Effects of eicosapentaneoic acid on innate immune responses in an Atlantic salmon kidney cell line in vitro.

Studies of the interplay between metabolism and immunity, known as immunometabolism, is steadily transforming immunological research into new understandings of how environmental cues like diet are affecting innate and adaptive immune responses. The aim of this study was to explore antiviral transcriptomic responses under various levels of polyunsaturated fatty acid. Atlantic salmon kidney cells (ASK cell line) were incubated for one week in different levels of the unsaturated n-3 eicosapentaneoic acid (EPA) resulting in cellular levels ranging from 2-20% of total fatty acid. These cells were then stimulated with the viral mimic and interferon inducer poly I:C (30 ug/ml) for 24 hours before total RNA was isolated and sequenced for transcriptomic analyses. Up to 200 uM EPA had no detrimental effects on cell viability and induced very few transcriptional changes in these cells. However, in combination with poly I:C, our results shows that the level of EPA in the cellular membranes exert profound dose dependent effects of the transcriptional profiles induced by this treatment. Metabolic pathways like autophagy, apelin and VEGF signaling were attenuated by EPA whereas transcripts related to fatty acid metabolism, ferroptosis and the PPAR signaling pathways were upregulated. These results suggests that innate antiviral responses are heavily influenced by the fatty acid profile of salmonid cells and constitute another example of the strong linkage between general metabolic pathways and inflammatory responses.

Toxicological evaluation of a fish oil concentrate containing Very Long Chain Fatty Acids.

Very long chain fatty acids (VLCFA) have a chain length ≥24 carbons. Fish contain low levels of these fatty acids. A commercial oil called EPAX® Evolve 05 with an up-concentration of VLCFAs of approximately 10 times, has been developed as a dietary supplement by Epax Norway AS. A series of toxicological studies were performed using mice and rats to determine the safety and toxicity of repeat dosing with a gavage administered VLCFA formulation. The results suggest transient lipid accumulation in kidneys and liver. Lipid accumulation was seen with the test item and with the soya control but was not dose related. Liver and kidney lipid accumulation, whilst present in 14- day repeat dose study, was absent in a 90-day rat study. No treatment-effect was seen in urine analysis in any of the studies. No treatment-related effects were seen with a functional observation battery, ophthalmological examination, haematology, urine analysis, oestrus cycle, thyroid hormones, organ weight, or histopathology. In the 90-day study the liver enzymes ALP, AST and ALT were statistically significantly increased with test item but within control values. There were no associated histological findings in the liver suggesting there was no toxic effect and the normalisation of values for all liver enzymes in the recovery groups suggests an adaptive response rather than a prevailing toxic response. The no-observed-adverse-effect level (NOAEL) was determined as 1200 mg VLCFA/kg b.w./day.

Different Dietary Ratios of Camelina Oil to Sandeel Oil Influence the Capacity to Synthesise and Deposit EPA and DHA in Zucker Fa/Fa Rats.

Plant-based food provides more ALA (α-linolenic acid) and less EPA (eicosapentaenoic acid) and DHA (docosahexanoic acid) than marine food. Earlier studies indicate that cetoleic acid (22:1n-11) stimulates the n-3 pathway from ALA to EPA and DHA. The present study aimed to investigate the dietary effects of camelina oil (CA) high in ALA and sandeel oil (SA) high in cetoleic acid on the conversion of ALA to EPA and DHA. Male Zucker fa/fa rats were fed a diet of soybean oil (Ctrl) or diets of CA, SA, or a combination of CA and SA. Significantly higher levels of DPA (docosapentaenoic acid) and DHA in blood cells from the CA group compared to the Ctrl indicate an active conversion of ALA to DPA and DHA. Increasing the uptake and deposition of EPA and DHA meant that a trend towards a decrease in the liver gene expression of Elovl5, Fads1, and Fads2 along with an increase in the dietary content of SA was observed. However, 25% of the SA could be exchanged with CA without having a significant effect on EPA, DPA, or DHA in blood cells, indicating that bioactive components in SA, such as cetoleic acid, might counteract the inhibiting effect of the high dietary content of DHA on the n-3 biosynthetic pathway.

Investigation of the functions of n-3 very-long-chain PUFAs in skin using in vivo Atlantic salmon and in vitro human and fish skin models.

