205 Evaluating bovine in vitro oocyte maturation, H4K16 acetylation rates, and in vitro embryo production with nicotinic acid and resveratrol addition to the oocyte maturation medium of Angus breed cows

Oxidative stress may impair oocyte and embryo development competence, as well as epigenetic modifications that occur during oocyte maturation. Therefore, the aim of this study was to evaluate the effects of adding resveratrol (Re) and nicotinic acid (NA) to the IVM medium of bovine oocytes from Angus breed cows on oocyte IVM, histone 4 lysine 6 (H4K16) acetylation, as well as in vitro embryo production (IVEP). Bovine ovaries were obtained from the abattoir and transported to the laboratory. Oocytes were aspirated from 6–8 mm follicles and selected for IVM using six different media: (1) control (without additives), (2) 0.5 µM Re (Re), (3) 0.5 mM NA (0.5 NA), (4) 1 mM NA (1 NA), (5) Re plus 0.5 NA, and (6) Re plus 1 NA. The oocytes were matured for 24 h in 5% CO₂ at 38.5°C. After IVM, the oocytes (n = 669, 9 replicates) were denuded and stained with Hoechst 33342 to verify the presence of the metaphase plate and the extrusion of the first polar body. To evaluate the acetylation of H4K16 after maturation, the denuded oocytes were incubated with a rabbit polyclonal antibody against H4K16ac overnight. They were then washed and incubated with a goat antibody anti-rabbit conjugated with immunoglobulin G Alexa Fluor 488 for 1.5 h. The oocytes were finally stained with Hoechst 33342 to evaluate metaphase II and acetylation using a confocal microscope. In vitro embryo production was achieved by fertilizing the matured oocytes in vitro and culturing the resulting presumptive zygotes for 7 days. The hatching rates were recorded 7 days post-insemination (Day 7). A mixed model was used to analyse the maturation rate per treatment, considering the treatment effect as fixed and the replicates as random. For IVEP and oocyte acetylation characteristic analysis, analysis of variance was employed, considering treatment and replicate effects. The means were compared using the Tukey test. Normality and homoscedasticity were assessed using the Shapiro-Wilk and Bartlett tests, respectively. The significance level was set at 5%. The analyses were performed using R 4.2.1 software (R Core Team, 2022). No differences were observed in IVM (mean: 75.2%), H4K16 acetylation rates of matured oocytes (mean: 49.5%), and IVEP rate (mean: 31%) among the treatments (P > 0.05). However, the highest hatching rate in relation to cleavage was observed with 0.5 NA (6.9%), Re plus 1 NA (3.8%), Re plus 0.5 NA (1.8%), and Re (2%) compared with the control (0%, P < 0.05). In conclusion, the addition of NA and Re, either in combination or individually, to the IVM medium, particularly 0.5 NA, increased the hatching rate and may therefore be beneficial for improving bovine IVEP in Angus breed cows.