Aquaculture nutrition, tập 19, số 5, 2013

Aquaculture Nutrition 2013 19; 641–650 doi: 10.1111/anu.12078 NIFES, National Institute of Nutrition and Seafood Research, Bergen, Norway The present paper gives an overview on the use of plant protein and plant oils as replacers for fish meal and fish oil in diets for Atlantic cod Gadus morhua L In focus are effects on growth, feed utilization, digestibility, gut health, muscle and liver uptake and retention of nutrients, and muscle quality Plant oil can replace fish oil without affecting growth provided that the requirement of marine long chain (LC) n-3 fatty acids is met, but the altered dietary fatty acid profile in diet will be reflected in both muscle and liver This can reduce the value of cod liver as an oil source for cod liver oil production For the fish itself, there are more challenges replacing fish meal than fish oil, due to the amount of fibre and antinutrients in plant protein meals However, A cod seems to tolerate a wide range of plant types and their inclusion levels provided that the amino acids requirements are met It is our view that there is sufficient knowledge to be able to design an A cod diet based on a mixture of plant and marine ingredients and be able to predict performance such as growth, feed utilization, digestibility, liver size and fish health in general KEY WORDS: A cod, plant protein, plant oil, growth, protein utilization, digestibility, gut health Received 30 November 2012; accepted 27 March 2013 Correspondence: G.-I Hemre, NIFES, National Institute of Nutrition and Seafood Research, Box 2029 Nordnes, 5817 Bergen, Norway E-mail: ghe@nifes.no Until recently, the protein and lipid in farmed fish diets have been based on fish meal and fish oil Currently, most marine resources used in fish meal and fish oil production ª 2013 John Wiley & Sons Ltd are exploited to the highest maximum level, simultaneously as the global production of farmed fish is increasing (FAO 2010) It has therefore been essential to evaluate the potential for utilizing plants as protein and oil sources in diets for farmed fish In Northern Europe, interest in farming of A cod (Gadus morhus L.) has increased steadily over the past decade stimulated by the decline in landings from fisheries and a more predictable supply of hatchery reared juveniles for ongrowing From 2000 to 2009, the production quantity of farmed A cod in Norway increased from 169 to 18 052 tonnes (FAO-Fisheries and Aquaculture Information and Statistics Service), and in 2010, 19 700 tonnes were slaughtered (Mugaas Jensen et al 2010) However, due to economic problems, disease problems, increased landings of wild A cod and low prices (Torskenettverksmøte 2011, Bergen) in 2012, there was only one main producer left in Norway This means that the cost of feed must be as low as possible, simultaneously as fish health and performance must be acceptable A cod is a carnivorous lean fish species containing 5–10 g lipid kgÀ1 muscle It has relatively low potential to utilize dietary lipid for energy (500 g kgÀ1) (Rosenlund et al 2004) Lipids are stored in the liver which often exerts 12–17% of the body weight The intestine is long with an intestinal length/fork length ratio of 0.72 (Buddington & Diamond 1987) and has up to 200 pyloric ceca located in the proximal intestine adjusted to the pyloric sphincter Buddington & Diamond (1987) found that 69% of the gut area was represented by the pyloric ceca giving a gut length/fork length ratio of 10, being 2.5 times higher than, for example, rainbow trout, increasing the absorptive surface of the intestine considerably Compared to the front and mid sections of the intestine, the distal part of the cod intestine has a clear distinguished appearance with a thick intestinal wall and a valve like structure The distal chamber has also a high colonization of bacteria in the brush border and is therefore probably a fermentation chamber, fermenting indigestible intestinal content (Refstie et al 2006b; Seppola et al 2006) This characteristic of A cod intestine can affect the ability to cope with plant ingredients, as its appearance is similar to the intestine of herbivorous fish species (Krogdahl et al 2005) This review aims to summarize the research carried out in replacing fish meal and oil with plant ingredients in diets for A cod The dietary lipids constitute an important energy source as well as being a source of essential fatty acids in A cod diets Fish oil is characterized by high level of the marine long chain n-3 fatty acids docosahexaenoic acid (DHA; 22:6n-3) and eicosapentaenoic acid (EPA; 20:5n-3), while plant oils are characterized being high in the n-6 [mainly linoleic acid (LA; 18:2n-6)] and n-9 fatty acids [mainly oleic acid (OA; 18:1n-9)] and the saturated fatty acids palmitic acid (16:0) and stearic acid (18:0), and does not contain DHA and EPA Replacement of fish oil with plant oil is widely studied in fatty fish like salmonids that have a higher lipid utilization capacity than the lean A cod (Turchini et al 2009) There are a few studies reported for A cod, where fish oil is used exclusively or partly as the dietary oil source (Lie et al 1986, 1992; Waagbø et al 1995; Bell et al 2006; Mørkøre 2006; Mørkøre et al 2007; Jobling et al 2008) The only negative effect on growth and feed intake in A cod was seen with use of dietary peanut oil (Lie et al 1986) Total or partial substitution of fish oil with linseed oil, echium oil or soybean oil in the diet gave no negative growth effects (Lie et al 1992; Bell et al 2006; Mørkøre 2006; Mørkøre et al 2007; Jobling et al 2008) The trial with peanut oil was also the only trial where squid mantle, not fish meal, was used as the dietary protein source and had the lowest levels of marine n-3 fatty acids (Table 1) Essential fatty acid deficiencies are unlikely when the diet contains a relatively large amount of fish meal (Turchini et al 2009), as fish meal usually contains 50–130 g kgÀ1 lipid, whereas 20–35% of the lipid is marine LC n-3 fatty acids (NRC 2011) When comparing A cod trials that gave no effect on the growth of dietary DHA level ranged from 7.7 to 9.9 g kgÀ1 and the EPA level ranged from 4.2 to 7.8 g kgÀ1 (Bell et al 2006; Mørkøre 2006), giving LC n-3 levels of 9.4–17.7 g kgÀ1 diet (Table 1) This indicates that these levels of DHA and EPA were sufficient to support growth This was in line with what was reported to be the requirement for other marine fish species (5.0–25.0 g LC n-3 kgÀ1 feed) (Turchini et al 2009) In the diet with peanut oil, the level of LC n-3 was only 3.4 g kgÀ1 (Lie et al 1986) and was probably deficient in LC n-3, as the first Table Level of long chain (LC) n-3 fatty acids in Atlantic cod diets with different plant oil sources Oil source Inclusion (% of oil) DHA (% of fatty acids) EPA (% of fatty acids) Sum LC n-3 (% of fatty acids) Echium oil 100 7.7 4.2 11.9 Rapeseed oil Linseed oil Fish oil Palm oil Linseed oil Fish oil Soybean oil Fish oil 40 20 40 40 20 40 40 60 9.5 6.8 16.3 9.5 7.0 16.5 9.9 7.8 17.7 Soybean oil 100 4.7 4.7 9.4 Peanut oil 100 2.5 0.9 3.4 Main findings Reference No effect on growth Changed tissue FA profile Reduced prostaglandin F2a production and macrophage activity in gill cells No effect on growth Changed tissue FA profile Bell & Waagbø (2008) No effect on growth Changed tissue FA profile Increased HIS No effect on growth Effects on muscle quality post mortem No effect on growth Changed tissue FA profile Reduced growth Changed tissue FA profile Jobling et al (2008) Jobling et al (2008) Mørkøre (2006) Lie et al (1992) Lie et al (1986) Aquaculture Nutrition, 19; 641–650 ª 2013 John Wiley & Sons Ltd 25.0 sign of deficiency normally is reduced growth, which was reported in this study (Lie et al 1986) In this trial, feed intake was reduced by 20% compared to a diet with cod liver oil Lipid digestibility was also reduced, which may partly explain the reduced growth (ibid) There have been some concerns regarding technical quality of fillet due to altered fatty acid profile of muscle when using diets high in plant oils As A cod muscle only contains 5–10 g kgÀ1 lipid, most of the lipid is in the form of phospholipids in the cell membranes, and changes in the fatty acid profile of the membranes can change the characteristics of the membrane Changes in dietary lipid level did not result in changes in muscle lipid level in A cod, however, it affected muscle vitamin E and C concentrations, Aquaculture Nutrition, 19; 641–650 ª 2013 John Wiley & Sons Ltd 10.0 Comp 1(18.0%) In Atlantic cod, Jobling et al (2008) found a tendency to increased HSI and liver lipid level when fed a diet with rapeseed or palm oil compared to fish oil (equal lipid level in all diets) However, this was not seen with use of soybean or echium oil (Bell et al 2006; Mørkøre 2006; Mørkøre et al 2007) The HSI was even reduced with the use of only soybean oil compared to fish fed a diet with only capelin oil in one trial (Waagbø et al 1995) The variable results between studies might be due to different fatty acids stimulating the b-oxidation capacity and thereby lipid storage