Host genetic variation in SPINK5 regulates rumen epithelial barrier function and shapes heritable microbial communities through SCFA-mediated mechanisms in dairy cattle

Background : Despite standardized feeding and management protocols, substantial variations in rumen microbial composition and fermentation profiles among dairy cattle suggest significant host genetic influence. Although genome-wide association studies have identified genetic polymorphisms linked to microbial abundances, the physiological mechanisms through which host genetics regulate rumen microecology remain largely unexplored. This knowledge gap limits our understanding of host-microbiome interactions and hinders the development of precision breeding strategies for optimizing rumen function. Results: We integrated host genomic, rumen microbiomic, and metabolomic data from 603 lactating Holstein dairy cows across five commercial farms to elucidate genetic mechanisms governing rumen function. Population structure analysis revealed four distinct ancestral clusters with clear genetic differentiation. Genetic clustering significantly influenced rumen microbial β-diversity across bacteria, methanogens, anaerobic fungi, and protozoa, explaining 4-22% of variation while controlling for farm-specific environmental effects. Species-level heritability analysis identified 54 heritable microbial taxa (h² ≥ 0.2) concentrated among keystone species, collectively representing 23% of bacterial abundance and 44% of methanogenic abundance. Fermentation parameters showed high heritability, with propionate exhibiting the highest genetic control (h² = 0.670). Genome-wide association analysis identified a chromosomal hotspot containing 13 significant SNPs, with fine-mapping localizing strongest associations to SPINK5 (serine peptidase inhibitor Kazal type 5). Functional validation revealed that SPINK5 , expressed specifically in the spinous layer of rumen stratified squamous epithelium, controls epithelial keratinization and barrier permeability. SPINK5 knockdown accelerated keratinization by upregulating nine keratin genes and desmosomal markers, strengthening epithelial barriers and reducing SCFA absorption, while overexpression increased epithelial permeability. Co-occurrence network analysis demonstrated that moderate SCFA concentrations promoted maximum connectivity among heritable microbes, indicating optimal conditions for microbial syntrophic relationships. Conclusions: This study reveals an innovative physiological mechanism whereby host genetic variants regulate rumen microbiota through SPINK5 -mediated epithelial barrier control. SPINK5 functions as a molecular rheostat that modulates epithelial permeability and paracellular SCFA absorption, thereby controlling intraluminal fermentation environments that shape heritable microbial communities. These findings provide actionable genetic markers for incorporating microbiome-associated traits into genomic selection programs, offering new strategies for enhancing rumen fermentation efficiency, reducing methane emissions, and improving dairy productivity through precision breeding approaches.