ISSN 1580-4003 THE SCIENTIFIC JOURNAL OF THE VETERINARY FACULTY UNIVERSITY OF LJUBLJANA SLOVENIAN VETERINARY RESEARCH SLOVENSKI VETERINARSKI ZBORNIK Volume t 49 1 V £ ' rl s I- ; ^^■Hfirrri ti , 91 8BT?- " V "je ; g ^^ tIt ■v ^ jfc . || y|| ^^HTfvfTi^l ■n ^Ä'^t};'. v* jsmbfc ^-V* Jji V ' _ ' Slov Vet Res • Ljubljana • 2012 • Volume 49 • Number 1 • 1-58 THE SCIENTIFIC JOURNAL OF THE VETERINARY FACULTY UNIVERSITY OF LJUBLJANA SLOVENIAN VETERINARY RESEARCH SLOVENSKI VETERINARSKI ZBORNIK Volume 49 Slov Vet Res • Ljubljana • 2012 • Volume 49 • Number 1 • 1-58 The Scientific Journal of the Veterinary Faculty University of Ljubljana SLOVENIAN VETERINARY RESEARCH SLOVENSKI VETERINARSKI ZBORNIK Previously: RESEARCH REPORTS OF THE VETERINARY FACULTY UNIVERSITY OF LJUBLJANA Prej: ZBORNIK VETERINARSKE FAKULTETE UNIVERZA V LJUBLJANI 4 issues per year / izhaja štirikrat letno Editor in Chief / glavni in odgovorni urednik: Gregor Majdič Technical Editor / tehnični urednik: Matjaž Uršič Assistants to Editor / pomočnici urednika: Valentina Kubale Dvojmoč, Klementina Fon Tacer Editorial Board / uredniški odbor: Frangež Robert, Polona Juntes, Matjaž Ocepek, Zlatko Pavlica, Modest Vengušt, 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) Reviewing Editorial Board / ocenjevalni uredniški odbor: Ivor D. Bowen, Cardiff School of Biosciences, Cardiff, Wales, UK; Antonio Cruz, Departement of Clinical Studies, Ontario Veterinary College, Guelph, Ontario, Kanada; Gerry M. Dorrestein, Duch Research Institute for Birds and Exotic Animals, Veldhoven, The Netherlands; Wolfgang Henninger, Veterinärmedizinische Universität Wien, Austria; Simon Horvat, Biotehniška fakulteta, Univerza v Ljubljani, Slovenia; Nevenka Kožuh Eržen, Krka, d.d., Novo mesto, Slovenia; Louis Lefaucheur, INRA, Rennes, France; Bela Nagy, Veterinary Medical Research Institute Budapest, Hungary; Peter O'Shaughnessy, Institute of Comparative Medicine, Faculty of Veterinary Medicine, University of Glasgow, Scotland, UK; Milan Pogačnik, Veterinarska fakulteta, Univerza v Ljubljani, Slovenia; Peter Popelka, University of Veterinary Medicine, Košice, Slovakia; Detlef Rath, Institut für Tierzucht, Forschungsbericht Biotechnologie, Bundesforschungsanstalt für Landwirtschaft (FAL), Neustadt, Germany; Hans-Peter Sallmann, Tierärtzliche Hochschule Hannover, Germany; Marko Tadic, Veterinarski fakultet, Sveučilište u Zagrebu, Croatia; Frank J. M. Verstraete, University of California Davis, Davis, California, US Slovenian Language Revision / lektor za slovenski jezik: Viktor Majdič 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: Agencija za raziskovalno dejavnost Republike Slovenije ISSN 1580-4003 Printed by / tisk: Birografika Bori d.o.o., 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 Slov Vet Res 2012; 49 (1) Original Scientific Articles Cvetkovikj I, Dodovski A, Naletoski I, Mrenoski S, Mitrov D, Krstevski K, Dzadzovski I, Cvetkovikj A. No evidence of avian influenza viruses in backyard poultry fram risk areas in R. Macedonia (Virological and molecular surveillance 2009)..............................................................5 Olechnowicz J, Jaskowski JM. Somatic cell counts and total bacterial count in bulk tank milk of small ruminants..........13 Pelin Can O. The efect of thyme oil on the shelf life of chicken balls during storage period.................................19 Snoj T, Čebulj-Kadunc N, Nemec Svete A, Kobal S. Determination of sex hormones in rat hair after administration of testosterone proprionate and estradiol valerate......................................................27 Satué K, Felipe M, Mota J, Muñoz A. Blood leukocytes in Carthusian mares with endometriosis or endometritis in comparison with healthy mares........................................................................35 Čeh K, Ježek D, Žužek MC, Čebulj-Kadunc N, Majdič G. Effects of organophosphate Chlormephos on development and function of testes and brain.....................................................................41 Klinc P, Rath D. Improvement of sperm sorting efficiency and fertilizing capacity employing two variations of a new bull semen extender (Sexcess®)..................................................................51 Slov Vet Res 2012; 49 (1): 5 -12 UDC 636.5.09:616.921.5-07:577.21:57.083.2 Original Scientific Article NO EVIDENCE OF AVIAN INFLUENZA VIRUSES IN BACKYARD POULTRY FROM RISK AREAS IN R. MACEDONIA (Virological and molecular surveillance 2009) Iskra Cvetkovikj1*, Aleksandar Dodovski1, Ivanco Naletoski2, Slavco Mrenoski1, Dine Mitrov1, Kiril Krstevski1, Igor Dzadzovski1, Aleksandar Cvetkovikj1 1Faculty of Veterinary Medicine in Skopje, Lazar Pop Trajkov 5-7, 1000 Skopje, Republic of Macedonia; international Atomic Energy Agency, Animal Production and Health section, Joint FAO/IAEA Division, Viena, Austria 'Corresponding author, E-mail: iskra@fvm.ukim.edu.mk Summary: Until 2009 no data are available for the circulation of avian influenza viruses (AIV) in backyard poultry in Republic of Macedonia. In 2009 virological and molecular surveillance was conducted in backyard poultry in the risk areas of the country according the National Annual Program for AI and Commission Decision 2007/268/EC. A total number of 2151 samples were analyzed. Two thousand and twenty cloacal swabs (sampled from backyard poultry - poultry sector 4 from the risk areas in Republic of Macedonia i.e., areas where migratory waterfowl gathers) and 131 organs/cloacal swabs (for daily routine diagnostics) were tested by virological and molecular methods. The virological diagnosis was performed by isolation on embryonated chicken eggs, followed by macro hemagglutination, hemagglutination on microtiter plate and inhibition of hemagglutination. The molecular diagnosis was performed by RRT-PCR for M-gene. All samples were negative for AIV. However, 4 samples were positive on avian paramyxovirus 1 (Newcastle disease virus). The wild birds, as a reservoir for AIV were not included in the surveillance which on the other hand does not ensure complete information for the AIV circulation in R. Macedonia and should be further investigated. This fact imposes the need for overall national action to ensure the concept of early detection and response in case of emergence of avian influenza. Key words: avian influenza viruses; backyard poultry; cloacal swabs; Republic of Macedonia; surveillance Introduction In the last 10 years the field of avian influenza (AI) has undergone something of a revolution. According to Alexander [1], this happened not just because of the actual zoonoses and the potential threat of a pandemic emerging, which has highlighted public awareness, but also because many aspects of the epidemiology of AI infections in poultry and other birds appear to have changed dramatically. The number of outbreaks of the highly pathogenic avian influenza (HPAI) disease has increased Received:15 March 2011 Accepted for publication: 21 January 2012 alarmingly in the last 10 years and, even more noticeably, the impact in terms of the number of birds involved and the costs of disease control have dramatically escalated [2]. In addition, the apparently unprecedented emergence and spread of the HPAI H5N1 virus in Asia and beyond has brought AI to the forefront of important animal diseases [1]. Influenza viruses are spherically or longitudinally shaped enveloped particles with an up to eight-fold segmented, single-stranded RNA genome of negative polarity. Influenza viruses hold generic status in the Orthomyxoviridae family and are classified into types A, B or C based on anti-genic differences of their nucleo- and matrix proteins. AIV belong to type A [3]. 6 I. Cvetkovikj , A. Dodovski, I. Naletoski, S. Mrenoski, D.Mitrov, K. Krstevski, I. Dzadzovski, A. Cvetkovikj The main antigenic determinants of influenza A and B viruses are the hemagglutinin (H or HA) and the neuraminidase (N or NA) transmembrane glycoproteins, capable of eliciting subtype-specific and immune responses which are fully protective within, but only partly protective across, different subtypes. Because of the antigenicity of these glycoproteins, influenza A viruses currently cluster into 16 H (H1 - H16) and nine N (N1 - N9) subtypes. These clusters are substantiated when phylogenetically analysing the nucleotide and deducted amino acid sequences of the HA and NA genes, respectively [4]. To date, only viruses of the H5 and H7 subtypes have been shown to cause HPAI. It appears that HPAI viruses arise by mutation after low pathogenic avian influenza (LPAI) viruses have been introduced into poultry by several mechanisms [5]. AIV have been shown to infect birds and mammals. The main factor that influences susceptibility to infection is the receptor conformation on host cells (AIV bind preferentially to SA-a2,3-Gal and human IV to SA- a2,6-Gal terminated saccharides [5]. Influenza viruses have been shown to infect a great variety of birds [6, 7], including free-living birds, captive caged birds, domestic ducks, chickens, turkeys and other domestic poultry. All available evidences suggest that in the conventional situation the primary introduction of LPAI viruses into a poultry population is a result of wild bird activity, usually waterfowl [5]. The greatest threat of spread of AIV is by mechanical transfer of infective faeces in which virus may be present at concentrations as high as 107 infectious particles/g and may survive for longer than 44 days [8]. The fact that AI represents one of the greatest concerns for public health that has emerged from the animal reservoir in recent times [5], leads to the considerable public and media attention having in mind its zoonotic potential. This situation has given rise to the fear that the virus might acquire the capacity of sustained human-to-human transmission and thus cause global influenza pandemic. This fear has been the main driving force behind the international commitment to global HPAI control, to date amounting to an estimated US$1.02 billion disbursed funds and US$1.68 billion committed funds as of June 2007 [9], which in turn has significantly influenced national responses to outbreaks of H5N1. The latter have involved destruction of birds on a massive scale, regulation of poultry production and trade and, in some countries, large-scale vaccination campaigns. These responses often come at considerable cost to poultry producers. HPAI, like other highly contagious animal diseases, affects animal production via three main pathways: direct losses, public impact and market reactions [10]. Firstly, HPAI causes direct losses to producers and other actors interlinked to the production and marketing of poultry through morbidity and mortality and the private costs associated with ex-ante risk mitigation (e.g. investment in animal housing) and/or ex-post coping measures during periods of downtime (e.g. bridging loans if the enterprise carries significant borrowings) and the need to reinvest in replacement birds. Secondly, animal diseases that are 'notifiable' can have severe impacts through government intervention, which carries a cost borne by the public at large and affects producers (and associated up- and downstream actors), irrespective of the disease status of their flocks. These costs include public investment in animal health infrastructure and epidemic preparedness. Thirdly, disease impacts arise through market reactions, which can be particularly severe on the demand-side for diseases that are associated with a public health risk. Analogous to disease control measures affecting producers even if their flocks have not contracted HPAI, market reactions can occur, irrespective of whether or not avian influenza has actually occurred in the country. Geographically, R. Macedonia belongs to the Black sea - Mediterranean migratory flyway, surrounded by Albania, Greece, Bulgaria, Serbia and Kosovo. Three of these neighboring countries at some point have reported to OIE outbreaks of HPAI H5N1 (Albania 2006 - 1 case, Greece 2006 -1 case, Bulgaria 2006 - 1 case and 2010 - 1 case). In the wider region, Romania has reported 109 cases of H5N1 in 2006, 1 in 2007 and 2 cases in 2010. Turkey has also reported 2 cases in 2005, 200 in 2006, 17 in 2007 and 7 cases in 2008. As pointed by the claims of EU, surveillance of AIV for poultry and wild birds is mandatory for every Member state and comprises the implementation of surveillance programs according the EU legislative guidelines. The National Annual programme for AI surveillance includes conducting surveillance programs No evidence of avian influenza viruses in backyard poultry from risk areas in R. Macedonia 7 in poultry and wild birds. Unfortunately, until now according to the National Annual program no surveillance has been conducted. This is a great disadvantage giving the fact that there is no available data for the previous years and it is anybody's guess if there is circulation of AIV or not. Although knowing the fact that 3 of the surrounding countries have reported outbreaks of HPAI H5N1, the question of AIV circulation in R. Macedonia remained unanswered and thus emphasized the need of investigation due to the possibility of spreading the disease to Macedonia. Therefore, the aim of this study was to assess the AIV situation in the poultry sector 4 in R. Macedonia, i.e., to affirm the presence and distribution and/or to confirm the absence of AIV in backyard poultry. Materials and methods The cloacal swabs were sampled from backyard poultry (poultry sector 4) from the risk areas in R. Macedonia i.e., areas where migratory waterfowl gathers. The poultry sector 4 defines backyard poultry production in villages with minimal biosecurity measures (birds are kept out most of the day with high possibility of contact with other chicken, ducks, domestic birds and wildlife). Those characteristics define the poultry sector 4 as the most prone sector for AI emergence, especially in locations near lakes and flooding areas where migratory waterfowl gathers. The distribution of the sampled villages in the risk areas is given in Table 1, and their location is given in Figure 1. The distribution of samples per poultry species is given in Table 2. As pointed in the Annual program for AI surveillance [11] and Commission Decision 2007/268/ EC [12], the sampling was representative for the whole state. A total number of 213 holdings in 59 villages (1-6 holdings per village) were sampled. According to the previously defined representative number for cloacal swabs per holding (n-11), 11 cloacal swabs were sampled wherever possible. In smaller holdings, the number of sampled cloacal swabs varied from 1 to 11 depending on the number of poultry reared in the holding. Besides these samples, the survey included 131 samples from domestic and wild birds submitted for daily routine diagnosis in the laboratories of the Faculty of veterinary medicine in Skopje (FVMS) (Table 3). Sam- ple processing was performed according to the guidelines in Commission decision 2006/437/EC [13] and OIE [14]. The extraction of RNA was performed with RNEasy Mini kit according to the producer's manual [15]. Detection of M-gene was conducted on Bio-Rad's IQ5 RRT-PCR. The method was performed as pointed in the protocol of the Waybridge reference laboratory [16]. The following reagents were used: 1. Primers and probes: • Sep 1 AGA TGA GTC TTC TAA CCG AGG TCG (Operon) • Sep2 TGC AAA AAC ATC TTC AAG TCT CTG (Operon) • SePRO FAM-TCA GGC CCC CTC AAA GCC GA-TAMRA (Operon) 2. Real time PCR master mix: Qiagen On- estep RT-PCR kit The virological methods (virus isolation on em-bryonated chicken eggs - ECE and identification by hemagglutination inhibition - HI test) were performed according to the Commission decision 2006/437/EC [13] and OIE [14]. These methods are not specific for AIV and are used for detection of other avian viruses that can be propagated on ECE and identified by HI test. The HI test was performed with specific antisera for avian paramyxovirus 1, AIV H5, AIV H7 and avian adenovirus. Table 1: Distribution and number of sampled poultry in risk areas Risk area Sampled villages N° of samples Artificial lake Mavrovo 6 188 "Sini virovi" Ohrid 4 164 Ohrid lake 20 690 Prespa lake 7 270 Fishery Bel Kamen 3 124 Fishery Bukri 2 68 Dojran Lake 4 139 Flooding area Monospitovsko blato 5 209 Flooding area Katlanovsko blato 5 103 Artificial lake Veles 3 65 TOTAL 59 2020 8 I. Cvetkovikj , A. Dodovski, I. Naletoski, S. Mrenoski, D.Mitrov, K. Krstevski, I. Dzadzovski, A. Cvetkovikj Figure 1: Location of the sampled villages in the risk areas of R. Macedonia Table 2: Sampled poultry species Poultry species No of samples Chickens 1847 Ducks 124 Geese 21 Turkeys 23 Guinea fowl 2 Pheasants 3 Table 3: Samples for daily routine diagnosis Species Sample No of samples Chickens (Holding with high rate mortality) Carcass 12 Gulls, herons, mallard ducks (Unspecified number per each species) Cloacal and tracheal swabs 37 Mallard ducks Organs 11 Accipiter Organs 2 White stork Organs 1 Ostrich Organs 2 Common quail Carcass 2 Geese Organs 1 Pigeons Carcass 12 Chickens Carcass 51 TOTAL 131 No evidence of avian influenza viruses in backyard poultry from risk areas in R. Macedonia 9 Results The 2020 analyzed cloacal swabs gave negative result for the presence of AIV by both virological and molecular diagnosis. The 131 analyzed samples for daily routine diagnosis also gave negative result for the presence of AIV by VI and PCR. Four samples (three from pigeons and one from poultry) were positive on avian paramyxovirus 1 (Newcastle disease virus). Discusion Until 2009, no molecular or virological surveillance was conducted to determine the presence and circulation of AIV i.e., no data were available for assessing the AI situation in R. Macedonia. The research of Dodovski et al. [17] from September 2008 till March 2009, showed seropositive flocks by ELISA but negative on hemagglutination inhibition test for H5/H7, which may be due to circulation of non H5/H7 viruses. So, this fact imposed the question: If seropositive flocks are present - is there a circulation of the virus? Following this fact, Cvetkovikj et al. [18] in 2009 performed virological and molecular surveillance in the commercial poultry sector resulting with negative results for circulation of AIV. According the 2009 surveillance there is no evidence of AIV subtypes circulating among backyard poultry population, which of course does not represent a constant result and imposes the need of further permanent surveillance. So, what are the possible reasons for the negative results? There are several possible issues that may lead to this situation and are addressed below. R. Macedonia is defined by the World Bank [19] as a country with low or moderate risk for AI outbreak, which contributes the fact that the surveillance yielded with negative result. According to World Animal Health Information Database (WAHID) Interface [20] in 2009, in the Black Sea-Mediterranean flyway AI was reported only in 4 countries with 6 outbreaks - 3 for Low Pathogenic Notifiable Avian Influenza (LPNAI) and 3 for Highly Pathogenic Notifiable Avian Influenza (HPNAI). On the other hand, in 2008, 9 countries with total number of 141 outbreaks for HPNAI were reported and no LPNAI was reported. This fact shows the trend of decreasing prevalence of Notifiable Avian Influenza (NAI) in the countries belonging to the Black Sea - Mediterranean flyway. The poultry sector 4 in R. Macedonia is defined as a sector with low biosecurity level and high risk for AI occurrence. In R. Macedonia the backyard poultry is reared mostly for egg production and kept in closed and covered fences, thus minimizing the possibility of contact with the wild birds or their faeces. Besides that, R. Macedonia does not possess large water areas attractive for the migratory waterfowl - representing the potential danger of input and spread of AIV in poultry. This surveillance was based on sampling cloacal swabs from backyard poultry. So, did the cloacal swabs influence the negative result? For most bird species it is prudent to collect both tracheal/oropharingeal and cloacal swabs for testing, or only tracheal/oropharingeal when gallinaceous birds are subject of the surveillance. However, usage of cloacal swabs in surveillance of AI in backyard poultry is not uncommon. Buscaglia et al. [21] in the surveillance in Argentina from 1998 to 2005, demonstrated no positive result in both serological and virological surveillance in backyard poultry using sera and 18.000 tracheal and cloacal swabs (only 10% tracheal swabs per year and 90% cloacal). After 2006, (isolation of H5N1 virus from mallard duck near Zagreb) Croatia has a regular monitoring for avian influenza in wild birds and poultry (especially in the backyard flocks using cloacal swabs). All samples were HPAI virus negative but LPAI viruses (H2N3, H3N8, H5N3 and H10N7) were isolated from wild birds [22]. Racnik et al. [23] also demonstrated negative result in backyard poultry using cloacal swabs as samples in the first Slovenian surveillance for avian influenza in migratory birds and backyard flocks. However, to estimate the ecology of AIV in non commercial poultry, most surveillance strategies are based on sampling both tracheal and cloacal or only tracheal swabs. Knowing the fact that gallinaceous birds typically shed AI viruses in respiratory secretions, a tracheal or oropharyngeal swab is the primary source of virus detection from chickens and turkeys. As pointed in the Diagnostic manual for avian influenza (2006/437/EC), for investigation of a holding suspected of being infected with the AI virus, the standard set of samples for virological testing is a combination of cloacal and tracheal/ oropharyngeal swabs. 10 I. Cvetkovikj , A. Dodovski, I. Naletoski, S. Mrenoski, D.Mitrov, K. Krstevski, I. Dzadzovski, A. Cvetkovikj This point could easily take us to the answer that maybe the negative result was influenced by sampling only cloacal and not both cloacal and tracheal/oropharyngeal swabs. Avian influenza has had an enormous economic and social impact on affected countries and the risk of this animal disease situation evolving into a global human influenza pandemic is of great public concern. Therefore, it is of interest of all countries, developed or developing, to invest in the containment of AI because there is continuing risk of AI spreading that no poultry-keeping country can afford to ignore. [24] The establishment of efficient preparedness and control of AI has to be substantiated by activities at national, regional and global level [24]. 1. At national level, each state has to strengthen Veterinary Public Health Services to better manage national surveillance systems, to improve early detection and response, define strategies and monitor their implementation. Likewise, disease prevention and control programmes have to be implemented including stamping out, biosecurity and vaccination as appropriate as well as developing and implementing better diagnostic tools. 2. At regional level, activities should be aimed to strengthen regional co-operation for harmonization of the surveillance and control strategies as well as promoting the regional diagnostic and surveillance networks etc. 3. Globally, the activities are aimed at donor support to provide the scientific and technical advice required by countries. In this context, R. Macedonia needs to implement surveillance programmes mostly aiming on strengthening the national preparedness plan that can only be efficient with collaboration of veterinary services, national reference laboratory, ornithologists, etc. Acknowledgements The survey was conducted as a part of the FP6 Project" Upgrading and introduction of sustainable diagnostic tools for the major transbound-ary diseases of animals in F.Y.R. of Macedonia" (TRANSB_DIS_ANIM_MK) - project coordinator: Prof. Dr. Ivancho Naletoski. References 1. Alexander DJ. An overview of the epidemiology of avian influenza. Vaccine 2007; 25: 5637-44. 2. Capua I, Alexander DJ. Avian influenza infections in birds - a moving target. Influenza Other Respir Viruses 2007; 1: 11-8. 3. Werner O, Harder TC. Avian influenza. In: Kampus BS, Hoffmann C, Preiser W, eds. Influenza report 2006. Paris: Flying Publisher, 2006: 49-86. 4. Fouchier R, Munster V, Wallensten A, et al. Characterization of a novel influenza A virus he-magglutinin subtype (H16) obtained from black-headed gulls. J Virol 2005; 79: 2814-22. 5. Capua I, Alexander DJ. Ecology, epidemiology and human health implications of avian influenza viruses: why do we need to share genetic data? Zoonozes Public Health 2008; 55: 2-15. 6. Alexander DJ. A review of avian influenza in different bird species. Vet Microbiol 2000; 74: 3-13. 