The effect of sperm concentrations during preincubation and at insemination on fertilization of rat eggs in vitro

Rat epididymal spermatozoa, at a concentration of 40-65 X 10(4) cells/ml, were preincubated for 5-5, 5 hr and then added to eggs from superovulated, immature rats. By 10-12 hr after insemination, 86% of the eggs had undergone fertilization, but many of them (41%) were polyspermic. When sperm were preincubated at almost the same concentration and for the time stated above, but then diluted to 1-3 X 10(4) cells/ml before insemination, the number of fertilized eggs and the incidence of polyspermy were both reduced, being 56% and 5%, respectively. When a sperm concentration of 1-3 X 10(4) cells/ml was used both during preincubation and at insemination, the fertilization rate of 60% or more, with polyspermy not in excess of 10%, a sperm concentration of 30-100 X 10(4) cells/ml during preincubation and then diluted to approximately 1-3 X 10(4) cells/ml at insemination appears to be optimal.