ISSN: YAZIŞMA ADRESİ (ADDRESS FOR CORRESPONDENCE)

Transkript

1

2

3 This journal is indexed and abstracted by Thomson Reuters Services beginning with Volume 13 (1) 2007 in the followings: Science Citation Index Expanded (also known as SciSearch ) Journal Citation Reports/Science Edition This journal is also indexed and abstracted in: CAB Abstracts ULAKBIM-TUBITAK YAZIŞMA ADRESİ (ADDRESS FOR CORRESPONDENCE) Kafkas Üniversitesi Veteriner Fakültesi Dergisi Editörlüğü Kars / TÜRKİYE Phone: /1244 Fax: vetdergi@kafkas.edu.tr ELEKTRONİK BASKI (ELECTRONIC EDITION) ISSN: ONLINE MAKALE GÖNDERME (ONLINE SUBMISSION)

4 Bu dergi Kafkas Üniversitesi Veteriner Fakültesi tarafından iki ayda bir yayımlanır. This journal is published bi-monthly, by the Faculty of Veterinary Medicine, University of Kafkas. Kafkas Üniversitesi Veteriner Fakültesi Adına Sahibi (OWNER) Prof.Dr. Şahin ASLAN Dekan (Dean) EDİTÖR (EDITOR-IN-CHIEF) Prof.Dr. İsa ÖZAYDIN YABANCI DİL EDİTÖRLERİ (ENGLISH EDITORS) Prof.Dr. H. Metin ERDOĞAN Doç.Dr. Ahmet ÜNVER EDİTÖR YARDIMCILARI (ASSOCIATE EDITORS) Doç.Dr. Mehmet ÇİTİL Doç.Dr. Özgür AKSOY Yrd.Doç.Dr. Erdoğan UZLU Yrd.Doç.Dr. Oktay ÖZKAN YÜRÜTME KURULU (EXECUTIVE BOARD) Prof.Dr. Nurhayat GÜLMEZ Prof.Dr. Abdullah DOĞAN Prof.Dr. Kemal IRMAK Prof.Dr. Sami ÖZCAN Prof.Dr. Yavuz ÖZTÜRKLER Prof.Dr. İsmail KAYA Doç.Dr. Hakan KOCAMIŞ SEKRETERYA (SECRETARY) Fahri ALTUN DİZGİ (COMPOSITION) Dr. Pınar DEMİR Dr. Asuman KIZILTEPE BASKI (PRINT) ESER OFSET MATBAACILIK Tel: ERZURUM Prof.Dr. Kemal AK Prof.Dr. Belma ALABAY Prof.Dr. Mustafa ALİŞARLI Prof.Dr. Feray ALKAN Prof.Dr. Çiğdem ALTINSAAT Prof.Dr. Metin BAYRAKTAR Prof.Dr. Burhan ÇETİNKAYA Prof.Dr. Hüdaverdi ERER Prof.Dr. Gül ERGÜN Prof.Dr. Ayhan FİLAZİ Prof.Dr. O. Sacit GÖRGÜL Prof.Dr. Ekrem GÜREL Prof.Dr. Tolga GÜVENÇ Prof.Dr. Ali İŞMEN Prof.Dr. Hakkı İZGÜR Prof.Dr. Zafer KARAER Prof.Dr. Arif KURTDEDE Prof.Dr. Erdoğan KÜÇÜKÖNER Prof.Dr. Kamil ÖCAL Prof.Dr. Metin PETEK Prof.Dr. Engin SAKARYA Prof.Dr. Berrin SALMANOĞLU Prof.Dr. Nesrin SULU Prof.Dr. Ş. Doğan TUNCER Prof.Dr. Halis YERLİKAYA DANIŞMA KURULU (ADVISORY BOARD) İstanbul Üniversitesi Veteriner Fakültesi Ankara Üniversitesi Veteriner Fakültesi Ondokuz Mayıs Üniversitesi Veteriner Fakültesi Ankara Üniversitesi Veteriner Fakültesi Ankara Üniversitesi Veteriner Fakültesi Fırat Üniversitesi Veteriner Fakültesi Fırat Üniversitesi Veteriner Fakültesi Selçuk Üniversitesi Veteriner Fakültesi Hacettepe Üniversitesi Fen Fakültesi Ankara Üniversitesi Veteriner Fakültesi Uludağ Üniversitesi Veteriner Fakültesi Abant İzzet Baysal Üniversitesi Fen Edebiyat Fakültesi Ondokuz Mayıs Üniversitesi Veteriner Fakültesi Çanakkale Onsekiz Mart Üniversitesi Su Ürünleri Fakültesi Ankara Üniversitesi Veteriner Fakültesi Ankara Üniversitesi Veteriner Fakültesi Ankara Üniversitesi Veteriner Fakültesi Süleyman Demirel Üniversitesi Mühendislik Mimarlık Fakültesi Adnan Menderes Üniversitesi Veteriner Fakültesi Uludağ Üniversitesi Veteriner Fakültesi Ankara Üniversitesi Veteriner Fakültesi Ankara Üniversitesi Veteriner Fakültesi Ankara Üniversitesi Veteriner Fakültesi Ankara Üniversitesi Veteriner Fakültesi Fırat Üniversitesi Veteriner Fakültesi

