Relative Effects of Dietary Administration of a Competitive Exclusion Culture and a Synbiotic Product, Age and Sampling Site on Intestinal Microbiota Maturation in Broiler Chickens

Abstract

In this research, the effects of early post-hatch inoculation of a competitive exclusion product (Br) and the continuous feeding of a synbiotic supplement (Sy) containing probiotic bacteria, yeast, and inulin on the production traits and composition of ileal chymus (IC), ileal mucosa (IM), and caecal chymus (CC) microbiota of broiler chickens were evaluated. The dietary treatments had no significant effects on the pattern of intestinal microbiota or production traits. The digestive tract bacteriota composition was affected mostly by the sampling place and age of birds. The dominant family of IC was Lactobacillaceae, without change with the age. The abundance of the two other major families, Enterococcaceae and Lachnospiraceae decreased with the age of birds. In the IM, Clostridiaceae was the main family in the first three weeks. Its ratio decreased later and Lactobacillaceae became the dominant family. In the CC, Ruminococcaceae and Lachnospiraceae were the main families with decreasing tendency in the age. In IC, Br treatment decreased the abundance of genus Lactobacillus, and both Br and Sy increased the ratio of Enterococcus at day 7. In all gut segments, a negative correlation was found between the IBD antibody titer levels and the ratio of genus Leuconostoc in the first three weeks, and a positive correlation was found in the case of Bifidobacterium, Rombutsia, and Turicibacter between day 21 and 40.

Keywords: Broilact; IBD antibody titre; caecal chymus; competitive exclusion; gut microbiota development; ileal chymus; ileal mucosa; synbiotic.

Figure 1

Effects of dietary treatments (C—Control, Br—Broilact^®, Sy—Synbiotic feed additive) and age of birds on the alpha diversity of ileal chymus (IC), ileal mucosa (IM), and caecal chymus (CC) microbiota in broiler chickens. Boxplots representing alpha diversity by Chao1 estimator, Shannon, and Simpson indexes. Boxes represent the median, the 25th, and the 75th percentiles of the groups. Alpha diversity indices at different taxonomical levels and in different intestinal samples (IC, IM, and CC) were compared using two-way ANOVA test with Tukey’s HSD multiple group comparison’s post hoc test, using the dietary treatments (C, Br and Sy) and the age of birds (7, 14, 21, and 40 day) as main factors. The differences were considered significant at a level of p ≤ 0.05. Dots represent the outlier values.

Figure 2

Principal coordinate analysis (PCoA) based on Bray—Curtis dissimilarity matrix on sampling sites: (A) Ileal chymus age effect; (B) Ileal chymus dietary treatment effect at d 7; (C) Ileal mucosa age effect; (D) Caecal chymus age effect. Dietary treatments were C—Control, Br—Broilact^®, Sy—Synbiotic feed additive. The percentage of variation explained by each PCoA is indicated on the axes with Bray—Curtis dissimilarity. To verify the significance of bacterial community analysis of similarities (ANOSIM) calculations were performed with 999 permutations. The differences were considered significant at a level of p ≤ 0.05.

Figure 3

The relative abundance of microbiota at phylum level in the ileal chymus (IC), ileal mucosa (IM), and caecal chymus (CC) as affected by the age of chickens and dietary treatments (C—Control, Br—Broilact^®, Sy—Synbiotic feed additive). Microbial composition at different taxonomical levels and in different intestinal samples (IC, IM and CC) were compared using two-way ANOVA test with Tukey’s HSD multiple group comparison’s post hoc test, using the dietary treatments (C, Br and Sy) and the age of birds (7, 14, 21, and 40 day) as main factors. The differences were considered significant at a level of p ≤ 0.05.

Figure 4

The relative abundance of microbiota at family level in the ileal chymus (IC), ileal mucosa (IM), and caecal chymus (CC) as affected by the age of chickens. Microbial composition at different taxonomical levels and in different intestinal samples (IC, IM, and CC) were compared using two-way ANOVA test with Tukey’s HSD multiple group comparison’s post hoc test, using the dietary treatments (C, Br, and Sy) and the age of birds (7, 14, 21, and 40 day) as main factors. The differences were considered significant at a level of p ≤ 0.05.

Figure 5

Boxplots showing the abundances of the taxonomic groups at genus level in the ileal chymus (IC) and ileal mucosa (IM) as affected by the dietary treatments and age of birds. Statistical significance was determined by one-way ANOVA and the Tukey multiple-comparisons test. Results with an adjusted p-value below 0.05 (* p < 0.05; ** p < 0.01; were considered statistically significant and results between 0.05 and 0.1 (0.05 < p < 0.10) were considered a trend (T). Dots represent the outlier values.

Figure 6

Relative abundance of potentially pathogenic bacteria according to BugBase analysis based on the 16S rRNA gene sequencing dataset. The outcome was grouped according to the gut segments and age. Gut segments were ileal chymus (IC), ileal mucosa (IM), and caecum chymus (CC). The relative abundance is presented on the y-axis, which is calculated by closed/open OTU picking ratio. Differences between groups were assessed using Mann–Whitney–Wilcoxon test, with Benjamini–Hochberg false discovery rate (FDR) correction. FDR-corrected p-values below 0.05 were considered significant. a, b: Averages with different letter marks differ significantly (FDR p < 0.05).

Figure 7

Infectious bursal disease (IBD) virus ELISA antibody titres in different treatment groups (mean ± SD). C—Control, Br—Broilact^®, Sy—Synbiotic feed additive; All p-values were calculated using two-sided tests and corrected with BH-FDR correction. FDR p-value less than 0.05 was considered statistically significant. a, b, c, d: Averages with different letter marks differ significantly (p < 0.05).