Untargeted Metabolomics Analysis Reveals Dynamic Shift in Follicular Fluid Metabolites After GnRH Induced Ovulation...

Oocyte and granulosa cells undergo dramatic molecular alterations prior to ovulation, which are critical for acquisition of oocyte developmental competence. These events are orchestrated by a complex bidirectional communication system mediated by the surrounding follicular cells, with follicular fluid reflecting the metabolic output of this system. The composition of follicular fluid is highly dynamic during folliculogenesis, especially following an ovulation-inducing surge of luteinizing hormone, as granulosa cells and theca cells respond and synthesize new metabolites. In the present study, untargeted metabolomics was performed on follicular fluid collected from synchronized Bos taurus cattle at 0, 6, and 22 h after gonadotropin releasing hormone-induced ovulation. High-performance liquid chromatography-mass spectrometry revealed a temporal shift in metabolite abundance across the periovulatory window. Partial least squares discriminant analysis demonstrated clear separation between 0- and 22-h samples, while 6-h samples displayed an intermediate metabolite profile. A total of 489 features were retained after variance-based filtering (interquartile range threshold of 25%), and a covariate-adjusted linear model identified 327 features as significantly altered across time points (p ≤ 0.05). Lipid-associated metabolites, including polyunsaturated fatty acids, oxylipins, and steroid-derived metabolites were enriched prior to induced ovulation while amino acids and related metabolites were progressively enriched from 0 to 22 h. Functional analysis of features that were progressively enriched across the periovulatory window revealed nine significantly affected pathways (false discovery rate < 0.1) associated with amino acid metabolism and biosynthesis, as well as galactose metabolism. These results enhance our understanding of the periovulatory follicular environment and have potential applications for improving in vitro oocyte maturation protocols.