Tracking pollutants in dietary fish oil: From ocean to table.

Dietary fish oil used in aquafeed transfers marine pollutants to farmed fish. However, the entire transfer route of marine pollutants in dietary fish oil from ocean to table fish has not been tracked quantitatively. To track the entire transfer route of marine pollutants from wild fish to farmed fish through dietary fish oil and evaluate the related human health risks, we obtained crude and refined fish oils originating from the same batch of wild ocean anchovy and prepared fish oil-containing purified aquafeeds to feed omnivorous lean Nile tilapia and carnivorous fatty yellow catfish for eight weeks. The potential human health risk of consumption of these fish was evaluated. Marine persistent organic pollutants (POPs) were concentrated in fish oil, but were largely removed by the refining process, particularly dioxins and polychlorinated biphenyls (PCBs). The differences in the POP concentrations between crude and refined fish oils were retained in the fillets of the farmed fish. Fillets fat content and fish growth were positively and negatively correlated to the final POPs deposition in fillets, respectively. The retention rates of marine POPs in the final fillets through fish oil-contained aquafeeds were 1.3%-5.2%, and were correlated with the POPs concentrations in feeds and fillets, feed utilization and carcass ratios. The dietary crude fish oil-contained aquafeeds are a higher hazard ratio to consumers. Prohibiting the use of crude fish oil in aquafeed and improving growth and feed efficiency in farmed fish are promising strategies to reduce health risks originating from marine POPs.

A comparative study: Difference in omega-6/omega-3 balance and saturated fat in diets for Atlantic salmon (Salmo salar) affect immune-, fat metabolism-, oxidative and apoptotic-gene expression, and eicosanoid secretion in head kidney leukocytes.

The aim of this study was to compare how different dietary vegetable oil n-6/n-3 ratios affect gene responses involved in inflammation, signaling pathways, fatty acid synthesis and oxidation, oxidation and apoptosis as well as eicosanoid production in salmon head kidney tissues and isolated head kidney leukocytes. Salmon smolts (200 g) were fed four different diets where the main lipid components were palm oil (n-6/n-3 ratio = 0.7), rapeseed oil (n-6/n-3 ratio = 0.9), and soybean oil (n-6/n-3 ratio = 2.4) and a high soybean oil diet with an n-6/n-3 ratio = 4. Both head kidney tissue and leukocytes isolated from head kidneys were sampled from the four diets, but from different fish. Leukocytes isolated from the head kidneys were seeded into culture wells and added lipopolysaccharide (LPS) to induce inflammatory responses. Controls without LPS were included. Head kidney leukocytes and the tissues should have the same phenotype reflecting the different diets. Interleukin 1ß (IL-1ß) transcription was elevated in head kidney tissue and especially in LPS treated leukocytes isolated from soybean oil (n-6/n-3 = 2.4) fed salmon, which confirmed the suitability of the in vitro model in this experiment. Leukocytes, treated with LPS, and isolated from salmon fed the soybean oil diet (n-6/n-3 = 2.4) also upregulated tumor necrosis factor alpha (tnf-α), cyclooxygenase (cox2), prostaglandin D and E synthase (ptgds, ptges), fatty acyl synthase (fas), 5 and 6 desaturases (5des, 6 des) and a fatty acid translocase protein (cd36) when compared to the other diets. The results suggest that diets with a specific n-6/n-3 ratio influence the transcription of pro-inflammatory genes and may be cross-linked to transcription of selected fatty acid metabolism genes. Salmon fed the palm oil diet (n-6/n-3 = 0.7) showed a lower expression of inflammatory genes. Instead, peroxisome proliferator activated receptor ß1 (pparß1), acyl coenzyme A (aco), apoptosis regulator (bax) and superoxide dismutase (sod) were upregulated in leukocytes in vitro, while head kidney tissue transcription of a dendritic marker (cd83) was lower than measured in tissues from fish fed the other diets. The concentration of LTB4 (10-20 ng/mL) were relatively constant in leukocyte supernatants, all diets. Head kidney leukocytes from soybean oil (n-6/n-3 = 2.4) fed fish produced LPS induced PGE2 (mean 0.5 ng/mL) while leukocytes isolated from palm oil diet (n-6/n-3 = 0.7) secreted very high amounts of LTB5 (50-70 ng/mL). In addition, equal amounts of LPS induced PGE2 and PGE3 (mean 0, 5 ng/mL) were produced, indicating that the n-6/n-3 ratio of this saturated fatty acid may have a specific impact on eicosanoid production in the head kidney of salmon.

Lens metabolomic profiling as a tool to understand cataractogenesis in Atlantic salmon and rainbow trout reared at optimum and high temperature.

