Sperm viability/vitality was evaluated using the eosin-nigrosin staining method. First, a 5 µL diluted semen sample was placed in a test tube. Then, 5 µL of each eosin and nigrosin staining solution was added and mixed well, followed by a 30 s wait. After 30 s, 5 µL of solution was placed on the slide, spread evenly with coverslip and left for 15 min to dry. Observed under 40× microscopic lenses, at least 300 sperm were analyzed to determine the sperm viability/vitality percentage estimation.
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