Alterations in the amino acid profile of follicular fluid of cows following ovarian stimulation and its association with oocyte quality and embryonic development.

Ovarian stimulation using exogenous follicle-stimulating hormone may enhance follicular recruitment; however, in the context of artificial insemination, it does not substantially improve pregnancy rates. Moreover, such physiological stimulation may impair oocyte developmental competence. To investigate the underlying metabolic mechanisms, the present study employed targeted metabolomics to compare the amino acid profiles in the follicular fluid of dairy cows during the natural estrous cycle (control group) and following ovarian stimulation (treated group). In total, 21 differentially abundant amino acids were identified: 7 were significantly upregulated, and 14 were significantly downregulated in the treatment group. Among the downregulated amino acids, L-serine was identified as a candidate hub metabolite via network analysis. Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis revealed that these differentially abundant amino acids were primarily enriched in the metabolic pathways of glycine, serine, and threonine, as well as in monocyclic lactam and arginine biosynthesis. Supplementation with 25-μM L-serine in the in vitro maturation medium significantly improved the oocyte maturation rate, cleavage rate, and blastocyst rate of oocytes from dairy cows undergoing ovarian stimulation. Additionally, supplementation promoted cumulus cell expansion and reduced the apoptosis rate of the formed blastocyst cells. The in vitro culture of bovine granulosa cells further demonstrated that L-serine could significantly promote cell proliferation and increase the estradiol to progesterone ratio (E2/P4). Real-time quantitative PCR analysis revealed that L-serine significantly upregulated the mRNA expression of key functional genes, including aromatase (CYP19A1), steroidogenic acute regulatory protein (STAR), and proliferating cell nuclear antigen (PCNA). These findings suggest a potential role for L-serine in supporting oocyte and embryo development, offering metabolic insights for further optimization of ovarian stimulation protocols.