Modulation of microbial communities and mucosal gene expression in chicken intestines after galactooligosaccharides delivery In Ovo

Abstract

Intestinal mucosa is the interface between the microbial content of the gut and the host's milieu. The goal of this study was to modulate chicken intestinal microflora by in ovo stimulation with galactooligosaccharides (GOS) prebiotic and to demonstrate the molecular responses of the host. The animal trial was performed on meat-type chickens (Ross 308). GOS was delivered by in ovo injection performed into the air cell on day 12 of egg incubation. Analysis of microbial communities and mucosal gene expression was performed at slaughter (day 42 post-hatching). Chyme (for DNA isolation) and intestinal mucosa (for RNA isolation) from four distinct intestinal segments (duodenum, jejunum, ileum, and caecum) was sampled. The relative abundance of Bifidobacterium spp. and Lactobacillus spp. in DNA isolated from chyme samples was determined using qPCR. On the host side, the mRNA expression of 13 genes grouped into two panels was analysed with RT-qPCR. Panel (1) included genes related to intestinal innate immune responses (IL-1β, IL-10 and IL-12p40, AvBD1 and CATHL2). Panel (2) contained genes involved in intestinal barrier function (MUC6, CLDN1 and TJAP1) and nutrients sensing (FFAR2 and FFAR4, GLUT1, GLUT2 and GLUT5). GOS increased the relative abundance of Bifidobacterium in caecum (from 1.3% to 3.9%). Distinct effects of GOS on gene expression were manifested in jejunum and caecum. Cytokine genes (IL-1β, IL-10 and IL-12p40) were up-regulated in the jejunum and caecum of the GOS-treated group. Host defence peptides (AvBD1 and CATHL2) were up-regulated in the caecum of the GOS-treated group. Free fatty acid receptors (FFAR2 and FFAR4) were up-regulated in all three compartments of the intestine (except the duodenum). Glucose transporters were down-regulated in duodenum (GLUT2 and GLUT5) but up-regulated in the hindgut (GLUT1 and GLUT2). In conclusion, GOS delivered in ovo had a bifidogenic effect in adult chickens. It also modulated gene expression related to intestinal immune responses, gut barrier function, and nutrient sensing.

Fig 1. The relative abundance of Bifidobacterium spp.

(A) and Lactobacillus spp. (B) in chyme from different sections of GIT in chickens supplemented in ovo with GOS prebiotic. GOS was delivered in ovo on day 12 of egg incubation. Samples of intestinal content were collected from 42-days-old chickens. Intestinal content was sampled from four distinct intestinal segments (duodenum, jejunum, ileum, and caecum) (n = 10). Bacteria quantification was done with qPCR based on bacterial DNA isolated from chyme. Statistical analysis was performed with two-way ANOVA with a Tukey HSD post hoc test. Significant differences found at P < 0.05.

Fig 2. A heat map of hierarchically clustered gene expression in different segments of intestinal mucosa in chicken treated with GOS in ovo.

Intestinal segments: DUO–duodenum, JEJ–jejunum, ILE–ileum, and CEC–caecum. In ovo injection was carried out on day 12 of egg incubation. Intestinal samples (n = 10) collected from chickens on day 42 post-hatching. RT-qPCR data were generated with custom-designed primers used for amplification with SYBR green dye; Glucose-6-phosphate dehydrogenase (G6PDH) and beta-actin (ACTB) were used as reference genes; relative gene expression (fold change) calculated as 2^–ΔΔCt. A Multiexperiment Viewer version 4.9 (MeV) was used for constructing a Hierarchical Cluster Tree based on fold change. Colours (red-black-green) show relative gene expression changes in GOS vs. C (red: down-regulated, green: up-regulated genes).

Fig 3. Relative mRNA expression of intestinal immune-related genes in different segments of intestinal mucosa in chickens injected in ovo with GOS.

The panel includes genes encoding cytokines, (A) IL-1β, (B) IL10 and (C) IL12p40; and host defence peptides, (D) AvBD1 and (E) CATHL. Intestinal segments: DUO–duodenum, JEJ–jejunum, ILE–ileum, and CEC–caecum. In ovo treatment groups: control (white bars), injected in ovo with physiological saline; GOS (black bars)–injected in ovo with galactooligosaccharides. In ovo injection was carried out on day 12 of egg incubation. Intestinal samples (n = 10) were collected from chickens on day 42 post-hatching. RT-qPCR data were generated with custom-designed primers used for amplification with SYBR green dye; Glucose-6-phosphate dehydrogenase (G6PDH) and beta-actin (ACTB) were used as reference genes; relative gene expression calculated as 2^–ΔΔCt; Two-way ANOVA with post hoc HSD Tukey test was used to compare the groups. Asterisk indicates pair-wise significant differences (P < 0.05). The results of fold induction less than 1 have been transformed by the formula -1/fold induction.

Fig 4. Relative mRNA expression of barrier function and nutrient sensing genes in different segments of intestinal mucosa in chickens injected in ovo with GOS.

The panel includes genes encoding mucin, (A) MUC6; tight junctions components, (B) CLDN1 and (C) TJAP1; free fatty acid receptors, (D) FFAR2 and (E) FFAR4; and glucose transporters, (F) GLUT1, (G) GLUT2 and (H) GLUT5. Intestinal segments: DUO–duodenum, JEJ–jejunum, ILE–ileum and CEC–caecum. In ovo treatment groups: control (white bars)–injected in ovo with physiological saline; GOS (black bars)–injected in ovo with galactooligosaccharides. In ovo injection was carried out on day 12 of egg incubation. Intestinal samples (n = 10) were collected from chickens on day 42 post-hatching. RT-qPCR data were generated with custom-designed primers used for amplification with SYBR green dye; Glucose-6-phosphate dehydrogenase (G6PDH) and beta-actin (ACTB) were used as reference genes; relative gene expression calculated as 2^–ΔΔCt; Two-way ANOVA with a post hoc HSD Tukey test was used to compare the groups. Asterisk indicates pair-wise significant differences (P < 0.05). The results of fold induction less than 1 have been transformed by the formula -1/fold induction.