Long-term feeding of Atlantic salmon in seawater with low dietary long-chain n-3 fatty acids affects tissue status of the brain, retina and erythrocytes.

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.

Effect of marine omega 3 fatty acids on methylmercury-induced toxicity in fish and mammalian cells in vitro.

Methylmercury (MeHg) is a ubiquitous environmental contaminant which bioaccumulates in marine biota. Fish constitute an important part of a balanced human diet contributing with health beneficial nutrients but may also contain contaminants such as MeHg. Interactions between the marine n-3 fatty acids eicosapentaenoic acid (20:5n-3, EPA) and docosahexaenoic acid (22:6n-3, DHA) with MeHg-induced toxicity were investigated. Different toxic and metabolic responses were studied in Atlantic salmon kidney (ASK) cell line and the mammalian kidney-derived HEK293 cell line. Both cell lines were preincubated with DHA or EPA prior to MeHg-exposure, and cell toxicity was assessed differently in the cell lines by MeHg-uptake in cells (ASK and HEK293), proliferation (HEK293 and ASK), apoptosis (ASK), oxidation of the red-ox probe roGFP (HEK293), and regulation of selected toxicological and metabolic transcriptional markers (ASK). DHA was observed to decrease the uptake of MeHg in HEK293, but not in ASK cells. DHA also increased, while EPA decreased, MeHg-induced apoptosis in ASK. MeHg exposure induced changes in selected metabolic and known MeHg biomarkers in ASK cells. Both DHA and MeHg, but not EPA, oxidized roGFP in HEK293 cells. In conclusion, marine n-3 fatty acids may ameliorate MeHg toxicity, either by decreasing apoptosis (EPA) or by reducing MeHg uptake (DHA). However, DHA can also augment MeHg toxicity by increasing oxidative stress and apoptosis when combined with MeHg.

Ghrelin is involved in voluntary anorexia in Atlantic salmon raised at elevated sea temperatures.

Due to global and local climate changes, farmed salmon may experience periods of elevated sea temperatures. An experiment was conducted to examine endocrine and dietary effects of high sea temperatures in adult (2.0 kg) and sexually immature Atlantic salmon, Salmo salar L. Groups of salmon were exposed to 19 °C while others were kept as controls at 14 °C. The experiment lasted for 56 days, and fish were given iso-nitrogenous diets with either a normal (335 g kg(-1); L34) or a lower lipid level (298 g kg(-1); L30). Fish held at 19 °C had a reduction in the daily feed intake, growth and feed utilization of more than 50% compared to the controls. Fish at 19 °C retained little ingested fat, and high maintenance cost lead to depleted endogenous energy body reserves. Circulating ghrelin concentration and stomach ghrelin-1 and hypothalamus growth hormone secretagogue receptor 1a-like receptor (GHSR1a-LR) mRNA levels were significantly reduced in salmon at 19 °C. An increasing number of fish kept at 19 °C had empty gastrointestinal tract after 21 days (11-67%) and 56 days (56-100%), with the highest numbers in fish fed the L34 diet. We suggest that lower circulating ghrelin during negative energy homeostasis induce down-regulation of GHSR1a-LR, neuropeptide Y, and anorexigenic factors at transcriptional levels in the hypothalamus, which over time lead to a voluntary anorexia development in adult salmon held at 19 °C. Reduction of feed intake and growth may be an important coping strategy for salmon during elevated temperatures.

Toxicokinetics and carry-over model of α-hexabromocyclododecane (HBCD) from feed to consumption-sized Atlantic salmon (Salmo salar).

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.

Susceptibility of Atlantic salmon lenses to hydrogen peroxide oxidation ex vivo after being fed diets with vegetable oil and methylmercury.

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.

Dietary fatty acids and inflammation in the vertebral column of Atlantic salmon, Salmo salar L., smolts: a possible link to spinal deformities.

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.

Reducing persistent organic pollutants while maintaining long chain omega-3 fatty acid in farmed Atlantic salmon using decontaminated fish oils for an entire production cycle.

