ABSTRACT

Background and Aim: Subclinical ketosis (SCK) and subclinical mastitis (SCM) are common disorders of early lactation dairy cows, but their concurrent metabolic effects remain poorly characterized. This study aimed to characterize plasma amino acid profiles associated with SCK, SCM, and concurrent SCK+SCM, evaluate metabolite-based discrimination among disease states, and investigate associations between ketosis status and mastitis pathogens.

Materials and Methods: Fifty-two Holstein cows at 5–40 days in milk and parity 2–5 were classified into four groups of 13 cows each: healthy controls, SCK, SCM, and concurrent SCK+SCM. Plasma concentrations of 25 amino acids and related metabolites were quantified using high-performance liquid chromatography. Group differences were evaluated using non-parametric analyses, followed by collinearity-aware multinomial logistic regression and receiver operating characteristic analyses to identify metabolites associated with disease classification and discriminatory performance.

Results: Twelve metabolites differed significantly among groups (p < 0.05). Tryptophan showed the strongest association with disease status (χ² = 17.956, p < 0.001), decreasing progressively from 35.5 ± 27.6 μmol/L in healthy cows to 23.9 ± 14.7, 19.4 ± 21.5, and 14.5 ± 16.4 μmol/L in the SCK, SCM, and SCK+SCM groups, respectively. Conversely, isoleucine and leucine increased across the disease continuum and were highest in cows with concurrent SCK+SCM (173.6 ± 63.0 and 212.0 ± 75.3 μmol/L, respectively). Multinomial logistic regression retained nine metabolites associated with disease classification: glutamic acid, alanine, citrulline, isoleucine, tyrosine, ornithine, tryptophan, arginine, and hydroxyproline. Serine (area under the curve = 0.811) and tryptophan (area under the curve = 0.760) showed the strongest individual discrimination between healthy cows and cows with concurrent SCK+SCM. No significant associations were detected between SCK status and specific mastitis pathogens.

Conclusion: SCK, SCM, and their concurrent occurrence were associated with distinct plasma amino acid profiles, with concurrent disease showing the greatest metabolic alterations. Tryptophan, serine, citrulline, and isoleucine emerged as candidate components of a multimetabolite signature. Larger longitudinal studies are required to validate their diagnostic and predictive utility.

Keywords: amino acid metabolomics, dairy cattle, early lactation, metabolic biomarkers, subclinical ketosis, subclinical mastitis, targeted metabolomics, tryptophan.