Slovenian Veterinary Research Slovenski veterinarski zbornik TH E SC IEN TIFIC JO U RN A L O F TH E V ETERIN A RY FAC U LTY U N IV ERSITY O F LJU BLJA N A Volume 60, Number 1, Pages 1-44 ISSN 1580-4003 Table of Content 5 Editorial Veterinary Illustration: Science and Art Telling a Story TogetherKubale V 9 Review Article Suitability of Alternatives to Rectal Temperature Measurements in Pet Rodents, Rabbits and Ferrets: A Literature Review Stans J 15 Original Research ArticleCharacterisation of the Haematological Profile in the Posavje Horse BreedMesarič M, Nemec M, Čebulj Kadunc N 25 Original Research Article The Effect of a Specific Chicken Based Renal Diet as Monotherapy on Clinical, Biochemical, Urinary and Serum Oxidative Stress Parameters in Cats with CKD Stage 1 and 2 Krofič Žel M, Nemec Svete A, Jakovac Strajn B, Pavšič Vrtač K, Vovk T, Kejžar N, Pavlin D 37 Case Report Computed Tomography and Magnetic Resonance Imaging of a Rhinosinusitis Secondary to a Dental Abscess in a Crested Porcupine (Hystrix cristata) Encinoso M, Morales D, Déniz S, Guerra JV, Jaber JR SloVetRes_naslovnica.indd 1 16/05/2023 09:46 Slovenian Veterinary Research Slovenski veterinarski zbornik TH E SC IEN TIFIC JO U RN A L O F TH E V ETERIN A RY FAC U LTY U N IV ERSITY O F LJU BLJA N A Volume 60, Number 1, Pages 1-44 SloVetRes_NOTRANJOST ver2.indd 1 09/05/2023 10:14 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 Volume 60, Number 1 / Letnik 60, Številka 1 Editor in Chief / Glavna in odgovorna urednica Klementina Fon Tacer Co-Editors / Sourednici Valentina Kubale Dvojmoč, Sara Galac Technical Editor / Tehnični urednik: Matjaž Uršič Assistant to Editor / Pomočnica urednice Metka Voga Published by / Založila: University of Ljubljana Press / Založba Univerze v Ljubljani For the Publisher / Za založbo Gregor Majdič, Rector of the University of Ljubljana / Rektor Univerze v Ljubljani Issued by / Izdala Veterinary Faculty University of Ljubljana / Veterinarska fakulteta Univerze v Ljubljani For the Issuer / Za izdajatelja Breda Jakovac Strajn, Dean of the Veterinary Faculty / Dekanja Veterinarske fakultete Editorial Board / Uredniški odbor Vesna Cerkvenik Flajs, Robert Frangež, Polona Juntes, Tina Kotnik, Alenka Nemec Svete, Matjaž Ocepek, Joško Račnik , Jože Starič, Nataša Šterbenc, Marina Štukelj, Tanja Švara, Ivan Toplak, Modest Vengušt, Milka Vrecl Fazarinc, Veterinary Faculty / Veterinarska fakulteta, Tanja Kunej, Jernej Ogorevc, Tatjana Pirman, Janez Salobir, Biotechnical Faculty / Biotehniška fakulteta, Nataša Debeljak, Martina Perše, Faculty of Medicine / Medicinska fakulteta, University of Ljubljana / Univerza v Ljubljani; Andraž Stožer, Faculty of Medicine University of Maribor / Medicinska fakulteta Univerze v Mariboru; Cugmas Blaž, Institute of Atomic Physics and Spectroscopy University of Latvia / Inštitut za atomsko fiziko in spektroskopijo Univerze v Latviji Editorial Advisers / Svetovalca uredniškega odbora Gita Grecs-Smole for Bibliography (bibliotekarka), Luka Milčinski for Electronic media (za elektronske medije) Reviewing Editorial Board / Ocenjevalni uredniški odbor Breda Jakovac Strajn, Gregor Majdič, Ožbalt Podpečan, Gabrijela Tavčar Kalcher, Nataša Tozon, Jelka Zabavnik Piano, Veterinary Faculty University of Ljubljana / Veterinarska fakulteta Univerze v Ljubljani; Alexandra Calle, John Gibbons, Laszlo Hunyadi, Howard Rodriguez-Mori, Texas Tech University, School of Veterinary Medicine / Šola za veterinarsko medicino Univerze Texas Tech; Jovan Bojkovski, Faculty of Veterinary Medicine, University of Belgrade / Fakulteta za veterinarsko medicino Univerze v Beogradu; Antonio Cruz, Justus Liebig University of Giessen / Univerza Justus Liebig v Giessnu; Gerry M. Dorrestein, Dutch Research Institute for Birds and Special Animals / Nizozemski raziskovalni inštitut za ptice in eksotične živali; Zehra Hajrulai-Musliu, Faculty of Veterinary Medicine, University Ss. Cyril and Methodius, Skopje / Fakulteta za veterinarsko medicino Univerze Ss. Cirila in Metoda v Skopju; Wolfgang Henninger, Diagnostic Centre for Small Animals, Vienna / Diagnostični center za male živali, Dunaj; Aida Kavazovic, Faculty of Veterinary Medicine University of Sarajevo / Fakulteta za veterinarsko medicino Univerze v Sarajevu; Nevenka Kožuh Eržen, Krka d.d, Novo mesto; Eniko Kubinyi, Faculty of Sciences, Eövös Loránd University Budapest / Fakulteta za znanosti Univerze Eövös Loránd v Budimpešti; Louis Lefaucheur, French National Institute for Agriculture, Food, and Environment (INRAE) / Francoski nacionalni inštitut za kmetijstvo, prehrano in okolje; Peter O’Shaughnessy, University of Glasgow / Univerza v Glasgowu; Peter Popelka, University of Veterinary Medicine and Pharmacy in Košice / Univerza za veterinarsko medicino in farmacijo v Košicah; Uroš Rajčević, Novartis, Lek Pharmaceuticals d.d., Ljubljana; Dethlef Rath, Federal Research Institute for Animal Health, Friedrich-Loeffler-Institut, Germany / Zvezni raziskovalni inštitut za zdravje živali, Inštitut Friedrich-Loeffler, Nemčija; Alex Seguino, University of Edinburgh / Univerza v Edinburgu; Ivan-Conrado Šoštarić-Zuckermann, Faculty of Veterinary Medicine University of Zagreb / Fakulteta za veterinarsko medicino Univerze v Zagrebu; Henry Staempfli, Ontario Veterinary College, Canada / Veterinarska visoka šola Ontario, Kanada; Frank J. M.Verstraete, University of California Davis / Univerza v Kaliforniji, Davis; Thomas Wittek, University of Veterinary Medicine Vienna / Univerza za veterinarsko medicino na Dunaju Address Veterinary Faculty, Gerbičeva 60, 1000 Ljubljana, Slovenia Naslov Veterinarska fakulteta, Gerbičeva 60, 1000 Ljubljana, Slovenija Tel.: +386 (0)1 47 79 100, Fax: +386 (0)1 28 32 243 E-mail slovetres@vf.uni-lj.si Sponsored by The Slovenian Research Agency Sofinancira Javna agencija za raziskovalno dejavnost Republike Slovenije ISSN 1580-4003 Printed by / tisk DZS, d.d., Ljubljana, March 2023 Number of copies printed / Naklada 220 Indexed in / Indeksirano v Agris, Biomedicina Slovenica, CAB Abstracts, IVSI Urlich’s International Periodicals Directory, Science Citation Index Expanded, Journal Citation Reports – Science Edition https://www.slovetres.si/ This work is licensed under a Creative Commons Attribution-ShareAlike 4.0 International License / To delo je ponujeno pod licenco Creative Commons Priznanje avtorstva-Deljenje pod enakimi pogoji 4.0 Mednarodna licenca SloVetRes_NOTRANJOST ver2.indd 2 09/05/2023 10:14 Table of Content 5 Editorial Veterinary Illustration: Science and Art Telling a Story Together 9 Review Article Suitability of Alternatives to Rectal Temperature Measurements in Pet Rodents, Rabbits and Ferrets: A Literature Review 15 Original Research ArticleCharacterisation of the Haematological Profile in the Posavje Horse Breed 25 Original Research Article The Effect of a Specific Chicken Based Renal Diet as Monotherapy on Clinical, Biochemical, Urinary and Serum Oxidative Stress Parameters in Cats with CKD Stage 1 and 2 37 Case Report Computed Tomography and Magnetic Resonance Imaging of a Rhinosinusitis Secondary to a Dental Abscess in a Crested Porcupine (Hystrix cristata) The cover illustration by Pšenica Kovačič, represents the Slovenian indigenous Posavje Horse breed. SloVetRes_NOTRANJOST ver2.indd 3 09/05/2023 10:14 Kubale V Stans J Mesarič M, Nemec M, Čebulj Kadunc N Krofič Žel M, Nemec Svete A, Jakovac Strajn B, Pavšič Vrtač K, Vovk T, Kejžar N, Pavlin D Encinoso M, Morales D, Déniz S, Guerra JV, Jaber JR SloVetRes_NOTRANJOST ver2.indd 4 09/05/2023 10:14 Slov Vet Res | Vol 60 No 1 | 5 DOI 10.26873/SVR-1723-2023 UDC 636.09:655.533:75.04 Pages: 5–7 Editorial Veterinary Illustration: Science and Art Telling a Story Together At first glance, Science and Art seem to be very different, as Science is supposed to be objective and guided by data, while Art is subjective and strongly influenced by feelings and opinions. While the results of Science and Art are quite different, both processes have undeniable similarities. Both put ideas about the world into a form that allows the viewer to connect with the idea. Observing a cell, a snake, or hu- man nature is what natural sciences and art depend on, albeit for different purposes. Veterinary and medical illus- trations capture visual representations of the animal or hu- man body or individual parts for documentation purposes, for teaching, or for veterinary/medical practice. They are closely related to historical, social, and technological devel- opments that have influenced and have been influenced by the content of illustrations, the artists themselves, and the techniques used to produce, reproduce, and disseminate them from prehistoric and historical times to the present. Since prehistoric times, people have illustrated the natural world through art. The extensive collection of Paleolithic cave paintings found around the world testifies to this deep- ly rooted habit. One of the best-preserved examples, which I would like to point out because I was fascinated by them last year in Marseille, France, are the Paleolithic depictions of nature and animals, dated between 33,000 to 30,000 years ago. They were discovered in 1985 by Henri Cosqueri, a professional diver in Parc National les Canaques. Since the entrance to the Cosquer Cave is now below sea level, Na prvi pogled se zdi, da sta si Znanost in Umetnost zelo raznoliki, saj naj bi bila Znanost objektivne narave ter podpr- ta z pridobljenimi rezultati, medtem ko naj bi bila Umetnost bolj subjektivne narave ter močno pod vplivom občutkov in mnenj. Medtem, ko so rezultati, ki jih prinašata Znanost in Umetnost precej različni, sta hkrati po drugi strani obe zelo podobni, saj predstavita svoje ideje na način, da se opazo- valec z njimi poveže. Znanost in Umetnost temeljita na opa- zovanju, bodisi celice, kače ali človeške narave, pa čeprav z različnim namenom. Veterinarske in medicinske ilustracije so bistvenega pomena za vizualno predstavljane zgradbe živalskega ter človeškega telesa oz. njegovih posameznih delov za namene dokumentiranja, poučevanja ali za name- ne veterinarske medicine/medicine v praksi. Tesno so pove- zane z zgodovinskim, družbenim in tehnološkim razvojem, na katerega je vplivala vsebina ilustracij, umetniki sami in tehnike, ki so jih uporabili za njihovo izdelavo, reprodukcijo in širjenje od prazgodovine do danes. Že od prazgodovine naprej so ljudje skozi oči umetnosti upodabljali naravni svet. O tej globoko zakoreninjeni navadi priča obsežna zbirka paleolitskih jamskih slikarij, najdenih po vsem svetu. Eden najbolje ohranjenih primerkov, ki bi jih želela izpostaviti, ker so me lani navdušili v Marseillu v Franciji, so paleolitske upodobitve narave in živali, ki segajo v obdobje med 33,000 do 30,000 let pred našim štetjem. Leta 1985 jih je med potapljanjem odkril Henri Cosqueri v nacionalnem parku les Canaques. Ker je vhod v jamo Ilustracija v veterini: Znanost in Umetnost skupaj pripovedujeta zgodbo Valentina Kubale Institute of Preclinical Sciences, Veterinary Faculty, University in Ljubljana, 1000 Ljubljana, Slovenia Accepted: 8 March 2023 SloVetRes_NOTRANJOST ver2.indd 5 09/05/2023 10:14 6 | Slov Vet Res | Vol 60 No 1 the images are very well preserved. Today we can all share this great experience by exploring the ancient art in the arti- ficial cave in the Villa Mediterannea at the port of Marseille. Throughout history, various forms of visual media have played an important role in communicating and explain- ing scientific concepts. The use of illustrations and other visual methods to convey scientific information is called scientific illustration. The main purpose of scientific illus- tration is to help the target audience better understand sci- entific concepts, whether they are researchers, students, or the general public. Therefore, scientific illustration is an important aspect of science communication at all levels. It tells a story. The distinctive feature of scientific illustration is the demand for accuracy and objectivity in presenting the concept as much as possible. A scientific illustration is certainly a form of an art, but art with the specific goal of communicating science. Veterinary science illustrations are valuable visual repre- sentations for student and client education, publications, teaching, presentations, and many other purposes. Artists collaborate in all areas, whether illustrating histology text- books, creating prosthetics, or designing artwork for phar- maceutical companies. Collaboration is key; researchers and clinicians, from veterinary to human medicine, micro- biology, and pharmaceutical sciences, work with artists. Science is a language, and veterinary and medical illustra- tors translate that language for a wide audience using a vi- sual language. Veterinary professionals can use veterinary science illustration to communicate complex and impor- tant information in clearly and concisely, in a way that is easy to understand. For example, illustrations can be used to show an animal‘s internal structures, such as its organs and bones, which can be difficult to visualize from the out- side. They can also be used to demonstrate the progres- sion of a disease or injury, helping clients understand what is happening to their animals and what treatment options are available. In addition, illustrations can be used to help veterinarians plan surgical procedures, by providing a de- tailed look at the anatomy of the affected area. They can also be used to document the progress of a treatment or procedure, allowing for more accurate tracking of changes over time. Today, there are careers in veterinary or human medical il- lustration that combine scientific knowledge with artistic skill. A medical illustrator is a professional artist with ad- vanced training in both life sciences and visual commu- nication, who translates complex information into visual images, often in collaboration with scientists, physicians, veterinarians, and other experts. However, medical illustra- tion is a small field in which there are not many trained pro- fessionals worldwide. Therefore, collaboration with artists and illustrators who share the same interest in this field is of great importance. Cosquer pod morsko gladino, so slike zelo dobro ohranje- ne. Danes lahko vsi delimo to veliko izkušnjo z raziskova- njem starodavne umetnosti v umetno ustvarjeni jami v vili Mediterannea v pristanišču Marseilla. Skozi zgodovino so imele različne oblike vizualnih medijev pomembno vlogo pri sporočanju in razlagi znanstvenih konceptov. Znanstvena ilustracija se nanaša na uporabo ilustracij in drugih vizualnih načinov z namenom posre- dovanja znanstvenih informacij različnim javnostim. Njen glavni namen je pomagati ciljnemu občinstvu bolje razume- ti znanstvene koncepte, ne glede na to, ali so raziskovalci, študenti ali splošna javnost. Zato je znanstvena ilustraci- ja pomembna na vseh ravneh v komunikaciji znanosti, saj pripoveduje njeno zgodbo. Posebnost znanstvene ilustra- cije je čim večja natančnost in objektivnost pri predstavitvi zamisli. Znanstvena ilustracija je vsekakor oblika umetno- sti, vendar umetnosti s posebnim ciljem komuniciranja znanosti. Veterinarske znanstvene ilustracije so dragoceni vizualni pripomočki za izobraževanje študentov in strank, pripravo raznovrstnih publikacij, poučevanje, predstavitve in števil- ne druge namene. Umetniki lahko sodelujejo z veterinar- sko stroko na vseh področjih, bodisi pri ilustriranju histo- loških učbenikov, ustvarjanju protetičnih pripomočkov ali pri oblikovanju umetniških del za farmacevtska podjetja. Sodelovanje je pri tem ključnega pomena; med raziskovalci in kliniki, od veterinarske do humane medicine, mikrobio- logije in farmacevtskih ved. Vsem je skupno sodelovanje z umetniki. Znanost je jezik, ki ga ilustratorji na področju veterinarske medicine in medicine prevajajo v za širše ob- činstvo razumljiv jezik in pri tem uporabijo svoj vizualni je- zik. Veterinarski strokovnjaki lahko uporabljajo ilustracije s področja veterinarske znanosti za sporočanje zaplete- nih in pomembnih informacij na jasen in jedrnat način, ki omogoča lažje razumevanje vsebine. Ilustracije lahko na primer uporabimo za prikaz notranjih struktur živali, kot so njeni organi in kosti, ki si jih je od zunaj težko predstavljati. Uporabijo se lahko tudi za prikaz napredovanja bolezni ali poškodbe, kar strankam pomaga razumeti, kaj se dogaja z njihovimi živalmi in kakšne možnosti zdravljenja so na voljo. Poleg tega so lahko ilustracije uporabne za pomoč veterinarjem pri načrtovanju kirurških posegov, tako, da jim omogočijo podrobnejši vpogled v anatomijo prizadetega območja. Uporabljajo se lahko tudi za dokumentiranje na- predka zdravljenja ali postopka, kar omogoča natančnejše sledenje spremembam skozi čas. Danes celo obstajajo na področju veterinarske ali humane medicinske ilustracije poklici, ki združujejo oboje - določe- no znanstveno znanje in umetniške spretnosti. Eden od takšnih je poklic medicinskega (znanstvenega) ilustratorja, ki je poklicni umetnik z daljšim usposabljanjem na podro- čju znanosti in vizualne komunikacije, ki interpretira kom- pleksne informacije znanosti v vizualne podobe, pogosto v sodelovanju z znanstveniki, zdravniki, veterinarji in drugimi strokovnjaki. Vendar je medicinska (znanstvena) ilustracija SloVetRes_NOTRANJOST ver2.indd 6 09/05/2023 10:14 Slov Vet Res | Vol 60 No 1 | 7 With this issue, in the light of Science and Art we welcome our first artistic collaboration with renewed artist Pšenica Kovačič, who has been working with the Veterinary faculty in Ljubljana for some time, enabling various researchers and academics to tell their stories to clients, students, and the scientific audience. She will be part of our team as an Art editor, designing and producing