Moderate Dietary Protein Restriction Optimized Gut Microbiota and Mucosal Barrier in Growing Pig Model

Abstract

Appropriate protein concentration is essential for animal at certain stage. This study evaluated the effects of different percentages of dietary protein restriction on intestinal health of growing pigs. Eighteen barrows were randomly assigned to a normal (18%), low (15%), and extremely low (12%) dietary protein concentration group for 30 days. Intestinal morphology and permeability, bacterial communities, expressions, and distributions of intestinal tight junction proteins, expressions of biomarkers of intestinal stem cells (ISCs) and chymous bacterial metabolites in ileum and colon were detected. The richness and diversity of bacterial community analysis with Chao and Shannon index were highest in the ileum of the 15% crude protein (CP) group. Ileal abundances of Streptococcaceae and Enterobacteriaceae decreased respectively, while beneficial Lactobacillaceae, Clostridiaceae_1, Actinomycetaceae, and Micrococcaceae increased their proportions with a protein reduction of 3 percentage points. Colonic abundances of Ruminococcaceae, Christensenellaceae, Clostridiaceae_1, Spirochaetaceae, and Bacterodales_S24-7_group declined respectively, while proportions of Lachnospiraceae, Prevotellaceae, and Veillonellaceae increased with dietary protein reduction. Concentrations of most bacterial metabolites decreased with decreasing dietary protein concentration. Ileal barrier function reflected by expressions of tight junction proteins (occludin, zo-3, claudin-3, and claudin-7) did not show significant decrease in the 15% CP group while sharply reduced in the 12% CP group compared to that in the 18% CP group. And in the 15% CP group, ileal distribution of claudin-3 mainly located in the cell membrane with complete morphological structure. In low-protein treatments, developments of intestinal villi and crypts were insufficient. The intestinal permeability reflected by serous lipopolysaccharide (LPS) kept stable in the 15% CP group while increased significantly in the 12% CP group. The expression of ISCs marked by Lgr5 slightly increased in ileum of the 15% CP group. Colonic expressions of tight junction proteins declined in extremely low protein levels. In conclusion, moderate protein restriction (15% CP) can optimize the ileal microbiota structure via strengthening beneficial microbial populations and suppressing harmful bacterial growth and altering the function of ileal tight junction proteins as well as epithelial cell proliferation.

Keywords: dietary protein restriction; growing pigs; intestinal microbiota; intestinal stem cell; mucosal barrier.

Figure 1

Effects of low-protein diets on the richness and composition of ileal microbiota in growing pigs at the phylum and family levels. (A) Venn of the operational taxonomic units (OTUs) among different dietary treatments in the ileum. (B) Chao index of the bacterial community in the ileum. (C) Shannon index of the bacterial community in the ileum. (D) Distribution of ileal bacteria at the phylum level with relative abundance higher than 1%. (E) Distribution of ileal bacteria at the family level with relative abundance higher than 1%.

Figure 2

Effects of low-protein diets on the composition of ileal microbiota in growing pigs at the genus level. (A) Distribution of the top 15 ileal bacteria at the genus level. (B) Fisher' exact test bar plot on the genus level between the top 15 ileal bacteria of the 12 and 18% CP groups. (C) Fisher' exact test bar plot on the genus level between the top 15 ileal bacteria of the 15 and 18% CP groups.

Figure 3

Effects of low-protein diets on the richness and composition of colonic microbiota in growing pigs at the phylum and family levels. (A) Venn of the operational taxonomic units (OTUs) among different dietary treatments in the colon. (B) Chao index of the bacterial community in the colon. (C) Shannon index of the bacterial community in the colon. (D) Distribution of colonic bacteria at the phylum level with relative abundance higher than 1%. (E) Distribution of colonic bacteria at the family level with relative abundance higher than 1%.

Figure 4

Effects of low-protein diets on the composition of colonic microbiota in growing pigs at the genus level. (A) Distribution of the top 20 colonic bacteria at the genus level. (B) Fisher' exact test bar plot on the genus level between the top 15 colonic bacteria of the 12 and 18% CP groups. (C) Fisher' exact test bar plot on the genus level between the top 15 colonic bacteria of the 15 and 18% CP groups.

Figure 5

Effects of low-protein diets on intestinal mucosal morphology and permeability of growing pigs (the stained sections were photographed at 200 × magnification). (A) Ileal morphology observation of different dietary protein groups. (B) Colonic morphology observation of different dietary protein groups. (C) Villi height and crypt depth of the ileum. (D) Villi height to crypt depth ratios in the ileum. (E) Colonic crypt depth. (F) Area of colonic epithelial cells. (G) Lipopolysaccharide (LPS) level in the serum. Values are means ± standard deviations (n = 6). Star indicates a significant difference (P < 0.05).

Figure 6

Effects of low-protein diets on the expression of intestinal tight junction proteins and biomarkers of intestinal stem cells of growing pigs. (A) Expression of occludin, ZO-3, claudin-3, and claudin-7 in ileal tissue. (B) Expression of occludin, ZO-3, claudin-1, and claudin-7 in colonic tissue. (C) Expression of Lgr5 and Bmi1 in ileal tissue. (D) Expression of Lgr5 and Bmi1 in colonic tissue. Values are means ± standard deviations (n = 3). Star indicates a significant difference (P < 0.05).

Figure 7

Effects of low-protein diets on staining of claudin-3 in the ileum and colon in growing pigs. (A) Distribution and intensity of claudin-3 staining in ileal tissue. (B) Distribution and intensity claudin-3 in colonic tissue. Values are means ± standard deviations (n = 3). Scale bar: 20 μm. Star indicates a significant difference (P < 0.05).