Oil from transgenic Camelina sativa as a source of EPA and DHA in feed for European sea bass (Dicentrarchus labrax L.).

Aquaculture, the fastest growing food production sector cannot continue to rely on finite stocks of marine fish as the primary source of the omega-3 (n-3) long-chain polyunsaturated fatty acids (LC-PUFA), eicosapentaenoic acid (EPA; 20:5n3) and docosahexaenoic acid (DHA; 22:6n-3), for feeds. A four-month feeding trial was conducted to investigate the impact of a de novo oil, with high levels of EPA and DHA, obtained from transgenic Camelina sativa on growth performance, tissue fatty acid profiles, and expression of lipid metabolism genes when used as a replacement for fish oil in feed for European seabass (Dicentrachus labrax). Triplicate groups of 50 juvenile fish (initial weight 16.7 ± 0.92 g) per tank were fed for 4 months with one of three isolipidic and isoproteic experimental diets consisting of a standard diet containing a commercial blend of fish oil and rapeseed oil (CFO), a diet containing transgenic Camelina oil (TCO), or a blend of fish oil and rapeseed oil with enhanced levels of EPA and DHA (EFO) formulated to match the n-3 LC-PUFA profile of the TCO feed. Final weight of fish fed the GM-derived oil was not different to fish fed either CFO or EFO. Slight lower growth performance of fish fed TCO at the beginning of the trial was related to transient reduced feed intake, possibly caused by glucosinolates in the raw Camelina sativa oil. The GM-derived oil improved the nutritional quality of the fish fillet by enhancing total n-3 PUFA levels compared to the fish fed the other two feeds, and maintained flesh EPA and DHA at the same levels as in fish fed the diets containing fish oil. The metabolic response in liver and intestine was generally relatively mild although diets TCO and EFO seemed to trigger a metabolic response consisting of an up-regulation of both ß-oxidation (cpt1a) and fatty acid transport (fabp1), possibly reflecting higher levels of LC-PUFA. Overall, the present study indicated that an oil of terrestrial origin, Camelina sativa, when engineered to contain high levels of EPA and DHA can replace fish oil in feeds for European seabass with no detrimental impact on growth or feed efficiency, while also maintaining or increasing tissue n-3 LC-PUFA contents.

Effects of dietary arachidonic acid in European sea bass (Dicentrarchus labrax) distal intestine lipid classes and gut health.

The use of low fishmeal/fish oil in marine fish diets affects dietary essential fatty acids (EFAs) composition and concentration and, subsequently, may produce a marginal deficiency of those fatty acids with a direct impact on the fish intestinal physiology. Supplementation of essential fatty acids is necessary to cover the requirements of the different EFAs, including the ones belonging to the n-6 series, such as arachidonic acid (ARA). ARA, besides its structural role in the configuration of the lipid classes of the intestine, plays an important role in the functionality of the gut-associated immune tissue (GALT). The present study aimed to test five levels of dietary ARA (ARA0.5 (0.5%), ARA1 (1%), ARA2 (2%), ARA4 (4%), and ARA6 (6%)) for European sea bass (Dicentrarchus labrax) juveniles in order to determine (a) its effect in selected distal intestine (DI) lipid classes composition and (b) how these changes affected gut bacterial translocation rates and selected GALT-related gene expression pre and post challenge. No differences were found between distal intestines of fish fed with the graded ARA levels in total neutral lipids and total polar lipids. However, DI of fish fed with the ARA6 diet presented a higher (P < 0.05) level of phosphatidylethanolamine (PE) and sphingomyelin (SM) than those DI of fish fed with the ARA0.5 diet. In general terms, fatty acid profiles of DI lipid classes mirrored those of the diet dietary. Nevertheless, selective retention of ARA could be observed in glycerophospholipids when dietary levels are low (diet ARA0.5), as reflected in the higher glycerophospholipids-ARA/dietary-ARA ratio for those animals. Increased ARA dietary supplementation was inversely correlated with eicosapentaenoic acid (EPA) content in lipid classes, when data from fish fed with the diets with the same basal composition (diets ARA1 to ARA6). ARA supplementation did not affect intestinal morphometry, goblet cell number, or fish survival, in terms of gut bacterial translocation, along the challenge test. However, after the experimental infection with Vibrio anguillarum, the relative expression of cox-2 and il-1ß were upregulated (P < 0.05) in DI of fish fed with the diets ARA0.5 and ARA2 compared with fish fed with the rest of the experimental diets. Although dietary ARA did not affect fish survival, it altered the fatty acid composition of glycerophospholipids and the expression of pro-inflammatory genes after infection when included at the lowest concentration, which could be compromising the physical and the immune functionality of the DI, denoting the importance of ARA supplementation when low FO diets are used for marine fish.

