**Background:** High-altitude environments (>2,500 m) pose physiological challenges including hypoxia, low pressure, and cold, which can alter gut microbiota and contribute to metabolic and digestive disorders. Sanhe cattle are a dual-purpose breed from Inner Mongolia with strong adaptability, but their gut microbiome and metabolic responses to high altitude had not been studied. This study aimed to evaluate differences in gut microbiota and fecal metabolomics between Sanhe heifers raised at low and high altitudes.
**Methods:** Twenty Sanhe heifers (15-month-old, mean body weight 334.82 ± 13.22 kg) were selected from two regions: Xiertala Cattle Breeding Farm in Hulunbeier, Inner Mongolia (approximately 700 m altitude, LA group) and Zhizhao Dairy Cow Farm in Lhasa, Tibet (approximately 3,650 m altitude, HA group), with 10 animals per group. Fecal samples were collected per rectum. Volatile fatty acid (VFA) concentrations were measured using gas chromatography. Gut microbial DNA was extracted and the V3–V4 region of the 16S rRNA gene was amplified and sequenced on an Illumina MiSeq. Sequences were clustered into operational taxonomic units (OTUs) at 97% similarity. Metabolites were extracted from fecal samples using cold methanol/acetonitrile/water and analyzed via ultra-high-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (LC-MS). Differential metabolites were identified using VIP >1.0 and p < 0.05 from OPLS-DA models. KEGG pathway enrichment was assessed using Fisher's exact test. Statistical comparisons used t-tests, Wilcoxon rank tests, and Spearman's rank correlations.
**Key Results:** Fecal VFA concentrations (acetate, propionate, butyrate, total VFAs) did not differ significantly between groups (p > 0.05), but the acetate-to-propionate ratio was significantly higher in the HA group (2.13 vs. 1.77, p = 0.002). A total of 868,445 raw sequences were generated (mean 43,422 ± 4,392.71 per sample), with 2,054 ± 133.75 OTUs per sample. Good's coverage was 0.97. PCoA based on Bray–Curtis dissimilarity showed significant separation between groups (R² = 0.58, p = 0.001). At the phylum level, Proteobacteria and Actinobacteria were significantly lower in HA, while Spirochaetae was significantly higher (p < 0.05). At the family level, Peptostreptococcaceae, Christensenellaceae, Erysipelotrichaceae, Family_XIII, Acidaminococcaceae, Peptococcaceae, Enterobacteriaceae, Spirochaetaceae, and Coriobacteriaceae were significantly lower in HA, while Lachnospiraceae, Clostridiales_vadinBB60_group, Bacteroidales_S24-7_group, Bacteroidales_RF16_group, and Porphyromonadaceae were significantly higher (p < 0.05). At the genus level, 43 genera were significantly higher and 15 significantly lower in HA. Notable fold-changes included Butyrivibrio (20.59-fold decrease, p = 0.002), Escherichia-Shigella (213.31-fold increase, p < 0.001), and Domibacillus (increasing from 0.00 to 0.027, p < 0.001). Metabolomics identified 1,727 total metabolites; 368 differed significantly between groups (231 in positive, 137 in negative ion mode). Ten KEGG pathways were enriched, including nicotine addiction, central carbon metabolism in cancer, mineral absorption, protein digestion and absorption, ABC transporters, neuroactive ligand-receptor interaction, cAMP signaling pathway, aminoacyl-tRNA biosynthesis, pyrimidine metabolism, and purine metabolism. Spearman correlation analysis showed that OTUs from Romboutsia, Paeniclostridium, and unclassified_f_Lachnospiraceae were strongly associated with 28 differential metabolites (|r| > 0.8, p < 0.05).
**Clinical Implications:** This study demonstrates that high-altitude environments significantly alter the gut microbial composition and fecal metabolome of Sanhe heifers, even when nutrient intake is standardized via total mixed ration. The observed shifts in bacteria involved in fiber degradation, protein digestion, and inflammation (e.g., reduced Christensenellaceae and Peptostreptococcaceae, increased Lachnospiraceae and Spirochaetaceae) suggest that high altitude may impair digestive efficiency and increase disease susceptibility. The enrichment of pathways related to nucleotide metabolism, mineral absorption, and signaling (cAMP, ABC transporters) indicates broad metabolic reprogramming. These findings provide a foundation for developing feeding management strategies to improve high-altitude adaptation in dairy cattle. However, the study is limited by its small sample size (n=10 per group) and cross-sectional design, which cannot establish causality.