7. Alexander DJ. Ecology of avian influenza in domestic birds. In: Proceedings of the International symposium on Emergence and Control of Zoonotic Ortho-and Paramyxovirus Diseases. Pa-dova: 2001: 25-34. 8. Utterback W. Update on avian influenza through February 21, 1984 in Pennsylvania and Virginia. In: Proceedings of the 33rd Western Poultry Disease Conference. Davis: University of California, 1984: 4-7. 9. Response to avian influenza and state of pandemic readiness: third global progress report. New York ; Washington: UN System Influenza Coordinator, World Bank, 2007: 93 str. 10. Otte J, Hinrichs J, Rushton J, Roland-Holst D, Zilberman D. Impacts of avian influenza virus on animal production in developing countries. CAB Rev Perspect Agric Vet Sci Nutr Nat Resour 2008; 3 (080): 1-18. 11. Annual programme for AI surveillance. PE Official Gazette of the Republic of Macedonia, 2007; 82: 31-48. 12. EC 2007. Commission decision 2007/268/ EC of 13.4.2007 on the implementation of surveillance programmes for avian influenza in poultry and wild birds to be carried out in the Member States and amending Decision 2004/450/EC. Off J EU L 2007; 115(3): 003-017. (3.5.2007) 13. EC 2006. Comission decision 2006/437/ No evidence of avian influenza viruses in backyard poultry from risk areas in R. Macedonia 11 EC of 4.8.2006 approving a Diagnostic Manual for Avian influenza as provided for in Council Directive 2005/94/EC. Off J EU L 2006; 237(237): 1-27. (31.8.2006). 14. Avian influenza. In: OIE Manual of diagnostic tests and vaccines for terrestial manual. 6th ed. Paris: Office International des Epizoot-ies, 2009: 1-20, chapter 2.3.4. http://www.oie. int/fileadmin/Home/eng/Health_standards/ tahm/2.03.04_AI.pdf (25 May 2010). 15. Qiagen. RNeasy® Mini handbook. 4th ed. Qiagen, 2006: 88 str. http://www.scribd.com/ ojbillions/d/36597674-RNeasy-Mini-Handbook (25 May 2010). 16. Avian Influenza Community Reference Laboratory. Detection of influenza A matrix gene by realtime Taqman® RT-PCR. SOP VI 493, 2nd ed. London: Veterinary Laboratory Agency, 2007: 1-10. 17. Dodovski A., Naletoski I., Mitrov D. Sero-logical surveillance of avian influenza in commercial flocks in Macedonia. In: 16th Congress of the World Veterinary Poultry Association: book of abstracts Marrakesh, Morocco, 2009: 268. 18. Cvetkovikj I., Dodovski A., Naletoski I., et al. Surveillance of the avian influenca viruses in farmed poultry in 2009 in Republic of Macedonia. Macedonian Vet Rev 2011; 33 (1): 19-24. 19. World Bank. Avian and Human Influenza: Financing needs and gaps, 2005: 39. 20. World Animal Health Information Database. WAHID Interface. http://web.oie.int/wahis/public. php?page=disease_immediate_summary&selected_ year=2009 (25 May 2010). 21. Buscaglia C, Espinosa C, Terrera MV, De Benedetti R. Avian influenza surveillance in backyard poultry of Argentina. Avian Dis 2007; 51: 467-9. 22. Prukner-Radovcic E, Gottstein Z, Savic V. Avian influenza in Croatia: current status. In: FAO/IAEA International Symposium on Sustainable Imporvement of Animal Production and Health. Vienna, 2009: 404-4. 23. Racnik J, Krapez U, Trilar T, Dovc A, Bar-lic - Maganja D, Zorman - Rojs O. Avian influenza surveillance in backyard flocks and migratory birds in Slovenia. Rad Hrv Akad Znan Umjet Med Znan 2006; 30: 81-6. 24. Jutzi SC. FAO address. In: Proceedings of the OIE/FAO International Scientific Conference on Avian influenza. Paris, 2005. Dev Biol (Basel) 2006; 124: 5-9. 12 I. Cvetkovikj , A. Dodovski, I. Naletoski, S. Mrenoski, D.Mitrov, K. Krstevski, I. Dzadzovski, A. Cvetkovikj NI DOKAZOV O OBSTOJU VIRUSA PTIČJE GRIPE PRI DVORIŠČNI PERUTNINI NA OGROŽENIH OBMOČJIH V R. MAKEDONIJI (Virološki in molekularni nadzor 2009) I. Cvetkovikj, A. Dodovski, I. Naletoski, S. Mrenoski, D. Mitrov, K. Krstevski, I. Dzadzovski, A. Cvetkovikj Povzetek: Do leta 2009 nimamo podatkov o razširjenosti virusa ptičje gripe (AIV) pri dvoriščni perutnini v Republiki Makedoniji. V skladu z nacionalnim letnim programom nadzora AI in odločbe Komisije 2007/268/ES je bil v letu 2009 izveden virološki in molekularni nadzor dvoriščne perutnine na ogroženih območjih države. Skupno smo analizirali 2151 vzorcev. Dva tisoč in dvajset kloakalnih brisov (vzorci dvoriščne perutnine perutninskega sektorja 4 iz ogroženih območij v Republiki Makedoniji, med njimi iz območij, kjer so zbirališča vodnih ptic selivk) in 131 organskih/kloakalnih brisov (za vsakodnevno rutinsko diagnostiko) je bilo testiranih z virološkimi in molekularnimi metodami. Virološka diagnoza je bila izvedena z izolacijo oplojenih kokošjih jajc, kateri je sledila makrohemaglutinacija, nato hemaglutinacija na mikrotiterski plošči in zaviranje hemaglutinacije. Molekularna diagnostika je bila izvedena z RRT-PCR za M-gen. Vsi vzorci so bili AIV negativni, medtem ko so bili 4 vzorci pozitivni na ptičji paramiksovi-rus 1 (virus Newcastle). Divje ptice kot primarni gostitelj AIV niso bile vključene v nadzor, kar ne zagotavlja popolne informacije o razširjenosti AIV v R. Makedoniji in jo je treba še dodatno raziskati. To pomeni, da se je treba lotiti izdelave koncepta nacionalnih ukrepov za zgodnje odkrivanje in odziv ob pojavu ptičje gripe. Ključne besede: virus ptičje gripe; dvoriščna perutnina; kloakalni bris; R. Makedonija; nadzor Slov Vet Res 2012; 49 (1): 13 - 8 UDC 636.3.09:579.62:637.1:616-078 Original Scientific Article SOMATIC CELL COUNTS AND TOTAL BACTERIAL COUNT IN BULK TANK MILK OF SMALL RUMINANTS Jan Olechnowicz*, J^drzej Maria Jaskowski Department of Veterinary Medicine, Faculty of Animal Breeding and Biology, Poznan University of Life Sciences, Wojska Polskiego 52, 60-625 Poznan, Poland Corresponding author, E-mail: olejanko@up.poznan.pl Summary: Total bacterial count (TBC) is a good tool for monitoring the hygienic environment of small ruminants. TBC is significantly correlated with the number of somatic cells in bulk milk (BMSCC). Programs to improve the hygienic quality of bulk milk are moreeffective ifyou take into account bothvariables(TBCand BMSCC). Asignificant impact on BTSCCin ewebulk milkwas found for the following factors: herd/flock, sampling month, dry therapy practice, breed of sheep, and type of milking. Factors associated with the mean standard plate count (SPC) included the month of bulk milk sampling and the number of milkings contained in bulk tank milk, as well as the adopted milking technique. In fresh sheep chesses the casein fractions (as1-1-CN and |3-CNs) were significantly lower, when BTSCC was high. These cheeses have lower fat contents and as a result an increased pH and fat acidity. Yoghurts made from milk with a high BTSCC were characterized by a higher protein content and the highest level of syneresis. To improve the udder health status of small ruminants it is necessary to ensure hygienic conditions of animal maintenance and optimization of milking machine standards and parlor systems. Key words: small ruminants; somatic cell count; total bacterial count; bulk tank milk Introduction Total bacterial count (TBC) can serve as a tool in monitoring hygiene of dairy sheep flocks and can also provide a basis for payment systems for milk (4, 8, 15). A statistically significant correlations was found between TBC and the number of somatic cells in bulk milk (BMSCC). Programs to improve the hygienic quality of bulk milk are more effective if you take into account both variables (8). More recent results indicate that predictors of quality milk for cheese production include effectiveness of lactose, casein % and somatic cell Received:24 October 2011 Accepted for publication: 1 February 2012 count (SCC) of bulk milk (15). Milk microbiological standard stipulated by the European Council Directive 92/46/EEC may be used as a basis to develop specific quality parameters in the standard system for assessing milk of small ruminants, as it has been the case in Switzerland (27). In recent years, the Single Strand Conformation Polymorphism (SSCP) method proved to be a powerful tool for distinguishing milks and classifying them on the basis of the balance between different bacterial populations (26). In goats apocrine secretion mechanisms cause in milk the presence of cytoplasmic particles (CP), which are mostly anuclear and part of physiological secretions (22). Application of flow cytometry facilitates a rapid differentiation of somatic cells; however, it is necessary 14 J. Olechnowicz, J. M. Jaákowski to use DNA-specific fluorescent dyes to avoid overlapping of SCC and CP (2). This new method allows a differentiation between viable and nonviable polymorphonuclear neutrophils (PMN), macrophages, and lymphocytes. The aim of the study is to review the scientific literature in the field of hygienic and microbiological quality of bulk milk and the quality of products (cheese and yogurt) made from the milk of small ruminants. Bulk milk somatic cell counts related to bulk milk total bacterial counts The base level of SCC in bacteria free udders of goats (~ 300,000 cells/ml) and sheep (~ 200,000 cells/ml) is generally higher than in dairy cows (~ 70,000 cells/ml); hence, the level of SCC in animals with udder infection is usually much higher in small ruminants than in dairy cows (24). In-tramammary infection (IMI) is a major cause of an increased SCC in milk of dairy ruminants (7, 8, 9, 13, 14, 21, 22). The innate immune system plays a major role, especially polymorphonuclear neu-trophils (PMN), that appear in the mammary gland in response to invading pathogens (21). However, non-infectious factors are the cause of the variability of SCC in goat milk and some species of bacteria cause different levels of somatic cells as well as varying degrees of mastitis (22). Similarly in sheep, the increase in SCC is related to the stage of lactation and parity, but mainly results from intramammary infections (21). Many authors emphasize that the use of SCC is a good indicator in the assessment of sanitary conditions in herds/ flocks, especially in herds with maintained control of IMI (8, 9, 22). In herds of Murciano-Granadina goats several animals infected with mycoplasma do not cause an increase in BTSCC, provided that basic rules of hygiene are kept in the herd, and infected animals are rapidly removed from the herd (6). From the 1068 collected goat milk bulk-tank samples, 84 (7.9%) were positive for the presence of Mycoplasma species (4). Most of the isolated species were Mycoplasma agalactiae, which were found in 69 samples of milk (82%), and in 14 samples (17%) was detected Mycoplasma mycoides subsp. Mycoides large colony type (MmmLC) in the herd. Higher BTSCC levels (1,176,000 cells/ml) were recorded in bulk-tank samples containing mycoplasmas in comparison to the count recorded for samples testing negative (875,000 cells/ ml), and these differences were statistically significant. Bulk goat milk generally has a high SCC level; however, less than 50% of milk supplied by producers comply with the standards, i.e. 1 x 106 cells/ml (7). In a high percentage of milk samples neutrophils and variable bacterial counts, and a lack of correlation with SCC indicate problems with milk contamination. These problems may be caused by residual lactation and low milk production, but further research needs to be carried out on the high percentage of neutrophils reported in goat milk. In a study by the year 2010 in 53 herds of dairy goats 3 bulk milk samples were taken at 2-week intervals to assess how different bacterial groups in bulk milk are related to BTSCC, TBC and bulk milk standard plate counts (SPC) (13). It was found that the staphylococcal count was correlated to SCC (r = 0.40), TBC (r = 0.51) and SPC (r = 0.53). Coliform count was correlated only to TBC (r = 0.33); however, Staphylococcus au-reus was not correlated to SCC. The share of the staphylococcal count in the SPC was 31%, while coliform count was only 1% of the SPC. Somatic cells in goat bulk milk showed a moderate correlation to the bacterial counts, and Koop et al. (14) hypothesized that IMI is an important factor affecting SCC in bulk milk. However, many other factors associated with the management of the herd, as well as nutritional factors and diseases, of which an important role is played by mastitis, affect SCC in bulk milk. In the literature there is little information showing an association between bulk tank total bacterial count (TBC) and bulk tank SCC (BTSCC) in dairy sheep (8, 10, 18). Dry therapy practice and milking the ewes in parlor systems were associated with low TBCs (8). A study on milk quality in Assaf ewes indicated that antibiotic residue (AR), SCC and TBC in bulk tank milk can be used as variables to monitor mammary health, milk hygiene, and safety in dairy ewes (10). High SCCs were associated with an increased AR occurrence. It was confirmed that the prevalence of AR in the milk of sheep is inversely proportional to milk yield and the highest degree of AR incidence was in the autumn. The milk from small ruminants, similarly as milk of other species in the EU countries cannot contain antibiotic residues (5). In raw goat and ewe bulk tank milk in Switzerland a wide distribution of subclinical infection with Mycobacterium avium ssp. Paratu-berculosis (MAP) was found, amounting to 23% and 24%, respectively (18). Somatic cell counts and total bacterial count In bulk tank milk of small ruminants 15 Risk factors associated with a deterioration of bulk milk quality A basic condition for good quality milk from small ruminants, especially in case of small farms, is the observance of hygiene on the farm, mainly to maintain hygiene equipment and utensils for milking in good condition (17, 20). The IMI that cause subclinical and clinical mastitis has been estimated at 5 - 30% and 5%, respectively (5, 16). In problematic flocks mastitis incidence is much higher than 30 - 50% and may reach up to 70%. Many pathogens can cause mastitis; however, Staphylococcus spp. are the most commonly diagnosed microorganism of IMI in goats and sheep (5). Manual milking was associated with a 62% risk of bacterial infection of milk compared to mechanical milking, while mechanical milking with portable devices was associated with a 40% higher risk of bacterial infection compared to mechanical milking with fixed plants (16). In Switzerland for goat milk median SPC was 4.68 log cfu/ml and mean SPC 6.92 log cfu/ml, whereas for ewe milk median SPC was 4.79 log cfu/ ml and mean SPC was 6.05 log cfu/ml, respectively (27). Factors associated with SPC included the month of bulk milk sampling, while among the four evaluated months (April, May, June and July) the highest median SPC (5.24 log cfu/ml) and mean SPC (7.13 log cfu/ml) were found in June. Differences in SPC results between the months of sample collection were significant. The number of milkings contained in bulk tank affected SPC (log cfu/ml). The highest median value of the SPC (5.70 log cfu/ ml) and mean SPC (7.24 log cfu/ml) were found in bulk tank milk containing four milkings. According to Zweifel et al. (27) also milking technique affected SPC value, the highest median SPC (5.06 log cfu/ ml) and mean SPC (6.90 log cfu/ml) were found for bucket milking without a parlor; however, the hand milking technique showed the lowest SPC results (median SPC 4.48 log cfu/ml, and mean SPC 6.87 log cfu/ml). Flock size also affected SPC value, as the highest median SPC (4.85 log cfu/ ml) and mean SPC (6.86 log cfu/ml) were found in larger flocks (> 25 animals). In case of BTSCC in ewe bulk milk a significant impact was found for the following factors: flock/herd, sampling month, dry therapy practice, breed of sheep (Spanish As-saf, Awassi, Churra, Castel), type of milking (hand vs. machine, and type of installations used for machine milking (milking buckets and parlors: looped type milkline, dead-milkline ended, midlevel and low-level systems) (9). Effects of somatic cell counts and contamination on quality products from goat milk and sheep milk Bulk milk from ewes of three breeds, i.e. Churra, Castelana, and Assaf, with different levels of somatic cells was used to produce hard cheese (23). Chesses were produced from bulk milk of three classes: low (< 500,000 ml-1), medium (1,000,000 - 1,500,000 ml-1) and high (>2,500,000ml-1) SCCs. A significant increase has been shown in proteolysis with an increase in SCC levels. In fresh cheeses the casein fractions (as1-1-CN and p-CNs) were significantly lower as SCC values increased. In cheeses made from milk with a high SCC a reduced fat content was recorded, and it followed an increase of pH and fat acidity. Yoghurts were made from milk of the Baluchi breed with two SCCs, i.e. <200,000 ml-1 and >750,000 ml-1 (19). Different levels of SCC are associated with changes in milk composition and properties of yoghurt; the SCC had no effect on acidity, while for pH of the yoghurt after 24 h this effect was significant after 168 h of storage. Somatic cell count had no effect on total solids and fat content of the yoghurt. A high SCC was associated with a higher protein content and the highest level of syneresis. In Norway the presence of potential-poisoning bacteria was evaluated during the process of small scale cheese production using caprine and bovine raw milk (12). The prevalence of Staphylococcus aureus was varied during the manufacturing process of cheese from the milk of both species. The prevalence of Staphylococcus aureus in caprine cheese ranged from 91.8% in milk at 0 h and 95.9% at 24 h, and reduced to 42.9% after 30 days. The prevalence of Staphylococcus aureus in bovine cheese was 47.2% at 0 h, peaked to 80.8% after 5 - 6 h, and amounted to 24.7% after 30 days. The highest contamination levels of Staphylococcus aureus were observed in both caprine and bovine cheese after 5-6 h. Somatic cell count in bulk goat milk below the admissible limit according to the current Pasteurized Milk Ordinance regulation (PMO) amounting to 1,000,000 cells/ml in grade. A goat milk, did not affect milk composition and semisoft cheese yield (3). However, the high SCC in goat milk resulted in a lower texture score as well as a 16 J. Olechnowicz, J. M. Jaákowski lower total sensory score. Cheeses made with high SCC milk exhibited higher lipolysis during ripening compared with cheeses made from low SCC milk. The high levels of SCC in goat and ewe milk are associated with milk yield losses, changes in milk composition end affect the quality and stability of cheese (22). Control and prevention strategies In a study by Berthelot et al. (1) was demonstrated that in ewe milk individual SCC (iSCC) represent a useful tool for the detection of sub-clinical mastitis in dairy ewes. The authors propose a classification as a healthy udder (specificity = 75%), if throughout lactation iSCC are lower than 0.500 x 106 cells/ml, and infected (sensitivity = 82%) if at least two iSCC are higher than 1 or 1.2 million cells/ml. However, the decision should be adapted to different control strategies for SCC in bulk milk. Antibiotic dry-off therapy was found to significantly reduce IMI incidence in small ruminants (5, 9, 11, 25). This treatment significantly decreased BTSCC from log BTSCC 6.24 and the geometric mean of 1,725 x 103 cells/ml in the pretreatment lactation to log BTSCC of 6.15 and 1.422 x 103 cells/ml in the subsequent lactation (11). Some authors are of a different opinion that the dry treatment did not result in reduced SCC in the subsequent lactation (25). Ewes with a history of high SCC during the previous lactation (i.e. more than 3 monthly tests > 400,000 cells/ ml) were more likely to have IMI in the post parturition period. Treatment with antibiotics requires veterinary supervision to ensure proper hygiene, because some mastitis outbreaks originated from contamination of the syringe (5). Dry therapy is generally associated with improved milking hygiene in the subsequent lactation, thus reducing IMI and improving milk quality. This practice and milking in parlor systems were associated with low TBC, and a significant reduction of BTSCC (8, 9, 10). More pulsations (180 cycles/min) and low vacuum levels (34 to 36 kPa) are optimal for udder health during milking of ewes (9). Conclusion BTSCC of small ruminants depends mainly on TBC, that cause infection of mammary glands. Factors that may reduce the risk of IMI include the ability to keep milking hygiene and adherence to correct milking parameters. Cheeses made from milk with a high BTSCC had a reduced fat content and increased pH and fat acidity. In yoghurt high BTSCC was associated with a higher protein content and the highest level of syneresis. Further research is needed to determine the exact thresholds of BTSCC in the milk of sheep and goats related to dairy products. References 1. Berthelot X, Lagriffoul G, Concordet D, Barillet F, Bergonier D. Physiological and pathological thresholds of somatic cell counts in ewe milk. Small Ruminant Res 2006; 62: 27-31. 2. Boulaaba A, Grabowski N, Klein G. Differential cell count of caprine milk by flow cytometry and microscopy. Small Ruminant Res 2011; 97: 117-23. 3. Chen SX, Wang JZ, Van Kessel JS, Ren FZ, Zeng SS. Effect of somatic cell count in goat milk on yield, sensory quality, and fatty acid profile of semisoft cheese. J Dairy Sci 2010; 93: 1345-54. 4. Contreras A, Miranda RE, Sánchez A, et al. Presence of Mycoplasma species and somatic cell counts in bulk-tank goat milk. Small Ruminant Res 2008; 75: 247-51. 5. Contreras A, Sierra D, Sánchez A, et al. Mastitis in small ruminants. Small Ruminant Res 2007; 68: 145-53. 6. Corrales JC, Sánchez A, Luengo C, Poveda JB, Contreras A. Effect of clinical contagious agalactia on the bulk tank milk somatic cell count in Murciano-Granadina goat herds. J Dairy Sci 2004; 87: 3165-71. 7. Droke EA, Paape MJ, Di Carlo AL. Prevalence of high somatic cell counts in bulk tank goat milk. J Dairy Sci 1993; 76: 1035-9. 8. Gonzalo C, Carriedo JA, Beneitez E, Juárez MT, De La Fuente LF, San Primitivo F. Bulk tank total bacterial count in dairy sheep: factors of variation and relationship with somatic cell count: short communication. J Dairy Sci 2006; 89: 549-52. 9. Gonzalo C, Carriedo JA, Blanco MA, et al. Factors of variation influencing bulk tank somatic cell count in dairy sheep. J Dairy Sci 2005; 88: 969-74. 10. Gonzalo C, Carriedo JA, Garcia-Jimeno MC, Pérez-Bilbao M, De La Fuente LF. Factors influencing variation of bulk milk antibiotic residue occurrence, somatic cell count, and total Somatic cell counts and total bacterial count In bulk tank milk of small ruminants 17 bacterial count in dairy sheep flocks. J Dairy Sci 2010; 93:1587-95. 11. Gonzalo C, Linage B, Carriedo JA, et al. Effect of dry therapy using an intramammary infusion on bulk tank cell count in sheep: short communication. J Dairy Sci 2009; 92:156-9. 12. Jakobsen RA, Heggeb0 R, Sunde EB, Skjervheim M. Staphylococcus aureus and Listeria monocytogenes in Norwegian raw milk cheese production. Food Microbiol 2011; 28: 492-6. 13. Koop G, Dik N, Nielen M, Lipman LJA. Repeatability of differential goat bulk milk culture and associations with somatic cell count, total bacterial count, and standard plate count: short communication. J Dairy Sci 2010; 93: 2569-73. 14. Koop G, Nielen M, van Werven T. Bulk milk somatic cell counts are related to bulk milk total bacterial counts and several herd-level risk factors in dairy goats. J Dairy Sci 2009; 92: 4355-64. 15. Leitner G, Merin U, Silanikove N. Effects of glandular bacterial infection and stage of lactation on milk clotting parameters: comparison among cows, goats and sheep. Int Dairy J 2011; 21: 279-85. 16. Marogna G, Rolesu S, Lollai S, Tola S, Leori G. Clinical findings in sheep farms affected by recurrent bacterial mastitis. Small Ruminant Res 2010; 88:119-25. 17. Millogo V, Sjaunja KS, Ouedraogo GA, Agenäs S. Raw milk hygiene at farms, processing units and local markets in Burkina Faso. Food Control 2010; 21: 1070-4. 18. Muehlherr JE, Zweifel C, Corti S, Blanco JE, Stephan R. Microbiological quality of raw goat's and ewe's bulk-tank milk in Switzerland. J Dairy Sci 2003; 86: 3849-56. 19. Najafi MN, Koocheki A, Valibaigy S. Effects of somatic cell counts on the physicochemical and rheological properties of yoghurt made from sheep's milk. Int J Food Sci Tech 2010; 45: 713-8. 20. Oliveira CJB, Hisrich ER, Moura JFP, Givisiez PEN, Costa RG, Gebreyes WA. On farm risk factors associated with goat milk quality in Northeast Brazil. Small Ruminant Res 2011; 98: 64-9. 21. Paape MJ, Wiggans GR, Bannerman DD, et al. Monitoring goat and sheep milk somatic cell counts. Small Ruminant. Res 2007; 68: 114-25. 22. Raynal-Ljutovac K, Pirisi A, de Crémoux R, Gonzalo C. Somatic cells of goat and sheep milk: analytical, sanitary, productive and technological aspects. Small Ruminant Res 2007; 68: 126-44. 