4. Sperm DNA Fragmentation

The percentage of sperm apoptosis was determined by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay using a commercially available kit (In Situ Cell Death Detection Kit, fluorescein, Roche, USA). Spermatozoa with normal DNA show the background fluorescent color, while sperm with high DNA fragmentation has many 3-OH ends, resulting in a strong fluorescent color. Firstly, the smears were fixed in methanol solution for 4 minutes. The slides were washed with PBS for 5 minutes three times. Later, they were incubated with blocking solution for 15–20 minutes at 15°C–25°C in a dark room. Samples were incubated with 0.1% (v/v) Triton X-100 containing 0.1% (w/v) sodium citrate for 10 minutes on ice. Slides were again washed three times with PBS for 5 minutes and were stained with 50 μL of TUNEL reaction mixture for 1 hour at 37°C in a dark and humidified atmosphere. Then, they were examined under a fluorescent microscope at ×1,000 magnification (BX51; Olympus, Tokyo, Japan) (Figure 1C) [20].