5 Bu Sayının Hakem Listesi (alfabetik sıra) The Referees List of This Issue (in alphabetical order) AKSOY Melih AKSOY Özgür AKYÜZ Bilal ALAÇAM Erol ALDEMİR Adnan ALTUNATMAZ Kemal ALTUNOK Vahdettin ARSLAN Cavit ARSLAN Mükremin Özkan ATABAY H. İbrahim BAYTOK Erol BOLAT Duran BUMİN Ali CİHAN Mete COŞKUN Behiç ÇAKMAK Ayşe ÇELEBİ Selahattin ÇİÇEK Hatice ÇOLAK Armağan DENİZ Suphi DİK Bilal DİLER İbrahim EREN Hasan EYDURAN Ecevit GICIK Yunus GÜNEŞ Vehbi GÜNGÖR Örsan HOŞTÜRK Gülhan Turkay IRMAK Kemal KAÇAR Cihan KAHRAMAN Recep KAR Sırrı KARAKAŞ Asuman KURAR Ercan KÜÇÜKERSAN Kemal NALBANTOĞLU Serpil NUR Zekeriya ÖZEN Abdullah ÖZEN Rahşan ÖZTÜRKLER Yavuz PANCARCI Şükrü Metin SAATCI Mustafa SAÇAKLI Pınar SAĞIRKAYA Hakan SARIERLER Murat SEL Tevhide SUR Emrah ŞEN İsmail UYSAL Ongun VURAL Rıfat YARDIMCI Hakan YILDIZ Gültekin YILDIZ Hüseyin YÜKSEK Nazmi Adnan Menderes Üniversitesi Veteriner Fakültesi Dölerme ve Suni Tohumlama Anabilim Dalı Kafkas Üniversitesi Veteriner Fakültesi Cerrahi Anabilim Dalı Erciyes Üniversitesi Veteriner Fakültesi Zootekni Anabilim Dalı Ankara Üniversitesi Veteriner Fakültesi Doğum ve Jinekoloj Anabilim Dalı Kafkas Üniversitesi Fen Edebiyat Fakültesi Biyoloji Bölümü İstanbul Üniversitesi Veteriner Fakültesi Cerrahi Anabilim Dalı Selçuk Üniversitesi Veteriner Fakültesi Biyokimya Anabilim Dalı Kafkas Üniversitesi Veteriner Fakültesi Hayvan Besleme ve Beslenme Hastalıkları Anabilim Dalı Kafkas Üniversitesi Veteriner Fakültesi Parazitoloji Anabilim Dalı İzmir Yüksek Teknoloji Enstitüsü Erciyes Üniversitesi Veteriner Fakültesi Hayvan Besleme ve Beslenme Hastalıkları Anabilim Dalı Yüzüncü Yıl Üniversitesi Veteriner Fakültesi Hayvan Besleme ve Beslenme Hastalıkları Anabilim Dalı Ankara Üniversitesi Veteriner Fakültesi Cerrahi Anabilim Dalı Kafkas Üniversitesi Veteriner Fakültesi Cerrahi Anabilim Dalı Selçuk Üniversitesi Veteriner Fakültesi Hayvan Besleme ve Beslenme Hastalıkları Anabilim Dalı Ankara Üniversitesi Veteriner Fakültesi Parazitoloji Anabilim Dalı Atatürk Üniversitesi Tıp Fakültesi Mikrobiyoloji Anabilim Dalı Afyon Kocatepe Üniversitesi Veteriner Fakültesi Parazitoloji Anabilim Dalı Atatürk Üniversitesi Veteriner Fakültesi Doğum ve Jinekoloji Anabilim Dalı Yüzüncü Yıl Üniversitesi Veteriner Fakültesi Hayvan Besleme ve Beslenme Hastalıkları Anabilim Dalı Selçuk Üniversitesi Veteriner Fakültesi Parazitoloji Anabilim Dalı Süleyman Demirel Üniversitesi Eğirdir Su Ürünleri Fakültesi Su Ürünleri Yetiştiriciliği Adnan Menderes Üniversitesi Veteriner Fakültesi Parazitoloji Anabilim Dalı Iğdır Üniversitesi Ziraat Fakültesi Zootekni Bölümü Kafkas Üniversitesi Veteriner Fakültesi Parazitoloji Anabilim Dalı Erciyes Üniversitesi Veteriner Fakültesi İç Hastalıkları Anabilim Dalı Mehmet Akif Ersoy Üniversitesi Veteriner Fakültesi Doğum ve Jinekoloji Anabilim Dalı İstanbul Üniversitesi Veteriner Fakültesi Biyokimya Anabilim Dalı Kafkas Üniversitesi Veteriner Fakültesi İç Hastalıkları Anabilim Dalı Kafkas Üniversitesi Veteriner Fakültesi Doğum ve Jinekoloji Anabilim Dalı İstanbul Üniversitesi Veteriner Fakültesi Hayvan Besleme ve Beslenme Hastalıkları Anabilim Dalı Namık Kemal Üniversitesi Fen Edebiyat Fakültesi Biyoloji Bölümü Uludağ Üniversitesi Yenişehir MYO Gıda Teknolojisi Programı Selçuk Üniversitesi Veteriner Fakültesi Genetik Anabilim Dalı Ankara Üniversitesi Veteriner Fakültesi Hayvan Besleme ve Beslenme Hastalıkları Anabilim Dalı Ankara Üniversitesi Veteriner Fakültesi Parazitoloji Anabilim Dalı Uludağ Üniversitesi Veteriner Fakültesi Dölerme ve Suni Tohumlama Anabilim Dalı Fırat Üniversitesi Veteriner Fakültesi Veteriner Hekimliği Tarihi ve Deontoloji Fırat Üniversitesi Veteriner Fakültesi Veteriner Hekimliği Tarihi ve Deontoloji Kafkas Üniversitesi Veteriner Fakültesi Dölerme ve Suni Tohumlama Anabilim Dalı Kafkas Üniversitesi Veteriner Fakültesi Doğum ve Jinekoloji Anabilim Dalı Mehmet Akif Ersoy Üniversitesi Veteriner Fakültesi Zootekni Anabilim Dalı Ankara Üniversitesi Veteriner Fakültesi Hayvan Besleme ve Beslenme Hastalıkları Anabilim Dalı Uludağ Üniversitesi Veteriner Fakültesi Dölerme ve Suni Tohumlama Anabilim Dalı Adnan Menderes Üniversitesi Veteriner Fakültesi Cerrahi Anabilim Dalı Ankara Üniversitesi Veteriner Fakültesi Biyokimya Anabilim Dalı Selçuk Üniversitesi Veteriner Fakültesi Histoloji ve Embriyoloji Anabilim Dalı Selçuk Üniversitesi Veteriner Fakültesi İç Hastalıkları Anabilim Dalı Ankara Üniversitesi Veteriner Fakültesi Dölerme ve Suni Tohumlama Anabilim Dalı Ankara Üniversitesi Veteriner Fakültesi Doğum ve Jinekoloji Anabilim Dalı Ankara Üniversitesi Veteriner Fakültesi Mikrobiyoloji Anabilim Dalı Ankara Üniversitesi Veteriner Fakültesi Hayvan Besleme ve Beslenme Hastalıkları Anabilim Dalı Uludağ Üniversitesi Veteriner Fakültesi Anatomi Anabilim Dalı Yüzüncü Yıl Üniversitesi Veteriner Fakültesi İç Hastalıkları Anabilim Dalı

6 İÇİNDEKİLER (CONTENTS) ARAŞTIRMA MAKALELERİ (RESEARCH ARTICLES) Comparison of Two Different Media for in vitro Production of Dog Embryos EVECEN M, CIRIT U, DEMIR K, KARAMAN E, BAKIRER G, HAMZAOGLU AI, BIRLER S, PABUCCUOGLU... Use of GnRH Agonist (Desloreline) in Combination with PGF2α on the Termination of Pregnancy in Bitches GUNGOR O, KAYA M, GURBULAK K, ORAL H, KAYA S, KACAR C... Tropikal Theileriozisli Sığırlarda Klinik, Hematolojik ve Bazı Biyokimyasal Parametreler ile Serum Kobalt ve B12 Vitamin Düzeyleri ISSI M, GUL Y, BASBUG O, SAHIN N... Karadeniz Alabalığı (Salmo trutta labrax PALLAS, 1811) Larvalarında Canlı ve Granül Yem Kullanımı: Büyüme Performansının Karşılaştırılması BASCINAR NS, CAKMAK E... Leptin Gene Polymorphisms in Native Turkish Cattle Breeds OZTABAK K, TOKER NY, UN C, MENGI A, KARADAG O, SOYSAL D... Vejetasyonun Farklı Dönemlerinde Biçilen Macar Fiği Buğday Karışımının Besin Madde Kompozisyonu, Rumende Yıkılım Özellikleri, in vitro Sindirilebilirlik ve Rölatif Yem Değerinin Belirlenmesi AKSOY I, NURSOY H... Farklı Kekik Yağı Dozlarının Mısır Silajının Sindirimi ve Rumen Fermantasyonu Üzerine Etkileri CANBOLAT O, KARAMAN S, FILYA I... Efficacies of Formic, Oxalic and Lactic Acids Against Varroa destructor in Naturally Infested Honeybee (Apis mellifera L.) Colonies in Turkey GIRISGIN AO, AYDIN L... Effect of Oocyte Diameter on in vitro Embryo Production in Dogs EVECEN M, CIRIT U, DEMIR K, KARAMAN E, BAKIRER G, HAMZAOGLU AI, BIRLER S, PABUCCUOGLU... Postpartum Farklı Günlerdeki İneklere Uygulanan Double-Ovsynch Programının Bazı Reprodüktif Parametrelere Etkileri BILGEN O, EZENC K... Keçilerde Deneysel Oluşturulan Septik Artritisin Farklı Dönemlerinde, Artroskopik Tanı ve Sağaltımında Artroskopik Lavaj ve İntraartiküler Antibiyotik Uygulanmasının Karşılaştırılması GORGUL OS, SALCI H, OZAKIN C, DALYAN CİLO B, SEYREK-INTAS D, CELIMLI N, CECEN G... Electroretinographic Evaluation of Photoreceptor Cells in Turkish Shepherd Dogs SAROGLU M, BASOGULLARI EKICI A... Detection of Cryptosporidium spp. in Humans and Calves Through Nested PCR and Carbol Fuchsin Staining Methods in Ankara, Turkey SAKARYA Y, KAR S, TANYUKSEL M, KARAER Z, BABUR C, VATANSEVER Z... A Survey on Animal Welfare Attitudes of Veterinary Surgeries, Veterinary Students, Animal Owners and Society in Turkey IZMIRLI S, YASAR A... Değişik Yem Bitkileri ve Karışımlarından Hazırlanan Silajlarda İnokulant Kullanımının Flieg Puanı ve Ham Besin Maddeleri Üzerine Etkileri KARAKOZAK E, AYASAN T... Genetic Parameters and Correlations for Lactation Milk Yields According to Lactation Numbers in Jersey Cows ANALAN A, CANKAYA S... Tarsal plate: Protective Structure Peculiar to Buzzard s (Buteo buteo) Palpebra Inferioris GULTIKEN ME, ONUK B, KARAYIGIT O, YILDIZ D... Sayfa (Page)