23. Revilla I, Rodríguez-Nogales JM, Vivar-Quintana AM. Effects of somatic cells on the protein profile of hard ovine cheese produced from different breeds. J Dairy Res 2011; 78: 279-86. 24. Silanikove N, Leitner G, Merin U, Prosser CG. Recent advances in exploiting goat's milk: quality, safety and production aspects. Small Ruminant Res 2010; 89: 110-24. 25. Spanu C, Berger YM, Thomas DL, Ruegg PL. Impact of intramammary antimicrobial dry treatment and teat sanitation on somatic cell count and intramammary infection in dairy ewes. Small Ruminant Res 2011; 97:139-45. 26. Verdier-Metz I, Michel V, Delbés C, Montel M-Ch. Do milking practices influence the bacterial diversity of raw milk? Food Microbiol 2009; 26: 305-10. 27. Zweifel C, Muehlherr JE, Ring M, Stephan R. Influence of different factors in milk production on standard plate count of raw small ruminant's bulk-tank milk in Switzerland. Small Ruminant Res 2005; 58: 63-70. 18 J. Olechnowicz, J. M. Jaákowski ŠTEVILO SOMATSKIH CELIC IN SKUPNO ŠTEVILO BAKTERIJ V ZBIRALNICAH MLEKA DROBNICE J. Olechnowicz, J. M. Jaskowski Povzetek: Skupno število bakterij (TBC, angl. total bacterial count) v mleku je dober pokazatelj higienskih razmer pri reji drobnice. TBC je povezan s številom somatskih celic v zbiralnicah mleka (BMSCC, angl. number of somatic cells in bulk milk), če pa upoštevamo obe spremenljivki, TBC in BMSCC, dobimo še zanesljivše rezultate. Pomemben vpliv na BMSCC v zbiralnicah ovčjega mleka imajo naslednji dejavniki: čreda, mesec vzorčenja, uporaba suhe terapije, pasma ovc in način molže. Na povprečno vrednost celic na plošči (MSPC, angl. mean standard plate count) pa vplivajo: mesec vzorčenja mleka in število molž v zbiralnici kakor tudi način molže. V svežem ovčjem siru so bile kazeinske frakcije (as1-1-CN in beta-CNS) precej nižje, medtem ko je bila vrednost BMSCC visoka. Ti siri vsebujejo manj maščob in imajo zato višji pH in maščobno kislost. Jogurti, narejeni iz mleka z visoko vrednostjo BMSCC, imajo značilno višjo vsebnost beljakovin in visoko stopnjo sinereze. Potrebno je zagotoviti ustrezne higienske pogoje pri ravnanju z živalmi in določiti higienske standarde za molzne stroje in molzišča, da bi izboljšali zdravje vimen malih prežvekovalcev. Ključne besede: drobnica; število somatskih celic; skupno število bakterij; zbiralnice mleka Slov Vet Res 2012; 49 (1): 19 - 26 UDC 614.31:579.67:637.5'65:664.3 Original Scientific Article THE EFFECT OF THYME OIL ON THE SHELF LIFE OF CHICKEN BALLS DURING STORAGE PERIOD Ozlem Pelin Can University of Cumhuriyet, Faculty of Engineering, Department of Food Engineering, Sivas, Turkey Corresponding author, E-mail: opcan@cumhuriyet.edu.tr Summary: The effects of thyme essential oil on the shelf life of chicken balls during refrigerated storage for 12 days were examined. Treatments examined in the present study were as follows: A (control samples, untreated), B (final concentration of thyme essential oil; 0.4% w/w, added to ball), C (final concentration of thyme essential oil; 0.4% w/w, on surface of ball). The shelf-lives of samples were determined by means of microbiological, chemical, sensory, colour and texture analyses. Microbial populations in B group samples were determined to be reduced at the end of the storage period. Lactic acid bacteria (LAB) and En-terobacteriaceae were the most sensitive groups among the microorganisms examined. B group treatment samples resulted in lower microbiological counts in comparison to the control (A) and C group samples, but LAB was found to be lower in group C samples. Salmonella spp. and Staphylococcus spp. were not detected in all samples. TBA values for C group samples remained lower than 1 mg MDA/kg throughout the 12 day storage period. The pH values varied between 6.4 (day 0) and 5.9 (day 12). The C group samples were found to be desirable (organoleptically acceptable) following sensory analysis. Based primarily on sensory data (taste attribute) B and C groups samples extended the product's shelf-life by ca. 4 and 6 days, respectively, as compared to the control sample. The results of the analyses of chicken balls showed no significant differences (p>0.05) in colour and texture among all samples. Key words: thyme oil; chicken ball; microbiology; chemical and sensory quality; colour and texture analysis Introduction Poultry meat is very popular in the world and nowadays it is consumed increasingly in many countries. Chicken meat has a low cost of production, low fat content and high nutritional value. However, poultry products are pathogenic and spoilage microorganisms (Salmonella, Yersinia enterocolitica, such as) and remains a significant concern for consumers and public health officials worldwide. High consumption of poultry products leads to concerns in the industry about shelf-life extension. Escherichia coli and Staphylococcus au- Received:28 October 2011 Accepted for publication: 31 January 2012 reus have been used in poultry products to assess microbiological safety, sanitation conditions during processing and the retention of the quality of the product (1, 2). Natural food preservatives are used to ensure protection from both spoilage and pathogenic microorganisms (3). Recently, herb and spice extracts have been a particular focus. These extracts have been used to improve sensory characteristics and to extend the shelf-life of foods (4). There has been a great interest within the food industry during the last decade regarding the antimicrobial substances used in foods, such as thyme oil (5). Thyme (Thymus vulgaris L.) is used both as a spice and a condiment. The essential oil of thyme (Thymus vulgaris L.) has a significant rate of fungal and antibacterial activity with 20 O. Pelin Can strongly inhibited lipid peroxidation and high off radical scavenging (6, 7). This study was carried out to investigate the effects of thyme oil (on the surface of and as an additive to chicken balls) on the quality of chicken balls during storage. Material and methods Preparation of chicken balls Chickens were purchased from a local market. After washing, the skin and the apparent fat and bone tissues were removed. The meat was treated using a kitchen food processor with a pore size of 5 mm. The chicken ball included 85% chicken mince, 2% salt, 0.5% cumin, 0.5% red pepper, 10% onion and 2% garlic. The ingredients were homogenized with a kitchen blender. The spices were purchased from Bagdat Company (Istanbul, Turkey). The chicken meat batter was shaped into balls using stainless steel equipment (approximately 20 g). Preparation of experimental groups The following lots of samples were prepared: The first lot of samples comprised the controls (aerobic packaging, group A). Lot two consisted of samples with thyme oil (Sigma, Germany) 0.4% (B and C group). The B group which thyme oil %0.4 added batter chicken meatball (considering amount of batter meat ball). The preparation of C group, thyme oil was surface balls (final con-centr ations equal to 0.4% w/w). Thyme oil was added undiluted using a micropipette. The thyme oil was massaged onto the product, so as to get even distribution of the oil using gloved fingers (to avoid cross-contamination of samples and also transmission of food poisoning organisms). After three groups of chicken balls were produced, they were refrigerated at 4 ± 1 °C in straphor trays covered with aluminium foil for testing microbiological, chemical, sensory, colour measurement and texture analysis on 0, 3, 6, 9 and 12 days of the storage. Microbiological analysis Approximately 25 g of the chicken meat balls were sampled using sterile scalpels and forceps, immediately transferred into a sterile stomacher bag, containing 225 ml of 0.1% peptone water (pH 7.0), and homogenized for 60 s in a Lab Blender 400 Stomacher at room temperature. Microbiological analyses were conducted using standard microbiological methods (8). The amount of 0.1 ml of these serial dilutions of chicken homogenates was spread on the surface of dry media. Total viable counts (TVC) were determined using Plate Count Agar (PCA, Merck code 1.05463), after incubation for three days at 30 °C. Pseudomonads were determined on cetrimide fusidin cephalorid-ine agar (Oxoid code CM 559, supplemented with SR 103) after incubation at 25 °C for 2 days (9, 10). For members of the family Enterobacteriaceae, 1.0 ml sample was inoculated into 5 ml of molten (45 °C) Violet Red Bile Glucose Agar (Oxoid code CM 485). After setting, a 10 ml overlay of molten medium was added and incubation was carried out at 37 °C for 24 h. The large colonies with purple haloes were counted. Lactic acid bacteria (LAB) were determined on de Man Rogosa Sharpe Medium (Oxoid code CM 361) after incubation at 25 °C for 5 days. Yeasts and moulds were enumerated using Rose Bengal Chloroamphenicol Agar (RBC, Merck code 1.00467) after incubation at 25 °C for 5 days in the dark. Isolation of Salmonella spp. was carried out in four stages. After incubation at 35-37 °C for 16-20 h for the pre-enrichment step, 0.1 and 1 ml of the homogenate was transferred to RV (Rappaport Vassiliadis, Merck) and Selenite Cystein Broth (Merck) for selective enrichment with an incubation period of 42 and 35 °C for 24 h, respectively. After incubation, a loop-ful from each tube was streaked on Brillant Green Phenol Red Lactose Agar (Merck) and Bismuth Sulfite Agar (Merck). These plates were incubated for 20-24 h at 35 °C and checked for typical colonies. Five colonies were selected for biochemical tests and were grown in Nutrient Agar (Oxoid) at 35 °C for 18-24 h (11). Baird Parker Agar (Merck) was used for the estimation of Staphylococcus spp. counts. After incubation at 35 °C for 45-48 h, typical colonies were tested for the detection of coagulase production. (12). Duplicate plates were spread from each dilution of 0.1 ml. All plates were examined for typical colony types and morphology characteristics associated with each growth medium. Chemical analysis The pH value was recorded using a pH meter. Chicken samples were thoroughly homogenized The effect of thyme oil on the shelf life of chicken balls during storage period 21 with 10 ml of distilled water and the homogenate used for pH determination (13). Thiobarbituric acid (TBA) was determined according to the method proposed by Pearson (14). Sensory analysis The door and taste of cooked chicken balls were evaluated by a panel of seven judges, experienced in chicken ball evaluation on each day of sampling. Chicken ball samples were cooked individually in an oven, for 5 min and were then immediately presented to the panellists. The scale points were: excellent, 5; very good, 4; good, 3; acceptable, 2; poor (first off-odour, off-taste development), 1; a score of acceptability (15). Colour measurement Colorimetric measurements of cooked chicken balls were determined in triplicate using a Colorimeter (Minolta spectrophotometer CM 3500d, Japan). The colour reading includes lightness (L), redness (a) and yellowness (b). The equipment was standardized with a white colour Standard. Five replicate measurements were taken for each sample, following the guidelines for colour measurements from American Meat Science Association (16). Texture profile analysis (TPA) Texture measurement of cooked chicken balls were used a Texture Analyzer (TA.XTPlus Stable Micro Systems, UK). Texture Profile Analysis (TPA) was used to determine hardness, cohesiveness, chewiness and springiness (17). This test was carried out by using compression plate with a diameter of 75 mm. The mean of five measurements was taken for each hardness, cohesiveness, chewiness and springiness. Statistical analysis Analysis of the data was conducted using a Statistical Analysis System (SAS) packages software. Values between groups and within groups-between days were compared. Data were subjected to variance analysis in accordance with 3 x 5 x 3 x 2 factorial design and in terms of fix effects and inter-variable interactions so that "repetition number x sampling time x test groups x number of samples examined at one instance from each test group". According to the General Linear Models (GLM) procedure, the Fisher's smallest squares average (LSD) test was used. Standard deviation figures of all averages were calculated (18). The alpha value was determined as 0.05. Result Microbiological changes are given in Fig. 1. The thyme oil treated groups had a lower count TVC. The control samples reached a TVC count of upper 7 log cfu/g storage day. Listeria spp. and Salmonella spp was not detected in any of the groups. Whereas on the 3rd day of storage, the number of Pseudomonads in group B was 1 log cfu/g, no reduction was determined in the Pseudomonads values of the C group (P> 0.05). On day 9 of storage, group B was 3.11 log cfu/g (P<0.05), while groups C and A were determined as 5.11, 7.37 log cfu/g. Initial populations of LAB were ca. 3.42 and increased to reach counts of 7.13 log cfu/g on day 9 of storage, group A samples (P<0.05). However, significantly lower LAB counts (P < 0.05) were recorded for C samples during the entire storage period under refrigeration. The Enterobacteriaceae counts were determined by 7.56 log cfu/g, 2.13 log cfu/g and 2.59 log cfu/g group A, B and C on day 9, respectively. The yeast and mould counts were initially (0 day) determined as 2.78 log cfu/g all samples. In the A group of samples, the yeast and mould count was shown to have increased steadily during storage time (2.78, 4.26, 4.6 and 5.13 log cfu/g, 0, 3, 6 and 9 day, respectively). B and C group samples were found to contain a yeast and mould count of <10 log cfu/g, on the third and sixth days of storage. B group samples were not observed on day 9 while the yeast and mould counts were found to be 2.56 log cfu/g, at the end of storage. C group samples were found to be 2.19, 2.43 log cfu/g, on days 9 and 12, respectively. Changes of pH values are shown in Fig. 2a. Values of pH initial were recorded for groups A, B and C as 6.17, 6.21 and 6.19, respectively. The TBA values for all chicken ball treatments are given in Fig. 2b. The TBA values for the control group varied between 0.3 and 1.8 mg MDA/kg. A very low lipid oxidation value of between 0.3 and 0.9 mg MDA / kg was determined for the treatment group. 22 O. Pelin Can 4 6 8 10 12 Storage Time (Day) 4 6 8 10 12 Storage Time (Day g 2 02 4 6 8 10 Storage Time (Day) g 2 - 12 14 4 6 8 10 Storage Time (Day) 9 8 6 3 2 0 2 14 0 2 4 8 8 6 4 4 0 0 0 2 2 14 Figure 1: Changes (log cfu/g) in (a) Total Viable Counts (TVC); (b) Pseudomonas spp.; (c) LAB and (d) Enterobacte-riaceae, control (A), batter with added thyme EO 0.4% (B) and ball surface thyme EO 0.4% (C). Each point is the mean of three samples taken from two replicate experiments (n: 3x2: 6). Error bars show SD. » A^O^- B T C Storage Time (Day) Storage Time (Day) Figure 2: Changes of pH (a) and TBA (b) values of chicken balls, control (A), batter with added thyme EO 0.4% (B) and ball surface thyme EO 0.4% (C). Each point is the mean of three samples taken from two replicate experiments (n: 3 x 2: 6). Error bars show SD. * 0 T C The sensory properties (odour and taste) of the cooked chicken balls are given in Fig. 3a-b. Sensory analysis was carried out until the 6th day of storage for control group samples and until the 12th day of storage for treatment group samples. Due to the microbiological quality of the control group samples, exceeded limit values were not subject to sensorial evaluation. Taste attribute has usually been a more sensitive parameter than odour; therefore, taste attribute was used for the sensory evaluation of the thyme products and the determination of sensorial shelf-life in the present study. On day 0 of storage, the cooked chicken balls had a pleasant taste and odour. The presence of thyme oil (on ball surface) in the samples produced a very pleasant taste (P<0.05). The effect of thyme oil on the shelf life of chicken balls during storage period 23 Storage Time (Day) 5,5 -5,0 -4,5 -4,0 -3,5 -3,0 -2,5 - 0 2 4 6 8 10 12 14 Storage Time (Day) Figure 3: Changes in the taste (a) and odour (b) of chicken balls, control (A), batter with added thyme EO 0.4% (B) and ball surface thyme EO 0.4% (C). Each point is the mean of three samples taken from two replicate experiments (n: 3 x 2: 6). Error bars show SD. —*— B T C Table 1 shows the colour measurement results of chicken balls. All chicken balls varied insignificant in their L, a and b values (p>0.05). Table 1: Colour properties of experimental chicken balls Group Storage time (day) Analysis 0 3 6 9 12 A L* 75.42±0.46 az 75.42±0.33 az 75.33±0.56 az 75.56±0.41 az 75.33±0.39 az a* 0.90±0.09 az 0.90±0.06 az 0.91±0.4 az 0.93±0.21 az 0.90±0.36 az b* 16.77±0.25 az 16.77±0.21 az 16.81±0.56 az 16.85±0.43 az 16.77±0.21 az B L* 75.33±0.19 az 76.16±0.26 az 76.01±0.31 az 75.19±0.18 az 75.96±0.16 az a* 0.91±0.04 az 0.90±0.02 az 0.96±0.05 az 0.91±0.04 az 0.93±0.04 az b* 17.13±0.22 az 17.09±0.19 az 17.33±0.24 az 17.13±0.19 az 17.21±0.21 az C L* 76.01±0.56 az 76.81±0.54 az 76.16±0.42 az 76.21±0.36 az 76.33±0.29 az a* 0.93±0.01 az 0.91±0.06 az 0.94±0.02 az 0.90±0.01 az 0.92±0.03 az b* 16.36±0.12 az 16.22±0.21 az 16.43±0.23 az 16.21±0.19 az 16.23±0.21 az Texture analysis results for chicken balls are shown as in Table 2. Results regarding hardness, co-hesiveness, springiness and chewiness value during storage time were determined to be similar for all groups, therefore, the mean values have been given. Table 2: Textural properties of experimental chicken balls. Group Hardness (kg) Cohesiveness(mm/mm) Springiness (mm/mm) Chewiness (kg/mm) M ± SD A 3.73±0.22 0.67±0.01 12.56±0.42 31.33±1.56 B 3.96±0.19 0.65±0.04 12.93±0.46 35.21±1.41 C 3.52±0.28 0.69±0.02 12.87±0.52 32.56±1.82 24 O. Pelin Can Discussion The changes in total viable count (TVC) of the chicken meat balls are given Fig. 1a. The initial value of TVC (day 0) for fresh chicken meat was ca. 4.3 log cfu/g, indicative of good quality chicken meat (19). TVC reached a value of 7 log cfu/g, considered as the upper microbiological limit for good quality fresh poultry meat, as defined by the ICMSF (2), on day 6 for the control samples. Samples treated with thyme oil in groups never reached the limit of 7 log cfu/g during the 12 day storage period. The group B samples were determined to be 4.83 log cfu/g 0 day. This value decreased about 2 log cfu/g on day 3 of storage and reached 5.21 log cfu/g at the end of storage. There was no significant change to the C groups sample during storage. Thyme oil in the additive ball was more effective than in the surface ball on reducing TVC. Thyme oil resulted in a shelf-life extension of six days as compared to the control samples. In related studies, Giatrakou (7) mentioned a shelf-life extension of 2 days after the application of thyme oil 0.2% v/w on chicken, while Ergezer (20) reported that TVC in chicken balls was reduced with whey powder stored at -18 °C for three months. Listeria spp. and Salmonella spp were found in one of the samples. They are also in agreement with those reported by Yava§ et al. (21), who reported no determination in ground chicken balls after 21 days of storage under modified atmosphere pressure (MAP). Pseudomonads (Fig. 1b) are Gram-negative bacteria, comprising the main spoilage microorganisms in meat (22). In other studies, the combined use of thyme oil on chicken products stored aerobically at 4 °C, resulted in about 8.0 log cfu/g of the final population of pseudomonas (6). Deans and Richie (23) showed that thyme oil was very effective against Pseudomonas aeruginosa, in a study where the inhibitory properties of ten plant essential oils were tested using an agar diffusion technique. This is in agreement with Elgayyar et al. (24) who reported that oregano essential oil was less effective in inhibiting P. aeruginosa compared to other microorganisms. Lactic acid bacteria (LAB) (Fig. 1c) facultative anaerobic species were found to be members of the microbial flora of the chicken product. Of all the antimicrobial treatments in this study, thyme oil on surface of ball treatment proved to be the most effective in inhibiting the growth of LAB, almost stable during the storage period. For Geor-gantelis et al. (25), the combined use of rosemary essential oil and chitosan on fresh pork sausages stored aerobically at 4 °C, resulted in a 2.0 log cfu/g reduction of the final population of LAB. In another study, LAB count reduced by 0.2% in the chicken balls with thyme oil during storage (6). With respect to Enterobacteriaceae (Fig. 1d), considered as a hygiene indicator (26), the initial counts of 2.33 log cfu/g were indicative of good quality chicken meat. Enterobacteriaceae grew under essential oil applications at a slower rate than others. This is in agreement with the results of Choularaia et al. (27), who reported that oregano oil had a strong effect in the reduction of Entero-bacteriaceae counts. On day 9 of storage, the use of thyme oil had practically no effect on Enterobacteriaceae counts (p> 0.05). On the same day, the Enterobacteriaceae counts were determined by 7.56 log cfu/g, 2.13 log cfu/g and 2.59 log cfu/g in groups A, B and C, respectively. Giatrokau et al. (7) reported that Enterobacteriaceae of chicken products were inhibited in the presence of thyme oil packaging. As a result, the initial population of yeasts and moulds were very low (2.78 log cfu/g) and no population was determined on day 3 and 6 of storage for B and C group samples. The balls with added thyme oil were a lot more effective in reducing yeast populations in comparison to surface added balls (P<0.05). Yeast and mould were not determined in B and C group samples on day 9 of storage, whereas 5.3 log cfu/g was determined for the A group samples on the same day. Conner and Beuchat (28) reported a strong inhibitory action of oregano and thyme oils on the growth of yeast. The above results are in agreement with those of Giatrakou et al. (7), who reported that thyme oil 0.2% significantly lower yeasts-moulds counts than the control samples during the storage. The microbiological analysis was performed on B and C groups for day 12, while group A was done until day 9. Kayi§oglu et al. (29) reported a pH value of 6.1 for raw chicken doner kebab, this situation is similar to our finding. The addition of thyme oil resulted in a slight increase in pH values. The group A samples increased pH value during storage (P<0.05). Georgantelis (25) reported higher pH values for Greek-style sausages, containing chi-tosan and rosemary extract. The effect of thyme oil on the shelf life of chicken balls during storage period 25 Choluaria et al. (27) who reported TBA values of 0.1-0.9 mg MDA / kg meat for chicken product after 25 days of storage. Thyme oil significantly affects the degree of lipid oxidation under present experimental conditions, given the anti-oxidant properties of this essential oil. Kim et al.(5) reported TBA values of 0.13-0.68 mg MDA/ kg meat for turkey and pork after seven days of storage, and these figures are similar to our findings. The thyme oil application of samples showed slightly higher values of TBA. Botsoglou et al. (4), who reported a threefold reduction in the degree of lipid oxidation (0.6-0.2) in turkey meat treated 200 mg/kg oregano oil, this is in contrast our results. According to Choluaria et al.(27) the addition of thyme oil to chicken products results in a more acceptable odour and flavour as compared to the untreated samples. Colour parameters (L*, a*, and b*) and TPA values in samples containing the thyme oil were not affected during storage. Changes in colour parameters and TPA values were statistically insignificant (P>0.05) in all samples. As a result, it was determined in this study that thymol prolongs the shelf life of chicken meat balls. The effect of essential oil is already known. However, thyme oil added to chicken balls is as an effective method as applying it to the surface. Moreover, the taste of chicken balls with added thyme oil on the surface was probably better. This method may be preferred because it is safe, practical and a positive sensory feature. This method may be used to protect other foods as well. References 1. Bean NH, Griffin M. Foodborne disease outbreaks in the United States, 1973-1987: pathogens, vehicles, and trends. J Food Prot 1990; 53: 804-16. 2. ICMSF. International Commission on Microbiological Specifications for Foods. Sampling for microbiological analysis: principles and scientific applications. University of Toronto Press, 1986. 3. Chouliara E, Kontominas MG. Combined effect of thyme essential oil and modified atmosphere packaging to extend shelf life of fresh chicken meat. In: Govil JN, Singh VK, Almad K, Sharma RK, eds. Recent progress in medicinal plants: natural product I. Vol. 15. Houston : Studium Press LLC, USA2006: 423-42. 