Screening and characterization of Limosilactobacillus reuteri L16 and its probiotic properties in Hy-Line Brown chicks.

With the increasingly restricted use of antimicrobial growth promoters (AGPs) in poultry production, host-derived probiotics have attracted growing attention as green feed additives for improving growth performance and intestinal health. In this study, 84 lactic acid bacteria (LAB) strains were isolated from feces of healthy Hy-Line Brown laying hens using MRS agar supplemented with 2 % CaCO3. After screening for acid and bile salt tolerance, strains L16 and L20 were selected based on antibacterial activity, cell surface properties, safety, and genomic features. Average nucleotide identity (ANI) and whole-genome phylogenetic analyses identified both strains as Limosilactobacillus reuteri (L. reuteri). Genomic characterization revealed relatively complete metabolic pathways, genetic information processing systems, and gene clusters associated with probiotic functionality and antimicrobial potential. In vivo, L16 significantly improved growth performance in Hy-Line Brown chicks, as evidenced by increased body weight and average daily gain, reduced feed conversion ratio, and enhanced thymus and bursa of Fabricius (BF) indices. Moreover, L16 increased jejunal villus height (VH) and the villus height-to-crypt depth ratio (VH/CD), upregulated tight junction proteins (Occludin and ZO-1), decreased IFN-γ, and increased IL-10 levels, indicating enhanced intestinal barrier function and immunomodulatory capacity. 16S rRNA gene sequencing further showed that L16 modulated gut microbiota composition by enriching beneficial taxa, including Levilactobacillus brevis and Luoshenia tenuis. Overall, L16 exerted growth-promoting and gut health-improving effects comparable to those of bacitracin methylene disalicylate (BMD), while producing more favorable changes in body weight, average daily gain, and immune organ development. These findings highlight the potential of L16 for further development and application as a probiotic feed additive in poultry production.