Effects of Dietary Protein Levels on Sheep Gut Metabolite Profiles during the Lactating Stage

Diet-associated characteristics such as dietary protein levels can modulate the gut's primary or secondary metabolites, leading to effects on the productive performance and overall health of animals. Whereas fecal metabolite changes are closely associated with gut metabolome, this study aimed to see changes in the rumen metabolite profile of lactating ewes fed different dietary protein levels. For this, eighteen lactating ewes (approximately 2 years old, averaging 38.52 ± 1.57 kg in their initial body weight) were divided into three groups (n = 6 ewes/group) by following the complete randomized design, and each group was assigned to one of three low-protein (D_I), medium-protein (D_m), and high-protein (D_h) diets containing 8.58%, 10.34%, and 13.93% crude protein contents on a dry basis, respectively. The fecal samples were subjected to untargeted metabolomics using ultra-performance liquid chromatography (UPLC). The metabolomes of the sheep fed to the high-protein-diet group were distinguished as per principal-component analysis from the medium- and low-protein diets. Fecal metabolite concentrations as well as their patterns were changed by feeding different dietary protein levels. The discriminating metabolites between groups of nursing sheep fed different protein levels were identified using partial least-squares discriminant analysis. The pathway enrichment revealed that dietary protein levels mainly influenced the metabolism-associated pathways (n = 63 and 39 in positive as well as negative ionic modes, respectively) followed by protein (n = 15 and 8 in positive as well as negative ionic modes, respectively) and amino-acid (n = 14 and 7 in positive as well as negative ionic modes, respectively) synthesis. Multivariate and univariate analyses showed comparative changes in the fecal concentrations of metabolites in both positive and negative ionic modes. Major changes were observed in protein metabolism, organic-acid biosynthesis, and fatty-acid oxidation. Pairwise analysis and PCA reveal a higher degree of aggregation within the D-h group than all other pairs. In both the PCA and PLS-DA plots, the comparative separation among the D_h/D_m, D_h/D_I, and D_m/D_I groups was superior in positive as well as negative ionic modes, which indicated that sheep fed higher protein levels had alterations in the levels of the metabolites. These metabolic findings provide insights into potentiated biomarker changes in the metabolism influenced by dietary protein levels. The target identification may further increase our knowledge of sheep gut metabolome, particularly regarding how dietary protein levels influence the molecular mechanisms of nutritional metabolism, growth performance, and milk synthesis of sheep.