This research was carried out to define in vitro conditions that would enable ejaculated rabbit sperm to fertilize ova in vitro. During 20 min exposure of sperm cells to defined media of increasing NaCl concentrations, progressive removal or alteration of sperm surface seminal plasma antigenic components was initiated and completed as reflected by an immunological assay at approximately 300 and 380 mOsm/kg, respectively. Hypertonic (380 mOsm/kg) medium effected complete removal or alteration of these components as determined by the immunological assay during the first 5 min of sperm treatment. Isotonic (305 mOsm/kg) treatment was shown to have effected removal or alteration of some but not all of the antigenic sperm coating seminal plasma components detectable by this means during 20 min of exposure.

Sperm within the perivitelline space, presence of pronuclei, and 2- and 4-cell cleavage stages were taken as evidence for fertilization. None of 34 ova incubated for 27 h in vitro with once-washed sperm showed evidence of fertilization. Following treatment of sperm with hypertonic (380 mOsm/kg) medium prior to in vitro insemination, proportions of ova showing evidence of fertilization ranged from 8.1 percent (12 to 148 ova) to 72.2 percent (52 of 72 ova); and, following treatment of sperm with isotonic (305 mOsm/kg) medium, fertilization of ova ranged from 0 percent (0 to 19 ova) to 73.9 percent (51 of 69 ova) when results were considered according to individual male sperm donors. No large differences were found by the immunological assay that could be linked to variability of fertilizing ability of sperm from different bucks. Differences in metabolic characteristics of sperm cells was suggested as a likely reason for differences in fertilizing ability of sperm from different bucks.

In experiments using sperm and ova from the same sources, no differences in fertilization results were apparent following 305 and 380 mOsm/kg sperm treatments. Initiation, but not completion, of removal or alteration of sperm surface seminal plasma antigenic components, then, was a necessary prerequisite to in vitro insemination for successful achievement of fertilization in vitro. Sperm penetration into ova occurred in some cases before 7–10 h after insemination (4 of 54 ova penetrated), but actively motile sperm were found within the perivitelline space of other ova as late as 25 h after insemination.

One hundred and seventy-six 2- and 4-cell stage embryos were transferred to 14 recipient does. Twenty-four embryos (13.1 percent) implanted in 6 recipients but most were resorbed. Three embryos resulting from in vitro fertilization with in vitro capacitated ejaculated rabbit sperm were carried successfully to term by 2 recipients and 2 of the offspring were males. This provides documentation for the authenticity of in vitro capacitation of ejaculated sperm of a mammalian species and represents the initial successful combination of in vitro sperm capacitation with in vitro fertilization and embryo transfer in the rabbit.