visual content such as illustrations for the cover of the journal and helping us com- municate our stories to a wider audience. This year’s first issue contains four interesting and various peer-reviewed articles, a review article on alternatives to rectal temperature measurement in rats, two original sci- entific articles, on the hematological profile of the Posavje horse breed and on the influence of diet on oxidative stress parameters in cats and a case report on magnetic imaging of the crested porcupine. Since the Posavje horse is an autochthonous “cold-blood- ed“ horse typical of the southeastern part of Slovenia and Croatia, it was an easy decision to feature it on the first illus- trated cover of the Slovenian Veterinary Research journal. The breed originated from unintentional crosses with vari- ous horse breeds, especially Belgian cold-blooded horses. It was selected mainly for heavy draft work, especially in steep forest areas. Today these horses are the smallest coldblooded breed in Europe and their pedigree was intro- duced in 1993. This breed is characterized by a small, thin head, a straight profile, a medium-length neck with a short back, and a short, very broad, and moderately restrained lower back. They have relatively large and strong hooves. The legs are covered with a protective coat. They have a robust constitution, are very fertile and their sexual dimor- phism is pronounced. The breed is known to be good-na- tured and peaceful. I would like to thank our new Editor-in-Chief Dr. Klementina Fon Tacer for inviting me to write this Editorial and for pav- ing the way to combine Art and Science in the field of vet- erinary illustration in our journal. During the preparation of this Editorial, a lot of material and ideas were collected on this topic, so there will be more to report in one of the future issues of the Journal. I would like to conclude with a quote from American sur- geon and medical illustrator Frank H. Netter. “Draw what can’t be seen, watch what‘s never been done, and tell thousands about it without saying a word.” Frank H. Netter, M.D. zelo specifično in ne preveč razširjeno področje ustvarjanja, z le nekaj izobraženimi strokovnjaki po vsem svetu. Zato je zelo pomembno sodelovanje z umetniki in ilustratorji, ki jih to področje zanima. V prvi letošnji številki v luči Znanosti in Umetnosti pozdravlja- mo naše prvo umetniško sodelovanje s priznano umetnico Pšenico Kovačič, ki že nekaj časa sodeluje z Veterinarsko fakulteto v Ljubljani in omogoča večim raziskovalcem in akademikom, da pripovedujejo svoje zgodbe naročnikom, študentom, kakor tudi znanstvenem občinstvu. Postala je del naše uredniške ekipe kot umetniška urednica, ki bo oblikovala in pripravljala vizualne vsebine, kot so ilustracije za naslovnico revije, ter nam pomagala posredovati naše zgodbe širšemu občinstvu. Letošnja prva številka vsebuje štiri zanimive in raznolike recenzirane članke, in sicer pregledni članek o alternativah rektalnemu merjenju temperature pri podganah, dva origi- nalna znanstvena članka o hematološkem profilu avtohto- ne slovenske pasme posavskega konja oz. posavca in vpli- vu prehrane na parametre oksidativnega stresa pri mačkah ter študijo primera o magnetnem slikanju afriškega ježevca. Ker je posavski konj kot avtohtoni »hladnokrvni« konj, zna- čilen za jugovzhodni del Slovenije in Hrvaške, je bila odlo- čitev, da prav to pasmo upodobimo na prvi ilustrirani na- slovnici revije Slovenskega Veterinarskega Zbornika, hitro sprejeta. Pasma je nastala z naključnimi križanji različnih pasmah konj, predvsem belgijskimi hladnokrvnimi konji. Selekcioniran pa je bil zlasti za težka vlečna dela, predvsem v strmih gozdnih predelih. Danes pasma velja za najmanjšo hladnokrvno pasmo konj v Evropi. Rodovnik so uvedli leta 1993. Za pasmo je značilna majhna, tanka glava, z ravnim profilom, srednje dolgim vratom, kratkim hrbtom ter krat- kim, zelo širokim in zmerno omejenim spodnjim delom hrbta. Njihova kopita so relativno velika in močna. Noge so pokrite z zaščitno dlako. Imajo robustno konstitucijo, so zelo plodni in imajo izrazit spolni dimorfizem. Pasma je znana kot dobrodušna in miroljubna. Naši novi glavni urednici dr. Klementini Fon Tacer se zahva- ljujem za povabilo k pisanju tega uvodnika in za tlakovanje poti združevanja Umetnosti in Znanosti na področju veteri- narske ilustracije v naši reviji. Med pripravo uvodnika se je nabralo veliko gradiva in idej na to tematiko, zato bo sledilo podrobnejše nadaljevanje v eni od prihodnjih številk revije. Zaključujem pa s citatom ameriškega kirurga in medicin- skega ilustratorja Franka H. Netterja. “Rišite, česar se ne vidi, glejte, kar še ni bilo narejeno, in na tisočim povejte o tem, ne da bi rekli besedo.” Dr. Frank H. Netter SloVetRes_NOTRANJOST ver2.indd 7 09/05/2023 10:14 8 | Slov Vet Res | Vol 60 No 1 SloVetRes_NOTRANJOST ver2.indd 8 09/05/2023 10:14 Slov Vet Res | Vol 60 No 1 | 9 Suitability of Alternatives to Rectal Temperature Measurements in Pet Rodents, Rabbits and Ferrets: A Literature Review Key words rectal temperature, rodents, rabbits, ferrets, alternatives Jelle Stans Institute for Globally Distributed Open Research and Education, Beringen, Belgium *Corresponding author: jelle.stans@igdore.org Abstract: Body temperature is a vital parameter to assess the health of exotic animals. Rectal thermometry is a common way to measure body temperature in rodents, rabbits and ferrets and often considered the gold standard. However, taking a rectal tempera- ture often involves restraint and can lead to stress in these animals. To avoid the stress of rectal temperature measurements, alternative (often less invasive) techniques have been utilized in several species. These methods include tympanic thermometry, axillary thermometry and infrared thermography. It is however important to establish whether these strategies yield comparable readings to the gold standard. Therefore, a literature review was performed using the MedLine and Google Scholar databases. Base terms referring to rectal temperature and thermometry were combined with species-specific search terms. Relatively few studies were identified about alternatives to rectal tempera- ture measurements in rodents, rabbits and ferrets. In general, it can be noted that only transponder measurements have repeatedly been described to be a valid alternative to rectal temperature measurement. Further research should be conducted. Received: 14 November 2022 Accepted: 8 March 2023 DOI 10.26873/SVR-1640-2023 UDC 612.56:536.5:636.045:636.932 Pages: 9–14 Review Article Introduction Body temperature is a vital parameter to assess the health of exotic animals (1). An elevated body temperature can signal an infection or systemic inflammation (2) while hy- pothermia may, for example, arise as a complication as a complication of general anaesthesia and surgery (3). In ro- dents and rabbits, body temperature was shown to be of prognostic value in a clinical setting (4, 5). Rectal thermometry is a common way to measure body temperature in rodents (6), rabbits (7) and ferrets (8) and of- ten considered the gold standard. However, taking a rectal temperature often involves restraint and can lead to stress in these animals (9, 10). This can, amongst others, impact the readings (11). To avoid the stress of rectal temperature measurements, alternative (often less invasive) techniques have been uti- lized in several species. These methods include tympanic thermometry (12), axillary thermometry (13) and infrared thermography (14). It is however important to establish whether these strategies yield comparable readings to the gold standard. In cats and dogs, such studies have been conducted suggesting that alternative methods are not always a good replacement for rectal thermometry (14, 15). In rodents, rabbits and ferrets, the body of literature comparing temperature measurement seems to be limited while this is a particularly important topic because of the stress-inducing consequences of rectal thermometry. Therefore, the aim of the current review article is to sum- marize the non-invasive temperate measure methods in- vestigated in pet rodents, rabbits and ferrets and assess whether they are a suitable alternative to rectal tempera- ture measurement. Additionally, suggestions for further re- search are formulated. SloVetRes_NOTRANJOST ver2.indd 9 09/05/2023 10:14 10 | Slov Vet Res | Vol 60 No 1 Search strategy and inclusion Between the 3rd and 5th of September 2022, the MedLine database was searched through Pubmed. The base term “(“rect*”) AND ((“temp*”) OR (“therm*”))” was combined with species-specific terms: “AND ((“guinea pig*”) OR (“cavy”) OR (“cavies”))”, “AND ((“mouse*”) OR (“mice”))”, “AND ((“rat”) OR (“rats”))”, “AND (“hamster*”)”, “AND (“ger- bil*”)”, “AND (“degu*”)”, “AND (“chinchilla*”)”, “AND (“rab- bit*”)” and “AND (“ferret*”)”. Google Scholar was searched during the same period with the base term “rectal tempera- ture” combined with the following species-specific terms: “guinea pig*”, “mice”, “rat*”, “hamster*”, “gerbil*”, “degu*”, “chinchilla*”, “rabbit*” and “ferret*”. The titles of the publications in the search results were screened for papers that could be eligible for inclusion. The abstracts of potentially eligible publications were read and included if (1) they described a comparison between rectal temperature measurement and at least one non-invasive method, (2) described agreement between rectal measure- ment and a non-invasive method and (3) the study was conducted in at least one eligible species (rodent, ferret or rabbit). Characteristics of included studies The searches yielded eligible studies for guinea pigs (3 studies, 16-18), mice (5 studies; 16, 19-22), rats (4 studies; 9, 19, 23-24), chinchillas (1 study, 25), rabbits (3 studies; 7, 16, 26) and ferrets (3 studies; 8, 27, 28). No eligible studies were identified for hamsters, gerbils and degus. The included studies and important characteristics are shown in table 1. All publications described prospective studies of multiple animals with sample sizes ranging from 6 to 48. Studies were conducted in both healthy animals and patients. In selected studies, there was a focus on laboratory animals. Publication dates ranged from 1997 and 2021, but it is clear that a significant number was published over 15 years ago. Suitability of alternative methods per species In Guinea pigs, two studies (16,17) investigated the use of transponders to measure body temperature, with mixed re- sults. Both studies were conducted in an experimental set- ting using animals raised as laboratory animals. One study mentioned it to be a valid alternative to rectal temperature (17) while the other mentioned it was not (16). Other meth- ods compared to rectal temperatures were tympanic, laser, axillary and inguinal thermometry (4, 17, 18). None of these methods were mentioned to be a valid alternative for rec- tal temperature measurement. The authors of the second study (17) mentioned that the transponder system they used made sounds that may be disturbing to the guinea pigs. Additionally, they stated that due to the hand-held na- ture of non-contact thermometers, it is difficult to obtain measurements from a comparable distance. In mice, microchip transponders were also investigated, both subcutaneously and intraperitoneally (16, 19). One of these studies reported subcutaneously and intraperitoneal transponders to be a valid alternative to rectal temperature measurement (19). One study also mentioned infrared ther- mometry of the ear and back skin to be a valid alternative (20). They also stated that this technique allows skin tem- perature to be measured easily at these sites. Other strate- gies were not deemed a suitable alternative in all cases (20, 21, 22). All of these studies were performed in laboratory animals. In rats, microchip transponders (intraperitoneally and sub- cutaneously) and (temperature-sensitive) telemetry were deemed to be usable alternatives to rectal thermometry (9,19, 23, 24). However, as stated above, it is important to assess whether the specific detection method does not disturb the animals. Additionally, the telemetry was only in- vestigated in a research setting (9, 23). This means it should be assessed whether these results can be translat- ed to the clinic. Finally, all of these studies were performed in laboratory rats. Human and veterinary thermometers were investigated as an alternative to rectal temperature measurement in chin- chillas (25). Unfortunately, both methods were deemed un- satisfactory. In this case, the studies were also conducted in an experimental setting. The animals were sourced from breeding facilities. Thermography was assessed in the eye, inner ear, external ear and nose of rabbits (26). The publica- tion mentioned that this was an effective tool to measure the temperature of several regions. However, this is not the same as being a reliable alternative for body temperature measurement in a clinical setting. Implantable microchip transponders were mentioned as a suitable alternative (7). Noncontact infrared thermometer (ear and thigh) and tympanic thermometer (human and veterinary) were not a replacement for rectal temperature measurement (7). All of these studies were performed in a research setting with laboratory animals. In ferrets, microchip transponder thermometry was men- tioned as an alternative to rectal temperature measurement (8). Paediatric and veterinary auricular, axillary, dorsal skin, inguinal, noncontact infrared and tympanic thermometry were not deemed to be alternatives (27, 28). One study (27) was conducted in animals presented within a clinical set- ting. The other studies were conducted in an experimental setting in laboratory animals. Discussion Relatively few studies have been published about alterna- tives to rectal temperature measurements in rodents, rab- bits and ferrets. The internal and external validity of the pub- lished studies also leaves room for improvement. In general, it can be noted that only transponder measurements have SloVetRes_NOTRANJOST ver2.indd 10 09/05/2023 10:14 Slov Vet Res | Vol 60 No 1 | 11 repeatedly been described to be a valid alternative to rectal temperature measurement. It is clear that there is a difference in number of studies per species. One potential reason for the higher number of stud- ies in mice and rats is that they are often used as laboratory animals. Temperature measurements are often performed in animal experiments (6) and need to be reliable and not be impacted by stress-induced responses. This may stimulate research into this area, which can be translated into clinical practice. Further research is needed to address the lack of studies in hamsters, gerbils and degus. Table 1: Study design of includes studies Reference Title Study design Sample size Database Guinea pigs Hartinger et al., 2003 Suitability of temperature-sensitive transponders to measure body temperature during animal experiments required for regulatory tests Prospective 10 PubMed Devalle, 2005 Comparison of tympanic, transponder, and noncontact infrared laser thermometry with rectal thermometry with rectal thermometry in strain 13 Guinea pigs (Cavia porcellus) Prospective 28 PubMed Google Scholar Levy et al., 2020 Comparison of axillary and inguinal body temperature to rectal temperature in healthy guinea pigs (Cavia porcellus) Prospective 40 Google Scholar Mice Kort et al., 1997 A microchip implant system as a method to determine body temperature of terminally ill rats and mice Prospective 10 PubMed Google Scholar Hartinger et al., 2003 Suitability of temperature-sensitive transponders to measure body temperature during animal experiments required for regulatory tests Prospective 12 PubMed Saegusa and Tabata, 2003 Usefulness of infrared thermometry in determining body temperature in mice Prospective 6 PubMed Newsom et al., 2004 Comparison of body surface temperature measurement and conventional methods for measuring temperature in the mouse Prospective 12 PubMed Google Scholar Fiebig et al., 2018 Evaluation of Infrared thermography for temperature measurement for temperature measurement in adult male NMRI nude mice Prospective 10 PubMed Rats Dilsaver et al., 1992 Measurement of temperature in the rat by rectal probe and telemetry yields compatible results Prospective 12 PubMed Google Scholar Kort et al., 1997 A microchip implant system as a method to determine body temperature of terminally ill rats and mice Prospective 30 PubMed Google Scholar Eshraghi et al., 2005 Cochlear temperature correlates with both temporalis muscle and rectal temperatures. Application for testing the otoprotective effect of hypothermia Prospective 6 PubMed Google Scholar Dangarembizi et al., 2017 Measurement of body temperature in normothermic and febrile rats: Limitations of using rectal thermometry Prospective 31 PubMed Google Scholar Hamsters Gerbils Degus Chinchillas Ozawa et al., 2017 Comparison of rectal and tympanic thermometry in chinchillas (Chinchilla lanigera) Prospective 47 PubMed Google Scholar Rabbits Hartinger et al., 2003 Suitability of temperature-sensitive transponders to measure body temperature during animal experiments required for regulatory tests Prospective 10 PubMed Chen and White, 2006 Comparison of rectal, microchip transponder, and infrared thermometry techniques for obtaining body temperature in the laboratory rabbit (Oryctolagus cuniculus) Prospective 46 PubMed Google Scholar Jaén-Téllez et al., 2021 Relationship between rectal temperature measured with a conventional thermometer and the temperature of several body regions measured by infrared thermography in fat- tening rabbits. Influence of different environmental factors Prospective 48 