Differential shaping of equine gut microbiota structure and function by breed and feeding regimen

Background The gut microbiota plays an essential role in host energy metabolism and immune function. Horses are non-ruminant herbivores that rely heavily on hindgut microbial fermentation to meet their energy requirements. However, the relative contributions of host genetic background (breed) and environmental factors (feeding regimen and geographical location) to shaping the equine gut microbiota remain poorly understood. Methods In this study, 16S rRNA gene sequencing and functional prediction analysis were performed on 139 equine fecal samples to systematically investigate the differential effects of breed and feeding regimen on the gut microbiota. Samples were collected from 30 Thoroughbreds (TH), 31 stabled hybrid horses (HH1), 30 grazing hybrid horses (HH2) (with HH1 and HH2 sired by Thoroughbreds out of Mongolian mares), 32 Mongolian horses (MH), and 16 Warmblood horses (WBH1 and WBH2). Alpha and beta diversity analyses, taxonomic profiling, and PERMANOVA were used to assess microbial composition and the contributions of different factors. Results Alpha diversity analysis revealed that the richness and diversity of the TH, HH1, HH2, and MH groups were significantly higher than those of the Warmblood horses ( p < 0.001), with Mongolian horses exhibiting the highest diversity and the hybrids showing intermediate levels between their parental breeds. Regarding taxonomic composition, the TH, HH1, HH2, and MH groups shared a microbial structure dominated by Firmicutes and Bacteroidota, yet each possessed distinct characteristics: Thoroughbreds were enriched with Treponema; Mongolian horses harbored the highest abundances of Rikenellaceae_RC9_gut_group and NK4A214_group; and the grazing hybrid horses developed a fiber-degrading bacterial community centered on Ruminococcus and Fibrobacter , demonstrating breed-specific microbial features. In contrast, the Warmblood horses exhibited a gut microbiota with distinct features characterized by significantly reduced microbial diversity and core fiber-degrading genera, concomitant with an enrichment of environmental-associated bacteria from the phylum Proteobacteria (e.g., Acinetobacter , Stenotrophomonas ) and other genera (e.g., Comamonas , Brevundimonas ). PERMANOVA analysis further quantified the contributions of different factors: breed explained 44.8% of the total variation (R² = 0.448, p < 0.001), followed by feeding regimen (10.3%, p < 0.001) and geographical location (2.7%, *p* < 0.01), confirming breed as the predominant factor. Conclusion This study provides evidence that breed establishes the foundational framework of the gut microbiota, while feeding regimen performs fine-tuning functions. We also systematically characterized the unique microbial composition of Warmblood horses, offering a scientific basis for breed-specific health management, precision nutritional interventions, and future disease risk monitoring in horses. Although all horses appeared clinically healthy, the distinct microbial composition observed in Warmblood horses warrants further investigation to determine its biological significance.