Integrative analysis of blood biochemistry and sperm metabolome profiles reveals metabolic determinants of cryotolerance in Bali bulls under standardized nutritional conditions

Abstract

Background: ). Cryotolerance, or post-thaw variability in semen quality, limits AI results and probably reflects systemic and cellular metabolic variations. To determine the metabolic determinants of cryotolerance under standardized nutrition and supervision, this study combined blood biochemistry with the sperm metabolome. Methods: Ten healthy breeding bulls at the regional artificial insemination center (RAIC) were maintained on a uniform forage-concentrate diet. Blood biochemical parameters were measured using ethylenediaminetetraacetic acid (EDTA) plasma. For metabolomics, washed sperm pellets were prepared from frozen-thawed semen straws and profiled using untargeted gas chromatography-mass spectrometry (GC-MS). Multivariate analyses (hierarchical clustering, K-means, and partial least squares-discriminant analysis (PLS-DA) with variable importance in projection (VIP) scores) summarized the global patterns. Spearman correlations were used to integrate blood indices, sperm metabolites, and post-thaw traits (motility, viability, plasma membrane integrity (%PMI), and morphological abnormalities). Results: Eighteen intracellular metabolites were identified, predominated by fatty acyls. Unsupervised clustering and PLS-DA revealed clear inter-individual separation, with palmitic and stearic acids being among the most discriminant features (VIP ≥ 1.0). Systemic markers of lipid carriage and ionic tone aligned with sperm lipid composition: albumin and potassium were associated with higher intracellular palmitate levels and related metabolites. Functionally, lipid and short-chain fatty acid features, 1-monopalmitin, nonadecanoic acid, caproic acid, and valeric acid, were positively associated with viability and/or PMI%, whereas dodecanoic acid and glycerol monostearate were inversely related to morphological abnormalities. However, no robust association was detected with motility. Discussion: . The prioritized metabolites constitute practical biomarker candidates for sire screening and provide a mechanistic basis for refining extenders and cryopreservation protocols at AI centers. Targeted tandem mass spectrometry, membrane-focused lipidomics, and mitochondrial functional assays offer immediate paths to translation, with the potential to improve reproductive efficiency and, ultimately, bolster sustainable beef production and food security.

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