Metabolic lipid alterations in subclinical ketotic dairy cows: A multisample lipidomic approach

ABSTRACT: Subclinical ketosis (SCK) is a prevalent metabolic disorder in dairy cows during the transition period, characterized by elevated BHB levels without overt clinical symptoms. This study employed a multicompartment lipidomic approach to investigate the systemic and localized lipid alterations associated with SCK across serum, rumen fluid, and feces. Twelve multiparous Chinese Holstein cows (3.3 ± 1.4 lactations, mean ± SD), between 2 and 21 DIM were selected from a commercial dairy farm. Based on blood BHB concentrations, cows were categorized into subclinical ketotic (SCK; n = 6; BHB >1.4 and ≤2.6 mmol/L) and nonketotic (NK; n = 6; BHB ≤0.8 mmol/L) groups, with parity and DIM closely matched. Using liquid chromatography–tandem MS, we identified significant disruptions in lipid metabolism, particularly in glycerophospholipids and sphingolipids, across all biological matrices. Multivariate analysis revealed distinct lipidomic profiles between SCK and NK cows, with the most pronounced changes observed in serum. Key differential lipid species were significantly altered, including phosphatidylcholine, phosphatidylethanolamine, and ceramides, indicating potential links between lipid metabolism, inflammatory responses, and oxidative stress. Serum metabolic analysis further revealed that SCK cows exhibited elevated levels of nonesterified fatty acids (NEFA) and BHB, accompanied by lower glucose and insulin concentrations, indicative of disrupted energy metabolism. Additionally, SCK cows showed increased markers of oxidative stress and inflammation, suggesting an association between lipid dysregulation and systemic metabolic stress. Pathway enrichment analysis identified glycerophospholipid and sphingolipid metabolism as the most affected pathways in SCK cows. Correlation analysis further highlighted associations between altered lipid species and metabolic indicators such as BHB, NEFA, and inflammatory cytokines. These findings provide novel insights into the metabolic adaptations associated with SCK, emphasizing the role of lipid dysregulation, oxidative stress, and inflammation in disease pathophysiology. The identified lipidomic alterations may serve as potential biomarkers for early diagnosis and intervention in SCK-affected dairy cows.