7 Determination of Ibuprofen in Rabbit Plasma by High-Performance Liquid Chromatography YILMAZ B, ASCI A, PALABIYIK SS... Cloning and Expression of Thermostable β-amylase Gene of Thermoanaerobacterium thermosulfurogenes in Escherichia coli and Bacillus subtilis BR151 OZCAN BD, OZCAN N... Kınalı Kekliklerde (Alectoris chukar) Uçmanın Egellenmesi Amacıyla Kullanılan Tenektomi, Kapsülektomi veya Tenektomi + Kapsülektomi Tekniklerinin Etkinliğinin Karşılaştırılması DEMIRKAN I, ALTIN S, CEVIK DEMIRKAN A, KORKMAZ M... Molecular Identification of Bacteria Isolated from Dairy Herds with Mastitis TURKYILMAZ S, YILDIZ O, ORYASIN E, KAYNARCA S, BOZDOGAN B... Türkiye de Yetiştirilen Altı Yerli Koyun Irkında BMPR-IB (Booroola Geninde FecB Allel Varlığının PCR-RFLP Yöntemiyle Araştırılması KARSLI T, BALCIOGLU MS... Fare in vitro Merkezi Sinir Sistemi Kültürlerinde Yeni Bir Metot: Etkinlik Araştırması ERDOGAN E, OZTURK G, RAGBETLI MC... A Research Based Evaluation of the Natural Grasslands within the Aspect of Sustainable Livestock Production Systems in Highlands of the Eastern Turkey BOZKURT Y, KAYA I... Tahirova Tarım İşletmesindeki Siyah Alaca İneklerin Süt ve Döl Verimi Özelliklerinin Genetik Parametreleri SAHİN A, ULUTAS Z... Trakya Bölgesi nde (Türkiye) Culicoides Latreille, 1809 (Diptera: Ceratopogonidae) Tür Kompozisyonu DENIZ A, ONCEL T, PATAKAKIS MJ... Sayfa (Page) KISA BİLDİRİ (SHORT COMMUNICATION) Antifungal Effects of Lactobacillus spp. Bacteria on Candida Yeast KARIPTAE E, TULUMOGLU S, ERDEM E OLGU SUNUMU (CASE REPORT) Is It Possible to Treat Equine Papillomatosis with Ivermectin Given by Orally Route? ASLAN O, ORUC E... Bir Gazella gazella da (Hatay Dağ Ceylanı) Ultrasonografİ ile Gebelik Tanısı ERGUN Y, DOGRUER G, SARIBAY MK DERLEME (REVIEW) Allografting and Xenografting of Testis Tissue and Using Possibilities in the Future YILMAZ A, GOKDAL O

8 Kafkas Univ Vet Fak Derg 16 (6): , 2010 RESEARCH ARTICLE Comparison of Two Different Media for In vitro Production of Dog Embryos [1] Mithat EVECEN * Ümüt CİRİT ** Kamber DEMİR * Elif KARAMAN * Gül BAKIRER * Asiye İzem HAMZAOĞLU * Sema BİRLER * Serhat PABUCCUOĞLU * [1] * ** The present work was supported by the Research Fund of Istanbul University (Project No: 1145) Department of Reproduction and Artificial Insemination, Faculty of Veterinary Medicine, Istanbul University, TR Avcilar, Istanbul - TURKEY Department of Reproduction and Artificial Insemination, Faculty of Veterinary Medicine, Dicle University, TR Diyarbakir - TURKEY Makale Kodu (Article Code): KVFD Summary Embryo production via in vitro fertilization and nuclear transfer has been accomplished in the dog, and the transfer of the cloned embryos has recently resulted in the birth of puppies. However, the efficiency of these technologies is still very limited. Until now, only two morulas and single blactocyst have been achieved in vitro. Therefore, the aim of the present study was to examine the effects of two different media (msof and TCM 199) on in vitro maturation (IVM), in vitro fertilization (IVF) and in vitro culture (IVC) of immature dog oocytes. The study was performed in two steps. At the first step, the effects of two different media, on IVM of dog oocytes were investigated. At the end of the IVM period, the nuclear maturation rates were evaluated by aceto-orcein staining method. At the second step, after the IVM period the oocytes inseminated with fresh spermatozoa for 24 h and left for IVC for 7 d. At the end of the IVC period, embryonic development was assessed by microscopic observation at 24 h intervals and then fixed and stained by the same method. Consequently, maturation rates of oocytes in msof medium were significantly higher than those of the TCM 199 (P<0.001). After 7 d of the IVC period, only one oocyte was cleaved in the TCM 199, while eight oocytes cleaved and one of them developed to morula stage in msof medium group (P=0.037). Keywords: Dog, Oocyte, In vitro, Maturation, Fertilization Özet İn Vitro Köpek Embriyosu Üretimi Üzerine İki Farklı Medyumun Etkisi Köpeklerde in vitro fertilizasyon yöntemi ile embriyo üretilebilmiş ve nükleer transfer yöntemiyle klonlanmış embriyolardan canlı bir doğum elde edilebilmiş olmasına karşın, bu alandaki teknolojilerin başarısı oldukça sınırlı düzeydedir. Bu güne kadar yapılan in vitro çalışmalarda, sadece iki adet morula ve bir adet blastosist elde edilebilmiştir. Sunulan çalışmanın amacı, iki farklı medyumun (msof ve TCM 199) köpek oositlerinin in vitro maturasyon (İVM), in vitro fertilizasyon (İVF) ve in vitro kültürü (İVK) üzerine etkilerini araştırmaktır. Çalışma iki aşamada gerçekleştirildi. Birinci aşamada, iki farklı medyumun köpek oositlerinin İVM u üzerine etkisi araştırıldı. İVM sürecinin sonunda fikze edilen oositlerin nükleer olgunlaşma değerlendirmesi, aseto-orsein ile boyanarak yapıldı. Çalışmanın ikinci aşamasında ise, İVM sonrasında oositler taze sperma ile 24 saat İVF a tabii tutuldular ve ardından yedi gün boyunca in vitro kültüre edildiler. Embriyoların gelişimsel kontrolleri her 24 saatte bir mikroskop bakısıyla kontrol edildikten sonra, yedinci günün sonunda aynı metotla fikze edilip boyanarak değerlendirildiler. Sonuç olarak, İVM oranları açısından msof medyum grubunda bulunan oositlerin TCM 199 grubundakilere nazaran daha başarılı olduğu belirlendi (P<0.001). İVK sürecinin sonunda TCM 199 medyum grubunda sadece bir adet oosit yarıklanma gösterirken, msof grubunda sekiz adet oositin yarıklandığı ve bunlardan birisinin de morulaya ulaştığı saptandı (P=0.037). Anahtar sözcükler: Köpek, Oosit, İn vitro, Olgunlaştırma, Fertilizasyon İletişim (Correspondence) /17262 evecen@istanbul.edu.tr

