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The present study evaluated the effect of supplementing  in vitro  culture medium with  J .  insularis  compared to FSH on isolated secondary follicles and  in vitro  maturation of oocytes from those follicles. Secondary follicles were isolated from sheep ovaries and individually cultured for 18 days in α-MEM +  ( Control ), α-MEM +  supplemented with 100 ng/mL recombinant bovine follicle stimulating hormone (FSH) or with 0.3, 1.25, or 2.5 mg/mL of  J .  insularis  extract (JI0.3, JI1.25, and JI2.5, respectively). Culture medium collected every 2 days was used to measure ROS levels. At the end of the culture period, cumulus oocytes complex (COCs) were collected and matured  in vitro . Follicular walls were used for mRNA quantitation. JI0.3 led to a higher (P < 0.05) percentages of intact follicles than other groups after 18 days of culture. While follicular diameter remained unchanged from Day 6 onwards with JI0.3 and FSH, percentages of antral cavity formation were higher (P < 0.05) with JI0.3 at Day 6 than in all other treatments. No differences were observed between controls and treatment groups regarding ROS levels and mRNA expression of genes. Viability of resulting oocytes was higher (P < 0.05) in JI0.3 compared to FSH. Interestingly, in control experiment, supplementation of maturation medium with JI0.3 led to higher (P < 0.05) percentages of metaphase II compared to controls. Although more validations will be needed, it seems that this natural extract could be used as a cheap and easily available alternative to commercial FSH.

Supplementation of in vitro culture medium with FSH to grow follicles and mature oocytes can be replaced by extracts of Justicia insularis