The purpose of this study was to investigate the effect of dietary n-3 very-long-chain PUFA (n-3 VLC-PUFA) on the maturation and development of skin tissue in juvenile Atlantic salmon (Salmo salar) in vivo, as well as their effects on skin keratocyte and human skin fibroblast cell migration in vitro. Atlantic salmon were fed different dietary levels of n-3 VLC-PUFA from an initial weight of 6 g to a final weight of 11 g. Changes in skin morphology were analysed at two time points during the experiment, and the effects on skin tissue fatty acid composition were determined. Additionally, in vitro experiments using human dermal fibroblasts and primary Atlantic salmon keratocytes were conducted to investigate the effect of VLC-PUFA on the migration capacity of the cells. The results demonstrated that increased dietary levels of n-3 VLC-PUFA led to an increased epidermis thickness and more rapid scale maturation in Atlantic salmon skin in vivo, leading to a more mature skin morphology, and possibly more robust skin, at an earlier life stage. Additionally, human skin fibroblasts and salmon skin keratocytes supplemented with n-3 VLC-PUFA in vitro showed more rapid migration, indicating potentially beneficial effects of VLC-PUFA in wound healing. In conclusion, VLC-PUFA may have beneficial effects on skin tissue development, function and integrity.

Increasing dietary levels of the n-3 long-chain PUFA, EPA and DHA, improves the growth, welfare, robustness and fillet quality of Atlantic salmon in sea cages.

The present study evaluated the effects of increasing the dietary levels of EPA and DHA in Atlantic salmon (Salmo salar) reared in sea cages, in terms of growth performance, welfare, robustness and overall quality. Fish with an average starting weight of 275 g were fed one of four different diets containing 10, 13, 16 and 35 g/kg of EPA and DHA (designated as 1·0, 1·3, 1·6 and 3·5 % EPA and DHA) until they reached approximately 5 kg. The 3·5 % EPA and DHA diet showed a significantly beneficial effect on growth performance and fillet quality compared with all other diets, particularly the 1 % EPA and DHA diet. Fish fed the diet containing 3·5 % EPA and DHA showed 400-600 g higher final weights, improved internal organ health scores and external welfare indicators, better fillet quality in terms of higher visual colour score and lower occurrence of dark spots and higher EPA and DHA content in tissues at the end of the feeding trial. Moreover, fish fed the 3·5 % EPA and DHA diet showed lower mortality during a naturally occurring cardiomyopathy syndrome outbreak, although this did not reach statistical significance. Altogether, our findings emphasise the importance of dietary EPA and DHA to maintain good growth, robustness, welfare and fillet quality of Atlantic salmon reared in sea cages.

Low Omega-3 Levels in the Diet Disturbs Intestinal Barrier and Transporting Functions of Atlantic Salmon Freshwater and Seawater Smolts.

Due to a limited access to marine raw materials from capture fisheries, Atlantic salmon feeds are currently based on mainly plant ingredients (75%) while only 25% come from traditional marine ingredients including marine fish meal and fish oil. Thus, current feeds contain less of the essential omega-3 fatty acids. The aim of the study was to assess the impact of different omega-3 levels in fish feed on intestinal barrier and transporting functions of Atlantic salmon freshwater and seawater smolts. Atlantic salmon were fed three levels of omega-3 (2, 1 and 0.5%) and fish performance was followed through smoltification and the subsequent seawater acclimation. Intestinal barrier and transporting functions were assessed using Ussing chamber methodology and combined with transcript analysis of tight junction related proteins and ion transporters. A linear decrease in growth was observed with decreasing omega-3 levels. Low (0.5%) inclusion of omega-3 impaired the barrier function of the proximal intestine compared to 2% inclusion. Further, low levels of omega-3 decrease the transepithelial electrical potential across the epithelium indicating disturbed ion transport. It can be concluded that low dietary levels of omega-3 impair somatic growth and intestinal function of Atlantic salmon.

Long-term feeding of Atlantic salmon with varying levels of dietary EPA + DHA alters the mineral status but does not affect the stress responses after mechanical delousing stress.

