RIA yöntemiyle kanda ovPAG ölçümüne dayalı koyunlarda erken gebelik teşhisi
Date
2019-11-11
Authors
Yavuz, Ahmet
Journal Title
Journal ISSN
Volume Title
Publisher
Bursa Uludağ Üniversitesi
Abstract
Koyunlar ülkemizin coğrafi konumundan dolayı mevsime bağlı poliöstrik hayvanlardır. Koyunlarda, çiftleşme sezonu içinde gebe kalmadıkları sürece 17-20 günde bir ovulasyon şekillenir. İdeal bir sürü yönetiminde, çiftleştirilmiş ya da tohumlanmış koyunların gebelikleri 17 gün içinde tespit edilerek, gebe kalmamış olanların bir sonraki östrus siklusunda gebe bırakılması önemlidir. Bu yüzden gebelik teşhisinin çiftleşmeden sonraki 17 gün içinde yapılması üretim planlaması açısından büyük önem arz etmektedir. Bu sebeple, koyunlarda, kanda gebelikle ilişkili glikoprotein (Pregnancy Associated Glycoprotein, PAG) konsantrasyonlarının radyoimmunoassay (RIA) yöntemiyle ölçülerek gebelik teşhisinin mümkün olan en kısa sürede yapılması amaçlanmıştır. Bu tezde PAG konsantrasyonları, serum örneklerinden RIA yöntemi ile doku örneklerinden real-time PCR yöntemi ile ölçüldü. Materyal olarak Türkiye' de Bursa ili Nilüfer ilçesine bağlı Görükle mahallesinde bulunan bir (40⁰ 23' N, 28⁰ 85' E) özel koyun çiftliğinden, T.C. Gıda Tarım ve Orman Bakanlığı'na bağlı Bursa İl Tarım ve Orman Müdürlüğü kayıtları esas alınarak 2-4 yaş aralığındaki multipar özellikte olan ve en son 4 ay önce doğum yapmış koyunlar kullanıldı. Kan örnekleri için 20 koyun (Gebe:10, Gebe olmayan:10, n=20), doku örnekleri içinde 12 adet koyun (n=12) kullanıldı. Örneklerin toplanması için gerekli olan koyunların östrus senkronizasyonuna 2017 yılı mayıs ayında başlandı. Koyunlarda erken dönemde gebelik teşhisi için 6 farklı RIA yöntemi test edildi. Bu amaçla koyunlar çiftleştirildikten sonra 0., 10., 15., 16., 17., 18., 19. ve 20. günlerde kan örnekleri alınarak kan serumunda ovPAG konsantrasyonu RIA-497, RIA-706, RIA-780, RIA-860, RIA-srPool ve RIA-wbPool yöntemleriyle ölçüldü. Koyunlarda gebelik teşhisi için RIA-860 ve RIA-wbPool yöntemleri ilk defa bu tez çalışmasında test edildi. Ayrıca uterus dokusunda ovPAG ekspresyonunu ortaya koymak için real-time PCR ölçümü yapıldı. Doku örnekleri çiftleştirmeyi takiben 0., 14., 20. ve 30. günlerde toplandı. Ortalama PAG konsantrasyonları, RIA-780'de 17. günden, RIA-706'da 18 günden, RIA-srPool ve RIA-wbPool'da 19. günden itibaren arttığı görüldü (P <0.001). Real-time PCR'da ölçülen PAG1 ekspresyonunun, upregüle veya downregüle olmadığı tespit edildi. Periferal kan serumu örneklerinde 17. günden itibaren ovPAG konsantrasyonunun ölçülebiliyor olması, RIA yönteminin hem güvenilir hem de erken dönemde gebelik teşhisi yapabildiğini göstermiştir.