Periods of high or fluctuating seawater temperatures result in several physiological challenges for farmed salmonids, including an increased prevalence and severity of cataracts. The aim of the present study was to compare cataractogenesis in Atlantic salmon (Salmo salar L.) and rainbow trout (Oncorhynchus mykiss) reared at two temperatures, and investigate whether temperature influences lens metabolism and cataract development. Atlantic salmon (101±2 g) and rainbow trout (125±3 g) were reared in seawater at either 13°C (optimum for growth) or 19°C during the 35 days experiment (n = 4 tanks for each treatment). At the end of the experiment, the prevalence of cataracts was nearly 100% for Atlantic salmon compared to ~50% for rainbow trout, irrespective of temperature. The severity of the cataracts, as evaluated by slit-lamp inspection of the lens, was almost three fold higher in Atlantic salmon compared to rainbow trout. The global metabolic profile revealed differences in lens composition and metabolism between the two species, which may explain the observed differences in cataract susceptibility between the species. The largest differences were seen in the metabolism of amino acids, especially the histidine metabolism, and this was confirmed by a separate quantitative analysis. The global metabolic profile showed temperature dependent differences in the lens carbohydrate metabolism, osmoregulation and redox homeostasis. The results from the present study give new insight in cataractogenesis in Atlantic salmon and rainbow trout reared at high temperature, in addition to identifying metabolic markers for cataract development.

Antioxidant nutrition in Atlantic salmon (Salmo salar) parr and post-smolt, fed diets with high inclusion of plant ingredients and graded levels of micronutrients and selected amino acids.

The shift from marine to plant-based ingredients in fish feeds affects the dietary concentrations and bioavailability of micronutrients, amino acids and lipids and consequently warrants a re-evaluation of dietary nutrient recommendations. In the present study, an Atlantic salmon diet high in plant ingredients was supplemented with graded levels of nutrient premix (NP), containing selected amino acids, taurine, cholesterol, vitamins and minerals. This article presents the results on the antioxidant nutrients vitamin C, E and selenium (Se), and effects on tissue redox status. The feed ingredients appeared to contain sufficient levels of vitamin E and Se to cover the requirements to prevent clinical deficiency symptoms. The body levels of α-tocopherol (TOH) in parr and that of Se in parr and post-smolt showed a linear relationship with dietary concentration, while α-TOH in post-smolt seemed to be saturable with a breakpoint near 140 mg kg-1. Ascorbic acid (Asc) concentration in the basal feed was below the expected minimum requirement, but the experimental period was probably too short for the fish to develop visible deficiency symptoms. Asc was saturable in both parr and post-smolt whole body at dietary concentrations of 190 and 63-89 mg kg-1, respectively. Maximum whole body Asc concentration was approximately 40 mg kg-1 in parr and 14 mg kg-1 in post-smolt. Retention ranged from 41 to 10% in parr and from -206 to 12% in post-smolt with increasing NP supplementation. This indicates that the post-smolts had an extraordinarily high consumption of Asc. Analyses of glutathione (GSH) and glutathione disulphide (GSSG) concentrations and the calculated GSH based redox potentials in liver and muscle tissue, indicated only minor effects of diets on redox regulation. However, the post-smolt were more oxidized than the parr. This was supported by the high consumption of Asc and high expression of gpx1 and gpx3 in liver. Based on the present trials, the recommendations for supplementation of vitamin C and E in diets for Atlantic salmon are similar to current practices, e.g. 150 mg kg-1 of α-TOH and 190 mg kg-1 Asc which was the saturating concentration in parr. Higher concentrations than what would prevent clinical deficiency symptoms are necessary to protect fish against incidents of oxidative stress and to improve immune and stress responses. There were no indications that the Se requirement exceeded the current recommendation of 0.3 mg kg-1.

Fish pre-acclimation temperature only modestly affects cadmium toxicity in Atlantic salmon hepatocytes.

An emerging focus in environmental toxicology is how climate change will alter bioavailability and uptake of contaminants in organisms. Ectothermic animals unable to adjust their temperature by local migration, such as farmed fish kept in net pens, may become more vulnerable to contaminants in warmer seas. The aim of this work was to study cadmium (Cd) toxicity in cells obtained from fish acclimated to sub-optimal growth temperature. Atlantic salmon hepatocytes, harvested from fish pre-acclimated either at 15°C (optimal growth temperature) or 20°C (heat-stressed), were exposed in vitro to two concentrations of Cd (control, 1 and 100µM Cd) for 48h. Cd-induced cytotoxicity, determined with the xCELLigence system, was more pronounced in cells from fish pre-acclimated to a high temperature than in cells from fish grown at optimal temperature. A feed spiked with antioxidants could not ameliorate the Cd-induced cytotoxicity in cells from temperature-stressed fish. At the transcriptional level, Cd exposure affected 11 out of 20 examined genes, of which most are linked to oxidative stress. The transcriptional levels of a majority of the altered genes were changed in cells harvested from fish grown at sub-optimal temperature. Interaction effects between Cd exposure and fish pre-acclimation temperature were seen for four transcripts, hmox1, mapk1, fth1 and mmp13. Overall, this study shows that cells from temperature-stressed fish are modestly more vulnerable to Cd stress, and indicate that mechanisms linked to oxidative stress may be differentially affected in temperature-stressed cells.