differently, as found for other species of fish (Stubhaug et al 2007) The fatty acid profile of the diet influences both muscle and liver fatty acid profiles of A cod, where soybean, rapeseed and palm oil fed fish had higher levels of LA and sum of n-6 fatty acids in muscle and liver than fish oil–fed fish (Lie et al 1992; Mørkøre 2006; Jobling et al 2008) Feeding A cod full-fat soybean meal or a combination of fullfat soybean, corn gluten and wheat gluten meal resulted in muscle and liver reflecting the fatty acid profile of the diet (Hemre et al 2004; Albrektsen et al 2006; Karalazos et al 2007) (Fig 1) The liver reflecting the fatty acid profile of the diet can have consequences for the use of farmed A cod liver in the production of cod liver oil, as the level of DHA and EPA will decrease when dietary LC n-3 decrease This can be solved by feeding a finishing diet high in LC n-3 fatty acids; however, reduction of LA in fish earlier fed plant oil is found to be low (Mørkøre et al 2007; Jobling et al 2008) FM diet FM liver 22:1 22:6n-3 20:1n-9 20:5n-3 16:1n-7 14:0 18:4n-3 18:1n-7 18:0 22:5n-3 24:1n-9 20:4-n-3 22:5n-6 20:4n-6 20:2n-6 18:3n-3 –5.0 16:0 18:1n-9 36% SBM liver 36% SBM diet 18:2n-6 20.0 35.0 –20.0 –5.0 10.0 25.0 40.0 Comp 2(79.1%) Figure PCA showing the correlation between diet and liver fatty acid composition in Atlantic cod fed an all FM diet and a diet with 360 g kg-1 full-fat soybean meal (SBM) (modified from Karalazos et al 2007) and which showed a negative correlation with EPA and DHA and the ratio n-3/n-6 in muscle (Hemre et al 2004) Changes in dietary fatty acid profile not affect muscle pH, liquid losses or texture after frozen storage, however, soybean oil fed fish had significant lower degree of gaping (Mørkøre 2006; Mørkøre et al 2007) A trained test panel could taste a difference between soybean oil and fish oil fed Atlantic cod and preferred no one over the other (Mørkøre et al 2007) There have also been some concerns regarding fish health, when the levels of DHA and EPA in the diet are reduced due to inclusion of plant oil This concern is connected to the positive effects of DHA and EPA seen in mammals on coronary heart disease, but also to general disease resistance (Turchini et al 2009) Waagbø et al (1995) showed lower specific antibody response against V Anguillarum in soybean oil fed A cod compared to marine oil fed fish, indicating reduced disease resistance On the other hand, Bell et al (2006) indicated a positive effect on some immune parameters with use of dietary echium oil, which resulted in reduced prostaglandin F2a production in gill cells together with reduced macrophage activity This was probably connected to the increases of ARA/EPA ratio in the fish when fed echium oil, as ARA is the primary eicosanoid precursor in fish, and macrophages are activated by eicosanoids (Tocher 2003) Changing the LC n-3 level in Atlantic salmon diets has shown both positive and negative effects on the immune system (Turchini et al 2009), indicating immune effects will be dependent on how large and which changes in fatty acid composition the plant oil causes In conclusion, there is great potential to replace fish oil with plant oil without compromising performance; however, this will reduce the value of the A cod liver as a marine LC n-3 source in, for example, cod liver oil production, if not using a ‘finishing’ diet There is a wide variety of plant protein ingredients being good alternatives to fish meal as protein sources in fish feeds This includes oilseeds (soybean, sunflower, cottonseed and rapseed/canola), legumes (lupins, beans, peanut and pea) and grails (corn and wheat glutens) There are several different products and qualities of those plant protein ingredients, varying in protein, fat and antinutrient contents (NRC 2011) To obtain acceptable growth and feed utilization, simultaneously avoiding large liver sizes, A cod needs diets with 500–600 g kgÀ1 protein, 130–200 g kgÀ1 lipid and moderate carbohydrate levels (Lied & Braaten 1984; Jobling 1988; Lie et al 1988; Hemre et al 1989; Dos Santos et al 1993; Morais et al 2001; Rosenlund et al 2004; GrisdaleHelland et al 2008; Hansen et al 2008) Lower dietary protein levels not result in lowered growth, but increased FCR, as A cod is found to be able to compensate with higher feed intake to reach its optimal level of protein and energy intake to meet its growth potential (Hemre et al 1989; Lekva et al 2010) To achieve a high dietary protein level in plant-based diets (above 500 g kgÀ1), processed plant ingredients like wheat and corn gluten and soy protein concentrate need to be used A cod, like other animals, has a requirement for indispensable amino acids (IAA), for optimal growth and protein utilization The requirement for IAAs has been shown to highly correlate with the amino acid pattern of the fish (Wilson & Poe 1985; Mambrini & Kaushik 1995), and this pattern is similar between fish species (Mambrini & Kaushik 1995) High-quality fish meal is therefore regarded as a good protein source that covers the need for all amino acids On the other hand, plant proteins differ from fish meal in several IAAs Further, some plant proteins have high levels of undesirable components like ANFs and fibre, which can alter intestinal function and influence digestibility coefficients in fish (Francis et al 2001) Fish meal has been replaced by plant protein in several trials with A cod (Table 2) (von der Decken & Lied 1993; Albrektsen et al 2006; Refstie et al 2006a; Hansen et al 2007a,b; Karalazos et al 2007; Walker et al 2010) Effects on growth are dependent on the plant ingredients, which mixture is used, and the inclusion level Replacing fish meal protein with 30% protein from full-fat soybean meal in diets for A cod induced reduced growth (von der Decken & Lied 1993; Karalazos et al 2007) This was due to reduced feed intake, maybe a consequence of reduced palatability or ANFs, but it was also speculated to be caused by the shortage of one or several amino acids triggering a metabolic mechanism of food intake regulation (von der Decken & Lied 1993) On the other hand, no effects on growth and feed intake were observed by Albrektsen et al (2006) using a mixture of full-fat soybean meal and corn gluten up to 54% of total dietary protein Similarly, no effect on growth was found using 24% (of total protein) solvent-extracted or bioprocessed soybean meal (Refstie et al 2006a), a combination of solvent-extracted soybean meal and corn gluten meal (70–310 g kgÀ1) or where 58% of protein came from a 1:1 combination soy protein concentrate and wheat gluten (Hansen et al 2007b) Also, no negative effects on growth were seen when 100% of the fish meal was replaced with soy protein concentrate (Walker et al 2010) Using a regression design from an all fish meal to an all plant protein diet (500 g kgÀ1 wheat gluten + 140 g kgÀ1 bioprocessed soybean meal + 360 g kgÀ1 soy protein concentrate) resulted in reduced growth at a plant protein inclusion level of 50% and higher, with a mean reduction in SGR of 16% when given 75% plant protein, and 43% when given 100% plant protein (Hansen et al 2007a) Increased feed intake was reported for A cod when including plant protein (Albrektsen et al 2006; Refstie et al 2006a; Hansen et al 2007a) This is in contrast to what is seen with total (de Francesco et al 2004; Espe et al 2006) or partial (Gomes et al 1995; Refstie et al 1998; Torstensen et al 2008) replacement of fish meal for salmonids, where a reduction in feed intake was seen especially in the first period of the trials In the trial by Hansen et al (2007b), the plant protein inclusion in cod diets also resulted in reduced protein utilization The increased feed intake registered could therefore be due to a compensation for the lowered protein utilization, as it has Aquaculture Nutrition, 19; 641–650 ª 2013 John Wiley & Sons Ltd Table Studies where plant proteins replace fish meal in diets for Atlantic cod Plant ingredient Extracted soybean Full-fat soybean Bioprosessed soybean Soy protein concentrate g kg-1 of diet % of protein 246 54.2 Amino acid supplemented Growth Feed utilization Protein retention Met No effect Increased No effect 40–150 4–16 No No effect No effect No effect 80–240 16–22 No Reduced Reduced Not reported 120–360 45–56 Lys, met (all diets) Met Reduced Reduced Not reported No effect Increased No effect No effect No effect Not reported No effect No effect No effect 214 53.9 145–580 28–100 Corn gluten 60–240 7–31 Lys, met, tau (all diets) No Full-fat soybean + corn gluten Extracted soybean + corn gluten Soy protein concentrate + wheat gluten Bioprosessed soybean + soy protein concentrate + wheat gluten 60–420 32–59 Lys (all diets) No effect No effect No effect 100–400 11–44 Met (44% of pro.) No effect No effect No effect 220–440 32–58 Met (58% of pro.) No effect No effect No effect 170–730 25–100 Lys, met (100% of pro.) Gradually reduced Gradually reduced Gradually reduced been found that A cod increase its feed intake to meet dietary demands, for example, for protein and energy (Hemre et al 1989; Lekva et al 