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

Abstract

In the present study attempts were made to detect and quantify the generation of superoxide anion (O(2)( * -)) and hydrogen peroxide (H(2)O(2)) by capacitating buffalo spermatozoa. Ejaculated buffalo spermatozoa were suspended in sp-TALP medium at 50x10(6)mL(-1) and incubated at 38.5 degrees C with 5% CO(2) 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 6h. Production rate of O(2)( * -) and H(2)O(2) by spermatozoa at different hours of capacitation were measured by cytochrome c reduction and phenol red oxidation assays, respectively. Spermatozoa generated both O(2)( * -) and H(2)O(2) spontaneously and following stimulation with heparin and a significant increase of O(2)( * -) production was observed in the presence of NADPH. However, DPI inhibited this NADPH-induced O(2)( * -) production and suggested for existence of putative NADPH-oxidase that constitute a specific O(2)( * -) generating systems in buffalo spermatozoa. Results of our study indicated that buffalo spermatozoa generate O(2)( * -) and H(2)O(2) and production of these free radicals is induced during capacitation.