) compared to their conspecifics in GH treatment. On the other hand, GH mice showed higher levels of ingestive behaviour compared to SH mice (F3,60= 10.851, P= 0.01) (Fig. 4). Main effect of strain Several behaviour patterns recorded in this study showed an effect of mouse strain: Enrich ment-directed behaviour and sleep were higher in DBA/2J mice in both housing conditions compared to C57BL/6J (F3,60= 30.654, P= 0.001) (Fig. 1) (F3,60= 44.075.94, P= 0.001) (Fig. 5) respectively, in contrast, exploration (F3,60= 54.327, P< 0.001) (Fig. 6) and locomotion (F3,60= 37.623, P< 0.001) (Fig. 7) were higher in C57BL/6J . Housing*strain interaction effects SH mice of the DBA/2J strain displayed higher levels of grooming (F3,60= 9.204, P= 0.01) (Fig. 2). Whereas bedding-directed behaviours in SH treatment (F3,60= 9.661, P< 0.009) (Fig. 3) were more frequently performed by C57BL/6J mice than those of DBA/2J. Furthermore, feeding behaviour were highly significant in GH treatment in DBA/2J mice (F3,60= 11.083, P= 0.006) (Fig. 4). Table 1 : Ethogram for behavioural elements recorded Behavioural category Description Feeding behaviour Grooming Sleep Exploratory behaviour (investigation of food and environment) Bedding-directed behaviours Locomotion In-the-crawl ball Rears up to gnaw at food pellets through the bars of the food hopper. Lick its fur, groom with the forepaws, or scratch with any limb. Lying un alert with both eyes closed, curled up on its side, with its face tucked into its body and motionless. Investigate food; investigate a non-food object in the environment (Sniffing cage wall, cage top and cage floor, (air-in) sniffing air inside the cage). Digging, sniffing bedding, bedding manipulation (pushing bedding material forwards or backwards with nose, forepaws or hind legs) and burrowing. Movement in the cage. Mice were encapsulated by the crawl ball in which the whole body of the animal- with or without its tail- was inside the ball. Effect of housing system on C57BL/6 and DBA/2 mice behaviour 43 90 n: -Q 80 "i ar 70 .c -Q Q. V so OS 70 60 50 40 30 20 10 □ D2G ■ B6G □ D2S □ B6S D2G B6G D2S Treatment B6S Figure 5: Average % of scans sleeping by the mice of the two inbred strains in the two experimental treatments. Data represent mean ± SEM, ** P<0.01, *** P<0.001. D2G: GH- DBA/2; B6G: GH-C57BL/6J; D2S: SH-DBA/2; B6S: SH- C57BL/6J > a -C e -Q o t a r _o tt X e a 0.10) -0.58080 (P > 0.10) -0.35475 (P > 0.10) German Shepherd 1 ± 0.034 0.01158 7.561 21 0.38302 (P > 0.10) 0.37188 (P > 0.10) 0.16720 (P > 0.10) Table 5: Haplotype diversity and power of discrimination Parameter Egyptian Baladi dogs German Shepherd dogs Total number of animals 46 42 No. of haplotypes 22 12 Power of discrimination 0.908318 0.851474 SNPs 34 22 Total number of singleton mutations 13 6 Number of parsimony informative sites 20 15 Transitional Pairs (si) 8 7 Transversional Pairs (sv) 0 0 R = si/sv 31.1 24.0 Genetic diversity among two common populations of Canis lupus familiaris in Egypt by using mitochondrial DNA ... 81 Figure 1: The second Maximum likelihood tree based on kimura-2 parameter method (+G+I) of 34 haplo-types of the two dog breeds included in this study. The number of bootstrap replications=1000. The evolutionary history was inferred by using the Maximum Likelihood method and Kimura 2-parameter model. The tree with the highest log likelihood (-23500.98) is shown Figure 2: The second Maximum likelihood tree based on kimura-2 parameter method (+G+I) of 34 haplo-types of the two dog breeds and U96639 included in this study. The number of bootstrap replications=1000. U96639 is the first published dog mitochondrial genome. G refers to the most common ancestral line 82 MA Elmadawya, S El-Kassasb, SE Abdoc, A Nagaid, Y Bunaid U96639 Hap_l: 2 [GYSD12 EGYBD22] Hap_2: 3 [GYSD11 EGYBD16 EGYBD09] Hap_3: 2 [GYSD10 EGYBD15] Hap_4: 1 [GYSD09] Hap_5: 2 [GYSD08 EGYBD08] Hap_6: 1 [GYSD07] Hap_7: 1 [GYSD06] Hap_8: 1 [GYSD05] Hap_9: 2 [GYSD04 EGYBD20] 1 [GYSD03] 1 [GYSD02] 1 [GYSD01] 1 [EGYBD21] 1 [EGYBD19] 1 [EGYBD18] 1 [EGYBD17] 1 [EGYBD14] 1 [EGYBD13] 1 [EGYBD12] 1 [EGYBD11] 2 [EGYBDIO EGYBDOl] 1 [EGYBD07] 1 [EGYBD06] 1 [EGYBD05] 1 [EGYBD04] 1 [EGYBD03] 1 [EGYBD02] 1[U96639] ■ German Haplotypes I [ Egyptian Haplotypes Figure 3: Median-joining network (£ = 0) depicting Genetic relationships among dog breed mtDNA HVR1 haplotypes from this study using Network v5.0.1.0. Circled areas are proportional to the corresponding haplotype frequency Figure 4: Mitochondrial DNA haplogroup distributions in the most common Egyptian dog breeds Genetic diversity among two common populations of Canis lupus familiaris in Egypt by using mitochondrial DNA ... 83 Discussion German shepherd and Egyptian Baladi are widely distributed and the most common dog breeds in Egypt. These dogs are kept mainly for guarding and have a crucial role in the forensic cases (2). However, up to date, there is missing information on the genetic diversity between these two breeds. Also, there are no effective controlled breeding programs for these breeds. So, for many purposes, such as forensic cases, it is highly recommended to differentiate between these two breeds. The canine mtDNA, especially HVR1, is highly polymorphic that can be used effectively as a DNA marker for the breed identification (22). However, little is known regarding using HVR! in dog breeds identification in Egypt. As the haplotype diversities are informative tools about the history of animals, and for the breed identification. So, the high diversities reported in the current study probably explain the lack of differentiation between the Egyptian Baladi and German shepherd dogs in Egypt (23). These results agreed with the previous studied (11, 24) which reported high haplotype diversities although using different dog breeds and nu-cleotide numbers. Moreover, the Egyptian Baladi dogs had high haplotype diversity almost, as high as that noticed in the case of German shepherd dogs. This might imply that Egyptian dogs were developed from several breeds over their breeding history. In addition, the high genetic variability among the two studied breeds probably indicates that the dog populations in Egypt have been undergoing a rapid expansion in recent history (25). Plus, an increased effective population size, and a reduced genetic drift might be inferred from the identified high genetic diversity (26). Likewise, the reported high nucleotide diversities for the two breeds might elucidate high genetic differentiation and presence of large differences between haplotypes (27). The rate of genetic diversity is determined effective population size, and rate of mutation. Where, the larger the population size, the higher the genetic diversity (28). So, it is perhaps occurred due to a relatively large long-term effective population size rather than any severe bottleneck during dog evolution (29). Also, the detected higher haplotype and nucleotide diversities probably indicate that the studied populations recently divergent from each other (30). Additionally, the detected high haplotype and nucleotide diversities could be a signature of a rapid population expansion (31). This was confirmed by the genetic distant from the most frequent haplotypes and the ancestral haplotype (U96639) (25). Another evidence of the rapid population expansion was concluded from calculating the Tajima's D test and Fu's Fs tests that are usually used to find out the population expansion. For the Egyptian Baladi dogs, the Tajima's D test and Fu's Fs values were non-significantly negative. The overall negative values of these neutrality tests perhaps designated an excess of the rare mutations in populations, which might suggest a recent population expansion as well as an evidence of a selective sweep (32). While for the German shepherd dogs, the positive non-significant values of Tajima's D test and Fu's Fs probably implied low levels of both low and high frequency polymorphisms and a balancing selection (32). These non-significant results observed in case of Egyptian and German dogs indicated non-significant variations and the absence of a clear population structure (33). The phylogenetic analysis of the different haplotypes found in the two dog breeds under study and the reference dog mtDNA genome (U96639), was performed. The analysis revealed the presence of two main clades subdivided to many subclades. Also, most of the Egyptian and German haplotypes as well as the reference dog mtDNA genome (U96639) were clustered in the same clade indicating absence of clear demarcations and a strong relationship between the haplotype sequences of the present study and the reference dog mtDNA genome (U96639). This might indicate that two studied breeds are descended from the same ancestral line, recently originated, and mutations that account for their differences have yet to be reported and become fixed (23). Additionally, similarities among the Egyptian Baladi and German shepherd dogs were revealed from the 84 MA Elmadawya, S El-Kassasb, SE Abdoc, A Nagaid, Y Bunaid haplogroup distribution in which the major hap-logroup (A) was identified in the two breeds. Conclusion In conclusion, this study aimed to use the mtDNA HVR1 to differentiate between the most common dog breeds in Egypt; German shepherd and Egyptian Baladi. The calculated high genetic diversity indicates a lake of differentiation between the two breeds. The most frequent haplotypes of the two breeds was belonged to haplogroup A followed by haplog-roup B and C. The negative neutrality tests imply recent population growth of the Egyptian Baladi dogs. The results of this study would be helpful in advancement of Egyptian forensics and animal genetic studies. Disclosure statement No conflicts of interest, financial, or otherwise, are declared by the authors. Acknowledgements This work was supported by a fund given to M.A. Elmadawy from Science and Technology Development Fund (STDF) (grant no. 25431). We thank the Science and Technology Development Fund (STDF) for their help. References 1. Verscheure S, Backeljau T, Desmyter S. Dog mitochondrial genome sequencing to enhance dog mtDNA discrimination power in forensic casework. Forensic Science International: Genetics 2014; 12: 60-8. 2. Coppinger L, Coppinger R. 14 Dogs for Herding and Guarding Livestock. Livestock Handling and Transport: Theories and Applications 2014: 245-9. 3.Wang GD, Zhai W, Yang HC, Wang L, Zhong L, Liu Y-H, et al. Out of southern East Asia: the natural history of domestic dogs across the world. Cell research 2016; 26: 21-7. 4. Wang GD, Xie HB, Peng MS, Irwin D, Zhang YP. Domestication genomics: evidence from animals. Annu Rev Anim Biosci. 2014; 2: 65-84. 5. Akey JM, Ruhe AL, Akey DT, Wong AK, Connelly CF, Madeoy J, et al. Tracking footprints of artificial selection in the dog genome. Proceedings of the National Academy of Sciences 2010;107:1160-5. 6. Osbom DJ, Osbornova J. The mammals of ancient Egypt: Aris & Phillips Warminster; 1998. 7. Himmelberger AL, Spear TF, Satkoski JA, George DA, Garnica WT, Malladi VS, et al. Forensic utility of the mitochondrial hypervariable region 1 of domestic dogs, in conjunction with breed and geographic information. Journal of forensic sciences 2008; 53: 81-9. 8. Imes DL, Wictum EJ, Allard MW, Sacks BN. Identification of single nucleotide polymorphisms within the mtDNA genome of the domestic dog to discriminate individuals with common HVI haplotypes. Forensic Science International: Genetics 2012; 6: 630-9. 9. Dadi H, Lee S-H, Jung KS, Choi JW, Ko MS, Han Y-J, et al. Effect of population reduction on mtDNA diversity and demographic history of Korean cattle populations. Asian-Australasian journal of animal sciences 2012; 25: 1223--9. 10. Achilli A, Olivieri A, Soares P, Lancioni H, Kashani BH, Perego UA, et al. Mitochondrial genomes from modern horses reveal the major haplogroups that underwent domestication. Proceedings of the National Academy of Sciences 2012;109:2449-54. 11. Sugiyama S, Chong YH, Shito M, Kasuga M, Kawakami T, Udagawa C, et al. Analysis of mitochondrial DNA HVR1 haplotype of pure-bred domestic dogs in Japan. Legal Medicine 2013; 15: 303-9. 12.Vila C, Savolainen P, Maldonado JE, Amorim IR, Rice JE, Honeycutt RL, et al. Multiple and ancient origins of the domestic dog. Science 1997;276: 1687-9. 13. Picornell A, Gomez-Barbeito L, Tomas C, Castro JA, Ramon MM. Mitochondrial DNA HVRI variation in Balearic populations. American journal of physical anthropology 2005; 128: 119-30. 14. Kim KS, Lee SE, Jeong HW, Ha JH. The complete nucleotide sequence of the domestic dog (Canis familiaris) mitochondrial genome. Molecular phylogenetics and evolution 1998; 10: 210-20. 15. Savolainen P, Arvestad L, Lundberg J. A novel method for forensic DNA investigations: repeat-type sequence analysis of tandemly repeated mtDNA in domestic dogs. Journal of Forensic Science 2000; 45: 990-9. 16. Ausubel F, Brent R, Kingston R, Moore D, Seidman J, Smith J, et al. Phenol extraction and ethanol precipitation of DNA. Short Protocols in Molecular Biology, 3rd Ed; John Wiley and Sons, Inc: Canada 1995: 2-3. Genetic diversity among two common populations of Canis lupus familiaris in Egypt by using mitochondrial DNA ... 85 17. Kumar S, Stecher G, Li M, Knyaz C, Tamura K. MEGA X: Molecular Evolutionary Genetics Analysis across Computing Platforms. Molecular biology and evolution 2018; 35: 1547-9. 18. Okonechnikov K, Golosova O, Fursov M, Team U. Unipro UGENE: a unified bioinformatics toolkit. Bioinformatics 2012; 28: 1166-7. 19. Tamura K, Nei M. Estimation of the number of nucleotide substitutions in the control region of mitochondrial DNA in humans and chimpanzees. Molecular biology and evolution 1993; 10: 512-26. 20. Librado P, Rozas J. DnaSP v5: a software for comprehensive analysis of DNA polymorphism data. Bioinformatics 2009; 25: 1451-2. 21. Bandelt H-J, Forster P, Röhl A. Median-joining networks for inferring intraspecific phylogenies. Molecular biology and evolution 1999; 16: 37-48. 22. Maruyama S, Komuro T, Izawa H, Tsutsumi H. Analysis of human mitochondrial DNA polymorphisms in the Japanese population. Biochemical genetics 2013; 51: 33-70. 23. Pires AE, Ouragh L, Kalboussi M, Matos J, Petrucci-Fonseca F, Bruford MW. Mitochondrial DNA sequence variation in Portuguese native dog breeds: diversity and phylogenetic affinities. Journal of Heredity 2006; 97: 318-30. 24. Gundry RL, Allard MW, Moretti TR, Honeycutt RL, Wilson MR, Monson KL, et al. Mitochondrial DNA analysis of the domestic dog: control region variation within and among breeds. Journal of forensic sciences 2007; 52: 562-72. 25. Zhao Z, Yu N, Fu Y-X, Li W-H. Nucleotide variation and haplotype diversity in a 10-kb noncoding region in three continental human populations. Genetics 2006; 174 (1); 399-409. 26. Ostrander EA, Wayne RK. The canine genome. Genome research 2005; 15: 1706-16. 27. De Jong MA, Wahlberg N, Van Eijk M, Brakefield PM, Zwaan BJ. Mitochondrial DNA signature for range-wide populations of Bicyclus anynana suggests a rapid expansion from recent refugia. PloS one 2011; 6: e21385. 28. Gasser R, Samson-Himmelstjerna Gv. Haemonchus contortus and haemonchosis-past, present and future trends: Academic Press; 2016. 29. Li W-H, Sadler LA. Low nucleotide diversity in man. Genetics 1991; 129: 513-23. 30. Song J, Hou F, Zhang X, Yue B, Song Z. Mitochondrial genetic diversity and population structure of a vulnerable freshwater fish, rock carp (Procypris rabaudi) in upper Yangtze River drainage. Biochemical Systematics and Ecology 2014;55: 1-9. 31. Low VL, Adler PH, Takaoka H, Ya'cob Z, Lim PE, Tan TK, et al. Mitochondrial DNA markers reveal high genetic diversity but low genetic differentiation in the black fly Simulium tani Takaoka & Davies along an elevational gradient in Malaysia. PLoS One 2014; 9: e100512. 32. Schmidt D, Pool J. The effect of population history on the distribution of the Tajima's D statistic. J Endocrinol. 2002; 33: 211-22. 33. Amiri Ghanatsaman Z, Adeola AC, Asadi Fozi M, Ma Y-P, Peng M-S, Wang G-D, et al. Mitochondrial DNA sequence variation in Iranian native dogs. Mitochondrial DNA Part A 2018; 29: 394-402. Slov Vet Res 2019; 56 (Suppl 22): 87-98 DOI 10.26873/SVR-746-2019 Original Research Article IMPACT OF BIOMOS AND AGRIMOS DIETARY SUPPLEMENTATION ON GROWTH PERFORMANCE, FEED UTILIZATION AND IMMUNOLOGICAL PARAMETERS OF NILE TILAPIA (Oreochromis niloticus) FINGERLINGS Fawzy I. Magouz 1, Mahmoud F. Salem2, Eman M. Moustafa3*, Salwa A. Elkhamy1 department of Animal Production, Faculty of Agriculture, Kafrelsheikh University, Postal code: 33516, Egypt, 2Sakha, Aquaculture Research Unit, Central Lab for Aquaculture Research, Ab-basa, Sharkia- ARC, Egypt, department of Fish Diseases and Management, Faculty of Veterinary Medicine, Kafrelsheikh University Corresponding author, E-mail: emantarek2002@yahoo.com Abstract: This study aimed to evaluate the potential benefits of Biomos® and Agrimos® as prebiotics in Nile tilapia diets. Seven experimental treatments were formulated from 30% protein basal diet to contain Biomos® and Agrimos® at levels of 0.1, 0.2 and 0.3 % for each, in addition to the control diet without any additives. Three hundreds and fifteen fingerlings of Nile Tilapia (O. niloticus) with average initial weight (7 ± 0.5 g), were randomly allocated into 7 treated groups allotted into 21 glass aquaria (three replicates of 15 fish / each treatment). Each aquarium measured 60* 35* 40 cm2. The fish were fed at 3% fish biomass along the experiment which lasted for 15 weeks. The results revealed significant improvements in growth and all feed utilization parameters in the prebiotic supplemented groups. The diets containing Biomos® (0.1%) and Agrimos® (0.2%) revealed the highest growth and protein utilization parameters values. Experimental fish carcass composition was relatively affected by the different dietary treatments. The hematological, biochemical and immunological parameters of the experimented groups indicated significant increase in Biomos® and Agrimos® treated groups. The achieved results demonstrated that Biomos® and Agrimos® at levels of 0.1% and 0.2%, respectively could be used in Nile tilapia diets without negative effects on growth, feed utilization, blood and immunological parameters. Hence, Biomos® and Agrimos® could be added to commercial diets to improve tilapia fingerlings immune response. Key words: agrimos; biomos; growth; immunity; Oreochromis niloticus Introduction Aquaculture in Egypt is the main source of fish production. It represents 77% of the absolute fish production of which 85% were delivered by the developed pond-based aquaculture around the Nile Delta lakes (1). Tilapia is the most generally developed species in these ponds which speaks to over 65% of the all out aquaculture production (2) because of the expanded dimension of intensification and cultured regions (3). Heightening of aquaculture production frameworks exposes fish to various ecological stressors like; poor water quality, Received: January 2019 Accepted for publication: February 2019 88 F. Magouz, M. Salem, E. Moustafa, S. Elkhamy over-crowdness, improper handling and transport which may contrarily influence their growth and health, as well as limiting the outcome of aquaculture systems (4-6). Nutrition assumes an essential job in the growth, development and wellbeing upkeep of fish (7). Few years ago, fish meal was utilized as the primary protein source in tilapia diets. Because of the expanding cost and insecure supply of this ingredient, many attempts have been attempted to enhance the growth performance and decline the production expenses of cultivated tilapia. This was done through utilizing probiotics or prebiotics as safe supplements which have neither residues in the farmed fish nor harmful effects for the consumers (8-10). Prebiotics are non-digestible dietary carbohydrates which get away from the assimilation in the upper gastrointestinal tract. Prebiotics advantageously influence the host by specifically invigorating the growth and/or activating the metabolism of health-promoting bacteria in the gastrointestinal tract (11, 12). Likewise, they modify the gut bacterial composition through changing the kind of substrate gave to the current gut microbiota (13, 14). Mannan oligosaccharide (MOS) is a yeast cell wall derived feed ingredient. It works locally in the gut enhancing assimilation and gut wellbeing in animals through diminishing the colonization of undesirable bacteria. Furthermore, MOS functions as a prebiotic, favoring development of helpful bacteria in the gut (15, 16). AGRIMOS® is a particular mix of MOS and B-glucans separated from the yeast cell walls of Saccharomyces cerevisiae; that are especially critical to help the non-specific immune system of the animal (17). Although few investigations revealed the dietary prerequisites of biomos and Agrimos to maintain growth, while, immunological responses still not very much archived particularly on the fish resistance level (17). The current study was directed to evaluate the potential benefits of Biomos® and Agri-mos® dietary supplementation on growth performance, feed utilization, body chemical composition, internal organs indices, hematological parameters, immunological response and economical efficiency of Nile Tilapia (O. niloticus) fingerlings under Egyptian conditions. Material and methods This study was conducted in fish Aquaculture Research Unit in Kafr El-Sheikh Gover-norate. All handlings of fish were directed according to the guidelines for animal care and use for scientific purposes built up by the Ethics Committee of the Faculty of Agriculture, Kafrelsheikh University, Egypt (Approval Date: 18-03-2018). Diet preparation A basal diet was formulated from commercial ingredients including fish meal, soybean meal, yellow corn, wheat bran, vitamins, minerals mix and fish oil. The dry ingredients were grounded utilizing a feed processor into little size particles. Seven diets were formulated from the basal diets by adding the prebiotics at different concentrations. Contents and chemical composition of each diet were exhibited in Table (1; A & B). The ingredients were weighed and blended by a mixture blender for 20 minutes. A constant Biomos and Agrimos levels was added for all diets except control diets. After homogenous blending, every hundred gram diet was gradually added to the blend as indicated by (18). The diets were cooked on water exaporator for 20 minutes. The diets were pelleted through grain machine and the pellets were dried at room temperature for 24 h before utilized. The pellets were gathered and spared in plastic bags and stored in a refrigerator at 40C through the experimental period to dodge nutrients deterioration. The utilized feed additive, Biomos® and Agrimos®, were commercial natural enhancers blend; (Bio-Mos®; Alltech, Inc., Nicholasville, KY, USA), (Agrimos®; LALLEMAND ANIMAL NUTIION, FRANCE). Experimental design The experiment was performed using 315 Nile Tilapia (O. niloticus) fingerlings (weighing on average 7 ± 0.5 g). They were collected from a private fish farm in Al Reyad, tolompate Impact of biomos and agrimos dietary supplementation on growth performance ... 89 7, Kafr El-Sheikh Governorate. All collected fish were kept in a fiberglass tank, for three weeks for accommodation; where fish were fed a commercial diet (containing 30% dietary protein level (CP). After the accommodation period, the fingerlings were randomly divided into 7 groups of 45 fingerlings / each group allotted into three replicates of 15 fingerlings / each replicate). Fingerlings were put in glass aquariums of 60 x 35 x40 cm in size contained 70 L of water, (15 fish/ aquarium) and were equipped with effective aeration system. The seventh Groups 1 (control group) were fed a commercial diet, Group 2to 4 were fed diets supplied with 1, 2, 3 kg/ ton) of Biomos. While, groups 5 to 7 were fed diets supplemented with 1, 2, 3 kg/ ton of Agrimos. Fish were fed the experimental diets for 12 weeks at a rate of 3% of the all-out stocking bi-omass/aquarium. Diets were applied twice a day (at 8:00 am & 14:00 pm). Fish were weighed at fortnightly intervals along the experimental period and the feed amounts were corrected by the change in live body weight. Fish excreta and feeding wastes were expelled by siphoning and about half of water in every aquarium was day by day replaced by dechlo-rinated new water. PER=Live weight gain (g)/protein intake (g). (21). Determination of diet proximate analysis Dry matter, crude protein, ether extract, crude fiber and ash contents of the experimental diets and the whole body of fish at the end of the experiment were performed according to AOAC (22). Hematological investigations Toward the finish of the experiment, twelve fishes from every group (4 fishes /every replicate) were randomly sampled and weighed. Anti-coagulated blood samples were taken from the caudal vein for blood analysis and differential leukocyte count. Due to the small fish size, blood samples collected from 3-4 fish were pooled according to Urbinate & Carneiro (23). Red blood cells count (RBCsx106/mm) and white blood cells count (WBCsx103/mm) were determined according to the method described by Stoskopf, (24). Hemoglobin concentration (Hb g/dl) was estimated according to the method of Zinkl (25). Packed cell volume (PCV %) was estimated by the micro-haematocrite method described by Decie & Lewis (26). Determination of internal organs indices Determination of fish growth parameters The fish were totally weighed (15 fish/each repliToward the finish of the experiment, four cate) using an electronic balance. Total weight gain (TWG), average daily gain (ADG), specific growth rate (SGR), survival rate (SR %), feed conversion ratio (FCR), and protein efficiency ratio (PER), were calculated according to following equations: fishes from every treatment were slaughtered and the abdominal cavity was directly opened to evacuate liver, kidney, spleen and gonads then weighed separately. Liver index (HSI), kidney index (KSI), spleen index (SSI) and gonads index (GSI) were calculated as follows: Total weight gain (TWG) (g) = last body weight -HePato s°matic index (^ %) = 100 x weight (g) / body weight (g)] (27). Kidney somatic index (KSI %) =100x [kidneys weight (g) /body weight (g)] (28). n somatic index (SSI %) = 100x [spleen weight (g) / body weight(g)] (29). Gonado somatic index (GSI %) =100x [gonads weight (g) / body weight (g)] (30). Immunological parameters Phagocytic activity (PA) and index (PI) were determined according to Kawahara et al., (31). beginning body weight (19). Average Daily Gain (g/fish/day) = TWG (g)/trial period (d). Specific growth rate (SGR % / day) = [Ln last body weight- Ln introductory body weight] x 100/trial period (d) (20). SR= Total number of fish at the end of the experiment x 100/ absolute number of fish at the start of the experiment. FCR= feed consumption (g)/Live weight gain. (21). 90 F. Magouz, M. Salem, E. Moustafa, S. Elkhamy The nitro blue tetrazolium assay was used to investigate the respiratory burst activity as previously described (32). The lysozyme activity was examined by the technique described by Demers and Bayne (33) depending on the ability of lysozyme to lyses Gram positive lysozyme delicate bacterium; Micrococcus lyso-deikticus. Biochemical parameters Total antioxidant capacity (TAC) of liver tissue was performed by the technique described by Prieto et al., (34). Catalase (CAT) activity was performed utilizing spectrophotometric assurance of hydrogen peroxide (H2O2) which framed stable complex with ammonium molyb-date (35). Statistical analysis The obtained data were statistically analyzed utilizing general direct models technique adjusted by SPSS (36) for users guide, with a restricted ANOVA. Means were statistically compared for the significance (P < 0.05) using Duncans multiple range test (37). Results In the current study, the physiological responses of O. niloticus fingerlings to Biomos® and Agrimos® were researched through assurance of fish development and hematological parameters. It was observed that there was a huge increase in absolute weight gain (TWG) and average daily gain (ADG) in all prebiotics treated groups except in T7 group compared with control group (T1). The most elevated qualities were noticed in the T2 and T6 groups. However, the specific growth rate (SGR) was fundamentally expanded in T2 group only. Survival rate percent (SR%) was significantly increased in T2, T3, T5, T6 groups with most noteworthy qualities reported in the case of T2 & T5 groups as shown in table (2: A). The impacts of the two utilized prebiotics on feed intake, food conversion rate (FCR) and protein efficiency ratio (PER) were summarized in table (2: B). It was noticed that the best food conversion rate and protein efficiency ratio values were in T2 and T6 groups. Chemical composition of the experimental fish body, average dry matter (DM), crude protein (CP), ether extract (EE), ash and nitrogen free extract (NFE) were determined and summarized in table 3. There was a critical difference in the DM in case of T2, T3, T6 and T7 groups with most astounding increment found in the T3 group. While, CP% was fundamentally expanded in T2, T5 followed by T3 group contrasted with other treated groups. On the other hand, ether extract was altogether diminished in all treated groups contrasted with control one. The most astounding estimation of ash content was recorded in T3 and T5 groups and the least incentive in T7 group. RBCs, and WBCs count, Hb, and PCV demonstrated noteworthy increases in all prebi-otics supplemented groups contrasted with the control one (Table 4). The highest level of RBCs count was recorded in T3 and T6 groups. The results of serum total proteins, albumin and globulin, showed non-significant increases in all probiotic treated groups contrasted with the control group except T4 group which expanded essentially. The T4 group demonstrated a huge decline in AST level. No noteworthy changes were observed in ALT in all groups. Regarding immunological parameters, phag-ocytic activity and index, respiratory burst activity, lysozyme, total antioxidant and catalase activities were altogether expanded and increased in all groups with most extreme dimensions on account of group 2 and group 6, respectively (Table 5). Discussion In aquaculture, probiotics can be admitted either as feed added substances or as added substances to the water (38, 39). The shape and span of prebiotic and probiotic administration can impact their viability on fish health (8). The dietary supplementation of pre- and probiotics has been archived as a superior strategy of guaranteeing the effectiveness of the probiotic bacterial colonization in the fish gastrointestinal tract (7, 14, 40). Aqua feeds industry are focusing mainly on getting double advantages of both upgraded development and resistant reaction of vast majority Impact of biomos and agrimos dietary supplementation on growth performance ... 91 Table 1A: Composition of the experimental diets offered for each group Ingredients Dietl con- Diet2 Diet3 Diet4 Diet5 Diet6 Diet7 trol Fish meal 10 10 10 10 10 10 10 (72% CP) Yellow corn 22 22 22 22 22 22 22 Soybean 42 42 42 42 42 42 42 meal (45% CP) Wheat bran 20 20 20 20 20 20 20 Fish Oil 5 5 5 5 5 5 5 Vit&Min 1 1 1 1 1 1 1 Biomos - 0.1 0.2 0.3 - - - Agrimos - - - - 0.1 0.2 0.3 Table 1B: Chemical analysis of the experimental diets (% on DM basis). Ingredients Diet 1 Diet 2 Diet 3 Diet 4 Diet 5 Diet 6 Diet 7 control (0.1% Bio- (0.2% Bio- ■ (0.3% Bio- (0.1% (0.2% Ag- (0.3% Ag- mos) mos) mos) Agrimos) rimos) rimos) Dry matter 92.78 92.38 92.22 92.07 92.56 92.60 92.66 Crude protein 31.08 31.08 31.08 31.08 31.08 31.08 31.08 Ether extract 6.32 5.62 4.71 4.94 6.71 6.02 6.10 CF 1.10 2.21 1.92 1.93 2.13 2.09 2.21 Ash 6.73 6.65 6.45 9.37 6.49 6.61 6.14 NFE 54.77 54.44 55.84 52.68 53.59 54.2 54.47 Gross energy(GE) 458.265 445.622 448.879 437.775 459.625 453.861 454.254 (Kcal/lOOg)1 Digestible energy(DE) 321.82 318.4 321.32 307.37 327.94 319.51 317.83 (Kcal/lOOg) 2 P/E ratio3 67.82 72.39 71.35 72.61 71.21 69.60 67.38 1 Gross energy (Kcal/100g), based on 5.6Kcal/g protein, 9.44 Kcal/g lipid, 4.1 Kcal/g carbohydrate. 2 Digestible energy (Kcak/100g), based on 5.0Kcal/g protein 9.0Kcal/g lipid, 2.0Kcal/g carbohydrate. According to (Wee & shu, 1989). 3 P/E (protein to energy ratio)= mg crude protein/Kcal of gross energy. 92 F. Magouz, M. Salem, E. Moustafa, S. Elkhamy Table 2A: Effect of Used prebiotics on growth parameters and survival rate of Nile tilapia Treatment I.W. F.W. T.W.G A.D.G S.G.R S.R.% (g/fish) (g/fish/day) (%/day) T1 7.02±.02 24.93±0.06b 18.10±0.20d 0.16±0.03c 1.3±0.03b 95.5±2.2c T2 6.97±.02 29.46±0.78a 23.50±0.36a 0.27±0.01a 1.8±0.16a 100±0.00a T3 7.00±.00 27.36±0.08ab 20.86±0.84ab 0.21±0.01ab 1.5±0.03ab 97.7±2.2b T4 7.00±.00 26.96±1.00ab 20.13±0.98b 0.18±0.04b 1.43±0.03b 95.5±4.4c T5 7.32±.32 27.00±1.01ab 20.86±1.2ab 0.21±0.01ab 1.6±0.18ab 100±0.00a T6 7.00±.00 28.86±0.57a 21.86±0.59ab 0.23±0.01ab 1.7±0.15ab 97.7±2.2b T7 7.01±.01 25.06±0.86b 18.26±0.84cd 0.16±0.03c 1.40±0.05b 95.5±2.2c T1=Diet 1 (control group), T2= Diet 2 (0.1% Biomos), T3= Diet 3 (0.2% Biomos), T4=Diet 4 (0.3% Bio-mos), T5= Diet 5 (0.1% Agrimos), T6= Diet 6 (0.2% Agrimos), T 7= Diet 7 (0.3% Agrimos), I.W. = Initial Weight, F.W. = Final Weight, T.W.G = Total weight gain, A.D.G = Average daily gain, S.G.R = Specific growth rate, S.R.% = Survival Rat Table 2B: Feed intake, feed conversion rate and protein efficiency ratio of Nile tilapia in response to prebiotics supplementation Treatment FI FCR PER T1 42.16±.16c 2.33±.03a 1.26±.08b T2 48.90±1.50a 1.97±.08b 1.53±.03a T3 47.43±.51a 2.10±.17ab 1.43±.06ab T4 46.33±.83ab 2.22±.13a 1.30±.15b T5 45.33±1.51abc 2.13±.08ab 1.46±.03ab T6 46.16±1.63ab 1.99±.04b 1.53±.03a T7 42.70±1.40bc 2.20±.26a 1.33±.08b T1=Diet 1 (control group), T2= Diet 2 (0.1% Biomos), T3= Diet 3 (0.2% Biomos), T4=Diet 4 (0.3% Biomos), T5= Diet 5 (0.1% Agrimos), T6= Diet 6 (0.2% Agrimos), T 7= Diet 7 (0.3% Agrimos), FI: Feed Intake, FCR: Feed conversion rate, PER: Protein efficiency ratio Impact of biomos and agrimos dietary supplementation on growth performance ... 93 Table 3: Composition Analysis of fish body fed graded levels of Biomos and Agrimos Treatment DM CP EE ASH GE Kcal/100g T1 24.52±0.01c 58.92±0.01c 17.54±0.01a 17.84±0.02b 518.89 T2 24.34±0.02d 64.32±0.01a 13.33±0.01b 18.42±0.01a 502.140 T3 25.16±0.01a 61.52±0.01b 13.57±0.01c 19.16±0.01a 496.187 T4 24.52±0.01c 57.52±0.01c 13.33±0.02c 18.42±0.01a 491.940 T5 24.55±0.01c 64.32±0.01a 15.07±0.01b 18.71±0.00a 510.242 T6 24.65±0.00b 58.66±0.01c 17.17±0.01a 18.17±0.00a 515.180 T7 24.10±0.00e 59.72±0.01b 15.33±0.01b 17.74±0.00b 508.708 T1= Diet 1 (control group), T2= Diet 2 (0.1% Biomos), T3 Diet 5 (0.1% Agrimos), T6= Diet 6 (0.2% Agrimos), T 7= crude protein, EE= ether extract, GE= gross energy = Diet 3 (0.2% Biomos), T4= Diet 4 (0.3% Biomos), T5= Diet 7 (0.3% Agrimos). DM= Averege dry matter, CP= Table 4: Effect of Biomos and Agrimos on haematological parameters and serum biochemical analysis in Oreochromis niloticus T1 T2 T3 T4 T5 T6 T7 RBCs (x10/mm3) Hb (g/100ml) PCV (%) WBCs (x103/mm3 ) Total protein (g/dl) Albumin (g/dl) Globulin (g/dl) ALT (U/I) AST (U/I) 2.8±.02c 2.9±.02bc 3.9±.05a 3.2±.12abc 3.3±.07abc 3.8±.09a 3.5±.53ab 8.1±.11a 9.2±.70a 10.57±.67a 10.51±1.04 9.5±.26a 8.3±.21a 10.9±1.6a a 24±0.00b 26±2.0ab 30.5±2.5a 30±1.00ab 27.5±.50a 26.5±.50a 32±3.00a b b 31.9±5.01 37.7±7.9a 41.3±10.1a 38.7±.7a 44.4±4.4a 32.2±1.5a 36.3±7.3a ab 4.8±.10b 5.2±.20ab 5.1±.24 5.9±.11a 5.6±.24ab 5.7±.39ab 5.8±.38 2.9±.08a 3.06±.06a 3.02±.21a 3.4±.08a 3.3±.05a 3.4±.22a 3.2±.19a 1.9±.02b 2.14±.14a 2.11±.03ab 2.49±.03a 2.3±.18ab 2.41±.17a 2.41±.19' 5.10±.28a 5.11±.02a 5.11±.30a 4.5±.32a 5.3±.10a 4.8±.04a 5.11±.19a 77±.93a 76.6±.4a 75.1±4.01a 69.8±.04b 78±.90a 72.8±.57a 62.8±.40c T1=Diet 1 (control group), T2= Diet 2 (0.1% Biomos), T3= Diet 3 (0.2% Biomos), T4=Diet 4 (0.3% Biomos), T5= Diet 5 (0.1% Agrimos), T6= Diet 6 (0.2% Agrimos), T 7= Diet 7 (0.3% Agrimos). RBCs = Red Blood Cells, HB = Haemoglobin, PCV = Packed Cell Volume, WBCs = White Blood Cells, ALT = Alanine aminotransferase, AST = Aspartate amninotransferase a b b b b b 94 F. Magouz, M. Salem, E. Moustafa, S. Elkhamy Table (5): Effect of prebiotics supplementation on immunity and biochemical parameters Treatment Phagocytic activity phagocytic index Respiratory burst activity lysozyme activity Total antiox-idant activity Catalase activity T1 16.47±1.02c 4.27±0.08a 2.41±0.03Da 0.04±0.001Da 0.38±0.002Fa 2.05±0.15Ea T2 42.55±1.66ab 19.33±0.41ab 8.72±0.022Aa 0.83±0.002Aa 1.95±0.006Aa 7.78±0.08Aa T3 31.35±1.73a 13.26±0.27ab 5.37±0.021Ba 0.34±0.003Ba 1.21±0.023Ca 3.56±0.05Ca T4 29.63±3.11bb 11.47±0.74a 4.68±0.044Cb 0.39±0.003Cb 1.54±0.002Ab 5.31±0.08Ab T5 28.27±1.64abc 11.37±0.44a 4.23±0.032Ba 0.025±0.005Ba 1.11±0.002Da 2.14±0.17Db T6 39.93±1.27ab 16.25±0.36b 7.29±0.027Aa 0.76±0.003Aa 1.52±0.002Ba 5.13±0.13Ba T7 25.87±3.47bc 9.62±0.89a 3.85±0.052Cb 0.31±0.004Cb 1.12±0.002Bb 4.33±0.02Bb T1=Diet 1 (control group), T2= Diet 2 (0.1% Biomos), T3= Diet 3 (0.2% Biomos), T4=Diet 4 (0.3% Biomos), T5= Diet 5 (0.1% Agrimos), T6= Diet 6 (0.2% Agrimos), T 7= Diet 7 (0.3% Agrimos) Aqua feeds industry are focusing mainly on getting double advantages of both upgraded development and resistant reaction of vast majority of farmed fish species, as well as preventive medicinal services by means of numerous healthful procedures to guarantee its manageability in the aquaculture system (41). The nutritional status is the main key that affects the immune status of cultured fish species so as to finally get a superior assurance (42). In the current study, the increased growth parameters among prebiotic treated groups may be attributed to improved feed utilization in fish. The results are similar to those reported by some authors (43-47); but in contrast to those reported by Genc et al., (48). The improvement of food conversion rate, the best was recorded in T2 and T6 groups, may be due to the effect of used prebiotics in the current study which led to decreased amount of feed necessary for producing one unit of fish leading consequently to production cost reduction. The results are in agreement with some authors (45, 46, 49). The dietary MOS could altogether build the intestinal microvilli length or potentially thickness prompting expanded nutrient absorptive capacity (43, 46, 49). The results of chemical composition of the experimental fish body, average dry matter, crude protein, ether extract, ash and nitrogen free extract, are in a partial consent to Orban et al., (50), where they recorded that body composition was strongly affected by their feed composition. In the current study, there were no critical contrasts recorded in body composition among groups; however, an expansion was noticed in protein level of carcass prawn with expanding the Agrimos® incorporation level in the diet. These outcomes are like those announced by numerous authors (45, 48, 51, 52). The elevated number of WBCs may be attributed to the improved defense response as a result of Biomos and/or Agrimos feed supplementation. The obtained result is similar to those reported by some authors (53-55). However, it is in contrast to other authors (56, 57). The variety in the results of haematological parameters might be due to the probiotic type and dose, fish physiological status, species, size, age, ecological conditions and dietary routine (58). The increased level of total proteins in all prebiotic treated groups may be attributed to improved body defense and stronger innate response of fish as a result of prebiotic supplementation (53, 59, 60). The results were in agreement to some authors (57, 61); however, in contrast to Andrews et al. (53). Fluctuations in hematological and serum biochemical factors may be species-related and rely upon the Impact of biomos and agrimos dietary supplementation on growth performance ... 95 incorporation rates of MOS, diet ingredients and/or the raising time frame period (62). This study demonstrated that the immune response of O. niloticus fingerlings was fundamentally influenced and expanded in all groups with the maximum level in group 2 and 6. Ly-sozyme is a standout amongst the most fundamental safe reactions of fish. It is originating from neutrophils and macrophages emitted into blood and mucus to apply bacteriolytic impacts (63) helping organisms to oppose bacterial, viral and parasitic diseases (64). The present outcomes demonstrated that the dimensions of ly-sozyme action were fundamentally modified due to Biomos and Agrimos supplementation. The highest serum lysozyme activity was observed in fish encouraged eating diet with 0.1% Biomos diet and 0.2% Agrimos, respectively. The expanded lysozymal action might be due to the immune-stimulatory effects of dietary Biomos and Agrimos (42). Respiratory burst activity, a key for innate immunity, could be estimated utilizing nitro-blue tetrazolium (NBT) (65, 66). It shows the oxidative capability of reactive oxygen species (ROS) like; hydrogen peroxide, superoxide anions, and hydroxyl radicals (67), delivered by initiated phagocytic cells and in charge of killing or degrading engulfed materials, including microbes (67). In the present study, group 2 (0.1% Biomos) and group 6 (0.2% Agrimos) demonstrated a huge upgrade of NBT toward the finish of the investigation. It implies that both Biomos and Agrimos assumes a critical job in activating antioxidant defense systems including superoxide dismutase (SOD), cata-lase (CAT), and glutathione peroxidase (GPx) (68). The expanded bactericidal, lysozyme, and antioxidant activities in the present investigation might be attributed to the resistant stimulatory impacts of dietary Biomos and Agrimos. Conclusion It could be concluded that prebiotics supplementation of Biomos® and Agrimos® are highly beneficial in O. niloticus fingerlings diets resulting in an increased nutrient utilization and improving growth rate, hematological, bi- ochemical parameters, immunological responses and survival rate. From the obtained results, it is preferable to use Biomos® and Agrimos® as feed additives at levels of 0.1% and 0.2%, respectively, with commercial feeds to improve tilapia fingerlings immune response. References 1. GAFRD. General Authority for Fishery Resources Development. Fish Statistics Year- book 2014. Egypt: Ministry of Agriculture and Land Reclamation. 2016. 2. Dickson M, Nasr-Allah A, Kenawy D, Kruijs-sen F. Increasing fish farm profitability through aquaculture best management practice training in Egypt. Aquacult 2016; 465:172-8. 3. Tri nhu N. The utilization of soybean products in tilapia feed-A review. Proceeding of (ISTA). 8th Int Symp Tilapia Aquac 2008; (1): 53-67. 4. Gatesoupe FJ. The use of probiotics in aquaculture. Aquacult 1999; 180: 147-65. 5. Plumb JA. Health maintenance and principal microbial diseases of cultured fishes. Ames: The Iowa State University Press. 1999. 6. Sakai M. Current research status of fish im-munostimulants. Aquacult. 1999;172: 63-92. 7. Merrifield DL, Dimitroglou A, Foey A, Da-vies SJ, Baker RTM. The current status and future focus of probiotic and prebiotic applications for salmonids. Aquacult. 2010; 302: 1-18. 8. Welker TL, Lim C. Use of probiotics in diets of tilapia. J Aquac Res develop. 2011; S1, 014. 9. Hai NV. Research findings from the use of probiotics in tilapia aquaculture: a review. Fish Shellfish Immunol 2015; 45: 592-7. 10. Ibrahem MD. Evolution of probiotics in aquatic world: Potential effects, the current status in Egypt and recent prospectives. J Adv Res 2015; 6: 765-91. 11. Gibson GR Roberfroid MB. Dietary modulation of the human colon microbiota: introducing the concept of prebiotics. J Nut 1995; 125: 140112. 12. Manning T, Gibson G. Prebiotics. Best Practice & Research: Clin. Gastroent 2004;18: 287-98. 13. FAO (Food and Agriculture Organization). "FAO technical meeting on prebiotics." FAO: Rome. September 15—6. 2007 14. Mei GY, Carey CM, Tosh S, Kostrzynska M. Utilization of different types of dietary fibers by potential probiotics. Canad J Microb 2011; 57: 857-65. 96 F. Magouz, M. Salem, E. Moustafa, S. Elkhamy 15. Staykov Y, Spring P, Denev S and Sweet-man J. Effect of amannan oligosaccharide on the growth performance and immune status of rainbow trout (Oncorhynchus mykiss). Aquacult Int 2007; 15: 153-61. 16. Refstie S, Baeverfjord G, Seim RR, Elvebe O. Effects of dietary yeast cell wall ß-glucans and MOS on performance, gut health and salmon lice resistance in Atlantic salmon (Salmo salar) fed sunflower and soybean meal. Aquacult 2010; 305: 109-16. 17. Mohamed Kh, Megahed ME, Ali MM. Effect of dietary supplementation of Agrimos® on growth performance, feed utilization and immuno-logical parameters of Macrobrachiumrosenbergii juveniles. Aquacult Int 2017; 25:1441-52. 18. Shimeino S, Masumoto T, Hujita T, Mima T, Ueno SI. Alternative protein sources for fish meal diets of young yellowtail. Nipp Suis Gakk 1993;59:137-43. 19. Annet CS. A model to facilitated optimal aquaculture production by quantitatively relating fish growth to feed and other environmental resources. Ph.D., Thesis, Michigan. State University, U.S.A. 1985. 20. Pouomonge V, Mbonglang M. Effect of feeding rate on the growth of tilapia (O. niloticus) in earthen ponds. Isr J aquac 1993; 45: 147-53. 21. De Silva SS, Anderson TA. Fish Nutrition in Aquaculture, Ed., Champman and Hall, 2-6 Bou-day Raw, London SEI8 FIN, UK. 1995. 22. AOAC. Official methods of Analysis. In: Association of Official Analysis of Chemists, Washington D.C. 15th Ed. 1990. 23. Urbinate EC, Carneiro PCF. Sodium chloride added to transport water and physiological responses of Matrinxä Brycon amazonicus (Teleost: Caracidae). Acta Amaz 2006; 36: 569-72. 24. Stoskopf MK. Fish medicine Ed., W.B. Sainders Company, London. 1993. 25. Zinkle JG. Avian hematology. In: Jain NC (Ed. Schalms) Veterinary Hematology, Philadelphia, Pai hea and Febiger. 1986. Pp 256-60. 26. Decie SIV, Lewis SM. Practical Haematol-ogy. Pp.736. Churchill Livingstone, London. ISBN: 13:978-0-443-06660-3. 10th Ed. 2006. 27. Jangaard PM, Ackman RG, Spois AJ. Seasonal studies of the fatty acids composition of liver, flesh, roe and milt lipid. J. Fish. Res. Biol. Boar. Canada. 1967; 24: 613-27. 28. Angelescu V, Gnri FS, Nani A. La merluza del mar argentine (Biologia e taxonomia). Secr. Mar. Serv. Hidrog. Nav. Publico. H1004. 1-224. 1956. 29. Abdelhamid AM, Salem MFI, Tolan AE. Evaluation of sesame meal as a dietary protein source for Nile tilapia (Oreochromis niloticus) fingerlings. J Agri Sci, Mansoura Univ. 2004; 29: 6887-97. 30. Tseng WY, Chan KL. The reproductive biology of the rabbit fish in Hong- Kong. J world Aquac Soc 1982; 13: 31-321. 31. Kawahara E, Ueda T, Nomura S. In vitro phagocytic activity of white spotted shark cells after injection with Aeromonas salmonicida extracelluar products. Gyobyo Kenkyu, Japan, 1991; 26(4): 213-4. 32. Anderson DP, Siwicki AK. Basic haematol-ogy and serology for fish health programs, Shariff M, Arthur JR, R.P. Subasinghe (Eds.), Diseases in Asian aquaculture II. Manila: Philippines fish health section, Asian Fisheries Soc, p. 185. 1995. 33. Demers NE, Bayne CJ. The immediate effect of stress on hormones and plasma lysozyme in rainbow trout. Developmental and Comparative Immunology, 1997; 21: 363-73. 34. Prito P, Pineda M, Anguilar M. Spectropho-tometric quantitation of antioxidant capacity through the formation of a Phosphomolybdenum Complex: Specific application to the determination of Vitamin E. Anal. Biochem., 1999; 269: 337-41. 35. Goth L. A simple method for determination of serum catalase activity and revision of reference range. Clin Chim Acta 1991; 196(2):143-51 36. SPSS. Statistical package for the social sciences, Revisions 6, spss Inc, Chicago, USA. 1997. 37. Duncan DB. Multiple Ranges and Multiple F - test. Biometerics 1955; 11: 1-42. 38. Moriarty DJW. Control of luminous Vibrio species in penaeid aquaculture ponds. Aquacult 1998;164:351-8. 39. Taoka Y, Maeda H, Jo JY, Kim SM, Park S, Yoshikawa T, Sakata T. Use of live and dead pro-biotic cells in tilapia, Oreochromis niloticus. Fish. Sci. 2006; 72: 755-6. 40. Rurangwa E, Laranja JL, Van Houdt R, Delaedt Y, Geraylou Z, Van de Wiele T, Van Loo J, Van-Craeyveld V, Courtin CM, Delcour JA, Ollevier F. Selected non-digestible carbohydrates and prebiotics support the growth of probiotic fish bacteria mono-cultures in vitro. J App Microb 2009;106: 932-40. 41. Kiron V. Fish immune system and its nutritional modulation for preventive health care. Anim Feed Sci Technol 2012; 173(1): 111-3. 42. Dawood MAO, Koshio S, Abdel-Daim MM, Van Doan H. Probiotic application for sustainable Impact of biomos and agrimos dietary supplementation on growth performance ... 97 aquaculture. Rev Aquac 2018; https://doi.org/10. 1111/raq.12272 43. Sang HM, Fotedar R. Effects of dietary beta-1,3-glucan on the growth, survival, physiological and immune response of marron, Cherax tenui-manus (Smith, 1912). Fish Shellfish Immunol. 2010a; 28:957-60. 44. Sang HM, Fotedar R. Effects of mannan ol-igosaccharide dietary supplementation on performances of the tropical spiny lobster juvenile (Pan-ulirus ornatus). Fish and Shellfish Immunol. 2010b;28: 483-9. 45. Mazlum Y, Güner Ö, Sirin S. Effects of feeding interval on growth, survival and body composition of narrow clawed crayfish, Astacus lepto-dactylus Eschscholtz, 1823 juveniles. Turk J Fish Aquat Sci 2011; 11: 283-89. 46. Zhang J, Liu Y, Tian L, Yang H, Liang G, Xu D. Effects of dietary mannan oligosaccharide on growth performance, gut morphology and stress tolerance of juvenile Pacific white shrimp (Li-topenaeus vannamei). Fish Shellfish Immunol. 2012;33:1027-32. 47. Samuel A, Adel AC, Malachi AW, Donald AD. Effects of Bacillus subtilis strain and the prebi-otic previda on growth, Immune parameters and susceptibility to Aeromonas hydrophila infection in Nile Tilapia. Aquac Res 2017; 1-13. 48. Genc MA, Aktas M, Genc E, Yilmaz E. Effects of dietary mannan oligosaccharide on growth, body composition and hepatopancrease histology of Penaeus semisulcatus (de Haan 1844). Aquac Nut 2007; 13:156-61. 49. Daniels C, Merrifield D, Boothroyd D, Da-vies S, Factor J, Arnold K. Effect of dietary Bacillus spp. and mannan oligosaccharide (MOS) on European Lobester (Homarus gammarus L.) larvae growth performance, gut morphology and gut mi-crobiota. Aquacult 2010; 304: 49-57. 50. Orban E, Nevigato T, Masci M, Lena DG, Casini I, Caproni R, Gambelli L, Angeli DP, Ramp-cci M. Nutritional Quality and safety of European Perch (Perca fluviatilis) from three lakes of central Italy. Food Chem 2007; 100: 482-90. 51. Shankar R. Efficacy of nucleotides in enhancing immune response and disease resistance against white muscle disease in giant freshwater prawn, Macrobrachium rosenbergii (de man). PhD Thesis submitted to Karnataka University, Animal and fisheries Sciences Uniiversity, Bidar, Karna-taka. 2008. 52. Jagadeesh TD. Evaluation of mannan oligo-saccharide (MOS) on growth, survival and immune response challenged to Aeromonas hydrophila in Indian major carp, Labeo rohita. (M. F. Sc. Thesis submitted to Karnataka Veterinary, Animal and Fisheries Sciences University, Bidar, Karnataka, India). 2010. 53. Andrews SR, Sahu NP, Pal AK, Kumar S. Hematological modulation and growth of Labeo rohita fingerlings: effect of dietary mannan oligosaccharide, yeast extract, protein hydrolysate and chlorella. Aquac Res 2009; 41: 61-9. 54. Sado RY, Bicudo AJA. Dietary levamisole influenced hematological parameters of juvenile pacu, Piaractus mesopotamicus (Holmberg 1887). J World Aquac Soc 2010; 41: 66-75. 55. Ha N, Gonc.alves AFN, Sousa LC, Biller-Takahashi JD, Takahashi LS. Dietary carbohydrates and protein of yeast modulate the early stages of innate immune response in tilapia (Oreochromis niloticus) primarily after LPS inoculation. Aquac Int 2017; 25:755-76. 56. Welker TL, Lim C, Yildirim-Aksoy M, Shelby R, Klesius PH. Immune response and resistance to stress and Edwardsiella ictaluri, fed diets containing commercial whole cell yeast or yeast subcomponents. J World Aquac Soc 2007; 38: 24-5. 57. Ali SR, Ambasankar K, Praveena E, Nan-dakumar S, Syamadayal J. Effect of dietary mannan oligosaccharaide on growth, body composition, he-matology and biochemical parameters of Asian sea-bass (Lates calcarifer). Aquac Res 2017; 48: 899908. 58. Osuigwe DI, Obiekezie AI, Onuoha GC. Some hematological changes in hybrid catfish (Het-erobranchus longifilis x Clarias gariepinus) fed different dietary levels of raw and boiled jack bean (Canavalia ensiformis) seed meal. Afr J Biotech 2005; 4:1017-21. 59. Thomas JS. Overview of plasma proteins. In: Feldman BF, Zinkl JG, Jain NC (eds) Schalm's Veterinary Hematology. pp 891-989. Lippincott Williams, Wilkins, Philadelphia,. 5th ed. 2000. 60. Sahoo PK, Mukherjee SC. Effect of dietary 1,3 b-glucan on immune responses and disease resistance of healthy and aflatoxin B1-induced immune compromised rohu (Labeo rohita Hamilton). Fish Shellfish Immunol. 2001; 11: 683-95. 61. Razeghi-Mansour M, Akrami R, Ghobadi SH, Amani-Denji K, Ezatrahimi N, Gharaei A. Effect of dietary mannan oligosaccharide (MOS) on growth performance, survival, body composition, and some hematological parameters in giant sturgeon juvenile (Huso huso) Linnaeus, 1754. Fish Physiol Biochem 2012; 38: 829-35. 98 F. Magouz, M. Salem, E. Moustafa, S. Elkhamy 62. Ta'ati R, Soltani M, Bahmani M, Zamini AA. Growth performance, carcass composition and immune physiological indices in juvenile great sturgeon (Huso huso) fed on commercial prebiotic, Im-munoster. Iran J Fish Sci 2011; 10: 324-35. 63. Saurabh S, Sahoo P. Lysozyme: an important defense molecule of fish innate immune system. Aquac Res 2008; 39: 223-239. 64. Yano T. the nonspecific immune system: humoral defense. Fish Physiol 1997; 15:105-57. 65. Dawood MAO, Koshio S, Ishikawa M, Yokoyama S. Effects of dietary inactivated Pedio-coccus pentosaceus on growth performance, feed utilization and blood characteristics of red sea bream, Pagrus major juvenile. Aquac Nut 2016; 22(4):923-32 66. Song SK, Beck BR, Kim D, Park J, Kim J, Kim HD, Ring0 E. Prebiotics as immunostimulants in aquaculture: a review. Fish Shellfish Immunol 2014; 40(1):40-48. 67. Atencio L, Moreno I, Jos Ä, Prieto AI, Mo-yano R, Blanco A, Cameän AM. Effects of dietary selenium on the oxidative stress and pathological changes in tilapia (Oreochromis niloticus) exposed to a microcystinproducing cyanobacterial water bloom. Toxico 2009; 53(2):269-82 68. Köhrle J, Brigelius-Flohe R, BöckA, Gärtner R, Meyer O, Flohe L. Selenium in biology: facts and medical perspectives. Biol Chem 2000; 381(9-10):849-64. Slov Vet Res 2019; 56 (Suppl 22): 99-108 DOI 10.26873/SVR-747-2019 Original Research Article DIGESTIVE ENZYMES, IMMUNITY AND OXIDATIVE STATUS OF NILE TILAPIA (Oreochromis niloticus) REARED IN INTENSIVE CONDITIONS Mahmoud A.O. Dawood1*, Mustafa Shukry2, Mohamed Mamdouh Zayed3, Amira Alaa El-Dein Omar4, Amr I. Zaineldin5, Mohammed F. El Basuini6 department of Animal Production, Faculty of Agriculture, Kafrelsheikh University, 33516, Egypt, 2Department of Physiology, Faculty of Veterinary Medicine, Kafrelsheikh University. 3Department of Aquaculture, Faculty of Aquatic and Fisheries Sciences, Kafrelsheikh University. 4Department of Fish Diseases and Management, Faculty of Veterinary Medicine Kafrelsheikh University. 5An-imal Health Research Institute (AHRI-DOKI), Egypt, 6Department of Animal Production, Faculty of Agriculture, Tanta University, 31527, Egypt Corresponding author, E-mail: mahmouddawood55@gmail.com Abstract: High stocking density is significantly disturbing the growth and productivity of aquatic animals. Digestive enzymes, immunity and oxidative status of Nile tilapia were investigated in case of culturing in several densities. Fish (14.3±0.03g) were stocked in 12 aquaria (60 L) at four densities of 10 (SD10), 20 (SD20), 30 (SD30) and 40 (SD40) fish per aquarium for 30 days. Fish growth, feed efficiency ratio, digestive enzyme activity and dissolved water oxygen significantly (P<0.05) decreased, while the total ammonia increased with increasing stocking density. Immunoglobulin and NBT levels decreased significantly (P<0.05) in SD40 set compared to SD20 set without no differences with the other two groups. Lysozyme activity reported the highest significant (P<0.05) values in SD10 and SD20 groups over the high stocking density group (SD40) without no difference with SD30 group. Bactericidal, phagocytic activities and phagocytic index reported significantly (P<0.05) lower values in fish reared in SD30 and SD40 groups than fish reared in SD10 and SD20 groups. Peroxidase activity also showed significantly (P<0.05) low values in SD40 and SD30 groups with the weakest activity in SD40 group. Total serum protein lowered relatively in SD30 and SD40 groups without no differences with the other groups. Furthermore, fish reared at high stocking densities resulted in significantly (P<0.05) decreased superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPX) activities as well as increased malonaldehyde (MDA) activity in blood of tilapia suggesting suppressed antioxidant response. In conclusion, intensive conditions depressed the growth, digestive enzyme activity, immunity and oxidative status of Nile tilapia. Key words: digestive enzyme activity; growth; immunity; nile tilapia; oxidative status; stocking density Introduction Intensive aquaculture conditions can affect the water quality negatively; thus, can markedly threaten the fish health and productivity. It has Received: January 2019 Accepted for publication: February 2019 100 M. Dawood, M. Shukry, M. Zayed, A. Omar, A. Zaineldin, M. El Basuini been found that increased stocking led to high ammonia accumulation, hydromineral balance and mortality in different fish species including, tambaqui, winter flounder, Nile tilapia and Japanese flounder (1-4). The low growth performance is concretely affected by the intensive conditions due to decreased feed intake. The feed utilization is related to the activity of microbiota and digestive enzyme activity in the gastrointestinal tract (GIT), which increases feed digestibility and utilization and ultimately improves the growth and health status of fish (5). Protease, lipase and amylase are the major digestive enzymes, which play the main roles in feed digestion and absorption. If the activity of these enzymes increases, overall body metabolism may increase (6). The activities of digestive enzyme were depressed in Nile tilapia and Japanese flounder when reared in intensive conditions (3,4). Additionally, oxidative stress and immunosuppression are the direct features occurring during the intensive conditions, which either caused by the physiological stress (7) or water quality deterioration, such as a decrease in dissolved oxygen and an increase in ammonia levels (8). Due to intensive rearing conditions, the fish respiration rate increases leading to hydromineral imbalance (9, 10). Although not studied, high stocking densities may suppress immune responses of the fish, because physiological stress (11) and low water quality (12) are sources of immunosuppression. As a result, fish become more susceptible to infectious diseases. Nile tilapia (Oreochromis niloticus) is one of the most important cultured fresh water species in the world. Tilapia is usually farmed in Egypt using intensive culture system which often caused stressful circumstances and de-pressingly disturb their growth and wellbeing (13). Low growth performance was observed in tilapia reared in intensive conditions as reported by Liu et al. (3) and Wu et al. (14). For Nile tilapia, there are enough information about the growth performance and feed utilization of fish reared under intensive conditions. However, there is little data on the effect of stocking density on the digestive enzyme activity, immunity and oxidative status of Nile tilapia. Moreover, available information on the relationship between physiological changes with the growth of Nile tilapia stocked at different densities was limited. Therefore, this study aims to investigate the effect of intensive conditions on growth performance, digestive enzyme activity, immunity and oxidative status of Nile tilapia. Material and methods Fish, diet and experimental protocol Tilapia fingerlings were obtained from a private farm located in Kafrelsheikh, Egypt, and transported to Animal Production Department, Faculty of Agriculture, Kafrelsheikh University, Egypt. After 2 weeks acclimation, 300 tilapia (14.3±0.03 g) were put into 12 glass aquaria (60 L) and distributed in four stocking densities; at 10 (SD10), 20 (SD20), 30 (SD30) and 40 (SD40) fish per aquarium. Each aquarium was provided with an air stone for aeration. Feeding rate was fixed at 2 to 3 % of body weight per day with two feeding times 8:00 and 15:30 hr for 30 days. Fish fed diets prepared as described by Dawood et al., (13). The nutritional profile for each diet was confirmed by AOAC (15). The leftover feed was siphoned out after 3 h and 50 % of water was replaced daily with fresh, dechlorinated water of similar temperature. Lighting in the culture unit was set at 12:12 light: dark cycle throughout the study. Water quality parameters were monitored regularly throughout the experimental period. Water temperature, pH and dissolved oxygen (DO) were measured using thermometer, portable digital pH meter (Martini Instruments Model 201/digital) and Waterproof Portable Dissolved Oxygen (model Hanna waterproof IP67). Total ammonia-nitrogen was measured calorimetrically. Sampling schedule All fish were fasted for 24 hr prior to final sampling. Fish were individually measured for final body-weight. Then, the intestine sampled for digestive enzymes analysis from 9 fish per experimental group. Fish were randomly caught and euthanized by "diluted tricaine me-thanesulfonate (MS-222; 400 ppm ratio; Sigma-Aldrich, Egypt)". Intestine aseptically Digestive enzymes, immunity and oxidative status of Nile tilapia (Oreochromis niloticus) reared in intensive 101 taken, washed with PBS (pH 7.5; 1 g per 10 mL), homogenized and centrifuged for 5 min at 8000 rpm. The supernatant was then kept at 4°C. The total protein content was measured using diluted homogenates following the method of Lowry et al. (16) using bovine serum albumin as a standard. Protease activity was evaluated according to Anson (17) using Folin phenol reagent, and amylase activity was measured according to Jiang (18) and Worthington (19) using iodine solution to reveal non-hydrolysed starch. Protease and amylase activities were both expressed as "specific activity" (units per mg of protein). Specific activity of lipase was assessed based on the protocol described by Borlongan (20) and Jin (21) with olive oil as a substrate. Fatty acid, derived from enzymatic hydrolysis of triglyceride on stabile emulsion of olive oil, was titrated with NaOH. One unit of specific activity of lipase was determined as the volume of NaOH 0.05 N needed to neutralize fatty acid released after 6 hours-long incubation with substrate. Lipase activity was expressed as "units per gram" of intestine content. Blood collection and immunological assays Blood was collected from the caudal vein of 9 anaesthetized fish per group and quickly put into 1 ml EDTA coated vials for whole blood and non-coated vials for serum collection. The blood samples were left for 30 minutes till blood clotting then serum separation by centrif-ugation at 3000 rpm for 10 minutes. Serum samples were stored at -20 °C until further analysis. Blood total serum protein was carried out by RA-50 chemistry analyzer (Bayer) using readymade chemicals (kits) supplied by Spinre-act Co. Spain, following manufacturer's guidelines. Immunoglobulin M (IgM) was measured by an ELISA assay using a commercial kit (Cusabio; Wuhan, Hubei, China). The result of IgM was expressed as mg per dl. Respiratory burst activity of the whole blood was quantified by the nitro-blue-tetrazolium (NBT) assay according to Secombes (22). The NBT reduction was measured using the micro- plate reader (Optica, Mikura Ltd, UK) at 630 nm. Lysozyme activity was measured following Parry et al. (23). The result was expressed as "a reduction in absorbency of 0.001/min". Serum bactericidal activity against Aeromonas hy-drophila was detected by following Rainger and Rowley (24). The results were recorded as survival index (SI). Values were calculated as follows: "SI = CFU at end / CFU at start x100". The total peroxidase activity of serum was also assessed using the spectrophotometer at 540 nm as described by Quade and Roth (25) and partially modified by Sahoo et al. (26). Phagocytic activity and phagocytic index were determined following Kawahara et al. (27). The number of phagocytized cells was counted in the phagocytic cells to calculate the phagocytic index according to the following equations: "Phagocytic activity (PA) = Macrophages containing yeast/Total number of Macrophages x100; Phagocytic index (PI) = Number of cells phagocytized/Number of phagocytic cells". Oxidative status Superoxide dismutase (SOD), malonalde-hyde (MDA), catalase (CAT), and glutathione peroxidase (GPX) in fish serum were measured using the diagnostic reagent kits following the manufacturer's (Cusabio Biotech Co., Ltd; China) procedure. Growth performance calculations During the final sampling, all fish per tank were weighed separately. Growth performance and feed utilization were evaluated using weight gain (WG), specific growth rate (SGR) and feed efficiency ratio (FER). Calculations were made using the following formulae: WG (%) = (FBW- IBW) x100/IBW; SGR (%BW/day) = 100((lnFBW -lnIBW)/T); FER = WG /FI. Where FBW = body weight final (g), IBW= body weight initial (g), T = duration of the trial in days, WG = wet weight gain (g) and FI = estimated feed intake (g). 102 M. Dawood, M. Shukry, M. Zayed, A. Omar, A. Zaineldin, M. El Basuini Statistical analysis Shapiro-Wilk and Levene tests confirmed normal distribution and variance homogeneity. All statistical differences were assessed by oneway ANOVA tests (SPSS version 22, SPSS Inc., Il, USA) with Duncan's as post-hoc test where differences in experimental groups occurred. The level of significance was accepted at P<0.05. All data are presented as means ± standard error (SE). Results Water quality values Water physicochemical characteristics are shown in Table 1. Intensive conditions led to significant (P<0.05) decrease in dissolved oxygen (DO) and increase in total ammonia levels of rearing water. The lowest DO and the highest total ammonia values were detected in SD30 and SD40 groups. No significant (P>0.05) differences were detected among all the groups in terms of rearing water temperature and pH levels. Growth and feed efficiency Growth performance of fish (FBW, WG and SGR) decreased significantly in SD40 group than the other groups (Table 2). Also, feed efficiency ratio decreased significantly (P<0.05) in SD40 compared to SD10, while no significant (P>0.05) differences were reported among the other groups (Table 2). Survival rate lowered significantly (P<0.05) in SD30 and SD40 groups than the low stocking density group (SD10) without no differences with SD20 group. Digestion enzymes Amylase, lipase and protease enzymes showed significantly (P<0.05) higher activities in fish reared in low stocking densities (SD10 and SD20 groups) over the high stocking density (SD40) without no significant difference (P>0.05) with SD30 group (Fig. 1). Immune response High stocking density led to significant im-munosuppression in tilapia as declared by the blood immunity in Figure 2. Immunoglobulin and NBT levels decreased significantly (P<0.05) in SD40 group compared to SD20 group without no differences with the other two groups. Lysozyme activity reported the highest significant (P<0.05) values in SD10 and SD20 groups over the high stocking density group (SD40) without no difference with SD30 group. Bactericidal, phagocytic activities and phago-cytic index reported significantly (P<0.05) lower values in fish reared in intensive conditions (SD30 and SD40 groups) than fish reared in low stocking density (SD10 and SD20 groups). Peroxidase showed significantly (P<0.05) lower value in SD40 and SD30 groups compared to the other groups with the lowest level in SD40 group. Total serum protein lowered relatively in SD30 and SD40 groups without no differences with the other groups. Oxidative status Oxidative and antioxidative enzymes activities are shown in Figure 3. SOD and CAT decreased significantly (P<0.05) in SD30 and SD40 groups compared to SD10 and SD20 groups. Similarly, GPX exhibited significantly (P<0.05) lower activity in SD30 and SD40 groups than SD10 and SD20 groups with the highest level in SD10 group. However, MDA increased significantly (P<0.05) in fish reared in intensive conditions (SD30 and SD40 groups) over fish reared in low stocking density (SD10 and SD20 groups). Discussion Aquaculture is based on the culture of fish in an optimal environmental and culture conditions. High stocking density is among the rearing strategies of aquatic animals. In this system the water components are utilized efficiently in order to get higher fish production per unit of rearing water. Nevertheless, over stocking density can be a risky stress which suppress the growth, survival, immune response and oxida-tive status (28, 29). Digestive enzymes, immunity and oxidative status of Nile tilapia (Oreochromis niloticus) reared in intensive 103 Table 1: Water quality parameters of Nile tilapia reared under different stocking densities Test group SD10 SD20 SD30 SD40 Temperature (°C) 27.07±0.15 27.1±0.24 27.2±0.2 27.4±0.13 pH 7.2±0.06 7.23±0.12 7.17±0.09 7.23±0.07 Dissolved oxygen (mg L-1) 5.43±0.09c 4.83±0.15b 4.4±0.15a 4.2±0.06a Total ammonia (mg L-1) 0.57±0.03a 1.22±0.2b 1.71±0.09c 1.86±0.2c "Values expressed as means ± SE (n = 3). Different superscript letters indicate significant differences for each pair-wise comparison between treatments Table 2: Growth and feed efficiency of Nile tilapia reared under different stocking densities Test group SD10 SD20 SD30 SD40 IBW 14.43±0.23 14.2±0.17 14.23±0.34 14.37±0.33 FBW 31.18±3b 34.6±2.67b 32.83±2.25b 28.28±2.31a WG (%) 115.06±20.78b 138.61±18.4b 126.42±15.53b 95.01±16a SGR 1.26±0.15b 1.44±0.13b 1.35±0.12b 1.1±0.14a FER 0.85±0.03b 0.79±0.07ab 0.77±0.06ab 0.73±0.02a Survival 100±0b 96.67±1.67ab 92.22±1.11a 92.5±1.44a "Values expressed as means ± SE (n = 3). Different superscript letters indicate significant differences for each pairwise comparison between treatments ■ Amylase ■ Lipase ■ Protease SD10 SD20 SD30 SD40 Test group Figure 1: Digestive enzymes activities of Nile tilapia reared under different stocking densities. Values are expressed as mean ± SE from triplicate groups. Bars with an asterisk are significantly different from those of control group (P<0.05) 104 M. Dawood, M. Shukry, M. Zayed, A. Omar, A. Zaineldin, M. El Basuini 5 ^ 4 43 3 m vü 2 i1 ^0 ab ab 0,3 o 02 m 0,2 vo ta Q O 0,1 SD10 SD20 SD30 Test group SD40 SD10 SD20 SD30 SD40 Test group 400 S300 J3200 e £100 iy N I 0 C°60 ox ^50 >40 t o « 30 "53 1220 0 'gi0 1 0 m S 3 8 r " b b 2 ' SD10 SD20 SD30 Test group SD40 SD10 SD20 SD30 SD40 Test group 60 •5; 50 1 40 1 30 a 120 o re10 Ph SD10 SD20 SD30 SD40 Test group SD10 SD20 SD30 SD40 Test group ^ 60 - 50 40 'I 30 a .2 20 It 10 o oga 0 Ph 3,5 SD10 SD20 SD30 SD40 Test group SD10 SD20 SD30 SD40 Test group b 0 c c b 0 Figure 2: Activity of blood immune responses in Nile tilapia reared under different stocking densities. Values are expressed as mean ± SE from triplicate groups. Bars with an asterisk are significantly different from those of control group (P<0.05) Digestive enzymes, immunity and oxidative status of Nile tilapia (Oreochromis niloticus) reared in intensive 105 ■ SOD l CAT ■ GPX ■ MDA SD10 SD20 SD30 SD40 Test group Figure 3: Oxidative status [SOD (IU L-1), CAT (IU L-1), GPX (IU L-1) and MDA (nmol ml-1)] of Nile tilapia reared under different stocking densities. Values are expressed as mean ± SE from triplicate groups. Bars with an asterisk are significantly different from those of control group (P<0.05) The current study illustrated that the growth performance of tilapia reared in intensive conditions (SD40) was slower than the other densities indicating that growth was influenced by the unreasonable stocking density. Parallel results also were obtained in tilapia and other fish species (14, 28, 30). Wendelaar (31) concluded that the low growth of fish reared in intensive conditions is due to the high level of required energy level to deal with stress which resulted in low available energy for fish growth. Survival rate is a significant parameter to express the welfare and health status of fish (32). In agreement with our study, fish reared in intensive conditions showed depressed survival rates (14, 30, 33). In this study, compared with the low stocking density (SD10), fish reared in intensive conditions (SD30 and SD40) showed lower feed efficiency ratio (FER). Low FER is related to energy metabolism rate (30, 32). Fish reared in intensive conditions are usually exposed to se-vere-stressor circumstances that increase the required energy (9, 29). The extra energy requirements were probably met by mobilizing body resources, resulting in lower growth and feed utilization (34). The decreased growth and feed efficiency of Nile tilapia in this study might be related to decreased activity of digestive enzymes. Major digestive enzymes produced by fish are protease, lipase and amylase to play its role in feed digestion and utilization. If these enzymes increase the overall body metabolism may increase (3,6). It is possible that intensive rearing conditions can weaken the digestion and utilization process of feed by affecting the activity of digestive enzymes. Further microbiome and proteomic studies are required to reveal the effect of intensive aquaculture conditions on the intestinal digestive enzymes and microbes. Water quality is one of the main factors which affect the growth and feed utilization of fish (32, 35). The present study showed that high stocking density causes higher stress in Nile tilapia, leading to higher oxygen consumption and an increased ammonia level. There are many studies on the effects of stocking density on water quality with almost similar results. Similarly, increased stocking density led to lower DO and higher CO2 levels (1, 14, 33). Randall and Tsui (36) reported that fish under stressful conditions excrete more ammonia and the present results suggest that the fish in high stocking densities experienced more severe stressed status. This is supported by the results of immune response and oxidative status. 106 M. Dawood, M. Shukry, M. Zayed, A. Omar, A. Zaineldin, M. El Basuini It is well known that stress causes immunosuppression in fish (3). High stocking stress clearly declined soluble immune components in Nile tilapia with similar results in Senegalese sole and Rainbow trout (28). It has been proposed that, stressful conditions resulted in high produced corticosteroid levels which significantly inhibits the production of cytokines and immune responses (11). In this study, fish reared at high stocking densities exhibited suppressed immune responses "e.g. NBT, lyso-zyme activity, IgM, bactericidal activity and phagocytosis". Immunoglobulins are "heterodi-meric glycoproteins that play a vital role in recognizing natural antigens and exist in the skin, gill and gut mucus, bile as well as systemically found in the plasma of fish" (37). Low serum IgM was noticed in tilapia of SD40 group. The respiratory burst activity (NBT) is a reliable parameter used to detect oxidative radical production which reflect the immunity of cultured fish to show its ability to resist the infectious diseases and environmental stressors (22, 38). The lysozyme activity can breakdown the polysaccharide walls of pathogenic bacteria and offers stronger innate immune defense in fish against stressors (32). The lysozyme activity depends on the leucocyte counts which produce lysozymes that catalyse the glycosidic bonds of pathogenic bacterial cell walls resulted in enhanced complement system and phagocytosis (39). In agreement with our results, high stocking density reduced lysozyme and peroxidase activities, suggesting some degree of immuno-suppression in Solea senegalensis, Gilthead seabream, Rainbow trout and Nile tilapia reared in intensive conditions (29, 40, 41, 42). Phagocytosis is one of the significant cellular immune system components in fish (43). Its role is to guarantees that fish can avoid pathogen attacks efficiently by recognize the pathogens and to bound their spread and progress (44). Our study demonstrated decreased bactericidal and phagocytosis, suggesting weakening immune response and tolerance against high stocking density. These results suggested that the resistance to the pathogenic bacteria could be weakened because of the intensive conditions. The reactive oxygen species (ROS) is produced by animal cells in the presence of several antioxidant defense mechanisms. The oxidative stress normally happens when the production and removal of ROS is unbalanced, since the oxidative damage of cultured species is directly related to the quality of rearing environment (45). Among the antioxidant enzymatic defenses SOD, GPX and CAT enzymes (46, 47). In this study, fish reared in intensive conditions resulted in decreased SOD, CAT and GPX activities as well as increased MDA activity in blood of tilapia suggesting suppressed antioxi-dant response. The depression was a response to the continuous stresses of stocking density and might reflect the limited abilities for antioxidant systems in tilapia to wholly remove these harmful SOD, finally leading to oxidative damage (28, 40, 48). Usually, the antioxidant system is activated to control the ROS which resulted in oxidative stress (49). High level of lipid peroxidation is a result of excessive ROS production which ends by the production of MDA. High MDA levels finally leading to oxidative damage to DNA, protein and cytoplasm (50). The obtained results revealed increased MDA in fish at high stocking level indicated cell damage. Earlier reports also revealed decreased antioxidant enzyme (SOD, CAT and GPX) (40, 48) and increased oxidative enzyme (MDA) levels in fish reared in intensive conditions (28). Conclusion It can be concluded that, the intensive rearing conditions can impair the welfare of tilapia fingerlings and depress the growth, digestive enzyme activity, immune response and oxida-tive status. References 1. Gomes LC, Roubach R, Araujo-Lima CA, Chippari-Gomes AR, Lopes NP, Urbinati EC. Effect of fish density during transportation on stress and mortality of juvenile tambaqui Colossoma mac-ropomum. J World Aquac Soc 2003;34(1):76-84. 2. Sulikowski JA, Fairchild EA, Rennels N, Huntting Howell W, Tsang PC. The effects of Digestive enzymes, immunity and oxidative status of Nile tilapia (Oreochromis niloticus) reared in intensive 107 transport density on cortisol levels in juvenile winter flounder, Pseudopleuronectes americanus. J World Aquac Soc 2006;37(1): 107-12. 3. Liu G, Ye Z, Liu D, Zhao J, Sivaramasamy E, Deng Y, Zhu S. Influence of stocking density on growth, digestive enzyme activities, immune responses, antioxidant of Oreochromis niloticus fingerlings in biofloc systems. Fish Shellfish Immunol. 2018;81:416-22. 4. Bolasina S, Tagawa M, Yamashita Y, Tanaka M. Effect of stocking density on growth, digestive enzyme activity and cortisol level in larvae and juveniles of Japanese flounder, Paralichthys oliva-ceus. Aquaculture 2006;259 (1-4):432-43. 5. Dawood MAO, Koshio S. Recent advances in the role of probiotics and prebiotics in carp aquaculture: a review. Aquaculture 2016;454:243-51. 6. Dawood MAO, El-Dakar A, Mohsen M, Ab-delraouf E, Koshio S, Ishikawa M, Yokoyama S. Effects of using exogenous digestive enzymes or natural enhancer mixture on growth, feed utilization, and body composition of Rabbitfish, Siganus rivu-latus. Journal of Agricultural Science and Technology. B. 2014;4(3B). 7. Birnie-Gauvin K, Peiman KS, Larsen MH, Aarestrup K, Willmore WG, Cooke SJ. Short-term and long-term effects of transient exogenous cortisol manipulation on oxidative stress in juvenile brown trout. J Exp Biol 2017:jeb-155465. 8. Foss A, Kristensen T, Âtland Â, Hustveit H, Hovland H, 0fsti A, Imsland AK. Effects of water reuse and stocking density on water quality, blood physiology and growth rate of juvenile cod (Gadus morhua). Aquaculture 2006; 256 (1-4): 255-63. 9. Lupatsch I, Santos GA, Schrama JW, Verreth JA. Effect of stocking density and feeding level on energy expenditure and stress responsiveness in European sea bass Dicentrarchus labrax. Aquaculture 2010;298(3-4):245-50. 10. Liu B, Jia R, Han C, Huang B, Lei JL. Effects of stocking density on antioxidant status, metabolism and immune response in juvenile turbot (Scophthalmus maximus). Comp Biochem Physiol C Toxicol Pharmacol 2016;190:1-8. 11. Tort L. Stress and immune modulation in fish. Dev Comp Immunol 2011;35(12): 1366-75. 12. Li M, Gong S, Li Q, Yuan L, Meng F, Wang R. Ammonia toxicity induces glutamine accumulation, oxidative stress and immunosuppression in juvenile yellow catfish Pelteobagrus fulvidraco. Comp Biochem Physiol C Toxicol Pharmacol 2016;183:1-6. 13. Dawood MAO, Eweedah NM, Moustafa EM, Shahin MG. Synbiotic Effects of Aspergillus oryzae and P-Glucan on Growth and Oxidative and Immune Responses of Nile Tilapia, Oreochromis niloticus. Probiotics Antimicrob Proteins 2019;8:1-2. 14. Wu F, Wen H, Tian J, Jiang M, Liu W, Yang C, Yu L, Lu X. Effect of stocking density on growth performance, serum biochemical parameters, and muscle texture properties of genetically improved farm tilapia, Oreochromis niloticus. Aquacult Int 2018:1-3. 15. AO AC. Method 2007-04. Association of Official Analytical Chemists. Washington, DC. 2007. 16. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with the Folin phenol reagent. J Biol Chem 1951;193(1):265-75. 17. Anson ML. The estimation of pepsin, trypsin, papain and cathepsin with hemoglobin. J Gen Physiol 1938;22:79-89. 18. Jiang CK. Activity measuring for imple-mental enzyme. Science and Technology Press, Shanghai.1982. 19. Worthington V. Worthington enzyme manual: enzymes and related biochemicals. Worthington Chemical, Lakewood, New Jersey. 1993. 20. Borlongan IG. Studies on the digestive lipases of Milkfish, Chanos chanos. Aquaculture 1990; 89:315-325. 21. Jin ZL. The evaluation principle and method of functional food. Beijing Publishers, Bei-jing.1995. 22. Secombes CJ. Isolation of salmonid macrophages and analysis of their killing activity. Tech. Fish Immunol 1990;1:137-154. 23. Parry, R. M., Chandon, R. C. & Shahani, K. M. (1965) Proc. Soc. Exp. Biol. 119, 384-386. 24. Rainger GE, Rowley AF. Antibacterial activity in the serum and mucus of rainbow trout, On-corhynchus mykiss, following immunisation with Aeromonas salmonicida. Fish Shellfish Immunol 1993;3(6):475-82. 25. Quade MJ, Roth JA. A rapid, direct assay to measure degranulation of bovine neutrophil primary granules. Vet Immunol Immunopathol 1997;58: 239-248. 26. Sahoo PK, Kumari J, Mishra BK. Nonspecific immune responses in juveniles of Indian major carps. J Appl Ichthyol 2005;21:151-155. 27. Kawahara E, Ueda T, Nomura S. In vitro phagocytic activity of white-spotted char blood cells after injection with Aeromonas salmonicida extracellular products. Fish Pathol 1991;26(4):213-4. 28. Andrade T, Afonso A, Pérez-Jiménez A, Oliva-Teles A, de las Heras V, Mancera JM, Serra-deiro R, Costas B. Evaluation of different stocking 108 M. Dawood, M. Shukry, M. Zayed, A. Omar, A. Zaineldin, M. El Basuini densities in a Senegalese sole (Solea senegalensis) farm: implications for growth, humoral immune parameters and oxidative status. Aquaculture 2015;438:6-11. 29. Montero D, Izquierdo MS, Tort L, Robaina L, Vergara JM. High stocking density produces crowding stress altering some physiological and biochemical parameters in gilthead seabream, Sparus aurata, juveniles. Fish Physiol Biochem 1999; 20(1):53-60. 30. Garcia F, Romera DM, Gozi KS, Onaka EM, Fonseca FS, Schalch SH, Candeira PG, Guerra LO, Carmo FJ, Carneiro DJ, Martins MI. Stocking density of Nile tilapia in cages placed in a hydroelectric reservoir. Aquaculture 2013;410:51-6. 31. Wendelaar BSE. The stress response in fish. Physiol Rev 1997;7:591-625. 32. Ellis T, North B, Scott AP, Bromage NR, Porter M, Gadd D. The relationships between stocking density and welfare in farmed rainbow trout. J Fish Biol 2002;61:493-531. 33. Jia R, Liu BL, Han C, Huang B, Lei JL. Influence of Stocking Density on Growth Performance, Antioxidant Status, and Physiological Response of Juvenile Turbot, Scophthalmus maximu, Reared in Land-based Recirculating Aquaculture System. J World Aquacult Soc 2016;47(4):587-99. 34. Vijayan MM, Ballantyne JS, Leatherland JF. High stocking density alters the energy metabolism of brook charr, Salvelinus fontinalis. Aquaculture 1990;88(3-4):371-81. 35. Sloman KA, Gilmour KM, Taylor AC, Metcalfe NB. Physiological effects of dominance hierarchies within groups of brown trout, Salmo trutta, held under simulated natural conditions. Fish Physiol Biochem. 2000;22(1):11-20. 36. Randall D, Tsui T. Ammonia toxicity in fish. Mar Pollut Bull 2002;45:17-23. 37. Magnadottir B. Immunological control of fish diseases. Mar Biotechnol 2010;12(4):361-379. 38 Uribe C, Folch H, Enriquez R, Moran G. Innate and adaptive immunity in teleost fish: a review. Vet Med 2011;56(10):486-503. 39. Cecchini S, Terova G, Caricato G, Saroglia M. Lysozyme Activity in Embryos and Larvae of Sea Bass (Dicentrarchus labrax L.), Spawned by Broodstock Fed with Vitamin C Enriched Diets. Bull Eur Assoc 2000;20(3): 120-4. 41. Telli GS, Ranzani-Paiva MJ, de Carla Dias D, Sussel FR, Ishikawa CM, Tachibana L. Dietary administration of Bacillus subtilis on hematology and non-specific immunity of Nile tilapia Oreo-chromis niloticus raised at different stocking densities. Fish Shellfish Immunol 2014;39(2):305-11. 42. Yarahmadi P, Miandare HK, Hoseinifar SH, Gheysvandi N, Akbarzadeh A. The effects of stocking density on hemato-immunological and serum biochemical parameters of rainbow trout (Onco-rhynchus mykiss). Aquacult Int 2015;23(1):55—63. 43. Zhang XY, Fan HP, ZHONG QF, ZHUO YC, LIN Y, ZENG ZZ. Isolation, identification and pathogenicity of Streptococcus agalactiae from tilapia. Journal of Fisheries of China 2008;5:772-9. 44. Harikrishnan R, Kim JS, Kim MC, Bal-asundaram C, Heo MS. Prunella vulgaris enhances the non-specific immune response and disease resistance of Paralichthys olivaceus against Uronema marinum. Aquaculture 2011;318(1-2):61-6. 45. Apel K, Hirt H. Reactive oxygen species: metabolism, oxidative stress, and signal transduction. Annu Rev Plant Biol 2004;55:373-99. 46. David M, Munaswamy V, Halappa R, Marigoudar SR. Impact of sodium cyanide on cata-lase activity in the freshwater exotic carp, Cyprinus carpio (Linnaeus). Pest Biochem Physiol 2008;92(1): 15-8. 47. Gaetani GF, Ferraris AM, Rolfo M, Mangerini R, Arena S, Kirkman HN. Predominant role of catalase in the disposal of hydrogen peroxide within human erythrocytes. Blood 1996;87(4): 1595-9. 48. Braun N, de Lima RL, Baldisserotto B, Dafre AL, de Oliveira Nuner AP. Growth, biochemical and physiological responses of Salminus brasili-ensis with different stocking densities and handling. Aquaculture 2010;301(1-4):22-30. 49. Lushchak VI. Environmentally induced oxi-dative stress in aquatic animals. Aquat Toxicol 2011; 101(1): 13-30. 50. Yao J, Wang JY, Liu L, Li YX, Xun AY, Zeng WS, Jia CH, Wei XX, Feng JL, Zhao L, Wang LS. Anti-oxidant effects of resveratrol on mice with DSS-induced ulcerative colitis. Arch Med Res 2010;41(4):288-94. Slov Vet Res 2019; 56 (Suppl 22): 109-17 DOI 10.26873/SVR-748-2019 Original Research Article UTILIZATION OF SUGAR BEET BY PRODUCTS IN FEEDING GROWING RAHMANI LAMBS Mohamed AN1, Nabil Eweedah1, Hamed Gaafar2*, Metwally Abd El-Salam2 department of Animal Production, Faculty of Agriculture, Kafrelsheikh University, Egypt. 2Animal Production Research Institute (APRI), Agricultural Research Center, Ministry of Agriculture, Egypt. Corresponding author, E-mail: hamedgaafar@gmail.com Abstract: Twenty female Rahmani lambs with an average initial body weight 31.01±0.68 kg were randomly divided into four similar groups (5 in each group). To receive one of the tested rations, Ration1 containing 50% concentrate feed mixture 1 plus 50% berseem hay as a control ration. In rations 2 and 4, 50% of berseem hay was replaced by dry sugar beet tops. While, in rations 3 and 4, 50% energy ingredients (yellow corn grains, wheat bran and rice bran) were replaced by dry sugar beet pulp in concentrate feed mixture 2. Ration 3 had significantly (P<0.05) the highest digestibility coefficient of all nutrients followed by rations 4 and 2, while, while ration 1 had the lowest values. Digestible crude protein had nearly the same trend, but total digestible nutrients and digestible energy were significantly higher in ration 3 compared with the other rations. A logarithmic scale of acidity or basicity of rumen liquor were nearly similar for all the different groups. Dry sugar beet pulp in rations 3 and 4 showed significantly the higher concentrations of total volatile fatty acids and ammonia nitrogen compared to the other rations. Dry sugar beet pulp increased dry matter, total digestible nutrients, digestible crude protein and digestible energy, but tended to decrease with dry sugar beet tops compared with control group. Growth rate and feed conversion were significantly higher (P<0.05) for lambs fed ration 3 compared with the other rations. Lambs fed ration 3 recorded significantly the highest net revenue and economic efficiency. Key words: growing lambs; dry sugar beet pulp; dry sugar beet tops; growth performance Introduction In Egypt, there is a shortage of available fed for ruminant. So that a growing attention is being focused on the use of crop by-products such as sugar beet by-products. Using of these byproducts will decrease the fed cost as well as limitation the environmental pollution. In 2017 a large area was (about 545000 fed-dans) cultivated with sugar beet in Egypt, which produced about 650000 tons of dried sugar beet pulp (DSBP) as a by-product of' sugar beet manufacture. It also produced about 6.8 million tons of fresh sugar beet tops (FSBT) as an agricultural by-product (3). Sugar beet pulp may be used as a partial or whole replacement of energy sources in mixed rations of ruminants (21, 17). Also, dried sugar beet tops (DSBT) was used as good roughage in feeding ruminants with appreciable reduction Received: January 2019 Accepted for publication: February 2019 110 M. Ali, N. Eweedah, H. Gaafar, M. Abd El-Salam in feeding cost without any health troubles (6, 22, 4 and 12). The objectives of this study were to investigate the effect of feeding female Rahmani lambs on ration containing DSBP as partial replacement 50% of energy sources (Yellow corn grains, wheat bran and rice bran) and/or DSBT as partial replacement 50% of berseem hay (BH) on feed intake, digestibility, rumen fermentation activity, some bloods parameters, growth performance, fed conversion and economic efficiency. Material and methods The present study was carried out during summer 2017 at private farm, El-Hamol, Kafr El-Sheikh Governorate, Egypt in cooperation with the Animal Production Department, Faculty of Agriculture, Kafrelsheikh University, Egypt. Sugar beet tops (SBT) were collected after harvesting sugar beet crop and spread on ground, then turning it from time to time (before sunrise and after sun down) till drying (the drying period was about 7 days) then material was collected and stored. While DSBP was obtained from the Dakahlia Company of Sugar, Dakahlia Governorate as a by-product of sugar beet industry. Twenty female Rahmani lambs weighted 31.01±0.68 kg were used in feeding trial that lasted 90 days. Animal were divided into four similar groups (5 lambs each). Animals were randomly assigned in to four experimental ratios. R1 containing 50% concentrate feed mixture (CFM1) plus 50% berseem hay BH (control ration). In R2 and R4 50% of BH was replaced by DSBT, while, in R3 and R4 50% of energy sources (yellow corn grains, wheat bran and rice bran) in CFM1 were replaced by DSBP in CFM2. Crude protein in CFM2 was adjusted by adding 0.6 urea (46% N) to compensate the lower protein content in DSBP. Each 1 ton of CFM1 contained 110 kg soybean meal, 300 kg yellow corn grains, 300 kg wheat bran, 250 kg rice bran, 10 kg common salt, 15 kg limestone, 7.5 kg premix, 5 kg sodium bicarbonate and 2.5 kg yeast culture. Whereas, CFM2: 50% from sources of energy (yellow corn, wheat bran and rice bran) were replaced by DSBP with addition 4.2 kg urea over the 1 ton to adjust protein content. Animals were weighed before morning feeding on 2 consecutive days at the beginning and at the end of the experimental and once biweekly during the experimental period. At the end of the feeding trial, three lambs from each group were used in three digestion trials to evaluate the previous rations by using acid insoluble ash (AIA) technique (29). Samples of feed ingredients, ration and feces were taken for chemical analysis according (1). Digestible energy was calculated as follows: DE= TDN 0.04409 (24). At the last day of the digestibility trail, rumen liquor samples were taken 3 hr after the morning feeding by a rubber stomach tube. The samples were filtered through a double layer of cheese cloth. The rumen pH value was determined directly by HANNA pH meter (HI-8424 Sophisticated micro-processor, pH meter). The rumen samples were transferred into covered plastic tubes with two drops of formalin for stopping microbial activity and kept in a deep freezer until analysis. Total volatile fatty acids (TVFA's) concentration was estimated by using steam distillation method (30). While Am-monia-N (NH3-N) concentration was determined using magnesium oxide (Mgo) as described by (1) Blood samples taken at the same time of rumen liquor collection from the jugular vein in clean tubes and then were centrifuged at 2500 rpm for 20 minutes to separate blood serum using serological pipettes. Samples of whole blood were collected using EDTA as anticoagulant and immediately directed to hematologi-cal determination. Different items of the blood picture were carried out, the counts of red blood cells (RBC's) and white blood cells (WBC's) (20), hemoglobin (Hb) (9), total protein (Tp) (8), albumin (AL) (14) and GOT and GPT (26). The data were analyzed using IBM SPSS Statistics general linear models procedure (15). Results and discussion Results in Table 1, revealed that a variation in chemical composition among the different Utilization of sugar beet by products in feeding grow-ing rahmani lambs 111 feed ingredients. The CF content was higher, whereas the contents of CP and NFE were lower in DSBP compared to the average of other energy sources (yellow corn grains, wheat and rice bran). It reflects the differences in CF and NFE contents between CFM1 and CFM2, while CP content was adjusted in CFM2 by adding urea to become nearly the same for both concentrate feed mixtures. Also, CP content was nearly similar for both BH and DSBT. While, CF content was higher, but NFE and ash contents were lower in BH compared with DSBT. The calculated composition of experimental rations showed that CP content was nearly similar for different rations. Moreover, CF content increased with DSBP (R3), but it decreased with DSBT (R2). However, NFE content had opposite trend, which decreased with DSBP (R3 and R4) and increased with DSBT (R2). The EE content decreased with DSBP R3 and R4. Ash content increased with DSBT (R2). Similar results found that introducing DSBP in rations of buffaloes and sheep resulted in an increase in CF content (10, 25). Also, replacing BH by DSBT in rabbit's ration decreased CF content and increased NFE and ash contents (13). The digestibility coefficients and nutritive values of experimental rations are shown in Table (2). The digestibility coefficient off all nutrients in R3 (containing DSBP) had the highest significantly values (P<0.05) followed by R4 (containing DSBP + DSBT) and R2 (containing DSBT) while, the R1 (control) had the lowest values. The DCP% had the same trend nearly but, the TDN and DE values were significantly higher in R3 compared with the other rations. The improvements of digestibility and nutritive values with DSBP might be due to their higher fiber content (Table 1), which reduce the outflow rate of feed in the rumen as well as urea nitrogen supplemented with DSBP may be stimulate the rumen microorganisms. Although, the digestibility coefficients of all nutrients improved with DSBT compared to BH. These results agreed with those obtained by (23) indicated that using DSBP at level 25 and 50% in growing Angora goats rations increased the digestibility of DM, OM, CP and CF as well as nutritive value as TDN than control ration. On the other hand, (18) feeding Rahmani rams on complete pelleted rations with replacing 50 and 100% of BH by DSBT significantly increased the digestibility of DM, OM and NFE but, the digestibility of CP and EE and the values of TDN and DCP were decreased significantly higher. (4) concluded that DSBT could be used successfully as a replacer to 50% from BH in ration of growing lambs without any detritus effects on productive and reproductive performance. Rumen liquor parameters presented in Table (3) showed that pH values were nearly similar for the different groups. The pH values of all rations were between 6.47 and 6.60 which were with the normal range (27). No significant differences in rumen pH values were detected while, the concentration of total VFA's and NH3-N significantly increased between Rah-mani rams consuming rations containing different levels of DSBP (0, 25 and 50%) as replacement of concentrate mixture, (5). Rations 3 and 4 contained DSBP showed significantly (P<0.05) higher concentrations of TVFA's and NH3-N while, the lower TVFA's concentration was in R1 and NH3-N concentration was in R2. Adding urea with concentrate feed mixture containing DSBP (CFM2) led to increase ruminal NH3-N concentration, which simulate rumen microorganisms utilizing resulting in more fermentation of soluble carbohydrates and structure fiber producing VFA's. (23) indicated that the total VFA's concentration significantly increased as the proportion of DSBP increased in the Angora goats ration. (11) didn't find any significant differences in the ruminal pH between Rahmani rams fed different rations containing 100% BH as control or rations contained 50 and 100% of BH were replaced by DSBT. Results of blood hematology in Table (3) revealed significant differences (P<0.05) in the counts of white and red blood cells among the different groups, which R4 had the highest values followed by R3 and R2, while R1 had the lowest values. However, hemoglobin concentration and hematocrit percentage were nearly similar for different groups. The values of blood 112 M. Ali, N. Eweedah, H. Gaafar, M. Abd El-Salam hematology obtained in this study were within the normal range for sheep being 5-14.5 x 103/|l for WBC's, 8.2-12.3 x 106/|l for RBC's, 9-14 g/dl for hemoglobin and 25-38% for hematocrit (28). Serum parameters in Table (3) showed that the concentrations of total protein and globulin were significantly (P<0.05) higher in R3 and R4 followed by R2, while R1 had the lower values. On the other side, the concentrations of glucose and total lipids as well as the activity of GOT and GPT were significantly (P<0.05) higher in R1 followed by R2, while both R3 and R4 had the lower values. These results revealed that DSBP was more effective in whole blood and serum parameters than that DSBT. While, (4) found that no significant differences on blood parameters when lambs fed ration contained DSBT as replacement of BH at rate 50 and 100 % compared with control group (BH). (25) declared that incorporation SBP in sheep ration decreased plasma concentrations of triglyceride, cholesterol, urea and uric acid compared to control ration (CFM). Finally, all serum blood parameters values were within the normal range. Table 1 : Chemical composition of ingredients, concentrate feed mixtures and experimental rations DM % Composition of DM % Item OM CP CF EE NFE Ash Ingredients Soybean meal 92.46 89.70 44.04 5.38 2.09 38.19 10.30 Sources of energy Yellow corn grains 91.42 98.68 9.49 1.74 4.23 83.22 1.32 Wheat bran 90.49 95.18 13.89 9.04 3.76 68.49 4.82 Rice bran 90.41 87.87 13.97 9.52 15.29 49.09 12.13 Average 90.77 93.91 12.45 6.77 7.76 66.93 6.09 Dry sugar beet pulp 89.28 95.91 9.70 24.98 2.18 59.05 4.09 Berseem hay 89.45 89.70 13.45 27.64 2.45 46.16 10.30 Dry sugar beet tops 87.65 79.18 13.78 12.74 2.28 50.38 20.82 Concentrate feed mixtures CFM1 91.21 90.22 15.48 6.22 6.45 62.07 9.78 CFM2 90.60 90.96 15.49 13.97 4.25 57.25 9.04 Experimental rations R1 (control) 90.33 89.96 14.46 16.93 4.45 54.12 10.04 R2 89.43 84.70 14.63 9.48 4.37 56.22 15.30 R3 90.03 90.33 14.42 20.80 3.35 51.76 9.67 R4 89.58 87.70 14.53 17.08 3.31 52.78 12.30 Utilization of sugar beet by products in feeding grow-ing rahmani lambs 113 Table 2: Digestibility coefficients and nutritive values of experimental rations Item R1 R2 R3 R4 SEM Digestibility coefficients, % DM 65.25c 66.42bc 68.13a 67.70ab 0.38 OM 66.30c 67.03b 69.28a 67.50b 0.35 CP 66.53c 67.03bc 69.09a 67.45b 0.31 CF 61.48c 63.60b 66.30a 64.25b 0.55 EE 76.50c 78.90bc 82.73a 80.03b 0.74 NFE 64.05c 67.48b 69.28a 67.65b 0.58 Nutritive values, % TDN 62.35b 61.45b 65.85a 62.44b 0.56 DCP 9.62c 9.82b 9.96a 9.80b 0.04 DE, Mcal/kg DM* 2.75b 2.71b 2.90a 2.75b 0.02 a, b, c: Means in the row with different superscripts differ significantly (P<0.05) Table 3: Rumen liquor, whole blood and serum parameters of growing lambs in experimental groups Item R1 R2 R3 R4 SEM Rumen liquor pH value 6.47 6.50 6.60 6.55 0.03 TVFA's (meq/100 ml) 12.11b 13.19ab 14.82a 14.46a 0.38 NH3-N (mg/ 100 ml) 16.32ab 15.28b 19.50a 18.60ab 0.72 Whole blood WBC's (x 103/|l) 8.12b 8.57ab 9.05ab 9.50a 0.23 RBC's (x 106/|l) 9.74b 10.39ab 10.68ab 11.10a 0.24 Hb (g/dl) 9.67 9.97 10.57 10.97 0.27 HTC (%) 26.77 26.83 27.20 27.37 0.37 Blood serum Total protein 7.13b 7.60ab 8.07a 8.13a 0.15 Albumin 3.83 3.88 3.97 4.00 0.05 Globulin 3.30b 3.72ab 4.10a 4.17a 0.15 Glucose 51.00a 48.67a 45.33b 45.33b 0.82 Total lipids 153.00a 149.00a 135.33b 133.00b 3.11 GOT 46.78a 44.67ab 43.56b 41.89b 0.65 GPT 16.67a 15.67ab 14.83b 14.50b 0.32 a, b: Means in the row with different superscripts differ significantly (P<0.05) 114 M. Ali, N. Eweedah, H. Gaafar, M. Abd El-Salam Table 4: Average daily feed intake, live body weight, total and daily weight gain of growing lambs in experimental groups Item R1 R2 R3 R4 SEM Average daily feed intake: As fed basis, kg/head/day: CFM1 0.718 0.696 - - CFM2 - - 0.740 0.725 Berseem hay 0.718 0.348 0.725 0.362 Dry sugar beet tops - 0.348 - 0.362 Total 1.436 1.392 1.480 1.449 As dry matter basis, kg/head/day: DM 1.297 1.245 1.332 1.298 TDN 0.809 0.765 0.877 0.810 DCP 0.125 0.122 0.133 0.127 DE, cal/head/day* 3.56 3.37 3.86 3.57 Live body weight, total and daily weight gain: Duration (day) 90 90 90 90 Initial weight (kg) 31.38 30.58 30.83 31.24 0.68 Final weight (kg) 43.16ab 42.37b 44.06a 43.23ab 0.98 Total weight gain (kg) 11.78b 11.79b 13.23a 11.99b 0.47 Average daily gain (g) 130.89b 131.00b 147.00a 133.22b 5.23 Relative growth rate, % 37.54 b 38.55 b 42.91a 38.38b 0.97 a, b: Means in the row with different superscripts differ significantly (P<0.05) Feed intake as dry matter, TDN, DCP and DE was nearly similar for all groups (Table 4). (4) and (2) showed that no significant differences (P>0.05) in total dry matter and crude protein intake by sheep fed rations containing DSBP. Live body weight, total and daily weight gain of growing lambs in different experimental groups are shown in Table (4). While, final live body weight, total and average daily body weight gain were significantly (P<0.05) higher for lambs fed R3 contained DSBP compared with the other groups. The relative growth rate of lambs fed R1, R2, R3 and R4 were 37.54, 38.55, 42.91 and 38.38%, respectively. These results showed that DSBP achieved the higher growth rate of growing lambs, which confirmed with higher digestibility and nutritive values (Table 2) and rumen fermentation activity (Table 3). These results agreed with those obtained by (16) who found that increasing average daily gain of lambs fed diet containing 25 or 50% DSBP as a replacer of CFM. (12) showed that average daily gain of lambs was significantly higher with replacing yellow corn by DSBP. (18) didn't find any significant differences in average daily gain of growing lambs fed rations containing DSBT at rate 0, 50 and 100% in complete pelted rations as replacement of BH. (11) reported that no significant differences for the average daily gain of crossbred ewe lambs fed rations containing DSBT as a replacement of BH (0, 50 and 100% DSBT). Feed conversion of growing lambs fed the experimental rations presented in Table (5). Incorporation of dry sugar beet pulp and tops in rations of growing lambs significantly (P<0.05) improved feed conversion. Lambs in control group fed R1 showed the highest amounts of DM, TDN, DCP and DE required per one kg live weight gain, whereas those fed R3 containing DSBP had the lowest values of DM and DCP and those fed R2 containing DSBT had the lowest TDN and DE values. The improvement of feed conversion with DSBP attributed to the improvement of ADG (Table, 5). Utilization of sugar beet by products in feeding grow-ing rahmani lambs 115 Table 5: Feed conversion and economic efficiency of growing lambs in experimental groups. Item R1 R2 R3 R4 SEM Feed conversion: DM, kg/kg gain 9.91a 9.50ab 9.06b 9 74ab 0.15 TDN, kg/kg gain 6.13a 5.84b 5.97ab 6.08ab 0.06 DCP, kg/kg gain 0.955a 0.931ab 0.905b 0.953a 0.03 DE, Mcal/kg gain 27.20a 25.73b 26.26ab 26.80ab 0.23 Economic efficiency: 3.40b 3.31b Feed cost (LE/day) 4.00a 3.03c 0.07 Feed cost LE/ kg gain 30.56a 23.23b 23.13b 24.85b 1.26 Output of weight gain (LE/day) 7.20b 7.21b 8.09a 7.33b 0.29 Net revenue (LE/day) 3.20c 4.18ab 4.69a 4.02b 0.29 Net revenue improvement % 0.00c 30.63ab 46.56a 25.63b 0.65 Economic efficiency 1.80c 2.38a 2.38a 2.21b 0.07 a, b, c: Means in the row with different superscripts differ significantly (P<0.05) Moreover, feed intake decreased with feed DSBT (Table, 4). These results are illustrated with those obtained by (19) who found that efficiency of feed utilization was markedly improved in cows fed beet pulp as compared with those given corn. (16) reported improvement in feed efficiency of lams by feeding in rations containing DSBP at rate 25 or 50% as replacing of CM. (11) showed that feed efficiency (kg feed DM/kg gain) was nearly similar when fed crossbred ewe lambs on rations containing DSBT as a replacement of HB (0, 50 and 100% DSBT) and were no significant differences for all groups. Results of economic efficiency in Table (5) showed that feeding rations contained DSBP and DSPT significantly (P<0.05) reduced average daily feed cost compared to control group and the lowest value was with DSPT in R2. In addition, feed cost per one kg weight gain was significantly (P<0.05) higher with control group compared to the other groups containing DSBP and/or DSPT. This may be due to the lower price of DSBP than those of corn gain, wheat and rice bran as well as lower price of DSBT than that of BH. Meantime, R3 contained DSBP had significantly (P<0.05) the higher output of daily weight gain compared with the other groups, which attributed to higher ADG of lambs fed R3 (Table, 5). Moreover, lambs fed R3 recorded significantly (P<0.05) the highest net revenue followed by R2 and R4, while R1 had the lowest values. Furthermore, lambs fed R2 and R3 had significantly (P<0.05) the highest economic efficiency followed by R4, while R1 had the lowest value. These results agreed with those obtained by (7) who found that feed cost per kg live-weight gain decreased with increasing amounts of DSBP in the rations. (25) reported that replacing CFM with SBP supplemented with 10% soybean meal (SBM) in sheep ration led to decrease total daily feeding cost and Feed cost LE/ kg gain and improved daily profit by 38.07% compared to control ration. (4) and (11) reported that feed cost as LE/kg gain decreased significantly with improved the economic efficiency for crossbred ewe lambs on rations containing DSBT as a replacement of HB (50 and 100% DSBT) compared with control Conclusion From these results it could be concluded that replacing 50% of energy sources (yellow corn, wheat bran and rice bran) by dried sugar beet pulp and replacing berseem hay by dried sugar beet tops in rations of growing lambs improved their performance concerning digestibility, rumen fermentation activity, blood parameters, growth rate, feed conversion and economic efficiency. Conflict of interest Authors declare that no conflict of interest. 116 M. Ali, N. Eweedah, H. Gaafar, M. Abd El-Salam References 1. A.O.A.C. (1995). Association Official Analytical Chemists: Official Methods of Analysis. 16th Ed., Official Agriculture Chemistry, Washington DC, USA. 2. Abd-El Galil, E. R.; A.R. Khattab; H.M. Khat-tab; H.M. El Sayed and Faten F. Abou-Ammou (2016). Influence of Using Different Energy Sources on Growth Rate, Digestion Coefficients and Rumen Parameters in Sheep. Asian J. Anim. Vet. Adv., 11 (3): 190-7. 3. Agriculture Economics and Statistics Institute, Ministry of Agriculture (2017). Agricultural Economics, part 2 pull. By Agric. Res. Center, Ministry of Agriculture, Egypt. 4. Ali, M.F. and B E. El-Saidy (2003). The effect of feeding dried sugar beet tops on the productive and reproductive performance of ram lambs. J. Agric. Sci. Mansoura Univ., 28 (8): 5969 -83. 5. Ali, M F.; M.K. Mohsen; M.I. Bassiouni and M.M. Khalafalla (2000). The influence of using dried sugar beet pulp on sheep performance. J. Agric. Res. Tanta Univ., 26(2): 132 -44. 6. Bendary, M.M.; M.M. Mohamed; G.H.A. Ghenim and I.M. Abou-Selim (1996). Nutritional studies on using sugar beet tops in animal feeding performance of lactating Friesian cows fed dried sugar beet tops and its silage. Egyptian J. Anim. Prod., 33, Suppl. Issue: 199 -206. 7. Bouaque, C.H.V.; B.G. Cottyn; J.V. Aerts and F.X. Buysse (1976). Dried sugar beet pulp as a high energy feed for beef cattle. Anim. Feed Sci. Tech-nol., 1: 643-53 8. Doumas, B. I.; W. Watsn and H. Biggs (1971). Albumin standards and the measurements of serum albumin with bromocresol green. Clin. Chem. Acta., 31: 87 -92. 9. Drabkin D.L. and H. Austin (1932). spectro-photometric studies: I. spectrophotometric constants for common hemoglobin derivatives in human, dog and rabbit blood. J. Biol. Chem., 98: 71932. 10. El-Ashry, M.A.; Zeba A. Motagally and Y.A. Maareck (2000). Effect of dried sugar beet pulp in dairy buffalo rations on colostrums, milk yield and composition. Egyptian J. Nutrition and Feeds, 3(1): 15-22. 11. Eweedah, N.M; A.A. Sallam; B E. El-Saidy; M.S. Saleh; M.F. Ali and M.G. Gabr (2004). Productive and reproductive performance on ewe lambs fed dried sugar beet tops. J. Agric. Sci. Tanta Univ., 30(4): 812-24. 12. Eweedah, N.N.; M.F. Ali; M.S. Saleh and M.K. Mohsen (2001). Comparing between dried sugar beet pulp and yellow corn as source of energy with berseen in groing lambs rations. J. Agric. Sci. Mansoura Univ., 26(6): 3478-95. 13. Gaafar, H.M.A.; A.I.A. Abd El-Lateif and Salwa B. Abd El-Hady (2010). Effect of partial replacement of berseem hay by ensiled and dried sugar beet tops on performance of growing rabbits. Researcher, 2(9):10-5. 14. Hill, P.G. and T.N. Wells (1983). Bromocresol purple and the measurement of albumin. Falsely high plasma albumin concentrations eliminated by increased reagent ionic strength. Ann Clin Bio-chem, 20: 256 15. IBM SPSS Statistics (2014). Statistical package for the social sciences, Release 22, SPSS INC, Chicago, USA. 16. Khalafall, M.M.E. (1999). The effect of partial replacing concentrate mixture by sugar beet byproducts on the performance of ruminants. M.Sc. Thesis. Fac. of Agric., Kafr El-Sheikh, Tanta University. 17. Mahmoud A.E.M. and N.E. El-Bordeny (2016). The Nutritive Value of Sugar Beet Pulp-substituted Corn for Barki Lambs. Pakistan J. Zool., vol. 48(4), pp. 995-1002. 18. Mahmoud, S.A.; M.F. Ali; M.A. Helmy and A.A.H. Khalek (2001). Productive performance of growing lambs fed complete rations containing different levels of dried sugar beet tops. J. Agric. Sci. Mansoura Univ., 26(6): 3579 -89. 19. Mansfield, H.R.; M.D. Stern and D.E. Otter-bye (1994). Effects of beet pulp and animal byproducts on milk yield and in vitro fermentation by rumen microorganisms. J. Dairy Sci. 77, 205-16.Masuda (1995). 20. Millar, S.E. and J.M. Waller (1971). Test book of Clinical Pathology. 8th Ed. The Williams and Wilking Co., Baltimore Scientific Book, Agency Calcutta. 21. Mohamed, Sherien H. (2005). Biological treatment of sugar beet pulp and its use in sheep ration. M.Sc. Thesis, Fac. of Agric., Cairo Univ. 22. Mohi El-Din, A.M.A. (1998). Studies on cattle production "nutritional studies on the use of sugar beet by-products in feeding lactating cows". Ph.D. Thesis, Fac. Of Agric. Mansoura Univ., Egypt. 23. Mohsen, M.K.; M.F. Ali and M.I. Basiouni (1999). The effect of partial replacing concentrate mixture by dried sugar beet pulp on performance of growing Angora goats. Prod. of the 7th Sci. Conf. of Anim. Nutri., 19 - 21 Oct. El-Arish, Egypt: 309-18. Utilization of sugar beet by products in feeding grow-ing rahmani lambs 117 24. NRC (1988). Nutrition Requirements of Sheep. 7th Edn., National Academy Press, Washington, DC., USA. 25. Omer, H.A.A; Soha S. Abdel-Magid; AY. El-Badawi; I.M. Awadalla; M.I. Mohamed and Mona S. Zaki (2013). Nutritional impact for the whole replacement of concentrate feed mixture by dried sugar beet pulp on growth performance and carcass characteristics of Ossimi sheep. Life Sci. J., 10(4): 1987-1999. 26. Ritman, A. and S. Frankel (1957). A colour-imeteric Methods of Determination of S. GOT and S. GPT. American J. of Clinical Pathology, 28: 56. 27. Sung, H.G.; Y. Kobayashi; J. Chang; A. Ha; H. Hwang and J.K. Ha (2007). Low Ruminal pH reduces dietary fiber digestion via reduced microbial attachment. Asian-Aust. J. Anim. Sci., 20(2):200-7. 28. UCDAVIS, United States Department of Agriculture (2011). "Sugar: World Markets and Trade". FAS Information. 29. Van Keulen, J.V. and B.A. Young. (1977). Evaluation of Acid Insoluble Ash as a Natural Marker in Ruminant Digestibility Studies. J. Anim. Sci., 44: 282. 30. Warner, A.C.J. (1964). Production of volatile fatty acids in the rumen. Methods of measurements. Nutr. Abst. Rev., 34: 339. Slov Vet Res 2019; 56 (Suppl 22): 119-27 DOI 10.26873/SVR-749-2019 Original Research Article GROWTH AND REPRODUCTION PERFORMANCE OF JAPANESE QUAILS (Coturnix coturnix japonica) UNDER VARIOUS ENVIRONMENTS OF LIGHT COLORS Hassan E. Elkomy, Ayman E. Taha*, Heba A. Basha, Magda I. Abo-Samaha, Mohamed M. Sharaf Department of Animal Husbandry and Animal wealth Development, Faculty of Veterinary Medicine, Alexandria University, Egypt Corresponding author, E-mail: ayman_soma2007@yahoo.com Abstract: Light colors are important environmental factors affecting growth and reproductive performance of Japanese Quails. In the current study, a total number of 1060 of Japanese quail chicks were used to assess the effect of white, red, and green light color on body weight, age of sexual maturity, laying parameters as well as responses of reproductive organs. Results revealed that Japanese quails reared under red color light had higher (P<0.05) body weight at five weeks of age, relative growth rate (RGR), the first 42 days of egg production, relative ovaries and testicle weights, sperm motility and fertility and hatchability percentages (243.28 g, 184.50 %, 79.76 %, 4.33 %, 4.05 %, 90.50 %, 83.47 % and 83.47 %; respectively) than those raised under green and white light colors. Also, quails subjected to red color light during growth reached sexual maturity earlier (39.34 d) than birds subjected to white and green light colors (44.87 and 48.45 d). We conclude that using red light color during the period of growth and laying in Japanese quails improves growth and reproduction performances. Key words: Japanese quails; light colors; growth; reproduction performances Introduction Light is an environmental factor affecting quail production, light stimuli vary by intensity, wavelength, duration and color, all of which aggregate to influence the physiological, production, reproduction as well as behavioral parameters of the birds (1). Poultry have retinal photo-receptors that are responsible for vision, while the non-visual photoreceptors (extra-retinal) are responsible for detecting photoperiod and adapting their physiology to the environment (2). Light stimulates directly the GnRH pathway that increases the amount of gonadotropins, which enhancing the ovarian development. Simultaneously, the increase in day length decreases the amount of melatonin and GnRH, and removing the inhibition on the stimulatory axis. Increased photoperiod enhances the sexual maturity of chicks due to induction of gonadotropin and sex steroids secretion with subsequent increment in production (3). Interestingly, light color and its wavelength represent important factors that judge the performance of birds. It has been concluded that the red light diminishes cannibalism and feather pecking; blue light has a calming effect; however, orange-red and blue green color light have Received: January 2019 Accepted for publication: February 2019 120 H. E. Elkomy, A. E. Taha, H. A. Basha, M. I. Abo-Samaha, M. M. Sharaf enhancing chicken reproduction and growth (4), respectively. There were variations among the performance parameters of broiler chicks reared under various light intensities (5). However, no improvement was reported in broiler chicks exposed to various intensities of light, although green light color enhanced growth performance of such chicks as compared to orange, red and yellow light (6). However, Kim et al. (7) found that birds performed better when reared under red light without affecting feed conversion ratio (FCR). Hatchability of quail eggs collected from birds exposed to green bulbs was higher than those subjected to other light treatments (8, 9). Meanwhile, quails reared under red and incandescent lights had higher body weight than those reared under blue and green lights (8). The increased photoperiod to enhance the earlier sexual maturity, which may be altered according to the spectral output, Japanese quails exposed to red light reached to sexual maturity two weeks earlier than those exposed to blue or green light (8). Hens reared under red light laid eggs significantly earlier than those under incandescent light and blue light. The mean age at first egg of chicken pullets in red light was earlier by two to eleven days compared to pullets in all other light treatments (6). Similarly, laying hens maintained under red and white lights had early sexual maturity than hens maintained under green light (10). Moreover, light color affects egg numbers of laying hens (7, 10, and 11). This study aimed to investigate effects of light color on growth and reproduction parameters of Japanese quails. Material and methods Experimental design and birds management This study was performed using 1060 one day old Japanese quail chicks using rearing pens at the Department of Animal Husbandry and Animal Wealth Development, Faculty of Veterinary Medicine, Alexandria University, Egypt. Chicks were randomly distributed into three light treatments (white light n=426, red light n=320 and green light n=315). Pens were cleaned, disinfected and fumigated using formaldehyde gas. The chicks were brooded at a temperature of 35°C using automatic gas heaters as a source of heat and illumination. Temperature was gradually reduced 3°C weekly till reach room temperature 24 °C at the fifth week of rearing. Birds were placed in environmentally controlled lightproof rooms separated from each other by a wooden chip board. At the fifth week, Japanese quails were moved to wire cages and grouped to sire families. Each family consisted of one male and three females (1:3 sex ratios) according to Karousa et al. (12). Cages were separated by lightproof plastic sheets to prevent transmittance of light. Additionally, all windows were blacked out by light proof plastic sheets. Birds were fed add-libitum commercial starter diets containing 22% crude protein and 3100 kcal/kg of metabolizable energy meeting (13) requirements. While at the laying period, laying diet had calcium content of 3.5%; the birds also had free access to water. Animals were exposed to 24 h lighting during the brooding and growing periods. At laying period, the duration of light was adjusted by using a timer to 16 hours light and 8 hours darkness (16 L: 8 D). All light sources were equalized to 9W. The following light treatment regimens were used. Treatment 1 (White light): A total number of 426 Japanese quail chicks were reared in separate pens under white LED light (W) with wavelength 380 to 770 nm from hatching till five weeks; the birds were then moved to laying batteries containing 20 sire families illuminated with the same light. Treatment 2 (Red light): 320 Japanese quail chicks were reared in a separate pen illuminated with red LED bulbs (R) with wavelength 618 to 635 nm. At the fifth week, Japanese quails were moved to separate battery containing 28 sire families illuminated with same light. Treatment 2 (Green light): 314 Japanese quail chicks were reared in a pen illuminated green LED bulbs (G) with wavelength 515 to 535 nm. At the fifth week, Japanese quails were moved into laying batteries (14 sire families illuminated with same light). Bird eggs were collected from each treatment (white, red and green) daily and stored for 7 days then disinfected and incubated in previously cleaned and fumigated incubators by spraying TH4® (2 Growth and reproduction performance of japanese quails (Coturnix coturnixjaponica) under various 121 ml/liter water) to assess the effect of continuous use of different light colors on hatching performance (hatchability and fertility percentages) of Japanese quail eggs. Hatching performance Fertility % = (Number of fertile eggs/ Total number of eggs set) x 100 Hatchability of the total eggs% = (Number of hatched chicks/ Total number of eggs set) x 100 Hatchability of fertile eggs % = (Number of hatched chicks/ Number of fertile eggs set) x 100 Growth and reproductive performance Birds were weighed at hatch and every week till five weeks of age to the nearest gram using digital scale with accuracy 0.001g (WANT®), and then the differences between two successive weights were calculated to estimate the relative growth rate according to Brody (14). Relative growth rate = (W2 - W1)/ 0.5 (W1+W2) x 100, Where: W1= is the weight at any week and W2= is the weight at next week. Age of sexual maturity (age at the first egg) was expressed in days from hatching day till the day of the first egg of the quail hens. Egg production was recorded for each sire family of quails for the first 42 days after complete sexual maturity (15) with the estimation of hen day egg production (HDEP) for the same period. HDEP%= (Total of eggs produced/ Number of live hens) x 100 Male and female reproduction organs A random sample of three males and three females per treatment were selected at 98 days of age and weighed then slaughtered and dissected for removal of testes and ovaries. The right and left testes were immediately weighed to calculate the gonadosomatic index (GI = testes weight /body weight x 100). Semen was also collected from vas deferens immediately after slaughtering of males. Sperm motility was then evaluated by two trained evaluators in samples placed on glass slides (37 °C) at 400x magnifications. Sperm motility was expressed as the percentage of mobile cells. The ovary was immediately weighed and relative weight to live body weight (ovary weight / body weight x100) was calculated. Statistical analysis Data of body weight, weight gain and relative growth rate were analyzed by general linear model (GLM) procedure using SAS (16) according to the following model: Xijk= p + Ti + Sj + Di*j+ eijk ; p= population mean, Ti= treatment effect (dark, green and red light), Sj= sex effect (M= male and F= female), Di*j = the interaction between treatment and sex, eijk= random errors, Data of hatchability, fertility, egg quality parameters, egg production, age at sexual maturity and reproductive organ parameters were analyzed by analysis of variance (ANOVA) using SAS (16) and the significance between different treatment groups were assessed using Duncan's test. Prior to analysis, percentages of embryonic mortality were arcsine transformed to corresponding value according to Snedecor and Cochran (17). Reults and discussion Effect of light color on Growth The effects of different light colors on body weight of Japanese quails from hatch day till five weeks of age were showed in Table 1. The overall hatch weight was not significantly different between treatment groups. Similarly, this parameter did not differ between the sexes in each group. During the first week, the overall body weight in red light treated birds (55.25 g) was significantly higher than green and white light groups (49.92 and 30.62 g, respectively). Birds reared under red light also had significantly higher overall body weight at the 2nd, 3rd, 4th and 5th week of age (101.61, 160.26, 202.38 and 243.28 g, respectively) than those reared under green and white lights. Females reared under red and white colors recorded significantly higher body weight than males at the 2nd, 3rd, 4th and 5th week of age. In contrast, gender effect was not apparent for green light until 5th week of age. 122 H. E. Elkomy, A. E. Taha, H. A. Basha, M. I. Abo-Samaha, M. M. Sharaf Table 1: LS Means ± SE of body weight for Japanese quails subjected to different treatments of light color Light Sex Body weight (g) Group Hatch W1 W2 W3 W4 W5 F WMe mm 9.35±0.06a 31.25±1.27a 75.62±0.61a 120.00±0.89a 179.41±1.14a 221.85±1.32a 9.29±0.09a 29.98±1.86a 73.38±0.90b 114.48±1.30b 170.64±1.67b 206.41±1.93b Overall 9.32±0.05A 30.62±1.12C 74.50±0.54C 117.24±0.79C 175.02±1.01B 214.13±1.17C Green ,F, M 9.77±0.11a 50.04±2.30a 92.23±1.11a 154.28±1.61a 206.45±2.07a 246.18±2.39a 9.63±0.10a 49.81±2.19a 91.23±1.06a 150.80±1.53a 201.18±1.97a 222.89±2.28b Overall 9.70±0.08A 49.92±1.59B 91.73±0.77B 152.54±1.11 B 203.82±1.43A 234.54±1.65B Red F M 9.48±0.12a 57.06±2.47a 104.46±1.19a 164.78±1.73a 208.43±2.22a 263.19±2.56a 9.53±0.12a 53.44±2.54a 98.76±1.23b 155.75±1.78b 196.33±2.29b 223.39±2.64b Overall 9.50±0.08A 55.25±1.77A 101.61±0.85A 160.26±1.24A 202.38±1.60A 243.28±1.89A Overall means carrying different capital letters within the same column are significantly different (P < 0.05) Means carrying different small letters within group within same column are significantly different (P < 0.05) F= Female, M= Male, W= Week, LS Mean = least square mean Table 2: LS Means ± SE of relative growth rates for Japanese quails subjected to different treatments of light color Light _Relative growth rate (%) Sex Group R1 R2 R3 R4 R5 R6 F 107.29±0.50a 82.83±0.73a 45.49±0.46a 39.70±0.35a 21.18±0.41a 183.70±0.12a White 104.80±0.74b 43.69±0.67b 19.14±0.60b 182.71±0.18b M 84.06±1.07a 39.42±0.52a Overall 06.04±0.45C 83.45±0.65A 44.59±0.41B 39.56±0.31A 20.16±0.36A 183.21±0.11B F 129.13±0.91a 59.17±1.31a 50.44±0.83a 29.04±0.64a 17.70±0.74a 183.24±0.21a Green 10.19±0.71b 181.73±0.21b M 131.37±0.87a 58.75±1.26a 49.26±0.80a 28.76±0.61a Overall 130.25±0.63B 58.96±0.91B 49.85±0.57A 28.90±0.44B 13.94±0.51B 182.48±0.15C F 139.65±0.98a 59.00±1.42a 44.89±0.90a 23.41±0.69a 23.85±0.80a 185.72±0.23a Red 13.86±0.82b 183.26±0.24b M 137.26±1.01a 59.64±1.46a 44.87±0.92a 23.13±0.71a Overall 138.45±0.70A 59.32±1.02B 44.88±0.64B 23.27±0.49C 18.85±0.57A 184.50±0.17A Overall means carrying different capital letters within the same column are significantly different (P < 0.05) Means carrying different small letters within group within same column are significantly different (P < 0.05) F= Female, M= Male, W= Week, LS Mean = least square mean Table 3: Means ± standard errors for sexual maturity of Japanese quails subjected to different treatments of light color Treatment/Light Color Age at first egg (days) White 44.87±0.36b Green 48.45±0.40a Red 39.34±0.21c Means carrying different litters within the same column are significantly different (P<0.05) Growth and reproduction performance of japanese quails (Coturnix coturnixjaponica) under various 123 Table 4: Means ± standard errors for hen day egg production (HDEP %) of Japanese quails subjected to different treatments of light color Weeks Treatment White Green Red Wk1 70.47±4.07a 64.63±2.53a 73.13±1.63a Wk2 69.52±3.23b 56.12±2.13c 81.29±1.54a Wk3 77.38±2.39a 74.15±1.63ab 79.25±2.42a Wk4 61.19±3.13b 60.89±2.70b 79.25±1.38a Wk5 78.57±2.43a 66.33±2.42b 80.27±1.92a Wk6 80.48±1.54b 74.83±1.69c 85.37±1.27a Total 72.94±1.61b 66.16±1.44c 79.76±1.00a Means carrying different litters within the same row are significantly different (P<0.05) Table 5: Means ± SE for egg weight in grams, external and internal egg quality parameters of Japanese quail eggs subjected to different treatments of light color Parameters _Light treatment White Green Red Egg weight (g) 14.52 ± 0.16 a 13.72± 0.15 b 13.26 ± 0.29 b Egg shape index 78.07 ± 0.40 a 78.19 ± 0.52 a 78.50 ± 0.51 a Egg volume 12.34 ± 0.31 a 11.52 ± 0.27 b 11.63 ± 0.53 b Yolk+albumen % 78.92 ± 0.50 b 82.99 ± 0.29 a 82.23 ± 0.42 a Shell % 21.08 ± 0.98 a 17.01 ± 0.65 b 17.77 ± 0.90 b Yolk index 0.46 ± 0.08 c 0.52 ± 0.07 a 0.49 ± 0.09 b Haugh unit 93.02±0.72 b 95.44±0.44 a 96.44±0.60 a Means carrying different litters within the same row are significantly different (P<0.05) Table 6: Means ± standard errors for relative ovaries, testes and percentage of sperm motility weight of Japanese quails subjected to different treatments of light color for 14 weeks Parameters (%) Light Color / Treatment White Green Red Relative Ovaries weight 3.43±0.41a 4.27±0.96a 4.33±0.18a Relative testes weight 3.53±0.30 a 3.47±0.24 a 4.05±0.20 a Sperm motility 75.00±2.89b 84.33±7.22ab 90.50±1.26a Means carrying different litters within the same row are significantly different (P<0.05). Table 7: Means ± standard errors for fertility, scientific hatchability and commercial hatchability percentages of the first generation of Japanese quail eggs subjected to different treatments of light color Light Color / Treatment Fertility (%) Hatchability (%) Fertile eggs Total eggs White 81.17±0.44 b 84.80±0.35 a 77.40±1.25 b Green 85.37±0.56 a 86.93±0.91 a 80.53±0.62 a Red 90.07±1.13 a 92.53±1.27 a 83.47±0.58 a Means carrying different litters within the same column are significantly different (P<0.05). 124 H. E. Elkomy, A. E. Taha, H. A. Basha, M. I. Abo-Samaha, M. M. Sharaf Figure 1 : Means for hen day egg production (HDEP %) of Japanese quails subjected to different treatments of light color W= Quails reared under white light from G= Quails reared under green light from Data on relative growth rates of Japanese quails subjected to different light colors for five weeks are presented in Table 2. Birds reared under red light grew at significantly higher rate (R6) from zero to five weeks of age (184.50%) than those reared under white and green light colors (183.21 and 182.48, respectively). Moreover, the overall growth rate of females was significantly higher than that of males in all light treatments. These results are similar to those reported by others who found that Japanese quails reared under red lights had higher body weight than those reared under blue and green lights after five weeks (8). They speculated this was due to higher weight of reproductive organs as testes weight of male Japanese quail at five weeks of age with red light was double that of green light. Similarly, Li et al. (18) and Reddy et al. (19) reported growth-stimulating effect of red light in laying hens and documented increased weights and activity of reproductive organs (higher weight of the ovary and oviduct and more yellow yolk follicles). In contrast, Baxter et al. (10) found that hens reared under Green light had significantly higher body weight gain than birds under Red and White lights after 23 weeks of age. The sex effect on body weight of Japanese quail was also reported by (20) and (21) who found that female weights were significantly higher than those of males. Age at sexual maturity The onset of sexual maturity of Japanese quail subjected to different treatments of light color, (based on the age at first egg) was presented in Table 3. Females of Japanese quail reared under red light treatment reached sexual maturity significantly earlier (39.34 d) than those exposed to white light (44.87 d), while significantly late sexual maturity was recorded by females reared under green light (48.45 d). The result agreed with (22) who found that red light induced sexual maturity of broiler breeder birds 7 and 14 days earlier than green and blue light treated respectively. Also, laying hens maintained under red and white light had early sexual maturity than hens maintained under green light. Red light resulted in higher steroid and gonadotropin concentrations and higher neuropeptide mRNA expression (10). Similar results recorded in Japanese quail exposed to Red light reached sexual maturity two weeks earlier than those exposed to blue or green light (8). Growth and reproduction performance of japanese quails (Coturnix coturnixjaponica) under various 125 Egg production Table 4 and Figure 1 were illustrated the hen day egg production (HDEP %) of Japanese quails subjected to different light color treatments for six weeks after complete sexual maturity. There was no difference in HDEP among different light treatments during the first week of egg production. However, red light treatment birds had significantly higher HDEP percentage from the 2nd week till the 6th week of egg production, followed by white color treated group while the lowest HDEP% was recorded for green light treated group. The total HDEP percentage was significantly higher in red light treatments than white and green light treatments where the highest HDEP percentage recorded for red light (79.76%) and the lowest HDEP percentage recorded for green light (66.16%). These results are similar to those reported by Baxter et al. (10) and Hassan et al. (11) who found that red monochromatic light improved egg production of the laying hens more than blue or green treatments whereas the white light had an intermediate effect. Moreover, Yang et al. (22) reported that highest cumulative egg production values of laying hens occurred in the red-light treated birds. The same results found in Japanese quails brooded and kept for the 16-week production period under red or white light laid significantly more eggs than those brooded and kept under green or blue light (8). The present study revealed that long-wavelength light (red and white) expedited the age of sexual maturation (ASM) and increased level of egg production in birds, while short wavelength light (green) delayed sexual maturation and reduced egg production. These observations are similar to those of Mobarkey et al. (23), who found that long wavelengths (red light) contain more energy so they are able to penetrate through the skull and brain tissue and stimulate the hypothalamus to produce gonad-otropin releasing hormone. As such, hypotha-lamic photoreceptors might be stimulated by lower wavelength but high intensity of blue/green light (24). As well, it was suggested that stimulation of retinal photoreceptors by green light might be able to inhibit reproduction (25); the possible mechanisms for such inhibition still unclear; however, contribution of serotonin is suggested due to its synthesis in hypothalamus and retina (23). Egg quality Some external and internal egg quality parameters of Japanese quails subjected to different treatments of light color were presented in Table 5. Egg weights of white light treated hens were heavier (P<0.05) than those treated with green and red light colors (14.52, 13.72 and 13.26 g, respectively). The same results were reported by Er et al. (26), who found that egg weight was significantly higher in white and green light than Red one. In addition, Hassan et al. (11) found higher egg weight under green light than that under red light. However, other reports found that light color did not affect the average egg weight during the laying period (8, 27). Non-significant differences for egg shape index among different light colors. However, there were significant differences in egg length and width. Also, non-significant difference of the egg shape index between light treatments was recorded (22). On the other hand, white treated hens showed the highest egg volume (12.34 cm3) and shell % (21.08%). The results agreed with those reported by Pyrzak et al. (28), who found shell quality and percent of the shell did not affect by red, green and blue light treatments of the White Leghorns laying hens. Hens treated with green and red light colors produce eggs with higher albumen plus yolk, yolk index and Haugh unit (HU) than hens exposed to white light color. The result agreed with others who found non-significant difference in HU between red, green and blue light (11, 29), but disagreed with Yang et al. (22), who found that HU of the eggs in the white-light treated group was significantly higher than red, green and yellow light. Moreover, non-significant differences for yolk and albumin percentage among different light colors were recorded (30). 126 H. E. Elkomy, A. E. Taha, H. A. Basha, M. I. Abo-Samaha, M. M. Sharaf Male and female reproductive organs Relative ovary weight of female Japanese quails subjected to different light colors for 14 weeks were presented in Table 6. Japanese quail females reared under red and green light recorded higher non-significant (P>0.05) relative ovary weights (4.33 and 4.27%, respectively) than those reared under white light color (3.43%). Moreover, Japanese quail males reared under red light had higher non-significant relative testicle weights than those reared under green and white light colors, in addition sperm motility of males subjected to red color were significantly higher (P<0.05) than those subjected to white color but not significant with those exposed to green light color. These results agreed with Kim et al. (7), who found nonsignificant difference in ovary weight at 20 weeks of age among the different light treatments. Also, Carson et al. (31) and Pyrzak et al. (32) found non-significant difference in the rate of gonadal maturation in pullets illuminated with blue, green, or red monochromatic light incandescent light. On the other hand, Hassan et al. (11) found that laying chickens exposed to red LED light presented heavier ovaries compared with those exposed to green and blue LED lights. Moreover, Woodard et al. (8) found that testes weight of male Japanese quail at five weeks of age with red light was double that of green light. While, Retes et al. (33) found that white and red LED bulbs caused higher weights of testes and higher sperm mo-tility in the quails at 35 days of age. Hatching parameters Fertility and hatchability percentages of Japanese quail eggs reared under different light colors were presented in Table 7. Quails reared under red and green light colors had higher (P<0.05) fertility and hatchability of the total eggs than those reared under white light colors, on the other hand, non-significant differences for hatchability of fertile eggs were recorded between different treated groups. Yang et al. (22) reported better fertility percentages for the eggs produced in the green-light treated breeders, while higher hatchability was recorded for birds reared under white light color. On the other hand, Retes et al. (33) found that fertility of quail eggs produced in green light treated birds higher than white, red and blue LED treatments. While, Non-significant differences for hatchability percentages of Japanese quail eggs produced in different light colors were recorded (8). Conflict of interest None of the authors have any conflict of interest to declare. References 1. Parvin R, Mushtaq MH, Kim MJ, Choi HC. Light emitting diode (LED) as a source of monochromatic light: a novel lighting approach for behavior, physiology and welfare of poultry. World's poult sci 2014; 70 (3): 543-56. 2. Kumar V, Rani S. Light sensitivity of the photoperiodic response system in higher vertebrates: wavelength and intensity effects. Indian J Experim Biol (IJEB), 1999; 11:1053-64. 3. Bedecarrats GY, Mcfarlane H, Maddineni SR, Ramachandran R. Gonadotropin-inhibitory hormone receptor signaling and its impact on reproduction in chickens. General and comparative endocrinology 2009;163:7-11. 4. Rozenboim I, Biran I, Uni Z, Robinzon B, Halevy O. The effect of monochromatic light on broiler growth and development. Poult sci 1999; 78, 135 -8. 5. Manser CE. Effects of lighting on the welfare of domestic poultry: A review. Anim Welfare 1996; 5:341-60. 6. Heshmatollah K. Preference of broiler chicks for color of lighting and feed. Jpn Poult Sci 2007; 44:213-9. 7. Kim MJ, Hossan SM, Akter N, Na JC, Bang T, Kang HK et al. Effect of monochromatic light on sexual maturity, production performance and egg quality of laying hens. Avian Biol. Res 2012; 5: 16. 8. Woodard A, Moore J, Wilson W. Effect of wavelength of light on growth and reproduction in Japanese quail (Coturnix coturnix japonica). Poult Sci 1969; 48(1): 118-23. 9. S hafey T, Al-Mohsen T. Embryonic growth, hatching time and hatchability performance of meat breeder eggs incubated under continuous green light. Asian Aust J Anim Sci 2002; (15): 1702-7. Growth and reproduction performance of japanese quails (Coturnix coturnixjaponica) under various 127 10. Baxter M, Joseph N, Osborne V, Bedecar-rats G. Red light is necessary to activate the reproductive axis in chickens independently of the retina of the eye. Poult sci 2014; (93): 1289-97. 11. Hassan MR, Sultana S, Choe HS, Ryu KS. Effect of monochromatic and combined light color on performance, blood parameters, ovarian morphology and reproductive hormones in laying hens. Italian J Anim Sci 2013; 12:3, DOI: 10.4081/ ijas.2013.e56. 12. Karousa MM, Ahmed SA, Elaithy SM, El-gazar EA. Effect of housing system and sex ratio of quails on egg production, fertility and hatchability. Benha vet med 2015; (28): 241-7. 13. NRC. National Research Council: Nutrient requirements of poultry. 9th Ed. National Academy Press. Washington, DC 1994. 14. Brody S. Bioenergetics and growth. Reinhold Publishing Corporation 1945; New York. 15. Taha AE. Laying performance of Japanese quails divergently selected for body weight under different rearing and lighting systems. PhD Thesis, 2009; Fac. Of Vet. Med. Alex. Univ. Egypt. 16. SAS. Statistical user's Guide' Statistical Analysis System. 2004; INT., Cary, NC. USA. 17. Snedecor GW, Cochran WG. Statistical method, 7th Edition, The Iowa State University, Press Ames Iowa, 1981; U.S.A. 18. Li D, Zhang L, Yang M, Yin H, Xu H, Trask JS, et al. The effect of monochromatic light-emitting diode light on reproductive traits of laying hens. J Appl Poult Res 2014; 23: 367-75. 19. Reddy IJ, David CG, Selvaraju S, Mondal S, Kiran GR. GnRH-1 mRNA, LH surges, steroid hormones, egg production, and intersequence pause days alter in birds exposed to longer wavelength of light in the later stages of production in Gallus gallus domesticus. Trop anim health prod 2012; 44: 1311-7. 20. Tarhyel R, Tanimomo BK, Hena SA. Effect of sex, color and weight group on carcass characteristics of Japanese quail. Scientific Journal of Animal Science 2012; 1(1): 22-7 21. Olawumi SO. Effects of Housing and Sex on Growth Performance of Coturnix Quails in the Derived Savannah Zone of Nigeria. International Journal of Agriculture, Forestry and Fisheries 2015; 3 (6): 227-31. 22. Yang YF, Jiang JS, Pan JM, Ying YB, Wang XS, Zhang ML et al. The relationship of spectral sensitivity with growth and reproductive response in avian breeders (Gallus gallus). Sci reports 2016; 14 (6): 19291. doi: 10.1038/srep19291 23. Mobarkey N, Avital N, Heiblum R, Ro-zenboim I. The role of retinal and extra-retinal photostimulation in reproductive activity in broiler breeder hens. Domestic animal endocrinology 2010; 38: 235-43. 24. Pang SF, Ralph C, Reilly DP. Melatonin in the chicken brain: its origin, diurnal variation, and regional distribution. General and comparative en-docrinology1974; 22: 499-506. 25. Mobarkey N, Avital N, Heiblum R, Ro-zenboim I. The Effect of Parachlorophenylalanine and Active Immunization Against Vasoactive Intestinal Peptide on Reproductive Activities of Broiler Breeder Hens Photostimulated with Green Light. Biology of reproduction2013; 88 (83): 1-7. 26. Er D, Wang Z, Cao J, Chen Y. Effect of monochromatic light on the egg quality of laying hens. J Appl Poult Res 2007; 16: 605-12. 27. Rozenboim I, Zilberman E, Gvaryahu G. New monochromatic light source for laying hens. Poult Sci 1998; 77: 1695-8. 28. Pyrzak R, Snapir N, Goodman G, Perek M. The effect of light wavelength on the production and quality of eggs of the domestic hen, Theri-ogenology 1987; 28: 947-60. 29. Nunes KC, Garcia RG, Nääs IA, Eyng C, Caldara FR, Sgavioli S et al. Effect of Led Lighting Colors for Laying Japanese Quails. Revista Bra-sileira de Ciencia Avícola 2016; 18(SPE): 51-56. 30. Borille R, Garcia RG, Nääs IA, Caldara FR, Santana MR. Monochromatic light-emitting diode (LED) source in layers hens during the second production cycle. Revista Brasileira de Engenharia Agrícola e Ambiental 2015; 19(9): 877-81. 31. Carson JR, Junnila WA, Bacon BF, Sexual maturity and productivity in the chicken as affected by the quality of illumination during the growing period. Poult Sci 1958; 37: 102-12. 32. Pyrzak R, Siopes T. The effect of light color on egg quality of turkey hens in cages Poult Sci 1986; 65:1262-7. 33. Retes PL, Espósito M, das Neves DG, Viana AG, Coelho LM, Bobadilla-Mendez MF et al. Influence of different types of lamps on the reproductive development of male Japanese quail (Coturnix coturnix japonica). Theriogenology 2017; 94: 5963. Slov Vet Res 2019; 56 (Suppl 22): 129-38 DOI 10.26873/SVR-750-2019 Original Research Article INFLUENCE OF DIETARY VITAMIN C SUPPLEMENTATION ON GROWTH PERFORMANCE, BLOOD BIOCHEMICAL PARAMETERS AND TRANSCRIPT LEVELS OF HEAT SHOCK PROTEINS IN HIGH STOCKING DENSITY REARED BROILER CHICKENS Ramadan S. Shewita1, Karima El-Naggar1, Walaa S. H. Abd El Naby2* 1Department of Nutrition and Veterinary Clinical Nutrition, Faculty of Veterinary Medicine, Alexandria University, Egypt, 2Genetics and Genetic Engineering, Department of Animal Husbandry and Animal Wealth Development, Faculty of Veterinary Medicine, Alexandria University, Egypt *Corresponding author, E-mail: walaa.slouma@yahoo.com Abstract: This study was conducted to evaluate the effects of dietary supplementation of different levels of vitamin C (VC) in broiler chicken reared under high stocking density on the growth performance, blood biochemical parameters and the expression of heat shock protein genes. A total of 150, one day old mixed sex broiler chicks (Cobb 500) were randomly distributed in five equal groups. Group1 was reared in normal stocking density (10.6 birds/m2) and fed on the basal diet (BD) without VC (control). While, chicks in other groups (2, 3, 4 and 5) reared in high stocking density (15.6 birds/m2) and fed on BD supplemented with VC at 0, 200, 400 and 600 mg / kg diet, respectively. Birds reared in high stocking density showed a reduction of the final body weight and total feed intake, with high mortality (6.6%). Moreover, they revealed a significant up-regulation of HSP70 mRNA and elevated HSP90 and HSF1 mRNA expression in heart and liver tissue. Graded dietary levels of VC provided variable protection against the hazard of high density through improved final body weight and total feed intake, decreased the mortality % and downregulated liver HSP70 expression level. However, the best performance was observed in birds supplemented with 200 mg/kg VC (group 3). Key words: chicks' growth performance; gene expression; stocking density; vitamin C Introduction Broiler production facing many forms of stressor including high ambient temperature, nutritional stress (imbalance or deficiency in the nutrient requirements) and vaccination programs stress as well as diseases. It is known that birds will perform better when grown in more space, hence, high stocking density (SD) is considered one of these stressful factors which have undesirable effects on broiler performance, livability, health and immune system response, as it reduces bird access to feed and water (1). It also leads to raising the environmental temperature dangerously where more metabolic heat will be added to the house air than was planned (2). Different feed additives have a role in reducing stress in broiler feed are included in several studies (3-5). The ameliorative effects of ascorbic acid or vitamin C (VC) in many forms of Received: January 2019 Accepted for publication: February 2019 130 R. S. Shewita, K. El-Naggar, W. S. H. Abd El Naby stressors have been documented (6, 7). The heat shock proteins (HSPs) family is highly homologous chaperone proteins contributing to cellular protection, protein homeostasis and cell survival against a variety of environmental and metabolic stresses. HSP70 is one of the most conserved and important protein which plays a deep role in enhancing tolerance to various stressors in broiler chickens (8,9). Different studies indicated that HSP70 has an importance not only at high ambient temperatures but also in cell death mediated by free radicals and reactive oxygen species (10). Furthermore, in both physiological and stress conditions HSP90 is a molecular chaperone involved in maturation and stabilization of a wide range of proteins to maintains cellular homeostasis and function (11). We hypothesized that VC could modulate the negative impacts of high stocking density as a stress factor in broilers farms Therefore, this study aimed to investigate the effect of dietary VC supplementation with different concentrations on growth performance, some blood biochemical parameters, and heat shock protein genes expression in broiler chicks reared under high stocking density. Material and methods This experiment was approved by the local ethical committee of animal use from Faculty of Veterinary Medicine, Alexandria University. Birds, experimental design andfeeding Program One hundred and fifty, one-day-old mixed sex Cobb 500 broiler chicks were used in this study. The chicks were weighed at one day old and then randomly distributed into five groups (30 chicks/ group), each treatment has three replicates (10 chicks/replicate) in 15 compartments, every compartment was provided by a suitable feeder and waterer. All groups received their experimental diet for 6 weeks. The bird's compartments were bedded with fresh, clean chopped wheat straw forming a deep litter of 4 cm depth. The room temperature was adjusted on 33oC in the first week of age after that decreased 3oC/week until reaching 21oC at the fifth week of age and the relative humidity was kept at 70 %. The chicks were vaccinated according to a normal regime (vaccination against Newcastle disease on days 7, 18 and 28 and infectious bursal disease (Gumboro) on day 12 of chicks age). The broiler chicks were fed on the basal diet (BD) prepared from a corn-soybean meal based diet and were formulated to meet the nutrient requirements of poultry (12). Starter diet was given from 0-2 weeks of age, followed by grower diet (3-4 weeks) and finally finisher diet from 5-6 weeks of age. Chicks of group 1 (G1), were fed on BD without VC supplementation and reared in normal stocking density (10.6 birds/m2), while the chicks of groups G2, G3, G4 and G5 distributed to be in high stocking density (15.6 birds/m2) (13) and fed on the BD supplemented with VC (Introvit-C WS, Interchemie Co., Netherland) at levels of 0, 200, 400 and 600 mg/kg diet, respectively. The composition of experimental diet and its calculated analysis were presented in table (1). Data for final body weight (BWT) and feed intake (FI) were recorded. Feed conversion ratio (FCR) and body weight gain (BWG) were calculated. Sample collection At the end of the experiment, three blood samples from each replicate (n=9) were collected for analysis of some biochemical parameters, separation of the serum was done using centrifuge adjusted at 3000 rpm for 10 minutes then stored in -20°C. The serum was used for measuring serum total cholesterol, high density lipoprotein (HDL), low density lipoprotein (LDL), triglycerides, glucose, total protein, albumin, globulin, serum glutamic pyruvic transaminase (SGPT) and serum glutamic oxaloacetic amino-transferase (SGOT) using commercial kits (Biodiagnostic company, Egypt). The analysis was done by using a spectrophotome-ter according to the manufacture instructions. The liver and heart tissues from the same slaughter birds were collected and homogenized then snap frozen in liquid nitrogen immediately and stored at -80°C. Influence of dietary vitamin C supplementation on growth performance, blood biochemical parameters and. 131 Table 1 : Composition of experimental starter, grower and finisher diets (gm/kg diet) and calculated chemical analysis of the basal diet Ingredients Diet Starter Grower Finisher Yellow corn 542 558.8 606 Soybean meal (44%) 319 281 253.3 Corn gluten meal (60%) 71 81 48.1 Vegetable oil 29.8 41 54.4 Limestone 1 15 15 15 Monocalcium phosphate 14 14 14 Common salt 3 3 3 Mineral Premix2 1.5 1.5 1.5 Vitamin Premix2 1.5 1.5 1.5 Methionine 3 1 1 1 Lysine 4 1 1 1 Anti Coccidial5 0.2 0.2 0.2 Antimold6 1 1 1 Calculated Analysis Crude protein (CP) % 23.1 22.18 19.39 Metaboliziable 3053 3160.7 3252.6 Energy (ME) Kcal / kg diet 7 Calorie / protein ratio 8 132.16 142.5 167.7 1Limestone (contain 36% calcium). Monocalcium phosphate: contain 22 % Phosphorus and 16 % Calcium. 2Mineral and Vitamin premix produced by Heropharm and composed (per 3 kg) of vitamin A 12000000 IU, vitamin D32500000 IU, vitamin E 10000 mg, vitamin K3 2000 mg, vitamin B1 1000 mg, vitamin B2 5000 mg, vitamin B6 1500 mg, vitamin B1210 mg, niacin 30000mg, biotin 50 mg, folic acid 1000 mg, pantothenic acid 10000 mg, manganese 60000 mg, zinc 50000 mg, iron30000 mg, copper 4000 mg, iodine 300 mg, selenium 100 mg and cobalt 100 mg. 3 DL-Methionine (Produced by Evonic Co and contain 99 % methionine), 4 Lysine = lysine hydrochloride (contain 98 % Lysine). 5 Kill cox, Produced by Arabian company for pharmaceutical industries, 6 Produced by EL TOBA CO. For Premixes & Feed El-Sadat city Egypt. 7 ME calculated according to NRC (1994), 8 Calorie /protein ratio = ME Kcal /CP% Table 2: Primers used for quantitative real-time PCR Gene and ID Primer Sequence (5'-3') References HSP70 (EU747335) F: CCAAGAACCAAGTGGCAATGAA R: CATACTTGCGGCCGATGAGA (15) HSP90 (NM 206959) F: GAGTTTGACTGACCCGAGCA R: TCCCTATGCCGGTATCCACA (15) HSF1 (L06098.1) F :CAGGGAAGCAGTTGGTTCACTACACG R: CCTTGGGTTTGGGTTGCTCAGTC (15) GAPDH (NM_204305) F: GGGCACGCCATCACTATCTTC R: ACCTGCATCTGCCCATTTGA (16) 132 R. S. Shewita, K. El-Naggar, W. S. H. Abd El Naby Table 3: Effect of different dietary VC supplementation on growth performance of broiler chickens Parameters G1 G2 G3 G4 G5 W0 (Initial wt.) 45.64±0.48 45.64±0.54 45.63±0.52 45.61±0.56 45.69±0.46 W6 (Final wt.) 2549.82±72.73 2470.45±80.91 2643.50±73.00 2575.18±64.14 2540.00±55.58 TBG (g) 2504.18±72.27 2424.82±80.39 2597.87±72.50 2529.57±63.60 2494.31±55.14 TBG, RTG2 103.27 100 107.14 104.32 102.87 TFI (g) 4747.17 4494.32 4590.43 4604.81 4507.83 TFI, RTG2 105.63 100 102.14 102.46 100.30 FCR 1.90 1.85 1.77 1.82 1.81 FCR, RTG 102.70 100 95.68 98.38 97.84 Mortality % 3.3 6.6 3.3 0 0 TBG = Total body gain, TFI= Total Feed intake, FCR=Feed conversion ratio (Feed intake/ body gain), RTG2= Relative to group 2 Table 4: Effect dietary VC supplementation on some blood biochemical parameters of broiler chickens Parameters G1 G2 G3 G4 G5 Total protein (g/dL) 6.12±0.02 6.14±0.14 6.04±0.10 6.14±0.08 6.12±0.14 Albumin (g/dL) 5.23±0.08 5.27±0.02 5.23±0.05 5.24±0.02 5.27±0.02 Globulin (g/dL) 0.89±0.10 0.87±0.13 0.81±0.13 0.90±0.10 0.85±0.13 Cholesterol (mg/dL) 200.6±5.01 198.27±2.33 189.37±6.08 191.00±1.60 190.83±5.40 Triglyceride (mg/Dl) 200.43±2.42a 200.70±0.64a 199.73±0.65a 201.07±1.85a 184.53±8.48b HDL (mg/dL) 54.50±0.99a 55.93±0.18a 51.80±1.10ab 52.87±0.94b 50.80±1.38b LDL (mg/dL) 106.01±6.44 107.19±2.30 97.62±5.67 106.92±2.24 103.13±5.76 Glucose (mg/dL) 204.07±0.94b 212.97±1.09a 209.70±1.66ab 206.93±3.90ab 206.53±2.07ab SGOT (U/100 mL) 39.67±4.67ab 43.67±1.76a 39.33±4.67ab 39.67±4.63ab 32.33±1.33b SGPT(U/100 mL) 88.33±2.03 90.33±±3.18 88.671.20 88.33±2.33 88.33±2.96 Means with different letters in the same row differ significantly (P<0.05). HDL= High density lipoprotein, LDL= Low density lipoprotein, SGOT= Serum glutamic pyruvic transaminase and SGPT = Serum glutamic oxaloacetic amino-transferase Influence of dietary vitamin C supplementation on growth performance, blood biochemical parameters and. 133 Figure 1: The mRNA expression level of heat shock proteins (HSP90, HSP70 and HSF1) genes in heart tissue (a) and liver (b). Asterisks on the data bars indicated when P<0.05 (*), P<0.005 (**), and P<0.0005 (***). G1 (chicks were reared in normal stocking density and fed on the basal diet), G2 (chicks were reared in high stocking density and fed on the basal diet), G3, G4, and G5 (chicks were reared in high stocking density and fed on the basal diet with VC supplementation (200, 400, 600 mg/kg diet) respectively Total RNA isolation and cDNA synthesis Total RNA of liver and heart tissues was isolated using the Biozol (Bioflux, Japan) according to the manufacturer instructions. The cDNA was synthesis from isolated RNA using the SensiFAST™cDNA Synthesis Kit (Bioline, United Kingdom) according to the manufacturer instructions. Briefly, 4 pl of total RNA mixed with 4 pl 5X buffer, 1 pl reverse transcriptase and 11 pl RNase\ DNase free H2O was added. The reaction was incubated at 25°C for 10 min, 42°C for 15 min (reverse transcription) and 4°C hold. The obtained synthesis cDNA was checked by glyceraldehyde 3-phos-phate dehydrogenase (GAPDH) gene and stored at -20°C until further use. Quantitative real-time PCR and data analysis and liver tissues using specific primers for each gene (Table 2) and SensiFAST™Syber green master mix with low Rox (Bioline, United Kingdom) in the Mx3000P®System (Strata-gene, USA). The amplification reaction was 20 pl consisting of 2 pl cDNA, 0.8 pl primers (50 nm), 10 pl Syber green master mix and up to 20 pl RNase\ DNase free H2O. The PCR thermal program started as 95°C for 10 min followed by 40 cycles of 95°C for 15 sec then 60°C for 60 sec. The dissociation curve was carried at the end of the last cycle. The housekeeping gene (GAPDH) used to normalize threshold cycle value (Ct). The relative expression values were determined using comparative threshold cycle method 2-AACT(14) and the results were reported as fold change differences relative to the control group (G1). Statistical analysis Quantitative Real-Time PCR (qRT-PCR) was performed for detecting the expression levels of HSP70, HSP90 and HSF1 genes in heart The obtained data were analyzed using the statistical analysis system (17). The significance designated at P<0.05 for the differences 134 R. S. Shewita, K. El-Naggar, W. S. H. Abd El Naby among the different experimental groups for all studied parameters. The significance designated as (P<0.05) using one-way ANOVA between all genes in studied groups. Results Final BWT was improved in all groups which reared under high SD and fed diet supplemented with different levels of VC compared to G2 (Table 3). The G3 which received VC (200 mg/kg diet) showed the highest final BWT compared to G2. The G5, which received the highest level of VC (600 mg/kg) showed lower final BWT. The total body gain (TBG) of birds was improved in all studied groups supplemented with VC when compared with G2 with the highest TBG of chicks was found in G3 which subjected to high SD and supplemented with VC at 200 mg/kg diet. The lowest TBG was found in G2 that was in high SD without VC supplementation. Additionally, High SD in G2 decreased total feed intake (TFI) compared to G1. While the addition of VC in G3, G4, and G5 increased the TFI when compared with G2. The overall chick mortality percentage during the experiment was the highest in G2 (6.6 %), but it was decreased in G3 (3.3 %) with 200 mg/kg diet VC supplementation. Also, no mortality was observed with increased VC supplementation in G4 and G5 (400 and 600 mg/kg diet) (Table 3). The addition of VC in G3, G4 and G5 alleviated the stress effect induced by high SD through decreasing the level of serum glucose, cholesterol, SGOT and SGPT compared with those reared in high SD without VC supplementation (Table 4). In the present study, rearing broiler chicken in high SD leads to an alteration in the expression of HSPs (HSP70 and HSP90), and HSF1 in heart and liver tissue (Figure 1a and b). The expression level of HSP70 mRNA in both heart and liver tissue revealed a significant up-regulation (P<0.05) in the G2 (2.35 ± 0.12 and 6.76 ± 0.66 fold) relative to the control. While HSP70 mRNA expression in both tissues markedly lowered in chicken reared in high SD and supplemented with different concentration of VC than G2. The G2 showed an increase in expression level of HSP90 and HSF1 (2.07 ± 0.10 and 4.45 ± 0.28) in the heart tissue than G3, G4, and G5 supplemented with VC (200, 400 and 600 mg/kg ration, respectively). The HSP90 expression level also showed a significant increase (P<0.05) in G3 than G5 (Fig. 1a). In the liver tissue, HSP90, and HSF1 showed nearly similar expression pattern where they were higher in all groups especially G2 relative to the control (Fig. 1b). Discussion Managemental factors like SD had a significant effect on bird growth performance, health and welfare. In the present study, high SD in G2 decreased the final BWT compared with birds reared in normal SD. Similarly, Dozier et al. (18) showed that increasing the density produced some negative effects on the live performance of broilers. Addition of VC (200 mg/kg diet) to birds reared under high SD ameliorated these negative impacts on growth while failed with the higher levels of VC (600 mg / kg diet). Likewise, Elagib-Hind and Omer (19) reported that BWT was improved by the low and moderate levels (150 and 350 mg/kg) of VC compared with the higher level. Also, SabahElkheir et al. (20) observed that VC supplementation at higher doses (500 mg and 750 mg/kg) resulted in lower final weight and weight gain. Previous studies reported that VC supplementation increased TBG (21, 22). On the other hand, others found that broiler feed intake was not affected by the VC supplementation (23, 24). The FCR was improved in all groups reared in high SD with VC supplementation (G3, G4, and G5) compared to G2, and G1. McKee and Harrison (25) also, observed an improvement in FCR of broilers as result of VC supplementation during heat stress. Decreased mortalities with VC supplementation may confirm the relationship between adding VC and its protective effect as reported by other studies (26, 27) which noticed that VC under stress and disease conditions protects the birds by improving the immune response. Blood serum biochemical parameters are important diagnostic indicators especially under Influence of dietary vitamin C supplementation on growth performance, blood biochemical parameters and. 135 stress conditions such as high SD. In the present study, high SD altered the activity of liver function enzymes (SGPT and SGOT), glucose, and, HDL serum concentrations (increased). The increased serum SGPT and SGOT activity in birds reared under high SD indicate that high SD might cause oxidative lesions which are in accordance with the findings of Simsek et al. (28). Dietary inclusion of VC improved these negative impacts of high SD by lowering the levels of the above-mentioned serum parameters. Similarly, Al-Darajji et al. (29) showed that plasma glucose and cholesterol concentration and plasma SGOT and SGPT activities were significantly lowered in both male and female broiler breeder reared under hot climate and supplementation with ascorbic acid. Moreover, Kucuk et al. (30) reported that supplementation with VC decreased MDA, glucose, cholesterol, and triglyceride concentrations in laying hen reared under cold stress. The significant effect of high stocking density on HSP70 and a1-acid glycoprotein (AGP) indicated that it was physiologically stressful condition to broiler chickens (31). Furthermore, Beloor et al. (32) reported that the expression of HSP70 mRNA could be proper biomarkers to evaluate the stress induced by increased SD. As, HSPs help the stressed cells to manage the stressors, especially those affecting the protein machinery (33). Similarly, the previous study in other species (rainbow trout and sea bass) showed that HSP70 expression level elevated in high SD (34, 35). Furthermore, Higher levels of HSP70 gene in different tissues of birds after exposure to environmental stressors is important in the acquisition of stress tolerance (36, 37). As other study demonstrated that supplementation of VC plays an important role in the prevention of the heat stress in poultry and improvement of their performance (38). Moreover, there is a significant (P<0.05) difference in HSP70 mRNA expression in heart tissue between G2 versus G4 and G5 which fed on basal diet with 400 and 600 mg/kg ration VC supplementation. Also, a highly significant difference (P<0.0005) between G2 versus G4 and G5 and (P<0.005) between G2 versus G3 in the liver tissue were observed (Fig.1b). Similarly, Mahmoud et al. (39) demonstrated that chickens fed a diet supplemented with VC and exposed to cyclic high temperatures showed a significant decreased the expression level of HSP70 compared with control chickens. Furthermore, Jang et al. (40) reported that the mRNA expression of HSP70 in the liver of birds fed a diet containing VC significantly decreased compared with those birds fed basal diet under summer diurnal heat stress. In quail, HSP70 expression in ovary and brain was decreased as the dietary VC or vitamin E supplementation increased in stressed groups (41). Hence, we suggest that VC supplementation act as an antioxidant and helps to prevent the growth of free radicals, which damage cells and subsequently reduced expression of HSPs in chicken reared under high SD. As the HSF1 is a master regulator of the heat shock genes, through activating the HSPs transcription by binding to heat shock element in the upstream promoter region of HSP genes (42). It has the ability to mediate up-regulation of HSP70 and HSP90 which act a critical role in survivability providing to the organisms subjected to stress (43, 44). In both heart and liver tissue in the present study, the HSP1 expression showed more increased in G2 whish reared under high SD than G3, G4 and G5 relative to control. Its expression showed the same pattern of HSP70 and HSP90 expression in heart tissue and HSP90 expression in liver tissue. Moreover, Beloor et al. (32) demonstrated that the expression levels of HSP90 in the liver samples were higher in high density group compared with the low and standard groups but, didn't showed significant differences. The current study also showed that stress-induced due to high stocking density increases the expression of HSP70 and HSP90, which play essential protective roles in maintaining the metabolic and structural integrity of the cells and organs against stress-induced injury (45-47). Conclusions Increasing the stocking density from 11.6 to 15.6 birds/m2 caused stress in broiler chicks 136 R. S. Shewita, K. El-Naggar, W. S. H. Abd El Naby which tended to reduce their performance, increase mortality rate, affect liver function enzymes (SGPT and SGOT) and change the expression level of heat shock protein genes. Supplementation of VC especially at 200 mg/kg chick's diet may offer a suitable nutritional strategy to overcome the disadvantageous effects of increased stocking density. Conflicts of Interest The authors declare no conflicts of interest References 1. Thaxton JP, Dozier WA, 3rd, Branton SL, Morgan GW, Miles DW, Roush WB, et al.Stocking density and physiological adaptive responses of broilers. Poult Sci 2006; 85(5): 819-24. 2. Pandurang LT, Kulkarni GB, Gangane GR, More PR, Ravikanth K, Maini S, et al.Overcrowd-ing Stress Management in Broiler Chicken with Herbal Antistressor. IJAS 2011; 1(1): 49-55. 3. Whitehead CC, Keller T.An update on ascorbic acid in poultry. World's Poult Sci J 2003; 59(2): 161-84. 4. Zhang AW, Lee BD, Lee SK, Lee KW, An GH, Song KB, et al.Effects of yeast (Saccharomy-ces cerevisiae) cell components on growth performance, meat quality, and ileal mucosa development of broiler chicks. Poult Sci 2005; 84(7): 1015-21. 5. Campo JL, Davila S.Changes in heterophil to lymphocyte ratios of heat-stressed chickens in response to dietary supplementation of several related stress agents. Arch Fur Geflugelk 2002; 66: 80-4. 6. Ipek H, Avci M, Yerturk M, Iriadam M, Ay-dilek N.Effects of ascorbic acid and vitamin E on performance and hematological parameters of Japanese quails under high ambient temperature in San-liurfa. Arch Fur Geflugelk 2007; 71: 130-4. 7. Lin H, Jiao HC, Buyse J, Decuypere E.Strate-gies for preventing heat stress in poultry. World's Poult Sci J 2007; 62(1): 71-86. 8. Al-Aqil A, Zulkifli I.Changes in heat shock protein 70 expression and blood characteristics in transported broiler chickens as affected by housing and early age feed restriction. Poult Sci 2009; 88(7): 1358-64. 9. Zulkifli I, Liew PK, Israf DA, Omar AR, Hair-Bejo M.Effects of early age feed restriction and heat conditioning on heterophil/lymphocyte ratios, heat shock protein 70 expression and body temperature of heat-stressed broiler chickens. J Therm Biol 2003; 28(3): 217-22. 10. Jacquier-Sarlin MR, Fuller K, Dinh-Xuan AT, Richard MJ, Polla BS.Protective effects of hsp70 in inflammation. Experientia 1994; 50(11-12): 1031-8. 11. Jackson SE.Hsp90: structure and function. Top Curr Chem 2013; 328: 155-240. 12. NRC, Nutrient requirements of poultry. 9th Ed. Washington, DC:National Academic Press,1994. 13. Houshmand M, Azhar K, Zulkifli I, Bejo MH, Kamyab A.Effects of prebiotic, protein level, and stocking density on performance, immunity, and stress indicators of broilers. Poult Sci 2012; 91(2): 393-401. 14. Rao X, Huang X, Zhou Z, Lin X.An improvement of the 2A(-delta delta CT) method for quantitative real-time polymerase chain reaction data analysis. Biostat, Bioinforma Biomath 2013; 3(3): 71-85. 15. Xie J, Tang L, Lu L, Zhang L, Xi L, Liu HC, et al.Differential expression of heat shock transcription factors and heat shock proteins after acute and chronic heat stress in laying chickens (Gallus gallus). PloS one 2014; 9(7): e102204. 16. Nerren JR Swaggerty CL, MacKinnon KM, Genovese KJ, He H, Pevzner I, et al.Differential mRNA expression of the avian-specific toll-like receptor 15 between heterophils from Salmonella-susceptible and -resistant chickens. Immunogenetics 2009; 61(1): 71-77. 17. SAS, Statistical Analysis System North Car-olina:Users Guide Statistics. As. Institute Cary,1996. 18. Dozier WA, 3rd, Thaxton JP, Branton SL, Morgan GW, Miles DM, Roush WB, et al.Stocking density effects on growth performance and processing yields of heavy broilers. Poult Sci 2005; 84(8): 1332-8. 19. Elagib-Hind AA, Omer HM.Effect of Dietary Ascorbic Acid on Performance and Immune Response of Heat Stressed Broiler Chicks. Pakistan J Nutr 2012; 11(3): 216-20. 20. SabahElkheir MK, Mohammed AMM, Ab-del-Gadir SM.Effect of Feed Restriction and Ascorbic Acid Supplementation on Performance of Broiler Chicks Reared under Heat Stress. Res J Ani & Vet Sci 2008; 3: 1-8. 21. Sahin K, Kucuk O.Effects of vitamin E and selenium on performance, digestibility of nutrients, and carcass characteristics of Japanese quails reared under heat stress (34 degrees C). J anim Physiol Anim Nutr 2001; 85(11-12): 342-8. 22. Lohakare JD, Ryu MH, Hahn TW, Lee JK, Chae BJ.Effects of Supplemental Ascorbic Acid on Influence of dietary vitamin C supplementation on growth performance, blood biochemical parameters and. 137 the Performance and Immunity of Commercial Broilers. J Appl Poult Res 2005; 14(1): 10-19. 23. Blaha J, Kroesna K.Effect of vitamin and electrolytes supplements on broiler performance, slaughter value and chemical composition of meat during the heat stress. Universitäs Agriculturäe Praga Press 1997; 30: 103-13. 24. Jaffar GH, Blaha J.Effect of ascorbic acid supplementation in drinking water on growth rate, feed consumption and feed efficiency of broiler chickens maintained under acute heat stress conditions. Universitas Agriculturae Praga Press 1996; 41: 485-90. 25. McKee JS, Harrison PC.Effects of supplemental ascorbic acid on the performance of broiler chickens exposed to multiple concurrent stressors. Poult Sci 1995; 74(11): 1772-85. 26. Amady V, Tayb I, Yokhana J. Effects of supplemental ascorbic acid on humeral immune response in broilers reared under heat stress conditions. Res Opinions Anim Veter Sci. 2011; 1: 45962. 27. Amakye-Anim J, Lin TL, Hester PY, Thia-garajan D, Watkins BA, Wu CC.Ascorbic acid supplementation improved antibody response to infectious bursal disease vaccination in chickens. Poult Sci 2000; 79(5): 680-8. 28. Simsek UG, Dalkilic B, Ciftci M, Yuce A.The Influences of Different Stocking Densities on Some Welfare Indicators, Lipid Peroxidation (MDA) and Antioxidant Enzyme Activities (GSH, GSH-Px, CAT) in Broiler Chickens. J Anim Vet Adv 2009; 8(8): 1568-72. 29. Al-Darajji H, Al-Mashhadanp E, Al-Atharp A.Effect of ascorbic acid supplementation in the diets on haematological traits of Fawbro broiler breeders reared under hot climate Indian J Anim Sci. 2001; 71(9): 857-9. 30. Kucuk O, Sahin K, Sahin N, Gursu M, Gulcu F, Ozcelik M, et al. Egg production, egg quality, and lipid peroxidation status in laying hens maintained at a low ambient temperature (6 deg C) and fed a vitamin C and vitamin E-supplemented diet. Vet Med 2003; 48: 33-40. 31. Najafi P, Zulkifli I, Jajuli NA, Farjam AS, Ramiah SK, Amir AA, et al. Environmental temperature and stocking density effects on acute phase proteins, heat shock protein 70, circulating corti-costerone and performance in broiler chickens. Int J Biometeorol 2015; 59(11): 1577-83. 32. Beloor J, Kang HK, Kim YJ, Subramani VK, Jang IS, Sohn SH, et al.The Effect of Stocking Density on Stress Related Genes and Telomeric Length in Broiler Chickens. Asian-Aust J Anim Sci 2010; 23(4): 437-43. 33. Basu N, Todgham AE, Ackerman PA, Bibeau MR, Nakano K, Schulte PM, et al.Heat shock protein genes and their functional significance in fish. Gene 2002; 295(2): 173-83. 34. Gornati R, Papis E, Rimoldi S, Terova G, Sa-roglia M, Bernardini G.Rearing density influences the expression of stress-related genes in sea bass (Dicentrarchus labrax, L.). Gene 2004; 341: 111-8. 35. Aksakal E, Ekinci D, Erdogan O, Beydemir §, Alim Z, Ceyhun SB.Increasing stocking density causes inhibition of metabolic-antioxidant enzymes and elevates mRNA levels of heat shock protein 70 in rainbow trout. Livest Sci 2011; 141(1): 69-75. 36. Craig EA, Gross CA.Is hsp70 the cellular thermometer? Trends in Biochem Sci 1991; 16: 135-40. 37. Parsell DA, Lindquist S.The function of heat-shock proteins in stress tolerance: degradation and reactivation of damaged proteins. Annu Rev Genet 1993; 27: 437-96. 38. Sahin K, Sahin N, Onderci M, Gursu MF, Issi M.Vitamin C and E can alleviate negative effects of heat stress in Japanese quails. Food , Agric Envir 2003; 1(2): 244--9. 39. Mahmoud KZ, Edens FW, Eisen EJ, Ha-venstein GB.Ascorbic acid decreases heat shock protein 70 and plasma corticosterone response in broilers (Gallus gallus domesticus) subjected to cyclic heat stress. Comp Biochem Physiol B Biochem Mol Biol 2004; 137(1): 35-42. 40. Jang IS, Ko YH, Moon YS, Sohn SH.Effects of Vitamin C or E on the Pro-inflammatory Cytokines, Heat Shock Protein 70 and Antioxidant Status in Broiler Chicks under Summer Conditions. Asian-Australas J Anim Sci 2014; 27(5): 749-56. 41. Sahin N, Tuzcu M, Orhan C, Onderci M, Er-oksuz Y, Sahin K.The effects of vitamin C and E supplementation on heat shock protein 70 response of ovary and brain in heat-stressed quail. Br poult sci 2009; 50(2): 259-65. 42. Khassaf M, McArdle A, Esanu C, Vasilaki A, McArdle F, Griffiths RD, et al.Effect of vitamin C supplements on antioxidant defence and stress proteins in human lymphocytes and skeletal muscle. J Physiol 2003; 549(Pt 2): 645-52. 43. Fangue NA, Hofmeister M, Schulte PM.In-traspecific variation in thermal tolerance and heat shock protein gene expression in common killifish, Fundulus heteroclitus. J Exp Biol 2006; 209(Pt 15): 2859-72. 44. Copat C, Brundo MV, Arena G, Grasso A, Oliveri Conti G, Ledda C, et al.Seasonal variation 138 R. S. Shewita, K. El-Naggar, W. S. H. Abd El Naby of bioaccumulation in Engraulis encrasicolus (Linneaus, 1758) and related biomarkers of exposure. Ecotoxicol Environ Saf 2012; 86: 31-7. 45. Yu J, Bao E, Yan J, Lei L.Expression and localization of Hsps in the heart and blood vessel of heat-stressed broilers. Cell stress chaperones 2008; 13(3): 327-35. 46. Kim M, Ahn IY, Kim H, Cheon J, Park H.Molecular characterization and induction of heat shock protein 90 in the Antarctic bivalve Laternula elliptica. Cell stress chaperones 2009; 14(4): 36370. 47. Gu XH, Hao Y, Wang XL.Overexpression of heat shock protein 70 and its relationship to intestine under acute heat stress in broilers: 2. Intestinal oxidative stress. Poult Sci 2012; 91(4): 790-9. Slov Vet Res 2019; 56 (Suppl 22): 139-48 DOI 10.26873/SVR-751-2019 Original Research Article EFFECTS OF FEEDER SHAPE ON BEHAVIORAL PATTERNS, PERFORMANCE AND EGG QUALITY TRAITS OF JAPANESE QUAIL Hesham H. Mohammed1*, Ibrahim F. Rehan2, Ahmed F. Abou-Elnaga3, Radi A. Mohamed4 department of Veterinary Public Health, Faculty of Veterinary Medicine, Zagazig University, 44511, Egypt. 2Department of Animal Behavior and Husbandry, Faculty of Veterinary Medicine, South Valley University, 83523, Egypt, 3Department of Husbandry and Development of Animal Wealth, Faculty of Veterinary Medicine, Mansoura University, 35516, Egypt. 4Department of Aquaculture (Animal and Fish Behavior and Welfare), Faculty of Aquatic Sciences and Fisheries, University of Kafrelsheikh, 33511, Egypt Corresponding author, E-mail: heshamvet_hosny@yahoo.com Abstract: Offering the food to Japanese quails may help to reorganize their behavioral needs. However, limited data was reported on the effect of feeder shape on birds' behaviors, welfare, and productivity. Herein, the main objective was to assess the effects of the most available feeder shapes in the Egyptian market on the birds' behaviors. The shape of feeders were longitudinal, vertical-narrow, vertical-wide and round. All the used feeders were made from plastic, marked with a grey color and enriched with a net. A total of 180 one-day old Japanese quail (Coturnix japónica) were divided into four experimental groups (three replicates of each). Behavioral patterns of birds were investigated by direct observation and video recording for four days per week using scan sampling technique. The results indicated that there were feeder shape-dependent-differences in quail behaviors demonstrating the highest significant (P<0.001 ) levels of activity behaviors in a round feeder compared to other groups. Moreover, the highest significant (P<0.001) levels of resting behaviors within a vertical-wide feeder group. Likewise, flying behavior was recorded in the above mentioned group. However, the fear associated responses and alertness were the highest significant level (P<0.001 each) in longitudinal and vertical-wide feeders. Noticeably, the aggressive pecking was the highest significant (P<0.001) levels in birds dealt with a longitudinal feeder. Meanwhile, almost of a quail's egg quality traits demonstrated the highest significant values (P<0.001) during offering round containers for the birds. The results therefore suggested that quails might have a preference to deal more with a round feeder shape than other shapes. These findings may also have great implications to researchers, veterinarians and stakeholders to decide the most economic feeder shape of quails for greater performance and productivity. Key words: feeder shape; welfare; egg quality; quail Introduction Today, the poultry industry is the most popular and enriched sources of animal protein in the form of eggs. Therefore, poultry welfare is Received: January 2019 Accepted for publication: February 2019 140 H. H. Mohammed, I. F. Rehan, A. F. Abou-Elnaga, R. A. Mohamed essential to be deeply understood. Welfare of poultry is mainly regulated by basic principles, among which feeding plays as the most important input for intensive poultry production and has superseded effect on the financial viability of the production cycle (1). Previous and new studies have been done to maximize the feeding efficiency of chickens in areas like ingredients' selection and feed processing methods (2), the effect of feed particle size on flock performance (3), and gut development (4). However, a little data informed the impact and efficiency of different feeder shapes on birds' feeding behavior and/or productivity. Hence, Japanese quails were inexpensive to keep and had a high immunity against common poultry diseases, we should pay attention to the tool/feeder by which offered the food to realize the birds' behavioral needs toward welfare and productivity (5). Feeding of quail has been studied in several aspects, even though few have compared the different quail feeder design in a collective study. It was reported that design way, such as size, place, geometry, spacing and/or angle of feeders can change the behavior of animals (6). Moreover, nets or partition grids covering feed trays were widely used in the poultry farms to promote a better collection of the birds around the feeders and to reduce feed competition, fighting and wastage (7). The feeder body might be in the form of a channel having a substantially C-, V- or U-shape in cross-section and comprising a passageway extending between the first and the second openings (8). Moreover, the same author stated that the body of animal feeding device may have any suitable cross-sectional shape like circular, triangular, square or oval. Likewise, the most available feeder shapes are linear, vertical and round shape in the Egyptian market. It was given by the fact that the body of the animal/bird feeding device may be designed of any size, type, shape or configuration, and it will be understood that the size of the body will be linked to the animal with which the animal feeding device is intended to be used. Therefore, the patterns of brain activation might be regulated by particular neuropeptides and/or gene expressions like immediate-early gene toward the targeted preferences (9). Egg weight, shell weight, shell thickness, weight of albumen and yolk are the most important traits affecting egg quality under a good managemental condition (e.g., feeder shape) and fertility (10). Positive correlations among egg weight, shell weight and shell thickness have also been studied (11). However, poor egg quality results substantial economic losses to the worldwide egg industry (12). Together, it seems likely that the feeder shapes of quails may help to understand their behavioral needs towards the well-being and egg productivity. Therefore, the current study aims to check the influences of four different feeder shapes (longitudinal, vertical-narrow, vertical-wide and round) in relation to the quails' behaviour, performance and egg quality traits. Our finding may give a merit to decide the most attractive feeder shape of quail for economic purposes in quail farms. Material and methods This experiment was done after the approval by Ethics and Animal welfare committee of the poultry Research Unit, Faculty of Veterinary Medicine, Zagazig University, Egypt (ANWD-206). Birds and housing A total of 180 one-day-old Japanese quail chicks (Coturnixjaponica), weight 7.43 ± 0.28 g purchased in one batch from Faculty of Agriculture, Zagazig University. The chicks were divided into four groups (45 quails/each) and each group was divided into three replicates (each 15 birds/0.563m2) kept in brooder house in the same home pen. The experimental groups were categorized according to feeder's shapes (longitudinal, vertical-narrow, vertical-wide and round), as shown in Figure (1). All the used feeders were made of plastic type, marked with a red color and enriched with net to avoid the food wastage. Quail chicks were provided with 35°C ambient temperature during the 1st week of brooding and then gradually decreased by about 3.5°C/week till chicks were entirely feathered at 3-4 weeks. Each pen supplied with Effects of feeder shape on behavioral patterns, performance and egg quality traits of Japanese quail 141 10 cm thickness of sawdust (deep litter system) and hydrated lime was firstly sprinkled before spreading the well dried new litters. Available area per quail was 0.038m2/bird. Quails were supplied with food and water as ad-libitum source in a synchronization program twice daily, 7 am and 5 pm, throughout the study. The basic commercial quail's starter diet through the rearing phase contained 24% crude protein and 12.45 MJ/kg metabolizable energy (13). Laying hens in all groups were fed commercial feed mixtures (Table 1), which was formulated to give the typical nutrient requirements (14) of quail contained 20% crude protein, 2.5% calcium and 11.93 MJ/kg metabolizable energy. The lighting system was 16 h of light for the period of rearing and subsequently from the six weeks of age altered to 14 h light with 10 h dark until the end of the study. The group housed quails were marked on their back by using a permanent marker pen to permit individual identification and these marks were refreshed every week throughout the experimental period. Behavioral observation Quails behavior was recorded by direct observation through using scan sampling technique (15), where each group was observed 3 times daily (20 min each) for 4 days weekly for the duration of the rearing period at 7 am, 12 pm and 4 pm for reporting the different behavioral patterns (see Table 2). The same individual recorded the behavioral patterns in all experimental groups through standing directly in front of each group and waiting 10 min prior the recording data to avoid any disturbance in the behaviors and to minimize the error factors (16). After scanning, the numbers of quails were counted in the observed pens to calculate the frequencies of behaviors per 1 h. These numbers were important to be used in recording the activities of quails in all treated groups (17). Growth performance Growth performance was recorded previously (5), where the quails were weighed on 1st day of age as one-day-old live weight and then live body weights was subsequently estimated weekly until the 4th week of age using digital balance (Sartorius 1202 MP balance, GmbH, Gottingen, Germany). Also, the average feed intake was recorded daily after calculating the feed residues. Body weight gain and feed conversion ratio were also calculated. Egg quality traits 60 quail's eggs (from the 7th until the 10th weeks of age) were collected (15 eggs from each group). The Sartorius 1202 MP balance measured the weight (g) of whole egg, albumen, yolk and egg shell weight, while electronic digital caliper was used for calculating the whole egg length, its width and shell thickness (mm) (18-21). Data analyses Data were tested for distribution normality, linearity and homogeneity of variance. Data were analyzed using SAS statistical system, Package v9.2, version (22). Data were reported as means ± SEM, compared by one-way ANOVA and the Duncan's multiple range tests was used as a post hoc test. The behavioral variables did not meet the requirements of parametric tests even after transformation, therefore the equivalent non-parametric Kruskal-Wallis test was used to compare between different groups. While, the significant differences among groups were estimated by Mann-Whitney test. Results Effect of feeder shape on behavioral patterns The results showed that there was an effect of feeder shapes on the behavioral patterns and performance of quails (Table 3). The highest levels of eating behavior were recorded in the group dealt with a round feeder compared to the other treatment groups. Moreover, the highest levels of activity (e.g., walking, preening, body shaking, dust pecking, leg stretch and wing stretch) were observed in the same above group (P<0.001). Furthermore, the highest level of resting period (e.g., sitting, dust-bathing and sleeping) was recorded in the group dealt with a vertical-wide feeder (P<0.001). However, the 142 H. H. Mohammed, I. F. Rehan, A. F. Abou-Elnaga, R. A. Mohamed highest fear response (e.g., stand idle and elimination) were found in the group dealt with a longitudinal feeder (P<0.001, P<0.001) and the quail vices (e.g., feather pecking, loudly sounds) were the highest recoding in the same feeder shape, with a significant level P<0.001 each. Meanwhile, alertness (e.g., drinking, standing) were the highest observations in the group dealt with a vertical-narrow feeder, P<0.001 each, compared to the other treatment groups. Effect offeeder shape on bird's performance There were significant effects of the different feeder shape on quail performance (Table 4). In particular, quail group dealt with a vertical-wide feeder in the 1st and 3rd week (P<0.05) had the highest body weight in comparison with the other groups. However, the rest of weeks had no significance differences. Moreover, there was no significant difference in feed intake, weight gain and feed conversion ratio between different feeder shapes. While the longitudinal feeder showed the highest weight gain and the lowest feed conversion ratio, and vertical-narrow feeders recorded the lowest feed intake. Effect of feeder shape on egg quality traits There were statistical effects of feeder shapes on the external quality (egg weight, width, length, shell weight, and shell thickness) also on the internal quality (albumen and yolk weight) of quail's eggs (see Table 5). The highest levels of egg quality traits (e.g., egg weight, egg width, shell thickness and albumen weight) represented in the group dealt with a vertical-wide feeder (P<0.001) compared to the other treatment groups. Moreover, the highest level of shell weight demonstrated in the group dealt with a round feeder (P<0.001) compared to the other groups. However, the high levels of egg length and yolk weight shown in the group dealt with a longitudinal feeder (P=0.004, P<0.001, respectively) compared to the other groups. Relationship between external and internal egg quality traits There were correlation coefficients among the external and internal egg quality traits in Japanese quail dealt with the analyzed feeder shapes (Table 6). Albumin weight was positive correlated with all external traits except shell weight, which was negatively correlated with yolk weight. Furthermore, there was negative correlation between yolk weight and shell quality (shell weight and thickness), while yolk weight correlated positively with other external egg quality. Table 1: Ingredients of the experimental diet (kg/100kg) Ingredients Kg Calculated analysis Yellow corn 65 Metabolized energy 11.93 MJ/kg Soybean meal (44%) 20 Crude protein 20% Corn gluten 5.2 Calcium 2.5% Calcium carbohydrate & phosphate 2.1 Soybean oil 0.2 Premix and common salts 0.7 Other feed additives 0.6 Effects of feeder shape on behavioral patterns, performance and egg quality traits of Japanese quail 143 Table 2: Definition of recorded behaviors Behaviors Definition Eat Head extended towards available feed resources while beak in or above the drinker appears to be manipulating or ingesting feed Drink Beak in contact with water in or above the drinker and appears to be drinking water Walk Moves forward taking one or more steps Sitting Head rested on something (litter or another bird) while sitting Dust bathing Bathing the dust with the use of wings, head, neck and legs performing vertical wing-shaking Preen Beak related behavior that beak touches the plumage of the bird itself Stand The abdomen is not touching the litter and the bird is motionless with no apparent movement of legs Idl Standing with motionless Sleeping Bird's neck is fully recumbent and the eyes permanently closed while lying Body shaking Raise feathers and shake body Elemination Dropping of fecal materials Dust pecking Peck floor with feet usually associated with eating behavior Leg stretching Extending one leg at the same side of the body Wing stretching Extending one wing at the same side of the body Fly By forcing wings displacement from one place to another Feather pecking Birds pecks the feather of another birds Sound Call or vocalization given by the bird Table 3: Means (±SEM) of some behavioral patterns of Japanese quail using different shapes of feeders (Numbers of quails/hour) Behavioral patterns Shapes of feeders Longitudinal feeder Vertical narrow feeder Vertical wide feeder Round feeder P-Values Eat 356.58±23.98 343.50±33.89 369.67±33.86 372.17±55.53 0.36 Drink 122.17±1.27 b 134.17±1.50 a 130.50±2.17 a 105.83±1.01 c 0.000 Walk 300.00±3.78 c 318.67±3.24 b 325.83±1.96 ab 328.33±2.03 a 0.000 Sit 658.67±16.14 b 654.83±14.31 b 769.75±13.57 a 716.75±12.91 b 0.000 Dust bath 15.33±1.35 b 16.92±1.12 b 28.83±0.90 a 14.08±0.70 b 0.000 Preen 277.08±8.22 b 271.00±3.42 b 268.63±5.11 b 321.00±3.43 a 0.000 Stand 332.00±1.95a 336.17±2.47 a 229.25±3.26 c 275.42±5.56 b 0.000 Idle 142.08±3.38a 139.09±2.06a 94.33±1.69c 120.25±2.98b 0.000 Sleep 353.06±8.37c 356.42±3.80c 444.33±2.17 a 391.83±3.25 b 0.000 Body shaking 25.58±1.93a 14.66±0.56b 17.58±0.76b 25.66±1.26 a 0.000 Elimination 3.15±0.36a 2.08±0.28b 0.58±0.22c .33±0.03 c0 0.000 Dust peck 49.66±3.99b 35.58±1.94c 44.66±1.57b 59.00±0.94a 0.000 Leg stretch 47.5±2.36b 51.58±1.82b 46.92±1.31b 60.66±2.15 a 0.000 Wing stretch 76.08±2.06b 74.00±1.47b 68.00±2.23c 103.17±1.73 a 0.000 Fly 15.50±0.64c 22.16±0,71b 26.5±0.95a 21.5±0.87b 0.000 Feather peck 4.25±0.52 4.00±0.36 3.66±0.33 4.08±0.35 0.77 Sound 3.25±0.56 2.58±0.41 2.00±0.42 2.66±0.48 0.33 abc Means within the same row having different superscripts are significantly different at P<0.05 144 H. H. Mohammed, I. F. Rehan, A. F. Abou-Elnaga, R. A. Mohamed Table 4: Means (± SEM) of growth performance of Japanese quail using different shapes of feeders Shape of feeders Growth performance Longitudinal Vertical narrow Vertical wide Round P-Values feeder feeder feeder feeder Initial body weight (g) 6.76±0.57 7.37±0.32 7.27±0.66 8.32±0.56 0.25 Body weight in 1st week (g) 55.5±2.38ab 54.41±2.08ab 61.58±2.76 a 50.16±2.70 b 0.02 Body weight in 2nd week (g) 102.68±3.54 96.23±4.41 104.89±3.57 108.19±1.82 0.11 Body weight in 3rd week (g) 138.34±5.06 ab 135.72±4.36 ab 148.01±4.25a 129.84±4.21 b 0.05 Body weight in 4th week (g) 192.20±4.93 186.06±6.92 185.98±5.19 180.11±5.49 0.52 Feed intake (g) 15.58±0.72 a 15.11±0.64 a 15.37±0.65 a 15.91±0.53 b 0.83 Weight gain (g) 185.40±5.00 178.70±6.81 178.70±5.60 171.80±5.50 0.43 Feed conversion ratio 2.38±0.15 2.39±0.13 2.44±0.17 2.62±0.13 0.60 ab Means within the same row having different superscripts are significantly different at P<0.05. g= gram Table 5: Means (± SEM) of egg quality traits of Japanese quail using different shapes of feeders Egg quality Shape of feeders Longitudinal feeder Vertical narrow feeder Vertical wide feeder Round feeder P Values Egg weight (g) 11.48±0.18 a 10.59±0.23 b 11.61±0.15 a 10.36±0.16 b 0.000 Egg width (mm) 25.53±0.41 24.79±0.22 26.01±0.26 25.26±0.49 0.128 Egg length (mm) 32.20±0.66 a 30.64±0.46 b 32.09±0.26 a 30.06±0.43 b 0.004 Shell weight (g) 1.03±0.02 c 1.41±0.04 b 1.58±0.03 a 1.60±0.05 a 0.000 Shell thickness (mm) 0.21±0.007 c 0.26±0.017 ab 0.27±0.004 a 0.23±0.009 bc 0.001 Albumin weight (g) 6.12±0.07 a 5.82±0.16 a 6.24±0.12 a 5.12±0.22 b 0.000 Yolk weight (g) 4.33±0.16 a 3.42±0.15 b 3.78±0.09 b 3.64±0.12 b 0.000 abc Means within the same row having different superscripts are significantly different at P<0.05. g= gram mm= millimeter Table 6: Correlation coefficients among the external and internal egg quality traits in Japanese quails Egg quality traits Albumin weight (g) Yolk weight (g) Egg weight _(g_ Egg width Egg length (mm)_(mm) Shell weight _(g_ Shell thickness (mm) 0.786*** 0.610*** 0.312* 0.333*** 0 449*** 0.375*** -0.242 -0.362*** 0.296* -0.291* The asterisk (*) showed a significance level, P<0.05 and the asterisk (***) showed a significance level, P<0.001. G= gram mm=millimeter Figure 1: Different shape of feeders from left to right longitudinal feeder, vertical-narrow feeder, vertical wide feeder and round feeder. Discussion The behavioral patterns reflect a serious of the activities of endocrine and exocrine character for assessing the animal's response to its environment that consequently impairs its welfare and production (23). Herein, for the first time, we examine the influence of feeder shape on the CNS stimuli of quail hens to be targeted on their behavioral responses and production. As a result shown in Table 3, the eating behavior had no significant difference among all groups. It means that the feeder shapes have no Effects of feeder shape on behavioral patterns, performance and egg quality traits of Japanese quail 145 clear influences on feed intake of quails. Meanwhile, the quail hens have been dealt with round shape feeders were the best group to perform several behaviors other than eating, such as, activity behaviors (e.g., walking, preening, leg/wing stretching, pecking and body shaking) and resting behaviors (e.g., sitting, sleeping and dust-bathing). All these behaviors are considered to be 'natural behaviors', and good indicators of welfare of birds (24). However, the quail hens have been dealt with longitudinal/vertical-narrow shape feeders were the groups clearly performed the fear associated responses (e.g., elimination and idle) as well as alertness behaviors (e.g., drinking and standing). By which, these behaviors are considered poor indicators of welfare of birds. Moreover, it's well-known that quails/birds can stay rested up to 70-80% of their time (25). It can be understood that the birds using a vertical-wide feeder somehow felt more comfortable by staying/resting nearby this feeder shape than others. Changing of the management condition in quail farm might affect the performance of the birds. The result in Table 4 showed that quails dealt with a vertical-wide feeder showed significantly the best body weight, particularly in the 1st and 3rd week of age comparable to the other groups. In spite of the non-significant effects of different feeder shapes on quails' weight in the 4th week, quail dealt with a round feeder showed the best body weight and feed intake compared to the other groups. These results agreed with two other researchers who stated that some management condition can affect the growth performance of quails (26, 27), such as body weight (28) and feed consumption (29). Our results indicated that the vertical-wide and round feeder shapes can positively affect the performance of quails and it can show the good managerial conditions applied in our experiment. This preference of these feeder shapes towards the increasing the activities and performance of quails might relate to the centrally induced appetite by neuropeptide Y (30). Moreover, it is probably regulated by the high genetic expression of immediate-early gene Zenk in Pallial brain structure (9). In this study, the average values of external quality traits of quail's eggs (e.g., egg weight, its width, its length & shell weight and its thickness) in Table 5, indicated similarities with the finding of most researchers (11, 31-35). Likewise, the average of internal quality traits of quail's eggs (e.g., albumen weight and yolk weight) indicated similarities by the finding of above researchers. It seems likely that the slight differences between the results of egg quality traits of this research and the results of the other researchers might have resulted from the genetic structure, health condition, flock age, use of different content diet in feeding, and the differences in the care and management condition of the quails (e.g., feeder shape). We reached the point that offering feeder shapes might change the behavioral patterns of quails and remain an important question whether feeder shape can affect the egg quality traits. In this study (Table 6), statistically significant correlation (M±SEM) was obtained among the average of egg weight, its width, shell weight, and shell thickness, especially in the group dealt with a vertical-wide feeder compared to the other treatment groups. Therefore, the egg weight had an indirect relation with the shell quality of the egg. It has been stated by most of the researchers reported that the shell thickness had a direct relation to egg weight (36, 37), also had a positive correlation to the shell weight (36, 38). It's well-known that shell ratio in the total egg had an opposite relation to the egg weight. It comes from the fact that the increase depending on the egg weight on the shell weight and the shell thickness was less than the increase of other components that formed the egg (35). However, by offering a vertical-wide shape and a round shape feeder to quail hens, the shell weight (1.58±0.03, 1.60±0.05, respectively) and shell thickness (0.27±0.004, 0.23±0.009, respectively) have not been clearly affected by the egg weight. Therefore, the rounded feeders have the best economic value among the other groups. It means that the egg shell quality would be evaluated by using the egg weight values due to the positive relation determined between the egg weight and the shell thickness, and the shell weight. In this study, statistically a significant 146 H. H. Mohammed, I. F. Rehan, A. F. Abou-Elnaga, R. A. Mohamed negative correlation was present between the albumen and yolk weight of the egg particularly in the group dealt with a vertical-wide shape feeder (6.24±0.12, 3.78±0.09, respectively) and the other group dealt with a round shape feeder (5.12±0.22, 3.64±0.12, respectively). It was reported that the housing system can affect egg quality and concentration of cholesterol in egg yolk in hens (39), Thusly, increasing the yolk weight in the whole egg is not preferable for human health and marketing. In this study, the group dealt with a longitudinal feeder had the significant highest yolk weight (4.33±0.16) than the other groups. However, the improvement of the albumen weight indicated the dense albumen quality to perfectly estimate the internal egg quality traits (35). In our study, almost all internal quality traits of the egg were significantly influenced based on the influence occurred in the egg weight with respect to the external quality traits. However, the yolk weight and egg length were negatively correlated to the albumen weight. This case was found to be in conformity with the findings of some researchers (40). The results were in contrast with the results mentioned that there were positive significant differences among egg weight and egg length (41). Our finding has evidence that the quails had a more prefer to deal with rounded feeder than longitudinal and vertical ones indicating good behaviors and welfare. It has great implications for researchers, veterinarians and stockholders to decide the most economic feeder shape of quails for the maximum performance and productivity. Conclusion The different feeder shapes influenced the quails' behavior, welfare and productivity. Herein, the birds have demonstrated significantly the highest behavioral patterns when offering round shape feeder. However, the significant lowest activities have been detected when offering longitudinal/vertical shape feeder. It seems likely that the birds were familiarities to the round shape feeder. The quails' performance and egg quality traits had significant effects due to changes in shapes of feeder types. Therefore, for maintaining a successful and profitable quail farming business, the round feeder container as a recommended. Contributions Hesham H. Mohammed and Ibrahim F. Rehan are mutually contributed to this study, designed the survey protocol, supervised data collection procedures and drafted the final version of the manuscript; Ahmed F. Abou-Elnaga and Radi A. Mohamed analyzed the data and shared in experimental protocol. All authors have finalized the experimental design and revised the manuscript and then contributed to, edited, and approved the final manuscript as submitted. Competing financial interests The authors declare that they have no competing financial interests and non-financial interests. Acknowledgments We thank the staffs at the Department of Veterinary Public Health, Faculty of Veterinary Medicine, Zagazig University, Egypt for providing materials used in this study. References 1. Skinner JT, Waldroup AL, Waldroup PW. Effects of dietary nutrient density on performance and carcass quality of broilers 42 to 49 days of age. Journal of Applied Poultry Research 1992; 1(4): 36772. 2. Rodgers NJ, Choct M, Hetland H, Sundby F, Svihus B. Extent and method of grinding of sorghum prior to inclusion in complete pelleted broiler chicken diets affects broiler gut development and performance. Animal Feed Science and Technology 2012; 171(1): 60-7. 3. Amerah AM, Ravindran V, Lentle RG, Thomas DG. Feed particle size: Implications on the digestion and performance of poultry. World's Poultry Science Journal 2007; 63(3): 439-55. 4. Neves DP, Mehdizadeh SA, Tscharke M, de Alencar NI, Banhazi TM. Detection of flock movement and behaviour of broiler chickens at different feeders using image analysis. Information Processing in Agriculture 2015; 2(3-4): 177-82. 5. Mohammed HH, Said EN, Abdel-Hamid SE. Impact of different litter materials on behaviour, growth performance, feet health and plumage score Effects of feeder shape on behavioral patterns, performance and egg quality traits of Japanese quail 147 of Japanese quail (Coturnix japonica). European Poultry Science 2017; 81: 719-27. 6. Wolter BF, Ellis M, Curtis SE, Parr E, Webel DM. Feeder location did not affect performance of weanling pigs in large groups. Animal Science 2000; 78(11): 2784--9. 7. Neves DP, Nääs IA, Vercellino RD, de Moura DJ. Do broilers prefer to eat from a certain type of feeder? Revista Brasileira de Ciencia Avícola 2010; 12(3): 179-87. 8. Craig L. Animal feeder." U.S. Patent Application 2018: 15/556,819. 9. Voigt C, Hirschenhauser K, Leitner S. Neural activation following offensive aggression in Japanese quail. Biology open 2018 Jan 1:bio-038026. 10. Khurshid A, Farooq M, Durrani FR, Sar-biland K, Chand N. Predicting egg weight, shell weight, shell thickness and hatching chick weight of Japanese quails using various egg traits as regres-sors. International Journal of Poultry Science 2003; 2(2): 164—7. 11. Farooq M, Aneela K, Durrani FR, Muqarrab AK, Chand N, Khurshid A. Egg and shell weight, hatching and production performance of Japanese broiler quails. Sarhad Journal of Agriculture 2001; 17(3): 289-93. 12. Baylan M, Canogullari S, Ayasan T, Copur G. Effects of dietary selenium source, storage time, and temperature on the quality of quail eggs. Biological Trace Element Research. 2011; 143(2): 95764. 13. National Research Council. Nutritional requirements of poultry. 1994. 14. AO AC. Official Methods of Analysis. Association official analytical chemists, Gaithersburg, USA. 2002. 15. Fraser AF, Broom DM. Farm animal behaviour and welfare. BailliereTindall Publ., London, UK.1990. 16. Mohammed HH, Grashorn MA, Bessei W. The effects of lighting conditions on the behaviour of laying hens. Archiv für Geflügelkunde 2010; 74(3): 197-202. 17. Senaratna D, Samarakone TS, Madusanka AA, Gunawardane WW. Performance, behaviour and welfare aspects of broilers as affected by different colours of artificial light. Tropical Agricultural Research and Extension. 2012; 14(2): 38-44. 18. El-Tarabany MS, Abdel-Hamid TM, Mohammed HH. Effects of cage stocking density on egg quality traits in Japanese quails. Kafkas Univ Vet Fak Derg 2015; 21:13-8. 19. Ahmed AM, Rodríguez-Navarro AB, Vidal ML, Gautron J, García-Ruiz JM, Nys Y. Changes in eggshell mechanical properties, crystallographic texture and in matrix proteins induced by moult in hens. British Poultry Science 2005; 46(3): 268-79. 20. Sezer M. Heritability of exterior egg quality traits in Japanese quail. Journal of Applied Biological Sciences 2007; 1(2): 37-40. 21. Englmaierova M, Tumova E, Charvatova V, Skrivan M. Effects of laying hens housing system on laying performance, egg quality characteristics, and egg microbial contamination. Czech Journal Animal Science 2014; 59(8): 345-52. 22. SAS. SAS statistical system Package-Jmp 8 User's Guide.2nd Cary, NC, SAS Institute Inc. USA. ISBN 2009. 23. Uzunova K. Study of behaviour of broiler chickens subjected to biotic stressors. Trakia Journal of Sciences 2007; 5(3-4): 16-8. 24. S0rensen P, Su G, Kestin SC. Effects of age and stocking density on leg weakness in broiler chickens. Poultry Science 2000; 79(6): 864-70. 25. Bizeray D, Estevez I, Leterrier C, Faure JM. Effects of increasing environmental complexity on the physical activity of broiler chickens. Applied Animal Behaviour Science 2002; 79(1): 27-41. 26. Senaratna D, Atapattu NS, Belpagodaga-mage DU. Saw dust and refuse tea as alternative litter materials for broilers. Tropical Agricultural Research 2007; 19: 283-9. 27. El-Deek AA, Al-Harthi MA, Khalifah MM, Elbanoby MM, Alharby T. Impact of newspaper as bedding material in arid land on broiler performance. Egyptian Poultry Science 2011; 31: 715-25. 28. Toghyani M, Gheisari A, Modaresi M, Tabeidian SA, Toghyani M. Effect of different litter material on performance and behavior of broiler chickens. Applied Animal Behaviour Science 2010; 122(1): 48-52. 29. Abreu VM, Abreu PG, Coldebella A, Jae-nisch FR, Silva VS. Evaluation of litter material and ventilation systems in poultry production: I. overall performance. Revista Brasileira de Zootecnia 2011; 40(6): 1364—71. 30. McConn BR, Gilbert ER, Cline MA. Appetite-associated responses to central neuropeptide Y injection in quail. Neuropeptides 2018; 69: 9-18. 31. Yannakopoulos AL, Tserveni-Gousi AS. Quality characteristics of quail eggs. British Poultry Science 1986; 27(2): 171-6. 32. Uluocak AN, Okan F, Efe E, Nacar H. Exterior and interior quality characteristics of eggs and their variation according to age in Japanese quail. Turkish Journal of Veterinary and Animal Sciences 1995;19:181-5. 148 H. H. Mohammed, I. F. Rehan, A. F. Abou-Elnaga, R. A. Mohamed 33. Altan Ö, Ismail OG, Akba§ Y. Effects of selection for high body weight and age of hen on egg characteristics in Japanese quail (Coturnix coturnix japonica). Turkish Journal of Veterinary and Animal Sciences 1998; 22(6): 467-74. 34. Nazligül A, Türkyilmaz K, Bbrdak?ioglu HE. A study on some production traits and egg quality characteristics of Japanese quail. Turkish Journal of Veterinary and Animal Sciences 2001; 25(6): 1007-13. 35. Özcelik M. The phenotypic correlations among some external and internal quality characteristics in Japanese quail eggs. Veterinary Journal of Ankara University 2002; 49: 67-72. 36. Choi JH, Kang WJ, Baik DH, Park HS: A study on some characteristics of the fractions and shell quality of the chicken egg. Korean Animal Science 1983;25:651-5. 37. Stadelman WJ. The preservation of egg quality in shell eggs. In egg science and technology. Stadelman. WJ and Cotteril. OJ Avi Publishing Com. Inc. Westport. Connecticut. 1986. 38. Poyraz Ö. Kabuk kalitesi ile ilgili yumurta özellikleri arasindaki fenotipik korelasyonlar. Lalahan Zootekni Araçtirma Enstitüsü Derg 1989; 29(1-4): 66-9. 39. Zita L, Jenikovâ M, Härtlovä H. Effect of housing system on egg quality and the concentration of cholesterol in egg yolk and blood of hens of native resources of the Czech Republic and Slovakia. The Journal of Applied Poultry Research 2018; 27(3): 380-8. 40. Içcan KM, Akcan A. Broiler parent yumur-talarinda yumurta agirligi, yumurta özgül agirligi ve bazi yumurta kisimlari arasindaki iliçkiler. Hay Ara§ Derg 1995; 5(1-2): 49-52. 41. Camci Ö, Erensayin C, Aktan S. Relations between age at sexual maturity and some production characteristics in quails. Archiv für Geflügelkunde 2002; 66(6): 280-2. Slov Vet Res 2019; 56 (Suppl 22): 149-56 DOI 10.26873/SVR-752-2019 Original Research Article EFFECT OF BEDDING MATERIALS ON DUCK'S WELFARE AND GROWTH PERFORMANCE Hesham H. Mohammed1*, Asmaa I. Abdelaty1, Al-Sadik Y. Saleem1, Mohamed I. Youssef1, Shereen EL. Abdel-Hamid1 1 Department of Veterinary Public Health, Faculty of Veterinary Medicine, Zagazig University, 44511, Egypt Corresponding author, E-mail: heshamvet_hosny@yahoo.com Abstract: Ducks spend their entire life in contact with the litter material, thus the management strategies should focus on what is the best for ducks welfare and growth performance. Herein, the main object of the present study was assessing the effects of different litter materials on the ducks' welfare and growth performance. Four groups of ducks (24 duckling for each) were kept on four types of bedding materials, sawdust, plastic slatted floor, sand and without. The present study revealed several changes in duck's behaviors due to different bedding materials, where the most frequencies of maintenance behaviors (feeding, drinking, idling, preening) were significantly (P<0.05) higher in plastic slatted in comparing to other materials the highest significantly in plastic slatted litter and the lowest in non-bedding litter. Moreover, the ducks reared under sawdust floor revealed the highest frequency of foraging behavior (11.48 bouts/hour). While, feather pecking and aggression were the highest in non-bedding material (74.4, 5.51 bouts/hour, respectively). The hygienic conditions inside the non-bedding floor was the worst due to increase levels of ammonia and carbon dioxide (12, 0.97 Cm3/Litter, respectively). The ducks reared in plastic slatted floor were the best in growth performance with good signs of soundness. In non-bedding floor, the environmental stress revealed in the increase the level of plasma corticosterone hormone with bad signs of soundness. This study confirms importance of bedding materials in rearing of ducks, especially under plastic slatted floor. Key words: duck; bedding material; behavior; performance; welfare Introduction Poultry has an important role amongst agricultural industries in many countries. Poultry sector in Egypt is one of the major sources of animal protein supply (1). Duck production is an aspect of the poultry industry, which is very popular in many parts of the world. Ducks represent the second largest poultry production in Africa after chicken, beside pure Egyptian breeds there were some local developed strains that established for both meat and egg production (2). Duck used for meat production, which may partly compensate the increasing demand for animal protein, where duck meat is highly appreciated as it combines the characteristics of a red meat and the dietetic characteristics of poultry meat (3). From an economic standpoint, management strategies should focus on what is Received: January 2019 Accepted for publication: February 2019 150 H. Mohammed, A. Abdelaty, A. Saleem, M. Youssef, S. Abdel-Hamid the best for poultry welfare and growth performance (4). To increase the income from ducks rearing, producers are capable of modulating the management of ducks. The well-being of poultry and stress largely influence the poultry production (5). There are many factors which can decrease the performance and increase abnormal behavior of poultry such as management and housing (6). Deep-litter floor housing is most common when raising birds used for meat production (7).In this system, better litter management is crucial for providing good litter quality and for controlling the ammonia level inside the poultry houses. Distinct mixtures of different materials have been proposed as bedding for poultry (8). Duck litter is a mixture of duck excreta, spilled feed, spontaneously fallen feathers and farm bedding material (9). Wood sawdust is the most common used bedding material, however, there were many alternative materials that may be used such as rice and wheat straw (10), soya bean straw (11) and other dry, absorbent, low-cost organic materials. Moreover, the sand is occasionally used as a bedding material (12). The ducks spend their entire life in contact with the litter material. Therefore, its quality is considered a crucial factor of poultry welfare (13), where the good litter should be characterized by good absorption property with a reasonable drying time (14), fast drought, low price and acceptable as a fertilizer. Litter quality may play an important role in the activity levels of broilers through encouraging normal behaviors that require energetic movements such as leg exercise (walking, foraging and dusting bathing) (15, 16). Moreover, behavior is a part of an animal's interaction with its environment. Poor litter quality is considered a welfare problem in modern poultry production. For that reason, the objective of this study was to assess the effect of different bedding material on duck welfare. Material and methods This experiment was done after the approval by Ethics and Animal welfare committee of the poultry Research Unit, Faculty of Veterinary Medicine, Zagazig University, Egypt (ANWD-206). Experimental animals and management A total of ninety six one day old of Moulard duckling was collected on one batch from Faculty of Agriculture, Zagazig University. The ducks were divided randomly after one week of the brooding period into four groups (each of 24 ducklings) as according to bedding materials (17) into saw dust, plastic slatted, sand and without floor. Each group was subdivided into three replicates (each 8 birds) following identifying with wing rings and kept in brooder house in the same home pen. Each group was kept in a pen with a floor area of 3.5 m length X 3 m width X 3 m height. Each pen had provided with one incandescent lamp of 100 watts at height 2.5 m above the level of ducks. During the experimental period, ducks were provided with full light for 1st week, then decrease gradually until reach 8h /day. Newly hatched duckling should have a proper brooding temperature, where it was measured by thermometer at the level of a bird's back and maintained at about 32-34 °C in 1st week, then decreased 3-5 °C per week until it reached 1920 °C at 4 weeks (fully feathered ducks) (18). Ducks were provided with ad libitum basic commercial duck's starter diet throughout the rearing phase that contained 22% crude protein until 5 weeks old. Then, they were fed on a grower diet with 18% of crude protein until the end of experiment (19). Behavioral observation It was conducted in the home pen to record different behavior for 5hrs weekly from 6 am to 6 pm by focal sample technique. Observation was done by one person standing directly in front of each group and waiting 10 minutes for acclimation of ducks (6). An observation sheet, a stop watch and photographing camera were used during the observation time for recording the behavioral pattern (20, 21). After observation, the total frequencies of normal and abnormal behavior were counted and calculated, as mentioned in Table (1) in all bedding materials. Other welfare indicator At the end of the study, the ducks were captured and measured the physical condition (22). Effect of bedding materials on duck's welfare and growth performance 151 The condition of eye, nostril, feather, foot and gait was scored as the welfare indicators. The other welfare score was ranged from zero to two, where zero was the best and one was the worst. Growth performance parameters It was recorded previously (23), where, initial body weight (IBW) of ducks was weighed at the beginning of experiment (2nd weeks age) and weekly until 10th weeks age, also feed residues and thus average feed intake (FI) were recorded weekly. Average body weight gain (ABWG) was calculated by subtracting body weight between two successive weeks. Relative growth rate (RGR) was calculated by ABWG/ (initial BW+final BW) *0.5. Furthermore, feed conversion ratio (FCR) was calculated (feed intake/weight gain) over a period of experiment. Blood sampling and cortisol level At the end of the experimental period, blood samples had been collected randomly from 15 ducks/group, at morning to overcome the circa-dian variation in hormone level (23). Blood samples were obtained from wing vein into heparinized tubes, centrifuged at 4000 rpm for 15 min to obtain blood plasma, which stored on -80°C for evaluating the cortisol level, as one of stress indicating hormones (24). Air quality hygiene Carbon dioxide and total ammonia levels in air of each group were detected (25) to deter- Table 1: Definition of recorded behaviors mine the hygienic level inside all experimental groups. Statistical analysis All statistical procedures were performed using the SAS statistical system Package V9.2 (26). One-factorial analysis of variance (ANOVA) was performed. The analysis of data distribution suggested that all traits analyzed followed a normal distribution (P>0.05). Pearson correlations were performed to compute the relationship of the abnormal behavior and performance parameters. Results The results as shown in Table (2) revealed a significant differences in the most of the duck's behaviors, where the most of normal behaviors were the highest in plastic slatted and wood shaving, respectively. While, the abnormal behaviors (feather pecking and aggression) were significantly higher (P<0.05) in the non- bedding floor than other groups. Likewise, the growth parameters in Table (3) had notable differences among experimental groups with significant differences, where final body weight, body weight gain, growth rate were higher in plastic slatted floor than other groups. The hygienic conditions inside the non-bedding floor was the worst due to increase levels of ammonia and carbon dioxide, as shown in Table (4). The results in Table (5) showed that ducks reared in non-bedding floor bedding materials had the worst signs of health with significant increase in the level of plasma corticosterone hormone. Observed behavior Definition Eating Drinking Foraging Idling Activity Laying Feather preening Feather pecking Aggression_ Number of eating bouts on the troughs Number of eating bouts on the drinkers Number of pecking and scratching on ground, floor or other parts of pen Standing not engaged in any activity Either walk or run Laying or sitting on the floor Clean and care about their plumage with their peak using short and repeated action while standing or sitting Number of pecks at the feathered parts The ducks make hostile acts toward other birds 152 H. Mohammed, A. Abdelaty, A. Saleem, M. Youssef, S. Abdel-Hamid Table 2: Impact of different bedding material on behavior (mean±SE) of ducks Behavioral patterns Different bedding material wood shaving Plastic slatted Sand No bedding Feeding frequency /hour 10.1±0.54 ab 11.97±0.99 a 8.74±0.79 b 8.37±0.45 b Drinking frequency/hour 14.4±4.80b 29.31±6.37 a 9±1.58c 5.41±.98 c Foraging frequency/hour 11.48 ±0.35 a 2.45±0.75 c 4.80±0.97 b 1.54±0.75 c Idling frequency/hour 20.54±0.71 a 23.22±1.57 a 20.45±0.81 a 17±1.27 b laying frequency/hour 7.62±2.39 8.60±2.48 6.97±1.18 6.42±2.18 Activity frequency/hour 23.37±1.80 19.71±1.81 23.02±1.79 20.62±1.28 Feather preening frequency/hour 11.87±2.25 a 12.20±1.16 a 10.62±0.96 ab 8.17±0.61 b Feather pecking frequency/hour 49±13.58 b 21.03±2.44 c 56.41±19.8b 74.40±14.82 a Aggression frequency/hour 2.60±1.14 b 1.98±0.91 b 3.68±1.73 ab 5.51±2.04 a abc Means in the same row with different superscripts are significantly different at (P < 0.05) Table 3: The means (±SE) of growth parameters in ducks reared under different bedding materials Growth parameters Different bedding material wood shaving Plastic slatted Sand No bedding Initial body weight (g) Final body weight Total body weight gain (g) Weekly body weight gain (g/bird/week) Feed intake (g/bird/week) Relative growth rate Feed conversion rate 449.62±18.71 3175.4±74.89 b 2725.75±62.94 ab 340.72±7.87 ab 968.75±78.12 1.503±0.01 a 2.84±0.82 435.88±15.91 3260.6±65.97 a 2824.75±57.87 a 353.09±7.23 a 964.84±73.86 1.529±0.01 a 2.73±0.83 438.12±20.85 3044.3±71.17 b 2606.18±61.91 b 325.77±7.74 b 955.5±71.12 1.497±0.02 a 2.93±0.69 438.88±14.05 2785.5±76.03 c 2346.62±67.12 c 293.33±8.39 c 861.72±68.05 1.455±0.01 b 2.94±1.63 Means in the same row with different superscripts are significantly different at (P < 0.05). g= gram Table 4: Levels of ammonia and carbon dioxide in air of different bedding materials groups Environmental indicators Different bedding material wood shaving_Plastic slatted Sand No bedding Total ammonia (Cm3) Carbon dioxide (Cm3) 7.76±.43b 0.05±.005 d 10±1.15 ab 0.13±.005 b 8.40±.23 b 0.10±.011 c 12±1.15 a 0.97±.005 a abcd Means in the same row with different superscripts are significantly different at (P < 0.05) Table 5: The level of corticosterone hormone (pg/dl) and mean rank of physical condition of duck under different bedding material _Different bedding material_ wood shaving Plastic slatted Sand No bedding Eye Nostril Feather cleanliness Feather quality Foot pad Gait Corticosterone level (pg/dl) 1.25 1.16 1.04 1.08 1.21 1.21 8.475±1.90bc 1.25 1.16 0.87 1.08 1.21 1.21 4.82±.58 c 1.25 1.33 1.29 1.25 1.45 1.21 13.17±.2.52 ab 1.66 1.66 1.91 1.83 1.54 1.70 14.55±1.16 a Means in the same row with different superscripts are significantly different at (P < 0.05) Discussion The concerning on how to manage duck's dropping under the intensive system, leading to the discovery several absorbents that were generally referred to bedding material (27). Behavioral study is considered the chief indicator to assess poultry welfare and poultry's response to the surrounding environment (17). In this study, there were several changes in duck's behaviors due to different bedding materials, as mentioned before in previous studies (17, 28-31).The bedding materials had significant effects on inestive behavior including eating, Effect of bedding materials on duck's welfare and growth performance 153 drinking and foraging, where the frequencies of eating and drinking behaviors were significantly higher in plastic slatted in comparing to other bedding materials. While foraging bouts were the highest significance in the bedding from sawdust material. These results agreed with Mohammed et al. (17), who found significant correlation between ingestive behavior and different bedding materials. While, the present study did not agree with Karousa et al. (31), who cited that litter types had no significant effect on feeding and drinking behaviors. As mentioned before in Table (2), the frequencies of ingestive behavior (eating, drinking and foraging) were the lowest significantly in no bedding materials, which confirm the importance of bedding materials to improve the ingstive behavior. The observed differences in ingestive behavior may be due to the properties of the bedding materials affected the quality of the ration and water (30).Furthermore, Toghyani et al., (32) mentioned that there were significant difference in ingestive behavior among different bedding materials. In this study, the laying frequency was the highest in plastic slatted and the lowest in non- bedding floor, but the difference did not reach the significance. This result may be attributed to the absence of comfortable media for the resting in no bedding group. These results agree with Anisuzzaman and Chowdhury (33) and disagreed with Stub and Vestergaard (34). The ducks reared in on non-bedding material was standing less significantly without any activities in comparison to other groups. This result was agreed with Mohammed et al., (17), while was disagreed with Toghyani et al., (32), who found that standing behavior was slatted but the differences did not reach the significance. Those changes in idling and laying behaviors, as comfortable behavior (5) may be due to the variations in cleanliness, odor and other characters of different litters (35). The bouts of activity (walking and running) were higher in sawdust and sand bedding than plastic slatted and non-bedding material which may be attributed to the changes in particle size of the litter (17). The difference in duck's activity in the present study was supported by Oliveira and Carvalho (36), who cited that the locomotion of birds was affected by different type of litter. The activity in plastic slatted was the lowest which may be due to the negative correlation between activity and the rate of food consumption (37). Regarding to feather preening, the ducks reared under plastic slatted floor and sawdust were more performance of feather preening with significant difference in comparing to other groups. This result may be due to the ducks more comfort on these floors. This result was agreed with Mohammed et al. (17), and disagreed with Waitt et al. (38) and Rice et al. (39), who found that the floor types hadno significance difference on preening behaviour of ducks. Sameh et al., (40) did not record any significant differences in preening behavior among different litter materials. Moreover, the abnormal behaviors representing in feather pecking and aggressive were significantly affected by the bedding, where it was the highest in non-bedding material and the lowest in plastic slatted floor. These results agreed with Mohammed et al. (17) and disagreed with Sameh et al. (40). Likewise, the bedding materials had significant effects on the most parameters of growth performance, where the plastic slatted was the highest and non-bedding group was the lowest. The improvement in growth performance in a plastic slatted litter may be attributed to the increase of all maintenance behaviors in this litter. The ducks reared in plastic slatted group revealed a significant increase in final body weight, total body weight gain, weekly body weight gain and growth rate by comparing to other experimental groups. These results were comparable to other studies, who mention that litter materials had a significant effect in growth parameters (29, 32, 41). While, other researchers did not find a significant effect of bedding materials on growth performance (17). The feed consumption and feed conversion ratio did not reveal significant differences among the experimental group. Likewise, Davis et al., (42) and Mendes at el., (43) stated that different litters did not influence growth performance. While, other previous studies mention that bedding materials has a significant effect on feed consumption (44) and 154 H. Mohammed, A. Abdelaty, A. Saleem, M. Youssef, S. Abdel-Hamid feed conversion ratio (45). The date as mentioned before in Table (4) revealed the hygienic conditions in the study, where the levels of ammonia and carbon dioxide were significantly higher in non-bedding litter in compare to others. These results may attributed to accumulation of fecal matter and increase the humidity in non-bedding litter (46). These results go hand by hand with results obtained by Tasistro et al. (47), who stated that there was a significant interaction between bedding materials and level of ammonia. Also, Almeida et al. (28) found that the use of perforated plastic flooring can improve the air quality (less CO2 and NH3 concentration) and bird cleanliness. Nevertheless, Fraley et al. (48) mentioned that there were no differences for any of the environmental data between flooring systems. Lien et al., (49) reported that the litter absorbs moisture, reduces ammonia production and finally may affect the total body weight gain. The signs of soundness in eye, nostril, feather, feet and gait were better in presence of bedding materials, especially in plastic slatted and sawdust, respectively. While the non- bedding litter was the worst in the previous signs. These results may attributed to ability of bedding materials to absorb moisture and may reflect the good managerial conditions (49). These results were comparable to Buhr et al. (50); Waitt et al. (38); Fraley et al. (48); Karcher et al. (22). Garcia et al. (30) cited that litter material had no effect on the physical condition of birds. The level of plasma corticosterone was the highest significance in the non-bedding group, while the ducks reared in plastic slatted group revealed the lowest level of plasma cortisol. This result may be due to the ducks in plastic slatted floor had good signs of soundness. This result disagreed with that obtained by Asaniyan et al (27) and Sameh and El-Khloya (51), who noted that litter depth and litter type had no significant influence on hematological values and welfare indices. Conclusion In the present study, there were several changes in duck's behaviors due to different bedding materials, where the most of maintenance behaviors were the highest significantly in plastic slatted litter and the lowest in the non-bedding litter. Furthermore, growth parameters were the better in plastic slatted and sawdust floor, respectively, while the lowest growth parameters were in non-bedding floor. The hygienic conditions inside the non-bedding floor was the worst due to increased levels of ammonia and carbon dioxide. The ducks reared under non-bedding floor revealed the worst signs of soundness, while it was the best in plastic slatted floor. The plasma corticosterone is a useful indicator for acute and environmental stress, which was the highest in non-bedding litter. This study confirms importance of bedding materials in rearing of ducks, especially under plastic slatted floor. Conflict of interests None of the authors have any conflict of interest of declare. Acknowledgments We thank the staff at the Department of Veterinary Public Health, Faculty of Veterinary Medicine, Zagazig University, Egypt for providing materials used in this study. Also, we would like to thank the anonymous referees for their helpful comments on the manuscript. References 1. Abdel GaiedS, BakriH. An economic evaluation for the impacts of spreading of bird flu on poultry sector in Egypt. World Agricultural Science 2009; 5(3): 264-9. 2. Taha A, Abd El-Ghany F, Sharaf M. Strain and sex effects on productive and slaughter performance of developed local Egyptian and Canadian chicken strains. Egyptian Poultry Science 2010; 30: 1059-72. 3. Witak B. Tissue composition of carcass, meat quality and fatty acid content of ducks of a commercial breeding line at different age. Archives Animal Breeding 2008; 51(3): 266-75. 4. Mohammed HH, Shereen EA, Enas N. Impact of different light color in behavior, welfare parameters and growth performance of Fayoumi broiler chickens strain. Hellenic Journal of Veterinary Association 2018; 69(2): 951-8. Effect of bedding materials on duck's welfare and growth performance 155 5. Mohammed HH, Badawi ME, Walaa MA, Ali MA, Abd El-Aziz RM. The influence of chromium sources on growth performance, economic efficiency, some maintenance behaviour, blood metabolites and carcass traits in broiler chickens. Global Veterinaria 2014; 12: 599-605. 6. Mohammed HH, Grashorn M, Bessei W. The effects of lighting conditions on the behaviour of laying hens. European Poultry Science 2010; 74: 197-202. 7. Aviagen A. guide to managing broilers in open-sided housing, available at: AVIAEnvMgtO-penSidedHseBroiler-EN-2016. 8. Al Homidan A, Robertson J. Effect of litter type and stocking density on ammonia, dust concentrations and broiler performance. British Poultry Science 2003; 44: S7-S8. 9. Seidavi A, Taherparvar G, Leila A, Pares-Casanova P. Effect of different litters supplemented with chemical agents on broiler carcass components. Journal of Fisheries and Livestock Production 2015;3:1-4. 10. Benabdeljelil K, Ayachi A. Evaluation of alternative litter materials for poultry. Applied Animal Research 1996; 5(3): 203-9. 11. De Avila V, De Oliveira U, De Figueiredo E, Costa A, Abreu M, Rosa P. Alternative material to replace wood shavings as broiler litter. Revista Bra-sileira Zootec 2008; 37: 273-7. 12. Shields S, Garner J, Mench J. Effect of sand and wood-shavings bedding on the behavior of broiler chickens. Poultry Science 2005; 84(12): 1816-24. 13. Savory C. Broiler welfare: Problems and prospect. Special edition of OECD-Worksh"Growth and quality in broiler production" 1995. 14. Bilgili S, Montenegro G, Hess J, Eckman M. Sand as litter for rearing broiler chickens. Journal of Applied Poultry Research 1999; 8(3): 345-51. 15. Arnould C, Bizeray D, Faure J, Leterrier C. Effects of the addition of sand and string to pens on use of space, activity, tarsal angulations and bone composition in broiler chickens. Animal Welfare 2004; 13(1): 87-4. 16. Shields S, Garner J, Mench J. Dust bathing by broiler chickens: a comparison of preference for four different substrates. Applied Animal Behavior Science 2004; 87: 69-82. 17. Mohammed HH, Enas S, Shereen EA. Impact of different litter materials on behaviour, growth performance, feet health and plumage score of Japanese quail (Coturnix japonica). European Poultry Science 2017; 81: 719-27. 18. Sari M, Onk K, Isik S, Tilki M, Tufan T. Effects of housing system, slaughter age, and sex on slaughter and carcass traits of native Turkish ducks. Turkish Journal of Veterinary and Animal Sciences 2013; 37(6): 694-700. 19. AOAC. Official Methods of Analysis. Association official analytical chemists, Gaithersburg, USA 2002. 20. Amado M, Xavier D, Boere V, Torres-Pe-reira C, McManus C, Bernal F. Behaviour of captive ostrich chicks from 10 days to 5 months of age. Revista Brasileira Zootec 2011; 40 (7): 1613-8. 21. Shimmur T, Hirahara S, Eguchi Y, Uetake K, Tanaka T. Behaviour and Physiology, performance and physical condition of layers in conventional and large furnished cages in a hot environment. Animal Science 2007; 78: 314-22. 22. Karcher D, Makagon M, Fraley G, Fraley S, Lilburn M. Influence of raised plastic floors compared with pine shaving litter on environment and Pekin duck condition. Poultry Science 2013; 92(3): 583-90. 23. Mohammed HH, Mohamed I, Al Sadik S. Effect of different light intensities on performance, welfare and behaviour of turkey poults. Journal of Advance Veterinary and Animal Research 2016; 3(1): 18-23. 24. Abdel-Rahman M. Study on the effect of stocking density and floor space allowance on behaviour, health and productivity of turkey broilers. Journal of Assiut Veterinary Medicine 2005; 51: 113. 25. Houping H, Shih-Ger C. Method of regenerate ammonia for the capture of carbon dioxide. Energy Fuels 2002; 16(4): 904-10. 26. SAS. SAS statistical system Package-Jmp 8 User's Guide.2nd Cary, NC, SAS Institute Inc. USA. ISBN 2009. 27. Asaniyan EK, Agbede JO, Laseinde EA. Impact assessment of different litter depths on performance of broiler chickens raised on sand and wood shaving litters. World Journal of Zoology 2007; 2(2): 67-72. 28. Almeida EA, Sant'Anna AC, Crowe TG, Macari M, Furlan RL. Poultry rearing on perforated plastic floors and the effect on air quality, growth performance, and carcass injuries-Experiment 2: Heat stress situation. Poultry Science 2010; 12(3): 189-195. 29. El-Deek AA, Al-Harthi MA, Khalifah MM, Elbanoby MM, Alharby T. Impact of newspaper as bedding material in arid land on broiler performance. Egyptian Poultry Science 2011; 31: 715-25. 156 H. Mohammed, A. Abdelaty, A. Saleem, M. Youssef, S. Abdel-Hamid 30. Garcia RG, Almeida Paz IL, Caldara FR, Naas IA, Bueno LF, Freitas LW, Sim S. Litter materials and the incidence of carcass lesions in broilers chickens. Revista Brasileira de Ciencia Avícola 2012; 14(1): 27-32. 31. Karousa MM, Meneeh IS, Ahmed SA, Ahmed EA, Youseif HA. Effect of litter materials on broiler behaviour and performance. Benha Veterinary Medical Journal 2012, 23(1): 142-9. 32. Toghyani M, Gheisari A, Modaresi M, Tabeidian SA. Effect of different litter material on performance and behavior of broiler chickens. Applied Animal Behaviour Science 2010; 122(1): 4852. 33. Anisuzzaman M, Chowdhury SD. Use of four types of litter for rearing broilers. British Poultry Science 1996; 37(3): 541-5. 34. Stub C, Vestergaard KS. Influence of zinc bacitracin, light regimen and dust bathing on the health and welfare of broiler chickens. British Poultry Science 2001; 42(5): 564-8. 35. Villagrá A, Olivas I, Althaus RL, Gómez EA, Lainez M, Torres A. Behavior of broiler chickens in four different substrates: a choice test. Revista Brasileira de Ciencia Avícola 2014; 16(1): 6775. 36. Oliveira MC, Carvalho ID. Rendimento e lesSes em carcasa de frangos de cortecriadosem diferentes camas e densidades populacionais. Cien-cAgrotec 2002; 26: 1076-81. 37. Hocking PM, Hughes BO, Keer S. Comparison of food intake, rate of consumption, pecking activity and behavior in layer and broiler breeder males. British Poultry Science 1997; 38: 237-40. 38. Waitt C, Jones T, Dawkins MS. Behaviour, synchrony and welfare of Pekin ducks in relation to water use. Applied Animal Behaviour Science 2009; 121(3): 184-9. 39. Rice M, Meelker A, Fraley SM, Fraley GS. Characterization of Pekin duck drinking and preening behaviors and comparison when housed on raised plastic versus pine litter flooring. Journal of Applied Poultry Research 2014; 23(4): 735-41. 40. Sameh GA, Hamada DH, Mohamed AH, Khaled MG. Behaviour, welfare and performance of broiler chicks reared on different litter materials. Assiut Veterinary Medicine 2013; 59: 9-18. 41. Senaratna D, Atapattu NS, Belpagodaga-mage DU. Saw dust and refuse tea as alternative lit- ter materials for broilers. Tropical Agricultural Research 2007; 19: 283-9. 42. Davis JD, Purswell JL, Columbus EP, Kiess AS. Evaluation of chopped switch grass as a litter material. International Journal of Poultry Science 2010; 9(1): 39-42. 43. Mendes AS, Paixâo SJ, Restelatto R, Reffatti R, Possenti JC, De Moura DJ, De Carvalho TR. Effects of initial body weight and litter material on broiler production. Revista Brasileira de Ciência Avícola 2011; 13(3): 165-70. 44. Abreu VM, Abreu PG, Coldebella A, Jae-nisch FR, Silva VS. Evaluation of litter material and ventilation systems in poultry production: I. overall performance. Revista Brasileira Zootec 2011; 40: 1364-71. 45. Asaniyan EK, Agbede JO, Laseinde EA. Comparative influence of sand and wood shavings litter replacement frequency on the performance of broiler chickens. Journal of Animal and Veterinary Advances 2006; 5(12): 1080-7. 46. Jones TA, Dawkins MS. Environment and management factors affecting Pekin duck production and welfare on commercial farms in the UK. British Poultry Science 2010; 51(1): 12-21. 47. Tasistro AS, Ritz CW, Kissel DE. Ammonia emissions from broiler litter: response to bedding materials and acidifiers. British Poultry Science 2007; 48(4): 399-405. 48. Fraley SM, Fraley GS, Karcher DM, Mak-agon MM, Lilburn MS. Influence of plastic slatted floors compared with pine shaving litter on Pekin Duck condition during the summer months. Poultry Science 2013; 92(7): 1706-11. 49. Lien RJ, Hess JP, Conner DE, Wood CW, Shelby RA. Peanut hulls as a litter source for broiler breeder replacement pullets. Poultry Science 1998; 77: 41-6. 50. Buhr RJ, Cason JA, Dickens JA, Hinton JA, Ingram KD. Influence of flooring type during transport and holding on bacteria recovery from broiler carcass rinses before and after defeathering. Poultry Science 2000, 79(3): 436-41. 51. Sameh GA, El-Khloya SZ. Do alternative litter Materials Affect performance, Welfare and Immune Response of Broiler Chicks?. Alexandria Journal for Veterinary Sciences 2017; 52(1): 133-41. Slov Vet Res 2019; 56 (Suppl 22): 157-66 DOI 10.26873/SVR-753-2019 Original Research Article PESTICIDES AND TOXIC METALS RESIDUES IN MUSCLE AND LIVER TISSUES OF SHEEP, CATTLE AND DROMEDARY CAMEL IN SAUDI ARABIA Ahmed M.A. Meligy12, Abdallah Y. Al-Taher3, Mohamed Ismail3, Abdelmohsen A. Al-Naeem4, Sabry M. El-Bahr35*, Waleed R. El-Ghareeb67 1Central laboratory, College of Veterinary Medicine, King Faisal University, Saudi Arabia, 2Plant Protection Research Institute, Agricultural Research Center, Egypt, 3Department of Physiology, Biochemistry and Pharmacology, College of Veterinary Medicine, King Faisal University, Saudi Arabia, 4Department Clinical Studies, College of Veterinary Medicine, King Faisal University, Saudi Arabia, 5Department of Biochemistry, Faculty of Veterinary Medicine, Alexandria University, Egypt, 6Food Control Department, Faculty of Veterinary Medicine, Zagazig University, 44519, Egypt, 7Department of Veterinary Public Health and Animal Husbandry, College of Veterinary Medicine, King Faisal University, Saudi Arabia Corresponding author, E-mail: sabryelbahr@hotmail.com; selbahar@kfu.edu.sa Abstract: Data regarding determination of pesticide residues and toxic elements in edible tissues of different animals particularly in the camels in Eastern region of Saudi Arabia are scarce. Therefore, current study aimed to evaluate levels of forty-five pesticides and heavy metals (lead (Pb); cadmium (Cd); Arsenic (As)) in muscle and liver tissue samples that collected from forty-two animals; sheep (n=21), cattle (n=11) and dromedary camels (n=10) admitted to Al-Ahsa abattoir, Eastern province, Saudi Arabia. Seven of forty-five pesticides residues were detected in tissues by gas chromatographic mass spectrometry (GC/MS). The detected pesticides were organophosphorus (diazinon, profenofos and coumaphos), pyrethroid (cypermethrin, permethrin and lambda-cyhalothrin) and acari-cide (amitraz). Diazinon has been detected in both muscle and liver tissues of all examined carcasses. However, other detected residues distributed according to species. All toxic metals have been detected in both muscle and liver tissues of all tested animals. The heavy metal loads were species independent. The concentrations of Pb and As in liver tissues were comparable to that of muscle tissues of all tested animals. However, the concentrations of Cd in liver were significantly higher than that in muscles of all tested animals. All values of detected pesticide residues and heavy metals were below the maximum residue limits (MRLs). In conclusion, pesticide residues and toxic metals were detected in meat and liver tissues of sheep, cattle and dromedary camels that admitted to Al-Ahsa abattoir, Saudi Arabia. The detected pesticides residues and toxic metals were well below MRLs; thereby no risks are associated with its human consumption. Key words: chromatography; residues; toxic elements; meat; pesticides; animals Introduction The use of pesticide increased steadily during 1996-2013 in Saudi Arabia (1). In 2013, the Received: January 2019 Accepted for publication: February 2019 158 A. Meligy, A. Al-Taher, M. Ismail, A. Al-Naeem, S. El-Bahr, W. El-Ghareeb total usage of insecticide was 3130.5 tons of active ingredients compare to that during 2012 (2889 tons of active ingredients). The organo-phosphate ranked first of pesticide used during this period (1). The Date Palme still represents the most important crop cultivated in Al-Ahsa, Eastern Province, Saudi Arabia and an element contributing in economy of the beautiful oasis, Al-Ahsa. Pesticides used in Al-Ahsa to fight the red date Palme weevil (2) and other pests that attack different varieties of vegetable crops cultivated in Al-Ahsa region (1). Contamination of the environment with pesticide residues has been documented earlier (3). Based on the reports of the toxicity and detrimental effects of pesticides, their use has been restricted worldwide. Although the use of most of pesticides has been restricted, its residues in food were detected (4). Water, plants and grasses that contaminated by pesticides residues may ingested by herbivorous animals and secreted in meat and/or milk (4). Pesticides residues contamination has been reported in processed food (5, 6) and eggs (7, 8). In addition, organochlorine pesticides have been detected in edible tissues of camel, cattle and sheep carcasses slaughtered in Sharkia Province of Egypt (9), in meat of sheep slaughtered in Jaipur area of India (10) and in fish and poultry meat in India (11). The dromedary camels are one of the important sources of meat in the semiarid and arid areas of Asia and Africa (12). Camel meat characters are near to that of other red meat animal species (12). In Al-Ahsa region, camel meat is preferable along with the other meats of sheep and cattle. However, data reporting the detection of pesticides residues in edible tissues of these animals reared in Al-Ahsa region of eastern province of Saudi Arabia are scarce. The inorganic arsenic is more toxic than organic one. However, it is reported in literatures as total arsenic (13). The exposure of animal to lead increased when feed materials mixed with contaminated soil (14). Regarding lead toxicity, cattle and sheep are the most sensitive animal species. The elimination of lead is incomplete and slow. Its half-life is approximately 250 days in ewes, between 95 and 760 days in cattle (15, 16), and 2 to18 years in humans (17, 18). Like lead, animal exposed to cadmium through plants mixed with contaminated soil (19). Cadmium classified as a human carcinogen (Group 1) by International Agency of Cancer Research particularly cancer in lungs, endometrial tissue, bladder, and breast (20). Only one report (21) originated from our laboratory demonstrated the detection of metal contents in the edible tissues of camel and sheep slaughtered in Saudi Arabia. However, in this report (21), cattle meat and liver tissues were not investigated. GC-MS used for high speed, excellent separation and quantitative analysis of many volatile pesticides (22-29). The current study aimed to evaluate levels of forty-five pesticides and heavy metals (lead (Pb); cadmium (Cd); Arsenic (As)) in muscle and liver tissue samples that collected from forty-two animals; sheep (n=21), cattle (n=11) and dromedary camels (n=10) admitted to Al-Ahsa abattoir, Eastern province, Saudi Arabia. Material and methods Animals and samples Specimens of muscle (n=42) and liver (n=42) tissues have been collected from 42 animals of three species namely sheep (n=21), cattle (n=11) and dromedary camels (n=10) that have been admitted to Al-Ahsa abattoir, Eastern province, Saudi Arabia (25°22'18.1"N 49°26'21.1'E) directly after slaughter. Age of animals was determined by visual examination of teeth during the ante-mortem inspection (25). The average age of the slaughtered camel and cattle was estimated to be 5 ± 2 years, respectively while for sheep was and 8 ± 3 months. All animals were apparently healthy, active, and free from any disease. Sampled tissues for pesticide and heavy metal residues analysis were stored in plastic falcon tubes and stored at -20°C until the time of extraction and measurement of 45 pesticide and heavy metal residues. The experiments and procedures were done according to the regulations and guidelines of ethical research committee, King Faisal University, Saudi Arabia. All chemicals were purchased from Merck, Darmstadt, Germany and were of HPLC grade or the highest quality available. Specifications for pesticides standards used in the study are illustrated at Table 1. Pesticides and toxic metals residues in muscle and liver tissues of sheep, cattle and dromedary camel 159 Analytical procedure for pesticide residues The QuEChERS EN 15662 method for pesticide residue extraction was used (30) and the obtained extracts were transferred to an auto sampler vial of GC-MS for analysis of pesticide residues.Pesticides residues have been detected by GC/MS system (Shimadzu GC/MS-QP2010 Plus, Japan) equipped with a capillary column DB-5MS (30m length, 0.25 mm thickness, 0.25m diameter). The temperature of injector was 250°C. The temperature program of oven started with 60°C. Hold 6 min at 7°C/min to 200 °C, at 5 °C/min to 280 °C. Hold 20 min; carrier gas, helium; purity, >99.999%; flow rate, 1.2 ml/min; injection port temperature, 250 °C; injection volume, 1ul; injection mode, splitless, purge on after 1.5 min; ionization voltage, 70 eV; ion source temperature, 220 °C; GC/MS interface temperature, 250°C. The analyses were done at two modes, Selective Ion Monitoring (SIM mode) and full Scan mode for enhanced sensitivity and selectivity. The limits of detection (LOD) and limit of quantification (LOQ) for all pesticides are shown in Table 2. Analysis of heavy metals (Pb, Cd and As) The digestion of muscle and liver samples and the GFA-EX7 graphite furnace atomic absorption spectrophotometeic (Shimadzu, Ko-yoto, Japan) determination of Pb, Cd and As were done as described earlier (31, 32). Preparation of standard stock solutions of Cd, Pb and As and calibration procedures were the same of that discussed in our previous works (31, 32). The LOD for the three investigated heavy metals was 1 pg/kg. The absorbance was obtained by adjusting the hollow cathode lamps at the operation conditions shown in Table 3. Analytical quality control In the present study, to check the efficiency of digestion procedures and the subsequent recovery of the pesticides and metals, homogeneous mixtures of three samples of each of muscle and liver were spiked with multi-elements and multi-pesticides solutions that contain standard solutions of all pesticides and metals considered in the present study. Solutions were spiked in a manner to attain final concentrations of 3pg/g. A mixture without any pesticides and metals was used as control. All mixtures were then subjected to the digestion procedure. The resulting solutions were analyzed three times for pesticides and metal concentrations according to the same procedures as the samples to establish confidence in the accuracy and reliability of data generated. The amount of spiked pesticides and metals recovered after the digestion of the spiked samples were used to calculate percentage recovery as described earlier (33). The pesticide recovered from both tissues in a percentage over 89% as indicated at Table 2. All heavy metals recovered in a percentage ranged between (89.6-94.2 %) and (85-89.4 %) in liver and muscle tissues, respectively (Table 4). Specificity The specificity was confirmed based on the presence of the transition ions (quantifier and qualifier) at the correct retention times (that was defined formerly by using one sample from each type that spiked at 10ng/g to check the retention time of pesticides) corresponding to that of the precursor ion presented in Table 1. The measured peak area ratios of qualifier/quantifier were within the range defined in EU Commission Decision 2002/657/EC when compared to the standards. Statistical analysis The statistical analysis was performed using the SPSS 6.1.3 software package (SAS, Cary, NC, USA). All data were analyzed for variance using ANOVA test. Results and discussion Data summarized in Table 1 and Figure 1 indicated that, the total number of investigated pesticides standards were 45 standards. However, gas chromatographic analysis indicated the existence of only 7 types of pesticides residues (Diazinon, Lambda-Cyhalothrin-II, Profenofos, Cis-Permethrin, Cypermethrin, Amitraz, coumaphos). The aim behind the screening of large number of pesticides was to cover all existing chemical classes that includes, organophosphates, organo-chlorine, carbamates, synthetic pyrethroids, triazines, py-rimidines, triazoles, dicarboximides, bezimid- 160 A. Meligy, A. Al-Taher, M. Ismail, A. Al-Naeem, S. El-Bahr, W. El-Ghareeb azole, dithiocarbamates, nicotinoids, dinitro-phenol, pesticides aliphatic nitrogen, pesticides morpholine, pesticides from natural product derivatives, and pesticides of substituted urea class. However, only seven of screened pesticides have been detected in muscle and liver tissues of all tested animals (Table 1). The detected pesticides residues are of listed pesticides residues monitored in Saudi agricultural soils (34). Diazinon has been detected in muscle and liver tissues of all examined carcasses. This may attributed to the extensive use of diazinon for eradication of Ticks that affected animals related to all areas of Al-Ahsa governorate as said by the habitants of that region. Other detected pesticides residues were species related (Table 5 and 6). Profenofos residues has been detected in examined tissues of sheep and camels whereas, amitraz and permethrin have been detected only in muscle and liver of cattle carcass (Table 5 and 6). Coumaphos detected only in examined tissues of sheep carcass while Lambda-Cyhalothrin residues were detected only in muscle and liver tissues of camel carcass (Tables 5 and 6). The variation of pesticides contamination among tested animals may attribute to different contaminated areas because animals who admitted to Al-Ahsa abattoir were from different areas of Al-Ahsa gov-ernorate. The area dependent pesticides contamination has been confirmed in other study in Egypt (35). The data described in Tables 5 and 6 indicated that, all values of detected pesticide residues were below the international permissible limits (14). Parallel to the current findings, earlier report (9) demonstrated that, the organo-chlorines residues detected in muscle and liver tissues of camel, sheep and cattle were well below the respective MRLs set by local or international organizations. Data summarized in Tables 7 indicated that, the investigated toxic metal residues have been detected in muscle and liver tissues of all examined carcasses. The heavy metal loads were species independent (Tables 7). The concentrations of Pb and As in liver tissues were comparable to that of muscle tissues of all tested animals. However, the concentration of Cd in liver was significantly higher than its concentration in muscle in all tested animals (Tables 7). This may attribute to the fact that, the liver is main organ for detoxification in the live organism (36). The current findings come in accordance with recent work (21) reported that, liver tissues had a higher loads of heavy metals (Cd, Pb and As) than that of muscle tissues in camel and sheep slaughtered in the same area of Saudi Arabia. All values of detected heavy metals were well below the international permissible limit as described in Tables 7 (4, 13, 14, 20, 21, 37). The highest detected average of Arsenic, lead and cadmium residues either in muscle or in liver tissues were 11.6 ± 2.0pg/kg, 10.5 ± 1.8pg/kg and 0.9 ± 0.1 pg/kg whereas the arsenic, lead and cadmium MRLs are 1000pg/kg, 500pg/kg and 1000pg/kg, respectively. This indicated that, the contaminated tissues with all toxic elements are negligible and safe for human consumption. The detected values of Arsenic, lead and cadmium residues were much lower than that reported in liver and kidney bovine tissues (27.42-30.55 pg/kg; 231-226.96 pg/kg; 207.5197 pg/kg; (38), respectively. The wide range between detected elements in the current study compared with earlier report (38) may attribute to different detection limits and sensitivity. Eelier detection limits (38) were 100pg/kg for Cd, 150pg/kg for Pb and 30pg/kg for As. However, in the current study the detection limits were 1 pg/kg for the three investigated elements. The detected values of Cd and As in liver of camel (1.3 ± 0.1 pg/kg; 1.4 ± 0.4 pg/kg) and sheep (11.6 ± 2.0 pg/kg; 8.0 ± 2.0 pg/kg), respectively were near to that reported recently (21) in liver of camel (2.18 ± 0.71 pg/kg; 1.79 ± 0.66 pg/kg) and sheep (38.41 ± 25.15 pg/kg; 30.29 ± 22.57 pg/kg), respectively. The values of Cd and As in the muscle of camel (0.9 ± 0.1 pg/kg; 0.6 ± 0.1 pg/kg) and sheep (8.0 ± 1.1 pg/kg; 6.4 ± 1.2 pg/kg) respectively were near to that reported recently (21) in muscle of camel (0.29 ± 0.27 pg/kg; 0.44 ± 0.17 pg/kg) and sheep (12.89 ± 4.24 pg/kg; 10.05 ± 3.77 pg/kg), respectively. In the current study, the detected values of Pb in liver and muscle of camel and sheep were exactly comparable to that reported earlier in the same tissues of the same tested animals (21). Pesticides and toxic metals residues in muscle and liver tissues of sheep, cattle and dromedary camel 161 Table 1: Specifications for pesticides standards used in the study No Name of pesticide Retention time Quantifying ion Quali- Qualifying ion 1 fying ion2 No Name of pesticide Retention time Quantifying ion Quali- Qualifying ion1 fying ion2 1 Demeton-S-methyl 10.129 263 169 127 26 Propargite-I 31.003 135 350(7) 173(16) 2 a-HCH 11.108 219 183(98) 221(47) 27 Profenofos* 31.382 339 374 (39) 297 (37) 3 Diazinon * 15.670 304 179(192) 137(172) 28 Bifenthrin 32.39 181 166 (25) 165 (23) 4 HCB 17.567 284 282(51) 286(81) 29 Methoxychor 32.672 277 228(16) 212(4) 5 Phorate 18.271 260 121(160) 231(156) 30 Captan 32.782 264 149(104) 150(70) 6 P-HCH 19.954 219 217(78) 181(94) 31 Formothion 32.884 170 224(97) 257(63) 7 S-HCH 20.108 219 217(80) 181(99) 32 p,p'-DDT 33.108 235 237(65) 246(7) 8 Heptachlor 20.503 272 237(40) 337(27) 33 p,p'-DDD 33.232 235 237(64) 199(12) 9 Aldrin Lambda- 20.743 263 265(65) 293(40) 34 Cis-Perme-thrin * 33.759 183 184(14) 255(1) 10 Cyhalothrin-II * 22.533 181 241(205) 243(152) 35 Captafol 34.565 149 150(79) 152(79) 11 a-Endosulfan 23.087 241 265(66) 339(46) 36 p.p'-DDE 36.154 318 316(80) 246(139) 12 chlorothalonil 23.995 264 266(109) 268(62) 37 O',p'-DDD 36.337 235 237(65) 165(39) 13 Methylpara-thion 25.056 263 233(66) 246(8) 38 Cyperme-thrin* 36.457 181 152(23) 180(16) 14 Endosulfan sulfate 25.313 241 265(66) 339(46) 39 Amitraz* 36.613 293 162(113) 132(104) 15 Dieldrin 27.196 263 277(82) 380(30) 40 coumaphos* 37.106 362 226(56) 364(39) 16 Endrin 27.432 263 317(30) 345(26) 41 Trans-Perme-thrin 37.231 183 184(14) 255(1) 17 O,p'-DDE 27.683 246 318(34) 176(26) 42 Fenvalerate 37.317 167 225(53) 419(37) 18 Dicofol 28.104 139 141(72) 250(23) 43 Deltamethrin 37.969 181 172(25) 174(25) 19 Malathion 28.418 173 158(36) 143(15) 44 Cyfluthrin 41.512 206 199(63) 226(72) 20 Fenithrothion 29.053 277 260(52) 247(60) 45 Esfenvalerate 46.085 419 225(158) 181(189) 21 Bromopropy-late 29.452 341 183(34) 339(49) 22 penconazol 30.101 248 250(33) 161(50) 23 Pirimiphos-methyl 30.281 290 276(86) 305(74) 24 Procymidone 30.448 283 285(70) 255(15) 25 o,p'-DDT 30.767 235 237(65) 246(7) *Tissues detected pesticides Table 2: Limit of Detection (LOD), Limit of Quantification (LOQ) and recovery percentages of pesticide measured using the ions for each pesticide molecule No Name of pesticide RT LOD (pg/g) LOQ (pg/g) Recovery 1 Demeton-S-methyl 10.129 0.02 0.06 89.2 2 a-HCH 11.108 0.02 0.06 90.2 3 Diazinon * 15.670 0.006 0.017 95.0 4 HCB 17.567 0.02 0.06 92.1 5 Phorate 18.271 0.03 0.12 89.9 6 P-HCH 19.954 0.01 0.03 90.5 7 S-HCH 20.108 0.01 0.03 90.6 8 Heptachlor 20.503 0.002 0.007 92.4 9 Aldrin 20.743 0.02 0.05 89.5 10 Lambda-Cyhalothrin-II * 22.533 0.004 0.01 91.7 11 a-Endosulfan 23.087 0.003 0.008 90.9 12 chlorothalonil 23.995 0.006 0.015 93.3 13 Methylparathion 25.056 0.004 0.012 94.5 14 Endosulfan sulfate 25.313 0.002 0.006 93.4 15 Dieldrin 27.196 0.02 0.008 93.1 16 Endrin 27.432 0.01 0.04 92.2 162 A. Meligy, A. Al-Taher, M. Ismail, A. Al-Naeem, S. El-Bahr, W. El-Ghareeb Table 2: Continuation No Name of pesticide RT LOD fog/g) LOQ fog/g) Recovery 17 O,p'-DDE 27.683 0.02 0.06 90.5 18 Dicofol 28.104 0.003 0.009 90.7 19 Malathion 28.418 0.005 0.012 90.0 20 Fenithrothion 29.053 0.004 0.013 91.9 21 Bromopropylate 29.452 0.005 0.015 92.9 22 penconazol 30.101 0.004 0.012 93.0 23 Pirimiphos-methyl 30.281 0.006 0.018 90,1 24 Procymidone 30.448 0.01 0.030 90,7 25 o,p'-DDT 30.767 0.009 0.016 91.7 26 Propargite-I 31.003 0.009 0.015 91.4 27 Profenofos* 31.382 0.004 0.010 92.5 28 Bifenthrin 32.39 0.002 0.006 90.4 29 Methoxychor 32.672 0.006 0.012 93.7 30 Captan 32.782 0.01 0.03 94.0 31 Formothion 32.884 0.004 0.012 90.2 32 p,p'-DDT 33.108 0.002 0.006 92.9 33 p,p'-DDD 33.232 0.003 0.013 91.9 34 Cis-Permethrin * 33.759 0.004 0.010 93.7 35 Captafol 34.565 0.001 0.004 91,4 36 p.p'-DDE 36.154 0.004 0.011 89.8 37 O',p'-DDD 36.337 0.004 0.012 89.7 38 Cypermethrin* 36.457 0.003 0.009 93.9 39 Amitraz* 36.613 0.002 0.006 89.6 40 coumaphos* 37.106 0.002 0.006 94.0 41 Trans-Permethrin 37.231 0.003 0.009 90.9 42 Fenvalerate 37.317 0.02 0.06 90.8 43 Deltamethrin 37.969 0.002 0.007 94.3 44 Cyfluthrin 41.512 0.003 0.009 90.1 45 Esfenvalerate 46.085 0.01 0.03 90.2 *Tissues detected pesticides Table 3: Programs of heating method of graphite furnace atomic absorption (GFAAS) for Arsenic (As), lead (Pb) and cadmium (Cd) Steps Temperature (°C) Ramp(s) Hold(s) Argon flow rate (ml min-1) As Pb Cd Drying 1 150 150 150 5 20 250 Drying 2 200 200 200 5 15 250 Pyrolysis 1200 800 500 10 20 250 Atomization 2000 2000 1800 0 5 0 Clean-out 2000 2200 2200 1 3 250 Pesticides and toxic metals residues in muscle and liver tissues of sheep, cattle and dromedary camel 163 Table 4: The recovery of heavy metals (As, Pb and Cd) from digested liver and muscle samples:- Heavy metals Added Heavy metals (pg/kg) Recovered from liver (pg/kg) Recovery (%) Recovered from muscle (Mg/kg) Recovery (%) As 5 4.47 89.6 4.39 89.4 Pb 5 4.69 94.2 4.44 88.8 Cd 5 4.66 93 4.25 85 AS: (Arsenic); Pb: Lead; Cd: Cadmium Table 5: Pesticides residues (mg/kg) detected in muscle tissues (n=42) of sheep, cattle and dromedary camels admitted to Al-Ahsa Abattoir, Saudi Arabia No. of Animals tested No of Res- No of de- Concentration Animal No. of idues con- tected Types of detected of detected Permissible Species samples taining pesticides pesticides residues pesticides limit (13, 14) samples residues residues Diazinon 0.005 0.02 Sheep 21 21 3 4 Profenofos 0.015 0.05 Cypermethrin Caumaphos Diazinon 0.009 0.018 0.017 0.20 0.01 0.02 Cattle 10 10 3 4 Cypermethrin 0.035 0.20 Amitraz Permethrin Diazinon 0.033 0.029 0.018 0.02 0.50 0.02 Camel 11 11 2 3 Profenofos Lambda-Cyhalothrin 0.019 0.033 0.05 0.50 Table 6: Pesticides residues (mg/kg) detected in Liver tissues (n=42) ofsheep, cattle and dromedary camels admitted to Al-Ahsa Abattoir,Saudi Arabia. Animal Species No. of Animals tested No. of samples No of Residues containing samples No of detected pesticides residues Types of detected pesticides residues Concentration of detected pesticides residues Permissible limit (13, 14) Diazinon 0.012 0.02 Sheep 21 21 3 4 Profenofos 0.022 0.05 Cypermethrin 0.015 0.05 Coumaphos 0.024 0.01 Diazinon 0.034 0.02 Cattle 10 10 3 4 Cypermethrin 0.041 0.02 Amitraz 0.023 0.20 Permethrin 0.014 0.05 Diazinon 0.027 0.02 Camel 11 11 2 3 Profenofos 0.029 0.05 Lambda-Cyhalothrin 0.053 0.50 164 A. Meligy, A. Al-Taher, M. Ismail, A. Al-Naeem, S. El-Bahr, W. El-Ghareeb Table 7: Heavy metals (pg/kg) detected in muscleand liver tissues (n=42) of sheep, cattle and dromedary camels admitted to Al-Ahsa Abattoir, Saudi Arabia. Animal Species No. of Animals tested No. of samples Heavy metals concentration (ppb) *Pb in muscle tissues Pb in liver . in Cd in liver i As in mus-muscle tis- , tissues tissues cle tissues sues As in liver tissues Sheep 21 21 4.98 ± 1.44aw 7.8 ± 1.2aw 0.6 ± 0.1ax 1.4 ± 0.4ay 6.4 ± 1.2aw 8.0 ± 2.0aw Cattle 10 10 7.40 ± 2.00aw 7.9 ± 1.5aw 0.7 ± 0.2ax 1.3 ± 0.2ay 8.7 ± 2.0aw 11.1 ± 2.5aw Camel 11 11 7.30 ± 1.20aw 10.5 ± 1.8aw 0.9 ± 0.1ax 1.3 ± 0.1ay 8.0 ± 1.1aw 11.6 ± 2.0aw 'Permissible limit of Pb (500pg/kg) (38). tPermissible limit of Cd (1000pg/kg) (38). {Permissible limit of As (1000pg/kg) (38). Values are expressed as Mean ± SD. Means within the same column with different superscripts are significantly different (P < 0.05). Means within the same row with different superscripts are significantly different (P < 0.05). Figure 1 : GC/MS chromatogram of investigated pesticides standards Conclusion The current study concludes the existence of pesticide residues and toxic metal residues in muscle and liver tissues of sheep, cattle and dromedary camel carcasses at Al-Ahsa gover-norate. Diazinon contaminated muscle and liver tissues of all examined carcasses. Other pesticide residues differed according to the areas that animals originated. The heavy metal loads were species independent. The concentrations of Pb and As in liver tissues were comparable to that of muscle tissues of all tested animals. However, the concentration of Cd in liver was significantly higher than its concentration in muscle in all tested animals. Consumption of carcasses is safe to human health because the detected pesticide and toxic metal residues were well below international permissible limit. Pesticides and toxic metals residues in muscle and liver tissues of sheep, cattle and dromedary camel 165 Acknowledgments The authors thank the Deanship of Scientific Research in King Faisal University, Saudi Arabia, for their support of this study (Project# DSR130025). References 1. Saggu S, Rehma H, Alzeiber FMA, Aziz A. Current situation of pesticide consumption and poisoning in Saudi Arabia. Journal of Entomology and Zoology Studies 2016; 4(3): 153-8. 