A study was conducted to evaluate the potential of rescuing immature oocytes from the ovaries of an endangered wild bovid, the gaur (Bos gaurus). Recovered, immature gaur oocytes (n = 59) placed in culture were evaluated for: (1) nuclear maturation after 22 h of culture, (2) fertilization with either thawed homologous (gaur) or heterologous (Bos taurus) spermatozoa 18 h after insemination and (3) embryo development. Gaur oocytes (n = 6) evaluated by fixation and staining at 22 h had all matured to metaphase II in vitro. Insemination of gaur oocytes in vitro resulted in normal fertilization (defined as the presence of spermatozoa head or two pronuclei) and embryo development to the two- and four-cell stage of 53.6% (15 of 28) and 50.0% (9 of 18), respectively, using homologous spermatozoa. The incidence of normal fertilization of in vitro matured (IVM) gaur oocytes with heterologous spermatozoa was 53.8% (7 of 13). Insemination of domestic cow oocytes in vitro resulted in normal fertilization and embryo development of 41.7% (45 of 108) and 60.0% (12 of 20), respectively, using heterologous spermatozoa. Two of four gaur embryos (50%) developed to the blastocyst stage by day 7. Embryo transfer of these two conspecific gaur blastocysts into two Holstein recipients resulted in one confirmed pregnancy. One live-born calf was delivered by Caesarean section 308 days after embryo transfer. These results demonstrate the potential of combined IVM and IVF for recovering immature germplasm from an endangered species. Specifically, immature gaur ovarian oocytes are capable of in vitro maturation and fertilization with thawed homologous spermatozoa.(ABSTRACT TRUNCATED AT 250 WORDS)

Bovine ovaries (paired by cow) were obtained from a local abattoir and cumulus oocyte complexes were aspirated within six hours of slaughter. Two methods for activation [(1) calcium ionophore (ionomycin) alone (n = 191); and (2) ionomycin followed by the protein synthesis inhibitor cycloheximide (n = 207)] were evaluated for production of bovine parthenogenones. Activation with ionomycin alone resulted in a development rate of 33%, while activation with ionomycin and cycloheximide sequentially resulted in a development rate to two-cell stage of 49%. A procedure was developed to expedite accurate evaluation of activated oocytes for uniformly haploid development. Uniformly haploid parthenogenones that cleaved at least once in four days of in vitro culture were individually prepared for genetic analysis. Three techniques: (1) phosphate buffered saline; (2) TL-HEPES with 0.2% ovine serum albumin; and (3) TL-HEPES with 0.2% polyvinyl pyrrolidone were compared to harvest parthenogenones for genetic analysis. The only effective method that did not create spurious results during later genetic analysis was TL-HEPES with 0.2% polyvinyl pyrrolidone. Based on the results of this study, we estimate that an average of 5-7 uniformly haploid bovine parthenogenones can be realized from each donor (using pairs of ovaries). These parthenogenones, when maintained as family units, will be valuable for accomplishment of female-specific genetic linkage analysis.

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