VITAMIN D₃ SUPPLEMENTATION ON ANTIOXIDANT CAPACITY AND FERROPTOSIS IN JUVENILE GIBEL CARP (CARASSIUS AURATUS GIBELIO VAR. CAS V) AT DIFFERENT STOCKING DENSITIES

High-density crowding stress during the initial feeding stage poses severe challenges to fish health, promoting lipid peroxidation. This study aimed to assess the protective effects of vitamin D₃ (VD₃) against crowding stress and investigate its underlying mechanisms. A two-factor design was employed using juvenile gibel carp (Carassius auratus gibelio var. CAS Ⅴ)(0.47±0.03) g/fish for a 71-days feeding trial, three experimental diets with different VD₃ concentrations VD0 IU/kg (no VD₃), VD1000 IU/kg (low concentration), and VD5000 IU/kg (high concentration) were applied under two different rearing densities (L: 70 fish/tank, H: 210 fish/tank). Although macroscopic growth parameters showed no significant differences, hepatic biochemical and molecular profiles indicated a severe underlying metabolic burden. Specifically, at the metabolic level, hepatic GPT activity was significantly decreased under high density, while GPx4 activity and GSH content were abnormally elevated in a compensatory manner. Furthermore, the unsupplemented high-density group exhibited substantial accumulation of lipid hydroperoxide (LPO) and labile iron (Fe²⁺). Notably, dietary VD₃ supplementation significantly reduced both hepatic LPO and Fe²⁺ contents, effectively attenuating these ferroptosis-related markers. Transmission electron microscopy (TEM) examination revealed significantly shrunken mitochondria and vanished cristae, indicating typical ferroptosis, which were remarkably mitigated by VD₃ supplementation. Transcriptomic analysis further demonstrated that differentially expressed genes in the VD₃-treated group were enriched in ferroptosis, cysteine and methionine metabolism, and fatty acid biosynthesis pathways. qPCR validation confirmed the significant upregulation of key antioxidant genes (nrf2, gpx4a, and prdx6) and the downregulation of the pro-ferroptosis gene (acsl4a) by VD₃, aligning with transcriptomic trends. In conclusion, high-density rearing triggered pronounced hepatic ferroptosis and metabolic dysregulation in juvenile C. auratus gibelio, whereas dietary VD₃ supplementation effectively ameliorated lipid peroxidation and restored mitochondrial ultrastructure. These findings offer mechanistic insights into nutritional interventions for alleviating crowding stress and inform early-life nutritional programming strategies in this species.