Evaluate at least 200 spermatozoa, tally the numbers of live and dead spermatozoa using a laboratory counter, and calculate the percentage of live spermatozoa (see Note 10). The lower reference limit for vitality assessed by eosin–nigrosin stain (membrane-intact spermatozoa) is 58% (5th percentile, 95% CI 55–63) (1).
