Counting mammalian spermatozoa in biological fluids containing particulate matter

Accurate counting of spermatozoa in biological fluids by particle counters requires elimination of competing background. This was accomplished by dissolving cell organelles and other lipid and proteinaceous material with sodium dodecyl sulfate to leave the sperm nucleus. Progress of selective dissolution of interfering background versus sperm nuclei was monitored by phase contrast microscopy. Rabbit semen was diluted in 2.5% sodium dodecyl sulfate (wt/vol) and then 1:1 (vol/vol) with .5 M sodium hydroxide. Suspensions were incubated, diluted 1:200 in .1 M sodium citrate-.1% Triton-X, and counted after 5, 10, and 20 min with a Coulter Counter. All treatment times resulted in similar mean counts, ranging from 367 to 369 X 10(6) sperm/ml. These means were slightly higher than the 350 X 10(6) sperm/ml for hemocytometer counts, but the correlation with hemocytometer counts was r greater than or equal to .98. Bull semen was diluted in either 1) citrate-Triton-X, 2) .25 M Tris-20% egg yolk, or 3) heated whole milk and further treated with 10% sodium dodecyl sulfate and .5 M sodium hydroxide. After 15 min, treated samples were diluted 1:200 in citrate-Triton-X and counted. Following treatment, estimated sperm concentration in the three diluents was not different from that of untreated sperm in citrate-Triton-X. The procedure was successfully applied to bull sperm diluted in milk and packaged in .5-ml French straws used routinely for artificial insemination.