Transcriptome and microbiota analysis reveal differences in the cecum of weaning pigs in response to different dietary crude protein levels

Abstract

Objective: This study explored the effects of different dietary crude protein (CP) levels on the cecal transcriptome and microbial composition in weaning pigs.

Methods: Ninety-six weaning pigs were randomly assigned into three groups designated as H, M, and L groups. The H, M, and L groups were administered 20% and 18%, 18% and 16%, and 16% and 14% CP during the early (1-14 days) and late phases (15-28 days), respectively.

Results: The final body weight and average daily gain in the L group were significantly lower than those in the other groups (p = 0.008). The feed conversion ratio was lower in the H and M groups than in the L group (p = 0.01). Cecal transcriptome analysis revealed that heatmap and principal component analysis of differentially expressed genes indicated the presence of distinct clusters among the groups. Genes associated with cell proliferation and differentiation and inflammation were down-regulated in the M and L groups, compared with corresponding genes in the H group (p<0.001). Pathway enrichment analysis suggested that genes related to IL-17 signaling pathway was down-regulated in the M and L groups (p<0.05). Beta diversity analysis and heatmap for microbial composition and function indicated the presence of distinct clusters among the groups. Carbohydrate-fermenting bacteria, such as Megasphaera elsdenii DSM 20460 and Blautia luti DSM 14534, exhibited higher levels in the M and L groups compared with the H group (p≤0.05). The abundance of Lactobacillus amylovorus DSM 20531 was significantly greater in the M group than in the other groups (p≤0.05). The abundance of L. amylovorus DSM 20531 was positively correlated with growth performance. Integrated multi-omics analysis suggested significant similarities between the cecal transcriptome and microbiota (p<0.01).

Conclusion: Reducing CP levels modulates cell growth and alleviates inflammation in the cecum. A low CP diet causes cecal microbiota composition shift and promotes the proliferation of carbohydrate-fermenting bacteria. Overall, 18% CP in an early phase and 16% CP in a late phase can substantially improve growth and gut health in weaning pigs.

Keywords: Crude Protein; Growth; Microbiota; Transcriptome; Weaning Pig.

Figure 1

Gene expression in the cecum of weaning pigs fed diets containing different CP levels. (A) Volcano plot for differentially expressed genes in the cecum. The x-axis indicates the fold change in gene expression, whereas the y-axis indicates the statistical significance of the difference. (B) Heatmap for differentially expressed genes. Red and green indicate values greater and lower than the mean (average Z score) value, respectively. (C) Principal component analysis plot showing intra- and intergroup variations. H: supplementation of 20% CP in the early phase (1–14 days) and 18% CP in the late phase (15–28 days); M: supplementation of 18% CP in the early phase and 16% CP in the late phase; L: supplementation of 16% CP in the early phase and 14% CP in the late phase. Data were analyzed from 4 replicate pens per treatment. CP, crude protein.

Figure 2

Microbial composition in the cecal digesta of weaning pigs fed diets containing different CP levels. (A) Microbial alpha diversity of cecal digesta. Each bar represents the mean (n = 4). * p<0.05. (B) Venn diagram for the distribution of microbiota (at the species level) in the cecal digesta. The value for each region represents the number of bacteria corresponding to the region. Results of the (C) principal coordinate analysis and (D) nonmetric multidimensional scaling of cecal bacterial communities at the species level. H: supplementation of 20% CP in the early phase (1–14 days) and 18% CP in the late phase (15–28 days); M: supplementation of 18% CP in the early phase and 16% CP in the late phase; L: supplementation of 16% CP in the early phase and 14% CP in the late phase. Data were analyzed from 4 replicate pens per treatment. CP, crude protein.

Figure 3

Correlations and integrated multi-omics analysis. (A) Correlation between growth performance and dominant bacteria. (B) Correlation between cecal short-chain fatty acid levels and dominant bacteria. (C) Correlations among dominant bacteria. (D) Procrustes analysis of the principal component analysis plot, (E) Procrustes analysis of the principal coordinate analysis plot, (F) Procrustes analysis of the nonmetric multidimensional scaling plot. Positive correlations are indicated in blue, whereas negative correlations are indicated in red. Correlation coefficients ranged from 1.0 to −1.0. H: supplementation of 20% CP in the early phase (1–14 days) and 18% CP in the late phase (15–28 days); M: supplementation of 18% CP in the early phase and 16% CP in the late phase; L: supplementation of 16% CP in the early phase and 14% CP in the late phase. Data were analyzed from 4 replicate pens per treatment. ADFI, average daily feed intake; ADG, average daily gain; BW, body weight; FCR, feed conversion ratio; DI, diarrhea incidence; FS, fecal score; CP, crude protein.