Multi-omics and functional validation reveal that Methanobrevibacter-derived L-3-aminoisobutyrate alleviates subclinical mastitis in dairy goats via the HSPA1B-p65 signaling pathway.
BACKGROUND Subclinical mastitis (SCM) is prevalent in dairy livestock and compromises milk quality and lactation performance. Although often attributed to bacterial infection, many cases lack identifiable pathogens, suggesting alternative mechanisms. While evidence supports a gut-mammary gland axis, the microbial drivers and microbiota-derived metabolites linking gut dysbiosis to SCM remain unclear. Here, we aimed to identify SCM-associated gut microbial markers, prioritize candidate therapeutic metabolites and define the underlying mechanism. RESULTS Based on differences in somatic cell count (SCC) and inflammatory phenotypes across a cohort of 167 mid-lactation Saanen dairy goats, we selected 6 healthy and 6 SCM goats for downstream analyses. By integrating metagenomics, metabolomics, cross-species fecal microbiota transplantation (FMT) and functional validation in vitro and in vivo, we found that SCM was accompanied by reduced milk yield and heightened inflammatory signatures. Compared with the Healthy group, SCM goats exhibited marked remodelling of the gut microbiota, with enrichment of opportunistic taxa (Eubacterium and Blautia) and a pronounced depletion of archaeal Methanobrevibacter spp. Notably, FMT from SCM donors recapitulated mammary inflammatory phenotypes in mice, supporting a causal contribution of gut dysbiosis to mammary inflammation. Joint metagenomic functional profiling and metabolomics further identified the branched-chain amino-acid-derived metabolite L-3-aminoisobutyrate (BAIBA) as significantly enriched in the gut of healthy goats. Moreover, Methanobrevibacter spp. harboured key enzyme genes (vorA, vorB and vorD) implicated in BAIBA biosynthesis. In an LPS-challenged MAC-T model, BAIBA attenuated mammary epithelial inflammation by activating endoplasmic reticulum protein quality control programmes and restoring HSPA1B expression, thereby suppressing NF-κB activation and reducing pro-inflammatory cytokine production. Finally, in naturally infected goats, intramammary administration of BAIBA lowered SCC, highlighting translational potential. CONCLUSIONS This study identifies BAIBA as a microbiota-derived metabolite that protects against SCM by restraining mammary inflammation via the HSPA1B-NF-κB axis, establishing a mechanistic gut-mammary link and highlighting a potential non-antibiotic intervention strategy. Video Abstract.