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Several feeding trials with Atlantic salmon fed naturally high phytosterol concentrations due to dietary rapeseed oil inclusion have shown changes in lipid metabolism and increased hepatic lipid storage in the fish. An in vitro trial with Atlantic salmon hepatocytes was, therefore, performed to study the possible direct effects of phytosterols on lipid storage and metabolism. The isolated hepatocytes were exposed to seven different sterol treatments and gene expression, as well as lipid accumulation by Oil Red O dyeing, was assessed. Fucosterol, a sterol found in many algae species, had an effect on the size of individual lipid droplets, leading to smaller lipid droplets than in the control without added sterols. A sterol extract from soybean/rapeseed led to an increase in the percentage of hepatocytes with visible lipid droplets at 20× magnification, while hepatocytes of both the sterol extract-treated groups and fucosterol-treated groups had a larger proportion of their area covered with lipids compared to control cells. Brassicasterol, a sterol characteristic of rapeseed oil, was the only sterol treatment leading to a change in gene expression, affecting the expression of the nuclear receptors, peroxisome proliferator-activated receptor gamma (pparg) and retinoid X receptor (rxr). The current study thus shows that phytosterols can have direct, although subtle, effects on both hepatic lipid storage and gene expression of Atlantic salmon in vitro.

The increasing use of plant-based ingredients in aquafeeds for Atlantic salmon has led to increasing feed concentrations of plant sterols (phytosterols) combined with decreasing concentrations of cholesterol. Questions have been raised regarding how this may affect performance, health and lipid metabolism in a carnivorous fish species adapted to a high cholesterol diet in nature, and whether phytosterols could be involved in the liver lipid accumulation commonly observed in salmon fed diets with high content of vegetable oils. A feeding trial with 9 diet groups with varying additions of phytosterols and cholesterol was conducted at two temperatures (6 and 12 °C). Fish grew from an initial weight of ~ 250 g to a final weight of ~ 900 g. Overall, the addition of phytosterols and/or cholesterol within the range tested (1353–3147 mg kg− 1 phytosterols, 867–3153 mg kg− 1 cholesterol and phytosterol:cholesterol ratios of 0.7–3.2) exhibited few effects on fish performance, lipid metabolism and health. The main hypothesis regarding phytosterols or phytosterol:cholesterol ratio affecting liver lipid accumulation appears to be false. However, dietary cholesterol did increase muscle pigmentation at both temperatures, while fillet dry matter and hepatosomatic index were also increased by dietary cholesterol at 12 °C. Dietary cholesterol also had some minor effects on the fatty acid composition of liver polar lipids, probably mediated by LXR. In general, more effects were seen from variations in dietary cholesterol compared to phytosterols. The current trial is the first to feed salmon graded levels of phytosterols, cholesterol and their ratio without changing the dietary fatty acid composition, and thereby isolating the effects of dietary sterols.

The black soldier fly (Hermetia illucens) is one of the most promising insect species for use in animal feed. However, studies investigating feed and food safety aspects of using black soldier fly as feed are scarce. In this study, we fed black soldier fly larvae feeding media enriched with seaweed, which contains naturally high concentrations of heavy metals and arsenic. The aim of this study was to investigate the potential transfer of such undesirable substances from the feeding media to the larvae.; Results: The larvae accumulated cadmium, lead, mercury and arsenic. Concentrations of these elements in the larvae increased when more seaweed was added to the feeding media. The highest retention was seen for cadmium (up to 93%) and the lowest for total arsenic (up to 22%). When seaweed inclusion exceeded 20% in the media, this resulted in larval concentrations of cadmium and total arsenic above the current European Union maximum levels for these elements in complete feed.; Conclusion: Our results confirm that insect larvae can accumulate heavy metals and arsenic when present in the feeding media. A broader understanding of the occurrence of these undesirable substances in processed larvae products is needed to assess feed and food safety. © 2017 Society of Chemical Industry.; © 2017 Society of Chemical Industry.

In-depth metabolic profiling, also termed metabolomics, provides detailed information about the biochemical phenotype of an organism. Besides improving our understanding of biochemical processes, metabolomics is used for environmental monitoring, natural product discovery, or even chemotaxonomy, among others. However, for marine macroalgae, comparative large-scale metabolomics studies are lacking, even though seaweeds belong to the most important aquatic primary producers. In this study, we present via a broad scale systematic metabolomics survey 391 metabolites from 21 seaweeds species, representing brown, red and green algae. We demonstrate clear differences in metabolite composition of these seaweeds, reflecting their taxonomic classification. We highlight these differences for amino acid, amino acid derivative and peptide metabolites, energy and carbohydrate metabolites, for lipid, fatty acid and sterol metabolites and for secondary metabolites, including selected metabolic pathways such as the urea cycle, the citrate cycle and the glyconeogenesis/glycolysis, besides others. Additionally, we link selected seaweed biochemical properties to potential pharmaceutical and nutraceutical applications.

Oily fish, a source of long-chain omega-3 polyunsaturated fatty acids (LC n-3 PUFAs), may contain persistent organic pollutants (POPs), including α-hexabromocyclododecane (α-HBCD). In experimental studies, marine LC n-3 PUFAs ameliorate fatty liver development while HBCD exposure was found to cause liver fatty acid (FA) changes. The present study investigated interactions of FAs and α-HBCD in juvenile female BALB/c mice using a factorial design. Mice (n = 48) were exposed for 28 days to a low (100 μg*kg body weight (BW) −1 *day −1 ) or high dose (100 mg*kg BW −1 *day −1 ) of α-HBCD in diets with or without LC n-3 PUFAs. High dose α-HBCD affected whole body lipid metabolism leading to changes in body weight and composition, and pathological changes in hepatic histology, which surprisingly were aggravated by dietary LC n-3 PUFAs. Hepatic FA profiling and gene expression analysis indicated that the dietary modulation of the hepatotoxic response to the high dose of α-HBCD was associated with differential effects on FA β-oxidation. Our results suggest that in a juvenile mouse model, marine FAs accentuate hepatotoxic effects of high dose α-HBCD. This highlights that the background diet is a critical variable in the risk assessment of POPs and warrants further investigation of dietary mediated toxicity of food contaminants.

The Norwegian seaweed industry is expanding and there is a need for accurate estimates of protein content of seaweed species from Norwegian waters. A solid method to calculate protein content is through the sum of the proteomic amino acids; however, it can be expensive and beyond the capacities of many laboratories. The most commonly used method to quantify protein is based on the assessment of crude protein from overall nitrogen content, using the traditional nitrogen-to-protein conversion factor of 6.25. However, this approach can be inaccurate when applied to seaweeds, often resulting in an overestimation of their protein content. Specific nitrogen-to-protein conversion factors, calculated from amino acid composition and total nitrogen, give a more reliable protein quantification in seaweeds. However, no such factors are available for species from Norwegian waters. This study was designed to characterize the amino acid composition of 21 seaweed species from Norwegian waters and use the amino acid data to estimate protein contents of the seaweeds. Crude protein analysis (nitrogen × 6.25) was performed and resulted in overestimation (18–44 %) of the protein content compared to the sum of proteomic amino acids. Specific nitrogen-to-protein conversion factors, calculated for each species, ranged from 3.53 ± 0.1 to 5.13 ± 0.1. This study provides nutritional data on Norwegian seaweeds, covering a relatively wide range of species. Moreover, it is the first study to assess nitrogen-to-protein conversion factors on such species.

