Thymol Regulates the Expression of Antioxidant-Related Messenger RNAs and Improves Bovine Secondary Follicles Development and Antrum Formation In Vitro

This study investigated the effects of 1, 10, or 100 μg/mL thymol on bovine secondary follicles cultured in vitro for 18 days at 38.5 °C with 5% CO2. After culture, the following parameters were evaluated: (1) the follicular growth, antrum formation, viability, and ultrastructure, (2) the levels of malondialdehyde, reduced glutathione, and nitrite in the culture medium, and (3) mRNA levels of catalase (CAT), glutathione peroxidase 1 (GPX1), superoxide dismutase (SOD), peroxiredoxin 6 (PRDX6), proliferating cell nuclear antigen (PCNA), and growth differentiation factor 9 (GDF-9). The results showed that 10 μg/mL thymol increased cell viability, antrum formation and follicular development. At day 18, nitrite levels were higher in medium of follicles cultured with 10 μg/mL thymol than those found in control medium alone or with 1 μg/mL thymol. Gene expression analysis revealed that 10 μg/mL thymol increased catalase and glutathione peroxidase 1 mRNA levels, while the expression of other transcripts was unaffected. Transmission electron microscopy showed that granulosa cells exhibited rounded nuclei, elongated mitochondria, and sparse cytoplasmic vacuoles in all treatments, except for those cultured in control medium that had signs of degeneration. In conclusion, 10 μg/mL thymol increased mRNA expression of catalase and glutathione peroxidase 1, stimulated antrum and preserved granulosa cells of bovine secondary follicles in vitro.