Dietary supplementation with Chinese herb ultrafine powder improves intestinal morphology and physical barrier function by altering jejunal microbiota in laying hens

Abstract

Introduction: Chinese medicinal herbs play important roles in anti-inflammatory, antioxidant, and antibacterial activities. However, the effects of Chinese herb ultrafine powder (CHUP) on laying hens still need to be elucidated. Therefore, this study aimed to evaluate the effects of dietary CHUP supplementation on jejunal morphology, physical barrier function, and microbiota in laying hens.

Methods: A total of 576 Xinyang black-feather laying hens (300 days old) were randomly assigned into eight groups, with eight replicates per group and nine hens per replicate. The hens were fed a basal diet (control group) and a basal diet supplemented with 0.5% Leonuri herba (LH group), 0.25% Ligustri lucidi fructus (LF group), 0.25% Taraxaci herba (TH group), 0.5% LH + 0.25% LF (LH-LF group), 0.5% LH + 0.25% TH (LH-TH group), 0.25% LF + 0.25% TH (LF-TH group), and 0.5% LH + 0.25% LF + 0.25% TH (LH-LF-TH group), respectively, for 120 days.

Results: The results showed that dietary LH-LF and LH-LF-TH supplementation increased (p < 0.05) the jejunal villus height to crypt depth ratio of laying hens. Dietary LF-TH supplementation up-regulated jejunal claudin-5 expression, while LH supplementation up-regulated jejunal claudin-1 expression and increased the jejunal abundances of potentially beneficial bacteria related to short-chain fatty acids and bacteriocins production, such as Blautia, Carnobacterium, Clostridiales, and Erysipelotrichales (p < 0.05). In addition, dietary LH supplementation enriched (p < 0.05) the tetracycline biosynthesis, butirosin/neomycin biosynthesis, and D-arginine/D-ornithine metabolism, whereas steroid biosynthesis and limonene/pinene degradation were enriched (p < 0.05) in the LH-LF and LH-LF-TH groups. Moreover, Spearman's correlation analysis revealed the potential correlation between the abundance of the jejunal microbiota and jejunal morphology and the physical barrier function of laying hens.

Discussion: Collectively, these findings suggest that dietary CHUP supplementation could enhance the beneficial bacteria abundance, physical barrier function, and metabolic function associated with short-chain fatty acids and bacteriocins production. Moreover, combined supplementation of dietary CHUP showed better effects than the sole CHUP supplementation.

Keywords: Chinese herb; Xinyang black-feather hens; intestinal barrier function; microbiota; ultrafine powder.

Figure 1

Effects of dietary Chinese herb ultrafine powder supplementation on jejunal morphology of laying hens (HE staining). Scale bars, 500 μm (magnification 20×). Control group, fed a basal diet; LH group, basal diet supplemented with 0.5% Leonuri herba; LF group, basal diet supplemented with 0.25% Ligustri lucidi fructus; TH group, basal diet supplemented with 0.25% Taraxaci herba; LH-LF group, basal diet supplemented with 0.5% LH + 0.25% LF; LH-TH group, basal diet supplemented with 0.5% LH + 0.25% TH; LF-TH group, basal diet supplemented with 0.25% LF + 0.25% TH; LH-LF-TH group, basal diet supplemented with 0.5% LH + 0.25% LF + 0.25% TH.

Figure 2

Effects of dietary Chinese herb ultrafine powder supplementation on physical barrier function-related gene expressions in the jejunum of laying hens (n = 8). ^a–c Different superscript letters in the histogram indicate significant differences among different groups (p < 0.05). MUC-2, mucin-2; ZO-1, zonula occludens-1. CON, control group, fed a basal diet; LH group, basal diet supplemented with 0.5% Leonuri herba; LF group, basal diet supplemented with 0.25% Ligustri lucidi fructus; TH group, basal diet supplemented with 0.25% Taraxaci herba; LH-LF group, basal diet supplemented with 0.5% LH + 0.25% LF; LH-TH group, basal diet supplemented with 0.5% LH + 0.25% TH; LF-TH group, basal diet supplemented with 0.25% LF + 0.25% TH; LH-LF-TH group, basal diet supplemented with 0.5% LH + 0.25% LF + 0.25% TH.

Figure 3

Effects of dietary Chinese herb ultrafine powder supplementation on alpha diversity indices of jejunal microbiota of laying hens (n = 7–8). *p < 0.05, **p < 0.01. CON, control group, fed a basal diet; LH group, basal diet supplemented with 0.5% Leonuri herba; LF group, basal diet supplemented with 0.25% Ligustri lucidi fructus; TH group, basal diet supplemented with 0.25% Taraxaci herba; LH-LF group, basal diet supplemented with 0.5% LH + 0.25% LF; LH-TH group, basal diet supplemented with 0.5% LH + 0.25% TH; LF-TH group, basal diet supplemented with 0.25% LF + 0.25% TH; LH-LF-TH group, basal diet supplemented with 0.5% LH + 0.25% LF + 0.25% TH.