Atlantic salmon (Salmo salar) were fed five graded levels of eicosapentaenoic acid (EPA, 20:5n-3) and docosahexaenoic acid (DHA, 22:6n-3), from 1.4 to 5.2% of total fatty acids (FA, 5–17 mg kg−1 feed), and grew from ~160 g to ~3000 g, with the period from 1450 g onwards conducted both at 6 °C and at 12 °C. All fish appeared healthy, and there were no diet-related differences in haematological or plasma parameters, as well as intestinal histological or gut microbiota analysis. Fish reared at 6 °C had higher accumulation of storage lipids in the liver compared to fish reared at 12 °C. Liver lipids also increased with decreasing dietary EPA + DHA at 6 °C, while there was no such relationship at 12 °C. Gene expression of SREBP1 and 2, LXR, FAS and CPT1 could not explain the differences in liver lipid accumulation. In liver polar lipids, DHA was found to be reduced when dietary EPA + DHA was

This study investigated whether retention of n-3 fatty acids (FAs) is influenced by the levels of dietary monounsaturated (MUFA)-, saturated (SFA)- and n-6 FAs. The feeding trial used a mixture design, with rapeseed-, palm (PO)- and soybean oil (SO), providing high levels of MUFA, SFA and n-6 FAs, respectively, while 18:3n-3, eicosapentaenoic acid (20:5n-3) and docosahexaenoic acid (DHA, 22:6n-3) were kept constant. Furthermore, a diet group with high SO and reduced 18:3n-3 was included in addition to the mixture design. There were no differences in growth or proximate composition, but the PO diet gave reduced FA digestibility and increased feed conversion ratio. All diet groups had a net production of DHA, shown by retention values >100% (133%–177%). High dietary n-6 FAs of up to 43% of FAs (18:2n-6/18:3n-3 ratio 8.6) did not negatively affect DHA retention, but rather had a small positive impact. High levels of substrate (dietary 18:3n-3 at ~10% of FAs) did not enhance DHA retention compared to 5% 18:3n-3. This is a highly significant finding for the aquaculture industry, allowing for greater flexibility in the choice of lipid sources to replace fish oil in salmon feeds.

The present study was conducted to evaluate the effects on Atlantic salmon hepatic lipid metabolism when fed diets with increasing substitution of fish oil (FO) with a vegetable oil (VO) blend. Four diets with VOs replacing 100, 90, 79 and 65 % of the FO were fed for 5 months. The levels of eicosapentaenoic acid (EPA; 20:5n-3) and docosahexaenoic acid (DHA; 22:6n-3) in the experimental diets ranged from 1.3 to 7.4 % of fatty acids (FAs), while cholesterol levels ranged from 0.6 to 1.2 g kg(-1). In hepatocytes added [1-(14)C] α-linolenic acid (ALA, 18:3n-3), more ALA was desaturated and elongated to EPA and DHA in cells from fish fed 100 % VO, while in fish fed 65 % VO, ALA was elongated to eicosatrienoic acid (ETE; 20:3n-3), indicating reduced Δ6 desaturation activity. Despite increased desaturation activity and activation of the transcription factor Sp1 in fish fed 100 % VO, liver phospholipids contained less EPA and DHA compared with the 65 % VO group. The cholesterol levels in the liver of the 100 % VO group exceeded the levels in fish fed the 65 % VO diet, showing an inverse relationship between cholesterol intake and liver cholesterol content. For the phytosterols, levels in liver were generally low. The area as a proxy of volume of lipid droplets was significantly higher in salmon fed 100 % VO compared with salmon fed 65 % VO. In conclusion, the current study suggests that suboptimal dietary levels of cholesterol in combination with low levels of EPA and DHA (1.3 % of FAs) can result in minor metabolic perturbations in the liver of Atlantic salmon.

Increased plant oil inclusion in aquaculture feeds has led to higher dietary phytosterol concentrations and speculation about whether this affects the metabolism and health of the fish. The mechanisms of cholesterol absorption and how phytosterols may affect this is unknown in fish. Zebrafish (Danio rerio) were used to study the effects of phytosterols on the uptake and organ distribution of dietary cholesterol in fish. One meal of diets containing a constant addition of cholesterol (cold and [4-14C] cholesterol) and varying types and concentrations of phytosterols were fed to fish in individual compartments. The fish were not previously conditioned on the experimental diets. Activity of 14C was then measured in water and fish tissues to quantify the tissue distribution and excretion of cholesterol. There were no effects of the moderate dietary concentrations of phytosterols on the excretion or tissue distribution of dietary cholesterol 24 hours after the meal.

Request from the Norwegian Food Safety Authority (NFSA) In September 2013, the Norwegian Food Safety Authority requested VKM to update relevant parts of the benefit-risk assessment of fish in the Norwegian diet published by VKM in 2006. The background for the request was new knowledge and data on the content of some nutrients and contaminants both for wild and farmed fish since 2006. The proportion of vegetable ingredients used in farmed fish feed has in recent years increased, and new national dietary surveys for adults and children have been conducted. The Norwegian Food Safety Authority referred to VKM’s report from 2006, which pointed out that the positive impact of fish consumption on public health was especially due to the content of polyunsaturated fatty acids and vitamin D in fish. Further, VKM concluded that the contaminants that could pose a potential risk to public health through fish consumption mainly were methylmercury, dioxins and dioxin-like PCBs (dl-PCBs). Grey Literature Skare et al.; EJNFS, 5(4): 260-266, 2015; Article no.EJNFS.2015.023 261 The request included a reassessment of fish consumption in Norway with focus on specific nutrients; n-3 fatty acids (eicosapentaenoic acid (EPA), docosapentaenoic acid (DPA), docosahexaenoic acid (DHA), vitamin D, and the minerals iodine and selenium, and on specific contaminants; mercury, dioxins and dl-PCBs. VKM was asked to address the main changes in the use of raw materials in farmed fish feed and how these affect the levels of nutrients, mercury, dioxins and dl-PCBs and in fish feed. Further, VKM was asked to address to what extent levels of nutrients and contaminants in fish have changed since 2006, to describe these changes and estimate the human intake of the substances in question on the basis of recent dietary data. VKM was also requested to consider the benefits of eating fish with regard to the intake of nutrients and the risks associated with the intake of mercury, dioxins and dl-PCBs and comment on whether this change the conclusions from the report in 2006. Additionally, on the basis of updated knowledge, VKM was asked to comment whether other substances, like pesticide and residues of veterinary medicinal products, could affect the conclusions with regard to the impact on public health. The Norwegian Food Safety Authority and the Directorate of Health will use the updated assessment as a basis for public recommendations concerning the consumption of fish and fish products. How VKM has Addressed the Request The VKM appointed a working group consisting of VKM members and external experts to answer the request. Several of the scientific panels of VKM reviewed the report during its preparation. The Scientific Steering Committee of VKM has given their final assessment and approval of the current report. In the current report, VKM has mainly used data from national surveillance and monitoring programs for nutrient and contaminant concentrations in fish feed, farmed fish and wild caught fish, but occurrence data have also been derived from peer reviewed articles. VKM has estimated fish consumption in three population groups (2-year-olds, adults and pregnant women). The estimated fish consumption was compared to national dietary guidelines. To assess health effects of fish consumption, the current estimated fish intakes were also compared with assessments done by recognised international bodies and results from epidemiological studies addressing possible associations between fish consumption and specific health outcomes. Literature searches were done to identify relevant epidemiological studies. VKM has not systematically assessed reviews or meta-analyses nor individual studies for weight of evidence, but merely summarised the studies retrieved from the literature search. It was considered being beyond the scope of this assessment to review individual studies included in reviews or meta-analyses. Furthermore, based on current fish consumption in the various population groups, intake of nutrients and exposure to contaminants from fish were estimated. For benefit characterisation of the specific nutrients the estimated nutrient intake was compared with national recommendations of nutrients intake and for EPA and DHA a comparison was also done with European recommendations. For risk characterization of contaminant exposure from fish, VKM used health based guidance values set by international risk assessment bodies (WHO, EFSA). VKM noted that the request from NFSA was restricted to fish, whereas the VKM report in 2006 included both fish and other seafood. VKM focused on specific nutrients and contaminants as requested by the NFSA. In addition, VKM also commented on other substances that could affect the risk assessment, such as residues of veterinary medicinal products including residues of antibiotics, new contaminants from fish feed like the pesticide endosulfan, polycyclic aromatic hydrocarbons (PAHs), mycotoxins, the synthetic antioxidants ethoxyquin, butylhydroxyanisole (BHA) and butylhydroxytoluene (BHT), as well as environmental contaminants like brominated flame retardants and perfluorated organic compounds. Skare et al.; EJNFS, 5(4): 260-266, 2015; Article no.EJNFS.2015.023