Google Scholar Ferrets Maxwell et al., 2016 Comparison of digital rectal and microchip transponder thermometry in ferrets (Mustela putorius furo) Prospective 16 PubMed Google Scholar Aguilar et al., 2018 Comparison of body temperature acquired via auricular and rectal methods in ferrets Prospective 27 Google Scholar Keeney et al., 2020 Comparison of body temperature using digital, infrared, and tympanic thermometry in healthy ferrets (Mustela putorius furo) Prospective 20 Google Scholar SloVetRes_NOTRANJOST ver2.indd 11 09/05/2023 10:14 12 | Slov Vet Res | Vol 60 No 1 Table 2: Data extracted from included studies Reference Alternative temperature measurement method Agreement metric with rectal measurement Valid alternative for rectal measurement according to publication? Guinea pigs Hartinger et al., 2003 Implanted temperature-sensitive transponders Only graphically No Devalle, 2005 Tympanic thermometer 0.3956 intraclass correlation coefficient No Devalle, 2005 Laser 0.1229 intraclass correlation coefficient No Devalle, 2005 Transponder 0.5880 intraclass correlation coefficient Yes Levy et al., 2020 Axillary difference of mean -0.39 (95% CI -0.54 - -0.23) No Levy et al., 2021 Inguinal difference of mean was -0.73 (95% CI -0.94 - -0.52) No Mice Kort et al., 1997 Microship transponder (subcutaneous) differences within ± 0.5°C Yes Kort et al., 1997 Microship transponder (intraperitoneally) differences within ± 0.5°C Yes Hartinger et al., 2003 Implanted temperature-sensitive transponders Only graphically No Saegusa and Tabata, 2003 Infrared thermometry (ear) correlation r = 0.95 Yes Saegusa and Tabata, 2003 Infrared thermometry (back skin) correlation r = 0.96 Yes Saegusa and Tabata, 2003 Infrared thermometry (tail skin) correlation r = 0.59 No Saegusa and Tabata, 2003 Infrared thermometry (sole skin) correlation r = 0.59 No Newsom et al., 2004 Surface temperature measurements correlation r = 0.9773 No Newsom et al., 2004 Telemetry correlation r = 0.9699 No Fiebig et al., 2018 Infrared Thermography/Camera mean difference of 0.56 °C Yes* (in nude mice) Rats Dangarembizi et al., 2017 Temperature-sensitive radiotelemeters (intraperitoneally) rectal 0.5°C lower or 0.7°C greater than radiotelemeter Yes* (but investigated for research setting) Eshraghi et al., 2005 Cochlear temperature Correlation r = 0.959 No Kort et al., 1997 Microship transponder (subcutaneous) differences within ± 0.5°C Yes Kort et al., 1997 Microship transponder (intraperitoneally) differences within ± 0.5°C Yes Dilsaver et al., 1992 Telemetry after salicylate r = +0.83, after oxotremorine r = +0.93 Yes* (but investigated for research setting) Hamsters Gerbils Degus Chinchillas Ozawa et al., 2017 Human tympanic thermometer margin of error (combined human/veterinary) 1.7°C No Ozawa et al., 2017 Veterinary tympanic thermometer margin of error (combined human/veterinary) 1.7°C No Rabbits Hartinger et al., 2003 Implanted temperature-sensitive transponders Only graphically No Chen and White, 2006 Implantable microchip transponder 95% agreement limit: ±1.48 Yes Chen and White, 2006 Noncontact infrared thermometer (ear) Not calculated due to systematic deviations from avg temp No Chen and White, 2006 Noncontact infrared thermometer (thigh) Not calculated due to systematic deviations from avg temp No Chen and White, 2006 Human tympanic thermometer Not calculated due to systematic deviations from avg temp No Chen and White, 2006 Animal tympanic thermometer Not calculated due to systematic deviations from avg temp No Jaén-Téllez et al., 2021 Thermography (eye) Coefficient of determination: 0.15 Unclear Jaén-Téllez et al., 2021 Thermography (inner ear) Coefficient of determination: 0.22 “Best” Jaén-Téllez et al., 2021 Thermography (external ear) Coefficient of determination: 0.24 “Inefficient” Jaén-Téllez et al., 2021 Thermography (nose) Coefficient of determination: 0.20 Unclear Ferrets Maxwell et al., 2016 Microchip transponder thermometry 95% Agreement limits (°F): −1.82 to +1.96 (comp. to calibrated rectal) −2.19 to +0.84 (comp. to common rectal) Yes Aguilar et al., 2018 Pediatric auricular thermometers correlation thermometer 1: 0.4726 correlation thermometer 2: 0.5388 No Aguilar et al., 2018 Veterinary auricular thermometers correlation 0.6311 No Keeney et al., 2020 Axillary 95% CI of Mean Difference -1.32, -0.67 No Keeney et al., 2020 Dorsal Skin 95% CI of Mean Difference -0.14, 0.24 No* (possibly after further studies) Keeney et al., 2020 Inguinal 95% CI of Mean Difference -1.15, -0.63 No Keeney et al., 2020 Noncontact Infrared 95% CI of Mean Difference -8.37, -6.43 No Keeney et al., 2020 Tympanic 95% CI of Mean Difference -0.78, -0.38 No SloVetRes_NOTRANJOST ver2.indd 12 09/05/2023 10:14 Slov Vet Res | Vol 60 No 1 | 13 A further limitation is that most of the studies were con- ducted in an experimental setting and may therefore not fully resemble the clinical setting. Additionally, only one study was performed with animal patients while the rest were conducted with laboratory animals. These animals may have different relevant characteristics than patients, including different stress sensitivity. For now, it seems that rectal temperature measurement should remain the golden standard until further research has been performed. References 1. Lichtenberger M, Lennox M. Critical Care of the Exotic Companion Mammal (With a Focus on Herbivorous Species): The First Twenty- Four Hours. Journal of Exotic Pet Medicine 2012; 21(4): 284–92. 2. Mota-Rojas D, Wang D, Titto C, et al. Pathophysiology of Fever and Application of Infrared Thermography (IRT) in the Detection of Sick Domestic Animals: Recent Advances. Animals (Basel) 2021; 11(8): 2316. 3. Pottie RG, Dart CM, Perkins N, et al. Effect of hypothermia on recovery from general anaesthesia in the dog. Aust Vet J. 2007; 85(4):158–62. 4. Levy IH, Di Girolamo N, Keller KA. Rectal temperature is a prognostic indicator in client-owned guinea pigs. J Small Anim Pract. 2021; 62(10): 861–5. 5. Di Girolamo N, Toth G, Selleri P. Prognostic value of rectal temperature at hospital admission in client-owned rabbits. J Am Vet Med Assoc. 2016; 248(3): 288–97. 6. Meyer CW, Ootsuka Y, Romanovsky AA. Body Temperature Measurements for Metabolic Phenotyping in Mice. Front Physiol. 2017; 31(8): 520. 7. Chen PH, White CE. Comparison of rectal, microchip transponder, and infrared thermometry techniques for obtaining body temperature in the laboratory rabbit (Oryctolagus cuniculus). J Am Assoc Lab Anim Sci. 2006; 45(1): 57–63. 8. Maxwell BM, Brunell MK, Olsen CH, et al. Comparison of Digital Rectal and Microchip Transponder Thermometry in Ferrets (Mustela putorius furo). J Am Assoc Lab Anim Sci. 2016; 55(3): 331–5. 9. Dangarembizi R, Erlwanger KH, Mitchell D, et al. Measurement of body temperature in normothermic and febrile rats: Limitations of using rec- tal thermometry. Physiol Behav. 2017; 179: 162–7. 10. Clark DL, DeBow SB, Iseke MD, et al. Stress-induced fever after post- ischemic rectal temperature measurements in the gerbil. Can J Physiol Pharmacol. 2003; 81(9): 880–3. 11. Veening JG, Bouwknecht JA, Joosten HJ, et al. Stress-induced hy- perthermia in the mouse: c-fos expression, corticosterone and tem- perature changes. Prog Neuropsychopharmacol Biol Psychiatry. 2004; 28(4): 699–707. 12. Hall E, Carter A. Establishing a reference range for normal canine tym- panic membrane temperature measured with a veterinary aural ther- mometer. Veterinary Nursing Journal 2017; 32(12): 369–73. 13. Smith VA, Lamb V, McBrearty AR. Comparison of axillary, tympanic membrane and rectal temperature measurement in cats. J Feline Med Surg. 2015; 17(12): 1028–34. 14. Kunkle GA, Nicklin CF, Sullivan-Tamboe DL. Comparison of body tem- perature in cats using a veterinary infrared thermometer and a digital rectal thermometer. J Am Anim Hosp Assoc. 2004; 40(1): 42–6. 15. Sousa MG, Carareto R, Pereira-Junior VA, et al. Comparison between auricular and standard rectal thermometers for the measurement of body temperature in dogs. Can Vet J. 2011; 52(4): 403–6. 16. Hartinger J, Külbs D, Volkers P, et al. Suitability of temperature-sensi- tive transponders to measure body temperature during animal experi- ments required for regulatory tests. 2003; 20(2): 65–70. 17. Stephens JM. Comparison of tympanic, transponder, and noncontact infrared laser thermometry with rectal thermometry in strain 13 guinea pigs (Cavia porcellus). Contemp Top Lab Anim Sci. 2005 Sep; 44(5): 35–8. 18. Levy I, Allender M, Keller K. Comparison of axillary and inguinal body temperature to rectal temperature in healthy guinea pigs (Cavia porcel- lus). Journal of Exotic Pet Medicine 2020; 34: 1–5. 19. Kort WJ, Hekking-Weijma JM, TenKate MT, et al. A microchip implant system as a method to determine body temperature of terminally ill rats and mice. Lab Anim. 1998; 32(3): 260–9. 20. Saegusa Y, Tabata H. Usefulness of infrared thermometry in determin- ing body temperature in mice. J Vet Med Sci. 2003; 65(12): 1365–7. 21. Newsom DM, Bolgos GL, Colby L, et al. Comparison of body surface temperature measurement and conventional methods for measuring temperature in the mouse. Contemp Top Lab Anim Sci. 2004; 43(5): 13–8. 22. Fiebig K, Jourdan T, Kock MH, et al. Evaluation of Infrared Thermography for Temperature Measurement in Adult Male NMRI Nude Mice. J Am Assoc Lab Anim Sci. 2018; 57(6): 715–24. 23. Dilsaver SC, Overstreet DH, Peck JA. Measurement of temperature in the rat by rectal probe and telemetry yields compatible results. Pharmacol Biochem Behav. 1992; 42(3): 549–52. 24. Eshraghi AA, Nehme O, Polak M, et al. Cochlear temperature correlates with both temporalis muscle and rectal temperatures. Application for testing the otoprotective effect of hypothermia. Acta Otolaryngol. 2005; 125(9): 922–8. 25. Ozawa S, Mans C, Beaufrère H. Comparison of rectal and tympanic thermometry in chinchillas (Chinchilla lanigera). J Am Vet Med Assoc. 2017; 251(5): 552–8. 26. Jaén-Téllez J, Bartolomé E, Sánchez-Guerrero M, et al. Relationship between rectal temperature measured with a conventional thermom- eter and the temperature of several body regions measured by infrared thermography in fattening rabbits. Influence of different environmental factors. World Rabbit Science 2021; 29(4): 263–73. 27. Aguilar L, Chávez J, Watty A. Comparison of body temperature ac- quired via auricular and rectal methods in ferrets. Journal of Exotic Pet Medicine 2019; 28: 148–53. 28. Keeney C, Hung C, Harrison T. Comparison of body temperature using digital, infrared, and tympanic thermometry in healthy ferrets (Mustela putorius furo). Journal of Exotic Pet Medicine 2021; 36: 16–21. SloVetRes_NOTRANJOST ver2.indd 13 09/05/2023 10:14 14 | Slov Vet Res | Vol 60 No 1 Ustreznost alternativnih tehnik rektalnemu merjenju temperature pri hišnih glodavcih, kuncih in belih dihurjih - pregled literature J. Stans Izvleček: Telesna temperatura je pomemben parameter za oceno zdravja eksotičnih živali. Rektalno merjenje tempera- ture je običajen način merjenja telesne temperature pri glodavcih, kuncih in belih dihurjih in pogosto velja za zlati stan- dard. Vendar je merjenje rektalne temperature pri teh živalih pogosto povezano z omejevanjem gibanja in povzročanjem stresa. Da bi se izognili stresu pri merjenju rektalne temperature, so bile pri več vrstah živali uporabljene alternativne (pogosto manj invazivne) tehnike. Te metode vključujejo infrardečo termografijo ter merjenje temperature timpanično in aksilarno. Vendar pa je pomembno ugotoviti, ali te strategije dajejo primerljive rezultate z zlatim standardom. Zato smo opravili pregled literature z uporabo podatkovnih zbirk MedLine in Google Scholar. Osnovni izrazi, ki se nanašajo na rektalno temperaturo in merjenje temperature, so bili združeni z iskalnimi izrazi, značilnimi za posamezne vrste. Pri glodavcih, kuncih in belih dihurjih je bilo najdenih razmeroma malo študij o alternativah rektalnim meritvam temperature. Na splošno lahko ugotovimo, da so bile le meritve s transponderjem večkrat opisane kot veljavna alternativa rektalnemu merjenju temperature. Potrebne so nadaljnje raziskave. Ključne besede: rektalna temperatura; glodavci; kunci; beli dihurji; alternative SloVetRes_NOTRANJOST ver2.indd 14 09/05/2023 10:14 Slov Vet Res | Vol 60 No 1 | 15 Characterisation of the Haematological Profile in the Posavje Horse Breed Key words autochthonous breeds, Posavje horse, haematology, age, sex Matjaž Mesarič1*, Marija Nemec1, Nina Čebulj Kadunc2 1Clinic for Reproduction and Large Animals, University of Ljubljana, Veterinary Faculty, Cesta v Mestni log 47, 1000 Ljubljana, 2Institute for Preclinical Sciences, University of Ljubljana, Veterinary Faculty, Gerbičeva 60, 1000 Ljubljana, Slovenia *Corresponding author: matjaz.mesaric@vf.uni-lj.si Abstract: The aim of this study was to investigate the influences of sex and age on hae- matological values in the Posavje Horse breed. A total of 163 healthy Posavje horses (30 foals, 94 mares and 39 stallions) were used in this study; their complete blood counts and a leucogram were obtained with a haematological analyser. The horses were classi- fied into five groups: foals (1 to 6 months, n = 30), 3 to 6 years (n = 8 stallions/21 mares), 7 to 9 years (n = 9 stallions/22 mares), 10 to 13 years (n = 8 stallions/20 mares), 14 to 15 years (n = 6 stallions/10 mares) and 16 and over (n = 8 stallions/21 mares). The results obtained show an influence of sex on haematological parameters, with red blood cell count (RBC), haematocrit (HCT) and haemoglobin concentration (HGB) being higher in stallions (P < 0.001) and white blood cell count (WBC) being higher in mares. Differences between the age groups of the Posavje horses examined indicate a decrease in RBC and HGB with a compensatory increase in mean corpuscular volume and mean corpuscu- lar haemoglobin, a decrease in WBC and platelet counts (PLT) and proportion of lym- phocytes, and an increase of neutrophil to lymphocyte ratio (N/L) with age (P < 0.001). Although the Posavje horse is classified as a draft horse breed, its haematological pa- rameters show characteristics common to warm-blooded breeds, with the exception of the N/L ratio. One of the most important findings of this study is a higher neutrophil count in reproductively active breeding stallions. Higher levels of RBC, HGB, HCT and neutrophil count in the Posavje stallions suggest an effect of androgens (testosterone), which may be an effective mechanism to prevent infections, that can affect the survival of the stallions and thus the evolution of the species. Received: 10 May 2022 Accepted: 15 March 2023 DOI 10.26873/SVR-1508-2023 UDC 636.1.09:616.15-074 Pages: 15–23 Original Research Article Introduction Intensive artificial and natural selection have shaped sub- stantial variation among horse breeds, which are reflected in the differences of haematological and biochemical pa- rameters. In addition to geographical origin, horse breeds can be divided into phenotypic or performance categories. Due to the great diversity of breeds, horses are most often classified as “warm-blooded” breeds including light hors- es of Arabian descent (such as Arabians, Thoroughbreds, Standardbreds and Quarter Horses), and “cold-blooded” breeds essentially including heavy draft horses (such as the Belgian Horse, the Slovenian Cold Blooded Horse and the Posavje Horse). Several differences in haematological parameters were found between these two groups, such as a lower haematocrit in cold-blooded horses and higher erythrogram values in warm-blooded horses, which should be considered when determining reference values of blood parameters and interpreting blood tests (1-4). In contrast, light horse breeds have a higher red blood cell count (RBC), haemoglobin concentration (HGB), haematocrit value (HCT) and blood volume than draft horses (2, 5, 6). In ad- dition to horse type, haematological parameters may also vary due to numerous internal and external factors, includ- ing breed, sex, age, reproductive status, fitness and train- ing status, exercise load, feeding and, circadian variations. SloVetRes_NOTRANJOST ver2.indd 15 09/05/2023 10:14 16 | Slov Vet Res | Vol 60 No 1 Moreover, handling procedures during blood withdrawal, operating conditions, criteria for selecting healthy subjects, preparation of the subjects for the procedures, level of ex- citement and health status are also important factors that affect haematological values in horses (1, 6, 7, 8). Differences in the cellular constituents of the blood are the result of specific changes in an organ or organ system, or a general response of the individual to certain physiologi- cal or pathological conditions (6). For example, the total leukocyte count (WBC) and differential leukocyte count in healthy horses are dependent on age, which is associated with a steady decline in leukocyte counts (9-11) while the absolute and relative leucocyte counts, especially neutro- phils and lymphocytes, vary considerably (11) to reach a neutrophil to lymphocyte (N/L) ratio of 2:1 in older horses (2). Minor differences in leukocyte counts have been found between the different breeds of horses, with warm-blood- ed horses having higher WBC counts than to cold-blooded horses (6, 12). The horse population investigated in this study was the autochthonous Posavje Horse breed, originating from the Lower Sava River flatlands of the southeastern part of Slovenia (especially in the districts of Krsko and Brezice) and in Croatia, this breed resulted from the crossing of local warmblood mares with Norik stallions. In addition, Ardene stallions were used to improve the Posavje horses’ abilities for heavy draft work (13). The Posavje horse is the smallest cold-blooded breed in Europe, characterised by a good-na- tured