Broodstock nutrition in Atlantic salmon is crucial for reproductive success, yet modern plant-based feeds differ from traditional fish meal-based diets, raising questions about whether inclusion of feed additives meets the cumulative nutritional needs during maturation and spawning. This study investigated the effects of elevated dietary vitamin D3 on tissue storage, reproductive physiology, and offspring quality. The experiment was divided into three phases: Phase 1 involved feeding Atlantic salmon (~1200 g) control and test diets containing 5 or 15 IU/g vitamin D3, respectively, in duplicate sea cages; Phase 2 followed with broodstock diets containing 12.5 and 23.2 IU/g vitamin D₃, respectively, with all diets exceeding established vitamin D₃ requirements for Atlantic salmon. The Phase 2 diets were fed for six months prior to freshwater transfer, followed by Phase 3, in which spawning results from early-, mid- and late-season spawners with egg incubation until hatching were evaluated. Biometric data, tissue samples, blood metabolites, and reproductive parameters were collected to evaluate the impact of vitamin D₃ on maturation and reproductive outcomes. Fish reached up to 14 kg by the end of Phase 2, with males consistently heavier than females. Elevated dietary vitamin D₃ increased vitamin D3 concentrations in liver, muscle, gonads, and plasma, but did not affect bone mineral content in operculum or vertebrae. Gene expression analyses revealed diet-related upregulation of vitamin D metabolism genes in liver and sex-specific differences in gonadal expression of vitamin D receptors, calcium-binding proteins, and steroidogenesis-related genes. Fecundity, egg survival, and hatching rates were similar across diets, suggesting that additional vitamin D3 supplementation did not improve reproductive performance under the conditions tested. Additional dietary vitamin D3 increased vitamin D3 concentrations in yolk sac larvae, indicating maternal transfer of vitamin D3 to the offspring.