Supplementation of arachidonic acid rich oil in European sea bass juveniles (Dicentrarchus labrax) diets: effects on growth performance, tissue fatty acid profile and lipid metabolism.

The aim of this study was to evaluate the effects of increasing dietary arachidonic acid (ARA) levels (from 1 to 6% of total fatty acids) on European sea bass (Dicentrarchus labrax) juveniles' growth performance, tissue fatty acid profile, liver morphology as well as long-chain polyunsaturated fatty acids (LC-PUFA) biosynthesis, triglyceride and cholesterol synthesis and lipid transport. A diet with total fish oil (FO) replacement and defatted fish meal (FM) containing a 0.1-g ARA g-1 diet was added to the experimental design as a negative control diet. Dietary ARA inclusion levels below 0.2 g ARA g-1 diet significantly worsened growth even only 30 days after the start of the feeding trial, whereas dietary ARA had no effect on fish survival. Liver, muscle and whole body fatty acid profile mainly reflected dietary contents and ARA content increased accordingly with ARA dietary levels. Tissue eicosapentaenoic acid (EPA), docosapentaenoic acid (DPA) and docosahexaenoic acid (DHA) levels were positively correlated among them. Hepatic lipid vacuolization increased with reduced dietary ARA levels. Expressions of fatty acyl desaturase 2 and 3-hydroxy-3-methylglutaryl-coenzyme genes were upregulated in fish fed the negative control diet compared to the rest of the dietary treatments denoting the influence of ARA on lipid metabolism. Results obtained highlight the need to include adequate n-6 levels and not only n-3 LC-PUFA levels in European sea bass diets.

Microbial and genetically engineered oils as replacements for fish oil in aquaculture feeds.

As the global population grows more of our fish and seafood are being farmed. Fish are the main dietary source of the omega-3 (n-3) long-chain polyunsaturated fatty acids (LC-PUFA), eicosapentaenoic (EPA) and docosahexaenoic (DHA) acids, but these cannot be produced in sufficient quantities as are now required for human health. Farmed fish have traditionally been fed a diet consisting of fishmeal and fish oil, rich in n-3 LC-PUFA. However, the increase in global aquaculture production has resulted in these finite and limited marine ingredients being replaced with sustainable alternatives of terrestrial origin that are devoid of n-3 LC-PUFA. Consequently, the nutritional value of the final product has been partially compromised with EPA and DHA levels both falling. Recent calls from the salmon industry for new sources of n-3 LC-PUFA have received significant commercial interest. Thus, this review explores the technologies being applied to produce de novo n-3 LC-PUFA sources, namely microalgae and genetically engineered oilseed crops, and how they may be used in aquafeeds to ensure that farmed fish remain a healthy component of the human diet.

Bone development of the skull, pectoral and pelvic fins in Seriola rivoliana (Valenciennes, 1833) larvae.

Skull, pectoral and pelvic fin bone structures in longfin yellowtail Seriola rivoliana were studied from 3.43 ± 0.15 to 16.20 ± 0.73 mm standard length (SL) specimens. The S. rivoliana skull started to mineralize with the appearance of the parasphenoid and maxillary by 3.43 ± 0.15 mm SL at the neurocranium and jaw regions, respectively. The first pectoral structure to mineralize was the cleithrum at 3.75 ± 0.14 mm SL shortly followed by the supracleithrum and posttemporal. The pelvic fin started by 6.16 ± 0.32 mm SL with the spine and continued with the soft rays and basipterygium. The present study determined the onset of the skull, pectoral and pelvic fin mineralization. These results might be used as a reference for future studies in S. rivoliana or related species.

Assessment of a land-locked Atlantic salmon (Salmo salar L.) population as a potential genetic resource with a focus on long-chain polyunsaturated fatty acid biosynthesis.