The black soldier fly (Hermetia illucens) is one of the most promising insect species for use in animal feed. However, studies investigating feed and food safety aspects of using black soldier fly as feed are scarce. In this study, we fed black soldier fly larvae feeding media enriched with seaweed, which contains naturally high concentrations of heavy metals and arsenic. The aim of this study was to investigate the potential transfer of such undesirable substances from the feeding media to the larvae.The larvae accumulated cadmium, lead, mercury and arsenic. Concentrations of these elements in the larvae increased when more seaweed was added to the feeding media. The highest retention was seen for cadmium (up to 93%) and the lowest for total arsenic (up to 22%). When seaweed inclusion exceeded 20% in the media, this resulted in larval concentrations of cadmium and total arsenic above the current European Union maximum levels for these elements in complete feed.Our results confirm that insect larvae can accumulate heavy metals and arsenic when present in the feeding media. A broader understanding of the occurrence of these undesirable substances in processed larvae products is needed to assess feed and food safety. © 2017 Society of Chemical Industry.

Consumption of fatty acids from fishes is widely regarded as beneficial for preventing cardiovascular disorders. Nevertheless, salmonids themselves are victims of vascular diseases. As the pathogenesis and nature of these changes are elusive, they are here addressed using novel morphological and transcriptional approaches. Coronary arteries of wild Atlantic salmon Salmo salar L., (n = 12) were investigated using histological and immunohistochemical techniques, and RT-qPCR was employed to investigate expression of stretch-induced genes. In an experimental trial, fish were fed diets with different fatty acids composition, and histological features of the coronary arteries (n = 36) were investigated. In addition, the heart fatty acid profile (n = 60) was analysed. There were no differences in morphological or immunological features between wild fish and groups of experimental fish. Arteriosclerotic lesions consisted of smooth muscle cells in dissimilar differential stages embedded in considerable amounts of extracellular matrix in a similar fashion to what is seen in early stages of human atherosclerosis. No fat accumulations were observed, and very few inflammatory cells were present. In affected arteries, there was an induction of stretch-related genes, pointing to a stress-related response. We suggest that salmon may have a natural resistance to developing atherosclerosis, which corresponds well with their high investment in lipid metabolism.

The effects on Atlantic salmon (Salmo salar L.) metabolic health of including modern processed land animal by-products (LAP) to a plant- and marine-based diet (50% marine and 50% plant ingredients) were investigated. Three experimental diets were made with systematic replacements of both marine and plant ingredients with LAP as a source of protein (poultry meal and porcine blood meal) and fat (poultry oil) to fit a two-way factorial design. A 16-week feeding trial was performed with postsmolts in seawater (initial weight 372 g). The diet with both protein and lipids from LAP reduced liver triacylglycerols more than fourfold compared to the diet without LAP. This was confirmed by histological examinations showing reduced fatty degeneration in the liver of fish fed the high LAP diet. No severe negative effects on gut or tissue health were seen by histological examinations or by measuring genetic markers with qPCR, although a trend in the histological results indicated an improved gut health by including LAP in the diets. Clinical analyses of plasma and lipoprotein fractions showed no differences between dietary groups.

The suitability of land animal by-products (ABPs) in feed for Atlantic salmon postsmolts (initial weight 372 g) in sea water was studied in a feeding experiment, using poultry by-product meal (PBM) and porcine blood meal (BM) as protein sources and poultry oil as fat source. Four extruded feeds were tested in a 2 * 2 factorial model, with or without ABP protein sources and with or without poultry oil. The control feed contained a mix of marine and plant ingredients. Initial feed intake was highest in the ABP protein-based diets, whereas poultry oil had a weak opposite effect. No differences were seen in growth rate or body weight. Addition of PBM and BM led to increased FCR, and lower retention of crude protein and energy. This could be explained by lower digestibility of amino acids and crude protein, and a slightly lower energy level in these diets. Reduced igf1 mRNA levels in liver and muscle were seen in fish fed dietary ABP protein and oil. Despite lower protein digestibility of ABP protein, this study confirms the suitability of ABP protein and lipid in combination with plant ingredients in feed for Atlantic salmon growers.

In two long-term feeding trials in seawater, Atlantic salmon were fed EPA+DHA in graded levels, from 1·3 to 7·4 % of fatty acids (FA, 4–24 g/kg feed) combined with approximately 10 % 18 : 3n-3, at 6 and 12°C. Dietary EPA appeared to be sufficient in all diet groups, as no differences were seen in polar lipid tissue concentrations of either the brain, retina or erythrocytes. For DHA, a reduction in tissue levels was observed with low dietary supply. Effects on brain DHA at ≤1·4 % EPA+DHA of dietary FA and retina DHA at ≤2·7 % EPA+DHA of dietary FA were only observed in fish reared at 6°C, suggesting an effect of temperature, whereas tissue levels of n-6 FA increased as a response to increased dietary n-6 FA in both the brain and the retina at both temperatures. DHA levels in erythrocytes were affected by ≤2·7 % EPA+DHA at both temperatures. Therefore, DHA appears to be the limiting n-3 FA in diets where EPA and DHA are present in the ratios found in fishmeal and fish oil. To assess the physiological significance of FA differences in erythrocytes, the osmotic resistance was tested, but it did not vary between dietary groups. In conclusion, ≤2·7 % EPA+DHA of FA (≤9 g/kg feed) is not sufficient to maintain tissue DHA status in important tissues of Atlantic salmon throughout the seawater production cycle despite the presence of dietary 18 : 3n-3, and effects may be more severe at low water temperatures.

Dietary intake of linoleic acid (LA) has increased dramatically during the twentieth century and is associated with a greater prevalence of obesity. Vegetable oils are recognised as suitable alternatives to fish oil (FO) in feed for Atlantic salmon (Salmo salarL.) but introduce high amounts of LA in the salmon fillet. The effect on fish consumers of such a replacement remains to be elucidated. Here, we investigate the effect of excessive dietary LA from soyabean oil (SO) on endocannabinoid levels in Atlantic salmon and mice, and study the metabolic effects in mice when SO replaces FO in feed for Atlantic salmon. Atlantic salmon were fed FO and SO for 6 months, and the salmon fillet was used to produce feed for mice. Male C57BL/6J mice were fed diets of 35 % of energy as fat based on FO- and SO-enriched salmon for 16 weeks. We found that replacing FO with SO in feed for Atlantic salmon increased LA, arachidonic acid (AA), decreased EPA and DHA, elevated the endocannabinoids 2-arachidonoylglycerol (2-AG) and anandamide (AEA), and increased TAG accumulation in the salmon liver. In mice, the SO salmon diet increased LA and AA and decreased EPA and DHA in the liver and erythrocyte phospholipids, and elevated 2-AG and AEA associated with increased feed efficiency, weight gain and adipose tissue inflammation compared with mice fed the FO salmon diet. In conclusion, excessive dietary LA elevates endocannabinoids in the liver of salmon and mice, and increases weight gain and counteracts the anti-inflammatory properties of EPA and DHA in mice.

