Optimal isoleucine level improves growth, antioxidant capacity, and disease resistance of triploid rainbow trout (Oncorhynchus mykiss) fed a low-fishmeal diet via enhancing intestinal health

This study systematically investigated the effects of graded supplementation of isoleucine (Ile) in low-fishmeal diets on the growth performance, body composition, digestive enzyme activity, intestinal morphology, serum biochemistry, antioxidant capacity, immune-related gene expression, and tight junction (TJ) protein expression in triploid Oncorhynchus mykiss to determine the optimal dietary requirement. Five isonitrogenous and isolipidic low-fishmeal diets (10% fishmeal content) were formulated with graded Ile levels: 0.81% (Group G1), 1.76% (G2), 2.78% (G3), 3.73% (G4), and 4.67% (G5). Triploid O. mykiss with an initial body weight of (12.25 ± 0.30) g were subjected to a 56-day feeding trial. The results showed that weight gain rate (WGR) and specific growth rate (SGR) followed a quadratic curve, initially increasing and then decreasing ( P < 0.05), peaking in the G3 group. Body composition and muscle amino acid profile indicated enhanced protein deposition and an improved amino acid composition in the G3 group. In terms of digestive physiology, protease, amylase, and lipase activities in the intestine and liver were significantly enhanced in the G3 groups ( P < 0.01). Intestinal morphology revealed significantly increased villus length, width, and goblet cell count in the G3 group, while structural damage was observed in the G5 group. Serum analysis showed the highest levels of total protein, globulin, complement, and alkaline phosphatase activity in the G3 group, along with improved liver function indicators. Molecular mechanisms indicated that the G3 groups significantly upregulated key genes in the intestinal antioxidant pathways ( Nrf2 , AMPK , FOXO3 , Trx ), while suppressing the NF-κB pathway-mediated inflammatory response (downregulation of IL-1β and TNF-α , upregulation of IL-10 and IκB-α ). TJ protein expression ( claudin-3 , -7 , occludin , ZO-1 ) was also significantly enhanced. Under the experimental conditions, a quadratic regression model estimated the optimal dietary Ile requirement to be 2.53% – 2.84% of the diet (accounting for 6.33% – 7.10% of dietary protein). This optimal level synergistically optimizes amino acid metabolism, activates digestive enzyme secretion, and enhances the function of the " Nrf2 / AMPK – NF-κB –TJ" molecular network, thereby comprehensively improving the growth performance, digestive capacity, antioxidant status, immune level, and intestinal barrier integrity of triploid O. mykiss . Excessive supplementation exhibited inhibitory effects. This study provides a solid theoretical and practical foundation for the precise application of isoleucine in low-fishmeal diets for O. mykiss .