Sheep are seasonal polyestric animals due to the geographical location of our country. Ovulation occurs in every 17-20 days unless they become pregnant during the mating season in sheep. In an ideal herd management, it is important to identify pregnancies of sheep within 17 days after mating, and to make them ready to mate or inseminate before next oestrus cycle. Therefore, it is of great importance for production planning that pregnancy diagnosis is made within 17 days after mating. For this reason, it is aimed to make pregnancy diagnosis as soon as possible by measuring the Pregnancy-associated Glycoprotein (PAG) concentrations in sheep by radioimmunoassay (RIA) method. In this thesis, PAG concentrations were measured by RIA method from serum samples and real-time PCR method from tissue samples. As the materials, multiparous sheep who was 2-4 year old and had given birth 4 months ago were used. They were bought from a private farm located in Gorukle (40⁰ 23 'N, 28⁰ 85' E) in Bursa in Turkey. Sheep were selected based on the records of the Bursa Provincial Directorate of Agriculture and Forestry of the Ministry of Food related Agriculture and Forestry. Twenty sheep (pregnant: 10, non-pregnant: 10, n = 20) and 12 sheep (n = 12) were used for serum and tissue samples respectively. The oestrus synchronization of the ewes required for the collection of samples was begun in May 2017. Six different RIA methods were tested for early pregnancy diagnosis in sheep. Blood samples were taken on days 0, 10, 15, 16, 17, 18, 19 and 20 post-mating and ovPAG concentrations in blood serum were measured by using RIA-497, RIA-706, RIA-780, RIA- 860, RIA-srPool and RIA-wbPool methods for early pregnancy diagnosis. In this thesis, RIA-860 and RIA-wbPool methods were tested for pregnancy diagnosis in sheep, for the first time. In addition, real-time PCR was performed to determine ovPAG expression in uterine tissue samples. The tissue samples were collected from sheep on days 0, 14, 20 and 30 post-mating. The mean PAG concentrations were determined being increased from day 17, 18, 19 and 19 in RIA-780, RIA-706, RIA-srPool and RIA-wbPool, respectively (P <0.001). PAG1 gene expression was not upregulated or downregulated acording to real-time PCR results. The fact that ovPAG concentration can be measured in serum samples from day 17 post coitum has shown that the RIA method is both reliable and able to diagnoe early pregnancy.
Sheep are seasonal polyestric animals due to the geographical location of our country. Ovulation occurs in every 17-20 days unless they become pregnant during the mating season in sheep. In an ideal herd management, it is important to identify pregnancies of sheep within 17 days after mating, and to make them ready to mate or inseminate before next oestrus cycle. Therefore, it is of great importance for production planning that pregnancy diagnosis is made within 17 days after mating. For this reason, it is aimed to make pregnancy diagnosis as soon as possible by measuring the Pregnancy-associated Glycoprotein (PAG) concentrations in sheep by radioimmunoassay (RIA) method. In this thesis, PAG concentrations were measured by RIA method from serum samples and real-time PCR method from tissue samples. As the materials, multiparous sheep who was 2-4 year old and had given birth 4 months ago were used. They were bought from a private farm located in Gorukle (40⁰ 23 'N, 28⁰ 85' E) in Bursa in Turkey. Sheep were selected based on the records of the Bursa Provincial Directorate of Agriculture and Forestry of the Ministry of Food related Agriculture and Forestry. Twenty sheep (pregnant: 10, non-pregnant: 10, n = 20) and 12 sheep (n = 12) were used for serum and tissue samples respectively. The oestrus synchronization of the ewes required for the collection of samples was begun in May 2017. Six different RIA methods were tested for early pregnancy diagnosis in sheep. Blood samples were taken on days 0, 10, 15, 16, 17, 18, 19 and 20 post-mating and ovPAG concentrations in blood serum were measured by using RIA-497, RIA-706, RIA-780, RIA- 860, RIA-srPool and RIA-wbPool methods for early pregnancy diagnosis. In this thesis, RIA-860 and RIA-wbPool methods were tested for pregnancy diagnosis in sheep, for the first time. In addition, real-time PCR was performed to determine ovPAG expression in uterine tissue samples. The tissue samples were collected from sheep on days 0, 14, 20 and 30 post-mating. The mean PAG concentrations were determined being increased from day 17, 18, 19 and 19 in RIA-780, RIA-706, RIA-srPool and RIA-wbPool, respectively (P <0.001). PAG1 gene expression was not upregulated or downregulated acording to real-time PCR results. The fact that ovPAG concentration can be measured in serum samples from day 17 post coitum has shown that the RIA method is both reliable and able to diagnoe early pregnancy.
Description
Keywords
Koyun, Gebelikle ilişkili glikoprotein, Pregnancy-associated glycoprotein (PAG), Radyoimmunoassay (RIA), Real-time PCR (Polimeraz Zincir Reaksiyonu), Polymerase chain reaction, Sheep
Citation
Yavuz, A. (2019). RIA yöntemiyle kanda ovPAG ölçümüne dayalı koyunlarda erken gebelik teşhisi. Yayınlanmamış doktora tezi. Bursa Uludağ Üniversitesi Sağlık Bilimleri Enstitüsü.