9 898 Comparison of Two Different Media... INTRODUCTION Biotechnological studies continue for the protection of genetic materials of both domestic and endangered exotic animals and these technologies are essential for genetic salvage program in endangered canid species 1. The oocyte of the domestic dog is unique from that of other mammalian species. Ovulation occurs with the oocyte released at the germinal vesicle stage, and then completing nuclear and cytoplasmic maturation within the oviduct. In vivo meiotic maturation of the bitch oocyte is completed within h after ovulation 2. The first study on in vitro maturation (IVM) and fertilization (IVF) of dog oocytes was reported by Mahi and Yanagimachi 3. In that study, 25% of oocytes obtained from bitches were able to resume meiosis and developed to metaphase I (MI) or to MII stages after being cultured for h. In vitro insemination of cultured oocytes resulted in 70% of oocytes penetrated by spermatozoa and 20% containing decondensed sperm head(s). Since that report, little progress has been made on in vitro maturation and in vitro fertilization of dog oocytes in vitro 2. Although the IVM and cleavage rates ranging between 20-40% and 8-37% respectively have been reported 4,5 the results of the studies on IVF and IVC are still very low. Until now, only two morulas 6,7 and a single blactocyst 8 have been achieved. Although domestic dog puppies were recently produced from somatic cell nuclear transfer of in vivo matured oocytes 9 and in another study a single, non full-term pregnancy after transferring close to 100 presumptive zygotes to recipient bitches 10 was described, there has been no report on the production of live young from transferring IVM/IVF-derived embryos. The low rates of canine embryo development testify to the inefficient developmental competence of oocytes matured in vitro and justify the main focus of research in the dog to be IVM and IVF 4. Due to these inherently novel traits, in vitro culture systems developed for maturing oocytes of other species have been found inadequate for the maturation of dog oocytes 2. The rates of meiotic completion, IVF and IVC rates of canine oocytes have varied and still been very low, because the oocytes used for experiments have been collected from different sources and cultured with a variety of different culture systems and media 11. Therefore, the aim of the current study was to examine the effects of two different media (modified synthetic oviductal fluid and tissue culture medium 199) on IVM, IVF and IVC of canine oocytes in vitro. MATERIAL and METHODS All chemicals and reagents were purchased from Sigma Chemical Company (St.Louis, MO, USA), unless otherwise indicated. Ovaries were harvested from 55 mixed breed bitches (1-5 years of age) at the time of routine ovariohysterectomy. Bitches were at various stages of the estrous cycle. The ovaries were placed in Dulbecco s phosphate buffered saline (PBS) at 4 C and transported to the laboratory within 2-3 h 12. After keeping at room temperature for min, the ovaries were minced and rinsed in a washing medium (heparin supplemented hepes modified TCM 199) at 37 C in order to obtain cumulus oocyte complexes (COCs) 12. The oocytes with darkly pigmented ooplasm and complete surrounded by at least one layer of cumulus cells were classified as Grade I and were selected for IVM. A total of 775 Grade I COCs were used for IVM (Table 1). The in vitro maturation media employed in this study were, 1: Synthetic Oviduct Fluid (SOF) 13 supplemented with 10 μg/ml follicle stimulating hormone (FSH), 10 μg/ml luteinizing hormone (LH), 4% BSA (Fraction V), antibiotics, essential (MEM Amino Acids Solution, Biological Industries, ) and non essential (MEM Non-Essential Amino Acids Solution, Biological Industries, ) amino acids and 20 μg/ml Epidermal Growth Factor (EGF; Sigma, E 4127) (msof; 270 mosm, ph:7.2), and 2: TCM 199 (Sigma, M 5017) supplemented with 4% BSA (Fraction V), 2.2 g/l NaHCO3, 0.23 mm Na Pyruvate, FSH (10 μg/ml; Sigma, F-2293), LH (10 μg/ml; Sigma L-5269), antibiotics and 20 μg/ml Epidermal Growth Factor (EGF; Sigma, E 4127) (288 mosm, ph:7.3). COCs were selected and transferred into four-well petri dishes (NUNC, Denmark) containing 500 μl maturation medium under mineral oil in well for each maturation medium for maturation at 37 C for 72 h. Incubation atmosphere for IVM was humidified 5% CO2, 5% O2, and 90% N2. Depending upon the number of obtained Grade I oocyte per replication, COCs were left in each well for all experimental groups. A healthy 3 years aged Turkish Shepherd Dog (Kangal) was used as a semen donor. Second fractions of the ejaculates were collected by manual stimulation. Semen were transferred into a 15 ml conical tube onto a twolayer discontinuous gradient formed by layering 1ml of the 45% Percoll solution on top of 1ml of 90%, and centrifuged at 500 g for 20 min at room temperature. The supernatant was removed and the sperm pellet

10 899 EVECEN, CIRIT, DEMIR, KARAMAN, BAKIRER HAMZAOĞLU, BIRLER, PABUCCUOĞLU was washed with 5 ml of hepes supplemented SOF medium (hsof) by centrifugation at 500 g for an additional 10 min. The pellet was then recovered after aspiration of the supernatant, and the spermatozoa were resuspended to give a final concentration of 1x10 6 cells/ml in hsof medium supplemented with 0.56 mg/ml heparin to induce capacitation 14. For each oocyte groups, that had been previously matured for 72 h and placed in 500 μl culture medium, were co-incubated with the sperm suspension in 37 C humidified 5% CO2, 5% O2, and 90% N 2 atmosphere for 24 h. A total of 550 Grade I COCs were used for IVF (Table 2). At 24 h after IVF, presumptive zygotes were transferred into four-well petri dishes (NUNC, Denmark) containing 500 μl culture medium under mineral oil in well (msof and TCM 199), for IVC at 37 C for 7 days. Incubation atmosphere for IVC was the same environmental conditions used for IVM and IVF. The culture media were changed every 48 h. In vitro embryonic development was assessed by microscopic observation at 24 h intervals for 7 days. Following the IVM, oocytes were denuded completely by gentle pipetting in 0.2% (w/v) hyaluronidase and left in KCl solution (0.7% w/v) for chromatin dispersal for another 3-5 min at room temperature. Oocytes were positioned as described by Hewitt and England 15 and fixed for 2-4 days in acetic acid/ethanol fixative (1/3, v/v). Nuclear structures were visualized by staining with aceto-orcein (2% orcein in 45% acetic acid). Nuclear morphology was classified as germinal vesicle intact (GV), germinal vesicle breakdown (GVBD), metaphase I (MI), metaphase II (MII) and undetermined nuclear materials (UDNM) under a phase contrast microscope (400 original magnification). At seven days after insemination, oocytes/embryos was denuded using small glass pipettes, than fixed and stained according to the Hewitt and England s 15 method, and were evaluated by phase contrast microscope. Chi-square test was applied in SPSS 13.0 program to compare the IVM rates of oocytes and IVF rates of embryos between msof and TCM 1999 groups. RESULTS In vitro meiotic maturation results are summarized in Table 1. A total of 381 and 394 oocytes were matured in msof and TCM 199 media respectively. Oocytes matured in msof medium had significantly higher maturation rates (GVBD, MI, MII and MI+MII) than the oocytes in the TCM 199 medium (P<0.001). In vitro fertilization and IVC results are summarized in Table 2. A total of 300 oocytes in msof and 250 oocytes in TCM 199 medium were inseminated. After the IVF and IVC period, although only one two cell embryo found in TCM 199 group, there were 2 2 cell, cell, cell, cell and 1 morula stages embryos in msof medium group (P=0.037, Table 2 and Fig. 1). Table 1. In vitro maturation results of dog oocytes cultured in two different media Tablo 1. İki farklı medyumda kültüre edilen köpek oositlerinin in vitro olgunlaştırma sonuçları Medium Group Oocyte Number msof 381 TCM GV (%) GVBD (%) M I (%) M II (%) M I+M II (%) UDNM (%) (5.2) a (28.9) b (33.1) b (9.2) b (42.3) b (23.6) (42.9) b (12.7) a (11.9) a (3.3) a (15.2) a (29.2) Values with different superscripts in the same column are significantly different (P<0.001) GV: germinal vesicle, GVBD: germinal vesicle breakdown, M I: metaphase I, M II: metaphase II, UDNM: undetermined nuclear material Table 2. In vitro fertilization and development of canine embryos in two different media Tablo 2. İn vitro fertilizasyon ve köpek embriyolarının iki farklı medyumdaki in vitro gelişimleri Medium Group No of Oocytes Inseminated 2 Cell (%) 2 (0.7) 2-4 Cell (%) 2 (0.7) 4-8 Cell (%) 2 (0.7) 8-16 Cell (%) 1 (0.3) Morula (%) 1 (0.3) Total Cleaved Embriyo* (%) 8 (2.7) b msof 300 TCM (0.4) (0.4) a * Statistical analysis were done only for total values column Values with different superscripts in the same column are significantly different (P<0.05)