temperament and a pronounced sexual dimorphism. It was mainly selected for heavy draft work, especially in steep forest areas, but it has also been used for meat pro- duction (13). In 1993, a Slovenian breeding and conserva- tion programme was established for this horse breed. Since then, the breed has been bred according to the principles of conservation genetics: narrow relation of breeding stallions (sires) to mares with balanced breeding using different sires and moderate selection (13). Currently, reference values of haematological parameters are widely available for horses in general and for the most common and popular breeds (2, 5, 14). However, literature data for endemic breeds are sparse and only a few reports address haematology in au- tochthonous draft horse breeds (15-17). The aim of this study was therefore to investigate the characteristics of the haematological parameters in the Posavje Horse breed and to test the hypothesis that age, sex and reproductive status cause some haematological changes in horses. In addition to variations in haematological parameters in the Slovene Posavje Horse breed, the study was particularly focused on the characteristics of differential leucocyte counts and their variations with age gain. The measured haematological val- ues and their variations could serve as guideline values for further haematological investigations and as a basis for the development of an approach to determine haematological reference values for the Posavje breed (1,2, 6, 18). Materials and methods The study was conducted as a part of the routine annual breeding and registration procedures of the Slovenian breeding and conservation programme for the autoch- thonous Posavje horse breed at different locations in the region of south-eastern Slovenia during July and August. The stallions were located as sires in breeding stations and separated from the mares and foals kept on the farms of local breeders. Regardless of category and location, the horses were kept on pasture in natural environmental con- ditions during the day and stabled in individual boxes dur- ing the night. While stabled, they were fed hay, considering the needs of each category, and had free access to water. They were dewormed regularly, clinically sound on the day of sampling and did not receive medication in the last 3 months before blood sampling. The mares included in the study were not pregnant. As the horses were familiar with humans and accustomed to different handling procedures, no restraint was required during sampling. The study included 39 stallions aged 3 to 22 years (aver- age 11.3 years), 94 mares aged 4 to 22 years (average 11.1 years) and 30 foals aged 30-180 days (average 102 days). The grouping of horses by age and sex is presented in Table 1. Blood samples were collected from the jugular vein with double-ended needles and evacuated tubes contain- ing K2EDTA as an anticoagulant (Vaccuette; Greiner Labortechnik GmbH, Kreimsmünster, Austria) and stored at 4 °C for haematological analyses (6, 18), which were per- formed within the next 6 hours at the Laboratory for Clinical Pathology of the Clinic for Reproduction and Large Animals at Veterinary Faculty, University of Ljubljana. Routine hae- matological analyses included the following: Red blood cell tests (red blood cell count (RBC), haematocrit (HCT), haemoglobin concentration (HGB), red blood cell indices (mean cell haemoglobin concentration (MCHC), mean cell Table 1: Arrangement of horses to age groups Age group Age Sex Total (n)Mares (n) Stallions (n) Foals 30–180 days / / 30 Adults 3–22 years 94 39 134 Group A 3–6 years 21 8 29 Group B 7–9 years 22 9 31 Group C 10–13 years 20 8 28 Group D 14–15 years 10 6 16 Group E 16 and more 21 8 29 SloVetRes_NOTRANJOST ver2.indd 16 09/05/2023 10:14 Slov Vet Res | Vol 60 No 1 | 17 volume (MCV), mean cell haemoglobin (MCH)) and red blood cell distribution width (RDW)), white blood cell tests (total white blood cell count (WBC), absolute differential leu- kocyte count), platelet count (PLT) and mean platelet vol- ume (MPV) were performed using an automated veterinary haematology analyser (Scil Vet abc Plus+, Horiba, Japan), validated for equine samples and following original instruc- tions for use. The relative differential leukocyte count (neu- trophils - NEU, eosinophils - EOS, basophils - BAS, mono- cytes - MON, lymphocytes - LYM) was measured under the microscope using blood smears stained with the commer- cial staining kit Hemacolor (Merck Cat. No. 1.11661, Merck KGaA, Darmstadt, Germany). The neutrophil/lymphocyte ratio (N/L ratio) was calculated by dividing the neutrophil proportion by the lymphocyte proportion. Statistical calculations were performed using the Statistical Package for Social Sciences (SPSS for Windows, release 8.0.0). The normality of the data distribution was assessed using a Shapiro–Wilk test and significance was determined using all pairwise multiple comparisons (Tukey’s test). Differences between values calculated for horses grouped by age or sex were statistically analysed by one-way analy- sis ANOVA. When significant differences were found, a posthoc analysis was performed (Bonferroni–Holm test) to clarify the groups between which these differences existed. Differences were considered significant at P≤0.05. The val- ues measured are presented as the mean ± standard devia- tion in the text (x ± SD) and as mean ± error of the mean (x ± SE) in the figures. Results Red blood cell tests The mean values of haematological parameters for all ex- amined foals, stallions and mares of Posavje horses are shown in Table 2. Statistically significant differences be- tween mares and stallions were found for RBC, HCT and haemoglobin concentrations (P < 0.001). In foals, RBC lev- els were significantly higher (P < 0.001) than in stallions and mares, but HCT, MCV and MCH levels were significantly lower (P < 0.001). The mean HGB concentration was sig- nificantly lower in foals than in stallions (P < 0.001) and higher than in mares (P < 0.01). The differences between foals, mares and stallions were not significant for MCHC and RDW (Table 2). Age-dependent variations in RBC and indices in mares and stallions of the Posavje breed are presented in Fig. 1. The RBC values (Fig.1A) were highest in foals and signifi- cantly decreased thereafter in both sexes with age gain (P < 0.001). With the exception from foals and 3 to 4-year-olds, the RBC values in mares were lower than those in stallions (P < 0.001). The lowest MCV (Fig.1 B) and MCH (Fig.1 D) were measured in foals and increased significantly with age in both sexes (P < 0.001 for both parameters). MCV was significantly lower in mares aged 3 to 6 years than in older animals (P < 0.001). The differences between stallions and mares of all ages were insignificant for MCV and MCH. HCT (Fig. 1C) and HGB (Fig. 1E) values in mares decreased with age (P < 0.001), reaching the lowest values in the 14- to 15-year-old Table 2: Haematological parameters in Posavje horse foals, stallions and mares in total (x ± SD) Variable (unit) Foals (n=30) Stallions (n=39) Mares (n=94) RBC (× 1012/L) 9.84 ± 1.26a,b 8.79 ± 0.99c 7.45 ± 1.01 HGB (g/L) 124.27 ± 12.82 a,b 138.41 ± 14.93c 117.08 ± 12.82 HCT (L/L) 0.35 ± 0.04a,b 0.40 ± 0.05c 0.33 ± 0.04 MCV (fL) 36 ± 1.86a,b 45.18 ± 2.76 44.79 ± 3.35 MCH (pg) 12.68 ± 0.66a,b 15.78 ± 1.01 15.83 ± 1.25 MCHC (g/L) 351.93 ± 12.49 349.54 ± 8.26 353.52 ± 15.16 PLT (× 109/L) 350.93 ± 101.08a,b 229.54 ± 67.12 242.65 ± 67.62 MPV (fL) 7.48 ± 0.79a,b 6.36 ± 0.38c 6.61 ± 0.37 RDW (%) 17.86 ± 1.11 17.94 ± 0.74 17.99 ± 0.71 Legend: RBC - red blood cell count; HGB - haemoglobin concentration; HCT - haematocrit; MCV - mean cell volume; MCH - mean cell haemoglobin; MCHC - mean cell haemoglobin concentration; PLT – platelet count; MPV – mean platelet volume; RDW – red cell distribution width. Values in a row with the same superscript show significant differences (adifferences between foals and mares; bdifferences between foals and stallions; cdifferences between mares and stallions; P < 0.05) SloVetRes_NOTRANJOST ver2.indd 17 09/05/2023 10:14 18 | Slov Vet Res | Vol 60 No 1 5 6 7 8 9 10 11 12 RB C [× 10 12 /L ] Age [years] Mares Stal lions 30 35 40 45 50 foals 3 to 6 7 to 9 10 to 13 14 to 15 16 < M CV [f L] Age [years] Mares Stal lions 0,25 0,30 0,35 0,40 0,45 0,50 foals 3 to 6 7 to 9 10 to 13 14 to 15 16 < PC V [1 /1 ] Age [years] Mares Stal lions 12 13 14 15 16 17 18 foals 3 to 6 7 to 9 10 to 13 14 to 15 16 < M CH [p g/ L] Age [years] Mares Stal lions 100 110 120 130 140 150 160 foals 3 to 6 7 to 9 10 to 13 14 to 15 16 < HG B [g /L ] Age [years] Mares Stal lions 345 350 355 360 365 foals 3 to 6 7 to 9 10 to 13 14 to 15 16 < M CH C [g /L ] Age [years] Mares Stal lions Figure 1: Age-dependent changes of red blood cell number (RBC; Panel A), mean cell volume (MCV; Panel B), Haematocrit (HCT; Panel C), mean cell haemoglobin (MCH; Panel D), haemoglobin concentration (HGB; Panel E) and mean cell haemoglobin concentration (MCHC; Panel F) in Posavje mares and stallions (mean ± SE) group, while in stallions, a decrease was observed with peak values for HCT and HGB in the 7- to 9-year-old age group, followed by a gradual decrease in both values with age. Stallions aged 7 to 9 years or more had significantly higher HCT and HGB values than mares in the same age group (P < 0.001, respectively). The MCHC value (Fig. 1F) in stallions decreased slightly and reached the lowest val- ues at the age of 7 to 9 years and increased thereafter. In mares, MCHC increased with age, peaking at 10 to 13 years of age and decreasing thereafter; the differences in MCHC between mares and stallions of all ages were not signifi- cant (P > 0.05). RDW values remained stable with age in both mares and stallions and differences between sexes were not signifi- cant (hence, the changes are not shown graphically). The PLT count and MPV were significantly higher in foals than in stallions and mares (P < 0.001). The mean platelet volume (MPV) was higher in mares than in stallions (P < 0.001). SloVetRes_NOTRANJOST ver2.indd 18 09/05/2023 10:10 foals 3 to 6 7 to 9 10 to 13 14 to 15 16 < C D A B E F Slov Vet Res | Vol 60 No 1 | 19 White blood cell tests The mean total WBC and the relative and absolute differen- tial leucocyte counts in foals, stallions and mares are shown in Table 3. The mean total WBC and LYM counts (Table 3) were significantly (P < 0.001) higher in foals than in mares and stallions, while the NEU, MON, EOS and BAS counts were significantly lower in foals than in adults (P < 0.05). The highest WBC value (Fig. 2A) was found in foals of both sexes and then gradually decreased in mares to reach the lowest value in the age group of 16 years and over (P < 0.001). A significant decrease in WBCs was observed in stallions at 3 to 6 years of age (P < 0.01), followed by an increase in WBCs at 10 to 13 years of age and a gradual decrease thereafter. WBC counts were also significantly lower in stallions aged 3 to 6 years (P < 0.01) than in mares of the same age. In mares, WBC counts were significantly higher in the 3- to 6-year-old group than in the older group (P < 0.001). The NEU count increased and the LYM count decreased significantly (P < 0.001 and P < 0.001, respec- tively) with increasing age in stallions and mares (Fig. 2B). Mares aged 3 to 6 years had a significantly higher NEU count (4.61) compared to stallions of the same age (3.36, P < 0.05; Fig.2B). The N/L ratio was significantly lower in the foals than in Groups B (7 to 9 years), D (14 to 15 years) and E (16 and more years) (P < 0.001) (Table 4). The interactions between age and sex of NEU and LYM are shown in Fig. 2. MON did not significantly differ between Posavje foals, stal- lions and mares (Table 3). The BAS counts in foals were statistically lower than those in mares and stallions (P < 0.001 and P < 0.05, respectively). Discussion Red blood cell tests The mean RBC of Posavje stallions and mares was at the lower end of the normal range for warm-blooded horses (19) but at the upper end of the normal range for draft hors- es (16, 17) and slightly lower than in warmbloods (9, 20). In general, erythrogram values in this study were mostly com- parable to those for warmblooded horses (5, 14, 21), which is surprising considering that the Posavje horse is a cold- blooded breed, although some warmblood characteristics are still present. In Posavje foals, the mean RBC was higher than in adult horses, consistent with the literature (2, 9). However, MCV and MCH were lower than in adult horses, as in warm-blooded foals (19). The erytrogram values determined in our study were also related to the age of horses as described recently (14, 5, 22), with the RBC decreasing with age, followed by a Table 3: Total WBC count and relative/absolute differential leukocyte count (x ± SD) in foals, stallions and mares Variable Unit Foals(n=30) Stallions (n=39) Mares (n=94) WBC ×109/L 11.20 ± 2.03a,b 8.76 ± 1.81 9.14 ± 2.03 NEU % 39 ± 11.42 56.26 ± 12.93 51.9 ± 11.65 ×109/L 4.55 ± 1.57 4.99 ± 1.84 4.71 ± 1.45 LYM % 52.67 ± 10.39a,b 37.26 ± 12.38 39.2 ± 11.62 ×109/L 5.78 ± 1.66a,b 3.20 ± 1.11 3.63 ± 1.54 MON % 0.93 ± 1.27 1.23 ± 1.37 0.85 ± 0.97 ×109/L 0.12 ± 0.15 0.11 ± 0.13 0.08 ± 0.10 EOS % 3.52 ± 3.20 4.10 ± 3.14 5.66 ± 3.62 ×109/L 0.37 ± 0.25 0.36 ± 0.29 0.50 ± 0.32 BASO % 0.11 ± 0.42a 0.38 ± 0.63 0.79 ± 1.07 ×109/L 0.01 ± 0.04a 0.03 ± 0.06 0.07 ± 0.09 Ratio N/L 1/1 0.88 ± 0.54a,b 1.86 ± 1.23 1.55 ± 0.84 Legend: WBC – white blood cell count; NEU – neutrophil; LYM – lymphocyte, MON –monocyte; EOS – eosinophil; BAS – basophil; N/L –neutrophil/ lymphocyte ratio. Values in a row with the same superscript indicate significant differences (adifferences between foals and mares; bdifferences between foals and stallions; cdifferences between mares and stallions; P < 0.05) SloVetRes_NOTRANJOST ver2.indd 19 09/05/2023 10:14 20 | Slov Vet Res | Vol 60 No 1 compensatory increase of MCV and MCH in both sexes (5, 14, 18, 21, 22, 23, 25, 26). A gradual increase in MCV appears to be a common finding associated with equine ageing (22-24) causing changes in the dynamics of eryth- rocyte maturation (24). The MCV values of the Posavje horses studied were lower than those in the Przewalski and Kathiawari horse breeds (27, 28) but higher than those in the Zemaitukai horses (21) of comparable ages, while in foals they were consistent with those reported in many other breeds (29, 30). HGB concentrations differed between sex and age groups of Posavje horses in the present study and were consis- tent with (15, 31) or higher (32) literature data. In contrast to Lahora working horses (33) and Lipizzans (5), a sig- nificant increase in HCT and HGB was observed only in Posavje stallions up to the age of 7 to 9 years, followed by a gradual decrease in older groups, while the values in mares decreased with age. In general, the mean total RBC, HCT and HGB levels were significantly higher in Posavje stal- lions than in mares, as common for horses (15, 33, 34, 35). This trend is most likely due to the effect of testosterone, which is also known to increase circulating HGB, HCT and RBC in humans (36) stimulating haematopoietic tissue and erythropoiesis in men more than in women (37). The role of testosterone in haematopoiesis was also supported by the study in castrated goats (38). The mean RDWs of Posavje horses did not differ between age groups and sexes and were lower than (7, 39, 40) or sim- ilar to reported values (17, 19, 41, 42, 43, 44). A significant de- crease in RDW was previously observed in stallions after ex- ercise (7), but this decrease was not observed in our study. Platelet count in Posavje horses decreased with age, which is consistent with literature data (21, 22, 23), although no dif- ferences between age groups were reported (24). MPV was higher in Posavje mares than in stallions; in both sexes, the values were higher than in Shetland ponies (41) and lower than in Holstein horses (7, 44). Elevated MPV has been pro- posed as an indicator of platelet activation in humans, but the lack of defined limits to distinguish between activated and nonactivated platelets and the failure of platelet aggre- gation inhibitors to reverse a high MPV limit its utility as a platelet activation marker in human medicine (45). White blood cell test In the present study of horses of the Posavje breed, the highest mean WBC value was found in foals, followed by mares, and the lowest in stallions, with the later exhibiting the lowest value (14, 21, 25). In contrast, the WBC levels of Thoroughbred (9) and Lipizzan (46) stallions were higher in than in mares, and some studies failed to find signifi- cant differences between the sexes (47). In all age groups of Posavje horses WBC values in mares were higher than those in stallions, although this difference was significant 6,00 7,00 8,00 9,00 10,00 11,00 12,00 foals 3 to 6 7 to 9 10 to 13 14 to 15 16 < W BC [* 10 9 / L] Age [years] Mares Stal lions 0 1 2 3 4 5 6 7 8 foals 3 to 6 7 to 9 10 to 13 14 to 15 16 < N EU a nd L YM [* 10 9 / L] Age [years] Figure 2: Changes of total white blood cell number (x ± SE) (A), and neutrophil (NEU) and lymphocyte (LYM) counts (B) in Posavje mares and stallions with age Table 4: Neutrophil/lymphocyte ratio (N/L) (x ± SD) in mares and stallions of various age groups (A: 3–6, B: 7–9, C: 10–13, D: 14–15, E: 16 and more years old) Sex Age group Foals Group A Group B Group C Group D Group E Mares 0.84 ± 0.54 0.94 ±0.35 1.14 ±0.53 1.78 ±1.08 1.91 ±0.55 2.20 ±0.82 Stallions 0.75 ± 0.55 0.96 ±0.57 1.43 ±0.47 2.50 ±1.47 2.84 ±1.82 1.86 ± 0.81 SloVetRes_NOTRANJOST ver2.indd 20 09/05/2023 10:10 A B NEU mares NEU stallions LYM mares LYM stallions Slov Vet Res | Vol 60 No 1 | 21 only at the age