Effects of Dietary Protein Level on the Microbial Composition and Metabolomic Profile in Postweaning Piglets

Abstract

Since the human and porcine digestive systems have similar anatomical structures and physiological functions, pigs are a useful animal model for studying human digestive diseases. By investigating intestinal metabolites in piglets after weaning, this study attempted to identify the inherent connection between dietary protein levels and changes in the intestinal microbiota of piglets. Casein was employed as the only source of protein for the piglets in this study to avoid the influence of other protein sources. 14 weaning at 28-day-old piglets (6.9 ± 0.19 kg) formed into two dietary groups: 17% casein fed group (LP) and 30% casein fed group (HP). Piglets were allowed to free food and water during the 2-week experiment. Throughout the trial, the piglets' diarrhea index (1: no diarrhea and 3: watery diarrhea) and food intake were noted during the experiment. We discovered piglets fed a high-protein diet developed diarrhea throughout the duration of the research, whereas piglets fed a normal protein diet did not. In addition, the HP group had lower feed intake and body weight than the control group (P < 0.05). The HP diet influenced the content of short-chain and branched-chain fatty acids in the colon, including acetate and isovaleric acid. The ileal microbiota's 16S rRNA gene was sequenced, and it was discovered that the relative abundance of gastrointestinal bacteria differed between the HP and control groups. Dietary protein levels influenced bile acid biosynthesis, alpha-linolenic acid metabolism, phospholipid biosynthesis, arachidonic acid metabolism, fatty acid biosynthesis, retinol metabolism, arginine and proline metabolism, pyrimidine metabolism, tryptophan metabolism, and glycine and serine metabolism, according to gas chromatography-mass spectrometry analysis. Furthermore, a correlation analysis of the pooled information revealed a possible link between intestinal metabolites and specific bacteria species. These findings demonstrate that weaned piglets' microbiota composition and metabolites are modified by a high-protein diet and thus inducing severe postweaning diarrhea and inhibiting growth performance. However, the potential molecular mechanism of this regulation in the growth of piglets remains unclear.

Figure 1

High casein concentration reduced the growth performance of weaned piglets. (a) Body weight change. (b) Food intake. (c) ADG. (d) ADFI. (e) ADFI/ADG. Data are expressed as the mean ± SEM, n = 7.

Figure 2

High casein concentration decreased the concentration of SCFA in weaned piglets. Colon SCFA relative abundance (mmol/l) in postweaning piglets (n = 7). Data are shown as the mean ± SEM, n = 7.

Figure 3

High casein concentration influenced gut microbial diversity of piglets. (a) Observed species; (b) phylogenetic diversity (PD); (c) Shannon H index; (d) Chao1 index. (a) represents the LP group; (b) represents the HP diet group. (e) Genus and (f) phylum in the colon of piglets (n = 7).

Figure 4

High casein concentration influenced biomarker metabolites in piglets. PLS-DA and OPLS-DA of microbial metabolites in colonic contents from piglets fed LP and HP. The (a) PLS-DA score plot is in negative ion mode. The (b) OPLS-DA score plot is in negative ion mode. The (c) PLS-DA score plot is in negative ion mode, and (d) the OPLS-DA score plot is in positive ion mode in different protein treatments. Validation plot of OPLS-DA model in (e) negative ion model and in (f) positive ion mode in the HP protein group. (g) Analysis of metabolic pathway enrichment. An overview of the metabolites that were shown to be abundant in piglets fed the HP diet compared to the LP diet.

Figure 5

The ileum microorganism and the intestinal metabolome correlation analysis in piglets. The color scheme is based on the distribution of Pearson correlation coefficients.