11 900 Comparison of Two Different Media... Fig 1. Photomicrographs (x 200) of primer oocytes and in vitro derived dog embryos at various stage of development. (A) primer oocytes, (B) 2-cell, (C) 4-cell, (D) 8-16 cell, (E) morula and (F) morula fixed and stained by aceto orcein at 7 days post insemination (x400) Şekil 1. Primer oositler ile in vitro üretilmiş farklı gelişim dönemlerindeki kopek embriyolarının mikroskobik fotoğrafları (x 200). (A) primer oositler, (B) 2-hücreli, (C) 4- hücreli, (D) 8-16 hücreli, (E) morula ve (F) İVF sonrası 7. Günde fikze edilip aseto-orseinle boyanmış morula (x400) DISCUSSION Only 15-20% of ovarian dog oocytes could have been developed to the M II stage, and 30-40% IVF rates seem satisfactory. However, serious limitations are encountered in embryonic development (IVC) rate of fertilized oocytes and reaching morula-blastocyst stage is almost impossible 2. Until to date, only two morulas 6,7 and one blactocyst 8 have been achieved, and no offspring have been produced in the dog or other canids by transferring embryos derived from in vitro matured oocytes 2. For these reasons, the most important finding of our study was the development of one embryo to the morula stage which was the third morula obtained so far. The influence of various factors on the in vitro maturation of canine oocytes such as age of oocyte donors 15, reproductive statuses 16, follicle diameter 2, oocyte diameter 17, ovary transport temperature 18 and in vitro maturation period 6 has been evaluated. However, the rates of meiotic completion, IVF and IVC rates of canine oocytes have still been very low 11. In vitro culture systems developed for maturing oocytes of other species have been found inadequate for the maturation of dog oocytes 5. It is difficult to identify the components lacking in the medium, or which compounds may be detrimental or suppressive 19. Different media and/or various amount of protein, vitamin, hormone or growth factor addition to the medium were used to increase the IVM, IVF and IVC rates 5,11. However, it is still not known how supplements added to medium affect dog embryo development in vitro. It is generally accepted that the process of keeping the medium as simple as possible is the most reliable manner of knowing to what oocytes are being subjected. But, despite the complexity TCM 199 has emerged as a commonly used medium for various species, including canids 20. Some researchers stated that no difference was found in nuclear maturation of canine oocytes cultured in SOF versus TCM 199 1,21. Contrary, we found that the percentage of dog oocytes reaching MII was higher with msof than with TCM 199 (P<0.001). Moreover, the rate of unmatured oocytes (GV stage) was lower in the msof than the TCM 199 medium group (P<0.001). It is stated that maturation of COCs is probably the most critical in vitro step affecting subsequent embryo development to the blactocyst stage 22. It is known that a parameter routinely used as a marker of oocytes maturation is the expansion of cumulus cells. Expansion and mucification are wellestablished mechanisms derived from hyaluronic acid and protein production through LH secretion and camp increase in cumulus cells 19. It is reported that epidermal growth factor (EGF) added to the medium induced meiosis and facilitated cumulus cell expansion of dog oocytes in vitro 23. However, it has been suggested

12 901 EVECEN, CIRIT, DEMIR, KARAMAN, BAKIRER HAMZAOĞLU, BIRLER, PABUCCUOĞLU that cumulus cell expansion which is a sign of oocytes maturation in other mammals, was rare and seemed non essential for normal in vitro maturation and in vitro cleavage in dogs 19. At the end of 72 h in vitro maturation period, cumulus expansions were observed in all the oocytes in both media groups of the study. However, IVM rates in the TCM 199 group were lower than those of msof group (P<0.001). Also, in our study, total cleavage rate was higher in msof than TCM 199 (P=0.037). After seven day of the IVC period, only one oocyte was cleaved in the TCM 199, while eight oocytes cleaved, and one of them developed to morula stage in msof medium group. Synthetic oviduct fluid was originally formulated for use in the sheep 13 and it is not known how similar are the composition of canine oviductal content 1. Synthetic oviduct fluid has been used successfully to improve in vitro development of bovine 24, ovine 13 and feline 25 embryos. Our results suggests that in spite of its complex composition and bearing some vitamins, TCM 199 medium was not as successful as msof medium which is simpler in composition for IVM, IVF and further embryonic development of dog oocytes. However in this study from the eight zygotes produced at msof medium group, four embryos were arrested at 2-8 cell stage. An in vitro block to development has been described for many species in which IVF has been attempted 11. It is stated that arrested embryos enter a senescence-like state and that the stress-related protein p66 shc, which is responsible for the onset of senescence in somatic cells, is involved in the early embryonic arrest 26. On the other hand, the maternal embryonic transition constitutes a critical phase of embryo development. In foxes and dogs, structural studies and cultivation with 3 H-uridine indicate that activation of the genome occurs at the 6 to 8-cell stage in foxes and the 8-cell stage in dog embryos 27. The scarcity of reports in literature attempting to modify the culture conditions in vitro for IVM-derived embryos after the 8-cell stage may indicate that some difficulties have been encountered in propagating development past this stage, but too little information is available to conclude that such an in vitro block exists in dog oocytes 11. Relatively low cleavage rates in the present study could be attributed to inadequate in vitro culture environment. In conclusion, it could be concluded that msof medium could be more efficient than the TCM 199 on maturation, fertilization and further developmental stages of immature dog oocytes in vitro. In dogs, reliable systems for in vitro embryo production and further development of embryos are yet to be developed and further studies are required. REFERENCES 1. Hewitt DA, England GCW: Synthetic oviductal fluid and oviductal cell coculture for canine oocyte maturation in vitro. Anim Reprod Sci, 55, 63-65, Songsasen N, Wildt DE: Size of the donor follicle, but not stage of reproductive cycle or seasonality, influences meiotic competency of selected domestic dog oocyte. Mol Reprod Dev, 72, , Mahi CA, Yanagimachi R: Maturation and sperm penetration of canine ovarian oocytes in vitro. J Exp Zool, 196, , Luvoni GC, Chigioni S, Beccaglia M: Embryo production in dogs: From in vitro fertilization to cloning. Reprod Dom Anim, 41, , Songsasen N, Wildt DE: Oocyte biology and challenges in developing in vitro maturation systems in the domestic dog. Anim Reprod Sci, 98, 2-22, Otoi T, Shin T, Kraemer D, Westhusin ME: Influence of maturation culture period on the development of canine oocytes after in vitro maturation and fertilization. Reprod Nutr Dev, 44, , Hatoya S, Sugiyama Y, Torii R, Wijewardana V, Kumagai D, Sugiura K, Kida K, Kawate N, Tamada H, Swada T, Inaba T: Effect of co-culturing with embryonic fibroblasts on IVM, IVF and IVC of canine oocytes. Theriogenology, 55, , Otoi T, Murakami M, Fujii M, Tanaka M.Ooka A, Une S, Suzuki T: Development of canine oocytes matured and fertilized in vitro. Vet Rec, 146, 52-53, Lee BC, Kim M.K., Jang G, Oh HJ, Yuda F, Kim HJ, Shamin MH, Kim JJ, Kang SK, Schatten G, Hwang WS: Dogs cloned from adult somatic cells. Nature, 436, 641, England GWW, Verstegen JP, Hewitt DA: Pregnancy following in vitro fertilisation of canine oocytes. Vet Rec, 148, 20-22, Farstad W: Current state in biotechnology in canine and feline reproduction. Anim Reprod Sci, 60-61, , Evecen M, Cirit Ü, Demir K, Karaman E, Hamzaoğlu Aİ, Bakırer G: Developmental competence of domestic cat oocytes from ovaries stored at various durations at 4 o C temperature. Anim Reprod Sci, 116, , Tervitt HR, Whittingham DG, Rowson LEA: Successful culture in vitro of sheep and cattle ova. J Reprod Fertil, 30, , Rodrigues BA, Santos DLC, Rodrigues JL: Embryonic development of in vitro matured and in vitro fertilized dog oocytes. Mol Reprod Dev, 67, , Hewitt DA, England GCW: The effect of oocyte size and bitch age upon oocyte nuclear maturation in vitro. Theriogenology, 49, , Evecen M, Cirit Ü, Demir K, Özdaş ÖB, Taş M, Birler S, Pabuccuoğlu S: Effects of estrous cycle stage and transport temperature of ovaries on in vitro maturation of canine oocytes. Anim Reprod Sci, 117, , Otoi T, Fujii M, Tanaka M, Ooka A, Suzuki T: Canine oocyte diameter in relation to meiotic competence and sperm penetration. Theriogenolog, 54, , 2000.

