216 Metabolomic and proteomic profiles of bovine follicular fluid during the window of in vivo oocyte maturation

Bovine follicular fluid (bFF) is the microenvironment that supports oocyte maturation and competence. Research has shown that in vivo-matured, yet in vitro-fertilized and -cultured, bovine embryos reach the blastocyst stage at 75% efficiency compared with their in vitro-matured counterparts at only 20%–30%, suggesting a deficiency of IVM that needs to be investigated. The hypothesis states that bFF proteins and unique metabolites undergo drastic changes during in vivo oocyte maturation. Thus, we conducted the first bovine study to systematically characterise bFF unique metabolites and proteins under precise experimental timing throughout in vivo oocyte maturation. Holstein heifers (n = 24) were synchronized in a modified 7-day CIDR protocol and ovarian development was determined by daily ultrasonography. Animals were randomly assigned to one of 4 groups (n = 6), and bFF was collected at 24 h, 48 h, 60 h, and 72 h post-PG. For the metabolomics study, bFF samples were prepared by protein removal, and organic solvent removal, Quality Control (QC), pooled and quantified, Waters ACQUITY (UPLC), final analysis by LC-MS and (Q-Exactive HP-Orbitrap MS), and bioinformatic data analysed using Metabolon Platform. For the proteomics study, bFF samples were proteolytic trypsin digested, TMT labelled, samples pooled and quantified, fractionation, final analysis by LC-MS (Q-Exactive HP–Orbitrap MS), and bioinformatic data analysed by ThermoFisher™ PD 3.0. For statistical analysis, a one-way analysis of variance is performed on the metabolites- and peptides-level abundance across time points after PG and Fisher’s (l.s.d.) for pairwise comparisons. False discovery rate (FDR) analysis was used to be the metric for global confidence assessment before analysis. Here, we identified a total of 784 unique metabolites in bFF, 703 of known biochemical identity, and 81 compounds of unknown structural identity. Among the 784 metabolites, 432, 94, and 258 were low, medium, and high FDR, respectively. The 30 top-ranking biochemicals in bFF were amino acid, lipid, carbohydrate, and nucleotide metabolism. Fold-change analysis of bFF metabolites were shown as upregulated: downregulated as follows: 34: 106, 75: 152, and 67: 89, for 48-h, 60-h, and 72-h groups, respectively, while fold-change between 48-h and 72-h groups showed 111: 63 upregulated: downregulated metabolites, respectively. Four vitamins (A, B₅, C, and β-carotene), and three antioxidants (nicotinate, xanthosine, and allantoic acid) were identified as significant in bFF (P < 0.05). Prostaglandins E₂ and F_2α were identified in bFF with similar concentrations across time points but exhibited a huge spike at 72 h (P < 0.05). For the proteomics results, a total of 366 proteins were commonly present in all groups, however, 16, 41, 33, and 48 proteins have shown to be exclusively present in the 24-h, 48-h, 60-h, and 72-h groups, respectively. Fold change has shown as highest in the 72-h group with 231 upregulated peptides and 50 downregulated. The 37 and 33 proteins commonly present across groups were significantly expressed in 24-h and 72-h groups (P < 0.05), respectively. In conclusion, follicular metabolites and proteins undergo substantial changes during in vivo oocyte maturation.