In vitro Buffalo sperm capacitation and assessment of acrosome reaction by using Nigrosin-Eosin-Gemisa(NEG) staining technique were performed. Sperm capacitation was done by TALP capacitation media with different concentrations of heparin. During capacitation process the environmental condition was 5% CO2 with maximum humidity at 39°C as long as 6h incubation period. Acrosome reaction, livability and motility was evaluated after each hour of incubation. The event of sperm capacitation was finally evaluated through in vitro oocytes penetration test. Buffalo oocytes were collected from fresh ovaries of slaughtered animals, then prepared and matured in vitro in the same incubation condition for 24h. Minimal Essential tissue culture media (MEM) with 15% follicular fluid was used as a maturation media. The obtained results recorded a maximum value of 55.5% for alive sperm with reacted acrosome after 2h incubation with heparin concentration of 200 ug/ml TALP. This value was significantly (P<0.001) higher than the other heparin concentrations Moreover, the percentage of sperms with reacted acrosome was significantly (P<0.01) higher in the heparinized samples rather than in the non heparinized controls. A non significant difference was recorded in alive and dead sperm percentages between Nigrosin-Eosin(NE) and NEG stains for the same sample. Therefore, NEG staining technique have a powerful performance to identify alive and dead sperm beside acrosome reaction. In vitro oocyte maturation recorded that 75.2% of the selected oocytes were matured. After 48h of insemination, 30.1% of the matured oocytes showed sperm penetration..