Viability

To measure sperm viability, the Annexin V-FITC kit (IQP, Groningen, the Netherlands) was used for the determination of externalization of phosphatidyl serine as an indicator of apoptotic-like changes in the semen. Sperm samples were washed in calcium buffer, and the concentration was readjusted to 1 × 10⁶ sperm/mL, and 10 µL of Annexin-V was added to 100 µL of the sample. After the incubation of the samples for 15 min at room temperature, 1 μL of Propidium Iodide (PI) was added to each sample, and the sample was evaluated using a flow cytometer (Lotfi et al., 2017).