Combined Dietary Supplementation of Bacillus subtilis DSM 29784 and a Phytogenic Blend Modulates the Microbiome-Gut-Bone Axis in Broilers

Disruptions within the microbiome–gut–bone axis are increasingly recognized as key contributors to impaired bone metabolism and leg disorders in broiler chickens. This study investigated the effects of a combined dietary additive containing Bacillus subtilis DSM 29784 and a phytogenic blend of garlic and essential oil components (BsP) on the modulation of microbial communities, intestinal integrity, mineral utilization, and bone-associated immune–osteogenic pathways. Five hundred and sixty-one-day-old male Ross 308 broilers were randomly assigned to a basal control diet or the same diet supplemented with BsP for 42 days, with eight replicates per treatment. Growth performance, cecal microbiome composition, jejunal tight junction expression, pro-inflammatory cytokines, ileal calcium–phosphate transporters, and femoral inflammatory and osteogenic gene expression were evaluated. The results demonstrated that BsP supplementation significantly improved body weight, weight gain, and feed conversion ratio while enhancing intestinal barrier function. Birds receiving BsP displayed upregulated expression of tight junction–related genes (CLDN-1, OCLD-1, TJP-1, MUC-2) and reduced jejunal inflammatory markers (TNF-α, NF-κB). Improved mineral transport capacity was indicated by increased ileal CaSR and NaPi-IIb expression. Microbiome profiling revealed higher species richness (Chao1 and Shannon indices; P< 0.05) and diversity (Bray-Curtis, PERMANOVA; P < 0.001) on days 21, 35, and 42, with enrichment of beneficial taxa such as Clostridium butyricum, Enterococcus faecium, Lactobacillus salivarius, L. crispatus, and Bifidobacterium longum, accompanied by reduced Escherichia coli, and Enterococcus cecorum. Functional predictions suggested activation of serotonin-, melatonin-, and L-tryptophan–related pathways, indicating engagement of the microbiome–gut–brain axis. At the skeletal level, BsP reduced femoral expression of IL-6, IL-17, TNF-α, and NLRP3 and enhanced BMP-2, SMAD-1, RUNX-2, and SPARC, aligning with improved mineral deposition. Network analysis revealed distinct inflammation-, bone-, and microbiota-dominant modules, highlighting the structured interactions linking microbial signals to osteoimmunological responses. Overall, BsP effectively modulated the microbiome–gut–bone axis, supporting intestinal homeostasis, mineral absorption, and bone formation. These findings underscore the potential of BsP as a functional feed additive to promote both intestinal and skeletal health in broilers.