215 Effects of serum-free maturation medium and resveratrol supplementation on ovine oocyte maturation and quality

Oocyte in vitro maturation (IVM) allows for rescuing immature oocytes to be used for in vitro production of embryos. However, IVM is lagging behind in vivo maturation regarding post-maturation developmental competency. A downside to relying on IVM is the inclusion of animal serum, which introduces confounding variables and may contribute to growth abnormalities. Additionally, the effects of resveratrol IVM supplementation on ovine oocyte condition are of interest due to observed improvements in matured oocyte quality and embryonic development of several species. This study aimed to investigate the effect of supplementation of resveratrol in IVM medium with or without serum on oocyte maturation and oocyte quality. Sheep cumulus–oocyte complexes (COCs) were incubated in one of four IVM conditions; in-house IVM medium (CTRL, M199), CTRL with 1 mM resveratrol (CT-RES), BO-IVM medium (BO, serum-free medium from IVF Biosciences), and BO with 1 mM resveratrol (BO-RES) for 21 h. After maturation, COCs from each group were denuded and examined for nuclear maturation by observation of an extruded polar body. To assess the quality of oocytes, matured oocytes were analysed for glutathione (GSH) and reactive oxygen species (ROS) by incubating in 20 mM 4-chloromethyl-6,8-difluoro-7-hydroxycoumarin (CellTracker Blue CMF2HC) and 10 mM 2′,7′-dichlorodihydrofluorescein diacetate (H2DCFDA) for 25 minutes, respectively. Stained oocytes were imaged by a fluorescent microscope. Fluorescence intensity was analysed by ImageJ software to quantify oocyte and background fluorescence. Corrected oocyte fluorescence intensity was obtained by subtracting out the average background fluorescence. A generalized linear model was used to analyse maturation rates and the levels of ROS and GSH in the oocytes from different IVM groups (Jamovi). All the results were represented as mature/total; mean ± s.d. At least 4 replicates were performed for each experiment. There were no significant differences in the maturation rate between the control medium and commercial serum-free medium (255/352; 73.7 ± 10.1%; vs 278/362; 75.8 ± 6.0%; P > 0.05). Moreover, compared with CTRL and BO groups, no differences were observed in maturation in both resveratrol supplemented groups (CT-RES: 177/262; 69.5 ± 8.8% and BO-RES: 255/355; 71.6 ± 12.5%; P > 0.05). The GSH level was significantly lower in the CTRL group than that in the BO group (P < 0.001), but there were no differences observed between groups with or without resveratrol supplementation. For ROS, there were no significant differences within all four groups. In conclusion, compared with standard serum-containing maturation medium, BO-IVM commercial medium increases oocyte GSH levels but does not decrease ROS or improve the maturation rate. In future studies, the use of serum-free media and resveratrol supplementation in IVM will be investigated for their effects on embryo development.