The natural food for Atlantic salmon (Salmo salar) in freshwater has relatively lower levels of omega-3 (n-3) long-chain polyunsaturated fatty acids (LC-PUFA) than found in prey for post-smolt salmon in seawater. Land-locked salmon such as the Gullspång population feed exclusively on freshwater type lipids during its entire life cycle, a successful adaptation derived from divergent evolution. Studying land-locked populations may provide insights into the molecular and genetic control mechanisms that determine and regulate n-3 LC-PUFA biosynthesis and retention in Atlantic salmon. A two factorial study was performed comparing land-locked and farmed salmon parr fed diets formulated with fish or rapeseed oil for 8 weeks. The land-locked parr had higher capacity to synthesise n-3 LC-PUFA as indicated by higher expression and activity of desaturase and elongase enzymes. The data suggested that the land-locked salmon had reduced sensitivity to dietary fatty acid composition and that dietary docosahexaenoic acid (DHA) did not appear to suppress expression of LC-PUFA biosynthetic genes or activity of the biosynthesis pathway, probably an evolutionary adaptation to a natural diet lower in DHA. Increased biosynthetic activity did not translate to enhanced n-3 LC-PUFA contents in the flesh and diet was the only factor affecting this parameter. Additionally, high lipogenic and glycolytic potentials were found in land-locked salmon, together with decreased lipolysis which in turn could indicate increased use of carbohydrates as an energy source and a sparing of lipid.

Evaluation of a high-EPA oil from transgenic Camelina sativa in feeds for Atlantic salmon (Salmo salar L.): Effects on tissue fatty acid composition, histology and gene expression.

Currently, one alternative for dietary fish oil (FO) in aquafeeds is vegetable oils (VO) that are devoid of omega-3 (n-3) long-chain polyunsaturated fatty acids (LC-PUFAs). Entirely new sources of n-3 LC-PUFA such as eicosapentaenoic (EPA) and docosahexaenoic (DHA) acids through de novo production are a potential solution to fill the gap between supply and demand of these important nutrients. Camelina sativa was metabolically engineered to produce a seed oil (ECO) with > 20% EPA and its potential to substitute for FO in Atlantic salmon feeds was tested. Fish were fed with one of the three experimental diets containing FO, wild-type camelina oil (WCO) or ECO as the sole lipid sources for 7 weeks. Inclusion of ECO did not affect any of the performance parameters studied and enhanced apparent digestibility of individual n-6 and n-3 PUFA compared to dietary WCO. High levels of EPA were maintained in brain, liver and intestine (pyloric caeca), and levels of DPA and DHA were increased in liver and intestine of fish fed ECO compared to fish fed WCO likely due to increased LC-PUFA biosynthesis based on up-regulation of the genes. Fish fed ECO showed slight lipid accumulation within hepatocytes similar to that with WCO, although not significantly different to fish fed FO. The regulation of a small number of genes could be attributed to the specific effect of ECO (311 features) with metabolism being the most affected category. The EPA oil from transgenic Camelina (ECO) could be used as a substitute for FO, however it is a hybrid oil containing both FO (EPA) and VO (18:2n-6) fatty acid signatures that resulted in similarly mixed metabolic and physiological responses.

Effects of dietary fatty acids on mitochondrial phospholipid compositions, oxidative status and mitochondrial gene expression of zebrafish at different ages.

Mitochondrial decay is generally associated with impairment in the organelle bioenergetics function and increased oxidative stress, and it appears that deterioration of mitochondrial inner membrane phospholipids (PL) and accumulation of mitochondrial DNA (mtDNA) mutations are among the main mechanisms involved in this process. In the present study, mitochondrial membrane PL compositions, oxidative status (TBARS content and SOD activity) and mtDNA gene expression of muscle and liver were analyzed in zebrafish fed two diets with lipid supplied either by rapeseed oil (RO) or a blend 60:40 of RO and DHA500 TG oil (DHA). Two feeding trials were performed using zebrafish from the same population of two ages (8 and 21 months). Dietary FA composition affected fish growth in 8-month-old animals, which could be related to an increase in stress promoted by diet composition. Lipid peroxidation was considerably higher in mitochondria of 8-month-old zebrafish fed the DHA diet than in animals fed the RO diet. This could indicate higher oxidative damage to mitochondrial lipids, very likely due to increased incorporation of DHA in PL of mitochondrial membranes. Lipids would be among the first molecules affected by mitochondrial reactive oxygen species, and lipid peroxidation could propagate oxidative reactions that would damage other molecules, including mtDNA. Mitochondrial lipid peroxidation and gene expression of 21-month-old fish showed lower responsiveness to diet composition than those of younger fish. Differences found in the effect of diet composition on mitochondrial lipids between the two age groups could be indicating age-related changes in the ability to maintain structural homeostasis of mitochondrial membranes.