2. Mozib M, El-Shafie HA. Effect of red palm weevil, Rhynchophorusferrugineus (Olivier) infestation on temperature profiles of date palm tree. Journal of Entomology and Nematology 2013; 5(6): 77-3. 3. Jian-Ying H, Takako A, Yasumoto M. Analysis of pesticides in water with liquid atmospheric pressure chemical ionization mass spectrometry. Water Research 1999; 33(2): 417-25. 4. WHO (World Health Organization). Health risks of heavy metals from long-range trans boundary air pollution, 978 92 890 71796 WHO Regional Office for Europe, Copenhagen, Denmark, 2007. 5. Miyahara M, Saito Y. Effects of the processing steps in tofu production on pesticide residues. Journal of Agricultural and Food Chemis-try1994; 42(2): 369-73. 6. Gilbert L, Gracia B, Mezcua M, Ramos N, Fernandez AR, Molina A. Multi-residue determination of pesticides in fruit-based soft drinks by fast liquid chromatography time-of-flight mass spectrometry. Talanta 2010; 81: 1310-1321. 7. Herdman, RC, Pahrham, WE, Shen S. Pesticide Residues in Food: Technologies for Detection. Government Printing Office, Washington, 1988. 8. Tao S, Liu, WX, Li XQ, Zhou DX, Li X, Yang YF, Yue DP, Coveney RM. Organochlorine pesticide residuals in chickens and eggs at a poultry farm in Beijing, China. Environmental Pollution 2009; 157: 497-502. 9. Sallam KI, Morshedy AMA. Organochlorine pesticide residues in camel, cattle and sheep carcasses slaughtered in Sharkia Province, Egypt. Food Chemistry 2008; 108: 154-64. 10. Shinde AK, Karim SA. Screening of pesticide residues in meat and milk samples of sheep maintained in institute and field flocks. Indian Journal of small ruminants 2009; 15(1): 924. 11. Jadhav VJ, Waskar VS. Public Health Implications of Pesticide Residues in Meat. Veterinary World 2011; 4(4): 178-82. 12. Elgasim EA, Alkanhal MA. Proximate composition, amino acids and inorganic mineral content of Arabian camel meat: comparative study. Food Chemistry 1992; 45(1): 1-4. 13. European Food Safety Authority - EFSA. Scientific opinion on arsenic in food. EFSA panel on contaminants in the food chain (CONTAM). European Food Safety Authority 2009a; 1351: 1-199. 14. European Food Safety Authority - EFSA. Opinion of the Scientific Panel on Contaminants in the Food Chain on a request from the Commission related to lead as undesirable substance in animal feed. European Food Safety Authority 2004; 72: 1-24. 15. Mehennaoui, S, Charles E, Joseph-Enriquez B, ClauwM, Milhaud GE. Indicators of lead, zinc and cadmium exposure in cattle I and II. Veterinary and Human Toxicology 1988; 30: 550-5. 16. Rumbeiha, WK, Braselton, WE, Donch D. A retrospective study on the disappearance of blood lead in cattle with accidental lead toxicosis. Journal Veterinary Clinical Investigations 2001; 13: 373-8. 17. Nilsson U, Attewell R, Christoffersson JO, Schultz A, Ahlgren L, SkerfVing S, Mattsson S. Kinetics of lead in bone and blood after end of occupational exposure. Pharmacological and Toxicolog-ical 1991; 68: 477-84. 18. Rabinowitz MB. Toxicokinetics of bone lead. Environmental Health Perspectives 1991; 91: 33-7. 19. Nakamori T, Kaneko N. Biomarker responses reveal that food quality affects cadmium exposure in soli collembolan Folsomia candida. Environmental Pollution 2013; 176: 165-70. 20. European Food Safety Authority - EFSA. Cadmium in food. Scientific opinion of the panel on contaminants in the food chain. European Food Safety Authority 2009b; 980: 1-139. 21. El-Ghareeb WR, Darwish WS, Meligy AMA. Metal contents in the edible tissues of camel and sheep: human dietary intake and risk assessment in Saudi Arabia. Japanese Journal of Veterinary Research 2019; 67(1): in press 22. Stan HJ. Pesticide residue analysis in foodstuffs applying capillary gas chromatography with mass spectrometric detection state-of-the-art use of modified DFG-multimethod S19 and automated data evaluation. Journal of Chromatography A 2000; 892: 347-77. 23. Albero B, Sanchez-Brunete C, Tadeo JL. Determination of organopho- sphorous pesticides in fruit juices by matrix solid-phase dispersion and gas chromatography. Journal of Agricultural and Food Chemistry 2003; 51: 6915-21. 166 A. Meligy, A. Al-Taher, M. Ismail, A. Al-Naeem, S. El-Bahr, W. El-Ghareeb 24. Pugliese P, Molto' JC, Damiani P, Maine R, Cossignani L, Manes J. Gas chromatographic evaluation of pesticide residue contents in nectarines after non-toxic washing treatment. Journal of Chro-matography A 2004; 1050:185-91. 25. Rial-Otero R, Arias-Estevez M, Lopez-Peri-ago E, Cancho-Grande B, Simal- Gandara J. Variation in concentrations of the fungicides tebucona-zole and dichlofuanid following successive applications to greenhouse-grown lettuces. Journal of Agricultural and Food Chemistry 2005; 53 (11): 4471-5. 26. Balinova A, Mladenova R, Shtereva D. Solid-phase extraction on sorbents of different retention mechanisms followed by determination by gas chromatography-mass spectrometric and gas chromatography-electron capture detection of pesticide residues in crops. Journal of Chromatography A 2007;1150:136-44. 27. Pose-Juan E, Cancho-Grande B, Rial-Otero R, Simal-Gandara J. The dissipation rates of cypro-dinil, fludioxoni, procymidone and vinclozoline during storageofgrapejuice. Food Control 2006; 17(12): 1012-7. 28. Berrada H, Fernandez M, Ruiz MJ, Molto' JC, Manes J, Font G. Surveillance of pesticide residues in fruits from Valencia during twenty months (2004/05). Food Control 2010; 21: 36-44. 29. Menezes FA., Neves F, Afonso de Paula Pe-reira P. Development, validation and application of a methodology based on solid- phase micro extraction followed by gas chromatography coupled to mass spectrometry (SPME/GC-MS) for the determination of pesticide residues in mangoes. Talanta 2010;81: 346-54. 30. Lazartigues A, Fratta C, Baudot R, Wiest L, Feidt C, Thomas M, Cren-Olivé C. Multiresidue method for the determination of 13 pesticides in three environmentalmatrices: water, sediments and fish muscle. Talanta 2011; 85(3):1500-7. 31. El-Bahr SM, Abdelghany AM. Heavy metal and trace element contents in edible muscle of three commercial fish species, and assessment of possible risks associated with their human consumption in Saudi Arabia. Journal of Advanced Veterinary and animal Research 2015; 2: 271-8. 32. Meligy AMA. Comparative study of element contents in seven isolates of entomopathogenic nematodes. Egyptian Journal of Biological Pest Control 2018; 28:1 33. Guo FP, Yan ZC, Jin JZ, Chun LF, Yong ML, Xue ML, Guang QJ, Zeng YL, Yu QS, Yan PW, Tong TG. Validation study on 660 pesticide residues in animal tissues by gel permeation chro-matography cleanup/gas chromatography-mass spectrometry and liquid chromatography-tandem mass spectrometry. Journal of Chromatography A 2006;1125: 1-30. 34. Al- Wabel, MI, El- Saeid, MH, Al- Turki AM, Abdel- Nasser G. Monitoring of Pesticide Residues in Saudi Arabia Agricultural Soils. Research Journal of Environmental Sciences 2011; 5: 269-78. 35. Mansour SA, Belal MH, Abou-Arab AAK, Gad MF. Monitoring of pesticide and heavy metals in cucumber fruits produced from different farming system. Chemosphere 2009; 75: 601-9. 36. Murray RK, Bender DA, Botham, KM, Ken-nelly PJ, Rodwell VW, Weil PA. Harper's Illustrated Biochemistry (Harper's Biochemistry). 28th ed., McGgraw-Hill, 2012. 37. Korsrud G, Meldrum J, Salisbury C. Trace element levels in liver and kidney from cattle, swine and poultry slaughtered in Canada. Canadian Journal of Comparative Medicine 1985; 49: 159-63. 38. Filho JFA, Germano A, Dibai WLS, Vargas EA, Melo MM. Heavy metals investigation in bovine tissues in Brazil. Food Science and Technology (Campinas) 2014; 34(1): 110-5. Slov Vet Res 2019; 56 (Suppl 22): 167-78 DOI 10.26873/SVR-754-2019 Original Research Article GREEN TEA MITIGATES STREPTOZOTOCIN-INDUCED DIABETIC MICE THROUGH ANTI-FIBROTIC ACTIVITY AND MODULATION OF PROINFLAMMATORY CYTOKINES Hend M. Salah1, Eman A. Moussa1, Samaa M El. Bakr1, Mustafa S. Atta2*, Foad A. Farrag3, Ahmed S. Hafez4 department of Zoology, Faculty of Science, Kafrelsheikh University, 33516, Egypt, department of Physiology, Faculty of Veterinary Medicine, Kafrelsheikh University, department of Anatomy and Embryology, Faculty of Veterinary Medicine, Kafrelsheikh University, department of Pharmacology, Faculty of Veterinary Medicine, Aswan University, Egypt Corresponding author, E-mail: mostafa.ataa@vet.kfs.edu.eg Abstract: This research was planned to assess the protective effect of green tea extract (GTE) in STZ-induced diabetes in mice. Seventy-five female albino mice were used. Mice randomly allocated into five groups of 15 mice in each as follows: group 1 (control), group 2 (STZ, diabetic), group 3 (green tea + STZ), group 4 (protective group with green tea), and group 5 (green tea only). Oral administration of the green tea extract for three weeks to diabetic mice resulted in significant regaining in body weight, decreased blood glucose, cholesterol, triglyceride, free fatty acid and low-density lipoprotein cholesterol (LDL-C) levels and lowered malondialdehyde (MDA) contents and increased total protein and high-density lipoprotein cholesterol (HDL-C) as compared to untreated diabetic group. Histopathological changes were compatible with these biochemical findings. Diabetic mice pre- or co-treated with green tea also exhibited a significant downregulation in TGFfil and TNFa genes expression. Green tea extracts had protective and antidiabetic effect in controlling blood glucose level in addition to improving lipid metabolism and body weight in diabetic mice. Key words: diabetes; green tea; liver; TNFa; TGF-B1; mice Introduction Diabetes mellitus (DM) is one of the major intimidations to health in the world. It was expected to be more prevalent in 2025 (1). Hyper-glycemia is associated with vascular complications and kidney diseases (2). There are several other factors that play a great role in diabetes for instance, obesity and oxidative stress which lead to more ferocious complications (3), with decreased humoral immunity (4). DM type 1 is a consequence of the annihilation of P-cells of the pancreas (5). Deficiency of insulin primes to hyperglycemia. STZ is a structural analog of glucose (Glu) and N- acetyl glucosamine is reserved up by P-cells via the GLUT 2 transporter subsequently lead to P-cell loss via DNA destruction (6, 7). Moreover, cells that express GLUT 2 transporter like liver and kidney cells are also vulnerable to STZ. This clarifies kidney and liver complications (8, 9). Received: January 2019 Accepted for publication: February 2019 168 H. M. Salah, E. A. Moussa, S. M. El. Bakr M. S. Atta, F. A. Farrag, A. S. Hafez Oxidative anxiety is an inequity among the reactive oxygen species (ROS) and antioxidant protection of the body, which play a great role in diabetic complications (10). Since many studies stated that oxidative anxiety, facilitated by diabetes-induced production of ROS, lead to progress of diabetes, it noticed that improving oxidative anxiety via antioxidants might be a successful approach for demoting diabetic complications (11, 12). Green tea (Camellia sinensis) is prevalent nutraceutical antioxidant especially in Asian countries (13). Green tea has an important role in decreasing blood pressure, low-density lipo-protein cholesterol, and oxidative stress (14). Many studies proved the valuable effects of green tea on diabetic complications (15, 16) as well as it has antihyperglycemic effect in STZ diabetic animals (17). Moreover, green tea shows antioxidants and free radicals scavenger properties (18). This study was done to evaluate the protecting pathway by which green tea alleviate strep-tozotocin-induced diabetes in mice. Material and methods Chemicals We utilized a pharmaceutical-grade of STZ from Sigma (St. Louis, MO, USA). The STZ solution prepared by dissolving STZ in citrate buffer (0.01 M, pH 4.5) (19, 20). Chemicals consumed were of analytical grade. Total cholesterol, high-density lipoprotein (HDL)-cho-lesterol and triglyceride (TG) standard kits were purchased from Erba Diagnostics Mannheim Gambh, Germany. Green tea extract The green tea tablets, each tablet contain green tea extract 200 mg manufactured by El Obour City pharmaceutical industries. Following grinding, the obtained green tea powder dissolved in distilled water. This solution was provided to mice orally by using a stomach tube.(21) Experimental animals Seventy-five female albino mice, (9 - 11 weeks old, weighing about 25-30 gm was used. They obtained from Medical Technology Center, University of Alexandria, Egypt. Animals were managed according to the rules and regulations of our university committee. Female albino mice were housed in stainless steel pens of ambient temperature 23±2°C and light (12 h light/12 h dark) and free access to food and water. The mice were fed a standard diet according to (22) . Initiation of experimental diabetes After 2 weeks of accommodation, the mice were exposed to a 12 h fast. The mice were I/P injected with a single dose of 200 mg/kg STZ (7, 19). STZ was freshly dissolved in 0,1M cold sodium citrate buffer, pH 4.5(19, 20).after 2h from injecting we put 5% glucose and food to injected mice to avoid death(20) .we replace 5%glucose with water after 2h.The urine glucose level was checked three days after STZ injection to check the progress of diabetes. The diabetic mice were steadied for 5 days and then the experiment beginning. Mice which showed glucose levels >250 mg/dL was considered diabetic. Experimental design After acclimatization, mice were alienated randomly into five groups of 15 mice each: Group 1: healthy control mice received distilled water Group 2 (diabetic group): will be used as control diabetic mice. The mice were injected I/P with one dose of 200 mg/kg STZ (7, 19). In group 3 (treated group with green tea) diabetic mice were administrated green tea at a dose of 200 mg/kg bw orally by stomach tube daily for 21 days. In group4 (protective group with green tea) mice were given green tea at dose of 200 mg/kg by stomach gavage for 21 days and then diabetes was induced with a single dose of 200 mg/kg streptozotocin with continuous treatment with green tea at dose of 200 mg/kg orally for 21days. In group 5 (control green tea group) non-diabetic mice received green tea (200 mg/Kg /day)dissolved in distilled water orally by stomach tube for 21 days according to (23). Green tea mitigates streptozotocin-induced diabetic mice through anti-fibrotic activity and modulation of 169 Sampling Mice were sacrificed by cervical decapitation. Body weights of all the animals were recorded prior to the treatment and sacrifice. Weight change % = Initial weight - Final weight x 100/Initial weight Blood samples were collected in either EDTA coated tubes for the hematological investigation or plain tubes for serum separation (24) for biochemical assays. Kidneys, liver, spleen, and pancreases after decapitation were quickly removed and washed by cold saline to remove extraneous materials. Each organ was divided into 3 specimens. The first was snap frozen with liquid N2 and kept at -80°C until use for RNA extraction. The second was fixed in 10% neutral buffered formalin for the histo-logical examination. The third was used for transmission electron microscopy (TEM) handled using standard techniques. Blood analysis The serum samples for measurement of blood glucose level was determined based on glucose oxidase method (25), insulin concentrations were determined according to (26) using an insulin-ELISA kit (Morinaga Seikagaku, Tokyo, Japan). Malondialdehyde (MDA) was quantified by the method of Ohakawa et al. (27), HbAlc was appraised using DCA 2000 analyzer (Bayer, Elkhart, IN)(28, 29). Total protein concentration in serum was assayed col-orimetrically using commercial kit (Diamond, Egypt) and according to Lowry et al. (1951). Serum levels of total cholesterol, triglycerides, and high-density lipoproteins cholesterol (HDL-C) were estimated by quantitative color-imetric assay (Stanbio Laboratory, Inc., Texas, USA) according to (30) and low-density lipo-protein cholesterol (LDL-C) concentration assessed according to the formula of Friedewald et al; (31). LH, FSH, and estrogen were assessed using IMMULITE chemiluminescent assay kits (DPC, Glyn Rhonwy, Llanberis, Gwynedd, UK)(32). The Horiba ABX 80 Diagnostics (ABX pentra Montpellier, France) was used for hematological examination (33). Histopathological and transmission electron microscope examination The histopathology was carried out according to (34) using hematoxylin and eosin staining technique. Preparation od samples for transmission electron microscope (TEM) using collagen-coated nickel grids was applied as previously described (35). Molecular investigation Total RNA was extracted from tissue samples using RNA extraction kit (easy-REDTM, iNtRON Biotechnology, #17063, South Korea). cDNA synthesis was performed using reverse transcription kits (Thermo Scientific, Fermentas, #EP0451) which include the following: Revert Aid H Minus Reverse Transcriptase enzyme, Oligo dT,5X Reaction Buffer, RiboLock RNase Inhibitor, and dNTP Mix. Concentration of RNA and cDNA quantify using a Nanodrop (Q5000 Uv-Vis spectrophotometer, USA). Real-time PCR with SYBR Green (2X Maxima SYBR Green/ROX qPCR Master Mix) used to determine the expression of the target genes in the liver, with GAPDH as a housekeeping gene, according to the manufacturer protocol (Thermo Scientific, USA, # K0221). The primers used in the amplification are shown in Table 1. The web-based tool, Primer 3 (http://www-genome.wi.mit.edu/cgibin/pri-mer/primer3_www.cgi) used to design these primers. To confirm primer sequence is unique for the template sequence; we check similarity to other known sequences with BLAST. Calculation of fold changes in gene expression was done using2-AACt method. Statistical analysis Data was analyzed using GraphPad Prism 5 (GraphPad Software, San Diego, CA, USA). Results subjected to Tukey's multiple comparisons post-hoc test. Values are statistically significant when p < 0.05. Results Growth weight and Biochemical analysis Diabetic mice had a significant drop in body weight in relation to other groups.as shown in 170 H. M. Salah, E. A. Moussa, S. M. El. Bakr M. S. Atta, F. A. Farrag, A. S. Hafez figure 1.The diabetic mice showed a significantly increased level of serum glucose, triglycerides, cholesterol, LDL-C, VLDL-C relative to the control group and other treated groups and green tea protective groups. Serum insulin, total protein and HDL-C statistically decreased in diabetic mice as compared to the control group, treated and green tea protective groups (Table 2). Serum HbA1C pointedly increased in diabetic mice relative to the control group (Table 2). Diabetic mice showed a noteworthy increase in MDA compared to their equivalent levels in control animals. However, treated groups with green tea showed a significant diminution in the levels of MDA in relation to diabetic mice. Protective groups treated with green tea exhibited a significant reduced MDA levels as compared to diabetic mice (Table 2). LH, FSH and estrogen levels did not show any significant difference among all groups (Table 3). Hematological analysis RBC and WBC count, PCV, neutrophil % and platelets levels in diabetic mice decreased significantly, while MCV, MCH, and MCHC increased significantly in diabetic mice compared to the control mice. Green tea extract significantly normalized RBC and WBC count, PCV, neutrophil % and platelets in diabetic mice as well as MCV, MCH, and MCHC of diabetic mice to the control level (Table 4). Histopathological studies All results of histopathology were displayed in figure 2. Histopathological observation revealed that liver of the control animal showing normal hepatocytes arranged in cords around the central vein (arrowhead) whereas that of STZ-treated animal showing single cell necrosis (arrowhead) associated with active apopto-sis (arrow). Green tea treated diabetic animal showing slight hepatic vacuolation (arrowhead) adding that liver of green tea group showing normal hepatocytes around the central vein (arrow) as well as liver of protective group pre-treated with green tea showing normal hepato-cytes arranged around the central vein (arrow). Kidney of control animal showing normal renal glomeruli (arrowhead) and tubules (arrow) while kidney (cortex) of STZ-treated animal showing marked glomerular congestion (arrowhead) and degeneration within renal tubules (arrow) while that of green tea treated diabetic animal showing normal renal glomeruli (arrowhead) and patent normal tubules (arrow). Green tea group showing normal renal glomeruli (arrowhead) and tubules (arrow). Kidney of protective group with green tea-treated animal showing renal glomeruli (arrowhead) and tubules (arrow) within the normal limits. Pancreas of control animal showing normal glandular acini (arrow) and P islets (arrowhead) while that of STZ-treated animal showing degeneration of both glandular acini (arrow) and P islets (arrowhead). Green tea treated diabetic animal showing normal glandular acini (arrow) and slight vacuolation of P-cells (arrowhead) as well as pancreas of green tea-treated animal showing normal glandular acini and P islets (arrowhead). Diabetic animal pre-and post-treated with green tea-treated animal showing normal glandular acini (arrow) and P cells (arrowhead). Electron microscope studies All results of transmission electron microscope were displayed in figure 3. Liver of control group showing normal hepatocytes M indicates mitochondria and N indicates nucleus. While that liver of diabetic group showing mul-tilocular cytoplasmic fat vacuoles, decreased mitochondrial number and shrinkage of nuclear membrane. As well as, liver of green tea treated diabetic animals showing presence of small fat vacuoles, glycogen granules and multiple au-tophagic vacuoles. besides green tea group showing three adjacent hepatocytes separated with thin connective tissue layer and mostly normal. M indicates mitochondria, bar=500 pm., liver of protective group treated with green tea group showing moderate degree of hepatic steatosis, F indicates fat vacuoles. Pancreas (endocrine portion) of control group showing presence of large number of B secretary granules within the B cells (arrowhead), BC indicates blood capillary, pancreas of diabetic group showing exocrine pancreatic cell with noticeable decrease the number of secretory granules Green tea mitigates streptozotocin-induced diabetic mice through anti-fibrotic activity and modulation of 171 and mostly immature (arrowhead) with corrugated nuclear membranes (arrow), while that Green tea treated diabetic group showing exocrine pancreatic cell revealing normal nucleus (N) and with normal network of rough endo-plasmic reticulum (RER), pancreas of green tea group showing two adjacent exocrine cells with normal nuclei and surrounded with network of rough endoplasmic reticulum, in addition, pancreas (exocrine cell) of protective group pre-treated with green tea showing normal nucleus surrounded with network of rough endoplasmic reticulum and revealing large number of mature secretory vesicles (arrowhead). kidney of control group showing normal lining epithelial cells of the distal renal tubules with normal mitochondria (M) with mild condensation. Adding, kidney of STZ treated group showing the lining epithelial cells of the proximal tubules with slightly condensed mitochondria and some of them showing presence of fat vacuoles (arrowhead) and with normal brush border covering (arrow). as well as, green tea treated diabetic group showing lining epithelial cells of the distal renal tubules and mostly revealing lysis of the most intracellular organelles (arrow), kidney of green tea group showing the proximal renal tubules lining epithelial cells with normal brush border and mitochondria (M), bar=500 |im., while that of protective group pretreated with green tea group showing lining epithelial cells of the distal renal tubules and mostly revealing lysis of the most intracellular organelles (arrows). Table 1: Sequences of primers used in qPCR Outcome of streptozotocin, green tea on the relative expression of TGFfilgene and TNFa gene The results of qPCR were presented in Figure 4. A considerable upregulation in the expression of TGFfil gene in liver was found following administration of Streptozotocin as compared to the control group and other treated groups. Green tea treated diabetic mice showed a noteworthy decrease TGFfil gene expression as compared to the diabetic group. Furthermore, pre-treatment by green tea led to a significant decrease in TGFfil expression. Green tea alone led to insignificant downregulation of TGFfil gene expression as compared to the control group. TNFa upregulation was found following administration of Streptozotocin as matched to normal one and other treated groups. Administration of green tea with Strep-tozotocin resulted in a significant decrease in TNFa gene expression as compared to diabetic one. Furthermore, pre-treatment by green tea led to a significant reduction of TNFa expression. Administration of green tea alone led to momentous decreased of TNFa gene expression in relation to control group. Gene Reverse primer (/5------/3) Forward primer (/5------/3) Size (bp) Accession number TGFpi GACGTCAAAAGA-CAGCCACTCA GCAACATGTGGAACTC-TACCAGA 106 M13177 TNFa CTGATGA-GAGGGAGGCCATT GCCTCTTCTCATTCCTG CTTG 115 NM 00127860 1 GAPDH CCTGCTTCACCACC TTCTTGA TGTGTCCGTCGTG-GATCTGA 99 M32599 172 H. M. Salah, E. A. Moussa, S. M. El. Bakr M. S. Atta, F. A. Farrag, A. S. Hafez Table 2: Effect of green tea on serum glucose, HbAlC, insulin, MDA, total protein levels in STZ-induced diabetic female albino mice Control STZ STZ+Green tea pretreated with green tea Green tea Glucose (mg/dl) 120.5±13 c 407±49.6 a 186±27b 115±23c 143±4.7 bc HbAlC (%) 5.00±0.0 c 13.0±0.44 a 8.2±0.58 b 5.0±0.0 c 8.2±0.58 b Insulin pg/ml 1024±0.0 a 299.0±0.0 c 606±21.7 b 1024±0.0 a 826±50.8 b MDA nmol/ml 0.557±0.0 c 1.90±0.0 a 1.0±0.06 b 0.57±0.0 c 0.8±0.01 b Total protein (mg/dl) 6.21±0.17 a 4.44±0.18c 6.175±0.4 a 6.14±0.12 a 5.30±0.46 b Data are expressed as mean ± SEM.Superscript of different letters in each column differ significantly (P<0.05) from each other Table 3: Effect of green tea on LH, FSH and estrogen level levels in STZ-induced diabetic female albino mice Control STZ STZ+Green tea pretreated with green tea Green tea Estrogen(pg/ml) 24.2±0.3 22.8±0.5 23.0±0.4 23.6±0.24 22.8±0.37 FSH (pg/ml) 0.18±0.0 0.17±0.0 0.15±0.00 0.18±0.0 0.17±0.0 LH (pg/ml) 0.36±0.0 0.36±0.0 0.35±0.0 0.36±0.0 0.36±0.0 Data are expressed as mean ± SEM. Superscript of different letters in each column differ significantly (P<0.05) from each other Table 4: Effect of green tea on Haemogram in STZ-induced diabetic female albino mice Control STZ STZ+Green tea pretreated with green tea Green tea RBC (106/ul) 8.88±0.6a 7.45±0.13 c 8.31±0.18 b 8.6±0.04 ab 8.07±0.05 b Hb (g/dl) 15.4±0.18a 12.9±0.24c 14.5±0.2ba 14.3±0.21 b 14.5±0.2 ab HCT % 29.48±0.3 c 37.0±0.42 a 31.2±0.33 b 31.0±0.48 b 29.2±0.20 c MCV(fl) 40.7±0.28 c 46.0±0.28 a 43.0±0.35 b 43.1±0.23 b 40.7±0.35 c MCH(pg) 13.2±0.09 c 17.5±0.21 a 14.1±0.13 c 13.9±0.05 c 13.65±0.1 c MCHC (g/dl) 30.3±0.23 c 41.4±0.25 a 33.4±0.34 b 32.83±0.1 b 31.1±0.13 c WBC (103/ul) 17.0±0.07 a 3.54±0.39 c 7.07±0.22 b 7.82±0.44 b 6.15±0.42 c Lymphocyte % 65.0±0.16 a 11.94±4.1 c 45.65±4.1 b 44.59±3.3 b 61.21±0.6 a Monocyte % 17.8±0.11 a 5.345±0.4 c 14.95±0.0 b 13.83±0.2 b 17.08±0.1 a Granulocytes % 70.1±2.50 a 29.48±1.5 c 52.00±1.5 b 56.18±1.3 b 69.75±1.9 a Platelets (103/ul) 637.5±54 a 349.3±17 c 422±13.9 bc 543.0±13 b 486±14.6 b Data are expressed as mean ± SEM. Superscript of different letters in each column differ significantly (P<0.05) from each other Green tea mitigates streptozotocin-induced diabetic mice through anti-fibrotic activity and modulation of 173 a> TO 1 1' o £ 0-to £ -i -2- a I 7 —r ab nl b <2. ov Figure 1: Effect of green tea on body weight, food intake and water intake grade in STZ-induced diabetic female albino mice. Data are expressed as mean ± SEM. Superscript of different letters in each column differ significantly (P<0.05) from each other Control STZ STZ + GT GT GT+STZ + GT Figure 2: Histomicrograph of liver, kidney and pancreas of control group, STZ (diabetic) group, STZ+GT (green tea-treated STZ) group, GT (green tea) group and GT+STZ+GT (green tea-protective STZ) group. H&E, X200 174 H. M. Salah, E. A. Moussa, S. M. El. Bakr M. S. Atta, F. A. Farrag, A. S. Hafez Control STZ STZ + GT GT GT + STZ + GT Figure 3: Electron micrograph of liver, kidney and pancreas of the control group, STZ (diabetic) group, STZ+GT (green tea-treated STZ) group, GT (green tea) group and GT+STZ+GT (green tea-protective STZ) group. Scale bar = 500 ^m Figure 4: A. Effect of streptozotocin, green tea on the relative expression of TGFpi gene. Means within the same column carrying different superscript letters are significantly different (P< 0.05). B. Effect of Streptozotocin, green tea on the relative expression of TNFa gene. Means within the same column carrying -different superscript letters are significantly different (P< 0.05) Green tea mitigates streptozotocin-induced diabetic mice through anti-fibrotic activity and modulation of 175 Discussion Diabetes is a metabolic ailment characterized by hyperglycemia. Its consequences from faults in insulin creation and this leads to disturbance in the metabolism followed by worsening of muscles which leads diabetic complications (36). Our result revealed that diabetic mice had a significant drop in body weight as shown in figure 1. This result was consistent with (37-39) who reported momentous reduction in body weight in diabetic mice and attributed this effect to the highly muscle wasting and damage of tissue proteins. Diabetic mice have a marked rise in the levels of serum glucose, triglycerides, cholesterol, LDL-C, VLDL-C when compared with control groups. The obtained data were parallel to that reported by (40) who found that diabetes induces substantial surges in the levels of these parameters and attributed this effect to insulin deficiency that hinder lipoprotein lipase triggering hypertriglyceridemia. On the other hand, serum levels of insulin, total protein and HDL-C statistically decreased in STZ mice as compared to the control groups and these results agreed with (41-43) who reported similar reduction in these parameters in diabetic animals. Serum total protein in diabetic animals is obviously related to loss of structural proteins and drop in body weight which induced as a result of deficiency of insulin in diabetic mice. Result revealed that, green tea extract in diabetic mice resulted in noteworthy regaining in body weight, decreases serum glucose in diabetic one, decrease cholesterol, triglyceride, and LDL-C levels, and improved serum total protein and HDL-C. In parallel, (44) also reported that administration of green tea extract in diabetic mice resulted in similar improvement and attributed them to the antihyperglyce-mic and hypolipidemic activity of green tea (45). Moreover, daily administration of green tea to diabetic mice decreased glucose levels and HbA1c levels and this result was an in consistence with (47). In the present study, diabetic mice showed a noteworthy rise in MDA relative to control animals. However, pre and post-treatment of diabetic animals with green tea lead to a marked decrease in MDA level. These results was compatible with that obtained by (46) who reported that increase in MDA can be the result of diabetic complications. MCV, MCH, and MCHC markedly increased in diabetic mice as compared to control mice and suggesting occurrence of anemia in diabetic mice. This result agreed with (50) who reported that anemia in diabetes was due to the increased non-enzymatic glycosylation of RBC membrane proteins, which associated with hy-perglycemic oxidation of this glycosylated membrane. In contrast, green tea normalized the elevated MCV, MCH, and MCHC of diabetic mice. These results were consistent with those obtained by (50) who attributed this effect to the decreased lipid peroxide in RBC membrane which decreases hemolysis. The disrupted body defense mechanism of the diabetic animal against infections was distressed due to the neutrophil role in diabetes (48). The obtained results were similar to those obtained by (49) who reported that diabetic mice had a decreased WBC count and attributed this to Strep-tozotocin ability to suppress the immune system through hindrance of leukocytosis in the bone marrow. We also found a decrease in platelets levels in diabetic mice. In parallel, (51) also reported that platelet accumulation ability in diabetic mice could be due to deficiency of insulin. In contrast, green tea can return this reduced level to a level comparable to that of the control animals. These results are consistent with (52) who reported this effect to green tea ability to increase the biosynthesis of clotting factors. Similar to results obtained by (53), we found no significant change in LH, FSH and estrogen levels among the groups. Green tea treatment led to a significant down-regulation of TGFfil gene expression in liver. These results agreed with (54) who stated that green tea decreased hepatic expression of TGFfil and this accountable for reduction collagen synthesis and decrease fibrosis. Administration of green tea also led to significant 176 H. M. Salah, E. A. Moussa, S. M. El. Bakr M. S. Atta, F. A. Farrag, A. S. Hafez downregulation of TNFa gene expression. In inconsistence, (55) established that green tea persuaded growth inhibition and apoptosis by decrease TNFa expression. TNFa expression may also have a valuable effect on diabetes since TNFa is intricate in developing diabetes. Histopathological study and transmission electron microscope study revealed that administration of green tea can normalize the liver, kidney, pancreas and spleen that were deteriorated by STZ. In consistence, (56) reported that polyphenols (main ingredients in green tea) have anti-diabetic effects (57). Conclusion From the data found in this study, we can conclude that green tea has antidiabetic action though down-regulation of TGFfil and TNFa gene expression in liver, improvement of lipid metabolism and body weight in streptozotocin -induced diabetic mice. This study verified the protective action of green tea on experimentally induced diabetic mice. Conflict of interest All authors declare that they have no conflict of interest. References 1. Casqueiro J, Casqueiro J, Alves C. Infections in patients with diabetes mellitus: A review of pathogenesis. Indian journal of endocrinology and metabolism 2012; 16(Suppl1):S2-36. 2. Loghmani E. Diabetes mellitus: type 1 and type 2. Guidelines for adolescent nutrition services. 2005:167-82. 3. Kangralkar V, Patil SD, Bandivadekar R. Oxidative stress and diabetes: a review. Int J Pharm Appl. 2010; 1 (1): 38-45. 4. Peleg AY, Weerarathna T, McCarthy JS, Davis TM. Common infections in diabetes: pathogen-esis, management and relationship to glycaemic control. Diabetes/metabolism research and reviews 2007; 23 (1): 3-13. 5. Bukonla A, Benson OK, Akinsola AR, Aribigbola C, Adesola A, Seyi A. Effect of Type 1 Diabetes On Serum Electrolytes(Sodium and Potassium) Levels and Testosterone Hormone in Human Male Subjects. Webmed Central Biochemistry 2012; 3 (9): WMC003698. 6. Tesch GH, Allen TJ. Rodent models of strep-tozotocin-induced diabetic nephropathy (Methods in Renal Research). Nephrology. 2007; 12 (3): 2616. 7. Ventura-Sobrevilla J, Boone-Villa V, Aguilar C, Román-Ramos R, Vega-Avila E, Campos-Sepúlveda E, et al., editors. Effect of varying dose and administration of streptozotocin on blood sugar in male CD 1 mice. Proc West Pharmacol Soc; 2011; 54 :5-9 8. Eleazu C, Iroaganachi M, Eleazu K. Ameliorative potentials of cocoyam (Colocasia esculenta L.)and unripe plantain (Musa paradisiaca L.) on the relative tissue weights of streptozotocin-induced diabetic rats. Journal of diabetes research. 2013; 2013: 1 -9 9. RERUP CC. Drugs producing diabetes through damage of the insulin secreting cells. Phar-macologicalReviews. 1970; 22 (4): 485-518. 10. Pham-Huy LA, He H, Pham-Huy C. Free radicals, antioxidants in disease and health. Int J Bi-omed Sci. 2008; 4 (2): 89-96. 11. Giugliano D, Ceriello A, Paolisso G. Oxidative stress and diabetic vascular complications. Di-abetescare. 1996; 19 (3): 257-67. 12. Ceriello A. New insights on oxidative stress and diabetic complications may lead to a "causal" antioxidant therapy. Diabetes care. 2003; 26(5): 1589-96. 13. Cabrera C, Artacho R, Giménez R. Beneficial effects of green tea—a review. Journal of the American College ofNutrition. 2006; 25 (2): 79-99. 14. Nantz MP, Rowe CA, Bukowski JF, Percival SS. Standardized capsule of Camellia sinensis lowers cardiovascular risk factors in a randomized, double-blind, placebo-controlled study. Nutrition. 2009;2 5 (2): 147-54. 15. Iso H, Date C, Wakai K, Fukui M, Tama-koshi A. The relationship between green tea and total caffeine intake and risk for self-reported type 2 diabetes among Japanese adults. Annals of Internal Medicine. 2006; 144 (8): 554-62. 16. Wu CH, Lu FH, Chang CS, Chang TC, Wang RH, Chang CJ. Relationship among habitual tea consumption, percent body fat, and body fat distribution. Obesity research. 2003;11(9):1088-95. 17. Tsuneki H, Ishizuka M, Terasawa M, Wu JB, Sasaoka T, Kimura I. Effect of green tea on blood glucose levels and serum proteomic patterns in diabetic (db/db) mice and on glucose metabolism in healthy humans. BMC pharmacology. 2004;4 (1): 4-18. 18. Crespy V, Williamson G. A review of the health effects of green tea catechins inin vivo animal Green tea mitigates streptozotocin-induced diabetic mice through anti-fibrotic activity and modulation of 177 models. The Journal of nutrition. 2004; 134 (12): 3431S-40S. 19. Shinde UA, Sharma G, Xu YJ, Dhalla NS, Goyal RK. Insulin sensitising action of chromium picolinate in various experimental models of diabetes mellitus. Journal of Trace Elementsin Medicine and Biology. 2004; 1 8(1): 23-32. 20. Graham ML, Janecek JL, Kittredge JA, Hering BJ, Schuurman H-J. The streptozotocin-induced diabetic nude mouse model: differences between animals from different sources. Comparative medicine. 2011; 61 (4): 356-60. 21. Abolfathi AA, Mohajeri D, Rezaie A, Nazeri M. Protective effects of green tea extract against hepatic tissue injury in streptozotocin-induced diabetic rats. Evidence-Based Complementary and Alternative Medicine. 2012; 2012: 740671-81. 22. Reeves PG, Nielsen FH ,Fahey Jr GC. AIN-93 purified diets for laboratory rodents: final report of the American Institute of Nutrition ad hoc writing committee on the reformulation of the AIN-76A rodent diet. J nutr. 1993; 123 (11): 1939-51. 23. Maity S, Vedasiromoni JR, Ganguly DK. Role of glutathione in the antiulcer effect of hot water extract of black tea (Camellia sinensis). The Japanese Journal of Pharmacology. 1998; 78 (3): 285-92. 24. Yakubu M, Adesokan A, Akanji M. Biochemical changes in the liver, kidney and serum of rat following chronic administration of cimetidine. African Journal of Biomedical Research. 2006; 9(3): 213-8 25. Barham D, Trinder P. An improved colour reagent for the determination of blood glucose by the oxidase system. Analyst. 1972; 97 (1151):142-5. 26. Temple R ,Clark P, Hales C. Measurement of insulin secretion in type 2 diabetes: problems and pitfalls. Diabetic medicine. 1992; 9 (6): 503-12. 27. Ohkawa H, Ohishi N, Yagi K. Assay for lipid peroxides in animal tissues by thiobarbituric acid reaction. Analytical Biochemistry. 1979; 95 (2): 351-8. 28. Arsie M, Marchioro L, Lapolla A, Giacchetto G, Bordin M, Rizzotti P, et al. Evaluation of diagnostic reliability of DCA 2000 for rapid and simple monitoring of HbA1c. Acta diabetologica. 2000; 37 (1): 1-7. 29. Guerci B, DurainD, Leblanc H, Rouland J, Passa P, Godeau T, et al. Multicentre evaluation of the DCA 2000 system for measuring glycated haemoglobin. DCA 2000 Study Group. Diabetes & metabolism. 1997;23 (3): 195-201. 30. Allain CC, Poon LS, Chan CS, Richmond W, Fu PC. Enzymatic determination of total serum cholesterol. Clinical chemistry. 1974; 20 (4): 4705. 31. Friedewald WT, Levy RI, Fredrickson DS. Estimation of the concentration of low-density lipoprotein cholesterol in plasma, without use of the preparative ultracentrifuge. Clinical Chemistry. 1972;18(6):499-502. 32. Darne F, McGarrigle H, Lachelin GC. Diurnal variation of plasma and saliva oestrogen, progesterone, cortisol and plasma dehydroepi-androsterone sulphate in late pregnancy. European Journal of Obstetrics & Gynecology and Reproductive Biology. 1989; 32 (2): 57-66. 33. Dacie J, Lewis S. Practical Textbook of Haematology 7th Edition Edinburgh. Church Livingstone. 1991; 7:54-79. 34. Lillie RD. Histopathologic technic and practical histochemistry. Histopathologic technic andpractical histochemistry. 1954. 27(1): 73-6 35. Braet F, De Zanger R, Baekeland M, Crabbe E, Van Der Smissen P, Wisse E. Structure and dynamics of the fenestrae-associated cytoskeleton of rat liver sinusoidal endothelial cells. Hepatology. 1995; 21 (1): 180-9. 36. HallV, Thomsen RW, Henriksen O, Lohse N. Diabetes in Sub Saharan Africa 1999-2011: epidemiology and public health implications. A systematic review. BMC public health. 2011; 14 : 11-564. 37. Balamurugan R, Ignacimuthu S. Antidia-betic and hypolipidemic effect of methanol extract of Lippia nodiflora L. in streptozotocin induced diabetic rats. Asian Pacific Journal of Tropical Biomedicine. 2011; 1 (1): S30-S6. 38. Kumar R, Pate DK, Prasad SK, Sairam K, Hemalatha S. Antidiabetic activity of alcoholic leaves extract of Alangium lamarckii Thwaites on streptozotocin-nicotinamide induced type 2 diabetic rats. Asian Pacific journal of tropical medicine. 2011; 4 (11): 904-9. 39. Swanston-Flatt S, Day C, Bailey C, Flatt P. Traditional plant treatments for diabetes. Studies in normal and streptozotocin diabetic mice. Diabetologia. 1990;33(8):462-4. 40. Kasetti RB, Rajasekhar MD, Kondeti VK, Fatima SS, Kumar EGT, Swapna S, et al. Antihyperglycemic and antihyperlipidemic activities of methanol: water (4: 1) fraction isolated from aqueous extract of Syzygium alternifolium seeds in streptozotocin induced diabetic rats. Food and Chemical Toxicology. 2010;48(4):1078-84. 178 H. M. Salah, E. A. Moussa, S. M. El. Bakr M. S. Atta, F. A. Farrag, A. S. Hafez 41. Surana S, Gokhale S, Jadhav R Sawant R Wadekar JB. Antihyperglycemic activity of various fractions of Cassia auriculata Linn. in alloxan diabetic rats. Indian journal of pharmaceutical sciences. 2008, 70 (2): 227-9 42. Yokozawa T, Yamabe N, Kim HY, Kang KS, Hur JM, Park CH, et al. Protective effects of morroniside isolated from Corni Fructus against renal damage in streptozotocin-induced diabetic rats. Biological and Pharmaceutical Bulletin. 2008; 31 (7): 1422-8. 43. Gupta R, Gupta RS. Effect of Pterocarpus marsupium in streptozotocin-induced hypergly-cemic state in rats: comparison with glibenclamide. Diabetologia Croatica. 2009; 3 (2) : 945 44. Anandh Babu P, Sabitha K, Shyamaladevi C. GREEN TEA Extract Impedes Dyslipidaemia And Development Of Cardiac Dysfunction In Streptozotocin-Diabetic Rats. Clinical and Experimental Pharmacology and Physiology. 2006; 33 (12): 1184-9. 45. Kobayashi Y, Suzuki M, Satsu H, Arai S, Hara Y, Suzuki K, et al. Green tea polyphenols inhibit the sodium-dependent glucose transporter of intestinal epithelial cells by a competitive mechanism. Journal of Agricultural and Food Chemistry. 2000;48(11):5618-23. 46. Goycheva P, Gadjeva V, Popov B. Oxidative stress and its complications in diabetes mellitus. Trakia J Sci. 2006; 4 (1): 1-8. 47. Fukino Y, Ikeda A, Maruyama K, Aoki N, Okubo T, Iso H. Randomized controlled trial for an effect of green tea-extract powder supplementation on glucose abnormalities. European journal of clinical nutrition. 2008;62(8):953-60. 48. Berkowitz DB, Kryspin-S0rensen I. Transgenic fish: Safe to eat? Nature Biotechnology. 1994; 12 (3): 247-52. 49. Oyedemi S, Yakubu M, Afolayan A. Effect of aqueous extract of Leonotis leonurus (L.) R. Br. leaves in male Wistar rats. Human & experimental toxicology. 2010; 29 (5): 377-84. 50. Kennedy L, Baynes J. Non-enzymatic glycosylation and the chronic complications of diabetes: an overview. Diabetologia. 1984; 26 (2): 93-8 51. Jarald E, Joshi SB, Jain DC. Diabetes and herbal medicines. Iran J Pharmacol Ther. 2008; 7(1): 97-106. 52. Adebayo JO, Adesokan AA, Olatunji LA, Buoro DO, Soladoye AO. Effect of ethanolic extract of Bougainvillea spectabilis leaves on haemato-logical and serum lipid variables in rats. 2005; 17 (1):-45-50. 53. Johnson LM, Sidman RL. A reproductive endocrine profile in the diabetes (db) mutant mouse. Biology of reproduction. 1979; 20 (3): 552-9. 54. Abe K, Suzuki T, Ijiri M, Koyama Y, Isemura M, Kinae N. The anti-fibrotic effect of green tea with a high catechin content in the galactosamine-injured rat liver. Biomedical Research. 2007; 28 (1): 43-8. 55. Fujiki H, Suganuma M, Kurusu M, Okabe S, Imayoshi Y, Taniguchi S, et al. New TNF-a releasing inhibitors as cancer preventive agents from traditional herbal medicine and combination cancer prevention study with EGCG and sulindac or tamoxifen. Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis. 2003; 523: 119-25. 56. Li Y-W, Zhang Y, Zhang L, Li X, Yu J-B, Zhang H-T, et al. Protective effect of tea polyphenols on renal ischemia/reperfusion injury via suppressing the activation of TLR4/NF-kB p65 signal pathway. Gene. 2014 ;542 (1): 46-51. 57. Yang Y-S, Wang C-J, Huang C-N, Chen ML, Chen M-J, Peng C-H. Polyphenols of Hibiscus sabdariffa improved diabetic nephropathy via attenuating renal epithelial mesenchymal transition. Journal of agricultural and food chemistry. 2013; 61(31): 7545-51. Slov Vet Res 2019; 56 (Suppl 22): 179-86 DOI 10.26873/SVR-755-2019 Original Research Article BIOMARKERS VERSUS DUPLEX ULTRASONOGRAPHY FOR EARLY DETECTION OF ACUTE KIDNEY INJURY IN DOGS: AN EXPERIMENTAL STUDY Mohamed A. Donia, Naglaa A. Gomaa*, Mohamed Abdelmegeid, Midhat N. Nassif Department of Animal Medicine, Faculty of Veterinary Medicine, Kafrelsheikh University, 33516, Egypt Corresponding author, E-mail: naglaagomaa505@yahoo.com Abstract: Acute kidney injury (AKI) is a common problem in dogs and is associated with high mortality. Early recognition of AKI is lifesaving and could help in clinical management. The purpose of this experimental study was to identify the accurate biomarkers for early detection of AKI in dogs. Ten healthy mongrel dogs (7 females, 3 males) were used in a crossover experimental study. Cisplatin (5 mg/kg BW) was used for induction of AKI. Serum urea and creatinine, as well as serum Sodium, Potassium, Calcium, Phosphorus, and Bicarbonate, were measured spectrophotometrically using commercial kits. However, urinary Neutrophil Gelatinase-Associated Lipocalin (NGAL) were determined using ELISA. Renal Resistive Index (RI) was calculated using Doppler ultrasound (duplex). All examinations were carried out on the same day of induction and for other 4 consecutive days. Post-mortem and histopathological examinations were carried out on two dogs. Using the repeated measure MANOVA and student T-test, the data were statistically analyzed. There were significant increases of both serum urea and creatinine beginning from the second day of infusion, while renal RI was significantly increase beginning from the third day of infusion. There are marked changes in serum electrolytes including hyperkalemia, hyperphosphatemia, hyponatremia with metabolic alkalosis. However, urinary NGAL showed a non- significant increase during the experiment. It can be concluded that renal RI can be used for early diagnosis of acute renal injury in dogs. Key words: urinary biomarkers; renal resistive index; acute kidney injury; dogs Introduction Acute kidney injury is a common problem in dogs. It is costive either for diagnosis or for treatment and correlated with high mortality (1). The earlier diagnosis of such condition is regarded as lifesaving (2). There are many methods for detection of kidney injury such as estimation of glomerular filtration rate (GFR) (3). The routine technique used for detection of GFR is to measure serum creatinine concentration, but it is relatively insensitive with a delayed release in serum (4). Novel serum and urine biomarker that can differentiate between both acute and chronic glomerular and tubular renal injury is the Neu-trophil Gelatinase-Associated Lipocalin (NGAL) (5). It is a derived protein which increases in plasma and urine in acute ongoing renal injury after few hours (6). In human, NGAL Received: January 2019 Accepted for publication: February 2019 180 M. A. Donia, N. A. Gomaa, M. Abdelmegeid, M. N. Nassif was investigated in cases of AKI secondary to cardiac surgery, contrast-induced nephropathy and in critical illnesses. It was reported that elevated NGAL concentrations are a marker for AKI in such cases (7). Urinary NGAL is more sensitive and specific for predicting AKI and it was superior to creatinine and other markers of renal diseases (8). One of the most advanced methods for the detection of AKI is the Doppler-based renal resistive index (RI) measurement. This is a rapid and non-invasive investigative tool that was used for early detection of AKI in the intensive care unit patients (9). Moreover, it can differentiate transient from persistent AKI in critically ill patients (10, 11). Recently, it was reported that an increase in the renal RI is an accurate and sensitive tool for early prediction of AKI in rats (12). This study aims to compare the efficiency of selected serum biochemical parameters and the renal RI for early diagnose of AKI in dogs and to determine the most accurate, rapid, and applicable method in clinical practice. Material and methods Animals Ten healthy mongrel dogs (7 females, 3 males) were used in this a cross over experimental study. Their weight ranged from 8 to 20 Kgs and their age ranged from 9 to 15 months. They considered healthy based on physical, he-matological, biochemical and ultrasonographic examinations, in addition to a urine analysis. They were housed restless in separate boxes with plenty of food and water. The dogs were used as a control and treated group. In the control group, each dog was infused with normal saline at a dose rate of 5ml/kg BW. After two weeks, the same dogs were used as a treated group in which they were infused once with Cisplatin (Mylan, France) at a concentration of 5 mg/kg for induction of AKI. Physical, hema-tological, serum biochemical profiles, urine analysis, and ultrasonographic examinations were carried out on each dog of the treated group on the same day of induction and for other 4 consecutive days. All examinations were done after approval of the Ethics committee in Kafrelsheikh University. Samples Two blood samples were collected from each dog from the cephalic vein in the control and treated group. The first blood sample was about 5ml that collected in a heparinized test tube for hematological examination using Vet hematological analyzer (Mythic18, France). The second blood sample was collected in a plain test tube to obtain serum for biochemical analysis. The serum was collected by centrifu-gation of the clotted blood at 3000 rpm for 10 min. It was examined for blood urea nitrogen (BUN) using the colorimetric method by (BTS-302 Spectrophotometer) and (Urea Biosystem kit, Germany). Serum creatinine was estimated using the fixed time method by (BTS-302Spec-trophotometer) and (Creatinine Biosystem kit, Germany). Some blood electrolytes such as Sodium, Potassium, Chloride, Calcium, Phosphorus and the Bicarbonate were measured in the serum using (Cobas C & Cobas E, France). Urine samples were assembled by cystocen-tesis under ultrasonographic guide from each dog. Urine was examined macroscopically for colour and consistency. The specific gravity was measured by a standard Refractometer. The chemical examination for urine samples was accomplished using (Combi screen urine strips, Germany). Urine samples were examined microscopically for the presence of pus, cells, casts, and others. The investigation of Neutro-phil Gelatinase-Associated Lipocalin (NGAL) was carried out using Sandwich enzyme-linked immunosorbent assay ELISA- kit (Canine NGAL Elisa Kit (Bioporto® Diagnostic) (13). Ultrasonographic examination Ultrasonographic examination was performed on unsedated dogs in both groups for detection of any changes in the tissue architecture, kidneys dimensions using 2-D mode. Renal (RI) for each kidney was measured using Doppler ultrasound (duplex), (Mindray Z5 ultrasound machine, China), with a linear transducer 5-10 MHZ.A renal interlobar or arcuate Biomarkers versus duplex ultrasonography for early detection of acute kidney injury in dogs: 181 artery was distinguished firstly by color Dop-pler then switched on the pulsed wave Doppler using a gate of 1.5 mm width over the selected artery. The obtained image showed the blood flow without any aliasing. Multiple pulses from one artery in three poles for each kidney right and left in each dog was recorded (14). The renal RI was measured atomically by the ultrasound machine from the selected artery. Post mortem examination It was performed on two dogs, one of them dead at the 5th day of the experiment and the second has been euthanized by a large dose of anesthetics and kidney specimens were picked in formalin 4% for histopathological examination. Histopathology Kidneys were removed, fixed in 4% buffered paraformaldehyde, embedded in paraffin with usual manner, sections were about 2.5 mm thick), and stained with periodic acid-schiff and periodic acid-methenamine silver for histologic examination under light microscope (15). Statistical analysis A doubly repeated measure MANOVA was used to test the significant effect of cisplatin on the parameter; urea, creatinine, urinary NGAL, and the renal RI among the examined animals at P value < 0.05. This followed by univariate analysis for each parameter to examine its significant difference per day. Student T-test was used for comparison between serum electrolytes before and after the drug infusion and P value < 0.05. All analyses applied using SPSS version 23.0 (IBM, New York, NY, USA). Results The physical examination of examined animals revealed; non-significant changes in all digital body parameters such as body temperature, heart rate respiratory rate and pulse. The mucous membranes and superficial lymph nodes were normal. Only signs of vomiting and diarrhea appeared after half hour of the drug infusion in all dogs. A degree of lethargy was clear on 5 dogs that obviously increased with time. The physical examination of the urine samples showed changes in its colour beginning from the 3rd day of Cisplatin infusion in most dogs. The appearance of the urine changed gradually from the amber yellow colour to the turbid deep yellow then reddish bloody in two dogs. By chemical analysis, the urine samples showed traces of proteins and glucose beginning from the second day then increased gradually to three or four pluses at the end of the experiment using strips kits. The microscopical examination of urine sediments revealed a marked increase of epithelial cells, pus cells, RBCs, urate and phosphate crystals within the time of the experiment. There were no obvious changes in the 2D ultrasonographic measurements between before and after the Cisplatin infusion. The renal dimensions (length, width, and height) were between (5.64-6.30 mm, 2.79-4 mm and 2.56-3.3 mm), respectively. On the other hand, there was a significant increase in renal RI of the interlobar arteries beginning from3rd day after infusion at P value < 0.022 as in table 1 and (Fig. 1). Concerning the blood serum biochemical analysis (Table1), revealed a significant increase in both BUN and creatinine at (P-value < 0.000 and 0.002), respectively from the second day after cisplatin infusion. On the other hand, the urinary NGAL concentration showed non-significant during all the days of this study. Regarding serum electrolyte analysis (Table 2) showed insignificant (P=0.058) decrease in serum sodium and insignificant (P =0.09) increase in the serum ionized calcium. While there was a significant increase in serum inorganic phosphorus, potassium, and bicarbonate ions at (P< 0.01, 0.01 and 0.000), respectively. PM examination of the first dog revealed some congestion in both kidneys and intestine, while the second showed no abnormalities except slight congestion of kidneys (Fig. 2 A). Histopathological examination showed numerous apoptotic renal epithelial cells and thickening of the glomerular membranes, peri-glomerular fibrosis and marked renal tubular damage (Fig. 2 B). 182 M. A. Donia, N. A. Gomaa, M. Abdelmegeid, M. N. Nassif Table 1 Serum urea, serum creatinine, urinary NGAL and renal RI (mean and slandered deviation) after intravenous infusion of Cisplatin in dogs Control Time post-infusion (day) P value 1 2 3 4 5 Urea (mg/dl) 24.25±5.56 26±3.16 37.25±2.17** 81.25±31.19** 119.25±46** 327.5±54.65** 0.001 Creatinine (mg/dl) 0.94±0.06 0.91±0.07 1.35±0.07** 1.71±0.33** 2.72±0.28** 6.71±0.91** 0.000 Urinary NGAL (ng/ml) 0.82±0.25 2.23±1.09 1.67±0.94 2.03±1.18 2.17±0.97 2.15±1.26 0.131 Renal RI 0.62±0.03 0.62±0.02 0.64±0.03 0.68±0.03** 0.7±0.03** 0.74±0.04** 0.001 *Indicates a significant difference at different time points (p<0.05) **Indicates a significant difference at different time points (p<0.01) Table 2: Serum sodium, potassium, ionized calcium, phosphorus and bicarbonate (mean and slandered deviation) before and after 5th day of intravenous infusion of Cisplatin in dogs Before infusion After infusion Sodium (mEq/l) 138 ± 2.58 126.25 ± 12.61 Potassium (mEq/l) 4.18 ± 0.23 5.65 ± 0.9* Ionized calcium (mg/dl) 4.78 ± 0.25 5.28 ± 0.64 Phosphorus (mg/dl) 4.98 ± 0.13 5.83 ± 0.59* Bicarbonate (mmol/L) 18.48 ± 0.66 30.5 ± 4.20** *Indicates a significant difference at p<0.05 **Indicates a significant difference at p<0.01 mindray mumohium-toct tuwuiuiiii J$; mindrdy johmimiwimbcj Mm/ma h.imi »»"•"• WWWWVPk' I I » II i .... i .... I ... i .... i !. ... i .!... i .... i H . I . . . I .... I ..i.l..!,! *-1-i, on o.7 Figure 1: Pulsed wave Doppler ultrasonographic image of the interlobar artery before cisplatin infusion (A) and at the fifth day after the infusion (B) Biomarkers versus duplex ultrasonography for early detection of acute kidney injury in dogs: 183 Figure 2: A sagittal section of the kidney showing congestion (A). Histopathological section of the kidney (B), shows marked necrotic changes with glomerular tufts which filled with eosinophilic materials (arrowhead) and marked degenerative and necrotic changes within the renal tubular epithelium (arrow), H&E, bar= 40 pm Discussion In this study, acute renal injury was induced using cisplatin 5 mg/kg BW which is a widely used chemotherapeutic agent for different malignancies. It has a marked nephrotoxic effect as it causes renal tubular cell necrosis due to the presence of platinum in its structure (16, 17). Some of the treated dogs showed vomiting and diarrhea after infusion of the drug. This could be a side effect of the cisplatin due to its irritating effect on the mucosa of gastrointestinal tract (18, 19). Days after the infusion, there was ataxia or muscle weakness appeared on the dogs and it may be contributed to electrolytes disturbance and dehydration. After two days of the infusion, urine samples revealed an obvious increase of glucose and this may due to the failure of reabsorption of the glucose from the proximal convoluted tubules (20). Beginning from the 3rdday after infusion, there was a patent increase in proteins and RBCs in urine samples. This increase may be related to the decrease of glomerular filtration ate which follows the proximal tubular damage as an initial toxic effect of the cisplatin (17). After two days of infusion, there was a highly significant increase in serum urea level compared with the control group. However, serum creatinine was significantly increased after three days of the infusion and this may be attributed to the impaired kidney function (15). Although the estimation of urea and creatinine level in serum is routine work in the interpretation of kidney disease in the clinical practice, they have some limitations such as serum urea level may be increased in other abnormalities rather than kidney diseases such as liver dysfunction or increased dietary protein (21). It may also increase in cases of gastrointestinal bleeding as a consequence of increased absorption of nitrogenous compound (22). In addition, the magnitude of creatinine concentration cannot predict the origin and severity of renal dysfunction (23). Moreover, its concentration is reduced in sepsis and so limits its use as a marker of kidney injury in case of bacterial infections (24). NGAL is a neutrophil-derived protein related to lipocalin protein family (25). Its expression is upregulated by injured epithelia of renal tubules, trachea, bronchi, and also increase in the acute infections and different types of neo-plasia (26, 27). NGAL is markedly upregulated in damaged proximal tubules in order to induce re-epithelization (28). NGAL was reported as 184 M. A. Donia, N. A. Gomaa, M. Abdelmegeid, M. N. Nassif an early urinary biomarker for acute renal injury in dogs (29). Besides, it can be used as a real-time indicator of progressive kidney damage (30). In this study, NGAL was insignificantly increasing after the 5th day of infusion of cisplatin. This insignificant increase of NGAL may be attributed to several causes such as the lower dose of cisplatin used in this study and the low sensitivity of the test in the detection of mild renal injury (28, 31). Therefore, the measurement of urinary NAGL is not efficient for the detection of mild kidney injury. Moreover, it increases in septic condition, so it gives a false positive result in septic cases (32). Electrolyte disturbance usually occurs with kidney dysfunction especially those related to the renal tubules. The results of this study showed insignificant hyponatremia which may be attributed to reduced sodium conservation from damaged renal tubules (33). The significant hyperkalemia and hyperphosphatemia may have referred to the leakage of potassium and phosphate from the intracellular fluids to the extracellular fluids (43, 35). The highly significant increase of bicarbonate is unusual in such cases but, it may be due to vomition which develops an extra-renal cause of metabolic alka-losis. Alkalosis enhances calcium reabsorption in the distal nephron and this clarifies the insignificant increase in ionized calcium (36, 37). Ultrasonography is very useful in the diagnosis of all aspects of renal diseases in clinical practice (38). In this study, each kidney was evaluated for any changes in echogenicity, length, width, height and volume by the 2 D mode. There were non-significant changes neither in the echogenicity nor in kidney dimensions when compared with the control group (39). The Doppler ultrasonography can be used for assessing the renal hemodynamic which is very useful in the diagnosis of renal arteries and veins disorders such as thrombosis (40). The renal RI was significantly (p=0.022) increase beginning from the 3rd day of the infusion. It may have referred to vasoconstriction resulting from renin release in response to the decreased blood flow in the renal arteries secondary to renal injury (41). This result coincided with other studies on dogs with interstitial nephritis or tubular degeneration (42). In the present study, the increase of RI was parallel with the increase of serum creatinine after the third day of infusion. In another study on mice, the renal RI precedes the serum creatinine in a diagnosis of AKI after cisplatin infusion (12). However, there are some limitations for using RI in the diagnosis of all kidney diseases because it is affected by other hemodynamic disorders rather than in kidney as well as the difficulty of measuring RI without sedation of dogs which affected on its measurement (10, 12). The histopathological picture supported the clinical and biochemical findings which indicating tubular renal injury with secondary glomerular affection. This result was similar to that described in mice kidney after using cisplatin for induction of renal failure (43). Conclusion The results indicate that increasing renal RI go parallel with increasing serum urea and cre-atinine in dogs with AKI. Although renal RI alone is not sensitive for predicting the developing of AKI, the high increase of renal RI can be used as an early signal for Acute renal injury because of its high specificity. Conflict of interest None of the authors have any conflict of interest to declare. References 1. Langston C. Acute uremia In Ettinger S,Feld-man E, eds. Textbook of Veterinary Internal Medicine. Philadelphia: Saunders WB, 2010; 19552115. 2. Cowgill L, Langston C. Acute kidney insufficiency In Nephrology and Urology of small animals 2011;472-523. 3. Kerl M, Cook C. Glomerular filtration rate and renal scintigraphy. Clinical techniques in small animal practice 2005; 20:31-8. 4. Cobrin A, Blois S, Kruth S, et al. Biomarkers in the assessment of acute and chronic kidney diseases in the dog and cat. Journal of Small Animal Practice 2013; 54:647-55. 5. Urbschat A, Obermuller N, Haferkamp A. Biomarkers of kidney injury. Biomarkers 2011; 16: S22-S30. Biomarkers versus duplex ultrasonography for early detection of acute kidney injury in dogs: 185 6. Haase M, Haase-Fielitz A, Bellomo R, et al. Neutrophil gelatinase-associated lipocalin as a marker of acute renal disease. Current opinion in hematology 2011; 18:11-8. 7. Hirsch R Dent C, Pfriem H, et al. NGAL is an early predictive biomarker of contrast-induced nephropathy in children. Pediatric Nephrology 2007; 22:2089. 8. Nickolas T, O'Rourke M, Yang J, et al. Sensitivity and specificity of a single emergency department measurement of urinary neutrophil gelatinase-associated lipocalin for diagnosing acute kidney injury. Annals of internal medicine 2008; 148:810-9. 9. Darmon M, Schortgen F, Vargas F, et al. Diagnostic accuracy of Doppler renal resistive index for reversibility of acute kidney injury in critically ill patients. Intensive care medicine 2011; 37:6876. 10. Platt J, Rubin J, Ellis J. Acute renal failure: possible role of duplex Doppler US in distinction between acute prerenal failure and acute tubular necrosis. Radiology 1991; 179:419-23. 11. Schnell D, Deruddre S, Harrois A, et al. Renal resistive index better predicts the occurrence of acute kidney injury than cystatin C. Shock 2012;38:592-7. 12. Fisch S, Liao R Hsiao L, et al. Early Detection of Drug-Induced Renal Hemodynamic Dysfunction Using Sonographic Technology in Rats. Journal of visualized experiments: JoVE 2016; 109:52409-4145 13. Steinbach S, Weis J, Schweighauser A, et al. Plasma and Urine Neutrophil Gelatinase-Associ-ated Lipocalin (NGAL) in Dogs with Acute Kidney Injury or Chronic Kidney Disease. Journal of veterinary internal medicine 2014; 28:264-9. 14. Lin G, Cher T. Renal vascular resistance in normal children-a color Doppler study. Pediatric Nephrology1997; 11:182-5. 15. Abinaya A, Karu P, Karunakaran R, et al. Influence of age on blood biochemical profile of obese dogs. International Journal of Conservation Science 2018; 6:991-3. 16. Lim C, Han J, Kim S, et al. Effect of granu-locyte colony-stimulating factor administration on renal regeneration after experimentally induced acute kidney injury in dogs. American journal of veterinary research 2016; 77:199-207. 17. KintzelP. Anticancer drug-induced kidney disorders. Drug safety2001; 24:19-38. 18. Sharma R, Tobin P, Clarke S. Management of chemotherapy-induced nausea, vomiting, oral mucositis, and diarrhea. The lancet oncology2005; 6:93-102. 19. Florea A, Büsselberg D. Cisplatin as an antitumor drug: cellular mechanisms of activity, drug resistance, and induced side effects. Cancers2011; 3:1351-71. 20. Bagshaw S, Langenberg C, Wan L, et al. A systematic review of urinary findings in experimental septic acute renal failure. Critical care med-icine2007; 35:1592-8. 21. Sherlock S, Dooley J. Assessment of liver function In Diseases of the liver and biliary system: John Wiley & Sons, 2008; 19-38. 22. Prause L, Grauer G. Association of gastrointestinal hemorrhage with increased blood urea nitrogen and BUN/creatinine ratio in dogs: a literature review and retrospective study. Veterinary clinical pathology1998; 27:107-11. 23. Perrone R Madias N, Levey A. Serum cre-atinine as an index of renal function: new insights into old concepts. Clinical chemistry 1992; 38:1933-53. 24. Doi K, Yuen P, Eisner C, et al. Reduced production of creatinine limits its use as a marker of kidney injury in sepsis. Journal of the American Society of Nephrology 2009; 20:1217-21. 25. Flower D. The lipocalin protein family: structure and function. Biochemical journal.1996; 318:1-14. 26. Bolignano D, Donato V, Lacquaniti A, et al. Neutrophil gelatinase-associated lipocalin (NGAL) in human neoplasias: a new protein enters the scene. Cancer letters 2010; 288:10-6. 27. Fjaertoft G, Foucard T, Xu S, et al. Human neutrophil lipocalin (HNL) as a diagnostic tool in children with acute infections: a study of the kinetics. Acta Paediatrica2005; 94:661-6. 28. Mishra J, Mori K, Ma Q, et al. Neutrophil gelatinase-associated lipocalin: a novel early urinary biomarker for cisplatin nephrotoxicity. American journal of nephrology2004; 24:307-15. 29. Kai K, Yamaguchi T, Yoshimatsu Y, et al. Neutrophil gelatinase-associated lipocalin, a sensitive urinary biomarker of acute kidney injury in dogs receiving gentamicin. The Journal of toxico-logical sciences 2013; 38:269-77. 30. Mori K, Nakao K. Neutrophil gelatinase-as-sociated lipocalin as the real-time indicator of active kidney damage. Kidney international 2007; 71:96770. 31. Steinbach S, Weis J, Schweighauser A, et al. Plasma and Urine Neutrophil Gelatinase-Associ-ated Lipocalin (NGAL) in Dogs with Acute Kidney Injury or Chronic Kidney Disease. Journal of veterinary internal medicine2014; 28: 264-9. 186 M. A. Donia, N. A. Gomaa, M. Abdelmegeid, M. N. Nassif 32. Yilmaz A, Sevketoglu E, Gedikbasi A, et al. Early prediction of urinary tract infection with urinary neutrophil gelatinase-associated lipocalin. Pediatric Nephrology 2009; 24:2387. 33. Denis C, Jadot V, Bouquegneau A, et al. The Case Acute renal failure and refractory hyponatremia. Kidney international2016; 90:713-4. 34. Tanrikut C, McDougal W. Acid-base and electrolyte disorders after urinary diversion. World journal of urology 2004; 22:168-71. 35. Haycock G. Management of acute and chronic renal failure in the newborn. Seminars in neonatology 2003; 325-34. 36. Jeon U, Pressure B. Kidney and calcium homeostasis. Electrolyte and Pressure, Blood 2008; 6:68-76. 37. Felsenfeld A, Levine B. Milk-alkali syndrome and the dynamics of calcium homeostasis. Clinical Journal of the American Society of Neph-rology2006; 4:641-54. 38. O'Neill W. Sonographic evaluation of renal failure. American journal of kidney diseases 2000; 35:1021-38. 39. Adams W, Toal R, Breider M. Ultrasonographic findings in ethylene glycol (antifreeze) poisoning in a pregnant queen and 4 fetal kittens. Veterinary Radiology 1991; 32:60-2. 40. Ricci M, Lloyd D. Renal venous thrombosis in infants and children. Archives of Surgery1990; 125:1195-9. 41. Chang Y, Chan I, Cheng F, et al. Relationship between age, plasma renin activity, and renal resistive index in dogs. Veterinary radiology and ultrasound 2010; 51:335-7. 42. Morrow K, Salman M, Lappin M, et al. Comparison of the resistive index to clinical parameters in dogs with renal disease. Veterinary Radiology and Ultrasound 1996; 37:193-9. 43. Ramesh G, Reeves W. TNFR2-mediated apoptosis and necrosis in cisplatin-induced acute renal failure. American Journal of Physiology-Renal Physiology2003; 285: F610-F8. Slov Vet Res 2019; 56 (Suppl 22): 187-94 DOI 10.26873/SVR-756-2019 Original Research Article THERAPEUTIC EFFICACY OF ZINC OXIDE NANOPARTICLES IN DIABETIC RATS Mohammed A. Kamal 1, Mohamed H. Khairy 1, Nada A. ELSadek 1, Mohamed M.A. Hussein 2* 1Department of Pharmacology, Faculty of Veterinary Medicine, Zagazig University, 2Department of Biochemistry, Faculty of Veterinary Medicine, Zagazig University, 44519, Egypt Corresponding author: Email: hamza_vet@yahoo.com Abstract: This study attempted to scrutinize the potential efficacy of zinc oxide nanopar-ticles (ZnONPs) and the standard oral hypoglycemic drug glibenclamide in streptozotocin (STZ) -nicotinamide induced diabetic rats. Forty male Wistar rats were divided into four equal groups. Group 1 rats received saline orally and considered as a control group. Other groups were experimentally exposed to diabetes via intraperitoneal injection of STZ and group 2 was left as diabetic non-treated. Group 3 was orally administrated with ZnONPs at a low dose (1mg/kg), and group 4 orally received glibenclamide (600 Mg/kg) for 30 days (duration of the experiment). Blood glucose, insulin levels, lipogram profile and pancreatic antioxidant status were improved following ZnONPs or glibenclamide administration as compared to the diabetic group. Moreover, histopathological examination revealed a better outcome in the ZnONPs and glibenclamide treated groups. Although oral treatment of ZnONPs at a low dose 1 mg/kg body weight for 30 consecutive days had antidiabetic effect, this effect was less superior to glibenclamide. Therefore, further studies regarding increasing the dose of ZnONPs would be encouraging to get better anti diabetic effect. Key words: Zinc oxide nanoparticles; antioxidant; diabetes; glibenclamide Introduction Diabetes mellitus (DM) is a metabolic disorder with chronic hyperglycemic and hypertri-acylglycerolemic condition caused mainly due to defects in insulin secretion and/or action. The prevalence of diabetes is increasing rapidly worldwide (1). Zinc (Zn) is an essential micro-nutrient for pancreatic function through its effect on insulin stability and disturbance in Zn metabolism associated with diabetic complications (2). It is also a paramount player in the intracellular antioxidant machinery through its participation in the main antioxidant enzymes with free radical scavenging effect such as cat-alase, superoxide dismutase and metallothi-onein which attracted much attention in diabetes studies (3). Lower level of zinc in pancreatic tissues associated with lower insulin synthesis through p cells (4). Elevated ROS can induce oxidative damage in pancreatic tissues with subsequent increased hyperglycemia (5). The field of nanotechnology is one of the every foremost active analysis areas in fashionable materials science and biology (6, 7). Nano- Received: January 2019 Accepted for publication: February 2019 188 M. Kamal, M. Khairy, N. ELSadek, M. Hussein particles come with new properties and biomedical applications which owed to its size, distribution and morphology (8).On the other hand, the use of nanoparticles has a dark side through their generation of reactive oxygen species (ROS), resulting in oxidative damage and inflammation (9,10). Zinc oxide nanoparticles (ZnONPs) are widely used in paint, pharmaceutical industry, and cosmetic industries; furthermore, they have antimicrobial action (11). The harmful effects of ZnONPs could be detected through increasing the expression of adhesion molecules in endothelial cells, resulting in inflammation (12, 13). High dose of ZnONPs was used as a new anti diabetic agent (14) but at the same time this higher dose led to release of ROS (15, 16). However, the potential effect of ZnONPs low dose on diabetes has not been elucidated yet. Therefore, the present study was designed to investigate the potential therapeutic efficacy of ZnONPs at low dose, relative to glibenclamide as a standard oral hypoglycemic drug, on diabetic rats with regard to lipogram profile and antioxidant status in addition to the histopatho-logical picture of the pancreas. Material and methods Drugs and chemicals Zinc oxide nanoparticles (ZnONPs) were obtained from the Faculty of Science, Department of Physics, Zagazig University in the form of dispersion. The average nanoparticle size was less than 50 nm as detected by transmission electron microscope (Fig.1). The ZnONPs distribution was detected using dynamic light scattering (DLS) technique, pH 7±0.1 for aqueous systems and density 1.7 g/ml± 0.1 at 25 °C. The standard anti diabetic drug glibenclamide was used under a trade name of Daonil®' (Sanofi Avents Co. for Pharmaceutical Industries, Egypt, 5 mg active ingredient per tablet). Strep-tozotocin (STZ) was purchased from Sigma Chemicals Co., St. Louis, MO, USA. Animals Forty male Wistar rats, 2 months old and average body weight of 150± 20gm, were housed in metal cages at (23± 2°C) with a light-dark (12:12 h) cycle and food and water ad libitum. Animals were kept two weeks before starting the experiment to be accommodated under laboratory condition. The guidelines and ethical rules of Zagazig Veterinary Medicine have been followed. Experimental design Animals were allocated into 4 equal groups (10 animals per group). The 1st group was considered as a control and received only saline solution. Second group was diabetic, non-treated while the 3rd and 4th groups were diabetic rats and orally received 1mg/kg body weight ZnONPs (17) and 600 mg/kg body weight glibenclamide (18) for 30 days using stomach tube. Type 2 diabetes was induced by single intra-peritoneal injection of a freshly prepared solution of streptozotocin (60 mg/kg dissolved in citrate buffer pH 4.5) then after fifteen minutes, nicotinamide (95 mg/ kg) dissolved in saline was intraperitoneally injected (19). Rats were allowed to drink 10% glucose solution over night to overcome drug-induced hypoglycemia. Rats were considered diabetic when their blood glucose reached above 250 mg/dl. The animals were considered diabetic when their blood glucose levels became above 250 mg/dl on the 3rd day after STZ injection. Blood biochemical parameters At the end of the experiment (after 30 days), blood samples were collected from the medial canthus of the eye in either EDTA coated tubes (for determination of blood glucose) or plain tubes (for serum biochemical analysis). Blood glucose values were recorded using commercially available kits following manufacturer's instructions (20). Serum insulin levels were evaluated using commercially available ELISA kit. Serum triglyceride, total cholesterol, High-density-Lipoprotein cholesterol (HDL-c), low density lipoprotein cholesterol (LDL-c), and very low density lipoproteins (VLDL) were determined by the methods described by (21-24). Therapeutic efficacy of zinc oxide nanoparticles in diabetic rats 189 Antioxidant status and oxidative stress assay After euthanization, the spleen was quickly excised, rinsed with saline and tissue homoge-nate was prepared as previously described (25). The obtained supernatant from pancreatic ho-mogenate was used for determination of antiox-idant enzymes activities [glutathione peroxi-dase (GPx), catalase (CAT), and superoxide dismutase (SOD)] and lipid peroxidation (LPO) contents as previously described (26-28). Histopathological examination of pancreas Samples from the pancreas of all groups were collected and fixed in 10% neutral formalin for 24 h prior to routine processing in paraffin wax. Samples were cut into 5 pm sections. Slides were stained with Hematoxylin & Eosin (H&E) and examined microscopically. For morphometric analysis of the Islets diameter, Islets were isolated by a modification of the automated method described by (29). In each animal one slide was stained and 25 images/group were taken using Am Scope 5.0 MP microscope digital camera at 400 X magnification. Islets diameters were measured by using Mitocam® plus 2.0 (Motic Images plus 2.0, china) and Islets cells were counted using Image 1.45 s software (National Institute of Health USA). Data analysis Data were expressed as mean ± standard error (SE). Difference between groups was detected using one way ANOVA followed by Duncan's Multiple Range Test using SPSS version 21. Values at P < 0.05 were considered statically significant. Results Effect ZnONPs and glibenclamide on blood glucose and serum insulin levels Blood glucose and serum insulin levels were significantly elevated in STZ treated animals as compared to the control animals (Table1). Both ZnONPs and glibenclamide treated animals exerted a significant decrease in blood glucose levels as compared to STZ-treated animals. However, among the treated rats, glibenclamide treatment showed better results than ZnONPs. Effect ZnONPs and glibenclamide on lipid profile parameters STZ treated animals exhibited a significant higher serum levels of total cholesterol (TC), triglyceride (TG), LDL-c and VLDL and a significant lower HDL-c level relative to the control group (Table 1). In contrast, ZnONPs and glibenclamide treated groups exerted a significant decrease in TC, TG, LDL-c and VLDL and a marked increase in HDL-c compared to STZ treated animals. Glibenclamide evoked a better result compared to ZnONPs treated rats. Effect ZnONPs and glibenclamide on antiox- idant/oxidative status Antioxidant scavenging potentials for ZnONPs and glibenclamide treated animals were figured out in Table 2. The diabetic group showed a marked decrease in antioxidant enzymes (SOD, GPx, and CAT) activities and a significant increase in lipid peroxidation marker MDA. However, ZnONPs and glibenclamide treated groups exerted a significant increase in antioxidants (SOD, GPx, and CAT) activities and decrease in MDA levels as compared to diabetic animals. Histopathological examination Examined sections revealed normal size, population and structures of the pancreatic cells, normal Langerhans islet components in the control group (Fig., 2A). However, pancreas of diabetic rats showed degenerative changes, cytoplasmic vacuolation, apoptosis and hypo-cellularity of most of P-cells of islets of Langerhans, but alpha cells were normal in most parts (Fig., 2B). Pancreas of rats treated with glibenclamide showed normal histologic appearance with normal size, population and structures with mild congestion of islets capillaries (Fig., 2C). Pancreas of rats treated with ZnONPs showed apparently normal islet cells with preserved size and cellular population, with a few cells either apoptotic or hypertro-phied (Fig. 2D). 190 M. Kamal, M. Khairy, N. ELSadek, M. Hussein Table 1: The Effect of ZnONPs and glibenclamide on lipid profile, blood glucose and insulin level on healthy and diabetic rats Group Total cholesterol (mg/dl) Triglyceride (mg/dl) HDL-c (mg/dl) LDL-c (mg/dl) VLDL (mg/dl) Blood glucose (mg/dl) Insulin (^Iu/ml) Control 195.66± 2.33c 120.33± 3.17d 57.33± 4.48a 137.64± 4.09d 24.06± .64d 101.8± 10.29 c 2.82± 0.067 a Diabetic 295.28± 6.87a 186.85± 2.38a 26.71± 1.64c 284.20± 5.06a 37.37± .48a 335.6± 6.15 a 0.90± 0.045 d Diabetic treated with ZnONPs 241.66± 4.40b 154.66± 2.90b 34.66± 2.60c 214.96± 7.04b 30.93± .34c 231.8± 6.15 b 1.24± 0.035 c Diabetic treated with glibenclamide 226.25± 4.09b 137.50± 1.70c 47.250± 2.3b 181.54± 5.68c 30.93± .58b 221.2± 3.70 c 1.95± 0.054 b Means within the same column carrying different superscripts are significantly different at P<0.05 Table 2: The Effect of ZnONPs and glibenclamide on pancreatic antioxidant/oxidative stress status on healthy and diabetic rats Group CAT MDA SOD GPx (U/gm tissue) ( nmol/gm tissue) (nmol/gm tissue) (U/gm tissue) Control 199.79± 5.681a 5.623± 0.253c 21.040± 1.185a 116.33± 1.789a Diabetic 125.165± 3.200c 22.667± 1.015a 4.468± 0.322d 65.200± 2.279d Diabetic, ZnONPs treated 161.48± 4.015b 15.323± 1.433b 9.656± 1.258c 87.69± 4.614c Diabetic, glibenclamide treated 193.43± 4.768a 8.837± 1.236c 15.017± 0.933b 101.590± 3.398b Means within the same column carrying different superscripts are significantly different at P<0.05 Table 3: Lesion scores of different changes related to islets of Langerhans among experimental groups ö .g 2 _ O U *H us m m "CP O & O o Ö o T3 O O ng-SRZ-D3 V-ll AIFMV1 -ChHJ 3A-Roakln-4l3 -L.-II APMV1^CH/Jiapan/l3hll/G2/L/l - NDV-chicfceniN. ire4ananjister/67 ■ AKM't iol I 69 I wee to ■ AP M V1 -QUM-DE land-V4.-L HI I vectonnune ND strain _I-AFMV1 -aus-tralla ldctorlarVHlll Sé I-APMVI-c.h-Japan-aato-30-V-lll -APiMl V1 Htallen-W-IV -APM V/HertsJ33/ ÍV g-a i NDV/chlckerVMexlco/37S21.'3e/V I-iNDvraam«mnwi/u.s.(CA/V) NDV/nnixedi species/uaAflFL.yLairga/71/v -AFMV/arihlnoaflJ.3.CF«y440S3/33JV -IN OV ,'t u ikey / U SA (N D V4 3D S4 ,'S 2/V Genotype H tO -NDV/chlcken/Egrypti'3^2Q0S NDWclilckerVU-S.íCAyi083íFontaria}/72A/l ñ NDV/ABTW74ÍVI APMV 1 fc hi cken.' Japan.'C-hlba,' B I A/I arm V1 fc nickervjapan/cnitpa/e^rvi 33 . APMV l/chlcken."Japan." lbarafcl.'S5,!VIII -—A APMV l/clilcken/Japan/Tochlgll/OS/VllI I APMV i/píieasaní/JaparVTochigiresrvill i AP MV l/chlc ken.'Japa n'-Chlba-'GB.VI I AP MV l/Japanesebluemagle/Japan/Nara&hlno/SB/V1 APMTV1 /chicken/ Ja pa n/Shltzuofca/SS/VIII A APIJTVI/chicken/Japan/Yamanashl/BS/VIII A P BJiv ii/c h ick en/Japan/saft am &CS SfV III AF W l.*pheasant.*Japan-'Qunma/SSWIII ■ NDV-AH-02-12-Ch fijslon protein gene - CWSD/YQ/2013 fUsfton protein (F) gene - NDV-CWaD758/12 ftjstan protein 0.05) and aspartate aminotransferase (r=-0.361, P<0.01). Concisely, body condition score loss before and after calving may have significant consequences for blood metabolites, oxidative stress and body condition score profile in dairy cows. Key words: BCS; calving; blood metabolites; Friesian cow Introduction The transition period extends from 21 days ante partum to 21 days postpartum and can be described as the alteration from a gestational non-lactating state to a non-gestational lactating stage (1, 2, 3). The body condition score is a reliable, simple way of estimating the nutritional status, body reserves and energy balance in dairy cows. Its evaluation is centered on the outer appearance of cows that interrelates with its body fat reserves and so, influenced by the Received: January 2019 Accepted for publication: February 2019 210 M. E. El-Sharawy, I. M. Mashaly, M. S. Atta, M. Kotb, I. S. El-Shamaa energy balance (4). Lowman and Somerville (5) were the first to use a BCS Scale (4- points), even so, the scale used to measure the BCS (5-, 6-, 8-, and 10- point scales) differs between the countries. But, collectively, the low values always reflect the emaciation while the high value equate to obesity (6). Back fat thickness (BFT) related to other approaches of body condition scoring because its objective and precise. Nearby parturition, the adipose tissue begins to decompose to create fatty acids and glycerol (li-polysis) afford energy to the body. Non-esteri-fied fatty acid (NEFA) in blood is a respectable pointer of adipose tissue mobilization (7). NEFA and P hydroxy butyrate (BHBA) are important energy metabolites that are traditionally used as indicator of negative energy balance (NEB) during transition stage (8). The determination of malonaldehyde (MDA) and activity of superoxide dismutase (SOD) during transition period as an index of oxidative stress status. The aim of the present study was to explore the relationship between body condition score from 60 days' pre- partum until 60 days' postpartum and BCS change, BW, BFT and some metabolic blood parameters in pregnant Frie-sian dairy cows. Materials and methods The experimental protocol was approved by the Animal Care and Use Committee of Faculty of Agriculture, Kafrelsheikh University. Animals A total of 90 animals formed a herd of 350 Friesian dairy cows selected for experiment depend on their body weight (BW) and body condition score (BCS) 0 to 5 point scale according to the North American BCS (9), and divided into three groups (each of 30 cows) on 60th days pre-partum as fairly low (group 1, < 2.58 BCS,<490 kg BW), medium (group 2, < 3.12 BCS, 600 kg) and obesity (group 3, <4.75 BCS, <713 kg BW). The body condition score was performed by one person at -60, -21, -14, -7 pre-partum, at day of calving, at days 7, 14 and 60 post-partum. Animals were housed in a free stall barns, had a free access to water and fed twice daily (8 am and 4 pm) with a ration formulated according to the recommendation of NRC (10) (Tables 1 and 2). Measurement of back fat thickness (BFT) The investigation site is positioned in the sacral region among the caudal quarter and fifth connection line extending from the dorsal portion of the tuber ischia (pins) to the tuber coxae and is evaluated by ultrasound (4) and the data was converted to BCS. Biochemical assessment Blood samples were taken from the jugular vein of all cows on fixed time before morning feeding at -60, -21, -14, -7, pre- partum, at day of calving and at days 7, 14 and 60 post-partum. Serum were stored at -20 °C till essayed for NEFA, BHBA, haptoglobin (HP), and other biochemical analysis. NEFA and BHBA quantified using commercially kits (Randox laboratories Ltd, Crumlin Co., Antrim, UK), Serum Hp measured calorimetrically using a commercial kit (Phase HP kit, Tridelta Ltd., Ireland) according to the constructor's guidelines. MDA and SOD assessed using the commercially kit (Bio-diagnostic, Egypt) according to (11). Albumin, triglycerides, glucose, cholesterol and aspartate aminotransferase assessed spectrophotometrically using autoanalyzer (MCC-3000, China) (Biomed diagnostics Germany). Statistical analysis Mean and standard error were calculated. The obtained data were subjected to two-way factorial analysis of variance according to the procedures out lined by Snedecor and Cochran (12). The mean value of treatments was compared according to Duncan's multiple range test (DMRT) (13). Multiple correlation coefficient analyses were used. The data was analyzed using CoStat software for windows (version 6.3). Results Change of BCS, BW and BFT from pre- to post- partum in dairy cows Fluctuations of BCS in each group were presented in figure l. The obesity BCS cows Influence of body condition score on blood metabolites and oxidative stress in pre- and post-calving of ... 211 showed significantly higher BCS reduction (1.17) 60 d pre-partum to 60 d post-partum than thinner (-0.5) and medium BCS cows (-0.67). Changes of BW and BFT were statistically significant (P<0.05) among the 3 groups (Fig. 2 and 3). The 3rd group showed higher BW and BFT reduction from d 60 before calving to d 60 after calving (106 kg and 1.14, respectively) compared to thinner (70 kg and 0.28, respectively) and medium groups (103 kg and 0.68, respectively). During the experimental time, highly significant positive correlations were found among reduction of BCS and both of BW (r=0.86, P<0.01) and BFT (r=0.98, P<0.01). Thus, cows with greater BCS losses had also higher BW and BFT losses (Table 3). Metabolic parameters Serum concentrations of BHBA, NEFA and HP in high BCS cows were significantly (P<0.05) higher than the other two groups before and after calving (Figs, 4-6). The differences in HP among groups were not significant. The concentrations of BHBA and NEFA increased from -7d before calving and reached the high levels on 7d post- calving and then decreased with high values as compared to those observed before parturition. Oxidant and antioxidant status SOD gradually increased (P<0.05) in the last 21 days of pregnancy (at 21 d before calving) and reached the maximum at 7d earlier calving. After parturition, SOD dropped to reach the levels recorded before calving (Fig. 7). Serum concentration of MDA was relatively steady before calving (P>0.05) at parturition and one week after calving (Fig. 8). The differences among groups were highly significant (P<0.01). Serum biochemical parameters The concentrations of serum glucose were gradually increased from -60 d before calving and reached the highest values at calving and then dropped sharply at 7d post-calving and back increased to the levels registered 60 d before calving (Fig. 9). Serum triglyceride was not significantly differed among groups (Fig. 10). The serum cholesterol started to increase from -60 d before calving and reached the higher values on -7d before calving and then decreased to reach the low values on 60 d post-calving (Fig. 11), while the triglyceride concentration peaked at calving and dropped 7d and 14 d post-calving and then increased 60 d post-calving. Mean serum albumin content dropped to the lowest levels at calving followed by a subsequent increase to highest levels (P<0.05) at 7 d post-calving then dropped to levels 60d pre-calving and followed by an increase at 60 d post-calving. The differences among groups did not significantly differed (Fig. 12). AST concentrations steady increased (P<0.05) from 60 d before calving to parturition and followed by a subsequent increase at one wk., 2 wk and 2 months post-calving and reached maximum level (P<0.05) at 60 d post- calving (Fig. 13). Correlation study BCS, BW and BFT were negatively (P<0.01) correlated with both BHBA (r=-0.416, r=-0.355, r=-0.361, respectively), NEFA (r=-0.457, r=-0.470 and r=-0.448, respectively), and AST (r = -0.361, r = -0.407 and r = -0.341, respectively) and were definitely (P<0.01) linked with cholesterol (r=0.342, r=0.423 and r=0.323, respectively), (Table 3). 212 M. E. El-Sharawy, I. M. Mashaly, M. S. Atta, M. Kotb, I. S. El-Shamaa Table 1 : Chemical composition of ingredients rations during the pre- and the post- partum period Item Composition of DM% Ingredients DM% OM CP CF EE NFE Ash CFM 89.71 91.58 15.62 9.63 2.92 63.41 8.42 Corn silage 35.10 94.89 9.39 23.18 2.19 60.13 5.11 Hay 89.89 92.23 14.29 27.5 3.12 47.32 7.77 Rice straw 91.08 83.58 2.98 34.89 1.36 44.39 16.42 Rations Pre-partum 78.97 91.58 12.84 18.44 2.66 57.64 8.42 Close up 76.24 91.74 12.54 19.12 2.62 57.46 8.26 Post- partum 73.5 91.91 12.23 19.79 2.59 57.30 8.09 DM (dry matter); OM (organic matter); CP (crude protein); CF (crude fiber); EE (ether extract); NFE ( Nitrogen free extract); Ash (Ash) and CFM (concentrate feed mixture) Table 2: Average daily feed intake (Kg/head/day) during pre- and post-partum period Item Pre- Partum Close up Post- Partum CFM (concentrate feed 9.5 8.5 7.6 mixture) Corn silage 9.7 12.1 14.5 Hay 3.8 3.8 3.8 Rice straw 1.9 1.9 1.9 Table 3: Correlation coefficient between body condition score, body weight and back fat thickness with metabolic blood parameters and oxidative stress Items BCS BW BFT Body Condition Score 1 Body Weight 0.860(**) 1 Back Fat Thickness 0.977(**) 0.884(**) 1 ß hydroxy butyrate -0.416(**) -0.355(**) -0.361(**) Non-esterified fatty acid -0.457(**) -0.470(**) -0.448(**) Haptoglobin -0.232(*) -0.215(*) -0.184 Superoxide dismutase 0.123 -0.019 0.054 Malonaldehyde 0.445(**) 0.383(**) 0.465(**) Blood Serum Glucose 0.133 0.208 0.146 Blood Serum Triglyceride 0.040 0.017 -0.009 Blood Serum Albumin -0.133 -0.145 -0.137 Aspartate aminotransferase -0.361(**) -0.407(**) -0.341(**) Blood Serum Cholesterol 0.342(**) 0.423(**) 0.323(**) BCS (body condition score), BW (body weight), BFT (back fat thickness) **Correlation is significant at the 0.01 level (1-tailed) * Correlation is significant at the 0.05 level (1-tailed) Influence of body condition score on blood metabolites and oxidative stress in pre- and post-calving of ... 213 Figures 1-8: Effect of body condition score during pre- and post- calving on body condition score changes (BCS); body weight changes (BW); back fat thickness changes (BFT); P hydroxy butyrate concentrations (BHBA); non-esterified fatty acid (NEFA); haptoglobin (HP); superoxide dismutase concentrations (SOD) and malonaldehyde concentrations (MD), (Means ± SEM) 214 M. E. El-Sharawy, I. M. Mashaly, M. S. Atta, M. Kotb, I. S. El-Shamaa -21 -7 0 7 Dav& Pre and Fast Calving •LBCS MBCS —O— HBCS Aspartate aminotransferase (AST) 63 61 59 57 rJ 55 ^ 53 51 ■19 ■17 ■15 -21-7 0 7 Days Pre and Post Calving -LBCS MBCS —O- Figures 9-13: Effect of body condition score during pre- and post- calving on concentrations of blood serum glucose (G); triglycerides (TG); cholesterol (CL); albumin (AL) and aspartate aminotransferase (AST), (Means ± SEM) Discussion Cows with high body condition score showed higher (P<0.05) BCS loss pre and post-calving period than medium and thinner BCS cows. Greater loss of BCS in high BCS cows was expected and has been reported previously by Treacher et al. (14), who concluded that transition cows with great BCS waste more body weight and body condition than thinner one. In the present study, cows with high BCS had the greatest decline in BW and BFT when compared with the other groups, which is an indication of mobilizing greater body fat reserves than medium and thinner BCS cows. These results are in agreement with previous reports (6, 15, 16). In the present study, cows with high BCS had a sharp increase in plasma NEFA and BHBA concentration, which reached the highest values at 1 week after calving, while the lowest levels were found in LBCS cows. The highest plasma NEFA and BHBA concentrations for cows with high BCS were in accordance with other studies (17) and could be explained by the high fat mobilization. Results of the present study indicated that serum glucose increased in all groups reaching the peak at parturition then a sharp decrease 7 d post-calving, which may be attributed to cortisol and estrogen (17) or inherent hypoinsuline-mia which reduce glucose uptake by tissue sensitive to insulin (18). On the other hand, Garcia et al. (19) found that low glucose level was during calving and explained these results due to sever NEB suffered by the animals in this group that delivered high milk during the experimental time. Moreover, others found that glu- Influence of body condition score on blood metabolites and oxidative stress in pre- and post-calving of ... 215 cose concentration remained stable and increase slightly at calving reflecting an increase in gluconeogenesis in response to calving stress (20, 21). Cheng et al. (22) reported that cows in negative EB have low rates of glucose and high levels of BHBA. In the present study, triglycerides were relatively stable at range from 43.6 to 46.9 mg/dl and no significant differences were noticed among groups. This finding is consistent with previous report by García et al. (19) and González et al. (23) who attributed this to the excess of fatty acids mobilized to liver to be as energy resource. The reasons for such case are triglycerides accumulation in the liver and triglycerides taking up by the mammary gland for milk fat synthesis and secretion (24). Reduced cholesterol concentrations starting from the time at calving and post-calving weeks were found in all groups, which could be associated with reduced DM intake (25, 26). Conversely, Mouf-fok et al. (20) found that cholesterol was significantly elevated in post-partum which indicates good energy nutrition (27). Total cholesterol was significantly lower on week around parturition with the lowest value one week prior to calving (28, 29). Alternatively, García et al. (19) described that cholesterol level had a gradual upsurge as the lactation advanced. Reduced albumin concentration reaching the lowest values at the time of parturition in all groups and increase to the highest values one-week post calving. Our results are in disagreement with findings of Soca et al. (26), who found that albumin concentrations decreased after calving in low and moderate BCS cows and this may be associated with increasing of NEFA. Our results were in agreement with finding of Gheise et al. (16), who noted that the highest albumin levels at 1 d after calving. Albumin is synthesized via the liver and it's the core source of plasma thiol groups which are considered as an element of extracellular antioxidant defense against oxidative stress (30). In the present study, the activity of aspartate amino transferase (AST) was not significantly changed by BCS on the calving day in all experimental groups and increased gradually at 7, 14 and 60 d post-calving. No significant different was found among groups pre and post-calving. The higher level of AST in dairy cattle are allied with the fatty liver syndrome, lower dry matter consumption and ketosis marks (32). Haptoglobin (HP) concentration in the present study was significantly higher at calving day and post-calving days than before parturition. The present finding was in agreement with the finding of others (31, 32), who found that the highest value of HP was recorded in cows 3wk postpartum. This finding supported the previous results of Montagner et el. (33) who found the trend for higher HP concentrations in cows' pre-partum and the enhanced concentration in the post-partum were consistent with impaired hepatic function. Highest MDA concentration was recorded on the day of calving in all treated groups, then continuously decreased after calving on 7, 14 and 60 d post-calving that was in agreement with the finding of others (34). Castillo et al. (35) reported that MDA is the last product of lipid peroxidation, therefore changes of MDA concentrations can be used as a biomarker of oxidative stress. SOD enzyme is the major antioxidant defense competent in protecting the cells against increased ROS (36). The increase of SOD around calving (-7 d to +7 d pre and post- calving) because of a possible homeo-static control (37). Conclusion This study showed that lost body condition in 8 wks before calving had an adverse metabolic status, with increased serum concentrations of NEFA, BHBA, MDA and AST post calving. In addition, high BCS cows (obesity, G3) had highest BHBA profiles that suggested they had a higher risk of subclinical ketosis, in addition, highest serum concentrations of NEFA in all cows after calving, indicated the highest mobilization of body reserves after calving. Conflict of interest The authors state no conflicts of interest. 216 M. E. El-Sharawy, I. M. Mashaly, M. S. Atta, M. Kotb, I. S. El-Shamaa Acknowledgements Honest appreciation to Sakha agricultural research station, Kafrelsheikh Governate, for they kind and helpfulness during this study. References 1. Contreras GA, Sordillo LM. Lipid mobilization and inflammatory responses during the transition period of dairy cows. Comp Immunol Micro-biol Infect Dis. 2011;34(3):281-9. 2. Van Saun RJ. Indicators of dairy cow transition risks: Metabolic profiling revisited. Tierarztl Prax Ausg G Grosstiere Nutztiere. 2016;44(2): 11826. 3. Drackley JK. Biology of dairy cows during the transition period: The final frontier? Journal of Dairy Science. 1999;82(11):2259-73. 4. Schröder UJ, Staufenbiel R. Invited review: Methods to determine body fat reserves in the dairy cow with special regard to ultrasonographic measurement of backfat thickness. Journal of Dairy Science. 2006;89(1): 1—14. 5. Mulvany P. 6.5 Dairy cow condition scoring. BSAP Occasional Publication. 1981; 4:349—53. 6. Roche JR Friggens NC, Kay JK, Fisher MW, Stafford KJ, Berry DP. Invited review: Body condition score and its association with dairy cow productivity, health, and welfare. Journal of Dairy Science. 2009;92(12):5769—801. 7. Bell AW. Regulation of organic nutrient metabolism during transition from late pregnancy to early lactation. J Anim Sci. 1995;73(9):2804—19. 8. Duffield TF, Lissemore KD, McBride BW, Leslie KE. Impact of hyperketonemia in early lactation dairy cows on health and production. J Dairy Sci. 2009;92(2):571—80. 9. Edmonson A, Lean I, Weaver L, Farver T, Webster G. A body condition scoring chart for Holstein dairy cows. Journal of Dairy Science. 1989;72(1):68—78. 10. NRC. Nutrient Requirements of Dairy Cattle: Seventh Revised Edition. The National Academies. Press, Washington, DC. 2001;92(12):5770—95. 11. Ohkawa H, Ohishi N, Yagi K. Assay for lipid peroxides in animal tissues by thiobarbituric acid reaction. Analytical Biochemistry. 1979;95(2):351—8. 12. Snedecor GW, Cochran WG. Statistical methods, 8thEdn. Ames: Iowa State Univ Press Iowa. 1989; 6(4):329-35. 13. Duncan DB. Multiple range and multiple F tests. Biometrics. 1955;11(1): 1—42. 14. Treacher R, Reid I, Roberts C. Effect of body condition at calving on the health and performance of dairy cows. Animal Science. 1986;43( 1): 1—6. 15. Pires J, Delavaud C, Faulconnier Y, Pomies D, Chilliard Y. Effects of body condition score at calving on indicators of fat and protein mobilization of periparturient Holstein-Friesian cows. Journal of Dairy Science. 2013;96(10):6423-39. 16. Jamali Emam Gheise N, Riasi A, Zare Shah-neh A, Celi P, Ghoreishi SM. Effect of pre-calving body condition score and previous lactation on BCS change, blood metabolites, oxidative stress and milk production in Holstein dairy cows. Italian Journal of Animal Science. 2017;16(3):474-83. 17. Weber C, Hametner C, Tuchscherer A, Los-and B, Kanitz E, Otten W, et al. Variation in fat mobilization during early lactation differently affects feed intake, body condition, and lipid and glucose metabolism in high-yielding dairy cows. Journal of Dairy Science. 2013;96(1):165-80. 18. Bauman D. Regulation of nutrient partitioning during lactation: homeostasis and homeorhesis revisited. Ruminant physiology: digestion, metabolism, growth and reproduction. 2000; 1:311-28. 19. García A, Cardoso FC, Campos R Thedy DX, González FH. Metabolic evaluation of dairy cows submitted to three different strategies to decrease the effects of negative energy balance in early postpartum. Pesquisa Veterinária Brasileira. 2011;31:11-7. 20. Mouffok C-E, Madani T, Semara L, Ayache N, Rahal A. Correlation between body condition score, blood biochemical metabolites, milk yield and quality in Algerian Montbéliarde cattle. Pakistan Vet J. 2013;33(33): 191-4. 21. Kaewlamun W, Okouyi M, Humblot P, Remy D, Techakumphu M, Duvaux-Ponter C, et al. Effects of a dietary supplement of B-carotene given during the dry period on milk production and circulating hormones and metabolites in dairy cows. Revue Méd Vét. 2012;163(5):235-41. 22. Cheng X, Zhe W, Li Y-F, Niu S-L, Chuang X, Zhang C, et al. Effect of hypoglycemia on performances, metabolites, and hormones in periparturient dairy cows. Agricultural Sciences in China. 2007;6(4):505-12. 23. González F, Muiño R, Pereira V, Campos R, Castellote J. Blood indicators of lipomobilization and hepatic function in high yielding dairy cows during early lactation. Ciencia Animal Brasileira. 2009;10(Supplement 1):64-9. 24. Bernard L, Leroux C, Chilliard Y. Expression and nutritional regulation of lipogenic genes in the Influence of body condition score on blood metabolites and oxidative stress in pre- and post-calving of ... 217 ruminant lactating mammary gland. Bioactive components of milk: Springer; 2008. p. 67-108. 25. Remppis S, Steingass H, Gruber L, Schenkel H. Effects of energy intake on performance, mobilization and retention of body tissue, and metabolic parameters in dairy cows with special regard to effects of pre-partum nutrition on lactation-a review. Asian-Australasian Journal of Animal Sciences. 2011;24(4):540-72. 26. Soca P, Carriquiry M, Claramunt M, Gestido V, Meikle A. Metabolic and endocrine profiles of primiparous beef cows grazing native grassland. 1. Relationships between body condition score at calving and metabolic profiles during the transition period. Animal Production Science. 2014;54(7):856-61 . 27. Civelek T, Aydin I, Cingi CC, Yilmaz O, Kabu M. Serum non-esterified fatty acids and beta-hydroxybutyrate in dairy cows with retained placenta. Pakistan Veterinary Journal. 2011;31(4): 341-4. 28. Ling K, Jaakson H, Samarutel J, Leesmae A. Metabolic status and body condition score of Estonian Holstein cows and their relation to some fertility parameters. Veterinarija ir zootechnika. 2003;26:24. 29. Turk R, Podpecan O, Mrkun J, Kosec M, Fle-gar-Mestric Z, Perkov S, et al. Lipid mobilisation and oxidative stress as metabolic adaptation processes in dairy heifers during transition period. Animal reproduction science. 2013;141(3 -4): 109-15. 30. Celi P, Gabai G. Oxidant/antioxidant balance in animal nutrition and health: the role of protein oxidation. Frontiers in Veterinary Science. 2015;2:48. 31. Gymnich S, Knura-Deszczka S, Wimmers K, Bidlingmaier M, Schellander K, Petersen B. Hapto-globin as an indicator for animal welfare: Effects of different hygienic conditions and transport stress on haptoglobin plasma concentration. Acta Veterinaria Scandinavica. 2003;44(1):P33. 32. Vargova M, Hromada R Veszelits Lakticova K, Posivakova T, Benculak J, Kovac G. Correlations between acute phase proteins and the body condition score. Acta Veterinaria Brno. 2018;86(4):339-44. 33. Montagner P, Tavares Krause AR, Schwegler E, Menoncin Weschenfelder M, Stein Maffi A, Gu-larte Xavier E, et al. Relationship between pre-partum body condition score changes, acute phase proteins and energy metabolism markers during the peripartum period in dairy cows. Italian Journal of Animal Science. 2017;16(2):329-36. 34. Colakoglu HE, Yazlik MO, Kaya U, Colakoglu EC, Kurt S, Oz B, et al. MDA and GSH-Px activity in transition dairy cows under seasonal variations and their relationship with reproductive performance. Journal of Veterinary Research. 2017;61(4):497-502. 35. Castillo C, Hernandez J, Bravo A, Lopez-Alonso M, Pereira V, Benedito J. Oxidative status during late pregnancy and early lactation in dairy cows. The Veterinary Journal. 2005;169(2):286-92. 36. Abd Ellah MR. Oxidant and antioxidants during the transition period in dairy cows. Journal of Advanced Veterinary Research. 2016;6(4):130-3. 37. Bernabucci U, Ronchi B, Lacetera N, Nar-done A. Influence of body condition score on relationships between metabolic status and oxidative stress in periparturient dairy cows. Journal of Dairy Science. 2005;88(6):2017-26. Slov Vet Res 2019; 56 (Suppl 22): 219-28 DOI 10.26873/SVR-760-2019 Original Research Article EFFECTS OF DIET'S ACIDIFYING ADDITIVES ON THE INTESTINAL HISTOMORPHOLOGY IN DUCKS Ahmed Abdelfattah-Hassan*, Hanaa M. El-Ghazali Department of Anatomy and Embryology, Faculty of Veterinary Medicine, Zagazig University, 44519, Egypt Corresponding author, E-mail: aabdelfattah@vet.zu.edu.eg Abstract: This study aimed at evaluating the effect of acidifying additives as formic and propionic acids on weight gain and on small intestinal macroscopic, microscopic and SEM morphology in ducks. A total of 30 Mulard ducklings were used in this study and they were randomly assigned to control (given standard commercial duck diets with no acidi-fiers added), 1 % acidifiers added to the standard diet or 2% acidifiers added to the standard diet. After rearing the ducks for 52 days, their weight was recorded and then the length of the different parts of the small intestine was measured. Then, samples were taken for histological and scanning electron microscopical (SEM) examination of the small intestine. The present results found that addition of acidifying mixture to the diet significantly improved duck's weight (P<0.05), also there were significant changes in length, histology and SEM characteristics of the small intestine between different duck groups. The observed effects were dose dependent, i.e. with more organic acids in the diet, the more the changes were noticeable. Our results indicate that, the addition of organic acids to duck's diet increased small intestine's length and increased the length of intestinal villi, possibly resulting in more surface area for digestion and absorption of the diet, and, therefore, greatly improved the final body weight of the study ducks. Key words: acidified diets; organic acids; histology; intestinal villi; SEM Introduction Poultry meat is a good source of proteins and other valuable nutrients for human consumption, and it is considered an essential supply to meet the growing human population. The unsupervised use of antibiotics in poultry industry is still a serious problem and it poses great risks to human health; when improperly used as growth promotors. Antibiotics use as growth promotors is illegal now in Europe, since the European Union has banned their use in 2006. Other alternatives are currently still being studied in poultry industry and one modern feeding strategy is changing the pH of the diet through addition of acidifying substances; as organic acids. These acids will slightly change the pH of the intestine, and also has direct antibacterial effects (1-3). So, it will reduce the intestinal bacterial load, improve production and eliminate the need for antibiotics use as growth promotors. Several commercial mixtures of organic acids and inorganic acids are currently in Received: January 2019 Accepted for publication: February 2019 220 A. Abdelfattah-Hassan, H. M. El-Ghazali the market in the European Union as better replacements to growth promoting antibiotics. Being acids, organic acids improve the digestibility of dietary proteins and amino acids in the stomach, by enhancing the digestion of proteins (4). In addition, they improve the utilization of feed and result in better feed conversion and growth rates. Moreover, an additional advantage of organic acids is their use in the preservation of broiler feed stuff (5). Therefore, the addition of organic acids to the feed ingredients preserves its properties for longer time, result in better growth in reared poultry and increase the profitability for poultry producers. In fact, the addition of acidifying substances was described previously in poultry; to control infections such as campylobacter or to improve the weight gain of reared poultry. Several acids were used, such as, lactic acid in the drinking water (1), organic acids in drinking water (6), formic acid in feed (7) along with other additives reviewed in (8). Their use resulted in improved weight gain in poultry, because these organic acids enhance diet's protein and energy use and reduce microbial competition for these nutrients in bird's intestine (reviewed in (3)). Moreover, the addition of these acidifying substances was not harmful and did not cause damage in the intestinal epithelium, on the contrary it increased chicken-flock's biosecurity and prevented infections that could be harmful to human consumers (6, 9). In humans, dietary additives or high fat diets were also described to change intestinal adaptation and enhanced the growth of the intestinal villi after removal of parts of the bowl in resection operations (10), and it could be a useful strategy in treating humans with short gut syndrome (11-13). The possible macroscopic and microscopic changes to the intestine and intestinal villi upon feeding acidified diets has not been studied in detail in ducks. Therefore, the present study aimed at studying the small intestine's morphology, histology and scanning electron microscopy in response to feeding acidified diets (through addition of organic acids to the standard diet) in ducks. Material and methods All procedures in the study were following animal care and use committee of the Faculty of Veterinary Medicine, Zagazig University. Birds and study design Mulard Ducklings (n = 30); a sterile breed of domestic ducks raised for meat production and is a hybrid between male Muscovy (Cairina moschata) and female Pekin (Anas platyrhyn-chos domestica) ducks, were purchased from a commercial duck hatchery. Upon arrival, the ducklings were weighed and randomly assigned to one of three groups (each group contained 10 ducklings) and reared for 52 days in Bird Nutrition Research Unit (Nutrition and Clinical Nutrition Department, Faculty of Veterinary Medicine, Zagazig University). Ducklings feeding program consisted of standard starter diet (up to 21 days) and finisher diet (22-52 days). A commercial acidifier mixture was purchased, and it contained mainly formic acid and propionic acid. The three study groups received no (Control), 1% acidifier mixture in diet (1AM) or 2% acidifier mixture in diet (2AM). No forced feeding was done in this study and ducks had free access to water. After completing the rearing period, all ducks were weighed and then slaughtered, and samples were taken immediately. Gross anatomical examination Directly after slaughtering the study ducks, the intestine was removed and visually inspected for any apparent differences, then carefully straightened so the length of each small intestine segment (duodenum, jejunum and il-eum) was easily measured. Duodenum was defined starting from its beginning after the gizzard (ventriculus) till the beginning of the mesojejunum (the jejunal mesentery), the jejunum was measured from the end of the duodenum till the end of the mesojejunum, while the ileum was measured from the end of the mesojejunum till the junction between the ileum and the two ceca. Also, the length of the two ceca (part of the large intestine) was recorded. Effects of diet's acidifying additives on the intestinal histomorphology in ducks 221 Histological examination After performing the anatomical examination, samples from the middle of each part of the small intestine was taken, emptied from its contents by flushing with warm physiological saline solution and, then, immediately fixed in freshly prepared 10% neutral buffered formalin solution for at least 48 hours. Care was taken to obtain the samples from the same region in all study ducks. Afterwards, standard histological procedure was performed (dehydration in ascending grades of alcohol, clearing in xylene and embedding in paraffin wax). Finally, 5 pm sections were obtained, mounted, stained with Hematoxylin and Eosin stain (H&E) and examined under light microscopy. The general structure (mucosa, submucosa and muscular layer) of each part of the small intestine was examined and the height (including the crypt of Lieberkühn) and width of the intestinal villi, and the thickness of the muscularis mucosa were measured using S-view computer software provided with the microscope camera (EHC10, S-VIEW Technology Co., Ltd, Zhejiang, China). Scanning Electron Microscopy For the Scanning Electron Microscopy (SEM), a two cm long intestine sample (from the part next to the samples obtained for histo-logical examination) was cut open longitudinally, the cut was made very carefully not to touch the intestinal mucosa, and the mucosa was washed again with warm phosphate buffered saline. The SEM procedure followed previously published protocols for birds (14, 15). Briefly, the samples were immersed in 3% glu-taraldehyde (in 0.1M cacodylate buffer, pH 7.4) at room temperature and transported to the SEM unit (Al-Azhar University, Cairo, Egypt). Then, the samples were cut into smaller pieces (approx. 5 mm2) with sharp razor blades and fixed for an additional one hour in the same buffer. Then, the pieces were washed with 0.1M sodium cacodylate buffer (pH 7.4) and post-fixed with 1% osmium tetroxide (in ice-cold 0.1M sodium cacodylate buffer, pH 7.4) for two hours. Afterwards, the samples were washed with pure distilled water and dehydrated in ascending concentration of ethanol (50% to 100%, 2 times in each concentration for at least 20 min each). following this, the samples were critical point dried using liquid carbon dioxide (Autosamdri®-815, Tousimis, Maryland, USA), mounted on aluminum stubs with electrically conducting carbon mounting tabs and finally sputter-coated (SPI-Module™ Sputter Coater, Pennsylvania, USA). The arrangement of the villi and their surface epithelium were observed using JOEL scanning electron microscope (JSM-5500LV, JEOL Ltd., Tokyo, Japan) at 17-20 kV accelerating voltage. Statistical analyses The data collected was evaluated using ANOVA procedure, post hoc comparisons were applied, whenever appropriate, using Tukey's HSD test. All statistical procedures were performed using PASW statistics v.18 (SPSS Inc., USA). Statistical significance was considered at P < 0.05. Results Effects of acidifying mixtures on duck's final body weight The addition of acidifying mixture to duck's diet (starter and finisher), greatly improved the final body weight of the ducks during the 52 days study period (P<0.05, Table 1). With highest duck's weight obtained in the group supplied with 2% organic acids, followed by 1% organic acids, and the least weight belonged to duck's that did not have organic acids in their diets. Gross anatomical findings After measuring the length of the different parts of the small intestine (duodenum, jejunum and ileum), the addition of acidifying mixtures (1% or 2%) resulted in longer duodenum, jejunum, ileum and ceca compared to the control group with no additives (P<0.05, Table 1). In addition, only in the ileum, the 2% acids mixture resulted in significant longer intestinal length than in the 1% group (P<0.05). 222 A. Abdelfattah-Hassan, H. M. El-Ghazali Histological findings The addition of organic acids to the duck's diet resulted in significantly different length of duodenal, jejunal and ileal villi (P<0.05, Table 1). The shape and arrangement of the intestinal villi in duodenum, jejunum and ileum were also microscopically changed at 40X (Plate 1). At higher magnifications (100X, plate 1, and 400X, not shown) there were no observable difference in the lamina propria or the Crypts of Lieberkuhn with H&E stain. The addition of organic acids to the diet increased villus length (P<0.05) and the villi were less wide than in ducks fed no organic acids in their diets. The longest and thinnest duodenal villi were observed in the 1% group, while the longest and thinnest jejunal and ileal villi were observed in the 2% group (Table 1). The intervillous space was similar in duodenum in all groups, and it was wider in the 1% and 2% groups compared to the control group with no additives in their diet (Plate 1, Figs. H, K, I, L, N, Q, O and R). Table 1: Showing the final body weight, length of different parts of the small intestine (including villus length, width and muscularis thickness) and length of the ceca of studied Mulard ducks given no (control), 1% organic acids or 2% organic acids in their diets Control 1% organic acids in diet 2% organic acids in diet Final body weight (gm) 2407±96.21 c 2690±84.23 b 2895±93.39 a Duodenum length (cm) 29±2.58 b 33.33±2.17 a 34.67±2.26 a Jejunum length (cm) 98.33±5.89 b 112.33±6.38 a 116.33±8.71 a Ileum length (cm) 22.33±3.20 c 26.67±4.36 b 30.33±5.21 a Cecum length (cm) 9.67±1.45 b 12.67±1.33 a 13.33±1.67 a Duodenum villus height (^m) 1184.33 ±98.52 b 1613.00 ±131.55 a 1579.75 ±82.45 a Jejunum villus height (^m) 883.00 ±84.21 c 863.25 ±110.98 b 901.00 ±174.27 a Ileum villus height (^m) 393.00 ±40.05 c 671.88 ±107.03 b 797.43 ±97.03 a Duodenum villus width (^m) 145.00 ±15.89 a 116.89 ±39.68 c 134.83 ±16.52 b Jejunum villus width (^m) 225.44 ±49.02 a 206.00 ±38.05 b 171.67 ±16.50 c Ileum villus width (^m) 176.00 ±18.69 a 162.00 ±16.81 b 143.71 ±21.41 c Duodenum muscularis thickness (^m) 469.67 ±91.31 a 419.80 ±32.91 b 373.83 ±75.77 c Jejunum muscularis thickness (^m) 319.50 ±58.52 b 347.67 ±54.38 a 364.00 ±46.25 a Ileum muscularis thickness (^m) 233.50 ±25.32 b 254.33 ±28.08 a 258.33 ±39.55 a Different superscript letters denote statistical significance (P<0.05). Values shown as mean ± standard deviation Effects of diet's acidifying additives on the intestinal histomorphology in ducks 223 Scanning Electron Microscopical findings Results of the scanning electron microscopy showed the differences in shape and arrangement of the villi in different parts of the duck's small intestine in response to acidified diets (shown in Plates 2, 3, 4). Compared to the control group the duodenal villi were thinner, more abundant and interwoven between each other in the 1% group (Plate 2, Fig. D). While, in the 2% group they appeared thin, taller and less abundant (Plate 2, Fig. G). At higher magnifications (800X and 1500X) there were no obvious differences in the surface at the tip or sides of the duodenal villi between the study groups (Plate 2, Figs. B, C, E, F, H, I). In case of the jejunum, differences were more difficult to be noticed with the SEM (Plate 3). Only the sides of jeju-nal villi in the 2% group showed corrugations (Plate 3, Fig. H), these were less prominent in the 1% group and difficult to be observed in the control group. Differences in the ileum, between groups, were more pronounced than in the duodenum or jejunum. The 2% group showed more numerous ileal villi which are thinner and intermingled (Plate 4, Fig. G) with fairly corrugated sides at higher magnification (Plate 4, Fig. H). Whereas in the 1% group the ileal villi appeared less numerous than the 2% group and showed corrugated sides at higher magnification (Plate 4, Figs. D & E). At higher magnification (>1500X) the surface of the ileal villi in different groups showed no obvious differences (Plate 4, Figs. C, F, I). Plate 1 : Photomicrographs A-R, showing the H&E sections of duodenum, jejunum and ileum in the study groups given no (Control), 1% organic acids (1% OA) or 2% organic acids (2% OA) in their diet for 52 days. Different length and arrangement of intestinal villi were noticed in response to the organic acids' administration is evident at 40X and 100X magnification 224 A. Abdelfattah-Hassan, H. M. El-Ghazali Discussion Knowledge on duck's intestinal macro and microscopic anatomy is much less than the chicken in the literature, so far it has been given less attention. The histochemical developmental changes of the small intestine in ducks was described for the first time in 2011 (16). We here showed normal macroscopic and microscopic anatomy of the adult duck's small intestine, and possible changes that could arise from using modern feed additives; such as organic acids. The present results showed that with changing the acidity of the diet, by incorporation of organic acids, the ducks weight improved (Table 1) while their feed intake was not changed significantly (data not shown). Similar improvements were detected in Aigamo ducks fed bamboo charcoal with vinegar (17) or natural zeolite including plant extract (18), in chicken fed organic acids (4) and pigs fed organic acids, reviewed in (19). Whilst, some studies in broiler chickens didn't find changes in final body weight; mainly because feed intake was not calculated and no normalization of bird's weight to its feed intake was done (6) or due to the use of single organic acid at a much lower concentration in the diet (7). No damage was observed in the epithelium of the villi by SEM, nor visual differences was found, this underlines the safety of using up to 2% organic acids in the diet of ducks without causing any adverse effects. In chicken, organic acids use was also found to be safe on the intestine and did not cause damage to the villi (6). Plate 2: Photomicrographs A-I, showing the SEM scans of duodenum in the study groups given no (Control), 1% organic acids (1% OA) or 2% organic acids (2% OA) in their diet for 52 days. Different arrangement of intestinal villi was noticed in response to the organic acids' administration were noticed Effects of diet's acidifying additives on the intestinal histomorphology in ducks 225 Plate 3: Photomicrographs A-I, showing the SEM scans of jejunum in the study groups given no (Control), 1% organic acids (1% OA) or 2% organic acids (2% OA) in their diet for 52 days. Different arrangement of intestinal villi was noticed in response to the organic acids' administration were noticed Plate 4: Photomicrographs A-I, showing the SEM scans of ileum in the study groups given no (Control), 1% organic acids (1% OA) or 2% organic acids (2% OA) in their diet for 52 days. Different arrangement of intestinal villi was noticed in response to the organic acids' administration were noticed 226 A. Abdelfattah-Hassan, H. M. El-Ghazali The length of the intestine in ducks, and poultry in general, is directly proportional to the absorption and utilization of digested feed (20). Consequently, our results showed that by adding organic acids in duck's diet, longer intestine with longer villi were obtained which resulted in substantially increasing the surface area of absorption, and therefore, increased feed utilization with achieving higher final body weight. Longer intestinal villi with organic acids administration were also recently reported in broiler chickens (4). The ileum in poultry; ducks is no exception, has a characteristic ileal digestion (21), it was described to increase with addition of a single organic acid to the diet (7). The current results showed longer ileum and ileal villi with more organic acids in the diet, which implies that the utilization of feed is at its maximum, and this was reflected on the weight of reared ducks; as observed in our 2% organic acids group compared to the other groups. In addition, a higher dose of organic acids ensures that their favorable effects reach to the jejunum and ileum, thus guaranteeing maximum benefit from its actions throughout most of the small intestine; similar observations were previously reported in pigs (19). From our results, the 2% group ducks had the highest final body weight and this group had longer duodenum, jejunum and ileum, also with longer villi, than the remaining groups. Although, it is difficult in this study to clearly determine whether the body weight was increased due to longer intestinal parts or the longer intestinal parts are a consequence to higher body weight, either ways the result is higher duck's weight and thus profitability in duck farming. In the 1% group these findings were also present but to a lower extent compared to the 2% group. This also highlights that the observed weight improvement and histomorphological changes in this study are dose dependent. The arrangement of the villi was also different between groups with addition of organic acids to the diet. In the duodenum the villi became interwoven or zigzag-like and it was difficult to obtain a single straight villus in the H&E section (Plate 1, Figs. B & E) this was very clear with the 1% group and less clear in the 2% group compared to control ducks. While in the SEM the 2% group showed zigzag arrangement of jejunal villi (Plate 3, Fig. G). This could mean that different concentrations of organic acids can change the intestinal morphology. This interwoven or zigzag pattern of villus arrangement was mentioned in broiler chickens after giving organic acids in their diet (4); however, the authors failed to show any pictures of such arrangement in the published paper. The interwoven or zigzag arrangement of intestinal villi reported in the present study and in others suggests a more efficient nutrient absorption; as nutrients will have more contact time with the intestinal villi epithelium (22). Possible explanation to these changes in the arrangement of the villi could be due to either increased villus length (as seen in the present H&E results) or changes in the number of villi per square cm of intestine (as seen in the present SEM results). This was evident in both H&E stained sections and SEM images of the different intestinal parts; however, the exact mechanism needs further investigation. It has been previously noticed that the intestinal villi shape and length changed in response to feed additives. For example, changes in vil-lus height and cell mitosis rate was seen in response to dietary bamboo charcoal with vinegar (17) and to dietary natural zeolite including plant extract (18) in Aigamo ducks. Also, similar intestinal villi changes were seen in broiler chickens in response to addition of formic acid (7, 23), organic acid salts (24), organic acids (4) and aflatoxins (25) in their diet. In addition, similar differences were observed by comparing village chickens to commercial broilers in Malaysia (26). Finally, from the previous studies and our results, one can hypothesize that any changes or even small amounts of additives to bird's diet can greatly alter the intestinal length and villus morphology. An important question could arise from this point, as changing a single constituent of the diet (addition of organic acids or simply changing the pH of the diet) caused changes in intestine's anatomy, histology and SEM within the same animal species. Therefore, could the Effects of diet's acidifying additives on the intestinal histomorphology in ducks 227 anatomical differences in the digestive system (macro and microscopic anatomy) between different species of birds be attributed to its feed habits rather than species-related differences? Answers to this question are unavailable in the literature and this needs further investigation. Conclusions The present results indicate that the addition of up to 2% organic acids to duck's diet increased small intestine's length and increased the length of intestinal villi without adverse effects on their microscopic structure. This evidently led to larger surface area for digestion and absorption of the feed, and, therefore, greatly improved the final body weight of the study ducks. It is, therefore, recommended to use 2% organic acids in duck's diet. Conflict of interest The authors declare no conflict of interest. Acknowledgments This work was funded by personal funds from the present authors. We would like to thank Ass. Prof. Dr. Doaa Ibrahim Mohammed at the Bird Nutrition Research Unit (Nutrition and Clinical Nutrition Department, Faculty of Veterinary Medicine, Zagazig University) for help with feeding and rearing the study ducks. Supplementary data Supplementary data is available upon reasonable request by e-mailing the corresponding author. The file contains detailed methodology used herein in addition to supplementary plates and tables. References 1. Byrd JA, Hargis BM, Caldwell DJ, Bailey RH, Herron KL, McReynolds JL, Brewer RL, Anderson RC, Bischoff KM, Callaway TR, Kubena LF. Effect of lactic acid administration in the drinking water during preslaughter feed withdrawal on Salmonella and Campylobacter contamination of broilers. Poultry Science. 2001; 80:278-83. 2. Chaveerach P, Keuzenkamp DA, Urlings HA, Lipman LJ, van Knapen F. In vitro study on the effect of organic acids on Campylobacter jejuni/coli populations in mixtures of water and feed. Poultry Science. 2002; 81:621-28. 3. Dibner JJ, Buttin P. Use of organic acids as a model to study the impact of gut microflora on nutrition and metabolism. Journal of Applied Poultry Research. 2002; 11:453-63. 4. Samanta S, Haldar S, Ghosh TK. Comparative efficacy of an organic acid blend and bacitracin methylene disalicylate as growth promoters in broiler chickens: Effects on performance, gut histology, and small intestinal milieu. Veterinary Medicine International. 2010:18. 5. Samli HE, Sirci G, Lorenzo Bermejo J, Koc F, Agma Okur A, Senkoylu N. Effect of addition of a commercial organic acid mixture on feed microbiology according to duration and conditions of storage. Acta Agriculturae Scandinavica A: Animal Sciences. 2008; 58:186-90. 6. Chaveerach P, Keuzenkamp DA, Lipman LJA, Van Knapen F. Effect of Organic Acids in Drinking Water for Young Broilers on Campylobacter Infection, Volatile Fatty Acid Production, Gut Microflora and Histological Cell Changes. Poultry Science. 2004; 83:330-34. 7. Hernández F, García V, Madrid J, Orengo J, Catalá P, Megías MD. Effect of formic acid on performance, digestibility, intestinal histomorphology and plasma metabolite levels of broiler chickens. British Poultry Science. 2006; 47:50-56. 8. Yamauchi K. Review on Chicken Intestinal Villus Histological Alterations Related with Intestinal Function. The Journal of Poultry Science. 2002; 39:229-42. 9. Jansen W, Reich F, Klein G. Large-scale feasibility of organic acids as a permanent preharvest intervention in drinking water of broilers and their effect on foodborne Campylobacter spp. before processing. Journal of Applied Microbiology. 2014; 116:1676-87. 10. Choi PM, Sun RC, Guo J, Erwin CR, Warner BW. High-Fat Diet Enhances Villus Growth During the Adaptation Response to Massive Proximal Small Bowel Resection. Journal of Gastrointestinal Surgery. 2014; 18:286-94. 11. Park JH, Grandjean CJ, Hart MH, Baylor JM, Vanderhoof JA. Effects of Dietary Linoleic Acid on Mucosal Adaptation after Small Bowel Resection. Digestion. 1989; 44:57-65. 12. Sukhotnik I, Lerner A, Sabo E, Krausz MM, Siplovich L, Coran AG, Mogilner J, Shiloni E. Effects of Enteral Arginine Supplementation on the Structural Intestinal Adaptation in a Rat Model of Short Bowel Syndrome. Digestive Diseases and Sciences. 2003; 48:1346-51. 228 A. Abdelfattah-Hassan, H. M. El-Ghazali 13. Tappenden KA. Mechanisms of Enteral Nutrient-Enhanced Intestinal Adaptation. Gastroenterology. 2006; 130:S93-99. 14. Bohorquez D V., Bohorquez NE, Ferket PR. Ultrastructural development of the small intestinal mucosa in the embryo and turkey poult: A light and electron microscopy study. Poultry Science. 2011; 90:842-55. 15. Fischer ER, Hansen BT, Nair V, Hoyt FH, Dorward DW. Scanning Electron Microscopy. Curr. Protoc. Microbiol., vol. 25, Hoboken, NJ, USA, John Wiley & Sons, Inc. 2012; 25vol., p. 2B.2.1-2B.2.47. 16. Ding BA, Pirone A, Lenzi C, Xiaoming N, Baglini A, Romboli I. Histochemical features of the Muscovy duck small intestine during development. Tissue and Cell. 2011; 43:190-95. 17. Ruttanavut J, Yamauchi K, Goto H, Erikawa T. Effects of Dietary Bamboo Charcoal Powder Including Vinegar Liquid on Growth Performance and Histological Intestinal Change in Aigamo Ducks. International Journal of Poultry Science. 2009; 8:229-36. 18. Khambualai O, Ruttanavut J, Kitabatake M, Goto H, Erikawa T, Yamauchi K. Effects of dietary natural zeolite including plant extract on growth performance and intestinal histology in Aigamo ducks. British Poultry Science. 2009; 50:123-30. 19. Partanen KH, Mroz Z. Organic acids for performance enhancement in pig diets. Nutrition Research Reviews. 1999; 12:117-45. 20. Noy Y, Sklan D. Digestion and Absor- ption in the Young Chick. Poultry Science. 1995; 74:366-73. 21. Jamroz D, Wiliczkiewicz A, Orda J, Wertelecki T, Skorupinska J. Aspects of development of digestive activity of intestine in young chickens, ducks and geese. Journal of Animal Physiology and Animal Nutrition. 2002; 86:353-66. 22. Yamauchi KE, Isshiki Y. Scanning electron microscopic observations on the intestinal villi in growing white leghorn and broiler chickens from 1 to 30 days of age. British Poultry Science. 1991; 32:67-78. 23. Garcia V, Catala-Gregori P, Hernandez F, Megias MD, Madrid J. Effect of Formic Acid and Plant Extracts on Growth, Nutrient Digestibility, Intestine Mucosa Morphology, and Meat Yield of Broilers. The Journal of Applied Poultry Research. 2007; 16:555-62. 24. Paul SK, Halder G, Mondal MK, Samanta G. Effect of Organic Acid Salt on the Performance and Gut Health of Broiler Chicken. The Journal of Poultry Science. 2007; 44:389-95. 25. Witlock DR, Wyatt RD, Ruff MD. Morphological changes in the avian intestine induced by citrinin and lack of effect of aflatoxin and T-2 toxin as seen with scanning electron microscopy. Toxicon. 1977; 15:41-44. 26. Kadhim KK, Bakar MZA, Noordin MM, Babjee MA, Saad MZ. Light and scanning electron microscopy of the small intestine of young Malaysian village chicken and commercial broiler. Pertanika Journal of Tropical Agricultural Science. 2014; 37:51-64. Slov Vet Res 2019; 56 (Suppl 22): 229-38 DOI 10.26873/SVR-761-2019 Original Research Article THE IMPACT OF DIFFERENT STOCKING DENESITIES AND DIETARY PROTEIN LEVELS ON THE PERFORMANCE OF AFRICAN CATFISH (Clarias gariepinus) FINGERLINGS Fawzy I. Magouz1; Malik M. Khalafalla2, Eman M. Moustafa3* Amir S. Sadek1 department of Animal Production, Faculty of Agriculture, Kafrelsheikh University, 2Department of Aquaculture, Faculty of Aquatic and Fisheries Sciences, Kafrelsheikh University, 3Department of Fish Diseases and Management, Faculty of Veterinary Medicine, Kafrelsheikh University, Egypt Corresponding author, E-mail: emantarek2002@yahoo.com Abstract: The current experiment was randomly designed as a 3^2 factorial design to investigate the effects of two fixed factor (stocking density & dietary protein level) on growth performance, feed utilization, survival rate and physiological response of African catfish (Clarias gariepinus). Fishes were allotted in 18 concrete (8^3^0.5 m3; LxWxH) tanks at three different stocking density 30, 20 and 10 catfish fingerlings / m3 and fed on two different protein diets (25 and 30%). Each treatment was applied in three replicates. Fishes were fed 2.5% of biomass body weight twice/day. Results showed that growth performance, feed utilization, survival rate and hematological parameters were significantly improved with increased dietary protein level with low stocking density. The sixth treatment (high protein level 30% and lowest stocking density; 10 fish/m3) exhibited the highest growth performance with no mortalities. Based on the results of the current study, it could be recommended to use protein level diet of 30% or more and stocking density of 10 catfish fingerlings/ m3 to obtain high productivity in a short time with consequent decreased cost. Key words: African catfish; Clarias gariepinus; dietary protein; growth parameters; Stocking density Introduction Fish is one of the most important alternative sources of dietary protein required to meet the increased human population demand in Egypt together with deceased other sources of animal protein (1). Aquaculture is considered a main key for bridging the national fish demand-supply gap (2). Increasing aquaculture production is clearly needed to meet this demand in the third millennium, because capture fisheries is showing precipitous decline due to habitat destruction, over fishing and pollution (3). The utmost aquaculture goal is the production of high quality, high quantity, and low cost fish in short possible time. This can be achieved by careful species selection, good water quality, appropriate feeding and suitable stocking density (4).That is why it is important to determine the best carrying capacity of an aquatic environment in which we can keep certain numbers of fish in a given volume of the culture media resulting in the highest fish yield without negative Received: January 2019 Accepted for publication: February 2019 230 F. I. Magouz, M. M. Khalafalla, E. M. Moustafa, A. S. Sadek effect on growth rate depending mainly on the amount and the quality of available food (5) African catfish (Clariasgariepinus) is an endemic popular aquaculture species largely distributed in most African and Asian countries (610). It is widely cultured in freshwater ponds because of its high growth rate with excellent quality meat, ability to accept a wide variety of feed, resistance to diseases, easiness in reproduction and its ability to tolerate adverse environmental conditions, and high stocking densities under poly-culture conditions (7, 10-13). The most critical aspect of aquaculture is to rear the early life stages of fish (fry and fingerlings) (14, 15) because fish at these stages are very sensitive to the different factors or determinants of production. Inadequate stocking densities and poor quality feed have a major role in poor growth and low survival rate of fry and/or fingerling stages resulting consequently in lower production African catfish are carnivorous as well as predatory; having good special appetite for high protein diet. Therefore, dietary protein levels must be adjusted to meet their requirements. Moreover, stocking densities should be optimized to ensure that the available food is utilized more efficiently with maximum space utilization (16). The present work was conducted to evaluate the effect of two different levels of dietary protein together with varying degrees of stocking density on the growth parameters, feed efficiency, nutrient utilization, hematological parameters and survival rate of African catfish fingerlings in concrete tanks. Materials and methods The present work was carried out in one of the commercial fish farms located at Kafr El-Sheikh Governorate, Egypt, in co-operation with the Animal Production Department, Faculty of Agriculture, Kafrelsheikh University, during the year 2017. Fish A number of 4320 African catfish fingerlings (Clarias gariepinus) with an average initial weight of (50 ± 2 g) were obtained from a private hatchery in Kafr El-Sheikh governorate. The fish were treated with potassium permanganate solution (3ppm) and acclimatized on the new environment (experimental conditions) for two weeks before the beginning of the experiment in concrete tanks. During the adaptation period, the fish were supplied with natural feeding (trash fish). Experimental design The experiment was randomly designed in a 3^2 factorial design. Three stocking densities were used with two levels of dietary protein. Thus, a total of six treatments run in triplicate were allotted into18tanks. Treatments (1-3) comprised of fingerlings fed with 25% CP and stocked at 30, 20, 10 fingerlings/m3, respectively. However, Treatments (4-6) comprised of fingerlings fed with 30%CP and stocked at 30, 20, 10 fingerlings/m3, respectively. Catfish fingerlings (50 ± 2 g) were randomly stocked in 18 concrete tanks each was (8^3x0.5 m3) capacity. All tanks were supplied with drainage water from drain El-Mohet through inlet PVC pipes (2 inch in diameter). Water outlet was found to be at a rate of 15 liter per minute. The fish were fed at a level of 2.5% of live body weight for about 90 days (experimental period). Diets Two tested diets containing 25 and30% protein levels were formulated from the local ingredients including fish meal, soybean meal, yellow corn, wheat bran, rice bran, wheat middling, sun flower oils, vitamins and minerals mixture, and Di-calcium phosphate to obtain control diet. The premix was added to all the experimental diets. The composition of the experimental diets was showed in table (1, A). The diets were prepared by thoroughly mixing the dry ingredients at first then mixed with oil. The diets were analyzed at The Central Laboratory of Food & Feed (CLFF), Agricultural Research Center, Ministry of Agriculture. Chemical composition and calculated gross energy of different experimental diets are presented in Table (1, B). First experimental diet (Diet1) consist of 25% crude protein and 408.14 kcal/100g The impact of different stocking denesities and dietary protein levels on the performance of African catfish 231 gross energy and the second experimental diet (Diet2) consist of 30% crude protein and 420.73 kcal/100g gross energy. Feeding regime Catfish were fed natural feeding for two weeks during the acclimatization period in the experimental tanks. After the two weeks of acclimatization, the catfish were fed the experimental diets at 2.5% from the total biomass daily for 90 days, and were applied twice a day (at 10:00 am & 14:00 pm). The feed amount was adjusted every seven days according to the new weight of the fish. Proximate chemical analysis Samples of the experimental diets as well as the experimental fish at the start and at the end of the experiments were obtained and then force dried, milled and deep frozen until determination the chemical analysis according to the methods described by (17). Water quality management Water quality parameters, such as temperature, dissolved oxygen, pH, alkalinity, hardness, ammonia, nitrate, nitrite and water salinity were estimated throughout the experiment. Water temperature was measured using thermometer. Oxygen level was measured daily at 8 o'clock by using oxygen meter (Model FE 247, EDT Instruments LTD. Dover Kent, UK). The pH was monitored using pH meter (Model Digi-sense, Cole-Parmer Instruments Co. Vernon Hills, IL. USA, Figure 1). Alkalinity was determined using test kit (Model WAT-DR. Code 4491-DR, LaMotte CO. Chester-town, Maryland, USA). Hardness was determined using test kit (Model HA-DT Cat. 20636-00, Hach Co. Loveland, Colorado, USA). Ammonia-N was estimated using test kit (Model NI, Cat. No. 22669-00, Hach Co.). Nitrite and nitrate were measured using test kits (Model NI-16 Cat. No. 20596-00 and Model NI-14 Cat.No. 14161-00, Hach Co., respectively). Determinations were carried out weekly according to APHA (18). Determination of fish growth parameters Growth and feed utilization were assessed by calculating average weight gain, average daily gain, specific growth rate, protein efficiency ratio, feed intake, feed conversion ratio, feed efficiency and survival rate: Live body weight (LBW) was assessed in g for each individual group of each experimental treatment and recorded every 2 weeks (14 days).Total weight gain (TWG) (g/fish) = final body weight - initial body weight (19). Specific growth rate (SGR %/day) = [Ln final body weight- Ln initial body weight]*100/experi-mental period(d) (20). Average daily gain (ADG) = (W2 - W1) / t; where: W2 is the final weight, W1 initial weight and t is the time in days. Feed conversion ratio (FCR) = feed intake (g)/weight gain (g) (21). Protein efficiency ratio (PER) = weight gain (g)/protein intake (g). Protein productive value (PPV %) =100* (protein gain (g)/protein intake (g)). Survival rate (SR %) = total number of fish at the end of the experiment * 100 / total number of fish at the start of the experiment. Hematological investigations At the end of the experiment, fish in each group (3-4 fish) were weighed and blood samples were taken randomly from the caudal vein for blood analysis and differential leukocyte count. Anti-coagulated blood samples were prepared immediately for counting red and white blood cells, etc. Red blood cells count (RBCs*106/mm) and white blood cells count (WBCs*103/mm): were determined according to the method described by Stoskopf, (22). Hemoglobin concentration (Hbgm/dl) was estimated according to the method of Zinkl (23) and Packed cell volume (PCV%): was estimated by the microhaematocrite method described by Decie& Lewis (24). Statistical Analysis The obtained numerical data were statistically analyzed using SPSS (25) for two-way analysis of variance at 5% level of significance. When F-test was significant, least significant difference was calculated according to Duncan (26). Results and discussion Growth is the principal key of energy loss and/or gains in the aquatic environment; which 232 F. I. Magouz, M. M. Khalafalla, E. M. Moustafa, A. S. Sadek can be measured mainly by determination of the weight gain. Stocking density is a main factor significantly affecting the growth, feed utilization (FCR) and survival rate (SR) of the catfish fingerlings. High stocking densities act as stressors, fish aggregate for the same amount of feed and some of them cann't eat resulting in growth retardation (27) and/or decrease of feed utilization (28). Moreover, in high stocking densities, there is increased oxygen demand needed either for respiration or metabolism. Therefore, it is very important to adjust the stocking density with the carrying capacity of the aquaculture system. Growth performance and survival rate As shown in table 2, catfish fed on 30% protein level diet showed better final weights compared to those fed g on 25% protein level diet at the different stocking densities. The final weight was significantly increased with increased protein in diet (P < 0.05). The final weight of Treatment 1 (T1) was the lowest weight (214.3± 10.12) compared with the other treatments. While, final weight of treatment six (T6) was the highest weight (284.00± 14.32) (P<0.05) compared with the other treatments. The average weight gain (AWG) was improved especially in groups fed on high protein level. While, the first treatment (T1) showed the lowest AWG (164.30±8.24) compared with other treatments. Similarly, average daily weight gain (ADG) of treatments (4-6) feeding on 30% protein was better than treatments (1-3) feeing on 25% protein diet in all variant stocking densities; with the highest value recorded in treatment 6 (2.60±0.63) and the lowest value in treatment 1 (1.83±0.42). Besides, the highest SGR (1.71±0.41) was observed in treatment 6. The results are similar to those reported by some authors (29-33), where they reported that increased protein concentration in the diet has a positive correlation with the final weight gain expressed as AWG, ADG and SGR. On the other hand, results of the present study showed that the decreasing stocking density, the increased final weight. Decreased stocking density significantly affected the final weight gain, the highest stocking density in treatment 1 (30 fish/m3)(214.30±10.12) gave the worst final body weight compared with treatment 3 (10 fish/m3) (256.30±12.54), although both treatments feed on the same protein level diet 25%. These results also could be observed in fingerlings fed on 30% protein level diet, where treatment 6 (10 fish/m3) showed the best result of all growth parameters compared with other groups. This means that decreasing the stocking density gave a positive effect (P < 0.05) of final weight gain. These results agreed with many authors (34-38), where they found that increased stocking density resulted in less AWG, ADG and SGR. Survival rate of the experimental fish was recorded. It was 100% in treatments 2, 3, 5 and 6 (low stocking density), but treatment 1 and 4 (highest stocking density) lost some fingerlings in the first days of experiment. This may be attributed to increased oxygen demand in high stocking treatments which may result in fish may succumb to suffocation. Survival rate increases with increased dietary protein level, this may be attributed to that low protein diets might have not met the nutrition requirements of catfish fingerlings and thereby leading to nutrient-deficient related mortalities (39, 40). Water quality parameters Water quality parameters of the experimental ponds were insignificantly affected by different treatments during the experimental period (90 days). As summarized in table 3, all water parameters for all experimental ponds showed that, dissolved oxygen not least 4 mg/ litter, toxic ammonia (NH3) no more than 0.6 mg/ litter, pH values between 8.2 and 8.5 degree, nitrate (NO2) no more than 0.2 mg/ litter, alkalinity 320, hardiness 150, salinity between 2000 and 4000 ppm (source of water was brackish water) and temperature about 27°C. Water quality parameters observed in the current study were within the normal ranges required for normal growth of African catfish (9, 33, 39, 41-45); consequently any changes in the growth parameters may be attributed either protein level diet and/or stocking density. The impact of different stocking denesities and dietary protein levels on the performance of African catfish 233 Feed intake and nutrient utilization As shown in table 4; holding all factors including the different stocking density of the present study, results showed that dietary protein has a marked influence on feed utilization. Results showed that increasing the level of protein in the diet increased significantly (P < 0.05) feed conversion ratio of fish. Diet 30% protein gave the best results (P < 0.05) of feed conversion ratio compared with diet 25% protein with different stocking densities; and the sixth treatment (1.00±0.09) gave the best feed conversion ratio (FCR) compared with the third treatment (1.13±0.11) ( same of stocking density)while the first treatment (1.40±0.24) was the worst treatment compare with other treatments. Besides, increasing protein level in the diets affected significantly (P < 0.05) protein efficient ratio (PER) of fish. The same trend was observed with protein productive value (PPV); these results of feed utilization may be due to the impact of different protein levels together with different stocking densities. Protein efficiency ratio (PER) and protein productive value (PPV) were better generally with low stocking densities and high protein level diets. These results are in full agreement with some authors (35, 46-50). Body composition of African catfish as affected by the experimental diets Data concerning body composition responses of African catfish presented in table 5 revealed that the dry matter, crude protein, ether extract contents were increased by the time from the start to the end of experimental period (90) day, but the opposite was true for the ash percentage. Increasing protein level in the diet affected crude protein of body composition significantly (P < 0.05). Fingerlings in treatment 1 (25% protein) had low crude protein (66.92±5.36) (P > 0.05) as compared with the other treatments, while the sixth treatment (30% protein) (67.39±5.47) was the best in crude protein (CP) content (P > 0.05) compared with other treatments. However, dry matter (DM) in was low in treatment 1 (28.78±1.58) as compared with the other treatments, while sixth treatment (29.21±1.75) was the best treatment compared with the different treatments. Results also revealed that increasing level of protein in diet affected positively (P < 0.05) ether extract (EE) and gross energy (GE) of fish body. Hematological investigation As summarized in table (6), there was a significant increase in RBCs, Hb, PCV and WBCs in all treatments; the sixth treatment (30% protein and low stocking density) showed the best hematological parameters and treatment 1(25% protein and high stocking density) showed the worst hematological results. These results suggest that the physiological response of catfish fingerlings is improved when fed high protein level diet together with existing low stocking density. In the present study, Growth depression observed in higher stocking densities groups may be attributed to reduced amount of adequate oxygen, followed by slowed down metabolism resulting in lower growth than in lower stocking density (27, 51). The decreased FCR at high stocking density (30 fish/m3) may confirm that high stocking density reduced feed utilization efficiency. Due to competition for feed, there is low feed intake and consequently lower energy levels necessary for metabolism which is responsible to convert nutrients into fish flesh. Growth depression observed in lower protein level diets has been observed in various fish species under captivity; this might be due to reduction in the available energy for growth (52, 53). Fish usually obtain energy from chemical breakdown of proteins than large animals that is why high protein is highly important in fish diet (54). Lowest growth in 25% protein level diet may be due to that most of protein was used for maintain life but unavailable for growth; however 30% protein level diet resulted in higher growth rate and better feed utilization in catfish fingerlings. High focusing to body protein is of a great importance to meet the dietary requirements for tissue building, metabolism and repair; as a result of poor growth and poor feed utilization in fish group fed low protein level in the current study (55).This means the fingerlings survived in low stocking density and fed on 30% protein 234 F. I. Magouz, M. M. Khalafalla, E. M. Moustafa, A. S. Sadek level diet could use dietary protein more efficiently than fish survived in higher stocking densities and fed on 25% protein level diet. Table 1A: Composition of the experimental diets Raw Ingredients Composition (%) experimental diets Diet 1 (25% Protein)_Diet 2 (30% Protein) Fish mail 62% 10 15 Soya bean 44% 27.5 35 Yellow Corn 10 10 Rice bran 24 21.5 Wheat middling 11 10 Wheat bran 14 5 Sun flower oil 2 2 Premix 1 1 1 Di calcium phosphate 0.5 0.5 Total 100 100 (1)Premix Composition:- Each 3 kg contains , Vit A (1200000 i.u.), Vit D (300000 i.u.), Vit E (700 mg,) Vit K3 (500 mg,) Vit B1 (500 mg), Vit B2 200mg, Vit B6 (600mg), Vit B12 (3mg), Vit C 450mg, Niacin 3000mg, Methionine3000mg, Cholin chloride 10000mg, Folic acid 300mg, Biotin 6mg, Panthonic acid 670mg, Magnesiam salphate 3000mg, Copper sulphate 3000mg, Iron sulphate 10000mg, Zinc sulphate , 1800mg, Cobalt sulphate 300mg, Carrier upto 3000mg. Table 1B: Proximate analysis of the experimental diets Composition (%) Diet 1 (25% CP) Diet 2 (30% CP) Dry matter 90.30 90.90 Organic matter 78.30 80.50 Crude Protein 25.45 30.40 Ether extract 5.40 5.00 Crude Fiber 6.60 6.40 Ash 12.00 10.40 Nitrogen free extract 50.55 47.80 Calculated energy value: *GE (kcal/100g) 408.14 420.73 **DE(kcal/g) 306.11 315.55 *GE (gross energy) was calculated according to NRC (1993) by factors of 5.65, 9.45 and 4.22 kcal per gram of protein, Lipid and carbohydrate, respectively. **DE (digestible energy) was calculated by applying the coefficient of 0.75 to convert gross energy to digestible energy. Table 2: Growth performance parameters of African catfish fed different experimental diets Treatment Protein Levels % Stocking density fish/ m3 Initial BW(g) Final BW(g) G) W )g( 4 NO2 Nil Nil Nil Nil Nil Nil < 0.2 NH3 (mg /l) 0.6 :0.9 0.6 :0.9 0.6 :0.9 0.6 :0.9 0.6 :0.9 0.6 :0.9 < 0.6 Alkalinity 320 320 320 320 320 320 <500 Hardiness 150 150 150 150 150 150 <500 Salinity ppm 2000 :4000 2000 :4000 2000 :4000 2000 :4000 2000 :4000 2000 :4000 0 - 5000 Table 4: Average feed intake, feed conversion ratio, protein efficiency ratio (%), feed efficiency and protein productive value of African catfish fed different experimental diets Treatment Protein Levels % Stock density fish/ m3 Feed intake g/fish Average weight gain (g) FCR PER Add 1 30 230.56±11.24 164.30±8.24c 1.40±0.24c 2.80±0.35c 19.09±2.15b 2 25 20 231.25±11.35 177.00±9.54c 1.31±0.18bc 3.01±0.57b 20.56±2.22b 3 10 233.33±11.57 206.30±10.25ab 1.13±0.11a 3.47±0.64a 23.79±2.11a 4 30 230.56±10.98 183.60±8.98bc 1.26±0.17b 2.62±0.38 17.96±1.98c 5 30 20 231.25±10.89 197.00±9.68b 1.17±0.12ab 2.80±0.37c 19.20±2.08b 6 10 233.33±11.47 234.0±11.23a 1.00±0.09a 3.30±0.55ab 22.56±2.34a A,b and c mean the column bearing different letters differ significantly at 0.05 level Table 5: Body composition of African catfish as affected by the experimental diets (% on dry matter basis) Treat. % On Dry matter basis No DM CP EE Ash GE** (Kcal/100g) At the start of the experiment 25.00+1.15 At the end of the experiment T1 28.78+1.58 T2 29.12+1.64 62.80+4.56 66.92+5.36 67.13+4.87 14.10+1.22 17.08+1.54 17.02+1.36 20.95+0.38 13.90+0.15 13.91+0.21 497.14 +11.5 548.34+15.24 548.31+ 14.28 T3 29.11+1.52 67.36+5.24 16.94+1.54 13.74+0.32 548.94+13.47 T4 29.09+1.66 67.33+5.11 16.77+1.74 13.94+0.22 547.16+13.65 T5 29.19+1.87 67.36+5.22 16.77+1.64 13.87+0.19 547.50+13.78 T6 29.21+1.75 67.39+5.47 16.79+1.77 13.78+0.24 548.03+14.55 A,b and c mean the column bearing different letters differ significantly at 0.05 level. **Gross energy was calculated according to NRC (1993) by using factors of 5.65, 9.45 and 4.22 Kcal per 1 gram of protein, lipid and carbohydrate, respectively 236 F. I. Magouz, M. M. Khalafalla, E. M. Moustafa, A. S. Sadek Table 6: Effect of protein levels and stocking denesity on the haematological parameters in catfish fingerlings Haematological parameters Treatment Protein Levels % Stocking density(fish / m3) m s m ^ S £2 s m P ^ > o S Hb (g/100ml) O ^ Ph w T1 25 30 2.48±0.05d 75.86±1.12b 7.62±0.41c 21.00±0.5a T2 20 2.58±0.2b 73.45±0.81d 7.63±0.15c 22.50±0.5a T3 10 2.68±0.14b 74.23±1.3c 7.64±0.33c 22.00±0.5a T4 30 30 2.63±0.4c 76.07±2.1b 7.70±0.41a 23.50±1.0a T5 20 2.72±0.2a 72.51±0.5e 7.79±0.5ab 23.50±1.0a T6 10 2.83±0.08a 67.23±0.59f 7.82±0.28a 24.00±0.19a RBCs= Red Blood cells; WBCs= White Blood cells; Hb = Hemoglobin; PCV = Packed Cell Volume Conclusion Both Stocking density and Protein level in fish diet have a significant effect on growth rate, feed utilization, physiological responses and survival rate Clarias gariepinus fingerlings in concrete tanks. Based on the results of the current study, it could be recommended to use protein level diet of 30% or more in combination with stocking density of 10 fish/ m3 to obtain higher production in a short time. Conflict of interest The authors declare that they have no conflict of interest. References 1. Omotoyin BO. Introduction to Fish Farming in Nigeria. Ibadan University Press, Publishing House, University of Ibadan, Ibadan, Nigeria. 2007; 60. 2. Nwipie GN, Erondu ES, Zabbey N. Influence of Stocking Density on Growth and Survival of Post Fry of the African Mud Catfish, Clarias gariepinus. Fish Aquac J 2015; 6(1) DOI: 10.4172/2150-3508.1000116 3. World Bank. Marketing Africa's High-value Foods: Comparative Experiences of an Emergent Private Sector. Steven J. and John M (eds). 1995: 117. 4. Barua G. Gonadal development and fry rearing of Clarias batrachus. 1990. 5. Zonneveld N, Fadholi R. Feed intake and growth of red tilapia at different stocking densities in ponds in Indonesia. Aquaculture 1991; 99: 83- 94. 6. Goda AM, El-Haroun ER, Chowdhury MA. Effect of totally or partially replacing fish meal by alternative protein sources on growth of African catfish, Clarias gariepinus (Burchell. 1822) reared in concrete tanks. Aquac Res 2007; 38: 279-87. 7. Nyina-wamwiza L, Wathelet B, Kestemont P. Potential of local agricultural by-products for the rearing of African catfish Clarias gariepinus in Rwanda: effects on growth, feed utilization and body composition. Aquac Res 2007; 38:206-14. 8. Osman AGM, Wuertz S, Mekkawy IAA, Exner H, Kirschbaum F. Embryo-toxic effects of lead nitrate of the African catfish Clarias gariepinus (Burchell, 1822). J. Ichthyol. 2007; 23: 48 -58. 9. Abdelhamid AM. Recent Trends in Fish Culture. New Universal Office, Alexandria. 2009a. 10. Khan MA, Abidi SF. Dietary arginine requirement of Heteropneustes fossilis fry (Bloch) based on growth, nutrient retention and hematolog-ical parameters. Aquac Nut 2011; 17:418-28. 11. Richter CJJ, Eding EH, Verreth JAJ, Fleuren WLJ. African catfish (Clarias gariepinus). In: N.R. Bromage & R.J. Roberts (eds). Broodstock Management and Nutrition and Egg and Larval Quality. Blackwell Scientific Publications, Oxford, 1995; pp 42. 12. Dada AA, Wonah C. Production of exotic C. gariepinus at varying stocking density in outdoor ponds. J aqua Sci 2003;18(1): 21-4. 13. Chor WK, Lim LS, Shapawi R. Evaluation of feather meal as a dietary protein source for African Catfish fry, Clarias gariepinus. J Fish Aquac Sci 2013; 8:697-705. The impact of different stocking denesities and dietary protein levels on the performance of African catfish 237 14. Madu CT, Udodike EBC, Ita EO. Food and feeding habits of hatchlings of mudfish Clarias an-guillaris (L). Afr J Aquat Sci 1990; 5: 27-31. 15. Ibrahim MSA, Mona HA, Mohammed A. Zooplankton as live food for fry and fingerlings of Nile Tilapia (Oreochromis niloticus) and Catfish Clarias gariepinus in Concrete ponds. Central Laboratory for Aquaculture Research. (CLAR), Ab-bassa, Sharkia, Egypt. 8th Int Symp Tilapia in Aq-uac2008; 757-69. 16. Sirakov I, Invacheva E. Influence of stocking density on the growth performance of rainbow trout and brown trout grown in re- circulatory system. Bulg J Agric Sci 2008; 14(2): 150-4. 17. AOAC. Official methods of Analysis. In: Association of Official Analysis of Chemists, Washington DC. 15th Ed. 1990 18. APHA (American Public Health Association, American Water Works Association, and Water Pollution Control Federation). Standard Methods for the Examination of Water and Wastewater. 17th edition. APHA. Washington, DC. 1989 19. Annet CS. A model to facilitated optimal aquaculture production by quantitatively relating fish growth to feed and other environmental resources. Ph.D., Thesis, Michigan. State University, U.S.A. 1985. 20. Pouomonge V, Mbonglang M. Effect of feeding rate on the growth of tilapia (O. niloticus) in earthen ponds. Isr J aquac 1993; 45: 147-53. 21. De Silva SS, Anderson TA. Fish Nutrition in Aquaculture, Ed., Champman and Hall, 2-6 Bouday Raw, London SEI8 FIN, UK. 1995. 22. Stoskopf MK. Fish medicine Ed., W.B. Sainders Company, London. 1993. 23. Zinkle JG. Avian hematology.In: Jain NC (Ed. Schalrias) Veterinary Hematology, Philadelphia, Paihea and Febiger. 1986; 256-60. 24. Decie SIV, Lewis SM. Practical Haematol-ogy. Pp.736. Churchill Livingstone, London. ISBN: 13:978-0-443-06660-3. 10th Ed. 2006. 25. SPSS. Statistical package for the social sciences, Revisions 6, spss Inc, Chicago, USA. 1997. 26. Duncan DB. Multiple Ranges and Multiple F- test. Biometerics 1955; 11:1-42. 27. Rahman MA, Mazid MA, Rahman MR, Khan MN, Hossain MA. Effect of stocking density on survival and growth of critically endangered mashseer, Torputitora (Hamilton), in nursery ponds. Aquaculture 2005; 249: 275-84. 28. Suziki N, Kondo M, Günes E, Özongun M, Ohno A. Age and growth of Turbot Psetta maxima in the Black Sea, Turkey. Turk J Fish Aquat Sci 2001;1: 43-53. 29. Phonekhampheng O. On-farm Feed Resources for Catfish (Clarias gariepinus) Production in Laos: Evaluation of Some Local Feed Resources. Dissertation submitted as a partial fulfillment of the requirements for the degree of Doctor of Philosophy, Swedish University of Agricultural Sciences. 2008; Pp 65. 30. Diyaware MY, Modu BM, Yakubu UP. Effect of different dietary protein levels on growth performance and feed utilization of hybrid catfish (Heterobranchus bidorsalis^Clarias anguillaris) fry in north- east Nigeria. Afr J Biotechnol 2009; 8: 3954-7. 31. Kiriratnikom S, Kiriratnikom A. Growth, feed utilization, survival and body composition of fingerlings of slender walking catfish, Clarias nieuhofii, fed diets containing different protein levels. Songklanakarin J. Sci. Technol 2012; 34 (1):37-43. 32. Ahmad M, Qureshi TA, Singh AB, Manohar S, Kamlesh B, Salman RC. Effect of dietary protein, lipid and carbohydrate contents on the growth, feed efficiency and carcass composition of Cyprinus carpio communis fingerlings. Int J Fish Aquac 2012; 4(3):30-40. 33. Hecht T. A Review of on-farm feed management practices for North African catfish (Clarias gariepinus) in sub-Saharan Africa. In: Hasan M. Rand New M.B(ed.),On-farm Feeding and Feed Management in Aquaculture, FAO Fisheries and Aquaculture Technical Paper No. 583, Rome, Italy, 2013; pp: 463-479, 282-9. 34. Edward A, Ladu BMB, Elihu A. Growth, survival and production economics of Clarias gariepinus fingerlings at different stocking densities in concrete tanks. Afr J Gen Agric 2010; 6(2):59-66. 35. Gonçalves De Oliveira EG, Pinheiro AB, de Oliveira VQ, da Silva únior ARM, de Moraes MG, Rocha IRCB, de Sousa RR, Costa FHF. Effects of stocking density on the performance of juvenile pi-rarucu (Arapaima gigas) in cages. Aquaculture 2012; 370-371:96-101. 36. Jiwyam W, Nithikulworawong N. Stocking density-dependent growth and survival of Asian red-tailed catfish (Hemibagrus wyckioides) fries: early nursing in cages. Int Aquat Res 2014; 6:24550 37. Josiah AS, Mwatete MC, Njiru J. Effects of greenhouse and stocking density on growth and survival of African catfish (Clarias gariepinus 238 F. I. Magouz, M. M. Khalafalla, E. M. Moustafa, A. S. Sadek Burchell 1822) fry reared in high altitude Kenya regions. Int J Sci Res 2014; 3(9):558-63. 38. Abdur Rahim AG, Ferrando S, Gallus L, Abdul Ghaffar A, Hafeez-ur-Rehman M, Waryani B. Effects of varying dietary protein level on growth, nutrient utilization and body composition of juvenile black fin sea bream, Acanthopagrus berda (Forsskal,1775). Pakistan J Zool 2016; 48(4): 108997. 39. Barton BA, Morgan JD, Vijayan MM. Physiological and condition-related indicators of environmental stress in fish. In: Biological Indicators of Aquatic Ecosystem Health, Adams S.M. (ed.), American Fisheries Society, Bethesda, M.D, 2002; pp:111-48. 40. Mohanta KN, Subramanian S, Korikanthi-math VS. Effect of dietary protein and lipid levels on growth, nutrient utilization and whole-body composition of blue gourami, Trichogaster trichop-terus fingerlings. J Anim Physiol Anim Nut 2013; 97:126-36. 41. Boyd CE. Water Quality in warm water fish ponds. Anburn University, Alabama. 1979; p 359. 42. Abdelhamid AM. Field and Laboratorial Analysis in Animal Production. 1st Edn., Dar Al-nashr for Universities, Cairo, Egypt. 1996. 43. Abdelhamid AM. Fundamentals of Fish Production and Culture. New Universal Office, Alexandria. 2009b 44. FAO. Fisheries and food security, Documentation issued on the occasion of the world food summit in Rome (1996), Food and Agriculture organization of the United Nations, Rome, Italy, 1996; p: 2. 45. Abdelhakim NF, Baker MN, Soltan MA. Aquatic Environment for Fish Culture. Faber and Faber, Cairo. 2002. 46. Dai W, Wang XM, Guo YJ, Wang Q, Ma JH. Growth performance, hematological and biochemical responses of African catfish (Clarias gariepinus) reared at different stocking densities. Afr J Agric Res 2011; 6(28): 6177-82. 47. Florence ON, Harrison TO. Impact of Stocking Density on the Polyculture of Clarias gariepi-nus and Oreochromis niloticus. J Agric Sci Technol 2012; 2: 1018-23. 48. Kyoung DK, Kang WK, Bong JL, Maeng HS, Hyon SH, Jin DK. Dietary protein requirement for young far eastern catfish Silurus asotus. Fish Aquat Sci 2014; 17(4):455-9. 49. Suharmili R, Kamarudin MS, Saad CR, Ina-Salwany MY, Ramezani-Fard E, Mahmud MH. Effects of varying dietary protein level on the growth, feed efficiency and body composition of lemon fin barb hybrid fingerlings. Iran J Fish Sci 2015; 14(2):425-35. 50. Xia S, Sun Z, Feng S, Zhang Z, Rahman MM, Rajkumar M. Effects of dietary protein level on growth and ammonia excretion of Leopard coral grouper, Plectropomus leopardus (Lacepede, 1802). Sains Malaysiana, 2015; 44(4):537-43. 51. Jha P, Barat S. The effect of stocking density on growth, survival rate and number of marketable fish produced of Koi carp, Cyprinus carpio vr. Koi; in concrete tanks. J Appl Aquac 2005; 17: 84-102. 52. Kim KW, Wang XJ, Bai SC. Optimum dietary protein level for maximum growth of juvenile olive flounder, Paralichthys olivaceus (Temminck and Schlegel). Aquac Res 2002; 33: 673-9. 53. Farhat KMA, Khan MA. Growth, feed conversion and nutrient retention efficiency of African catfish, Clarias gariepinus (Burchell) fingerling fed diets with varying levels of protein. J Appl Aquac 2011; 23:304-16. 54. Kpogue D, Gangbazo H, Fiogbe E. A preliminary study on the dietary protein requirement of Parachanna obscura (Günther, 1861) larvae. Turk J Fish Aquat Sci 2013; 13:111-7. 55. Aliyu-Paiko M, Hashim R, Shu-Chien AC. Influence of dietary lipid/protein ratio on survival, growth, body indices and digestive lipase activity in snakehead (Channa striatus, Bloch1793) fry reared in re-circulating water system. Aquac Nut 2010; 16: 466-7. Slov Vet Res 2019; 56 (Suppl 22): 239-48 DOI 10.26873/SVR-763-2019 Original Research Article TRANSRECTAL ULTRASONOGRAPHY AND RECTAL PALPATION FOR JUDGING UTERINE AND CERVICAL INVOLUTIONS IN BUFFALO: A COMPARATIVE STUDY Adel A. Ramoun1*, Essam A. Almadaly1, Hamdy A. Hattab2, Samy A. Darwish3, Ismail I. EL- Kon1 department of Theriogenology, Faculty of Veterinary Medicine, Kafrelsheikh University, 33516, Egypt, 2General Authority for Veterinary Services, Kafr EL-Sheikh, Egypt, 3Mahallet Mousa Buffalo Research Station, Animal Production Research Institute, Agriculture, Research Center, Egypt Corresponding author: E-mail: ramoun2004@yahoo.com Abstract: Uterine and cervical involutions were judged by transrectal ultrasonography (US) versus rectal palpations (RP) in buffaloes (n = 26). The diameters of the pregravid uterine horn (PGUHD) and cervix (CvD) were estimated by both transrectal US and RP every three days until gross uterine or cervical involution. Also, the US-measured PGUHD and CvD were recorded on the day of gross uterine or cervical involution on the basis of RP. The combined thickness of the myometrium and perimetrium of the pregravid uterine horn (PGUHMPT) and cervical wall thickness (CvWT) were parallelly estimated with US-measuring of PGUHD and CvD. The intervals to gross uterine (P < 0.05) and cervical (P < 0.01) involutions, on the basis of RP, were shorter than those on the basis of US. Both US-measured PGUHD and PGUHMPT, on the day of involution on the basis of US, were less than their US-measured counterparts on the day of gross involution on the basis of RP. Both US-measured CvD and CvWT, on the day of cervical involution on the basis of US, were less than their US-measured counterparts on the day of involution on the basis of RP. It is concluded that US- measuring of the pregravid uterine horn diameter and the combined thickness of myometrium and perimetrium as well as CvD and CvWT, is more accurate than RP-measuring of diameters of pregravid uterine horn and cervix for assessing the involution of the reproductive tract in buffaloes. Key words: buffalo; involution; ultrasonography; cervical; combine thickness Introduction Uterine involution together with the earlier resumption of postpartum ovarian activity is prerequisites for having a successful reproductive performance in buffaloes (1). Days open is shorter in cows with smaller than those with the larger diameter of the pregravid uterine horn (2). Normal cervical and uterine involutions are essential for resuming ovarian activity (3). During the involution period, a crosstalk was detected between cervix and health status of the endometrium (4) from one side and with the resumption of the ovarian cyclicity from the other side such that the larger cervical diameter (CvD) associated with endometritis can result Received: January 2019 Accepted for publication: February 2019 240 A. Ramoun, E. Almadaly, H. Hattab, S. Darwish, I. EL- Kon in abnormal follicular selection and abnormal ovarian cyclicity (5). Before a cow is likely to conceive, it has to undergo cervical and uterine involutions (6). For the establishment of a new pregnancy in dairy cows, the uterus must return to its normal prepartum condition (7) and the cervix should close and regain its normal size and structure quickly (8). Although rectal palpation (RP) remains the most commonly used method for assessing uterine involution in cows, it is less accurate than transrectal ultrasonography due to its high level of subjectivity (2) and variation among individual palpators (9,10). Trans-rectal US is an alternative method for monitoring the dynamic changes in the reproductive system after calving which allows rapid, safe and accurate assessment of uterine involution and resumption of ovarian activity in cattle (10, 11). It gives information about cervical and uterine sizes, endometrial thickness and uterine contents, which are variables closely related to uterine involution (12). While the decrement in the diameter of the involuting uterus and cervix is expressed in < 0.5 cm at least in the case of RP, it is estimated by millimeters in case of the transrectal US (13). The transrectal US is utilized not only in the follow up of puer-perium but also shares in making reproductive management decision in many reproductive areas such as selection of animals to be kept in or culled from the herd, increasing the likelihood of the reproductive success and pregnancy diagnosis (14). The hypothesis of the work was that the US examination would be expected to be more accurate than RP for judging uterine involution thereby help to optimize reproductive management, ensuring higher future fertility, therefore the aim of the present work was to test the efficiency of transrectal US versus RP in judging cervical and uterine involutions in buffaloes. Material and methods Animals and management The study was carried out in Mahallet Mousa Buffalo Research Station affiliated to Animal Production Research Institute, Agriculture, Research Centre, Egypt during the period extending from March to August 2017. Twenty-six healthy, 5-8 years old Murrah buffaloes, were enrolled in the present study. Their body condition score averaged 3.3 ± 0.4 (on a scale; 1 = lean to 5 = fatty). They had normal parturition and spontaneous placental expulsion within the first 12 hours after parturition. The calves were isolated after they had received colostrum and the buffaloes were milked twice daily. The animals were fed on a ration that met their maintenance and milk production requirements according to the requirements of Animal Production Research Institute (APRI 1997, unpublished data). Fresh water was available ad libitum. The Buffaloes were kept indoors in open yards, whereas half of the area was sheltered. This study was carried out under the Animal Welfare according to the regulations of Egyptian guidelines with approval granted by the Animal Ethics Committee of the Faculty of Veterinary Medicine, Kafrelsheikh University, Kafr EL-Sheikh, Egypt. The judgment of uterine and cervical involutions Rectal palpation RP of the cervix and uterus was conducted by the same examiner every three days beginning on the third day postpartum. Uterine involution was considered complete when the diameters of the two uterine horns, at their bases, became nearly symmetrical and no further changes in the diameter of the pregravid uterine horn (PGUHD) could be detected during two successive RP of the uterus [6]. The cervix was considered involuted when the CvD, at three cm cranial to external OS, became stable in two successive RP (15). The PGUHD and CvD were recorded every three days and on the day of gross uterine or cervical involution. The interval to gross uterine or cervical involution on the basis of RP was recorded. Transrectal ultrasonography and rectal palpation for judging uterine and cervical involutions in buffalo 241 Ultrasonography All transrectal US examinations were conducted by digital Ultrasonic Diagnostic Imaging System Model: D-P-30Vet. 10-2012 equipped with a linear probe, 7.5 MHz. All examinations were conducted by one examiner. Cross-sectional images of PGUH, at its base, and cervix, at three cm cranial to external OS, were obtained by the transrectal US. When the image, cross section of either uterine horn or cervix, was not spherical, the diameter was estimated by averaging two 90° dimensions (2). Uterine involution was considered complete when uterine dimensions, PGUHD and the combined thickness of the myometrium and perimetrium of the pregravid uterine horn (PGUHMPT), became nearly stable in two successive transrectal US [16]. Cervical involution was considered complete when no further reduction in CvD/cervical wall thickness (CvWT) was detected in two successive transrectal US. The US-measured PGUHD and PGUHMPT were recorded when the uterine involution was considered on the basis of RP or US. Both the CvD and CvWT were measured and recorded every three days and on the day of a gross cervical involution on the basis of RP or US.The interval to gross uterine or cervical involution on the basis of transrectal US was recorded. The rationale for measuring PGUHMPT rather than the thickness of either the whole of the uterine wall or endometrium by transrectal-US was due to the great folding of the endometrium into the uterine lumen and presence of still enlarged caruncles, especially during the early pu-erperium. The US-measuring of the endome-trial thickness is more presumably beneficial for the diagnosis of endometritis rather than assessing the uterine involution. The involution rate The involution rate of PGUH The involution rate (cm/ 3 days) of PGUH as indicated by the rates of the reduction in the PGUHD on the basis of RP or transrectal US examination was calculated by subtracting thePGUHD recorded on the days 6, 12, 18, 24, 30, 33...etc. from those recorded on the days 3, 9, 15, 21, 27, 30...etc. respectively, each of its previous one: e.g. (PGUHD on day 3- PGUHD on day 6), and so on until complete uterine involution. Also, the involution rate of PGUH as indicated by the rate of reduction in the PGUHMPT was calculated in the same manner as in the case of calculating the rate of reduction in the PGUHD. The involution rate of the cervix The involution rate (cm/ 3 days) of the cervix as indicated by the rates of reduction in the CvD on the basis of RP or the transrectal US was calculated by subtracting the CvD recorded on Days 6, 12, 18, 24, 30, 36, 42...etc. from those recorded on Days 3, 9, 15, 21, 27, 33, 39...etc. respectively, each of its previous one e.g. (CvD on day 3- CvD on Day 6). Also, the involution rate of the cervix as indicated by the rates of reduction in the CvWT was calculated in the same manner as in the case of calculating the rates of reduction in the CvD. Statistical analysis Data were analyzed with a statistical software program (GraphPad Prism version 5.0; GraphPad Software, San Diego, CA, USA).The means±SEM of the interval to gross uterine or cervical involutions on the basis of RP were compared to those recorded on the basis of transrectal US by using t-test. Also, by using t-test, the US-measured PGUHD and PGUH-MPT or US-measured CvD and CvWT on the Day that either of the pregravid uterine horn or the cervix was considered involuted on the basis of RP were compared with their counterparts measured by transrectal US on the Day of uterine or cervical involutions on the basis of transrectal US. The involution rate of PGUH as indicated by the rate of reduction in either of PGUHD or PGUHMPT was examined by repeated measures ANOVA followed by Bonfer-roni's multiple comparison tests. Also, the involution rate of the cervix as indicated by the rates of reduction in CvD or CvWT was examined by repeated measures ANOVA. 242 A. Ramoun, E. Almadaly, H. Hattab, S. Darwish, I. EL- Kon Results Intervals to gross uterine and cervical involutions on the basis of RP and US The interval to gross uterine involution as being detected by RP was shorter (P < 0.05) than its counterpart detected by the transrectal US. The interval to gross cervical involutions as being detected by RP was shorter (P < 0.01) than its counterpart detected by transrectal US (Table 1). The US- measuredPGUHD and CvD on the day of gross uterine and cervical involutions on the basis of RP and US The US-measured PGUHD and PGUHMPT, recorded on the day of a gross uterine involution on the basis of RP, showed increases at P < 0.01 and P < 0.001 respectively compared with their counterparts recorded on the day of a gross uterine involution on the basis of transrectal US. Also, the US-measured CvD and CvWT recorded on the day of a gross cervical involution on the basis of RP showed increases (P < 0.05) compared with their counterparts recorded on the day of a gross cervical involution on the basis of transrectal US (Table 2). The Involution rate of pre-gravid uterine horn The involution rate of PGUH as being indicated by the rates of reduction in RP-measured PGUHD throughout involution period showed a decrease (P < 0.05) among interval: 3 to 6, 6 to 9 and 9 to 12 and no difference (P > 0.05) among intervals: 9 to 12, 12 to 15, 15 to 18, 18 to 21, 21 to 24, 24 to 27 and 27 to 30 (Fig. 1). The involution rate as being indicated by the rates of reduction in the US-measured PGUHD throughout the involution period showed a decrease (P < 0.05) among intervals: 3 to 6, 6 to 9,9 to 12, 12 to 15, 15 to 18, 18 to 21, 21 to 24, and no difference among intervals: 24-27, 27 to 30, 30 to 33 and 33 to 36 (Fig. 1).The involution rate of PGUH on the basis of rates of reduction in PGUHMPT showed significantly (P < 0.05) decrease among intervals: 3 to 6, 6 to 9, 9 to 12, 12 to 15, 15 to 18, 18 to 21, 21 to 24 and 24 to 27 days and no difference among intervals: 24 to 27, 27 to 30 and 30 to 33 respectively (Fig. 3). The involution rate of the cervix The involution rate of the cervix as being detected by the rates of reduction in the RP- measured CvD showed a difference (P < 0.05) among interval 3 to 6, 6 to 9 and 9 to 12, and no difference among intervals: 9 to 12, 12 to 15, 15 to 18, 18 to 21, 21 to 24, 24 to 27, 27 to 30 and 30 to 33. On the other hand, the involution rate as being detected by the rates of reduction in the US-measured CvD showed differences (P < 0.05) among intervals: 3 to 6, 6 to 9, 9 to 12, 12 to 15, 15 to 18, 18 to 21, 21 to 24, 24 to 27, 27 to 30, and 30 to 33 and no difference (P > 0.05) among intervals: 33 to 36, 36 to 39 and 39 to 42 (Fig. 2). The involution rate of the cervix on the basis of the rates of reduction in CvWT showed a significant decrease (P < 0.05) among postpartum intervals: 3 to 6, 6 to 9, 9 to 12, 12 to 15, 15 to 18, 18 to 21, 21 to 24, 24 to 27, 27 to 30, 30 to 33 and 33 to 36 and no difference (P > 0.05) between intervals from 36 to 39 and 39 to 42 days (Fig. 4). Table 1: Intervals to gross uterine and cervical involutions detected on the basis of rectal palpation and trans-rectal ultrasonography Parameter Method of Judgment Rectal palpation (RP) Ultrasonography (US) Interval to gross uterine involution Interval to gross cervical involution 24.8 ± 0.96 30.5 ± 0.92 29.3 ± 0.89* 37.0 ± 0.10** Means within the same row and bearing different superscripts were significantly different at * P < 0.05;** P < 0.01. The interval to gross uterine involution as being detected by RP was shorter (P < 0.05) than its counterpart detected by transrectal US. The interval to gross cervical involutions as being detected by RP was shorter (P < 0.01) than its counterpart detected by transrectal US. Transrectal ultrasonography and rectal palpation for judging uterine and cervical involutions in buffalo 243 Table 2: The ultrasound- measured PGUHD and CvD as well as PGUHMPT and CvWT on the days of gross uterine and cervical involutions on the basis of RP and US Day of involution Uterine dimensions Cervical dimensions PGUHD PGUHMPT CvD CvWT Based on RP 2.34 ± 0.06** 0.64 ± 0.02*** 2.01 ± 0.06* 0.52 ± 0.02* Based on US 2.09 ± 0.04 0.47 ± 0.01 1.72 ± 0.08 0.43 ± 0.02 Means within the same column and bearing different superscripts are significantly different at * P < 0.05;** P < 0.01; *** P < 0.001. PGUHMPT means ultrasound -measured combine thickness of both myometrium and perimetrium of the pregravid uterine horn; PGUHD means the diameter of the pregravid uterine horn; RP means rectal palpation, US means ultrasonography. Both the US-measured PGUHD and PGUHMPT, recorded on the day of gross uterine involution on the basis of RP, showed increases at P < 0.01 and P < 0.001 respectively compared with their counterparts recorded on the day of gross uterine involution on the basis of transrectal US. Both the US-measured CvD and CvWT recorded on the day of gross cervical involution on the basis of RP showed increases (P< 0.05) compared with their counterparts recorded on the day of gross cervical involution on the basis of transrectal US. O PL, a 0 1 0.05) among the rest of intervals from 3rd (9-12) till 8th interval (24-27). On the other hand, the rate of reduction in the US-measured PGUHD shows a decrease (p<0.05) among intervals from 1st (3-6) till the 7th (21-24) but no difference are observed among intervals from 7th till 10th (30-33). 244 A. Ramoun, E. Almadaly, H. Hattab, S. Darwish, I. EL- Kon o fl 0 1 « i-H tH o « C3 & 1.5 1.4 1.3 1.2 1.1 1.0 0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0.0 RP US j d d d d d d d k 3 6 9 12 15 18 21 24 27 30 33 36 39 42 Days of RP and US e Figure 2: Rate of reduction in the CvD in buffaloes throughout the involution period on the basis of RP and US examinations. CvD = Cervical diameter; RP = Rectal palpation; US = Ultrasound examination. For rectal palpation, values carrying different letters from a - d differ at P < 0.05. For ultrasound examination, values carrying different letters from e - o differ at P < 0.05. The rate of reduction in the CvD on the basis of RP shows a decrease (p<0.05) among intervals: 3-6, 6-9 and 9-12 and no difference (p > 0.05) among intervals from 3rd (9-12) till 9th (27-30). On the other hand, the US-measured CvD shows among 1st (3-6) till the 10th (30-33) intervals and no difference (p>0.05) among intervals from 10th till 13rd (39-42). Figure 3: The rate of reduction in the PGUHMPT per 3 days throughout the involution period on the basis of US in buffaloes. US = Ultrasound examination; PGUHMPT= combine thickness of the myometrium and perimetrium. *Values carrying different letters differ at P < 0.05. The PGUHMPT shows a decrease (p < 0.05) among intervals from the 1st (3-6) till the 8th (24-27) and no difference among the intervals from 8th till 10th (30-33). Transrectal ultrasonography and rectal palpation for judging uterine and cervical involutions in buffalo 245 0.5 -i 0.4 S= -—) .2 S t3 S 0.3 3 "—' "S ^ " 0.2 e 0.1 0.0 "3 6 9 12 ^ 2*ï 214 27 Days of US 30 33 36 39 Figure 4: Rate of reduction in CvWT per 3-days throughout involution period by transrectal US in buffaloes. US = Ultrasound examination, CvWT= Cervical wall thickness. Values carrying different letters differ at P < 0.05. The CvWT shows a significant decrease (P < 0.05) among postpartum intervals: 3 - 6, 6 - 9, 9 - 12, 12 -15, 15 -18, 18 - 21, 21 - 24, 24 - 27, 27 - 30, 30 - 33 and 33 - 36 and no difference (P > 0.05) was detected between intervals from 36 - 39 and 39 - 42 days. Discussion The aim of the present study was to compare the efficiency of RP and the transrectal US in judging cervical and uterine involutions in buffaloes. On the basis of RP, the interval to gross uterine involution (24 days) may come in concurrence with previous studies (17, 18) in buffaloes. On the other hand, longer intervals to gross uterine involution, 24-56 days (19); 24-44 days (20) and 21-74 days (21), were recorded in buffaloes. Also, shorter intervals, 14-29 days (22) and 20 days (23) were recorded in cows on the basis of RP. On the basis of transrectal US, the interval to gross uterine involution (30 days) in the present study, more or less, coincides with those recorded in previous studies: 30 days (24); 28 days (11); 31 days (25) and 27 days (17). Matching the interval to gross uterine involution either on the basis of RP or the transrectal US, recorded in the present study, with their counterparts in previously mentioned studies herein revealed that while greater variations were observed in case of RP, lesser variations were noted in case of transrectal US. This observation indicates the higher accuracy of transrectal US compared with RP for judging uterine involution. However, the wide variations between the interval to gross uterine involution on the basis of RP in the present study and intervals recorded in the previous studies might be due to the variations among the criteria used to evaluate the uterine involution and/or variations among individual palpators during measuring the dimensions of the involuting uterus (9). Also, Okano and Tomizuka. (1987) (16) attributed the inaccuracy in estimating the uterine dimension by RP to the variations in fingers width among palpators and nature of the post-partum uterus which is being relaxed and flat especially during early puerperium. However, the previously mentioned limitations, that affect the efficiency of RP in estimating uterine dimensions, are not present in case of transrectal US. The interval to gross cervical involution on the basis of RP coincides with that (31.1) recorded byEl-Fouly et al. (1976) (26)and Usmani et al. (2001) (27) in buffaloes but was being longer than that, 25, recorded by Atansov et al. (2012) (10)in buffaloes. However, the disagreement between the current study and the study of Atansov et al. (2012) (10) may be attributed to individual variations among palpators in the two studies. The increase in the interval to gross uterine or cervical involution on the basis of transrectal US compared with RP is believed to a b c d g h k k k 0 246 A. Ramoun, E. Almadaly, H. Hattab, S. Darwish, I. EL- Kon be expended in estimating the lesser differences (in terms of mm) in diameter of either pregravid uterine horn or cervix which would be supposed to still undergo involution on the basis of transrectal US. On the other hand, the reductions in the diameters (in terms of < 0.5 cm) between successive RP were greater thus required a shorter time to reach stability. Moreover, the US scanning of both uterus and cervix during assessing the involution gives more diagnostic criteria that cannot be accurately detected by RP such as the wall thickness of cervix and pregravid uterine horn as well as smaller amounts of intrauterine fluids (Lopez-Helguera et al. 2012) (12). Doubtless, the combining between the US- measured PGUHMPT or CvWT and corresponding PGUHD or CvD as diagnostic criteria, for assessing uterine and cervical involutions will be better than using either of them solely especially in case of assessing uterine involution. However, US measuring of the PMGHMPT may be more accurate than measuring PMGHD because the PGUH in some cases may be compressed under linear probe while it is being positioned over it giving false results. Thus, measuring PMGHMP could be efficiently used as a complementary parameter to PMGHD for assessing uterine involution. The benefit of assessing the involution rate of PGUH and cervix on the basis of detecting the rate of reduction in their diameters by RP or transrectal US was to check the efficiency of RP and transrectal US in determining the time when the greatest reduction in the uterine or cervical size had occurred as well as the normalcy of the involution process at any time throughout the involution period. Cengic et al. (13) reported that the speed of uterine involution could be monitored by detecting the decrease in the uterine horn diameter and its wall thickness. The results of the present study indicated that the greatest rate of reduction in the PGUHD had occurred during the intervals 0-9 days on the basis of RP and 0-15 days on the basis of transrectal US. Afterward, the rates of reduction in the successive RPs or US examinations became relatively smaller. This indicates that the higher efficiency of US compared with RP in determining the time in which the greatest reduction in the size of PGUH had occurred. However, the pattern of reduction in US-measured PGUHD throughout involution period may come in coincidence with that described by Atansov et al. (10). Matching the involution rates between RP and transrectal US methods revealed that although there were no differences among rates of involution beyond 9th day postpartum in the case of RP, there were differences among all transrectal US examinations conducted from 3rd until 27th day except during the interval from 18th to 21st day indicating the higher efficiency of transrectal US in assessing involution rates throughout the involution period. Matching the involution rate of the cervix as indicated by the rates of reduction in the CvD on the basis of RP with their counterparts on the basis of transrectal US revealed that although the involution rate detected by RP and the transrectal US on the Day 9 became similar, it abruptly decreased in case of RP to 0.29 cm/3 days on the 12th day and remained so until the end of involution period. On the other hand, in the case of transrectal US, it gradually decreased significantly until reaching comparable value, 0.30 cm/ 3days, on the 18th day. However, these results indicated the subjectivity of RP and higher efficiency of transrectal US in the follow up of the cervical involution. The higher reduction rates (1.40 and 1.25 cm/3 days recorded during postpartum intervals 3 to 6 and 6 to 9 respectively) in US-measured CvD indicate that the greatest cervical involution occurs within the first postpartum week. These results partially agree with Atansov et al. (10) who found that the mean CvD decreased by more than 45% between the first and seventh days postpartum. Also, the results of the present study agree with the same authors beyond day 19 whereas CvD regressed slowly until the 25 th day while non-significant differences were detected in CvD between the 25th and 34th day. However the overall pattern of the rates of reduction in the US-measured CvD may be comparable with that recorded by Pariksh et al. (1) in Buffalo who reported that the reduction in the Transrectal ultrasonography and rectal palpation for judging uterine and cervical involutions in buffalo 247 CvD was faster until the 15th day, marginal from the 16th to the 25th day and became negligible beyond the 25th day postpartum. Regarding the involution rate of the PGUH on the basis of rates of reduction in PGUHMPT, it was noted that the greatest rate of reduction (0.88 cm/3 days) was recorded in the period from the 3rd to the 6th day postpartum indicating that the greatest reduction in the PGUHMPT had occurred during the first week. Afterward, it abruptly decreased to 0.32 cm between 6th and 8th day. Later on, it was regularly decreasing (at 0.04 cm) from 0.32 between 6th and 8th to 0.16 cm in the interval between 18th and 20th day. This indicates that the involution rate became slower as the time became farther from parturition. However, beyond day 21, the involution rate was similar and became nearly negligible. Regarding the involution rate of the cervix on the basis of the rates of reduction in CvWT, it was noted that the greater rates of reduction were recorded in the intervals: 3 to 6 and 6 to 9 days. Afterward, although the differences between the rates of reduction among succeeding the transrectal US were significant, the decrease in the CvWT was slow. Beyond day 30, the rates of reduction among 3 successive transrectal US became similar and nearly negligible. This may be explained in light of the structural components of the cervix, which is mainly formed from connective tissues rather than smooth muscles as in the case of the uterus. It is concluded that US- estimation of the diameter and the combined thickness of myome-trium and perimetrium of the pregravid uterine horn as well as CvD and CvWT thickness is more accurate than RP-measured diameters of pregravid uterine horn and cervix for assessing the involution of the reproductive tract in buffaloes. Acknowledgments The authors would like to thank all colleagues in the Animal Production Research Institute for their help during conducting the practical part of the current work. Conflict of interest The authors declare that they have no conflict of interest. References 1. Parikh S, Suttar B, Sutaria T, Savaliya B, Makwana, R. Ultrasonographic evaluation of uterine involution in postpartum Mehsana buffaloes. Bull. Environ Pharmacol and Life Sci 2017; 6: 3845. 2. Sheldon I and Dobson H. Effect of administration of eCG to postpartum cows on folliculo-genesis in the ovary ipsilateral to the previously gravid uterine horn and uterine involution. J Reprod Fertil 2000; 119: 157-63. 3. Sheldon I, Williams E, Miller A, Nash D, Herath S. Uterine diseases in cattle after parturition. Vet J 2008; 176: 115-21. 4. Morrow D, Robert S, Mckntee K. A review of Postpartum ovarian activity and involution of uterus and cervix in dairy cattle. III. Days nongravid and services per conception. The Corn Vet 1969;59: 190-8. 5. Willams E, Fischer D, Noakes D, England G, Rycraft A, Dobson H, Sheldon I. The relationship between uterine pathogen growth, density and ovarian function in the postpartum dairy cow. Theri-ogenology 2007; 68: 549-59. 6. Kask K, Kindahl H, Gustafsson H. Resumption of uterine and ovarian functions following dex-amethasone-induced parturition in dairy cows. Acta Veterinaria Baltica 2000; 3: 11-21. 7. Stephen C. Effect of ecbolic therapy on uterine involution, disease and reproductive performance in dairy cows. 2010; Ph.D. Thesis, Faculty of graduate studies, the University of Guelph. 8. Van Engelen E, Taverne M, Everts , Van der weijden G, Doornenbal A, Breeveld Dwarkasing, V.Cervical diameter in relation to uterine and cervical EMG activity in earlypostpartum dairy cows with retained placentas after PGF2alpha induced calving. Theriogenology 2007; 68: 213-22. 9. Usmani R, Lewis G. Cervical and uterine involutions in Nilli-Ravi buffaloes. Buffalo bullet. 1984; 4: 3-7. 10. Atansov A, Dineveh J, Yotova S. Ultrasonographic evaluation of uterine involution in Bulgarian Murrah buffalo after administration of oxytocin. Anim Reprod Sci 2012; 133: 71-6. 11. Yindee M, Techakumphu M, Lohachit C, Sirivaidyapong S, Chiangmai A, Colenbrander B. Ovarian activity and sexual behavior in the postpartum swamp buffaloes (Bubalus bubalis). Ital J Anim Sci 2007; 6: 632-5. 12. Lopez-Helguera I, Lopez-Gatius F, Gracia-Ispierto I. The influence of genital tract status in the 248 A. Ramoun, E. Almadaly, H. Hattab, S. Darwish, I. EL- Kon postpartum period on subsequent reproductive performance in high producing dairy cows. Theri-ogenology 2012; 77: 1334-42. 13. Cengic B, Varatanovic N, Mutevelic T, Katica A, Mlaco N, Cutuk, A. Normal and abnormal uterine involution in cow monitored by ultrasound. Biotechnol Anim Husb 2012; 28: 205-17. 14. Perry G and Cushman R. Invited Review: Use of ultrasonography to make reproductive management decisions. The Prof Anim Sci 2016; 32: 154-61. 15. Okano A and Tomizuka T. Ultrasonic observation of postpartum uterine involution in cows. Theriogenology 1987, 27: 369-76. 16. Hajurka J, Macak V, Hura V. Influence of health status of reproductive organs of uterine involution in dairy cows. Bull Vet Inst, Pulawy 2005; 49: 53-8. 17. Usmani R, Ahmad M, Inskeep E, Dailey R Lewis P, Lewis G. Uterine involution and postpartum ovarian activity in Nili-Ravi buffaloes. Theriogenology 1985;24:435-48. 18. Tiwari S, Pathak M, Patel A. Study of ovarian steroids during the postpartum period of Surti buffaloes in relation to suckling and milking practices. Ind J Anim Reprod 1995; 16: 5-8. 19. Khattab, R, Abou-Ela M, Barkawi A, AbdelAal A. Physiological causes influencing open periods in Egyptian buffaloes of hot season calvers. Buffalo .J 1990; 2: 153-60. 20. El-Sabbagh K. A study on some biochemical constitutes of blood female buffalo during late pregnancy and puerperium. M.V.Sci. Thesis 1993; Faculty of Veterinary Medicine, Zagazig University, Egypt. 21. Qureshi M, Samad H, Nazir Ahmad Habib G, Anjum A, Siddiqui M. Reproductive performance of dairy buffaloes under peri-urban commercial farming in NWFP, Pakistan. Pakist Vet J 1998; 18: 197-2. 22. Purbey L. A composite study of Dangi herd of cattle with special reference to certain aspects of the physiopathology of reproduction. 1965; M.V.Sci. Dissertation, Bombay University. 23. Pandey S and Pandit R. Studies on the efficacy of involon in the treatment of retention of the placenta and subsequent reproductive performance in frieswal cows. Pashudhan 2003; 8: 4-8. 24. Kranthi K. Ultrasonographic evaluation of uterine involution and postpartum cyclicity in graded Murrah buffaloes (Bubalus bubalis). MV Sci Thesis 2016 SRI VENKATESWARA Veterinary University, TIRUPATI-517502 (A.P) India. 25. Khasatiya C, Kavani F, Dhami A, Derashri H, Panchal M, Desai P, Panchal M, Desai P. Studies on puerperal events and reproductive efficiency following hormone therapy at day 42 postpartum in Surti buffaloes. Int J Agri Biol 2006; 8: 132-7. 26. El-Fouly M, Kotby E, El-Sobhy H. Effect of suckling on uterine and cervical involution in post-partum Egyptian buffaloes. Ind J Anim Sci 1976; 46: 221--9. 27. Usmani R Ahmad N, Shafiq P, Mirza M. Effect of subclinical uterine infection on cervical and uterine involution, estrous activity and fertility in postpartum buffaloes. Theriogenology 2001; 55: 563-71. Slov Vet Res 2019; 56 (Suppl 22): 249-56 DOI 10.26873/SVR-764-2019 Original Research Article METHICILLIN RESISTANT Staphylococcus aureus (MRSA) IN CAMEL MEAT: PREVALENCE AND ANTIBIOTIC SUSCEPTIBILITY Waleed R. El-Ghareeb12*, Faisal S. Almathen1, Mahmoud M. Fayez3, Raed A. Alsultan4 1Department of Veterinary Public Health and Animal Husbandry, College of Veterinary Medicine and Animal Resources, King Faisal University, 400 Al-Hasa, 31982, Saudi Arabia, 2Food Control Department, Faculty of Veterinary Medicine, Zagazig University, 44519, Egypt, 3Veterinary Serum and Vaccine Research Institute, Cairo, Egypt, 4Ministry of Environment, Water and Agriculture, Saudi Arabia Corresponding author, E-mail: welsaid@kfu.edu.sa Abstract: This study is an initiative study intended to investigate the prevalence and antibiotic susceptibility of Staphylococcus aureus and MRSA obtained from fresh camel meat retailed in Al-Hasa, Saudi Arabia. The survey has been conducted for five months where fresh camel meat were microbiologically analyzed to isolate S. aureus using culture media and VITEK2 technique. The Identification of S. aureus was done by DNA expression of gene specific 16S rRNA, while mecA gene expression was recognized in the identified MRSA isolates using PCR. The recorded prevalence of S. aureus was 10.7 % (20/187) from raw camel meat. Three isolates out of twenty S. aureus isolates were confirmed to be MRSA. Susceptibility to 10 antimicrobials was estimated using the disc diffusion method. These results suggested that MRSA is found in camel meat retailed at AlHasa, Saudi Arabia. We recommend that, surveillance protocol should be embraced in meat safety and public health programs. Key words: camel meat, S. aureus, MRSA, antimicrobial resistance Introduction Staphylococcus aureus is an important pathogen causing food-borne illness worldwide and causes serious diseases in human being (1). The main sources of infection could be the inadequate personnel hygiene, unhygienic handling and storage of food infected with staphylococci. Frequently associated foods are meat, dairy products, cream-filled bakery items and salads (2). Methicillin-resistant S. aureus (MRSA) appeared as a dangerous agent for different patients and especially in those with impaired im- mune system. MRSA strains are actually widespread human isolate but are unusual animal isolates (3). MRSA firstly recognized as nosocomial infection. Later in the community and livestock. It has a continuous and dynamic epidemiology. Human may acquire MRSA infection via contact with contaminated environment or contaminated marketed meat (4, 5). Resistance in MRSA is mediated by the gene mecA, which located on the chromosome in staphylococcal cassette chromosome mec (SCCmec), and this encodes penicillin-binding protein (PBP) 2a with a low affinity for beta-lactams (6).Transmission of MRSA from food Received: January 2019 Accepted for publication: February 2019 250 W. R. El-Ghareeb, F. S. Almathen, M. M. Fayez, R. A. Alsultan to people causes a serious problem especially for the immunocompromised people. S. aureus was identified in 42 out of 176 raw meat (beef, poultry and pork) over a one-year survey (7) and they did not find any evidence indicating vancomycin, or methicillin-resistance. S. au-reus also isolated from the wild animal carcasses (2.0% prevalence) and authors found no methicillin-resistance (8). Several studies in Saudi Arabia were conducted to investigate the prevalence, risk factors and genetic distribution of MRSA isolated from healthy and clinical human and animal cases (9-14). However, rare reports were recorded with regard to the prevalence of MRSA in food especially camel meat. To the best of our knowledge, few studies were conducted to isolate and identify MRSA from camel meat allover Arabian countries (15, 16).This study was done to investigate the MRSA prevalence in camel meat samples retailed to the public at Al-Hasa, Saudi Arabia. mec-A gene expression was carried out in identified S. aureus isolates using PCR. Antimicrobial resistance profile for the S. aureus isolates were further analyzed. This study is one of the very few studies regarding the prevalence and genetic characters of MRSA from camel meat in Saudi Arabia. Materials and methods All experiments followed the ethical and scientific principles adopted by King Faisal University, Saudi Arabia. Collection of samples A total of 187 minced camel meat samples were collected randomly from supermarkets in Al-Hasa province, Saudi Arabia in a 5 months period. The collected samples were taken directly in sterile polyethylene bags (icebox) to the Meat Hygiene laboratory at the Veterinary Public Health and Animal Husbandry Department, College of Veterinary Medicine, King Faisal University for the subsequent microbial culturing and identification. Isolation and identification of coagulase positive S. aureus For investigation of coagulase positive S. aureus, after sample digestion in a Stomacher® 400 Circulator, an inoculum of 0.1 ml of serial dilution 10-1&10-2 was evenly surface distributed on Baird Parker agar base supported with egg yolk tellurite emulsion Agar (Oxoid CM0275) according to (17). After incubation at 37°C for 48 hours, counting of all typical colonies using colony counter was performed. For confirmation, five typical colonies were selected and transferred to brain heart infusion broth tubes (BHIB) (Oxoid CM1135) for subsequent culture and preservation. Presumptive colonies were transferred and subcultured on Mannitol Salt Agar (Oxoid CM0085) and then incubated for 24 hrs at 37°C. Gram stain, cata-lase, mannitol fermentation, DNAs and coagulase tests were applied on suspected colonies to identify coagulase S. aureus (18). In addition, VITEK 2-compact was used to identify and differentiate staphylococci species (BioMérieux, Rev 03, 2004). Screening for MRSA Screening of All S. aureus isolates for their methicillin resistance was done by the standard disc diffusion procedure described by Clinical and Laboratory Standards Institute (19) using cefoxitin (30 pg). DNA Extraction Staphylococcus aureus isolates were grown in BHIB at 37o C for 18 hours. The cells were harvested by centrifugation at 8000 rpm for 20 minutes at 4o C and washed twice by phosphate-buffered saline. The pellet was suspended in 1 ml of 10 mMTris-HCl (pH 8) containing lyso-zyme (2.5 mg/ml) and incubate at 37o C for 2 hours. Genomic DNA was extracted using QI-Aamp DNA Mini Kit (QIAGEN) according to the instructions from manufacturer. DNA amplification by Polymerase Chain Reaction (PCR): Detection of Staphylococcus 16S rRNA PCR was used for detection of Staphylococcus 16S rRNA using primers in (Table 1), according to (20).The PCR was performed in a 25ul volume, where 2 ul of the extracted DNA (100-ng) was added to 12.5 ul of oasig ™2X Methicillin resistant Staphylococcus aureus (MRSA) in camel meat: prevalence and antibiotic susceptibility 251 qPCR Master mix , 1 ul (0.5 mM concentration) of each primer set and 8.5 ul PCR grade H2O.PCR amplifications were performed in a MyGenie 32 Thermal Block (BIONEER as follows: Initial denaturation at 94°C for 5 min followed by 35 cycles of 94°C for 1 min, 57°C for 1 min, and 72°C for 2 min and ended with final extension for 10 min at 72°C and finally maintained at 4°C. Detection of mecA-gene The polymerase chain reaction (PCR) was used for detection of mecA-gene in antibiotic resistance strains using primers in (Table 1), according to (21). In a final volume of 50 ul, three ul of DNA template was added to 25 ul of oasig ™2X qPCR Master-mix; 1 ul (0.25 mM concentration) of each primer set and 20 ul PCR grade H2O. DNA amplifications were carried out in a MyGenie 32 Thermal Block (BIONEER as follows: Initial denaturation at 94°C for 5 min followed by 36 cycles of denaturation of 94°C for 30 s, annealing at 55°C for 30 s, and extension at 72°C for 1 min with a final extension at 72°C for 5 min. Strain MF01 (GenBank, KY647024.1) was used as a positive control for mecA gene.Ten microliters of PCR products (16S rRNA and mecA- genes) was analyzed by 1% agarose gel electrophoresis. A 100 bp molecular weight DNA ladder (Gel Pilot Plus, QI-AGEN) was used for the validation of length of the amplified products. Antimicrobial susceptibility All isolates were tested using 10 antimicrobial agents: Penicillin G (10U), ampicillin (10 pg), tetracycline (30 |ig), amoxicillin-clavu-lanic acid (20/10 pg), ciprofloxacin (5 pg), cefoxitin (30 |ig), trimethoprim/sulfamethoxa-zole (1.25/23.75 pg), erythromycin (15 pg), clindamycin (2 pg), gentamicin (10 pg), Antimicrobial sensitivity was monitored with the standard disk diffusion assay .The zone of inhibition was interpreted according to CLSI guidelines (19). S. aureus ATCC 25923 was used as reference strain. Results Primers for both staphylococcus 16S rRNA and mecA-gene were tabulated in table (1). Identification of S. aureus was done by DNA expression of gene specific 16S rRNA gene (figure 1). Figure (2) clarified that, S. aureus was present in 20 out of the 187 samples (10.7%). Of the 20 S. aureus isolates, three (15 %) were found to be MRSA, therefore, Figure 2 shows the prevalence of MRSA strains among total samples was only three (3/187) (1.6%). identification of MRSA was done by DNA expression of gene specific mec-A gene (figure 3). Table (2) showed different patterns of Antimicrobial susceptibility of S. aureus isolates. 35% were resistant to P-lactam, 50% were resistant to penicillin, 65% were sensitive to ciprofloxacin, 90% were sensitive to Erythromycin and Clindamycin, 85% were sensitive to Cefoxitin, 80% sensitive to Gentamicin, 60% were resistant to Penicillin G, 75% were sensitive to Trimethoprim and finally 75% were sensitive to tetracycline. Figure (4) declared the percentages of S. aureus resistant strains among the identified isolates using the disk diffusion method according to Clinical and Laboratory Standards Institute guidelines. Results showed a higher resistant level for both penicillin and Ampicillin (60% and 50%, respectively). As shown in table (3), only two isolates (10%) out of all analyzed strains exhibited single resistance to penicillin. MRSA isolates presented antimicrobial multi resistance. 252 W. R. El-Ghareeb, F. S. Almathen, M. M. Fayez, R. A. Alsultan Table 1: Primers for both Staphylococcus 16S rRNA and mecA-gene DNA target Primer pair Size (bp) Reference Staphylococ- 5'GTT ATT AGG GAA GAA CAT ATG TG-3' 750 Jaffe et al., cus 16S 5'CCA CCT TCC TCC GGT TTG TCA CC-3' 2000 rRNA mecA-gene 5' AAAATCGATGGTAAAGGTTGGC-3' 5' AG TTCTGCAGTACCGGATTTGC-3' 530 Murakami et al, 1991 Figure 1: Agarose gel electrophoresis of amplified 750-bp DNA fragment. Lanes: 1 DNA ladder molecular weight marker (GelPilot), 2-6 staphylococcus isolates, 7 ATCC 12600 Total samples Figure 2: Prevalence (%) of S. aureus and MRSA in camel meat samples Methicillin resistant Staphylococcus aureus (MRSA) in camel meat: prevalence and antibiotic susceptibility 253 X 234567 a 1000 700 500 •KO 300 200 100 Figure 3: Agarose gel electrophoresis of amplified 533-bp DNA fragment. Lanes: 1 DNA ladder molecular weight marker (GelPilot), 2-6 MRSA isolates, 7 Strain MF01, 8 Negative control. Table 2: Antimicrobial susceptibility of S. aureus isolates (n= 20): % S % I % R Antibiotic class Antimicrobial agent 65 0 35 ß-lactam+Inhibitors Amoxicillin/Clavulanic acid (AMC) 50 0 50 Penicillins Ampicillin (AMP) 65 5 30 Quinolones Ciprofloxacin (CIP) 90 0 10 Lincosamides Clindamycin (CLI) 90 0 10 Macrolides Erythromycin (ERY) 85 0 15 Cephems Cefoxitin (FOX) 80 0 20 Aminoglycosides Gentamicin (GEN) 40 0 60 Penicillins Penicillin G (PEN) 75 0 25 Folate pathway inhibitors Trimethoprim/Sulfamethoxazole (SXT) 75 0 25 Tetracyclines Tetracycline (TCY) R= Resistant, T=Intermediate, S=Sensitive Tetracycline (TCY) Trimethoprim v.1111,11 [ i ' 111 > ■. i. o I e (SXT) Penicillin G (PEN) Gentamicin (GEN) Cefoxitin (FOX) Erythromycin (ERY) Clindamycin (CLI) C ipr ofloxacin (CIP) Ampicillin (AMP) Amoxicillin/Clavulank: acid (AMC) Figure 4: Percentages of S. aureus resistant strains among the identified isolates using the disk diffusion method according to Clinical and Laboratory Standards Institute guidelines 254 W. R. El-Ghareeb, F. S. Almathen, M. M. Fayez, R. A. Alsultan Table 3: Antibiotic resistance profile (number of S. aureus isolates= 20) % N. of isolates Resistance profile 40 8 10 2 PEN I 5 1 PEN AMP GEN II 5 1 PEN AMP CIP III 5 1 PEN AMP CIP GEN TCY IV 10 2 PEN AMC AMP V 5 1 PEN AMC AMP CIP SXT VI 5 1 PEN AMC AMP CIP CLI GEN SXT TCY VII 5 1 FOX PEN AMC AMP CIP GEN SXT TCY VIII 5 1 FOX PEN AMC AMP ERY CIP SXT TCY IX 5 1 FOX PEN AMC AMP ERY CIP CLI SXT TCY X Discussion As a major meat species marketed in Saudi Arabia, camel meat was used for determination of the prevalence of S. aureus& MRSA and antimicrobial susceptibility profile of isolates. In a survey conducted for 5 months, 187 raw camel meat samples (retailed in Al-Hasa, Saudi Arabia) were analyzed. Identification of S. aureus was done by DNA expression of gene specific 16S rRNA (figure 1). Figure (2) clarifies that, S. aureus was present in 20 out of the 187 samples (10.7%). The prevalence of S. aureus in different foodstuffs and meats varied in the previous studies. They were reported 20.5% S. aureus from beef at USA (22). MRSA has been already isolated and identified from retail meat worldwide, and the possible human transmission existed (23-26 and 22). The reported prevalence varied which indicated that contamination by MRSA in different types of meat varied in the different localities. Of the 20 S. aureus isolates, three (15 %) were found to be MRSA, therefore, Figure 2 shows the prevalence of MRSA strains among total samples was only three (3/187) (1.6%) and this result is very low in comparison with that obtained by (16) who found that the prevalence of MRSA in camel meat retailed in Riyadh was (20%) while the prevalence of MSSA was (28%). This difference may be due to variations in hygienic levels, preparation and handling of meat. While our result was nearly similar to (22) and (27) who reported that 1.3% and 1.2%, respectively were positive for MRSA from meat retailed in USA. Meat play a significant role in transmission of antimicrobial resistance from livestock and food animals to human being, and antibiotic resistance usually has a great concern in hospital infection (nosocomial infections). Antimicrobial resistance may transfer via 3 ways: Through antimicrobial residues in food and meat, or consumption of resistant parts of original food microflora and transfer resistance to pathogenic microorganisms (28-31). The antimicrobial resistance was higher for Penicillin G and Ampicillin (60 and 50%, respectively). In addition, 15% of tested S. aureus strains were methicillin-resistant while 85% were sensitive to cefoxitin (table 2). Another study showed antibiogram sensitivity differences in S. aureus isolated from food (7). It was clear that MRSA isolates were resistant to all antimicrobials used. Figure 4 showed a higher resistant level for both penicillin and Ampicillin and this result is similar to that stated by (32) in chicken giblets. Only two isolates (10%) out of all analyzed strains exhibited single resistance to penicillin. MRSA isolates presented antimicrobial multi resistance (7 and 32); similar result was found in our study (table 3). Methicillin resistant Staphylococcus aureus (MRSA) in camel meat: prevalence and antibiotic susceptibility 255 Conclusion From the above, it could be concluded that 20 S. aureus positive isolates were identified out of 187 camel meat samples (10.7% prevalence). Three were resistant to methicillin (1.6 % prevalence), 35% of S. aureus isolates were resistant to P-lactam antibiotic. MRSA isolates were resistant to all antimicrobials tested, and 15 % of tested S. aureus strains were methicil-lin-resistant. Our results indicated low MRSA prevalence in camel meat, which pointed to the limited risk of transmission via meat. However, attention should be paid to the safety along food chain. Conflict of interest The authors declare that they have no conflict of interest. Acknowledgment The authors thank the Deanship of Scientific Research in King Faisal University, Saudi Arabia, for their support of this study. Many thanks is due to Prof. Dr. Mohammed M. Abdel Mohsen for his valuable reviewing and proof editing. References 1. Atanassova V, Meindl A, Ring C. Prevalence of Staphylococcus aureus and staphylococcal enter-otoxins in raw pork and uncooked smoked ham: a comparison of classical culturing detection and RFLP-PCR. Int J Food Microbiol 2001; 68:105-13. 2. Jett M, Ionin B, Das R, Neill R. The staphylo-coccal enterotoxins. In: Sussman M, editor. Molecular medical microbiology. San Diego, CA, USA: Academic Press2001; 1089:116. 3. Seguin JC, Walker RD, Caron JP, Kloos WE, George CG, Hollis RJ, Jones RN, Pfaller MA. Methicillin-resistant Staphylococcus aureus outbreak in a veterinary teaching hospital: potential human-to-animal transmission. Journal of Clinical Microbiology 1999; 37: 1459-63. 4. Petinaki E, Spiliopoulou I. Methicillin-re-sistant Staphylococcus aureus among companion and food chain animals: impact of human contacts. Clin Microbiol Infect 2012; 18(7):626-34. 5. Greeson K, Suliman GM, Sami A, Alowaimer A, Koohmaraie M. Frequency of antibiotic resistant Salmonella, Escherichia coli, Enterococcus and Staphylococcus aureus in meat in Saudi Arabia. Afr. J. Microbiol. Res 2013; 7: 309-16. 6. EFSA (European Food Safety Authority). Scientific opinion of the panel on biological hazards on a request from the European Commission on Assessment of the Public Health Significance of methicillin resistant Staphylococcus aureus (MRSA) in animals and foods. EFSA J. 993; 1-73. 7. Pesavento G, Ducci B, Comodo N, Lo Nostro A. Antimicrobial resistance profile of Staphylococ-cus aureus isolated from raw meat: A research for methicillin resistant Staphylococcus aureus (MRSA). Food Control 2007; 18: 196-200. 8. Traversa GR, Gariano S, Gallina DM, Bian-chi, Orusa R, Domenis L, Cavallerio P, Fossati L, Serra R, Decastelli L. Methicillin resistance in Staphylococcus aureus strains isolated from food and wild animal carcasses in Italy. Food Microbiol. 2015;52: 154-8. 9. Austin TW1, Austin MA, McAlear DE, Coleman BT, Osoba AO, Thaqafi AO, Lamfon MA. MRSA prevalence in a teaching hospital in Western Saudi Arabia. Saudi Med J 2003; 24(12):1313-6. 10. El Amin NM, Faidah HS. Methicillin-re-sistant S. aureus in the western region of Saudi Arabia: prevalence and antibiotic susceptibility pattern. Ann Saudi Med. 2012; 32(5):513-6. 11. Iyer AP, Baghallab I, Albaik M, Kumosani T. Nosocomial Infections in Saudi Arabia Caused by Methicillin Resistance Staphylococcus aureus (MRSA). Clin Microbial 2014; 3: 146. 12. Dagunton N, Salman K, Al-Saif S, Balkhy H. MRSA outbreak at a neonatal ICU in Saudi Arabia. Journal of Infection and Public Health 2015; 8 (4), 402-3. 13. Al Yousef SA, Taha EM. Methicillin-Re-sistant Staphylococcus aureus in Saudi Arabia: Genotypes distribution review. Saudi J Med Med Sci 2016; 4:2-8 14. El-Deeb W, Fayez M, Elmoslemany A, Kan-deel M, Zidan K. Methicillin resistant Staphylococ-cus aureus among goat farms in Eastern province, Saudi Arabia: Prevalence and risk factors. Prev Vet Med. 2018; 156:84-0. 15. Quddoumi SS, Bdour SM, Mahasneh AM. Isolation and characterization of methicillin-re-sistant Staphylococcus aureus from livestock and poultry meat. Annals of Microbiology 2006; 56 (2): 155-61. 16. Raji MA, Garaween G, Ehricht R, Monecke S, Shibl AM and Senok A. Genetic Characterization of Staphylococcus aureus Isolated from Retail Meat in Riyadh, Saudi Arabia. Front. Microbiol. 2016; 7: 911. 256 W. R. El-Ghareeb, F. S. Almathen, M. M. Fayez, R. A. Alsultan 17. American Public Health Association (APHA). Compendium of methods for the microbiological examination of food (4th ed.) 2001. Washington, DC: Author. 18. Quinn PJ, Markey BK, Leonard FC, Fitzpatrick ES, Fanning S, Hartigan P. Veterinary microbiology and microbial disease (2nd ed.) 2011; Oxford: Wiley-Blackwell. 19. Clinical and Laboratory Standards Institute (CLSI). Wayna, pa: Performance Standards for Antimicrobial Susceptibility Testing; Twenty-Fourth Informational Supplement 2014. Approved M100-S24. 20. Jaffe RI, Lane JD, Albury SV, Niemeyer DM. Rapid extraction from and direct identification in clinical samples of methicillin-resistant staphylococci using the PCR. J Clin Microbiol. 2000; 38:3407-12. 21. Murakami K, Minamide W, Wada K, Naka-mura E, Teraoka H. Identification of methicillin-re-sistant strains of staphylococci by polymerase chain reaction. J Clin Microbiol 1991; 29: 2240-4. 22. Bhargava K, Wang X, Donabedian S, Zervos M, da Rocha L, Zhang Y. Methicillin-resistant Staphylococcus aureus in retail meat, Detroit, Michigan,USA [letter].Emerg Infect Dis. 2011;1-4. 23. Pu S, Han F, Ge B. Isolation and characterization of methicillin-resistant Staphylococcus aureus strains from Louisiana retail meats. Appl Environ Microbiol. 2009; 75:265-7. 24. Weese JS, Avery BP, Reid-Smith RJ. Detection and quantification of methicillin-resistant Staphylococcus aureus (MRSA) clones in retail meat products. Lett ApplMicrobiol. 2010; 51:33842. 25. Lim SK, Nam HM, Park HJ, Lee HS, Choi MJ, Jung SC. Prevalence and characterization of-methicillin-resistant Staphylococcus aureus in raw meat in Korea. J Microbiol Biotechnol. 2010; 20:775-8. 26. de Boer E, Zwartkruis-Nahuis JT, Wit B, Huijsdens XW, de Neeling AJ, Bosch T. Prevalence of methicillin-resistant Staphylococcus aureus in meat. Int J Food Microbiol. 2009; 134:52-6. 27. Hansona BM, Dresslera AE, Harpera AL, Scheibel RP, Wardyna SE, L.K. Roberts LK, Kroegera JS, Smitha TC. Prevalence of Staphylo-coccus aureus and methicillin-resistant Staphylo-coccus aureus (MRSA) on retail meat in Iowa. Journal of Infection and Public Health 2011; (4): 16974. 28. Kruse H, Sorum H. Transfer of multiple drug resistance plasmids between bacteria of diverse origins in natural microenvironments. Applied and Environmental Microbiology 1994; 60(11): 4015-21. 29. Klein G. Food as a potential vector for antibiotic resistance. Berline Munch Tierarztl Woch-enschr 1999; 112: 365-9. 30. Teuber M. Spread of antibiotic resistance with food-borne pathogens. Cellular and Molecular Life Sci. 1999; 56: 755-63. 31. Mayrhofer S, Paulsen P, Smulders FJ, Hilbert F. Antimicrobial resistance profile of five major food-borne pathogens isolated from beef, pork and poultry. Int. J. Food Microbiol. 2004; 97: 23-9. 32. Darwish WS, Atia AS, Reda LM, Elhelaly AE, Thompson LA, Saad Eldin WF. Chicken giblets and wastewater samples as possible sources of methicillin-resistant Staphylococcus aureus: Prevalence, enterotoxin production, and antibiotic susceptibility. J Food Saf. 2018; 12:1-7. Slov Vet Res 2019; 56 (Suppl 22): 257-67 DOI 10.26873/SVR-765-2019 Original Research Article CORTICOSTEROID-INDUCED OSTEOPOROSIS AND OSTEONECROSIS: ROLE OF OXIDATIVE STRESS Reham H. Alattar1*, Abdel Alim F. Abdel Alim2, Sabry M.A. Abdelmetal2, Sayed A. Abdel Aziz2 1Zagazig University student's Hospital, Zagazig University, Egypt, 2Pharmacology Department, Faculty of Veterinary Medicine, Zagazig University, 44511, Egypt Corresponding author, E-mail: reham_attar2013@yahoo.com Abstract: Glucocorticoids (GC) play a significant role in body metabolism. In the last few years, advances and highlights have been made to understand the role of oxidative stress induced by corticosteroids in the pathogenesis of osteonecrosis (ON) and osteoporosis (OP) and the door for digging in GC mechanistic has been opened by the newly detection of high-affinity receptors for glucocorticoids and calcitriol in bones. The harmful free radicals produced by corticosteroid administration are strong emitters of many regulatory cytokines such as tumor necrosis factor (TNF), nuclear factor kappa p (NF-kB) and interleukins. For this, a great attention has been directed toward the possibility of using a novel free radical scavenger like natural antioxidant, e.g. ginseng, that can be helpful in the management of ON and OP. The use of antioxidants for the management of osteoporosis characterized by many improvements in the way of control the incidence rate of ON and OP. Many antioxidants have an anti-osteoporotic effect, with an overall redox state maintenance. Also, the lipid peroxides are alleviated and the intraosseous vascular integrity within the bone marrow are repaired. Moreover, the oxidative damage of DNA is contoured. The objective of this review is to highlight the role of oxidative stress in the pathogenesis of corticosteroid-induced osteonecrosis (ON) and osteoporosis (OP) and studying the possibility of using a novel free radical scavenger, a natural antioxidant, e.g. Ginseng, that can be helpful in the management of OP & ON. Key words: osteoporosis; osteonecrosis; corticosteroids; oxidative stress; ginseng Introduction Osteoporosis (OP) which is a widespread disease characterized by an abnormal mass, tissue, fragility and fracture risk of bones (1). Although, it is well known that impairment in the blood supply of the bone and administration of corticosteroids can lead to osteonecro-sis, the actual mechanisms of steroid hormone action on bone and how glucocorticoid- induced osteoporosis (GIOP) and how the cellular composition of bone is altered is still obscure. Secondary osteoporosis and fracture may occur in patients receiving corticosteroids either in high-doses or chronic therapy, which is considered as one of the major risk factors for ON (2). The oxidative stress was found to inhibit the differentiation and mineralization of bone and consequently induces necrosis of osteoblasts (3), and increases the expression of Received: January 2019 Accepted for publication: February 2019 258 R. Alattar, A. Abdel Alim, S. Abdelmetal, S. Abdel Aziz cytokines in the bone and thus induces osteoporosis. The effects of free radicals on the bone can be countered by the use of a proper antioxidant in a way that can prevent osteoporosis. The main topics which will be discussed in the review are: 1. Glucocorticoids. 2. Free radicals involved in osteoporosis and osteonecrosis and the possible role of an-tioxidants. 3. Involvement of oxidative stress in bone pathophysiology: Apoptosis and Caspa-ses. 4. Steroid-induced OP- animal models. 5. Osteoporotic biomarkers. 1. Glucocorticoids Owing to the potent anti-inflammatory effects of glucocorticoids, they are widely used as an essential mendicant (4). The prolonged use of these drugs may provide adverse effects not only on the metabolism of proteins, lipids and carbohydrates, but also has immunosup-pressive effects. Hyperglycemia is one of the most important effects that may play a role in the stress response and osteoporosis induced by glucocor-ticoids. The age-associated loss of bone mass and strength or the aging skeleton can be contributed to the inflammatory cytokines, but without clear molecular declare (5). In human subjects, GIO occurs in two phases: a rapid early phase in which bone mineral density falls, occurs 5-15% per year, probably due to excessive bone resorption. A slower phase, characterized by a more slowly decline of bone mineral density, and occur 2% per year, which mainly attributed to bone formation defect (6). 1.1 Glucocorticoids mechanism The mechanism of glucocorticoids action is found to be mediated by glucocorticoid receptors (GCR), belong to the nuclear steroid hormone receptor family, expressed in bone cells (7). In spite of the recent success in the molecular biological techniques, the precise molecular mechanism of steroid hormone action has remained obscure. These receptors include cytosolic glucocorticoid receptor (cGCR) for both the classical genomic and non-genomic mechanism of glucocorticoid action and membrane-bound glucocorticoid receptor (mGCR) for the mediated non-genomic effects. While, the interactions with cellular membranes result in a non-specific, non-genomic effects caused by direct interaction with the cell membranes (8). Seven members of steroid receptors are recognized: estrogen receptor a & b, estrogen-related receptors 1 & 2, and the receptors for mineralocorticoids, androgens and progesterone (9, 10). The gene encoding the human GR (hGR) is located on chromosome 5 in loci 3132 (5q31-32) (11). Three main functional domains are included in each receptor; N-terminal domain, DNA-binding domain (DBD), and ligand-binding domain (LBD). Two distinct receptor forms for glucocorti-coids in human, arise from alternative splicing of exon 9: hGR-a and hGR-p. While the classical signaling and modulation of gene expression are attributed to a- receptor form, while P-form may function only as a dominant negative inhibitor of a-signaling (8). The effects of glucocorticoids on bone differ according to the developmental stage i.e. from the beginning stage to the late stage, from human to animal species and moreover in a cell-type-specific manner (3). While GCs appear to be essential for the early osteoblastic stage, responsible for the differentiation of osteoblasts into bone-forming osteoblasts, act as inhibitory at the very late stages of osteo-blast differentiation and osteocytes (12). In bone, local activation of the inactive cortisone into the active Cortisol by 1ip-hydroxysteroid dehydrogenase is a pivot point, is responsible for function of osteoblasts, differentiation, proliferation and. One of the most important factors that cause increases in the bone turnover, loss of bone mass and fracture risk is the changes in the levels of steroid hormone as occurs in the adulthood of either sex. In osteoblasts and osteoclasts, estrogen receptor a (ERa) has a protective effect on bone (14, 15). Corticosteroid-induced osteoporosis and osteonecrosis: role of oxidative stress 259 Figure 1 : Pathophysiology of glucocorticoid-induced osteoporosis, adapted from (12, 13) 2. Free radicals involved in osteoporosis and osteonecrosis The proper bone modeling is controlled mainly by the function of two types of bone cells, osteoclasts (bone formation) and osteoblasts (bone resorption), and OP occurs when the balance between both functions is disrupted (16-18) . This balance is affected by many risk factors including high-doses of cortico-steroids, alcohol abuse, genetic, race, hormonal, mechanical and nutritional factors. 2.1 Free radicals The presence of unpaired electron(s) in atomic or molecular orbitals makes the molecule unstable and highly reactive and considered a free radical. As shown in table (1), free radicals are usually, but not always oxygen, which include reactive oxygen species (ROS) and reactive nitrogen species (RNS) (19) . Numerous agents and factors can induce significant generation of ROS, including drugs as anticancer, diseases as diabetes and environmental as UV irradiation, pollutants and toxicity (12). As mitochondria is the major generator of ROS. In cells, the main source of O2- is the leakage of electron(s) from the mitochondrial ETC, and therefore the damage occurs mainly for mtDNA leading to apoptosis (20). Under physiological conditions, H2O2, but not O2'" is produced by the peroxisomes (21). Oxygen free radicals can also be produced by NADPH oxidase on osteoclasts, and xanthine oxidase and NOS (22, 23). Also, treatment with interferon (IFN)-y, which act as a stimulator of the activity of NADPH-oxidase enzyme lead to liberation of free radicals. 2.3 Hyperglycemia-induced oxidative stress Various mechanisms were adopted to describe the hyperglycemia-induced oxidative stress. One mechanism is the increased production O2" by the disrupted ETC, which is increased according to diabetic complication state. Also, the oxidative stress is increased in diabetes because of the accelerated poly-ol/sorbitol pathway and the decrease in NADPH availability and thereby GSH depletion (24-26). 2.4 Oxidative stress Although, free radicals have been shown to play beneficial roles in biology, especially when present in physiological concentrations, may act as a second messenger in some of the signal transduction pathways. When the production of these free radicals is greater than the ability of the cell to detoxify, an oxidative stress is developed and oxidative damage of many biological molecules, especially proteins and nucleic acids occurs (27, 28). The levels of G-SH, is decreased, while the lipid peroxides (LPO) and advanced glycation end product (AGE) are accumulated (28-30). 260 R. Alattar, A. Abdel Alim, S. Abdelmetal, S. Abdel Aziz Table 1: Different reactive species Reactivity / Remarks Half life ( in sec) Symbol Reactive species I. Reactive oxygen species Generated in mitochondria , in cardiovascular system 10-4 s and others, very highly reactive , generated during iron overload and such conditions in our body 10-9 s Formed in our body by large number of reactions and Stable yields potent species like +OH Reactive and formed from lipids, proteins, DNA, sug- S ars etc. During oxidative damage Reacts with transient metal ions to yield reactive spe- Stable cies Highly reactive , formed during photosensitization 10-6 S and chemical reactions Present as an atmospheric pollutant , can react with S various molecules, yielding 1O2 II. Reactive nitrogen species Neurotransmitter and blood pressure regulator, can S yield potent oxidants during pathological states Formed from NO2 and 10-3 s O2- ■OH H2O2 ROO. ROOH O3 - Superoxide - Hydroxyl Radical - Hydrogen Peroxide - Peroxyl radical - Organic hydroperoxide - Singlet oxygen - Ozone NO. - Nitric oxide ONOO" - Peroxynitrite !O 2 The induction of OP and/or ON by the accumulated free radicals can be explained by several axes. An important axis occurs by inhibition the differentiation of two vital cell lines, the marrow stromal M2-10B4 cell line, and the pre-osteoblastic cell line. In this mechanism, the early differentiation marker, alkaline phos-phatase (ALP) was found to be markedly increased and the mineralization in these cell lines is lowered (31). Another one, is the foundation that metallothionein (MT) has a protective effect against H2O2-induced inhibition of the differentiation of osteoblasts and has the ability to scavenge 'OH & O2'" free radicals by mechanisms similar to G-SH and superoxide dismutase, respectively (32-34). It has been reported that the free radicals is a strong emitter of the expression of many cytokines and many of these cytokines are involved in bone osteoporosis including TNF-a, NF-kB, IL6 and IL-1 (35). The production of these signals is regulated by steroids. While TNF-a and IL-1 are a powerful bone resorption effectors by increasing the number of osteoclasts, IL-6 is an osteotropic (36, 37). NF-kB is involved in the regulation of cell growth, differentiation, death and development of bone and is a downregulator of the differentiation of osteoblasts (38-40). 2.5 Role of antioxidants in osteoporosis Oxidative stress arise when the production of the radicals oxygen and non-oxygen free radicals exceed the capacity of the antioxidants to overcome. A proper antioxidant (enzymes and non-enzymes) is essential to protect against free radicals (41, 42). Enzyme antioxi-dants include: superoxide dismutases, gluta-thione peroxidases, catalase, glutathione S-transferase, glutathione reductase, peroxire-doxins, glutaredoxin and thioredoxin Nonenzyme antioxidants include: Reduced gluta-thione (G-SH), tocopherols and P-carotenes (43). Figure 2: Possible mechanisms of antioxidants adapted from (44) Corticosteroid-induced osteoporosis and osteonecrosis: role of oxidative stress 261 In the post-menopause, the bone mineral density is decreased, the susceptibility for OP is increased and the antioxidants are depleted. Such condition can be alleviated by regular intake of exogenous antioxidant (45). The metabolism of calcium is dramatically affected, because of a decrease in the gastrointestinal calcium absorption and increase in the renal calcium loss. These manifestations are recorded in glucocorticoid administration. It was reported that co-administration of the antioxi-dants tocopherol or ascorbate can withstand the development of corticosteroid-induced ON (46-48). In addition, a secondary hyperparathyroidism has been suggested as a determinant effector of bone (49). 3. Osteoporotic biomarkers It have been suggested that osteoporosis is associated with several biochemical markers that can be taken as a monitor for the oxidative stress-induced OP and ON to follow-up and the possible ameliorative effect of a natural antioxidant. 4. Antioxidants 4.1 Genistein (Giensing or Ginseng) Ginseng, one of the most popular oriental medicinal herbs, widely used as an herbal remedy for various physiological and pathological disorders. White and red ginseng have been used as an excellent stimulant for the immune and antimicrobial defenses (51). Genistein is not only an effective scavenger of free radicals in vitro, but also a strong inhibitor of cell-derived H2O2 formation in vivo. It has an antioxidant capacity that may exceed that of acrobate and tocopherols (52). 4.2 Ginseng as a hypoglycemic effector An proposed and accepted anti-osteoporetic effect of ginseng is the hypoglycemic effect. It has been reported that the blood glucose level and the glycogen content are improved and controlled in ginseng-supplemented diabetics. One possible mechanism of this hypoglycemic effect of ginseng is the antioxidant scavenging effect, enhancement of the aerobic glycolysis through stimulation of P-adrenoreceptor and increase of various rate-limiting TCA enzyme activities (53). Ginseng supplementation significantly reduce the incidence of ON in the most studied corticosteroid- induced ON animal models, characterized by a marked increase in the GSH content and MDA levels decreased. Thus providing a fact that oxidative stress and ON was inhibited by ginseng (16, 28). 5. Involvement of oxidative stress in bone pathophysiology The pathophysiology of the oxidative stress varies according to the load of the stress and the provided antioxidant capacity. Oxidative stress in low levels may function as a signal mediator for cell growth and cellular signaling (54). In contrast, higher oxidative stress is harmful, causing cellular injury and involved in various disease pathogenesis. The damage include enzymes, structural proteins, lipids, and DNA are seriously affected in a way can lead to apoptosis (55). Bone architecture and osteoblast / osteoclast balance is also altered (28). 6. Apoptosis The programmed cell death (PCD), apopto-sis, is actually essential for the normal development and homeostasis and the removal of the damaged, infected and potentially neo-plastic cells (56). Glucocorticoids are potent inducers of apoptosis in many cells(30). GCs have proapoptotic effects on osteoblasts and osteocytes due to activation of caspase-3, the key mediator of apoptosis (56). As previously mentioned, apoptosis-induced by intake of corticosteroids varies according to the form of the receptor present; glucocorticoid receptors a-C (GRa-C) is a potent apoptotic inducer than GRa, while GRa-D is less (57). 