Haematological and plasma parameters are used in veterinary medicine as a diagnostic tool, allowing for the early detection of potential health and welfare issues. For the implementation of monitoring strategies, reference intervals (RIs) are needed to know when deviations in those parameters become clinically relevant. Here, haematological and plasma biochemical RIs of Norwegian Atlantic salmon (Salmo salar Linnaeus) are presented for 29 biomarkers. Two subgroups were defined from the reference population based on water type, referred to as pre‐adult (freshwater, 166.8 ± 118.4 g, N = 979) and adult (seawater, 2655 ± 2883 g, N = 4941), collected at 93 fish farms along the Norwegian coast. While the influence of various pre‐analytical variables on RIs is well documented, aquaculture practitioners cannot always control those variables in the field. Here, RIs were calculated from blood samples collected based on local pre‐analytical practices and then further processed under standardised conditions (analytical phase). RIs are therefore considered highly relevant for the application in the field. Finally, when taking blood samples in the field, it is advised to centrifuge whole blood as soon as possible, while plasma parameters investigated were stable for 168 h at 4°C or 72 h at room temperature.

Fish are believed not to synthesize vitamin D through UV exposure but to meet their requirements from dietary sources. The high vitamin D levels found in many fish species are thought to originate from UV‐induced synthesis in plankton, with vitamin D subsequently accumulating through the aquatic food chain. Atlantic salmon is a rich dietary source of vitamin D, but limited data exist on circulating vitamin D levels. To address this, plasma levels of the three main vitamin D metabolites were measured in Atlantic salmon from Norwegian fish farms using mass spectrometry. Here, we show that salmon reared in open sea pens have significantly higher vitamin D levels than those raised indoors. Monitoring an outdoor farm over 18 months revealed a distinct seasonal pattern between vitamin D levels and day length. In a follow‐up experiment, indoor‐raised fish were divided into two groups: one remained indoors, while the other was transferred outdoors for 52 days. Both groups received the same commercial feed, yet the outdoor group exhibited a fivefold increase in whole‐body vitamin D content. These surprising findings provide field evidence of endogenous vitamin D synthesis in fish induced by natural sunlight. This discovery could have important implications for aquaculture, emphasizing the risk of suboptimal vitamin D levels in farmed fish when sunlight exposure is limited.

Vitamin D ₃ (cholecalciferol) is an essential nutrient for vertebrate calcium homeostasis, skeletal integrity, and immune function. In aquaculture, declining use of fishmeal and fish oil has reduced dietary vitamin D ₃ levels in salmon feeds, raising questions about tissue deposition and nutritional quality of farmed fish. This study aimed to investigate the metabolic fate of elevated dietary vitamin D ₃ in farmed Atlantic salmon ( Salmo salar ) under commercial production conditions in southern Norway. Fish were fed either a control diet (~4000 IU/kg vitamin D ₃ ) or a test diet with a 10‐fold higher inclusion (~40,000 IU/kg vitamin D ₃ ) for 32 weeks from November to July 2025. Plasma, liver, and muscle samples were analyzed for vitamin D ₃ and its metabolites [25(OH)D ₃ and 1,25(OH) ₂ D ₃ ], alongside plasma biomarkers of health. Salmon tolerated the high dietary vitamin D ₃ without observed adverse effects on growth and mortality. Elevated dietary inclusion significantly increased vitamin D ₃ concentrations in plasma, liver, and muscle, with less pronounced effects on hydroxylated metabolites, compensating for lower levels during winter months. However, seasonal variation was evident in all tissues investigated. Overall, the results demonstrate that high dietary vitamin D ₃ supplementation increases tissue concentrations in Atlantic salmon without compromising health, thereby improving the nutritional profile of farmed salmon fillets for human consumption.