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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., This work was funded by Aker Biomarine Antarctic AS, Oslo, Norway

The supplemental effect of Antarctic krill meal (KM) into a low fish meal (FM) diet was evaluated for olive flounder (Paralichthys olivaceus). A 56% FM‐based diet was regarded as a high FM inclusion diet (HFM), and a low‐FM diet (LFM) was prepared by replacing 50% FM from the HFM. Four other diets were prepared by supplementing 3%, 6%, 9% and 12% KM into the LFM diet gradually replacing soy protein concentrate and tankage meal (designated as KM3, KM6, KM9 and KM12 respectively). Quadruplicate groups of fish were fed one of the diets for 12 weeks. The growth performance and feed utilization efficiency were improved by the dietary KM supplementation. Digestibility of dietary protein and dry matter was increased by the KM3‐9 diets. Haematocrit and haemoglobin were increased by KM supplementation. The innate immunity and antioxidant capacity assessed by Ig, antiprotease, lysozyme, GPx and SOD and the condition factor of fish were significantly increased by KM3‐9 diets. Moreover, goblet cell counts, villi length and fillet yield of fish were significantly improved by all the KM‐containing diets (KM3‐12). A 25‐day‐long challenge test with the Edwardsiella tarda pathogen showed that the cumulative mortality was higher in fish fed the LFM diet than in fish fed the HFM or KM‐supplemented diets. The results indicate that dietary KM supplementation in a LFM diet can increase growth performance and feed utilization efficiency, diet digestibility, intestinal development and functions, innate immunity and disease resistance of olive flounder. The recommended level of KM inclusion in a LFM diet seems to be 6.6% according to quadratic regression analysis.

A sustainable growth of the aquaculture sector implies the use of sustainable novel raw materials as replacers of the traditional fishmeal (FM) and fish oil (FO) ingredients. This fact has led to the development of sustainable and functional diets as part of a management strategy to reduce the effects on fish growth performance and health derived from low FM/FO dietary contents. In this sense, Antarctic krill (Euphausia superba) is considered a potential candidate in dietary inclusions to potentiate fish growth and health status. In this study, European sea bass (Dicentrarchus labrax) were fed a practical diet with either a 15% fishmeal content (KM0; control diet) or the same diet substituted by 30% (KM5; 50 g KM/kg diet) or 50% (KM7.5; 75 g KM/kg diet) Antarctic krill meal (KM) for 12 weeks in triplicates. At the end of the feeding trial, growth performance, liver morphology, liver proximate composition, lipid classes and fatty acid profiles, as well as the expression of hepatic genes related with lipid metabolism were evaluated. Fish fed KM-based diets presented higher (p 0.05) biochemical composition and fatty acid profile. However, smaller hepatocellular area and lower grade of cytoplasm vacuolization as well as a better alignment around sinusoidal spaces were found. The analyses of liver lipid classes revealed a positive correlation between the level of dietary KM and the pigmented material such as astaxanthin and free fatty acid content, as well as a negative correlation with the cholesterol levels. The expression of hepatic genes studied demonstrated a downregulation of 3-hydroxy-3-methylglutaryl-coenzyme A reductase (hmgr) and delta-6-desaturase (fads2) expression levels in fish fed KM-based diets. Besides, gene expression levels of fatty acid binding protein 7 (fabp7) and lipoprotein lipase (lpl) were significantly correlated with KM dietary levels. Altogether, these results profile KM as a potential promoter of growth and liver health in European sea bass fed low fish meal and oil diets.

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This study aimed to determine the effects of increasing docosahexaenoic acid (DHA) levels in Sparus aurata diets supplemented with antioxidants vitamins (vitamins C and E). Six isoproteic and isolipidic formulated diets, which varied in the DHA content (38–78 g kg−1 TFA), were fed to 19 dph S. aurata larvae. No significant differences were found in survival rates or growth, which indicates that all the diets covered the minimum essential fatty acid requirements of this species. Stress resistance was not negatively affected by dietary DHA contents. In agreement, two biomarkers of the oxidative stress of the fish and the gene expression of superoxide dismutase and glutathione peroxidase were not affected by the elevation of dietary DHA, denoting that fish were sufficiently protected from oxidative damage even at high dietary DHA contents. The elevation of dietary DHA did affect neither the occurrence of skeletal malformations nor the expression of osteological markers such as run-related transcription factor 2 or alkaline phosphatase. The results of this study have confirmed that increase of the dietary DHA levels in gilthead sea bream diets does not negatively affect larval performance if the larvae are protected from peroxidation risks. Despite not being harmful, increase of DHA over the recommended requirements does not have any significant improvement in larval performance.