To investigate effects on glucose and amino acid (AA) metabolism and neuroendocrine regulation of energy homoeostasis and GH-IGF system growth regulation, Atlantic salmon were fed high levels of plant protein (PP) and vegetable oil (VO) for 1 year and compared to fish fed intermediate levels or a control diet based on fish meal and fish oil. All plant-fed groups had reduced liver glucokinase mRNA and increased muscle glycogen compared to the control group. As digestible starch was highest in the control diet, the increased muscle glycogen might be related to other factors such as lower levels of lysine or higher levels of glucogenic AAs. There were minor effects on transcription of genes related to leptin, ghrelin and GH-IGF system. However, muscle growth hormone receptor was elevated in fish fed high PP, probably related to lipolytic action in these fish. Fish fed high levels of both PP and VO simultaneously had elevated plasma alanine aminotransferase and glucose and of pentose phosphate pathway enzymes. Furthermore, this group had elevated concentrations of some free AAs, especially proline. These results support interaction effects when both high levels of PP and VO are used together, which needs to be considered in the formulation of aquafeeds.

Adult Atlantic salmon (Salmo salar; approximately 800 g start weight) were fed diets with a high replacement of fish meal (FM) with plant proteins (70% replacement), and either fish oil (FO) or 80% of the FO replaced by olive oil (OO), rapeseed oil (RO) or soybean oil (SO) during 28 weeks in triplicate. Varying the lipid source only gave non-significant effects on growth and final weight. However, a significantly reduced feed intake was observed in the SO fed fish, and both feed utilization and lipid digestibility were significantly reduced in the FO fed fish. Limited levels of dietary 18:3n-3, precursor to EPA and DHA, resulted in no net production of EPA and DHA despite increased mRNA expression of delta-5-desaturase and delta-6-desaturase in all vegetable oil fed fish. Net production of marine protein, but not of marine omega-3 fatty acids, is thus possible in Atlantic salmon fed 80% dietary vegetable oil and 70% plant proteins resulting in an estimated net production of 1.3 kg Atlantic salmon protein from 1 kg of FM protein. Production of one 1 kg of Atlantic salmon on this diet required only 800 g of wild fish resources (Fish in - Fish out

Fish based diets have been linked to the amelioration of methylmercury (MeHg) induced symptoms in several epidemiological studies, particularly due to their contents of marine n-3 fatty acids. It has been suggested that n-3 fatty acids may mask the detrimental effects of MeHg due to their beneficial effect on the same biological functions which are negatively affected by MeHg. However, in vitro studies have implied that there may be direct interactions between the marine n-3 FAs and MeHg, which ameliorates MeHg toxicity through interactions at a biological level. To understand how marine n-3 FAs and MeHg interact in fish as a biological system, we wanted to investigate molecular interaction in a fish cell system. Atlantic salmon kidney (ASK) cells were pre-incubated with the marine n-3 FAs docosahexaenoic acid (22:6n-3, DHA) and eicosapentaenoic acid (20:5n-3, EPA) before exposing them to MeHg. Modulating effects of the marine FAs on MeHg toxicity were subsequently assessed using the exploratory technique of proteomics, in a factorial design. Thirty-four differentially regulated proteins were identified. From these; twenty-seven were shown to be differentially regulated by MeHg, twelve were regulated by the fatty acids, and another eight showed interaction effects between MeHg and the FAs. Several of the proteins were concomitantly affected by MeHg- and FA-main effects, as well as interaction effects. Functional annotations and pathway analysis of the proteins revealed that marine n-3 FAs and MeHg concurrently affected the abundance of protein markers relating to such molecular mechanisms as: cell signaling, calcium homeostasis, structural integrity, apoptosis, and energy metabolism. In conclusion, both marine n-3 FAs and MeHg can differentially affect the abundances of the same proteins, indicating modulating effects of EPA and DHA on MeHg metabolism, and possibly on its toxicity.

This experiment aimed to study the molecular toxicity of methylmercury (MeHg) in liver, brain and white muscle of Atlantic salmon fed a diet based on fish oil (FO, high dietary n-3/n-6 ratio) compared to an alternative diet mainly based on vegetable oil (VO, low dietary n-3/n-6 ratio). Juvenile salmon were fed decontaminated diets or the FO and VO diets enriched with 5 mg Hg/kg (added as MeHg) for three months. The dietary lipid composition affected the fatty acid composition in the tissues, especially in liver and white muscle. After 84 days of exposure, the liver accumulated three times as much MeHg as the brain and white muscle. Vitamin C content and heme oxygenase, tubulin alpha (TUBA) and Cpt1 transcriptional levels all showed significant effects of MeHg exposure in the liver. TBARS, α-tocopherol, γ-tocopherol, and the transcriptional levels of thioredoxin, heme oxygenase, TUBA, PPARB1, D5D and D6D showed an effect of dietary lipid composition in liver tissue. Effects of dietary lipids were observed in brain tissue for MT-A, HIF1, Bcl-X and TUBA. Interaction effects between MeHg exposure and dietary lipid composition were observed in all tissues. Our data suggest that dietary fats have modulating effects on MeHg toxicity in Atlantic salmon.

A two-compartmental model for the kinetics of carry-over of the brominated flame retardant α-hexabromocyclododecane (HBCD) from feed to the fillet of farmed harvest-sized Atlantic salmon (Salmo salar L.) was developed. The model is based on a fat compartment for storage of the lipophilic α-HBCD and a central compartment comprising all other tissues. Specific for this model is that the salmon has a continuous growth and that fillet contaminant levels are explained by both the fat and the central compartments. The uptake and elimination kinetics are obtained from experimental data where consumer sized (start weight approximately 1 kg) Atlantic salmon was fed α-HBCD spiked feed (280 ± 11 µg kg−1) for 2 months followed by a depuration period of 3 months. The model was used to simulate the HBCD feed-to-fillet transfer in Atlantic salmon under realistic farming conditions such as the seasonal fluctuations in feed intake, growth and fillet fat deposition. The model predictions gave fillet concentrations of 0.2–1.8 µg kg−1 depending on the level of fish oil inclusion in the salmon diets when using fish oil with high POP background levels. Model simulations show that currently farmed Atlantic salmon can contribute to a maximum of 6% of the estimated provisional food reference dose for HBCD.

The present study investigates the effects of genotype on responses to alternative feeds in Atlantic salmon. Microarray analysis of the liver transcriptome of two family groups, lean or fat, fed a diet containing either a fish oil (FO) or a vegetable oil (VO) blend indicated that pathways of cholesterol and lipoprotein metabolism might be differentially affected by the diet depending on the genetic background of the fish, and this was further investigated by real-time quantitative PCR, plasma and lipoprotein biochemical analysis. Results indicate a reduction in VLDL and LDL levels, with no changes in HDL, when FO is replaced by VO in the lean family group, whereas in fat fish fed FO, levels of apoB-containing lipoproteins were low and comparable with those fed VO in both family groups. Significantly lower levels of plasma TAG and LDL-TAG were measured in the fat group that was independent of diet, whereas plasma cholesterol was significantly higher in fish fed the FO diet in both groups. Hepatic expression of genes involved in cholesterol homeostasis, β-oxidation and lipoprotein metabolism showed relatively subtle changes. A significantly lower expression of genes considered anti-atherogenic in mammals (ATP-binding cassette transporter A1, apoAI, scavenger receptor class B type 1, lipoprotein lipase (LPL)b (TC67836) and LPLc (TC84899)) was found in lean fish, compared with fat fish, when fed VO. Furthermore, the lean family group appeared to show a greater response to diet composition in the cholesterol biosynthesis pathway, mediated by sterol-responsive element-binding protein 2. Finally, the presence of three different transcripts for LPL, with differential patterns of nutritional regulation, was demonstrated.

