Rutin can replace the use of other antioxidants in culture medium for sheep isolated secondary follicles

Rutin acts as a potent antioxidant in different cells types (KAUR and MUTHURAMAN, Life Sciences, 150, 89-94, 2016). However, there is no information about the effect of the antioxidant rutin on the in vitro culture of ovine secondary follicles. Thus, the aim of this study was to evaluate the effect of rutin as the only antioxidant added to the base medium on the in vitro culture of ovine isolated secondary follicles. Sheep (n = 90) secondary follicles (n = 55-61), approximately 200 µm, were individually cultured in 100 μL drops, during 12 days, at 39°C, in α-Minimal Essential Medium (α-MEM) supplemented with 3.0 mg/mL BSA, 10 ng/mL insulin, 2 mM glutamine and 2 mM hypoxanthine (antioxidant free-medium, called α-MEM-) or in this medium also added by 5.5 μg/mL transferrin, 5.0 ng/mL selenium and 50 μg/mL ascorbic acid (α-MEM+). To evaluate the effect of rutin, different concentrations of this antioxidant (0,1; 1 ou 10 µg) were added to the different base media, with or without antioxidant (α-MEM- or α- MEM+). All chemicals were purchased from Sigma Chemical Co. (St. Louis, USA). At each 6 days, normal follicles, antrum formation and follicular diameter were analysed. At the end of culture, the percentage of fullygrown oocytes (oocytes >110 μm) was evaluated. In treatments that had the best results of morphology, other parameters were analyzed, such as follicular viability through the fluorescent markers calcein-AM and ethidium homodimer 1, and intracellular levels of reactive oxygen species (ROS), glutathione (GSH) and active mitochondria. The rates of normal follicles, antrum formation, viability and oocytes >110 μm were compared by Qui-square test. Data from follicular diameter, growth rate, ROS, GSH and active mitochondria levels were submitted to ShapiroWilk followed by Kruskal-Wallis; data from diameter and growth rate were compared by Student Newman Keuls (P 0.05) in the diameter and growth rate. Moreover, α-MEM+ alone and α-MEM- added by 0,1 µg rutin showed similar (P > 0.05) percentages of follicular viability, antrum formation, fully-grown oocytes, levels of ROS and active mitochondria. However, treatment α-MEM- added by 0,1 µg rutin showed higher GSH levels (157 pixels/oocyte) than α-MEM+ (110 pixels/oocyte). In conclusion, at a concentration of 0,1 µg, rutin can be used as the only antioxidant present in base medium for in vitro culture of ovine secondary follicles, maintaining follicular viability and increasing GSH levels.