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The cells were then washed once in ice-cold PBS, trypsinized, pelleted, and then homogenized in mitochondrial isolation buffer (0.21 M mannitol, 0.07 M sucrose, 0.1 mM EDTA, 1 mM EGTA, 10 mM Tris HCl, 0.5% BSA, pH 7.4 using KOH)

ROS are mainly produced in spermatozoa via two pathways including the NADPH oxidase in the plasma membrane and the NADH oxidoreductase system in the mitochondria [13]