The development of cataract in farmed Atlantic salmon (Salmo salar L.) has been related to changes in feed composition resulting in sub-optimal lens nutrition. The present study was performed to investigate the ability of Atlantic salmon lenses to withstand oxidative stress ex vivo, with focus on the nutritional lipid history and exposure to methylmercury (MeHg) as a relevant dietary contaminant. Since dietary histidine has been shown to have a mitigating effect on the prevalence of cataract in farmed salmon, the antioxidative abilities of histidine and NAH, a major imidazole in the salmon lens, was also investigated ex vivo. Lenses from Atlantic salmon prefed diets based on either fish oil (FO) or vegetable oil (VO) as lipid source, with or without addition of 5 mg MeHgkg(-1) feed, were cultured for 96 h in normal medium (control), medium added 5 mM H(2)O(2) or in histidine enriched medium. Lipid class composition of the lenses was not affected by the dietary lipids; while VO fed fish had a decrease in lens n-3/n-6 fatty acid ratio due to minor but significant increase in the concentration of 18:2 n-6 and 20:4 n-6, and decrease in 20:5 n-3 fatty acids compared to FO fed fish. The lenses accumulated mercury in response to dietary levels, but neither the oxidative status nor any physiological responses were affected. The cultured lenses responded to H(2)O(2) exposure with loss of transparency, accumulation of auto-fluorescent compounds, volume increase and reduced glutathione concentration similarly and irrespective of the dietary history. Lenses extracted histidine from the media, and synthesised NAH during the culture period. The innate antioxidative defence system appeared to be influenced both by the dietary lipid history and histidine enrichment on a transcriptional level. Catalase and SPARC were expressed higher in lenses from FO fed fish, and glutaredoxin showed elevated expression levels in FO lenses cultured in histidine enriched medium, suggesting that histidine is related to the innate antioxidant defence in salmon lenses. Further, the concentration of NAH was significantly reduced in oxidatively stressed lenses. Based on the results from this study it is suggested that NAH has a novel role as antioxidant in the Atlantic salmon lens.

The aim of the present study was to examine whether a combined plant protein and vegetable oil replacement (P diet) of fish-based marine feed ingredients (F diet) imposes oxidative stress on Atlantic salmon after short- and long-term feeding. Oxidative stress was assessed by measuring a suit of hepatic biomarkers, including TBARS, enzymatic and transcriptional levels of a set of antioxidant proteins as well as antioxidant vitamins. Antioxidants assessed regularly in the long-term study were influenced by the dietary compositions, mostly influencing liver astaxanthin and tocopherols. In the short-term crossover study, fish were sampled initially, at Day 2 and at Day 17 after transfer to new diet. The results showed that fish relocated from the F diet to the P diet had significantly lower transcriptional levels of CuZn SOD (Day 2, P < 0.006) and Mn SOD (Day 17, P < 0.003), in addition to lower levels of catalase protein (Day 17, P < 0.05) and total glutathione (Day 17, P < 0.05). At Day 17, both groups of fish relocated to new diets had significantly increased transcriptional levels of GSH-Px (F-P: P < 0.05, P-F: P < 0.007). This study showed that switching from a marine-based to a plant-based diet affected Atlantic salmon gene expression and the antioxidative defence in the fish.

Atlantic salmon were fed one of four diets with increasing levels (11–58%of total fatty acids (FAs)) of n-3 highly unsaturated FAs (HUFAs) in order to investigate the effect on muscle and mitochondrial membrane lipids and their susceptibility to oxidative stress. The high n-3 eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) groups contained lower percentages of sphingomyelin and cardiolipin in total muscle than the intermediate n-3 HUFA group. Cardiolipin and sphingomyelin are particularly susceptible to peroxidation, and a reduced percentage of these lipids showed that mitochondrial membranes had been damaged by oxidation. The intermediate n-3 HUFA group had the highest level of mitochondrial integrity and tendencies of lower thiobarbituric acid reactive substances (TBARS) level than the other dietary groups. The high caspase-3 activity, in addition to casp3a and bax gene expression levels, in the n-3 DHA group also suggests that some degree of oxidative stress had occurred. Electron microscopy images showed a higher degree of myofibre–myofibre detachment in fish fed the high HUFA diets than in fish fed the intermediate n-3 HUFA diet. Our findings show that intermediate levels of n-3 HUFAs in salmon diets gave the best protection against oxidative damage of mitochondrial membranes and muscle structure.

Fish oil is the main contributor of persistent organic pollutants (POPs) in fish feed. A combination of active carbon filtration and steam deodourization can remove most of the POPs. However, other fat soluble compounds are also removed, thus possibly affecting the nutritional quality of decontaminated fish oils. Sea water–adapted Atlantic salmon were fed 18 months a commercially relevant diet based on either decontaminated or non-treated fish oil until market size. The development of production-related diseases (fin/skin erosion, bone deformity, cataract) and fillet quality parameters (gutted weight, fillet fat soluble vitamin levels and fatty acid composition, colour, gaping, texture and sensory quality) were assessed. No significant differences in growth performances, feed conversion ratio or quality parameters between the two dietary groups were found. The fillet levels of fat soluble vitamins in market size fish remained unaltered, and only marginal differences were observed in fatty acid profiles. There was a significantly lower percentage of deformed vertebrae in the tail region of fish fed the decontaminated fish oil diets, indicating a positive effect of the use of decontaminated fish oil. No apparent negative effects of the use of decontaminated fish oil in Atlantic salmon diets were reported in this study.

Vegetable oils (Vo) are an alternative to fish oil (Fo) in aquaculture feeds. This study aimed to evaluate the effect of dietary soybean oil (Vo diet), rich in linoleic acid, and of dietary fish oil (Fo diet) on the development of spinal deformities under bacterial lipopolysaccharide (LPS)-induced chronic inflammation conditions in Atlantic salmon, Salmo salar L. Fish [25 g body weight (BW)] were fed the experimental diets for 99 days. On day 47 of feeding (40 g BW), fish were subjected to four experimental regimes: (i) intramuscular injections with LPS, (ii) sham-injected phosphate-buffered saline (PBS), (iii) intraperitoneally injected commercial oil adjuvant vaccine, or (iv) no treatment. The fish continued under a common feeding regime in sea water for 165 more days. Body weight was temporarily higher in the Vo group than in the Fo group prior to immunization and was also affected by the type of immunization. At the end of the trial, no differences were seen between the dietary groups. The overall prevalence of spinal deformities was approximately 14% at the end of the experiment. The Vo diet affected vertebral shape but did not induce spinal deformities. In groups injected with LPS and PBS, spinal deformities ranged between 21% and 38%, diet independent. Deformed vertebrae were located at or in proximity to the injection point. Assessment of inflammatory markers revealed high levels of plasma prostaglandin E₂ (PGE₂) in the Vo-fed and LPS-injected groups, suggesting an inflammatory response to LPS. Cyclooxigenase 2 (COX-2) mRNA expression in bone was higher in fish fed Fo compared to Vo-fed fish. Gene expression of immunoglobulin M (IgM) was up-regulated in bone of all LPS-injected groups irrespective of dietary oil. In conclusion, the study suggests that Vo is not a risk factor for the development of inflammation-related spinal deformities. At the same time, we found evidence that localized injection-related processes could trigger the development of vertebral body malformations.