2010) Plant proteins in fish diets may lead to reduced protein retention (Kikuchi 1999; Regost et al 1999; Carter & Hauler 2000; Refstie et al 2000; Opstvedt et al 2003; de Francesco et al 2004; Lim et al 2004) This is possible due to deficiency of one or several amino acids Espe et al (2007) showed that 95% replacement of fish meal with plant protein could be used without compromising growth in diets for A salmon, provided that the amino acid levels mimicked that of a fish-meal-based control diet and equal feed intake This shows that differences in amino acid levels may explain the reduction in growth when exceeding 50% plant protein in diets for A cod (Hansen et al 2007a) In this latter trial, the plant-based diets methionine was limiting when compared to fish meal Methionine concentration in diet also highly correlated with plasma and muscle free methionine concentrations when sampled h after the last meal At 75% and 100% plant protein replacement of fish meal protein, free methionine was not detected in the muscle free pool at all, showing that the free amino acid Aquaculture Nutrition, 19; 641–650 ª 2013 John Wiley & Sons Ltd Reference Refstie et al (2006a) Hansen et al (2007a,b) von der Decken & Lied (1993) Karalazos et al (2007) Refstie et al (2006a) Walker et al (2010) Hansen et al (2007a,b) Albrektsen et al (2006) Hansen et al (2007a) Hansen et al (2007a) Hansen et al (2007a) (b) (a) G G MV MV Figure Hind gut from Atlantic cod fed a fish meal diet (a) and a 100% plant protein diet (b) MV, micro villi; G, goblet cells (Olsen et al 2007) pool was emptied of methionine (Fig 2) Further, a decrease in muscle free lysine of 67% was seen comparing the fish meal group and the 100% plant group, correlating well with dietary levels of this amino acid However, lysine and methionine supplementation to diets holding 650 g kgÀ1 plant protein did not result in improved total growth, feed intake or protein retention (Hansen et al 2011) This indicates that plant protein-based diets for A cod need not be added lysine or methionine, or both, to maintain total growth, foreseen that diets hold 19.2 g lysine kgÀ1 diet and 9.4 g methionine kgÀ1 diet Grisdale-Helland et al (2011) tested a dietary lysine level of 13.7–28.3 g kgÀ1 diet and did not find any effects on growth, but the best lysine gain was achieved with the diet containing 26.2 g lysine kgÀ1 diet Lysine requirement for A cod is in line with requirements reported for other marine species (17–26 g lysine kgÀ1 diet) (NRC 2011) At the same time, increased lysine intake affected lipid storage resulting in reduced lipid retention, HSI and plasma TAG (Hansen et al 2011) On the other hand, this was not observed in other trials with plant protein in A cod diets (Hansen et al 2007a,b), but a tendency of reduced HSI when including plant protein was reported in some trials (Albrektsen et al 2006; Refstie et al 2006a) Microarray and qPCR data comparing fish meal fed A cod with fish fed 75% plant protein of total protein, showed alteration in hepatic expression of genes involved in protein turnover, affecting both protein degradation and anabolic pathways (Lie et al 2011) Diets with plant protein can reduce digestibility of nutrients in fish (Hilton et al 1983; Francis et al 2001) Krogdahl et al (2003) showed reduced mucosal enzyme activities in Atlantic salmon fed up to 300 g kgÀ1 solventextracted soybean meal, mirroring reduced macronutrient digestibility Plant protein can contain soluble fibres and antinutrients that can interfere with nutrient digestibility (Krogdahl et al 2005) Soluble fibres increase viscosity of gut content, which potentially can reduce digestible enzyme activities, and negatively affect nutrient digestion and absorption (Storebakken 1985; Leenhouwers et al 2006) Of the antinutrients protease inhibitors, tannins and lectins are known to affect protein utilization and digestion (Francis et al 2001) Protease inhibitors, especially high levels in soybean, act by blocking the activity of trypsin and chymotrysin Lectins, on the other hand, bind to receptors on the intestinal cells and can cause damage to the villi Tibbetts et al (2006) investigated the digestibility of a number of plant protein ingredients in A cod and found ADC values in the same range for plant protein ingredients, like soybean meal, lupin meal, corn gluten and wheat gluten meal as for fish meal However, other studies have reported reduced ADC for protein and lipid in studies with A cod when including solvent-extracted soybean meal (Førde-Skjærvik et al 2006; Hansen et al 2006), and Hansen et al (2006) observed reduced ADC of protein, and fat and starch when including corn gluten meal Some of the discrepancy between studies may be due to different ingredient qualities and variable inclusion levels, for example, replacing 100% of the fish meal with plant protein results in a fibre level increase from 30 to 80 g kgÀ1 (calculated amount) (Hansen et al 2007a) The fibre fraction is shown to disturb lipid micelle formation in the intestine and to increase the viscosity of fish gut content, both factors that can explain a reduced fat digestion (Krogdahl et al 2005; Refstie et al 2006b; Olsen et al 2007) The reduced lipid digestion may also be linked to alcohol-soluble components from soybean, like saponins, that have been found to result in altered gut function and reduced lipid digestibility in A salmon (Olli & Krogdahl 1995; Knudsen et al 2007) and Japanese flounder (Chen et al 2011) Total saponin concentration increased from 0.42 to 0.86 mg gÀ1 (DW) when increasing plant protein inclusion from 50% to 100% of total protein, in A cod diets, and unfortunately the fish was not able to digest these saponins (Olsen et al 2007) In the gastrointestinal tract of A cod, you find a high number of bacteria (Hansen et al 2006; Refstie et al 2006a,b; Seppola et al 2006), which changed dependent on soybean inclusion in the diet (Ringø et al 2006) So, the reduced digestibility of protein and starch observed in the cod studies (Førde-Skjærvik et al 2006; Hansen et al 2006) may also be a consequence of changed intestinal microflora Morphological changes in the intestine have been shown for salmonids when including plant proteins, especially soybean (van den Ingh et al 1991; Baeverfjord & Krogdahl 1996; Storebakken et al 2000) Typically, extensive endocytotic activity and high numbers of intracellular vacuoles are found in cells of the distal part of the gut Damages are often characterized by increases in number of mucus-producing goblet cells, intracellular absorptive vacuoles, changed cellular structure of the lamina propria, amount of connective tissue, degree of mucosal folding and infiltration of the ephithelium or lamina propia by inflammatory cells In extreme cases, massive necrosis occurs, a condition referred to as soybeaninduced enteritis (van den Ingh et al 1991; Baeverfjord & Krogdahl 1996) In A cod, some effects in gut morphology were induced by plant protein, especially when dietary plant inclusion exceeded 75% of protein, but the alterations were moderate and involved mostly goblet cells (Olsen et al 2007) The main plant protein ingredient in those diets was, however, highly refined bioprosessed soybean meal with low levels of ANFs One would therefore not expect any major changes caused by bioprocessed soybean meal in A cod Aquaculture Nutrition, 19; 641–650 ª 2013 John Wiley & Sons Ltd gastrointestinal (GI) tract This was supported by Refstie et al (2006b) where an inclusion of 240 g kgÀ1 biopresessed soybean meal did not cause any alterations in intestinal morphology In A cod fed diets, where 100% of the fish meal was replaced with soy protein consentrate, no gut damages were seen (Walker et al 2010) In salmonids, soybeaninduced gut damage is usually related to distal parts of the GI-tract affecting cell types with endocytotic activity and high levels of intracellular vacuoles A cod does not have the same differentiation of cell types, and most of the intestine contains cells that not appear to be endocytotic (Odense & Bishop 1966) The difference between A cod and A salmon may have implications related to the gut sensitivity to ANFs Little research has been carried out regarding micronutrient requirements in on-growing A cod There are some concerns when replacing fish meal with plant proteins regarding the water-soluble vitamins riboflavin, niacin, pantothenic acid and vitamin B12 (Bell & Waagbø 2008) Hansen et al (2007a) showed a reduction in vitamin B12 concentration from 0.12 to 0.01 mg kgÀ1 in diets, when replacing 100% of fish meal with a plant protein blend Because the requirements of these vitamins are not established for A cod, addition levels have to be based on what is known for other marine species (NRC 2011) The only water-soluble vitamin studied in juvenile A cod is vitamin C, showing that 1.5 g A cod had a qualitative requirement for vitamin C (no vitamin C versus 500 mg vitamin C KgÀ1 diet) (Sandnes et al 1989), but the exact level needed to be present to avoid deficiency was not determined Major shifts in fish feed ingredient profiles, especially