Folic acid supplementation alleviates donor cattle’s heat stress and improves in vitro embryo production efficiency in summer

To mitigate the detrimental effects of heat stress (HS) on the production efficiency of Wagyu cattle oocyte pick-up (OPU)-in vitro fertilization (IVF), this study conducted a 20-d folic acid (VB9) supplementation trial in Wagyu cattle under HS conditions. Using a staged repeated measurements design, OPU-IVF production procedures were performed on Wagyu cattle (12-mo-old, n = 5 out of 20 total) at six time points during the supplementation period (days 0, 4, 8, 12, 16, and 20). ELISA-based biochemical assays were employed to evaluate the effects of VB9 supplementation on HS-related indicators in serum and follicular fluid of Wagyu cattle under HS conditions. Untargeted metabolomics was further utilized to analyze the impact of VB9 on metabolic profiles in the follicular fluid. The results demonstrated that supplementation with VB9 for 20 d significantly improved both oocyte quality and developmental competence obtained via OPU (n = 5), markedly enhancing cleavage rate (58.14% vs. 24.08%; P < 0.05) and blastocyst rate (35.9% vs. 10.58%; P < 0.05). VB9 supplementation significantly reduced HS-related indicators in both serum and follicular fluid, including levels of heat shock protein 70 (HSP70), heat shock protein 90 (HSP90), interleukin-1 (IL-1), interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-α), malondialdehyde (MDA), and reactive oxygen species (ROS). Concurrently, total antioxidant capacity (T-AOC) was significantly increased, and the activities of superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GSH-PX) were markedly elevated. Estradiol (E2) and progesterone (P4) levels were significantly increased, while the P4-to-E2 ratio exhibited a significant decrease. Furthermore, untargeted metabolomic analysis revealed 1,557 differentially expressed metabolites (DEMs) in follicular fluid following VB9 supplementation. These DEMs were primarily enriched in metabolic pathways, including purine metabolism, phenylalanine and tyrosine metabolism, steroid hormone biosynthesis, and ABC transporters. In conclusion, VB9 supplementation enhances OPU-IVF production efficiency under HS conditions and offers novel insights into the molecular mechanisms underlying VB9-mediated HS mitigation.