PSIV-11 The Effects of Vanillic Acid Supplementation During in Vitro Oocyte Maturation in Pigs

Abstract

Abstract In vitro fertilization (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 vanillic acid supplementation during porcine oocyte maturation on reactive oxygen species (ROS) generation, IVF, early embryonic development success rates, membrane lipid peroxidation, and catalase activity. Oocytes were matured for 40 to 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, membrane lipid peroxidation (n = 360), catalase activity (n = 724), or fertilized using frozen-thawed boar semen (1.0 x 105 sperm cells/mL) and co-incubated for 6 to 8 h followed by embryo culture for 144 h. After IVF, presumptive zygotes were fixed, permeabilized, 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 indicating ROS of individual oocytes, lipid peroxidation, catalase activity, and were analyzed using GLM, and means were compared using LSD. The IVF and embryo development data were reported as the percent observed/drop, and mean percentages, and means were analyzed using a GLM with differences being compared using Tukey’s test. Supplementation of 6 g/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, 84.80 ± 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. Supplementation of 6 mg/mL vanillic acid decreased (P < 0.05) membrane lipid peroxidation (0.46 ± 0.06) and catalase activity (0.01 ± 0.01) compared with no vanillic acid supplementation (0.79 ± 0.06, 0.03 ± 0.01; respectively). 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. Supplementation of 6 mg/mL decreases catalase activity and membrane lipid oxidation following oocyte maturation.