Multi-compartment omics identifies a reproducible association between circulating 5-androstene-3β,17β-diol and mastitis clinical status in dairy cows.

Introduction: Bovine mastitis is generally viewed as a localized intramammary infection, but the extent to which gastrointestinal, circulating, and mammary tissue features covary across clinical phenotypes remains unclear. We aimed to characterize concurrent alterations across the rumen, hindgut, serum, milk, and mammary gland in healthy control (HC), subclinical mastitis (SCM), and clinical mastitis (CM) cows and to evaluate circulating 5-androstene-3β,17β-diol (5-AED) as a correlate of mastitis phenotype. Methods: In a discovery cohort, fecal and rumen microbiota were characterized by 16S rRNA gene sequencing, and untargeted metabolomics was performed across four biological matrices. Mammary tissue was analyzed using tandem mass tag (TMT)-based proteomics, parallel reaction monitoring (PRM), and histology, while circulating inflammatory and metabolic mediators were measured in serum. A later cohort from the same herd was analyzed independently to replicate the serum 5-AED findings using targeted analysis. Results: Systemic inflammatory and lipid mediators differed in SCM cows despite the absence of overt clinical signs. Fecal and rumen microbial community structures differed among HC, SCM, and CM groups, with lower rumen microbial richness in mastitis cows. 16S rRNA-based functional predictions indicated differences in carbohydrate- and amino acid-related functional potential. Serum 5-AED showed the highest distribution in HC, an intermediate distribution in SCM, and the lowest distribution in CM, and this group ordering was reproduced in the later cohort. Mammary tissue exhibited lower abundance of claudin-8, fatty acid synthase, acetyl-CoA carboxylase, and ABCG2, together with enrichment of complement and coagulation pathways and annotation of a neutrophil extracellular trap-associated pathway. Targeted proteomics supported the direction of selected barrier-, lipid-, complement-, and fibrinogen-related differences. Hydroxysteroid 17β-dehydrogenase abundance also differed among groups; however, enzyme activity and a direct biochemical relationship with 5-AED were not established. Discussion: These findings reveal coordinated differences across gastrointestinal, circulating, and mammary compartments associated with distinct mastitis phenotypes, including subclinical disease. However, the cross-sectional design does not establish temporal progression, mastitis specificity, or a causal gut-mammary pathway. The association between circulating 5-AED and mastitis phenotype warrants prospective evaluation in independent herds using an independently validated analytical assay before its potential clinical utility can be assessed.