Responses of Intestinal Antioxidant Capacity, Morphology, Barrier Function, Immunity, and Microbial Diversity to Chlorogenic Acid in Late-Peak Laying Hens

Abstract

This study examined the influence of chlorogenic acid (CGA) on gut antioxidant status, morphology, barrier function, immunity, and cecal microbiota in late-peak laying hens. A total of 240 Hy-Line Brown hens, aged 43 weeks, were randomly assigned to four groups, the basal diet +0, 400, 600, and 800 mg/kg CGA, for 12 weeks. The results revealed that CGA significantly reduced ileal H₂O₂ and malondialdehyde levels; increased duodenal height, ileal villus height, and villus height-to-crypt depth ratio; while decreasing jejunal crypt depth. The 600 and 800 mg/kg CGA significantly upregulated the duodenal, jejunal, and ileal ZO-1 and occludin gene expression; increased IgG levels in serum and ileum; and upregulated ileal IgA gene expression. The 600 mg/kg CGA significantly upregulated CD3D and CD4 gene expression, while downregulating IL-1β gene expression in duodenum, jejunum, and ileum. Moreover, CGA changed the gut microbiota structure. The SCFA-producing bacteria unclassified_f__Peptostreptococcaceae, unclassified_f_Oscillospiraceae, Pseudoflavonifractor, Lachnospiraceae_FCS020_group, Oscillospira, Elusimicrobium, Eubacterium_ventriosum_group, Intestinimonas, and norank_f_Coriobacteriales_Incertae_Sedis were significantly enriched in the 400, 600, and/or 800 mg/kg CGA groups. The bacteria Lactobacillus, Bacillus, and Akkermansia were significantly enriched in the 600 mg/kg CGA group. Conclusively, dietary CGA (600-800 mg/kg) improved intestinal antioxidant status, morphology, barrier and immune function, and beneficial microbiota growth in late-peak laying hens.

Keywords: barrier function; cecal microbiota; chlorogenic acid; immune response; laying hen.

Figure 1

Effects of CGA supplementation on the intestinal morphology in laying hens. Scale bar = 500 μm. CGA: chlorogenic acid.

Figure 2

Effects of CGA on intestinal barrier function-related gene expression in laying hens. Data are expressed as the mean value accompanied by SEM (n = 12). ^a–c Bar charts annotated with distinct superscript letters indicate statistically significant differences (p < 0.05). Abbreviation: ZO-1: zonula occludens-1.

Figure 3

Effects of CGA on AHR/IL-22/STAT3 signaling pathway in the intestinal tract of laying hens. Data are expressed as the mean value accompanied by SEM (n = 12). ^a–c Bar charts annotated with distinct superscript letters indicate statistically significant differences (p < 0.05). Abbreviation: IL-22: interleukin-22; AHR: aryl hydrocarbon receptor; STAT3: signal transducer and activator of transcription 3.

Figure 4

Effects of CGA on immune factor levels in serum (A) and ileum (B) of laying hens. Data are expressed as the mean value accompanied by SEM (n = 12). * p < 0.05 and ** p < 0.01 indicate a significant difference compared to the control group; ## p < 0.01 indicates a significant difference compared to the group of 400 mg/kg CGA.

Figure 5

Effects of CGA on intestinal immune-related gene expression in laying hens: (A) duodenum; (B) jejunum; (C) ileum. Data are expressed as the mean value accompanied by SEM (n = 12). ^a–c Bar charts annotated with distinct superscript letters indicate statistically significant differences (p < 0.05). Abbreviation: IgA: immunoglobulin A; IL-1β: interleukin-1 beta; INF-γ: interferon-gamma; NF-κB: nuclear factor-kappa B.

Figure 6

Effects of CGA on microbial diversity in the cecum of laying hens: (A–C) Alpha diversity index analysis boxplot. (D) PCoA and (E) NMDS analysis of the cecum microbiota based on the bary_surtis metric. Venn diagrams of ASV (F,G) and genus (H,I) distribution in different groups. n = 7 hens per group (at least 1 hen per replicate). Con: control; CGA 400: 400 mg/kg chlorogenic acid; PCoA: principal coordinate analysis; NMDS: non-metric multidimensional scaling; ASV: amplicon sequence variant.

Figure 7

Bar graph illustrating the relative abundance of species at both the phylum (A) and genus levels (B). (C) Heatmap of species relative abundance clustering. n = 7 hens per group (at least 1 hen per replicate). Con: control group; CGA 400: 400 mg/kg CGA group; CGA 600: 600 mg/kg CGA group; CGA 800: 800 mg/kg CGA group.

Figure 8

The species with differences in abundance between the control group and CGA addition groups ((A) 400 mg/kg CGA, (B) 600 mg/kg CGA, (C) 800 mg/kg CGA). n = 7 hens per group (at least 1 hen per replicate). Con: control group; CGA 400: 400 mg/kg CGA group; CGA 600: 600 mg/kg CGA group; CGA 800: 800 mg/kg CGA group. * p  < 0.05, ** p  < 0.01.

Figure 9

Taxonomic cladogram obtained from LEfSe analysis (A). Displayed are taxa exhibiting an LDA score exceeding 2.5 (B). n = 7 hens per group (at least 1 hen per replicate). CGA 400: 400 mg/kg CGA group; CGA 600: 600 mg/kg CGA group; CGA 800: 800 mg/kg CGA group.