Parity-dependent plasma and milk metabolomic remodeling across hyperketonemia in Holstein cows

Parity is a major determinant of transition-cow metabolic load and disease susceptibility, yet hyperketonemia screening commonly applies uniform β-hydroxybutyrate (BHB) cut-points regardless of parity. The objective of this study was to determine whether hyperketonemia-associated metabolomic remodeling in plasma and milk differs among early-lactation Holstein cows of different parities. Cows at 5 to 7 d in milk without apparent clinical disease at routine farm observation were stratified by parity [1 (P1), 2 (P2), or 3 (P3)] and by whole-blood BHB status as control (CON; BHB <1.2 mmol/L), subclinical hyperketonemia (SCK; 1.2 ≤ BHB ≤3.0 mmol/L), or biochemical hyperketonemia (HK; BHB >3.0 mmol/L), resulting in 9 groups (n = 9 per group; n = 81 total). Paired plasma (morning) and milk (same day) were profiled using untargeted LC-MS/MS metabolomics. Within each parity, differential analyses across SCK vs. CON, HK vs. CON, and HK vs. SCK were combined with parity × status interaction screening to quantify parity-conditioned responses and to summarize stage-to-stage trajectories (e.g., early, late, progressive, or reversal patterns). Untargeted profiling detected 5,259 plasma and 8,423 milk features. At the global level, groups defined by BHB status (CON, SCK, and HK) separated more clearly than parities, indicating that BHB status dominated broad variance; however, the timing and magnitude of metabolomic remodeling were strongly dependent on parity. The timing and magnitude of BHB-associated changes in plasma and milk metabolomic features differed by parity and matrix. P2 cows showed relatively limited changes at the SCK stage but more extensive remodeling at the HK stage, whereas P3 cows showed more pronounced early and progressive changes in several summary measures. Plasma pathway enrichment highlighted ovarian steroidogenesis and primary bile acid biosynthesis, suggesting parity-conditioned engagement of endocrine-hepatic modules during early lactation. In milk, hyperketonemia was associated with parity-dependent remodeling of complex lipids and secretion-related metabolism, with enrichment of sphingolipid metabolism, choline metabolism, pantothenate and CoA biosynthesis, and pyrimidine/nucleotide pathways. Plasma-milk association patterns differed by parity: P1 had more cross-matrix correlations meeting the predefined screening criteria, whereas P2 and P3 had fewer such correlations and a greater contribution of lipid-related hub features. Collectively, these findings indicate that similar BHB concentrations can be associated with distinct parity-dependent metabolic signatures. These results suggest that parity should be considered when interpreting BHB-associated metabolomic profiles, particularly candidate plasma and milk features related to acylcarnitine metabolism, bile acid metabolism, complex lipid remodeling, and nucleotide metabolism.