Although dietary marine phospholipids are able to improve culture performance of marine fish larvae in a further extend than soybean lecithin, both types of phospholipids (PL) markedly increase oxidative risk. The inclusion of a fat-soluble antioxidant such as the vitamin E α-tocopherol could allow a better control of oxidative stress. The objective of this study was to determine the combined effect of graded levels of α-tocopherol with different levels and sources of krill phospholipids (KPL) and soybean lecithin (SBL) on growth, survival, resistance to stress, oxidative status, bone metabolism-related genes expression and biochemical composition of sea bream larvae. Sea bream larvae were completely weaned at 16 dph and fed for 30 days seven microdiets with three different levels of PL (0, 40 and 80 g kg−1 diet) and two of α-tocopherol 1500 and 3000 mg kg−1 diet. Sea bream larvae fed diets without PL supplementation showed the lowest survival, growth and stress resistance, whereas increase in PL, particularly KPL, markedly promoted larval survival and growth. However, feeding SBL markedly increased TBARs and GPX gene expression increasing the peroxidation risk in the larvae. Besides, KPL inclusion improved incorporation of n-3 HUFA and, particularly, EPA into larval tissues, these fatty acids being positively correlated with the expression of BMP-4, RUNX 2, ALP, OC and OP genes and to bone mineralization for a given larval size class. The increase in dietary α-tocopherol tends to improve growth in relation to the n-3 HUFA levels in the diet, denoting the protective role of this vitamin against oxidation. Indeed, dietary α-tocopherol decreased the oxidative stress in the larvae as denoted by the reduction in larval TBARs contents and gene expression of SOD and CAT, but not GPX. Thus, increase in dietary α-tocopherol effectively prevented the formation of free radicals from HUFA, particularly EPA, but did not affect the incidence of bone anomalies or the expression of genes related to osteogenetic processes.

The aim of the present study was to determine the optimum dietary levels of krill phospholipids (KPL) for sea bream (Sparus aurata) larvae, and its influence on larval development and digestive enzymes activity. Larvae were fed five formulated microdiets with five different levels of KPL. Complete replacement of live preys with the experimental microdiets for seabream larvae produced high survival and growth rates, particularly in fish fed the highest levels of KPL. In the present study, increase in dietary KPL up to 120 g kg−1 (100 g kg−1 total PL) significantly improved larval survival and growth, whereas further increase did not improve those parameters. An increase in alkaline phosphatase, trypsin and lipase activity with the elevation of KPL up to 120 g kg−1 was also found denoting a better functioning of digestive system. Besides, there was a linear substrate stimulatory effect of dietary KPL on phospholipase A2 activity. Finally, increasing dietary KPL lead to better assimilation of n-3 HUFA especially eicosapentaenoic acid, reflected in the higher content of these fatty acids in both neutral and polar lipids of the larvae. In summary, KPL were found to be an excellent source of lipids for seabream larvae. Optimum inclusion levels of this ingredient in microdiets to completely substitute live preys at this larval age were found to be 120 g kg−1 KPL.

The objective of this study was to determine the effect of dietary vitamin E on gilthead seabream (Sparus aurata) growth and survival, at two different highly unsaturated fatty acids (HUFAs) levels. Eighteen days old gilthead seabream larvae were fed four formulated experimental diets combining two different dietary levels of HUFAs (M: medium 2.5 + 1.5, DHA + EPA, H: high 5 + 2.5 DHA + EPA g per 100 g) with two different levels of vitamin E (M: medium 540 mg kg−1, H: high 2900 mg kg−1): MM, MH, HM, HH (HUFA/vitamin E). After 2-week feeding trial, the average survival rate was 52.6% and there were no significant differences found among treatments. Increase in vitamin E up to high level markedly improved larval growth, particularly when dietary HUFA levels were lower, suggesting a higher protection value when these fatty acids are more limiting. At medium dietary HUFA levels, increase in vitamin E from medium to high level enhanced larval growth performance in terms of total length. Moreover, increase in vit E enhanced HUFAs content in the larval polar lipids denoting the anti-oxidative effect of vitamin E.