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.

Dietary n-3 HUFA affects mitochondrial fatty acid beta-oxidation capacity and susceptibility to oxidative stress in Atlantic salmon.

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.

Development of intimal thickening of coronary arteries over the lifetime of Atlantic salmon, Salmo salar L., fed different lipid sources.

The objective of the present study was to investigate the development of intimal changes of coronary arteries over the lifetime of farmed Atlantic salmon, Salmo salar L., fed either a 100% fish oil or a 100% vegetable oil blend. The study was performed as a randomized observer blinded controlled trial with parallel group design. At the start of the project, the fish were divided in two groups and sampled at five different time points throughout their life span. The total study sample consisted of 259 healthy fish. Serial sections were taken from the coronary artery lying on the bulbus arteriosus for histopathological evaluation and for area measurements using semi-quantitative and quantitative methods. The earliest onset of vascular changes was detected in fish from both groups in the freshwater stage prior to smoltification. The mean range lesion (MRL), used to describe the severity of the lesions observed, increased significantly for both groups from sea transfer throughout the study period. Comparison of the two groups based on the overall material corrected for time of sampling did not show any difference (P = 0.20) between the two groups with regard to MRL. The percentage lumen loss (PLL) measured by a quantitative method and used as a measure to indicate lesion severity showed an incremental, non-significant increase from week 72 to week 92 and further to week 115 in both diet groups during the seawater phase. Comparison of the groups corrected for time of sampling indicated a difference of PLL in favour of VO (P = 0.02). Heart weight, body weight and body length were all positively and significantly correlated to Log MRL. The partial correlation analysis indicated that heart weight was the most dominant variable in the set. Early vascular changes were found in the major bifurcation of the coronary artery at the apex and beyond the flow divider into the daughter branches. The latter represented the dominant changes and were found throughout the entire lifecycle of the fish. Increasing in size over time they formed pads or cushions that were regularly located close to the outer walls of the bifurcation. The origin of the cells forming the intimal thickening has not been conclusively determined, but immunohistochemical findings indicate a smooth muscle cell origin, possibly of a myointimal type. Our findings suggest there is no correlation between diet and intimal changes. The severity of the changes, MRL and PLL, of the coronary vessels correlate with heart weight and fish weight growth and growth rate and mechanical factors are implicated in intimal development, but rather than being induced by external injury due to the location of the coronaries, haemodynamic factors and low shear stress are proposed as the main mechanism behind these changes.

Lipid metabolism and tissue composition in Atlantic salmon (Salmo salar L.)--effects of capelin oil, palm oil, and oleic acid-enriched sunflower oil as dietary lipid sources.

Triplicate groups of Atlantic salmon (Salmo salar L.) were fed four diets containing different oils as the sole lipid source, i.e., capelin oil, oleic acid-enriched sunflower oil, a 1:1 (w/w) mixture of capelin oil and oleic acid-enriched sunflower oil, and palm oil (PO). The beta-oxidation capacity, protein utilization, digestibility of dietary fatty acids and fatty acid composition of lipoproteins, plasma, liver, belly flap, red and white muscle were measured. Further, the lipid class and protein levels in the lipoproteins were analyzed. The different dietary fatty acid compositions did not significantly affect protein utilization or beta-oxidation capacity in red muscle. The levels of total cholesterol, triacylglycerols, and protein in very low density lipoprotein (VLDL), low density lipoprotein (LDL), high density lipoprotein (HDL), and plasma were not significantly affected by the dietary fatty acids. VLDL, LDL, and HDL fatty acid compositions were decreasingly affected by dietary fatty acid composition. Dietary fatty acid composition significantly affected both the relative fatty acid composition and the amount of fatty acids (mg fatty acid per g tissue, wet weight) in belly flap, liver, red and white muscle. Apparent digestibility of the fatty acids, measured by adding yttrium oxide as inert marker, was significantly lower in fish fed the PO diet compared to the other three diets.