of 3 to 6 years. The in WBC count of Posavje horses decreased with age gain (11, 24, 44) which could be attributed to the gradual decline in immunocompetence and cannot be considered as leukopenia (24, 44). In Posavje horses, slightly higher NEU and lower LYM counts were measured in stallions than in mares (48). A significant age-related decrease in LYM count, proportional to the decrease in WBC, was observed in both sexes of the Posavje horse, as also reported in other horse breeds (2, 15, 16, 46). This decrease may be the reason for the de- creased immunocompetence in older horses (10). The ab- solute NEU count has been reported to be higher in foals than in adult horses and remains stable with age gain (9, 25, 46), whereas it increased significantly in Posavje horses of both sexes. This increase was also the reason for a steady increase in N/L ratio with age, ranging from 0.84 to 2.01 in mares and from 0.75 to 1.68 in stallions. Similar changes in the N/L ratio in Andalusian horses (11) indicate a natural state reflecting a decreased bone marrow response. The predominance of NEU in the Posavje horses studied re- flects the cold-blooded origin of this breeds (12). In stallions aged 3 to 6 years the NEU count decreased sig- nificantly in parallel to the WBC count; however, it remained within physiological limits. In all other age groups, NEU counts were higher in stallions than in mares, as also re- ported for other horse breeds (32, 48, 49, 50); this difference could be attributed to increased testosterone production in reproductively active stallions. The plasma levels of tes- tosterone are an important regulator of NEU function and the associated inflammatory response in humans (51, 52) which represents the first line of defence against invading pathogens and tissue injury (53, 54). Therefore, the physi- ological increase in NEU in the blood of the stallions studied could be an evolutionary adaptation to prevent infections caused by injuries of stallions, fighting for mares within a harem. Surprisingly, that the described changes in the NEU counts of stallions have thus far gone unnoticed. Modest increases in NEU counts within the normal range may have been ignored and the stallions in studies that addressed this issue (14, 21, 34, 46) were not sufficiently old or were reproductively inactive. Another reason for leucocytosis in horses with increased NEU and decreased LYM numbers could be increased plasma cortisol levels under stress (53). We can exclude this cause in the Posavje stallions, as all age groups of the examined stallions were housed under similar environmental conditions and treated in the same manner. Neither age nor sex affected the EOS and MON, confirm- ing the results of previous studies in horses (46, 49). Higher BAS values in older Posavje stallions were likely the result of altered immunological load (14). Conclusions In conclusion, our study indicates breed-related differenc- es in haematological parameters of horses, and we have shown that haematological parameters vary with age and sex in the Posavje breed. The haematological traits iden- tified in our study represent interesting breed-, age- and sex-specific adaptations/responses but are of limited di- agnostic value. In general, the values of the haematologi- cal parameters in our study most closely matched those of warm-blooded horses, although the Posavje horse is a cold-blooded breed. The leucogram values and the N/L ratio determined in our study corresponded to those of cold-blooded horses. Furthermore, the results confirm and extend previous reports on age- and sex-related changes in haematological variables. 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Red blood cell distribution width in Quarter horses: a comparison between healthy and hospitalized animals. J Equine Vet Sci 2019; 73: 127–30. 44. Kisadere I, Bayraktar M, Salykov R. Some hematological and bio- chemical reference values of the thoroughbred Appaloosa horse breeds reared in Kyrgyzstan. Comp Clin Pathol 2019; 28: 1651–60. 45. Tsiara S, Elisaf M, Jagroop IA, Mikhailidis DP. Platelets as predictors of vascular risk: is there a practical index of platelet activity? Clin Appl Thromb Hemost 2003; 9: 177–90. 46. Čebulj-Kadunc N, Kosec M, Cestnik V. The variations of white blood cell count in Lipizzan horses. J Vet Med A Phys Pathol Clin Med 2003; 50(5): 251–3. 47. Lacerda L, Campos R, Sperb M, et al. Hematologic and biochemıcal parameters in three high performance horse breeds from southern Brazil. Arch Vet Sci 2006; 11(2): 40–4. 48. Ono T, Inoue Y, Hisaeda K, et al. Effect of seasons and sex on the physical, hematological, and blood biochemical parameters of Noma horses. J Equine Sci. 2021; 32(1): 21–5. 49. Romić S. Krvna slika noniusa. Zagreb : Poljoprivredni nakladni zavod, 1973: 289–98. (Poljoprivredna znanstvena smotra; 16/2) 50. Nidl C, Merino JO, López EA, et al. Effect of age, gender, and season on hematological pa-rameters in Quarter horses. J Vet Sci Med Diagn 2017; 6(2). doi: 10.4172/2325-9590.1000224 51. Marin DP, Bolin AP, dos Santos Rde C, Curi R, Otton R. Testosterone suppresses oxidative stress in human neutrophils. Cell Biochem Funct 2010; 28(5): 394–402. 52. Gagliano-Jucá T, Pencina KM, Guo W, et al. Differential effects of testosterone on circulating neutrophils, monocytes, and platelets in men: findings from two trials. Andrology 2020; 8(5): 1324–31. 53. Davis AK, Maney DL, Maerz JC. The use of leukocyte profiles to mea- sure stress in vertebrates: a review for ecologists. Funct Ecol 2008; 22: 760–72. 54. Hines MT, Schott HC, Bayly WM, Leroux AJ. Exercise and immunity: a review with emphasis on the horse. J Vet Intern Med 1996; 10: 280–9. 55. Krumrych W. Variability of clinical and haematological indices in the course of training exercise in jumping horses. Bull Vet Inst Pulawy 2006; 50: 391–6. Karakterizacija hematološkega profila pri posavskem konju M. Mesarič, M. Nemec, N. Čebulj Kadunc Izvleček: Cilj raziskave je bil proučiti vpliv spola in starosti na hematološke parametre pri pasmi posavski konj. V razis- kavo je bilo vključenih 163 konj posavske pasme (30 žrebet, 94 kobil in 39 žrebcev), pri katerih smo v vzorcih krvi določali hematološke parametre s hematološkim analizatorjem. Diferencialna bela krvna slika in razmerje med nevtrofilci in lim- fociti (N/L) je bilo določeno na krvnih razmazih. Konje smo razdelili v pet starostnih skupin: žrebeta (od 1 do 6 mesecev, n = 30), 3 do 6 let (n = 8 žrebcev/21 kobil), 7 do 9 let (n = 9 žrebcev/22 kobil), 10 do 13 let (n = 9 žrebcev/20 kobil), 14 do 15 let (n = 6 žrebcev/10 kobil) ter 16 in več let (n = 8 žrebcev/21 kobil). Rezultati naše raziskave kažejo vpliv spola na preis- kovane hematološke parametre; pri žrebcih so število rdečih krvnih celic (RBC), hematokrit (HCT) in koncentracija hemo- globina (HGB) značilno višji (P < 0,001), pri kobilah pa je višje število belih krvnih celic (WBC). Med starostnimi skupinami posavskih konj smo ugotovili zmanjšanje RBC in HGB in posledično kompenzacijo s povečanjem povprečnega volumna in hemoglobina eritrocitov, zmanjšanjem števila levkocitov, trombocitov (PLT) in limfocitov ter povečanjem razmerja med nevtrofilci in limfociti (N/L) s starostjo (P < 0,001). Posavski konj po zunanjosti spada med hladnokrvne konje, v raziskavi ugotovljeni hematološki profil pa kaže značilnosti, ki so skupne toplokrvnim pasmam konj, z izjemo razmerja N/L. Ena od pomembnejših ugotovitev te študije je večje število nevtrofilcev pri aktivnih plemenskih žrebcih. Višje vrednosti RBC, HGB, HCT in števila nevtrofilcev pri posavskih žrebcih kažejo učinek androgenov (testosterona), kar bi lahko bil učinkovit mehanizem za preprečevanje okužb, ki lahko vplivajo na preživetje žrebcev in s tem na evolucijo vrste. Ključne besede: avtohtone pasme; posavski konj; hematologija; starost; spol SloVetRes_NOTRANJOST ver2.indd 23 09/05/2023 10:14 SloVetRes_NOTRANJOST ver2.indd 24 09/05/2023 10:14 Slov Vet Res | Vol 60 No 1 | 25 The Effect of a Specific Chicken Based Renal Diet as Monotherapy on Clinical, Biochemical, Urinary and Serum Oxidative Stress Parameters in Cats With CKD Stage 1 and 2 Key words clinical parameters, symmetric dimethylarginine, oxidative stress, renal diet, cats, chronic kidney disease, urinary protein electrophoresis Martina Krofič Žel1, Alenka Nemec Svete1, Breda Jakovac Strajn2, Katarina Pavšič Vrtač2, Tomaž Vovk3, Nataša Kejžar4, Darja Pavlin1* 1Small Animal Clinic, 2Institute of Food Safety, Feed and Environment, Veterinary Faculty, University of Ljubljana, Gerbičeva 60, 1000 Ljubljana, 3Chair of Biopharmacy and Pharmacokinetics, Faculty of Pharmacy, University of Ljubljana, Aškerčeva 7, 1000 Ljubljana, 4Institute for Biostatistics and Medical Informatics, Faculty of Medicine, University of Ljubljana, Vrazov trg 2, 1000 Ljubljana, Slovenia *Corresponding author: darja.pavlin@vf.uni-lj.si Abstract: The aim of the study was to investigate the effect of a therapeutic renal diet on selected clinical, biochemical, and urinary parameters and on selected parameters of oxidative stress in cats with early stages of chronic kidney disease (CKD). A prospective study of a 3-month duration was conducted to evaluate the effect of renal diet on se- lected clinical and laboratory parameters in client-owned cats with early stages of CKD. Of a total of 29 enrolled client-owned cats, nineteen (19) cats completed the study, ten receiving renal diet and nine receiving a diet of the owner’s choice. A clinical examina- tion was performed, and blood and urine samples were collected on the day of presen- tation and at regular check-ups after 3-4, 7-8, and 10-12 weeks. Serum creatinine and symmetric dimethylarginine (SDMA) concentrations and selected parameters of oxida- tive stress (plasma glutathione peroxidase (GPX) activity and plasma malondialdehyde (MDA) and serum selenium concentrations), were measured and electrophoresis of uri- nary proteins was performed. At inclusion, a significant positive correlation (p < 0.001) was found between serum selenium concentration and plasma GPX activity (Pearson correlation coefficient 0.83 (95% CI: [0.65 - 0.92]) and a significant negative correlation (p < 0.001) between serum SDMA and urine specific gravity (Pearson correlation coeffi- cient -0.70 (95% CI: [-0.87 - (-0.38)]). At the end of the 3-month feeding trial no significant difference was found in SDMA and creatinine concentrations. Received: 11 July 2022 Accepted: 6 February 2023 DOI 10.26873/SVR-1515-2023 UDC 636.8.09:616.61:613.24:637.54:616.152/.153 Pages: 25–35 Original Research Article Introduction Chronic kidney disease (CKD) in cats has an overall preva- lence of 2-3% of the feline population (1, 2). The prevalence is higher in older cats: 10% in cats older than ten years (3) and 28% in cats older than 12 years (4). A prevalence of 28% and a 67% is reported in the overall population of cats and in those older than 18 years old, respectively, based on the serum concentration of symmetric dimethylarginine (SDMA) (5). Chronic kidney disease is characterized by progressive loss of functional renal tissue, leading to renal fibrosis, which can cause uremic crisis and death (6). Early recognition of CKD is essential for prompt management of such patients, leading to a better long-term prognosis (7,8). Proteinuria is an important and independent predictor of worsening of CKD (9,10,11). In contrast to dogs, chronic in- terstitial nephritis is predominantly found in cats. Therefore, tubular proteinuria is more common than glomerular pro- teinuria (12). SloVetRes_NOTRANJOST ver2.indd 25 09/05/2023 10:14 26 | Slov Vet Res | Vol 60 No 1 Oxidative stress aids progression of CKD in human patients (13). Plasma glutathione peroxidase (GPX), synthesized by renal tubular cells, is the major reactive oxygen species scavenger in the kidneys (14). In human CKD patients, plas- ma GPX activity decreases as the disease progresses and its activity is already reduced in patients with mild chronic uremia (15,16). On the other hand, a significantly higher plasma GPX activity was found in cats with CKD IRIS stage 4 compared with healthy cats (17). Plasma malondialdehyde (MDA) is one of the most popular and reliable markers of the extent of lipid peroxidation and thus oxidative stress (18). Selenium is an integral part of selenoproteins, one of which is plasma GPX. This micro- element is present in protein-rich foods, and its excess is excreted via the kidneys (19). Unlike human uremic patients, uremic cats do not have a selenium deficiency (17). According to evidence-based veterinary medicine, renal diet is the therapy of choice in both feline and canine CKD patients from the International Renal Interest Society (IRIS) stage 2 (20,21,22). Hall and colleagues (23) reported that cats with IRIS CKD stage 1 and 2 benefit from a diet with increased caloric density and enhanced concentrations of carnitine and essential amino acids. The biomarkers of kid- ney function, body weight and lean muscle mass were sta- ble in cats consuming such a diet. However, a recent study reported that feeding a highly phosphorus-restricted diet to cats with early-stage CKD may lead to hypercalcemia and urolithiasis, while a diet moderately restricted in protein and phosphorus may be beneficial (24). The authors are aware of only a few studies that deal with oxidative stress in feline CKD (17, 25, 26, 27, 28, 29, 30). However, the results of these studies are inconclusive about the role of oxidative stress in the pathophysiology of CKD. Furthermore, there is a lack of data on the effects of renal diet on oxidative stress parameters in CKD cats. The aim of the present study was to investigate/explore the effect of a therapeutic renal diet on selected clinical, bio- chemical, and urinary parameters and on selected param- eters of oxidative stress in cats with early stages of CKD and to provide credible insight with monitoring of clinical and laboratory parameters. Materials and methods This prospective study was conducted on client-owned cats with early stages of CKD and lasted for three months. The inclusion criterion was CKD stage 1 or 2, according to the IRIS guidelines (31), with no previous treatment recorded. Cats with acute kidney injury, prerenal or postrenal azo- temia, nephropathy of toxic or infectious origin within the last 28 days, urinary tract obstruction, acute systemic in- flammation, liver disease, chronic heart failure, cancer or serologically positive for feline leukemia or feline immuno- deficiency were excluded from the study. All owners signed a consent form before enrolling the cats in the study. All procedures complied with the relevant Slovenian governmental regulations (Animal Protection Act, Official Gazette of the Republic of Slovenia, No. 43/2007). The cats were randomly divided into two groups: cats in the control group, which received a regular diet and cats in the experimental group, which received a renal diet. Simple randomization method with sequentially numbered sealed envelopes was used to group the patients (32). The cats in both groups received their diet ad libitum ac- cording to their habitual regime. Clinical examination in- cluding body weight monitoring, blood pressure measure- ment, routine hematological and biochemical analyses, and urinalysis with UPC (urine protein to creatinine ratio) were performed on the day of presentation and regular check- ups after 3-4, 7-8, and 10-12 weeks. In addition to routine laboratory parameters, measure- ments of SDMA concentration, and selected parameters of oxidative stress (GPX, MDA, selenium) were also performed at each check-up and will be described below. Composition of the diet The cats in the experimental group were fed Vet Life Feline Renal Formula (Farmina Pet Foods, Naples, Italy). The composition of the renal diet is shown in Table 1. The re- nal diet used in the study had the same lot number for all cats. The cats in the control group continued to receive the maintenance diet to which they were accustomed to prior to participation in the study. Blood and urine sample collection, processing, and analysis Blood samples were taken from the jugular vein and trans- ferred into serum separator tubes (Vacuette, Greiner Bio- One, Kremsmunster, Austria) for the determination of se- rum biochemical profiles, including SDMA, and antigen detection of feline leukemia virus (FeLV) and specific an- tibody against feline immunodeficiency virus (FIV). The tubes were stored for 30 minutes at room temperature to clot and then centrifuged at 1300 × g for 10 minutes at room temperature to separate the serum. Serum samples for the determination of routine biochemical parameters (urea, creatinine, alanine aminotransferase, alkaline phosphatase, total proteins, albumins, total calcium, inorganic phosphate, electrolytes (sodium, potassium, chloride)) were analysed on the day of blood collection. For measurement of SDMA concentration in serum, an aliquot of the serum sample was prepared and immediately stored at -80°C until anal- ysed in batch. SloVetRes_NOTRANJOST ver2.indd 26 09/05/2023 10:14 Slov Vet Res | Vol 60 No 1 | 27 Blood samples for hematological analysis were collected into 0.5 ml EDTA-containing tubes (BD Microtainer Tubes, Becton, Dickinson and Company, Franklin Lakes, New Jersey, USA). Urine samples were collected by cystocentesis and ana- lyzed within 1 to 2 hours. Biochemical profiles (urea, creatinine, alanine aminotrans- ferase, alkaline phosphatase, total proteins, albumins, to- tal calcium, inorganic phosphate), except electrolytes and SDMA, were determined with an automated biochemistry analyser RX Daytona (Randox, Crumlin, UK). The electro- lytes were determined with an Ilyte electrolyte analyzer (Instrumentation