Induction of PCO

After laboratory adaptation, all animals had undergone daily vaginal smears for 7 consecutive days to evaluate ovarian cyclicity. Animals with regular oestrous cycles were selected to be included in the study. The experimental rats were given letrozole 1 mg/kg via oral gavage for 21 consecutive days every morning at 10 AM according to Reddy et al. [28]. Vaginal smears were performed every morning from day 14 and during treatment, using cotton buds immersed in saline. Never introduce deeply into the vagina to avoid cervical stimulation with subsequent pseudo-pregnancy state and persistence of the diestrus phase up to 14 days. Afterwards, the collected vaginal smears were placed on glass slides, dried, and stained with Giemsa [8].