The aim of the study was to evaluate the effects of single and double inseminations on the inflammation of the uterus in mares. Nine mix breed mares with endometrial biopsy scores grade I and IIA, aged 5-15, were used in the study. Two experiments were performed over two estrous cycles: (1) single insemination, (2) double insemination. All mares were used in both insemination group. In the single insemination cycles one insemination was performed 24 h after hCG injection, while in the double

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... emination cycles two inseminations were carried out 24 h and 48 h after the hCG injection. Endometrial biopsies were collected in both cycles immediately before (=0), and 6, 30 and 54 h after the first insemination. Gene expressions of the inflammatory markers IL-1, IL-6, IL-8, iNOS, SAA, COX-2 and CASP-3 and histopathological alterations of the endometrium were determined. The 6 h expression levels of IL-6 were higher when compared with 30 h and 54 h (P<0.05). CASP-3 expression levels in single insemination cycles were higher at 0 h and 6 h when compared with 54 h (P<0.05). In conclusion expression levels of the IL-1,-6,-8, iNOS, SAA and COX-2 were not different between single or double insemination cycles. This study provides preliminary evidence to further characterize the changes in the expression of relevant genes in response to fresh semen. Abstract The aim of the study was to evaluate the effects of single and double inseminations on the inflammation of the uterus in mares. Nine mix breed mares with endometrial biopsy scores grade I and IIA, aged 5-15, were used in the study. Two experiments were performed over two estrous cycles: (1) single insemination, (2) double insemination. All mares were used in both insemination group. In the single insemination cycles one insemination was performed 24 h after hCG injection, while in the double insemination cycles two inseminations were carried out 24 h and 48 h after the hCG injection. Endometrial biopsies were collected in both cycles immediately before (=0), and 6, 30 and 54 h after the first insemination. Gene expressions of the inflammatory markers IL-1β, IL-6, IL-8, iNOS, SAA, COX-2 and CASP-3 and histopathological alterations of the endometrium were determined. The 6 h expression levels of IL-6 were higher when compared with 30 h and 54 h (P<0.05). CASP-3 expression levels in single insemination cycles were higher at 0 h and 6 h when compared with 54 h (P<0.05). In conclusion expression levels of the IL-1β,-6,-8, iNOS, SAA and COX-2 were not different between single or double insemination cycles. This study provides preliminary evidence to further characterize the changes in the expression of relevant genes in response to fresh semen. Özet Çalışmada kısraklarda tek ve çift tohumlama sonrası immun yanıt değerlendirildi. Farklı ırktan, 5-15 yaşlarında ve endometriyal biyopsi skoru I ve IIA olan 9 adet kısrak kullanıldı. İki östrus siklusunda iki uygulama yapıldı: (1) tek tohumlama (2) çift tohumlama. Herbir kısrak tek ve çift tohumlama olmak üzere iki deney grubunda da yer aldı. Tekli uygulamalarda hCG uygulaması sonrası 24. saatte tohumlama yapılıyorken; çiftli uygulamalarda hCG uygulaması sonrası 24. ve 48. saatte tohumlama yapıldı. Tohumlamadan hemen önce (0.) ve tohumlamadan sonraki 6., 30. ve 54. saatte uterus biyopsi örnekleri alındı. Yangı parametrelerinden IL-1β, IL-6, IL-8, iNOS, SAA, COX-2 ve CASP-3 mRNA ekspresyonları ve endometriumdaki histopatolojik değişimler değerlendirildi. Tekli gruptaki kısraklarda 6. saatdeki IL-6 mRNA expresyonları 30. ve 54. saatlerdeki değerlere göre yüksek bulundu (P<0.05). 0. ve 6. saatlerdeki CASP-3 ekspresyonları ise 54. saatdeki değerlere göre yüksek bulundu (P<0.05). Sonuç olarak tekli ve çiftli gruplar arasında IL-1β,-6,-8, iNOS, SAA ve COX-2 expresyonları açısından fark bulunmadı. Taze semen ile yapılan tohumlamalardan sonra bahsi geçen gen expresyonlarına bakarak immun yanıt hakkında ön fikir elde edilebileceği anlaşıldı. Troedsson MHT, Liu IK, Ing M, Pascoe J, Thurmond M: Multiple site electromyography recordings of uterine activity following an intrauterine bacterial challenge in mares susceptible and resistant to chronic uterine infection. 25. Moares CN, Maia L, Lima PF, Dias MC, Raposo-Ferreira, TMM, Sudano MJ, Junior JB, Oba E: Temporal analysis of prostaglandin F2α receptor, caspase 3, and cyclooxygenase 2 messenger RNA expression and prostaglandin F2α receptor and cyclooxygenase 2 protein expression in endometrial tissue from multiparous Nelore (Bos taurus indicus) cows treated with cloprostenol sodium during puerperium. Theriogenology, 83, 276-284, 2015. 26. Troedsson MH, Liu IK, Thurmond M: Function of uterine and blood-derived polymorphonuclear neutrophils in mares susceptible and resistant to chronic uterine infection: Phagocytosis and chemotaxis.