6.1 Caspases Caspases are major player in the cell death by apoptosis. These cysteine-dependent caspa-ses are affected by many factors especially the redox state of the cell. As shown in figure (3): The function of glucocorticoid receptors can propagate signals either for pro-apoptotic genes or for anti-apoptotic genes that can either lead to apoptosis or cell survival, respectively depending on the cell type or tissue (58, 59). 262 R. Alattar, A. Abdel Alim, S. Abdelmetal, S. Abdel Aziz 2. Assessment of bone resorption 3. Biochemical determinations Table 2: Biomarkers can be used to follow-up the oxidative stress-induced osteoporosis, adapted from (50). _Biomarkers_ 1. Markers of -ALP: An osteoblastic differentiation marker. bone formation -Serum measurement of total and bone-specific alkaline phosphatase (BALP) - Osteocalcin (OC), -Type I collagen extension peptides. - Urine specimens were assayed for markers of bone resorption: N - telopeptides (NTX), free pyridinolines (Pyr), free deoxypyridinoline. - Measurement of fasting urinary calcium and hydroxyproline, -Urinary hydroxylysine glycosides, urinary excretion of the pyridinium cross-link and bone - specific alkaline phosphatase. - Osteoclast enzymes: .Tartarate-resistant acid phosphatase (TRACP) andCathepsin K. -The lipid peroxide marker, MDA. -Calcitonin. -DNA damage marker, 8-OHdG. -Antioxidant status: Reduced blood glutathione (G-SH). -The antioxidant enzymes: SOD, Catalase and Glutathione peroxidase (GPX). -Endothelin (ET) in plasma. -PTH, Calcium & Phosphorus. -The presence/absence of advanced glycation end-product expression. -Proinflammatory cytokines: (IL-1a, ip, 6, TNF- a). -Anti-inflammatory: (IL-4, 10). -Bone-marrow-specific cytokines: (IL-7 & 11). -Caspase: 1, 3, 7 & 9. -Caspase-activated DNase (CAD). -Adipogenic transcription factor (PPARy). -TGF-P2. - Vascular endothelial growth factor (VEGF). - Tumor necrosis factor-a (TNF- a). - Tumor necrosis factor-receptor-1 (TNF-R1). -The pro-apoptotic Bcl-2 (Bim). -The pro-apoptotic proteins (Bax/Bak). - NF-kB: For osteoblastic differentiation of BMSCs. -Sphingomyelin Synthase-2. -PTH receptor-1. 4. Immunohisto-chemical investigations 5. Determination the gene expression_ Table 3: The protective role of different antioxidants in osteoporosis, adapted from (44). Antioxidant Effect Reduced glutathione (G-SH) Vitamin E Vitamin C Vitamin A Genistein Ginkgo biloba Reduced glutathione is important for the maintenance of the cytoskeleton, stabilizes the lysosomal membrane and suppresses injury to the vascular endothelium and wall by inhibiting an increase of lipoperoxides. Vitamin E deficiency impairs calcium transport via the intestine and reduces bone density, free radical scavenger and has been shown to offer protection against free radical-associated diseases, such as atherosclerosis, cancer and osteoporosis. Enhances collagenogenesis, possible effect on bone formation, the antioxidant effect on osteoporosis is under investigation Excess vitamin A intake has been associated with accelerated bone loss, but this requires further investigation. Free radicals scavenger, inhibitor of cell-derived H2O2 formation. Free radical scavenger. Corticosteroid-induced osteoporosis and osteonecrosis: role of oxidative stress 263 Intrinsic as well as extrinsic apoptotic pathways are suggested as possible mechanisms of GC-induced apoptosis. The activation of the intrinsic apoptotic pathway is the classic mechanism (60). Mitochondrial-dependent or mitochondrial-independent mechanisms are involved in the extrinsic apoptotic pathway. Glucocorticoid signaling increases the expression of the pro-apoptotic Bcl-2 family member Bim, which can activate the pro-apoptotic proteins Bax/Bak to disrupt mitochondrial membrane potential, resulting in the release of cy-tochrome c and other apoptogenic proteins. This leads to caspase 9 activation and subsequent effector caspase 3 activation and apopto-sis occurs. Other factors that may influence the intrinsic pathway during GC-induced apopto-sis include up-regulation of other pro-apoptotic proteins such as Bad and Puma, or down regulation of anti-apoptotic proteins such as Bcl-2 or Bcl-xL. A major checkpoint in this pathway is the ratio between pro-apoptotic (Bax) to anti-apoptotic (Bcl-2) members, which is something unique for apoptosis. Caspase-3 and caspase-7 cleave this ICAD, resulting in the release of active CAD (61). 6.2 Steroid-induced OP- animal models Several animal models are proposed to describe the pathogenesis of corticosteroid-induced ON and OP, differing according to the animal used (rabbits or mice or rats), the steroid drug, the dose used either acute or chronic doses and the period of the experiment (38). Natural antioxidants could be applied as a functional material to prevent diseases caused by oxidative stress. The ginseng can significantly decrease the incidence of osteoporosis and osteonecrosis in the steroid-treated animal models e.g. rabbit. The oxidative stress is inhibited and the vascular endothelial dysfunction is ameliorated. The precise mechanism still requires a further in vivo study. Figure 3: Glucocorticoid signaling through GR, adapted after (29) 264 R. Alattar, A. Abdel Alim, S. Abdelmetal, S. Abdel Aziz Figure 4: Schematic model of mammalian cell death pathway, adapted from (56) Table 4: Experimental animal models for steroid-induced OP Animal Drug Dose Exp. Period Biomarkers Male NewZea- Methylprednisolone 4 mg/kg 14 days ref (36) GSH and LPO land white rabbits B.Wt. Female Japanese Methylprednisolone 4 mg/kg, Sampling at days: Histopathology, Bio- white rabbits I.M. once 3rd , 5 th & 14th, Ref chemical and immuno- (36) histothemisty Female Japanese Methylprednisolone 40 Sampling at days: Histopathology, Bio- white rabbits mg/kg, 1st, 3rd , 5 th & 14th chemical and immuno- I.M. once Ref (18) histothemisty Male Japanese Methylprednisolone 20 -2- 10 weeks , Ref Histopathology, hemato- white rabbits acetate mg/kg, (62) logical and immunohis- I.M. once tothemisty Male adult Methylprednisolone 20 Sampling at days: Histopathology, Bio- Newzealand mg/kg, 3rd , 5 th , 7th & 14th chemical and immuno- white rabbits I.M. once ,Ref63). histothemisty Rats Slow-release Pred- 1.5 mg/ 6 months Ref(60) Histopathology, Bio- nisone pellets, S.C. Kg B. chemical and immuno- implanted Wt. histothemisty Conclusion Finally, we can conclude that, the causes of osteonecrosis and osteoporosis are multifactorial. Both in vitro and in vivo data have recently shown that steroid-associated oxidative in- jury exerts a crucial role in the development of OP and ON. Several antioxidants have been reported to effectively suppress the development of this illness. Soon after steroid administration, attenuation of the protective antioxi-dative capability occurs, leading to tissue and Corticosteroid-induced osteoporosis and osteonecrosis: role of oxidative stress 265 protein modifications, especially in bone tissues, vascular endothelial dysfunction, which contributes to local intravascular thrombosis and circulatory disturbances, concomitantly with the development of osteoporesis. It is of a great clinical significance if natural products without side effects on the human body were able to indirectly increase the intravital antiox-idant defense system and directly eliminate excessive ROS. Conflict of interest None of the authors have any conflict of interest to declare. References 1. Rao, L., Will tomatoes prevent osteoporosis. Endocrinology Rounds, 2005; 5: 118-35. 2. Bouvard, B., et al., Ultrastructural characteristics of glucocorticoid-induced osteoporosis. Osteoporosis international, 2009; 20: 1089--92. 3. Boyle, W.J., W.S. Simonet, and D.L. Lacey, Osteoclast differentiation and activation. Nature, 2003; 423: 337. 4. Jilka, R.L., et al., Increased bone formation by prevention of osteoblast apoptosis with parathyroid hormone. The Journal of clinical investigation, 1999;104:439-46. 5. Cole, T.J., et al., Targeted disruption of the glucocorticoid receptor gene blocks adrenergic chromaffin cell development and severely retards lung maturation. Genes & development, 1995; 9: 1608-21. 6. Schmidt, S., et al., Glucocorticoid-induced apoptosis and glucocorticoid resistance: molecular mechanisms and clinical relevance. Cell death and differentiation, 2004; 11: S45. 7. O'brien, C.A., et al., Glucocorticoids act directly on osteoblasts and osteocytes to induce their apoptosis and reduce bone formation and strength. Endocrinology, 2004; 145: 1835-41. 8. Moudgil, V., Steroid receptors in health and disease An Oakland University-Serono Symposia, USA Conference, September 20-23, 1987, Meadow Brook Hall, Rochester. MI 48309-9908, USA. FEBS letters, 1988; 226: 213-6. 9. Jondal, M., A. Pazirandeh, and S. Okret, A role for glucocorticoids in the thymus? TRENDS in Immunology, 2001; 22: 185-6. 10. Laudet, V. and H. Gronemeyer, The nuclear receptor factsbook. 2002: Gulf Professional Publishing. 11. Bai, X.-c., et al., Oxidative stress inhibits osteoblastic differentiation of bone cells by ERK and NF-kB. Biochemical and biophysical research communications, 2004; 314: 197-7. 12. Briot, K., et al., 2014 update of recommendations on the prevention and treatment of glucocorticoid-induced osteoporosis. Joint Bone Spine, 2014; 81: 493-1. 13. Nishimura, J. and S. Ikuyama, Glucocorti-coid-induced osteoporosis: pathogenesis and management. Journal of bone and mineral metabolism, 2000;18: 350-2. 14. Provvedini, D.M., et al., 1, 25-dihydroxyvitamin D3 receptors in human leukocytes. Science, 1983; 221: 1181-3. 15. Tsoukas, C.D., D.M. Provvedini, and S.C. Manolagas, 1, 25-dihydroxyvitamin D3: a novel immunoregulatory hormone. Science, 1984; 224: 1438-40. 16. Manolagas, S.C., Steroids and osteoporosis: the quest for mechanisms. The Journal of clinical investigation, 2013; 123: 1919-21. 17. Yang, S.Y., et al., Antioxidant and Anti-Osteoporosis Activities of Chemical Constituents of the Stems of Zanthoxylum piperitum. Molecules, 2018; 23: 457. 18. Ichiseki, T., et al., The initial phase of oxi-dative stress in a steroid-induced osteonecrosis rabbit model. Advances in Bioscience and Biotechnology, 2012; 3: 978. 19. Okazaki, S., et al., Oxidative Stress In The Corticosteroid-induced Osteonecrosis Of The Femoral Head Rat Model. 20. Roux, C., Osteoporosis in inflammatory joint diseases. Osteoporosis International, 2011; 22: 421-33. 21. Halliwell, B. and J.M. Gutteridge, Free radicals in biology and medicine. 2015: Oxford University Press, USA. 22. Valko, M., et al., Free radicals and antiox-idants in normal physiological functions and human disease. The international journal of biochemistry & cell biology, 2007; 39: 44-84. 23. Orrenius, S., V. Gogvadze, and B. Zhivotovsky, Mitochondrial oxidative stress: implications for cell death. Annu. Rev. Pharmacol. Toxicol., 2007; 47: 143-83. 24. Farrugia, G. and R. Balzan, Oxidative stress and programmed cell death in yeast. Frontiers in oncology, 2012; 2: 64. 25. Matés, J.M., et al., Oxidative stress in apoptosis and cancer: an update. Archives of toxicology, 2012;86:1649-65. 26. Das, J., et al., Mangiferin exerts hepato-protective activity against D-galactosamine in- 266 R. Alattar, A. Abdel Alim, S. Abdelmetal, S. Abdel Aziz duced acute toxicity and oxidative/nitrosative stress via Nrf2-NFKB pathways. Toxicology and applied pharmacology, 2012; 260: 35-7. 27. Valko, M., H. Morris, and M. Cronin, Metals, toxicity and oxidative stress. Current medicinal chemistry, 2005; 12: 1161-1208. 28. Abdollahi, M., et al., Pesticides and oxidative stress: a review. Medical Science Monitor, 2004; 10: RA141-7. 29. Gruver-Yates, A.L. and J.A. Cidlowski, Tissue-specific actions of glucocorticoids on apop-tosis: a double-edged sword. Cells, 2013; 2: 20223. 30. Anderson, K., et al., Free radicals and reactive oxygen species in programmed cell death. Medical hypotheses, 1999; 52: 451-63. 31. Andreyev, A.Y., Y.E. Kushnareva, and A. Starkov, Mitochondrial metabolism of reactive oxygen species. Biochemistry (Moscow), 2005; 70:200-14. 32. Pratt, W.B. and D.O. Toft, Steroid receptor interactions with heat shock protein and immuno-philin chaperones. Endocrine reviews, 1997; 18: 306-60. 33. Oakley, R.H., M. Sar, and J.A. Cidlowski, The human glucocorticoid receptor isoform expression, biochemical properties, and putative function. Journal of Biological Chemistry, 1996; 271: 9550-9. 34. Finkel, T. and N.J. Holbrook, Oxidants, oxidative stress and the biology of ageing. Nature, 2000; 408: 239. 35. Li, G.-Y., et al., Edaravone, a novel free radical scavenger, prevents steroid-induced osteonecrosis in rabbits. Rheumatology, 2012; 52: 438-47. 36. Ichiseki, T., et al., Oxidative stress and vascular permeability in steroid-induced osteonecrosis model. Journal of Orthopaedic Science, 2004; 9: 509-15. 37. Mody, N., et al., Oxidative stress modulates osteoblastic differentiation of vascular and bone cells. Free Radical Biology and Medicine, 2001; 31: 509-19. 38. Key Jr, L., et al., Oxygen derived free radicals in osteoclasts: the specificity and location of the nitroblue tetrazolium reaction. Bone, 1990; 11: 115-9. 39. Steinbeck, M.J., et al., NADPH-oxidase expression and in situ production of superoxide by osteoclasts actively resorbing bone. The Journal of Cell Biology, 1994; 126: 765-772. 40. Suda, N., et al., Participation of oxidative stress in the process of osteoclast differentiation. Biochimica et Biophysica Acta (BBA)-General Subjects, 1993; 1157: 318-23. 41. Basu, S., et al., Association between oxidative stress and bone mineral density. Biochemical and biophysical research communications, 2001; 288: 275-9. 42. Lee, Y.-S., X. Chen, and J.J. Anderson, Physiological concentrations of genistein stimulate the proliferation and protect against free radical-induced oxidative damage of MC3T3-E1 osteo-blast-like cells. Nutrition Research, 2001; 21: 1287-98. 43. Baker, P.J., The role of immune responses in bone loss during periodontal disease. Microbes and Infection, 2000; 2: 1181-92. 44. Abdollahi, M., et al., Role of oxidative stress in osteoporosis. Therapy, 2005; 2: 787-96. 45. Garnero, P., et al., Markers of bone turnover predict postmenopausal forearm bone loss over 4 years: the OFELY study. Journal of Bone and Mineral Research, 1999; 14: 1614-21. 46. Iotsova, V., et al., Osteopetrosis in mice lacking NF-kB1 and NF-kB2. Nature medicine, 1997; 3: 1285. 47. Deyama, Y., et al., Inactivation of NF-kB involved in osteoblast development through inter-leukin-6. Biochemical and biophysical research communications, 2001; 282: 1080-4. 48. Kurokouchi, K., et al., TNF-a increases expression of IL-6 and ICAM-1 genes through activation of NF-kB in osteoblast-like ROS17/2.8 cells. Journal of Bone and Mineral Research, 1998; 13: 1290-9. 49. Polidori, M.C., et al., Profiles of antioxi-dants in human plasma. Free Radical Biology and Medicine, 2001; 30: 456-62. 50. Meier, C., et al., Supplementation With Oral Vitamin D3 and Calcium During Winter Prevents Seasonal Bone Loss: A Randomized Controlled Open-Label Prospective Trial. Journal of Bone and Mineral Research, 2004; 19: 1221-30. 51. Ricci, C., et al., Mitochondrial DNA damage triggers mitochondrial-superoxide generation and apoptosis. American Journal of Physiology-Cell Physiology, 2008; 294: C413-22. 52. Mikami, T., et al., Prevention of steroid-induced osteonecrosis by intravenous administration of vitamin E in a rabbit model. J Orthop Sci, 2010; 15: 674-7. 53. Martindale, J.L. and N.J. Holbrook, Cellular response to oxidative stress: signaling for suicide and survival. J Cell Physiol, 2002; 192: 1-15. 54. Zhou, J.-Y., et al., Corticosterone exerts immunostimulatory effects on macrophages via en- Corticosteroid-induced osteoporosis and osteonecrosis: role of oxidative stress 267 doplasmic reticulum stress. BJS, 2010; 97: 281-93. 55. Colavitti, R., et al., Reactive oxygen species as downstream mediators of angiogenic signaling by vascular endothelial growth factor recep-tor-2/KDR. J Biol Chem, 2002; 277: 3101-8. 56. Kannan, K. and S.K. Jain, Oxidative stress and apoptosis. Pathophysiology, 2000; 7: 153-63. 57. Angeli, A., et al., High prevalence of asymptomatic vertebral fractures in post-menopausal women receiving chronic glucocorti-coid therapy: a cross-sectional outpatient study. Bone, 2006; 39: 253-9. 58. Salvesen, G.S. and V.M. Dixit, Caspase activation: the induced-proximity model. Proc Natl Acad Sci U S A, 1999; 96: 10964-7. 59. Cao, Y., et al., Glucocorticoid receptor translational isoforms underlie maturational stage-specific glucocorticoid sensitivities of dendritic cells in mice and humans. Blood, 2013: blood- 2012-05-432336. 60. Kerachian, M.A., et al., New insights into the pathogenesis of glucocorticoid-induced avascular necrosis: microarray analysis of gene expression in a rat model. Arthritis Research & Therapy, 2010; 12: R124. 61. Sinha, K., et al., Oxidative stress: the mitochondria-dependent and mitochondria-independent pathways of apoptosis. Arch Toxicol, 2013;87:1157-80. 62. Yamamoto, T., et al., Effects of pulse methylprednisolone on bone and marrow tissues. Corticosteroid-induced osteonecrosis in rabbits. Arthritis & Rheumatism: Official Journal of the American College of Rheumatology, 1997; 40: 2055-64. 63. Lu, B.-B. and K.-H. Li, Lipoic acid prevents steroid-induced osteonecrosis in rabbits. Rheumatology international, 2012; 32: 1679-83. Slov Vet Res 2019; 56 (Suppl 22): 269-79 DOI 10.26873/SVR-766-2019 Original Research Article ALLEVIATION OF CHRONIC HEAT STRESS IN BROILERS BY DIETARY SUPPLEMENTATION OF NOVEL FEED ADDITIVE COMBINATIONS Saad I. Al-Sultan1, Sherief M. Abdel-Raheem1, 2*, Sherief M.S. Abd-Allah3, Abobakr M. Edris14 1Department of Veterinary Public Health and Animal Husbandry, College of Veterinary Medicine, King Faisal University, 400 Al-Ahsa ,31982, Saudi Arabia, 2Department of Animal Nutrition and Clinical Nutrition, Faculty of Veterinary Medicine, Assiut University, 71526, Egypt, 3Department of Food Hygiene (Meat Hygiene), Faculty of Veterinary Medicine, Assiut University, 71526, Egypt, 4Food hygiene and control Department, Faculty of Veterinary Medicine, Benha University, Egypt Corresponding author, E-mail: sdiab@kfu.edu.sa Abstract: Heat stress causes economic losses via decreasing feed intake, nutrient digestion, weight gain, feed conversion, immunity, carcass quality and increasing mortality in broilers. The aim of the study was to assess the ability of dietary additive combinations to ameliorate the detrimental effect caused by chronic heat stress (32 ± 2°C for 24 h per d) on performance, carcass traits, metabolic status and economic efficiency of broiler chickens. A total of 420, one-day old Cobb-500 broiler chicks were assigned into seven treatment groups (n=60) of five replicates. Each replicate contained 12 unsexed chicks. The control groups did not receive any supplementation to the basal diet (thermoneutral control (TNC) and heat stress control (HSC) groups). The other groups received control diet supplemented with 1 % cumin plus 1 % turmeric powders (T1); 1.5 g/kg potassium chloride plus 2 g/kg sodium bicarbonate (T2); 1000 ppm propolis plus 15000 IU vitamin A (T3); 1200 ppb chromium plus 500 ppm vitamin C (T4) ; 1200 ppm betaine plus 500 ppm vitamin E (T5). The results indicated that at 42 d of age, all dietary additive combinations improved the growth performance indices, carcass traits, concentrations of serum antioxidant enzyme biomarkers, stress biomarkers and economic efficiency in comparison to HSC group. Based on the obtained results, it could be concluded that dietary supplementation with betaine and vitamin E followed by chromium and vitamin C combinations offers a good management practice for alleviating heat stress related depression in the performance of broiler chickens. Key words: heat stress; broilers; vitamin C; betaine; chromium; propolis; turmeric Introduction The most obvious constraint on poultry production in Saudi Arabia, especially during summer months is high temperature with high humidity, which causes severe stress on birds and reduces performance. The continuous challenge for the poultry production in tropical and subtropical region all over the world remains heat stress. Hot environments making several alterations in blood hormones, glucose levels, leukocytes, electrolytes and organ functions (1). Received: January 2019 Accepted for publication: February 2019 270 S. I. Al-Sultan, S. M. Abdel-Raheem, S. M. S. Abd-Allah, A. M. Edris Heat stress causes economic losses via decreasing feed intake, nutrient digestion, weight gain (WG), egg production, feed conversion (FC), immunity, carcass quality and increasing mortality (2). Heat stress trigger the hypothalamo-hypo-physeal adrenocortical axis to secrete cortico-steroids (3). The catabolic effects of higher blood corticosteroids elevate the free radicals through changing oxidative metabolism, producing huge damage of cellular functions and cell membrane, which causing muscle degeneration and growth retardation (3). The greatest part of the energy production is directed to thermoregulation mechanisms, which develop stress-induced immunosuppression. The immune suppressed birds are predisposed to numerous dangerous diseases associated with high mortality rates (4) Dietary modifications by the use of commercial feed additives are considered the most favored and practical ways to mitigate the effect of heat stress in poultry (5). Therefore, dietary inclusion of some selected feed additives could also be effective for the preservation of growth and immunity for broilers during periods of heat stress. Many previous studies illustrated the beneficial effect different dietary supplements on the performance of broilers under heat stress independently (6, 7, 8, 9, 10, 11, 12). These studies revealed that Vitamin A, E, C, play important roles in the performance and immune function of poultry under heat stress. Betaine is a methyl donor and it has methionine sparing effect, help in maintaining cellular water balance, protect the integrity of cell membrane and improve broiler performance and to overcome stress. In addition, the supplementation of dietary electrolytes increase tolerance to heat stress improve performance of broilers under heat stress. Electrolyte prevent acid base balance disturbances and decrease the occurrence of respiratory alkalosis during heat stress. Herbal additive like cumin, turmeric and propolis very rich in flavonoid and aromatic acids and they have antioxidant, anti-inflammatory, antibacterial, antifungal activity and have a positive effect on broiler performance under heat stress. However, few studies have been conducted to detect the best combination at the recommended concentrations suggested by previous studies to mitigate the decline in performance caused by heat stress in broiler chickens. Therefore, the aim of the current study was to detect the best commercial additive combinations to ameliorate the detrimental effect caused by chronic heat stress on performance, carcass traits, metabolic status, antioxidant capabilities and economic efficiency of broiler chickens. Material and methods Experimental animals and protocol The current experiment was accomplished according to King Faisal University Animal Care and Use Committee and the experimental protocol was approved by the Deanship for Scientific Research with reference number (Decision No: KFU-REC/2017-04-01). The experiment was carried out on 420 one-day-old broiler chickens (Cobb - 500) purchased from a marketable hatchery (Al-Ahsa, KSA). Birds were housed in pens with wood shavings floor with 10 birds/m2at the Research and Agriculture Station, King Faisl University, Al-Ahsa, Saudi Arabia from 1 to 42 d old. Water and feed provided ad libitum during 24 hours. Constant observation for health status and behavior usually carried out for broiler chickens. The temperature was kept at 32 ± 2 °C with 65 % relative humidity (RH) for 24 h for all treatment groups except thermoneutral control (TNC). For maintaining the desired temperature and relative air humidity air conditioners, heaters with thermostats, and air circulators were applied. The thermoneutral control group (TNC) were held at thermoneutral chamber with normal ambient temperature (22 ± 4 °C) and relative humidity (RH) of commercial management program (55±3 %, RH) at the same experimental place. The experimental design A total of 420 one-day-old unsexed broiler chickens (Cobb - 500) randomly allocated to 7 experimental groups (60 chick/each). Each group consisted of 5 replicate pens with 12 Alleviation of chronic heat stress in broilers by dietary supplementation of novel feed additive combinations 271 chicks each. The control groups did not receive any supplementation to the basal diet (ther-moneutral control (TNC) and heat stress control (HSC) groups). The other groups received control diet supplemented with 1 % cumin plus 1 % turmeric powders (T1); 1.5 g/kg potassium chloride plus 2 g/kg sodium bicarbonate (T2); 1000 ppm propolis plus 15000 IU vitamin A (T3); 1200 ppb chromium (Cr) plus 500 ppm vitamin C (T4) ; 1200 ppm betaine (Bet) plus 500 ppm vitamin E (VE) (T5). According to NRC (13) guidelines, fourteen experimental diets, seven in each feeding phase (starting and growing) were formulated. The basal diets contained 23% and 20 % protein and 3200 kcal of ME/kg for starter and grower diets, respectively. Composition and chemical analysis of broiler basal diets are presented in (Table 1). Proximate composition of feed ingredients were performed according to AOAC (14) and the experimental diets were formulated based on these values. The selected feed additives were thoroughly mixed with small amount of feed on the expense of corn, then homogenized with the total amounts and added to the diet. Prophylactic vaccination against viral diseases was carried out. All chicks were immunized with eye drops by weak Newcastle virus vaccine at age of 7 days and with a live attenuated freeze-dried vaccine (Nobilis® Ma5 + Clone 30) at the age of 20 days. Broiler performance Feed consumption and body weight for each pen were recorded weekly during the whole experiment. Average daily gain, feed intake and feed conversion ratio were calculated during starter (1 to 21 d), grower (22 to 42 d), and overall (1 to 42 d) phases. Blood Sampling Ten birds from each group were used for collection of blood samples at days 21 and 42.The samples were collected into two tubes, one contained EDTA (as anticoagulant) and the other had no anticoagulant. Samples were centri-fuged at 3500 g for 15 min for gathering plasma and serum, respectively. Determination of New castle serum antibody titre The log10 of serum antibody titer of new castle disease was determined by using commercial ELISA kits (IDEXX® Laboratories, B.V., Netherlands) according to the recommendation of the supplier. Determination of blood antioxidant enzymes and oxidative stress biomarkers The activities of antioxidant biomarkers (glutathione peroxidase (GSHPx), superoxide dismutase (SOD) and malondialdehyde (MDA) were analyzed by spectro-photometrical method with commercial kits obtained from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). All procedures were achieved according to manufacturer's instructions. Plasma corticosterone (CS) levels was assayed by the use of ELISA kit (DetecteX® Corticosterone, EIA kit, Arbor Assays®, USA), according to procedures of Quintero-Filho et al. (3). Determination of blood biochemical profile Serum glucose, total protein, albumin, total cholesterol and globulin levels were analyzed spectrophotometrically by using commercial kits from Spinreact company ® (Spain) and according to manufacturer's instructions. Heterophil/lymphocyte (H/L) ratio At the end of the experiment d 42, 10 birds from each group were used for determination of H/L ratio. Smears were prepared using the standard two-slide wedge procedure for differential leukocyte counts. Subsequently, blood films were air dried and stained with Wright-Giemsa Quick stain. The H/L ratio was calculated according to method adopted from (15) Carcass characteristics and relative organ weight At 42 day of age, 10 bird from each treatment (2 from each pen) were randomly selected and humanely slaughtered according to Islamic method for carcass traits characteristics. The gizzard, bursa of Fabricius, liver, spleen, ab- 272 S. I. Al-Sultan, S. M. Abdel-Raheem, S. M. S. Abd-Allah, A. M. Edris dominal fat, heart, thymus, pancreas, and stomach were removed and blot dry weighed. Relative organ weight was calculated as a percentage of body weight. Economic efficiency Economic efficiency was estimated according to Wealleans et al., (16). Economic efficiency (EE) = (Net revenue/ total costs) xioo. Relative economic efficiency (REE) = Economic efficiency/economic efficiency of the positive control (17) Statistical analysis Data analysis was accomplished by one-way ANOVA using the procedure of SPSS software version 19 (18). Data were tested for normality before analysis by the use of the Kolmogorov-Smirnov test. Duncan is multiple range test (19) was used to detect the differences among means and significance level was set at P<0.05. Results The effect of diet supplementation with novel feed additives on growth performance of 42-day broilers was presented in (Table 2). The results presented in (Table 2) indicated the presence of statistically significant differences (P< 0.05) in the broiler growth performance indices such as body weight, feed intake, body weight gain and feed conversion ratio between the different treatment groups because of the dietary inclusion of all supplements. All dietary additive combinations induced a significant improvement in body weight gain, feed intake, feed conversion rate in birds of treatment groups during the entire period of the study (d 0 -42). On the other side, chronic heat stress (HSC group) induced a significant reduction in body weight, weight gain, feed intake, feed conversion efficiency and mortality during the entire period of the study. It is interesting to note that, dietary supplementation with the combination of 1200 ppm betaine and 500 ppm vitamin E followed by 1200 ppb chromium and 500 ppm vitamin C showed the most favorable and the best results regarding all growth performance indices of broilers subjected to heat stress in comparison with other treatment groups. All the dietary supplements significantly increased (P<0.05) the carcass weight, dressing weight, dressing percentage or carcass yield and relative liver, heart, gizzard weights in comparison with the HSC group (Table 3). The greater relative organ weights were recorded in T5, T4, T3 when compared with T1 and T2. The major impact of the heat stress on the carcass traits was showed in the increase of the relative abdominal fat in the HSC to about 1.98 %, which decreased to 1.03 and 1.12 % in birds receiving both the combination of betaine and vitamin E and the chromium and vitamin C, respectively. The highest dressing percentage value (70.27%) was observed in birds of T5 group in comparison with other treatments groups. The lowest dressing percentage was recorded in the heat stressed control group (68.42 %). The greater response of the dietary supplements on carcass traits of broilers under chronic heat stress was obtained by dietary supplementation of betaine and vitamin E combination. Heat stressed broilers had higher (P<0.05) levels of blood glucose, total cholesterol and albumen and lower (P< 0.05) level of total protein and globulin when compared with all treated groups and TNC (Table 4). The blood levels of triglycerides and total cholesterol were significantly reduced (p <0.01) in all treated groups in comparison with heat stressed control (HSC). In comparison with the heat stressed control, the dietary inclusion of all supplement combinations in broiler diets significantly increased (P < 0.001) the activities of antioxidant enzymes (SOD, GSH-Px,) and TAOC but reduced (P < 0.001) the MDA content in the serum of chicks at the ages 42 d (Table 4). Heat stress reduce blood antioxidant status in broilers and increased the oxidative stress indicators. It was noted that dietary supplementation with selected additive combinations had decreased the concentrations of both H: L ratio and corti-costerone significantly (P<0.001) of broilers under heat stress (Table 4) in comparison with HSC group. The concentrations of ND antibodies titer increased significantly (P<0. 001) at 21 and 42 d of age by dietary additives in treatment groups in comparison with heat stressed Alleviation of chronic heat stress in broilers by dietary supplementation of novel feed additive combinations 273 control and the highest values were noticed in birds of T5, T4 and T3 groups, respectively. This research extends our knowledge that dietary supplementation of betaine and vitamin E, chromium and vitamin C, propolis and vitamin A combinations are better option for enhancing serum antioxidant status and immune response (ND antibodies titer) for broilers exposed to chronic heat stress. The economic efficiency of 6 weeks old broiler chicks fed diet fortified with different supplement combinations under heat stress are shown on (Table 5). The dietary additive supplementation increased (P <0.001) the net return and economic efficiency in all treated groups in comparison with HSC group. Concerning the economic feed efficiency and the relative economic efficiency, the highest values were recorded for the treatment groups (T5, T4, T3 and T2) in comparison with T1 and HSC groups. Table 1 : Ingredients and analyzed chemical composition of basal diets Item* (% unless noted) Starter diet Grower and finisher diet Ingredient, % Corn 54.64 62.55 Corn gluten meal (60%) 4.11 3 Soybean meal, 44% 32.5 26.4 Sunflower oil 4.4 3.7 Limestone 1.5 1.5 Dicalcium phosphate 1.8 1.8 Salt 0.4 0.4 Vitamin - mineral premix 0.3 0.3 DL-methionine 0.2 0.2 L- lysine 0.1 0.1 Antioxidant 0.05 0.05 Total 100 100 Chemical analysis Dry matter 90.11 89.94 Crude protein 23.01 20.05 ME(Kcal/kg diet) b 3200 3200 Calcium 1.08 1.06 Non-phytae phosphorus 0.45 0.43 Na 0.20 0.17 Lysine 1.25 1.07 DL-Methionine 0.58 0.53 Methionine+cystine 1.05 0.87 Crude fiber 2.96 2.95 Crude fat 6.38 6.04 *Ingredient and nutrient composition are reported on as-fed basis. a The vitamin and mineral premix provided per kg of diet: vitamin A, 4000000 IU; vitamin D3, 667000 IU; vitamin E 3334 mg; vitamin K3, 1167 mg; vitamin B1, 334 mg; vitamin B2, 1667 mg; vitamin B3, 3334 mg; B6, 500 mg; vitamin B12 33.4 mg, Folic acid, 334 mg; Biotin, 17 mg; Iron, 10 ; Copper, 2.167; Zinc, 18.334; Manganese 20.0; Iodine, 0.167; Cobalt, 0.034 and Selenium, 0.034. b ME Calculated based on NRC (1994) feed composition tables 274 S. I. Al-Sultan, S. M. Abdel-Raheem, S. M. S. Abd-Allah, A. M. Edris Table 2: Effect of dietary treatments on body weight (BW), feed intake (FI) and feed conversion ratio (FCR) of boilers under thermoneutral (TN) and heat stress (Mean± Pooled SE) Item TNC HSC T1 T2 T3 T4 T5 SEM P Initial BW (day1) 40.56 41.23 40.86 41.25 42.07 41.33 40.07 0.23 0.332 0 to 21 day BW (day 21 W3) 904.19a 417.82d 621.73 b 608.06b 599.33b 572.40c 625.31b 13.39 <0.001 Feed intake (g/bird) 1191.04a 866.2e 1077.37b 1013.98cd 1027.17c 1001.76d 1019.69cd 9.05 <0.001 BW gain (g/bird) 863.62a 376.59e 580.86bc 566.81bc 557.27c 531.07d 585.31b 13.41 <0.001 FCR (g/g) 1.38d 2.32a 1.86bc 1.83bc 1.84bc 1.89b 1.74c 0.02 <0.001 22-42 day Feed intake (g/bird) 3475.15a 2431.59c 2612.92b 2478.13c 2512.65c 2486.72c 2479.62c 34.7 <0.001 BW gain (g/bird) 1740.81a 1045.52d 1142.93c 1151.5c 1169.67c 1217.33b 1177.81c 21.07 <0.001 FCR (g/g) 1.99d 2.33a 2.29a 2.16b 2.15b 2.05cd 2.11bc 0.01 <0.001 0-42 day Final BW (day 42) 2645.00a 1463.35d 1664.66c 1759.56c 1769.00b 1789.73bc 1803.13bc 21.64 <0.001 Feed intake (g/bird) 4666.18a 3297.79d 3690.28b 3492.11c 3539.81c 3488.49c 3499.31c 42.27 <0.001 BW gain (g/bird) 2604.43a 1422.11d 1723.8c 1718.31c 1726.93c 1748.4bc 1763.13b 33.33 <0.001 FCR (g/g) 1.79d 2.32a 2.14b 2.03c 2.04c 1.99d 1.98d 0.01 <0.001 Mortality, % 1.75 12.77 4.44 3.88 4.88 3.33 2.22 1.09 0.08 BW, body weight, FCR, feed conversation ratio = feed intake/ body weight gain a, b, c, d, e means with different letters in the same row are significantly different at P<0.05. TNC, thermoneutral control, HSC, heat stress control; basal diet supplemented with:1 % Cuminandl % turmeric powder (Tl) ; 1.5 g/kg potassium chloride and 2 g/kg sodium bicarbonate (T2) ; 15000 IU vitamin A and 1000 ppm propolis (T3); 500 ppmvitamin C and 1200 ppb chromium (T4); 1200 ppm betaine and 500 ppm Vitamin E (T5). Table 3: Effect of dietary additives on carcass characteristics and relative weight (% of live weight) of internal organs of broilers under thermoneutral (TN) and heat stress (Mean± Pooled SE) Item TNC HSC T1 T2 T3 T4 T5 SEM P Preslaughter weight 2665.00a 1621e 1722d 1730d 1788.00b 1798.00bc 1831.2b 56.21 <0.001 Carcass weight 1872.6a 1109e 1206d 1210d 1247c 1258c 1287b 40.29 <0.001 Dressing, % 70.28a 68.42c 70.08ab 69.94ab 69.74b 69.97ab 70.27a 0.33 <0.001 Liver, % 2.78a 2.15c 2.62b 2.46b 2.5b 2.68b 2.74a 0.04 0.04 Heart % 0.76a 0.45d 0.71b 0.65c 0.62c 0.72b 0.75b 0.11 0.001 Gizzard, % 1.73a 1.35c 1.54b 1.52b 1.55b 1.58b 1.61b 0.02 0.01 Abdominal fat, % 1.65b 1.98a 1.23d 1.48bc 1.39cd 1.12d 1.03e 0.05 <0.001 TNC, thermoneutral control, HSC, heat stress control; basal diet supplemented with: 1 % Cumin and 1 % turmeric powder (T1) ; 1.5 g/kg potassium chloride and 2 g/kg sodium bicarbonate (T2) ; 15000 IU vitamin A and 1000 ppm propolis (T3); 500 ppm vitamin C and 1200 ppb chromium (T4); 1200 ppm betaine and 500 ppm Vitamin E (T5) Alleviation of chronic heat stress in broilers by dietary supplementation of novel feed additive combinations 275 Table4: Effect of dietary additives on blood biochemical profile, oxidative stress biomarkers, and ND antibody titre (Mean± Pooled SE) Item TNC HSC T1 T2 T3 T4 T5 SEM P Glucose, mg/dl 221.67d 281.69a 240.27c 251.16b 230.48cd 224.61d 226.81d 3.533 <0.001 Total cholesterol, mg/dl 126.83e 174.92a 153.08b 146.17c 138.08 d 135.25d 121.25e 2.888 <0.001 Total protein, g/dl 3.79a 2.79e 3.24 cd 3.37 c 3.47bc 3.62 b 3.94a 0.062 <0.001 Albumen, g/dl 1.84a 1.86a 1.72 ab 1.63 b 1.62 b 1.62b 1.53b 0.028 0.004 Globulin, , g/dl 1.95b 0.93 1.52d 1.73c 1.85bc 2.00 b 2.41 a 0.075 <0.001 Triglycerides, mg/dl 99.17a 94.92a 86.72 b 82.29 bc 77.02 d 85.96b 80.86 bc 1.59 <0.001 SOD, U/ml 152.37bc 127.80 d 152.60bc 154.4abc 148.4c 160.60a 155.60ab 1.77 <0.001 GSH-PX, n mol/ml 11.02a 5.98e 7.48 d 8.08 cd 8.62 c 9.52 b 8.19 c 0.266 <0.001 MDA, nmol/ml 6.90e 11.77a 9.91b 9.10 c 7.98 d 7.06 e 8.92c 0.273 <0.001 TAOC, U/ml 8.02e 8.42e 9.64d 10.32 c 10.69bc 12.05a 10.85 b 0.233 <0.001 Heterophils 21.51e 28.9 a 23.88 ab 24.5 b 22.8 bcde 23.1 abcd 22.02 de 23.75 <0.001 lymphocytes 77.23abc 71.34d 76.30cd 76.94 abc 77.56 ab 76.60 bc 77.80 a 0.365 <0.001 H/L ratio 0.28d 0.41a 0.31 b 0.32a 0.29cd 0.30 bc 0.28d 0.006 <0.001 Corticosterone, ng/ml 10.92a 10.46b 9.57c 9.51c 9.52c 9.13d 9.07d 0.11 <0.001 ND titer (log 10) 21 days 1.95 g 2.27 f 2.47 e 2.58 d 2.76 c 2.87b 3.11a 0.06 <0.001 42 days 3.14e 3.51d 3.67c 3.74 bc 3.86b 4.44a 4.48a 0.08 <0.001 ^ k ^ 4 e means with different letters in the same row are significantly different at P<0.05.ND= Newcastle disease GSHPx, glutathione peroxidase; SOD, total superoxide dismutase and; MDA, malondialdehyde; TAOC, total antioxidant capacity, H/L heterophile/lymphocyte TNC, thermoneutral control, HSC, heat stress control; basal diet supplemented with: 1 % Cumin and 1 % turmeric powder (T1) ; 1.5 g/kg potassium chloride and 2 g/kg sodium bicarbonate (T2) ; 15000 IU vitamin A and 1000 ppm propolis (T3); 500 ppm vitamin C and 1200 ppb chromium (T4); 1200 ppm betaine and 500 ppm Vitamin E (T5) Table5: Economic analysis of6-week broilers fed different dietary treatments (Mean± Pooled SE) Item TNC HSC T1 T2 T3 T4 T5 SEM P Cumulative feed intake (g/bird) 4666.18a 3297.79d 3690.28b 3492.11c 3539.81c 3488.49c 3499.31c 42.27 <0.001 Feed cost / bird (SR) 5.6a 3.96d 4.42b 4.19c 4.25c 4.18c 4.19c 0.02 <0.001 Total Cost per bird (SR)* 8.6 a 6.96d 7.42b 7.19c 7.24c 7.18c 7.19c 0.02 <0.001 Total return (SR/bird) 31.25a 17.06 d 20.68 c 20.61 c 20.72 c 20.98 bc 21.16b 0.4 <0.001 Net profit (SR/bird) 22.65a 10.11 13.26 13.42 13.47 13.79 13.95 0.35 >0.05 Economic feed efficiency 263.47a 145.32e 178.45d 186.78c 185.94c 191.94b 193.91b 3.23 <0.001 Relative economic efficiency 1a 0.56e 0.68d 0.72c 0.71 c 0.73 b 0.75b 0.01 <0.001 SR, Saudi riyal TNC, thermoneutral control, HSC, heat stress control; basal diet supplemented with: 1 % Cumin and 1 % turmeric powder (T1) ; 1.5 g/kg potassium chloride and 2 g/kg sodium bicarbonate (T2) ; 15000 IU vitamin A and 1000 ppm propolis (T3); 500 ppm vitamin C and 1200 ppb chromium (T4); 1200 ppm betaine and 500 ppm Vitamin E (T5). *Total cost include (chick price, light, electricity, workers, vaccinations and bedding) 276 S. I. Al-Sultan, S. M. Abdel-Raheem, S. M. S. Abd-Allah, A. M. Edris Discussion The current study confirmed the detrimental impacts of heat stress on broilers performance, carcass traits, blood metabolites, antioxidant capabilities and economic efficiency. The harmful effects of heat stress on growth performance indices detected in the current experiment were consistent with previous studies (8, 10, 11). Heat stress reduces feed intake as an adaptation defense mechanism started by birds to reduce metabolic heat production (20). Heat stress increases free radical generation and lipid peroxidation, which impair growth performance. The improved broiler performance by the selected additive combination in the current studies are matching the findings of earlier studies (6, 7, 8, 9, 10, 11, 12). Antioxidant vitamins (C and E) had a beneficial effect in relieving heat stress in broilers through their antioxidant effects (21). Betaine is crucial to be supplied during heat stress, as poultry cannot synthesizes sufficient amount and poultry, feed considers as a poor source (22). This mixture vitamin E and betaine (Bet with VE) resulted in complete reduction of the negative effect of chronic heat stress (CHS) on broiler performance. The synergetic effect of Bet and vitamin E may reveal the different mode of action of both. The supplementation of betaine and vitamin E elicit the best significant effect on growth performance and this finding could have important implications in poultry production in subtopic and tropical regions. The potential effect of betaine represented by improving the digestibility of specific nutrients as energy and protein through enhancing the structural and functional characteristics of intestinal epithalia (23). Moreover, dietary supplementation of betaine and vitamin E combination provides a more potent antioxidant effect, and spares essential amino acids like choline and methionine (12, 23). A positive effect was detected between the dietary combination vitamin C and Cr as evidenced by their potent antioxidant property against oxidative stress in the current study and confirm the finding of previous studies (5, 24, 25). There are several explanation for this result, vitamin C and chromium supplementation increased serum Vit C, Cr, Vit E, Fe, Zn, Mn and improved nutrient digestibility in laying hens under cold stress (24). The rate of ascorbic acid uptake into the cells was decreased during heat stress because of the decrease in insulin level and hyperglycemia (25). Chromium indirectly increases the intracellular availability of Vit C by intensifying the action of insulin (24). The positive impact of the combination of vitamin A and propolis could be due to the role played by vitamin A as an effective radical-trapping antioxidant (26). Heat stress reduce the conversion of carotene to vitamin A and the concentration of vitamin A decreases during stress conditions so much vitamin A needed during heat stress (26). The observed beneficial effects of cinnamon and turmeric in the current study could be attributed to their stimulation of appetite, feed intake, digestive enzyme secretions, immune response and the antioxidant actions (27). Increased growth performance and carcass traits by KCl supplementation could be ascribed to decreased body temperature by more water ingestion after electrolyte supplementation that caused divert more energy toward BW gain. In accordance with the present results, previous studies have demonstrated that, the addition of KCl and NaHCO3 during heat stress, at levels of 1.5% and 0.5% respectively, improved final weight, feed efficiency and improved (P<0.05) serum potassium and bicarbonate level through providing of potassium and bicarbonate ions (2). It has been established that heat stress alter the carcass composition of birds through increasing fat deposition and reducing body protein content (6). The improved carcass characteristics due to the dietary additive in the current study are in line with the finding of previous studies (6, 28, 29) who reported that supplementation of propolis, ascorbic acid, chromium, and turmeric significantly increased the dressing percentage and improved crass characteristics in chicken reared under heat stress. The increase in dressing percentage in bird receiving vitamin E and Bet may be due to the osmotic effects of Bet, which increases water re- Alleviation of chronic heat stress in broilers by dietary supplementation of novel feed additive combinations 277 tention and improving the digestibility of energy and protein through enhancing the structure and function of intestinal epithalia and sparing essential amino acids (23). The present findings seem to be consistent with other researches which found that dietary inclusion of vitamin C (5, 24), or Cr (24) improved all carcass characteristics in broilers. Another study illustrated that Cr supplementation increased carcass yield and decreased abdominal fat in heat stressed chickens (28). The increase in glucose concentration in the current study could be ascribed to the increase in glucocorticoids that can result from heat stress as previously explained by (3). Glucocor-ticoids stimulate gluconeogenesis from muscle tissue proteins. Heat stress increased the plasma level of glucose and cholesterol and decreased total protein levels (30). Both Ascorbic acid and chromium supplementation enhance the utilization of corticosteroids released during stress (24), thus playing a crucial role in response to stress. Vitamin C mitigates the negative effects of stress related depression in poultry performance through reducing synthesis and secretion of corticosteroids (26). The increase in blood li-pids under heat stress caused by compensation of energy needed due to the reduction in feed intake through lipolysis of body lipid which resulting in increasing the blood cholesterol and triglyceride levels (30). The improved in blood oxidant and antioxi-dant biomarkers caused by dietary additives was in line with the results of previous studies (4, 7, 10, 21). These studies reported that dietary supplementation of chromium, betaine, vitamin E, C, betaine and propolis significantly improved primary and secondary immune response (P<0.01), and improved H/L ratio (P<0.05). This finding supports previous research of Niu et al. (4) dietary supplementation of Vitamin improved performance and immune competence of broilers under HS. The improvement in economic efficiency in all treated groups after dietary supplementation of different additive combinations may be attributed to the ability of these additives to increase broiler performance, feed efficiency and their ability to stimulate bird immunity and to reduce mortality rate. This result match those observed in earlier studies (7, 31). Conclusion Based on the obtained results, it could be concluded that dietary supplementation of broiler under chronic heat stress with selected additive combinations improved the growth performance, carcass traits, concentrations of serum antioxidant enzyme biomarkers and economic efficiency. From the tested dietary additive combinations, the use of betaine and vitamin E or chromium and vitamin C together gave the best results in terms of improved growth performance, feed efficiency, immune response, antioxidant status and profitability of broilers under chronic heat stress. From the findings of the current study, it could be recommend the use of dietary either betaine and vitamin E or chromium or vitamin C combinations as a good management practice for mitigating heat stress related depression in the performance of broiler chickens. Conflict of interest The authors declare that they have no conflict of interest Acknowledgments The authors would like to thank the Dean-ship of Scientific Research, King Faisal University, Al-Ahsa, Saudi Arabia, for financial support of this study (Research Project No. 170007). References 1. Mitchell MA, Kettlewell PJ. Physiological stress and welfare of broiler chickens in transit: solutions not problems! Poultry science. 1998; 77(12): 1803-14. 2. Naseem MT, Naseem S, Younus M, Iqbal Ch Z, Ghafoor G, Aslam A, Akhter, S. Effect of potassium chloride and sodium bicarbonate supplementation on thermos-tolerance of broilers exposed to heat stress. International Journal of Poultry Science 2005; 4 (11): 891-5. 3. Quinteiro-Filho WM, Rodrigues MV, Ri-beiro A, Ferraz-de-Paula V, Pinheiro ML, Sa LR, Ferreira AJ, Palermo-Neto J. Acute heat stress impairs performance parameters and induces mild intestinal enteritis in broiler chickens: Role of acute 278 S. I. Al-Sultan, S. M. Abdel-Raheem, S. M. S. Abd-Allah, A. M. Edris hypothalamic-pituitary-adrenal axis activation. Journal of Animal Science 2012; 90(6):1986-94. 4. Niu ZY, Wei FX, Liu FZ, Qin XG, Min YN, Gao YP. Dietary vitamin A can improve immune function in heat-stressed broilers. Animal 2009; 3 (10): 1442-8. 5. Sahin K, Onderci M, Sahin N, Gursu MF, Kucuk O. Dietary vitamin C and folic acid supplementation ameliorates the detrimental effects of heat stress in Japanese quail. The Journal of Nutrition 2003; 133(6): 1882-6. 6. Huang Y, Yang J, Xiao F, Lloyd K, Lin X. Effects of supplemental chromium source and concentration on growth performance, carcass traits, and meat quality of broilers under heat stress conditions. Biological Trace Element Research 2016; 170(1):216-23. 7. Hossain MM, Howlader AJ, Islam MN, Beg MA. Evaluation of locally available herbs and spices on physical, biochemical and economical parameters on broiler production. International Journal of Plant, Animal and Environmental Sciences. 2014; 4 (1) :317-23. 8. Yoo J, Yi YJ, Koo B, Jung S, Yoon JU, Kang HB, Lee DH, Heo JM. Growth performance, intestinal morphology, and meat quality in relation to alpha-lipoic acid associated with vitamin C and E in broiler chickens under tropical conditions. Revista Brasileira de Zootecnia 2016; 45(3): 113-20. 9. Guil-Guerrero JL, Ramos L, Paredes JZ, Carlosama-Yepez M, Moreno C, Ruales P. Effects of turmeric rhizome powder and curcumin in poultry production. A review. J. Anim. Feed Sci. 2017; 26(4):293-302. 10. Mahmoud UT, Amen OA, Applegate TJ, Cheng HW. Brazilian propolis effects on growth, productivity performance, gut characteristics and physiological changes in broiler chickens. Int. J. Poult. Sci. 2017; 16:169-79. 11. Chegini S, Kiani A, Rokni H. Alleviation of thermal and overcrowding stress in finishing broilers by dietary propolis supplementation. Italian Journal of Animal Science 2018; 17(2):377-85. 12. Egbuniwe IC, Ayo JO, Kawu MU, Mohammed A. Ameliorative effects of betaine and ascorbic acid on erythrocyte osmotic fragility and malondial-dehyde concentrations in broiler chickens during the hot-dry season. Journal of Applied Animal Research 2018; 46(1):380-5. 13. NRC. "National Research Council" Nutrient requirements of poultry. 9th. Rev. (Edn.). National Academy Press, Washington, DC., USA.1994. 14. AO AC. 1995. Official methods of analysis 16th Ed. Association of Official Analytical Chemists. Washington DC, USA. 15. Gross WB, Siegel HS. Evaluation of the heterophil/lymphocyte ratio as a measure of stress in chickens. Avian Diseases 1983; 27:972-9. 16. Wealleans AL, Li W, Romero LF, Mathis G, Lumpkins B. Performance and cost-benefit improvements following supplementation with a combination of direct-fed microbials and enzymes to broiler chickens raised with or without ionophores. Journal of Applied Poultry Research 2017; 27(1):23-32. 17. Zeweil HS. Evaluation of substituting ni-gella seed meal for soybean meal on the performance of growing and laying Japanese quails. Egyptian Poultry Science 1996; 16: 451-77. 18. SPSS, Statistical Packages for the Social Sciences. Statistical software for windows version 19.0, Microsoft, IBM Corp. Released 2010. IBM Chicago, IL., USA.2010. 19. Duncan DB. Multiple range tests for correlated and heteroscedastic means. Biometrics 1957; 13(2): 164-76. 20. Sahin K, Sahin N, Kucuk O, Hayirli A, Prasad AS. Role of dietary zinc in heat-stressed poultry: A review. Poultry science 2009; 88(10): 217683. 21. Attia YA, Al-Harthi MA, El-Shafey AS, Rehab YA, Kim WK. Enhancing tolerance of broiler chickens to heat stress by supplementation with vitamin E, vitamin C and/or probiotics. Annals of Animal Science 2017; 17(4):1155-69. 22. Attia YA, Hassan RA, Qota EM. Recovery from adverse effects of heat stress on slow-growing chicks in the tropics 1: Effect of ascorbic acid and different levels of betaine. Tropical animal health and production 2009; 41(5):807-18. 23. Sakomura NK, Barbosa NA, da Silva EP, Longo FA, Kawauchi IM, Fernandes JB. Effect of betaine supplementation in diets for broiler chickens on thermoneutral environment. Revista Bra-sileira de Zootecnia 2013; 8(2):336-41. 24. Sahin K, Sahin N, Yaralioglu S, Onderci M. Protective role of supplemental vitamin E and selenium on lipid peroxidation, vitamin E, vitamin A, and some mineral concentrations of Japanese quails reared under heat stress. Biological Trace Element Research 2002; 85 (1):59-70. 25. Perai AH, Kermanshahi H, Moghaddam HN, Zarban A. Effects of chromium and chro-mium+ vitamin C combination on metabolic, oxida- Alleviation of chronic heat stress in broilers by dietary supplementation of novel feed additive combinations 279 tive, and fear responses of broilers transported under summer conditions. International journal of bi-ometeorology 2015; 59 (4):453-62. 26. McDowell LR. Comparative aspects to human nutrition. Vitamin A and E. Vitamins in Animal Nutrition. Academic Press, London. 1989:93131. 27. Karami M, Alimon AR, Sazili AQ, Goh YM, Ivan M. Effects of dietary antioxidants on the quality, fatty acid profile, and lipid oxidation of longissimus muscle in Kacang goat with aging time. Meat Science 2011; 88(1):102-8. 28. Toghyani M, Toghyani M, Shivazad M, Gheisari A, Bahadoran R. Chromium supplementation can alleviate the negative effects of heat stress on growth performance, carcass traits, and meat li-pid oxidation of broiler chicks without any adverse impacts on blood constituents. Biological Trace Element Research 2012; 146(2):171-80. 29. Wang D, Huang H, Zhou L, Li W, Zhou H, Hou G, Liu J, Hu L. Effects of dietary supplementation with turmeric rhizome extract on growth performance, carcass characteristics, antioxidant capability, and meat quality of Wenchang broiler chickens. Italian Journal of Animal Science 2015; 14(3):3870. 30. Rashidi AA, Ivari YG, Khatibjoo A, Vakili R. Effects of dietary fat, vitamin E and zinc on immune response and blood parameters of broiler reared under heat stress. Research Journal of Poultry Science 2010; 3 (2):32-8. 31. Chand N, Naz S, Maris H, Khan RU, Khan S, Qureshi MS. Effect of betaine supplementation on the performance and immune response of heat stressed broilers. Pakistan Journal of Zoology 2017; 49(5): 1857-62. Slov Vet Res 2019; 56 (Suppl 22): 281-91 DOI 10.26873/SVR-767-2019 Original Research Article TREATMENT AND OUTCOME OF HORSES WITH CUTANEOUS PYTHIOSIS, AND META-ANALYSIS OF SIMILAR REPORTS Hoda Elkhenany1f*, Shady Nabil1, 2f, Howaida Abu-Ahmed1, Hassan Mahmoud3, Ahmed Korritum1, Hoda Khalifa4 department of surgery, Faculty of veterinary medicine, Alexandria University, Alexandria, Egypt, 2Brooke hospital for animals, Mansoura, Egypt, 3Department of Information systems, Faculty of computers and informatics, Banha University, Egypt, 4Department of Histology, Faculty of medicine, Alexandria University, Alexandria, Egypt t Equal contributor *Corresponding author, E-mail: hoda.atef@alexu.edu.eg Abstract: Pythiosis is reported to be one of the most life-threatening infections of people and animals in tropical, subtropical and temperate areas worldwide. Infection can result from ingesting zoospores of Pythium insidiosum organism or from contact with water contaminated with zoospores. The ingestion of zoospores may result in enteric pythiosis, whereas cutaneous contact with zoospores/oomycete may result in cutaneous pythiosis. Here, we reported the clinical details of 10 horses with pythiosis introduced to our clinic. These horses were treated by excision of the lesion and application of an antifungal cream to the resulting wound. Also, we performed a meta-analysis of 214 horses, of 18 reports, affected with cutaneous pythiosis and analysed the influence of the horse's sex, age, lesion site, and treatment on outcome. Treatment of horses in these 18 reports varied from surgical excision, topical, regional or systemic administration of an antifungal drug, immunization, or a combination of treatments. Horses with the most successful outcome were those treated by excision of the lesionc oupled with systemic or topical administration of an antifungal drug. Horses treated by immunization responded the poorest. Key words: pythiosis; skin lesions; equine; surgery; Pythium insidiosum Introduction Equine cutaneous pythiosis (ECP) is a granulomatous ulcerative disease caused byPythium insidiosum, an aquatic fungus-likepathogen (1, 2). The cutaneous lesion caused bythis pathogen has been described by some cliniciansas tumor-like, rounded, large, nodular granuloma-tous containing necrotic tissue and "irregularly shaped yellow-tan to grey, gritty, coral-like masses commonly calledkunkers or "leeches" (1). Kunkers, or "leeches"are composed of sequestered vessels, dead eosinophils, and Pythium hyphae and range in size from that of a grain of rice to several centimeters wide and several centimeters long (1). Kunkers can sometimes be found in dressings, when bandages are removed (2, 3). An epidemiological survey found the incidence of mortality of horses caused by pythiosis to be 1.3% in the Brazilian Pantanal region and 2.3% in the Brazilian Cerrado region. The case fatality rate for infected horses was 23- 45.5 (4). Received: January 2019 Accepted for publication: February 2019 282 H. Elkhenany, S. Nabil, H. Abu-Ahmed, H. Mahmoud, A. Korritum, H. Khalifa Because the cell wall of the oomycete of Pythium insidiosum is comprised primarily of cellulose and B-glucan, an essential component of the cell wall, researchers have suggested that administration of an inhibitor of b-glucan synthesis, such as caspofungin, could be an effective treatment of animals for pythiosis (5, 6). The effectiveness of caspofungin in resol-vingcutaneous pythiosis was tested by intrape-ritoneal injection in an experimentally induced pythiosis rabbit model, and resultsr evealed reduction in the growth of the lesions, but the lesions reappeared after treatment was stopped (7). Radical surgery, including amputation of anaffected limb, is a common and effective treatment for pythiosis of human patients and animals(3, 8-10). Immunotherapy has been effective in treatment of human patients and animals for pythiosis, but results of immunotherapy vary (3, 11, 12). Combining surgical therapy with immunotherapy is more effective than is immunotherapy alone or surgery alone (4, 13). The aims of this study were to evaluate the effectiveness of topic antifungal cream application after surgical management in 10 horses suffering from cutaneous infection of Pythium insidiosum. We also, performed a meta-analysis of 214 horses, in 18 reports, affected with cutaneous pythiosis to evaluate the effectiveness of different methods of treatment. This analysis also examined the influence of age, sex, pregnancy and the site of the lesion on the outcome of the infected horses. Materials and methods Case report history and clinical signs Ten horses, all of which were from the Delta region of Egypt, were presented to the Brooke Hospital for Animals (BHA) between July and September 2017 for treatment for cutaneous lesions of pythiosis. These horses had one or more exudative, ulcerative, edematous, granu-lomatous lesions (Fig. 1A). The presumptive diagnosis of pythiosis, based on the appearance of the lesion, was confirmed by histological examination of tissue excised from the lesion, using H&E and Masson's trichrome staining. The surface area (A) of each lesion was calculated using following equation: (A= n x r(a) x r(b), where r(a) was the half (%) of the longitudinal diameter and r(b) was the ^ of transverse diameter of the lesion. The age, sex, pregnancy status of females, site, and size of the lesion of each horse were recorded. Also recorded the date where the horse was first examined, the date of 1st surgery, date of recurrence, and the date at which the disease was considered to be resolved. Consent forms for treatment were signed by all owners. Surgical excision The horses were sedated with xylazine HCl (1.0 mg/kg, IV; Xylaject, Adwia, Egypt) and anesthetized with propofol (2 mg/kg, IV; Deprivan, Astra Zeneca, UK). The horses were then positioned in lateral or dorsal recumbency, depending on the site of lesion. The lesion was excised with a scalpel, after preparing the lesion and surrounding skin for surgery, then we used curette to remove the superficial layer of the skin in case of large size lesions. An antifungal cream containing terbinafine HCl (Lamisil AT 1% Cream; No-vartis, Switzerland) was applied topically after surgery once daily for one week and covered with bandage. All horses were administered metronidazole (20 mg/kg, IV; Amrizole, Am-riya pharm, Egypt), flunixin meglumine 0.50 mg/kg, IV; Flunix, Norbrook, U.K) and Procaine penicillin G (4 mg /100 kg, IM; Norocil-lin, Norbrook, U.K) for 5 days. Medline database analyses Our meta-analysis of studies of various treatments for human patients and animals for pythi-osis included 37 studies. We divided these studies into in vitro and in vivo preclinical studies (n=19) and clinical studies (n=18). Twelve preclinical studies tested the sensitivity of P. insid-iosum to various antifungal drugs, minerals, and herbal either in vitro alone (n=7), in vivo a lone (n=1) or both in vitro and in vivo (n=4). Sev en preclinical studies were conducted to identify the molecular, proteomic, and genomic makeup of P. insidiosum. Treatment and outcome of horses with cutaneous pythiosis, and meta-analysis of similar reports 283 We computed the significance between each treatment group, 95% confidence interval and sample odd ratio using Fisher exact test statistics using R program, ver.3.5.1 Results Surgical removal followed by application of an antifungal drug The 10 horses presented because of lesions caused by P. insidiosum were adults 3 to 11 years old (mean, 6.5±2.6 years), weighing 300 to 360 kg (mean, 324±18.3 kg). Three were male and 7 were mares. Four of the mares were pregnant. The cutaneous lesions were located on abdomen (n=7; the lesion of one extended caudally to include the mammary glands), shoulder (n=1), thigh (n=1), and fetlock (n=1). The cutaneous lesions were ranged in size from 27 to 297 mm2 (mean, 147.64± 123.1; Fig. 1). The results of surgical excision and topical antifungal cream application showed that this protocol was effective in 7 out of 8 cases (87.5%). One of the horses was lost to follow-up, and another one died after the second surgery as the lesion extended to the udder and animal was emaciated with very big lesion at the time of introduction. Four of the horses recovered after excision of the lesion, but for the other four horses, signs of recurrence of the lesion appeared after surgery. These 4 horses again underwent excision of the lesion 12 to 45 days (mean 22 days) after the first surgery. The average total cure time was 56.57± 19.3 days from the time of first surgery until the day of full recovery. Microscopic examination of histological sections stained with H&E or Masson's Tri-chome stain revealed fragments of hyphae surrounded by well-developed fibro-cellular capsule, connective tissue rich in collagen fibers, fibroblasts surrounded by esosinophils and radiating, esosinophilic material creating the histological appearance of the Splendore-Hoeppli phenomenon (Fig. 2). The outcome of Medline database analysis of preclinical studies The antifungal drug, terbinafine HCl, was used to treat the horses for pythiosis, which is a fungal-like disease. In one in vitro study of the effects of various antifungal drugs on P. in-sidosum, miconazole was found to be the drug most effective against P. insidiosum, followed by ketoconazole (14). In other in vitro studies, some antifungal drugs were found to be ineffective against P. insidiosum (15). In one study, the antifungal drug caspofungin (lipopeptide) was found to have a limited fungistatic effect against P. insidiosum (7). In another study, the fungistatic effect of caspofungin on P. insidio-sum was found to be similar to that resulting from immunotherapy but that treating a horse with caspofungin was more expensive than was treating a horse using immunotherapy (16). The efficacy of caspofungin against P. insidiosum was compared with that of other antifungal drugs in two in vitro studies, and in neither study was caspofungin found to be as effective as the other drugs (17, 18). Mefenoxam was found to have the highest fungal eradication (< 90%) in a study comparing the efficacy of cas-pofungin in resolving infection caused by P. in-sidiosum with that of itraconazole, posacona-zole, voriconazole, terbinafine, and mefenoxam, Brown et al. (17). Argenta et al. (18) used caspofungin in combination with terbinafine, itraconazole, ibuprofen, or fluvas-tatin, and found the best combination for inhibiting growth of P.insidiosum to be terbinafine, itraconazole, and fluvastatin. Valente et al. (19) recently reported that plant-derived essential oils (EOs) and itraconazole worked synergisti-cally to resolve infection caused by P. insidio-sum in vitro. Some herbal extracts, such as propolis and garlic, which are known for their antifungal and antibacterial effect, have shown efficacy against P. insidiosum in vitro (20, 21). The results of Zanette et al. (2013) study showed that an iron chelator, deferasirox, could ameliorate anemia caused by thalassemia but could not resolve lesions of pythiosis (22). They also reported that administering deferasirox in combination with immunotherapy resulted in dissemination of pythiosis to the lung. Zanette et al. (2015) treated rabbits for pythiosis with deferasirox in combination with the antifungal drug, micafungin, and reported and incidence of resolution of 88.2% (23). 284 H. Elkhenany, S. Nabil, H. Abu-Ahmed, H. Mahmoud, A. Korritum, H. Khalifa Sekhon et al. (1992) reported that the polyene antibiotics, like amphotericin B, were not effective against P. insidiosum (14), whereas another class of antibiotics as Macrolides and tetracycline were reported to be effective against P. insidiosum (24). These antibiotics exert their effect on P. insidiosum by inhibiting protein synthesis and inhibition of amino acid transport (25). Another study examining the effect of various antibiotics with antifungal activity against P. insidiosum found that gentamicin, neomycin, paromomycin, and streptomycin each had an inhibitory and fungicidal effect on at least 62.5% of all P. insidiosum isolates from equine pythiosis; tigecycline inhibited growth of 70.8% of the isolates (26). In vitro studies examining the effect of various drug on P. insidiosum isolated from equine lesions are summarized in Table 1. The outcome of Medline database analyses of clinical studies Eighteen clinical studies involving 214 horses infected with P. insidiosum from 7 countries, including Australia (n=5 horses), Brazil (n=106), Colombia (n=1), Costa Rica (n=39), Egypt (n=43), Thailand (n=1), and the USA (n=19), were evaluated. The youngest adult horse (above one year old) in these studies was 2 years old, and oldest was 25 years old (mean, 11.45±6.3) and foals (up to one year old) at ages ranged from 3 to 4 months with susceptibility rate only 6% in comparison to 94% for adults. The number of mares in the study group was ab out 2.5 times greater than the number of males. Twenty one of 59 mares (38%) for which information about pregnancy status was available were pregnant (Fig. 3). Cutaneous lesions of pythiosis were most commonly found on the limbs (54.12%) followed by abdomen (25.88%). Less common locations of lesions included the mammary glands, the thorax, including the withers. Some horses had lesions of pythiosis in the nasal cavity or intestine. Horses in these 18 clinical studies were treated by surgery, immunotherapy, injection of an antifungal drug into the lesion, or combination these treatments. In a report of 33 horses treated for pythiosis by surgery alone, 73% of horses had complete resolution of the lesions (27). Surgical excision of the lesion, coupled with oral administration sodium iodide for 2 weeks resulted in complete resolutions of lesions on the back and prepuce of a 3-monthold foal, but the time of follow-up for this foal was only 3 months (28). Statistical comparison between surgical approach alone and the empirical methods (developed by nonveterinarian where they used acids and another drugs combination) revealed a significant difference (P<0.05, 95% Confidence interval= 1.09-23.59 and odd ratio= 4.75) between treatments (Figure 3D). Eighty percent of horses in our study had complete resolution of cutaneous pythiosis after they were treated by surgical excision of the lesion and by topical application of an anti-fungal cream to the resulting wound. In another study of horses affected with cutaneous pythio-sis reported that 8 0% of horses had complete resolution of lesion after local injection with amphotericin B (8). When the antifungal drug, amphotericin B, administered by intravenous regional limb infusion (IV-RLP) after surgical excision to resolve lesions of pythiosis located on the distal portion of the limb, 92% of horses had complete resolution of infection (29). When amphotericin B administered in combination with 10% DMSO, 100% of horses had complete resolution of infection (30). The complete infection resolution was significantly higher among horses that received surgery and I V-RLP with amphotericin B than among horses that received surgery alone (P<0.0067, 95% confidence interval=1.39-24.06 and odd ratio=5.14). The complete resolution of infection was significantly higher among horses that received IV-RLP with amp hotericin B, with or without DMSO, than among horses that were treated empirically (P<0.00001, 95% confidence interval= 4.35- 154.2 and odd ra-tio=22.47) (Fig. 3). Immunotherapy alone was used to treat 36 horses for pythiosis in 3 studies (4, 12, 13). Immunotherapy combined with surgery was used to treat 24 horses for pythiosis in 4 studies (3, 4, 11, 31). There was no significant difference in incident of resolution between those Treatment and outcome of horses with cutaneous pythiosis, and meta-analysis of similar reports 285 horses that were treated with immunotherapy alone and those horses treated with surgery in addition to immunotherapy (P = 1, 95% confidence interval=0.246-3.80, and odd ratio=1). The outcome of horses in the group that received only immunotherapy was significantly better than the outcome of 15 horses treated empirically (P<0.001, 95% confidence inter-val=1.78-42.87 and odd ratio=7.84). The outcome of horses were treated with a combination of immunotherapy and surgery was also significantly higher than the outcome of horses treated empirically (P<0.01, 95% confidence interval=0.00-116.7 and odd ratio=0) (Fig. 3B). One report described using photoablation with a neodymium: yttrium-aluminum garnet laser after excising the lesion of two horses to induce thermal necrosis of infected tissue that surgeons failed to remove at time of surgery (32). The authors reported that the lesions had not recurred after one year. The results of all the published studies of horses undergoing treatment for pythiosis, including the number of horses in each study, age, sex, methods of treatment, and the incidence of resolution are summarized in Table 2. Figure 1: Representative clinical cases diagnosed as cutaneous pythiosis, before surgical intervention (A, B and C) and after complete recovery (D, E and F). Each case has a brief information about the case as the gender, weight, age and the surface area of the lesion on the lower left corner of the image (A, B and C) also the day of the recovery at the lower right corner in (D, E and F) images 286 H. Elkhenany, S. Nabil, H. Abu-Ahmed, H. Mahmoud, A. Korritum, H. Khalifa Figure 2: Representative histological section of skin lesion stained with H&E (A) and Trichome Stain (B) showing the characteristic Splendore-Hoeppli like reaction. Scale bar = 500 pm Figure 3: MEDLINE database analysis of clinical cases. (A) Publications around the world, bars represent no. of animals in all studies published/country (B) Gender susceptibility, (C) Age susceptibility and (D) MEDLINE database analysis comparing between different treatment approaches that have been used to treat equine pythiosis, *P<0.05, ***P<0.001, ****P<0.000001 Treatment and outcome of horses with cutaneous pythiosis, and meta-analysis of similar reports 287 Table 1: Summary of published preclinical studies in vitro and in vivo using different antifungal drugs, minerals and herbals to eradicate P. insidiosum Drugs Drug under investigation In vitro/ In vivo Animal M/F /Age Results RE F Amphotericin B, hamycin, 5-fuo-rocytosine, fluoconazole, itraconazole, ketoconazole and miconazole In vitro P. insidiosum were sensitive to fluconazole, ketoconazole, and miconazole and miconazole was the strongest one. (14) m ? Terbinafine, itraconazole, caspofun-gin, ibuprofen and Fluvastatin In vitro and In vivo Rabbit (M/3m) Terbinafine, itraconazole and Fluvastatin combination resulted in the lowest hyphae number. (18) t3 "ö3 un «a Caspofungin In vitro and In vivo Rabbit (M&F/3 m) The results showed that caspofungin has limited fungistatic activity against P. insidiosum. (7) ü 90% inhibition rate (17) ron chelator Immunotherapy, Iron chelator de-ferasirox and combination of both In vitro and In vivo Rabbit (F/3m) Deferasirox alone did not result in any significant enhancement for the lesion from the control. However, it could ameliorate the anemia. The combination between the immunotherapy and iron were the worse than the immuno-therapy alone. (22) Micafungin with or without defer-asirox In vitro and In vivo Rabbit (F/3m) Micafungin alone is not effective against P. insidiosum but effective when combined with deferasirox cure rate 88.2% however, disease dissemination in the lung were reported. (23) Vï o actr Terbinafine or itraconazole with Melaleuca alternifolia, Mentha piperita and Origanum vulgare essential oils (EOs) In vitro The antimicrobial combinations using EOs with terbinafine or itraconazole can be an attractive therapeutic option for controlling P. insidiosum infections. (19) ■e 0.05). Similar results were obtained by (16), who obtained that, the dry matter percentage was 93.16 and 94.23; crude protein was 70.44 and 52.12; crude fat was 7.36 and 23.47 and ash was 11.18 and 18.34 in FM and PBM, respectively. The opposite studies showed no significant variation in the case of crude protein between FM and PBM values (17). Moreover, (18) indicated that PBM is a suitable replacement of FM in practical formulation diets for tilapia. Generally, the composition of PBM depends on processing conditions and the source of raw materials (19). So, there are some PBM have very high protein content (75-90%) with low contents of ash (less than 10%) and fat (less than 15%). Opposite values were reported by (20), who showed that the low quality of PBM had protein content (55-75%) higher levels of ash (up to 15%) and fat (more than 15% and up to 30%). The present data revealed that, the essential amino acids (EAA) profile was significantly higher in the basal diet (control) except both threonine and arginine were higher in ABM and KWM, respectively. The lowest level of EAA found in KWM (Table 3). The chemical score of the free essential amino acid of the experimental FM, PBM, ABM and KWM were low for methionine, iso-leucine, and phenylalanine, while the highest value recorded for arginine and lysine. Similar results were obtained by (21). Additionally, (22) showed that the EAAs in FM and PBM were 5.97 and 6.09 for arginine; 2.06 and 1.98 for histidine; 3.73 and 3.89 for isoleucine, 6.78 and 7.04 for leucine; 12.78 and 12.17 for lysine; 2.92 and 2.74 for methionine; 4.05 and 4.17 for phenylalanine; 3.88 and 3.65 for threonine; 0.43 and 0.42 for tryptophan and 4.59 and 4.84 g /kg diet for valine, respectively. However, PBM is deficient in one or more essential amino acids (16). But, the level of amino acid profiles of PBM is relatively like FM (23) making the ingredient a valuable protein source for many species. However, at replacing of FM with PBM may need to be supplemented with some essential amino acid such as lysine, methionine and threonine to make the best growth performance and body composition of fish. The deficiency of EAAs results in poor utilization of dietary CP and reduces growth and feed efficiency. By estimating the level of EAAs in the tested ingredients, it was clear that, the level of all the essential amino acids was higher than the required level for fish feeding. As shown in Table (3), the highest values of non-essential amino acids in tested different ingredients were recorded in FM except glycine and tyrosine. In contrast, the lowest value of non-essential amino acids was found in KWM and PBM had the highest value of tyrosine and aspartic acids while glycine showed the highest value in ABM. Similarly, (24) who found that amino acids of Nile tilapia fed FM, PBM or porcine by-product meal had similar amino acids profile. The present results showed lower value of non-essential amino acid than those reported by (22), who reported that the non-essential amino acids value in FM and PBM were for alanine 5.98 and 5.85; aspartic 8.36 and 7.67; cystine 0.95 and 0.94; glycine 8.54 and 8.37; glutamate 13.37 and 13.53; proline 5.35 and 5.80; serine 3.75 and 3.68; tyrosine 2.80 and 2.70 g /Kg, re- 296 M. Khalafalla, A. Mohamed, M. Zayed, A. Metwally spectively. In opposite, FM could only be replaced with PBM at a level which did not exceed 50%. Table 1: Ingredients and chemical composition (%) of the experimental diets Ingredients Treatments FM PBM ABM KWM Fish meal 10. 00 00.00 00.00 00.00 Poultry by-product meal 00.00 5.00 2.50 2.50 Animal by -product meal 00.00 2.50 5.00 2.50 Kitchen west meal 00.00 2.50 2.50 5.00 Soybean meal 50 .56 50.50 50.60 50.75 Yellow com 32.50 32.35 32.20 32.00 Com oil 4.38 4.50 4.43 4.38 Limestone, ground 1.52 1.60 1.71 1.80 Methionine 0.24 0.25 0.26 0.27 Premix* 0.30 0.30 0.30 0.30 Chromic oxide 0.50 0.50 0.50 0.50 Total 100 100 100 100 Proximate analysis of the experimental diets (% DM) Dry matter (DM) 91.57±0.11 91.69±0.07 91.60±0.07 91.48±0.12 Crude protein (CP) 35.20±0.15 35.03±0.14 35.16±0.25 34.67±0.45 Crude fat (EE) 10.17±0.21 10.33±0.14 9.85±0.08 10.57±0.65 Ash 12.18±0.16 12.54±0.55 12.70±0.28 12.48±0.21 Crude fiber (CF) 4.95±1.50 5.13±1.48 5.59±0.82 5.75±0.92 Nitrogen-free extract (NFE) 37.50±0.32 36.97±0.61 36.70±0.59 36.53±0.50 *Each 1 kg contains , Vitamin A, 200,00000 IU: Vitamin D3, 400,000 IU; Vitamin E, 5,00 mg; Vitamin C, 20,000 mg; Vitamin B1, 800 mg; Vitamin B2, 1,000 mg; Vitamin B6, 2,400 mg; Vitamin B 12, 40 mg; L-lysine, 3,000 mg; DL Methionine, 2,000 mg; Choline chloride, 5,000 mg; Niacinamide, 10,000 mg; Magnesium sulphate, 24,000 mg; Cobalt Sulphate, 80 mg; sodium selenite, 20 mg; potassium iodide, 240 mg; Calcium d pantothenate, 2,000 mg; Biotin, 150 mg, ferrous sulphate, 28,000 mg; Copper sulphate, 24,000 mg; Zinc sulphate, 24,000 mg; Manganese sulphate, 6,800 mg; Inositol, 5,000 mg. Using of animal by products as an alternative protein source in Nile tilapia (Oreochromis niloticus) diets 297 Table 2: Chemical composition of ingredients waste meal used in experimental diets Treatments Items FM PBM ABM KWM Dry matter Crude protein Crude Fat Ash 96.08± 0.15 72.61± 2.35 a 9.61± 0.82c 12.09± 1.45 c 95.68± 0.08 58.31± 2.55 b 22.61± 1.05b 17.49± 2.15 b 96.71± 0.85 51.98± 3.05 c 25.06± 1.12 a 19.28± 1.90ab 96.11± 0.90 52.98± 2.60 c 24.26± 0.90 a 21.63± 3.45a Means on the same row with different superscripts are significantly different (P<0.05). Table 3: Essential and nonessential amino acids of FM, PBM, ABM and KWM used in experimental diets (%, wet/ wt.) Treatments Items FM PBM ABM KWM Threonine 2.79±0.18b 2.08±0.16 c 3.25± 0.21 a 2.00±0.21c Valine 3.72±0.21 a 2.80±0.21b 3.06±0.30 b 2.56±0.19c Methionine 1.17±0.11 a 1.05± 0.01 a 0.52± 0.08 b 0.68±0.01 b Isoleucine 2.71±0.23 a 1.97± 0.12b 2.26±0.21 b 1.59±0.15 c Leucine 5.53±0.31 a 3.74± 0.25c 4.24±0.31b 2.78±0.21 d Phenylalanine 2.76±0.17 a 2.08± 0.14b 2.62±0.15 a 1.67±0.14c Lysine 5.03±0.26 a 3.43± 0.22b 3.87±0.23 b 3.67±0.31b Histidine 3.15± 0.20 a 2.92± 0.11 a 2.15±0.17 c 2.67±0.19 b Arginine 3.56± 0.25b 2.32±0.19 c 2.59±.020c 4.08±0.31 a Taurine 2.28± 0.14 a 1.95± 0.26b 2.00± 0.16b 1.61± 0.14 c Aspartic 1.84± 0.21 a 1.96± 0.11 a 1.34± 0.08 b 1.05± 0.07 b Serine 2.26± 0.14 a 1.82± 0.24b 1.41± 0.10c 1.68± 0.18 b Glutamic 12.20± 0.41 a 8.98± 0.31 b 7.97± 0.31b 11.07± 0.53 a Glycine 2.37± 0.19b 1.70± 0.17c 3.90± 0.26 a 1.92± 0.24 c Alanine 7.79± 0.31 a 5.29± 0.21c 7.20± 0.34 a 6.31± 0.44b Cystine 0.12±0.01a 0.07±0.002c 0.09±0.001b 0.07±0.001c Tyrosine 2.58± 0.24 b 2.93± 0.21 a 1.51± 0.22c 2.12± 0.19b Proline 3.05± 0.31 a 2.65± 0.18b 1.87± 0.13d 2.24± 0.21c Means on the same row with different superscripts are significantly different (P<0.05). Table 4: Apparent digestibility coefficients of the different diets Treatments Items FM PBM ABM KWM Dry matter (%) 83.2±0.34a 82.4±0.26a 78.2±0.32c 80.3±1.62b Organic matter (%) 85.3±0.14a 85.2±0.35a 80.9±1.27c 83.1±1.24b Crude Protein (%) 89.8±0.24a 88.9±0.52a 81.2±0.65c 84.3±1.52b Ether Extract (%) 96.6±0.45a 96.5±0.23a 87.2±0.65c 93.4±0.65b Means on the same row with different superscripts are significantly different (P<0.05) 298 M. Khalafalla, A. Mohamed, M. Zayed, A. Metwally Table 5: Initial body weight, diets (mean ± SD) nutrient utilization and economical evaluation of Nile tilapia fed different Treatments Items FM PBM ABM KWM Initial body weight (g) 5.72±0.11a 5.92±0.22a 5.60±0.14a 5.92±0.22a Final body weight (g) 99.25±0.07a 96.82±0.23 a 86.00±0.48b 83.32±0.06c Body weight gain (g) 93.53±0.21a 90.90±0.32b 80.40±0.21c 77.40±0.10d Gain in weight (g fish) 0.78±0.004a 0.76±0.010a 0.67±0.006b 0.65±0.004b Specific growth rate 2.38±0.15a 2.33±0.05a 2.28±0.08b 2.20±0.05b Total feed intake (g/fish) 180.9± 3.0 a 178.5±3.0 b 162.6 ±3.0c 157.2±3.0d Av. Daily feed intake(g/fish) 1.51± 0.15 a 1.49± 0.15 a 1.36±0.15b 1.31± 0.15c Feed conversion ratio 1.93 ±0.14a 1.96±0.07a 2.02±0.09a 2.03±0.31a Crude protein (%) 32.23±0.15a 32.12±0.14 a 32.21±0.25 a 31.72±0.45 a Protein intake (g/fish) 58.30±1.35 a 57.33±2.10 a 52.37±1.45b 49.86±2.15 c Protein efficiency ratio 1.60±0.01a 1.59±0.05a 1.54±0.21a 1.55±0.07a Survival ratio (SR %) 100 a 100 a 100 a 100 a Economical evaluation2 Costs (L.E)/ton 9000 7175 7275 7150 Relative to control (%) 100 79.72 80.83 79.44 Decrease in feed costs 00.00 20.28 19.17 20.56 Feed costs * (L.E)/kg WG 17.41 14.09 14.71 14.52 Relative to control (%) 100 80.93 84.25 83.16 Decrease in feed costs* (L.E)/kg WG 00.00 19.07 15.75 16.84 Kg Feed /kg weight 1.934 1.964 2.022 2.031 kg Weight / Kg feed_0.517_0.509_0.495_0.492 Values are the mean ± S.E. of triplicate groups of each treatment. 1Live body weight (LBW) in g of individual group of each experimental treatment was recorded every 2 weeks (14 days); Weight gain (WG) = final weight (g) - initial weight (g); Specific growth rate (SGR % /day) =100(lnW2-lnW1) / T; Average daily gain (ADG) = (W2 - W1) / t; Feed conversion ratio (FCR) = feed intake (g)/weight gain (g); Protein efficiency ratio (PER) = weight gain (g)/protein intake (g); Survival rate (SR %) = total number of fish at the end of the experiment x 100 / total number of fish at the start of the experiment. Where: W2 is the final weight, W1 initial weight and t is the time in days; ln = the natural log; T = period. 2Local price (L.E./Kg) for feed ingredients used FM (25); PBM (6); ABM (10) and KBW (5). Apparent digestibility coefficients (ADC) of the different experimental tilapia fish diets are shown in (Table 4). The current data illustrated that the ADC of all studied parameters recorded the highest value in FM and PBM without any significant variation between them followed by ABM and KWM. These results are supported by (25), who demonstrated that, the ADC of commercial FM replacement diets with different level of PBM (0, 25, 50 and 100%) were organic matter 65.4, 67.5, 62.4 and 69.1 1; crude protein 97.2, 97.2, 96.7 and 97.2; lipid 85.3, 87.2, 87.0 and 89.9 %, respectively. In the same way, (26) reported that the ADC of DM, EE, CP and NFE were not altered by the inclusion of PBM. The present results showed higher values than those reported by (27) where the ADC of diets containing mixture of ABM 25 (83.6% ± 1.15%), mixture of ABM 50 (79.21% ± 1.01%) and mixture of ABM 75 (78.7%± 1.57%) were not significantly different (P>0.05) compared Using of animal by products as an alternative protein source in Nile tilapia (Oreochromis niloticus) diets 299 with the control diet (83.4% ± 5.21%). The high ash content in PBM could reduce protein digestibility in fish diet (28, 29). As shown in Table (5) the average initial weight of experimental Nile tilapia fish was ranged between 5.60 and 5.92 g/fish without any significant differences between the different treatments. The highest final weight was measured in FM (99.25±0.07 g) followed by PBM (96.82±0.23 g) and ABM (86.00±0.48 g), and KWM (83.32±0.06 g). This means that the final weight in FM increased about 2.45, 13.35 and 16.05 % when compared with PBM, APM and KWM, respectively. In the same trend, ADG was significantly different between different diets and take the same direction of previous results whereas the highest value of ADG noted in T1 followed by T2, T3 and T4, respectively. Similarly, (26) reported that the highest growth performance was recorded in tilapia fish fed PBM. (30) showed that the higher final growth weight and ADG of fish may be due to the type of diet and its composition, level of essential amino acids, the higher initial weight of the stocked fish or to higher rates of the supplemental food offered to the cultured fish. On the contrary, the present results of the ADG was lower than reported by (31) who found that the ADG values was between 1.6 and 3.04 g d-1 after feeding the cultured fish. PBM is rich of protein (65%) with ridiculous the essential amino acids (EAA) profile which resulted in increased growth in fish (32,33. Improved growth rate and daily gain may be attributed to improve protein composition and essential nutrients in the test diets (34). Moreover, (16) reported that the growth performance increased in fish fed diets in which up to 50% of the FM was replaced by PBM, similar to results in Tilapia zilli (35). As well as, average feed utilization in terms of total feed intake (TFI), daily feed intake (DFI), feed conversion ratio (FCR), total protein intake (TPI) and protein efficiency (PE) are presented in Table (5). The present results showed that DFI and TPI recorded the highest values in FM and PBM without significant differences between them and significantly higher when compared with APM and KWM. While, there are no significant difference in FCR and PE between treatments. Also, TFI recorded the highest quantity in FM followed by PBM, ABM and KWM. Similarly, (36) reported that the average daily intake of fish fed FM diet did not differ from those of fish fed diets FM with 20 and 40% PBM (P> 0.05) due to the protein source, fish species and size, experimental period and culture systems. The major problem in feeding PBM is limited content of essential amino acids especially methionine, phenylala-nine, and lysine (37). (31) reported that, the FCR was ranged from 1.17 to 1.6 for sex-reversed tilapia fed on 30% crude protein. In contrary, high FCR value (5.56 -7.77) were obtained by (38) in Nile tilapia. In the present study, the overall survival rate was 100% during the experimental period. These results harmonized with those reported by many researchers (25;39; and 22). Moreover, the one ton feed cost in the present study was reduced in all replacing treatments of FM by 20.28; 19.71 and 20.56 % for PBM; ABM and KWM, respectively and decreased feed costs/kg weight gain by19.07; 15.75 and 16.84%, respectively . In this trend, (40) reported that, replacement of FM by a mixture of plant protein sources significantly reduced incidence costs, as well as being of immediate importance for feed production in Egypt. Conclusion From the present study, it could be concluded that using waste protein sources especially containing high PBM as a replacement of FM would be a helping tool to reduce feed cost per Kg fish production. References 1. MahfouzM, Hegazi M, El-Magd M, Kasem E. Metabolic and molecular responses in Nile tilapia, Oreochromis niloticus during short and prolonged hypoxia. Marine and Freshwater Behaviour and Physiology 2015; 48: 319-40. 2. Tacon A and Forster I. Biotechnology in aquatic feeds. In: Biotechnology-Aquaculture Interface: National Conservation and Training Center, 300 M. Khalafalla, A. Mohamed, M. Zayed, A. Metwally U.S. Fish and Wildlife Center, Shepherdstown, West Virginia, March 2001; 5-7. 3. Dawood MAO, Eweedah NM, Moustafa EM, Shahin MG. Synbiotic Effects of Aspergillus oryzae and P-glucan on Growth, Oxidative and Immune Responses of Nile Tilapia, Oreochromis niloticus. Probiotics and Antimicrobial Proteins 2019: 1-12. 4. GAFRD. General authority of fish resource department, one of the authorities of the Ministry of Agriculture and Land Reclamation. Fish statistics year book. Egypt, 2015. 5. Wilson R. Amino acids and protein. In: Halver, J.E. and Hardy, R.W.,(Eds.) Fish Nutrition 2002; 143-179. San Diego, CA, USA. 6. Sogbesan O, Ugwumba A and Madu C. Nutritive potentials and utilization of garden snail (Li-micolaria aurora) meat meal in the diet of Clarias gariepinus fingerlings. African J. Biotech. 2006; 4:124 -45. 7. Zhang Y, Ji W, Wu Y, et al. Replacement of dietary fish meal by soybean meal supplemented with crystalline methionine for Japanese seabass (Lateolabrax japonicus). Aquaculture Research 2016; 47:243-52. 8. Naylor R, Hardy R, Bureau D, et al. Feeding aquaculture in an era of finite resources. Proceedings of the National Academy of Sciences 2009; 106:15103-10. 9. Shapawi R, Ng W, and Mostafa S. Replacement of fish meal with poultry by-product meal in diets formulated for the humpback grouper (Cromileptes altivelis). Aquaculture 2007; 273: 118-389. 10. NRC. National Research Council). Nutritional requirement of fish. National Academy press, Washington DC. 1993, 114. 11. AOAC (2006). Official Methods of Analysis. Horwitz W. 18th edition 2006, Washington, DC: 1018 12. Duncan D. Multiple range and multiple F tests. Biometrics 1955; 11:1-42. 13. SAS. SAS User's Guide 1986; Version 6 Edition SAS Institute, Cary, NCUSA. 14. Olvera-Novoa M, Pereira-Pacheco F, Olivera-Castillo L. et al,. Vigna unguiculata protein concentrate as a replacement for fishmeal in diet for tilapia (Oreochromis nitloticus) fry. Aquaculture 1997;158:107-16. 15. Sugiura S, Dong F, and Hardy R. Primary responses of rainbow trout to dietary phosphorus concentration. Aqua. Nutrition 2000; 6: 235-45. 16. Aydin B, and Gumus E. Replacement of Fish Meal by Poultry By-product Meal with Lysine, Methionine and Threonine Supplementation to Practical Diets for Nile Tilapia Fry (Oreochromis niloticus), Isr. J. Aquaculture - Bamidgeh, IJA 2013; 65:.885 -92. 17. Cruz-Suarez L, Tapia -Salazar M, Villareal -Cavazos D, et al. Apparent dry matter, energy, protein and amino acid digestibility of four Soybean ingredients in white shrimp Litopenaeus vannamei juveniles .Aquaculture 2009; 292: 87-94. 18. Yones A, and Metwalli A. Effects of Fish Meal Substitution with Poultry By-product Meal on Growth Performance, Nutrients Utilization and Blood Contents of Juvenile Nile Tilapia (Oreochromis niloticus). J. Aquac. Res. Development 2016; 7:389-96. 19. Johnson M, and Parsons C. Effects of raw material source, ash content and assay length on protein efficiency ratio and net protein ratio values for animal protein meals. Poultry Science 1997; 76: 1722—7. 20. Prado, J.P.S.; Cavalheiro, J.M.O.; Caval-heiro, T. B. and Silva, F.V.G. (2016). Degradation of amino acids by leaching in feeds for shrimp,. Gaia Scientia,10: 361-71. 21. Willingham, W.T.; Colt, J. E .; Fava, J.A. ; Al Hillaby, B. ; Ho, C.L.; Katz,M.; Russo, R.C. and Wood, D.L. ( 2004). Dogmas and controversies in the handling of nitrogenous wastes: is exogenous ammonia a growth stimulant in fish? J. Exp. Biol., 207: 2043-54. 22. Silvâo,C.F. and Nunes, A.J. (2017). Effect of dietary amino acid composition from proteins alternative to fishmeal on the growth of juveniles of the common snook, Centropomus Undecimalis, Res. Bras. Zootec., 46:569-75. 23. NRC (2011). Nutrient Requirements of Fish and Shrimp - Books & Reviews 24. Hernandez, C.; Osuna, L.O.; Hernandez, A.B.; Gutierrez ,Y.S. and Rodriguez ,B.G. (2014). Replacement of fish meal by poultry by-product meal, food grade, in diets for juvenile spotted rose snapper (Lutjanusguttatus). Latin American Journal of Aquatic Research, 42:111-20 25. Rawles, S.D.; Richie, M.; Gaylord, T.G.; Webb, J. and Freeman, D.W. (2006) .Evaluation of poultry by-product meal in commercial diets for hybrid striped bass (Morinechrysops x Moronesaxat-ilis) in recirculated tank production .Aquaculture, 259: 377-89. Using of animal by products as an alternative protein source in Nile tilapia (Oreochromis niloticus) diets 301 26. Yones, A.M.M. and Metwalli, A.A. (2016).Effects of Fish Meal Substitution with Poultry By-product Meal on Growth Performance, Nutrients Utilization and Blood Contents of Juvenile Nile Tilapia (Oreochromis niloticus). J. Aquac. Res. Development, 7:389. 27. Jamil,K.; Abbas,G.; Akhtar,R.; Hong, L.and Zhenxing ,L. (2007). Effects of replacing fishmeal with animal by-products meal supplementation in diets on the growth and nutrient utilization of mangrove red snapper, J. Ocean Univ. China, 6:292-98. 28. Kureshy, N., Davis, D. A. and Arnold, C. D. (2000): Partial replacement of fish meal with meat-and-bone meal, flash dried poultry by product meal and enzyme digested poultry by-product meal in practical diets for juvenile red drum. North American Journal of Aquaculture, 62: 266 -72. 29. Lee, H. Y. M.; Cho, K. C.; Lee, J. E. and Yang, S. G. (2001). Dietary protein requirement of juvenile giant croaker, Nibea japonica, Temminck and Schlegel. Aquaculture Research, 32:112-8. 30. Yi, M.; Yu, Q. and Huang, L. (2002). An idio gram on pachytene bivalents with high resolution multiple bands of zebra fish. Sci. China (C), 45:370-8. 31. Diana, J.S.; Lin, C. K .and Yi, Y. (1995). Timing of supplemental feeding for tilapia production. J. World Aqua. Society (submitted).PP.117-25. 32. NRC (2011). Nutrient Requirements of Fish and Shrimp - Books & Reviews 33. Badillo ,D.; Herzka, S.Z. and Viana ,M.T. (2014). Protein Retention Assessment of Four Levels of Poultry By-Product Substitution of Fishmeal in Rainbow Trout (Oncorhynchus mykiss) Diets Using Stable Isotopes of Nitrogen (d15N) as Natural Tracers. PLoS ONE 9(9): e107523. 34. Chitmanat,C.; Tipin, A.; Chaibu, P. and Traichaiyaporn, S.(2009). Effects of replacing fish-meal with wastes derived from local fisheries on the growth of juvenile tilapia, Oreochromis niloticus, J. Sci. Technol., 31: 105-10. 35. Yildirim, O. ; Turker, A. and Senel, B.( 2009). Effects of natural zeolite (Clinoptilolite) levels in fish diet on water quality, growth performance and nutrient utilization of Tilapia (Tilapia zillii ) fry. Fresenius Environ. Bulletin, 8:1567-71. 36. Wu, Y.; Wang, Y.; Ren, G.; Qin, J.G. and Kim, S.H. (2016). Improvement of fish meal replacements by soybean meal and soy protein concentrate in golden pompano diet through y-ray irradiation. Aqua. Nutrition, 22:873-80. 37. Dong, F.M.; Hardy, R.W.; Haard, N.F.; Borrows, F. and Forster, I.P. (1993). Chemical composition and protein digestibility of poultry by-product meals for salmonid diets. Aquaculture, 116: 14958. 38. Essa, M.A. (1997). Utilization of some aquatic plants in diets for Nile tilapia, Oreochromis niloticus, fingerlings. Egypt J. Aquat. Biol. Fish, 1: 19-34. 39. Tabinda, A.B. and Butt, A. (2012). Replacement of fish meal with PBM meal (Chicken intestine) as a protein source in carp (grass carp) fry diet. Pakistan Journal of Zoology, 44:1373-81. 40. El-Saidy, D.M.S. and Gaber, M.M.A. (2003). Replacement of fish meal with a mixture of different plant protein sources in juvenile Nile tilapia Oreochromis niloticus (L.), diets. Aquaculture Research, 34: 1119-27. Slov Vet Res 2019; 56 (Suppl 22): 303-12 Original Research Article DOI 10.26873/SVR-769-2019 MYCOLOGICAL EXAMINATION OF FISH FEED STUFF WITH SPECIAL REFERENCE TO MYCOTOXIN PRODUCTION Maha M.Kholife1, 2*, Amgad A.Moawad1, Amany M.Diab3, El-keredy M.S. Abeer4 1Department of Bacteriology, Mycology and Immunology, Animal Health Research Institute, Kafr El Sheikh, Egypt, 2Department of Bacteriology, Mycology and Immunology, Faculty of Veterinary Medicine, Kafrelsheikh University, Egypt, 3Aquatic Microbiology, Faculty of Aquatic and Fisheries sciences, Kafrelsheikh University. 4Department of Biochemistry, Nutritional deficiency diseases and Toxicology, Animal Health Research Institute, Kafr El Sheikh, Egypt *Corresponding author, E-mail: maha mahmod1988@gmail.com Abstract: Mycotoxin contamination is considered as one of the most economic problems for livestock and feed industries. A total of thirty fish feedstuffs were collected for isolation and identification of fungi from fish farms in Kafr El Sheikh Governorate, Egypt. Five fungal genera were isolated from all examined feedstuffs. The most frequent isolated fungi was Aspergillus spp. (86.66%) followed by Penicillium spp. (23.33%), and Fusariam spp. (10%), Mucor spp. (6.66%) then Rhizopus spp. (3.33%). Aspergillus niger was the most prevailing genus (43.33%) followed by Aspergillus flavus ((30%), and Aspergillus fumiga-tus (6.66%), Aspergillus versicolor (3.33%) then Aspergillus terrus (3.33%). Aspergillus ochraceus was not found despite the presence of ochratoxin A (OTA) in low concentrations. Some mycotoxins were produced by more than one fungal species. Mycotoxins determination using HPLC revealed that 23.33% (7/30) of examined fish feed samples were positive, while 23 samples (76.66%) were mycotoxins free. In positive samples of mycotoxins, aflatoxins (AF) and OTA represented 23.33%, 10%, respectively. The estimated carcinogenic aflatoxins were AFB1, AFB2, AFG1 with a percentage of 23.33%, 13.34%, 6.67%, respectively, but AFG2 was not found. About 42.86% of the AFs producing fish feeds was higher than the permissible limit of aflatoxins (permissible limit is 20 ppb). The aflatoxigenic ability of the recovered nine A. flavus referred to 6 out of 9 (66.67%) were aflatoxin producers. Polymerase chain reaction (PCR) was carried out using the norsolonic acid reductase (nor), versicolorin A (ver-1) and O-methylsterigmato-cystin (omtA) as aflatoxin producing genes of the isolated A. flavus strains. From the AF producing A. flavus, 50% was above the permissible limit. Therefore, the high contamination of fish feedstuffs with fungi, AFB1 and low OTA in fish farms indicated potential risks to fish liveliness, derived products and the health of fish consumers. Key words: fish feedstuff; fungi; aflatoxins; ochratoxin A Introduction Fish serve as a substantial source of human dietary protein worldwide, especially in African countries (1). Fish feed is considered the Received: January 2019 Accepted for publication: February 2019 304 M. Kholife, A. Moawad, A. Diab, M. El-keredy enormous cost item in the aquaculture industry and represents 40-50% of the total production costs in intensive culture systems (2). The low quality feed intake have adverse effects on animal health and productivity (3). Presence of mold in fish feeds indicates contamination probably due to improper selection of ingredients for manufacturing or an inadequate storage. Mold increase over a temperature range of 10-40 °C, pH range of 4-8 and humidity levels greater than 62% as well as more than 12-13% moisture. Fungal growth and production of their metabolites (mycotoxins) are related to extremes weather, improper storage conditions causing low feedstuff quality and bad feeding conditions (4). The most repeatedly isolated genus of fungi in feed was Aspergillus spp. followed by penicillium spp. and Aspergillus fla-vus is the most prevalent isolated fungi species (5). Mycotoxins are a structurally diverse group of mostly small molecular weight compounds, produced by the secondary metabolism of fungi that grow in feeds, from the harvested products to the consumers. Mycotoxins occur sporadically both seasonally and geographically (6). Many reports indicated an economic losses from mycotoxicosis in fish from chronic infection as well as increasing feed conversion ratios and unforeseen outbreaks of fish mortality (7, 8). The remarkable mycotoxin types of concern produced by fungal genera Aspergillus, Penicillium and Fusarium include the aflatoxins, ochratoxin A, trichothecenes and fumonisins (9). Aflatoxin was considered to have a great importance in aquaculture. Aflatoxin exerts carcinogenic effects in fish as in all animal species (10). The intake of moderate to high doses of aflatoxin by fish develop an acute intoxication, called acute aflatoxicosis, that generally leads to poor heath, fertility, productivity loss, weight gain reduction and immunosuppression (11). The main types of aflatoxins are B1, B2, G1, and G2 based on their fluorescence under UV light blue or green and relative chromato-graphic mobility during thin-layer chromatog-raphy (12). The main sources of AFB1 contamination in aquaculture represented in pelleted fish feed due to the isolation and identification of fungi Aspergillus spp., Penicillium spp. in feed samples, as well as other several genera (13). Ochratoxin A (OTA) is produced by Aspergillus and Penicillium species (14). For fungal examination, the slide culture technique is carried out for some mould species identification , which characterized by their restricted growth making difficulty to be identified using the wet mount slide method (15). The prepared samples were analyzed using a validated method by reversed-phase HPLC separation and fluorescence detection after post-column derivatization (16). Herein, this investigation was conducted to examine and identify the most prevalent fungi in fish feeds as well as to determine aflatoxins and OTA in examined fish feed samples moreover the aflatoxigenic ability of isolated Asper-gillus flavus. Materials and methods Fish feed samples collection A total of 30 fish feedstuff samples were collected from ten different fish farms in Kafr El Sheikh Governorate in Egypt. Each representative fish feedstuff sample was thoroughly ground and mixed. The samples were examined for mycotic contamination and mycotoxin production. The samples after the dilution were inoculated into plates containing prepared media for isolation Sabouraud's dextrose agar (SDA) with chloramphenicol (0.05g/l) (17). Lactophe-nol cotton blue stain for fungal microscopic examination prepared as previously described (18). The examined samples of fish feeds and nine isolates of A. flavus were sent to laboratory for mycotoxins determination. Isolation and purification of fungi The dilution of the samples was carried out (19). Approximately 10 g of each sample were homogenized in a sterile mortar, diluted in 90 ml distilled water, and then 1 ml was transferred to tube containing 9 ml sterile distilled water. The tube was shaken and 1ml was removed into a sterile petri dish containing SDA with chloramphenicol (0.05g/l) using surface spread method and incubated at 25-28oC for 7-10 days Mycological examination of fish feed stuff with special reference to mycotoxin production 305 under complete aseptic conditions. After incubation, the plates were examined visually and microscopically by making films. All the positive fungal cultures were purified by subcultur-ing on SDA plates and incubated at 25-28 oC for 3-5 days. Stock isolates maintained in SDA slopes in refrigerator for further identification. Identification of fungal isolates The identification of the isolated fungi recovered from the examined fish feedstuffs included the macroscopical and microscopical examination. The identification of mold genera and species was carried out (18, 20) for genus Aspergillus and (21- 24) for the other mold genera. The macroscopical examination shed light on the rate and pattern of growth as color, texture, basal and surface mycelia. The microscopical examination of fungal colonies was carried out using both wet mount slide method and the micro slide culture technique. The slide culture method (23) was carried out in case of some mold species, which characterized by their restricted growth and failed to be detected using the wet mount slide method. Estimation of mycotoxins in the examined fish feedstuffs Qualitative and quantitative estimation of aflatoxins (AF) Determination of aflatoxins in fish feed samples by high-performance liquid chromatog-raphy (HPLC) (16). The preparation of chemicals, standard Aflatoxin (AF) solutions of B1, B2, G1 and G2 were prepared (25). Extraction and clean-up procedures for HPLC analysis of the prepared samples (16) using a validated method by reversed-phase HPLC separation and fluorescence detection after post-column derivatization. Qualitative and quantitative estimation of ochratoxin A The samples were extracted (26) with few modifications. The sample (15 g) was blended (15 min) in 50 ml of acetonitrile - water (45:05, v/v), using high speed blending and then the extract was filtered through filter paper. About 5 ml of the filtrate was mixed with 50 ml of phosphate buffer saline (PBS) and filtered through a glass microfiber. Then 10 ml of the filtrate was passed through immunoaffinity columns. OTA was eluted from the column by passing 1.5 ml of methanol (HPLC grade) and collected in a vial. The eluate was evaporated until dryness at 40°C and residues were re-dissolved in 1 ml of mobile phase i.e. acetonitrile: water: acetic acid (47/51/2, v/v/v) for HPLC analysis. Stock standard solutions of each sample were prepared then the method was validated using The European Commission (27) as for guidelines. Selectivity was determined from retention time, ion ratios, and identification-points (IP) for each analyte. The permissible limit of aflatox-ins and ochratoxin A were determined as previously described (30). Screening of aflatoxigenicity testfor isolated Aspergillus flavus strains The technique using fluorescence of agar medium under U/V light (28) was performed for detection the toxigenic strains of A. flavus including the cultivation, the observation of fluorescence and the extraction of A. flavus toxins. In the cultivation, the identified isolates of A. flavus were inoculated at the center of a solidified fluorescence agar medium in glass Petri-dishes then the plates were incubated at 25oC for 10 days. The plates were examined under U/V illumination at 360 nm, starting from the 7th day of incubation up to 10th day for the detection of the fluorescence in the agar surrounding the colonies. Finally, the extraction of A. flavus toxins was carried out (29) whereas the toxic strains of A. flavus that illuminated in the fluorescence agar medium were inoculated in rice medium and incubated at 25oC for 15 days. At the end of incubation period, 25 ml of chloroform were added and the mixture was thoroughly homogenized for one minute. The ho-mogenate was centrifuged at 3000 rpm for 10 minutes and the chloroform layer was decanted. The chloroform extraction was repeated only once. Furthermore, 1 ml ethanol, 3 g copper, 111 g hydroxide carbonate and 5 g anhydrous sodium sulphate were added to the chloroform extract, mixed well and filtered. The filtrate was 306 M. Kholife, A. Moawad, A. Diab, M. El-keredy evaporated in rotatory vacuum evaporator to obtain the produced aflatoxins. Polymerase Chain Reaction (PCR) for demonstration of aflatoxigenic strains of A. flavus The demonstration of virulence factors including norsolonic acid reductase (nor), versi-colorin A (ver-1) and O-methylsterigmatocys-tin (omtA) as aflatoxin producing genes of the isolated A. flavus strains was carried out (31) using the primers in (Table 1). The strains of identified A. flavus were grown for 72 hours under continuous shaking conditions in the Potato Dextrose Broth (32). The mycelium was then harvested by filtration, transferred to a mortar, frozen in liquid nitrogen and ground to a powder which was resus-pended in a lysis buffer (50 mmol/L EDTA, 0.2% SDS, pH =8.5) and heated immediately at 68°C for 15 min. After centrifugation for 15 min at 15000 rpm, 10 ml volume of the supernatant fluid was transferred to a new centrifuge tube and 1 ml 4 mol/L sodium acetate was added. This solution was placed on ice for 1 h and centrifuged for 15 min at 15000 rpm. After centrifugation, the supernatant fluid was transferred to a fresh tube and extracted by genomic DNA extraction Kit. The amplification was performed (33) on a Thermal Cycler (Master cycler, Eppendorf, Hamburg, Germany). All of the isolated A. fla-vus were examined for the presence of three important aflatoxin genes (nor-1, ver-1 and omtA) enclosed in the aflatoxin biosynthesis pathway by multiplex PCR using the above mentioned 3 primer sets. PCR reaction was performed in 25 pL containing 2.5 pL 1 X PCR buffer, 0.75 pL 50 mM MgCl2, 0.5 pL 10 mM dNTPs, 2 pL of each primer, 0.2 pL Taq DNA polymerase (1 U/pL), 5 pL extracted DNA as template and 8.05 pL sterile distilled water. The PCR cycling protocol was applied as following: a total of 35 cycles was started with heating at 94°C for 5 min, and continued by denaturation for 30 sec at 94°C, annealing for 30 sec at 67°C, elongation for 30 sec at 72°C and a final extension of 10 min at 72°C. The amplified DNA fragments were analyzed by 1% of agarose gel electropho-resis (Applichem, Germany, GmbH) in 1x TBE buffer stained with ethidium bromide and visualized on UV transilluminator. A 100 bp DNA ladder was used as a marker for PCR products. Determination of crude protein in fish feed samples The protein content of fish feed samples was determined (34) using the Kjeldhal method in an attempt to find a relation between the incidence of mycotoxins produced by fungi in fish feedstuffs and the crude protein (C.P) content. Results Isolation and identification of fungi in fish feedstuff The mycological analysis of examined fish feedstuffs demonstrated presence of five genera of fungi. The Aspergillus species was the most predominant fungi (86.66%), followed by Penicillium spp. (23.33%), and Fusariam spp. (10%), Mucor (6.66%), and then Rhizopus (3.33%) (Table 2). Percentages had been calculated in relation to the total number of examined samples (30 samples). Aspergillus species in fish feed samples Aspergillus species was the most prevalent isolated mold. Aspergillus niger was the most frequent (43.33%), followed by A. flavus (30%), and A. fumigates (6.66%), A. versicolor (3.33%) and then A. terrus (3.33%) (Table 2). The results also showed that nine isolates (30%) of Aspergillus flavus were recovered from the examined samples (Tables 2 and 3). Mycotoxins analysis Determination of the mycotoxins by HPLC in showed higher prevalence rate for aflatoxins in 7 samples (23.33%) out of 30 examined samples (Table 3). The negative samples to mycotoxins contamination were 23 (76.67%) in spite of showing fungal growth on the culture. OTA was found in three samples (10%). From the aflatoxins producing samples, 42.86% were higher than the permissible limit of aflatoxins in fish feeds (permissible limit is 20 ppb). OTA producing samples were found in Mycological examination of fish feed stuff with special reference to mycotoxin production 307 low percentages. 66.67% of the detected OTA was higher than the permissible limit of OTA (permissible limit of OTA is 5 ppb). The estimated carcinogenic types of aflatoxins AFB 1, AFB2 , AFG1 were (23.33% , 13.34%, 6.67%), respectively and AFG2 was not found. AFB 1 was the predominant type of detected aflatoxins (23.33%) in 7 fish feed samples (Table 3). On the other hand, 3 samples (10%) of the examined fish feedstuffs contained aflatoxins but did not give Apergillus flavus on culture (Table 3). However, 3 samples (10%) contained OTA but did not produce Aspergillus ochraceus on culture. The negative OTA producing samples of fish feedstuff were 27 samples (90%). OTA was (10%) of total detected mycotoxins in the all examined fish feedstuffs but was higher than the permissible limit (5 ppb). Crude protein determination of fish feedstuffs The protein content in each fish feedstuff was determined (Table 3). The variations of c.p % were recorded pointing to the difficulty to demonstrate the relation between estimated my-cotoxins produced by the isolated fungi and the protein content of fish feedstuffs. Toxigenic ability of isolated Aspergillus fla- vus As shown in (Table 4), six isolates of A.fla-vus (66.67%) produced aflatoxins at rate of 50% above the permissible limits while 3 isolates of A.flavus (33.33%) were negative to af-latoxins. The multiplex PCR of nor-1 at 400 bp, ver-1 at 537 bp and omtA at 797 bp virulence genes was used for demonstration of aflatoxigenic strains of isolated Aspergillus flavus as shown in (Fig.1). PCR of A.flavus isolates screened that 3 isolates were negative for the aflatoxin producing genes and 6 isolates were positive. The percentages were calculated regarding to the total number of A.flavus isolated from the examined fish feedstuffs (9 isolates). Table 1: Primers' sequences for PCR identification of aflatoxin producing genes of the isolated A. flavus strains Primer Target Oligonucleotide sequence (5' ^ 3') gene Product References size (bp) nor-1 nor-1 ver-1 ver-1 omtA omtA aflD aflM aflP F-ACCGCTACGCCGGCACTCTCGGCAC R-GTTGGCCGCCAGCTTCGACACTCCG F-GCCGCAGGCCGCGGAGAAAGTGGT R-GGGGATATACTCCCGCGACACAGCC F-GTGGACGGACCTAGTCCGACATCAC R-GTCGGCGCCACGCACTGGGTTGGGG 400 537 797 (31) (31) (31) 308 M. Kholife, A. Moawad, A. Diab, M. El-keredy Table 2: Incidence of isolated mold in the examined fish feedstuffs Fungal species Number of fungi in examined samples Frequency (%) Aspergillus spp 26 86.66% Aspergillus niger 13 43.33% Aspergillus flavus 9 30% Aspergillus fumigates 2 6.66% Aspergillus terrus 1 3.33% Aspergillus versicolor 1 3.33% Penicillium spp 7 23.33% Fusarium spp 3 10% Mucor 2 6.66% Rhizopus 1 3.3% Percentages were calculated in relation to the total number of examined samples (30). Table 3: Determination of aflatoxins (AF) and ochratoxin A(OTA) in fish feedstuff using (HPLC), crude protein (C.P%) and the isolated fungi in each examined fish feed sample Serial Aflatoxins (microgram/kg"ppb") Ochratoxin No. Isolated fungi A C.P% (ppb) AFB1 AFB2 AFG1 AFG2 1. 28.09% A.flavus,A.niger 29.4 15.1 6.6 0.0 10.7 2. 30.89% A.niger 4.6 0.0 0.0 0.0 0.0 3. 30.54% A.flavus,Rhizopus 1.8 0.0 0.0 0.0 0.0 4. 25.375% A.niger,Mucor 13.9 7.2 1.3 0.0 3.5 5. 26.022% A.niger 2.0 0.0 0.0 0.0 0.0 6. 28.875% A.flavus,A.niger 18.7 7.9 0.0 0.0 6.1 7. 24.1% A.flavus 3.5 1.0 0.0 0.0 0.0 8. 25.03% A.fumigatus,A.niger 0.0 0.0 0.0 0.0 0.0 9. 26.78% A.niger 0.0 0.0 0.0 0.0 0.0 10. 28.22% A.niger,penicillium 0.0 0.0 0.0 0.0 0.0 11. 33.51% A.niger 0.0 0.0 0.0 0.0 0.0 12. 23.63% Penicillium 0.0 0.0 0.0 0.0 0.0 13. 27.74% A.niger 0.0 0.0 0.0 0.0 0.0 14. 21.96% A.niger 0.0 0.0 0.0 0.0 0.0 15. 30.63% A.niger 0.0 0.0 0.0 0.0 0.0 16. 30.89% A.niger 0.0 0.0 0.0 0.0 0.0 17. 23.19% A.versicolor 0.0 0.0 0.0 0.0 0.0 18. 26.25% Fusarium 0.0 0.0 0.0 0.0 0.0 19. 24.5% Penicillium 0.0 0.0 0.0 0.0 0.0 20. 25.38% Fusarium 0.0 0.0 0.0 0.0 0.0 21. 25.38% Penicillium 0.0 0.0 0.0 0.0 0.0 22. 29.3% Mucor 0.0 0.0 0.0 0.0 0.0 23. 26.69% A.flavus 0.0 0.0 0.0 0.0 0.0 24. 28.26% Penicillium, A. fumigatus 0.0 0.0 0.0 0.0 0.0 25. 24.23% Penicillium, Fusarium 0.0 0.0 0.0 0.0 0.0 26. 25.31% Penicillium 0.0 0.0 0.0 0.0 0.0 27. 24.76% A.flavus 0.0 0.0 0.0 0.0 0.0 28. 30.19% A.flavus 0.0 0.0 0.0 0.0 0.0 29. 31.4% A.flavus 0.0 0.0 0.0 0.0 0.0 30. 31.24% A.flavus 0.0 0.0 0.0 0.0 0.0 Mycological examination of fish feed stuff with special reference to mycotoxin production 309 Table 4: Determination of Aflatoxins using (HPLC) produced by A.flavus isolates (microgram/K "ppb") Serial No. A. flavus isolates A. flavus isolates 1 -ve AFBi AFB2 AFG1 AFG2 2 +ve 0.0 0.0 0.0 0.0 3 -ve 16.9 9.2 4.5 0.0 4 +ve 0.0 0.0 0.0 0.0 5 +ve 5.2 0.0 0.0 0.0 6 +ve 14.6 5.7 0.0 0.0 7 +ve 23.1 12.9 7.3 0.0 8 -ve 9.4 0.0 0.0 0.0 9 +ve 0.0 0.0 0.0 0.0 1 2 3 C+ M C- 4 5 6 1000 100 Figurel: Agarose gel electrophoresis of multiplex PCR of nor-1 (400 bp), ver-1 (537 bp) and omtA (797 bp) virulence genes for demonstration of aflatoxigenic strains ofAspergillus flavus Lane M: 100 bp ladder as molecular size DNA marker Lane C+: Control positive strain for nor-1, ver-1 and omtA genes Lane C-: Control negative Lane 4: Positive A.flavus strain for nor-1, ver-1 and omtA genes Lanes 1, 3 & 5: Positive A.flavus strains for nor-1 and omtA genes Lanes 6: Positive A.flavus strain for ver-1 and omtA genes Lane 2: Positive A.flavus strain for omtA gene 310 M. Kholife, A. Moawad, A. Diab, M. El-keredy Discussion The significance of fish as an imperative wellspring of human protein, particularly in African nations is taken into consideration. The nearness of molds in fish feedstuff is a guide of tainting likely because of an unseemly feed preparing for assembling or insufficient stockpiling of fish feeds motivating the mycotoxin output. Mycotoxins are created by fungal growth causing injurious consequences for human. In the present study, the isolation rate of fungi from the all 30 fish feedstuffs collected from different fish farms in Kafr El Sheikh gov-ernorate involved Aspergillus spp., Penicillium spp., Fusariam spp., Mucor spp. and Rhizopus spp. (86.66%), (23.33%), (6.66%), (10%), (3.33%), respectively .This is nearly similar to (35) in Brazil who demonstrated that Aspergillus spp. was the most frequent followed by Penicillium spp. in fish feed intended for fish farms. Similarly, (36) detailed that Aspergillus spp. prevailed alternate types of fungi in fish feedstuffs pursued by Penicillium and Fusarium. Morever, Nunes et al. (37) in Piaui, Brazil declared a higher rate of Penicillium spp. (83.3%) and Rhizopus spp. (23.3%) in the fish feed. In between the fungal species, Fusarium spp. was not recovered in fish feed by (35) while was isolated in the examined fish feedstuffs (10%). The rate of isolation of Penicillium spp. converted with Embaby et al. (38) who isolated Penicillium spp. in a frequency of (24.4%). In between Aspergillus species, A .niger was the most predominant nearly agreed with (35, 39) who isolated A. niger (36%),(40%) from fish feeds in Brazil and Qena in Egypt, respectively. Marijani et al. (40) isolated A. niger in a lower percentage (6%) from fish feed in Kenya, Tanzania, Rwanda and Uganda. Nunes et al. (37) did not isolate A. niger from the finished fish feed. Many previously investigations reported that the variations in mold species growth and their frequencies might be attributed to different weather conditions in combination with variant storage measures and manufacturing of fish feeds. Mycotoxins detected by HPLC in the examined samples revealed the incidence of high af-latoxin contamination and low OTA. AFB1 was found in all mycotoxin producing fish feed samples and AFG2 were not found. Nearly similar results were reported by (37, 41) as AFB1 in fish feed was (16.7%) and (28.5%) respectively. Also (39) recorded low concentrations of AFB1, AFB2 and AFG2 differing with this study about AFG2 which not found. The high frequency of AFB1 in fish feed (55%) reported by (35) and (40). However, it is substantial to be taken in consider that fish aflatoxicosis has been mentioned in spite of the low concentration of 20 ppb of AFB1 in feed as reported by (44) causing a high risk to consumers through mycotoxins residues in fish musculature fed on aflatoxins especially AFB1 and ochratoxins containing fish feed stuffs. OTA recorded in low rate (10%) while (42) revealed that OTA containing samples were (25%). This may be attributed to (14) who reported that A. niger is less important OTA producers while Aspergil-lus ochraceus is considered a big producer of OTA but not found in this investigation while Aspergillus niger was the most frequent. The aflatoxiginicity of isolated nine A.flavus from the examined fish feedstuffs showed that 66.67% of A.flavus isolates produced aflatoxins at rate of 50% above the permissible limit (6 isolates out of 9). This nearly agrees with (43) who reported that 55.5% of isolated A.flavus (10 isolates out of 18) from fish feed produced aflatoxins. It has been elucidated that a higher rate of aflatoxiginicity of A. flavus in fish feedstuffs produced by (39) that 83% (10 isolates out of 12) of isolated A.flavus were af-latoxigenic. Accordingly, PCR assay was performed in this study to detect the presence of aflatoxin genes produced by the isolated A. fla-vus from the examined fish feedstuff using specific primers indicating the aflatoxigenic ability. Conclusion All examined fish feedstuffs intended for fish farms in Kafr El Sheikh governorate showed the presence of fungal growth especially the mycotoxigenic fungi as well the incidence of carcinogenic mycotoxins AFB1, AFB2, AFG1 and OTA were estimated. HPLC Mycological examination of fish feed stuff with special reference to mycotoxin production 311 and PCR techniques used for mycotoxins detection providing an accurate results. Either improper processing of fish feed ingredients concerning in their manufacturing or inadequate storage are catalyzers for growth of fungi so more adequate measures are required for manufacturing and storage. More reports are needed to provide a new vital insights into AFBi and OTA levels regarding to fish feeds and their bioaccumulation in fish flesh for human consumption. The relation between protein content of fish feedstuff and the mycotoxin incidence requires more investigations to limit mycotoxin production introduced by fungi. References 1. Sharawy ZZ, Thiele R, Abbas EM, El-Magd MA, Hassaan MS, Peter C, Schmidt J, Saborowski R, Goda AMA-S, Slater MJ. Antioxidant response, body composition of whiteleg shrimp litopenaeus vannamei co-cultured with nile tilapia oreochromis niloticus in recirculating aquaculture. Aquaculture Environment Interactions 2017;9:257-268. 2. Enyidi U, Pirhonen J, Kettunen J, Vielma J. Effect of feed protein: lipid ratio on growth parameters of African catfish Clarias gariepinus after fish meal substitution in the diet with bambaranut (Voandzeia Subterranea) meal and soybean (Glycine max) meal. Fishes 2017; 2,1. doi: 10.3390/fishes2010001. 3. Greco MV, Pardo AG, Ludemann V, Martino PE, Pose GN. Mycoflora and natural incidence of selected mycotoxins in rabbit and chinchilla feeds. Sci World J 2012; 1-9. 4. Doerr JA, Campbell JML, Huff W E. Interaction between dietary citrinin and Ochratoxin A in broiler chickens. Poult ScL 1982; 61: 1453. 5. Ariyo LA, Anthony HM, Lami HM. Survey of Mycotoxigenic Fungi in Concentrated Poultry Feed in Niger State, Nigeria. JFR 2013; 2(2)128-35. 6. Pestka JJ. Deoxynivalenol toxicity, mechanisms and animal health risks. Anim Feed Sci Tech-nol 2011; 137: 283-98 7. Fuchs R, Appelgren L and Hult K. Distribution of 14C-Ochratoxin A in the rainbow trout Sal-mogaidneri. Acta Pharmacol. Et Toxicol J P 1986; 220-7. 8. El-Shaboury FA. Fungal flora of Brolus lake fish at Kafr El-Sheikh Province Alex J Vet Sci 1998; 14(3): 17-28. 9. Danicke T , Goyarts S, Valenta H, Ueber-schar KH. Carry-over of Fusarium toxins (deoxynivalenol and zearalenone) from naturally contaminated wheat to pigs. Food Addit Contam 2011; 25:389-420. 10. Abdelhady D, El-Abasy M, Abou-Asa S, Elbialy Z, Shukry M, Hussein A, Saleh A, El-Magd M. The ameliorative effect of aspergillus awamori on aflatoxin bl-induced hepatic damage in rabbits. World Mycotoxin Journal 2017;10:363-73. 11. Stewart D, Larson E. Aflatoxicosis in wildlife. Information Sheet 1582. Mississippi State Univ. Extension Service, Cooperating with U.S. Dept. of Agriculture 2002. 12. D'Mello JPF, MacDonald AMC. Mycotoxins. Anim Feed Sci Technol 1997; 69: 155-66. 13. Santacroce M P, Narracci M, Acquaviva M I, Cavallo R A, Zacchino V, Centoducati G. New Development in Aflatoxin Research: From Aquafeed to Marine Cells, Aflatoxins - Detection, Measurement and Control, Dr Irineo Torres-Pacheco (Ed), ISBN 2011; 978-953-307-711-6. 14. Benford D, Boyle C, Dekant W, Fuchs E, Gaylor D W , Hard G et al. Ochratoxin A Safety Evaluation of Certain Mycotoxins in Food. WHO Food Additives Series 47. FAO Food and Nutrition Paper, WHO, Geneva, Switzerland, 2001; 74: 281415. 15. Samson RA, Stalk A, Hadlok R. Revision on the subsection fasiculata of Penicillium and some allied species. Studies in Mycology 1976; 2: 75-. 16. Mushtaq M, Sultana B, Anwar F, Khan M, Ashrafuzzaman M. Occurrence of aflatoxins in selected processed foods from Pakistan. Int J Mol Sci 2012; 13(7): 8324-37. 17. Cruickshank R, Maimon D, Swain RHA. Medical microbiology 12thED Vol II Churchil Livingstone, 1975. 18. Raper KB, Fennel DI. The genus Aspergil-lus.1st Ed. Williams and Wilkins Company, Baltimore, 1965. 19. Dalcero A, Magnolt C, Luna M, Ancast G, Reynoso MM, Chiacchiera S, Miazzo R, Palacio G. Mycoflora and naturally occurring mycotoxins in poultry feeds in Argentina. Mycopathologia 1998;141 (1): 37-43. 20. Samson RAA. Complication of Aspergilli. Studies in Mycology 1979; 3: 198-202. 21. ARX JA, Pilzkunde J, Cramer inder A R Canter Verlag, Kommanditge sellschaft. FL - 9490 Vaduz 1967. 22. Raper KB, Thom CA. Manual of the penicil-lia, Williams and Wilkins Company, Baltimore, 1969. 312 M. Kholife, A. Moawad, A. Diab, M. El-keredy 23. Samson RA, Stalk A, Hadlok R. Revision on the subsection fasiculata of Penicillium and some allied species. Studies in Mycology 1976; 2: 75-8. 24. Zycha H, Siepman R, Linnemann G. Mu-corales Eine Besebreibung alter Gattunger und Arten diesser pilzgruppe ver lag Von. J Carmer 1969. 25. Association of Official Analytical Chemists "AOAC" Official Methods of the AOAC International Analysis. 13th Ed, Horwitz W, (Editor), Academic Press, Washington D C, USA, 2000. 26. Toscani T, Moseriti A, Dossena A, Dallasta C, Semoncini N, Virgili R. Determination of ochra-toxin A in dry-cured meat products. J Chromatogr B Analyt Technol Biomed Life Sci 2007; 855(2):242-8. 27. European Council "EC" Commission Regulation No. 401/2006 of 23 February 2006 laying down the methods of sampling and analysis for the official control of the levels of mycotoxins in foodstuffs. Official Journal of the European Union, L70 pp 2006;12-34. 28. Hera S, Fennel D, Hesseltine C. Aflatoxin producing strains of A. flavus detected by fluorescence of agar medium under U/V light. Appl Microbiol 1994;47 (6): 118-23. 29. Pestka J. Fungal toxins in raw and fermented meats. Fermented meat, A textbook, (Eds. G Campbell and P E Cook), Blakie Academic and Professional Press, London, UK 1995. 30. Schuller PL, Van Egmond HP, Leonard Stoloff. Limits and regulation on mycotoxins. Proc Int Symp Mycotoxins PP 1983;111-29. 31. Criseo G, Bagnara A, Bisignano G. Differentiation of aflatoxin-producing and non-producing strain of Aspergillus flavus group. Lett Appl Microbiol 2001; 33(4):291-5. 32. Erami M, Hashemi S, Pourbakhsh S, Shah-savandi S, Mohammadi S, Shooshtari A, Jahanshiri Z. Application of PCR on detection of aflatoxino-genic fungi. ARI 2007; 62 (2): 95-100. 33. Davari E, Mohsenzadeh M , Mohammadi G , Shahsavandi S, Doloei R. Characterization of afla-toxigenic Aspergillus flavus and A. parasiticus strain isolates from animal feedstuffs in Northeastern Iran. Iran J Vet Res 2015; 16(2): 150-5. 34. Randhir S, Pradhan K. Forage evaluation. First Published, Printox, New Dalhi, Dhawan Printing works 1981. 35. Barbosa TS, Pereyra CM, Soleiro C, Dias EO, Oliveira A, Keller KM, Silva PP, Cavaglieri LR, Rosa CA. Mycobiota and mycotoxins present in finished fish feeds from farms in the Rio de Janeiro State, Brazil. Int Aquat Res 2013; 5: 3. 36. Cardoso F, F D C. Monitoramento de fungos toxigenicos e aflatoxinas em ra^oes utilizadas na piscicultura em Teresina, Piauí, Brasil. Master's Thesis, Universidade Federal do Piauí, Teresina, Brasil. 2011; In Portuguese. 37. Nunes EMCG, Maria M, Gomes-Pereira, Amilton P Raposo-Costa et al. Screening of afla-toxin B1 and mycobiota related to raw materials and finished feed destined for fish. Lat Am J Aquat Res 2015;43(3): 595-600. 38. Embaby EM , Ayaat NM., Abd El-Galil MM , Allah Abdel-Hameid N, Gouda MM . Mycoflora and mycotoxin contaminated chicken and fish feeds. Mid East J Appl Sci 2015; 5(4): 1044-54. 39. Mohamed HMA, Emiesh WFA, Braeuning A, Hammad S. Detection of aflatoxin -producing fungi isolated from Nile Tilapia and fish feed. EX-CLI J 2017;16:1308-18. 40. Marijani E, Wainaina James M, Charo-Ka-risa H, Nzayisenga L, Munguti J, Gnonlonfin BJG, Kigadye M, Okoth S. Mycoflora and mycotoxins in finished fish feed and feed ingredients from smallholder farms in East Africa. Egy J Aquat Res 2017; 43 (2): 169-76. 41. Hashimoto EH, Santos MA, Ono EYS, Hayashi C, Bracarense APFRL, Hirooka EY. Bro-matologia e contaminado com fumonisina e afla-toxina em ra^oes utilizadas na piscicultura da regiäo de Londrina, Estado do Paraná, Brasil. Seminario: Ciencias Agrárias 2003; 24(1):123-32. 42. Greco M, Pardo A, Pose G. Mycotoxigenic fungi and natural co-occurrence of mycotoxins in rainbow trout (Oncorhynchus mykiss) feeds. Toxins (Basel) 2015; 7(11):4595-609. 43. Hassan AA, Hassan AM, ElShafei MH, El Ahl RMH, Abd El-Dayem RH. Detection of afla-toxigenic moulds isolated from fish and their products and its public health significance. Mater Sci 2011; 9 (9): 106-14. 44. Mahfouz EM, Sherif HA. A Multiparameter investigation into adverse effects of aflatoxin on Oreochromis niloticus health status. J Basic Appl Zool 2015; 71:48-59. Slov Vet Res 2019; 56 (Suppl 22): 313-19 DOI 10.26873/SVR-770-2019 Original Research Article ANTIBACTERIAL AND ANTI-OXIDANT ACTIVITIES OF LAUREL OIL AGAINST Staphylococcus aureus AND Pseudomonas fluorescence IN Oreochromis niloticus FILLETS Ebeed A. Saleh1*, Ibrahim I. Al-Hawary2, Mayada M.Elnajar3 1Department of Food Hygiene, Faculty of Veterinary Medicine, Damanhour University, Egypt, 2Department of Fish Processing and Biotechnology, Faculty of Aquatic and Fisheries Science, Kafrelsheikh University, Egypt, 3Department of Food Hygiene, Faculty of Veterinary Medicine, Damanhour University *Corresponding author, E-mail: ebeed.saleh@yahoo.com Abstract: Laurus nobilis L. commonly known as Bay belonging to the family Lauraceae, is one of the most useful plant used in foods as a spice in Mediterranean cookery. This study was designed to evaluate the antibacterial and anti-oxidant activities of different concentrations of laurel essential oil (0.5%, 1%, 1.5%) against two bacterial strains "Staphylococcus aureus as food poisoning bacteria and Pseudomonas fluorescence as food spoilage bacteria" in order to enhance fish safety and increase shelf life. A total of 80 samples of freshly dead Oreochromis niloticus fish were collected after experimental infection with the two bacterial strains followed by treatment with different concentrations of laurel essential at different storage time at chilling temperature. The results showed that laurel essential oil was efficient against the tested bacteria, but varied in its antibacterial activity as follows; the highest reduction percent of S. aureus and P. fluorescence at rate of 99.97% and 94.5% was detected 6 days after laurel oil treatment, respectively, and reached 100% 9 day after oil treatment at the same concentration. While the lowest value was obtained with 0.5% concentration. Laurel oil (1.5%) also showed a significant decrease in pH, TNB-N and TBA. Based on the obtained data, it could be concluded that, laurel essential oil has strong antibacterial and antioxidant activity against the tested bacteria, so that it can be used as natural food preservatives alternatives to other synthetic agents to keep quality and increase fish shelf life. Key words: Laurus nobilis; essential oil; antibacterial activity; fish shelf-life; food safety Introduction Fish is an important source of protein to humans which rich of many sources of therapeutically important polyunsaturated fatty acids, vitamins and various other micro nutrients. Seafood has a limited shelf life due to it is highly perishable, microbial spoilage is the main cause of fish decomposition (1). It affects both the quality and the safety of fish and leads to food poisoning, so that the concepts of enhancing its shelf life and quality became needful in the recent years (2). Due to the highly potential health hazard of synthetic preservatives and its toxicological effects, natural preservatives have been explored Received: January 2019 Accepted for publication: February 2019 314 E. A. Saleh, I. Al-Hawary, M. M. Elnajar by the researchers for controlling the microbial and chemical mechanisms responsible for spoilage in fish (3). Plant extracts are safe, effective and successful treatments with the potential to extend the shelf life of foods. Plant extracts and its essential oils have antimicrobial and anti-oxidant properties against food borne pathogens, today they have been considered as natural preservatives or food additives in food industry for raw and processed food preservation (4). Bay laurel (Laurus nobilis L.) is an evergreen shrub native to the Mediterranean region, being the only European representative of the Lauraceae family. Its dried leaves and essential oil are used in Italy France, Turkey, Algeria, Morocco, Spain, Portugal and Mexico as a valuable spice in the culinary and food industry (5). Thus, the aim of this study was to evaluate the antibacterial and antioxidant activities of laurel essential oil against two bacterial strains "Staphylococcus aureus as food poisoning and Pseudomonas fluorescence as food spoilage bacteria " for enhancing shelf life of fish. Materials and methods Collection and preparation of samples A total of 80 samples of freshly dead Ore-chromis niloticus fishes were collected from local market at Kafrelsheikh Governorates and transferred quickly to the Lab of "Animal Health Research Institute" for bacteriological and chemical examination. Then fishes were swabbed with ethyl alcohol 70% and scorched with flame, the fish flesh taken from the back muscle (6). Fillets cut with a sterile scalpel to (100 g), put under the UV light for 20 minutes to reduce number of the microorganisms attached to its surface. Preparations of inocula Staphylococcus aureus and Pseudomonas fluorescence strains were obtained from Food Analysis Center, Faculty of Veterinary Medicine, Benha University, Egypt. Bacteria were sub-cultured on Brain-Heart Infusion (BHI) broth and harvested by centrifugation (3000*g, 15 min), and resuspended with saline (NaCl, 0.85%, w.v) (7). For inoculation of the Oreochromis niloticus fillets, 1 ml of the dense suspension 3*106 /g for each strains was employed. Essential oil extraction Extraction of the plant material was applied by immersion in absolute methanol for three days using automatic shaker. Then twice filtra-tions were applied to remove solid and fine plant particles. The enriched extract was concentrated by evaporation with heating in water bath at 65 ° C until solid mass was obtained. Finally, spreading under shaded area till complete dryness and stored in the refrigerator until using. Each extract was used for preparation of 1% (w/v) solution (8), then was inoculated into Oreochromis niloticus fillets. Bacteriological examination To 25 g of the sample, 225 ml of sterile peptone water were added and mixed, for preparation tenfold serial dilutions for the further examinations (9). A volume of 0.1ml of this serial dilution was spread over Baired Parker agar plate, then the plate was incubated at 37°C for 48 h. S.aureus count /g was calculated (10). A volume of 0.1 ml of each sample homogenate was separately inoculated into duplicate petri-dishes of Pseudomonas selective agar medium and was evenly spread, then the developed colonies (greenish yellow colonies) were counted (11). Chemical examination Ten grams of sample were blended in 10 ml of neutralized distilled water. The pH value was determined by using an electrical pH meter (Bye model 6020, USA) (12). The technique applied for determination of total volatile nitrogen (TVN) was recommended by Food and Agriculture Organization "FAO" (1980) (13) . Determination of thiobarbituric acid number (TBA) (mg malonaldehyde/kg) was recommended by Pikul et al. (1989) (14). Antibacterial and anti-oxidant activities of laurel oil against fish pathogens 315 the lowest reduction showed at 0.5% concentration by 40%, 75.26% and 86.9% after 3,6 and 9 day of treatment (Table 1). In the case of P.fluorescence inoculated fillets, it displayed a lower reduction percent compared to S.aureus inoculated fillets also, it showed significant reduction percent with different concentration, at 0.5% concentration the reduction was 21.33%, 62% and 69.13% after the 3, 6 and 9 day of treatment, at 1% concentration the reduction was 48.67%, 70.12% and 93.89% after 3,6, and 9 day of treatment, the higher reduction percent was at 1.5% concentration by 82.43%, 94.15% , 100% after 3, 6 and 9 day of treatment with laurel oil (Table 2). Table 1: Antibacterial activity of essential oil of laurel on S.aureus inoculated into Oreochromis niloticus fillets by intensity of 3x106 /g (n=5) Treatment Control 0.5 % Laurel oil 1 % Laurel oil 1.5 % Laurel oil Storage time Count %%* Count R % Count R % Count R % 3.0x106± 3.0x106± 3.0 x106± 0.2 x106 4.11 x104± 0.7 x104 9.0 x102± 1.6 x102 ND Zero time 0.2x106 2.89x106± 0.1 x106 3 67 3.0x106± 0.2x10s 1.80x106± 0.2 x106 40 0.2x106 9 64x105± 2.0x105 6 7 86 98.63 3 days 266x106± 1133 0.3 x106 7.42x105± 1.5x105 3.93x105± 1.1x105 . 5.37x104± 0.8x104 98 22 99.97 6 days 9 days 2.59x106± 0.2x106 . 86.90 2 50x103± 2n0 10r 99.91 0.4x103 R %*= Reduction %; ND= Not detected; Reduction %= count before - count after / count before x100 Table 2: Antibacterial activity of essential oil of laurel on P. fluorescence inoculated into Oreochromis niloticus fillets by intensity of 3*106 Ig (n=5) Treatment Control 0.5 % Laurel oil 1 % Laurel oil 1.5 % Laurel oil Storage time Count R %* Count R % Count R % Count R % Zero time 3.0x106± 0.2x106 2.95x106± 0.24 x106 2.87x106± 0.17x106 3.0x106± 0.2x106 . ,7 2.36x106± 1 0.19x106 1 14x 106± 4 33 j..14 10 ± 4 0.08x106 3.0x106± 0.2x106 1 54 x106± 21 33 1 10 ± 21 0.12 x106 8.97 x105± 62 1.65 x105 ------ 3.0 x106± 0.2 x106 3 days 48.67 5.27 x105± 1.03 x105 82.43 6 days 70.12 1.65 x104± 0.21 x104 93.89 ND 94.50 9 days 2.84x106± 0.22 x106 9.26x105± 1.84x105 ,Q1, 1.81 x105± 69 13 0.25 x105 R %*= Reduction %; ND= Not detected Statistical Analysis The obtained results were statistically evaluated by application of Analysis of Variance (ANOVA) test according to Feldman et al. (15). Results Antibacterial activity S. aureus inoculated fillets showed a significant reduction following addition of different Laurel oil concentration at different storage time. The highest reduction percent was at 1.5% concentration of oil by 98.63%, 99.97% and 100% after 3, 6 and 9 day of treatment, while Different concentration of laurel oil showing significant reduction percent of S. aureus and P.fluorescence at different storage time; the highest reduction % was 1.5% laurel oil by 100% reduction after the 9 day of treatment. 316 E. A. Saleh, I. Al-Hawary, M. M. Elnajar Figure 1: Showing pH values of S.aureus and P.fluorescence inoculated Oreochromis niloticus fillets for control and Laurel oil (0.5%, 1%, 1.5%) concentration at different storage time Figure 2: Showing TVB-N values of S.aureus and P.fluorescence inoculated Oreochromis niloticus fillets for control and Laurel oil (0.5%, 1%, 1.5%) concentration at different storage time Figure 3: Showing TBA values of S.aureus and P.fluorescence inoculated Oreochromis niloticus fillets for control and Laurel oil (0.5%, 1%, 1.5%) concentration at different storage time Antibacterial and anti-oxidant activities of laurel oil against fish pathogens 317 Chemical evaluation The mean pH values of the examined Oreo-chromis niloticus fillets inoculated with S. aureus and P.fluorescence showed gradually decrease after addition of laurel oil in compared to the untreated group. Differences associated with the examined samples of the pH values between different concentration of oil and different storage time were highly significant (P < 0.05) (Fig.1). Different concentration of laurel oil showed significant decrease in the mean TVB_N (mg %) value at different storage time; the most significant record was at 1.5% laurel oil after the 9 day of treatment by 17.99 ± 0.01 for S. aureus inoculated fillets and 16.85 ± 0.01 for P.fluorescence, compared to untreated fillets. Showed in (Fig. 2). Also, laurel oil showed significant decrease in the mean value of TBA (mg/Kg) at different storage time, but the highest significant value was at 1.5% concentration after the 9 day of treatment by 4.02 ± 0.01 and 4.27 ± 0.01 for S. aureus and P. fluorescence respectively compared to un treated fillets that was 6.28 ± 0.01 and 6.51 ± 0.01 after the 9 day (Fig. 3). Data is presented as means ± SE. Mean values followed by different small (lowercase) letters indicates significant effect of the time factor with the same treatment. While, mean values followed by different uppercase letters indicates significant effect of time factor between the different treatments (P < 0.05). Discussion The present study reported that, treatment of Oreochromis niloticus fillets, experimentally infected with S. aureus by intensity 3x106 \g with Laurel essential oil with different concentration (0.5%,1%,1.5%) at different storage time, it showed various reduction percent as follows; at 0.5% concentration it showed reduction by 40%, 75.26% and 86.9% after 3,6 and 9 day of treatment At 1% concentration the reduction percent was 67.86%, 98.22% and 99.91% after the 3 ,6 and 9 day of treatment, While the concentration 1.5% of Laurel oil showed the higher reduction percent was 98.63%, 99.97% at 3 and 6 day of treatment and 100% after the 9 day, as showed in (Ta-ble1). While, fillets infected with P.fluorescence displayed different reduction percent with laurel oil , it was 21.33%, 62% and 69.13% after the 3, 6 and 9 day of treatment respectively ,at 0.5% concentration while at 1% concentration it showed 48.67%, 70.12% and 93.89% reduction percent after 3, 6 and 9 day of treatment, the higher reduction percent was at 1.5% concentration of oil by 82.43%, 94.15% and 100% after 3,6 and 9 day of treatment, as showed in (Table 2). Another study has been found, that Eucalyp-tol that is the main component of laurel essential oil inhibit growth of S. aureus at the concentrations of 0.7%, 0.6% and 1% (16). while other, reported that Pseudomonas spp. were very sensitive to the essential oil of Laurel even very diluted (17). The present study concluded that, S. aureus and P. fluorescence are sensitive to different concentration of laurel oil due to the great number of different chemical compounds found in essential oil (18). The major constituents were eucalyptol (27.2%), alpha terpinenyl acetate (10.2%), linalool (8.4%), methyleuge-nol (5.4%), sabinene(4.0%) and carvacrol (3.2%) (5). Eucalyptol is known to possess a strong antioxidant activity and antibacterial action against Staphylococcus aureus, Linalool is an aliphatic alcohol with strong to moderate antibacterial activity against several bacteria, such as Shigella sonnei, S. flexneri , Pseudomonas spp. and L. monocytogenes (16). S. aureus showed more reduction than P. fluorescence, this may be due to sensitivity of different Pseudomonas towards different antibiotics this sometimes paradoxical and it displays an intrinsic resistance to wide variety of essential oils (17). Measuring of pH is used as indicator to evaluate the shelf life and quality of the fish fillets. Lipid and protein oxidation consider the main cause of reducing meat quality leading to off-flavor and rancidity, increasing value of TVN indicate incidence of spoilage of fish after different periods storage reaching 20mg/ 100g if 318 E. A. Saleh, I. Al-Hawary, M. M. Elnajar increased the meat decomposing occur , otherwise TBA number was related to the oxidation in meat causing rancidity (19). The pH value was measured for control and different treatment of oil at different storage time, the results revealed that; the mean pH value of untreated groups showed marked change to alkalinity (7.69, 7.97) after the 9 day, as showed in (Fig.1). this indicated that the fish fillets was rapidly deteriorated, then after treatment with laurel oil the values of pH decreased gradually for maintaining the border line pH, also, ANOVA analysis indicated that there is low significance effect of the time factor within the same treatment, while there was a great significant of time factor for different oil treatment in compared with untreated group (P < 0.05). By measuring TVB-N it was found that, different concentration of laurel oil showed significant decrease in the (TVB_N) value at different storage time in compared to control; The most significant record of mean TVN was 1.5% laurel oil after the 9 day of treatment, followed by 1% concentration, followed by 0.5%, as showed in (Fig.2). ANOVA indicated that there was high significance (++) between different storage time of the same treatment group, and also, high significance of time factor between different treatment "control, 0.5%, 1%, 1.5%". TBA number was measured for control and oil treated fillets and the results showed in (Fig.3), revealed that; different concentration of Laurel oil showed significant decrease in TBA value at different storage time in compared to control ; The most significant record of TBA was 1.5% Laurel oil after the 9 day of treatment, followed by 1% concentration, followed by 0.5%. The ANOVA indicated that there was a high significance (++) between the time factor within both the same treatment and between different treatment. Laurel essential oil with different concentration (0.5%, 1%, 1.5%) maintain value of TBA that indicate decreasing of oxidative rancidity causing spoilage, thus Laurel oil suggested to has anti-oxidant properties. another matching study, reported that essential oil from laurel, eu-genol and methyl eugenol may be considered the main mediators of antioxidant activity (20). The antioxidant activity of eugenol has been reported in several studies and there are few reports regarding the radical scavenging activity of the constituents of bay laurel leaves. It was concluded from the present study that Laurel essential oil has strong antibacterial activity against Staphylococcus aureus and Pseudomonas fluorescence and it can inhibit growth of these bacteria with its different concentrations; also, treatment of Oreochromis niloticus fillets with laurel oil, help in keeping quality of fish fillets and prolong fillets shelf life, in addition to the anti-oxidant activity of laurel oil that maintain fillets freshness and prevent lipid pe-roxidation, so that enhance shelf life for a period of time Conflict of interest statement None of the authors have any conflict of interest to declare. Acknowledgment This research has been done with the support of Food and Feed Safety Laboratory, Faculty of Veterinary Medicine, Damanhour University and Faculty of Aquatic and Fisheries Science, Kafrelsheikh University. References 1. Ghaly AE, Dave D, Budge S, Brooks M. Fish spoilage mechanisms and preservation techniques. American Journal of Applied Sciences. 2010; 7: 859 2. Vaclavik VA, Christian EW. Food Preservation. Essentials of food science: Springer; 2014. p. 323-42. 3. Seely KA, Lapoint J, Moran JH, Fattore L. Spice drugs are more than harmless herbal blends: a review of the pharmacology and toxicology of synthetic cannabinoids. Progress in Neuro-psychophar-macology and biological psychiatry. 2012; 39: 23443. 4. Tajkarimi M, Ibrahim SA, Cliver D. Antimicrobial herb and spice compounds in food. Food control. 2010; 21: 1199-218. 5. Ramos C, Teixeira B, Batista I, Matos O, Serrano C, Neng N, et al. Antioxidant and antibacterial activity of essential oil and extracts of bay laurel Laurus nobilis Linnaeus (Lauraceae) from Portugal. Natural product research. 2012; 26: 518-29. 6. Stoskopf MK. Fish medicine1993. Antibacterial and anti-oxidant activities of laurel oil against fish pathogens 319 7. Tassou C, Drosinos E, Nychas G. Effects of essential oil from mint (Mentha piperita) on Salmonella enteritidis and Listeria monocytogenes in model food systems at 4 and 10 C. Journal of Applied Bacteriology. 1995; 78: 593-600. 8. Tandon S, Rane S. Decoction and hot continuous extraction techniques. Extraction technologies for medicinal and aromatic plants. 2008; 93. 9. González R, Tamagnini L, Olmos P, De Sousa G. Evaluation of a chromogenic medium for total coliforms and Escherichia coli determination in ready-to-eat foods. Food microbiology. 2003; 20: 601-4. 10. Aycicek H, Cakiroglu S, Stevenson TH. Incidence of Staphylococcus aureus in ready-to-eat meals from military cafeterias in Ankara, Turkey. Food control. 2005; 16: 531-4. 11. Hassan M, Shaltout F, Maarouf A, El-Shafey W. Psychrotrophic bacteria in frozen fish with special reference to Pseudomonas species. Benha Veterinary Medical Journal. 2014; 27: 78-83. 12. Chan ST, Yao MW, Wong Y, Wong T, Mok C, Sin DW. Evaluation of chemical indicators for monitoring freshness of food and determination of volatile amines in fish by headspace solid-phase microextraction and gas chromatography-mass spec-trometry. European Food Research and Technology. 2006; 224: 67. 13. Howgate P. A critical review of total volatile bases and trimethylamine as indices of freshness of fish. Part 1. Determination. Electronic Journal of Environmental, Agricultural & Food Chemistry. 2010; 9. 14. Pikul J, Leszczynski DE, Kummerow FA. Evaluation of three modified TBA methods for measuring lipid oxidation in chicken meat. Journal of Agricultural and Food Chemistry. 1989; 37: 1309-13. 15. Nabil M, Edris A, Shaltout F, Zakaria I. Effect of some preservatives on bacterial load of some poultry meat products. Benha Vet Med J. 2014; 26: 94-103. 16. Zengin H, Baysal AH. Antibacterial and antioxidant activity of essential oil terpenes against pathogenic and spoilage-forming bacteria and cell structure-activity relationships evaluated by SEM microscopy. Molecules. 2014; 19: 17773-98. 17. Bennadja S, Kaki YTA, Djahoudi A, Hadef Y, Chefrour A. Antibiotic activity of the essential oil of laurel (Laurus nobilis L.) on eight bacterial strains. Journal of Life Sciences. 2013; 7: 814. 18. Lahlou M. Methods to study the phytochem-istry and bioactivity of essential oils. Phytotherapy Research: An International Journal Devoted to Pharmacological and Toxicological Evaluation of Natural Product Derivatives. 2004; 18: 435-48. 19. El-Deen G, El-Shamery M. Studies on contamination and quality of fresh fish meats during storage. Academic journal of biological science. 2010; 2: 65-74. 20. Politeo O, Jukic M, Miloš M. Chemical composition and antioxidant activity of free volatile aglycones from laurel (Laurus nobilis L.) compared to its essential oil. Croatica chemica acta. 2007; 80: 121-6. Slov Vet Res 2019; 56 (Suppl 22): 321-31 DOI 10.26873/SVR-771-2019 Original Research Article SEASONAL SCREENING OF THE MYCOTIC INFECTIONS OF CULTURED FRESHWATER FISHES IN KAFR EL-SHEIKH GOVERNORATE Nadia B. Mahfouz1, Eman M. Moustafa1*, Mohamed Kassab2, Wesam H. Marzouk3 department of Fish Diseases and Management, Faculty of Veterinary Medicine, Kafrelsheikh University, Egypt, 2Department of of Cytology and Histology, Faculty of Veterinary Medicine, Kafrelsheikh University, 3Food safety specialist in Directorate of Health in Gharbia, Egypt Corresponding author, E-mail: emantarek2002@yahoo.com Abstract: The present study was carried out to screen the predominant mycotic infections among freshwater fishes (Oreochromis niloticus and Clarias gariepinus) in Kafrelsheikh fish farms; with special focus on the seasonal incidence; as well as, the histopathological changes induced by the detected fungi. 500 specimens of freshwater fishes (400 O. niloticus and 100 C. gariepinus) were investigated for seasonal incidence of mycotic diseases. Mycological examination revealed the isolation of 2148 fungal isolates from 375 diseased and 125 apparently healthy fish samples (1828 mould and 320 yeast isolates), of which 1258 were isolated from O. niloticus and 890 isolates from C. gariepinus. Sapro-legnia was the predominant among diseased fishes with highest prevalence in late autumn (10.68%, 6.96%) and winter (6.81%, 7.87%) in O. niloticus and C. gariepinus, respectively. However, Pencillium sp. and Aspergillus sp. were the most predominant fungi isolated from apparently healthy fishes. The highest prevalence of Pencillium sp. were recorded in winter, whileas Aspergillus showed variations between species; Aspergillus flavus, Aspergillus niger were more prevalent in summer (25.44%, 23.22%) and (26.9%, 37.44%) and Aspergillus terrus, Aspergillus fumigatus were more prevalent in autumn (5.98%, 5.67%) and (7.69%, 8.23%) in O. niloticus and C. gariepinus, respectively. Moreover, the highest prevalence of Fusarium species was recorded in spring (11.8%, 5.91%) from O. niloticus, C. gariepinus, respectively. Mucor recorded the highest prevalence in autumn (20.09%) in O. niloticus and winter (29.21%) in C. gariepinus; whileas Rhizopus was highest in summer (7.89%, 5.21%) in O. niloticus and C. gariepinus, respectively. Four genera from yeast were isolated; Candida sp. (28.44%, 36.27%), Rhodotorula sp. (36.24%, 24.51%), Cryptococcus sp. (16.97%, 20.59%) and Tricho-sporon Sp. (18.35%, 18.63%) in O. niloticus and C. gariepinus, respectively. The histo-pathological findings revealed severe degenerative changes in skin and gills with presence of fungal hyphae and spores. Key words: Clarias gariepinus; moulds; mycotic diseases; Oreochromis niloticus; yeast Introduction Fish serves as an important source of human dietary protein worldwide, especially in Afri- Received: January 2019 Accepted for publication: February 2019 322 N. Mahfouz, E. Moustafa, M. Kassab, W. Marzouk can countries (1). To compensate animal protein deficiency resulting from the increased interest for fish as human nourishment, fish farming is rapidly extending all over the world (2). In Egypt, the aquaculture industry provides about 77% of the total national fish production (3, 4). Nile Tilapia (O. niloticus) is viewed as a standout amongst the most prevalent freshwater fishes in Egypt. It is widely cultured because of its palatability, cheap price, high growth rate, capacity to withstand pressure and infections, ability to spawn effectively and the minimal prerequisites with regard to management and energy inputs (5). With expanding freshwater fish production movement around the world for farming, alongside enhanced ecological observing of fungal and fungal-like infections that are full degree of the effect of these pathogens on wild fish populations will soon rise as a noteworthy danger to freshwater biodiversity (6). Serious aquaculture conditions can advance the transmission of fish diseases, particularly contagious fungal sicknesses, causing economic losses. Fungal infections are one of the primary drivers for mortalities and extraordinary financial misfortunes in cultured fishes. (7). The significance of fungal diseases in freshwater fish not halted just for frequency of mortalities but rather additionally as financial significance, such as decline growth rate, hatcha-bility in choronic infection or by mycotoxins production by tainted organism in case of bad stockpiling feed. Fungal infections in fish are viewed as auxiliary to some other pathogen, water quality issues, poor conditions, injury (unpleasant taking care of or hostility), bacterial disease and/or parasites (8). Numerous fungi influencing fishes are considered opportunistic, assaulting the fishes only if they are stressed or immune-compromised as a result of troublesome natural conditions, or optional to bacterial or viral diseases, or when they have lost their bodily mucus protection due to trauma or excessive handling (9). Disregarding the fungal infections significance our insight about them is still poor for two fundamental reasons: difficult distinguishing proof of pathogenic fungi and the productive development of saprophytic fungi once the fish is dead (10). Fungi is mostly attacked due to temperature change and bad water conditions which allow excessive zoospores to grow and the ammonia which is formed by rottening of fish waste wears away the mucus that protects the skin (11). Moreover, fungi can assault fishes of all the ages and it can also forestall fruitful hatching when it invades fish eggs (12). The most widely recognized fungal infection was sapro-legniosis which is the real oceanic mycotic winter freshwater fish disease, frequently impacts wild and cultured fishes (13). Saprolegnia sp. taints the fishes because of sudden drop of water temperature and was regularly influencing fishes exhibiting fungal skin lesions which, unmistakable as cottony-white development on the epidermis of the influenced fishes (14). As-pergillus sp. causes systematic diseases with high death rates in fish, whereby the infections mostly occur through contamination of fish feed (15) and the pathogenesis of Aspergillus fumigatus and Aspergillus niger had been accounted in fresh water fishes by Chauhan (16). The current study was carried out to screen the predominant mycotic infections with special focus on the seasonal incidence among freshwater fishes (O. niloticus and C. gariepi-nus) in Kafrelsheikh fish farms; as well as, detect the histopathological changes induced by the detected fungi. Materials and methods A- Materials 1. Fish A total number of 500 examined cultured freshwater fishes; 400 O. niloticus and 100 C. gariepinus, were collected alive from different freshwater fish farms at Kafr El Sheikh Gover-norates along the four seasons of the year 2017. The samples were collected with an average body weight of (40±5 & 150±10 gm) for O. ni-loticus and C. gariepinus, respectively. The alive collected fishes were transferred to the wet lab., Fish Diseases and Management Department, Faculty of Veterinary Medicine, Kafr El-Sheikh University, Egypt, held in well- Seasonal Screening of the Mycotic Infections of Cultured Freshwater Fishes in Kafr El-Sheikh Governorate 323 prepared glass aquaria supplied with sufficient amounts of dechlorinated water with continuous aeriation (17). B- Methods 1. Clinical examination The collected fish were examined clinically according to the methods described by Mc Vicar (18) to detect any external changes or clinical abnormalities. 2. Postmortem examination Postmortem examination of the internal organs was carried out on sacrificed and freshly dead fish according to Austin & Austin (19). 3. Mycological examination A. Isolation of the fungus from diseased fishes Mycological examination was done according to (18). Samples were taken from fish showing skin lesions using sterile dissecting needle from the skin, gills and internal organs (liver, kidney). Gathered specimens were inoculated into duplicate plates of SDA media with 500mg of cyclohexamid and 50 mg of chloramphenicol dissolved in 3 ml ethanol 95% were added to the media after autoclaving. The inoculated plates were incubated at 25°C- 30°C. For 3-5 days (20). Negative plates were not disposed before 2 weeks (21). All the positive moulds cultures examined for gross and micro morphological characteristics (22). B. Identification of different fungi B.i.Identification of moulds Recognization of moulds was completed according to Refai (23). Preliminary recogniza-tion utilizing wet mount preparation of fish samples made in 10% KOH. The confirmatory test of identification was carried out using sou-letip technique (24). Slide culture technique was carried out on those isolates whose identification was inconclusive after staining with lactophenol cotton blue (25). B. 2. Identification of yeast Plates of suspected specimens were analyzed microscopically for the presence of chlamydo-spores, arthrospores and blastospores (20, 23) and the plan of recognizable proof of yeasts given by Terrence (26). Rice agar media was used for identification of yeasts especillay Candida albicans by production of characteristic chlamydospores (23). The confirmatory distinguishing proof was carried out by germ tube test (27). Biochemical reaction using urease test was also conducted (28). 4. Histopathological Examination Tissue specimens from the skin, gills and kidney of the infected fish samples were taken for histopathological examination (29). Results and Discussion The present work was applied to investigate the seasonal incidence of mycotic diseases among some freshwater fishes (O. niloticus & C. gariepinus) in Kafrelsheikh Governorate. Clinical examination The external gross lesions of the examined O. niloticus revealed darkened skin, pale body coloration, scale detachment, fins erosion, and eye opacity as shown in (Plate 1: Fig. 1, 2). These results agree with (24, 30). However, in fish naturally infected with Saprolegnia, there was scattered grayish white cotton wool-like growth on various parts of the body as well as presence of ulcerative areas in some cases as shown in (Plate 1: Fig. 3, 4); with unilateral eye cloudiness or opacity (Plate 1: Fig. 5); the result is in accordance with that of El-Atta, (31). Fish death may be due to either blindness which consequently disable fish to feed or due to the fungal growth over gills causing suffocation. The ulcerative areas over the skin may be attributed to the lytic action of primary bacterial infection as all fungal infections are considered as secondary invader pathogen; these results agreed with many authors (8, 23, 32-36). On the other side, the infected C. gariepinus showed skin ulceration and scattered hemor-rhagic patches on the ventral abdomen and mouth (Plate 1: Fig. 6). These symptoms may be attributed to the toxins secreted by moulds and yeasts causing severe symptomatic changes that appear on the fish in the form of haemorrhagic patches, ascitis and destruction and degeneration of the gills; the result agreed with (16, 24). 324 N. Mahfouz, E. Moustafa, M. Kassab, W. Marzouk P.M. lesions The main observed postmortem lesions were liver enlargement with moderate petechial hemorrhage (Plate 1: Fig. 6& 7). This result may support that the saprolegnia is a secondary invader following systemic bacterial infection which is responsible mainly for this internal lesions due to toxins produced by fungi and yeasts that interfere with function of liver causing congestion in internal organs especially liver (2, 31). Mycological examination Mycological examination revealed the isolation of 2148 fungal isolates from 375 diseased and 125 apparently healthy freshwater fish samples; 1258 fungal isolates from O. niloticus and 890 isolates from C. gariepinus (from skin, gills, liver and kidney). Recognizable proof of fungi into mould and yeast revealed that the incidence of mould and yeast was marginally higher in O. niloticus (56.89%, 68.12%) in contrast with that in C. gariepinus (43.11%, 33.88%). The high frequency of mould isolates in O. niloticus agree with some authors (9, 24) and disagree with the incidence in C. gariepi-nus; as both of the two authors recorded high incidence of yeast isolates from catfish. This might be attributed to variable host susceptibility due to geographical distribution. Morphological identification of isolated moulds The colonies of Saprolgnia sp. appeared as white cotton-wool like growth on the petri dish (Plate 2: Fig. A) while, microscopically appeared as long, branched, un-septated hyphae (Plate 2: Fig. B, C). This result is in accordance with some reports (8, 31). Pencillium sp. Colonies were white and fluffy then, turned into greenish blue in colour (Plate 2: Fig D), while microscopically, there were septated hyphe with un branched condio-phores possessing metule with flask- shaped strigmata forming brush appearance. (Plate 2: Fig E, F). Nonetheless, Fusarium sp. Colonies were cottony or wooly in texture, snow white, pink-violet or blushing red in shading, with dis- semination of hued colors into the switch reverse surface of the medium (Plate 2: Fig G) and microscopically, they seemed long, extended and septated hyphae from which short conidiophores climbed and sometimes branched. Two kinds of conidia were watched, a huge banana shaped, septated macroconidia and a little, round, non septated microconidia. (Plate 2: Fig H). These results are in accordance with some authors (9, 37). On the other side, Aspergillus sp. demonstrated few varieties within the same genus. A. flavus seemed smooth with various aerial growths; the shading changes from yellow to yellowish green by aging (Plate 2: Fig I), and microscopically the conidiophores were long and thick, the vesicles were globose and the strigmata were biseriate and radiate (Plate 2: Fig J). Colonies of A. niger had black color with radiated edges with wooly texture (Plate 2: Fig K), while microscopically had extremely long, smooth and the strigmata were biseriate, minimized and radiate and the conidia were globes and smooth (Plate 2: Fig L). Colonies of A. ter-rus were velvety cinnamon buff to dark brown (Plate 2: Fig M), and microscopically, little hemispherical vesicle with long and smooth co-nidiophore (Plate 2: Fig N). Colonies of A. fu-migatus have unmistakable edge with a few shades of green, surface has a powder appearance with a white overskirt was seen at the edge in the zone of dynamic development (Plate 2: Fig O), and microscopically portrayed by hyaline and particularly septated hyphae, conidio-phores were long with club-molded vesicle, round conidia were conceived from single row of strigmata (Plate 2: Fig P). These results agree with some authors (9, 37). Colonies of Mucor sp. appeared fast-growing, white-to-gray cotton candy, became dark with time and fills the petri dish with fluffy mycelium and microscopically, non-septate broad hyphae, Sporangiophores are long, might be expanded and end with bear terminal round sporangia. The spores scattered and no rhizoids are formed. Rhizopus sp. colonies were deeply cottony; white turned to gray-brown on surface with aging. Microscopically, broad hyphae Seasonal Screening of the Mycotic Infections of Cultured Freshwater Fishes in Kafr El-Sheikh Governorate 325 could be observed, Sporangiophores are un-branched and connect to each another by sep-tated hyphae, large sac-like sporangia that contain sporangiospores. These results agree with some authors (9, 37). Morphological identification of yeast isolates The isolates were cultivated on Rice agar media after culturing on SDA. In the current study, four genera were identified (Candida, Rhodotorula, Trichosporon and Cryptococcus). All genera reacted positively with urease test except Candida. Rhodotorula sp. was identified on SDA by formation of carotenoid pigments; that vary from orange to red (light pink flat colonies). Microscopically, revealed budding of round, oval large cells when stained with Gram's stain. On Rice agar media, showed large round blasto-conidia with absence of pseudohyphae. Cryptococcus sp. appeared rapidly on SDA as flat or slightly heaped shiny moist mucoid colonies with smooth edges. Its color changed from creamy at first to brown later. Microscopically, the colonies were ovoid, spherical with thick wall and mostly showed capsule with budding. On Rice agar media, no pseudo-hyphae but appeared as budding cells. Trichosporon sp. appeared on SDA firstly as smooth flat, or wrinkled white to creamy colonies that turned waxy with central folds surrounded by wrinkled furrows. Microscopically, appearance of hyaline mycelium which is separated and fragmented into rectangular arthro-spores. On Rice agar media, Trichosporon sp. appeared as septated hyphae, pseudohyphae and arthrospores. Candida species colonies on SDA appeared creamy colored pasty colonies within 48-72hrs. On Rice agar media, C. albi-cans showed terminal chlamydospores, blasto-condia and pseudo-hyphae. Other Candida sp. fails to produce pseudohyphae. This result is in accordance with some authors (9, 37- 39). Incidences of moulds and yeast among different seasons and different organs As shown in table (1 & 2), mycological examination of 400 O. niloticus and 100 C. gariepinus revealed an incidence of several moulds including Saprolegnia sp. (53, 32) isolates, Penicillium sp. (152, 131), Fusarium sp. (78, 38) A. flavus (233, 149), A. niger (247, 223), A. terreus (35,22), A. fumigatus (29, 15) Mucor (148, 136) and Rhizopus (65, 42) in O. niloticus and C. gariepinus, respectively. Saprolegnia sp. showed the highest prevalence in late autumn (10.68%, 6.96%) and winter (6.8%, 7.8%) in O. niloticus and C. gariepi-nus, respectively. This may be attributed to that seasonal variation play an important role in spreading of the Saprolegnia infection among freshwater fishes where the water temperature was low. These results agree with some authors (9, 24, 37) where they mentioned that sapro-legniasis occurred during the winter season and colder months of the year. The highest incidence within organs was observed to be from the skin and fins (77.4%, 65.63%) followed by the gills (22.6%, 34.4%) in O. niloticus and C. gariepinus, respectively but, not isolated from liver and/or kidney. The results are in accordance with those of many authors (8, 9, 14, 30, 31) as shown in table (2). Pencillium sp. was isolated with the highest prevalence in winter (18.98%& 23.22%) from O. niloticus, C. gariepinus respectively. The highest incidence within organs was observed to be from the skin and fins (36.2%, 47.3%) followed by gills (30.9%, 20.6%) in O. niloticus and C. gariepinus, respectively. These results are similar to those reported by Ali, (37). Besides, Penicillum sp. could be isolated also from liver (22.4%, 13.7%) and kidney (10.5%, 18.3%). These results agree with some authors (9, 30). Different species of Pencillium were isolated with high incidence from apparently healthy fishes rather than diseased one, therefore members of this genus can be considered as saprophytes (9, 37). Fusarium sp. was isolated with the highest prevalence in spring (11.8%, 5.91%) from O. niloticus, C. gariepinus respectively. The highest incidence within organs was observed to be in gills (37.18%, 34.2%) in O. niloticus and C. gariepinus, respectively. This might be attributed to the high affinity of fungal spores to high oxygen tension (37). It could be isolated from skin, fins, liver and kidney as well. 326 N. Mahfouz, E. Moustafa, M. Kassab, W. Marzouk Aspergillus sp. showed some variation according to species. A. flavus was recorded all over the year and more prevalent during hot weather with high incidence during summer (25.44%, 23.22%) followed by spring (16.24%, 22.08%) in O. niloticus, C. gariepinus. The result is similar to some previous papers (9, 37, 40). A. niger was more prevalent during hot weather with high incidence during summer (26.9%, 37.44%) in O. niloticus, C. gariepinus, respectively. These results are similar to some previous papers (9, 37, 40). A. terrus was more prevalent during autumn (5.98%, 5.69%) in O. niloticus and C. gariepinus, respectively. These results are similar to some previous papers (9, 37, 40). A. fumigatus was more prevalent during autumn (7.69%, 8.23%) in O. niloticus, C. gariepinus. These results are similar to some previous papers (9, 37, 40). In the current study, the highest incidence within organs was observed to be in liver in most of Asperigillus sp. This may support the fact that Asperigillosis is a systemic disease. Zygomycetes (Mucor and Rhizopus) are the most common fungi isolated from apparently healthy fish and diseased O. niloticus and C. gariepinus with high incidence during autumn season and these results agree with Ali, (37). Mucor species were isolated with the highest prevalence in autumn (20.09%) from O. niloticus and winter (29.21%) from C. gariepinus, respectively. The highest incidence within organs was observed to be from the skin and fins (45.3%, 46.3%) followed by gills (29.7%, 27.9%) in O. niloticus and C. gariepinus, respectively. The result is similar to those reported by Ali (37). It could be isolated also from liver (15.5%, 16.9%) and kidney (9.46%, 8.8%) in O. niloticus and C. gariepinus, respectively. These results agree with many authors (9, 37). On the other side, Yeast isolates revealed 4 genera; Candida (62, 37), Rhodotorula (79, 52), Trichosporon (40, 19) and Cryptococcus (37, 21) isolated from O. niloticus and C. gariepinus, respectively. Yeast was also isolated with high frequency from diseased fishes rather than apparently healthy; these results came in agreement with those recorded (9). Candida sp. accounted for (28.44%, 36.27%) of the isolates and Rhodotorula sp. (36.24, 24.51%) from O. niloticus and C. gariepinus. The current results came in agreement with those recorded by Tartor et al., (39). Samples collected from skin, gills, liver, and kidney revealed that C. albicans and Rhodotorula sp. were the highest yeast isolates. These findings were supported by the view reported by (9). Cryptococcus sp. in the present study was isolated with prevalence of (16.97%, 20.59%) from O. niloticus and C. gariepinus, respectively; nearly similar results were recorded by (38) but disagree with Tartor et al., (39). Trichosporon sp. was detected to be (18.35%, 18.63%) from O. niloticus and C. gariepinus, respectively; nearly similar results were recorded by (24, 38) but disagree with Tartor et al., (39). Histopathological findings Histopatholgical examination of naturally infected O. niloticus with Saprolrgnia sp. revealed severe degenerative changes in the skin. Necrosis of dermis and hypodermis, the underling dermis was edematous with degenerative changes of muscle fibers containing fragments from the fungal hyphae (Plate 3: Fig. A). Ulceration, loss of epidermis and loss of texture of scales and sometimes ulcer can be observed (Plate 3: Fig. B, C). Gills showed severe hyper-plasia and hypertrophy of the epithelial lining of secondary lamellae with congestion of branchial blood vessels (Plate 3: Fig. D, E). Kidney revealed necrosis in some tubules together with peritubular fibrosis (Plate 3: Fig. F). These results agreed with many authors (11, 31, 34). Seasonal Screening of the Mycotic Infections of Cultured Freshwater Fishes in Kafr El-Sheikh Governorate 327 Table 1: Seasonal Prevalence of Mould and yeast in O. niloticus and C. gariepinus Oreochromis niloticus (100 fish/season) Winter Spring Summer Autumn Winter Clarias garipinus (25 fish/season) Spring Summer Autumn No. % No. % No. % No. % No. % No. % No. % No. % 28 6.81 0 0 0 0 25 10.68 21 7.87 0 0 0 0 11 6.96 78 18.98 11 4.06 39 11.40 24 10.26 62 23.22 29 11.42 23 10.90 17 10.76 17 4.14 32 11.81 20 5.85 9 3.85 8 2.99 15 5.91 10 4.74 5 3.16 65 15.82 44 16.24 87 25.44 37 15.81 10 3.75 56 22.05 49 23.22 34 21.52 83 20.19 50 18.45 92 26.90 22 9.40 37 13.86 82 32.28 79 37.44 25 15.82 0 0 0 0 11 3.22 18 7.69 0 0 0 0 9 4.27 13 8.23 7 1.70 10 3.69 4 1.17 14 5.98 0 0 6 2.36 0 0 9 5.67 62 15.09 12 4.43 27 7.89 47 20.09 78 29.21 27 10.63 6 2.84 25 15.82 19 4.62 6 2.21 27 7.89 13 5.56 17 6.37 5 1.97 11 5.21 9 5.67 26 6.33 30 11.07 6 1.75 17 7.26 7 2.62 9 3.54 5 2.37 4 2.53 10 2.43 25 9.23 19 5.56 8 3.42 13 4.87 8 3.15 12 5.69 6 3.80 7 1.70 20 7.38 10 2.92 0 0 3 1.12 9 3.54 7 3.32 0 0 9 2.19 31 11.44 0 0 0 0 11 4.12 8 3.15 0 0 0 0 No. % No. % No. % No. % No. % No. % No. % No. % 411 32.67 271 21.54 342 1258 27.19 234 18.60 267 30 254 28.54 211 890 23.71 158 17.75 Mould Saprolegnia Pencillium Fusrium Asp.flavus Asp.Niger Asp.Fu-migtus Asp.terrus Mucor sp Rhizopus sp Yeast Rhodotorulla sp Candida sp Tricho-sporon sp Cryptococ-cus sp Total Total No. of isolates Table 2: Incidence of Mould and yeast in organs of O. niloticus and C. gariepinus No. Oreochromis niloticus (100 fish/season) Skin&Fins Gills Liver Kidney No. % No. % No. % No. % Clarias garipinus (25 fish/season) Skin&Fins Gills Liver Kidney No. No. % No. % No. % No. % 53 41 77.4 12 22.6 0 0 0 0 32 21 65.63 11 34.37 0 0 0 0 152 55 36.2 47 30.9 34 22.4 16 10.5 131 62 47.4 27 20.6 18 13.7 24 18.3 78 21 26.92 29 37.18 11 14.1 17 21.8 38 10 26.3 13 34.2 9 23.7 6 15.8 233 83 35.6 47 20.20 78 33.5 25 10.7 149 33 22.1 62 41.6 30 20.1 24 16.1 247 92 37.2 46 18.6 81 32.8 28 11.3 223 95 42.6 72 32.3 33 14.8 23 10.3 29 10 34.48 12 41.40 3 10.34 4 13.8 15 10 66.7 5 33.3 0 0 0 0 35 13 37.1 7 20 10 28.6 5 14.3 22 9 40.9 5 22.7 7 31.8 1 4.5 148 67 45.27 44 29.73 23 15.54 14 9.46 136 63 46.3 38 27.9 23 16.9 12 8.8 65 42 64.6 23 35.4 0 0 0 0 42 30 71.4 12 28.6 0 0 0 0 79 25 31.6 40 50.6 4 5.1 10 12.7 25 7 28 2 8 12 48 4 16 62 18 29 20 32.3 16 25.8 8 12.9 37 7 18.9 4 10.8 17 45.9 9 24.3 40 18 45 12 30 10 25 0 0 19 2 10.53 7 36.84 10 52.63 0 0 37 0 0 25 67.57 12 32.43 0 0 21 0 0 13 61.9 6 28.6 2 9.5 No. % No. % No. % No. % No. % No. % No. % No. % 485 38.55 364 28.93 282 22.42 127 10.01 349 39.21 271 30.45 165 18.54 105 11.79 Mould Saprolegnia Pencillium Fusrium Asp.flavus Asp.Niger Asp.Fu-migtus Asp.terrus Mucor sp Rhizopus sp Yeast Rhodotorulla sp Candida sp Tricho-sporon sp Cryptococ-cus sp Total Total No. of isolates 1258 890 328 N. Mahfouz, E. Moustafa, M. Kassab, W. Marzouk Plate 1: (1, 2) O. species showing skin darkening, scale detachment and erosion of membranous part of fins. (3, 4) O. species showing cotton wool-like growth on various parts of the body ,ulceration of skin. (5) O. species showing cloudy and opaque eye. (6) C. gariepinus showing skin ulceration, scattered hemorrhagic patches on the ventral abdomen and mouth. (7) Naturally diseased O.niloticus showing moderate petechial hemorrhage, dark liver enlargement (8) A naturally examined O. nilot-icus showing threads of congestion along the surface of live Plate 2: (A) Saprolegnia species with the characteristic cotton- wool like growth colony on SDA, (B, C) branched aseptic hyphe, (D, E) Penicillium sp. on SDA with different colour and texture, (E, F) Penicillium sp. showing brush- like arrangement, (G) a colony of Fusarium sp. on SDA with rose pigments on the center, (H) Fusarium under light microscope (I) A. flavus on SDA, (J) A. flavous showing characteristic typical head, (K) A colony of A. niger on SDA, (L) Aspergillus niger showing characteristic round head with black conidia, (M), Colonies of Aspergillus terreus on SDA, (N) A. terreus with small hemispherical vesicle, (O) A colony of A. fumigatus on SDA, (P) A. fumigatus with columnar head Seasonal Screening of the Mycotic Infections of Cultured Freshwater Fishes in Kafr El-Sheikh Governorate 329 Plate 3: Photomicrograph of skin, gills and kidney of Oreochromis niloticus infected with Saprolegnia sp. (A) Necrosis of dermis, hypodermis, edema and degenerative changes of muscle fibers (Arrow) with presence of hyphae of saprolegnia (Arrow head) x 400. (B) Ulceration & loss of epidermis and loss of texture of scales (Arrow). (C) Normal scale (Arrow) and Ulcer in neighboring (Arrow head). (D) Gills showing sever Hyperplasia & Hypertrophy of the epithelial lining of secondary lamellae with fusion in neighboring (Arrow). (E) Infection of saprolegnia at the tip of primary lamellae with congestion of blood vessles and hyperplasia& hypertrophy (Arrow head) while other part filament is normal (Arrow). (F) Infected kidney with peritubular fibrosis( Arrow) and necrosis in some tubule (Arrow head). Conclusion From the present study, it could be concluded that Saprolegnia was the predominant among diseased fishes with highest prevalence in late autumn (10.68%, 6.96%) and winter (6.8%, 7.8%) in O. niloticus and C. gariepinus, respectively. However, Pencillium sp. and Aspergillus sp. were the most predominant fungi isolated from apparently healthy fishes. The highest prevalence of Pencillium sp. were recorded in winter, whileas Aspergillus showed variations between species; A. flavus, A. niger were more prevalent in summer and A. terrus, A. fumigatus were more prevalent in autumn in O. niloticus and C. gariepinus, respectively. Moreover, the highest prevalence of Fusarium species were recorded in spring from O. niloticus and C. gariepinus, respectively and Zygomycetes (Mucor and Rhizopus) recorded the highest prevalence in autumn in O. niloticus and winter in C. gariepinus. Four genera from yeast were isolated; Candida sp., Rhodotorula sp., Cryptococcussp. and TrichosporonSp. The histopathological findings revealed severe degenerative changes in skin and gills with presence of fungal hyphae and spores. Conflict of interest The authors declare that no conflict of interest. 330 N. Mahfouz, E. Moustafa, M. Kassab, W. Marzouk References 1. Hussain SM, Javed M, Javid A, Javid T, Hussain N. Growth responses of Catla catla, Labeo rohita and Cirrhina mrigala during chronic exposure of iron. Pak J Agric Sci. 2011;(1);48:225-30. 2. Mohamed HMA, Walaa FAE, Braeuning A, Hammad S. Detection of aflatoxin -producing fungi isolated from tilapia and fish feed. Excil J. 2017; 16:1308-18. 3. El-Sayed AFM, Dickson MW, El-Naggar GO. Value chain analysis of the aquaculture feed sector in Egypt. Aquac 2015; 437: 92-101. 4. FAO. Fishery and Aquaculture Statistics Yearbook 2014. Rome, Italy: Food and Agriculture organization of the United Nations. ISSN 20706057 (2016). 5. Nandlal S, Pickering T. Tilapia fish farming in Pacific Island countries. Vol. 1: Tilapia hatchery operation. Noumea, New Caledonia: Secretariat of the Pacific Community, 2004. 6. Rodolphe EG, Marshall WL, Lilje O, Jes-sop CN, Gleason FH, Andreou D. Current ecological understanding of fungal-like pathogens of fish: what lies beneath?. HAL Id: hal-01321245 http://hal .upmc.fr/hal-01321245. 2014 7. Ashour AA, Mustafa SA, Yassein SN. His-topathological studies on common carp (Cyprinus carpio L.) infected with Saprolegnia sp. and treated with Virkon®. Mirror Res. Vet. Sci. Anim. 2017; 6: 19-30. 8. Noor El-Deen A, Hussein M O, Zaki M S and Abo-State H A Mass Mortality in Cultured Nile Tilapia Oreochromis niloticus due to Sapro-legniosis with Emphasis on Treatment Trials. J Biol Sci. 2018; 18(1):39-4 9. Refai MK, Laila K, Amany M, Shimaa ES. The assessment of Mycotic settlement of freshwater fishes in Egypt. J Am Sci, 2010; 6(11): 823--31. 10. Pickering AD, Willoughby LG. Sapro-legnia infections of salmonid fish. In Microbial Diseases of Fish, Edit Roberts, RJ Academic Press, London, 1982, 271-98. 11. Chauhan R, Lone S, Beigh AH. Pathogencity of three species of Aspergullis (A. fu-migatus, A. niger & A. sydowii) on some freshwater Fishes. Life Sci Leaflets, 2014; 14:65-72. (Online print). 12. West PV. Saprolegnia paracitica, an oomy-cyte pathogen with a fishy appetite: New challenges for an old problem, mycologist, 2006: 20(3):99-104. 13. Yanong RP. Fungal diseases of fish. Vet. Clin. North Am. Exot. Anim. Pract. 2003; 6: 377400. 14. Eissa AE, Abdelsalam M, Tharwat N, Zaki M. Detection of Saprolegnia parasitica in eggs of angelfish Pterophyllum scalare (Cuvier-Valenci-ennes) with a history of decreased hatchability. Int. J. Vet. Sci. Med. 2013; 1: 7-14. 15. Urquhart K, Collins C, Monte M, Sokolow-ska J, Secombes C, Collet B. Individual measurement of gene expression in blood cells from Rainbow trout On- corhynchus mykiss (Walbaum). JEAAS. 2016; 2(1):1-9. 16. Chauhan R. Studies on conidial fungi isolated from some fresh water fishes. International journal of Advanced Life Sciences (IJALS), 2013; 6 (4): 131—5. 17. Innes WT. Exotic Aquarium Fishes, 19th edition. Aquarium Incorporated, New Jersey 1966; 1(12):24-25,29-30 and530-3 18. McVicar AH. Ichthyophonus infections of fish. In: Roberts RJ (ed) Microbial diseases of fish. Academic Press. London, 1982: 243-69. 19. Austin B, Austin DA. Bacterial fish pathogens; diseases of farmed and wild fish. Springer, New York, London). 2012. 20. Taha M. Medical mycology "Atlas of medically important & dermatomycosis". 1st ed., Mecca printing house, Cairo, Egypt. 2011. 21. Feingold SM, Baron EJ. Bailey and Scoll's Diagnostic Microbiology. The Cv. Mosby co., St. Louis, 1986. 22. Ellis MB. Dematiaceous Hyphomycetes. Commonwealth Mycological Institute: Kew Surrey, UK, 1971. 23. Refai M. Isolation and Identification of fungi. Fac. Vet. Med. Cairo University. 1987 24. Diab AMA. Studies on the mycological affections in cultured fresh water fishes in KafrelSheikh governorate M.V.Sc Thesis (Fish diseases and management), Fac Vet. Med., Tanta University; 2006. 25. Domsch KH, Gams W, Anderson TH. Compendium of soil fungi. 2nd taxonomically revised edition by W. Gams. IHW, Eching. 2007. 26. Terrence CD. A practical approach to identification of yeast like organisms. Amer. Jour Microbiol 1971; 35(5):580-90. 27. Martin MV. Germ tube formation by oral strains of Candida albicans. J. Med. Microbiol. 1979; 12:187-193. 28. Cruickshank R, Duguid JP, Mariom BP, Swain RHA. Medical Microbiology the Practice of Medical Microbiology. 12th Edn., Churchill Livingstone, London, New York. 1975. Seasonal Screening of the Mycotic Infections of Cultured Freshwater Fishes in Kafr El-Sheikh Governorate 331 29. Bancroft JD, Gamble M. Techniques. 5th edition; Churchill Livingstone, London, UK., 2007: 125-38. 30. Marzouk MS, Rezeka SM, El Gamal MH. Some mycological investigations on cultured tilapia in Kafr El Sheikh Governorate. Kafr El Sheikh Vet. Med. J., 2003; 1: 97-111. 31. El-Atta MEA. Saprolegniosis in freshwater cultured Tilapia nilotica (Oreochromis niloticus) and trial for control by using Bafry D50/500. Proceedings of the 8th International Symposium on Tilapia in Aquaculture, October 12-14, 2008, Cairo, Egypt, 2008: 1403-18 32. Bruno DW, Wood BP. Saprolegnia and other Oomycetes. In: Fish Disease and Disorder, Viral, Bacterial and Fungal Infections, Woo, P.T.K. and D.W. Bruno (Eds.)., CABI Publishing, Wallingford, Oxon, United Kingdom, 1994: 599-659. 33. Hussein MM, Hatai K. Saprolegnia salm-onis sp. nov. isolated from sockeye salmon, Onchrhynchus nerka. Mycoscience, 1999; 40: 387--91. 34. El-Ashram AMM, Abd El-Rhman AM, Sakr SF. A contribution to saprolegniosis in cultured Nile tilapia (Oreochromis niloticus) with special reference to its control. Egypt J. Aquat. Biol. & fish. 2007; 11(3):943-55. 35. Chauhan R. Studies on some fresh water fishes found infected with dermatomycoses, collected from different water bodies in and around Bhopal, India. Indo Am. J. Pharm. Res. 2014; 4: 1591-6. 36. Ganguly S, Wakchaure R, Praveen PK, Para PA. Fungal infections in fishes: A brief review. Int. J. Pharm. Life Sci., 2016; 7: 5245-6. 37. Ali ShELM. Studies on the causative agents of mycotic diseases in fresh water fishes. M.V.Sc Thesis (Microbiology), Fac Vet. Med., Cairo University. 2007. 38. Abbas MS, Khalaf JM, Yassein SN. Isolation and identification of systemic mycological isolates from fishes samples that obtained from local markets in Baghdad, Iraq. World J Exp Biosci 2016; 4: 143-6. 39. Tartor Y, Taha M, Mahboub H, El Ghamery M. Yeast species associated with diseased fish: Occurrence, identification, experimental challenges and antifungal susceptibility testing. Aquac 2018; 40. Diab AMA. Studies on some systemic mycotic affection in cultured fresh water fish Ph.D Thesis (Fish diseases and hygiene), Fac Vet. Med. Alexandria university.2010. Slov Vet Res 2019; 56 (Suppl 22): 333-40 DOI 10.26873/SVR-772-2019 Original Research Article QUALITY CHARACTERISTICS OF BEEF HAMBURGER ENRICHED WITH RED GRAPE POMACE POWDER DURING FREEZING STORAGE Omima S. Abdelhakam, Essam M. Elsebaie*, Adel K. Ghazi, Mahmoud S. Gouda Department of Food Technology, Faculty of Agricultural, Kafrelsheikh University, 33516, Kafrelsheikh, Egypt Corresponding author, E-mail: Essam.Ahmed@agr.kfs.edu.eg Abstract: Integrating the consumption of agro-industrial residues with satisfactory consumers is an opposition that, may affect the financial dividend of the frozen meat manufacturing, aside from producing products with diversified nutritional values and satisfying technical characteristics. Thence, we aimed in this research to elaborate and appraise the physicochemical and microbiological characteristics of beef hamburger with the addendum 2 and 4 % of red grape pomace powder (RGPP) throughout its stockpiling at (18 ± 2°C) over a period 12 weeks. A significant influence on pH value was observed at the beginning (zero time), however the control sample, 2% and 4% had pH values of 6.68, 6.41 and 6.22, respectively. Meantime, there was a significant influence of (RGPP) on thiobarbituric acid (TBA) evidenced on 4th week. The results showed that the microbial growth increased as a function to storage period for all and the addendum of RGPP in beef burger caused a reduction in total bacterial count in comparison with control. Thus, in general, it can be concluded that addendum of RGPP is an efficacious mean to improve burger quality and increasing its storage stability. Key words: beef burger; red grape pomace; storage stability Introduction Meat products can be suffered from numerous negative changes throughout frozen and refrigerated storage as a result for the growth of microorganisms and oxidation of lipids, which result in quality shortage, meat deterioration, and economical damage (1). The growth of microorganisms in meat and its other products may be lead to slime forming, degrading structural constituents, reduction in water holding capability, bad odor, and alteration in appearance and texture which decrease nutritive value, quality, and shelf- life (2). Lipid oxidation is contingent on the composition of fatty acids, the concentration of vitamin E, and on oxidation-enhancing substances such as free iron in the muscles. Oxidation products can lead to lose color and nutritional value as a result to the breakdown of lipids, proteins, pigments, vitamins and carbohydrates (3). The oxidative rancidity may be cause a lot of alterations in meat and their products ranged from large changes in flavor, color loss and structural breakdown for proteins (freshness losing) that impede consumer's repeated purchase (4). Artificial anti-oxida-tive substances such as butylated hydroxyani-sole (BHA) and butylated hydroxytoluene (BHT) were utilized to hold up oxidation of li- Received: January 2019 Accepted for publication: February 2019 334 O. S. Abdelhakam, E. M. Elsebaie, A. K. Ghazi, M. S. Gouda pids in meat (5). Nonetheless, contemporary attention over its utilization has generated a necessity and induced research for alternate anti-oxidants, predominately from naturalistic origin. The usage of naturalistic preservatives with meat products increases the storage shelf life is a hopeful technology as numerous vegetative materials posse antimicrobial and antioxidant characteristics (6). Functional substances addendum into meat products might upgrade the health and nutritional attributes and extension their storage shelf-life (4). Vegetation extracts which are affluent with polyphenolic components are perfect candidates, because of its extraction easiness from the naturalistic origins and their efficacy in the prevention of lipid oxidizing in food (6). Grape seeds extract anti-oxidative efficacy has been assessed in a little number of meat sorts and was found to enhance the oxidative resistance in turkey patties (7), cooked beef (8) and nuggets (9). Dietary fibers are considered to be a functional component of meat products. Incorporating fiber can get the texture, enhancing cooking performance because of its water binding and fat retention characteristic and dietary fibers can also produce technical characteristics that enhance physical, chemical and sensory attributes. Naturalistic antioxidants and dietary fibers are considered as dietetic agents that participate in enhancing human health. Natural products with high levels of phenolic antioxidants and dietary fibers are named antioxidant dietary fibers (ADF) (10). The current investigation was intended to investigate the influence of red grape pomace powder (RGPP) integration in beef hamburgers on its physical, chemical, microbiological and sensory properties during frozen storage. Materials and methods Red grapes, variety Red Globe1, by products were obtained in August 2016 from Faragello factory for juice, Sixth of October City, Egypt. Red muscles beef, kidney fats and other components used for burger preparation were obtained from local market at Kafr El-Sheikh city, Egypt. The beef meat and fat tissue were transported to the food technology laboratory, faculty of agriculture, Kafrelshiekh University using an icebox. All chemicals used in this study of HPLC grade (99.9% purity) were obtained from Sigma Company of chemicals and drugs, St. Louis, MO, USA. Preparation of samples The pomace (skin and seeds) was sorted by hand for removing stems and debris, and thereafter lyophilized, and milled up to pass through 20 mesh screen sieve. The yielded powder samples were mixed, and stored in polyethylene bags, then kept at room temperature until use (11) . Burger preparation Beef burger meat and kidney fat was grounded separately in a meat grinder by passing meat through a plate having 6 mm holes. The beef burger was formulated to contain the following ingredients 80% red muscles beef meat, 20% kidney fat, 18% (w/w) water (ice), 1.5% (w/w) salt, ground black pepper (0.3%), red pepper (0.2%) and cumin (0.2%) according to Aleson-Carbonell, et al.(12).The minced beef was treated with two concentrations of RGPP (2 and 4%). Control without RGPP was used in all assays. The beef burgers were frozen at -18 °C until the start of analysis. Thermal treatment After the thawing at 4 °C for 12 hours, the beef burgers were stewed using an electrical grill (Genwex GW-066) at 220°C (the space between heat source and the samples was 4 cm) for 8 min (4 min for each side of beef burgers) (2) Chemical analyses Gross chemical composition of red grape pomace powder was analyzed according to A.O.A.C. (13). Where, total carbohydrates were calculated by difference. The amount of total polyphenols was estimated as mg Gallic Acid Equivalent/100gm using the method outlined by González-Centeno et al (11). Quality characteristics of beef hamburger enriched with red grape pomace powder during freezing storage 335 Identification of phenolic acids For HPLC analysis, one gram of the red grape pomace powder was mixed with acidified methanol (10 ml)for 4 min and centrifuged at 10000 rpm for10 min and the supernatant was filtered through a 0.2 um Millipore membrane. The filtrates were collected in a vial for injection into HPLC Hewllet packared (series 1050) equipped with auto-sampling injector, solvents (methanol and acetonitril) were degassed and ultraviolet (UV) detector was set at 280 nm and quarter HP pump. The column temperature was maintained at 35°C, where gradient separation was performed using the mobile phase (methanol and acetonitril) at flow rate of 1 ml/min following the method of Goupy, et al. (14). Physical analyses of burger The pH value of beef hamburgers was determined by using a pH meter (Jenwae model 3357, USA) according to the method described by (15). Water holding capacity (WHC) was determined by filter press method as described by (15). Beef burgers were weighted before and after cooking to determine cook loss according to the method of 15 by using the following equations: Cooking loss (%) = [(Raw weight - Cooking weight)/ Raw weight]*100 Chemical analyses of burger Thiobarbituric acid reactive substances (TBARS) of beef hamburger samples were estimated as reported by (16). Microbiological Analyses Total count of aerobic microorganisms and fungi and yeasts were carried out in triplicate and using the methodologies mentioned by (17). Statistical analysis General linear model of SPSS (Ver. 16.0, 2007) was used to conduct ANOVA for determination of differences between means. The probability levels o f P < 01and P < 0 .05 were considered to be significant for statistical procedures. All measurements and trials were done in triplicate. Results and discussion Chemical composition of red grape pomace There are many factors affecting the chemical composition of red grape pomace such as variation in organic compounds of the soil, varieties, fertilizers applied, climatic and environmental conditions (18). The data in Table (1) indicate that pomace is considered a good source of protein, crude fiber and total carbohydrates which are important from the nutritional point of view. The results revealed that the moisture, crude protein, ether extract, ash, crude fiber and total carbohydrate contents were 5.1, 12.5, 11.5, 1.8, 9.2 and 74.2%, respectively. These results were in harmony with those of (19). Identification of phenolic acids content of ethanolic red grape pomace powder extract HPLC procedure was utilized for fractionation and identification of the phenolic components extricated from the tested sample. The results were tabulated in Table (2). The results revealed that, red grape pomace ethanolic extract have 24 phenolic components. Pyrogallol was the highest compound of poly-phenolic compounds found in ethanolic pomace extract (38.38%) followed by Catechol (8.72%) then Epicatechin (7.87%), where Cinnamic (0.09%) and Resveratrol (0.15 %) were the lowest ones. These results are in the same line with others obtained by (20). Physical analyses of burger Meat products physical characteristics such as pH and WHC are highly remarkable because of its influence on the quality attributes like cooking loss, juiciness and tenderness. pH values The results in Table (3) revealed that there was an increment in beef hamburger pH values as a function of the storage time which on the same trend with (21) who stated that the inconsiderable increment in pH values throughout the storage period might be back to protein liberation metabolites as a consequence for bacterial efficiency. The results also appeared that 336 O. S. Abdelhakam, E. M. Elsebaie, A. K. Ghazi, M. S. Gouda the addendum of RGPP was performed to reduce beef hamburger pH values. Also, there was a decrement in pH values as a function for increasing the addendum levels of RGPP as compared to control treatment which has the highest pH values along storage period whereas beef hamburger containing 4 % RGPP treatment has the lowest pH values. This pH decrement might be lead to the low pH values of RGPP added (22). At the end of storage time, the pH value of the control sample was 6.93 which significantly different from the pH value of beef hamburger enriched with 4 % RGPP (6.51). Water holding capacity (WHC) The results given in Table 3 appeared that WHC (cm2) of beef hamburger has been reduced along the storage period. This decrement may be back to proteins denaturation which causing a reduction in its ability to hold water leading to lower WHC. The addendum of RGPP into beef hamburger negatively influence on WHC which was reduced with increasing the addendum level of RGPP, Beef hamburger enriched with 4 % RGPP has achieved the lowest WHC values (the highest area of squeezed water) during storage period whereas control treatments were recorded the highest values which appear that RGPP had lower water holding capacity. Table 1 : Proximate chemical composition of red grape pomace (on dry weight basis) Component % Moisture 5.1 Crude protein (N x 6.25) 12.5 Ether extract 11.5 Ash 1.8 Crude fiber 9.2 *Total carbohydrates 74.2 Total polyphenols (mg GAE/g) 4.92 *Total carbohydrates were calculated by difference. Table 2: Phenolic compounds in ethanolic extract of red grape pomace powder Compounds % of the Total Compounds % of the Total phenolics phenolics Gallic acid 3.64 Ferulic 0.62 4-Amino benzoic acid 0.20 Isoferulic 1.76 Protocatchuic 2.72 Resveratrol 0.15 Catechin 6.77 Ellagic 4.29 Chlorogenic 4.00 Alpha-Coumaric 1.18 Catechol 8.72 e-Vanillic 4.70 Epicatechin 7.87 Benzoic 7.58 Caffeine 0.68 3,4,5 Methoxycinnamic 0.38 Caffeic 1.14 Coumarin 0.31 Vanillic 1.96 Salicylic 1.56 ß-coumaric acid 0.34 Cinnamic 0.09 Pyrogallol 38.38 P-OH-benzoic 0.92 Quality characteristics of beef hamburger enriched with red grape pomace powder during freezing storage 337 Table 3: Changes in some physical characteristics of beef hamburger enriched with red grapes pomace powder throughout frozen storage Storage (months) period Control PH value 0 1 2 3 6.68a 6.74 a 6.85 a 6.93 a Water holding capacity (cm2) 0 1 2 3 Cooking loss (%) 0 1 2 3 3.80 c 3.86 c 3.98 c 4.20 c 3.17 a 6.34 a 7.62 a 7.93 a Beef hamburger treatments 2% red grapes pom- 4% red grapes pomace ace 6.41 6.49 6.61 6.75 3.96 4.10 4.16 4.33 1.27 3.92 4.55 5.98 6.22c 6.30 c 6.37 c 6.51 c 4.10 a 4.21a 4.29a 4.42 a 0.63 c 2.93 c 3.13 c 4.87 c Values followed by the same letter in the same row are not significantly different at P < 0.05 Table 4: Changes in TBA values (mg malonaldehyde / kg) of beef hamburger enriched with red grapes pomace powder during frozen storage Beef burger type treatments Control 2% red grapes pomace 4% red grapes pomace Storage period per months 0 0.367a 0.352b 0.338c 1 0.595a 0.470b 0.396c 2 0.709a 0.501b 0.412c 3 0.932a 0.600b 0.563c Table 5: Changes in microbiological properties of beef hamburger incorporated with red grapes pomace powder during frozen storage Storage period (months) control 2% red grapes pomace 4% red grapes pomace Plate count bacteria (log cfu/g) 0 3.21 3.21 3.21 1 3.89 3.00 2.93 2 4.96 2.88 2.68 3 6.42 2.61 2.44 Yeast and molds count (log cfu/g) 0 2.20 2.20 2.20 1 3.40 2.18 1.90 2 3.62 1.93 1.80 3 4.70 1.70 1.65 338 O. S. Abdelhakam, E. M. Elsebaie, A. K. Ghazi, M. S. Gouda Cooking loss According to data in Table (3), cooking loss percentage of beef hamburger is increased as a function of the storage period which on the line with the water holding capacity results. Also, the cooking loss of beef hamburger enriched with RGPP is decreased with increasing the addendum levels since beef hamburger enriched with RGPP had cooking loss values lower than control treatment along the storage period. Burger containing 4 % RGPP had the lowest cooking loss values at any storage period while, control treatment have the highest cooking loss value. This might be back to the fiber components of RGPP, which could influence on the cooking loss of the beef hamburger, since fibers could reduce the water loss during cooking by forming gels as reported by (22). Changes in chemical properties of beef hamburger during frozen storage Thiobarbituric acid (TBA) value Lipid oxidation is one of the main limiting factors for the quality and acceptability of meat and meat products. Thiobarbituric acid (TBA) value (malonaldehyde / kg) is used as an index for measuring oxidative rancidity which takes place in meat products during storage. Table (4) shows the effect of incorporation of RGPP on the oxidative stability of beef hamburger during frozen storage. The data show that the TBA value is increased as the storage time increased which in agree with (21). The TB A values of beef hamburger were decreased with increasing RGPP incorporation level. There are significant differences between control and the other red grapes pomace powder containing treatments exhibited lower TBA values than control treatments. These due to that polyphenols have been re-ported to have antioxidant activity in vitro (23). At the end of frozen storage period TBA values of control was higher than the spoilage limit of Egyptian Standard Specifications (ESS) (24) (0.9 mg malonaldehyde / kg) since it recorded 0.932 mg malonaldehyde / kg. On the other hand beef hamburger treatments contain-ing 2 and 4 % RGPP were reached to 62 -66 % of the ESS spoilage limit. These results may be due to the polyphenol content of red grapes pomace powder which play important role in reducing the oxidative reaction of lipids which reflects in low TBA values. The data also show that at any storage time the lowest TBA value was observed for beef hamburger containing 4% RGPP. Changes in microbiological load of beef hamburger incorporated with red grapes pomace powder during frozen storage Table (5) shows the effect of incorporation of red grapes pomace powder (RGPP) on the microbial growth of beef hamburger during frozen storage. The results show that as the storage time increase the microbial growth increase for all treatments. The incorporation of red grapes pomace powder in beef hamburger results in lowering the total bacterial count as compared to control, since it was gradual decreasing as the RGPP incorporation level increased. This may be due to the higher phenolic compounds content in beef hamburger incorporated with RGPP. The results show that there are significant differences between all treatments. At any storage time the lowest total bacterial count was observed for beef burger incorporated with 4 % RGPP while the highest bacterial co unt was observed for control treatment which reached the spoilage limit of ESS (24) (6 log cfu/g) for total bacterial count at 90 d ays of frozen storage whereas treatments incorporated with RGPP reached to 30 - 35 % o f this limit. Also, the molds and yeasts counts were decreased as the incorporation level increased, the lowest counts were observed for treatment incorpor ated with 4 % RGPP at all sto rage times. At the end of storage period there were significant differences between control and treatments incorporated with RGPP. Conclusion The results of this study demonstrated that red grape pomace ethanolic extract contains 24 phenolic components. These compounds helped to improve pomace powder extract as anti-oxidant agent that can be used to prepare functional foods which provide the physical, chemical and microbiological properties of beef hamburger. Quality characteristics of beef hamburger enriched with red grape pomace powder during freezing storage 339 Conflict of interest Authors declare that no conflict of interest. References 1. Essa RY, Elsebaie E. Effect of Using Date Pits Powder as a Fat Replacer and Anti-Oxidative Agent on Beef Burger Quality. J Food and Dairy Sci, Mansoura Univ 2018;9:91-6. 2. Oliveira RBSd, Lucia FD, Ferreira EB, et al. Quality of beef burger with addition of wet okara along the storage. Ciencia e Agrotecnologia 2016;40:706-17. 3. Dave D, Ghaly AE. Meat spoilage mechanisms and preservation techniques: a critical review. American Journal of Agricultural and Biological Sciences 2011;6:486-510. 4. Quevedo R, Pedreschi F, Valencia E, et al. Kinetic modeling of deterioration of frozen industrial burgers based on oxidative rancidity and color. Journal of Food Processing and Preservation 2018:e13655. 5. Pereira D, Pinheiro RS, HELDT LFS, et al. Rosemary as natural antioxidant to prevent oxidation in chicken burgers. Food Science and Technology (Campinas) 2017:0-0. 6. Nowak A, Czyzowska A, Efenberger M, et al. Polyphenolic extracts of cherry (Prunus cerasus L.) and blackcurrant (Ribes nigrum L.) leaves as natural preservatives in meat products. Food microbiology 2016;59:142-9. 7. Nardoia M, Ruiz-Capillas C, Herrero AM, et al. Effect of added grape seed and skin on chicken thigh patties during chilled storage. International Journal of Food and Nutritional Science 2017;4: 0. 8. Cosansu S, Juneja VK. Growth of Clostridium perfringens in sous vide cooked ground beef with added grape seed extract. Meat science 2018; 143:252-6. 9. Cagdas E, Kumcuoglu S. Effect of grape seed powder on oxidative stability of precooked chicken nuggets during frozen storage. Journal of food science and technology 2015;52:2918-25. 10. Mehta N, Ahlawat S, Sharma D, et al. Novel trends in development of dietary fiber rich meat products—a critical review. Journal of Food Science and Technology 2015;52:633-47. 11. González-Centeno M, Comas-Serra F, Femenia A, et al. Effect of power ultrasound application on aqueous extraction of phenolic compounds and antioxidant capacity from grape pomace (Vitis vinifera L.): experimental kinetics and modeling. Ultrasonics sonochemistry 2015;22:506-14. 12. Aleson-Carbonell L, Fernandez-Lopez J, Perez-Alvarez J, et al. Characteristics of beef burger as influenced by various types of lemon albedo. Innovative Food Science & Emerging Technologies 2005;6:247-55. 13. A.O.A.C. Association of Official of Analytical Chemists, Official Methods of Analysis. 18th Ed., Pub. By the A.O.A.C.: Arlington, Virginia, 2220 USA., 2010. 14. Goupy P, Hugues M, Boivin P, et al. Antioxidant composition and activity of barley (Hordeum vulgare) and malt extracts and of isolated phenolic compounds. Journal of the Science of Food and Agriculture 1999;79:1625-34. 15. Pinero M, Parra K, Huerta-Leidenz N, et al. Effect of oat's soluble fibre (P-glucan) as a fat replacer on physical, chemical, microbiological and sensory properties of low-fat beef patties. Meat science 2008;80:675--80. 16. Trindade R, Mancini-Filho J, Villavicencio A. Natural antioxidants protecting irradiated beef burgers from lipid oxidation. LWT-Food Science and Technology 2010;43:98-104. 17. Georgantelis D, Blekas G, Katikou P, et al. Effect of rosemary extract, chitosan and a-tocopherol on lipid oxidation and colour stability during frozen storage of beef burgers. Meat Science 2007;75:256-.64 18. Howard L, Talcott S, Brenes C, et al. Changes in phytochemical and antioxidant activity of selected pepper cultivars (Capsicum species) as influenced by maturity. Journal of Agricultural and Food Chemistry 2000;48:1713-20. 19. Silvia PM, loan PM. The Chemical Composition Assessment Of The Feteasc Neagr Grape Pomace And Its Fractions Obtained From Wine Industry In Different Years. Analele Stiintifice ale Universitatii" Al I Cuza" Din Iasi(Serie Noua) Sectiunea 2 a Genetica si Biologie Moleculara 2015;16:167. 20. Lafka T-I, Sinanoglou V, Lazos ES. On the extraction and antioxidant activity of phenolic compounds from winery wastes. Food Chemistry 2007;104:1206-14. 21. Kalaikannan A, Anjaneyulu A, Santhi D. Effect of egg proteins on the quality and refrigerated storage life of chicken patties made with broiler-spent hen meat and by-products. International journal of food science & technology 2007;42:579-86. 22. Garcia ML, Calvo MM, Selgas MD. Beef hamburgers enriched in lycopene using dry tomato peel as an ingredient. Meat science 2009;83:45-9. 340 O. S. Abdelhakam, E. M. Elsebaie, A. K. Ghazi, M. S. Gouda 23. Powell ZD, Lakesha C. Antioxidant capa- 24. ESS. Egyptian Standard Specifications, No. cityof lycopene-containing foods. International 439, 2005/Thermally treated.treated poultry meat journal of food sciences and nutrition 2001; products. 2005. 52:143-9. Slov Vet Res 2019; 56 (Suppl 22): 341-52 DOI 10.26873/SVR-773-2019 Original Research Article LAYING PERFORMANCE, FAT DIGESTIBILITY AND LIVER CONDITION OF LAYING HENS SUPPLEMENTED WITH VITAMIN B12 OR BIOTIN AND/ OR BILE ACIDS IN DIET Mohamed I. El-Katcha, Mosaad A. Soltan, Karima El-Naggar*, Set A. El-Shobokshy, Mohamed A. El-Erian Department of Nutrition and Veterinary Clinical Nutrition, Faculty of Veterinary Medicine, Alexandria University, 22758, Egypt Corresponding author: karima.muhammad@alexu.edu.eg; ORCID ID: 0000-0002-7847-7613 Abstract: For 8 weeks feeding trial, two hundred and seventy, 53 weeks old laying hens were used to investigate the effects of dietary supplementation of vitamin B12 or biotin and/or bile acids on performance, egg quality, fat digestibility and liver composition and histopathology. Birds were randomly divided into 6 groups (three replicates) and fed the experimental diets; group1 (G1) fed on the basal diet without additives as control while G2 and G3 supplemented with 0.02 ppm vitamin B12 and 0.3 mg biotin/kg diet respectively, groups 4-6 fed as the previous detailed design of G1 - G3 with the addition of 400 g of dried bile acid (DBA) /tone feed. Biotin supplementation significantly (P<0.05) increased body weight losses of laying hens, both vitamins significantly (P<0.05) decreased daily feed intake (FI) and improved FCR. DBA addition alone or with biotin reduced these weight losses along with significant (P<0.05) increase in daily FI. Vitamin B12 supplementation alone or with DBA increased average egg production (P<0.05) while was reduced with biotin supplementation. Fat digestibility was non-significantly improved (P>0.05) with both vitamins supplementation without or with DBA. Biotin significantly (P<0.05) reduced the average yolk relative weight, which was increased when mixed with DBA, while significantly increased average egg albumin relative weight. Vitamin B12 or biotin addition without or with DBA non-significantly increased blood serum GOT and GPT activities, non-significantly reduced (P>0.05) fat content of liver tissue (on dry matter or fresh basis) and serum lipid profile parameters except serum HDL concentration, was increased, with no histopathological changes in hepatic tissue. It could be summarized that vitamin B12 supplementation without or with dBa is recommended in layer diet as it gave the best performance, reduced serum lipid profile and improved fat digestibility and the hepatic health. Key words: layers; egg quality; liver; vitamin B12; biotin; bile acids Recently, laying hens facing many challenges influencing egg production industry resulting in significant economic losses. Such Received: January 2019 Accepted for publication: February 2019 342 M. El-Katcha, M. Soltan, K. El-Naggar, S. El-Shobokshy, M. El-Erian challenges as environmental changes, imbal-anced or poor-quality feed, frequent diseases affect the physiological condition of layers and bring out pathological burden to hen body especially to sensitive and important organs such as liver. Therefore, liver is in continuous overloaded condition, which disturbs its function. One of the common disorders influences liver in layers is the fatty liver hemorrhagic syndrome (FLHS), negatively affecting their performance. FLHS is a metabolic or nutritional disorder of layers characterized by excessive accumulation of fat depots in the hepatic tissue and abdominal cavity as liver become enlarged, pale and ends up with rupture and hemorrhage causing death of the affected birds (1).This syndrome occurs when the lipogenesis process exceeds the capacity of lipoprotein synthesis and secretion (2). The actual cause of FLHS has not been fully defined, but could be a combination of nutritional, genetic, environmental, and hormonal factors (3). Different studies have been done to follow up the actual factors inducing this problem in laying hens with the development of some managemental and nutritional approaches to reduce its occurrence. One of these strategies is dietary supplementation of some feed additives such as some lipotropic factors and antioxidants, which could control the lipid metabolism, reduce the production of free radicals and protect the liver from damage. Methionine and choline as lipotropic factors are used to treat or prevent this syndrome as their inadequate dietary levels increase oleic acid absorption and decreased triacylglycerol secretion (4, 5). Other nutrients deficiency such as vitamin B12 and vitamin E increased hepatic triacylglycerol accumulation in laying hens (6, 7), increasing the incidence of occurrence. Earlier studies reported that biotin deficiency is an important dietary factor involved in inducing fatty liver in birds (8, 9). Recently, the poultry production sector is continuously searching for new feed additives to improve feed efficiency and physiological condition of layers for optimal performance and egg production. Bile acids are natural components synthesized in human and animal body from cholesterol, which have a critical role in dietary fat digestion and absorption through the intestinal wall (10). Previous studies have been done investigating impacts of B-vitamins supplementation and emulsifiers as bile acids on broilers health and productivity (11-14), however, little literature is available regarding their effect on laying hens productive performance and egg quality parameters. We hypothesized that dietary inclusion of these additives could help in the digestion of fat, maintain the healthy condition of the liver of laying hens. Therefore, the main objectives of the present study to investigate the effects of vitamin B12 or biotin supplementation without or with dried bile acids on productive performance, egg quality, fat digestibility, serum biochemistry and liver histopathology of laying hens. Material and methods Birds, experimental design and feeding program Management procedures for all birds used during the course of experiment followed the guidelines approved by the Animal Care and Use Committee, Faculty of Veterinary Medicine, Alexandria University. A total of two hundred and seventy, 53 weeks old of Isa brown laying hens purchased from local company and used in this experiment. Hens were weighed separately, then randomly divided into 6 separate groups (45 bird /group) with three replicates each (15 bird/ replicate). Birds were kept in wire cages, and each cage was provided with feeder and water troughs. The control group (Group 1) was fed the basal diet (BD) without any additives, group 2 and 3 were fed the BD supplemented with 0.02 ppm vitamin B12 and 0.3 mg biotin/kg diet respectively, while group 4-6 were fed as the previous detailed design of the first three groups but, with the addition of 400 g bile acid/tone diet. BD were formulated to meet bird nutrient requirements according to NRC (15). BD composition and its chemical analysis is presented in table 1, which was analyzed according to AOAC (16). Additives used in this experiment included; vitamin B12 and biotin produced by Allgäu Vet. Co., China; the dried bile acids (DBA) (Shangdong Longchang Vitamin B12, biotin and bile acids supplementation in layer diet 343 Animal Health Product Co., Ltd, Jinan, China). All the experimental groups were fed the designated diets with free access to fresh water for 8 weeks experimental period. Hens were weighed individually at the beginning and end of experiment and the live body weight (BW) changes were taken with recording of feed intake (FI). Feed conversion ratio (FCR) was calculated based on feed consumed (g) and produced egg mass (g). Egg production and Egg quality Eggs produced from various laying hens were collected daily for calculating the average daily egg production % (EP) and egg weight (g) for each group for 14 days period for four successive periods. Egg mass was calculated by multiplying egg weight by egg production. At the end of the experiment, 20 eggs from each group were collected to estimate the egg quality parameters (yolk weight and relative weight to total egg weight, yolk index, albumin weight, and its relative weight, albumen index, shell thickness and weight as well as its relative weight were recorded) according to Card and Nesheim (17). Six samples of egg yolk from each group were collected for egg yolk cholesterol determination according to Rotenberg and Christensen (18). Fat digestibility During the last week of the feeding trail, the excreta were quantitatively collected for 5 successive days in addition to recording the amount of feed consumed. The excreta then dried in hot air oven, ground and stored until chemical analysis of fat. Ether extract of feed, fecal and liver samples was determined according to AOAC (16). Serum biochemical parameters Blood samples were collected (n= 6/group), then left to coagulate at room temperature. Separation of serum was done by centrifugation at 3000 rpm for 10 minutes. The clear serum samples were kept in freezer (-20 C) until analysis of the following blood serum indices; total cholesterol, high-density lipoprotein (HDL), low-density lipoprotein (LDL), triglyceride (TG), glutamic oxaloacetic transaminase (GOT) and glutamic pyruvic transaminase (GPT). All these parameters were determined by spectrophotom-eter using specific commercial kits produced by Biodiagnostic, Co. (Diagnostic and Research reagents). Liver Histopathology At the end of the experiment (61 week of hen age), three hens from each group (one hen from each replicate) were collected randomly and used for liver tissue collection. Samples were fixed in 10% formalin for at least 2 days then slides were prepared according to Bancroft et al. (19), and stained with Hematoxylin and Eosin (H&E) for general inspection. Statistical analysis Results were analyzed using analysis of variance (one-way ANOVA) using IBM SPSS Statistics 22 statistical package (SPSS Inc., Chicago, IL, USA) to measure the significant differences between the means of different variables. Differences among experimental groups were considered significant at (P<0.05). Results and discussion Layer Performance Biotin supplementation significantly (P< 0.05) increased BW losses of laying hens. Both vitamins significantly (P<0.05) decreased average daily FI, but, improved FCR compared with control. On the other hand, DBA addition alone or with biotin reduced these losses in BW along with significant (P<0.05) increase in daily FI compared with laying hens without DBA addition (table 2). Moreover, FCR was significantly improved with DBA inclusion with vitamin B12 and biotin compared with birds fed the same diet without DBA or control group. Unlike the obtained results, earlier studies examined the effects of vitamin B12 (20, 21), concluded that it had no clear effect on FI of commercial laying hens, while increased FI with Halle and Ebrahem (22). Improvement of FCR with vitamin B12 supplementation in the present study is in line with Halle and Ebrahem (22), while inconsistent with Kato et al. (20). Additionally, Abdel-Mageed and Shabaan (23) documented another different result as similar 344 M. El-Katcha, M. Soltan, K. El-Naggar, S. El-Shobokshy, M. El-Erian FI was obtained in laying hens groups fed bio-tin-supplemented diets while, improved FCR with increasing its level from 162.5 to 325.5 pg/kg. Additionally, Whitehead (24), found that biotin supplementation during lay did not have any beneficial effect upon FI and FCR of laying hens. Bile acids are the principal constituents of bile, playing a significant role in emulsification, digestion, and absorption of fat and lipid-solu-ble vitamins (25). Best FCR was obtained with birds fed BD contained DBA plus vitamin B12 (2.08), while the worst value with birds fed BD supplemented with DBA (2.49). Previous reports documented improvements of broiler performance (weight gain, higher FI and FCR) with the dietary inclusion of bile acids (13, 2628), improved FCR in quail laying hens (29). The variation in response may be related to different breeds considered in these trials. Regarding EP (table 2), vitamin B12 supplementation alone or with DBA increased average EP (P<0.05) with the highest EP (86.34%) in birds supplemented with the 2 additives suggesting that they could have a synergistic effect. On the other hand, biotin supplementation showed dissimilar result as it reduced EP (72.71%), even when added with DBA (78.22%), compared with control birds. Unlike the obtained result, Kato et al. (20) found that vitamin B12 supplementation had no effect on EP, attributing it to short experimental period. Additionally, Bunchasak and Kachana (30) reported that supplemental vitamin B12 didn't alter egg production or FI. However, Daryabari et al. (31) reported that 0.3g biotin/L water supplementation improved EP of breeding hens, as well as Abdel-Mageed and Shabaan (23) who found that biotin inclusion in diet enhanced it. Biotin concentration in the blood has been reported to be lower in young broiler breeder hens and increases linearly with age (32), therefore, the lower or unaffected EP in the biotin supplemented groups in the present study could be attributed to the higher level of biotin in birds. Response due to bile acids addition is inconsistent with the finding obtained with (29) as 1.2% supplemental ox bile addition in quail laying hens diet significantly improved EP %. Both vitamins supplementation non-signifi-cantly (P>0.05) reduced average egg weight (table 2). The same response was obtained when diet was supplemented with DBA alone or combined with vitamin B12 while, when DBA mixed with biotin showed slightly higher egg weight compared with birds fed on the same diet without DBA. On contrary, Kato et al. (20) found increased egg weight of laying hens with vitamin B12 supplementation. Fat digestibility Fat digestibility showed non-significant improvement (P>0.05) with vitamin B12 or biotin compared with control (table 2). The same result was obtained when DBA was supplemented alone or with both vitamins. Fat digestibility improvement especially in laying hens supplemented with DBA being the highest was supported by Alzawqari et al. (28), who found that supplemental increased levels of bile in diet linearly increased fat digestibility in broiler chicks. This improvement with the addition of DBA might be attributed to inadequate amount of bile salts secreted by bird, so more emulsification of fats occur by the exogenous bile acids or for replacement of the active catabolism of bile salts occur by gut microflora (26). Moreover, Lai et al. (12) indicated that supplementation with 60 and 80 mg/kg bile acids significantly increased the activity of intestinal lipase and lipoprotein lipase on day 21 and 42. The present study revealed a close relationship between dietary DBA supplementation and fat digestibility. Most of the previous studies reported, were concerned with investigation of the effects of these exogenous emulsifiers in broiler diet especially at younger age of chicks due to the physiological limitation of the digestive tract to produce endogenous emulsifiers. However, literature on their application in layers diets is scarce. Egg quality (External and internal quality) Average egg shell relative weight was non-significantly increased (P>0.05) with vitamin B12 or biotin compared with the reference group (table 3). DBA addition single or combined with vitamin B12 increased (P>0.05) egg shell Vitamin B12, biotin and bile acids supplementation in layer diet 345 relative weight, while, was reduced (P>0.05) when biotin was added to diet. Regarding egg shell thickness, no changes were found among different treatments. In support, Keshavarz (33) concluded that vitamin B12 supplementation without or with methionine improved egg shell quality, Rajalekshmy (34) reported that shell weight was decreased at higher levels of folic acid (4 ppm) when vitamin B12 was added (0.01 ppm). However, Kato et al. (20) found another different result as egg shell thickness and weight per unit of surface area were higher (p<0.01) for birds fed diet without vitamin B12 supplementation. In terms of internal egg quality (table 3), biotin supplementation significantly reduced (P<0.05) average egg yolk relative weight, but this effect was reversed when DBA was added with biotin. On the other hand, average egg albumin relative weight was significantly increased with biotin supplementation, though the lowest relative weight was recorded with birds received diet supplemented with DBA only. Regarding average yolk index, it was significantly reduced in birds supplemented with biotin and DBA, with no significant difference between the other experimental groups. The present data are in line with El-Husseiny et al. (35), who found that dietary supplementation with 0.01-0.02 ppm vitamin B12 did not have any significant effect on egg shell thickness, shell percentage, Haugh units and yolk index. Moreover, Whitehead (24), reported that albumen height was slightly improved with biotin supplementation. Additionally, Abdel-Mageed and Shabaan (23), showed that biotin inclusion at 325.5 pg/kg diet gave the highest increase in egg shape index, yolk index and Haugh unit. Furthermore, Soltan (29) concluded that ox bile addition in quail laying hens had no significant influence on internal and external egg quality. Egg yolk triglyceride and cholesterol content were non-significantly (P>0.05) reduced with BDA supplementation compared with birds fed the same diets without DBA addition. These finding could support that DBA has an important role in fat metabolism through enhancing its digestibility and utilization. The egg is a highly nutritious food for human consumption, however, its higher content of cholesterol associated with cardiovascular diseases restricts its consumption. So, previous studies were concerned with the production of egg with low cholesterol content through genetic selection; dietary interventions or using some drugs such as bile acids sequestrants in the diet of laying hens (36, 37). Serum biochemical parameters Blood serum lipid profile (table 4) was non-significantly altered with additives used. However, serum total cholesterol, TG, LDL, and VLDL concentrations were non-significantly (P>0.05) reduced with DBA addition single or in combination with vitamins used except for serum HDL concentration which was increased. The higher reduction was observed in birds supplemented with DBA. Serum TG, LDL, and HDL concentrations considered an important diagnostic indicators of lipid metabolism. The synthesis of adipose tissue, fat deposition, and formation of yolk in poultry is dependent on available serum TG. Most of fatty acids are produced in the liver and transported via LDL or chylomicrons for storage in adipose tissue as TG (2). On the other hand, HDL enhances the uptake and transport of cholesterol from extra hepatic tissues to hepatic tissue for catabolism (38), which was confirmed in the current study as it was non significantly increased especially with DBA addition. Therefore, these findings suggesting that the used feed additives (vitamin B12, biotin and DBA) could have a hypolipidemic effect by affecting lipid metabolism. On contrary, Alzawqari et al. (13), reported that dietary inclusion of DBA significantly increased serum components of cholesterol, TG, HDL and LDL concentrations at days 21 and 42 of age, however, serum cholesterol concentration was numerically decreased (28). Furthermore, Lai et al. (12) documented that bile acid inclusion in broiler diets showed no differences in serum TG, HDL, and LDL concentrations among all groups at days 21 and 42. Inconsistency in results between experiments maybe related to differences in breed, age of birds and feeding experimental design. 346 M. El-Katcha, M. Soltan, K. El-Naggar, S. El-Shobokshy, M. El-Erian Vitamin B12 or biotin supplementation without or with DBA non-significantly increased blood serum GOT and GPT activities compared with their reference birds (table 4). Serum GOT and GPT activity are traditional biochemical indices of liver function and are used clinically for diagnosis of liver injury. Yousefi et al. (5), documented that AST enzyme activity (GOT) could be used as diagnostic indicator for FLHS in laying hens. All liver function related parameters in the present study were within normal range (39), which could confirm that these supplemental additives in laying hen diet had no adverse effects on liver function. Liver composition and histopathology As presented in table 4, both vitamin supplementation without DBA addition non-signifi-cantly decreased liver moisture % while significantly increased (P<0.05) it when combined with DBA compared with their reference birds. On the other hand, the fat content of the liver tissues (on dry matter or fresh basis), was non-significantly reduced (P>0.05) with both vitamins supplementation without or with DBA. Fatty liver is associated with the reductions in egg production with poor egg shell quality (40), which is particularly a serious problem during the late period of laying. With the long-term accumulation of fat and various toxins in the hepatic tissue, hepatocytes are injured with impaired function, reducing absorption and utilization of fat-soluble vitamins which consequently affecting the assimilation and deposition of calcium resulting in poor egg shell quality. So, liver health is closely associated with egg production and egg shell quality. Dietary inclusion of bile acids enhances the clearance function of very low-density lipoproteins, which transports fat from liver to systemic tissues, and reduces fat deposition in hepatocytes. Additionally, it stimulates the thyroid hormone activity (41), increases energy expenditure, reducing excessive accumulation of fat inside the liver and other body tissues of laying hens. The examined liver of laying hens fed on the BD without any supplemental additives showed centrilobular vacuolation represented with clear vacuoles with round border consistent with fatty changes (Fig.1A). Most of the hepatic sinusoids were congested in addition to multifocal lymphocytic cells aggregation. Vitamin B12 or biotin supplementation markedly decreased these pathological vacuolations with nearly normal hepatocytes (Fig.2 B and C). DBA inclusion in the diet without or with vitamin B12 or biotin supplementation showed mild vacuolation of hepatocytes or even normal hepatocytes (Fig.1D, E, and F). The present findings are in line with Lai et al. (42), who concluded that liver of broiler chickens supplemented with bile acids was normal and no histological changes. Therefore, these findings revealed that these feed additives especially bile acids could help in maintaining liver health by reducing fat deposition in the hepatic tissue. Vitamin B12, biotin and bile acids supplementation in layer diet 347 Table 1: Ingredients composition and chemical analysis of experimental diet Ingredients % Yellow corn ground 56.83 Soybean meal (44% CP) 20.0 Corn gluten meal 5.0 Wheat bran 5.0 Vegetable oils (sunflower oil) 1.25 Ground lime stone1 9.80 Monocalcium phosphate2 1.30 Common salt 0.25 Vitamin premix 3 0.15 Mineral premix 4 0.15 Lysine5 0.02 Methionine6 0.10 Choline7 0.10 Mycotoxin adsorbent 0.05 Chemical analysis (%) Moisture 11.65 Crude protein 17.07 Ether extract 4.87 Ash 12.76 Crude fiber 4.43 NFE8 49.22 Calcium 3.72 Phosphorus 0.63 ME (Kcal/kg )9 2746.88 :Lime stone contains 37 % calcium and locally produced. 2Mono calcium phosphate contains 21% phosphorus and 17 % calcium. 3Vitamin premix: Each 1.5 kg contains: Vit. A (12000000 IU), vit. D (2000000 IU), vit. E (10 g), vit. K3 (2 g), vit. B1 (1 g), vit. B2 (5 g), vit. B6 (1.5 g), vit. B12 (10 g), nicotinic acid (30 g), pantothenic acid (10 g), folic acid (1g), biotin (50 mg), produced by Archar Daniels Midland Co., LL., USA. 4Mineral Premix: Each 1kg contain, Manganese 100000 mg, Zinc 600000 mg, Iron 30000mg, Copper 10000 mg, Iodine 1000 mg, Selenium 200 mg, Cobalt 100 mg. 5Lysine: 98% lysine hydrochloride, Shandong Longue Co., China. 6DL-methionine, Evonik Co. (guaranteed analysis 99.5% DL- methionine).7Cho-line: choline chloride 60 % with vegetable carrier (corn powder) produced by Shandong pharmaceutical Co., China. 8Nitrogen free extract was calculated by difference. 9ME were calculated according to NRC (1994). 348 M. El-Katcha, M. Soltan, K. El-Naggar, S. El-Shobokshy, M. El-Erian Table 2: Effect of dietary vitamin B12 or biotin supplementation without or with dried bile acids on performance and fat digestibility of laying hens Supplementation Parameters Control Vitamin B12 Biotin Control Vitamin B12 Biotin Without DBA1 With DBA Initial body weight (g/hen) 1851.82 1826.55 1815.66 1798.47 1844.27 1829.60 ±21.07a ±19.19a ±23.77a ±22.20a ±19.63a ±24.50a Final body weight (g/hen) 1827.74 1802.24 1731.11 1794.15 1815.27 1795.38 ±35.02a ±29.49ab ±31.14b ±30.66ab ±28.93ab ±26.79ab Weight changes (g/hen) -24.08 -24.31 -84.55 -4.32 -29.00 -34.22 ±20.02b ±16.06b ±19.26a ±17.40c ±17.35b ±10.38b Average Feed Intake 113.41 109.99 96.90 120.21 111.35 107.34 (g/hen/day) ±0.07b ±0.19d ±0.13f ±0.06a ±0.12c ±0.16e Average FCR 2.35 2.14 2.14 2.49 2.10 2.19 ±0.03b ±0.03c ±0.06c ±0.03a ±0.03d ±0.03c Average egg production 76.65 81.56 72.17 78.60 86.34 78.22 (%) 53 - 61 ±0.69bc ±0.74ab ±1.37c ±0.79b ±4.27a ±0.76b Average egg wt. (g) 62.83 62.74 62.48 61.38 61.75 62.65 ±0.22a ±0.15a ±0.15a ±0.16a ±0.14b ±0.14a Average egg mass (g/hen) 48.17 51.21 45.20 48.21 53.33 49.02 ±0.46bc ±0.50ab ±0.89c ±0.48bc ±2.63a ±0.50b Fat digestibility % 75.07 81.67 78.97 84.40 82.30 79.20 ±2.84a ±2.40a ±4.65a ±0.60a ±1.95a ±6.45a Values are means ± standard error. Means within the same row of different litters are significantly different at (P < 0.05).1 DBA: Dried bile acids Table 3: Effect of dietary vitamin B12 or biotin supplementation without or with dried bile acids on egg quality parameters Supplementation Parameters Control Vitamin B12 Biotin Control Vitamin B12 Biotin Without DBA1 With DBA External egg quality Average eggshell relative 12.86 13.25 12.90 13.43 12.97 12.23 weight ±0.49a ±0.20a ±0.39a ±0.34a ±0.13a ±0.29a Average eggshell thickness 38.78 37.56 37.04 37.19 37.94 37.33 (mm) ±0.36a ±0.47a ±0.70a ±0.64a ±1.42a ±0.94a Internal egg quality Average yolk relative wt. 29.99 29.82 29.22 30.06 30.74 31.47 ±0.89ab ±0.50ab ±0.66b ±0.58ab ±0.69ab ±0.76a Average yolk index 0.43 0.41 0.43 0.43 0.41 0.40 ±0.01a ±0.01ab ±0.00a ±0.00a ±0.01ab ±0.01b Average albumin relative wt. 57.15 57.17 58.24 54.84 56.51 55.17 ±0.60ab ±0.39ab ±0.64a ±2.02b ±0.50ab ±0.82ab Average albumin index 8.89 8.71 8.78 8.68 8.81 8.84 ±0.24a ±0.09a ±0.23a ±0.20a ±0.16a ±0.15a Yolk cholesterol (mg/g yolk) 9.27 8.93 10.11 8.85 8.85 8.89 ±0.41a ±0.89a ±1.44a ±0.25a ±0.33a ±0.34a (mg/whole yolk) 181.48 165.58 181.54 170.35 161.63 162.76 ±3.65a ±20.61a ±35.65a ±6.36a ±11.76a ±6.47a Yolk triglycerides (mg/g yolk) 10.11 10.31 9.74 9.75 9.95 9.56 ±0.49a ±1.55a ±0.13a ±0.52a ±0.66a ±0.48a (mg/whole yolk) 197.86 189.29 176.66 187.14 181.00 174.69 ±3.73a ±23.47a ±11.79a ±4.53a ±11.40a ±5.72a Values are means ± standard error. Means within the same row of different litters are significantly different at (P < 0.05). 1 DBA: Dried bile acids Vitamin B12, biotin and bile acids supplementation in layer diet 349 Table 4: Effect of dietary vitamin B12 or biotin supplementation without or with dried bile acids on blood serum parameters of laying hens Supplementation Control Vitamin B12 Biotin Control Vitamin B12 Biotin Without DBA6 With DBA Blood serum lipid profile parameters Cholesterol 198.15 191.23 196.30 195.53 190.69 190.77 (mg/dl) ±0.70 a ±6.12a ±2.37a ±3.43a ±2.31a ±2.75a Triglyceride 205.14 205.86 206.91 203.61 201.08 204.90 (mg/dl) ±1.14a ±3.62a ±1.70a ±3.06a ±2.29a ±0.58a HDL1 47.17 48.43 47.23 49.80 50.80 51.63 (mg/dl) ±2.06a ±2.10a ±1.57a ±1.42a ±2.60a ±1.49a LDL2 109.95 101.62 107.69 105.01 99.67 98.15 (mg/dl) ±1.13a ±5.46a ±3.61a ±3.24a ±0.75a ±3.06a VLDL3 41.03 41.17 41.38 40.72 40.22 40.98 (mg/dl) ±0.23a ±0.72a ±0.34a ±0.61a ±0.46a ±0.12a Blood serum parameters related to liver functions GOT4 (u/L) 65.33 96.33 71.33 94.00 82.50 77.00 ±17.9a ±3.18a ±16.51a ±2.08a ±12.50a ±10.50a GPT 5(u/L) 20.90 24.67 21.50 21.67 22.50 24.67 ±3.51a ±1.45a ±0.50a ±1.45a ±0.50a ±1.67a Liver tissue composition (%) Moisture 70.37 70.23 69.99 69.08 73.03 71.13 ±0.53ab ±0.43 ab ±2.54ab ±1.59b ±0.71a ±0.93 ab Fat (DM basis) 25.43 ±3.30a 22.78 ±2.47a 19.62 ±3.50a 22.86 ±4.18a 21.51 ±1.67a 23.61 ±0.99a Fat (fresh ba- 7.55 6.08 5.96 7.06 5.82 6.80 sis) ±1.05a ±0.81a ±1.27a ±1.72a ±0.59a ±0.12a Values are means ± standard error. Means within the same row of different litters are significantly different at (P<0.05). 1HDL: high-density lipoprotein, 2LDL: low-density lipoprotein (LDL), 3VLDL: very low-density lipoprotein, 4 GOT: glutamic oxaloacetic transaminase, 5GPT: glutamic pyruvic transaminase. 6 DBA: Dried bile acids 350 M. El-Katcha, M. Soltan, K. El-Naggar, S. El-Shobokshy, M. El-Erian Figure 1: Histopathological changes of hepatic tissue of laying hens fed on control (without DBA) showing centrilobular hepatic vacuolation with fatty changes (arrow) (A); or with DBA mild degree of hepatic vac-uolation consistent with fatty changes (arrow) (B); hepatic tissues of birds supplemented with vitamin B12 supplementation and without DBA or with DBA addition showing normal hepatocytes (C and D); Birds supplemented with biotin without or with DBA addition showing normal hepatocytes (E and F) Vitamin B12, biotin and bile acids supplementation in layer diet 351 Conclusion Under the conditions of the present study, vitamin B12 supplementation single or combined with dried bile acids is recommended in laying hen diet as it gave the best productive performance and improved fat digestibility, hepatic health and reduced the serum lipid profile. On the other hand, biotin supplementation is not recommended for laying hens except if combined with DBA. Conflict of interest All authors declared that no conflicts of interest, as they are responsible for the content and writing this article. References 1. Crespo R, Shivaprasad HL, Developmental, metabolic, and other noninfectious disorders, in: YME Saif (Ed.), Diseases of Poultry, Iowa State Press, Ames, Iowa, 2003, 1055-102. 2. Hermier D. Lipoprotein metabolism and fattening in poultry. J Nutr 1997; 127(5 Suppl): 805s--8s. 3. Hansen RJ, Walzem RL. Avian fatty liver hemorrhagic syndrome: a comparative review. Adv Vet Sci Comp Med 1993; 37: 451-68. 4. Rinella ME, Elias MS, Smolak RR, Fu T, Borensztajn J, Green RM. Mechanisms of hepatic steatosis in mice fed a lipogenic methionine choline-deficient diet. J Lipid Res 2008; 49(5): 1068-76. 5. Yousefi M, Shivazad M, Sohrabi-Haghdoost I. Effect of Dietary Factors on Induction of Fatty Liver-Hemorrhagic Syndrome and its Diagnosis Methods with Use of Serum and Liver Parameters in Laying Hens. Int J Poult Sci 2005; 4: 568-72. 6. Couch J. Fatty livers in laying hens - a condition which may occur as a result of increased strain. Feedstuffs 1956: 28-46. 7. Leeson S. Metabolic Challenges: Past, Present, and Future. J Appl Poult Res 2007; 16(1): 121-5. 8. Whitehead CC, Blair R, Bannister DW, Evans AJ, Jones RM. The involvement of biotin in preventing the fatty liver and kidney syndrome in chicks. Res Vet Sci 1976; 20(2): 180-4. 9. Whitehead CC, Bannister DW, Evans AJ, Siller WG, Wight PAL. Biotin deficiency and fatty liver and kidney syndrome in chicks given purified diets containing different fat and protein levels. Br J Nutr 1976; 35(1): 115-25. 10. Reshetnyak VI. Physiological and molecular biochemical mechanisms of bile formation. World J Gastroenterol 2013; 19(42): 7341-60. 11. Quarantelli A, Bonomi A, Righi F, et al. The effects of different levels of dietary biotin on the performances and on bone growth in the broiler. Ital J Anim Sci 2003; 2 (sup1): 453-5. 12. Lai W, Huang W, Dong B, et al. Effects of dietary supplemental bile acids on performance, carcass characteristics, serum lipid metabolites and intestinal enzyme activities of broiler chickens. Poult sci 2018; 97(1): 196-202. 13. Alzawqari MH, Al-Baadani HH, Alhidary IB, Al-Owaimer AN, Abudabos AM. Effect of taurine and bile acid supplementation and their interaction on performance, serum components, ileal viscosity and carcass characteristics of broiler chickens. S Afr J Anim Sci 2016; 46: 448-57. 14. Cengiz O, Hess JB, Bilgili SF. Dietary biotin supplementation does not alleviate the development of footpad dermatitis in broiler chickens. J Appl Poult Res 2012; 21(4): 764-9. 15. NRC, Nutrient requirements of poultry. 9th Ed. Washington, DC: National Academic Press, 1994. 16. AOAC, Official methods of analysis. (18th ed.) Washington, DC: AOAC: Association of Official Analytical Chemists, 2005. 17. Card LE, Nesheim MC, Poultry Production, 11th Edn. Phidelphia: Lea and febiger Press, 1972. 18. Rotenberg S, Christensen K. Spectro-photometric Determination of Total and Free Cholesterol in Egg Yolk and Animal Tissues. Acta Agric Sc and A 1976; 26(2): 94-8. 19. Bancroft JD, Layton C, Suvarna SK, Bancroft's Theory and Practice of Histological Techniques. 7th edition. Churchill Livingstone: Elsevier, 2013. 20. Kato R, Bertechini A, Fassani E, Santos C, Dionizio M, Fialho E. Cobalt and vitamin B12 in diets for commercial laying hens on the second cycle of production. Braz J Poult Sci 2003; 5: 4550. 21. Squires MW, Naber EC.Vitamin profiles of eggs as indicators of nutritional status in the laying hen: vitamin B12 study. Poult sci 1992; 71(12): 2075-82. 22. Halle I, Ebrahem M. Influence of Vitamin B12 and Cobalt on performance of laying hens Landbauforschung 2012; 3(62): 111-6. 352 M. El-Katcha, M. Soltan, K. El-Naggar, S. El-Shobokshy, M. El-Erian 23. Abdel-Mageed MAA, Shabaan SA. Effect of supplemental biotin on the performance of aged fayoumi hens and progeny performance Egypt Poult Sci 2012; 32: 895-8. 24. Whitehead CC. Biotin- New information on requirements and supplementation in poultry, In Proceedings of Roche symposium, London, 1980. 25. Russell DW, Setchell KDR. Bile acid biosynthesis. Biochemistry 1992; 31(20): 4737-49. 26. Maisonnier S, Gomez J, Bree A, Berri C, Baeza E, Carre B. Effects of microflora status, dietary bile salts and guar gum on lipid digestibility, intestinal bile salts, and histomo-rphology in broiler chickens. Poult sci 2003; 82(5): 805-14. 27. Parsaie S, Shariatmadari F, Zamiri MJ, Khajeh K. Influence of wheat-based diets supplemented with xylanase, bile acid and antibiotics on performance, digestive tract measurements and gut morphology of broilers compared with a maize-based diet. Br Poult Sci 2007; 48(5): 594-600. 28. Alzawqari M, Moghaddam HN, Kermanshahi H, Raji AR.The effect of desiccated ox bile supplementation on performance, fat digestibility, gut morphology and blood chemistry of broiler chickens fed tallow diets. J Appl Anim Res 2011; 39(2): 169-74. 29. Soltan MA. Effect of diet containing Nigella Sativa (Black seeds) and/or ox bile on growth and productive performance of Japanese quail. AJVS 1999; 15(3): 655-69. 30. Bunchasak C, Kachana S. Dietary folate and vitamin B12 supplementation and consequent vitamin deposition in chicken eggs. Trop Anim Health Prod 2009; 41(7): 1583. 31. Daryabari H, Akhlaghi A, Zamiri MJ, et al. Reproductive performance and oviductal expression of avidin and avidin-related protein-2 in young and old broiler breeder hens orally exposed to supplementary biotin. Poult sci 2014; 93(9): 2289-95. 32. Whitehead CC. Biotin intake and transfer to the egg and chick in broiler breeder hens housed on litter or in cages. Br Poult Sci 1984; 25(2): 28792. 33. Keshavarz K. Effects of reducing dietary protein, methionine, choline, folic acid, and vitamin B12 during the late stages of the egg production cycle on performance and egg shell quality. Poult sci 2003; 82(9): 1407-14. 34. Rajalekshmy PK, Effects of dietary choline, folic acid and vitamin B12 on laying hen performance, egg components and egg phospholipid composition(Ph.D.).University of Nebraska, Lincolin, 2010. 35. El-Husseiny OM, Soliman AZ, Omara II, El-Sherif HMR. Evaluation of Dietary Methionine, Folic Acid and Cyanocobalamin (B12) and Their Interactions in Laying Hen Performance. Int J Poult Sci 2008; 7: 461-9. 36. Mori AV, Mendonca JR Watanabe C. Effects of cholestyramine and lovastatin upon plasma lipids and egg yolk cholesterol levels of laying hens. Braz J Vet Res Anim Sci 2000; 37: 0000. 37. Ismail IB, Al-Busadah KA, El-Bahr SM. Effect of Dietary Supplementation of Canola Oil on Egg Production, Quality and Biochemistry of Egg Yolk and Plasma of Laying Hen. Int J Biol Chem 2013; 7: 27-37. 38. Miller NE, Nestel PJ, Clifton-Bligh P. Relationships between plasma lipoprotein cholesterol concentrations and the pool size and metabolism of cholesterol in man. Atherosclerosis 1976; 23(3): 535-47. 39. Marjanovic D, Kozic L, Petrovic M, Palic T, Litricin V. Serum transaminase activity in fowls. Vet Bull 1975; 45: 2890-6. 40. Trott KA, Giannitti F, Rimoldi G, et al. Fatty liver hemorrhagic syndrome in the backyard chicken: a retrospective histopathologic case series. Vet Pathol 2014; 51(4): 787-95. 41. Watanabe M, Houten SM, Mataki C, et al. Bile acids induce energy expenditure by promoting intracellular thyroid hormone activation. Nature 2006; 439(7075): 484-9. 