Laboratory, Lexington, Massachusetts, USA). Hematological analyses were performed with an au- tomated laser hematology analyzer ADVIA 120 (Siemens, Munich, Germany) using species-specific software. ELISA for the detection of antigen against FeLV and spe- cific antibody against FIV were carried out according to the instructions of the manufacturer (IDEXX, Lenexa, Kansas, USA) on the day of collection. Determination of the MDA concentration Blood samples for the determination of plasma MDA con- centration were collected into 2 ml EDTA-containing tubes (Vacuette, Greiner Bio-One, Kremsmunster, Austria). All samples were immediately centrifuged at 1500 × g for 15 min at 4°C. The plasma was separated and immediately frozen at -80°C until analysis. The total plasma concentration of MDA was determined by a gentle alkaline saponification and derivatization method (33). MDA was derivatized with 2,4-dinitrophenylhydrazine to a pyrazole derivative and determined with an Agilent 1200 series high performance liquid chromatography sys- tem (Agilent, Waldbronn, Germany). The derivatized sam- ples were separated on an Agilent Eclipse XBD-C18 column by gradient elution with acetonitrile, water and acetic acid and the MDA derivative was detected with the diode array detector. The plasma MDA concentration was expressed as µmol per L (µmol/L). Determination of the SDMA concentration All serum SDMA concentrations were measured in batch at the end of the study by IDEXX Laboratories in Germany (IDEXX SDMA Test, IDEXX Laboratories INC., Leipzig, Germany). Determination of GPX activity Plasma GPX activity was measured spectrophotometri- cally with an automated biochemistry analyzer RX-Daytona (Randox, Crumlin, UK) using the commercial Ransel kit (Randox Laboratories, Crumlin, UK) which is based on the Table 1: The composition of the renal diet Raw protein 26.00% Raw oils and fats 20.00% Raw fiber 2.40% Raw ashes 7.30% Calcium 0.80% Phosphorus 0.60% Sodium 0.35% Potassium 0.90% Magnesium 0.07% Omega 3 fatty acids 0.40% Omega 6 fatty acids 3.90% EPA 0.10% DHA 0.15% Energy value 3965 kcal/kg – 16.6 MJ/kg Nitrogen-free extract/1000 kcal 11.77 g/1000 kcal Selenomethionine 60 mg per kg corresponding to 13.5 mg selenium/kg dry matter) Legend: EPA eicosapentaenoic acid; DHA docosahexaenoic acid Composition: pea starch, potatoes, chicken fat, hydrolyzed fish proteins, dehydrated whole eggs, hydrolyzed chicken proteins, dehydrated chicken meat, quinoa seed extracted, dehydrated fish, fish oil, calcium carbonate, inulin, fructooligosaccharides, mannanoligosaccharides, potassium chloride, sodium chloride, glucosamine (500 mg/kg), Marigold extract (source of lutein) Additives per kg Nutritional additives: Vitamin A 15000 IU; Vitamin D3 600 IU; Vitamin E 550 mg; niacin 125 mg; pantothenic acid 42 mg; Vitamin B2 17 mg; Vitamin B6 7 mg; Vitamin B1 8 mg; Vitamin H 1.3 mg; folic acid 1.3 mg; Vitamin B 12 0.08 mg; choline chloride 2500 mg; beta-carotene 1.5 mg; zinc chelate of the analogous methionine hydroxylase 725 mg; manganese chelate of the analogous methionine hydroxylase 385 mg; ferrous chelate of glycine hydrate 185 mg; copper chelate of the analogous methionine hydroxylase 54 mg; selenomethionine 60 mg; calcium iodate anhydrous 2.4 mg; taurine 2000 mg; DL methionine 5000 mg; L-lysine HCl 2000 mg; L-tryptophan 2000 mg; L-carnitine 250 mg. Technological additives: potassium citrate 3000 mg. Antioxidants: tocopherol-rich extracts of natural origin 10 mg. SloVetRes_NOTRANJOST ver2.indd 27 09/05/2023 10:14 28 | Slov Vet Res | Vol 60 No 1 29 cats met the inclusion criteria and were randomised 15 cats in the experimental group 10 cats completed the study in the experimental group 5 cats excluded 1 cat: renal lymphoma 2 cats: urethral obstruction 2 cats: less than 3 sample collections due to uncompliant owner 14 cats in the control group 9 cats completed the study in the control group 5 cats excluded 1 cat: urethral obstruction 4 cats: less than 3 sample collections due to uncompliant owner Figure 1: The flow diagram of cats with CKD during the study Paglia and Valentine method (34). The activity of plasma GPX was expressed as units per L (U/mL). Determination of the selenium concentration The microwave digestion of serum samples was performed with a Start D Microwave Acceleration Reaction System (Milestone, Sorisole, Italy). 0.4 to 1 mL of the samples were transferred into a 100 mL Teflon vessel and 3 mL 65% nitric acid, 0.5 mL 30% hydrogen peroxide and 4.5 mL Milli-Q wa- ter were added. The samples were digested in a closed 10- vessel microwave system at 200°C for 30 min. After cooling to room temperature, the solutions were diluted with Milli-Q water, and the concentrations of selenium were determined by inductively coupled plasma mass spectrometry (Varian 820-MS, Mulgrave, Australia). Argon was used as the car- rier gas, and the isotope 78Se was selected as the analyti- cal mass in ICP-MS normal sensitivity mode. For measure- ments of selenium, a Collision Reaction Interface (CRI) was used to reduce common polyatomic interferences. Urinalysis Urinalysis included the measurement of specific grav- ity with a refractometer, the use of a standard multitest urine dipstick (Multistix 10SG, Siemens, Munich, Germany) and microscopic examination of the urine sediment. The urine samples were centrifuged at 800 × g for 10 minutes at room temperature. Urine supernatants were used to determine protein and creatinine concentrations to calcu- late the UPC. Protein and creatinine concentrations were measured with an automated biochemistry analyzer RX Daytona (Randox, Crumlin, UK) using the pyrogallol red and picric acid methods, respectively. Protein concentrations were not determined if the urine samples were grossly con- taminated with blood. Gel electrophoresis was performed routinely by a commercial laboratory Euregio Laboratory Services (Kerkrade, Netherlands) in batch at the end of the study. Statistical analysis Based on the sample size, five keynote parameters were selected for statistical analysis (body weight, creatinine, SDMA, MDA and GPX). The differences between the first and the last (4th) measurement time-point were compared. The remaining parameters were presented in the form of descriptive statistics (median and interquartile range (IQR)) and with boxplots over time (Supplementary material). Basic characteristics and baseline measurements of sys- tolic blood pressure selected hematological, biochemical, oxidative stress, and urinalysis parameters in both groups were compared using the Fisher exact test (categorical) and the Mann-Whitney U test (numerical variables). Since a slight deviance towards older cats in the experimental group was observed, the comparison of the difference in keynote parameters was adjusted for age by the use of linear regression. The P-values for the group comparison from the linear models were corrected by Holm procedure (5%). The Pearson product-moment correlation was used to in- vestigate the possible correlation between the parameters (scatter plots are presented in the Supplementary material). SloVetRes_NOTRANJOST ver2.indd 28 09/05/2023 10:14 Slov Vet Res | Vol 60 No 1 | 29 Results Patients’ baseline characteristics A total of 29 cats of different breeds were enrolled, all of which were neutered. Of 29 cats enrolled in the study, 19 cats (ten cats in the ex- perimental group and nine cats in the control group) com- pleted the study. They represented the sample that we used for all analyses. The flow diagram of cats included in the study is presented in Figure 1. Blood and urine samples were collected at all four sched- uled check-ups from most cats enrolled. The missing blood and urine samples were not obtained due to problems with owners’ compliance. In all animals the first and the last (10- 12 weeks later) check-up were performed. Baseline characteristics (demographic data) are shown in Table 2. At the point of randomization in terms of age, sex, and body weight, there were no significant differences be- tween the experimental group and the control group; how- ever, the cats in the experimental group that completed the study were slightly older. Clinical signs and baseline clinical and laboratory parameters Clinical signs at presentation were mild in all included cats; the owners usually reported polyuria/polydipsia. At the end of the study, most owners of cats in the experimental group reported an improvement in the clinical status of the cats with reduced vomiting. At inclusion, all cats of both groups were reported to have normal appetite, which remained un- changed during the whole observation period. On each occasion, the owners were asked to evaluate their cats’ appetite and acceptance; the information on the ap- petite and acceptance of the diet was recorded at each check-up. The cats were given the amount of the diet that was appropriate for their weight as recommended by the manufacturer. The new diet was accepted 100 % by all cats in the experimental group; and the diet change was achieved in two weeks. During the first two weeks, the new diet was mixed with the usual diet and the amount of the new diet was gradually increased until the meal consisted only of the new diet. The owners reported that the entire amount of the meal was eaten by all included cats both during the transition period and during the study. Furthermore, the amount of the diet eaten during the study remained the same as before being enrolled into the study. Medians (IQR) for baseline and final (4th) measurement of laboratory parameters are presented in Supplementary material. The distributions of none of the parameters mea- sured at baseline significantly differed between groups. Time monitoring of all measured parameters is also pre- sented graphically in Supplementary material. Serum creatinine concentration During the study, median serum creatinine concentrations decreased in both the experimental and control groups (Supplementary material). The age adjusted difference in mean serum creatinine concentration decrease between both groups was not significant (Figure 2 A, Table 3). With one exception, all the included cats had stable CKD and re- mained at the same IRIS stage during the study. However, one cat in the experimental group was reclassified from IRIS 2 to 3 at the end of the study. The exact cause of the increase in serum creatinine concentration in that cat was not found. Seven out of 19 cats with elevated serum SDMA and creatinine concentrations, abnormal renal imaging findings and pathological urinary sediment, consistent with CKD, that were classified to IRIS stage 2, retained their urine concentration ability (Supplementary material). Serum SDMA concentration After the 3-month feeding trial, the median serum SDMA concentration decreased numerically in the Table 2: Baseline demographic and laboratory characteristics of cats in the experimental and the control group Group Control group (n = 9) Experimental group (n = 10) p value F/M 4/5 3/7 0.65 Age (months) Median (IQR) 78 (64−107) 116 (94−166) 0.066 Body weight (kg) Median (IQR) 5.9 (3.3−6.5) 4.9 (3.6−6.2) 0.842 Creatinine Median (IQR) 140.2 (130.0−178.8) 168.9 (161.1−178.8) 0.441 UPC (unitless) Median (IQR) 0.14 (0.12−0.19) 0.17 (0.11−0.26) 0.755 Legend: F-female cats; M-male cats; IQR-interquartile range SloVetRes_NOTRANJOST ver2.indd 29 09/05/2023 10:14 30 | Slov Vet Res | Vol 60 No 1 experimental group but remained the same in the control group (Supplementary material). There was no significant difference in age-adjusted de- crease of serum SDMA concentration between both groups (Table 3). Plasma MDA concentration At inclusion there was no significant difference in median plasma MDA concentrations between the sampled cats in the control group and those in the experimental group (Supplementary material). After the 3-month feeding trial, the mean age-adjusted difference in MDA was negligible in both groups (Table 3). Serum selenium concentration and plasma GPX activity In sampled cats receiving renal diet, median values of plasma GPX activity and serum selenium concentra- tion did not differ from the median values in the control group (Supplementary material). There was a negligible mean age-adjusted difference in plasma GPX activity after 3-month feeding trial in both groups (Table 3). A significant positive correlation (Pearson correlation coef- ficient 0.83 (95% CI: [0.65 - 0.92]) was found between the selenium concentration and plasma GPX activity at the be- ginning of the study. Urinary Protein electrophoresis At the beginning of the study, four cats in the control group and three cats in the experimental group had microalbu- minuria. Furthermore, more cats in the experimental group had non-zero fractions of protein in the urinary sample. The leading protein fractions at the beginning of the study were alpha 1 and albumin in the control group and beta in the experimental group. At the end of the study, beta fraction predominated in both groups. The median per cent fraction of the urinary protein at the beginning of the study in com- parison to the end of the study is shown in Table 4. Moreover, at the beginning of the study, a significant nega- tive correlation (Pearson correlation coefficient -0.70 (95% Figure 2: The changes in the concentrations of serum creatinine, serum SDMA, plasma MDA, and plasma GPX activity from the beginning to the end of the study in cats receiving renal diet in comparison to the control group A – change in serum creatinine concentration (P = 0.92), B – change in serum SDMA (symmetric dimethylarginine) concentration (P = 0.85), C – change in plasma MDA (malondialdehyde) concentration (P = 0.18), D – change in plasma GPX (glutathione peroxidase) activity (P = 0.65); P-values stand for comparisons of age-adjusted differences SloVetRes_NOTRANJOST ver2.indd 30 09/05/2023 10:14 Slov Vet Res | Vol 60 No 1 | 31 CI: [-0.87 - (-0.38)]) between SDMA and USG in the sampled cats were found. Discussion Cats with CKD IRIS stage 1 and 2 were monitored during a prospective 3-month feeding trial. All cats tolerated the diet change well in the experimental group and had normal ap- petite. During the study, the SDMA concentrations did not significantly change in any of the groups studied. Previously published data in cats with CKD IRIS stage 1 and 2 report a gradual increase in serum SDMA concentration regardless of the diet used (23). In the mentioned study, serum SDMA concentration increased from baseline at the first check- up after one month and continued to increase after three months in both the experimental and control groups (23). According to the published data, SDMA has a lower index of individuality than creatinine in cats (35) and dogs (36). Furthermore, consecutive measurements were performed in our study, and an individual value for each cat deter- mined. A gradual increase in SDMA in successive measure- ments can therefore be due to a gradual decrease in kidney function (35,37). However, the difference in results of the mentioned studies might be ascribed to different renal pa- thologies of the patients that were included in both studies. Since the IRIS classification is applied to all patients suffer- ing from CKD regardless of their cause, a variety of patients can be included. The progression of CKD and its response to treatment may be more variable at early stages than later when the majority of nephrons are lost. Although the se- rum SDMA concentration is correlated with the glomerular filtration rate (GFR) (38), further studies are warranted to assess the effect of the renal diet on the GFR. Serum selenium concentration and plasma GPX activity measured in the present study were generally consistent with previously reported values (13, 39). No cat was seleni- um deficient at the beginning nor at the end of the 3-month feeding trial. In addition, a significant positive correlation between serum selenium concentration and plasma GPX activity was found, which contrasts with previously pub- lished data in cats (39). The study mentioned above found a correlation between serum selenium concentration and plasma GPX activity only in the case of selenium deficien- cy. When selenium concentrations continued to increase, plasma GPX activity reached its plateau, which was not ob- served in our study. Unlike in human patients, selenium is not a limiting factor in feline CKD (16, 17, 39). In addition, the correlation between the above-mentioned parameters is inconsistent in human CKD patients (16). The median plasma MDA concentrations measured in our study were slightly higher, but in general agreement with the previously published values in healthy and CKD cats (40). We found no significant difference in mean change (final measurement – baseline) of plasma MDA concentration or Table 3: Results of five linear regression models, modelling difference (measurement 4 – measurement 1) between control and experimental group; the model controls for the age of cats Linear regression coefficient at group P value Adjusted P value Weight (kg) 0.401 0.054 0.272 Creatinine (µmol/L) -2.907 0.919 > 0.999 SDMA (µg/dL) 0.373 0.854 > 0.999 MDA (µmol/L) 0.891 0.175 0.699 GPX (U/mL) 0.875 0.654 > 0.999 Legend: Adjusted P-values are adjusted by Holm procedure for multiple comparisons; SDMA-Serum symmetric dimethylarginine; MDA-Plasma malondialdehyde; GPX-Plasma glutathione peroxidase Table 4: Median per cent fraction of the urinary protein at the beginning of the study in comparison to the end of the study (after 3 months) albumin Alpha 1 Alpha 2 Beta gamma Beginning Control group 4.0 5.8 0 2.4 0 Experimental group 3.1 4.1 0 14.7 0 After 3-month diet Control group 3.8 7.3 0 54.1 0 Experimental group 1.9 9.4 0 12.7 0 SloVetRes_NOTRANJOST