Abstract
Two in vitro culture methods, an oil covered culture method and an open culture method, were used to determine the effects of fetal calf serum (FCS) and epidermal growth factor (EGF) on the development of bovine oocytes fertilized in vitro. Immature bovine oocytes were cultured to maturity as follows, fertilized in vitro and cultured by standard procedures. The first experiment compared the open culture method with the oil covered method. The open culture method involved using 500 μl of medium per well. The oil covered method involved using 100 μl droplets of medium covered by paraffin oil. In this experiment, 25 mM HEPES buffeted TCM-199 medium (TCM-199) supplemented with FSH (2 μg/ml), E2 (1 μg/ml) and FCS (10%) was used as the maturation medium. The differences between the two methods were not significant in both the cleavage rate (65.4 vs. 64.1%) and the development rate into blastocysts (22.8 vs. 17.6%). In the second experiment, development of immature oocytes was compared using three media, A) TCM-199 + FSH (2 μg/ml)+ E2 (1 μg/ml), B) TCM-199 + FSH + E2 + FCS (10%) and C) TCM-199 + FSH + E2 + EGF (10 ng/ml), and cultured using the open culture method. Neither the cleavage rate (68.9, 67.4 and 73.1%) nor the development rate (23.0, 22.7 and 25.9%) was significantly different among the three media. The oocytes matured in the serum-free medium can develop as well as oocytes matured in the medium supplemented with the serum. The development rate of oocytes matured in a serum-free medium in combination with the open culture method was not inferior to oocytes matured on the conventional system using a medium supplemented with serum and covered with oil. It appears from the present results that the serum-free medium under the open culture system is stable and can be used for mass production of bovine embryos.
