The effect of stage of maturation of the germinal vesicle of porcine oocytes at the time of in vitro maturation on subsequent developmental competence was examined. A large variation exists in the germinal vesicle morphology of oocytes at the time of collection of cumulus-oocyte complexes (COCs) and after culture in the absence of dibutyryl cAMP (dbcAMP) for 20 h. However, the morphology of the germinal vesicle was synchronized to a specific stage after culture in the presence of 1 mM dbcAMP for 20 h. There was no difference in germinal vesicle breakdown rate (total mean, 75.0 +/- 5.4%) or in maturation rate (total mean, 82.1 +/- 2.1 %) at 28 and 44 h of culture, respectively. However, differences in meiotic progress of oocytes were observed (p < 0.05) at 36 h of culture when COCs were exposed to dbcAMP for the first 20 h of maturation, as compared to controls. The incidence of embryos that developed to the blastocyst stage after in vitro fertilization was higher (p < 0.05) when COCs were exposed to dbcAMP (21.5 +/- 2.5%) as compared to controls (9.2 +/- 1.6%). After transfer of experimental embryos to four recipient gilts, the three pregnant recipients delivered 19 live piglets. These results indicate that exposure of COCs to dbcAMP for the first 20 h of culture for maturation increases the homogeneity of oocyte nuclear maturation and improves the efficiency of in vitro production of swine embryos.

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The effect of hormone supplements on cytoplasmic maturation in vitro was examined by incubating oocyte-cumulus complexes, in a medium with PMSG (10 iu ml-1), hCG (10 iu ml-1) and oestradiol (1 microgram ml-1) for various periods and then transferring them to medium without added hormones for the remainder of the maturation period. Exposure of oocyte-cumulus complexes to hormone supplements for 2 h improved only germinal vesicle breakdown and maturation rates compared with complexes not exposed to added hormones. The removal of hormone supplements at 20 h after the start of culture enhanced the ability of oocytes to form male pronuclei 10 to 12 h after insemination. Further, the effects of transfer of intact and oocytectomized oocyte-cumulus complexes to hormone-free medium at 20 h on cumulus expansion were examined. The diameter and morphology of the intact oocyte-cumulus complexes were improved after the removal of oocyte-cumulus cell complexes from hormonal exposure. The responses of oocytectomized oocyte-cumulus complexes to hormone were similar to those of intact oocyte-cumulus complexes with the exception of corona radiata expansion. The results suggest that the removal of hormone supplements from maturation media at 20 h after culture enhanced cytoplasmic maturation and cumulus expansion. Further, cumulus expansion does not appear to depend on intercellular communication between cumulus cells and oocytes. Oocytectomy did influence expansion of the corona radiata during culture.

A suitable chemically defined culture medium for 1-cell rat embryos (mR1ECM) was modified to obtain sperm penetration, and the developmental competence of oocytes fertilized in the medium was compared to that of oocytes fertilized in a traditional medium, modified Krebs-Ringer bicarbonate medium (mKRB). Sperm penetration was not observed when polyvinyl alcohol was replaced with BSA in mR1ECM (mR1ECM-BSA); the incidence was improved only when the osmolarity in mR1ECM-BSA was increased to that in mKRB (310 mOsm) by addition of NaCl. The proportion of oocytes penetrated in mR1ECM-BSA with NaCl increased (71.6 +/- 6.9%), which was not different compared to that in mKRB (76.7 +/- 13.7%). High incidences of sperm penetration (88.8 +/- 4.1% to 93.1 +/- 5.1%) were also observed when NaCl concentration in mR1ECM-BSA was increased from 76.7 mM to 100-130 mM. The incidence of embryos developing to the morula and blastocyst stages was higher when fertilized in mR1ECM-BSA containing 110-130 mM NaCl (91-94%) than in mKRB (70%). A total of 5 offspring were obtained after transfer of the morulae and blastocysts (69 embryos/7 females). These results demonstrate that a high developmental ability of rat embryos to the blastocyst stage is attained when the embryos have been fertilized in mR1ECM-BSA containing 110-130 mM NaCl and then cultured in mR1ECM.