from fish meal to plant protein meals, call for increased attention to mineral contents and availability in diets, due to fish meal being a good source of several minerals (NRC 2011) Diets high in plant ingredients are expected to increase the need for supplementation The two minerals with highest concentration in fish meal are phosphorus (P) and calcium (Ca) (NRC 2011) The P level in soybean is mostly in the form of phytate which is less bio-available, phytate is also regarded as an ANF Kousoulaki et al (2010) studied growth and tissue mineralization in A cod fed diets holding 4.7–10.4 g P kgÀ1 diet, supplemented with Ca giving a dietary Ca level of 4.1–11.9 g kgÀ1 The results showed that 7.6–10.4 g P kgÀ1 diet had positive effects on fish performance, growth and tissue mineralization A cod Aquaculture Nutrition, 19; 641–650 ª 2013 John Wiley & Sons Ltd diets containing 0.05) Compared with the CD, the fish fed D1 (378 g kgÀ1 of soybean meal) showed clear signs of inflammation in the distal intestine during the experimental period (Figs & 2) The mucosal folds, supranuclear vacuoles, goblet cells and lamina propria were all significantly affected by the inclusion of soybean meal; nevertheless, the subepithelial mucosa was affected by a delay of week The addition of LAB to the diet (D2) decreased the inflammation at day 28, and all histological parameters of inflammation were reduced However, signs of inflammation were still observed, and only the subepithelial mucosa dropped to a normal score In addition, the mucosal folds, lamina propria and subepithelial mucosa parameters of fish fed D2 reached a normal score by day 35 (b) Figure Epithelium of the distal intestine of Atlantic salmon (Salmo salar) (a) Fish fed the control diet (CD) containing 510 g kgÀ1 of fishmeal had normal distal intestinal morphology (b) Fish fed D1 containing 378 g kgÀ1 soybean meal showed clear signs of inflammation after days of feeding Haematoxylin and eosin staining (a and b): 10X MF, mucosal folds; SNV, supranuclear vacuoles; GC, goblet cells; LP, lamina propria; SM, subepithelial mucosa Aquaculture Nutrition 19; 827–836 ª 2013 John Wiley & Sons Ltd Mucosal folds Enteritis score Enteritis score 3 1 Supranuclear vacuoles 0 14 21 28 35 42 14 Enteritis score Enteritis score 35 42 35 42 28 Lamina propria Globet cells 21 Days of feeding Days of feeding 14 21 28 35 42 14 Days of feeding To our knowledge, this is the first report describing this intestinal enteropathology in Chile Recently, we analysed the effect of including 252 g kgÀ1 of vegetable meal (130 g kgÀ1 corn, 60 g kgÀ1 sunflower and 62 g kgÀ1 soybean meal) on the distal intestine of rainbow trout, and we did not observe any signs of inflammation after months of feeding (Navarrete et al 2012) In the present study, we increased the inclusion of soybean meal to 378 g kgÀ1, and we used Atlantic salmon (Salmo salar) to increase the chance of enteritis in the distal intestine of the salmonids Fortunately, we were able to reproduce the morphological alterations of the distal intestine, which were similar to those observed previously (Uran et al 2008b, 2009), although with a lesser magnitude of inflammation (Ur an et al 2009) Interestingly, we observed that some histological parameters normalized after 28 days of feeding, although signs of inflammation were detected until day 35 Previous studies have reported that rainbow trout and common carp can adapt to a diet containing soybean meal and that signs of inflammation in these fish are attenuated Aquaculture Nutrition 19; 827–836 ª 2013 John Wiley & Sons Ltd 28 Sub epithelial mucosa Enteritis score Figure Evaluation of the degree of inflammation using the semiquantitative method reported by Uran et al 2009 Fish fed the control diet (CD) containing 510 g kgÀ1 of fishmeal, D1 containing 378 g kgÀ1 soybean meal, or D2 (D1 with the addition of LAB) Intestinal samples were collected at days 0, 7, 14, 21 and 35 21 Days of feeding CD D1 D2 0 14 21 28 35 42 Days of feeding after a period of acclimatization to the diet (Refstie et al 1997; Ur an et al 2008b) This observation has not been reported in Atlantic salmon, except in cases in which the soybean meal was withdrawn from their diet (Baeverfjord & Krogdahl 1996) Genetic differences between the Chilean and Norwegian Atlantic salmon (Salmo salar) species could explain their different biological responses to soybean meal In the present study, the inclusion of LAB in D2 significantly attenuated the effect of soybean meal on lamina propria at day 28 Additionally, the mucosal folds, goblet cells and supranuclear vacuoles improved somewhat with the addition of LAB; however, this effect was not significant (P > 0.05) Using a commercial blend of micro-organisms (including yeast and LAB), one study observed a positive effect on the growth of rainbow trout fry fed 200 g kgÀ1 soybean meal (Sealey et al 2009); in contrast to our results, no detectable benefit of LAB supplementation was observed on the inflammatory status of the fish in their study Recently, the addition of 300 g kgÀ1 bacterial meal Table The total and viable bacterial counts (Log10) per gram of intestinal content were determined using epifluorescence microscopy and culture analysis in fish sampled during the experimental period Total count Viable count Sampling days (Fish sampled) (9) (21) 14 (30) 21 (30) 28 (30) 35 (22) Total = 142 Control diet (CD) Diet (D1) Diet (D2) P- value Control diet (CD) Diet (D1) Diet (D2) P value 7.56 7.53 8.05 8.43 8.66 8.33 8.01 7.58 8.14 8.17 8.17 8.10 7.98 7.63 7.88 8.27 8.25 8.21 >0.05 >0.05 >0.05 >0.05 >0.05 >0.05 5.01 5.41 5.87 6.79 6.51 5.92 5.23 4.95 6.31 6.83 5.42 6.88 5.45 5.68 5.99 6.40 5.43 5.22 >0.05 >0.05 >0.05 >0.05 >0.05 >0.05 from Methylococcus capsulatus has been shown to prevent the development of inflammation provoked by the inclusion of 200 g kgÀ1soybean meal in the diet of Atlantic salmon (Romarheim et al 2011) The authors suggested that the phospholipids and nucleic acids present in the M capsulatus meal may modulate the immune system of the fish; however, more studies will be needed to determine the precise mechanism involved The average numbers of total and viable bacteria in the intestinal content are detailed in Table Compared with the CD, D1 and D2 did not alter the total and viable bacterial counts found in the distal intestine (P > 0.05) (Table 3) Similarly, the bacterial counts of rainbow trout were not affected by the diet (Navarrete et al 2012) The specific composition of the microbiota in the intestine of fish fed the experimental diets was examined by describing the bacterial taxa and estimating the bacterial load (Table 4) The bacteria added to D2, L lactis sp lactis and C maltaromaticum were only recovered from the fish fed D2 at a bacterial load average of 4.93 and 5.12 log10 CFU gÀ1 of intestinal content, respectively (Table 4) An overall determination of the bacterial composition was performed by principal component analysis (PCA) (Fig 3a) The PCA revealed that the microbiota composition was grouped according to the diet, except for day 0, for which all of the intestinal samples shared the same bacterial composition Table The total viable counts (Log10) of identified bacterial species per gram of intestinal content in fish sampled during the experimental period Control diet (CD) Diet (D1) Diet (D2) Bacteria identified RFLP Pattern 14 21 28 35 14 21 28 35 14 21 28 35 Lactococcus lactis subsp lactis Carnobacterium Shewanella Lactococcus lactis sp cremoris Aeromonas Microbacterium Pseudomonas Citrobacter freundii Bacillus Aeromonas Sporosarcina aquimarina Staphylococcus Acinetobacter Alcaligenes I – – – – – – – – – – – – – 5.41 5.47 5.33 3.92 4.43 V II I – 4.99 2.19 – 5.41 1.20 – 5.64 4.67 – 6.79 3.08 – 6.07 4.38 – 5.87 – 5.01 3.56 – 4.72 – – 6.31 – – 6.56 – – 5.42 – – 6.65 4.16 – 5.23 3.20 5.02 5.05 – 4.99 5.75 – 5.09 5.93 – 4.99 5.20 – 3.41 5.13 – VIa III IVa VII 3.36 – – – – 2.90 2.08 – 3.79 5.39 – – – – – 2.12 6.31 – – – 4.76 – – – 4.80 – – – – – – – – – – – – – – – – – – – 3.37 – – – 5.04 – – – – – – – – – – – – – – – – – – – – – – IVb VIb VIIIa – – – – – – – – – – – – – – – – – – – – – 2.08 – – – – – – 6.49 2.22 – – – – 6.49 – – – – – – – – – – – – – – – – – – – X VIIIb IX – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – 2.26 – – – – – – – – – – – – – – – – 6.13 – – – – 2.82 – Aquaculture Nutrition 19; 827–836 ª 2013 John Wiley & Sons Ltd Variables (axes F1 y F2: 41.48%) (a) S.aquimarina Ae VIb 0.75 Diet-D1 Enteritis 0.5 Staphylo Bacillus F2 (15.15%) 0.25 Cf Ae VIa Shew Diet-D2 Acineto L.cremoris –0.25 no enteritis –0.5 L lactis Carno Alcaligenes Diet-C Microb Pseudo –0.75 –1 –1 –0.75 –0.5 –0.25 0.25 0.5 0.75 F1 (26.33%) Observations (axes F1 and F2: 41.48%) (b) D1day21 F2 (15.15%) D1day35 D1day28 D1day7 CDday35 D1day0 D1day14 D2day0 CDday0 CDday21 D2day7 D2day21 CDday28 D2day14 D2day35 D2day28 CDday14 –1 –2 –3 –4 –7 CDday7 –6 –5 –4 –3 –2 –1 F1 (26.33%) Figure Principal component analysis (PCA) (a) of the intestinal microbiota of fish fed the control diet (CD) containing 510 g kgÀ1 fishmeal, D1 containing 378 g kgÀ1 