Dietary leucine supplementation enhances growth performance, digestive enzyme activity, and intestinal barrier function in triploid rainbow trout (Oncorhynchus mykiss) fed plant-protein based diets

This study aimed to investigate the effects of different gradients of leucine (Leu) added to low fish meal feed (10 %) on the growth pattern, digestive enzyme function, antioxidant capacity, intestinal health, and serum physiological and biochemical parameters of triploid rainbow trout (Oncorhynchus mykiss). Five iso-nitrogenous and iso-energetic test feeds were designed, with leucine levels of 2.21 % (G1), 2.66 % (G2), 3.08 % (G3), 3.49 % (G4), and 4.07 % (G5). Triploid O. mykiss with an initial weight of 12.22 ± 0.20 g was fed for a period of 56 days. Results indicated that for growth performance, the 3.08 % Leu group showed significantly higher weight gain rate (WGR) and specific growth rate (SGR) than the 2.21 % and 4.07 % groups, with the lowest feed conversion ratio (FCR) and highest feed efficiency (FE) among all groups (P < 0.05). Whole-body crude protein content peaked in the 3.08 % group, while moisture, lipid, and ash contents showed no significant differences among groups. Regarding amino acid deposition, the contents of Val, Ile, Leu, Lys, Glu, Tyr, and Pro increased progressively with dietary Leu levels, reaching significant enrichment in the 4.07 % group. Digestive enzyme analysis revealed: intestinal protease activity was significantly higher in the 3.49 % group than in other groups; intestinal amylase activity peaked at 2.66 % and subsequently declined; and intestinal lipase activity in the 3.08 % group was significantly higher than that in the 2.21 % group. Morphologically, the 3.49 % group exhibited optimal intestinal villus height and the thickest muscular layer (P < 0.05). Serum biochemistry demonstrated significant elevations in total protein, globulin, complement C3/C4, and alkaline phosphatase (ALP); albumin content was highest in the 3.49 % group; while aspartate aminotransferase (AST), low-density lipoprotein cholesterol (LDL-C), and triglycerides (TG) levels were minimized in the 3.08 % group. Gene expression of antioxidant axis components (Nrf2, NQO1, AMPK, FOXO3, Trx) was significantly upregulated compared to the 2.21 % group. For immune markers, IL-1β, TNF-α, IL-8, NF-κB, and MLCK were significantly downregulated, whereas IL-10 and IκB-α were markedly upregulated. Peak expression of tight junction proteins occludin, ZO-1, and claudin-3 occurred in the 3.08 % group, while claudin-7 expression peaked in the 3.49 % group. Broken-line regression identified optimal dietary Leu levels at 3.03 % for SGR and 3.05 % for WGR. Dietary leucine supplementation at 3.03 – 3.05 %（7.57 % – 7.63 % of dietary protein） simultaneously optimizes growth, feed utilization, digestive enzyme activity, antioxidant capacity, immune response and intestinal barrier function in triploid O. mykiss fed low-fishmeal diets.