134 EFFECT OF REPLACING FETAL BOVINE SERUM BY DIFFERENT GROWTH FACTORS IN THE PRE-IMPLANTATION DEVELOPMENT OF BOVINE EMBRYOS GENERATED BY IN VITRO FERTILIZATION

Abstract

Different culture systems have been studied that support pre-implantation development of bovine embryos up to the blastocyst stage. However, the use of chemically defined culture systems has been less studied. The objective of the present study was to evaluate the effect, in the developmental potential of in vitro-produced bovine embryos, of replacing fetal bovine serum (FBS) by different growth factors in the maturation and embryo culture media. In experiment 1, oocytes collected by aspiration of ovaries from a local slaughterhouse were matured in standard TCM-199 culture medium at 38.5°C, 5% CO₂, and saturation humidity. The effect of insulin-like growth factor 1 (100 ng mL^–1), epidermal growth factor (10 ng mL^–1), and fibroblast growth factor 2 (500 ng mL^–1) was evaluated at 24 h by the presence of a polar body after removal of cumulus-oocyte complexes. In experiment 2, oocytes matured in vitro in the presence of FBS were fertilized by co-incubation with commercial sperm (mL) for 18 h in standard fertilization medium (Fert-TALP). The presumptive zygotes were denuded and randomly allocated in a chemically defined culture medium based on KSOM supplemented with polyvinyl alcohol (PVA), fructose, and each of the growth factors listed previously. Undefined cultured medium was based on KSOM supplemented with 5% FBS. Embryos were cultured at 38.5°C in a mixture of gases and saturation humidity. Cleavage and blastocyst rates were recorded on Days 3 and 7, respectively. Analysis of variance was used to test for statistically significant differences between groups (P < 0.05) using Stat Graphics Plus 2 Software. In cases where statistically significant differences were observed, a multiple comparison test was run using Tukey's test. In experiment 1, a similar maturation rate was observed in all treatments relative to the undefined maturation medium (range = 88–91%). In experiment 2, no differences were observed in the cleavage (79, 87, 85, and 85%) and the blastocyst rates (24, 25, 26, and 30%) for the epidermal growth factor, insulin-like growth factor 1, fibroblast growth factor 2, and FBS treatments, respectively. In conclusion, we demonstrated that maturation of bovine oocytes can be achieved in chemically defined conditions by replacing FBS by each of the growth factors evaluated herein. Furthermore, chemically defined KSOM medium supplemented by any of these growth factors can generate a similar rate of blastocyst than the undefined medium containing FBS. Analyses are under way to evaluate the effect of completely defined culture conditions (maturation and embryo culture) on the pre-implantation development of embryos produced in the presence of these growth factors.