There is limited information on the specific effects of long-chain PUFA (LCPUFA) on neuron development and functioning. Deficiency of those essential fatty acids impairs escape and avoidance behaviour in fish, where Mauthner cells (M-cells) play a particularly important role in initiating this response. Gilthead seabream larvae fed two different LCPUFA profiles were challenged with a sonorous stimulus. Feedingn-3 LCPUFA increased the content of these fatty acids in fish tissues and caused a higher number of larvae to react to the stimulus with a faster burst swimming speed response. This faster startle response in fish fedn-3 LCPUFA was also associated with an increased immune-positive neural response, particularly in M-cells, denoting a higher production of acetylcholine. The present study shows the first evidence of the effect ofn-3 LCPUFA on the functioning of particular neurons in fish, the M-cells and the behaviour response that they modulate to escape from a sound stimulus.

The objective of this study was to evaluate three products derived from krill as sources of essential fatty acids, protein and, particularly, phospholipids in microdiets for larval gilthead seabream (Sparus aurata). Their effect on larval performance, biochemical composition and histological development was investigated. The addition of krill phospholipids, rich in highly unsaturated fatty acids, improved larval sea bream growth in terms of weight and length, enhanced hepatic utilization of dietary lipids and reduced the incidence of enterocyte injuries. These results confirm the higher nutritional value of marine phospholipids for the early development of marine fish larvae in comparison with soybean phospholipids.

The objective of the present study was to investigate the combined effect of several dietary contents of vitamin E and polyunsaturated fatty acids (PUFA), mainly docosahexaenoic acid (DHA), on growth, survival, biochemical composition and tissue morphology of sea bass along early development. A feeding experiment was conducted in sea bass larvae using five different diets with the same proximate composition and different ratios of DHA concentrated fish oil [10, 30 and 50 g kg−1 dry weight (DW)] and vitamin E (α-tocopherol acetate) (1500 and 3000 mg kg−1 DW). DHA was readily deposited in fish tissues and associated with higher sea bass mortalities probably because of increased peroxidation risks. Besides, the elevation of dietary DHA contents up to 5% severely increased the incidence of muscular lesions and the presence of ceroid pigment within hepatocytes. However, elevation of dietary vitamin E levels markedly reduced the incidence of these symptoms in sea bass, increasing the tissue content in several PUFA and improving growth and stress resistance. Moreover, when sea bass was fed diets containing high vitamin E levels, fish showed a significant improvement in growth when dietary DHA was raised from 1% to 3%. Therefore, in sea bass larvae, a ratio of 30 g kg−1 DHA and 3000 mg kg−1 vitamin E seems to be adequate to achieve a good larval performance and to avoid muscular lesions.

The objective of this study was to determine the effect of mannan oligosaccharides derived from the outer cell wall of a select strain of Saccharomyces cerevisiae (Bio-Mos, Alltech Inc, USA) on mucus production, selected mucus immune parameters activity, gut morphology and in vivo and ex vivo gut bacterial translocation for European sea bass (Dicentrarchus labrax). Specimens were fed 4 g kg⁻¹ dietary MOS level of inclusion in a commercial sea bass diet for eight weeks. At the end of this period, anterior gut mucosal folds height, width and folds surface area were increased by MOS supplementation (P < 0.05, n = 240). Posterior gut presented shorter folds (P < 0.05, n = 240) but wider that those fed control diet (P < 0.05, n = 240) resulting in increased total surface area (P < 0.05, n = 240). For rectum, feeding MOS reduced fold length (P < 0.05, n = 240). Gut morphological analyses showed an enhancement in the number of cells secreting acid mucins by area unit, higher density of eosinophilic granulocytes (ECGs) in the mucosa for fish fed MOS together with an improvement in gut mucus lysozyme activity which could be related to the reduced in vivo and ex vivo gut bacterial translocation found. No differences were found for the skin mucus immune parameters evaluated.

