The objectives of present study were (a) validation of annexin V/PI assay for estimation of sperm apoptosis in buffalo (Experiment 1) and (b) determining the effect of stages of cryopreservation on sperm apoptosis and its correlation with sperm motility and plasma membrane integrity (Experiment 2). In Experiment 1, different levels of apoptosis were artificially induced in buffalo semen (100x10(6)sperm/aliquot) through graded doses of camptothecin (5, 10 and 20microM/aliquot). Higher concentrations of camptothecin (10 and 20microM) successfully (P<0.05) induced apoptosis as compared to the lower (5microM) dose and/or control. In Experiment 2, semen samples (n=9, three pooled semen samples from each of the three buffalo bulls separately) were cryopreserved using vapor freezing. The mean percentage of apoptotic, necrotic and viable sperm did not differ between fresh and before freezing stages. However, freezing and thawing increased (P<0.05) the percentage of apoptotic sperm (25.4+/-0.6 vs. 36.5+/-1.9) while decreased (P<0.05) the necrotic (35.1+/-1.2 vs. 29.7+/-0.7) and viable sperm (37.2+/-1.3 vs. 32.8+/-1.9, (P<0.07). Likewise, the mean percent motility and plasma membrane integrity decreased (P<0.05) (64+/-2.1 vs. 49.4+/-1.3) and (79.6+/-0.5 vs. 38.7+/-0.3) respectively, at post thaw compared to other stages. Coefficient of correlation, combined at all stages for each variable revealed that sperm apoptosis was inversely correlated with sperm motility and plasma membrane integrity. It is concluded that (a) the annexin V/PI assay can be used as a tool to determine the buffalo semen apoptosis and (b) freezing and thawing induces apoptosis in buffalo sperm.