Atlantic salmon were fed diets containing graded levels of EPA + DHA (1·0, 1·3, 1·6 and 3·5 % in the diet) and one diet with 1·3 % of EPA + DHA with reduced total fat content. Fish were reared in sea cages from about 275 g until harvest size (about 5 kg) and were subjected to delousing procedure (about 2·5 kg), with sampling pre-, 1 h and 24 h post-stress. Delousing stress affected plasma cortisol and hepatic mRNA expression of genes involved in oxidative stress and immune response, but with no dietary effects. Increasing EPA + DHA levels in the diet increased the trace mineral levels in plasma and liver during mechanical delousing stress period and whole body at harvest size. The liver Se, Zn, Fe, Cu, and Mn and plasma Se levels were increased in fish fed a diet high in EPA + DHA (3·5 %) upon delousing stress. Furthermore, increased dietary EPA + DHA caused a significant increase in mRNA expression of hepcidin antimicrobial peptide (HAMP), which is concurrent with downregulated transferrin receptor (TFR) expression levels. High dietary EPA + DHA also significantly increased the whole-body Zn, Se, and Mn levels at harvest size fish. Additionally, the plasma and whole-body Zn status increased, respectively, during stress and at harvest size in fish fed reduced-fat diet with less EPA + DHA. As the dietary upper limits of Zn and Se are legally added to the feeds and play important roles in maintaining fish health, knowledge on how the dietary fatty acid composition and lipid level affect body stores of these minerals is crucial for the aquaculture industry.

Deposition and metabolism of dietary n-3 very-long-chain PUFA in different organs of rat, mouse and Atlantic salmon.

There is limited knowledge about the metabolism and function of n-3 very-long-chain PUFA (n-3 VLC-PUFA) with chain lengths ≥ 24. They are known to be produced endogenously in certain tissues from EPA and DHA and not considered to originate directly from dietary sources. The aim of this study was to investigate whether n-3 VLC-PUFA from dietary sources are bio-available and deposited in tissues of rat, fish and mouse. Rats were fed diets supplemented with a natural fish oil (FO) as a source of low dietary levels of n-3 VLC-PUFA, while Atlantic salmon and mice were fed higher dietary levels of n-3 VLC-PUFA from a FO concentrate. In all experiments, n-3 VLC-PUFA incorporation in organs was investigated. We found that natural FO, due to its high EPA content, to a limited extent increased endogenous production of n-3 VLC-PUFA in brain and eye of mice with neglectable amounts of n-3 VLC-PUFA originating from diet. When higher dietary levels were given in the form of concentrate, these fatty acids were bio-available and deposited in both phospholipids and TAG fractions of all tissues studied, including skin, eye, brain, testis, liver and heart, and their distribution appeared to be tissue-dependent, but not species-specific. When dietary EPA and DHA were balanced and n-3 VLC-PUFA increased, the major n-3 VLC-PUFA from the concentrate increased significantly in the organs studied, showing that these fatty acids can be provided through diet and thereby provide a tool for functional studies of these VLC-PUFA.

Genomic and Phenotypic Agreement Defines the Use of Microwave Dielectric Spectroscopy for Recording Muscle Lipid Content in European Seabass (Dicentrarchus labrax).

Recording the fillet lipid percentage in European seabass is crucial to control lipid deposition as a means toward improving production efficiency and product quality. The reference method for recording lipid content is solvent lipid extraction and is the most accurate and precise method available. However, it is costly, requires sacrificing the fish and grinding the fillet sample which limits the scope of applications, for example grading of fillets, recording live fish or selective breeding of fish with own phenotypes are all limited. We tested a rapid, cost effective and non-destructive handheld microwave dielectric spectrometer (namely the Distell fat meter) against the reference method by recording both methods on 313 European seabass (Dicentrarchus labrax). The total method agreement between the dielectric spectrometer and the reference method was assessed by Lin's concordance correlation coefficient (CCC), which was low to moderate CCC = 0.36-0.63. We detected a significant underestimation in accuracy of lipid percentage 22-26% by the dielectric spectrometer and increased imprecision resulting in the coefficient of variation (CV) doubling for dielectric spectrometer CV = 40.7-46% as compared to the reference method 27-31%. Substantial genetic variation for fillet lipid percentage was found for both the reference method (h 2 = 0.59) and dielectric spectroscopy (h 2 = 0.38-0.58), demonstrating that selective breeding is a promising method for controlling fillet lipid content. Importantly, the genetic correlation (r g) between the dielectric spectrometer and the reference method was positive and close to unity (r g = 0.96), demonstrating the dielectric spectrometer captures practically all the genetic variation in the reference method. These findings form the basis of defining the scope of applications and experimental design for using dielectric spectroscopy for recording fillet lipid content in European seabass and validate its use for selective breeding.

