242 Ovum pickup and in vitro embryo production (OPU-IVEP) in Indian riverine buffaloes: can stimulation before OPU improve efficiency?

Faster multiplication of superior germplasm can be attained through ovum pickup and in vitro embryo production (OPU-IVEP) in buffaloes, but it is limited by low oocyte recovery and poor embryo production rate compared to cattle. In order to address this, we conducted a pilot study on buffaloes at farmers’ doorsteps. Considering the short breeding season (Dec–March) and small number of participating farmers, we could perform 33 OPUs on 12 donors (2 OPUs/donor in 9 Banni buffaloes and 5 OPUs/donor in 3 Murrah buffaloes). A structured study was difficult, as all the donors were not available at the same time. Initially, a few OPUs were performed on the available donors (n = 9) on a random day without any hormonal intervention (nonstimulated [NS]). As we observed a poor blastocyst rate, stimulation protocol 1 (SP1) was subsequently performed in available donors (n = 6). In SP1, ovulation of donors was synchronised using two PGF_2α injections im 11 days apart (Day 0 on first injection) and a GnRH injection (10 µg, im) on Day 14. On Days 16 and 17, donors received a total dosage of 200 mg of FSH im in four tapering doses at a 12 h interval (57, 57, 43, and 43 mg). There was an improvement in the embryo production rate; however, to reduce the number of visits for injections, available donors were then subjected to stimulation protocol 2 (SP2; n = 17). Under SP2, donors received an intravaginal progesterone device (CIDR) along with oestradiol benzoate (2 mg, im) on a random day of the oestrous cycle (Day 0 = CIDR-insertion day). On Days 4 and 5, donors were stimulated with FSH in the same manner as described for SP1. OPUs were performed after 52 h of coasting in both SP1 and SP2. The OPU-IVEP procedures were performed following standard procedures in the laboratory. Oocytes were subjected to IVM and IVF (22 h in 5% CO₂ in air with maximum humidity at 38.5°C) followed by IVC (7 days from IVF in 5% CO₂, 5% O₂ with maximum humidity at 38.5°C). Only blastocysts and expanded blastocysts of Grade I, as per IETS, were considered. Mean values of different parameters between the protocols were compared by one-way ANOVA using GLM, and means were considered to differ significantly if P < 0.05. The mean number of follicles available for aspiration/OPU was higher in SP1 and SP2 compared to NS (SP1 = 21.0 ± 5.7; SP2 = 18.2 ± 2.2; NS = 17.4 ± 5.0). The mean number of oocytes recovered/OPU was similar in NS (11.8 ± 5.1) and SP2 (11.4 ± 1.3), but it was lower in SP1 (7.8 ± 2.2). Oocyte recovery rates followed a similar trend, with a significantly lower rate in SP1 (SP1 = 36.87 ± 3.4%; SP2 = 65.35 ± 3.5%; NS = 64.26 ± 10.7%; P = 0.01). Higher cleavage and blastocyst rates were observed in both SP1 (68.8 ± 10.1% and 17.2 ± 7.0%; P = 0.02) and SP2 (58.8 ± 5.9% and 34.9 ± 5.2%; P = 0.01) than NS (35.2 ± 8.4% and 8.2 ± 4.0%). Blastocysts/OPU was higher in SP1 (1.5 ± 0.5%) and SP2 (3.5 ± 0.7%; P = 0.01) than NS (1.2 ± 0.5%). Therefore, inducing ovarian stimulation before OPU, particularly in the presence of exogenous luteal support (CIDR), enhances blastocyst rate and blastocysts/OPU and can aid in the multiplication of superior buffalo germplasm.