4. Botsoglou NA, Grigoropoulou SM, Botsoglou E, Govaris A, Papageorgiou G. The effects of dietary oregano essential oil and a-tocopheryl acetate on lipid oxidation in raw and cooked turkey during refrigerated storage. Meat Sci 2003; 65: 1193-200. 5. Kim J, Marshall MR, Wei CI. Antibacterial activity of some essential oil components against five foodborne pathogens. J Agr Food Chem 1995; 43: 2839-2845. 6. FSIS USDA. Nationwide raw ground chicken microbiological survey. Washington DC : US Department of Agriculture, Food Safety and Inspection Service, Science and Technology, Microbiology Division, 1996: 8 str. 7. Giatrakou V, Kykkidou S, Papavergou A, Kontominas MG, Savvaidis IN. Potential of oregano essential oil and MAP to extend the shelf-life of fresh swordfish: a comparative study with ice storage. J Food Sci 2008; 73: M167-73. 8. Harrigan WF. Laboratory methods in food microbiology. 3rd ed. London : Academic Press, 1998. 9. Speck ML, ed. Compendium of methods for the microbiological examination of foods. 2nd ed. Washington DC: American Public Health Press, 1984. 10. Mead GC, Adams BW. Selective medium for rapid isolation of Pseudomonas associated with poultry meat spoilage. Br Poultry Sci 1977; 18: 661-70. 11. Bilgehan H. Identifying methods in clinical microbiology. Bari§ Publications,1995. 12. Halkman AK. Merck Gida Mikrobiyolojisi Uygulamalari. Ed. Ba§ak Matbaacilik, 2005. 13. AOAC: Official methods of analysis of association of official chemists. 15th ed. Arlington : AOAC Inc, 1990. 14. Kirk RS, Sawyer R. Pearson's composition and analysis of foods. 9th ed. Harlow, UK : Longman Scientific & Technical, 1991. 15. Kurtcan Ü, Gönül M. Gidalarin duyusal degerlendirilmesinde puanlama metodu. Ege Univ Müh Fak Dergisi 1987; 5: 137-46. 16. Hunt MC, Kropf DH. Color and appearance. New York : Van Nostrand, 1987: 13-8. 17. Bourne MC. Texture profile analysis. Food Technol 1978; 33: 62-6. 18. SAS Institute. Statistical Analysis System (Version 6.1). Cary, North Carolina, USD : SAS Institute, 1996. 19. Dawson PL, Hon H, Vollet LM, Clardy LB, Martinez RM, Acton JC. Film oxygen transmission 26 O. Pelin Can rate effects on ground chicken meat quality. Poultry Sci 1995; 14: 1381-7. 20. Ergezer H, Akgun AA, Gok^e R. Vakum ambalajda donmu§ olarak depolanan kaplamali tavuk koftelerinde depolama suresince olu§an kimyasal, mikrobiyolojik ve duyusal degi§iklikler. Turkiye 10. Gida Kongresi. Erzurum, 2008. 21. Yava§ E, Bilgin B, Yilmaz i. Pili? etinden uretilen kaplamali urunlerde raf omru boyunca meydana gelen degi§imler. Turkiye 10. Gida Kongresi. Erzurum, 2008. 22. Jay JM. Microbial spoilage indicators and metabolites. In: Pierson MD, Sterm NJ, eds. Food-borne microorganisms and their toxins: developing methodology. Marcell Dekker : Basel Press, 1986:219-40. 23. Deans SG, Richie G. Antimicrobial properties of plant essential oils. J Food Microbiol 1987; 5: 165-180. 24. Elgayyar M, Draughon FA, Golden DA, Mount JR. Antimicrobial activity of essential oils from plants against selected pathogenic and saprophytic microorganisms. J Food Prot 2000; 64: 1019-24. 25. Georgantelis D, Ambrosiadis I, Katikou P, Blekas G, Georgakis SA. Effect of rosemary extract, chitosan and a-tocopherol on microbiological parameters and lipid oxidation of fresh pork sausages stored at 4 °C. Meat Sci 2008; 76: 172-81. 26. Zeitoun AAM, Debevere JM, Mossel DAA. Significance of Enterobacteriaceae as index organisms for hygiene on fresh untreated poultry, poultry treated with lactic acid and poultry stored in modified atmosphere. Food Microbiol 1994; 11: 169-76. 27. Chouliara I, Savvaidis I, Riganakos K, Kontominas MG. Shelf-life extension of vacuum-packaged sea bream (Sparus aurata) fillets by combined g-irradiation and refrigeration: microbiological, chemical and sensory changes. J Sci Food Agric 2005; 85: 779-84. 28. Conner DE, Beuchat LR. Effects of essential oils from plants on growth of food spoilage yeasts. J Food Sci 1984; 49: 429-34. 29. Kayi§oglu S, Yilmaz I, Demirci M, Yetim H. Chemical composition and microbiological quality of the doner kebabs sold in Terkidag market. Food Control 2003; 14: 469-74. VPLIV TIMIJANOVEGA OLJA NA ROK UPORABNOSTI PIŠČANČJIH KROGLIC V OBDOBJU SKLADIŠČENJA O. Pelin Can Povzetek: Ugotavljali smo učinke timijanovega eteričnega olja na rok uporabnosti piščančjih kroglic v 12-dnevnem obdobju skladiščenja. Uporabili smo naslednje vzorce: A (kontrolni vzorci, netretirani), B (0.4% koncentracija eteričnega olja timijana dodana kroglici), C (0.4% koncentracija eteričnega olja timijana, nanesena na površino kroglice). Obstojnost vzorcev smo ugotavljali s pomočjo mikrobioloških in kemičnih analiz ter z zaznavo barve in teksture. V skupini B se je mikrobna populacija na koncu obdobja skladiščenja zmanjšala. Med opazovanimi mikroorganizmi so mlečnokislinske bakterije (LAB, angl. lactic acid bacteria) in enterobakterije najbolj občutljive. V skupini B je bilo ugotovljenih manj mikroorganizmov kot v kontrolni skupini (A) in skupini C, vendar je bilo ugotovljeno, da je vrednost LAB nižja v skupini vzorcev C. Salmonella spp. in Staphylococcus spp. nista bili odkriti v nobenem vzorcu. Skupno število bakterij vzorcev C je ostalo pod 1 mg MDA / kg v celotnem 12 dnevnem obdobju shranjevanja. pH vrednosti so se gibale med 6.4 (dan 0) in 5.9 (dan 12). Po izvedeni senzorični analizi je bilo ugotovljeno, da je skupina vzorcev C organoleptično sprejemljiva. Na osnovi senzorične analize (okus) je bilo ugotovljeno, da se je vzorcem skupin B in C podaljšal rok uporabnosti za 4 oz. 6 dni v primerjavi s kontrolnimi vzorci. Analiza piščančjih kroglic vseh vzorcev ni pokazala značilnih razlik (p> 0,05) v barvi in teksturi. Ključne besede: timijanovo olje; piščančje kroglice; mikrobiologija; kemična in senzorična kakovost; analiza barve in teksture Slov Vet Res 2012; 49 (1): 27 - 34 UDC 577.175.6:57.083.33:591.478:599.323.41 Original Scientific Article DETERMINATION OF SEx HORMONES IN RAT HAIR AFTER ADMINISTRATION OF TESTOSTERONE PROPIONATE AND ESTRADIOL VALERATE Tomaž Snoj1*, Nina Čebulj-Kadunc1, Alenka Nemec Svete2, Silvestra Kobal1 institute of Physiology, Pharmacology and Toxicology, 2Clinic for Surgery and Small Animal Medicine, Veterinary Faculty, Gerblčeva 60, 1000 Ljubljana, Slovenia Corresponding author, E-mail: tomaz.snoj@vf.uni-lj.si Summary: 1713-testosterone (testosterone) and 1713-estradiol (estradiol), together with their synthetic analogues, have been banned in the EU as growth promoters for fattening purposes. A simple, reliable method for their detection in animals is therefore required. Levels of testosterone and estradiol in hair and plasma of Wistar rats have been determined, following intramuscular (i.m.) administration of testosterone propionate and estradiol valerate, using commercial ELISA kits. Experiments were performed on prepubertal female rats for the detection of hair testosterone and on prepubertal male rats for the detection of hair estradiol. A significant increase of testosterone in hair was observed 3, 6 and 9 days after the administration of testosterone propionate, with no significant change in plasma content. Estradiol valerate administration also resulted in a significantly increased concentration in hair on the 3rd and 9th days, and in plasma on the 9th day. Thus, detection of testosterone and estradiol in hair after i.m. testosterone propionate and estradiol valerate administration can be achieved using commercial ELISA kits. Up to 0.80 % of the administered testosterone and 0.06 % of the administered estradiol were present in hair. This method could therefore serve as the basis for establishing a pre-screening tool for detecting abuse of anabolic steroids in farm animals. Key words: testosterone; estradiol; hair; plasma; rats; ELISA Introduction 17p-testosterone (testosterone) is the principal endogenous androgen synthesized by Leydig cells in the testes. Small amounts are also produced by the adrenal cortex. 17p-estradiol (estradiol) is produced mostly in the ovary and placenta. Sex steroids are transported in the blood from the steroidogenic tissues to target cells by blood where they are bound to sex hormone-binding globulin. They diffuse readily across the cell membrane and bind to intracellular receptors in the cytoplasm. Their activity is achieved through interaction of Received:24 November 2011 Accepted for publication: 28 February 2012 the receptor-hormone complexes to the hormone response elements on DNA that act as transcription factors, activating or inhibiting gene expression (1). Testosterone has androgenic and anabolic activities. It regulates the male reproductive system by binding to androgen receptors present in particular in the reproductive organs, as well as interacting with receptors in muscles and fat tissue. Testosterone is also converted to estradiol and the more potent androgen dihydrotestoster-one. In veterinary medicine testosterone and its synthetic analogues have been used for treating aplastic anaemia in dogs and cats, myeloprolifera-tive disease and enhancing athletic performance in horses (1, 2). 28 T. Snoj, N. Čebulj-Kadunc, A. Nemec Svete, S. Kobal Estradiol plays an important role in the estrous cycle and in late pregnancy. Therapeutically it is used to prevent uterine infections and accelerate uterine involution in postpartum cows, for es-trus synchronization in heifers and for induction of parturition in mares (1). Treatment of perianal gland adenoma in dogs with estradiol has also been described (3). Although testosterone and estradiol, as well as their synthetic analogues, have been banned in the EU as growth promoters for fattening purposes (4, 5) they are still in use in many countries. According to The 2011 Prohibited List' of the World Anti-Doping Code (6), anabolic androgenic steroids belong to class S1.1 and are prohibited in and out of competition, including, notably, athletes and racing horses worldwide. For fattening and doping purposes steroids are usually administered as synthetic steroid esters. In the organism they are rapidly hydrolysed into natural steroids and free fatty acids. Therefore, exogenous steroid esters are difficult to detect in body fluids. Following oral intake of testosterone undecanoate, the unmetabolized ester could be detected in plasma of athletes only for 6 hours (7). Sex steroids of endogenous and exogenous origin are inactivated in the liver and excreted mostly in the bile and urine. Estimation of reproductive status in various species by measuring faecal an-drogens, estrogens and progestins has been described (8, 9, 10, 11). Besides their presence in urine and faeces, steroids are also present in saliva, milk, different tissues, and hair (12, 13, 14, 15). Steroids and steroid esters can be incorporated into the hair fibre from blood via the hair follicles (15, 16, 17) and via sweat and sebum excretion at the surface of the skin, followed by diffusion into the hair fibres (15, 18). Some individual differences in steroid metabolism rates also occur (18). Hair steroids can be detected and quantified by gas or liquid chromatography coupled to mass spectrometry (17, 18, 19, 20, 21), desorption elec-trospray ionization mass spectrometry (22) and enzyme-immunoassay (23). Although commercial ELISA kits are useful for determining steroids in various matrices, their detection in hair by this means has not been described. The aim of the present study was therefore to explore whether commercial ELISA kits can be used for detecting testosterone and estradiol in hair. The study was performed by measuring testosterone and estra-diol concentrations in rat hair after intramuscu- lar administration of testosterone propionate and estradiol valerate. Testosterone and estradiol concentrations were also determined in plasma. The differences in hair steroid content between treated and non-treated rats could serve as a base for establishing the use of ELISA for detecting steroids in farm animals, where abuse of anabolic steroids can occur. To avoid the influence of endogenous testosterone and estradiol, experiments were performed on prepubertal female rats for the detection of hair testosterone and on prepubertal male rats for the detection of hair estradiol. The cross-reactivity of testosterone propionate and estradiol valerate with commercial ELISA kits was also checked. Materials and methods Experimental design for testosterone detection in hair 15 female Wistar rats aged 46-48 days at the beginning of the experiment were used in the study. The animals were divided into a control group of 7 and an experimental group of 8 animals. The environmental conditions were: 21°C, 12h light-dark regime, standard rat feed and water ad libitum. Seven days after housing, the animals from the experimental group were treated i.m. with 20 mg of testosterone propionate (T1875, Sigma - Aldrich, St.Louis, USA) per kilogram of body weight. Testosterone propionate injection solution was prepared by dissolving testosterone propion-ate in sterile olive oil (20 mg/mL) one day before treatment. Rats from the control group were treated with placebo (0.2 mL of olive oil i.m.). Experimental design for estradiol detection in hair 15 male Wistar rats aged 47-48 days at the beginning of the experiment were used in the study. The animals were divided into a control group of 7 and an experimental group of 8 animals. The environmental conditions were the same as in the testosterone experiment. Seven days after housing, the animals from the experimental group were treated i.m. with 10 mg of estradiol valerate (E 1631, Sigma - Aldrich, St.Louis, USA) per kilogram of body weight. Solutions were prepared by dissolving estradiol valerate in sterile olive oil (10 mg/mL) one day before treatment. Rats from the Determination of sex hormones in rat hair after administration of testosterone propionate and estradiol valerate 29 control group were treated with placebo (0.2 mL of olive oil i.m.). The study protocol was approved by the Ministry of Agriculture, Forestry and Food, Veterinary Administration of the Republic of Slovenia; license number 34401-11/2011/4. Sampling regime Hair samples were collected by cutting with electric scissors from the rat's back. They were collected before treatment and on the 3rd, 6th and 9th day after treatment and stored at -20 0C until extraction. Blood samples were collected from the ophthalmic plexus using heparinized glass micro-haema-tocrit tubes. Plasma was prepared by centrifuga-tion at 2.500 g for 30 minutes at room temperature and stored at -20 0C until analysis. Blood samples were collected before and on the 9th day after the treatment. At the time of blood sampling the rats were anaesthetized with carbon dioxide. Steroid extraction from hair Approximately 0.15 g of hair was put in a mortar and 20 mL of liquid nitrogen was added to freeze the hair. The sample was pulverized by grinding for two minutes. 0.1 g of hair powder was weighed and placed into a plastic tube. 0.6 mL absolute methanol (Cat. No.1.06007., Merck, New Jersey, USA) and 0.5 mL bi-distilled water were added (24). The tube contents were shaken at 500 RPM for 30 minutes, centrifuged at 2500 g for 20 minutes at 4 0C and the supernatant decanted. The extract (supernatant) was stored in plastic tubes at -20 0C until analysis. ELISA procedure Testosterone and estradiol concentrations in intact plasma and in hair extracts were determined using commercial Testosterone ELISA and 17beta - estradiol ELISA kits (both IBL, Hamburg, Germany) following the instruction manuals. Ab-sorbances were measured with a microtiter plate photometer Multiskan FC (Thermo Fisher Scientific, Waltham, USA). The concentrations of testosterone and estradiol in hair extracts were expressed as ng per g of hair. Partial validation of the ELISA kits, which included determination of intra-assay and inter-as- say coefficients of variation (CV) for hair testosterone and estradiol, was performed. Samples with low (control group) and high (experimental group) concentrations of testosterone and estradiol in hair extracts were run in triplicate in each ELISA test and repeated in the next ELISA tests. Intra-assay CVs for hair testosterone were determined as 4.7 % and 4.3 %. Inter-assay CVs for testosterone were 8.2 % and 7.7 %. Intra- and inter-assay CVs for hair estradiol were 5.1 % and 9.3 % (intra-assay CVs) and 6.7 % and 11.4 % (inter-assay CVs). Commercial testosterone and estradiol ELISA kits were also tested for cross-reactivity with testosterone propionate and estradiol valerate. 100 ng of testosterone propionate (T1875, Sigma - Aldrich, St.Louis, USA) or estradiol valerate (E 1631, Sigma - Aldrich, St.Louis, USA) were dissolved in 1 ml of absolute methanol (Cat. No. 1.06007., Merck, New Jersey, USA). Serial dilutions with phosphate buffer were used for preparing 10 ng/mL and 1 ng/mL of testosterone propionate and estradiol valerate solutions respectively, which were used for testing the commercial testosterone ELISA kit for cross-reactivity with testosterone propionate and the commercial estradiol ELISA kit for cross-reactivity with estradiol valerate. Statistical analysis Data were analysed using SPSS 17.0 (Chicago, USA) commercial software. Means and standard errors of the mean (mean ± SE) were calculated for plasma and hair testosterone and estradiol concentrations. Repeated measures ANOVA with Bonfer-roni correction was used for testing for statistically significant differences of testosterone and estradiol concentrations in hair between different sampling times in the control and experimental groups of animals. At each sampling time, hair testosterone and estradiol concentrations between control and experimental group were compared by independent t-test, using Bonferroni correction for multiple tests. In the control and experimental groups, paired t-test was used to compare plasma testosterone and estradiol concentrations before and after treatment. Independent t-test was used to compare plasma estradiol concentrations in the control and experimental groups before and after treatment. Pearson's correlation coefficient analysis was performed to determine if there is a statistically significant correlation between plasma and hair testosterone and estradiol levels. A value of P < 0.05 was considered significant. 30 T. Snoj, N. Čebulj-Kadunc, A. Nemec Svete, S. Kobal Results Testosterone concentrations in hair and plasma Hair testosterone concentrations in the control and experimental group are presented in Figure 1. Before treatment the mean hair testosterone concentration (± SE) did not differ significantly between the control and experimental groups, 7.11 ± 1.09 and 10.17 ± 2.82 ng/g, respectively. In the control group testosterone concentrations remained unchanged throughout the experiment, while in the experimental group, mean hair testosterone concentration increased, reaching the highest value (182.47 ± 32.92 ng/g) three days after treatment. On the 6th day of the experiment, mean testosterone concentration (150.01 ± 23.78 ng/g) was lower than that on day 3, although the difference was not significant. At the end of experiment (on the 9th day) , the mean testosterone concentration (76.76 ± 14.33 ng/g) was significantly lower than on days 3 and 6 (P < 0.05); however, the value was significantly higher (P < 0.05) than that before treatment (10.17 ± 2.82 ng/g). The differences in hair testosterone concentrations between the control and experimental groups were statistically significant (P < 0.01) on days 3, 6 and 9 of the experiment. 0 3 6 9 (lay after treatment Figure 1: Testosterone concentrations in hair of the control and experimental groups *P<0.05 (comparison of control and experimental groups) HP<0.05 (comparison with value before treatment) Mean plasma testosterone concentrations (Table 1) before and on the 9th day after testosterone propionate or placebo administration were low, with no significant differences between and within the control and experimental group. Table 1: Plasma testosterone concentrations (mean ± SE) in the control and experimental groups plasma testosterone (ng/mL) before treatment after treatment control group (N = 7) 0.77 ± 0.18 0.48 ± 0.09 experimental group (N = 8) 0.65 ± 0.26 0.56 ± 0.14 Pearson's correlation coefficients between hair and plasma testosterone were r = 0.10 (control group) and r = 0.24 (experimental group) and were not found significant. Determination of cross-reactivity of the testosterone ELISA kit to testosterone propionate showed that measurements of testosterone propionate solutions in concentrations of 10 and 1 ng/mL had not achieved the detection limit (0.1 ng/mL). Estradiol concentrations in hair and plasma Hair estradiol concentrations in the control and experimental groups are presented in Figure 2. Before estradiol valerate or placebo administration, mean hair estradiol concentrations did not differ significantly between the control and experimental groups (0.40 ± 0.05 and 0.49 ± 0.09 ng/g, respectively). In the control group, estradiol concentrations remained unchanged throughout the experiment. In the experimental group, mean hair estradiol concentration increased significantly (P < 0.05) from 0.49 ± 0.09 ng/g to 2.75 ± 0.53 ng/g on the 3rd day after estradiol valerate administration. The peak value (6.07 ± 1.53 ng/g) was observed on the 6th day after treatment. On the 9th day after treatment there was a drop of mean hair estradiol (4.46 ± 0.75 ng/g). In the experimental group, no significant differences in estradiol concentrations between those measured at days 3, 6 and 9 were observed. Hair estradiol concentrations in the treated animals were significantly higher (P < 0.01) than on the control group on days 3, 6 and 9 after treatment. Determination of sex hormones in rat hair after administration of testosterone propionate and estradiol valerate 31 H control group (N = 7) □ experimental group (N = 8) 16" ^ 14- *ôî f 12" -E = 10-He W *n T *n T day after treatment Figure 2: Estradiol concentrations in hair of the control and experimental groups * P < 0.05 (comparison of control and experimental group) a P < 0.05 (comparison with value before treatment) P P = 0.055 (comparison with value before treatment) In plasma, before treatment, there were no significant differences in estradiol concentrations (Table 2) between the two groups. Nine days after estradiol valerate administration, mean plasma estradiol concentration was significantly higher (P < 0.01) than the mean value before treatment, as well as than the control group value (P < 0.01). Table 2: Plasma estradiol concentrations (mean ± SE) in the control and experimental groups plasma estradiol (ng/mL) before treatment after treatment control group (N = 7) 0.053 ± 0.018 0.062 ± 0.022 experimental group (N = 8) 0.091 ± 0.023 1.132 ± 0.133*n * P < 0.01 (comparison of control and experimental group) a P < 0.01 (comparison with value before treatment) Pearson's correlation coefficient between hair and plasma estradiol in control group (r = 0.20) was not found significant. In contrast, a significant positive correlation (r = 0.56; P < 0.05) was found between hair and plasma estradiol in the experimental group. Determination of cross-reactivity of the estradiol ELISA kit to estradiol valerate showed that measurement of estradiol valerate solutions in concentrations of 10 and 1 ng/mL had not reached the detection limit (0.025 ng/mL). Discussion Commercial ELISA kits are used for routine and research purposes in endocrinology; they are accurate and sensitive, relatively cheap and easy to handle. Although mass spectrometry is considered to offer more selectivity than ELISA, mainly due to variable extents of cross-reactivity of steroids against the antibody (21), our results suggest that ELISA could serve as a pre-screening or alternative method for detecting testosterone and estradiol in hair. Our results have demonstrated for the first time that testosterone and estradiol can be detected in hair extracts after intramuscular administration of testosterone propionate and estradiol valerate using commercial ELISA kits. In Wistar rats treated with testosterone propionate or estradiol valerate, testosterone and estradiol concentrations in hair were significantly higher than in control groups from the 3rd to the 9th day after treatment. The results indicate that testosterone propionate and estradiol valerate are hydrolysed (7) in the rat's body and that, during distribution and excretion, some amounts of free testosterone and estradiol are incorporated into rat hair. In animals treated with testosterone propion-ate, the content of testosterone in hair was, from the 3rd to the 9th day after treatment, significantly higher than in the control group. High testosterone values could