The aquaculture industry has become a sustainable source of food for humans. Remaining challenges include disease issues and ethical concerns for the discomfort and stress of farmed fish. There is a need for reliable biomarkers to monitor welfare in fish, and the stress hormone cortisol has been suggested as a good candidate. This study presents a novel method for measurement of cortisol in fish feces based on enzymatic hydrolysis, liquid–liquid extraction, derivatization, and finally instrumental analysis by liquid chromatography coupled with tandem mass spectrometry. Hydrolysis and extraction conditions were optimized. Cortisol appeared to be mostly conjugated to sulfate and less conjugated to glucuronic acid in the studied samples of feces from farmed Atlantic salmon. The method was suitable for quantification of cortisol after enzymatic deconjugation by either combined glucuronidase and sulfatase activity, or by glucuronidase activity alone. The limit of detection was 0.15 ng/g, the limit of quantification was 0.34 ng/g, and the method was linear (R² > 0.997) up to 380 ng/g, for measurement of cortisol in wet feces. Method repeatability and intermediate precision were acceptable, both with a coefficient of variation (CV) of 11%. Stress level was high in fish released into seawater, and significantly reduced after eight days.

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.

The aquaculture industry has become a sustainable provider of food to humans. Remaining challenges include disease issues, as well as ethical concerns for discomfort and stress among farmed fish. There is a need for reliable biomarkers to monitor welfare in fish, and the stress hormone cortisol has been suggested as a good candidate. This study presents a novel method for measurement of cortisol in fish feces based on enzymatic hydrolysis, liquid-liquid extraction, derivatization, and finally instrumental analysis by liquid chromatography coupled with tandem mass spectrometry. Hydrolysis and extraction conditions were optimized. Cortisol appeared to be mostly conjugated to sulfate and less conjugated to glucuronic acid in the studied samples of feces from farmed Atlantic salmon. The method was suitable for quantification of cortisol after enzymatic deconjugation by either combined glucuronidase and sulfatase activity, or by glucuronidase activity alone. The limit of detection was 0.15 ng/g and the limit of quantification was 0.34 ng/g, and the method was linear (R2>0.997) up to 380 ng/g, for measurement of cortisol in wet feces. Method repeatability and intermediate precision were acceptable, both with a CV of 11%. Stress level was high in fish released into seawater, and significantly reduced after eight days.

Cardiomyopathy syndrome (CMS) poses a significant threat to farmed Atlantic salmon (Salmo salar), leading to high mortality rates during the seawater phase. Given that controlled experimental challenge trials with PMCV do not reproduce the mortality observed in severe field outbreaks of CMS, field trials on natural CMS outbreaks are warranted. This field study explored the impact of a clinical nutrition intervention, specifically a diet enriched with eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), on a severe CMS outbreak in a commercial sea farm. CMS was diagnosed in a single sea cage with high mortality rates. Histopathological analysis, RT-qPCR in situ hybridization for virus detection, and fatty acid composition analysis were used to monitor the impact of disease and the inclusion of EPA and DHA in heart tissue. Following the implementation of clinical nutrition, a decline in mortality rates, regression of CMS-associated changes, and a significant reduction in piscine myocarditis virus (PMCV) RNA load were observed within the salmon population. Fatty acid composition analysis of heart samples demonstrated increased levels of EPA and DHA, reinforcing the association between dietary factors, viral load dynamics, and overall fish health. Although further validation is needed in future studies, as field trials may not be sufficient to establish causation, our results indicate that optimizing the EPA + DHA levels may prove beneficial in severe CMS outbreaks.

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.