Melatonin and betaine supplementation mitigate oxidative and endocrine dysregulation to restore ovarian activity in summer anestrus buffaloes.

Buffaloes are polyestrous but display a marked seasonal decline in reproductive efficiency during summer, primarily due to longer photoperiod, heat stress, altered endocrine profiles, and heightened oxidative stress, resulting in a high incidence of summer anestrus (36.6-59.6%). This study evaluated the effects of melatonin and betaine supplementation on ovarian function, endocrine dynamics, and oxidative status in buffaloes during the low-breeding season. Anestrus buffaloes (n = 22) with a history of up to 90 days postpartum anestrus were monitored for 21 days and allocated into three groups: Control (n = 7), melatonin-treated (T1; n = 7; sustained-release melatonin @ 18 mg/50 kg BW, S/C, once), and betaine-treated (T2; n = 8; 25 g/day orally for 30 days). Blood sampling and transrectal ultrasonography were performed at 10-day intervals to assess follicular activity, endocrine responses, and oxidative stress markers. Melatonin and betaine treatments significantly alleviated summer-induced stress, as evidenced by reduced prolactin and nitric oxide (NO) levels (P < 0.05) and by increased total antioxidant capacity (TAC) compared with controls. Melatonin administration caused a sustained increase in circulating melatonin concentrations and improved LH-dependent ovarian activity. Although dominant follicles developed in all animals, untreated controls failed to ovulate and exhibited follicular atresia, hyperprolactinemia, elevated NO, and reduced TAC. In contrast, treated groups exhibited increased TAC, reduced NO and prolactin from day 20 onward, and elevated progesterone following estrus induction, confirming ovulation. Estrus induction and conception rates were highest in the melatonin group (57.14% and 66.6%), followed by betaine (25% and 50%), whereas no conceptions occurred in controls. Overall, melatonin and betaine effectively mitigated oxidative and endocrine disruptions, enhanced follicular function, and facilitated reactivation of the hypothalamic-pituitary-ovarian axis, thereby improving estrus induction and ovulatory responses in buffaloes during the low-breeding summer season.