42. Lai W, Cao A, Li J, Zhang W, Zhang L. Effect of High Dose of Bile Acids Supplementation in Broiler Feed on Growth Performance, Clinical Blood Metabolites, and Organ Development. J Appl Poult Res 2018; 27(4): 532-9. Slov Vet Res 2019; 56 (Suppl 22): 353-63 DOI 10.26873/SVR-774-2019 Original Research Article INFLUENCE OF VITAMIN E AND SELENIUM SUPPLEMENTATION ON THE PERFORMANCE, REPRODUCTIVE INDICES AND METABOLIC STATUS OF OSSIMI EWES Sherief M. Abdel-Raheem1*, G. B Mahmoud2, Waleed Senosy3, Taymour M. El-Sherry4 1Department of Animal Nutrition and Clinical Nutrition, Faculty of Veterinary Medicine, Assiut University, 71526, Egypt, department of Animal production, Faculty of Agriculture, Assiut University, department of Theriogenology, Faculty of Veterinary Medicine, New Valley University, 51714, Egypt, department of Theriogenology, Faculty of Veterinary Medicine, Assiut University Corresponding author, E-mail: sherief.diab@aun.edu.eg Abstract: The objective of this study was to investigate the effects of pre and post-lambing intramuscular (i.m.) injections of vitamin E (VE) and selenium (Se) on the performance, reproductive indices and metabolic status of Ossimi ewes. A total of 20 pregnant ewes were divided randomly into two equal groups of ten animals. The control group (CG) was administered 3 ml of normal saline. The treated group (TG) was administered a combination of 5 mg of sodium selenite and 450 mg of VE twice weekly for 2 weeks pre and post lambing. The performance, reproductive indices, colostrum and milk mineral profiles, blood metabolites and antioxidant enzyme biomarkers were investigated in this experiment. VE and Se supplementation had a significant effect on ewe, lamb performance, colostrum and milk mineral profiles. The treated ewes lost less weight (0.7 kg) compared with the ewes in the control group (3.68 kg) on 30th day postpartum (p<0.001). Lambs born to treated ewes were heavier than those born to control ewes (4.47 kg vs 3.99 kg, respectively), gained more daily weight quickly (268.66 vs. 226.00 g, respectively, P<0.001) and weighed more on 30th day post-lambing (12.53 vs. 10.77 kg, respectively, P<0.001). Treated ewes had higher concentrations of blood glucose, total protein, total cholesterol, antioxidant enzyme biomarkers, and lower concentrations of serum non-es-terified fatty acids (NEFA). Ve and Se supplementation had a significant effect (P<0.05) on postpartum reproductive performance of ewes in terms of fast uterine involution, earlier resumption of ovarian function and ovulation with large size ovulatory follicles, and greater numbers of small, medium, and large-sized follicles. In conclusion, VE and Se supplementation during late gestation and early lactation could be considered an effective strategy for minimizing the weight loss of ewes, enhancing lamb performance, the antioxidant and metabolic status, which improves the reproductive performance of Ossimi ewes. Key words: vitamin E; selenium; lamb performance; reproductive performance; antioxidant status; Ossimi ewes Introduction Over the last decade, ewe fertility has been progressively deteriorating around the world. Received: January 2019 Accepted for publication: February 2019 354 S. Abdel-Raheem, G. Mahmoud, W. Senosy, T. El-Sherry Improving productivity by improving the peri-partum period has become an important problem. Ewes are often deficient in certain trace elements and vitamins important for lambing because they graze on dry pastures of poor-quality during pregnancy, which can influence lamb survival, birth weight, subsequent poor animal production and welfare (1). Vitamins and minerals play an important role in the growth, physiology, and reproductive performance of animals. Se deficiency is linked to several reproductive disorders of cattle including abortion, retained placenta, neonatal weakness, metritis, poor uterine involution, cystic ovaries, impaired fertilization of ova, impaired fertility, and repression of immunity (2, 3). Selenium (Se) and vitamin E (VE) both prevent oxidative degeneration of biological membranes. Vitamin E and the Se-containing enzyme glutathione peroxidase (GSHpx) are critical components of the antioxidant system that functions in all cells (4). Vitamin E and Se are integral parts of the antioxidant defense system and participate in critical enzymatic reactions, which directly influence growth performance, function of the immune system, and reproductive performance of animals (5, 6). Newborns are likely to experience VE deficiency; due to the insignificant amount of VE transmitted to the fetus in the uterus, it is important that colostrum must supply enough VE to the lamb (7). Ewes that were supplemented with VE and Se during late gestation gave birth to heavier lambs with reduced mortality as compared to lambs born to non-supplemented ewes (8, 9). Although fresh grass contains high amount of VE, the amount of VE decreases during storage and conservation and depends on forage species and time of harvest (7). The previous National Research Council (NRC, 1985) (10) recommendation of 15 IU of VE/kg dry matter (DM) was increased by approximately 17-times in the NRC's most recent set of recommendations (2007) (11) to increase the immunocompetence of animals. However, it has not been fully documented that VE and Se supplementation of ewes has a beneficial synergistic effect on reproductive and productive lamb performance. Therefore, the objective of this study was to investigate the effect of pre and post lambing intramuscular (i.m.) injections of VE and Se on the performance, reproductive indices and metabolic status of Ossimi ewes Material and methods The study was performed according to the regulations and procedures approved by the Ethics Committee on Animal Experimentation of Assiut University, Faculty of Veterinary Medicine and the Guide for the Care and Use of Laboratory Animals (National Institute of Health publication no. 8023, revised 1978). Experimental site The study period was the winter breeding season from December to February. The ambient temperature ranged from 21.6-26 °C while the relative humidity was ranged from 50-54%. Ewes were kept indoors at the Research and Production Farm, Department of Animal Production, Faculty of Agriculture, Assiut University (latitude 28 ° 07'N and 30° 33'E). Animals, management, and treatments A total of 20 pregnant Ossimi ewes (identified by ultrasonography) were used of nearly similar age (4-5 years), with 1-2 parity and their average weight of 53.86 kg. Two weeks prior to anticipated lambing, healthy ewes clinically free from external and internal parasites were selected for the present study. Each ewe had free access to food and water and was maintained in an individual pen. Ewes were fed a late pregnancy diet (Table 1) until a parturition lactation diet was offered. Ewes were divided into two equal groups of 10 animals. The control group (CG) received no supplementation and administered 3 ml of normal saline. The treated group (TG) received an injection of VE and Se (3 ml i.m. from Viteselen 15®, Adwia Company, Egypt). Each ewe in the TG was injected with 5 mg of sodium selenite and 450 mg VE twice weekly for two weeks pre and post lambing; lambing occurred over a two week period. Food was mixed daily and ewes were fed two times a day. All nutrients met or exceeded the requirements for 60 kg ewe at the last four weeks of gestation and ewes at the first 6-8 weeks of lactation, according to guidelines Selenium and vitamin E on productive and reproductive performance in ewes 355 from the National Research Council for sheep (NRC, 1985). The late pregnancy diet consisted of 70 % Berseem hay and 30 % concentrate mixture. The lactation diet consisted of 40 % Berseem hay and 60 % concentrate mixture along with wheat straw offered ad libitum (Table 1). For both groups, the experimental diet contained approximately 0.38 ppm Se and 23 mg VE/kg diet. Two meals of equally allotted feed were given to all ewes at 08:00 and 17:00 and leftover feed was removed and weighed. The amount of feed consumed daily was recorded and representative samples were subjected to chemical analysis. Ovarian and uterine examinations Sonographic examinations were performed on postpartum days 2, 4, 6, 8, 11, 14, 17, and 25 using a real-time B-mode ultrasound scanner (Pie Medical, 100 LC, Holland) with a line array transducer of 5.0 MHz. Ovarian examination was carried out by ultrasonography to monitor follicular growth, ovulation, and corpus lu-teum formation. Uterine involution was investigated by measuring the maximal transversal cross-sectional diameter and the lumen diameter of both the right and left uterine horns. Furthermore, the presence and echogenicity of uterine cavity contents were included in this investigation. Entire follicles of 3 mm in diameter or greater were recorded. The term wave was defined as one or more antral follicles growing 3 to > 5 mm in diameter before regression. Blood sampling and estimation of biochemical parameters and steroid hormones Blood samples were collected from the jugular vein parallel to sonographic examination. Duplicate blood samples were collected per animal, which were subjected to centrifugation at 3000 rpm for 20 min. Following centrifugation, plasma was harvested and stored at -20 °C prior to testing. A colorimetric method was used to evaluate the blood metabolites (glucose, total protein, triglycerides, total cholesterol, AST, and ALT) using kits from Spinreact ® Company (Spain). A direct ELISA was used to determine concentrations of progesterone (P4) (Diagnostic System Laboratory Co. (DSL), USA, catalogue no. 3900). The intra- and inter-assay coefficient of variation for estrogen and progesterone was 3.6% and 12.43%, respectively. The sensitivity of the assay for P4 was 0.12 ng. Determination of oxidant and antioxidant levels The levels of the oxidant (malondialdehyde (MDA); catalog no. MAK085, Sigma-Aldrich, USA), the antioxidant (reduced glutathione (GSH); catalog no. RS505, Randox Laboratories Ltd, UK) and total antioxidant capacity (TAC) (catalog no. NX2332, Randox Laboratories Ltd, UK) were determined by a colorimetric method using kits according to the instructions provided by the manufacturer's (Sigma-Aldrich, USA and Randox Laboratories Ltd, UK). Plasma and milk concentrations of trace elements A flame atomic absorption spectrophotome-ter (Shimadzu, AA-7000, Japan model) was used to measure Cu and Zn levels in colostrum and milk according to the procedure described by El-Bahr and Abdelghany (12). A graphite furnace (GFA-EX7) atomic absorption spectro-photometer (Shimadzu, Koyoto, Japan) was used to measure Se. Sample digestion was performed according to Official Methods of Analysis of AOAC (13). The graphite furnace program for measuring Se by GFA-EX7 and instrument settings were similar to those previously described by Meligy (14). Statistical analysis SPSS software (SPSS analytical program for windows version 16; SPSS) (15) was used to conduct the statistical analyses. Independent t-test was used to detect the significant differences in the treatment means of estrus onset, es-trus duration, time of ovulation, emergence of ovulatory follicles, maximum size of ovulatory follicles, and blood metabolites. The effect of time on each variable in each group was evaluated using analysis of variance with repeated measurements of the general linear model using the Bonferroni multiple comparison test. Prob- 356 S. Abdel-Raheem, G. Mahmoud, W. Senosy, T. El-Sherry ability values less than 0.05 (P <0.05) were considered significant. Results are presented as mean ± SE. Results Lamb and ewe performance There was a significant improvement in lamb performance of TG in terms of heavier birth weight and higher weight gain at 30 days post-partum (Table 2). Similarly, there was a significant improvement in ewe body weight of TG at the time of lambing and 30 days post-partum. Postpartum uterine involution The rate of uterine involution was significantly faster (P<0.05) in the TG compared with the control group at 6 and 11 days post-lambing (Figure 2). Uterine diameter was smaller in the TG than that of the CG on days 4, 6, 8, and 11 postpartum while there were no differences between both groups on day 14, 17, 20, and 25 postpartum (Figure 2). Postpartum ovarian resumption The first ovulation and ovarian resumption occur earlier in TG compared with CG. As confirmed by the recorded first elevation in progesterone (P4) postpartum that was coincided with ultrasonic lutein tissue on the ovarian surface was at 16.7 ± 1.0 days in treated ewes, which was significantly earlier (p<0.01) compared with the CG (25.7 ± 1.0 days). This result indicates that the first ovulation and ovarian resumption occurred earlier in the treated group compared with CG. The size of the first ovulatory follicle was larger (P<0.05) in the TG (6.6 ± 0.02 mm) in comparison with the CG (5.8 ± 0.03 mm). Moreover, progesterone concentration at that time was 1.4 ± 0.08 ng/ml and 1.1 ± 0.07 ng/ml for the TG and CG, respectively. The number of small follicles was higher (P<0.05) in the TG at different times during the postpartum period (Fig. 2, A). The overall number of small follicles during the study period was higher (P<0.001) in the TG (Fig. 2, D). The number of medium sized follicles fluctuated during the study period but was higher in the TG (Fig 2, B). In addition, the overall number of medium-sized follicles was high (P<0.01) in treated ewes compared with the CG (Fig. 2, D). The number of large follicles was higher in the TG (P<0.05) during different intervals of the postpartum period (Fig. 2, C). However, the overall number of large follicles did not differ between both groups during the study period (Figure 2, D). Metabolic and biochemical profiles The concentration of glucose in blood (mg/l) was higher in the treated ewes (P <0.05) in comparison with the control during the early postpartum period until day 20 postpartum (Figure 3, A). Similarly, total protein (g/l) was higher in the TG (P<0.05) on day 2, 4, 8, 14, 17, and 20 postpartum. Furthermore, the concentration of total cholesterol (mg/dl) was higher in the treated ewes during the postpartum period of the study. The concentration of non-esterified fatty acids (NEFA; mmol/l) significantly elevated (P<0.05) in the CG during the postpartum period except at day 6 postpartum as compared to the CG. The concentration of triglycerides (mg/dl) was higher (P<0.05) at day 2 and day 6 postpartum than control while it lowered during days 17, 20 and 25 postpartum in the treated ewes. Antioxidant enzyme biomarkers and selenium concentrations The MDA concentration (mmol/l) was higher (P<0.05) in control ewes compared with the treated ones during the postpartum period (Table 3). The blood concentration of TAC was significantly higher (P< 0.05) in the TG at days 2, 4 and numerically not statistically on days 14, 17, 20, and 25 compared to the CG. In addition, plasma levels of reduced GSH (U/g Hb) were significantly higher on days 4, 14, 17, 20, and 25 in the TG compared with the CG. The concentration of Se (pmol/L) in serum was significantly higher in the TG on days 2, 4, 14, 17, 20, and 25 compared with the CG. Trace element profile in colostrum and milk The data exhibited in table 4 revealed that the concentrations (ppm) of copper and Se in colostrum and milk were significantly (p<0.05) higher in the TG. However, the concentration Selenium and vitamin E on productive and reproductive performance in ewes 357 of zinc (ppm) in the colostrum and milk harvested from treated ewes was decreased signif icantly (P<0.05) than those of CG. Table 1: Ingredients and analyzed chemical composition of experimental diets during pregnancy and early lactation (as fed basis) Item Late pregnancy diet Early lactation diet Ingredient, % Concentrate mixture 30 60 Berseem hay 70 40 Wheat straw Ad libitum Ad libitum Total 100 100 Concentrate mixtures, % Ground yellow corn 75.7 75.7 Soybean meal (44 %) 3 15 Wheat bran 19 7 Vitamin and mineral Premix* 0.3 0.3 Limestone 1.5 1.5 Salt 0.5 0.5 Total 100 100 Chemical composition (%, unless stated) Dry matter 87.67 87.42 ME Mcal/head/day * * 3.67 5.47 Crude protein 13.00 14.00 Crude fiber 20.11 12.81 Crude Fat 2.46 2.38 Nitrogen free extract 41.02 49.74 Ash 11.08 8.49 Organic matter 76.59 78.93 Calcium 1.00 0.84 Phosphorus 0.30 0.30 Selenium, mg/kg 0.38 0.38 Vitamin E, mg/kg 23.00 23.00 *Vitamin and mineral premix each 3 kg contain: 1250000 IU Vit. A; 2500000 IU Vit. D3; 1000 mg Vit E; 80000 mg Mn; 60000 mg Zn; 50000 iron, 20000 copper, 5000 iodine, 250 Se, 1000 Co mg tell 3 kg caco3. **ME, metabolizable energy calculation based on NRC tables for sheep (1985). Table 2: Lamb and ewe performance of control and treatment groups (means ± SE) Item CG TG P Lamb performance Number of lambs/ewe 1.5 ± 0.17 Lamb birth weight, kg 3.99 ± 0.05b Lamb weight, kg (30 day post-lambing) 10.77 ± 0.04b Total weight gain, kg 6.78±0.06b Daily weight gain, g 226.00±1.84b 1.6 ± 0.16 4.47 ± 0.03a 12.53 ± 0.06a 8.06±0.07a 268.66±2.28a 0.67 <0.001 <0.001 <0.001 <0.001 Ewe performance DMI g/day (late pregnancy) Ewe BW, kg (d 130 pregnancy) Ewe BW, kg (d 140 pregnancy) DMI g/day (early lactation) BW after lambing (d 1) BW after lambing (d 30) Weight loss, kg 1699.92 ± 3.68 53.86 ± 0.11 54.86 ± 0.12 2296.74 ± 6.02 44.86 ± 0.14b 41.18 ± 0.25b -3.68 ± 0.23 1695.85 ± 3.88 54.02 ± 0.09 55.02 ± 0.1 2303.38 ± 5.60 46.02 ± 0.15a 45.32 ± 0.13a -0.70 ± 0.05 0.46 0.29 0.30 0.43 <0.001 <0.001 <0.001 *Means bearing different superscript letters in the same raw differ significantly (P < 0.05). BW, body weight; DMI, dry matter intake; d, day 358 S. Abdel-Raheem, G. Mahmoud, W. Senosy, T. El-Sherry Table 3: Activities of antioxidant enzymes and selenium concentrations of treated and control groups (means ± SE) Group TAC, mmol/L GSH U/g Hb MDA, ^mol/L Selenium, umol/L CG 42.33±3.28b 206±1.73b 15.44±0.18 a 0.96±0.04b Day 2 TG 73.81±3.26a 312.24±9.8a 14.27±0.09 b 1.74±0.05 a P 0.03 0.13 0.004 <0.001 CG 39.00 ± 3.6b 204.00±0.58b 15.4±0.12a 0.91±0.04b Day4 TG 70.63 ± 3.5a 592.33±1.45a 14.63±0.09 1.71±0.02 a P 0.03 <0.001 0.006 0.001 CG 70.91±5.75 205.67±1.2b 13.17±0.12a 0.97±0.07 b Day 14 TG 72.49±11.65 593.00±1.53a 11.90±0.26b 1.72±0.04b P 0.9 <0.001 0.01 0.001 CG 70.91±5.75 201.33±1.76b 10.8±0.17a 1.01±0.03b Day 17 TG 72.49±11.65 594.21±8.17a 9.53±0.22b 1.75±0.05a P 0.9 0.001 0.01 <0.001 CG 70.91±5.75 204.00±1.15b 8.33±0.09 a 1.00±0.06b Day 20 TG 72.49±11.65 595.33±0.88a 7.7±0.06b 1.84±0.02a P 0.9 <0.001 0.004 <0.001 CG 70.91±5.75 196.0±1.73b 7.4±0.15a 1.19±0.07b Day 25 TG 72.49±11.65 563.33±1.76a 6.73±0.09b 3.48±0.26a P 0.9 <0.001 0.02 0.001 *Means with different superscripts (a, b) in the same column (for each day separately) differ significantly (P<0.05). TAC, Total antioxidant capacity; MDA, Malondialdehyde; GSH, Glutathione peroxidase Table 4: Mineral profile (ppm) of colostrum and milk of control and treatment groups (means ± SE) _Item_CG_TG_P Colostrum Cu, 0.51 ± 0.03b 0.94±0.09 a 0.01 Se 0.08 ± 0.003a 0.17±0.001a <0.001 Zn 13.94±0.07a 10.59 ± 0.06b 0.02 Milk Cu 0.49 ± 0.05 b 1.14±0.22 a 0.04 Se 0.06 ± 0.003b 0.13±0.02 a 0.02 Zn 4.98 ± 0.24a 3.64±0.27 b 0.01 *Means bearing different superscript letters in the same raw differ significantly (P < 0.05) Figure 1: Pattern of uterine involution in treated and control group during postpartum period Selenium and vitamin E on productive and reproductive performance in ewes 359 —□—control SF —O—Treamtent SF « 5 el lc = 4 ol e 1 2 I 1 0 -1 2 4 6 8 11 14 17 20 25 Days postpratum A e -Q 5 4,5 4 3,5 3 2,5 2 1,5 1 0,5 0 —□—Control MF —O—Treatment MF 2 4 6 8 11 14 17 20 25 Days postpartum 3,5 3 wi el ■M 2,5 lol M- •5 2 r e £ 1,5 3 z 1 0,5 0 -0,5 -1 —□—Control LF —O—Treatment LF 14 12 10 e -Q m 4 6 8 11 14 17 20 25 Days postpartum □ Control group □ Treatment group MJ SF(<3mm) MF(3-5mm) LF(> 5mm) 3 B 8 6 4 2 0 C Figure 2: A, Number of small follicle (SF); B, medium follicle (MF); C, large follicle (LF); D, Overall number of small, medium and large follicles in treated and control ewes during postpartum period. Means are significantly different between groups in all parameters in comparison (P <0.05) 360 S. Abdel-Raheem, G. Mahmoud, W. Senosy, T. El-Sherry 68 66 ^ 64 ra 62 & 60 a) g 58 o O 56 54 52 ns Control group Treated group A 2 4 6 8 11 14 17 20 25 Days postpartum TJ CT E o r a) t s .Si o .c o 80 75 70 65 60 55 50 45 40 —O—Control group —O—Treated group 2 4 6 8 11 14 17 20 25 Days postpratum 8 7,8 7,6 7,4 7,2 7 6,8 6,6 6,4 ns ns ns Control group Treated group 2 4 6 8 11 14 17 20 25 Days postpartum o m m < u_ m 1,9 1,8 1,7 1,6 1,5 1,4 1,3 1,2 1,1 1 ns 2468 Days postpartum ^^ Control group —O—Treated group 11 14 17 20 25 D * B * C ct m s a) T3 'C a) o > 19 18,5 18 17,5 17 16,5 16 15,5 15 Control group Treated group 2 4 6 8 11 14 17 20 25 Days postpartum E Figure 3: Concentration of blood glucose (A), total proteins (B), total cholesterol (C), non-esterified fatty acids, NEFA (D) and triglycerides (E) in control and treated group during postpartum period (P < 0.05). Discussion Previous studies have suggested that supplementation with VE and Se can improve the function of the immune system, reproductive performance, and growth performance of goats and can increase the survival of Merino lambs (1, 5, 16). The improved productivity of Ossimi ewes was consistent with the previous studies in terms of higher lamb numbers, heavier birth Selenium and vitamin E on productive and reproductive performance in ewes 361 weight, and higher body weight gain on day 30 in the TG (9, 17). These studies showed that the supplementation or inclusion of VE and Se in the diet of the mother can improve the lamb performance. Lambs reared by ewes supplemented by VE and Se in the late gestation period showed better weight gain and heavier weaning weight. An explanation for that, VE and selenium supplementation increased their concentrations in colostrum and milk, which improve the immune function, feed efficiency and daily weight gain of suckling lambs (18, 19). The rate of uterine involution was significantly faster (P<0.05) in the TG in comparison to control ewes. This result is in agreement with the previous observations of Kiracofe (20) who reported that the diameter of uterine horns returned to their non-pregnant size by 21 days postpartum in ewes and that the uterus was similar in size to the non-pregnant uterus by the day 25. The earlier first ovulation with large-sized ovulatory follicles with large numbers of small, medium, and large-sized follicles in the treated ewes indicated the earlier resumption of ovarian activity and agreed with the results of previous investigations (9, 21). The Role of VE in the development of follicles and ovarian activity is unknown, but ovulation-induced oxidative damage to the ovarian epithelium of ewes can be prevented by supplementation of VE (22). Selenium is important for formation of the selenoprotein glutathione peroxidase 1(GPx1) which plays an important role in female reproductive function; i.e., involved in determining the follicle growth, maturation, and dominance in both cows and women (23). Potential role of antioxidant like vitamin E and Se in follicle dominance are protecting the dominant follicle from increasing levels of reactive oxygen species (ROS) (23). The significant increase of Se in serum and selenium and copper in colostrum, and milk is in agreement with earlier studies (24, 25). These previous studies indicated that VE and Se supplementation resulted in increasing the levels of VE and Se in the blood and consequently in the colostrum and milk of ewes during lactation. On the other hand, significant differences (P<0.05) in blood biochemical parameters were observed between the TG and the CG. Ewes showed a significant increase (P<0.05) in the levels of plasma total protein, glucose, total cholesterol, and a low level of NEFA after VE plus Se supplementation during the late gestation and suckling periods. These results consistent with previous studies (26, 27). A higher level of glucose (relative to control ewes) following the supplementation of Se and VE was observed in previous studies (28, 29). Although, Se and VE directly affect the metabolism of carbohydrates, it is not clear whether increased glucose level directly or indirectly through increasing thyroxin and triiodothyro-nine hormones in supplemented animals as reported in previous studies (26). In the pregnant ewes, the increased concentrations of total protein and albumin following VE and Se supplementation were reported by previous studies (28, 29). The exact mechanism that leads to a higher total protein in the serum of the TG was not elucidated in these studies but increased concentrations of y globulin could be a part of the mechanism. Another supporting research to our findings reported that the concentration of blood metabolites and the total antioxidant status in plasma were elevated in sheep received Se and VE supplements (27, 30). In this study, significant increase of GSH-Px and TAC was noticed after VE and Se supplementation compared with the control. This indicates that the maternal supplementation of Se and VE during late pregnancy and early lactation can improve the antioxidant status of Ossimi ewes. The increased levels of NEFA in CG were nearly similar with the findings observed by Avci and Kizil (31) in transition cows and are indicative of the increasing depletion of lipid reserves existed because of the increased energy demand. Also, the increased NEFA in the CG indicates the utilization of fat storage reserves to meet the rapid and dramatic energy responses in the ewes of this group. It can therefore be assumed that VE and Se supplementation may improve the energy balance in lactat-ing ewes and decrease the weight loss during lactation. 362 S. Abdel-Raheem, G. Mahmoud, W. Senosy, T. El-Sherry Conclusion Supplementation of pregnant ewes with VE and selenium (two weeks pre and post-lambing) had a significant effect (P<0.05) on the postpartum reproductive performance of ewes in terms of higher uterine involution, earlier ovarian resumption and first ovulation with large size ovulatory follicle, higher numbers of small, medium and large sized follicles. Furthermore, VE and Se supplementation had improved ewe and lamb performance, colostrum and milk mineral profiles, the metabolic and antioxidant status of Ossimi ewes. In conclusion, during the periods of late gestation and early lactation, VE and Se supplementation could be considered an effective strategy for minimizing the weight loss of ewes, enhancing lamb performance, the antiox-idant and metabolic status, which improves the reproductive performance of Ossimi ewes. Conflict of interest The authors declare that they have no conflict of interest Acknowledgments The authors would like to thank the staff of the Research and Production farm, Department of Animal Production, Faculty of Agriculture, Assiut University for their support and help during running the experiment. Author contributions S. M. A. designed the experiment, shared in running the experiment, sample collection and analysis, carry out statistical analysis of the data and wrote the manuscript. G.B. M. shared in running the experiment, sample collection and analysis and shared in revising the manuscript. W.S. shared in running the experiment, writing and revising the manuscript. T. M. E shared in sample collection, performed ultrasound measurements and revised the manuscript. All authors read and approved the final manuscript. References 1. Sterndale S, Broomfield S, Currie A, Hancock S, Kearney GA, Lei J, Liu S, Lockwood A, Scanlan V, Smith G, Thompson AN. Supplementation of Merino ewes with vitamin E plus selenium increases a-tocopherol and selenium concentrations in plasma of the lamb but does not improve their immune function. Animal 2018; 12(5):998-1006. 2. Harrison JH, Hancock DD, Conrad HR. Vitamin E and Selenium for Reproduction of the Dairy Cow1, 2. Journal of Dairy Science 1984; 67(1): 12332. 3. Pieczynska J, Grajeta H. The role of selenium in human conception and pregnancy. Journal of Trace Elements in Medicine and Biology 2015; 29:31-8. 4. Scherf H, Machlin LJ, Frye TM, Krautmann BA, Williams SN. Vitamin E biopotency: Comparison of various 'natural-derived'and chemically synthesized a-tocopherols. Animal Feed Science and Technology 1996; 59(1-3):115-26. 5. Aghwan ZA, Sazili AQ, Kadhim KK, Alimon AR, Goh YM, Adeyemi KD. Effects of dietary supplementation of selenium and iodine on growth performance, carcass characteristics and histology of thyroid gland in goats. Animal Science Journal 2016; 87(5):690-6. 6. Maraba KP, Mlambo V, Yusuf AO, Marume U, Hugo A. Extra dietary vitamin E-selenium as a mitigation strategy against housing-induced stress in Dohne Merino lambs: Effect on growth performance, stress biomarkers, and meat quality. Small Ruminant Research 2018; 160:31-7. 7. McDowell LR, Williams SN, Hidiroglou N, Njeru CA, Hill GM, Ochoa L, Wilkinson NS. Vitamin E supplementation for the ruminant. Animal Feed Science and Technology 1996; 60(3-4):273-96. 8. Kott RW, Thomas VM, Hatfield PG, Evans T, Davis KC. Effects of dietary vitamin E supplementation during late pregnancy on lamb mortality and ewe productivity. Journal of the American Veterinary Medical Association. 1998; 212(7):997-1000. 9. Koyuncu M, Yerlikaya H. Short Communication Effect of selenium-vitamin E injections of ewes on reproduction and growth of their lambs. South African Journal of Animal Science 2007;37(4):233-6. 10. NRC, National Research Council (US). Nutrient requirements of sheep. Sixth revised Edition, The National Academy Press, Washington, DC, 1985: 99. 11. NRC, National Research Council (US), Nutrient Requirements of Small Ruminants: Sheep, Goats, Cervids, and New World Camelids", The National Academies Press, Washington, DC", 2007. 12. El-Bahr SM, Abdelghany A. Heavy metal and trace element contents in edible muscle of three commercial fish species, and assessment of possible risks associated with their human consumption in Selenium and vitamin E on productive and reproductive performance in ewes 363 Saudi Arabia. Journal of Advanced Veterinary and Animal Research. 2015; 2(3):271-8. 13. AOAC. f International. 16th ed., vol. 1 (Cun-nif, P. Ed.), AOAC Int., Arlington, Virginia, USA., 1995. 14. Meligy AM. Comparative study of element contents in seven isolates of entomopathogenic nematodes. Egyptian Journal of Biological Pest Control 2018; 28(1):1. 15. SPSS. Statistical Packages for the Social Sciences. Statistical software for windows version 16.0, Microsoft, Chicago, IL., USA, 2007. 16. Song YX, Hou JX, Zhang L, Wang JG, Liu XR, Zhou ZQ, Cao BY. Effect of dietary selenome-thionine supplementation on growth performance, tissue Se concentration, and blood glutathione pe-roxidase activity in kid boer goats. Biological Trace element Research 2015; 167(2):242-50. 17. Rosales Nieto CA, Meza-Herrera CA, Ce-dillo FD, Najera MD, Vázquez HG, Pérez FD, Liu S. Vitamin E supplementation of undernourished ewes pre-and post-lambing reduces weight loss of ewes and increases weight of lambs. Tropical Animal Health and Production 2016; 48(3):613-8. 18. Macit M, Aksakal V, Emsen E, Esenbuga N, Aksu MI. Effects of vitamin E supplementation on fattening performance, non-carcass components and retail cut percentages, and meat quality traits of Awassi lambs. Meat Science 2003 May 1; 64(1): 1-6. 19. Moeini MM, Jalilian MT. Effect of selenium and vitamin E injection during late pregnancy on im- mune system and productive performances of Sanjabi ewes and their lambs. Global Journal of Animal Scientific Research 2014; 2(3):210-9. 20. Kiracofe GH. Uterine involution: its role in regulating postpartum intervals. Journal of Animal Science. 1980; 51(suppl II):16-28. 21. Musa SI, Bitto II, Ayoade JA, Oyedipe OE. Effects of Vitamin E and Selenium on Fertility and Lamb Performance of Yankasa Sheep. Open Journal of Veterinary Medicine 2018; 8(09):167-73. 22. Murdoch WJ, Martinchick JF. Oxidative damage to DNA of ovarian surface epithelial cells affected by ovulation: carcinogenic implication and chemoprevention. Experimental Biology and Medicine 2004; 229(6):546-52. 23. Ceko MJ, Hummitzsch K, Hatzirodos N, Bonner WM, Aitken JB, Russell DL, Lane M, Rodgers RJ, Harris HH. X-Ray fluorescence imaging and other analyses identify selenium and GPX1 as important in female reproductive function. Metallomics 2014; 7(1):71-82. 24. Capper JL, Wilkinson RG, Kasapidou E, Pattinson SE, Mackenzie AM, Sinclair LA. The effect of dietary vitamin E and fatty acid supplementation of pregnant and lactating ewes on placental and mammary transfer of vitamin E to the lamb. British Journal of Nutrition. 2005; 93(4):549-57. 25. Jalilian MT, Moeini MM, Karkodi K. Effect of selenium and vitamin E supplementation during late pregnancy on colostrum and plasma Se, Cu, Zn and Fe concentrations of fat tail Sanjabi ewes and their lambs. Acta Agriculturae Slovenica (Slovenia). 2012; 100(2): 125-9. 26. Mohri M, Ehsani A, Norouzian MA, Bami MH, Seifi HA. Parenteral selenium and vitamin E supplementation to lambs: hematology, serum biochemistry, performance, and relationship with other trace elements. Biological Trace Element Research 2011; 139(3):308-16. 27. Shi L, Ren Y, Zhang C, Yue W, Lei F. Effects of maternal dietary selenium (Se-enriched yeast) on growth performance, antioxidant status and haemato-biochemical parameters of their male kids in Taihang Black Goats. Animal Feed Science and Technology 2017; 231:67-75. 28. Pisek L, Travnicek J, Salat J, Kroupova V, Soch M. Changes in white blood cells in sheep blood during selenium supplementation. Veteri-narni Medicina Praha 2008; 53(5):255. 29. Avci M, Karakil?ik Z, Kanat R. Effects of vitamins A, E and selenium on reproductive performance and serum levels of some biochemical parameters in ewes, and birth weight and survival rates in their lambs. Turkish Journal of Veterinary And Animal Sciences 2000; 24(1):45-50. 30. Alhidary IA, Shini S, Al Jassim RA, Abuda-bos AM, Gaughan JB. Effects of selenium and vitamin E on performance, physiological response, and selenium balance in heat-stressed sheep. Journal of Animal Science 2015; 93(2):576-88. 31. Avci C, Kizil O. The effects of injectable trace elements on metabolic parameters in transition cow. Kafkas Universitesi Veteriner Fakultesi Dergisi 2013; 19 (Supplement A) ): A73--8. Slov Vet Res 2019; 56 (Suppl 22): 365-79 DOI 10.26873/SVR-775-2019 Original Research Article COMPARATIVE THREE DIMENSIONAL COMPUTED TOMOGRAPHY (CT) SCANS AND ANATOMICAL INVESTIGATION OF RABBIT (Oryctolagus cuniculus) AND CAT (Felis domestica) SKULL Attia A. A. Moselhy*, Eman A. A. Mahdy Department of Anatomy and Embryology, Faculty of Veterinary Medicine, Zagazig University, Egypt Corresponding author, E-mail: atiaanatomy@gmail.com Abstract: This study was conducted to elucidate the comparative anatomical features of the skull of rabbits and cats using CT scans. Adult healthy New Zealand rabbits and Domestic Baladi cats of both sexes, were prepared for X-ray and CT scan of the heads at different positions. The heads were also processed for bone preparation and were photographed at various views. Some measurements were taken to exhibit the main differences between the two animal species. There was a cranial pharyngeal canal in the ba-sisphenoid bone of rabbit, which was absent in cat. The retroarticular process situated caudal to the mandibular fossa in cat and absent in rabbit. The carotid foramen was large and situated only in rabbit ventromedial to the bulla tympanica. The supraorbital process of rabbit extended anteriorly and posteriorly forming rostral and caudal supraorbital fissures. The orbit was located laterally in rabbit rostrally in cat. The facial surface of the maxilla was perforated by several formina in rabbit. A large retroalveolar foramen was found only in the mandible of rabbit. The condyloid process of the mandible was large and present longitudinally in rabbit and transversally in cat. Also, the hyoid bones and the paranasal sinuses were compared at both animals. The hyoid bone located in the mandibular space in rabbit and caudal to this space in cat. The lingual process of the hyoid bone was absent in cat. The middle and great cornuae of the hyoid bone were absent in rabbit. The paranasal sinuses of rabbit were maxillary and ethmoidal sinuses. While in cat, there were frontal, sphenoidal and maxillary sinuses. The combination between the traditional gross morphology of the skulls, X-ray and scan aimed to clarify and confirm all the points of comparison between the two animal species used, which was not achieved by using only one method of them. Key words: rabbit; cat; skull; computed tomography; anatomy Introduction The skull is a highly constructed and integrated part of the axial skeleton. All breeds of domestic rabbits descend from European rabbit (Oryctolagus cuniculus), which is a member of the family Leporidae (rabbits and hares) (1). The skull is divided into two primary units, neu-rocranium and basicranium. The brain case provides protection for the brain and opening for cranial nerve connections, the bone of the face provide a location and protection for the organs of special senses. Received: January 2019 Accepted for publication: February 2019 366 A. Moselhy, E. Mahdy The domestic cat (Felis catus), a common buddy animal, is only one of the species in the family Felidae (2). The rabbit is utilized for numerous purposes, including biomedical research, meat and fur production. Rabbits and cats are often used as human substitutes in olfaction and inhalation tests, both of which require the knowledge of their anatomy (3, 4). There are many papers about the morpho-metric features of the head of cat and rabbit and the gross anatomical studies were required. (5, 6).The cat as carnivorous and the rabbit which is herbivores animal, they show certain anatomical peculiarities specially their skeleton. The cat has large brain case and anterior located orbit and the rabbit skull long, narrow and laterally situated orbit. The current study was conducted to elucidate the comparative anatomical features of the skull of a rabbit and cat as educational tools in veterinary studies. Materials and methods Animals The handling of animals in this study was followed the guidelines of the Institutional Animal Care and the Research Ethics Committee of the Zagazig University, with an ethical approval number of (ZU-IACUC/2/F /92/2018). Adult healthy of each New Zealand rabbits and domestic cats (n = 10 for each) of both sexes weighed about 2-4 kg, were used. Rabbits were obtained from a laboratory farm in Faculty of Agriculture, Zagazig University. The age of rabbit was 10 months and cat was 13 months. The cats were purchased from a pet animal's clinic in Zagazig city, Sharkia Governorate, Egypt. Rabbits were injected through the ear vein with Xylazine (3 mg/kg) followed by injection of Ketamine (3 mg/kg) (7). The dose in cats was 1 mg/kg of xylazine followed by 5 mg/kg of I.M ketamine for sedation and anesthesia (7). Radiography For radiography, the animal heads were photographed in dorsal and lateral position using Mobile Fischer X-ray machine H.G. Fischer, inc. Franklin Park, Illinois, USA, in Department of Surgery, Faculty of Veterinary Medicine, Zagazig University. Computed tomography For (CT), both species were used, at AL-Bayan Center of radiology and CT in Belbes, Sharkia Governorate, Egypt. CT images were taken without contrast medium using multi-slices CT system, which was capable of acquiring up to 32 slices per second with fast whole-body scan time of 0.5 seconds, 50 kW X-Ray Generator, Multiple kV and mA techniques and 5.0 MHU X-Ray Tube. TOSHIBA 600HQ (third generation) Japan (8). Anatomical dissection of heads The heads of both species were carefully separated and cleaned from all attached tissues. Also, the intact hyoid bones were carefully dissected. The skulls were prepared following the method of (9). The measurements were taken with a caliper to demonstrate the main differences between the two animal species. The obtained skulls were photographed using a Sony digital camera, Dsc W810 20.1 MP. The weight of animals obtained by using a digital scale. The nomenclatures were taken as a basis for the denomination on the skulls (10). The cranial and facial portions of the skull were described the most characteristic points of differences of both animals. Also, the hyoid bones and the paranasal sinuses were compared at both animals. Statistical analysis All statistical procedures were performed using the SAS statistical system Package V9.2 (SAS, 2009) (11). Differences between means due to different anatomical parameters were tested by student's t-test. Data were reported as mean ± SEM, and the differences were considered significant at P < 0.05 Results he apparent point of comparison between two species skull in the current study that, rabbit skull as a whole was long, narrow and compressed. But, that of a cat was short, broad and heavy in weight table 1. Anatomy and CT scans of rabbit and cat skull 367 The skull is composed of two main portions; cranial and facial. I- Bones of the cranium The occipital bone The occipital bone (Fig.1 A, B, C and D) was subdivided into three parts; supra-occipital, lateral part and basioccipital. In rabbit, the occipital bone, shared in the formation of the roof of the cranial cavity and the supra-occipital part was fenestrated and depressed dorsally. The external sagittal crest was sharper, higher and longer in cat than in rabbit. The nuchal crest of rabbit was low, curved and located caudolater-ally. However, in a cat, the latter crest was sharp and long. The external occipital protuberance appeared as a sharp elevated ridge in the rabbit and reached to the foramen magnum. While in cat, it was small and separated from the foramen magnum by the external occipital crest. The occipital condyles in a cat were larger and broader than that of the rabbit. The jugular processes were longer in rabbit and closely attached to bulla tympanica in cat. The hypoglos-sal foramen was double in rabbit and single in cat. The foramen magnum is diamond in shape and notched dorsally in rabbit and rounded in cat. The basilar part of rabbit was longer, wider and increased in thickness rostrally, although in the cat, it had the same width along its whole length. The interparietal bone The interparietal bone was larger and longer in cat than in rabbit (Fig. 1A and B). There was an intracranial extension of the interparietal bone termed osseous tentorium cerebeli, which was very large in cat and small in rabbit. The sphenoid bone The basisphenoid in rabbit was wedge shaped and directed rostodorsally with its narrow rostral part was lodged between the ptergy-oid bones. In cat, the bone was directed rostrally and quadrate in shape (Figs.1E and F and 2A, B, and C). The basisphenoid bone was grooved dorsally in rabbit at its middle; there was a cranial pharyngeal canal which was absent in cat. The wing of the sphenoid bone in cat contained four foramina; rotundum, orbital, optic and eth-moidal foramen. In rabbit, the optic foramen was sited. The presphenoid The presphenoid bone (Fig. 1 E and F) in a cat was large, wedge shape separated from the pterygoid bone by the openings of the pterygoid canal. While in rabbit, this bone was located anterior to basisphenoid and anterio- ventral to the optic foramen. The temporal bone The temporal bone (Figs. 1 A, B, E and F, 2 C and 3 A and B) composed of two parts; Pars petrosa and Pars squamosa. In rabbit, the temporal fossa was small, short, shallow (nearly flat) and it participated in the formation of the posterior part of the orbit. In cat, the fossa was longer, larger and deeper (concave) and located caudal to the orbit. The mandibular fossa articulated with the mandible forming temporomandibular joint. It was located more dorsally in rabbit than in cat (Figs. 2C and 3C and D). The retroarticular process situated caudal to the mandibular fossa in cat and absent in rabbit. The bulla tympanica of rabbit was smaller than in cat and its shape was seashell like but resembling an egg in cat. The external acoustic process was located at high level in rabbit and absent in cat. The external acoustic meatus positioned dorsal to the level of bulla tympanica in rabbit, and it placed rostodorsal to bulla tym-panica in cat. The jugular and lacerum anterior foramina were larger in cat than that of rabbit. The carotid foramen was large and situated only in rabbit ventromedial to bulla tympanica (Fig. 1F). The internal acoustic meatus was larger and deeper in rabbit than in cat. The parietal bone The parietal bones participated in the formation of the roof of the cranial cavity in rabbit. But in cat, they were large and shared in the lateral boundary in addition to the roof of the cranial cavity (Fig. 1A and B). 368 A. Moselhy, E. Mahdy The frontal bone The main parts of the frontal bone were Squama, nasalis and Pars orbitalis. In rabbit, the frontal bone (Figs. 1A, B, 3A, B, C and D and 3A, B, E and F) was large, narrow, depressed dorsally and tapered anteriorly, forming inverted V-shape between the nasal bones. In cat, it was broad, short and lodged rostrally between the nasal bones and maxilla. The supraorbital process of rabbit was large, prominent. It extended anteriorly and posteriorly forming rostral and caudal supraorbital processes. It constituted the dorsal margin of the orbit. In cat, the supraorbital process was one part, long and it formed the caudal half of the orbital boundary. The orbit The orbit of rabbit was located laterally, slightly oval in shape and it contained one large optic foramen in the interorbital septum (Fig. 3A and C). The ethmoidal foramen was present just craniodorsal to the latter foramen. The orbit of cat was situated rostrally, rounded in shape and incomplete caudally, but closed by the orbital ligament (Figs. 3B and D and 3G and H). The ethmoid bone The ethmoid bone (Fig. 4 A and B) was consisted of cribriform plate and perpendicular plate in both species. The olfactory fossa of the cribriform plate was deep and small in rabbit but, long dorsovental and shallow in cat. The perpendicular plate in cat was longer, larger and situated in the caudal two third of the short nasal cavity, it was lodged in the sulcus vomeris ventrally. In rabbit, the forementioned plate was small, it did not reach to the level of vomer bone and it was restricted to the most caudal part of the long nasal cavity. So, the bony nasal septum was longer in cat than in rabbit which it was mostly cartilaginous. II- Bones of the face The lacrimal bone The lacrimal bones (Figs. 1 B and 3 A and B) can be seen from lateral view of the skull and formed the rostromedial part of the orbit. In cat, it was smaller than that of rabbit. In both species, the bone had an opening for the lacrimal canal, which it located between this bone and the maxilla in rabbit. The nasal bone The nasal bones (Figs. 1 A and B and 3) were larger and longer in rabbit than that of cat. In rabbit, they had the same width along its whole length with notched rostral end. The nasal bones of cat were short, broad rostrally and narrow caudally with the rostral end was curved C -shaped. In both animals, the caudal end was fitted between the frontal bones. The nasal bone of rabbit was separated from the maxilla by the nasal process of the premaxilla. But in cat, the caudal two third of the bone was related to the maxilla and only the rostral third correlated to the nasal process of the premaxilla. The nasal surface of the nasal bone had a dorsal turbinate crest for attachment of dorsal turbinate bone. In rabbit, the latter crest was long and present in the middle of this surface of the bone, while in cat, the crest was short and ventrally situated. The premaxilla The premaxilla (Figs. 1E and F, 2, A, B, C, and D 3 and4 A and B) of rabbit was large, long and it constituted the rostral part of the lateral wall of the nasal cavity. The premaxilla of cat was short, small and it represented the lateral and ventral boundary of the rostral osseus nasal aperture. In rabbit, the body of the premaxilla had two pairs of upper incisors teeth, the interdental space was very long and the interincisive canal was absent. In cat, it had three pairs of upper incisors teeth and the interdental space was very short or absent. The nasal process of premaxilla of rabbit was long, narrow and it extended caudodorsally until it reached to the frontal bone and making deep nasoincisive notch. While in cat, it was short and extended slightly between the nasal bone and maxilla, forming a shallow nasoincisive notch. In rabbit, the palatine process of premaxilla was long, extended caudally and formed the rostral part of the basis of the hard palate. It united with its followed and surrounded its lateral and caudal sides by long palatine fissures. Meanwhile, the palatine process of premaxilla of cat was very Anatomy and CT scans of rabbit and cat skull 369 short, joined the other side and the palatine fissures were small and short. The premaxilla formed most the basis of the hard palate in rabbit while in cat it formed only one quarter. The maxilla The maxilla consisted of body and two processes; zygomatic and palatine. The body of the maxilla (Figs. 1E and F, 2 and 3) formed the lateral wall of the nasal cavity. In rabbit, the facial surface of the bone was perforated by several formina and had a facial tuberosity laterally and alveolar bulla appeared from the rostroven-tral part of the orbit. Three foramina were present medial to the maxillary tuberosity; maxillary, sphenopalatine and posterior palatine foramen. In cat, there were no facial nor maxillary tuberosities. The infraorbital canal was very short in cat and extended dorsoventrally but, it was long and extended in a craniocaudal manner in rabbit. The facial tuberosity prolonged caudally into zygomatic process and reached to the malar bone. The dorsal border of the maxilla related to the nasal process of premaxilla in rabbit and to the nasal bone in cat. The alveolar border of the maxilla carried two premolar, three molar and a small molar tooth present cau-dally in rabbit. However, in cat, this border had one pair of canine, three pairs of premolar and one molar teeth. The zygomatic process of maxilla directed upward and backward joined the zygomatic bone. The palatine process of maxilla of cat was larger and broader than that of rabbit. This process of rabbit had a pointed rostral end and it formed the caudal boundary of the palatine fissure. In both species, the nasal surface of the maxilla contained maxillary sinus and ventral turbinate crest. In rabbit, the bony lacrimal canal was long and crossed the whole length of latter surface of the maxilla at its middle part. In cat, the bony lacrimal canal was very short and situated ventrally in the caudal part of the nasal surface of the maxilla. The turbinate bones In rabbit, the ethmoturbinate bone was narrower than cat and it was restricted to the caudal half of the nasal cavity. In both species, it was situated below the dorsal turbinate bone and caudal to the ventral turbinate bone. The dorsal turbinate bone in rabbit was long, narrow and it had a middle bulged part while in cat; it was broad, short and placed dorsal to the ethmotur-binate. The ventral turbinate bone in rabbit was larger than cat and situated rostral to the ethmo-turbinate and ventral to the dorsal turbinate bone. While in cat, it was very small and positioned ventral to the ethmoturbinate (Fig. 4A and B). The cavities of the skull The cranial cavity in general was larger in cat than rabbit. The rostral end of this cavity in cat was wide and separated from the frontal bone by the frontal sinus. But in rabbit, it was narrow and located in higher level just below the frontal bone due to the absence of the frontal sinus. The nasal cavity was longer in rabbit than cat. In cat, it was about half the length of the cavity in rabbit (Fig. 4 A and B). The palatine bone It consisted of Lamina horizontalis and per-pendicularis (Fig. 1E and F). In rabbit, the horizontal part of the palatine bone represented eighth the bony part of the hard palate. In cat, it symbolized half of the latter. The bone contained the rostral palatine foramen at the level of the 3rd upper premolar tooth in rabbit and located between the level of the 2nd and 3rd upper premolar teeth in cat. The perpendicular part of the palatine bone was narrow in rabbit and broad in cat. In both species the perpendicular part formed the lateral boundary of the posterior nares. The pterygoid bone In rabbit, the pterygoid bone (Figs. 1E and F, 2A and 2C) was larger than that of cat. It was very thin, transparent bone. Also, it had two pterygoid processes, each of them enclosed a triangular cavity termed pterygoid fossa. At the base of the latter fossa, there was alar foramen. In cat, the pterygoid bone was small connected rostrally with the perpendicular plate of the palatine bone by pterygopalatine suture. Its ventral border carried long pointed hamulus ptery-goideus. There were two foramina between the 370 A. Moselhy, E. Mahdy pterygoid bone and the body of the sphenoid bone (foramina of pterygoid canal). The zygomatic bone In rabbit, the zygomatic bone (Figs. 1A and B, 2B, C and D and 3A and B) was smaller, shorter and narrower than that of a cat. It formed a small ventral part of the rabbit orbit and all the ventral boundary of the cat orbit. The zygomatic process of this bone in rabbit extended more caudally than the level of the same process of the temporal bone. In cat, the caudal end of this bone was bifid into dorsal frontal and ventral zygomatic processes. The zygomatic process was longer, narrower and joined the zygomatic process of the temporal bone. The vomer bone The vomer bone (Figs. 1E, 3G and H and 4A and B) was short, straight and located more ros-trally in cat. It was long and curved rostroven-trally in rabbit. The bone was broader in rabbit than cat while, the sulcus vomeris was larger in cat. The vomer bone ended at the level of the rostral end of the nasal bone in cat. However, in rabbit it was parallel to the caudal two third of the nasal bone. In rabbit, the bone can be seen from the large palatine fissure. While in cat, it is not seen from the short palatine fissure. In both species, the vomer bone not divided the posterior nares. The mandible The mandible (Figs. 4 C and D and 5A) composed of body and two rami. In rabbit, the body was longer, narrower and represented by two parts; incisive and molar. While in cat, the body was short, broad and thick. The lower jaw in rabbit carried one pair of lower incisor, two premolar and three molar teeth. But in cat, there were two pair of incisor, one pair of canine, two premolar and one molar tooth. The rabbit had no canine teeth so, there was long inter-dental space (diastema). The latter was very short or nearly absent in cat. In rabbit, Symphysis man-dibulae was long and clear ventrally than dor-sally. However, in cat it was short and well distinct dorsally and ventrally. The lateral surface of the body of the mandible of rabbit was perforated by several formina resemble that of the maxilla. The mental foramen of rabbit was only one and present laterally on the body, away from the incisor teeth, just in front the first premolar tooth. In cat, the latter foramen was two in number; rostral and caudal. These foramina located more forward than in the rabbit. A large retroalveolar foramen was found only in rabbit, just caudal to the last molar tooth. The ramus of the mandible in rabbit was thin, transparent plate of bone its lateral surface had sallow mas-setric fossa and the medial one contained deep pterygiod fossa and fovea pterygoidea. At the cranial margin of the latter fossa, the mandibular foramen was located. The ramus of the mandible of cat was thick and compact. It had deep massetric fossa and the mandibular foramen present caudally than in the rabbit. The angle of the mandible and the vascular notch were clear in rabbit. The condyloid process was large and present longitudinally in rabbit and transverslly in cat. The mandibular notch in rabbit was shallower than in cat and it is located rostral to the condyloid process. In rabbit, the coronoid process was thin and it bent medially above a deep groove on the rostral border of the ramus. While in cat, the coronoid process was thick and elevated dorsally. Distal to the condyloid process, positioned Collum mandibulae in rabbit and angular process in both species. The madibular space was narrow in rabbit and wide in cat. The hyoid bone In rabbit, the hyoid bone (Figs. 2A and C, 3D and 5B, C, D and E) located in the mandibular space. While in cat, it situated caudal to the mandibular space. The hyoid bone of rabbit was smaller than that of a cat. It consisted of four parts; body (basihyoideum), small cornua (ceratohyoideum), somewhat long thyrohy-oideum and short lingual process. In cat, the bone formed of five parts. The lingual process was absent in cat. In addition to the parts of the bone in rabbit present middle (epihyoideum) and great (stylohyoideum) cornuae. Basihy-oideum of rabbit was large and quadrilateral in shape. While in cat, it was narrow and small plate of bone (Fig. 5A, B, C, D and E). There was no articulation between the hyoid bone and the skull of rabbit, but in cat, the great cornua Anatomy and CT scans of rabbit and cat skull 371 Paranasal sinuses: The paranasal sinuses of rabbit were two in number; maxillary and ethmoidal sinuses. While in cat, there were three paranasal sinuses; frontal, sphenoidal and maxillary sinuses (Figs. 4A and B and 5F, G, H and I). Table 1: Anatomical measurements of the skull and mandible between rabbits and cats Anatomical measurements Rabbits Cats P-Values Skull length (cm) 8.98±0.18 10.05±0.13 0.003 Skull width (at the middle of the orbit) 4.02±0.11 6.47±0.12 0.000 Cranium length 4.27±0.17 4.52±0.11 0.281 Nasal cavity length 4.07±0.11 3.05±0.16 0.002 Nasal bone length 3.97±0.13 1.20±0.10 0.000 Orbit Height 1.47±0.08 2.07±0.11 0.005 Breadth 2.25±0.11 2.97±0.10 0.004 Base of the skull (length) 1.05±0.11 2.05±0.13 0.001 Foramen magnum Height 0.40±0.4 0.50±0.04 0.157 Breadth 0.62±0.03 0.70±0.01 0.111 External acoustic meatus Height 0.39±0.13 0.49±0.16 0.004 Breadth 0.39±0.02 0.78±0.16 0.000 Zygomatic arch length 4.10±0.12 5.52±0.28 0.008 Mandible length 7.02±0.16 6.57±0.13 0.081 Ramus of the mandible height 4.00±0.15 3.02±0.11 0.002 Mandibular space 3.05±0.17 4.97±0.17 0.000 Mandibular symphysis (length) 2.22±0.12 1.45±0.11 0.008 Weight of the skull (without mandible) (gm) 15.07±0.11 40.12±0.41 0.000 Weight of mandible (two rami) (gm) 8.02±0.13 10.00±0.12 0.000 had tympanohyoideum which articulate with the mastoid process of the temporal bone (Figs. 3D and 5C). 372 A. Moselhy, E. Mahdy Figure 1: Photomacrographs of the dorsal aspect of the rabbit skull (A) and cat (B): 1- Squama occipitalis 2- Os interparietale 3-Os parietale 4- Crista sagittalis externa 5- Processus zygomaticus os temporal 6- Os frontale 7- Processus supraorbitalis 8- caudal branch of (7) 9- rostral branch of (7) 10- Incisura supraorbitalis caudalis 11- Incisura supraorbitalis rostralis 12- Os lacrimale 13- Foramen lacrimale 14- Maxilla 15- Foramen maxillare 16- Tuber faciale 17- Processus zygomaticus Maxilla 18- Os zygomaticum 19- Processus frontalis os zygomaticum 20- Os nasale 21-Processus nasalis os incisivum. Photomacrographs of the caudal aspect of the skull rabbit (C) and cat (D): 1 - Foramen magnum 2- Condylus occipitalis 3- Fossa condylaris 4-Processus jugularis 5- Protuberantia occipitalis externa 6-Crista occipitalis externa 7- Crista nuchae. Photomacrographs of the ventral aspect of the skull of rabbit (E) and cat (F) 1- Corpus ossis incisivi 2- Dentes incisivi 3- Processus palatinus os incisivum 4- Fissura palatines 5- Vomer 6-Processus palatines Maxilla 7- Dentes premolars 8- Dentes molars 9- Lamina horizontalis os palatinum 10-Foramen palatinum majus 11- Os zygomaticum 12- Lamina perpendicularis os palatinum 13- Choanae 14- Os presphenoidale 15- Canalis craniopharyngeus 16- Ala presphenoidale 17- Os pterygoideum 18- Os basisphenoidale 19- Pars basilaris os occipital 20- Bulla tympanica 21- Canalis caroticus 22-Foramen jugulare 23- Processus jugularis 24- Protuberantia occipitalis externa 25- Condylus occipitalis 26- Foramen magnum 27- Alveoli incisivi 28- Alveoli caninus 29- Alveoli molars 30- Fossa mandibulae 31- Processus retroarticularis 32- Hamulus pterygoideus 33- Foramen alare caudalis 34-Foramen alare cranialis Anatomy and CT scans of rabbit and cat skull 373 Figure 2: CT scan (A) and X- ray (B) images of the rabbit head (ventral view): 1- Os incisivum 2- Os zygomaticum 3- Os occipitale 4- Os basisphenoidale 5- Mandibula 6- Processus pterygoideus 7- Os hyoideum. CT (C) and X- ray (D) images of the cat head (ventral view): 1- Os incisivum 2- Os zygomaticum 3- Os occipital 4- Articulatio temporo-mandibularis 5- Mandibula 6- Processus frontalis os zygomaticum 7- Processus zygomaticus os frontale 8- Hamulus pterygoideus 9- Bulla tympanica. lO-Os basisphenoidale 11- Os presphenoidale 12- Os hyoideum 374 A. Moselhy, E. Mahdy Figure 3: Photomacrographs of the lateral aspect of the rabbit skull (A) and cat (B): 1-Os nasale 2-Os incisivum 3-Maxilla 4-Os lacrimale 5-Orbita 6-Os frontale 7-Os zygomaticum 8-Processus supraorbitalis 9- Processus zygomati-cus os frontale 10-Processus frontalis os zygomaticum 11-Os parietale 12-Os interparietale 13-Os occipitale 14-Crista nuchae 15-Processus zygomaticus os temporale 16-Bulla tympanica 17-Processus jugularis 18-Meatus acusticus ex-ternus 19- Pars squamosa os temporale 20-Processus retroarticularis 21- Dentes premolares 22- Dentes molars 23-Hamulus pterygoideus 24- Canalis opticus 25- Fissura orbitalis 26- Dentes incisivi 27- Processus nasalis os incisivum 28- Tuber maxillae29- (Tuber faciale 30- Sinus maxillaries. CT images of the rabbit (C) and cat (D) heads (lateral view) : 1- Maxilla (fenestrated in rabbit) 2- Articulatio temporomandibularis 3- Mandibula (fenestrated in rabbit) 4-Os hyoideum 5- Site of attachment of Os hyoideum with the skull. X- ray images of rabbit (E) and cat (F) heads (lateral view) demonstrating: 1- Os occipitale 2- Os parietale 3- Os frontale 4- Maxilla 5- Os nasale 6- Os incisivum 7- Dentes incisivi (Alveoli incisivi) 8- Margo interalveolaris 9- Dentes premolares (Alveoli premolars) 10- Fossa masseterica 11- Sinus frontalis 12- Processus angularis 13- Meatus acusticus externus 14-Bulla tympanica. Photomacrographs of the rostral aspect of the skull of a rabbit (G-a) and cat (G-b) showing: 1- Os nasale 2- Os incisivum 3-Processus nasalis os incisivum 4- Dentes incisivi (Alveoli incisivi) 5- Alveoli caninus 6- Maxilla 7- Foramen infraorbitale 8- Lamina perpendicularis os ethmoidale (Septum nasi osseum) 9- Vomer 10- Os conchae nasalis 11- Orbita. CT image of the cat head (H) (rostral view) demonstrating the orbit (1) which closed with orbital ligament (arrow) and vomer bone (2) Anatomy and CT scans of rabbit and cat skull 375 Figure 4: Photomacrographs of the sagittal section of the skull of rabbit (A) and cat (the perpendicular part of the ethmoid bone removed) (B): 1- Os incisivum 2- Os nasale 3- Maxilla 4- Os frontale 5- Os parietale 6- Os interparietale 7- Os occipital 8- Os temporal 9- Vomer 10- Ethmoturbinalia 11- Os conchae nasalis dorsalis 12- Os conchae nasalis ventralis 13- Cvaum crania 14-Tentorium cerebelli osseum 15- Meatus acusticus internus 16- Crista nuchae 17- Processus jugularis 18- Canalis hypoglossi 19- Bulla tympanica 20- Hamulus pterygoideus 21- Pars basilaris os occipital 22- Os basisphenoidale 23- Os presphenoidale 24- Sinus sphenoidalis 25- Sinus frontalis 26- Septa sinuum frontalium 27- Lamina perpendicularis os ethmoidale 28- Lamina cribrosa os ethmoidale 29- Processus pterygoideus. Photomacrographs of the mandible of rabbit (C) and cat (D) lateral (a) and medial (b) surfaces: 1 - Corpus mandibulae 2-Ramus mandibulae 3- Symphysis mandibulae 4- Incisura vasorum facialium 5- Dentes incisivi 6- Alveoli caninus 7-Margo interalveolaris 8- Foramina mentalia 9- Dentes premolares 10- Dentes molars 11- Angulus mandibulae 12-Fossa masseterica 13- Fossa pterygoidea 14- Fovea pterygoidea 15- Foramen mandibulae 16- Processus angularis 17-Collum mandibulae 18- Processus condylaris 19- Incisura mandibulae 20- Processus coronoideus 376 A. Moselhy, E. Mahdy Figure 5: Photomacrographs of the mandible (rostral view) of rabbit (A-a) and cat (A-b) 1- Corpus mandibulae 2-Symphysis mandibulae 3- Dentes incisivi 4- Alveoli caninus 5- Dentes premolares 6- Dentes molars 7- Foramen retroalveolaris 8- Processus coronoideus 9- Processus condylaris 10- Mandibular space. Photomacrographs of the hyoid bone of rabbit (B-a) and cat (B-b) 1- Basihyoideum 2- Processua lingualis 3- Thyrohyoideum 4- Ceratohy-oideum 5- Epihyoideum 6- Stylohyoideum 7- Tympanohyoideum. CT image of the rabbit head (D) (ventrolateral view) demonstrating the absence of articulation between the hyoid bone and the skull (arrow). CT images of the rabbit (D) and cat (E) heads (ventral view) demonstrating the hyoid bone at different levels (arrows) and Mandibula (1). CT image of the rabbit head (F) sinus maxillaries at anterior level (1) and septum nasi (2). CT image of the rabbit head (G) sinus maxillaries at posterior level (1) and sinus ethmoidales (2). CT images of the cat head (H) sinus maxillaries (a-arrow) and sinus frontalis at anterior level (b- arrow). CT images of the cat head (I) sinus frontalis at posterior level (a-1) which divided by septa sinuum frontalium, Sinus sphenoidalis (a-2), nasopharynx (a-3) and Sinus sphenoidalis at posterior level (b- arrow) which was divided by a median longitudinal partition Anatomy and CT scans of rabbit and cat skull 377 Discussion The cranium was much longer than the facial region in leopard cat. The orbit and the cranial cavity were large in the domestic cat, which agreed with the same result in leopard cat (12). The head of Persian cats was characterized by a short face and open orbits (13, 14) which was in the same line with the current study. The shape of the skull was differed in the same species as quadrate in domestic rabbits and elongated flat in wild rabbits (15). In agreement with (16), the parietal bones in cat were large and they cover the most of the roof of the cranial cavity. The features of the occipital bone in rabbit were confirmed by (17). The caudal aspect of the skull of cat was formed by the occipital bone, which it was triangular in shape, this finding correlated with (16) in Jungle cat. The jugular process was short and small and the foramen magnum was rounded in cat, this finding agreed with (18). The paracondylar process was sharper in leopard cat than in domestic cat (12). The supraorbital process of rabbit extended anteriorly and posteriorly forming rostral and caudal parts, which was similar to that observed by (17, 19, 20). The middle depression of the frontal bone in domestic cat was not observed in Jungle cat (16). In the present study, the bulla tympanica of the rabbit was smaller than in the cat and its shape was a seashell like but an egg like in the cat. On the other hand (17, 20) described the tympanic bulla of the rabbit as a rounded structure. (21) added that the tympanic bulla of dog and cat extended in a lower level than the occipital bone. The mastoid process was short in cat and the jugular processes were longer in rabbit. The external acoustic process in rabbit appeared as a long tube, but in cat it was nearly absent or took the shape of a bony ring that came in accordance with (21) in cat. The bulla tympanica of cat was very large and was lodged caudolaterally which was similar to the leopard cat (12) meanwhile, the external acoustic meatus found dorsally but more rostrally in the present work and at the dorsal border of bulla tym-panica in leopard cat. The zygomatic process of the zygomatic bone in rabbit extended more caudally than the level of the same process of the temporal bone in addition to the absence of the retroarticular process that made a difference in the tem-peromandibular joint from that of dog and cat that agreed with the results of (17,19). There were two optic canals in cat and single optic foramen in rabbit, similar observation recorded by (17, 19). The incisive bones were large, long in the rabbit and characterized by very long nasal process, had two pairs of upper incisor teeth, long interdental space and absence of the interincisive canal and the canine tooth. This observation was characteristic to the skull of the rabbit as an herbivorous animal and simulated the results of (17, 19). In rabbit, the palatine processes and fissure of the premaxilla were long, so the former composed most the basis of the hard palate. On the other hand, the palatine processes in cat were very short formed only one quarter of the basis of the hard palate and the palatine fissure was extremely short and small. Similar descriptions were evidenced by (17). In contrast to the current study, (15) in cat demonstrated that, the horizontal part of the palatine bone showed two elevations on either side of the median plane. The infra-orbital foramen in rabbit was situated at the level of the first premolar tooth, similar result was recorded by (22, 23). The facial crest was absent in domestic cat and the infraorbital canal was very short as in dog and cat (24, 25) in contrary to the other domestic animal, this crest was clear (18). The basisphenoid bone of rabbit was characterized by the presence of cranial pharyngeal canal, this foundation agreed with (17). This canal had different names in the previous studies, foramen caver-nosum (19). In the present study, the mandible of the rabbit was characterized by lack of canine teeth, long diastema and symphysis mandibu-lae, perforated lateral surface, presence of ret-roalveolar foramen and peculiar condyloid and coronoid processes. This result was similar to that recorded by (17, 26). The lateral surface of the mandibular ramus located had masseric fossa and there were several mental foramina at the rostrolateral aspect of the mandible in, it 378 A. Moselhy, E. Mahdy correlated (16). The hyoid bone of rabbit lacked epihyoideum and stylohyoideum as reported by (17, 27, and 28). So, there was no articulation between the hyoid bone and the skull of rabbit. The latter authors found muscular attachment between the bone and the skull. On the contrary, (27) in rabbit referred to the articulation between the ceratohyoideum and the occipital bone. The paranasal sinuses of rabbit were maxillary and ethmoidal sinuses, meanwhile, (29, 30) stated that maxillary and dorsal conchal sinuses were existed. On the contrary, (31, 32, 33) recorded only the maxillary paranasal sinus. The ethmoidal sinus of the rabbit in the present work was confirmed by the observations of (34, 35, 36). The paranasal sinuses of the cat were frontal, sphenoidal and maxillary sinuses, this finding was agreed with that of (36). This study can be applied in a veterinary clinic of cat and rabbit such as regional anesthesia during the treatment of head injury and dental extraction. Conclusion There were great differences between rabbit and cat in the bones forming the cranial cavity, nasal cavity, orbit, hard palate and jaws. Also, numerous variations recorded in the hyoid bone of the two animals; in its position, parts and articulation between this bone and the skull. The paranasal sinuses varied in rabbit than cat; in its number and bones contained them. So, the skull of each animal gave it complete adaptation with the nature of rabbit as herbivores and cat as carnivorous animals. Conflict of interest The authors declare that they have no conflict of interest. Acknowledgments The authors thank Professor Mervat Kon-sowa, Head of Anatomy and Embryology Department, Faculty of Veterinary Medicine, Zag-azig University, Egypt for beneficial comments on the manuscript. References 1. Nathan R. Biology of the rabbit. J of the American Association for Lab Anim Sci, 2006; 45: 8-24. 2. Pitakarnnop T, Buddhacha K, Euppayo T, Kriangwanich W, Nganvongpanit K. Feline (Felis catus) skull and pelvic morphology and morphometry: gender-related difference. Anat Histol Em-bryol. 2017; 46 : 294-303. 3. Norris Reinero CR, Decile KC, Berghaus RD, Williams KJ, Leutenegger CM, Walby Schelegle WF, Hyde DM, Gershwin LJ. An Experimental Model of Allergic Asthma in Cats Sensitized to House Dust Mite or Bermuda Grass Allergen. Int Arch Allergy Immunol. 2004; 135: 11731. 4. Xi J, Si XA, Kim J, Zhang Y, Jacob RE, Ka-bilan S, Corley RA. Anatomical Details of the Rabbit Nasal Passages and Their Implications in Breathing, Air Conditioning, and Olfaction. Anat Rec. 2016; 299: 853-8. 5. Saber AS, Caceci T, Gummow B, Johns K. Morphometric Studies on the Skull of the Australian Domestic Cat (F. catus) and its Clinical Implications for Regional Anesthesia. J. Vet. Anat. 2016; 9 (1) : 1 -24. 6. Gurbuz I, DemiraslanY, Asian K. Morphometric Analysis of the Skull of New Zealand Rabbit (Oryctolagus cuniculus L.) According to Gender. A RC J Anim and Vet Sci. 2015;1 (1): 27-32. 7. Hall LW, Clarke KW, Trim CM. Veterinary Anaesthesia. 10th ed. WB Saunders. Harcourt Publishers Limited. 2001; 441 -66. 8. Bohler A, Henninger W. Computed tomography of the rabbit head without general anaesthe-sia.Vet. Med. Austria / Wien. Tierjrztl. Mschr. 2008; 95: 116 -20. 9. Simoens R, Poles R, Lauwers H. Morphomet-ric analysis of foramen magnum in Pekingese dogs. Am J Vet Res. 1994; 55: 33-9. 10. Nomina Anatomica Veterinaria. 5 th ed, prepared by the International Committe on Veterinary Gross Anatomical Nomenclature (I.C.V.G.A.N.) and authorized by the General assembly of the World Association of Veterinary Anatomists (W.A.V.A.), konxville,T.N (USA). Published by the Editorial Committee, Hannover, Columbia, Ghent and Sapporo. 2012; 11-9. 11. SAS. SAS statistical system Package-Jmp 8 User's Guide.2nd Cary, NC, SAS Institute Inc. USA. 2009, ISBN 978-1-60764-301-2. Anatomy and CT scans of rabbit and cat skull 379 12. Sarma K, Nashiruddullah K, Islam S. Anatomy of the skull of a leopard cat (Felis bengalensis). Indian J. anim sci, 2001; 71 (11): 1011-3. 13. .Künzel, W, Breit S, Oppel M. Morphomet-ric investigations of breed-specific features in feline skulls and considerations on their functional implications. Anat Histol Embryol, 2003; 32 (4): 218-23. 14. Monfared AL. Anatomy of the Persian Cat's Skull and its Clinical Value During Regional Anesthesia. Global Veterinaria. 2013 10 (5): 551-5. 15. Böhmer C, Böhmer E. Shape Variation in the Craniomandibular System and Prevalence of Dental Problems in Domestic Rabbits: A Case Study in Evolutionary Veterinary Science. Vet. Sci, 2017; 4, 5. 16. Nameer PO, Nasser PO, Ipe MO, Ommar PA. Anatomy of Jungle cat skull (Felis chaus, Schreber). Zoos' Print J, 2003; 18 (2): 1011-8. 17. Farag FM, Daghash SM, Mohamed EF, Hussein MM, Hagrass SM. Anatomical studies on the skull of the domestic rabbit (Oryctolagus cunicu-lus) with special reference to the hyoid apparatus. J. Vet. Anat, 2012; 5 (2): 49-70. 18. Nickel R, Schummer A, Seiferle E. Anatomy of the domestic animals. Vol . 1986, Verlag Paul Parey, Berlin and Hamburg. 19. Brewer NR. Historical Special Topic Overview on Rabbit Comparative Biology. J Am Assoc Lab Anim. 2006; 45 (1): 8-24. 20. Salih KM. Gross Anatomical And Moro-phmetrical Studies To The Skull Bones Of The Local Rabbit (Oryctohguscuniculus). Bas j vet Res. 2013; 12 (2), 267-77 21. King AM, Cranfield F, Hall J, Sullivan M. Anatomy and ultrasonographic appearance of the tympanic bulla and associated structures in the rabbit. Vet J. 2007; 173, 512-21. 22. Popesko P, Rajtova V, Horak J. 1992 A colour atlas of the anatomy of small laboratory animals, Vol.1, Rabbit and guinea pig. Wolfe Publishing, Bratislava. 23. Monfared AL. Applied Anatomy of the Rabbit's Skull and its Clinical Application During Regional Anesthesia. Global Veterinaria, 2013; 10 (6): 653-7. 24. Getty R. Sisson and Crossman: Carnivore Osteology. Skull. In the anatomy of the domestic animals. 1975, 5th Ed. W.B. Saunders Company. 25. Miller ME. Guide to the dissection of the dog, 2004, fourth edition W.B. Saunders company. 26. Salih KM. Morphological and morph metric study of the mandible in the local Rabbits (Oryctolagus Cunicuius). Iraqi J. Vet. Sci. 2016; 40(1): 25-9. 27. Barone R Pavaux C, Blin PC, Cuq P. 1973, Atlas of Rabbit Anatomy (Masson et Cie, Paris), 11-50. 28. Little CJ, Lane JG. The surgical anatomy of the feline bulla tympanica. J Small Anim Pract, 1986; 27, 371-8. 29. Casteleyn C, Cornillie P, Hermens A, Van, LD, Hoorebeke LV, Broeck W, Simoens P. Topography of the rabbit paranasal sinuses as a prerequisite to model human sinusitis. Rhinology. 2010; 48: 300-4. 30. Varga M. Textbook of the rabbit medicine. 2014, Second edition. Butterworth Heinemann Elsevier inc. 31. Kara CO. Animal Models of Sinusitis: Relevance to Human Disease. Curr Allergy Asthma Rep. 2004; 4:496-9. 32. Pereira ME, Macri NP, Creasy DM. Evaluation of the Rabbit Nasal Cavity in Inhalation Studies and a Comparison with Other Common Laboratory Species and Man. Toxicol Pathol. 2011; 39: 893900. 33. Özkadif S, Eken E. Three-dimensional reconstruction of multidetector computed tomography images of paranasal sinuses of New Zealand rabbit. Turk J Vet Anim Sci. 2013; 37: 675-81. 34. Ozcan KM, Akdogan O, Kopara S, Ozcan I, Gurgen SG, Ozogul C, Selcuk A, Deren T, Dere H. Comparison of Histopathological and CT Findings in Experimental Rabbit Sinusitis. Indian J Otolaryngol Head Neck Surg. 201; 63: 56-9. 35. Hadad S. Some anatomical studies on the development of the nasal cavity and its related structures in the white New Zeeland. PhD thesis. 2015. 36. Ragab SA, Abouelela YS, Daghash SM, Khattab MA, Tahon RR. Comparative morpho-log-ical and histochemical characteristics of the nasal cavity and paranasal sinuses of adult rabbits and domestic cats in Egypt. Inter J Vet Sci. 2018; 7 (2): 97-105. Slov Vet Res 2019; 56 (Suppl 22): 381-90 DOI 10.26873/SVR-776-2019 Original Research Article IMPACT OF ALTERING DIETARY OMEG 6 TO OMEGA 3 FATTY ACIDS RATIO ON GROWTH PERFORMANCE, CARCASS COMPOSITION, HEMATO-BIOCHEMICAL PARAMETERS AND ABSORPTIVE CAPACITY OF THE INTESTINE OF NILE TILAPIA Eldsokey Nassef1*, Elsayed Hegazi1, Ahmed Sherif2, Mohamed Elshamy2, Abdelnasser Bakr1, Tarek k. Abouzed3 1Nutrition and Clinical Nutrition Department, Faculty of Veterinary Medicine, Kafrelsheikh University, Kafrelsheikh, Egypt, 2Agriculture Research Center, Animal Health research Institute, Kafrelsheikh, Egypt, 3Biochemistry Department, Faculty of Veterinary Medicine, Kafrelsheikh University, Kafrelsheikh, Egypt. Corresponding author, E-mail: dsokeynassef@yahoo.com Abstract: The present study investigated the influence of altering omega 6 to omega 3 fatty acids (FA) ratio on growth, hemato- biochemical parameters and absorptive capacity of Nile tilapia. One hundred and fifty fingerlings (25.1±0.3 g), were assigned into 3 groups (5 replicates each) and fed on iso-caloric and iso-nitrogenous diets. Diets were supplemented with fish and soy oil to create 3 different ratio of omega 6 to omega 3 FA, namely, 0.91, 2.85 and 9.3 parts of omega 6 to 1 part of omega 3 (R1, R3 and R9, respectively). Increasing the ratio from R1 to R9 significantly decreased average daily gain and increased feed conversion ratio. Also this alteration decreased ash content in the carcass on the expense of ether extract. Number of erythrocytes, packed cell volume, hemoglobin concentration were significantly increased in fish fed R1. Feeding R9 increased heterophils and decreased lymphocytes as compared with R1 and R3. Fish fed R1 significantly had high serum protein and serum antibacterial activity (47.6%) than R9 (43.5%) and R3 (44.9%). Feeding R1 significantly decreased serum triglycerides, cholesterol and creatinine. Feeding R3 and R1 enhanced absorptive capacity of jejunum villi more than the group fed R9. Increasing the ratio of omega 6 to omega 3 fA from R1 to R9 decreased growth performance through decreasing absorptive capacity of jejunum villi. However, Feeding R3 had a positive effect on growth performance and absorptive capacity of the intestine of Nile tilapia. Key words: fish oil; growth performance; intestinal villi; Nile tilapia; soybean oil Introduction Fish oil is the main lipid source used in the formulation of commercial aqua feeds. The continuous little global production of fish oil into 2030 (1), the highly variable cost have forced intensive research activities to evaluate alternative lipid sources (2). Vegetable oils are important lipid sources with low cost and wide availability. To our knowledge, there was scarce information on the assessment impact of altering omega 6 to omega 3 fatty acids (FA) Received: January 2019 Accepted for publication: February 2019 382 E. Nassef, E. Hegazi, A. Sherif, M. Elshamy, A. Bakr, T. Abouzed ratio on antibacterial activity and absorptive capacity of the intestine of Nile tilapia. Tilapia is the most common type of fish used in aquaculture where its production reaches to 66.8% of total aquaculture production (3). Fish meals and oils are essential part of diet composition in tilapia as a source of protein and lipid (2). Dietary lipids are important for regular growth and modulating immune responses in mammals, fish, sea cucumber, and shrimps (4). Because the expansion of aquaculture, it is expected that the requirements of fish meals and oils by the aquaculture sector will increase, but there are troubles of the high cost and limiting availability of fish meals and fish oils (1; 4). We hypothesized that vegetable oils could replace fish oil due to its low cost and wide availability. However, the difference in the ratio of omega 6 to omega 3 FA can impact fish health and disease resistance. Lipids affect on the immune system by influencing the phos-pholipids of immune cell membranes, membrane-associated signaling molecules (eico-sanoids) and receptor sites (5). Many previous studies used fish meal as a protein supplement when evaluating vegetable oils which supplied a certain amount of long chain poly unsaturated fatty acids. This study aimed to investigate the influence of altering omega 6 to omega 3 FA on growth performance, hematological and biochemical parameters, carcass composition and antibacterial activity of Nile tilapia and assessment of the absorptive capacity of duodenum, jejunum and ilium. Materials and methods Fish and experimental design One hundred and eighty fingerlings of Nile Tilapia (25.1±0.3 g) were purchased from commercial farm in Kafrelsheikh governorate and were acclimatized to tank conditions for 2 weeks. One hundred and fifty apparently healthy fingerlings of homogenous size were selected and randomly distributed into 3 groups (5 replicates each). Each replicate contained 10 fish per tank of 50-L which was equipped with continuous aeriation, inlet and outlet. The procedures have been approved by the Institutional Aquatic Animal Care and Use Committee, Faculty of Aquatic and Fisheries Sciences, Kafrelsheikh University. Three experimental diets were supplemented with fish and soy oils to create 3 different ratio of omega 6 to omega 3 FA. The first ratio (R1) was of 0.91 parts of omega 6 to 1 part of omega 3 FA. The second ratio (R3) was of 2.85 parts of omega 6 to 1 part of omega 3 FA. The final ratio (R9) was of 9.3 parts of omega 6 to 1 part of omega 3 FA. The diets were nearly similar in protein and energy (Table 1). The ingredients were ground to pass through a sieve of 1 mm and mixed for 20 min. the oil was added with the continuous mixing. Distilled water was added to the diets till forming soft dough. The diets were pelleted in a laboratory pellet mill through 2 mm diameter die. Pellets were dried in an oven at 60 °C for 2 h and freshly used. The experimental period lasted for 8 weeks. The fish were fed twice daily at 8.00 and 15.00 h at a level of 4% of body weight for 2 weeks; 3.5% from 3 to 4 weeks then 3% from 5-8 weeks. Feed refusal was recovered after feeding and dried using oven at 60 °C for 2 h then subtracted from the offered feed. Growth trial Growth parameters were determined according to the following equations: Body weight gain (BWG, g fish-1) = Final BWT - Initial BWT; Average daily gain (ADG, g) = BWG/dura-tion of the experiment (56 days); Feed intake (FI, g fish-1, 56 days) = (offered feed-feed refusal recovered and dried)/no of fish Feed conversion ratio (FCR) = FI (g)/ BWG (g); Protein efficiency ratio (PER) = BWG (g)/dry protein intake (g); Protein retention (PR, %) = (protein gain, g/protein intake, g) x100 Energy retention (ER, %) = (energy gain, kcal)/energy intake, kcal) x100 Chemical analysis of feed and fish Representative feed samples were ground through 1mm screen (Cyclotec, Foss Sweden). The ground samples were analyzed for crude Omega-3 FA and fish performance 383 protein based on Kjeldahl method and ether extract (Ankom Technology method), according to (6). NFE was calculated according to the following formula: NFE= 100- (moisture + crude protein + ether extract + ash + crude fiber). Lysine, methionine, calcium and available phosphorus were calculated based on feed composition tables in (2). At the end of the experiment, 3 fish were randomly collected from each replicate; dried in oven at 60 °C for 48 h. Dry matter and moisture were determined. The dried samples were analyzed for crude protein, ash and ether extract according to (6). Blood analysis and immune response assay At the end of the experiment, pooled blood sample was collected from 5 fish per tank via the caudal vein into heparinized disposal syringe for complete blood count (CBC). Further blood sample without anticoagulant was collected, centrifuged at 3000 rpm for 15 min to obtain the serum and stored at -20 °C for the biochemical analysis. Blood film was prepared according to (7). Differential leukocyte count was calculated according to (8). Hematocrit, erythrocytes, white blood cell (WBCs) and hemoglobin (HB) were examined according to (9). Serum protein and albumin were calorimet-rically measure based on (10, 11) respectively. Serum globulin was determined by subtracting the concentration of albumin from protein. The activity of alanine amino transaminase (ALT) and aspartate amino transaminase (AST) was determined as previously described (12). Histopathological analysis Intestinal tissue specimen were obtained at the end of experiment to examine absorptive capacity of villi. The samples were immersed in formalin 10%, embedded in paraffin, stained with haematoxylin and eosin (H & E) and documented photographically with a digital camera (DCM 130E/1.3 megapixels, CMOS Software Scopephoto, China) connected to a light microscope (Leica). Serum antibacterial test Serum bactericidal activity was done following the procedure of (13). Equal volumes (100pl) of serum and A. hydrophila bacterial suspension 2x108 (CFU) were mixed and incubated at 25 oC for 1 h. Blank control was made by replacing serum with sterile phosphate buffer saline (PBS). The mixture was then diluted with sterile PBS at a ratio of 1:10. The diluted mixture (100pl) was plated on blood agar and incubated at 37 oC for 24 h. The number of viable bacteria was determined by counting the colonies grown on the agar plates. Statistical analysis Analysis of variance (ANOVA) was done for all data using the SPSS program (14). Duncan's multiple range was used to determine the significant difference among means at P<0.05. Results Growth performance As shown in Table 2, the final weight, BWG and ADG were significantly higher in fish fed R1 than those fed R9. But, fish fed R3 had nearly similar growth parameters to fish fed R1. In sum, ADG of fish fed R1 was the greatest followed by R3 then R9, There was no significant difference in feed intake of fish fed either R1 or R3 or R9. But, FCR was significantly improved in fish fed R1 followed by R3 then R9. PER and PR were greater in fish fed R1 than those fed the other diets. In contrast, ER was significantly greater in fish fed R9 followed by R3 then R1. Body chemical composition As illustrated in Table 3, there is no significant differences between different groups in dry matter, moisture and crude protein. Feeding omega 3 FA (R1) significantly increased mineral density in the skeleton as compared to R9. In contrast, feeding R1 decreased fat deposition in the whole body of fish. Whereas, Feeding R3 still had the same effect of R1 on ash and ether extract concentrations. 384 E. Nassef, E. Hegazi, A. Sherif, M. Elshamy, A. Bakr, T. Abouzed Hematological parameters As illustrated in Table 4, feeding R1 increased RBCs count, Hb concentration and PCV as compared to R3 or R9. Feeding R1, R3 increased WBCs than R9. However feeding R9 increased heterophil but decreased lymphocyte when compared to the other groups, while mon-ocytes, eosinophil and basophil still unchanged among the dietary treatments. Biochemical analysis As shown in Table 5, although the ALT and AST were significantly lower in fish fed R1. The values of further groups were within the acceptable range. The same trend was recorded in creatinine analysis. Serum globulin was markedly high in fish fed R1. Subsequently, it elevated serum protein because the serum albumin was nearly similar among all treatment groups. Serum triglyceride and cholesterol were significantly lesser in fish fed R1 as compared to R9 or R3 groups. No significant difference in cholesterol concentration between R9 and R3 groups was detected. Feeding R1 increased HDL and decreased LDL than the other two groups. On the other hand, fish fed R3 had a higher HDL and a lower LDL than R9 group. Antibacterial activity As shown in Table 6, the result indicated a significant enhancement of the immune status of group fed R1 but differences in other groups were in acceptable range which showed no threat on fish life. Table 1 : Physical and chemical composition (%) of the diets for Nile tilapia. Items Dietary treatments a R1_R3_R9 Corn gluten meal 10 10 10 Soybean oil 0 2.5 5 Fish oil 5 2.5 0 Corn grain 30.0 30.0 30.0 Dehulled soybean meal 51.6 51.6 51.6 Monocalcium phosphate 1.57 1.57 1.57 Limestone 0.68 0.68 0.68 Salt 0.35 0.35 0.35 Methionine, DL 0.2 0.2 0.2 Premix b 0.1 0.1 0.1 Antimycotoxin 0.2 0.2 0.2 Vitamin C 0.1 0.1 0.1 Binder 0.2 0.2 0.2 Chemical composition of dried pelleted diets Digestible energy, Kcal/kg c 3325 3313 3302 Crude protein % 32.23 32.23 32.23 Lysine % 1.73 1.73 1.73 Methionine % 0.75 0.75 0.75 Crude fat % 6.93 6.93 6.93 Calcium % 0.7 0.7 0.7 Available phosphorus % 0.45 0.45 0.45 a Treatments represent the ratio between omega 6 to omega 3 FA in the diet. R1 is a ratio of 0.91 parts of omega 6 to 1 part of omega 3; R3 is a ratio of 2.85 parts of omega 6 to 1 part of omega 3; R9 is a ratio of 9.3 parts of omega 6 to 1 part of omega 3. b Premix: Magnesium 40mg ; Manganese 10mg ; Zinc150mg ; Iron 30mg ; Copper 5mg ; Iodine 5mg ; Cobalt 0.005mg ;Selenium 0.1mg; vit A 5500 UI; vit D3 1000UI; vit E 50mg ; vit B1 20mg ; vit B2 20mg ; vit B6 20mg ; vit B12 0.02 mg ; niacin 100mg; vit K3 10mg ; biotin 0.1mg folic acid 5mg. c Calculated according to (2). Omega-3 FA and fish performance 385 Table 2: Impact of altering omega 6 to omega 3 FA ratio on growth performance and feed utilization Dietary treatments 1 Items R1 R3 R9 Initial weight 25.23±0.18 25.17±0.03 25.40±0.10 Final weight 54.96±0.6 a 51.12±2.84 ab 50.27±3.1 b Body weight gain 29.3±0.90 a 25.96±2.87 ab 24.87±1.1 b Average daily gain 0.52±0.02 a 0.47±0.05 ab 0.44±0.06 b Feed intake 57.16±0.72 55.2±1.41 54.4±1.10 Feed conversion ratio 1.95±0.04 a 2.1±0.17 ab 2.24±0.21b Protein efficiency ratio 1.59± 0.01a 1.45± 0.12 b 1.42b± 0.13 Protein retention 25.8± 1.5 a 23.55± 0.1 b 23.19± 1.05 b Energy retention 45.6± 2.28 c 48.78± 0.16 b 50.05± 0.6 a Means ± SE with different letter within the same raw are significantly different at P<0.05. 1 Treatments represent the ratio between omega 6 to omega 3 FA in the diet. R1 is a ratio of 0.91 parts of omega 6 to 1 part of omega 3; R3 is a ratio of 2.85 parts of omega 6 to 1 part of omega 3; R9 is a ratio of 9.3 parts of omega 6 to 1 part of omega 3. Table 3: Impact of altering omega 6 to omega 3 FA ratio on body chemical composition (on dry matter basis) Dietary treatments 1 Items Dry matter Moisture Crude protein Ether extract Ash R1 R3 R9 28.37±0.56 71.63±0.58 55.73±0.15 21.8±1.25 b 19.6± 1.98 a 29.01±0.29 71.0±0.29 55.7±1.7 23.1±1.1 a 17.98±1.9 b 28.97±0.88 71.03±0.88 55.9±0.11 23.8±0.4 a 16.46±0.78 c Means ± SE with different letter within the same raw are significantly different at P<0.05. 1 Treatments represent the ratio between omega 6 to omega 3 FA in the diet. R1 is a ratio of 0.91 parts of omega 6 to 1 part of omega 3; R3 is a ratio of 2.85 parts of omega 6 to 1 part of omega 3; R9 is a ratio of 9.3 parts of omega 6 to 1 part of omega 3. Table 4: Impact of altering omega 6 to omega 3 FA ratio on hematological parameters Dietary treatments 1 Items R1 R3 R9 RBCS 2.57±0.09 a 2.01±0.08 b 1.88±0.1 b WBCS 121.9±0.3 a 119±0.7 a 116.3±1.4 b Heterophil 25±0.6 b 26.3±0.6 b 30±0.6 a Monocyte 4.7±0.3 3.7±0.9 5.3±0.7 Lymphocyte 66.7±0.3 a 65.7±1.2 a 60.7±0.7 b Eosinophil 3.7±0.3 4.3±0.6 4±0.6 Basophil 0.3±0.3 0.3±0.3 0.7±0.3 Hb 8.7±0.26 a 7.14±0.14 b 6.66±0.1 b PCV 26.1±0.8 a 22.1±0.4 b 21±0.03 b Means ± SE with different letter within the same raw are significantly different at P<0.05. 1 Treatments represent the ratio between omega 6 to omega 3 FA in the diet. R1 is a ratio of 0.91 parts of omega 6 to 1 part of omega 3; R3 is a ratio of 2.85 parts of omega 6 to 1 part of omega 3; R9 is a ratio of 9.3 parts of omega 6 to 1 part of omega 3. 386 E. Nassef, E. Hegazi, A. Sherif, M. Elshamy, A. Bakr, T. Abouzed Table 5: Impact of altering omega 6 to omega 3 FA ratio on biochemical parameters and antibacterial activity Dietary treatments 1 Items R1 R3 R9 ALT (p/l) 34.3±1.3 c 41.2±0.8 b 47.3±0.9 a AST (p/l) 106.3± 2.96 b 112.5±1.3 b 130±6.4 a Creatinine (mg/dl) 0.89±0.05 c 1.47±0.09 b 1.86±0.14 a Total protein (g/dl) 6.6±0.15 a 5.7±0.2 b 5.3±0.09 b Albumin (g/dl) 4.1±0.06 4.07±0.02 4±0.03 Globulin (g/dl) 2.5±0.15 a 1.6±0.3 b 1.2±0.1 b Triglycerides (mg/dl) 149.5±1.9 c 295.5±1.45 b 303.3±2 a Cholesterol (mg/dl) 98.5±1.6 b 118.2±0.7 a 123.5±2.4 a HDL (mg/dl) 56.2±0.7 a 51.3±0.9 b 48.6±0.6 c LDL (mg/dl) 2.87±0.3 c 11.5±3.3 b 20±0.8 a Serum antibacterial (%) 47.6 ±0.6 a 44.9 ±0.4 b 43.5 ±0.8 b Means ± SE with different letter within the same raw are significantly different at P<0.05. treatments represent the ratio between omega 6 to omega 3 FA in the diet. R1 is a ratio of 0.91 parts of omega 6 to 1 part of omega 3; R3 is a ratio of 2.85 parts of omega 6 to 1 part of omega 3; R9 is a ratio of 9.3 parts of omega 6 to 1 part of omega 3 Table 6: Impact of altering omega 6 to omega 3 FA ratio on mucosal and villi parameters of the intestine Dietary treatments Duodenum Jejunum Ilium 1 length width length width length width R1 541±33 141±13 938±35 a 115±4b 570±37a 149±23 R3 510±30 139±13 769±27 b 113±6 b 187±23 b 133±12 R9 539±31 150±10 901±37 a 142±10a 405±23c 140±10 Means ± SE with different letter within the same column are significantly different at P<0.05. treatments represent the ratio between omega 6 to omega 3 FA in the diet. R1 is a ratio of 0.91 parts of omega 6 to 1 part of omega 3; R3 is a ratio of 2.85 parts of omega 6 to 1 part of omega 3; R9 is a ratio of 9.3 parts of omega 6 to 1 part of omega 3 Figure 1: Jejunum of fish supplemented with diet containing fish oil showing normal long villi lined with normal epithelium, (H&E, X200) Omega-3 FA and fish performance 387 .'.-a. I — Figure 2: Jejunum of fish supplemented with diet containing vegetable oil showing normal villi, (H&E, X200) Figure 3: Jejunum of fish supplemented with diet containing a mix of fish and soybean oils showing most of villi length was similar to group 1 with increase branches (arrow), (H&E,X200) Histopathological analysis capacity of intestinal villi as similar to the group fed R1. Jejunum of fish fed R1 showing normal Increasing ratio of omega 6 to omega 3 FA long villi lined with normal epithelium (Fig. 1). from R1 to R9 decreased length of j ejunum and Jejunum of fish fed R9 showing normal villi but ilium villi. Feeding R3 improved the absorptive 388 E. Nassef, E. Hegazi, A. Sherif, M. Elshamy, A. Bakr, T. Abouzed was shorter than the other groups (Fig. 2). Jejunum of fish fed R3 showing most of villi length was similar to fish fed R1 with increase branches and width (Fig. 3). The effect of dietary treatments on duodenum histopathology was not significant (Table 6). Discussion The result of the present study revealed that the fish fed a diet containing a blend of omega 6 and omega 3 FA (R3) had similar BWG to the group fed more omega 3 FA (R1) but higher than that fed omega 6 FA (R9). Similarly, (15) found that the final weight was lower when increasing dietary omega 6 FA by using 6% soy oil in fish diet for 6 weeks. Also, a recent study denoted that increasing omega 6 to omega 3 FA ratio for 8 weeks depressed growth of tilapia (16). (17) who reported that a moderate increase of omega 6 FA by using 4.5% SO in replacement of FO in hybrid tilapia diets for 10 weeks, found that all groups had similar final weight. Also, (18) investigated a moderate omega 6 to omega 3 ratio in red hybrid tilapia diets for 5 months and detected no significant difference in either the growth performance or feed efficiency. Some studies have designated that both omega-3 and omega-6 FAs are indispensable for tilapia (2). These results implied that the presence of moderate dietary levels of omega 6 FA may spare a part of the requirement for omega 3 FA series. This study revealed that feeding R1 reduced body fat content. This might be due to the high phospholipid content of fish oil than the soy oil. Lipid rich in omega 3 PUFA is reported to impede the fat synthesis and to reduce the deposition of these FAs in the liver and whole body of fish (19). With regard to phospholipids, (20) found that it decreased total lipids of whole body of rainbow trout as compared to a mixture of olive and linseed oils, also, (21) stated that omega 3 FA decreased lipid concentration in rainbow trout than the omega 6 FA. On the other hand, feeding R1 increased mineral deposition in the skeleton. The relation between type of FA and mineral metabolism in fish is less investigated. In some studies on European sea bass, (22) demonstrated that EPA and DHA of marine origin supported vertebral and cephalic growth with less deformities. In terrestrial animals, phospholipid facilitated cartilage mineralization and stimulated insulin like growth factor production which activate bone formation and matrix production. On the other hand, pros-taglandins E2 derived from arachidonic acid is a powerful stimulator of bone resorption (23). These results clarify that feeding R1 improved general health condition of Nile tilapia as indicated by increasing HB concentration, PCV and RBCs count which positively affected the growth performance. Blood cells reflect dietary changes due to their fast renovation (24). Feeding omega 3 FA increases DHA incorporation in cell membrane of erythrocytes which maintains osmotic pressure and nutrient transport across the cell membrane. On the other hand, feeding omega 6 FA to channel catfish increased C18 PUFA and decreased LC-PUFA which appeared to increase erythrocyte fragility (25). In another study on salmon, vegetable oils (blend of rapeseed and linseed oils), which are rich in omega 6 FA, decreased eryth-rocyte counts and hematocrit (26). Fish fed more omega 3 FA or a mixture of omega 6 and omega 3 FA (R3) had high lymphocyte and low heterophil count than those fed omega 6 FA (R9). However, total count of WBCs was high in fish fed R1 than the other groups. These results differed from the previous reports which documented that omega 3 PUFA inhibit lymphocyte proliferation (27). The current results revealed that feeding R1 or R3 enhanced liver function as indicated by lowering serum ALT and AST levels than fish fed R9. This may be due to the associative influence of dietary lipid types on the histological structure of the liver (28). Increasing omega 6 FA in gilt head sea bream diets increased fat accumulation in hepatocyte (29) which subsequently reduced hepatocyte activity leading to metabolic imbalance. Omega 6 FA had a lipo-genic effect and lowered oxidation capacity of fish (30). More certainly, feeding omega 6 FA to Atlantic salmon increased molecular expression of adipophilin in the liver (31) which is a marker for lipid accumulation. Omega-3 FA and fish performance 389 Altering the ratio of omega 6 to omega 3 FA modified immune status (32). Our result of serum antibacterial test indicated that the group fed R1 had significant enhancement of antibacterial activity of fish over the other groups. The use of a moderate omega 6 to omega 3 FA ratio did not alter the health conditions (33). Results of the histopathology confirmed the beneficial effect of partial replacement of omega 3 with omega 6 FA on absorptive capacity of jejunum villi. Increasing ratio of omega 6 to omega 3 FA from R1 to R9 had a harmful effect on the absorptive capacity of the intestine, subsequently it decreased the growth performance of Nile tilapia fish. To our knowledge, there was scarce information on the effect of altering omega 6 to omega 3 FA ratio on the absorptive capacity of the intestine. Fish oil is rich in omega 3 FA which maintain integrity of cell membrane of the enterocytes which might show the higher length of the jejunum villi than soy oil. Conclusion Increasing the ratio of omega 6 to omega 3 FA from R1 to R9 decreased growth performance and feed utilization of the Nile tilapia. Also, it cleared that feeding more omega 3 and less omega 6 FA was very essential for blood and liver cells. Feeding R9 weakened immune response against bacterial infection and decreased absorptive capacity of jejunum villi. However, it could feed R3 without adverse effect on growth performance, feed utilization and absorptive capacity of the intestine. Acknowledgments The authors would like to thank the stuff members of Feed, Water and Food of animal origin analysis lab, Faculty of Veterinary Medicine, Kafrelsheikh University for their practical provision. Conflict of interest There is no conflict of interest. References 1. Food and Agriculture Organization. Fish to 2030 Prospects for Fisheries and Aquaculture. World bank report number 83177-GLB. December 2013. 2. National Research Council. Nutrient requirements of fish and shrimp. National Academies Press, Washington 2011. 3. El-Adawy M, El-Aziz MA, El-Shazly K, Ali NG, El-Magd MA. Dietary propionic acid enhances antibacterial and immunomodulatory effects of ox-ytetracycline on Nile tilapia, Oreochromis niloticus. Environmental Science and Pollution Research, DOI: 101007/s11356-018-3206-5 2018. 4. Turchini G, Torstensen B, Ng W. Fish oil replacement in finfish nutrition. Reviews in Aquaculture 2009; 1:10-57. 5. Montero D, Socorro J, Tort L, Caballero M, Robaina L, Vergara J, Izquierdo M. Glomerulone-phritis and immunosuppression associated with dietary essential fatty acid deficiency in gilthead sea bream, Sparus aurata L., juveniles. Journal of Fish Diseases 2004; 27: 297-6. 6. Association of Official Analytical Chemists. Official Method of Analysis 2010; 18th ed. AOAC, Gaithersburg, MD. 7. Lucky Z. Methods for Diagnosis of Fish Disease 1977. Amerind Publishing Co. New York, 1977. 8. Schalm O. Veterinary hematology 1986. Lea and Febiger Philadeiphia. 4th ed. 9. Barros M, Lim C, Klesius P. Effect of soybean meal replacement by cottonseed meal and iron supplementation on growth, immune response and resistance of channel catfish (Ictalurus puctatus) to Edwarsiella ictaluri challenge. Aquaculture 2002; 207: 263-79. 10. Weichselbaum C. An accurate and rapid method for the determination of proteins in small amounts of blood serum and plasma. American journal of clinical pathology 1946; 16: 40-9. 11. Doumas B, Waston W, Biggs H. Albumin standards and the measurements of serum albumin with Bro-mocresol Green. Clinica Chimica Acta 1971; 31: 87-6. 12. Abdelhady D, El-Abasy M, Abou-Asa S, Elbialy Z, Shukry M, Hussein A, Saleh A, El-Magd M. The ameliorative effect of Aspergillus awamori on aflatoxin b1-induced hepatic damage in rabbits. World Mycotoxin Journal 2017;10:363-73. 13. Kajita Y, Sakai M, Atsuta S, Koba-yash M. The immunostimulatory effects of levamisole on rainbow trout, On-corhnchus mykiss. Fish Pathol-ogy1990; 25: 93-8. 14. SPSS. Statistical and package for social science, SPSS for windows release14.0.0, 19 June, 390 E. Nassef, E. Hegazi, A. Sherif, M. Elshamy, A. Bakr, T. Abouzed 2004." Standard version, copyright SPSS Inc., 19892004. 15. Szabo A, Mezes M, Hancz C, Molnar T, Varga D, Romvari R, Febel H. Incorporation dynamics of dietary vegetable oil fatty acids into the triacylglycerols and phospholipids of tilapia (Oreo-chromis niloticus) tissues (fillet, liver, visceral fat and gonads). Aquaculture Nutrition 2011; 17: 132— 47. 16. Peng X, Li F, Lin S, Chen Y. Effects of total replacement of fish oil on growth performance, lipid metabolism and antioxidant capacity in tilapia (Ore-ochromis niloticus). Aquaculture International 2016; 24:145-56. 17. Han C, Zheng Q, Feng L. Effects of total replacement of dietary fish oil on growth performance and fatty acid compositions of hybrid tilapia (Ore-chromis niloticus 9 O.aureus). Aquaculture International 2013; 21:1209-17. 18. Ng W, Chong C, Wang Y, Romano N. Effects of dietary fish and vegetable oils on the growth, tissue fatty acid composition, oxidative stability and vitamin E content of red hybrid tilapia and efficacy of using fish oil finishing diets. Aquaculture 2013; 372-375: 97-110. 19. Ribeiro P, Logato P, Paula D, Costa A, Murgas L, Freitas R Effect of different oils in the diet on lipogenesis and the lipid profile of Nile tilapias. Rev Bras Zootec 2008; 37 (8):1331-7. 20. Rinchard J, Czesny S, Dabrowski K Influence of lipid class and fatty acid deficiency on survival, growth, and fatty acid composition in rainbow trout juveniles. Aquaculture 2007; 264:363-71. 21. Guler M, Yildiz M. Effects of dietary fish oil replacement by cottonseed oil on growth performance and fatty acid composition of rainbow trout (Oncorhynchus mykiss). Turk J Vet Animal Science 2011; 35 (3): 157-67. 22. Villeneuve L, Gisbert E, Le Delliou H, Cahu C, ZamboninoInfante J. Intake of high levels of levels of vitamin A and polyunsaturated fatty acids during different developmental periods modifies the expression of morphogenesis genes in European sea bass (Dicentrachus labax). Br J Nutrition 2006; 95: 677-87. 23. Watkins B, Li Y, Lippman H, Seifert M. Omega-3 polyunsaturated fatty acids and skeletal health. Exp Biol Med 2001; 226:485-97. 24. Caballero M, Izquierdo M, Kjorsvik E, Fernandez A, Rosenlund G Histological alterations in the liver of sea bream, Sparus aurata L., caused by short or long term feeding with vegetable oils: recovery of normal morphology after feeding fish oil as the sole lipid source. J Fish Diseases 2004; 27:531-41. 25. Klinger R, Blazer V, Echevarria C Effects of dietary lipid on the hematology of channel catfish, Ictalurus punctatus. Aquaculture 1996; 147, 22533. 26. Bell J, McGhee F, Campbell P, Sargent J. Rapeseed oil as an alternative to marine fish oil in diets of post-molt Atlantic salmon (Salmo salar): changes in flesh fatty acid composition and effectiveness of subsequent fish oil "wash out." Aquaculture 2003; 218:515-28. 27. Calder P. Polyunsaturated fatty acids and inflammation. Prostaglandins Leukotrienes Essential Fatty Acids 2006; 75: 197-202. 28. Genc E, Yilmaz E, Akyurt I. Effects of dietary fish oil, soy-acid oil, and yellow grease on growth and hepatic lipidosis of hybrid tilapia fry. Israeli JAquaculture-Bamidgeh 2005; 55: 90-6. 29. Robaina L, Izquierdo M, Moyano F, Socorro J, Vergara J, Montero D. Increase of the dietary n3/n6 fatty acids ratio and addition of phosphorus improves liver histology alterations induced by feeding diets containing soybean meal in gilthead seabream (Sparus aurata). Aquaculture 1998; 161: 281-93. 30. Menoyo D, Izquierdo M, Robaina, L, Gines R, Lopez-Bote C, Bautista J Adaptation of lipid metabolism, tissue composition and flesh quality in gilthead sea bream (Sparus aurata) to the replacement of dietary fish oil by linseed and soybean oils. Br J Nutrition 2004; 92:41-52. 31. Leaver M, Villeneuve L, Obach A, Jensen L, Bron J, Tocher D, Taggart J. Functional genomics reveals increased cholesterol and highly unsaturated fatty acid biosynthesis after dietary substitution of fish oil with vegetable oils in Atlantic salmon (Salmo salar). BMC Genomics 2008; 9:299. Doi: 10.1186/1471-2164-9-299. 32. Kiron V. Fish immune system and its nutritional modulation for preventive health care. Animal Feed Science Technology 2012; 173:111-33. 33. Mourente G, Good J, Bell J. Partial substitution of fish oil with rapeseed, linseed and olive oils in diets for European sea bass (Dicentrarchus labrax L.): effects on flesh fatty acid composition, plasma prostaglandins E-2 and F-2 alpha, immune function and effectiveness of a fish oil finishing diet. Aquaculture Nutrition 2005, 11:25-40. Slov Vet Res 2019; 56 (Suppl 22): 391-7 DOI 10.26873/SVR-777-2019 Original Research Article MONITORING OF PATHOLOGICAL LESIONS IN NON-GRAVID OVINE UTERUS Karam H. Al-Mallah1*, Mariem M. Hussien2 department of pathology and poultry diseases, college of Veterinary Medicine, University of Mosul, Iraq, 2Veterinary hospital, Ninavah directorate of agriculture, Iraq Corresponding author, E-mail: karamalmallah@yahoo.com Abstract: A total of 108 non-gravid ovine uteri were collected from slaughtered ewes at Mosul slaughter house and private butcheries to monitor and identify the uterine pathological lesions. The uteri were grossly examined for observation of gross pathological lesions. Tissues samples were collected for histopathological studies. The incidence of different pathological lesions was calculated. Diffuse or nodular endometrial hyperplasia, endometrial degeneration and desquamation and endometrial coagulative necrosis at ratios 36.11%, 20.37% and 16.66% respectively. Endometritis and subendometrial fibrosis were noticed in 7.40% and 3.70% of the examined samples. Also vascular hyperemia and vascular wall thickening were observed in 12.96% and 5.55% of total examined samples. It is concluded that most of the detected lesions in nongravid uteri appears to be related with previous infections or pregnancies. Key words: Nongraviduteri; lesions; ewes Introduction Uterus is the largest part of female genital tract which is formed from perimetrium, my-ometrium and the endometrium. Endometrium is essential for fetal implantation and placenta-tion. The uterine glands in the endometrium secrets uterine milk necessary for nutrition of the conceptus during the preimplantation period. Also the uterine glands secrete PGF2 a necessary for luteolysis of the CL periodicum with subsequent initiation of a new oestrous cycle (1). The uteri in farm animals including ewes can be affected with a wide range of pathogens. Hyperemia, hemorrhage and hydrometra were recorded as circulatory disturbances in the uterus (2, 3) .Uterine atrophy may arise due to ovarian dysfunction or nutritional deficiency (4). Physical affections or trauma may result in uterine torsion, uterine prolapse and uterine rupture especially when occur near the time of parturition (2). Metritis and endometritis are common uterine affections in farm animals which may be acute or chronic resulting in infertility (5).They may develop more complicated sequels including pyometra and muco-metra (2). Parasitic infestation like Hydatid cysts, Echinococcusgranulosus, was recorded in the uterus (6). Uterine tumors including leiomyoma and lymphosarcoma(2) squamous cell carcinoma (7) and hemangioma (8) were also recorded in cattle and sheep. The present study aimed to monitor and identify the types and incidence of uterine lesion in the ovine uterus in the Mousl region. Received: January 2019 Accepted for publication: February 2019 392 K. Al-Mallah, M. Hussien Materials and methods Directly after slaughtering 108 non-pregnant uteri were collected from ewes at Mosul abattoir and several private butcheries in the city for a period extended from November 2012 to May 2013. The uteri were identified and transported in ice tank to the Department of pathology and poultry disease, College of Veterinary Medicine, University of Mosul. The uteri were macroscopically examined for presence of gross pathological lesions. These lesions were recorded and some of them were photographed. Tissues samples were collected from two uterine horns (apex, middle part and base of each horn as well as from uterine body. The samples were preserved in 10% neutral buffered formalin for two days. The samples were prepared for histopathological examination and stained with H&E according to Khoda-karm-Tafti and Davari (4). The prepared slides were examined under light microscope, Kruss, Germany, and photographed using digital camera (SONY Japan). The pathological changes were recorded and classified as has been mentioned in table, 1. Also the incidence of each specific pathological lesion was calculated in relation to the total number of the examined uteri. Results Mild to moderate cellular adaptations lesions as being manifested by endometrial hyperplasia and endometrial atrophy were recorded at the percentages of 36.11% and 1.85% respectively (Table, 1 and Figs. 1, 2). Disturbances in cell metabolism as being represented by endometrial degeneration and desquamation (20.37%) and endometrial coag-ulative necrosis (16.66%) were recorded (Table, 1 and Figures, 3, 4).Inflammations and repair were recorded as endometritis (7.4%), sub endometrial fibrosis (3.7%), placental retention and adhesions (0.93%), pyometra (1.85%). Also endometrial scarification, endometrial ulceration, myometrial abscesses and granuloma were recorded at the rate of 0.93% for each (Ta-ble1 and Figs. 5-8). The circulatory disturbances including hy-peremia, hemorrhage and vascular wall thickening were recorded at the rates of 12.96%, 2.77% and 5.55% respectively (Table1 and Fig. 9). Disturbances of pigmentation were observed as hemosiderosis and jaundice at the rates of 1.85% and 0.93% respectively. A single case of uterine tumor was diagnosed as metastatic squamous cell carcinoma in the uterus at a rate of 0.93% (Figs. 10, 11, Table 1). Discussion The aim of the present study was to screen the incidence and the types of pathological lesions in the ovine uterus in the Mousl region. The incidence of endometrial hyperplasia (36.11%) is higher than that (2.3%) recorded in Basarha region (10). This may be attributed to the variations in the environmental condition and the in the type and incidence of diseases producing such conditions between the two regions. The endometrial hyperplesia may arise as result of oestrogen level. Reynolds et al, (2009) (9) observed s similar conditions in overectomized ewes infected with higher doses of oestrogen. Also endometrial hyperplasic condition was recorded in ewes fed on estrogen rich clover (11). Endometrial atrophy was recorded in the bovine uterus in Al-qisysia province (12). It was mainly detected in the uteri affected with chronic endometritis (4, 13). Disturbances in cell metabolism appeared as endometrial degeneration and necrosis. The degenerative changes may be confused with physiological apoptosis normally occurring during estrus cycle (14). However the pathological endometrial degenerations and necrosis usually accompany abortions, endometritis and prolonged dystocia as mentioned by (5). Endometrial necrosis and desquamation were diagnosed in case of chlamydial infections. Also stated endometrial degenerations and fibrinonecrotic endometritis were induced in pregnant ewes after experimental intravenous injection of Trypano-somaevansi (16). Melo et al, (2017) (17) found that feeding of pregnant goats on green leaves of the plantMultiglandulosa terapterys resulted in abortion, focal placentitis and coagulative necrosis of endometrium. Monitoring of pathological lesions in non-gravid ovine uterus 393 Table 1: The incidence of pathological lesions in the non-pregnant uteri of ewes collected from Al-Mousl region Type of lesion Classification of the lesion Incidence Rate Percentage Cellular adaptations Endometrial hyperplasia (Nodular or diffused ) 39/108 36.11% Endometrial atrophy 2/108 1.85% Disturbances in cell metabolism Endometrial degeneration and desquamation. 22/108 20.37% Endometrial coagulative necrosis 18/108 16.66% Inflammation and repair Endometritis 8/108 7.4% Sub endometrial fibrosis 4108 3.7% Placental retention 1/108 0.93% Pyometra 2/108 1.85% Endometrial scarification 1/108 0.93% Ulceration 1/108 0.93% Myometrial abscess 1/108 0.93% Granuloma 1/108 0.93% Circulatory disturbances Hyperemia 14/108 12.96% Hemorrhage 3/108 2.77% Vascular wall thickening 6/108 5.55% Disturbances of pigmentation Hemosiderosis 6/108 1.85% Jaundice 1/108 0.93% Tumors Squamous cell carcinoma 1/108 0.93% A B B Figure 1: Photomicrograph of a section in ovine uterus showing nodular hyperplasia in endometrium (A). H&E, 40X Figure 2: Photomicrograph of a section in ovine uterus showing endometrial atrophy manifested by shrinking and reduction in the size of the uterine glands (A) and subendometrial. H&E, 115 X 394 K. Al-Mallah, M. Hussien Figure 3: Photomicrograph of a section in ovine uterus showing vacuolar degeneration in endometrial cells (A) Sloughing of endometrium (B). H&E, 40X Figure 5: Photomicrograph of a section in ovine uterus showing endometritis manifested by infiltration of inflammatory cells (A) and edema (B). H&E, 165 X. Figure 4: Photomicrograph of a section in ovine uterus showing coagulative necrosis in endometrial cells (A) Sloughing of endometrium (B). H&E, 115X Figure 6: Photomicrograph of a section in ovine uterus showing subendometrial fibrosis (A). H&E, 40X. Figure 7: Photomicrograph of a section in ovine uterus showing scarification being obvious by fibrous tissue deposition (A) extending through endo-metrium and myometrium. H&E, 68X Figure 8: Photomicrograph of a section in ovine uterus showing microabscesses (A) within myome-trium. H&E, 40X B A B r A A A • -V Monitoring of pathological lesions in non-gravid ovine uterus 395 1 A B B C A B Figure 9: Photomicrograph of a section in ovine uterus showing hyperemic artery (A) thickening of vascular wall (B) hemosiderosis of vascular musculature (C). H&E, 68X_ B B A A Figure 11: Photomicrograph of a section in ovine uterus showing sequamous cell carcinoma appeared as actively mitotic sequamous cell sheathes (A) and keratin nest structures (B). H&E, 200 X The incidence of the endometritis in the current study (7.4%) was higher than those recorded in Ireland (2.3%) (18), Iran (2.3%) (4) and India (24.8%) (20) and lower than that (24.8%) in Al-Basraha Iraq (19). The variations in rate may be attributed to the differences in environment and animal administration systems but still common affections worldwide. Many pathogens causing endometritis such as Brucella abortus (21), Salmonella, Klebsiella , E.coli , Staphylococcus aureous and Proteus, were isolated from genital tract (22) suffering from endometritis . Viral endometritis accompanies diseases like FMD (23). Figure 10: Photomicrograph of ovine uterus showing sequamous cell carcinoma appeared as actively mitotic cell sheathes (A) and hyperchromatic multi mitotic figure cells invades uterine glands (B). H&E, 256 X Subendometrial fibrosis indicates chronic metritis which is usually associated with extensive deposition of collagen fibers. Charlotta et al, (200) (24) stated that fibrosis was observed in the uteri of mares with the advancement of age. Retained placental tissue was recorded at lower rate (0.93%) in the current study. The etiology may be mechanical, nutritional also bacterial, viral and mycotic infections (25). Pyometra was recorded at rate of 1.85% of examined uteri. It was higher than that (0.15%) recorded by Khodakarm (2013) (4) and comparable to that recorded by Sharma et al, (2014) (20) in ewes. It was realized that pyometra may be related with cystic ovaries in affected animals (26). Endometrial scarification (0.93%), as indicated by scar granulation tissue extending through endometrium and myometrium can be explained as previous healing process from previous injuries of parturition (27). This lesion was recorded in 3 out of 30 ewes experimentally infected with Mycoplasma capricolum capripneumoniae (28). The presence of Microabscesses in myometrium resembles the results of (29) in goats who confirmed the lesion , also (30) suggested that systemic pyogenic diseases like tuberculosis may induce that form of lesions in the genital system .granulomatous reaction was identified at 0.93% of samples , it may be expressed by bacterial , viral , fungal or parasitic infection of 396 K. Al-Mallah, M. Hussien uterus (31), Also (8) recorded cases of granulomatous metritis in cattle and classified as military tuberculosis and (29) stated that granulomatous endometritis in goat was recorded at 1.29% and explained as brucellosis . Circulatory disturbances manifested by hy-peremia at 12.96% of samples mostly accompanied physiological changes as increasing estrogen level. Ali and Madboli (2013) (32) stated that increasing alpha and /or beta oestrogen receptors and decreasing norepinephrine in uterus causes hyperemia in guinea pig. Hemorrhage in myometrium and sub endometrium was noticed in three samples at rate of 2.77%. It has been thought that it accompanies sever constrictions of the myometrium during delivery or abortion which is called postpartum atonic haemorrhage (33). Thickening of the vascular walls of uterus was described by (34) as morphological changes in the myometrial arteries resulting from accumulation of collagen and mucopoly-saccharides in subendothelium with focal fi-brotic changes in smooth muscle layers. It is usually seen in women after multiple pregnancies. .Hemosiderosis is a precipitation of hemosiderin pigment in tissue and it is reported by Beena et al, (2015) (29) in the caprine uteri in India at 25.32% of the examined samples. Jaundice stains body organs and tissues yellow by bilirubin and not specific to genital system. Squamous cell carcinoma was diagnosed in only one uterus sample at ratio 0.93%. It may arise outside the uterus and metastasized to the uterus because this type of tumors is not specific to the genital system. This suggestion may be supported by the observations of (35, 36) who recorded this type of tumors in human uteri Conflict of interest The authors declare that they have no conflict of interest. References 1. Pineda, M H. Female Reproductive system. Pineda M H, Dooley M P. McDonald's veterinary Endocrinology and reproduction. 5th Ed. Iowa State Press, Blackwell publishing Company. Iowa, USA. 2003:295-300. 2. Schlafer D H, Miller R B. Female genital system. Maxie G M. Jubb, Kenned and Palmers Pathology of Domestic animals, Vol3, 5th Ed. Saunders ELSEVIER. China. 2007. 3. Azawi O I, Ali A J, Lazim E H. pathological and anatomical abnormalities affecting buffalo cows reproductive tract in Mousl. Iraq Journal of veterinary science. 2008. 22(2):159-67. 4. Khodakarm-TaftiA, Davari A. congenital and acquired abnormalities of Reproductive tract of non-pregnant ewes slaughtered in fars province. Iranian journal of Veterinary Research. 2013. 14:1404. 5. Brozos C N , Lazariads L, Karagainnis I, Kiossis E, Tsousis G, psychas V, Giadinis N D. Prolonged dystocia, uterine necrosis and ovariohyster-ectomy in Chios ewe. Turk. J. Vet. Anim. Sci. 2012. 36(2):211-3. 6. Bickers W M. Hydatid disease of the female pelvis. AM. J. best. Gynecol. 1970. 107:477-483. 7. Ferren M L, Lacasta D , Ramos J J , Jalon J G , Arcaute R. M , Condet, T. Squamous cell Carcinoma of the vagina ANO Cervix in Sheep Case Report. Aetaveterinaria Hungarica. 2011. (1): 125-9. 8. Hatipoglu F, Qrtatali M, Kirin M M, Erer H, CiftCi M K. An Abattoir study of Genital pathology in cows: n. Uterus, Cervix and Vagina. Re-vuMedVet. 2002. 153(2):93-100. 9. Reynolds L P, Kiresch J D, Kraft K C, Knut-son D L, McClaflin W J, Redmer, D A. Time-Course of the Utrine Response to Estradiol-17p in Ovariectomized Ewes : Uterine Growth and Micro-vascular Development. Biology of Reproduction. 1998. 59: 606-12. 10. Adams N R. Pathological changes in the tissue of infertile ewes with clover disease .j.comp.Pathol.1976 .86: 29-35. 11. Abdul-kareem, K F, Al-Mayah A A, Dawood, K. Abnormalities of reproductive organs of ewes, A prospective Histopathological study. Bas. J, vet. Res. 2007. 6(1):97-109. 12. Rhyaf, A. G. Histopathological Study of En-dometritis of the cows AL-Qadisya Journal of Vet. Med. Sci. 2010. 9(1). 13. Dogan I , Sonmez G , Sagirkaya H . Histo-pathological investigation of endometritis in report breeder cow .Indian journal of animal sciences. 2002. 72 (3): 223-6. 14. Hammond J. The physiology of reproduction in the cow. 2nd Ed. Cambridge University Press. 2014. 15. Sanderson T P. Pathology and diagnosis of chlamydial abortion in ewes Retrospective Theses and Dissertations. 1992. Monitoring of pathological lesions in non-gravid ovine uterus 397 16. Mostafa D S, Amer H A, Rwaida M R. Pathological and Some Biochemical Studies on Pregnant Ewes Experimentally Infected with Tryp-anosomaevansi. Egypt. I. Comp. path. & Clinic. Path. 2008. 21(1): 372-400. 17. Melo M M , Vasconcelos A C, Dants G C, Serakides R, Alzamora-Filho F. Expermintal intoxication of pregnant goats with Tetrapterysmultiglan-dulosa A. Juss. (Malpighiaceae). Arq. Bras. Med. Vet. Zootec. 2001. 53(1). 18. Alsota R A, Vaugnan L, Collins J D. An abattoir survey of Ovine Reproductive tracts in Ireland. Theriogenology. 1998. 50 (3), 457-64. 19. Dawood, K. E. Pathological abnormalities of the reproductive tract of ewes in Basra, Iraq Vet Rec. 2010. 166 (7):205-7. 20. Sharma A, Kumar P, Singh M, Vasishta N. K. Reproductive health Status of North Western Himalayan Gaddi Sheep : An abattoir Study : Open Veterinary Journal. 2014. 4(2):103-6. 21. Abd El-Razik A, Desouky H M , Ahmed W M. Investigation on brucellosis in Egyptian Baladi Dose with emphasis on evalution of dignostic techniques. Pakistan J. Biol. Sci. 2007. 10:342-8. 22. AL-Zubaidi S F. Antibiotic Susceptibility of Bacteria Isolated from The Genital System of Cows in AL-hilla, Iraq: Malaysian. j of Vet. Rec. 2015. 6 (1):53-59. 23. Ali, S M, Madboli A A. Further Immuno-pathological Studies on the Female Genital System and Some Visceral Organs of Sheep and Goats Naturally Infected with Foot and Mouth Disease Virus Global Vet. 2013. 11(4):472-80. 24. Charlotta, O. Development of endometrial fibrosis in the mare: factors involved in tissue remodeling and collagen deposition. 2008. http://hdl. handle. net/1842/4218 25. Han Y K, Kim I H . Risk factors for retained placenta and the effect of retained placenta on the occurrence of postpartum diseases and sub-sequent reproductive performance in dairy cows. 2005. J. Sci. 6:53-59. 26. Iqbal A, Jafri S A, Khan S A. Incidence of various Gross Reproductive disorders in Female Teddy Goats in different Age Groups. Pakistan J. Agri. Res. 1992. 13(3). 27. Dhaliwal G S, Murray R D. Some aspects of immunology of the bovine uterus related to treatments for endometritis. Anim. Reprod. Sci. 2001. 67:135-52. 28. Madboli AA. Histopathological and Im-munohistochemical Studies on Field Samples of Uterus, Placenta and Some Visceral organ Collected from Ewes and Goat Naturally Infected with Mycoplasma Capricolum Capripneumoniae. Global Verterinaria. 2015. 15(4):351-6. 29. Beena P, Shivasharanappa N, Gurnraj K , Gupta V K , Neeraj K , Gangwar D D , Singh M , Karikalan A K , Kumar H, Kumar A , Singh, R. Occurrence of pathological conditions in the finial genitalia of goat. Indian. J. Vet. Pathol. 2015. 39(3): 197-201. 30. Regassa F , Mengesha D , Dargie M , Tolosa T. Abattoir evidence on association between uterine and ovarian abnormalities in Ethiopian highland ewes . Anim.Reprod.Sci. 2009. 111: 384-90. 31. Almoujahed M O, Briski L E, Prysak M, Johnson L B, Khatib R. Uterine granuloma: clinical and pathological features. Am j clinpathol. 2002. 117:771-5. 32. Garris D R, Ingenito AI, Mconnaughey M M, Dar M S. Regulation of estrogen-induced uterine hyperemia and contractility in the guinea pig: cho-linergic modulation of an alpha-adrenergic response .Biol. Reprod. 1984. 30 (4): 803-8. 33. Hayashi M , Mori Y , Nogami K , Takagi Y, Yori M , Ohkura T. A hypothesis to explain the occurrence of inner myometrial laceration causing massive postpartum hemorrhage. Acta obstet Gynecol scand. 2000. 79 (2):99-106. 34. Obimbo M M ,Ogengo J A , Saidi H. Comparative regional morphometric changes in human uterine artery before and during pregnancy. Pan African Medical Journal. 2012. 13:30. Available online at: http\\ www.panafrican-med-Journal. 35. Barnett H. Squamous cell Carcinoma of the body of uterus. I Clin. Path. 1965. 18, 715. 36. Bagga P K , Jaswal T S , Datta U , Mahajan N C. Primary endometrial squamous cell carcinoma with extensive squamous metaplasia and dysplasia . 2008. 51(2): 267-86. Slov Vet Res 2019; 56 (Suppl 22): 399-408 DOI 10.26873/SVR-778-2019 Original Research Article COMPARATIVE HISTOPATHOLOGICAL CHANGES OF LIVER, KIDNEY AND APPENDIX OF RABBITS TREATED WITH INORGANIC NANO CHROMIUM TO AMELIORATE HEAT STRESS EFFECT Sahar H. El-Nagar1*, Mohamed A. Helal1, Shawky Mahmoud2 , Leanne Dillard3 department of Animal Wealth Development, Faculty of Veterinary Medicine, Kafrelsheikh University, 33516, Egypt, 2Department of Physiology, Faculty of Veterinary Medicine, Kafrelsheikh University, 3Department of Animal Sciences, Auburn University, Auburn, 36849 USA Corresponding author, E-mail: sahr_hamdy@vet.kfs.edu.eg Abstract: The objective of the current experiment was to describe the effects of nano-chromium chloride (CrCl3) on chronic hyperthermia (32.8 ± 1.5 °C) on rabbit liver, kidney, and appendix in comparison with rabbits at room temperature (24.5 ± 1.3 °C) and treated with the same concentration of nano-chromium chloride. For this study, 108 rabbits of two different breeds (New Zealand White and Rex) were used and randomly allocated into 12 groups. The study was conducted as a completely randomized 2 * 2 * 3 factorial (n = 9) design. Treatments were temperature, breed, and concentration of nano-chromium chloride (0, 1, or 2 mg/L) the results showed that heat stress caused granular hepatic vacuolation, severe congestion of the central vein, and sinusoids in the liver. As well as degenerative changes within the epithelial lining of tubules in the kidney and lymphoid depletion in the appendix. The liver tissue of the New Zealand rabbits was affected more by heat stress than Rex Rabbits, but no difference was observed in the kidney or appendix tissues. The addition of 2 mg/L nano-chromium was more effective than 1 mg/L on the heat stressed rabbit tissues, but it caused hepatic vacuolation with glycogen infiltration in liver tissue and mild vacuolation in the renal tubular epithelium. Key words: rabbit; nano-chromium; heat stress; histopathology Introduction The thermal comfort zone for rabbits is 210C. Any elevation from this temperature is considered heat stress (1). Heat stress is divided into two types acute and chronic heat stress. This division depends on the period of exposure and the presence of tissue damage including: pyknosis, apoptosis, necrotic areas, and an increase in melanomacrophage centers in liver tissues through, DNA digestion and cell mem- brane destruction (2, 3). Previous studies (4-6) observed vacuolated hepatic degeneration with dilation and congestion of sinusoid, widespread necrosis, and infiltration of leukocyte in some parts of the liver and kidney in heat stressed rabbits. The authors also reported damage to the glomeruli of the kidneys. As a consequence, Ondruska et al. (7) reported high animal mortality rates, leading to economic losses for rabbit producers during summer season. Received: January 2019 Accepted for publication: February 2019 400 S. El-Nagar, M. Helal, S. Mahmoud, L. Dillard More recent studies have tried to reduce heat stress through different management and nutritional methods. Nano-chromium is one of the most important nutritional supplements that can be used to decrease and possibly eliminate heat stress damage. However, low doses must be used as higher doses may lead to adverse animal effects such as degenerative changes and necrosis in liver as well as hyaline casts in kidney and tubules and glomeruli (8). Chromium is an essential trace mineral required for most vital metabolic processes. It is used in carbohydrate, protein, and lipid metabolism (9); therefore it has anabolic effects during periods of stress, including heat stress. Research has shown that nano-chromium has an anabolic effect when used in low concentrations and enhances the nucleic acid synthesis in the liver of the mouse (10). The objective of the current study was to determine what dosage of nano-chromium chloride is needed to ameliorate the degenerative changes in the vital tissues of heat stressed rabbits. Materials and methods This study was carried out at the Department of Animal Wealth Development in the Faculty of Veterinary Medicine at Kafrelsheikh University in Kafrelsheikh, Egypt. The experiment was approved by the guiding of committee on Animal Welfare and Ethics of the Faculty of Veterinary Science, Kafrelsheikh University, in accordance with Egyptian national laws regarding animal welfare. One hundred eight weaning aged rabbits of two different breeds (Rex and New Zealand White; 35 ± 3 and 33 ± 2 d weaning age, respectively) were used to evaluate the effect of nano-chromium chlordie on liver, kidney, and appendix tissues. Experimental design Each breed had an initial body weight of 602 ± 9.5 and 531 ± 6.5 g for Rex and New Zealand Breed, respectively. Animals of each bred were divided equally into 6 groups with 9 rabbits in each group. Three groups of each breed were subjected to severe heat stress (32.8 ± 1.5 °C; HT) and the other three groups were reared under room temperature conditions (24.5 ± 1.3 °C; RT) (11). Each group contained 3 replicates with 3 rabbits in each replicate. Each replicated group was reared on wire cages 50 x 50 x50 cm and fed commercial rabbit pellets (18% crude protein) ad libitum. The nano-chromium particles were prepared by chemical precipitation method of chromium chloride salt (12), and then characterized by transmission electron microscope (TEM) at the Nanotechnology Institution of Kafrelsheikh University, Egypt. The resulting particles were 40 - 60 nm. These particles were then added to the drinking water at 3 different concentrations (0, 1 or 2 mg/L; C, 1, and 2, respectively) for the duration of the 8 week study. At the end of the experiment rabbits were slaughtered for tissue collection. The liver, kidney and appendix tissues were collected in 10% formalin, sectioned, and stained with H&E stain for microscopic examination (13). Results Liver The liver of the control groups from both breeds and untreated with nano-chromium showed the normal arrangement of the hepato-cytes in cords around the central vein and normal sinusoids (Figure 1- A and C). However, the liver of the groups that were exposed to heat stress but untreated with nano-chromium showed that the New Zealand breed was more susceptible to heat stress than the Rex breed. This is due to the appearance of severe congestion of the central vein and sinusoids, as well as granular hepatic vacuolation (Figure 1-B and D). Mild hepatic valuation in New Zealand rabbits treated with 1 mg/L nano-chromium at room temperature was observed (Figure 2). This is consistent with excessive glycogen storage in the liver. This was also seen when New Zealand rabbits received the same concentration of nano-chromium, but that were also exposed to heat stress (Figure 2-C). However, when Rex Rabbits were treated with 1 mg/L of nano-chromium under room temperature conditions, they appeared to have Histopathological changes of liver, kidney and appendix and inorganic nano chromium 401 normal hepatocyte and liver tissue as seen in Figure 2-B. When exposed to heat stress with the same concentration of nano-chromium, the Rex rabbits responded the same as the New Zealand rabbits, and also appeared to have congestion of the sinusoids. The addition of 2 mg/L nano-chromium caused hepatic vacuola-tion with glycogen infiltration that was severe in New Zealand breeds (Figure 3-A) and mild in Rex Rabbits (Figure 3-B). While in animals exposed to heat stress, the same effect was observed in both breeds which appears as marked decrease in hepatic vacuolation. Kidney There was no observable difference in kidney tissues between breeds under the same temperature conditions. Under room temperature conditions, the two breeds showed normal renal glomeruli and tubules (Figure 4-A and C). However under heat stress, they showed degenerative changes within the lining epithelium of the renal tubules (Figure 4-B and D). In regards to renal tissues, there was no observable difference in the response between the breeds when treated with 1 mg/L nano-chromium and under the same temperature (Figure 5-A and C). The tissue appeared to be normal under room temperature conditions. Mild degenerative changes in the lining of the epithelium of the renal tubules appeared in heat stressed animals (Figure 5-B and D). In rabbits treated with 2 mg/L nano-chromium, there was no observable difference in response between two breeds under room temperature. Rex rabbits showed normal renal glomeruli and tubules (Figure 6-A). The New Zealand rabbits showed normal renal glomeruli, but showed mild vacuolation of the renal tubular epithelium (Figure 6-B). There was also no difference between the two breeds under heat stress compared with New Zealand under room temperature. Appendix Under room temperature conditions and treated with 0 mg/L nano-chromium, the two breeds showed normal lymphoid follicles (Figure 7-A and C), but heat stress caused a mild lymphoid depletion in the germinal center of the follicle of Rex rabbits and on the basal follicle of the New Zealand rabbits (Figure 7-B and D). When rabbits were treated with 1mg/L nano-chromium under room temperature conditions, they were not different from the untreated rabbits under the same temperature (Figure 8-A and C). However under heat stress, the addition of 1mg/L of nano-chromium ameliorated the lymphoid depletion that resulted from the heat stress in untreated rabbits (Figure 8-B and C). Figure 9 from A to D showed that there was no difference between breeds treated with 2 mg/L nano-chromium under different temperature treatment. Discussion Liver The granular hepatic vacuolations resulting from exposure to heat stress observed in Figure 1 may be due to the activation of mito-chondrial reactive oxygen species (ROS; 14) which can lead to cytotoxicity, apoptotic cell death, and necrosis (15). This degenerative condition appears as vacuoles in hepatocytes. However, the severe congestion of the central vein was likely due to the fact that the liver has the densest concentration of mitochondria, which are overloaded during stress. This can lead to increased blood supply to this organ as compensatory mechanism (16). Figure 2-A and B represent the liver tissue unexposed to heat stress and treated with 1mg/L nano-chromium. The New Zealand breed showed a better response to nano-chromium treatments than the Rex breed. This is evident in the mild hepatic vacuolation that is consistent with excessive glycogen storage. This is in agreement with Huskisson et al. (17) that illustrated that Cr III enhanced glucose uptake by the liver cells. On the other hand, Muthulingam et al. (18) observed that Cr lead to decreased glycogen in the gills, liver, and kidneys of fish. This was observed in Figure 3, where the degenerative changes and congestion of the liver caused by heat stress disappeared and were replaced by marked hepatic vacuolation due to the increased dose of nano-chromium. 402 S. El-Nagar, M. Helal, S. Mahmoud, L. Dillard Figure 1: A) Liver of room temperature (R) Rex rabbits showing normal hepatocyte arranged in cords around the central vein (arrow), H&E, bar = 40 pm. B) Liver of heat stressed (HS) Rex breed showing granular hepatic vacuolation (arrow), H&E, bar = 40 pm. C) Liver of New Zealand-R rabbits showing normal hepatocytes arranged in cords around the central vein (arrow), H&E, bar = 40 pm. D) Liver of HS-New Zealand rabbits showing severe congestion of the central vein and sinusoids (arrowheads) and granular hepatic vacuolation (arrow), H&E, bar = 40 pm Figure 2:A) Liver of room temperature (R) Rex treated with 1 mg/L nano-chromium (T1) showing normal hepatocytes (arrow), H&E, bar = 40 pm. B) Liver of HS Rex rabbits T1 showing sinusoidal congestion (arrowhead) and mild hepatic vacuolation consistent with hydropic changes (arrow), H&E, bar = 40 pm. C) Liver of R -New Zealand rabbits T1 showing mild hepatic vacuolation consistent with over glycogen storage (arrow), H&E, bar= 40 pm. D) Liver of heat stressed (HS) New Zealand rabbits T1 showing hepatic vacuolation consistent with over glycogen infiltration (arrow), H&E, bar = 40 pm Histopathological changes of liver, kidney and appendix and inorganic nano chromium 403 Figure 3:A) Liver of room temperature (R) Rex rabbits treated with 2mg/L nano-chromium (T2) showing mild hepatic vacuolation consistent with glycogen storage (arrow), H&E, bar= 200 pm. B) Liver of Rex heat stressed (HS) rabbits T2 showing marked decrease of hepatic vacuolation (arrow), H&E, bar = 40 pm. C) Liver of R New Zealand rabbits T2 showing marked hepatic vacuolation consistent with glycogen infiltration (arrow), H&E, bar= 200 pm. D) Liver of HS New Zealand rabbits T2 showing marked decrease of hepatic vacuolation (arrow), H&E, bar= 40 pm Figure 4:A) Kidney of room temperature (R) Rex rabbits showing normal renal glomeruli and tubules (arrowhead and arrow respectively), H&E, bar = 40 pm. B) Kidney of heat stressed (HS) Rex rabbits showing marked degenerative changes within the lining epithelium of the tubules (arrows), H&E, bar= 40 pm. C) Kidney of R New Zealand rabbits showing normal renal glomeruli and tubules (arrowhead and arrow respectively), H&E, bar= 40 pm. D) Kidney of HS New Zealand rabbits showing marked degenerative changes within the lining epithelium of the tubules (arrows), H&E, bar= 40 pm 404 S. El-Nagar, M. Helal, S. Mahmoud, L. Dillard hsmi WiBM Sreiwl ^mmMBSBm :SnK?' Ok'-MiI ^mi w tmi ¡§g| llilE^iiMi iTHBlfTlMIMl Figure 5:A) Kidney of room temperature (R) Rex rabbits with 1 mg/L nano-chromium (T1) showing normal renal glomeruli and tubules (arrowhead and arrow respectively), H&E, bar = 40 ^m. B) Kidney of HS Rex rabbits T1 showing mild degenerative changes within the lining epithelium of the renal tubules (arrow), H&E, bar = 40 ^m. C) Kidney of R New Zealand rabbits T1 showing normal renal glomeruli and tubules (arrowhead and arrow respectively), H&E, bar= 40 ^m. D) Kidney of HS New Zealand rabbits T1 showing mild degenerative changes within the lining epithelium of the renal tubules (arrow), H&E, bar = 40 jim Figure 6:A) Kidney of room temperature (R) Rex rabbits with 2 mg/L nano-chromium (T2) showing normal renal glomeruli and tubules (arrowhead and arrow respectively), H&E, bar= 40 ^m. B) Kidney of heat stressed (HS) Rex rabbits T2 showing normal renal glomeruli and mild vacuolation of the renal tubular epithelium (arrow), H&E, bar = 40 ^m. C) Kidney of R-T2 New Zealand rabbits showing normal renal glomeruli (arrowhead) and mild vacuolation of the renal tubular epithelium (arrow), H&E, bar = 40 ^m. D) Kidney of HS-T2 New Zealand rabbits showing normal renal glomeruli (arrowhead) and mild vacuolation of the renal tubular epithelium (arrow), H&E, bar = 40 ^m Histopathological changes of liver, kidney and appendix and inorganic nano chromium 405 Figure 7: A) Appendix of room temperature (R) Rex rabbits showing normal lymphoid follicles covered with epithelial covering (arrow), H&E, bar = 200 pm. B) Appendix of heat stressed (HS) Rex rabbits showing mild degree of lymphoid depletion of the germinal center of the follicles (arrow), H&E, bar= 200 pm. C) Appendix of R New Zealand rabbits showing normal lymphoid follicles covered with epithelial covering (arrow), H&E, bar = 200 pm. D) Appendix of HS New Zealand rabbits showing lymphoid depletion of the germinal center of the basal follicles (arrow), H&E, bar= 200 pm Figure 8: A) Appendix of room temperature (R) rabbits treated with 1 mg/L nano-chromium (T1) showing normal lymphoid follicles (arrow), H&E, bar = 200 pm. B) Appendix of heat stressed (HS)-T1 Rex rabbits showing normal lymphoid follicles (arrow), H&E, bar = 200 pm. C) Appendix of R-T1 New Zealand rabbits showing normal lymphoid follicles (arrow), H&E, bar = 200 pm. D) Appendix of HS-T1 New Zealand rabbits showing normal lymphoid follicles (arrow), H&E, bar = 200 pm. 406 S. El-Nagar, M. Helal, S. Mahmoud, L. Dillard Figure 9: A) Appendix of room temperature (R) rabbits treated with 2 mg/L nano-chromium (T2) showing normal lymphoid follicles (arrow), H&E, bar = 200 pm. B) Appendix of heat stressed (HS) rabbits T2 showing normal lymphoid follicles (arrow), H&E, bar = 200 pm. C) Appendix of New Zealand R-T2 rabbits showing normal lymphoid follicles (arrow), H&E, bar = 200 pm. D) Appendix of HS-T2 New Zealand rabbits showing normal lymphoid follicles (arrow), H&E, bar = 200 pm Kidney When both breeds were exposed to room temperature conditions and untreated with nano-chromium they showed normal glomeruli and tubules (Figure 4-A and C). Kidneys of animals exposed to heat stress, but also untreated with nano-chromium particles showed degenerative changes within the lining of the epithelium of the renal tubules. This is likely due to the cytolytic response of the tissues by the heat followed by endotoximia that enhanced the release of inflammatory cytokines and consequently caused vascular endothelium injury (19). This appeared as degenerative changes in tubular lining epithelium in this study. Figure 5-A and C showed normal renal and glomerular structures when 1mg/L of nano-chromium was administered. This is interrupted to mean that this concentration of nano-chromium is safe and does not alter the renal structure in rabbits housed in room temperature conditions. This level of nano-chromium also decreased, but did not remove, the degenerative changes resulting from heat stress in both breeds. While the addition of 2mg/L nano-chromium did ameliorate the degenerative changes in the heat stressed rabbits, this was replaced by mild vacuolation in lining epithelium. This may be due to glucose uptake by the cell increased by addition of this concentration (17) rather than toxicity. Appendix Animals of both breeds that were not treated with nano-chromium and kept under room temperature conditions appeared to have normal lymphoid follicles, as they were covered with epithelium. This indicates that this temperature was comfortable for the immune system of the rabbits. On the other hand, the groups that were exposed to heat stress and untreated with nano-chromium appeared to have a lymphoid depletion of the germinal center of the follicle (Figure 7-B and D). This may be due to the heat stress enhanced the production of ROS (20) that lead to death of Histopathological changes of liver, kidney and appendix and inorganic nano chromium 407 most of cells (21), including immune cells and resulted in lymphoid depletion. The addition of both 1 and 2mg/L of nano-chromium had the same effect on appendix tissues during heat stress. The addition of nano-chromium prevented the lymphoid depletion resulting from this stress (Figure 8-B and D; Figure 9-B and D). These results may be due to the catalytic effect of Cr III on the H2O2 inactivating enzyme, consequently decreasing the cell apoptosis (22). The structure of the appendix was not altered in groups treated with 2mg/L of nano-chromium, but not exposed to heat stress. This in the agreement with Yucesoy et al. (23), that illustrated that Cr salts do not have an effect on the immune system cells. Conclusion In conclusion, addition of 2 mg/L nano-chromuim particles to the drinking water of the heat stressed rabbits could be used to alleviate the granular degenerative changes and the sinusoidal congestion of the rabbit liver. As well as to ameliorate the effects of the previous concentration on the degenerative changes in the epithelium lining renal tubules. It is also able to reduce the lymphoid depletion in the lymphoid follicles of the rabbit appendix. References 1. Fayez I, et al., Thermoregulation in rabbits. Cahiers Options Mediterraneennes (CIHEAM) 1994. 2. Hernandez-Lopez JR, et al., Thermal Effect of Acute and Chronic Stress on Hepatic and Renal Tissue of the Pacific Sardine, Sardinops sagax caeruleus (Jenyns, 1842)/Efecto del Estres Termico Agudo y Cronico Sobre el Tejido Hepatico y Renal de la Sardina del Pacifico, Sardinops sagax caeruleus (Jenyns, 1842). International Journal of Morphology 2018; 36(1): 212-21. 3. Barry MA, Behnke CA, and Eastman A, Activation of programmed cell death (apoptosis) by cisplatin, other anticancer drugs, toxins and hyperthermia. Biochemical pharmacology 1990; 40(10): 2353-62. 4. Zhang HJ, et al., Heat-induced liver injury in old rats is associated with exaggerated oxidative stress and altered transcription factor activation. The FASEB journal 2003; 17(15): 2293-5. 5. Ando M, et al., Age-related effects of heat stress on protective enzymes for peroxides and microsomal monooxygenase in rat liver. Environmental health perspectives 1997; 105(7): 726. 6. Aengwanich W, and Simaraks S, Pathology of heart, lung, liver and kidney in broilers under chronic heat stress. Pathology 2004; 26(3): 418. 7. Ondruska L, et al., Influence of elevated ambient temperature upon some physiological measurements of New Zealand White rabbits. Veterinarni Medicina 2011; 56(4): 180-6. 8. Raut S, et al., Effect of trivalent chromium on haemato-biochemical and histopathological parameters at higher doses in broilers. Indian Journal of Veterinary Pathology 2012; 36(1): 73-9. 9. Berenjian A, et al., Effect of Chromium Nanoparticles on Physiological Stress Induced by Exogenous Dexamethasone in Japanese Quails. Biological trace element research 2017; 1-8. 10. Okada S, Suzuki M, and Ohba H, Enhancement of ribonucleic acid synthesis by chromium (III) in mouse liver. Journal of inorganic biochemistry 1983; 19(2): 95-103. 11. Pei Y, Wu Y, and Qin Y, Effects of chronic heat stress on the expressions of heat shock proteins 60, 70, 90, A2, and HSC70 in the rabbit testis. Cell Stress and Chaperones 2012; 17(1): 817. 12. Iqbal T, Tufail S, and Ghazal S, Synthesis of Silver, Chromium, Manganese, Tin and Iron Nano Particles by Different Techniques. International Journal of Nanoscience and Nanotechnology 2017; 13(1): 19-52. 13. Luna LG, Manual of histologic staining methods of the Armed Forces Institute of Pathology. 1968. 14. Kikusato M, and Toyomizu M, Heat stress-induced overproduction of mitochondrial reactive oxygen species is down-regulated in laying-type chickens, in Energy and protein metabolism and nutrition in sustainable animal production. 2013; Springer 267-8. 15. Orrenius S, Reactive oxygen species in mitochondria-mediated cell death. Drug metabolism reviews 2007; 39(2-3): 443-5. 16. Pitts J, What Causes a Congested Liver? Healthguidance for better health, 2018. 17. Huskisson E, Maggini S, and Ruf M, The role of vitamins and minerals in energy metabolism and well-being. Journal of international medical research 2007; 35(3): 277-89. 408 S. El-Nagar, M. Helal, S. Mahmoud, L. Dillard 18. Muthulingam M, et al., Impact of heavy metal, chromium on glycogen contents in gill, liver and kidney of freshwater fish, Channa striatus (BLOCH) International Journal of Current Research and Development 2017; 5(1): 1-9. 19. Bouchama A, and Knochel JP, Heat stroke. New England journal of medicine 2002; 346(25): 1978-88. 20. Yang L, et al., Effects of acute heat stress and subsequent stress removal on function of hepatic mitochondrial respiration, ROS production and lipid peroxidation in broiler chickens. Comparative Biochemistry and Physiology Part C: Toxicology & Pharmacology 2010, 151(2): 204-8. 21. Circu ML, and Aw TY, Reactive oxygen species, cellular redox systems, and apoptosis. Free Radical Biology and Medicine 2010. 48(6); 74962. 22. Blasiak J, and Kowalik J, A comparison of the in vitro genotoxicity of tri-and hexavalent chromium. Mutation Research/Genetic Toxicology and Environmental Mutagenesis 2000; 469(1): 135-45. 23. Yücesoy B, et al, In vitro effects of various metals on natural killer cell activity in cultured human lymphocytes. Immunopharmacology and immunotoxicology 1999; 21(3): 599-7. Slov Vet Res 2019; 56 (Suppl 22): 409-19 DOI 10.26873/SVR-779-2019 Original Research Article EFFECTIVENESS OF EXOGENOUS DIGESTIVE ENZYMES SUPPLEMENTATION ON THE PERFORMANCE OF RABBITFISH (Siganus rivulatus) Mahmoud A. O. Dawood1*, Mohamed Mohsen1, Ashraf El-dakar2, Elsaid Abdelraouf1, Eman M. Moustafa3, Hamada A. Ahmed4 1Animal Production Department, Faculty of Agriculture, Kafrelsheikh University, 33516 Kafrelsheikh, Egypt, 2Aquaculture Department, Faculty of Fish Resources, El-Arish, Suez University, Egypt, 3Department of Fish Diseases and Management, Faculty of Veterinary Medicine, Kafrelsheikh University, 33516, Kafrelsheikh, Egypt, 4Department of Nutrition and Veterinary Clinical Nutrition, Faculty of Veterinary Medicine, Damanhour University, Damanhour, Egypt *Corresponding author, E-mail: mahmouddawood55@gmail.com Abstract: The current study was established to test the possibility of using exogenous digestive enzymes (EDE) in rabbitfish (Siganus rivulatus) diets. Five experimental diets containing 34% soybean meal were prepared and supplemented with EDE at 0, 1, 2, 3, and 4 g kg-1 diet. Fish with average initial weight (± 1.1 g) were fed the test diets for 74 days. Rabbitfish offered the diets with EDE supplements exhibited significantly (P<0.05) higher growth performance, feed intake, protein efficiency ratio, protein gain, and protein retention over the basal diet. But, no significant (P>0.05) differences were observed in carcass composition and somatic parameters index with EDE supplementation. The physiological condition of fish fed diets with EDE showed that the hematocrit (%), total plasma protein and total plasma globulin were significantly (P<0.05) higher in fish fed EDE than the control group. It can be concluded that the addition of EDE in rabbitfish diets improved the growth performance, feed efficiency and health condition. Key words: blood parameters; exogenous digestive enzymes; feed efficiency; growth; rabbitfish Introduction Aquaculture is one of the most economically important food sectors, representing the cheapest animal protein source for the increased population around the globe (1). Aquafeeds is contributing by around 70 to 80% from the total cost of the fish aquaculture (2). Thus, high quality feeds with balanced composition should be formulated to present the nutritional requirement and welfare for aquatic animals (3). Fish meal has been served as the major protein source in aquafeed of carnivorous fish which tend to possess higher dietary protein requirements than omnivorous and herbivorous due to its balanced amino acid profile and palatability (4-7). However, the resource shortages and rising price of fish meal seriously restrict the use of fish meal in aquaculture. In Received: January 2019 Accepted for publication: February 2019 410 M. Dawood, M. Mohsen, A. El-dakar, E. Abdelraouf, E. Moustafa, H. Ahmed this light, much efforts have been conducted to seek the sustainable supplies of protein sources to substitute fish meal. The plant protein sources have been limited used due to their relative low protein content and palatability, the presence of anti-nutritional factors and unbalanced amino acids profiles (4, 8, 10, 11). Digestive enzymes are helping in the food digestion process by transforming the non-digestible ingredients into more absorbable nutrients (9). Using of digestive enzymes is also recommended to facilitate the digestion process of fibrous ingredients existed in plant proteins such as: starch, cellulose and protein into simpler substances (3). Amylases, lipases, cellulases, xylanases, phytases and proteases are different forms of exogenous digestive enzymes which has been applied successfully in animal nutrition and aquafeed (10, 11). Since specific enzyme preparations have become valuable tools for economically manipulating digestive processes in animals (12), there are also considerable interests in using enzymes as supplements in fish feeds (3,9,13). Rabbitfish species have long been identified as appropriate candidates for aquaculture in the Indo-Pacific and the Middle East area (14). Although they are herbivorous and could potentially be fed soy bean meal-based diets, non-fish meal feeds might reduce the digestibility and assimilation of this species. Under the circumstances, fish diets should be supplemented by using digestive enzymes to further utilize the prepared feeds efficiently (13, 15, 16). The present study was performed to examine the possibility of using exogenous digestive enzymes "cellulases, xylanases, a-amylase and proteases mixture" on growth, feed efficiency, carcass composition and health condition of rabbitfish (Siganus rivulatus). Materials and methods Experimental system Rabbitfish (S. rivulatus) juveniles were obtained from the coast of Mediterranean Sea, El-Arish, Egypt. The collected fish were carried carefully in well prepared tanks to the "Mariculture Research Center, Faculty of Environmental Agricultural Sciences, Suez Canal University, El-Arish, Egypt", then acclimatized for 1 week before starting the trial. A total of 150 fish were distributed in 15 glass aquaria (50 l) where each aquarium was stocked with 10 fish with similar size (initial weight 1.1 g). The trial was conducted in triplicates. Each aquarium was provided with submerged air diffusers for aeration and about 30-35% of water was changed daily. The water quality parameters were measured daily and remained at 34 g kg-1, 25±1°C, 8.9 and 7 mg kg-1, for salinity, temperature, pH and DO, respectively. Diet preparation The basal diet (32.2% crude protein and 8.5% lipids) was supplemented with exogenous digestive enzymes (EDE) (ZADO®, patent No. 22155 of Egypt) at 0, 1, 2, 3 and 4 g kg-1. The basal diet was prepared to contain fish meal (65%) and soybean meal (45%) as protein sources and other ingredients were added by following El-Dakar et al. (13) (Table 1). The pellets size was 1.5-mm die. Pellets were air dried and stored at -20C. Each diet was offered to three randomly chosen aquaria at 2-3% of biomass daily at 8.00, 12.00 and 16.00 h for 74 days. Fish were weighed at 2-week intervals and ration was adjusted according to new fish weight per cage. Sample collection and analysis After 74 days, all fish in each aquarium were weighed, counted and total length was measured to calculate the specific growth rate (SGR), feed conversion ratio (FCR), protein efficiency ratio (PER), protein gain (PG), protein retention (PR), survival and condition factor (CF) using the following formulae: SGR = 100 *(ln FBW -ln IBW)/t, FCR = FI (g)/WG (g), PER = WG/protein intake, PG = {(FBW (g) xfrnal whole-body protein content (%)/100) - (IBW (g) xinitial whole-body protein content (%)/100)}/ (WG (g)) xi000, PR= (protein gain (g kg WG-1) xl00)/ protein intake (g kg WG-1), CF = 100 *[body weight (g)/length3 (cm)] and survival= (final number/initial number)*100. Where FBW is final body weight (g), IBW is initial body Effectiveness of exogenous digestive enzymes supplementation on the performance of rabbitfish 411 weight (g), WG is weight gain, FI is feed intake (g) and t is 74 days. Fish for initial carcass composition and diet samples were taken at the beginning of the trial and stored at -20 °C until analysis. 3 fish were randomly taken from each aquarium and killed, then the liver was dissected and weighed to calculate the hepatosomatic index (HSI) where HSI = 100 *(liver weight/body weight). Three fish from each aquarium were collected, pooled and stored at -20 °C for carcass analysis. Using heparinized syringes, blood was collected from the caudal vein of 3 fish in each replicate aquarium and pooled. A small fraction of the heparinized blood was used to analyze the hematocrit and hemoglobin levels. Plasma samples were obtained by centrifugation at 3000*g for 15 min using a high-speed refrigerated micro centrifuge and kept at -20°C. Hematocrit was determined by using micro hematocrit-heparinized capillary tubes and a micro hematocrit centrifuge (10,000 g for 10 min.) (17). Hemoglobin concentrations were determined by the cyan hemoglobin method, at 540 nm. Total plasma protein and albumin were determined using commercial kits produced by Pasteur labs (Egypt). However, the total plasma globulin was calculated by subtracting the total plasma protein from total plasma albumin according to Coles (18). Chemical analysis of feed and fish were performed according to the methods described in AOAC (19). Statistical analysis Data were analyzed using one-way analysis of variance (Package Super-ANOVA 1.11, Abacus Concepts, Berkeley, California, USA). Significance differences between means were evaluated using Duncan's new multiple range test at a probability level of 0.05 when t-test was significant. Results Growth, survival and feed efficiency Survival rate of all fish groups fed varied levels of EDE ranged between 90 to 100 %. Using of EDE resulted in improved FBW, WG and SGR significantly (P<0.05) than the control. Group 4 which supplemented with 3 g kg-1 EDE recorded the highest FBW and WG significantly (P<0.05) than other groups. Fish gained 2025%,2181, 2307, 2453 and 2212 % of their initial weight for the control, 1, 2, 3 and 4 g kg-1 groups than their weights at the start of the experiment (Table 2). Feed efficiency (FI, PG, and PR) of fish fed EDE is presented in Table 2. Results showed that rabbitfish fed diet contains different levels of EDE gave higher values significantly (P<0.05) than EDE free group. Moreover, those fed 3g kg-1 EDE gave significantly the higher values than the other groups (P<0.05). Although, FCR recorded no significant differences (P>0.05) among all experimental groups, rabbitfish fed 3g kg-1 recorded the lowest value numerically comparing the other experimental groups. PER of fish fed diets supplemented with EDE at 1, 2, and 3 g kg-1 was significantly (P<0.05) higher than the non-supplemented group. Carcass composition No significant differences (P>0.05) were observed in the analyzed carcass compositions for fish collected at the end of the trial in comparison with the values before the feeding trial (Table 3). Further, no difference was detected in CF and HSI values of fish fed different test diets (Table 3). Blood parameters Hematological parameters were presented in Figure 1A; hematocrit of rabbitfish was significantly (P<0.05) improved in fish fed either 3 or 4 g kg-1 EDE than the other experimental diets. Although, hemoglobin value was not affected by EDE supplementation among tested groups, diets supplemented with 3 and 4 g kg-1 EDE recorded higher hemoglobin value significantly (P<0.05) than the EDE free group (Fig. 1B). Blood chemical parameters in rabbitfish were presented in Figure 2. Fish fed diets with EDE had significantly (P<0.05) higher levels of total plasma protein compared to fish fed EDE free diet (Fig. 2A). Total plasma albumin recorded the lowest value significantly 412 M. Dawood, M. Mohsen, A. El-dakar, E. Abdelraouf, E. Moustafa, H. Ahmed (P<0.05) in fish fed 2 g kg-1 EDE among the plasma globulin significantly (P<0.05) than other experimental groups (Fig. 2B). Fish fed other experimental groups (Fig. 2C). EDE at 2, 3, and 4 g kg-1 recorded higher total Table 1 : Formulation and chemical proximate composition of the experimental diets (g kg-1 dry weight basis). Ingredients EDE supplementation level (g kg"1) 0 1 2 3 4 Fish meal 120 120 120 120 120 Soybean meal 340 340 340 340 340 Wheat bran 100 100 100 100 100 Wheat milling by product 170 169 168 167 166 Corn starch 50 50 50 50 50 Rice bran 150 150 150 150 150 Oil mixture1 50 50 50 50 50 Vitamin and mineral premix 2 20 20 20 20 20 Digestive enzyme mixture 3 0 1 2 3 4 Total 1000 1000 1000 1000 1000 Proximate composition and energy content (% dry matter basis) Crude protein 32.9 31.5 31.8 32.5 32.2 Crude lipid 9.7 7.3 7.5 8.3 8.8 Crude fiber 2.5 2.5 2.3 2.4 2.4 Crude ash 9.8 8.5 9 9 8.6 Nitrogen free extract 45.1 50.2 49.4 47.8 48 GE (kJ g-1 diet)4 19.34 19.00 19.01 19.19 19.35 DE (kJ g-1 diet)5 16.69 16.41 16.40 16.55 16.72 P/E ratio (mg CP kJ-1 DE) 19.71 19.20 19.39 19.64 19.26 1Mixture of Sunflower oil and linseed oil with a ratio of 1:1. 2Commercial Vitamin (Super Vit, Arab veterinary Industrial Co., Jordan). 15.000 IU vitamin A, 0.7 g vitamin C (Stay C®, 35% active), 15.000 IU vitamin D3, 2 mg vitamin E, 2.5 mg vitamin B2, 2 mg vitamin K3, 10 mg nicotineamide, 3 mg vitamin B6, 5 mg vitamin B12, 2 mg vitamin B1, 2 mg folic acid, 5.5 mg Ca-D-pantothenate, and mineral premix (Eco Vit, Egyptian veterinary produced and feed a additives Co., Demyatta, Egypt). 200 g calcium, 90 g phosphate, 40 g sodium, 2.5 g copper, 48 g magnesium, 3.6 g manganese, 23.5 g zinc, 8 g iron, 450 mg cobalt, 200 mg iodine and 20 mg selenium. 3Exogenous digestive enzymes (EDE), ZADO® (enzymic preparation containing cellulases, xylanases, a-amylase and proteases from anaerobic bacterium, patent No. 22155 of Egypt). 4Gross energy was calculated by using factors of 23.62, 39.5 and 17.56 kJ g-1 for protein, lipid and carbohydrate, respectively (20). 5Digestible energy content was estimated as 16.72, 16.72 and 37.62 kJ g-1 for protein, carbohydrate and lipid, respectively (21). Effectiveness of exogenous digestive enzymes supplementation on the performance of rabbitfish 413 Table 2: Growth performance, survival and feed efficiency utilization of rabbitfish fed diets supplemented with EDE for 74 days* Parameters EDE supplementation level (g kg-1) 0 1 2 3 4 INW 1.13±0.02 1.14±0.01 1.09±0.01 1.1±0.03 1.14±0.03 FBW 24.01±0.07a 26.01±0.46b 26.24±0.17b 28.07±0.19c 26.41±0.35b WG (g) 22.88±0.07a 24.87±0.46b 25.15±0.18b 26.97±0.21c 25.26±0.36b WG (%) 2025.42±31a 2181.12±33.6b 2307.29±26.6bc 2453.55±56c 2212.88±71. 7b SGR (%) 4.13±0.02a 4.23±0.02b 4.3±0.02bc 4.38±0.03c 4.24±0.04b Survival (%) 100 95 90 100 95 FI 55.01±0.29a 59.25±0.14b 59.38±0.28b 62.26±0.09c 59.76±0.32b FCR 2.4±0.01 2.38±0.05 2.36±0.02 2.31±0.02 2.37±0.04 PER 1.26±0.01a 1.34±0.03b 1.33±0.01b 1.33±0.1b 1.31±0.02ab PG 142.18±0.00a 146.41±0.01b 147.04±0.04c 147.52±0.01d 146.45±0.08b PR 25.73±0.14a 27.33±0.07b 27.76±0.13c 29.85±0.05e 28.18±0.17d "Values are means of triplicate groups' ±S.E.M. Within a row, means with different alphabet are significantly different (P<0.05), means with the same letters are not significantly different (P>0.05). Absence of letters indicates no significant difference between treatments. Table 3: Whole body proximate analysis (%) and somatic parameters of rabbitfish fed diets supplemented with EDE for 74 days* Items EDE supplementation level (g kg-1) Initial fish 0 1 2 3 4 Moisture 70.89±0.65 69±0.4 69.6±0.32 69.3±0.24 69.6±0.06 69.15±0.06 Crude protein 14.18±0.15 14.22±0.29 14.62±0.21 14.68±0.15 14.73±0.1 14.64±0.1 Crude lipid 10.18±0.16 11.06±0.22 11.79±0.45 11.81±0.47 11.79±0.45 11.46±0.75 Crude ash 4.58±0.15 4.62±0.15 4.43±0.33 4.22±0.34 4.27±0.09 4.67±0.09 CF 3 - 1.98±0.04 1.86±0.01 1.82±0.07 1.76±0.11 1.93±0.03 HSI4 - 1.39±0.09 1.45±0.18 1.65±0.09 1.64±0.09 1.54±0.07 "Values are means of triplicate groups ±S.E.M. Within a row, Absence of letters indicates no significant difference between treatments, Initial values are not included in the statistical analysis. 414 M. Dawood, M. Mohsen, A. El-dakar, E. Abdelraouf, E. Moustafa, H. Ahmed (A) •G o o ta (U e 33 32 31 30 29 28 27 26 25 1 2 3 EDE supplemented (g kg-1) (B) 9 8 7 6 5 4 m3 e E 2 1 0 d g O lg o 1 2 3 EDE supplemented (g kg-1) b b 0 4 b b 0 4 Figure 1: Average percentage of hematocrit (A) and hemoglobin values (B) in the blood of rabbitfish fed diets supplemented with different levels of EDE for 74 days. Values are means ± pooled SEM from triplicate groups. Means with different alphabet are significantly different (P<0.05) Effectiveness of exogenous digestive enzymes supplementation on the performance of rabbitfish 415 (A) (B) (C) T3 w C '53 o !-h a ' S « 03 £ 4 3 2 1 0 0 12 3 EDE supplemented (g kg-1) w S3 S £ m dl s pl t £ 1,5 1,4 1,3 1,2 1,1 1 0123 EDE supplemented (g kg-1) d g u o eS S s pl t £ 2,5 2 1,5 1 0,5 0 0 1 2 3 EDE supplemented (g kg-1) Figure 2: Average percentage of total plasma protein (A), total plasma albumin (B) and total plasma globulin (C) for rabbitfish fed diets supplemented with different levels of EDE for 74 days. Values are means ± pooled SEM from triplicate groups. Means with different alphabet are significantly different (P<0.05) c 4 c 4 b 4 416 M. Dawood, M. Mohsen, A. El-dakar, E. Abdelraouf, E. Moustafa, H. Ahmed Discussion Nowadays, EDE are extensively used all over the world as additives in fish feeds to improve the nutritional value of diets, especially with the increase of plant proteins (22). In this study, FBW, survival, WG and SGR of fish fed diet containing 3 g kg-1 EDE were significantly improved. It means that the enzymatic treatment with EDE improved the digestion process to be more suitable for increasing anaerobic bacteria and exogenous enzymes activity. EDE was used as an exogenous enzyme extracted from anaerobic bacteria, would provide cellulases, xylanases, a-amylase and proteases in fish feed. Diets containing EDE at level of 3 g kg-1 showed significantly higher SGR than other experimental diets. Similarly, the effects of dietary supplementation of exogenous enzymes increased the growth performance in several fish species including, Channel catfish (23, 24), Clarias catfish (25), Nile tilapia (26-28), Rainbow trout (29), and Common carp (30), respectively. However, Yigit and Keser (9) reported that, using of digestive enzymes in canola-based diets had no positive effect on the growth of Rainbow trout. The discrepancy with the present study was probably due to the differences in species and of plant protein meal used. In the present study, EDE supplementation in rabbitfish feeds improved feed intake (FI). Fish fed diet containing 3 g kg-1 EDE showed increased FI (62.26 g per fish) compared to 55.01 g per fish in the control diet. These findings might be due to the action of EDE, which may be caused by better utilization of some dietary fibre. As a result, the growth rates improved when fish fed the diets containing dietary EDE, reflecting that the lowest FCR value was detected in case of presence EDE in diets at level of 3 g kg-1 EDE. In this connection, McAllister et al. (31) reported that proposed modes of action for direct-fed enzymes include "solubilisation of dietary fibre before ingestion, provision of readily fermentable substrate for micro-organisms and/or enhancement of microbial enzyme activity in the gut". Shalaby et al. (32) reported that rabbitfish could utilize efficiently carbohydrates in their feeds. The results indicated that the diets with EDE improved PER, PG, and PR values. This may be ascribed to stimulating the digestion of the fibrous components by increasing the rate of fibre digestion. Singh et al. (30) reported that the protease enzyme can hydrolyses proteins to short peptides in food which in turn increased the protein digestibility in the gastrointestinal tract and accordingly enhance the feed efficiency and growth performance of fish (33). The enzyme efficiency can be affected by the level of inclusion and species of organism (31). Chakrabarti et al. (34) demonstrated that the amylase activity was enhanced when the formulation contains 13-25% corn starch. Rabbitfish showed a strong activity of P-glucuronidase which is responsible about the digestion of carbohydrate (35, 36). Rainbow trout also exhibited increased levels of absorbed protein as a result of increasing the activity of amylase (37). The amylase activity is affected by several factors including: feeding habit, structural of the carbohydrate, temperature and season of rearing (20, 38). The activity of amylase enzyme is higher in herbivorous and omnivorous fish than the carnivores (36). However, use of EDE in fish feeds will need to be more investigated on the basis of feeding habits, fish size and dietary composition. It is suggested that smaller fish may give better results in growth rate when received EDE containing diets than bigger ones. Body compositions of rabbitfish were not affected by dietary supplementations of EDE. The present work confirmed the results obtained by Lara-Flores et al. (39). They found that no differences were observed in the whole-body moisture, protein, lipid, and ash contents. On the other hand, Goda et al. (3) reported that Nile tilapia carcass protein, lipid, and ash contents were affected by EDE addition. Results showed that the somatic indices (CF and HSI) were not affected by the inclusion of EDE in rabbitfish diets. Similar results were obtained by El-Dakar et al. (13) who reported that the HSI was not affected by EDE feeding. Effectiveness of exogenous digestive enzymes supplementation on the performance of rabbitfish 417 The CF of the fish fed EDE was lower than that of control, which gave a CF similar to fish fed EDE at 4 g kg-1. These results reflect the small differences in weight together with length of the harvested fish. The general health status of fish can be evaluated by measuring the blood indices which reflect the physiological response of fish towards different feeding strategies (40-42). In this study, high values of hematocrit were observed in fish fed EDE. However, EDE-free group exhibited lower hematocrit levels, which may trigger anaemia in fish (3). Results of the present study also revealed that all fish fed EDE gave higher levels of total plasma protein and total plasma globulin. This may be attributed to fast metabolism which in turn resulted in better total plasma protein and globulin contents. Conclusion In conclusion, our results showed that using of EDE for rabbitfish is recommended to enhance the growth, nutrient utilization and health condition. Improved performances were observed in fish fed diets supplemented with 3 g kg-1 EDE for 74 days. Acknowledgements The first author would like to acknowledge the head and staff of "the Mariculture Research Center, Faculty of Environmental Agricultural Sciences, Suez Canal University, El-Arish, North-Sinai, Egypt", for technical assistance throughout the course of the study and providing of fish. We are grateful to E.S.H. Lab., El-Arish, North-Sinai, Egypt members for their technical assistance during the blood analysis. Conflict of interest The authors declare that they have no conflict of interest. References 1. Dawood MAO, Koshio S. Recent advances in the role of probiotics and prebiotics in carp aquaculture: a review. Aquaculture 2016; 454:243— 51. 2. Dawood MAO, El-Dakar A, Mohsen M, Abdelraouf E, Koshio S, Ishikawa M, Yokoyama S. Effects of using exogenous digestive enzymes or natural enhancer mixture on growth, feed utilization, and body composition of Rabbitfish, Siganus rivulatus. Journal of Agricultural Science and Technology. B. 2014;4(3B). 3. Goda AM, Mabrouk HA, Wafa MA, El-Afifi TM. Effect of using baker's yeast and exogenous digestive enzymes as growth promoters on growth, feed utilization and hematological indices of Nile tilapia, Oreochromis niloticus fingerlings. Journal of Agricultural Science and Technology. B. 2012;2(1B). 4. Dawood MA, Koshio S, Ishikawa M, Yokoyama S. Effects of partial substitution of fish meal by soybean meal with or without heat-killed Lactobacillus plantarum (LP20) on growth performance, digestibility, and immune response of amberjack, Seriola dumerili juveniles. BioMed Res Int2015;2015. 5. Dossou S, Koshio S, Ishikawa M, Yokoyama S, Dawood MAO, El Basuini MF, El-Hais AM, Olivier A. Effect of partial replacement of fish meal by fermented rapeseed meal on growth, immune response and oxidative condition of red sea bream juvenile, Pagrus major. Aquaculture 2018; 490:228-35. 6. Dossou S, Koshio S, Ishikawa M, Yokoyama S, Dawood MAO, El Basuini MF, Olivier A, Zaineldin AI. Growth performance, blood health, antioxidant status and immune response in red sea bream (Pagrus major) fed Aspergillus oryzae fermented rapeseed meal (RM-Koji). Fish Shellfish Immunol 2018; 75:253-62. 7. Dossou S, Koshio S, Ishikawa M, Yokoyama S, El Basuini MF, Zaineldin AI, Mzengereza K, Moss A, Dawood MAO. Effects of replacing fishmeal with fermented and non-fermented rapeseed meal on the growth, immune and antioxidant responses of red sea bream (Pagrus major). Aquacult Nutr 2019; https://online library.wiley.com/doi/abs/10.1111/anu.12876 8. Nordrum S, Bakke-McKellep AM, Krogdahl Ä, Buddington RK. Effects of soybean meal and salinity on intestinal transport of nutrients in Atlantic salmon (Salmo salar L.) and rainbow trout (Oncorhynchus mykiss). Comp Biochem Physiol B Biochem Mol Biol 2000; 125(3):317-35. 9. Yigit NO, Keser E. Effect of cellulase, phytase and pectinase supplementation on growth performance and nutrient digestibility of rainbow trout (Oncorhynchus mykiss, Walbaum 1792) fry fed diets containing canola meal. J Appl Ichthyol 2016;32(5):938-42. 418 M. Dawood, M. Mohsen, A. El-dakar, E. Abdelraouf, E. Moustafa, H. Ahmed 10. Feord JC. Exogenous enzymes improve performance of carp and tilapia when fed diets containing high levels of soybean meal, in: VII International Symposium on Nutrition and Feeding of Fish. 1996. 11. Forster I, Higgs DA, Dosanjh BS, Rowshandeli M, Parr J. Potential for dietary phytase to improve the nutritive value of canola protein concentrate and decrease phosphorus output in rainbow trout (Oncorhynchus mykiss) held in 11 °C fresh water. Aquaculture 1999; 179(1-4):109-25. 12. Johnson R Williams P, Campbell R. Use of enzymes in pig production. Proc. 1st Symp. On Enzymes in Animal Nutrition (Ed. C. Wenk and M. Boessinger). 1993: 49-60. 13. El-Dakar AY, Shalaby SM, Saoud IP. Assessing the use of a dietary probiotic/prebiotic as an enhancer of spinefoot rabbitfish Siganus rivulatus survival and growth. Aquacult Nutr 2007;13(6):407-12. 14. Pillay TVR, Kutty MN. Aquaculture: Principles and Practices, 2nd edn. Blackwell Publishing, Ames, IA, USA, pp 2005:458-60. 15. Dawood MAO, Koshio S, Abdel-Daim MM, Van Doan H. Probiotic application for sustainable aquaculture. Rev Aquacult 2018. https://onlinelibrary.wiley.com/doi/abs/10.1111/ra q.12272 16. Dawood MAO, Koshio S, Esteban MA. Beneficial roles of feed additives as immunostimulants in aquaculture: a review. Rev Aquacult 2018; 10(4):950-74. 17. Dawood MAO, Koshio S, Ishikawa M, Yokoyama S. Effects of heat killed Lactobacillus plantarum (LP20) supplemental diets on growth performance, stress resistance and immune response of red sea bream, Pagrus major. Aquaculture 2015; 442:29-36. 18. Coles EH. Plasma Proteins, Veterinary Clinical Pathology, 2nd ed., W. B. Saunders Co., London. pp. 1974:558-560. 19. AO AC. Official Methods of Analysis of Association of Official Analytical Chemists, 15th edn. Published by the Association of Analytical Chemists, Arlington, VA, USA.1990. 20. NRC. Nutrition Requirements of Fish. National Academy Press, Washington, DC, USA. 1993. 21. Garling Jr DL, Wilson RP. Optimum dietary protein to energy ratio for channel catfish fingerlings, Ictalurus punctatus. J Nutr 1976; 106(9):1368-75. 22. Kolkovski S, Tandler A, Izquierdo MS. Effects of live food and dietary digestive enzymes on the efficiency of microdiets for seabass (Dicentrarchus labrax) larvae. Aquaculture 1997; 148(4):313-22. 23. Jackson LS, Li MH, Robinson EH. Use of Microbial Phytase in Channel Catfish Ictalurus punctatus Diets to Improve Utilization of Phytate Phosphorus 1. J World Aquacult Soc 1996; 27(3):309-13. 24. Debnath D, Sahu NP, Pal AK, Jain KK, Yengkokpam S, Mukherjee SC. Mineral status of Pangasius pangasius (Hamilton) fingerlings in relation to supplemental phytase: absorption, whole-body and bone mineral content. Aquacult Res 2005;36(4):326-35. 25. Giri SS, Sahoo SK, Sahu AK, Meher PK. Effect of dietary protein level on growth, survival, feed utilisation and body composition of hybrid Clarias catfish (Clarias batrachusx Clarias gariepinus). Animal Feed Sci Tech 2003;104(1-4): 169-78. 26. Ali HM, Ghazalah AA, Gehad EA, Hammouda YA, Abo-State HA. Practical aspects and immune response of probiotics preparations supplemented to Nile tilapia (Oreochromis niloticus) diets. Nature & Science 2010; 8:39-45. 27. El-Haroun ER, Goda AS, Kabir Chowdhury MA. Effect of dietary probiotic Biogen® supplementation as a growth promoter on growth performance and feed utilization of Nile tilapia Oreochromis niloticus (L.). Aquacult Res 2006; 37(14): 1473-80. 28. Lin S, Mai K, Tan B. Effects of exogenous enzyme supplementation in diets on growth and feed utilization in tilapia, Oreochromis niloticus x O. aureus. Aquacult Res 2007;38(15):1645-53. 29. Irianto A, Austin B. Use of probiotics to control furunculosis in rainbow trout, Oncorhynchus mykiss (Walbaum). J Fish Dis 2002;25(6):333-42. 30. Singh P, Maqsood S, Samoon MH, Phulia V, Danish M, Chalal RS. Exogenous supplementation of papain as growth promoter in diet of fingerlings of Cyprinus carpio. International Aquatic Research 2011; 3:1-9. 31. McAllister TA, Hristov AN, Beauchemin KA, Rode LM, Cheng, KJ. Enzymes in ruminant diets. In: Bedford, M., Partridge, G. (Eds.), Enzymes in Farm Animal Nutrition. CABI Publishing, Oxon, UK, 2001; 273-98. 32. Shalaby SM, El-Dakar AY, Ghoneim SI. Protein sparing effect by carbohydrate in diets of rabbitfish, Siganus rivulatus. Egyptian Journal of the Aquatic Biology and Fisheries 2001; 5:87-98. Effectiveness of exogenous digestive enzymes supplementation on the performance of rabbitfish 419 33. Wong MH, Tang LY, Kwok FS. The use of enzyme-digested soybean residue for feeding common carp. Biomedical and environmental sciences: BES. 1996;9(4):418-23. 34. Chakrabarti I, Gani MA, Chaki KK, Sur R, Misra KK. Digestive enzymes in 11 freshwater teleost fish species in relation to food habit and niche segregation. Comp Biochem Physiol A Mol Integr Physiol 1995 Sep; 112(1):167-77. 35. Moraes G, Bidinotto PM. Induced changes in the amylohydrolytic profile of the gut of Piaractus mesopotamicus (Holmberg, 1885) fed different levels of soluble carbohydrate: its correlation with metabolic aspects. Revista de Ictiologia. 2000;8(1/2):47-51. 36. Lundberg B, Lipkin Y. Natural food of the herbivorous rabbitfish (Siganus spp.) in northern Red Sea. Botanica Marina 1979; 22(3):173-82. 37. Kawai S. Studies on digestive enzymes of fishes-III. Development of digestive enzyme of rainbow trout after hatching and the effect of dietary change on the activities of digestive enzyme in the juvenile stage. Bull Jpn Soc Sci Fish 1973; 39:81927. 38. Sabapathy U, Teo LH. A quantitative study of some digestive enzymes in the rabbitfish, Siganus canaliculatus and the sea bass, Lates calcarifer. J Fish Biol 1993;42(4):595-602. 39. Lara-Flores M, Olvera-Novoa MA, Guzmân-Méndez BE, Lopez-Madrid W. Use of the bacteria Streptococcus faecium and Lactobacillus acidophilus, and the yeast Saccharomyces cerevisiae as growth promoters in Nile tilapia (Oreochromis niloticus). Aquaculture 2003;216(1-4): 193-201. 40. Dawood MAO, Koshio S, Ishikawa M, Yokoyama S. Interaction effects of dietary supplementation of heat-killed Lactobacillus plantarum and P-glucan on growth performance, digestibility and immune response of juvenile red sea bream, Pagrus major. Fish Shellfish Immunol 2015; 45(1):33-42. 41. Dawood MAO, Koshio S, Ishikawa M, Yokoyama S. Immune responses and stress resistance in red sea bream, Pagrus major, after oral administration of heat-killed Lactobacillus plantarum and vitamin C. Fish Shellfish Immunol 2016;54:266-75. 42. Dawood MAO, Koshio S, Ishikawa M, Yokoyama S, El Basuini MF, Hossain MS, Nhu TH, Dossou S, Moss AS. Effects of dietary supplementation of Lactobacillus rhamnosus or/and Lactococcus lactis on the growth, gut microbiota and immune responses of red sea bream, Pagrus major. Fish Shellfish Immunol 2016; 49:275-85. Slov Vet Res 2019; 56 (Suppl 22): 421-31 DOI 10.26873/SVR-780-2019 Original Research Article HEMATO-BIOCHEMICAL AND MOLECULAR MARKERS (IS900) OF CATTLE INFECTED WITH JOHNE'S DISEASE IN EGYPT Ahmed M. Abdelaal1, Magdy M. Elgioushy2*, Shaimaa M.Gouda1, Mohamed M. El-Adl3, Emad A. Hashish4, Shimaa A. Elgaml4, Mayar O. Ali5, Attia A El-Gedawy6 1Department of Animal Medicine, Faculty of Veterinary Medicine, Zagazig University, Zagazig 44511, Egypt,2Department of Animal Medicine, Faculty of Veterinary Medicine, Aswan University, Aswan 37916, Egypt, 3Department of Biochemistry and Chemistry of Nutrition, Faculty of Veterinary Medicine, Mansoura University, Mansoura 35516, Egypt, 4Department of Clinical Pathology, Faculty of Veterinary Medicine, Zagazig University, Zagazig 44511, Egypt, 5Department of Animal welfare and Husbandry, Faculty of Veterinary Medicine, Mansoura University, Mansoura 35516, Egypt, 6Department of Bacteriology, Animal Research Institute, Egypt Corresponding author, E-mail: magdy.gioushy@aswu.edu.eg Abstract: In Egypt, limited studies were performed to investigate Jhon's disease (JD) caused by Mycobacterium avium subspecies paratuberculosis (MAP). This study was concerned with diagnosis of MAP in cattle dairy farms using indirect-ELISA technique and amplification of IS900 using PCR technique for confirmation of infection and investigated adequate diagnostic markers for MAP through determining hemato-biochemical profile in cattle dairy farms. Screening of MAP was done by indirect ELISA between the periods from January 2017 to June 2018. A total of 200 cows suffered from persistent diarrhea (n=17) and apparently healthy cows in contact with them (n=183) were used in this study. Positive JD was reported in 62 cases (31%) and negative JD was reported in 138 cases (69%). Out of the positive JD, 17 cases were clinically affected by persistent diarrhea and weight loss (clinical JD), where 45 cases were apparently healthy (sub-clinical JD). Typical colonies of MAP were isolated from the fecal samples of clinically affected cows with positive staining of MAP bacilli. Amplification of IS900 using PCR technique confirmed the presence of MAP infection. Phylogenetic analysis revealed identical similarities with MAP strains in India and Egypt. Hemato-biochemical investigations revealed significant decrease in RBCs, PCV, total protein, globulin and glucose with non-significant change in oxidative stress markers in clinically affected cows. This study is considered one of the limited researches that highlight the role of MAP infection in dairy farms in Egypt. Key words: dairy cattle; ELISA; IS900; Johne's disease; MAP Introduction Johne's disease is a chronic infectious enteric disease of ruminant where MAP is the causative agent of the disease. It is a slow growing, gram-positive and acid-fast bacterium that requires several months of incubation (1). Severe economic losses in the cattle industry were reported in farms infected with MAP (2). Ingestion of colostrum, water and feed contaminated with fecal matters via the fecal oral route can predispose to JD infection (3). Received: January 2019 Accepted for publication: February 2019 422 A. Abdelaal, M. Elgioushy, S. Gouda, M. El-Adl, E. Hashish, S. Elgaml, M. Ali, A. El-Gedawy The disease has a characteristic cross infection between species without restrictions, thus it is hard to be controlled (4). Vaccination may be effective in controlling infection and reduction shedding of MAP in the fecal matter with no complete elimination of infection (5). During the clinical stage of JD, the infection causes excessive diarrhea and wasting of the affected animal. Sub-clinically affected animals may, additionally, shed the bacterium with colostrum, milk and feces, particularly in the last stage of the disease (6). Furthermore, JD has zoonotic importance as MAP existed in the pathogenesis of Crohn's disease (CD) (7). Several methods might be used for diagnosis of JD. Identification of MAP through fecal subculture consumes a long time and therefore it is not considered an appropriate method of diagnosis. Additionally, ELISA can be used for the detection of the immune reaction of the microorganism (8). Polymerase chain reaction (PCR) can be used in amplification of certain genes in MAP (9). Several specific genetic sequences for MAP have been identified, such as IS900, F57element, and hsp X gene. The IS900 is considered the target of choice and the most widely used for MAP detection (10). The aim of the current study was to determine hemato-biochemical profile of cattle herds infected with MAP with the aid of indi-rect-ELISA technique and amplification of IS900 using PCR technique for confirmation of infection as well as to construct a phylogenetic analysis for determining the genetic difference between current infection and other infected strains in other localities. Materials and methods Animals and clinical examination Two hundred dairy cows were selected from three dairy farms in Sharqia and Ismailia Gov-ernorates in Egypt during the period from January 2017 to June 2018. The selection of cows was based on the existence of persistent diarrhea in addition to all cows over 3 years of age in the same farms were also selected. Cows were classified into three categories based on the ELISA screening test. Clinically affected cows were classified based on positive ELISA results in association with persistent diarrhea (n=17). The sub-clinically infected cows with Johne's disease was classified based on positive ELISA results without any clinical signs (n=45). Out of the remaining negative cows (n=138), 15 cows were randomly selected as negative control cows. All cows enrolled in this study received a thorough clinical examination via methods that have been previously described in cattle (11). Blood and fecal samples collection Blood samples were collected from the jugular vein of two hundred selected cows for the initial ELISA screening test. Seventy seven blood samples were collected from the clinically, sub-clinically and control cows for hemato-biochemical profile. Fecal samples were collected from clinically affected cows from the rectum for the bacteriological isolation, identification and amplification of IS900 using PCR technique. MAP antibodies titration Serum from all selected cows was examined for anti-MAP antibodies in ELISAs. To titrate anti-MAP antibodies, we used Mycobacterium paratuberculosis (MAP) test Kit for dairy cattle PARACHEK2 (Prionics, Switzerland) according to (12). Examination of fecal samples Fecal samples were collected from 17 clinically affected cows which were positive by ELISA and associated with persistent diarrhea. Two grams of fecal sample were collected from each animal and kept into a sterile mortar and decontamination of the fecal sample was done using 30 ml of 0.9% Hexadecylpyridinium chloride solution (HPC), to 3g of aliquot for 24 h through vortexing and shaking (13). After 30 min, at room temperature, 20 ml of the supernatant was transferred into another sterile tube and placed for 24 h at room temperature in a dark place in a vertical position, and then the tubes were centrifuged for 10 min by 3000 rpm (14). Three hundred pl of the decontaminated pellet was used after discarding of the supernatant and inoculated with Herrold's egg yolk medium (HEYM) slants and supplementation with Hemato-biochemical and molecular markers (IS900) of cattle infected with Johne's disease in Egypt 423 mycobactin J. One week incubation of slants was done in a horizontal position at 37oC, and then the tubes were kept in a vertical position for 8-16 weeks incubation period. Later the tubes were being checked for the growth of typical MAP colonies. Then smears were taken, from the suspected samples for microscopic examination. Ziehl-Neelsen staining of fecal smears Prepared fecal smears were air dried for 10 minutes, followed by a heat fixation (60-70°C), for 2 h. The smears were dipped in carbol fuchsin 1%, and then gently heated until a small portion of steam rises without reaching the boiling point. All slides were kept for 5-10 minutes, and then rinsed with tap water followed by addition of acid alcohol (3% Hcl in 70% methyl alcohol) for 1-2 minutes then thoroughly rinsed with tap water again. Slides were immersed in Methylene blue 5%, and left for 1-2 minutes, then rinsed with tap water and dried in air. Finally, the slides were examined under oil immersion lens. Molecular detection of MAP using IS900 Extraction of DNA from isolated MAP was carried out, using QIAamp DNA Mini kit (Qi-agen, Germany), according to manufacturer's instruction. Amplification of IS900 MAP was performed on Bio-Rad T100 Thermal cycler (Hercules, CA, USA) with a 60 ng of DNA template, 12.5 pl of Emerald master mix (Takara, Japan) and 2 pl of forward and reverse primer for MAP IS900 marker (F: CCG CTA ATT GAG AGA TGC GAT TGG; R: AAT CAA CTC CAG CAG CGC GGC CTC G) (15). PCR was started with an initial denaturation step at 94°c for 10 minutes, then followed by 35 cycles of denaturation for 60 seconds at 94 °c, annealing for 90 seconds at 61 °C and extension for 60 seconds at 72 °C to amplify the fragment of DNA of 298 bp for IS900. PCR products were then run on gel electrophoresis (2%) for visualization of the amplified PCR product. The required product size was optimized using a 100bp DNA ladder (Jena bioscience, Germany) and was cut with a clean scalpel for purification of DNA fragment with Gene JET Gel extraction kit (Thermo Scientific, Lithuania). The purified DNA products were then sent for sequencing in 3130 X DNA Sequencer (Genetic Analyzer, Applied biosystem, Japan) with both forward and reverse primer used in PCR. Phylogenetic analysis The obtained nucleotide sequence was aligned, trimmed with the aid of ClustalW that was integrated with MEGA 6 software (16). The trimmed DNA samples were checked on Genbank for determining similarity index between other species of MAP on Genbank. Hematological analysis Two ml of blood samples were collected into EDTA tubes for the investigation of hematological parameters. Total RBCs count was manually done by using Neubauer-ruled hemocytom-eter. Hemoglobin (Hb) concentration was determined using the method of cyanomethemo-globin (J.T. Baker, London, England). Packed cell volume (PCV) was determined by micro-hematocrit centrifugation. The value of PCV was determined by using a built-in reader. The erythrocytic indices, include mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH) and mean corpuscular hemoglobin concentration (MCHC), were calculated from the value of total RBCs count, Hb concentration and PCV by using the standard formulas (17). Biochemical analysis Five ml of blood was collected into plain test tubes without anticoagulant. It was used for the separation of serum samples for the biochemical investigations. Blood left at room temperature for 2 h to be clotted, and then centrifuged at 3000 rpm for 10 min. All biochemical parameters were analyzed colorimetrically by commercial kits (BioMerieux, Marcy, L'Etoile, France). The serum glucose (18), serum cholesterol (19), triglycerides (20), serum total protein (21) and albumin (22) were measured. Globulin was calculated based on subtracting serum albumin from serum total protein. Liver transam-inases (ALT & AST) (23) and alkaline phos-phatase (24) were determined. Creatinine (25) and urea (26) were also measured. 424 A. Abdelaal, M. Elgioushy, S. Gouda, M. El-Adl, E. Hashish, S. Elgaml, M. Ali, A. El-Gedawy Antioxidant status and oxidative stress markers Several parameters were evaluated in the serum samples to measure the antioxidant status and stress markers. The total antioxidant capacity (TAC) was calculated (27), serum glutathi-one-S-transferase (GST) activity was estimated using Chloro dinitro benzene derivative (28). Reduced glutathione (GSH) concentration was colorimetrically determined using dithio dini-trobenzene derivatives (29). Hydrogen peroxide (H2O2), malondialdehyde (MDA) and nitric oxide (NO) concentrations were determined colorimetrically according to (30-32), respectively. Statistical analysis Differences between subclinical, clinical JD and control cows were analyzed using the Krus-kal-Wallis with post hoc Dunn's multiple comparison tests because of the non-normal distribution of markers. Results were presented as medians ± standard errors (S.E.). The significance was determined at P< 0.05. All statistical analysis was performed using the statistical software (SPSS Inc., Chicago, IL, USA). Results Clinical signs Signs of clinically affected cows with JD were recorded, (Table 1). Clinically affected cows showed normal systemic reactions (body temperature, respiratory rate and heart rate). Persistent diarrhea, chronic loss of body weight, pronounced decrease in milk yield and dehydration (sunken eyes) were the most pronounced clinical signs observed in 100% of cases. Reduced ruminal sound, right ping sound and sub-mandibular edema were observed in 29.4% of clinically affected cows. Rectal palpation revealed corrugated rectal mucosa in 70.6% of cases. Diarrhea was watery, contains air bubbles. Marked reduction was observed in milk production in clinically affected cows when compared with sub-clinically infected cows and control (average milk production was 10±2.4 kg/day in clinically affected cows, 18±3.1 kg/day in sub-clinically infected cows and 21± 5.3 kg/day in control cows). Detection of MAP antibodies The MAP antibodies were detected in the serum samples of clinically affected and sub-clinically infected dairy cows of two hundred investigated dairy cows for the presence of MAP IgG antibodies for in serum of cattle. Specific anti-MAP IgG antibodies were detected from the serum samples of the clinically infected cows 8.5% and sub-clinically affected cows 22.5%, but no IgG antibodies were detected from the serum samples of the control cows. Isolation and microscopic examination of MAP MAP was usefully isolated from 4 out of 17 fecal samples of clinically affected cows which diagnosed by indirect ELISA. After 16 weeks of incubation on Herrold's media containing egg yolk slope, typical colonies of MAP were observed and appeared very small, convex (hemispherical), soft, non-mucoid and initially colorless and translucent. Staining of direct fecal smears revealed 3 of 17 fecal samples were positive. Microscopic appearance of MAP showed red acid alcohol-fast Ziehl-Neelsen staining bacilli. The amplification of MAP DNA samples was sequenced, matched and deposited on Genbank with accession number of MH663496 and named as Mycobacterium avium subsp. paratu-berculosis Egyl. The aligned MAP species are KY587112, KT075353, KT075351, KT075350, MH663496, KJ173784, JQ837281, EU130943, EF514831, EF536056, EF536046, S74401, AF416985, AF305073, AJ250018, AJ011838, AB052552, AJ250023, AJ251434, AJ250022, X16293, EF536047, AY974347, KT275243, EF536041, FJ775182, AY974346, JN983503, EU232747, EF514825, AF455252, EF536048, EU714039, EF514828, KJ173781, EU232756, EU714041, EU232748, HQ830160, EU057153, EU057170, EU714040, EU232755, KJ882903, HM172613, GQ144322. It showed a 100% identity with MAP strain KVAFSU_S196. Phy-logenetic tree (Fig. 1) was divided into two clades (clade A and B) where our isolated species of MAP was located in subclade B among other related species of M.avium (MAP strain Hemato-biochemical and molecular markers (IS900) of cattle infected with Johne's disease in Egypt 425 KVAFSU_S196, MAP strain 25, MAP strain steroyl ACP desaturase, MAP strain B42, MAP strain 25C) that was isolated from India. Moreover, pairwise distance showed 0.0009 with our isolated strain. The Indian strain and our Egyptian strain were shown a close genetic distance with high node support. The highest pairwise difference was observed in JQ837281 (1.3 EU130943 (1.4), S74401 (1.3), AJ011838 (1.3) and KT275243 (1.4). Those species were located in different localities in Australia, India, and United Kingdom and in Egypt. Hematological analysis Clinically affected cows showed a significant (P<0.05) decrease in the RBCs, PCV and the level of MCV with a significant (P< 0.05) increase in the level of MCHC, meanwhile the Hb concentration revealed a non-significant changes when compared with the control and sub-clinically infected group. The blood indices in this group reflect microcytic hyperchromic anemia. There was a non-significant change in the RBCs, Hb, PCV, MCV, and MCHC level in sub-clinically infected cows when compared with the control ones. The blood indices were close to the level of normal in this group, (Table 2). Biochemical parameters Clinically affected cows showed a non-significant change in the levels of cholesterol, triglycerides, albumin, serum hepatic marker enzymes (ALT, AST, ALP) and the markers of renal damage (urea and creatinine), meanwhile the levels of serum total protein, globulin and glucose were significantly (P< 0.05) decreased when compared with the control and sub-clinically infected group. All these parameters revealed a non-significant change in the sub-clinically infected cows when compared with the control, (Table 3). Antioxidant status and oxidative stress markers Clinically affected cows showed non-significant changes in total antioxidant capacity (TAC), glutathione S-transferase (GST), glutathione (GSH), Hydrogen peroxide (H2O2), malondialdehyde (MDA) and nitric oxide (NO) when compared with the control and sub-clinically infected group. All these oxidative and li-pid peroxide markers revealed a non-significant change in sub-clinically infected cows, meanwhile There was a significant (P< 0.05) decrease in the level of Hydrogen peroxide (H2O2) when compared with the control, (Table 4). Table 1: Clinical picture of 17 cows affected by Johne's disease Clinical signs Clinically affected cows by Johne's (n=17) Number of cows % Normal vital parameters* 17 100 Normal ruminal motility** 12 70.6 Hypomotile rumen 5 29.4 Right ping sound 5 29.4 Chronic weight loss 17 100 Persistent diarrhea 17 100 Dehydration (sunken eye) 17 100 Decrease in milk production 17 100 Submandibular edema 5 29.4 Corrugated rectal mucosa by rectal palpa- 12 70.6 tion *Normal vital parameters mean normal reference range of temperature (38.2-39.2° C), heart rate (70- 90/ minute) and respiratory rate (20- 30/minute) (11). ** Normal ruminal motility (3-5 ruminal contraction/2minute) (11). 426 A. Abdelaal, M. Elgioushy, S. Gouda, M. El-Adl, E. Hashish, S. Elgaml, M. Ali, A. El-Gedawy Table 2: Hematological picture in control, sub-clinically and clinically affected cows Control Sub-clinical Johne's Clinical Johne's Parameter (n=15) (n=45) (n=17) RBCs 1012/L 5.8±0.19 a 5.85 ±0 .16a 3.97±0.18b Hb g/dl 6.6±0.45a 6.9±0.27a 7±0.29a PCV % 38±1.17a 35.1±0.85a 21 ± 1.5b MCV fl 65.32±0.9a 60.4±0.32a 53.03±0.8b MCH pg/ cell 12.1±0.77b 11.8 ±0.55b 17.64 ± 1a MCHC % 17.37±.02b 19.64±0.12b 33.33 ±.01a Medians with different superscripts indicate significant difference at P <0.05 Table 3: Biochemical picture in control, sub-clinically and clinically affected cows Control Sub-clinical Johne's Clinical Johne's Parameter (n=15) (n=45) (n=17) Glucose mg/dl 48±4.5a 45.45±1.9a 38±4.1b Cholesterol mg/dl 157±11.2a 165±10.6a 169±21.2a Triglycerides mg/dl 49±3.17a 55±2.55 a 58±4.7a Total protein g/dl 8±0.3a 8.64±0.28a 5.15 ± 0.48b Albumin g/dl 3.52±0.28a 3.8±0.15 a 3.15±0.31a Globulin g/dl 4.48±0.38a 4.84 ± 0.27a 2 ± 0.37b ALT u/l 10±0.4a 13±0.66a 11±0.62 a AST u/l 19±0.81a 16±0.85a 18±0.77a ALP u/l 112±30.4a 89±33.7a 99±24.67a Urea mg/dl 25.06±2. 83a 20.14± 2.97a 17.17±2.76a Creatinine mg/dl 0.87±0.068a 0.8±0.041a 0.6±0.067a Medians with different superscripts indicate significant difference at P <0.05 Table 4: Oxidative stress biomarkers in control, sub-clinically and clinically affected cows Control Sub-clinical Johne's Clinical Johne's Parameter (n=15) (n=45) (n=17) GST u/L 163.7±11.76a 160.7±54.1a 168.7±37.8a GSH mg/d 9.73±1.04a 11.7±0.5a 11±1.17a TAC mM/L 4.1±2.28a 3.8±2.3a 3.9±0.21a H2O2 mM/L 0.3±0.012a 0.26 ± 0. 01b 0.28±.01ab MDA nmol/ml 6.6±1.3a 6.2±1.2a 5.6±0.9a NO umol/L 18±3.6a 23±4.2a 29± 6.06a Medians with different superscripts indicate significant difference at P <0.05 Hemato-biochemical and molecular markers (IS900) of cattle infected with Johne's disease in Egypt 427 Figure 1: Phylogenetic analysis of different strains of MAP infection with our isolated strain"blue rectangle". phylogenetic tree was divided into two clades; clade A and clade B. Clade A was subdivided into two sub clades A and B where our isolated sequence (Mycobacterium avium subsp paratuberculosis EGY1) was located (sub-clade B) Discussion MAP has severe hazard to animal health. It causes significant economic losses to livestock animals worldwide. Limited studies were carried out to investigate MAP infection in Egypt, which directs our attention to this disease. Identification of MAP requires either culturing organisms in specific media or detecting the antibodies in sera of infected animals (33). The use of diagnostic markers to study the response of the host toward MAP infection is crucial for diagnosis of Johne's disease. Generally, ELISA is considered the most widely used test that assigned to detect MAP infection in animals. ELISA has been used in this study for the differentiation of the clinically affected cows from the sub-clinically infected cows. This based on the immune reaction of the microorganism in correlation to the presence or absence of the clinical signs in animals. Alterations in the general health condition were observed. The heart rate, respiratory rate, and temperature remain normal which coincided with the previously reported data (4). The most common clinical signs reported in clinically affected cows were persistent diarrhea, chronic loss of body weight and dehydration which were nearly similar to those previously reported(34). The associated diarrhea may be due to the release of histamine as a result of antigen antibody reactions in the intestine (35). The characteristic thickened corrugated intestine by rectal examination might be attributed to the associated inflammatory process which involves the lymph node draining the intestine (36). The chronic loss of body weight might be 428 A. Abdelaal, M. Elgioushy, S. Gouda, M. El-Adl, E. Hashish, S. Elgaml, M. Ali, A. El-Gedawy due to the inflammation which resulted in thickening of the intestinal wall, leading to poor nutrient absorption in affected animals (4). Submandibular odema was observed in some cows, it might attribute to protein loss from the blood stream into the digestive tract (37). Generally, the use of IS900 had been used extensively for studying MAP in domestic animals (38, 39). PCR positive result of MAP for IS900 can be used correctly in the identification of the causative agent for Johne's disease (40). Different sequences for IS900 were deposited in Genbank, besides the original sequence isolated from United Kingdom (X16293) (33) with a product size of around 1500 bp which showed a change in a nucleotide sequence of our obtained sequence with a pair wise genetic difference of 0.009. Our obtained sequence of IS900 was identical to a sequence that was isolated from India (KY587112), while it was showing a higher difference when compared with other strains from Egypt (KJ173784 and KJ173784), although they were isolated from the fecal matter of cattle. In the two strains from Egypt, our isolated strain of MAP showed 98% similarity index, but was named as transposase sequence where it is responsible for transposition of gene in pro-karyotes. Insertion sequence regulates gene transposition (41). On the contrary, another isolated sample from Egypt (JN983503) was isolated from fecal matter of bovine species would find to share higher than 99% similarity index with our obtained sequence. Johne's disease is reported to induce some hematological alterations. Significant decrease in the RBCs count and the PCV was reported in clinically affected cows when compared with sub-clinically infected and control ones. The significant decrease in the RBCs count is similar to that observed in JD clinically affected camels (42). Similar results of a significant decrease in PCV were also reported (43). These findings indicated the presence of anemia among the clinically affected cows, which is similarly reported before (44). Anemia is an important common extra intestinal complication associated with Crohn's disease in human. The microcytic hyperchromic anemia was reported in our study, where Crohn's disease show the same type of anemia which characterized by microcytic hyperchromic cells (45). The use of serum biochemical parameters can be used as a diagnostic marker for certain diseases(46). Changes in the serum biochemical parameters were noticed. Significant decrease in the total protein in clinically affected cows was reported compared with sub-clinically infected and control cows. This was previously reported in the serum samples of positively infected cows with MAP (47). The hypo-proteinemia might attributed to protein-losing enteropathy which might be due to the impaired integrity of the mucosa of the gastrointestinal tract (48) or due to the increase in the gut motility with a decreased in the absorption results in an increase in the intestinal loss of protein (43). Most of the cows suffer from clinical Johne's disease showed a low level of albumin with a normal level of globulin (49). Significant decrease in globulin was reported in our study. Hypoglobulinemia was previously reported in Crohn's disease which attributed to the decrease in globulin synthesis or increased catabolism of immunoglobulins (50). Hypoglycemia was reported in clinically affected cows when compared with sub-clinically infected and control cows. The presented findings matched with other researchers who reported hypoglycemia in response to gastrointestinal inflammatory disorders resulted from infection (51, 52). While serum biochemical parameters (cholesterol, triglycerides, ALT, AST, ALP, urea and creatinine) were reported to be non-significant changed. The non- significant change in the serum creatinine was similar to that observed in camel infected with JD (43). With concern to oxidative and antioxidative markers, the non-significant changes in antioxidative markers (GST, GSH and TAC) in clinical and subclinical groups compared to the control group is nearly similar (53) who noted nonsignificant changes in serum superoxide dis-mutase and glutathione perioxidase activities of M. paratuberculosis positive cows. Non-significant changes in oxidative markers (H2O2, NO Hemato-biochemical and molecular markers (IS900) of cattle infected with Johne's disease in Egypt 429 and MDA) in clinically affected cows are indicative they are not associated with oxidative stress. It could be concluded that the infection with the clinical form has a serious significant impact on the health of cows and the production of Egyptian dairy farms. A more continuous exact evaluation of its economic impact might provide a greater impact with an insight into the cost efficiency of detection and screening for subclinical infections, which could help in prevention the new infections and also improve the health and productivity of the herd. The parameters related with hemato-biochemical parameters such as glucose, total protein and globulin level in the present study may be considered in the differential diagnosis of clinical and subclinical M. paratuberculosis infection in dairy farms and waiting for further confirmation of the JD by definitive tests as culture, ELISA and PCR. Still more deep investigation on a large scale is needed to identify the number of cases affected with JD in different localities in Egypt. References 1. Norton S, Johnson WO, Jones G, Heuer C. Evaluation of Diagnostic Tests for Johne's Disease (Mycobacterium Avium Subspecies Paratuberculosis) in New Zealand Dairy Cows. Journal of Veterinary Diagnostic Investigation 2010; 22 (3):341—51. 2. Pieper L, Sorge US, DeVries TJ, Godkin A, Lissemore K, Kelton DF. Evaluation of the Johne's disease risk assessment and management plan on dairy farms in Ontario, Canada. Journal of dairy science 2015 ; 98 (10):6792-800. 3. Lombard JE. Epidemiology and economics of paratuberculosis. Veterinary Clinics: Food Animal Practice 2011; 27 (3):525-35. 4. Salem M, Heydel C, El-Sayed A, Ahmed SA, Zschock M, Baljer G. Mycobacterium avium subspecies paratuberculosis: an insidious problem for the ruminant industry. Tropical Animal Health and Production 2013; 45 (2):351-66. 5. Tewari D, Hovingh E, Linscott R, Martel E, Lawrence J, Wolfgang D, Griswold D. Mycobacterium avium subsp. paratuberculosis (MAP) Antibody Response, Fecal shedding and Antibody Cross-reactivity to Mycobacterium bovis in MAP Infected Cattle Herds Receiving Johne's Vaccination. Clinical and Vaccine Immunology 2014; 00032-00014. 6. Beaudeau F, Belliard M, Joly A, Seegers H. Reduction in milk yield associated with Mycobacterium avium subspecies paratuberculosis (Map) infection in dairy cows. Veterinary Research 2007; 38 (4):625-34. 7. Chiodini RJ, Chamberlin WM, Sarosiek J, McCallum RW. Crohn's disease and the mycobacterioses: a quarter century later. Causation or simple association? Critical Reviews in Microbiology 2012; 38 (1):52-93. 8. Gilardoni LR, Paolicchi FA, Mundo SL. Bovine paratuberculosis: a review of the advantages and disadvantages of different diagnostic tests. Rev Argent Microbiol 2012; 44 (3):201-15. 9. Bölske G, Herthnek D. Diagnosis of Paratuberculosis by PCR. Paratuberculosis: Organism, Disease, Control 2010; 267. 10. Sohal J, Singh S, Subodh S, Shoran N, Narayanasamy K, Singh P, Singh A, Maitra A. Mycobacterium avium subspecies paratuberculosis diagnosis and geno-typing: Genomic insights. Microbiological Research 2009; 164 (3):330-37. 11. Rosenberger G. Die Klinische Untersuchung des Rindes. 3rd ed. Paul Parey, Berlin,München. InTech 1990. 12. Manual OT. Paratuberculosis (Johne" s disease) Section 2.1. Chapter 2008. 13. Kalis C, Hesselink J, Barkema H, Collins M. Culture of strategically pooled bovine fecal samples as a method to screen herds for paratuberculosis. Journal of Veterinary Diagnostic Investigation 2000;12: 547-51. 14. Fernández-Silva J, Abdulmawjood A, Bülte M. Diagnosis and molecular characterization of Mycobacterium avium subsp. paratuberculosis from dairy cows in Colombia. Veterinary Medicine International 2011; 352561. 15. Vary P, Andersen P, Green E, Hermon-Taylor J, McFadden J. Use of highly specific DNA probes and the polymerase chain reaction to detect Mycobacterium paratuberculosis in Johne's disease. Journal of Clinical Microbiology 1990; 28 (5):933-37. 16. Tamura K, Stecher G, Peterson D, Filipski A, Kumar S. MEGA6: molecular evolutionary genetics analysis version 6.0. Molecular Biology and Evolution 2013; 30: 2725-9. 17. Dacie JV. Dacie and Lewis practical haematology. Elsevier Health Sciences 2006. 18. Trinder P. Determination of glcose in blood using glucose oxidase with an alternative oxygen acceptor. Annals of Clinical Biochemistry 1969; 6 (1):24-27. 430 A. Abdelaal, M. Elgioushy, S. Gouda, M. El-Adl, E. Hashish, S. Elgaml, M. Ali, A. El-Gedawy 19. Allain CC, Poon LS, Chan CS, Richmond W, Fu PC. Enzymatic determination of total serum cholesterol. Clinical Chemistry 1974; 20 (4):470-75. 20. Fossati P, Prencipe L. Serum triglycerides determined colorimetrically with an enzyme that produces hydrogen peroxide. Clinical Chemistry 1982; 28(10):2077-80. 21. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with the Folin phenol reagent. Journal of Biological Chemistry 1951; 193 (1):265-75. 22. Doumas B, Biggs H. Determination of Serum albumin: In standard methods of Clinical Chemistry (Cooper GA) Academic press Inc. New York 1972; 1:175 23. Reitman S, Frankel S. A colorimetric method for the determination of serum glutamic oxalacetic and glutamic pyruvic transaminases. American Journal of Clinical Pathology 1957; 28 (1):56-63. 24. Hausamen T-U, Helger R Rick W, Gross W. Optimal conditions for the determination of serum alkaline phosphatase by a new kinetic method. Clinica Chimica Acta 1967; 5 (2):241-45. 25. Heinegard D, Tiderstrom G. Determination of serum creatinine by a direct colorimetric method. Clinica Chimica Acta 1973; 43 (3):305-10. 26. Marsh WH, Fingerhut B, Miller H. Automated and manual direct methods for the determination of blood urea. Clinical Chemistry 1965; 11 (6):624-27. 27. Koracevic D, Koracevic G, Djordjevic V, Andrejevic S, Cosic V. Method for the measurement of antioxidant activity in human fluids. Journal of Clinical Pathology 2001; 54 (5):356-61. 28. Habig WH, Pabst MJ, Jakoby WB. Glutathione S-transferases the first enzymatic step in mercapturic acid formation. Journal of Biological Chemistry 1974; 249 (22):7130--9. 29. Beutler E. Improved method for the determination of blood glutathione. J Lab Clin Med 1963; 61:882-88. 30. Frew JE, Jones P, Scholes G. Spectrophotometric determination of hydrogen peroxide and organic hydropheroxides at low concentrations in aqueous solution. Analytica Chimica Acta 1983; 155:139-50. 31. El-Bayomi K M, Saleh A A, Awad A, El-Tarabany M S, El-Qaliouby H S, Afifi M, El-Komy S, Essawi W M, Almadaly E A, El-Magd M A. Association of CYP19A1 gene polymorphisms with anoestrus in water buffaloes, Reproduction, Fertility and Development 2018; 30(3): 487-97. 32. Nims RW, Darbyshire JF, Saavedra JE, Christodoulou D, Hanbauer I, Cox GW, Grisham MB, Laval F, Cook JA, Krishna MC. Colorimetric methods for the determination of nitric oxide concentration in neutral aqueous solutions. Methods 1995; 7 (1):48-54. 33. Green E, Tizard M, Moss M, Thompson J, Winterbourne D, McFadden J, Hermon-Taylor J. Sequence and characteristics or IS 900, an insertion element identified in a human Crohn's disease isolate or Mycobacterium paratuberculosis. Nucleic Acids Research 1989; 17 (22):9063-73. 34. Harris NB, Barletta RG. Mycobacterium avium subsp. paratuberculosisin veterinary medicine. Clinical Microbiology Reviews 2001; 14 (3):489-512. 35. Tiwari A, VanLeeuwen JA, McKenna SL, Keefe GP, Barkema HW. Johne's disease in Canada: Part I: Clinical symptoms, pathophysiology, diagnosis, and prevalence in dairy herds. The Canadian Veterinary Journal 2006; 47 (9): 874-82. 36. Clarke C. The pathology and pathogenesis of paratuberculosis in ruminants and other species. Journal of Comparative Pathology 1997; 116 (3):217-61. 37. Manning E, Collins M. Mycobacterium avium subsp. paratuberculosis: pathogen, pathogenesis and diagnosis [Johne's disease, paratuberculosis]. Revue Scientifique et Technique de l'OIE (France) 2001; 20(1):133-50. 38. Hulten K, Karttunen T, El-Zimaity H, Naser S, Collins M, Graham D, El-Zaatari F. Identification of cell wall deficient forms of M. avium subsp. paratuberculosis in paraffin embedded tissues from animals with Johne's disease by in situ hybridization. Journal of Microbiological Methods 2000; 42:185-95. 39. Bauerfeind R Benazzi S, Weiss R Schliesser T, Willems H, Baljer G. Molecular characterization of Mycobacterium paratuberc-ulosis isolates from sheep, goats, and cattle by hybridization with a DNA probe to insertion element IS900. Journal of Clinical Microbiology 1996;34: 1617-21. 40. Motiwala AS, Strother M, Amonsin A, Byrum B, Naser SA, Stabel JR, Shulaw WP, Bannantine JP, Kapur V, Sreevatsan S. Molecular epidemiology of Mycobacterium avium subsp. paratuberculosis: evidence for limited strain diversity, strain sharing, and identification of unique targets for diagnosis. Journal of Clinical Microbiology 2003; 41: 2015-26. Hemato-biochemical and molecular markers (IS900) of cattle infected with Johne's disease in Egypt 431 41. Mahillon J, Chandler M. Insertion sequences. Microbiology and Molecular Biology Reviews 1998; 62: 725-74. 42. El-Deeb W, Fouda T, El-Bahr S. Clinico-biochemical investigation of paratuberculosis of Dromedary camels in Saudi Arabia: Proinflammatory cytokines, acute phase proteins and oxidative Stress Biomarkers. Pakistan Veterinary Journal 2014; 34 (4): 484-88. 43. Tharwat M, Al-Sobayil F, Ali A, Hashad M, Buczinski S. Clinical, ultrasonographic, and pathologic findings in 70 camels (Camelus dromedarius) with Johne's disease. The Canadian Veterinary Journal 2012; 53 (5):543-48. 44. Weiss G, Goodnough LT. Anemia of chronic disease. New England Journal of Medicine 2005; 352 (10): 1011-23. 45. Park BS, Park S, Jin K, Kim YM, Park KM, Lee J-N, Kamesaki T, Kim YW. Coombs-Negative Autoimmune Hemolytic Anemia in Crohn's Disease. The American Journal of Case Reports 2014; 15:550-53. 46. Moore RE, Kirwan J, Doherty MK, Whitfield PD. Biomarker discovery in animal health and disease: the application of post-genomic technologies. Biomarker insights 2007; 2: 185-96. 47. Donat K, Erhardt G, Soschinka A, Brandt H. Decreased serum protein associated with Mycobacterium avium subspecies paratuberculosis shedding in German Holstein cows. Vet Rec 2014; 174 (16): 408. 48. Sweeney R, Collins M, Koets A, McGuirk S, Roussel A. Paratuberculosis (Johne's disease) in cattle and other susceptible species. Journal of Veterinary Internal Medicine 2012; 26 (6): 123950. 49. Brady C, O'Grady D, O'Meara F, Egan J, Bassett H. Relationships between clinical signs, pathological changes and tissue distribution of Mycobacterium avium subspecies paratuberculosis in 21 cows from herds affected by Johne's disease. Veterinary Record 2008; 162 (5):147-52. 50. Elson CO, James SP, Graeff AS, Berendson RA, Strober W. Hypogammaglobulinemia due to abnormal suppressor T-cell activity in Crohn's disease. Gastroenterology 1984 ; 86 (3):569-6. 51. .Kamal AM. Some biochemical, heatological and clinical studies of selected ruminal and blood constituents in camels affected by various diseases. Res J Vet Sci 2008; 1 (1): 16-27. 52. Salem M, El-Sayed A, Fayed A, Abo El-Hassan D. Subclinical infection of paratuberculosis among camels in Egypt. Journal of American Science 2012; 8:1141-47. 53. Cenesiz M, Ciftci G, Dalgin D, Kilic Y, Gul FY, Cenesiz S. Evaluation of Oxidant and Antioxidant Capacity in Paratuberculosis Positive Cattle. Pakistan Journal of Zoology 2016; 48 (5): : 1603-06. Slov Vet Res 2019; 56 (Suppl 22): 433-43 DOI 10.26873/SVR-781-2019 Original Research Article EFFECT OF FEEDING DIFFERENT CONCENTRATE ROUGHAGE RATIO ON GROWTH, REPRODUCTIVE PERFORMANCE AND BEHAVIOR OF SHEEP Anaam E. Omar 1*, Heba S. Gharib2, Enas N. Said2 department of Nutrition and Clinical Nutrition, Faculty of Veterinary Medicine, Zagazig University, 44511, Egypt, department of Veterinary Public Health, Faculty of Veterinary Medicine, Zagazig University, Egypt Corresponding author, E-mail: dr.anaamomar85@gmail.com, madamreda72@yahoo.com Abstract: The goal of this study was to examine the influence of feeding different concentrate roughage ratios on growth, reproductive parameters and behavior of sheep. The study design involved the use of a 30 sexually mature ewes assigned to three groups consisting of three ratios of concentrate (C) to roughage (R), Group1. 80%C: 20%R, Group2. 70%C: 30%R and Group3. 60%C: 40%R. The variables of the studies were live weight changes and Body condition score (BCS) of ewes during pregnancy and postpartum till weaning and behavior of ewes in addition to the birth weight and average daily gain of neonatal lambs and subsequent changes till weaning. The conception rate of ewes and their overall reproductive performance are under investigation. The results reveled that, different ratios of concentrate and roughage feeding had no significant effect on body weight, BCS and average dry matter intake of ewes during pregnancy and postpartum till weaning. Lambs birth weight and subsequent weights till weaning and average daily gain were significantly (P<0.05) increased by feeding on ration contain 80%C: 20% R, followed by those feeding on 70% C: 30% R and lowest in those feeding on 60% C: 40%R. Rumination was significantly increased in Group3 lambs than other groups. Higher concentrate ratios had positive effect on reproductive performance of ewes and body weight, daily body weight gain and neonatal behavior of lambs till weaning. Key words: concentrate; roughage; ewe; lamb; growth performance; behavior Introduction There are about 5.6 and 4.13 million heads of sheep and goats in Egypt (1). Sheep and goat production constitute a major part of Bedouins income but in extensive system of production, quality and quantity of pastures affected by weather and environmental changes which would affect feeds availability and quality for sheep (2). These changes had an effect on the nutrition and performance of lambs and their dams. Above pasture conditions would have an impact on birth weight, on quantity of milk production and on weaning weight of lambs. Behavioral understanding is essential for the performance enhancement of sheep on grazing, mainly due to intense climate changes which influence animal physiology. However, it is important to enhance the utilization of pastures Received: January 2019 Accepted for publication: February 2019 434 A. Omar, H. Gharib, E. Said and applying strategies for nutritional management achieving the system highly productive (3).A study in India by (4) also reported significant effect of raising lambs on ADG under intensive system where lambs feed on complete rations compared with extensive system. Feed quality and quantity are the main factors affecting ruminant productivity under tropical conditions, providing large quantities of concentrated feeds in the diet increase energy density of the diet that may improve feed efficiency and animal performance (5) however, feeding on high concentrate diet for long time may be result in decreased ruminal pH due to accumulation of volatile fatty acids and lactic acid and cause chronic digestive disorder (6). Therefore, feeding on proper concentrate level is one of the most important factors to ensure the growth and reproduction of ewes and their lambs. Nutrition play important role on reproduction performance of ruminant animals (7) also affect the onset of postpartum ovarian cyclicity of ruminant animals (8) but there is lack of information regarding the effects of diet on the lambing rate and overall reproductive efficiency of the fat-tailed sheep. In a study, (9) showed that high concentrated feed caused higher ovulation rate and less ova viability. The survival of a neonatal is mainly depending on vigor (the ability to stand up and suck milk during a few hours after birth), maternal behavior and colostrum production. These factors are influenced by the ewe's level of nutrition during late pregnancy (10, 11). The objectives of our study are to define appropriate concentrate roughage ratio that ensure high growth and reproduction performance and behavior of ewes and their lambs. Materials and methods This study was conducted at private farm belonging to Sharkia governorate, Egypt. Animals, housing, diets and experimental design A total of thirty Baladi ewes were purchased locally and selected on the basis that they had lambed at least once before. Their body weights ranged between 40 to 50 Kg and their ages varied between 2.5 to 3 years. They were identified by ear tags, vaccinated against pasteurellosis, sheep pox, colostridial, pestedespetits ruminant (PPR) and enterotoxaemia potential infections. The ewes were exposed to the feedlot housing management and feeding as confined animals (zero grazing). Ewes were randomly divided considering age and body weight in to 3 experimental groups (10 ewes/ group) with 10 replicates of 1 ewe in each (Group1. 80%C: 20%R, Group2. 70%C: 30%R and Group3. 60%C: 40%R) in different stages of the reproduction of ewes. Daily observation was carried out on animals for any health disorders. No mortalities were occurred among different groups during the whole experimental period. The sheep sheds were respectively cleaned thoroughly and disinfected to eliminate any possible infection that transferred from previous sheep flocks. The water and feeding troughs were also cleaned. Individual housing for each animal in individual pens with feed and water troughs. All animals were reared under the same management condition. Ewes lambed indoor and new born lambs live together with their dams in individual pens. Lamb weight was calculated at birth and fortnightly till weaning. The experimental diets were formulated according to specific requirements of ewes (12) as shown in Table 1. Experimental feeds were analyzed for (DM, Moisture, CP, CF, EE and Ash) according to (13). The animals were fed individually twice daily, equally dividing concentrate and roughage components, once in the morning between 6.00 and 7.00 AM, and again between 5.00 and 6.00 PM. The left-over feed was collected on a daily basis in the morning. Actual intake of the concentrate mixture and roughages were recorded. Growth performance of ewes and lambs The ewes were individually weighed at beginning of experiment to obtain the average initial body weight then the body weight was Effect of feeding different concentrate roughage ratio on growth, reproductive performance and behavior of ... 435 recorded monthly during pregnancy period and biweekly after lambing till weaning of lambs to calculate the average body weight of the animals in each group. Feed intake of each ewe was recorded as the difference between weight of the feed offered and residues left. The birth weight of new born lambs was taken within one hour after birth. The subsequent weight of lambs was recorded fortnightly in the morning and before feeding up to weaning. The lambs weight gain was measured as the difference between final body weight at the intended period and primary body weight at same period. Body Condition Scoring Procedures The BCS evaluates the body fat reserve of the sheep and was determined by examining the tail head and the loin areas according to (14). The BCS of sheep was measured by feeling the backbone and the transverse processes of vertebrae in the loin region by the finger. Reproductive performance The ewes were treated with progestogen-impregnated (30 mg) intravaginal sponges for 14 days. After 14 d, the sponges were withdrawn and 400 I.U. of Pregnant Mare Serum Gonadotropin (PMSG) was injected intramuscularly. The ewes were then introduced to rams for natural mating. The number of conceived ewes and number of lambs born a live and number of abortion and still birth were recorded. Litter size was calculated as total number of lambs born / total number of ewes. The live weight of lambs was measured within one hour after lambing. Behavior measurement Focal sampling technique was used to record the time spent by ewes with each behavior. The behavioral activities were recorded through 10 hours (from 7.00 am till 17.00 pm) per week at 10 minutes interval. Behavioral traits recorded are eating, drinking, rumination, standing, walking, lying, urination and defecation. Eating: the mean time (min) spent in eating ration per 10h observation, Drinking: The mean time (min) spent in drinking water per 10 hours observation, Rumination: the mean time (min) spent in rumination /10h observation. Standing: The time (min.) spent in standing without doing any activities during 10hr, observation, Lying: time (min) spent in lying on the ground during 10hr observation, Defecation: when ewe makes voiding of food and water that are not retained in the body for metabolic processes as a solid material from the anus (faeces), Urination: when ewe makes voiding of water that are not retained in the body for metabolic processes as a liquid. While the data for neonatal lambs were collected for the first 2 hours following lambing. The observer recorded the duration in minutes that the neonate spent to be able to stand up without help, to reach udder (approach udder and nudge the inguinal region), time to suck (holding the teat with its mouth and appears to be sucking for more than 5 seconds) and the suckling attempts as described by (15,16). Behavior was evaluated by an observer stood up 2 meter from the pen using an observation sheet, a stop watch and a photograph during the observation period. Statistical analysis The collected data were subjected to analysis of variance using one-way ANOVA using SPSS 24.0 for Windows (SPSS Inc., Chicago, IL, USA) and the differences between experimental groups were calculated by Duncan's multiple-range test at (P < 0.05)(17). Results and discussion In this study three ratio of concentrate and roughage diets were considered and hypothesized as main contributing factors affecting the productive and reproductive performances of ewes under the feedlot (zero grazing) management system. The effect of C: R ratios assigned to 80%C: 20%R, 70%C: 30%R and 60%C: 40%R on the live weight and BCS of ewes during pregnancy and after parturition till weaning are presented in Table 2. No significant (P>0.05) influence of C: R ratios on live weight and BCS of ewes are observed. Our data disagree with results of previous studies where the final live weight and total live weight gain of does increased with higher level of concentrate supplementation (18,19,20), moreover (21) reported that ewes 436 A. Omar, H. Gharib, E. Said under sub-feeding level (0.500, 0.750 and 1.00 kg concentrate /ewe) during first 15 weeks of pregnancy, last 6 weeks of pregnancy and 60 days from parturition till weaning, respectively had lower final weight and BCS than those fed on normal (0.750, 1.00 and 1.25 kg/ewe) and high concentrate level (1.00,1.25 and1.5 kg /ewe). The lack of our effect on growth performance of ewes might be related to the management practice, genetic factor, composition of basal diets (diets are isonitrogenous) and constant feed intake (22). Average daily DM intake of ewes for the entire duration of feeding periods covering the pregnancy and lactation periods was shown in Table 3. The results showed that different ratio of concentrate and roughage had no effect on average dry matter intake/ewe through advanced weeks of experiment from 1-28 weeks. The lack of effect on DM intake may be due to the diets were isonitrogenous (22). Our results were consistent with previous researches in goats were DM intake was not affected with increasing concentrate percentage in diet from 30% to 70% (23) and (24) in Holstein cow were increasing forage concentrate ratios (47:53, 54:46, 61:39 and 68:32) had no effect on dry matter intake. In contrast feeding high level of concentrate increase dry matter intake of goat (18) moreover, (25) found that increasing concentrate percentage in diet from 30% to 60% increasing dry matter intake of dairy goats. Similarly, cow fed on 30% forage: 70% concentrate diet had significantly higher dry matter intake than those fed on 50% forage: 50% concentrate diet (26). New born lamb performance It is important that farmers feed their animals cheap, effective feed ingredients to remain gainful in the face of increasing feed prices. How quickly and efficiently the lambs grow is most important agents affecting economic capability in livestock production. High growing rate of lambs reduce the time needed to reach market weight, which in turn reduces the employments and the feed costs associated with raising of lambs. Composition and quantity of milk are essential for newborn lambs as the milk supplies the main nutrients needed (27), so growth rate of suckling lambs increase if the quantity or nutritive composition of the milk increase.There are also non-genetic factors can affect the lamb growth rate, one of them is system of production, the results showed that the feed lot rations were related with improved lamb growth rates, bigger carcass fatness and better dressing percentage (28). The birth weight and biweekly changes of live weight of new born lambs till weaning are shown in Table 4. The mean lambs birth weight was significantly (P < 0.05) increased by greater concentrate level feeding. The average BW and ADG at week 2 were significantly (P < 0.05) increased in groupl in comparison with group 2&3. The average body weight and ADG at week 4 were significant (P < 0.05) the highest in lambs fed on 80% C: 20% R followed by lambs fed on 70% C: 30% R and lowest in lambs fed on 60% C: 40 % R. The lambs fed on 80%C: 20%R had (P < 0.05) significant higher average BW and ADG at week 6 and 8 followed by lambs fed on 70%C: 30%R while lambs fed on 60%C: 40%R had the lowest. Overall ADG was significantly (P < 0.05) increased by feeding higher levels of concentrate and it is the highest in group 1 and lowest in group 3. Our results of increasing ADG of lambs with increasing concentrate level in diets are consistent with results of (29,30,31). Also, (32,33) indicated that the ADG and FCR of lambs were better in high concentrate diets than low C diets under intensive system of production. Similar types of observations were also recorded by (34) in lambs, (18,35) in kids. However, our data disagree with result of (21) who observed no significant effect on lamb birth weight by feeding different levels of concentrate (0.750, 1.00 and 1.25 kg/ewe). Growth rate of kids feeding different levels of concentrate did not significant different (20). There was no effect of increasing levels of concentrate on feed intake, final live weight, ADG, FCR in lambs (36). Effect of concentrate: roughage ratio on behavior of pregnant and lactating ewes Effect of feeding different concentrate roughage ratio on growth, reproductive performance and behavior of ... 437 Data in the Table 5 revealed that the concentrate percent in ration had no significant impact on most of maintenance behavior of pregnant and lactating ewes. Regarding ingestive behavior, it is observed that ewe fed on Group 3(60%C: 40%R) spent longer eating time than other rations (Group 1and Group 2). These results may be explained to the fact that rapid consumption of concentrates by animals. Our results are in accordance with (22) who found that the eating time was decreased linearly with increasing the concentrate level. The time spent ruminating was significantly higher in pregnant and lactating ewes of group fed on 60%C: 40%R (12.26±0.38 and 11.26±0.4 min/h respectively) than other groups. This may explained to more time needed to ruminate the forage. In a reverse trend, (22) noted that the level of concentrates had no effect on time spent in rumination. Diets rich with concentrate prefered time spent in ruminating during the day while time spent in resting through the night (37). Effect of concentrate: roughage percent on neonate lamb behavior The impact of different ratio of concentrate and roughage during pregnancy on some neonatal lamb behavior are listed in Table 6 generally Group 1, Group 2 and Group 3 lambs showed significant behavioural differences. Lambs' response time to stand was shorter in Group 1 lambs, search for the udder and also response times to first suckling were significantly shorter at Group 1 lambs. Supplementation of high concentrate in peripartum period has an impact on lamb vigour. Lambs' response time to stand up without help, search for the udder and also response time to first suckling were significantly shorter in Group 1 neonates in comparison with other groups. These results were comparable with the results described in other sheep breeds (38). Beginning of suckling could achieve after a longer time because Group 2 and Group 3 neonates tend to tumble more times comparing to Group 1 neonates. Postpartum Group 1 ewes moved and cleaned their neonates more frequently, and depending on their maternal experience which can stimulate lamb activity (39). Feeding supplies at birth had an impact on lamb strength. Generally, latency to standing and suckling was slower in low-birth weight lambs than heavier lambs. Lamb vigor is affected by amount of adipose tissue and locomotor ability. Brown adipose tissue provides the neonate lamb with energy needed for thermoregulation and nutrition from birth till colostrum intake (40). Development of CNS of lamb, and subsequently locomotor activity, may be impacted by the nutrition of dam through pregnancy (41). Reproductive performance of Ewes In sheep production operations, the producers are interested in the number of ewes becoming pregnant, lamb born alive and lambs survive till weaning and then finishing for the markets. These important variables were summarized showing dietary influences in Table 7. About 80%, 50% and 40% of ewes fed groups 1, 2 and 3 respectively conceived producing a total of 8, 5 and 4 lambs from 10 ewes joined. No abortion or still birth has been reported. All births were for single lamb. The conception rate for ewes was higher in ewes fed on 80%C: 20%R followed by ewes fed on 70%C: 30% R then ewes fed on 60%C: 40%R. No twin birth was observed. The average litter size was higher in group 1 compared to groups 2 and 3. Group 1 was found to have more lambs born/ewe than other two groups. Reproductive performance of the sheep was known to be one of the major factors determining the efficiency of their production (42). Seasons of production, nutrition, breed, age, management and environment are some factors that affect reproductive activities on sheep (43, 44). Our results are consistent with results of (19) where the concentrate supplementation had positive effect on litter size and birth weight of kids moreover, concentration feeding had positive effect of reproductive traits of goat (20). (21) reported no effect of feeding different levels of concentrate on lambing, fecundity or prolificacy rates. In earlier work, (45) observed that the level of feeding or energy in the diet is 438 A. Omar, H. Gharib, E. Said a major influencing factor on the birth weight of the lambs. The ewes receiving high energy diet during the last trimester of pregnancy gained more live weight than those receiving low energy diets. (46) found that the ovulation rate of induced estrus increased with improved nutritional regime of ewes, therefore the conception rate and litter size were significantly and positively influenced by the interaction of nutrition and induced estrus. . However, (47) reported no effect of nutrition on estrus and ovulation rate of ewes. Table 1 : Proximate and chemical composition of Experimental diets Ingredients Group 1 (80% C: 20% R) Group 2 (70% C: 30% R) Group3 (60% C : 40% R) A. Concentrates Wheat Bran, kg Corn, kg Soyabean Meal (48%),kg *Vit. & Min. Premix, kg Limestone, kg Calcium dibasic Phosphate, kg Salt, kg Total A, kg B. Roughages Alfalfa Hay, kg Wheat Straw, kg Total B, kg Total A + B, kg_ 32.4 34.5 11 0.3 100 0.3 0.5 80.00 10.00 10.00 20.00 100 23 33 12 0.3 0.6 0.6 0.5 70.00 15.00 15.00 30.00 100 9.00 35.5 13.5 0.3 0.2 1.00 0.5 60.00 20.00 20.00 40.00 100 Chemical composition DE, Kcal/Kg CP, % EE, % CF, % Ca, % Available phosphorus % 2545 14.07 2.23 9.31 0.66 0.53 2529.2 14.09 2.03 9.24 0.67 0.52 2554.1 14.05 2.00 10.44 0.69 0.51 * Vitamins and minerals premix: Phosphorus 5.00%, Calcium 18 %, Sodium 5 %, Magnesium 5 %, Manganese 500 mg/kg, Cobalt 100 mg/kg, Zinc 2000 mg/kg, Iodine 125 mg/kg, Selenium 10 mg/kg, Vitamin A 400000 IU/kg, and Vitamin E (Alpha-Tocopherol) 400IU/kg. C: Concentrate, R: Roughage, DE: Digestible energy, CP: Crude protein, EE: Ether extract, CF: Crude fiber, Ca: Calcium. Effect of feeding different concentrate roughage ratio on growth, reproductive performance and behavior of ... 439 Table 2: Effect of feeding different concentrate roughage ratio on live weight (kg) and body condition score (BCS) of ewes during pregnancy and after parturition till weaning. Period Variable Group 1 Group 2 Group3 P-value (80%C:20%R) (70%C:30%R) (60%C:40%R) During pregnancy First month Weight 46.30 ± 3.16 44.71 ± 4.16 44.29 ± 4.28 0.498 BCS 3.00 ± 0.00 a 3.00 ± 0.00 a 2.78 ± 0.26 b 0.006 Second month Weight 48.43 ± 2.10 47.27 ± 3.96 46.46 ± 5.04 0.566 BCS 3.00 ± 0.00 3.00 ± 0.00 2.89 ± 0.22 0.123 Third month Weight 49.10 ± 3.82 48.96 ± 3.96 48.68 ± 4.76 0.976 BCS 3.10 ± 0.21 3.06 ± 0.18 3.06 ± 0.17 0.857 Fourth month Weight 51.50 ± 3.11 51.21 ± 6.28 50.57 ± 3.98 0.903 BCS 3.40 ± 0.21 3.25 ± 0.27 3.17 ± 0.25 0.123 Fifth month Weight 57.01 ± 5.78 56.13 ± 2.25 55.68 ± 3.78 0.801 BCS 3.65 ± 0.24 3.56 ± 0.32 3.44 ± 0.18 0.227 Postpartum to Weaning At Birth Weight 51.86 ± 2.76 55.44 ± 7.36 49.50 ± 2.38 0.226 BCS 3.50 ± 0.35 3.58 ± 0.38 3.25 ± 0.29 0.355 At week 2 Weight 49.86 ± 3.56 53.42 ± 7.91 47.70 ± 1.99 0.297 BCS 3.30 ± 0.27ab 3.58 ± 0.20a 3.12 ± 0.25b 0.032 At Week 4 Weight 50.16 ± 4.25 52.25 ± 7.67 48.10 ± 0.96 0.599 BCS 3.30 ± 0.27 3.33 ± 0.41 3.00 ± 0.00 0.345 At Week 6 Weight 48.44 ± 3.45 49.67 ± 5.33 48.33 ± 1.15 0.857 BCS 3.00 ± 0.00 3.17 ± 0.26 3.00 ± 0.00 0.251 At Week 8 Weight 46.84 ± 4.07 49.67 ± 4.56 48.67 ± 1.26 0.519 BCS 3.10 ± 0.22 3.17 ± 0.26 3.17 ± 0.29 0.895 ab Means within the same row carrying different superscripts were significantly different at (P < 0.05). C: Concentrate R: Roughage BCS: Body condition score Table 3: Effect of feeding different concentrate(C) roughage (R) ratio on daily DM intake of ewes. Advancing weeks (pregnancy and lactation) Group 1 (80%C:20%R) Group 2 (70%C:30%R) Group 3 (60%C:40%R) P-value 1 993.25 ± 8.16 985.17 ± 11.86 991.38 ± 9.36 0.181 2 998.57 ± 1.21 997.71 ± 2.49 999.14 ± 1.00 0.187 3 1095.00 ± 4.54 1097.36 ± 6.60 1099.00 ± 3.16 0.214 4 1097.86 ± 5.63 1097.64 ± 4.30 1100.00 ± 0.00 0.376 5 1094.50 ± 9.83 1097.78 ± 5.48 1100.00 ± 0.00 0.182 6 1100.00 ± 0.00 1099.57 ± 1.35 1100.00 ± 0.00 0.381 7 1100.00 ± 0.00 1100.00 ± 0.00 1100.00 ± 0.00 — 8 1144.88 ± 10.02 b 1156.07 ± 9.24 a 1151.67 ± 10.27 ab 0.053 9 1233.18 ± 17.10 1221.50 ± 25.09 1225.86 ± 17.91 0.442 10 1224.53 ± 17.83 1228.71 ± 14.02 1229.50 ± 17.60 0.773 11 1240.27 ± 12.95 1239.01 ± 9.53 1238.54 ± 8.67 0.931 12 1232.56 ± 20.15 1241.86 ± 6.23 1240.72 ± 9.11 0.249 13 1244.43 ± 6.21 1243.86 ± 6.84 1239.64 ± 6.03 0.204 14 1244.50 ± 5.55 1245.64 ± 3.82 1245.14 ± 5.77 0.883 15 1244.78 ± 6.94 1245.93 ± 3.91 1245.07 ± 5.56 0.894 16 1245.50 ± 5.31 1244.50 ± 4.69 1244.78 ± 4.49 0.894 17 1246.57 ± 5.21 1245.14 ± 3.94 1243.07 ± 7.29 0.392 18 1246.00 ± 4.94 1245.07 ± 4.72 1244.86 ± 6.25 0.879 19 1244.21 ± 6.86 1244.86 ± 5.63 1242.71 ± 8.42 0.787 20 1245.07 ± 6.28 1243.07 ± 7.89 1242.14 ± 8.89 0.693 21 1243.07 ± 10.16 1240.14 ± 15.33 1245.00 ± 5.17 0.617 22 1245.71 ± 6.60 1244.50 ± 7.65 1244.86 ± 6.26 0.921 23 1247.00 ± 3.65 1243.00 ± 9.17 1245.43 ± 6.73 0.437 24 1246.71 ± 3.61 1245.43 ± 6.86 1245.50 ± 4.97 0.832 25 1247.71 ± 2.95 1244.00 ± 5.94 1245.86 ± 4.06 0.200 26 1245.64 ± 3.76 1244.00 ± 6.67 1247.36 ± 2.18 0.280 27 1243.07 ± 10.16 1240.14 ± 15.33 1245.00 ± 5.17 0.617 28 1245.71 ± 6.60 1244.50 ± 7.65 1247.36 ± 2.18 0.280 ab Means within the same row carrying different superscripts were significantly different at (P < 0.05). 440 A. Omar, H. Gharib, E. Said Table 4: Effect of feeding different concentrate roughage ratio on body weight and body weight gain of newborn lambs till weaning (Mean ±SD) Variable Group 1 (80%C:20%R) Group 2 (70%C:30%R) Group 3 (60%C:40%R) P- value BW at Birth (kg). 5.133 ± 0.37 a 4.817 ± 0.73 ab 4.337 ± 0.50 b 0.027 BW at week 2 (kg). 9.017 ± 0.80 a 8.06 ± 0.83 b 7.519 ± 0.46 b 0.002 BW at week 4 (kg). 13.650 ± 1.06 a 12.250 ± 0.50 b 10.887 ± 0.64 c 0.000 BW at week 6 (kg). 17.893 ± 1.29 a 16.125 ± 0.25 b 14.625 ± 0.69 c 0.000 BW at week 8 weaning (kg). 21.87 ± 1.50 a 19.63 ± 0.25 b 17.63 ± 1.22 c 0.000 ADG From birth to week 2 (g). 277.45 ± 38.60 a 231.79 ± 21.42 b 227.23 ± 36.77 b 0.033 ADG From week 2 to week 4 (g). 330.92 ± 33.58 a 299.11 ± 26.79 a 240.63 ± 43.74 b 0.001 ADG From week 4 to week 6 (g). 303.06 ± 17.91 a 276.79 ± 17.86 ab 266.94 ± 26.43 b 0.019 ADG From week 6 to week 8 weaning (g). 284.18 ± 40.82 a 250.00 ± 29.16 ab 214.29 ± 50.51 b 0.023 Overall ADG from birth till week 8 (kg) 16.739 ± 1.23 a 14.807 ± 0.75 b 13.288 ± 1.22 c 0.000 BW: Body weight; ADG: Average daily gain. abc Means within the same row carrying different superscripts were significantly different at (P < 0.05). Table 5: Means (±SE) of time of some maintenance behavior of pregnant and lactating ewes in relation to different ratio of roughage and concentrate feeding (min/h) Behavior Pregnant ewes Lactating ewes Group 1 Group 2 Group 3 P-value Group 1 Group 2 Group 3 P-value Eating 19.0±4.8 15.1±5.93 27.0±2.05 0.217 19.6±4.01 16.9±5.56 25.8±1.8 0.264 Drink 6.4±1.2 5.2±1.39 5.4±1.12 0.771 7.0±0.83 5.60±1.28 7.2±0.80 0.489 Rumination 6.4±1.77 6.88±1.54 12.26±0.38 0.012 6.4±1.63 7.28±1.16 11.26±0.4 0.009 Lying 12.86±3.6 18.40±7.4 14.2±6.7 0.807 13.26±3.51 21.8±6.43 16.4±6.51 0.574 Standing 5.2±0.95 7.06±1.06 2.54±0.5 0.011 5.4±0.91 7.6±1.33 2.02±0.72 0.007 Walking 3.7±0.24 3.1±1.32 0.90±0.40 0.072 8.8±1.06 3.9±1.33 1.10±0.33 0.386 Urination 0.62±0.19 0.44±0.20 0.41±0.17 0.717 0.82±0.21 0.52±0.18 0.41±0.17 0.333 Defecation 2.02±0.6 1.16±0.47 0.61±0.24 0.138 2.42±0.5 1.48±0.36 1.08±0.0 0.058 Table 6: Effect of feeding different concentrate roughage ratio during gestation period on some neonatal behavior Group 1 (80%C:20%R) Group 2 (70%C:30%R) Group 3 (60%C:40%R) P-value Birth to stand(min) 19.75±0.64 21.20±0.58 23.25±0.85 0.014 Birth to udder(min) 23.75±0.75 27.40±0.74 28.5±1.70 0.009 Birth to suck (min) 27.00±0.70 32.40±0.92 32.75±0.85 0.000 Suckling attempt 4.25±0.36 5.20±0.37 6.75±0.47 0.003 Effect of feeding different concentrate roughage ratio on growth, reproductive performance and behavior of ... 441 Table 7: Effect of feeding different concentrate roughage ratio on reproductive performance of ewes Variables Group 1 (80%C:20%R) Group 2 (70%C:30%R) Group 3 (60%C:40%R) No. of ewe mated 10 10 10 No. conceived 8 5 4 Abortions/stillbirths 0 0 0 No. of lambs born alive 8 5 4 No. of lambs aborted/stillborn 0 0 0 Total number of Lambs produced 8 5 4 Conception rate% 80 50 40 Lambing rate % (Lambs produced/ewe lamb mated) 80 50 40 Average litter size 0.8 0.5 0.4 C: Concentrate R: Roughage Conclusion It could be concluded that feeding higher concentrate roughage ratio (80C%: 20%R) and (70%C: 30%R) improved body weight, body weight gain and vigour of newborn lambs and reproductive performance of ewes. References 1. Egyptian Ministry of Agricultural Economic statistical book 2009. 2. Momoh OM, Rotimi EA, Dim NI. Breed effect and non-genetic factors affecting growth performance of sheep in a semi-arid region of Nigeria. Journal of Applied Biosciences 2013; 67:5302-7. 3. Silva TP, Marques CA, Torreao JN, Bezerra RN, Araújo MJ, Lima LA. Effect of concentrate supplementation and time scales of evaluation on behavioral and physiological responses of pregnant ewes on grazing system. Acta Scientiarum. Animal Sciences 2016; 38 (1):77-86. 4. Malisetty V, Yerradoddi RR, Devanaboina N, Mallam M , Mitta P. Effect of feeding sorghum straw based complete rations with different roughage to concentrate ratio on dry matter intake, nutrient utilization, and nitrogen balance in Nellore ram lambs. Tropical Animal Health and Production 2014; 46(5):759-64. 5. Missio RL, Brondani IL, Alves Filho DC, Restle J, Arboitte MZ, Segabinazzi LR. Características da carcasa e da carne de tourinhos terminados em confinamento, recebendo diferentes niveis de concentrado na dieta. Revista Brasileira de Zootecnia 2010; 39(7):1610--7. 6. Chen Y, Oba M, Guan LL. Variation of bacterial communities and expression of Toll-like receptor genes in the rumen of steers differing in susceptibility to subacute ruminal acidosis. Veterinary Microbiology 2012; 159:451-9. 7. Maurya VP, Sejian V, Kumar D, Naqvi SMK. Effect of induced body condition score differences on sexual behavior, scrotal measurements, semen attributes, and endocrine responses in Malpura rams under hot semi-arid environment. Journal of Animal physiology and Animal Nutrition 2010; 94: 308-17. 8. Zarazaga LA, Guzman JL, Dominguez C, Perez MC, Prieto R. Effect of plane of nutrition on seasonality of reproduction in Spanish Payoya goats. Journal of Animal Reproduction Science 2004; 87: 253-67. 9. Al-Khozam NM. Effect of peri-ovulatory diet composition on the subsequent development of fertilized sheep ova in vivo and during in vitro culture. M.Sc. thesis, 1994, Aberdeen University, UK. 10. Dwyer C. Maternal behaviour and lamb survival: from neuroendocrinology to practical application. Animal 2014; 8:102-12. 11. Nowak R and Poindron P. From birth to colostrum: early steps leading to lamb survival. Reproduction Nutrition Development 2006; 46:431-46. 12. NRC. Nutrient Requirements of Dairy Cattle, 7th ed. Washington, D.C.: The National Research Council, National Academy Press 2001. 442 A. Omar, H. Gharib, E. Said 13. AO AC. Official Method of Analysis. Association of Official Agricultural Chemists. Washington. D.C. and New York: Academic Press 2002:1-350. 14. Russel AJF, Doney JM, Gunn RG. Subjective assessment of body fat in live sheep. Journal of Agriculture Sciences 1969; 72: 451-4. 15. Dwyer CM, Calvert SK, Farish M, Donbav J, Pickup HE. Breed, litter and parity effects on placental weight and placentome number, and consequences for the neonatal behaviour of the lamb. Theriogenology 2005; 63:1092-110. 16. Pedernera M, Pérez-Sánchez LA and Romero-Aguilar LD et al. Effects of high concentrate supplementation of Saint Croix sheep during peripartum on neonatal lamb behaviour. Journal of Applied Animal Research 2017; 46(1): 720-4. 17. Duncan DB. Multiple range and multiple F. tests. Biometrics 1955; 11: 1-42. 18. Ferdous MR, Khan MJ, Rashid MA and Kamruzzaman M. Effect of different levels of concentrate supplementation on the performance of Black Bengal goat. Bangladesh Journal of Animal Science 2011; 40: 40-5. 19. Mahfuz SU, Islam MSD, Chowdhury MR, Islam S, Hasan MK, Uddin MN. Influence of concentrate supplementation on production and reproduction performance of female Black Bengal goat. Indian Journal of Animal Research 2018; 52(5): 735-9. 20. Sultana S, Khan MJ, Hassan MR, Khondoker MAMY. Effects of concentrate supplementation on growth, reproduction and milk yield of Black Bengal goats (Caprahircus). The Bangladesh Veterinarian 2012; 29(1): 7 - 16. 21. El-Shakhret KJ, Harb MY, Abu-Zanat M, Tabbaa MJ. Effect of different feeding levels of concentrate on voluntary intake of straw and on productive and reproductive performance of Awassi sheep in Jordan Valley. Agricultural Sciences 1996; 23(2). 22. Nunes PH, Maia PM and Silva RMG et al. Increasing levels of concentrate digestibility, performance and ingestive behavior in lambs. Revista Brasileira de Saúde e Produ^ao Animal 2016; 17(2): 186-94. 23. Cantalapiedra-Hijar C, Yáñez-Ruiz DR, Martín-García AI, Molina-Alcaide E. Effects of forage: concentrate ratio and forage type on apparent digestibility, ruminal fermentation, and microbial growth in goats. Journal of Animal Science 2014; 87:622-31. 24. Aguerre MJ, Wattiaux MA, Powell JM, Broderick GA, and Arndt C. Effect of forage-to-concentrate ratio in dairy cow diets on emission of methane, carbon dioxide, and ammonia, lactation performance, and manure excretion. Journal of Dairy Science 2011; 94:3081-93. 25. Desnoyers M, Duvaux-Ponter C, Rigalma K, Roussel S, Martin O, Giger-Reverdin S. Effect of concentrate percentage on ruminal pH and timebudget in dairy goats. Journal of Animal Science 2008; 2:1802-8. 26. Murphy M, Akerlind M, Holtenius K. Rumen fermentation in lactating cows selected for milk fat content fed two forage to concentrate ratios with hay or silage. Journal of Dairy Science 2000; 83:756-4. 27. Hegarty M, Montello DR, Richardson AE, Ishikawa T, Lovelace K. Spatial abilities at different scales: individual differences in aptitude test performance and spatial layout learning. Intelligence 2006; 34:151-76. 28. Cloete JJE, Hoffman LC, Cloete SWP. A comparison between slaughter traits and meat quality of various sheep breeds: wool, dual purpose and mutton. Meat Science 2012; 91:318-24. 29. Carvalho S, Brochier MA, Pivato J, Teixeira RC, Kieling R. Desempenho e avalia^ao económica da alimentado de cordeiros confinados com dietas contendo diferentes relates volumoso: concentrado. Ciencia Rural 2007; 37(5):1411-7. 30. Karim SA and Rawat PS. Growth performance and carcass characteristics of lambs raised on varying proportions of roughage and concentrate. Indian Journal of Animal Sciences 1997; 67: 902-5. 31. Noel A. Claffey, Alan G. Fahey, Vasiliki Gkarane, Aidan P. Moloney, Frank J. Monahan, and Michael G. Diskin. Effect of forage to concentrate ratio and duration of feeding on growth and feed conversion efficiency of male lambs. Translation Animal Science 2018; 2: 419-27. 32. Santra A and Karim SA. Effect of dietary roughage and concentrate ratio on nutrient utilization and performance of ruminant animals. Animal Nutrition and Feed Technology 2009; 9: 113-35. 33. Venkateshwarlu V, Venkateswarlu M, Ramana Reddy Y, Harikrishna CH. Effect of feeding maize stover based complete rations on growth and carcass characteristics in Nellore x Deccani ram lambs. Indian Journal of Animal Nutrition 2014; 31: 36-43. 34. Santra A and Karim SA. Nutrient utilization and growth performance of defaunated and faunated Effect of feeding different concentrate roughage ratio on growth, reproductive performance and behavior of ... 443 lambs maintained on complete diets containing varying proportion of roughage and concentrate. Animal Feed Science and Technology 2002; 101:87-99. 35. Singla M, Sirohi AS, Lamba JS, Ahuja CS. Effect of soaked and boiled concentrate feeding on growth, tvfa's production and blood biochemistry of stall-fed beetal kids. Indian journal of Animal Research 2014; 48 (5): 448-51. 36. Sudheer Babu A, Srinivasa Rao D, Nagalakshmi D, Ramana Reddy Y. Effect of feeding various roughage based processed complete rations on growth performance and carcass characteristics in growing ram lambs. Livestock Research for Rural Development 2013; 25(5). 37. Minervino AHH, Kaminishikawahara CM and Soares FB et al. Behaviour of confined sheep fed with different concentrate sources. Brazilian Journal of Veterinary and Animal Science 2014; 66(4): 1163-70. 38. Dwyer C. Behavioral development in the neonatal lamb: effect of maternal and birth-related factors. Theriogenology 2003; 59:1027-50. 39. Dwyer CM. The welfare of the neonatal lamb. Small Ruminant Research 2008; 76:31-41. 40. Symonds M, Sebert S, Budge H. Nutritional regulation of fetal growth and implications for productive life in ruminants. Animal 2010; 4:107583. 41. Alamy M and Bengelloun WA. Malnutrition and brain development: an analysis of the effects of inadequate diet during different stages of life in rat. Neuroscience and Biobehavioral reviews Journal 2012; 36:1463-80. 42. Ibarra D, Laborde D, Van-Lier E. Repeatability and relationship with field mating performance of a serving capacity pen test in rams. Small Ruminant Research 2000; 37: 156-69. 43. Amer HA and Hazzaa AM. The effect of different progesterone protocols on the reproductive efficiency of ewes during the nonbreeding season. Veterinarski Arhiv 2009; 79: 19-30. 44. Ozyurtlu N, Kucukaslan I and Cetin Y. Characterization of Oestrous Induction Response, Oestrous Duration, Fecundity and Fertility in Awassi Ewes During the Non-Breeding Season Utilizing both CIDR and Intravaginal Sponge Treatments. Reproduction in Domestic Animals 2010; 45:464-7. 45. Khalaf AM, Doxy DL, Baxter JT. A study of some reproductive and productive trails in a flock of awassi sheep. World Review of Animal Production 1979;3:29-33. 