Integrated proteomic profiling of serum, follicular fluid and ovarian tissue in dairy cows with inactive ovaries.

BACKGROUND: Inactive ovaries (IO) are a common reproductive disorder in early lactating dairy cows. This condition significantly reduces reproductive efficiency and economic returns. Although many studies have explored the metabolic characteristics of postpartum IO, systematic proteomic analyses using multiple biological samples are still lacking. This study used data-independent acquisition (DIA) proteomics to systematically analyze protein expression profiles in serum, follicular fluid (FF), and ovarian tissues of dairy cows. The goal was to identify potential molecular regulatory mechanisms and key biological pathways involved in IO. Serum, FF, and ovarian tissue samples were collected from six healthy and six IO multiparous Holstein cows at 63 (± 3) days postpartum. These samples were analyzed using DIA-based quantitative proteomics. RESULTS: In total, 155, 171, and 157 differentially expressed proteins (DEPs, P < 0.05 and FC ≥ 1.2 or ≤ 0.83) were identified in serum, FF, and ovarian tissues, respectively. Among these, 67 DEPs were upregulated and 88 were downregulated in serum, 38 were upregulated and 133 downregulated in FF, and 71 were upregulated and 86 downregulated in ovarian tissue. Integrated analysis revealed seven consistently altered proteins (APCS, KLKB1, HRG, TTR, CTSS, RBP4, and CUTA), of which CTSS was upregulated, while the others were downregulated across all sample types. Functional enrichment analysis indicated that most metabolic pathways, particularly glycolysis/gluconeogenesis, the tricarboxylic acid cycle, carbon metabolism, starch and sucrose metabolism, and amino acid biosynthesis and degradation (including branched-chain amino acid pathways), were markedly downregulated in IO cows, suggesting metabolic suppression. In contrast, cholesterol metabolism and complement and coagulation cascades showed a general upregulation trend. CONCLUSIONS: This study, for the first time, systematically characterizes coordinated alterations in key metabolic and reproductive pathways of IO cows through integrated multi-tissue proteomics. The findings provide insight into the molecular mechanisms of IO and suggest potential biomarkers and targets for improving ovarian function.