Rearing system and immune status influence the small intestinal microbiota of IPB-D3 chickens: A full-length 16S rRNA metagenomic approach.

Background and Aim: The small intestinal microbiota plays a pivotal role in poultry digestion and immune function. Rearing systems can influence their composition, thereby affecting the overall health and performance of the birds. This study aimed to investigate how rearing systems (intensive [IN] vs. free-range [FR]) and immune status, reflected by leukocyte profiles, influence the small intestinal microbiome of IPB-D3 chickens, a genetically improved Indonesian local breed. Materials and Methods: Ninety IPB-D3 chickens were reared for 12 weeks under either IN or FR systems. Hematological profiling was conducted to assess health status, with leukocyte counts used to stratify birds. Microbiota samples from the small intestine were analyzed using full-length 16S ribosomal RNA (V1-V9) sequencing on the Oxford Nanopore platform. Taxonomic identification was performed using the SILVA database. Statistical comparisons were made using t-tests, and microbial diversity was assessed through alpha and beta diversity metrics. Results: While most hematological parameters did not differ significantly between rearing systems, total leukocyte counts were higher in intensively reared chickens (p = 0.002). FR chickens exhibited significantly greater microbial diversity (p < 0.05) across multiple alpha diversity indices. A total of 1,294 unique species were identified in FR birds versus 720 in the IN group, with 1,761 shared species. Leukocyte level further influenced microbial profiles; chickens with high leukocyte (HL) counts were dominated by Ligilactobacillus aviarius, whereas low-leukocyte chickens had a higher abundance of Bacteroides caecigallinarum. Gallibacterium anatis, a potential pathogen, dominated in IN systems with elevated leukocytes. Conclusion: This study demonstrates that both the rearing environment and immune status substantially influence small intestinal microbial composition in IPB-D3 chickens. FR systems promoted richer, more beneficial microbial communities, while IN systems, especially with HL levels, were associated with opportunistic pathogens. Leukocyte profiling may serve as a non-invasive biomarker for gut health, supporting future development of precision poultry management strategies and immune-responsive probiotics.