46. Lassoued N, Rekik M, Mahouachi M, Ben Hamouda M. The effect of nutrition prior to and during mating on ovulation rate, reproductive wastage, and lambing rate in three sheep breeds. Small Ruminant Research 2004; 52:117-25 47. Henniawati H and Fletcher ICX. Reproduction in Indonesian sheep and goats at two levels of nutrition. Animal Reproduction Science 1986; 12: 77-84. Slov Vet Res 2019; 56 (Suppl 22): 445-55 DOI 10.26873/SVR-782-2019 Original Research Article IMMUNOLOGICAL RESPONSE OF A NEW TRIVALENT CAPRIPOX-VIRUS VACCINE IN PREGNANT EWES AND DOES Eman B. Abd-Elfatah1, Mamdouh F. El-Mekkawi1, Emad A. Aboul-Soud2, Elshaima M. Fawzi1*, Sherif A. El-Soally3 1Department of Animal Medicine, Division of Infectious Diseases, Faculty of Veterinary Medicine, Zagazig University, Sharkia , 44511, Egypt, 2Veterinary Serum and Vaccines Research Institute, Department of Pox, Abbasia, Cairo, Egypt, 3 Military Veterinary Hospital, Cairo, Egypt Corresponding author, E-mail: elshaimafawzi@yahoo.es, a.fawzy@zu.edu.eg Abstract: This study was conducted as a field trial of using a new candidate trivalent Capripoxvirus (CaPVs) vaccine in pregnant ewes and does to detect safety degree and level of immunity, in comparison with commercial Romanian sheep pox viral (RSPPV) vaccine. Monitoring the post-vaccinal reaction and measurement of cellular and humoral immunity by evaluating the lymphocyte proliferation assay (LPA) and serum neutralization test (SNT), respectively, in addition to detect the acquired maternal immunity in new-born lambs and kids .The post-vaccine reaction in animals vaccinated with trivalent CaPVs vaccine was more noticeable than that vaccinated with RSPPV vaccine without significant difference (P-Value>0.05). In vitro, the evaluation of cellular and humoral immunity revealed that the animals vaccinated with trivalent vaccine can significantly induce lymphocyte proliferation response in addition to of high level of antibodies (Abs) in comparison to that vaccinated with RSPPV vaccine (P-Value <0.05) with peak titre of lymphocyte proliferation and Abs titre at 10th and 28th, respectively. Furthermore, the evaluation of maternal immunity in new-born lambs and kids from previously vaccinated dams with trivalent CaPVs and RSPPV vaccines by SNT was shown that the peak level of receiving immunity was appeared at 1st week after birth with increasing its level in lambs and kids of dams vaccinated with trivalent CaPVs vaccine than those of dams vaccinated with RSPPV vaccine with a significant difference in between (P-Value <0.05). The result of this study provide a good profile about a high immune response with a long level of maternal immunity provided with trivalent CaPVs vaccine in comparison to the RSPPV vaccine so toward about the preferable use of new vaccine to control infection with sheep and goat pox diseases. Trivalent CaPVs vaccine could be candidate to be used against lumpy skin disease (LSD) due to antigenic relationship between genus CaPVs strains. Key words: does; ewes; monovalent RSPPV; safety; trivalent CaPVs Introduction Sheep and goat pox (SGP) diseases are one of the contagious viral diseases affecting small ruminants caused by sheep pox virus (SPPV) and goat pox virus (GTPV), belonged to genus Capripoxvirus (CaPVs) along with lumpy skin disease virus (LSDV) of cattle, within family Received: January 2019 Accepted for publication: February 2019 446 E. Abd-Elfatah, M. El-Mekkawi, E. Aboul-Soud, E. Fawzi, S. El-Soally Poxviridae (1). CaPVs strains are antigenically and serologically identical but can be differentiated at the genetic level (2). In adequate management, bad hygiene and a route of CaPVs diseases transmission corroborate the diseases spreading particular in the poor farmers and this adversely affects on central economy that relay on the production of livestock (3). SPPV and GTPV were considered to be host specific viruses but some strains as Yemen, Kenya and Oman isolates infect both goats and sheep while most SPPV and GTPV isolates show distinct host preferences with more virulence in one of the two species (1). Cross protection between SPPV and GTPV or vice versa is partial so a homologous vaccine was recommended for an optimum protection (4, 5). In the most countries at least two different vaccines containing the isolates of either GTPV or SPPV are necessary to protect small ruminants against both viruses (6, 7). In endemic countries with CaPVs, control of the diseases depend mainly on a laboratory diagnosis that followed by a quarantine application, in addition to a vaccination is considered the most easy and effective protection way against all CaPVs diseases (8, 9,10). Live attenuated tissue culture vaccines in a lyophilized form provided a good protection and able to control an outbreaks of CaPVs diseases (11). CaPVs strains share a major neutralizing site; consequently it is possible to protect cattle against LSD using strains of CaPVs derived from either sheep or goats as used in Egypt by using Romanian sheep pox (RSPPV) vaccine. Furthermore SPPV and GTPV do not occur in Southern Africa so only attenuated LSD vaccine are used, whereas in northern, central Africa and in the Middle East where the distribution of SPP, GTP and LSD viruses overlap, an attenuated SPP vaccines such KSGP O-240, RM65 and Romanian SPPV strains have been used against LSD (12,13). Kenyan SGP O108 vaccine used to immunize both small ruminant and cattle against CaPVs with remarkable success (11, 14, 15). In Egypt, it was reported that the live attenuated RSPPV vaccine did not provide cattle with complete protection against LSD and reoccurrence of an outbreaks in vaccinated animals (16-18), goats vaccinated with RSPPV vaccine are fully unsuccessful (6,19). In Egypt, three CaPVs (SPPV, GTPV, LSDV) were endemic and appeared in sporadic and an outbreak cases, trying to use a trivalent vaccine of SPPV strains (Romanian, Kenyan 0180) and GTPV vaccine with an equal volume and nearly equal titre used as abroad-spectrum vaccine candidates against all CaPVs diseases is a good trail with a remarkable high immunity compared with commercial RSPPV vaccine alone (20-22). SPPV and GTPV vaccines do not replicate in small animals models to evaluate its efficacy, an evaluation of the vaccine in its preferable and specific host is the first step to detect the efficacy of the vaccine experimentally and in field application. This study was done to evaluate the safety and efficacy of trivalent CaPVs vaccine in pregnant animals by evaluating both cell-mediated immunity (CMI) and humoral immunity, in comparison with positive control group of vaccinated pregnant small ruminants with commercial RSPPV vaccine. In addition to evaluate the passive maternal immunity in new-born lambs and kids to determine the suitable time to start vaccination in both types of vaccines. Materials and methods The experimental protocol was approved by Zagazig University Institutional Animal Care and Use Committee (ZU-IACUC/2/F/98/2018) Animals Pregnant small ruminants (ewes, does) Eighteen ewes and seventeen does at last third of pregnancy and free from antibodies (Abs) against SGPV in which 5 of these animals (3ewes and 2 does) were applied to check the safety test of new candidate trivalent CaPVs vaccine, other thirty ewes (15 ones) and does (15 ones) were divided into 3 groups: Group 1 (G1) composed of 12 ewes and does, divided equally, were vaccinated with trivalent vaccine; (G2) composed of 12 ewes and does, divided equally, were vaccinated with RSPPV vaccine Immunological response of a new trivalent Capripoxvirus vaccine in pregnant ewes and does 447 (control positive) and (G3) composed of 6 un-vaccinated animals (control negative). New-born lambs and kids Thirteen lambs and thirteen kids with good healthy, well-body condition and have adequate amount of colostrums directly after parturition were chosen in this experiment. The new-born lambs and kids were divided into 3 groups: (G4) 10 lambs and kids from dams vaccinated with trivalent vaccine, (G5) 10 lambs and kids from dams vaccinated with RSPPV vaccine (control positive), (G6) 6 from unvaccinated dams (control negative). Vaccines and animals vaccination program Experimentally trivalent vaccine composed of three strains (KSPP 0108, RSSP and Held GTP) with a titer (104 75 TCID50) and monovalent RSPPV vaccine (10525TCID 50) that was obtained kindly from the veterinary serum and vaccine research institute (VSVRI), Egypt. The titer of each virus vaccine was titrated according to Reed and Muench (23). Reconstituted vaccines with sterile phosphate-buffered saline were kept on ice, protected from direct sunlight and used within 1hour (h). Pregnant dams were vaccinated at last 2 months of pregnancy with 1 ml /intradermal or/and subcutaneous according to manufacture instructions of the reconstituted vaccines contain (1025TCID50/ml) according to the manufacturer's instructions, while the control group kept un-vaccinated. Safety test The new candidate trivalent CaPVs was tested to check its safety by inoculation 5 pregnant small ruminants (3 ewes and 2 does) with 20x field dose I/D (14). Samples Peripheral blood mononuclear cells (PBMCs) Whole blood collected from both vaccinated and control animals regularly at 0, 2, 5, 10, 20, 30 days post-vaccination from jugular vein, PBMCs separated by collection 3 ml of peripheral blood from each animal on Na heparin, blood was overlaid on the surface of lymphocyte separation medium by lymph prep (1:1) in sterile centrifuge tubes then centrifuged at 2500 rpm for 30 minutes, PBMCs aspirated by pipette from interphase layer and washed 3 times by heparinized PBS after centrifuged at 2000 rpm for 10 min (24), cells re-suspended in RPMI-1640 with antibiotic and 10% FCS. Serum Serum samples were collected from pregnant sheep and goats to monitoring the immune response Abs for 3 months at day 0 before vaccination then every week during 1st month post vaccination then every 2 weeks during 2nd and 3rd months post vaccination (0, 1, 2, 3, 4, 6, 8, weeks...etc). Good healthy new-born lambs and kids were selected for assessment of maternal immunity which were clinically examined for well-weight and good general body condition, serum samples were collected weekly at 1st month then every two weeks at 2nd and at 3rd months of age (1, 2, 3, 4, 6, 8, weeks.. .etc), to determine the level maternal Abs against the two types of vaccines which transferred from vaccinated dams to their new-lambs and kids and the best time of immunization, all the serum samples were tested for the presence of Abs by using SNT. Lymphocyte proliferation assay (LPAj LPA was done in 96-well flat-bottom plates using XTT assay according to (25, 26), the assay was carried on PBMCs according to the instructions KitX6493 purchased from ther-mofisher scientific company, briefly PBMCs cells grown in a 96-well plate at a density of 104-105 cells/well in 100 |L of culture medium, the plate was incubated in a C02 incubator for 24-48 h, 10 |L of the prepared PMS solution in the prepared 4 mL of XTT solution created immediately before labeling cells, 25 |L of XTT/PMS solution directly to each well then the plate incubate for 2 h at 37°C in a CO2 incubator and read absorbance at 450 nm. Serum neutralization test (SNTj The test was applied to detect Abs level before and after vaccination of pregnant ewes and does and passive maternal Abs in new- born 448 E. Abd-Elfatah, M. El-Mekkawi, E. Aboul-Soud, E. Fawzi, S. El-Soally lambs and kids to detect the best time to start vaccination. SNT was applied according to the standard protocol of VSVRI following the OIE manual (14), the neutralization index (NI) was calculated according to Reed and Muench (23). Statistical analysis In the present study, the obtained data was analyzed by two-way repeated measures ANOVA procedures of the Statistical Package for Social Sciences version 21.0 (SPSS for Windows 21.0, Inc, Chicago, IL, USA). Results were documented as means ± standard error (SE), P-value< 0.05 was used to indicate statistical significance and the difference among the means were determined by Duncan multiple range test (DMRT). Results Safety test and the post-vaccine reaction The safety test of new trivalent CaPVs vaccines was checked before field administration of the vaccine, the vaccine was safe without abortion case recorded, after that the trivalent CaPVs vaccine was applied in the field trial in comparison with RSPPV. Both vaccinated groups (G1, G2) were put under a closed observation for 14 days to detect the post-vaccine reaction particularly measuring of rectal temperatures. Clinical examination reveal that there is no clinical signs of CaPVs infection were appeared except rise of temperature was observed in G1 after 24-72h post- vaccination while in G2 slight rise of temperature was appeared after 48- 72h post-vaccination (Table 1) without significant difference between both group (P-value>0.05), this rise of temperature was accompanied by slight loss of appetite and slight increase of respiratory rate without any abortion. Moreover all animals were still in complete alert state with good body condition. Mild local reaction at the inoculation site in both vaccinated groups was appeared in the form of slight redness and swelling as shown in (Fig. 1) that disappeared within 4-5 days from vaccination. Evaluation of cellular immunity by lymphocyte proliferation response Lymphocyte response was calculated as optical density (O.D) by using sensitive XTT substance that varies from time to time and from animal to animal in each group. PBMCs of vaccinated groups were shown higher lymphocytes proliferation than un-vaccinated group with significant difference (P-value <0.05). Mean lymphocytes proliferation of G1 (trivalent vaccinated group) were higher than G2 (monovalent vaccinated group) with significant difference (P-value <0.05) at all time of PBMCs samples collection, along the time of experiment O.D of lymphocytes proliferation in sheep was higher than goats in response to vaccines. Peak level of lymphocytes proliferation in both G1 and G2 was appeared at 10th DPV then decreased thereafter (Fig. 2). Evaluation of humoral immunity by SNT Serum samples were collected to detect Abs titer using SNT in vaccinated and un-vaccinated groups, the mean protective neutralization index (NI) was detectable after 2st WPV in sheep and goat vaccinated with trivalent CaPVs vaccine (1.750±0.102) then increased gradually till reached to the peak level at 4th WPV with average means (2.6250±0.143, 2.1563±0.105) in G1 and G2 respectively with significant difference (P<0.05) in between. Sheep and goats in G1 were shown higher mean NI than sheep and goats in G2 with significant difference (P<0.05) between weeks of samples collection (Fig. 3). Evaluation of maternal immunity in newborn lambs and kids using SNT Peak protective (NI>1.5) was recorded at 1st week in lambs and kids of G4 and G5 compared with non-protective NI in G6 with significant difference (P<0.05) between groups, average means of NI in each group differ between weeks of serum samples collection with significant difference (P<0.05). In G4 (lambs and kids from trivalent vaccinated dams) the level of NI of Abs remain till 10th week after birth protective with a relatively high level in lambs and kids, on the other side Abs of lambs and Immunological response of a new trivalent Capripoxvirus vaccine in pregnant ewes and does 449 kids from monovalent vaccinated dams (G5) results lambs were received higher level of Abs remain protective till 6th and 4th weeks after than kids. birth respectively (Table 2). From the obtained Table 1 : Physical parameters of vaccinated groups *Days post Parameters vaccination Body temperature (0C) Habitus (DPV) G1 G2 G1 G2 0 39.5±0.092 39.48±0.037 Alert Alert 1 39.74±0.302 39.5±0.031 Slight decrease in appetite and animals Slightly decrease of movement with animals movement slightlyincreased with good respiratory rate respiratory rate 2 39.82±0.102 39.64±0.051 Slightly decrease in Mild decrease in appetite and animals appetite movement 3 39.86±0.103 39.64±0.024 Animals appetite is Animals became acceptable with good alert with good movable movable 5 39.66±0.093 39.5±0.032 Alert Alert 7 39.62±0.073 39.42±0.012 Alert Alert 14 39.5±0.051 39.4±0.03 Alert Alert *Significant difference (P-Value<0.05) between days of post-vaccinal reaction Figure 1: Post- vaccinal reaction in form of slight redness and swelling under the tail of sheep (A) and goat (B) 450 E. Abd-Elfatah, M. El-Mekkawi, E. Aboul-Soud, E. Fawzi, S. El-Soally Cellular immunity (LPA test) in vaccinated sheep with trivalent CaPVs and monovalent RSPPV vaccines 3.0 0,0 -.-.-.-.-. 0 2 5 10 20 30 Days post vaccination Control —•— Monovalent — ■— Trivalent Cellular immunity (LPA test) in vaccinated goat with trivalent CaPVs and monovalent RSPPV vaccines 2.5 0.0 -.-.-.-.-. 0 2 5 10 20 30 Days post vaccination Control —•— Monovalent — ■— Trivalent Figure 2: Lymphocyte proliferation index of vaccinated sheep and goats. PBMCs in response to trivalent CaPVs vaccine and monovalent RSPPV vaccine at days 0 (before vaccination), 2, 5, 10, 20 and 30 postVaccination of sheep (A) and goat (B). Protective O.D> 1, values were means ± SEM Immunological response of a new trivalent Capripoxvirus vaccine in pregnant ewes and does 451 Neutralization index (Ml) in pregnant sheep vac ciliated with trivalent CaPVs and monovalent RSPPV vaccines x A 13 C c 0 1 N z fll c z a t> E 3.0 2.5 2.0 1,5 1.0 0.5 0.0 3 A 1.....-I ....... x ^f—I / y ^ir--i 1 2 3 4 6 8 10 12 Weeks post vaccination Conlril -Monovalent Trivalent N e utrafi zaticn i nde x {Nl} in pre gn am does vac c inat e d wilh trivalent CaPVs and monovalent RSPPV vaecines 3.0 Si 2,5 v c I 2.0 <5 N 1 1-5 5 a; i ™ ® 0.5 0.0 M r \ --1 s J * / j / * / t Jr i—......j ^a y r y * t t t V _ _I_ r ¿¿ci "r"— 2 3 4 6 8 Weeks p ost vaccinal io n 10 12 B — Control Monovalent —■— Trivalent Figure 3: Average NI of vaccinated sheep and goats with trivalent CaPVs vaccine and monovalent RSPPV vaccine from 0 week (Pre-vaccination) till 12 WPV. Sheep (A) and goat (B).Values were means ± SEM. *8 week (time of parturition) 452 E. Abd-Elfatah, M. El-Mekkawi, E. Aboul-Soud, E. Fawzi, S. El-Soally Table 2: Mean neutralization index of antibodies in serum of lambs and kids. The means with different superscript are statistically difference interaction based on Duncan test , ,, Lambs Kids Weeks after birth Trivalent Monovalent Trivalent Monovalent 1 2.38±.0.0161ab 1.81±.0.063ef 2.50±0.102a 2.07±0.063cd 2 2.25±.0.102ac 1.69±.0.063fgh 2.19±0.119bcd 1.63±0.072fgh 3 2.19±.0.1196bcd 1.63±.0.072fgh 2.06±0.063cd 1.62±0.068fgh 4 2.00±.0.102de 1.63±0.072fgh 1.81±0.063ef 1.56±0.063ghl 6 2.00±.0.102de 1.50±0.102hlJ 1.8±0.068ef 1.13 ±0.072lm 8 1.81±0.063ef 1.31±0.063jkl 1.69±0.063fgh 1.11 ±0.066lm 10 1.75±0.001fg 1.31±0.063jkl 1.63±0.072fgh 1.06±0.063m 12 1.38±0. 063ljk 1.07±0.063m 1.25±0.102klm 1.04 ±0.075m Discussion Vaccines have an important role in protection against endemic infectious diseases in Egypt. Vaccination protects the welfare of farm animals and reduces the pain associated with illness, present study was intended to detect the safety of a candidate trivalent CaPVs vaccine in pregnant small ruminants (specific and preferable host) and evaluate the immunity of this vaccine compared with commercial RSPPV vaccine (positive control) as well as the passive immunity in the new-born lambs and kids to determine the most suitable age for the first immunization. Post-vaccinal reaction in the form of accepted increases in rectal temperature with redness and swelling at the inoculated site are the common characteristic feature of genus CaPVs due to its dermotrophic nature, this reaction was observed exactly in G1 than G2 without statistically significant difference in between (P>0.05), this indicate initial immunogenic response of the vaccines, similar results were recorded by (27, 28, 21). Gari et al. (29) said that when the local reactions at inoculation site of vaccine were very low or not observed this indicate that the vaccine viruses was over-attenuated and therefore failed to produce an effective CMI. The close observation of vaccinated groups revealed that both vaccines were found to be safe without adverse reaction on animal's health according to parameters of vaccine safety recorded by (14). Protective immunity of genus CaPVs depend on both CMI and the humoral immunity, CMI response considered the predominant way to eliminate the infection against CaPVs. In recent years, the immunological studies focused on CMI role in defense mechanism (30). PBMCs after activation with CaPVs antigens were shown higher lymphocyte proliferation than un-activated PBMCs (31). In the present study, cultured PBMCs of both (trivalent vaccinated group) G1 and G2 (monovalent vaccinated group) were analyzed by using the sensitive XTT substance and were shown high proliferation than G3 (un-vaccinated group) with a significant difference (P-value >0.05), lymphocytes proliferation of sheep and goats in G1 were shown higher optical density level than sheep and goats in G2 at all-time points of experiment, these results were in agreement with (21,22,32) who reported that increase of lymphocyte proliferation at 3rd DPV after CaPVs vaccines strains activation till reached its peak on day 10th day then decreased thereafter. Immunological response of a new trivalent Capripoxvirus vaccine in pregnant ewes and does 453 The protective immune system against CaPVs infection not only depends on the cellular immunity but also the specific Abs that related to the durability of protection (33). SNT is a reliable and accurate method for specific Abs detection (34). In this study, (trivalent vaccinated group) G1 and (monovalent vaccinated group) G2 were able to produce Abs in response to CaPVs vaccine strains with significant difference (P-value<0.05). Protective NI> 1.5 against genus CaPVs according to (14) reach its peak level at 4th WPV agreement with (28) with increase its level in sheep and goats of G1 than sheep and goats of G2 all weeks of samples collection till end of experiment (12 weeks). Christine et al. (21) evaluated bivalent (RSPPV and Held GTPV) vaccines in cattle and reported that the bivalent vaccine is effective than RSPPV vaccine with a good protective index till 20 weeks (end of experiment) that could be still later. Trivalent CaPVs vaccine was evaluated in cattle by (22) who recorded that trivalent CaPVs vaccine provide highly protective Abs compared with monovalent RSPPV vaccine with 66% and 0% till 9 month post- Vaccination, respectively. Increases level of LPA and SNT in ruminants animals vaccinated with trivalent CaPVs vaccine than monovalent RSPPV vaccine was explained by (35, 36) who reported that GTPV vaccine induce a good immuno-genic response than SPPV vaccine with high level of lymphocyte proliferation and Abs titer. In the present research, the peak level of O.D in vaccinated sheep and goats appeared at 10th day post-vaccination in comparison with the neutralizing protective Abs that reached its peak at 4th weeks post-vaccination this showed that the immunity against pox disease depend at first days after acquired immunity on T- lymphocytes. Immunoglobulin G (IgG) is transferred through the colostrums and it's the main Ig responsible for neutralizing viruses through binding pathogenic agents and induces the humoral immune response (37), so in this study we should evaluate the new candidate trivalent CaPVs vaccine in pregnant dams and measure the level of protective immunity compared with monovalent RSPPV vaccine. In the actual study, the peak protective Abs was measured in G4 (lambs and kids of trivalent vaccinated dams) and G5 (lambs and kids of monovalent vaccinated dams) after 1st week after birth these results were agreed with (38) who reported that SPPV and GTPV vaccines were effective in producing hyperimmune colostrum that providing new-born lambs and kids during their first seven days. NI titer of SNT was still protective with a relatively good level till 10th weeks after birth in lambs and kids of G4 (1.75±0.001, 1.63±0.072, respectively) while at 12 week of experiment the level of NI is slightly decreased under protective NI of lambs and kids (1.38± 0. 063, 1.25±0.102, respectively) so it's advisable to start the vaccination program at 2.5 months after birth before the protective Abs decline with CaPVs vaccine while lambs and kids from monovalent vaccinated dams (G5) Abs still protective till (6th, 4th weeks after birth, respectively) with average means (1.500±.0.102, 1.563±0.063, respectively). Effective vaccination of young animals at the appropriate time is cost effective practice for eradication of genus CaPVs diseases in Egypt. Little recent researches display the duration of maternal immunity of SPPV vaccine in sheep and goats, according to (39, 40) who found that maternal immunity in new-born lambs and Kids from vaccinated dams with SPPV vaccine last for 3 and 2 months respectively but in the present study the data obtained that the pregnant dams vaccinated with RSPPV vaccine provide its newborn lambs and kids with Abs protection last for 45 days and 30 days, respectively in comparison with trivalent CaPVs vaccine that provided a relatively protective Abs last for 3 months in both lambs and kids. It is unknown whether the low titre of circulating maternal are sufficient to block vaccine responsiveness, so early onset of lambs and kids vaccination with a safe trivalent CaPVs vaccine will provide more efficient protection by inducing both CMI and humeral immunity. The major mention of using trivalent CaPVs can be used for protection cattle against LSDV due to its induction of a long duration of immunity without generalized post-vaccinal reaction (21, 22). 454 E. Abd-Elfatah, M. El-Mekkawi, E. Aboul-Soud, E. Fawzi, S. El-Soally Conclusion From this investigation, we concluded that the trivalent CaPVs vaccine gave a good immune response based on both cellular and humoral immunity which represented by the induction of higher level of lymphocytes proliferation and Abs titer respectively, in addition to a high safety degree on pregnant dams and ability to provide relative long protective passive immunity to new-born lambs and kids than the commercial RSPPV vaccine. Trivalent vaccine should be used in vaccination of lambs and kids at 2.5 month of age to avoid the re-occurrence of outbreak. Due to the close antigenic relationship between genus CaPVs strains so we recommended the use of this trivalent vaccine as new candidate in Egypt to control reoccurrence of LSD in cattle with further study to evaluate its efficacy in cattle is also advisable. Conflict of interest The authors declare that they have no conflict of interest. References 1. Kitching RP, Taylor WP. Clinical and antigenic relationship between isolates of sheep and goatpoxviruses.Trop Anim Health Prod 1985; 17: 64-74. 2. Babiuk S, Bowden TR, Boyle DB, Wallace DB, Kitching RP. Capripoxviruses: An Emerging Worldwide Threat to Sheep, Goats and Cattle. TransbounEmerg Dis 2008; 55(7):263-72. 3. Buller RM, Arif BM, Black DN, Dumbell KR, Esposito JJ. Virus Taxonomy: Classification and Nomenclature of Viruses. Eighth Report of the International Committee on Taxonomy of Viruses. Elsevier Academic Press, 2005;117-33. 4. Rao TVS, Bandyopadhyay SK. A comprehensive review of Goatpox and Sheeppox and their diagnosis.Anim Health Res Rev 2000; 1: 127-132. 5. Bhanuprakash V, Moorthy ARS, Krishnappa G, SrinivasaGowda RN, Indrani BK. An epidemio-logical study of sheeppox infection in Karnataka state, India. Revue scientiet technique (Inter Office of Epizo) 2005; 24 (3): 909-20. 6. Abu-Elzein E, Housawi F, Ramadan O. Observations on natural and experimental infection of sheep and goats with a virulent field Capripoxvirus with high affinity to goat. Vet Arhiv 2003; 73(3): 119-31. 7. Iran Veterinary Organization. survey analysis on sheeppox and goatpox in IRAN during 20102014. Pox National CommiteeBultin of Iran Vet Organization, Tehran, Iran 2013. 8. Kitching RP. The control of sheep and goatpox. Revue Scientiet Technique de l'OIE (France). 1986; 5:503-11. 9. Rweyemamu MM, Roeder PL, Taylor WP. Towards the global eradication. In: Barret T, Edn.). Cambridge: Institute of Animal Health, Biology of Animal Infection Series, Academic Press, 2006; 99-322. 10. Bhanuprakash V. Prospects of control and eradication of capripox from the Indian subcontinent: A perspective. Antiviral Res 2011; 91: 22532. 11. Kitching RP. Vaccines for lumpyskindisease, sheeppox and goatpox. Develop in biolog. 2003; 114: 161-7. 12. Brenner J, Bellaiche M, Gross E, Elad D, Oved Z, Haimovitz M, Wasserman A, Friedgut O, Stram Y, Bumbarov V, Yadin H. Appearance of skin lesions in cattle populations vaccinated against lumpyskindisease: statutory challenge. Vaccine J 2009; 27:1500-3. 13. Somasundaram MK. An outbreak of lumpyskindisease in a Holstein dairy herd in Oman: a clinical report. Asian J of Anim and Vet Ad-van 2011; 6(8):851-9 14. OIE (Office International des Epizooties. Sheeppox and goatpoxManual of Diagnostic Tests and Vaccines for Terrestrial Animals, Paris.Chapter 2. 2012; 7(14): 1-12. 15. Gelaye E, Belay A, Ayelet G, Jenberie S, Yami M, Loitsch A, Tuppurainen E, Grabher R, Di-allo A, Lamien CE. Capripox disease in Ethiopia: Genetic differences between field isolates and vaccine strain, and implications for vaccination failure. Antiviral Res 2015; 119: 28-35. 16. Salib FA, Osman AH. Incidence of lumpyskindisease among Egyptian cattle in Giza Governorate, Egypt. Vet World. 2011; 4: 162-7. 17. Elhaig MM, Selim A, Mahmoud M.Lumpyskindisease in cattle: Frequency of occurrence in a dairy farm and a preliminary assessment of its possible impact on Egyptian buffaloes. Onder-stepoort J of Vet Res 2017; 2219-35. 18. Fatma M Abdallah, Hend M El Damaty, Gamilat F Kotb. Sporadic cases of lumpyskin disease among cattle in Sharkia province, Egypt: Genetic characterization of lumpyskindisease virus isolates and pathological findings. Vet World. 2018; 2231-0916. Immunological response of a new trivalent Capripoxvirus vaccine in pregnant ewes and does 455 19. Agrawal SK, Soman JP. Assessment of immune efficacy of attenuated live goatpox vaccine against sheeppox and contagious ecthyma.Indian Vet J 1997; 74(12): 1016-18. 20. Christine A Mikhael. Trialsfor preparation of improved bivalent vaccine for sheeppoxand goatpox viruses. PhD Cairo university 2012. 21. Christine A Mikhael, Olfat E. Nakhla,Namaa A. Mohamed. Study on the capability of a dual Capripoxvaccine in protection of cattle against LSD infection. J of vet medicine res 2017; 24 (1): 22433. 22. Aboul-Soud EA, Kafafy MH, El-Suall SA, Zaghloul MA, Christine A Mikhael. Preparation of trivalent vaccine against lumpy skin disease using different capripox viral strain. International J. of Vet. Science and Medicine 2018 (inpress). 23. Reed LJ, Muench H. Simple methods of estimating fifty percent end point .American J. of Hygi 1938; 27:493-7. 24. Ting A, Morris PJ. A technique for lymphocyte preparation from stored heparinized blood. Vox.Sang.1971; 20:561-3. 25. Roehm NW, Rodgers GH, Hatfield SM. An improved colorimetric assay for cell proliferation and viability utilizing the tetrazolium salt XTT. J of immuno methods. 1991; 142:257-65. 26. Berridge MV, Herst PM, Tan AS. Tetrazo-lium dyes as tools in cell biology: New insights into their cellular reduction. Biotech Annual Rev 2005; 11:127-52. 27. Fakri F, Ghzal F, Daouam S, Elarkam A, Douieb L, Zouheir Y, Tadlaoui K, Fassi-Fihri O. Development and field application of a new combined vaccine against Peste des Petits Ruminants and SheepPox. Trials in Vaccin.2015; 4: 33-7. 28. Zeidan SM, NamaaAM, Hanan MSE, Saad MAA, Olfat EN, Afaf AAW, Abeer AT, El- Dakhly AT. Preliminary study for preparation of combined attenuated vaccine against sheeppox and PPR viruses. Inter. J. of Adva. Res 2016;4: 212-9. 29. Gari G, Abie G, Gizaw D, Wubete A, Kidane M, Asgedom H. Evaluation of the safety, immuno-genicity and efficacy of three capripoxvirus vaccine strains against lumpy skin disease virus. Vaccine J 2015; 33(28):3256-1. 30. Green S, Ennis FA, Mathew A. Long term recall of memory CD8T cells in mice to first and third generation smallpox vaccines. Vaccine J 2011; 29(8): 1666--76. 31. Ahmed AM, Mukhtar MM, El Hussein AM, Tageidin AM, Fadol MA. Immune response of sheep vaccinated with Capripox vaccine. Vet. Res 2007; 1(1):12—16. 32. Nermeen G Shafik, Ibrahim MM, Sonia A Rizk, Ali AM. Use of Different Immune Response Assays for Evaluation of Live Attenuated Sheep pox Vaccine as Alternatives to Challenge Test. Global J of Medical Res 2017; 17:2(1)0975-5888. 33. Barmana D, Chatterjee A, Guhaa C, Biswasa U, Sarkarb J, Royc TK, Roya B, Baidyad S. Estimation of post-vaccination antibody titre against goatpox and determination of protective antibody titre. Small Rum Res 2010; 93(2): 76-8. 34. Gelagay A, Nigussu F, Shiferaw J, Gedlu M, Teshale S, Haileleul N. Study on immunogenicity of combined sheep and goatpox and peste des petitis ruminants vaccines in small ruminants in Ethiopia. Afri J of Microbio Res 2012; 6: 7212-7. 35. Norian R, AfzalAhangran N, Varshovi HR, Azadmehr A. Comparative efficacy of two heterologous capripox vaccines to control lumpy skin disease in cattle. Bulg J of Vet Med 2017; 1311-477. 36. Varshovi HR, Norian R, Azadmehr A, AfzalAhangranN.Immune response characteristics of Capripoxvirus vaccines following emergency vaccination of cattle against lumpy skin disease virus. Iranian J of Vet Sci and Tech 9(2) 33-40. 37. Atkinson DE, Boyd RDH,Sibley CP. Placental Transfer. Elsevier, Manchester press, 2000; 2787-846. 38. Burezq HA, Razzaque MA. Effects of immunizing pregnant ewes and does on the humoral immune response of secreted colostrums. J of Anim and Plant Sci 2018; 28 (1): 341-7. 39. Kitching P. Passive protection of sheep against Capripoxvirus. Res in vet sci 1986; 41: 24750. 40. AbuElzein EME, Housawi F.M.T, Al-Afaleq AI, Ibrahim AO. Protection of Goats, with a Sheeppox Vaccine, Against a Virulent Field Capripoxvirus with High Affinity to Goats.Sci J of King Faisal Univ 2004; 5 (2): 1425. Slov Vet Res 2019; 56 (Suppl 22): 457-64 DOI 10.26873/SVR-783-2019 Original Research Article ISOLATION OF WHITE SPOT SYNDROME VIRUS (WSSV) IN EGYPTIAN SHRIMP USING CONVENTIONAL PCR AND REAL TIME PCR (QPCR) TECHNIQUES Yaser M. Hafez1, Nader Y. Moustafa2, Asmaa F. Magouz3, Naglaa F. Al-Maria13* 1EPCRS Excellence Center &Plant Pathology and Biotechnology Lab., Faculty of Agriculture, Kafrelsheikh University, Egypt, 2Food Control Department, Faculty of Veterinary Medicine, Kafrelsheikh University, 33516, Egypt, 3Virology Department, Faculty of Veterinary Medicine, Kafrelsheikh University Corresponding author, E-mail: naglaafikry5@yahoo.com Abstract: Shrimp aquaculture industry threated by high mortality rates and severe economic losses as a result of white spot syndrome virus (WSSV) infection. Early-screening and diagnosis of WSSV are great strategies to decrease the economic losses of the disease on shrimp aquaculture. Therefore, this study was carried out to detect of white WSSV infected shrimp under using two molecular based methods, conventional PCR and qPCR. A total number of 90 samples of red (Aristeus antennatus) and gray (Penaeus latisclcatus) shrimp were collected from Kafr El-Sheikh and Alexandria governorates. External examination of shrimps collected from Kafr El-Sheikh Governorate revealed typical WSSV clinical signs (including loose and easily detached cuticle with appearance of small white spots (3 mm in diameter) and /or larger patches in the external surface of carapace and cephalothorax. The internal examination showed yellowish white, fragile and swollen hepatopancreas and swollen or shrunken lymphoid tissue. Red shrimp showed slightly obvious white spots without any internal lesions. PCR results confirmed the clinical investigation and postmortem (PM) examination and revealed presence of WSSV partial sequences with a size of 190 bp in shrimp samples from Kafr El-Sheikh Governorate. In contrast, samples collected from Alexandria (Borg El-Arab) gave negative results. The result of qPCR confirmed that obtained by conventional PCR and showed that all positive results of WSSV by conventional PCR gave cycle threshold (Ct) values ranged from 34.81 to 40.06. Our results concluded that, WSSV Diseases of shrimp attack shrimp markets of Kafr El-Sheikh Governorate. The conventional PCR and qPCR based methods for isolation and identification of shrimp WSSV, provided accurate results. Key words: WSSV; PCR; qPCR; shrimp Introduction Shrimp are one of the greatest important food sources for human consumption because shrimp have high levels of omega-3 fatty acids (1). The industry income related to shrimp species is about 50 billion dollars annually (2). Production declining of shrimp was observed Received: January 2019 Accepted for publication: February 2019 458 Y. M. Hafez, N. Y. Moustafa, A. F. Magouz, N. F. Al-Maria from 1994 to 1997 in India and from 1997 to 1998 in Asia (3-5). In Egypt, the white spot syndrome virus (WSSV) caused high losses in shrimp fields since 2009 (6). WSSV infects shrimp, lobsters, crayfish and crabs belonging to freshwater and marine crustaceans (2, 7). WSSV is the only member of Whispovirus genus (belong to Nimaviridae family) (8). It is enveloped double stranded circular DNA virus with ovoid to bacilliform shape and a tail like end (9, 10). Shrimp aquaculture industry threated by high mortality rates and severe economic losses as a result of WSSV infection. Early-screening and diagnosis of WSSV are great strategies to decrease the economic losses of the disease on shrimp aquaculture. Conventional polymerase chain reaction (PCR), in situ PCR, quantitative PCR, nested PCR as well as loop-mediated isothermal amplification (LAMP) (11-16) has been established for detection of WSSV. The WSSV causes serious economic losses because of high percent of mortality which leads to total crop losses through ten days or less under certain farming conditions (17). The host range of WSSV is at least 78 species, mostly to decapod crustaceans particularly shrimp (18). In coastal area of Egypt, industry depends on shrimp are gradually proceeded to cover the market needs, nevertheless the high risk shrimp farms infected with virus could be disturb the production (19). To the best of our knowledge, only few researches conducted about WSSV in Egyptian shrimp. Consequently, the goal of this research article was early identification of WSSV in shrimp under Egyptian conditions using of molecular based methods (PCR and qPCR). Materials and methods Shrimp samples A total of 90 shrimp samples were collected from two Egyptian Governorates located on Mediterranean Sea; Kafr El-Sheikh and Alexandria. The red shrimp (Aristeus anten-natus) and gray shrimp (Penaeus latisclcatus) obtained from Kafr El-sheikh Governorate as well as gray shrimp (Penaeus latisclcatus) obtained from Borg El-Arab farm (Alexandria Governorate). Conventional PCR assay Shrimp samples were prepared for DNA extraction. Carapace and abdominal segments of declining shrimp as well as the control shrimp were split using a scalpel. A part of gills, internal organs, muscle and cuticle are preserved in -20oC for extraction of DNA. Total DNA was extracted according to the instructions of Gene-spin TM Viral Extraction Kit (Intron, South Korea, Cat. No. 1715) and as previously described (20). The PCR reaction mixture was 25 pl which consisted of 12.5pl of 2X master mix (0.1U/pl Taq polymerase, 500 pM dNTP, 20mM Tris-HCl (pH8.3), 100mM KCl, 3mM MgCl2 and Stabilizer and enhancer), 1 pl of 10 pmol of each primer, and 2 pL of template DNA (50 pg/ml). The primers were designed as previously described (19). Amplification was performed in a thermocycler (Bio-Rad, C - 1000). The amplified products were examined on 1.5% agarose gel. Ten pL of amplified product, negative control were injected into the well then run with 50 bp DNA ladder in 1X TAE electrophoresis buffer (5 volts/cm2 for 45min.). At the end of the run of electrophoresis, the gel was captured by a gel documentation system. The expected DNA fragments were 190 base pairs (bp) in length. Real time PCR assay A single tube qPCR reaction was adjusted according to the kit manual instruction. In brief, the reaction mixture was 25pl which consisted of 12.5pl of 2X SYBR Green qPCR Master Mix, 1 pl of 10 pmol of each primer (5-AATGGTCCCGTCCTCATCTCA-3) as well as (5-GCTGCCTTGCCGGAAATT-3) specific for WSSV (15), and 2 pL of template DNA (50 pg/ml). PCR was conducted in an eppendorf thermal cycler amplification was performed in a thermocycler (Real time PCR-Agilent Technologies - Stratagene MX300P). Early denaturation at 94 0C for 5 min, then cyclic condition was 35 cycles at 94 0C for 30 sec, annealing at 54 0C for 1 min as well as extension at 72 0C for 1 min. The final extension at 72 0C for 10 min (15, 20). The melting temperature for all obtained products was 80°C indicating Isolation of white spot syndrome virus (WSSV) in Egyptian shrimp using conventional PCR and real time 459 the specificity of primers annealing to the template. Results Results of gross pathology The collected shrimps collected from Kafr El-Sheikh Governorate were suspected to be infected with White Spot Syndrome virus (WSSV) based on the following main symptoms: loose and easily detached cuticle with appearance of white spots (3 mm in diameter) in external surface carapace and cephalothorax (Fig.1). These spots were not easy to be removed and in some region, they collected forming large patches of different sizes with whitish circular spots. The internal examination of these shrimps revealed yellowish white, fragile and swollen hepatopancreas and swollen and shrunken lymphoid tissue (Fig.1). In addition to accumulation of the fluids that caused swelling of bronchiostegites. On the other hand, red shrimp showed slightly obvious white spots without any internal lesions (Fig.1). Results of conventional PCR PCR results confirmed the clinical investigation and postmortem (PM) examination and revealed presence of WSSV Table 1: Incidences of isolated WSSV among exam partial sequences with a size of 190 bp in shrimp samples which had WSSV gross lesions obtained from Kafr El-Sheikh Governorate (Fig. 2). In contrast, samples collected from Alexandria (Borg El-Arab) gave negative results. PCR products of infected shrimp samples of different species revealed that 54 out of 60 shrimp samples from Kafr El-sheikh Governorate were PCR positive. Meanwhile, 30 samples collected from Borg El-Arab Alexandria Governorate gave negative (-ve) results fFig. 2). Our results cleared that, there was a significant differences of the incidences of WSSV (P < 0.01) among the type of shrimp and among different regions. The results cleared that, the degree of WSSV infected shrimp from Kafr El-sheikh markets showed high incidences to WSSV infection however, no disease incidence were recorded in Alexandria gray shrimp (Table 1). qPCR results of WSSV in shrimp samples The 90 samples were examined with real time PCR using specific primers for WSSV. The result showed that all positive results of WSSV by conventional PCR gave cycle threshold (Ct) values ranged from 34.81 to 40.06 (Table 2, Fig. 3). ;d shrimp after conventional PCR Number of sample Number of positive sample Locality Site of DNA extraction Appearance of symptoms Results % of infection 15 14 Kafr-Elsheikh Cuticle of Have +ve 93.3 % Kafr-Elsheikh red shrimp symptoms (WSSV) 15 13 Cuticle of Have +ve 92.9 % Kafr-Elsheikh gray shrimp symptoms (WSSV) 15 13 Internal Have +ve 92.9 % organs of symptoms (WSSV) Kafr-Elsheikh gray shrimp 15 14 Internal Have +ve 93.3 % organs of symptoms (WSSV) red shrimp 15 0 Alexandria Cuticle of No -ve (WSSV) 0 gray shrimp symptoms 15 0 Alexandria Internal No -ve (WSSV) 0 organs of symptoms gray shrimp Chi2 = 11.14 ** ** = Significant at (P < 0.01) 460 Y. M. Hafez, N. Y. Moustafa, A. F. Magouz, N. F. Al-Maria Table 2: Mean of Cycle Threshold of samples examined for WSSV using qPCR Samples NO. Site of DNA extraction Mean of Cycle Threshold 1 Cuticle of red shrimp from Kafr-Elsheikh 40.06 2 Cuticle of gray shrimp from Kafr-Elsheikh 36.01 3 Internal organs of gray shrimp from Kafr-Elsheikh 34.81 4 Internal organs of red shrimp from Kafr-Elsheikh 37.60 5 Cuticle of gray shrimp from Alexandria 0.0 6 Internal organs of gray shrimp from Alexandria 0.0 A: Loose and easily detached cuticle B: White spots that appear on carapace C White spots observed on external surface. D: White spots that appear on carapace and _ céphalothorax_ E: Swelling of bronchiostegites . F: whitish patches and circular spots of variable sizes Figure 1: White spot syndrome virus (WSSV) infection gross lesion on shrimp. A, B: from red shrimp. C- F: from gray shrimp Isolation of white spot syndrome virus (WSSV) in Egyptian shrimp using conventional PCR and real time Ml 2 3 4 5 6 461 Figure 2: PCR results of WSSV on agarose gel. M= 50 bp ladder, 1= (sample from cuticle of red shrimp), 2= (sample from cuticle of gray shrimp), 3= (sample from internal organs of gray shrimp), 4= (sample from internal organs of red shrimp), 5= (sample from cuticle of gray shrimp), 6= (sample from internal organs of gray shrimp). 1-4 samples collected from Kafr EL-sheikh, 5-6 samples collected from Alexandria Discussion WSSV is one of the most serious shrimp disease which does not only attack shrimp farms in Egypt and causes economic losses but also infects other freshwater and marine crustaceans, mostly crayfish and crabs (1, 9). The virus is so dangerous so that can lead to 100% mortalities during only 2-10 days from appearance of symptoms (2, 21). Little information and knowledges about the incidences and prevalence of this virus in shrimp aquaculture have been reported in Egypt. Although the shrimp aquaculture 462 Y. M. Hafez, N. Y. Moustafa, A. F. Magouz, N. F. Al-Maria became more advanced in Egypt, the infected shrimp threaten shrimp farming. Thus, control WSSV is required to avoid shrimp losses. The aim of this study was to throw the light on the occurrence and incidences of WSSV in some two species of the shrimp present in Egypt. Our results revealed that 54 out of 60 shrimp samples collected from Kafr El-sheikh Governorate showed positive results for WSSV however, no positive results were obtained from the 30 total shrimp samples collected from Alexandria Governorate. These results supported by the results obtained by Megahed et al 2019, Eissa et al 2009 and Salama et al 2008 (6, 19, 22) and confirming WSSV identification among Egyptian shrimps. Herein, gross pathological examination revealed presence of white spots on the shrimp body after removal of cephalothorax cuticle. Our results agreed with those obtained by (2, 23-26) who also found cuticle chromophores and calcium deposition. Although we did not find softening of exoskeleton, in many epizootics of this disease, this softening could be observed. Changes in the structural integrity of the exo and pro cuticle could be the most vital reason for WSSV syndrome (28-30). The obtained internal examination results agreed with (2, 27, 31) and proved that white spot virus infected hepatopancreatic sheath. PCR (both conventional and real time) confirmed presence of WSSV in shrimps collected from Kafr El-sheikh Governorate. In the present study, 90 shrimp samples were examined with conventional PCR using specific primers of WSSV. The result showed that 54 out of 60 gray and red shrimp samples of Kafr El-sheikh Governorate were PCR positive. Meanwhile 30 gray shrimp samples collected from Borg El-Arab, Alexandria Governorate gave negative (-ve) results. Results from conventional PCR were further confirmed by qPCR which showed amplification curves with Ct values ranged from 34.81 to 40.06 in all conventional PCR positive samples. PCR has been applied for the diagnosis of WSSV infections in clinical samples and shown to be rapid, sensitive and specific diagnostic method (32-35). Conclusion To the best of our knowledge, this may be the first study to detect shrimp WSSV in Egypt using qPCR. The conventional PCR and qPCR based methods are successful methods for early identification of WSSV in clinical samples of infected shrimp that delivers accurate tool for identification of this virus. Acknowledgment This research article was conducted in accredited Plant Pathology and Biotechnology Lab. (PPBL) and certified EPCRS Excellence Centre, Faculty of Agriculture, Kafrelsheikh University. We thank staff members at PPBL, EPCRS and Department of Fish Diseases and Management, Faculty of Veterinary Medicine, Kafrelsheikh University. References 1. Shahidy MS, El-Gamal RM, Dessouki A. et al. Detection of white spot syndrome in cultured penaeid shrimp in Egypt: histopathological observation and polymerase chain reaction. MENA Science Journal 2015; 1(1): 5-14. 2. Tao M, Zhou H, Luo K, Lu J, Zhang Y, Wang F. Quantitative serum proteomics analyses reveal shrimp responses against WSSV infection. Dev Comp Immunol. 2019; 93:89-92. 3. Sharawy ZZ, Thiele R, Abbas EM, El-Magd MA, Hassaan MS, Peter C, Schmidt J, Saborowski R, Goda AMA-S, Slater MJ. Antioxidant response, body composition of whiteleg shrimp Litopenaeus vannamei co-cultured with Nile tilapia Oreochromis niloticus in recirculating aquaculture. Aquaculture Environment Interactions 2017; 9:257-68. 4. Pillai, N.G.K.; Katiha, P.K. Evolution of fisheries and aquaculture in India. Central Marine Research Institute, Kochi, India 2004; p240. 5. FAO. Fishery and Aquaculture Statistics. FAO Year Book 2006. Food and Agriculture Organization of the United Nations, Rome 2008; p57. 6. Megahed ME, Kanrar S, Dhar AK . Complete Genome Sequence of White Spot Syndrome Virus Isolated from Indian White Prawn (Fenneropenaeus indicus) in Egypt. Microbiol Resour Announc 2019; 3(1): 8-14. 7. Tsai Y, Lina Y, Choua P, et al. Detection of white spot syndrome virus by polymerase chain reaction performed under insulated isothermal Isolation of white spot syndrome virus (WSSV) in Egyptian shrimp using conventional PCR and real time 463 conditions. Journal of Virological Methods 2012; 181(1): 134—7. 8. Mayo, M. A. A summary of taxonomic changes recently approved by ICTV. Archives of Virology2002; 147, 1655-63. 9. Van Hulten M, Witteveldt J, Peters S. et al. The white spot syndrome virus DNA genomic sequence. Virology 2001; 286: 7-22. 10. Yang F, He J, Lin X, et al. Complete genome sequence of the shrimp white spot bacilliform virus. Journal of Virology 2001; 75: 11811-20. 11. Vaseeharan B, Jayakumar R, and Ramasamy, A. PPCR-based detection of white spot syndrome virus in cultured and captured crustaceans in India. Lett. Appl. Microbiol 2003; 37: 443-7. 12. Jian X, Lu L, Chen Y, et al. Comparison of a novel in situ polymerase chain reaction (ISPCR) method to other methods for white spot syndrome virus (WSSV) detection in Penaeus vannamei. Dis. Aquat. Organ 2005; 67: 171-6. 13. Durand S, and Lightner D. Quantitative realtime PCR for the measurement of white spot syndrome virus in shrimp. J. Fish Dis.2002; 25: 381-9. 14. Nunan, L, and Lightner D. Optimized PCR assay for detection of white spot syndrome virus (WSSV). J. Virol. Methods 2011; 171: 318-21. 15. Chou P, Lin Y, Teng P, et al. Real-time target-specific detection of loop-mediated isothermal amplification for white spot syndrome virus using fluorescence energy transfer-based probes. J. Virol. Methods 2011; 173: 67-74. 16. Kono T, Savan R, Sakai M, Itami T. Detection of white spot syndrome virus in shrimp by loop-mediated isothermal amplification. J. Virol. Methods 2004; 115: 59-65. 17. Pradeep B, Karunasagar I and Karunasagar I. Fitness and virulence of different strains of white spot syndrome virus. Journal of Fish Diseases 2009; 32: 801-5. 18. Flegel TW. Detection of major penaeid shrimp viruses in Asia, a historical perspective with emphasis on Thailand. Aquaculture 2006; 258: 133. 19. Eissa I, Badran A, Diab A, et al. Diagnosis of white spot syndrome virus (WSSV) among shrimp for the first time in Egypt. SCVMJ, VIX 2009; (1). 20. Orabi A, Hussein A, Saleh A A, El-Magd M A, Munir M. Evolutionary insights into the fusion protein of Newcastle disease virus isolated from vaccinated chickens in 2016 in Egypt, Archives of Virology 2017; 162(10): 3069-79. 21. Lightner DV. A handbook of pathology and diagnostic procedures for diseases of penaeid shrimp. Baton Rouge, Louisiana, USA: World Aquaculture Society1996. 22. Salama A, Diab A, Abd El-Samie, A, and Abdel-Wahab A. Isolation and Identification of White Spot Syndrome Virus. Zag. Vet. J. 2008; (ISSN. 1110-1458) Vol.36, No. 4 pp.105-13. 23. Wang Y, Lee K, Najiah M. et al. A new bacterial white spot syndrome (BWSS): in cultured tiger shrimp Penaeus monodon and its comparison with white spot syndrome (WSS): caused by virus. Dis. Aquat. 0rg.2002; 41 (1): 9-18. 24. Lotz J, and Soto M A. Model of white spot syndrome virus (WSSV) epidemics in Litopenaeus vannamei. Diseases of Aquatic 0rganisms 2002; 50:199-209. 25. Wu W, Wang L and Zhang X. Identification of white spot syndrome virus (WSSV) envelope proteins involved in shrimp infection. Virology 2005;332:578-83. 26. Chen L, Leu J, Huang C. et al. Identification of a nucleocapsid protein (VP35) gene of shrimp white spot syndrome virus and characterization of the motif important for targeting VP35 to the nuclei of transfected insect cells. Virology 2002; 293: 4453. 27. Wang Q, White B, Redman R and Lightner D. Per os challenge of Litopenaeus vannamei postlarvae and Farfantepenaeus duorarum juveniles with six geographic isolates of white spot syndrome virus. Aquaculture 1999; 170: 179-194. 28. Kasornchandra J, Boonyaratpalin S, and Itami T. Detection of white spot syndrome in cultured penaeid shrimp in Asia: microscopic observation and polymerase chain reaction. Aquaculture 1998; 164: 243-51. 29. Peng S, Lo C, Ho C et al. Detection of white spot baculovirus (WSBV) in giant fresh water prawn; Macrobrachium rosenbergii using polymerase chain reaction. Aquaculture 1998; 164: 253-62. 30. Karunasagar I, 0tta S and Karunasagar I. Histopathological and bacteriological study of white spot syndrome of Penaeus monodon along the west coast of India. Aquaculture 1997; 153: 913. 31. Chang P, Lo C, Wang Y, and Kou G. Identification of white spot syndrome virus associated baculovirus (WSBV) target organs in the shrimp Penaeus monodon by in situ hybridization. Diseases of Aquatic Organisms 1996; 27: 131-9. 32. Lo C, Leu J, Ho C, et al. Detection of baculovirus associated with white spot syndrome 464 Y. M. Hafez, N. Y. Moustafa, A. F. Magouz, N. F. Al-Maria (WSBV) in penaeid shrimps using polymerase chain reaction. Dis. Aquat. Org.1996; 25: 133-41. 33. Wang Y, Lo C, Chang P and Kou G. Experimental infection of white spot baculovirus in some cultured and wild decapods in Taiwan. Aquaculture 1998; 164: 221-31. 34. Tang KFJ. Lightner, D.V. Quantification of white spot syndrome virus DNA through a compe- titive polymerase chain reaction. Aquaculture 2000; 189: 11-21. 35. Kathy Tang-Nelson and Donald V. Lightner. Development of real-time PCR assays for detection of white spot syndrome virus, yellow head virus, Taura syndrome virus, and infectious hypodermal and hematopoietic necrosis virus in penaeid shrimp 2001, NA06FD0448. Slov Vet Res 2019; 56 (Suppl 22): 465-74 DOI 10.26873/SVR-784-2019 Original Research Article OCCURRENCE AND RISK FACTORS OF DIGITAL DERMATITIS IN EGYPTIAN DAIRIES El-SayedA.El-Shafaey1,3, Mohamed Z. Sayed-Ahmed24, Sherif M. Shoeib5, Sabry A. El-khodery2*, department of Surgery, Anesthesiology and Radiology, Faculty of Veterinary Medicine, Mansoura University, Mansoura 35516, Egypt, 2Department of Internal Medicine and Infectious Diseases, Faculty of Veterinary Medicine, Mansoura University, Mansour 35516, Egypt, 3Depart-ment of Veterinary Medicine, College of Agriculture and Veterinary Medicine, Qassim University, Buraydah 51452, Qassim, Saudi Arabia, 4Department of Clinical Pharmacy, College of Pharmacy, Jazan University, Jizan45142, Saudi Arabia, 5Veterinary Teaching Hospital, Faculty of Veterinary Medicine, Mansoura University, Mansoura 35516,Egypt *Corresponding author, E-mail: khodery@mans.edu.eg Abstract: In the present study, the prevalence of digital dermatitis (DD) and the related risk factors in dairy cows was investigated. For 12 months, information about the establishment of DD lesions and other claw disorders in dairy cows was collected. A questionnaire was designed to get data about age, milk yield, season, parity, transition period, concurrent diseases and biosecurity. The hind claws were more affected than fore claws (95.24 vs 4.76%) particularly the left hind claws (56.67%). However, The DD more prevalent during the second lactation than the third lactation (42.85 vs 36.50%) and first lactation (42.85 vs. 19.06%) which gradually decreased reaching the lowest incidence in heifer cows (42.85 vs 1.59%). Furthermore, DD was more prevalent during summer and autumn. Based on cow level, the final step of logistic regression analysis revealed a significant relationship between DD and cow' age (P <0.05; Odds Ratio [OR]: 1.01; 95% confidence interval [CI]: 0.67-1.47), parity (P <0.05; OR: 1.01.11; 95% CI: 0.47- 401.93), season (P < .05; OR: 1.36; 95% CI: 1.01-1.81), and transition period (P <.05; OR: 1.53; 95% CI: 0.48-4.83). The recognition of factors which influence occurrence of DD may permit changes in herd management or breeding policies which can be applied to diminish the prevalence of DD, and can establish the appropriate preventive procedures. Key words: digital dermatitis; dairy cows; risk factors; epidemiology Introduction Digital dermatitis (DD) is a global emerging digital skin disease of dairy cows, causes lameness with subsequent economic losses (1-3). Digital dermatitis decreases reproductive performance, milk production, as well as produc- tive lifespan and animal welfare (4, 5). The lesions of DD are mainly oval or circular, with strawberry-like appearance, with presence of a circumscribed erosive purulent epidermitis, which can develop to an ulcerative granulomatous lesion. The predilection site of typical lesions of DD are the posterior part of digits, , and Received: January 2019 Accepted for publication: February 2019 466 E. El-shafaey, M. Sayed-Ahmed, S. Shoeib, S. El-khodery the mid-way between the heel bulbs, and adjacent to the coronet (6,7). The precise cause and factors which predispose to DD occurrence in herds are still unclear, but it seems a multifactorial disease (8). Environmental, farm-management and individual animal factors are the suggested risks for this disease (9, 10). These risk factors can be split into two groups; cow-level factors (breed, parity, stage of lactation, calving season, transition period and other claw affections) (11-13). The transition period in dairy cows is the time extending from three weeks pre-calving, to three weeks post-calving. Minor alteration in the management system of dairy cows induces a dramatic effect on feed intake, health condition, locomotion and final profitability (14). Prompt alteration in both metabolic and hormonal systems occur during this period induces a stress in the cow, which is a critical determinant of the high risk of diseases in a dairy herd. Lameness may be triggered during the transition period. Physiological and behavioral changes during transition can damage the co-rium. This damage is not immediately apparent but results in poor hoof growth inducing DD, sole ulcers and hemorrhage (15). The pathogen-esis and clinical findings of DD lesions have been well presented. However, there are no standard features of classifying the lesions, while trials have been made to characterize the lesions based on the stage and severity (16-18). Scoring system has been presented to reflect the stages of DD (6, 19). Scores 1 and 2 are the initial stage of lesions, which has intact epidermis. But the score 3 is characterized by presence of ulcerative lesion. Scores 4 and 5 are the late lesions in the course of healing (19). In a study, it has been suggested that, animals could be classified based on their DD to no lesions, single lesions or recurring lesions (20, 21). Reducing the occurrence of via early diagnosis and treatment is an crucial management system (22, 23). There are a lot of treatment options for DD; systemic antibiotics, individual topical treatment, and mass topical therapy using a footbath (24). Topical treatment of DD worldwide includes antibiotic and non-antibiotic preparations (sprays or pastes) applied with/without bandage (25, 26, 27). Furthermore, when treatment of DD is in progress, thorough claw trimming should be done (28). In this study and epidemiological investigation on the occurrence and factors associated with digital Dermatitis in Egyptian dairies. Materials and methods Area The area of study is present in the northeastern part of Egypt, at the Damietta, along with branch, of the River Nile, fifteen kilometers from the Mediterranean Sea. The study region is 200 kilometers north to Cairo. It represents approximately 5% of the total area of Delta, but 1% of the total area of Egypt. Study animals For 12 months, an epidemiological investigation was achieved in one dairy herd in Dam-ietta governorate. We constructed a questionnaire to include age, milk yield, season, parity, transition period, concurrent diseases and bi-osecurity was provided by veterinary medical officer. A total of 63 (14%) of 450 dairy cows were randomly selected and examined weekly for DD lesions using risk factors scores for subjective assessment of risk parameters (Table 1). Management system The animals were housed in a cubicle (free-stall/feedlot) barn with straw-bedded stalls, and a slatted floor which was automatically scraped. The cows were tied in a long-stall barn with a daily replaced chopped straw was used as bedding on the top of rubber mats. The feeding system was a total mixed ration (TMR) for all cows, and milking was twice/day. The intake of concentrates was increased 4 weeks before calving and peaked 3 weeks after. The annual milk production per cow in the farm averaged 8500 kg energy-corrected milk during the year. A professional hoof trimmer trimmed the cows, feet routinely twice a year, in autumn and spring using a transportable hydraulic trimming chute. Footbaths were not used on this dairy farm during the study. Based on the approval of the Committee of Animal Welfare and Ethics, Occurrence and risk factors of digital dermatitis in Egyptian dairies 467 Mansoura University, this study was carried out. Case identification The clinical signs of DD in the 63 Holstein-Friesian dairy cows were recorded weekly and over 4 week after treatment. At each examination, cows were placed in a hoof trimming chute, the feet were washed manually with a water hose and a brush, and a detailed examination of the feet of each cow was performed for observation of DD lesions. Visual inspection and digital palpation were applied to evaluate the lesions of DD according to Dopfer et al. and Laven et al. (29,30). On the basis of clinical presentation, location, shape, depth, color, the diameter of the lesions and lesion maturity score (stage) were noted. The lesion diameter was measured at the widest part of the lesion in the examination stanchion using measuring tape. Statistical analyses For analysis of data, a commercial software program was used (SPSS version 21.0, SPSS Inc, USA). As an initial step, the descriptive statistics were presented and the distribution of risk factors among cows with DD was identified. The Association between the incidence of DD and the related factors was assessed by logistic regression on cow level. Two levels of statistics were performed, the univariate then multivariate. In univariate step, the status of cows with DD was the dependent, but the risk factor was the independent. When the significance level was at (P<.1), further backward stepwise multiple logistic regression was conducted. The standard parameters of logistic regression were presented finally. The association between DD and variables of clinical symptoms was assessed by chi-square test. In all steps, at P < .05, the outcome was recorded as significant. Results Prevalence Sixty-three (33.2%) Egyptian dairy cows were proved to be infected with DD. The hind claws were significantly higher than fore claws (95.24 vs. 4.76%) particularly the left hind claws (56.67%). DD was significantly higher in young age 3-5 years than older cows (>5 years old) (33; 52.4 vs. 10; 15.9%) (P < .05; OR: 1.01; 95% CI: .67-1.47). It was significantly higher during the second lactation than third lactation (27; 42.85 vs. 23; 36.50%) (P<.05; OR: 8.01; 95% CI: 6.5-6.5) and first lactation (12; 19.06%), which gradually decreased reaching the lowest incidence in heifer cows (1;1.59%) as showed in Fig.1A.There was a decrease in milk production in cows with DD than control cows. DD prevalence was affected by the transition period of the affected cows. It was progressively increased from calving till 3 weeks after calving. However, cows at time 0 of calving showed a significant (P<0.04) retraction of the lesion score. On the other hand, after calving, a significant increase (P<0.05), particularly during the first 3 month after calving was recorded. The seasonal prevalence of DD was summer (26, 41.27%); autumn (21;33.34%); winter (9;14.29%); spring (7;11.12%) as showed in Fig.1B. Univariate statistics The results of univariate analysis were presented in Tables 2, with the distribution of suggested risk factors associated with DD on cow levels. Multivariable analysis The results of this final step indicated that Hosmer and Lemeshow's goodness of fit model adequately fits the data on cow level (x2, 7.89; P= 0.20). On cow level, age, parity, season and transition period were the potential risk factors (Table 3). Clinical findings A total of 63 of 190 Holstein-Frisian dairy cows were diagnosed. Sixty lesions were present on the hind claws and three on the fore claws, especially on the plantar skin over the bulb of the heels (45; 71.43%), and occasionally on the anterior aspect of the interdigital cleft (10; 15.87%) or adjacent to the dorsal coronet (8;12.70%).Thirty-four lesions were recorded on left hind claws, 26 on the right hind claws, 2 left fore claws and1right fore claw 468 E. El-shafaey, M. Sayed-Ahmed, S. Shoeib, S. El-khodery cows. while, the DD lesions were located in a cow on both hind claws. In 63 examined claws, the skin lesion appeared as strawberry (33; 52.38%), oval (18; 28.57%) and circular spots (12; 19.05%) on the plantar aspect of the feet (Fig.2D). Most of them were distal, but adjacent to the bulbar skin-horn junction. Small lesions (< 1 cm) were (16; 25.39%)and lesions that were assumed to be early cases were located at the skin-horn junction (commonly in direct proximity to the interdigital cleft/plantar commissure). While, the medium lesions (1-2cm) were (21, 33.33%) and larger lesions (>2cm) were (26; 41.27%) were situated anywhere along the junction and sometimes merged to cover the entire bulbs. Lesions located exclusively on the skin of the interdigital cleft were elongated with a layered appearance. The pili in the area bordering the skin-horn junction were erected, elongated approximately 3 to 4 times their normal length, and matted. There were 5 M1 lesions (7.94%), 54 M2 lesions (85.71%), 3 M3 lesions 4.76%),and1M4le- sions(1.59%)asshowedinFig.2A.TheM2lesion-swerelocated over the bulb of the heel while, M1 lesions were found within the interdigital space. The M2 lesions were circumscribed areas of ulcerative, erosive dermatitis which often painful, and easily bleed when they were manipulated than other stages. The surrounding skin was reddened and the follicles appeared to be hypertrophied. Severely affected feet had a massive soft-tissue swelling in the bulbar area. There was as strongest association between M1 and M2 DD lesion. The M1 lesion has the liability to be M2 than negative one (M0). While, M2 lesions usually becomes M3-M4 in their way for recovery. There was a close link between the stage and color of lesion. Therefore, that 40 (63.49%) cows showed pink/red (erosive) lesions were observed during the clinical examination. While, 10(15.87%) cows showed brown/gray lesion and creamy/yellow (granu-lomatous) lesions were identified in 13 (20.63%) cows asshowedinFig.2C.The lesion depth was varied between shallow to deep lesions. Most of the evaluated feet showed proliferative lesion 37 (58.73%) cows and deep lesion in9(14.29%) cows. However, 15 (23.81%) cows showed shallow lesions (Fig.2B). Table 1: The risk factors scores for subjective assessment of risk parameters in 63 Holstein-Fri-sian Dairy cows with digital dermatitis Risk factors scores Score and Description Age (Year) 0 = 1-3 1 = >3-5 2 = >5-7 Milk yield (kg/Y) 0 =2100-4000 1 = 4200 2 = 6300 3 = 6000- 8500 Season 0 = Winter 1 = Spring 2 = Summer 3 = Autumn Lactation number/parity 0 = Heifer 1 = 1st 2 = 2nd 3 = 3rd 4 = 4th and over Transition period/wk 0 = Before calving 1 = After calving Concurrent disease 0 = Negative 1 = Positive (mastitis, metritis, claw affections) Affected limb 0 = Left fore claw 1 = Right fore claw 2 = Both fore claw 3 = Left hind claw 4 = Right hind claws 5 = Both hind claws = Means equal Occurrence and risk factors of digital dermatitis in Egyptian dairies 469 Table 2: Odds ratios (OR; including 95% confidence interval), frequencies, and digital dermatitis (DD) prevalence for the variables associated with DD among 63 cows in Egypt Category Description Frequency Prevalence OR CI P Value Age 1- 3 Years 20 31.7 > 3 - 5 Years 33 52.4 3.86 .7- 21.2 .1 > 5 -7 Years 10 15.9 2.86 .5- 14.01 .1 Milk yield 2100-4000 Kg/Y 27 42.9 4200 Kg/Y 25 39.7 1.08 0.1- 0.8 1. 6300 Kg/Y 11 17.5 0.56 0.0- 0.7 1. Season Winter 9 14.29 Spring 7 11.12 .035 .001- 1.2 .06 Summer 26 41.27 17.7 17.6- 17.7 .50 Autumn 21 33.34 .44 .04- 4.6 Parity Heifer 1 1.59 1st lactation 12 19.06 5.6 2nd lactation 27 42.85 8.1 6.5- 6.5 1.0 3rd lactation 23 36.50 6.5 Concurrent Diseases Negative Positive 0 63 0 100 Limb Left fore claw 20 31.7 Right fore claw 3 3.73 3.5 Right hind claw 33 56.67 3.5 Left hind claw 5 7.9 Abbreviations: CI, confidence interval at 95%; OR, odds ratio Table 3: Final logistic regression model the risk factors associated with digital dermatitis in Egyptian dairy cattle on cow level Variable ß SE P OR Cl Age .006 0.20045 .98 1.01 0.67-1.47 Parity .051 0.36224 .89 1.01 0.47-1.93 Season .304 0.14894 .04 1.36 1.01-1.81 Transition period .423 0.58770 .47 1.53 0.48-4.83 Constant .271 0.75946 .72 56.65 ------ Abbreviation: B, regression coefficient; Cl, confident interval at 95%; OR, odds ratio; P, P value; SE, stander error 470 E. El-shafaey, M. Sayed-Ahmed, S. Shoeib, S. El-khodery Figure 1: Effect of Parity and season on digital dermatitis in Egyptian dairies Occurrence and risk factors of digital dermatitis in Egyptian dairies 471 <5 P = 0.22 Figure 2: Effect of management system on digital dermatitis in Egyptian dairies Discussion Proper understanding of the predispositions factors of digital dermatitis in cattle may aid the improvement of management strategies to avoid or diminish the disease and its economic outcome (31). In our study, cow level risk factors were studied. The breed predisposition is one of the exciting causes of DD in dairy cows. Holstein cows are more susceptible to DD than other dairy breeds (32,33). The present study revealed that Holstein cows were vulnerable to DD. Moreover, DD is recognized to be more common in the pelvic feet than in forefeet (34,35). In our results, the lesions of DD were observed exclusively in the hind claws more affected than fore claws (95.24 vs.4.76%) particularly the left hind claws (56.67%). This could be attributed to longer periods of cows recumbency on such side with their left hind leg positioned under the abdomen in a somewhat contaminated, moist, and warm site (35). The current results provide an idea about the occurrence and the important associated factors of DD in dairy cows. The disease predominance in the current study was 32.2% in Egyptian dairy cows. Such finding is in concurrence with that of earlier studies in dairy cows (31,36).In Swedish dairy cows, the rate of the disease is 472 E. El-shafaey, M. Sayed-Ahmed, S. Shoeib, S. El-khodery higher (41%) than that of the current findings (27). However, in Netherlands, DD were found less prevalent (20%) than in our study (3). Parity was significantly higher during the second lactation than 3rd lactation (27; 42.85 vs 23; 36.50%) and these results agree with Holzhauer et al. (3), who reported that there was a negative relationship between prevalence of DD and parity. At 30-60 days in milk and at the third parity cow, the odds ratio is higher than other times. The presence of concurrent claw affection were increased the prevalence of DD in dairy cows up to 100%. This results was agree with Holzhauer et al. (3), who found that other claw diseases, as interdigital phlegm on, hyperplasia, and heel horn erosion predispose to the disease. Incidence of DD was higher in dairy cows in the second and third lactation number than in subsequent lactations or heifer. This is in agreement with results presented by (12,3,37) who stated that, DD is an infectious disease and decreased incidence in older cows might be related to increased local immunity. Limited research has focused specifically on the relationships among DD prevalence and the transition period. The results of the present study showed that, throughout the transition period, the cows are more liable to develop lesions of DD than in other lactating months. At transition period, the period of high risk for DD, the cows are commonly liable to external influences, and clinical diseases (38).Abrupt environmental and metabolic changes during the transition period may explain the increased probability of hoof and horn diseases during this period. The seasonal changes in calving-related management and environment are the main stressors, which play a significant role in the occurrence and severity of DD. The seasonal prevalence of DD in the present study was higher in summer and autumn in comparison with winter and spring. Interestingly, the housing system has been also found a main risk factor for DD (33,35). The housing system of selected farm for this study was free-stall with the cows tied in a long-stall barn, so there might be an association with prolonged housing periods and the increased prevalence of DD. There is a strong association between wet/dirty claw environments and the occurrence of DD. It has been observed that housing on concrete floor has higher odds than on a slatted floor with manure scraper (40). In this study, dairy cows were housed in a cubicle barn with mattress-bedded stalls, and a slatted floor which was automatically scraped. Frequent removal of cows manure would diminish the undesirable effects of the unhygienic and wet claw environment, via increasing the resistance of the claw capsule to erosions and subsequent bacterial infection (40,41). In this study, most of the DD lesion definition such as diameter, shape, color, depth and location were evaluated as shown in table/figure. In dairy cows, M1, M2have been reported the major types of lesions. The M2 lesions are circumscribed areas of ulcerative, erosive dermatitis. Due to involvement of the dermis, the lesions are painful and affected animals are lame. Digital dermatitis lesions were assed everywhere on the solar aspect of the claws of the affected dairy cow included in this study. The heel bulbs are close together which more prone to continually being moist, which favors the development of DD especially M2 lesions (42). However, the M1 lesions were located in the interdigital space which has some aeration and dryness than the heel bulbs. The rate of development and resolution of DD lesions were evaluated along the weekly observation of the affected claws. Moreover, the changes in the lesion from stage to another in the same claw were also assessed weekly. The shift among various stages of DD can progress within one month. Therefore, weekly observation would be beneficial in understanding the changes in DD lesions stages without missing even small lesions. The majority of transitions between different stages of DD have steady order, where the transitions between M2 and M4 are the most common, though transitions between M4 and M1 have also been recorded. These present findings coincide with those reported in previous studies (43,44,45). Occurrence and risk factors of digital dermatitis in Egyptian dairies 473 Conclusions The results of this study provide an idea about the prevalence and risk factors of digital dermatitis in dairy cows in Egypt, Because of the interrelation between infectious causes of claw diseases, an effective interference against DD should directed to an integrated approach to the control of all infectious claw diseases. Furthermore, the recognition of factors which influence occurrence of DD may permit changes in herd management or breeding policies which can be applied to diminish the prevalence of DD, and can establish the appropriate preventive procedures. Conflict ofinterests None of the authors of this article has a financial or personal relationship with other people or organizations that could appropriately influence or bias the content of the article. There are no funding sources for this study. References 1. Laven RA, Proven MJ. Use of an antibiotic footbath in the treatment of bovine digital dermatitis. Vet Rec 2000; 147: 503-6. 2. Manske T, Hultgren J, Bergsten C. Topical treatment of digital dermatitis associated with severe heel-horn erosion in a Swedish dairy herd. Prev Vet Med 2002; 53: 215-31. 3. Holzhauer M, Hardenberg C, Bartels CJM, Frankena K. Herd and cow-level prevalenceofdig-italdermatitisinTheNetherlandsandassociatedrisk-factors.J Dairy Sci. 2006; 89:580-8. 4. Capion N. Animale Welfare and digital dermatitis. Cattle Consultancy Days: 2008;23-6. 5. Andrew P Fidler, Mark L Alley, Geof W Smith. Evaluation of a Serpens speciesbacterin for treatment of digital dermatitis in dairy cattle. Research in Vet Sci 2012;93: 1258-60. 6. Dopfer D, Koopmans A, Meijer FA, Szakall I, Schukken, YH, Klee W, Bosma RB, Cornelissevan JL,Asten AJ, ter Huurne A A H M. Histopathologi-caland bacteriological evaluation of digital dermatitis in cattle, with special reference to spirochaetes and Campylobacter faecalis. Vet Rec 1997; 140: 620-3. 7. Shearer JK, Hernandez J: Efficacy of two modified nonantibiotic formulations (Victory) for treatment of papillomatous digital dermatitis in dairy cows. J Dairy Sci 2000; 83: 741-5. 8. Dirksen G, Gründer H.D, Stober M: InnereMedizin und Chirurgie des Rindes: Parey in MVS Medizinverlage: Stuttgart, Germany 2005; 5. 9. Read DH, Walker RL: Papillomatous digital dermatitis (footwarts) in California dairy cattle: clinical and gross pathologic findings. J Vet Diagnostic Invest 1998; 10: 67-6. 10. Rodriguez-Lainz A, Hird DW, Walker RL, Read DH. Papillomatous digital dermatitis in 458 dairies. J American Vet Med Ass 1996; 209: 14647. 11. Rodriguez-Lainz A, Melendez-Retamal P, Hird DW, Read DH, Walker RL. Farm-and hostlevel risk factors for digital dermatitis in Chilean dairy cattle. Prev Vet Med 1999; 42: 87-97. 12. Somers JGCJ, Frankena K, Noordhuizen-Stassen EN, Metz JHM. Risk factors for digital dermatitis in dairy cows in cubicle housing systems in The Netherlands. Prev Vet Med 2005a; 71: 11-21. 13. Holzhauer M, Hardenberg C, Bartels CJM, Frankena K: Herdand cow-level prevalence of digital dermatitis in The Netherlands and associated risk factors. J Dairy Sci 2006; 89: 580--8. 14. Emanuelson U and Oltenacu PA. Incidences and effects of diseases on the performance of Swedish dairy herds stratified by production. J Dairy Sci 1998; 81:2376-82. 15. Grant R J, and Albright JL: Feeding behavior and management factors during the transition period in dairy cattle. J AnimSci 1995; 73:2791-803. 16. Dopfer D, Willemen MA: Standardization of infectious claw diseases. In: Lischer, Ch., Ossent, P. (Eds.), Proceedings of the 10th International Symposium on Lameness in Ruminants, Lucerne, Swit-zerland.1998; 244--54. 17. Vink DW. Investigating the epidemiology of Bovine Digital Dermatitis: Causality, transmission and infection dynamics. Thesis, Doctorate of Philosophy, University of Liverpool, 2006. 18. Dopfer D, Anklam K, Mikheil D, Ladell P. Growth curves and morphology of three Treponema subtypes isolated from digital dermatitis in cattle. Vet J 2012; 193: 685-93. 19. Manske T, Hultgren J, Bergsten C: Topical treatment of digital dermatitis associated with severe heel-horn erosion in a Swedish dairy herd Prev Vet Med 2002; 53: 215-31. 20. Dopfer D, van Boven MR, de Jong, MCM: A mathematical model for the dynamics of digital dermatitis in groups of cattle to study the efficacy of group-based therapy and prevention strategies. In: Joshi, N.P., Herdt, T.H. (Eds.), Proceedings of the 12th International Conference for Production Diseases in Farm Animals. 2004; 138. 474 E. El-shafaey, M. Sayed-Ahmed, S. Shoeib, S. El-khodery 21. Holzhauer M, Dopfer D, de Boer J, van Schaik G. Effects of different intervention strategies on the incidence of papillomatous digital dermatitis in dairy cows. Vet Rec 2008a; 162: 41-6. 22. Read DH, Walker RL: Papillomatous digital dermatitis (footwarts) in California dairy cattle. clinical and gross pathological finding. J Vet Diag Invest 1998;10:67-76 23. Dirksen G, Gründer HD, Stöber M: InnereMedizin und Chirurgie des Rindes: Parey in MVS edizinverlage: Stuttgart, Germany: 2005; 5. 24. Shearer JK, Elliot JB. Papillomatous digital dermatitis: treatment and control strategies-part 1. Compend Contin Educ Pract Vet 1998; 20:158-65. 25. Rodriguez-Lainz A, Melendez-Retamal P, Hird DW, Read DH, Walker RL. Farm and host-level risk factors for digital dermatitis in Chilean dairy cattle. Prev Vet Med 1999; 42: 87-97. 26. Moore Dale A, Steven L. B, Marla L. T, Vasyl K: Efficacy of a nonantimicrobial cream administered topically for treatment of digital dermatitis in dairy cattle. J Am Vet Med Ass 2001; 219: 1435-8. 27. Manske T, Hultgren J, Bergsten C: Prevalence and interrelationships of hoof lesions and lameness in Swedish dairy cows. Prev Vet Med 2002; 54: 247-63. 28. Kofler J, Pospichal M, Hofmann-Parisot, M Efficacy of the Non-antibiotic Paste Protexin Hoof-Care for Topical Treatment of Digital Dermatitis in Dairy Cows. J Vet Med A 2004; 51: 447-52. 29. Döpfer D, Koopmans A, Meijer FA, Szakall I, Schukken YH: Histological and bacteriological evaluation of digital dermatitis in cattle, with special reference to spirochaetes and campylobacter fae-calis. Vet Rec1997; 140: 620-3. 30. Laven R: Control of digital dermatitis in cattle. In Practice, 2001; 23: 336-34. 31. Ettema JF, 0stergaard O. Economic decision making on prevention and control of clinical lameness in Danish dairy herds. Livestock Sci 2006; 102, 92-106. 32. Rodriguez-lainz A, David WH, Carpenter TE: Case-control study of digital dermatitis in southern California dairy farms. Prev Vet Med 1996;28:117-31. 33. Murray RD, Downham DY, Demirkan I, Carter SD Some relationships between spirochaete infections and digital dermatitis in four UK dairy herds. Res Vet Sci 2002; 73: 223--30. 34. Murray RD, Clarkson MJ, Downham DY, Faull WB, Hughes JW, Manson FJ, Merritt JB, Russell WB, Sutherst JE, Ward WR. Epidemiology of lameness in dairy cattle: description and analysis of foot lesions. Vet Rec 1996; 138: 586-91. 35. Cramer G, Lissemore KD, Guard CL, Leslie KE, Kelton DF. Herd-and cow-level prevalence of foot lesions in Ontario dairy cattle. J. Dairy Sci 2008; 91:3888-95. 36. Somers JG, Frankena CJ, Noordhuizen-Stas-sen K, Metz EN. Risk factors for digital dermatitis in dairy cows in cubicle housing systems in The Netherlands. Prev Vet Med 2005a; 71: 11-21. 37. Nielsen B, Thomsen P, Sorensen J: A study of duration of digital dermatitis lesions after treatment in a Danish dairy herd. Acta Vet Scand 2009; 51: 27-31. 38. Argaez-Rodriguez F, Hird DW, de Anda JH, Read DH, Rodriguez-Lainz A: Papillomatous digital dermatitis on a commercial dairy farm in Mexi-cali, Mexico: incidence and effect on reproduction and milk production. PrevVet Med 1997; 32: 39. Hultgren J, Bergsten C. Effects of a rubber -slatted flooring system on cleanliness and foot health in tied dairy cows .Prev Vet Med. 2001; 52: 75-89. 40. Wells SJ, Garber LP, Wagner BA. Papillo-matous-digital dermatitis and associatedrisk factors in US dairy herds. Prev Vet Med 1999; 38: 11-24. 41. Somers JG, Frankena K, Noordhuizen-Stas-sen EN, Metz JH: Prevalence of clawdisorders in Dutch dairy cows exposed to several floor systems. J Dairy Sci 2003; 86: 2082-93. 42. Read DH, Walker RL. Papillomatous digital dermatitis (footwarts) in Californiadairy cattle: clinical and gross pathologic findings. J Diag Invest 1998; 10:67-76. 43. Berry SL, Dopfer D, Burgi K, Cook N. A longitudinal study on the transition ofdigital dermatitis lesions for 11 months after topical treatment with lincomycin H Clusing 4 scoring systems. In: Proceedings of 15th International Symposium on Lameness in Ruminants, Kuopio, Finland 2008; 913: 302-3. 44. Dopfer D, Bennett TB, Cook NB. Dynamics of digital dermatitis infection spread in a large free stall housed Wisconsin dairy herd. In: Proceeding from The 15thInternational Symposium and the 7th Conference on Lameness in Ruminants,Kuopio, Finland 2008; 9-13. 45. Holzhauer M, Bartels CJM, Dopfer D, van Schaik G. Clinical course of digital dermatitis lesions in an endemically infected herd without preventive herd trategies. Vet J 2008b; 177: 222-30. Slov Vet Res 2019; 56 (Suppl 22): 475-85 DOI 10.26873/SVR-785-2019 Original Research Article OCCURRENCE AND MOLECULAR CHARACTERIZATION OF EXTENDED SPECTRUM BETA-LACTAMASE PRODUCING Enterobacteriaceae IN MILK AND SOME DAIRY PRODUCTS Walaa Gaffer1, Mayada Gwida2*, Randa Abo Samra3, Maha Al- Ashmawy1 1Department of Food Hygiene and Control, Faculty of Veterinary Medicine, Mansoura Universit, Mansoura 35516, Egypt, 2Department of Hygiene and Zoonoses, Faculty of Veterinary Medicine, Mansoura University, Mansoura 35516, Egypt, 3Food Inspection Laboratory, Animal Health Research Institute, Damietta Port, Egypt Corresponding author, E-mail: mayada_gwida@mans.edu.eg Abstract: In the present study a total of one hundred samples were randomly purchased from different supermarkets in Damietta city, Egypt, including 25 samples each of raw milk, ice cream, Kareish and Domiati cheese. The collected samples were screened initially for cefotaxime-resistant bacteria using selective enrichment. The suspected colonies were further characterized by analysis of resistance genes in ESBL-producing isolates. A total of 59 ESBLs producers colonies (24 from raw milk, 15 from kareish cheese, 13 from Domiati cheese and 7 from ice cream) were recovered and identified as Escherichia coli (n = 29), Klebsiella pneumonia (n = 8), Klebsiella oxytoca (n = 5), Enterobacter aerogenes (n =8), Citrobacter diversus (n = 8) and Serratia liquefaciens (n = 1). Resistance to two or more antibiotics was observed among the recovered isolates. E. coli isolates showed high resistance pattern against cefaclor (100%), cefoxitin (100%), cefal-axim (96.5%), ceftazidime (93%), pencillin (93%) and 79% for Pipercillin/Tazobactam. All the recovered Klebseilla spp. isolates were resistant to Cefepime and Pipercillin/Tazobactam (84.6% each) and exhibited 100% resistant to other antimicrobials agents. Meanwhile, all the recovered isolates were sensitive to imipenem and meropenem. Gene encoding blaCTX-M1 was mostly predominant among screened genes, being present in 13 (48%) dairy samples for blaTEM and blaSHV, they were detected in 12 (44%) and 4 (14.8%) isolates, respectively. In conclusion, there is clear evidence of circulation of antibiotic-resistant food borne ESBL producing Enterobacteriaceae in the examined dairy samples. The concern about increasing the risk of dissemination of such multi-drug resistant pathogens rises with a potential asymptomatic colonization and complication of systemic infection in human subjects. There is also a possible interface for the exchange of resistance genes within and across species and with commensal bacteria of the human and animals. Key words: dairy products; ESBL producing Enterobacteriaceae; Egypt; public health Introduction The emergence of antimicrobial resistance foodborne zoonotic bacteria, particularly Enter- Received: January 2019 Accepted for publication: February 2019 476 W. Gaffer, M. Gwida, R. Abo Samra, M. Al- Ashmawy obacteriaceae that carry ESBLs, has been recognized as one of the most important global problems in both veterinary and human medicine (1). As a matter of growing concern, livestock and animal-derived foods especially milk and its products are the most incriminated foods of animal origin that is linked to disease outbreaks around the word and represent important reservoirs for ESBLs-producing Enterobacteri-aceae (2). Escherichia coli and Klebsiella pneumoniae are considered the most predominant species of ESBLs producing Enterobacte-riaceae worldwide and are being the causal of urinary tract infections, pneumonia and sepsis in human patients (3). In most of developing countries involving Egypt, there are no restricted rules for the use of antibiotics for the treatments of dairy ailments particularly third and fourth generations of cephalosporin due to their therapeutic effectiveness or their short withdrawal periods. Hence, massive and indiscriminate use of these antibacterial agents is critically important to dairy farming and could have the potential to the spread of ESBLs-pro-ducing bacteria or even multi drug resistance (MDR) pathogens (4). Resistance to P- lac-tamase in the family Enterobacteriaceae has been reported to be linked to the production of class A and C P-lactamase enzymes which able to hydrolyze and inactivate the P-lactam ring and confer different degrees of resistance to various P- lactam classes (5). ESBLs are bacterial enzymes that degrade oxyiminocephalo-sporins with the most clinically important class A P-lactamase enzymes and plasmid-mediated Temoniera (TEM), sulfhydryl variable (SHV), and Cefotaxime-Munich (CTX-M) types are the three main families of ESBLs (6). Raw milk can be contaminated with ESBLs producing Enterobacteriaceae in several entities such as mastitis, directly by animal feces or indirectly during milking (7). Unfortunately, the vast majority of the population in Egypt's, especially in rural families, still consume raw dairy products without pasteurization including traditional Egyptian cheese as Kareish and Domiati cheese (most popular soft white cheese) with a general believe that pasteurization would drastically affect the milk quality (8). To date, very limited information existing regarding ESBLs producing Enterobacteriaceae isolated from dairy cattle (3). Therefore, it is of utmost significance to address the potential occurrence of ESBLs producing Enterobac-teriaceae from raw milk and some dairy products which marketed in different localities in Damietta Governorate, Egypt and to highlight the threats to human health posed by consumption of raw milk and dairy products. Materials and methods Sampling and sample preparation A total of 100 samples (25 each of market raw milk, Kareish cheese, Domiati cheese and small scale ice cream) were randomly purchased from groceries, retail outlets, supermarkets in Damietta city, Egypt in March 2018. All the collected samples were transported immediately in coolers in its original package under aseptic conditions to the laboratory for bacteriological examinations which were started quickly after receiving the samples according to the previously described protocol (9). Isolation and identification of cefotaxime- resistant bacteria Twenty five ml or g of each dairy samples were diluted with 225 ml of buffered peptone water (BPW). In case of cheese, samples were homogenized in a stomacher for through mixing. All the prepared samples were immediately plated onto MacConkey agar plates (Oxoid, Ba-singstoke, UK) supplemented with 1 mg /L cefotaxime (Sigma-Aldrich) and incubated for 24 h at 37 °C. At least three grown colonies were selected and sub-cultured onto Mac-Conkey agar supplemented with 1 mg/L cefotaxime at 37 °C for 24 h to obtain pure cultures for subsequent analysis. Bacterial characterization was performed using Gram staining, and conventional biochemical testing including Oxidase, indole, methyl red, vogus-prauskaur, citrate, and urease. Serotyping of identified E. coli Serotyping of biochemically confirmed E. coli isolates were performed by agglutination tests by using rapid diagnostic E .coli antisera Occurrence and molecular characterization of extended spectrum beta-lactamase producing ... 477 sets according to the previously described protocol (10) (Denka Seiken Co., Japan) at Food Analysis Center, Faculty of Veterinary Medicine, Benha University, Egypt. Antimicrobial susceptibility testing and ESBLs detection All recovered isolates were screened for susceptibility test using 13 antimicrobial agents on Mueller-Hinton agar using disc diffusion method and evaluated according to CLSI criteria (11). The following antimicrobial discs were applied: Penicillin G. (P 10 pg), Ampicillin (AM10 pg), Imipenem (IPM 10 pg), Mero-penem (MEM 10 pg), Cefalaxin (CL 30 pg), Cefaclor (CEC30 pg), Cefoxitin (FOX 30 pg), Ceftazidime (CAZ 30 pg), Cefotaxime (CTX 30 pg), Cefepime (FEP 30 pg), Azteonam (ATM 30 pg), Amoxicillin-clavulinic (AMC 20/10 pg), Piperacillin-tazobactam (Tpz 100/10). Each isolate that exhibited resistant to one or more of the third and fourth-generation cephalosporins were confirmed for ESBL production by Double Disk Synergy Test (DDST) according to CLSI guidelines. Characterization of ft-lactamases Genomic bacterial DNA was extracted from the identified ESBLs producers E. coli (n= 17) and Klebseilla spp (n= 10) using the QIAamp® DNA Mini Kit Qiagen according to the manufacturers guidelines. The purified DNA was further analyzed by PCR targeting ESBL encoding genes including blaTEM, blaSHV and blaCTX-M using specific oligonucleotide primers sequences and PCR conditions as described previously (Table 1). Results Out of hundred dairy samples, 30 yielded bacterial growth on MacConkey agar supple mented with cefotaxime : 8 (32%) from raw milk, 8 (32%) from Domiati cheese, 9 (36%) from kareish cheese and 5 (20%) from ice cream. Overall, 59 ESBLs producers colonies (24 came from raw milk, 15 from kareish cheese, 13 from Damietta cheese and 7 from ice cream) and were biochemically identified as E. coli (n = 29, 49%), Klebsiella pneumonia (n = 8, 13.5%), Klebsiella oxytoca (n = 5, 8.5%), Enterobacter aerogenes (n =8, 13.5%), Citrobacter diversus (n = 8, 13.5% ) and Serratia lique-faciens (n = 1, 1.7% ) (Table 2&3). The most prevalent phenotype was E.coli (n = 17, 17%), Klebsiella pneumonia (n = 6, 6%), 5 (5%) for each of Enterobacter aerogenes and Citrobac-ter diversus, 4 (4%) for Klebsiella oxytoca and 1 (1%) for Serratia liquefaciens (Table 3). The pattern of antimicrobial drug susceptibility for the 59 ESBLs producers are presented in Table 4&5. Briefly, E. coli isolates showed high resistance pattern against cefaclor (100%), cefoxitin (100%), cefalaxim (96.5%), ceftazidime (93%), pencillin (93%) and 79% for Pipercillin/Tazobactam. All the recovered Klebseilla spp. isolates (n =13) were resistant to Pencillin, Ampicillin, Cefalaxim, cefaclor, Cefoxitin and Ceftazidime (100% each) and to Cefepime and Pipercillin/Tazobactam (84.6% each). On the other side, all recovered isolates were sensitive to imipenem and meropenem. Gene encoding blaCTX-M1 was mostly predominant among screened genes (fig 1&2), being present in 13 (48%) dairy samples. for blaTEM and blaSHV, they were detected in 12 (44%) and 4 (14.8%) isolates, respectively. Three isolates harbored the three screened ß-lactamase genes; blaCTX-M1 and blaTEM were detected in four isolates; while blaSHV and blaCTX-M1 were observed in one isolate. Table 1: Oligonucleotide primers sequences used for amplification of B-lactamase resistance genes Target gene_Oligonucleotide sequence (5' ^ 3')_Product size (bp) References blaCTX-M1 (F) 5' TTAGGAAGTGTGCCGCTGTA '3 655 12 blaCTX-M1 (R) 5' CGGTTTTATCCCCCACAAC'3 blaSHV (F) 5' AGCCGCTTGAGCAAATTAAAC '3 13 blaSHV (R) 5' ATCCCGCAGATAAATCACCAC '3 713 blaTEM (F) 5' CATTTCCGTGTCGCCCTTATTC '3 13 blaTEM (R)_5' CGTTCATCCATAGTTGCCTGAC '3 800_ 478 W. Gaffer, M. Gwida, R. Abo Samra, M. Al- Ashmawy Table 2: Distribution of ESBLs producers Enterobacteriacea spp. from the examined dairy samples Sample Origin Number of isolated Enterobacteriacea spp. from the positive samples number E.coli K. penumonia K. oxytoca Enterobac-ter Citrobacter Serratia Milk 1 Pos n= 8/25 (32%) 2 6 3 1 10 3 1 11 3 12 2 1 1 13 1 1 14 3 21 2 Damietta chesses 23 Pos n= 8/25 (32%) 1 27 1 1 29 2 31 1 35 1 41 1 1 43 1 49 1 Kariesh cheese 51 Pos n= 9/25 (36%) 2 54 1 58 2 59 1 1 61 2 69 1 70 3 74 2 75 2 Ice Cream 79 Pos n= 5/25 (20%) 1 80 2 83 2 94 1 97 1 Total pos n = 30/100 (30%) 29 8 5 8 8 1 Occurrence and molecular characterization of extended spectrum beta-lactamase producing ... 479 Table 3: Occurrence and distribution of different ESBLs producers Enterobacteriaceae spp. in examined dairy samples Enterobacte-riacae spp. Milk n =25 Kariesh cheese n = 25 Damietta cheese n = 25 Ice Cream n =25 Total Dairy samples Pos samples N of colonies Pos samples N of colonies Pos samples N of colonies Pos samples N of colonies Pos samples N of colonies E.coli 4 (16%) 9 6 (24%) 10 4 (16%) 5 3 (12%) 5 17 (17%) 29 Klebseilla .pneumoniae 1 (4%) 2 1 (4%) 2 3 (12%) 3 1 (4%) 1 6 (6%) 8 Klebseilla .oxy-toca 2 (8%) 2 1 (4%) 2 1 (4%) 1 0 0 4 (4%) 5 Enterobacter aerogenes 3 (12%) 6 0 0 1 (4%) 1 1 (4%) 1 5 (5%) 8 Citrobacter di-versus 2 (8%) 4 2 (8%) 3 1 (4%) 1 0 0 5 (5%) 8 Serratia lique-faciens 1 (4%) 1 0 0 0 0 - 0 1 (1%) 1 Total number of ESBLs n=59 24 17 11 7 59 480 W. Gaffer, M. Gwida, R. Abo Samra, M. Al- Ashmawy Table 4: Characteristics of ESBL-positive Enterobacteriaceae identified from dairy samples Sample number Origin Species b-lactam antibiotic resistances P AM IPM MEM CL CEC FOX CAZ CTX FEP ATM AMC TPZ 1 Milk E.col (O128:H2) R R S S R R R R R S R R R 1 Milk E.coli (O91:H21) R R S S R R R R R S S R R 6 Milk E.coli (O55:H7) R S S S R R R S R S S R R 6 Milk E.coli (O121:H7) R R S S R R R R R R S S R 6 Milk E.coli (O146:H21) R S S S R R R R R R S S R 10 Milk E.coli (O111:H2) R R S S R R R R R R S S S 10 Milk E.coli (O111:H2) R S S S R R R R R S S S R 10 Milk E.coli (O114:H4) S S S S R R R R R S S S S 13 Milk E.coli ( O78) R S S S R R R R R S S S S 27 Domiate cheese E.coli (O111:H2) R R S S R R R R R R R R R 29 Domiate cheese E.coli (O121:H7) R R S S R R R R R R R R R 29 Domiate cheese E.coli ( O26:H11) R S S S R R R R R R S S R 41 Domiate cheese E.coli (O26:H11) R S S S R R R R R S S S R 43 Domiate cheese E.coli (O114:H4) R R S S R R R R R R S R R 54 kareish cheese E.coli (O124) R R S S R R R R R R S R R 59 kareish cheese E.coli (O111:H2) R R S S R R R R R R S R R 61 kareish cheese E.coli (O91:H21) R S S S R R R R R S S S R 61 kareish cheese E.coli (O121:H7) R S S S S R R R R S S S S 69 kareish cheese E.coli (O91:H21) R S S S R R R R R S S S R 70 kareish cheese E.coli (O126:H21) R S S S R R R R R R S S R 70 kareish cheese E.coli (O128:H2) R R S S R R R R R S R R R 70 kareish cheese E.col i (O111:H2) R R S S R R R R R S R R R 74 kareish cheese E.coli (O127:H6) R R S S R R R R R S R S R 74 kareish cheese E.coli(O146:H21) R R S S R R R R R R S S S 80 Ice cream E.coli (O26H11) R R S S R R R R R R S S S 80 Ice cream E.coli (O91:H21) R R S S R R R R R R S S R 83 Ice cream E.coli (O111:H2) R R S S R R R R R R S R R 83 Ice cream E.coli (O121:H7 R R S S R R R R R R R S R 94 Ice cream E.coli ( O26:H11) R S S S R R R R R R S S S Occurrence and molecular characterization of extended spectrum beta-lactamase producing ... 481 Table 4: Continuation Sample number Origin Species b-lactam antibiotic resistances P AM IPM MEM CL CEC FOX CAZ CTX FEP ATM AMC TPZ 21 Milk k. pneom R R S S R R R R R R R S S 21 Milk k. pneom R S S S R R R R R S S R R 27 Domiate cheese K.pnemo R R S S R R R R R R S S R 31 Domiate cheese K.pnemo R R S S R R R R R S S S S 41 Domiate cheese k.pnemo R R S S R R R R R R S S R 75 kareish cheese k.pnemo R R S S R R R R R R R S R 79 Ice cream k.pnemo R R S S R R R R R R S S S 10 Milk k. oxy R R S S R S R R R R S S R 13 Milk k. oxy R R S S R S R R R R S R R 23 Domiate cheese K.oxy R R S S R S R R R S S R R 51 kareish cheese k. oxy R R S S R R R R R R S S R 51 kareish cheese k.oxy R R S S R R R R R R S S R 6 Milk Enterobacter R R S S R R R R R R R R R 12 Milk Enterobacter S S S S R R R R R S S S S 12 Milk Enterobacter R R S S R R R R R S S S R 14 Milk Enterobacter R R S S R R R R R S S R R 14 Milk Enterobacter R S S S R R R R R S S S R 14 Milk Enterobacter R S S S R R R R R S S S R 35 Domiate cheese Enterobacter R R S S S S R R R R S S R 97 Ice cream Enterobacter R S S S R R R R R S S S R 11 Milk Citerobac-ter R R S S R R R R R R S S R 11 Milk Citerobac-ter R R S S R R R R R S S R R 11 Milk Citerobac-ter R S S S R R R R R S S S R 12 Milk Citerobac-ter R R S S R R R R R S R R R 49 Domiate cheese Citerobac-ter R R S S R R R R R S S R R 58 kareish cheese Citerobac-ter R R S S R R R R R R S S R 58 kareish cheese Citerobac-ter R R S S R R R R R S R S R 59 kareish cheese Citerobac-ter R S S S R S R R R S S S S 12 Milk Serratia R S S S R R R R R S S R R P= Pencillin , AM = Ampicillin, IPM = Impipenem, MEM = Meropenem, CL=Cefalaxim, CEC=Cefaclor, FOX = Cefoxitin, CAZ = Ceftazidime , CTX= Cefotaxime, , FEP = Cefepime, ATM=Azteronam, AMC=Amoxycil-lin/clavulinic, TPZ = Pipercillin/Tazobactam, 482 W. Gaffer, M. Gwida, R. Abo Samra, M. Al- Ashmawy Table 5: Antimicrobial resistance profiles in the identified Enterobacteriaceae spp. Used anti- Raw milk Damietta Cheese Kareish Cheese Ice cream biotic E.coli K. P k.O E C S E.coli K. P k.O E C E.coli K. P k.O C E.coli K. P E p 88.88 100 100 83.33 100 100 100 100 100 100 100 90 100 100 100 100 100 100 (8/9) (2/2) (2/2) (5/6) (4/4) (1/1) (5/5) (3/3 (1/1) (1/1) (1/1) (9/10) (2/2) (2/2) (3/3) (5/5) (1/1) (1/1) AM 66.66 100 100 50 75 0 60 100 100 100 100 50 100 100 66.66 80 100 0 (6/9) (2/2) (2/2) (2/4) (3/4) (0/1) (3/5) (3/3 (1/1) (1/1) (1/1) (5/10) (2/2) (2/2) (2/3) (4/5) (1/1 (0/1) CL 100 100 100 100 100 100 100 100 100 0 100 90 100 100 100 100 100 100 (9/9) (2/2) (2/2) (6/6) (4/4) (1/1) (5/5) (3/3 (1/1) (0/1) (1/1) (9/10) (2/2) (2/2) (3/3) (5/5) (1/1) (1/1) CEC 100 100 0 100 100 100 100 100 0 0 100 100 100 100 100 100 100 100 (9/9) (2/2) (0/2) (6/6) (4/4) (1/1) (5/5) (3/3 (0/1) (0/1) (1/1) (10/10) (2/2) (2/2) (3/3) (5/5) (1/1) (1/1) FOX 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 s (9/9) (2/2) (2/2) (6/6) (4/4) (1/1) (5/5) (3/3 (1/1) (1/1) (1/1) (10/10) (2/2) (2/2) (3/3) (5/5) (1/1) (1/1) CAZ 77.77 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 (7/9) (2/2) (2/2) (6/6) (4/4) (1/1) (5/5) (3/3 (1/1) (1/1) (1/1) (10/10) (2/2) (2/2) (3/3) (5/5) (1/1) (1/1) CTX 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 (3/3) 100 100 100 (9/9) (2/2) (2/2) (6/6) (4/4) (1/1) (5/5) (3/3 (1/1) (1/1) (1/1) (10/10) (2/2) (2/2) (5/5) (1/1) (1/1) FEP 33.33 100 100 16.66 25 0 80 66.66 0 100 0 40 100 100 33.33 100 100 0 (3/9) (2/2) (2/2) (1/6) (1/4) (0/1) (4/5) (2/3 (0/1) (1/1) (0/1) (4/10) (2/2) (2/2) (1/3) (5/5) (1/1) (0/1) ATM 11.11 50 0 16.66 25 0 40 0 0 0 0 30 50 0 33.33 20 0 0 (1/9) (1/2) (0/2) (1/6) (1/4) (0/1) (2/5) (0/3 (0/1) (0/1) (0/1) (3/10) (1/2) (0/2) (1/3) (1/5) (0/1) (0/1) AMC 33.33 0 50 33.33 50 100 60 0 100 0 0 40 100 0 0 20 0 0 (3/9) (0/2) (1/2) (2/6) (2/4) (1/1) (3/5) (0/3 (1/1) (0/1) (0/1) (4/10) (2/2) (0/2) (0/3) (1/5) (0/1) (0/1) TPZ 66.66 50 100 83.33 100 100 100 66.66 100 100 100 80 100 100 66.66 80 0 100 (6/9) (2/2) (2/2) (5/6) (4/4) (1/1) (5/5 (2/3 (1/1) (1/1) (1/1) (8/10) (2/2) (2/2) (2/3) (4/5) (0/1) (1/1) 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 Figure 1: Agarose gel electrophoresis of multiplex PCR amplification of blaCTX-M1, blaSHV and blaTEM in E. coli with expected amplicon size ~ 655, 713, 800 bp respectively. Lane 11 is DNA ladder; lane 10 is positive control while lane12 is negative control Occurrence and molecular characterization of extended spectrum beta-lactamase producing ... 483 10 11 12 13 Figure 2: Agarose gel electrophoresis of multiplex PCR amplification of blaCTX-M1, blaSHV and blaTEM in Klebseilla spp. with expected amplicon size ~ 655, 713, 800 bp respectively. Lane 7 is DNA ladder; lane 6 is positive control while lane 8 is negative control Discussion In Egypt, there is a lack of data regarding ESBLs producing Enterobacteriaceae from milk and dairy product with a limited report about the surveillance of ESBLs producing E. coli in dairy cattle (3). Therefore, the present study was set to characterize ESBLs producing Enterobacteriaceae in some dairy products sold in Damietta city. In this study, the overall detection rate of ESBL producer E. coli in the examined dairy samples was 17%. A nearly similar detection rate was previously reported from bulk tank milk samples from German dairy farms (1); while a low frequency of ESBLs (0.7%) was reported in raw milk from the Czech Republic (14). In contrast, a higher detection rate 42.8% (114/266) of ESBL-produc-ing E.coli was previously reported in Egypt from dairy cattle (3). It is really difficult to compare studies from Egypt and other countries due to lack of reports and different methods being used for determining the presence of ESBL producing Enterobacteriaceae. In one study, the authors failed to determine ESBL-producing isolates in bulk tank milk from 100 different Swiss dairy farms (15). In another study from Turkey, the authors identified ESBL-producing Enterobacteriaceae in 100 raw milk with the percentage of 43.6% (16). In a recent study from Sudan, the authors detected ESBL production in 17 out of the 22 isolated E. coli (29.3%) from 70 raw cow milk samples collected from different villages in Aljazira state (17). A prevalence of 42.22% ESBL-positive E. coli isolates out of 45 E. coli strains was identified from 24 typical Slovak cheeses made from raw milk (18). In the present study, 13.5% of the identified isolates and 6% of the examined samples were classified as ESBL producing Klebsiella pneumonia. Different detection rate was reported from recent studies in India (19) and Sudan (17). In those studies, detection rate of 1.5% and 44.8% of K. pneumonia were identified as ESBL producing Klebsiella pneumonia from raw milk. ESBLs producing Enterobacteri-aceae has been described for the first time from 484 W. Gaffer, M. Gwida, R. Abo Samra, M. Al- Ashmawy hospitalized humans but recently, several researchers from different countries reported its dissemination in the community and also in healthy food producing animals worldwide (1, 3. 5, 15). Resistance to antimicrobial agents represents a real challenge in Egypt, with high burden on the Egyptian health care system (20). In that study, the authors found that 151 out of 355 (42.5%) of the Klebsiella spp. and 47 of 87 (54%) of Escherichia coli from the patient in intensive care units were identified as ESBL producers. Several studies have discussed the resistance spectra of different ESBL producers regardless their origin (i.e. human, animal or food origins) (5,15-16). Globally, ESBL-pro-ducing microorganisms are one of the most severe problematic multi-resistant and are being identified with increased frequency (4). Our results showed that resistance to two or more antibiotics was frequently encountered among the recovered isolates. As the production of most ESBLs is plasmids encoding, co-resistance to other groups of antibiotics is a common sequel (15). Remarkably, the range of resistance in our study was sufficiently worrisome. For instance, if an infection caused by any identified strains (as that observed in this study) it could represent a great concern due to limited therapeutic options. According to latest report of Infectious Diseases Society of America, ESBL-producing E coli and Klebsiella spp. were recognized as one of the six drug-resistant microbes that urgently required new remedies (21). Our geno-typic characterization of the positive dairy samples by PCR assays revealed that 48% of the positive dairy samples were possessed blaCTX-M1encoding CTX-M broad-spectrum P-lac-tamases, while other genes like blaTEM and blaSHV were detected in 44% and 14.8%, respectively. This finding was in agreement with that previously reported (22, 24, 15, 23, 16). Unlike to our finding, several authors reported the absence of the blaCTX-M gene but identified only blaTEM and blaSHV genes in 15 and 4 isolates (18). The blaSHV-110, blaSHV-111, and blaCMY-41 genes have been identified from Domiati cheese from small producers in El Behera and Alexandria governments, Egypt (5). The production of broad-spectrum lactamases could provoke a significant clinical consequences with a resultant threaten the successful treatment of infectious diseases resulting in exacerbate public-health concerns (18). Conclusion In conclusion, there is clear evidence of circulation of antibiotic-resistant food borne ESBL producing Enterobacteriaceae in the examined dairy samples. As the majority of Egyptian population still consume raw milk and its product like soft cheese e.g. Domiati and Ka-reish cheese which contained ESBL producing Enterobacteriaceae , the concern about increasing the risk of a spread of such multi-drug resistant pathogens raises with a potential asymptomatic colonization and complication of systemic infection in human subjects and this could be achieved with safe use of antibiotics, reinforced sanitary measures, and a continues investigation of the phenomenon of antibiotic resistance in food producing animals. Conflict of interest The authors declare that they have no conflict of interest. References 1. Odenthal S , Akineden Ö &Usleber E. Extended-spectrum ß-lactamase producing Enterobacteriaceae in bulk tank milk from German dairy farms. International journal of food microbiology 2016; 238: 72-8. 2. Horigan V , Kosmider R D , Horton R A , Randall L & Simons R. R. L. . An assessment of evidence data gaps in the investigation of possible transmission routes of extended spectrum ß-lac-tamase producing Escherichia coli from livestock to humans in the UK. Preventive veterinary medicine 2016;124:1-8. 3. Braun SD, Ahmed MFE, El-Adawy H, Hot-zel H, Engelmann I, Weiß D, Monecke S &Ehricht R . Surveillance of Extended-Spectrum Beta-Lac-tamase-Producing Escherichia coli in Dairy Cattle Farms in the Nile Delta, Egypt. Front. Microbiol. 7:1020. doi: 10.3389/ fmicb. 2016; 01020. 4. OIE .Terrestrial Animal Health Code. Risk Analysis for Antimicrobial Resistance Arising from the Use of Antimicrobials in Animals. Version7 2014. Occurrence and molecular characterization of extended spectrum beta-lactamase producing ... 485 5. Hammad A M , Ishida Y & Shimamoto T. Prevalence and molecular characterization of ampi-cillin-resistant Enterobacteriaceae isolated from traditional Egyptian Domiati cheese. Journal of food protection 2009; 72 (3): 624-30. 6. Seiffert S N , Hilty M , Perreten V & Endimi-ani A. Extended-spectrum cephalosporin-resistant Gram-negative organisms in livestock: an emerging problem for human health? Drug Resistance Updates 2013; 16 (1-2): 22-45. 7. Dahmen S, Métayer V, Gay E, Madec J Y & Haenni M. Characterization of extended-spectrum beta-lactamase (ESBL)-carrying plasmids and clones of Enterobacteriaceae causing cattle mastitis in France. Veterinary microbiology 2013; 162 (2-4): 793-99. 8. Zeinhom M M & Abdel-Latef G. K. Public health risk of some milk borne pathogens. Beni-Suef University Journal of Basic and Applied Sciences 2014; 3(3): 209-15. 9. Roberts D& Green wood M. Practical Food Microbiology, 3rd. Section 10: confirmatory biochemical testes. Blackwell 2003. 10. Kok T ,Worswich D & Gowans E . Some serological techniques for microbial and viral infections. In Practical Medical Microbiology (Collee J , Fraser A , Marmion B and Simmons A , eds.) 14th ed., Edinburgh, Churchill Livingstone, UK 1996. 11. CLSI. Performance Standards for Antimicrobial Susceptibility Testing. 27th ed. CLSI supplements M100. Wayne, PA: Clinical and Laborat ory Standards Institute 2017. 12. Ogutu J, Zhang Q, Huang Y, Yan H, Su L, Gao B, Zhang W, Zhao J, Cai W, Li W, Hong Zhao H, Chen Y, Song W, Chen X, Fu Y & Zhang F. Development of a multiplex PCR system and its application in detection of blaSHV, blaTEM, blaCTX-M-1, blaCTX-M-9 and blaOXA-1 group genes in clinical Klebsiella pneumoniae and E. coli strains. The J. of Antibiotics 2015 ; 68: 725-33. 13. Perez F, Jones H, Hanson N & Geyer C. Global challenge of multidrug-resistant Acineto-bacter baumannii. Antimicrob. Agents Chemother. 2007;51: 3471-84. 14. Skockovâ A , Bogdanovicoâ K , Kolâckovâ I & Karpiskovâ R . Antimicrobial-Resistant and Extended-Spectrum P-Lactamase-Producing Escherichia coli in Raw Cow's Milk. Journal of food protection 2015; 78(1): 72-7. 15. Geser N , Stephan R & Hâchler H. Occurrence and characteristics of extended-spectrum P-lactamase (ESBL) producing Enterobacteriaceae in food producing animals, minced meat and raw milk. BMC veterinary research 2012; 8(1): 21. 16. Tekiner i H & Özpinar H. Occurrence and characteristics of extended spectrum beta-lac-tamases-producing Enterobacteriaceae from foods of animal origin. brazilian journal of microbiology 2016 ; 47(2): 444-51. 17. Badri A M , Ibrahim I T , Mohamed S G , Garbi M I , Kabbashi, A S & Arbab M. H. Prevalence of extended spectrum beta lactamase (ESBL) producing Escherichia coli, and Klebsiella pneu-moniae isolated from raw milk samples in Al Jazirah state, Sudan. Mol. Biol, 2017: 7(1000201), 10-4172. 18. Vrabec M , Lovayova V , Dudrikova K , Gallo J & Dudrikova, E .Antibiotic resistance and prevalence of Enterococcus spp. and Escherichia coli isolated from bryndza cheese. Italian Journal of Animal Science 2015; 14(4): 3968. 19. Koovapra S , Bandyopadhyay S , Das G , Bhattacharyya D , Baneijee J , Mahanti A & Dimri, U. Molecular signature of extended spectrum ß-lac-tamase producing Klebsiella pneumoniae isolated from bovine milk in eastern and north-eastern India. Infection, Genetics and Evolution 2016; 44: 395402. 20. Talaat M , El-Shokry M , El-Kholy J , Ismail G , Kotb S , Hafez S & Lessa F. C. National surveillance of health care-associated infections in Egypt: developing a sustainable program in a resource-limited country. American journal of infection control 2016 ; 44(11): 1296-301. 21. Pitout J D& Laupland KB . Extended-spectrum ß-lactamase-producing Enterobacteriaceae: an emerging public-health concern. The Lancet infectious diseases 2008; 8(3): 159-66. 22. Amador P , Fernandes R , Prudencio C & Brito L. Resistance to ß-lactams in bacteria isolated from different types of Portuguese cheese. International Journal of Molecular Sciences 2009; 10 (4): 1538-155. 23. Warjri I , Dutta T K , Lalzampuia H & Chandra, R. Detection and characterization of extended-spectrum ß-lactamases (blaCTX-M-1 and blaSHV) producing Escherichia coli, Salmonella spp. and Klebsiella pneumoniae isolated from humans in Mi-zoram. Veterinary world 2015; 8(5): 599. 24. El-Domany R, Emara M, El-Magd M, Moustafa W, Abdeltwab N. Emergence of imipenem-resistant Pseudomonas aeruginosa clinical isolates from Egypt coharboring vim and imp carbapenemases. Microbial Drug Resistance 2017; 23:682-6. Slov Vet Res 2019; 56 (Suppl 22): 487-98 DOI 10.26873/SVR-786-2019 Original Research Article SESAME OIL MITIGATES INITIATION STAGE OF DIETHYNITROSAMINE HEPATOCARCINOGENESIS IN RATS AbdAllah A. Mokh1, Doaa H. Abdelhady1, Emad W. Ghazy1, Hadeer Aboumosalem1*, Wael M.Goda2 1Faculty of Veterinary Medicine, Department of Clinical Pathology, Kafrelsheikh University, 2Faculty of Veterinary Medicine, Department of Clinical Pathology, Damanhour University, Egypt *Corresponding author, E-mail: hadeeraboumosalem@gmail.com Abstract: Diethylnitrosamine (DEN) induced hepatocarcinogenesis in experimental animals through triggering reactive oxygen species (ROS) release and subsequent induction of oxidative stress dependant liver damage. This study was conducted to estimate the protective role of sesame oil (SO) in the initial phase of DEN induced hepatocarcinogenesis. Forty five male Wistar rats were randomly divided into five groups groups (n = 9 each). In the first group (control), rats were orally administrated normal saline. Rats of second group (DEN) were intraperitoneally (i.p) injected with a single dose of (200mg/kg body weight, DEN) at the 8thday of the experiment. The third, fourth, fifth groups orally administrated SO at a dose (2.5, 5, 10 mL/kg b.w), respectively 1 week before i.p injection of DEN and continued for 4 successive weeks. DEN- induced hepatotoxicity as detected by normocytic normochromic anemia with marked increase in white blood cells and significant increase in hepatic damage enzymatic markers (alanine transaminase (ALT), aspartate aminotransferase (AST), Y-glutamyl Transferase (yGT) and alkaline phosphatase (ALP)) with significant decrease in serum total protein. Hepatic malondialdehyde (MDA) was increased significantly while hepatic antioxidant biomarkers superoxide dismutase (SOD), catalase (CAT) and hepatic reduced glutathione (GSH) were significantly decreased. Histological examination of hepatic tissue of DEN treated rats proved centrol-obular necrosis associated with bile duct and oval cell proliferation. This was accompanied with over expression of CYP2E1 and down regulation in BAX gene expression in liver. Administration of SO minimized the harmful effects of DEN on hematological, biochemical, antioxidant and histopathological parameter as well as on gene expression. The degree of improvement was in dose dependant manner. Our findings revealed that SO supplementation can mitigate the toxic effects of DEN via their potent antioxidant and free radical-scavenging activities. Key words: diethylnitrosamine, sesame oil, antioxidant, gene expression, rats Introduction Liver cancer considers one of the most frequent causes of death. Hepatocellular carcinoma (HCC) is a malicious tumor of liver cell originating from hepatocyte and considered as Received: January 2019 Accepted for publication: February 2019 488 A. Mokh, D. Abdelhady, E. Ghazy, H. Aboumosalem, W. Goda the most common third cause of cancer death worldwide (1, 2). The main risk factors in liver cancer include hepatitis C virus, feed additives as (BHA BHT, nitrates/nitrites), mycotoxins, air and water pollutants (3). In Egypt, The prevalence of liver cancer has been growing in latest years (4). Nitrosamines are potent environmental carcinogens because of their mutagenic and carcinogenic abilities. Nitrosamines found in water, industrial product, tobacco, cured cheese and smoked meats (5). Diethylnitrosamine (DEN) is commonly used to instigate hepato-cellular carcinoma in experimental rat models (6, 7) probably through induction of oxidative stress, resulting in liver damage with increased deleterious free radicals formation (8). Chemo-prevention may help to decrease the incidence or severity of carcinogenic insult. They can be used as approaches for liver cancer treatment with less toxic effects. Sesame oil (SO) extracted from Sesamum in-dicum seeds which has phenolic lignans as ses-amol, sesamin, sesamolin and tocopherol (vitamin E) all of which give sesame oil the significant free radical-scavenging ability (9). Sesame oil has the ability to minimize ROS production and lipid peroxidation in various animal model through its antioxidant ability (10). Thus, the current work aimed to estimate the hepatopro-tective effect of sesame oil on initial stage of HCC induced by DEN in rat models. Materials and methods Preparation of DEN and sesame oil DEN (purcashed from Sigma Aldrich) was dissolved in normal saline. Sesame oil was obtained from Harraz Company (Cairo, Egypt 100% pure) in a solution form, given at different concentrations (2.5, 5, 10 ml/kg b.w) by stomach tube according to animal body weight. Animals Forty-five healthy male Wistar rats (weighing 90±20 g/rat and at age of 1 month old) were obtained from the Alexandria Organization for Biological Products. The rats were housed in metallic cages with thermally controlled tem- perature ranged from 22to25 °C, relative humidity 50-60%, with12 h photoperiods and12 h dark. During the entire period of study, the rats were provided with a semi-purified basal diet and water ad libitum. The experiment protocol followed the Guide for the Care and Use of Laboratory Animals at Kafrelsheikh University. All safety measures had been taken to minimize animal stress. Experimental design After two weeks acclimatization period, random classification of rat into 5 different equal groups (9/each) was done. Rats in the1st group were kept as control and were only given normal saline by gastric intubation during the whole period of the experiment. A single ip injection of normal saline was also given to these rats at the 8th day of the experiment to initiate hepatocarcinogenesis (11). Rats of the 2nd group (DEN) were i.p injected with a single dose of DEN (200 mg/kg b.w) at the 8 th day of the experiment (11). The 3rd group rats orally administrated sesame oil (2.5 mL/kg b.w) 1 week before i.p injection of DEN (at the first day of the experiment) and continued for 4 successive weeks. The 4th group orally received sesame oil at a dose of (5 mL/kg b.w) 1 week before i.p injection of DEN (at the first day of the experiment) and continued for 4 successive weeks (10). The 5th group received sesame oil at a dose of (10 mL/kg b.w, orally) 1 week before i.p injection of DEN (at the first day of the experiment) and continued for 4 successive weeks (12). Blood and liver sampling Blood samples were collected from rats eyes by retro-orbital venus plexus bleeding under effect of mild ether anesthesia by using of clean capillary tubes and immediately grouped into two groups the first with anticoagulant for he-matological parameters determination, the second group without anticoagulant for serum biochemical estimation. Later, rats were slaughtered and livers were rapidly removed then trimmed from excess tissues and washed by normal saline and distilled water, cut apart into three parts; The 1st part was cut into slices and Sesame oil mitigates initiation stage of diethynitrosamine hepatocarcinogenesis in rats 489 directly put in liquid nitrogen, then stored at-80 °C for molecular analysis. The 2nd portion used to prepare tissue homogenate for antioxidant examination as previously described (13). The last portion was directly put in 10% formalin for histopathological analysis. Hematological examination Blood samples collected in tubes coated with EDTA (1mg/ml blood) were used for measuring of complete blood count (RBCs, Hb, PCV, MCV, MCHC, WBCs, and platelets count) using exigoautomated cell counter (Exigo BM800, USA) Biochemical parameters The serum level of (ALT) and (AST) enzymes were kinetically determined as previously described (14), while the serum level of (yGT) and(ALP) enzymes were Calorimetri-cally estimated by (15). Serum total protein and glucose were calorimetrically detected as previously detailed (16, 17). MDA level and activities of catalase, SOD and GSH were determined in liver homogenate as method described by (18-22). Histopathological analysis Liver tissue specimens were preserved in 10% neutral formalin, fixed in paraffin, sectioned and stained by H&E (23). Molecular investigation of BAX and CYP2E1 genes Total RNA was extracted from liver with Trizol reagent (total RNA isolation reagent, IN-TRON Biotechnology, Inc). Complementary DNA (cDNA) was synthesized using cDNA synthesis kits as previously described (24). PCR tubes containing SYPR green master mix (Enzynomic company, Cat number RT500), cDNA, primers (Table1) and RNase free water were inserted in BloRad IQ2 real time thermal cycler. P- actin was used as a housekeeping gene. Thermal cycling conditions were: incubation at 94 °C for 15 min then 94 °C for15 s (40 cycles) followed by 60 °C for 30s and 70 °C for 30s. The data reported at extension step. Melting curve analysis used to determine specificity of PCR products. IQ5 software was used to detect amplification curves and Ct values. Gene expression variation of different samples determined through comparing between the Ct values of all groups by using "2AACt" method. The PCR products were confirmed by using 1.5% agarose gel. Data statistical analysis Graph pad prism version 5.0 was used to analyze the resulted data. Differences in values were analyzed by one-way analysis of variance (ANOVA), then Tukey's-compare all pairs of columns. All data were exposed as mean ± standard error of the mean (SEM) with citation of significance level at p < 0.05. Results Hematological finding Data explored in table (2) showed deleterious impacts of (DEN) and the ameliorative effects of Sesame oil on the erythrogram. A significant decrease in hematological parameters (RBCs, Hb, PCV and platelets) without significant change in the values of (MCV, MCH and MCHC) was noticed in DEN treated group as compared to the control group. Opposing to these results WBCs count was significantly increased. SO treated group showed a marked (P < 0.05) decrease in hematological parameters (RBCs, Hb, PCV and platelets) as compared to DEN-treated group, but with a significant (P < 0.05) decrease in WBCs count. Serum Biochemical assays Data demonstrated in (Fig. 1A) showed toxic effects of DEN and the protective role of sesame oil on serum biochemical measurements. Group II (DEN) showed significant increases in the activity of hepatic damage enzymatic markers (ALT, AST, ALP, yGT) and serum glucose when compared with the control group. Group III, IV, V which supplied by sesame oil and (DEN) showing significant decrease in the activity of serum liver function marker enzymes (AST, ALT, ALP, yGT) and serum glucose when compared with the DEN group. The degree of improvement was in dose dependant manner. 490 A. Mokh, D. Abdelhady, E. Ghazy, H. Aboumosalem, W. Goda Data illustrated in table (2) showed deleterious effects of (DEN) and the ameliorative effects of sesame oil on Serum proteins. Group II (DEN) showed significant decreases in total proteins, albumin and globulins concentration when compared with control group. Group III, IV, V which supplied by sesame oil and DEN showing significant increase in total proteins, albumin and globulins concentration when compared with DEN group. Data demonstrated in (Fig. 2) revealed the harmful effects of DEN as well as the challenge effects of Sesame oil on lipid peroxidation and antioxidant biomarkers. Group II (DEN) showed significant increase in hepatic MDA content, while liver CAT, GSH, and SOD activities were statically decreased when compared with control group. Group III, IV, V (DEN -SO) showed significant decreases in hepatic MDA content but liver CAT, GSH, SOD activities had increased markedly in comparing to DEN group. Histopathological finding Effect of DEN and Sesame oil on histopathological features of liver is presented in (Fig. 3). Histological examination of the hepatic tissue sections of control negative rats revealed normal hepatocellular architecture mainly consisting of normal hepatocytes with normal cytoplasm and small uniform vesicular-shaped nuclei which arranged in a radial pattern around the central vein of the hepatic lobules (Fig. 3A). Liver of control positive animal (DEN) revealed centrolobular necrosis associated with severe ballooning of hepatocytes. Moreover, severe ballooning of hepatocytes were associated with bile duct and oval cell proliferation (Fig. 3B). Liver of diseased animal treated with (2.5 ml Sesame oil) showed decreased hepatic vacuolation, bile duct hyperplasia and centrol-obular necrosis (Fig. 3C).The fourth and fifth groups treated with (5-10 ml sesame oil) respectively, showing reduction of hepatic vacu-olation with subsequent marked reduction the altered hepatocytes as well as the number of oval cells and the necrobiotic changes associated with DEN treatment appearing the hepatic tissues mostly within normal limits (Fig.3D, E), respectively. Molecular analysis The expression of CYP2E1 and BAX genes was determined by Real time PCR that reveals the transcription levels changes of these genes in liver of rats after i.p injection of DEN alone or in combination with Sesame oil in three doses (2.5, 5, 10 ml/kg b.w). DEN injection showed marked (P<0.05) increase in CYP2E1 gene expression and significant decrease in BAX gene expression in comparison with the control, while supplying of Sesame oil before DEN injection at a dose of (2.5, 5, 10 ml/kg b.w) decreased the CYP2E1 gene expression and increased the expression of BAX gene when compared with animals treated with DEN only as showed in (Fig. 4). The degree of improvement was dose dependent. Table 1 : Primers used in qPCR Gene Primer sequence _(5'-3') Rat B-actin F 5' -TCCTCCTGAGCGCAAGTACTCT -3' R 5' -GCTCAGTAACAGTCCGCCTAGAA -3' BAX F 5' -CACCAGCTCTGAACAGATCATGA - 3' R 5' -TCAGCCCATCTTCTTCCAGATGGT -3' CYP2E1 F 5' R 5' -CTCCTCGTCATATCCATCTG -3' -GCAGCCAATCAGAAATGTGG -3' Sesame oil mitigates initiation stage of diethynitrosamine hepatocarcinogenesis in rats 491 Table 2: The impact of SO supplementation against DEN on hematological parameters and Serum protein profile. RBCs 106/ pl HB g % HCT% MCV/fl MCHC% Pl(x103/ pl ) WBCs 103/ pl Total potien Albumin gm/dl gm/dl Globulin gm/dl Control DEN DEN+ Sesame oil (2.5ml/kg) DEN+ Sesame oil (5 ml/kg) DEN+ Sesame oil ( 10ml/kg) 6.9± 0.10a 13.4±0.36a 46.6± 1.14a 5.9± 0.05b 10.3± 0.04b 36.9± 0.10b 66.9±1.63 28.8±0.40 714.8±6.5a 62.2±0.67 27.8 ± 0.13 303.8±8.5d 10.2 ±1.12dc 6.86 ± 0.1a 3.86 ± 0.08a 7.3± 0.31a 13.7± 0.10a 48.3 ±0.98a 64.9±0.31 28.7± 0.36 455.8±5.3c 24.0 ±0.85a 5.14± 0.12c 3 ± 0.07c 2.28± 0.14b 20.5 ±0.51ab 6.1 ± 0.08b 3.4 ± 0.07b 2.78 ± 0.03a 7.0± 0.21a 13.0 ±0.26a 47.1 ±0.66a 7.2 ± 0.10a 13.5 ± 0.18a 48.0± 1.32a 67.4±1.72 27.6± 0.23 571.2±4.6b 66.4±1.33 27.7± 0.61 700.2±3.2a 14.8 ±0.62c 6.58± 0.03a 12.5± 1.10c 6.7± 0.05a 3.66 ± 0.04ba 2.92 ± 0.02a 3.7 ± 0.07a 3 ± 0.1a Values are means ± standard error. (n=9) Mean values with different letters at the same column differ significantly at (p <0.05). RBCs: red blood cells, Hb: hemoglobin, HCT%: hematocrite, MCV: mean corpuscular volume, MCHC: mean corpuscular hemoglobin concentration. WBCs: white blood cells, PI: platelets. Figure 1: Effect of different concentrations of sesame oil on serum levels of liver enzymes (ALT, AST, ALP, GGT) of DEN-intoxicated rats. Values are means ± standard error. (n=9). Mean values with different letters in each graph significantly differ at (p <0.05) 3 ±0.05 492 A. Mokh, D. Abdelhady, E. Ghazy, H. Aboumosalem, W. Goda □ CONTROL E3 DEN ^ DEN+Sesame oi (Z5ml/kg) ED DEN+Sesame oil (5ml/kg) Effl DEN+Sesame oil (10mIfkg) Figure 2: Effect of different concentrations of sesame oil on hepatic level of lipid peroxidation marker MDA, and endogenous antioxidants (GSH, SOD, CAT) of DEN-intoxicated rats. Values are means ± standard error. (n=9). Mean values with different letters in each graph significantly differ at (p <0.05) « - r u «5« i' •' Sfflft ■ ^ is; Mi rrriiffinrnrifTrTiiiy ^ "V'v: Figure 3: A. Liver of control animal showing normal hepatocytes arranged in cords around the central vein, H&E, X 200. B. Liver of control positive animal showing centrolobular necrosis (arrows) associated with severe ballooning of hepatocytes (arrowheads), H&E, X20 Sesame oil mitigates initiation stage of diethynitrosamine hepatocarcinogenesis in rats 493 fle: ill WBt ■ Fig.3. C. Liver of animal treated with DENA and Sesame oil (2.5 ml) showing decrease hepatic vacuolation (arrowhead) and centrolobular necrosis (arrow). D. Liver of animal treated with DENA and Sesame oil (5 ml) showing marked decrease hepatic vacuolation (arrowhead). E. Liver of animal treated with DENA and Sesame oil (10 ml) showing significant decrease hepatic vacuolation (arrowhead). H&E, X 200 Figure 4: Effect of different concentrations of sesame oil on relative expression of CYP2E1 and BAX genes in liver of DEN-intoxicated rats. Values are means ± standard error. (n=9). Mean values with different letters in each graph significantly differ at (p <0.05). 494 A. Mokh, D. Abdelhady, E. Ghazy, H. Aboumosalem, W. Goda Discussion T Hepatocellular carcinoma induced by several factors such as food supplement(25), endogenous or environmental stimuli (26). It happens stepwise through initial stage alterations, proliferation resulting in malignant transformation (27). DEN could be metabolized by cytochrome P450 to produce active ethyl radical, which can interact with DNA causing mutagen-icity and consequent carcinogenesis (28, 29). Research on plants helped in the detection of compounds (30)with antitumor activity from non-traditionally useful plants that clinically used as effective drugs(31). The current study has been initiated to investigate whether sesame oil supplementation could inhibit the initiation stage of hepaticcarcinogenesis induced by DEN in rats. Hematopoietic system is a very sensitive systems to estimate the danger impacts of drugs and toxins on our health (32). The current work revealed that i.p injection of DEN caused marked bad effect on the eyrthrogram of DEN rats. SO pretreatment had improved the disturbances of hematological parameters caused by DEN in an effective manner. The damaging consequences of DEN on hemogram was proved by significant reduction in RBCs, Hb%, HCT and platelets counts without significant changes in MCV or MCHC. Opposing to these results, a significant elevation in TLC count was also detected in control positive group (DEN-treated). These results explained the etiological relationship between anemia (normo-cytic normochromic anemia) and DEN treatment which might be produced as a result of different mechanisms including either bone marrow cells damage or increase osmotic fragility of RBCs and damage of cell membrane (33). furthermore, (34)reported that DEN not only resulted in reduction in RBCs and platelets count but also induced an increase in the TLC count. The reduction in platelets count could be due to either inhibition of bone marrow activity by DEN which consequently decrease its production or increased its consumption(35). On the other hand, elevation in the total leukocytic count could be due to inflammation occurs dur- ing DEN treatment and bioactivation in the experimental animals. SO has a good role in counteracting DEN - hazard effects on the erythrogram by increasing RBCs and Hb count. SO has antioxidant ability to improve erythrocyte deformability markedly (10). The liver condition is indirectly examined through the determination of serum ALT, AST, ALP and yGT. This study reported that The elevations of these enzymes could be attributed to their leakage from damaging cell membrane which induced as aresult of several pathological conditions as hepatic cellular injury (36, 37). DEN treated group resulted in increase in ALT, AST, ALP and yGT serum activities which proved hepatic cell damage. These results were reported elsewhere(38, 39).In the current experiment treatment with sesame oil caused a significant reduction in ALT, AST, ALP and yGT activities which indicate significant recovery of hepatic cell function. The hepatoprotective role of sesame oil may be due to its antioxidant active componentas (sesamin, sesamol, sesa-molin, andtocopherol). Which reduce ROS production and lipid peroxidation, by this means the membrane permeability was alleviated and the leakage of these enzymes into the blood was minimized (40). Sesame oil treated group caused a significant reduction in level of blood glucose when compared with DEN treated group as sesame oil contains monounsaturated fatty acids and bio-active compounds which enhanced P cells to produce insulin and regulate the blood glu-cose(40). The fat-soluble lignans (sesamin, ses-amolin, and sesamol) help in decreasing of the hepatic oxidative destruction as a result the blood sugar level was reduced (41). Concerning to evaluation of serum proteins such as albumin and globulin which concern a good criteria for assessing the synthetic function of the liver(42). In the present study it was noticed that the reduction of total protein level indicate diseased and bad health condi-tion(43).This present study showed considerable decrease in blood serum total protein, albumin, and globulin concentrations in rats with DEN induced hepatopathy that lead to severe liver damage correlated with tissue histoarchi-tecture. When sesame oil was administrated at Sesame oil mitigates initiation stage of diethynitrosamine hepatocarcinogenesis in rats 495 different doses with i.p injection of DEN result in significant elevation in total protein, albumin and globulin concentrations which proved the recovery of synthetic function of the liver, These results agree with (44, 45). DEN injection enhances the oxidative damage through elevation of hepatic MDA and depleted enzymatic (SOD and CAT) and non enzymatic (GSH) antioxidant markers(46, 47). ROS have a critical role in the initiation of lipid peroxidation(48). Which produced in high amount as a result of cellular damage (49). generation of ROS and LPO help in initiation of tu-morigenesis (50). DEN enhances hepatic oxidative destruction resulted in HCC formation(51). This study agreed with(46, 47). The administration of sesame oil resulted in marked decrease in MDA level and elevation in the activities of (SOD, catalase and GSH) when compared with the DEN-treated rats. The antioxidant effect of SO is due to the non-fat antioxidants content as (sesamol, sesamin, sesamolin and tocopherol (vit E) (52), which protect the cells from oxida-tive damage. The histopathological changes in liver tissues caused by DEN revealed the increased percentage of degenerated hepatocytes manifested as areas of perivascular inflammatory infiltrates with diffuse ballooning degeneration, severe ballooning of hepatocytes associated with bile duct and oval cell proliferation. This may be due to the hepatotoxic effect of DEN which causes oxidative stress and liver tissue damage. Hepatic cell degeneration induced by DEN administration is in harmony with those obtained, (25, 53, 54). Sesame oil administration decreased oxidative damage in HCC rats through reducing production of ROS and LPO leading to significant improvement of hepatic tissue which evidenced as marked decrease in hepatic vacuolation, inflammatory infiltrates, oval cell proliferation and centrolobular necrosis. Molecular gene expression (CYP2E1 and BAX) are considered important genes expression in cancer. The cytochromes P450 (CYPs) are main enzymes in development and treatment of cancer. They enhance the metabolic activation of several carcinogenic substances as (benzene, CCL4,chloroform, styrene, V-nitro-sodimethylamine, NNK)(55). Thus, CYP2E1 might be an essential gene in detection toxicity and carcinogenicity susceptibility to human from environmental and industrial chemicals (54, 56). CYP2E1 is one of CYPs Class one which activated during pre-carcinogens and drugs metabolism. In this study liver of DEN-treated rats produced an over expression of CYP2E1gene opposing to control group re-sults,(57) supported these findings. CYP2E1 has an important role in metabolism and activation a number of chemicals, solvents, cancer producing agents. Sesame oil treated animals showed a significant down regulation in CYP2E1 gene expression when compared with DEN-treated group. Sesame oil has a meth-ylenedioxypheny compounds which are potent inhibitors or inactivators of CYP isoforms. These compounds could interact with the CYP450 isozymes and affect the drug metabolisms resulting in inhibition in the activity of this gene(58). BAX is a pro-apoptotic gene that regulates cell death. It is an important indicator of mito-chondrial dysfunction and one of the essential pro-apoptotic members of the Bcl-2 family proteins. It manages the apoptosis process within normal and cancer cells. Apoptosis Dysfunction makes the cancer treatment more difficult and helps tumorigenesis to progress. Activation of BAX gene increases permeability of the mi-tochondrial membrane; result in releasing of apoptotic factor cytochrome c which causes cancer cell death. In the current study DEN-treated rats showed significant down regulation in the expression of BAX gene when compared with control negative group. DEN inhibits apoptosis, promoting the proliferation of cancer cell and increase cell survival. These results agreed with(59).Sesame oil treated rats revealed a significant over expression of BAX gene in comparing with DEN-treated animals as Sesame oil enhanced cytochrome releasing from mitochondria leading to promote caspase-3 cleavage (the initiator- and important caspa-ses in the intrinsic pathway of apoptosis)induce apoptosis (60), which subsequent arrest proliferation of cancer cell and cause death to it. 496 A. Mokh, D. Abdelhady, E. Ghazy, H. Aboumosalem, W. Goda Conclusion The data in the current study conclusively demonstrated that oral administration of sesame oil exert significant protective effects against DEN induced oxidative and liver damage by increasing host antioxidant defense mechanisms. This could be attributing to the improvement of anemia, decrease in serum liver enzyme activity, reduced the degree of hepatic vacuolation and necrosis of by DEN, down regulation of CYPE21and up regulation of BAX gene which enhance cancer treatment. References 1. Tabor E. Hepatocellular carcinoma: global epidemiology, Digestive and Liver Disease 2001; 33(2): 115-7. 2. Alzahrani F A, El-Magd M A, Abdelfattah-Hassan A, Saleh A A, Saadeldin I M, El-Shetry E S, Badawy A A, Alkarim S. Potential Effect of Exo-somes Derived from Cancer Stem Cells and MSCs on Progression of DEN-Induced HCC in Rats, Stem Cells International 2018; 2018: 17 https://doi.org/10.1155/2018/8058979. 3. Jemal A, Siegel R, Ward E, Hao Y, Xu J, Thun M J. Cancer statistics, 2009, CA: a cancer journal for clinicians 2009; 59(4): 225-49. 4. Ferlay J, Shin H R, Bray F, Forman D, Mathers C, Parkin D M. Estimates of worldwide burden of cancer in 2008: GLOBOCAN 2008, International journal of cancer 2010; 127(12): 2893917. 5. Ciemniak A. A comparison of N-nitrosodi-methylamine contents in selected meat products, Roczniki Panstwowego Zakladu Higieny 2006; 57(4): 341-6. 6. Al-Rejaie S S, Aleisa A M, Al-Yahya A A, Bakheet S A, Alsheikh A, Fatani A G, Al-Shabanah O A, Sayed-Ahmed M M. Progression of diethyl-nitrosamine-induced hepatic carcinogenesis in car-nitine-depleted rats, World journal of gastroenterology: WJG 2009; 15(11): 1373. 7. Elkeiy M, Khamis A, El-Gamal M, Abo Ga-zia M, Zalat Z, El-Magd M. Chitosan nanoparticles from Artemia salina inhibit progression of hepato-cellular carcinoma in vitro and in vivo, Environ Sci Pollut Res Int. doi: 10.1007/s11356-018-3339-6 2018. 8. Valko M, Rhodes C, Moncol J, Izakovic M, Mazur M. Free radicals, metals and antioxidants in oxidative stress-induced cancer, Chemico-biologi-cal interactions 2006; 160(1): 1-40. 9. Chandrasekaran V R M, Wan C-H, Liu L-L, Hsu D-Z, Liu M-Y. Effect of sesame oil against ac-etaminophen-induced acute oxidative hepatic damage in rats, Shock 2008; 30(2): 217-21. 10. Abdel-Daim M M, Taha R, Ghazy E W, El-Sayed Y S. Synergistic ameliorative effects of sesame oil and alpha-lipoic acid against subacute dia-zinon toxicity in rats: hematological, biochemical, and antioxidant studies, Canadian journal of physiology and pharmacology 2015; 94(1): 81-8. 11. Sayed-Ahmed M M, Aleisa A M, Al-Rejaie S S, Al-Yahya A A, Al-Shabanah O A, Hafez M M, Nagi M N. Thymoquinone attenuates diethylnitros-amine induction of hepatic carcinogenesis through antioxidant signaling, Oxidative medicine and cellular longevity 2010; 3(4): 254-61. 12. Saleem M T, Chetty M C, Kavimani S. Anti-oxidants and tumor necrosis factor alpha-inhibiting activity of sesame oil against doxorubicin-induced cardiotoxicity, Therapeutic advances in cardiovascular disease 2014; 8(1): 4-11. 13. Abdelhady D H, El-Magd M A, Elbialy Z I, Saleh A A. Bromuconazole-induced hepatotoxicity is accompanied by upregulation of PXR/CYP3A1 and downregulation of CAR/CYP2B1 gene expression, Toxicol Mech Methods 2017; 27(7): 544-50. 14. Adnan F, Sadiq M, Jehangir A. Anti-hyper-lipidemic effect of Acacia honey (desi kikar) in cholesterol-diet induced hyperlipidemia in rats, Bio-medica 2011; 27(13): 62-7. 15. Belfield A, Goldberg D. Colorimetric determination of alkaline phosphatase activity, Enzyme 1971; 12(5): 561-8. 16. Burtis C A, Ashwood E R, Bruns D E, Tietz textbook of clinical chemistry and molecular diagnostics-e-book, Elsevier Health Sciences2012. 17. Nagy F M, Taha N M, Mandour A E W A, Lebda M A, Hashem A S. The Biochemical Effects of Berberine on Hyperlipidemia and Insulin Resistance in Rats Fed High Fat Diet, Alexandria Journal for Veterinary Sciences 2016; 51(2): 142-7. 18. Esterbauer H, Cheeseman K, Dianzani M, Poli G, Slater T. Separation and characterization of the aldehydic products of lipid peroxidation stimulated by ADP-Fe2+ in rat liver microsomes, Biochemical Journal 1982; 208(1):129-35. 19. Sinha A K. Colorimetric assay of catalase, Analytical biochemistry 1972; 47(2): 389-94. 20. Packer L. Antioxidant action of carotenoids in vitro and in vivo and protection against oxidation of human low-density lipoproteins, Annals of the New York Academy of Sciences 1993; 691(1): 4860. Sesame oil mitigates initiation stage of diethynitrosamine hepatocarcinogenesis in rats 497 21. Beutler E. Improved method for the determination of blood glutathione, J. lab. clin. Med. 1963; 61: 882—8. 22. Glazer A N, Packer L, Oxygen Radicals in Biological Systems: Oxygen radicals and antioxi-dants, Academic Press1990. 23. McDonald J, Pilgram T. Nuclear expression of p53, p21 and cyclin D1 is increased in bronchio-loalveolar carcinoma, Histopathology 1999; 34(5): 439-46. 24. El-Magd M A, Abbas H E, El-kattawy A M, Mokhbatly A. Novel polymorphisms of the IGF1R gene and their association with average daily gain in Egyptian buffalo (Bubalus bubalis), Domestic animal endocrinology 2013; 45(2): 105--10. 25. Chen B, Ning M, Yang G. Effect of paeonol on antioxidant and immune regulatory activity in hepatocellular carcinoma rats, Molecules 2012; 17(4): 4672-83. 26. Valko M, Izakovic M, Mazur M, Rhodes C J, Telser J. Role of oxygen radicals in DNA damage and cancer incidence, Molecular and cellular biochemistry 2004; 266(1-2): 37-56. 27. Hassan H A, Serag H M, Abdel-Hamid N M, Amr M M. Synergistic curative effect of chicory extract and cisplatin against thioacetamide-induced hepatocellular carcinoma, Hepatoma Res 2015; 1: 147-54. 28. Chakraborty T, Chatterjee A, Rana A, Dhachinamoorthi D, Kumar A, Chatterjee M. Carcinogen-induced early molecular events and its implication in the initiation of chemical hepatocarcin-ogenesis in rats: Chemopreventive role of vanadium on this process, Biochimica et Biophysica Acta (BBA)-Molecular Basis of Disease 2007; 1772(1): 48-59. 29. Verna L, Whysner J, Williams G M. N-nitro-sodiethylamine mechanistic data and risk assessment: bioactivation, DNA-adduct formation, muta-genicity, and tumor initiation, Pharmacology & therapeutics 1996; 71(1-2): 57-81. 30. Ramakrishnan G, Raghavendran H R B, Vinodhkumar R, Devaki T. Suppression of N-nitro-sodiethylamine induced hepatocarcinogenesis by si-lymarin in rats, Chemico-biological interactions 2006; 161(2): 104-14. 31. Kinghorn A D, Su B-N, Jang D S, Chang L C, Lee D, Gu J-Q, Carcache-Blanco E J, Pawlus A D, Lee S K, Park E J. Natural inhibitors of carcino-genesis, Planta medica 2004; 70(08): 691-705. 32. Liju V B, Jeena K, Kuttan R. Acute and sub-chronic toxicity as well as mutagenic evaluation of essential oil from turmeric (Curcuma longa L), Food and chemical toxicology 2013; 53: 52-61. 33. Yuan G, Dai S, Yin Z, Lu H, Jia R, Xu J, Song X, Li L, Shu Y, Zhao X. Toxicological assessment of combined lead and cadmium: acute and sub-chronic toxicity study in rats, Food and chemical toxicology 2014; 65: 260-68. 34. Markovic S D, Žižic J B, Dačic D S, Obra-dovic A D, Curčic M G, Cvetkovic D M, Dordevic N Z, Ognjanovic B I, Štajn A. Alteration of oxidative stress parameters in red blood cells of rats after chronic in vivo treatment with cisplatin and selenium, Archives of Biological Sciences 2011; 63(4): 991-9. 35. Sirag H. Biochemical and hematological studies for the protective effect of oyster mushroom (Pleurotus ostreatus) against glycerol-induced acute renal failure in rats, J Biol Sci 2009; 9(7):746-52. 36. Kadir F A, Kassim N M, Abdulla M A, Ye-hye W A. Hepatoprotective role of ethanolic extract of Vitex negundo in thioacetamide-induced liver fibrosis in male rats, Evidence-Based Complementary and Alternative Medicine 2013; 2013. 37. Nyblom H, Berggren U, Balldin J, Olsson R. High AST/ALT ratio may indicate advanced alcoholic liver disease rather than heavy drinking, Alcohol and alcoholism 2004; 39(4): 336-9. 38. Hemieda F, Serag H, El-Baz E, Ramadan S. Therapeutic efficacy of licorice and/or cisplatin against diethylnitrosamine and carbon tetrachlo-ride-induced hepatocellular carcinoma in rats, J Am Sci2016;12:10-19. 39. Kumar V R. Protective potential of Hesperi-din against Diethylnitrosamine induced Hepatocar-cinogenesis in rats, Journal of Drug Discovery and Therapeutics 2014; 2.(15) 40. Hsu D-Z, Su S-B, Chien S-P, Chiang P-J, Li Y-H, Lo Y-J, Liu M-Y. Effect of sesame oil on ox-idative-stress-associated renal injury in endotoxe-mic rats: involvement of nitric oxide and proinflammatory cytokines, Shock 2005; 24(3): 276-80. 41. Ramesh B, Saravanan R, Pugalendi K. Influence of sesame oil on blood glucose, lipid peroxida-tion, and antioxidant status in streptozotocin diabetic rats, Journal of medicinal food 2005; 8(3): 377-81. 42. Yakubu M T, Bilbis L S, Lawal M, Akanji M A. Evaluation of selected parameters of rat liver and kidney function following repeated administration of yohimbine, Biokemistri 2003; 15(2): 50-6. 43. Ibrahim S S, Nassar N N. Diallyl sulfide protects against N-nitrosodiethylamine-induced liver tumorigenesis: role of aldose reductase, World Journal of Gastroenterology: WJG 2008; 14(40): 6145. 498 A. Mokh, D. Abdelhady, E. Ghazy, H. Aboumosalem, W. Goda 44. Andri^oiu C V, Andri^oiu V, Cuciureanu M, Nica-Badea D, Bibire N, Popa M. Effect of apither-apy products against carbon tetrachloride-induced toxicity in Wistar rats, Romanian journal of morphology and embryology= Revue roumaine de morphologie et embryologie 2014; 55(3): 835-47. 45. Mansour H H, Hafez H F, Fahmy N M. Si-lymarin modulates cisplatin-induced oxidative stress and hepatotoxicity in rats, Journal of biochemistry and molecular biology 2006; 39(6): 65662. 46. Zhang C-L, Zeng T, Zhao X-L, Yu L-H, Zhu Z-P, Xie K-Q. Protective effects of garlic oil on hepatocarcinoma induced by N-nitrosodiethylamine in rats, International journal of biological sciences 2012; 8(3): 363-7. 47. Singh R, Sharma J, Goyal P. Prophylactic role of Averrhoa carambola (star fruit) extract against chemically induced hepatocellular carcinoma in Swiss albino mice, Advances in pharmacological sciences 2014; 2014. 48. Droge W. Free radicals in the physiological control of cell function, Physiological reviews 2002; 82(1): 47-95. 49. Valko M, Leibfritz D, Moncol J, Cronin M T, Mazur M, Telser J. Free radicals and antioxidants in normal physiological functions and human disease, The international journal of biochemistry & cell biology 2007; 39(1): 44-84. 50. Kensler T W, Trush M A. Role of oxygen radicals in tumor promotion, Environmental muta-genesis 1984; 6(4): 593-616. 51. Qi Y, Chen X, Chan C y, Li D, Yuan C, Yu F, Lin M C, Yew D T, Kung H F, Lai L. Two-dimensional differential gel electrophoresis/analysis of diethylnitrosamine induced rat hepatocellular carcinoma, International journal of cancer 2008; 122(12): 2682-8. 52. Fukuda Y. Food chemical studies on the antioxidants in sesame seed, Nippon Shokuhin Kogyo Gakkaishi 1990; 37(6): 484-92. 53. Imamoto R, Okano J I, Sawada S, Fujise Y, Abe R, Murawaki Y. Null anticarcinogenic effect of silymarin on diethylmtrosamine-induced hepatocar-cinogenesis in rats, Experimental and therapeutic medicine 2014; 7(1): 31-8. 54. Rajan B, Ravikumar R, Premkumar T, Devaki T. Carvacrol attenuates N-nitrosodiethyla-mine induced liver injury in experimental Wistar rats, Food Science and Human Wellness 2015; 4(2): 66-74. 55. Rodriguez-Antona C, Ingelman-Sundberg M. Cytochrome P450 pharmacogenetics and cancer, Oncogene 2006; 25(11): 1679-84. 56. Rooseboom M, Commandeur J N, Ver-meulen N P. Enzyme-catalyzed activation of anticancer prodrugs, Pharmacological reviews 2004; 56(1): 53-102. 57. Oyama T, Kagawa N, Kunugita N, Kitagawa K, Ogawa M, Yamaguchi T, Suzuki R, Kinaga T, Yashima Y, Ozaki S. Expression of cytochrome P450 in tumor tissues and its association with cancer development, Front Biosci 2004; 9: 1967-76. 58. Gokbulut C. Sesame oil: potential interaction with P450 isozymes, Journal of Pharmacology and Toxicology 2010; 5(8): 469-72. 59. Zhu H-Z, Ruan Y-B, Wu Z-B, Zhang C-M. Kupffer cell and apoptosis in experimental HCC, World journal of gastroenterology 2000; 6(3): 405. 60. Hsieh P F, Hou C-W, Yao P-W, Wu S-P, Peng Y-F, Shen M-L, Lin C-H, Chao Y-Y, Chang M-H, Jeng K-C. Sesamin ameliorates oxidative stress and mortality in kainic acid-induced status ep-ilepticus by inhibition of MAPK and COX-2 activation, Journal of neuroinflammation 2011; 8(1): 57. Slov Vet Res 2019; 56 (Suppl 22): 499-508 DOI 10.26873/SVR-787-2019 Original Research Article EFFECT OF SOME FEED-ADDITIVES ON THE GROWTH PERFORMANCE, PHYSIOLOGICAL RESPONSE AND HISTOPATHOLOGICAL CHANGES OF RABBITS SUBJECTED TO OCHRATOXIN-A FEED CONTAMINATION Maha S. A. Salama1, Wael A. M. Morsy1, Radi A. Mohamed*2, Samy A. El-Midany3 1Animal Production Research Institute, Kafr El-Sheikh branch, Ministry of Agriculture, Dokki-Egypt 2Department of Aquaculture, Faculty of Aquatic Sciences and Fisheries, Kafrelsheikh University, El-Geish Street, 33516 Kafrelsheikh, Egypt, 3Hygiene and Preventive Medicine Department, Faculty of Veterinary Medicine, Kafrelsheikh University, (P.O. 33516) Kafr El-Sheikh, Egypt Corresponding author, E-mail: r.mohamed.vet@gmail.com Abstract: This experiment aimed to evaluate the toxic influence of ochratoxin- A (OTA) feed contamination and the effectiveness of some feed-additives (Humic acids, Bio-Plus 2B, Bio-Mos) in detoxifying ochratoxicosis. Thirty five day old weaned Animal Production Research Institute (APRI) rabbits were selected and were allocated to four groups. Group 1 (control, OTA group): rabbits were fed basal diet (ochratoxin- A level: 19 ^g /kg diet). Group 2: rabbits were fed a basal diet containing 0.2 % humic acid. Group 3: rabbits were fed a basal diet containing 0.4 mg/kg diet Bacillus subtilis and Bacillus licheniformis (BioPlus 2B). Group 4: rabbits were fed a basal diet containing 0.1 % Manna oligosaccharides (Bio-Mos). The control group showed significantly reduced feed intake, daily weight gain and growth performance index. Furthermore, there was a significant increase in AST, ALT, malondialdehyde, creatinine, and urea. Moreover, Bio-Mos group revealed significantly increased serum Hemoglobin, PCV, RBCs, MCV and RBCs/ lymphocyte ratio, and significantly decreased serum SOD and CAT activities. Additionally, internal organs (liver, kidney and intestine) of groups received feed additives revealed less deteriorative change in histopathological investigations in comparison with the control group. In conclusion, supplementation of contaminated feed with ochratoxin-A by feed-additives improves feed intake and final body weight and reduces mortality rate of rabbits. In addition, it improves liver and kidney functions and reduces its pathological changes, and restores antioxidant to its normal level. Key words: rabbits; ochratoxin-A; growth performance; anti-oxidant Introduction Animal feed and feed ingredients are commonly contaminated with different types of my-cotoxins which produced by some fungal species (1). One of the most dangerous mycotoxins types is ochratoxin-A. It created by some species of Aspergillus and Pénicillium. Presence of ochratoxin-A (OTA) in animal feeds raises concerns in poultry, rabbits and livestock industry due to subclinical intoxications and poor growth in animals (2). Contamination of animal Received: January 2019 Accepted for publication: February 2019 500 M. Salama, W. Morsy, R. Mohamed, S. El-Midany feed with OTA is very come and its toxic consequences are focused on the internal organs in form of hypertrophy, increase weight and sever destruction in active cells (3). In case of rabbits and some other poultry, the most affected organ is kidney (39). However, the other organs (liver, intestine, muscle, bone, bone marrow, testes, ovaries and lymphatic organs) may also be influenced by OTA toxicity (4). (3) found that rabbits suffering from OTA toxicity showed lower consumption of feed, reduced weight gain, higher feed conversion ratio, lower profit gain and consequently reduced overall production of rabbit (5). Due to the severity of OTA toxicity, many trials were done to overcome its bad effects. Using of feed additives is a novel method used for reducing the destructive effects of OTA by reducing its absorption from animal intestine. (6). Humate is a substance originated from the decayed animal and pants in soil and contains many active ingredients such as humic acid, humus, fulvic acid, and some micro-minerals that have strong binding and absorption activity (7). Therefore, humic acid was used as therapeutic agents in the field of veterinary and human medicine for example as a coating agent in diarrhea treatment, antibacterial agent and immune stimulator (8). Some Bacillus species have a strong powerful capability in toxins removal especially in the field of food production (9). (10) found that most of toxins produced by sever pathogenic fungi are biologically controlled by using Bacillus subtilis. Moreover, the growth of Asper-gillus species could be inhibited under effect of Bacillus subtilis (11) and the aflatoxin produced by A. flavus and A. parasiticus and could be prevented under the effect of Bacillus stea-rothermophilus (12, 13). Another example for feed additives is manna oligosaccharides that produced by yeast (Saccharomyces cerevisiae) cell wall. It characterized by its strong binding capacity with microbes, chemical agents and toxins preventing its accumulation and absorption from intestine (14). The aim of this experiment was to evaluate the influence of OTA and the antitoxic efficacy of some feed-additives on feed utilization, performance, hemato-biochemical parameters and histopathological changes of APPRI growing rabbits. Materials and methods Chemical analysis of OTA in rabbit feed The feed was prepared to supply experimental animals with their basic requirements for the growth (Table 1) according to (15). Analysis of feed ochratoxin-A was performed using florometer- antibody column method in Vet. Med. Laboratory, Kafrelsheikh University, Egypt and the value of OTA in basal diet of rabbit was 19 pg/kg diet. Experimental design The experiment was conducted in a Sakha Station rabbits farm), Animal Production Research Institute, Agriculture Research Center, Egypt. Animals used in the experiment were weaned and 35-day old APRI-line rabbits. Each animal was housed in a separate box of a larger cage. Animals were arbitrarily allocated to four groups (20 rabbit each), two replicate each. For acclimatization, all rabbits were fed the same feed for a week before starting the experiment. At 35 days of age, each experimental group received its specific feed as follow: Group 1 (control): rabbits were fed only on basal diet (ochra-toxin- A level: 19 pg /kg diet). Group 2 (Humic acids): rabbits were fed a basal diet contain 0.2 % humic acids. Group 3 (PLUS): rabbits were fed a basal diet contain 0.4 mg/kg diet Bacillus subtilis and Bacillus licheniformis (Bio-Plus 2B). Group 4 (MOS): rabbits were fed a basal diet contain 0.1% Manna oligosaccharides (Bio-Mos) (8 - 11). Feed additives were mixed with pelleted feed daily by spraying of feed with molasses mixed with water as a binder for additives and then additives mixed carefully with feed. In case of group 1 (control, OTA group), feed was mixed only with molasses mixed with water. All groups received continuous supply of feed and water. Rabbits feed consumption was recorded on daily bases. Whereas, body weight (BW), weight gain (WG) and feed conversion ratio (FCR) were Effect of some feed-additives on the growth performance physiological response and histopathological 501 measured weekly. Mortality rate (%) was recorded along the experimental period. Economic efficiency (16), relative growth rate [(W2 - W1) x100]/ [1/2 (W2+W1)] and performance index [(final body weight (kg)/ feed conversion ratio) x 100] were calculated for each group (17).Whereas: W1is the initial weight, and W2 is the final body weight. Blood was aspirated from the marginal ear vein at the end of the experiment. Blood (3 ml) was aspirated and mixed gently in heparinized tubes. Whole blood was used for hematological assay. Plasma was obtained by centrifugation of 2 ml whole blood at 3000 rpm for 15 min. Then, plasma was stored at -20 °C until used for biochemical assay. At the end of experiment (91 day), six rabbits were randomly selected from group 1, 2, 3 and 4and were slaughtered using sharp knife to obtain internal organs (liver, kidney and intestine) for histopathological examination. The Ethical Committee, Faculty of Veterinary Medicine, University of Kafrelsheikh and the Agricultural Administrative Authority approved the conduct of this experiment. Hematological examination Heparinized whole blood was analyzed after collection by 2 hours for estimation of red blood cells (RBCs) count, packed cell volume (PCV), haemoglobin concentration, mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), white blood cells (WBCs) according to (18). On two clean microscope slides, thin blood smears were prepared from each blood sample. Slides were left to dry in room temperature. Slides were stained with a modified Wright's stain, and covered. One hundred cells were counted under x 100 lense and the number of neutrophils count, lymphocytes count, monocyte, eosinophil and basophil were calculated. Biochemical examination Plasma samples were used for calorimetric estimation of total protein (TP), globulin, albumin (ALB), triglyceride (TG), total cholesterol (TC) , HDL-C, LDL-C, VLDL-C, cholesterol/ HDL ratio, LDL/ HDL ratio, glucose, urea, creatinine (CREA), alanine aminotransferase (ALT), aspartate aminotransferase (AST) by using commercial kits (Bio-Diagnosis Co., Cairo, Egypt). Antioxidant parameters and lipid peroxida- tion biomarker Lipid peroxidation biomarker used in this study was malondialdehyde (MDA). The anti-oxidant indicators used in this study were superoxide dismutase (SOD) and catalase activity that were estimated as previously described (19), (20) and (21). Histopathological changes Kidneys, liver and intestine tissues specimens were fixed in 10% formalin immediately after slaughtering. The fixed tissues were paraffin embedded, sectioned (3 pm) and stained by hematoxylin and eosin (H&E) (22). Statistical analysis Data was tested for distribution normality. Data were analyzed using SAS® statistical system, Package v9.2, version 2002. Data were reported as means and SEM, compared by oneway ANOVA and the Duncan's multiple range tests was used as a post hoc test (23) except for mortality rate that was analyzed using Chi-square test. The level of significance was at (P<0.05). Results Feed consumption and rabbits performance Feeding rabbits on diet contaminated with OTA (control group) led to a significant (P<0.05) decrease in feed consumption, WG, final BW and performance index as compared to the other groups (Table 2). On the other hand, administration of MOS, plus and humic groups reduced the negative influence of OTA and significantly (P<0.05) enhanced feed consumption, final BW, relative growth rate, and performance index in comparison with group 1 (control). Rabbits in the control group showed higher FCR and mortality (30 %) rate than other groups (Table 2). 502 M. Salama, W. Morsy, R. Mohamed, S. El-Midany Biochemical parameters Although TP, ALB and globulin concentration showed no significant differences (Table 3), glucose concentration reached highest level significantly (P<0.05) in MOS and plus groups. While, the TG and TC recorded the lowest significant levels (P<0.05) in MOS group matched to other groups (Table 3). The LDL and VLDL show lower significant (P<0.05) differences in MOS group, while HDL shown higher significant different in Bio-Plus 2B (PLUS) group. The MOS and plus groups revealed a significant reduction (P<0.05) in blood serum creati-nine and urea compared to humic and control groups. Meanwhile, AST and ALT activities significantly (P<0.05) increased in control group compared to other groups (Table 3). Hematological parameters Supplementation of MOS and Bio-Plus 2B improved (P<0.05) blood hemoglobin, PCV, RBCs, MCV and RBCs/ lymphocyte ratio (Table 4). However, the MCH, WBCs, neutrophils and lymphocyte percentages were significantly (P<0.05) higher in the control group (Table 4). While, MCHC, monocyte and eosinophil revealed absence of significant effect of all groups. Lipid peroxidation and antioxidants enzymes activity Plasma MDA level was significantly higher in rabbits supplied with basal diet (control group) and was lowered following supplementation with MOS, PLUS and humic acid (Table 5). Plasma CAT and SOD activities were increased significantly (P<0.05) with MOS and PLUS supplementation compared to the control group. Histopathological changes Histopathological findings of the control group showed marked swelling of hepatocytes with granular vacuolated cytoplasm (Fig. 1a), tubular degeneration and necrosis of kidney (Fig. 1b) and intestinal sub-epithelial lympho-cytic infiltration (Fig. 1c). While, liver of the humic (Fig. 2a) fed rabbits showed slightly swollen hepatocytes with granular vacuolated cytoplasm, mild to moderate degree of renal tubular epithelium degeneration of kidney (Fig. 2b) and mild degree of intestinal villi atrophy associated with sub-epithelial infiltration of lymphocytes (Fig. 2c). Yet, in group 3 the liver showed normal hepatocytes (Fig. 3 a), only mild degree of renal tubular degeneration of the kidney (Fig. 3b) and slight degree of mononuclear inflammatory cell within the lamina propria and hyperplasia of the covering epithelium of intestine (Fig. 3c). Meanwhile, liver (Fig. 4a), kidneys and intestine (Fig. 4b, c), sections of the MOS group 4 showed nearly normal morphological appearances. Table 1 : Composition and chemical analysis of basal diet Ingredients % Chemical analysis (% as DM): % Berseem hay Barley grain Wheat brain Soybean meal (44% CP) Molasses Limestone Di-calcium phosphate Sodium chloride Mineral-vitamin premix(1) DL-Methionine Total 30.05 24.60 21.50 17.50 3.00 0.95 1.60 0.30 0.30 0.20 100 Dry matter (DM) Crude protein (CP) Organic matter (OM) Crude fiber (CF) Ether extract (EE) Digestable energy(DE, kcal/kg)(2) Calcium(2) Phosphorus(2) Methionine(2) Lysine (2) 85.81 17.36 91.42 12.37 2.230 2412 1.243 0.808 0.454 0.862 (1) One kilogram of mineral-vitamin premix provided: Vitamin A, 150,000 UI; Vitamin E, 100 mg; Vitamin K3, 21mg; Vitamin B1, 10 mg; VitaminB2, 40mg; Vitamin B6, 15mg; Pantothenic acid, 100 mg; Vitamin B12, 0.1mg; Niacin, 200 mg; Folic acid, 10mg; Biotin, 0.5mg; Choline chloride, 5000 mg; Fe, 0.3mg; Mn, 600 mg; Cu, 50 mg; Co, 2 mg; Se, 1mg; and Zn, 450mg Effect of some feed-additives on the growth performance physiological response and histopathological 503 Table 2: Effect of experimental diets on growth performance of growing APRI-line rabbits from 5 to 13 weeks of age Parameters Control Humic acids PLUS MOS SEM P-value Initial body weight (g) 674.6 671.7 672.9 676.2 11.53 0.9953 Final body weight (g) 2052.5d 2134.0c 2201.0b 2332.0a 20.75 0.0001 Daily weight gain (g) 24.6c 26.1b 27.3b 29.6a 0.421 0.0001 Feed intake (g/d) 83.9c 85.8bc 87.2ab 89.2a 0.824 0.0007 Feed conversion ratio 3.421a 3.295ab 3.199b 3.025c 0.057 0.0005 Relative growth rate 101.1c 104.3bc 106.3ab 110.1a 1.396 0.0018 Performance index (%) 60.3c 65.1bc 69.0b 77.4a 1.703 0.0001 Mortality rate (%) 30a 20b 10c 10c - - SEM = Standard error of means a, b, c, d, Means in the same row with different superscripts are significantly different (P<0.05) Table 3: Effect of experimental diets on some blood parameters of APRI-line rabbits Items Control Humic acids PLUS MOS SEM P-value Total protein (g /dl) 5.19 5.57 5.71 5.88 0.143 0.1921 Albumin (g /dl) 3.50 3.72 3.75 3.85 0.127 0.4375 Globulin (g /dl) 1.69 1.85 1.95 2.03 0.113 0.2178 Glucose (mg/ dl) 89.3b 93.0b 102.3ab 114.7a 4.631 0.0259 Triglycerides (mg/ dl) 97.3a 86.7a 86.0a 70.0b 3.844 0.0048 Cholesterol (mg /dl) 67.0a 53.0b 47.0bc 38.0c 3.055 0.0007 HDL (mg /dl) 13.9b 13.0b 18.0a 14.5b 0.888 0.0089 LDL (mg /dl) 26.3a 18.3b 17.2b 9.47c 2.210 0.0019 VLDL (mg /dl) 27.7a 21.3b 18.7bc 14.0c 2.517 0.0063 Kidney function: Creatinine (mg/ dl) 1.37a 1.27a 1.20ab 1.08b 0.061 0.0346 Urea (mg/ dl) 33.7a 31.3a 29.6ab 26.7b 1.167 0.0213 Liver function: AST (U/L) 125.3a 101.3b 83.3b 86.3b 6.960 0.0076 ALT (U/L) 71.0a 69.7a 62.3ab 51.3b 3.464 0.0138 SEM = Standard error of means, a, b, e, Means in the same row with different superscripts are significantly different (P<0.05) Table 4: Effect of experimental diets on blood hematological values of APRI-line rabbits Items Control Humic acids PLUS MOS SEM lvalue Hemoglobin (g/ dl) 10.5c 11.7b 12.1ab 12.4a 0.200 0.0005 PCV (1) (%) 32.7c 35.7bc 37.1b 38.8a 0.623 0.0007 RBCs (x106/ pl) 5.28b 5.49ab 5.60ab 6.20a 0.240 0.0748 MCV(2) (fl) 60.1c 61.3bc 63.8ab 66.1a 0.800 0.0030 MCH(3) (pg) 20.4a 19.6ab 19.3bc 18.5c 0.300 0.0152 MCHC(4) (g/ l) 32.3 32.0 32.1 31.5 0.384 0.4456 WBCs (x103/ pl) 9.23a 9.03a 7.67b 6.30c 0.351 0.0011 Neutrophils (%) 52.0a 51.7a 32.0b 35.7b 3.283 0.0016 Lymphocyte (%) 53.7a 44.3b 39.3bc 34.3c 2.333 0.0012 RBCs/ Lymphocyte ratio 0.10c 0.13bc 0.14b 0.18a 0.012 0.0041 Monocyte (%) 9.00 8.67 9.67 8.33 1.155 0.8640 Eosinophil (%) 5.33 5.33 4.67 3.67 0.333 0.1617 Basophil (%) 0 0 0 0 - - SEM = Standard error of means, a, b, e, means in the same row with different superscripts are significantly different (P<0.05).1 Packed cell volume, 2Mean corpuscular volume, 3Mean Corpuscular Hemoglobin 4Mean Corpuscular Hemoglobin Concentrations- 504 M. Salama, W. Morsy, R. Mohamed, S. El-Midany Table 5: Effect of experimental diets on anti-oxidant parameters of APRI-line rabbits Items Control Humic acids PLUS MOS SEM .P-value MDA (N/ mol) 2.45a 2.05ab 1.90ab 1.65b 0.250 0.0903 CAT (U/ l) 1.75b 1.87ab 2.05a 2.09a 0.087 0.0585 SOD (U/ ml) 17.5c 19.5bc 21.6ab 22.4a 0.722 0.0034 SEM = Standard error of means, a, b,.. ..e, Means in the same row with different superscripts are significantly different (P<0.05) i ïdfcH-'tyf ■ af? < Mtt /fy Vf yiyryr^ifffffffiT^lirr^*^ ^ ^ iSà a WrwifABTk 11 èilK lllâ iPÉC»^ v ss^uir1 Figure 1: Control group 1 showing (a) liver showing marked swelling of hepatocytes with granular vacuolated cytoplasm (arrow), (b) the kidney showing tubular degeneration and necrosis (arrow), and (c) the Intestine showing marked sub-epithelial lymphocytic infiltration. (H&E, X 200) f BTarxV -ri^r^^TJifilyfTri a L I ri C ems&x n.Wr. ... V-l ^ * a .'A, Js A s- 'j.nLPI . - ■ ■ gMMVKVn • 7 w/m' J¥vji iffii «fil ^WMwÊ flJÈ^WW ■PB SWlsISr '¡¡¡r wGmÊ. ^SsSWs ySSr.MVj&âiSkWtiiPm* Figure 2: Humic group 2 showing (a) liver showing swelling of hepatocytes and with granular vacuolated cytoplasm (arrow), (b) the kidney showing mild to moderate degree of renal tubular epithelium degeneration (arrow), and (c) the Intestine showing mild degree of intestinal villi atrophy associated with sub-epithelial infiltration of lymphocytes. (H&E, X 200) dTftffJJMMM tm MM/ M ' Mfm r ; JUkî^I M fié £&§ w>; t- m yjTvsr jU i„ • y, r, ¿A a «r® m MR* w'Jc r* H * ■//-'.?/ fcl kl fi \ JB t ^■'^mXÊm1 kWiWm*H ' Tli frJ'lm i* nblVr " M I(>,-..r. it' I Figure 3: Plus, group 3 showing (a) liver showing normal hepatocytes (arrow), (b) the kidney showing mild degree of renal tubular degeneration (arrow), and (c) the Intestine showing slight degree of mononuclear inflammatory cell within the lamina propria and hyperplasia of the covering epithelium. (H&E, X 200) Effect of some feed-additives on the growth performance physiological response and histopathological 505 mSKSSL¿¡¡¿mMM lMS- « M^ ro /I" \ /I i i V rvrAiin I / o i tiAnnil hAnotA/>\ t+ac orrotii Figure 4: MOS group 4 (a) normal hepatocytes arranged in cords around the portal area (b) the kidney showing normal renal glomeruli and tubules, and (c) the Intestine showing normal branched intestinal villi with normal pseudo stratified epithelium with goblet cells. (H&E, X 200) Discussion To Feeding rabbits on feed contain OTA reduced final BW, WG and daily feed intake and performance index observed in the control group. These findings are in agreement with earlier studies recording the adverse effects of OTA on feed utilization and performance (3). These adverse effects might be attributable to phenylalanine moiety that present in OTA, which inhibit synthesis of phenylalanine-t-RNA and consequently reduce protein synthesis (24). Furthermore, OTA reduce carbohydrate metabolism, mainly glucogenolysis through inhibition of DNA, RNA and protein synthesis. While, feed additives especially MOS improve feed consumption, WG, final BW and decrease FCR. These results agreed with (25) who observed that using of MOS increase the height of intestinal villi. Accordingly, the surface area of intestinal villi increased and the absorption capacity improved that may improve feed consumption and performance. Rabbits in control group showed higher levels ALT and AST. These findings are in concordance with the previous studies conducted by (26, 27). Higher level of ALT indicates changes in the hepatocyte membrane permeability due to the vascular congestion induced by OTA toxicity and finally hepatic destruction (28). While, higher level of AST secretion implies skeletal muscle destruction (29). In present study, the control group showed an increase in the concentration of creatinine and urea that may give an indication about kid- ney damage due to OTA toxicity. These findings are similar to that stated in rabbits and some domestic animals (26, 28). (30) reported that toxicity by OTA is a direct cause of ne-phrotoxicity. Furthermore, due to nephrotoxi-city urea and creatinine are accumulated in de-structed nephrons resulting in further damage and kidney failure. Supplementation of feed additives especially MOS and PLUS improved the reduced blood hemoglobin, PCV, RBCs, MCV and RBCs/ lymphocyte ratio those were elevated in the control group. These results are in consistence with (31) who reported internal bleeding, hemorrhages or bruising, stomach ulcers in ochratoxicosis rabbits that resulted in lower hemoglobin and RBCs. Control group appeared in exhausted condition due to the signs of anemia that may be due to increased anaerobic metabolism and reduced aerobic one. (32) reported that during OTA toxicity, rabbits showed lower RBCs count, haemoglobin content and PCV which might lead to an increase in anaerobic metabolism and decrease in oxygen supply to tissue and cells. Furthermore, RBCs cytotoxicity that may resulted from destruction of RBCs and may be occur in case of OTA toxicity (33). Glucose concentration reached the lowest level in the control group. This result agreed with (34) who reported ochratoxicosis induced hypoglycemia; this finding may be due to intestinal villi destruction and subsequently poor absorption. In addition, it may be due to destruction in the membrane of hepatocytes. On the other hand, kidney failure and nephrotoxicity may lead to sever loss of glucose while using 506 M. Salama, W. Morsy, R. Mohamed, S. El-Midany of feed additives protects the intestinal villi, hepatocytes membrane and kidney nephrons from destruction (35, 26). In some cases of OTA toxicity, low glucose levels may be the cause of nervous manifestations observed in rabbits before death (36). Triglycerides and cholesterol are significantly increased in control group compared to other groups.it may be due to destruction of hepatocytes and changes in metabolism of lipid which was prevented by using feed additives. (37) observed similar results in broiler subjected to ochratoxicosis. Rabbits in the control group showed high oxidative destruction and changes in the plasma biochemical levels. While, rabbits supplied with feed additives showed high resistance to the OTA induced oxidative damage. Toxicity with OTA causes destruction in liver, kidney, intestine and muscle cell membranes that may be due to the increased lipid peroxidation (38). Therefore, activities of antioxidant were investigated due to its protective action in form of superoxide anion production, which protect cell membranes from OTA induced destruction that may be due to the effect of feed additives (38). Kidney is the primary target organ for ochra-toxin-A (39). The control group showed kidney tubular degeneration and necrosis while, Kidney histopathological lesions included mild to moderate degree of renal tubular epithelium degeneration of plus and humic groups. Liver his-topathological lesions showed marked swelling of hepatocytes with granular vacuolated cytoplasm of control group. Meanwhile, intestine showing slight degree of mononuclear inflammatory cell within the lamina propria and hy-perplasia of the covering epithelium, and mild degree of intestinal villi atrophy associated with sub-epithelial infiltration of lymphocytes while control group marked sub-epithelial lym-phocytic infiltration. These indicate the feed additives improve liver, kidney and intestinal histology and function. The results of histo-pathological examination in kidney, intestine and liver confirmed by biochemical profile of examined rabbits were consistent with those reported by (40). Toxicity with OTA resulted in impaired feed utilization and performance of rabbits. In addition, it caused high mortality rate. Consequently, the outcomes of this study represented by higher relative growth rate and performance index in grouped received feed additives may be reflected on the rabbit's economic production. Therefore, using feed additives especially MOS might improve feed utilization, WG, FCR and performance resulting in high economic revenue. Conclusions Supplementation of contaminated feed with ochratoxin-A by feed-additives especially MOS improves feed intake and final body weight and reduces mortality rate of rabbits. In addition, it improves liver and kidney functions and reduces its pathological changes, and the antioxidant biomarkers. Acknowledgements The authors are highly thankful to the Department of Hygiene and Preventive Medicine, Faculty of Veterinary Medicine, Kafrelsheikh University, Egypt for carrying out this research work. Conflict of interest The authors declare that there is no conflict of interest. References 1. Bennett JW, Klich M. (2003) Mycotoxins. Clin Microbiol Rev 2003; 16: 497-516. 2. Zia MA, Rahman KU, Sheikh MA, Khan IA. Chemically treated strain improvement of Aspergillus niger for enhanced production of glucose oxidase. Int J Agric Biol2010; 12: 964-6. 3. Stoev SD. Studies on carcinogenic and toxic effects of ochratoxin A in chicks. Toxins 2010; 2:649-64. 4. Paterson RR, Lima N. Toxicology of mycotoxins. EXS 2010; 100: 31-63. 5. Greco MV, Pardo AG, Ludemann V, Martino PE, Pose GN. Mycoflora and natural incidence of selected mycotoxins in rabbit and chinchilla feeds. Sci World J 2012; ID956056: 1-6. 6. Ramos-Cormenzana A, Juarez-Jimenez B, Garcia-Pareja MP. Antimicrobial activity of olive Effect of some feed-additives on the growth performance physiological response and histopathological 507 mill waste-waters (alpechin) and biotransformed olive oil mill wastewater. Int Biodeterior Biodegradation 1996; 38(3-4): 283-90. 7. Islam KMS, Schumacher A, Gropp JM. Humic acid substances in animal agriculture. Pak. J. Nutr.2005; 4: 126-34. 8. Rath NC, Huff WE, Huff GR. Effects of humic acid on broiler chickens. Poult Sci 2006; 85: 410-4. 9. Patrcia C, Anna L, Dionz M. Prevention of ochratoxin A contamination of food and ochratoxin A detoxification by microorganisms - A review. Czech J Food Sci 2010; 28: 465-74. 10. Thakaew R, Niamsup H. Inhibitory activity of Bacillus subtilis BCC 6327 metabolites against growth of aflatoxigenic fungi isolated from bird chili powder. Int J Biosci Biochem Bioinform 2013; 3: 27-32. 11. Foldes T, Banhegyi I, Herpai L, Varga L, Szigeti J. Isolation of Bacillus strains from the rhi-zosphere of cereals and in-vitro screening for antagonism against phytopathogenic, food-borne pathogenic and spoilage microorganisms. J Appl Micro-biol 2000; 89: 840-6. 12. Faraj MK, Smith JE, Harran G. Aflatoxin biodegradation: effects of temperature and microbes. Mycol Res 1993: 97:1388-92. 13. Sommartya T. Peanut Disease. Bangkok: Kasetsart University Publishing 1997. 14. Devegowda G, Raju MVLN, Swamy M. Mycotoxins: noval solution in their counteraction. Foodstuff 1998: 7:12-5. 15. DE Blas C, Mateos GG. Feed formulation. In the Nutrition of the Rabbit (eds de Blas C. and Wiseman J.), Editor CABI Publishing 1998; 24153. 16. Raya AH, Abbas AM, Hussein MAA. Comparative studies on the efficiency of some plant protein sources to replace soybean meal in rations for broiler chicks performance and economic efficiency. J. Agric. Sci., Mansoura Univ.1991; 16: 2514-27. 17. North MO. Commercial Chicken. Production Annual. 2nd Edition, Production 6th Ed. Interstate Printers and Publishers. INC., USA 1981. 18. Jain NC. Essentials of Veterinary Hematol-ogy, Fifth ed. Lea and Fibiger, Philadelphia, USA 1993. 19. Beuge JA, Aust SD. 1978. Microsomal lipid peroxidation. In: In: Colowick, S.P., Kaplan, N.O. (Eds.), Methodes in Enymology 52. Academic Press, New York 1978;302-10. 20. Nishikimi M, Roa NA, Yogi K. The occurrence of superoxide anion in the reaction of reduced phenazine methosulfate and molecular oxygen. Biochem Biophys Res Commun 1972; 46:849-54. 21. Abdelhady D, El-Abasy M, Abou-Asa S, Elbialy Z, Shukry M, Hussein A, Saleh A, El-Magd M. The ameliorative effect of Aspergillus awamori on aflatoxin Bl-induced hepatic damage in rabbits, World Mycotoxin Journal 2017; 10(4): 363-73. 22. Bancroft JD, Gamble M. Theory and practice of histological techniques, 5th ed. Churchill Livingstone, Edinburgh, UK 2007; 800. 23. Duncan DB. Multiple range and multiple F-Test, Biometrics 1955; 11: 1-42. 24. Konrad I, Röschenthaler R. Inhibition of phenylalanine tRNA synthetase from B. subtilis by ochratoxin A. FEBS LeU.1977; 83: 341-7. 25. Mourao JL, Pinheiro V, Alves A, Guedes CM, Pinto L, Saavedra MJ, Spring P, Kocher A. Effect of mannan oligosaccharides on the performance, intestinal morphology and cecal fermentation of fattening rabbits. Anim. Feed Sci. Technol. 2006;126:107-20. 26. Kumar M, Dwivedi P, Sharma AK, Telang AG, Patil RD, Singh ND, Sanga M. Ochratoxin-A and citrinin induced biochemical changes in New Zealand White rabbits. Indian J. Vet. Pathol. 2007; 31: 135-9. 27. Kumar A, Jindal N, Shukla CL, Pal Y, Le-doux DR, Rottinghaus GE. Effect of ochratoxin A on Escherichia coli - challenged broiler chicks. Avian Dis 2003; 47: 415-24. 28. Agawane, S.B., Lonkar, P.S. Effect of probi-otic containing Saccharomyces on experimental ochratoxicosis in broilers: Hematobiochemical studies. J. Vet. Sci. 2004; 5: 359-67. 29. Thacker HL, Carlton WW. Ochratoxin-A mycotoxicosis in the guinea pigs. Fd. Contaqm. Toxicol.1977; 15: 563-74. 30. Pfohl-Leszkowicz1 A, Manderville RA. Ochratoxin A: An overview on toxicity and car-cinogenicity in animals and humans. Mol. Nutr. Food Res.2007; 51: 61-99. 31. Aziz NH, El-Aziz AMA, Omran RMA. Effects of T-2 mycotoxin on histopathological changes in rabbits. Biomed. Lett. 1995; 51: 271-81. 32. Shalini M. Studies on ochratoxin contamination and ochratoxicosis in rabbits. Ph.D. Thesis, Bhavnagar University, Bhavnagar, India, 1996. 33. Zofair SM, Shalini M, Verma RJ. Ochra-toxin induced hemolysis in rabbits. Indian J Exp Biol 1996; 34: 592-3. 34. Raina JS, Roy KS, Balwant S. Biochemical and Histochemical studies in experimental mycotoxicosis in chicks. Indian J. Anim. Sci. 1991; 61: 1276--81. 508 M. Salama, W. Morsy, R. Mohamed, S. El-Midany 35. Mir MS, Dwivedi P, Charan K. Ochratoxin - A induced acute toxicity in rabbits. Indian J. Vet. Pathol. 1999; 23: 8-13. 36. Mir MS, Dwivedi P. Clinical studies on experimental sub acute ochratoxicosis in rabbits. Indian J. Vet. Pathol. 2000; 24: 99-101. 37. Anitha N. Pathology of ochratoxicosis and its amelioration with herbal oil and toxin binder in broiler chicken. M.V.Sc. thesis. Univ. KVAFSU,Bangalore, 2007. 38. Amézqueta S, González-Peñas E, Murillo- Arbizu M, López de Cerain A. Ochratoxin A decontamination A review. Food Control 2009; 20:32633. 39. Bayman P, Baker JL. Ochratoxins: A global perspective. Mycopathologia 2006; 162(3): 21523. 40. Manoj K, Prabhaker D, Anil KS, Nittin DS, Rajendra DP. Ochratoxin A and citrinin nephrotox-icity in New Zealand White rabbits: an ultrastructural assessment. Mycopathologia 2007; 163: 2130. Slov Vet Res 2019; 56 (Suppl 22): 509-14 DOI 10.26873/SVR-788-2019 Original Research Article EFFECT OF USING CHICORY ROOTS POWDER AS A FAT REPLACER ON BEEF BURGER QUALITY Tamer El Zeny, Rowida Y. Essa*, Badia A. Bisar, S. M. Metwalli Food Technology Department, Faculty of Agriculture, Kafrelsheikh University, Egypt Corresponding author, E-mail: rowida.eisa@agr.kfs.edu.eg Abstract: Chicory roots powder is considered as a rich source of fiber and polyphones. It can be interfered in the formation of many functional foods that play an important role in maintaining human health from many diseases such as obesity and diabetes. So, this research was carried out to determine the chemical composition of the chicory roots and to evaluate assess the quality properties of reduced- fat burger as influenced with chicory roots powder. The prepared burger samples contained chicory roots powder as fat replacer with substitution ratio of 25, 50 and 75% of animal fat. Cooking quality and sensory evaluation were measured in burger samples. The results revealed that adding Chicory roots powder lead to an improvement in burger nutritional value and cooking properties. In burger contained chicory roots powder, there was an increment in cooking yield meanwhile, shrinkage and feeder number were decreased. Adding chicory roots powder to burger as a fat replacer does not cause any negative effects on its sensory properties. Key words: fat replacer; burger; dietary fiber Introduction Consumers now have good knowledge about their health and food. Therefore, healthy processed meat product must have low fat, cholesterol, and calories (1). Burger is considered as one of the highest popularity food in Egypt and all over the world (2). It has a high acceptability and consuming rates because it is a cheap quick meal (3). On the other hand, it has some harmful effect due to high content of saturated acids (20-30%) (4). Fat has an important role in burger increasing emulsion stability of meats as well as water holding capacity; decreasing loss during cooking process and improving organo-leptic characteristics (5). However, the presence of fats in meat products leads to a high content of cholesterol and saturated fatty acids (6). The increment of saturated fats intake levels causes harmful diseases such as, cardiovascular disease, stroke, obesity and cancer (7). Obesity is one of the most serious diseases in the world, especially as it is linked to other diseases such as heart disease and diabetes (8). According to the American Heart Association (2002), fat should be shared with about 15% to 30% of the total calories taken daily. WHO also recommended that saturated fat should not exceed 10% of daily supplemented calories (9, 11). Therefore, many studies have been conducted to produce healthy meat products and reducing the proportion of fat. On the other hand, it must be borne in mind that this may lead to some problems with the acceptance of the prod- Received: January 2019 Accepted for publication: February 2019 510 T. El Zeny, R. Y. Essa, B. A. Bisar, S. M. Metwalli uct, because fat is a main component that effects on meat product properties such as sensory attributes, texture and flavor (12, 13). Reducing the proportion of fat by adding substances that are based on non-meat proteins, carbohydrate and dietary fiber is a scientific way to solve this problem (14). Dietary fiber is a part of plant food that is not fully digested by digestive enzymes, and it is very important for human health. On the other hand, human consume dietary fiber less than recommended by the WHO (23-38 g/day) (15, 16). In processed foods, part of the fat is replaced by dietary fiber (17). In meat processing, crude fiber has good applications in minimizing formula cost cooking yield enhancement, and texture improvement (18). Several studies have shown the importance of fiber to human health, it helps to reduce cholesterol, also reduce high blood pressure and reduce the chances of colon cancer as well as obesity (19, 20). Cichorium intybus L. (chicory) is a Mediterranean plant species belonging to the Aster-aceae family. Cichorieae tribe includes approximately hundred genera and many hundreds species of which some genera are used as salad vegetables (21). On the other hand, the word 'Chicory'is likely to be derived from the Egyptian word 'Ctchorium'. All parts of this plant are pharmacologically useful due to the presence of a number of medicinally and nutritionally important compounds such as inulin, flavonoids, caffeic acid derivatives, terpenoids, sesquiter-pene, vitamins, steroids oils, lactones, volatile compounds, and coumarins, It possesses antibacterial, antioxidant and anti-inflammatory (22). There is not enough information about using chicory as a fat replacer in meat products; therefore, the main aim of this study was to evaluate addition of chicory powder at different levels as fat replacer on burger quality attributes. Materials and methods Chicory (Cichorium intybus) was obtained in January, 2017 from the local field of Kafr El-shiekh governorate, Egypt. The chicory roots were free of physical damage and injury of insects and fungi infection. Beef meat and other components used for burger preparation were obtained from local market at Kafr El-shiekh city, Egypt. Chemicals All chemicals and reagents used in this study were obtained from Sigma Aldrich Chemical Co. (St. Louis, M.O, USA). All other chemicals and solvents were of analytical grade. Preparation of Chicory roots powder The plant of chicory were transferred directly to the laboratory of food technology department, Faculty of Agriculture, Kafrelshiekh University then it was washed with tap water to removeremaining soil and other impurities. The chicory roots were cut into small pieces and dried at 40°C for 2 days in an air oven. The dried roots were crushed using a laboratory mill, then, sieved (100 meshes) and finally, the powder was stored in sealed bags at 4± 2°C (23). Burger preparation The beef burger was formulated to contain the following ingredients 80% red beef meat, 20% kidney fat, 18% (w/w) water (ice), 1.5% (w/w) salt, ground black pepper (0.3%), red pepper (0.2%) and cumin (0.2%) according to Aleson-Carbonell, Fernandez-Lopez (24). Aforementioned ingredients were used to prepare the control sample while 25, 50 and 75% of control fat content were replaced by chicory roots powder to prepare burger supplemented with chicory roots powder as a fat replacer. Meat burgers weighed approximately 30 g each. The beef burgers were stewed using an electrical grill (Genwex GW-066) at 220°C (the space between heat source and the samples was 4 cm) for 8 min (4 min for each side of beef burgers). Chemical analyses Gross chemical composition of chicory roots and burger was analyzed according to A.O.A.C. (25). Cooking yield Cooking weight x (%) = Raw weight 100 Raw diameter - Cooking diame-Shrinkage ter x (%) = Raw diameter 100 Effect of using chicory roots powder as a fat replacer on beef burger quality 511 Cooking loss (%) = ((Raw weight - Cooking weight)/ Raw weight)*100 Feeder num- Moisture content % ber = organic nonfat content % Where, % organic non fat = 100 - (fat % + ash content + moisture %) Sensory evaluation Sensory evaluation of twenty panelist have been assessment burger samples for their sensory properties (taste, color, odour, texture, tenderness and overall acceptability) using a he-donic scale of 1-10 according to the method of Badr and El-Waseif (28) Statistical analysis General linear model of SPSS (Ver. 16.0, 2007) was used to conduct ANOVA for determination of differences between means. The probability levels of P < 0.01and P < 0.05 were considered to be significant for statistical procedures. All measurements and trials were done in triplicate. Results and discussion Chemical composition of chicory roots powder Moisture and protein content of chicory roots powder were 6.84±0.89 and 9.01±0.93 (Table1) .On the other hand, data in Table (1) showed that, chicory roots had a high ash content (5.60%) and ether extract (1.60%), crude fiber (5.92%) and antioxidants (78.02). These results in the same trend as the results obtained previously by (29). From the previous by results obtained, one can record that chicory roots could be considered as a good source of fiber, so it can be used as an alternative ingredient to fats in many processed foods. Chemical composition of prepared beef burger Table (2) showed that chemical composition of the cooked burger formulated with chicory roots powder, the chemical analysis of cooked burger showed that the percentage of moisture, protein, ash, crude fiber and available carbohydrates content were increased by increasing the amounts of chicory roots powder replacement in the burger. The increment of moisture content may be due to the capability of chicory roots powder rich with fiber to hold more water via preparation and cooking process. Meanwhile, the increment in other constituents may be a reflection of the quantity of these constituents in chicory roots. On the other hand, the data in the same table revealed that ether extract content values in cooked burger were decreased significantly with chicory roots powder addition (p<0.05) in comparison with the control sample. Maximum ether extract content percentage was noticed in control sample while, treatment contained (CRP) with 75% of animal fat showed the minimum percent. These obtained results were in harmony with those reported by Gok, Akkaya (7), Kihn99eker and Kurt (26) and Yousefi, Zeynali (27) who stated that beef burger integrated with different types of fat replacers were highly in some constituents such as moisture, ash, protein, fiber and available carbohydrates contents and lower in fat than in the control group. Burger cooking properties Moreover, data in Table (3) revealed that burger samples which replaced by chicory roots have cooking loss percentages lower than control. This decrement is due to the ability of chicory roots fiber to hold a large amount of water. There was a significant decrement in the loss via cooking process as a function of the increment in fat substitution level with chicory roots. Also, the results declared that adding chicory roots showed a positive influence on burger cooking yield. These results are in agreement with Kassem and Emara (30) and Namir, Siliha (31) who stated that there was a decrement in the cooking loss values of low fat burger when the levels of high fiber substances was increased. Preventing shrinkage considered as one of the most important factors to maintain the quality levels of burgers because some consumers related to shrinkage and adding a high amount of water. Control beef burger sample had a high percentage of shrinkage after cooking process in a comparison with burger integrated with 512 T. El Zeny, R. Y. Essa, B. A. Bisar, S. M. Metwalli chicory roots powder. These results are in conformity with the finding stated by Namir, Siliha (31). Feeder number is applied to assess the meat products physical characteristics. Feeder number was 0.70 for control samples, which decreased gradually with the increment of chicory roots powder level. It was for all laboratory samples was lower than 4.0. as stated by Pearson (32) who recorded that feeder number in good products should be lower than 4.0. Sensory evaluation In the present study, the sensory evaluation of cooked burgers containing Chicory roots powder with 25%, 50% and 75% of control sample fat were recorded in Table (4). Concerning the data in Table (4), one can be noticed that there were a slightly difference be- Table 1: Gross chemical composition of chicory roc tween control sample and that contained chicory roots powder with percentages of 25%, 50% and 75% of animal fat for all sensory characteristics. While the sensory scores of burger contained date chicory roots powder with ratio of 75% of fat were low compared with control sample, however they were in the acceptable limits (more than 6). Conclusion Chicory roots powder is considered as a rich source of fiber so that it can be used as fat replace in beef burger. Also, this study revealed that substituting 75% of animal fat in burger with chicory roots powder showed manufacture no negative effects on physical and cooking quality of processed burger. powder (% on dry weight basis) Component Chicory roots powder Moisture (%) 6.84±0.89 Protein (%) 9.01±0.93 Ash (%) 5.60 Ether extract (%) 1.60 Crude fiber (%) 5.92 Table 2: Proximate chemical composition of burger with different concentrations of chicory roots powder as a fat replacer (on dry weight basis) ^\^Treatments Component% Cooked burger Control CRP 25% of fat CRP 50% of fat CRP 75% of fat Moisture 32.09b 30.15d 31.33c 35.12 Crude protein 26.17d 28.00c 28.27b 28.52 Ether extract 20.01a 16.54b 11.97c 7.63d Ash 1.97c 1.99c 2.10b 2.19a Crude fiber 1.23d 2.98c 3.78b 4.21a available carbohydrates 50.62c 50.49cd 53.88b 57.451 CRP means chicory roots powder, Values followed by the same letter in the same column are not significantly different at P < 0.05. a a a Effect of using chicory roots powder as a fat replacer on beef burger quality 513 Table 3: Cooking properties of burger with different concentrations of chicory roots powder as a fat replacer Treatments Control CRP 25% of CRP CRP 75% of Properties fat 50% of fat fat Cooking yield (%) 47.14d 53.08c 57.82b 61.90a Cooking loss (%) 52.46a 46.92b 42.18c 37.70d Shrinkage (%) 25.15a 21.86b 18.89c 15.89d Feder number 0.70a 0.59b 0.57b 0.64b CRP means chicory roots powder Values followed by the same letter in the same row are not significantly different at P < 0.05. Table 4: Effect of Chicory roots powder percentage as a fat replacer on the sensory properties of burger* Treatments Control CRP 25% of fat CRP CRP 75% of fat Sensory 50% of fat Taste 8.0a 7.23b 7.05b 6.99bc Colour 8.04a 7.20b 7.11c 6.95c Odour 8.13a 7.14b 7.09c 6.96cd Texture 8.12a 7.10b 7.07c 6.88d Tenderness 8.21a 7.05b 7.01b 6.70c Total acceptability 8.1a 7.14b 7.06c 6.89d *All data are the mean±SD of twenty replicates. Mean followed by different letters in the same row differs significantly (P<0.05) References 1. Pinero M, Parra K, Huerta-Leidenz N, De Moreno LA, Ferrer M, Araujo S, Barboza Y. Effect of oat's soluble fibre (P-glucan) as a fat replacer on physical, chemical, microbiological and sensory properties of low-fat beef patties. Meat science 2008; 80(3): 675-80. 2. Eldemery ME. Effect orange albedo as a new source of dietary fiber on characteristics of beef burger. In The 5th Arab and 2nd International Annual Scientific Conference on: Recent Trends of Developing Institutional and Academic Performance in Higher Specific Education Institutions in Egypt and Arab World. Mansoura University-Egypt 2010. 3. Colmenero FJ. Relevant factors in strategies for fat reduction in meat products. Trends in Food Science & Technology 2000; 11(2): 56-66. 4. Selani MM, Shirado GA, Margiotta GB, Rasera ML, Marabesi AC, Piedade SM, Carmen JC, Solange GC. Pineapple by-product and canola oil as partial fat replacers in low-fat beef burger: Effects on oxidative stability, cholesterol content and fatty acid profile. Meat science 2016; 115: 9-15. 5. Rather SA, Masoodi F, Akhter R, Gani A, Wani S, Malik A. Xanthan gum as a fat replacer in goshtaba-a traditional meat product of India: effects on quality and oxidative stability. Journal of food science and technology 2015; 52(12): 8104-12. 6. Pappa I, Bloukas J, Arvanitoyannis I. Optimization of salt, olive oil and pectin level for low-fat frankfurters produced by replacing pork backfat with olive oil. Meat Science 2000; 56(1): 81-8. 7. Gok V, Akkaya L, Obuz E, Bulut S. Effect of ground poppy seed as a fat replacer on meat burgers. Meat science 2011; 89(4): 400--4. 8. Cobb LK, Appel LJ, Franco M, Jones-Smith JC, Nur A, Anderson CA. The relationship of the local food environment with obesity: a systematic review of methods, study quality, and results. Obesity 2015; 23(7): 1331-44. 9. Missmer SA, Chavarro JE, Malspeis S, Ber-tone-Johnson ER, Hornstein MD, Spiegelman D, Robert LB, Walter CW, Susan EH. A prospective study of dietary fat consumption and endometriosis risk. Human Reproduction 2010; 25(6): 1528-35. 10. Kratz M, Baars T, Guyenet S. The relationship between high-fat dairy consumption and obesity, cardiovascular, and metabolic disease. European journal of nutrition 2013; 52(1): 1-24. 11. Sullivan EL, Nousen EK, Chamlou KA. Maternal high fat diet consumption during the perinatal period programs offspring behavior. Physiology & behavior 2014; 123: 236-42. 12. Weiss J, Gibis M, Schuh V, Salminen H. Advances in ingredient and processing systems for meat and meat products. Meat science 2010; 86(1): 196-213. 514 T. El Zeny, R. Y. Essa, B. A. Bisar, S. M. Metwalli 13. Youssef M, Barbut S. Fat reduction in comminuted meat products-effects of beef fat, regular and pre-emulsified canola oil. Meat Science 2011; 87(4): 356-60. 14. Brewer MS. Reducing the fat content in ground beef without sacrificing quality: A review. Meat Science 2012; 91(4): 385-95. 15. Ng S, Tan C, Lai O, Long K, Mirhosseini H. Extraction and characterization of dietary fiber from coconut residue. J Food Agric Environ 2010; 8(2): 172-7. 16. McGill R, Anwar E, Orton L, Bromley H, Lloyd-Williams F, O'Flaherty M, Taylor-Robinson D, Guzman-Castillo M, Gillespie D, Moreira P, Allen K. Are interventions to promote healthy eating equally effective for all? Systematic review of socioeconomic inequalities in impact. BMC public health 2015; 15(1): 457. 17. Verma AK, Banerjee R. Dietary fibre as functional ingredient in meat products: a novel approach for healthy living—a review. Journal of food science and technology 2010; 47(3): 247-57. 18. Choe JH, Kim HY, Lee J-M, Kim YJ, Kim CJ. Quality of frankfurter-type sausages with added pig skin and wheat fiber mixture as fat replacers. Meat science 2013; 93(4): 849-54. 19. Slavin J. Fiber and prebiotics: mechanisms and health benefits. Nutrients 2013; 5(4): 1417--35. 20. Essa, RY and Elsebaie EM. Effect of using date pits powder as a fat replacer and anti-oxidative agent on beef burger quality. . Food and Dairy Sci., Mansoura University 2018; 9 (2): 91 -6. 21. Carazzone C, Mascherpa D, Gazzani G, Papetti A. Identification of phenolic constituents in red chicory salads (Cichorium intybus) by high-performance liquid chromatography with diode array detection and electrospray ionisation tandem mass spectrometry. Food Chemistry 2013; 138(2-3): 1062-71. 22. Minaiyan M, Ghannadi AR, Mahzouni P, Abed AR. Preventive effect of Cichorium intybus L. two extracts on cerulein-induced acute pancreatitis in mice. International journal of preventive medicine 2012; 3(5): 351-7. 23. Bouaziz MA, Amara WB, Attia H, Blecker C, Besbes S. Effect of the addition of defatted date seeds on wheat dough performance and bread quality. Journal of texture studies 2010; 41(4): 511-31. 24. Aleson-Carbonell L, Fernandez-Lopez J, Perez-Alvarez J, Kuri V. Characteristics of beef burger as influenced by various types of lemon albedo. Innovative Food Science & Emerging Technologies 2005; 6(2): 247-55. 25. A.O.A.C. Association of Official of Analytical Chemists, Official Methods of Analysis. 18th Ed., Pub. By the A.O.A.C.: Arlington, Virginia, 2220 USA; 2010. 26. Kilin^eker O, Kurt §. Effects of inulin, carrot and cellulose fibres on the properties of raw and fried chicken meatballs. South African Journal of Animal Science 2018; 48(1). 27. Yousefi N, Zeynali F, Alizadeh M. Optimization of low-fat meat hamburger formulation containing quince seed gum using response surface methodology. Journal of food science and technology 2018; 55(2): 598-4. 28. Badr S, El-Waseif M. Influence of Caper (Capparis spinosa L) Seeds Powder Addition as Source of Bioactive Phytochemicals on Quality Attributes and Shelf Life Extension of Beef Burger Patties. Middle East Journal 2017; 6(4): 1243-58. 29. Mona IM, Wafaa AA, Elgindy A. Chemical and technological studies on Chicory (Cichorium Intybus L) and its applications in some functional food. Journal of Advanced Agricultural Research 2009; 14(3): 735-42. 30. Kassem MG, Emara M. Quality and acceptability of value-added beef burger. World Journal of Dairy and Food Sciences 2010; 5(1): 14-20. 31. Namir M, Siliha H, Ramadan MF. Fiber pectin from tomato pomace: characteristics, functional properties and application in low-fat beef burger. Journal of Food Measurement and Characterization 2015; 9(3): 305-12. 32. Pearson D. The chemical analysis of Food National College of Food Technology. University Of Reading, Weybridge Surry T and A Churchill 1970. Slov Vet Res 2019; 56 (Suppl 22): 515-22 DOI 10.26873/SVR-789-2019 Original Research Article ASSESSTMENT OF MOULD CONTAMINATION OF TILAPIA NILOTICA AND Mugil cephalus FISH AND TRIALS TO REDUCE USING NATAMYCIN Nader Y. Mostafa1, Ghada A. K., Kirrella1, Hoda A. M. Aideia2, Jeena M. M. Abo Shaisha3* 1Food Control Department, Faculty of Veterinary Medicine, Kafrelsheikh University, 2Microbial Toxin Unit, Animal Health Research Institute, Dokki, Giza, 3Food Control Department, Animal Health Research Institute, Kafrelsheikh *Corresponding author, E-mail: jeena.aboshaisha@yahoo.com Abstract: Contamination of fish by fungi is considered one of the most serious causes of losses in aquaculture. Therefore, our study was firstly aimed to screen the fungal status of two commonly consumed fish species in Egypt, Tilapia nilotica and Mugil cephalus. Secondly, an experimental trial to investigate the antifungal effect of natamycin on Tilapia nilotica. A total of 60 fish samples including Tilapia nilotica and Mugil cephalus (30 of each) were randomly collected from various retail markets and shops at dissimilar sanitation levels at Kafrelsheikh Governorate, Egypt during the winter season, 2018. All samples were examined for fungal contamination. Tilapia nilotica and Mugil cephalus samples showed average mould counts of 3.63*102 and 1.65*102 CFU/g, respectively. Nine fungal species were isolated from two fish species. Seven and five species were isolated from Tilapia nilotica Mugil cephalus, respectively. The highest prevalent fungal species isolated from the two fish was Aspergillus flavus. Natamycin showed significant antifungal properties in a concentration- dependent manner. Thus, efficient hygienic handling, rapid cooling of fish can reduce the fungal contamination of fish. In addition, we highly recommend soaking or spraying fish with natamycin solution as an efficient strategy in reducing the fungal load of raw fish. Key words: Tilapia nilotica; Mugil cephalus; fungi, natamycin Introduction Fish is a significant source of animal protein, vitamins, minerals and omega-3-fatty acids, however, on the same time, fish is a rapidly decomposed food if not properly handled and stored. With the growing demand for fish as human food, fish farming is rapidly extending all over the world to recover the need for animal protein. Tilapia nilotica and Mugil cephalus are considered as two of the most popular fishes in Egypt. Fish diseases and hypoxic stress play an important role in lowering fish production in aquacultures (1). Source of fungal infection in aquaculture may be contributed to the utilization of contaminated feed as revealed by positive correlation between decomposition of such feed and fungal infections (2). Fungal diseases consider a serious cause of lowering Received: January 2019 Accepted for publication: February 2019 516 N. Mostafa, G. Kirrella, H. Aideia, J. Abo Shaisha fish production. Most fungi affecting fish considered opportunistic which infect fish when they are under stress or reduced immunity due to unfavorable ecological conditions. Fungal infections occur secondarily to viral or bacterial infections, or at mucosal loss resulting from trauma or severe handling (3). Intensive aquaculture conditions may cause transmission of fish diseases, especially fungal diseases, resulting in drastic economic losses. Assessment of fungal contamination of fish by using mould count could evaluates the quality and shelf life of fish (4). Contamination of fish by different fungal species as As-pergillus glaucus, A. sulpheus, A. ochraceus, A. candidus, A. versicolor, A. wentii, A. ory-zae, A. melleus, Mucor racemosus, Torula fulignea and Cladosporium herbarum was reported (5). Additionally Aspergilli observed to be the most widely recognized soil growth found in high numbers in water connected with biofilms and residue (6). Thus, there is a great need for proper handling, storage and preservation of this precious food source. Using food preservatives to prolong the shelf life of fish and to control fungal contamination is another alternative way for fish preservation. Natamycin is a natural poly-ene macrolide antifungal compound generated from fermentation of bacteria such as Strepto-myces natalensis. It had an antimicrobial effect, considerably fungicidal and active against all food spoilage moulds and yeasts with most strains. Despite its long-term use, resistance does not naturally occur unlike the chemical organic propionate and acid sorbate as preservative for which a number of resistant moulds and yeasts have been detected (7). Furthermore, natamycin is applied, in over 150 countries, in the food industry as a preservative (8). It can be added in a different ways: as an aqueous suspension (such as mixed into brine) sprayed on the product or into which the product is dipped, or in powdered form (along with an anticaking agent such as cellulose) sprinkled on or mixed into the product (9). Therefore, this study was conducted to check the mycological contamination of fresh Tilapia nilotica and Mugil cephalus and to find out the most suitable methods for decreasing of the fungal contamination of Tilapia nilotica using natamycin with different concentrations. Materials and methods Samples Collection Sixty random fish samples of Tilapia nilotica and Mugil cephalus (30 each) were collected from different retail markets and shops at various sanitation levels at Kafrelsheikh Gov-ernorate, Egypt. The collected samples were packed in a sterile polyethylene bags, closed and cooled in an insulated box contained crushed ice, then immediately transported to the laboratory for further examination. The fish samples were subjected to mycological examinations. Mycological examinations Preparation of samples All fish samples were prepared under complete aseptic conditions, 25 g of each sample cut-off, weighed and homogenized with 225 ml of 0.1% of sterile buffered peptone water (LAB104 , LAB M , UK) for 1-2 minutes at 2000 rpm using sterile homogenizer (type M-p3- 302, mechanic, precyzina, Poland), such homogenate represents the dilution of 10-1. The homogenate was kept for 5 minutes at room temperature. One ml from the original dilution (10-1) was aseptically transferred to another sterile tube containing 9 ml of sterile buffered peptone water 0.1% and further tenfold decimal serial dilutions were prepared (10). Determination of the total mould counts The total mould counts were determined by culturing duplicate plates of Sabouraud's dextrose agar media (Oxoid, Basingstoke, UK) supplemented with chloramphenicol 100 mg/L followed by dark incubation at 25°C for 5-7 days. During incubation time, the plates were examined daily for the fungal growth. Estimation of total mould was obtained by direct counting of the cultured agar plates (10). Assesstment of mould contamination of Tilapia nilotica and Mugil cephalus fish and trials to reduce 517 Identification of some isolated moulds Colonies of different fungal species inspected under microscope. Macroscopic and microscopic characteristics of the developing cultures were utilized (11, 12, 13 and 14). Experimental work In a trial for reduction of mould contamination of Tilapia nilotica fish using diluted solutions of natamycin (food grade- China), different concentrations were used under 0.5, 1and 2.5g/l. A total number of 40 Tilapia nilotica fish (weigh about 200 g/each) were divided to 4 groups (n=10 fish). The first group was soaked in sterile distilled water for 30 min and kept as a control (non- treated) group. The second, third and fourth groups were soaked with repeated shaking for 30 min at room temperature in 0.5g/l ,1g/l and 2.5 g/l natamy-cin solutions, respectively. Total mould count was conducted as mentioned before. Statistical analysis All results were expressed as (means ± SE). The values were analyzed statistically by Graph Pad Prism5 software. Results and discussion Evaluation of fungal status of Tilapia nilotica and Mugil cephalus Contamination of fish by fungi may be due to presence of microorganisms on their skin surfaces, intestine and in the gills. In many parts of Egypt and other developing countries, fish is sold in the fish markets in open air, kept in wooden boxes and covered with ice. Poor handling of the fish might lead to rupture of the intestinal tract and spoilage of the fish body with the fish intestinal contents. Thus, in the first part of this study, the fungal status of the examined fish species was investigated via estimation of total mould counts. During catching, handling, transportation and processing, contamination may occur which leads to introduction of pathogens into the fish meat. The sources of these pathogens may be from the surrounding environment (15). Many factors affect the microbiological characters of different sea food types like species differences, environment, methods of catching, on board, handling, fishing vessels, sanitation, processing, preservatives and packaging (16). Tilapia nilotica is commonly contaminated with mould and yeast than other types of fish which may fed on feed contaminated with moulds and yeasts (17). Also, the presence of high number of fish in limited areas encourages the growth, multiplication of mould and enhances spread of food borne outbreak infections. In the present study, Tilapia nilotica had higher total mould count than Mugil cephalus. The mean values of the total mould counts were 3.63x102 ± 8.75x10 and 1.65x102 ± 4.78x10 CFU/g in the examined Tilapia nilotica and Mugil cephalus, respectively (Table 1 and Fig. 1). This may be attributed to the higher moisture content of Tilapia nilotica than Mugil cephalus which may cause a higher contamination with mould (18). These results were in agreement with (19) who recorded higher mould counts in Tilapia nilotica sold in Ed Dueim, Sudan. Furthermore, some studies (20, 21) revealed higher mould counts in Tilapia nilotica and Mugil cephalus collected from different fish farms and natural water channels in Kafrelsheikh and Dakahlia Gover-norate, Egypt. This fungal contamination may be indicate inadequate sanitary measures performed starting from the point of catching. The conditions of the environment in the refrigerators, cooling boxes, anglers' hands and clothes are very suitable for the development of mould spores (22 and 23). Fungal contamination of fish may lead to their spoilage and production of mycotoxins with potential health hazards to human due to their carcinogenic effects, liver diseases and organ damage (24). Fungal species isolation from fish Moreover, to verify the most common fungi in both Tilapia nilotica and Mugil cephalus phenotypic identification of fungal isolates was carried out. Results in table (2), showed 9 fungal species were isolated from two fish species. Seven species was isolated from Tilapia nilotica while five species was isolated from Mugil cephalus. 518 N. Mostafa, G. Kirrella, H. Aideia, J. Abo Shaisha Figure (2) showed that Tilapia nilotica had the highest value of isolation percentage of Aspergillus flavus (86.95%) followed by A. niger (73.91%), however, the lowest values were obtained for A. ochraceus, A. parasiticus and Alternaria species as (8.69%). Mugil cephalus had A. flavus as the highest value of isolation percentage (78.94%) followed by Cladosporium species (47.36%), while the lowest values were obtained for A. niger (10.52%) (Fig. 3). These findings were in concurrence with detailed information by (2). It was found that aspergillomycosis in African Tilapia (Oreochromis species) is caused by A. flavus, A. japonicas and A. terreus (25). Also, we found Cladosporium species at a lower rate as described beforehand (26). The incidence of contamination of fish feed by Aspergillus species, especially A. flavus at high moisture levels lead to increase fungal growth during the storage period of feed at increased dampness levels (27-29). The polluted water supply, worker's hands and feeds, represent very important role on the health status of fish (30, 31). In agreement with our results, (32) isolated A. niger, A .flavus, A. versicolor, A. parasiti-cus, Rhizopus spp., Mucor spp., Phoma herbarum and Trichoderma hamatum from Tilapha nilotica. Moreover, (9) isolated 14 and 10 fungal species from Tilapia and Mullet, respectively. The highest was A. niger (100%) in Tilapia, while A. flavus was the highest (83.3%) in mullets. (33) also isolated 7 genera of mould from different types of fish. Variety in the results are most added to contrasts in land territory, temperature, dampness, and hygienic conditions (34). Therefore, the fungal contamination of fish could be due to incorrect sanitation during transportation, handling, catching, storage, marketing and manufacturing of fish (35 and36). Reduction of mould contamination in Tilap- ia nilotica fish using natamycin Fungal contamination of fish had several implications starting from organoleptic changes may lead to fish spoilage, which is an unacceptable metabolic process which causes foods to be rejectable and undesirable for human consumption due to changes in sensory characteristics. Fish is also very liable to spoilage due to enzymatic and chemical activities, the breakdown of protein, carbohydrates and fat of fish result in the progress of off-odor, off flavor and formation of slime which make the fish rejectable for human consumption. Thus, one major task of the food hygienists and microbiologists is to find ways to prolong the shelf life of fish and to decontaminate or decrease fungal contamination levels of fish. Thus, in the second part of this study, trials to control the outgrowth of moulds contamination of Tilapia nilotica fish using natamycin was evaluated. Natamycin could significantly reduce fungal contamination of Tilapia nilotica fish and extend the shelf life of fish in a concentration-dependent manner (Table 3 and Fig. 4). For instances, natamycin 2.5g/l significantly reduced total mould count (77.09%). Similarly, (37) also declared the anti-fungal activity of natamycin in Saloio cheese. Fungal infections in fish have expanded probably due to an absence of a strong anti-fungal therapy, and for the development of fungicidal resistant strains (38, 39) and the poisoning of the most relevant anti-fungal components (40). So, there is a mandatory need to look for new strategies against fungal contamination in fish (40 and 41). As natamycin is approved for surface treatment of cheese, sausages, yoghurt and many foods all over the world as a food additive and its use is considered to be safe, it could be used to control mould growth on fish. Assesstment of mould contamination of Tilapia nilotica and Mugil cephalus fish and trials to reduce 519 Table 1: Total mould count (cfu/g) of the examined fish samples (n=60) Fish type No.of positive samples No. % Min. Max. Mean±SE.M Tilapia nilotica Mugil cephalus 23 19 76.66 63.34 3.4x10 1.8x10 9.8x102 2.6x102 3.63x102±8.75x10 1.65x102±4.78x10 Table 2: Incidence of fungal species in examined fish samples Identified mould spp Tilapia nilotica Mugil cephalus No. of positive samples(23) % No. of positive samples(19) % A. flavus 20 86.95 15 78.94 A. niger 17 73.91 2 10.52 A.ochraceus 2 8.69 5 25.31 A.parasiticus 2 8.69 0 0 A.versicolor 0 0 6 31.57 Alternaria spp. 2 8.69 0 0 Cladosporium spp. 0 0 9 47.36 Rhizopus spp. 8 34.78 0 0 Mucor spp. 4 17.39 0 0 The table expressed as the percentage was calculated in relation to the number of positive examined fish samples Table 3: Effect of natamycin with different concentrations in Tilapia nilotica fish Concentration Total mould count Reduction% Control(nontreated) 3 ,63> < 102±8 .75 >10 0 Natamycin 0.5g/l 2 ,73> < 102±3 7> 10 24.83 Natamycin 1g/l 1 ,76> <102±1 45 >10 51.4 Natamycin 2.5g/l 0 ,83> < 102±3 71 >10 77.09 Reduction % = Control-After treatment/ Controlx100 Figure 1: Total mould count (cfu/g) of the examined samples 520 N. Mostafa, G. Kirrella, H. Aideia, J. Abo Shaisha ^ 90 i80 I 170 I *60 !: 1" a: I I Figure 2: Percent of the isolated mould from Tilapia nilotica fish Figure 3: Percent of the isolated mould from Mugil cephalus fish Figure 4: Effect of natamycin with different concentrations in Tilapia nilotica fish Assesstment of mould contamination of Tilapia nilotica and Mugil cephalus fish and trials to reduce 521 Conclusion Efficient hygienic handling, rapid cooling of fish, using of clean water during fish wash could reduce the initial fungal load of fish. It is critical that the proprietors of the fish's ranches and administrative specialists ought to adjust a superior technique to counteract fungal contamination of fishes amid developing periods and amid taking care of, preparing, transportation and processing. In addition, we highly recommend soaking or spraying of fish with natamycin solution as an efficient strategy for reduction of the fungal load of raw fish. Acknowledgments We would like to thank assistance provided from all staff members of Food control Department. Faculty of Veterinary Medicine, Kafrelsheikh University, Egypt, and Food control Department, at Animal Health Research Institute, Dokki, Giza and Kafrelsheikh branch, Egypt. Conflict of interest The authors declare that they have no conflict of interest. References 1. Mahfouz M, Hegazi M, El-Magd M, Kasem E. Metabolic and molecular responses in Nile tilapia, Oreochromis niloticus during short and prolonged hypoxia, Marine and Freshwater Behaviour and Physiology 2015; 48(5): 319-0. 2. Iqbal Z, Saleemi S. Isolation of pathogenic fungi from fresh water and commercial fish catlacatla .Hamilton.Sci Int (Lahore). 2013; 25:851-5. 3. Quiniou SMA, Bigler S, Clem LW, Bly JE. Effects of water temperature on mucous cell distribution in channel cat fish epidermis: a factor in winter saprolegniasis.Fish Shellfish Immunol. 1998; 8:1-11. 4. Grigorakis K, Taylor KDA, Alexis MN. Or-ganoleptic and volatile aroma compounds comparison of wild and cultured gilthead sea bream (Spa-rus aurata): sensory differences and possible chemical basis. Aquaculture. 2003; 225(2):109-19. 5. Kita,G., Some Japanese fungi Zentralbi Bac-teriol. 1914; 11:351:363. 6. Goncalves, A.B .,R.R.M. Paterson and N. Lima. Survey and significance of filamentous fun- giform tap water. Int. J. Hyg. Environ. Health. 2006; 209: 257-64. 7. Van Hee, P., Van Santen, L. C. A., & Fara-gher, J. M. U.S.2015; 9: 018,178. Washington, DC: U.S. Patent and Trademark Office. 8. Branen, A. Larry; Davidson, P. Michael; Salminen, Seppo; Thorngate, John. Food Additives. CRC Press.2001; pp:599-600. ISBN 9780824741709 9. Hashem, M. Isolation of mycotoxin-producing fungi from fishes growing in aquacultures. Research Journal of Microbiology, 2011; 6(12), 862. 10. American Public Health Association (APHA). Compendium of methods for the microbiological examination of food, 4th Ed. American Public Health Association, Washington,2001. D.C 11. Moubasher,A.H.,Soil Fungi in Qatar and other Arab countries. Center of Scientific and Applied Research, University of Qatar, Doha, Qatar, 1993. ISBN-13:9992121025, PP:566. 12. Raper, K.B. and D.I. Fennell. Thegenus As-pergillus.Williams and Wilkins Company, Baltimore, MD.1965. 13. Pitt JI. The genus penicellium and its telemorphic states Eupenicellium and Talaromy-ces. London: Academic Press, 1979. 14. Pitt JI, Hocking AD. Fungi and food spoil-age.1997; 2nd ed. London: Springer. 15. Jha P, Barat S, Nayak CR .Fish production, water quality and bacteriological parameters of koi carp ponds under live food and manure based management regimes. ZoolRes. 2008; 29(2) 165-73. 16. Ward DR, Baj ND. Factors affecting microbiological quality of sea foods. Food Technol. 1988; 42(3):85-9. 17. Yanong, R.P.E. Fungal diseases of fish. The Veterinary Clinics of North America: Exotic Animal Practice.2003;6(2):377-400. 18. Cyperian O. Optimal storage conditions for fresh farmed tilapia. M.Sc. Thesis. Department of Food Science andNutrition, Faculty of Science, University of Iceland. 19. Goja, A. M. Microbiological assessment of three types of fresh fish (Tilapia niloticus, Labeo niloticus and Hydrocynus spp.) sold in Ed Dueim, Sudan. New York Science Journal. University of Bakht Alruda, Ed Dueim, Sudan, 2013. 6, 49-54. 20. Aman, I.M., Ali, Y.E.S., Moustafa, N.Y., et al. Quality assessment of Tilapia nilotica and Mu-gil cephalus fish from Egypt. J Vet Med Allied Sci.2017.,1(2): 1-6.. 21. El-Moaty, A., Amany, M., Gad, T. M., El-Shafei, R. A., & Ibrahim, N. A. Microbiological 522 N. Mostafa, G. Kirrella, H. Aideia, J. Abo Shaisha assessment and the potential antimicrobial activities of nisin and\or garlic extract in commercial fish. Alexandria Journal for Veterinary Scienc-es,2018; 57(1):66-73. 22. Mizakova, A., Pipova, M., Turek, P. The occurrence of moulds in fermented raw meat products. Czech J. Food Sci.,2002. 3: 89-94. 23. Reij, M.W. Den Aantrekker, ED. Recontamination as a source of pathogens in processed foods International Journal of Food Microbiology, 2004.: 91, 1-11. 24. Darwish, W.S., Ikenaka, Y., Nakayama, S., Ishizuka, M. An overview on mycotoxin contamination of food: African Scenario. J. Vet. Med. Sci.2014. 76(6): 789-97. 25. Willoughby LG. Fungi and fish diseases. Stirling, UK:Pisces Press, 1994. 26. Hasballah AF, Abd El-Hamid HT. Detection of aflatoxins in water, sediment and fishes of a brackish waterlake in the Nile Delta, Egypt. J Adv Biol Biotechnol. 2016;6(3):1-6. 27. Almeida IF, Martins HM, Santos SM, Freitas MS, da Costa JM, D Almeida Bernardo FM. Mycobiota and aflatoxin B1 in feed for farmed sea bass, Dicentrarchuslabrax.Toxins (Basel). 2011;3:163-71. 28. Hassan EMF, El-Tabiy AA, Hassan HM. Assessment of ochratoxin A and aflatoxin B1 levels in the smoked fish with special reference to the moisture and sodium chloride content. Res J Mi-crobiol. 2011b; 6:813-25. 29. Kumar P, Mahato DK, Kamle M, Mohanta TK, Kang SG. Aflatoxins: a global concern for food safety, human health and their management. Front Microbiol.2017; 7:2170. 30. Hassan, A.A. and Abdel- Dayem, R. H. Prevalence of fungi and mycotoxins in fresh and salted fish. J. Egypt. Vet. Med. Assoc. ;64, (1): 1-11-and 59-68. 31. Hassan A.A.; Hammad, A.M; El Barawy, A.M. and Manal,A.H. Incidence of aflatoxigenic fungi in frozen and canned fishes and trials to inhibit aflatoxin production by use of some minor elements and lupinustermis seeds. Egypt. J. Appl. Sciences, 2007;22, (10B): 351-60. 32. Ali, E.H.,M.Hashem and M.B.AlSalahy. Pathogenicity and oxidative stress in Nile Tilapia caused by Aphanomyces l aevis and Phomaherba-rum isolated from farmed fish. Dis. Aquat. Org., 2011;94:17-28. 33. Atef, A. H., Manal, A. H., Howayda, M. E., Rasha, M. H. S., & Abdel-Dayem, R. H. Detection of aflatoxigenic moulds isolated from fish and their products and its public health significance. Nature and Science, 2011.9(9), 106-14. 34. Milicevic DR, Skrinjar M, Baltic T. Real and perceived risks for mycotoxin contamination in foods and feeds:challenges for food safety control. Toxins (Basel) 2010; 2:572-92. 35. Ward, D.R. and Baaji, N.J.: Factors affecting microbiological quality of sea foods.Food Technol,1998.42:58. 36. Hassan, A.A.: Detection of some mycotox-ins and mycotoxins producing fungi in both macro-and microenvironment of diseased animals. 7th Sci. Cong. Egyptian Society for Cattle Diseases. Assiut, Egypt. 2003; pp. 112 -19, 37. Fajardo, P., Martins, J. T., Fucinos, C., Pastrana, L., Teixeira, J. A., & Vicente, A. A. Evaluation of a chitosan-based edible film as carrier of natamycin to improve the storability of Saloio cheese. Journal of Food Engineering, 2010; 101: (4), 349-56. 38. Yanong RP. Fungal diseases of fish. Vet Clin North Am Exot Anim Pract. 2003; 6:377-400. 39. Nowakowska D, Gaj Z, Sobala W, Wilczynski J. Evaluation of susceptibility to anti-fungal agents of fungal strains isolated from pregnant women with diabetes and healthy pregnant women. Ginekol Pol. 2009; 80:274-9. 40. Gupta A.K., Tomas E., New antifungal agents. Dermatol Clin. 2003; 21: 565-76. 41. Wormley, F. L. J. r. and Perfect, J. R. Immunology of infection caused by Cryptococcus neoformans .Methods Mol Med.,2005. 118: 193-8. Slov Vet Res 2019; 56 (Suppl 22): 523-30 DOI 10.26873/SVR-790-2019 Original Research Article EFFECT OF SUPPLEMENTATION OF AROMATIC PLANTS OILS ON IMMUNITY, UDDER HEALTH AND MILK PRODUCTION OF FRIESIAN COWS Atef Y. Salem1*, Hassan G. El-Awady1, Mohamed A. Tag EL-Dein2, Dina A. Eisa 1Department of Animal Production, Faculty of Agriculture, Kafrelsheikh University 33516, Egypt, 2Animal Production Research Institute, Egypt Corresponding author, E-Mail: dinaeisa2030@gmail.com Abstract: Eight weeks prior to expected calving date, 32 Friesian cows were assigned according to parity to four homogenous groups (8 animals each). Cows were fed a basal ration alone as a control group 1 (CON G1), or basal ration supplemented with 25 mg black seed oil per kg body weight, (BSO G2), 11 mg chamomile flower oil (CFO G3), or 25 mg oregano leaves oil (OLO G4). Biochemical and hematological parameters were measured and there were within the normal ranges of cattle. Relative to the control group, aromatic plant oils significantly reduced plasma lipids and increased plasma concentration of IgG. Feed intake was similar among groups. Economic evaluations were in favor of BSO group. BSO significantly (P<0.05) improved daily milk yield (DMY), fat and protein, but reduced lactose content of milk. CFO increased (P<0.05) daily milk yield. Only fat-corrected DMY was increased with OLO supplementation. The three supplements reduced (P<0.05) milk electric conductivity (EC) and somatic cell count (SCC). Overall, our results indicated that supplementing diets of Friesian cows with aromatic plant oils, particularly black seed oil, improved milk yield, udder health, and some immune parameters. Key words: aromatic oils; cows; blood parameters; milk production and immunity Introduction Essential oils are a diverse group of secondary plant metabolites that contain naturally occurring volatile components that support smell and taste of plants (1). The volatile aromatic compounds have an oily appearance and they are extracted from plants (2). As a product, they are extracted from a plant or some parts through hydrodistillation, steam distillation, or dry distillation, without heating by a mechanical process (3). Essential oils have unique properties and offer a huge potential benefit for animal performance. One potential benefit that gained a lot attention is the antibacterial properties of these compounds. There is interest that essential oils could be a potential approach to improve feed efficiency, nutrient utilization, and animal health as an alternative to antibiotics. However, at this point we still need further research to provide information for practical feeding recommendations. Fatty acids, volatile oils and trace elements combination contributes black seed effectiveness. Little is known about the activity of volatile oil in N. sativa (4). Black seed oil (Ni-gella sativa) contains a substance called Ni-gellone which is a natural antioxidant that can modulate and regulate the early activation steps in the acquired immune response (5). Received: January 2019 Accepted for publication: February 2019 524 A. Salem, H. El-Awady, M. Tag EL-Dein, D. Eisa Oregano (Origanum vulgare L.) is an herb with high antioxidant capacity-compared to several other medicinal herbs (6, 7). Oregano oil can be defined as plant extract that contains mainly carvacrol and thymol, which are the major phenols constitute about 78-82% of essential oil. Moreover, it has antifungal (8) and antimicrobial properties (9) which make it an appropriate organic alternative for antibiotic as well as a promising feed additive in order to prevent meat lipid oxidation (10). Furthermore, 50% of essential oil of chamo-mile flowers (Chamomila recutita) contains alphabisabolol (trepenoid) and azulene these compounds showed anti-inflammatory, antibacterial, antimycotic and ulcer protective properties (11). Electrical conductivity (EC) of milk was indicator parameter for mastitis and is measured by the presence of ions (12). The current work was amid to effects of black seed, oregano and chamomile oil supplementation on blood parameters, immunity, udder health, and yield and composition of milk of lactating Friesian cows. Materials and methods This study was carried out at Sakha Animal Production Research Station, belonging to Animal Production Research Institute (APRI), Agricultural Research Center, Ministry of Agriculture, Egypt. Animals and experimental groups Thirty-two Friesian cows with live body weight (LBW) of 547.50±15.75 kg at eight weeks before parturition were classification into four groups (8 each) there to LBW and season of lactation (2nd to 8th) continued until four months postpartum. Cows fed a basal ration contained (on DM basis) 40% concentrate feed mixture (CFM), 25% fresh berseem (FB), 20% corn silage (CS) and 15% rice straw (RS) without any supplement in G1, which was served as control. The ration was supplemented with 25 mg black seeds oil (5) per kg LBW in G2 (13), 11 mg chamomile flower oil (14) per kg LBW in G3 (15) and 25 mg oregano leaves oil (16) per kg LBW in G4 (17). Composition of feedstuffs and calculated composition were show in Table (1). The CFM composed of 20% soybean meal, 24% wheat bran, 34% yellow corn, 12% rice bran, 5% linseed, 3% molasses, 0.5% premix and 1.5% common salt. Chemical analysis of samples of feedstuffs, were carried out according to the methods A.O.A.C (18). Management Cows housed in semi-open backyards under sheds and were fed their rations to cover their recommended requirements (19). The CFM was offered two parts at 8 a.m. and 4 p.m. daily. All cows had free access to fresh drinking water throughout the day. Samples of milk yield (MY) Animals were machine milked twice daily at 6 a.m. and 5 p.m. Daily milk yield was recorded individually and corrected for 4% fat contact (FCM): 4% FCM = 0.4 x MY (kg) + 15 x fat yield (kg) (20). Milk samples were taken from cows every two weeks and were analyzed for milk composition by Milko-Scan (model 133B). Milks of EC were measured for all quarter using "Dramtnski Electronic in agriculture, Mastitis detection, Italy". Furthermore, SCC analyzed milk sample with a Fossomatic 5000 "Foss Electric A S 69, Slangerupgade DK 3400 Hilleroed, Denmark Comp". Samples of blood Samples of blood were taken all cows before partition at days 45, 30 and 15 and after partition at 7th day. Blood samples were taken from the jugular vein by clean sterile needle in the clean dry plastic tube after 4 hours from the morning feeding in two parts. First part centrifuged at 4000 r.p.m for 15 min and stored at -20oC. Some boichemical parameters of blood were determined calorimetri-cally by using commercial kits (Diagnostic System Laboratories, Inc., USA). Second part used for determining hematological parameters in whole blood samples with EDTA (anticoagulant). Hematological analysis was performed by Medici Vet. (21). Hematological Effect of aromatic plants oils on immunity, udder health and milk production of Friesian cows 525 analysis (Medonic CA 620, Sweden) was performed within 1-2 hrs after samples collection. Hematological variables were red blood cells (RBC's), hemoglobin (HGB), hematocrit (HCT), platelet (PLT). Leucocyte variables were white blood cells (WBC's) and differential white cells (lymphocytes, monocytes and neutrophils). The concentration of immunoglobulin G (IgG) in the blood serum samples determined using the quantitative ELISA Bovine (IgG), ELISA Quantitation Kit, Bethyl laboratories, UK. Economic evaluation Economic efficiency calculated between the price of milk yield 4% FCM and the cost of daily feeding consumed. The prices in Egyptian pound (LE) per ton were 4750 LE for CFM, 320 LE for fresh berseem, 690 LE for corn silage and 250 LE for rice straw. Also, the prices per kg were 110 LE for black seed oil, 90 LE for chamomile oil, 90 LE for oregano oil and 5 LE for 4% FCM produced during year 2017. Statistical analysis The obtained data were statistically analyzed system using SAS (22). One way ANOVA was used. The significant differences among treatment groups were using Duncan's New Multiple Ranges Test (23). Table 1: Chemical composition and calculated feeding values of feed ingredients and basal ration. Item DM % Composition of DM % Calculated % OM CP CF EE NFE Ash TDN DCP CFM 91.01 91.48 16.52 8.37 2.46 64.13 8.52 68.9 12.8 Fresh berseem 20.01 91.19 15.63 24.54 2.88 48.21 8.83 64.4 12.6 Corn silage 33.60 90.97 9.45 17.32 2.55 61.65 9.03 69.9 8.4 Rice straw 90.30 83.59 2.56 31.79 1.09 48.15 16.41 48.2 0.5 Basal ration 61.67 90.12 12.79 17.72 2.37 57.24 9.88 64.87 10.03 DM: dry matter; OM: organic matter CP: crude protein; CF: crude fiber; EE: ether extract; NFE: nitrogen free extract; TDN: total digestible nutrients; DCP: digestive protein. CFM: Concentrate feed mixture Table 2: Blood serum biochemicals of cows supplemented with aromatic plant oils. Experimental groups Item G1 G2 G3 G4 Total protein (g/dl) 5.90±0.43b 7.55±0.32a 6.83±0.15ab 6.82±0.33ab Albumin (g/dl) 3.78±0.21a 3.54±0.11b 3.56±0.11ab 3.64±0.14a Globulin (g/dl) 2.50±0.16b 4.10±0.28b 3.27±0.21b 3.06±0.35b Total lipids (mg/dl) 221.0±0.46a 191.0±0.29b 184.0±0.43c 183.0±0.40c Cholesterol (mg/dl) 71.6±0.32a 68.9±0.17b 65.5±0.24c 64.0±0.21d Triglyceride (mg/dl) 93.0±0.21a 87.8±0.21b 84.8±0.18c 83.7±0.18c IgG (mg/ml) 37.4±0.93b 50.9±3.96a 46.5±3.70a 45.7±1.45a Values are represented as the mean ± SE. abcWithin-row different superscript letters denote significant difference (P<0.05). G1: basal diet. G2: basal diet supplemented with 25 mg black seed oil per kg body weight.G3: basal diet supplemented with 11 mg chamomile oil per kg body weight.G4: basal diet supplemented with 25 mg oregano oil per kg body weight. 526 A. Salem, H. El-Awady, M. Tag EL-Dein, D. Eisa Table 3: Blood hematological parameters of cows supplemented with aromatic plant oils Item Experimental groups G1 G2 G3 G4 WBC's (103/pl) 9.63±0.36c 13.7±0.89a 11.6±0.41b 11.2±0.92b Lymphocytes % 52.02±0.44c 58.1±0.88a 54.7±1.72b 52.7±1.72c Monocytes % 6.96±0.17b 8.93±0.58a 7.33±0.81ab 7.63±0.49b Neutrophils % 26.9±0.32c 31.5±1.84a 29.7±2.01b 28.2±2.25b PLT (103/pl) 108.0±2.72d 185.0±6.48a 168.0±6.75b 152.0±3.07c RBCs (106/pl) 6.34±0.32 6.46±0.19 6.32±0.40 6.10±0.36 Hgb (g/dl) 10.7±0.72 12.7±0.72 12.5±1.02 12.2±0.32 HCT (%) 28.9±0.53b 31.5±0.53a 30.1±0.87ab 29.5±0.80ab Values are represented as the mean±SE. a Different superscript letters within the same row denote significant difference (P<0.05). G1: basal diet. G2: basal diet supplemented with 25 mg black seed oil per kg body weight. G3: basal diet supplemented with 11 mg chamomile oil per kg body weight. G4: basal diet supplemented with 25 mg oregano oil per kg body weight. WBCs: PLT: platelets; RBCs: Hgb: Hemoglobin; HCT: hematocrit Table 4: Average actual yield, milk composition, electric conductivity and somatic cells count of cow's milk supplemented with aromatic plant oils Experimental groups Item G1 G2 G3 G4 Actual yield, kg/day 12.9±0.25b 15±0.35a 14.5±0.24a 14.1±0.30ab Fat corrected milk yield, kg/day 12.3±0.28c 15.9±0.34a 14.8±0.53ab 14.2±0.48b Fat% 3.66±0.11 b 4.42±0.13 a 4.15±0.19 ab 4.03±17 ab Protein, % 3.01±0.03 b 3.11±0.11 a 3.03±.05 b 3.06±.07 ab Lactose, % 4.85±0.09 a 4.19±0.12 b 4.20±0.14 ab 4.32±0.19 a Electrical conductivity, MS/ml 5.59±0.02a 5.21±0.11b 5.26±0.06b 5.26±0.07b Somatic cells count, 103/ml 393±3.65a 162±5.96c 178±2.61b 183±2.01b Values are represented as the mean±SE. Different superscript letters within the same row denote significant difference (P<0.05). G1: basal diet. G2: basal diet supplemented with 25 mg black seed oil per kg body weight.G3: basal diet supplemented with 11 mg chamomile oil per kg body weight.G4: basal diet supplemented with 25 mg oregano oil per kg body weight. Table 5: Feed intake and economic efficiency of cows in different experimental groups Experimental groups Item G1 G2 G3 G4 Total DM intake (kg/day) 17.64±0.32 17.73±0.35 17.77±0.34 17.75±0.41 Feed cost (LE/day) 51.69±0.56 51.94±0.54 52.06±0.57 52.00±0.50 Feed cost (LE/kg FCM) 4.24±0.03a 3.26±0.04c 3.53±0.07b 3.66±0.05b Total revenue (LE/day) 60.85±0.74c 79.65±0.68a 73.80±0.65b 71.05±0.75b Net revenue (LE/day) 9.16±0.10c 27.71±0.08a 21.74±0.11b 19.05±0.09b Economic efficiency 1.18±0.02b 1.53±0.03a 1.42±0.04ab 1.37±0.03ab Relative EE % 100.00±2.54b 129.66±1.89a 120.34±2.15ab 116.10±2.28ab Different superscript letters within the same row denote significant difference (P<0.05). G1: basal diet. G2: basal diet supplemented with 25 mg black seed oil per kg body weight.G3: basal diet supplemented with 11 mg chamomile oil per kg body weight.G4: basal diet supplemented with 25 mg oregano oil per kg body weight. Effect of aromatic plants oils on immunity, udder health and milk production of Friesian cows 527 Results and discussion Blood parameters Blood serum biochemical of cows in experimental groups Table (2) showed that G2 had the higher concentrations of total protein and globulin in serum (7.55- 4.10 g/dl) followed by G3 (6.83-3.27 g/dl) and G4 (6.823.06 g/dl), while G1 (5.90- 2.50g/dl) had the lowest values. However, G1 recorded the higher concentrations of albumin, total lipids, cholesterol and triglycerides, but G2 had the lowest albumin and G4 had the lowest total lipids, cholesterol and triglycerides concentrations. Results cleared that BSO supplementation increased the concentrations of total protein and globulin in serum. Oregano oil (OLO) decreased the concentrations of total lipids, cholesterol and triglycerides. The increasing of total protein and lipids in treatments may be due to that aromatic oil increased digestibility coefficients of crud protein and lipids. Blood total protein and their fractions can be used as indicators to evaluate the ruminant nutritional status and physiological changes (24). Moreover, immunoglobu-lin G (IgG) was higher significantly (P<0.05) in G2 (50.9 mg/ml), G3 (46.5 mg/ml) and G4 (45.7 mg/ml), whereas G1 (37.4mg/ml) had the lowest concentration which attributed to contains of minerals and vitamins, which may have a role in improvement immunoglobulin and immunity response (22). Concentrations of total protein, albumin, globulin and glucose in blood serum were higher of animals received chamomile flower, garlic and fenugreek seeds comparison with the control one (26). Plasma total protein, albumin, and plasma immunoglobulin tended to higher (P<0.05) in lactating buffaloes received 10 ml BSO/head/day (13). Polyherbal supplementation at the rate of200-250 mg/kg body weight improved immunity (27). Blood hematological parameters of Frie-sian cows in experimental groups are presented in Table (3). Cows in G2 supplemented with black seed oil showed highest (P<0.05) total WBC's count and relative percentages of lymphocytes, monocytes and neutrophils (13.7, 58.1, 8.93 and31.5 %), followed by G3 (11.6, 54.7, 7.33 and 29.7 %) and G4 (11.2, 52.7, 7.63, 28.2 %), whereas G1 (9.63, 52.02, 6.96 and 26.9 %) had the lowest values. The increase of immunoglobulin could be credited to B-lymphocyte stimulation (28). The WBCs are soldiers of body and their high counts may be due to increase or development of the immune systems of the animals and the lymphocytes constituted majority of WBCs counts and the cells increase with age in early life of animals (23). Also, G2 had the higher PLT count and HCT (185 103/|l and 31.5%) followed by G3 (168 103/|l and 30.1 %) and G4 (152 103/|l and 29.5%), but the lowest values were in G1 (108 103/|l and 28. 9%). While RBC's and Hgb was nearly similar for the different groups and not significantly affected by oil supplementation. It is clear that black seed oil (G2) was more effective in blood hematological parameters than chamomile oil (G3) and oregano oil (G4). Blood hematological profile reflects the physiological response of the cow to its external environments and internal with by feeding and feeds (29). Package cell value significantly increase (P>0.05) with 10 ml (BSO)/head/day of lactating buffaloes (13). Addition of medicinal plant CF significantly increased total RBC's and Hb concentration in lamb's blood (30). Black seed (Nigella sativa) contains minerals and vitamins which may have a vital role improving immune system and improve of formation and maturation of RBC's count (13, 25). Milk production Actual and 4% FCM yield (Kg/day) for cows of the experimental groups (Table 4) significantly (P<0.05) among groups, which have higher in G2 (15, 15.9) followed by G3 (14.5, 14.8) and G4 (14.1, 14.2), while were lower in G1 (12.9, 14.2). Superiority of G2, G3 and G4 compared to G1 in milk production and fat corrected milk as affected by the galactogoetic, which have the positive effect of the active components that aromatic oils may contain (30). Oregano leaf had little increase 3.5% FCM yield in dairy cattle (16). 528 A. Salem, H. El-Awady, M. Tag EL-Dein, D. Eisa Milk compositions of cows in experimental groups were presented in Table (4). Results indicated that G2 significantly higher (P<0.05) contents of fat and protein (4.42 and 3.11 %), followed by G3 (4.12 and 3.03 %) and G4 (4.03 and 3.06 %), but G1 (3.66 and 3.01%) had the lowest contents. The increasing of total protein and fat in milk due to the high contain of them on blood plasma; this declares the positive correlation between them. While, lactose mean was nearly similar in each group. In general, aromatic plant oils supplementation especially BSO improved milk composition of Friesian cows, which might due to improve the udder health. Treatment with NS was improvement milk yield and lactose in buffaloes than in the control and recorded slight increase in milk protein percentage (31). The use of CF and NSS 10 g/ewe/day as natural feed additives increased (P<0.05) total protein (TP). But, lactose not effects in CF or NSS supplementation (30). Udder health Aromatic plant oils led to significantly lowest (P<0.05) in EC and SCC than in control Table (4). Black seed oil (BSO) supplement showed the lowest values of electrical conductivity and somatic cells count. The EC of milk has a positive correlation with somatic cell count. The infection with clinical mastitis increased in Friesian cows with the increase of somatic cell count levels (33). The values of EC in our study ranged (5.59 - 5.21 MS/ml) and are within EC of healthy cow milk is 4.0-5.5 milisenses (MS)/cm (12, 34). EC is used as routine test for sub clinical mastitis diagnosis (35) The EC determined anions and cations (Na+ and Cl ) ions concentrations increased and K+ concentration and lactose decreased when the cows and buffalos were suffering with mastitis due to inflammation of udder hence increase the EC. Although EC is also affected by some other factors such as bread, lactation stage and milking interval (36). Polyherbal treated at the rate of 200-250 mg/kg live weight improved udder health (27). Economic efficiency Feed intake and economic efficiency of cows in experimental groups were presented in Table (5). Total DM intake from the different rations was nearly similar for the different groups indicating that aromatic plant oils supplementation don't have any adverse effect on feed intake. Also, the costs of feed intake were similar for all groups, whereas G2 only significantly (P<0.05) lower feed cost per kg 4% FCM compared to G1 had the higher value, while G3 and G4 were intermediate between them. However, G2 recorded significantly (P<0.05) highest values of total revenue of 4% fat corrected milk, net revenue, economic efficiency and relative economic efficiency, followed by G3 and G4, but G1 had the lowest values. The improvements in economic efficiency with aromatic plant oils supplementation might be due to the increase of both actual fat and 4% fat corrected milk yield. Black seed and pumpkin oils supplementation for growing rabbits didn't affected feed cost, but significantly (P<0.05) increased total and net revenue (37). Conclusion These results concluded that aromatic plant oils additives to diets of dairy cows improve production and milk composition, udder health and immunity as well as economic efficiency and that best results obtained was with black seeds oil. Disclosure statement The authors declare that they have no conflict of interest. References 1. Calsamiglia S, Busquet M, Cardozo PW, Castillejos L, Ferret A. Invited Review: Essential oils as modifiers of rumen microbial fermentation. J. Dairy Sci., 2007; 90: 2580-95. 2. Tassoul MD, Shaver RD. Effect of a mixture of supplemental dietary plant essential oils on performance of periparturient and early lactation dairy cows. J. Dairy Sci. 2009; 92: 1734-1740. 3. Miguel MG. Antioxidant and anti-inflammatory activities of essential oils: a short review. Molecules, 2010; 15: 9252-87. Effect of aromatic plants oils on immunity, udder health and milk production of Friesian cows 529 4. El-Kamali HH, Ahmad AH, Mohammad AS, Yahia AAM. Antibacterial properties of essentials oils from Nigella sativa. Fitoterapia, 1998; 69: 77-8. 5. Abd El-Hafeez AM, Ali MAE, Abu El-Hamd MA, Wahba AA And Kamla M. El-Sayed. Productive Performance, Immune Status And Metabolic Activity Of Suckling Bovine Calves Treated With Nigella Sativa Oil. Egypt. J. Agric. Res., 2014; 92 (4). 6. Dragland S, Senoo H, Wake K, Holte K, Blomhoff R. Several culinary and medicinal herbs are important sources of dietary antioxidants. J. Nutr., 2003; 133: 1286-90. 7. Matsuura H, Chiji H, Asakawa C, Amano M, Yoshihara T, Mizutani J. DPPH radical scavengers from dried leaves of oregano (Origanum vulgare). Biosci. Biotechnol. Biochem., 2003; 67: 2311-6. 8. Daouk R, Dagher SM, Sattout E. Antifungal activity of the essential of Driganum syriacum L. Journal of Food Protection, 1995; 58(10): 11479. 9. Dorman HJD, Deans SG. Antimicrobial agents from plants: antimicrobial activity of plant volatile oils. J. Appl. Microbiol., 2000; 88: 30816. 10. Simitzis PE, Deligeorgis SG, Bizelis JA, Dardamani A, Theodosiou I, Feggeros K. Effect of dietary oregano oil supplementation on lamb meat characteristics. Meat Sci, 2008; 79: 217-23. 11. Yarosh DB, Galvin JW, Nay SL, Pena AV, Canning MT, Brown DA. Anti-inflammatory activity in skin by biomimetic of evodia rutaecarpa extract from traditional Chinese medicine. J. Der-matol. Sci. 2006; 42: 13-21. 12. Ilie LI, Tudor L, Galis AM. The electrical conductivity of cattle milk and the possibility of mastitis diagnosis in Romania. Lucrari Stiintifice Medicina Veterinara, XLIII2010; (2), 220-7. 13. Khattab HM, El-Basiony AZ, Wakwak SM, Marwan BA. Immune response and productive performance of dairy buffaloes and their offspring supplemented with black seed oil. Iranian Journal of Applied Animal Science, 2011; 1(4): 227-34. 14. Salamon I, Ghanavati M, Khazaei H. Chamomile biodiversity and essential oil qualitative-quantitative characteristics in Egyptian production and Iranian landraces Emir. J. Food Agric. 2010. 22 (1): 59-64 15. Mokhber Dezfouli MR, Mohammadi HR. Nadalian MG, Nazem Bokaee Z, Hadjiakhoondi A, Nikbakht Borujeni GR, Tajik P, Jamshidi R.2011. Influence of parenteral administration of chamomile (Matricaria recutita L.) extract on co-lostral IgG absorption in neonatal calves. 16. Tekippe JA, Hristov AN, Heyler KS, Cas-sidy TW, Zheljazkov VD, Ferreira JFS, Karnati SK, Varga GA. Rumen fermentation and production effects of Origanum vulgare L. in lactating dairy cows. J. Dairy Sci., 2011; 94: 5065-79. 17. Yuan Liguo,Tong Hengmin,Shen Jianzhong. Study on Application of Oil origanum and Flavomycin in Dairy Production. 2004; Feed Industry. 18. AOAC, 2012 Association of Official Analytical Chemists, 19th Ed. Official Methods of Analysis, Washington, DC, USA. 19. NRC (Nutrient Requirements of Dairy Cattle).7th Rev. Ed. National Academy Press, Washington, 2001; DC. USA. 20. Gains WL. 1928. The energy basis of measuring milk yield in dairy cows. University of Lllinois. Agriculture Experiment Station. Bulletin. No. 308. 21. Drew P, Harles CRJS, Trevor B, John L. 2004. Oxford Handbook of Clinical Hematology. 2th Edition, Oxford University Press, USA. 22. SAS. 2004. Statistical Analysis System. SAS statistics. Guide release, version 8.00 TS level OOMO, SAS Institute Inc., Cary, NC. 23. Duncan DB. Multiple range and multiple F tests. Biometrics1955; 11:1-42. 24. Kummer N, Snigh UB, Verma DN. Effect of different levels of dietary protein and energy on growth of male buffalo calves. India J. Anim. Sci., 1981; 51:513. 25. Ismail AM, Sedki AA, Abdallah AG. Influence of black seed, garlic and onion supplementation on reproductive performance in rabbits. Egyptian. J. Agr. Res. 2003; 81, 1193-207. 26. Khattab HM, Abo El-Nor SAH , Kholif SM , El-Sayed HM , Abd El-Shaffy OH, Saada M. Effect of different additive sources on milk yield and composition of lactating buffaloes. Livestock Sci., 2010; 131(1): 8-14. 27. Sharma A, Prasad S, Singh Y, Bishisth R. Effect of polyherbal preparation supplementation on immunity and udder health of periparturient Karan-Fries crossbred dairy cows. J. Appl. Anim. Res., 2014; 42(2): 217-21. 28. Tizard I. Veterinary Immunology: An Introduction. 4th ed. London: WB Saunders Co, Philadelphia PA, El-Gaafrawy AM, Ahmed N, ElBanna MK, Ibrahim IL. 2000. Effect of Selenium and vitamin E supplementation on immue response and performance of Baladi calves. Proc. 530 A. Salem, H. El-Awady, M. Tag EL-Dein, D. Eisa Conf. Anim. Prod. In the 21th Century, Sakha, 1820 April 1992; 267-76. 29. Esonu BO, Fmenalom OO, Udedibie ABI, Herbert U, Ekpor CF, Okolie IC, Iheukwumere FC. Performance and blood chemistry of weaner pigs fed raw mucuna (velvet bean). Tropical animal Production Investigations.2001; 4: 49-54. 30. EL-Ghousein S. Safaa. Effect of some medicinal plants as feed additives on lactating Awassi ewe performance, milk composition, lamb growth and relevant blood items. Egyptian J. Anim. Prod., 2010; 47(1):37-49. 31. Abo El-Nor SAH, Khattab HM, Al-Alamy HA, Salem FA, Abdou MM. Effect of some medicinal plants seeds in the rations on the productive performance of lactating buffaloes. Int. J. Dairy Sci.2007; 2: 348-55. 32. Singh N, Kumari R, Yadav RS, Akbar MA, Sengupta BP. Effect of some commonly used galactagogue on milk production and bio-genic amines in buffaloes. Indian Vet. Med. J., 1991; 15: 20-4 33. Dina A Eisa. Evaluation of somatic cell count and it is relationships with milk yield traits and mastitis in Friesian cow.2007; MSc. Faculty of Agriculture, Kafer El-Shiekh University. 34. Hamann J, Gyodi P. Somatic cells and electrical conductivity in relation to milking frequency. Milchwissensch.2000; 55: 303-7. 35. Milner PKL, Page AW, Hillerton JE. Detection of clinical mastitis by changes in electrical conductivity of fore milk before visible changes in milk. Journal of Dairy Science.1996; 79: 83-6. 36. Kamal RM, Bayoumi MA, Abd El Aal SFA.Correlation between Some direct and indirect test for screen detection of subclinical mastitis. International food research journal, 2014; 21(3), 1249-54. 37. Gaafar HMA, Ayat A, Ragab, El-Reidy KFA. Effect of diet supplemented with pumpkin (Cucurbita moschata) and black seed (Nigella sativa) oils on performance of rabbits: 2- Productive and reproductive performance of does and their offspring. Report Opinion, 2014; 6(1):60-8. Slov Vet Res 2019; 56 (Suppl 22): 531-39 DOI 10.26873/SVR-791-2019 Original Research Article DIAGNOSIS AND PHYLOGENETIC ANALYSIS OF THE CIRCULATING PESTE DES PETITS RUMINANTS VIRUS IN Al-SHARQIA GOVERNATE Hend E. Elsheikh1*, Mamdouh F. El-Mekkawi1, Abd Allah A. Abou-zaid1, Amal M. Abd El Raof2 1Department of Animal Medicine (Infectious Disease), Faculty of Veterinary Medicine, Zagazig University, Zagazig 44511, Egypt, 2Tissue Culture Unit - Virology Department Animal Health Research Institute Dokki, El-Giza Corresponding author, E-mail: hendelsheikh91@yahoo.com Abstract: Peste des petits ruminants (PPR) is a Morbillivirus within the Paramyxoviridae family which characterized by highly contagious nature with high morbidity and mortality rates in domestic small ruminants. The aim of this study was to investigate an outbreak of P PRV in a flock of sheep and goats in Belbes city, AL-Sharqia governorate in 2018 by virus isolation and conventional RT-PCR. Phylogenetic analysis of N gene sequence of PPRV isolate. Also, compare it with other isolate from previous outbreak in Zagazig city, Al-Sharqia governorate in 2017 as a measurement of the infection status in Egypt. The current study applied on a flock of 55 small ruminants (42 goats and 13 sheep) in Belbes city which suspected to be infected by PPRV. All infected animals were not vaccinated against PPRV and randomly move from place to place. The morbidity rate was 100% and mortality rate was (23.8% in goats and 7.69% in sheep). Diseased animals suffered from fever, mucopurulent ocular discharge, nasal discharge, dyspnea, diarrhea, necrotic tissue and diphtheritic membrane in oral cavity. The ten samples (6 tissue scraping from oral lesions and 4 oculo-nasal swabs) were tested by conventional r everse transcription PCR which revealed 100% sensitivity compared to VI (virus isolation) 70%. Comparative of N gene sequence of both PPRV isolates revealed that homogenous population of PPR virus isolates up to 99%. Also, the PPRV isolate of the current study is related to Ethiopian strain and the previous Egyptian strains (Ismailia 3/ Egy/2010 isolate, El-Kalubeya isolate and Ismailia 1 -2014 isolate). The PPRV isolate of present study and all previous Egyptian isolates belong to lineage IV in phylogenetic analysis. The results emphasize the importance of molecular methods for a broader understanding of the epidemiology and development of the virus in the country. Key words: PPRV; Conventional RT- PCR; phylogenetic analysis; Al-Sharqia Introduction Sheep and goats contribute significantly to the economy of farmers in African and Asian nations. Sheep and goats are a source of meat, milk, and wool similarly to their speedy growth and reproduction. Poor man considers goats as cows in developing international locations (1). The primary isolation of PPRV became recorded in Egypt in 1987 (2). The causative agent Received: January 2019 Accepted for publication: February 2019 532 H. Elsheikh, M. El-Mekkawi, A. Abou-zaid, A. Abd El Raof of PPRV is a member of the genus morbillivirus underneath the circle of relatives of the para-myxoviridae (3). The genome of PPRV is a linear, single stranded, non-segmented, negative sense RNA encoding six structural proteins: nu-cleoprotein (N), phosphoprotein (P), matrix (M), fusion (F), haemagglutinin (H) and a large polymerase (L) and two nonstructural (C and V) proteins (4, 5). The incubation period of PPRV is ranged from 2-7 days (6) and the disease is manifested clinically by fever, presence of vesicles, ulcers on tongue and inside oral cavity, ocular discharge, leukopenia, profuse watery diarrhea, and respiratory manifestation (7, 8). PPRV cause high morbidity up to 100% and mortality up to 90% (6, 9). The most extensively used cell culture for virus isolation are Vero cells (10). The cytopathic effects (CPE) produced by PPRV in Vero cells are giant cells, cell rounding, formation of grape-like clusters, and small syncytia (10, 11). Reverse transcription polymerase chain reaction (RT-PCR) accompanied by nucleotide sequencing is the maximum diagnostic method used for PPRV identity (12, 13). Although PPRV is consider as one strain or serotype(14) , genetic type, based at the fusion (F) protein gene and the nucleoprotein (N) gene (15), has diagnosed four wonderful lineages (lineages I, II, III and IV) and is considered a powerful tool for the worldwide spread of virus. Lineage I is disbursed in West African strains in the 1970s and in Central African strains in those years; Lineage II in west Africa in Guinea, Ivory coast, and Burkina Faso strains; Lineage III in Sudan, east African, Oman and Yemen strains and lineage IV in The Arabian Peninsula, south east Asian, middle east, northern and central African strains (16) . Thus, the aim of this study was to investigate the outbreak of PPRV in a flock of sheep and goats in Belbes city, AL-Sharqia governorate in 2018 by virus isolation and conventional RT-PCR. Phylogenetic analysis of N gene sequence of PPRV isolate and compare it with o ther isolate from previous outbreak in Zagazig city, Al-Sharqia governorate in 2017 as a measu rement of the infection status in Egypt.. Materials and methods The experimental design was approved by Zagazig University's Animal Care and Use Committee (ZU-IACUC/2/F/19/2019). Field examination A flock of 55 small ruminants (42 goats and 13 sheep) in Belbes city In Al-Sharqia gov-ernate in August 2018 was suspected to be infected by PPRV. All infected animals were not vaccinated against PPRV. The case history showed that these infected animals were randomly move from place to place. Ten samples of clinically diseased animals (6 tissue scraping from oral lesions and 4 oculo-nasal swabs) were collected. Until laboratory testing, the samples were kept at-20 ° C. Samples preparation Preparation of oculo-nasal swabs (four samples) Oculo Nasal swabs were obtained on saline containing 10% pen-strep- amphotericin B to avoid bacterial and fungal contamination. Mixing well by pulse vortexing. The mixture was pipetted into a plain tube. Centrifugation at 2000 rpm for 10 minutes. The supernatant was pipetted into sterilized epindorf tube and stored at - 20°C till laboratory examination. Preparation of tissue scraping from oral lesions (six samples) Tissue samples were homogenized using tissue homogenizer. Nine milliliters of phosphate buffer saline was added to make 10 % tissue suspension. Centrifugation at 2000 rpm for 10 minutes was carried out and supernatant was taken into sterilized tube and stored at - 80°C till laboratory examination. Isolation of PPRV on tissue culture PPRV grown in Vero cell-culture supplied by Tissue Culture Unit in Animal Health Research Institute according to (17). The cells should be examined daily for 7-14 days for evidence of cytopathic effect (CPE). Infected cells developed a characteristic CPE consisting of retraction of the cell membrane from surrounding cells, and eventually rounding of cells. The cells with typical CPE were freeze thawed three Diagnosis and phylogenetic analysis of the circulating peste des petits ruminants virus in Al-Sharqia governate 533 times to take the virus. If cytopathic effect were not evident after 4-7 days of the third passage, the samples were declared negative. Purification of PPRVRNA It was carried out using QIAamp viral RNA minikit (Qiagen, Germany) according to manufacturer instructions. Conventional RT-PCR It was carried out for confirmation of PPRV in suspected diseased animals by detecting the viral nucleic acid using Qiagen one step RT-PCR kit (Qiagen, Germany) and primers directed to the highly conserved sequence of nu-cleoprotein gene of PPR virus as described by (12) and according to instructions of the manufacturer. PPRVisolates sequence PPRV, isolate of the current study was compared with PPRV isolate from mesenteric lymph node of dead carcass of goat within a flock infected by PPRV in Zagazig city, Al-Sharkia governorate in 2017 (18). The N gene specific PCR amplicons were sequenced and assessed for sequence variations. A purified RT-PCR product was sequenced in the forward direction on the automated DNA sequencer Applied Biosystems 3130 (ABI, 3130, USA). Use the Bigdye Terminator V3.1 cycle sequencing kit ready for reaction. (Per-kin-Elmer / Applied Biosystems, Foster City, CA), with Cat. No. 4336817. A BLAST ® (Basic Local Alignment Search Tool) (19) was initially performed to determine the identity of the sequence for GenBank accessions. The sequence reaction was done according to the manufacturer's instructions. Phylogenetic analysis A comparative sequence analysis was performed using the CLUSTAL W multiple sequence alignment program, version 1.83 of MegAlign of the Lasergene DNAStar software module Pairwise, which was designed by(20) and phylogenetic analyses were performed using maximum likelihood, neighboring joining and maximum parsimony in MEGA6 (21). Results Field examination Clinical examination of diseased animals showed fever, congested mucus membrane, normal lymph nodes, increase heart and respiratory rates. The clinical signs observed were mucopuru-lent ocular and nasal discharges, conjunctivitis, stomatitis, ulcers and diphtheritic membrane inside the oral cavity, dyspnea and diarrhea (Fig. 1). The morbidity rate was 100% and mortality was (23.8% in goats and 7.69% in sheep) (Table 1). Isolation of virus on Vero cell culture Out of 10 clinical samples, 7 (70%) samples showed CPE after three successive passage of virus on Vero cell culture as cells exhibited cell detachment, vacuolation and cell rounding (Fig. 2). Conventional RT-PCR The result of Conventional RT-PCR revealed all tested 10 samples (6 tissue scraping from oral lesions and 4 oculo-nasal swabs) were positive. (Table 2). PPRV Sequence and phylogenetic analysis Sequence analysis of the N gene of the current study (PPRV isolate from infected animals in Belbes City 2018) and isolate from the mes-enteric lymph node of dead goat carcass in a flock infected with PPRV in Zagazig City, Al-Sharkia governorate in 2017 showed that the two isolates shared a 99 percent homology. The percentage of our isolate identity to Ethiopian strain was 98% applied from gene bank. Furthermore, the nucleotide isolate identity to the previous Egyptian isolates (Ismailia 3/Egy/ 2010 isolate, El-Kalubeya isolate and Ismailia 1-2014 isolate) were 99%, 98% and 98% respectively (Fig. 3). The PPRV isolate of the present study belong to lineage IV in phylogenetic analysis. 