Oily fish are an important source of health promoting nutrients such as the very long chain marine omega-3 (VLC-n3) fatty acids and simultaneously a source of potentially hazardous contaminants. Fish oils that are used in fish feed are the main source for both contaminants and VLC-n3. Decontamination techniques have recently been developed to effectively remove persistent organic contaminants from fish oils. The aim of the present study was to assess the level of potentially hazardous contaminants and the health beneficial fatty acids in Atlantic salmon reared on novel decontaminated feeds. Atlantic salmon were fed for 18 months (an entire seawater production cycle) on diets based on decontaminated or non-treated (control) fish oils until market size (approximately 5 kg). The level of known notorious persistent organic pollutants (POPs, i.e. dioxins, dioxin-like polychlorinated biphenyls (DL-PCBs), non dioxin-like PCBs, poly brominated diphenyl ethers (PBDE), and organochlorine pesticides), as well as fatty acid composition were analysed in fish oils, the two diets, and Atlantic salmon fillet. The oil decontamination process was a two-step procedure using active carbon and short path distillation. The fillet levels of POPs in market size fish were reduced by 68-85% while the concentration of very long chain omega-3 fatty acids was reduced by 4-7%. No differences in biomarkers of dioxin-like component exposures, such as hepatic gene expression of CYP1A or AhR2B, CYP1A protein expression and 7-ethoxyresorufin O-deethylase (EROD) activity, were observed between salmon raised on normal or decontaminated feeds, thus indicating that the difference in POPs levels were of no biological significance to the fish. Atlantic salmon reared on decontaminated feeds had sum polychlorinated dibenzodioxins/furans (PCDD/Fs) and DL-PCB concentrations that were comparable with terrestrial food products such as beef, while the level of marine omega-3 fatty acids remained as high as for commercially farmed Atlantic salmon.

The composition of the feed may alter the cellular composition of an organism and thus has the potential to influence a xenobiotic response. The main aim of this study was to see if the fatty acid composition of primary hepatocytes isolated from Atlantic salmon (Salmo salar L.) obtained from fish fed either a fish oil or a vegetable oil based diet, influenced the response to endosulfan exposure in vitro. The primary cultures were exposed to six different concentrations of endosulfan (0.001, 0.01, 0.1, 1, 10 and 100 µM) for 48 h. Cell morphology as well as a molecular toolbox of 16 genes encoding stress responsive and biotransformation proteins was examined. Endosulfan exposure caused moderate cytotoxicity and steatosis in a dose-dependent manner in the hepatocytes. In general, endosulfan hepatoxicity seems to be unaffected by the fatty acid composition of the hepatocytes. Exceptions were general stress (HSP70) and markers for estrogen exposure (ZP and VTG), which appeared to be slightly less responsive in hepatocytes isolated from the vegetable oil fed fish.

The study of CCAAT/enhancer binding proteins (C/EBPs) is important in the understanding of adipogenesis, but little is known about their regulation in fish. Here, we report three Atlantic salmon orthologs of c/ebp, and their expression in different tissues and in adipogenesis in vitro. During differentiation the expression of c/ebpα and fatp1 were upregulated in early differentiation stage with continuing high expression level in mature adipocytes, whereas c/ebpδ and fabp11 expression were elevated in mature adipocytes. Furthermore, the fatty acids eicosapentaenoic (EPA) and docosahexaenoic (DHA), suppressed the expression of the c/ebps, pparβ, and fatty acid transport protein (fatp1) during terminal adipocyte differentiation. The study indicates that C/EBPs are induced upon the differentiation of primary-cultured adipocytes from Atlantic salmon and that marine n-3 highly unsaturated fatty acids (HUFAs) affect the c/ebps expressions in mature adipocytes. Therefore, the established cell model described here appears to be valuable for studying modulation of fat content in farmed Atlantic salmon.

Relative gene expression pattern of fatty acid transport proteins (FATP and cd36), intracellular fatty acid-binding proteins (FABP3, FABP10 and FABP11), β-oxidation-related genes [carnitine palmitoyl transferase II (CPTII), peroxisome proliferator-activated receptor β (PPARβ), acyl-CoA oxidase (AOX), long-chain fatty acyl-CoA synthetase (FACS), acyl-CoA dehydrogenase (dehydrogenase)] and uncoupling protein 2 (UCP2) was assessed by RT-qPCR in Atlantic salmon muscle (red and white), liver, heart, myosepta and visceral fat. FABP11, a FABP isoform not previously described in Atlantic salmon, was highly expressed in visceral fat and myosepta and at the lower level in red muscle, white muscle, myosepta and heart. Furthermore, Atlantic salmon were fed either a diet containing fish oil (FO) or a complete replacement of FO with a vegetable oil blend (55% rapeseed oil, 30% palm oil and 15% linseed oil; VO) for the production cycle (27 months from start of feeding and until ∼4.5 kg mean weight). The expression of genes related to β-oxidation, fatty acid uptake and transport in the white muscle indicate (n = 3) significant down-regulation in VO fed Atlantic salmon and correlated with previously reported white muscle triacylglycerol stores and β-oxidation. FABP11 in visceral fat and myosepta was also down-regulated in VO fed fish.

This study had two main objectives: first, to evaluate the impact of different types and levels of dietary n-6 and n-3 fatty acids (FAs) on Atlantic salmon muscle structure integrity; second, to highlight a possible role of lysosomes and lysosomal degrading enzymes, cathepsins, in fish muscle structure integrity, in relation to dietary fatty acids. Four groups of Atlantic salmon (90 g starting weight) in fresh water tanks were fed one of four diets containing 23% crude lipids, with 100% of the added oils as either fish oil (FO), rapeseed oil (RO), eicosapentaenoic acid (EPA) enriched-oil or docosahexaenoic acid (DHA) enriched-oil. The RO diet was characterised by low levels of EPA + DHA (10% of total FAs), whereas the EPA and DHA diets were characterised by very high levels of EPA + DHA (>50% of total FAs). Fatty acid composition of the muscle crude lysosomal fraction (CLF) generally reflected the diets. Salmon fed the RO diet presented a muscle CLF FA composition close to the one of the FO group, showing moderate PUFA levels, and comparable cathepsin B and cathepsin L activities, relative gene expression of cathepsin B and cathepsin L in the muscle and rate of myofibre–myofibre detachments post-mortem. Salmon fed the EPA and DHA-enriched-oil diets presented a fairly similar muscle CLF FA composition, but different from the FO and RO groups. In the EPA and DHA groups, the percentage of PUFAs in the muscle CLF, the rate of myofibre–myofibre detachments and the relative gene expression of cathepsin B were higher than in the FO and RO groups. Cathepsin B and cathepsin L total activities in the muscle were however lower in the EPA and DHA groups 0 h post-mortem. Dietary lipids influenced the level of lysosomal degrading enzyme activity cathepsin B and cathepsin L as well as the relative gene expression of cathepsin B. Feeding Atlantic salmon with rapeseed oil and extreme levels of EPA + DHA highlighted the impact of fatty acid composition of the diet on salmon muscle integrity and the complexity of the process involving muscle lysosomes and cathepsins in relation to these dietary fatty acids.

