Alternative feed ingredients may alter intestinal structure and function. We examined jejunal, ileal, and cecal responses in Large Diqing Tibetan pigs fed a control diet or one in which walnut cake replaced 50% of soybean meal. Two pigs were sampled from each of three pens per treatment. Intestinal morphology was assessed histologically, and RNA sequencing data from 8 jejunal, 12 ileal, and 10 cecal samples retained after screening were analyzed by voom–dream, differentially expressed gene (DEG) enrichment analysis, and gene set enrichment analysis (GSEA). The 50W group showed higher villus-related indices in the jejunum and ileum and greater cecal mucosal thickness and crypt depth. The screened-sample analysis identified 103, 67, and 1979 DEGs, respectively. Pathway analyses associated jejunal responses with nutrient metabolism, ion transport, and epithelial organization; ileal responses with DNA replication, cell cycle, and epithelial renewal; and cecal responses with extracellular matrix organization and cell adhesion. Although jejunal and cecal DEG counts were sensitive to sample inclusion, principal pathway-enrichment directions were broadly maintained in full-sample and leave-one-pen-out analyses. RT-qPCR results for five selected genes showed concordant directions of expression change with the corresponding RNA-seq results.

Feed scarcity constrains livestock production, particularly on the Qinghai-Tibet Plateau. The effects of green alfalfa (GA) on Diqing Tibetan pig performance remain unclear. This study aimed to evaluate GA effects on Diqing Tibetan pig performance and to explore the potential underlying mechanisms through integrated metagenomic, transcriptomic, and metabolomic analyses. Thirty-six Diqing Tibetan pigs were randomly assigned to two groups and fed either a basal diet or a diet containing 90% basal diet and 10% GA. GA did not adversely affect growth performance but reduced 6–7 rib backfat thickness and muscle water loss rate by 19.79% (FDR = 0.027) and 17.80% (FDR = 0.036), while increasing muscle moisture content by 3.51% (FDR = 0.036). GA increased cecal microbial alpha diversity, Bacteroidota-related taxa, and functional genes related to lipid and vitamin metabolism, while decreasing Bacillota and Lactobacillus johnsonii. In the longissimus dorsi, TNNI1, MYL2 and MYL3 were upregulated, whereas FOS and FOSB were downregulated; GA increased vanillyl alcohol, L-histidine, LPE (0:0/22:5), and licochalcone B, but decreased glyceryl monostearate, benzaldehyde, cortisol, tryptamine, 4-ethyloctanoic acid, 8-methylnonanoic acid, and purine. Overall, 10% GA reshaped gut microbial, muscle transcriptomic, metabolomic profiles and collectively influenced 6–7 rib backfat thickness and muscle water-holding capacity in Diqing Tibetan pigs.