result from hydrolysis of testosterone propionate and excretion of free testosterone onto the hair (15, 16, 18). Higher testosterone hair level on the 3rd day than on days 6 and 9 after testosterone propionate administration indicates intensive testosterone excretion via sweat, sebum and saliva immediately after administration. The slight decrease in testosterone concentrations on the 6th and 9th days after treatment could be caused by less intensive excretion onto the hair surface and eventual incorporation of previously excreted testosterone into hair fibre (15, 18). Blood plasma testosterone concentrations before treatment and on the 9 th day of the experiment were low, with no significant differences between control and experimental groups. These results indicate that, by the 9th day of the experiment, all 32 T. Snoj, N. Čebulj-Kadunc, A. Nemec Svete, S. Kobal administered testosterone propionate has been hydrolyzed and all free testosterone excreted from the blood, which could also explain the gradual decrease of testosterone levels in hair from the 3rd to the 9th day after treatment. Non-significant correlation between hair and plasma testosterone might also be caused by the same reasons. Similarly to testosterone, estradiol concentrations in hair increased but reached their highest mean value on the 6 th day after administration although the value was not significantly higher (P = 0.055) than that before treatment. Some individual differences in metabolism and excretion rate of steroids are possible (18), which could be the reason for the wide dispersion of the hair estradiol measurements on the 6th day following treatment and consequent high standard deviation that resulted in a P value close to statistical significance. In contrast to plasma testosterone concentrations, plasma estradiol increased significantly after estradiol valerate administration, resulting in a significant difference in estradiol concentration between the control and experimental groups. This suggests slower hydrolysis of estradiol valerate and prolonged persistence of estradiol in blood than of testosterone in the corresponding experiment. Estradiol is thus excreted onto the hair fibre over a longer period than that for testosterone, which could be the reason for the highest concentration being reached not earlier than on the 6th day after estradiol valerate administration. Additionally, the significant positive correlation between hair and plasma estradiol suggests that increased hair estradiol levels are the consequence of high plasma estradiol concentration. The transport of steroids into hair is mediated via the hair follicle or by diffusion into the hair fibre from sweat, sebum and saliva (16, 17, 18, 25). Significantly higher hair testosterone and estradiol values on the 3rd, 6th and 9th days than in control groups confirmed that it is possible to detect prior use of testosterone and estradiol esters by measuring testosterone or estradiol concentrations in hair. According to our results of cross-reactivity testing, testosterone propionate and estradiol valerate cannot be detected by the commercial testosterone and estradiol ELISA kits used in the present study, which indicates the reliability of the chosen method, since possible contamination of the hair during administration is not detectable. Additionally, the low intra- and inter-assay CVs in testosterone and estradiol measurements, the significant correlation between hair and plasma estradiol, and the fact that the concentrations of testosterone and estradiol measured in the samples of hair extracts were above the detection limit indicate the repeatability, sensitivity and accuracy of the described method. Comparison of the intramuscularly administered doses of testosterone propionate and estra-diol valerate and the testosterone and estradiol hair concentrations indicates that up to 0.80 % of the former and up to 0.06 % of the latter is incorporated in hair. Due to their prolonged presence in hair, high levels could indicate previous use of testosterone and estradiol esters. For fattening purposes in farm animals, steroids are used in lower doses (26) than those used in our study; the described method therefore cannot be applied directly to detect hair testosterone and estradiol in farm animals. Because species-specific kinetic characteristics must also be considered, the present study can only serve as a base for establishing the ELISA method for the detection of steroids in hair in farm animals. Although detection of steroids and steroid esters is widely performed by the more selective gas and liquid chromatography coupled to mass spectrometry (16, 17, 19, 20), commercial ELISA kits could potentially serve as a pre-screening tool for the detection of testosterone and estradiol in hair after parenteral administration of testosterone and estradiol esters. Acknowledgements The study was financially supported by the Veterinary Administration of the Republic of Slovenia and Slovenian Research Agency grant P4-0053. Authors are grateful to Mrs Katarina Babnik and Mr Boštjan Drolc for their technical assistance and to Professor Roger H. Pain for review of English. References 1. Reddy DS, Gadsby JE. Hormones affecting reproduction. In: Riviere JE, Papich MG, eds. Veterinary pharmacology and therapeutics. Ames: Wiley - Blackwell, 2009: 717-33. 2. Guan F, Uboh CE, Soma LR, You Y, Liu Y, Li X. High-throughput UHPLC-MS/MS method for the detection, quantification and identification of fifty-five anabolic and androgenic steroids in equine plasma. J Mass Spectr 2010; 45: 1270-9. 3. Elling H, Ungemach FR. Demonstration of estrogen receptors in perianal tumors in dogs. Determination of sex hormones in rat hair after administration of testosterone propionate and estradiol valerate 33 Zentralbl Veterinarmed A1980, 27: 758-64. 4. Council Directive 1996/22/EC. 5. Council Directive 2003/74/EC. 6. Anabolic agents. Substances and methods prohibited at all times. International standards. Montreal: World ant-doping program, 2012. 7.http: //www.wada-ama.org/en/World-Anti-Doping-Program/Sports-and-Anti-Doping-Organizations/International-Standards/ Prohibited-List/The-2011-Prohibited-List/ Prohibited-at-All-Times/ (12. okt. 2011) 8. Peng SH, Segura J, Farré M, González JC, de la Torre X. Plasma and urinary markers of oral testosterone undecanoate misuse. Steroids 2002; 67: 39-50. 9. Hogan LA, Phillips CJ, Lisle A et al. Non-invasive methods of estrus detection in captive southern hairy-nosed wombats (Lasiorhinus latifrons). Anim Reprod Sci 2010; 119: 293-304. 10. Lueders I, Hildebrandt TB, Pootoolal J, Rich P, Gray CS, Niemuller CA. Ovarian ultrasonography correlated with fecal progestins and estradiol during the estrous cycle and early pregnancy in giraffes (Giraffa camelopardalis rothschildi). Biol Reprod 2009; 8: 989-95. 11. Bamberg E, Schwarzenberger F. Fecal steroid assay for monitoring of estrus cycle and pregnancy. In: Proceedings of 4th congress of International society of animal clinical biochemistry. Davis, 1990: 95-6. 12. Snoj T, Cebulj-Kadunc N, Cestnik V, Pardubsky T. Determination of faecal gestagens in sows by commercial progesterone EIA kit. Acta Vet Brno 1998, 67: 21-5. 13. Moriyoshi M, Tamaki M, Nakao T, Kawata K. Early pregnancy diagnosis in the sow by saliva progesterone measurement using a bovine milk progesterone qualitative test EIA kit. J Vet Med Sci 1996; 58: 737-41. 14. Gao Y, Short RV, Fletcher TP. Progesterone concentrations in plasma, saliva and milk of cows in different reproductive states. Br Vet J 1988; 144: 262-8. 15. Kaklamanos G, Theodoridis G, Dabalis T. Determination of anabolic steroids in muscle tissue by liquid chromatography-tandem mass spectrometry. J Chromatogr 2009; 1216: 8072-9. 16. Anielski P, Thieme D, Schlupp A, Grosse J, Ellendorff F, Maueller RK. Detection of testosterone, nandrolone and precursors in horse hair. Anal Bioanal Chem 2005; 383: 903- 8. 17. Stolker AAM, Groot MJ, Lasaroms JP et al. Detectability of testosterone esters and astradiol benzoate in bovine hair and plasma following pour-on treatment. Anal Bioanal Chem 2009; 395: 1075-87. 18. Gaillard Y, Vayssette F, Balland A, Pépin G. Gass chromatographic tandem mass spectrometric determination of anabolic steroids and their esters in hair. Application in dopping control and meat quality control. J Chromatogr B Biomed Sci Appl 1999; 735: 189-205. 19. Anielski P. Hair analysis of anabolic steroids in connection with doping control - results from horse samples. J Mass Spectrom 2008; 43: 1001-8. 20. Nielen M, Lasaroms J, Mulder P, van Hende J, van Rhijn J, Groot M. Multi residue screening of intact testosterone esters and boldenone undecylenate in bovine hair using liquid chromatography electrospray tandem mass spectrometry. J Chromatogr B 2006; 830: 126-34. 21. Boyer S, Garcia P, Popot M, Steiner V, Lesieur M. Detection of testosterone propionate administration in horse hair samples. J Chromatogr B 2007; 852: 684-8. 22. Scarth J, Akre C, van Ginkel L et al. Presence and metabolism of endogenous androgenic-anabolic steroid hormones in meat-producing animals: a review. Food Addit Contam Part A 2009; 26: 640-71. 23. Nielen HWF, Nijrolder AWJM, Hooijerink H, Stolker AAM. Feasibility of desorption electrospray ionization mass spectrometry for rapid screening of anabolic steroid esters in hair. Anal Chim Acta 2011; 700: 63-9. 24. Gleixner A, Meyer HHD. Detection of estradiol and testosterone in hair of cattle by HPLC/EIA.. Fresenius J Anal Chem 1997; 357: 1198-201. 25. Mostl E, Lehmann H, Wenzel U. Gestagens in the faeces of mink and cats for monitoring corpus luteum activity. J Reprod Feril 1993; 47 (Suppl): 540-1. 26. Cefalu WT, Pardridge WM, Chaudhuri G, Judd HL. Serum bioavailability and tissue metabolism of testosterone and estradiol in rat salivary gland. J Clin Endocrinol Metab 1986; 63: 20-8. 27. Velle W. The use of hormone in animal production. Agriculture and Consumer Protection Department. Rome: FAO, 1982: 21 str. http:// www.fao.org/D0CREP/004/X6533E/X6533E01. htm (12. okt. 2011) 34 T. Snoj, N. Čebulj-Kadunc, A. Nemec Svete, S. Kobal UGOTAVLJANJE SPOLNIH HORMONOV V DLAKI PODGAN PO APLIKACIJI TESTOSTERON PROPIONATA ALI ESTRADIOL VALERATA T. Snoj, N. Čebulj-Kadunc, A. Nemec Svete, S. Kobal Povzetek: Uporaba 17 13-testosterona (testosteron) in 17 13-estradiola (estradiol) ter podobnih sintetičnih snovi z namenom, da bi se izboljšal prirast pri farmskih živalih, je v EU prepovedana. Za določanje teh snovi pri živalih bi bila uporabna enostavna in zanesljiva metoda. Z uporabo komercialnih ELISA kompletov smo v raziskavi ugotavljali vsebnost testosterona in estradiola v dlaki in plazmi podgan pasme wistar po predhodni intramuskularni (i.m.) aplikaciji testosteron propionata in estradiol valerata. Poskus ugotavljanja vsebnosti testosterona v dlaki je bil izveden na mladih samicah, estradiol v dlaki pa se je ugotavljal pri mladih samcih. Statistično značilno višjo koncentracijo testosterona v dlaki smo ugotovili tretji, šesti in deveti dan po aplikaciji testosteron propionata, medtem ko statistično značilnih razlik v krvni plazmi nismo ugotovili. Po aplikaciji estradiol valerata smo ugotovili statično značilno višjo koncentracijo estradiola v dlaki tretji in deveti dan, višja vrednost pa je bila deveti dan ugotovljena tudi v plazmi. Na podlagi rezultatov sklepamo, da je z uporabo komercialnih ELISA kompletov možno ugotavljati vsebnost testosterona in estradiola v dlaki po predhodni i.m. aplikaciji testosteron propionata in estradiol valerata. V dlaki je do 0,80 odstotka apliciranega testosterona in do 0,06 odstotka apliciranega estradiola. Opisana metoda bi se lahko uporabila kot osnova za uvedbo presejalne metode ugotavljanja zlorabe anaboličnih steroidov pri rejnih živalih. Ključne besede: testosteron; estradiol; dlaka; plazma; podgane; test ELISA Slov Vet Res 2012; 49 (1): 35 - 40 UDC 636.1.09:616.15:618.14-002 Original Scientific Article BLOOD LEUKOCYTES IN CARTHUSIAN MARES WITH ENDOMETRIOSIS OR ENDOMETRITIS IN COMPARISON WITH HEALTHY MARES Katy Satué1*, Manuel Felipe2, Javier Mota2, Ana Muñoz3 department of Animal Medicine and Surgery, Faculty of Veterinary Medicine, CEU-Cardenal Herrera University, Avd. Seminary, 46113 Moneada, Valencia; 2Carthusian farm "La Cartuja-Hierro of Bocado", Road Medina el Portal, Km 6.5, Jerez de la Frontera (Cádiz);3Department of Animal Medicine and Surgery, Faculty of Veterinary Medicine, Córdoba University, Campus of Rabanales, Córdoba, Spain Corresponding author, E-mail: ksatue@uch.ceu.es Summary: Significant changes in total blood white blood cells (WBC) and populations are found in many generalized and localized pathological conditions in veterinary medicine. Endometritis is an inflammatory process that might lead to infertility in mares and it is associated with an influx of polymorphonuclear neutrophils (PMN) into the uterine lumen. Endometriosis is a chronic degenerative endometrial disease that causes severe alterations in the connective tissues of the uterus. We took blood samples from 6 Carthusian mares with endometritis (EM1) and 8 with endometriosis (EM2) in order to check whether these changes lead to changes in hematology. Further, 6 healthy mares of the same breed and similar age were also sampled (control group, CG). An analysis of variance was used to evaluate the differences between the three groups of mares. In comparison with CG, mares of group EM1 had significantly higher hemoglobin concentration, hematocrit, mean corpuscular volume, mean corpuscular hemoglobin concentration, segmented PMNs and neutrophils/lymphocytes ratio, and lower total white blood cells and lymphocytes. Mares of group EM2 presented lower red blood cell and white blood cell counts. No morphological changes were detected in the different types of WBC when examined in the blood smear. It is concluded that some hematological parameters differ in mares with endometritis and endometriosis in comparison to healthy mares. Even though the hematological study is not useful in the diagnosis of these diseases, the results of the present research are important in order to diagnose other clinical conditions that could coexist with these reproductive diseases. Key words: endometritis; endometriosis; hematology; horse; mare Introduction Leukocytes (WBC) are vital for host defense, and for the initiation and control of inflammation and immunity. Although generally protective or supportive of the host tissue, WBCs are also involved in host-harmful allergic and immunemediated diseases. Significant changes in blood WBC numbers and/or of their different populations, with or without morphological changes are observed in many diseases and they are of pivotal help in the diagnosis and prognosis in veterinary Received:29 November 2011 Accepted for publication: 20 February 2012 medicine. The pattern of the WBC response depends on many factors, such as animal species, susceptibility of the patent, virulence of the pathogenic agent involved, immun status of the patient and extension of the inflammation and/or infection (localized vs. generalized) (1, 2). The WBC response to different reproductive diseases has been reported in small animals. Dogs with localized infections, such as pyometra exhibits leukocytosis with neutrophilia and sometimes, toxic changes in neutrophils and left shift, with increased band neutrophil numbers (3, 4, 5, 6). Endometritis encompasses endometrial changes associated with acute or chronic inflammation and it is a major cause of mare infertility aris- 36 K. Satué, M. Felipe, J. Mota, A. Muñoz ing from incapacity or failure to remove bacteria, spermatozoa and inflammatory exudates. Defects in genital anatomy, myometrial contractions, lymphatic drainage, mucociliary clearance, cervical function vascular degeneration and ageing underlie endometritis (7). It should be promptly diagnosed and resolved in order to avoid chronic-ity. Endometritis might be difficult to identify, because clinical signs, ultrasonographic and labo-ratorial findings can vary according to the cause. Some cases are associated with an influx of inflammatory cells, mainly polymorphonuclear neu-trophils (PMN) and fluid into the uterine lumen (8). Although affected mares do not show clinical signs associated with systemic disease, it is plausible to think that an acute inflammation might exist. Recently, it has been reported that mares with endometritis experimentally induced by intrauterine infusion of E. coli had increased serum concentrations of some acute-phase proteins (e.g. amyloid A and fibrinogen). These results indicated that endometritis in mares is linked to a systemic acute phase response (9). Endometriosis is a chronic degenerative en-dometrial disease involving severe alterations in the uterine connective tissues. It is characterized by destruction of the uterine gland, including cystic dilation, hyperplasia and periglandular fibrosis, glandular epithelial hypertrophy, lymphatic lacunae or lymphangiectasia, angiosis (a vascular pathological condition with associated venous congestion) and, subsequently resulting in the development of a stromal fibrosis (10, 11). Briefly, the term 'endometritis' is used to describe inflammatory changes, whereas 'endometriosis' reflects chronic changes associated with age and parity (12, 13). When an acute phase response appears, the number and percentage and/or the morphological characteristics of the different blood WBC populations might change. The current study aims to compare the hematological profile in three groups of mares: healthy, mares with endometritis and with endometriosis. It is hypothesized that mares with endometritis will show modifications in the leukogram in response to the inflammation. Further, both of them could be chronic processes, and therefore, it is also plausible to evidence a leukogram of chronic disease. Material and methods This research was approved by the Ethic Committee for Animal Experimentation of the Cardenal Herrera-CEU University. Jugular venous blood samples were withdrawn from a total of 20 adult Carthusian mares, divided into three groups: healthy (n=6), considered as control group, CG, mares with endometritis (n=6), EM1 and with endometriosis (n=8), EM2. The 20 mares had previously foaled, although the rate of success was different. The reproductive history of the studied mares is presented in table 1. The mares were considered healthy (CG) when the echography of the reproductive system was unremarkable, physical examination did not provide any clinical indication of disease, all of them were pregnant without problems, have viable foals and had hematological parameters within the reference range established for this breed (14, 15). The diagnosis of mares EM1 was made with ultrasound (Pie Medical 480®), cytology and microbiological cultures. Mares of EM1 presented pus or fluid in the uterine lumen and endometrial edema and cytology showed predominant numbers of PMN. Diagnosis of mares of group EM2 was made by echography and endometrial biopsy. The echography showed increased echo-texture in endometrial mucosa and endometrial Table 1: Age and reproductive history of the 20 Carthusian mares included in this research (CG, control group; EM1, with endometritis; EM2, with endometriosis) Mare groups Age (mean±SD) Number of previous successful foaling Previous foal CG (n=6) 8.345±2.45 3 foals (n=6) Previous mating (n=6) EM1 (n=6) 10.54±2.12 1 foal (n=3) 2 foals (n=2) 3 foals (n=1) 1 season without successful mating (n=4) 2 seasons without successful mating (n=2) EM2 (n=8) 14.96±1.23 1 foal (n=4) 2 foals (n=4) At least 3 seasons without successful mating Blood leukocytes in carthusian mares with endometriosis or endometritis in comparison with healthy mares 37 biopsy revealed a chronic endometriosis degree III (system of Kenney and Doig; 12, 13). The mares of the three groups were sampled in the morning, before feeding. The samples were extracted in the follicular phase, 2-3 days before ovulation. Immediately after blood extraction, and directly from the syringe, without anticoagulant, a blood smear was carried out. The rest of the blood was poured into tubes with EDTA-3K for hematol-ogy and into glass tubes in order to obtain serum to measure total proteins. In all the cases, the samples remained refrigerated during the transportation to the laboratory and the analyses were performed within the first 12 hours after extraction. The following hematological parameters were included in this research: red blood cells (RBC), hemoglobin concentration (HB), hematocrit (HT), mean corpuscular volume (MCV), mean corpuscular hemoglobin concentration (MCHC), white blood cells (WBC) and platelets (PLT). These measurements were made using a semiautomatic counter (System F820®). The differential WBC count was performed in the blood smear, fixed with ethanol and stained with May-Grunwald-Giemsa technique. The absolute number of the WBC populations was quantified by microscope (Olympus CX 21®): lymphocytes (LYMP), polymorphonuclear neutrophils (PMNs), segmented PMN (SPMN) and in band PMN (BPMN), eosinophils (EOS), monocytes (MON), basophils (BAS) and neutrophil/lym-phocyte ratio (N/L). In the blood smear, we also assessed the presence of morphological changes in the PMNs, such as Döhle's bodies, basophilia, cytoplasmatic vacualization, nuclear edema and/ or hypersegmentation. They were subjectively scored over 5 points (0: abscense of changes; 5: maximum morphological changes). Finally, the concentration of total serum proteins (TSP) was assessed by spectrophotometry with specific reagents (Metrolab, RAL®). Data are presented as means±SD. Differences between the three groups were analyzed with an analysis of variance ANOVA. Statistical significance was set at p<0.05. The statistical program Statistica v. 7.0 for windows was used (Statsoft®, Inc, 2001. Tulsa, Oklahoma, USA) Results The hematological parameters for the three groups of mares (CG, EM1 and EM2) are presented in table 2. Mares of group EM1 in comparison to CG had higher HB, HT, MCV, MCHM, SPMN and N/L ratio and lower WBC and LYMP. Mares of Parameter and unit CG EM1 EM2 RBC (106/pl) 9.43±1.58 9.49±2.29 8.90±2.34* HB (g/dl) 12.60±1.40 13.50±2.06* 13.06±2.77 HT (%) 46.39±6.21 49.85±12.93* 47.51±13.76 MCV (fl) 50.39±2.33 52.43±2.86* 52.82±5.54 MCHC (g/dl) 26.86±5.23 27.82±3.84* 29.55±13.65 PLT (103/pl) 199.1±116 143.2±104 164.1±118 TSP (g/dl) 7.35±0.30 7.72±0.46 7.50±0.49 WBC (103/pl) 12.44±2.28 11.40±5.97* 10.46±8.01* SPMN (103/pl) 4.92±0.15 5.45±1.46* 5.09±2.17 BPMN (103/pl) 0.21±0.01 0.24±0.12 0.24±0.28 LYMP (103/pl) 5.06±0.12 3.18±0.59* 4.55±2.30 EOS (103/pl) 0.67±0.04 0.69±0.48 0.65±0.12 MON (103/pl) 0.22±0.01 0.24±0.10 0.24±0.08 N/L 1.02±0.03 1.84±0.69* 1.21±0.64 Table 2: Mean±standard deviation of the hematological parameters in three groups of Carthusian mares (CG, control group; EM1, with endometritis; EM2, with endometriosis). Significant differences from the CG are indicated with an asterisk. Level of significance p<0.05 (RBC, red blood cells, HB, hemoglobin concentration, HT, hematocrit; MCV, mean corpuscular volume; MCHC, mean corpuscular hemoglobin concentration; PLT, platelets; TSP, total serum proteins; WBC, white blood cells; SPMN, segmented polymorphonuclear neutrophils; BPMN, band neutrophils; LYMP, lymphocytes; EOS, eosinophils; MON, monocytes; N/L, neutrophil/lymphocyte ratio) 38 K. Satué, M. Felipe, J. Mota, A. Muñoz group EM2 showed lower RBC and WBC (Table 2). No morphological changes in PMN neither in LYMP were found when assessing the blood smears. Discussion In the present research, we have found significant differences when comparing the hema-tological profile in mares with localized diseases (endometritis and endometriosis) with healthy mares. Both conditions are common causes of reproductive failure in mares and they are not accompanied by systemic clinical signs. However, they can cause changes in the uterine tissues that could lead to changes in the blood profile. Thus, endometritis is associated with an infiltration of inflammatory cells, mainly PMN in the stratum compactum (16, 17). Recently, it has been demonstrated that endometritis induces a systemic acute phase reaction, with increased serum amyloid (9). Traditionally, fever and changes in WBC numbers and/or morphology have been considered to be the hallmarks of inflammation and infection, even though many reports lastly have confirmed that they are less sensitive that acute phase proteins (18, 19). Despite these ideas, it is plausible to think that endometritis could induce some variations in the hematological profile of the mare. The higher HT of the EM1 mares seemed to result from the higher size of the erythrocytes (higher MCV), as RBC were similar in both groups. The reasons of these results are unknown. Firstly, this result was not associated with dehydration, as TSP did not exhibit significant differences between both groups. This was an unexpected finding, as chronic diseases lead to a normocytic normochro-mic anemia or do not exert any significant effect on the number of circulating RBCs (20, 21). Increased PMN occurs during infectious and non-infectious inflammatory conditions (20, 21). Immature PMN, i.e. BPMN is the most sensitive differentiator of infectious inflammatory disease, but they are not seen as frequently in horses as in other animal species (21). In equids, BPMN are most commonly seen in severe acute bacterial infections or septicemic processes. This can be the explanation for the similar values for BPMN found in CG and EM1. The higher number of SPMN in EM1 is consistent with inflammation. It has been demonstrated that PMN increased in uterus exu- dates at 30 min after experimental endometritis, induced by infusion of Streptococcus zooepidemi-cus (16). The lower LYMP in group EM1 could be the reflex of stress of the disease. Other causes of decreased LYMP, such as viral infections or glu-cocorticoid administration do not appear to be important in our mares. Probably both facts i.e. increased SPMN and decreased LYMP led to the higher N/L of the EM1 group in comparison with CG. Endometriosis is a chronic degenerative en-dometrial disease associated with age and parity and with the presence of fibrosis (10, 11). The lower RBC in the mares of group EM2 in our study is consistent with the chronicity and degenerative condition of the process. Surprisingly, the other types of WBC, mainly MON, were not different between groups. Increased MON are found during disease processes with increased tissue demand for phagocytosis of particles, such as tissue necrosis and infection (20, 21), as happen in endometritis. Finally, it is interesting to indicate that HT, MCV, TSP WBC, SPMN, BPMN, LYMP, EOS and N/L ratio had greater SD in both diseased groups (EM1 and EM2) than in CG. This result has two implications. Firstly, it could have limited the evidence of statistical significance when comparing the three groups and secondly, it might indicate the clinical differences between groups. Our results indicate that some hematological parameters are different in Carthusian mares with endometritis and with endometriosis in comparison with a control group of mares of the same age and breed. Although the hematological profile is not used for the diagnosis of these diseases, it is important to establish whether the hematological parameters vary in mares with these reproductive problems in order to diagnose or to assess other clinical conditions that could co-exist with these conditions. References 1. Schultze AE. Interpretation of canine leukocyte response. In: Weiss DJ, Wardrop KJ, eds. Schalm's veterinary hematology. Ames: Wiley-Blackwell, 2010: 321-34. 