Transcriptome and functional responses to absence of astaxanthin in Atlantic salmon fed low marine diets.

High content of carotenoids in tissues of salmonid species suggests possible functional importance, which has so far remained unclear. The objective of this study was to investigate the effect of astaxanthin on performance and gene expression of sea water adapted Atlantic salmon (Salmo salar) fed diets with low content of marine ingredients (7.5% fishmeal and 5% fish oil). Salmon with start weight 197 g were fed two diets with identical proximate composition except for the content of astaxanthin (<1 and 48 mg/kg, respectively) for 84 days. Absence of dietary astaxanthin caused significant transcriptome changes revealed with DNA microarray. The growth rate was not optimal for the two diet groups but was not affected by dietary astaxanthin concentration. Accumulation of lipid in the intestine and liver was found in salmon fed both diets, indicating malabsorption of lipid. Salmon fed the diet without astaxanthin had larger livers and higher fat content in liver due to accumulation of triglycerides, but the difference in fat content was not significant. Transcriptome responses in different organs suggested that lack of dietary astaxanthin may have functional consequences in salmon fed low marine diets. In the intestine of astaxanthin deprived salmon, decreased expression was observed in a suite of immune genes including genes of innate antiviral immunity, transporters and enzymes of glycan metabolism. Transcriptome responses in liver suggested effect of absence of astaxanthin on lipid metabolism and especially on increased biosynthesis of terpenoids and steroids and only minor effects on immune genes. The greatest transcriptome changes were observed in skeletal muscle in the absence of astaxanthin, with an up-regulation of immune-related genes (119) and multiple genes with well-established association with stress. The condition resembled a mild inflammation of the muscle. Small or moderate scale of gene expression changes were in concordance with equal growth performance of fish fed both diets, however their character may indicate potential risk of absence of dietary carotenoids.

The heritable landscape of near-infrared and Raman spectroscopic measurements to improve lipid content in Atlantic salmon fillets.

BACKGROUND: Product quality and production efficiency of Atlantic salmon are, to a large extent, influenced by the deposition and depletion of lipid reserves. Fillet lipid content is a heritable trait and is unfavourably correlated with growth, thus genetic management of fillet lipid content is needed for sustained genetic progress in these two traits. The laboratory-based reference method for recording fillet lipid content is highly accurate and precise but, at the same time, expensive, time-consuming, and destructive. Here, we test the use of rapid and cheaper vibrational spectroscopy methods, namely near-infrared (NIR) and Raman spectroscopy both as individual phenotypes and phenotypic predictors of lipid content in Atlantic salmon. RESULTS: Remarkably, 827 of the 1500 individual Raman variables (i.e. Raman shifts) of the Raman spectrum were significantly heritable (heritability (h2) ranging from 0.15 to 0.65). Similarly, 407 of the 2696 NIR spectral landscape variables (i.e. wavelengths) were significantly heritable (h2 = 0.27-0.40). Both Raman and NIR spectral landscapes had significantly heritable regions, which are also informative in spectroscopic predictions of lipid content. Partial least square predicted lipid content using Raman and NIR spectra were highly concordant and highly genetically correlated with the lipid content values ([Formula: see text] = 0.91-0.98) obtained with the reference method using Lin's concordance correlation coefficient (CCC = 0.63-0.90), and were significantly heritable ([Formula: see text] = 0.52-0.67). CONCLUSIONS: Both NIR and Raman spectral landscapes show substantial additive genetic variation and are highly genetically correlated with the reference method. These findings lay down the foundation for rapid spectroscopic measurement of lipid content in salmonid breeding programmes.

Modulation of hepatic miRNA expression in Atlantic salmon (Salmo salar) by family background and dietary fatty acid composition.

