Effects of Anethole on Meiotic Resumption and Oxidative Balance During in Vitro Maturation of Ovine Oocytes

The aim of this study was to evaluate the effect of anethole addition to the ovine oocyte in vitro maturation (IVM) medium on viability and chromatin configuration, intracellular production of reactive oxygen species (ROS), enzymatic activity of superoxide dismutase (SOD) and catalase (CAT), as well as the levels of reduced thiols in IVM medium, in addition to its antioxidant potential (DPPH and ABTS). Cumulus-oocyte complexes (COCs) were randomly matured in IVM medium in the absence (control group, CTRL) or presence of anethole at concentrations of 30 (AN30), 300 (AN300), and 2000 μg/mL (AN2000). COCs were matured in groups under mineral oil at 39°C in a humidified atmosphere containing 5% CO₂ in air for 24 h. At the end of IVM, part of the COCs was used to assess viability and chromatin configuration using Hoechst 33,342. The remaining COCs were used to assess intracellular ROS levels using the fluorescent probe H2DCFDA. Furthermore, the IVM medium was collected to evaluate antioxidant status (SOD, CAT, and reduced thiols). Finally, the antioxidant potential of different anethole concentrations was evaluated using DPPH and ABTS assays. It was observed that AN300 promoted higher meiotic resumption rates compared to the other treatments (p < 0.05) and a lower rate of oocyte degeneration compared to AN30 (p < 0.05). However, regarding the proportion of oocytes at MII, it was observed that only AN2000 was lower than AN300 (p < 0.05). Moreover, AN30 resulted in lower ROS levels compared to AN300 and AN2000 (p < 0.05). SOD activity was lower in AN2000 compared to the other treatments (p < 0.05). AN2000 also showed the highest antioxidant capacity in the DPPH and ABTS assays among the anethole concentrations, although it was lower than the control group. In conclusion, anethole supplementation during IVM positively affected oocyte meiotic progression in sheep.