ver2.indd 31 09/05/2023 10:14 32 | Slov Vet Res | Vol 60 No 1 plasma GPX activity between the experimental group and the control group. Our results suggest that the renal diet had no significant effect on the parameters of oxidative stress measured in our study. In contrast to previously published studies (39, 41), we found that three out of ten cats staged to IRIS 1 with elevated se- rum SDMA concentration, abnormal renal imaging findings as well as pathological urinary sediment, consistent with CKD, had a normal urinary specific gravity. The loss of the ability to concentrate urine is one of the first clinical signs of CKD and occurs when two-thirds of the nephrons are not functional. Apparently, the serum SDMA concentration increased before the ability of the kidneys to concentrate urine was impaired and proved valuable in the clinical evalu- ation of feline CKD patients at risk of developing CKD. Furthermore, it was observed that some cats (seven out of 19) with elevated serum SDMA and creatinine concen- trations, abnormal renal imaging findings and pathological urinary sediment, consistent with CKD, that were classified to IRIS stage 2, retained their urine concentration ability. The USG in these cats was up to 1.070 without showing clinical signs of heart failure, dehydration or hypovolemia. The literature data on this topic are scarce; Watson (42) reported that in contrast to dogs, USG values may remain normal (up to 1.045) in some cats with CKD and azotemia and that kidney disease may therefore still be suspected in a cat if these values are accompanied by persistent azo- temia. Furthermore, cats often retain some concentrating ability in IRIS stages 2 and 3 CKD (43). The authors assume that the high USG in the CKD cats included in the present study could partially be caused by eating dry food. Further research is warranted to address this topic. Cats of both groups had early kidney disease. Most of them exhibited borderline proteinuria, some were non-protein- uric. Except for one cat in the experimental group, the kid- ney disease was stable and did not deteriorate during the study. Two cats in the experimental group ended the study with a marked decrease of UPC. However, in one of these cats the serum creatinine and SDMA concentration rose to such extent that the cat was restaged from IRIS 2 to 3. The reason for this progression remained unknown. As serum SDMA and creatinine concentrations are negatively corre- lated to glomerular filtration, it might be assumed that sub- clinical dehydration, lower glomerular filtration rate as well as lower glomerular pressure may have led to a decreased UPC in that cat. Though not expected, we found a negative correlation between SDMA and USG in the sampled cats. A similar finding has already been reported in dogs with decreased glomerular filtration rate (44). Cats with CKD have and im- paired GFR; SDMA in such patients is increased (38). With a concurrent impairment of urine concentration ability, a de- crease in USG is observed. A negative correlation between SDMA and USG in the sampled cats that all had CKD might therefore reflect the pathogenesis of CKD or it might be a consequence of a stochastic chance. At the beginning of the study, four cats in the control group and three cats in the experimental group had microalbu- minuria. According to Giraldi and colleagues (45), micro- albuminuria is found in cats at risk for developing CKD. Overall, urinary protein electrophoresis and the UPC val- ues indicate that tubular processes rather than glomerular disease were present in the cats that were enrolled in the study. Furthermore, we observed more cats in the experi- mental group to have non-zero fractions of protein in the urinary sample which might be partially ascribed to a higher systolic blood pressure or to a different predominant renal pathology. At the end of the study, beta fraction predomi- nated in cats of both groups of our sample. However, when compared to the beginning of the study, there is an increase in the median percent beta fraction in cats in the control group, while it remained similar in the experimental group. Furthermore, the per cent albumin fraction decreased in cats in the experimental group while it remained similar in the control group. The presence of a leading beta frac- tion at the end of our study in both groups suggests tubu- lar kidney damage, although the cats were non-azotemic, non-proteinuric or borderline proteinuric. Thus, we may as- sume that the tubular inflammatory processes progressed in both groups of cats, but to a different extent, although the UPC values remained grossly unchanged (45). Furthermore, the results of our sample show no effect of renal diet on USG and UPC. The results are similar to the study published by Hall and colleagues (23), where no change in UPC or USG are reported. Therefore, we may conjecture that the tested renal diet had no effect either on electrophoretic pattern of urinary proteins or on halting the progression or development of proteinuria. Urine protein electrophoresis seems to be a valuable tool in assessing the progression of CKD. In order to provide better insight into the dynamics of CKD, we suggest urine protein elec- trophoresis to be added into monitoring scheme of feline CKD. Furthermore, recent recommendations in dogs with CKD include urinary electrophoresis, especially in those where renal biopsy is not indicated or not possible to be performed. The same recommendations may also be pro- posed in cats (46). The main limitation of our study was the small number of patients who completed the study. In addition, the study lasted for a relatively short period of time, which may be an additional reason for the lack of significant differences in the measured parameters between the groups. Further studies with greater number of animals and with the as- sessment of GFR and urinary protein typization including the LMW (low molecular weight) spectrum are needed to get a thorough insight of the effect of renal diet on renal pathology. SloVetRes_NOTRANJOST ver2.indd 32 09/05/2023 10:14 Slov Vet Res | Vol 60 No 1 | 33 Another limitation of the study is the fact that the diet of the cats in the control group was not standardized. As some cats do not tolerate any changes in their feeding regime including the diet change, the data gathered in this study give insight into the natural progression of early CKD where no medical intervention is possible. Moreover, some nu- tritional studies in human medicine follow similar design, where only experimental group receives diet, and the con- trol group consists of individuals who continue with their habitual diet (47). The study was performed on client-owned cats. Due to this fact, some cats were not brought to every scheduled check- up and some samples could therefore not be collected. The compliance of the owners in clinical studies like the present one tends to be a common problem. Conclusions After the 3-month feeding trial, no significant change in dif- ference of body weight, serum creatinine or serum SDMA concentrations between experimental and control group was observed. Renal diet did not significantly increase the level of lipid peroxidation and decrease the activity of GPX, indicative of increased oxidative stress. Furthermore, our study demonstrated a significant positive correlation be- tween serum selenium concentration and plasma GPX ac- tivity and a significant negative correlation between SDMA and USG in all CKD cats at inclusion. Acknowledgements The study was supported by Farmina Pet Foods. The funders were not involved in the study design, sample collection, analysis, data interpretation, or writing the manuscript. References 1. Conroy M, Brodbelt DC, O’Neill D, Chang YM, Elliott J. Chronic kid- ney disease in cats attending primary care practice in the UK: a VetCompass (TM) study. Vet Rec 2019; 184(17): e526. Available from: doi: 10.1136/vr.105100 2. O’Neill DG, Church DB, McGreevy PD, et al. Prevalence of disorders recorded in cats at-tending primary-care veterinary practices in Eng- land. Vet J 2014; 202: 286–91. 3. 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Holben DH, Smith AM. The diverse role of selenium with selenopro- teins: a review. J Am Diet Assoc 1999; 99: 836–43. 20. Polzin DJ. Evidence-based step-wise approach to managing chronic kidney disease in dogs and cats. J Vet Emerg Crit Care 2013; 23(2): 205–15. 21. Elliott J, Rawlings JM, Markwell PJ, et al. Survival of cats with natu- rally occuring chronic renal failure: effect of dietary management. J Small Anim Pract. 2000; 41: 235–42. 22. Ross SJ, Osborne CA, Kirk CA, Lowry SR, Koehler LA, Polzin DJ. Clinical evaluation of dietary modification for treatment of spontane- ous chronic kidney disease in cats. J Am Vet Med Assoc 2006; 229: 949–57. 23. Hall JA, Fritsch DA, Jewell DE, Burris PA, Gross KL. Cats with IRIS stage 1 and 2 chronic kidney disease maintain body weight and lean muscle mass when fed food having increased caloric density, and enhanced concentrations of carnitine and essential amino acids. 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J Vet Intern Med 2017; 31(2): 449–56. doi: 10.1111/jvim.14634 28. Yu S, Paetau-Robinson I. Dietary Supple-ments of vitamins E and C and β-carotene reduce oxidative stress in cats with renal insufficien- cy. Vet Res Commun 2006; 30: 403–13. 29. Granick M, Leuin AS, Trepanier LA. Plasma and urinary F2-isoprostane markers of oxidative stress are increased in cats with early (stage 1) chronic kidney disease. J Feline Med Surg 2021; 23(8): 692–9. 30. Valle E, Prola L, Vergnano D, et al. Investigation of hallmarks of cat- bonyl stress and for-mation of end products in feline chronic kidney disease as markers of uraemic toxins. J Feline Med Surg 2019; 21(6): 465–74. 31. Intenational Renal Interest Society. IRIS staging of CKD 2019 [inter- net] http://www.iris-kid-ney.com/pdf/IRIS_Staging_of_CKD_modi- fied_2019.pdf. 32. Ferreira JC, Patino CM. Randomization: beyond tossing a coin. J Bras Pneumol 2016; 42 (5): 310. doi: 10.1590/S1806-37562016000000296 33. Czauderna M, Kowalczyk J, Marounek M. The simple and sensitive measurement of malondialdehyde in selected specimens of biologi- cal origin and some feed by reversed phase high performance liquid chromatography. J Chromatogr B Analyt Technol Biomed Life Sci 2011; 879(23): 2251–8. 34. Paglia DE, Valentine WN. Studies on the quantitative and qualitative characterization of erythrocyte glutathione peroxidase. J Lab Clin Med 1967; 70: 158–69. 35. Prieto JM, Carney PC, Miller ML, et al. Biologic variation of symmet- ric dimethylarginine and creatinine in clinically healthy cats. Vet Clin Pathol 2020; 49(3): 401–6. 36. Kopke MA, Burchell RK, Ruaux CG, Burton SE, Lopez-Villalobos N, Gal A. Variability of symmetric dimethylarginine in apparently healthy dogs. J Vet Intern Med 2018; 32(2): 736−42. doi: 10.1111/jvim.15050 37. Mack RM, Hegarty E, McCrann DJ, Michael HT, Grauer GF. Longitudinal evaluation of symmetric dimethylarginine and con- cordance of kidney biomarkers in cats and dogs. Vet J 2021; 276: e105732. doi: 10.1016/j.tvjl.2021.105732. 38. Braff J, Obare E, Yerramilli M, Elliott J, Yerramilli M. Relationship be- tween serum symmetric dimethylarginine concentration and glo- merular filtration rate in cats. J Vet Intern Med 2014; 28: 1699–701. 39. Foster DJ, Thoday KL, Arthur JR, et al. Selenium status of cats in four regions of the world and comparison with reported incidence of hyperthyroidism in cats in those regions. Am J Vet Res 2001; 62(6): 934–7. 40. Todorova I, Simeonova G, Kyuchukova D, et al. Reference values of oxidative stress parameters (MDA, SOD, CAT) in dogs and cats. Comp Clin Pathol 2005; 13(4): 190–4. 41. Hall JA, Yerramilli M, Obare E, Yerramilli M, Yu S, Jewell DE. Comparison of serum concentrations of symmetric dimethylarginine and creatinine as kidney function biomarkers in healthy geriatric cats fed reduced protein food s enriched with fish oil, L-carnitine, and me- dium-chain triglycerides. Vet J 2014; 202: 588–96. 42. Watson ADJ, Lefebvre HP, Elliott J. Using urine specific gravity, 2015 [internet] http://www.iris-kid-ney.com/education/urine_specific_ gravity.html 43. Bradley R, Tagkopoulos I, Kim M, et al. Predicting early risk of chronic kidney disease in cats using routine clinical laboratory tests and ma- chine learning. J Vet Intern Med 2019; 33(6): 2644–56. 44. Pelander L, Häggström J, Larsson A, et al. Comparison of the diag- nostic value of symmetric dimethylarginine, cystatin C, and creati- nine for detection of decreased glomerular filtration rate in dogs. J Vet Intern Med 2019; 33(2): 630−9. doi: 10.1111/jvim.15445 45. Giraldi M, Paltrinieri S, Scarpa P. Electro-phoretic patterns of protein- uria in feline spontaneous chronic kidney disease. J Feline Med Surg 2019; 22(2): 114–21. doi: 10.1177/1098612X1 46. Dornelas L, Orozco A, Ramírez López C, et al. Application of serum and urinary electrophoresis as an aid tool for evaluating dogs at dif- ferent stages of chronic kidney disease. Comp Clin Pathol 2021; 30: e1–11. doi: 10.1007/s00580-021-03231-2 47. Markhus MW, Kvestad I, Midtbø LK, et al. Effects of cod intake in pregnancy on iodine nutrition and infant development: study protocol for Mommy’s Food - a randomized controlled trial. BMC Nutr 2018; 4: e7. doi: 10.1186/s40795-018-0215-1 SloVetRes_NOTRANJOST ver2.indd 34 09/05/2023 10:14 Slov Vet Res | Vol 60 No 1 | 35 Učinek monoterapevtske ledvične diete na klinične, biokemijske, urinske in serumske parametre oksidativnega stresa pri mačkah s KLB stopnje 1 in 2 M. Krofič Žel, A. Nemec Svete, B. Jakovac Strajn, K. Pavšič Vrtač, T. Vovk, N. Kejžar, D. Pavlin Izvleček: Namen študije je bil raziskati učinek terapevtske ledvične diete na izbrane klinične, biokemijske in urinske parametre ter parametre oksidativnega stresa pri mačkah v začetnih stopnjah kronične ledvične bolezni (KLB). Raziskava je bila zasnovana kot prospektivna, tri mesece trajajoča klinična študija, v katero je bilo vključenih 29 lastniških mačk. Devetnajst mačk je zaključilo študijo, od teh jih je deset prejemalo ledvično dieto, devet pa vzdrževalno dieto po izboru lastnika. Pri vseh mačkah smo izvedli klinični pregled in odvzem krvnih ter urinskih vzorcev na dan vključitve v študijo in pri treh kontrolnih pregledih, ki so bili izvedeni 3–4, 7–8 in 10–12 tednov kasneje. Določili smo serumsko koncentracijo kreatinina, simetričnega dimetilarginina (SDMA) in izbrane parametre oksidativnega stresa (aktivnost plazemske gluta- tion peroksidaze (GPX) in plazemsko koncentracijo malondialdehida (MDA) ter serumsko koncentracijo selena). Poleg tega smo izvedli elektroforezo urinskih proteinov. Ob vključitvi mačk v raziskavo smo ugotovili značilno pozitivno kore- lacijo (p < 0,001) med serumsko koncentracijo selena in aktivnostjo plazemske GPX (Pearsonov korelacijski koeficient 0,83, 95 % CI: [0,65–0,92]) ter značilno negativno korealcijo (p < 0,001) med koncentracijo SDMA in specifično težo urina (Pearsonov korelacijski koeficient –0,70 (95 % CI: [–0,87–(–0,38)]). Trimesečno hranjenje s terapevtsko hrano ni privedlo do značilnih sprememb v serumski koncentraciji SDMA in kreatinina pri vključenih mačkah. Ključne besede: klinični parametri; simetrični dimetilarginin; oksidativni stres; ledvična dieta; mačke; kronična ledvična bolezen; elektroforeza urinskih proteinov SloVetRes_NOTRANJOST ver2.indd 35 09/05/2023 10:14 SloVetRes_NOTRANJOST ver2.indd 36 09/05/2023 10:14 Slov Vet Res | Vol 60 No 1 | 37 Computed Tomography and Magnetic Resonance Imaging of a Rhinosinusitis Secondary to a Dental Abscess in a Crested Porcupine (Hystrix cristata) Key words porcupine, dental abscess, computed tomography, magnetic resonance imaging Mario Encinoso1, Daniel Morales1, Soraya Déniz1, Jose V Guerra2, Jose Raduan Jaber3* 1Hospital Universitario, Facultad de Veterinaria, Universidad de Las Palmas de Gran Canaria, Trasmontaña, Arucas, 35413 Las Palmas, 2Rancho Texas Lanzarote Park, Puerto del Carmen, Tías 35510, Lanzarote, 3Departamento de Morfologia. Facultad de Veterinaria, Universidad de Las Palmas de Gran Canaria. Trasmontaña, Arucas, 35413 Las Palmas, Spain *Corresponding author: joseraduan.jaber@ulpgc.es Abstract: A captive crested porcupine (Hystrix cristata) adult male was imaged due to reduced food intake, anorexia, fever, nasal discharge, changes in fecal quantity and size, and respiratory difficulties. Advanced imaging diagnostic techniques such as comput- ed tomography and magnetic resonance imaging were performed to evaluate the ani- mal. These techniques were very helpful to delineate the dental abscess, as well as the extension of the process to other locations such as the nasal cavity and the tympanic bulla. This is the first description of rhinosinusitis secondary to a dental abscess in a crested porcupine. Received: 24 March 2022 Accepted: 21 February 2023 DOI 10.26873/SVR-1499-2023 UDC 599.324.32:616.314-002:616-073 Pages: 37–43 Case Report Introduction In recent years, the introduction of modern diagnostic imag- ing techniques has improved the visualization of diseases in exotic mammal medicine. Traditionally, standard radiog- raphy has been used by clinicians (1). Nonetheless, results of previous works have proposed that computed tomog- raphy (CT) and magnetic resonance imaging (MRI) may provide more information to improve diagnostic accuracy, prognosis, and treatment of diseases (2, 3, 4). These tech- niques avoid the superimposition of adjacent anatomical structures and depict the anatomic detail of specific tissue densities more finely, which improves its interpretation (2). Moreover, the refinements in CT technology involve the ap- plication of computer software for the generation of