Grape pomace, a by-product of grape winemaking and processing, is rich in polyphenols, dietary fiber, and other bioactive compounds, and has attracted increasing attention as a potential feed resource for ruminants. Previous studies have shown that appropriate supplementation with grape pomace can improve animal growth performance, antioxidant capacity, digestive function, and intestinal microbial structure. However, its effects on intestinal amino acid metabolism in ruminants remain unclear. As the ileum is a key site for amino acid digestion, absorption, and host-microbe interactions, this study used integrated metagenomic and metabolomic analyses to investigate how grape pomace regulates amino acid metabolism in the sheep ileum.

In this experiment, a total of 40 Australian Merino × Hu crossbred sheep were randomly allocated into five groups and fed experimental diets with grape pomace replacing roughage at levels of 0, 30, 60, 90, and 120 g/kg for 60 days. Ileal digesta and liver samples were collected for integrated metagenomic and metabolomic analyses. Differences in ileal microbial composition, functional enzyme profiles, and amino acid-related metabolites among groups were compared. Combined differential and correlation analyses were conducted to investigate the potential pathways by which grape pomace regulates amino acid metabolism in sheep.

Partial replacement of roughage with grape pomace significantly affected local changes in amino acid metabolism in the sheep ileum. Compared with the control group, the GP9 group showed decreased levels of L-lysine, its downstream product pipecolic acid, and L-tryptophan, accompanied by a decrease in hepatic serotonin. Meanwhile, the ileal microbiota of the GP9 group was altered, with the genera Prevotella and Bacteroides being significantly increased. In addition, the changes in ileal microbial genera in sheep fed diets containing 90 g/kg grape pomace replacing part of the roughage were accompanied by alterations in the abundances of functional genes such as glycine cleavage system P protein subunit 2, glycine cleavage system T protein, and homoserine dehydrogenase, reflecting changes in glycine cleavage, one-carbon metabolism, and aspartate-family amino acid metabolism, which in turn affected the utilization of small-molecule amino acid substrates such as glycine in the ileum. These results indicate that the alterations in ileal microbial composition and functional enzymes induced by diets in which grape pomace partially replaced roughage were highly associated with local amino acid metabolic processes in the sheep ileum and the characteristic changes in gut-liver axis-related metabolites.

Metabolomics, Metagenomics, Grape pomace, amino acid, differential functional enzymes