This study finds significant differences in hepatic fatty acid composition between four groups of Atlantic salmon (Salmo salar) consisting of offspring from families selected for high and low capacities to express the delta 6 desaturase isomer b and fed diets with 10% or 75% fish oil. The results demonstrated that hepatic lipid metabolism was affected by experimental conditions (diet/family). The fatty acid composition in the four groups mirrored the differences in dietary composition, but it was also associated with the family groups. Small RNA sequencing followed by RT-qPCR identified 12 differentially expressed microRNAs (DE miRNAs), with expression associated with family groups (miR-146 family members, miR-200b, miR-214, miR-221, miR-125, miR-135, miR-137, miR_nov_1), diets (miR-203, miR-462) or both conditions. All the conserved DE miRNAs have been reported as associated with lipid metabolism in other vertebrates. In silico predictions revealed 37 lipid metabolism pathway genes, including desaturases, transcription factors and key enzymes in the synthesis pathways as putative targets (e.g., srebp-1 and 2, Δ6fad_b and c, hmdh, elovl4 and 5b, cdc42). RT-qPCR analysis of selected target genes showed expression changes that were associated with diet and with family groups (d5fad, d6fad_a, srebp-1). There was a reciprocal difference in the abundance of ssa-miR-203a-3p and srebp-1 in one group comparison, whereas other predicted targets did not reveal any evidence of being negatively regulated by degradation. More experimental studies are needed to validate and fully understand the predicted interactions and how the DE miRNAs may participate in the regulation of hepatic lipid metabolism.

Sensitivity to Dietary Wheat Gluten in Atlantic Salmon Indicated by Gene Expression Changes in Liver and Intestine.

Feed safety is a necessity for animal health and welfare as well as prerequisite for food safety and human health. Wheat gluten (WG) is considered as a valuable protein source in fish feed due to its suitability as a feed binder, high digestibility, good amino acid profile, energy density and most importantly, due to its relatively low level of anti-nutritional factors (ANFs). The main aim of this study was to identify the impact of dietary WG on salmon health by analysing growth, feed efficiency and the hepatic and intestinal transcriptomes. The fish were fed either control diet with fishmeal (FM) as the only source of protein or diets, where 15% or 30% of the FM were replaced by WG. The fish had a mean initial weight of 223 g and approximately doubled their weight during the 9-week experiment. Salmon fed on 30% WG showed reduced feed intake compared to the 15% and FM fed groups. The liver was the less affected organ but fat content and activities of the liver health markers in plasma increased with the inclusion level of WG in the diet. Gene expression analysis showed significant changes in both, intestine and liver of fish fed with 30% WG. Especially noticeable were changes in the lipid metabolism, in particular in relation to the intestinal lipoprotein transport and sterol metabolism. Moreover, the intestinal transcriptome of WG-fed fish showed shifts in the expression of a large number of genes responsible for immunity and tissue structure and integrity. These observations implied that the fish receiving WG-containing diet were undergoing nutritional stress. Overall, the study provided evidence that a high dietary level of WG can have a negative impact on the intestinal and liver health of salmon with symptoms similar to gluten sensitivity in humans.

DHA Modulates Immune Response and Mitochondrial Function of Atlantic Salmon Adipocytes after LPS Treatment.

Adipocytes play a central role in overall energy homeostasis and are important contributors to the immune system. Fatty acids (FAs) act as signaling molecules capable to modulate adipocyte metabolism and functions. To identify the effects of two commonly used FAs in Atlantic salmon diets, primary adipocytes were cultured in the presence of oleic (OA) or docosahexaenoic (DHA) acid. DHA decreased adipocyte lipid droplet number and area compared to OA. The increase in lipid load in OA treated adipocytes was paralleled by an increase in iNOS activity and mitochondrial SOD2-GFP activity, which was probably directed to counteract increase in oxidative stress. Under lipopolysaccharide (LPS)-induced inflammation, DHA had a greater anti-inflammatory effect than OA, as evidenced by the higher SOD2 activity and the transcriptional regulation of antioxidant enzymes and pro- and anti-inflammatory markers. In addition, DHA maintained a healthy mitochondrial structure under induced inflammation while OA led to elongated mitochondria with a thin thread like structures in adipocytes exposed to LPS. Overall, DHA possess anti-inflammatory properties and protects Atlantic salmon against oxidative stress and limits lipid deposition. Furthermore, DHA plays a key role in protecting mitochondria shape and function.

Lipid Deposition and Mobilisation in Atlantic Salmon Adipocytes.

