Granulosa cells from prehierarchical follicles (3–5 and 6–8 mm) and preovulatory follicles (9–12 mm) were dispersed into single-cell suspension and treated with 0, 1, 10, or 20 mM metformin for 60 min in the presence or absence of rhFSH (10 ng/ml) at 41°C. Cell lysates were prepared following treatment, and a Western blot analysis was performed under reducing conditions to quantify the abundance of pERK1/2, ERK1/2 and β-actin. Control received no rhFSH and no metformin treatments. β-actin is used as a protein loading control. A,B,CP < 0.001 by ANOVA; n=4 replicates of 4 broiler breeder hens each. Inset: Photograph of prehierarchical follicles and the most-recently recruited preovulatory follicle to denote the source of granulosa cell (open box).