2. Welles EG. Interpretation of equine leukocyte response. In: Weiss DJ, Wardrop KJ, eds. Schalm's veterinary hematology. Ames: Wiley-Blackwell, 2010: 314-20. Blood leukocytes in carthusian mares with endometriosis or endometritis in comparison with healthy mares 39 3. Fukuda S. Incidence of pyometra in colony-raised beagle dogs. Exp Anim 2001; 50: 325-49. 4. Hagman R. Serum a1-acid glycoprotein concentrations in 26 dogs with pyometra. Vet Clin Pathol 2001; 40: 52-9. 5. Plavec T, Celinsek B, Dolinar K, Pecar J, Nemec, Butinar J. Haemostatic impairment in bitches with piometra. Acta Vet (Beograd) 2006; 56: 529-40. 6. Nakamura M, Takahashi M, Ohno K, et al. C-reactive protein concentration in dogs with various diseases. J Vet Med Sci 2008; 70: 12-31. 7. LeBlanc MM, Causey RC. Clinical and subclinical endometritis in the mare: both threats to fertility. Reprod Domest Anim 2009; 44: 10-22. 8. LeBlanc MM. Advances in the diagnosis and treatment of chronic infections and post-mating-induced endometritis in the mare. Reprod Domest Anim 2010; 45: 21-7. 9. Christoffersen M, Baagoe CD, Jacobsen S, Bojesen AM, Petersen MR, Lehn-Jensen H. Evaluation of the systemic acute phase response and endometrial gene expression of serum amyloid A and pro- and anti-inflammatory cytokines in mares with experimentally induced endometritis. Vet Immunol Immunopathol 2010; 138: 95-105. 10. Walter I, Helmreich M, Handler J, Aurich C. Mineralised deposits in the uterine glands of mares with chronic endometrial degeneration. Vet Rec 2003; 153: 708-10. 11. Hoffman C, Ellenberger C, Mattos RC, Aupperle H, Dhein S, Schoon HA. The equine endometriosis: new insights into the pathogenesis. Anim Reprod Sci 2009; 111: 261-78. 12. Kenney RM, Doig PA. Equine endometrial biopsy. In: Morrow DA, ed. Current therapy in theriogenology: diagnosis, treatment and prevention of reproductive diseases in small and large animals. London: WB Saunders, 1986: 723-9. 13. Holyoak GD, Ley WB. Management regimes for uterine cysts. In: Samper JC, Pycock JF, McKinnon AO, eds. Current therapy in equine reproduction. Philadelphia: Saunders Elsevier, 2007: 121-5. 14. Satué K, Blanco O, Muñoz A. Age-related differences in the hematological profile of Andalu-sian broodmares of Carthusian strain. Veterinarni Med 2009; 54: 178-82. 15. Satué K, Muñoz A, Blanco O. Pregnancy influences the hematological profile of Carthusian broodmares. Pol J Vet Sci 2010; 13: 393-4. 16. Pycock JF, Allen WF. Inflammatory components in uterine fluid from mares with experimentally induced bacterial endometritis. Equine Vet J 1990; 22: 422-5. 17. Overbeck W, Witte TS, Heuwieser W. Comparison of three diagnostic methods to identify subclinical endometritis in mares. Theriogenoloy 2011; 75: 1311-8. 18. Jacobsen S, Jensen JC, Frei S, Jensen AL, Thoefner MB. Use of serum amyloid A and other acute phase reactants to monitor the inflammatory response after castration in horses: a field study. Equine Vet J 2005; 37: 552-6. 19. Jacobsen S, Nielsen JV, Kjelgaard-Hansen M, et al. Acute phase response to surgery of varying intensity in horses: a preliminary study. Vet Surg 2009; 38: 762-9. 20. Barrelet A, Ricketts S. Haematology and blood biochemistry in the horse: a guide to interpretation. In Pract 2002; 24: 318-27. 21. Muñoz A, Riber C, Trigo P, Castejón F. Hematology and clinical pathology data in chronically starved horses. J Equine Vet Sci 2010; 30: 581-9. 40 K. Satué, M. Felipe, J. Mota, A. Muñoz LEVKOCITI V KRVI KARTUZIJSKIH KOBIL Z ENDOMETRIOZO ALI ENDOMETRITISOM V PRIMERJAVI Z ZDRAVIMI KOBILAMI K. Satue, M. Felipe, J. Mota, A. Munoz Povzetek: Pri številnih splošnih in lokaliziranih patoloških stanjih ugotavljamo znatne spremembe vseh belih krvničk (WBC) kot tudi njihovih populacij. Endometritis je vnetni proces, ki lahko vodi do neplodnosti pri kobilah in je povezan z vdorom poli-morfonuklearnih nevtrofilcev (PMN) v svetlino maternice. Endometrioza je kronična degenerativna endometrična bolezen, ki povzroča resne spremembe v vezivnem tkivu maternice. Odvzeli smo vzorce krvi šestih kartuzijskih kobil z endometritisom (EM1) in osmih z endometriozo (EM2), da bi preverili, ali te spremembe povzročijo spremembe v krvni sliki. Poleg tega smo odvzeli vzorce šestim zdravim kobilam iste pasme in podobne starosti (kontrolna skupina, CG). Z analizo variance smo ocenjevali razlike med tremi skupinami kobil. Kobile iz skupine EM1 so imele bistveno višjo vsebnost hemoglobina, višji hematokrit, višjo povprečno prostornino krvnih celic, višjo povprečno vsebnost hemoglobina v krvnih celicah, več polimorfonuklearnih celic in višje razmerje nevtrofilci/limfociti ter manjšo količino belih krvničk in limfocitov v primerjavi s CG. Kobile iz skupine EM2 so imele manj rdečih in belih krvničk v krvi. V krvnih brisih pri različnih vrstah WBC nismo zaznali morfoloških sprememb. Ugotovili smo, da se nekateri hematološki parametri razlikujejo pri kobilah z endometritisom in endometriozo v primerjavi z zdravimi kobilami. Čeprav krvna študija ni uporabna pri diagnosticiranju teh bolezni, so rezultati raziskave pomembni za diagnosticiranje drugih kliničnih stanj, ki so lahko tudi prisotna pri omenjenih reproduktivnih boleznih. Ključne besede: endometritis; endometrioza; hematologija; konj; kobila Slov Vet Res 2012; 49 (1): 41- 9 UDC 615.9:661.164.2:612.616:612.82:591.5: 599.323.4 Original Scientific Article EFFECTS OF ORGANOPHOSPHATE CHLORMEPHOS ON DEVELOPMENT AND FUNCTION OF TESTES AND BRAIN Katerina Čeh1, Davor Ježek4, Monika C. Žužek2, Nina Čebulj-Kadunc2, Gregor Majdič1,3* 1Center for Animal Genomics, institute of Physiology, Pharmacology and Toxicology, Veterinary Faculty, University of Ljubljana, Gerbičeva 60, 1000 Ljubljana; institute of Physiology, Medical Faculty, University of Maribor, Slomškov Trg 5, 2000, Maribor, Slovenia; 4Department of Histology and Embryology, School of Medicine, University of Zagreb, Šalata 3, 10000 Zagreb, Croatia Corresponding author, E-mail: gregor.majdic@vf.uni-lj.si Summary: Several chemicals, known as endocrine disrupting chemicals have the capacity to interfere with hormone action in the mammalian body. The aim of our study was to establish whether long term exposure to low doses of organophosphorus compound Chlormephos affects the development and function of reproductive tract and endocrine parts of the brain. Adult male and female mice were exposed to 3.5|jg/ml and 0.35|jg/ml of Chlormephos in the drinking water during mating, pregnancy and lactation until weaning of the offspring. Testes development was studied in the offspring of exposed mice by monitoring daily sperm production, number of apoptotic cells and spermatogenesis. Immunoexpression of antimüllerian hormone and 313-hydroxysteroid dehydrogenase was assessed qualitatively in testes from 9 and 19 days old offspring, respectively. No statistically significant differences (or qualitative differences in assessment of immunoexpression) were found in any of the observed parameters, suggesting that low dose of Chlormephos does not act as an endocrine disruptor in reproductive tract. To examine whether exposure to low doses of Chlormephos affect brain development, offspring of the exposed mice were tested for anxiety like behaviour in the elevated plus maze. Mice exposed neonatally to the higher dose of Chlormephos spent significantly less time in the open arms of the elevated plus maze in comparison to mice from control group, suggesting an increase in the anxiety like behaviour. Ultrastructural analyses did not reveal any changes in brain ultrastructure, in particular in blood-brain barrier which has been reported before to be a target for organophosphorous compounds. Key words: mice; testis; brain; organophosphorus; daily sperm production; electron microscopy; apoptosis; anxiety-like behavior Introduction Endocrine disruptor chemicals (EDCs) in the environment could possibly interfere with the endocrine system and mimic or depress the function of endogenous hormones. EDCs usually interfere with the endocrine system through hormone receptors such as estrogen and androgen receptors. In recent years, several studies have shown that environmental contaminants such as isomers of dichlorodiphenyltrichloroethane (DDT), some polychlorinated biphenyls (PCBs), the plasticizer bis-phenol A, and some detergents such as p-no- Received:12 January 2012 Accepted for publication: 24 April 2012 nylphenol, can exhibit estrogenic or anti-andro-genic activity (1). Organophosphates (OPs) are a large class of chemicals, which act as irreversible inhibitors of acetylcholinesterase (AChE). OPs cause accumulation of neurotransmitter acetylcholine and they are often used in farming as insecticides and an-tiparasitics (2, 3). Chlormephos (S-chloromethyl 0,0-diethyl phosphorodithioate) is an organophos-phorous (OP), insecticide that was introduced to the market in 1973 to control soil-dwelling pests (4). Chlormephos was in use until 2006 when it was withdrawn from all EU markets. OP could act as endocrine disruptors in reproductive system, brain development and behavior. Sarkar et al. reported that OP pesticide quinalphos 42 K. Čeh, D. Ježek, M. C. Žužek, N. Čebulj-Kadunc, G. Majdič disrupts hypothalamo-pituitary-gonadal axis. Sublethal chronic administration of quinalphos resulted in decreased testicular mass and AChE activity in central and peripheral organs; increased serum LH, FSH, prolactin and testosterone concentrations; decreased pituitary and increased testicular activity and severe disruption of sper-matogenesis with increasing doses of pesticide (5). Verma and Mohanthy (6) showed that in utero and lactational exposure of mice to low doses of dimethoate caused reduced testis weight, reduced sperm count, histopathological changes in testes and epididymidis, lower plasma LH and testosterone and reproductive dysfunction of adult mice. Study by Umzucu et al. (7) demonstrated that early postnatal exposure of rats to metoxyclor resulted in reduced ovarian weight, inhibition of folliculogenesis and stimulation of anti-mullerian hormone production. Several studies investigated effects of low-level exposure to OPs pesticides during pregnancy on neurodevelopment and behavior of newborns. Prenatal exposure of children to higher doses of chlorpiryfos resulted in Psychomotor Development Index and Mental Development Index delays, attention problems, attention-deficit/hyperactivity disorder problems, and pervasive developmental disorder problems at 3 years of age (8). Developing blood-brain barrier is highly vulnerable to single or repeated exposure to low doses of certain pesticides such as organophosphate quinalphos, pyre-throid cypermethrin and organochlorine lindane although there seems to be some species differences in vulnerability of blood-brain barrier to the pesticides (9, 10). The aim of our study was to determine whether long term exposure to low levels of organophos-phorus compound Chlormephos can affect the development and functioning of testes and brain in mice. Although there are no data about human exposure to Chlormephos, the data for exposure to other organophosphorous pesticides show that human dietary or environmental exposure is similar to doses used in our study (11, 12). Material and methods Animals Sexually mature (60 - 70 days old) BALB/c male and female mice (all together 18 control females, 16 females exposed to 3.5 Mg/ml and 12 females exposed to 0.35 Mg/ml Chlormephos) were breed in standard conditions at 20 - 25° C, humidity 5565%, dark : light cycle 12:12, with chow without phytoestrogens (2916, Harlan Tekland, England) ad libitum. All animal experiments were approved by the Veterinary commission of Slovenia (approval number 3440-176/2006) and were done according to the EU directive and NIH guidelines. Mice were supplied either with drinking water containing 3.5 Mg or 0.35 Mg of Chlormephos per ml (10 and 100 times lower dose of LD50 for mammals) (13). As Chlormephos is insoluble in water, it was first dissolved in ethanol at concentration 1 mg/ml, and this stock solution was used for preparing final drinking water solution. Final concentration of ethanol in the drinking water was 3.5 Ml per ml in group exposed to 3.5 Mg of Chlormephos per ml and 0.35 Ml per ml in group exposed to 0.35 Mg of Chlormephos per ml. Control group received drinking water without Chlormephos, containing 3.5 Ml of ethanol per ml (0.35 %, vol/vol). The effects of Chlormephos were monitored in the offspring of exposed animals. Sexually mature male and female mice were randomly divided into control or treated groups; weighted and exposed to Chlormephos/Ethanol for seven days. After seven days, one male and one female mouse were joined in a single cage. Mice were exposed to Chlormephos/Ethanol throughout pregnancy and lactation period until offspring weaning at 21 days. Litter size was carefully monitored; pups were always counted on the day of delivery. Male pups from different litters were randomly assigned to groups for sacrifice at different postnatal age (in each age group of mice, pups from at least three different litters were included). Pups were sacrificed at 9, 19, 48 and 70 days of age, 48 and 70 days old mice were exposed to Chlormephos/Ethanol only until weaning on day 21. Nine and 70 days old mice were sacrificed by CO2 exposure followed by cervical dislocation. Mice 19 and 48 days old were anaesthetized by a mixture of ketamine (Vetoquinol Biowet, Gorzowie, Poland; 100 Mg/g BW), xylazine (Chanelle Pharmaceuticals Ltd., Loughrea, Ireland; 10 Mg/g BW) and acepromazine (Fort Dodge Animal Health, Fort Dodge, IA, USA; 2 Mg/g BW) and perfused with Bouin's solution. Testes were isolated and postfixed in Bouin's solution for 4 to 20 hours depending on size. Subsequently, testes were processed into paraffin wax using standard procedures. At the time of sacrifice, whole mice as Effects of organophosphate Chlormephos on development and function of testes and brain 43 well as testes and seminal vesicles were weighted. Testis weight (average of left and right testis) was expressed as a percentage of total body weight (= relative testis weight). Some offspring of exposed animals were breed in adulthood, number of pups was monitored. Anxiety tests were performed on offspring of exposed mice. Effects of Chlormephos on daily sperm production (DSP) Daily sperm production was examined in 70 days old male offspring of treated and control mice as described before (14). Briefly, testes were dissected, weighed and homogenized in 5ml of physiological saline containing 0.05% Triton X-100. Spermatozoa were counted in Bürker - Türk chamber under 400x magnification. Ten visual fields per sample were counted and daily sperm production was calculated using formula N x 25 x 5000 (N = number of counted cells, 25 and 5000 dilution factors), divided by testes weight and this result was divided by 4.48, thus obtaining average DSP. Immunohistochemistry Immunohistochemistry was performed as described before (15) using antibodies against an-timüllerian hormone (AMH) in concentration 1: 200 and 3ß-hydroxysteroid dehydrogenase (3ß-HSD) in concentration 1:1000 using peroxidase anti-peroxidase system. Specific rabbit antibodies against AMH were gift from Natalie Josso (France), polyclonal rabbit antibodies against 3ß-HSD from dr. Ian Mason (United Kingdom). Apoptosis detection Apoptotic cells were detected by ApopTag® Peroxidase In Situ Apoptosis Detection Kit (Millipore, Billerica, MA, USA)) following manufacturer's protocol. Apoptotic cells were counted in testicular sections in 40 visual fields for each sample in testes from 48 old male mice exposed neonatally to Chlormephos. Results are presented as average number of apoptotic cells in one visual field. Anxiety-like behavior test In 70 to 80 days old mice of both sexes, standardized test for anxiety-like behavior using elevated plus maze was performed (16). Tests lasted 5 minutes during the beginning of the dark cycle with dim red light. Time in opened and closed arms of the maze as well as latency to enter closed or opened arms was recorded using Stopwatch+ program (version 1.5.1, Center for behavioral neuroscience, Atlanta, USA). Electron microscopy For electron microscopy, offspring from control and higher concentration Chlormephos treated group were sacrificed by carbon dioxide at 70 days of age. Part of the brain cortex from the frontal lobe was immediately fixed in 4% of glutaraldehyde for 2 hours. Samples were prepared by a standard procedure for electron microscopy with Durcupan (Fluka, Electron Microscopy Sciences, Hatfield, Pennsylvania, USA) embedding medium. Briefly, the brain tissue fixed in glutaraldehyde was rinsed several times in a 0.05M phosphate buffer (pH=7.1-7.4, 800 mOsm), postfixed with 1% OsO4 and dehydrated in a series of ascending alcohols. Tissue was then incubated in a mixture of absolute alcohol and acetone (1:1, 30 min.), absolute acetone (15 min.) and mixture of absolute acetone and Durcupan (Fluka). This step was followed by embedding the brain tissue into 100% Durcupan. After a polymerisation of the embedding medium in a thermostat (at 60°C, 72 hours), tissue blocks were trimmed and prepared for sectioning. Semithin sections (section thickness = 0.9 ^m) were made by Power-Tome XL ultramicrotome (Bal Tec/RMC) and stained with 1% toluidine blue. The sections were analysed by Nikon binocular microscope (Eclipse E200). Based on the bright filed microscopic analysis of semi-thin sections, ultra-thin sections were made (section thickness = 70 nm), placed on copper grids, and contrasted with lead citrate and uranyl acetate. The sections were then examined by transmission electron microscope Zeiss 902A (Centre for Electron Microscopy, Medical School University of Zagreb). Statistical analyses Differences between groups were analyzed by two-way analysis of variance with sex and treatment as independent variables, followed by Bonferroni posthoc test to determine differences between groups. Statistical significance was determined at p < 0.05. 44 K. Čeh, D. Ježek, M. C. Žužek, N. Čebulj-Kadunc, G. Majdič Results Litter size, body size and testis size in control and treated groups We have reported before in our report of preliminary data (17) that there were no differences in the litter size between control and Chlormephos exposed groups (mice that received Chlormephos through drinking water). There were also no differences in body size, absolute testis weight, relative testis weight and seminal vesicle weight between control mice and mice exposed to either concentration of Chlormephos during neonatal period through mothers (17). Similarly, when adult mice, exposed to Chlormpehos in neonatal period were mated, no statistical differences in litter size were observed with average 4.5 ± 0.9 (n=8 litters) pups in control group, 5.5 ± 0.8 (n= 4 litters) and 4.8 ± 1.2 (n=4 litters) in groups exposed neonatally to 0.35 and 3.5pg per ml Chlormephos, respectively. Daily sperm production (DSP) Similarly to our previously reported preliminary data (17), analysis of daily sperm production at 70 days old mice did not show any statistical differences between control and treated groups. In control group, DSP was 32,259.999 ± 1,602.126 (n=8) while it was 36,207.131 ± 539.750 (n=4) and 30,876.956 ± 5,151.737 (n=5) in groups exposed to high or low concentration of Chlormephos, respectively. Testes histology, number of apoptotic cells and 3fi-HSD and antimullerian hormone expression Tissue sections stained with haematoxylin and eosin from testes of all age groups of offspring were examined to determine any irregularity in spermatogenesis. No differences between control and treated groups were noted. Similarly, statistical analysis of apoptotic cells labeled with TUNEL method in 48 days old testes did not reveal any differences in the number of apoptotic cells between groups (n was 5 for each group). Qualitative assessment of immunohystochemi-cal staining for antimullerian hormone (testes from 9 days old mice) and 3p-hydroxysteroid dehydrogenase (19 days old offspring) did not reveal any differences in the immunoexpression of these two proteins (Figure 1). Figure 1: Histological analyses did not reveal any differences in general testis histology at 48 days of age between control (a) and groups exposed to Chlormephos through their mothers (b - 0.35 pg/ ml Chlormephos; c - 3.5 pg/ ml Chlormephos). Similarly, there were no obvious differences in intensity or pattern of antimullerian hormone (d, e, f) immunoexpression in Sertoli cells in testes from 9 days old pups or 3p-HSD immunoexpression (g, h, i) in Leydig cells from 19 days old pups between control (d, g) and Chlormephos exposed mice (e, h - 0.35 pg/ ml Chlormephos; f, i - 3.5 pg/ml Chlormephos). Arrows in panels g, h and i represent newly developed adult Leydig cells. Bar = 50 pm a b c d e f . .. * 9 h *' ,5 '"/¿'v ' ■>],'« Effects of organophosphate Chlormephos on development and function of testes and brain 45 Anxiety-like behavior test Elevated-plus maze tests at 70 days of age results revealed statistically significant differences between control mice and mice neonatally exposed to higher dose of Chlormephos. There was a significant effect of treatment in both time spent in open arms (p < 0.05) and time spent in closed arms (p < 0.05). Posthoc bonferroni test revealed significant difference between control groups and group exposed neonatally to 3.5pg per ml of water, but no difference between control group and group exposed neonatally to lower dose of Chlo-rmephos. However, there was no significant difference between control and both Chlormephos exposed groups in latency to enter open arms or number of entrances into the open arm of elevated plus maze (Table 1). Table1: Statistical analysis of behavior of mice in elevated plus maze revealed significant difference (* = p < 0.05) between control group and group