Malfunctioned inflammatory response and serotonin metabolism at the microbiota-gut-brain axis drive feather pecking behavior in laying hens

Abstract

Feather pecking (FP) is a multifactorial abnormal behavior in laying hens where they display harmful pecks in conspecifics. FP has been associated with the altered functioning of the microbiome-gut-brain axis affecting host emotions and social behavior. The altered levels of serotonin (5-HT), a key monoaminergic neurotransmitter at both terminals of the gut-brain axis, affect the development of abnormal behavior, such as FP in laying hens. However, the underlying mechanism involving reciprocal interactions along the microbiota-gut-brain axis, particularly about the metabolism of 5-HT, remains unclear in FP phenotypes. This study examined the microbiota diversity, intestinal microbial metabolites, inflammatory responses, and 5-HT metabolism in divergently selected high (HFP; n = 8) and low (LFP; n = 8) FP hens to investigate the possible interconnections between FP behavior and the examined parameters. The 16S rRNA analysis revealed that compared to LFP birds, the gut microbiota of HFP birds exhibited a decrease in the abundance of phylum Firmicutes and genera Lactobacillus, while an increase in the abundance of phylum Proteobacteria and genera Escherichia Shigella and Desulfovibrio. Furthermore, the intestinal differential metabolites associated with FP phenotypes were mainly enriched in the tryptophan metabolic pathway. HFP birds had higher tryptophan metabolites and possibly a more responsive immune system compared to the LFP birds. This was indirectly supported by altered TNF-α levels in the serum and expression of inflammatory factor in the gut and brain. Moreover, HFP birds had lower serum levels of tryptophan and 5-HT compared to LFP birds, which was consistent with the downregulation of 5-HT metabolism-related genes in the brain of HFP birds. The correlation analysis revealed that genera Lactobacillus and Desulfovibrio were associated with differences in intestinal metabolites, 5-HT metabolism, and inflammatory response between the LFP and HFP birds. In conclusion, differences in the cecal microbiota profile, immune response and 5-HT metabolism drive FP phenotypes, which could be associated with the gut abundance of genera Lactobacillus and Desulfovibrio.

Keywords: 5-HT metabolism; feather pecking; inflammatory response in laying hen; microbiome-gut-brain axis.

Figure 1

Microbial composition changes in cecal samples from LFP and HFP hens. (A) Alpha diversity analysis of gut microbiota by ACE, Chao-1, Shannon, and Simpson indices. (B) Principle coordinate analysis (PCA) plot was generated using OTU metrics based on unweighted uniFrac distance similarity for the samples in different groups highlighted with different colors. PC1 and PC2 represent the potential factors that caused a microbial composition shift in the cecal microbiota. Asterisk indicates a statistically significant difference (P < 0.05). (C) The mean percentage of the total population at phylum level in the cecal microbiota of LFP and HFP birds (n = 8 per group). Differences were assessed by the Mann-Whitney U test and significance was established at P < 0.05. (D) Linear discriminant analysis (LDA) effect size method was performed to compare taxa between LFP (green; n = 8) and HFP birds (red; n = 8). The bar plot shows the significantly differential taxa based on effect size (LDA score (log 10) >3). (E) Heatmap cluster analysis of the top 10 bacteria at the genus level; the heatmap is color-coded based on row z-scores.

Figure 2

Differences in intestinal microbial metabolites (A) Orthogonal projections from latent structure-discriminant analysis (OPLS-DA) score plots in LFP and HFP birds (R2X = 0.407, R2Y = 0.999, Q2Y = 0.659) and permutation test of OPLS-DA. (B) KEGG pathways enriched by differential metabolites between LFP and HFP birds. The color of circles represents the magnitude of the significant difference, and the sizes of the circles indicate the number of differential metabolites between LFP and HFP birds. (C) A heatmap describing significant differences in intestinal microbial metabolites between the 2 groups. n = 8 birds/group. The color of the respective spot in the heatmap corresponds to the normalized and log-transformed raw abundance of the metabolites in that sample. The metabolites were organized according to their order in the phylogenetic tree generated by their representative sequences.

Figure 3

Correlations between altered gut microbiota genera, FP, physiological indicators, and intestinal metabolites. (A) The Spearman correlation coefficient for GFP, SFP, 5-HT metabolism, and immune cytokines, and different bacterial genera. (B) The Spearman correlation coefficient for metabolite type with the highest VIP values and differential bacterial genera. Heatmaps exhibit notable statistical correlation values (r). Red squares imply significant positive correlations (0 < r ≤ 1), white squares imply no correlations (r = 0), and blue squares indicate significant negative correlations (−1 ≤ r < 0); the deeper color means the greater correlation, respectively; * P < 0.05, ** P < 0.01. Only significant values (P < 0.05/0.01 after FDR adjustment) are shown.

Figure 4

Differences in the inflammatory response and serotonin signaling in the brain and gut. (A) qRT-PCR analysis of hypothalamic and ileal TNF-α and IL-10 expression in HFP birds compared with LFP birds. (B) Relative expression of 5-HTR1A, TPH1, TPH2, MAO-A, MAO-B, and SERT genes in the hypothalamus and ileum of LFP and HFP birds. Data are shown as means ± SEM, n = 8 birds/group. Differences were assessed by the Mann-Whitney U test and significance was established at P < 0.05 (*P < 0.05, **P < 0.01).