534 H. Elsheikh, M. El-Mekkawi, A. Abou-zaid, A. Abd El Raof Table 1: The morbidity and mortality rates of infected animals with PPRV Speaches Total number of animals Number of diseased animals Number of dead animals Morbidity rate Mortality rate Goats Sheep Total 42 13 55 42 13 55 10 1 11 100% 100% 100% 23.81 7.69 20% Table 2: Percentage of positive samples in virus isolation and conventional RT-PCR Samples tissue scraping from oral lesions Number of Samples 6 Viral isolation Conventional RT-PCR Number of Percentages Number of Percentages +ve samples % +ve samples % 4 66.66% 6 100% Oculo-nasal swabs 75% 100% Total 10 70% 10 100% Figure 1: Diseased animals showed (A) Mucupurulant occular discharge. (B) Errosion and diphtheritic membrane of hard palate. (C) Necrotic tissue and ulceration of tongue. (D) Soiling of hind quarters revealing profuse watery diarrhea 4 3 4 7 Diagnosis and phylogenetic analysis of the circulating peste des petits ruminants virus in Al-Sharqia governate 535 Figure 2: (A) Control Vero cell culture, spindle shape of cells. (B) Cytopathic effect of PPRV on Vero cell culture showed cell detachment. (C) Cytopathic effect of PPRV on Vero cell culture showed cell rounding Figure 3: Phylogenetic relationship and comparative of PPR virus isolate of the current study (PPR 2018/goat Egypt ■) with other virus isolate, from previous study in Zagazig city 2017 (PPR 2017/goat Egypt A) based on partial sequences of N gene. Also, Neighbor-joining tree of PPRV N gene showing phylogenetic relationships of the PPRV isolates of present study with ather closely releated Egyption isolates available from GenBank 536 H. Elsheikh, M. El-Mekkawi, A. Abou-zaid, A. Abd El Raof Discussion Peste des petits ruminants (PPR) is a Mor-billivirus within the Paramyxoviridae family which characterized by highly contagious nature with high mortality rates of domestic small ruminants (22). The disease is manifested by sudden onset of depression, fever, mucopurulent ocular and nasal discharges, oral lesions, dyspnea, diarrhea and death (23). The Food and Agriculture Organization (FAO) reported that approximately 63 percent of small ruminants are highly susceptible to PPRV infection specially in South Africa, Central Asia and Southern Europe (24). In Egypt, PPR was first recorded in 1987(2). PPR has been diagnosed with polymerase chain reactions in various locations in Egypt over the past 10 years (13, 25, 26 and 27). Therefore, PPRV considered one of the most seriously contagious viral disease of small ruminant causing huge economic losses in Egypt. The present study applied on a flock of 55 small ruminants (42 goats and 13 sheep) in Belbes city in Al-Sharkia governorate in 2018, suspected to be infected with PPRV as infected animals showed fever, depression, off food, mucopurulent ocular discharge, mucopurulent nasal discharge, dyspnea, diarrhea, necrotic tissue and diphtheritic membrane in oral cavity. These symptoms agree with those described by (7, 8). The present study revealed that the morbidity rate was 100% in both goats and sheep and mortality rate was (23.8% in goats and 7.69% in sheep). The high mortality rate percentage in goats may be due to severe immunosuppression of infected goats (28) as PPRV downregulate CD46 (29), inhibit the leukocyte proliferation (30), and/or apoptosis of mononuclear cells in peripheral blood (31) .These finding were supported by the previous studies of (32) and (33) who observed that the PPRV symptoms in sheep were less severe than goats. Furthermore, mild infection in sheep has been reported in previous studies of (25, 27, 34, and 35). On the contrary, (36) observed significantly higher incidence rate of PPRV in sheep 39.1% than in goats 23%. The ten samples (6 tissue scraping from oral lesions and 4 oculo-nasal swabs) were tested by conventional reverse transcription PCR which revealed 100% sensitivity compared to VI (virus isolation) 70%. The CPE produced after three successive passage were cells detachment, cell rounding, and formation of grapelike clusters. Our result supported by(10, 11) who noted that (CPE) produced in Vero cells by PPRV consist of giant cells, cell rounding, formation of typical grape-like clusters, and formation of small syncytia. Despite low sensitivity, virus isolation is the most valuable method for detecting PPRV (37). Low-quality samples, poor transport, poor storage, aging cells and virus isolation in suboptimal cell culture (without the lymphocyte activation molecule (SLAM) that helps to isolate them) are predisposing factors for low VI sensitivity (38). Viral isolation also takes long time and requires further confirmation (39). This study reported that all tested 10 samples 100% were positive by conventional RT-PCR. These findings endorsed those obtained by (13) who found that the positive samples tested by conventional RT-PCR and real-time PCR were 90% and 100% respectively. (37, 40) observed that conventional RT-PCR could detect virus in eye swabs in experimentally infected goats four days after infection. PCR is the most favorable and highly sensitive tool for virus identification and molecular epidemiological studies among the various techniques used for PPRV detection (39). Phylogenetic analysis of N gene sequence is the key method for differentiation between circulating PPRV lineages and help in molecular epidemiology of the disease (41). The comparative of N gene sequence of the current study (PPRV isolate from Belbes City 2018) and the isolate from the previous study in Zagazig City 2017 showed that the two isolates had a 99 percent homology suggesting that there are no rapid genetic changes to circulating viruses. Nucleotide sequences were aligned with other PPRV sequences available in GenBank and phylogenetic tree were used to determine the genetic lineage of the circulating virus. Diagnosis and phylogenetic analysis of the circulating peste des petits ruminants virus in Al-Sharqia governate 537 Circulating strains have been classified as lineage IV. The high percent homology between the two isolates may be due to uncontrolled movement of sheep and goats within Al-Sharqia gov-ernorate. Also PPRV outbreaks are more common around festivals periods. In addition, the PPRV isolate of the current study related to Ethiopian strain and the previous Egyptian strains (Ismailia 3/Egy/2010 isolate, El-Kalubeya isolate and Ismailia 1-2014 isolate). The origin of the circulating viruses may be from Ethiopia. Egypt imports small ruminants from different African countries including Ethiopia because it has meat production deficit. The similar result demonstrated by (13) who reported that, the cause of outbreak in Ismailia and Suez in period 2014 and 2016 was Ethiopian origin. Another study revealed that the Sudanese strain caused PPRV outbreaks in Ismailia, Egypt during the 2010-2012 (27). Also, this study confirmed that all Egyptian isolates classified as PPRV lineage IV strains circulate extensively from governorate to other without controlled measures. Conclusion The study confirms that PPRV has been detected and circulated in a flock of goats and sheep in the city of Belbes, Al-Sharqia. The N gene sequence and phylogenetic analysis confirm that the PPR virus circulating in this region of the country is of lineage IV and closer to PPRV isolated in Zagazig in 2017. Also PPRV isolate of present study revealed high level of homology with majority of the previous Egyptian isolates and Ethiopian strain. The movement of unbarred animals and their trade could be one of the reasons for the transmission of this virus. The results emphasize the importance of molecular methods for a broader understanding of the epidemiology and development of the virus in the country. The information from these studies will help to achieve the goal of the disease control and eradication. Conflict of interest There is no conflict of interest. References 1. Chakraborty S, Kumar N, Dhama K, Verma AK, Tiwari R, Kumar A, et al. Foot-and-mouth disease, an economically important disease of animals. Adv Anim Vet Sci 2014; 2:1-18. 2. Ismail IM, House J. Evidence of identification of peste des petits ruminants from goats in Egypt. Arch Exp Veterinaries 1990; 44(3):471-4. 3. Gibbs EP, Taylor WP, Lawman MJ, Bryant J. Classification of peste des petits ruminants virus as the fourth member of the genus Morbillivirus. In-tervirology 1979 ;11(5):268-74. 4. Bailey D, Banyard A, Dash P Ozkul A, Barrett T. Full genome sequence of peste des petits ruminants virus, a member of the Morbillivirus genus. Virus research 2005; 110(1-2): 119-24. 5. Munir M, Zohari S, Berg M. Genome Organization of Peste des Petits Ruminants Virus.In Molecular Biology and Pathogenesis of Peste des Petits Ruminants Virus 2012:1-22. 6. Kumar A, Rahal A, Chakraborty S, Verma AK, Dhama K. Mycoplasma agalactiae, an Etiolog-ical Agent of Contagious Agalactia in Small Ruminants: A Review. Veterinary Medicine International 2014; 2014:13. 7. Lefevre PC, Diallo A. Peste des petits ruminants. Revue scientifique et technique (International Office of Epizootics) 1990; 9(4):935-81. 8. Mariner JC, Jones BA, Rich KM, Thevasaga-yam S, Anderson J. The Opportunity to Eradicate Peste des Petits Ruminants 2016; 196(9):3499-506. 9. Baron MD, Diallo A, Lancelot R, Libeau G. Peste des Petits Ruminants Virus. Advances in virus research 2016; 95:1-42. 10. Hamdy FM, Dardiri AH. Response of white-tailed deer to infection with peste des petits ruminants virus. Journal of wildlife diseases 1976; 12(4):516-22. 11. Kawther SZ, Wahid MA. Isolation and Identification of Field Isolate of Peste des petits ruminants Virus in Egypt. Global Veterinaria 2014; 12(5):667-72. 12. Couacy-Hymann E, Roger F, Hurard C, Guillou JP, Libeau G, Diallo A. Rapid and sensitive detection of peste des petits ruminants virus by a polymerase chain reaction assay. Journal of virolog-ical methods 2002; 100(1-2):17-25. 13. Elhaig MM, Selim A, Mandour AS, Schulz C, Hoffmann B. Prevalence and molecular characterization of Peste des Petits Ruminants virus from Ismailia and Suez, Northeastern Egypt, 2014-2016. Small Ruminant Research 2018; 169:94-8. 538 H. Elsheikh, M. El-Mekkawi, A. Abou-zaid, A. Abd El Raof 14. Shaila MS, Shamaki D, Forsyth MA, Diallo A, Goatley L, Kitching RP, et al. Geographic distribution and epidemiology of peste des petits ruminants virus. Virus research 1996; 43(2): 149—53. 15. Kwiatek O, Minet C, Grillet C, Hurard C, Carlsson E, Karimov B, et al. Peste des petits ruminants (PPR) outbreak in Tajikistan. Journal of comparative pathology 2007; 136(2-3): 111-9. 16. Banyard AC, Parida S, Batten C, Oura C, Kwiatek O, Libeau G. Global distribution of peste des petits ruminants virus and prospects for improved diagnosis and control. The Journal of general virology 2010; 91(12):2885-97. 17. Office International des Epizooties (OIE): OIE disease information 1999; 12:137. 18. Ayman Sa. Studies on Peste Des Petits Ruminants In Goats And Sheep With Special Emphasis Recent Techniques Used For Diagnosis: Faculty of Veterinary Medicine, Zagazig University 2017. 19. Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ. Basic local alignment search tool. Journal of molecular biology 1990; 215(3):403-10. 20. Thompson JD, Higgins DG, Gibson TJ. CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic acids research 1994;22(22):4673-80. 21. Tamura K, Stecher G, Peterson D, Filipski A, Kumar S. MEGA6: Molecular Evolutionary Genetics Analysis version 6.0. Molecular biology and evolution 2013;30(12):2725-9. 22. Barrett T, Banyard AC, Diallo A. Molecular biology of the morbilliviruses. In: Molecular biology of the morbillivirus. Barrett, T, Pastoret, PP, Taylor, WP (Eds), Academic Press, Elsevier, Amsterdam 2005:31-67. 23. Ozmen O, Kale M, Haligur M, Yavru S. Pathological, serological, and virological findings in sheep infected simultaneously with Bluetongue, Peste-des-petits-ruminants, and Sheeppox viruses. Tropical animal health and production 2009;41(6): 951-58. 24. Libeau G, Diallo A, Parida S. Evolutionary genetics underlying the spread of peste des petits ruminants virus. Anim Front 2014;4:14-20. 25. Abd El-Rahim IH, Sharawi SS, Barakat MR, El-Nahas EM. An outbreak of peste des petits ruminants in migratory flocks of sheep and goats in Egypt in 2006. Revue scientifique et technique (International Office of Epizootics 2010;29(3):655-62. 26. Sharawi SSA, Abd-El-Rahim IHA. Nucleotide Sequencing and Phylogenic Analysis of Fusion (F) Epitope for Egyptian Pestes Des Petit Ruminants Virus (PPRV) Predicting Unique Criteria Stated as Egypt 2009. International Journal of Virology 2011;7:204-9. 27. Soltan MA, Abd-Eldaim MM. Emergence of peste des petits ruminants virus lineage IV in Is-mailia Province, Egypt. Infection, genetics and evolution : journal of molecular epidemiology and evolutionary genetics in infectious diseases 2014;28:44-7. 28. Rajak K, Sreenivasa B, Madhusudan H, Singh RP, Singh SK, Singh RK, et al. Experimental studies on immunosuppressive effects of Pestte Des Petits Ruminants (PPR) virus in goats 2005;28: 287-96 . 29. Galbraith SE, Tiwari A, Baron MD, Lund BT, Barrett T, Cosby SL. Morbillivirus downregu-lation of CD46. Journal of virology 1998; 72(12): 10292-7. 30. Heaney J, Barrett T, Cosby SL. Inhibition of in vitro leukocyte proliferation by morbilliviruses. Journal of virology 2002; 76(7):3579-84. 31. Mondal B, Sreenivasa BP, Dhar P, Singh RP, Bandyopadhyay SK. Apoptosis induced by peste des petits ruminants virus in goat peripheral blood mononuclear cells. Virus research 2001; 73(2): 113-9. 32. Khan H, Siddique M, Arshad M, M K, S. U. Rehman Q. Sero-prevalence of peste des petits ruminants (PPR) virus in sheep and goats in Punjab Province of Pakistan. Pakistan Veterinary Journal 2007; 27(3): 109-12. 33. Abubakar M, Jamal SM, Hussain M, Ali Q. Incidence of peste des petits ruminants (PPR) virus in sheep and goat as detected by immuno-capture ELISA (Ic ELISA). Small Rumin Res 2008; 75:256-9. 34. Abubakar M, Khan HA, Arshed MJ, Hussain M, Ali Q. Peste des petits ruminants (PPR): disease appraisal with global and Pakistan perspective. Small Ruminant Res 2011; 96:1-0. 35. Kivaria FM, Kwiatek O, Kapaga AM, Swai ES, Libeau G, Moshy W, et al. The incursion, persistence and spread of peste des petits ruminants in Tanzania: epidemiological patterns and predictions. The Onderstepoort journal of veterinary research 2013; 80(1):593. 36. Shanker H, Gupta VK, Singh N. Occurrence of pest des petitis ruminants like disease in small ruminants in Uttar Pradesh. Indian J Anim Sci 1998; 68:38-40. 37. Balamurugan V, Hemadri D, Gajendragad MR, Singh RK, Rahman H. Diagnosis and control Diagnosis and phylogenetic analysis of the circulating peste des petits ruminants virus in Al-Sharqia governate 539 of peste des petits ruminants: a comprehensive review. Virusdisease 2014; 25(1):39-56. 38. Adombi CM, Lelenta M, Lamien CE, Shamaki D, Koffi YM, Traore A, et al. Monkey CV1 cell line expressing the sheep-goat SLAM protein: a highly sensitive cell line for the isolation of peste des petits ruminants virus from pathological specimens. Journal of virological methods 2011; 173(2):306—13. 39. Couacy-Hymann E. Current Advances in Genome Detection of Peste Des Petits Ruminants Virus. Peste Des Petits Ruminants Virus. Springer 2015:155-69. 40. Abubaker M, Mehmood F, Jeelani A, Arshed JM. application of PCR in Diagnosis of pest des petits ruminants. INTECT. 2012. 41. Kerur N, Jhala MK, Joshi CG. Genetic characterization of Indian peste des petits ruminants virus (PPRV) by sequencing and phylogenetic analysis of fusion protein and nucleoprotein gene segments. Research in veterinary science. 2008; 85(1): 176-83. Slov Vet Res 2019; 56 (Suppl 22): 541-51 DOI 10.26873/SVR-792-2019 Original Research Article EFFECT OF DIFFERENT CRYOPROTECTANTS ON THE POST-THAW SPERM CHARACTERISTICS AND IN VIVO FERTILITY OF BUFFALO (Bubalus bubalis) BULL SEMEN Essam A. Almadaly1*, Fady S. Tawfik2, Ismail I. El-Kon1, Bassiouni A. Heleil1, El-Sayed M. Fattouh1 department of Theriogenology, Faculty of Veterinary Medicine, Kafrelsheikh University, El-Geish Street, 33516 Kafrelsheikh, Egypt, 2Artificial Insemination Center, Veterinary Medicine Directorate, Ministry of Agriculture, Al-Gharbia, Egypt Corresponding author, E-mail: dr_essam_2005@yahoo.com; essam.mostafa@vet.kfs.edu.eg Abstract: This study aimed to investigate the effect of different cryoprotectants, glycerol (GLY) or ethylene glycol (EG) or dimethyl sulfoxide (DMSO) on sperm characteristics, and in vivo fertility of frozen-thawed buffalo-bull semen. A total of 85 ejaculates collected by artificial vagina from buffalo-bulls of proven fertility were used in this study. The collected ejaculates were examined for volume, motility, viability, morphology, and sperm cell concentration. The qualifying ejaculates (> 3 mass motion, > 70% progressive motility and viability, < 15% abnormal morphology and > 1 x 109 sperm cells/mL) were pooled and diluted with Tris-based diluent containing either 7% GLY or 5% EG or 5% DMSO. After 4 h equilibration time, the diluted semen was loaded in 0.5 mL straws, labeled, sealed and frozen stored until analysis. Frozen straws were thawed and evaluated for progressive motility, viability, hypo-osmotic swelling test (HOST), acrosomal membrane integrity, and acrosome reaction (AR) in response to calcium ionophore A23187. Moreover, in vivo fertility was calculated after artificial insemination (AI) of 75 buffalo-cows (25 female/cryoprotectant) treated with double doses of prostaglandin F2a (PGF2a) 11 days interval. The proportions of progressive motility, viability and intact-acrosome were higher (p < 0.05) in extender containing 7% GLY compared to 5% EG and 5% DMSO. The proportion of intact-plasma membrane was comparable (p > 0.05) between GLY and EG but higher than that of DMSO. A time-dependent increase in the % AR and % relative AR was recorded in the three cryoprotectants with clear significant (p < 0.01) difference among them at 30 and 60 min incubation, respectively. Moreover, GLY yielded higher pregnancy rate (52%) than EG (32%) and DMSO (16%). In conclusion, GLY is recommended for preservation of buffalo-bull semen in order to maintain sperm plasma membrane integrity and improve in vivo fertility of frozen-thawed buffalo-bull semen. Key words: buffalo-bull; frozen-thawed semen; cryoprotectant; estrus synchronization; In vivo fertility. Introduction Buffalo (Bubalus bubalis) is the most important and popular livestock animal for milk Received: January 2019 Accepted for publication: February 2019 542 E. Almadaly, F. Tawfik, I. El-Kon, B. Heleil, El. Fattouh production in Egypt. It was introduced to Egypt from India, Iran and Iraq around the middle of the 7th century. Its lactation season ranged from 210-280 day with annual average of 915 kg milk/animal. The total population of buffaloes in Egypt was decrease from 4.16 million in 2012 to 3.64 million in 2016 (1) with decline of approximately 11% within four years, most of them reared in small herds (1-3 animals). Despite it is the best milk, meat and leather producing animal in our country but it has not received sufficient consideration regarding the improvement of its breeding strategies. Improvement of livestock production can be achieved using one of the assisted reproductive technologies such as artificial insemination (AI) (2) which is an essential tool for the quick dispersion of valuable germplasm and the feasible method to improve the genetic quality of farm animals (3). Cryopreservation is an essential method for conservation of germ cells and is suitable for the proper genetic management (4). Semen quality and fertility is clearly reduced by freezing-thawing damage compared with fresh semen and it can be compensated by the insemination of larger number of sperm cells. Generally, 40 - 50% of sperm cells die after cryopreservation even with optimized methodology (5) due to an inevitable chemical, osmotic, thermal, and mechanical stresses which lead to lower post-thaw motility and viability (6). Intracellular ice crystallization is implicated in this inevitable damage of spermatozoa during cryopreservation (7). Moreover, buffalo-bull spermatozoa are more vulnerable to freezing-thawing damage compared with other mammalian species (8). The fertility rate of frozen-thawed buffalo-bull spermatozoa is ranged from 33% (9) to 45% (10). Sudden cooling of diluted semen from 30 °C toward 0 °C result in a lethal stress called cold shock in some of the sperm cells which was proportional with the cooling rate, the temperature interval and range (11). This sharp variation in temperature induces damage in sperm cell membranes. It is possible that these effects are related to phase change in li-pids and altered functional state of membranes (6). The composition of semen extender containing proper cryoprotectants is critical for successful semen cryopreservation (12) where the cryoprotectant minimize the intracellular ice crystals formation and/or damage. The penetrating cryoprotectants such as glycerol (GLY), ethylene glycol (EG) and dimethyl sulfoxide (DMSO) have the ability to pass through the sperm plasma membrane. Accordingly, it acts both intra and extra-cellular, rearrange the membrane proteins, reduce intracellular ice crystals formation and thus, protect sperm cells from freeze-thaw damage (13). Addition of penetrating cryoprotectants to sperm cell suspension, the cells first shrink due to water efflux then swell as the cryoprotectant get intracellular with water to maintain chemical potential. During removal of cryoprotectant the cell will initially swell due to water influx and then return to isosmotic size due to efflux of cryoprotectant with water. This osmotic stress adversely affects sperm motility, viability and function (14). Conventionally, GLY is used at concentration ranged from 5 to 8% to cryopreserve bovine sperm. However, it has both osmotic and toxic effects on the plasma membrane and metabolism of frozen-thawed semen with subsequent reduced motility and fertility (12). Also, higher concentration of GLY may lead to cell death (15). Recently, the high membrane permeability of EG was shown to reduce the plasma membrane damage of bovine sperm compared with GLY (16). Thus, apparently EG has less detrimental effect on viability and mo-tility and offer protective effect to sperm acro-some than GLY (17). Nowadays, biotechnology of cryoprotectants play substantial role in improving the quality of frozen-thawed semen. Incorporation of efficient and less toxic cryo-protectant within semen extender can make a substantial impact in improving the quality of frozen-thawed buffalo-bull spermatozoa. Therefore, the present study aimed to investigate the effect of different cryoprotectants (GLY, EG and DMSO) on sperm characteristics and in vivo fertility of frozen-thawed buffalo-bull semen. Effect of cryoprotectants on in vitro semen quality and in vivo fertility of buffalo (Bubalus bubalis) bull semen 543 Materials and methods Animals The present study was carried out at Me-halet-Mousa Research Station, Kafrelsheikh, Egypt. Five healthy Egyptian buffalo bulls (56 years-old) of proven fertility, acceptable libido and of good semen quality were used for semen collection. Seventy five Egyptian buffalo-cows (4-5 years-old) having history of normal parturition was used for in vivo fertility experiment. All animals were kept indoors all over the year and fed on ration that met both maintenance and production requirements. Buffalo-cows were fed on concentrates plus corn silage and straw in summer and concentrates plus green clover and straw in winter. Chemicals, media and reagents Unless otherwise stated, all chemicals used were of high purity and procured from Sigma-Aldrich Chemical Company (St. Louis, MO, USA). Freshly prepared Tris-based extender consists of 3.028 g Tris, 1.678 g citric acid and 1.0 g fructose, 20% (v/v) egg yolk, 1000 IU/mL penicillin G sodium, 1 mg/mL streptomycin sulfate and the same extender containing either 7% GLY (Sigma-Aldrich, USA) or 5% EG (SDFCL, India) or 5% DMSO (Fluka, Germany) was used for semen dilution and freezing (5). Saline medium consists of 142 mM NaCl, 2.5 mM KOH, 10 mM glucose and 20 mM Hepes and adjusted to pH 7.55 at 25 °C with NaOH (18) was used for dilution and incubation of spermatozoa. Saline medium containing 275 mM sucrose in place of NaCl was used for washing spermatozoa and designated as sucrose medium (18). Both media contained 0.1% (w/v) polyvinyl alcohol (PVA, molecular weight 30,000-70,000) and 0.1% (w/v) polyethylene glycol (PEG). A fixative used was 2% Glutar-aldehyde in 0.165 M of sodium cacodylate buffer, adjusted to pH 7.3 at 25 °C. Sperm counting medium [(SCM) 0.9% (w/v) NaCl, 0.1% (v/v) formaldehyde, 0.1% (w/v) PEG and 10 mM ethylenediaminetetraacetic acid in H2O] was used for counting of sperm cells. Calcium ionophore A23187 (free acid; Calbiochem-Novabiochem Corp., San Diago, USA) was prepared at 100 mM in DMSO, and frozen stored at - 30 °C in small aliquots wrapped from light. Immediately before use, an aliquot was thawed and used at a 1:100 dilution in saline medium to give a final concentration of 1 mM. Semen collection Ejaculates were collected from well-trained buffalo-bulls using an artificial vagina of inner sleeve temperature 40 °C according to Rasul et al. (19). Ejaculates were collected twice a week early morning with 10 min interval. Immediately, after collection the collected ejaculates were kept in water bath of 37 °C during initial semen evaluation. Semen evaluation As soon as possible the collected ejaculates were observed for color, consistency, hygienic quality and volume. An aliquot (200 pL) from each ejaculate was used for evaluation of the following sperm characteristics: 1. Sperm motility 1.1. Mass motility As quick as possible the fresh semen samples were examined on pre-warmed glass slide on a warm stage (38.5 °C) under phase contrast microscope (Olympus BX20, Tokyo, Japan) at 100 x magnification. The mass motility was scored from 0-5 grade depending upon the speed and intensity of waves. All ejaculates of mass motility less than 3 were excluded. 1.2. Individual motility An aliquot (5 pL) of fresh semen was placed on warm glass slide and diluted with 20 pL isotonic sodium citrate dihydrate 2.9%. The diluted semen was covered with a warm cover slip and examined under phase contrast microscope at 200 and 400 x magnifications. At least, five microscopic fields were examined to calculate the average in increments of 5%. Ejaculates of less than 70% progressive motility were excluded. 2. Sperm viability Alive sperm percentage was determined in Eosin-Nigrosin (EN) stained semen smear according to Bjoerndahl et al. (20). Briefly, 5 pL of fresh semen was mixed with 20 pL pre warmed EN stain and smeared on warm glass 544 E. Almadaly, F. Tawfik, I. El-Kon, B. Heleil, El. Fattouh slide, dried and examined at 400 and 1000 x magnification of phase contrast microscope. At least, two microscopic fields containing 200 spermatozoa were examined. The proportion of unstained spermatozoa was expressed as viabil-ity%. 3. Sperm cell concentration Sperm cell concentration was determined by direct cell count using Neubauer haemocytom-eter (Marienfeld, Germany) (21). Exactly, 10 pL of fresh semen was diluted with 3000 pL SCM and thoroughly mixed. An aliquot (12.5 pL) of sperm suspension was spotted into counting chamber and count sperm cells under phase contrast microscope at 400 x magnification. Semen dilution and cryopreservation Immediately before dilution, Tris-based extender was centrifuged at 3310 x g for 20 min and the upper portion of supernatant and sediment were discarded (22). Extenders (intermediate portion after centrifugation) were prepared one day before semen collection and kept overnight at 4 °C. The ejaculates of at least 3 score mass motility, 70% progressive motility and 800 x 106/mL sperm cell concentration were diluted. Both, ejaculate and extender were maintained in water bath of 37 °C. The selected ejaculates were pooled together to avoid the bull effect before being diluted with cryopro-tectant free extender stepwise at room temperature. The semen was diluted gradually 1:10 (each straw had at least 20 x 106 motile sperm). Diluted semen was cooled to 4 °C for 2 h and further diluted (1:1) with cryoprotectant containing extender (23). The cryoprotectant was either 7% GLY (24) or 5% EG (3) or 5% DMSO (19). Diluted semen was equilibrated at 4 °C for at least 4 h (25) before being loaded in 0.5 mL polyvinyl French straws (IMV, France) with a suction pump at 4 °C in a cold cabinet (Minitub, Germany). The filled straws were plunged above liquid nitrogen vapor (-120 °C) for at least 10 min then immersed in liquid nitrogen (-196 °C) for storage. Evaluation of frozen-thawed semen Two straws for each treatment were thawed in water bath of 39 °C for exactly 1 min, thoroughly dried and gently evacuated in 1.5 mL vial which kept on a warming plate (38.5 °C) during evaluation. Frozen-thawed semen was examined for the following sperm characteristics: 1. Progressive motility The proportion of progressive motility was determined in wet mount using 10 pL of frozen-thawed semen on pre-warmed glass slide covered with warm cover slip (18 x 18 mm), and examined at 400 x magnification under phase contrast microscope. Five microscopic fields were examined for the proportion of progressive motile sperm according to Chutia et al. (26). 2. Sperm viability The proportion of live sperms was determined in EN-stained smears similarly as mentioned before in fresh semen analysis. At least, 200 spermatozoa were examined for each semen sample to calculate the proportion of unstained spermatozoa which expressed as viability %. 3. Sperm plasma membrane integrity Plasma membrane integrity was determined by using hypo-osmotic swelling test (HOST) (27). Hypo osmotic solution was prepared by dissolving 0.73 g sodium citrate and 1.35 g fructose in 100 mL Milli-Q water (osmotic pressure ~190 mOsmol/kg). For evaluation, 50 pL of frozen-thawed semen was suspended in 500 pL of pre-warmed HOST solution and incubated at 37 °C for 30 - 60 min. After incubation, 50 pL of HOST exposed sperm suspension was fixed with an equal volume of 2% Glutar-aldehyde in sodium cacodylate buffer for at least 30 min at room temperature. Two wet mount (2 pL/each) were prepared and examined for the proportion of sperm cells showing tail swelling under (1000x) phase contrast microscope. In total, 200 spermatozoa per each wet mount were examined for their swelling characterized by coiled tail. The proportion of HOST-positive sperm cells (% intact-membrane) was determined from the following equation (HOST-positive % = Y - X/400 x Effect of cryoprotectants on in vitro semen quality and in vivo fertility of buffalo (Bubalus bubalis) bull semen 545 100), where X was spermatozoa having tail abnormalities and Y was spermatozoa having swelled tails according to Fukui et al. (28). 4. Acrosomal membrane integrity Acrosomal membrane integrity was determined in EN-stained semen smears according to the method described by Bamba [29]. At least, 200 spermatozoa were examined in each semen sample under phase contrast microscope at 1000 x magnification. Sperm cells having crescent shape apical ridge considered intact-acrosome otherwise consider damaged-acro-some. 5. Stimulation of frozen-thawed spermatozoa with Ca2+ and Ca2+ iono-phore A23187 Spermatozoa were incubated with Ca2+ and Ca2+ ionophore A23187 as previously described by Almadaly et al. (18). Two straws were thawed at 39 °C for 1 min and evacuated in 1.5 mL vials. The two vials were centrifuged at 830 x g for 6 min at room temperature and remove the supernatant. The sperm pellets were resuspended in saline medium then was overlay on sucrose medium containing 0.1% PVA and 0.1% PEG and centrifuged at 400 x g for 5 min then at 1000 x g for 10 min at room temperature. After centrifugation the supernatant was discarded and sperm cell concentration was adjusted to 6 x 106/mL. Spermatozoa were resus-pended in saline medium containing 3 mM CaCl2 and incubated in the presence and absence (DMSO vehicle, control) of 1 mM Ca2+ ionophore A23187 at 37 °C for up to 60 min in air. At 0, 5, 10, 15, 30, and 60 min subsamples were collected and fixed with 1% Glutaralde-hyde in 0.165 M cacodylate buffer at room temperature for at least 30 min. After incubation, wet mounts (2 pL/each) were prepared and examined under oil immersion lens of phase contrast microscope. Sperm having a clear, dense apical ridge on the head were considered acrosome-intact, but sperm cells with ruffled or vesiculated acro-some or without acrosome were considered acrosome reacted. The raw data expressed as % acrosome reaction (AR) was transformed to percentage of spermatozoa that were acrosome- intact at the onset of stimulation but subsequently have an AR in response to Ca2+ and Ca2+ A23187 (% relative AR) with the following formula: (%AR at each time point of stimulation - percentage at 0 min) / (100 - percentage at 0 min) x 100 (30). Estrus synchronization and artificial insemination This experiment was applied on 75 pluripar-ous buffalo-cows maintained at the same farm of buffalo-bulls and of more than 90 days post-partum with normal genitalia. Before the onset of experiment all buffalo-cows were examined by rectal palpation and/or ultrasound (Mindry, Germany) for judging the ovarian activity which indicated by the presence of follicles and/or corpus luteum. Cyclic buffalo-cows received double dose of prostaglandin F2a (PGF2a) with 11 days interval (31) where each animal received 750 pg PGF2a analogue (Clo-prostenol sodium, Estrumate, Berkhamsted, England) intramuscularly on days 0 and 11. Exactly, 48 h after the second dose of PGF2 a animals were carefully observed for the signs and/or the reflexes of estrus throughout the day and night. Estrus buffalo-cows were inseminated with frozen-thawed straws (two straws/animal) containing either GLY (n = 25) or EG (n = 25) or DMSO (n = 25). All animals were intrauterine inseminated (recto-vaginal technique) by the same inseminator using am-pm regime. Pregnancy diagnosis All inseminated buffalo-cows were rectally palpated and/or ultrasound examined for positive findings of pregnancy on day 45 post-insemination. Presence of either amniotic vesicle or fetal membrane slip was considered a reliable sign for the pregnant buffalo-cow. Statistical analysis The results were presented as mean ± standard error of mean (SEM). All analyses were achieved using a statistical software program (Graphpad Prism Version 6.0; Graphpad Software, San Diego, CA, USA). Analysis of variance (ANOVA) with Turkey's multiple com- 546 E. Almadaly, F. Tawfik, I. El-Kon, B. Heleil, El. Fattouh parison test was used subsequently for comparison of means at a significance level ofp < 0.05 (32). The proportion of pregnant females was analyzed by Chi-square test atp < 0.05. Results Sperm characteristics of fresh semen The obtained results revealed that all ejaculates of the five buffalo-bulls used for processing of frozen semen had acceptable mass motility (4.5 ± 0.2), progressive motility (78.7 ± 1.3), viability (79.5 ± 0.8) and sperm cell concentration (1.6 ± 0.1 x 106/mL) as well as % abnormal morphology (9.6 ± 0.7, Table 1). Sperm characteristics of frozen-thawed semen 1. Sperm motility The mean ± SEM of % progressive motility of frozen-thawed buffalo-bull spermatozoa frozen-stored in the presence of one of the three cryoprotectants have been presented in Table 2. The progressive motility were extremely (p < 0.001) different among the three cryoprotectant where it was greater in GLY (41.3 ± 1.2) followed by EG (31.4 ± 0.9) and lower (25.4 ± 1.1) for DMSO. 2. Sperm viability Regarding the results of sperm viability (Table 2), GLY yielded the greater (p < 0.001) sperm viability (47.7 ± 1.1) than both EG (42.3 ± 0.8) and DMSO (39.2 ± 1.2) without significant difference between EG and DMSO. 3. Plasma membrane integrity The proportion of intact-plasma membrane of spermatozoa frozen-stored in the presence of GLY or EG (Table 2) were greater (p < 0.001) than that of DMSO (46.3 ± 0.9) without significant difference between GLY (58.4 ± 1.5) and EG (58.7 ± 0.8). 4. Acrosomal membrane integrity The proportion of intact-acrosome was significantly (p < 0.001) different among the three cryoprotectant with greater % (61.7 ± 1.2) in GLY than EG (58.4 ± 0.8) and DMSO (48.4 ± 0.5) as shown in Table 2. 5. Induction of AR with Ca2+ and Ca2+ ionophore A23187 Incubation of spermatozoa with Ca2+/ A23187 resulted in a time-dependent increase in the % AR (Table 3), in the three cryoprotect-ants without significant (p > 0.05) difference at 0 and 5 min but with significant (p < 0.01) difference at 10, 15, 30 and 60 min. Regarding % relative AR (Table 4) it was similar among the three cryoprotectants at 5, 10 and 15 min but was extremely different among them at 30 and 60 min. Almost in all incubation times GLY was predominant in both % AR and % relative AR (Tables 3, 4). Pregnancy rate The pregnancy rate was different (x2 = 7.32, p < 0.05) among the three cryoprotectants as depicted in Figure 1, where it was greater (52%) with GLY than EG (32%) and DMSO (16 %). □ Pregnant □ Non-Pregnant GLY EG Cryopreotectants DMSO Chi-square = 7.32 P = 0.025 Figure 1: Effect of GLY, EG and DMSO in Tris-based extender on the in vivo fertility of frozen-thawed buffalo bull spermatozoa GLY = Glycerol, EG = Ethylene glycol, DMSO = Dimethyl sulfoxide. Effect of cryoprotectants on in vitro semen quality and in vivo fertility of buffalo (Bubalus bubalis) bull semen 547 Table 1: Fresh semen quality (mean ± SEM) of buffalo-bulls* No. of Mass motility replicates (0 - 5) Progressive motility (%) Sperm viability (%) Abnormal Sperm cell concentration morphology (%) (x 109/mL) 12 4.5 ± 0.2 78.7 ± 1.3 79.5 ± 0.8 9.6 ± 0.7 1.6 ± 0.1 *For each replicate ejaculates were collected and examined in duplicate for each semen parameter. Table 2: Characteristics (mean ± SEM) of frozen-thawed buffalo-bull spermatozoa frozen-stored with different cryoprotectants* No 0f Progressive Cryoprotectant ' ^ motility replicates (%) Viability (%) Plasma membrane integrity (%) Acrosomal membrane integrity (%) GLY 17 41.3 ± 1.2a 47.7 ± 1.1a 58.4 ± 1.5a 61.7 ± 1.2a EG 17 31.4 ± 0.9b 42.3 ± 0.8b 58.7 ± 0.8a 58.4 ± 0.8b DMSO 17 25.4 ± 1.1e 39.3 ± 1.2b 46.3 ± 0.9b 48.4 ± 0.5c P value - < 0.001 < 0.001 < 0.001 < 0.001 *Two straws were thawed and pooled together for each replicate and examined in duplicate for each sperm characteristic. Means bearing different superscript within the same column were significantly different at p < 0.05. Table 3: The % AR (mean ± SEM) of frozen-thawed buffalo-bull spermatozoa frozen-stored with different cryoprotectants in response to Ca2+ and Ca2+ ionophore A23187* Cryoprotectant No. of Incubation time (min) replicates 0 5 10 15 30 60 GLY 6 21.2 ± 0.7a 28.8 ± 0.6a 36.2 ± 1.2a 46.0 ± 1.3a 62.7 ± 1.4a 76.3 ± 1.1a EG 6 19.3 ± 0.6a 30.2 ± 0.8a 33.7 ± 0.8ab 42.5 ± 0.8ab 52.2 ± 1.1b 66.7 ± 1.5b DMSO 6 19.2 ± 0.5a 28.3 ± 0.7a 31.7 ± 0.7b 41.5 ± 0.8b 47.5 ± 0.8e 59.2 ± 0.8e P value - 0.06 0.13 0.008 0.01 <0.001 <0.001 *Two straws were thawed and pooled together for each replicate and examined for the % AR in response to calcium ionophore. Values bearing one similar superscript within the same time point were similar (p > 0.05, repeated measures ANOVA followed by Tukey's multiple comparison test). Table 4: The % relative AR (mean ± SEM) of frozen-thawed buffalo-bull spermatozoa frozen-stored with different cryoprotectants in response to Ca2+ and Ca2+ ionophore A23187* Cryoprotectant No. of Incubation time (min) replicates 5 10 15 30 60 GLY 6 10.0 ± 0.7a 20.0 ± 2.1a 31.4 ± 1.9a 52.6 ± 2.0a 72.1 ± 1.5a EG 6 13.4 ± 1.4a 17.7 ± 1.0a 28.7 ± 0.9a 40.6 ± 1.4b 58.6 ± 1.9b DMSO 6 11.2 ± 1.3a 16.2 ± 1.0a 27.6 ± 1.3a 34.9 ± 1.1b 49.5 ± 1.0c P value - 0.06 0.16 0.17 <0.001 <0.001 * The proportion of relative AR = %AR at each time point of stimulation - percentage at 0 min / (100 - percentage at 0 min x 100. Values bearing one similar superscript within the same time point were similar (p > 0.05, repeated measures ANOVA followed by Tukey's multiple comparison test). 548 E. Almadaly, F. Tawfik, I. El-Kon, B. Heleil, El. Fattouh Discussion It is imperative to mention that buffalo-bull sperm are more susceptible to freeze-thaw damage than other domestic animals. This poor freezability of buffalo-bull sperm might be related to the lower content of membrane phos-pholipid and/or its loss during freezing and thawing procedures (33). Also, intracellular ice crystal formation during cryopreservation was the main cause of sperm cell damage. Thus, the composition of diluent containing proper cryo-protectants is critical factor for successful semen preservation (34) as the cryoprotectant minimizes intracellular ice formation (12). Moreover, the ideal cryoprotectant must have low molecular weight, high water solubility, permeated cell quickly and of low toxicity (35). On the basis of existing information on freezing of buffalo-bull sperm in the presence of different cryoprotectants, GLY improved the post-thaw sperm characteristics including motility, viability, plasma membrane integrity, acrosomal membrane integrity, % AR and % relative AR in response to calcium ionophore A23187 as well as yielded higher pregnancy rate in comparison with EG and DMSO. These findings were regular with the findings of Tasdemir et al. (34) who reported that there was no advantage in using EG or DMSO spermatozoa where GLY yielded the best post-thaw sperm characteristics in comparison with EG and DMSO. Also, Guerrero (36) found that the post-thaw progressive motility and % intact-membrane of bull sperm frozen in 7% GLY-Tris-based extender were significantly higher than those of sperm frozen in 7% EG-Tris-based extender. On contrary, % intact-acro-some was similar between 7% GLY and 7% EG. Moreover, the findings of this study were in harmony with Rasul et al. (19) who concluded that GLY is still an efficient cryoprotectant for buffalo-bull sperm and DMSO antagonized the action of GLY and thus reduced the post-thaw quality of buffalo-bull sperm. Moreover, 6% GLY added at 37 °C, provided better cryopro-tection to the motility and plasma membrane integrity of buffalo-bull sperm (19). Our findings were in agreement with Rohilla et al. (25) who reported that 6.8% GLY greatly improve post-thaw buffalo-bull sperm motility, viability, and intact-acrosome in comparison with 5% EG. Furthermore, Gabr (37) reported that higher concentration of cryoprotectant for instance, > 7% GLY resulted in gradual reduction in motility. Post-thaw sperm motility with GLY and DMSO didn't differ significantly and there is a beneficial effect of 7% GLY on improving sperm motility of buffalo-bull semen extended in Tris-based extender in comparison with different (5, 7, 10%) concentrations of DMSO and EG (37). Noteworthy, our findings were partially inconsistent with the findings of Swelum et al. (3) who reported that 5% EG resulted in greater post-thaw intact-acrosome, intact-plasma membrane and conception rate of buffalo-bull spermatozoa than 7% GLY. On contrary, the post-thaw sperm motility was lower with 5% EG than 7% GLY which was in agreement with our results but sperm viability didn't affected with the different cryoprotectants either GLY or EG. El-Harairy et al. (38) found that post-thaw motility and the conception rate of bull spermatozoa frozen in lactose-yolk-citrate extender containing 7% GLY or 7% DMSO was similar. This discrepancy might be due to different animal species, breed, and semen extender as well as different cooling and freezing protocols. For instance, permeability of sperm plasma membrane to cryoprotectant and the consequent osmotic damage was species-dependent because each animal species has different structure and composition of sperm plasma membrane. It is well known that the sperm plasma membrane considered being the foremost site of freeze-thaw damage due to its high content of poly unsaturated fatty acids that render it more vulnerable to the intracellular ice crystals formed during the freezing process. This membrane damage lead to loss of lipids and phos-pholipids (39), cholesterol and consequently, the ratio of poly unsaturated fatty acids and saturated fatty acids is disturbed. On the basis of results obtained in the present study that GLY provide higher proportion of intact-plasma membrane, % AR and % relative AR post-thaw Effect of cryoprotectants on in vitro semen quality and in vivo fertility of buffalo (Bubalus bubalis) bull semen 549 thus, GLY have utmost cryoprotective effect on buffalo-bull sperm plasma membrane in comparison with EG and DMSO. Incorporation of GLY in semen diluent used for frozen storage of buffalo-bull semen significantly improved its post-thaw quality and function than both EG and DMSO might be due to EG has toxic effect on sperm cells where it badly affects the motility of fresh and frozen-thawed semen (40). Furthermore, EG could not reverse the capacitation like changes (cryocap-citation) of frozen-thawed sperm cells (41) which might be the plausible explanation for the improved function (in terms of % AR and % relative AR) of sperm cells frozen in the presence of GLY compared to those frozen in the presence of EG. Since the true test to evaluate a semen sample is to estimate the fertility rate after its in vivo insemination in a routine AI program under field conditions (42). Then GLY was efficient in cryopreservation of buffalo-bull sperm as the higher in vivo fertility was recorded with extender having 7% GLY compared to 5% EG and 5% DMSO. This high fertility is a logical result for the improved sperm characteristics viz; progressive motility, plasma and acrosomal membranes integrity, viability, % AR and % relative AR suggesting that all the sperm characteristics used to assess the semen quality do have substantial role in the fertilization process (43). In conclusion, incorporation of 7% GLY in the semen extender significantly improves in vitro quality and in vivo fertility of buffalo-bull semen. Conflict of interest All the authors declare no conflict of interest. Acknowledgments The authors thank all staff members of Me-halet-Mousa Research Station, and to staff of International Livestock Management training center, Sakha, Kafrelsheikh, Egypt for their great assistance during collection and processing of semen. References 1. Food and Agricultural Organization (FAO). FAOSTAT, Global livestock production and health atlas. Animal Production and Health Division, Roma, Italy, 2018. Available from http: //www .fao. org/faostat/en/#data. 2. Andrabi SMH. Factors affecting the quality of cryopreserved buffalo (Bubalus bubalis) bull spermatozoa. Reprod Domest Anim 2009; 44: 552-69. 3. Swelum AA, Mansour HA, Elsayed AA, Amer HA. Comparing ethylene glycol with glycerol for cryopreservation of buffalo bull semen in egg-yolk containing extenders. Theriogenology 2011; 76: 833-42. 4. Singh P, Kumar D, Kumar P, Singh I, Yadav PS. Cryopreservation and quality assessment of buffalo bull semen collected from farmer's doorstep. Agri Res 2013; 2: 148-52. 5. Lei SU, Xilong LI, Jiexia Quan SY, Yahui LI, Xiechao HEX, Ianghui T. A comparison of the protective action of added egg yolks from five avian species to the cryopreservation of bull sperm. Anim Reprod Sci 2007; 104: 212-9. 6. Watson PF. The causes of reduced fertility with cryopreserved semen. Anim Reprod Sci 2000; 60-61: 481-92. 7. Mazur P. Freezing of living cells: mechanisms and implications. Am J Physiol 1984; 247: 125-42. 8. Ansari MS, Rakha BA, Andrabi SMH, Ullah N, Iqbal R, Holt WV, Akhter S. Glutathione supplemented tris-citric acid extender improves the post-thaw quality and in vivo fertility of buffalo (Bubalus bubalis) bull spermatozoa. Reprod Biol 2012; 12: 271-6. 9. Chohan KR, Iqbal J, Asghar AA, Chaudhry MA. Fertility of liquid and frozen semen in Nili Ravi buffaloes. Pak Vet J 1992; 12: 4-5. 10. Pandey AK, Dhaliwal GS, Ghuman SPS, Agarwal SK. Impact of pre-ovulatory follicle diameter on plasma estradiol, subsequent luteal profiles and conception rate in buffalo (Bubalus bubalis). Anim Reprod Sci 2011; 123: 169-74. 11. Watson PF. The roles of lipid and protein in the protection of ram spermatozoa at 5 oC by egg-yolk lipoprotein. J Reprod Fertil 1981; 62: 483-92. 12. Hammerstedt RH, Graham JK, Nolan john P. Cryopreservation of mammalian sperm: what we ask them to survive. J Androl 1990; 11: 73-88. 13. Holt WV. Basic aspects of frozen storage of semen. Anim Reprod Sci 2000; 62: 3-22. 14. Gao DY, Mazur P, Critser JK. Fundamental cryobiology of mammalian spermatozoa. In: Karow AM, Critser JK eds. Reproductive Tissue Banking. 550 E. Almadaly, F. Tawfik, I. El-Kon, B. Heleil, El. Fattouh Scientific Principles. San Diego: Academic Press, 1997, 263-328. 15. Wündrich K, Paasch U, Leicht M, Glander HJ. Activation of caspases in human spermatozoa during cryopreservation - an immunoblot study. Cell Tissue Bank 2006; 7: 81-90. 16. Guthrie HD, Liu J, Critser JK. Osmotic tolerance limits and effects of cryoprotectants on motility of bovine spermatozoa. Biol Reprod 2002; 67: 1811-6. 17. Ball BA, Vo A. Osmotic tolerance of equine spermatozoa and the effects of soluble cryoprotect-ants on equine sperm motility, viability, and mito-chondrial membrane potential. J Androl 2001; 22: 1061-9. 18. Almadaly E, Hoshino Y, Ueta T, Mukoujima K, Shukry M, Farrag F, El-Kon I, Kita K, Murase T. Desalted and lyophilized bovine seminal plasma delays induction of the acrosome reaction in frozen-thawed bovine spermatozoa in response to calcium ionophore. Theriogenology 2015; 83: 175-85. 19. Rasul Z, Ahmed N, Anzar M. Antagonist effect of DMSO on the cryoprotection ability of glycerol during cryopreservation of buffalo sperm. Theriogenology 2007; 68: 813-9. 20. Bjoerndahl L, Soederlund I, Kvist U. Evaluation of the one-step eosin-nigrosin staining technique for human sperm vitality assessment. Hum Reprod 2003;18:813-6. 21. Graham, JK. Analysis of stallion semen and its relation to fertility. Vet Clin North Am Equine Pract 1996; 12: 119-29. 22. Gil J, Soderquist L, Rodriguez-Martinez H. Influence of centrifugation and different extenders on post-thaw sperm quality of ram semen. Theriogenology 2000;54:93-108. 23. Masuda H. Preservation of semen in liquid and frozen state, In: Artificial Insemination Association of Japan (Eds.), Textbook for the Course of Artificial Insemination in Domestic Animals., Fuji Planning. 2006, 343-52. 24. Abbas A, Andrabi MSH. Effect of different glycerol concentrations on motility before and after freezing, recovery rate, longevity and plasma membrane integrity of Nili Ravi buffalo bull spermatozoa. Pak Vet J 2002; 22: 1-4. 25. Rohilla RK, Tuli RK, Goyal RL. Comparative study of the effects of cryoprotective agents in freezing Murrah buffalo bull semen. Ind J Vet Res 2005;14: 37-43. 26. Chutia T, Biswas RK, Tamuli MK, Deka BC, Sinha S, Goswami J, Banik S, Kayastha RB. Effect of holding of semen and washing of seminal plasma on quality and fertility of Hampshire boar semen preserved at liquid state. Anim Reprod Sci 2014; 45:141-9. 27. Waheed S, Ahmad N, Rahman NU, Jamil-ur-Rahman H, Younis M, Iqbal S. Evaluation of duck egg yolk for the cryopreservation of Nili-Ravi buffalo bull semen. Anim Reprod Sci 2012; 131: 95-9. 28. Fukui Y, Togawa M, Abe N, Takano Y, Asada M, Okada A, Iida K, Ishikawa H, Ohsumi S. Validation of the sperm quality analyzer and the hypo-osmotic swelling test for frozen-thawed ram and minke whale (Balanenoptera bonarensis) spermatozoa. J Reprod Dev 2004; 50: 147-54. 29. Bamba K. Evaluation of acrosomal integrity of boar spermatozoa by bright field microscopy using an eosine-nigrosine stain. Theriogenology 1988; 29:1245-51. 30. Murase T, Mukohjima K, Sakaguchi S, Ohtani T, Tsubota T, Kita I. Characterization of frozen-thawed Japanese black bull spermatozoa by standard semen analysis, mucus penetration test and the ability to undergo the acrosome reaction in response to calcium and the calcium ionophore A23187. J Reprod Dev 2001; 47: 237-43. 31. Barile VL. Technologies related with the artificial insemination in Buffalo. J Buffalo Sci 2012; 1: 139-46. 32. Snedecor, GW, Cochran, WG. Statistical Methods, 9th edition. Iowa State University Press Ames, Iowa, United States 1994. 33. Galli A, Bornaghi V, Balduzzi D, Buttazzoni L, Aleandri R. Sexual behavior and semen quality relating to italian buffalo. Proc 3rd World Buffalo Congr, Varna, Bulgaria 1993; 1: 562-70. 34. Tasdemir U, Buyukleblebici S, Tuncer PB, Coskun E, Ozgurtas T, Aydin FN, Buyukleblebici O, Gurcan IS. Effects of various cryoprotectants on bull sperm quality, DNA integrity and oxidative stress parameters. Cryobiology 2013; 66: 38-42. 35. Harrison RAP, Ashworth PJC, Miller NGA. Assessment of sperm function under fertilizing conditions. Reprod Dom Anim 1996; 31: 25-30. 36. Guerrero CA. Cryopreservation and intra-cytoplasmic sperm injection with bovine epididy-mal spermatozoa (PhD Thesis). Fac Agric and Mechanical College, Louisiana State University; Mexico, 2006. 37. Gabr AA. Assessment of in vitro and in vivo fertilizing capacity of buffalo sperm treated with various cryoprotectants (PhD Thesis). Fac Agric, Mansoura University, Egypt, 2009. 38. El-Harairy MA, Eid LN, Zeidan AEB, Abd El-Salaam AM, El-Kishk MAM. Quality and fertility of the frozen-thawed bull semen as affected by Effect of cryoprotectants on in vitro semen quality and in vivo fertility of buffalo (Bubalus bubalis) bull semen 551 the different cryoprotectants and glutathione levels. J Am Sci 2011; 7: 791-801. 39. Sarmah BC, Kaker ML, Razdan MN. Effect of cold shock and freezing on loss of total lipids and phospholipids of buffalo spermatozoa (Bubalus bubalis). Theriogenology 1984; 22: 621-4. 40. Alvarez JG, Storey BT. Taurine, hypotau-rine, epinephrine and albumin inhibit lipid peroxidation in rabbit spermatozoa. Biol Reprod 1983; 29: 548-55. 41. Rota A, Milani C, Cabianca G, Martini M. Comparison between glycerol and ethylene glycol for dog semen cryopreservation. Theriogenology 2006; 65:1848-58. 42. Ansari MS, Rakha BA, Akhter S. Cryopres-ervation of Nili-Ravi buffalo (Bubalus bubalis) semen in AndroMed® extender; in vitro and in vivo evaluation. Reprod Dom Anim 2017; 52: 992-7. 43. Layek SS, Mohanty TK, Kumaresan A, Parks JE. Cryopreservation of bull semen: Evolution from egg yolk based to soybean based extenders. Anim Reprod Sci 2016; 172:1-9. Slov Vet Res 2019; 56 (Suppl 22): 553-69 DOI 10.26873/SVR-793-2019 Original Research Article SYNERGISTIC AMELIORATIVE EFFECT OF Lactobacillus AND Spirulina platensis AGAINST EXPERMINTAL COLITIS IN ALBINORATS: ANTIOXIDANT, HISTOPATHOLOGICAL AND MOLECULAR STUDIES Emad W. Ghazy1, Abd-Allah A. Mokhbatly1, Samah S. Keniber1*, Khaled M. Shoghy2. 1Clinical Pathology Department, Faculty of Veterinary Medicine, Kafrelsheikh University, Egypt, 2Anatomy & embryology Department, Faculty of Veterinary Medicine, University of Sadat city, Egypt *Corresponding author, E-mail: samoha.saber@yahoo.com Abstract: Ulcerative colitis (UC) considers one of inflammatory disorders which affect colon mucosa cause a substantial burden on human day life. In the past, treatment of UC depended on aminosalicylates and antibiotics but due to their adverse side effects and incomplete effectiveness, antioxidant anti-inflammatory agents are used nowadays to ameliorate UC. The aim of this work is to evaluate the modulatory effect of Lactobacillus and/or Spirulina oral administration in acetic acid induced colitisin albino rats. Rats were divided randomly into (6) groups. 1stgroup (negative control), 2nd group (acetic acid), 3rdgroup (Mesalazine) at dose 20mg/kg orally was used as positive drug control. 4th group (Lactobacillus) at dose1 *109 CFU/rat daily. 5th group (Spirulina) at dose500mg/kg daily. 6thgroup (Lactobacillus at dose 1 *109 CFU/rat + Spirulina at dose 500mg/kg). Results revealed that the experimental colitis group showed significant increase in DAI, macroscopic damage, colon weight, colonic MDA, NO, molecular expressions (iNOS and COX-2) and significant decrease in colon length, GSH level and CAT activity. Lactobacillus and/or Spirulina supplementation revealed significant improvement in macroscopic and microscopic finding, increase antioxidant biomarkers, significant inhibitions of MDA and nitric oxide. Furthermore, significant decline in COX-2 and iNOS expressions were reported. In conclusion, the protective effects of Lactobacillus and/or Spirulina in UC are due to their ability to reduce iNOS and COX-2 expressions, increase antioxidant biomarkers and significant inhibition of lipid peroxidations. Furthermore, Lactobacillus and Spirulina have synergistic protective effect on colon tissue and could be used in combination to ameliorate UC. Key words: colitis; acetic acid; Lactobacillus; Spirulina platensis; Mesalazine; antioxidant, anti-inflammatory. Introduction Inflammatory bowel disease (IBD) is a an inflammatory disorder of the gastro intestinal tract, including ulcerative colitis (UC) and Crohn's disease(CD) (1). Ulcerative colitis is restricted to the colon mucosa, while any part of the whole gastrointestinal tract can be af- Received: January 2019 Accepted for publication: February 2019 554 E. Ghazy, A. Mokhbatly, S. Keniber, K. Shoghy fected in Crohn's disease (2). It's occurrence and prevalence are common in any age and different area around the world, that means its emergence global disease(3). Ulcerative colitis pathogenesis is not completely understood. There are many factors affected these diseases, including immune genetic factors (4), abnormal micro biota (5), epithelial barrier disruption, broken of intestinal microbiota and other environmental factor (6). The most clinical signs of colitis are abdominal tenderness, bloody mucous stool, purulent stool, and relapse. Furthermore, diarrhea, interrupted digestion, loss of body weight and an extensive burden on daily life (7). These clinical manifestation were established by scoring of disease activity index (DAI) (5). Acetic acid is considered one of the main chemical widely used animal model to induced ulcerative colitis (8). Pathogenesis, histopatho-logical features and inflammatory mediator profile to this type of colitis is phenotypically more identical to human IBD (9). Specialized intestinal epithelial cells (IECs) are considered the physical barrier of luminal microbiota and has important role in maintaining intestinal homeostasis. So, any disturbance in the epithelial layer and intestinal permeability lead to dysregulated intestinal immune ho-meostasis and lead to IBD (2). Ulcerative colitis induced an increase reactive free radicals inductions and pro-inflammatory cytokines production. Furthermore, it showed significant intestinal epithelial cells apoptosis which disintegrate intestinal mucosal and barrier function. At the same time, inducible nitric oxide synthase (iNOS) (10) and cyclooxygenase-2 (COX-2) production are increased that playing a critical function in the incidence of this disease (11). Probiotics known as "live microorganisms when supplemented in sufficient numbers, they induce health benefits to the host "(12). Lactobacillus is considered one of probiotics species which improving the component of intestinal micro flora(13), preventing action on IBD, and relieving colic symptoms(14). Furthermore, Lactobacillus has pronounced antimicrobial effect (15) and may change gut pH by producing some acids which prevent growth of another pathogenic bacteria(16). Spirulina platensis is one of marine blue green microalgae with high nutritional values(17). It is rich source for many minerals, vitamins, protein and antioxidant anti-inflammatory compounds such as carotenoids, and phycocyanin pigment (18).Regarding to several effects of spirulina, it includes anticancer (19), antiviral (20), anti-allergic (21), antimutagenic (22), cytoprotective (23) and cardioprotective effects (24). Moreover it induces blood vessel-relaxing effect (25), hypo-cholesterolemic effect(26), hypolipidemic actions (27). In addition, hepatoprotective (28), neuroprotective, reduced concentrations of tumor necrosis factor (TNF-alpha) (29)and immune-enhancing action (30) were also reported. In the past, treatment of UC depended on aminosalicylates, antibiotics, steroids, and immune modulators but incomplete effectiveness and their adverse side effects, natural an-tioxidant anti-inflammatory agents are used nowadays to ameliorate UC (10). For that reason, the present work aimed to judge the modulatory effects of oral administration of Lactobacillus and/or Spirulina platensis in experimental colitis models in rats by estimating antioxidant parameters as well as molecular and histopathological investigations. Materials and methods Chemicals Lactéal fort (Lactobacillus LB) capsules purchased from Tenth of Ramadan for pharmaceutical industries &diagnostic reagents (Rameda), Egypt. Spirulina purchased from the Algal unit of Biotechnology (National Research Center, Dokki, and Cairo, Egypt). Mar-salaz tablet purchased from Marci pharmaceutical industries El- Obour City, Egypt. Acetic acid obtained from El-Nasr Pharmaceutical Chemicals Company (Cairo, Egypt). Diethyle ether obtained from (spinreact) Spin. EDTA from (Salix). Malondialdehyde (MDA), Cata-lase (CAT), Nitric oxide (NO) and Glutathione reduced (GSH) purchased from BIO- Synergistic ameliorative effect of Lactobacillus and Spirulina platensis against expermintal colitis in albinorats: 555 DIGNOSTIC Company kits-Egypt. All chemicals utilized in this study was of analytical grade. Animals and feed management Forty-nine male albino rats of average weight (100 g/rat) were purchased from Animal House Colony of the Tanta Center. The rats were adapted to standard laboratory conditions (temperature 22-25°C, relative humidity 50-60%), rats were fed a balanced diet and water ad libitum. Experimental design Rats after adaptation period (2 weeks) were distributed randomly into six (6) groups (8 rats in each except control positive 9 rats). The 1st group (negative control), received saline orally for 10 days then followed by normal saline instillation at tenth day per rectum. The 2nd group (positive control), received saline orally for 10 days followed by 2ml of acetic acid (4%) intrarectally (10)at tenth day. The 3rd group (Mesalazine group) received 2ml acetic acid (4%) rectal instillation on day 10, then given Mesalazine orally at dose (20mg/kg) (31)for 5days after induction of colitis. The 4th group (Lactobacillus group) received daily Lactobacillus at dose (1*109CFU) orally/rat (14)for 10 days, then rectal instillation of 2ml acetic acid (4%) at tenth day. The 5 th group (Spirulina group) received Spirulina daily at dose (500mg/kg) orally(32)for 10 days, then rectal instillation of 2ml acetic acid (4%) at tenth day. The 6th group (Spirulina + Lactoba-cillus) (combination group) oral received both Spirulina (500mg/kg) and Lactobacillus (1x109CFU) daily for 10 days, then rectal instillation of 2ml acetic acid (4%) at tenth day. All groups were observed daily for clinical signs, mortality rate, body weights, food and water intakes of the rats from each group. The rats were sacrificed at 5thday from induction of colitis, rats were slaughter under diethyl ether anesthesia. Colon segments were dissected, removed adipose tissue, washed with normal saline, for macroscopic and microscopic examination. Colon samples were preserved imme- diately at-80°C for molecular and oxidative stress analysis. Induction of colitis Colitis was produced by intra-colonic administration of 4% acetic acid (2ml)(33). Through a lubricant rectal pediatric urinary catheter under low-dose of ether anesthesia. Briefly, animals were restricted from food for 24h (34). The instillation site was about 8cm from the anal margin into the rectum. After instillation directly rats were maintained in vertical position for about 1 min to prevent acid leakage (35). Animals in the group one exposed to the same practice with saline instead of the acetic acid solution. Assessment of disease activity index According to (36), clinical signs in colitis were determined by evaluating the disease activity index (DAI)(table 1). The disease activity index includes (stool consistency, presence of rectal hemorrhage and decrease in body weight), DAI in all the tested groups were observed daily after induction of colitis by 4% acetic acid. Assessment of scoring severity of colitis For macroscopic damage score (Table 2), the colon was examined visually either for adhesions or gross morphological alterations immediately after death (37). Determination of colon weight and colon length After separated the colon from adipose tissue and remind intestine must be determine the colon weight (g) and colon length (cm). Tissue sample for histopathology and anti- oxidant biomarkers Tissue specimens of colon were rapidly taking and equally divided into three parts : proximal part for molecular investigation (rapidly stored at -80oC) (38), middle part used for histopathology, and distal part of colon stored at -20oC and used for oxidative stress and antioxi-dant parameters. 556 E. Ghazy, A. Mokhbatly, S. Keniber, K. Shoghy Estimation of antioxidant biomarkers of colon tissue homogenates Prior to dissection, distal part of colon were rinsed with phosphate buffered saline (PBS) solution, pH 7.4, containing 0.16 mg/ml heparin to get rid of any red blood cells and clot. Then tissue homogenize in 5-10 ml buffer (i.e. 50 Mm potassium phosphate pH 7.4, 1Mm EDTA and 1 ml/l triton x-100 /g. tissue and Centrifuge at 4.000 rpm for 15 min. at 4oC. The supernatant was separated into an Eppendorf tube and was preserved at -80oC into ali-quots for the spectrophotometric analysis of lipid peroxidation content by Malondialdehyde (MDA)(39), nitric oxide (NO) (40), Catalase (CAT)(41), and reduced Glutathione (GSH) estimation (42). Detection of gene expression quantitatively by real time PCR Forward and reverse primers sequence for iNOS, COX2 and fi-actin genes are presented in Table 3. Tissue RNA was extracted with Trizol (total RNA isolation reagent, iNtRON Biotechnology, Inc). Complementary DNA (cDNA) was synthesized by using Oligo (dT) primer HiSenSripte TMRH cDNA synthesis kits (IntROn) as described by the manufacturer's directions. The SYBR green was performed using BIoRad IQ2 (Japan) and the following protocol was used (43).The mRNA expression levels were normalized using P-actin. Histopathological studies Histopathological tissues preparation and examination was done according to (44)using H&E. Statistical analysis Data were presented as means ± S.E. using one-way ANOVA followed by Newman-keuls multiple comparisons using graph pad prism 7 software. Statistical significance was acceptable to a level of P <0.05 Results Mortality rate Control negative group, fed on standard diet, showed no abnormal clinical signs or mortality during the whole period of experimental. Mortality rate showed in table 4. Generally, the control positive group revealed obvious increase in mortality rate (4/9) (44.4%). Meanwhile, administration of Lactobacillus alone decreased mortality rate (2/8) (25%). Spirulina alone also decrease mortality rate (2/8) (25%). Furthermore, the co-administration of Lactobacillus with Spirulina led to pronounced decrease in mortality rate (1/8) (12.5%), similar to Mesalazine group which used for ulcerative colitis treatment. Disease activity index The effect of Lactobacillus and/or Spirulina in experimental colitis on DAI was explained in figurel. Data demonstrated significant (p < 0.05) elevation in DAI in acetic acid group as compared with control one. Mesalazine group revealed insignificant decrease in DAI in comparison with control positive group. Similarly, Lactobacillus group caused pronounced decrease in DAI but this reduction was still insignificant as contrasted with control positive group. Meanwhile, Spirulina alone or in combination with Lactobacillus revealed significant (p<0.05) improvement in DAI as matched with control positive group. Scoring severity of colitis The effect of Lactobacillus and/or Spirulina on macroscopic damage score was illustrated in figure 2. Data explored that significant (p <0.05) increase in damage score in acetic acid positive colitis as compared with the negative none treated one. Whereas, all other treated groups reported significant (p<0.05) reduction in score damage in comparison with the positive control group. Colon weight and length The effect of Lactobacillus and/or Spirulina on colon weight and colon length was showed in table 4 and figure 1. Concerning to colon Synergistic ameliorative effect of Lactobacillus and Spirulina platensis against expermintal colitis in albinorats: 557 weight, the obtained data illustrated significant (p<0.05) increase in the control positive group as contrasted with control negative one. On the other hand, Mesalazine group showed a decrease in colon weight but still insignificant as matched with control positive group. At the same time, simultaneous supplementation of Lactobacillus and/or Spirulina in acetic acid (4%) induced colitis revealed a significant (p<0.05) improvement in colon weight as compared with control positive group. Furthermore, control positive group showed significant (p<0.05) decrease in colon length in comparison with control negative group. Meanwhile, Mesalazine group revealed a significant (p<0.05) increase in colon length as contrasted with control positive group. Similarly, a significant (p<0.05) increase in colon length was detected in Lactobacillus and/or Spirulina groups as matched with control positive group. Lipid peroxidation and antioxidant bi- omarkers: The effect of Lactobacillus and/or Spirulina on Lipid peroxidation and antioxidant bi-omarkers were portrayed in figure 2. The data illustrated a significant (p<0.05) increase in MDA and nitric oxide in colon tissue homoge-nate in the control positive group as compared with control negative one. Meanwhile, control positive group showed marked decrease in GSH and CAT but this decrease still statically insignificant in comparison with control negative group. Nevertheless, Mesalazine group revealed a significant (p<0.05) decrease in MDA and NO content as contrasted with control positive group. Meanwhile, treatment by Mesalazine revealed increase in GSH and CAT but still insignificant as matched with control positive group. Regarding, Lactobacil-lus or Spirulina supplementation in acetic acid (4%) induced colitis group showed a significant (p<0.05) decrease in colon tissue content of MDA and NO as compared with control positive group. Meanwhile, increase in GSH and CAT were observed but still insignificant in contrast with control positive one. Similarly, the co-administration of Lacto-bacillus with Spirulina revealed a significant (p<0.05) decline in MDA and NO as contrasted with control positive group. On the other hand, obvious enhancement in GSH and CAT were observed as matched with control positive group. Molecular investigation The effect of Lactobacillus and/or Spirulina in acetic acid (4%) induced colitis on the quantitative gene expression by real time PCR were illustrated in figure 2. The data reflect a significant (p <0.05) increase iniNOS and COX2expressionin the control positive group as matched with the control negative one. On the contrary, Mesalazine group showed a significant (p<0.05) reduction in iNOS and COX2 expression as compared with control positive group. Gastric intubations of Lactoba-cillus or Spirulina showed a significant (p<0.05) improvement in iNOS and COX2expressionin comparison with control positive group. At the same time, simultaneous administration of Lactobacillus with Spirulina showed a significant (p<0.05) decline in iNOS and COX2 expression as contrasted with control positive group. Histopathological findings The effect of Lactobacillus and/or Spirulina in acetic acid (4%) induced colitis on histo-pathological features was showed in figure 3. Colon of rats in control negative group demonstrated normal epithelium, intestinal glands and intestinal lumen. Colon of rats in none treated positive group (severe colitis) revealed necrosis of surface epithelial and en-terocytes, deposition of irregular collagen fibers, crypt distortion, and loss of intestinal glands, mononuclear cell infiltration and apop-tosis of mononuclear cell. Colon of rats in the Mesalazine group (colitis) revealed patchy mucosal necrosis, proliferation of submucosal lymphoid elements such as, lymphocytes, macrophages and plasma cells with submuco-sal edema and necrosis together mononuclear cell infiltration. Colon of rats in the Lactoba-cillus group (moderate colitis) showed mono- 558 E. Ghazy, A. Mokhbatly, S. Keniber, K. Shoghy nuclear cell infiltration in the mucosa and submucosa, in addition to, congestion of sub-mucosal blood vessels and submucosal edema. Meanwhile, Lactobacillus group showed cell infiltration between the mucosal glands with necrosis of the surface enterocytes. Colon of rats in Spirulina group (mild colitis) showed mononuclear cell infiltration in the mucosa Table 1 : Scoring of disease activity index (DAI) Score Weight loss % Stool consistency Occult/gross bleeding 0 0 Normal Normal 1 1-5% - - 2 5-10% Loose stools Occult blood 3 10-15% - - 4 > 20% diarrhea Gross bleeding+ mucous Table 2: Primers used for qPCR Gene primer sequence (5/------3/) Reference iNOS F:CCTCCTCCACCCTACCAAGT R: CACCCAAAGTGCTTCAGTCA (Villaran et al., 2010) COX2 F:TGCGATGCTCTTCCGAGCTGTGCT R: TCAGGAAGTTCCTTATTTCCTTTC (Bhatia et al., 2008) ß-actin F: TGTGATGGTGGGAATGGGTCAG R: TTTGATGTCACGCACGATTTCC (Villaran et al., 2010) Table 4: colon lesion parameters (mean ± SE) in control and different treated groups C-ve C+ve M L S L+S Mortality rate% 0%_44.4% 12.5%_25%_25%_12.5% Body weight change 104.2±2.392a 81.6±1.97b 103±2.43a 102.9±1.96a 93±2.77a 95.5±3.6a Colon weight 1.28±0.09b 1.7±0.15a 1.45±0.08ab 1.36±0.08ab 1.43±0.13ab 1.4±0.04ab Colon length 14.54±0.20a 12±0.58c 13.2±0.2b 13.75±0.48ab 13.67±0.33ab 13.8±0.37ab weight/length ratio 0.09±0.01b 0.15±0.02a 0.11±0.0b 0.10±0.01b 0.11±0.02b 0.10±0.01b C-ve: Control negative, C+ve: Control Positive, (M): Mesalazine group, L: Lactobacillus group, SP: Spirulina group, L+SP: Lactobacillus +Spirulina. Data were statistically analyzed as mean ± SEM. Rows carrying different superscript letters are significantly different at p<0.05. and between the mucosal glands, edema in tunica muscularis, and normal surface epithelium. Colon of rats in Lactobacillus with Spirulina group (mild colitis) showed only mono-nuclear cell infiltration between the mucosal glands with normal surface epithelium. roups parameters Synergistic ameliorative effect of Lactobacillus and Spirulina platensis against expermintal colitis in albinorats: 559 Figure 1: A) refer body weight change (g), B) disease activity index, C) Macroscopic damage in control and different treated groups, D) Colon length in control and different treated groups 560 E. Ghazy, A. Mokhbatly, S. Keniber, K. Shoghy Figure 2: A, B) Lipid peroxidation, C,D) antioxidant biomarkers and E,F) molecular gene expression in control and different treated groups. Data were statistically analyzed as mean ± SEM. Rows carrying different superscript letters are significantly different at p<0.05 Synergistic ameliorative effect of Lactobacillus and Spirulina platensis against expermintal colitis in albinorats: 561 Figure 3: Histopathological feature in control and different treated groups. Microscopic features of colon rats by H&E stain. 1. Normal group, normal histologic architectures. 2. Acetic acid group (acute chemical colitis): showing necrosis of epithelial surface with deposition of irregular collagen fibers, crypt distortion, and loss of intestinal glands. 3. Mesalazine treated group, colitis: showing patchy mucosalnecrosis, proliferation of submucosal lymphoid elements (lymphocytes, macrophages and plasma cells), and sub-mucosal edema.4. Lactobacillus treated group moderate colitis: showing mononuclear cell infiltration in the mucosa and submucosa, congestion of submucosal blood vessels and edema. 5. Spirulina treated group, mild lymphocytic colitis: showing mononuclear cell infiltration in the mucosa, edema with normal surface epithelium. 6. Lactobacillus+Spirulina treated group, mild lymphocytic colitis: showing mononu-clear cell infiltration in the mucosa, with normal surface epithelium 562 E. Ghazy, A. Mokhbatly, S. Keniber, K. Shoghy Discussion Ulcerative colitis is an inflammatory condition which cause abdominal pain, bloody diarrhea and mucous in stool(7). Furthermore, weight loss and other many symptoms that differ from person to person and it may be lead to colorectal cancer (45). The study designed to evaluate the effect of some natural agents (Lactobacillus and/or Spirulina) to ameliorate the clinical manifestation of acetic acid (4%) induced colitis in rats. The present study exhibited significant increase in DAI and macroscopic damage in control positive rats as compared with the normal negative one. This increase could be referred to loss of appetite, decrease food consumption and feverish conditions which resulted in body weight reduction(46). Others referred this increase in DAI due to high inflammatory response and occasional ulceration that induced bloody diarrhea as confirmed by elevated mortality rate percent (44.4%), histo-pathological lesions and high macroscopic damage score and these results parallel with (9, 47). At the same time, rats of control positive group showed significant increase of colon weight with significant reduction in colon length as compared with non treated one that might be referred to cellular swelling which resulted from shift of extracellular water into the cells associated with neutrophils and macrophage infiltration. Furthermore, cells turgor, submucosal edema, vascular dilatation and goblet cell hyperplasia. Similar result was obtained by(48, 49). Oxidative stress and lipid peroxidation play acritical role in pathogenesis of ulcerative colitis (50, 51). Control positive animals revealed marked reduction in CAT and GSH, meanwhile, a significant elevation of MDA and NO was recorded as contrasted with control negative group. These results run parallel with those obtained by (52) Generation of ROS and free radicals from migrated neutrophils attack the cellular mac-romolecules and lead to epithelial cell disruption with extensive colon damage(53, 54). Furthermore, ROS lead to massive oxidation of cell membrane phospholipids, proteins, and DNA. This oxidation cause further stimulates of more neutrophils and macrophage infiltration to damaged tissue (49).Consequently, intestinal mucosa to regulate ROS levels, have enzymatic and non-enzymatic complex antiox-idant defenses mechanism such as reduced glutathione (GSH) and catalase (CAT), which try to heal and repair the damaged cells. Moreover, GSH and CAT were consumed by inflamed colon tissues to neutralize oxidative stress(55, 56). Malondialdehyde (MDA) considered the end result of lipid peroxidation which caused by ROS. Based on that, the elevated MDA in acetic acid (4%) induced colitis rats referred to the increased lipid peroxidation and high cell damage(57, 58). Nitric oxide (NO) is produced by some inflammatory cells such as granular leukocytes (neutrophils) or granular leukocytes (mono-cytes, and macrophages) as well as extravascu-lar compartment as epithelial cells from inflamed colon tissue. Thereby, elevated NO content in experimental colitis considered as an index of inflammation(59). Molecular genes expressions (iNOS and COX-2) are considered important gene expressions in acetic acid induced colitis, and they have synergistic effect to augmented the inflammatory reaction(60). Furthermore, recent investigations reported close interrelationship existing between iNOS and COX-2 expressions at sites of inflammation, and leading to excessive induction of inflammatory mediators which may causing the development of intestinal damage(61). In this study, colon tissues exposed to acetic acid produced an over expression of iNOS and COX-2messenger as compared with the control negative one, these results were supported by findings of (62, 63). Concerning to inducible nitric oxide syn-thase (iNOS), it is an enzyme usually expressed during inflammatory reactions(64). Meanwhile, synthesis of large amounts of nitric oxide (NO) content is demonstrated in acute or chronic inflammation, and it produced largely when iNOS expression is increased. Synergistic ameliorative effect of Lactobacillus and Spirulina platensis against expermintal colitis in albinorats: 563 Overall, elevated of iNOS expression may be an indicator of inflammation progression(10). Enhancement of COX-2 (inducible cy-clooxygenase enzyme) expressions from inflamed colon tissues play an integral role in the pathogenesis of ulcerative colitis (65, 66). Moreover, excessive over expressions of COX-2 lead to prostaglandin (PGE) releasing (67), and ROS production which cause further cell injury (65). Histopathological finding go side by side with the obtained macroscopic damage score, oxidative and anti-oxidative biomarkers in addition to molecular gene expressions, these data were in harmony with those obtained by(68). Concerning to Mesalazine, it considered positive drug control which inhibit oxidative stress and ROS releasing from inflamed colon tissue. Moreover, its antioxidant (31) antiinflammatory effects (69)reduced tissue damage and modulated mortality rate (12.5%). The current data declared that, gastric intubation of Lactobacillus on acetic acid induced colitis showed marked decrease in DAI in comparison with control positive group. This improvement of DAI could be owed to either increase body weigh by increasing energy efficiency (70) or decrease bloody diarrhea and these results were confirmed by decrease mortality rate percent (25%). On the other hand, Lactobacillus supplementation revealed significant decline in macroscopic damage score which was confirmed by reduce colon weight associated with significant enhancement of colon length as contrasted with control positive group (71). The damage score was ameliorated due to the ability of Lactobacillus to modulate inflammatory response of colon tissue through interferes with innate immune system and adaptive immunity. Therefore, it enhances anti-inflammatory cytokine production (72). Others attributed the reduction of inflammation by Lactobacillus to bacteriocins secretions which act as broad spectrum antimicrobial substances and protect against secondary infections, Consequently decreasing the duration of diarrhea (73). In the other hand, Lactobacil- lus may changepH of the gut flora which leading to reduce inflammatory state of colon(74). At the same time, lipid peroxidation and antioxidant biomarkers evaluate ability of Lactobacillus to reduce ROS and free radicals from inflamed colon (75). In this study, supplementation of Lactobacillus revealed an improvement of antioxidant biomarkers (GSH and CAT) and decreased in oxidative stress parameters (MDA and NO) as matched with control positive group, these facts were confirmed by result of (76). Multiple experimental studies demonstrated the antioxidative activities of Lactobacillus by secreting enzymatic and non enzymatic anti-oxidant substance and promoting its release from the inflamed colon tissue (77). Moreover, Lactobacillus enhance the production of particular antioxidant biomolecules, for example, exopolysaccharides (EPSs) which probably useful for elimination of oxidative stress from intestine (78). Consequently decrease lipid peroxidation (MDA) and NO marker from inflamed colon tissue. Finally, it exhibited metal chelating activities which get together with the pathogenesis of most chronic diseases(79). These results were confirmed by significant decrease in iNOS andCOX-2 expression in Lactobacillus supplemented group as matched with control positive group, these data are in harmony with those obtained by(80). Consistent with this mechanism, histo-pathological findings of Lactobacillus group were showed moderate colitis. This finding agrees with (12). Therefore, Lactobacillus had many therapeutic benefits and was used as vehicles for treatment of gastrointestinal dis-eases(81) In this experiment, Spirulina supplementation played great role in relieving the incidence of induced colitis. Significant enhancement in DAI and macroscopic damage score were recorded in Spirulina group as compared with control positive group, this effect could be explained by the high protein content, ami-no acids, vitamins (vitamin B complex) and folic acid which induce an increase in nutri- 564 E. Ghazy, A. Mokhbatly, S. Keniber, K. Shoghy tional value of this algae and promote weight gain (27). Similarly, a significant decline in bloody diarrhea was found in Spirulina group reach to (0%) may be due to mucopolysachharids content which makes building blocks of colon cell membranes and accelerates healing of colon tissue (30) These results were confirmed by decreased mortality rate to 25% and histo-pathological findings. Furthermore, gastric intubation of Spirulina in acetic acid induced colitis showed a marked decline in colon weight with significant increase in colon length as contrasted with control positive group. This improvement could be referred to ability of Spirulina to manage the inflammatory conditions and oxidative damages (82). Phycocyanin present in Spirulina considers anti-inflammatory ingredients of it, which decrease production of intracellular ROS and histamine from mast cell, inhibit inflammatory cell infiltration specially neutrophil and reduced edema index in the induced inflamma-tion(83, 84). Besides that, Spirulina has excellent antioxidative properties and preservative effects to structural integrity of colon tissue(85). In present study, Spirulina supplementation showed improvement in CAT and GSH and significant decrease in NO and MDA in comparison with control positive group. These results confirmed by molecular RNA expression of iNOS and COX-2 which showed significant decrease in contrast with control positive group. The antioxidant anti-inflammatory effects of Spirulina could be attributed to its content of phycocyanin and P-carotene which have capability to scavenge free radicals and ROS from inflamed colon tissue (86). In the same way, Spirulina decreased inflammatory mediators and inflammatory cyto-kines (IL-1^, IL-6, and IL-12) releasing from inflamed colon (87). Therefore, it plays a fundamental role in UC improvement. Moreover, histopathological finding of Spirulina showed mild colitis, and this result supported by finding of (32). Noteworthy, the combination of Spirulina and Lactobacillus in acetic acid induced colitis afforded a higher protection and more effectiveness than each one alone, this result was confirmed by recording the lowest mortality rate (12.5%), molecular expressions and histo-pathological findings similar to positive drug control (Mesalazine), that owed to an improvement growth performance and health condition (88). Spirulina not only modulate inflammatory response of acetic acid induced colitis, but also it consider as importance nutritional point for Lactobacillus due to rich source of protein content, amino acids, vitamins etc which could be needed to nourishment of Lactobacillus and improve intestinal colonization (89, 90). Over all, Spirulina has growth promoting effect on Lactobacillus to reduce the inflammatory effect of ulcerative colitis. Conclusion In conclusion, the protective effect of Lactobacillus and /or Spirulina against experimental colitis in rats could be directly attributed to scavenging ROS, inhibiting lipid peroxi-dation and suppressing NO releasing. Furthermore, Lactobacillus and Spirulina have synergistic protective effect on colon tissue and could be used in combination to ameliorate ulcerative colitis. Conflict of interest The authors declare that they have no conflict of interest. References 1. Shiina T, Shima T, Naitou K, Nakamori H, Sano Y, Horii K, et al. Actions of probiotics on trinitrobenzenesulfonic acid-induced colitis in rats. BioMed Research International. 2015;2015. 2. Coskun M. Intestinal epithelium in inflammatory bowel disease. Frontiers in medicine. 2014; 1: 24. 3. Molodecky NA, Soon S, Rabi DM, Ghali WA, Ferris M, Chernoff G, et al. Increasing incidence and prevalence of the inflammatory bowel diseases with time, based on systematic review. Gastroenterology. 2012;142(1): 46-54. e42. 4. Laroui H, Ingersoll SA, Liu HC, Baker MT, Ayyadurai S, Charania MA, et al. Dextran sodium sulfate (DSS) induces colitis in mice by forming nano-lipocomplexes with medium-chain-length Synergistic ameliorative effect of Lactobacillus and Spirulina platensis against expermintal colitis in albinorats: 565 fatty acids in the colon. PloS one. 2012; 7(3): e32084. 5. Oliveira Lgd, Cunha Ald, Duarte Ac, Casta-ñon Mcmn, Chebli Jmf, Aguiar Jakd. Positive correlation between disease activity index and matrix metalloproteinases activity in a rat model of colitis. Arquivos de gastroenterologia. 2014;51(2): 10712. 6. Silveira ALM, Ferreira AVM, de Oliveira MC, Rachid MA, da Cunha Sousa LF, dos Santos Martins F, et al. Preventive rather than therapeutic treatment with high fiber diet attenuates clinical and inflammatory markers of acute and chronic DSS-induced colitis in mice. European journal of nutrition. 2017; 56(1): 179-91. 7. Najafi A, Motaghi E, Hosseini MJ, Ghasemi-Pirbaluti M. The effect of sodium valproate on acetic acid-induced colitis in rats. Inflammophar-macology. 2017;25(1): 137-45. 8. Ashry EE, Abdellatief RB, Mohamed AE, Kotb HI. Protective Effect of Ketamine against Acetic Acid-Induced Ulcerative Colitis in Rats. Pharmacology & Pharmacy. 2016;7(01): 9. 9. Guerra GC, Araújo AA, Lira GA, Melo MN, Souto KK, Fernandes D, et al. Telmisartan decreases inflammation by modulating TNF-a, IL-10, and RANK/RANKL in a rat model of ulcerative colitis. Pharmacological Reports. 2015;67(3): 5206. 10. Bezerra GB, de Souza LdM, dos Santos AS, de Almeida GKM, Souza MTS, Santos SL, et al. Hydroalcoholic extract of Brazilian red propolis exerts protective effects on acetic acid-induced ulcerative colitis in a rodent model. Biomedicine & Pharmacotherapy. 2017;85: 687-96. 11. Ali AA, Al Haleem ENA, Khaleel SA-H, Sallam AS. Protective effect of cardamonin against acetic acid-induced ulcerative colitis in rats. Pharmacological Reports. 2017; 69(2): 268-75. 12. Toumi R, Soufli I, Rafa H, Belkhelfa M, Biad A, Touil-Boukoffa C. Probiotic bacteria lac-tobacillus and bifidobacterium attenuate inflammation in dextran sulfate sodium-induced experimental colitis in mice. International journal of im-munopathology and pharmacology. 2014;27(4): 615-27. 13. Wang Y-h, Huang Y. Effect of Lactobacil-lus acidophilus and Bifidobacterium bifidum supplementation to standard triple therapy on Helico-bacter pylori eradication and dynamic changes in intestinal flora. World Journal of Microbiology and Biotechnology. 2014;30(3): 847-53. 14. Lorén V, Manyé J, Fuentes MC, Cabré E, Ojanguren I, Espadaler J. Comparative effect of the I3. 1 probiotic formula in two animal models of colitis. Probiotics and antimicrobial proteins. 2017;9(1): 71-80. 15. Kaur IP, Kuhad A, Garg A, Chopra K. Probiotics: delineation of prophylactic and therapeutic benefits. Journal of medicinal food. 2009;12(2): 219-35. 16. Islam SU. Clinical uses of probiotics. Medicine. 2016;95(5). 17. Kostyukevich Y, Vlaskin M, Vladimirov G, Zherebker A, Kononikhin A, Popov I, et al. The investigation of the bio-oil produced by hydrothermal liquefaction of Spirulina platensis using ultrahigh resolution Fourier transform ion cyclotron resonance mass spectrometry. European Journal of Mass Spectrometry. 2017; 23(2): 83-8. 18. González-Torres L, Vázquez-Velasco M, Olivero-David R, Bastida S, Benedí J, González RR, et al. Glucomannan and glucomannan plus spirulina added to pork significantly block dietary cholesterol effects on lipoproteinemia, arylesterase activity, and CYP7A1 expression in Zucker fa/fa rats. Journal of physiology and biochemistry. 2015; 71 (4): 773-84. 19. Zhang L-X, Cai C-E, Guo T-T, Gu J-W, Xu H-L, Zhou Y, et al. Anti-cancer effects of polysac-charide and phycocyanin from Porphyra yezoensis. Journal of Marine Science and Technology. 2011; 19(4): 377-82. 20. Murugan T. Screening for antifungal and antiviral activity of C-phycocyanin from Spirulina Platensis. 2011. 21. Pankaj PP, Varma M. Potential role of Spirulina platensis in maintaining blood parameters in alloxan induced diabetic mice. Int J Pharm Pharm Sci. 2013;5: 450-6. 22. Basha OM, Hafez RA, El-Ayouty YM, Mahrous KF, Bareedy MH, Salama AM. C-Phycocyanin inhibits cell proliferation and may induce apoptosis in human HepG2 cells. Egypt J Immunol. 2008; 15(2): 161-7. 23. Elzawahry ZA, Abass MA, El-Haleem MRA, Hamid RAA, Atteia HH. Spirulina protects against tacrolimus-induced hepatic and renal tox-icity in rats: A biochemical and histological study. Journal of Toxicology and Environmental Health Sciences. 2016;8 (7): 46-56. 24. Khan M, Shobha JC, Mohan IK, Naidu MUR, Sundaram C, Singh S, et al. Protective effect of Spirulina against doxorubicin-induced car-diotoxicity. Phytotherapy Research. 2005; 19(12): 1030-7. 25. Gur CS, Erdogan DK, Onbasilar I, Atilla P, Cakar N, Gurhan ID. In vitro and in vivo investiga- 566 E. Ghazy, A. Mokhbatly, S. Keniber, K. Shoghy tions of the wound healing effect of crude Spirulina extract and C-phycocyanin. Journal of Medicinal Plants Research. 2013; 7(8): 425-33. 26. Nagaoka S, Shimizu K, Kaneko H, Shiba-yama F, Morikawa K, Kanamaru Y, et al. A novel protein C-phycocyanin plays a crucial role in the hypocholesterolemic action of Spirulina platensis concentrate in rats. The Journal of nutrition. 2005; 135(10): 2425-30. 27. Deng R, Chow TJ. Hypolipidemic, antioxi-dant, and antiinflammatory activities of microalgae Spirulina. Cardiovascular therapeutics. 2010;28(4). 28. Kuriakose GC, Kurup MG. Hepatoprotec-tive effect of Spirulina lonar on paracetamol induced liver damage in rats. Asian J Exp Biol Sci. 2010;1(3): 614-23. 29. Benedetti S, Benvenuti F, Pagliarani S, Francogli S, Scoglio S, Canestrari F. Antioxidant properties of a novel phycocyanin extract from the blue-green alga Aphanizomenon flos-aquae. Life sciences. 2004;75(19): 2353-62. 30. Somchit MN, Mohamed NA, Ahmad Z, Zakaria ZA, Shamsuddin L, Omar-Fauzee M, et al. Anti-inflammatory and anti-pyretic properties of Spirulina platensis and Spirulina lonar: a comparative study. Pakistan journal of pharmaceutical sciences. 2014;27(5): 1277-80. 31. Moura RM, Hartmann RM, Licks F, Schemitt EG, Colares JR, do Couto Soares M, et al. Antioxidant effect of mesalazine in the experimental colitis model induced by acetic acid. Journal of Coloproctology (Rio de Janeiro). 2016;36(3): 139-48. 32. Abdel-Daim MM, Farouk SM, Madkour FF, Azab SS. Anti-inflammatory and immuno-modulatory effects of Spirulina platensis in comparison to Dunaliella salina in acetic acid-induced rat experimental colitis. Immunopharmacology and immunotoxicology. 2015;37(2): 126-39. 33. Al-Rejaie SS, Abuohashish HM, Al-Enazi MM, Al-Assaf AH, Parmar MY, Ahmed MM. Protective effect of naringenin on acetic acid-induced ulcerative colitis in rats. World Journal of Gastroenterology: WJG. 2013;19(34): 5633. 34. Fattahian E, Hajhashemi V, Rabbani M, Minaiyan M, Mahzouni P. Anti-inflammatory effect of amitriptyline on ulcerative colitis in normal and reserpine-induced depressed rats. Iranian journal of pharmaceutical research: IJPR. 2016;15(Suppl): 125. 35. Dodda D, Chhajed R, Mishra J. Protective effect of quercetin against acetic acid induced inflammatory bowel disease (IBD) like symptoms in rats: Possible morphological and biochemical al- terations. Pharmacological Reports. 2014;66(1): 169-73. 36. Murthy S, Cooper HS, Shim H, Shah RS, Ibrahim SA, Sedergran DJ. Treatment of dextran sulfate sodium-induced murine colitis by intraco-lonic cyclosporin. Digestive diseases and sciences. 1993;38(9): 1722-34. 37. Motta J-P, Flannigan KL, Agbor TA, Beatty JK, Blackler RW, Workentine ML, et al. Hydrogen sulfide protects from colitis and restores intestinal microbiota biofilm and mucus production. Inflammatory bowel diseases. 2015;21(5): 1006-17. 38. Nagy ZT. A hands-on overview of tissue preservation methods for molecular genetic analyses. Organisms Diversity & Evolution. 2010;10(1): 91-105. 39. Ohkawa H, Ohishi N, Yagi K. Assay for lipid peroxides in animal tissues by thiobarbituric acid reaction. Analytical biochemistry. 1979;95(2): 351-8. 40. Ingram G, Montgomery H, Dymock JF, Henneberry G, Baker B, Forbes J, et al. Notes. Analyst. 1961;86(1023): 411-22. 41. Saleh A A, El-Magd M A. Beneficial effects of dietary silver nanoparticles and silver nitrate on broiler nutrition, Environmental Science and Pollution Research 2018; 25(27): 2703127038. 42. Owens C, Belcher R. A colorimetric micro-method for the determination of glutathione. Biochemical Journal. 1965;94(3): 705. 43. Kim YH, Kwon H-S, Kim DH, Cho HJ, Lee HS, Jun J-G, et al. Piceatannol, a stilbene present in grapes, attenuates dextran sulfate sodium-induced colitis. International immunopharmacolo-gy. 2008;8(12): 1695-702. 44. Dries DJ. Histological and histochemical methods: theory and practice. Shock. 2008;30 (4): 481. 45. Kim ER, Chang DK. Colorectal cancer in inflammatory bowel disease: the risk, pathogene-sis, prevention and diagnosis. World Journal of Gastroenterology: WJG. 2014;20(29): 9872. 46. Minaiyan M, Hajhashemi V, Rabbani M, Fattahian E, Mahzouni P. Evaluation of anti-colitic effect of fluvoxamine against acetic acid-induced colitis in normal and reserpinized depressed rats. European journal of pharmacology. 2015;746: 293300. 47. Adjadj M, Boumerfeg S, Charef N, Baghia-ni A, Khennouf S, Arrar L, et al. Protective Effect of Paronychia argentea L. on Acetic Acid Induced Ulcerative Colitis in Mice by Regulating Antioxi-dant Parameters and inflammatory Markers. Synergistic ameliorative effect of Lactobacillus and Spirulina platensis against expermintal colitis in albinorats: 567 48. D'Argenio G, Mazzone G, Tuccillo C, Ri-becco MT, Graziani G, Gravina AG, et al. Apple polyphenols extract (APE) improves colon damage in a rat model of colitis. Digestive and Liver Disease. 2012;44(7): 555-62. 49. Krishnan M, Jayaraj RL, Megala J, Elango-van N. Antioxidant mediated antiulcer effect of Eupatorium triplinerve Vahl against acetic acid induced ulcerative colitis in mice. Biomedicine & Aging Pathology. 2014;4(2): 153-60. 50. Prabhu V, Guruvayoorappan C. Protective effect of marine mangrove Rhizophora apiculata on acetic acid induced experimental colitis by regulating anti-oxidant enzymes, inflammatory mediators and nuclear factor-kappa B subunits. International immunopharmacology. 2014;18(1): 124-34. 51. El Morsy EM, Kamel R, Ahmed MA. Attenuating effects of coenzyme Q10 and amlodipine in ulcerative colitis model in rats. Immunopharma-cology and immunotoxicology. 2015;37(3): 24451. 52. Pasala PK, Alluri R, Mavulati SC, Mailavaram RP, Shaik K, Konduri P. Protective Effect of A. 53. Hasko G, Linden J, Cronstein B, Pacher P. Adenosine receptors: therapeutic aspects for inflammatory and immune diseases. Nature reviews Drug discovery. 2008;7(9): 759. 54. Mohamed A, Metwally N, Mohamed S, Hassan E. Protective capacity of butanolic extract of myoporum laetum against oxidative stress and immune disorder induced tissue damage in profenofos intoxicated rats. International Journal of Academic Research. 2010;2(2). 55. Subudhi U, Chainy GB. Expression of hepatic antioxidant genes in l-thyroxine-induced hyperthyroid rats: regulation by vitamin E and curcumin. Chemico-Biological Interactions. 2010;183(2): 304-16. 56. Kandhare AD, Raygude KS, Ghosh P, Ghule AE, Gosavi TP, Badole SL, et al. Effect of hydroalcoholic extract of Hibiscus rosa sinensis Linn. leaves in experimental colitis in rats. Asian Pacific journal of tropical biomedicine. 2012;2(5): 337-44. 57. Karbiner MS, Sierra L, Minahk C, Fonio MC, de Bruno MP, Jerez S. The role of oxidative stress in alterations of hematological parameters and inflammatory markers induced by early hypercholesterolemia. Life sciences. 2013;93(15): 5038. 58. Kumar CS, Reddy KK, Reddy AG, Vinoth A, Boobalan G, Rao G. Protective effect of Lactobacillus plantarum 21, a probiotic on trinitroben- zenesulfonic acid-induced ulcerative colitis in rats. International immunopharmacology. 2015;25(2): 504-10. 59. Gillberg L, Varsanyi M, Sjostrom M, Lo-rdal M, Lindholm J, Hellstrom PM. Nitric oxide pathway-related gene alterations in inflammatory bowel disease. Scandinavian journal of gastroenterology. 2012;47(11): 1283-98. 60. Sakthivel K, Guruvayoorappan C. Amen-toflavone inhibits iNOS, COX-2 expression and modulates cytokine profile, NF-kB signal transduction pathways in rats with ulcerative colitis. International immunopharmacology. 2013;17(3): 90716. 61. Ya Sklyarov A, Panasyuk N, Fomenko I. Role of nitric oxide-synthase and cyclooxygen-ase/lipooxygenase systems in development of experimental ulcerative colitis. Journal of Physiology and Pharmacology. 2011;62(1): 65. 62. Sadar SS, Vyawahare NS, Bodhankar SL. Ferulic acid ameliorates TNBS-induced ulcerative colitis through modulation of cytokines, oxidative stress, iNOs, COX-2, and apoptosis in laboratory rats. EXCLI journal. 2016;15: 482. 63. Wu X, Song M, Gao Z, Sun Y, Wang M, Li F, et al. Nobiletin and its colonic metabolites suppress colitis-associated colon carcinogenesis by down-regulating iNOS, inducing antioxidative enzymes and arresting cell cycle progression. The Journal of nutritional biochemistry. 2017;42: 1725. 64. Naito Y, Takagi T, Handa O, Ishikawa T, Nakagawa S, Yamaguchi T, et al. Enhanced intestinal inflammation induced by dextran sulfate sodium in tumor necrosis factor-alpha deficient mice. Journal of gastroenterology and hepatology. 2003;18(5): 560-9. 65. Takedatsu H, Taylor KD, Mei L, McGovern DP, Landers CJ, Gonsky R, et al. Linkage of Crohn's disease-related serological phenotypes: NFKB1 haplotypes are associated with anti-CBir1 and ASCA, and show reduced NF-kB activation. Gut. 2009;58(1): 60-7. 66. Carroll IM, Andrus JM, Bruno-Barcena JM, Klaenhammer TR, Hassan HM, Threadgill DS. Anti-inflammatory properties of Lactobacillus gasseri expressing manganese superoxide dis-mutase using the interleukin 10-deficient mouse model of colitis. American Journal of Physiology-Gastrointestinal and Liver Physiology. 2007;293 (4): G729-G38. 67. Rogler G, Andus T. Cytokines in inflammatory bowel disease. World journal of surgery. 1998;22(4): 382-9. 568 E. Ghazy, A. Mokhbatly, S. Keniber, K. Shoghy 68. Baumgart D, Baumgart DC. Crohn's Disease and Ulcerative Colitis: Springer; 2017. 69. Zeng C, Xiao J-H, Chang M-J, Wang J-L. Beneficial effects of THSG on acetic acid-induced experimental colitis: involvement of upregulation of PPAR-y and inhibition of the Nf-Kb inflammatory pathway. Molecules. 2011;16(10): 8552-68. 70. Million M, Angelakis E, Paul M, Armou-gom F, Leibovici L, Raoult D. Comparative meta-analysis of the effect of Lactobacillus species on weight gain in humans and animals. Microbial pathogenesis. 2012;53(2): 100-8. 71. Fernandez EM, Valenti V, Rockel C, Hermann C, Pot B, Boneca IG, et al. Anti-inflammatory capacity of selected lactobacilli in experimental colitis is driven by NOD2-mediated recognition of a specific peptidoglycan-derived muropeptide. Gut. 2011: gut. 2010.232918. 72. Dos Santos T, Melo T, Santos D, Rezende R, Dias J, Romano C. Efficacy of oral administration of lactic acid bacteria isolated from cocoa in a fermented milk preparation: reduction of colitis in an experimental rat model. Genet Mol Res. 2016;15(3). 73. Ciszek-Lenda M, Nowak B, Srottek M, Gamian A, Marcinkiewicz J. Immunoregulatory potential of exopolysaccharide from Lactobacillus rhamnosus KL37. Effects on the production of inflammatory mediators by mouse macrophages. International journal of experimental pathology. 2011;92(6): 382-91. 74. AFRC RF. Probiotics in man and animals. Journal of Applied Microbiology. 1989;66(5): 36578. 75. Motterlini R, Foresti R, Bassi R, Green CJ. Curcumin, an antioxidant and anti-inflammatory agent, induces heme oxygenase-1 and protects endothelial cells against oxidative stress. Free Radical Biology and Medicine. 2000;28(8): 1303-12. 76. Spyropoulos BG, Misiakos EP, Fotiadis C, Stoidis CN. Antioxidant properties of probiotics and their protective effects in the pathogenesis of radiation-induced enteritis and colitis. Digestive diseases and sciences. 2011;56(2): 285-94. 77. de LeBlanc AdM, LeBlanc JG, Perdigon G, Miyoshi A, Langella P, Azevedo V, et al. Oral administration of a catalase-producing Lactococcus lactis can prevent a chemically induced colon cancer in mice. Journal of Medical Microbiology. 2008;57(1): 100-5. 78. Kodali VP, Sen R. Antioxidant and free radical scavenging activities of an exopolysaccha-ride from a probiotic bacterium. Biotechnology journal. 2008;3(2): 245-51. 79. Lee J, Hwang K-T, Heo M-S, Lee J-H, Park K-Y. Resistance of Lactobacillus plantarum KCTC 3099 from Kimchi to oxidative stress. Journal of medicinal food. 2005;8(3): 299-304. 80. Peran L, Sierra S, Comalada M, Lara-Villoslada F, Bailón E, Nieto A, et al. A comparative study of the preventative effects exerted by two probiotics, Lactobacillus reuteri and Lactobacillus fermentum, in the trinitrobenzenesulfonic acid model of rat colitis. British Journal of Nutrition. 2007;97(1): 96-103. 81. Csutak O, Sarbu I. Genetically Modified Microorganisms: Harmful or Helpful? Genetically Engineered Foods: Elsevier; 2018. p. 143-75. 82. Abdel-Daim MM. Pharmacodynamic interaction of Spirulina platensis with erythromycin in Egyptian Baladi bucks (Capra hircus). Small Ruminant Research. 2014;120(2): 234-41. 83. Gonzalez R, Rodriguez S, Romay C, González A, Armesto J, Remirez D, et al. Anti-inflammatory activity of phycocyanin extract in acetic acid-induced colitis in rats. Pharmacological research. 1999;39(1): 55-9. 84. González R, González A, Remirez D, Romay C, Rodriguez S, Ancheta O, et al. Protective effects of phycocyanin on galactosamine-induced hepatitis in rats. Biotechnology Aplicada. 2003;20: 107-10. 85. Sharma K, Upreti N, Sharma S, Sharma S. Protective effect of Spirulina and tamarind fruit pulp diet supplement in fish (Gambusia affinis Baird & Girard) exposed to sublethal concentration of fluoride, aluminum and aluminum fluoride. 2012. 86. Vo T-S, Ryu B, Kim S-K. Purification of novel anti-inflammatory peptides from enzymatic hydrolysate of the edible microalgal Spirulina maxima. Journal of Functional Foods. 2013;5(3): 1336-46. 87. Katsuura S, Imamura T, Bando N, Yamanishi R. P-Carotene and P-cryptoxanthin but not lutein evoke redox and immune changes in RAW264 murine macrophages. Molecular nutrition & food research. 2009;53(11): 1396-405. 88. Ramakrishnan CM, Haniffa M, Manohar M, Dhanaraj M, Arockiaraj AJ, Arunsingh S. Effects of probiotics and spirulina on survival and growth of juvenile common carp (Cyprinus carpio). 2008. 89. Parada JL, de Caire GZ, de Mule MCZ, de Cano MMS. Lactic acid bacteria growth promoters from Spirulina platensis. International journal of food microbiology. 1998;45(3): 225-8. 90. Varga L, Szigeti J, Kovács R, Foldes T, Buti S. Influence of a Spirulina platensis biomass Synergistic ameliorative effect of Lactobacillus and Spirulina platensis against expermintal colitis in albinorats: 569 on the microflora of fermented ABT milks during storage (R1). Journal of dairy science. 2002;85(5): 1031-8. 91. Villarán RF, Espinosa-Oliva AM, Sarmiento M, De Pablos RM, Argüelles S, Delgado-Cortés MJ, et al. Ulcerative colitis exacerbates lipopolysaccharide-induced damage to the nigral dopaminergic system: potential risk factor in Parkinsons disease. Journal of neurochemistry. 2010; 114(6): 1687-700. 92. Bhatia HS, Candelario-Jalil E, de Oliveira ACP, Olajide OA, Martínez-Sánchez G, Fiebich BL. Mangiferin inhibits cyclooxygenase-2 expression and prostaglandin E2 production in activated rat microglial cells. Archives of biochemistry and biophysics. 2008;477(2): 253-8. Slov Vet Res 2019; 56 (Suppl 22): 571-7 DOI 10.26873/SVR-794-2019 Original Research Article HEMATOLOGICAL AND BIOCHEMICAL PROFILE IN FEMALE CAMELS (CAMELUS DROMEDARIUS) DURING THE TRANSITION PERIOD Eman A.Ebissy1, Ahmed A. El-Sayed1*, Ragab Hassan Mohamed2 1Department of Animal health and Poultry, Animal and Poultry Production Division, Desert Research Center (DRC), Matariya, Cairo, Egypt, 2Theriogenology Department, Faculty of Veterinary Medicine, Aswan University, Aswan, Egypt Corresponding author, E-mail: decernes@yahoo.com Abstract: The aim of the present study was to evaluate the hematological and biochemical alterations in female camels during the transition period. Ten apparently healthy camels were randomly selected and subjected to clinical examination. The investigated camels demonstrated no detectable clinical illness and kept under veterinary supervision throughout the study period. A blood sample was collected from each camel for laboratory investigation before and after 2 and 4 weeks of parturition and at time of parturition. Our findings revealed a significant (p=0.001) high values of neutrophils, MCH and MCHC at +14, and a significant (p=0.001) low values of lymphocyte count at -14 and at time of parturition. The globulin concentration increased significantly (p=0.029) at +28. For serum phosphorus and magnesium concentrations, their values were significantly (P< 0.05) elevated at -14 and +28, respectively, while enzymatic activity of liver enzymes including aspartate aminotransferase (AST), Y-glutamyl transferase (GGT) and alkaline phosphatase (ALP) were significantly (P< 005) elevated at +14, while serum activity of GGT and ALP continued at the same pattern at +28. For serum glucose and cortisol concentrations, their values were significantly elevated at (0), while serum creatinine concentrations were significantly (p=0.023) decreased at +14. Estrogen concentration increased significantly at -14 and 0, but decreased significantly at +14 and +28. Progesterone concentrations increased significantly at -14 and decreased at 0, +14 and +28. Other tested parameters did not differ significantly pre- and post-partum. The data presented in this study could be used as a reference guide for female camels during the transition period. Key words: Biochemistry; camel; hematology; transition period Introduction Dromedary camels are characterized by a seasonal activity and induced ovulators. Reproductive physiology of Arabian dromedary camels has gotten little interest compared to other animal species, especially in relation with changes in blood constituents. Changes in several biochemical parameters have been reprimanded for reproductive disappointments (1). Severe economic losses can result from suboptimal transition of pregnant animal from the late-pregnancy to lactation; this could probably to impaired production and reproductive performance (2, 3). Hence, optimal transition Received: January 2019 Accepted for publication: February 2019 572 E. Ebissy, A. El-Sayed, R. Mohamed requires a comprehensive understanding of biochemical alterations that occurs during the transition period (4). The transition period, defined as three weeks around the time of calving, is characterized by distinct endocrine alterations that are much more dramatic than at any other time stage of lactation-gestation cycle (2, 5). In Egypt, only limited data on serum biochemistry and hema-tology of one humped camel are available in the literature (6) and most of these literatures encompass limited laboratory measurements (7, 8). Therefore, the present study is an attempt to provide an insight into the dynamics of selected haematobiochemical alterations in the transitional one humped camel in order to provide potentially new and useful information about the guidelines for the management strategies during different physiological phases. Materials and methods Animals The present study included ten apparently healthy female dromedary camels reared at Mariut Research Station, Desert Research Center, El-Amria, Alexandria, Egypt. Their mean body weight was 415 kg (range: 313 - 590 kg) and their mean ages were 12.6 years (range: 10 -15 years). All procedures were performed in accordance with the guidelines of Desert Research Center (Egypt) and approved by its Ethical Committees. The camels were considered clinically sound on the basis of physical examination of heart, lungs, rumen and intestine and other vital signs (9, 10) in tandem with the preliminary findings of hematological examination. Camels were housed in an open yard and fed on a maintenance ration composed of a concentrate mixture including 50% corn, 47% barley, 2% minerals, 1% salt which given at rate of 3 kg/head/day, while Egyptian clover hay (Tri-folium alexandrinum) and fresh water were offered ad libitium. Blood sampling Ten milliliter of blood was collected from each animal at -28,-14, 0, +14 and + 28 days of expected date of delivery via jugular vein puncture using vacutainer tube containing anticoagulant (EDTA or sodium fluoride) and without anticoagulant to yield whole blood or serum, respectively. The tubes containing EDTA were used for prompt hematologic examination. The hematological indices included total leucocytes (WBC) and differential leukocyte count (lymphocytes, neutrophils, monocytes, and esin-ophils), red blood cells (RBC), hemoglobin (HGB), hematocrit, mean erythrocyte volume (MCV), mean corpuscular hemoglobin (MCH), and mean corpuscular hemoglobin concentration (MCHC) which were carried out on automatic blood cell counter (Exigoeos veterinary Hematology system, Boule Medical AB, Sweden). On the other side, tubes containing sodium fluoride were used for quantifying concentrations of glucose which were measured spectrophotometrically using a commercial test kit supplied by Spectrum Egypt (Ref: 250-001).The plain tubes were kept at room temperature overnight to be centrifuged at 3000 rpm for 15 minutes. Only clear sera were collected then aliquoted and kept frozen at -20 0C for subsequent biochemical analyses using commercial test kits according to the standard protocols of suppliers. For the total protein, albumen, calcium, phosphorus, magnesium and cre-atinine, commercial test kits supplied by Bio-Med Egypt were used (Ref: ALB100250; TP116250; CAL103100; PH123100, MG122050 and CRE 106100), respectively. For AST, ALP, j GT and cholesterol, commercial test kits supplied by Spectrum Egypt were used (Ref: AST260001; ALP214001;j GT246001 and CH0L230001), respectively. For BUN, commercial test kits supplied by Bi-oScien Egypt were used (Ref: BSU117100). For cortisol and estradiol, commercial test kits supplied by Parameter™ USA were used (Ref: KGE008B and KGE014), respectively. For Progesterone, commercial test kit supplied by Oxford Biomedical Research USA was used Hematological and biochemical profile in female camels (Camelus dromedarius) during the transition period 573 (Ref: EA 74). For creatinine kinase, commercial test kit supplied by Bio Chain USA (Ref: Z5030048) was used. Statistical analysis Statistical analyses were carried out using a statistical software program (SPSS, ver.20, Inc., Chicago, USA). Descriptive statistics were performed for all parameters. Repeated measures ANOVA was used to test the effect of physiological status during the transitional period on blood metabolic profile. Results were considered statistically significant at P < 0.05. Results An overview of serial measurements of hematological and serum biochemical profile in the studied camels during the transition period was illustrated in Tables 1 &2. Clinically, the investigated animals showed no detectable clinical alterations throughout the study period and remained clinically healthy. All camels demonstrated normal laboring and delivered a single calf without obvious clinical illness. Table 1 illustrates the hematological variables in examined camels. The total white blood cells revealed non-significant differences (P = 0.296) through the different time points (-28, -14, 0, +14 and +28). Lymphopenia was observed at - 14 and 0 time points (P = 0.001) and neutrophilia at +14 and +28 postpartum (P = 0.001). There was no significant difference between Monocyte and eosinophils during the periparturient period (P = 0.730 and P=0.447, respectively). There was no significant difference between the red blood cells, haemoglobin, hematocrit and MCV (P>0.05) throughout the periparturient period. On the other side, the MCH and MCHC increased significantly at+14 after parturition (P=0.001). Table 2 summarizes the biochemical variables in examined camels. Serum concentrations of the total protein and albumin concentrations did not differ significantly among all the tested time points pre- and post-partum (P>0.05). However, globulin concentrations were significantly (P=0.029) increased at +28. Calcium concentration did not differ significantly among all the tested time-points pre- and post-partum (P=0.201). While concentrations of serum inorganic phosphorus and magnesium were significantly (P=0.004 and 0.023) increased at -14 and +28 respectively. For serum activity of AST, GGT and ALP, their values showed a significant increase (P>0.05) at +14. The concentration of ALP and GGT remained also at a significant (P = 0.001) high pattern at +28 after parturition. In contrast the serum activity of creatinine kinase did not differ significantly pre- and postpartum (P = 0.125). Concentrations of serum glucose were significantly (P = 0.001) elevated at the time of parturition. The serum concentration of BUN did not show any significance at the tested time points (P = 0.370). However, creatinine concentrations were significantly (P = 0.023) decreased at +14 post-partum. Serum concentration of cortisol showed a significantly (P = 0.001) elevation at the time of parturition. hand the concentration of serum estrogen was increased significantly at -14 and 0, but decreased significantly at +14 and +28 (P = 0.001). The serum progesterone level increased significantly at -14 and decreased at 0, +14 and +28 (P = 0.001). 574 E. Ebissy, A. El-Sayed, R. Mohamed Table 1: Hematological parameters (mean ± SEM) in female dromedary camels during the periparturient period (n=10) - 28 - 14 0 + 14 + 28 P value WBC (x109/L) 11.24 ± 0.49 8.52 ± 1.29 9.96 ± 1.22 11.75 ± 1.65 9.50 ± 0.68 0.296 RBC (x 1012/L) 10.17 ± 0.71 11.65 ± 0.25 10.74 ± 0.42 9.09 ± 1.23 10.90 ± 0.44 0.218 Hb (g/dl) 10.98 ± 0.82 11.64 ± 0.46 10.62 ± 0.55 12.30 ± 0.45 11.64 ± 0.96 0.414 PCV% 33.70 ± 1.24 30.56 ± 1.31 32.10 ± 1.44 33.00 ± 1.22 29.98 ± 1.64 0.319 MCV (fL) 31.42 ± 1.06 30.36 ± 1.71 30.06 ± 1.53 30.66 ± 1.25 28.14 ± 1.50 0.583 MCH (pg) 9.00 ± 0.61 12.90 ± 0.78 8.98 ± 0.54 14.74* ± 0.53 10.58 ± 0.46 0.001* MCHC (g/dl) 31.95 ± 3.04 36.74 ± 2.19 26.65 ± 0.95 44.38 *± 0.24 32.45 ± 1.71 0.001* Lymph (x109/L) 6.69 ± 0.32 3.01* ± 0.66 1.92* ± 0.31 3.69 ± 0.50 6.16 ± 0.52 0.001* Monocyte (x 109/L) 0.28 ± 0.04 0.31 ± 0.08 0.25 ± 0.04 0.23 ± 0.07 0.19 ± 0.07 0.730 Neutrophil (x109/L) 3.68 ± 0.31 3.18 ± 0.74 5.31 ± 0.15 8.20* ± 0.96 5.67* ± 1.14 0.001* Eosinophil (x109/L) 0.25 ± 0.05 0.19 ± 0.02 0.26 ± 0.12 0.33 ± 0.07 0.21 ± 0.01 0.447 *Values with an asterisk within the same raw are statistically significant (P<0.05). WBC: White blood cells; RBC: Red blood cells; Hb: Hemoglobin; PCV: Packed cell volume; MCV: Mean corpuscular volume; MCH: Mean corpuscular hemoglobin; MCHC: Mean corpuscular hemoglobin concentration; Lymph: Lymphocyte Table 2: Biochemical parameters (mean ± SEM) in female dromedary camels during the transition period (n=10) -28 -14 0 + T4 +28 P value Ca (mmol/l) 1.99 ± 0.09 2.18 ±0.11 2.22 ± 0.08 2.26 ± 0.05 2.16 ± 0.03 0.201 P (mmol/l) 1.76 ± 0.14 2.07* ± 0.05 1.70 ± 0.11 1.38 ± 0.07 1.76 ± 0.09 0.004* Mg (mmol/l) 1.02 ± 0.06 1.02 ± 0.04 1.03 ± 0.03 1.00 ± 0.03 1.18* ± 0.02 0.023* AST (U/l) 102.5 ± 4.11 109.75 ± 0.85 114.25 ± 2.39 124.25* ± 2.17 111.5 ± 4.99 0.005* ALP (U/l) 33.75 ± 4.21 39.5 ± 4.99 49.5 ± 5.8 81.50* ± 4.09 65.75* ± 4.71 0.001* GGT (U/l) 17.45 ± 0.77 16.45 ± 0.84 26.50 ± 1.71 39.90* ± 0.95 33.25* ± 3.92 0.001* CK (U/L) 107.25 ± 3.3 104.75 ± 2.78 113.75 ± 1.49 116.00 ± 1.47 110.75 ± 2.17 0.125 Glucose (mg/dl) 194.5 ± 3.4 162.0 ± 12.86 228.0* ± 5.21 155.75 ± 14.6 160.0 ± 5.18 0.001* TP (g/l) 5.62 ± 0.14 5.51 ± 0.18 5.92 ± 0.17 5.66 ± 0.06 6.07 ± 0.10 0.156 Alb (g/l) 2.43 ± 0.09 2.82 ± 0.04 2.65 ± 0.19 2.52 ± 0.04 2.39 ± 0.11 0.186 Globulin (g/l) 32.25 ± 0.81 32.73 ± 2.19 29.53 ± 2.10 31.38 ± 0.58 40.68* ± 3.89 0.029* BUN (mmol/l) 29.15 ± 5.04 30.25 ± 6.95 17.09 ± 0.46 26.50 ± 4.34 28.28 ± 5.41 0.370 Creatinine (mg/dl) 1.00 ± 0.06 1.12 ± 0.04 1.05 ± 0.11 0.69* ± 0.11 0.97 ± 0.07 0.023* Cortisol (ug/dl) 2.59 ± 0.32 1.76 ± 0.13 5.05* ± 0.66 2.80 ± 0.59 1.53 ± 0.14 0.001* Progesterone(ng/ml) 3.86 ± 0.10 4.73* ± 0.17 2.76* ± 0.24 2.41* ± 0.25 2.41* ± 0.26 0.001* Estrogen (pg/ml) 202.8 ± 22.5 303.3* ± 22.6 657.5* ± 36.4 126.8* ± 13.3 102.5* ± 17.2 0.001* *Values with an asterisk within the same raw are statistically significant (P<0.05). Ca: Calcium; Ph: Phosphorus; Mg: Magnesium; AST: aspartate aminotransferase; TP: Total protein; Alb: Albumen; ALP: Alkaline phosphatase; GGT: Gamma Glutamyl Transferase; BUN: Blood Urea Nitrogen; CK: Creatinine Kinase Discussion The transition period in dromedary camels has gained very little attention relative to that of dairy cattle although a limited report has exist in guanacos (Lama guanicoe) and in the llama (Lama glama) (7, 8). Indeed, laboratory profiling, can detect sick animals, and help identify herds at a potential risk of having metabolic and reproductive ailments. To fulfill this research gap, we sampled ten female pregnant dromedary camels at four weeks prior to the anticipated time of parturition to four weeks after calving. In this study, the most noticeable hematological changes in the female camels during the transition period were neutrophilia which was occurred at +14 and +28. This finding was in accordance with that reported previously in transition camel (11) and in cattle during the periparturient period (12). The authors Hematological and biochemical profile in female camels (Camelus dromedarius) during the transition period 575 attributed such finding to stress being associated with parturition and the beginning of lactation stage. The current study revealed that the serum concentration of calcium did not show significance pre- and post-partum. This finding was in agreement with that previously given in camel (11, 13) but away from that reported by other researchers (14, 15) who found lower calcium levels at the end of pregnancy in dromedary camel and buffaloes, respectively. The authors attributed such findings to the impairment of absorption of these metabolites from the alimentary tract and to the excessive losses through urine, colostrum. In the current study, there was a significant increase in the serum phosphorus level prepar-tum and declined thereafter. This finding was in constant with that given by (11) in camel and that reported in buffalo (15) who found lower phosphorus levels during early stage of lactation in buffaloes. The authors attributed such finding as a result of the important role of phosphorus in the colostrums synthesis (16) and enhanced carbohydrate metabolism. Magnesium plays a vital role during the metabolism of carbohydrates, lipids, nucleic acids and proteins. In present investigation, there was a significant increase in the serum magnesium at +28 days postpartum. Our finding was similar to that obtained by (11) in transition camel and by (17) in Ongole cows but away from that, reported by (18) who recorded lowered magnesium mean values in cattle and buffalo during early lactation. The alterations of serum globulin, but not albumin, could be resulted from the formation of immunoglobulins. This finding was noticed to be in harmony with that given in transition camel (11) and in dairy cows (5), while the decrease of serum albumin levels could be associated with hepatic diseases. This finding is in accordance with previous reports in transition camel (11, 21). However, in this study its reduction was not significant. In the present study, there was a significant decrease in the serum creatinine level at +14 days postpartum. This finding is in constant with that reported previously in periparturient camel (11) and in transition cattle (12). The cre-atinine level (marker of kidney function) is dependent on total body content of creatinine, which in turn depends on the dietary intake, rate of synthesis of creatinine, and muscle mass (19). The creatinine concentrations in the prepartum period were higher than those in postpartum. This finding is line with that reported previously (20) at late gestation camel who found high creatinine levels in dromedary camels during late gestation. The authors attributed such finding to high requirement of protein and lower rate of kidney elimination associated with late stage of pregnancy (13). In the current study, there was a significant increase in the serum activity of AST, GGT and ALP at +14. The serum activity of GGT and ALP remained also at a significant (P=0.001) high pattern at +28 after parturition. Our findings were similar to that obtained in periparturient camel (11) and in dairy cows (5) but unlike to that given in late pregnant dromedary camels (22) and in pregnant mares (23) who reported a significant decrease in the GGT activity in the pregnant camels and in pregnant mare. It has been suggested that GGT is involved in the metabolism of glutathione to maintain antioxida-tive status of the entire body (24). The increased concentration of glucose at parturition (0) may be due to the stress of parturition and coincide with elevation of cortisol at this period. This finding was in agreement with that given previously in transition camel (11) and in dairy cows (5) but unlike to that given by other researchers (25, 26) in peri-parturient dromedary camel who had found a significant decrease of glucose concentration before parturition compared to the period after. In the former study, the authors attributed such finding to the effect of gestational state on glucose level. The current study revealed that, the serum level of BUN did not show any significance pre- and post-partum changes. This finding was in agreement with that given in transition camel (11) but in contrary with that reported in pregnancy of dromedary camels (22). The authors reported a significant increase of serum BUN in pregnant camels. The authors attributed such 576 E. Ebissy, A. El-Sayed, R. Mohamed finding as a result of protein catabolism and high requirement for energy by pregnant camels during the late pregnancy period resulting to an increase in BUN level. Our findings demonstrated that concentration of serum cortisol was significantly increased at parturition (0) which may be due to stress of parturition due to the increase in the concentration of ACTH secretion from the foetal pituitary (11, 27, 28,29). The serum activity of estrogen was elevated at -14 and reached its maximum peak at the time of parturition and declined thereafter. Such elevation of serum estrogen is considered as physiological response to potentiate uterine contraction during parturition. These findings were in agreement with that given by (11, 30, 31, 32) in camel and (33, 34) in goat. The authors attributed the dropped of maternal estrogen postpartum to that the estrogen measured in pregnant camel was fetoplacental in origin. On the other side, serum activity of progesterone was lowered at the time of parturition and the time onward (i.e. 0 to +28) which was considered as a physiological response in order to remove the corpus luteum of pregnancy. These findings were in agreement with that given by (11, 33, 34). Conclusion The data herein demonstrated a profound haematobiochemical alteration which could be considered as a feature of transition period in peripartum camels. Such alterations are not necessarily indicative of disease but could reflect a physiological variation and could be used as a reference guide for she-camels during the periparturient period. Conflict of interest The authors declare that they have no conflict of interest. Acknowledgments The authors acknowledge the staff members of Animal Health and Poultry Department, Desert Research Center, Egypt. References 1. Zarrouk A, Souilem O, Beckers JF. Actuali-téssur la reproduction chez la female dromedary (Camelusdromedarius): Revue Elev Med Vét Pays Tropi 2003; 56: 95-102. 2. Drackley JR. Biology of dairy cows during the transition period: The final frontier. J Dairy Sci 1999; 82:2259-73 3. Overton TR, Waldron MR. Nutritional management of transition dairy cows: Strategies to optimise metabolic health. J Dairy Sci 2004; 87 (E Suppl.), E105-19. 4. Guo J, Peters RR, Kohn RA. Effect of a transition diet on production performance and metabolism in periparturient dairy cows. J Dairy Sci2007;90:5247-58. 5. Tharwat M, Oikawa S ,Buczinski S. Ultrasonographic prediction of hepatic fat content in dairy cows during the transition period. J Vet Sci Technol 2012; 3:1. 6. Osman TE, Al-Busadah KA. Effects of age and lactation on some biochemical constituents of camel blood in Saudi Arabia.Camel Practice and Research 2000; 2: 149-52. 7. Leon JB, Smith BB, TimmKI, LeCren G. Endocrine changes during pregnancy, parturition and the early post-partum period in the llama (Lama glama). Journal of Reproduction and Fertility 1990; 88: 503-11. 8. Riveros JL, Urquieta B, Bonacic C, Hoffmann B, Bas F, Schuler G. Endocrine changes during pregnancy, parturition and post-partum in guanacos (Lama guanicoe). Animal Reproduction Science 2009;116: 318-25. 9-Rosenberger G. Die KlinischeUntersuchung des Rindes. 3rd edn. Berlin and Munich, Paul Parey 1990 10. Radostits OM, Mayhew IG, Houston DM. Veterinary Clinical Examination and Diagnosis. W.B. Saunders, London 2000 11. Tharwat M, Ali A, Al-Sobayil F, Selim L,Abbas H.Hematobiochemical profile in female camels (Camelusdromedarius) during the peripar-turient period. Journal of Camel Practice and Research. 2015; 22 (1): 101-6. 12. El-Ghoul W, Hofmann W, Khamis Y,Has-sanein A. Relationship between claw disorders and the peripartal period in dairy cows. PraktTierarzt 2000; 81:862-8. 13. Omidi A, Sajedi ZH, MontazerTorbati MB, Mostafai M. Metabolic profile of pregnant, non- Hematological and biochemical profile in female camels (Camelus dromedarius) during the transition period 577 pregnant and male two humped camels (Camelus-bactrianus) of Iran. Iran. J. Vet. Med 2014; 8: 23542. 14. Eltohamy MM, Salama A, Yousef AA. Blood constituents in relation to the reproductive state in she-camel (Camelusdromedarius). Beitr Trop. LandwirtschVeterinarmed. 1986; 24:425--0 15. Hagawane SD, Shinde SB, Rajguru DN. Haematological and Blood Biochemical Profile in Lactating Buffaloes in and around Parbhani city. Veterinary World, 2009; 2(12):467-69 16. Rook JAF, Thomas PC. Nutritional physiology of farm animals Ed 1.Longman Inc. New York 1983. 17. Rao DG. Studies on some biochemical constituents of blood in Ongole cows. Ind. Vet. J. 1981; 870-3. 18. Gupta GC, Rai P. A note on biochemical profile in pre and post-partum states in cattle and buffaloes. Indian J Vet Med. 1987; 7: 45-6 19. Patel SS, Molnar MZ, Tayek JA, Noori N, Benner D, Heymsfield S, Kopple JD, Kovesdy CP, Kalantar-Zadeh K. Serum creatinine as a marker of muscle mass in chronic kidney diseases: results of a cross- sectional study and review of literature. J Ca-chexiaSarcopenia Muscle.2013;4: 19-29. 20. Yousef FA, Karima GMM, Mohamed MMK, Nawito MF, AbdelRazik AMA. Histomor-phometry aspect of thyroid gland and biochemical profile in pregnant and non-pregnant dromedary camels. African Journal of Biotechnology. 2016; 15 (10): 370--5. 21. Nehra V, Angulo P, Buchman AL, Lindor KD. Nutritional and metabolic considerations in the etiology of nonalcoholic steatohepatitis.Digestive Diseases and Sciences2001; 46:2347-52. 22. Omidi A, Fathi MH,Asiaban M. Elevated levels of blood urea nitrogen and creatinine in the last trimester of pregnancy of dromedary camels (Camelusdromedarius). Iranian Journal of Veterinary Medicine.2015;9 (4): 249-55. 23. Milinkovic-Tur S, Peric V, Stojevic Z, ZdelarTuk M, Pirsljin J. Concentrations of total proteins and albumins, and AST, ALT and GGT activities in the blood plasma of mares during pregnancy and early lactation. Veterinarski Arhiv 2005; 75: 195-202. 24. Chen Y, Dong H, Thompson DC, Shertzer HG, Nebert DW, Vasiliou V.Gluta- thione defense mechanism in liver injury: in- sights from animal models. Food ChemToxi- col. 2013;60: 38-44 25. Kelanmer R, Antoine-Moussiaux N, Abu-Median AAK, Hanzen CH, Kaidi R. Effect of nutrition on reproductive performance during the peri-partum period of female camel (Camelus-dromedar-ius) in Algeria. J Anim Vet Advan 2018; 14 (7): 192-6. 26. Saeed A, Khan IA, Hussein MM. Change in biochemical profile of pregnant camels (Camelusdromedarius) at term. Comparative Clinical Pathology.2009; 18: 139-43. 27. Suganya G, Gomathy VS. Hormone profile of Tellicherry goats during periparturient period.Ta-milnadu. Journal of Veterinary and Animal Sciences 2009;5: 211-21. 28. Suganya G, Leela V, Viswanathan S, David BP. Circulating levels of cortisol during pregnancy, parturition and post-partum periods in goats. Indian Veterinary Journal 2000;77: 202-4. 29. Arthur GH, Noakes DE, Pearson H. Veterinary Reproduction and Obstetrics, 6th edn, ELBS Publishers, London, UK. pp 133.1989. 30. Ayoub A, EL-Khouly AA, Mohamed TM. Some hematological and biochemical parameters and steroid hormone in the one- humped camel during the different physiological conditions. Emir J Agric. Sci. 2003; 15 (1): 44-55. 31. Zhao XXY, Chen BX. Serum progesterone and 17p estradiol concentration during pregnancy of Bactrian camel (Camelusbactrianus). Theriogenol-ogy.1998;50: 595-604. 32. Skidmore JA, Billah M, Allen WR. Pattern of hormone secretions throughout pregnancy in the one- humped camel (camels dromedaries). Reprod. Fertil.Dev.1996; 8:863-9. 33. Alwan AF, Amin FAM, Ibrahim NS. Blood progesterone and oestrogen hormones level during pregnancy and after birth in Iraqi sheep and goat. Basrah Journal of Veterinary Research 2010; 10: 153. 34. Khan JR, Ludri RS. Hormone profile of crossbred goats during the periparturient period. Tropical Animal Health and Production 2002; 34:151-62. Slov Vet Res 2019; 56 (Suppl 22): 579-88 DOI 10.26873/SVR-795-2019 Original Research Article ASSESSMENT THE SYNCHRONY BETWEEN UTERINE STATUS AND HORMONAL PROFILES IN MODIFIED OVSYNC PROTOCOLS IN RELATION TO FERTILITY IN BUFFALOES Adel A. Ramoun1*, Ismail I. EL- Kon1, Essam A. Almadaly1, Aly M. Aly2, Hasballah A. Abo EL- Ghate3 department of Theriogenology, Faculty of Veterinary Medicine, Kafrelsheikh University, Kafrelsheikh, 33516, Egypt, 2General Authority for Veterinary Services, Kafr EL-Sheikh, Egypt, 3Mahallet Mousa Buffalo Research Station, Animal Production Research Institute, Agriculture, Research Center, Egypt Corresponding author, E-mail: ramoun2004@yahoo.com Abstract: A total of 36 pluriparous buffaloes were used to study the uterine morphometry in response to serum estradiol concentrations and associated estrogen receptors (ERs) expression in modified ovsync protocols. The buffaloes were assigned to St-ovsync, CIDR-sync and Insulin-sync (n=12 for each). The St-ovsync consisted of two IM injections of 20|jg buserelin on the Day 0 (GnRHI) and on the Day 9 (GnRH2) and an IM injection of 500 jg of Cloprostenol sodium (Estrumate) on Day 7. Buffaloes in CIDR-sync and Insulin-sync protocols were treated as in the St-Ovsync protocol in addition to intravaginal insertion of CIDR from Day 0 to 7 in CIDR-sync and SC injection of Insulin at a dose of 0.25 IU/kg body weight on Days 7, 8 and 9 in Insulin-sync. Blood samples were collected and the uterine wall thickness (UWT) was simultaneously measured by transrectal ultrasonography on Days 0, 3, 5, 7, 8 and 9. Endometrial biopsies samples were collected from five buffaloes in each group to quantify the abundance of estrogen receptors. The UWT on Days 3 and 5 decreased significantly (P < 0.05) while serum P4 concentration on Day 3 was significantly (P < 0.05) increased in eventually diagnosed pregnant (EDP) buffaloes in CIDR-sync compared with their counterparts in either St-Ovsync or Insulin-sync. On Day 9, although there was a decrease in serum E2 concentration in CIDR-sync compared with either St-Ovsync or Insulin-sync in EDP buffaloes, there was an increase (P < 0.05) in the ER mRNA expression in CIDR-sync compared with St-Ovsync. It could be concluded that modifying the St-Ovsync by P4 supplementation through intravaginal insertion of CIDR from Day 0 to 7 or by SC injection of insulin on the Days 7, 8 and 9, could modulate uterine morphometry such conductive to proper fertility response. Key words: Buffaloes, ERa; uterine wall thickness; Ovsync Introduction Optimal uterine environment at the time of FTAI, especially in cows induced to ovulate immature follicles in ovsync programs, is critical to establish and maintain pregnancy (1). The variation in the fertility status of cows kept under the same nutritional and environmental conditions may result from the different response Received: January 2019 Accepted for publication: February 2019 580 A. Ramoun, I. EL- Kon, E. Almadaly, A. Aly, H. Abo EL- Ghate of the uterus to steroid hormones during the periovulation time (2). Adequate rise in the proestrous estradiol (E2 ) following preexposure to progesterone (P4 ) prepares the uterus for embryonic development and establishment of pregnancy (3, 1, 2, and 4). The growth and regression of endometrium are synchronized through changes in the circulating and/or local levels of E2 and P4 (5). Endometrial thickness increases during proestrous and reach maximal thickness on the day before ovulation and decreases throughout diestrous (6, 7). Suboptimal uterine environment induced by low level of estrogen during proestrous and progesterone during diestrous decreases infertility in dairy cows (8). Infertility in cows induced to ovulate immature follicles especially in Ovsync-TAI program may be attributed to suboptimal uterine function caused by low steroids at the periovulation time (9, 10, and 1). Not only the availability of steroid hormones with optimal levels in the peripheral blood is adequate to produce the desired effect on the uterus but binding to their specific nuclear receptors is critical (11). The effect of steroid hormones on the uterus depends on tissue and cell specific expression of steroid receptors (12) which reach maximum levels around the time of heat (13, 14). E2 modulates the expression of its receptors and their function at the cellular level in such way that increase the rate of mitosis and tissues edema (15). Ovsync-TAI programs either GnRHbased or estradiol-based, are used nowadays to improve the reproductive performance of dairy cows (16, 17). In estradiol - based TAI programs, the exogenous E2, in addition to inducing GnRH/LH surges, it modulates uterine environment. However, the relatively small follicles that might be present prior to second GnRH in case of GnRH-based Ovsync-TAI program may not produce adequate amount of estradiol sufficient to modulate uterine environment such compatible to support pregnancy (1). The US-measured uterine wall thickness (UWT) and horn diameter could be utilized to test if the uterus has been exposed to adequate concentration of steroid hormones compatible with optimal fertility (18). Scoring the uterine status prior to the breeding time could be useful for predicting fertility in many species including human. Amongst the methods used to evaluate uterine status are cytology of uterine secretion, ultrasonographic (US) examination and uterine biopsy (19). The current study tests the hypothesis that if the US-measured UWT together with the uterine biopsy could be used to predict fertility of buffaloes subjected to modified ovsync programs. Souza et al (2014) (4) utilized endometrial thickness as a predictor of fertility in high producing dairy cows. They concluded that an en-dometrial thickness less than 7 mm is predictive to failure of conception in dairy cows. However, the associated steroid hormones profile together with estrogen receptors were quantified to correlate between hormonal profiles and uterine morphometry. Thus, the current study was designed to match US-measured UWT with steroid hormones profiles together with estrogen receptors expression in the endometrial biopsy sample in an attempt to utilize UWT as a predicting measure of fertility following application of modified CIDR- and Insulin- ovsyncs in buffaloes. Materials and methods The animals The study was performed in Mahallet Mousa Buffalo Research Station, affiliated to Animal Production Research Institute, present in Kafrelsheikh province in the northern of the Nile Delta, Egypt. The experiment was per-formedfrom April to October which coincides withlow breeding season in the Egyptian buffaloes. A total of 36 pluriparous cyclic Murrah buffaloes having a parity of 2- 4 and an average body condition score of 2.75 to 3.50 (Scor-ingsystem was 1 = thin to 5 = fat) were used to carry out this study. Buffaloes were kept indoors throughout the year in yards where 50% of the yard area was sheltered. They hadfree access to water. They were milked twice daily and received a diet that covered both maintenance and production requirements according to the Recommendation of Animal Production Research Institute (APRI, 1997unpublished data). Assessment the synchrony between uterine status and hormonal profiles in modified Ovsync protocols 581 All animals were cyclicand had healthy genital tract on the basis oftransrectal US scanning of the reproductive system. The cyclic activity was assessed onthe basis of detecting a corpus luteum in eitherof two transrectal US examinations of ovariesdone at 10 days' interval (from Day -10 to Day 0). Day 0 was the day of the first GnRH injection. Experimental design Buffaloes (N=36) were randomly assigned to three treatment protocols (12 each): standard ovsync (control group, St-Ovsync), modified CIDR-sync (CIDR-sync) and modified Insulin-sync (Insulin-sync). Each buffalo in the St-Ovsync group received IM injections of 20 pg buserelin acetate (GnRH agonist,5 ml Re-ceptal®, Intervet Company, Holland) on the Day 0 (GnRHl), 500 pg of Cloprostenol sodium (PGF2a analogue, 2 ml Estrumate®, Coopers, Schering Plough Company, England) on the Day 7 and a 2nd dose of GnRH agonist similar to GnRH1, on the Day 9 (GnRH2). Buffaloes in the CIDR-syncgroup were treated as in St-ovsync in addition to intravaginal inser-tionof CIDR (Controlled Internal Drug Release, it contains 1.38 gm of progesterone, Pfizer Company, New Zealand) from the Day 0 to the Day 7. Buffaloes in Insulin-sync were also treated as St-Ovsync in addition to daily s/c injection of insulin (biphasic isophane insu-lin,0.25 IU / kg of B.W, Mixtard 30 HM®, Nova, Nordisk, Bagsvared, Denmark) on the Days 7, 8 and 9. Each 1 ml of Mixtard 30 HM® contain 30 IU of soluble and 70 IU of isophane biosynthetic human insulin. Buffaloes in all groupswere bred at 16 hr after the second GnRH treatment. Ultrasonographic scanning US examination was conducted by using portable ultrasound device (ULTRASCAN MODEL DP 30 VET, Shanghai International Holding Crop GmbH, Europe) equipped with multifrequency (3-10 MHz) linear probe. For transrectal scanning of the uterus, the probe while being carried in the palm of the hand was guided into the rectum. The uterus was identified then the probe was transversely placed on the base of each uterine horn at 3 cm cranial to the intercornual ligament. The UWT was measured by electronic calipers in the cross section of the frozen image. The UWT was measured as the distance between the edge of uterine lumen to the external edge of the perimetrium. The UWT was estimated for each uterine horn then the average value of the two horns was calculated for each cow. The UWT was measured on Days 0, 3, 5, 7, 8 and 9. Collection of endometrial tissues On Day 9, endometrial tissue samples were obtained by an endometrial biopsy knife as described by Nielson (7). The endometrial tissues samples were removed by fine forceps and immersed in a microcentrifuge tube containing 30 mm of the lysis buffer supplemented with mer-captoethanol to quantify the expression of mRNAs of estrogen receptor alpha (ERa) gene in the endometrium. RT-qPCR assay for ERa gene expression Total RNA was extracted from endometrial biopsy (30 mg) using RNA isolation kit (Thermo-scientific, fermentas Ko731) according to the manufacturer's instructions. The concentration of the total RNA was measured using Nanodrop Spectrophotometer. To obtain cDNA, the total RNA (1 pg) was reverse transcribed using reverse transcription kits (Thermo-scientific, fermentas # EP0451 according to the manufacturer's instructions). Quantifying of mRNA for ERa in the three protocols was determined by quantative RTPCR using SYBR Green with GAPDH as an internal control reference. The isolated cDNA was amplified using 2x Maxima SYBER Green/Rox qPCR Master Mix (Thermo-scien-tific, USA, # K0221) and gene specific primers according to the manufacturer's instructions. The primers for ERa gene were; 5'GAAGTGGGCATGATGAAAGG-3' forward and AAGGTTGGCACGTCTCATGT reverse and for GAPDH gene were 5'- CCTG-GAGAAACCTGCCAAGT-3' forward and 5'GGTAGAAGAGTGAGTGTCGCT-3' reverse. The primer for ERa receptor gene was 582 A. Ramoun, I. EL- Kon, E. Almadaly, A. Aly, H. Abo EL- Ghate designed using web based tool (http://www.ge-nome.wi.mit.edu/egiin/primer/primer3www. egi) on the basis of published buffalo sequences to ensure that the primer sequence is unique for the template sequence. Amplicon sequence identity was confirmed with NCBI Blast tool software (Blast http://www.Blast.ncbi.nlm. nih.gov/Blast.egi). The reactions were conducted in a final volume of 25|L using 12.5|L of 2x Maxima SYBER Green/ Rox qPCR Master MIX, 1|L of each primer (forward and reverse), 3|L of cDNA (10-20 ng/|L) and 7.5|L of water nuclease free. The protocol conditions included initial denaturation at 95°c for 10 minutes and 40 cycles with denaturation at 95°c for 15sec, annealing at 60°c for 30sec and extension at 72°c for 30 sec. The relative expression levels of target gene (ERa receptors) were calculated by using the - AACT method (20). The house keeping gene (GHPDH) was used as the normalizing reference gene. The cycle threshold (Ct) values calculated for target gene were normalized against reference gene. The St-ovsync (control) group was used as calibrator, while CIDR-sync and Insulin- were considered as test groups for both target and references genes. The mean cycle threshold (Ct) values were used to calculate ACT for both target and reference genes in each of test and control groups by the following equation: ACT (test) = Ct (target in test groups) - Ct (ref in test groups) ACT (calibrator) = Ct (target in control) -Ct (ref. in control). Then ACt of the test genes were normalized to the ACt of the calibrator: AACt = ACt (test) - ACt (calibrator). The fold change of the relative gene expression was calculated as follows: Fold change = (2-AACt). Blood sampling and hormonal assay: Blood sampling Blood samples were collected by jugular vein puncture on the Days 0, 3, 5 and 9. The samples were centrifuged at 3000 rpm for 15 min. The harvested sera were stored at - 20°C until estrogen and progesterone assays. Serum progesterone assay The serum P4 concentrations were measured by radioimmunoassay using RIA kit (Beckman coulter RIA progesterone IMMUTECH, S.r.o Radiova 1-10227 Prague - Czech Republic) according to the manufacturer's instructions described in the catalog enclosed with the kit. The inter- and intra- assay coefficients of variations were 8.66 and 8.15 respectively. The average sensitivity was 9.58 pg/ml. Serum estradiol assay The serum E2 concentrations were estimated by radioimmunoassay using estradiol kit (Beck-man coulter RIA Estradiol; IMMUNTECH, s.r.o Radiova 1-10277 Prauge - Czech Republic) according to manufacturer's instructions described in the catalog enclosed with the kit. The inter- and intra- assay coefficients of variations were 14.5 and 14.4 respectively. The average sensitivity was 9.58 pg/ml. Serum insulin assay The serum insulin concentrations were estimated using an IMMUNORADIOMETRIC kit (Insulin (e) IMRA kit; IMMUNOTECH, s.r.o Radiova 1-10227 praque- Czech Republic) according to the manufacturer's instructions described in the catalog enclosed with kit. The inter- and intra- assay coefficients of variations were 8.3% and 5.6% respectively. The average sensitivity was 4.55ng/ml. Reproductive management Buffaloes were inseminated at 16 h after the 2nd GnRH treatment (FTAI) with frozen-thawed semen. On Day 30 post TAI, buffaloes were examined by transrectal US of their uteri for pregnancy diagnosis. Conception rates were calculated by dividing the number of buffaloes gets pregnant on the total number of buffaloes submitted to applied ovsync protocols TAI programs in the current study. Assessment the synchrony between uterine status and hormonal profiles in modified Ovsync protocols 583 Statistical analysis All data, except conception rates, were presented as means ± SEM. The statistical significance of differences was tested by the analysis of variance (ANOVA) followed by Bonfer-roni's multiple comparison test using Graphpad prism ver. 6. 0 for Mac (Graphpad software. San Dieogo, USA). Chi-Square analysis was used to compare the conception rates among the three protocols. Results Ultra sound - Measured UWT On Day 0, the US-measured UWT did not differ (P > 0.05) among St-ovsync, CIDR-sync and Insulin-sync in either eventually diagnosed pregnant (EDP) buffaloes or eventually diagnosed non pregnant (EDnP) buffaloes. It was observed that while the UWT decreased (P<0.05) in the CIDR-sync compared with either St-ovsync or Insulin-sync in EDP buffaloes, it did not differ among the 3 protocols in EDnP buffaloes on either of the 3rd or 5th day. Within each protocol, the UWT did not differ (P > 0.05) between EDP and EDnP buffaloes in all the investigated days (Table, 1). Serum progesterone concentrations on the Days 0, 3 and 5 On the Days 0 and 5, the serum P4 concentrations did not differ among the three protocols neither in EDP nor EDnP buffaloes. In contrary, on Day 3, it increased (P < 0.05) in EDP and EDnP buffaloes in CIDR-sync compared with either St-ovsync or Insulin-sync (Table 1). The uterine wall thickness on Day 9 The UWT did not differ (P < 0.05) between either EDP or EDnP buffaloes on Day 9 as well as among EDnP buffaloes on Day 7. However, the UWT decreased (P < 0.05) in EDP buffaloes in CIDR-sync compared with either St-ovsync or Insulin-sync group on Day 7 (Table, 2). Serum estradiol concentration on the Day 9 In EDP buffaloes, serum E2 concentration on Day 9 showed a decrease (P < 0.05) in CIDR-sync compared with either Stovsync or Insulin-sync. In EDnP buffaloes, it showed nonsignificant (P > 0.05) variations among the three protocols. Serum insulin concentration on Day 9 The serum insulin concentration on Day 9 increased (P < 0.05) in both EDP and EDnP buffaloes in Insulin-sync compared with either St-ovsync or CIDR-sync group (Table 2). Estrogen receptor (ER) gene expression The expression of ERa gene was upregulated (P < 0.05) in insulin-sync protocol compared with St-ovsync. Also the expression was upregulated in CIDR-sync compared with St-ovsync (Figure 1) regardless the buffaloes were EDP or EDnP. There was nonsignificant difference between insulin-sync and CIDRsync. Relative to the St-ovsync, the fold change of 2.81 and 2.45 were recorded for the upregulation of ERa gene expression in the Insulin-sync and CIDR-sync respectively (Figure 1). The fertility response Pregnancy diagnosis on Day 30 post- TAI revealed that 5/12 (41.66 %); 6/12 (50%) and 8/12 (66.67 %) buffaloes were diagnosed pregnant in St-ovsync, CIDR-sync and Insulin-sync protocols respectively (Figure 2). 584 A. Ramoun, I. EL- Kon, E. Almadaly, A. Aly, H. Abo EL- Ghate Table 1: Ultrasound-measured UWT and serum P4 concentrations in EDP and EDnP buffaloes in St-ovsync, CIDR-sync and Insulin-sync protocols on Days 0, 3 and 5 Parameter Synchronization protocols St-Ovsync CIDR-Sync Insulin-Sync EDP EDnP EDP EDnP EDP EDnP UWT (mm) Day 0 42 ± 0.02 a 42 ± 0.01 d 41 ± 0.02 a 41 ± 0.03 d 42 ± 0.01 a 45 ± 0.01 d Day 3 45 ± 0.02 a 40 ± 0.01 d 34 ± 0.01 b 39 ± 0.02 d 41 ± 0.01 a 38 ± 0.02 d Day 5 42 ± 0.03 a 42 ± 0.01 d 32 ± 0.01 b 38 ± 0.03 d 41 ± 0.01 a 45 ± 0.02 d Serum P4 concentration (ng/ml) Day 0 3.41 ± 0.83 b 1.91 ± 0.29 " 3.84 ± 0.66 b 2.55 ± 0.28 " 3.73 ± 0.98 b 2.00 ± 0.34 " Day 3 3.58 ± 0.62 b 3.26 ± 0.67e 6.11 ± 0.39 a 6.39 ± 1.01 d 3.49 ± 0.51 b 3.26 ± 0.52 <= Day 5 3.25 ± 0.15 b 4.05 ± 0.17 " 5.95 ± 1.39 b 5.14 ± 0.57 <= 3.04 ± 0.64 b 3.98 ± 0.30 <= Within the same row, values carrying small letters from a to c in case of EDP and from (d to f) in case of EDnP buffaloes are different at P<0.05. Within the same treatment group, values carrying asterisk are different at P<0.05 between EDP and EDnP buffaloes. Table 2: Ultrasound - measured UWT on Days 7, 8 and 9 and serum concentrations of E2 and insulin on Day 9 in St-ovsync, CIDR-sync and Insulin-sync protocols Parameter St-Ovsync EDP EDnP Treatment protocol CIDR-Sync Insulin-Sync EDP EDnP EDP EDnP UWT (mm) Day7 42 ± 0.02 a 42 ± 0.01 d Day 8 43 ± 0.04 a 44 ± 0.01 d Day 9 34 ± 0.02 1 50 ± 0.03 Serum estradiol concentration (pg/ml) Day 9 17.3 ± 1.55 a 9.43 ± 1.23 d Serum insulin concentration (ng/ml) Day 9_7.18 ± 1.85 b 5.79±2.56 e 33 ± 0.01 b 35 ± 0.02 a 34 ± 0.02 b 12.1 ± 1.45 6.00±0.72 e 35 ± 0.3 d 36 ± 0.02 d 49 ± 0.04 d 9.65 ± 2.11 d 6.00±0.72 e 43 ± 0.02 a 45 ± 0.02 1 43 ± 0.03 a 44± 0.05 d 41 ± 0.04 41 ± 0.04 d 22.26 ± 1.31 a 12.13 ± 1.90d 32.13±2.64 ' 15.13±2.21 d EDP = eventually diagnosed pregnant; EDnP = eventually diagnosed non-pregnant. Within the same row, values bearing different letters from a to b in case of EDP buffaloes and from d to e in case of EDnP buffaloes were different at (P<0.05). Within the same protocol, values bearing asterisk were different at P < 0.05 between EDP and EDnP buffaloes. Figure 1: Oestrogen receptor alpha (ERa) gene expression in insulin-sync and CIDR-sync groups relative to St-ovsync shown as fold changes (2-AAct). Relative to the St-ovsync (fold change (Fc)=1), the fold changes of 2.81 and 2.45 represented the upregulation of ERa gene in Insulin-sync and CIDR-sync groups respectively Figure 2: The conception rates on Day 30 post-TAI in buffaloes treated with St-ovsync, CIDR-sync and In su-lin-sync. The conception rate was 5/12 (41.66 %); 6/12 (50 %) and 8/12 (66.67 %) in St-ovsync, CIDR- sync and Insulin-sync respectively Assessment the synchrony between uterine status and hormonal profiles in modified Ovsync protocols 585 Discussion The present work aimed to study the uterine morphometry in terms of us-measured UWT in relation to steroid hormones profiles and endo-metrial oestrogen receptors in modified ovsync programs in buffaloes. Since endometrial growth and regression are synchronized with ovarian function through changes in circulating and/or local levels of oestrogen and progester-one(21, 4), ultrasound measuring of the uterine wall thickness, simple and reproducible technique, can be used to evaluate steroid hormone induced uterine environment changes (22). On the day 3 and 5, the decrease in the UWT in the buffaloes eventually diagnosed pregnant (EDP) in CIDR-Sync compared with either St-Ovsync or Insulin-sync may be attributed to the increase in the serum progesterone concentrations at p < 0.05 and p > 0.05 on the days 3 and 5 respectively. Taking into account the similarity between the diestrous phase in the estrous cycle and short luteal phase, 2-7 days, in an ovsync protocol (Ref), the reduced UWT, recorded on the days 3 and 5 in the current study, came in line with (23) who reported that the uterine horns had minimal thickness, minimal luminal fluid and maximal horn curl during the period extending from 3rd to 16th day of the oes-trous cycle. Sajjan (2014) (23) reported that the thickness of the endometrium, (most responsive layer of the uterine wall to the changes in the serum steroid hormones concentrations) decreased from 5.47 to 5.06 mm with the increase in the serum P4 concentration from 0.76 to 1.08ng/ml in cows. In the same respect, Jimenez-Krassel et al (2009) (24) attributed the decrease in the endometrial thickness to the increase in the circulating P4 concentration The non-significant variations in the UWT among EDP buffaloes on Day 0 as well as among EDnP buffaloes may be explained in the light of non-significant variations in the serum P4 concentrations on the respective days. On Day 9, matching the serum E2 concentrations with UWT in case of EDP buffaloes revealed that the serum E2 concentrations, being high, were nearly consistent with respective UWT in case of either ST-ovsync or Insulin- sync but not in CIDR-sync. Conversely in EDnP buffaloes, it was inconsistent with respective UWT in all of three groups. In the EDP buffaloes, then insignificant increase in the UWT in the insulin-sync protocol compared with either ovsync or CIDR- sync may be explained in the light of increase in the respective serum E2 concentration in the insulin-sync compared with its counterpart value in case of either St-ovsync or CIDR - sync protocol. However, the increase in E2 receptors in CIDR-sync compared with St- ovsync may increase the response of the endo- metrium, which is the main responder part of the uterine wall, to relatively low serum E2 levels in CIDR- sync thereby increasing UWT that became comparable to its counterpart value in case EDP buffaloes in St-ovsync. In line with this explanation, Xia and Goff (25) reported that the number of endometrial receptors determines the sensitivity of the uterus to the steroid stimulation. The increase in the concentration of serum E2 in Insulin-sync compared with the other two groups may be due to stimulatory effect of insulin on the steroidogenic activity of the largest follicle. The exogenous insulin increase both follicular growth and steroidogenic activity in cows (26), buffaloes (27) and goats (28). In CIDR-sync, the lower serum E2 concentration in EDP buffaloes on Day 9 compared with either St-ovsync or Insulin- sync may be attributed to the presence of persistent follicles that bypassed the effect of the first GnRH and continued to grow and produce E2 at lower rates under the effect of low tonic level of LH induced by the negative feedback mechanism of P4 released from CIDR until Day 7 (Day of CIDR removal). Some of these persistent follicles continued to produce E2 at the previous lower level until Day 9. However, an interval of 48 h (from Day 7 to 9) is not sufficient for the persistent follicle to survive P4-induced low tonic level of LH during the previous 7 days, thus their steroidogenic activity became compromised. In line with this explanation, Cerri et al (2010) (29) found that the tonic levels of LH were lower in cows having high serum P4 concentrations on Day 5 of CIDR-sync protocol. 586 A. Ramoun, I. EL- Kon, E. Almadaly, A. Aly, H. Abo EL- Ghate On the other hand, Cerri et al, (29) reported that the decreased concentrations of P4 during synchronization protocol resulted in high basal LH concentrations, faster growth of dominant follicle (DF) and higher circulating E2. In spontaneous estrous, a proestrous period of 3-4 days may be sufficient for resumption of proper tonic level of LH that stimulates proliferative and steroidogenic activity of the DF. In accordance with this suggestion, Bridges et al, (2010) (30) reported that reducing the duration of proes-trous in cows, induced to ovulate small follicles, resulted in decreased serum preovulatory E2 (30, 31). Nonetheless, Cerri et al. (29) found concentration was lesser from day 5 to 9 of CIDR-sync protocols in cows having high compared with those having low serum P4 that the serum E2 concentrations. The benefit of studying the abundance of ERa in these three Ovsync protocols comes from the fact that the changes in the uterine morphometry in response to E2 are an outcome of the interaction of the local/peripheral E2 concentration with its receptors in the uterine tissues. Okumu et al. (11) reported that not only the availability of a steroid hormone in the peripheral circulation is adequate to produce the desired effect on the uterus but binding to their specific nuclear receptors is critical. In the current study, the expression pattern of ERa in the endometrial biopsy is compatible with serum E2 concentrations in either Insulin-sync or St-ovsync but not with CIDR- sync to some extent. The higher E2 levels in case of Insulin-sync and St-ovsync came in agreement with Sunderland et al (1994) (32) who reported that the expression pattern of ERa is consistent with the circulating concentration of E2 during oestrous cycle. In the same respect, Clark et al and Mann et al (33, 34) detected the highest level of ERa and PRs mRNA at the time of oestrous and concluded that the stimulatory effect of E2 on sex hormone receptors expression. Also Spencer and Bazer (35) reported that E2 as well as those for P4 and the functions at the cellular level to increase the rate of mitosis and tissue oedema (15), a finding which explained the increased UWT in either of Insulin-sync or St-ovsync compared with CIDR-sync. However, the reduced UWT in the CIDR-sync may result from the delayed recovery of UW from the effect of high P4 concentration during period of CIDR insertion. However, the abundance of ERa was higher in CIDRsync compared with St-ovsync but the E2 concentration was higher (P > 0.05) in St-ovsync compared with CIDR-sync. Although the E2 concentration was lower (P > 0.05) in CIDR-sync compared with St-ovsync, the abun- dance of ERs in CIDR-sync was higher than St-Ovsync. This may be attributed to the pre exposure of the uterus in case of CIDR-sync to higher P4 concentration during the period of CIDR insertion. This explanation may be supported by the results of Shimizu et al (36) who reported that E2 and P4 provoke transcriptome changes within the endometrium, with the response to E2 being greater when the uterus was preexposed to higher P4. Conclusion It is concluded that modifying the St-ovsync by including P4 through CIDR-insertion from Day 0 to 7 or treatment with insulin on Days 7, 8 and 9 could modulate uterine morphometry, thereby improving fertility outcome of ovsync protocols. Further studies are required in the future to study the effects of the P4 or insulin-induced transcriptome changes in the uterus of cows treated with modified P4 or insulin-modified ovsyncs on the uterine histotrophs secretion that may explain its beneficial effects on the animal fertility. Conflict of interest The authors declare that they have no conflict of interest. References 1. Bridges G A, Mussard M L, Hessler L A, Day M L. Comparison of follicular dynamics and hormone concentration between 7-day and 5-day co-sync + CIDR program in primiparous beef cows. Theriogenology2014; 81: 632-8. 2. Mesquita F S, Pugliesi G, Scolari S C, Franca M R, Ramos R S, Olivera M, Papa P C, Bressan F F, Meirelles F V, Silva L A, Nogueira G P, Mem-brive C M B, Binelli M. Manipulation of sex steroi- Assessment the synchrony between uterine status and hormonal profiles in modified Ovsync protocols 587 dal mileu affects endometrial but not luteal gene expression in early diestrous Nelore cows. Theri-ogenology 2015; 81: 861-9. 3. Araujo E R, Sponchiado M, Pugliesi G, Hoeck V V, Mesquita F S, Membrive C M B, Binelli M. Spatio-specific regulation of endocrine responsive gene transcription by preovulatory profiles in the bovine reproductive tract. Reprod Fertil Dev 2015; 28: 1533-44. 4. Souza A H, Narciso C D, Batista E O S, Car-valho P D, Wiltbank M C. Effect of uterine environment on embryo production and fertility in cows. AnimReprod2014; 11: 159-67. 5. Hericks D M, Hill J R Dickey J F, Lamond D R. Plasma hormone levels in beef cows with induced multiple ovulation. J Reprod Fertil 1973; 35: 225-33. 6. Pierson R A, and Ginther, O J. Ultrasonographic appearance of the bovine uterus during the estrous cycle. J A M Vet Assoc 1987; 190: 9951001. 7. Bonafos L D, Kot K, Ginther O J. Physical characteristics of the uterus during the bovine es-trous cycle and early pregnancy. Theriogenology 1995; 43:713-21. 8. Wiltbank M, Lopez H, Sartori R, Sangsritavong S, Gumen A. Changes in reproductive physiology of lactating dairy cows due to elevated steroid metabolism. Theriogenology2006; 65: 17-29. 9. Perry G A, Smith M F, Lucy M C, Green J A, Parks T E, Mac-Neil, M D, Roberts J A, Geary T W. Relationship between follicular size at insemination and pregnancy success. Proc Natl Acad Sci USA 2005;102: 5268-73. 10. Bridges G A, Mussard M L, Pate J L,Ott T L, Hansen TR, Day M L. Impact of preovulatory estradiol concentration on conceptus development and uterine gene expression. Anim Reprod Sci 2012; 133: 16-26. 11. Okumu L A, Forde N, Fahy A G, Fitz Patrick E, Roche J F, Crowe MA, lonergan P. The effect of elevated progesterone and pregnancy status on m RNA expression and localization of progesterone and oestrogen receptors in the bovine uterus. Reproduction 2010 ;104:143-3. 12. Kimmins S, Maclaren L A. Oestrous cycle and pregnancy effects on the distribution of oestrogen and progesterone receptors in bovine endometrium. Placenta 2001; 22: 742-8. 13. Ing N H, Tornesi M B. Etrogen Upregulates estrogen receptors and progesterone receptor gene expression in specific ovine uterine cells. Biol Reprod 1997;56:1205-15. 14. Meikle A, Sahlin L, Ferraris A, Masironi B, Blanc J E, Rodriguez-Irazoqui M, Rodriguez-Pinon M, KindahiH, Forsberg M. Endometrial mRNA expression of estrogen receptors and insulin-like growth factor-1 (IGF-1) throughout the bovine oes-trous cycle. Anim Reprod Sci 2001; 68: 45-56. 15. Tibbetts T A, Conneely O M, O'Malley B W. Progesterone via its receptors antagonizes the pro-inflamatory activity of estrogen in the mouse uterus. Biol Reprod 1999; 60: 1158-65. 16. Souza A H, Ayres H, Ferreira R M, Wiltbank M C. A new pre synchronization system (Double-ovsync) increases fertility at first postpartum timed AI in lactating dairy cows. Theriogenology 2008; 70: 208-15. 17. Herlihy M M, Giordano J O, Souza A H, Ayres H, Ferreira R M, Keskin A, Nascimento A B, Guenther J N, Gaska J M, Kacuba S J, Crowe M A, Butler S T, Wiltbank M C. Pre synchronization with double ovsync improves fertility at first postpartum artificial insemination in lactating dairy cows. J Dairy Sci 2012; 95: 7003-14. 18. Souza A H, Silva E P B, Cunha A P, Gumen A, Ayres H, Brusveen D J, Guenther J N, Wiltbank M C. Ultrasonographic evolution of endometrial thickness near timed A.I as a predictor of fertility in high producing dairy cows. Theriogenology 2011; 75: 722-33. 19. Baerwald A R Pierson R A. Endometrial development in association with ovarian follicular waves during the menstrual cycle. Ultrasound Obstet Gynec 2004; 24:453-60. 20. Livak K J, Slimittgen T D. Analysis of relative gene expression data using real time quantative PCR and 2-AActmethod. A new mathematical method. Methods 2001; 25: 402-8. 21. Johnson M L, Redmer D H, Reynolds L P. Effect of ovarian steroids on uterine growth, morphology and cell proliferation in ovariectomized, steroid-treated ewes. Biol Reprod 1997; 57: 58896. 22. Delisle M F, Villeneuve M, Boulvain M. Measurement of endometrial thickness with transvaginal ultrasonography: is it reproducible J Ultrasound Med 1998; 17: 781-4. 23. Sajjan S. Studies on the ultrasonographic characterization of endometrial thickness in estrus cycle of cows. M V Sci thesis, Univ BIDAR India 2014. 24. Jimenez-Krassel F, Folger J K, Ireland J L H, Smith G W, Hou X, Davis J F, Longergan P, Evans A d, Ireland J J. Evidence that high variation in ovarian reserves of healthy young adult has a negative impact on the corpus luteum and endometrium 588 A. Ramoun, I. EL- Kon, E. Almadaly, A. Aly, H. Abo EL- Ghate during oestrous cycles in cattle. Biol Reprod 2009; 80:1272-81. 25. Xiao C W, Goff A K. Hormonal regulation of oestrogen and progesterone receptors in cultured bovine endometrial cells. J Reprod Fertil 1999; 115: 101-9. 26. Simpson R D, Chase J R C C, Spicer L J, Vernon, R K I, Hammond A C X, Rae D O. Effect of exogenous insulin on plasma and follicular insulin-like growth factors-1, growth factor binding protein activity, follicular estradiol and progesterone and follicular growth in super ovulated Angus and Brahman cows. J Reprod Fertil 1994; 102: 483-2. 27. Ramoun A A, Osman K T, Darwish S A, Karen A M, Gamal M H. Effect of pretreatment with insulin on the response of buffaloes with inactive ovaries to gonadotropin releasing hormone against treatment in summer. Reprod Fertil Dev 2007; 19: 351-5. 28. Suguna K, Mehorta S, Agarwal S K, Hoqueh M, Shanker U, Singh S K, Varshney V P. Effect of exogenous insulin administration on ovulation function, embryo/fetal development during pregnancy in goats. Anim Reprod Sci 2009; 111:202-13. 29. Cerri R L A, Chebel R C, Rivera F, Narciso C D, Olivera R A, Amstalden M, Baez-Sandoval G M, Olivera L G, Tahatcher W W, Santos J E. Concentration of progesterone during the development of the ovulatory follicle: II. Ovarian and uterine responses. J Dairy Sci 2011; 94: 3352-65. 30. Bridges G A, Mussard M L, Burke C R, Day M L. Influence of the length of proestrous on fertility and endocrine function in female cattle. Anim-ReprodSci2010; 117:208. 31. Mussard M L, Burke C R, Behlke K J, Gasser C L, Day M L. Influence of premature induction of a luteinizing hormone surge with gonadotrophin releasing hormone on ovulation, Luteal function and fertility in cattle. J Anim Sci 2007; 85:937-3. 32. Sunderland S J, Crowe M A, Boland M P, Roche J F, Ireland J J. Selection, dominance and atresia of follicles during the oestrous cycle of heifers. Journal of Reprod Fertil 1994; 101:547-56. 33. Clark J H, Schrader W T, O'Malley B W. Mechanisms of steroid hormones action. In: Wilson J D, Foster DW(Eds.), Williams Textbook of Endocrinology, 8th Edition Saunders, Philadelphia pp 1992;35-90. 34. Mann G E, Lamming G E, Robinson R S, Wathes D C. The regulation of interferon-produc-tion and uterine hormone receptors during early pregnancy. J Reprod Fertil 1999; 54: 317-28. 35. Spencer T E and Bazer F W. Temporal and spatial alterations in uterine estrogen receptor and progesterone receptor gene expression during the estrous cycle and early pregnancy in the ewe.Biol-ogy of Reproduction 1995; 53: 1527-43. 36. Shimizm T u s, Krebs S, Bauersachs H, Blum E, Wolf, Miyamoto A. Actions and interaction of progesterone and estrogen on transcriptome profiles of the bovine endometrium. Physiol Genomics 2010; 42:290-00. Slov Vet Res 2019; 56 (Suppl 22): 589-94 DOI 10.26873/SVR-797-2019 Original Research Article HYPOMAGNESEMIC TETANY IN CAMEL CALVES (CAMELUS DROMEDARIUS): CLINICAL CONSEQUENCES AND TREATMENT OUTCOMES Sherif M. Shoeib1, Mohamed Z. Sayed-Ahmed23, Sabry A. El-khodery2* 1VeterinaryTeachingHospital,FacultyofVeterinaryMedicine,MansouraUniversity, Mansoura 35516, Egypt, 2Department of Internal Medicine and Infectious Diseases, Faculty of Veterinary Medicine, Mansoura University, Mansour 35516, Egypt, 3Department of Clinical Pharmacy, College of Pharmacy, Jazan University, Jizan45142, Saudi Arabia *Corresponding author, E-mail: khodery@mans.edu.eg Abstract: Camels are the top herds wealth in Saudi Arabia, since is considered as heavy animal population, breeding resist more dangerous diseases. This clinical study was delineated to explain the clinical presentation of hypomagnesaemia in camel calves and to evaluate the efficacy of treatment with magenesium therapy. The investigation was performed in central Saudi Arabia during the period from November 2017 to October 2018. Twenty-seven dromedary camels, 3-6months of age were included in this study on the basis of clinical and treatment outcomes during the period from November 2017 to October 2018. Animals were allocated to two groups, the first group (n = 5) was considered as healthy (control) animals. The second group (n = 22) was clinically showing signs of hypomagnesemis. Allanimals were subjected to biochemical analysis of serum and rum-inal fluid. Therapeutic response of the clinically affected animals was assessed after intravenous injection of 100 ml calcium-magnesium preparation intramuscularly. injection of 10ml Vitamin B complex, and oral administration of 2gm Mg sulphate orally for five consecutive days. Clinically affected camels revealed tetany, star gazing, episodes of convulsions with erected ears and tail, extension of head and neck, widening and dilatation of nares. Serum and ruminal fluid analysis revealed a significant decreased (p<0.05) in Mg, Ca, and Phosphorus (P) in clinically affected cases compared to control and treated groups.Total protein (TP), globulin, albumin, , urea and creatinine were significantly higher in clinical cases compared to treated cases. In conclusion, the hypomagnesemic tetany had a high occurrence in camel calves 3-6 month of age . Furthermore, the treatment regieme of hypomagnesemic tetany had agood threrapeutic response. Key words: biochemical tests; camel calves; hypomagnesemic tetany; Saudi Arabia Introduction Hypomagnesemic tetany is a metabolic dis- disorders in ruminants (1). Magnesium defi-order that occurs in a wide range of nutritional ciency results from a reduced ability of the Received: January 2019 Accepted for publication: February 2019 590 S. Shoeib, M. Sayed-Ahmed, S. El-khodery calves to absorb dietary magnesium as exogenous dietary supply (2). Magnesium homeostasis mainly depends on its absorption from gastrointestinal tract, bioa-vailability and requirement for milk, but not on a hormonal feedback system. Any extra Mg is excreted via urine. Reduced Mg absorption from the rumen may explain the insufficient inflow (3, 4).Consequently, the level of magnesium in the blood and extracellular fluid depends onthe balance between input and output (4). The calves affected have concurrent hy-pocalcemia, as they fed milk with low magnesium (Mg) concentration. The camel calves fed with only milk, will be insufficient to maintain the balance of Mg and calcium (Ca). In lactat-ing mothers and their camel calves, higher serum values of aluminum (Al) and lower values of Mg were observed compared to other groups. It is suggested that the increase in serum Al in calves might be due to feeding Al-rich milk and that the decline in Mg concentration might be a consequence of Al and increased secretion of parathyroid hormone (5).The efficiency of absorption during intestinal transit is reduced in cases of diarrhea (4). Mild neurological signs with increased excitability occur when there is a critical decrease in the serum Mg level. However, animal may not show clinical signs, although there is low serum Mg level (6). Ingestion of green grasses caused high rumen pH, with subsequent reduction of Mg absorption (7). The most famous clinical signs of hypomag-nesemic tetany are represented by excitations and muscle cramps, which are strongly interrelated with the concentration of Mg in the cere-brospinal fluid. It is suggested that the neurological signs of hypomagnesaemia are induced by activation of neurons in the cerebrospinal fluid (3). Therefore, the present study describes clinical consequences and successful management of hypomagnesemic tetany in camel calves. Materials and methods Study area This clinical study was carried out in central Saudi Arabia (Al-Qassim, Hermla and Shaqra). Each farm was visited twice monthly to explore the presence of clinical disease. Animals and study design A total of 27 camel calves with age range from 2-6 months were investigated clinically and biochemically from November 2016 to April 2017. 22 camels had history of nervous signs such as tetany, convulsion, ataxia, staggering gait, muscular tremors and inability to stand up. Most of the clinical cases were recorded in winter and spring seasons. All the clinically affected animals were treated with Mg therapy as mentioned below. Clinical examination All calves in the control, and clinical groups were clinically examined as previously documented (4). Serum sampling and biochemical analysis Blood samples from control and diseased calves were obtained from the jugular vein and collected into sterile tubes without anti-coagulant. Serum was obtained and stored at -80 °C till analysis. Serum biochemistry was conducted using commercial kits (Bio- Mureix) for spectrophotometric determination of Mg, P, albumin , calcium, potassium, Aluminium, urea nitrogen, and creatinine (8-15), Globulin and Albumin/Globulin ratio were calculated.The parathyroid hormone was measured by radio-immunoassay (16). Ruminal fluid collection and analysis Ruminal samples were collected by stomach tube from each animal. The samples were cen-trifuged at 3000 rpm for 15 minutes. The supernatant layer was transferred to a clean tube and keep in deep freezer for spectrophotometric determination of Mg, Ca, and P using commercial kits (Bio-morieux) and spectrophotometer (spin lab, Spinreact S.A. Model 2003) as previously described (17). Hypomagnesemic tetany in camel calves (Camelus dromedarius): clinical consequences and treatment outcomes 591 Assessment of therapeutic response Therapeutic response of the clinical cases was assessed after a five day treatment with IV injection of 100 ml calcium magnesium borogluconate (Cal-Bor-Mag, kela Pharamceu-ticals, belgium) daily. In addition, 10 ml of Vitamin B complex (corobral, Vetoqunol, france) was given IM and 2gm Mg sulphate were given orally for 5 days (18). Statistical analysis Statistical assessment of the clinical parameters, biochemical findings of the control group and of hypomagnesemic calves before and after treatment were done using SPSS Ver. 16.0 package program with Student's paired-t test as previously described (14). All data were given as Mean ± SEM, and results were considered significant at P < 0.05. Results Table 1: Serum biochemical parameters (Mean± SE) of camel calves with hypomagnesemic tetany before and after treatment Parameter Control Diseases Treated Calcium (mmol/L) 2.6±0.18 2.31 ±0.3 2.5 ±0.4 Chloride (mmol/L) 110.9±1.3 121.5±1.4 112.2±1.5 Cupper (mmol/L) 82.5±1.5 67.2±2.1 76.6±1.5 Phosphorus (mmol/L) 2.44±0.14 2.25± 0.3 5.7±0.2 Magnesium (mmol/L) 1.27±0.17 0.89±0.09* 1.3±0.2 Aluminium 2.4±0.17 5.4 ± 0.23 2.8±0.07 Zink (mmol/L) 57.8±1.6 62.6±2.1 52.6±1.4 Total protein (g/L) 66.4±0.2 80.1±0.28 76±0.2 Albumin (g/L) 38.4±0.08 38.1±0.13 34 ±0.1 Globulin (g/L) 28±0.2 42±0.15 42±0.16 Urea(mg/dl) 24.9±0.2 22.07±0.5 22.07±0.4 Creatinine (mg/dl) 1.8±0.1 1.3±0.05 1.3±0.07 *: means significantly different at p<0.05 Table 2: Calcium, phosphorus and magnesium in ruminal fluid analysis (Mean± SE) of camel calves with hypomagnesemic tetany befor and after treatment Parameter Control Before treat After treat Mg (mmol/L) 1.23±0.4 0.98 ± 0.40* 1.2±0.5 Ca (mmol/L) 2.8±0.17 1.6±0.21* 2.84±0.4 P (mmol/L) 1.81±0.12 1.5±0.05* 1.8±0.03 *: means significantly different at p<0.0 All the twenty -two animals from twelve different herds in the area were found to express nervous manifestations such as tetany, convulsion, ataxia, staggering gait, muscular tremors and inability to stand up (Fig.1). There was also tachypnea, tachycardia and mild elevation of rectal temperature. Clinicopathological findingsshowed a significant^ < 0.05) decrease in the serum values of Mg in camels calves before treatment (0.89±0.09mmol/L) compared with treated camel calves (1.3±0.2 mmol/L). The other examined parameters remained within the normal average in both diseased and normal camels (P > 0.05). Ruminal fluid analysis showed a significant reduction (P<0.05) in Mg, Ca and P in hypomagnesemic tetany camel calves compared to control animals (Table 1 & 2). 592 S. Shoeib, M. Sayed-Ahmed, S. El-khodery Figure 1: A camel calves suffered from hypomagnesemictetany showing nervous signs (A), tetanic convulsion and recumbency (B), and star gazing and staggering gait (C) Discussion Diseases that cause neurological signs in animals are many and are usually caused by a variety of agents including viruses, parasites, bacteria, neuro-toxic chemicals and plant poisons as well as genetic abnormalities and nutritional deficiencies (19).Clinical findings of clinical hypomagnesemia in camel calves were tetanic convulsions, opisthotonos, star gazing, episodes of convulsions with erected ears, extension of head and neck, erected tail, widening and rotation of the eye ball and dilatation of nares, similar findings were recorded previously (18,20). It has been demonstrated that slow type of hypomagnesemic tetany is diagnosed in cows for several months, especially during cold season. Critical reduction of Mg levels in the plasma is usually accompanied by clinical signs of hyperirritability and incoordination. Hypo-magnesemic tetany is termed the winter tetany, associated with low dietary Mg, low quality of feeds and environmental stresses (21).The clinical parameters showed a significant increase (P<0.05) in the heart and respiratory rates in hy-pomagnesemic cases than control. Body temperature showed mild increased in hypomag-nesemic cases compared to control and treated cases. However, ruminal motility was significantly decreased than control. These results were similar to those recorded in previous stud- ies (20, 22, 23). The imbalance between calcium and magnesium ratio in extra and intracel-lular fluid might induce the abnormal function of nerve fibers in peripheral and/or central nervous system, which lead to increase in heart rate and rapid respiration (24). The reduction of ruminal motility in diseased cases could be attributed to the biological role of Mg in maintaining the ruminal contractions (3).The clinical identification of hypomagnesaemia is usually made by a combination of clinical history, signs and response to Mg therapy. The clinical signs rapidly progress and death occurs within few hours, but most of animals with hypomagnesae-mia may found dead (25). In most cases, low dietary magnesium intake not only produces clinical signs, but also may need other predisposing factors. In the present study the Mg level was reduced to 0.89mg/dl in clinical tetanic cases. The hyperesthesia, the tetany and convulsion in hypomagnesaemia could be attributed to the central effect or the disturbance in the neuromuscular junction due to Mg/Ca deficiency (26).This study showed marked increase in aluminum in camel calves with tetany. It has been demonstrated that increase in serum Al in calves might be due to feeding Al-rich milk and that the decline in Mg concentration might be a consequence of Al and increased secretion of parathyroid hormone (5).Moreover, release of adrenal glucocorticoid as a result of stress reaction, causes increased circulating level of K and decrease Mg transport across the Hypomagnesemic tetany in camel calves (Camelus dromedarius): clinical consequences and treatment outcomes 593 choroidal plexus, with subsequent appearance of the neurological signs (27). Another pathway is that, the decrease of energy intake inhibits the rumen fermentation process, with subsequent decrease of volatile fatty acids and CO2 concentrations and increase of ammonia concentration. The decrease of energy intake can also increase the rumen pH due to high ammonia and low volatile fatty acid with eventual reduction of the mg absorption (3). The increased urea level in diseased cases has been observed also in other studies (29). Anorexia and starvation associated with the disease causes protein catabolism and consequently retention of nitrogenous wastes (6). In ruminants, it is known that when there is low protein intake, animals can diminish their urinary urea loss, and increase the availability of nitrogen required for protein synthesis in the rumen. (30). Ruminal fluid showed a significant reduction (P<0.05) in Mg, Ca and P in hypomag-nesemic tetany camel calves compared to control These results were comparable to those recorded in goats with experimentally-induced hy-pomagnesaemia (29). The Mg concentration in the diet is a major determinant of it concentration in the ruminal fluid (31). Therefore, the low Mg in ruminal fluid could be attributed to low Mg intake, which consequently leads to low serum Mg levels. In young calves, it has been known that Mg is absorbed efficiently from the small intestine. But, when the rumen and retic-ulum are well developed, they are the main site of absorption. (32). The absorption of Mg through ruminal wall depends on its concentration in the ruminal fluid and efficacy of Mg transport action (31). There are 2 mechanisms for Mg absorption from rumen epithelium, the active and passive mechanisms. Active transport of Mg across the rumen wall is necessary when Mg in supplement in diet is little, active transport of the Mg crosswise the wal of rumen is of great importance. However, when there is high rumen fluid Mg, passive transport occurs. Passive transport of Mg allow decrease of concentration gradient into the extracellular fluid (3).The minimum ruminal fluid Mg level that activates the passive mechanism is 9.2 mg/dl (33). Because the Mg concentration in ru-minal fluid of clinical hypomagnesemia was lower than that level, the passive transport mechanism of Mg is inactivated. In conclusion, the results of our investigation indicate that hypomagnesaemia exists in camel calves and it should be considered when confronted with cases showing nervous manifestations. Conflict of interest There is no conflict of interest References 1. Smith RA and Edward WC. Hypomanesem-ictetany of ruminants.Vet Clin North Am Food An-imPract.1988; 4(2):365-77. 2. Smith RH. Importance of magnesium in the control of plasma calcium in the calf. Nature,1961; 191: 181-2. 3. Martens H and Schwigel M. Pathophysiology of grass tetany and other hypomagnesiumias.Impli-cation for clinical management Vet Clin North Am Food AnimPract2000; 16 (2):339-8. 4. Radostits OM, Gay CC, Hinchcliff KW, Constable PD. Veterinary Medicine: a textbook of the diseases of cattle, horses, sheep, pig and goats.2010; 10thEd. B. Saunders, London, NewYork, Philadelphia, Sydney and Toronto. 5. Khaled A Al-Busadah. Serum Concentration of Aluminum, Calcium, Magnesium and Phosphorous in Camels Scientific Journal of King Faisal University (Basic and Applied Sciences) 2010; 11 (1): 1431. 6. Hoff B, Rognmo A, Havre G, Morberg H. Seasonal Hypomagnesemia in reindeer on Kau-tokeino winter pasture in Finnmark County, Norway. Rangifer1993; 13 (3): 133-6. 7. Parkinson TJ, Vermunt JJ, Malmo J. Disorders of magnesium metabolism. Diseases of cattle in Australasia. New Zealand Veterinary Association Foundation for Continuing Learning2010; 532-41. 8. Fischbach FT, Dunning MB: Manual of Laboratory and Diagnostic Tests, 8th ed. Philadelphia: 2009; Lippincott Williams and Wilkins. 9. Burtis C, and Bruns DTietz Fundamentals of Clinical Chemistry. 6th ed. WB. Saunder Co., Philadelphia, USA 2007. 10. Henry RF, Cannon DC and Winkelman JW. Clinical Chemistry Princepals 2nd. Ed.harper and Roe, Hagerstown, MD. Hoff, B.; Rognmo, A.; Havre, G. and Morberg, H. (1993): Seasonal Hypo- 594 S. Shoeib, M. Sayed-Ahmed, S. El-khodery magnesemia in reindeer on Kautokeino winter pasture in Finnmark County, Norway. Rangifer1974; 13 (3): 133-136. 11. Young DS. Effect of drugs on clinical Laboratory tests, 3rd Edition. AACC Press, Washington, D.C.1990; pp. 3122-31. 12. Chernecky CC and Berger BJ: Laboratory Tests and Diagnostic Procedures, 5thed. St.Louis: Saunders2008. 13. Thefeld W: Annuals of Clinical Biochemistry. Cited by: Wooton and Freemon1974;pp.79. 14. Ozkan C, Altug N, Yuksek N, Kaya A, Akgul Y. Assessment of lectrocardiographic findings, serum nitric oxide, cardiac troponins and some enzymes in calves with hyperkaliemia related to neonatal diarrhea. Revue MedVet 2011; 162 (4): 171176. 15. Pagana KD and Pagana TJ. Mosby's Manual of Diagnostic and Laboratory Tests 4thed. St. Louis: 2010; Mosby Elsevier. 16. Mayer GP, Hurst JG, Barto JA, Keaton JA, Moor MP. Effect of epinephrine" on PTH hormone secretion in calves. Endocrinol1979; 104: 1181-7. 17. Reynolds CK, Bell MC, Sims MH. Changes in Plasma, Red Blood Cell and cere- brospinal fluid Mineral Concentrations in Calves during Magnesium Depletion Followed by Reple- tion with Rec-tally Infused Magnesium Chloride. J Nutr.1984; 114: 1334-41. 18. Naik SG, Ananda KJ, Rani BK. Magnesium deficiency in young calves and its management. Veterinary World.2010;3(4): 192-3. 19. Al-Dubaib MA, Al-SwailemA, Al-GhamdiG, Al-YamaniE, Al-NaeemAA, Al-MejaliAM, ShehataM, HashadM, El-LithyDA, MahmoudOM.Dubduba syndrome: A new neurological disease of camels with a possible viral etio-logic agent. J Camel Prac Res 2008; 15: 147-2. 20. Attia MT. Some studies on hypomagnesemia in calves under natural and experimental condi-tions.MVSc1999; Thesis, Faculty of Veterinary Medicine, Zagazig University, Egypt. 21. Matsui T. Significance of Magnesium in Animals. New Perspectives in Magnesium Res 2007; 13: 381-91. 22. Mills J. Hypomagnesemia in calves. Vet. Rec. 131: 3. Morris, M.E.: Brain and CSF magne sium concentrations during magnesium deficit in animals and humans: neurological symptoms. Magnes Res 1992; 5(4): 303-13. 23. Jayanthi M, Sundar SN, Karunandhi PS, Chaudhuri PC. Serum biochemical findings in experimental hypomagnesemia in buffaloe calves. Indian Vet J 1997; 14 (9): 752-4. 24. Shiga A, Keino M, Fujio O. Experimental studies on hypomagnesemia in ruminants: effects of varying calcium content in low-magnesium diets in serum concentration of magnesium and calcium and heart rate in lactating ewes. Jpn J Vet Sci1983; 45 (4): 435-42. 25. McCoy MM.Hypomagnesemia and new data on vitreous humour magnesium concentration as a post-mortem marker in ruminants. Magnesium Res2004; 17 (2): 137-45. 26. Morris ME. Brain and CSF magnesium concentrations during magnesium deficit in animals and humans: neurological symptoms. Magnes Res1992; 5(4): 303-13. 27. Robson AB, Sykes AR, McKinnon AE, Bell ST. A model of magnesium metabolism in young sheep: transactions between plasma, cerebrospinal fluid and bone. Br J Nutr 2004; 91: 73-9. 28. El-Sangary FH, El-Barawy, AM, Faris A, El-Hamied SSA.Hypomagnesemia in beef calves at its relation with parathyroid hormonelevel.Assiut Vet Med J 2011; 57 (131): 300-11. 29. Washia NM. Study on Hypomagnesaemic-tetany in goats. MVSc2007; Thesis, Faculty of Vet. Medicine, Alexandria University, Egypt. 30. Kaneko JJ. Clinical Biochemistry of Domestic Animals.1989; Ed. J. J. Kaneko, Academic Press Inc., New York. 31. Goff JP. The monitoring, prevention, and treat- ment of milk fever and subclinical hypocalcemia in dairy cows. Vet J 2008; 176: 50-7. 32. Dua K and Care AD. Impaired absorption of magnesium in the aetiology of grass Tetany. British Vet J1995;151:413-6. 33. Ram L, Schonewille, JT, Martens H, van'tKlooster AT Beynen AC. Magnesium absorption by wethers fed potassium bicarbonate in combination with different dietary magnesium concentrations. J Dairy Sci. 1998; 81: 2485-92. Slov Vet Res 2019; 56 (Suppl 22): 595-606 DOI 10.26873/SVR-798-2019 Original Research Article POTENTIAL AMELORATIVE EFFECT OF PANAX GINSENG ON HEPATORENAL DAMAGE OCUURED IN HYDROXUREA-TREATED RATS Samar S. Elblehi*, Dina R. S. Gad El-Karim Department of Pathology and Clinical Pathology, Faculty of Veterinary Medicine, Alexandria University, 11511, Egypt Corresponding author, E-mail: samarsaad30@yahoo.com Abstract: Hydroxyurea (HU) is an anti-neoplastic drug which is used for treatment of many types of tumors; however, its wide range of deleterious effects may limit its medical usage. This study was conducted to assess the possible protective role of Panax ginseng extract (Ginseng) against HU- induced hepato-renal damage in rats. Forty male Wistar albino rats were randomly divided into four equal groups (10 rats each). Group I (control) received 1ml purified water. Group II (Ginseng-treated) received 200 mg/kg body weight (b.wt) of Panax ginseng extract. Group III (HU-treated) received HU at a dose of 500 mg/ kg b.wt. Group IV (HU plus ginseng) received HU and Panax ginseng extract in similar doses of group II and III. All the treatments were administrated orally. Treatment with Panax ginseng failed to ameliorate the hematological alterations caused by HU, but, its ability to alleviate hepato-renal cytotoxic effect was proved through significant reduction in plasma level of hepato-renal injury biomarkers (AST, ALT, GGT, urea and creatinine) which were elevated in HU-treated group. Also, this effect was affirmed by decreased level of plasma pro-inflammatory cytokines (TNF-a and IL-6) and enhanced anti-oxidant state (decreased MDA content and increased GPX activity) of hepato-renal tissues upon Panax ginseng extract administration with HU. Histopathologically, HU induced impairments in both hepatic and renal tissues of the treated rats. Conversely, concurrent administration of ginseng with HU partially improved these alterations. Key words: hydroxyurea; hepato-renal toxicity; Panax ginseng; rats Introduction Hydroxyurea (HU) as an anti-neoplastic drug is used in humans and dogs for the treatment of chronic myelogenous leukemia, poly-cythemia, ovarian cancer, melanoma, and squa-mous cell carcinoma of the head and neck. Also, it has been used for treatment of renal cell carcinoma, transitional carcinoma of the urinary bladder, prostatic carcinoma, and carci- noma of the uterine cervix in addition to thrombocytosis (1). Moreover, HU has been used for treatment of mast cell tumor and hypereosino-philic syndrome in dogs (2,3). It is the only approved option to be used in humans with sickle-cell disease as it reduces vaso-occlusive crises frequency, blood transfusions, the number of days spent in hospital and mortality of these patients (4). That's because administration of HU to sickle-cell disease patients increases the production of erythrocytes fetal hemoglobin which Received: January 2019 Accepted for publication: February 2019 596 S. S. Elblehi, D. R. Gad El-Karim can carry more oxygen than adult hemoglobin (5), decreases circulating neutrophils, enhances the deformability of erythrocytes, decrease adhesion of erythrocytes to endothelium, promotes vasodilation through production of nitric oxide and increasing adenosine deaminase activity in circulating monocytes leading to lower adenosine levels (6, 7).The mechanism of action of HU as an anti-neoplastic drug depends on its ability to arrest rapidly dividing cells at S phase of cell division (8) through inhibition of the class I ribonucleotidereductase enzyme (9) which is responsible for the formation of de-oxyribonucleotides from ribonucleotides (10) by inactivating the tyrosyl radical required for enzyme activity leading to massive reduction in the production of deoxyribonucleotides that are necessary for DNA replication (11,12). HU is generally tolerated however it has a low safety margin due to myelosuppression, as the earliest and most common reported adverse findings in human patients were pan-leukope-nia, anemia and thrombocytopenia (3, 5, 13). Additionally, growth retardation spermatogenic arrest, hematopoietic, lymphoid, cardiovascular, and gastrointestinal toxicity associated with hemosiderosis of spleen and liver have been observed in dog toxicity studies (13, 14). In rats administration of HU at high doses induced reduction in the body weight gain, anemia, leuko-cytopenia, thrompocytopenia associated with hypocellularity of lymphoid organs and bone marrow; epithelial degeneration and/or dyspla-sia of the stomach and small intestine (14) and decreased fertility in males as itinduced testicular atrophy and decreased sperm production (15), also, it can induce hepatic damage with fatty changes (13). Cardiovascular effects and slight methemoglobinemia were detected in some species ''at doses higher than clinical levels'' (13, 16), skin and nail lesions have been reported in human and canine patients (2,17,18). In addition, HU can induce lung tox-icity with pulmonary edema, lung infiltration and dyspnea (19), and pulmonary fibrosis in a minority of patients (20). Traditional herbal medicine has become the point of attraction in the clinical and experi- mental research. Panax ginseng (Korean ginseng), commonly known as ginseng is a famous medicinal plant that has been used extensively in traditional Chinese medicine for more than 2,000 years (21-24). Bioactive constituents of ginseng include ginsenosides, polysaccharides, phenolics, flavonoids, and polyacetylenes (25). In particular, the main active components of ginseng are the ginsenosides (or triterpenoid-saponins), aswell as about 38 more types of gin-senosides have been identified approximately (26). These compounds are proved to be responsible for most of the favorable effects of ginseng, which has a wide range of protective or therapeutic effects against many diseases. Ginsenosides have been studied intensively as the main active components of ginseng that have a variety of pharmacological effects, including anti-diabetes (27), anti-oxidant and anti-inflammatory (28), anti-aging (29), anti-obesity (30), immune modulator and anti-tumor effect (31, 32). To the pest of our knowledge, very few detailed reports of HU induced tox-icity in lab animals have been published and the published reports were insufficient to demonstrate the hepato-renal effect of HU administration. In this context, this study was conducted to assess the adverse effects of HU administration to male albino rats up to 1 month and the ability of Panax ginseng to ameliorate these effects. Materials and methods Chemicals Commercially available formulation of hy-droxyurea capsules (Hydrea®500mg, E R Squibb & Sons Ltd., England) was used. Panax ginseng extract were obtained in form of capsules containing 550 mg of Korean red ginseng extract, 8% ginsenosides (Nature's Way, USA). Animals and experimental design: Forty male Wistar albino rats, 6-8 weeks old and 180-200 g body weight were obtained from the closed bred colony in Faculty of Agriculture, Alexandria University, Egypt. Rats were housed in metal cages under controlled environmental conditions (24-27 °C temperature, 55% RH, and 12 h light/dark cycle) for 2 weeks. Potential amelorative effect of Panax ginseng on hepatorenal damage ocuured in hydroxurea-treated rats 597 They were observed carefully during this period to make sure that they were free from any apparent health problem. All animals fed on a standard laboratory diet and received water ad libitum during the experiment. This study was approved by committee of Care and Use of Laboratory Animals of Alexandria University. After the acclimatization period, rats were allocated into four groups (10/each). All groups received daily oral intubation using stomach tube and the treatment lasted for 30 days as the following: Group I (control) received 1ml pu-rifled water (the vehicle used for HU). Group II (ginseng-treated) received 200 mg/kg b.wt of Panax ginseng extract (33). Group III (HU-treated) received hydroxyurea at a dose of 500 mg/ kg b.wt (34), HU solution was freshly prepared by tablets dissolving in purified water. Group IV (HU + ginseng) received hy-droxyurea and ginseng extract in similar doses of group II and III. Twenty four hours after the last dose, the rats of each group were euthanized after light ether anesthesia to be subjected to the following studies. Hematological studies Anti-coagulated blood samples (in EDTA containing tubes) were collected from retro-orbital venous plexus for determination of eryth-rocytic (RBCs) count, and platelets count in addition to total and differential leukocytic count (Exigo® veterinary hematology system, Sweden) and for separation of plasma by samples centrifugation at 1000xg for 10 min. Biochemical analysis Plasma activities of AST, ALT and GGT, in addition to levels of urea and creatinine were determined using commercially available kits. Plasma level of IL-6 and TNF-alpha were detected by quantitative sandwich enzyme immu-noassay method using Rat spicific ELISA kits (abcam, USA). Antioxidant studies About 1 g of kidney and liver of each euthanized animal were separated, perfuse with phosphate buffer saline solution (PBS) solution (pH, 7.4) containing 0.16 mg/ ml heparin to remove any blood or clots, and homogenized in 9 ml PBS solution using tissue homogenizer (Glas-Col. Tissues homogenizer, China). Ho-mogenate aliquots were centrifuged at 10,000 x g for 30 min, supernatant were removed and stored at -80 till the time of assessment of concentration of malondialdehyde (MDA) and activity of glutathione peroxidase enzymes (GPX) using locally available kits (BiodiagnosticE-gypt; Biovision, USA) and as previously described (35). Protein content was detected using Bradford's reagent (Sigma-Aldrich, USA). Histopathological examination Following necropsy specimens of livers and kidneys of each rat were collected and rapidly fixed for at least 24h in10% neutral buffered formalin. The fixed specimens were processed through the conventional paraffin embedding technique, sectioned at 5 microns and stained with Mayer's haematoxylin and eosin (H&E). Stained sections were examined by light microscope and photographed using digital camera. Semiquantitative histopathological scoring system Semi quantitative histological scoring was carried out for the liver and kidneys slides. Five fields (x100) were randomly selected from each rat in each group, and the most important pathological alterations were selected and scored in each organ; the severity of lesion was estimated according to the percentage of affected area/entire section where in: 0 = absence of lesion, 1 = 5-25%, 2 = 26-50%, and 3 = >50% Statistical analysis The statistical analysis was carried-out using one way analysis of variance (ANOVA) for studying of the effect of different treatment groups on different parameters studied using SAS, (2004) (36). Results Hematological results As presented in Table 1, blood picture of HU-treated rats significantly showed a decrement in RBCs count which coupled by significant decrease in total leukocytic count (TLC), granulocytes, lymphocytes and monocytes 598 S. S. Elblehi, D. R. Gad El-Karim counts in addition to platelets count when compared to control group. Unfortunately, co- administration of ginseng with HU failed to increase the diminished values of the previously listed parameters of blood picture as compared to HU-treated rats. The sole treatment with ginseng did not enhance any of these hematologi-cal parameters compared to control group. Biochemical changes As shown in Table 2, treatment of rats with HU evoked significant elevation in plasma activity of AST, ALT and GGT enzymes when compared to control group. Administration of ginseng with HDU significantly decreases plasma activity of these enzymes if compared to HU-treated animals. In the same manner, treatment with HU led to significant increase in plasma concentration of urea and creatinine which accompanied by significant decrease in plasma albumin concentration compared to control group. Comparatively with HU-treated group, co-administration of ginseng with HU significantly ameliorated the changes in these parameters. While, treatment with ginseng alone significantly decreased plasma activity of AST and ALT without any significant effect on the previously elucidated plasma hepato-renal function biomarkers compared to control group. Plasma level of inflammatory cytokines Plasma level of TNF-alpha and IL6 revealed a significant increase in HU-treated rats in comparison with control group. Meanwhile, their values were significantly diminished following co-administration of ginseng as compared to HU-treated animals. Moreover, administration of ginseng alone did not produce any alteration in plasma level of previously detected pro-inflammatory cytokines as compared to control group. (Table 2). Oxidative changes The level of MDA recorded a significant increase in hepatic and renal tissues of HU-treated rats which was accompanied by significant decline in GPX activity in these tissues when compared to control group. These elevations were significantly ameliorated toward normal control level upon co-treatment with ginseng. Treatment with ginseng alone did not reveal any significant changes in oxidant/anti-oxidant parameters in relation to control group (Table 3). Histopathological examination The histopathological changesdetected in livers and kidneys slides were represented in Figs. (1 and 2) and Table (4) which summarized the lesions' incidence and severity reported in HU and HU+ Ginseng-treated groups. No his-topathological alterations were detected in either control or Ginseng-treated rats groups. Liver Control (Fig.1A) and Ginseng-treated rats' livers showed normal histological structures. Meanwhile, livers of HU -treated rats showed diffuse hepatocytic cytoplasmic vacuolation of both fatty type (sharply outlined vacuoles, Fig.1B, Table4) and hydropic type (hazy border vacuoles). Widening of the hepatic sinusoids, hypertrophy of Kupffer cells, widespread moderate to severe congestion and multifocal areas of hemorrhages (Fig1C) were noticed. Furthermore, multifocal areas of hepatocellularnecro-sis with mononuclear cells infiltrations were evident (Fig. 1D, Table 4).The Portal triads were thickened with edema and mononuclear cell infiltrations associated with atrophied hepatic cords (Fig1E, Table4). Conversely, livers of HU+ Ginseng- treated rats showed the previous described lesions but they were less in the severity and distribution (Fig.1F, Table4). Kidney Control (Fig.2A) and Ginseng-treated rat's kidneys exhibited normal histological limits of the glomeruli and renal parenchyma. HU -treated rats showed degenerated tubular epithelium with star-shaped tubular lumen. Also, epithelium attenuation and necrosis with desquamated necrotic debris and inter tubular hyaline casts (Fig.2B, Table 4) were noticed. Regarding to the glomerular lesions, compressed capillary tufts with increased urinary spaces and presence of necrotic remnants of capillary tufts (Fig. 2C, Table 4) were recorded. Throughout the renal parenchyma, the interstitial tissue exhibited Potential amelorative effect of Panax ginseng on hepatorenal damage ocuured in hydroxurea-treated rats 599 multifocal areas of interstitial nephritis with mononuclear cells infiltration, inter tubular edema and marked tubular dilatation (Fig. 2D). In addition, vascular congestion, moderate peri-vascular edema associated with lymphocytic cells infiltration and moderate fibroplasia (Fig. 2E, Table 4) were noticed. In contrast, the detected renal alternations in HU+ Ginseng-treated group were reduced in the severity and distribution with marked improvement of the renal tissue (Fig. 2 F, Table 4) as compared to HU -treated group. Table 1: Effect of HU and its combination with Ginseng on blood picture of male Wistar albino rats. Groups Control Ginseng HU HU+ginseng RBCs(x106/^L) 7.86±0.27a 7.85±0.27a 5.40±0.19b 5.28±0.24b TLC(X103/^L) 14.12±0.23a 14.96±0.28a 8.56±0.26b 8.31±0.25b Granulocyt(x103/^L) 4.18±0.26a 4.59±0.13a 1.82±0.05b 1.84±0.03b Lymphocyte(x103/^L) 8.46±0.21a 8.76±0.23a 5.78±0.27b 5.68±0.25b Monocyte(x103/^L) 1.47±0.16a 1.51±0.15a 0.95±0.07b 0.93±0.06b Platelets(x103/^L) 555.20±8.01a 552.80±9.32a 330.80±17.31b 329.00±17.42b All values are expressed as mean ± Std. Error. Means within the same raw of different litters are significantly different at (P < 0.05). H: Hydroxyurea, Ginsing: Panax ginseng extract Table 2: Effect of different treatments on different plasma hepatic and renal function biomarkers and plasma pro-inflammatory cytokins in male Wistar albino rats Groups Control Ginseng HU HU+ginseng AST(U/L) 51.40±2.60b 48.40±2.93c 84.70±2.01a 58.30±4.09b ALT(U/L) 34.20±2.99c 32.40±2.86d 72.60±5.18a 47.60±2.96b GGT(U/L) 31.00±3.30c 31.20±2.29c 57.00±2.57a 39.90±2.90b Urea(mg/dl) 54.20±2.51b 50.60±1.98c 80.40±2.94a 53.84±4.00b Creatinine(mg/dl) 0.54±0.01c 0.53±0.01c 0.76±0.01a 0.63±0.01b Albumin(g/dl) 6.02±0.06a 5.92±0.13a 4.09±0.08c 5.02±0.18b TNF(pg/ml) 27.76±2.17c 28.20±2.33c 236.10±17.65a 138.50±6.67b IL6 (pg/ml) 18.94±1.52c 17.50±1.50c 125.96±5.65a 64.58±8.42b All values are expressed as mean ± Std. Error. Means within the same raw of different litters are significantly different at (P < 0.05). 600 S. S. Elblehi, D. R. Gad El-Karim Table 3: Effect of different treatments on hepatic and renal MDA and GPX in male Wistar albino rats Groups Control Ginseng HU HU+ginseng Liver MDA (pmol/g protein) 8.98±0.93c 7.59±0.53c 26.96±1.44a 26.96±1.44a Kidney MDA (pmol/g protein) 5.03±0.87c 4.31±0.74c 22.46±2.06a 13.57±0.92b Liver GPX (U/mg. protein) 3.28±0.29a 3.66±0.37a 1.81±0.12b 2.84±0.28a Kidney GPX (U/mg protein). 3.04±0.42ab 3.44±0.26a 1.74±0.15c 2.37±0.19b All values are expressed as mean ± Std. Error. Means within the same raw of different litters are significantly different at (P < 0.05). Discussion Hydrxyurea is a potent anti-neoplastic drug which is widely used to prevent painful crises of sickle cell anemia (37). Most available toxicity data from animals are present only in product labels, which are incomplete, and are often found only in summary statements (reviewed by Liebelt et al. 2007) (38).This study aimed to evaluate the toxic side effects of HU on liver and kidney tissues of rats and the probable role of Panax ginseng in refinement of such effects. HU is a small molecule, which is rapidly taken up by water and distributed widely throughout the body; it may reach vulnerable areas rapidly causing prompt cytotoxicity (39,40). Myelosupression is one of the major associated complications of the treatment with hy-droxyurea (41), as HU can kill S-phase colony-forming unit (CFU) cells of bone marrow and retard the entry of surviving cells into S-phase of division (42) and this may explain the significant reduction in RBCs, leukocytic and platelets count of HU-treated group. Also, HU has the ability to induce damage to erythrocytes membranes due to erythrocytic membrane lipid peroxidation causing lysis of RBC (43) which can be considered as another illustration for the decrement of RBCs count. On the other hand, hydroxyurea inhibit DNA synthesis in germinal center of lymph follicle (44) which may explain the cause of lymphopenia in HU-treated animals. It was suggested that hydroxyurea can induce production of intracellular ROS (42-47) which are involved in cytotoxic effect of hy-droxyurea (48,49), as the mechanisms of action of HU depends on its rapid metabolism which produce carbomoylnitroso intermediate which then turned to nitroxide compounds (including nitric oxide) that are responsible for most of toxic effects of HU (50,51). These nitroxides act as free radicals which enhance formation of reactive oxygen species, creating oxidative stress state, interacting with nucleic acids and proteins to offset cellular functions, and altering cell signaling (51). This could be the reason for the elevated plasma activities of different liver enzymes (AST, ALT and GGT) due hepato cellular necrosis and membrane damage causes the release of these enzymes into the circulationin addition to increased plasma level of urea and creatinine. This Indicated oxidative hepato-ne-phrotoxic effect of HU which was affirmed by our histopathological findings. Potential amelorative effect of Panax ginseng on hepatorenal damage ocuured in hydroxurea-treated rats 601 Figure 1: Representitive photomicrograph of rat livers stained with HE: (A) Normal histoarchitecture of a control rat liver (X100). (B,C,D&E) livers of HD- treated rats: (B) Hepatocytes showing sharply outlined fat vacuoles (arrows, X400), (C) Replacement of the necrotic hepatocytes with RBCs and mononuclear cells (H) associated with congestion (C, X400), (D) Hepatocyte cytoplasm showing hazy border vacuoles (arrows) in and hepatocellular necrosis with mononuclear cells infiltrations (asterisks, X400), (E) Thickening of the portal areas with edema and mononuclear cells infiltrations (asterisks) with newely formed bile ductules and atophied hepatic cords (arrows, X100). (F) HD+ Ginseng -treated rats showed markrd improvement of the hepatic tissue with minute area of hepatocellular necrosis ( arrow, X100) 602 S. S. Elblehi, D. R. Gad El-Karim Figure 2: Representitive photomicrograph of rat kidneys stained with HE.X100: (A) Normal histoarchitecture of a control rat kidney. (B,C,D&E) kidneys of HU- treated rats: (B) Entertubular hyaline casts (black arrows), thining and attinuation of the tubular epitheium (blue arrows) and perivascular lymphocytic cells infiltration (asterisk), (C) Necrotic capillary tufts with widening of urinary space (arrows) associated with vascular congestion,(D) Interstitial mononuclear cells infiltrations in the renal cortex ( asterisk) with atrophied and necrotic glomerular tufts (black arrows) cystically dilated renal tubules (blue arrows), (E) Vascular congestion(c) and fibroplasia of interstitial tissue (F). (F) HU + Ginseng -treated rats showing marked improvement of the renal tissue. Potential amelorative effect of Panax ginseng on hepatorenal damage ocuured in hydroxurea-treated rats 603 Table 4: Incidence and Severityof histopathological lesions recorded in livers and kidneys tissyes of HU and HU+Ginseng- treated rats Incidence1 and Severity2 of histopathological Lesions HU-treated rats HU+ Ginseng-treated rats Scored lesions Ab- Mild Moder- Severe Ab- Mild Moder- Severe sent (+) ate (+ -++) sent (+) ate (+++) (-) (++) (-) (++) Liver a-Hepatocytic vac-ular and hydropic degeneration 0 0 4 6 0 0 8 2 b-Hepatocytic fat-tyvaculation 0 0 7 3 0 6 3 1 c-Congestion of central and portal veins. 0 1 3 6 1 6 4 0 d-Hepatocellular ne- 0 0 8 2 0 7 2 1 crosis. e-Periportal infilama- 0 0 6 4 0 5 3 2 tory cells infiltra- tions. a-Inter tubular cast 0 1 Kidneys 6 3 0 6 3 1 formation b-Tubular epitheium 0 0 3 7 0 7 2 1 necrosis c-Vascular conges- 0 0 4 6 0 6 4 0 tion (cortical or me- dullary) d-Glomerular lesions 0 0 7 3 0 8 2 0 e-Interstitial mononu- 0 0 8 2 0 6 4 0 clear cells infiltra- tions f-Interstitial fibrosis 0 0 6 4 3 5 2 0 1Number of rats with lesions per total examined (10 rats). 2Severity of lesions was graded by estimating the percentage area affected in the entire section. -Lesion scoring: (0) absence of the lesion=0%, (+) mild=5-25%, (++) moderate=26-50% and (+++) severe >50% of the examined tissue sections. HU(hydroxyurea) and Ginsing(Panax ginseng extract ). Plasma albumin level is considered as a good indicator for assessment of liver and kidney function as it synthesized by liver and reabsorbed by renal tubules after glomerular filtration, in addition, it is considered one of negative acute phase proteins as its level decreases in response to inflammation (52), so, its level decrement in HU-treated rats may be attributed to the pre-mentioned oxidative hepato-renal affection which could affect hepatic albumin production and increase urinary albumin loss or due to hepato-renal inflammatory state. Oxidative damage of HU was proved in our study by increased level of MDA (lipid peroxide) and decreased level of GPX in both of hepatic and kidney tissues of HU-treated animals which accompanied by an elevated level of IL6 and TNF alpha in serum of the same animal group. Parallel to this, HU-induced hepato-ne-phrotoxic damages were affirmed histopatho-logically by presence of different hepatic and renal lesions that were previously mentioned in HU-treated rat group. Similar lesions were re- 604 S. S. Elblehi, D. R. Gad El-Karim ported by Ahmad et al. (2018) (53). The detected hepato-renal structural distortion may be due to previously mentioned HU-induced oxidative damage. Fortunately, most of biochemical and histopathological alterations caused by HU administration were partially reversed upon co-administration with ginseng. The semiquantitative scoring system demonstrated the protective role of ginseng in HU treated- rats group which was displayed by decreasing the intensity and distribution of the recorded hepatorenal lesions. This effectuation may be attributed to antioxidant activity of Panax ginseng that was previously cleared in several studies (54-56). Hepato-renal protective effect of ginseng as an antioxidant was previously recorded in different studies (57-60). Conclusion Finally, we could conclude that hydroxyurea has an oxidative stress-related deleterious effect on liver and kidneys, in addition to blood cellular components, but, these effects can be partially ameliorated upon administration of ginseng extract as a dietary antioxidant. Conflict of interest The authors declare that they have no conflict of interest. References 1. FDA. Center for Drug Evaluation and Research. Hydrea® packet insert.2010 http://www.ac-cessdata.fda.gov/drugsatfda_docs/la-bel/2010/016295s040lbl.pdf. 2. Marconato L, Bonfanti U, Fileccia I. Unusual dermatologic toxicity of hydroxyurea in two dogs with spontaneously occurring tumors. J Sm An Pract2007;48: 514-17. 3.Rassnick KM, Al-Sarraf R, Bailey DB, Chretin JD, Phillips B Zwhalen CH. Phase II open-label study of single-agent hydroxyureafor treatment of mast cell tumours in dogs. Vet Comp Oncol 2010;8:103-11. 4. Bunn HF. Pathogenesis and treatment of sickle cell disease. N Eng J Med1997; 337: 762-69. 5.Charache S, Terrin ML, Moore RD, Dover GJ, Barton FB, Eckert SV, McMahon RP, Bonds DR. Effect of hydroxyurea on the frequency of painful crises in sickle cell anemia. N Eng J Med 1995; 332: 1317-22. 6. Venkayala SL, HargisJC, Woodcock HL.Un-lockingthebinding and reaction mechanism of hydroxyurea substrates as biological nitric oxide donors. J Chem Info Modeling 2012; 52: 1288-97. 7.Silva-Pinto AC, Dias-Carlos C, Saldanha-Araujo F, Ferreira FIS, Palma PVB, Araujo AG et al. Hydroxycarbamide modulates components involved in the regulation of adenosine levels in blood cells from sickle-cell anemia patients. Ann Hematol 2014; 93:1457-65. 8. Philips FS, Sternberg SS, Schwartz HS, Cro-nin AP, Sodergren JE, Vidal PM. Hydroxyurea I. Acute cell death in proliferating tissues in rats.Can-cer Res 1967; 27: 61-74. 9. Jiang J, Jordan SJ, Barr DP, Gunther MR, Maeda H, Mason RP. In vivo production of nitric oxide in rats after administration of hydroxyurea. MolPharmacol. 1997; 52(6):1081-6. 10. Jordan A., Reichard P. Ribonucleotide reductases. Annu. Rev. Biochem. 1998;67:71-98. 11. Shao J, Zhou B, Chu B, Yen Y. Ribonucleotide reductase inhibitors and future drug design. Curr Cancer Drug Targ 2006; 6:409-31. 12. Saban N, Bujak M. Hydroxyurea and hy-droxamic acid derivatives as antitumor drugs. Cancer ChemotherPharmacol 2009; 64: 213-21. 13. FDA. Center for Drug Evaluation and Research. Droxia® packet insert. 2012 http://www.ac-cessdata.fda.gov/drugsatfda_docs/la-bel/2012/016295s041s042lbl .pdf. 14. Morton D, Reed L, Huang W, Marcek JM, Austin-LaFrance R, Northcott CA et al. Toxicity of Hydroxyurea in Rats and Dogs.ToxicolPathol 2015;43(4):498-512. 15.Saalu LC, Osinubi AA, Akinbami AA., YamaOE, Oyewopo AO, Enaibe BU. Moringaoleif-era Lamarck (drumstick) Leaf Extract Modulates the Evidences of Hydroxyurea -Induced Testicular Derangement. Int J Appli Res Natur Products 2011; 4 (2): 32-45. 16. Wray JD. Methaemoglobinaemia caused by hydroxycarbamide (hydroxyurea) ingestion in a dog. J Sm An Pract 2008;49: 211-15. 17. O'Branski EE, Ware RE, Prose NS, Kinney TR. Skin and nail changes in children with sickle cell anemia receiving hydroxyurea therapy. J Am AcadDermato2001; l44: 859-61. 18. Vassallo C, Passamonti F, AdrigoM,Nolli G, Mangiacavalli S, Boroni G. Muco-cutaneous changes during long-term therapy with hy-droxyurea in chronic myeloid leukemia. ClinEx-pDermatol 2001; 26: 141-48. Potential amelorative effect of Panax ginseng on hepatorenal damage ocuured in hydroxurea-treated rats 605 19. Imai H, MatsumuraN, YamazakiY, Kanaya-maY, MasudaT, KuwakoT, KamideY, TomizawaT, Matsumoto S, MitsuiT, KairaK, OnoA, KogaY, ShibusawaN, SunagaN, HisadaT, Yokoo H, Yamada M. Hydroxyurea-induced Pneumonitis in a Patient with Chronic Myelomonocytic Leukemia: An Autopsy Case. Int Med 2015; 54: 3171-76. 20. Bargagl IE, Palazz IM, Perri F, TorricelliE, Rosi E, BindiA, PistolesiM, Voltolini L. Fibrotic Lung Toxicity Induced by Hydroxycarbamide. In Vivo 2017; 31(6): 1221-23. 21. Kiefer D., Pantuso T. Panax ginseng. Am Fam Physician. 2003; 68: 1539-1542. 22. Cho I.H. Effects of Panax ginseng in neurodegenerative diseases. J Ginseng Res. 2012; 36: 342-53. 23. Ernst E. Panax ginseng: an overview of the clinical evidence. J Ginseng Res. 2010; 34: 259-63. 24. Park HJ, Kim DH, Park SJ, Kim JM, Ryu JH. Ginseng in traditional herbal prescriptions. J Ginseng Res. 2012; 36:225-41. 25. Baek SH, Bae ON, Park JH. Recent methodology in ginseng analysis. J Ginseng Res. 2012; 36:119-34. 26. Choi KT. Botanical characteristics, pharmacological effects and medicinal components of Korean Panax ginseng CA Meyer. ActaPharmacol Sin. 2008; 29:1109-18. 27. Park SH, Oh MR, Choi EK, Kim MG, Ha KC, Lee SK, Kim YG, Park BH, Kim DS, Chae SW. An 8-wk, randomized, double-blind, placebo-controlled clinical trial for the antidiabetic effects of hy-drolyzed ginseng extract. J Ginseng Res. 2014; 38(4):239-43. 28. Cha KM, Kopalli SR, Han SY, Lee SH, Jeong MS, Cho JY ha KM, Rajendra KS, Yun HS, Lee SH, Jeong MS, Cho J, Han CG, Lee SH, Kim SN, Kim JC, Kim SK. Korean red ginseng attenuates doxorubicin-induced testicular dysfunction in rats by modulating inflammatory, oxidative, and autophagy responses, J Funct Food 2018; 40:73643. 29. Kim SJ, Kim AK. Anti-breast cancer activity of fine black ginseng (Panax ginseng Meyer) and ginsenoside Rg5. J Ginseng Res 2015; 39(2):125-34. 30.Chen G, Li H, Zhao Y, Zhu H, Cai E, GaoY,Liu S, Yang H1, Zhang L. Saponins from stems and leaves of Panax ginseng prevent obesity via regulating thermogenesis, lipogenesis and lipol-ysis in high-fat diet-induced obese C57BL/6 mice. Food ChemToxicol 2017; 106(A) 393-403. 31. Kang S, Min H. Ginseng, the 'Immunity Boost': the effects of Panax ginseng on immune system. J Ginseng Res 2012; 36:354-68. 32. Kim SJ, Kim AK. Anti-breast cancer activity of fine black ginseng (Panax ginseng Meyer) and ginsenoside Rg5, J. Ginseng Res 2015; 39(2):125-34 33.Kalkan Y, Kapakin KA, Kara A, Atabay T, Karadeniz A, Simsek N, Karakus E, Can I, Yildirim S, Ozkanlar S, Sengul E. Protective effect of Panax ginseng against serum biochemical changes and apoptosis in kidney of rats treated with gentamicin sulphate. J MolHist 2012; 43(5):603-13. 34. Nair AB, Jacob S. A simple practice guide for dose conversion betweenanimals and human J Basic Clin Pharma 2016; 7: 27-31. 35. El-Bayomi K M, Saleh A A, Awad A, El-Tarabany M S, El-Qaliouby H S, Afifi M, El-Komy S, Essawi W M, Almadaly E A, El-Magd M A. Association of CYP19A1 gene polymorphisms with anoestrus in water buffaloes, Reproduction, Fertility and Development 2018; 30(3): 487-97. 36. SAS. Statistical analysis system. SAS User's Guide, SAS incorpInst 2004. 37.Schecheter AN, Rodgers GP. Sickle cell anemia-basic researches reach the clinic. Eng J Med 1995;332: 1372-74. 38.Liebelt EL, Balk SJ, Faber W, Fisher JW, Hughes CL, Lanzkron S M. et al. NTP-CERHR expert panel report on the reproductive and developmental toxicity of hydroxyurea. Birth Defects Res 2007; 80: 259-366. 39. Ahmad ME, Shadab GG, Azfer, MA. Effect of oxymetholone on SCE frequency in human lymphocyte chromosomes in vitro. Teratogen Carcinog Mutagen 2003; 1: 267-72. 40. Johnson C, Telen MJ. Adhesion Molecules And Hydroxyurea In The Pathophysiology Of Sickle Cell Disease. Haematologica 2008; 93: 48185. 41.Iyamu EW, Fasold H, Roa D, del Pilar Agu-inaga M, Asakura T, Turner EA. Hydroxyurea-in-duced oxidative damage of normal and sickle cell hemoglobins in vitro: amelioration by radical scavengers. J Clinic Lab Anal.2001; 15: 1-7. 42.Vassort F, Winterholer M, FrindelE,Tubiana M. Kinetic Parameters of bone marrow stem cells Using in vivo Suicide by Tritiated Thymidine or by Hydroxyurea. Blood 1973; 41(6): 789-96. 43. Malec J, Przybyszewski MW, Grabarczyk M, Sitarska, E. Hydroxyurea has the capacity to induce damage to human erythrocytes which can be modified by radical scavengers. BiochemBiophys Res Commun1984; 120:566-73. 606 S. S. Elblehi, D. R. Gad El-Karim 44. Farber E, Baserga R. Differential Effects of Hydroxyurea on Survival of Proliferating Cells in Vivo.Cancer Res. 1969; 29(1):136-9. 45. Sakano k. Oikawa s. Hasegawa K. Ka-wanishi S. Hydroxyurea Induces Site-specific DNA Damage via Formation of Hydrogen Peroxide and Nitric Oxide. Jpn. J. Cancer Res. 2011; 92: 116674. 46. Nagai T, Tarumoto T, Miyoshi T, Ohmine K, Muroi K, Komatsu N, Sassa S, Ozawa K. Oxidative Stress is Involved in Hydroxyurea-induced Erythroid Differentiation. British J. of Haematol 2003; 121: 657-61. 47. Cokic VP, Smith RD, Beleslin-Cokic BB, Njoroge JM, Miller JL, Gladwin MT,Schechter AN. Hydroxyurea induces fetal hemoglobin by the nitric oxide-dependent activation of soluble guan-ylylcyclase. J clinic invest 2003; 111(2): 231-9. 48. Przybyszewski WM, Malec J. Protection against hydroxyurea-induced cytotoxic effects in L5178Y cells by free radical scavengers. Cancer Lett 1982;17: 223-8. 49. Grabarczyk M, Przybyszewski WM, Kwiat-kowska J, SitarskaE,Malec J. Alteration in lyso-some supravital staining as marker ofhydroxyurea-induced cytotoxicity and its modification by radical scavengers in L5178Y cells in culture. Neoplasma 1983; 30:541-6. 50. Yarbro JW. Mechanisms of action of hydroxyurea. SeminOncol 1992; 19:1-10. 51. Koviac P. Hydroxyurea (therapeutics and mechanism): Metabolism, carbamoylnitroso, ni-troxyl, radicals, cell signaling and clinical applications. Med Hypotheses 2011; 76: 24-31. 52. Latimer KS, Mahaffey EA, Prasse KW. Veterinary Laboratory Medicine, Clinical Pathology. 4th ed. Iwoa State Press. USA 2003. 53. Ahmad MF, Ansari MO, Jameel S, Wani AL, Parveen N, Siddique HR et al. Protective Role of Nimbolide against Chemotherapeutic Drug Hydroxyurea Induced Genetic and Oxidative Damage in an Animal Model. Environ Toxicol Pharmacol 2018; 60: 91-9. 54. Choe WCS, Chung W, Lee SKW, Leung AWN, Cheng CHK, Yue KKM. Ginsenoside Re of Panax Ginseng possesses significant antioxidant and antihyperlipidemic efficiencies in streptozotocin-in-duced diabetic rats. Euro J Pharma 2006; 550: 173-9. 55. Swairess FAR. Effect of Ginseng Extract Supplementation on Renal Functions in Diabetic Rats. J of Agri Sci 2011; 3(2): 17-31. 56. El-Denshary ES, Al-Gahazali MA, Mannaa FA, Salem HA, Hassan NS, Abdel-Wahhab MA. Dietary honey and ginseng protect against carbon tetra-chloride-induced hepato-nephrotoxicity in rats. Exp Toxicol Pathol 2012; 64(7-8): 753-60. 57. Zhu H, Long MH, Wu J, Wang MM, Li XY, Shen Het al. Ginseng alleviates cyclophosphamide-induced hepatotoxicity via reversing disordered homeostasis of glutathione and bile acid. Sci Rep. 2015;5:17536. 58. Qi ZL, Wang Z, Li W, Hou JG, Liu Y, Li XDet al. Nephroprotective Effects of Anthocyanin from the Fruits of Panax ginseng (GFA) on Cispla-tin-Induced Acute Kidney Injury in Mice. Phy-tother Res 2017; 31(9):1400-9. 59.Wang W, Wang S, Liu J, Cai E, Zhu H, He Zet al.Sesquiterpenoids from the root of Panax Ginseng protect CCl4-induced acute liver injury by anti-inflammatory and anti-oxidative capabilities in mice. Biomed Pharmacother2018; 102: 412-19. 60.Zhou YD, Hou JG, Liu W, Ren S, Wang YP, Zhang Ret al. 20(R)-ginsenoside Rg3, a rare sapo-nin from red ginseng, ameliorates acetaminophen-induced hepatotoxicity by suppressing PI3K/AKT pathway-mediated inflammation and apoptosis. In-tImmunopharmacol 2018; 59: 21-30. Slov Vet Res 2019; 56 (Suppl 22): 607-13 DOI 10.26873/SVR-799-2019 Original Research Article NIGHT FEEDING REGIME IMPROVES WATER QUALITY, GROWTH PERFORMANCE AND FEEDING BEHAVIOUR OF EUROPEAN EELS, Anguilla anguilla Abdel-Hay M. Abdel-Hay1, Sara A. Hamed1, Mohamed M. Zayd1, Fatma A. Abouelenien2, Ibrahim F. Rehan3, Seham El-Kassas4, Amany M. Deyab1, Radi A. Mohamed1* department of Aquaculture, Faculty of Aquatic Sciences and Fisheries, Kafrelsheikh University, 33516, Egypt, 2Department of Hygiene and Preventive Medicine, Faculty of Veterinary Medicine, Kafrelsheikh University, 3Department of Animal Behavior and Husbandry, Faculty of Veterinary Medicine, South Valley University, Qena (83523), Egypt, 4Department of Animal Wealth Development, Faculty of Veterinary Medicine, Kafrelsheikh University Corresponding author: r.mohamed.vet@gmail.com and RadiMohamed@fsh.kfs.edu.eg Abstract: This study was aimed to explore differences between day and night feeding of farmed European eels (Anguilla anguilla) on: (1) the growth and feeding behaviour of eels; and (2) the subsequent impact on the quality of the culture water. A 30 day field experiment was conducted in which 40 European eels (186.4 ± 9.26 g) were arbitrarily allocated into two replicate concrete ponds (200 x 100 x 125cm) that were divided into two treatment groups, one receiving feed by day (DF) and the other receiving feed at night (NF). Results revealed a significant improvement in eel's growth performance (larger final weight and faster growth) of the fish that were fed by night (NF). Moreover, feeding behaviour was also improved in the NF group with a significantly shorter latency to start feeding and shorter feeding duration. As a consequence of improved feeding behaviour, more favourable water quality conditions (higher dissolved oxygen concentrations and lower ammonia concentrations) were observed in the NF group. In conclusion, feeding eels by night could considerably enhance the culturing of eels by promoting enhanced growth and feeding behavior, and simultaneously improving water quality. Key words: feeding regime; European eel; behaviour; nocturnal feeding; growth; performance Introduction The European eel (Anguilla anguilla) is a commercially important fish species that is distributed worldwide. It is a catadromous fish which spawns in the Sargasso Sea. For reproduction and spawning, the eel's larvae migrate to the Atlantic coast at which larvae molt sev- eral times till reach silver eels stage then return to lake or liver habitat to feed and grow up until it reach partial maturation. At this stage of growth, eels migrate to the marine water for another cycle of proliferation and spawning (1). Due to a large demand in eels for consumption and a dwindling stock in the wild, the Received: January 2019 Accepted for publication: February 2019 608 A. M. Abdel-Hay, S. A. Hamed, M. M. Zayd, F. A. Abouelenien, I. F. Rehan, S. El-Kassas, A. M. Deyab, R. A. Mohamed farming of eels has become increasingly popular in recent years. Given that the activity is still relatively new, there are many aspects of eel culture that are to date unknown. For example, preferences in feeding time and variations in diurnal and nocturnal feeding patterns. Most fish species show daily rhythms in locomotion and feeding behaviour. These activities are carried out diurnally or nocturnally according to the species of fish in question (2). Previous studies have described the locomotion and feeding activities of the European eel as mainly nocturnal with increased activity around the time of light shifts; from dark to light and light to dark (1). Feeding at an optimal time of day can yield biological advantages to the species in question such as reducing energy loss (3). The European eel is thought to feed both diurnally and nocturnally and its feeding patterns are thought to be independent of locomotor activity (4). In eel culture, to avoid feed waste and its bad effects on water quality, animals are provided with feed in a single meal per day and uneaten feed was removed as soon as possible. The feed should be provided daily in the same time to improve the feeding utilization and digestion because fish prepare its digestive secretions just before the upcoming meal that giving the opportunity for better feed digestibility (5). From this perspective, little is known about the effect of feeding time on European eel (Anguilla anguilla) reared for commercial production. Consequently, this study was aimed to investigate the effect of feeding time on the growth performance and feeding behavior of the European eel (Anguilla anguilla) reared in concrete ponds in Egypt, as well as the subsequent impact on water quality. Materials and methods Animal, management and experimental design A total of 80 farmed animals were obtained from the General Authority for Fish Resources Development (GAFRD), Cairo, Egypt. The experiment was conducted in 4 equal-sized concrete tanks (200 x 100 x 125 cm). The study was conducted in two treatments; one receiving feed by day (DF) and the other receiving feed at night (NF), two replicates each with continuous air pumping as a source of oxygen. Each tank was supplied with a separate water inlet and outlet covered with mesh to prevent the entrance of undesirable fish and predators and to prevent the escape of the study animals. The bottom of each pond contains crockery and PVC pipes (Figure 1). The eels were placed in a quarantine tank for 30 days to allow them to recover from the stress of transport. The experimental tanks were first disinfected and filled with water before being stocked with 10 randomly selected eels of a similar body weight. The eels in the first two tanks were fed during daylight at 7 am (DF) and the eels in the second two tanks were fed during nighttime at 7 pm (NF). The eels were fed raw fish flesh for 30 day, with a feeding ratio of 10% of body weight. The eels received feed once every 24 hours on a mesh in the corner of each pond to prevent leakage of the feed to the pond bottom. Committee of Aquatic Animal Care and Use in Research, Faculty of Aquatic Sciences and Fisheries, Kafrelsheikh University, Egypt, approved the protocol and conduct of the study. Water quality analysis Water quality was analysed once per week at 10 am and 10 pm on the same day to evaluate the quality of pond water. The physico-chemical properties of water was assessed in three samples (250 ml each) in each treatment to determine total ammonia, unionized ammonia (UIA) using Martini MI 405 portable photometer. For estimation of temperature, pH, electrical conductivity (EC), total dissolved solids (TDS) and salinity a multiparameter meter was used (HI9829-03042-HANA). Dissolved oxygen (DO) was determined in each treatment at different water depth (20, 80 and 120 cm) using a specific DO meter (AQ 600 Milwaukee, Romania). Fish performance and growth indicators Eel feed intake (FI) was calculated everyday along the experiment (30 day). All 40 eels Night feeding regime improves water quality, growth performance and feeding behaviour of European eels, 609 were weighed at the beginning of the experiment (initial body weight, IBW). At the end of the experiment, the eels were collected from the treatment ponds using a suitable narrow net and retained in different clean plates. The eels were dried using a clean sterile filter paper to remove the excess water from the body before measuring and weighing of final body weight (FBW). Weight gain (WG) was calculated as (FBW-IBW), specific growth rate (SGR): 100[(FBW-IBW)/t]; where (t) is the culture period in days. Feed conversion ratio (FCR) equal to feed intake/ weight gain. Eel behaviour The behaviour of eels during feeding was recorded by the observer during the feeding process. Latency to start feeding (the time elapsed between provision of the feed on the mesh and the first eel starting to eat in seconds) and feeding duration (the time elapsed between starting of feed till the ending of feed in minutes) were calculated. Statistical analysis The data was subjected to distribution normality test before analysis. Data were analyzed using Graph Pad™ Prism 6. Data were compared by the Student's t-test with the signifi- cance level P<0.05. Results are presented as means ± SEM. Results The outcomes of water parameters analysis are presented in Table 1. Dissolved oxygen concentrations were significantly higher in the NF group both during daylight and night except at 10 am at 20 cm depth. Total ammonia, unionized ammonia and pH were significantly lower in the NF group than in the DF group. There were no significant differences in temperature, salinity, TDS and EC between the NF and DF group. The growth performance parameters are presented in Table 2. The growth performance parameters (FBW, WG, FI, FCR and SGR) were significantly improved by feeding eels at night in comparison to DF. Feeding behaviour of European eels (Anguilla anguilla) is shown in Figures 2 and 3. There was a significant difference in latency to start feeding and the feeding duration between the DF and NF group. The latency to start feeding was shorter in NF than DF group. While the feeding duration was prolonged in DF compared to NF group. Figure 1: Design of concrete pond used for rearing of European eels (Anguilla anguilla) 610 A. M. Abdel-Hay, S. A. Hamed, M. M. Zayd, F. A. Abouelenien, I. F. Rehan, S. El-Kassas, A. M. Deyab, R. A. Mohamed Table 1: Effect of feeding regime on water quality of European eels (Anguilla anguilla) Water parameter Monitoring time Day feeding Night feeding ^-value DO 10 am 20 cm depth 14.370± 0.186 15.030±0.606 0.388 80 cm depth 7.367±0.273 10.730±0.722 0.031* 120 cm depth 2.933±0.177 4.933±0.166 0.001* 10 pm 20 cm depth 8.533±0.291 9.900±0.208 0.023* 80 cm depth 5.233±0.203 6.900±0.300 0.014* 120 cm depth 1.633±0.203 3.967±0.491 0.028* Temperature 10 am 24.00±0.058 23.97±0.088 0.770 10 pm 20.17±0.219 20.20±0.265 0.928 PH 10 am 8.233±0.088 7.577±0.137 0.022* 10 pm 9.133±0.145 7.900±0.058 0.002* Total ammonia 10 am 0.027±0.003 0.013±0.004 0.047* 10 pm 0.034±0.003 0.022±0.001 0.047* UIA 10 am 0.00237 0.00077 0.001* 10 pm 0.0119 0.0001 0.001* Salinity (ppt) 10 am 1.900±0.057 1.900±0.058 0.999 10 pm 1.900±0.057 1.900±0.058 0.999 TDS 10 am 302.000±9.539 301.700±8.988 0.981 10 pm 300.000±9.210 300.700±7.951 0.981 EC 10 am 591.300±13.040 592.000±13.230 0.973 10 pm 590.300±13.100 590.000±13.410 0.974 Dissolved oxygen (DO), unionized ammonia (UIA), total dissolved salts (TDS), Electric conductivity (EC). Asterisks indicate significant differences between experimental groups (Student t-test *p<0.05) Table 2: Effect of feeding regime on performance of European eels (Anguilla anguilla). Day feeding Night feeding ^-value Initial body weight (g) 186.000±3.559 186.800±14.600 0.958 Final body weight (g) 189.100±3.609 203.100±5.258 0.042* Weight gain (g) 3.050±0.450 16.250±2.822 0.002* Feed intake (g) 16.600±1.024 66.200±5.017 0.001* Feed conversion ratio 6.473±0.483 4.632±0.259 0.004* Specific growth rate 0.09±0.004 0.542±0.003 0.001* Asterisks indicate significant differences between experimental groups (Student t-test *p<0.05) Night feeding regime improves water quality, growth performance and feeding behaviour of European eels, 611 Figure 2: Effect of feeding regime on feeding behaviour (latency to start feeding) of European eels (Anguilla anguilla). (Student t-testp-value =0.001) Discussion The present study showed that NF regime influenced water quality parameters, growth performance and feeding behaviour of European eels (Anguilla anguilla). Water quality is a key factor in the success of any aquaculture system. When stocking eels in high densities, a higher level of DO is needed to attain better growth and to maintain normal activity. Furthermore, pond water exchange by pumping of new water and drain the old one in a regular way is also required (6). Water samples should be analysed regularly for DO, TDS, UIA and temperature. The DO in eel's water should be not less than 3 ppm, UIA level not more than 0.2 mg/L and PH around 7 (7). Additionally many trials were done to improve the quality of pond water and decrease the load of pollutants (8) which if discharged will cause environmental hazard. The process of NF improved the water quality measurements in form of increased level of DO; which considered the most important determinant for the soundness of aquaculture, (9) both during daylight and night in the three points of monitoring (20, 80 and 120 cm from water surface). Moreover, it reduced the level of PH, total ammonia and UIA both during both monitoring. Generally, low pH 40 T Figure 3: Effect of feeding regime on feeding behaviour (feeding duration) of European eels (Anguilla anguilla). (Student t-testp-value =0.001) will decreased the level of toxic form of ammonia (UIA), which is the most dangerous form. On the other hand, the feeding time has no significant effect on water temperature, salinity, TDS and EC even during daylight and night monitoring. This improvement in the water quality in the NF group has a direct effect on enriching the rearing environmental resource (3). Water quality improvement due to NF will decrease the rate of water exchange, which is more economic and decrease the water pollution load (6). In this study, eels were reared in a temperature ranged from 20.2 to 24°Cwhich considered within the optimum range of rearing temperature in captivity (10). Not only this lower temperature was found to reduce bacterial growth in rearing tanks, but also improved water quality and enhanced survival rates of cultured eels (10). It also reduced the basal metabolic rate of cultured eels and saved the metabolic energy that may have a direct effect in form of improved growth and indirect effect in form of reduced eel's waste products and therefore improving water quality (11). These findings have coincided with Degani et al. (12) who found that the level of DO and UIA concentrations depend on stocking rate, fish metabolic products and pond water quality. 612 A. M. Abdel-Hay, S. A. Hamed, M. M. Zayd, F. A. Abouelenien, I. F. Rehan, S. El-Kassas, A. M. Deyab, R. A. Mohamed Herein the two groups of European eels displayed different patterns of growth performance. The NF eels showed higher growth performance in form of higher FBW, WG, FI and SGR than DF. While it showed a lower feed conversion ratio than DF. This may be attributed to the increased activity of eels during the night (1) that improved feed intake and consequently improved performance (3). For individual eel weight in the DF group, there was an extensive difference between individuals which may be due to differences in genetic makeup of the individuals within the same treatment or due to their different feeding habits. The same finding was observed by Lopez-Olmeda et al. (4). In addition, the improvement in water quality in the NF group especially high DO and low ammonia, UIA and pH found to have a direct impact on growth performance of eels. Degani et al. (12) reported that DO, total ammonia, temperature and pH are considered an important factors affecting eels performance. Interactions among these parameters greatly affect the rate of growth in eels. Lopez-Olmeda et al, (13) found that the feeding time may affect the cortisol level in eels. The lower level of cortisol was observed during the night. Feeding time may be affected by cortisol level and gastric secretion (e.g. gastric PH). Feeding eels once per day may improve digestive secretion that may be increased just before the next meal. This may be the explanation for improved digestion and performance (14). All of these factors may be interrelated with higher WG and FI, lower FCR and improved growth performance in NF group compared to DF group. Feeding behaviour of European eels was influenced by feeding time. Day feeding group showed prolonged latency to start feeding and feeding duration. This may be due to the reduction of European eel's activity during daylight (1). In addition, the high level of cortisol during daylight period may influence on eels behaviour and the eels become very nervous and stressed that might make differences in the feeding duration or FI which depend on the stress level (4). Furthermore, the prolonged latency to start feeding and feeding duration in the DF group may have a direct impact on the water quality of this group. It may lead to waste of unconsumed feed and then decomposed in pond water (10). This process may be the reason for higher pH, total ammonia and UIA in DF than NF group. Similar findings were observed by Degani et al. (12) and Tau-fiq et al. (6). Conclusion The obtained results suggested that manipulating both environmental and managemental factors can regulate healthy growth performance and feeding behaviour of European eel (Anguilla anguilla). Night feeding regime improved water quality, growth performance and feeding behaviour of European eels. Water quality improvement due to NF will reduce the rate of water exchange. Acknowledgement This study was conducted in Department of Aquaculture, Faculty of Aquatic Sciences and Fisheries, Kafrelsheikh University. The authors would like to thank Wasseem Emam, Institute of Aquaculture, University of Stirling, UK for helpful comments and revising the language of the article. Conflict of interest The authors declare that they have no conflict of interest. References 1. van Ginneken V, Maes GE. The European eel (Anguilla anguilla, Linnaeus), its lifecycle, evolution and reproduction: a literature review. Rev Fish Biol Fish 2005; 15:367-98. 2. Reebs SG. Plasticity of diel and circadian activity rhythms in fishes. Rev Fish Biol Fish 2002; 12:349-71. 3. DeCoursey PJ. The behavioral ecology and evolution of biological timing systems. In: Dunlap JC, Loros JJ, DeCoursey PJ. (eds) Chronobiology. Biological time keeping. Sunderland 2004; 27-65. 4. López-Olmeda JF, López-García I, Sánchez-Muros MJ, Blanco-Vives B, Aparicio R, Sánchez-Vázquez FJ. Daily rhythms of digestive physiology, metabolism and behaviour in the European eel (Anguilla anguilla). Aquacult Int 2012; 20:108596. Night feeding regime improves water quality, growth performance and feeding behaviour of European eels, 613 5. Montoya A, Lopez-Olmeda JF, Yu'fera M, Sa'nchez-Muros MJ, Sa'nchez-Va'zquez FJ. Feeding time synchronises daily rhythms of behaviour and digestive physiology in gilthead sea bream (Sparus aurata). Aquaculture 2010; 306:315-21 6. Taufiq-Spj N, Sunaryo S, Wirasatriya A, Sugianto DN. The use of water exchange for feeding rate and growth promotion of shortfin eel Anguilla bicolor bicolor in recirculating water system. In IOP Conference Series: Earth and Environmental Science 2017; 55: 012015. 7. Suzuki Y, Maruyamaa T, Numataa H, Sato H, Asakawa M. (2003) Performance of a closed recirculating system with foam separation, nitrification and denitrification units for intensive culture of eel: towards zero emission. Aquacult Eng 2003;29:165-82 8. Abouelenien F, Elsaidy N, Kirrella GA, Mohamed RA. Hygienic Effect of supplementing Oreochromis niloticus farm with fresh or fermented chicken manure on: water, fish quality and performance. Alexandria J Vet Sci 2015; 45: 79-90. 9. Franklin PA. (2014) Dissolved oxygen criteria for freshwater fish in New Zealand: a revised approach. New Zeal J Mar Fresh 2014;48: 112-26. 10. Okamura A, Yamada Y, Horita T, Horie N, Mikawa N, Utoh T, Tanaka S, Tsukamoto K. (2009) Rearing eel leptocephali (Anguilla japonica Temminck and Schlegel) in a plankton kreisel. Aquac Res2009;40:509-12. 11. Okamura A, Horie N, Mikawa N, Yamada Y, Tsukamoto K. (2014) Recent advances in artificial production of glass eels for conservation of anguillid eel populations. Ecol Freshw Fish 2014; 23:95-110. 12. Degani G, Horowitz A, Levanon D. Effect of protein level in purified diet and of density, ammonia and O2 level on growth of juvenile European eels (Anguilla anguilla L.). Aquaculture 1985;46:193-200. 13. Lopez-Olmeda JF, Sanchez-Vazquez FJ. Feeding rhythms in fish: from behavioral to molecular approach. In: Kulczykowska E, Popek W, Kapoor BG (eds) Biological clock in fish. Science Publishers, Enfield 2010; 155-84 14. Montoya A, Lopez-Olmeda JF, Garayzar ABS, Sa nchez-Va zquez FJ. Synchronization of daily rhythms of locomotor activity and plasma glucose, cortisol and thyroid hormones to feeding in Gilthead sea bream (Sparus aurata) under a light-dark cycle. Physiol Behav 2010; 101:101-7. Slov Vet Res 2019; 56 (Suppl 22): 615-21 DOI 10.26873/SVR-800-2019 Original Research Article USING REAL-TIME POLYMERASE CHAIN REACTION TO STUDY THE EFFECT OF SALICYLIC ACID AND SODIUM CHLORIDE ON THE PYOCYANIN PRODUCTION OF Pseudomonas aeruginosa Alaa A. Sherif, Hesham S. Nada, Amgad A. Moawad*, Ibrahim E. El- desouky, Salwa M. Helmy Department of Bacteriology, Mycology and Immunology, Faculty of Veterinary Medicine, Kafrelsheikh University, 33516, Egypt Corresponding author, E-mail: moawadamgad@gmail.com Abstract: Pseudomonas aeruginosa is a bacterium that seizes the opportunity to cause serious infections in both man and animals. Pyocyanin is a strong virulence factor produced by P. aeruginosa. The present study was performed to explain the effect of salicylic acid (aspirin) and sodium chloride (NaCl) on P. aeruginosa and Pyocyanin using concentrations of 1.5%, 3% and 6% for aspirin and 3%, 6% and 9% for NaCl. The effectiveness of aspirin and NaCl was detected by real time PCR (qPCR). The P. aeruginosa isolate was from broilers suffering from septicemia in Zagazig city, Egypt. The study revealed that 6% and 9% concentration of NaCl had a bactericidal effect on the bacterium but 3% concentration of NaCl has an inhibitory effect on both the growth and the expression of Pyocyanin (by 0.5-fold lesser than the untreated sample). Also, data revealed that 1.5%, 3% and 6% concentrations of aspirin inhibited the growth and reduced the expression of pyocyanin with slight various degrees (by 0.7955, 0.4234, 0.1948-fold lesser than untreated sample, relatively). The results of this study could be useful in pharmacological intervention and clinical therapy as aspirin and NaCl could be combined with antimicrobial agents to treat Pseudomonas infections. Key words: Pseudomonas aeruginosa; pyocyanin; sodium chloride; salicylic acid Introduction Pseudomonas aeruginosa (P. aeruginosa) is a broad, Gram-negative bacterium that seizes the opportunity to infect the immune-compromised hosts including people infected by HIV, patients exposed to chemotherapy and patients suffering from severe burns (1). The infection with P. aeruginosa gives rise to mortality in chickens and clinical symptoms including respiratory signs and septicemia (2). Proteases (elastase, protease, and alkaline protease), toxins (exoenzyme S and exotoxin A), hemolysins and phenazines are virulence factors in P. aeruginosa. They have been found to participate in P. aeruginosa pathogenicity in man and animals. Pyocyanin is a phenazine compound (5-N-methyl-1-hydroxy phenazine) considered as both a signal molecule and an important Received: January 2019 Accepted for publication: February 2019 616 A. A. Sherif, H. S. Nada, A. A. Moawad, I. E. El- desouky, S. M. Helmy virulence factor. In cystic fibrosis lungs in vitro, pyocyanin interferes with the ciliary function of the respiratory system and can change the host immune response. Also, pyocyanin is considered as the last signal in the quorum-sensing (QS) system, and organizes many genes in P. aeruginosa (3). Some compounds can inhibit the quorum-sensing system such as salicylic acid (aspirin) (4). Aspirin can inhibit not only biofilm forming of P. aeruginosa, but also it can decrease the pyocyanin production (5). It has been reported that salicylic acid cause changes in the membrane of P. aeruginosa. Salicylic acid is regarded as an antimicrobial agent in the treatment of Pseudomonas keratitis (6). Many environmental stimuli such as osmolality, pH, NaCl and the diversity of ions have a distinct influence on P. aeruginosa (7, 8). The swarming motility of the bacterium is affected by NaCl. Bacterial growth is inhibited by 6% NaCl solution. Bactericidal action occurs at 10% NaCl solution (8). 