43. Integrated Transcriptomic and Metabolomic Analysis of Immune and Metabolic Pathways in Broiler Chickens Supplemented with Lactobacillus Reuteri and Streptomyces Coelicolor

Abstract The overuse of antibiotics in poultry production has raised global concerns about antimicrobial resistance, creating a need for effective and sustainable alternatives such as probiotics. Previous studies have shown that Lactobacillus reuteri and Streptomyces coelicolor can improve growth and health performance in broiler chickens, but the molecular mechanisms behind these effects are still not well understood. This study aimed to assess the transcriptomic and metabolomic responses of broiler chickens fed diets supplemented with L. reuteri, S. coelicolor, or their combination. A total of 240-day-old broiler chicks were randomly assigned to four dietary treatments and raised under standard commercial conditions for eight weeks. Liver and intestine samples collected in weeks 4 and 8 were used for RNA sequencing and metabolomic analysis. Transcriptome profiling showed distinct gene expression patterns for each probiotic treatment and tissue type while functional enrichment analysis indicated that probiotics influenced various immune pathways such as cytokine signaling and lymphocyte activation, as well as metabolic pathways related to lipid metabolism and electrolyte balance. Notably, interleukin (IL) gene analysis revealed clear treatment effects. IL17F was upregulated in the intestine of chickens fed L. reuteri and S. coelicolor alone, while IL17RA and IL17RD were most upregulated in the combination group, suggesting improved mucosal immunity through IL-17 signaling. The combination group also showed increased expression of IL18 and IL18R1 in liver tissue, indicating enhanced innate immune activity. In addition, IL10R2 and IL22RA1 were more highly expressed in intestinal samples from the combination group highlighting stronger anti-inflammatory signaling and epithelial repair. These results show that the combined supplementation created a balanced immune response both locally and systemically. Metabolomic profiling in the positive ion acquisition mode revealed treatment-specific differences in metabolite diversity. Among the individual strains, L. reuteri and S. coelicolor yielded 250 and 240 unique metabolites, respectively, while the combination of both probiotics produced the highest (about 260) and richest metabolite diversity, therefore suggesting a potential synergistic effect of L. reuteri and S. coelicolor in modulating the metabolome of broiler chickens. Moreover, KEGG pathway analysis showed that most metabolites were involved in central metabolism, secondary metabolite biosynthesis, and cofactor and lipid metabolism. Some unannotated metabolites were also detected only in the negative ion mode, suggesting the presence of novel compounds that may have biological importance. Furthermore, shared and unique metabolite patterns confirmed that the combination treatment reflected features of both single strains while generating additional unique metabolites.Overall, the combined supplementation of L. reuteri and S. coelicolor produced a synergistic effect at both the transcriptomic and metabolomic levels. These findings provide insight into how probiotics influence gene expression and metabolism in broiler chickens and support the potential use of multi-strain probiotics as functional feed additives to enhance poultry health and productivity.