Production of superoxide anion and hydrogen peroxide by capacitating buffalo (Bubalus bubalis) spermatozoa

In the present study attempts were made to detect and quantify the generation of superoxide anion (O2−) and hydrogen peroxide (H2O2) by capacitating buffalo spermatozoa. Ejaculated buffalo spermatozoa were suspended in sp-TALP medium at 50 × 106 mL−1 and incubated at 38.5 °C with 5% CO2 in air in the absence or presence of heparin (a capacitation inducer) or reduced nicotinamide adenine dinucleotide phosphate (NADPH) or diphenyleneiodonium (DPI, a flavoprotein inhibitor) for 6 h. Production rate of O2− and H2O2 by spermatozoa at different hours of capacitation were measured by cytochrome c reduction and phenol red oxidation assays, respectively. Spermatozoa generated both O2− and H2O2 spontaneously and following stimulation with heparin and a significant increase of O2− production was observed in the presence of NADPH. However, DPI inhibited this NADPH-induced O2− production and suggested for existence of putative NADPH-oxidase that constitute a specific O2− generating systems in buffalo spermatozoa. Results of our study indicated that buffalo spermatozoa generate O2− and H2O2 and production of these free radicals is induced during capacitation.