Bovine oocytes matured in vitro were stimulated by a single pulse of direct current (DC), then cultured for 0.5-6 h and evaluated by light (LM) and transmission electron microscopy (TEM). At the light microscopic level, the beginning of oocyte chromatin decondensation was first observed 3 h post-activation (14%). A well-developed pronucleus with evenly granulated nucleoplasm surrounded by nuclear membrane was found in 12, 61 and 81% oocytes at 4, 5 and 6 h post-activation, respectively. The TEM evaluation revealed that nuclear membrane vesicules were first visible at 0.5 h post-activation and became even more prominent at 1 h. Based on these observations, it is concluded that a nuclear membrane starts to form immediately after oocyte activation, while a well-developed pronucleus appears at 4-6 h.

Using a monoclonal antibody (LIS-4) to an immunosuppressive factor isolated from boar vesicular gland secretion it was determined that this gland secretes a tissue-specific immunosuppressive molecule that is absorbed onto the acrosome of spermatozoa during ejaculation. Absorption of the immunosuppressive molecule onto murine embryos at the 2-, 4-, 8-cell, morula and blastocyst stages in vitro was evaluated by indirect immunofluorescence. In vivo absorption was detected on the zona pellucida of murine embryos obtained from oviducts injected with the immunosuppressive molecule. Immunofluorescence revealed that the immunosuppressive molecule was not absorbed onto murine embryos after solubilization of the zona pellucida. There was no effect of the antibody to the immunosuppressive molecule on the ability of boar spermatozoa to penetrate the porcine zona pellucida.

Pig zona-free and zona-intact ova were successfully penetrated in vitro by ejaculated boar spermatozoa. The oocytes were obtained from antral follicles (2-5 mm in diameter) of slaughtered gilts and matured in M-1 99 with porcine follicular fluid (50%) and follicle stimulating hormone (FSH) (0.25 IU ml-1) for 44 h at 38.5-degrees-C. In vitro fertilisation (IVF) was carried out with a spermatozoa-rich fraction of a fresh boar ejaculate which had been incubated in M-199 supplemented by 10% inactivated bovine serum. The penetration rate of zona-free oocytes was dependent on the duration of sperm incubation. IVF of zona-intact ova was designed to estimate the optimal spermatozoa concentration for insemination. The concentration of (1-1.5) X 10(6) spermatozoa ml-1 gave the best fertilisation results (87%), whereas the concentration below 1 X 10(6) spermatozoa ml-1 resulted in a lower fertilisation rate (62%) but in a higher number of monospermic eggs (36%) and male pronucleus formation (80%).

Studies were conducted to compare viability of immature and mature equine and bovine oocytes vitrified in ethylene glycol, Ficoll using open-pulled straws. Oocytes from slaughterhouse ovaries (N=50/group) with >2 layers of compact cumulus cells were vitrified immediately after collection (immature groups) or vitrified after 36 to 40 (equine) or 22 to 24 (bovine) h of maturation (mature groups). Immature oocytes were matured after thawing. Before vitrification, oocytes were exposed to TCM-199 + 10 PCS + 2.5 M ethylene glycol + 18% Ficoll + 0.5 M sucrose (EPS) for 30 sec and then to 5 M ethylene glycol in EFS for 25 to 30 sec at 37°C. Oocytes were loaded into straws in ~2 μL of cryoprotectant and plunged directly into LN2. Warming straws and dilution of cryoprotectant was at 37°C in TCM-199 + 10% FCS + 0.25 M sucrose for 1 min and then TCM-199 + 10% FCS + 0.15 M sucrose for 5 min. Non-vitrified oocytes undergoing the same maturation protocol for both species were used as controls. Oocytes were stained with orcein for nuclear maturation and live/dead status was determined using Hoechst 33342.