Unsustainable fishing practices have placed a heavy emphasis on aquaculture to meet the global shortfalls in the supply of fish and seafood, which are commonly accepted as the primary source of health-promoting essential omega-3 (n-3 highly unsaturated fatty acids). However, dietary fish oil is required for the production of omega-3-rich farmed fish and this commodity, in a vicious circle, is at present derived solely from wild fisheries. Decreasing global availability coupled with the highly variable price of this resource has forced the aquaculture industry to investigate the possibilities of alternative dietary lipid sources. This review attempts to compile all principal information available regarding the effects of fish oil replacement for the diets of farmed finfish, analysing the findings using a comparative approach among different cultured fish species. The review initially focuses on the present situation with regard to the production, availability and main nutritional characteristics of fish oil and the principal alternative lipid sources (such as vegetable oils and animal fats). Following this, the effects of fish oil replacement in finfish nutrition on feed quality, fish performance, feed efficiency, fish lipid metabolism, final eating quality and related economic aspects are presented and discussed.

We have investigated how n-3 highly unsaturated fatty acids (HUFAs) in the diet affect fatty acid (FA) utilization, fat storage and oxidative stress (OS) in Atlantic salmon (Salmo salar) white adipose tissue (WAT). Four groups of Atlantic salmon were fed for 21 weeks on one of the four diets supplemented with 23% (of dry matter) lipid. Docosahexaenoic acid (DHA; 22:6n-3) and eicosapentaenoic acid (EPA; 20:5n-3) levels increased from 10% of total FAs in the rapeseed oil (RO) diet, to 20% in the fish oil (FO) diet, and to 50% and 55% in the DHA-enriched and EPA-enriched diets, respectively. Increased dietary levels of n-3 HUFAs resulted in lower fat percentage in WAT. Furthermore, mitochondrial FA beta-oxidation activity was higher in the FO group than it was in the RO group. The relative levels of DHA and EPA in phospholipids (PLs) from WAT and mitochondrial membranes increased with the increasing dietary levels of these HUFAs. In general, the mitochondrial membrane PLs were characterised by lower relative levels of n-3 HUFAs and higher relative levels of linoleic acid (LA; 18:2 n-6) than WAT membrane PLs. The predominance of LA relative to n-3 HUFAs in mitochondrial membrane PLs may help to protect these PLs from peroxidation. Cytochrome c oxidase measurements revealed higher incidence of disrupted mitochondrial membranes in the DHA and EPA dietary groups than in the FO and RO dietary groups. This disruption further affected the mitochondrial function, resulting in a marked reduction in FA beta-oxidation capacities. The reduction in mitochondrial function and the increase in the activity of superoxide dismutase (SOD) in the DHA and EPA groups showed that high dietary dose of DHA and EPA resulted in oxidative stress (OS). The increased activity of caspase 3 in the high n-3 HUFA groups suggested the induction of apoptosis and increased incidence of cell death in WAT, which may be one of the factors explaining the lower fat percentage found in these groups.

The aim of the present study was to combine maximum replacement of fish meal and fish oil with plant ingredients in feed for Atlantic salmon, in order to gain a sound and sustainable net fish protein production. The design implied that all known nutrient requirements should be met. Atlantic salmon smolts with an initial weight of 0.3 kg were fed in triplicate either a fully marine control diet or one of three plant based diets through the seawater production phase for 12 months, until final weight of approximately 4 kg. In a maximum plant based diet, 80% of the fish meal was replaced with a mixture of plant protein ingredients and krill meal, while 70% the fish oil was replaced with a mixture of vegetable oils. Two intermediate replacement diets contained either one half of this fish meal replacement level and maximum fish oil replacement, or one half replacement level of fish oil and maximum fish meal replacement. Fish performance was assessed by measuring mortality, feed intake, growth, nutrient digestibility and nutrient utilisation. Specific growth rate was significantly lower in the combined high replacement group compared to the other experimental groups, both for the first 3-month period (12%) and for the complete 12 months (9%) of feeding. The final fish weights were 17% lower in the combined high replacement group and 9% lower in the high plant protein and intermediate vegetable oil group, compared to the marine control and the intermediate plant protein group. Significantly reduced feed intake during the first period and slightly reduced digestibility of 16:0 and starch were identified as possible causes for growth depression, since minor differences in protein or lipid digestibility, feed conversion ratio, and protein and lipid retention were observed. The maximum fish meal and fish oil replacement represented a net production of fish protein, with 2 kg salmon protein produced per kg fish meal protein fed. This being four-fold more efficient usage of fish meal in the 80% plant protein diets compared to the 100% fish raw material diet.

Lipids are the predominant source of energy for fish. The mechanisms by which fish allocate energy from lipids for metabolism, development, growth, and reproduction are critical for understanding key life-history strategies and transitions. Currently, the major lipid component in aquaculture diets is fish oil (FO), derived from wild capture fisheries that are exploited at their maximum sustainable limit. The increasing demand from aquaculture for FO will soon exceed supply and threaten the viability of aquaculture. Thus, it is essential to minimize FO use in aquaculture diets. This might be achieved by a greater understanding of lipid storage and muscle growth, or the identification of alternatives to FO in feeds. This review focuses on recent research applying molecular and genomic techniques to the study of fin-fish lipid metabolism from an aquaculture perspective. Accordingly, particular emphasis will be given to fatty acid metabolism and to highly unsaturated fatty acid (HUFA) biosynthesis and to the ...

Atlantic salmon (Salmo salar) (90 g) were fed four different diets for 21 weeks (final weight 344 g). The levels of n-3 highly unsaturated fatty acids (HUFA) ranged from 11% of the total fatty acids (FA) in the low n-3 diet to 21% in the intermediate n-3 diet, to 55 and 58% in the high n-3 diets. The high n-3 diets were enriched with either docosahexaenoic acid (DHA) or eicosapentaenoic acid (EPA). Increasing dietary levels of n-3 HUFA led to increasing percentages (from 31 to 52%) of these FA in liver lipids. The group fed the highest level of DHA had higher expressions of peroxisome proliferator-activated receptor (PPAR) beta and the FA beta-oxidation genes acyl-CoA oxidase (ACO) and carnitine palmitoyltransferase (CPT)-II, compared to the low n-3 groups. The high n-3 groups had reduced activity of mitochondrial cytochrome c oxidase and beta-oxidation capacity, together with increased activities of superoxide dismutase (SOD) and caspase-3 activities. In the group fed the highest level of n-3 HUFA, decreased percentages of major phospholipids (PL) in the mitochondrial and microsomal membranes of the liver were also apparent. The percentage of mitochondrial cardiolipin (Ptd(2)Gro) was 3.1 in the highest n-3 group compared to 6.6 in the intermediate group. These data clearly show an increased incidence of oxidative stress in the liver of fish fed the high n-3 diets.

The main aim of the current in vitro experiment was to search for makers for PFOS exposure in isolated hepatocytes from Atlantic salmon Salmo salar, based on genes responding to PFOS exposure in other animals. Primary cell cultures of hepatocytes were exposed to four concentrations of PFOS (2.1-6.2-15.1-25.0 mg/L) for 24 and 48 h and the transcriptional levels of 12 genes encoding proteins known to respond to PFOS were quantified with real-time RT-PCR. The 12 examined genes were caspase 3B (apoptosis), GSH-Px and HSP70 (cellular stress), CYP1A, CYP3A, GST and UGT (P450 and phase II enzymes), acyl-CoA oxidase, PPARalpha, PPARbeta and PPARgamma (lipid metabolism) and Na(+)-K(+)-ATPase (ion regulation). Most of the studied genes responded in a dose-dependent manner to PFOS exposure, although the transcriptional differences in general where small with regard to fold change. Our results clearly suggest that PFOS exposure enhanced cellular stress in the examined cells, even though the exact mechanisms behind this stress remain unknown. The results from this in vitro experiment showed that genes known to be affected by PFOS exposure in other species also were induced in hepatocytes of Atlantic salmon, giving us the rationale to expand to testing the actual in vivo magnitude of effect in Atlantic salmon exposed to PFOS at doses usually seen in nature/diets.

