176 Effect of epigallocatechin-3-gallate on bovine oocyte in vitro maturation, fertilization, and development

Abstract

Epigallocatechin-3-gallate (EGCG) is a major ingredient of catechin polyphenols, and a strong antioxidant compound. Huang et al. (2018 Asian-australas. J. Anim. Sci.) reported that adding 50μM EGCG can improve the bovine oocyte maturation rate. In this research, we investigated the effect of EGCG supplementation on different periods in bovine IVF. Cumulus-oocyte complex (COC) collected from ovaries of slaughtered cows were cultured in maturation medium (20 to 30 oocytes per 100-µL droplet), which consisted of TCM-199 with Earle’s salts and 25mM HEPES supplemented with 10% (vol/vol) fetal bovine serum (FBS), 1µg mL−1 oestradiol, 0.02mg mL−1 FSH, and antibiotics at 38.5°C in a humidified atmosphere of 5% CO2 in air for 24h (in vitro maturation, IVM). After IVM, COC were fertilized in the fertilization medium (modified Brackett-Oliphant media supplemented with 10 µgmL−1 heparin, 10mM caffeine, and 3mg mL−1 BSA) for 6h using semen of one bull at final sperm concentration of 1×107 mL−1 (IVF). After IVF, COC were denuded and cultured in culture medium [CR1aa supplemented with 10% (vol/vol) FBS and antibiotics] at 38.5°C in a humidified atmosphere of 5% O2, 5% CO2, and 90%N2 for 8 days (in vitro culture, IVC). The EGCG was supplemented at 10, 25, 50, and 100M in IVM medium; 25 and 50 µM in IVF medium; and 50 and 100 µM in IVC medium. After 24h in IVM medium, COC were denuded by pipetting, fixed in 3:1 ethanol:acetic acid for 24h and then checked for nuclear and polar body by using aceto-orcein stain. After 18h in IVF, the pronucleus in zygote was fixed in 3:1 ethanol:acetic acid for 24h and checked by aceto-orcein staining. Embryo development was evaluated by counting the total number of embryos that had reached compacted morula by 6 to 8 days after IVF. Significant differences were analysed by chi-squared test and residual analysis. A P-value<0.05 was considered statistically significant. When EGCG was added to IVM, there was no significant difference of oocyte maturation rate between all concentrations (0v. 10v. 25v. 50v. 100 μM: 73.9% v. 56.7% v. 76.7% v. 72.7% v. 63.5%). When EGCG was added to IVF, there was no significant difference of fertilized rate (0v. 25v. 50 μM: 59.4% v. 73.7% v. 64.9%). When EGCG was added to IVC, there was no significant difference in development rate (0v. 50v. 100 μM: 26.2% v. 15.7% v. 22.0%). In this research, EGCG addition did not affect bovine in vitro fertilization.