Functional amino acids in fish nutrition, health and welfare.

Protein is the most expensive part of fish diets and supplies amino acids (AA) for energy, growth, protein synthesis and as substrates for key metabolic pathways. Functional AA is a term used to describe AA that are involved in cellular processes apart from protein synthesis. A deficiency, or imbalance, in functional AA may impair body metabolism and homeostasis. Recent years have seen an increased interest in AA to increase disease resistance, immune response, reproduction, behavior and more. This has led to a boost of commercially available functional fish feeds that aim to optimize fish performance and quality of the product. This review aim to collect recent findings of functional AA and of how they may improve fish health and welfare. It will focus on functional properties of some of the most studied AA, namely arginine, glutamine, glutamate, tryptophan, sulfur amino acids (methionine, cysteine and taurine), histidine and branched chain amino acids. Where information is not available in fish, we will point towards functions known in animals and humans, with possible translational functions to fish.

Effect of temperature and diet on wound healing in Atlantic salmon (Salmo salar L.).

Compromised skin integrity of farmed Atlantic salmon, commonly occurring under low temperature and stressful conditions, has major impacts on animal welfare and economic productivity. Even fish with minimal scale loss and minor wounds can suffer from secondary infections, causing downgrading and mortalities. Wound healing is a complex process, where water temperature and nutrition play key roles. In this study, Atlantic salmon (260 g) were held at different water temperatures (4 or 12 °C) and fed three different diets for 10 weeks, before artificial wounds were inflicted and the wound healing process monitored for 2 weeks. The fish were fed either a control diet, a diet supplemented with zinc (Zn) or a diet containing a combination of functional ingredients in addition to Zn. The effect of diet was assessed through subjective and quantitative skin histology and the transcription of skin-associated chemokines. Histology confirmed that wound healing was faster at 12 °C. The epidermis was more organised, and image analyses of digitised skin slides showed that fish fed diets with added Zn had a significantly larger area of the epidermis covered by mucous cells in the deeper layers after 2 weeks, representing more advanced healing progression. Constitutive levels of the newly described chemokines, herein named CK 11A, B and C, confirmed their preferential expression in skin compared to other tissues. Contrasting modulation profiles at 4 and 12 °C were seen for all three chemokines during the wound healing time course, while the Zn-supplemented diets significantly increased the expression of CK 11A and B during the first 24 h of the healing phase.

Transcriptional effects of dietary exposure of oil-contaminated Calanus finmarchicus in Atlantic herring (Clupea harengus).

Suppression subtractive hybridization (SSH) cDNA library construction and characterization was used to identify differentially regulated transcripts from oil exposure in liver of male Atlantic herring (Clupea harengus) fed a diet containing 900 mg crude oil/kg for 2 mo. In total, 439 expressed sequence tags (EST) were sequenced, 223 from the forward subtracted library (enriched for genes putatively upregulated by oil exposure) and 216 from the reverse subtracted library (enriched for genes putatively downregulated by oil exposure). Follow-up reverse-transcription (RT) quantitative polymerase chain reaction (qPCR) analyses of gene transcription were conducted on additional herring exposed to food containing 9 (low), 90 (medium), and 900 (high) mg crude oil/kg feed for 2 mo. Chronic exposure of Atlantic herring to an oil-contaminated diet mediated upregulation of transcripts encoding antifreeze proteins, proteins in the classical complement pathway (innate immunity), and iron-metabolism proteins. Gene ontology (GO) analysis showed that "cellular response to stress," "regulation to biological quality," "response to abiotic stimuli," and "temperature homeostasis" were the most affected go at the biological processes level, and "carbohydrate binding," "water binding," and "ion binding" at the molecular function level. Of the genes examined with RT-qPCR, CYP1A, antifreeze protein, retinol binding protein 1, deleted in malignant brain tumor 1, and ovary-specific C1q-like factor demonstrated a significant upregulation. Myeloid protein 1, microfibrillar-associated protein 4, WAP65, and pentraxin were downregulated in liver of fish from the high exposure group. In conclusion, this study suggests that 2 mo of oil exposure affected genes encoding proteins involved in temperature homeostasis and possible membrane stability in addition to immune-responsive proteins in Atlantic herring.