16S rDNA-based PCR assays provide fast, plain and dependable identification of P. aeruginosa and its discrimination from other Pseudomonas species which are closely related in phylogenesis (9). Real-Time PCR (qPCR) was applied to study the fluorescence level in a particular selected gene. It is a highly sensitive technique that has remarkable potential for the high-volume analysis of gene expression in both research and routine medical diagnostics. This technique provides real-time quantitation of an initial template of DNA or RNA (10). The present study was designed to illustrate the effect of NaCl and aspirin on an Egyptian P. aeruginosa strain isolated from septicemic broilers. Material and methods The origin of the strain P. aeruginosa was isolated from broilers suffering from septicemia in Zagazig City, Egypt since 2017. P. aeruginosa was identified after examining for phenotypic and gen-otypic characteristics in the laboratory of microbiology department in Zagazig University. The isolate was maintained on Tryptone Soya Agar (Oxoid, CM0131, UK) (TSA) slopes at 5 °C and sub-cultured every month. P. aeruginosa was transferred on TSA slopes to Kafrelsheikh University at the laboratory of bacteriology. Determination of the effect of Sodium Chloride on P. aeruginosa P. aeruginosa subculture occurred in King A broth (Himedia M1544) supplemented with 30, 60, and 90g/l of NaCl (Bio Jet) to obtain 3%, 6%, and 9% concentrations respectively. Cultures were incubated in a shaker incubator (180 rpm at 30°C) for 48h. The growth was determined visually and compared with the untreated sample of P. aeruginosa (3). Determination of the effect of aspirin P. aeruginosa subculture was occurred in nutrient broth overnight (PO) at 37 °C. Aspirin (100 mg, BAYER) was used. 2-fold serial dilutions in nutrient broth were prepared resulting in 100, 50, 25, 12.5, 6, 3, 1.5 mg/ml concentrations. Seven tubes each one contains ml from nutrient broth. In the first tube, one tablet from aspirin (aspirin protect 100mg) was added. The concentration became 100 mg /ml in the first tube. 0.5 ml from the first tube was taken to second tube giving concentration 50 mg /ml in the second tube. Repeat the same in other tubes giving the concentrations 25, 12.5, 6, 3, 1.5 mg/ml). Then discard 0.5 ml from the last tube. The last three tubes No. 5(D),6(E) and 7(F) contained 6,3,1.5 % Aspirin respectively. Diluted aspirin (D, E, F) was inoculated with 100 Micron PO containing 6 _ 105 CFU/ml and incubated at 37 °C for 24 h. Minimum inhibitory concentration (MIC) (the lowest concentration of antibacterial agent that inhibit the apparent growth of the bacteria) was measured. PO was treated with sub-inhibitory concentrations (sub-MIC) of aspirin (1/4 MIC). The number of viable emerged bacterial cells was estimated and collated to the number of untreated PO using the plating Using real-time PCR to study the effect of salicylic acid and sodium chloride on the pyocyanin production 617 method (12). The growth of both PO treated with 1/4 MIC of aspirin and untreated PO was monitored. Inoculation of an overnight culture of PO into nutrient broth tubes treated with aspirin (1/4 MIC) and control ones without aspirin occurred. Incubation at 37 °C for 24 h. Every hour, Optical Density 600 nm for treated and untreated samples was measured (4). Molecular identification The extraction of DNA was done by QI-Aamp DNA Mini kit instructions (Qiagen, Germany, GmbH, Catalog no. 51304) with some changes. Incubation of 200 pl of the bacterial suspension occurred at 56°C for 10 min after addition of 10 pl of proteinase K and 200 pl of lysis buffer. Then, 200 pl of ethanol 100% (Applichem) were appended to the lysate. The sample was washed and cen-trifuged according to the manufacturer's index. Then, elution of the nucleic acid occurred with 100 pl of elution buffer presented in the kit. The reaction contained 25 pl of the following PCR master mix. Emerald Amp GT PCR master mix (2x premix) was 12.5 pl, PCR grade water was 4.5 pl. Forward primer (20 pmol) was 1 pl. Reverse primer (20 pmol) was 1 pl. Template DNA was 6 pl. The amplification of target gene phenazine (phzM) of Pseudomonas genus was performed using primer pair of phzM; 5'-ATGGAGAGCGGGATCGACAG- '3 and 5'- ATGCGGGTTTCCATCGGCAG- '3 amplifying 875 bp (13). The used primers were supplied from Metabion (Germany). Amplification cycles (n=35) were performed under these conditions; primary denaturation for 5 min at 94°C, secondary denaturation for 30 sec at 94°C, 1 min of annealing at 54°C and 1 min of extension at 72°C. Electrophoresis of amplified products in 1.5% agarose gel (ABgene) was done. The sizes of the amplified product were determined by a gene ruler 100 bp DNA Ladder (catalog No. SM0243) (Fermentas, Thermo Scientific, Germany). Photographing was done by Alpha imager (Innotech, Biomedia). A computerized analysis was performed. RNA extraction for qPCR was done following RNeasy Mini Kit instructions (Qiagen, Catalog no. 74104) with some modifications. To protect RNA from degradation, a double volume (1 ml) of the RNA protect Bacteria Reagent (Qiagen, Germany, GmbH Catalog no. 76506), was added to one volume (0.5 ml) of the broth of P. aeruginosa culture, then the mix was incubated at room temperature for 5min. Then add 200 pl of Tris EDTA buffer (TE buffer) (Thermo Fisher, Catalog no. 12090-015) containing 1 mg/ml Lyso-zyme (Biochemica, Applichem, Catalog no. A3711) and 700 pl of lysis buffer. Then, 500 pl of 96% ethanol (Applichem) were added to the lysate. Washing and centrifugation of the sample were performed according to the manufacturer's index. Then, elution of RNA by 50 pl RNase free water (Quantitect SYBR green PCR kit) (catalog. No .204141). The reaction contained 25 pl of the R master mix according to Quantitect SYBR green PCR kit. QuantiTect SYBR Green PCR master mix (2x premix) was 12.5 pl. RevertAid Reverse Transcriptase (Thermo Fisher, Catalog number: EP0441) was 0.25 pl. Forward primer (20 pmol) was 0.5 pl. Reverse primer (20 pmol) was 0.5 pl. Template RNA was 3 pl. RNase free water was 8.5 pl. The target genes: phenazine (phzM) of Pseudomonas genus was performed using primer pair of phzM (mentioned previously), a primer pair of 16S rRNA gene;5'- GGGGGATCTTCG-GACCTCA- '3 and 5'- TCCTTAGAG-TGCCCACCCG- '3 (14). The following cycling condition protocol was used: 30 min for reversed transcription at 50 °C, 5 min for primary denaturation at 94 °C, amplification cycles (n=40) were performed under the following conditions; 15 min of secondary de-naturation at 94°C, annealing (Optics on) at 52 °C for 45 min for 16S rRNA but at 54 °C for 45 min for phzM, 30 sec for extension at 72 °C, dissociation curve (one cycle) conditions; 1 min of secondary denaturation at 94 °C, 1 min of annealing at 52 °C for 16S rRNA but at 54 °C for 1 min for phzM, 1 min of final denaturation at 94 °C. The curves of amplification and cycle threshold (Ct) values 618 A. A. Sherif, H. S. Nada, A. A. Moawad, I. E. El- desouky, S. M. Helmy were evaluated by the Stratagene MX3005P qPCR software. To assign the difference in the gene expression on the RNA of the samples, a comparison between the Ct of each sample and that of the control sample occurred according to the "2-AACt" equation (15). Results The effect of NaCl Growth appeared at 3% concentration NaCl but no growth occurred at 6, 9%. Growth turbidity at 3% concentration of NaCl was less than that of untreated sample. The effect of Aspirin Initially, the minimum inhibitory concentration of aspirin against P. aeruginosa PO was revealed as 24 mg/ml. The sub-inhibitory concentrations of aspirin (1/4, 1/8 and 1/16 MIC) corresponding to 6, 3 and 1.5 mg/ml concentrations of aspirin respectively were applied to evaluate the inhibitory influence on both quorum sensing system and the virulence factors of P. aeruginosa PO. The treated P. aeruginosa PO with sub-MIC of aspirin was as similar as the untreated PO in its viability. Example for that, the bacterial number was 154 _ 104 CFU/ml at 1/4 MIC in treated PO but the bacterial number of the untreated PO was 160 _ 105 CFU/ml. Also, both treated and untreated PO attained a stationary phase after incubation for 9 h at 37 °C, signifying no impact on the growth. But the bluish metallic sheen OF P. aeruginosa (11) was changed to some extent. Turbidity degree was inversely proportional with the concentration of Aspirin. Treated Pseudomonas with 1/4 MIC showed the least turbidity. The turbidity of treated Pseudomonas with 1/16 MIC was lesser than the untreated sample. Polymerase Chain Reaction PCR reaction was done to diagnose P. aeruginosa using 16S rDNA. The results showed that one band 875 bp (product size) as a result of agarose gel compare with DNA marker (100 bp) (figure 1). Quantitative real-time PCR The fluorescence light emitted within qPCR is directly proportional to the formed DNA and can be assumed as an amplification plot. As a result of Ct values (table 1), the data revealed that 3% concentration of NaCl reduced the expression of PhzM (by 0.5-fold less than the untreated sample). The reduction percentage of PhzM was 50%. But for Aspirin, 1.5%, 3% and 6% concentrations caused decrease in the expression of pyocya-nin with various degrees (by 0.7955, 0.4234, 0.1948 -fold less than untreated sample relatively), i.e., the reduction in expression of PhzM with 80.52 %, 57.66% and 20.45% in sub MIC (1/4 (6mg/ml), 1/8 (3mg/ml) and 1/16 (1.5mg/ml)) of Aspirin respectively (chart1). The experiment had been repeated three times. The amplification curve of 16SrDNA (figure 2) but that of PhzM (figure 3) were showed. Table 1: The Ct values of 16SrDNA and PhzM for each sample with the fold change of them were analyzed. The Control sample was the untreated isolated P. aeruginosa Sample Sample ID 16S rDNA _phzM_ No. _CT_CT_Fold change B Control 19.78 22.86 -C NACL 3% 20.84 24.92 0.5000 D Aspirin 6% 20.39 25.83 0.1948 E Aspirin 3% 19.21 23.53 0.4234 F_Aspirin 1.5%_20.54_23.95_0.7955 Using real-time PCR to study the effect of salicylic acid and sodium chloride on the pyocyanin production 619 Figure 1: agarose gel electrophoresis of pyocyanin gene amplification of Pseudomonas aeruginosa strain. L: 100bp ladder. Lane (A): positive sample. Pos: positive control and neg: negative control. Positive and /or negative controls were represented by field sample that was previously confirmed to be positive or negative by PCR for the related gene in the Reference laboratory of Animal health research institute, Zagazig University. Amplicon size: 875 bp Chart 1: The inhibition percentage of PhzM expression in each sample. B: control (untreated sample). C: a sample containing NaCl 3%. D: a sample containing Aspirin 6%. E: a sample containing Aspirin 3%. F: a sample containing Aspirin 1.5% Figure 2: Amplification curve of 16SrDNA. X-axis: amplification cycles. Y-axis: distinctive fluorescence. The axis that was parallel to X axis was a threshold axis. Each point of each sample of the curve which crossed the threshold axis was the Ct value Figure 3: Amplification curve of PhzM. X-axis: amplification cycles. Y-axis: distinctive fluorescence. The axis that was parallel to X-axis is threshold axis. Each point of each sample of the curve which crossed the threshold axis was Ct value Discussion In the present study, the influence of NaCl and Salicylic acid on the P. aeruginosa growth and expression of pyocyanin virulence factor of it was investigated. Results demonstrated that NaCl and Aspirin had a significant impact on the P. aeruginosa growth and pyocyanin production. This study revealed that 6% and 9% concentration of NaCl had a bactericidal effect on the bacterium but 3% concentration of NaCl has an inhibitory effect on the growth 620 A. A. Sherif, H. S. Nada, A. A. Moawad, I. E. El- desouky, S. M. Helmy and also on the expression of Pyocyanin (by 0.5-fold lesser than the untreated sample). The reduction percentage of PhzM was 50%. But for Aspirin, 1.5%, 3% and 6% concentrations caused inhibition of growth and decrease in the expression of pyocyanin with slight various degrees (by 0.7955, 0.4234, 0.1948 -fold lesser than untreated sample relatively), i.e., the reduction in expression of PhzM with 80.52 %,57.66% and 20.45% in sub MIC (1/4 (6mg/ml),1/8(3mg/ml) and 1/16 (1.5mg/ml) of Aspirin respectively. Previous studies revealed that Pseudomonas influenced by a different concentration of salinity. Pyocyanin production increased proportionally with salinity concentration. The high amount of pyocyanin (29.57pg/ml) was obtained with 20g/L of NaCl (2% concentration). The result also indicated that salinity above 20g/L have totally affected pyocyanin production but did not affect the bacterium growth which was completely inhibited at 60 g/L of NaCl (6% concentration). The production of pyocyanin was salinity-dependent when P. aeruginosa had grown in King A medium supplemented with 2.5 to 20g/l of NaCl. The highest amount of pyocyanin was obtained at 20g/l (2% NaCl) and the growth of P. aeruginosa was completely inhibited at 50 g/l of NaCl (5 % NaCl) (3); However, another study showed that maximum of productivity was found in a medium salinity ranging from 5-10g/l. but at concentration 5 g /l or less, there was not significantly inhibition of the growth and the pyocyanin production. (16, 17). Other studies revealed that the normal growth of P. aeruginosa appeared on a medium which contained 0.25 up to 1.25% concentration of NaCl in comparison to control medium. Above 1.75% NaCl concentration in the medium, it can survive up to 2.75% NaCl but the survival number of Pseudomonas gradually reduced. The inhibition impact was observable at a solution containing 4% concentration of NaCl. A solution containing 6% concentration of NaCl could inhibit the growth of Pseudomonas after incubation for 24 h. The bactericidal effect on Pseudomonas occurred by 8% and 10% of NaCl (18, 8). For patients suffering from cystic fibrosis in the lung, aerosols containing hypertonic saline (7% NaCl) are useful in the treatment due to abolish both motility and growth of P. aeruginosa (19). Salicylic acid can inhibit production of pyocyanin (4,6). Distinct reduction in the virulence factors of P. aeruginosa treated with 6 mg/ml (1/4 MIC) and 3 mg/ml (1/8 MIC) concentrations of aspirin. Pyocyanin reduction was 78% due to treating with aspirin (1/4 MIC) (4). Growth in the sub-inhibitory concentrations of salicylic acid resulted in a significant reduction in bacterial cell number, cell density and in quorum-sensing signaling molecules (20). Salicylic acid plays a role in down-regulating some of the virulence factors (especially pyocyanin) in P. aeruginosa and attenuating the virulence of the bacterium on Caeno-rhabditis elegans and Arabidopsis thaliana. A 50% reduction of Pyocyanin production was resolved after treatment with 0.1 mM salicylic acid but an 80% reduction of pyocyanin production with no visible influence on the bacterial growth was determined after treatment with 1.0 mM SA (20, 21). Another study revealed that P. aeruginosa 1604 was still able to produce a considerable amount (0.44 pgml-1) of pyocyanin after a 72 hours lag period in the presence of 0.1 % aspirin. At 0.3 % concentration, the inhibitory effect was evident for 120 hours. For long-term inhibitions, a higher amount of applied salicylic compounds (0.3 and 0.4 %) are necessary (5). Salicylic acid can reduce bacterial numbers and its virulence during keratitis so it is effective as an antimicrobial substance to treat Pseudomonas keratitis (6). Conclusion In this study, P. aeruginosa isolate was from broilers suffering from septicemia in Zagazig city, Egypt. By using PCR and qPCR, the data revealed that Aspirin and Sodium chloride inhibit the P. aeruginosa growth and decrease pyocyanin production. This new therapeutic approach can be used in combination with antimicrobial agents to treat P. aerugino-sa cases. Using real-time PCR to study the effect of salicylic acid and sodium chloride on the pyocyanin production 621 Conflict of interest None of the authors disclose any conflict of interest. References 1. El-Domany R, Emara M, El-Magd M, Moustafa W, Abdeltwab N. Emergence of Imipenem-Resistant Pseudomonas aeruginosa Clinical Isolates from Egypt Coharboring VIM and IMP Carbapenemases, Microbial Drug Resistance 2017; 23(6): 682-6. 2. Satish S, Priti M. Pseudomonas aeruginosa Infection in Broiler Chicks in Jabalpur. International J Ext Res 2015; 6:37-9. 3. Hicham D, Rachid D, Saïd N. Antimicrobial, antioxidant and hemolytic effects of Pyocyanin produced by Pseudomonas aeruginosa isolated from saline soil of Mina river, Algeria. International J of Biosciences 2016; 9(5): 134-43. 4. Somaia A, Khaled H, Shahenda M, Mona I. Aspirin is an efficient inhibitor of quorum sensing, virulence and toxins in Pseudomonas aeruginosa. Microbial Pathogenesis J. 2014; 74:25-6. 5. Agnes D, Hajnalka F, Akos K, Petra S, Péter k. Inhibition of exopolysaccharide biopolymer and pyocyanin virulence factors produced by Pseudomonas aeruginosa 1604 by salicylic compounds, Periodica Polytechnica Chemical Engineering J. 2014;58:75-80. 6. Mahesh B, Padmaja S, Hua Z, Emma H, Mark W. Effect of Salicylic Acid on the Membrane Proteome and Virulence of Pseudomonas aeruginosa. Cornea J. 2016; 57:1213-20. 7. Rahul M, Sudhir A, Saroj S, Sanjay C and Kusum H. Urinary tract infections caused by Pseudomonas aeruginosa. Journal of Infection and Public Health 2009; 2: 105. 8. Anne-Laure M, Estelle JB, Raphae C, Brigitte L, He' le'ne M. Advances toward the Elucidation of Hypertonic Saline Effects on Pseudomonas aeruginosa from Cystic Fibrosis Patients. PLOS ONE J. 2014; 9(2): e90164. 9. Mohammed EA, Ismail HA, Abeer AM. Identification Pseudomonas aeruginosa by 16s rRNA gene for Differentiation from Other Pseudomonas Species that isolated from Patients and Environment. Baghdad Science J.2014; 11(2): 1028-33. 10. Crystal AC. Study of Secondary Metabolite Gene Expression in Marine Microbial Co-Cultures Using Quantitative Real-Time PCR. Nova Southeastern University Works. HCNSO Student Theses and Dissertations. Oceanographic Center 2010; 222:4-187. 11. Quinn PJ, Markey BK, Carter ME, Donnelly WJ, Leonard FC. Veterinary Microbiology and Microbial Disease. 2nd Ed, 2011:287. 12. Sanders ER, Aseptic laboratory techniques: plating methods. Journal of Visualized Experiment 2012 ;( 63): e3064. 13. Finnan S, Morrissey JP, Gara, FO, Boyd EF. Genome Diversity of Pseudomonas aerugino-sa Isolates from Cystic Fibrosis Patients and the Hospital Environment. Journal of Clinical Microbiology 2004; 42:5783-92. 14. Spilker T, Coenye T, Vandamme P, LiPuma JJ. PCR-Based Assay for Differentiation of Pseudomonas aeruginosa from Other Pseudomonas Species Recovered from Cystic Fibrosis Patients. Journal of Clinical Microbiology 2004; 42(5):2074-9. 15. Yuan JS. Reed A, Chen F, Stewart CN. Statistical analysis of real-time PCR data. BMC Bio-informatics J. 2006; 7:85. 16. Akpor OB, Morah BL, Adejobi O, Olonine-fa SD, Babalola OO. Tolerance of Pseudomonas aeruginosa to selected concentrations of metals and Sodium Chloride. CIBTech Journal of Microbiology 2015; 4(3):27-33. 17. Prabhakaran P, Puthumana J, et al. Antagonistic effect of Pseudomonas aeruginosa isolates from various ecological niches on Vibrio species pathogenic to crustaceans. Journal of Coastal Life Medicine 2014; 2(1):76-84. 18. Vishal KD, Punkaj K. Effect of Salinity on Growth and PGPR Activity of Pseudomonads. Journal of Academia and Industrial Research 2013; 2(6): 353-6. 19. Viktoria H, Eric S, et al. Inhibitory Effects Of Hypertonic Saline On P. Aeruginosa Motility. NIH Public Access. J Cyst Fibros 2008; 7(4): 267269. 20. Mahesh BK, Hua Z, Padmaja RS, Mark DP. Salicylic Acid Reduces the Production of Several Potential Virulence Factors of Pseudomonas aeru-ginosa Associated with Microbial Keratitis. Investigative Ophthalmology & Visual Sci J. 2006; 47(10):4453-60. 21. Prithiviraj B, Bais HP, et al. Down Regulation of Virulence Factors of Pseudomonas aeru-ginosa by Salicylic Acid Attenuates Its Virulence on Arabidopsis thaliana and Caenorhabditis ele-gans. Infection and Immunity. American Society for Microbiology J.2005; 73(9): 5319-28. Slov Vet Res 2019; 56 (Suppl 22): 623-32 DOI 10.26873/SVR-801-2019 Original Research Article PRENATAL DEVELOPMENT OF THE VOMERONASAL ORGAN IN RABBIT Saleh A. M. A. ALomaisi12*, Hanaa M. El-Ghazali2, Hamed M. Nosseur2, Salah El-dein A. Ahmed2, Mervat M. Konsowa2 departments of Animal Research Regional Research Station, Agriculture Research and Extension Authority, 87148 Thamar Yemen, 2Departments of Anatomy and Embryology, Faculty of Veterinary Medicine, Zagazig University, 44511, Egypt. Paper extracted from PH.D. thesis of Saleh Ahmed Mohammed Ali Al-Omaisi Corresponding author: elemesy18@vet.zu.edu.eg, alomisy78@yahoo.com Abstract: The vomeronasal organ (VNO) is the peripheral receptors, with the ability to detect pheromones, and so it has a role in social behavior, sexual reactions and reproduction. The aim of this study was to designing, categorize, define and demonstrate the normal explaining of the onset time of first appearance and origin as well as developmental changes of vomeronasal organ of the rabbit and its allied structures. This study was carried out on 116 rabbit embryos and fetuses of both sexes. The specimens were obtained, from 14 normal and apparently healthy adult female rabbits. At days (D) 9-11 of pregnancy the nasal placode was invaginated to form the nasal (olfactory) pits. The primordia of the VNO was formed at the ventromedial area of this nasal pit. At D13, the VNO appeared as bilateral un-differentiated epithelial thickenings of the rostroventral part of nasal septum. At D14, the dorsomedial part of the epithelium was about a twice as thick as its ventrolateral part. At D16the VNO opened into the nasal cavity the miniature VNO nerve fascicles were appeared. At D18, VNO immature glands appeared in the dorsolateral part of the VNO the chondral plate was differentiated into chondro-blasts. The lumen of the VNO was extremely increased in wide and closed to be oval lumen. At D20, the rostral opening of VNO duct opened directly into the floor of nasal cavity. The medial wall was thicker and had about 8-10 layers of stratified columnar and the lateral one consisted of 3-4 pseudostratified columnar cell layer. At D22, the of acini of the VNO glands determined at the dorsal commissure of the VNO duct. At D28, the lumen was lined by a thick medial mucosa with stratified olfactory like epithelium and thin ventrolateral respiratory epithelium. Key words fetus; vomeronasal organ; nerve; glands; olfactory epithelium; respiratory epithelium Introduction The mammalian vomeronasal organ (VNO) is the site for peripheral receptors of the acces- sory olfactory system. By detecting phero-mones, the VNO has an important role in social behaviour and reproductive or sexual reactions. In mammals, the latter occurs due to its Received: January 2019 Accepted for publication: February 2019 624 S. A. ALomaisi, H. El-Ghazali, H. Nosseur, S. Ahmed, M. Konsowa ability to detect pheromones, and so VNO has an important role in many social and sexual behaviour. The olfactory system is well developed in rabbit (1). The vomeronasal complex (vomeronasal organ, cartilage, vessels, glands and nerves) is the most outstanding of the peripheral sensory structures found in the nasal septum of mammals. Although recent findings suggest it could be involved in pheromone-mediated behavior (2), VNO subserved basic chemosensory functions in rodents, mainly related to sexual behavior (3). It also plays a major role in the perception of stimuli related to social and/or reproductive behavior in many species of vertebrates (4). The vomeronasal system (VNS), an olfactory neural network that participates in the control of reproductive physiology and behavior, is sexually dimorphic (5). The real function of VNO is still controversial, a further developmental studies on this may give some more useful information to explain its function. However, there have been so far published only a little reports and details of morphogenesis remain unknown (6). There were several papers discussed the development of the VNO in rabbit (7, 8), rat (9), mouse (10), hamster (11), pig (12-14), mammals (15), primates (16), human (17-19), bovine (20), goat (21), camel (22), and domestic animals (23). The aim of this study was to categorize, define and demonstrate the normal explaining of the onset time of first appearance and origin as well as developmental changes of VNO of the rabbit and its allied structures. Materials and methods This study was carried out on 116 rabbit embryos and fetuses of both sexes. The specimens were obtained from 14 normal and apparently healthy adult female rabbit. The rabbits were obtained from the rabbit farm of the Faculty of Agriculture, Zagazig University. They were housed for one week before experiment for acclimatization standard pellet ration (El-Nasr Chemical Company, Cairo, Egypt) and were given free accesses to water ad libitum. All animals managed according to Animal Ethical Committee of Faculty of Veteri- nary Medicine, Zagazig University approval number ZU-IACU/2/F/109 /2018. The female were served by natural mating and each female housed individually in stainless-steel cages in environmentally controlled rooms and each maternal rabbit was given about100g-day of certified rabbit nitration free access to water. The pregnant rabbit were tested at age 9-28th days of pregnancy. The age of embryo was estimated by the pregnancy records and age of pregnancy depended on the time of mating. Just after slaughtering, evisceration and evacuation of their uteri. The obtained embryos and fetuses were classified into two group representing the all ages of pregnancy. Group (A) were immersed as a whole in 10% neutral buffered formalin and the other group (B) were immersed as a whole in Bouin's solution for 3-24 hours and then washed carefully with distilled water and transferred to 70% ethyl alcohol. Then the specimens were subjected to the following techniques: Histological technique The heads of fetuses over 20 days were immersed in EDTA 5.5% buffered to 7.0 PH with sodium hydroxid and neutralized in 5% sodium sulphate. The time taken for decalcification depended on the age of fetuses according to (24). After all specimens assembled for normal histological technique, all specimens dehydrated in ascending grades of alcohols, cleared in three changes of benzene and embedded in paraffin wax. Paraffin sections of 5-7p thickness were obtained and stained by different histological stains such as: Hematoxylin and Eosin (H&E) stain for general histological demonstration and silver impregnation (24, 25). The slides were examined by using both light and stereo (Zeiss, Germany) microscopes and the observations were recorded. Scanning electron microscope The specimens were delivered at hourly post conception. Specimens were trimmed and fixed in glutaraldehyde for 12-24 hour and then post fixed in 1 % osmium tetroxide for 90-120 min (26). The palates were dehydrated Prenatal development of the vomeronasal organ in rabbit 625 through an ascending concentration of ethyl alcohol followed by 2.5 % buffered glutaral-dehyde + 2 % paraformaldehyde, in 0.1 M sodium phosphate buffer pH 7.4. The specimens were washed 3 x 15 min in 0.1 M sodium phosphate buffer + 0.1 M sucrose and re-fixed in 2 % sodium phosphate buffered osmium tetroxide pH 7.4 for 90 min. Following washing and dehydration, the specimens were incubated overnight in 70 % acetone + 0.5 % uranyl acetate + 1 % phosphotungstic acid (at 4° C for 15 min), 80 % ethanol (2 x 15 min), 90 % ethanol (2 x 15 min), 96 % ethanol (3 x 20 min), and 100 % ethanol. The specimens were coated with gold-palladium membranes and observed in a Jeol JSM-6510 L.V SEM, The microscope was operated at 30 KV at EM Unit, Mansoura University, Egypt. The nomenclature used in this manuscript was adopted by Nomina Anatomica Veterinaria (27), Nomina Embryologica Veterinaria (28) and Nomina Histologica Veterinaria (29). Results Rabbit embryo of 11 days old The primordia of VNO performed at the medial aspect of the nasal pit (olfactory pits) as a thickening of the epithelium. The epithelium lined the pit was similar to that of the plac-ode, and merged steadily with the general ectoderm contiguous the outside opening, which migrate towards the mesenchyme and causes a slight recess on the olfactory purse. Later on, cellular bud grew dorsally, caudally, and to the midline on both side and formed the primordia of the vomeronasal groove (Figs. 1A-C). Rabbit embryo of 13 days old The first appearance of VNO was in the arrangement of bilateral undifferentiating epithelial thickenings on rostroventral region of the nasal septum. The organ was enclosed via the immature vomeronasal cartilage. The ventral part of the primitive nasal cavity had an invagination of the epithelial covering the ventral part of the nasal septum giving rise to the future vomeronasal duct (Fig. 1D). Rabbit embryo of 14 days old The primordia of VNO appeared as narrow luminal tube at the base of nasal septum (Fig. 2A). The primordial vomeronasal tube was lined laterally by a layer of stratified columnar epithelium with darkly stained and basely located elongated nucleus. The medial lining epithelium was thicker than the lateral one (Fig. 2B). Rabbit embryo of 16 days old The duct of future VNO was in contact with the nasal cavity (Fig. 2C). The rostral part of the future vomeronasal duct was lined by stratified cuboidal to stratified columnar epithelium (Fig. 2D). The lumen of the middle part of the vomeronasal duct was slit like. The dor-somedial wall of this duct was thicker than the ventrolateral one. Vomeronasal cartilage appeared more condensed than in the previous stage (Fig. 3A). Rabbit fetus of 18 days old The lining epithelium of vomeronasal duct was differentiated into thick olfactory like epithelium and thin respiratory epithelium. The C-cartilage of the vomeronasal organ was built up of chondroblastic cellular aggregation (Fig. 3B). Rabbit fetus of 20 days old The rostral opening of vomeronasal organ opened directly into the floor of nasal cavity (Fig. 3C). While at the middle region, the future vomeronasal duct had slit-like lumen and its medial wall was thicker and had about 8-10 layers of stratified columnar and the lateral one consisted of 3-4 cell layer (Fig. 3D) Rabbit fetus of 22 days old The vomeronasal duct showed two types of epithelium, the medial one resembled the olfactory form, while the lateral one appeared to be formed of respiratory type. The Primordia of acini of the vomeronasal glands were determined at the dorsal commissure of the vo-meronasal duct (Fig. 4A). 626 S. A. ALomaisi, H. El-Ghazali, H. Nosseur, S. Ahmed, M. Konsowa Rabbit fetus of 28 days old VNO opened rostraly in the nasal vestibule (Fig. 4B). The mucous membrane was of stratified columnar, aggregations of the acini of vomeronasal glands were determined at the dorsal commissure, some of them were lumi-nized and others were still obliterated (Figs. 4C and D). Figure 1: (A) A photomicrograph of L. S. of the rabbit Embryo of 9 days old showing; Prominentia fron-tonasalis (PFN), Placoda lentis (PL), Vesicula optica (VOP), Placoda otica (PO), Tubus neuralis (TN) with existence of the Arcus pharyngei [branchiales] I- I I I (APh I, APh I I, APh I I I). (H.E. stain 40X).(B) A photomicrograph of scanning electron micrograph (SEM) of the head of rabbit Embryo of 11 days old showing; the nasal placodes as a Fovea nasal is (FN), Prominentia nasalis lateralis (PNL), Prominentia nasalis medialis (PNM), Prominentia frontonasalis (PFN), Processus maxillaris (PMax), Sulcus nasolacrimalis (SNL) and Sulcus nasomaxillaris (SNM). (C) A photomicrograph of L. S. of the rabbit Embryo of 11 days old showing; Fovea nasalis (FN), stomodaeum (Sto), Prominentia frontonasalis (PFN), Placoda otica (OP), Prominentia nasalis lateralis (PNL), Prominentia nasalis medialis (PNM), Tubus neuralis (TN), Vesicula optica (VO) (H.E. Stain 40X). (D) A photomicrograph of C. S. of the head of the rabbit Embryo of 13 days old showing; the primordia of Ductus vomeronaslis (DV), primor-dia Cartilago vomeronaslis (CaV), Cavum nasi (CN) and Septum nasi (SN). (H.E. stain 100X) Prenatal development of the vomeronasal organ in rabbit 627 Figure 2: (A) A photomicrograph of C. S. of the head of the rabbit Embryo of 14 days old showing; the primordia of the secondary Septum nasi (SSN), primordia Concha nasalis ventralis (CNV), Concha nasalis dorsalis (CND), Processus palatinus medianus (PPM), primordia of Organum vomeronasale (OVN) and lingua (L) (H.E. stain 40X). (B) A photomicrograph of C. S. of the head of the rabbit Embryo of 14 days old showing; the primordia of the Septum nasi (SN), Cavum nasi (CN), primordia Concha nasalis ventralis (CNV), Concha nasalis dorsalis (CND), primordia of Ductus nasolacrimalis (PDNL), primordia of Organum vomeronasale (OVN), Primordial of Meatus nasi dorsalis (PMND) and Capsula nasalis primitivae (CpN). (H.E. stain 100X). (C) A photomicrograph of L. S. of the head of the rabbit Embryo of 16 days old showing; the primordia of Cavum nasi (CN), Septum nasi (SN), primordia Concha nasalis ventralis (CNV), Concha nasalis dorsalis (CND), Ostium of Organum vomeronasale (OOVN), primordia of Capsula nasalis primitivae (CpN). Processus palatinus medianus (PPM), primordia secondary palate (SP) and lingua (L). (H.E. stain 40X). (D) A photomicrograph of L. S. of the head of the rabbit Embryo of 16 days old showing; Neurofibra vomeronasale (NFV), Organum vomer-onasale (OVN), Septum nasi (SN), Processus palatinus medianus (PPM), Cavum nasi (CN) and lingua (L). (Silver I. stain 40X) 628 S. A. ALomaisi, H. El-Ghazali, H. Nosseur, S. Ahmed, M. Konsowa Figure 3: (A) A photomicrograph of C. S. of the head of the rabbit Embryo of 16 days old showing; Ductus vomeronasalis (DVN) slit like opening was lined by thin epithelium ventrolaterally and thick epithelium dorsomedial, Organum vomeronasale (OVN), Cartilago vomeronasalis (CaV), Septum nasi (SN), Cavum nasi (CN). (H.E. stain 100X). (B) A photomicrograph of C. S. of head of the rabbit Fetus of 18 days old showing; the lining Epithelium of Ductus vomeronaslis (DVN), Cartilago vomeronaslis (CaV), Organum vomeronasale (OVN). Was differentiated into thick Tunica mucosa olfactoria vomeronaslis(O) and thin Tunica mucosa respiratoria vomeronaslis(R). (H.E. stain 400X). (C) A photomicrograph of C. S. of head of the rabbit Fetus of 20 days old showing; The rostral opening Organum vomeronasale (OOVN) opened directly into the floor of Cavum nasi (CN), Septum nasi (SN), Ductus vomeronasalis (DVN). (H.E. stain 100X). (D) A photomicrograph of C. S. of head of the rabbit Fetus of 20 days old showing; lumen of Ductus vomeronaslis (DVN), a thick medialTunica mucosa olfactoria vomeronaslis(O) and thin lateral Tunica mucosa respiratoria vomeronaslis(R). Cartilago vomeronaslis (CaV), (H.E. stain 400X) Prenatal development of the vomeronasal organ in rabbit 629 Figure 4: (A) A photomicrograph of C. S. of the head of the rabbit Fetus of 22 days old showing; lumen of Ductus vomeronaslis (DVN), a thick medialTunica mucosa olfactoria vomeronaslis(O) and thin lateral Tunica mucosa respiratoria vomeronaslis(R). Cartilago vomeronaslis (CaV), the Primordia Acinus glandula vomeronasalis (AGVN) were determined at the dorsal commissure of Organum vomeronasale (OVN). (HE. stain 100X). (B) A photomicrograph of C. S. of the head of the rabbit Fetus of 28 days old showing; Ductus vomeronaslis (DVN), a thick medialTunica mucosa olfactoria vomeronaslis(O) and thin lateral Tunica mucosa respiratoria vomeronaslis(R). Cartilago vomeronaslis (CaV), was opened into the rostral of ventral part of Septum nasi, the mucous membrane was of stratified columnar type and the rostral opening of the duct blended directly with that of Vestibulum nasi (VN). (H.E. stain 400X). (C) A photomicrograph of C. S. of the head of the rabbit Fetus of 28 days old showing; Ductus vomeronaslis (DVN), Tunica mucosa olfactoria vomeronaslis(O),Tunica mucosa respiratoria vomeronaslis (R). Cartilago vomeronaslis (CaV) and Acinus glandula vomeronasalis (AGVN). (H.E. stain 400X). (D) A photomicrograph of C. S. of the head of the rabbit Fetus of 28 days old showing; of Ductus vomeronaslis (DVN), a thick medialTunica mucosa olfactoria vomeronaslis(O) and thin lateral Tunica mucosa respiratoria vomeronaslis(R). Cartilago vomeronaslis (CaV), (H.E. stain 400X). Discussion The present investigation showed that, the thickened epithelium of the nasal placode was invaginated to make the nasal pits (olfactory pits) at 11 days old of rabbit embryo. The primordia of VNO was performed at the medial aspect of the nasal pit as a thickening of the epithelium a result which came in agreement with, (6) in hamster, (7) in rabbit and (30) in rat whose mentioned that, the vomeronasal organ was embryologically derived from the olfactory placode. In human, early during the fifth week, the ectoderm in the upper one-third of each enlarging nasal sac became thickened and developed into the olfactory epithelium (19, 32). On the contrary in mammals (15) mentioned that the vomronasal organ originated from the medial wall of the olfactory pit shortly after the middle of the embryonic period. In the present investigation, there was a clear invagination, the epithelium lined the pit was similar to that of the placode, and merged 630 S. A. ALomaisi, H. El-Ghazali, H. Nosseur, S. Ahmed, M. Konsowa steadily with the general ectoderm contiguous the outside opening, which majorities towards the mesenchyme and causes a slight recess on the olfactory purse at 11 days old of rabbit embryo. Later on, cellular bud grew dorsally, caudally, and to the midline on both sides and formed the primordia of the vomeronasal groove these results were in accordance with (32) in hamster whose compared between the vomeronasal sensory and the olfactory epithe-lia and noticed that, the both epithelia were divergently derived from the olfactory placodes. At 13 days old embryo VNO appeared as arrangement of bilateral undifferentiating epithelial thickenings on rostroventral region of the nasal septum. The organ was enclosed via the immature vomeronasal cartilage, which appeared as but densely arranged in small amorphous cells. The latter cells were suggestive of their future dispositions. The medial and lateral sides of the nostrils showed the miniature of nasal cartilages. These results were in disagreement with (33) who stated that, the vomeronasal organ appear as two blind epithelial like tubes in the ventral aspect of the nasal septum at nasal vestibule at sixteenth prenatal day of rat. While our findings were in accordance with (7, 8) in rabbit, (9,34) in rat, (21) in goat, (19, 31, 35) in human, (36) in mammals and (37) in animals. The present work revealed that, the (future) VNO was in contact with the nasal cavity at 13- 28 days old of the rabbit embryos and fetuses. This result could not met with the available literature in the rabbit or the other animals and human expect (16) in primates whose mentioned that, one exception occurred in the largest fetal Tarsius (25 mm crown-rump length), in which the vomeronasal organ communicated with the nasal cavity alone. Conclusion To the best of our knowledge, this may be the first description to the designing, categorize, define and demonstrate the normal explaining of the onset time of first appearance and origin as well as developmental changes of vomeronasal organ of the rabbit and its allied structures Conflict of interest The authors declare that they have no conflict of interest. References 1. Elgayar SAM, Eltony SA, Othman MA. Morphology of Non-Sensory Epithelium during Post-natal Development of the Rabbit Vomeronasal Organ. Anat. Histol. Embryol 2014. Blackwell Verlag GmbH; 43: 282-293. Doi: 10.1111/ahe.12073. 2. Vaccarezza OL, Sepich LN, Tramezzani JH. The vomeronasal organ of the rat. J. Anat.1981; 132,(2)167-85. 3. Tarozzo G, Cappello P, De Andrea M, Walters E, Margolis FL, Oestreicher B, Fasolo A. Prenatal differentiation of mouse vomeronasal neurons. European J. of Neuroscience 1998; 10:392-396. 4. Weiler E, Apfelbach R, Farbman AL. The development organ of the male ferret. Chem.senses 1999; 24: 127-36. 5. Segovia S, Garcia-Falgueras A, Carrillo B, Collado P, Pinos H, Perez-Laso C, Vinader-Caerols C, Beyerm C, Guillamon A. Sexual dimorphism in the vomeronasal system of the rabbit. Brain research 2006; 1102:52-62. doi: 10.1016/j .brainres.05.017. 6. Taniguchi k, Taniguchi k, Mochizuki k. Developmental studies on the vomeronasal organ in the golden hamster. Jpn. J. Sci. 1982a; 44:709-716. 7. Roongruangchai J, Pilakasiri K, Imjai S. Development of the Face, Siriraj Med J 2006; 58:716-9. 8. Seham-Gabr SH. Some anatomical studies on the development of the nasal cavity and its related structures in the white New Zeeland rabbit. Ph.D. Thesis. Anat. and Emb. Depart. Faculty of Vet. Med.- Sadat City University 2015. 9. Valverde F, Santacana M, Heredia M. Formation of an olfactory glomerulus: morphological aspects of development and organization. Neuroscience 1992; 49(2): 255 -75. 10. Key S, Wray S. Two olfactory placode derived galanin subpopulations: luteinizing hormone releasing hormone neurones and vomeronasal cells. Journal of Neuroendocrinology 2000; Blackwell Science Ltd., Bethesda, USA, 12: 53545. 11. Taniguchi K, Taniguchi K. Embryonic and Postnatal Differentiation of Olfactory Epithelium and Vomeronasal Organ in the Syrian Hamster. J. Vet. Med. Sci 2008; Towada Japan, 70(1): 57-64. Prenatal development of the vomeronasal organ in rabbit 631 12. Salazar I, Lombardcro M, Cifuentes JM, Quinteiro PS, Aleman N. Morphogenesis and growth of the soft tissue and cartilage of the vo-meronasal organ in pig. J. Anat. 2003; 202:503-14. 13. Salazar I, Quinteiro PS, Lombardcro M, Aleman N, De Troconiz PF. The Prenatal Maturity of the Accessory Olfactory Bulb in Pig. Chem. Senses 2004; 29: 3-11. Doi: 10.1093/chemse/bjh001. 14. Park J, Lee W, Jeong C, Hwangryong K, Taniguchi K, Shin T. Developmental changes affecting lectin binding in the vomeronasal organ of domestic pigs, Sus scrofa. Acta Histochemica 2012;114: 24-30. Doi: 10.1016/j.acthis.2011.02.001. 15. Garrosa M, Gayoso MJ, Esteban FJ. Prenatal development of the mammalian vomeronasal organ. Microscopy research and technique 1998; 41: 456-70. 16. Smith TD, Siegel MI, Bhatnagar KP. Observations on the vomeronasal organ of prenatal Tarsius bancanus borneanus with implications for ancestral morphology. J. Anat 2003; Blackwell Publishing Ltd. 203, pp473-81. 17. Smith TD, Bhatnagar KP. The human vomeronasal organ. Part II: prenatal develop. J. Anat. 2000;197:421-36. 18. Neskey D, Eloy JA, Casiano RR. Nasal, septal, and turbinate anatomy and embryology. Otolaryngol Clin 2009; N Am, Elsevier Inc.,42:193-205. Doi:10.1016/j.otc.2009.01.008. 19. Som PM, Naidich TP. Illustrated Review of the Embryology and Development of the Facial Region, Part 1: Early Face and Lateral Nasal Cavities. Ajnr. Am. J. 2013; Neuroradiol, 34:2233- 40. http://dx.doi.org/10.3174/ajnr.A3415. Ebook ISBN: 978-0-7020-5032-9. 20. Abdelaziz SE. Some anatomical studies on the nasal cavity of the buffalo in Egypt (Bos bubalis). M. V. Sc. Thesis. Anatomy and Embryology Department, Faculty of Veterinary Medicine, Zagazig University 1983. 21. Abdelaziz S, Ammar SM, Nosseur HM. The prenatal development of the vomeronasal organ in goat. The 14th Egyptian Anatomical Society, conference, Ein -Shams University 1991. 22. Ibrahim D, Taniguchi K, Yamamoto Y, Taniguchi, K, Nakamuta N. Immunohistochemical and Lectin Histochemical Studies on the Developing Olfactory Organs of Fetal Camel. Microscopy Research and Technique 2015; Wiley Periodicals, Inc, 78: 613-619. Doi 10.1002/jemt.22517. 23. Kostov DL. Vomeronasal organ in domestic an-imals (A short survey). BJVM 2007; 10 (1): 53-7. 24. Drury RAB, Wallington EA. Carleton's histological technique. Fifth Ed. Oxford University. USA. 1980. 25. Suvarna SK, Layton c, Bancroft JD. Bancroft's theory and practical of histological techniques. Eighth ed. Elsevier. China. 2019; ISBN: 978-0-7020-6864-5. 26. Cheville NF, Stasko J. Techniques in Electron Microscopy of Animal Tissue. Veterinary Pathology 2014; 51(1):28-41. Doi: 10.1177/0300985813505114. 27. Nomina Anatomica Veterinaria. International committee on veterinary gross anatomical Nomenclature. General assemble of the world association of veterinary anatomists, 6th Ed., Hannover (Germany), Columbia, MO (U.S.A.), Ghent (Belgium), Sapporo (Japan) 2017. 28. Nomina Embryologica Veterinaria. International Committee on Veterinary Embryological Nomenclature (I.C.V.E.N.). General Assembly of the World Association of Veterinary Anatomists (W.A.V.A.) Knoxville, TN (U.S.A.) 2003, 2th Ed., Ghent (Belgium) 2017. 29. Nomina Histologica Veterinaria. International committee on veterinary Histological Nomenclature (ICVHN). General assemble of the world association of veterinary anatomists, 2018 by the General Assembly of the WAVA. 1stEd., Hannover (Germany), Columbia, MO (U.S.A.), Ghent (Belgium), Sapporo (Japan) 2017. 30. Cuschieri A, Bannister LH. The development of the olfactory mucosa in mouse light microscopy. J. Anat 1975; 119(2):277-86. 31. Kumoi T, Nishimura Y, Shiota k. The em-bryologic development of the human anterior nasal apreture. Acta Otolaryngol 1993; 113(1): 93-7. 32. Taniguchi k, Taniguchi k, Mochizuki k. Comparative developmental studies on the fine structure of the vomeronasal sensory and olfactory epithelia in the golden hamster. Sci. Jap. J. Vet 1982b; 44(6): 881-90. 33. Vidic B, Greditzer HG, Litchy WJ. The structure and prenatal morphogenesis of the nasal septum in the rat. J. Morphol 1972; 137:131-148. 34. Vidic B. The prenatal morphogenesis of the lateral nasal wall in the rat (Mus rattus). J.Morphol. 1971; 133:303-18. 35. Müller F, O'Rahilly R. Olfactory structures in staged human embryos. Cells tissues organs 2004; karger AG, Basel,178:93-116. S. A. ALomaisi, H. El-Ghazali, H. Nosseur, S. Ahmed, M. Konsowa 632 36. Noden DM, De Lahunta A. The embryology of domestic animals developmental mechanisms and malformations. Williams and Wilkins Baltimore and London 1985; Pp. 161-70. 37. McGeady TA, Quinn PJ, Fitz Patrik ES, Ryan MT, Kilroy D, Lonergan P. Veterinary Embryology. John Wiley & Sons, Ltd, second edition, U.K. 2017; Pp, 232-240, SBN: 9781118940617. Slov Vet Res 2019; 56 (Suppl 22): 633-45 Original Research Article DOI 10.26873/SVR-802-2019 IMPACT OF DIETARY EICOSAPENTAENOIC AND DOCOSAHEXAENOIC FATTY ACIDS SUPPLEMENTATION ON INFLAMMATORY RESPONSE OF POST CALVING COWS DURING TRANSITION TO LACTATION Shaimaa Elbaz1, Eldsokey Nassef2*, Abdelnasser Bakr2, Elsayed Hegazi2, Abeer M. S. EL-Keredy1 department of Biochemistry, Nutritional deficiency diseases and Toxicology, Animal health Research Institute, Kafrelsheikh, Egypt, 2Nutrition and Clinical Nutrition Department, Faculty of Veterinary Medicine, Kafrelsheikh University, Egypt *Corresponding author, E-mail: dsokeynassef@yahoo.com Abstract: The aim of current study was to determine the effect of dietary omega 3 fatty acids (FA) mainly eicosapentaenoic (EPA) and docosahexaenoic (DHA) on inflammatory response of post calving cows during transition to lactation. Twenty Holstein lactating cows were assigned into two groups (10 each). The two groups fed on the basal diet while one of them was supplemented with 70 g of omega-3 FA per cow. The supplement was added to the diet from the 1ST day of calving till the 60th days of lactation. Blood samples were taken 3 times weekly from the 2nd to the 4th week post calving. The blood samples were analyzed for glucose, insulin, non-esterified fatty acids (NEFA), beta hydroxyl butyric acid (BHBA), C- reactive protein (CRP), haptoglobin, albumin and globulin. Feeding omega-3 FAs significantly increased (P<0.05) blood glucose level by 17.35%, 28.23% and 24.73% than the control at weeks 1, 2, 3 of the experiment, respectively. BHBA was lower in cows fed omega-3 at 1st and 2nd week then became nearly similar to the control at the end of experiment. At 1st and 2nd week, cows fed omega-3 FAs had significantly lower serum CRP than those fed the control diet and became nearly similar to control at the end of the experiment. Serum, globulin was significantly higher in cows fed omega 3 FAs at 2nd and 3rd week by 81.1% and 51.2%, respectively. Omega 3 FA improved energy status of fresh cow by increasing serum insulin and glucose levels while decreasing NEFA and BHBA parameters. Also, feeding omega 3 FA improved humoral immunity through increasing serum globulin and reduced inflammatory response by lowering CRP post calving. Key words: omega 3 fatty acids; lactating cow; inflammation; energy balance; serum protein Introduction The onset of lactation is one of the most crit- ical period affecting dairy cows in which massive changes and more metabolic stresses for Received: January 2019 Accepted for publication: February 2019 634 S. Elbaz, E. Nassef, A. Bakr, E. Hegazi, A. EL-Keredy dairy cows (1). In this period there is high demand for energy for milk synthesis and secretion accompanied by changes in blood metabolites (2). The impaired glucose production that occurs in early lactating cows would lead to an increase in the breakdown of adipose tissue, elevated plasma NEFA and increased ketones production by the liver. After parturition by few several weeks there is more incidence of diseases and morbidity on many dairy cows with especially mastitis, metritis, milk fever, ketosis and fatty liver. Postpartum dairy cow is suffering from systemic inflammatory state even with the absence of clinical disease (3). On the other hand, physiological changes that occurring in the fresh period after calving can affect cellular immune responses and modulate the influence of dietary fatty acids on immune functions. Also, the elevated NEFA leading to abnormal immune cell functions, increased inflammation and increased risk of metabolic and infectious diseases. The elevated plasma NEFA impaired cy-tokine production (4) and altered endothelial cell adhesion (5). One of the different nutritional approach to decrease inflammation post calving is the use of omega 3 polyunsaturated fatty acids. It is thought that the n-3 fatty acids attenuate inflammatory response in lactating dairy cows (6) and eicosapentaenoic (EPA) and docosahexaenoic (DHA) have more immunomodulatory activities (7). (8, 9) reported that EPA and DHA performing anti-inflammatory functions via direct or indirect inhibitory mechanisms. A reduction of inflammatory cytokine production with feeding of EPA and DHA to dairy cows and therefore they have the ability to modulate the inflammatory response this was reported by (6). One effect of cytokines is to activate production of acute phase proteins, that primarily produced by the liver, this class of proteins includes hap-toglobin, serum amyloid A and C-reactive protein. The importance of acute phase proteins in the response to infection is somewhat unclear, but they have gained wide spread acceptance as markers of inflammation. (10) stated that cell surfaces receptors were more directly characterized mediate anti-inflammatory effects of omega-3 fatty acids. An attenuated inflammatory response was observed by (11) due to increase intake of EPA and DHA fatty acids which resulted in increase the proportion of these FAs in the membrane phospholipids. (12) noticed an increased plasma glucose and decreased plasma ketones when feeding omega3 source compared to omega-6 sources in fresh cows in addition to anti-inflammatory effect of omega 3 due to increased phagocytic activity of circulating leukocytes. An increase in plasma glucose level was reported by (13) with feeding omega 3 polyunsaturated fatty acids. The improvement in metabolic profile with omega-3 fatty acids supplementation may be the key to beneficial effect on both immunity and inflammation. From the economic point of view, (14) reported that as the diet enriched in n-3 fatty acids, the nutrient expenditure would be reduced as omega-3 fatty acids attenuate the inflammatory responses. This study aimed to evaluate the anti-inflammatory effect of EPA and DHA fatty acids in the post calving cows during transition to lactation. Also, their effects on energy balance were determined. Materials and methods Cows and dietary treatment Twenty Holstein cows (10 multiparous and 10 primiparous) were assigned to two groups according to parity, body weight (BW) and previous milk yield (expected milk yield for primiparous cows). Cows averaged 680 kg of BW at the beginning of the experiment (post calving date). Cows had continuous access to fresh clean water. A basal postpartum TMR was formulated to meet or exceed (15) recommendation. Ingredients and nutrients composition are presented in Tables 1 and 2, respectively. Analysis of TMR on dry matter basis is presented in Table 3. Cows were fed three times daily at 4 am, 11 am and 4 pm for ad libitum intake. Treated group was top-dressed on the TMR and hand mixed at the time of feeding 70 gm/head/day with omega 3 FA supplement (STRATA G113) which its composition was fish oil 90%, calcium 9.95% and BHT (antioxidant ) 0.05%. Impact of dietary eicosapentaenoic and docosahex-aenoic fatty acids supplementation on inflammatory response 635 The supplement was added at a rate 40gm/cow at 4 am and 15 gm/cow for each feeding 11 am and 4 pm. The typical analysis of fatty acids using gas chromatograph for STRATA G113 were: palmitic acid (16:0), 39.4%; palmitoleic acid (16:1), 6.8%; stearic acid (18:0), 9.9%; oleic acid (18:1), 18.8%; linoleic acid (18:2), 6.1%; linolenic acid (18:3), 2.2%; eicosapentaenoic acid (20:5), 10.3%; docosahexaenoic acid (22:6), 6.5%. Experimental measures and samples analysis Daily feed intake and refusal were recorded daily. Feed and refusal samples were collected daily, subsamples weekly, and composite at the end of experiment. TMR and feed refusal were immediately frozen. The subsamples of TMR and feed refusal were dried at 60 °C in hot air circulations oven (Heraeus UT 20 model, Germany). The dried samples of TMR, corn silage and other feed ingredients used were ground through 1mm screen of grinder (Cyclotec 1093 Foss, Sweden). The ground samples were analyzed for final dry matter at 105 °C for 3 hour, crude protein (Kjeltec system 2100-Foss, Sweeden), ether extract (Soxtec system 2045 Foss-Sweeden), and ash (Furnace6000, Thermolyne USA) according to (16). Neutral detergent fiber without using sodium sulfite, acid detergent fiber (ADF) (17) and lignin were determined by digesting the ADF residue in 72% sulfuric acid, (18). Fiber fractions were determined with fibertec M61020-Foss Sweden. Neutral detergent insoluble crude protein (NDICP) was determined after NDF extraction by analyzing the residue for crude protein. The NFC concentration was calculated using the following equation: NFC= 100-(NDF%+CP%+EE%+Ash %). The values of TDN and NEL were determined according to (15) based on nutrient analysis (CP, NDF, ADF, NFC, EE, Lignin, NDICP and ADICP). Total aflatoxins (b1, B2, G1 and G2) level was analyzed fluorometrically using VICAM series 4 USA according to (19). Blood metabolites and hormones Blood samples were collected from all animals at 11:00 am, 3 times weekly at the 2nd, 3rd and 4th weeks postpartum. Ten ml of blood was collected from coccygeal vessel and divided into 2 tubes for each cow. One tube contained K2EDTA for plasma separation and the other tube was without anticoagulant for serum separation. Plasma was separated immediately after the collection of blood by centrifugation at 3000 rpm for 10 minutes. The clear plasma was obtained using sterilized pipettes and kept at -20 °C till analysis. Serum was separated by blood centrifugation at 3000 rpm for 15 minutes and then stored at -20 °C till analysis. Biochemical analysis The collected plasma was analyzed for glucose according to (20) based on glucose oxidase enzyme activity. Plasma NEFA concentration was measured according to (21) using diagnostic reagent for quantitative in vitro determination of NEFA in plasma on photometric systems. Concentration of plasma BHBA was determined according to (22) using quantitative determination of B-hydroxy butyrate in plasma for in vitro diagnostic use Pointe Scientific, INC. Serum was analyzed for total protein according to (23) using quantitative method based on the intensity of the color formed which is proportional to the total protein concentration in the sample. Albumin was measured in serum according to (24) based on bromocresol green binding method using available commercial albumin kits. Globulin was calculated according to (25) by subtraction of albumin from total protein. Haptoglobin was measured according to (26) based on determination of serum hemoglobin binding capacity. C reactive protein (CRP) was measured in serum according to (27) based on Rete Nephelometry. Insulin was measured in serum according to (28) based on a solid phase enzyme-linked immunosorbent assay (ELISA). Statistical analysis The obtained data were analyzed using statistical software (SPSS Version 16). Group means were compared using analysis of T-inde-pendent-Samples T Test. Levene's test for equality of variances was used to indicate the 636 S. Elbaz, E. Nassef, A. Bakr, E. Hegazi, A. EL-Keredy significance. The means were considered different at P<0.05. Results Results of blood insulin, glucose, NEFA and BHBA concentrations are shown in figures 1, 2, 3 and 4, respectively. Cows fed omega-3 FAs had significantly (P<0.05) higher blood insulin level (0.37 and 1.1 mIU/ml) than those fed the control diet (0.17 and 0.33 mIU/ml). This trend lasted to 2 weeks then declined to be non-significant at 3rd week of experiment (4th week post calving). Feeding omega-3 FAs increased blood glucose level (mg/dl) by 17.35%, 28.23% and 24.73% than the control at weeks 1, 2, 3 of the experiment, respectively. This was more significant at 2nd and 3rd weeks. Cows fed omega 3 FAs had significantly higher blood NEFA (mg/dl) at 1st week and then rapidly declined at 2nd week and became numerically lower but non-significant than the control at 3rd week of experiment. In contrast, BHBA (mg/dl) was lower in cows fed omega-3 at 1st and 2nd weeks then became nearly similar to the control at the end of experiment. Results of CRP, haptoglobin, albumin and globulin concentration in serum are shown in figures 5, 6, 7 and 8, respectively. At 1st and 2nd weeks, cows fed omega-3 FAs had significantly (P<0.05) lower serum CRP (0.27 and 0.21 mg/L) than those fed the control diet (0.37 and 0.36 mg/L) and became nearly similar to the control at the end of the experiment. Cows fed omega 3 FAs had no significant difference between the control and the experimental cows on serum haptoglobin and serum albumin along the experiment. In contrast, serum globulin was significantly higher in cows fed omega 3 FAs at 2nd and 3rd week which increased by 81.1% and 51.2%, respectively, than the control. While at the 1st week serum globulin was nearly similar in between the control and the experimental cows. Table 1: Ration ingredients used for early lactating cows during the experiment Ingredients DM% DM As fed For each cow_Kg/day_Kg/day Corn silage 28.0 7.41 26.45 Wheat straw 92.0 0.39 0.43 Corn grain ground dry 88.0 4.61 5.24 Wheat bran 88.8 0.76 0.86 Soybean meal 46% 90.0 3.26 3.62 Soy Best 89.0 0.97 1.09 Megalac 97.0 0.45 0.47 Optigen 99.0 0.04 0.04 Calcium carbonate 99.5 0.20 0.20 Salt white 99.5 0.08 0.08 Sodium bicarbonate 99.5 0.21 0.21 Vitamin mineral premix* 99.5 0.06 0.06 Premix provides 5500 IU vitamin A; 500 IU vitamin D; 50 IU vitamin E; 100 mg zinc; 60 mg iron; 40 mg manganese; 20 mg copper; 0.6 mg iodine; 0.3 selenium and 0.25 mg cobalt per kg dry matter. Impact of dietary eicosapentaenoic and docosahex-aenoic fatty acids supplementation on inflammatory response 637 Table 2: Nutrients composition of the basal diet Nutrient DM As fed Dry matter (%) 100.00 47.99 Forage (%) 41.62 19.97 Crude protein (%) 17.98 8.63 Rumen undegradable protein (% of CP) 37.01 17.76 Rumen degradable protein (% of CP) 62.99 30.23 Soluble protein (% of CP) 27.89 13.38 Metabolizable energy (Mcal/kg) 2.60 1.25 Net energy for maintenance (Mcal/Kg) 1.69 0.81 Net energy for gain (Mcal/kg) 1.08 0.52 Acid detergent fiber (%) 19.25 9.24 Neutral detergent fiber (%) 30.17 14.48 Forage neutral detergent fiber (%) 23.39 11.22 Lignin (%NDF) 8.44 4.05 Lignin (%DM) 2.55 1.22 Non fiber carbohydrate (%) 38.75 18.60 Table 3: Analysis of total mixed ration on dry matter basis Nutrients (%) As fed basis Dry matter basis Dry Matter 46.47975% 100% Moisture 53.52025% 0.00 Crude Protein 8.12% 17.47% Neutral detergent fiber 16.8% 36.14% Acid detergent fiber 10.1% 21.73% Cellulose 8.61% 18.53% Hemicellulose 6.7% 14.41% Lignin 1.48% 3.19% NDICP 1.44% 3.1% ADICP 0.61% 1.31% Crude Fat 1.48% 3.19% Ash 3.81% 8.21% Non fiber carbohydrate 16.27% 35% Total digestible nutrients 32.48% 69.8865% DE (Mcal/kg) 1.56 3.17 ME (Mcal/kg) 1.25 2.54 NEL (Mcal/kg) 0.79 1.6 ADICP, acid detergent insoluble crude protein; NDICP, neutral detergent insoluble crude protein. DE, digestible energy; ME, metabolizable energy; NEL, net energy for lactation 638 S. Elbaz, E. Nassef, A. Bakr, E. Hegazi, A. EL-Keredy Figure 1: Influence of dietary omega-3 fatty acids on plasma concentrations of insulin in fresh lactating cows Figure 2: Influence of dietary omega-3 fatty acids on plasma concentrations of glucose in fresh lactating cows Impact of dietary eicosapentaenoic and docosahex-aenoic fatty acids supplementation on inflammatory response 639 Figure 3: Influence of dietary omega-3 fatty acids on plasma non-esterified fatty acids (NEFA) concentration in fresh lactating cows Control Omega-3 fatty acids Weeks postpartum Figure 4: Influence of dietary omega-3 fatty acids on plasma beta hydroxyl butyric acid (BHBA) concentration in fresh lactating cows 640 S. Elbaz, E. Nassef, A. Bakr, E. Hegazi, A. EL-Keredy 0,4 0,35 0,3 0,25 0,2 0,15 0,1 Control Omega-3 fatty acids 1^0,36 0,27 0,26 0,21 - -0,2 0,5 1 1,5 2 Weeks postpartum 2,5 3,5 Figure 5: Influence of dietary omega-3 fatty acids on serum C reactive protein concentration in fresh lac-tating cows 12 11 M 10 ° 9 M o £ 8 a x 7 6 0,5 Control Omega-3 fatty acids ' 10,09 9,82 1.9,23 ; 8,67 H 8,73 1,5 2 Weeks postpartum 2,5 3,5 0 3 0 1 3 Figure 6: Influence of dietary omega-3 fatty acids on serum haptoglobin concentration in fresh lactating cows Impact of dietary eicosapentaenoic and docosahex-aenoic fatty acids supplementation on inflammatory response 641 5.5 5 4.5 -o u 4 c F 3. 5 M t 3 2.5 2 0.5 ^^^Control Ornega-3 fatty acids "4-22 4.38- 1 * 4 02 E 3.39 1.5 2 Weeks postpartum 2.5 3.5 Figure 7: Influence of dietary omega-3 fatty acids on serum albumin concentration in fresh lactating cows -a M .a _o «5 10 9 8 7 6 5 4 3 2 Control Omega-3 fatty acids 8,07 8,01 8,22 7,74 - ► 5,12 " 4,54 . i i i i i i i 0 0,5 1 1,5 2 Weeks postpartum 2,5 3 3,5 Figure 8: Influence of dietary omega-3 fatty acids on serum globulin concentration in fresh lactating cows Discussion changes, more metabolic stresses for dairy cows and also more incidence of diseases and Post calving cows suffering from many morbidity. Researchers reported that postpar-problems during the transition of these cows to tum dairy cows suffering from systemic inflam-lactation. In this period there is massive 642 S. Elbaz, E. Nassef, A. Bakr, E. Hegazi, A. EL-Keredy matory state (29). It is thought that the fatty acids of n-3 family improving metabolic profile of dairy cows and also attenuate measure of inflammatory response in lactating cows and so we conducted the present study to investigate their effects on inflammation and energy balance post calving. It is well known that insulin concentration reflecting the glucose availability to stimulate beta cells of pancreas to produce and release insulin. The concentration of blood glucose with omega-3 feeding increased during the 3 weeks of the experiment with significant difference in 2nd and 3rd weeks of the trial. But, no significant effect was detected in the 1st week of the trial between the control and the omega-3 groups. The concentration of insulin was significantly increased at 1st and 2nd weeks of the trial at P value 0.015 and 0.029 respectively, for the omega-3 group and also increased in the 3 rd week but with no significant at P value 0.1. (30) reported that omega-3 of fish oil decreased insulin response to glucose in rats and this finding may explain why the plasma glucose concentration increased with feeding of omega-3 of fish oil and this greater concentration of plasma glucose for cow fed omega-3 was the result of a better energy status. The result of glucose was in agree with (31,32) and disagree with (33) that reported a decrease in plasma concentration of glucose with feeding fish oil and also with (34) that observed no change in blood glucose concentration with feeding of 5% fish meal and n-3 fatty acids to early lactating cows. The past using fish meal or fish oil as a source of long chain omega 3 fatty acids for ruminants might expose these fatty acids to saturation by rumen microbes which led to loss of its functional importance in the metabolism. In contrast, our study used EPA and DHA which were protected from rumen microbes, passed to aboma-sum in which were hydrolyzed and absorbed without change through the intestinal tract. So the difference might be due to the dose of EPA and DHA which arrived the metabolic pool without saturation. The result of insulin in the current study is in agree with (34) and disagree with (35) that noticed a reduction in insulin concentration in blood of dairy cows when fed fish oil containing DHA. Most of the changes occurred in plasma concentration of NEFA in the early lactation as the result of changes in adipose tissue mobilization and energy status (36). In our study, the plasma concentration of NEFA was higher (49.91±3.56 mg/dl) in the 1st week in the omega-3 group. Then it rapidly declined in 2nd (44.97±2.45 mg/dl) and 3rd (38.27±4.09 mg/dl) week. In contrast to the control group which was the lowest concentration in 1st week (32.69±2.46 mg/dl) and then increased till reached 43.70±2.90 mg/dl. This was reflecting sever mobilization of fat from adipose tissue and also increasing the effect of negative energy balance while the decline of NEFA concentration in omega-3 group that reached to 38.27±4.09 mg/dl reflecting an improvement in the energy status and metabolic profile. The result in current study of NEFA concentration was in agree with (33) that noticed a decrease in NEFA concentration by 21 day post-partum with cows that fed fish oil, and also agreed with (37) that reported a high concentration of plasma NEFA in the 3rd week postpar-tum and then gradually decreased with omega-3 feeding. The result of plasma NEFA disagreed with (31) which reported a high plasma NEFA concentration with cows fed calcium soap of fatty acids and also stated that feeding them did not improve the energy status of early lactating cows. Also our study disagreed with (38) that reported high concentration of plasma NEFA in all groups that fed omega-3 compared to the control group. However, our findings agreed with (39) who observed a great increase in plasma NEFA concentration in the 2nd week post-partum and then decreased gradually. The differences among studies might be due to the type, dose and technology of protection of fatty acids used in the fat supplement. BHBA is one of ketone bodies that produced by the liver as a result of incomplete oxidization of fat. Elevated BHBA above 14 mg/dl indicate ketosis that had impact on both cow health and milk yield. In the current study, BHBA in plasma was lower in cows fed omega-3 FA at 1st week at P-value 0.016 and then slight increase with no clinical ketosis at level 11.7 Impact of dietary eicosapentaenoic and docosahex-aenoic fatty acids supplementation on inflammatory response 643 mg/dl. Then it decreased to become nearly the same to the control group at the end of experiment at P-value 0.576. In agreement, (37) and (40) also reported that cows fed flax seed were in positive energy balance that indicated by a lower BHBA, NEFA and high glucose concentration. In contrast, (33) stated that the concentration of BHBA in plasma in cows fed fish oil increased with the increasing days postpartum (P<0.02). The same result of the current study agreed with (12) who reported an increase in plasma concentration of glucose and decreased plasma ketones in fresh cows that fed flax seed (omega-3) compared to omega-6 and adding that the anti-inflammatory effect of omega-3 source resulted in increased phagocytic activity of leucocytes and thus omega-3 source could improve metabolic and immune function. Acute phase proteins (APP) are a group of glycoproteins produced by liver in respond to an inflammatory stimulus, infection or trauma in animals (41). APP stimulate immunoglobu-lin production, tissue repair and prevent more injury (42). Results of APP including C-reac-tive protein (CRP) and haptoglobin (HP) in the current study are found in figures 5 and 6 respectively. The mean of serum concentration of CRP ( mg/l)was lower in cows supplemented with omega-3 in the 1st and 2nd weeks of experiment (0.27 and 0.21 ) respectively than the control group, and at the 3rd week of the trial there were no significant difference between control and omega-3 groups. The reduction in serum CRP in the 1st and 2nd weeks in the cows supplemented with omega-3 indicated that omega-3 has an anti-inflammatory effect. The same results was reported by (6) that noticed an anti-inflammatory effect when cows fed n-3 fatty acids from fish oil illustrated by decreased acute phase proteins. The means of haptoglobin concentrations during the 3 weeks of experiment not significantly differ between control and omega-3 group and this in agree with (43) and dis agree with (44)that stated that haptoglobin was a sensitive marker of inflammation in cattle. A low levels of haptoglobin was documented by (6) when cows fed fish oil from 35 to 160 days postpartum. The difference of results in this study and other studies may be due to different level of addition of omega-3, different omega-3 sources or time of addition omega-3. Blood serum proteins are significant indicators of animal health. There was no significant difference in the mean of serum globulin level in the 1st week of trial between control and experimental groups while in the 2nd and 3rd weeks there was a significant increase in the means of serum globulin in the cows supplemented with omega-3. These results indicate that omega-3 fatty acids are very important for humoral immunity. However, no difference in serum albumin was observed between control and experimental groups. Further study will be conducted regarding with effect of omega 3 fatty acids on milk yield, persistency and reproductive performance of lactating cows. Also recording metabolic problems, cost of treatment and culling to evaluate economic importance of omega 3 fatty acids in lactating Conclusion Feeding omega-3 FA tended to increase blood glucose and insulin levels in lactating cows and decrease the plasma level of NEFA gradually in the blood from 2nd to 4th week post-partum. There was a significant decrease in serum C - reactive protein in the cows fed omega-3 FA. Also, omega 3 FA improved humoral immunity by increasing serum globulin. Finally feeding dairy cows with EPA and DHA fatty acids during transition to lactation minimized inflammatory response post calving and improved their energy status. Conflict of interest The authors declare that they have no conflict of interest. References 1. Piccione G, MessinaV, Marafioti S, Casella S, Giannetto C and Faziof. Changes of some haemato chemical parameters in dairy cows during late gestation, postpartum, lactation and dry periods. Vet. Med. Zoot. 2012; 58, 59-64. 2. Wathes DC, Cheng Z, Chowdhury W , Fen-wick MA , Fitzpatrick R, Morris DG, Patton J and 644 S. Elbaz, E. Nassef, A. Bakr, E. Hegazi, A. EL-Keredy Murphy JJ. Negative energy balance alter global gene expression and immune responses in the uterus of postpartum dairy cows. Physiol Genomics, T. 2009; 39:1-13. 3. Humblet MF, Guyot H,Boudry B, Mbayahi F, Hanzen C, Rollin F, and Godeau JM. Relationship between haptoglobin, serum amyloid A, and clinical status in a survey of dairy herds during a 6-month period.Vet. Clin. Pathol. 2006; 35:188-93. 4. Scalia D, Lacetera N, Bernabucci U, Demey-ere K, Duchateau L and Burvenich C. Invitro effects of nonesterified fatty acids on bovine neutrophils oxidative burst and viability.J. Dairy Sci.2006; 89: 147-54. 5. Contreras GA, Rephael WS, Mattmiller A, Gandy J, and Sordillo LM. Non esterified fatty acids modify inflammatory response and eicosanoid biosynthesis in bovine endothelial cells. J. Dairy sci.2012b; 95:5011-23. 6. Silvestre F, Carvalho PC, Crawford JE, Santos P, Staples CR, Jenkins T, and Thatcher WW. Effects of differential supplementation of fatty acids during the peri partum and breeding periods of Holstein cows: II .Neutrophil fatty acids and function, and acute phase proteins .J. Dairy sci.2011; 94:2285-301. 7. Calder PC, Yaqoop, Thies F, Wallace FA, Miles EA .Fatty acids and lymphocyte func-tions.Br.J.Nutr.2002; 87:S31-48. 8. Clarke SD. The multi-dimensional regulation of gene expression by fatty acids: poly unsaturated fats as nutrient sensors.Curr.opin.Lipid.2004; 15:13-8. 9. Lee JY, Zhao L, and Hwang DH. Modulation of pattern recognition receptor-mediated inflammation and risk of chronic diseases by dietary fatty ac-ids.Nutr.Rev.2010; 68:38-61. 10. Oh DY, Talukdar S, Bae EJ, Imamure T, Morinaga H, Fan W , Li P, Lu WJ, Watkins SM, and Olefsjy JM.GPR120 IS an omega-3 fatty acid Receptor Mediating Potent Anti-Inflammatory and insulin-sensitizing Effects. Cell. 2010; 142 (5):687-98. 11. Calder PC. Mechanisms of action of (n-3) fatty acids. J. Nutr. 2012; 142: 5925-95. 12. Gandra JR, Barletta RV , Mingoti RD, Ver-durico LC, Freitas JE , Oliveira L J ,Takiya CS, Kfoury JR, Wiltbank MC, and Renno FP. Effects of whole flaxseed ,raw soybeans ,and calcium salts of fatty acids on measures of cellular immune function of transition dairy cows .J. Dairy Sci. 2016a; 99:4590-606. 13. Dirabdeh E,Towhidi A, Zein oaldin IS, Ganj khanoul M,Ansaripirsaraei Z,Fouladi-Nashta A.Effects of different polyunsaturated fatty acids supplementations during the postpartum periods of early lactating dairy cows on milk yield ,metabolic responses, and reproductive performances. J. Anim.Sci.2013; 91:713-21. 14. Gifford CA, Holland BP, Mills RL, Maxwell CL, Farney JK, Terrill SJ, Step DL, Richards CJ, Burciaga Robles LO, and Krehible CR. Impacts of inflammation on cattle growth and Carcass merit. J. amin. Sci. 2012; 90:1438-51. 15. National Research Council .Nutrients of Dairy Cattle .7th Rev. Ed.Natl. ACAD.Sci.2001; Washington, DC. 16. Associations of Official Analytical Chemists. Official Methods of Analysis .16th Ed.AOAC1995; Arlington .VA. 17. Vansoest PJ, Robert son JB and Iewis BA. Methods for dietary fiber, neutral detergent fiber and non-starch polysaccharides in relation to animal nutrition .J. Dairy .Sci.1991; 74:3583-97. 18. Hintz RW and Mertens DR. Effects of sodium sulfite on recovery and composition of detergent fiber and lignin. J.AOAC Int.1996; 79: 16. 19. Association of Official Analytical Chemists. Official Methods of Analysis 1991; 74:81. 20. Abdellatif K R A, Fadaly W A A, Kamel G M, Elshaier Y A M M, El-Magd M A. Design, synthesis, modeling studies and biological evaluation of thiazolidine derivatives containing pyrazole core as potential anti-diabetic PPAR-y agonists and antiinflammatory COX-2 selective inhibitors, Bioor-ganic Chemistry 2019; 82: 86-99. 21. Smith and Wilson. Free fatty Acids and Atherosclerosis. J. Clin .Endocrinal Metab 2006; 91: 2506-8. 22. Murray CH, Blanchflower WJ, and Rice DA.Clinical chemistry.3013, 421; 1984. 23. Burtis CA Tietz Text book of clinical chemistry, 3rd ed, AACC 1999. 24. Reinhold RR. Determination of serum albu-min.clin.chem.1953; 21:1370-1372. 25. Coles EH: Vet. Clin.Path.1974; 211-213.W.B.Sounders Company Philadelphia, Lon-don,Toronto. 26. Chu CC, Lee SL and Fung HP. Purification, analysis and basal level of serum haptoglobin in cat-tle.P.88.In: Congress of the Taiwan Association of Veterinary Medicine and Animal Husbandry, Tai-wan.2002. 27. Burtis CA, Ashwood ER,Titez Text book of clinical chemistry. 2nd Edition, W.B. Saunders, Philadelphia, PA (1994). Impact of dietary eicosapentaenoic and docosahex-aenoic fatty acids supplementation on inflammatory response 645 28. Eatham RD: Biochemical Values in Clinical Medicine, 7th Ed. Bristol. England .John Wright&sons,Ltd: 1985. 29. Humblet MF, Guyot H,Boudry B, Mbayahi F, Hanzen C ,Rollin F,and Godeau JM. Relationship between haptoglobin, serum amyloid A,and clinical status in a survey of dairy herds during a 6-month period.Vet.Clin.Pathol.2006;35:188-93. 30. Pighin D, Kara batas L, Rossi A, Chicco A, Basabe JC, Lombardo YB,2003.Fish oil affects pancreatic fat storage, Pyruvate dehydrogenase complex activity and insulin secretion in rats fed a sucrose -rich diet.J.Nutr.2003;133:4095-101. 31. Juchem SO, Santos JEP, Cerri RLA, Chebel RC, Galvao KN, Bruno R, Depeters EJ, Scott T , Thatcher WW , Luchini D. Effect of calcium salts of fish and palm oils on lactational performance of Holstein cows .Ani.feed .Sci.2008;140:18-38. 32. Gandra JR, , Mingoti RD, Barletta RV , Takiya CS , Verdurico LC, Freitas JE ,Paiva PG, Jesus EF, Calomeni GD and Renno FP . Effects of flaxseed ,raw soybeans ,and calcium salts of fatty acids on apparent total tract digestibility, energy balance and milk fatty acid profile of transition cows .Animal.2016b;1:1-8. 33. Mattos R, Staples CR, Arteche A, Wiltbank MC, Diaz FJ, Jenkins TC and Thatcher WW. The effects of feeding fish oil on uterine secretion of PGF2a, Milk composition, and metabolic status of periparturient Holstein cows.J.Dairy .Sci.2004; 87:921 -32. 34. Heravi Moussavi AR, Gilbert RO, Verton TRO, Bauman DE and Butler WR. Effects of feeding fish meal and n-3fatty acids on milk yield and metabolic responses in early lactating dairy cows J.Dairy sci. 2007; 90:136-44. 35. Bilby TR, Sozzi A, Lopez MM, Silvestre FT, Ealy AD, Staples CR& Thatcher WW. Pregnancy , bovine somatotropin, and dietary n-3 fatty acids in lactating dairy cow: I. Ovarian, conceptus, and growth hormone - insulin -like growth factor system responses .Journal of dairy Sci. 2006b; 89: 3360-74. 36. Drackley JK. Biology of dairy cows during the transition period; the final frontier? J. Dairy Sci.1999; 82:2259-73. 37. Greco LF, Neves Neto JT, Pedrico A, Fer-raza RA, Lima FS, Bisinotto RS, Martinez N, Garcia M, Ribeiro ES, Gomes GC, Shin JH, Ballas MA, Thatcher WW, Staples CR, and Santos JEP. Effects of altering the ratio of dietary n-6 ton-3 fatty acids on performance and inflammatory responses to a lipopolysaccharide challenge in lactating Holstein cows.J.Dairy Sci.2015;98:602-17. 38. Gonthier C, Mustafa AF, Ouellet DR, Choui-nard PY, Berthiaume R, and Petit HV. Feeding Mi-cronized and extruded flaxseed to dairy cows. Effects on Blood parameters and milk fatty acid composition .J.Dairy Sci.2005; 88:746-56. 39. Elis S, Freret S, Desmarchais A, Maillard V, Cognie J, Briant E, Touze JL, Dupont M, Faverdin P, Chajes V, Uzbekova S, Monget P and Dupont J. Effect of a long chain n-3PUFA-enriched diet on production and reproduction variables in Holstein dairy cows. Animal Reproduction Sci.2016; 164:121-32. 40. Ulfina GG, Kimotni SP,Oberoi PS, Baithalu RK, Kumaresan A, Moohanty TK, Imtiwati P and Dang AK. Modulation of post-partum reproductive performance in dairy cows through supplementation of long-or short -chain fatty acids during transition period JPn.2015;99,1056-64. 41. Marinkovic S, Jahreis GP, Wong GG and Baumann H. IL-6 modulates the synthesis of a specific set of acute phase plasma proteins in vivo .J.Immunol.1989; 142:808-12. 42. Kent JE. Acute Phase Proteins: their use in Veterinary diagnosis .Br.vet.J.1992; 148:279-82. 43. Staples CR, Amaral B, Silvestre F, Caldari Torres C, Cullens FM, Badinga L, Arthington JD and Thatcher WW .2008.Immune system responses to diseases /Disorders in the dairy animal and potential effects of essential fatty acids Florida Ruminant Nut.symp.2008;F1 32611,352-92. 44. Nazifi S, Rezak hani A, Koohimoghadam M, Ansari-Lari M and Esmailnezhad Z. Evaluation of scrum haptoglobin in clinically healthy cattle and cattle with inflammatory diseases in Shiraz, a tropical area in Southern Iran. Bulg. J. vet. Med. 2008;11, No2, 95-101. Slov Vet Res 2019; 56 (Suppl 22): 647-55 DOI 10.26873/SVR-803-2019 Original Research Article PHENOTYPIC AND MOLECULAR IDENTIFICATION OF YEASTS ISOLATED FROM CULTURED TILAPIA (OREOCHROMIS NILOTICUS) IN KAFRELSHEIKH PROVINCE, EGYPT Amgad A. Moawad1*, Ahmed I. Noor El Deen2, Nadia B. Mahfouz3 Salwa M. Helmy1, Yasmen F. Hashem4 departments of Bacteriology, Mycology & Immunology Department, Faculty of Veterinary Medicine, Kafrelsheikh University, Egypt, 2Department of Hydrobiology, Veterinary Division, National Research Center, Egypt, 3Department of Fish Diseases and Management, Faculty of Veterinary Medicine, Kafrelsheikh University, Egypt, 4Veternarian in Directorate of Veterinary Medicine in Kafrelsheikh, Egypt Corresponding author, E-mail: Moawadamgad@gmail.com Abstract: The present study aimed to identify yeasts by phenotypic, biochemical and genotypic methods. Three hundred live Oreochromis niloticus samples were collected from different locations at Kafrelsheikh Governorate, Egypt and during different seasons. Yeast isolates were identified according to their phenotypical characterization in combination with polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) method. Two hundred and twenty-six yeast isolates from eighty diseased and two hundred and twenty apparently healthy O. niloticus samples. Isolated yeasts were Rhodotorula spp. (18.14%), Candida albicans (19.47%), Candida parapsilosis (16.37%), Candida guilliermondii (14.6%), Candida inconspicua (8.85%), Trichosporon asahii (17.25%), Geotrichum spp. (2.66%) and unidentified Ascus forming yeasts (2.66%). Yeasts most frequently isolated from gills and skin. Phenotypic methods are valuable in identification of yeasts into genera but these methods take more time. Molecular methods through the amplification of (internal transcribed spacer) ITS1-5.8S-ITS2 regions of fungal rRNA, followed by RFLP-PCR using Msp1 restriction enzyme allowed simple, rapid, cost-effective, sensetive and accurate identification of the phenotypically identified yeasts. Key words: Oreochromis niloticus; yeast; PCR; Msp1; PCR-RFLP Introduction Parasitic fungi are facultative saprophytic (1). Yeasts are identified according to the ability of sugars fermentation and assimilation of carbon and nitrogen compounds (2). Fungal reproduction is not simple and depends upon more than one method of propagation. Environmental conditions stimulate formation of special structures that can help in sexual and asexual reproduction by efficiently dispersing spores or spore-containing propagates (3). Unicellular reproduction can occur by budding, fission or by both processes, called bud fission. Yeasts that reproduce sexually are members of Received: January 2019 Accepted for publication: February 2019 648 A. Moawad, A. Noor El Deen, N. Mahfouz, S. Helmy, Y. Hashem two large phyla of the fungi and have different modes of sporulation. After meiosis ascomy-cetous yeasts produce different shapes of asco-spores inside an ascus that is not enclosed in a complex fruiting body or an ascocarp. Basidio-mycetous yeasts show a wide variety in the ba-sidium morphology, where the formation of the external meiotic spores (4). Phylogenetic and population genetic methods are used to identify species and pathogenic fungi and detect the modality of their reproduction in nature (5). Fish that infected by fungi was previously exposed to some other pathogens or stressors like bacterial, viral, parasitic diseases, poor water quality and trauma (6). Fungal can infect externally or internally and over the past decade fungi have received high attention all over the world (7). Many of fungi are considered opportunists because attacking fish under the effect of different stressors (8). Conventional identification of pathogenic yeasts depends on the morphological and biochemical characters using tests that need more time. Also, the available commercial methods frequently fail to identify the less common pathogens or to differentiate between closely related species. Molecular methods used for the identification are rapid and sensitive alternatives to conventional identification for yeasts (9). RFLP method was relatively more accurate and reliable for the detection of various yeasts compared PCR (10). The present study was conducted to isolate and identify yeast from freshwater fish by phe-notypic methods and study reproductive behavior on different culture media, rice agar for asexual spores while carrot media for detection of sexual spores. Molecular identification of yeast by RFLP-PCR was further applied to confirm phenotypic results. Materials and methods A total of 300 live O. niloticus comprised 80 clinically diseased and 220 apparently healthy fish were collected from private fish farms at Kafrelsheikh province. The Body weights were ranged from 150±10 g. The collected fish were examined clinically with paying attention to the behaviors in the ponds, changes in color and respiratory manifestations with special care to the external lesions according to the methods described by (11). The samples were collected aseptically transferred alive to the provisional laboratory of department of microbiology in faculty of veterinary medicine, Kafrelsheikh University. The live fish samples were transferred in a separate plastic bags one-third volume of water and provided with oxygen aeration by battery aerator (Beauty, Italy) and then subjected to mycological examination. Media Sabouraud's dextrose agar (SDA) (oxoid). It was used for isolation and preservation of fungi according to (13), Germ tube test (14) for detection of reproductive behavior, Rice agar media (15) for asexual reproduction of yeasts, Carrot agar (CA) (16) for induction of sexual sporulation of fungi, differential and selective chro-mogenic medium for the isolation and quick identification of Candida spp., Christensen's urea agar (Oxoid) for biochemical identification, Isolation of yeast The obtained samples were inoculated into duplicate plates of SDA with 500 mg of chloramphenicol dissolved in 3 ml ethanol 95% (that were added to the media after autoclav-ing). The inoculated plates were incubated at 30oC-37oC. A pure culture of each yeast colony was collected and the genus identified based on morphological characteristics (13). RFLP-PCR RFLP-PCR was carried out using primers (to amplify the ITS-1 and ITS-2 regions of yeast spp. using universal primers (internal transcribed spacer) ITS1 (5-TCC GT AG-GTGAACCTGCCGG-3') and ITS4 (5'-TC CTCCGCTTATTCATATGC-3') and Msp1 restriction enzyme for yeast spp. as previously described (17). Before RFLP-PCR, DNA extraction from yeast isolates was performed using DNA isolation kit (Jena Bioscience) and as previously described (18). Phenotypic and molecular identification of yeast isolated from cultured tilapia (Oreochromis niloticus) in 649 Results Phenotypic identification of yeast isolates Yeast isolates were identified according to microbiological characters (hyphae, pseudohy-phae, chlamydospores, and blastospores) into genera. Two hundred and twenty-six yeast isolates that yielded from the samples were identified into 4 genera (Candida, Rhodotorula, Tri-chosporon and Geotrichum). Identification of Candida species One hundred and thirty-four isolates of Candida spp. were subjected for identification according to the following (Fig. 1): A. Macromorphology on SDA C. albicans colonies were characterized by creamy colored pasty colonies within 48-72hrs, germ tube positive, grew well on media containing cycloheximide with light green colonies on chromogenic candida agar, while C. para-psillosis colonies were white and smooth colonies on both SDA and chromogenic Candida agar with no growth on media containing cyclo-heximide. C. guilliermondii colonies were dull, white, creamy or yellow, flat smooth colonies, while on chromogenic agar, they appeared pale pink to purple. Other Candida spp had the same cultural characters on SDA but can't grow on media containing cycloheximide; with light pink colonies on Chromogenic candida agar and showed negative germ tube test. B. Micromorphology on Rice agar media Isolates could be identified into C. albicans according to Rice agar media which showed terminal chlamydospores, blastocondia and pseudohyphae. Pseudohyphae of C. parapsil-losis were long and branched and carried clusters of elongated blastospores without chlamydospores. Other Candida spp. failed to produce pseudohyphae. C. guilliermondii appeared as short branched pseudohyphae bearing clusters of blastospores All Candida isolates were ure-ase negative. Identification of Trichosporon species Thirty-nine isolates of Trichosporon isolated on SDA appeared firstly as smooth flat or wrinkled white to creamy colonies that turned waxy with central folds surrounded by wrinkled furrows. They grew well at 300C, 370C, and also on media containing cycloheximide. Microscopically, hyaline mycelium separated and fragmented into rectangular arthrospores appeared. On Rice agar media, Trichosporon appeared as septated hyphae, pseudohyphae, and arthrospores. Trichosporon species were urease positive. Identification of Rhodotorula species Forty-one isolates Rhodotorula were identified on SDA as Rhodotorula spp. (light pink flat colonies). It grows well at 300C with faint growth at 370C, no growth on media containing cycloheximide. Microscopic examination of Gram's stained films revealed large round yeast cells, on Rice agar media showed large round blastoconidia with no pseudohyphae. Rhodotorula spp. was urease positive. Identification of Geotrichum species Six isolates Geotrichum spp. were identified on SDA as they grew rapidly as white to creamy, flat and smooth, yeast-like colonies which turned into mold-like in old colonies, grew at 300C but not at 370C, or on media containing cycloheximide. Micromorphology, hyaline septated branched hyphae break up into short chains or individual arthroconidia appeared. No pseudohyphae or blastoconidia were formed, a character which differentiated it from Trichosporon species. Arthroconidia produced a hyphal extension from one corner producing a hockey stick appearance. Geotrichum spp. was urease negative. Phenotypic identification of yeast isolates Molecular identification of yeast isolates Results obtained by preliminary conventional identification of yeast isolates combined with detection of characteristic asexual spore helping in identification of most of them into genera (Candida, Trichosporon, Rhodotorulla, Geotrichum), while using RFLP-PCR we found (C. albicans, C. parapsilosis, C. guilliermondii, C. inconspicua, Trichosporon asahii) (Figs. 2 and 3). Based on the results of RFLP-PCR we determined the incidence of yeast isolates. 650 A. Moawad, A. Noor El Deen, N. Mahfouz, S. Helmy, Y. Hashem The incidence of yeast spp. isolated from spp. isolated from different organs of O. nilot-diseased and apparently healthy O. niloticus icus fishes was shown in Table 2. fishes was shown in Table 1. Incidence of yeast Figure 1: Microscopical identification of isolated yeasts. (A) Candida albicans showed on RAT blastspores, pseudohyphase and chlamydoconidia. (B) Yeast showed on RAT budding cells (blastspores without peudohyphae). (C) Ascospore forming yeast on carrot agar Table 1: Incidence of yeast spp. isolated from diseased and apparently healthy O. niloticus fishes Isolated yeast No. of examined O. niloticus No. of diseased (80)_No. of apparently healthy (220) No. % No. % Rhodotorulla spp. 15 18.75% 30 13.63% Candida albicans 16 20% 22 7.27% Candida parapsilosis 5 6.25% 15 6.81% Candida spp. 4 5% 11 5% Candida guilliermondii 9 11.25% 15 6.81% Trichosporon spp. 6 7.5% 18 8.18% Ascus forming yeast 3 3.75% 7 3.18% Phenotypic and molecular identification of yeast isolated from cultured tilapia (Oreochromis niloticus) in 651 Table 2: Incidence of yeast spp. Isolated from different organs of O. niloticus fishes Isolated No. of iso- % Organs Yeast lates Skin Gills Liver Kidney No. % No % No. % No % Rhodotorulla spp. 41 18.14 12 29.3 22 53.7 2 4.9 5 12.2 C. albicans 44 19.47 13 29.5 15 34.1 10 22.7 6 13.6 C. parapsilosis 37 16.37 8 21.6 19 51.4 6 16.2 4 10.8 Candida spp. 20 8.85 4 20 10 50 4 20 2 10 Trichosporon spp. 39 17.25 21 53.8 9 23.1 9 23.1 0 0 Candida guilliermondii 33 14.6 0 0 18 54.5 15 45.5 0 0 Geotrichum spp. 6 2.66 2 33.3 3 50 1 16.7 0 0 Unidentified Ascus form- 6 2.66 2 33.3 3 50 1 16.7 0 0 ing yeasts Total 226 100 62 99 48 17 Figure 2: PCR product of yeast isolates, lane 1,12 - 100 bp ladder, lane2- C. albicans, lane3 - C. albicans, lane4 - C. guilliermondii 608bp and C. parapsilosis 520bp, lane5 - C. parapsilosis lane 6 - C. parapsilosis, lane 7 - C. inconspicua, lane 8 - c. parapsilosis lane 9 - Unidentified Candida spp. lane 10 - Unidentified Candida spp. lane 11 Unidentified Candida spp. ,Lane13 - Tr. asahii lane14 - Tr. asahii, lane15 -Unidentified Trichosporon spp. lane16 - Tr. asahii 652 A. Moawad, A. Noor El Deen, N. Mahfouz, S. Helmy, Y. Hashem 1500 • « 11 537 520 520 520 520 oeM — ... — mm 298 371 239 ',55 287 520 287 287 M I rnm mm 0 333 333 mm 180 180 333 180 Figure 2: RFLP-PCR of yeast isolates by MSP1 enzyme, lane 1,12 - 100 bp ladder, lane2- C. albicans, lane3 - C. albicans and C. parapsilosis, , lane4 - C.parapsillosis and C. guillerimondi, lane5 - C. para-psillosis and C. inconspicua lane 6 - C. parapsilosis, lane 7 - C. inconspicua, lane 8 - C. C. parapsillosis and C. inconspicua lane 9 - Unidentified Candida spp., lane 10 - Unidentified Candida spp, lane 11-Unidentified Candida spp. Lane13 - Tr. asahii lane14 -Tr. asahii, lane15 - Unidentified Trichosporon spp., lane16 - Tr. Asahi Discussion Yeasts play a key role in several ecosystem processes, establishing ecological relations with other organisms which have been identified as a part of wild caught as well as farmraised fish microbiota and display an ability to interfere with the fish's nutrition and sanitary conditions (19). 226 yeast isolates out of 300 (75.33%) were isolated, with 5 genera: Candida, Rhodotorula, Trichosporon, Geotrichum and unidentified ascus forming yeast from O. niloticus. Candida spp. and were isolated at an incidence of 59.29% (134/226) and 18.14% (41/226), respectively. These results agreed with those recorded by (20) who isolated Candida spp. and Rhodotorula spp. at incidence rate of 65.12% and 28.84%, respectively. C. albicans (19.47%) and Rhodotorula spp. (18.14%) were the highest yeast isolates collected from skin, gills, kidney, and liver and. These results coincided with (21) who found incidence of C. albicans, and Rhodotorula spp. at 35.9 % and 31.4%, respectively. Trichosporon spp. was isolated with higher incidence (17.25%) and lower rate for Geotrichum spp. (2.66%) and ascus forming unidentified yeast (2.66%). Nearly similar results were recorded by (22). But (20) recorded that Trichosporon spp. (4.18%), and also Geotrichum spp. and ascus forming unidentified yeast in a low percent 0.46%. C. guilliermondii and C. parapsilosis were isolated in higher number that in agreement with (23) who found a rising of these pathogens but in freshwater environment. In the present study, several species of genus Candida (such as C. parasitosis, C. guillier-mondii and C. inconspicua) were mostly isolated from gills nearly by 50%. There is an evidence that exposure to environmental stressors can predispose fish to infection according to Phenotypic and molecular identification of yeast isolated from cultured tilapia (Oreochromis niloticus) in 653 (24). The polluted feeds, water and worker hands play important role in fish health (25). We observed that many yeast species that affect human have been isolated from freshwater environments. Terrestrial environments and wastewater may be the origin of many of these species (26). Molecular approaches exhibit the potential to be a strong supplement for the phenotypic identification than the conventional methods (27). The most frequently used targets for yeast identification is the ITS region, which lies between the 18S and 28S rRNA gene and divided by 5.8S rRNA gene into ITS1 and ITS2 regions. In many studies the ITS2 showed considerable interspecies variability to identify 99.7% of yeasts and 100% of molds to species level, while ITS1 had identification accuracy of 96.8%-100% for yeasts and 100% for molds (28). Although ITS2 has been used in wide range for identification of fungi, in other study the ITS1 exhibit higher interspecies variability than ITS2 region (29). PCR-RFLP assays have been used to identify the isolated yeast species as was successfully done by (30). An advantage of this method is the stable and easy-to-read RFLP patterns. Unlike previous reports, this method involves only one or two DNA fragments. Also, it is easy and rapid to perform. In addition to time-saving restriction enzymes it takes few hours to be completed. It requires no complicated equipment except a traditional thermal cycler. Regarding that DNA sequencer may not be easily obtainable to most clinical laboratories, this molecular method is applicable for obvious identification and differentiation of different yeast species (31). In the present study, yeast samples that was molecularlt identified as Candida spp. by universal primers became specifically identified as C. inconspicua by RFLP either in samples mixed with C. parapsilosis or alone. All examined isolates showed comparable RFLP patterns to their respective reference strains. Regarding to genus Rhodotorula is commonly observed in fish microbiota, as Trichosporon that in agreement with (32). Also, Trichosporon asahii is the most frequently isolated species in these infections that agreed with this study as PCR-RFLP assure the present results (33). The remaining unidentified Candida spp or unidentified ascus forming yeasts need for sequencing for accurate defining of yeast species. Conclusion Yeasts isolated from healthy and diseased O. niloticus included Candida, Trichosporon, Rhodotorula, Geotrichum. Phenotypic methods succeed in the identification of yeasts into genera. Molecular methods using RELP PCR confirm the phenotypic identification and specifically identify the unknown species. The RFLP method follows a common protocol that can be done for identification of yeasts in few hours. So, it can be used substitutional to the traditional methods. References 1. Eli A, Briyai OF, and Abowei JFN. A review of Some Fungi Infection in African Fish Saprolegni-asis, Dermal Mycoses; Branchiomyces infections, Systemic Mycoses and Dermocystidium. Asian Journal of Medical Sciences 2013; 3(5): 198205. 2. Refai MK, Marouf S, Nermeen A, and Rasha HS. Monograph on Fungal Diseases of Fish A guide for postgraduate students 2016; Part 1. pp288. 3. Ali HH. Isolation and Identification of Pathogenic Fungi from Carp fish in Suliamania Province. Global Journal of Bio-science and Biotechnology 2015; 4(4): 356-63. 4. Lachance M. Yeasts. University of Western Ontario, Ontario, Canada. John Wiley & Sons, Ltd: Chichester 2011. Dol:10. 1002/9780470015902. a0000380. pub2. 5. Mousavi SA, Samira SS, Rezaie S, Nejad NS, Hadizadeh S, Kamyabi H, and Aghasi H. Identification of Candida Species Isolated From Oral Colonization in Iranian HIV-Positive Patients, by PCR-RFLP Method. undishapur Journal Microbiology 2012; 5(1): 336-40. 6. Noor El-Deen A, Mohamed HO, Mona SZ, and Hanan AA. Mass Mortality in Cultured Nile Tilapia Oreochromis niloticus due to Saprolegniasis with Emphasis on Treatment Trials. Journal of Biological Sciences. 2018; 18(1): 39-45. 7. Yanong RE, VMD. Fungal disease of fish. The Veterinary Clinics Exotic Animal Practice 2003; (6): 377-400. 654 A. Moawad, A. Noor El Deen, N. Mahfouz, S. Helmy, Y. Hashem 8. Quiniou SMA, Bigler S, and Clem LW. Effects of water temperature on mucous cell distribution in channel catfish epidermis: a factor in winter saprolegniasis. Fish Shellf Immunology 1998; 8:111. 9. Linton CJ, Borman AM, Grace Cheung, Holmes AD, Szekely A, Palmer MD, Bridge PD, Campbell CJ, and Johnson EM. Molecular Identification of Unusual Pathogenic Yeast Isolates by Large Ribosomal Subunit Gene Sequencing. Journal Clinical Microbiology 2007; 45(4): 1152-8. 10. Byung HO, Young CS, Yang WL, Yong BC, and Kyu JA. Comparison of Nested PCR and RFLP for Identification and Classification of Malassezia Yeasts from Healthy Human Skin Annals of Dermatology 2009; 21(4): 352-7. 11. Noga EJ. Text Book of Fish Disease: Diagnosis and treatment. 2nd ed. USA: Wiley-Blackwell 2010; pp: 519. 12. El-Adawy M, El-Aziz M A, El-Shazly K, Ali N G, El-Magd M A. Dietary propionic acid enhances antibacterial and immunomodulatory effects of oxytetracycline on Nile tilapia, Oreochromis ni-loticus, Environmental Science and Pollution Research. DOI: 10.1007/s11356-018-3206-5 2018. 13. Taha M. Medical mycology "Atlas of medically important & dermatomycosis". 1st ed., Mecca printing house, Cairo, Egypt. 2011. 14. Sheppard DC, Locas M, Christiane Restieri C, Laverdiere M. Utility of the Germ Tube Test for Direct Identification of Candida albicans from Positive Blood Culture Bottles. Journal of Clinical Microbiology 2008; 46(10): 3508-9. 15. Refai M. Isolation and identification of fungi. Faculty of Veterinary Medicine. Cairo University. 1987. 16. Leslie JF, and Summerell BA. The Fusarium Laboratory Manual. Blackwell Pub. 2006; 12-13. 17. Mohammadi R, Mirhendi H, Matehkolaei AR, Ghahr M, Shidfar MR, Jalalizand N, and Makimura K. Molecular identification and distribution profile of Candida species isolated from Iranian patients. Medical Mycology 2013; (51): 657-63. 18. El-Magd M A, Abbas H E, El-kattawy A M, Mokhbatly A. Novel polymorphisms of the IGF1R gene and their association with average daily gain in Egyptian buffalo (Bubalus bubalis), Domestic animal endocrinology 2013; 45(2): 105--0. 19. Navarrete P, and Tovar-Ramirez D. Use of yeasts as probiotics in fish aquaculture. In: M.P. HernandezVergara & C.I. Perez-Rostro (eds.). Agricultural and biological sciences-sustainable aquaculture techniques 2014; 135-72. 20. Tartor Y, Taha M, Mahboub H, El Ghamery M. Yeast species associated with diseased fish: Occurrence, identification, experimental challenges and antifungal susceptibility testing. Aquaculture 2018;488: 134-44. 21. Refai MK, Laila A. Mohamed, Amany M. Kenawy and Shimaa El-S M.A. The assessment of mycotic settlement of freshwater fishes. Journal of American Science 2010; 6(11): 595-602. 22. Maysoon SA, Jenan MK, and Shaimaa NY. Isolation and identification of systemic mycological isolates from fishes samples that obtained from local markets in Baghdad, Iraq. World Journal of Experimental Bioscience 2016; 4(2): 143 -6. 23. Medeiros AO, Kohler LM, Hamdan JS, Mis-sagia BS, Barbosa FAR, and Rosa CA. Diversity and antifungal susceptibility of yeasts from tropical freshwater environments in Southeastern Brazil. Water Research 2008; 42(14): 3921-9. 24. Wildgoose WH, Basva manual of ormamen-tal fish, (Text Book), 2nd. Edition, 1992. 25. Hassan AA, Hammad AM, El Barawy AM, and Manal AH. Incidence of aflatoxigenic fungi in frozen and canned fishes and trials to inhibit afla-toxin production by use of some minor elements and lupinustermis seeds. Egypt. Journal of Applied Sciences 2007; 22(10B) 351-60. 26. Nagahama, T. Yeast biodiversity in freshwater, marine and deep-sea environments. In: Rosa, C.A., Gabor, P. (Eds.), Biodiversity and Ecophysi-ology of Yeasts. Springer, Berlin 2006; 241-62. 27. Ecker DJ, Sampath R, Li H, Massire C, and Matthews HE. New technology for rapid molecular diagnosis of bloodstream infections Expert Review of Molecular Diagnostics 2010; 10(4): 399-415. 28. Leaw SN, Chang HC, Sun HF, Barton R, Bouchara JP, and Chang TC. Identification of medically important yeast species by sequence analysis of the internal transcribed spacer regions. Journal of Clinical Microbiology 2006; 44(3): 693-9. 29. Chen YC, Eisner JD, Kattar MM, Ras-soulian-Barrett SL, Lafe K, Bui U, Limaye AP, Cookson BT. Polymorphic internal transcribed spacer region 1 DNA sequences identify medically important yeasts. Journal of Clinical Microbiology 2011; 39(11): 4042-51. 30. Mousavi SA, Khalesi E, Shahidi Bonjar GH, Aghighi S, Shaifi F, Aram F. Rapid molecular diagnosis for Candida species using PCR-RFLP. Biotechnology 2007; 6(4): 583-7. 31. Ping YG, Wang L, Lu GX, Shen YN, and Lui WD. A simple and reliable PCR-restriction fragment length polymorphism assay to identify Candida albicans and its closely related Candida Phenotypic and molecular identification of yeast isolated from cultured tilapia (Oreochromis niloticus) in 655 dubliniensis. Brazilian Journal of Microbiology 2012; 43(3): 873-9. 32. Gatesoupe FJ. Live yeasts in the gut: natural occurrence, dietary introduction, and their effects on fish health and development. Aquaculture. 2007; 267: 20-30. 33. Suzuki K. Fatal Trichosporon fungemia in patients with hematologic malignancies. European Journal of Haematology 2010; 84: 441-7. Slov Vet Res 2019; 56 (Suppl 22): 657-63 DOI 10.26873/SVR-804-2019 Original Research Article PIVOTAL ROLE OF Lactobacillus STRAINS IN IMPROVEMENT OF SOFT CHEESE QUALITY AND INHIBITING THE GROWTH OF HARMFUL AND DANGEROUS BACTERIAL PATHOGENS Yaser M. Hafez1*, Azza M. Sobeih2, Nehal M. Mansour1, 2 1EPCRS Excellence Center &Plant Pathology and Biotechnology Lab., Faculty of Agriculture, Kafrelsheikh University, 33516, Egypt, 2Food Control Department, Faculty of Veterinary Medicine, Kafrelsheikh University, 33516, Egypt Corresponding author, E-mail: hafezyasser@gmail.com Abstract: This study was carried out to determine the effect of addition of Lactobacillus bacteria to improve the soft cheese quality and inhibiting the microbial growth on cheese. Lactobacillus acidophilus DSM 20079 and Lactobacillus casei ss. casei DSM 20011 were studied in vitro for their probiotic properties through their antimicrobial activity. These strains were able to inhibit the growth of Escherichia coli O157 ATCC51659, Staphylococcus aureus and Bacillus cereus. Low salt soft cheese was manufactured, with acceptable organoleptic characters and prolonged shelf-life by using L. acidophilus and L. casei as bio preservative. The results cleared that, the addition of L. acidophilus or L. casei to the soft cheese improved the quality of the soft cheese through improvement of organoleptic characters of soft cheese such as appearance, body texture, flavour and extended soft cheese shelf-life through microbiological characteristics of manufactured cheese. The effect of the L. acidophilus is higher than the effect of L. casei than the control group. So we can use these strains as food preservative. Key words: Lactobacillus spp.; cheese making; antibacterial activity Introduction Cheese is one of the essential milk products for human feed because it contains most of nutrients required for growth and body health (1). It considered as source of microbial contamination. It is commonly made from non pasteurized milk of poor bacteriological quality, under unhygienic conditions and final product kept uncovered. Thus, there is high threat of contamination with several pathogenic and spoilage microorganisms. Harmful pathogenic bacteria such as Coliform, Escherichia coli, Staphylo-coccus and Salmonella as well as yeasts and molds were recognized in Karish cheese which factory-made with traditional method (2, 3). These harmful microbes cause inflammation and allergic diseases (3). Consumers need natural and chemical-free cheese. Therefore, it became urgent to search for alternative methods for cheese bio-preservation. Lactobacillus is energetic for recent food technologies, because of their probiotic potential to replace antibiotics (4). Lactic acid bacteria used as a starter for cheese conservation, improving the organoleptic properties, source of fermentation industry and control of food borne Received: January 2019 Accepted for publication: February 2019 658 Y. Hafez, A. Sobeih, N. Mansour microbes (5, 6, 7). It enhanced the immune system and produce bacteriocins, organic acids, H2O2 as antimicrobial compounds as well as short chain fatty acids (8, 9). According to our knowledge not enough research were conducted on soft cheese bio-preservation in Egypt, therefore, the aim of this research was to study the antimicrobial activity of Lactobacillus strains against harmful bacteria such as E. coli Oi57,S. aureus and B. cereus as well as improving the hygienic quality of soft cheese. Materials and methods Bacterial Culture Identified Lactobacillus acidophilus DSM 20079 and Lactobacillus casei ss. casei DSM 20011 from Cairo-MIRCEN, Faculty of Agriculture, Ain-Shams University, Cairo, Egypt. Bacteria were cultured on MRS broth (9ml) media then, kept at 37°C for 24hours. After that, they were cultured in sterile skim-milk and incubated at 37°C for 24 hours to activate the bacterial strains and to increase the bacterial number to the target probiotic dose (107Colony forming unit/g) for soft cheese manufacture. Antimicrobial activity of Lactobacillus strains Antibacterial activity of Lactobacillus strains was evaluated by agar well diffusion assay (10).Culture media (15 ml), melted and tempered at 45°C, were inoculated with E. coli Oj57,S. aureus and B. cereus at 106 CFU/ml then poured into Petri dishes that had wells which made by cork borer. Wells were filled with 100 pl of 48 hours-culture-supernatants of the Lactobacillus strains, then, incubated at 37 °C for 24 hours. The bacterial activity seen by appearance of inhibition zone which measured by milli-meter ruler. Cheese manufacturing Soft cheese was made as by Mehanna and Rashed 1990 and El-Sheikh et al. 2001 (11, 12) with slight modification. Pasteurized half fat milk (12L, 1.5% fat,8.5% SNF) were heated to 40°C then, the total solid was standardized to 14% by adding 4% skimmed milk pow-der(<1.25% fat,< 32% protein and >53% lactose), Nacl 3% and CaCl2 0.02% also were added. The bulk volume of milk was divided into 3 groups (4 Kg each) after mixing and inoculated by the activated starter cultures the first group was considered as control, the second was inoculated with L. acidophilus culture and the third was inoculated with L. casei, then, 0.3g rennet was added to each group and incubated at 37°C until curd formation, the curd was kept to drain in previously sterilized stainless steal frames lined with cheese cloth. The produced cheese and their whey were packaged in pre-sterilized bottles and kept at4°C. The cheese was tested fresh (zero time) and at equal intervals of 7 days till the spoilage was appeared. The experiment was repeated in triplicates and average results for each group were tabulated. Cheese characters evaluation Organoleptic examination Cheese samples were tested for appearance, texture, body, and flavor (13). The identical samples were labeled using random numbers and presented to the judges in random manner by 10 examiners. Chemical examination Cheese samples were tested for pH (14). Microbiological examination Samples were homogenized using sodium citrate (2%) and 10 fold serial dilutions were made (15). Then, they were tested for total Col-iform count (15); Lactobacilli count (16); as well as mold and yeast count (17). Statistical analysis The statistical analysis was done using analysis of variance (ANOVA) to compare between the parameters studied among different treatments. Chi2-test was used also, to determine the incidences of bacterial isolates between different treatments (18). Pivotal role of Lactobacillus strains in improvement of soft cheese quality and inhibiting the growth of 659 Results Antibacterial effect of Lactobacillus strains Lactobacillus acidophilus and Lactobacillus casei ss casei inhibited the growth of harmful bacteria such as Echerichia coliOi57, Staphylo-coccus aureus and Bacillus cereus (Table 1,Fig. 1). Positive impact of lactobacilli on soft cheese quality Our results presented in table (2) cleared that, the Lactobacillus acidophilus and Lacto-bacillus casei improved the average appearance of the soft cheese and its body texture than the control group and the Lactobacillus acidophilus decreased the spoilage followed by the Lacto-bacillus casei than the control group. The results also, cleared that, the cheese still preserved its flavour in case of Lactobacillus aci-dophilus till 32 day from its storage and 30 day in case of Lactobacillus casei and in the control group the flavour still present till 14 day of preservation. The overall score due to addition of Lacto-bacillus acidophilus and casei improved the score of cheese especially in Lactobacillus aci-dophilus followed by Lactobacillus casei than the control group. Effect of Lactobacillus bacteria on pH level of soft cheese The observed results in table (3), exhibited a significant differences (P < 0.05) on the effect of lactobacillus bacteria on pH level of soft cheese. Lactobacillus acidophilus improved the pH level till 30day of cheese storage, followed by Lactobacillus casei and both of them improved the pH level than the control group. Effect of Lactobacillus level on the bacterio-logic aspect of the soft cheese Our results observed in table (4) cleared that, the level of Lactobacillus acidophilus increased gradually by storage time of soft cheese and reached its maximum level at 21 day of storage. By increasing the level of Lactobacillus aci-dophilus and Lactobacillus casei causes decreasing the level of coliforms bacterial growth but the effect of Lactobacillus acidophilus greater than the effect of Lactobacillus casei. Effect of lactobacillus bacteria on mycolog-ical aspect of the soft cheese Our results observed in Table (5), cleared that, there is a significant different of the effect of lactobacillus bacteria (P < 0.05) on the level of mould and yeast count. The results cleared that, the increasing level of Lactobacillus associated with decreasing level of moulds and yeast than the control group. The effect of Lac-tobacillus acidophilus higher than the effect of Lactobacillus casei in prevention of the growth of moulds and yeast. Table 1: Inhibition zones (mm) caused by Lactobacillus strains in agar diffusion assay (n = 5) Lactobacillus Species (Diameter of inhibition zones in mm) Escherichia coli O157 Bacillus cereus Staphylococcus aureus Lactobacillus casei Lactobacillus acidophi-lus Control 2.6±0.20B 2.9±0.3A 0 2.9±0.10A 2.57±0.50B 0 2.75±0.50AB 2.55±0.20B 0 Means within the same row of different litters are significantly different at (P < 0.05) 660 Y. Hafez, A. Sobeih, N. Mansour Table 2: Effect of Lactobacillus on organoleptic characteristics of soft cheese. Average Appearance (10) Body texture (60) Flavor (30) Overall score (100) Storage time Lacto- Lactoba- Lac- Lacto- Con- bacil- cillus Con- toba- bacillus trol lus aci- trol cillus aci- Lactoba- Lactobacil-Control cillus ca- lus aci-sei dophilus Lacto- Lactoba-Control bacillus cillus ac-casei idophilus Zero time 7 9 8 56 58 57 28 29 29 91 96 94 7 days 6 8 7.5 55 57 56.5 26 28 28 87 93 92 14 days 5 7 7 45 55 54 19 26 26 69 88 87 21 days S 5 5 S 50 53 S 20 20 S 75 78 30 days S S 4 S S 52 S S 19 S S 75 32 days S S S S S S S S S S S S Chi2 10.55** 11.22** 12.45** 9.24** S = spoiled Significant at (P < 0.01) Table 3: Effect of Lactobacillus on pH of the soft cheese. Storage time Average pH Control Lactobacillus casei Lactobacillus acidophilus Zero time 5.54±0.04A 5,78±0.02A 5.64±0.04a 7 days 5.54±0.05A 5.78±0.01A 5.64±0.04a 14 days 5.45±0.05A 5.76±0.03A 5.54±0.05A 21 days S 5.64±0.04b 5.50±0.05B 30 days S S 5.35±0.03B 32 days_S_S_S S = spoiled. Means within the same row with different litters are significantly different at (P < 0.05) Table 4: Effect of Lactobacillus on bacteriological aspect of the examined samples of soft cheese Average coliforms count(cfu/g) Average Lactobacilli count (cfu/g) Storage time Control Lactobacillus casei Lactobacillus aci- Lactobacillus casei Lactobacillus aci- dophilus dophilus Zero time** 9.9x106 ±9.0 X103 A 4.5x106±1.50 2.5x105±1.0 X103 D 1.4x107±3.0 X103 B 1.3x107±3.0 X103 B X103C 7 days 1.3x108±0.50X103A 2x107± 2.50 X103 B 3x105±1.0 X103 C 2x107±3.0 X103 B 1.8x107±3.0 X103 B 14 days 1.5x108±4.0 X103 A 4x106±3.0 X103 C 1x105±1.0 X103 D 2.8x107±3.0 X103 B 6.13x107±2.0 X103 B 21 days S 1x103±1.0 X103 D 11x102±1.0 X103 D 1.1x107±1.0 X103 D 5.2x107±1.0 X103 D 30 days S S 18x102±1.0 X103 B S 2.21x107±A1.0 X103 32days S S S S S S= spoiled. Means within the same row of different litters are significantly different at P < 0.05 Pivotal role of Lactobacillus strains in improvement of soft cheese quality and inhibiting the growth of 661 Table 5: Effect of Lactobacillus on mycological aspect of the soft cheese Storage time Average mould count (cfu/g) Average yeast count (cfu/g) Control Lactobacilus casei Lactobacillus acidophilus Control Lactobacillus casei Lactobacillus aci-dophilus Zero time 7 days 14 days <10D <10D 2x101D <10D <10D <10D <10D <10D <10D 1.3x107A 1x108A 1.3x108A 4.80x106B 2.5x107B 2.40x107B 1.50x105 C 2.20x106C 1.10x106C 21 days S 1.50x101C <10D S 2.17x104A 1.18x104B 30 days S S 2x101B S S 1.06x103A 32 days S S S S S S S= spoiled, Means within the same row of different litters are significantly different at (P < 0.05) Figure 1: Antibacterial activity of Lactobacillus casei ss. casei DSM 20011 against (1) Escherichia coli O157 ATCC51659 (2) Bacillus cereus (3) Staphylococcus aureus determined by appearance of inhibition zone(mm). Antibacterial activity of Lactobacillus acidophilus DSM 20079 against (4) Escherichia coli O157 ATCC51659 (5) Bacillus cereus (6) Staphylococcus aureus determined by appearance of inhibition zone (mm) Discussion Influence of Lactobacilli on cheese quality showed that both of Lactobacillus strains were able to improve cheese appearance, body texture, flavour, than the control group. Lactobacillus acidophilus decreased the spoilage incidence than Lactobacillus casei than the control. These results attributed to the increasing pH level which prevents the cheese spoilage with improvement of sensory characters. L. acidophilus improved the pH level till 30 days of cheese storage, followed by L. casei as compared to control group. The effect of Lactobacilli level on the bacteriologic aspect of the soft cheese cleared that, the level of L. acidophilus increased gradually by storage time of soft cheese and reached to its maximum level at 21 day of storage. The obtained data from microbial quality assessment of collected soft cheese samples indicated that, their inferior hygienic quality with a great chance of being a cause of food-borne disease. According to the Egyptian Standard ES 1008-2000; 50 % of cheese samples are not agreed due to the high counts of 662 Y. Hafez, A. Sobeih, N. Mansour coliform as fecal contamination indicator mostly in cheese variety (19). As a result of increasing the level of L. acidophilus and L. casei, the level of Coliforms bacterial growth was decreased however the effect of L. acidophilus greater than the effect of L. casei. These positive results attributed to the Lactobacillus ability to improve the pH level which prevents the growth of coliforms in soft cheese. High yeast count regularly indicates disused hygienic measures during production and management (2). The effect of Lactobacillus bacteria on mycological side of soft cheese proved that, there is a significant difference effect of the bacteria (P < 0.1) on yeast and moulds count. The increasing level of Lactobacilli correlated with decreasing level of yeast and moulds compared with control group. Interestingly, the effect of L. acidophilus higher than L. caseie effect in prevention of moulds and yeast growth. Lactobacilli as biotherapeutic agents serve as natural food preservative through the antimicrobial bacterial activity (3). Both Lacto-bacillus strains were able to inhibit the growth of Escherichia coli O157, Staphylococcus au-reus and Bacillus cereus as is seen by appearance of inhibition zone. The obtained results were in supported with that results in which proved the antagonistic activity of Lactobacil-lus against Staphylococcus spp. and Coliform sp. (20, 21). We were able to produce soft cheese with advanced procedure and reached the optimum hygienic conditions. Increased Lactobacillus counts might be relied on the absence of nutrients competition between Lactobacillus and other microorganisms, particularly in the individual culture treatments. Lactobacillus spp. compete with other microbes their metabolic end products (22). Molds absence as a result of treatments till the 21th day of cold storage, may translate the antimicrobial effect of those two promising strains which achieve the main aim of this study in better microbial quality and extended shelf life of produced cheese. These results agreed with (23) who stated that; Lactoba-cillus isolated from fermented foods produce organic acids and other antimicrobial agents, which are responsible for quality and palatabil-ity. Analysis of chemical and texture analyses, where less protein/ dry matter % decreased hardness and compacted structure of the new products causing softness. Keeping organolep-tic characteristics similar to conventional soft cheese with higher hygienic quality is encouraging. These results agreed with what reported that Lactobacilli are members of autochthonous non-starter lactic acid (24). The relation with the chemical properties where compact structure was a result of relatively higher protein/ dry matter % of control. The higher moisture content, the more soft cheese that coats the mouth during eating (25). Such characteristics improvements could increase consumer demand to the new cheese product. Conclusion It can be concluded that this research study cleared that, the two promising strains of Lac-tobacillus acidophilus and Lactobacillus casei ss. casei were able to inhibit the growth of harmful food pathogenic bacteria such as Esch-erichia coli O157, Staphylococcus aureus and Bacillus cereus. This perhaps the first study which conducted on Escherichia coli O157 as one of the dangerous and fetal human bacteria in Egypt. In addition to that both of Lactobacil-lus strains improved the quality of the soft cheese through improving the pH value, improvement of organoleptic characters such as appearance, body texture and flavour as well as improving the total score level of the soft cheese microbiological characters. The positive effect of the Lactobacillus aci-dophilus is higher than Lactobacillus casei effect than the control group. Acknowledgment This research was conducted in ISO/17025 accredited Plant Pathology and Biotechnology Lab. (PPBL) and certified EPCRS Excellence Centre, Fac. of Agric., Kafrelsheikh University. The authors would like to thank the staff members of PPBL., EPCRS and AFood Control Department, Fac. of Vet. Medicine, Kafrelsheikh University, Kafr-Elsheikh, Egypt. Pivotal role of Lactobacillus strains in improvement of soft cheese quality and inhibiting the growth of 663 Conflict of interest The authors declare that they have no conflict of interest. References 1. Allam MGM, Darwish AMG, Ayad EHE, Shokery ES, Darwish SM. Lactococcus species for conventional Karish cheese conservation. LWT -Food Sci. Technol. [Internet]. Elsevier Ltd.; 2017;79:625-31. 2. Soliman NSM, Aly SA. Occurrence and identification of yeast species isolated from Egyptian Karish cheese 2011;2:59-64. 3. Awad S. Microbial safety criteria and quality of traditional Egyptian Karish cheese. African J. Microbiol. Res. 2016;10: 804-12. 4. Bernardeau M, Guguen M, Vernoux JP. Beneficial lactobacilli in food and feed: Long-term use, biodiversity and proposals for specific and realistic safety assessments. FEMS Microbiol. Rev. 2006; 30:487-513. 5. Mahmoud SF, El-Halmouch Y, Montaser MM. Effect of probiotic bacteria on Karish Cheese production. Life Sci. J. 2013;10: 1279-84. 6. Darwish AMG, Ayad EHE, Shokry ES, Ma-shaly RE, Osman NA, Darwish SM. Wild Egyptian probiotic cultures for functional fermented dairy products. Egypt. Conf. Dairy Sci. Technol. 2010; 565-83. 7. Martinez RCR, Wachsman M, Torres NI, LeBlanc JG, Todorov SD, Franco BD. Biochemical, antimicrobial and molecular characterization of a noncytotoxic bacteriocin produced by Lactobacillus plantarum ST71KS. Food Microbiol. [Internet]. 2013;34:376-81. 8. Allam, M. G.; Darwish, A. M. G. and Ayad, H. E. (2017): Improve Hygienic Quality of Egyptian Karish Cheese Employing Isolated Antagonistic Lactobacilli Strains. ARRB. 2017; 19(2). 9. Allam MGM, Darwish AMG, Ayad EHE, Shokery ES, Mashaly RE, Darwish SM. In vivo evaluation of safety and probiotic traits of isolated Enterococcus feacium strain KT712. Res. J. Microbiol. 2016; 11. 0. Frizzo, L., Zbrun, M., Soto, L., Bertozzi, E., Signorini , M.,and Sequeira, G. Protective Effect of an Inoculum of Lactic Acid Bacteria from Bo vine Origin Against Salmonella Serotype Dublin in the Intestinal Tract of Mice. J Anim Vet Adv,2010; 9(16),2113-22. 11. Mehanna, A.S. and Rashed, M.A. An attempt to improve the keeping quality of Tallaga cheese by using milk treated with carbon dioxide. Egypt. J. Dairy Sci. 1990; 18:377-88. 12.EL-Sheikh,M.M.; Farrage, A.F.; Shahein, N. M. and EL- Shibiny, S. Low fat Domiati cheese with particulate whey protein concentrate (PWPC). Egypt. J. Dairy sci. 2001; 29:331-42. 13. Bodyfelt, F.W.; Tobias, J. and Trout, G. M. The sensory evaluation of dairy products. Chapter 8: sensory evaluation of cheese. An Avi book, publ. by Van Nostrand Reinhold, New York. 1988; PP:300-76. 14. Pearson, D. Chemical analysis of foods. 8 th ed., Churchill Livingston, Edinburgh, Lon-don.1984. 5. American Public Health Association (APHA) . Standard methods for the examination of dairy products. INC.,16th ed., New York.1992. 16. Dave, R.I. and Shah, N.P. Evaluation of media for selective enumeration of S. thermophilus, L. delbreukii ssp. Bulgaricus lactobacillus acidophilus and Bifidobacteria. J. Dairy sci. 1996; 79:1529. 17. Koburger, J.A. and Farahat, B.Y. Fungi in foods. I.A comparison of media to enumerate yeasts and molds. J. Milk and Food Technol. 1975; 38:466-8. 18. SAS Statistical analysis system. SAS User's Guide SAS Incorporation Institute.2004. 19. El Sayed MA, Hosny IM, El Kholy WI, El Dairouty RK, Mohamed HS. Microbiological evaluation of Egyptian white soft cheeses style. J. Am. Sci. 2011; 7:517-26. 20. El Soda M, Ahmed N, Omran N, Osman G, Morsi a. Isolation, identification and selection of lactic acid bacteria cultures for cheesemaking. Emirates J. Food Agric. 2003;15:51-71. 21. Soltis DE, Soltis PS. The role of phylogenet-ics in comparative genetics. Updat. Mol. Syst. 2003;132:1790-800. 22. Hitendra J, Prasad BDN, Gurumurthy H, Su-varna VC. Role of Lactic Acid Bacteria (LAB) in Food Preservation. 2016;5:255-7. 23. Mokoena MP, Mutanda T, Olaniran AO. African traditional fermented foods and beverages. Food Nutr. Res. 2016;60:1-12. 24. Poga T, Maillard M, Leclerc A. Lactobacillus and Leuconostoc volatilomes in cheese conditions. Appl. Microbiol. Biotechnol. 2016;100: 2335-46. 25. Bourne MC. Food texture and viscosity: Concept and measurement. 2nd Editio. Elsevier Science & Technology Books; 2002. Slov Vet Res 2019; 56 (Suppl 22): 665-72 DOI 10.26873/SVR-805-2019 Original Research Article IMPACT OF L-CARNITINE SUPPLEMENTATION ON GROWTH OF BROILER CHICKEN THROUGH DETERMINATION OF CHANGES IN THE EXPRESSION OF CAT2, MYOD AND MYF5 GENES Tarek K. Abouzed1*, Doaa A. Dorghamm 1, Khaled A. Kahilo1, Azza M. Elkattawy1, Eldsokey Nassef 2, Hanan B. El-sawy2 1Department of Biochemistry, Faculty of Veterinary Medicine, Kafrelsheikh University, 33516, Egypt, 2Nutrition and Clinical Nutrition Department, Faculty of Veterinary Medicine, Kafrelsheikh University, Egypt "Corresponding author, E-mail: dosouk2000@yahoo.com Abstract: This study explored the impact of supplementation of L- carnitine on the performance of broiler chicken, biochemical parameters and expression of cationic amino acid transporter (CAT2), myogenic determining factor {MYOD) and myogenic factor 5 (MYF5) genes. This study included two groups; the first one is the control group and the second one received L-carnitine at a dose 50 mg/kg/day in drinking water. After 35 day, the sera and muscle tissue were obtained for biochemical and real time polymerase chain reaction (qPCR) analysis. The obtained results revealed a significant increase in the body weight, total body weight gain and a significant decrease in food consumption, food conversion rate in L-carnitine-supplemented group as compared to the control group. Moreover, there was no significant changes in serum cholesterol, triacylglycerol, AST, ALT, total protein and uric acid levels which showed a slight decrease in L-carnitine-supplemented group in comparison with the control group. This study also revealed a significant up regulation for the expression of CAT2, MYOD and MYF5 genes in muscles of broiler chicken-supplemented with L- carnitine as compared to the control one. The present study concluded that supplementation of L- carnitine to broiler chicken was useful to increase the total body weight gain and muscle mass and this beneficial effect was associated with up regulation of CAT2, MYOD, and MYF5 genes. Key words: L-carnitine; broiler chicken; growth traits; CAT2; MYOD; MYF5 Introduction Poultry industry has assumed an imperative role in meeting the deficiency of animal protein in world. The poultry production, particularly broiler production, is the quickest way to increase the accessibility of high quality protein for human utilization (1, 2).The poultry production differs from animal production in a different ways. Broiler chicken production requires between fifty to sixty days, while the formation of red meats requires 9 to12 months. Additionally, chicken require around 3 kg of food (ration) to create 1 kg of meat, while animals should consume 7 kg of food to produce 1 kg of red meat (3). The problem in the poultry production is containing excess body fat. There are many Received: January 2019 Accepted for publication: February 2019 666 T. Kamal, D. Dorghamm, K. Kahilo, A. Elkattawy, E. Nassef, H. El-sawy factors, for example diets and hereditary factors that increase the tendency to aggregate excess body fat. In this manner, improvement of poultry composition by other additives is consider a primary focal point of nutritional research (4). Poultry food is fundamentally composed of soybean, maize and plant products which all have less carnitine content (5). The latter can be biosynthesized endogenously from methio-nine and lysine amino acids in the presence of folic acid and vitamin B3, B6, B12, and C, in addition to iron (6, 7). When diets are not provided with these two amino acids, the bird unable to form a sufficient amounts of car-nitine (5). Dietary L-carnitine addition induces the P-oxidation of unsaturated fats to produce aden-osine triphosphate (ATP) energy and enhance energy utilization (8-10). L-carnitine has two main functions. The first is to encourage the transfer of long-chain fatty acids across the inner mitochondrial membrane, and the second is to encourage the expulsion of short and medium-chain fatty acids from the mitochondria that aggregate as a result of typical and unusual metabolism (11, 12). It also has an impact on fat metabolism and reduction of protein formation to form energy in chick's body subsequently a greater amount of energy will be oxidized and won't be decomposed in abdominal tissues (13). Myogenic determining factor (MYOD) and myogenic factor 5 (MYF5) are a group of proteins belong to myogenic regulatory factors (MRFs). These basic helix-loop-helix (bHLH) transcription factors act consecutively in myo-genic differentiation. MYOD is a key tran-scriptional factor for muscle differentiation and growth. MYF5 is the first myogenic regulatory protein expressed in the skeletal muscle lineage. In mammals, both MYOD and MYF5 are required for the formation of skeletal muscle (14). In chickens, MYOD and MYF5 are associated with post-hatch chicken myogenesis (15). CATs are primary amino acid transport system used by tissues to concentrate lysine, arginine and ornithine into cellular amino acid pools to be used in nitrogen metabolism (16). This study was conducted to explore the potential effect of L- carnitine on the performance of broiler chicken, biochemical parameters and CAT2, MYOD, MYF5 genes expression. Materials and methods This study was conducted in the chicken experimental house at Faculty of Veterinary Medicine, Kafrelsheikh University, Egypt. A total number of 120 mixed sex Ross 308 broiler chicks at 1 day old age were obtained from a private poultry company at El-Gharbia Gov-ernorate, Egypt. All the chicks were immunized against both New Castle's disease on 7th and 18th days and Gumboro disease on 14th day. The experimental birds were offered feed and water ad libitum for 5 weeks. Birdfeeding A starter diet was available for the first 21 days then changed to a grower diet till the end of the experiment (35 days). Diets were formulated to meet the supplement recommendations for broilers by the National Research Council (17) with some modification according to updated nutrients specification of broiler chickens (Tables 1&2). Experimental design and treatment One hundred and twenty chicks were allocated into two groups (60 chicks per group). Each group was subdivided into 6 replicates (10 chicks/replicate).The first group: received drinking water only and basal diet (without any additive) and was considered as the control group; and the second group received L-carnitine (sigma Aldrich company in drinking water at dose of 50 mg/kg/day (18). Blood and tissue samples At the end of experiment, chickens were subjected to 12 hours feed withdrawal and then blood samples were collected from the wing veins in clean and dry Eppendorf tubes. After coagulation and centrifugation at 3000 rpm for 15 minutes clear serum samples were collected. Sera immediately kept frozen at -200C until biochemical analysis. Birds were slaughtered after mild anesthesia and dissec- L-carnitine supplementation on growth of broiler chichen through determination of changes in the expression ... 667 tion of skin under complete aseptic condition. Then muscle specimen was excised from breast muscle and quickly dunked in liquid nitrogen, then frozen at -80°C for RNA extraction. Performance parameters Body weight was recorded at the end of every week. Body weight gain was calculated weekly following this equation: body weight gain = final weight- start weight. Feed intake was recorded daily according to the following equation: Feed intake=feed allowed - feed refusal. Feed conversion ratio (FCR) was calculated according to the following equation: FCR = feed intake/body weight gain. Biochemical analysis Colorimetric estimation of glucose, cholesterol, triacyl glycerol, and uric acid was done as previously described (19). Serum total protein was evaluated by colorimetric procedure based on (20). Serum enzyme activities of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were spectrophoto-metrically measured as previously described (21). Quantitative determination of CAT2, MY- OD andMYF5 using real-time polymerase chain reaction (qPCR) Breast muscle tissue was used to isolate total RNA using TRIZOL (GeneZOl, Lot^:30117B07003, Genetix Biotech) following the manufacturer's protocol. The concentration of RNA was measured using a Nanodrop. cDNA was synthetized using HiSenScript™ cDNA synthesis kit (Intron Biotechnology, cat.no.25014,). This was done by mixing 10 pl 2X RT reaction buffer, 1pl enzyme mix solution, 1 pg of RNA and completed with RNas free water to 20 pl total volume. The mixture was incubated at 50 oC for 30 min and 85oC for 10 min. All PCR reactions were done by utilizing SYBR Green qPCR Master Mix (Enzynomics, cat. No. RT500) and specific primers (Table 3), at the following conditions: initial denaturation at 92 °C for 10 min, followed by 40 cycles of 15 s at 92 °C, 30 s at 60 °C and 30 s at 72 °C. The differences in gene expression among the different groups were estimated by using the 2-△△Ct method (22), standardized to GAPDH and expressed as relative mRNA levels relative to the control. Melting curve analysis was performed to verify the correct product. Statistical analysis All data were presented as mean ± standard error (SE). Statistical analysis was conveyed utilizing the T-independent test (Levene's test for equality of variances) to compare the significance among the groups. A value of P < 0. 05 were considered to be significant. Results Effect of L- carnitine on performance parameters Supplementation of L-carnitine group showed a significant increase in the final body weight as compared with control group .There were a significant increase in the body weight gain in the group received L-carnitine compared with control group. Feed intake in the control group was higher than the group treated with L-carnitine. However, feed conversion ratio was significantly lower in L-carnitine supplementation than group without supplementation (Table 4). Effect of L- carnitine supplementation on biochemical parameters As shown in (Table 5) the level of blood glucose, serum cholesterol, total proteins insignificantly change in chicken treated with L-carnitine relative to the control chicken. However, the levels of the remaining serum parameters (triacylglycerol, uric acid, ALT, and AST) showed an insignificant decrease as compared to the control group. Effect of L- carnitine on CAT2, MYOD and MYF5 expression As shown in figure 1, the L-carnitine supplementation significantly upregulated (P<0.05) the expression of CAT2, MYOD and MYF5 in comparison with the control group. 668 T. Kamal, D. Dorghamm, K. Kahilo, A. Elkattawy, E. Nassef, H. El-sawy Table 1: Ingredients composition of the diets (%) Ingredients (%) Starter diet Grower diet Yellow corn 53.55 58.2 Corn Gluten meal 6.9 6.0 Soybean meal, 44% 33.2 29.3 Soybean Oil 2.5 3 Di-calcium phosphate 1 1.8 1.6 Ground Limestone 2 1.2 1.0 Mineral and Vitamin premix3 0.3 0.3 Salt 0.3 0.3 DL- methionine4 0.1 0.1 Lysine 5 0.1 0.15 Choline chloride 0.05 0.05 Total 100 100 1 Dicalcium phosphate (contains 18% Phosphorus and 21% Calcium). 2 Limestone (contain 38 % calcium). 3 Premix provides vitamin A (12000 Iu), vitamin D (5000 Iu), vitamin E (50 mg), vitamin K3 (3 mg), vitamin B1 (3 mg), vitamin B2 (8 mg), nicotinic acid (30 mg), pantothenic acid (15 mg), vitamin B6 (4 mg), vitamin B12 (0.016 mg), folic acid (2 mg), biotin (0.2 mg), manganese (120 mg), zinc (100 mg), iron (40 mg), copper (16 mg), iodine (1.25 mg), selenium (0.3 mg) per 1 kg diet.4 DL-Methionine (Produced by Evonic Co. and contains 99 % methionine).5 Lysine = lysine hydrochloride (contains 70 % Lysine). Table 2: Chemical composition of the diets. Starter diet Grower diet Moisture (%) 9 9 Dry Matter (%) 91 91 Crude protein (%) 23.01 21.20 Crude fat (%) 5.02 5.62 Crude fiber (%) 3.67 3.78 Ash (%) 6.23 5.59 Calcium (%)* 1.07 0.95 Available Phosphorus (%)* 0.49 0.45 Lysine (%)* 1.28 1.20 Methionine (%)* 0.56 0.52 Nitrogen free extract (%) ** 53.07 54.81 ME (Kcal/kg) * 3043 3115 Calorie/protein ratio *** 132.3 146.9 * ME, lysine, methionine, calcium and phosphorus were calculated according to (17).** NFE= Nitrogen free extract was calculated by difference =100- (moisture% + CP% + EE % + CF% + ash %).*** Calorie/protein ratio = ME Kcal /CP Table 3: Sequences of primers used in qPCR. Gene GenBank no._Sequence_ CAT2 BU492799 Forward CAACTGGAGAAAGAGGTCAGGAA Reverse AATTAGGCCATGAAGCAGACAAG MYOD NM_204214 Forward ATCACCAAATGACCCAAAGC Reverse GGGAACAGGGACTCCCTTCA MYF5 NM_001030363 Forward TGAGGAACGCCATCAGGT Reverse GCGAGTCCGCCATCACAT GAPDH NM_204305 Forward ACATGGCATCCAAGGAGTGAG Reverse GGGGAGACAGAAGGGAACAGA L-carnitine supplementation on growth of broiler chichen through determination of changes in the expression ... 669 Table 4: Effect of L- carnitine supplementation on performance parameters Control L. carnitine Initial weight (g) 40.20±1.8 40.8±1.5 Final weight (g) 1689±3.5 b 1731±10.1 a Body weight gain (g) 1648±2.6 b 1690±8.3 a Average daily gain (g) 47.11±0.1 b 48.29±0.29 a Cumulative feed intake/bird 3297.9±28.6 3235.9±25.2 FCR 2.0±0.01 a 1.91±0.01 b Mean±SE (n=60/group) a,b Means in the same raw with different superscript are significantly different (P<0.05) Table 5: Effect of L- carnitine supplementation on serum biochemical parameters Control L- Carnitine Glucose (mg/dl) 158±12.14 160.8±5.46 Cholesterol (mg/dl) 530±53.5 539.2±74 Triacylglycerol (mg/dl) 48.2±3.67 39.63±7.10 Uric acid (mg/dl) 3.32±0.30 3.2±0.4 Total protein(g/dl) 4.34±1.74 4.762±0.56 ALT(U/I) 24.5±6.11 16.25±4.95 AST(U/I) 347±18.52 247±15.55 Mean±SE (n=18/group) ALT (alanine transaminase), AST (Aspartate transaminase) CAT2 MYOD .2 51 fl fl S 3. ai 3 Eg <( Z 2 n « MYF5 .2 6' " A U 4- <( Z Pi 2- Figure 1: Quantitative real-time PCR analysis of the levels of expression of genes (A) cationic amino acid transporter (CAT 2),(B) myogenic determining factor (MYOD), (C) myogenic factor 5 ( MYF5) *P <0.05 versus control group (n=18/group) g 1 D 670 T. Kamal, D. Dorghamm, K. Kahilo, A. Elkattawy, E. Nassef, H. El-sawy Discussion L-carnitine supplementation has a positive effect on growth rate as revealed by higher growth rate, body weight gain and lower feed consumption or feed conversion rate. These result agree with different studies which reported that, addition of L-carnitine enhance growth rates and feed potency (23). The breast or thigh meat yield were increased, and abdominal fat content was decreased by supplementation of L-carnitine (10, 23). Likewise, L-carnitine incorporation to broiler feed enhanced body gain, improved FCR , yielded a lot of breast meat, and lowered abdominal fat depositions (13). Increased growth by addition of carnitine is also partly related to its amino-acid-sparing function in addition to its role in fat metabolism. Theoretically, the external addition of carnitine can lower the necessity for bio synthesis of carnitine from essential amino acid so stinting essential amino acid for different biological functions (24). L- carnitine can be used as a potential protecting agent against stress induced by high environmental temperatures in boilers (25). In the contrary, some studies documented no change in growth rates or feed potency in L-carnitine-supplementation to broiler chickens (10, 26). Likewise, Leibetseder (27) reported that performance (BWG and FCR) and the content of abdominal fat of broilers weren't affected by dietary carnitine at a concentration of 200 mg/kg diet. These contradictory results may be attributed to variation in species, age, supplementary L-carnitine doses, dietary fat and protein intake. This clarification confirmed by Xu (10) who reported that L-carnitine at a dose of 25 mg/kg has no effects in daily body weight gain or feed conversion of birds. Whereas addition of L-carnitine at a dose higher than 25 mg/kg considerably exaggerated breast muscle yield and crude fat content of the muscles and lowered abdominal fat content. The obtained results showed that supplementation of L-carnitine have no vital modification in blood glucose level. This is consistent with other publications which docu- mented that orally administration of L-carnitine supplementation for 4 weeks did not effect of insulin sensitivity (28) or secretion (29). This study revealed no significant change of cholesterol by L-carnitine supplementation and non-significant decrease Triacylglycerol, this result not consistent with Maccari (30) who documented that the significant lowered plasma triacylglycerol, cholesterol, phospho-lipids, nonesterified fatty acid and very low density lipoprotein concentrations (VLDL) by supplementation of L-carnitine to rats. Likewise, Amin (31) reported that adding of external L-carnitine made significant lower of serum TG, T-cholesterol. Additionally, L-carnitine makes vital reductions in total serum cholesterol in skeletal muscles of weighty rats (32). These contradictory results may due to variation in species, age, supplementary L-carnitine doses, dietary fat and protein intake. Carnitine metabolism occurs mainly in the liver; therefore it is not surprising that car-nitine metabolism is failed in patients and experimental animals with certain types of chronic liver disease. L-carnitine will have a therapeutic effect on certain type of liver diseases (33). Our study revealed insignificant decrease of AST, ALT , total protein. Amin (31) reported that administration of L-carnitine make a significant lowering influence in AST and ALT activity in obese rats. Malaguarnera (34) also reported that oral L-carnitine supplementation improve liver functions and histological findings in patients with nonalcoholic steatohepatitis. The present study revealed insignificant change in uric acid, this finding agrees with Rajasekar (35) who reported L-carnitine has beneficial effects on renal function. The present study revealed upregulated expression of MYOD, MYF5 by L- carnitine, and this agrees with Bailey (36) who reported that MYOD, MYF5 and variety of MRFs initiate and maintain the expression of muscle-specific genes throughout embryogenesis and postnatal muscle growth. In rats, the loss of MRFs cause reduction in body size (37). This finding confirms that the expression of MYOD, L-carnitine supplementation on growth of broiler chichen through determination of changes in the expression ... 671 MYF is important for increase body weight. CATs are the basic amino acid transport system utilized by tissues to concentrate lysine, arginine and ornithine into cellular amino acid pools for use in nitrogen metabolism (16), is harmonious with our finding that revealed upregulation of CAT2 expression and this associated with increased body weight and growth. Conclusion The present study concluded that the supplementation of L-carnitine has appositive influence on growth performance through up-regulation of CAT2, MYOD and MYF5 genes. Conflict of interest The authors declare that they have no conflict of interest. References 1. Qureshi A. Selecting the best feeding and watering equipment, Misset World Poultry 1991; 7: 17-9. 2. El-Magd M A, Saleh A A, Abdel-Hamid T M, Saleh R M, Afifi M A. Is really endogenous ghrelin a hunger signal in chickens?: Association of GHSR SNPs with increase appetite, growth traits, expression and serum level of GHRL, and GH, General and Comparative Endocrinology 2016; 237(C): 131-9. 3. El Nagar A, Ibrahim A, Case study of the Egyptian poultry sector, Proceedings of the International Poultry Conference, 2007. 4. Khoshkhoo P H, Azad G A, Ila N, Moayer F, Nayeri H D, Effect of dietary L-carnitine supplementation on overall performance, carcass traits, serum components and immune response in broiler chicken, Verona, Italy: In: EPC European Poultry Conference, 2006, p. 167. 5. Arslan C. L-carnitine and its use as a feed additive in poultry feeding a review, Revue de médecine vétérinaire 2006; 157(3): 134. 6. Michalczuk M, Lukasiewicz M, Niemiec J, Wnuk A, Mroczek-Sosnowska N. Effect of L-carnitine on performance and dressing percentage of broiler chickens, Annals of Warsaw University of Life Sciences 2012; 51: 89-99. 7. Adabi S G, Cooper R, Ceylan N, Corduk M. L-carnitine and its functional effects in poultry nutrition., World's poultry science journal 2011; 67(2): 277-96. 8. Neuman S, Lin T, Heste P. The effect of dietary carnitine on semen traits of white Leghorn roosters, Poultry Science 2002; 81(4): 495-503. 9. Corduk M, Ceylan N, Ildiz F. Effects of dietary energy density and L-carnitine supplementation on growth performance, carcass traits and blood parameters of broiler chickens, South African Journal of Animal Science 2007; 37(2): 65-73. 10. Xu Z, Wang M, Mao H, Zhan X, Hu C. Effects of L-carnitine on growth performance, carcass composition, and metabolism of lipids in male broilers, Poultry Science 2003; 82(3): 40813. 11. Matalliotakis I, Koumantaki Y, Evageliou A, Matalliotakis G, Goumenou A, Koumantakis E. L-carnitine levels in the seminal plasma of fertile and infertile men: correlation with sperm quality, International journal of fertility and women's medicine 2000; 45(3): 236-40. 12. Buyse J, Janssens G, Decuypere E. The effects of dietary L-carnitine supplementation on the performance, organ weights and circulating hormone and metabolite concentrations of broiler chickens reared under a normal or low temperature schedule, British Poultry Science 2001; 42(2): 230-41. 13. Rodehutscord M, Abel H, Friedt W, Wenk C, Flachowsky G, Ahlgrimm H-J, Johnke B, Kühl R, Breves G. Consequences of the ban of byproducts from terrestrial animals in livestock feeding in Germany and the European Union: alternatives, nutrient and energy cycles, plant production, and economic aspects, Archives of Animal Nutrition 2002; 56(2): 67-91. 14. Rudnicki M A, Schnegelsberg P N, Stead R H, Braun T, Arnold H-H, Jaenisch R. MyoD or Myf-5 is required for the formation of skeletal muscle, Cell 1993; 75(7): 1351-9. 15. Day K, Paterson B, Yablonka-Reuveni Z. A distinct profile of myogenic regulatory factor detection within Pax7+ cells at S phase supports a unique role of Myf5 during posthatch chicken myogenesis, Dev Dyn 2009; 238(4): 1001-9. 16. Bröer S. Adaptation of plasma membrane amino acid transport mechanisms to physiological demands, Pflügers Archiv 2002; 444(4): 457-466. 17. NRC., Nutrient requirements of poultry, National Research Council. National Academy Press Washington eUSA USA, 1994. 18. Grandjean D, Valette J, Jouglin M, Gabillard C, Bacque H, Bene M, Guillaud J. Dietary supplementation with L carnitine, vitamin C and vitamin B12 in sport dogs. Experimental 672 T. Kamal, D. Dorghamm, K. Kahilo, A. Elkattawy, E. Nassef, H. El-sawy study with sledge dogs [cyanocobalamine], Recueil de Medecine Veterinaire (France) 1993. 19. Tietz N W, Clinical guide to laboratory tests, WB Saunders Co1995. 20. Gornall A G, Bardawill C J, David M M. Determination of serum proteins by means of the biuret reaction, Journal of biological chemistry 1949; 177(2): 751-66. 21. Elkeiy M, Khamis A, El-Gamal M, Abo Gazia M, Zalat Z, El-Magd M. Chitosan nanoparticles from Artemia salina inhibit progression of hepatocellular carcinoma in vitro and in vivo, Environ Sci Pollut Res Int. doi: 10.1007/s11356-018-3339-6 2018. 22. Yuan J S, Reed A, Chen F, Stewart C N. Statistical analysis of real-time PCR data, BMC bioinformatics 2006; 7(1): 85. 23. Rabie M H, Szilágyi M. Effects of L-carnitine supplementation of diets differing in energy levels on performance, abdominal fat content, and yield and composition of edible meat of broilers, British Journal of Nutrition 1998; 80(4): 391-400. 24. LaCount D, Drackley J, Weigel D. Responses of Dairy Cows During Early Lactation to Ruminal or Abomasal Administration of L-Carnitine1, Journal of Dairy Science 1995; 78(8): 1824-36. 25. Celik L, Oztürkcan O. Effects of dietary supplemental L-carnitine and ascorbic acid on performance, carcass composition and plasma L-carnitine concentration of broiler chicks reared under different temperature, Archives of Animal Nutrition 2003; 57(1): 27-38. 26. Lien T, Horng Y. The effect of supplementary dietary L-carnitine on the growth performance, serum components, carcase traits and enzyme activities in relation to fatty acid P-oxidation of broiler chickens, British Poultry Science 2001; 42(1): 92-5. 27. Leibetseder J. [Effects of L-carnitine in poultry], Arch Tierernahr 1995; 48(1-2): 97-108. 28. González-Ortiz M, Hernández-González S O, Hernández-Salazar E, Martínez-Abundis E. Effect of oral L-carnitine administration on insulin sensitivity and lipid profile in type 2 diabetes mellitus patients, Annals of Nutrition and Metabolism 2008; 52(4): 335-8. 29. Musser R, Goodband R, Tokach M, Owen K, Nelssen J, Blum S, Dritz S, Civis C. Effects of L-carnitine fed during gestation and lactation on sow and litter performance, Journal of Animal Science 1999; 77(12): 3289-95. 30. Maccari F, Arseni A, Chiodi P, Ramacci M, Angelucci L, Hulsmann W. L-carnitine effect on plasma lipoproteins of hyperlipidemic fat-loaded rats, Lipids 1987; 22(12): 1005-8. 31. Amin K A, Nagy M A. Effect of Carnitine and herbal mixture extract on obesity induced by high fat diet in rats, Diabetology & metabolic syndrome 2009; 1(1): 17. 32. Rajasekar P, Anuradha C V. Effect of L-carnitine on skeletal muscle lipids and oxidative stress in rats fed high-fructose diet, Journal of Diabetes Research 2007; 2007. 33. Krahenbuhl S. Carnitine metabolism in chronic liver disease, Life sciences 1996; 59(19): 1579-99. 34. Malaguarnera M, Gargante M P, Russo C, Antic T, Vacante M, Malaguarnera M, Avitabile T, Volti G L, Galvano F. L-carnitine supplementation to diet: a new tool in treatment of nonalcoholic steatohepatitis—a randomized and controlled clinical trial, The American journal of gastroenterology 2010; 105(6): 1338. 35. Rajasekar P, Viswanathan P, Anuradha C. Renoprotective action of l-carnitine in fructose-induced metabolic syndrome, Diabetes, Obesity and Metabolism 2008; 10(2): 171-80. 36. Bailey P, Holowacz T, Lassar A B. The origin of skeletal muscle stem cells in the embryo and the adult, Current opinion in cell biology 2001; 13(6): 679-689. 37. Knapp J R, Davie J K, Myer A, Meadows E, Olson E N, Klein W H. Loss of myogenin in postnatal life leads to normal skeletal muscle but reduced body size, Development 2006; 133(4): 601-10. Slov Vet Res 2019; 56 (Suppl 22): 673-9 DOI 10.26873/SVR-806-2019 Original Research Article PROTECTIVE EFFECT OF GLYCYRRHIZC ACID AGAINST CARBON TETRACHLORIDE-INDUCED LIVER FIBROSIS IN RATS: ROLE OF INTEGRIN SUBUNIT p LIKE 1 (ITG B L1) Amal Ragab, Nasr Elsayed Nasr*, Khalid Kahilo Biochemistry Department, Faculty of Veterinary Medicine Kafrelsheikh University Corresponding author, E-mail: nasr_157@yahoo.com Abstract: Glycyrrhizc acid (GA) is one of the herbal plants with a proved hepato-protec-tive effect. The current study was carried out to estimate the hepato-protective effect of GA against liver fibrosis and to disclose its mode of action. Thirty two male albino rats were randomly distributed into 4 groups (n=8), i.e., control group, GA group, CCl4 group and CCl4 + GA group. Liver enzymes alanine aminotransferase (ALT), aspartate aminotransferase (AST), gamma-glutamyl transferase (GGT), liver histopathology, malondialdehyde (MDA), superoxide dismutase (SOD), catalase (CT) and gene expression of integrin subunit p like 1 (ITGBL1) were analyzed. The obtained data revealed that GA remarkably protected CCl4-induced liver injury as reflected by reduced AST, ALT, GGT, and fibrosis compared with the CCl4-only group. Moreover, gA significantly reduced the levels of MDA, as well as increased the activity of SOD and CT. Consequently, GA prevents CCl4-induced fibrosis in rats. The prophylactic action of GA against liver fibrosis was mediated through its antioxidant and anti-inflammatory activities. Additionally, GA downregulated fibrogenic ITGBL1. Key words: glycyrrhizc acid; ITGBL1; liver fibrosis; integrins Introduction Liver fibrotic diseases are generally resulted from chronic liver injury, leading to chronic inflammation and fibrosis with obliteration of the normal hepatic tissue construction and eventually loss of liver function (1). As a result of chronic liver diseases, cirrhosis is the sequelae of advanced liver fibrosis. Recently, it is reported that cirrhosis could be reversible in its early stages if the underlying liver diseases have been properly treated (2). However, advanced stages of cirrhosis are considered to be irreversible. Cirrhosis is believed to be the main source of a variety of serious complications, which lead to highly fatal systemic disorders (3). Therefore, cirrhosis the 8th deadliest disease and is responsible for high percent of mortality worldwide. Worryingly, the prevalence of cirrhosis is increasing in numerous nations, including well developed ones (4). Chronic liver diseases such as alcoholic liver disease, HCV, HBV, haemochromatosis and non-alcoholic fatty liver disease (NAFLD) are the most frequent causes of cirrhosis worldwide. Despite their lower incidence rate, a wide range of other diseases can end with cirrhosis (5). Unfortunately, there is a shortage of an effective remedy for liver fibrosis to date. Therefore, there Received: January 2019 Accepted for publication: February 2019 674 A. Ragab, N. Nasr, K. Kahilo is an urgent requisite for potent anti-fibrotic remedies. Fibrogenesis is a highly complicated process employing a variety of different cells, including hepatocytes, hepatic stellate cells (HSCs) and immunocytes. The key cellular elements in development of liver fibrosis is the activation of HSC and their trans-differentiation into myofi-broblast-like cells (6). HSC activation is mainly initiated by growth factors and fibrogenic cyto-kines released by activated epithelial cells (including hepatocytes and cholangiocytes) and inflammatory cells, which among them the TGFP 1 is the key regulator (7). Wide range genes take part in fibrogenesis through controlling the TGFP signaling pathway, among which integrins have crucial regulatory role. Integrins are cellular receptors that consist of an a and a P subunit and form at least 24 different dimers that mediate cell-cell and cell-ECM interactions (8). Moreover, integrins also response to ECM-induced extracellular changes during pathological processes, initiating cellular responses, which manipulate ECM alteration (9). It has previously showed that integrins manage pivotal roles in fibrogenesis (7). Throughout biliary fibrosis development, integ-rin avP6 is highly upregulated in cholangio-cytes and stimulate fibrogenesis through TGFP 1 activation (10). Several genes and signaling pathways highly connected to fibrotic progression were discovered, among which integrin subunit P like 1 (ITGBL1) was recognized as a key factor (11). The pathological pathway of ITGBL1 were clearly demonstrated by using in vitro experiments, which revealed that ITGBL1 encourages HSC activation and the subsequent liver fibrosis by upregulating TGFP1. These observations secure essential base for further research on liver fibrosis which may propose the discovery of new antifibrotic agents. Through ancient eras, medicinal herbs have long been prescribed to prevent and treat liver diseases and have recently gained wider attention due to their availability, long term effectiv-ity and benign side effects (12). Generally, hepatoprotective effects of medicinal herbs is conducted via mechanisms including hindering fibrogenesis, defeating tumorigenesis, eradicating viruses, and suppressing oxidative tissue damage (13). Glycyrrhizae Radix et Rhizoma, also known as licorice root, is commonly consumed to treat viral hepatitis (14). Licorice root main constituents include glycyrrhizc acid (GA), P-sitosterol, flavonoids, and hydroxycou-marins. GA improves CCL4-induced liver damages by down-regulating proinflammatory mediators (15), as well as its antioxidative action via upregulation of catalase and glutathi-one-S-transferases (16). The current study aimed to define the underlying mechanism of anti-fibrotic action of GA on the sub-molecular level by investigating the effects of GA on ITGBL1 binding activity, one of the major controllers of fibrosis, in the CCl4 rat model of liver cirrhosis. Materials and methods Animals and ethics statement This research was approved by the Ethics Committee of faculty of veterinary medicine, Kafrelsheikh University. Experimental design Thirty male albino rats weighing 180-200 g were recruited after 7 days for adaptation to the animal house circumstances (12-hour light/dark cycle). Water and food were supplied ad libitum. The rats were arbitrarily distributed to 4 groups: the control group (n = 8), the CCl4 group (n = 8), the CCl4 + GA (n = 8) and the GA 150 mg/kg BW group (n = 8). The liver fibrosis was induced by intraperitoneal (i.p.) injection with CCl4 mixed with olive oil as vehicle in 1:1 ratio (0.2 mL/100 g BW) twice weekly for 2 weeks followed by i.p. injection of reduced dose (0.1 mL/100 g BW) twice weekly for 6 weeks as described by Constandinou (17). GA was given by oral gavage once daily for 8 weeks. Three days after the last CCl4 injection, rats were sacrificed, and blood samples were taken in the plain tubes and EDTA tubes. Samples in plain tubes were left to clot then centrifuged at 3,000 g, 4°C for 15 min, to separate serum. The serum samples were stored at -20°C until analyzed. The liver was immediately excised from Protective effect of glycyrrhizc acid against carbon tetrachloride-induced liver fibrosis in rats: 675 each animal, washed by saline and divided to 2 parts: one part was prepared for histopatholog-ical examination, while the other part was preserved frozen at -80°C for both oxidative stress and genetic analysis. Biochemical analysis Activities of liver enzymes ALT, AST, GGT, levels of total protein, albumin and total bilirubin in plasma were measured using commercial kits (Spinreact, Spain) according to the manufacturer's directions. Hepatic SOD, CT and MDA were determined in the hepatic tissue homogenate using (Biodiagnostic, Egypt) kits following the manufacturer's guideline and as previously described (18, 19). RNA extraction and real time PCR RNA was extracted out by using Trizol (Invitrogen Co., Carlsbad, CA, USA) and real time PCR performed following the manufacturer's instructions using M-MLV reverse transcriptase (Takara Shuzo Co., Ltd, Japan) and real time PCR Master Mix (SYBR Green) Kit (Toyobo Co., Ltd. Japan). The sequences of ITGBL1 primers were forward 5'TTTGTGA-GAAAGGATGGTTTGGT3' and reverse 5'TGCTTTGTTCTTCGGTCATATTA CA3'. GAPDH was used as an internal control. The PCR conditions were 95°C for 10 min, and then 40 cycles of 95°C for 20 s, 54°C for 30 s and 72°C for 30s. Each experiment was carried out thrice in triplicate. The fold-change in mRNA of target gene relative to that of GAPDH was calculated according to previously described (20). Histopathological examination Sections of liver tissue 3 pm thickness were obtained from each animal under investigation and fixed in 10 % neutral buffered formalin, then dehydrated in ascending concentration of ethyl alcohol (70: 100%) followed by staining according to standard protocol of Hematoxylin an Eosin stain (H&E) as described by Bancroft e al., (21) Statistical Analysis Statistical analysis was carried out using Graphpad prismV5 software package. Results were obtained as means ± standard deviations (SD). Statistical analysis was performed using one way analysis of variance (ANOVA) comparisons. Values showing p <0.05 was considered as statistically significant. Results Biochemical parameters Serum ALT, AST, and GGT activities were determined as indicators of liver damage. As displayed in table 1, significant high levels in the activities of these marker enzymes were recorded in CCl4-intoxicated rats. In contrast, GA supplement significantly reversed those enzyme activities. Histopathological Findings The liver of control animal (Fig.1A) showed normal hexagonal shape lobule with centrally located blood vessels (central vein) while the peripheral area revealed normal portal areas which contained hepatic artery , portal vein and bile duct. Hepatocytes (large round to polygonal cell with eosinophilic cytoplasm and vesicular nucleus) mostly arranged in radiating manner around the central vein. Similarly, animal treated with GA were with normal limits (Fig.1B). Animals treated with CCU (Fig.1C) showed remarkable hepatic distortion associated with centro-lobular hepatic vacuolation and necrosis. Obvious periportal hepatic fibro-sis accompanied with noticeable periportal, interlobular and intralobular proliferating fibrous connective tissue (arrowhead) that given the nodular appearance of the hepatic tissue. While diseased animal treated with GA showed distinct decrease of hepatic necrosis and fibrosis (Fig.1D). Effect of GA treatment on ITGB1 expression The obtained qPCR results revealed a significant (P<0.05) upregulation of the fibrosis marker ITGB1 gene in liver of CCl4-intoxicated rats as compared to the control group (Table 1). This elevated expression was significantly downregulated following treatment by GA, but still higher than that in the control group. Additionally, no significant difference was noticed between the two control groups (G1, G2). 676 A. Ragab, N. Nasr, K. Kahilo Table 1: Effect of GA on biochemical parameters. Normal GA CCl4 GA + CCl4 AST (IU\L) 62.8 ± 3.58 c 64.63 ± 3.18 c 346.16 ± 11.15a 222.20 ± 8.93 b ALT (IU\L) 59.7 ± 1.82 c 63.10 ± 3.95 c 253.23 ± 22.65 a 118.31 ± 7.56 b GGT( IU \ L) 67.5 ± 1.77 c 67.44 ± 3.96 c 114.26 ± 4.54 a 90.18 ± 3.26 b Total protein (g\dl ) 6.54 ± 0.12 a 6.01 ± 0.16 a 3.03 ± 0.24 c 4.41 ± 0.17 b Albumin (g\dl ) 4.27 ± 0.20 a 4.02 ± 0.26 a 2.20 ± 0.20 c 2.92 ± 0.10 b Total bilirubin (mg\dl ) 0.91 ± 0.06 c 0.90 ± 0.06 c 1.56 ± 0.05 a 1.15 ± 0.04 b SOD (u\g tissue) 13.03 ± 0.57 a 13.16 ± 0.52 a 4.56 ± 0.46 c 7.19 ± 0.33 b Catalase (u\g tissue) 41.43 ± 1.94 a 39.78 ± 1.65 a 20.25 ± 0.92 c 27.14 ± 0.93 b MDA (nmol\g tissue) 4.00 ± 0.36 c 4.40 ± 0.49 c 10.51 ± 0.63 a 7.84 ± 0.56 b ITGB1 gene (fold change) 1.00 ±0.09 c 1.51±0.24 c 11.16 ± 0.43 a 5.78 ± 0.29 b Data were presented as mean ± SEM. Means carrying different superscript letters are significantly different at P<0.05. Figure 1: Liver sections of control (A), GA (B), CCL4 (C), and GA+CCL4 (D) groups. H&E, X200 Discussion Inflammation is considered the native defense system of the body against harmful factors with playing a vital role in healing the potential injury. Consistently, liver is highly related to inflammation being highly vascular and vulnerable to serious hepatic injurious factors including toxin metabolites, viruses, fat rich diet and excessive alcohol intake. Prolonged exposure to those agents leads to chronic hepatitis accompanied by fibrosis and subsequent cirrhosis with loss of liver function (22). Accordingly, liver fibrosis in the current study was induced by repeated doses of CCU which is the most popular procedure between the liver research laboratories. However, it differs from one laboratory to another in terms of CCU dose, route of administration, treatment duration and the expected changes to be studied. Protective effect of glycyrrhizc acid against carbon tetrachloride-induced liver fibrosis in rats: 677 Communally, CCl4 mediated liver fibrosis proceeds with elevated serum AST and triglycerides along with liver atrophy (23). Furthermore, these alterations are accompanied by a significant low value of serum albumin indicating advanced loss of hepatic function during extended fibrogenesis. These observations appear consistent with our recorded albumin levels in CCl4 intoxicated group supported by histo-pathological lesions. Cytochrome P450 superfamily of monooxy-genases process CCl4 to the trichloromethyl radical (CCb)(24). Subsequently, this radical damages the key cellular metabolic pathways resulting in altered lipid metabolism (fatty degeneration and steatosis) and decreased protein quantities. Moreover, CCb interacts with hepatocytic DNA leading to mutations and the development of HCC. Further oxygenation of CCl3 results in the formation of trichloro-methylperoxy radicals (CCl3OO*) initiating li-pid peroxidation by breakdown of polyunsatu-rated fatty acids with reduction of membrane permeability of the plasma membrane extended to mitochondrial and endoplasmic reticulum membrane ended by cellular death. The cellular death eventually develops as zonal or focal necrosis with destruction of normal hepatic tissue construction (22). Biochemically, this severe OS leads to exhaustion of antioxidant activity of SOD and release of high levels of MDA in agreement with our results of the same group. Inappropriately, Fibrosis develops as a healing course in response to inflammation and OS (25) , and can finally progress into HCC (26). Prolonged stimuli of liver injury leads to failure of the regenerative response and substitution of hepatocytes with massive ECM (27) formed mainly by Hepatic stellate cells (HSC) (28). Interestingly, integrins direct the development of fibrosis regulating inflammation, and by transforming hepatocytes injury into stimulus of matrix-producing mesenchymal cells [HSC/myofibroblasts (MFB)]. The expression of integrins by wide range of cells engaged in liver fibrosis course, as well as their ability to interact with growth factors and other signaling molecules render the concept of targeting integrins an interesting tactic for antifibrotic therapy. There is no typical treatment plan for hepatic fibrosis, however prophylaxis against liver injury, including minimizing of fat consumption and toxin exposure or administration of an efficient viral hepatitis treatment can resist fibrosis. Surprisingly, no efficient anti-fi-brotic drugs have yet to be developed although substantial progress has been made in exploring the pathogenesis of hepatic fibrosis over the last two decades. Medicinal herbs and their bioac-tive ingredients and extracts could prevent liver fibrosis by two means: through suppression of HSC activity and via inhibition of ECM expansion. HSCs activation are initiated when gene expression and phenotype changes render the inactive cells responsive to other cytokines and stimuli (29). Oral traditional Chinese herbal medicine has long been used as a non-invasive therapy. The therapeutic mechanisms of herbal medicines and their active compounds have been gradually uncovered and interpreted through in vivo studies. Recent studies have provided a greater understanding of the molecular mechanisms and new therapeutic approaches for liver fibro-sis, but it still requires an efficacious remedy. We believe that herbal medicines are sufficiently worthy as potential therapy agents for liver fibrosis if more profound studies about the underlying mechanisms of herbal medicines with improved methodological quality are undertaken. GA can prevent CCl4- related liver fibrosis effectively. This is evidenced in restoring the nearly normal hepatic lobule in GA+CCU animals. It could happen by less HSC proliferation, thus reduced levels of collagen, hyaluronic acid (HA), and laminin (LN). In an animal model, GA reduced the death rate of acetaminophen intoxicated mice via inhibition of acetaminophen-induced hepatotoxicity, and reduced the number and area of GGT positive foci, thus reserving liver function and preventing HCC from development (30). GA showed a highly effective chemopreventive action agent against lead acetate hepatic induced oxidative stress in rats because it chelates lead (31), which support the current data obtained from improved oxidative stress markers in the sera of the same group. In 678 A. Ragab, N. Nasr, K. Kahilo the molecular level, GA supplement accompanied by suppression of the profibrotic gene ITGBL1, which has a crucial role in management of ECM and the activity of HSC. Up to the published data, it is the first time uncover the role of GA in correlation to integrins during its anti-fibrotic effect. However in concanavalin A- (ConA-) mediated mouse model, GA attenuated ConA-induced hepatitis and fibrosis pgrowth in livers via supression of CD4+ T cell proliferation in response to ConA via the Jun N-terminal kinase (JNK), extracellular signal-regulated kinase (ERK), and phosphoinositide 3-kinase (PI3K)/AKT pathways(32). Conclusion Wide attention should be given to integrins as they are infirmly connected to the development of liver fibrosis. Integrins targeting therefore represents an interesting concept of therapeutic strategy, particularly because experimental data recommend potent efficiency of such trend. However, more investigations are needed to identify potentials to specifically antagonize integrins (including specific integrin antagonists, or small nonpeptidic molecules) to stop or reverse fibrosis and avoid detrimental effect of such inhibition. References 1. Anthony P P, Ishak K G, Nayak N C, Poulsen H E, Scheuer P J, Sobin L H. The morphology of cirrhosis. Recommendations on definition, nomenclature, and classification by a working group sponsored by the World Health Organization, J Clin Pathol 1978; 31(5): 395-414. 2. Buti M, Fung S, Gane E, Afdhal N H, Flisiak R, Gurel S, Flaherty J F, Martins E B, Yee L J, Dinh P, Bornstein J D, Mani Subramanian G, Janssen H L, George J, Marcellin P. Long-term clinical outcomes in cirrhotic chronic hepatitis B patients treated with tenofovir disoproxil fumarate for up to 5 years, Hepatol Int 2015; 9(2): 243-50. 3. Blachier M, Leleu H, Peck-Radosavljevic M, Valla D C, Roudot-Thoraval F. The burden of liver disease in Europe: a review of available epidemiological data, J Hepatol 2013; 58(3): 593608. 4. Disease G B D, Injury I, Prevalence C. Global, regional, and national incidence, prevalence, and years lived with disability for 328 diseases and injuries for 195 countries, 1990-2016: a systematic analysis for the Global Burden of Disease Study 2016, Lancet 2017; 390(10100): 1211-59. 5. Heidelbaugh J J, Bruderly M. Cirrhosis and chronic liver failure: part I. Diagnosis and evaluation, Am Fam Physician 2006; 74(5): 756-62. 6. Hernandez-Gea V, Friedman S L. Pathogenesis of liver fibrosis, Annu Rev Pathol 2011; 6: 425-56. 7. Pellicoro A, Ramachandran P, Iredale J P, Fallowfield J A. Liver fibrosis and repair: immune regulation of wound healing in a solid organ, Nat Rev Immunol 2014; 14(3): 181-94. 8. Luo B H, Springer T A. Integrin structures and conformational signaling, Curr Opin Cell Biol 2006; 18(5): 579-86. 9. Conroy K P, Kitto L J, Henderson N C. alphav integrins: key regulators of tissue fibrosis, Cell Tissue Res 2016; 365(3): 511-9. 10. Patsenker E, Popov Y, Stickel F, Jonczyk A, Goodman S L, Schuppan D. Inhibition of integrin alphavbeta6 on cholangiocytes blocks transforming growth factor-beta activation and retards biliary fibrosis progression, Gastroenterology 2008; 135(2): 660-70. 11. Wang M, Gong Q, Zhang J, Chen L, Zhang Z, Lu L, Yu D, Han Y, Zhang D, Chen P, Zhang X, Yuan Z, Huang J, Zhang X. Characterization of gene expression profiles in HBV-related liver fibrosis patients and identification of ITGBL1 as a key regulator of fibrogenesis, Sci Rep 2017; 7: 43446. 12. Elgazar A A, Selim N M, Abdel-Hamid N M, El-Magd M A, El Hefnawy H M. Isolates from Alpinia officinarum Hance attenuate LPS induced inflammation in HepG2: Evidence from In Silico and In Vitro Studies, Phytotherapy Research 2018; 32(7): 1273-88. 13. Del Prete A, Scalera A, Iadevaia M D, Miranda A, Zulli C, Gaeta L, Tuccillo C, Federico A, Loguercio C. Herbal products: benefits, limits, and applications in chronic liver disease, Evid Based Complement Alternat Med 2012; 2012: 837939. 14. Hanje A J, Fortune B, Song M, Hill D, McClain C. The use of selected nutrition supplements and complementary and alternative medicine in liver disease, Nutr Clin Pract 2006; 21(3): 255-72. 15. Feng C, Wang H, Yao C, Zhang J, Tian Z. Diammonium glycyrrhizinate, a component of traditional Chinese medicine Gan-Cao, prevents murine T-cell-mediated fulminant hepatitis in IL- Protective effect of glycyrrhizc acid against carbon tetrachloride-induced liver fibrosis in rats: 679 10- and IL-6-dependent manners, Int Immunopharmacol 2007; 7(10): 1292-8. 16. van Rossum T G, Vulto A G, de Man R A, Brouwer J T, Schalm S W. Review article: glycyrrhizin as a potential treatment for chronic hepatitis C, Aliment Pharmacol Ther 1998; 12(3): 199-205. 17. Constandinou C, Henderson N, Iredale J P. Modeling liver fibrosis in rodents, Methods Mol Med 2005; 117: 237-50. 18. Alzahrani F A, El-Magd M A, Abdelfattah-Hassan A, Saleh A A, Saadeldin I M, El-Shetry E S, Badawy A A, Alkarim S. Potential Effect of Exosomes Derived from Cancer Stem Cells and MSCs on Progression of DEN-Induced HCC in Rats, Stem Cells International 2018; 2018: 17 https://doi.org/10.1155/2018/8058979. 19. El-Bayomi K M, Saleh A A, Awad A, El-Tarabany M S, El-Qaliouby H S, Afifi M, El-Komy S, Essawi W M, Almadaly E A, El-Magd M A. Association of CYP19A1 gene polymorphisms with anoestrus in water buffaloes, Reproduction, Fertility and Development 2018; 30(3): 487-497. 20. Abd-Allah S H, Shalaby S M, Abd-Elbary E, Saleh A A, El-Magd M A. Human peripheral blood CD34+ cells attenuate oleic acid-induced acute lung injury in rats, Cytotherapy 2015; 17(4): 443-53. 21. Bancroft J D, Stevens A, Turner D R, Theory and practice of histological techniques, Fourth Edition ed., Churchill Livingstone, New York, 1996. 22. Chatterjee R, Mitra A. An overview of effective therapies and recent advances in biomarkers for chronic liver diseases and associated liver cancer, Int Immunopharmacol 2015; 24(2): 335-345. 23. Paquet K J, Kamphausen U. The carbon-tetrachloride-hepatotoxicity as a model of liver damage. First report: Long-time biochemical changes, Acta Hepatogastroenterol (Stuttg) 1975; 22(2): 84-8. 24. Li X X, Zheng Q C, Wang Y, Zhang H X. Theoretical insights into the reductive metabolism of CC14 by cytochrome P450 enzymes and the CC14-dependent suicidal inactivation of P450, Dalton Trans 2014; 43(39): 14833-40. 25. Chen P J, Huang C, Meng X M, Li J. Epigenetic modifications by histone deacetylases: Biological implications and therapeutic potential in liver fibrosis, Biochimie 2015; 116: 61-9. 26. Santos J C, Valentim I B, de Araujo O R, Ataide Tda R, Goulart M O. Development of nonalcoholic hepatopathy: contributions of oxidative stress and advanced glycation end products, Int J Mol Sci 2013; 14(10): 19846-66. 27. Elpek G O. Cellular and molecular mechanisms in the pathogenesis of liver fibrosis: An update, World J Gastroenterol 2014; 20(23): 726076. 28. Reeves H L, Friedman S L. Activation of hepatic stellate cells--a key issue in liver fibrosis, Front Biosci 2002; 7: d808-26. 29. Friedman S L. Mechanisms of hepatic fibrogenesis, Gastroenterology 2008; 134(6): 165569. 30. Wan X Y, Luo M, Li X D, He P. Hepatoprotective and anti-hepatocarcinogenic effects of glycyrrhizin and matrine, Chem Biol Interact 2009; 181(1): 15-9. 31. Rahman S, Sultana S. Chemopreventive activity of glycyrrhizin on lead acetate mediated hepatic oxidative stress and its hyperproliferative activity in Wistar rats, Chem Biol Interact 2006; 160(1): 61-9. 32. Tu C T, Li J, Wang F P, Li L, Wang J Y, Jiang W. Glycyrrhizin regulates CD4+T cell response during liver fibrogenesis via JNK, ERK and PI3K/AKT pathway, Int Immuno-pharmacol 2012; 14(4): 410-21. Slov Vet Res 2019; 56 (Suppl 22): 681-91 DOI 10.26873/SVR-807-2019 Original Research Article HEMOTOXIC AND GENOTOXIC EFFECTS OF LEAD ACETATE AND CHLORPYRIFOSE ON FRESHWATER CAT FISH (Calarias gariepinus) Shireen S. Abd El-satar1, Nasr E. Nasr2*, Khaled A. Khailo2, Hossam E. Sayour3 1Biochemistry Department, Animal Health Research Institute, Kafrelsheikh, Egypt, 2Biochemistry Department, Faculty of Veterinary Medicine, Kafrelsheikh University, Egypt, 3Biomedical Chemistry Unit, Department of chemistry, Animal Health Research Institute, Dokki, Giza, Egypt Corresponding author, E-mail: nasr_157@yahoo.com Abstract: This study was conducted to evaluate hemotoxicity and genotoxicity induced by lead acetate and chlorpyrifos in catfish (Clarias gariepinus) as a model for checking genotoxic pollutants in aquatic surroundings. Lead acetate was added by a dose of 24.4 mg/L (20% of the LC50) daily, chlorpyrifos was added by a dose of 1.65 mg/L (1/10 LC50) daily. Fish were kept in standard condition in which water temperature (25°C±2); pH (7.6 ±0.4) and dissolved oxygen (5.4±0.4 mg/L). Blood and liver were sampled after 4 weeks. The result revealed a significant reduction of RBCs count, Hb, hematocrit in fish exposed to lead and chlorpyrifos. Furthermore, MDA level and catalase activity showed significant increase and decrease, respectively in groups exposed to lead acetate and chlorpyrifos in comparison with the control fish. Toxic effect of lead acetate and chlorpyrifos confirmed by histopathological changes in liver sections which showed marked hepatic vacuolation and parenchymal hemorrhage. DNA damage detected by comet assay also revealed a significance increase in tail length, tail DNA% and tail moment at sublethal concentration of lead acetate and chlorpyrifos. This study concluded that lead acetate and chlorpyrifos have hemotoxic and genotoxic effect probably through, at least in part, induction of reactive oxygen species and chlorpyrifos has more hemotoxic and genotoxic effect than lead acetate. Key words: catfish; lead acetate; chlorpyrifos; DNA damage Introduction Pollution of the aquatic surroundings is a serious and growing problem, caused by increasing variety and quantity of industrial, agricultural and business chemicals (1-3). Many water pollutants are capable of inducing oxidative stress on fish (4) which consequently led to cellular and tissues damage in fish (5). Heavy metals and pesticides are poisonous to animals just as individuals (6). Heavy metals are considered as the most important pollutants, because of their notable impacts on the aquatic system equilibrium, bioaccumulation in life forms, long term persistence and ability to aggregate in sediments and water (7). Heavy metals, like mercury, cadmium, copper, lead and zinc are the most vital poisons which influence aquatic environment and fish. They are extremely unsafe for the health of fish. These metals can ef- Received: January 2019 Accepted for publication: February 2019 682 S. Shireen, E. Nasr, A. Khaled, E. Hossam fectively affect the vital operations and reproduction of fish; weaken the immune system, and lead to pathological changes (8) Lead (Pb) is a prevalent, cumulative and insidious environmental waste product that induces a broad range of physiological, biochemical and behavioral dysfunctions (9-11). With the increasing prevalence of lead pollution in the aquatic surroundings, the potential danger of lead to aquatic animals must be evaluated (12). Studies have demonstrated that lead can induce neurological, gastrointestinal, reproductive, circulatory, immunological, histopatho-logical and histochemical effects in the animals and human (13, 14). One of the best known impacts of lead is its interference with synthesis of heme (15). High Pb concentration in soft tissues inactivate alpha-amino levulinic acid dehydra-tase (ALAD) enzyme which associated with rise of alpha -aminolevulinic acid (ALA) level (16). Increase of ALA level leads to production of reactive oxygen species (17) Pesticides are used to control pests, insects, aquatic weeds, plant diseases, and Aquatic snails which transfer schistosomiasis. Pesticides have been observed to be very harmful not only to fish but also to the other organisms. (18). Widespread of pesticides and their improper use cause changes in the aquatic medium. Observing of different effects is wanted for good pesticides control (19). Two main types of pesticides; organophosphates and orga-nochlorines, cause serious harmful impact on the aquatic system and their residues present in water are toxic to aquatic organisms (20, 21). One such organophosphate, is chlorpyrifos (CPF) [O, O-diethyl-O-(3,5,6 -trichloro-2-pyridyl) phosphorothionate] which is broadly utilized for the management of domestic and agricultural pests. CPF utilized additionally for over 10 years to control foliar insects (arthrop-oda) that influence farming yields, and subterranean termites (22). Oxidative stress which induced by pesticide has been a focal point of tox-icological research for the last decade as a potential mechanism of toxicity (23, 24) . Toxicity of reactive oxygen species counteracted by an-tioxidant enzymes such as catalase (CAT), su- peroxide dismutase (SOD), and glutathione reductase (GR) (25). Antioxidants protect the cells and tissues from oxidative damage under normal conditions (26). The antioxidants in fish could be considered as biomarkers of exposure to aquatic contaminants (27). Exposure to pollutants causes several biochemical and physiological responses which may be adaptive or lead to toxicity. Thus, it is important that pollutant effects be detected and interpreted in biochemical terms, to show mechanisms of pollutant action, and possible ways to prevent there bad effects (28). African catfish (Clarias gariepinus) is very important fish because of its high growth rate, high consumer acceptability and high resistance to bad water quality and low oxygen (29). It is omnivorous feeder and a general scavenger with a marked tendency to feed on benthic organisms and this make it in continuous exposure to different pollutants which concentrated in the sediments. Biomagnification of pollutants throuth eating fish which already accumulate chemicals make this fish accumulate pollutants in greater amounts than other herbivorous fish (30). Catfish considered as an excellent model for toxicological studies and has been used in fundamental research (31, 32). Since fish are important as proteins and li-pids sources for humans and domestic animals, so health of them is very important for human beings (18). Therefore, this study was conducted to indicate the hemotoxic and genotoxic effect of both chlorpyrifose and lead acetate on African catfish Materials and methods Chemicals Lead acetate trihydrate (Mol.Wt. 379.33 g/mol, product No. 316512, purity 95%)and other chemicals used in this study were purchased from (Sigma-Aldrich), chlorpyrifos was obtained as a commercial product (Pyrifos EL NASR 48% EC) from (El Nasr Pharmaceutical Company, Cairo-Egypt). Experimental fish Catfish (Clarias gariepinus) of both sexes, average weight 150 were purchased from fish Hemotoxic and genotoxic effect of lead acetate and chlorpyrifose on freshwater cat fish (Clarias gariepinus) 683 farm in Kafrelsheikh, Egypt. Fish was transported in large plastic water containers and maintained in the glass aquaria in the laboratory of faculty of veterinary medicine, Kafrelsheikh University. Fish left without any treatment for two week for acclimation before starting the experiment. Fish were kept in standard condition during the period of acclimation and the experiment in which water temperature is 25°C± 2, pH is 7.6±0.4 and dissolved oxygen (5.4±0.4 mg/L). Water was supplied with oxygen by air pump, fish kept in 12/12hour light and dark, and were fed on commercial high protein diet (30 %) at ratio of 3% of body weight/day. The water was changed every 2 days to prevent the accumulation of fecal matter and to maintain the pollutants concentration. Experimental design and treatment A total of 120 fresh water catfish were divided into three groups, each group has two replicates (20 fish replicate), kept in (100x50x30 cm) glass aquaria contain 100L tap water (de-chlorinated); the first group (G1) was considered as control group, the second group (G2) was exposed to lead acetate by a dose of 24.4 mg/L (20% of the LC50) (33) and the third group(G3) was exposed to chlorpyrifos by a dose of 1.65 mg/L ( 1/10 LC50) (34), for 4 weeks, the water was changed every 2 days to avoid the accumulation of fecal matter and to maintain the toxicant concentration. Blood and tissue samples By the end of the experimental period at 4th week, whole blood was collected from caudal blood vessels in clean and dry Eppendorf tubes containing EDTA as anticoagulant, other blood samples were collected without anticoagulant and left at room temperature to coagulate then centrifuged at 3000 rpm for 15 min and clear sera were separated and kept in -20 0C until biochemical analysis. Fish were then killed by spinal rupture; the liver was rapidly excised and divided into three parts: the first part was kept in sterile Eppendorf tube, was immediately immersed in liquid nitrogen and then kept in -20 0C for anti-oxidant determination, the second part about (1g) was kept in PBS in sterile plastic tubes then was stored in -20 0C for comet assay analysis and the third part was stored in 10% formalin for histopathological analysis. Hematological analysis Total red blood cell (RBCs 106/pl) counts were determined by the Natt & Herrick (35) method (NH) using a Newbauer hemocytome-ter. The microhematocrit (PCV%) was estimated by the method of Hesser (36). Hemoglobin (Hb g/dl) values were assessed calorimetri-cally according to method of Wintrobe and Greer (37) by determining the formation of cy-anomethemoglobin. Biochemical analysis Malondialdehyde was spectrophotometri-cally measured in serum based on (38) at wave length of 534 nm, catalase activity was determined in liver homogenates , briefly obtained as follow (homogenize 1 gm. of liver tissue in 5 ml of cold phosphate buffer saline PH:7.4 centrifuge at 4,000rpm/ 15 minute at 4oC, then used supernatant for assay) according to Aebi (39). Histopathological analysis Fixed liver samples were dehydrated in ascending grades of ethanol, then embedded in paraffin wax, cut into sections of 5 pm thickness. Sections were stained using Mayer's hematoxylin and eosin (H&E) stains according to the method described by Bancroft (40) and were examined under a light microscope. Hepatic histological changes photographed by computer system with a digital camera (Nikon digital camera, Japan). DNA damage detection Comet assay (single cell gel electrophoresis, SCGE) was used to detect any prospective damage for DNA after treatments. DNA strand breaks and alkali labile sites detected by measuring the migration of damaged DNA from immobilized nuclear DNA. The comet assay was performed according to the protocol described by Singh et al., (41) and the calculations were done as previously described (42). 684 S. Shireen, E. Nasr, A. Khaled, E. Hossam Statistical analysis Data were analyzed by one way (ANOVA), followed by Bonferroni's Multiple Comparison Test to compare the significant differences between different groups. All the data were expressed as mean ±SE. A value of P < 0.05 was considered to be significant. Results Hematological parameters The hematological parameters are listed in Table (1). Exposure to lead acetate and chlorpyrifos led to a significant reduction in RBCs count as compared to the control fish. Furthermore, lead acetate and chlorpyrifos exposed group illustrated a significant decrease in Hb concentration (in comparison with the control group. Also, there was a significant reduction in PCV% in group exposed to lead acetate and chlorpyrifos when compared with the control group. Effect of lead acetate and chlorpyrifos on MDA level and catalase activity Exposure to lead acetate and chlorpyrifos led to a significant increase in serum MDA level and a significant decrease in hepatic catalase activity as compared with the control fish a (Fig. 1). Furthermore, chlorpyrifos led to a higher increased MDA level than lead acetate. Effect of lead acetate and chlorpyrifos on DNA (comet assay) DNA damage in liver of lead acetate, chlorpyrifos-intoxicated fish were detected by comet assay. The results of comet assay were shown in Fig. (2) and Table (2). Fish exposed to lead acetate showed asignificant (P<0.05) increase in DNA damage as revealed by increase in tail length, tail DNA% and tail moment. Fish exposed to chlorpyrifos showed a significant (P<0.05) increase DNA damage with higher damage in chlorpyrifos than lead acetate groups as compared to the control group (G1). Histopathological changes Normal fish (Fig. 3A) showed normal hepatocytes separated with blood sinusoids, while liver of lead acetate -intoxicated fish (Fig. 3B) showed hepatic vacuolation, parenchymal haemorrahge and focal leukocytic infiltration. Furthermore, liver of chlorpyrifos - intoxicated fish (Fig.3C) showed hepatic vacuolation and hepatic necrosis. Table 1: Effect of lead and chlorpyrifos on hematological parameters in cat fish Groups RBCs(106/pl) PCV% Hb(g/dl) Control 3.90±0.06a 39.06±0.6 a 9.26±0.07a Lead acetate 3.10±0.06 b 31.07±0.61b 7.76±0.08b Chlorpyrifos 2.91±0.06c 29.10±0.07c 7.27±0.05b Mean±SE (n=5/group) a,b Means in the same raw with different superscript are significantly different (P<0.05) Table 2: Comet assay parameters obtained by image analysis in all groups Group Tailed % Untailed % Tails length (pm) Tail DNA% Tail moment Normal 2 98 1.53±0.16 c 1.64 2.51 Lead acetate 19 81 6.13±0.49 b 5.48 33.59 Chlorpyrifos 29 71 9.34±0.70 a 8.02 74.91 Different superscript letters in the same column of tail length showed significance difference at P< 0.05 Hemotoxic and genotoxic effect of lead acetate and chlorpyrifose on freshwater cat fish (Clarias gariepinus) 685 Figure 1: Showing effect of lead acetate and Chlorpyrifos on MDA and catalase activity. Data expressed Mean±SE. (n=10/group). *P <0.05 versus control group Figure 2: Showing DNA damage (comet assay) in normal fish (A), lead acetate -intoxicated fish (B) and Chlorpyrifos - intoxicated fish (C) 686 S. Shireen, E. Nasr, A. Khaled, E. Hossam Figure 3: Showing Hematoxylin and eosin (H&E) stains of liver normal fish (A) ,lead acetate -intoxicated fish (B) and Chlorpyrifos - intoxicated fish (C) Discussion Because of over population and unintended civilization, many aquatic ecosystems are facing complicate problems of contamination (43). A few contaminates, for example detergents, household waste pesticides, insecticides, bacteria, parasites, metals, salts, acids, and other chemicals have been found in many aquatic surroundings (44). African catfish (Clarias gariepinus) is an excellent model for aquaculture, fundamental research, and environmental contamination studies (45, 46), So the present study try to illustrate, hemotoxicity and geno-toxicity of lead and chlorpyrifos as pollutant on the Clarias gariepinus fish. The result of present study revealed a significant decrease of RBCs count, Hb concentration and PCV values in lead acetate -intoxicated fish comparison with control fish. This was in harmony with (33) who reported that sub-chronic exposure of C. gariepinus to lead decreased RBCs count. Changes in Hb concentration and PCV values, were also compatible with Hemotoxic and genotoxic effect of lead acetate and chlorpyrifose on freshwater cat fish (Clarias gariepinus) 687 Adeyemo (47), who reported significant decreases in Hb, RBCs count and PCV values in C. gariepinus exposed to lead nitrate. Lead causes damage of hemopoietic organs , structural damage of RBC membranes resulting in hemolysis, impairment in hemoglobin synthesis, stress-related release of RBCs from the spleen and hypoxia (48). Lead causes early mortality of mature red blood cells and inhibition of hemoglobin synthesis through inactiva-tion of RBC alpha-amino levulinic acid dehydratase (17). Also, our results revealed that RBCs count, hemoglobin concentration and hematocrit a significantly less in CPF -intoxicated fish in comparison with the control group. This result was compatible with (34) who reported that exposure of freshwater fish Clarias batrachus to chlorpyrifos led to significant decrease in RBCs and Hb indicating a condition of erythropenia and hemolysis. This may be attributed to inhibition of erythropoiesis, he-moformation, osmoregulatory dysfunction or due to increased rate of RBCs destruction in hematopoietic organ by chlorpyrifos (49). Indeed, chlorpyrifos more toxic than lead acetate. Contaminants can possibly initiate oxidative stress in fish through creation of free radicals and reactive oxygen species (ROS) which lead to an imbalance between intracellular ROS levels and antioxidant defense (50, 51). ROS cause damage to proteins, lipids, carbohydrates and nucleic acids (52). This damage may alter cell functions, eventually leading to cell death (53). Our results showed that serum MDA was significantly increased and catalase activity was significantly decreased in fish exposed to lead as compared to the control fish. Similarly, other studies also reported that toxic action caused by lead might be due to its ability to produce ROS which cause oxidative damage in several tissues by increasing lipid peroxidation through Fenton reaction (54, 55). We also found a similar change following addition of CPF. Our study revealed that serum MDA was significantly increased and catalase activity was significantly decreased in tissue of CPF-exposed fish. Similar results obtained by Kaur and Jindal (56) who reported that CPF was very toxic to Ctenopharyngodon idellus even at very low concentration. Its administration increases MDA level and affected adversely the antioxi-dative defense system in different organs of the fish. This study indicated chlorpyrifos more toxic than lead acetate. Histopathological examination of liver tissue is important marker for exposure to environmental stressors or pollutants, as the liver is important organ of detoxification (57). This study revealed that liver tissue in fish exposed to lead acetate showed hepatic vacuolation, parenchymal haemorrahge and focal leukocytic infiltration, which agreed with Rubio, et al., (58) who reported that lead cause inhibition of mono amino oxidase and acetylcholine esterase, to cause pathological changes in tissue and organs. Additionally, CPF also resulted in vac-uolation and hepatic necrosis in hepatic tissue of cat fish, and this was in consistent with Deb and Das (59) who reported that common carp exposed to CPF revealed different degree of hydropic degeneration, vacuolization, pyknotic nuclei, and fatty infiltration in their liver tissue.. Although every metal have distinctive mechanism of toxicity however there are some common mechanism such as mimicry, adduct formation with DNA or protein and oxidative damage. Generation of ROS is caused by heavy metals in their ionic forms leading to oxidative changes in DNA, causing aberrant gene expression and carcinogenesis (60). The present study revealed induction of DNA damage by lead and CPF. In support, other studies also reported exposure to lead cause genotoxic effects, such as chromosome aberration, mutation, DNA breakage and DNA synthesis inhibition (61, 62). This result may due to lead can mimic the essential elements such as magnesium, iron, calcium and zinc, increased the production of inaccurate nu-cleotides in which it implicated as a co- carcinogen and effect on DNA repairing mechanisms (63). Similarly, Yin, et al., (64) reported that chlorpyrifos caused genotoxicity in RBCs and liver cells of Chinese toad (Bufo gargarizans) when the tadpoles were exposed to the sub lethal dose of chlorpyrifos. Indeed DNA damage was higher in chlorpyrifos than lead acetate. 688 S. Shireen, E. Nasr, A. Khaled, E. Hossam Conclusion This study illustrated that lead acetate and chlorpyrifos have hemotoxicity through decrease RBCs count, Hb concentration, and hematocrit, generate ROS and has genotoxicity through induction of DNA damage in cat fish after exposure to sub-lethal concentrations. Moreover chlorpyrifos is more toxic than Lead acetate Conflict of interest The authors declare that they have no conflict of interest. References 1. McGlashan D, Hughes J. Genetic evidence for historical continuity between populations of the Australian freshwater fish Craterocephalus stercusmuscarum (Atherinidae) east and west of the Great Dividing Range, Journal of Fish Biology 2001; 59: 55-67. 2. Ali F K, El-Shafai S A, Samhan F A, Khalil W K. Effect of water pollution on expression of immune response genes of Solea aegyptiaca in Lake Qarun, African Journal of Biotechnology 2008; 7(10). 3. Mahfouz M, Hegazi M, El-Magd M, Kasem E. Metabolic and molecular responses in Nile tilapia, Oreochromis niloticus during short and prolonged hypoxia, Marine and Freshwater Behaviour and Physiology 2015; 48(5): 319-40. 4. Ayadi I, Monteiro S M, Regaya I, Coimbra A, Fernandes F, Oliveira M M, Peixoto F, Mnif W. Biochemical and histological changes in the liver and gills of Nile tilapia Oreochromis niloticus exposed to Red 195 dye, RSC Advances 2015; 5(106): 87168-78. 5. Pandey S, Parvez S, Sayeed I, Haque R, Bin-Hafeez B, Raisuddin S. Biomarkers of oxidative stress: a comparative study of river Yamuna fish Wallago attu (Bl. & Schn.), Science of the Total environment 2003; 309(1-3): 105-15. 6. Gilliom R J, Hamilton P A, Pesticides in the nation's streams and ground water, 1992-2001-a summary, 2006. 7. Monroy M, Maceda-Veiga A, de Sostoa A. Metal concentration in water, sediment and four fish species from Lake Titicaca reveals a large-scale environmental concern, Science of the Total environment 2014; 487: 233-44. 8. Authman M M, Zaki M S, Khallaf E A, Abbas H H. Use of fish as bio-indicator of the effects of heavy metals pollution, Journal of Aquaculture Research & Development 2015; 6(4): 1-13. 9. Patra R, Swarup D, Dwivedi S. Antioxidant effects of a tocopherol, ascorbic acid and L-methionine on lead induced oxidative stress to the liver, kidney and brain in rats, Toxicology 2001; 162(2): 81-8. 10. Gurer H, Ercal N. Can antioxidants be beneficial in the treatment of lead poisoning?, Free Radical Biology and Medicine 2000; 29(10): 92745. 11. El-Magd M A, Kahilo K A, Nasr N E, Kamal T, Shukry M, Saleh A A. A potential mechanism associated with lead-induced testicular toxicity in rats, Andrologia 2016; 49(9): e12750. 12. Dai W, Liu S, Fu L, Du H, Xu Z. Lead (Pb) accumulation, oxidative stress and DNA damage induced by dietary Pb in tilapia (Oreochromis niloticus), Aquaculture Research 2012; 43(2): 20814. 13. Abdallah G M, El-Sayed E-S M, Abo-Salem O M. Effect of lead toxicity on coenzyme Q levels in rat tissues, Food and Chemical Toxicology 2010; 48(6): 1753-6. 14. Nordberg , Fowler , Nordberg , Friberg. Handbook on the Toxicology of Metals. 3rd Edn. Academic press, Amesterdam., ISBN 2007; -10: 0123694132, pp: 10-24. 15. Makino S, Tsuruta H, Takata T. Relationship between blood lead level and urinary ALA level in workers exposed to very low levels of lead, Industrial health 2000; 38(1): 95-8. 16. Flora S J, Pande M, Mehta A. Beneficial effect of combined administration of some naturally occurring antioxidants (vitamins) and thiol chelators in the treatment of chronic lead intoxication, Chemico-Biological Interactions 2003; 145(3): 267-80. 17. Ademuyiwa O, Ugbaja R, Ojo D, Owoigbe A, Adeokun S. Reversal of aminolevulinic acid dehydratase (ALAD) inhibition and reduction of erythrocyte protoporphyrin levels by vitamin C in occupational lead exposure in Abeokuta, Nigeria, Environmental Toxicology and Pharmacology 2005; 20(3): 404-11. 18. Sabra F S, Mehana E. Pesticides toxicity in fish with particular reference to insecticides, Asian Journal of Agriculture and Food Sciences (ISSN: 2321-1571) 2015; 3(01). 19. Lari S Z, Khan N A, Gandhi K N, Meshram T S, Thacker N P. Comparison of pesticide residues in surface water and ground water of agriculture intensive areas, Journal of Environmental Health Science and Engineering 2014; 12(1): 11. Hemotoxic and genotoxic effect of lead acetate and chlorpyrifose on freshwater cat fish (Clarias gariepinus) 689 20. Banaee M, Mirvagefei A, Rafei G, Majazi Amiri B. Effect of sub-lethal diazinon concentrations on blood plasma biochemistry, International Journal of Environmental Research 2008; 2(2): 189-8. 21. Wang Y, Qiu Y, Fei Y, Li L, Zhu Z, Zhao J, Yao E, Yao Y. Measurement and preliminary human health risk assessment of representative organochlorines in farmed Mandarin fish, Huan jing ke xue= Huanjing kexue 2011; 32(8): 2385-90. 22. Rao J V, Begum G, Pallela R, Usman P, Rao R N. Changes in behavior and brain acetylcholinesterase activity in mosquito fish, Gambusia affinis in response to the sub-lethal exposure to chlorpyrifos, International Journal of Environmental Research and public health 2005; 2(3): 478-83. 23. Akhgari M, Abdollahi M, Kebryaeezadeh A, Hosseini R, Sabzevari O. Biochemical evidence for free radicalinduced lipid peroxidation as a mechanism for subchronic toxicity of malathion in blood and liver of rats, Human & experimental toxicology 2003; 22(4): 205-11. 24. Cicchetti R, Argentin G. The role of oxidative stress in the in vitro induction of micronuclei by pesticides in mouse lung fibroblasts, Mutagenesis 2003; 18(2): 127-32. 25. Orbea A, Ortiz-Zarragoitia M, Solé M, Porte C, Cajaraville M P. Antioxidant enzymes and peroxisome proliferation in relation to contaminant body burdens of PAHs and PCBs in bivalve molluscs, crabs and fish from the Urdaibai and Plentzia estuaries (Bay of Biscay), Aquatic Toxicology 2002; 58(1-2): 75-8. 26. Sharawy Z Z, Thiele R, Abbas E M, El-Magd M A, Hassaan M S, Peter C, Schmidt J, Saborowski R, Goda A M A-S, Slater M J. Antioxidant response, body composition of whiteleg shrimp Litopenaeus vannamei co-cultured with Nile tilapia Oreochromis niloticus in recirculating aquaculture, Aquaculture Environment Interactions 2017; 9: 257-68. 27. Ahmad I, Hamid T, Fatima M, Chand H S, Jain S K, Athar M, Raisuddin S. Induction of hepatic antioxidants in freshwater catfish (Channa punctatus Bloch) is a biomarker of paper mill effluent exposure, Biochimica et Biophysica Acta (BBA)-General Subjects 2000; 1523(1): 37-48. 28. Narra M R, Regatte R R, Kodimyala R. Effects of chlorpyrifos on enzymes as biomarkers of toxicity in Fresh water field crab Barytelphusa guerini, International Journal of Environmental Sciences 2012; 2(4): 2015. 29. Karami A, Christianus A, Ishak Z, Courtenay S, Syed M, Azlina M N, Noorshinah H. Effect of triploidization on juvenile African catfish (Clarias gariepinus), Aquaculture international 2010; 18(5): 851-58. 30. Hassanain M, Abdel-Rahman E H, Abo-Hegab S, Tawfik M, Abbas W T. Induction of cytochrome P450 1A1 as a biomarker of Benzo-a-pyrene pollution in Egyptian fresh water fish, Pakistan journal of biological sciences: PJBS 2007; 10(8): 1161-9. 31. Mekkawy I A, Mahmoud U M, Osman A G, Sayed A E-D H. Effects of ultraviolet A on the activity of two metabolic enzymes, DNA damage and lipid peroxidation during early developmental stages of the African catfish, Clarias gariepinus (Burchell, 1822), Fish physiology and biochemistry 2010; 36(3): 605-26. 32. Sayed A E-D H, Hamed H S. Induction of apoptosis and DNA damage by 4-nonylphenol in African catfish (Clarias gariepinus) and the antioxidant role of Cydonia oblonga, Ecotoxicology and environmental safety 2017; 139: 97-101. 33. Alkahemal-Balawi H F, Ahmad Z, Al-Akel A S, Al-Misned F, Suliman E-A M, Al-Ghanim K A. Toxicity bioassay of lead acetate and effects of its sub-lethal exposure on growth, haematological parameters and reproduction in Clarias gariepinus, African Journal of Biotechnology 2011; 10(53): 11039--47. 34. Narra M R, Rajender K, Reddy R R, Rao J V, Begum G. The role of vitamin C as antioxidant in protection of biochemical and haematological stress induced by chlorpyrifos in freshwater fish Clarias batrachus, Chemosphere 2015; 132: 172-8. 35. Natt M P, Herrick C A. A new blood diluent for counting the erythrocytes and leucocytes of the chicken, Poultry Science 1952; 31(4): 735-738. 36. Hesser E F. Methods for routine fish hematology, The Progressive Fish-Culturist 1960; 22(4): 164—71. 37. Nwani C D, Somdare P O, Ogueji E O, Nwani J C, Ukonze J A, Nwadinigwe A O. Genotoxicity assessment and oxidative stress responses in freshwater African catfish Clarias gariepinus exposed to fenthion formulations, Drug and chemical toxicology 2017; 40(3): 273-80. 38. Ohkawa H, Ohishi N, Yagi K. Assay for lipid peroxides in animal tissues by thiobarbituric acid reaction, Analytical biochemistry 1979; 95(2): 351-8. 39. Aebi H, [13] Catalase in vitro, Methods in enzymology, Elsevier1984, pp. 121-6. 690 S. Shireen, E. Nasr, A. Khaled, E. Hossam 40. Bancroft J D, Cook H C, Manual of histological techniques and their diagnostic application, Churchill Livingstone1994. 41. Singh N P, McCoy M T, Tice R R Schneider E L. A simple technique for quantitation of low levels of DNA damage in individual cells, Experimental cell research 1988; 175(1): 184-91. 42. Badawy A A, El-Magd M A, AlSadrah S A. Therapeutic Effect of Camel Milk and Its Exosomes on MCF7 Cells In Vitro and In Vivo, Integrative Cancer Therapies 2018; 7(4): 1235--46. 43. El-Adawy M, El-Aziz M A, El-Shazly K, Ali N G, El-Magd M A. Dietary propionic acid enhances antibacterial and immunomodulatory effects of oxytetracycline on Nile tilapia, Oreochromis niloticus, Environmental Science and Pollution Research. DOI:10.1007/s11356-018-3206-5 2018. 44. Hayati A, Pratiwi H, Khoiriyah I, Winarni D, Sugiharto, Histopathological assessment of cadmium effect on testicles and kidney of Oreochromis niloticus in different salinity, AIP Conference Proceedings, AIP Publishing, 2017, p. 020014. 45. Osman A G, Mekkawy I A, Verreth J, Wuertz S, Kloas W, Kirschbaum F. Monitoring of DNA breakage in embryonic stages of the African catfish Clarias gariepinus (Burchell, 1822) after exposure to lead nitrate using alkaline comet assay, Environmental Toxicology: An International Journal 2008; 23(6): 679-87. 46. Nguyen L, Janssen C. Embryo-larval toxicity tests with the African catfish (Clarias gariepinus): comparative sensitivity of endpoints, Archives of environmental contamination and toxicology 2002; 42(2): 256-62. 47. Adeyemo O K. Haematological profile of Clarias gariepinus (Burchell, 1822) exposed to lead, Turkish Journal of Fisheries and Aquatic Sciences 2007; 7(2). 48. Shah S L. Hematological parameters in tench Tinca tinca after short term exposure to lead, Journal of Applied Toxicology: An International Journal 2006; 26(3): 223-8. 49. Jenkins F, Smith J, Rajanna B, Shameem U, Umadevi K, Sandhya V, Madhavi R. Effect of sub-lethal concentrations of endosulfan on hematological and serum biochemical parameters in the carp Cyprinus carpio, Bulletin of Environmental Contamination and Toxicology 2003; 70(5): 09937. 50. Toni C, Ferreira D, Kreutz L C, Loro V L, Barcellos L J G. Assessment of oxidative stress and metabolic changes in common carp (Cyprinus carpio) acutely exposed to different concentrations of the fungicide tebuconazole, Chemosphere 2011; 83(4): 579-84. 51. Xing H, Wang X, Sun G, Gao X, Xu S, Wang X. Effects of atrazine and chlorpyrifos on activity and transcription of glutathione S-transferase in common carp (Cyprinus carpio L.), Environmental Toxicology and Pharmacology 2012; 33(2): 23344. 52. Abdelhady D, El-Abasy M, Abou-Asa S, Elbialy Z, Shukry M, Hussein A, Saleh A, El-Magd M. The ameliorative effect of Aspergillus awamori on aflatoxin B1-induced hepatic damage in rabbits, World Mycotoxin Journal 2017; 10(4): 363-73. 53. Sharbidre A A, Metkari V, Patode P. Effect of methyl parathion and chlorpyrifos on certain biomarkers in various tissues of guppy fish, Poecilia reticulata, Pesticide Biochemistry and Physiology 2011; 101(2): 132-41. 54. Leonard S S, Vallyathan V, Castranova V, Shi X, Generation of reactive oxygen species in the enzymatic reduction of PbCrO 4 and related DNA damage, Oxygen/Nitrogen Radicals: Cell Injury and Disease, Springer2002, pp. 309-15. 55. El-Sokkary G H, Kamel E S, Reiter R J. Prophylactic effect of melatonin in reducing lead-induced neurotoxicity in the rat, Cellular & molecular biology letters 2003; 8(2): 461-70. 56. Kaur M, Jindal R. Oxidative Stress Response in Liver, Kidney and Gills of Ctenopharyngodon Idellus (Cuvier & Valenciennes) Exposed To Chlorpyrifos, MOJ Biol Med 2017; 1(4): 00021. 57. Abdelhady D H, El-Magd M A, Elbialy Z I, Saleh A A. Bromuconazole-induced hepatotoxicity is accompanied by upregulation of PXR/CYP3A1 and downregulation of CAR/CYP2B1 gene expression, Toxicol Mech Methods 2017; 27(7): 544-50. 58. Rubio R Tineo P, Torreblanca A, Del Ramo J, Mayans J D. Histological and electron microscopical observations on the effects of lead on gills and midgut gland of Procambarus clarkii, Toxicological & Environmental Chemistry 1991; 31(1): 347-52. 59. Deb N, Das S. Chlorpyrifos toxicity in fish: A Review, Curr. World Environ 2013; 8(1): 77-84. 60. Ballatori N. Transport of toxic metals by molecular mimicry, Environmental health perspectives 2002; 110 (Suppl 5): 689. 61. 61. Danadevi K, Rozati R, Banu B S, Rao P H, Grover P. DNA damage in workers exposed to lead using comet assay, Toxicology 2003; 187(2-3): 183-93. Hemotoxic and genotoxic effect of lead acetate and chlorpyrifose on freshwater cat fish (Clarias gariepinus) 691 62. Celik A, Ogenler O, Çomelekoglu U. The evaluation of micronucleus frequency by acridine orange fluorescent staining in peripheral blood of rats treated with lead acetate, Mutagenesis 2005; 20(6): 411-5. 63. Fracasso M E, Perbellini L, Soldà S, Talamini G, Franceschetti P. Lead induced DNA strand breaks in lymphocytes of exposed workers: role of reactive oxygen species and protein kinase C, Mutation Research/Genetic Toxicology and Environmental Mutagenesis 2002; 515(1): 159-9. 64. Yin X H, Li S N, Zhang L, Zhu G N, Zhuang H S. Evaluation of DNA damage in Chinese toad (Bufo bufo gargarizans) after in vivo exposure to sublethal concentrations of four herbicides using the comet assay, Ecotoxicology 2008; 17(4): 280-6. Slov Vet Res 2019; 56 (Suppl 22): 693-8 DOI 10.26873/SVR-809-2019 Original Research Article MOLECULAR DETECTION OF invA AND hilA VIRULENT GENES IN SALMONELLA SEROVARS ISOLATED FROM FRESH WATER FISH Sarah A. Allam1*, Nader Y. Mostafa1, Ghada A. K. Kirrella1, Nesreen Z. Eleiwa2, Mohammed A. El-Magd3 1Food Control Department, Faculty of Veterinary Medicine, Kafrelsheikh University, 33516, Egypt, 2Animal Health Research Institute, Dokki, Giza, Egypt, 3Anatomy Department, Faculty of Veterinary Medicine, Kafrelsheikh University Corresponding Author: sara.alam9912@yahoo.com Abstract: The present study was conducted to monitor the prevalence of salmonellae in fresh water fish in Gharbia governorate, Egypt. A total of 120 random samples of fresh water fish (Tilapia niloticus, Mugil cephalous and Clarias lazera, 40 of each) were analyzed bacteriologically for the presence of salmonella pathogens and were further identified using biochemical and serological tests. Positive samples were confirmed by polymerase chain reaction (PCR) through detection of common virulent genes invasion A (invA) and hyper-invasive locus (hilA). The obtained biochemical and serological results revealed presence of seven different serotypes including s. typhimurium which was the most frequent one followed by s. enteriditis, S. infantis , S. virchow, S. heidelberg, S. wingrove and s. tsevie with a prevalence of 33.3%, 23%, 12.8%, 12.8%, 10.25%, 5% and 2.5%, respectively. The obtained molecular results revealed presence of the two virulent genes in all the detected serovars with a total prevalence rate of 100% for invA gene and 66.6% for hila gene. These results reflected a high prevalence of salmonella species in fresh water fish which act as a potential source of food borne infection and constitutes a major concern for public health. Key words: fresh water fish; salmonella; serotypes; invA; hilA; PCR Introduction Fish are considered as an excellent cheap source of high quality protein with very low cholesterol levels and large quantities of poly-unsaturated fatty acids that cannot be synthesized by human metabolism (1, 2). However, fish are also considered as a major carrier for food borne pathogens as its natural habitat is extremely susceptible to pollution from domestic, industrial and agricultural discharges (3). Bacterial diseases in fish cause a serious threat not only to aquaculture industry but also on human health. Thus, it is not surprising to find that 12% of the food poisoning outbreaks associated with fish consumption are caused by bacteria including salmonella (4). Salmonella, as a member of family entero-bacteriacea, is a Gram negative rod shaped bacteria that contains more than 2500 serovars. Salmonella, which is considered as a common water-borne pathogen, apparently presents in the tissues of normal fish and represents no clinical signs. It can easily reach water through many Received: January 2019 Accepted for publication: February 2019 694 S. Allam, N. Mostafa, G. Kirrella, N. Eleiwa, M. El-Magd routes, especially through sewage discharges, and can concentrate in fish tissues. It also can reach fish outside water through fish handling without following strict hygienic measures. When these contaminated fish were eaten raw or with quick inappropriate heating, the consumers would suffer from enteritis and typhoid fever. Despite extensive public health measures over the past century, salmonella remains the second leading cause of food-borne diseases worldwide (5). Accordingly, providing a safe, high quality and acceptable food to a consumer is considered a major goal for food processing industry. This can be applied by prompt and accurate isolation and characterization of salmonella and other food-borne pathogens which are the cornerstone required for proper control of food-borne diseases. This can be achieved using molecular identification of these pathogens by polymerase chain reaction (PCR) technique. The latter, is highly sensitive and more accurate method for bacterial isolation and identification, especially for salmonella sp. (6). Thus, our study was planned to identify the prevalence of salmonella species in fresh water fish in Ghar-bia Governorate, Egypt and to detect the common virulent genes of salmonella serovars using PCR technique. Materials and methods Isolation of salmonellae A total of 120 random samples of fresh water fish including Tilapia niloticus, Mugil ceph-alous and Clarias lazera (40 of each) were collected from different markets in Gharbia gover-norate, Egypt. Samples were placed separately in sterile plastic bags in an ice box and transported immediately to the laboratory for bacteriological examination. According to ISO 6579 (2002) method [11]: 25 g of back muscles of each sample of fresh water fish were transferred to a homogenizer flask containing 225 ml of sterile buffered peptone (1%) and homogenized for 1 min, then incubated at 37 0C for 18 h. According to ISO 6579 (2002) method: 1 ml of each homogenate was inoculated separately into a tube of 9 ml selenite-f- broth and incubated at 370C for 18 h or 0.1 ml was transferred to 9 ml of Rappaport - Vassiliadis Soya broth (RVS) and incubated at 420C for 24 h. A loopful from selective enriched broth was streaked onto plates of Xylose Lysine Deoxycholate (XLD) and incubated at 37 0C for 24 h. Typical pink colonies with or without black center were picked up for further identification. Identification of salmonellae For microscopical identification, films from suspected colonies were prepared, fixed and stained with Gram stain showing Gram negative rod-shaped, motile bacilli that indicates salmonella pathogen. For biochemical identification, according to ISO 6579 (2002) method: purified isolates were examined by different biochemical reaction based on oxidase, urea hydrolysis, H2s production on tripple sugar iron, indole, methyl red, vogus-proskaur and citrate utilization tests. For biochemical identification, salmonella isolates were serotyped according to Kauffman (1974) method (7) for the determination of somatic (O) and flagellar (H) antigens using salmonella antiserum (DENKA SEIKEN Co., Japan). Molecular detection of salmonella virulent genes by multiplex PCR Bacterial DNA was extracted using QIA amp extraction kit following manufacturer's protocol and as previously described (8). Multiplex PCR was applied to amplify salmonella virulence genes invA and hila using specific primers as shown in table 1. PCR reaction mixture (25 pl) contained 5 pl of bacterial DNA, 2.5 pl of 10x Master mix (containing 1.25 U of Taq DNA polymerase, 1.5 mM MgCl2 and 2 pl 10mM dNTP mix), 1 pl of each primer (10 pmol) 15.5 pl DNase free water. The PCR cycling protocol was applied as following: an initial denaturation at 94°C for 60 sec, followed by 35 cycles of denaturation at 94°C for 60 sec, annealing at 64°C for 30 sec and extension at 72°C for 30 sec, followed by a final extension at 72°C for 7 min. Finally, 5 pl of each am-plicon was electrophoresed in 1.5 % agarose gel, stained with ethidium bromide and visualized and captured on UV transilluminator. A 100 bp DNA ladder was used as a marker for PCR products Molecular detection of invA and hilA virulent genes in salmonella serovars isolated from fresh water fish 695 Results The prevalence of salmonella species The total prevalence rate of isolated salmonella was 32.5% (39/120) in all fish with highest incidence rate in Mugil species (47.5%, 19/39) followed by Clarias lazera (30%, 12/39) and Tilapia niloticus (20%, 8/30). Serotyping Salmonella isolates (n = 39) were serotyped using "O" and "H" antisera and the results showed 7 different salmonella serotypes: S. typhimurium, S. enteritidis, S. infantis, S. vir-chow, S.Heidelberg, S. wingrove and S. tsevie with a total percentage of 33.3%, 23%, 12.8%, 12.8%, 10.25%, 5% and 2.5%, respectively. The prevalence of each serotype in each fish type was shown in Table 2. Incidence of virulence genes The incidence of invA and hilA virulent genes among the 7 salmonella serotypes as detected by multiplex PCR revealed prevalence of invA gene in all salmonella serotypes with a percentage of 100%, hilA gene in only 66.6% of all serotypes (Table 3 and Fig. 1). Table 1 : The primer sequence of salmonellae used for PCR Target gene Oligonucleotide sequence (5' ^ 3') Product size (bp) References invA (F) 5' GTGAAATTATCGCCACGTTCGGGCA '3 284 (9) invA (R) 5' TCATCGCACCGTCAAAGGAACC '3 hilA (F) 5' CTGCCGCAGTGTTAAGGATA '3 497 (10) hilA (R) 5' CTGTCGCCTTAATCGCATGT '3 Table 2: Serotyping of salmonellae isolated from the examined samples of fresh water fishes Fish species Serotypes Tilapia niloticus Mugil cephalus Clarias lazera Group Antigenic structure No (%) No (%) No (%) O H S. typhimurium 3(7.5) 6(15) 4(10) B 1,4,5,12 i :1,2 S. infantis 1(2.5) 1(2.5) 3(7.5) C1 6,7 r :1,5 S. virchow 2(5) 1(2.5) 2(5) C1 6,7,14 r :1,2 S. enteritiditis 2(5) 5(12.5) 2(5) D1 1,9,12 g,m S. heidelberg 0 3(7.5) 1(2.5) B 1,4,5,12 r :1,2 S.tsevie 0 1(2.5) 0 B 4,5 i:e,n,z15 S. wingrove 0 2(5) 0 C2 8,20 c:1,2 Total (120) 8(20) 19 (47.5) 12(30) Table 3: Incidence of virulence genes of different salmonella strains isolated from the examined samples of fish. Salmonella No. of invA hila serovars isolates No (%) No (%) S. typhimurium 4 4 (100) 3 (75) S. infantis 4 4 (100) 2 (50) S. virchow 4 4 (100) 2 (50) S. enteritidis 3 3 (100) 3 (100) S. heidelberg 1 1 (100) 1(100) S. tsevie 1 1 (100) 1(100) S. wingrove 1 1 (100) 0 696 S. Allam, N. Mostafa, G. Kirrella, N. Eleiwa, M. El-Magd 18 17 16 15 14 13 12 11 10 9 8 7 6 5 4 3 2 1 C-C+ M Figure 1 : Agarose gel electrophoresis of multiplex PCR of invA (284 bp) and hilA (497 bp) virulence genes for characterization of salmonella species. Lane M: 100 bp ladder; lane C+: Control positive strain for invA and hilA genes; lane C-: Control negative; lanes 1-18 isolated samples. Discussion In the present study, we found high incidence of salmonella pathogen among different fish species with highest rate in Mugil ceph-alous (47.5%), followed by Clarias lazera (30%), and then Tilapia niloticus (20%). Higher incidence in Mugil cephalous may be due to its feeding nature as omnivorous bottom feeders swim over sandy-muddy bottom were they greases the pond detritus, organic matter and sediments which are usually of a high bacterial count (11) and salmonella can survive (54 days in water and up to 119 days in sediment) (12). This also may be due to the higher exposure of mugil species to contamination during their long transportation at high temperature from far cities as the point of production (farms) is so far from the point of distribution (fish markets) in Gharbia governorate. On the other hand, tilapia mainly inhabited shallow streams which are usually of a low bacterial count as a result of higher speed of water flow which prevent exceeding contamination with microorganisms, indicating the possibility of occurring cross contamination during offloading, landing and marketing of tilapia fish besides applying of poor hygienic practice via using of unsanitary water, ice and containers (13). And is not surprising in cat fish species since they mostly bred in area near from sewage which are usually related to fecal contamination of the water. Collectively, our findings are in consistence with (14) who reported that the variation in salmonella percentage in different types of fish could be attributed to fish species, environments, and methods of catch, extend of handling during transportation, distribution and storage. Our results partially agreed with (15) who also isolated different salmonella serotypes from fresh water fish (tilapia, mugil and catfish) from another locality in Egypt (Zagazig). However, this latter study showed a slight different in prevalence order, with highest rate in mugil samples (26.66%), followed by Tilapia niloticus (20%) then cat fish (13.3%). This variations may be due to variety of factors such as geographical distribution, applying of poor personal hygine of fish handlers during sorting and selling, the degree of utensils contamination, the bacterial load of ice used for fish preservation and the time of sampling (16). The results obtained from serological identification of the examined salmonella isolates revealed presence of 7 different salmonella serovars which are S. typhimurium, S. enter-itidis, S. infantis, S. virchow , S.Heidelberg, S. wingrove and S. tsevie with a percentage of 33.3%, 23%, 12.8%, 12.8%, 10.25%, 5% and 2.5%, respectively. These results agreed with (17) who reported that Salmonella typhimurium was the most frequently serovar isolated from Molecular detection of invA and hilA virulent genes in salmonella serovars isolated from fresh water fish 697 Nile tilapia from Winam Gulf of lake victoria, kenia with a percentage of 14.3% regarding other salmonella serovars indicating its ability for survival and adaptation in the aquatic environment. This also was in consistence with WHO, (18) which reported that Salmonella typhimurium is the most common salmonella serotype isolated from cases of food poisoning and represents about 50-60% of such cases. Many of these incidents have been linked to consumption of raw or improperly cooked fish and fish products. It has been found that salmonella has a large number of genes which implicated in its virulence. Many of these virulent genes are chromosomal genes located on the pathogenicity islands referred to as salmonella pathogenicity island (spI) which is a part of type 3 secretion systems (T3SS) and encoded genes like invA, and hilA allowing some salmonella species to invade the host cells and induce infection. Our obtained PCR results revealed that invA gene was present in all the isolates with a percentage of 100%. Meanwhile, hila gene was present only in 66.6% of all the isolates. Other studies have reported similar results (19-22) indicating that invA gene can be used as a specific target for detection of salmonella as they are widely distributed among salmonella serovars reflecting the potential virulence of these isolates. These finding play a great role in determining the severity of the microbe since invA gene was found to be present and functional in most if not all salmonella serotypes responsible for the invasion of the cells of the intestinal epithelium allowing salmonella pathogen to enter and survive inside the eukaryotic cells with subsequent diseases in variety of hosts. In this regard, it has been used in determining the presence and metabolic activity of salmonella species (23). Meanwhile, hilA gene is required for regulation of type 3 secretion apparatus genes which secret proteins that are related to cell invasion [13]. And has been used in detecting of S.enterica serovars from fecal samples according to [19]. In recent studies, it has been used as a diagnostic target to validate alternative methods for food analysis and food-borne pathogen detection instead of the standard method of ISO 6579, 2004 which are time consuming and can take up to 4-6 days for confirmative results. Conclusion Presence of salmonella as enteropathogens in fresh water fish can reflects the poor hygienic conditions under which fish have been captured, handled and transported. The main finding in our study was the high prevalence of salmonella pathogen with a diversity of serotypes in fresh water fish in gharbia governorate creating a high level of attention towards the hazards of presence of these zoonotic pathogens in marketed fish and their bad impact on public health. so, our study recommends a monitoring and surveillance programs to be adopted against the food borne-salmonella by applying a strict hygienic measures under the supervision of the food controlling authorities in order to control the keeping quality of the locally consumed fish and to protect the consumer from the risk of food poisoning associated with consumption of contaminated fish with such dangerous pathogens. Acknowledgements We thank Dr. Seham Elkassas, Poultry & Fish Breeding & Production Department, Kafrelsheikh University and Dr. Reda Abdallah, Food Control department, Kafrelsheikh University for their help. References 1. Food and Agriculture Organization (FAO). 2014. The State of World Fisheries and Aquaculture 2014. Rome. 223. 2. El-Adawy M, El-Aziz M A, El-Shazly K, Ali N G, El-Magd M A. Dietary propionic acid enhances antibacterial and immunomodulatory effects of oxytetracycline on Nile tilapia, Oreochromis ni-loticus, Environmental Science and Pollution Research. DOI: 10.1007/s11356-018-3206-5 2018. 3. Raufu I, Lawan F, Bello H, et al. Occurrence and antimicrobial susceptibility profiles of Salmonella serovars from fish in Maiduguri, sub-Saharah, Nigeria. The Egyptian J. Aqua. Res. 2014; 40 (1): 59-63. 4. Aberoumand, A. Estimation of microbiological variations in Minced Lean fish products. World J. Fish Mar. Sci. 2010; 2 (3): 204-7. 698 S. Allam, N. Mostafa, G. Kirrella, N. Eleiwa, M. El-Magd 5. Wong M and Chen S. First detection of oqxAB in Salmonella spp. isolated from food. Antimicro. Agen. Chemothera. 2013; 57(1): 658-60. 6. McGuinness S, McCabe E and O'Regan E. Dolan, G. Duffy, C. Burgess, S. Fanning, T. Barry, and J. O'Grady. Development and validation of a rapid real-time PCR based method for the specific detection of Salmonella on fresh meat. Meat Sci. 2009; 83: 555-62. 7. Kauffman G. Kauffmann white scheme. J. Acta. Path. Microbiol. Sci. 1974; 61:385-393. 8. Shanmugasamy M, Velayutham T and Ra-jeswar J. Inv A gene specific PCR for detection of Salmonella from broilers. Vet. World, 2011; 4 (12): 562-4. 9. Singh S, Singh H, Tewari S, Prejit N and Agarwal R. Characterization of virulence factors among diverse Salmonella serotypes and sources. Adv. Anim. Vet. Sci., 2013; 1(2): 69-74. 10. Guo X, Chen J, Beuchat Land Brackett R. PCR detection of Salmonella enterica serotype Montevideo in and on raw tomatoes using primers derived from hilA. Appl. Environ. Microbiol. 2000; 66: 5248-52. 11. Abdelhamid A, Gawish M and Soryal K. Comparative study between desert cultivated and natural fisheries of mullet fish in Egypt, II-microbi-ological concern. J Agric Sci. 2007; 31(5):5681-7. 12. Moore B, Martinez E, Gay J, and Rice D. Survival of Salmonella enterica in freshwater and sediments, and transmission by the aquatic midge Chironomus tentans (chironomidae: diptera). Appl Environ Microbiol. 2003; 69:4556--60. 13. Food and Agriculture Organization (FAO). 2010. Expert Workshop on the application of biose-curity Measures to control Salmonella Contamination in Sustainable aquaculture FAO Fisheries and Aquaculture Report No. 937 ISSN 20706987, Mangalore, India, 19-21 January 2010. 14. Wang S, Chen J, and Fan J. Quality changes in fresh Tilapia and Milkfish during refrigerated (4 degree C) and frozen (-15 degree C) storage J. Food during Analysis, 1994; 2(4):311 -6. 15. El Bayomi R, Darwish W, Abd El-Moaty A, and Gad T. Prevalence, antibiogram, molecular characterization and reduction trial of Salmonella typhimurium isolated from different fish species. Japanese Journal of Veterinary Research 2016; 64(Supplement 2): S181-6. 16. Falcäo J, Dias A, Correa E, and Falcäo D. Microbiological quality of ice used to refrigerate food .J Food microb. 2002; 19(4): 269-76. 17. David O, Wandili S, Kakai R, and Waindi E. Isolation of Salmonella and Shigella from fish harvested from the Winam Gulf of Lake Victoria, Kenya. J. Infect. Dev. Ctries. 2014; 3: 99-104. 18. World Health Organization (WHO). 1997. Microbial aspects of food hygiene,technical Report Series, No. 598, pP.2l - 23. WHO, Geneva, Switzerland. 19. Bhatta D, Bangtrakulnonth A, Tishyadhigama P, et al. Serotyping, PCR, phage-typing and antibiotic sensitivity testing of Salmonella serovars isolated from urban drinking water supply systems of Nepal. Lett. Appl. Microbiol. 2007; 44: 588-94. 20. Maria A. Non typhoid Salmonella gastroenteritis in pediatric patients from urban areas in the city of Merida, Venezuela. J. Infect. Developing Countries 2009; 3: 28-34. 21. Oliveira S, Rodenbusch C, Michae G, et al. Detection of virulence genes in Salmonella Enter-itidis isolated from different sources. Braz. J. Microbiol. 2003;34:123-. 22. Tekale A. Detection and Virulence Gene Characterization of Salmonella Isolates from Fish by Conventional and Molecular Methods. J. Vet. Pub. Hlth. 2015; 13 (1): 43-6. 23. Fey A, Eichler S, Flavier S, et al. Establishment of a real-time PCR-based approach for accurate quantification of bacterial RNA targets in water, using Salmonella as a model organism. Appl Environ Microbiol. 2004; 70: 3618-23. 24. Lesnick M, Reiner N, Fierer J, and Guiney D. 2001. The Salmonella spvB virulence gene encodes an enzyme that ADPribosylates actin and destabilizes the cytoskeleton of eukaryotic cells. Mol Microbiol 39: 1464-70. 25. Pathmanathan S, Cardona-Castro N, Sanchez-Jimenez M, et al. Simple and rapid detection of Salmonella strains by direct PCR amplification of the hilA gene. J Med Microbiol. 2003; 52: 773-6. Slov Vet Res 2019; 56 (Suppl 22): 699-706 DOI 10.26873/SVR-810-2019 Original Research Article GROWTH AND ECONOMIC PERFORMANCE OF USING DRIED TOMATO POMACE FOR MALLARD DUCKS Mohamed A. Omar1, Tamer M. Abdel-hamid2, Sara Esam3, Annam E. Omar 1,3Veterinary Economics and Farm Management, Animal Wealth Development, Faculty of Veterinary Medicine, Zagazig University. 2Animal Breeding and Production, Animal Wealth Development, Faculty of Veterinary Medicine, Zagazig University. 4Animal Nutrition and Clinical Nutrition Department, Faculty of Veterinary Medicine, Zagazig University. Corresponding author, E-mail: omarkafay@yahoo.com Abstract: This study was aimed to economic evaluate the effect of Dried Tomato Pomace (DTP) on growth performance, biochemical profiles and economic efficiency of Mallard Ducks from 1 days old until 72 days (age of marketing). A total of 240 unsexed one - day old, Mallard ducklings were used in this study and were randomly and independently allocated to the four dietary treatments, each containing 60 ducks and divided in to four groups, T1 (control ), T2 ( 10 % DTP), T3 (15% DTP) and T4 (20% DTP). The diets were formulated in mash form fortified with vitamin and mineral premix and chemical analysis were applied for starter and finisher rations. Moreover, feed and water were provided for ad-libitum consumption. The results showed that, ducks fed 20 % DTP had higher live body weight with a coincident significant increase of the feed intake (P <0.05). In addition, total cholesterol, Triglycerides, high density lipoprotein (HDL) are decreased significantly (P <0.05) meanwhile low density lipoprotein (LDL), total protein profiles non-significant (P > 0.05). In terms of economic analysis, T4 ( 20% DTP) diet revealed the lowest cost per Kg live weight of ducks 77.57 L.E / duck, along with the highest economic return 91.38 L.E per duck and net profit 13.81 L.E per duck. In conclusion, Mallard duck can efficiently utilize diet containing high DTP (20%) diets, consequently, it can be used to optimize their growth performance and maintain the maximum economic return. Key words: Mallard duck; dried tomato pomace; growth performance; economics efficiency Introduction The poultry sector is considered the fast growing and flexible of all live stock sectors and Egyptian poultry industry has improved and become occupied a place of glory among the livestock enterprises as it is providing a great part of increasing demand for animal protein. Moreover, it is characterized by rapid monetary turnover, short production cycle and higher return on investment (1). The feed plays remarkable role in poultry production by sharing about 70% of total production costs. So, great efforts have been made to improve feed utilization and conversion to meat to minimize the feeding cost by feeding a well-balanced diet and supplementing diet with various feed additives (2, 3). Tomato (Lycopersicon esculentum) is one of the most popular vegetables used as salad in Received: January 2019 Accepted for publication: February 2019 700 M. A. Omar, T. M. Abdel-Hamid, S. Esam, A. E. Omar food preparation and as juice, soup, ketchup or paste. Commercial processing of tomato produce large amount of waste at various stages. Tomato pomace (TP) is mixture of tomato skin, pulp and crushed seed that remain after the processing of tomato for juice, paste and ketchup (4). From 1000 kg of fresh tomatoes, 100 to 300 kg wet tomato pulp are produced and normally disposed of being sold as animal feedstuffs (5), and its nutritional value is highly dependent on the tomato cultivars, growing conditions, degree of drying and processing method (6). Some by-products originated from food industry processing are utilized in animal feeding. However, the utilization of certain products is not generally applied in animal nutrition. One of these by-products is the peels and seeds of tomato called in common (tomato pomace) remaining usually from the processing of tomato. In Egypt, about 550.000 to 660.000 tons of tomato by-products are yearly produced from canning industry. Unfortunately, a great part of it is lost without utilization. These by-products remain from the squeeze of tomato; is rich in protein, energy and crude fiber (7, 8). Wet tomato pulp can be further dried to approximately 900 g/kg DM and, because of its chemical composition, which possesses nutritional value, can provide the poultry industry with an alternative feedstuff (9). The objective of this study was to economically evaluate substitution the corn with Dried Tomato Pomace (DTP) for mallard duck by study their effect on the productive and economic performance traits that included final body weight, feed conversion rate, feed efficiency, total costs, total returns and finally net returns. Also biochemical profiles of total proteins and lipids were measured for determine the effect of DTP on the quality of the meat for Mallard duck. Materials and methods This work was carried out during the period from January 2017 till March 2017. A total number of 240, unsexed one - day old, Mallard ducklings were used in this study. All ducks were weighed individually and distributed randomly among 4 treatments each treatment include 60 ducks were nearly similar in initial average body weights. Data collection Rations were formulated to contain approximately the same crude protein level, and energy (kcal/ kg). Four treatments were used: - T1 (controlled), corn - soy diet fed for group (1). T2, 10% Tomato Pomace replaces corn fed for group (2). T3, 15% Tomato Pomace replaces corn fed for group (3). T4, 20% Tomato Pomace replaces corn fed for group (4). Four experimental starter diets (from 1day to 42 days) and finisher diets (from 42 day to76days) were fed to ducklings and chemical analysis of experimental diets (10) Growth performance measurements Through the experiment, the following measurements were recorded: Live body weight (LBW) Ducks were weighted in each blocks every 7 days during experimental period. Total individual live weights in each blocks were divided by the number of ducks in the blocks to obtain the average live body weight. Feed intake Ducks in each treatment were provided with a weighed amount of feed every 1 week, the residual were obtained at the end of the 1 week of age and the amount consumed was calculated by the difference. The average amount of feed consumed per duck = amount of feed consumed per ducks / number of duck consuming feed. Feed conversion rate (FCR) Feed conversion rate = (feed intake per kilograms in week / body weight gain per kilograms in week) (11). Feed efficiency (FE) The feed efficiency is weekly or for total experimental period and calculated as follow: Feed efficiency = (Gain in live body weight in this period / Feed intake in certain period) (12,13). Blood parameters On a random basis, blood samples were collected from 5 ducks per group at slaughtering Growth and economic performance of using dried tomato pomace for Mallard ducks 701 and lipid profiles including triglycerides (TG) (14), total cholesterol (TC), high density lipoprotein (HDL) (15), low density lipoprotein (LDL) (16) were determined. Also total plasma proteins (TP) (17), albumin (ALB) (18), globulin (GLB) (19), were determined calorimetri-cally using the commercial kits (ELITech SEEPPIM S.A.S. Zone industrielle - 61500 France). Economic analysis At the end of experiment, the following indices were calculated per each groups of duck: Total fixed costs (TFC), total costs (TC) (15,20). Total return (NR), net profit (NP) and economic efficiency (Ee)(11, 12, 21, 22, 23, 24, 25, 26). Statistical Analysis Data handling and statistical analysis was carried out at the Dept. of Animal Wealth Development, Faculty of Vet. Medicine, Zagazig University. Analysis was done using SPSS/PCT, (Statistical Package for Social Sciences version 22.0) (IBM Corp., Armonk, NY, USA) software Results were reported in means ± SEM (Standard Error of Mean). The value of P < 0.05 was used to indicate statistical significance. The statistical method was ANOVA test (one way analysis of variance) to test the differences in productive and economic efficiency parameters of ducks according to different experimental diets. The Duncan multiple range test are also used (27, 28). Results Growth performance The effects of different levels of dietary DTP on final body weight, Total feed intake, FCR and FE were showed in Table 3. And results showed that the final body weight was significant (P < 0.05) where the highest in T4 and the lowest was in T1.also the total feed intake was significant (P < 0.05) different in different groups the highest was in T3. Meanwhile the feed efficiency and the feed conversion ratio are high with significant in T1 and T3 respectively. Biochemical analysis The different protein profiles are analyzed in table (4) where the total protein, globulin and A/G ratio are non-significant at (P > 0.05). Meanwhile the total albumin are high significant at (P < 0.05) for different groups where the value are high in control groups (5.66) and in T2, T3 and T4 the values were 5.27, 5.36 and 5.45 respectively. Table 5 shows the analysis for different lipid profiles, the mean values for total cholesterol, high density lipoprotein (HDL) , low density lipoprotein(LDL) and triglycerides all are significant at (P < 0.05). Economical analysis The different economic measures are shown in table 6 that shown highly significant (P < 0.05) for total variable costs (LE/ duck), total costs (LE/ duck), total returns (LE/ duck) and net profit (LE/ duck) for different groups and shown that T4 is the highest in terms of net returns and control group (T1) is the lowest in that term of net returns. Also this table shown that total fixed costs (LE/ duck) are non-significant at (P > 0.05) for all groups. The efficiency measures for economic analysis are shown in table 7 that includes percent of total returns to total costs, percent of total returns to total variable costs, percent of net profit to the total variable costs and finally percent of net profit to the total costs. All are significant at (P < 0.05). Discussion Considering our main interest was to establish a bio-economic optimum for feed formulation with DTP that meet the nutritional requirements of mallard duck and maintain maximum performance with least cost as well. Growth Performance The final body weight was highest in T4 with significant effect that indicate that the high percent of DTP (20%) results in increasing the body weight gain and this results are consistent with those of (4) who reported that DTP increase feed performance and final body weight. 702 M. A. Omar, T. M. Abdel-Hamid, S. Esam, A. E. Omar Table 1 : Chemical composition (%) of experimental diets used in the starter stage from 1 weeks to 7 weeksItem % Experimental diets for each 100 kg diet Control 10%Tomato 15%Tomato 20%Tomato pomace pomace pomace ME, Kcal/Kg 2891 2890 2883 2872 CP, % 22.41 22.25 22.24 22.22 EE, % 4.38 4.64 4.76 4.90 CF, % 3.5 5.4 6.43 7.29 Ca, % 0.92 0.92 0.92 0.92 Lysine, % 1.13 1.12 1.11 1.11 Methoinine, % 0.4 0.4 0.4 0.4 Available ph.% 0.58 0.58 0.58 0.58 ME: Meatabolizale Energy CP : Crude Protein CF: Crude Fiber Ca : Calcium Table 2: Chemical composition (%) of experimental diets used in the finisher stage from 7 weeks to 10 weeks Item % Experimental diets for each 100 kg deit Control 10% Tomato 15% Tomato 20% Tomato pomace pomace pomace ME, Kcal/Kg 3000 2994 2987 2981 CP, % 19.83 19.76 19.74 19.71 EE, % 6.8 7.07 7.21 7.34 CF, % 3.37 5.37 6.37 7.43 Ca, % 0.95 0.95 0.95 0.95 Lysine, % 1.18 1.17 1.16 1.15 Methoinine, % 0.50 0.49 0.48 0.48 Available ph.% 0.45 0.45 0.45 0.45 ME: Meatabolizale Energy CP : Crude Protein CF: Crude Fiber Ca : Calcium Table 3: Economic parameters of Mallard duck performance that affected by dietary treatments Items Different treatments that supplied with dried tomato pomace Treatment 1 (T1) Treatment 2 (T2) Treatment 3 (T3) Treatment 4 (T4) Number of ducks 60 60 60 60 Initial body 57.53±0.18a 57.37±0.18a 57.46±0.20a 57.36±0.19a weight (g) Final body weight 4020.93± 5.78c 4108.11± 8.42b 4146.68±3.44a 4154.03±3.03a (g) Total Feed intake 8082.73± 6.18c 8513.60± 54.17b 8786..27± 40.84a 8716.85± 39.18a (g) Feed Conversion 2.01± 0.003c 2.07± 0.10b 2.11± 0.009a 2.09± 0.009a rate (FCR) Feed Efficiency 0.49± 0.008a 0.48± 0.002b 0.47± 0.002c 0.47± 0.002c (FE) Means within the same row in each category carrying different litters are significant at (P < 0.05) Growth and economic performance of using dried tomato pomace for Mallard ducks 703 Table 4: Effect of experimental diets on protein profiles of Mallard ducks (Mean ± SE) Groups No. Treatment 1 (T1) Treatment 2 (T2) Treatment 3 (T3) Treatment 4 (T4) Total 5 5.66±0.05a 5.27±0.03a 5.36±0.07a 5.45±0.01a protein (g/dl) Albumin 5 2.49±0.03a 2.16±0.03b 2.17±0.05b 2.27±0.05b (g/dl) Globulin 5 3.17±0.07a 3.10±0.04a 3.19±0.03a 3.18±0.06a (g/dl) A/G ratio 5 0.78±0.01a 0.69±0.01a 0.68±0.02a 0.71±0.01a Means carrying different superscripts in the same row are sig. different at (P<0.05) Table 5: Effect of experimental diets on Lipid profiles of Mallard ducks (Mean ± SE) Groups No. Treatment 1 (T1) Treatment 2 (T2) Treatment 3 (T3) Treatment 4 (T4) Total 5 196.6± 1.36a 172.2±1.04b 172.9±0.81b 173.6±0.82b cholesterol (mg/dl) LDL- 5 77.21± 1.04b 77.16±0.43b 77.76±1.07a 77.85±0.87a cholesterol (mg/dl) HDL- 5 83.52±0.90a 66.93±2.91b 66.00±0.79b 67.65±0.75b cholesterol (mg/dl) Triglycerides 5 218.69±2.29a 200.08±3.15b 198.92±1.61b 201.84±1.52b (mg/dl) Means carrying different superscripts in the same row are sig. different at (P<0.05) Table 6: Economic parameters of duck performance that affected by dietary treatments Items Number of ducks Total Variable cost (LE/Duck) Total Fixed cost (LE/Duck) Total cost (LE/Duck) Total Returns (LE/Duck) Different treatments that supplied with dried tomato pomace Treatment 1 (T1) Treatment 2 (T2) Treatment 3 (T3) Treatment 4 (T4) 60 60 70.04±0.15a 11.65±0.11a 81.69± 0.19a 88.46± 0.12c 60 67.78±0.21b 11.65± 0.12a 79.43± 0.24b 90.37± 0.18b 60 67.04±0.14c 11.64±0.11a 78.69± 0.18c 91.22±0.07a 66.06±0.15c 11.51±0.10a 77.57± 0.17c 91.38± 0.06a Net Profit (LE/Duck) 6.76± 0.20c 10.94±0.37b 12.53± 0.20a 13.81±0.20a Means within the same row in each category carrying different litters are significant at (P < 0.05) 704 M. A. Omar, T. M. Abdel-Hamid, S. Esam, A. E. Omar Table 7: Economic efficiency of Mallard duck performance that affected by dietary treatments Different treatments that supplied with dried tomato pomace Items Number of ducks Total Return / total cost (%) Total Return / total variable cost (%) Treatment 1 (T1) 60 1.08 ±0.002c 1.26± 0.002c Treatment 2 (T2) 60 1.13±0.005b 1.33± 0.006b Treatment 3 (T3) 60 1.15±0.002a 1.36±0.003a Treatment 4 (T4) 60 1.17±0.002a 1.37±0.003 a Net Return / Total variable cost 0.09± 0.003c 0.16± 0.005b 0.18± 0.003a 0.20± 0.003a Net Returns / Total cost (%) 0.08± 0.002c 0.13± 0.005b 0.15± 0.002a 0.17± 0.002a Means within the same row in each category carrying different litters are significant at (P < 0.05) The total feed intake (g/duck), feed conversion ratio and feed efficiency are the highest in T4 and T3 that results are agreement with (7) who found that dried tomato pomace can be used in broiler chicken diets up to 20 %. However, in general it seems dried tomato pomace can be used in poultry diets as a feed ingredient any level and results in increasing total feed intake, FE and FCR. These results were in contrary with (29) who reported that increased TP level (10, 20%) in both starter and finisher broiler chicken diets resulted lower live weight (P <0.01). However there was no significant difference between control and 5% DTP supplemented diet groups. Biochemical analysis The total protein, globulin and A/G ratio are non-significant at (P > 0.05). The observed result was agreed with (30) who recorded that total protein, globulin and albumin: globulin ratio levels of broilers not affected by different levels of DTP. Table 5 shows the analysis for different lipid profiles, the result revealed significant (P<0.05) decrease in total cholesterol, triglycerides, HDL and LDL levels. The total cholesterol of control group was 196.6 mg/dl while that of the groups fed diet contained 5%, 10% and 20% DTP were 172.2, 172.9, and 173.6 mg/dl respectively. The triglycerides of control group was 281.69 mg/dl while that of the groups fed diet contained 5%, 10% and 20% DTP were 200.08, 198.92, and 201.84 mg/dl respectively. Also the HD of control group was 83.52 mg/dl and that T2, T3 and T4 were 66.93, 66.00 and 67.65 mg/dl respectively. The LDL level of control group was 77.21 mg/dl and for T2, T3 and T4 were 77.16, 77.76 and 77.85mg/dl respectively The observed result was agreed with (30) who recorded that the serum cholesterol content, LDL and HDL of poultry fed on diet contained 8, 16% DTP were lower as compared with other groups (P<0.05). In the same line (31) reported that tocopherols and tocotrienols in DTP lowers serum cholesterol by supperess-ing the posttranscriptional action of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA)re-ductase, the rate -limiting enzyme in the meva-lonate pathway of endogenous cholesterol synthesis by the liver Economical analysis Table (6): showed non significant difference (P > 0.05) among all groups for the total fixed cost where at the control groups it was 11.65 Growth and economic performance of using dried tomato pomace for Mallard ducks 705 LE/duck and for T2,T3 and T4 total fixed costs were 11.65, 11.64 and 11.51 LE/duck. The total variable costs are significant difference (P < 0.05) among all groups where the large total variable costs was at T1 (70.04 LE/duck) and the lowest one was at T4 (66.06LE/duck). Also in Table (6): showed significant difference (P < 0.05) among all groups for the total returns where at the control groups it was 88.46 LE/duck and for T2,T3 and T4 the total returns were 90.37 91.22, and 91.38 LE/duck respectively. This results indicates the significance using Tomato pomace at 20% for the group four more than 10% and 15%. And the highly total returns in this group may be due to the high final growth weight in this group. Table (6): showed significant difference (P< 0.05) among all groups for the net profit where at the control groups it was 6.76 LE/duck and for T2,T3 and T4 the for the net profit were 10.94, 12.53, and 13.81 LE/duck respectively. This results indicates the significance difference among all groups in the net profit and as showed the higher body weight gain in T4 are due to the high feed intake and consequently high total returns and finally higher net profit. Table (5): showed significant difference (P< 0.05) between all groups for the different economic efficiency measures. The percent of total returns to the total cost for the control groups it was 1.08 and for T2,T3 and T4 were 1.13, 1.15 and 1.17 respectively. Meanwhile The percent of total returns to the total variable cost for T4 groups was 1.37 and for T1, T2 ,and T3 were 1.26, 1.33 and 1.36 respectively. Also in table (5), The percent of net returns to the total cost is higher in T4 group ( 0.17) and for T1, T2 ,and T3 were 0.08, 0.13 and 0.15 respectively. These economic results agree with (32,20). Conclusion Using of Tomato pomace as a percentage of corn has no side effect on the final growth weight of the mallard ducks and through this research work the 20% is more economic than 15% and 10% so we concluded that using the tomato pomace with percentage of 20% of the corn to the ration of the mallard duck and suggested that new research are needed to study the effect of percentage more than 20% as percentage of corn in ration of mallard duck. Conflict of interest The authors declare that they have no conflict of interest. References 1. Ahmed A. A comparative study on the effect of some growth promoters in chickens. Ph.D. Thesis, Pharmacology Dept., Fac. Vet. med., Zagazig University Egypt 2014. 2. Ayhan V, Aktan S. Using Possibilities of Dried Tomato Pomace in Broiler Chicken Diets. Hayvansal Uretim 2004; 45(1): 19-22. 3. Kannan D, Viswanathan K, Mohan B. The effect of feeding Virginiamycin and Lactobacillus sporogenes on broiler production performance characters. Tamilnadu Journal of Veterinary and Animal Sciences 2007; 3 (2): 106 -8. 4. Nobakht A, Safamehr A. The effect of inclusion different levels of dried tomato pomace in laying hens diets] on performance and plasma and egg yolk cholesterol content. J.of Anim. & Vet.Ad-vances 2007; 6(9):1101---6. 5. Ben-Gera I, Kramer, A The utilization of food industries wastes. Advanced Food Research 1969; 17: 77-135. 6. Persia M, Parsons C, Schang M, Azcona J. Nutritional evaluation of dried tomato seeds. Poult. Science 2003; 82: 141-6. 7. El-Hassan M. Effect of some agricultural industrial byproducts on the performance of broiler chicks. Ph.D. Thesis Fac Agric. Zagazig, Univ. Egypt.1999. 8. Soliman A, Abd El-Baset A, Amany A. Utilization of tomato by-products in growing rabbit diets. J. Agric. Sci. Mansoura University 2005; 8: 4469-83. 9. Jafari M, Pirmohammadi R, Bampidis V. The use of dried] tomato pulp in diets of laying hens. Int. J. Poult. Sci. 2006; 5: 618-22. 10. National Research C. Nutrient requirements of domestic animal (Poultry) 4th edition Nat. Acad. Sci. Washinton. D.C.12 1994. 11. Omar M. Economic and productive efficiency of poultry farms in relation to veterinary inputs. M.V.Sc. Thesis, Fac. Vet. Med., Zagazig University 2003. 706 M. A. Omar, T. M. Abdel-Hamid, S. Esam, A. E. Omar 12. Omar M. Economic study on the productive and reproductive efficiency of dairy farms in relation to veterinary management. Ph.D. faculty of Veterinary Medicine Zagazig University 2009. 13. Rosegrant M, Msangi C, Ringler T, Sulser T, Zhu S. International Model for Policy Analysis of Agricultural Commodities and Trade (IMPACT) Model description. Washington, D.C. International Food Policy Research Institute 2008. 14. Buccolo, G. Quantitative determination of serum triglycerides by use of enzymes. Clin Chem 1973; 19 (5): 476-82 15. Naito H, Kaplan A. High density lipoprotein (HDL) Cholesterol. Clin Chem. Toronto. Princeton 1984; 1207-13. 16. Saleh A A, Amber K, El-Magd M A, Atta M S, Mohammed A A, Ragab M M, Abd El-Kader H. Integrative effects of feeding Aspergillus awamori and fructooligosaccharide on growth performance and digestibility in broilers: promotion muscle protein metabolism, Biomed Res Int 2014; 2014: 946859. 17. Abdelhady D H, El-Magd M A, Elbialy Z I, Saleh A A. Bromuconazole-induced hepatotoxicity is accompanied by upregulation of PXR/CYP3A1 and downregulation of CAR/CYP2B1 gene expression, Toxicol Mech Methods 2017; 27(7): 544-50. 18. Doumas B, Watson W, Biggs H, Albumin standards and the measurement of serum albumin with bromocresol green Clin. Chem.Acta 1973; 31:83-7. 19. Doumas B, Biggs H, Determination of serum globulin standard methods of clinical chemistry Edited by cooper, New York, Academic press 1972; (7). 20. Nasser A. Economic Evaluation Of Some Unconventional Rations in Duck Farms Master degree thesis . Faculty of Veterinary Medicine .Zaga-zig University 2014. 21. Atallah S. Effect of cattle diseases on reproductive, productive and economic efficiency of dairy farms. Minufiya Vet. J. 2004; 99-114. 22. Atallah S. Economic and productive efficiency of veterinary management in dairy farms. Ph.D. Degree, Animal Husbandry Department, Faculty of Veterinary Medicine Alexandria University 1997. 23. El-Tahawy A. Cattle diseases and their effects on economic and productive efficiency of dairy farms. Ph.D. Faculty of Veterinary Medicine, Alexandria University 2007. 24. Gilson W, Analysis of mastitis costs. Annual report. UGA Animal and Dairy Science 1995; 182185. 