Mitochondrial function and reactive oxygen species action in relation to boar motility

Flow cytometric assays of viable boar sperm were developed to measure reactive oxygen species (ROS) formation (oxidization of hydroethidine to ethidium), membrane lipid peroxidation (oxidation of lipophilic probe C11-BODIPY581/591), and mitochondrial inner transmembrane potential (ΔΨm; aggregation of mitochondrial probe JC-1) during hypothermic liquid storage and freeze-thawing of boar semen and to investigate relationships among ROS, motility, ΔΨm, and ATP production. Basal ROS formation and membrane lipid peroxidation were low in viable sperm of both fresh and frozen-thawed semen, affecting ≤4%. Sperm in fresh, liquid-stored and frozen-thawed semen appeared to be equally susceptible to the activity ROS generators xanthine/xanthine oxidase, FeSO4/ascorbate, and hydrogen peroxide (H2O2). Of the ROS generators tested, FeSO4/ascorbate was specific for membrane lipid peroxidation, whereas menadione, xanthine/xanthine oxidase, and H2O2 were specific for oxidization of hydroethidine. Menadione (30 μM) and H2O2 (300 μM) decreased (P < 0.05) motility by 90% during 60 min of incubation. Menadione decreased (P < 0.05) the incidence of sperm with high ΔΨm by 95% during 60 min of the incubation, although ATP content was not decreased (P > 0.05) until 120 min. In contrast, H2O2 did not affect ΔΨm or ATP at any time. The formation of ROS was not associated with any change in viability (90%) for either menadione or H2O2 through 120 min. Overall, the inhibitory affects of ROS on motility point to a mitochondrial-independent mechanism. The reduction in motility may have been due to an ROS-induced lesion in ATP utilization or in the contractile apparatus of the flagellum.