Dietary Supplementation With Citrus Extract Altered the Intestinal Microbiota and Microbial Metabolite Profiles and Enhanced the Mucosal Immune Homeostasis in Yellow-Feathered Broilers

Abstract

The present study aimed to investigate the effects of citrus extract (CE) on intestinal microbiota, microbial metabolite profiles, and the mucosal immune status in broilers. A total of 540 one-day-old yellow-feathered broilers were randomly allotted into three groups and fed a basal diet (control group), or a basal diet containing 10 mg/kg of zinc bacitracin (antibiotic group), or 10 mg/kg of CE (CE group). Each treatment consisted of six replicates, with 30 broilers per replicate. After 63-day feeding, two broilers per replicate were randomly selected and slaughtered, and their ileal and cecal digesta and ileal tissue were collected for microbial composition, microbial metabolites, and gene expression analysis. The results showed that CE significantly increased the abundance of Barnesiella and Blautia than did the antibiotic group (adjusted P < 0.05), whereas it decreased the abundance of Alistipes and Bacteroides (adjusted P < 0.05). Meanwhile, the CE group also increased the numbers of Bifidobacterium and Lactobacillus than did the control and antibiotic groups (P < 0.05), whereas it decreased the number of Escherichia coli (P < 0.05). For microbial metabolites, dietary supplementation with CE increased the concentrations of lactate, total short-chain fatty acids, acetate, and butyrate in the cecum than did the control and antibiotic groups (P < 0.05), whereas it decreased the concentrations of amino acid fermentation products (ammonia, amines, p-cresol, and indole) (P < 0.05). Additionally, supplementation with CE up-regulated (P < 0.05) the mRNA expression of intestinal barrier genes (ZO-1 and Claudin) in the ileum than did both the control and antibiotic groups. However, antibiotic treatment induced gut microbiota dysbiosis, altered the microbial metabolism, and disturbed the innate immune homeostasis. In summary, these results provide evidence that dietary supplementation with CE can improve the intestinal barrier function by changing microbial composition and metabolites, likely toward a host-friendly gut environment. This suggests that CE may possibly act as an efficient antibiotic alternative for yellow-feathered broiler production.

Keywords: citrus extract; immune homeostasis; intestinal microbial community; microbial metabolites; yellow-feathered broilers.

FIGURE 1

(A) Principal coordinates analysis (PCoA) of bacterial communities in the cecal digesta of Chinese yellow-feathered broilers (based on the Bray–Curtis distance). Circles with dashed or solid line indicate that groups were significantly distinct using AMOVA (P < 0.05). (B) Phylum-level relative abundance of 16S rRNA gene sequences from the cecal digesta of Chinese yellow-feathered broilers. CE, citrus extract; AMOVA, analysis of molecular variance.

FIGURE 2

Significantly changed bacteria genera by citrus extract treatment. The values were expressed as the medians (n = 6). Asterisks indicate statistical differences between different groups (Kruskal–Wallis test): ^∗FDR-adjusted P value < 0.05; ^∗∗FDR-adjusted P value < 0.01. CE, citrus extract; FDR, false discovery rate.

FIGURE 3

Significantly changed bacterial OTUs in the cecal digesta by citrus extract treatments, as revealed by LEfSe analysis. Only an LDA score of >2 was considered significant. CE, citrus extract; OTUs, operational taxonomic units; LDA, linear discriminant analysis; LEfSe, LDA effect size.

FIGURE 4

Response of the copy numbers (Log 10 gene copies/g digesta sample) of major bacterial taxonomic groups in the ileal (A) and cecal (B) digesta of Chinese yellow-feathered broilers toward citrus extract treatment. The values are means ± SEM (n = 6). Asterisks indicate statistical differences between different groups (one-way ANOVA with a Tukey post hoc test): ^∗P < 0.05. CE, citrus extract.

FIGURE 5

Effect of the dietary supplementation with citrus extract on the carbohydrate metabolites in the ileal and cecal digesta of Chinese yellow feathered broilers: (A) Ileal lactate, (B) Ileal SCFAs, (C) Cecal lactate, and (D) cecal SCFAs. The values are means ± SEM. Asterisks indicate statistical differences between different groups (one-way ANOVA with a Tukey post hoc test): ^∗P < 0.05. Total SCFA, total short-chain fatty acid; BCFA, branched-chain fatty acid.

FIGURE 6

Effect of the dietary supplementation with citrus extract on the concentrations of amino acid fermentation products in the cecal digesta of Chinese yellow-feathered broilers: (A) ammonia, (B) phenolic and indole compounds, and (C) biogenic amines. The values are means ± SEM. Asterisks indicate statistical differences between different groups (one-way ANOVA with a Tukey post hoc test): ^∗P < 0.05. CE, citrus extract.

FIGURE 7

Effect of the dietary supplementation with citrus extract on the relative mRNA expression of genes related to TLR signaling pathway, cytokines, and barrier function in the ileal tissue of Chinese yellow-feathered broilers. Asterisks indicate statistical differences between different groups (one-way ANOVA with a Tukey post hoc test): ^∗P < 0.05, ^∗∗P < 0.01. CE, citrus extract; TLR, toll-like receptor.

FIGURE 8

Pearson’s correlation analysis between the abundance of ileal microbiota (qPCR), microbial metabolites, and mucosa gene expression level affected by dietary treatment. Cells are colored based on the correlation coefficient between the significantly changed bacteria (the numbers of bacteria), metabolites (concentrations), and mucosal gene expression level. The intensity of the colors represents the degree of association. Red represents a significant positive correlation (P < 0.05), blue represents significantly negative correlation (P < 0.05), and white shows that the correlation was not significant (P > 0.05). Total SCFA, total short-chain fatty acid.