Dietary Clostridium butyricum and Alanyl-Glutamine Modulate Low-Fishmeal-Induced Growth Reduction, Intestinal Microbiota Disorders, Intestinal Inflammatory Injury, and Resistance Against Aeromonas salmonicida in Triploid Oncorhynchus mykiss

Low-fishmeal feed is increasingly being adopted across the global aquaculture industry. This study evaluated dietary Clostridium butyricum and alanyl-glutamine (Ala-Gln) supplementation in juvenile triploid rainbow trout (Oncorhynchus mykiss) with a low-fishmeal diet. Four diets were tested: basal diet (SBM, 15% fishmeal and 21.6% soybean meal), SBM + 0.5% C. butyricum (CB), SBM + 1.0% Ala-Gln, and SBM + 0.5% C. butyricum + 1.0% Ala-Gln (CB-AG). Fish were fed in 500 L tanks in recirculating aquaculture systems for 8 weeks (62.52 ± 0.47 g). Each group comprised three tanks, with each tank housing 30 fish. Then 10 fish per tank were challenged with Aeromonas salmonicida. CB-AG showed significantly higher weight gain and specific growth rates than the SBM group (p < 0.05). Mortality was significantly lower in CB-AG and AG than in SBM after A. salmonicida challenge. Histomorphology revealed significant differences (p < 0.05) between CB-AG and SBM in muscularis thickness, villus width, and height. SBM sections showed inflammatory infiltration and border damage were attenuated in supplemented groups. Serum malondialdehyde (MDA) and dioxygenase (DAO) were significantly lower in CB-AG than SBM (p < 0.05), while serum and hepatic lysozyme (LZM) and hepatic superoxide dismutase (SOD) were higher. Digestive enzymes indicated significantly higher trypsin and lipase activities in CB-AG (p < 0.05). CB-AG upregulated intestinal tight junction proteins and PepT1 and downregulated pro-inflammatory mediators. Combined 0.5% C. butyricum and 1.0% Ala-Gln inclusion effectively preserved growth performance, antioxidant capacity, gut microbiome homeostasis, and intestinal health in rainbow trout on low-fishmeal diets.