soybean meal, and D2 (D1 supplemented with LAB) The intestinal samples were collected at days 0, 7, 14, 21 and 35 (b) Correlation circle of the PCA Ae VIa: Aeromonas RFLP pattern VIa; Ae VIb: Aeromonas RFLP pattern VIb; S aquimarina, Sporosarcina aquimarina; Staphylo, Staphylococcus; Shew, Shewanella; Acineto, Acinetobacter; L lactis, Lactococcus lactis sp lactis; Carno, Carnobacterium maltaromaticum; Pseudo, Pseudomonas; Microb, Microbacterium; L cremoris, Lactococcus lactis sp cremoris Some bacterial groups were recovered exclusively from the guts of fish fed with the three diets (Table and Fig 3b) The correlation generated from PCA results (Fig 3b) showed a projection of the variables in the factor space In this projection, when two variables are far from the centre, the following correlations are observed: if these variables are close to each other, they are significantly positively correlated (r close to 1); if these variables are orthogonal, they are not correlated (r close to 0); and if these variables are on the opposite side of the centre, they Aquaculture Nutrition 19; 827–836 ª 2013 John Wiley & Sons Ltd are significantly negatively correlated (r close to -1) In this context, the main observation was that no bacterial group was significantly correlated with the development of enteritis (Fig 3b) In contrast, the presence of the Microbacterium, Pseudomonas, L lactis sp cremoris and Aeromonas VIa RFLP patterns, which were positively correlated with the CD, was negatively correlated with the development of enteritis Figure 3b confirms that specific bacterial groups are correlated with some diets, as follows: Aeromonas VIb RFLP pattern and S aquimarina are positively correlated with D1, whereas Alcaligenes, Acinetobacter, L lactis sp lactis and C maltaromaticum are positively correlated with D2 Interestingly, Shewanella was not affected by the diet and was present in all of the intestinal samples (Table 4) Bacillus and Staphylococcus were only recovered from the fish fed D1; however, these species were poorly correlated with this diet (Fig 3b) These results are consistent with those of previous studies, which showed that the inclusion of soybean meal produced differences in the cultivable intestinal microbiota (Heikkinen et al 2006; Ringo et al 2006; Bakke-McKellep et al 2007; Merrifield et al 2009) As described in previous studies in salmonids, we found Shewanella to be a dominant bacterium of the intestinal microbiota, regardless of the diet (Romero & Navarrete 2006; Navarrete et al 2010) This result may be due to the wide range of metabolic capabilities of this bacterial genus, which has been recently proposed as a probiotic in fish and shrimp (Zadeh et al 2010; Garcıa de la Banda et al 2012) In contrast, Lactococcus has previously been described as a common component of the intestinal content of salmonids (Hovda et al 2007; Navarrete et al 2010); however, this genus was mainly detected in the CD Interestingly, Lactococcus was present at the beginning of the treatment with D1 and D2, but became undetectable by day 7, at which point inflammation was also observed Strains of Lactococcus have been proposed to be probiotic; however, some studies have suggested that the protection and stimulation of the immune system conferred by this strain are limited compared that of the Lactobacilli strains (Perez-S anchez et al 2011) Aeromonas and Pseudomonas have also been thought to be important in salmonids and other cultivated fish (Romero & Navarrete 2006; Wu et al 2010), and some strains have also been proposed to be probiotic based on their ability to stimulate the innate immune system of zebrafish (Rawls et al 2004, 2007; Kiron 2012) As previously described, several RFLP profiles of the aeromonas group may be present in the fish gut (Navarrete et al 2008) and may have the ability to adapt to changes in the fish diet Sporosarcina has been previously assigned to the genus Bacillus (Yoon et al 2001) The genus Sporosarcina is widely distributed and has been isolated from sea water in Korea (Yoon et al 2001) and the Arabian Gulf (El-Tayeb & Abed 1999), as well as the digestive tracts of fish (Ringo et al 2008; Silva et al 2011) However, limited information is known about its contribution to host nutrition and health To date, no specific relationship has been established between salmon enteritis and the presence of a particular bacterial group in the intestine However, the relationship between bacteria and inflammation is clearer in mammals The presence of intestinal bacteria is required to reproduce intestinal disorders in almost all rodent models of intestinal inflammation; inflammation does not occur in germfree conditions (Mueller & Macpherson 2006) Moreover, in a rodent model of colitis, the use of antibiotics has been shown to attenuate the inflammation (Madsen et al 2000) This latter observation has not been reproduced in fish, as the addition of the antibiotic oxytetracycline to a fish diet containing soybean meal was not found to have any protective effect against the development of enteritis in the Atlantic salmon, despite a significant decrease in the adherent bacterial counts (Bakke-McKellep et al 2007) In conclusion, some bacterial groups were correlated with a specific diet, but none was correlated with soybean meal–induced inflammation in the Atlantic salmon The addition of LAB to the soybean diet (D2) decreased this inflammation, but did not abolish it The role of the intestinal microbiota in the enteritis aetiology of fish therefore will require further study This study was supported by FONDECYT N°1110253 and N°3100075 We thank EWOS for experimental diets formulation and Universidad de los Lagos for diets elaboration Baeverfjord, G & Krogdahl, A (1996) Development and regression of soybean meal induced enteritis in Atlantic salmon, Salmo salar L, distal intestine: a comparison with the intestines of fasted fish J Fish Dis., 19, 375–387 Bakke, A (2011) Pathophysiological and immunological characteristics of soybean meal-induced enteropathy in salmon: contribution of recent molecular investigations In: Avances en nutrici on acuıcola XI - Memorias del Decimo Primer Simposio Internacional de Nutrici on Acuıcola (Cruz-Suarez, L.E ed.) XI International Symposium on Aquaculture Nutrition, Mexico Bakke-McKellep, A.M., Penn, M.H., Salas, P.M., Refstie, S., Sperstad, S., Landsverk, T., Ringo, E & Krogdahl, A (2007) Effects of dietary soyabean meal, inulin and oxytetracycline on intestinal microbiota and epithelial cell stress, apoptosis and proliferation in the teleost Atlantic salmon (Salmo salar L.) Brit J Nutr., 97, 699–713 Balcazar, J.L., De Blas, I., Ruiz-Zarzuela, I., Vendrell, D., Calvo, A.C., Marquez, I., Girones, O & Muzquiz, J.L (2007) Changes in intestinal microbiota and humoral immune response following probiotic administration in brown trout (Salmo trutta) Brit J Nutr., 97, 522–527 Bates, J.M., Mittge, E., Kuhlman, J., Baden, K.N., Cheesman, S.E & Guillemin, K (2006) Distinct signals from the microbiota promote different aspects of zebrafish gut differentiation Dev Biol., 297, 374–386 Buttle, L.G., Burrells, A.C., Good, J.E., Williams, P.D., Southgate, P.J & Burrells, C (2001) The binding of soybean agglutinin (SBA) to the intestinal epithelium of Atlantic salmon (Salmo salar) and rainbow trout (Oncorhynchus mykiss), fed high levels of soybean meal Vet Immunol Immunop., 80, 237–244 Carter, C.G & Hauler, R.C (2000) Fish meal replacement by plant meals in extruded feeds for Atlantic salmon, Salmo salar L Aquaculture, 185, 299–311 Chikwati, E.M., Venold, F.F., Penn, M.H., Rohloff, J., Refstie, S., Guttvik, A., Hillestad, M & Krogdahl, A (2012) Interaction of soyasaponins with plant ingredients in diets for Atlantic salmon, Salmo salar L Brit J Nutr., 107, 1570–1590 Corthesy, B., Gaskins, H.R & Mercenier, A (2007) Cross-talk between probiotic bacteria and the host immune system J Nutr., 137, 781S–790S Dunn, O.J & Clark, V.A (2009) Nonparametric statistics In Basic Statistics, pp 189–200 John Wiley & Sons, Inc., Hoboken, NJ, USA El-Tayeb, S.M & Abed, K.F (1999) The effects of certain nutritional additives on the activity of Sporosarcina ureae in biodegrading oil in the Arabian Gulf water Arab Gulf J Sci Res., 17, 303–321 Francis, G., Makkar, H.P.S & Becker, K (2001) Antinutritional factors present in plant-derived alternate fish feed ingredients and their effects in fish Aquaculture, 199, 197–227 Garcıa de la Banda, I., Lobo, C., Chabrill on, M., Le on-Rubio, J.M., Arijo, S., Pazos, G., Marıa Lucas, L & Mori~ nigo, M.A (2012) Influence of dietary administration of a probiotic strain Shewanella putrefaciens on senegalese sole (Solea senegalensis, Kaup 1858) growth, body composition and resistance to Photobacterium damselae subsp piscicida Aquacult Res., 43, 662–669 Gatesoupe, F.J (2008) Updating the importance of lactic acid bacteria in fish farming: natural occurrence and probiotic treatments J Mol Microb Biotech., 14, 107–114 Gatlin, D.M., Barrows, F.T., Brown, P et al (2007) Expanding the utilization of sustainable plant products in aquafeeds: a review