exposed to 3.5 pg/ml Chlormephos in both time spent in open arms and time spent in closed arms. There was no significant difference between groups in latency to enter open arms or number of entries to open arms, and no difference in any of parameters between control group and group exposed to 0.35 pg/ml Chlormephos. All data are presented as mean ± SEM Time spent in closed arms (s) Time spent in open arms (s) Number of entries into open arms Latency to enter open arms (s) Male control (n=10) 73.12 + 40.18 177.34 + 38.63 4.11 + 1.38 28.16 + 21.75 Female control (n=13) 113.19 + 22.31 124.16 + 23.18 5.13 + 1.06 53.53 + 20.84 Male Cl0.35 (n=8) 71.50 + 21.36 170.37 + 38.79 7.67 + 2.11 22.92 + 7.26 Female Cl0.35 (n=8) 113.08 + 29.87 148.65 + 37.50 5.83 + 1.54 53.68 + 32.03 Male Cl3.5 (n=10) 96.08 + 22.66* 105.19 + 29.25* 5.31 + 1.62 47.98 + 32.83 Female Cl3.5 (n=13) 162.44 + 16.88* 50.66 + 14.16* 3.45 + 0.88 84.10 + 30.67 Electron microscopy Detailed analysis of brain - blood barrier in the cortex region from offspring of control mice and mice treated with higher concentration of Chlormephos (3 females and 3 males in each group) did not reveal any structural changes. In brains from control and treated mice, brain capillaries displayed normal morphology: endothelial cells had a flattened nucleus and an elongated cytoplasm. The euchro-matin was located in the central region of the nucleus, whereas a NP NP Figure 2: Blood vessel ultrastructure appeared to be intact in the brain from 70 days old mice exposed neonatally to 3.5 ^g/ml Chlormephos. np> i. NP - neuropil; e - erythrocyte; ec - endothelial cell; bl - basal lamina; m - mitochondria; arrows - junctional complexes (2 - magnification 3000x; 2a - magnification 7000x) 46 K. Čeh, D. Ježek, M. C. Žužek, N. Čebulj-Kadunc, G. Majdič thick area of heterochromatin was arranged along the nuclear membrane. Within endothelial cells, one could occasionally observe a number of endo-cytotic vacuoles that were located either in the cell cytoplasm or associated with the cell membrane. Neighboring endothelial cells were joined by well-developed junctional complexes and laid on a continuous basement lamina. There was no damage or disruption of the basement membrane and/or endothelial cell cytoplasm. Capillaries displayed a normal perivascular space in all investigated animals. Neurons and the accompanying glial cells and neuropil preserved normal fine structure in the Chlormephos treated animals when compared to controls (Figure 2). Discussion Endocrine disrupting chemicals (EDC) are a group of chemicals that could potentially interfere with an endogenous hormonal system. Most EDCs could interfere with endocrine system by binding to estrogen or androgen receptors although other mechanisms such as interference with expression of activity of steroidogenic enzymes or interactions with aryl-hydrocarbon receptor were described (18, 19). Exposure of animals and human to EDCs could disturb development and function of reproductive system, and possibly of central nervous system (20). The aim of our study was to determine if Chlormephos, an organophosphorous compound, could acts as endocrine disruptor. Several studies suggested that different organo-phosphorus compounds can act as EDCs. Naraya-na et al. (2005) reported, that treatment with metyl parathion in doses relevant for human exposure (0.5-1 mg/kg) resulted in decreased number of spermatozoa and higher number of morphological defective spermatozoa in treated mice, although this treatment did not affect the number of offspring in treated rats. Several other studies also reported effects of certain organophosphorus compounds can affect endocrine system in humans (21, 22) and animals (23, 24, 25, 26). Recio et al. evaluated relationship between OP metabolites in urine and serum levels of pituitary and sex hormones of agricultural workers exposed to mixture of different OP (methylparathion, metamidophos, endosulfan, dimethoate and diazinon) and reported that OP disrupted FSH and LH levels (22). Okahashi et al. (2005) studied effects of orga-nophosphorus insecticide fenithrotion in rats. Fe- nithrotion in high doses decreased the activity of brain cholinesterase in parental generation, but did not affect reproduction, organ weight, his-topathology and semen parameters in rats treated with sub-lethal doses of fenithrotion. Furthermore, offspring of exposed mice did not differ from control groups in anogenital distance, beginning of puberty, organ weights and histopathology suggesting that fenithrotion did not act as endocrine disruptor (27). These results are similar to the results from our study where we could not find any differences between control and treated groups in any reproductive parameters studied. Our results therefore suggest, that Chlormephos does not act as endocrine disruptor and therefore does not pose a risk for endocrine system even if it would be released into the environment. In addition to effects on reproductive organs, some endocrine disruptors could also affect central nervous system development and/or function. Some studies have suggested that different chemicals such as bisphenol A and metoxychlor, can affect sexual and non-sexual behaviors (28, 29, 30, 31). Exposure to malathion, chlorpyrifos and other OP during development can result in long-term impacts on intellectual function and delayed effects on the functioning of the central nervous system (8, 32, 33). Blood cholinesterase was also decreased in Wistar rats exposed to low doses of dichlorvos, relevant for human exposure (24). Animals exposed to higher dose of Chlorme-phos in our study showed increased anxiety-like behavior in comparison to the control group. Previous studies have shown that organophosphates could negatively affect growth and terminal differentiation of nerve axons in the developing brain, thus causing long lasting changes in the brain that could be expressed only in the adult life (34, 35, 36). Organophosphates are liposoluble, so they can pass across the blood-brain barrier and come into direct contact with nerve cells, causing lasting damage to these cells. Several studies also revealed that low doses of organophosphates could have deleterious, long lasting effect on the blood-brain barrier at least in rodents (9, 10, 37). Interestingly, Song et al. (37) have shown structural damage in the blood-brain barrier in adult rats, exposed neonatally to paroxon, however, when paroxon was given to adult rats, no effect on blood-brain barrier was observed, suggesting that blood-brain barrier might be especially sensitive to paroxon during neonatal period. We exam- Effects of organophosphate Chlormephos on development and function of testes and brain 47 ined cerebral cortex in control and treated mice using electron microscopy, but could not detect any defects in the structure of the brain tissue at the ultrastructural level. In particular, there were no differences in capillaries' walls that are part of blood-brain barrier. However, only small part of the brain was examined by electron microscopy and therefore, it is not possible to firmly conclude that blood-brain barrier was not affected perhaps in some other specific area. In conclusion, our results showed that Chlo-rmephos treatment of parents did not disturb development of testes and reproductive tract in the offspring of treated mice. Therefore, our study suggest that Chlormephos does not act as an endocrine disruptor in the reproductive system. However, treatment with higher dose of Chlormephos (3.5 pg/ml) produced long lasting effects on behavior as evident by increased anxiety like behavior in mice exposed to Chlormephos only in neonatal period through mothers. This study, therefore, suggests that Chlormephos as a representative organophosphorous compound could affect brain development in experimental animals and further studies are needed to examine possible long lasting effects of organophosphorous compounds on brain development in humans. References 1. Crews D, McLachlan JA. Epigenetics, evolution, endocrine disruption, health, and disease. Endocrinology 2006; 147: S4-10. 2. Weinbroum AA. Pathophysiological and clinical aspects of combat anticholinesterase poisoning. Br Med Bull 2004; 72: 119-33. 3. Gupta RC. Veterinary toxicology. London, New York: Academic Press, Elsevier, 2007: 477-88. 4. Hrzenjak R, Zidar P, Virant Celestina T, Kozuh Erzen N, Kolar L. Toxicity of organophosphorus insecticide chlormephos to the earthworm Eisenia andri and the terrestrial isopo Porcellio scaber. Acta Biol Slov 2009; 52: 85-94. 5. Sarkar R, Mohanakumar KP, Chowdhury M. Effects of an organophosphate pesticide, quinal-phos, on the hypothalamo-pituitary-gonadal axis in adult male rats. J Reprod Fertil 2000; 118: 29-38. 6. Verma R, Mohanty B. Early-life exposure to dimethoate-induced reproductive toxicity: evaluation of effects on pituitary-testicular axis of mice. Toxicol Sci 2009; 112: 450-8. 7. Uzumcu M, Kuhn PE, Marano JE, et al. Early postnatal methoxychlor exposure inhibits folliculogenesis and stimulates anti-Mullerian hormone production in the rat ovary. J Endocrinol 2006; 191: 549-58. 8. Rauh VA, Garfinkel R, Perera FP, et al. Impact of prenatal chlorpyrifos exposure on neurodevelopment in the first 3 years of life among innercity children. Pediatrics 2006; 118: e1845-59. 9. Gupta A, Agarwal R, Shukla GS. Functional impairment of blood-brain barrier following pesticide exposure during early development in rats. Hum Exp Toxicol 1999; 18: 174-9. 10. Sinha C, Shukla GS. Species variation in pesticide-induced blood-brain barrier dysfunction. Hum Exp Toxicol 2003; 22: 647-52. 11. Coronado GD, Vigoren EM, Thompson B, et al. Organophosphate pesticide exposure and work in pome fruit: evidence for the take-home pesticide pathway. Environ Health Perspect 2006; 114: 999-1006. 12. Katz JM, Winter CK. Comparison of pesticide exposure from consumption of domestic and imported fruits and vegetables. Food Chem Toxicol 2009; 47: 335-8. 13. University of Hertfordshire. Pesticide Properties Data Base. Chlormephos http://sitem.herts. ac.uk/aeru/footprint/en/index.htm. 9.8.2011. 14. Thayer KA, Ruhlen RL, Howdeshell KL, et al. Altered prostate growth and daily sperm production in male mice exposed prenatally to subclinical doses of 17alpha-ethinyl oestradiol. Hum Reprod 2001; 16: 988-96. 15. Kovačevič K, Budefeld T, Majdič G. Reduced seminiferous tubule diameter in mice neo-natally exposed to perfume. Slov Vet Res 2006; 43: 177-83. 16. Schneider P, Ho YJ, Spanagel R, et al. A novel elevated plus-maze procedure to avoid the one-trial tolerance problem. Front Behav Neurosci 2011; 5: e43 (7 str.) 17. Ceh K, Cebulj-Kadunc N, Majdic G. Vpliv nizkih doz organofosfatov na razvoj in delovanje spolnih organov pri miših. In: Kozuh-Erzen N, ed. Proučevanje škodljivih učinkov in mehanizmov dolgotrajnega delovanja organofosfornih snovi na ljudi, živali in okolje in načrtovanje ustreznih protiukrepov. Ljubljana: Oikos, 2009: 51-8. 18. McLachlan JA. Environmental signaling: what embryos and evolution teach us about endocrine disrupting chemicals. Endocr Rev 2001; 22: 319-41. 48 K. Čeh, D. Ježek, M. C. Žužek, N. Čebulj-Kadunc, G. Majdič 19. Mnif W, Hassine AI, Bouaziz A, et al. Effect of endocrine disruptor pesticides: a review. Int J Environ Res Public Health 2011; 8: 2265-303. 20. Rasier G, Toppari J, Parent AS, et al. Female sexual maturation and reproduction after prepubertal exposure to estrogens and endocrine disrupting chemicals: a review of rodent and human data. Mol Cell Endocrinol 2006; 254/255: 187-201. 21. Guven M, Bayram F, Unluhizarci K, et al. Endocrine changes in patients with acute organophosphate poisoning. Hum Exp Toxicol 1999; 18: 598-601. 22. Recio R, Ocampo-Gomez G, Moran-Martinez J, et al. Pesticide exposure alters follicle-stimulating hormone levels in Mexican agricultural workers. Environ Health Perspect 2005; 113: 1160-3. 23. Narayana K, Prashanthi N, Nayanatara A, et al. Effects of methyl parathion (o,o-dimethyl o-4-nitrophenyl phosphorothioate) on rat sperm morphology and sperm count, but not fertility, are associated with decreased ascorbic acid level in the testis. Mutat Res 2005; 588: 28-34. 24. Okamura A, Kamijima M, Shibata E, et al. A comprehensive evaluation of the testicular tox-icity of dichlorvos in Wistar rats. Toxicology 2005; 213: 129-37. 25. Okamura A, Kamijima M, Ohtani K, et al. Broken sperm, cytoplasmic droplets and reduced sperm motility are principal markers of decreased sperm quality due to organophosphorus pesticides in rats. J Occup Health 2009; 51: 478-87. 26. Maitra SK, Mitra A. Testicular functions and serum titers of LH and testosterone in methyl parathion-fed roseringed parakeets. Ecotoxicol Environ Saf 2008; 71: 236-44. 27. Okahashi N, Sano M, Miyata K, et al. Lack of evidence for endocrine disrupting effects in rats exposed to fenitrothion in utero and from weaning to maturation. Toxicology 2005; 206: 17-31. 28. Palanza P, Gioiosa L, vom Saal FS, et al. Effects of developmental exposure to bisphenol A on brain and behavior in mice. Environ Res 2008; 108: 150-7. 29. Palanza P, Morellini F, Parmigiani S, et al. Ethological methods to study the effects of maternal exposure to estrogenic endocrine disrupters: a study with methoxychlor. Neurotoxicol Teratol 2002; 24: 55-69. 30. Panzica GC, Viglietti-Panzica C, Mura E, et al. Effects of xenoestrogens on the differentiation of behaviorally-relevant neural circuits. Front Neuroendocrinol 2007; 28: 179-200. 31. Gioiosa L, Fissore E, Ghirardelli G, et al. Developmental exposure to low-dose estrogenic endocrine disruptors alters sex differences in exploration and emotional responses in mice. Horm Behav 2007; 52: 307-16. 32. Eskenazi B, Marks AR, Bradman A, et al. Organophosphate pesticide exposure and neurodevelopment in young Mexican-American children. Environ Health Perspect 2007; 115: 792-8. 33. Eskenazi B, Rosas LG, Marks AR, et al. Pesticide toxicity and the developing brain. Basic Clin Pharmacol Toxicol 2008; 102: 228-36. 34. Slotkin TA, Levin ED, Seidler FJ. Comparative developmental neurotoxicity of organophosphate insecticides: effects on brain development are separable from systemic toxicity. Environ Health Perspect 2006; 114: 746-51. 35. Howard AS, Bucelli R, Jett DA, et al. Chlorpyrifos exerts opposing effects on axonal and dendritic growth in primary neuronal cultures. Toxicol Appl Pharmacol 2005; 207: 112-24. 36. Qiao D, Seidler FJ, Tate CA, et al. Fetal chlorpyrifos exposure: adverse effects on brain cell development and cholinergic biomarkers emerge postnatally and continue into adolescence and adulthood. Environ Health Perspect 2003; 111: 536-44. 37. Song X, Pope C, Murthy R, et al. Interactive effects of paraoxon and pyridostigmine on blood-brain barrier integrity and cholinergic toxicity. Toxicol Sci 2004; 78: 241-7. Effects of organophosphate Chlormephos on development and function of testes and brain 49 VPLIV ORGANOFOSFATA KLORMEFOSA NA RAZVOJ IN DELOVANJE MOD IN MOŽGANOV K. Čeh, D. Ježek, M. C. Žužek, N. Čebulj-Kadunc, G.Majdič Povzetek: Različne kemične snovi, pogosto imenovane hormonski motilci, lahko motijo delovanje hormonskega sistema v sesalskem telesu. Namen naše raziskave je bil ugotoviti, ali dolgotrajna izpostavljenost organofosfornemu insekticidu klor-mefosu lahko vpliva na normalen razvoj spolnega sistema in endokrinega dela možganov. Odraslim mišim obeh spolov smo preko pitne vode dodajali klormefos v koncentracijah 3,5 Mg/ml in 0,35 |jg/ml. Pari miši so bili izpostavljeni klormefosu 2 tedna pred paritvijo, ves čas brejosti in nato do odstavitve mladičev pri 3 tednih starosti. Razvoj mod pri potomcih izpostavljenih miši smo proučevali s spremljanjem teže mod, dnevne proizvodnje semenčic, apoptoze v modih ter izraženosti beljakovin an-timulerjevega hormona in 3-beta hidroksisteroidne dehidrogenaze. Pri nobenem od preiskovanih parametrov nismo ugotovili statistično zanesljivih razlik med kontrolno skupino in skupinama, izpostavljenima klormefosu, kar kaže, da klormefos ne deluje kot hormonski motilec spolnega sistema. Za ugotavljanje morebitnih škodljivih učinkov klormefosa na možgane v času razvoja, smo pri potomcih izpostavljenih miši ugotavljali obnašanje, podobno anskioznemu s testiranjem v dvignjenem labirintu. Miši, ki so bile med razvojem izpostavljene višji koncentraciji klormefosa, so kazale statistično značilno poudarjeno obnašanje, podobno anksioznemu (več časa so preživele v zaprtih krakih labirinta), v primerjavi s kontrolno skupino. Preiskava možganov z elektronskim mikroskopom pa ni pokazala nikakršnih razlik v ultrastrukturi možganov in krvno-možganske pregrade, čeprav nekatera poročila kažejo, da naj bi bila prav krvno-možganska pregrada prizadeta ob zgodnji izpostavljenosti organofosfornim snovem. Ključne besede: miš; modo; organofosforne snovi; dnevna proizvodnja semenčic; apoptoza; elektronska mikroskopija; anksioznemu podobno obnašanje. Slov Vet Res 2012; 49 (1): 51- 8 UDC 636.2.082.4:591.463.1 Original Scientific Article IMPROVEMENT OF SPERM SORTING EFFICIENCY AND FERTILIZING CAPACITY EMPLOYING TWO VARIATIONS OF A NEW BULL SEMEN ExTENDER (SExCESS®) Primož Klinc1*, Detlef Rath2 1Clinic for Reproduction and Horses, Veterinary Faculty, Gerbičeva 60, 1000 Ljubljana, Slovenia; 2Friedrich-Loeffler-Institut, Institute of Farm Animal Genetics Hoeltystr. 10, 31535 Neustadt, Germany Corresponding author, E-mail: primoz.klinc@vf.uni-lj.si Summary: The aim of the present study was to analyse the effect of temporary chemical inhibition of sperm motility before and during flow cytometrical sorting on the quality and fertilizing capacity of sex sorted and frozen/thawed spermatozoa. The quality and fertilizing capacity of temporarily immobilized spermatozoa sorted in the presence of sodium fluoride (S-AO-NF) was compared with sorted semen using a standard protocol as developed in our laboratory (S-AO) and with unsorted control (C). Semen from two bulls (Holstein Friesian and Limousine) was used and in total 283 first inseminations with 2 million live, frozen/thawed sex sorted and unsorted spermatozoa were performed on 197 farms. Motility of spermatozoa was completely inhibited after addition of sodium fluoride and after sorting and centrifugation returned to a similar degree as before. Motility after thawing and incubation at 37°C did not differ between groups. After further incubation at 37°C for 12 h and 24 h motility was significantly higher (P<0.001) in both sorted groups compared to unsorted semen. The percentage of spermatozoa with acrosome abnormalities in frozen/ thawed samples was significantly higher (P<0.05) in S-AO and C group when compared to S-AO-NF group. The same difference was found for morphological sperm abnormalities. The percentage of acrosome reacted and viable spermatozoa was analysed by FITC-PNA/PI staining. The percentage of acrosome reacted spermatozoa was significantly lower (P<0.001) in both groups of sorted semen when compared to unsorted control. The percentage of viable spermatozoa was significantly higher (P<0.05) in the S-AO-NF group when compared to groups S-AO and C. Pregnancy rates after artificial insemination with the frozen/thawed semen were not significantly different between sorted spermatozoa and controls (72.7 % vs. 73.3 % vs. 79.2 % for S-AO-NF, S-AO and C respectively). Key words: semen; spermatozoa; flow cytometry; sorting; sex; freezing / thawing; artificial insemination; pregnancy Introduction Like in other domestic animal species, a broader utilisation of sexed sperm would be very beneficial for cattle industry. So far, only flow cytometry allows producing a significant shift of sex ratios. However, the technique still has its limitations in sorted sperm output (1, 2), although many improvements have helped in the recent years to start first commercial applications as shown in UK and Texas, USA. Received: 17 January 2012 Accepted for publication: 22 March 2012 Especially introduction of high speed flow cy-tometry (3), better orientation of spermatozoa in front of the Laser beam (4) and improvements in sperm preparation before and after sorting by special media components (unpublished data) allow to minimize the sperm concentration and use liquid as well as frozen/thawed spermatozoa. However, fertilizing potential of sorted frozen/thawed spermatozoa has not reached the quality of unsorted analogues (5). The lifetime after sorting and freezing/ thawing is limited and even with adapted insemination protocols, the number of calves born per AI or number of AI necessary to get the same fertility rates are critical (5). A potential solution might be to 52 P. Klinc, D. Rath safe as much energy as possible during processing and sorting. This might be achieved with reversible, chemical inhibition of the sperm metabolism. Ornidazone (6), bromoydroxypropanone (7), nitric oxide (8), alfa-chlorhydrin (9), gossypol (10), Cytochalasin B (11), formaldehyde (12) and sodium fluoride (13, 14) are examples for chemicals, known to have an inhibiting effect on sperm motility. Dott et al. (12) were able to achieve pregnancies after artificial insemination in ewes and sows with formaldehyde-immobilized spermatozoa. As the toxicity of formaldehyde is high (15), specific inhibition of sperm motility may be performed by sodium fluoride (13). Sodium fluoride is a conventional inhibitor of protein-thyrosine-phosphatase (16), which is included in the process of sperm capacitation and motility (17, 18) and was reported as immobilizing substance for bull spermatozoa (19). Flow cytometrical sorting could benefit from temporarily inhibition of sperm activity not only for the prolongation of sperm lifetime, but also to get a better orientation of spermatozoa in front of the Laser beam and to reduce individual bull effects during staining and sorting. In the present study, we analysed the effect of temporary sperm immobilization with sodium fluoride on sorting efficiency, quality and fertility of frozen thawed bull spermatozoa. Materials and methods L-a-Lysophosphatidylcholine (L 5004); Lectin, FITC labelled from Arachis hypogaea (peanut) (L7381); Propidium iodide (P4170), 6-Carboxy-fluorescein diacetate (C5041); Bisbenzemide H 33342 (B2261), Pyruvic Acid (P-3662) and Cata-lase (C-1345) were purchased from Sigma-Aldrich (Taufkirchen, Germany) all the other chemicals, if not specially stated, were purchased from Carl Roth GmbH + Co (Karlsruhe, Germany). Methods Semen ejaculates were collected from fertility proven and performance tested Holstein Friesian bull (Bull 1, 11 ejaculates) with high fertility rate and a young Limousine bull (Bull 2, 4 ejaculates) in performance test. Ejaculates that were included into research had to reach minimum criteria of at least 70% motility and 80% of morphological normal spermatozoa. Ejaculates were kept in a water bath at 27°C and motility was estimated under phase contrast microscope (Olympus BX 60) at 37°C. Morphology was analysed after fixation in Hancock fixative (2.784 g Tri-Natriumcitrat-Dihydrat, 4 mL 37 % formaldehyde solution and bi-distillated water to 100 mL), within 4 hours after semen collection. Concentrations of spermatozoa in ejaculates were determined with a haemocytometer (Coulter counter®). Raw semen was divided into two parts and diluted to 1x108 spermatozoa/mL either with the commercially available extender Sexcess® AX (Masterrind Verden, Germany), supplemented with antioxidants and BSA fraction V (group S-AO), or Sexcess® FX additionally containing sodium fluoride (group S-AO-FX). Spermatozoa were labelled with 15, 20 and 25 ^L of a 8.12 mM Hoechst 33342 solution and incubated for 1 hour at 34°C. Thereafter, labelled semen samples were kept at 22°C in the dark and sorted within 7 hours after onset of incubation. The labelled semen samples were pre-tested for maximal separation with a flow cy-tometer and the concentration of Hoechst 33342 stain giving the best resolution of two sperm populations was used for daily sorting. The other part of the ejaculate, which was not used for sorting, was frozen following a routine protocol of a commercial AI center and served as control. Briefly, semen was gradually diluted with TRIS egg-yolk freezing extender I to the concentration of 26.4x106 spermatozoa/ml, cooled to 5°C within two hours and further diluted with TRIS egg-yolk freezing extender II containing 0.75 % detergent (Equex STM®, Nova Chemical sales, Inc., Scituate, USA) and 12.8 % (v/v) of 87 % Glycerol to a concentration of 13.2x106 spermatozoa / mL or 3.3x106 spermatozoa / straw, filled into 0.25 mL straws (Minitub, Tiefenbach, Germany), and frozen in liquid nitrogen (LN2). Briefly, straws were placed on metal holder in nitrogen vapour 3-5 cm above LN2 for 15 minutes. Frozen samples were then plunged into LN2 and kept in the semen container (in LN2) until used for analysis or artificial insemination. Processing of the sorted spermatozoa Sperm sorting was performed according to the Beltsville Sperm Sorting Technology (3). Sorted spermatozoa of group S-AO were collected into collection fluid (max. 8 million spermatozoa per tube) and then centrifuged at 838xg for 20 minutes. Centrifugation of sorted spermatozoa in group S- Improvement of sperm sorting efficiency and fertilizing capacity employing two variations of a new bull semen extender (Sexcess®) 53 AO-NF allowed lower force (500xg for 15 minutes) because cells were immobilized and gave a more distinct pellet. Supernatant was discharged and the remaining sperm pellet of both groups was diluted with extender Sexcess I® (Masterrind Verden, Germany) to a concentration of 41x106 spermatozoa /mL and cooled to 5°C within 2 hours. Once 5°C was reached, semen samples were further diluted with extender Sexcess II® (Masterrind Verden, Germany) to a concentration of 20.5x106 spermatozoa/mL. Plastic straws (0.25 mL) (Min-itub, Tiefenbach, Germany) were pre-filled with a first segment (50 mL of a mixture made from extender I and II), and with a second segment, filled with 160 mL sorted semen (3.3 millions spermatozoa in total, equivalent to approx. 