Establishment of pregnancy in cattle by reconstituted eggs produced by fusion of an in vitro matured oocyte and a blastomere from a 16-cell embryo produced in vivo

Abstract

The techniques of in vitro maturation (IVM) and in vitro fertilization (IVF) of bovine oocytes, with development of the resulting embryos to advanced preimplantation stages in vitro, have gained widespread popularity in recent years because of the potential for obtaining information about regulatory mechanisms, and for inexpensively mass-producing embryos for research or for transfer. However, the efficiency of the techniques (blastocyst yield) is unsatisfactory, due to inadequate information about the requirements of bovine embryos for development in culture, and of oocytes for achieving normal maturation. An experiment was designed to determine effects of using different media for bovine oocyte maturation on subsequent embryo development. Five of seven media tested on oocyte maturation resulted in higher levels of fertilization and/or first cleavage division. In addition, the data indicated that culture conditions for oocyte maturation also affect development to the morula/blastocyst stages. A second experiment evaluated culture conditions for embryo development, using two media (HECM-1 and TCM-199) with or without supplementation by serum and/or oviduct cell conditioning, in a 2×2×2 factorial design. Neither serum supplementation nor conditioning nor the two together increased morula/blastocyst formation (35–46% in all treatments). Serum had a biphasic effect, suppressing the first cleavage division but enhancing morula compaction. No block was observed at the 8- to 16-cell stage with TCM-199 + serum, contrary to many other studies. The data support the hypothesis that the reported stimulation of bovine IVM/IVF embryo development by somatic cell conditioning is due to removal of inhibitory influences from the culture environment. The ability to support development of IVM/IVF embryos to the morula and blastocyst stages in defined media (i.e., without serum or somatic cell conditioning) will help to elucidate the metabolic and nutritional requirements of bovine oocytes and embryos, and to formulated improved culture media. These advances will increase the utility of bovine IVM/IVF for both basic and applied resarch, and for commercial embryo production.