Iron, a vital micro-mineral for fish, plays essential roles in numerous biological processes. However, the impacts of dietary iron supplementation on American eels remain poorly explored. This study mainly aimed to evaluate the effects of different dietary iron supplementation levels on growth performance, iron utilization, and intestinal health of juvenile American eels during a 10–week feeding trial. Five experimental diets were formulated using FeSO4·H2O as iron source, with iron supplementation levels in the diets being 0, 150, 300, 450, and 600 mg kg−1, respectively. In comparison to American eels fed control diet, those fed a diet supplemented with 150 mg kg⁻¹ of iron displayed enhanced growth performance, better feed efficiency, increased whole–body iron retention, fortified immune function, strengthened intestinal structural integrity, elevated intestinal antioxidant capacity, and increased probiotic colonization. Conversely, dietary supplementation with 600 mg kg⁻¹ of iron induced growth retardation, inhibited feed utilization, intestinal epithelial damage and oxidative stress, and pathogen proliferation. Additionally, the contents of whole–body iron and serum iron were up–regulated, but the contents of whole–body calcium, phosphorus, zinc, and manganese were down–regulated, as dietary iron supplementation levels increased. Quadratic regression analysis indicated 190 mg kg⁻¹ as the optimal dietary iron supplementation level for maximal weight gain rate of American eels. These results offer a practical guidance for dietary iron supplementation and clarify the optimal dietary iron supplementation level for American eels.
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