Effects of dietary chlorogenic acid on cecal microbiota and metabolites in broilers during lipopolysaccharide-induced immune stress

Abstract

Aims: The aim of this study was to investigate the effects of chlorogenic acid (CGA) on the intestinal microorganisms and metabolites in broilers during lipopolysaccharide (LPS)-induced immune stress.

Methods: A total of 312 one-day-old Arbor Acres (AA) broilers were randomly allocated to four groups with six replicates per group and 13 broilers per replicate: (1) MS group (injected with saline and fed the basal diet); (2) ML group (injected with 0.5 mg LPS/kg and fed the basal diet); (3) MA group (injected with 0.5 mg LPS/kg and fed the basal diet supplemented with 1,000 mg/kg CGA); and (4) MB group (injected with saline and fed the basal diet supplemented with 1,000 mg/kg CGA).

Results: The results showed that the abundance of beneficial bacteria such as Bacteroidetes in the MB group was significantly higher than that in MS group, while the abundance of pathogenic bacteria such as Streptococcaceae was significantly decreased in the MB group. The addition of CGA significantly inhibited the increase of the abundance of harmful bacteria such as Streptococcaceae, Proteobacteria and Pseudomonas caused by LPS stress. The population of butyric acid-producing bacteria such as Lachnospiraceae and Coprococcus and beneficial bacteria such as Coriobacteriaceae in the MA group increased significantly. Non-targeted metabonomic analysis showed that LPS stress significantly upregulated the 12-keto-tetrahydroleukotriene B4, riboflavin and mannitol. Indole-3-acetate, xanthurenic acid, L-formylkynurenine, pyrrole-2-carboxylic acid and L-glutamic acid were significantly down-regulated, indicating that LPS activated inflammation and oxidation in broilers, resulting in intestinal barrier damage. The addition of CGA to the diet of LPS-stimulated broilers significantly decreased 12-keto-tetrahydro-leukotriene B4 and leukotriene F4 in arachidonic acid metabolism and riboflavin and mannitol in ABC transporters, and significantly increased N-acetyl-L-glutamate 5-semialdehyde in the biosynthesis of amino acids and arginine, The presence of pyrrole-2-carboxylic acid in D-amino acid metabolism and the cecal metabolites, indolelactic acid, xanthurenic acid and L-kynurenine, indicated that CGA could reduce the inflammatory response induced by immune stress, enhance intestinal barrier function, and boost antioxidant capacity.

Conclusion: We conclude that CGA can have a beneficial effect on broilers by positively altering the balance of intestinal microorganisms and their metabolites to inhibit intestinal inflammation and barrier damage caused by immune stress.

Keywords: broilers; chlorogenic acid; gut metabolites; gut microbiota; immune stress.

Figure 1

Effect of dietary chlorogenic acid supplementation on cecal microbiota diversity in lipopolysaccharide-challenged broilers. (A) Rarefaction curve. (B) Species accumulation curves. (C) Community diversity and richness. (D) Two-dimensional OTU abundance-based principal coordinate analysis (PCoA) of cecal microbiota. (E) Analysis of bacterial community structure between groups based on permanova test. *Means significant difference between groups (p < 0.05). **Means statistically significant difference between groups (p < 0.01).

Figure 2

Effect of dietary chlorogenic acid on cecal microbiota composition of lipopolysaccharide-challenged broilers. (A) Venn diagram of the composition of bacterial OTUs. (B) Microbial composition at the phylum level. (C) Microbial composition at the genus level. Differences between the cecal microbiota of MS and MB groups of broilers (D), (E) MS and ML groups, and (F) ML and MA groups were determined by linear discriminant analysis effect size (LEfSe).

Figure 3

Prediction of microbial function in the broiler cecum. The second level of the KEGG pathway is shown in the extended error bar.

Figure 4

Partial least squares-discriminant analysis (PLS-DA) scores of cecum metabolites. Includes positive and negative ion modes. (A, B) Model parameters for MS and MB groups (positive ions, R2Y = 0.992, Q2 = 0.643; negative ions, R2Y = 0.997, Q2 = 0.796). (C, D) Model parameters for the MS and ML groups (positive ions, R2Y = 0.999, Q2 = 0.488; negative ions, R2Y = 0.97, Q2 = 0.309). (E, F) Model parameters for the ML and MA groups (positive ions, R2Y = 0.959, Q2 = −0.00296; negative ions, R2Y = 0.999, Q2 = 0.68).

Figure 5

Volcano plots of differential metabolites in the cecum of broilers. Each point represents a kind of metabolite, and the scatter color indicates the final screening result. Significant upregulated metabolites are indicated in red (Up), downregulated metabolites are indicated in green (Down), and nonsignificant differences in metabolites are gray (Nodiff). (A, B) Differential metabolite changes in MS and MB groups in positive and negative ion modes. (C, D) Differential metabolite changes in MS and ML groups in positive and negative ion modes. (E, F) Differential metabolite changes in ML and MA groups in positive and negative ion modes.

Figure 6

KEGG pathway enrichment analysis of differential metabolites. The enrichment factor is the ratio of the number of differential metabolites annotated to the pathway to all the metabolites annotated to the pathway. The color of the dots represents the p-value of the hypergeometric test. KEGG enrichment analysis of differentially expressed metabolites in (A) MS and MB groups, (B) MS and ML groups, and (C) ML and MA groups.