Apoptotic changes
The translocation of phosphatidylserine (PS) from the inner to the outer leaflet of the plasma membrane was detected using Annexin V and PI following the manufacturer’s instruction. Sperm cells were resuspended in 100µL of annexin-binding buffer and mixed with 5µL of Alexa Fluor® 488 annexin V and 1µLofPI working solution. After 15 min of incubation 400µL of EK diluent was added, and the samples were analyzed using flow cytometry. The sperm cell population was separated into three groups as live (low level of fluorescence), apoptotic (green fluorescence) and dead (both red and green fluorescence).