SPINK5 variation regulates epithelial barrier function and shapes heritable rumen microbiota in dairy cattle

Abstract Host genetics contributes to inter-individual variation in rumen microbiota and fermentation, but the underlying physiological mechanisms remain unclear. This study integrated genomic, rumen microbiome, and fermentation parameter data from 603 lactating Holstein cows across five farms. Genetic clustering significantly influenced ruminal microbial community structure and diversity. We identified 54 heritable microbial taxa and found that moderate SCFA concentrations were associated with maximal connectivity among heritable microbial networks. Genome-wide association analysis of fermentation traits revealed a chromosome 7 hotspot associated with butyrate and propionate concentrations, with fine-mapping prioritizing SPINK5 as the strongest candidate gene. SPINK5 was specifically expressed in the rumen stratified squamous epithelium and functionally regulated epithelial keratinization and barrier permeability. Knockdown of SPINK5 promoted keratinization-related gene expression and strengthened epithelial barrier properties, whereas overexpression increased epithelial permeability. This reveals a novel mechanism whereby host SPINK5 variants regulate rumen microecology via the epithelial barrier-SCFA homeostasis axis, providing actionable markers for microbiome-targeted genomic selection to enhance fermentation efficiency, mitigate methane emissions, and improve dairy performance.