Sperm analysis

The epididymides and vas deferens isolated from 3-month-old mice were cut into small pieces in 2 ml of the M16 medium (Sigma-Aldrich) and sperm allowed to swim into the medium, which was maintained at 37°C and 5% CO₂. After 30 min, the medium was collected and diluted for sperm number and motility assessment. For evaluating sperm motility, sperm samples (sperm number>200) were used. “Progressive motility” of sperms is characterized by “spermatozoa moving actively, either linearly or in a large circle, regardless of speed” [33]. For evaluating the sperm morphology, the sperm were collected through centrifugation at 300× g for 5 min and were then resuspended in PBS. Sperm were spread onto coated slides and air-dried. One hundred spermatozoa per mouse were examined per group through staining with Mito-tracker and DAPI under a microscope at 400X magnification. All results were obtained from experiments performed on at least 6 mice per genotype group and 100 sperm per mouse. Data were analysed using the Student's t test to determine significant differences between the two groups. Error bars have been presented as the mean ± SEM. A p-value of <0.05 was considered statistically significant.