Lipid peroxidation
Lipid peroxidation was evaluated using a fluorescent lipid probe C11-BODIPY581/591 as we described before[31]. This probe emits orange fluorescence in its non-oxidized state, shifting to green fluorescence when peroxidized. Diluted samples were incubated with 1µL of 2 mM C11-BODIPY581/591 in ethanol and incubated for 30 min at 37°C in the dark. Samples were then centrifuged at 500g for 3 min and the sperm pellets were resuspended in 500µL of EK. To determine viability sperm was loaded with PI and incubated further for 5 min at room temperature before cytometric analysis.