Integrated Microbiome and Metabolomics Analysis Reveals Altered Aggressive Behaviors in Broiler Chickens Showing Different Tonic Immobility.

Tonic immobility (TI) serves as an indicator of innate stress response recovery in poultry. Broilers with different TI phenotypes exhibit varying levels of aggressive behavior, which can significantly impact their welfare. However, the influences of TI phenotypes on broiler aggression remain largely unexplored. In this study, broiler chickens were stratified into two distinct phenotypic groups based on the TI duration: short TI (STI) and long TI (LTI). The impacts of TI phenotypes on broiler aggression were investigated by analyzing cecal intestinal morphology, cecal bacteria, plasma metabolites, and corticosterone levels. Compared to LTI broilers, STI broilers showed significantly reduced plasma corticosterone (CORT) levels (p < 0.05) and a decreased frequency of aggressive behaviors, including dominant and subdominant types (p < 0.01). Histological analysis revealed that STI broilers have an increased duodenal villus height and villus-height-to-crypt-depth ratio (p < 0.01), a decreased jejunal crypt depth with an increased villus-height-to-crypt-depth ratio (p < 0.01), and a reduced ileal crypt depth and villus height (p < 0.01) compared to LTI broilers. 16S rDNA sequencing and Linear discriminant analysis effect size (LefSe) identified differential cecal bacterial abundance, notably in the genus cc115 belonging to Firmicutes. Specific microbiota in LTI broilers exhibited significant positive correlations with aggressive behavior and plasma corticosterone, while those in STI broilers showed significant negative correlations. Untargeted plasma metabolomics revealed 21 downregulated and 17 upregulated metabolites between TI phenotypes. Correlation analysis showed that the genus cc115 and 10 plasma metabolites were positively correlated with aggressive behavior, whereas 8 metabolites were negatively correlated. LTI broilers have higher plasma corticosterone content and more intense aggressive behavior than STI broilers. The distinct behavioral and physiological profiles observed in broilers with different TI phenotypes are strongly correlated with their specific gut microbiota and differential plasma metabolite profiles. The identified gut microbial signatures serve as key biomarkers for regulating aggressive behavior in broilers, while the differential plasma metabolites represent potential early indicators for detecting stress and behavioral issues in poultry farming.