three- dimensional (3D) reconstruction of an area of anatomic interest (5). These advantages have demonstrated great value for the diagnosis of several diseases in exotic mam- mal species (2 ,3, 4, 5). Some of these species, such as the crested porcupine (Hystrix cristata), appear in worrisome categories of the IUCN red list since are regionally or lo- cally threatened and thus require appropriate conservation policies in regional and local contexts. It is a species of ro- dent in the family Hystricidae native to Italy and Sicily, and a broad central strip ranging from Senegal and west to Somalia and east to Kenya and Tanzania (6, 7). Porcupine presents strong and pointed quills that cover their tail, sides and top of the body. Concerning its head, it is large and robust with an enlarged infraorbital foramen so that portions of the masseter extend through it and arise from the frontal side surface of the snout (8). The anato- my and physiology of their oral cavity can produce several dental disorders. The teeth of rodents grow continuously (incisors in all species and molars in some species), there- fore, any disease affecting the positioning of teeth within this cavity and disrupting normal attritional movements will lead to overgrowth and malocclusion (2, 3). Nonetheless, their oral cavity is very difficult to examine because of its anatomic configuration; these features typically allow the evaluation of superficial changes (9). For these motives, new imaging modalities are of tremendous importance for SloVetRes_NOTRANJOST ver2.indd 37 09/05/2023 10:14 38 | Slov Vet Res | Vol 60 No 1 the assessment of teeth and surrounding structures (10). Therefore, the use of CT and MRI allows an accurate diag- nosis of dental disorders since it is highly effective in visual- izing soft tissues and, unlike conventional radiology, can be used to diagnose even small dental abscesses, as well as osteoarthritis of the temporomandibular joint and odonto- genic tumours (2, 11). Case Presentation An adult male crested porcupine (Hystrix cristata) weigh- ing 7,8 kg from Rancho Texas Lanzarote Park (Lanzarote, Canary Islands, Spain) was admitted to the Veterinary Hospital of Las Palmas de Gran Canaria University (Canary Islands, Spain) to be imaged due to medical history of weight loss, reduced activity and food intake, anorexia, fe- ver, mucopurulent nasal discharge, reduced stool produc- tion, and respiratory difficulties. The blood collection re- vealed mild anemia, marked lymphopenia, hypoglycaemia, hypoalbuminaemia, slightly high serum urea concentra- tions, and low potassium levels. To perform the imaging study and evaluate the animal, we sedated the porcupine using a combination of dex- medetomidine (0,25 mg/kg IM, DEXDOMITOR®, Ecuphar, Barcelona, Spain) and ketamine (25 mg/kg, Imalgene®, Boehringer Ingelheim, Barcelona, Spain). No physical ab- normalities were observed when we checked the head, However, the inspection of its oral cavity revealed an ab- scess of the right maxillary molar tooth and halitosis. The images were obtained using a 16-slice helical CT scanner (Toshiba Astelion, Toshiba Medical System, Madrid, Spain). The animal was positioned symmetrically in sternal recum- bency on the CT couch, and a standard clinical protocol (120 kVp, 80 mA, 512 X 512 acquisition matrix, 1809 x 858 field of view, a spiral pitch factor of 0,94, and a gantry rota- tion of 1,5 s) was used to acquire sequential transverse CT images of 1 mm thickness slice. To optimize the CT ap- pearance of the head structures, two CT algorithms (bone/ Figure 1: A) Dorsal MPR image of the porcupine head, bone window. The image displays an increase of the alveolar space corresponding to the third right maxillary molar tooth (white arrows). B) Volume-rendered reconstruction image of the porcupine head, displaying the dentary abscess (black arrows) SloVetRes_NOTRANJOST ver2.indd 38 09/05/2023 10:14 Slov Vet Res | Vol 60 No 1 | 39 pulmonary algorithm), and two windows were applied by adjusting the window widths (WW) and window levels (WL): a bone window setting (WW=1500; WL=300) and a soft-tissue window setting (WW=350; WL=40). Multiplanar reconstruction (MPR) of CT images was performed to im- prove the visualization of the normal anatomy of the af- fected area as well as greater diagnostic accuracy of the extension of the disease. In addition, the original data were used to generate volume-rendered reconstructed images after manual editing of the transverse CT images to remove soft tissues using a standard Dicom 3D format (OsiriX MD, Geneva, Switzerland). The CT and volume rendering reconstruction images dis- played an increase in the alveolar space corresponding to the third right maxillary molar tooth (Figure 1). This alveolar space was hypoattenuating concerning the subjacent soft tissue. The rest of the oral cavity appeared unremarkable without any sign of disease. The nasal cavity and paranasal sinuses showed a large amount of fluid collection affecting both sides (more marked on the right side) that were hyper- attenuated in the transverse and MPR images (Figure 2, 4a). No other remarkable findings were observed. The MRI study was conducted with a 1.5-Tesla magnet (Toshiba, Vantage Elan, Japan) with the animal placed in ventral recumbence. A standard MRI protocol was used to generate spin-echo (SE) T1-weighted, and T2-weighted images in sagittal, transverse, and dorsal planes. SE T1- weighted transverse images were acquired with the follow- ing settings: Echo time (TE), 10 ms, repetition time (TR), 800 ms, acquisition matrix of 536 x 384, and 4,5 mm slice thickness with 4 mm spacing between slices. For SE T2- weighted transverse images, the TE 120 ms, TR 10541 ms, acquisition matrix 624 x 448, and 3 mm slice thickness with 3 mm interslice spacing. For SE T2-weighted sagit- tal images, the TE 120 ms, TR 7529 ms, acquisition matrix 512 x 804, and 2,8 mm slice thickness with 2 mm interslice spacing. For SE T2-weighted dorsal images, the TE 120 ms, TR 8282 ms, acquisition matrix 468 x 512, and 3,4 mm slice thickness with 3 mm interslice spacing. We used a medical imaging viewer (OsiriX MD, Geneva, Switzerland) to evalu- ate the images of the study. Dorsal, transverse, and sagittal MR images of the porcu- pine head are presented (Figures 3, 4B). These images showed an increase of the alveolar space corresponding Figure 2: A) Transverse CT image of the porcupine head, bone window. The image shows an increase of the alveolar space corresponding to the third right maxillary molar tooth (black arrow). This increased space produced deviation to the right midline of the right wall of the nasopharynx (white arrow). In addition, there was a fluid collection in the paranasal sinuses (white arrow). B) Transverse CT image of the porcupine head, bone window. The image shows fluid collection in the dorsal and ventral nasal conchae (white arrows) SloVetRes_NOTRANJOST ver2.indd 39 09/05/2023 10:14 40 | Slov Vet Res | Vol 60 No 1 to the third right maxillary molar tooth. The increase of the alveolar space was hyperattenuated in T2W images when compared with the subjacent soft tissue. The dorsal and sagittal T2W images depicted abundant fluid collection in the nasal cavity and paranasal sinuses (Figures ·3A, 4B). In addition, the tympanic bullas showed a slight amount of fluid that was hyperintense in the T2W transverse images (Figure 3B). No other Imaging findings were identified. We submitted samples of the fluid collection for aerobic and anaerobic bacterial culture. These were performed us- ing sterile cotton swabs in a transport medium (Eurotubo®, Rubi, Barcelona, Spain). The swab was introduced 2-4 cm into the medial aspect of each nare and was stored and kept at 4 0C until further processing in the laboratory. Later, samples were inoculated on Blood agar, MacConkey agar, Baird parker agar, and sabouraud agar. Plates were incu- bated at 37 0C for 24 hours. There was bacterial growth on blood agar and Baird parker agar. We also performed a Gram stain, resulting in gram-positive cocci in pure cul- ture. Subsequently, the API 20 Staph gallery confirmed the presence of Staphylococcus aureus. The antimicrobial re- sistance was tested to select effective drugs for treatment with those antimicrobials used in people against staphy- lococcal infection (12). The antimicrobial sensitivity discs (Oxoid, England) were cephalexin, enrofloxacin, ciprofloxa- cin, and amoxicillin-clavulanic acid. With this result, we recommend enrofloxacin (15 mg/kg s.c. q24h) for 14 days. Unfortunately, medical treatment was ineffective so surgi- cal therapy was performed to extract the affected tooth and instilment the surgical site with antibiotic preparations (doxycycline-containing polymer gel). Later, the porcupine was maintained with amoxicillin–clavulanate 7,5 mg/kg q48h s.c. for six weeks. Further evaluation of the animal revealed no further complications. Discussion To the authors’ knowledge, the present study is the first to characterize CT and MRI findings of a dental abscess in a crested porcupine. Different reports have postulated that rodents can develop dental diseases in their lifetime and be Figure 3: MRI of the porcupine head. A) T2W, dorsal image, displaying the fluid collection in the nasal cavity and paranasal sinuses (black arrows). B) T2W, transverse image. The image shows the tympanic bullas with a slight amount of fluid that is hyperintense (white arrows) SloVetRes_NOTRANJOST ver2.indd 40 09/05/2023 10:14 Slov Vet Res | Vol 60 No 1 | 41 a cause of morbidity (13, 14) since these diseases may lead to infection of teeth and surrounding structures. Among these diseases, we highlighted dental abscesses, which are common in lagomorphs and rodents (10, 11, 15, 16). However, the singular aspects of its dental anatomy, physi- ologic characteristics, and host response make abscess di- agnosis and treatment quite difficult (17). The aetiology of dental abscesses implies food and microorganisms being able to track up a loosened or broken tooth into the peri- odontal tissues and alveolar socket, resulting in the genera- tion of an abscess associated with the maxilla or mandible, which can extend to the nasal cavity (17, 18). In this study, CT and MR images supported the diagnosis of rhinosinus- itis secondary to dental associated infection. Information concerning rhinosinusitis in captive and free-ranging wild- life species is sparse. Thus, only a few reports have de- scribed this finding in rodents such as ground squirrels (19) or an orange-spined hairy dwarf porcupine (20), and as in our case, the nasal cavity inflammation was associated to Staphylococcus spp infection. Diseases of incisors and cheek teeth result in clinical signs that could require appropriate imaging techniques to obtain a definitive diagnosis, formulate a prognosis, and develop a treatment plan. The use of radiographic imaging can be contemplated as a primary diagnostic tool to evaluate these processes. Unfortunately, the small size of rodents and the overlapping of the dental quadrants make radio- graphic evaluation quite arduous (18). Advanced diagnos- tic imaging such as CT and MRI have become popular in exotic mammal medicine since these techniques improve anatomic identification and lesion detection that allows ac- curate assessment, detailed prognosis, the diagnosis of un- derlying lesions and treatment choice (10, 18). CT has been widely used in rabbits to evaluate acquired dental disease and its associated problems, such as deformities and os- teomyelitis, as well as the extension of the infection pro- cess to different bone cavities of the skull as the nasal or the paranasal cavities and the tympanic bullae (10, 11, 12). Therefore, the clinical signs are commonly related to the primary dental problem or complications connected with dental disease. The clinical signs observed in our animal such as anorexia, reduced food intake, excessive salivation, or nasal discharge were similar to those described in previ- ous reports (10, 11, 12). Additionally, mild anemia, marked lymphopenia, and hypoglycaemia have also been reported in other animals with dental diseases (3, 14, 15). In this study, a third-generation CT scan provided trans- verse and three-dimensional reconstructed images that gave an adequate overview of head morphology, dis- playing a good depiction of the affected areas. Thus, the transverse CT images were helpful to delineate the dental abscess, as well as the extension of the process to other locations, such as the nasal cavity and the tympanic bulla. Interestingly, other studies performed on rabbits showed similar Imaging findings (2, 3, 15, 16). Three-dimensional CT reconstruction is a helpful procedure to evaluate the extension of bony lesions with excellent detail by cropping part of the volume to evaluate deeper anatomic structures (5, 21). Hard and soft tissues can be added virtually or sub- tracted to different extents and degrees of density, provid- ing a comprehensive relationship between soft and hard tissues. Shaded surface displays present a contoured sur- face map of the entire image volume, converting CT data into an image very similar to the depiction of an anatomic specimen (10). Despite these arguments, this technique has been infrequently used in exotic veterinary medicine. Three-dimensional CT reconstruction may be of critical importance for diagnostic accuracy and selecting the best Figure 4: A) Sagittal MPR image of the porcupine head displaying the tooth abscess and the fluid collection in the paranasal sinuses (white arrows). B) T2W, sagittal image, displaying the abundant fluid collection in the nasal cavity and paranasal sinuses (black arrows) SloVetRes_NOTRANJOST ver2.indd 41 09/05/2023 10:14 42 | Slov Vet Res | Vol 60 No 1 surgical approach, depending on the nature and extension of the process. Thus, the use of this technique in rabbits has been quite helpful evaluating osteomyelitis, or dental and skull abnormalities (10). In our study, the images ob- tained by three-dimensional CT reconstruction displayed excellent detail of the dental abscess and the extension of the process to the nasal cavity, providing additional infor- mation to the transverse CT images. In recent years, the contribution of MRI to the knowledge of exotic animals has increased (10, 11, 16). This Imaging technique displays soft tissues with excellent resolution. Therefore, an MRI of the head for reasons other than stud- ies of the brain is a very helpful tool in pet rabbits and ro- dents to diagnose the presence and the extent of abscess- es (10, 11, 16). In our study, we used a magnet of 1.5 T that provided T2W images with high resolution. These images displayed abundant fluid collection affecting the nasal cav- ity and the tympanic bulla, thus, diagnosing the presence and extension of the abscess. Anaerobic and aerobic bacteria have been cultured from dental abscesses when pertinent techniques are used (17). Specific bacteria such as Staphylococcus Aureus, previ- ously described to be important etiologic in rabbit dental infections (22), was isolated in our study. It is a versatile opportunistic pathogen that causes a wide spectrum of pathologies. It is also a mammalian commensal and op- portunistic pathogen that colonizes niches such as skin, nares, and diverse mucosal membranes. The prevalence in animals varies from host species but colonization and infection have only been superficially investigated in small rodent wild animals (23) such as beavers, ground squirrels, red squirrels, or wood mice (24). To summarize, advanced imaging diagnostic techniques such as computed tomography and magnetic resonance imaging were helpfully delineating the dental abscess and the extension of this process to other locations such as the nasal cavity and the tympanic bulla. 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Jaber Izvleček: Odraslega samca afriškega ježevca (Hystrix cristata) v ujetništvu smo slikali zaradi zmanjšanega vnosa hrane, anoreksije, nosnega izcedka, sprememb v količini in velikosti iztrebkov ter težav z dihanjem. Uporabili smo napredne tehnike slikovne diagnostike, kot sta računalniška tomografija in magnetna resonanca. Te tehnike so bile zelo koristne pri opredelitvi zobnega abscesa in razširitve procesa na druga mesta, kot sta nosna votlina in timpanični del temporalne kosti. To je prvi opis rinosinuzitisa, ki je posledica zobnega abscesa pri afriškem ježevcu. Ključne besede: ježevec; zobni absces; računalniška tomografija; magnetna resonanca SloVetRes_NOTRANJOST ver2.indd 43 09/05/2023 10:14 SloVetRes_NOTRANJOST ver2.indd 44 09/05/2023 10:14 Slovenian Veterinary Research Slovenski veterinarski zbornik TH E SC IEN TIFIC JO U RN A L O F TH E V ETERIN A RY FAC U LTY U N IV ERSITY O F LJU BLJA N A Volume 60, Number 1, Pages 1-44 ISSN 1580-4003 Table of Content 5 Editorial Veterinary Illustration: Science and Art Telling a Story TogetherKubale V 9 Review Article Suitability of Alternatives to Rectal Temperature Measurements in Pet Rodents, Rabbits and Ferrets: A Literature Review Stans J 15 Original Research ArticleCharacterisation of the Haematological Profile in the Posavje Horse BreedMesarič M, Nemec M, Čebulj Kadunc N 25 Original Research Article The Effect of a Specific Chicken Based Renal Diet as Monotherapy on Clinical, Biochemical, Urinary and Serum Oxidative Stress Parameters in Cats with CKD Stage 1 and 2 Krofič Žel M, Nemec Svete A, Jakovac Strajn B, Pavšič Vrtač K, Vovk T, Kejžar N, Pavlin D 37 Case Report Computed Tomography and Magnetic Resonance Imaging of a Rhinosinusitis Secondary to a Dental Abscess in a Crested Porcupine (Hystrix cristata) Encinoso M, Morales D, Déniz S, Guerra JV, Jaber JR SloVetRes_naslovnica.indd 1 16/05/2023 09:46