Assays based on spem-zona pellucida binding have been developing as diagnostic tests to predict the fertilizq/ion potential of bull spermatozoa. in this study a similar assay for buffalo sperm was used. The results showed a significant variation (P<0.01) in accessory sperm number between the fresh semen of three bulls, in spite of the same physical characters. Also three bulls, in their physical characters sand number of accessory sperm. The overall mean of accessory sperm was significantly increased (P<0.01) by fresh semen than by frozen semen (181.0 ± 3.0 Vs 30.7 ±7 ± 2.2). The effect of (2mM) caffeie and/or (10 pg/m1) heparin on the number of accessory sperm was significantly obvious (P<0.01) when compared with the control, while, there was no significant variations between them. Addition of both caffeine and heparin in the incubation media improved the number of accessory sperm than in fertilization media, but without significant variations. The number of the accessory sperm was insigificantly increased when the oocytes co-cultured in sperm droplets than when the sperm co-cultured with oocytes in fertilization medium.