Ovsynch-based Estrus Synchronization and Pregnancy Monitoring in Buffaloes

All animals (n = 101) were treated with a modified Ovsynch program [1st GnRH, (400 μg, intramuscular (IM); Ningbo Sansheng Pharmaceutical (NSP), China) at day −10, PGF₂α (0.5 mg, IM; NSP) on the −3 day, 2nd GnRH injection (200 μg) and mifepristone (0.4 mg/kg BW, IM, Hubei Yun Cheng Sai Technology, China) on day −1, AI after 24 h of the 2nd GnRH using frozen-thawed semen and 3rd GnRH on the day 5th day of AI; Figure 1]. Ultrasonography (Desktop B-type veterinary ultrasound scanner, WED-9618-v, LV2-3/6.5 MHz rectal probe; Shenzhen Well D Medical Electronics, Guangdong, China) was used to monitor follicle development (from the day before PGF₂α treatment until 72 h of the 2nd injection of GnRH), CL dynamics (at day 20 and 21 of AI). Prior to AI, the buffaloes were also monitored for estrus signs (vaginal mucous discharge, bellowing, milling, swollen vagina, and head butting) twice daily (06:00 and 18:00). Buffaloes were also observed for estrus signs between days 18 to 24 of AI to record the non-return rate in synchronized buffaloes.
The experimental buffaloes were further divided into four groups (GnRH-0; n = 26, GnRH-100; n = 22, GnRH-150; n = 25 and GnRH-200; n = 28) according to the dose of 4th GnRH injection (0, 100, 150, and 200 μg) at day 20 of AI. The buffaloes were monitored for pregnancy losses at day 30, 40, 60, and 90 days after AI using ultrasonography. Milk samples were collected at day 20 of AI for milk P4 level (Quanzhou Ruixin Biological Technology Co., LTD) to predict the pregnancy in synchronized buffaloes. In addition, blood samples were obtained from synchronized buffaloes at days 20, 40, and 60 to observe deviation in the P4 level of buffaloes with pregnancy or embryonic loss.