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 50 μM 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 25 mM HEPES supplemented with 10% (vol/vol) fetal bovine serum (FBS), 1 µg mL⁻¹ oestradiol, 0.02 mg mL⁻¹ FSH, and antibiotics at 38.5°C in a humidified atmosphere of 5% CO₂ in air for 24 h (in vitro maturation, IVM). After IVM, COC were fertilized in the fertilization medium (modified Brackett-Oliphant media supplemented with 10 µgmL⁻¹ heparin, 10 mM caffeine, and 3 mg mL⁻¹ BSA) for 6 h 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 100 M in IVM medium; 25 and 50 µM in IVF medium; and 50 and 100 µM in IVC medium. After 24 h in IVM medium, COC were denuded by pipetting, fixed in 3:1 ethanol:acetic acid for 24 h and then checked for nuclear and polar body by using aceto-orcein stain. After 18 h in IVF, the pronucleus in zygote was fixed in 3:1 ethanol:acetic acid for 24 h 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 (0 v. 10 v. 25 v. 50 v. 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 (0 v. 25 v. 50 μM: 59.4% v. 73.7% v. 64.9%). When EGCG was added to IVC, there was no significant difference in development rate (0 v. 50 v. 100 μM: 26.2% v. 15.7% v. 22.0%). In this research, EGCG addition did not affect bovine in vitro fertilization.