13 902 Comparison of Two Different Media Taş M, Evecen M, Özdaş ÖB, Cirit Ü, Demir K, Birler S, Pabuccuoğlu S: Effect of transport and storage temperature of ovaries on in vitro maturation of bitch oocytes. Anim Reprod Sci, 96, 30-34, Rodrigues BA, Rodrigues JL: Responses of canine oocytes to in vitro maturation and in vitro fertilization outcome. Theriogenology, 66, , Rodrigues BA, Santos, DLC, Rodrigues JL: Effect of maturation medium on in vitro cleavage of canine oocytes fertilized with fresh and cooled homologous semen. Zygote, 15, 43-53, Rota A, Cabianca G: In vitro maturation rates of canine oocytes from anoestrous bitches in simple media. Reprod Nutr Dev, 44, , Massip A, Mermillod P, Van Langendonct A, Touze JL, Dessy F: Survival and viability of fresh and frozen-thawed in vitro bovine blastocyst. Reprod Nurt Dev, 35, 3-10, Cui XS, Jin YX, Shen XH, Lee JY, Lee HS, Yin XJ, Kong IK, Kim NH: Epidermal growth factor enhances meiotic resumption of canine oocytes in the presence of BSA. Theriogenology, 66, , Keskintepe L, Brackett BG: In vitro developmental competence of in vitro-matured bovine oocytes fertilized and cultured in completely defined media. Biol Reprod, 55, , Evecen M, Şahin BE, Baran A, Ak K, İleri İK: In vitro fertilization of cat oocytes. J Fac Vet Univ Istanbul, 29, , Favetta LA, Robert C, St John ET, Betts DH, King WA: P 66shc but not p 53 is involved in early arrest of in vitro produced bovine embryos. Mol Human Reprod, 10, , Farstad W, Hyttel P, Grondahl C, Monval MM, Smith AJ: Fertilization and early embryonic development in the blue fox (Alopex lagopus). J Reprod Fertil, 47,

14 Kafkas Univ Vet Fak Derg 16 (6): , 2010 RESEARCH ARTICLE Use of GnRH Agonist (Desloreline) in Combination with PGF2 on the Termination of Pregnancy in Bitches [1] Örsan GÜNGÖR * Mehmet KAYA ** Kutlay GÜRBULAK *** Hasan ORAL **** Semra KAYA **** Cihan KAÇAR **** [1] * ** *** **** This study was financially supported by the TÜBİTAK (The Scientific and Technological Research Council of Turkey) - Agriculture, Forestry and Veterinary Sciences Research Grant Group - Turkey (Project No. 108 O 009) Mehmet Akif Ersoy University, Faculty of Veterinary Medicine, Department of Obstetrics and Gynaecology, TR Burdur - TURKEY Ondokuz Mayıs University, Faculty of Veterinary Medicine, Department of Physiology, TR Kurupelit, Samsun - TURKEY Erciyes University, Faculty of Veterinary Medicine, Department of Obstetrics and Gynaecology, TR Kocasinan, Kayseri - TURKEY Kafkas University, Faculty of Veterinary Medicine, Department of Obstetrics and Gynaecology, TR Pasacayiri, Kars - TURKEY Makale Kodu (Article Code): KVFD Summary Ten bitches whose sexual cycle periods were determined with vaginal cytology to be mated at appropriate time were used in this study. Desloreline implant (Suprelorin ) was inserted under the dorsal skin of bitches that were determined to be pregnant using ultrasonography 20 days after the first day of mating. Ten days after the implant insertion, single dose PGF2 (cloprostenol, 20 μg/kg, IM) was administered. The pregnancy was terminated on all bitches that received this procedure. It was determined that the level of progesterone decreased from an average of 28.87±11.86 ng/ml on the day of implant insertion (day of pregnancy) to 0.43±0.31 ng/ml when the abortion was completed. It was observed that after the implant insertion, the abortion was completed in an average of 3.9±0.7 days following PGF2 administration. It has been observed that abortions was completed in an average of 2,3±0.48 days after the first vaginal discharge was seen. In conclusion, it was determined that Desloreline, a GnRH agonist, successfully could terminate pregnancy when it is combined with a single dose of PGF2 administration in bitches. Keywords: Bitch, Pregnancy termination, Desloreline, PGF2 Özet Köpeklerde Gebeliğin Sonlandırılması Amacıyla GnRH Agonisti (Desloreline) ile PGF2 Kombinasyonunun Kullanımı Araştırmada seksüel siklus evresi vaginal sitoloji ile belirlenerek en uygun zamanda çiftleştirilen on dişi köpek kullanıldı. İlk çiftleştirme gününü takiben 20. günden itibaren yapılan ultrases muayenesi ile gebe olduğu belirlenen köpeklere Suprelorin boyun derisi altına yerleştirildi. İmplantın yerleştirilmesini takiben 10. günde bir kereye mahsus olmak üzere PGF2 (cloprostenol, 20 μg/kg,) kas içi yolla uygulandı. Uygulama yapılan köpeklerin tümünde gebelik sonlandı. Progesteron değerinin implantın uygulandığı gün (gebeliğin günü), ortalama 28.87±11.86 ng/ml iken, abortion tamamlandığında ortalama 0.43±0.31 ng/ml ye düştüğü belirlendi. İmplant uygulamasından 10 gün sonra enjekte edilen PGF2 yı takiben ortalama 3.9±0,7 günde köpeklerde abortionun tamamlandığı görüldü. Abortionun vajinal akıntının ilk kez görülmesinden sonra 2.3±0.48 gün içinde tamamlandığı belirlendi. Sonuç olarak, uzun etkili GnRH anoloğu olan Deslorelin in köpeklerde tek doz PGF2 uygulaması ile birlikte kombine edildiğinde gebeliği başarılı olarak sonlandırabildiği belirlendi Anahtar sözcükler: Köpek, Gebeliğin sonlandırılması, Desloreline, PGF2 İletişim (Correspondence) gungororsan@hotmail.com

15 904 Use of GnRH Agonist (Desloreline)... INTRODUCTION Pregnancy in bitches lasts an average of 63 days after ovulation. However, this period varies based on the number of puppies, the time of mating and the breed 1. In bitches, the corpus luteum (CL) is the primary source of the progesterone (P4) hormone, which is necessary for sustaining pregnancy 2, and the placenta does not secrete P4 2,3. In order to sustain pregnancy in bitches, P4 must maintain a level of at least 2 ng/ml, otherwise pregnancy will be terminated 4. The level of progesterone begins to increase before ovulation and peaks about 15 to 25 days after estrus, reaching a level of ng/ml 5. P4 concentrations increases a second time at days. If pregnancy develops, P4 concentrations tend to drop in the last week of pregnancy 6. All procedures carried out to terminate unwanted pregnancies in bitches depend on either directly or indirectly eliminating the influence of the P4 and prolactin (PRL) hormones. The abortive agents that are most frequently used to terminate pregnancy are prostaglandins (dinoprost, cloprostenol), dopamine agonists (cabergoline, bromocriptine), corticosteroids (dexamethasone, prednisolone), antiprogestins (mifepristone, aglepristone) and agents that inhibit protein synthesis (epostan) 3,6-12. Because the use of those drugs are more difficult due to their side effects such as breast tumors, pyometra, acromegalic changes, diabetes, adrenocortical suppression, masculinity in female fetuses, local skin reactions, bradycardia, hypersalivation and vomiting, GnRH agonists (desloreline, nafareline), which indirectly suppress the secretion of LH and FSH, are frequently used to suppress cyclic activity in bitches In bitches, LH is necessary as a luteotropic hormone for the luteal secretion of P4 throughout pregnancy 9. For this reason, it is thought that slowly released GnRH agonists could be used to terminate pregnancy because it suppresses the LH. It has been thought that desloreline could eliminate P4 secretion for the maintenance of pregnancy by suppressing the secretion of LH. Short-acting and long-acting GnRH agonists have been available in implant form 16. Long-acting GnRH agonists have been tested to control the reproduction of wild animals with positive results 13. Moreover, desloreline, a slow-release GnRH agonist with no side effects, has been used to delay the estrous cycle in bitches. However, there is no study with regard to use of long-acting GnRH agonists for the termination of pregnancy in bitches. Therefore, the objective of this study is to investigate whether previously untested GnRH agonists could be used to terminate unwanted pregnancies in bitches. MATERIAL and METHODS This study was carried on ten bitches whose stages of sexual cycle was determined with vaginal cytology to be mated at a suitable time. Ethical Board approval of this investigation has taken from Erciyes University Veterinary Faculty (date:26/12/2006, number:041). During the study, the bitches were kept at the department of clinical sciences of the Kafkas University, Faculty of Veterinary Medicine. A schema of the experiments design of the study has been shown in Fig. 1. Pregnancy diagnosis was carried out by using transabdominal ultrasonography (Pie Medical, 5 MHz) days after the first day of mating. After the dog was found to be pregnant with ultrasound (20-25 days after mating), desloreline (Suprelorin, 4.7 mg [6-D-tryptophan-9 (N-ethyl-L-prolinamide)-10 deglycinamde)], GnRH, Peptech Animal Health, Australia) was inserted under the skin on the back between the two scapula. In order to record changes in P4 levels, 3 ml of blood was taken immediately prior to the insertion of the implant and then once every 2 days until the termination of pregnancy. Ten days after the implant insertion, a single IM dose of PGF2α (Cloprostenol sodium, 20 μg/ kg, Estrumate, DIF, Turkey) was administered after taking blood. The blood plasma was separated with a centrifuge (Nuve NF-615) at 3000 rpm for 20 min and then the samples were stored at -20 C until to be Fig 1. A schematic of the experiment s design Şekil 1. Deney dizaynının şematik sunumu

