336 SHEEP OOCYTES MATURED IN A SIMPLEX MEDIUM (HECM-1), AN ANSWER TO IN VITRO FERTILIZATION

Abstract

In vitro maturation and in vitro fertilization (IVM and IVF) of mammalian oocytes still show unsatisfactory results when applied to the study of embryo development. This is probably due to inadequate information about the use of media components and supplements for oocyte maturation and to a discrepancy between results obtained by focusing strictly on oocyte maturation and those that are focused on IVF. A conventional medium that provides adequate results in studies of oocyte maturation (TCM-199) contains hypoxanthine, phosphate ions, and glucose, all known to inhibit embryo development in vitro in some species. In contrast, it has been shown that a simpler medium (HECM-9) increases embryo development in bovine although its use for oocyte maturation has not been defined. This medium contains taurine (an amino acid that reduces intracellular peroxide content) and is supplemented with polyvinyl alcohol (PVA) instead of using protein components, making it a simple defined medium that reduces variability in embryo development. Adding sodium panthothenate to media also confers cell protection against reactive oxygen species. Finally, supplements such as epidermal growth factor (EGF) increase the number of oocytes that complete maturation (Metaphase II, MII) and facilitate embryo development. An adequate combination of our knowledge about in vitro maturation and fertilization of oocytes, together with the requirements for embryo development, is important for the preparation of culture media to study regulatory mechanisms for embryo development and to increase the number of viable and normal term individuals. In this study we compared the effects of HECM-9 (containing panthothenate) vs. TCM-199 (both media supplemented with PVA, EGF, and FSH/LH) on the integrated processes involving IVM and IVF. No significant differences were found between the results obtained with these media in relation to oocyte maturation (65% MII for HECM-9 vs. 71% for TCM-199); however, those oocytes matured in HECM-9 showed a highly significant difference in in vitro fertilization using a conventional IVF medium (SOFm) (25% in HECM-9 vs. 6% in TCM-199). Maturation results were relatively low but in accordance with those reported by other groups, whereas IVF results are lower than those reported in the literature, perhaps because we have been using frozen and thawed samples and do not have complete control over the sperm quality. At present, we are extending our investigation using fresh semen samples.