25. Lotfollahian H, Hosseini S. Evaluation of metabolizable energy values of some feeding stuffs Pak.,J.Biol. Sci 2007; 10: 995-7. 26. Lundholm M . Cost-benefit analysis and the marginal cost of public funds. Department of Eco-nomics.Stockholm University 2005. 27. Cooper W, Seiford L, Zhu J , Hand book on Data Envelopment Analysis Springer, Kluwer Academic 2004. 28. Duncan D. Multiple range and multiple F. tests. Biometrics 1955; (11): 1-42. 29. Squires M, Naber E, Toella V. The effect of heat, water, acid and alkali treatment of tomato can-nary waste on growth, metabolizable energy value and nitorgen utilization of broiler chicks. Poult. Sci., 1992; 71: 522--9. 30. Rahmatnejad E, Bojarpour, A, Mirzadeh, K, Chaji A, Ashayerizadeh O. The effect of different levels of dried tomato pomace on broiler chicken haematological indices. Journal of Animal and Veterinary Advances 2009; 8(10): 1989-92 31. Song-Hae B, Sung-Heui L, Yong-Bok P, Ki-Hwan B, Kwang-Hee S, Tae-Sook J, Myung-Sook C. Plasma and hepatic cholesterol and hepatic activities of 3-hydroxy-3-methyl-glutaryl-CoA lower in rats fed citrus peel extract or mixture of citrus bioflavonoids J.Nutr. 1999; 129: 1182-5. 32. Mohamed A, Abdel-Hamid T. Economic Evaluation of using Dried Tomato Pomace (DTP) for Ducks. Proc. of the 6th Animal Wealth Research Conf. in the Middle East & North Africa MENAJAS) ISSN online 2013; 20: 2356-6302. Slov Vet Res 2019; 56 (Suppl 22): 101-16 DOI 10.26813/SVR-811-2019 Original Research Article GROWTH AND ECONOMIC PERFORMANCE, SOME HEMATOLOGICAL, BIOCHEMICAL AND OXIDATIVE STRESS PARAMETERS, AND CARCASS TRAITS OF SUMMER STRESSED CALIFORNIAN AND CROSSBRED RABBITS AS AFFECTED BY DIETARY SUPPLEMENTATION OF FORMIC ACID Tamer M. Abdel-hamid1, Mohamed A. Omar2 1Animal Breeding and Production, Animal Wealth Development, Faculty of Veterinary Medicine, Zagazig University, Egypt, 2Veterinary Economics and Farm Management, Animal Wealth Development, Faculty of Veterinary Medicine, Zagazig University Corresponding author, E-mail: Omarkafafy@yahoo.com Abstract: The aim of present study was to examine the effect dietary supplementation of formic acid on growth performance, some heamatological, biochemical and oxidative stress parameters as well as carcass traits of summer stressed Californian (CAL) and Californian * Rex (CAL * RX) crossbred rabbits. A total of 60 rabbits 4 weeks old were allotted into 2 * 3 completely randomized design (two genotypes; CAL and CAL * RX and three dietary treatment; 0, 0.3 and 0.5% formic acid).Rabbits were subjected to a temperature of 32±1°C during the experimental period, they were slaughtered at 10 weeks of age and blood samples were collected at slaughtering. Rabbits fed diet with 0.5% formic acid had the higher final body weight (p<0.001), body weight gain (BWG) (p<0.001), average daily gain (ADG) (p<0.001) and the lowest feed to gain ratio (p<0.001) compared with those fed diet with 0.3% formic acid and the control rabbits. Rabbits fed diet supplemented with 0.3% formic acid had higher significant total protein (p<0.001) and globulin (p<0.001) than the control rabbits whilst, the highest significant red blood cell (RBCs), white blood cell (WBCs) count and catalase activity have been recorded in 0.5% formic acid group. Carcass traits significantly affected (p<0.05) by dietary supplementation of formic acid were; hot and reference carcass weights, and dressing-out, skin, stomach, intestine, liver, hind part proportions. The effect of genotype * dietary supplement of formic acid was significant for average daily feed intake (ADFI), (p=0.028), cholesterol (p=0.001), Hb (p=0.002), haematocrit percentage (p=0.006) glutathione peroxidase (p=0.026), and dressing-out% (p=0.028). Net profit for CAL and CAL * RX were 8.68 and 10.34 (LE/rabbit) and for 0, 0.3 and 0.5% formic acid were 4.87, 10.11 and 13.72 LE/rab-bit. In conclusion, Formic acid can be used as a dietary supplement at 0.5% of diet to improve economic efficiency, growth traits and health, immune parameters in the blood and improved the antioxidant status without any harmful effect on carcass traits. Key words: formic acid; blood; oxidative stress; carcass; gross income Received: January 2019 Accepted for publication: February 2019 708 T. M- Abdel-hamid, M. A. Omar Introduction Rabbits can be used as a good alternative source of animal protein for humans in the developing countries (1). They characterized by high reproductive output and can digested high fibers diet such as forages and agriculture byproduct to produce high quality meat (2) These meat high in protein and low in fat (3). The problem of bacterial antibiotic resistance arises from use of antibiotics as a feed additive attracts a public health concern (4, 5). This fact encourages the European Union to ban the use of antibiotic as a growth promoters since 2006 (6). So other safe, natural, efficient and economic feed additives that exert the same purpose becomes very interesting. Organic acids can be used as replacement for antibiotics in rabbits' diet (7). There are many benefits of using organic acids as a feed additives such as antifungal property, antibacterial activity against anaerobic pathogens (8), reduced mortality caused by gastrointestinal tract disease (9-11) and stimulate gastrointestinal mucosal growth (12). Also (13) observed that dietary treatment with 0.5% acetic acid or 0.5% lactic acid had a positive effect on body weight gain and feed conversion ratio. In addition to, plasma cholesterol and total lipids have been lowered in summer stressed growing New Zealand White rabbits. (14,15) found that rabbits fed diet supplemented with a blend of microencapsulated formic and citric acids 0.2% in the finishing period (56-77 d) had higher significant ADG compared with control. The diet supplementation with organic acids had no effect on carcass traits (13-18) detected that inclusion of organic acids, butyric acid, calcium formate or its salts in the diet had no effect on internal organs and skin weights, but hot carcass weight and dressing-out percentage were significantly affected. The objective of this study was to study the effect dietary supplementation with formic acid on growth performance, haematological, biochemical and oxidative stress parameters as well as carcass traits in rabbit of different genotype exposed to heat stress. In addition to this, to evaluate the economics of formic acid inclusion in rabbits' feed. Materials and methods Animals, diet and management A total of 60 CAL and CAL x RX of equal sex rabbits about 4 weeks old at the start of this investigation nearly homogenous with average initial body weight of 434.33±17.43 have been subjected to 2 x 3 completely randomized design (two genotypes; 30 of each CAL and CAL x RX, and threes dietary treatments; 20 of each 0, 0.3 and 0.5% of diet formic acid) and ten replicates. The ingredients of the basal diet was firstly prepared then divided into three equal parts. The formic acid was added to the last two parts at 0.3 and 0.5% of the diet. The proximate chemical composition of the basal diets were determined according to AOAC (19). The experimental rabbits were ear tagged, housed in well ventilated house in flat deck arranged metal cages (two rabbit of the same genetic group per cage; one male and one female), the dimensions of the cage were (45 x 45 x 35 cm). The manure was regulatory disposed on a daily bases and a metal feeder and a drip nipple drinker have been provided for each cage. Ration and water were offered for ad libitum consumption. This experiment has been performed during summer months (July and August) of Egypt and inside temperature has been maintained to be averaged 32±1°C. A fourteen hours of day light has been maintained. The fattening period was 6 weeks (from 4 to 10 weeks of age). Growth performance The initial and the final body weights were recorded on individual bases while feed intake were recorded on cage bases and the feed to gain ratio were calculated. Carcass traits All the experimental rabbits were slaughtered at 10 weeks of age in the same day and all the slaughtered procedures follow the world rabbit science association recommendations (20). The rabbits were fasted to about 12 hours. Firstly, the live weights were recorded, then rabbits were stunned and the two jugulars were served. Blood, skin including distal paws and Growth and economic performance, some hematological, biochemical and oxidative stress parameters 709 tail, intestine, stomach were weighed, the remaining was the hot carcass, then chilled in ventilated room at 4°C for 24 hour and then head, liver, heart, lungs, esophagus, trachea, thymus gland, and kidney free of perirenal fat were removed as reference carcass. Finally, the reference carcass was divided into three anatomical parts (fore, mid and hind parts). Blood haematological, biochemical and oxidative stress parameters while slaughtering rabbits two blood samples per rabbit have been collected, on ethylenediaminetetra-acetic acid (EDTA) for haematological studies and one without anticoagulants for plasma separation for biochemical and oxidative stress parameters. Red blood cell count (RBC), white blood cell count (WBC), h aemoglobin (Hb) concentration and haematocrit% were performed as previously described (21-23). For serum separation the blood samples were centrifuges 3000g for 15 min at a temperature of 15-24°C and the samples were stored at -20°C until they were assayed. Total proteins, albumin, triglycerides and cholesterol were determined photometrically by using commercial kits and as previously described (24). The serum level of glu-tathione peroxidase, superoxide dismutase, cat-alase activity and lipid peroxidase were measured as previously detailed (25, 26). Statistical analysis A general linear mode (GLM) of (27) was utilized to analyze the data of growth performance, blood parameters, carcass traits and economic efficiency parameters. The following model used: Yijk = p + Gi + Tj + (GT)ij + eijk Where Yijk is an observation on nth rabbit of each trait, p is the overall mean, Gi is the fixed effect of ith breed (i = 1and 2, i.e. CAL and CAL x rx), Tjis the fixed effect of dietary treatment with formic acid (j = 1, 2 and 3, i.e. 0, 0.3 and 0.5% of diet), GTij is the effect of interaction between genotype and dietary treatment with formic acid level and eijk is the random residual effect. The comparisons between means were performed using (28) at P<0.05. Economic parameters The following indices were calculated per each groups of rabbits: Total fixed costs (TFC), total costs (TC)(30,31). Total return (NR), net profit (NP) and economic efficiency (EE)(29-32). Results Growth performance CAL rabbits had heavier final body weight than CAL x RX (P=0.009) (Table 1).Rabbits fed diet with 0.5% formic acid had the highest significant final body weight (P<0.001), BWG (P<0.001), ADG (P<0.001) and the lowest feed to gain ratio (P<0.001) compared with those fed diet with 0.3% formic acid and the control rabbits (Table 1). The effect of genotype x dietary supplementation was non-significant on all growth traits except for ADFI (Table 1). Blood heamatological, biochemical and ox- idative stress parameters The CAL genotype had higher significant serum triglycerides concentration (P=0.003) than CAL x RX, however, CAL x RX rabbit had higher significant WBC count (P<0.001) and catalase activity (P<0.001) than their CAL counterparts (Table 2). Regardless the rabbits' genetic group, those fed diet with 0.3% formic acid had higher significant total protein (P<0.001) and globulin (P<0.001) than the control rabbits. In contrast, they had lower significant albumin/globulin ratio (P<0.001) than the 0% formic acid group (Table 3). The control rabbits had the highest significant albumin (P<0.001) albumin/globulin ratio, whilst they recorded the lowest significant total protein, globulin, RBC (P<0.001), WBC count (P<0.001) and catalase activity compared with those fed diet supplemented with 0.3% and 0.5% formic acid. The highest significant RBC, WBC count and catalase activity have been determined in 0.5% formic acid group (Table 2). The only blood parameters showed a significant genotype x dietary supplementation with formic acid were; cholesterol (P=0.001), Hb (P=0.002), haematocrit percentage (P=0.006) and Glutathione peroxidase (P=0.026) (Table 710 T. M- Abdel-hamid, M. A. Omar 3). CAL rabbits supplemented with 0.3% formic acid had the highest cholesterol and Glutathione peroxidase concentration whereas, the same genetic group fed diet with 0.5% formic acid had the highest Hb concentration. Rabbits of CAL x RX genetic group had received 0.5% acid had depicted the highest haematocrit percentage. Carcass traits Heavier hot and reference carcasses (P=0.001and P=0.003) have been recorded in CAL rabbits compared with CAL x RX (Table 4). Rabbits supplemented with 0.5% formic acid had the highest significant hot and reference carcass weights (P=0.001), and hind part percentage (P=0.018). However, those fed diet supplemented with 0.3% formic acid had the highest stomach (P=0.001), intestine (P<0.001), liver (P=0.005) and periscapular fat percentages (P=0.008). On the other hand, the control rabbits had the highest skin% (P=0.016) (Table 4). The effect of genotype x dietary supplementation was non-significant on all carcass traits with the exception of dressing out % (P=0.028) (Table 4). Economic parameters Significant genotype x dietary treatment interaction with formic acid had been detected on total feed intake and total feed cost per rabbit (P<0.05), but if the fixed effects of dietary formic acid level and breed considered separately, it is cleared that rabbits fed on diet with 0.5% formic acid revealed lower total feed intake and total feed cost per rabbit than either control or 0.3% formic acid rabbit groups, but not significant. The effect of genotype x dietary treatment interaction on total feed intake and total feed cost per rabbit was non-significant (P>0.05) and different economic measures are presented in (Table 5). Where Net profit for CAL and CAL x RX were 8.68 and 10.34 (LE/rabbit) and for 0, 0.3 and 0.5% formic acid were 4.87, 10.11 and 13.72 LE/rabbit Table 1: Effects of genotype and formic acid on growth performance of rabbits Genotype (G) Variable 'Dietary treatment with formic acid (T) P-value CAL CAL x RX sem 0% 0.3% 0.5% sem G T G x T Rabbits, No. Initial body weight, g Final body weight, g Body weight gain, g Average daily gain, g/d Average daily feed intake, g/d Feed to gain ratio 30 30 20 415.12 453.53 25.26 408.78 1718.03 1676.80 10.82 1528.74c 1276.69 1250.05 27.85 1113.22c 30.39 29.764 0.66 26.50c 118.62 4.077 117.29 2.05 4.01 0.104 119.66 4.58a 20 446.29 1716.43b 1275.13b 30.36b 118.28 4.02b 20 447.91 1847.09a 1401.76a 33.37a 115.928 3.531c 30.93 0.287 0.602 0.987 13.26 0.009 <0.001 0.180 34.11 0.502 <0.001 0.753 0.81 0.502 <0.001 0.754 2.51 0.650 0.573 0.028 0.12 0.673 <0.001 0.862 Means within the same row within the dietary treatment category not sharing the same superscript letter were significantly different at P< 0.05. 1Dietary treatment with formic acid at 0, 0.3 and 0.5% of diet; CAL = Californian; CAL x RX = Californian x Rex; G x T = genotype x dietary treatment interaction; sem = standard error of mean Growth and economic performance, some hematological, biochemical and oxidative stress parameters 711 Table 2: Some blood biochemical, haematological and oxidative stress parameters of Californian and Cal-ifomian x Rex rabbits at 10 weeks of age as affected by formic acid. Results of significant genotype x dietary treatment interaction were summarized in Table 4 Genotype (G) Variable 'Dietary treatment with formic acid (T) P-value CAL CAL x RX sem 0% 0.3% 0.5% sem G T G x T Rabbits, No. 30 30 20 20 20 Total protein (g/dl) 5.70 5.67 0.045 5.30b 5.93a 5.82a 0.055 0.626 <0.001 0.355 Albumin (g/dl) 3.48 3.46 0.028 3.61a 3.42b 3.38b 0.034 0.534 <0.001 0.075 Globulin (g/dl) 2.21 2.21 0.048 1.68b 2.51a 2.43a 0.059 0.922 <0.001 0.482 Albumin / Globulin ratio 1.67 1.62 0.055 2.16a 1.37b 1.41b 0.067 0.549 <0.001 0.391 Triglycerides (mg/dl) RBCs (x106 /mm3) 16.78 16.73 0.013 16.73 16.76 16.75 0.017 0.003 0.378 0.901 4.04 4.07 0.059 3.79b 4.12a 4.26a 0.072 0.709 <0.001 0.555 White blood cells (x109/L) 8.95 9.59 0.11 6.96c 9.14b 11.70a 0.13 <0.001 <0.001 0.485 Lipid peroxidase (nmol/mol xl06) 15.27 15.30 0.15 15.13 15.40 15.34 0.19 0.880 0.577 0.199 superoxide dis-mutase (m/mg) 84.62 85.73 1.69 85.95 86.17 83.41 1.98 0.629 0.554 0.749 Catalase (mg/g) 393.74 578.14 3.21 428.13c 484.09b 545.61a 3.93 <0.001 <0.001 0.228 Means within the same row within the dietary treatment category not sharing the same superscript letter were significantly different at P< 0.05. RBCs = red blood cell count (x106/mm3);1dietary treatment with formic acid at 0, 0.3 and 0.5% of diet CAL = Californian; CAL x RX = Californian x Rex; G x T = genotype x dietary treatment interaction; sem = standard error of mean Table 3: The effects due to interactions between genotype and formic acid on some blood biochemical, haematological and oxidative stress parameters at 10 weeks of age Variable CAL CAL x RX sem P-value 1Control 20.3% 30.5% Control 0.3% 0.5% G T G xT Cholesterol (mmol/L) Hb (g/dL) 0.867c 0.962a 0.910b 0.926b 0.940ab 0.937ab 0.010 0.012 <0.001 0.001 11.283a 10.901b 11.476a 10.908b 11.354a 11.252a 0.117 0.612 0.051 0.002 Haemato- crit percent- 29.36b 30.11a 30.04a 30.11a 29.86a 30.20a 0.148 0.079 0.037 0.006 age GP (mg protein) 110.002ab 120.531a 98.028ab 103.461ab 95.453b 112.881ab 7.156 0.343 0.939 0.026 Means within the same row not sharing the same superscript letter were significantly different at P< 0.05. CAL = Californian; CAL x RX = Californian x Rex; 1 2 3dietary treatment with formic acid at 0, 0.3 and 0.5% of diet; Hb (g/dL) = haemoglobin concentration measured by gram in deciliter; G x T = genotype x dietary treatment interaction; sem = standard error of mean. GP = Glutathione peroxidase. Number of rabbits per group is 10 (5 males and 5 females). 712 T. M- Abdel-hamid, M. A. Omar Table 4: The carcass traits of Califomian and Califomian * Rex rabbits at 10 weeks of age as affected by formic acid level Variable Genotype (G) 1Dietary treatment with formic acid (T) P-value CAL x RX sem sem CAL 0% 0.3% 0.5% G T G xT Rabbits, No. 30 30 20 20 20 Live body weight, g 1718.03 1676.80 10.82 1528.74c 1716.43b 1847.09a 13.26 0.009 <0.001 0.180 Hot carcass, g 2Reference carcass, g 1106.65 1036.40 11.66 945.85c 1056.32b 1212.40a 14.28 0.001 0.001 0.674 881.74 846.35 7.94 757.41c 882.71b 952.02a 9.73 0.003 0.001 0.221 3Dressing-out, % 51.23 50.45 0.82 49.54 51.41 51.56 1.01 0.152 0.005 0.028 % of Live weight Blood 3.07 3.00 0.107 3.06 3.16 2.89 0.131 0.665 0.336 0.821 Skin 17.71 17.46 0.32 18.46a 17.52ab 16.77b 0.39 0.599 0.016 0.264 Stomach 5.67 5.43 0.167 5.77a 6.01a 4.88b 0.20 0.314 0.001 0.264 Intestine 10.50 10.49 0.23 10.65a 11.26a 9.59b 0.28 0.974 <0.001 0.945 % of hot carcass Liver 4.59 4.76 0.18 4.13b 5.21a 4.70ab 0.22 0.509 0.005 0.966 Heat & lung 1.62 1.65 0.04 1.56 1.72 1.62 0.05 0.727 0.087 0.667 Kidney 1.06 1.04 0.03 1.04 1.08 1.02 0.04 0.689 0.610 0.629 Head 10.45 10.49 0.13 10.65 10.42 10.33 0.16 0.835 0.368 0.464 % of reference carcass PSF 0.44 0.44 0.01 0.41b 0.50a 0.42b 0.02 0.936 0.008 0.856 PRF 2.18 2.19 0.06 2.10 2.28 2.17 0.08 0.924 0.326 0.437 Fore part 37.14 37.29 0.42 37.03 37.40 37.21 0.51 0.793 0.879 0.699 Mid part 21.21 21.32 0.39 21.91 21.16 20.73 0.47 0.844 0.223 0.893 Hind part 38.59 38.24 0.25 38.06b 38.01b 39.17a 0.31 0.347 0.018 0.815 Means within the same row within the dietary treatment category not sharing the same letter were significantly different at P< 0.05. CAL = Califomian; CAL * RX = Californian * Rex; 1dietary treatment with formic acid at 0, 0.3 and 0.5% of diet; G * T = genotype * dietary treatment interaction;2Reference carcass = chilled carcass minus head, liver, heart, lungs, esophagus, trachea, thymus gland, and kidney free of perirenal fat;3calculated in relation to reference carcass; PSF = periscapular fat; PRF = Perirenal fat. Growth and economic performance, some hematological, biochemical and oxidative stress parameters 713 Table 5: Effects of genotype and formic acid level on some economic parameters and efficiency for Cal-ifornian and Californian x Rex rabbits Genetic group (G) Variable 1Dietary treatment with formic acid (T) P-value CAL CAL x RX sem 0% 0.3% 0.5% sem G T G xT 30 30 20 20 20 4982.13 4926.36 86.36 5025.69 4968.09 4868.95 105.78 0.650 0.573 0.028 22.42 22.16 0.38 22.61 22.35 21.91 0.47 0.650 0.573 0.028 54.97 53.65 0.34 48.92c 54.92b 59.10a 0.42 0.009 <0.001 0.180 56.01 54.50 0.25 50.20c 56.30b 60.30a 0.78 0.002 <0.001 0.170 40.42 39.16 0.23 40.33 41.19 41.58 0.47 0.530 0.523 0.122 5.00 5.00 0.14 5.00 5.00 5.00 0.02 0.11 0.321 0.180 47.42 44.16 0.22 45.33 46.19 46.58 0.33 0.221 0.273 0.128 8.68 10.34 0.03 4.87c 10.11b 13.72a 0.22 0.03 0.001 0.008 0.140 0.234 0.001 0.107 0.218 0.294 0.02 0.12 0.101 0.11 0.154 0.189 0.021 0.097c 0.179b 0.227a 0.002 0.001 0.002 0.028 Rabbits, No. Total feed intake per rabbit, g 2Total feed cost per rabbit, LE 3Price per rabbit, LE 4Total Returns 5Total variable cost 6Total fixed costs Total costs(LE/rabbit) Net profit (LE/rabbit) Net Profit/ total costs Net Profit / total returns Means within the same row within the dietary treatment category not sharing the same superscript letter were significantly different at P< 0.05. 1Dietary treatment with formic acid at 0, 0.3 and 0.5% of diet; CAL = Californian; CAL x RX = Californian x Rex; LE = Egyptian pound; 2calculated as total feed intake per rabbit multiplied by price per kg of feed (4.5 LE); Calculated as final body weight multiplied by price per kg of live rabbit sold (32 LE); G x T = genotype x dietary treatment interaction; sem = standard error of mean.4 Calculated as price of rabbit plus price of litter. 5Calculated as price of rabbit and all medicaments (LE/ rabbit). 6 Calculated as rent, employments and other fixed costs (LE/ rabbit). Discussion The improvement in body weights, BWG, and ADG and the worseness in the feed to gain ratios after supplementation with organic acids (formic acid) has been seen in this study are in agreement with those reported previously (33,34). The first authors found feed intake to decrease and consequently, improvement feed conversion with the supplementation of diet with citric acid from 0.5% and up to 2.0% whereas, the second author recorded improvement of body weight gains with 0.5% fumeric, citric, and malic acids. However, no response has been recorded on weight gain, but feed intake was lowered and thus the feed conversion was improved in rabbits fed citric acid at g/kg diet (35). Dietary treatment with 0.5% acetic acid or 0.5% lactic acid had a positive effect on body weight gain and feed conversion ratio of summer stressed growing New Zealand White rabbits (13). Similarly, positive effects of organic acids on growth traits have been reported by (36). The addition of 1.5% fumaric acid to rabbits' diet improved daily gain and feed efficiency (37,34). This positive effect of formic acid on growth performance could be attributable to the unfavorable environment to pathogens in the cecum and ileum that the formic acid exert by lowering the ph. Also, it acts as substrates in the intermediary metabolites, increase gastric protein digestion and availability of some elements that complex with (17). There is no doubt that the average final body weights had been recorded in the present study were below the average expected for these animal and for these ages and this could be attributed to the higher temperature to which the rabbits were exposed and this previously documented (38,39). Genetic group x dietary treatment was non-significant on final body weight (40).In a study on New Zealand x Californian mixed-sex rabbits, rabbits fed diet supplemented with a blend of microencapsulated formic and citric acids 0.2% in the finishing period (56-77 d) had higher significant (P=0.019) ADG (48.0 g) compared with control (43.9 g) (8) Rabbits fed diet with organic acids at 1.0 g/kg depicted higher significant body weights at 14 weeks of age and daily body weight gains 714 T. M- Abdel-hamid, M. A. Omar at 7-14 weeks age interval and lower significant feed conversion ratios compared with control. Blood heamatological, biochemical and oxidative stress parameters The significant differences between purebred CAL and CAL* RX crossbred rabbits in the serum level of triglycerides are in accordance with the findings of (41). Whereas, other authors reported that genetic group to have a non-significant effect on blood haematological and biochemical parameters (40, 36). In agreement with (13) they found that dietary supplementation with organic acid had a significant effect on serum globulin and cholesterol concentration. However, (13) observed that dietary supplementation of 0.5% acetic acid or 0.5% lactic acid had a non-significant effects on plasma total protein, albumin, globulin and total lipids, but they significantly lowered plasma cholesterol concentration compared with Control rabbits. The dietary treatment of rabbits with organic acids at 1.0 g/kg had no effect on blood biochemical parameters. Also, blood biochemical parameters not significantly differed in rabbits fed diet supplemented with mixture of formic and propionic acids and those on the basal diet only (16,41). The exposure of rabbits to high temperature leads to stress on these rabbits, leading to increased free radicals and lipid oxidation and these free radicals can lead to irreversible damage to cells if not treated (42).Antioxidant enzymes have an important role in the animals in getting rid of free oxygen radicals that are released as a result of heat stress (43). The rabbits fed diet supplemented with Mucuna Pruriens leaf meal had higher and significant glutathione peroxidase and catalase in the blood these enzymes help these rabbit to cope with stress-induced oxidative destruction (1) Carcass traits The higher significant hot and reference carcass weights of CAL than that of CAL * RX are consistent with those reported by (38), they detected significant differences among genetic groups for the majority carcass traits. Also, the commercial and the reference carcass weights were higher in purebred rabbits than crossbreds at 30°C. The higher periscapular in rabbits fed on diet with 0.3% formic acid collaborate those reported previously (13) who found higher abdominal fat in rabbits supplemented with 0.5% lactic acid than control but not significant. The diet supplementation with organic acids had no effect on carcass traits (13,16,17).Inclusion of organic acids, butyric acid, calcium formate or its salts in the diet had no effect on internal organs and skin weights, but hot carcass weight and dressing out percentage were significantly affected. Any contrasting results with previous studies regarding the effect of organic acid on carcass traits could be explained by different experimental animal, methods of calculations, slaughter age and/or experimental procedures. The non-significant effect of genetic group x dietary treatment on carcass traits are in agreement with those reported by (40). Economic parameters The results showed significant effect of formic acid at 0.5% where the total returns CAL and CAL x RX were 56.01 and 54.5 (LE/rabbit) and for 0, 0.3 and 0.5% formic acid were 50.2, 56.3 and 60.30 LE/rabbit respectively. Meanwhile Net profit for CAL and CAL x RX were 8.68 and 10.34 (LE/rabbit) and for 0, 0.3 and 0.5% formic acid were 4.87, 10.11 and 13.72 LE/rabbit on the side of economic efficiency measurements the Net profit/ total returns were significant at p < 0.05 for CAL and CAL x RX were 0.15 and 0.18 and for 0, 0.3 and 0.5% formic acid were 0.09 , 0.17 and 0.22 that indicated the economic importance of using formic acid at 0.5% for rabbits. Conclusion The dietary supplementation with formic acid at 0.5% of diet has been associated with an improvement in growth traits, health, immunity and antioxidant blood parameters of summer stressed CAL and CAL x RX rabbits without any negative effect on carcass traits. In addition to, the economic efficiency was found to be higher in rabbits fed diet with 0.5% formic acid. Growth and economic performance, some hematological, biochemical and oxidative stress parameters 715 Significant genotype x dietary supplementation with formic acid interactions have been detected for ADFI, cholesterol, Hb, haematocrit percentage, glutathione peroxidase, dressing-out% ,total feed intake, total feed cost, net profit per rabbit and net profit/ total return. Conflict of interest The authors declare that they have no conflict of interest. References 1. Lukefahr S, Cheeke P. Rabbit project development strategies in subsistence farming systems: 1. Practical considerations World Animal Review 1991;68: 60-70 2. McNitt J, Lukefahr S, Cheeke P, PattonN. Rabbit production. 9th edn. (British Library: London) BMC Veterinary Research 2013; 7: 17466148. 3. Dalle Zotte A. Perception of rabbit meat quality and major factors influencing the rabbit carcass and meat quality. Livestock Production Science 2002; 75(1): 11-32. doi: 10.1016/S0301-6226(01)00308-6. 4. Allen S, Boerlin P, Janecko N. Antimicrobial resistance in generic Escherichia coli isolates from wild small mammals living in swine farm, residential, landfill, and natural environments in Southern Ontario, Canada. Applied and Environmental Microbiology 2011;882-8. 5. Azza M, Kamal T, Abd El-Hakim , Amal M. Influence of some organic acids supplementation on growth performance and some biochemical parameters in growing Rabbits. Egypt. J. Comp. Path. & Clinic. Path. 2008; 21: 174 -89 6. Hassanin K, Youssef I. The effect of dietary supplementation of some antioxidants on performance, oxidative stress, and blood parameters in broilers under natural summer conditions. J. World's Poult. Res., 2014; 4: 10-19. 7. Falcao-e-Cunha L, Castro-Solla L, Maertens L, Marounek M, Pinheiro V, Freire L, Mourao J. Alternatives to antibiotic growth promoters in rabbit feeding: a review. World Rabbit Sci. 2007;15: 12740. 8. Rotruck J, Pope A, Ganther H, Hafeman D, Hoekstra W.Selenium: Biochemical role as a component of glutathione peroxidase. Science 1973; 179: 588-90. 9. Huber H, Ziegler D, Pfluger V. Prevalence and characteristics of methicillin resistant coagu-lase-negative staphylococci from livestock, chicken carcasses, bulk tank milk, minced meat, and contact persons 2011. 10. El-Adawy M, El-Aziz M A, El-Shazly K, Ali N G, El-Magd M A. Dietary propionic acid enhances antibacterial and immunomodulatory effects of oxytetracycline on Nile tilapia, Oreochromis niloticus, Environmental Science and Pollution Research. DOI: 10.1007/s11356-018-3206-5 2018. 11. Kishawy A, Amer S, Osman A, Elsayed S, Abd El-Hack M, Swelum A, Ba-Awadh H, Saa-deldin I. Impacts of supplementing growing rabbit diets with whey powder and citric acid on growth performance, nutrient digestibility, meat and bone analysis, and gut health. AMB Express 2018; 8(1) : 86-94. 12. Cardinali R, Rebollar P, Dal Bosco A, Cag-iola M, Crotti S, Scicutella N, Rutili D, Castellini C. Integrazione alimentare di acidi organici ed oli es-senziali microincapsulati nel controllo delle in-fezioni enteriche del coniglio. In Proc.: Giornate di Coniglicoltura ASIC, Forli, Italy, 2007; 137. 13. Scapinello C, de-Faria H, Furlan A, Pedro M. Influence of different levels of fumaric acid and acetic acid on the growing rabbits performance. Revista Brasileira de Zootecnia, 1998; 27: 945-50. 14. Huber H, Ziegler D, Pflüger V. Prevalence and characteristics of methicillin resistant coagu-lase-negative staphylococci from livestock, chicken carcasses, bulk tank milk, minced meat, and contact persons 2011. 15. Michelan A, Scapinello C, Natali M, Furlan A, Sakaguti E, Faria H, Santolin M. Hernandes A.B. Utilizado de probiotico, ácido orgánico e antibiótico em dietas para coelhos em crescimento: ensaio de digestibilidade, avalia^ao da morfometria intestinal e desempenho. Rev. Bras.Zootec., 2002; 31: 2227-37. 16. Dorra T, Ismail F, Sherif K, Rabie M. Growth performance of fattening rabbits as affected by stocking density and added dietary organic acids. J. Animal and Poultry Prod. 2013; 4(5): 249-62. 17. Coles E. Veterinary Clinical Pathology. WB Saunders, Philadelphia, USA, 4th 1986 18. Kirchgessner M, Roth Fx. Ergotrope effekte durch organisch sauren in der ferkelaufzucht und schweinemast.Ubersichten zur Tierenahrung, 1988; 16 (93): 1-8. 19. AOAC. Association of Official Agricultural Chemists. Official methods of analysis. Washington. DC. 1990 20. Blasco A, Ouhayoun J. Harmonization of criteria and terminology in rabbit meat research: revised proposal. World Rabbit Science 1993; 4: 939. 716 T. M- Abdel-hamid, M. A. Omar 21. El-Magd M A, Khamis A, Nasr Eldeen S K, Ibrahim W M, Salama A F. Trehalose enhances the antitumor potential of methotrexate against mice bearing Ehrlich ascites carcinoma, Biomedicine & Pharmacotherapy 2017; 92: 870-8. 22. Singh K. Effect of heat stress on blood constituents in crossbred heifers. The Indian Journal of Animal Sciences 1983; 53: 353-5 23. Bauer J. Numerical evaluation of red blood, white blood cells and platelets, part III, hematology. In 'Clinical laboratory methods and diagnosis'. 7th edn. (Eds S Frankel, S Reitman, AC Somen Wirth) Vol. 1, pp. 1970; 399-402. 24. Saleh A A, Amber K, El-Magd M A, Atta M S, Mohammed A A, Ragab M M, Abd El-Kader H. Integrative effects of feeding Aspergillus awamori and fructooligosaccharide on growth performance and digestibility in broilers: promotion muscle protein metabolism, Biomed Res Int 2014; 2014: 946859. 25. Abdelhady D, El-Abasy M, Abou-Asa S, Elbialy Z, Shukry M, Hussein A, Saleh A, El-Magd M. The ameliorative effect of Aspergillus awamori on aflatoxin B1-induced hepatic damage in rabbits, World Mycotoxin Journal 2017; 10(4): 363-73. Ohkawa H, Ohishi N, Yagi K. Assay for lipid peroxides in animal tissues by thiobarbituric acid reaction. Anal. Biochem. 1979; 95: 351-8. 26. SAS .Statistics analysis system user's guide, release 9.2.' SAS Institute Inc.: Cary, NC 2008. 27. Duncan D. The multiple range and F. tests. Biometrics 1955; 11: 1-45. doi:10.2307/3001478. 28. Atallah S. Economic and productive efficiency of veterinary management in dairy farms. Ph.D. Degree, Animal Husbandry Department, Faculty of Veterinary Medicine Alexandria University 1997. 29. El-Tahawy A. Cattle diseases and their effects on economic and productive efficiency of dairy farms. Ph.D. Faculty of Veterinary Medicine, Alexandria University 2007. 30. Mohamed A, Abdel-Hamid T. Economic Evaluation of using Dried Tomato Pomace (DTP) for Ducks . Proc. of the 6th Animal Wealth Research Conf. in the Middle East & North Africa MENAJAS) ISSN online 2013; 2356 -6302 31. Omar M. Economic and productive efficiency of poultry farms in relation to veterinary inputs. M.V.Sc. Thesis, Fac. Vet. Med. 2003. 32. El-Kerdawy D. Acidified feeds for growing rabbits. Egyptian J. Rabbit Sci. 1996; 6: 143-56. 33. Scapinello C, de-Faria H, Furlan A, Pedro M. Influence of different levels of fumaric acid and acetic acid on the growing rabbits performance. Revista Brasileira de Zootecnia, 1998; 27: 945-50. 34. El-Allawy H. The use of bio-feed additives to improve rabbit performance. Ph.D. Thesis, Cairo Univ. 2001. 35. Khalil M, El-Zarie M.Genetic groups comparisons for growth, carcass, meat quality and blood parameters in program of synthesizing new lines of rabbits. In 'Proceeding of 10th world rabbit congress, 3-6 September,Sharm El-Sheikh, Egypt, World Rabbit Science Association. pp. 2012; 2419. 36. Michelan A, Scapinello C, Natali M, Furlan A, Sakaguti E, Faria H, Santolin M. Hernandes A.B. Utilizado de probiotico, ácido orgánico e antibiótico em dietas para coelhos em crescimento: ensaio de digestibilidade, avalia^áo da morfometria intestinal e desempenho. Rev. Bras.Zootec., 2002; 31: 2227-37. 37. Okab A, El-Banna S. Physiological and biochemical parameters in New-Zealand white male rabbits during spring and summer seasons. Egyptian Journal of Basic and Applied Physiology 2003; 2: 289-300. 38. Okab A, El-Banna S, Koriem A. Influence of environmental temperatures on some physiological and biochemical parameters of male New-Zealand rabbits. Slovak Journal of Animal Science 2008; 41: 12-19. 39. Al-Dobaib S, Khalil M, Hashad M, Al-Saef A.Growth, carcass and caecal traits in V-Line and crossbred rabbits fed diets containing discarded dates. World Rabbit Science 2007; 15: 81-90. 40. Abdel-Azeem A, Abdel-Azim A, Darwish A, Omar E. Haematological and biochemical observations in four pure breeds of rabbits and their crosses under Egyptian environmental conditions. World Rabbit Science.2010; 18:103-10. 41. Meineri G, Giacobini M, Forneris G. Evaluation of physiological parameters of the plasma oxidative status in rabbits. J. Appl. Anim. Res. 2017; 45: 315-9 42. Li S, Zhao M, Jiang T, Lv W, Gao S, Zhou Y, Miao Z. Growth performance and antioxidant status of growing rabbits fed on diets supplemented with Eucommia ulmoides leaves. World Rabbit Sci. 2018; 26: 35-41. Slov Vet Res 2019; 56 (Suppl 22): 717-23 DOI 10.26873/SVR-812-2019 Original Research Article THE ROLE OF L. METHIONINE, L. CARNITINE, CHOLINE AND/OR SILYMARIN IN HEPATOPROTECTION AGAINST PARACETAMOL INTOXICATION AND OXIDATIVE STRESS IN BROILERS Mustafa A. Aziz1, Abu Elnasr A. Zahra1, Zaghloul A. Kheder2, Hend M. Fikry2* department of Pharmacology, Faculty of Veterinary Medicine, Kafrelsheikh University, Egypt, 2Animal Health Research Institute, Mansoura Provinical Lab, Egypt Corresponding author, E-mail: hendfikry999@gmail.com Abstract: Paracetamol (Acetaminophen) was used for long time in poultry as an antipyretic drug and as a growth stimulator. However, high doses of paracetamol cause unpleasant side effects such as hepatorenal toxicity as mirrored by depletion of glutathione reserve, increase lipid peroxidation and increase liver enzymes or even sudden death with lethal doses. Therefore, this study was designated to evaluate the ameliorative effect of L. methionine, L. carnitine, choline and/or silymarin on the hepatotoxic effect induced by high doses of paracetamol. The study was applied on 80 chicks (from 1 till 33 days old) in special cages divided into 8 groups each one contained 10 chicks. The 1st group was used as a control, while the next 2-5 groups were supplemented with L. methionine, L. carnitine, choline and silymarin, respectively with doses as recommended by NRC, the 5th group was supplemented with silymarin (1 g/kg diet), the 6th group was supplemented with a mixture of the 4 supplements, the 7th group (hepatic intoxicated group) was given paracetamol (650 mg/kg diet for 7 days), and the 8th group was administrated paracetamol with a mixture of the 4 supplements. Serum samples were collected to determine levels of lipid profile [triglycerides (TG) and total cholesterol (TC)], liver damage enzymes [alanine transaminase (ALT), aspartate transferase (AST)], lipid peroxidation marker malondialdehyde (MDA), and activity of antioxidant enzymes [glutathione reductase (GR), superoxide dismutase (SOD)]. The obtained results revealed that there was a significant improvement in all measured serum biochemical parameters and final body weight gain in the combined group (4 supplements + paracetamol) as compared to the paracetamol group. These data conclude that supplementation of poultry diets with L. methionine, L. carnitine, choline and silymarin can improve the negative effect of paracetamol through increasing the body weight gain and antioxidant activity of glutathione and superoxide dismutase and decreasing malondialdehyde, liver enzymes, cholesterol and triglycerides. Key words: L. methionine; choline; L. carnitine; silymarin; hepatoprotection; broilers Introduction Paracetamol was used for long time as an antipyretic drug and as a growth stimulator. There was unpleasant side effects of paracetamol with Received: January 2019 Accepted for publication: February 2019 718 M. Aziz, A. Zahra, Z. Kheder, H. Fikry high doses as hepatorenal damage (1). Toxic effect of paracetamol is caused by its toxic metabolite N-acetyl-P-benzoquinone imine which is normally conjugated with glutathione in liver and converted into mercapturic acid which is not toxic and excreted by kidney. High doses of paracetamol cause toxic metabolite accumulation which leads to depletion of glutathione reserve , increases liver enzymes levels in blood, lipid peroxidation and consequently leads to hepatic necrosis (2). There are ongoing trends of improving poultry performance and productivity in ways that are different from antibiotics (3). Supplementing poultry diets with different amino acids improve health status of productive animals especially in organic poultry systems (4). Rapid growth, higher feed intake, lower feed conversion ratio and higher final body weight could be recently achieved by many natural supplements to poultry diets (5). Methionine is an essential sulphur containing amino acid which is important to the health and growth of broilers either when given in natural or synthetic form (6). Poultry performance and body weight are affected by methionine supply but not by the methionine source. While, deficiency of methionine in poultry diets increased the redness value of post mortem tissue (7). High methionine supplemented poultry diet positively affects broiler growth performance and meat quality (8). On the other hand, broiler fed methionine supplemented diet for days then slaughtered and processed showed high meat quality as fresh smell and fresh red color during storage (9). Choline is an important amino acid in poultry nutrition due to its stimulatory effect on growth. Higher choline concentration in poultry diets results in higher growth rate and average daily feed intake (10). Another study by Saun-derson and Mackinlay (11) evaluated the effect of methionine and choline supplementation to poultry diets and found that low methionine level significantly decreased the growth rate and cystathionine beta-synthase enzyme aciv-ity, while high choline level increased significantly choline oxidase enzyme activity. L. carnitine is the main abdominal fat lowering supplementer in poultry diets beside its growth enhancement effect (12). The same results were obtained by Leibetseder (13) who found that feeding chickens with 500 mg L. car-nitine lead to a reduction in the abdominal fat and serum and yolk cholesterol levels and feed conversion ratio but with increased body weight gain and poultry performance. L. car-nitine lowers subcutaneous fat deposition through reducing fat metabolism enzymes activity such as glucouse -6- phosphate dehydro-genase, malic dehydrogenase, isocitrate dehy-drogenase and lipo protein lipase (14). It is well known that silymarin has a prominent hepatocyte protective effect in hepatic intoxication caused by ochratoxin A or paracetamol, via its anti-oxidative and anti-apoptotic effect as well as through increasing superoxide dismutase (SOD) and glutathione peroxidase (GPx) activity and decreasing lipid peroxida-tion product malondialdehyde (15). Moreover, it has a performance enhancement effect as revealed by increasing daily feed intake and final weight gain (16). This study was planned to investigate the potential ameliorative effect of L. methionine, choline, L. carnitine and silymarin on liver intoxication induced by paracetamol high doses through measuring serum AST, ALT, lipid peroxidation product (malondialdehyde), cholesterol and triglycerides levels, SOD and GPx activity. The effect of these 4 supplements on growth performance was also investigated. Materials and methods The experimental protocol was approved by the Animal Care and Use Committee of Faculty of Veterinary Medicine, Kafrelsheikh University. A total number of 80 mixed sex Ross 308 broiler chicks at 1 day old age were obtained from a private poultry company at Kafrelsheikh Governorate, Egypt. All the chicks were immunized against both New Castle's disease on 7th and 18th days and Gumboro disease on 14th day. The experimental birds were offered feed and water ad libitum for 33 days. A starter diet was available for the first 21 days then changed The role of L. methionine, L. carnitine, choline and/or silymarin in hepatoprotection against paracetamol 719 to a grower diet till the end of the experiment (33 days). Diets were formulated to meet the supplement recommendations for broilers by the National Research Council with some modification according to updated nutrients specification of broiler chickens. Chicks were divided into 8 equal groups. The 1st group was used as a control, while the next 2-5 groups were supplemented with L. methionine, L. carnitine, choline and silymarin, respectively with doses as recommended by NRC, the 5th group was supplemented with silymarin (1 g/kg diet), the 6th group was supplemented with a mixture of the 4 supplements, the 7th group (hepatic intoxicated group) was given paracetamol (650 mg/kg diet for 7 days, orally by gastric tube), and the 8th group was administrated paracetamol with a mixture of the 4 supplements. The doses were given each first 3 days of each week till 33 days age. Body weight was recorded at the end of every week. Final body weight gain was calculated following this equation: final body weight gain = final weight- start weight. Blood samples were collected from wing vein. Serum was prepared as previously described (17) and was used to determine SOD, GR, MDA, cholesterol, triglycerides, ALT and AST levels using commercially available kits and as previously described (18-20). Data were analyzed using One Way ANOVA test with post Hoc Tukey test was used to compare between groups using IBM SPSS software package version 20.0. Quantitative data were described using mean ± standard error. Significance of the obtained results was judged at p< 0.05. Results and discussion Effect of the 4 supplements on final body weight gain The present study showed that paracetamol group showed a significant decrease in the final body weight gain as compared to the control group. While L. methionine, L. carnitine, cho-line and the 4 supplements combined groups showed a significant increase in the final body weight compared to the control group. Moreover, chicken administrated the 4 supplements and paracetamol showed a significant increase in final body weight compared to the paracetamol group without statistical change relative to the control group (Table 1). This means that these 4 supplements had the ability to improve the reduced body weight induced by paracetamol. In consistence with our findings, Jahanian and Khalifeh-Gholi (21) also reported that supplementation of broiler diets with methionine at NRC recommendation levels Ross 308 broiler chicks led to increasing feed conversion ratio and final body weight. Effect of the 4 supplements on triglycerides and total cholesterol The present study reported that paracetamol group showed a significant increase (p<0.001) in TG and TC levels as compared to the control group (Table 1). L. methionine, choline, L. car-nitine and combined groups showed a significant decrease in TG and TC levels, with best effect for combined group, as compared to the control group. Moreover, chicken administrated the 4 supplements and paracetamol showed a significant decrease (p<0.002) in TG and TC levels as compared to the paracetamol group without statistical change relative to the control group (Table 1). This means that these 4 supplements had the ability to reduce the elevated TG and TC levels triggered by paracetamol. In agreement, Jahanian and Ashnagar (22) reported that chicks fed diet supplemented with choline and L. carnitine had a decreased feed conversion ratio and leg fat content and total plasma lipid level. Similarly, Khajali and Khajali (23) also found a decreasing in total plasma cholesterol and abdominal fat deposition in chicks fed diet supplemented with 200 mg /kg of L. carnitine. Effect of the 4 supplements on ALT and AST The paracetamol group showed a significant increase (p<0.001) in ALT and AST levels as compared to the control group. While chicks fed on diet supplemented with the 4 supplements each alone or in combination, with best effect for combined group, showed a significant decrease level of these two liver enzymes as compared to the control group. Moreover, 720 M. Aziz, A. Zahra, Z. Kheder, H. Fikry chicken administrated the 4 supplements and paracetamol showed a significant decrease (p<0.001) in ALT and AST as compared to the paracetamol group without statistical change relative to the control group (Table 2). This indicates that these 4 supplements had the ability to decrease the elevated AST and ALT elevated by paracetamol. In support, Tedesco, et al. (24) also reported a similar anti-hepatotoxic effect for silymarin (600 mg/kg) as revealed by reduction in AST and ALT level in chicks. Additionally Selvan et al. (25) reported that broiler diets deficient in choline resulted in elevation of liver enzymes and liver histopathology showed many abnormalities and fatty liver. Table 1: Effect of the 4 supplements on final body weight gain and lipid profile parameter Final body weight gain (g) Triglyceride fog/dl) Cholesterol (Mg/dl) Control 1802.0± 39.6 b 17.04± 0.77 b 31.56± 2.29 b L. methionine 1971.0± 29.7 a 13.88± 0.48 c 24.92± 2.05 c Choline 1946.0± 59.4 a 12.54± 1.24 c 23.72 ±3.73 c L. carnitine 1932.6± 24.6 a 12.82± 0.94 c 25.34± 2.31 c Silymarin 1924.0± 22.2 a 16.32± 0.78 b 28.08± 3.45 b 4 supplements 1994.0± 50.3 a 12.10± 1.68 c 20.44± 2.33 Paracetamol 1578.0± 25.9 c 21.08± 0.86 a 41.92± 2.69 a 4 supplements + paracetamol 1741.0 ±46.7 b 16.66± 0.79 b 31.02 ±2.18 b Data are presented as mean ± SEM. Means carrying different superscript letters are significantly different at p < 0.05 Table 2: Effect of the 4 supplements on liver damage enzymes and oxidant/antioxidant status ALT (U/L) AST (U/L) GR (U/L) SOD (U/L) MDA (nm/ml) Control 36.58± 3.39 b 132.81± 3.37 b 51.86± 1.7 b 65.30± 1.63 b 6.24± 0.75 b L. methionine 28.68± 4.16 c 118.28± 5.54 c 61.54± 2.8 a 69.90± 1.88 a 4.84± 0.36 c Choline 28.16± 3.72 c 117.32 ±5.18 c 59.08± 2.03 a 72.48± 2.85 a 6.06± 0.27 b L. carnitine 27.14± 4.74 c 113.26± 5.04 c 58.26±2.89 a 69.90± 3.15 a 6.18± 0.36 b Silymarin 28.68± 4.46 c 118.82± 3.80 c 59.12± 3.21 a 70.32± 2.09 a 6.08± 0.30 b 4 supplements 16.12± 4.27 d 83.96± 4.89 d 61.5 ±3.11 a 71.98± 3.29 a 5.16 ±0.65 c Paracetamol 69.78± 3.42 a 174.74± 5.8 a 40.34 ±3.16 c 47.56± 6.80 c 8.96 ±0.69 a 4 supplements + paracetamol 40.46± 4.43 b 135.04± 4.73 b 49.0± 1.8 b 62.08± 4.79 b 6.72 ±0.48 b Data are presented as mean ± SEM. Means carrying different superscript letters are significantly different at p < 0.05 The role of L. methionine, L. carnitine, choline and/or silymarin in hepatoprotection against paracetamol 721 Effect of the 4 supplements on glutathione reductase and superoxide dismutase Chick fed diet supplemented with paracetamol exhibited a significant decrease in serum level of glutathione reductase (GR) and superoxide dismutase (SOD) as compared to the control group (Table 2). While L. methionine, choline, silymarin and combined groups showed a significant increase in these two enzymes, with best effect for combined group, as compared to the control group. Moreover, chicken administrated the 4 supplements and paracetamol showed a significant increase in GR and SOD as compared to the paracetamol group without statistical change relative to the control group. This indicates that these 4 supplements had the ability to increase the activity of antioxidant enzymes that was increased by paracetamol. Consistent with these results, Zhang, et al. (26) denoted that increasing methionine level above NRC recommendation for 500 male broiler chickens from one day old to 26 days of age led to increasing total glutathione and reduced glu-tathione in blood and breast muscle. Also, Jan-kowski, et al. (27) reported that increasing me-thionine level in broiler diets resulted in a significant increase in glutathione concentration and decreasing both malondialdehyde and plasma triglycerides levels. Furthermore, Ruan, et al. (28) reported that 120 one day old broilers supplemented with methionine deficient diet resulted in decreasing SOD, catalase and glutathione peroxidase. Similarly, Wang, et al. (29) reported that adding L. carnitine at a dose of 100 mg/kg to broilers diet decreased MDA level in heart tissue, triglycerides content and increased SOD and GR. Effect of the 4 supplements on malondialde- hyde The paracetamol group showed a significant increase in malondialdehyde (MDA) level as compared to the control group. Only L. methi-onine and combined groups showed a significant decrease in MDA relative to the control group. Moreover, chicken administrated the 4 supplements and paracetamol showed a significant decrease in MDA as compared to the para- cetamol group without statistical change relative to the control group (Table 2). In agreement, Park, et al. (30) also reported that increasing methionine level during first 28 days of age of chicks resulted in decreasing plasma MDA and increasing total plasma glutathione level. In support, Mohammadi, et al. (31) also recorded that methionine supplementation in broilers at 1-42 days of age at a concentration of 80 mg/kg of diet decreased plasma MDA level, reduced abdominal fat and plasma cholesterol level. Conclusion The present study reported that supplementation of poultry diets with L. methionine, L. carnitine, choline and silymarin improved chicken final body weight gain and enhance the negative impact of paracetamol through increasing the activity of glutathione reductase and superoxide dismutase and decreasing malondialdehyde, liver enzymes, cholesterol and triglycerides. Conflict of interest The authors declare that they have no conflict of interest. References 1. Savita Marmat, Taj N Qureshi and Rathore H. Lethal doses of acetaminophen (paracetamol)for young broiler chicks. Indian Journal of pharmaceutical science and research 2015; 23-6. 2. Joulideh Pour Amin Reza. Effects of Acetaminophen as toxic drug on the liver function in poultry. Journal of cancer prevention and current research 2016; 4(4): 100-28. 3. Gadde U, Kim W, Oh S and Lillehoj H. Alternatives to antibiotics for maximizing growth performance and feed efficiency in poultry. Animal Health Research Review 2017;18(1):26-45. 4. Mehri U, Herbert U and Mercier A. Addition of amino acids in poultry. Animal Health Research Review 2016; 12(1):23-35. 5. Saleh A A, Amber K, El-Magd M A, Atta M S, Mohammed A A, Ragab M M, Abd El-Kader H. Integrative effects of feeding Aspergillus awamori and fructooligosaccharide on growth performance and digestibility in broilers: promotion muscle protein metabolism, Biomed Res Int 2014; 2014: 946859. 6. Hayat Z , Rehman A , Akram K , Farooq U and Saleem G. Evaluation of a natural methionine 722 M. Aziz, A. Zahra, Z. Kheder, H. Fikry source on broiler growth performance. Journal of Science and Food Agriculture 2015; 95 (12): 2462-6 7. Conde-Aguilera J, Cholet J, Lessire M, Mercier Y, Tesseraud S, and van Milgenj. The level and source of free-methionine affect body composition and breast muscle traits in growing broilers. Poultry Science 2016; 95(10):2322-31. 8. Wen C, Jiang X , Ding L , Wang T and Zhou Y. Effects of dietary methionine on growth performance, meat quality and oxidative status of breast muscle in fast- and slow-growing broilers. Poultry Science 2017; 96(6):1707-14. 9. Albrecht A , Herbert U, Miskel D, Heinemann C, Braun C, Dohlen S, Zeitz J, Eder K , Saremi B and Kreyenschmidt J . Effect of methionine supplementation in chicken feed on the quality and shelf life of fresh poultrymeat. Poultry Science 2017; 96(8):2853-61. 10. Siegert W, Ahmadi H, Helmbrecht A and Rodehutscord M. A quantitative study of the interactive effects of glycine and serine with threonine and choline on growth performance in broilers. Poultry Science 2015; 94(7):1557-68. 11. Saunderson C and Mackinlay J. Changes in body-weight, composition and hepatic enzyme activities in response to dietary methionine, betaine and choline levels in growing chicks. British Journal Nutrition 1990; 63(2):339-49. 12. Rehman Z , Chand N and Khan R . The effect of vitamin E, L-carnitine, and ginger on production traits, immune response, and antioxidant status in two broiler strains exposed to chronic heat stress. Environment Science Poultry Research Institute 2017; 24(34):26851-7. 13. Leibetseder J. Effects of L-carnitine in poultry. Arch Tierernahr 1995; 48(1-2):97-108. 14. Xu Z , Wang M , Mao H , Zhan X and Hu C. Effects of L-carnitine on growth performance, carcass composition, and metabolism of lipids in male broilers. Poultry Science; 2003 82(3):408-13. 15. Yu Z, Wu F, Tian J, Guo X and An R. Protective effects of compound ammonium glycyr-rhizin, L-arginine , silymarin and glucurolactone against liver damage induced by ochratoxin A in primary chicken hepatocytes. Mol Med Rep 2018; 18(3):2551-60. 16. Jahanian E, Mahdavi A , Asgary S and Jaha-nian R . Effects of dietary inclusion of silymarin on performance, intestinal morphology and ileal bacterial count in aflatoxin-challenged broiler chicks. Journal of Animal Physiology and Animal Nutrition 2017; 101(5):e43-54. 17. El-Magd M A, Khamis A, Nasr Eldeen S K, Ibrahim W M, Salama A F. Trehalose enhances the antitumor potential of methotrexate against mice bearing Ehrlich ascites carcinoma, Biomedicine & Pharmacotherapy 2017; 92: 870-8. 18. Saleh A A, Amber K, El-Magd M A, Atta M S, Mohammed A A, Ragab M M, Abd El-Kader H. Integrative effects of feeding Aspergillus awamori and fructooligosaccharide on growth performance and digestibility in broilers: promotion muscle protein metabolism, Biomed Res Int 2014; 2014: 946859. 19. Alzahrani F A, El-Magd M A, Abdelfattah-Hassan A, Saleh A A, Saadeldin I M, El-Shetry E S, Badawy A A, Alkarim S. Potential Effect of Exo-somes Derived from Cancer Stem Cells and MSCs on Progression of DEN-Induced HCC in Rats, Stem Cells International 2018; 2018: 17 https://doi.org/10.1155/2018/8058979. 20. Elkeiy M, Khamis A, El-Gamal M, Abo Ga-zia M, Zalat Z, El-Magd M. Chitosan nanoparticles from Artemia salina inhibit progression of hepato-cellular carcinoma in vitro and in vivo, Environ Sci Pollut Res Int. doi: 10.1007/s11356-018-3339-6 2018. 21. Jahanian R and Khalifeh-Gholi M. Marginal deficiencies of dietary arginine and methionine could suppress growth performance and immuno-logical responses in broiler chickens 2018; 102(1):e11-20. 22. Jahanian R and Ashnagar M. Effects of dietary supplementation of choline and carnitine on growth performance, meat oxidative stability and carcass composition of broiler chickens fed diets with different metabolisable energy levels. 2018; 59(4):470-6. 23. Khajali F and Khajali Z. L-Carnitine supplementation decreases lipid peroxidation and improves cardiopulmonary function in broiler chickens reared at high altitude .Acta Vet Hung 2014;62(4):489-99. 24. Tedesco D, Steidler S, Galletti S, Tameni M, Sonzogni O and Ravarotto L. Efficacy of silymarin-phospholipid complex in reducing the toxicity of af-latoxin B1 in broilerchicks. Poultry Science 2004; 83 (11): 1839-43. 25. Selvan R, Saravanakumar M, Suresh S, Chandrasekeran C and Prashanth D. Evaluation of polyherbal formulation and synthetic choline chloride on choline deficiency model in broilers: implications on zootechnical parameters, serum biochemistry and liver histopathology. Asian-Australas Journal of Animal Science 2018; 31(11):1795-806. The role of L. methionine, L. carnitine, choline and/or silymarin in hepatoprotection against paracetamol 723 26. Zhang S, Gilbert E , Saremi B and Wong E. Supplemental methionine sources have a neutral impact on oxidative status in broiler chickens. Journal of Animal Physiology and Animal Nutrition 2018; 102(5): 1274-83. 27. Jankowski J, Ognik K, Kubinska M, Czech A, Juskiewicz J and Zdunczyk Z. The effect of DL, L-isomers and DL-hydroxy analog administered at 2 levels as dietary sources of methionine on the metabolic and antioxidant parameters and growth performance of broilers. Poultry Science 2017; 96(9):3229-38. 28. Ruan T, Li L, Lyu Y, Luo Q, Wu B. Effect of methionine deficiency on oxidative stress and apoptosis in the small intestine of broilers. Acta Vet Hung 2018; 66(1):52-65. 29. Wang Y, Ning D, Peng Y and Guo Y. Effects of Dietary L-carnitine Supplementation on Growth Performance, Organ Weight, Biochemical Parameters and Ascites Susceptibility in Broilers Reared Under Low-temperature Environment Asian-Aus-tralas Journal Animal Science 2013; 26(2):233-40. 30. Park I, Pasquetti T, Malheiros R , Ferket P and Kim S . Effects of supplemental L-methionine on growth performance and redox status of broilers compared with the use of DL-methionine. Poultry Science 2018; 97(1):102-9. 31. Mohammadi V, Ghazanfari S, Mohammadi-Sangcheshmeh A and Nazaran M. Comparative effects of zinc-nano complexes, zinc-sulphate and zinc-methionine on performance in broiler chickens. British Poultry Science 2015; 56(4):486-93. Slov Vet Res 2019; 56 (Suppl 22): 725-34 DOI 10.26873/SVR-813-2019 Original Research Article CHARACTERIZATION OF CLASS 1 INTEGRONS AND SOME ANTIMICROBIAL RESISTANCE GENES IN Salmonella SPECIES ISOLATED FROM POULTRY IN EGYPT Safaa M. Shabana1*, Salwa M. Helmy2, Abd El-Halem M. Hegazy3 1Department of Microbiology, Animal Health Research Institute, Kafr El Sheikh branch, Agriculture research center, Giza, Egypt, department of Bacteriology, Mycology and immunology, Faculty of Veterinary Medicine, Kafrelsheikh University, Egypt, department of Poultry Disease, Animal Health Research Institute, Kafr El Sheikh branch, Agriculture research center, Giza, Egypt *Corresponding author, E-mail: sofy.shabana@yahoo.com Abstract: Salmonella enterica includes serotypes that were implicated as a food borne pathogens crucially affecting public health and the economic organization. This study was directed to isolate and identify of Salmonella strains from 222 different species and ages of poultry (broiler, chick, ducks, pigeon, quails) from Kafr El Sheikh governorate. The Salmonella isolation rate was (4.5%) as (0.9%) from apparently healthy, (3.6%) from diseased birds. The outer membrane protein F gene was used as promising tool for detection of Genus Salmonella, after that four isolates were identified serologically as two Salmonella enterica serovar Enteritidis and two Salmonella enterica serovar Typhimurium. The resistance pattern of positive Salmonella isolates showed multidrug resistance pheno-types and qnrS for quinolone resistant genes was recorded in one isolate while blaTEM for p-lactam resistant isolates, aacC for aminoglycosides were recorded in all four Salmonella isolates using PCR technique. Also, Class 1 integrons detected with a percentage of (100%) in examined isolates. Sequencing of the class 1 integrons cassettes showed genes encoding resistance specified to streptothricin acetyltransferase (sat) gene, aminoglycoside acetyltransferase (aac3-Id) and aminoglycoside adenyltransferase (aadA7). Class 1 integrons harbored gene encoding domains unfunction protein (duf) in one s. enterica serovar Typhimurium isolate. This study spotlights the significant role of the drug-resistance genes and Class 1 integrons in Salmonella as zoonotically important pathogens of public health importance. Key words: poultry; Salmonella; drug- resistance genes; integrons gene cassettes Introduction Salmonella include approximately 2500 serovars. Salmonella enterica represents the most of the Salmonella serovars and Salmonella enterica serovar Enteriditis was the most popu- lar serovar with a zoonotic effect, then Salmonella enterica serovar Typhimurium (1). Globally, Salmonella enterica subsp. Enterica included serotypes that have economically and public health significantly effects (2). The most non-typhoidal salmonellosis (NTS) cases related to consuming of contaminated animal Received: January 2019 Accepted for publication: February 2019 726 S. Shabana, S. Helmy, A. E. Hegazy origin foods, especially fowl, meat and in some cases vegetables (3). Poultry considered an important reservoir of many zoonotically important pathogens, such as Salmonella, which acted as a prime importance (4). The pore-forming proteins of Salmonella and other Gram negative bacteria outer membrane (OM) called porins (5). Among OMPs (outer membrane proteins), the outer membrane protein F (ompF) and outer membrane protein C (ompC were the most types porins that represented 2% of the total porins, and ompF was the most ideal structural and functional characterization porin protein (6). Also, the ompF gene was used as a promising tool for detection of Salmonellae where it could discriminate genus Salmonella from other non-Salmonella organisms in clinical samples (7). Multidrug resistant (MDR) non-typhoidal Salmonella (NTS) might be transmitted from the poultry to human through the food series, whilst the antimicrobial resistance (AMR) could be carried among bacteria throughout the resistance genes associated with integrons and another mobile genetic elements as plasmids and transposons (8). Avian Salmonella showed resistance against many antimicrobial groups such as P-lactam, aminoglycosides and quin-olones (9). Salmonella species associated with qnr genes were isolated from the poultry field might cause a harmful effect on the public health because these could be transmitted to humans via poultry products or by contact with poultry and could rapidly increase fluoroquinolone resistance in various bacterial species through the transfer of plasmids harboring qnr genes. (10). The resistance to aminoglycosides as Gentami-cin could confer using the aminoglycoside acetyltransferase (aac) genes which were detected in numerous isolates of Salmonella (11). The class 1 integrons played a character in the presence of AMR in Salmonella enterica which might isolate from broilers, meat and hogs products (12). Class 1 integrons, the most communal integron located on Salmonella ge-nomic island 1 (SGI 1), was found in various Salmonella serovars, including S. enterica serovar Typhimurium; S. enterica serovar Newport and S. enterica serovar Oslo. (13). The classes of integrons which might be recovered from GenBank were nine, but the first four categories had been sustained only. Cass 1 integrons was widely distributed among the family Enterobacteriaceae organisms (14). These integrons include two conserved segments (5' CS and 3' CS) separated by a variable region that normally comprises one or more gene cassettes. Integrons encompass three important parts: an integrase gene (IntI1); an adjacent attachment site (attI1) and a promoter region (PC) (15). An open reading frame (ORFs) where a specific site containing a modular structure called gene cassettes (16). The collection of gene cassettes (up to nearly half a dozen) had related the integrons with MDR (17). This study highlighted the importance of the strains of Salmonella, which isolated from different species and age poultry as zoonotically important pathogen, some antimicrobial drug resistance genes of Salmonella species and class 1 integrons gene cassettes in this public health importance organism. Materials and methods Collection of samples and isolates characterization In this work, which was conducted from April 2017 to April 2018, a total of 222 apparently healthy (56) and diseased (166) from different species and ages of poultry (Broiler, chick, ducks, pigeon, quails) from the Kafr El Sheikh governorate. The internal organs (liver, spleen, gall bladder, ileocaecal tonsil, yolk sac), cloacal swabs and the pooled samples. Samples were transferred to the laboratory in an ice tank within 2hours for bacteriological isolation and identification (18), then confirmed biochemically by the API 20E system. Serological identification Biochemically suspected isolates were sero-typed according to Kauffman (19) at Serological unit in Institute of Animal Health Re-search,Giza, Egypt. Characterization of class 1 integrons and some antimicrobial resistance genes in Salmonella species 727 Identification of genus Salmonella using ompF gene Programming of PCR to amplify ompF gene was used as promising tool for detection of genus Salmonella was done according to Ta-tavarthy and Cannons (20) using oligonucleotide primers in Table 1. Antimicrobial susceptibility The susceptibility test of samples were done as Finegold and Martin, (21). A total of 11 antimicrobial discs was used for sensitivity (Ox-oid) were Amoxicillin-clavulanic acid (AMC), 30 pg; Cefotaxime (CTX), 30 pg; Ceftazidime (CAZ), 30 pg; Chloramphenicol (C), 30 pg; Ciprofloxacin (CIP), 5 pg; Gentamicin (CN), 10 pg; Nalidixic acid (NA),30 pg; Spectinomy-cin (SH), 10 pg; Colistin (CT), 10 pg; Norfloxacin (NOR), 10 pg and Doxycycline (DO), 30 pg. Interpretation as resistant, moderately susceptible or susceptible as recorded in the Clinical and Laboratory Standards Institute CLSI (22). Molecular analysis of antimicrobial resistance genes The DNA extraction was done using QI-Aamp DNA Mini Kit (Catalogue no. 51304) according to manufactures' guidelines. The primer sequences for detection of aacC gene (encoded for aminoglycoside resistance) (23), qnrS gene (encoded for quinolones resistance) (24), blaTEM gene (encoded for P-lactams resistance) (25) and class 1 integrons gene cassettes (26) (Table 1). Sequencing screen for class 1 integrons gene cassettes QIAquick kit. (Qiagen Inc. Valencia, CA): It was used for purification of the PCR product from 1.5 % agarose gels. Applied Biosystems 3130 automated DNA Sequencer (ABI, 3130, USA). Identification similarity of nucleotide and amino acid sequences between Salmonella strains and other Enterobacteriacae recorded in GenBank was done using (National Center for Biotechnology Information "NCBI"). Using the BioEdit sequence alignment editor for compar- isons of the nucleotide sequences (27). Phylo-genetic analysis was done using ClustalW (http://www.ebi.ac.uk/clustalw/). Results The incidence of Salmonellae from different samples The obtained results of Salmonella isolation revealed that 10 (4.5%) were positive for Salmonella identified biochemically out of 222 examined birds, The isolation rates from chicken, ducks and quails were 8 of 156 samples (5.1%), 1 of 35 samples (3.2%) and 1 of 2 samples (50%), respectively, while could no isolation of Salmonella from chick and pigeon samples. The positive biochemically Salmonella isolates from different samples represented in 4 out of 100 (4%), 2 out of 51 (3.9%), 2 out of 36 (5.5%), 1 out of 19 (5.2%) and 1 out of 2 (50 %) from cloacal swabs, pooled samples, liver, gall bladder and yolk sac samples respectively, while the isolation from the spleen and ileoce-cal tonsil samples was negative for Salmonellae (Table 2). Serotyping of Salmonella isolates Four isolates from ten biochemically positive suspected Salmonella isolates were classified under two different serotypes, including two Salmonella enterica serovar Enteritidis were isolated from cloacal swab of chicken and duck and Salmonella enterica serovar Typhi-murium isolated from the quail yolk sac and chicken liver samples. Antimicrobial susceptibility Salmonella isolates showed resistance to Gentamycin, Ciprofloxacin, Doxycycline, Spectinomycin and Colistin with (50%), however, showed sensitive to Ceftazidime with (100%), followed by Cefotaxime by (75%), amoxicillin clavulinic acid, Nalidixic acid, Chloramphenicol and Norofloxacine with (50%) (Table 3). Two non-typhoidal Salmonella isolates showed multidrug resistant (MDR) phenotypes to five different antibiotic classes (Table 3). 728 S. Shabana, S. Helmy, A. E. Hegazy Detection of genus Salmonella using OmpF gene by PCR All examined Salmonella isolates was positive at 519 bp of ompF using the PCR technique with a percentage of (100%) (Fig. 1). Antimicrobial resistance encoding genes The phenotypic antimicrobial resistant Salmonella isolates was analyzed by PCR technique to key out some resistance coding genes. The positive percentage of qnrS gene for quin-olone resistant was (25%), where blaTEM for P-lactam resistant gene, aacC for aminoglyco-sides resistant gene and Class 1 integrons were (100%) (Fig. 1). Class 1 integrons sequencing of the variable amplicons showed the gene cassettes containing streptothricin acetyltransferase (sat) gene encoding resistance against Streptothricin (an early aminoglycoside) in two Salmonella serovars isolated in the current work, but ami-noglycoside acetyltransferase (aac(3)-Id) and aminoglycoside adenyltransferase (aadAl) genes which encoding resistance against Gen-tamycin and to streptomycin and spectinomy-cin, respectively in isolate of S. enterica serovar Typhimurium only. One S. enterica serovar Typhimurium isolate Class 1 integrons harbored gene encoding domains of unknown function protein (duf). Table 1: Oligonucleotide primers used for detection of ompF , antimicrobial resistance coding genes (aacC, qnrS and blaTEM) and class 1 integrons cassettes Gene Primer sequence (5'-3') Length of amplified product Reference ompF Forward- CCTGGCAGCGGTGATCC 519 bp Tatavarthy and Can- Reverse- TGGTGTAACCTACGCCATC nons,(20) aacC Forward- GGCGCGATCAACGAATTTATCCGA 448 bp Lynne et al., (23) Reverse- CCATTCGATGCCGAAGGAAACGAT qnrS Forward- ACGACATTCGTCAACTGCAA 417 bp Robicsek et al., (24) Reverse- TAAATTGGCACCCTGTAGGC blaTEM Forward- ATCAGCAATAAACCAGC 516 bp Colom et al., (25) Reverse- CCCCGAAGAACGTTTTC class 1 integron Forward- GGC ATC CAA GCA GCA AG Variable Sow et al., (26) cassettes Reverse- AAAG CAG ACT TGA CCT GA Table 2: The incidence of Salmonellae isolated from different organs and identified biochemically Poultry Organs samples Samples positive sam- species No. ples Cloacal swab *Pooled samples liver Spleen Gall bladder Yolk sac iliocecal tonsil No. % Chicken 67 34 29 7 17 - 2 156 8 5.1% Chicks 6 5 2 2 - 1 3 19 - 0% Duck 19 10 4 - 2 - - 35 1 3.2% Pigeon 8 2 - - - - - 10 - 0% Quails - - 1 - - 1 - 2 1 50% Total 100 51 36 9 19 2 5 222 10 4.5% (4%) (3.5%) (5.5%) (0%) (5.2%) (50%) (0%) *Pooled samples from different organs of poultry submitted to Kafr El Sheikh lab. For examination Characterization of class 1 integrons and some antimicrobial resistance genes in Salmonella species 729 Table 3: Antimicrobial resistance patterns, resistance genes and class 1 integron profiles of Salmonella serotypes in this study NO Serovars Resistance pattern **MDR Resistance Integron Genes Accession (source of isolates) isolates N (%) genes amplicon size (bp) cassettes numbers 1 S. enterica serovar Enteritidis (duck) CT - blaTEM, aacC + - - 2 S. enterica serovar Enteritidis (chicken) CN, DO, C, CT, CIP + blaTEM, aacC 600 sat MK335377 3 S. enterica serovar SH - blaTEM, 800 duf gene MK359461 4 Typhimurium (quail) S. enterica serovar CN, NOR, DO, + aacC blaTEM, 650 sat MK349001 Typhimurium (chicken) AMC, CIP, SH aacC, qnrS 1600 1800 aac3-Id, aadA 7 aadA7 MK349002 MK359462 Total_2(50%)_ **Multidrug resistant (MDR) Salmonella isolates were 2(50%) to five different antibiotic classes Figure 1: PCR amplification of the different genes in this study; "Pos" stands for positive control, "Neg": Negative control; L: 100 bp DNA ladder; Lane (1, 2, 3, 4) examined Salmonella isolates. A. ompFgene(519 bp). Resistance associated genes, B. aacC gene (448bp). C. qnrS gene (417bp). D. blaTEM gene (516 bp). E. Class 1 integrons at variable sizes in Salmonella isolates 730 S. Shabana, S. Helmy, A. E. Hegazy Discussion Salmonella represents a critical problem to livestock in countries where not interest to the control measures or in those where the environmental conditions help in the development of these microorganisms (28). In the present study, Salmonella spp. were isolated and identified from different species and ages of poultry and molecular characterized for many important antimicrobial resistance genes and class 1 integrons of Salmonella species. The results indicated that 10 (4.5%) isolates out of 222 examined bird suspected to be Salmonella isolates from 166 diseased birds and 56 apparently healthy birds with the percentage of (3.6%) and (0.9%), respectively by phenotypic and biochemical characterization that agree with report in Egypt where 4.4% were positive for Salmonella isolated from poultry farms (7), but higher than those of Salmonella isolation from small poultry farms with (1.6%) in California (29), and lower than (8.65%) of Salmonella isolated from poultry (30) . The consequence of isolation appears to be high from the diseased bird than apparently healthy bird 8vs 2, although the samples were gathered up from each of diseased and apparently healthy birds together. These variations in the overall prevalence of Salmonella may be related to several factors such as environment, hygienic conditions of the farm and health status of the examined bird (31, 32) which leading to the bird become weaker and therefore are easily infected by Salmonella. Similarly, Salmonella was isolated from apparently healthy chickens lower than from diseased chickens in Shanghai and in Egypt (32, 33). Currently, the isolation percentages from chicken, ducks and quails were 8 of 156 samples (5.1%), 1 of 35 samples (3.2%) and 1 of 2 samples (50%), respectively, were positive for Salmonella strains while the chick and pigeon samples were negative for Salmonellae which are not compatible with (7.25%) Salmonella incidence from chickens and (15.55%) of pigeons (30) and also with percentage (6%) in ducks in Egypt (34). The high Salmonella isolation rate of liver and gallbladder samples 2 of 36 samples (5.5%), 1 out of 19 samples (5.2%), respectively agrees with the highest rate of Salmonella isolation from liver samples (35, 36). All spleen and ceca samples were negative for Salmonel-lae that agree with another study on Salmonella was not isolated from spleen samples (37) but also, disagree with those isolated the highest Salmonella percentage from spleen samples in Egypt (38). It is common knowledge that the cloacal swab is considered a particular signal of incessant intestinal colonization in poultry, but its diagnostic accuracy is minimized where the Salmonella infected birds are intermittent shedding via feces (39). In present the study the four isolated Salmo-nellae were classified under two different serovars, Salmonella enterica serovar Enter-itidis and Typhimurium with a percentage of 2 of 4 (50%) for each. The difference between the results of sero-logical and bacteriological examination to identify Salmonella assigned to Salmonella give identical colony morphology on S.S agar and biochemical reactions with the other members of the family Enterobacteriaceae and this difference consistent with the opinion of there are problems in the biochemical identification reactions (40). Similarly, there were differences in the identification of Salmonella spp. as used conventional techniques was (10.5%), the API 20E system was (9%) and by serotyping was (7.8%) (35). The serological identification result referred to an isolation of two serotypes, Salmonella enterica serovar Typhimurium and Enteritidis, similar that reported in central Ethiopia (41) and Egypt (42) where they isolated only Salmonella enterica serovar Enteritidis and Typhi-murium, but disagrees with a previous study on S. enterica serovar Enteritidis isolated from commercial layer farms (43). The phenotype antimicrobial resistance result was resistant to (CN), (CIP), (DO), (SH) and (CT) with a percentage (50%). Moreover, the isolates were sensitive to (CAZ) with (100%), followed by (CTX) with (75%) then Characterization of class 1 integrons and some antimicrobial resistance genes in Salmonella species 731 (AMC), (NAL), (C) and (NOR) with (50%). These results concur with study reported that the resistance to Gentamycin was observed in (39.58%) (44) and those reported that the resistance to Tetracycline, Ciprofloxacin and Spectinomycin was (51.9%), (48.7%) and (34.4%), respectively (45), but disagree with those reported that Gentamycin inhibited to all Salmonella strains and resistance of Ciprofloxacin with a percentage (10%) (46). The high sensitivity to Ceftazidime (100%), followed by Cefotaxime (75%) in the present work agrees with the previous reports described a low Cephalosporin resistance prevalence of S. enterica serovar Enteritidis in Kohat and Egypt (47, 48). Two non-typhoidal Salmonella isolates (50%) showed multidrug resistant (MDR) phenotypes to at least five various antibiotic types which similar with another study reported that the multidrug resistant Salmonella isolates represented 55% in Malaysia (49). The outer membrane protein F (ompF) gene detected in the examined isolates in this current study with a percentage of (100%) using the PCR technique. The ompF gene considers a good tool for fast identification of Salmonella, so ompF mutation or loss might lead to mistakes in the identification analyze of Salmonella strains (20). Similarly, using the ompF gene as a tool for detection of Salmonella genus in Egyptian poultry farms (7). Poultry acts as a carrier of multidrug resistant Salmonella and this no related to resistance genes presence, so other acquiring resistance mechanisms might be present (50). The detection result of resistance coding genes (blaTEM and aacC) was (100%) and this disagrees with a previous report detected blaTEM in S. enterica serovar Typhimurium isolates only in Japan (51), also with another report detected aacC gene with (30%) of Salmonella isolates in broiler in Egypt (52). The result of the current study similar to another study detected the blaTEM gene with (93.3%) in Salmonella isolates obtained from commercial layers in Egypt (31). The qnrS gene, a gene quinolone resistant was reported in the present work with the percentage of (25%) that parallel with the result of another study in Egypt (31). The differences in phenotypic-genetic antibiotic resistance results recorded in this study of Salmonella isolates was also registered in other reports (53), and was usually mentioned to either existence of resistance alternative mechanisms or defect in the resistant genes expression . The result of Class 1 integrons detection was (100%) of this work, similarly, the result of Class 1 integrons detection of Salmonella isolated from Egypt (54). The sat gene was detected within class 1 integrons of S. enterica serovar Enteritidis (chicken, 600bp) and S. enterica serovar Typhi-murium (chicken, 650bp) in this investigation was preceding identity in S. Typhimurium (KT449570) in Egypt (54), S. Choleraesuis (EU834941) in southern Taiwan (55), other family Enterobacteriaceae organisms class 1 integrons as, Shigella sonnei from western Ireland (AY090896) (56), E.coli plasmid (CP022735) (57), and other bacteria as, in Vibrio alginolyticus plasmid (KU160531) (58). The aac (3)-Id and aadA 7 genes had been identified in class 1 integrons gene cassettes of (S. Typhimurium, chicken) showed a preceding identity in class1 integrons of S. Derby (KT427378), S. enterica (KT581256) in Egypt (54). In the current investigation, the detection of sat, aac (3)-Id and aadA7 genes within class1 integrons of isolated Salmonella may be related to the extensive using the aminoglycoside antibiotics group in poultry farms. The domains of unknown function protein (duf) gene was identified in class 1 integrons gene cassettes of (S. Typhimurium, quail) in the current work, which difficult to decide its function due to lack of its protein sequences identity with interpreted biochemical function. The duf gene represents more than (20%) of all protein domains (59). The class 1 integrons cassettes sequencing of the two isolated Salmonella serovars in this current investigation were documented into the GenBank with accession numbers (MK335377); (MK349001); (MK349002), (MK359462) and (MK MK359461). 732 S. Shabana, S. Helmy, A. E. Hegazy Conclusion Poultry acts as the important reservoir of many zoonotically important pathogens, such as Salmonella and detection of resistance genes related to significant antimicrobial drugs which used in the medical establishments. Integrons cassettes carrying antimicrobial resistance genes in Salmonella have an important role in the spreading of AMR so, the strategy used to control of using of antimicrobial drugs against this organism as well as other emerging pathogens of public health importance should be improved. Acknowledgments The authors would like to thank the staff members of Microbiology Department, Faculty of Veterinary Medicine, Kafrelsheikh University, Egypt and Bacteriology unit, Kafr El Sheikh Animal Health Research Institute, Egypt who supported this investigation and facilitated this work. Conflict of interest The authors declare that they have no conflict of interest. References 1. Amagliani G, Brandi G, Schiavano GF. Incidence and role of Salmonella in seafood safety. F Res Int 2012; 45:780-8. 2. Scallan E, Hoekstra RM, Angulo FJ, Tauxe RV, Widdowson MA, Roy SL, Jones JL, Griffin PM. Foodborne illness acquired in the United States-major pathogens. Emerg Infect Dis 2011; 17(1):7-15. 3. Antunes P , Mourao J, Campos J, Peixe L. Salmonellosis: The role of poultry meat. Clin Microbiol Infect 2016; 22:110-21. 4. European Food Safety Authority. The European Union summary report on trends and sources of zoonoses, zoonotic agents and food-borne outbreaks in 2010. EFSA J 2012; 10(3): 2597. 5. Baalaji NS, Mathew MK, Krishnaswamy S. Functional assay of Salmonella Typhi ompC using reconstituted large unilamellar vesicles: a general method for characterization of outer membrane proteins. Bio chemie 2006; 88: 1419-24. 6. Williams KM, Bigley EC, Raybourne RB. Identification of murine B-cell and T-cell epitopes of Escherichia coli outer membrane protein F with synthetic polypeptides. Infect Immun 2000; 68:2535-45. 7. Abd El- Tawab AA, Ahmed MA, Amany IE, Mai ME. A study of outer membrane protein (OMPs) genes for detection of salmonella organisms in poultry farms. BVMJ 2016; 30 (1): 231-7. 8. Ben Salem R Abbassi MS, Garcia V, Garcia-Fierro R Fernandez J, Kilani H, Jaouani I, Khayeche M, Messadi L, Rodicio MR. Antimicrobial drug resistance and genetic properties of Salmonella enterica serotype Enter-itidis circulating in chicken farms in Tunisia. J Infect Public Health 2017; 10 (6): 855-60. 9. Taddele MH, Rathore R, Dhama K. Antibio-gram assay of S.Gallinarum and other S. Enteric serovars of poultry origin in India. J Anim Vet Adv 2012; 7: 309-17. 10. Kim JH, Cho JK, Kim KS. Prevalence and characterization of plasmid-mediated quinolone resistance genes in Salmonella isolated from poultry in Korea. Avian Pathol 2013; 42(3): 221-9. 11. Frye JG, Jackson CR . Genetic mechanisms of antimicrobial resistance identified in Salmonella enterica, Escherichia coli, and Enteroccocus spp. isolated from U.S. food animals. Front Microbiol 2013;4: 135. 12. Trongjit S, Angkititrakul S, Tuttle RE, Poungseree J, Padungtod P, Chuanchuen R. Prevalence and antimicrobial resistance in Salmonella enterica isolated from broiler chickens, pigs and meat products in Thailand-Cambodia border provinces. Microbiol Immunol 2017; 61(1): 23-33. 13. Kakatkar AS, Pansare LS, GautamRK, Shashidhar R, Karani M, Bandekar JR. Molecular characterization of antibiotic resistant Salmonella isolates from Indian foods. Food Res Int 2011; 44(10):3272-5. 14. Phongpaichit S, Wuttananupan K, Samasanti W. Class1 integrons and multidrug resistance among Escherichia coli isolates from human stools. Southeast Asian J Trop Med Public Health 2008; 39 (2): 279-87. 15. Carattoli A. Importance of integrons in the diffusion of resistance. Veterinary Research, Bio-Med Central 2001; 32 (3-4): 243-59. 16. Labbate M, Case RJ, Stokes HW. The in-tegron gene cassette system: an active player in bacterial adaptation. Methods Mol Biol 2009; 532: 103-25. 17. Leverstein-van MA, Blok HE, Donders AR, Paauw A, Fluit AC, Verhoef J. Characterization of class 1 integrons and some antimicrobial resistance genes in Salmonella species 733 Multidrug resistance among Enterobacteriaceae is strongly associated with the presence of integrons and is independent of species or isolate origin. J Infect Dis 2003; 187 (2): 251-9. 18. International Standards Organization (ISO 6579). General guidance on methods for the detection of Salmonella, Geneva, Switzerland, 2002. 19. Kauffman G. Kauffmann white scheme. J Acta Path Microbiol Sci 1974; 61: 38. 20. Tatavarthy A, Cannons A. Real-time PCR detection of Salmonella species using a novel target: the outer membrane porin F gene (ompF). Lett Appl Microbiol 2010; 50 (6): 645-52. 21. Finegold SM, Martin WJ. Diagnostic microbiology. 6th Ed., The C.V. Mosby Company, St. Louis, Toronto, London, 1982. 22. CLSI. Performance Standards for Antimicrobial Susceptibility Testing; Twenty- Fifth Informational Supplement (M100 - S25), Clinical and Laboratory Standards Institute. Pennsylvania 19087, USA, 2015; 44-50J 23. Lynne AM, Rhodes-Clark BS, Bliven K, Zhao S, Foley SL. Antimicrobial Resistance Genes Associated with Salmonella enterica serovar Newport isolates from Food Animals. Antimicrob Agents Chemother 2008; 52(1): 353-6. 24. Robicsek A, Strahilevitz J, Jacoby GA, Macielag M, Abbanat D, Park CH, Bush K, Hooper DC. Fluoroquinolone modifying enzyme: a new adaptation of a common aminoglycoside acetyltrans-ferase. Nat MED 2006; 12 (1): 83 -88. 25. Colom K, Perez J, Alonso R, Fernandez-Aranguiz A, Larino E, Cisterna R. Simple and reliable multiplex PCR assay for detection of blaTEM, blaSHV and blaOXA-1 genes in Enterobacteriaceae. FEMS Microbiol Lett 2003; 223 (2):147-51. 26. Sow AG, Wane A, Diallo MH, Boye CS, Ai'dara-Kane A. Genotypic characterization of antibiotic-resistant Salmonella Enteritidis isolates in Dakar, Senegal. J Infect Developing Countries 2007; 1 (3): 284-8. 27. Hall A. BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nuc Acids Symp Ser 1999; 41: 95-8. 28. Barrow PA, Freitas Neto OC. Pullorum disease and fowl typhoid new thoughts on old diseases. Avian Pathol 2011; 40 (1): 1-13. 29. Clothier KA, Kim P, Mete A, Hill AE. Frequency, serotype distribution and antimicrobial susceptibility patterns of Salmonella in small poultry flocks in California. J Vet Diagn Invest 2018; 30 (3): 471- 475. 30. Akbarmehr J. Isolation of Salmonella spp. from poultry (ostrich, pigeon, and chicken) and detection of their hilA gene by PCR method. A J Microbiol Res 2010; 4 (24): 26782681. 31. Lebdah MA, Waffa MM, Samah E, Rehab IH. Molecular detection of some Antimicrobial Resistance Genes in Salmonella species isolated from commercial layers in Egypt. Zagazig Vet J 2017; 45(1): 29-38. 32. Liu W B, Chen J, Huang YY, Liu B, ShiX M. Serotype, genotypeand antimicrobial susceptibility profiles of Salmonella from chicken farms in Shanghai. J Food Prot 2010; 73(3): 562-567. 33. Abd El-Ghany WA, Soumaya SA, El-ShafiiHatem ME. A survey on Salmonella species isolated from chicken flocks in Egypt. AJAVA 2012; 7 (6): 489-501. 34. Ibrahim MA, Emeash HH, Nahed HG, Ab-del-Halim MA. Seroepidemiological Studies on Poultry Salmonellosis and its Public Health Importance. J World's Poult Res 2013; 3 (1): 18-23. 35. AL-Iedani AA, Khudor MH, Oufi NM. Isolation and identification of Salmonella spp. from poultry farms by using different techniques and evaluation of their antimicrobial susceptibilities. Bas J Vet Res 2014; 13(1): 246-259. 36. Dhaher FH, Awni MDH, Mahmood NR, Jamil MM. Isolation and diagnosis of Salmonella in Animal origin food. Iraq Acad Sci J 2011; 3(5): 1-19. 37. Marwa RA. Molecular characterization of some Antibiotic Resistance Genes in Salmonella Species isolated from diseased poultry. MVSc thesis, Fac of Vet Med, kafrelsheikh Univ, Egypt, 2016. 38. Orady M R, Salwa MH, Ahmed MAA, Wafaa MH, Etab MA, Azza S E. Molecular Characterization of Class 1 Integrons and Antibiotic Resistance Genes in Salmonella enterica isolated from Chicken. Global Veterinaria 2017; 18 (5): 322-31. 39. Gast R K. Paratyphoid infections. In D. E. Swayne (Ed.), Diseases of Poultry, Ames, IA: Wiley-Blackwell Publishing 2013; 693-733. 40. Neubauer H, Hensel A, Aleksic S, Meyer H. Identification of Yersinia enterocolitica within the genus Yersinia. Syst Appl Microbiol 2000; 23(1): 58-62. 41. Mohamed NS. A survey of Salmonella contamination in chicken carcass and giblets in central Ethiopia. Revue de Med Vet 2003; 154(4):267-70. 734 S. Shabana, S. Helmy, A. E. Hegazy 42. Ahmed AM, Shimamoto T. Genetic analysis of multiple antimicrobial resistance in Salmonella isolated from diseased broilers in Egypt. Microbiol Immunol 2012; 56 (4): 254-61. 43. García C, Soriano JM, Benítez V, Catalá-Gregori P. Assessment of Salmonella spp. in feces, cloacal swabs, and eggs (eggshell and content separately) from a laying hen farm. Poult Sci 2011; 90 (7): 1581-5. 44. Ren D, Chen P, Wang Y, Wang J, Liu H, Liu H. Phenotypes and antimicrobial resistance genes in Salmonella isolated from retail chicken and pork in Changchun, China. J Food Saf 2017; 37 (2): e12314. 45. Zhu Y, Lai H, Zou L, Yin S, Wang C, Han X, Xia X, Hu K, He L, Zhou K, Chen S, Ao X, Liu S. Antimicrobial resistance and resistance genes in Salmonella strains isolated from broiler chickens along the slaughtering process in China. Int J Food Microbiol 2017; 259:43-51. 46. Begum K, Mannan SJ, Ahmed A. Antibiotic Resistance, Plasmids and Integron Profile of Salmonella Species Isolated from Poultry Farm and Patients. Dhaka Univ. J Pharm Sci 2016; 15(2): 20914. 47. Asif M, Rahman H, Qasim M, Khan TA, Ul-lah W, Jie Y. Molecular detection and antimicrobial resistance profile of zoonotic Salmonella Enteritidis isolated from broiler chickens in Kohat, Pakistan. J Chin Med Assoc 2017; 80(5):303-6. 48. Abdel-Maksoud M, Abdel-Khalek R, El-Gendy A, Gamal RF, Abdelhady HM, House BL. Genetic characterisation of multidrug-resistant Salmonella enterica serotypes isolated from poultry in Cairo, Egypt. Afr J Lab Med 2015; 4 (1): 158-65. 49. Abatcha MG, Mohd EE, GulamR. Prevalence, antimicrobial resistance, resistance genes and class 1 integrons of Salmonella serovars in leafy vegetables, chicken carcasses and related processing environments in Malaysian fresh food markets. Food Control 2018; 91: 170-80. 50. Álvarez- Fernandez E, Alonso-Calleja C,García-Fernández C, Capita R. Prevalence and antimicrobial resistance of Salmonella serotypes isolated from poultry in Spain: comparison between 1993 and 2006. Int J food Microbiol 2012; 153 (3): 281-7. 51. Ahmed AM, Younis EE, Ishida Y, Shimamoto T. Genetic basis of multidrug resistance in Salmonella enterica serovars Enteritidis and Typhimurium isolated from diarrheic calves in Egypt. Acta Trop 2009; 111(2): 144-9. 52. Elmonir W, Hegazym AM, El-Tras WF, Shohiep A. Extremely drug-resistant Salmonella in broiler production chain in Egypt. Life Sci J 2017; 14(9): 82-7. 53. Zishiri OT, Mkhize N, Mukaratirwa S. Prevalence of virulence and antimicrobial resistance genes in Salmonella spp. isolated from commercial chickens and human clinical isolates from South Africa and Brazil. Onderstepoort J Vet Res 2016; 83(1): 1-11. 54. Gharieb RM, Tartor YH, Khedr MH. Non-Typhoidal Salmonella in poultry meat and diar-rhoeic patients: prevalence, antibiogram, virulotyp-ing, molecular detection and sequencing of class I integrons in multidrug resistant strains. Gut Pathog 2015;7:34. 55. Lee MF, Chen YH, Peng CF. Molecular characterisation of class 1 integrons in Salmonella enterica serovar Choleraesuis isolates from southern Taiwan. Int J Antimicrob Agents 2009; 33(3): 216-22. 56. De Lappe N, O'Halloran F, Fanning S, Cor-bett-Feeney G, Cheasty T, Cormican M. Antimicrobial resistance and genetic diversity of Shigella sonnei isolates from western Ireland, an area of low incidence of infection. J Clin Microbiol 2003; 41(5): 1919-24. 57. Alghoribi MF, Balkhy H, Doumith M, Al Jo-hani SM, Upton M, Woodford N, Ellington MJ. Molecular Epidemiology, Virulence Potential and Antibiotic Susceptibility of the Major Lineages of Uro-pathogenic Escherichia coli. Ph.D. thesis, Faculty of Medical and Human Sciences, Manchester Univ, 2015. 58. Ye L, Li R, Lin D, Zhou Y, Fu A, Ding Q, Chan EW, Yao W, Chen S. Characterization of an IncA/C Multidrug Resistance Plasmid in Vibrio al-ginolyticus. Antimicrob Agents Chemother 2016; 60(5):3232-5. 59. Goodacre NF, Gerloff DL, Uetz P. Protein domains of unknown function are essential in bacteria. mBio 2013; 5(1):e00744-13. Slov Vet Res 2019; 56 (Suppl 22): 735-45 DOI 10.26873/SVR-814-2019 Original Research Article THE ROLE OF THYMOQUINONE IN AMELIORATING THE HEPATOXIC EFFECT OF DIAZINON IN MALE RATS Emad W. Ghazy1, AbdAllah A. Mokh1, Doaa H. Abdelhady1, Wael M. Goda2, Eman M. Hashem1* department of Clinical Pathology, Faculty of Veterinary Medicine, Kafrelsheikh University, Egypt, 2Department of clinical Pathology, Faculty of Veterinary Medicine, Damanhur University, Egypt "Corresponding author, E-mail: eman.amr123.eh@gmail.com Abstract: Diazinon (DZN) is one of most dangerous hepatotoxic organophosphorous insecticides used in veterinary practices which induces oxidative stress. The present study aimed to evaluate the ameliorative role of thymoquinone (TQ) in diazinon toxicity. Forty nine male albino rats and were divided into seven groups (n =7 for each). The first group (c-ve group) orally received saline daily all over the experiment (8 weeks). The second group (c+ve1) orally received DZN (15 mg/kg body weight, b-w) for the first four weeks and saline for the next four weeks. The third group (c+ve2) orally received saline for the first four weeks and DZN (15 mg /kg b-w) for the following four weeks. The fourth group (DZN-TQ 10 mg group) received DZN (15 mg/kg b-w) daily for the first four weeks then TQ (10 mg/kg b-w) for the next four weeks. The fifth group (TQ 10 mg-DZN) orally treated with TQ (10 mg/kg b-w) and DZN (15 mg/kg b-w) for the next for weeks. The sixth group (DZN-TQ 5 mg group) received DZN (15 mg/kg b-w) for first four weeks and TQ (5 mg/kg b-w) for the following four weeks 5 mg. The seventh group treated by TQ (5 mg/kg b-w) daily for the first four weeks and DZN (15 mg/kg b-w) for the following four weeks (TQ 5 mg-DZN group). DZN intoxicated groups showed macrocytic hypochromic anemia and serum biochemical alteration related to liver injury, including elevation of alanine aminotransferase (ALT), aspartate aminotransferase (AST) and a significant decrease in total protein and albumin. Furthermore, these groups also exhibited a significant increase in liver tissue malondialdehyde (MDA), significant decrease in total antioxidant capacity (TAC) and catalase (CAT) activities and over-expression of the two apoptotic hepatic genes Bax and caspase 3. Administration of DZN also resulted in hepatic vacuolation, necrosis and congestion of hepatic sinusoids. Thymoquinone ameliorated the most deleterious effect of diazinon on hematological, biochemical, antioxidant, molecular and his-topathological parameters in a dose dependent manner and a prophylactic strategy is better than therapeutic one. Key words: diazinon; thymoquinone; oxidative stress; hepatotoxicity Introduction Organophosphorous are still used as pesticides in agriculture, industry and in animal care as insecticides to control external parasites on Received: January 2019 Accepted for publication: February 2019 736 E. Ghazy, A. Mokh, D. Abdelhady, W. Goda, E. Hashem animals all over the world. However, they are the most toxic pesticides to all animal species and human (1). The improper use of these pesticides is dangerous on human being and environment (2, 3). Organophosphorus insecticide residues are detectable in grains, crops, soil and some food products. Diazinon (DZN) considered as one of the most important organophos-phorous compounds used in agriculture practices to control external parasites and flying insects of food crops and ornamental plants (4). It is also used to control the external parasites in veterinary practices. It was found that the main mechanism of action of DZN is inhibition of acetyl choline esterase enzyme (5). Furthermore DZN increased the level of malondialde-hyde and lipid peroxidation biomarker in male rat (6). Nowadays, the interest for using natural products for pharmacological purposes was increased. As this natural products gaining acceptance from the public, due to their useful effect on treatment of many disease without side effects which caused by many chemical products (7-9). Nigella sativa commonly known as black seed and black cumin. The essential oil and fixed oil of Nigella sativa showed powerful action in treatment of respiratory and gastrointestinal diseases (10). Furthermore, N. sativa has anti-oxidant, anti-inflammatory and hepato-protective activities (11). Thymoquinone (TQ) is the most potent active constituent in the volatile oil of seeds of N. sativa. The possible mechanisms of TQ action including suppression of protein kinase B activation (Akt/PKB) by dephosphorylation (12), induction of apop-tosis in cancerous cells by increasing apoptotic genes expression and decreasing anti-apoptotic gene expression (13, 14), induction of cytokines production (15) and activation of anti-oxidant enzymes (16). As result of these different beneficial effects of thymoquinone, TQ has various therapeutic effects such as anti-inflammatory effect (17), immune modulatory effect (18), free radical scavenger, antioxidant effect (19), anti-carcinogenic effect (20), apoptotic effect (21) and hepatoprotective effect by inhibiting lipid peroxidation (22). For this reasons, the present study aimed to assess the role of thymoquinone in alleviating the harmful effect of diazinon toxicity in male albino rats. Materials and methods Chemicals Ectodat 60 EC, commercial formulation containing 60% of active ingredient (diazinon), was purchased from Kemet Company which imported the product from Jorden Company ATI. Thymoquinone was obtained from Sigma-Aldrich Company (product of china origin), CAS 490-9-5C10H12O2 MW 164.201 g/mol. Animals and treatments Forty nine male albino rats, purchased from Animal House Colony of Alexandria Center institute, weighting average 70±5 g/rat. The rats were kept in well ventilated animal house with a controlled dark -light cycle (12 h light - 12 h dark) and constant temperature (26±2 0C) food and water were provided ad libitum. Rats were allowed to accommodate two weeks before the start of the experiment. They were randomly divided into seven groups (n=7 for each group). The first group (c-ve group) orally received saline daily all over the experiment (8 weeks). The second group (c+ve1) orally received DZN (15 mg/kg body weight, b-w) (23) daily for the first four weeks and saline for the next four weeks. The third group (c+ve2) orally received saline for the first four weeks and DZN (15 mg/kg b-w) for the following four weeks. The fourth group (DZN-TQ 10 mg) received DZN (15 mg/kg b-w) for first four weeks then TQ (10 mg/kg b-w) (24) for the next four weeks. The fifth group (TQ 10 mg-DZN) orally treated by TQ (10 mg/kg b-w) daily and DZN (15 mg/kg b-w) for the next four weeks. The sixth group (DZN-TQ 5 mg) received DZN (15 mg/kg b-w) daily and TQ (5 mg/kg b-w) (20) for the following four weeks. The seventh group (TQ 5 mg-DZN) treated by TQ (5 mg/kg b-w) for first four weeks then treated daily by DZN (15 mg/kg b-w) for the next four weeks. Twenty four hours, after the end of the experiment, the blood samples were taken from retro-orbital venous plexus under effect of Di Ethyl Ether anesthesia and taken in aliquots The role of thymoquinone in ameliorating the hepatoxic effect of diazinon in male rats 737 with and without anti-coagulant for hematological and biochemical analysis, respectively. Blood samples without anticoagulant were left at room temperature to clot, then were centri-fuged at 3000 rpm for 15 min and the obtained were collected for biochemical analysis. Then rats were sacrificed by decapitation. The liver of each animal was rapidly removed, trimmed from excess tissues and washed by saline and distilled water. The liver samples divided into three parts; the first part used to preparation of tissue homogenate for determination antioxi-dant activities. The second part was cut into slices of about 0.1 g and directly put in liquid nitrogen then stored at -80°C for molecular analysis. The last part was kept in 10% formalin to be used for histopathological examination. Animal rearing, handling and all experimental designs were approved by the Research Ethical Committee of faculty of veterinary medicine, Kafrelsheikh University, Egypt. Hematological examinations Aliquot containing EDTA (1mg/ml) were used for determination of complete blood count (RBC, WBC, MCH, MCHC, PCV, platelets count, and differential leukocytic count) by using Sysmex-Xp 300 automatic hematological analyzer (25). Biochemical analysis Serum levels of liver damage marker (ALT and AST) were determined as previously described (26). Determination of total protein and albumin were determined according to (27) and (28) methods respectively using Genway spec-trophotometer instrument. Determination of glucose was done by (29) method. Determination of oxidative stress/antioxi- dants The dissected tissues were washed by sodium phosphate buffered saline (50 mmol/l) with EDTA (0.1 mmol/l) in ice to remove any RBCs and clots. The tissues were homogenized in 5 ml cold buffer /g of tissue then were centri-fuged at 2000 rpm for 20 minutes. The resulting supernatant was collected and preserved in 80°C for estimation of MDA (30), catalase (CAT) (31) and total antioxidant capacity TAC (32). Histopathological examination Tissue preparation and histopathological examination were performed as previously described (33). Molecular investigation RNA extraction from hepatic tissue was applied using QIAamp RNeasy Mini kit (Qiagen, Germany, GmbH) GmbH) following manufacture protocol. A 25 |l mixture containing 12.5 |il of the 2x Quantitect SYBR Green PCR Master Mix (Qiagen, Germany, GmbH), 0.25 |l of Revert Aid Reverse Transcriptase (200 U/|L) (Thermo Fisher), 0.5 |l of each primer of 20 pmol concentration (Table 1), 8.25 |l of water, and 3 |l of RNA template was put in a Strata-gene MX3005P real time PCR machine. Amplification curves and Ct values were determined by the Stratagene MX3005P software. To estimate the variation in gene expression, the Ct of each sample was compared with that of the positive control group according to the 2-AAct method (34). Statistical analysis For data analysis, Graph pad prism statistical package version 5.0 for windows was used. Differences in values were analyzed by one-way analysis of variance (ANOVA), followed by Tukey's multiple range test. All data expressed as mean ±SEM, and the level of significance were cited at p< 0.05. Results Hematological findings Data explored in table (2) showed a significant ()p< 0.05 decrease in hematological parameters (RBCs, HGB and MCHC) and an insignificant decrease in (HCT, MCH and PLT) in DZN treated group (c+ve1). DZN c+ve2 group showed marked but not a significant decrease in hematological parameters (RBCs, HGB and PLT) and a significant decrease in HCT, MCH and MCHC. Significant (p< 0.05) increase in MCV and WBCs was noticed in 738 E. Ghazy, A. Mokh, D. Abdelhady, W. Goda, E. Hashem DZN treated groups as compared with the non-treated group (c-ve). DZN-TQ 10 mg group showed insignificant decrease in WBCs and a significant (p< 0.05) increase in hematological parameters (RBCs, HCT, MCH and MCHC), similarly a marked increase in PLT but still in significant was also observed in DZN-TQ 10 mg group as compared with the DZN treated group (c+ve1). TQ prophylactic group (TQ 10 mg-DZN) showed marked increase in hematological parameters (RBCs, HCT, HGB, MCHC and PLT). Meanwhile, insignificant decrease in WBCs count was observed in this group as compared to the c+ve2 group. TQ therapeutic group (DZN-TQ 5 mg) showed a significant increase in RBCs and MCHC and a marked increase but still insignificant in HCT, HGB, MCH and PLT as compared with the c+ve1 group. TQ prophylactic group (TQ 5 mg-DZN) showed a marked increase in hematological parameters (HCT, HGB, MCH, MCHC and PLT) and a marked increase but still insignificant in RBCs as compared with the c+ve2 group. Serum biochemical parameters The harmful effect of diazinon toxicity and the useful effect of TQ on some biochemical parameters illustrated in table (2). Significant increase in serum level of (ALT and AST) in DZN treated groups (c+ve1 and c+ve2) as compared with non-treated group (c-ve). In contrast to this result, there was a significant decrease in total protein and albumin in DZN treated groups in comparison with c-ve group. Significant increase of glucose level in DZN treated groups as compared with c-ve group. DZN-TQ 10 mg showed a significant decrease in serum level of ALT, AST, glucose and a significant increase in TP and albumin as compared with c+ve1 group. In TQ prophylactic group (TQ 10 mg-DZN) showed marked decrease in serum ALT, AST, glucose and a significant increase in TP and albumin as compared with c+ve2group. TQ therapeutic group (DZN-TQ 5 mg) showed a significant decrease in ALT, AST and glucose. Meanwhile, significant increase in TP and albumin as compared with DZN treated group (c+ve1). TQ prophylactic group (TQ 5 mg-DZN), showed a significant decrease in serum biochemical parameters (ALT, AST and glucose) as compared with DZN treated group (c+ve2). Antioxidants biomarkers on liver tissues The antioxidant effect of TQ on DZN toxicity was shown in (Fig.2). DZN toxic groups (c+ve1 and c+ve2) illustrated significant decrease in TAC, Catalase activities and significant increase in MDA as compared with c-ve group. TQ therapeutic group (DZN-TQ 10 mg) showed a significant increase in TAC, Catalase activities and significant decrease in MDA as compared by c+ve1. TQ prophylactic group (TQ 10 mg-DZN) showed a significant increase in TAC, Catalase activities and a significant decrease in tissue MDA as compared with (c+ve2). TQ therapeutic group (DZN-TQ 5 mg) showed marked decrease of MDA but still insignificant and a significant increase in TAC and Catalase activities as compared with c+ve1 group. TQ prophylactic group (TQ 5 mg-DZN) showed significant decrease in MDA and a significant increase in TAC, Catalase activities and as compared with c+ve2. Significant increase in TAC and CAT activities in TQ10 mg groups (prophylactic and therapeutic groups) as compared with TQ 5 mg groups. TQ prophylactic groups (TQ 10 mg-DZN and TQ 5 mg-DZN) showed a significant decrease in MDA as compared by TQ therapeutic groups (DZN-TQ 10 mg and DZN-TQ 5 mg). Role of thymoquinone in ameliorating the histopathological alteration induced by dia- zinon Data explored in (Fig.2) showed the histo-pathological alteration caused by DZN. Normal hepatocytes arranged in cords was observed in non-treated group (Fig.2A). DZN intoxicated group (c+ve1) showed marked congestion of hepatic sinusoids associated with atrophy of hepatocytes (Fig.2B), focal hepatic necrosis (Fig.2Cm D). DZN intoxicated group (c+ve2) showed marked periportal inflammatory cells infiltration (Fig.2E), diffuse hepatic vacuola-tion (Fig.2F) and focal hepatic necrosis The role of thymoquinone in ameliorating the hepatoxic effect of diazinon in male rats 739 (Fig.2J). TQ reversed histopathological changes in liver tissues caused by DZN. (DZN-TQ 10 mg) showed mild degree of granular hepatic vacuolation (Fig.2H) as compared with (c+ve1) group. (TQ 10 mg-DZN) group showed mild degree of centrolobular vacuolation (Fig.2K) as compared with c+ve2 group. TQ therapeutic group (DZN-TQ 5 mg) showed periorbital inflammatory cells infilteration (Fig.2M), mild to moderate degree of granular hepatic vacuolation (Fig.2N) as compared with c+ve1. TQ prophylactic group (TQ 5 mg-DZN) showed mild to moderate degree of granular vacuolation (Fig.2O) as compared with c+ve2. Effects of thymoquinone on Bax and caspase3 gene expression in liver of DZN intoxicated rats The expression of caspase3 and Bax genes were significantly increased in liver of DZN in- toxicated groups (c+ve1 and c+ve2) as compared to non-treated group (c-ve) as showed in Fig.3. TQ therapeutic group (DZN-TQ 10 mg) illustrated a significant decrease in the levels of caspase3 and Bax gene expression compared with DZN intoxicated group (c+ve1). The mRNA levels of caspase3 and Bax were significantly downregulated in TQ prophylactic group (TQ 10 mg-DZN) as compared with the c+ve2 group. The gene expression of caspase3 and Bax were significantly attenuated in TQ therapeutic group (DZN-TQ 5 mg) as compared with c+ve1 one. TQ prophylactic group (TQ 5 mg-DZN) showed a significant decrease of caspase3 and Bax genes expression as compared with the c+ve2 group. The caspase and Bax genes expression was significantly decrease in (TQ 10 mg) groups as compared by (TQ 5 mg) groups. Table 1 : Primers used in qPCR Gene Primer sequence (5'. .3') references ß-actin F:TCCTCCTGAGCGCAAGTACTCT R:GCTCAGTAACAGTCCGCCTAGAA BAX F: CACCAGCTCTGAACAGATCATGA R:TCAGCCCATCTTCTTCCAGATGGT Caspase3 F:AGTTGGACCCACCTTGTGAG R:AGTCTGCAGCTCCTCCACAT (Banni et al., 2010) (Kinouchi, 2003) (Shi et al., 2009) Table 2: Hematological and biochemical parameters of control and treated groups Parameters c-ve c+vei c+ve2 DZN- TQi0mg- DZN- TQ5mg-DZN TQi0mg DZN TQ5mg RBCs(x106/^L) 7.6±0.22a 5.8±0.16b 6.4±0.5ab 6.85±0.3a 7.09±0.1a 7.17±0.14a 6.7±0.1ab HCT (%) 44.2±1.66a 40.5±0.9ab 40.1±0.9b 43.34±0.39a 45.2±0.56a 41.2±0.84ab 43.2±0.56a HGB(g/dl) 15.9±0.4a 11.8±0.4b 12.8±0.4ab 15.2±0.39a 15.1±0.13a 14.4±0.4ab 15.8±0.12a MCV(FL) 57.1±0.4bc 71.3±1.3a 66.5±1.6a 58.1±0.44b 59.3±1.07ab 61±0.12ab 59.3±1.07ab MCH(pg.) 21±0.48ab 20.4±0.3ab 19.6±0.3b 22.1±0.1a 21±0.12ab 20.08±0.14ab 23.5±0.12a MCHC(g/dl) 35.9±0.3a 29.7±1.3b 31.9±0.8b 35±0.06a 34.6±0.06a 34.9±0.12a 34.6±0.06a PLT(x10V) 1080±174a 741±63.6ab 710±28.4ab 1004.3±127ab 1133.6±36a 993±54ab 1077±116a WBCs(x10V) 10.5±1.7b 24.7±4.5a 22.3±4.7a 12.1±0.4ab 12.1±1.3ab 11.9±0.6ab 14.5±0.14ab ALT(U/l) 14.6±6.69cd 92.9±0.97a 73±4.6a 41±1.4b 43.o3±5.6bc 43±1.6b 39.5±2.2b AST(U/l) 36±8.1bc 94±11.24a 101±23a 52.5±2.6b 47.16±1.9bc 65.7±0.8b 57.8±8b TP(g/dl) 8.4±0.24a 5.7±0.088c 6.12±0.10c 7.32±0.26b 7.3±0.080b 7.38±0.27b 5.9±0.134c Albumin(g/dl) 4±0.1a 3.9±0.13b 3.3±0.05b 4.1±0.05a 4±0.01a 4.15±0.05a 3.7±0.05ab Glucose(mg/dl) 102±4.8c 145±7.6a 163±1.5a 121±1.3b 116±1.67b 128±0.2b 117.6±3.28b Notes; the data are expressed as ±SE (n=7). Different upper case letters within a row indicate different mean value (p<0.05). TQ, thymoquinone; DZN, diazinon; RBCs, red blood cells; HCT, hematocrit; HGB, hemoglobin; MCH, mean corpuscular hemoglobin; MCHC, mean corpuscular hemoglobin concentration; PLT, platelets; WBCs, white blood cells, ALT, alanine aminotransferase; AST, aspartate aminotransferase; TP, total protein. 740 E. Ghazy, A. Mokh, D. Abdelhady, W. Goda, E. Hashem 0 5-1 = 4- ra 3- o E E a 2- bc cde bcd ^ c-ve ^ c+ve1 B c+ve2 OH] DZN-TQ 10 mg ^ TQ 10 mg-DZN ^ DZN-TQ 5 mg EHJ TQ 5 mg-DZN IB 20-i 15- .5? 10- < o 5- d d WM rrm c-ve c+ve1 c+ve2 DZN-TQ 10 mg TQ 10 mg-DZN DZN-TQ 5 mg TQ 5 mg-DZN a b b b c c e 0 0 i 100-1 <1) 3 80- (A (A •i? 60- O £ c 40- < S 20- a ii bc abc ^ c-ve ^ c+ve1 ^ c+ve2 EI] DZN-TQ 10 mg ^ TQ 10 mg-DZN ^ DZN-TQ 5 mg EHJ TQ 5 mg-DZN Figure 1: the antioxidant biomarkers in liver tissues in treated and non -treated groups. TAC, Total Anti-oxidant Capacity; CAT, Catalase enzyme; MDA, malondialdehyde; DZN, diazinon; TQ, thymoqui-none. Different letters indicate significantly different mean values (p<0.05) a c c c 0 Figure 2: A; Liver of rat of non-treated group showing normal hepatocytes arranged in cords (arrow). B; Liver of animal from (c+ve1) showing marked congestion of the hepatic sinusoids (arrow). C; Liver of animal from (c+ve1) showing focal hepatic necrosis (arrowhead). D; Liver of animal from (c+ve1) showing focal hepatic necrosis (arrow). E; Liver of animal from (c+ve2) group showing marked periportal inflammatory cells infiltration with presence of few eosinophils (arrow). F; Liver of animal from (c+ve2) group showing diffuse vacuolation of hepatocytes (arrow). J; Liver of normal animal (c+ve2) showing focal hepatic necrosis (arrow). K; Liver of normal animal (DZN-TQ 10 mg) group showing mild degree of granular hepatic vacuolation (arrow). L; Liver of normal animal (TQ 10 mg-DZN) group showing mild degree of centrolobular vacuolation (arrow). M; Liver of normal animal (DZN-TQ 5 mg) group showing periportal inflammatory cells infiltration (arrow). N; Liver of normal animal (DZN-TQ 5 mg) group showing mild to moderate degree of granular vacuolation (arrow). O; Liver of normal animal (TQ 5 mg-DZN) group showing mild to moderate degree of granular hepatic vacuolation (arrow). H&E, bar= 100 pm The role of thymoquinone in ameliorating the hepatoxic effect of diazinon in male rats 741 □ m 5- CO cp Once/day cleaning1 0 1 1 66.7 0.7 1 practices Once/day cleaning 1 0 0 33.3 1 Acid rinse 0 0 1 33.3 0.04 -0.02 No acid rinse 1 1 0 66.7 +0.02 1 : cleaning of milking equipment after milking cycle. F: Fisher's Exact test R: Pearson's R correlation coefficient Ecology of Staphylococcus aureus and its antibiotic resistance genes in dairy farms: contributing factors and 751 14 13 12 11 10 9 8 7 6 5 4 3 2 1 M P N 1500 ZZZZ vanA 100 Figure 1: Molecular detection of antibiotic resistance genes (mecA and vanA) among S. aureus pathogens isolated from different samples in farm A. mecA gene: 533 bp, vanA gene: 1030 bp, Lanes 1-6: Cows' isolates, Lane 7: Milking equipment's isolates, Lanes 8-10: Barns' isolates, Lanes 11-14: Workers' isolates, M: 100 bp DNA marker, P: Positive control, and N: Negative control ■ MRSA ■ VRSA ■ MVRSA Figure 2: Frequency distribution of antibiotic resistance genotypes among S. aureus pathogens isolated from examined farms. MRSA: Methicillin resistant S. aureus (mecA+), VRSA: Vancomycin-resistant S. aureus (vanA+) and MVRSA: Methicillin-vancomycin-resistant S. aureus (mecA+ -vanA+). BTM: Bulk tank milk. All frequency results are represented as percent 752 W. Elmonir, H. Essa, W. El-Tras Farm workers Farm worker are at risk of occupational infection with livestock associated S. aureus infection. In addition, they may play a critical role in dissemination of infection to cows, equipments, farm environment, and milk (3, 10, 17). The overall prevalence of S. aureus in workers' samples was 81.5%, which was higher than a report (27%) in USA (10). S. aureus was detected in nostrils (88.9%), hand skin (100%), and stool (55.6%) samples from workers in this study (Table 1). Nostrils prevalence was higher than a report (29.3%) in Turkey (14). Lower rates for hand skin (3.3 - 32%) were reported in Brazil (3) and Ethiopia (6). Intestinal colonization and stool carriage of S. aureus in humans were previously reported (18). There was a significant association (P= 0.04) between hand skin and fecal carriage of S. aureus. Same association was recorded in USA (18). This could be attributed to lack of adequate personnel hygiene of examined workers as improper hand washing. No other association was recorded between workers samples. Barns environment Barns environment may act as a vehicle for S. aureus transmission to cows, workers, farm equipment and BTM (10, 13). S. aureus was detected in 88.9% of the examined barn samples (Table 1), which was higher than other reports (1-20%) in USA (10) and Norway (13). Milking equipment Milking equipment are critical vehicles for dissemination of S. aureus between individual dairy cows as well as from barns, cows and worker to milk (3, 17). In the present study, S. aureus was isolated from 40% of milking equipment (Table 1). This was higher than other reports (2.1-11.1%) in USA (10), Brazil (3) and Ethiopia (6). High rates of S. aureus detection in various sources in examined dairy farms highlight their role as critical points for S. aureus dissemination and may also refer to unsanitary dairy farm practices. This is an alarming threat for both dairy animals and public health. Differences in the prevalence rates may be due to variations in the farm sanitary conditions, animal breeds, animal health status (e.g. subclinical mastitis), sampling strategies, seasons and geographical locations. Factors contributing to milking equipment contamination with S. aureus in dairy farms Carriage of S. aureus via cows, workers and barns was significantly associated with milking equipment contamination (P= 0.01 - 0.02) (Table 2). Nostrils (cows and workers), udder milk (cows) and hand skin (workers) were the sources that contributed significantly to milking equipment contamination (P= 0.004 - 0.03) (Table 2). Our finding agreed with Zadoks et al. (17) who confirmed S. aureus transmission from hand skin and udder milk to the milking equipment. Open-sides parlors design positively associated with milking equipment contamination (P= 0.04) (Table 2). This could be linked to the significant effect of contaminated barns environment. With open-sides parlors walls, air drafts can introduce infection from contaminated barns to parlors during and in between milking cycles. Lacking of acid rinse was significantly associated with milking equipment contamination (P= 0.04) (Table 2). This finding agreed with Elmoslemany et al. (19) who reported the positive association between inadequate acid rinse and milk contamination within dairy farms. Acid rinse removes milk stones, which could act as niches for microbial growth within milking equipment (19). Also acid has antibacterial activity against broad range of bacteria (20). Both mechanisms may explain the significant effect of acid rinse in reducing S. aureus contamination of milking equipment. Teat dip was not associated with milking equipment contamination in this study (Table 1). However it may be contributed to the elimination of teat skin role in contaminating milking equipment as recorded in this study (P= 0. 4) (Table 1). Ecology of Staphylococcus aureus and its antibiotic resistance genes in dairy farms: contributing factors and 753 Frequency distribution of S. aureus antibiotic resistance genotypes in dairy farms Almost half (45%) of the S. aureus isolates carried at least 1 antibiotic resistance gene. MRSA (mecA+), VRSA (vanA+), and MVRSA (mecA+, vanA+) resistance genotypes represented 27.5%, 5%, and 12.5% of S. aureus isolates respectively (Fig. 2). MRSA isolates were detected in cows, workers, barns, and BTM. VRSA isolates were from workers and barns, while MVRSA isolates were from cows and workers. Hence, workers were the only source that harbored the 3 resistance genotypes in the examined farms. In agreement with our findings, MRSA isolates were detected in cows, workers, and environment of dairy farms in Korea (4) and Italy (5). VRSA isolates were reported in cows with mastitis in China (9). However, as far as we know, this the first report of MVRSA in dairy farms in Egypt. Detection of MVRSA isolates in dairy farms is alarming and their emergence requires further investigation. MRSA acquiring vanA gene by plasmid transfer was previously reported in human clinical cases (21). Same mechanism of gene transfer could explain the emergence of MVRSA in this study. High rate of intersources transmission and mixing of S. aureus isolates within same farm may facilitate this gene transfer. The detection of the 3 genotypes in 2 farms (A and B) and the findings of Loca-telli et al. (5), who reported MRSA genotypes exchange between cows, workers and environment within same dairy farm, support our hypothesis. Yet, further investigation is required to elucidate the ecology and molecular bases of MVRSA emergence in dairy farms. Knowledge, attitudes and practices offarm workers Among respondents, 47.8% lacked awareness regarding milk-borne zoonoses, which was lower than that (87%) reported in Ethiopia (6). Raw milk consumption was reported by 52.2% of the workers. This was higher than another report (35%) of raw milk consumption by dairy workers in Ethiopia (6). None of workers used PPE (gloves or masks) during work. Around half (47.8%) of workers don't wash their hands, which was lower than that reported in Ethiopia where none of the workers (100%) wash their hands (6). Finally, 82.6% and 73.9% of the worker would work with sore throat and diarrhea, respectively. Lack of awareness and unhygienic practices (raw milk consumption, lack of hand wash and PPE use) may pose an occupational zoonotic threat for the workers in these farms. In addition the unhygienic practices and willing to work with illness may contribute to the significant role of workers in disseminating S. aureus contamination in examined farms. Conclusion This study records high dissemination rate of S. aureuspathogens and their antibiotic resistance genes in dairy farms in Egypt, which may impact the health of dairy products consumers in Egypt. The study also highlights the critical points and practices associated with S. aureus dissemination in dairy farms, which will help in improving biosecurity planning and application in dairy farms in Egypt. References 1. Haran KP, Godden SM, Boxrud D, Jawahir S, Bender JB, &Sreevatsan S. Prevalence and Characterization of Staphylococcus aureus, Including Methicillin-Resistant Staphylococcus aureus isolated from Bulk Tank Milk from Minnesota Dairy Farms. Journal of Clinical Microbiology 2012; 50: 688-95. 2. Smith TC. Livestock-Associated Staphylococcus aureus: The United States Experience. PLoS Pathogens 2015; 11: e1004564. 3. Lee SH, Camargo CH, Gon?alves JL, Cruz AG, Sartori BT, Machado MB & Oliveira CA. Characterization of Staphylococcus aureus isolates in milk and the milking environment from small-scale dairy farms of Sao Paulo, Brazil, using pulsed-field gel electrophoresis. Journal of Dairy Science 2012; 95: 7377-83. 4. Lim SK, Nam HM, Jang GC, Lee HS, Jung SC & Kim TS. Transmission and persistence of methicillin-resistant Staphylococcus aureus in milk, environment, and workers in dairy cattle farms. Foodborne Pathogens and Diseases 2013;10:731-6. 5. Locatelli C, Cremonesi P, Caprioli A, Carfora V, Ianzano A, Barberio A, Morandi S, Casula A, Castiglioni B, Bronzo V &Moroni P. Occurrence of methicillin-resistant Staphylococcus aureus in dairy 754 W. Elmonir, H. Essa, W. El-Tras cattle herds, related swine farms, and humans in contact with herds. Journal of Dairy Science 2017; 100: 608-19. 6. Ayele Y, Gutema FD, Edao BM, Girma R, Tufa TB, Beyene TJ, Tadesse F, Geloye M &Beyi AF. Assessment of Staphylococcus aureus along milk value chain and its public health importance in Sebeta, central Oromia, Ethiopia. BMC Microbiology 2017; 17: 141. 7. Centers for Disease Control and Prevention (CDC). Staphylococcus aureus resistant to vanco-mycin--United States, 2002. Morbidity and Mortality Weekly Report (MMWR) 2002; 51: 565-7 8. Rahimipour F, Ghazvini K&Youssefi M. Reports of Vancomycin-Resistant Staphylococcus aureus from Middle East Countries, Archives of Clinical Infectious Diseases 2018;13: e59522. 9. Feng Y, Qi W, Xu-rong W, Ling W, Xin-pu L, Jin-yin L, Shi-dong Z, Hong-sheng L. Genetic characterization of antimicrobial resis-tance in Staphylococcus aureus isolated from bovine mastitis cases in Northwest China. Journal of Integrative Agriculture 2016; 15:2842-7. 10. Roberson JR Fox LK, Hancock DD, Gay JM &Besser TE. Ecology of Staphylococcus aureus isolated from various sites on dairy farms. Journal of Dairy Science 1994; 77: 3354-64. 11. Elmonir W, Abo-Remela EM &Sobeih A. Public health risks of Escherichia coli and Staphy-lococcus aureus in raw bovine milk sold in informal markets in Egypt. Journal of Infection in Developing Countries 2018; 12: 533-41. 12. Amghalia E, AL-Haj N, Shamsudin M, Radu S, Rosli R Neela V & Rahim R. Multiplex PCR assays for the detection of clinically relevant antibiotic resistance genes in Staphylococcus aureus isolated from Malaysian hospitals. Research Journal of Biological Sciences2009; 4: 444--8. 13. J0rgensen HJ, M0rk T &R0rvik LM. The occurrence of Staphylococcus aureus on a farm with small-scale production of raw milk cheese. Journal of Dairy Science2005; 88: 3810-7. 14. Garipcin M &Seker E. Nasal carriage of methicillin-resistant Staphylococcus aureus in cattle and farm workers in Turkey. VeterinarskiArhiv 2015;85:117-29. 15. Dimitracopoulos G, Kalkani-Boussiakou H &Papavassiliou J. Animal fecal carriership and biotypes of Staphylococcus aureus. Applied and Environmental Microbiology 1977; 34: 461-4. 16. Piccinini R, Cesaris L, Dapra V, Borromeo V, Picozzi C, Secchi C &Zecconi A. The role of teat skin contamination in the epidemiology of Staphylococcus aureus intramammary infections. Journal of Dairy Science 2009; 76: 36-41. 17. Zadoks RN, van Leeuwen WB, Kreft D, Fox LK, Barkema HW, Schukken YH & van BelkumA.Comparison of Staphylococcus aureus isolates from bovine andhuman skin, milking equipment, and bovine milk by phage typing,pulsed-field gel electrophoresis, and binary typing. Journal of Clinical Microbiology 2002; 40:3894-902. 18. Bhalla A, Aron DC &Donskey CJ. Staphylo-coccus aureus intestinal colonization is associated with increased frequency of S. aureus on skin of hospitalized patients. BMC Infectious Diseases 2007; 7: 105. 19. Elmoslemany AM, Keefe GP, Dohoo IR Wichtel JJ, Stryhn H &Dingwell RT. The association between bulk tank milk analysis for raw milk quality and on-farm management practices. Preventive Veterinary Medicine 2010; 95: 32-40. 20. Reinemann DJ, Wolters GMVH, Billon P, Lind O, Rasmussen MD. Review of practices for cleaning and sanitation of milking machines. 2000. Available athttps://milkquality.wisc.edu/wp-con-tent/uploads/sites/212/ 2011/10/review-of-prac-tices-for-cleaning.pdf (Accessed 10 November 2018). 21. Rossi F, Diaz L, Wollam A, Panesso D, Zhou Y, Rincon S, Narechania A, Xing G, Di Gioia TS, Doi A, Tran TT, Reyes J, Munita JM, Carvajal LP, Hernandez-Roldan A, Brandäo D, van der Heijden IM, Murray BE, Planet PJ, Weinstock GM & Arias CA. Transferable vancomycin resistance in a community-associated MRSA lineage. New England Journal of Medicine 2014; 370: 1524-31. Slov Vet Res 2019; 56 (Suppl 22): 755-63 DOI 10.26873/SVR-816-2019 Original Research Article ANTIMICROBIAL PHENOTYPES OF GEOGRAPHICALLY MATCHED Staphylococcus aureus ISOLATED FROM BUFFALO'S MILK AND CLINICAL HUMAN CASES IN EGYPT: POTENTIAL ZOONOTIC RISKS Walid Elmonir1*, Hader Aglan1, Ehab K.Elmahallawy2, Wael F. El-Tras3 1Hygiene and Preventive Medicine (Zoonoses) Department, Faculty of Veterinary Medicine, Kafrelsheikh University, Kafrelsheikh 33516, Egypt, 2ZoonosesDepartment, Faculty of Veterinary Medicine, Sohag University, Sohag82511 Egypt, 3Faculty of Aquatic and Fisheries Sciences (Zoonoses), Kafrelsheikh University, Kafrelsheikh33516, Egypt Corresponding author: walid.elmonir@gmail.com Abstract: Global emergence of antibiotic-resistant food-borne pathogens is a major public health problem. This study aimed to determine the potential zoonotic risk of Staphylococcus aureus in buffalo's milk in Egypt. A total of 102 raw buffalo's milk samples and 51 human nasal swabs were collected at Kafrelsheikh city, Egypt. All samples were examined for occurrence of s. aureus. Detected isolates were characterised based on DNase activity, mecA gene acquisition, and antibiotic resistance patterns. s. aureus was detected in 33.3% of buffalo's milk samples and29.4% of human nasal swabs. Multiple drug-resistant S.aureus (MDRSA) represented 88.2% and 90% of buffalo and human s. aureus isolates, respectively. Buffalo and human s. aureus isolates showed highest resistance rate for erythromycin (100%), and lowest resistance rate for gentamicin (22.2%). Interestingly, there was no significant difference in resistance patterns between methicil-lin-resistant s. aureus (MRSA) and methicillin-sensitive s. aureus (MSSA) (p<0.46 -0.97). One-third of the detected s. aureus phenotypes (5/15, 33.3%) were identical between buffalo and human isolates. Moreover, there was no significant difference in antibiotic resistance patterns between buffalo and human isolates (p<0.1 - 0.97).This study highlights the potential public health risk of MDRSA transmission via buffalo's milk. Key words: buffalo's milk, s. aureus, DNase, mecA, antibiogram, zoonotic risk Introduction The last few years have witnessed an alarming increase in the reports of antibiotic-resistant pathogens in human and veterinary practices. Special concern is given to the possibility of food contamination with multiple drug-resistant (MDR) bacteria or bacteria carrying antibiotic-resistant genes (1,2). Methicillin-re-sistant Staphylococcus aureus (MRSA) strains are P-lactam-resistant S. aureus that can easily acquire resistance to a wide spectrum of other antibiotics (3). Thus, the high rate of dissemination of these pathogens worldwide has created an additional infection control problem in both human and veterinary medicine (4). Multiple drug-resistant S. aureus (MDRSA) strains, including MRSA strains, have been isolated from bovine milk and different dairy products worldwide (5-7). Bovine MRSA strains have Received: January 2019 Accepted for publication: February 2019 756 W. Elmonir, H. Aglan, E .Elmahallawy, W. El-Tras emerged as zoonotic organisms based on several lines of evidence showing the possibility of direct transmission of MRSA between bovines and humans (4, 8). In Egypt, buffalo's milk and its derived dairy products are more popular than those derived from cows owing to the higher fat content, whiter colour and creamier flavour of buffalo's milk than of cow's milk (9). Buffalo's milk is the second most produced milk in Egypt, with over2 million tons produced annually (10), making Egypt the fourth largest buffalo's milk producer worldwide (10). Despite this important aspect, there is not sufficient data on the role of Buffalo's milk in maintaining the epidemiological foci of MDRSA at the national level. Therefore, the aim of this study was to elucidate the potential public health risk of S. aureus in buffalo's milk by reporting the prevalence and antibiotic resistance patterns of S. aureus isolates from buffalo's milk used for consumption by residents of Kafrelsheikh, Egypt. Furthermore, the phenotypes of buffalo S. aureus isolates were compared with those isolated from geographically matched human clinical cases to evaluate the potential zoonotic risk of buffalo's milk-borne S. aureus isolates. Materials and methods Sampling Buffalo's milk and human nasal samples were collected from Kafrelsheikh city, the capital of Kafrelsheikh Governorate, which is located in the northern region of the Nile Delta, Lower Egypt (31°06'42'N 30°56'45''E). Buffalo milk used for consumption in the study region was bought from either markets or livestock smallholders. A total of 102 milk samples (100 ml per sample) were collected from Kafrelsheikh between September and December 2016. The buffalo's milk samples were divided as follows: half of the samples (51 samples) were bought from various markets at different localities in the city, while the other half were collected directly from buffaloes owned by smallholders. Potential heat treatment of market milk samples was evaluated by the pe-roxidase (Storch) test (11), and only raw samples were included in this study. Milk samples from buffaloes were collected under aseptic conditions as composite milk samples from the 4 quarters. All sampled buffaloes were apparently healthy, and the collected milk samples showed no physical or organoleptic abnormalities. During the same study period, a total of 51 nasal swabs were collected from outpatients at Kafrelsheikh Chest Hospital. All human samples were collected by the medical staff of the hospital. Isolation and identification of S. aureus Isolation of S. aureus were conducted according to the guidelines of the Food and Drug Administration (12). Suspected S. aureus colonies were identified based on the following criteria: grape-like clusters of gram-positive cocci by Gram staining and yellow colonies on man-nitol salt agar (Oxoid, Hampshire, U.K.), beta haemolysis on sheep blood agar (Oxoid, Hampshire, U.K.), and firm coagulation in the tube coagulase test using rabbit plasma (12). Phenotyping of S. aureus isolates From the 49 detected S. aureus isolates, 27 (17 buffalo and 10 human) isolates were chosen for phenotyping based on biochemical profiles, mecA gene acquisition, and antibiotic resistance patterns. For biochemical profiles, the following tests were used (13): DNase, catalase, oxidase, Voges-Proskauer, growth in 10% NaCl, argi-nine decarboxylase, esculin hydrolysis, and sugar fermentation (lactose, maltose, ribose, arabinose, sorbitol, and raffinose). All S. aureus isolates showed the same biochemical profile except for the DNase test results; DNase activity was used as a marker for the biochemical profiles. For molecular detection of the mecA gene, S. aureus cultures incubated in broth overnight were used for DNA extraction by the QIAamp DNA Mini kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions. Amplification of the mecA gene was conducted according to Murakami et al. (14) using the following primers: forward mecA1-5 AAAATCGATGGTAAAGGTTGG 3' reverse mecA2-5'AGTTCTGCAGTACCGGATTTG Antimicrobial phenotypes of geographically matched Staphylococcus aureus isolated from buffalo's milk 757 3'. Five pL of DNA extracted from S. aureus-culture was added to a PCR mixture that contained 1 pL (20 pmol) of each primer, 12.5 pL of DreamTaq PCR Master Mix (Thermo Fisher Scientific, Waltham, USA) and distilled water up to the 25 pL reaction volume. PCR was performed in a Mastercycler (Eppendorf, Hamburg, Germany) using the following conditions: 95°C for 3 min followed by 35 cycles of 94°C for 30 s, 55°C for 30 s and 72°C for 1 min and a final extension at 72°C for 10 min. Positive control (mecA+ S. aureus isolate) was kindly provided by Prof. Mohamed Hassan, Prof. of Food Hygiene, Faculty of Veterinary Medicine, Benha University, Egypt. PCR products were examined by gel electrophoresis and UV illumination (Fig. 1). For antibiotic resistance patterns, a standard disk diffusion assay was used for antibiotic sensitivity testing according to the guidelines of the Clinical and Laboratory Standards Institute (15). The antibiotic disks (Oxoid, Hampshire, U.K.) used in this study are listed in table 2. S. aureus isolates resistant to cefoxitin were considered resistant to all ß-lactams and were designated as MRSA. S. aureus isolates sensitive to cefoxitin were designated as methicillin-sen-sitive S. aureus (MSSA). MSSA isolates were further tested using ampicillin (10 pg) disks. S. aureus isolates that showed resistance to ampi-cillin were tested by nitrocefin discs (Thermo Scientific, Lenexa, USA) for ß-lactamase activity. Statistical analysis The potential difference in antibiotic resistance patterns among the chosen 27 S. aureus isolates was assessed using a univariate logistic regression model to compare MRSA versus MSSA isolates or buffalo versus human isolates as the response variables. The model was built for all tested antibiotics with the exception of erythromycin and ampicillin. These antibiotics were removed because all 27 isolates were resistant to erythromycin and because ampicillin was used only for MSSA isolates. The statistical analyses were carried out using SAS 9.2 (SAS Institute Inc., 2008). Statistical significance was considered at P<0.05. Ethical approval The research details and methods were approved by the Ethics Committee of the Hygiene and Preventive Medicine Department, Faculty of Veterinary Medicine, Kafrelsheikh University, Egypt. For human samples, all nasal samples were collected by the medical staff of Kafrelsheikh Chest Hospital. The research details and risks of participation were explained to the participating outpatients, and written consent was obtained. Results and discussion In the present study, S. aureus was isolated from 33.3% of the examined buffalo's milk samples (table 1), which is similar to the results of a previous study (33.3%) in Turkey (5). However, our result was higher than another report (17.5%) in Iran (16). S. aureus was detected in 29.4% of nasal swabs from outpatients at Kafrelsheikh Chest Hospital (table 1). Likewise, Ungureanu et al. (17) isolated S. aureus from 35.38% of nasal exudates collected from hospitalized patients and outpatients in Romania. Bovine milk could act as a vehicle for MRSA transmission to humans (4, 8). Acquisition of the mecA gene defines MRSA isolates (4).A total of 35.5% of S. aureus isolates from buffalo's milk were MRSA (table 1). This value was higher than a report (9.2%) in Turkey (5). However, a higher rate of MRSA (56.1%) was reported in Uganda (6). The relatively high rates of MRSA reported in this study may be attributed to unhygienic milking and rearing of buffaloes in Egypt. The majority of buffalo populations in Egypt are household reared, and their milk is sold in informal markets (7). Manual milking and unhygienic milking equipment, milk storage conditions, and milk transportation are common practices in this production system. These practices may contribute to the high contamination rate observed in this study. MRSA represented 40% of human S. aureus isolates, which was higher than that in other studies (18.5-30%) reported elsewhere (17,18). MDR to more than 3 classes of antibiotics was observed in 88.2% and 90% of buffalo and 758 W. Elmonir, H. Aglan, E .Elmahallawy, W. El-Tras human S. aureus isolates, respectively (table 1). In agreement with our findings, high rate (95.5%) of MDRSA were previously reported in bovine milk in Egypt (7). In contrast, other studies reported high sensitivity of bovine milk S. aureus isolates to several antibiotics in Brazil (19) and in Pakistan (20). The majority (90%) of the human S. aureus isolates were MDRSA (table 1). This value was higher than that in a study in Romania (18.75 - 45.45%) by Ungure-anu et al. (17) but comparable to that in another report in Iran (61 - 93%) (18). All MRSA isolates in this study were cefox-itin-resistant (table 2). This result implies a broad-spectrum resistance to all B-lactams, with the exception of anti-MRSA cephalosporins (15). Interestingly, 47.1% of the MSSA isolates were resistant to ampicillin (table 2). Ampicil-lin-resistant MSSA is also resistant to penicilli-nase-labile B-lactams (15). Both buffalo and human MSSA isolates showed ampicillin resistance at rates of 45.5% and 50%, respectively (table 2). In line with our findings, several pre vious studies reported resistance of bovine MSSA (2, 5), and human MSSA (17, 18) to penicillinase-labile B-lactams at variable rates. MRSA isolates harboured the mecA gene, which mediates resistance to B-lactams by encoding penicillin-binding protein 2a (PBP2a). On the other hand, MSSA isolates in this study lacked the mecA gene but produced the fi-lac-tamase enzyme (100%); the filactamase enzyme is encoded by the blaZ gene (21). In agreement with this finding, Yokoyama et al. (21) attributed B-lactam resistance in MSSA to the production of the fi-lactamase enzyme. They also reported a higher prevalence of the blaZ gene among MSSA isolates than among MRSA isolates (21). All buffalo and human S. aureus isolates were resistant to erythromycin (100%) (table 2). High rates of erythromycin resistance among bovine milk S. aureus isolates (50.5 -77.3%) were also reported by Pamuk et al. (5) and Elmonir et al.(7). In contrast, Aires-deSousa et al. (19) and Asiimwe et al.(6) reported much lower erythromycin resistance rates (3.3 - 6.5%). Similarly, high rates of erythromycin resistance (61.3 - 93%) were shown in human S. aureus isolates in other studies (17, 18). In addition, buffalo and human S. aureus isolates showed high resistance rates (70.4 -88.9%) for kanamycin, sulphamethoxazole/tri-methoprim, and tetracycline (table 2). Previous reports recorded comparable resistances of bovine and human S. aureus isolates for kanamy-cin, sulphamethoxazole/trimethoprim, and tetracycline (6, 7, 17, 18). However, other reports showed high efficacy of sulphamethoxazole/trimethoprim and tetracyclines on bovine S. aureus isolates (6, 19, 20). All S. aureus isolates in this study showed low resistance for ciprof-loxacin (33.3%) and gentamicin (22.2%) (Table 2). High efficacy of ciprofloxacin was also reported elsewhere for bovine isolates (6, 20) and for human isolates (17). In contrast, Rahimi et al. (18)reported high resistance (95%) of human MRSA isolates for ciprofloxacin. Gen-tamicin is used in combination with vancomy-cin or cephalosporins as an alternative therapeutic choice for severe MRSA infections that show reduced susceptibility to vancomycin or daptomycin (22, 23). Hence, resistance to gentamicin limits treatment options and raises public health concerns. A total of 11.8% and 40% of buffalo and human isolates, respectively, showed resistance to gentamicin (table 2). In line with our findings, several studies reported resistance of bovine S. aureus isolates to gen-tamicin at rates ranging from 10% to 64.3% (5, 7, 20, 24).However, the findings of this study disagree with those of other studies that showed high rates of gentamicin efficacy (6, 19). High rates (18.5 - 59%) of gentamicin resistance among human S. aureus isolates were previously reported (17, 18), which agrees with our findings. The discrepancies in the present results compared with the results from various previous studies could be attributed to multiple factors, including differences in national policies for antibiotic administration to animals, animal production systems, sanitary measures of animal rearing and animal byproduct marketing, personal hygiene, and sampling methods as well as demographic and regional differences. Antimicrobial phenotypes of geographically matched Staphylococcus aureus isolated from buffalo's milk 759 Table 1: Frequency distribution of S. aureus isolated from buffalo's milk and human nasal swabs in this study Buffaloes Humans Isolates Market Household Total Milk Nasal Total Milk Milk swabs S. aureus 16/51 18/51 (35.3) 34/102 15/51 49/153 (31.4) (33.3) (29.4) (32.03) MRSA 3/8 3/9 6/17 4/10 10/27 (37.5) (33.3) (35.3) (40) (37.04) MSSA 5/8 6/9 11/17 6/10 17/27 (62.5) (66.7) (64.7) (60) (62.96) MDRSA 7/8 8/9 15/17 9/10 24/27 (87.5) (88.9) (88.2) (90) (88.9) MDR-MRSA 3/3 3/3 6/6 3/4 9/10 (100) (100) (100) (75) (90) MDR-MSSA 4/5 5/6 9/11 6/6 15/17 (80) (83.3) (81.8) (100) (88.2) Brackets: Percent; MRSA: Methicillin-resistant S. aureus; MSSA: Methicillin-sensitive S. aureus; MDRSA: multiple drugs resistant S. aureus Table 2: Antibiotic resistance diversity of S. aureus isolates detected in this study Antimicrobial agent Species Methicillin resistance Total No. (%) Buffalo No. (%) Human No. (%) MRSA No. (%) MSSA No. (%) Cephems FOX (30^g) 6 (35.3) 4 (40) 10(100) 0 (0) 10 (37.04) Penicillins AMP (10^g) 11 (64.7) 7 (70) 10 (100) 8 (47.1) 18 (66.7) Aminoglycosides CN (10Mg) 2 (11.8) 4 (40) 3 (30) 3 (17.6) 6 (22.2) K (30pg) 15 (88.2) 9 (90) 9 (90) 15 (88.2) 24 (88.9) Macrolides E (15pg) 17(100) 10(100) 10(100) 17 (100) 27 (100) Fluoroquinolones CIP (5^g) 4 (23.5) 5 (50) 4 (40) 5 (29.4) 9 (33.3) Nitrofurantoins F (300^g) 5 (29.4) 5 (50) 4 (40) 6 (35.3) 10 (37.04) Tetracyclines TE (30^g) 12 (70.5) 7 (70) 7 (70) 12 (70.6) 19 (70.4) Phenicols C (30pg) 9 (52.9) 6 (60) 6 (60) 9 (52.9) 15 (55.6) Sulfonamides SXT (25^g) 15 (88.2) 9 (90) 9 (90) 15 (88.2) 24 (88.9) FOX: cefoxitin; AMP: ampicillin; CN: gentamicin; K: kanamycin; E: erythromycin; CIP: ciprofloxacin; F: nitrofurantoin; TE: tetracycline; C: chloramphenicol; SXT: sulfamethoxazole/trimethoprim The main difference between MRSA and MSSA is the spectrum of antibiotic resistance. Previous studies showed that the majority of MRSA strains are multi-resistant to P-lactams and to a wide spectrum of other antibiotics, while MSSA strains show much lower rates of resistance to multiple antibiotics in both human and bovine infections (3, 17, 24). Unlike these studies, our study showed no significant difference (P<0.46 - 0.97) in resistance patterns between MRSA and MSSA isolates for 7 classes of antibiotics (table 3). This study recorded un-precedentedly high rate (81.8%) of MDR among MSSA isolates from buffaloes (Table 1). Reports of MDR-MSSA in bovine milk are mostly from developing countries (2, 7). This fact could be attributed to unhygienic milk production and misuse of antibiotic therapy in veterinary practices in these countries. This evidence also highlights the role of buffaloes in the carriage and dissemination of MDR-MSSA, 760 W. Elmonir, H. Aglan, E .Elmahallawy, W. El-Tras Table 3: Univariate regression model for the association of antibiotic resistance among MRSA vs. MSSA and Buffalo vs. Human S. aureus isolates MRSA vs. MSSA Buffal o vs. Humans Variable C. OR P< 95% CI C. OR P< 95% CI K MRSA - - - Buffalo - - - MSSA 0.83 0.89 0.1 - 10.6 Human 1.2 0.88 0.1 - 15.20 SXT MRSA - - - Buffalo - - - MSSA 0.83 0.88 0.1 - 10.6 Human 1.2 0.88 0.1 - 15.20 TE MRSA - - - Buffalo - - - MSSA 1.03 0.97 0.19 - 5.68 Human 0.97 0.97 0.18 - 5.37 C MRSA - - - Buffalo - - - MSSA 0.75 0.72 0.15 - 3.65 Human 1.33 0.72 0.27 - 6.0 F MRSA - - - Buffalo - - - MSSA 0.82 0.80 0.16 - 4.10 Human 2.4 0.3 0.48 - 12.13 CIP MRSA - - - Buffalo - - - MSSA 0.63 0.57 0.12 - 3.22 Human 3.25 0.17 0.61 - 17.28 CN MRSA - - - Buffalo - - - MSSA 0.5 0.46 0.08 - 3.15 Human 5.0 0.10 0.72 - 34.92 C.: Categories; OR: Odd ratio; CI: Confidence interval which raises concerns regarding potential hazards to the animal industry and safety of milk in these countries, including Egypt. MSSA is characterized by higher fitness and shorter generation time than MRSA; hence, MSSA causes a higher number of cases than MRSA does (3). If such strains attain MDR, which hinders the treatment and eases the dissemination of these pathogens, a serious public health hazard is predictable. Klein et al. (25) reported an increase in hospitalization costs associated with MSSA-related infections relative to MRSA-related infections between 2010 and 2014 in the USA. They hypothesized that ineffective treatment approaches for MSSA may be one of the reasons for this shift (25). We may expect a further increase in the burden of MSSA infections at the human-animal interface with the potential emergence of MDR-MSSA. Zoonotic transmission of S. aureus between bovines and humans is well documented (4, 8). Fifteen phenotypes of S. aureus were determined in this study (table 4). Approximately one-third of these phenotypes (5/15, 33.3%) were shared by 8/10 (80%) and 6/17 (35.3%) of the human and buffalo isolates, respectively. Additionally, none of the tested antibiotics showed a significant difference between buffalo and human isolates by univariate analysis (P<0.1 - 0.97), (table 3). In contrast, Jayaweera and Kumbukgolla (2) reported significantly higher odds of resistance to gentamicin and ciprofloxacin for animal S. aureus isolates than for human isolates; however, they included isolates from other species (e.g., poultry and pigs) in their analysis, which may explain the contradiction with our findings. The high phenotypic similarity of human isolates with buffalo isolates, especially in terms of antibiotic resistance patterns, highlights the potentially high rate of zoonotic interspecies transmission between humans and buffaloes and emphasizes the role of buffalo's milk as a vehicle of MDRSA for humans in the study region. Antimicrobial phenotypes of geographically matched Staphylococcus aureus isolated from buffalo's milk 761 Table 4: Phenotyping of isolated S. aureus isolates from buffalo's milk and human nasal swabs samples P* Dnase mecA Antibiogram profile Source No. P1 + FOX, E, K, SXT, TE, C, F, CIP, CN Human 2 Buffalo 1 P2 - FOX, E, K, SXT, TE, C, F, CIP Human 1 P3 + + FOX, E, K, SXT, TE, C Buffalo 2 P4 + FOX, E, K, SXT, TE Buffalo 1 P5 + FOX, E, K, SXT Buffalo 2 P6 + FOX, E Human 1 Total MRSA 10 P7 + AMP, E, K, SXT, TE, C, F, CIP, CN Human 2 Buffalo 1 P8 + AMP, E, K, SXT, TE, C, F, CIP Buffalo 2 P9 + AMP, E, K, SXT, TE, C, F Buffalo 1 P10 + AMP, E, K, SXT, TE, C Human 1 Buffalo 1 P11 + - E, K, SXT, TE, C Buffalo 1 P12 + E, K, SXT, TE Human 1 Buffalo 2 P13 + E, K, SXT Human 2 Buffalo 1 P14 + E Buffalo 1 P15 - E Buffalo 1 Total MSSA 17 P: Phenotype 762 W. Elmonir, H. Aglan, E .Elmahallawy, W. El-Tras (A) M P N 533 bp 1 2 3 4 5 6 7 8 9 10 500 ^m* Figure 1: Molecular detection of Methicillin resistance gene (mecA) among S. aureus pathogens isolated from Buffalo's milk and human nasal swabs. (A) Human nasal swabs isolates. (B) Buffalo's milk isolates. M: 100 bp DNA marker, P: Positive control, and N: Negative control. Conclusion Our study highlights the possibly high risk of MDRSA dissemination to humans via buffalo's milk in the study region. The high rates of buffalo's milk-borne MDR-MRSA and MDR-MSSA need further investigation to explore resistance mechanisms and risk factors that contribute to the emergence of these pathogens in the study region. This study also highlights the genuine need for hygienic production and marketing of buffalo's milk, restriction and supervision of antibiotic therapy in veterinary practices, and public awareness about the potential risks of raw buffalo's milk consumption and processing. Acknowledgements We thank Yamen Hegazy for assistance with statistics. Conflict of interest None declared. References 1. Laxminarayan, R., Matsoso, P., Pant, S., Brower, C., Rottingen, J.A., Klugman, K. and Davies, S. Access to effective antimicrobials: a worldwide challenge. Lancet 2016;387: 168-75. 2. Jayaweera, J. and Kumbukgolla, W.W. Antibiotic resistance patterns of methicillin-resistant Staphylococcus aureus (MRSA) isolated from livestock and associated farmers in Anuradhapura, Sri Lanka. Germs2017;7: 132-9. 3. Rozgonyi, F., Kocsis, E., Kristof, K. and Nagy, K. Is MRSA more virulent than MSSA? Clin Microbiol Infect 2007;13: 843-5. 4. Becker, K., Ballhausen, B., Kock, R. and Kriegeskorte, A. Methicillin resistance in Staphylococcus isolates: the "mec alphabet" with specific consideration of mecC, a mec homolog associated with zoonotic S. aureus lineages. Int J Med Microbiol 2014;304: 794-804. 5. Pamuk, S., Yildirim, Y., §EKER, E., Gürler, Z., Kara, R. A survey of the occurrence and properties of methicillinresistant Staphylococcus aureus and methicillin-resistant Staphylococcus intermedius in water buffalo milk and dairy products in Turkey. International Journal of Dairy Technology2012; 65: 416-22. Antimicrobial phenotypes of geographically matched Staphylococcus aureus isolated from buffalo's milk 763 6. Asiimwe, B.B., Baldan, R., Trovato, A. and Cirillo, D.M. Prevalence and molecular characteristics of Staphylococcus aureus, including methicillin resistant strains, isolated from bulk can milk and raw milk products in pastoral communities of South-West Uganda. BMC Infect Dis 2017;17: 422. 7. Elmonir, W., Abo-Remela, E.M. and Sobeih, A. Public health risks of Escherichia coli and Staphylococcus aureus in raw bovine milk sold in informal markets in Egypt. . Infect Dev Ctries 2018;12: 533-41. 8. Juhasz-Kaszanyitzky, E., Janosi, S., Somogyi, P., Dan, A., van der Graaf-van Bloois, L., van Duijkeren, E. and Wagenaar, J.A. MRSA transmission between cows and humans. Emerg Infect Dis 2007;13: 630-2. 9. Borghese, A. Recent developments of buffaloes in Europe and Near East. Proc of the Seventh World Buffalo Congress, Manila, Philippines 20 to 23 Oct 2004: 10-6. 10. FAOSTAT Food and agriculture organization statistics.2016available at: http://www.fao.org/faostat/en/#data/QL. (accessed in 10-10-2018). 11. Roberts, D.G., M. Practical food microbiology, 3rd edition. Oxford: Blackwell publishing Ltd. 2003, 206 p. 12. FDA, Bacteriological Analytical Manual Online. Chapter 12: Staphylococcus aureus (January 2001 update) Available: https://wwwfdagov/Food/FoodScienceResearch/La boratoryMethods/ucm071429htm (Accessed: 10 October 2018). 13. MacFaddin, J.F. Biochemical tests for identification medical bacteria. . Warery Press Inc, Baltimore, Md 21202,2000, USA. 14. Murakami, K., Minamide, W., Wada, K., Nakamura, E., Teraoka, H. and Watanabe, S. Identification of methicillin-resistant strains of staphylococci by polymerase chain reaction. J Clin Microbiol 1991;29: 2240-4. 15. Clinical and Laboratory Standards Institute [CLSI] Performance Standards for Antimicrobial Susceptibility Testing; twenty second Informational Supplement. CLSI document M100-S22, 2012,ISBN 1-56238-785-5. 16. Rahimi, E. and Alian, F. Presence of enterotoxigenic Staphylococcus aureus in cow, camel, sheep, goat, and buffalo bulk tank milk. Veterinarski Arhiv2013; 83: 23-30. 17. Ungureanu, A., Zlatian, O., Mitroi, G., Drocas, A., Tirca, T., Calina, D., Dehelean, C., Docea, A.O., Izotov, B.N., Rakitskii, V.N., Cioboata, R., Spandidos, D.A., Tsatsakis, A.M. and Gaman, A. Staphylococcus aureus colonisation in patients from a primary regional hospital. Mol Med Rep 2017;16: 8771-80. 18. Rahimi, F., Bouzari, M., Katouli, M. and Pourshafie, M. Antibiotic Resistance Pattern of Methicillin Resistant and Methicillin Sensitive Staphylococcus aureus Isolates in Tehran, Iran. Jundishapur J Microbiol 2012;6: 144-9. 19. Aires-de-Sousa, M., Parente, C.E., Vieira-da-Motta, O., Bonna, I.C., Silva, D.A. and de Lencastre, H. Characterization of Staphylococcus aureus isolates from buffalo, bovine, ovine, and caprine milk samples collected in Rio de Janeiro State, Brazil. Appl Environ Microbiol2007; 73: 3845-9. 20. Aqib, A.I., Ijaz, M., Anjum, A.A., Malik, M.A.R., Mehmood, K., Farooqi, S.H. and Hussain, K. Antibiotic susceptibilities and prevalence of Methicillin resistant Staphylococcus aureus (MRSA) isolated from bovine milk in Pakistan. Acta Trop 2017;176: 168-72. 21. Yokoyama, T., Honda, J., Kawayama, T., Kajimura, K. and Oizumi, K. Increased incidence of beta-lactamase-plasmid negative, high level methicillin-resistant Staphylococcus aureus (MRSA). Kurume Med J 1996;43: 199-206. 22. Nimmo, G.R. and Coombs, G.W. Community-associated methicillin-resistant Staphylococcus aureus (MRSA) in Australia. Int J Antimicrob Agents2008; 31: 401-10. 23. Unger, N.R., Osiyemi, O.O., Richardson, R.M., Jose Menajovsky, J. and Jennifer Kuretski, J. Ceftaroline and gentamicin for the treatment of daptomycin-non-susceptible meticillin-resistant Staphylococcus aureus bacteraemia and endocarditis in a pregnant patient. JMM Case Reports 2014; DOI 10.1099/jmmcr.0.002394. 24. Li, T., Lu, H., Wang, X., Gao, Q., Dai, Y., Shang, J. and Li, M. Molecular Characteristics of Staphylococcus aureus Causing Bovine Mastitis between 2014 and 2015. Front Cell Infect Microbiol 2017;7:127. 25. Klein, E.Y., Jiang, W., Mojica, N., Tseng, K.K., McNeill, R., Cosgrove, S.E. and Perl, T.M. National Costs Associated with Methicillin-susceptible and Methicillin-resistant S. aureus Hospitalizations in the United States, 2010-4. Clin Infect Dis2018; doi: 10.1093/cid/ciy399. Slov Vet Res 2019; 56 (Suppl 22): 765-72 DOI 10.26873/SVR-817-2019 Original Research Article EFFECT OF SUPPLEMENTATION OF OMEGA-3 FATTY ACIDS ON BLOOD PARAMETERS AND SEMEN QUALITY OF FRIESIAN BULLS Mohamed A. Abu El-Hamd1*, Abd Salam M. Metwally2, Mohamed M. Hegazy1, Zahya R. Ghallab2, Ola A. Elateeqy2 1Animal Production Research Institute, Agricultural Research Center, Dokki, Egypt, 2Department of Animal Production, Faculty of Agriculture, Kafrelsheikh University, 33516, Egypt Corresponding author E-mail: abuelhamd68@yahoo.com Abstract: The aim of this work was to evaluate the effects of flaxseed oil supplementation as a source of omega-3 fatty acids (OFA) on some blood parameters, semen quality and testosterone level in male Frisian bull. A total of 30 Friesian bulls (14.2 ± 0.16 month of age and 265±15 kg body weight) were divided into three equal groups according to age and live body weight. Bulls in the 1st group (G1) were supplemented with 2% dray matter intake (DMI) flaxseed oil as a source of OFA, while those in 2nd group were supplemented with 4% DMI flaxseed oil as a source of OTA at 14 to 21 months of age (G2). Bulls in the 3rd group were fed a basal diet and considered as a control group (G3). The obtained results revealed that OFA administrated animals had a significant (P< 0.05) inducing effect on serum levels of total protein, globulin, glucose, high density lipoprotein (HDL) and triglyceride compared to the control group. No significant changes were noticed in albumin levels among the three groups. However, the levels of total cholesterol, urea and low density lipoprotein (LDL) were significantly lower in bulls received OFA than the control group. Omega-3 fatty acids treatment has a significant positive effect on the semen characteristics and lower abnormality in G2 and G3 than in G1. G2 and G3 also showed a significant higher intact spermatozoa cell membrane than in G3 by about 9.68 and 10.84%, respectively. Omega-3 treatment significantly increased blood testosterone levels to be 23,78% in G2 and 31.82% in G3 higher than that of the control. In conclusion, dietary supplementation with omega-3 in male Frisian ration improved semen quality and reproductive potentiality as well as testosterone level. Key words: Frisian bull; omega-3 fatty acids; testosterone and semen quality Introduction Bull fertility has a high economic importance in cattle artificial fertilization (AI) industry as semen high quality is crucial for suc-cess-ful AI. Flaxseed oils are an excellent as a source of a- linolenic acid, a member of the omega-3 fatty acids (OFA) (1). The fraction of flaxseed oil is approximately 0.55 omega-3a-linolenicacid (2). OFAs are able to lower the risks of some diseases (1, 3). Alpha linolenic acid, an essential OFA, is a precursor of eicosa-pentaenoic acid (EPA), which in turn is a precursor for the formation of eicosanoids. Eico-sanoids are hormone-like compounds that play an essential role in immunity faction. Some Received: January 2019 Accepted for publication: February 2019 766 M. Abu El-Hamd, A. Metwally, M. Hegazy, Z. Ghallab, O. Elateeqy study reported that EPA can elongate further to docosahexanoic acid (DHA), an OFA that is essential for cell membrane integrity and brain health (4). Prostaglandins (PG) may play an important role in reproductive performance, especially semen quality (5). Arachidonic acid is a subsequent production of PG and is involved on synthesis of the steroid hormone (6). Total sperm number (7) and sperm motility (8) were improved following administration of fish oil to boars. On the other hand, semen characteristics were negatively affected after treatment with omega-6 in humans (9). High concentrations of polyunsaturated fatty acid (PUFA) in sperm membranes may improve semen quality after supplementation of long-chain omega-3 (5). The aim of this work was to study the effect of OFA on some blood parameters, testosterone level and semen quality in Friesian bull. Materials and methods The experiment was performed at Sakha Experimental Station, Kafr-Elsheikh Governorate located in the Northern part of the Nile delta, at Animal Production Research Institute (APRI), Agricultural Research Center, Ministry of Agriculture, Egypt. Animals A total number of 30 Friesian bulls averaging 14.2 ± 0.16 month of age and 265±15 kg body weight were used in this study. The bulls were randomly classified into three groups (10 each), according to their body wieght and age. Bulls in the 1st group (G1) were supplemented with 2% DMI flaxseed oil as a source of OFA, while the bulls in the 2nd group were supplemented with 4% DMI flaxseed oil (G2). Bulls in the 3rd group were fed a basal diet and considered as a control group (G3). All bulls were judged as free of diseases and physical defects genitalia. The experimental animals were kept freely under semi-open sheds and were fed according to the recommendations of Animal Production Research Institute (APRI, 10) throughout the experimental period. Feeding system and management The concentrate feed mixture (CFM) was used in bull feeding in all groups. It was composed of 37.5% yellow corn, 20% soybean meal, 15% corn gluten, 22.5% wheat bran, 3% molasses, 0.5% premix (one kg of premix contained 3.3 x 106 IU vit. A; 3.3 g vit. E; 3.3 x 106 IU vit. D3; 0.33 g vit. K; 0.33 g vit. B1; 1.33 g vit. B2; 6.67 g vit. B5; 0.50 g vit. B6; 3.3 g vit. B12; 3.3 g vit. pantothenic acid; 0.33 g folic acid; 16.67mg Biotin; 166.67 g Cholin; 1g Copper; 10 g Iron; 13.3g Mn; 15 g Zn; 0.1 g iodin; 0.03 g Se and carrier CaCO3 to 1kg) and 1.5% common salt. Bulls in all groups were fed equal amounts of diet containing CFM, rice straw and fresh berseem (during winter season) or berseem hay (during summer season) according to the recommendation of the APRI, (10). Allowances for growing dairy bulls based on live body weight. Chemical analysis of representative monthly samples of foodstuffs was analyzed for CP, CF, EE, NFE and ash on DM basis according to the official methods of the A.O.A.C (11). Chemical composition of CFM, rice straw, fresh berseem and berseem hay used in feeding bulls in both groups is shown in Table (1). Experimental procedures Throughout the experimental period, semen samples were collected on all animals twice weekly using an artificial vagina at 18 months of age bulls at time of collection up to 21 months of age. After semen collection, each ejaculate was evaluated for volume, sperm concentration (x106/ml) mass motility (%), live sperm (%), sperm abnormality (%) and sperm concentration/ejaculate (x106/ml). As well as, sperm cell concentration was directly evaluated according to Barth (12). The integrity of plasma membrane for the fresh spermatozoa was examined using hypo-osmotic swelling (HOS) test (13). The HOS solution at a concentration of 100 mOsm/kg was prepared by mixing 0.49 g of sodium citrate and 0.9 g fructose with 100 ml distilled water. In brief, 250 pi of diluted semen were added to Effect of supplementation of omega-3 fatty acids on blood parameters and semen quality of friesian bulls 767 1ml of the pre-warmed HOS solution and incubated for 60 min at 37°C. A volume of 5pl from each sample was put on clean and warm, microscope slide and were examined at x400. Spermatozoa were counted 200 per sample and the indicative of intact plasma membrane were also determined. Blood sampling Blood samples were monthly collected during the experimental period in clean test tubes via the jugular vein from all the experimental bulls and were centrifuged at 3000 rpm for 10 minutes. The obtained serum was keep at -20oC until determination of testosterone concentration. Testosterone assay Total and free serum testosterone assay was conducted by radio immune assay method (RIA) using Pontex 335 kit (I125). Total testosterone included free testosterone and that bound to sex steroid binding globulin hormone, albumin, corticosteroid binding globulin (CBG). The standard curve of testosterone ranged between 0.1 and 25.6 ng/ml. Biochemical assays Serum biochemical parameters (total protein, globulin, glucose, cholesterol, triglyceride, HDL and LDL) were done using commercial kits (Diagnostic System Laboratories, Inc., USA) and as previously described (14-16). Statistical analysis Statistical analyses of data were carried out applying the package of Snedecor and Cochran (17). A factorial design (3 groups x ages) was used and the statistical model was: Yijk = U + Ai + Bj + ABij + eijk. Where: Yijk = Observed values; U = Overall mean; Ai = group; Bj = age; ABij = Interaction due group x age; eijk = Random error The significant differences among means were tested using Duncan Multiple Range Test. Correlation analysis was carried out using computer programmer of SAS system. The percentage values of semen characteristics were tested by arcsine transformation so the means were presented after recalculated from the transformed values to percentages. Results and discussion Serum biochemical parameters in blood Flaxseed oil is essential polyunsaturated fatty acids work as constituent of many enzymes which involved of majority of metabolic pathways also was important for metabolism of protein and growth of organ and immunity response. In the current study, flaxseed oil (as a source of OFA) supplementation to Friesian bulls improved the serum content of total protein and lipid profile and immunity markers (Table 2). The addition of flaxseed oil to the bulls ration, significantly (P<0.05) increased serum HDL, albumin and globulin, and decreased the LDL, cholesterol, TG and blood urea as compared to the control. These results agree with those of other studies where flax-seed oil was reported to reduce total lipids concentration in calves' blood serum (18-22). Some studies suggested different ways by which PUFA can alter cholesterol concentration (23, 24). The synthesis of cholesterol is known to be increased with high PUFA intake (23, 24). However, in the current study, the lowering of serum cholesterol with supplementation of flaxseed oil as source of PUFAs could be attributed to the upregulation of LDL receptors (25) and/or the cholesterol redistribution between tissue pools and plasma (23). Flaxseed oil had high percentage of a-linolenic acid about 55% of oil's total fatty acids (26, 27). The diets which are rich in OFA decrease aggregation of platelet also, blood triglycerides and levels of cholesterol, blood clots formation, also, show both antithrombotic and anti-inflammatory effects (28, 29). 768 M. Abu El-Hamd, A. Metwally, M. Hegazy, Z. Ghallab, O. Elateeqy Table 1: Chemical analysis of different feedstuffs (on dry matter basis) used in feeding bulls Chemical composition (%) (on DM basis) Item Rice Fresh Berseem Straw Berseem Hay Dry matter, DM 90.42 89.24 15.26 88.23 Organic matter, OM 90.24 83.22 86.15 88.58 Crude protein, CP 16.04 1.59 14.71 14.41 Crude fiber, CF 10.96 37.21 24.9 24.67 Other extract, EE 4.91 1.47 2.90 6.04 Nitrogen free extract, NFE 56.38 42.85 43.64 43.16 Ash 9.76 16.78 13.85 11.42 Table 2: Concentration of biochemical parameters in serum as affected by flaxseed oil supplementation Item Experimental group G1 G2 G3 ±MSE Total protein (g/100 ml) 7.68a 7.55a 7.23b 0.03 Albumin (g/100 ml) 3.62 3.67 3.51 0.02 Globulin (g/100 ml) 4.06a 3.89ab 3.72b 0.01 Glucose (mg/100ml) 69.87a 71.03a 61.66b 2.54 Total cholesterol (mg/100ml) 166.2b 163.1b 187.3a 3.12 High density lipoprotein 99.50b 102.4a 77.39c 3.80 (mg/100ml) Low density lipoprotein 67.33b 61.39b 93.99a 4.62 ( mg/100ml) Triglyceride (mg/100ml) 37.03a 38.01a 28.9b 0.85 Urea-N (mg/dl) 26.45b 24.75c 31.56a 2.12 G1and G2: Bulls received flaxseed oil 2 and 4%/kg DMI, respectively. G3: Control Data in the raw followed by different letters are significant at P<0.05. Table 3: Semen quality in Friesian bulls as affected by flaxseed oil supplementation Item Experimental group G1 G2 G3 ±MSE Ejaculate volume (ml) 4.13a 4.24a 3.74b 0.03 Sperm cell concentration (x 106/ml) 1.372a 1.444a 1.138b 0.02 Mass motility (%) 75.76a 79.17a 68.06b 0.01 Live sperm (%) 77.00a 81.17a 71.17b 1.85 Sperm abnormality (%) 10.06b 9.39b 13.67a 3.12 Sperm cell concentration per ejaculate (x106/ml) 5.776a 6.240a 4.261b 0.32 Hypo-osmotic swelling test (%) 53.94a 54.51a 49.18b 1.24 G1and G2: Bulls received flaxseed oil 2 and 4%/kg DMI, respectively. G3: Control Data in the raw followed by different letters are significant at P<0.05. Table 4: Concentration of testosterone hormone(ng/ml) in blood serum as affected by flaxseed oil supplementation Time (month) Experimental group G1 G2 G3 ±MSE Overall means 15 1.507 1.960 1.189 0.12 1.552c 16 2.340 2.633 2.328 0.10 2.434ab 17 2.474 2.263 2.023 0.12 2.253b 18 2.323 2.339 1.949 0.11 2.204b 19 2.541 3.084 2.082 0.11 2.569a 20 2.732 2.860 1.753 0.15 2.448a 21 2.736 2.860 2.121 0.14 2.572a Overall means 2.774a 2.954a 2.241b 0.13 G1and G2: Cows received flaxseed oil 2 and 4%/kg DMI, respectively. G3: Control Data in the raw followed by different letters are significant at P<0.05. Effect of supplementation of omega-3 fatty acids on blood parameters and semen quality of friesian bulls 769 Semen quality In the current study, flaxseed oil as a source of omega-3 fatty acids treatment significantly (P<0.05) increased ejaculate volume, sperm cell concentrations, mass motility (%), live sperm (%) and sperm cell concentrations per ejaculate (1x106 ml) in G2 and G3 than in G (Table 3). Moreover, sperm abnormality (%) was significantly lower in treated groups than in the control group by 26.41% in G1 and 31.31% in G2, respectively. This improvement in all semen characters could be attributed to flaxseed oil supplementation with its high content of linoleic and linolenic acids as good antioxidants. Supplementation of flaxseed oil also produced a great improvement of all semen characters of rams (31). Moreover, different ratios of omega-3/omiga-6 PUFA were reported to improve semen characteristic by elevating omega-3/omega-6 PUFA that increased sperm concentration and motility and to reduce the deformity rate of the sperm (7). In the rats diet appropriate ratio of omega-3/omega-6 PUFA improved semen quality and changes in hormone metabolism due to improving reproductive performance (32). The progressive motility was reported to be higher in frozen-thawed semen in the flaxseed oil treated group than in the fish oil group (33). Dietary supplemented with PUFA improved reproductive performance, development of testis, spermatogenesis, sperm of motility and viability in fresh or freeze semen sample in ruminant (34). In the HOS test, incubation of sperm in hypo-osmotic media is necessary to estimate the plasma membrane covering the principle piece (35, 36). In the current study HOS test show that flaxseed oil supplementation to bulls significantly (P<0.05) increased the resistance of the sperm covering membrane to the hypo osmotic challenge in both G1 and G2 compared to the control G3 by about 9.68 and 10.84%, respectively. The incorporation of DHA may be increased with Omega-3 treatment in the principle piece, facilitating sperm membrane stability against hypo-osmotic media (37). The improvement of semen quality may be related to the supplementation with flaxseed oil with its PUFA the important molecules that serve as a source of energy and are critical components of the physical and functional structure of cells (38). Addition of OFA to animals diets improved sperm characteristics (39), increased sperm density and concentration per ejaculate (40, 41). Testosterone concentrations Testosterone is the key player in spermato-genesis and development reproductive tract in male (49). In this study, Friesian bulls serum testosterone concentrations were significantly increased by flaxseed oil treatments in G2 and G1 by 31.82 and 23.78% as compared to bulls in control (G3) (Table 4). The results agree with some study stated that omega-3 and 6 PUF may affect metabolism of important reproductive hormones. Testosterone concentration significantly increase in bulls supplemented with omega-3 may be due to the adequate amount of unsaturated fatty acids such as linoleic and lin-olenic acids. These unsaturated fatty acids especially lenolenic could be converted or involved in the synthesis of cholesterol which is considered the precursor materials for testosterone synthesis (43). It was indicated that spermatogenesis and steroidogenesis in the avian testis are increased with the omega-3 diets and this improvement dependent on the increase levels of FSH, LH and testosterone. However, O'Donnell et al. (45) reported that the concentrations of reproductive hormones and testosterone were positively higher related to presence of some important fatty acids and quality and morphology of sperm. Conclusion Dietary supplementation with omega-3 in male Frisian ration improved semen quality, testosterone level, lipid profile and immune function. Therefore the addition of flaxseed oil as a source of omega-3 is recommended to improve male animals semen quality and repro-ductively. Conflict of interest The authors declare that they have no conflict of interest. 770 M. Abu El-Hamd, A. Metwally, M. Hegazy, Z. Ghallab, O. Elateeqy References 1. Sinclair AJ, Attar-Bashi NM, Li D. What is the role of a-linolenic acid for mammals? Lipids 2002; 37(12): 1113-23. 2. Mustafa AF, McKinnon JJ, Christensen DA, He T. Effects of micronization of flaxseed on nutrient disappearance in the gastrointestinal tract of steers. Anim Feed Sci Technol 2002; 95: 123-2. 3. Parodi PW. Cow's milk fat components as potential anti-carcinogenic agents. J Nutr 1997; 127(6) 1055-60. 4. Conners WE. Importance of n-3 fatty acids in health and disease. Am J Clin Nutr 2000; 71: 171S--5S. 5. Wathes DC, Abayasekara DRE, Aitken RJ. Polyunsaturated fatty acids in male and female reproduction. Biol Reprod 2007; 77,190-201. 6. Abayasekara DR, Wathes DC. Effects of altering dietary fatty acid composition on prostaglandin synthesis and fertility. Prostaglandins Leukot Essent fatty acids 1999; 61: 275-87. 7. Estienne M, Harper A, Crawford R. Dietary supplementation with a source of omega-3 fatty acids increases sperm number and the duration of ejaculation in boars. Theriogenology 2008; 70: 706. 8. Mitre R, Cheminade C, Allaume P, Legrand P and Legrand AB. Oral intake of shark liver oil modifies lipid composition and improves motility and velocity of boar sperm. Theriogenology 2004; 62, 1557-66. 9. Safarinejad MR, Hosseini SY, Dadkhah F, Asgari MA. Relationship of omega-3 and omega-6 fatty acids with semen characteristics, and anti-oxidant status of seminal plasma: a comparison between fertile and infertile men. Clin Nutr 2010; 29:100-5. shark liver oil modifies lipid composition and improves 10. Animal Production Research Institute. Animal Nutrition Scientifically and Practically. 1st Ed. Animal Production Re-search Institute, Agricultural Research Center, Ministry of Agriculture, Dokki, Giza, Egypt 1997. 11. AOAC. Official Methods of Analysis. 18th ed. Assoc. Off. Anal. Chem., Gaithersburg, MD 2006. 12. Barth AD. Bull breeding soundness evaluation. The western Canadian association of bovine practitioners 2002. 13. Revell SG, Mrode RA. An osmotic resistance test for bovine semen. Anim Reprod Sci 1994; 36 77-86. 14. Abdelhady D, El-Abasy M, Abou-Asa S, Elbialy Z, Shukry M, Hussein A, Saleh A, El-Magd M. The ameliorative effect of Aspergillus awamori on aflatoxin B1-induced hepatic damage in rabbits, World Mycotoxin Journal 2017; 10(4): 363-73. 15. Almadaly E A, Farrag F A, Saadeldin I M, El-Magd M A, El-Razek I M A. Relationship between total protein concentration of seminal plasma and sperm characteristics of highly fertile, fertile and subfertile Barki ram semen collected by elec-troejaculation, Small Ruminant Research 2016; 144: 90-9. 16. El-Bayomi K M, Saleh A A, Awad A, El-Tarabany M S, El-Qaliouby H S, Afifi M, El-Komy S, Essawi W M, Almadaly E A, El-Magd M A. Association of CYP19A1 gene polymorphisms with anoestrus in water buffaloes, Reproduction, Fertility and Development 2018; 30(3): 487-97. 17. Snedecor GW, Cochran WG. Statistical Methods. 7th Ed. Iowa Univ. Press, Ames. Iowa, USA 1982. 18. Lee HJ, Khan MA, Lee WS, Kim HS, Ki KS, Kang SJ, Hur TY, Khan MS, Choi YJ. Growth, blood metabolites, and health of Holstein calves fed milk replacer containing different amounts of energy and protein. Asian-Australasian J Anim Sci 2008; 21(2): 198-203. 19. Abu El-Hamd MA, El-Diahy YM, El-Ma-ghraby MM, Elshora MA. Effect of flaxseed oil on digestibility, blood parameters, immuno-response and productive performance of suckling Friesian calves. J Anim Poult Prod Mansoura Univ 2015; 6(11): 663 -75. 20. Teama FEI, El-Tarabany AA. Physiological and biochemical response to Omega- 3 plus as a dietary supplement to growing goats under hot summer conditions. R Bras Zootech 2016; 45(4): 174-80. 21. Petit HV, Dewhurst RJ, Proulx JG, Khalid M, Haresign W, Twagiramungu H. Milk production, milk composition and reproductive function of dairy cows fed different fats. Can J Anim Sci 2001; 81: 263-71 22. Robinson, R.S.; P.G.A. Pushpakumara; Z. Cheng; A.R. Peters; D.R.E. Abayasekara and D.C. Wathes (2002). Effects of dietary polyunsaturated fatty acids on ovarian and uterine function in lactat-ing dairy cows. Reproduction 2002; 124: 119-31. 23. Matson FH, Grundy SM. Comparison of effects of dietary saturated, monounsaturated and pol-yunsaturated fatty acids on plasma lipids and lipo-proteins in man. J Lipid Res 1985; 26: 194-202. 24. Jones PJH, Ausaman LM, Croll DH, Feng JY, Schaefer EA, Lichtenstein AH. Validation of Effect of supplementation of omega-3 fatty acids on blood parameters and semen quality of friesian bulls 771 deuterium incorporation against sterol balance for measurement of human cholesterol biosynthesis. J Lipid Res 1998 39: 1111-7. 25. Fernandez ML McNamara DJ. Dietary fat-mediated changes in hepatic apoprotein B/E receptor in the guinea pig: effect of polyunsaturated, monounsaturated, and saturated fat. Metabolism 1989;38:1094-102. 26. Byers EM, Schelling GT. Lipids in ruminant nutrition. In: Church D.C. eds. The Ruminant Animal Digestion, Physiology and Nutrition. Prentice-Hall, Inglewood Cliffs NJ USA 1988 pp. 298-310. 27. Petit HV. Digestion, milk production, milk composition and blood composition of dairy cows fed formaldehyde treated flaxseed or sunflower seed. J Dairy Sci 2003; 86: 2637-46 28. Nash, DM, Hamilton RMG, Hulan HW. The effect of dietary herring meal on the omega-3 fatty acid content of plasma and egg yolk lipids of laying hens. Can. J Anim Sci 1995; 75: 247-53. 29. Simopoulos AP. Omega-3-fatty acids and public health. Proc. Flax Council of Canada Conf. Flax, the Next Decade. 1996 Dec. 3-4. Winnipeg, MB. pp. 5-28. 30. Khan, N.A., Farooq, M.W., Ahmad, N., Cone, J.W. and Hendriks, W.H., 2015. Effect of species and harvest maturity on the fatty acid content and composition of tropical forages. J. Anim. Pl. Sci., 25: 739-46 31. Speake, BK, P.F. Surai PF, Rooke JA. Regulation of avian and mammalian sperm production by dietary fatty acids. In: Male Fertility and Lipid Metabolism. De Vriese SR, Christophe AB (eds). The American Oil Chemists Society Press, Urbana, USA 2003; pp 96-117 32. Yan, L., X L. Bai; Z.F. Fang; L.Q. Che; S.Y. Xu and D. Wu. Effect of different dietary omega-3/omega-6 fatty acid ratios on reproduction in male rats. Lipids Health Dis. 2013; 12:33. 33. Moallem U, Neta N, Zeron Y, Zachut M, Roth Z. Dietary a-linolenic acid from flaxseed oil or eicosapentaenoic and docosahexaenoic acids from fish oil differentially alter fatty acid composition and characteristics of fresh and frozen-thawed bull semen. Theriogenology 2015; 83, 1110-20. 34. Tran, L.V.; B.A.; Malla; S. Kumar and A. K. Tyagi. Polyunsaturated fatty acids in male ruminant reproduction. A Review. Asian-Australas J. Anim. Sci. 2017; (5): 622-37. 35. Brito, L.F.C.; A.D. Barth; S. Bilodeau Goeseels; P.L. Panich and J.P. Kastelic. Compari- son of methods to evaluate the plasma lemma of bovine sperm and their relationship with in vitro fertilization rate. Theriogenology 2003; .60:1539 -51 36. Moce E, Graham JK. In vitro evaluation of sperm quality. Anim Reprod Sci 2008; 105:104 -18. 37. Gholami H, Chamani M, Towhidi A, Fazeli MH. Improvement of Semen Quality in Holstein Bulls during Heat Stress by Dietary Supplementation of Omega-3 Fatty Acids.Int J Fertil Steril. Jan 2011; 4(4): 160-7 38. Santos JEP, Bilby TR, Thatcher WW, Staples CR, Silvestre ET. Longc hain fatty acids of diet as factor influencing reproduction in cattle. Reprod. Dom Anim 2008; 43:23-30. 39. Robinson JJ, Ashworth CJ, Rooke JA, Mitchell LM, McEvoy TG. Nutrition and fertility in ruminant livestock. Anim. Feed Sci., Technol 2006; 126:259-76. 40. Rooke, JA, Shao CC, Speake BK. Effects of feeding tuna oil on the lipid composition of pig spermatozoa and in vitro characteristics of semen. Reprod 2001; 121:315-22. 41. Brinsko SP, Dickson DV, Charles CL, T.L. Blancharda TL, Dayc BC, Wilson ME. Effect of feeding a DHA-enriched nutriceutical on the quality of fresh, cooled and frozen stallion semen. Theriogenology 2005; 63:1519-27. 42. Gulliver CE, Friend MA, King BJ, Clayton ET. The role of omega-3 polyunsaturated fatty acids in reproduction of sheep and cattle. Anim Reprod Sci 2012; 131, 9-22. 43. Hafez ES. Reproduction in farm animals. 5 th ed Philadelphia lea & Febig 1987. 44. Vizcarra JA, Kirb JD, Kreider DL. Testis development and gonadotropin secretion in broiler breeder males. Poult Sci 2010; 89(2):328-34. 45. O'Donnell L, McLachlan RI, Wreford NG, Robertson DM. Testosterone promotes the conversion of round spermatids between stages VII and VIII of the rat spermatogenic cycle. Endocrinology 1994; 135(6):2608-14. 46. Petit HV, Twagiramungu H. Conception rate and reproductive function of dairy cows fed different fat sources. Theriogenology 2006; 66: 1316-24. 47. Dirandeh E, Towhidi A, Zeinoaldini S, Ganjkhanlou M, Ansari Pirsaraei Z, Fouladi Nashta A. Effects of different polyunsaturated fatty acid supplementations during the postpartum periods of early lactating dairy cows on milk yield, metabolic responses, and reproductive performances. J Anim Sci 2013; 91(2): 713-21. 772 M. Abu El-Hamd, A. Metwally, M. Hegazy, Z. Ghallab, O. Elateeqy 48. Hawkins DE, Niswender KD, Oss GM, Moeller CL, Odde KG, Sawyer HR et al. An increase in serum lipids increases luteal lipid content and alters the disappearance rate of progesterone in cows. J Anim Sci 1995; 73: 541-5. 49. McLachlan RI, O'Donnell L, Meachem SJ, Stanton PG, de Kretser DM, Pratis K, Robertson DM. Identification of specific sites of hormonal regulation in spermatogenesis in rats, monkeys and man. Rec. Prog. Horm. Res 2002; 57, 149-79. Slov Vet Res 2019; 56 (Suppl 22): 773-80 DOI 10.26873/SVR-818-2019 Original Research Article PRODUCTION OF CHITOSAN FROM SHRIMP SHELLS BY MICROWAVE TECHNIQUE AND ITS USE IN MINCED BEEF PRESERVATION Neama A. Omara, Essam M. Elsebaie*, H. E. Kassab, A. A. Salama Department of Food Technology, Faculty of Agricultural, Kafrelsheikh University, P. B. 33516, Kafrelsheikh, Egypt Corresponding author, E-mail: essam.ahmed@agr.kfs.edu.eg; http://orcid.org/0000-0002-8507-0694 Abstract: This work was undertaken to figure out the optimum conditions for transformation of chitin to chitosan via microwave and to determine chitosan physical properties, antioxidant and antimicrobial activities to evaluate the possibility of using such by-products in preparing some functional foods. The obtained results revealed that deacetylation degree (DA) 93 % attained by chitin remediation with 50% aqueous sodium hydoxide sol at 1:25 solid:liquid ratio for 10 min into a microwave using a power 800W. Purified chitosan was characterized for molecular weight (4200 KDa), solubility (94%), water holding capacity (505%), oil holding capacity (321 %) as well as intrinsic viscosity (13.2 dl/g). Samples of meat containing chitosan (0.02%) have the minimal experiment into the microbiological count, e.g. total count, yeasts and moulds. The anti-microbial effectiveness of chitosan was frequently higher than that of sorbistat-K or their blend by adding 0.01% chi-tosan and the same percent from sorbistat-K. Sensual attributes of samples contained chitosan, i.e. odour, texture and colour, were greater than other samples (sorbistat-K and control treatments). Though, chitosan nominated like a potent, eco-friendly and naturalistic substitution to reserve meat and protect its comprehensive quality. Key words: chitosan; microwave; antioxidant activity; natural preservative Introduction Artificial anti-oxidative substances like bu-tylated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT) were utilized to hold up oxidation of lipids in meat (1). Nonetheless, contemporary attention over their utilization has generated a necessity and induced research for alternate antioxidants, predominately from naturalistic origin. The usage of naturalistic preservatives with meat products increases the storage shelf life is a hopeful technology as nu- merous vegetative materials posse antimicrobial and antioxidant characteristics (2). Functional substances supplement into meat products might upgrade the health and nutritional attributes and extension their storage shelf-life (3). The meat industry is rottenly suffering from many critical challenges related to well-being and hygienic of the final product (4). Microbial pathogens transference to the products can be carried out during processing steps or from outer parts of the carcass (5). The removing of contamination or disinfection of the carcass Received: January 2019 Accepted for publication: February 2019 774 N. A. Omara, E. Elsebaie, H. Kassab, A. Salama body is hard to be realized by the use of a coadministered antimicrobial agent. In addition, commonly used antimicrobial and preservative substances possess artificial and alchemical properties, offering a lot of harmful effects and prospect hazards to consumer health (5). Shrimp considered as one of the world's leading fishery products, especially in Egypt (6). The product is mainly exported under freezing conditions, passing through the shells and heads removing process (7). The economic value of raw shrimp shell substances and heads is little and they are considered as biological wastes or sold to factories of animal feeds (8). In the processing of shrimp industry, shrimp produce a large amount of biological waste, accounting for 45-55% of the raw weight of shrimp (9). Also, these biological wastes are able to be utilized in producing high-added value substances like chitosan and chitin. Chi-tosan, consisted of D-glucosamine units linked with P-linkage, is a cationic amino-polysaccha-ride which produced by partially deacetylation of chitin (10). If the acetyl- glucosamine groups proportions are higher (above 50%), it is named chitin, however if the proportion is lower than 50%, the ingredient is called chitosan (11, 12). The conventional methods for chitin extraction from crustaceans are chemical processes which involve the use of strong acid for demineraliza-tion and strong base for deproteination. However, these methods reduce the quality and increase the costs and environmental problems (13, 14). Chitosan can be characterized in terms of its quality, intrinsic properties (purity, molecular weight, viscosity, and degree of deacetylation) and physical forms (15). In modern decades, microwave radiation has received considerable attention for its ability to speed up the reaction duration as compared to else procedures which transfer heat by conventional heating procedures, and can also perform heat transfer more uniformly in food compositions (16, 17). Therefore, the aim of the present study was to figure out the optimum conditions to get chitosan from chitin via microwave and to evaluate its physical properties, antioxidant and antimicrobial activities to determine the possibility of using this chitosan as preservatives in minced beef. Materials and methods Materials and preparation of samples All chemical used in this study of HPLC grade (99.9% purity) were obtained from Sigma company of chemicals and drugs, St. Louis, MO, USA. Wastes of shrimp (shells and heads) have been acquired from local restaurant in Kafr El-sheikh governorate, Egypt. The wastes were dried in an electric oven then were ground up to pass through 20 mesh screen sieve, the yielded powder samples were mixed, and stored in polyethylene bags, then kept at room temperature until use. Chemical composition Moisture, crude protein, ether extract, ash, crude fiber contents of investigated samples (shrimp waste, crude chitin and chitosan) were performed using the methods given in the (18). Isolation of chitin Various chitin extraction stages were carried out according to Synowiecki and Al-Khateeb (19). Demineralization process was operated by mixing 4 g shrimp waste with 40 ml hydrochloric acid solution 2% ( v / v) for 12 hrs at 30 ° C, and the mixture was centrifuged for 15 min at 4000 rpm, then the precipitate twice treated by distilled water. Deporteinization of the precipitate was operated by adding sodium hydroxide solution 4% (w / v) to precipitate at liquid to solid ratio (10:1 v/w) for 12 hrs at 30 ° C, and separation of different insoluble alkaline fractions by centrifugation for 15 min at 4000 rpm, then was twice treated with distilled water and was dried overnight at 40° C. The product obtained after drying was marked as pure chitin. Chitosan production via microwave route The various stages for chitosan production have been done as briefed via Sahu, et al. [13]. Chitosan was prepared from isolated chitin using various concentrations of sodium hydroxide aqueous solution (NaOH 30%, NaOH 40% and NaOH 50 %) at several sample to solvent ratios Production of chitosan from shrimp shells by microwave technique and its use in minced beef preservation 775 (1:10, 1:15, 1:20 and 1:25) at different microwave power (800W, 900W and 1000W) for various irradiation durations (5, 10, 15 and 20 minutes). chitosan yield recovered from each step was determined. Chemical, physical and functional attributes for chitosan Extracted and commercial chitosan deacety-lation grade was measured using the method of Qin et al., (20). Molecular weight and solubility of extracted and commercial chitosan were determined according to the method of (21). Water binding capacity and oil binding capacity of extracted chitosan sample were measured using the method of (22). Chitosan usage to preserve minced beef Fresh clean meat specimens were treated with the disinfectant sodium hypochlorite (100 g meat piece was soaked into 10 ppm aqueous solution of sodium hypochlorite for 60 min followed by twice washes in distilled water). Pieces of meat were finely minced, blended and split to four groups. The first group (G1) untreated samples, G2 treated with 0.02% chitosan, G3 treated with 0.02% sorbistat-K, and G4 treated with a mixture of 0.01% chitosan and 0.01% sorbistat-K. All samples were stored at 5°C for seven days. Microbiological analyses Total count of aerobic microorganisms and fungi and yeasts were carried out at zero time and the end of storage period in triplicate as previously described (23). Sensory assessment After storage period, twenty semi-trained panelists from food technology department, kafrelshiekh University evaluated texture, color, and odour according to the method described by Pohlman, et al. (4). Statistical analysis All data (except phenolic content) were done in triplicate independent analyses and expressed as mean ± standard deviation. Data of antibacterial activity were analyzed using one- way ANOVA according to (24) procedure. The significance level was set at p < 0.01. Results and discussion Chemical composition of shrimp waste Shrimp wastes were chemically analyzed for their contents of moisture, crude protein, ether extract, ash, crude fiber, total carbohydrates and the results were tabulated in Table (1). The data in Table (1) indicate that shrimp waste is considered a good source of protein, crude fiber and total carbohydrates which are important from the nutritional point of view. The results showed that the moisture, crude protein, ether extract, ash, crude fiber and total carbohydrate contents were 74.38, 11.55, 6.47, 32.21, 7.81 and 41.96%, respectively. These results were in harmony with those of (25, 26). Also, the data in the same table revealed that chitin content of shrimp wastes was 36.43% (on dry weight bases). According to the pervious results, shrimp wastes considered as a good source for chitin which can be modified to chitosan. Production of chitosan by microwave Effect of sodium hydroxide concentration (%) on chitosan yield Table (2) shows the effect of using different concentrations (%) of sodium hydroxide on the chitosan yield. The modification process by microwave heating was carried out using different concentrations of sodium hydroxide (30, 40 and 50%) at 800W microwave power for 5 min and solid : liquid ratio (1:10). It could be observed that, there was an increment in chitosan yield as a function for increasing sodium hydroxide concentration. The highest chitosan yield (53.2%) was obtained with NaOH concentration (50%). These results were in harmony with those of (17). Effect of solid : liquid ratio on chitosan yield In this experiment, chitin modification process was carried out at 800W microwave power for 5 min where, sodium hydroxide (50%) was used as a solvent. The effect of solid: solvent ratio on chitosan yield was studied and the results were presented in Table (2). The results 776 N. A. Omara, E. Elsebaie, H. Kassab, A. Salama revealed that chitosan yield was significantly increased with increasing solid: liquid ratio from 1:10 to 1:20. Meanwhile, chitosan yield was decreased when solid: liquid ratio reached up 1:20. The decrement in chitosan yield after reaching the optimum condition of solid: liquid ratio may be due to the degradation effect of alkali solution on chitosan (27). From such results, it could be noticed that, the highest chi-tosan yield was obtained at 1:20 of sample: solvent ratio .Where at this ratio, the percent of chitosan yield was 67.4%. These results are almost in agreement with those reported by (17). Effect of microwave power on chitosan yield In this experiment, chitin modification process was carried out using sodium hydroxide (50%) as a solvent at 1:25 solid: liquid ratio for 5 min. The effect of microwave power on chi-tosan yield was studied and the results were presented in Table (2). The results revealed that, chitosan decreased with increasing microwave power from 800 to 1000. From such results, it could be noticed that, the highest chitosan yield was obtained at microwave power (800 W). At this microwave power; the percent of chitosan yield was 67.4%. These results are in agreement with those found by (17). The obtained results may be due to thermal degradation of chitosan as a function to microwave power (28). Effect of using different modification periods on chitosan yield In this experiment, the modification process of chitin was carried out using 800 W as microwave power, sodium hydroxide (50%) as a solvent at 1:25 solid: liquid ratio for different periods (5, 10 and 15 min) and the results are illustrated in Table (2). The results indicated that, chitosan yield (%) increased gradually with extending the modification time from 5 to 10 min. This means that, the optimum period for chitin modification to chitosan was 10 min. These results are in agreement with those found by (17). The previous results may be explained by (29) who reported that, prolonging the modification time after reaching the optimum time leads to degradation of chitosan and lost into the reaction solvent. Gross chemical composition of chitosan Table (3) show that, the total carbohydrates content of chitosan prepared from shrimp shell wastes was 89.11%. It had low percentages of crude protein 0.98%, ether extract 1.30%, ash 0.31% and 2.21% crude fiber, respectively. Similar results were found by (30, 31). A high quality grade of chitosan should have less than 1% of ash content (32). Physicochemical and functional properties of chitosan The produced chitosan had a degree of deacetylation and molecular weight higher than commercial chitosan (Table 4). The deacetylation degree and molecular weight are important parameters for chitosan, as they affect its functional properties (33). The degree of deacetylation of chitosan is important for its application in the industry. Thus, certain researchers (34) suggested that the term chitosan should be used when the degree of deacetylation is above 70%. Solubility, water holding capacity, oil holding capacity and intrinsic viscosity values of the produced chitosan were 94, 5.05, 3.21 and 10.13, respectively. Moreover, produced chitosan has water holding capacity and oil holding capacity compared with commercial chitosan. Our results were in the same line with those obtained by (31, 32). Using chitosan in minced beef preservation Microbiological criteria The influence of adding chitosan and sorbi-stat-K into minced meat, on the amount of microorganisms, is presented in Figure 1. It was evidenced that the addition of 0.02% chitosan had the strongest effect for lowering the micro-bial count toward all examined microbial species. The combination of chitosan and sorbistat-K, at percentage of 0.01% from each, was the second strongest supplement, whereas the application of sorbistat-K was the weakest treatment to reduce microbial count. The total Production of chitosan from shrimp shells by microwave technique and its use in minced beef preservation 777 Table 1 : Proximate chemical composition of shrimp waste (on dry weight basis) --- Shrimp wastes Moisture 74.38 Crude protein (N x 6.25) 11.55 Ether extract 6.47 Ash 32.21 Crude fiber 7.81 Total carbohydrates 41.96 Chitin 36.43 Total carbohydrates were calculated by difference Table 2: Effect of process variables on chitosan producing by microwave from shrimp waste Type of Sodium hydroxide Ratio Time Microwave Chitosan yield experiment Concentration (%) (sample/solvent) (min) power (W) (%) Sodium hydroxide concentration 30 40 50 Sample/solvent ratio 50 50 50 50 Microwave power 50 50 50 Time 50 50 50 1:10 1:10 1:10 1:10 1:15 1:20 1:25 1:20 1:20 1:20 1:20 1:20 1:20 5 5 5 5 5 5 5 5 5 5 5 10 15 800 800 800 800 800 800 800 800 900 1000 800 800 800 52.40 52.80 53.20 53.20 60.40 67.40 63.10 67.40 60.40 54.80 67.40 69.83 61.09 Table 3: Gross chemical composition Isolated chitosan ~~ Samples Components % chitosan Moisture 8.30 Crude protein (N x 6.25) 0.98 Ether extract 1.3 Ash 0.31 Crude fiber 2.21 Total carbohydrates 89.11 778 N. A. Omara, E. Elsebaie, H. Kassab, A. Salama Table 4: Physicochemical properties of prepared chitosan ' ■—-—Samples Components % ——^^^ Produced chitosan Commercial chitosan Degree of deacetylation 93 85 Molecular weight (k Daltons) 4200 300 Solubility 94 99 Water holding capacity 5.05 3.12 Oil holding capacity 3.21 1.56 Intrinsic viscosity 10.13 15.92 Figure 1 : Effect of treating minced beef with chitosan and potassium sorbate on microbial count Figure 2: Effect of treating minced beef with chitosan and potassium sorbate on sensory characteristics Production of chitosan from shrimp shells by microwave technique and its use in minced beef preservation 779 Sensory evaluation There was a significant improvement in the values of sensual attributes of minced beef as a result for usage of chitosan as a preservative. After ending the storage duration, the sensory attributes (e.g. colour, odour and texture) values of untreated beef samples were highly poor, compared with other samples contained chitosan or sorbistat-K (Fig. 2). Chitosan get a higher efficiency than sorbistat-K in maintaining meat texture and odour quality, whilst, meat samples integrated with sorbistat-K had the highest values in colour. The average for texture, odour and colour were 93.1, 91.0 and 94.9% for 0.02% chitosan preserved meat; 87.1, 89.9 and 92.0% for 0.02% sorbistat-K and 9.9, 30.0 and 20.1 % for control, respectively. Lipid auto-oxidation and myoglobin-oxidation are the essential reason for meat discoloration and oxidative rancidity or another un acceptable odour or flavour components (36). With minimizing the fat auto-oxidation level, there is a prospective that the deterioration in red meat colour will be minimized as they are related reactions (37). Conflict of interest The authors declare that they have no conflict of interest. References 1. Pereira D, Pinheiro R S, Heldt L F S, Moura C d, Bianchin M, Almeida J d F, Ribeiro I S, Haminiuk C W I, Carpes S T. Rosemary as natural antioxidant to prevent oxidation in chicken burgers, Food Science and Technology (Campinas) 2017; 37(1): 17-23. 2. Nowak A, Czyzowska A, Efenberger M, Krala L. Polyphenolic extracts of cherry (Prunus cerasus L.) and blackcurrant (Ribes nigrum L.) leaves as natural preservatives in meat products, Food microbiology 2016; 59: 142-9. 3. Quevedo R, Pedreschi F, Valencia E, Díaz O, Bastías J, Muñoz O. Kinetic modeling of deterioration of frozen industrial burgers based on oxidative rancidity and color, Journal of Food Processing and Preservation 2018: e13655. 4. Pohlman F, Stivarius M, McElyea K, Johnson Z, Johnson M. Reduction of microorganisms in ground beef using multiple intervention technology, Meat Science 2002; 61(3): 315-22. 5. Siragusa G r. The effectiveness of carcass decontamination systems for controlling the presence of pathogens on the surfaces of meat animal carcasses Journal of Food Safety 1995; 15(3): 229-38. 6. Sharawy Z Z, Thiele R Abbas E M, El-Magd M A, Hassaan M S, Peter C, Schmidt J, Saborowski R, Goda A M A-S, Slater M J. Antioxidant response, body composition of whiteleg shrimp Litopenaeus vannamei co-cultured with Nile tilapia Oreochromis niloticus in recirculating aquaculture, Aquaculture Environment Interactions 2017; 9: 257-68. 7. Hirano K, Budiyanto E, Winarni S, Biological characteristics and forecasting outbreaks of the whitefly, Bemisia tabaci, a vector of virus diseases in soybean fields, ASPAC Food & Fertilizer Technology Center1993. 8. Suchiva K, Chandrkrachang S, Methacanon P, Peter M. Proceedinggs of the 5th Asia Pacific Chitin and Chitosan Symposium & Exhibition, Bangkok, Thailand 2002. 9. Lertsutthiwong P, How N C, Chandrkrachang S, Stevens W F. Effect of Chemical Treatment on the Characteristics of Shrimp Chitosan, Journal of Metals, Materials and Minerals 2002; 12(1): 11-8. 10. Elkeiy M, Khamis A, El-Gamal M, Abo Gazia M, Zalat Z, El-Magd M. Chitosan nanoparticles from Artemia salina inhibit progression of hepatocellular carcinoma in vitro and in vivo, Environ Sci Pollut Res Int. doi: 10.1007/s11356-018-3339-6 2018. 11. Kamboj S, Singh K, Tiwary A, Rana V. Optimization of microwave assisted Maillard reaction to fabricate and evaluate corn fiber gum-chitosan IPN films, Food Hydrocolloids 2015; 44: 260-76. 12. Thakur V K, Voicu S I. Recent advances in cellulose and chitosan based membranes for water purification: a concise review, Carbohydrate polymers 2016; 146: 148-65. 13. Sahu A, Goswami P, Bora U. Microwave mediated rapid synthesis of chitosan, Journal of Materials Science: Materials in Medicine 2009; 20(1): 171-5. 14. Younes I, Rinaudo M. Chitin and chitosan preparation from marine sources. Structure, properties and applications, Marine drugs 2015; 13(3): 1133-74. 15. Berger J, Reist M, Chenite A, Felt-Baeyens O, Mayer J, Gurny R. Pseudo-thermosetting chitosan hydrogels for biomedical application, International journal of pharmaceutics 2005; 288(2): 197-206. 780 N. A. Omara, E. Elsebaie, H. Kassab, A. Salama 16. Alishahi A, Mirvaghefi A, Tehrani M, Farahmand H, Koshio S, Dorkoosh F, Elsabee M Z. Chitosan nanoparticle to carry vitamin C through the gastrointestinal tract and induce the non-specific immunity system of rainbow trout (Oncorhynchus mykiss), Carbohydrate polymers 2011; 86(1): 142-6. 17. El-Mahdy S S, Hayam F, Abd El-Wanis N, Hamoud M. Comparative studies between different commercial types of live infectious bursal disease [IBD] vaccine strains in Egypt, American Journal of Research Communication 2013; 1(10): 113-29. 18. A.O.A.C. Association of Official of Analytical Chemists, Official Methods of Analysis. 18th Ed., Pub. By the A.O.A.C., Arlington, Virginia, 2220 USA., (2010). 19. Synowiecki J, Al-Khateeb N A A Q. Mycelia of Mucor rouxii as a source of chitin and chitosan, Food Chemistry 1997; 60(4): 605-10. 20. Qin C, Li H, Xiao Q, Liu Y, Zhu J, Du Y. Water-solubility of chitosan and its antimicrobial activity, Carbohydrate polymers 2006; 63(3): 367-74. 21. Fernandez-Kim S-O. Physicochemical and functional properties of crawfish chitosan as affected by different processing protocols, 2004. 22. Sciarini L, Maldonado F, Ribotta P, Pérez G, León A. Chemical composition and functional properties of Gleditsia triacanthos gum, Food Hydrocolloids 2009; 23(2): 306-13. 23. Georgantelis D, Blekas G, Katikou P, Ambrosiadis I, Fletouris D J. Effect of rosemary extract, chitosan and a-tocopherol on lipid oxidation and colour stability during frozen storage of beef burgers, Meat Science 2007; 75(2): 256-64. 24. Steel R G, Torrie J H, Dickey D A. Principles and procedures of statistics: A biometrical approach. McGraw-Hill, New York, Principles and procedures of statistics: A biometrical approach. 2nd ed. McGraw-Hill, New York. 1980: -. 25. Kim S E, Park J H, Cho Y W, Chung H, Jeong S Y, Lee E B, Kwon I C. Porous chitosan scaffold containing microspheres loaded with transforming growth factor-pi: implications for cartilage tissue engineering, Journal of Controlled Release 2003; 91(3): 365-74. 26. Ushakumari U N, Ramanujan R. Astaxanthin from shrimp shell waste, International journal of pharmaceutical chemistry research 2012; 1(3): 1-6. 27. Mourya V, Inamdar N N. Chitosan-modifications and applications: opportunities galore, Reactive and Functional polymers 2008; 68(6): 1013-51. 28. Luo J, Wang X, Xia B, Wu J. Preparation and characterization of quaternized chitosan under microwave irradiation, Journal of Macromolecular Science, Part A: Pure and Applied Chemistry 2010; 47(9): 952-6. 29. Al Sagheer F, Al-Sughayer M, Muslim S, Elsabee M. Extraction and characterization of chitin and chitosan from marine sources in Arabian Gulf, Carbohydrate Polymers 2009; 77(2): 410-19. 30. Khan T A, Peh K K, Ch'ng H S. Reporting degree of deacetylation values of chitosan: the influence of analytical methods, J Pharm Pharmaceut Sci 2002; 5(3): 205-12. 31. Kucukgulmez A, Celik M, Yanar Y, Sen D, Polat H, Kadak A E. Physicochemical characterization of chitosan extracted from Metapenaeus stebbingi shells, Food Chemistry 2011; 126(3): 1144-8. 32. No H K, Meyers S P. Preparation and characterization of chitin and chitosan—a review, Journal of aquatic food product technology 1995; 4(2): 27-52. 33. Rout S K. Physicochemical, Functional and Spectroscopic Analysis of Crawfish Chitin and Chitosan as Affected by Process Modification, 2001. 34. Li Q, Dunn E, Grandmaison E, Goosen M F. Applications and properties of chitosan, Journal of Bioactive and Compatible Polymers 1992; 7(4): 370-97. 35. Moussa S H, Tayel A A, Al-Hassan A A, Farouk A. Tetrazolium/formazan test as an efficient method to determine fungal chitosan antimicrobial activity, Journal of Mycology 2013; 2013. 36. Chan W K, Faustman C, Decker E A. Oxymyoglobin oxidation as affected by oxidation products of phosphatidylcholine liposomes, Journal of Food Science 1997; 62(4): 709-12. 37. Brand-Williams W, Cuvelier M-E, Berset C. Use of a free radical method to evaluate antioxidant activity, LWT-Food science and Technology 1995; 28(1): 25-30. Slov Vet Res 2019; 56 (Suppl 22): 781-8 DOI 10.26873/SVR-819-2019 Original Research Article ISOLATION AND IDENTIFICATION OF SOME POSSIBLE CAUSATIVE AGENTS OF SWOLLEN HEAD SYNDROME (SHS) IN BROILER CHICKENS IN EGYPT Nasif S. Abdelmoez1, M. M. Shawky2, Heba A. Abdelhady3, M. A. Lebdah2, Selim S. Salama1* Central Laboratory for Evaluation of Veterinary Biologies, Abbasia, Cairo - Egypt, 2Avian and Rabbit Medicine Dept. Faculty of Vet. Med., Zagazig University, Egypt, 3Animal Health Research Institute (AHRI), Alexandria branch, Egypt Corresponding author, E-mail: selimsalama2000@yahoo.com Abstract: The current study was designed to detect, isolate and identify the etiological agents of SHS in chicken flocks in Egypt. Clinical samples, including the trachea, lung and choanal cleft swabs, from different flocks, ages and localities were collected from non-vaccinated commercial broiler flocks with respiratory signs then transmitted directly to the laboratory for further examination. Avian metapneumovirus RNA (aMPVRNA) was extracted then amplified using one step RT-PCR using both subtype A and B (G gene) primers then differentiation occurred by real time PCR (qPCR). Swab samples from subcutaneous edema and exudates were directly cultured and the suspected bacterial colonies were biochemically identified then confirmed by PCR. Four samples were positive and one doubtful for aMPV using RT-PCR confirmed by qPCR. Bacteria isolation revealed that out of 40 samples, 16 samples were positive for E.coli, 4 samples were positive for P. aeruginosa, 6 samples were positive for P. mirabilis, 4 were mixed E. coli and P. aeruginosa, 8 were mixed E. coli with P. mirabilis and 2 samples were mixed P. mirabilis and P. aeruginosa. Depending on these findings, we could prescribe a preliminary guide to decrease SHS outbreaks by primary control of bacterial complication. Key words: Swollen Head Syndrome; PCR; bacterial complication Introduction The respiratory diseases considered one of the most serious problems affecting chicken flocks resulting in severe economic losses. Several etiological agents like viruses and bacteria have been incriminated. Among viruses infecting broiler flocks, avian metapneumovirus (aMPV), which cause a disease entitled as Turkey Rhinotracheitis (TRT) in turkey and swollen head syndrome (SHS) in chickens (1). In Egypt, aMPV infection was diagnosed through detection of the infected broiler chickens by RT-PCR that revealed circulation of both aMPV subtypes A and B in turkey flocks (2 and 3). Unfortunately till now, no identification to aMPV subtype circulating in broiler chickens in Egypt. Swollen head syndrome is a disease of upper respiratory tract affecting broilers and broiler breeders and characterized by swelling of head Received: January 2019 Accepted for publication: February 2019 782 N. S. Abdelmoez, M. M. Shawky, H. A. Abdelhady, M. A. Lebdah, S. S. Salama and facial edema which resulted from accumulation of inflammatory exudate beneath the skin of the head in response to secondary bacterial infection usually E. coli following the initial upper respiratory viral infection and it has become a problem in many countries in the last few years. SHS has been described as a multi-factorial disease. The initial lesion mainly caused by virus, while the clinical signs were a consequence of bacterial complications. The severity of the disease depends on presence of some environmental factors such as accumulation of ammonia, dust, overcrowding and bad ventilation. So, aMPV cause the initial damage of the respiratory tract, allowing invasion of bacteria, especially E.coli, which were responsible for the clinical disease (4). SHS is a disease of chickens of all ages mainly 4 - 6 weeks old with morbidity may reach to 10% and mortality about 2% (5). The affected birds showed depression, decreased feed intake, nasal exudate, sneezing, coughing and conjunctivitis followed by facial edema which start around eye extending over the head and descending to subman-dibular tissues (6). Therefore, the current study was designed to detect, isolate and identify the etiological agents of SHS in chicken flocks in Egypt. Materials and methods Virus isolation and identification Clinical Samples Samples were collected from non-vaccinated commercial broiler flocks with respiratory signs such as, sneezing, coughing, tracheal rales, nasal and ocular discharges, swollen infraorbital sinuses and foamy conjunctivitis then transmitted directly to the laboratory for further examination. Samples included the trachea, lung and choanal cleft swabs. Samples were collected from different flocks, ages and localities. Extraction of viral RNA Procedure adopted using QIAamp Viral RNA Mini Kit (Cat# 52906) according to manufacture instructions using reagents provided in the kit. Amplification of aMPV RNA The reaction was prepared according to QI-AGEN One Step RT-PCR (Cat# 204443) manufactures instructions. The reaction was performed for both subtype A and B (G gene) as following: reverse transcription (42°C /30 min), initial denaturation step (95°C /10 min), followed by 40 cycles of denaturation (95°C /1 min), annealing (54°C /1 min), extension (72°C /1 min) and final extension (72°C /10 min). The PCR product was then analyzed by electropho-resis in 1.5% agarose gel with 100 bp DNA ladder using TAE running buffer and 100 volts for 40-50 min. The ethidium bromide stained PCR bands were visualized in the agarose gel using UV transilluminator. Primers used for aMPV RT-PCR were designed as described by (7) Ga(gf)- forward CCGGGACAAGTATCTCTATGG for all subtypes, G2(gaf)-reverse CCACACTTG AAA-GATCTACCC for A subtype and G12(gb)- reverse CAGTCGCCTGTAATC TTCTAGGG for B subtype. Also probes used for aMPV real time PCR were designed following (8) as: SHf: TAGTTTTGATCTTCCTTGTTGC SHr: GTAGTTGTGC TCAGC TCTGATA MB-SH-A:HEXCGCGATCGTGGAC-CTCCT GCACTGTGGATCGCG-Iowa Black FQ MB-SH-B :FAMCGCGATCATT-GTGACAGC CAGCTTCACGATCGCG-Iowa Black FQ. qPCR was done using SuperScript® III Platinium ® One-Step qPCR system-Invitrogen (Cat# 11732-088) according to manufacture instructions. Sampling and bacterial isolation Swabs were collected aseptically from subcutaneous edema and sinuses exudate. Collected samples were inoculated in nutrient broth then 24 hr incubation at 37°C aerobically. Loopfull transferred onto nutrient agar, Mac-Conkey agar and blood agar media and incubated for 24 hr aerobically at 37°C. Suspected colonies were identified by cultural, morphologically (9), biochemically (API 20E) (10), and molecularly by PCR. Isolation and identification of some possible causative agents of Swollen Head Syndrome (SHS) in broiler ... 783 Polymerase Chain Reaction (PCR) Extraction of bacterial DNA from E.coli were propagated by inoculation on nutrient broth then injected subcutaneously in mice followed by re-isolation from heart and liver on MacConkey agar. A single bacterial colony was transferred into 2 ml of nutrient broth and incubated overnight at 37°C. Culture was centri-fuged and the bacterial pellet was re-suspended in 400pl and DNA was extracted using DNA Extraction Kit (PrestoTM Mini gDNA Bacteria Kit Cat# GBB101). E. coli isolates were screened for presence of 16srRNA gene using specific primers (Eco-1F, GACCTCGGTTTAGTTCACAGA and Eco-2R, CACACGCTGACGCTGACCA giving rise a specific PCR product at a size of 585 bp (11). The cyclic conditions were performed as previously described (12). Vitek2 compact system method for bacterial identification Bacterial identification by Vitek-2 compact system was done according to the manufacture's instruction (Biomeriux VITEK-2 Compact ref Manual - Ref-414532) Results Clinical signs The suspected samples were collected from birds showing coughing, snicking, wet or frothy eyes, conjunctivitis, sneezing, facial edema, unilateral or bilateral swelling of infraorbital sinuses and some chickens showed swelling of their entire face including wattles. At the same concern the postmortem lesions of suspected dead cases revealed often yellowish extensive gelatinous to purulent edema of subcutaneous tissues of head region, sinusitis, tracheitis, perihepatitis, pericarditis and few cases showed pneumonia and air saculitis. Detection of aMPV by RT-PCR The genomic RNA of aMPV was tested using Ga and G2 primers for subtype A that revealed all samples were negative while with using Ga and G12 primers for subtype B only 4 samples were positive giving rise to a product of 312 bp (Fig. 1). Detection, confirmation and subtyping with qPCR The positive, doubtful and negative samples were re-examined by qPCR for accurate confirmation, 5 samples out of 40 were positive and confirmed aMPV subtype B isolates (Table 1, Fig2). Colonial characteristics of isolated bacterial associated with SHS infection Several bacterial causative agents were isolated from suspected samples on different media as shown in Table (2). The suspected colonies of E. coli showed slight turbidity on nutrient broth, circular, smooth surface, mucoidy colonies, and greyish in color on nutrient agar while on MacConkey agar showed Pink colonies due to lactose fermentation. Meanwhile the suspected colonies of Pseudomonas spp. showed abundant growth and turbidity with bluish green color on nutrient broth and convex, glistening, translucent with bluish green color on nutrient agar while on MacConkey agar it showed pale colonies of non-lactose fermenter. Proteus spp. showed uniform turbidity with a slight powdery deposit and an ammonical odour on nutrient broth while on the nutrient agar made swarms intermittently in the characteristic step-like pattern across the agar surface but it looked like pale colonies with non-lactose fermentation on MacConkey agar. Bacterial isolation and biochemical identification Frequency of isolation as confirmed by Vi-tek2 assay and PCR was illustrated in Table (2). The identification results revealed that 28 samples out of 40 were E. coli (16 as single infection, 8 mixed infection with P. mirabilis and 4 mixed infection with P. aeruginosa). Also 16 samples out of 40 were P. mirabilis (6 as single infection, 8 mixed infection with E. coli and 2 mixed infection with P. aeruginosa). Finally 10 isolates out of 40 were P. aeruginosa (4 as single infection, 4 mixed infections with E. coli and 2 mixed infection with P. mirabilis). The amplified PCR product of suspected E. coli samples were 585 bp using the specific primers (Fig. 3). 784 N. S. Abdelmoez, M. M. Shawky, H. A. Abdelhady, M. A. Lebdah, S. S. Salama Table 1 : Frequency of positive samples as detected by qPCR AMPV type No. of tested No. of +ive No. of -ve Detection samples samples samples percentage aMPV Subtype A 40 0 40 0% aMPV Subtype B 40 5 35 12.5% % calculated according to the number of positive to tested samples. Table 2: Frequency and isolation rate of some bacterial isolates either single or mixed complication associated with AMPV infection Bacterial type Tested flocks No. Frequency of isolation Percent of isolation E. coli 40 16 40% P. aeruginosa P. mirabilis 40 40 4 6 10% 15% E.coli + P. aeruginosa 40 4 10% Mixed infec- E.coli + P. mirabilis 40 8 20% tions P. mirabilis + P. aeruginosa 40 2 5% % calculated according to the number of positive to tested samples. 7 6 5 4 3 2 1 L 312 bp 1500 / ----__ _ 1000 500 400 300 200 100 Figure 1: Ethidium bromide-stained 1.5% agarose gel showing RT-PCR amplification products of aMPV subtype B (312 bp). (L: 100 bp DNA Ladder; Lane 1: Negative control; Lane 2: Positive control; Lane 3: doubtful sample and Lanes 4, 5, 6 and 7 were positive sample Isolation and identification of some possible causative agents of Swollen Head Syndrome (SHS) in broiler ... 785 Amplification :...........................................Pos itive conti-ol^^^v / / / / / /y ... y......./......../ / / s / /¿LM / / / / /7 c77 //........... / ■ / / / y y y y \>oarivp cnn trol 0 10 20 30 40 Cycles Figure 2: Amplification curves of qPCR showing cycle threshold (Ct) values of aMPV isolates where red line: positive control; green lines: positive samples and black line: negative control Figure 3: Ethidium bromide-stained 1.5% agarose gel showing PCR amplification products of 16srRNA gene of E.coli (585 bp). M, marker; positive samples are 1, 2, 3, 5, 6, 7 and 9; while negative samples are 4, 8 and 10 Discussion Infection associated with respiratory tract has significant economic impact on poultry production worldwide. SHS is a disease of upper respiratory tract and considered as one of these problems in last few years. The disease affects broilers and broiler breeders which resulted in inflammatory exudate beneath the skin (13). SHS has been described as a multi-factorial disease where the initial lesion mainly caused by aMPV, while the clinical signs are a consequence of bacterial complications and the severity of the disease depends on environmental factors (4). This study was planned to detect and try to isolate some of the possible etiological agents of swollen head syndrome from different 40 broiler flocks showing swollen heads and respiratory manifestations with frothy conjunctivitis, snicking, wet or frothy eyes, sneezing, facial edema, unilateral or bilateral swelling of infraorbital sinuses. Samples included lung tissues, trachea and choanal cleft swabs and scrabs from sinuses and turbinates. These observations were parallel to the findings obtained by (5) who reported that, the first symptoms of swollen head syndrome in broiler chickens were sneezing, redness of conjunctiva with swelling of lacrimal glands followed by subcutaneous edema of head starting around eye. Similar findings had been reported by (14). At the same concern (4) reported that the severity of the SHS depends on environmental factors. The postmortem lesions of suspected freshly dead cases revealed often yellowish extensive gelatinous to purulent edema of subcutaneous 786 N. S. Abdelmoez, M. M. Shawky, H. A. Abdelhady, M. A. Lebdah, S. S. Salama tissues of head region and extended in some cases to include the submandibular area, sinusitis and tracheitis. On the other hand, few cases showed perihepatitis and pericarditis, pneumonia and airsaculitis. These findings were the same that recorded by (15). Also (16) observed other lesion in the form of various amounts of casious exudate in lacrimal gland. In addition to (17) found that, under field conditions gross pathology depends largely on the presence of secondary infections in form of airsaculitis, pericarditis, perihepatitis and pneumonia. Regarding detection of aMPV using conventional RT-PCR, only 4 samples out of 40 in a percentage of 10% were detected meanwhile, when the same 40 samples were examined using qPCR, 5 samples out of the collected 40 flock samples were detected, in a percentage of 12.5% and the detected virus isolate belonged to subtype B of aMPV. The difference between the two results could be attributed to the higher sensitivity of qPCR than conventional PCR. Parallel results were recorded in Egypt by (18) who detected TRT antibodies in broiler chickens from ten farms showing swollen heads and in other five healthy broiler farms in different Egyptian provinces using two different ELISA kits indicating seroprevalences of virus in Egypt. Also (3) detected the TRT virus (aMPV) subtype B in turkey flocks in Egypt. On the other hand, our results were in agreement with that of (19) who detected aMPV subtype B in 17 broiler flocks out of 133 examined flocks constituting about 12.5% of tested flocks in Jordan. Regarding bacterial complications in the examined 40 flocks regardless to presence or absence of aMPV infection, our results revealed that twenty eight samples out of 40 were positive for E. coli isolation (16 as single infection, 8 mixed infections with P. mirabilis and 4 mixed infection with P. aeruginosa), sixteen samples out of 40 were carried P .mirabilis (6 as single infection, 8 mixed infection with E. coli and 2 mixed infection with P. aeruginosa) and ten isolates out of 40 were infected with P. aeruginosa (4 as single infection, 4 mixed infection with E. coli and 2 mixed infection with P. mirabilis). Concerning the secondary bacterial infection accompanying the aMPV infection among the 5 infected flocks, the E. coli was the predominant secondary bacterial spp. in the 5 flocks either alone in 2 flocks or complicated with either P .mirabilis (2 flocks) or P. aeruginosa as in one flock. Similar results were recorded by (20) and (21) who isolated pure E. coli culture from cases of aMPV infection. In the same context many authors reported mixed bacterial infection complicating aMPV infection in broilers including (22) who had isolated E. coli, Pseudomonas spp. besides Moraxella spp. from SHS cases. Also, (23) who had isolated P. aeruginosa, E. coli, P. mirabilis and Staphylococcus spp. complicating viral infection from young chicken during 1992 with SHS. Furthermore, (24) could isolate Pneumovirus for the first time together with E. coli and P .mirabilis from a broiler flock with swollen head syndrome in Japan. Regarding the other 35 flocks that were negative for aMPV and showing SHS signs, our results revealed incrimination of E. coli alone or mixed with P. mirabilis and /or P. aeruginosa to be the causative agents of SHS in that chicken flocks besides the recorded bad environmental and/or managemental factors. These findings were parallel to those obtained by (25) who reported that TRT virus did not play a causal role in SHS in commercial broilers in Greece but other bacterial agents together with bad environmental factors seemed to be essential in the occurrence and severity of the disease. The lower detection rate of aMPV from the affected broiler flocks may be due to short period of presence of that virus in the tissues of affected birds, nearly not more than 4 days, in addition to presence of secondary bacterial infection in most of affected cases. From the above mentioned findings, we could prescribe a preliminary guide to subside and decrease SHS outbreaks by primary control of bacterial complication either by application of biosafety and biosecurity measures, optimization of environmental factors, application pf available vaccination programs or curative and prophylactic treatment will help and support the reduction of Isolation and identification of some possible causative agents of Swollen Head Syndrome (SHS) in broiler ... 787 prevalence of SHS infections among broiler and broiler breeder flocks in Egypt. Conclusion It could be concluded that these results confirming the prevalence of aMPV subtype B among poultry flocks and its detection for the first time among broiler chickens in Egypt, through isolation, identification of the aMPV virus using RT-PCR and qPCR. In addition to, isolation and identification of some bacterial co-infection as E. coli, P. mirabilis and P. aeruginosa using traditional methods, PCR and Vitek2 compact system. Conflict of interest The authors declare that they have no conflict of interest. References 1. Picault I P.; Giraud P.; Deouin P.; Guittet N.; Benjean H.; Lamande L.; Toquin D and Gueguen C. Isolation of turkey rhinotracheitis like virus from chickens with swollen head syndrome. 1987; Vet. Rec., 121: 135. 2. Abdel-Azeem A.A.S.; Franzo G.; Dalle Zotte A.; Drigo M.; Martini M. and Cecchinato M. First evidence of avian metapneumovirus subtype A infection in turkeys in Egypt. Trop Anim Health Prod., 2014; 46:1093-7. 3. Arafa A.; Mady W.; Hussein A.; Tamam S. and Madbouly H. Molecular Characterization of Vaccine-Derived Mutants of Avian Metapneu-moviruses Isolated from Turkeys in Egypt. Am. J. Virolo., 2015; 4 (1): 1.11. 4. Nakamura K.; Mase M.; Tanimura N.; Yama-guchi S. and Yuasa N. Attempts to reproduce swollen head syndrome in specific pathogen free chickens by inoculating with Escherichia coli and/or turkey rhinotracheitis virus. Avian Pathol., 1998; 27: 21-7. 5. Morley A.J. and Thomson D.K. Swollen head syndrome in broiler chickens. Avian Dis., 1984; 28: 238-43 6. Kuhne P. Ways to control avian metapneu-movirus. International poultry production, 2009; 17 (3): 7-9. 7. Bayon-Auboyer M.H.; Jestin V.; Toquin D.; Cherbonnel M. and Eterradossi N. Comparison of F-, G- and N-based RT-PCR protocols with conventional virological procedures for the detection and typing of turkey rhinotracheitis virus. Arch. Virol., 1999;144: 1091-109. 8. Cecchinato M.; Lupini C.; Pogoreltseva O.S.; Listorti V.; Drigo A.M. and Catelli E. Development of a real-time RT-PCR assay for the simultaneous identification, quantitation and differentiation of avian metapneumovirus subtypes A and B. Avian Pathol., 2013; 42 (3): 283-9. 9. Cruickshank R.; Duguid J.P.; Marmion B.P. and Swain R.H.A. Medical Microbiology. The Practice of Medical Microbiology. 12th Ed. Vol. II Churchill Livingstone, Edinburgh, London and New York.1975. 10. Ghanbarpour R.; Salehi M. and Oswald E. Virulence genotyping of E. coli isolates from avian cellulitis in relation to phylogeny. Comp. Clin. Pathol., 2010; 19:147-53. 11. Tonu NS, Sufian MA, Sarker S, Kamal MM, Rahman MH, and Hossain MM. Pathological study on colibacillosis in chickens and detection of E. coli by PCR. Bangl J Vet Med. 2011; 9: 17-25. 12. Khatun N.M.; Mahbub E.; Elahi A.T.M.; Ahmed S.; Parvej M.S.; Akhter S.; Ansari W.K. and Ali M.S. Frequency of drug resistant E. coli isolated from commercial chickens in Bangladesh. International journal of Natural and Social Sciences, 2015; 2 (4): 1-5. 13. Seifi S and Boroomand Z. The role of avian metapenumo-virus in respiratory complex disease circulating in broilers in Northern Iran. Trakia J. Sci., 2015; No 2, pp 175-9. 14. Homayounfar N.; Shoushtari H.; Charkhkar S. and Bozorgmehrifard M. Detection by reverse transcriptase-polymerase chain reaction and molecular characterization of avian metapneumovirus in Chicken flocks in Iran: WALIA J., 2015; 31 (S3): 170-4 15. Jirjis F.F.; Noll S.L.; Halvorson D.A.; Naga-raja K.V. and Shaw (D.P. 2002): Pathogenesis of avian pneumovirus infection in turkeys. Vet. Pathol., 39: 300-10. 16. Nunoya T.; Tajima M.; Izuchi T.; Takahashi K.; Otaki Y.; Nagaswa Y. and Hakogi E. Pathology of a broiler disease characterized by swollen head. J. Vet. Med. Sci., 1991; 53: 347-9. 17. Jones R.C. and Rautenschlein S. Avian metapneumovirus. In: Swayne, D.E., John R. Glisson, Larry R. Mcdougald, J.R. Glisson, Lisa K. Nolan, Suarez, D.L. and Nair, V. (eds.) Diseases of poultry. 13th ed. Ames: John Wiley and Sons, Inc.2013. 18. Aly M. M.; El-Zaher A.A. and Amin A. Studies on swollen head syndrome in Egypt. 1. Se-rological survey and comparison between two 788 N. S. Abdelmoez, M. M. Shawky, H. A. Abdelhady, M. A. Lebdah, S. S. Salama ELISA kits for detection of antibody. Veterinary Medical Journal Giza, 1997; 45 (2): 251-8. 19. Gharaibeh S.M. and Algharaibeh G.R. Sero-logical and molecular detection of avian pneu-movirus in chickens with respiratory disease in Jordan. Poult. Sci., 2007; 86: 1677-1681. 20. Hafez H.M. and Loehren U. Swollen head syndrome: clinical observations and serological examinations in West Germany. Dtsch. Tierarztl. Wochenschr., 1990; 97: 322-4. 21. Al-Ankari A.S.; Al-Ramadan A.M. and El-Demerdash Risk factors associated with prevalence of swollen head syndrome (SHS) in broiler chickens in eastern province-Saudi Arabia. Int. J. Poult. Sci., 2004; 3 (10): 646-50. 22. McDougall J.S. and Cook J.K. Turkey rhi-notracheitis: preliminary investigations. Vet. Rec., 1986;118:206-7. 23. Goodwin M.A. and Waltman W.D. Clinical and pathological findings in young Georgia broiler chickens with oculofacial respiratory disease (so-called swollen head). Avian Dis., 1994; 38: 376-8. 24. Tanaka M.; Takuma H.; Kokumai N.; Oishi E.O.B.; Hiramatsu K. and Shimizu Y. Turkey rhi-notracheitis virus isolated from broiler chicken with swollen head syndrome in Japan. J. Vet. Med. Sci., 1995;57: 939-41. 25. Georqiades G., Iordanidis P., Koumati M. Cases of S-H-S in broiler chickens in Greece. Avian dis. 2001; 45 (3): 745-50. Slov Vet Res 2019; 56 (Suppl 22): 789-95 DOI 10.26873/SVR-820-2019 Original Research Article FIELD APPLICATION FOR EXPERIMENTAL INACTIVATED MULTIVALENT P. multocida AND AVIAN INFLUENZA (H9N2) VACCINE IN POULTRY Selim S. Salama1*, Heba A. Abdelhady2, Lamiaa Atia3 1Central Laboratory for Evaluation of veterinary Biologics (CLEVB), Cairo, Egypt, 2Animal Health Research Institute (AHRI), Alexandria branch, Alexandria, Egypt, 3Animal Health Research Institute (AHRI), Banha branch, Qalyubia, Egypt "Corresponding author, E-mail: selimsalama2000@yahoo.com Abstract: Fowl cholera (FC) and Avian Influenza (AI) are two of the major economically important respiratory and septicaemic disease of poultry in Egypt and allover the world. A field trails was conducted to evaluate the immunogenicity of an experimentally multivalent inactivated Fowl cholera (P. multocida) and Avian Influenza (H9N2) vaccine. The In this study a combined inactivated Montanide ISA fowl cholera and AI vaccine was prepared then the immunization potency and protective efficacy were evaluated through experimental application on different poultry breeds including broiler breeder, broiler and turkey flocks.The peak of humeral immune response against P. multocida as measured by ELISA was at the 5th week post vaccination and at 3rd week post boostering all over the used poultry flocks. At the same time, AI immune response as measured by HI reached the peak at the 6th week post vaccination and at 2nd week post boostering. Regarding the challenge test, the protection levels were 76.6, 70 and 80% and 90, 86.6 and 93.3% against P. multocida while protection levels were 83.3, 80 and 86.6% and 93.3, 90 and 96.6% against AI virus after challenge with the used virulent strains post single and booster dose respectively. In conclusion the combined prepared fowl cholera and AI vaccine succeeded in eliciting protective antibody titres and full protection against both fowl cholera and AI diseases. Key words: Pasteurella multocida; AI; vaccine; ELISA; PHA; HI; challenge test Introduction Fowl cholera (1) and AI (2) are two of the major economically important respiratory and septicaemic diseases of poultry in Egypt and allover the world. They are highly contagious diseases causing devasting economic losses to the poultry industry through death, weight loss and condemnation of carcasses worldwide. Fowl Cholera caused by P. multocida which belongs to the family Pasteurellaceae and classified into five groups based on capsular antigens and into 16 serotypes based on LPS antigens (3). It occurs sporadically or enzootically as peracute, acute or chronic form all over the world (4). Mortality may range from only few percent to nearly 100% and recovered birds may remain as carriers even after 9 weeks after infection (1). Received: January 2019 Accepted for publication: February 2019 790 S. Salama, H. Abdelhady, L. Atia Avian influenza (AI) is an infectious respiratory disease of birds caused by avian influenza type A viruses that are members of the family Orthomyxoviridae (5). The H9N2 avian influenza virus (AIV) was reported to be of low pathogenicity in chickens (6, 7) causing minimal clinical signs other than a slight drop in egg production but greatly has immunosuppressive effect (8) confirmed that the control of H9N2 viruses in poultry is important. Combination of bacterial and viral vaccines which contain multiple antigens has many benefits for the manufacturer as it reduce production costs, for the administrator as it save time, effort and simplify the immunization schedule and for the animals as it minimize stress of multiple vaccinations (9). The aim of this study is to evaluate the usage of a combined vaccine against FC and AI diseases prepared with Montanide ISA 206 as adjuvant under field condition. Materials and methods Strains used a- Pasteurella multocida serotypes (1, 3, and 4): obtained from the Strain Bank, Central Laboratory for Evaluation of Veterinary Biologics (CLEVB), Cairo, Egypt. b- AI H9N2: isolated in the Central Laboratory for Evaluation of Veterinary Biologics (CLEVB), yearling 2015, Cairo, Egypt. Vaccine Preparation a- Preparation of inactivated Pasteurella multocida antigenic phase A virulent local strains of P. multocida Serotypes 1, 3, 4 and D2 were propagated in brain heart infusion broth (BHI) at 37 OC for 24 hours to obtain a dense culture containing approximately 2x109 CFU of each strain. After inacti-vation by addition of 0.1% formalin, each culture was tested for purity, safety and sterility as mentioned by (10). Finally, cultures were equally mixed together then preserved with 0.01% of thiomersal and stored at 4 C° until use. b- Preparation of inactivated AI antigenic phase Propagation and titration of AI H9N2 that locally isolated were done in SPF 9 -10 day old ECE according to (11) and (12). Its titer was 109 EID50/ml. Inactivation of AI virus was carried out using binary ethelenamine 0.1M with final concentration 0.01M. Inactivation and testing of inactivation was done according to (13). c- Preparation of combined inactivated FC and AI vaccine by using ISA 206 A combined vaccine of P. multocida and AI was prepared water in oil in water emulsion by mixing equal volumes of the inactivated P. multocida antigenic phase and inactivated AI anti-genic phase to form an aqueous phase and immersed in equal volume of Montanide ISA 206 oil adjuvant (SEPPIC, cosmetics, pharmacy division, Paris, France). Quality control of the prepared vaccine The vaccine was tested for sterility and safety following the standard international protocol as described by OIE (14) before the usage in the field trial. Experimental design A separate broiler breeder, broiler and turkey flock houses were used through the experimental work of this study. Birds were vaccinated I/M at the age of six weeks, 2 weeks and 4 weeks respectively with 0.5ml / bird with the prepared combined vaccine. Random 60 birds from each group were transferred to a specific isolator to be challenged 3 weeks post first vaccination. A group of the rest birds of the different flocks were then received booster dose and 3 weeks apart challenge test were done. Also a group of the broiler were kept to continue in a special house to complete the period of experiment. A group of bird per each flock were kept as non-vaccinated control group. Random serum samples were collected from all flocks all over the experimentation period. Enzyme Linked Immunosorbent Assay (ELISA) ELISA was performed according to the manufacturer instruction (ID-VET) for determining P. multocida antibody titer. Field application for experimental inactivated multivalent P. multocida and avian influenza (H9N2) vaccine 791 Haemagglutination inhibition (HI) test It was carried out according to OIE (14) for the evaluation of immune response against AI. Challenge test a- Challenge with virulent P. multocida strains. This test was done according to (15). Vaccinated birds (30 per each flock) as well as non-vaccinated (15 per each flock) were challenged by inoculation of 0.1ml of 2x102 CFU of P. multocida Serotypes 1, 3, and 4 (10 vaccinated and 5 non vaccinated birds for each serotype) via intramuscular route 3 weeks after both first and booster dose vaccination. All birds were observed for 14 days and mortalities were recorded. b- Challenge with AI H9N2 strain This was done according to (16). 30 vaccinated as well as 10 non-vaccinated birds per each flock were challenged by inoculation of 0.1ml of AI 106 EID50 strain via intranasal route 3 weeks after first and booster dose of vaccination then cloacal swaps were collected on 1, 3 and 5 days post challenge to check virus shedding and protection obtained. Results and discussion Control of fowl cholera and avian influenza are still subject of interest and usually attract the attention of researchers to know more about diseases, epidemiology and how to control in susceptible birds. Despite the prophylactic measures for protection against both diseases using various vaccines and different vaccination programmes, mortality and losses still occur due to respiratory diseases. Combination of bacterial and viral vaccines has many benefits for the manufacturer as it reduce production costs, save time, effort and simplify the immunization schedule. Also it minimize stress of multiple vaccinations on the animals. Vaccination is still considered one of the major tools for controlling both of the two diseases. Vaccine efficacy depend on many variables, such as the nature, the amount of antigen administrated and the presence of adjuvant to enhance immuno-genicity (17). FC and AI vaccine was prepared using Montanide ISA 206 and evaluated by measuring humeral immune response and vaccination challenge assays against both in this field study. Assessment of Quality control measures for the prepared inactivated vaccine indicated that, the prepared vaccine was completely sterile, haven't any bacterial or fungal contaminants when tested on specific bacteriologic and fungal media. In addition there was no local, systemic reaction or mortalities were recorded in inoculated chicks, and these denote to the safety of the prepared vaccine. Regarding the potency of the prepared vaccine, the serological tests carried out on serum samples obtained from vaccinated chickens with the combined FC and AI vaccine revealed that such birds exhibited detectable P. multo-cida antibodies from the first week post vaccination as shown in Table (1). The ELISA antibody titers increased gradually till reach its maximum level at the 5th week post first vaccination where it records 1247, 1197 and 1226 in case of broiler breeder, broiler and turkey flocks respectively. This is consistent with (18) who reported that the antibody titres significantly increased 3 weeks after primary vaccination. Booster doses caused a rapid shooting in the antibody titer and reached its highest level at the end of 3rd week post boostering. The highest antibody titers were 1832, 1578 and 1801, respectively. It is clear that all birds showed a secondary response greater than that seen in birds vaccinated once, these findings was in accordance with that recorded by (15) who stated that a significant immunological stimulus had been elicited by the 2nd exposure and (19) who reported that the immune response of secondary immunization was significantly higher than the primary immunization. On the other hand (20) evaluated the immune response induced by several adjuvants included in Pasteurella multo-cida vaccines and stated that there is no significant decrease of antibody titres was observed between 4 and 8 weeks after vaccination. The inoculation of booster dose of combined vaccine at the start of 4th week caused a rise of antibodies titer where reached the peak against 792 S. Salama, H. Abdelhady, L. Atia P. multocida at the 3rd week post boostering. From these investigations, it was clear that for longer term protection against FC, the vaccines should be given at two doses, at least three weeks apart. As regards to the HI test that carried out on serum samples obtained from vaccinated chickens with the prepared combined FC and AI vaccine, it revealed that there was an increase in AI antibodies with titer of 6.4, 5.8 and 6.4 log2 when measured at the 3rd week post single dose of vaccination which raised gradually up to its maximum level at the 5th week recording 7.8, 7.2 and 7.6 in broiler breeder, broiler chickens and turkey respectively as shown in Table (2). Meanwhile the inoculation of booster dose after 3wks caused a rapid rise of antibodies titer where the peak of AI antibody titer was 8.6, 8.0 and 8.2 log2 by the 3rd week post boostering respectively. The same results obtained by (21) who determined the efficacy of two oil emulsion (ISA 206, ISA70) HA subunit vaccine derived from H5N1 virus. The same results obtained by (22) who evaluated the quality of oil emulsion AI H9N2 vaccine in-vitro by HI test without boostering. They found that the antibody titer increased till reach its peak (6.24 log2) at 5th week post vaccination. On the other hand, (23) evaluated a reverse genetics H5N3 avian influenza virus cell cultured vaccine by single dose and the HI titer reached its peak (6.2 log2) at the 4th week. (21) Found that chickens which received one dose of rHA- H5 vaccine had a markedly poor response and priming by two dosages appeared to improve the antibody response. (24) evaluated inactivated H9N2 vaccine with gel-primed and mineral oil- boosted regimen in broiler breeders. They concluded that a single dose regimen of inactivated H9N2 vaccine provided not enough anti body level in the broiler breeder flock and recommended a two doses regimen than single dose. Also stated that a gel- primed and oil- boosted regimen might be an economical and effective vaccine strategy for poultry producers. Regarding the challenge test, the protection percentages of birds against P. multocida strains were 76.6, 70.0 and 80.0 %, among broiler breeder, broiler and turkey groups respectively when challenged 3weeks after single dose, while they were 90.0, 86.6 and 93.3 %, among the same groups when challenged 3 weeks after booster dose as shown in Table (3). The results obtained by (25) who reported that the protection % in ISA 206 combined FC and ND vaccine was 100%. Also (26) noticed that chickens vaccinated with FC vaccine containing ISA 206 showed 90% protection. From these data it was clear that the antibody titer was measured with ELISA test was highly correlated with protection against challenge with virulent organisms as reported by (15). For the protection against AI H9N2 virus, it was 83.3, 80.0 and 86.6% among the broiler breeder, broiler and turkey bird groups when challenged 3weeks after single dose as shown in Table (4). These protection percentages were raised up to 93.3, 90.0 and 96.6 % for the same bird groups when challenged 3 weeks post booster of vaccination. An inactivated ISA 70 H9N2 vaccine was evaluated by (27) and concluded to this vaccine induced protection 100% 3weeks post vaccination where the virus isolated from 0/7 cloacal samples collected on 1, 3 and 5 days post challenge. In contrast (23) concluded that ISA 206 adjuvant vaccine provides only 50% or even less protective efficiency to SPF chickens. Meanwhile (28) recorded that the protection levels were found to increase up to 90 % when the Montanide ISA 70 and Montanide ISA 206 formulations were used. The findings of this study indicated that the vaccine is valid and has a satisfactory result according to specifications of OIE (14). Conclusion Conclusively, it is very beneficial to apply the inactivated polyvalent Pasteurella multocida and AI H9N2 vaccine for the protection against both FC and AI outbreaks as it reduce production costs, for the administrator as it save time, effort and simplify the immunization schedule. Also for the birds, it minimizes stress of multiple vaccinations. Field application for experimental inactivated multivalent P. multocida and avian influenza (H9N2) vaccine 793 Table 1: P. multocida ELISA mean titer of different bird flocks vaccinated with the prepared FC and AI (H9N2) vaccine post single and booster dose vaccination Type of Weeks post vaccination Control Birds 1 2 3 4 5 6 7 Broiler Single dose 523 841 1011 1176 1247 1208 1185 36 breeder Booster dose 1381 1566 1832 1792 41 Broiler Single dose 469 719 904 1066 1191 1174 1081 39 Booster dose 1218 1466 1578 1511 38 Turkey Single dose 497 819 973 1118 1226 1211 1172 44 Booster dose 1341 1494 1801 1716 29 Table 2: AI H9N2 HI mean titer of different bird flocks vaccinated with the prepared FC and AI (H9N2) vaccine post single and booster dose vaccination Type of Birds Weeks post vaccination_ Control 1 2 3 4 5 6 7 Broiler Single dose 3.6 4.8 6.4 6.6 7.8 7.6 7.2 0.0 breeder Booster dose 7.2 8.2 8.6 8.2 0.0 Broiler Single dose 3.2 4.0 5.8 5.8 7.2 6.8 6.2 0.0 Booster dose 6.8 7.4 8.0 7.6 0.0 Turkey Single dose 3.6 4.6 6.4 6.2 7.4 7.2 6.8 0.0 Booster dose 7.2 7.6 8.2 7.8 0.0 Table 3: Protection percentages obtained in birds vaccinated with single and booster doses of the prepared FC and AI vaccine against virulent P. multocida strain Challenge 3 weeks post_ Control Type of Bird Single dose vaccination Booster tion dose vaccina- Bird No M. P % Bird No M. P % Bird No M. P % Broiler breeder Broiler Turkey 30 30 30 7 9 6 76.6 70.0 80.0 30 30 30 3 4 2 90.0 86.6 93.3 15 15 15 12 14 13 20.0 07.0 13.4 M = Mortalities P% = protection percent Table 4: Protection percentages obtained in birds vaccinated with single and booster doses of the prepared FC and AI vaccine against AI H9N2 virus HA Vaccination Type of bird ECE No. ——tt-——-- Protection % ___Positive Negative_ Broiler breeder 30 5 25 83.3 Single dose Broiler 30 6 24 80.0 Turkey 30 4 26 86.6 Broiler breeder 30 2 28 93.3 Booster dose Broiler 30 3 27 90.0 Turkey 30 1 29 96.6 794 S. Salama, H. Abdelhady, L. Atia Conflict of interest The authors declare that they have no conflict of interest. References 1. Glisson, J.R., C.L. Hofacre and J.P. Christensen. Fowl cholera. In: Diseases of poultry, Saif YM, Barenes HJ, Glisson JR, Fadly AM, McDou-gald LR and Swayne DE (Editors). Blackwell Publishing, Ames, Iowa, USA, 2008; 739-58. 2. Swayne, D.E. and D.A. Halvorson. Avian influenza. In: Saif, Y.M., Barnes, H.J., Fadly, A.M., Glisson, J.R., McDougald, L.R., Swayne, D.E. (Eds.). Diseases of Poultry. 11th ed. Iowa State Press, Iowa. 2003; pp. 135-60. 3. Adler, B., D. Bulach, J. Chung, S. Doughty, M. Hunt, K. Rajakumar, M. Serrano, A. Van Zan-den, Y. Zhang and C. Ruffolo. Candidate vaccine antigen and genes in Pasteurella multocida. J. Biotech. 1999; 73(2-3): 83-90. 4. Takai, H., Y. Hayakawa, E. Shintani, H. Komae, Y. Yoshida, H. Ide, G. Abe and T. Sawada. The first outbreak of P.multocida. infection in turkeys in Japan. J. Japan Vet. Med. Association, 1994; 47:923-7. 5. Voyles, B.A. Orthomyxoviruses. In: The biology of viruses 2nd Ed. New York, NY: McGraw-Hill, 147. 2002. 6. Alexander, D. J. Should We Change the Definition of Avian Influenza for Eradication Purposes? Avian Diseases. 2003; 47: 976-81. 7. Bano, S., K. Naeem and S.A. Malik. Evaluation of Pathogenic Potential of Avian Influenza Virus Serotype H9N2 in Chickens. Avian Diseases. 2003; 47:817-22. 8. Alexander, D. J. A review of avian influenza in different bird species. Vet. Microbiol. 2000; 74 (1 -2): 3-13. 9. Orabi A, Hussein A, Saleh A A, El-Magd M A, Munir M. Evolutionary insights into the fusion protein of Newcastle disease virus isolated from vaccinated chickens in 2016 in Egypt, Archives of Virology 2017; 162(10): 3069-79 10. Reddy, G.S.; K. Ananda and V.A. Sriniva-san. Performance of oil adjuvant combined vaccine cintaining FMD, Rabies, Pasteurella multocida and Clostridium chauvoei antigens. Indian Veterinary Journal, 2001; 78(11): 990-3. 11. Swayne, D.E.; D.A. Senne and C.W. Beard. Influenza, in: Swayne, D.E.; Glisson, J.R.; Jackwood, M.W.; Pearson, J.E. and Reed, W.M. (Eds), Isolation and identification of avian pathogens: Kennet Square: American Association of Avian Pathologist. 1998; 150-5. 12. Villegas, P. 1998. Titration of biological suspensions. In: Swayne, D.E. (Ed), A Laboratory Manual for the Isolation and Identification of Avian Pathogens, fourth ed. American Association of Avian Pathologists, Kennett Square, PA, 1998; 248-54. 13. Sarachai, C.; J. Sasipreeyajan, and N. Chansiripornchai. Avian Influenza virus (H5N1) in-activation by Binary Ethylenimine. Thai J. Vet. Med. 2010; 40(1): 41-6. 14. OIE. Manual of Standars of Diagnostic Tests and Vaccines for Terrestrial Animals Safety Test, 2017; chapter, 2.3.4., Avian influenza page no.446. 15. Jabbri, A. R. and G.R. Moazeni Jula. Fowl cholera: Evaluation of a Trivalent Pasteurella multocida Vaccine Consisted of Serotypes 1, 3 and 4. Arch. Razi Ins. 2005; 59: 103-11. 16. Kwon, J.S.; H.J. Lee, D.H. Lee, Y.J. Lee, I P. Mo, S.S. Nahm, M.J. Kim, J.B. Lee, S.Y. Park, I.S. Choi and C.S. Song. Immune response and pathogenesis in immunocompromised chickens in response to infection with the H9N2 low pathogenic avian influenza virus. Virus Research, 2008; 133: 187--94. 17. Stone, D.; M. Brugh, S R. Hophins, H.W. Yoder and C.W. Peard. Preparation of inactivated oil emulsion vaccines wiyh avian viral or Myco-plasma antigens. Avian Dis., 1978; 22: 666-47. 18. Parvin, M.S., M. P. Siddique and M. T. Islam. Humoral immune response to fowl cholera vaccine in different breeds of commercial birds Bangl. J. Vet. Med. 2011; 9 (2): 127 -31. 19. Iqbal, M.T.; M.H. Haque, S. Sarker, M.A. Islam and K.A. Choudhury. Determination of immune response against alum- precipitated fowl cholera vaccine in the quail, coturnix japonica. Univ. J. Zool. Rajshahi. 2010; 29: 57-60. 20. Belloc, C.; L. Dupuis, S. Deville, H. Aucou-turier and A. Laval. Evaluation of safety and immune response induced by several adjuvants included in Pasteurella multocida vaccines in chickens. Revue Med. Vet., 2008; 159(7): 371-5. 21. Lin, Y. J.; M. C. Deng, S. H. Wu, Y. L. Chen, H. C. Cheng, C. Y. Chang, M. S. Lee, M. S. Chien and C.C. Huang. Baculovirus-Derived He magglutinin Vaccine Protects Chickens from Lethal Homologous Virus H5N1 Challenge. J. Vet. Med. Sci. 2008; 70(11): 1147-52. 22. Rajabi, Z.; H.T. Nasrabadi and A.B. Syofi-Khojin. Evaluation the quality of oil-Emulsion Field application for experimental inactivated multivalent P. multocida and avian influenza (H9N2) vaccine 795 Avian Influenza subgroup H9N2 vaccines in invito. Vaccinology, 2010; 2: 22-5. 23. Liu, C.G.; Ming Liu, Fei Liu, Da F Liu, Yun Zhang, Wei Q Pan, Hao Chen, Chun H Wan, En C Sun, Hong T Li, and Wen H Xiang. Evaluation of several adjuvants in avian influenza vaccine to chickens and ducks. Virol J.; 2011; 8: 321. 24. Lee, D.H.; J.S. Kwon, H.J. Lee, Y.N. Lee, W. Hur, Y.H. Hong, J.B. Lee, S.Y. Park, I S. Choi and C.S. Song. Inactivated H9N2 avian influenza virus vaccine with gel-primed and mineral oil-boosted regimen could produce improved immune response in broiler breeders. Poultry Science, 2011; 90: 1020-2. 25. Elham, A. youssef; Eman, A. Hassan; Mervata, A. El-Koffy and A.M. Daoud. Trial for preparation of combined oil inactivated vaccine against Newcastle and Fowl Cholera diseases by using Montanide ISA 206 oil for improving the immune response in chickens. Vet. Med. J. Giza, 2005; 53 (2): 341-52. 26. Hala, A.F.; I.S. Ibrahim, H.G. Fawzy and S.M. Aboul Saoud. Preparation and evaluation of avian cholera oil emulsion vaccine using Montanide ISA 206. Egypt. Vet. Med. Assoc., 2002; 62(2). 27. Jun G. C.; J.L. Youn, J.K. Yong, K.L. Eun, O.M. Jeong, W S. Haan, H.K. Jae and H.K. Jun. An inactivated vaccine to control the current H9N2 low pathogenic avianinfluenza in Korea. J. Vet. Sci., 2008; 9 (1): 67-74. 28. Dungu, B.; B. Brett, R. Macdonald, S. Deville, L. Dupuis, J. Theron and R.R. Bragg. Study on the efficacy and safety of different antigens and oil formulations of infectious coryza vaccines containing an NAD-independent strain of Avibacterium paragallinarum. Onderstepoort Journal of Veterinary Research, 2009; 76:299-309. 29. Fatma Elzahraa G. AboElkhir, Afaf A. Khedr and Elseedy F R. Identity of Pasteurella multocida and Avian Influenza H9N2 Strains Used in Preparation of a Combined Inactivated Vaccine Using PCR. Global Veterinaria 2005; 14 (4): 535-8.