2 x106 life spermatozoa). Semen samples were frozen in liquid nitrogen as already described for control samples. Sperm analysis of frozen thawed samples Motility analysis Sperm motility of raw semen, of sperm samples before and after sorting and after thawing was analysed. Prior to analyses samples were pre-warmed to 37°C for 15 minutes and analysed under a phase-contrast microscope (OlympusBX60) at 100x magnification, equipped with heating plate to maintain 37°C. From each sample two 6 mL drops were transferred onto pre-wormed objective glass and covered with coverslip glass. At least three fields were evaluated per drop. Morphology analysis Morphology was analysed after fixation of the samples in Hancock solution under a phase-contrast microscope (Olympus BX 60) at 1000x magnification. At least 200 spermatozoa were examined per sample for morphological abnormalities (MAS) and acrosome integrity. Spermatozoa were divided into two groups. Spermatozoa that had any pathological change of acrosome were included into group named damaged acrosomes (DA). Group MAS corresponds to the percentage of spermatozoa with damaged acrosomes plus spermatozoa that had any other morphological abnormalities. Acrosome integrity and membrane stability of spermatozoa (FITC-PNA/PI) Acrosome integrity and membrane stability were analysed with FITC-PNA/PI as described previously (20). Pre-warmed Eppendorf cups were filled with 50 mL of semen and 1 mL FITC-PNA (2 mg FITC-PNA in 2 mL PBS) as well as 2 mL PI (1 mg propidium iodide in 10 mL physiological NaCl solution) were added. Samples were incubated at 38°C for 5 minutes and supplemented with 5 mL paraformaldehyde (1 % in PBS) immediately before microscopic examination. At least 200 spermatozoa were examined under a fluorescence and phase contrast microscope (Olympus BX 60; U-MNIB filter) at 400x magnification. Spermatozoa were divided into four groups: 1. PNA-nega-tive/PI-negative (viable spermatozoa with intact acrosome); 2. PNA-negative/PI-positive (spermatozoa with damaged plasma membrane and intact acrosome); 3. PNA-positive/PI-positive (spermatozoa with damaged plasma membrane and reacted acrosome); 4. PNA-positive/PI-negative (spermatozoa with intact plasma membrane and reacted acrosome). Mean percentages of viable spermatozoa with intact membranes (group 1) and acro-some reacted spermatozoa (group 3 and 4) are presented in the results. Artificial insemination and pregnancy control All the straws were coded in order to make it impossible for inseminators to know the content. Straws were randomly distributed to three well experienced AI technicians within 1.5 month after sorting. For AI, semen samples were thawed at 37°C for 20 sec. and inseminated 12-24 hours after onset of natural oestrus. Technicians were advised to insert a normal AI catheter under rectal control as deep as possible into the uterine horn but without extra force or strong manipulation. In cases where it was difficult to determine the location and size of the follicle, semen was either deposited into the uterine body or the content of the straw was split for AI in both uterine horns. Pregnancies were controlled 30-60 days after insemination by transrectal examination and transrectal ultrasonography (Aloka®; 5 MHz). 54 P. Klinc, D. Rath Statistical analyses Statistical analyses were performed with SIGMA STAT 2.03 for windows (Jandel Scientific Cooperation, San Ragael, CA, USA). Pregnancy rates were analysed with Chi-square test. Semen quality parameters were tested for normal distribution and either analysed with One-way ANOVA or ANOVA and Ranks and Tukey test. Data are expressed as percentages or means ± SD. Differences were considered to be significant at P<0.05. Results In the raw semen samples 72.1 ± 3.7 % spermatozoa were motile. After labelling and before sort- ing in the S-AO group 66.9 ± 4.6 % were motile, whereas all spermatozoa in the S-AO-NF group were immobilized. After sorting the percentage of motile spermatozoa in S-AO-NF group was 72.9 ± 4.9 %, whereas sperm motility in S-AO samples was 70.6 ± 5.6%. The difference was not significant (P<0.05). Sperm motility after thawing and incubation at 37°C for 6 h was not significantly different between groups. Further incubation of frozen thawed spermatozoa for 12 and 24 hours showed significant higher motility of spermatozoa in both sorted groups compared to unsorted standard processed semen (Table 1). Table 1: Sperm motility of sorted and control groups immediately after thawing (0 h) and after further incubation at 37°C for 6, 12 and 24 h 0 h (%) 6 h (%) 12 h (%) 24 h (%) Control S-AO S-AO-NF 61.5 ± 3.8 59.4 ± 4.2 59.0 ± 5.9 45.8 ± 9.3 45.6 ± 9.4 47.9 ± 7.0 7.5 ± 11.6a(L) 33.1 ± 6.5b 37.1 ± 4.9b 0.0 ± 0.0a 21.3 ± 10.3b(l) 22.9 ± 7.6b(L) Values with different superscripts differ significantly (P<0.001). Data are shown as mean ± SD. (L) - only local motile spermatozoa were observed. After thawing the percentage of spermatozoa with damaged acrosomes and morphologically abnormal spermatozoa were significant lower in S-AO-NF group compared to S-AO group and un-sorted control samples (Table 2). Table 2: Percentage of spermatozoa with damaged acro-somes (DA) and morphologically abnormal spermatozoa (MAS) DA (%) MAS (%) Control 22.3 ± 4.0a 29.0 ± 5.8a S-AO 23.0 ± 5.2a 30.0 ± 6.7a S-AO-NF 14.7 ± 7.5b 19.0 ± 8.1b Values with different superscripts differ significantly (P<0.05). Data are shown as mean ± SD. The percentage of acrosome reacted spermatozoa as seen after FITC-PNA staining was significantly lower in both sorted groups as compared to unsorted control samples. The percentage of viable spermatozoa was significantly higher in S-AO-NF group compared to S-AO group and unsorted control (Table 3). Table 3: Percentage of acrosome reacted (AR) and spermatozoa with intact membranes (viable) in frozen/thawed samples, according to FITC-PNA/PI staining AR (%) Viable (%) Control 19.1 ± 4.7a 61.4 ± 8.2c S-AO 10.6 ± 3.5b 56.9 ± 4.7c S-AO-NF 6.7 ± 2.6b 72.1 ± 5.0d Values with different superscripts are significantly different: a:b P<0.001; c:d P<0.05-Data are shown as mean ± SD. In total 283 inseminations were performed under field conditions on 197 Slovenian farms. Mostly it was one insemination per farm, than two inseminations were performed on 30 farms, 3 on 11 farms, 4 on 3 different farms and 5 inseminations on 1 farm. Table 4 shows the pregnancy results. No differences were found between sorted and un-sorted semen samples or among sorted samples. Reanalysis of semen after thawing revealed 92.5 % average purity for X-chromosome bearing spermatozoa. Improvement of sperm sorting efficiency and fertilizing capacity employing two variations of a new bull semen extender (Sexcess®) 55 Table 4: Pregnancy rates after artificial insemination with sorted frozen/thawed spermatozoa diluted in Sexcess® AX or Sexcess® FX Control Sexcess® AX (S-AO) Sexcess® FX (S-AO-NF) Bull 1(%) 75.5 % 73.5 % 62.1 % Pregnant/all (n/n) 37/49 36/49 36/58 Bull 2 (%) 79.2 % 73.3 % 72.7 % Pregnant/all (n/n) 42/53 32/44 22/30 Total (%) 77.5 % 73.1 % 65.9 % Total (n/n) 79/102 68/93 58/88 No differences in pregnancy rates were seen among AI technicians (69.1, 70.1 and 77.9 %). Place of the semen deposition was recorded in 260 cases. It had a significant effect (P<0.05) on the pregnancy rates and were significantly lower after semen deposition into the uterine body and into uterine horn ipsilateral to the ovulation site compared to those inseminations where the content of the straw was divided and inseminated into both horns. Table 5: Effect of place of semen deposition on pregnancy rates AI place all/pregnant % Uterine body 50/33 66.0a Ipsilateral to ovulation 130/88 67.7a both horns 80/66 82.5b Values with different superscripts are significantly different: a:b P<0.05 Discussion In the present study, we have demonstrated that optimal handling according to the modified Beltsville Sperm Sorting Technology (3) and protection during staining, sorting, centrifuga-tion, diluting and freezing of the sorted spermatozoa leads to high quality of sexed semen and to pregnancy rates that are comparable with those of unsorted controls after artificial insemination under normal field conditions. However, it has to be mentioned that all experiments were performed with semen from selected bulls. Selection criteria were that non return rates were above average and that staining and sorting was homogenous between collection days in order to speed up the sorting process. Previous studies demonstrated lower pregnancies rates after artificial inseminations with flow cytometrically sorted spermatozoa when compared to unsorted control. Probable reasons were induction of capacitation, membrane damages (21, 22, 23) and rapid loss of motility (24). In consequence, insemination protocols had to be changed and insemination had to be performed closer to ovulation. In the present study, lifetime of sorted spermatozoa was at least as long as for those in the control group. The percentage of motile spermatozoa was even higher in both groups of sorted semen compared to unsorted controls after incubation at 37°C for 12 and 24 h. This was probably caused by utilization of antioxidants, preventing generation of reactive oxygen species, oxidation of membrane lipids, and loss of motility due to oxidation (25). Addition of sodium fluoride caused a complete, reversible inhibition of sperm motility and seemed to reduce interference of mitochondria in the mid piece of the sperm tail with the high voltage electric field, necessary to separate both sperm populations. Sorted spermatozoa regained their motility after sorting and centrifugation completely, which shows the complete reversibility of motility inhibition with sodium fluoride. Chinoy and Naraya (26) reported toxic effects of sodium fluoride on human spermatozoa, causing irreversible damages like elongated heads, deflagel-lation, acrosome loss and coiling of the tails. However, in the present study, concentration of sodium 56 P. Klinc, D. Rath fluoride was much lower and enabled spermatozoa to completely regain motility. So far, no negative effects of sodium fluoride were seen. In opposite, the highest sperm membrane integrity was found in those samples that were diluted with Sexcess® FX. In addition, these samples also showed better sperm quality after sorting, which might mainly be due to shorter centrifugation time and force. Laboratory results were confirmed by pregnancy data that were obtained from an AI field trial in heifers. No significant differences were seen between inseminations with sorted vs. unsorted semen. The handling was as easy as with unsorted semen and no significant effect on the insemination results were found among AI technician. However, the site of semen deposition in the female genital tract had significant effect on pregnancy rates. Williams et al. (27) reported reduced fertility after cervical deposition of the semen compared to uterine body or bicornual insemination, and Senger et al. (28) saw significantly more pregnant animals after bicornual insemination compared to insemination into the uterine body. Accordingly, in the present study significantly more animals became pregnant after bicornual compared to uterine body insemination. This is probably because more spermatozoa were lost during transport to the fertilization site after they were deposited into the uterine body. It is also interesting that lower pregnancy rates were obtained after semen was deposited into the ipsilateral uterine horn compared to the bicornual insemination. We believe that the reason could be due to the failure of correct diagnosis of ovulation site and consequently more inseminations were performed into the wrong uterine horn. It is also possible that the ovary (follicle) was damaged when the inseminator diagnosed the site of follicle growth. In conclusion, the use of Sexcess® extender to optimize bull sperm quality has successfully been approved and improved the fertilizing ability of sexed spermatozoa to a comparable status of un-sorted frozen/thawed bull semen. Further studies will have to analyse more intensively bull effects to broaden the number of males to be included in a sperm sorting based breeding programme. Acknowledgements Authors thank Mrs. Sieg and Mrs. Struckmann and to Veterinary praxis Tenetise for their skilful technical assistance. References 1. Rath D, Ruiz S, Sieg B. Birth of female piglets following intrauterine insemination of a sow using flow cytometrically sexed boar semen. Vet Rec 2003; 152: 400-1. 2. Seidel GE Jr, Schenk JL, Herickhoff LA, et al. Insemination of heifers with sexed sperm. Theriogenology 1999; 52: 1407-20. 3. Johnson LA, Welch GR, Rens W. The Beltsville sperm sexing technology: high-speed sperm sorting gives improved sperm output for in vitro fertilization and AI. J Anim Sci 1999; 77(Suppl 2): 213-20. 4. Rens W, Welch GR, Johnson LA. A novel nozzle for more efficient sperm orientation to improve sorting efficiency of X and Y chromosome-bearing sperm. Cytometry 1998; 33: 476-81. 5. Seidel GE Jr, Garner DL. Current status of sexing mammalian spermatozoa. Reproduction 2002; 124: 733-43. 6. Bone W, Jones NG, Kamp G, et al. Effect of ornidazole on fertility of male rats: inhibition of a glycolysis-related motility pattern and zona binding required for fertilization in vitro. J Reprod Fertil 2000; 118: 127-35. 7. Porter LM, Jones AR. Inhibition of glycer-aldehyde 3-phosphate dehydrogenase in boar spermatozoa by bromohydroxypropanone. Reprod Fertil Dev 1995; 7: 107-11. 8. Rosselli M, Dubey RK, Imthurn B, et al. Effects of nitric oxide on human spermatozoa: evidence that nitric oxide decreases sperm motility and induces sperm toxicity. Hum Reprod 1995; 10: 1786-90. 9. Brown-Woodman PD, White IG. A comparison of the inhibition of the metabolism of ram and rabbit spermatozoa be alpha-chlorohydrin in vitro. Theriogenology 1976; 6: 29-37. 10. Poso H, Wichmann K, Janne J, et al. Gos-sypol, a powerful inhibitor of human spermatozoal metabolism. Lancet 1980; 1(8173): 885-6. 11. Peterson RN, Freund M. Reversible inhibition of the motility of human spermatozoa by cytochalasin B. Fertil Steril 1977; 28: 257-61. 12. Dott HM, Moor RM, Polge C. Artificial insemination with spermatozoa in formaldehyde. J Reprod Fertil 1976; 46: 277. 13. Foote RH. Inhibition of sperm motility does not affect live-dead separation of bull sperm by glass beads. Asian J Androl 2001; 3: 193-8. Improvement of sperm sorting efficiency and fertilizing capacity employing two variations of a new bull semen extender (Sexcess®) 57 14. Wang GL, Jiang BH, Semenza GL. Effect of protein kinase and phosphatase inhibitors on expression of hypoxia-inducible factor 1. Biochem Biophys Res Commun 1995; 216: 669-75. 15. Conaway CC, Whysner J, Verna LK, et al. Formaldehyde mechanistic data and risk assessment: endogenous protection from DNA adduct formation. Pharmacol Ther 1996; 71: 29-55. 16. Errasfa M, Stern A. Inhibition of epidermal growth factor-dependent protein tyrosine phos-phorylation by phorbol myristate acetate is mediated by protein tyrosine phosphatase activity. FEBS Lett 1994; 339: 7-10. 17. Baldi E, Luconi M, Bonaccorsi L, et al. Human sperm activation during capacitation and acrosome reaction: role of calcium, protein phosphorylation and lipid remodelling pathways. Front Biosci 1996; 1: d189-205. 18. Luconi M, Porazzi I, Ferruzzi P, et al. Tyrosine phosphorylation of the a kinase anchoring protein 3 (AKAP3) and soluble adenylate cyclase are involved in the increase of human sperm motility by bicarbonate. Biol Reprod 2005; 72: 22-32. 19. Schoff PK, Lardy HA. Effects of fluoride and caffeine on the metabolism and motility of ejaculated bovine spermatozoa. Biol Reprod 1987; 37: 1037-46. 20. Way AL, Killian GJ. Capacitation and induction of the acrosome reaction in bull spermatozoa with norepinephrine. J Androl 2002; 23: 352-7. 21. Knoeppel S. Lektinbindungsvermoegen von gesextem und ungesextem Sperma. Soest: GHS Padeborn, 2001. 22. Maxwell WM, Long CR, Johnson LA, et al. The relationship between membrane status and fertility of boar spermatozoa after flow cytometric sorting in the presence or absence of seminal plasma. Reprod Fertil Dev 1998; 10: 433-40. 23. Rath D, Johnson LA, Dobrinsky JR, et al. Production of piglets preselected for sex following in vitro fertilization with X and Y chromosome-bearing spermatozoa sorted by flow cytometry. Theriogenology 1997; 47: 795-800. 24. Hollinshead FK, Gillan L, O'Brien JK, et al. In vitro and in vivo assessment of functional capacity of flow cytometrically sorted ram spermatozoa after freezing and thawing. Reprod Fertil Dev 2003; 15: 351-9. 25. Baumber J, Ball BA, Gravance CG, et al. The effect of reactive oxygen species on equine sperm motility, viability, acrosomal integrity, mitochondrial membrane potential, and membrane lipid peroxidation. J Androl 2000; 21: 895-902. 26. Chinoy NJ, Narayana MV. In vitro fluoride toxicity in human spermatozoa. Reprod Toxicol 1994; 8: 155-9. 27. Williams BL, Gwazdauskas FC, Whittier WD, et al. Impact of site of inseminate deposition and environmental factors that influence reproduction of dairy cattle. J Dairy Sci 1988; 71: 2278-83. 28. Senger PL, Becker WC, Davidge ST, et al. Influence of cornual insemination on conception in dairy cattle. J Anim Sci 1988; 66: 3010-16. 58 P. Klinc, D. Rath IZBOLJŠANJE USPEŠNOSTI LOČEVANJA IN OPLODITVENE SPOSOBNOSTI PO SPOLU LOČENIH SEMENČIC Z UPORABO DVEH OBLIK NOVEGA RAZREDČEVALCA ZA BIKOVO SEME (SEXCESS®) P. Klinc, D. Rath Povzetek: V raziskavi smo ugotavljali vpliv začasne kemične zaustavitve gibljivosti semenčic na kakovost in oploditveno sposobnost sortiranih in zamrznjenih/odmrznjenih semenčic. Vpliv smo ugotavljali pred in med postopkom ločevanja semenčic po spolu s pomočjo pretočne citometrije. Kakovost in oploditveno sposobnost semenčic, ki so bile začasno imobilizirane s pomočjo natrijevega fluorida (S-AO-NF), smo primerjali z rezultati, pridobljenimi pri semenu, ki smo ga sortirali po standardnem postopku, razvitem v našem laboratoriju (S-AO), ter kontrolnim nesortiranim semenom (C). V poizkus smo vključili seme dveh bikov. Na 197 kmetijah smo opravili 283 prvih osemenitev z 2 milijonoma živih odmrznjenih sortiranih in nesortiranih semenčic. Gibljivost semenčic, ki se je popolnoma zaustavila po dodatku natrijevega fluorida, se je po sortiranju in centrifugiranju vrnila na predhodno stopnjo. Po tajanju in inkubaciji na 37 °C med skupinami ni bilo statistično značilne razlike v gibljivosti semenčic. Po nadaljnji 12- in 24-urni inkubaciji na 37 °C pa je bila gibljivost semenčic statistično značilno višja (P<0.001) pri sortiranem semenu v primerjavi s kontrolnim semenom. Pri odmrznjenem semenu je bil odstotek semenčic s poškodovanimi akrosomi statistično značilno višji (P<0.05) v skupinah S-AO in C v primerjavi s skupino S-AO-NF. Pri ugotavljanju skupnih morfoloških sprememb smo prav tako ugotovili statistično značilno (P<0.05) višji odstotek spremenjenih semenčic v skupinah S-AO in C v primerjavi s skupino S-AO-NF. S pomočjo barvanja FITC-PNA/PI smo ugotavljali odstotek semenčic z reagiranimi akrosomi in odstotek vitalnih semenčic. V obeh skupinah sortiranega semena smo ugotovili statistično značilno nižji (P<0.001) odstotek semenčic z reagiranimi akrosomi v primerjavi z ne sortiranim semenom. Odstotek vitalnih semenčic je bil statistično značilno višji (P<0.05) v skupini S-AO-NF v primerjavi s skupinama S-AO in C. Po umetni osemenitvi z zamrznjenim/odmrznjenim semenom nismo ugotovili statistično značilne razlike v oploditveni sposobnosti med posameznimi skupinami sortiranega semena in kontrolnim semenom (72.7 % proti 73.3 % proti 79.2 % za skupine S-AO-NF, S-AO in C). Kljub temu da je bil spol potomcev statistično značilno (P<0.05) spremenjen v obeh skupinah sortiranega semena, je bila sprememba statistično značilno (P=0.04) višja pri skupini S-AO-NF v primerjavi s skupino S-AO. Ključne besede: seme; semenčice; pretočna citometrija; ločevanje; spol; zamrzovanje / tajanje; umetno osemenjevanje; brejost © M D Svetovanje Finančne storitve www.vasefina nce. si Izterjava dolgov tn upravljanje s terjatvami Namen ustanovitve in delovanja podjetja MD svetovanje d. o. o. je pomagati podjetjem pri poslovanju z nudenjem produktov in storitev, ki ne spadajo v osnovno dejavnost podjetja. To dosegamo s celovito ponudbo predstavljenih produktov in storitev. Zato smo naš moto Skupaj bomo uspešnejši! nadgradili še z motom in sloganom Vse za Vas na enem mestu! Vizija Postati vodilna neodvisna družba s celotno ponudbo za podjetja in posameznike na enem mestu in na ta način prihraniti podjetjem in posameznikom čas in denar. Vse to nam bo uspelo s trdim delom in kakovostno izvedbo storitev in zaupanih nam nalog, predvsem če bomo sledili naslednjim načelom: zagotavljanje celovite ponudbe, vedno delo v dobro stranke, strokoven razvoj, organizacijsko izpopolnjevanje, zagotavljanje visoke stopnje kakovosti storitev z upoštevanjem predlogov naših strank, ustvarjanje novih delovnih mest, povečanje produktivnosti in dobičkonosnosti, visoko motiviran in usposobljen kader s primernim vodenjem, kar zagotavlja kakovost izvajanja storitev, postati vodilno podjetje, ki ponuja rešitve, ki podjetju omogočajo da si na enem mestu zagotovi vse dejavnosti, ki ne spadajo v njegovo osnovno dejavnost. Prednosti poslovanja z nami: vse svoje potrebe in vizije uresničite s klicem na eno telefonsko številko, razbremenite se ukvarjanja z obrobnimi zadevami, posvetite se svojemu strokovnemu delu, informacijska tehnologija, prilagodljivost, zanesljivost, povečanje dobičkonosnosti, zmanjšanje stroškov dela, MD svetovanje, poizvedbe in storitve d.o.o. Dunajska cesta 421, 1231 Ljubljana - Črnuče PE Ljubljana-Vič Cesta dveh cesarjev 403, 1102 Ljubljana 01 /620-47-01 01 /620-47-04 041 /614-090 www. mdsvetovanje. eu Zakaj M D Svetovanje d.o.o. visoka profesionalizacija, visoka strokovnost, visoka uspešnost, konkurenčne cene, vse na enem mestu. SLOVENIAN VETERINARY RESEARCH SLOVENSKI VETERINARSKI ZBORNIK Slov Vet Res 2012; 49 (1) Original Scientific Articles Cvetkovikj I, Dodovski A, Naletoski I, Mrenoski S, Mitrov D, Krstevski K, Dzadzovski I, Cvetkovikj A. No evidence of avian influenza viruses in backyard poultry fram risk areas in R. Macedonia (Virological and molecular surveillance 2009)..............................................................5 Olechnowicz J, Jaskowski JM. Somatic cell counts and total bacterial count in bulk tank milk of small ruminants..........13 Pelin Can O. The efect of thyme oil on the shelf life of chicken balls during storage period.................................19 Snoj T, Čebulj-Kadunc N, Nemec Svete A, Kobal S. Determination of sex hormones in rat hair after administration of testosterone proprionate and estradiol valerate......................................................27 Satué K, Felipe M, Mota J, Muñoz A. Blood leukocytes in Carthusian mares with endometriosis or endometritis in comparison with healthy mares........................................................................35 Čeh K, Ježek D, Žužek MC, Čebulj-Kadunc N, Majdič G. Effects of organophosphate Chlormephos on development and function of testes and brain.....................................................................41 Klinc P, Rath D. Improvement of sperm sorting efficiency and fertilizing capacity employing two variations of a new bull semen extender (Sexcess®)..................................................................51