Aquacult Res., 38, 551–579 Heikkinen, J., Vielma, J., Kemilainen, O., Tiirola, M., Eskelinen, P., Kiuru, T., Navia-Paldanius, D & von Wright, A (2006) Effects of soybean meal based diet on growth performance, gut histopathology and intestinal microbiota of juvenile rainbow trout (Oncorhynchus mykiss) Aquaculture, 261, 259–268 Hendriks, H.G.C.J.M., Vandeningh, T.S.G.A.M., Krogdahl, A., Olli, J & Koninkx, J.F.J.G (1990) Binding of soybean agglutinin to small intestinal brush-border membranes and brush-border membrane enzyme-activities in Atlantic salmon (Salmo salar) Aquaculture, 91, 163–170 Aquaculture Nutrition 19; 827–836 ª 2013 John Wiley & Sons Ltd Hovda, M.B., Lunestad, B.T., Fontanillas, R & Rosnes, J.T (2007) Molecular characterisation of the intestinal microbiota of farmed Atlantic salmon (Salmo salar L.) Aquaculture, 272, 581– 588 Kaushik, S.J., Cravedi, J.P., Lalles, J.P., Sumpter, J., Fauconneau, B & Laroche, M (1995) Partial or total replacement of fishmeal by soybean protein on growth, protein-utilization, potential estrogenic or antigenic effects, cholesterolemia and flesh quality in rainbow trout, Oncorhynchus mykiss Aquaculture, 133, 257– 274 Kiron, V (2012) Fish immune system and its nutritional modulation for preventive health care Anim Feed Sci Technol., 173, 111–133 Knudsen, D., Øyvind, R., Baardsen, G., Smedsgaard, J., Wolfgang, K & Frøkiæ, H (2006) Soyasaponins resist extrusion cooking and are not degraded during gut passage in Atlantic salmon (Salmo salar L.) J Agricult Food Chem., 54, 6428–6435 Knudsen, D., Jutfelt, F., Sundh, H., Sundell, K., Koppe, W & Frokiaer, H (2008) Dietary soya saponins increase gut permeability and play a key role in the onset of soyabean-induced enteritis in Atlantic salmon (Salmo salar L.) Brit J Nutr., 100, 120–129 Krogdahl, A., Penn, M., Thorsen, J., Refstie, S & Bakke, A.M (2010) Important antinutrients in plant feedstuffs for aquaculture: an update on recent findings regarding responses in salmonids Aquacult Res., 41, 333–344 Liu, Y.Y., Fatheree, N.Y., Mangalat, N., Dingle, B.M., Tran, D.Q & Rhoads, J.M (2011) Lactobacillus reuteri DSM 17938 reduces inflammation in experimental necrotizing enterocolitis and increases regulatory T cells in mensenteric lymph nodes Gastroenterology, 140, S848–S848 Madsen, K.L., Doyle, J.S., Tavernini, M.M., Jewell, L.D., Rennie, R.P & Fedorak, R.N (2000) Antibiotic therapy attenuates colitis in interleukin 10 gene-deficient mice Gastroenterology, 118, 1094–1105 Merrifield, D.L., Dimitroglou, A., Bradley, G., Baker, R.T.M & Davies, S.J (2009) Soybean meal alters autochthonous microbial populations, microvilli morphology and compromises intestinal enterocyte integrity of rainbow trout, Oncorhynchus mykiss (Walbaum) J Fish Dis., 32, 755–766 Mueller, C & Macpherson, A.J (2006) Layers of mutualism with commensal bacteria protect us from intestinal inflammation Gut, 55, 276–284 Navarrete, P., Mardones, P., Opazo, R., Espejo, R & Romero, J (2008) Oxytetracycline treatment reduces bacterial diversity of intestinal microbiota of Atlantic salmon J Aquat Anim Health, 20, 177–183 Navarrete, P., Espejo, R.T & Romero, J (2009) Molecular analysis of microbiota along the digestive tract of juvenile Atlantic salmon (Salmo salar L.) Microb Ecol., 57, 550–561 Navarrete, P., Magne, F., Mardones, P., Riveros, M., Opazo, R., Suau, A., Pochart, P & Romero, J (2010) Molecular analysis of intestinal microbiota of rainbow trout (Oncorhynchus mykiss) FEMS Microbiol Ecol., 71, 148–156 Navarrete, P., Magne, F., Araneda, C., Fuentes, P., Barros, L., Opazo, R., Espejo, R & Romero, J (2012) PCR-TTGE analysis of 16S rRNA from rainbow trout (Oncorhynchus mykiss) gut microbiota reveals host-specific communities of active bacteria PLoS ONE, 7, e31335 Panigrahi, A., Kiron, V., Satoh, S., Hirono, I., Kobayshi, T., Sugita, H., Puangkaew, J & Aoki, T (2007) Immune modulation and expression of cytokine genes in rainbow trout Oncorhynchus Aquaculture Nutrition 19; 827–836 ª 2013 John Wiley & Sons Ltd mykiss upon probiotic feeding Dev Comp Immunol., 31, 372– 382 Perez-Sanchez, T., Balcazar, J.L., Merrifield, D.L., Carnevali, O., Gioacchini, G., de Blas, I & Ruiz-Zarzuela, I (2011) Expression of immune-related genes in rainbow trout (Oncorhynchus mykiss) induced by probiotic bacteria during Lactococcus garvieae infection Fish Shellfish Immunol., 31, 196–201 Pusztai, A & Bardocz, S (1996) Biological effects of plant lectins on the gastrointestinal tract: metabolic consequences and applications Trends Glycosci Glyc., 8, 149–165 Rawls, J.F., Samuel, B.S & Gordon, J.I (2004) Gnotobiotic zebrafish reveal evolutionarily conserved responses to the gut microbiota P Natl Acad Sci USA, 101, 4596–4601 Rawls, J.F., Mahowald, M.A., Ley, R.E & Gordon, J.I (2006) Reciprocal gut microbiota transplants from zebrafish and mice to germ-free recipients reveal host habitat selection Cell, 127, 423–433 Rawls, J.F., Mahowald, M.A., Goodman, A.L., Trent, C.M & Gordon, J.I (2007) In vivo imaging and genetic analysis link bacterial motility and symbiosis in the zebrafish gut Proc Natl Acad Sci USA, 104, 7622–7627 Refstie, S., Helland, S.J & Storebakken, T (1997) Adaptation to soybean meal in diets for rainbow trout, Oncorhynchus mykiss Aquaculture, 153, 263–272 Ringo, E., Sperstad, S., Myklebust, R., Refstie, S & Krogdahl, A (2006) Characterisation of the microbiota associated with intestine of Atlantic cod (Gadus morhua L.): the effect of fish meal, standard soybean meal and a bioprocessed soybean meal Aquaculture, 261, 829–841 Ringo, E., Sperstad, S., Kraugerud, O.F & Krogdahl, A (2008) Use of 16S rRNA gene sequencing analysis to characterize culturable intestinal bacteria in Atlantic salmon (Salmo salar) fed diets with cellulose or non-starch polysaccharides from soy Aquacult Res., 39, 1087–1100 Romarheim, O.H., Overland, M., Mydland, L.T., Skrede, A & Landsverk, T (2011) Bacteria grown on natural gas prevent soybean meal-induced enteritis in Atlantic salmon J Nutr., 141, 124–130 Romero, J & Espejo, R (2001) The prevalence of noncultivable bacteria in oysters (Tiostrea chilensis, Philippi, 1845) J Shellfish Res., 20, 1235–1240 Romero, J & Navarrete, P (2006) 16S rDNA-based analysis of dominant bacterial populations associated with early life stages of coho salmon (Oncorhynchus kisutch) Microb Ecol., 51, 422– 430 Rumsey, G.L., Siwicki, A.K., Anderson, D.P & Bowser, P.R (1994) Effect of soybean protein on serological response, nonspecific defense mechanisms, growth, and protein utilization in rainbow trout Vet Immunol Immunopath., 41, 323–339 Sealey, W.M., Barrows, F.T., Smith, C.E., Overturf, K & LaPatra, S.E (2009) Soybean meal level and probiotics in first feeding fry diets alter the ability of rainbow trout Oncorhynchus mykiss to utilize high levels of soybean meal during grow-out Aquaculture, 293, 195–203 Silva, F.C., Nicoli, J.R., Zambonino-Infante, J.L., Kaushik, S & Gatesoupe, F.J (2011) Influence of the diet on the microbial diversity of faecal and gastrointestinal contents in gilthead sea bream (Sparus aurata) and intestinal contents in goldfish (Carassius auratus) FEMS Microbiol Ecol., 78, 285–296 Talpur, A.D., Memon, A.J., Khan, M.I., Ikhwanuddin, M., Danish, M.M & Daniel & Abol-Munafi, A.B., (2012) Inhibition of pathogens by lactic acid bacteria and application as water additive multi isolates in early stages larviculture of P Pelagicus (Linnaeus, 1758) J Anim Plant Sci., 22, 54–64 Ur an, P.A., Goncalves, A.A., Taverne-Thiele, J.J., Schrama, J.W., Verreth, J.A.J & Rombout, J.H.W.M (2008a) Soybean meal induces intestinal inflammation in common carp (Cyprinus carpio L.) Fish Shellfish Immun., 25, 751–760 Ur an, P.A., Schrama, J.W., Rombout, J.H.W.M., Obach, A., Jensen, L., Koppe, W & Verreth, J.A.J (2008b) Soybean mealinduced enteritis in Atlantic salmon (Salmo salar L.) at different temperatures Aquacult Nutr., 14, 324–330 Ur an, P.A., Schrama, J.W., Rombout, J.H.W.M., Taverne-Thiele, J.J., Obach, A., Koppe, W & Verreth, J.A.J (2009) Timerelated changes of the intestinal morphology of Atlantic salmon, Salmo salar L., at two different soybean meal inclusion levels J Fish Dis., 32, 733–744 V azquez, J.A., Gonzalez, M.P & Murado, M.A (2005) Effects of lactic acid bacteria cultures on pathogenic microbiota from fish Aquaculture, 245, 149–161 Vendrell, D., Balcazar, J.L., de Blas, I., Ruiz-Zarzuela, I., Girones, O & Muzquiz, J.L (2008) Protection of rainbow trout (Oncorhynchus mykiss) from lactococcosis by probiotic bacteria Comp Immunol Microb., 31, 337–345 Verschuere, L., Rombaut, G., Sorgeloos, P & Verstraete, W (2000) Probiotic bacteria as biological control agents in aquaculture Microbiol Mol Biol R., 64, 655–671 Wu, S., Gao, T., Zheng, Y., Wang, W., Cheng, Y & Wang, G (2010) Microbial diversity of intestinal contents and mucus in yellow catfish (Pelteobagrus fulvidraco) Aquaculture, 303, 1–7 Yoon, J.H., Lee, K.C., Weiss, N., Kho, Y.H., Kang, K.H & Park, Y.H (2001) Sporosarcina aquimarina sp nov., a bacterium isolated from seawater in Korea, and transfer of Bacillus globisporus (Larkin and Stokes 1967), Bacillus psychrophilus (Nakamura 1984) and