Figure 4

Effects of dietary Chinese herb ultrafine powder supplementation on β-diversity of jejunal microbiota of laying hens (n = 7–8). Principal coordinate analysis (PCoA) of microbiota based on Jaccard distances. CON, control group, fed a basal diet; LH group, basal diet supplemented with 0.5% Leonuri herba; LF group, basal diet supplemented with 0.25% Ligustri lucidi fructus; TH group, basal diet supplemented with 0.25% Taraxaci herba; LH-LF group, basal diet supplemented with 0.5% LH + 0.25% LF; LH-TH group, basal diet supplemented with 0.5% LH + 0.25% TH; LF-TH group, basal diet supplemented with 0.25% LF + 0.25% TH; LH-LF-TH group, basal diet supplemented with 0.5% LH + 0.25% LF + 0.25% TH.

Figure 5

Effects of dietary Chinese herb ultrafine powder supplementation on microbial community composition in the jejunum of laying hens at the phylum level (n = 7–8). (A) Taxonomic composition at the phylum level, and (B) taxonomic differences among different groups. ^a,b Different superscript letters in the histogram indicate significant differences among different groups (p < 0.05). CON, control group, fed a basal diet; LH group, basal diet supplemented with 0.5% Leonuri herba; LF group, basal diet supplemented with 0.25% Ligustri lucidi fructus; TH group, basal diet supplemented with 0.25% Taraxaci herba; LH-LF group, basal diet supplemented with 0.5% LH + 0.25% LF; LH-TH group, basal diet supplemented with 0.5% LH + 0.25% TH; LF-TH group, basal diet supplemented with 0.25% LF + 0.25% TH; LH-LF-TH group, basal diet supplemented with 0.5% LH + 0.25% LF + 0.25% TH.

Figure 6

Effects of dietary Chinese herb ultrafine powder supplementation on microbial community composition in the jejunum of laying hens at the family level (n = 7–8). (A) Taxonomic composition at the family level, and (B) taxonomic differences among different groups. ^a–d Different superscript letters in the histogram indicate significant differences among different groups (p < 0.05). CON, control group, fed a basal diet; LH group, basal diet supplemented with 0.5% Leonuri herba; LF group, basal diet supplemented with 0.25% Ligustri lucidi fructus; TH group, basal diet supplemented with 0.25% Taraxaci herba; LH-LF group, basal diet supplemented with 0.5% LH + 0.25% LF; LH-TH group, basal diet supplemented with 0.5% LH + 0.25% TH; LF-TH group, basal diet supplemented with 0.25% LF + 0.25% TH; LH-LF-TH group, basal diet supplemented with 0.5% LH + 0.25% LF + 0.25% TH.

Figure 7

Effects of dietary Chinese herb ultrafine powder supplementation on microbial community composition in the jejunum of laying hens at the genus levels (n = 7–8). (A) Taxonomic composition at the genus levels, and (B) taxonomic differences among different groups. ^a–c Different superscript letters in the histogram indicate significant differences among different groups (p < 0.05). CON, control group, fed a basal diet; LH group, basal diet supplemented with 0.5% Leonuri herba; LF group, basal diet supplemented with 0.25% Ligustri lucidi fructus; TH group, basal diet supplemented with 0.25% Taraxaci herba; LH-LF group, basal diet supplemented with 0.5% LH + 0.25% LF; LH-TH group, basal diet supplemented with 0.5% LH + 0.25% TH; LF-TH group, basal diet supplemented with 0.25% LF + 0.25% TH; LH-LF-TH group, basal diet supplemented with 0.5% LH + 0.25% LF + 0.25% TH.

Figure 8

Effects of dietary Chinese herb ultrafine powder supplementation on differential bacterial taxa in the jejunum of laying hens (n = 7–8). (A) Taxonomic cladogram and (B) histogram of the distribution of LDA values for different species identified by the LEfSe algorithm. Species with significant difference have an LDA score greater than the estimated value (2.0). CON, control group, fed a basal diet; LH group, basal diet supplemented with 0.5% Leonuri herba; LF group, basal diet supplemented with 0.25% Ligustri lucidi fructus; TH group, basal diet supplemented with 0.25% Taraxaci herba; LH-LF group, basal diet supplemented with 0.5% LH + 0.25% LF; LH-TH group, basal diet supplemented with 0.5% LH + 0.25% TH; LF-TH group, basal diet supplemented with 0.25% LF + 0.25% TH; LH-LF-TH group, basal diet supplemented with 0.5% LH + 0.25% LF + 0.25% TH.

Figure 9

Differences in metabolic functions based on PICRUSt2 analysis of jejunal microbiota of laying hens. (A) CON vs. LH, (B) CON vs. LF, (C) CON vs. TH, (D) CON vs. LH-LF, (E) CON vs. LH-TH, (F) CON vs. LF-TH, and (G) CON vs. LH-LF-TH. CON, control group, fed a basal diet; LH group, basal diet supplemented with 0.5% Leonuri herba; LF group, basal diet supplemented with 0.25% Ligustri lucidi fructus; TH group, basal diet supplemented with 0.25% Taraxaci herba; LH-LF group, basal diet supplemented with 0.5% LH + 0.25% LF; LH-TH group, basal diet supplemented with 0.5% LH + 0.25% TH; LF-TH group, basal diet supplemented with 0.25% LF + 0.25% TH; LH-LF-TH group, basal diet supplemented with 0.5% LH + 0.25% LF + 0.25% TH.

Figure 10

Spearman’s correlation analysis between jejunal morphology, gene expression level, and the microbiota abundances. Red represents a significant positive correlation, and blue represents a significant negative correlation. *p < 0.05. VH, villus height; CD, crypt depth; VCR, VH to CD ratio; MUC-2, mucin-2; ZO-1, zonula occludens-1.