The aim of this study was to test the effect of progesterone supplementation to Ovsynch protocol in cyclic and non-cyclic Mediterranean Italian buffaloes on conception rate after fixed time artificial insemination. From 169 pluriparous buffaloes, 2 groups were identified and subjected to: (1) Ovsynch protocol (OV; n = 83) and (2) Ovsynch protocol with the supplementation of progesterone from days 0 to 7 (OV + PROG.; n = 86). All cows were inseminated 16-20 h after the second GnRH administration. Within each group, non-cyclic buffaloes were identified (OV = 21 and OV + PROG. = 20). Overall conception rate was significantly higher in cyclic compared to non-cyclic buffaloes: 43.7% versus 17.0%, respectively, P = 0.001. A significant effect of progesterone supplementation on conception rate was observed in non-cyclic buffaloes (30% versus 4.7%, P = 0.04) but not in cyclic buffaloes (51.5% versus 35.7%, P = 0.077). Collectively, the presence of a large follicle (≥10 mm) detected at the beginning of the Ovsynch protocol by ultrasound significantly affected conception rate (44% versus 8%, P = 0.01). The findings of the present study suggest that (i) progesterone supplementation to the Ovsynch protocol in buffaloes increases conception rate in non-cyclic animals, (ii) the presence of a large follicle at the beginning of the Ovsynch protocol is a determining factor for a successful synchronization of ovulation and high conception rates and (iii) ultrasound monitoring can improve the overall efficiency by selectively identifying more suitable cycling animals carrying a responsive follicle at the time of first GnRH administration
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