Sustainable antibiotic reduction in poultry production with Pulsatilla saponins and herbal supplementation.

The prolonged use of antibiotics in poultry production promotes the accumulation and spread of antibiotic resistance genes (ARG), raising concerns for animal health and public safety. Developing effective antibiotic alternatives that support performance while limiting resistance risk is therefore a priority. Using broiler chickens as a model, this study evaluated the effects of Pulsatilla saponins, alone or combined with a compound herbal formulation, on growth performance, immune responses, cecal microbiota, and the intestinal resistome, with an antibiotic-treated group as reference. Growth and immune parameters were integrated with shotgun metagenomic sequencing to characterize microbial and ARG responses to dietary interventions. Compared with antibiotic supplementation, the combination of 0.5% herbal medicine and 0.6% Pulsatilla saponins (ZBZ) combination significantly enhanced immune traits, including spleen index and serum IgA and IgM levels, while increasing cecal microbial diversity and reshaping community composition. Metagenomic analyses showed that antibiotic treatment enriched efflux pump and target modification associated ARG, indicative of a multidrug resistance profile. In contrast, ZBZ markedly reduced the abundance and diversity of multidrug resistance-related ARG. Notably, ZBZ supplementation enriched short-chain fatty acid-producing taxa that were negatively correlated with multiple ARG classes, suggesting that improvements in the intestinal metabolic environment and colonization resistance constrained the expansion of resistant bacteria. Overall, the combined use of Pulsatilla saponins and a compound herbal formulation improved growth and immune performance while reducing intestinal ARG burden through coordinated modulation of the cecal microbiota-resistome axis, providing a sustainable nutritional strategy for antibiotic-reduced poultry production.