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

Enhancing the nation's beef supply and guaranteeing long-term food security requires increasing the effectiveness of artificial insemination (AI) in Bali cattle (Bos javanicus). 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. 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–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). 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. Under a controlled dietary baseline, a lipid-centric blood-to-sperm metabolic axis emerges as a key determinant of cryotolerance in B. javanicus. 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.

提升国家牛肉供应水平、保障长期粮食安全，需提高巴厘牛（Bos javanicus）人工授精（AI）的实施效率。冷冻耐受性（即精液解冻后品质的波动）是制约人工授精效果的关键因素，其大概率反映了机体与细胞的代谢差异。为明确标准化饲养与管理条件下冷冻耐受性的代谢决定因子，本研究将血液生化指标与精子代谢组学分析相结合。本研究选取区域人工授精中心（RAIC）的10头健康种公牛，统一采用粗饲料-精料混合日粮进行饲养。使用乙二胺四乙酸（EDTA）抗凝血浆检测血液生化指标。代谢组学分析方面，本研究从解冻后的精液细管中制备洗涤后的精子沉淀，并通过非靶向气相色谱-串联质谱（GC-MS）完成代谢物谱分析。本研究通过多变量分析（包括层级聚类、K均值聚类，以及带有投影变量重要性（VIP）得分的偏最小二乘判别分析（PLS-DA））对整体代谢模式进行了总结。采用斯皮尔曼相关性分析整合血液指标、精子代谢物与解冻后精液品质性状（包括活力、活率、质膜完整性（%PMI）及形态异常率）。本研究共鉴定出18种细胞内代谢物，其中以脂肪酰类物质占比最高。无监督聚类与PLS-DA分析显示个体间代谢谱存在显著分离，棕榈酸与硬脂酸为最具判别力的特征代谢物之一（VIP≥1.0）。机体脂质转运与离子稳态的相关指标与精子脂质组成具有显著关联：白蛋白与钾离子水平与细胞内棕榈酸酯及相关代谢物含量升高呈正相关。功能层面，脂质与短链脂肪酸类特征代谢物（包括1-单棕榈酸甘油酯、十九烷酸、己酸及戊酸）与精子活率和/或质膜完整性百分比呈正相关；而十二烷酸与单硬脂酸甘油酯则与形态异常率呈负相关。但未检测到其与精子活力存在显著关联。在标准化饲养条件下，以脂质为核心的血液-精子代谢轴是决定巴厘牛冷冻耐受性的关键因素。本研究筛选得到的优先代谢物可作为种公牛筛选的实用生物标志物候选对象，同时为人工授精中心优化精液稀释液配方与冷冻保存方案提供了机制层面的理论依据。靶向串联质谱、膜靶向脂质组学以及线粒体功能检测可为该研究成果的快速转化提供可行路径，有望提升繁殖效率，最终助力可持续牛肉生产与粮食安全保障。