16 905 GÜNGÖR, KAYA, GÜRBULAK ORAL, KAYA, KAÇAR assayed for P4. Commercially available kits were used to determine the plasma P4 level (Progesterone EIA kit, DRG International, Inc., USA). After administering Suprelorin, a vaginal examination and ultrasonographic examination were performed every other day. During the vaginal examination, the condition of the cervix and any abnormal discharge were recorded. At ultrasonographic examination, the size of the uterus and the viability of the fetus and the amount and quality of amniotic fluid were examined to monitore abortion. The descriptive analysis was performed by using the SPSS 11.0 statistics program. RESULTS It was found that all pregnancies were terminated in the dogs that were administered desloreline together with PGF2α. It was noted that the pregnancies of the dogs continued normally until ten days following implant insertion. The P4 level was in an average of 28.87±11.87 ng/ml on the day the implant insertion (day of the pregnancy), and it dropped to an average of 0.43±0.31 ng/ml when the abortion was completed. Following implant insertion, the P4 level initially increased for two days, and then it began dropping. It was in an average of 1 ng/ml on day of PGF2α administeration. In two dogs, P4 level dropped below 2 ng/ml eight days after the initiation of procedure. Two days after PGF2α administration, P4 dropped to an average of 0.6±0.4 ng/ml and it dropped to below 0.5 ng/ml on day four (Table 1, Fig. 2). An average of 1.6±0.5 days after PGF2α was administered, it was noted that the cervix of the dogs opened and a bloody reddish-green vaginal flow began to appear. Five min after PGF2α was administered, the dogs exhibited very pronounced salivation and vomiting, slightly pronounced sweating and pupillar dilation, and some of them exhibited diarrhea. However, these side effects of PGF2α disappeared within one hour. It was found that the abortion was completed 2.3±0.48 days after the first flow began to appear (Table 2, Fig. 3). Table 1. Plasma P4 (ng/ml) levels following induction of abortion (day 0) in bitches Tablo 1. Abortion indüklenmesini (0. gün) takiben köpeklerdeki plasma P4 değerleri (ng/ml) Dog s Name Day 0 Day 2 Day 4 Day 6 Day 8 Day 10 Day 12 Day 14 Odie Marsu Müdür Zalim Vetkopat Lessi Boncuk Ares Afro Zen Average 28.87±11.86 a 30.46±14.88 ab 18.50±14.89 bc 15.62±14.76 c 3.84±3.18 d 0.99±0.65 e 0.60±0.40 f 0.43±0.31 f a, b, c, d, e, f : Supercripts within a same row indicates significance (P<0.05) (Aynı satırdaki farklı harfler P<0.01 olduğunu gösterir) Fig 2. Average P4 levels during induction of abortion (Day 0) in bitches Şekil 2. Köpeklerde abort indüksiyonu (0.gün) süresince ortalama P4 değerleri

17 906 Use of GnRH Agonist (Desloreline)... Table 2. Initiation (day), completion (day) and duration (days) of abortion following Desloreline and PGF2α administrations in dogs Tablo 2. Köpeklere Desloreline ve PGF2α uygulamasını takiben abortun başlama, bitiş ve oluş süreleri (gün) Dog s Name Initiation of Abortion Following Desloreline Administration Initiation of Abortion Following PGF2α Administration Completion of Abortion Following Desloreline Administration Completion of Abortion Following PGF2α Administration Duration of Abortion Odie Marsu Müdür Zalim Vetkopat Lessi Boncuk Ares Afro Zen Average 11.6± ± ± ± ±0.48 Fig 3. Ultrasound images taken from the beginning of the induction of abortion until the end of the abortion in a bitch Şekil 3. Abortusu indüklenen bir köpeğin, uygulamanın başlangıcından abortus sonuçlanana kadar alınan ultrases görüntüleri

18 907 GÜNGÖR, KAYA, GÜRBULAK ORAL, KAYA, KAÇAR DISCUSSION Johnston et al. 5 reported that the P4 level begins to rise prior to ovulation in dogs and reaches its the highest level (15-80 ng/ml) days following estrus. In this study, the P4 level was found to be high (28.84 ng/ml) due to implant insertion; however, P4 levels were still in the range reported by Johnston et al. 5 following mating. After GnRH agonists are administered, there is a sudden increase (flare effect) in gonadotropic hormone levels, followed by drop to their lowest levels 17. GnRH agonists desensitize adenohypophis receptors via continous stimulation which stops the secretion of FSH and LH 16,18,19. It is thought that the increase in P4 levels following desloreline insertion could be due to the increase in gonadotropin levels. Subsequently, the secretion of gonadotropin could be ceased due probably to desensitization of the receptors in the adenohypophis following continous stimulation; all together it leads to drop in P4 concentrations in this study. The P4 level increased during the first two days after the desloreline implant was administered, and then it dropped below 2 ng/ml on days eight, ten and twelve of the procedure in two, seven and one bitch, respectively Note that Johnston et al. 4 and Alaçam 20 state that a level of 2 ng/ml is necessary for the maintenance of pregnancy. As a result, it is thought that desloreline could be the primary agent that could induce the abortion. As the pregnancy progresses, the puppies also naturally grow. When an abortion is induced after the pregnancy has progressed, the duration of the abortion is extended. This not only has various negative effects on the bitch, but it also causes the puppies to be born live and subsequently die, which has a further negative effect on the psychology of the dog s owner 7. For this reason, we used PGF2α, which is well known agent to stimulate the contractility of the uterus in order to terminate the pregnancy before it progresses 21,22. After a single dose of PGF2α, was administered, the abortion was completed in an average of 3.9±0.7 days, and it is thought that this procedure could speed up the progression of the abortion due to its positive effect on uterine contractility. This is because on the day on which PGF2α was administered, except for one dog, the P4 level was below the value necessary to sustain pregnancy, as reported by other researchers 4,20. For this reason, it is assumpted that the primary factor that induced the abortion was desloreline, and that PGF2α could help the abortion to occur more quickly by increasing the contractility of the uterus. Furthermore, it has been reported that the administration of a single dose of PGF2α is insufficient to induce an abortion in bitches, and two administrations were needed to achieve positive results Moreover, it would be helpful to administer PGF2α in repeated times until the abortion occurs in combination with the other abortion-inducing agents However, in our study, a single application of PGF2α was sufficient, and more benefit was obtained from its effect on uterine contractility than its luteolytic effect. The side effects that occurred a short time after administering PGF2α disappeared within a few hours, as it was also reported by Wanke et al. 3. In conclusion, administration of Desloreline, a longacting GnRH analog, in combination with a single dose of PGF2α lead to decrease in the P4 level below the minimum level required to sustain pregnancy, and termination of the pregnancy in bitches. Furthermore, since a large number of injections are not performed on the dog, it is our opinion that the method is easier, less expensive and causes less negative impact on the bitch than other abortion inducting procedures. Desloreline in combination with PGF2α could be an alternative method to terminate pregnancy because this procedure is easier, less expensive and with less negative impact on the bitch. Termination of pregnancy with use of desloreline warrants further research to be accepted for routine application in bitch. REFERENCES 1. Feldman EC, Nelson RW: Breeding, pregnancy and parturition. In, Feldan EC, Nelson RW (Ed): Canine and Feline Endocrinology and Reproduction. 1st ed., pp , W.B. Saunders Company, Philadelphia, Onclin K, Verstegen JP, Concannon PW: Time-related changes in canine luteal regulation: in vivo effects of LH on progesterone and prolactin during pregnancy. J Reprod Fertil, 118, , Wanke MM, Romagnoli S, Verstegen J, Concannon PW: Pharmacological approaches to pregnancy termination in dogs and cats including the use of prostaglandins, dopamine agonists, and dexamethasone. Accessed: 13 August, Johnston SD, Kustritz MVR, Olson PNS: Canine and Feline Theriogenology. 3rd ed., pp , W.B. Saunders Co, Philadelphia, Johnston SD, Kustritz MVR Olson PNS: Canine and Feline, Theriogenology. 3rd ed., pp , W.B. Saunders Co, Philadelphia, Wanke M, Loza M, Monachesi N, Concannon P: Clinical use of dexametasone for termination of unwanted pregnancy in dogs. J Reprod Fert Suppl, 51, , Palmer CW, Post K: Prevention of pregnancy in the dog with a combination of prostaglandin F2 and bromocriptine. Can Vet J, 43, , Eilts BE: Pregnancy termination in the bitch and queen. Clin Tech Small Anim Pract, 17, , Concannon PW: Contraception in dogs and cats. 29th World Congress of the World Small Anim Vet Assoc, Rodos, Greece,

