The magnitude of damage to buffalo spermatozoa during incubation with different levels of H2O2 was assessed. A total number of 24 ejaculates from four Murrah buffalo bulls were analysed in the study. Each ejaculate was split into two parts (part I and II). Part I was extended in Tris-egg yolk-citrate extender (20% egg yolk:7% glycerol), equilibrated (4 h at 5 degrees C) and cryopreserved in 0.5-ml French straws and stored in liquid nitrogen. The other part was utilized for fresh semen studies. The sperm in fresh, equilibrated and frozen-thawed semen was separated by centrifugation (1500 g; 15 min) and were washed with sperm TALP. The sperm cells were re-suspended in incubation TALP at the rate of 10(8) sperm cells per millilitre and incubated with 0, 10, 25, and 50 microm H2O2 per ml at 37 degrees C. Sperm motility, viability and intact acrosome percentages were assessed at 15-min intervals up to 60 min of incubation. Lipid peroxidation levels of sperm were assessed at 0 and 60 min of incubation. The results of the experiment revealed that sperm motility decreased drastically during incubation with H2O2. Among the different levels of H2O2, the 50-microm H2O2-incorporated group had significantly (p<0.05) higher malonaldehyde (MDA) level than the other groups. In the 50-microm H2O2-incorporated group, the MDA levels in fresh, equilibrated and frozen-thawed semen after incubation for 60 min were 961.6+/-12.7, 991.8+/-10.3 and 1234.9+/-9.6 nm per 10(9) spermatozoa respectively. An inverse relationship was observed between sperm motility, viability, intact acrosome percentages and concentration of H2O2 and duration of incubation. The decrease in sperm functions with duration of incubation and concentration of H2O2 was significantly (p<0.05) higher in frozen-thawed than fresh and equilibrated spermatozoa.