Follicular dynamics, Oocyte pick up-in vitro fertilization in swamp buffaloes (bubalus bubalis)

Abstract

The objectives of the studies were to elucidate ovarian follicular dynamics in swamp buffalo cows (Bubalus bubalis) following an estrous synchronization protocol (EXP 1), to evaluate the efficiency of Ovum Pick Up (OPU) in cycling and lactating postpartum swamp buffaloes without gonadotropin stimulation (EXP 2) and to develop the embryo production from oocytes retrieved by ovum pick up (EXP 3). EXP. 1. A total of 9 cyclic buffalo cows, received a progesterone ear implant for 10 days: and a single PGF2[alpha] at the day of ear implant removal. Daily ultrasound monitoring and blood collection were perfomed to determine serum concentration progesterone starting one day after implant removal. Data analysis was carried out for the first 5 days since the ear implant removal and at least two consecutive cycles in each buffalo with estrous sign. This study was performed during hot season (March to June 2004) and cool season (November 2004 to February 2005). The results showed that the estrous cycles of swamp buffalo presented the characteristic pattern of follicular growth waves. From 22 estrous cycles, 5(22.7%) presented one follicular wave; 17(77.3%) presented two follicular waves. The characteristics of the follicular dynamics in the swamp buffaloes; one follicular wave, the wave emerged on day 2.3 +- 0.5 and 1.8 +- 0.4 of the cycle in hot and cool respectively. The dominant follicle reached its maximum diameter on day 13.5 +- 1.2 versus 12.6 +-1.5 with the maximum diameter was 14.5 +- 2.1 versus 16.4+- 2.7 mm. In hot and cool seasons, respectively. No difference of follicular development between hot and cool season. EXP. 2. Cycling (n=5) and lactating postpartum (n=6) cows received hormonal stimulation were given a total of 400 mg, follicle stimulating hormone (FSH), together with 100[microgram] of GnRH, 24 h after the last FSH injection. Following a resting period of 1 month, the two groups of buffaloes, were subjected to the same OPU regimen, but without any hormonal treatment for an additional six OPU sessions. The number of aspirated follicles recorded from the hormonal stimulated, cycling animal and lactating, postpartum buffaloes was not significantly different, 7.2+-3.7 and 9.0+-3.2, respectively (P > 0.05). Recovered oocytes collected from the two groups of hormonally stimulated animals were also not statistically different: 3.7+-2.7 in the cycling and 5.9+-3.5 in the lactating postpartum group (P > 0.05). In the two groups of buffaloes not receiving hormonal stimulation, the number of aspirated follicles was not significantly different: 2.1+-1.4 and 1.4+-0.7 in cycling and lactating postpartum buffaloes respectively (P > 0.05). Recovered oocytes in the non-treated groups were also similar: 1.4+-1.3 vs 0.7+-0.8 in cycling and lactating buffaloes (P > 0.05). OPU can be performed successfully in swamp buffalo in different reproductive status and FSH administration was shown to increase the number of aspirated oocytes in both cycling and lactating, postpartum buffaloes. EXP. 3. The OPU was performed in 5 buffalo cows, administered with 400 mg (NIH unit) of follicle stimulating hormone. The recovered oocytes were washed in TCM 199 2.5 Hepes 2 times and then incubated in TCM 199 NaHCO[subscript3]+10% fetal calf serum for 24 h at 38.5 degrees Celsius, in 5% CO[subscript2] in air. Fertilization and culture with B2 (Menezo) + 2.5% fetal calf serum on vero cells for 7 days and observation the developmental of embryo. The COC and single layer cumulus oocytes were submmited for IVF. The results showed that the maturation rate were 62.8% and the cleavage rate of 40.5% (34/84), but only 1 oocyte(1.2%) was developed until blastocyst. In conclusion, it is possible to produce embryos from OPU in swamp buffalo, however the morula and blastocyst rates were poor due to the low percentage of good quality oocytes.