19 908 Use of GnRH Agonist (Desloreline) Kalender H, Beceriklisoy HB, Kanca H, Findik M, Erunal- Maral N, Handler J, Aslan S: Plasma concentrations of folic acid, vitamin B12 and progesterone of cyclic bitches, bitches during pregnancy and induced abortion and bitches with pyometra. Dtsch Tierarztl Wochenschr, 3, , Kutzler M, Wood A: Non-surgical methods of contraception and sterilization. Theriogenology, 66, , Gobello C: New GnRH analogs in canine reproduction. Anim Reprod Sci, 100, 1-13, Bertschinger HJ, Asa CS, Calle PP, Long JA, Bauman K, DeMatteo K, Jochle W, Trigg TE, Human A: Control of reproduction and sex related behaviour in exotic wild carnivores with the GnRH analogue deslorelin: Preliminary observations. J Reprod Fertil Suppl, 57, , Trigg TE, Wright PJ, Armour AF, Williamson PE, Junaidi A, Martin GB, Doyle AG, Walsh J: Use of a GnRH analogue implant to produce reversible long-term suppression of reproductive function in male and female domestic dogs. J Reprod Fertil Suppl, 57, , Herbert CA, Trigg TE: Applications of GnRH in the control and management of fertility in female animals. Anim Reprod Sci, 88, , Padula AM: GnRH analogues-agonists and antagonists. Anim Reprod Sci, 88, , Wright PJ, Verstegen JP, Onclin K, Jochle W, Armour AF, Martin GB, Trigg TE: Suppression of the oestrous responses of bitches to the GnRH analogue deslorelin by progestin. J Reprod Fertil Suppl, 57, , Padula AM, Macmillan KL: Restoration patterns for luteinizing hormone and ovarian function following treatment with GnRH agonist implants (deslorelin) for 7, 14 or 21 days in cycling dairy cows. Anim Reprod Sci, 87, 11-24, Schultze-Mosgau A, Griesinger G, Altgassen C, Von Otte S, Hornung D, Diedrich K: New developments in the use of peptide gonadotropin-releasing hormone antagonists versus agonists. Expert Opin Investig Drugs, 14, , Alaçam E: Köpek ve Kedilerde Üreme Süreci ve Sorunları. Birinci Baskı , Medisan Yayınevi, Ankara, Romagnoli SE., Cela M, Camillo F: Use of prostaglandin F2 alpha for early pregnancy termination in the mismated bitch. Vet Clin North Am: Small Anim Pract, 21, , Onclin K, Verstegen JP: Comparisons of different combinations of analogues of PGF2 alpha and dopamine agonists for the termination of pregnancy in dogs. Vet Rec, 144, , Schäfer-Somi S, Aksoy OA, Beceriklisoy HB, Einspanier A, Hoppen HO, Aslan S: Repeated induction of abortion in bitches and the effect on plasma concentrations of relaxin, progesterone and estradiol-17beta. Theriogenology, 68, , 2007.

20 Kafkas Univ Vet Fak Derg 16 (6): , 2010 RESEARCH ARTICLE Tropikal Theileriozisli Sığırlarda Klinik, Hematolojik ve Bazı Biyokimyasal Parametreler ile Serum Kobalt ve B12 Vitamin Düzeyleri Mustafa İSSİ * Yusuf GÜL * Onur BAŞBUĞ * Nurhan ŞAHİN ** * ** Fırat Üniversitesi, Veteriner Fakültesi, İç Hastalıkları Anabilim Dalı, TR Elazığ - TÜRKİYE Fırat Üniversitesi, Veteriner Fakültesi, Hayvan Besleme ve Beslenme Hastalıkları Anabilim Dalı, TR Elazığ - TÜRKİYE Makale Kodu (Article Code): KVFD Özet Bu çalışmada tropikal theileriozis teşhisi konan sığırların bazı klinik hematolojik ve biyokimyasal parametreler ile kan serumu kobalt ve B12 vitamin düzeylerinin belirlenmesi amaçlandı. On sağlıklı (Kontrol Grubu) ve 10 tropikal theileriozisli (Hasta Grubu) olmak üzere toplam 20 sığır araştırma materyalini oluşturdu. Kontrol grubuna göre hasta grubunda; vücut sıcaklığı, solumun frekansı, total bilirubin (P<0.001), aspartat amino transaminaz (AST), alanin amino transaminaz (ALT), alkalen fosfataz (ALP) (P<0.01) ve kalp frekansı, B12 vitamininde (P<0.05) artış belirlenirken, rumen hareketi, hemoglobin miktarı, hematokrit değer, total lökosit sayısı (P<0.001), eritrosit sayısı (P<0.01) ve serum kobalt seviyelerinde (P<0.05) azalma belirlendi. Sonuç olarak, tropikal theileriozisli sığırlarda serum kobalt düzeyi düşük, fakat serum B12 vitamin düzeylerinin yüksek olduğu tespit edildi. Anahtar sözcükler: Sığır, Tropikal theileriozis, B12 vitamini, Kobalt Clinical, Hematological and Some Biochemical Parameters with Serum Cobalt and Vitamin B12 Levels in Cattle with Tropical Theileriosis Summary It was aimed to determine some clinical, hematological and biochemical parameters along with serum cobalt and B12 vitamin levels in cattle with tropical theileriosis. A total of 20 cattle including 10 clinically healthy cattle (control group) and 10 cattle with tropical theileriosis (patient group) were used as materials. While body temperature, respiratory frequency, total bilirubin (P<0.001), aspartate amino transaminase (AST), alanine amino transaminase (ALT), alkaline phosphatase (ALP) (P<0.01), heart frequency and B12 vitamin levels (P<0.05) were increased in patient group compared with control group, rumen motility, hemoglobin amount, hematocrit value, total leukocyte number (P<0.001), erythrocyte number (P<0.01) and serum cobalt amount (P<0.05) were decreased in patient group compared with control group. In conclusion, it was determined that serum cobalt levels low, however B12 vitamin levels high in cows with tropical theileriosis. Keywords: Cattle, Tropical theileriosis, Vitamin B12, Cobalt GİRİŞ Theileria annulata tarafından meydana getirilen tropikal theileriozis özellikle kültür ırkı sığırlarda başta anemi olmak üzere önemli hematolojik değişikliklere 1-3 ve özellikle karaciğer olmak üzere çeşitli organ ve dokularda tahribata neden olmaktadır 3-5. Anemiyi önlemek için B12 vitamini ve kobalt verilebileceği, karaciğer tahribatına karşı ise destekleyici tedavilerin yapılabileceği 6,7 ifade edilmektedir. Normal büyüme ve eritrositlerin oluşumu için esansiyel bir vitamin olan B12 vitaminini monogastrik hayvanların dışarıdan alması gerekirken ruminatlar kobalt varlığında rumende bulunan bakteriler (Selemonas, Peptostreptococcus ve Butyrivibrio grubu) tarafından sentezleyebilirler 8,9. Emilebilmesi için mide salgısında bulunan ve intrinsik faktör adı verilen sialinik asitli bir İletişim (Correspondence) mustafaissi@hotmail.com