Effects of follicular fluid and fetal bovine serum on in vitro maturation of bovine oocytes

Abstract

The aim of this study was to investigate the effect of bovine follicular fluid (BFF) collected from 2-4 mm and 4-6 mm in diameter of bovine follicles, and to optimize different concentration of fetal bovine serum (FBS) on in vitro maturation (IVM) of bovine oocytes. In the first experiment, bovine oocytes-cumulus-granulosa cells complexes (OCGs) were cultured in maturation medium supplemented with concentration of 15% BFF that was collected from 2-4mm and 4-6mm in diameter of bovine follicles. The results showed the maturation rate of group contains 15% BFF collected from 4-6mm in diameter of bovine follicles was significantly higher than BFF collected from 2-4mm in diameter of bovine follicles. In second experiment, the effects of maturation medium containing with 15% BFF collected from 4-6mm in diameter of bovine follicles were supplemented with different concentrations of FBS (0%, 1%, 5%, 10% and 15%) on in vitro maturation rate of bovine oocytes. The results indicated that bovine oocytes cultured with concentration of10% FBS reached to 78.00% of maturation rate and significantly higher than the other groups. The groups with concentration of 1% FBS and 5% FBS obtained the maturation rate was similar to the group of control (60.37%, 61.00%, and 60.90%, respectively). Whereas the group with concentration of 15% FBS had significantly decreased maturation rate to 48.08%.The next experiment was carried out to test the effect of different floating drop culture systems. In the first floating drop system, 150μl of culture medium was drop in culture dish and cover with mineral oil (control group, without floating). In the second floating drop system, 30μl of culture medium was drop into culture dish and cover with mineral oil, then add more 120μl culture medium in to the first drop (30-120 group). In the third floating drop system, 50μl of culture medium was drop into culture dish and cover with mineral oil, then add more 100μl culture medium in to the first drop (50-100 group). In the last floating drop system, 100μl of culture medium was drop into culture dish and cover with mineral oil, then add more 50μl culture medium in to the first drop (100-50 group). The results showed the maturation rates after 24 hours culture were significant lower in group of control (46.60%) than in 30-120 group (59.48%), 50-100 group (59.57%), and in 100-50 group (62.40%). In conclusion, this study provides evidence that BFF collected from 4-6mm in diameter of bovine follicle was supported the high rate of in vitro bovine oocytes maturation than BFF collected from 2-4 mm in diameter. The highest proportion of maturate bovine oocytes were obtained when OCGs were cultured in maturation medium contain 15% BFF collected from 4-6 mm in diameter of bovine follicles and supplemented with 10% FBS in a floating drop culture system. Key words: Oocytes-cumulus-granulosa cell complexes (OCGs), In vitro maturation (IVM), Bovine follicular fluid (BFF), fetal bovine serum (FBS, floating drop