Fertilizability of in vitro capacitated riverine buffalo spermatozoa to in vitro matured swamp buffalo oocytes

Three studies were conducted to determine the most appropriate media for in vitro capacitation of buffalo spermatozoa and determine its fertilizing ability to in vitro matured swamp buffalo oocytes. In study 1, the capacitation characteristics of buffalo spermatozoa treated with varying levels of caffeine, heparin and calcium-ionophore in TCM [tissue culture medium] 199 was observed. The basis of evaluation includes a) percent motility, b) percent sperm passage to Sephadex filter and hyaluronidase activity. Caffeine concentration of 5 mmol/ml shows significant (P less than 0.01) signs of sperm capacitation. Similarly, 12.5 micro/ml of heparin and 0.15 micro Caionophore exhibited significant (P less than 0.01) positive effect that causes the sperm to capacitate and activate acrosome reaction. In study 2, the capacitation characteristics of buffalo spermatozoa was observed after treatment of different combination of caffeine and heparin in different media i.e., TCM 199, SPTL [sperm tyrode lactate] and mSOF [modified synthetic oviductal fluid]. On the basis of percent motility, percent sperm passage to Sephadex and percent acrosome reaction different combination of caffeine, heparin and mSOF in different media (TCM 199 and SPTL) shows different capacitation characteristics in vitro, that can successfully penetrate IVM [in vitro maturation] oocytes of swamp buffalo. However, combination of 5.0 microliter mSOF/ml + 4 microgram heparin/ml + 7 microgram caffeine/ml in SPTL and 4 microgram heparin/ml + 7 microgram caffeine/ml in TCM 199 resulted to a higher penetration rate of 38.09 and 33.13 percent, respectively. In study 3, cleavage and development of in vitro matured swamp buffalo oocytes fertilized with in vitro capacitated riverine buffalo spermatozoa was observed. In vitro fertilized swamp buffalo oocytes were observed at 24, 48, 72, 120, and 144 hours after insemination. The development of fertilized oocytes was observed to exhibit a cleavage rate of 47.86 percent after 24 hours. Morula and early blastocyst was observed at 120 hours after insemination, 39.29 percent of the oocytes that cleaved. But at 144 hours, the number increases to a total of 45 embryo (80.35 percent) at morula and blastocyst stage. This observation suggest that heparin, mSOF and caffeine are important component and it is suitable for successful fertilization of buffalo oocytes in vitro. The protocol herein developed indicated that in vitro capacitation of Murrah buffalo spermatozoa as described could fertilized swamp buffalo oocytes and cultured up to morula or blastocyst stage of embryo