Hydrophilic-hydrophobic imbalance is associated with alcohol-positive milk in Holstein cows: An untargeted metabolomics analysis

Alcohol-positive milk (APM) is a prevalent milk quality defect causing economic losses in dairy production. Using a longitudinal case-control design, this study characterized the metabolomic profile of APM and clarified the core molecular signatures underlying its occurrence in Holstein cows. Twenty milk samples from Holstein cows (10 normal milk [NM] vs. 10 APM) were collected for case-control comparison, and 7 paired samples from spontaneously recovered APM cows (recovered alcohol-negative milk, RAPM) were obtained for longitudinal analysis. Untargeted metabolomic profiling was performed using ultra-high performance liquid chromatography coupled with tandem high-resolution mass spectrometry (UHPLC-HRMS/MS). APM exhibited a distinct bidirectional metabolic dysregulation. On the hydrophilic side, glycolysis/gluconeogenesis and pentose phosphate pathways were inhibited. Key hydrophilic phospholipid precursors (3-phosphoglycerate, 2-phosphoglycerate) were downregulated, impairing de novo synthesis of phosphatidylcholine (PC) and phosphatidylethanolamine (PE). Levels of phosphorylcholine, phosphoethanolamine, and core structural phospholipids (e.g., PC(18:2/0:0), PE(18:2/0:0)) were all reduced. On the hydrophobic side, highly hydrophobic long-chain fatty acid derivatives (e.g., 9,10-epoxystearic acid) were markedly enriched. Excessive lysophosphatidylethanolamine (LPE) also accumulated due to enhanced phospholipid hydrolysis. In conclusion, the bidirectional imbalance between downregulated hydrophilic protective metabolites and upregulated hydrophobic detrimental metabolites is the core metabolic signature of APM. This imbalance disrupts the intrinsic hydrophilic-hydrophobic homeostasis of milk and drives APM occurrence.