Wanwan Zhu, Nan Zhang, Xuekai Wang, Yi Xiong, Fuyu Yang, Yongsheng Wang
Mulberry leaf is a promising plant feed resource for aquaculture, yet high inclusion limits its application due to anti-nutritional factors. Exogenous enzyme supplementation can improve the utilization efficiency of bioactive compounds in mulberry leaf-containing diets. This study aimed to evaluate the dose-dependent effects of mulberry leaf powder supplemented with a multi-enzyme premix (MLE) on growth, intestinal health, antioxidant and immune responses, as well as intestinal microbiota and metabolome in juvenile Acipenser schrenckii. Four diets containing 0%, 2%, 4% and 6% MLE were formulated for a 10-week feeding trial. Moderate MLE supplementation maintained normal growth without significantly improving growth performance relative to the control, and was associated with reduced serum triglyceride, total cholesterol and glucose, as well as improved intestinal morphology and digestive enzyme activity. Appropriate MLE supplementation was associated with changes in the Nrf2/Keap1 signaling pathway, enhanced antioxidant capacity, and upregulated anti-inflammatory gene il10. Multi-omics analysis revealed that MLE induced taxon-specific changes in the intestinal microbiota and was associated with altered metabolic profiles, enriching beneficial taxa including Priestia, Bacillus_P and Cetobacterium_A. These changes suggest a potential modulation of the microbiota-metabolite-immunity axis that may help sustain intestinal homeostasis. Nevertheless, the beneficial effects declined at the 6% inclusion level. Collectively, MLE was associated with dose-dependent protective effects on the intestinal barrier that may involve the microbiota-metabolite-immunity axis and the Nrf2/Keap1 pathway; the optimal supplementary range is preliminarily estimated at 3.5-4.5% for juvenile Acipenser schrenckii, although this estimate is based on four dietary levels and requires validation in future dose-response studies.