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A rapid, sensitive method for sex determination which uses nested, allele-specific amplification of bovine zfx and zfy was developed. Accuracy of the sex determination assay was verified with lymphocyte-derived DNA from 24 cattle of known gender. Reliability and sensitivity of the assay in embryonic sex determination were examined by typing duplicate bovine embryo biopsies of 2–8 cells. Sex determination was possible in 38 of 40 biopsies, and agreement between biopsy assays occurred in 17 of 18 cases. Applicability of the sex determination assay was further verified by examining gender of 12 fetuses derived from sexed embryos. Embryonic and fetal gender determinations agreed in 11 of 12 cases with the twelfth yielding an ambiguous result from fetal ultrasound examination.
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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 Caesarian 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. The resulting embryos are capable of advancing to blastocysts in culture and of producing live-born offspring after embryo transfer.