Bacillus pasteurii (Chester 1898) to the genus Sporosarcina as Sporosarcina globispora comb nov., Sporosarcina psychrophila comb nov and Sporosarcina pasteurii comb nov., and emended description of the genus Sporosarcina Int J Syst Evol Microbiol., 51, 1079– 1086 Zadeh, S.S., Saad, C.R., Christianus, A., Kamarudin, M.S., Sijam, K., Shamsudin, M.N & Neela, V.K (2010) Assessment of growth condition for a candidate probiotic, Shewanella algae, isolated from digestive system of a healthy juvenile Penaeus monodon Aquacult Int., 18, 1017–1026 Zhao, Y., Qin, G.X., Sun, Z.W., Che, D.S., Bao, N & Zhang, X.D (2011) Effects of soybean agglutinin on intestinal barrier permeability and tight junction protein expression in weaned piglets Int J Mol Sci., 12, 8502–8512 Aquaculture Nutrition 19; 827–836 ª 2013 John Wiley & Sons Ltd Aquaculture Nutrition 2013 19; 837–844 doi: 10.1111/anu.12048 Instituto de Acuicultura Torre de la Sal (IATS-CSIC), Ribera de Cabanes, Castell on, Spain This contribution was presented at the XI International Symposium on Aquaculture Nutrition in Mexico (Guest Editor Dr Luis H ector Hern andez Hern andez) Highly unsaturated fatty acids (HUFA), like the eicosapentaenoic (EPA) and docosahexaenoic (DHA) acids and polar lipids (essentially phospholipids, PL) have been identified as essential nutrients for common octopus (Octopus vulgaris) paralarvae However, they are not available in sufficient amounts in live preys as Artemia, making necessary a supplementation of these nutrients previous use A commercial emulsion, soya liposomes, and marine and soya lecithins were used to supply HUFA and PL to Artemia metanauplii, those being regarded as suitable size preys for octopus paralarvae Our results prove that a simultaneous enrichment in HUFA and PL is possible using enrichment diets combining HUFA- and PL-rich products in short-term (4 h) incubations Particularly interesting was the enrichment efficiency shown by the marine lecithin, which enabled the enhancement of the PL fraction of Artemia metanauplii and, importantly, also their HUFA with a remarkable 13% DHA of total fatty acids Marine lecithin arises as a novel enrichment diet for Artemia and more effective than some commercial products currently used in hatcheries worldwide KEY WORDS: Artemia metanauplii, enrichment, highly unsaturated fatty acids, Octopus vulgaris, phospholipids Received 20 August 2012; accepted 30 December 2012 Correspondence: F Hontoria, Instituto de Acuicultura Torre de la Sal (IATS-CSIC), 12595 Ribera de Cabanes, Castell on, Spain E-mail: hontoria@iats.csic.es The common octopus (Octopus vulgaris) is a promising candidate to diversify species in the Mediterranean aqua- ª 2013 John Wiley & Sons Ltd culture for its rapid growth, elevated food conversion index and its great commercial interest (Vaz-Pires et al 2004) Octopus culture has therefore become an attractive area of research, and extensive investigations devoted to understand diverse aspects of its biology have now made possible to on-grow wild-captured specimens in floating cages to commercial size (Iglesias et al 2006) However, the octopus life cycle in captivity has not been closed yet, as massive mortalities during metamorphosis of early pelagic life stages (paralarvae) to benthic stages occur (Iglesias et al 2007; Villanueva & Norman 2008) Nutritional studies have highlighted the importance that some dietary components, including proteins and amino acids (Villanueva et al 2004), essential and non-essential elements (Villanueva & Bustamante 2006) and vitamins (Villanueva et al 2009), have to alleviate paralarval mortalities (Villanueva et al 2004) Moreover, Navarro & Villanueva (2000, 2003) studied the lipid requirements of early stages of cephalopods to conclude that suboptimal levels of polar lipids (essentially phospholipids, PL) and highly unsaturated fatty acids (HUFA) like eicosapentaenoic (EPA) and docosahexaenoic (DHA) acids in live preys such Artemia might be responsible for the high mortalities encountered in paralarvae Newly hatched Artemia nauplii are arguably the most commonly used live prey in marine finfish and crustacean larviculture (Sorgeloos et al 2001) However, their suitability as diet for marine larvae has been often questioned due to their relatively low PL and HUFA contents in comparison with natural preys (Navarro et al 1993) Beneficial aspects derived from dietary PL and HUFA have been reported on survival, growth and development of marine larvae (Sargent et al 1997; Tocher et al 2008) To enhance the nutritional quality of Artemia nauplii, enrichment protocols have been optimized (Navarro et al 1999; Han et al 2001) Whereas enrichment of HUFA has been successfully achieved with the use of commercial products such as marine oil-based emulsions (Leger et al 1986; Han et al 2000; Copeman et al 2002) or spray-dried cells of Schizochytrium sp (Barclay & Zeller 1996), enhancement of PL contents in Artemia (McEvoy et al 1995; Monroig et al 2003, 2006) and also rotifers (Rainuzzo et al 1994) has been posed difficult due in part to rapid metabolic conversions inside the living preys to other lipid classes such as triacylglycerides (TAG) Thus, liposomes, lipid vesicles with high PL contents, produced modest increases in the PL fraction when used as enrichment diets for Artemia nauplii (McEvoy et al 1995; Monroig et al 2003, 2006) Barr et al (2005) also reported enhanced levels of PL in rotifers and Artemia nauplii that, after conventional HUFA enrichment procedures, were subsequently subjected to short-term incubations with liposomes formulated with soy phosphatidylcholine (PC) Whereas the abovementioned studies employed liposomes formulated with purified PL sources normally consisting of PC from either marine (McEvoy et al 1995; Monroig et al 2003, 2006) or terrestrial (Barr et al 2005) origin, the socalled lecithins, often referred to coarse products containing a mixture of phospholipid types or even non-polar lipid classes such as TAG, appear as a cheap alternative to expensive highly purified PC-based PL sources that can be utilized for live prey enrichments at larger scale On-grown (metanaupliar) stages of Artemia have been regarded as more adequate preys for octopus paralarvae (Iglesias et al 2006) Similarly to naupliar stages, Artemia metanauplii also have detrimental lipid profiles for octopus paralarvae, and experimental trials have partly solved the problem by on-growing Artemia in presence of marine phytoplankton (Iglesias et al 2004; Viciano et al 2011) While phytoplankton is not readily available and its use involved large-scale facilities, cheaper alternative protocols addressed to improve the nutritional value of Artemia metanauplii are required We here report a series of experiments aiming at establishing optimized protocols for the simultaneous enrichment of PL and HUFA contents of Artemia metanauplii for their potential use as live preys in common octopus paralarvae culture Artemia franciscana metanauplii were obtained from the hatching of Great Salt Lake cysts purchased to INVE Aquaculture Nutrition (Dendermonde, Belgium) Cysts were incubated for 24 h in L cylinder-conical tubes containing seawater at 28 °C and 37 g LÀ1 salinity, vigorous bottom aeration and continuous light After hatching, nauplii were placed in 90 L cylindrical methacrylate containers with seawater and maintained at room temperature and a density of 4000 individuals LÀ1 Nauplii were fed microalgae Tetraselmis suecica ad libitum (around 200 000 cells mLÀ1) Fresh microalgae cultures were daily added to maintain cell density Artemia metanauplii were grown for days, attaining a mean length of 1.47 mm and then used in the different enrichment experiments Multilamellar liposomes were formulated with soya PC from Avanti Polar Lipids Inc (Alabaster, AL, USA) and cholesterol (Sigma-Aldrich Quımica S.A., Alcobendas, Spain) included as a membrane stabilizer at a : w/w ratio (cholesterol:PC) Liposomes were prepared according to the method proposed by Bangham et al (1965), but using filtered seawater as the aqueous phase as previously described (Monroig et al 2003, 2006) Briefly, the lipid mixture was dried under nitrogen flux in a thin layer on the bottom of a flask and rehydrated with the aqueous phase for h by vortexing frequently until a homogenous suspension was achieved The commercial emulsion Easy DHA Selco (INVE Aquaculture Nutrition, Dendermonde, Belgium) with 15% DHA was self-dispersed in seawater to obtain a suspension following supplier’s instructions Marine lecithin LC60 (PhosphoTech Laboratories, St Herblain, France) containing 680 mg g wwÀ1 of lipids (~50% being PL) with 13% and 33% of total fatty acids (FA) as EPA and DHA, respectively, was used after dispersion in seawater with a domestic blender Soya lecithin (Korot SL, Alcoy, Spain), with 740 mg g wwÀ1 of lipids (~80% being PL) with 48% of total FA as linoleic acid (LOA) and lacking EPA and DHA, was prepared following the same procedure described for Marine lecithin LC60 A series of three experiments was conducted to explore practical strategies to enhance the contents