Roles of selenoprotein antioxidant protection in zebrafish, Danio rerio, subjected to dietary oxidative stress.

In vertebrates, selenium (Se) is an essential micronutrient for vertebrates that is involved in antioxidant protection and thyroid hormone regulation among other roles and functions through its incorporation into proteins, the selenoproteins. Long-chain polyunsaturated fatty acids, such as docosahexaenoic acid (DHA), are essential nutrients for fish although high dietary levels may lead to increased oxidative stress due to the high degree of unsaturation. The present study investigated the effects of Se supplementation on zebrafish, Danio rerio, oxidative status together with selenoprotein expression profiles when subjected to a high-DHA diet. Fish were fed for 8 weeks with one of the four experimental diets, containing high or low DHA in combination with or without organic Se (7 mg/kg). Fish performance, Se content, fatty acid composition and TBARS of zebrafish were determined, as well as gene expression of selected selenoproteins in liver and muscle. The Se levels in whole fish reflected dietary content. High dietary DHA increased oxidative stress as indicated by reduced growth and high TBARS content, although Se supplementation reduced oxidation. The expression patterns of selenoproteins varied between liver and muscle with only deiodinase type II displaying a transcriptional response when high dietary Se was supplied. High dietary DHA decreased selenoprotein W expression in muscle and sps2 expression in liver regardless of the dietary Se content. These data suggest that oxidative stress protection associated with a high dietary intake of Se may not be solely mediated by transcriptional changes in teleost selenoprotein expression.

A nutritionally-enhanced oil from transgenic Camelina sativa effectively replaces fish oil as a source of eicosapentaenoic acid for fish.

For humans a daily intake of up to 500 mg omega-3 (n-3) long-chain polyunsaturated fatty acids (LC-PUFA) is recommended, amounting to an annual requirement of 1.25 million metric tonnes (mt) for a population of 7 billion people. The annual global supply of n-3 LC-PUFA cannot meet this level of requirement and so there is a large gap between supply and demand. The dietary source of n-3 LC-PUFA, fish and seafood, is increasingly provided by aquaculture but using fish oil in feeds to supply n-3 LC-PUFA is unsustainable. Therefore, new sources of n-3 LC-PUFA are required to supply the demand from aquaculture and direct human consumption. One approach is metabolically engineering oilseed crops to synthesize n-3 LC-PUFA in seeds. Transgenic Camelina sativa expressing algal genes was used to produce an oil containing n-3 LC-PUFA to replace fish oil in salmon feeds. The oil had no detrimental effects on fish performance, metabolic responses or the nutritional quality of the fillets of the farmed fish.

Oxidative status and histological changes in sea bass larvae muscle in response to high dietary content of docosahexaenoic acid DHA.

In previous studies, we observed dystrophic alterations in muscle of 48-day-old sea bass fed imbalanced docosahexaenoic acid (DHA) and vitamin E diets. To understand the whole pathological process associated with oxidative stress, a histological study was performed by feeding 14-day-old sea bass larvae with microdiets containing different ratios of DHA/vitamin E (1/150, 5/150 and 5/300) for a period of 21 days. Larvae fed diet 1/150 showed no lesions in contrast to larvae fed diets 5/150 and 5/300 where the highest incidence of muscle lesions and thiobarbituric acid reactive substances (TBARS) content was observed. Semithin sections revealed focal lesions consisting of degenerated fibres with hypercontracted myofilaments and extensive sarcoplasm vacuolization affecting both red and white muscle. Ultrathin sections of degenerating muscle fibres showed diffuse dilatation of sarcoplasmic reticulum, disorganized myofilaments and autophagic vacuoles containing myelin figures and dense bodies. Additionally, some macrophages were observed among injured fibres as numerous satellite cells. Results from the study agree with those obtained from previous work, proving the pathological potential of free radicals in sea bass larvae musculature. Moreover, high vitamin E inclusion could not completely protect cell membranes from free radicals action.