Folic acid alleviates heat stress-induced ovarian dysfunction in dairy cows via modulation of follicular fluid metabolism and ABC transporter activity

Heat stress impairs follicular microenvironment and oocyte competence in dairy cows, reducing fertility and economic losses. This study investigated whether dietary folic acid (FA) could ameliorate heat stress-induced ovarian dysfunction by modulating antioxidant status, inflammation, and follicular fluid (FF) metabolism. Lactating Holstein cows were randomly assigned to three groups: (1) heat stress (HS, n = 20, THI > 72 for >15 days); (2) FA supplementation (VB9, n = 20, 7 g rumen-protected FA/day); and (3) thermoneutral control (TN, n = 20, THI < 72). Ovum pick-up-in vitro production (OPU-IVP) outcomes, Hormones, oxidative stress/inflammatory markers, and FF metabolomics were analyzed. Results showed FA improved oocyte quality, cleavage/blastocyst rates, serum/FF antioxidant capacity (CAT, GSH-Px, SOD), and reduced oxidative damage (MDA), inflammation (IL-1, IL-6, TNF-α), and heat shock proteins (HSP-70, HSP-90). Non-targeted metabolomics revealed distinct FF profiles, with FA restoring TN-like metabolite composition and enriching ABC transporter pathways. In vitro, FA promoted granulosa cell proliferation, reduced Apoptosis, and enhanced steroidogenesis; these effects were amplified by ABC transporter inhibition (KO143). Ex vivo, FA and KO143 improved oocyte maturation and embryonic development. Collectively, FA mitigates heat stress-induced ovarian impairment by modulating oxidative-inflammatory balance and ABC transporter activity, offering a nutritional strategy to enhance fertility in heat-stressed dairy cows.