Supplemented Morus nigra extract-based medium associated with FSH enables the survival and growth of isolated ovine secondary ovarian follicles.

The effects of Morus nigra ethanolic extract, without or with addition of supplements associated or not with FSH, on in vitro culture of ovine secondary follicles were evaluated. In experiment 1, isolated secondary follicles were cultured for 12 days in α-MEM alone (control) or in different concentrations of M. nigra extract (MN 0.025; 0.05 or 0.1 mg/ml). In experiment 2, culture media were α-MEM supplemented with BSA, insulin, transferrin, selenium, glutamine, hypoxanthine and ascorbic acid (α-MEM ) or this medium associated with FSH (α-MEM  + FSH), or 0.1 mg/ml M. nigra without supplements (MN 0.1) or supplemented (MN 0.1 ) without or with FSH (MN 0.1  + FSH). In experiment 1, 0.1 mg/ml M. nigra showed the highest percentages (p < .05) of normal follicles and fully grown oocytes, besides a higher follicular diameter than α-MEM and other M. nigra extract concentrations. Moreover, MN 0.1 showed lower (p < .05) glutathione (GSH) levels and similar (p > .05) mitochondrial activity compared to α-MEM. In experiment 2, MN 0.1  + FSH showed similar results (p > .05) to α-MEM  + FSH for all parameters evaluated, except for the daily growth rate, which was higher (p < .05) in MN 0.1  + FSH. The GSH levels were higher in MEM than all treatments. In addition, oocytes from follicles cultured in MN 0.1  + FSH showed ability to resume meiosis. In conclusion, M. nigra extract (0.1 mg/ml) added by supplements and FSH can be an efficient medium for ovine secondary follicle development.