Atlantic salmon (Salmo salar) preadipocytes, isolated from visceral adipose tissue, differentiate from an unspecialized fibroblast like cell type to mature adipocytes filled with lipid droplets in culture. The expression of the adipogenic gene markers peroxisome proliferated activated receptor (PPAR) alpha, lipoprotein lipase (LPL), microsomal triglyceride transfer protein (MTP), fatty acid transport protein (FATP) 1 and fatty acid binding protein (FABP) 3 increased during differentiation. In addition, we describe a novel alternatively spliced form of PPARgamma (PPARgamma short), the expression of which increased during differentiation. Eicosapentaenoic acid (20:5n-3, EPA) and docosahexaenoic acid (22:6n-3, DHA) lowered the triacylglycerol (TAG) accumulation in mature salmon adipocytes compared to oleic acid (18:1n-9, OA). This finding indicates that a reduced level of highly unsaturated n-3 fatty acids (HUFAs) in fish diets, when the traditional marine oil is exchanged for n-9 fatty acids (FAs) rich vegetable oils (VOs), may influence visceral fat deposition in salmonids. Moreover, major differences in the metabolism of EPA, DHA and OA at different stages during differentiation of adipocytes occur. Most of the EPA and DHA were oxidized in preadipocytes, while they were mainly stored in TAGs in mature adipocytes in contrast to OA which was primarily stored in TAGs at all stages of differentiation.

The nutritional requirement for n-3 long-chain PUFA in fast-growing Atlantic salmon (Salmo salar) during grow out in the sea is not well documented. Diets were formulated with levels of EPA (20 : 5n-3) and DHA (22 : 6n-3) ranging from 1·3 to 7·4 % of fatty acids (4–24 g/kg feed). Two long-term trials were conducted through the seawater phase, the first at 6 and 12°C, and the second at 12°C. In the first trial, growth at both temperatures was significantly lower in fish fed 1·4 % EPA+DHA of total fatty acids compared with the 5·2 % EPA+DHA group. In the second trial, growth was significantly lower in fish fed 1·3 and 2·7 % compared with 4·4 and 7·4 % EPA + DHA. Fatty acid composition in the fish reflected diet composition, but only after a 7-fold increase in body weight did the fatty acid profile of the fish stabilise according to dietary fatty acids (shown for EPA and DHA). The retention efficiency of DHA increased with decreasing dietary levels, and was 120–190 and 120–200 % in trials 1 and 2, respectively. The retention efficiency of EPA was lower (60–200 %), and values >100 % were only achieved at the lowest dietary levels in both trials. Temperature did not affect fatty acid retention efficiency. These results suggest that Atlantic salmon have a specific requirement for EPA + DHA >2·7 % of fatty acids for optimal long-term growth in seawater, and that short-term growth trials with less weight increase would not show these effects.

Extended use of plant ingredients in Atlantic salmon farming has increased the need for knowledge on the effects of new nutrients and contaminants in plant based feeds on fish health and nutrient-contaminant interactions. Primary Atlantic salmon hepatocytes were exposed to a mixture of PAHs and pesticides alone or in combination with the nutrients ARA, EPA, α-tocopherol, and γ-tocopherol according to a factorial design. Cells were screened for effects using xCELLigence cytotoxicity screening, NMR spectroscopy metabolomics, mass spectrometry lipidomics and RT-qPCR transcriptomics. The cytotoxicity results suggest that adverse effects of the contaminants can be counteracted by the nutrients. The lipidomics suggested effects on cell membrane stability and vitamin D metabolism after contaminant and fatty acid exposure. Co-exposure of the contaminants with EPA or α-tocopherol contributed to an antagonistic effect in exposed cells, with reduced effects on the VTG and FABP4 transcripts. ARA and γ-tocopherol strengthened the contaminant-induced response, ARA by contributing to an additive and synergistic induction of CYP1A, CYP3A and CPT2, and γ-tocopherol by synergistically increasing ACOX1. Individually EPA and α-tocopherol seemed more beneficial than ARA and γ-tocopherol in preventing the adverse effects induced by the contaminant mixture, though a combination of all nutrients showed the greatest ameliorating effect. Keywords: Interactions, Nutrients, PAH, Pesticides, Lipidomics, Metabolomics, RT-qPCR transcriptomics

Atlantic salmon Salmo salar were fed fish feeds based on 100% replacement of fish oil (FO) with plant oil (PO) as compared to a 100% FO-based diet. The transcript levels of eight genes’ encoding proteins involved in the cellular response to stressors [metallothionein-A isoform (MT-A), cytochrome P450 1A (CYP1A), heat-shock protein 70 (HSP70), catalase (CAT), glutathione S-transferase (GST), glutathione reductase (GR), proliferating cell nuclear antigen (PCNA) and caspase 6A] were quantified and compared to cell division (% PCNA-positive cells) and apoptosis [% terminal transferase mediated dUTP nick end labelling technique (TUNEL)-positive cells] in the mid intestinal (MI) section of the gastrointestinal tract. Five of these genes were also quantified in the pylorus caeca region (PR). Fish fed 100% PO-based diets had significantly lower density of both PCNA and TUNEL-positive cells in the MI section compared to fish fed a 100% FO-based diet. The transcription levels of GST and caspase 6A were found to be significantly lower in the MI section of individuals fed a PO-based diet compared to a FO-based diet, analysed by a t-test. In the PR, GR expression was significantly lower in the PO group compared to the FO group. The apoptosis markers PCNA and TUNEL were lower in the group fed a PO-based diet. The results suggest that the transcription levels of three of the studied genes, GST, caspase 6A and GR, can be used as indicators of the MI response to feeding S. salar a PO-based diet.

The effects of dietary oil source on lipid storage were evaluated in harvest size Atlantic salmon fed fish (FO) or vegetable oil (VO) based diets from start-feeding. Final weight and fat content were not significantly different for salmon fed FO and VO diets over a 27 month period. Fish fed these diets were scanned by computerized X-ray tomography (CT) and the whole body scans partitioned into two equal sections according to distance in longitudinal direction. Visible depot fat (area % of soft tissues) and mixed tissue fat levels were significantly higher in the anterior compared to posterior sections, but longitudinal fat distribution was not affected by diet. Differences in the fatty acid (FA) composition of the FO and VO diets were reflected in the tissues (white muscle, red muscle, white muscle myosepta, belly flap and visceral fat) of Atlantic salmon fed these diets. Lipid levels in these tissues were not significantly different for salmon fed FO and VO diets. No obvious differences were observed histologically in the lipid storage patterns of these tissues for salmon fed FO and VO diets. Visceral fat and belly flap tissues were predominated by adipocytes and contained the highest levels of total lipid and triacylglycerol (TAG) per g wet weight for the tissues tested. The red muscle contained higher lipid levels than the white muscle (> 4 fold for salmon fed the same FO or VO diet). Intracellular lipid droplets were observed by light microscopy in oil red O stained frozen sections of the red muscle but not observed to any extent in the white muscle (dorsal, lateral and ventral) for salmon fed FO and VO diets. Observations at higher magnifications, however, revealed small lipid droplets located in close association with mitochondria in the cytoplasm of white muscle cells. High densities of adipocytes were observed in the myosepta of red and white muscle for salmon fed FO and VO diets. White muscle myosepta had a significantly higher total lipid content and proportion of TAG than the white muscle. The belly flap, myosepta and visceral fat, (adipocyte-rich, lipid storage tissues) contained a higher proportion of TAG and monounsaturated FA as well as a lower proportion of polar lipid and n-3 polyunsaturated FA compared to the muscle tissues.