209 Supplementation of the in vitro maturation medium with Moringa oleifera extract: antioxidant effect on bovine oocytes

In vitro maturation (IVM) is a process that can be affected by the oxidative stress generated by the accumulation of reactive oxygen species (ROS) and the low concentrations of antioxidants in the culture media, which affects the quality of the oocytes. Moringa oleifera leaves have secondary metabolites, mainly phenolic compounds, that confer antioxidant properties. Therefore, its use has potential as a supplement in oocytes’ IVM, aiming to increase the success of the in vitro embryo production. Hence, the aim of this study was to evaluate whether supplementation with Moringa oleifera leaf extract during IVM improves the quality and competence of bovine oocytes. Ovaries were collected at a local abattoir and cumulus-oocyte complexes (n = 1311) were selected and matured in TCM-199-based medium supplemented with 0 (C), 50 (M1), 100 (M2), and 150 μg/mL (M3) of Moringa oleifera mature leaf extract. For positive control, maturation media was supplemented with 50 μg/mL of ascorbic acid. The rate of nuclear maturation and the levels of ROS and intracellular glutathione (GSH) in oocytes were evaluated by Hoechst 33342 staining solution (n = 161), 2,7-dichlorodihydrofluorescein diacetate staining (n = 109) and Cell Tracker Blue fluorescent stain (n = 111), respectively. Fluorescence intensities of oocytes were analysed by ImageJ software (Version 1.50v, National Institutes of Health) and normalised to control oocytes. Subsequently, oocytes matured (n = 930) were fertilised and the presumptive zygotes were cultured in vitro until Day 7, when the rate of blastocysts was assessed. Data were analysed in the Infostat Statistical Software (version 2018), and its normality verified through the Shapiro-Wilk test. Kruskal-Wallis analysis was performed for nonparametric data. Results are presented as percentage mean ± standard error of the mean (P < 0.05). ROS fluorescence levels in oocytes were significantly reduced (P < 0.05) with all concentrations of Moringa oleifera extract compared with control groups (M1: 0.52 ± 0.09, M2: 0.51 ± 0.09, M3: 0.43 ± 0.1, C: 1 ± 0.1, AA: 0.81 ± 0.09). GSH fluorescence levels were significantly reduced with the treatments M2 and M3 compared to control groups and M1 treatment (M1: 0.73 ± 0.07, M2: 0.59 ± 0.07, M3: 0.41 ± 0.04, C: 1 ± 0.08, AA: 0.93 ± 0.07). No significant difference was observed in the nuclear maturation or blastocyst rates. In conclusion, this study showed evidence that the extract of mature leaves of Moringa oleifera reduced the concentrations of ROS in bovine oocytes matured in vitro when supplemented in the maturation medium.