207 The effects of vanillic acid supplementation during oocyte maturation on in vitro fertilisation and embryonic development in pigs

In vitro fertilisation (IVF) typically has low success rates in pigs, partially due to the negative effects of oxidative stress on both the sperm and egg. Antioxidants, such as vanillic acid, are known to have protective effects on the oocyte in terms of maturation. The purpose of this study was to determine the effects of the vanillic acid supplementation during oocyte maturation on reactive oxygen species (ROS) generation, IVF, and early embryonic development success rates in pigs. Oocytes were matured for 40–44 h supplemented with vanillic acid (0, 3, 6, 12 mg/mL; Sigma-Aldrich Co.). After maturation, oocytes were denuded and either evaluated for ROS production (n = 183, r = 3) by measuring the fluorescent intensity from the oxidation of 2′,7′-dichlorodihydrofluorescein diacetate or fertilised using frozen-thawed boar semen (1.0 × 10⁵ sperm cells/mL) and co-incubated for 6–8 h followed by embryo culture for 144 h. After IVF, presumptive zygotes were fixed, permeabilised, and stained with bisBenzimide H 33342 trihydrochloride and evaluated for penetration, polyspermy, and pronucleus formation rates (n = 390), and embryos were evaluated for cleavage and blastocyst formation (n = 529) at 48 h and 144 h post-IVF, respectively. Fluorescent intensities of individual oocytes were analysed using a GLM and means were compared using l.s.d. The IVF and embryo development data were reported as the percent observed/drop and mean percentages using a GLM and were analysed with differences being compared using Tukey’s Test. Supplementation of 6 mg/mL vanillic acid or 12 mg/mL vanillic acid reduced (P < 0.05) the amount of ROS (0.68 ± 0.06, 0.63 ± 0.06; respectively) compared with no vanillic acid supplementation (1 ± 0.06). Vanillic acid supplementation did not have any effect on oocyte penetration rates, however both 6 mg/mL and 12 mg/mL vanillic acid decreased (P < 0.05) polyspermic penetration rates (23.79 ± 0.02, 24.03 ± 0.02; respectively), and increased (P < 0.05) pronuclear formation (83.76 ± 0.02, 24.03 ± 0.02; respectively). Supplementation of vanillic acid had no effect on cleavage rates (78.69 ± 0.02) by 48 h post-IVF or blastocyst formation (31.18 ± 0.03) by 144 h post-IVF. Based on these results, supplementation of 6 mg/mL vanillic acid or 12 mg/mL vanillic acid during oocyte maturation reduces the amount of ROS formed in vitro and improves IVF success rates.