Physiological and Transcriptomic Responses of Triploid Rainbow Trout (Oncorhynchus mykiss) to Different Stocking Densities in a Recirculating Aquaculture System

Background/Objectives: The level of stocking density serves as a critical modulator of both growth performance and physiological homeostasis in fish reared within recirculating aquaculture systems (RAS). Methods: In this study, an 8-week feeding trial was designed incorporating three density gradients specifically 30, 50, and 70 kg/m3 with the aim of systematically investigating the consequent alterations in growth rates, digestive functionality, antioxidative status, and hepatic transcriptional profiles of triploid rainbow trout (Oncorhynchus mykiss). Results: All treatment groups maintained their growth vigor, and there were no significant differences in final body weight or body length. The final stocking densities were 36.85 ± 0.73, 59.70 ± 2.73, and 70.97 ± 0.32 kg/m3 in the 30G, 50G, and 70G groups, respectively. However, survival rates decreased as stocking density increased. Digestive enzyme activities were generally higher at 50 kg/m3 but declined at 70 kg/m3, particularly intestinal amylase and trypsin activities. Hepatic MDA content increased with stocking density, reaching 5.47 ± 0.28 nmol/mg protein at 70 kg/m3, while T-AOC decreased significantly to 25.00 ± 1.52 μmol/g protein (p < 0.05), indicating enhanced oxidative stress and weakened antioxidant defense. Mild hepatocellular vacuolation was also observed at higher stocking densities. Transcriptomic analysis identified 558 differentially expressed genes between the 30 kg/m3 group and the 70 kg/m3 group; these genes were primarily enriched in pathways related to foreign body metabolism, ferroptosis, and cell adhesion molecules (p < 0.05). At 70 kg/m3, several antioxidant-related genes, including cat, gclc, gsr, gpx1, gpx4a, prdx1, and hsp70, were downregulated, whereas sod1, sod2, maf, me1, and gadd45a were upregulated. Conclusions: These results indicate that rainbow trout can maintain growth at stocking densities up to 70 kg/m3 under the tested RAS conditions, but higher density is associated with reduced digestive capacity and increasing hepatic oxidative stress. The 50 kg/m3 treatment showed a relatively favorable balance between growth, digestive function, and antioxidant status, suggesting its potential suitability for intensive RAS production.