The present study aimed to elucidate how Atlantic salmon adipocytes pre-enriched with palmitic (16:0, PA), oleic (18:1n-9, OA), or eicosapentaenoic (20:5n-3, EPA) acid respond to a fasting condition mimicked by nutrient deprivation and glucagon. All experimental groups were supplemented with radiolabeled PA to trace secreted lipids and distribution of radioactivity in different lipid classes. There was a higher content of intracellular lipid droplets in adipocytes pre-enriched with OA than in adipocytes pre-enriched with PA or EPA. In the EPA group, the radiolabeled PA was mainly esterified in phospholipids and triacylglycerols, whereas in the OA and PA groups, the radioactivity was mainly recovered in phospholipids and cholesterol-ester. By subjecting the experimental groups to nutrient-deprived media supplemented with glucagon, lipolysis occurred in all groups, although to a lower extent in the OA group. The lipids were mainly secreted as esterified lipids in triacylglycerols and phospholipids, indicating mobilization in lipoproteins. A significant proportion was secreted as free fatty acids and glycerol. Leptin secretion was reduced in all experimental groups in response to fasting, while the mitochondria area responded to changes in the energy supply and demand by increasing after 3 h of fasting. Overall, different lipid classes in adipocytes influenced their mobilization during fasting.

Dietary inclusion of Antarctic krill meal during the finishing feed period improves health and fillet quality of Atlantic salmon (Salmo salar L.).

There is an urgent need to find alternative feed resources that can further substitute fishmeal in Atlantic salmon diets without compromising health and food quality, in particular during the finishing feeding period when the feed demand is highest and flesh quality effects are most significant. This study investigates efficacy of substituting a isoprotein (35 %) and isolipid (35 %) low fishmeal diet (FM, 15 %) with Antarctic krill meal (KM, 12 %) during 3 months with growing finishing 2·3 kg salmon (quadruplicate sea cages/diet). Final body weight (3·9 (se 0·04) kg) was similar in the dietary groups, but the KM group had more voluminous body shape, leaner hearts and improved fillet integrity, firmness and colour. Ectopic epithelial cells and focal Ca deposits in intestine were only detected in the FM group. Transcriptome profiling by microarray of livers showed dietary effects on several immune genes, and a panel of structural genes were up-regulated in the KM group, including cadherin and connexin. Up-regulation of genes encoding myosin heavy chain proteins was the main finding in skeletal muscle. Morphology examination by scanning electron microscopy and secondary structure by Fourier transform IR spectroscopy revealed more ordered and stable collagen architecture of the KM group. NEFA composition of skeletal muscle indicated altered metabolism of n-3, n-6 and SFA of the KM group. The results demonstrated that improved health and meat quality in Atlantic salmon fed krill meal were associated with up-regulation of immune genes, proteins defining muscle properties and genes involved in cell contacts and adhesion, altered fatty acid metabolism and fat deposition, and improved gut health and collagen structure.

Dietary Lipid:Protein Ratio and n-3 Long-Chain Polyunsaturated Fatty Acids Alters the Gut Microbiome of Atlantic Salmon Under Hypoxic and Normoxic Conditions.

Researchers have adjusted dietary lipid:protein ratios and n-3 long-chain polyunsaturated fatty acids (LC-PUFA) to optimize the growth performance of Atlantic salmon. However, dietary impacts on the gut microbiome are lacking, especially under varying environmental conditions. To examine this response, post-smolt salmon (184 ± 5 g) were fed diets with lipid:protein ratios considered low (180, 570 g/kg) and high (230, 460 g/kg) along with low and high levels of n-3 LC-PUFA (7 or 14 g/kg) while fish were reared under low and high levels of dissolved oxygen (6.7 or 8.0 mg/L). At day 0, 35 and 116, digesta in the distal intestine were collected and analyzed for viable counts and 16S ribosomal RNA (rRNA) genes (V4 region) using Illumina MiSeq. The reduction in oxygen had negligible effects, except on viable plate counts of total bacteria and an initial effect on beta-diversity. In contrast, the high lipid (HL) diets had an increased alpha-diversity (e.g., Shannon and Chao-1) at day 0 and day 35 whereas high n-3 diets suppressed these indices at day 116. Generally, a reduction in alpha-diversity was observed over time and an interaction between lipid:protein ratio x n-3 was found. Between diets, beta-diversity and phyla abundance were similar as both Proteobacteria (44%) and Firmicutes (21%) dominated. However, at the genus level Aliivibrio, Streptococcus, Weissella, and Lactobacillus, were associated with low lipid (LL) diets while the high lipid diets were associated with less abundant bacteria, e.g., Chromohalobacter. At day 116, the relative abundance of the Tenericutes phylum increased 10-fold (36%). Fish fed the high lipid diet with high n-3 had reduced alpha-diversity, lowest abundance of lactic acid bacteria, and highest abundance of Mycoplasma, which may indicate a less healthy gut microbiome. Phylogenetic Investigation of Communities by Reconstruction of Unobserved States (PICRUSt) analysis revealed that saturated and unsaturated fatty acid biosynthesis pathways were several folds higher in fish fed the high lipid diet, possibly to compensate for the lack of dietary n-3. In summary, our results show that the viable plate counts, alpha-diversity, beta-diversity, and predictive function of gut bacteria in Atlantic salmon post-smolts are influenced by dietary lipid:protein ratio and n-3 LC-PUFA over several time points with little effect by dissolved oxygen.

