Effects of dustbathing environment on gut microbiota and expression of intestinal barrier and immune-related genes of adult laying hens housed individually in modified traditional cage

Abstract

Litters, the composition of sand and peat rich in microbiota, are essential to trigger the dustbathing behavior of chickens. To investigate the effects of a dustbathing environment (DE) on the intestinal health, gut microbiota, and immune responses of laying hens, a total of 72 healthy Hy-Line Brown laying hens at 69 wk of age (WOA) were housed individually in modified traditional cages and randomly divided into 2 groups: one group had free access to litters (CT), while the other one was restricted from litters (CC). The experiment lasted for 42 d. At the end of the experiment, the intestinal histomorphology and immune status of laying hens were determined, and the 16S rRNA sequencing method was used to assess the composition of the intestinal microbial community of birds. Intestinal histomorphology changed, including villus height and villus-to-crypt ratio significantly increased in the CT group (P < 0.01). DE reshaped the microbial community and increased the microbial richness with the higher indicators of Chao1 and observed species and the comparatively abundant beta diversity (P < 0.05). Ten genera, including Faecalibacterium and Coprococcus, declined in laying hens from the CT group (P < 0.05), while Alistipes increased in CT hens (P < 0.05) compared to those hens from the CC group. The expression levels of intestinal barrier-related genes of claudin-1, claudin-4, occludin, ZO-1, and ZO-2 and immune-related genes of IL-4, IL-6, IL-8, IFN-γ, IgA, TLR-2, and TLR-4 were significantly upregulated in the intestine of laying hens in CT group (P < 0.05). DE also increased the serum levels of IL-4, IL-6, IL-8, IFN-γ, and IgA (P < 0.01). The alteration of the gut microbiota by DE is closely related to host immune responses, including Lactobacillus positively correlated with IL-4 and IgA. Thus, a dustbathing environment can improve the welfare of laying hens by changing the intestinal histomorphology, immune response, and the gut microbial community.

Keywords: dustbathing environment; immune response; intestinal microbiota; laying hen; modified traditional cage.

Figure 1

A front view of a modified traditional cage.

Figure 2

Morphological alteration of jejunum in laying hens subjected to DE. (A) Magnification: 50×, CC group; (B) magnification: 50×, CT group; (C) and (D) is villus height and villus to crypt ratio of ileum in 2 groups, *, and ** for P < 0.05, and P < 0.01, respectively.

Figure 3

Venn diagram and rarefaction curve of different groups. (A) Venn analysis of ASVs from CC and CT groups. (B) The rarefaction curve of all the birds in 2 groups.

Figure 4

Comparison of the microbial diversity index between 2 groups. Alpha diversity analysis of the microbiota of the 2 groups was compared according to the microbial diversity index: (A) Chao1, (B) observed species, (C) Simpson, (D) Shannon, and (E) coverage between 2 groups, *, and ** for P < 0.05, and P < 0.01, respectively. The beta diversity of the microbiota of the 2 groups was analyzed by (E) principal coordinates analysis (PCoA) and (F) nonmetric multidimensional scaling (NMDS).

Figure 5

The microbial community structure. (A) Different hens at the phylum level. (B) Different hens at the genus level.

Figure 6

LEfSe analysis generated differences in the abundance of the bacterial taxa of CT group (red) and CC group (blue) hens (P < 0.05, LDA > 2).

Figure 7

Relative mRNA expression levels of intestinal barrier-related and immune-related genes in the ileum of laying hens. (A) Intestinal barrier-related genes. (B) Immune-related genes. (C) TLR genes. (D) Serum cytokine concentrations in chickens. * for P < 0.05, ** and *** for P < 0.01, and P < 0.001, respectively.

Figure 8

A heatmap showing correlations between predominant genera in the intestine and immune indicators in serum, * for P < 0.05, ** and *** for P < 0.01, and P < 0.001, respectively.