Effects of dietary lipid, vitamins and minerals on total amounts and redox status of glutathione and ubiquinone in tissues of Atlantic salmon (Salmo salar): a multivariate approach.

The hypothesis of the present study was that Atlantic salmon (Salmo salar) would respond to large variations in supplementation of dietary pro- and antioxidants, and marine lipid, with adjustment of the endogenously synthesised antioxidants, glutathione (GSH) and ubiquinone (UQ). An experiment with 2(7-3) reduced factorial design (the number of cases reduced systematically from 2(7) (full design) to 2(4) (reduced design)) was conducted, where vitamins, minerals and lipid were supplemented in the diet at high and low levels. For the vitamins and minerals the high levels were chosen to be just below anticipated toxic levels and the low levels were just above the requirement (vitamin C, 30 and 1000 mg/kg; vitamin E, 70 and 430 mg/kg; Fe, 70 and 1200 mg/kg; Cu, 8 and 110 mg/kg; Mn, 12 and 200 mg/kg). For astaxanthin, the dietary levels were 10 and 50 mg/kg and for lipid, 150 and 330 g/kg. The experiment was started with post-smolts (148 (sd 17 g)) and lasted for 5 months. The only effect on GSH was a minor increase ( < 10 %) in total concentration in the liver in response to high dietary lipid. GSH redox state was not affected. UQ responded to dietary lipid, astaxanthin and vitamin E, both with regard to total concentration and redox state. Except for an effect of Fe on plasma GSH, the trace elements and vitamin C had no effect on tissue levels and oxidation state of GSH and UQ. This shows that the endogenous redox state is quite robust with regard to variation of dietary pro- and antioxidants in Atlantic salmon.

Effect of plant-based feed ingredients on osmoregulation in the Atlantic salmon lens.

Lenses of adult Atlantic salmon fed with a plant oil and plant protein-based diet (plant diet) were compared to lenses of fish fed a diet based on traditional marine ingredients (marine diet) with respect to biochemical composition and functionality ex vivo. After 12 months of feeding, plant diet-fed fish had smaller lenses with higher water contents and lower concentrations of histidine (His) and N-acetylhistidine (NAH) than fish fed with the marine diet. Cataract development in both dietary groups was minimal and no differences between the groups were observed. Lens fatty acid and lipid class composition differed minimally, although a significant increase in linoleic acid was observed. The lenses were examined for their ability to withstand osmotic disturbances ex vivo. Culture in hypoosmotic and hyperosmotic media led to increase and decrease of lens volume, respectively. Lenses from plant diet-fed fish were less resistant to swelling and shrinking, released less NAH into the culture medium, and accumulated His and NAH at higher rates than lenses from marine diet-fed fish. Culture in hypoosmotic medium resulted in higher cataract scores than in control and hyperosmotic medium. mRNA expression of selected genes, including glutathione peroxidase 4 and SPARC (secreted protein acidic and rich in cysteine), was affected by diet and osmotic treatment. It can be concluded that lenses of farmed Atlantic salmon are affected by the diet composition, both in biochemical composition and physiological functionality in relation to osmoregulation.

The impact of different water gas levels on cataract formation, muscle and lens free amino acids, and lens antioxidant enzymes and heat shock protein mRNA abundance in smolting Atlantic salmon, Salmo salar L.

The present experiment was conducted to examine if freshwater (FW) oxygen and carbon dioxide regimes cause physiological responses that lead to cataract formation in Atlantic salmon (Salmo salar L.) smolt. Duplicate groups of 50 g Atlantic salmon smolts were exposed to three freshwater oxygen saturation regimes (95, 112 or 125% saturation), with or without addition of carbon dioxide (measured 17-18 and 2-3 mg L(-1), respectively), for six weeks before transfer to seawater (SW). The FW exposure groups were followed up for another six weeks under a common SW regime. Fish were screened for cataract and sampled accordingly, at start, after 6 weeks in FW and after 6 weeks in SW. Increased growth related cataract incidences and severities were recorded in SW, mainly in the groups previously exposed to normoxic and hyperoxic conditions in FW, as compared to the respective groups added carbon dioxide. The concentration of histidine compounds (imidazoles) in muscle and lens tissue, used as quantitative risk markers of cataract, were lower than observed in earlier studies, however, neither were affected by the present water gas regimes in FW nor after follow up in SW. Independently of water oxygenation in FW, muscle free amino acid profiles in salmon groups concomitantly exposed to elevated carbon dioxide indicated use of selected free amino acids for energy purposes. Significantly lower abundance of heat shock protein 70 mRNA and trends towards stepwise reduction of antioxidant enzymes mRNA in the lens from fish exposed to increased water oxygenation were recorded, probably linked to increased growth and/or external stress during smoltification. This represents a first communication on using early molecular markers to express reduced protection of the fish lens against external stress to explain cataract development.