Dietary Fish Oil Alters DNA Methylation of Genes Involved in Polyunsaturated Fatty Acid Biosynthesis in Muscle and Liver of Atlantic Salmon (Salmo salar).

Adequate dietary supply of eicosapentaenoic acid (20:5n-3) and docosahexaenoic acid (22:6n-3) is required to maintain health and growth of Atlantic salmon (Salmo salar). However, salmon can also convert α-linolenic acid (18:3n-3) into eicosapentaenoic acid (20:5n-3) and docosahexaenoic acid (22:6n-3) by sequential desaturation and elongation reactions, which can be modified by 20:5n-3 and 22:6n-3 intake. In mammals, dietary 20:5n-3 + 22:6n-3 intake can modify Fads2 expression (Δ6 desaturase) via altered DNA methylation of its promoter. Decreasing dietary fish oil (FO) has been shown to increase Δ5fad expression in salmon liver. However, it is not known whether this is associated with changes in the DNA methylation of genes involved in polyunsaturated fatty acid synthesis. To address this, we investigated whether changing the proportions of dietary FO and vegetable oil altered the DNA methylation of Δ6fad_b, Δ5fad, Elovl2, and Elovl5_b promoters in liver and muscle from Atlantic salmon and whether any changes were associated with mRNA expression. Higher dietary FO content increased the proportions of 20:5n-3 and 22:6n-3 and decreased Δ6fad_b mRNA expression in liver, but there was no effect on Δ5fad, Elovl2, and Elovl5_b expression. There were significant differences between liver and skeletal muscle in the methylation of individual CpG loci in all four genes studied. Methylation of individual Δ6fad_b CpG loci was negatively related to its expression and to proportions of 20:5n-3 and 22:6n-3 in the liver. These findings suggest variations in dietary FO can induce gene-, CpG locus-, and tissue-related changes in DNA methylation in salmon.

n-3 Canola oil effectively replaces fish oil as a new safe dietary source of DHA in feed for juvenile Atlantic salmon.

Limited availability of fish oils (FO), rich in n-3 long-chain (≥C20) PUFA, is a major constraint for further growth of the aquaculture industry. Long-chain n-3 rich oils from crops GM with algal genes are promising new sources for the industry. This project studied the use of a newly developed n-3 canola oil (DHA-CA) in diets of Atlantic salmon fingerlings in freshwater. The DHA-CA oil has high proportions of the n-3 fatty acids (FA) 18 : 3n-3 and DHA and lower proportions of n-6 FA than conventional plant oils. Levels of phytosterols, vitamin E and minerals in the DHA-CA were within the natural variation of commercial canola oils. Pesticides, mycotoxins, polyaromatic hydrocarbons and heavy metals were below lowest qualifiable concentration. Two feeding trials were conducted to evaluate effects of two dietary levels of DHA-CA compared with two dietary levels of FO at two water temperatures. Fish increased their weight approximately 20-fold at 16°C and 12-fold at 12°C during the experimental periods, with equal growth in salmon fed the FO diets compared with DHA-CA diets. Salmon fed DHA-CA diets had approximately the same EPA+DHA content in whole body as salmon fed FO diets. Gene expression, lipid composition and oxidative stress-related enzyme activities showed only minor differences between the dietary groups, and the effects were mostly a result of dietary oil level, rather than the oil source. The results demonstrated that DHA-CA is a safe and effective replacement for FO in diets of Atlantic salmon during the sensitive fingerling life-stage.