The effect of different arachidonic acid (ARA) dietary contents at several dietary eicosapentaenoic acid (EPA) levels on the growth, survival and biochemical composition of gilthead seabream larvae was studied to better define the importance of this fatty acid as a function of EPA. Larvae of 18 days were fed one of the five isonitrogenous and isolipidic microdiets with three different EPA (0.3%, 2% and 4%) and ARA amounts (0.1%, 0.6% and 1.2%). Although a dietary increase in either ARA or EPA alone did not improve survival significantly, the increase in both fatty acids significantly enhanced growth and survival, suggesting an optimum dietary value of EPA:ARA close to 4:1.2. Dietary ARA was more efficiently incorporated into larval tissues than EPA. Increased dietary EPA or ARA contents reduced the incorporation of ARA or EPA into larval lipids, indicating their competition as substrates for different enzymes. The possible negative effect of further elevation of dietary ARA and its competition with EPA for phospholipids synthesis deserves further studies in marine fish larvae.

Together with docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA), arachidonic acid (ARA) is being considered to be an essential fatty acid in marine fish larval diets. The objective of the present study was to determine the importance of dietary ARA levels for larval European sea bass performance, when EPA and DHA are also present in the diet. Eighteen-day-old larvae were fed, for 14 days, gelatine-based microdiets containing the following ARA levels: 0.3%, 0.6% or 1.2%. Elevation of dietary ARA up to 1.2% showed a positive correlation with larval survival and a significant improvement in the specific growth rates, body weight and total length. Arachidonic acid was efficiently incorporated into larval lipids, even at a higher proportion than that in the diets. Increased accumulation of ARA did not affect the incorporation of DHA or EPA from the diet into larval total lipids. A significant positive correlation was found between dietary ARA levels and survival after handling stress, indicating the importance of this fatty acid in sea bass larvae response to acute stressors. The results show the importance of ARA for sea bass larvae, but higher dietary levels should be tested to determine whether there is a negative effect of ARA in sea bass as reported for other species.

Two single cell heterotrophs were produced as alternative sources of docosahexaenoic acid (DHA; 22:6 n-3) to fisheries-derived oils. Schizochytrium G13/2S or Crypthecodinium cohnii biomasses, either homogenised (S H , C H , respectively) or non-homogenised (S, C), were tested in gilthead seabream ( Sparus aurata ) microdiets. In Trial 1 the biomases of both species algae were used in replacement of fish oil, whereas in Trial 2 all dietary lipids, including those derived from squid meal, were replaced by the algal biomass in all diets except for control diet. Larval dietary acceptance, overall survival, air-exposure stress survival, growth and disease resistance were not significantly affected by the source of DHA employed when only fish oil was substituted. However, complete substitution of all dietary lipids by the algal biomass, resulted in reduced growth and survival which was apparently related to other dietary imbalances besides simply DHA content, such as changes in the DHA:EPA (eicosapentaenoic acid; 20:5 n-3) ratio. After 15 and 21 d feeding the experimental diets to S. aurata larvae in Trial 1 (0.1 mg initial larval dry weight) and Trial 2 (0.15 mg initial larval dry weight), respectively, the algal biomass inclusion or homogenisation did not significantly affect the DHA deposition in fish body (14.4 ± 0.3% total fatty acids ± SEM and 28.4 ± 0.4, average values for Trials 1 and 2, respectively). Higher arachidonic acid levels were found in larvae fed Schizochytrium sp. diets in Trial 2 and were related to the retro-conversion from docosapentaenoic acid (DPA; 22:5 n-6) which was high in these diets. These results show the potential of single cell heterotrophs as alternative DHA sources for fish oil in microdiets for gilthead seabream but also point out the necessity of EPA sources to completely replace fisheries-derived oils.

Developmental changes in swimming speed were analysed in the seabream (Sparus aurata) larvae. Four feeding regimes using live preys (rotifer Brachionus plicatilis) enriched with fish oil, soybean oil, linseed oil and rapeseed oil, differing in fatty acid profile, were tested during the first weeks of larval life. There was an increase in burst swimming speed and cruise swimming speed during the visual stimulus experiment at day 16th of life in the present study in agreement with the better eye development in larvae of this age. Swimming activity before stimulus was significantly reduced when larvae were fed rotifers enriched with vegetable oils. Larvae fed with rotifers enriched with fish oil reacted with a higher burst swimming speed after a visual stimulus than after the sound stimulus (159.5 SL/s vs. 18.30 SL/s) denoting the importance of the vision during this period of development not only for predation but also for the burst. The reduction in dietary essential fatty acid contents, by the enrichment with vegetable oils, delays the appearance of response to visual stimulus, in agreement with the minor DHA content in eyes and brains of these larvae and suggesting a delay in the functional development of brain and vision.

