Icariin shapes post-withdrawal fecal resistome dynamics in layer hens

Abstract Background While the livestock industry actively seeks alternatives to antibiotics, residual low-dose exposures continue to drive the spread of antibiotic resistance genes (ARGs). Icariin, a plant-derived compound, is recognized for improving poultry growth and immunity. However, it remains unclear how this compound influences the environmental persistence of ARGs, mobile genetic elements (MGEs), and horizontal gene transfer (HGT) during the vulnerable recovery phase after antibiotic withdrawal. Results We designed a two-phase feeding trial with laying hens, using longitudinal metagenomic sequencing to track post-withdrawal resistance dynamics. Following initial exposure to a low-dose antibiotic mixture that established a baseline of elevated resistance, hens received either a basal diet, an icariin-supplemented diet, or a copper sulfate-supplemented diet. The data indicate that icariin supplementation consistently reduced the burdens of both ARGs and MGEs. It also suppressed the potential for HGT and restricted the diversity of microbial hosts harboring these resistance elements. Conversely, copper sulfate—a traditional metal-based additive—exacerbated resistance risks by expanding both the abundance and the host range of ARGs and MGEs. Across all treatments, the population of Escherichia and the prevalent ARG subtype bacA correlated strongly with total resistance loads, tracking the overall resistome burden. Conclusions Compared to conventional copper sulfate treatments, icariin facilitates a safer ecological recovery in the poultry gut by actively lowering ARG and MGE reservoirs after antibiotic withdrawal. These genomic insights, combined with its known physiological benefits, support icariin as a sustainable feed additive. Furthermore, the Escherichia - bacA correlation provides a reliable, streamlined indicator for monitoring resistance risks in farm environments. However, as these findings rely on short-term fecal metagenomic tracking, further validation through multi-environment studies is warranted.