Three methods were used to make differential counts of living and dead bull spermatozoa in samples of frozen semen in an egg-yolk citrate medium containing glycerol.

With two methods (‘phase’ and ‘nigrosin’ methods) dead spermatozoa were identified by their altered structure. With a third method (nigrosineosin) dead spermatozoa were identified by their staining affinity. With the phase method spermatozoa were immobilised by treatment with approximately M/40 sodium fluoride and were examined by phase-contrast microscopy in unfixed wet preparations. With the other two methods the spermatozoa were examined in smears stained either with nigrosin alone (nigrosin method) or with nigrosin and eosin (nigrosin-eosin method).

Analysis of variance of the results of a factorial experiment involving fluoride-treated and untreated samples from 6 bulls with the three methods showed that differences between semen samples contributed 66% of the variation. A defect of the phase method was that the variance between counts was greater than the theoretically expected value.

All spermatozoa in nigrosin-eosin stained preparations were stained with eosin within a few days of the smears being made, so that living and dead spermatozoa could not be distinguished by their differing affinities for eosin. Repeat counts on nigrosin-stained preparations did not differ significantly from counts made several days previously. Sodium fluoride in the concentration used here (M/40) tended to reduce the percentage of dead spermatozoa.