Docosahexaenoic acid (DHA) is an essential fatty acid necessary for many biochemical, cellular and physiological functions in fish. However, high dietary levels of DHA increase free radical injury in sea bass (Dicentrarchus labrax) larvae muscle, even when vitamin E (α-tocopherol, α-TOH) is increased. Therefore, the inclusion of other nutrients with complementary antioxidant functions, such as vitamin C (ascorbic acid, vitC), could further contribute to prevent these lesions. The objective of the present study was to determine the effect of vitC inclusion (3,600 mg/kg) in high DHA (5 % DW) and α-TOH (3,000 mg/kg) microdiets (diets 5/3,000 and 5/3,000 + vitC) in comparison to a control diet (1 % DHA DW and 1,500 mg/kg of α-TOH; diet 1/1,500) on sea bass larvae growth, survival, whole body biochemical composition and thiobarbituric acid reactive substances (TBARS) content, muscle morphology, skeletal deformities and antioxidant enzymes, insulin-like growth factors (IGFs) and myosin expression (MyHC). Larvae fed diet 1/1,500 showed the best performance in terms of total length, incidence of muscular lesions and ossification degree. IGFs gene expression was elevated in 5/3,000 diet larvae, suggesting an increased muscle mitogenesis that was confirmed by the increase in the mRNA copies of MyHC. vitC effectively controlled oxidative damages in muscle, increased α-TOH larval contents and reduced TBARS content and the occurrence of skull deformities. The results of the present study showed the antioxidant synergism between vitamins E and C when high contents of DHA are included in sea bass larvae diets.

The objective of the present study was to determine the effect of Se inclusion in high-DHA and vitamin E microdiets (5 g DHA/100 g dry weight and 300 mg vitamin E/100 g dry weight; 5 g DHA/100 g dry weight and 300 mg vitamin E/100 g dry weight supplemented with Se) in comparison with a control diet (1 g DHA/100 g dry weight and 150 mg vitamin E/100 g dry weight) on sea bass larval growth, survival, biochemical composition, malonaldehyde (MDA) content, muscle morphology and antioxidant enzymes (AOE), insulin-like growth factors (IGF) and myosin expression. For a given DHA and vitamin E dietary content, Se inclusion favoured larval total length and specific growth rate, and reduced the incidence of muscular lesions, MDA contents and AOE gene expression. In contrast, IGF gene expression was elevated in the 5/300 larvae, suggesting an increased muscle mitogenesis that was corroborated by the increase in mRNA copies of myosin heavy chain. The results of the present study denoted the beneficial effect of Se not only in preventing oxidative stress, as a glutathione peroxidase cofactor, but probably due to other as yet unknown physiological functions.

Dietary omega − 3 long chain polyunsaturated fatty acids (n − 3 LCPUFA), eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), have a marked effect on fish behavior. There is limited information on the mechanisms involved in this effect and its relation to neuron development and functioning. Deficiency of n − 3 LCPUFA reduces fish escape swimming. Mauthner cells (M-cell) are neurons responsible for initiating an escape response. The aim was to compare the effect of dietary DHA and EPA on escape behavior and neuronal activity of sea bream larvae. We studied burst swimming speed as a measure of behavior. M-cell activity was studied by ChAT immuno-fluorescence. Feeding the lowest n − 3 LCPUFA levels a lower burst swimming speed. Increase in dietary EPA did not significantly improve escape response. Elevation of dietary DHA was correlated with a higher burst speed denoting the importance of this nutrient for escape swimming. Incorporation of DHA into larval tissues was proportional to DHA dietary levels and significantly correlated with burst speed. In addition, a higher immunoreactivity to ChAT, associated to a higher neural activity, was found in M-cell of larvae fed higher dietary DHA contents. These results show first evidence of n − 3 LCPUFA on fish neuronal activity and their implications in behavior, denoting that DHA boosts escape swimming and this effect is at least partly mediated by the increase in neural activity of M-cell.

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.