of both HUFA and PL in Artemia metanauplii Experiment aimed to enrich metanauplii in HUFA or PL separately The objective of Experiment was to ascertain whether HUFA and PL could be supplied simultaneously Experiment was used to enrich metanauplii in HUFA and PL using the Aquaculture Nutrition 19; 837–844 ª 2013 John Wiley & Sons Ltd alternative cheaper products lecithins All the enrichment experiments were carried out by placing ~30 000 days old metanauplii in a L cylinder-conical tube containing 0.5 L of filtered seawater at 28 °C, strong aeration from the bottom and continuous light Experiment In Experiment 1, carried out to enhance either the PL or HUFA contents of Artemia metanauplii, two experimental treatments were established: Treatment 1A, consisting of an enrichment with the commercial emulsion Easy DHA Selco and aiming to provide HUFA to metanauplii; and Treatment 1B, consisting of liposomes formulated with soya PC and cholesterol and used to improve PL contents of metanauplii To establish the incorporation dynamics of both enrichment diets and, therefore, the optimal duration of the incubation, metanauplii samples were collected after 2, 4, and 24 h of exposure and immediately stored at À80 °C until further analysis Both enrichment products were dispensed at concentrations of 0.6 g LÀ1 Experiment The objective of Experiment was to produce metanauplii simultaneously enriched in PL and HUFA Three different enrichment treatments were assayed: Treatment 2A, consisting of the same commercial emulsion (Easy DHA Selco) dispensed at a concentration of 0.6 g LÀ1 during h; Treatment 2B, consisting of a first incubation with Easy DHA Selco for h dispensed at 0.6 g LÀ1, followed by a medium (filtered seawater) renewal and a subsequent 2-h enrichment with soya liposomes at 0.6 g LÀ1; and Treatment 2C, consisting of a mixture of liposomes (0.3 g LÀ1) and Easy DHA Selco (0.3 g LÀ1), both dispensed simultaneously at the beginning of the incubation and maintained for h Metanauplii samples were collected after h and stored at À80 °C for further analyses Experiment This experiment, while aiming at the simultaneous provision of PL and HUFA to Artemia metanauplii like Experiment 2, assessed the use of lecithins, readily available coarse materials with potential use beyond experimental scale Three enrichment treatments were established: Treatment 3A, consisting of a dispersion of Marine lecithin LC60 at 0.6 g LÀ1; Treatment 3B, being a mixture of dispersed marine lecithin LC60 (0.3 g LÀ1) and Easy DHA Selco (0.3 g LÀ1) dispensed at the beginning of the incubation; and Treatment 3C, consisting of a mixture of dispersed soya lecithin (Korot SL) (0.3 g LÀ1) and Easy DHA Selco (0.3 g LÀ1) dispensed at the beginning of the incubation Metanauplii samples were collected after h and immediately stored at À80 °C for further analyses Aquaculture Nutrition 19; 837–844 ª 2013 John Wiley & Sons Ltd Total lipids and FA were determined as described by Monroig et al (2006) Briefly, total lipids from liophylized metanauplii samples were extracted with chloroform/methanol (2 : v/v) according to the method of Folch et al (1957) FA determinations were carried out according to the methodology described by Christie (1982) FA methyl esters (FAME) were extracted with n-hexane/diethyl ether (1 : 1, v/v) and purified by thin-layer chromatography (Silica gel G 60; Merck, Darmstadt, Germany) using a mixture of n-hexane/diethyl ether/acetic acid (85 : 15: 1.5, v/v/v) as solvent system The analyses of FAME were performed with a Thermo (Thermo Trace GC Ultra; Thermo Electron Corporation, Waltham, MA, USA) gas chromatograph equipped with a fused silica 30 m 0.25 mm open tubular column (Tracer, TR-WAX, film thickness: 0.25 lm; Teknokroma, Barcelona, Spain) and a cold on-column injection system Helium was used as carrier, and a 50–220 °C thermal gradient was established during the running of samples A personal computer system equipped with Azur Datalys (St Martin d’Heres, France) software was used in the recording and processing the data proceeding from the flame-ionization detector Peaks were determined by comparison with known standards FA contents in the enrichment products and Artemia metanauplii were expressed as percentages of total FA Phospholipids were estimated through the quantification of the inorganic phosphorous (Pi) of the total lipid fraction according to Zhou & Arthur (1992) and with the following modifications Total lipid aliquots (50 lg) in duplicates were placed into assay glass tubes, and the solvents were evaporated off under nitrogen flux After addition of 0.2 mL of perchloric acid (37% purity), samples were heated at 180 °C for h After cooling, 0.2 mL of distilled water and mL of working solution containing malachite green were added Absorbance at 660 nm was then measured in a U-2001 Spectrophotometer (Hitachi, Tokio, Japan) Pi concentrations (lg g dwÀ1) were calculated according to calibration curves constructed with KH2PO4 standard solutions of 1, 2.5, 5, 7.5 and 10 lg mLÀ1 Statistical analyses were performed with the SPSS for Windows 15.0 statistical package (SPSS Inc., Chicago, IL, USA) Data are expressed as means Æ standard deviations One-way ANOVA were used to assess differences between treatments in Experiments 1–3 Moreover, two-way ANOVA were carried out to identify differences in Experiment where two distinct factors (time of incubation and enrichment diet) were established A posteriori mean comparison Tukey’s tests were utilized when appropriate Comparisons of the means with P-values less or equal than 0.05 were considered significantly different Lipids from days Artemia metanauplii prior to enrichment basically contained the saturates 16 : and 18 : (~10% each), the monoenes 18:1n-9 and 18:1n-7 (17.9% and 11.9%, respectively) and 18:3n-3 (13.7%) Interestingly, basal FA composition of non-enriched metanauplii included 7.2% of EPA and no DHA The exposure of Artemia metanauplii to the enrichment diets from Experiment notably modified their FA composition (Table 1) Differences between the treatments assayed in every FA were significant (two-way ANOVA, P 0.05) in all cases Interestingly, the EPA contents of Treatment 1A metanauplii were significantly higher than those of Treatment 1B metanauplii after only h of exposure (two-way ANOVA, P 0.05) Compared to EPA contents at h, no significant increases in Treatment 1A metanauplii were detected despite longer exposure times (4, and 24 h) (Table 1) Similarly, DHA was rapidly incorporated and only h were required to achieve maximum contents in Treatment 1A Artemia (Table 1) DHA/EPA ratios, calculated only for Treatment 1A as soya liposomes lack DHA, were significantly higher (one-way ANOVA, P 0.001) at 2, and h than at 24 h (Table 1), consistent with a steady decrease in the DHA contents of metanauplii from to 24 h These results indicate that h of exposure is sufficient for delivering EPA and DHA through conventional commercial emulsion into Artemia metanauplii PL enrichment of Artemia metanauplii was also shown to be a rapid process when using liposomes Thus, Treatment 1B metanauplii contained significantly more Pi than metanauplii from Treatment 1A after only h of enrichment (two-way ANOVA, P 0.05) (Fig 1a) The results obtained from Experiment showed that it is possible to simultaneously enrich Artemia metanauplii in PL and HUFA, but increases in one of these components occur in detriment of the other Whereas Treatment 2A metanauplii, treated exclusively with the commercial emulsion Easy DHA Selco, showed the highest EPA and DHA contents among treatments in Experiment (Table 2), their Pi content was significantly lower than those of liposomebased diets (Treatments 2B and 2C) (Fig 1b) However, the increase in Pi in metanauplii treated with liposomes Table Selected fatty acid content (percentage of total fatty acids) in the Artemia metanauplii total lipid fraction collected during Experiment Treatment 1A Time (h) 16:0 18:0 18:1n-9 18:1n-7 18:2n-6 18:3n-3 20:4n-6 20:5n-3 22:6n-3 DHA:EPA Ratio Σ% FAME mg of total lipids g dwÀ1 12.9 8.3 19.1 8.9 4.4 9.8 1.3 8.1 6.2 0.8 95.0 15.2 Treatment 1B Æ Æ Æ Æ Æ Æ Æ Æ Æ Æ Æ Æ 0.2a 0.1b 0.2a 0.1a,b 0.2a 0.3b 0.0a 0.1a 0.1b,c 0.0b 0.2a 0.2b 12.7 7.7 19.9 8.6 4.8 9.2 1.3 8.4 6.4 0.8 95.3 14.4 Æ Æ Æ Æ Æ Æ Æ Æ Æ Æ Æ Æ 0.5a 0.1a 0.2b 0.2a 0.1b 0.3a,b 0.0a,b 0.3a 0.1b 0.0b 0.4a 0.3b 12.7 7.7 20.6 8.6 5.1 9.0 1.4 8.5 6.1 0.7 95.5 17.3 24 Æ Æ Æ Æ Æ Æ Æ Æ Æ Æ Æ Æ 0.5a 0.1a 0.2c 0.3a 0.2b 0.3a,b 0.0b 0.3a 0.1c 0.0b 0.3a 0.6b 12.6 8.1 23.5 9.3 5.7 8.8 1.5 8.3 2.6 0.3 94.3 7.0 Æ Æ Æ Æ Æ Æ Æ Æ Æ Æ Æ Æ 0.0a 0.1b 0.0d 0.1b 0.0c 0.2a 0.0c 0.0a 0.2a 0.0a 0.1a 2.5a 11.0 Æ 7.4 Æ 14.7 Æ 7.6 Æ 31.9 Æ 10.9 Æ 0.6 Æ 4.1 Æ ND NA 96.1 Æ 16.4 Æ 0.0b 0.1b 0.0b 0.1c 0.7a 0.1d 0.0b 0.1c 0.2a 1.1a 10.3 Æ 6.8 Æ 14.3 Æ 6.9 Æ 36.8 Æ 10.4 Æ 0.5 Æ 3.7 Æ ND NA 96.7 Æ 18.1 Æ 0.1a 0.2a 0.0ab 0.0b 0.4b 0.1c 0.0a 0.0b,c 0.0b 0.4a 10.1 Æ 6.5 Æ 14.0 Æ 6.68 Æ 39.0 Æ 10.0 Æ 0.5 Æ 3.4 Æ ND NA 96.8 Æ 18.5 Æ 24 0.1a 0.1a 0.0a 0.0a,b 0.3b,c 0.2b 0.0a 0.1b 0.1bc 0.3a 11.1 Æ 7.5 Æ 14.0 Æ 6.5 Æ 40.0 Æ 8.5 Æ 0.5 Æ 3.0 Æ ND NA 97.2 Æ 18.2 Æ 0.3b 0.2b 0.4a 0.3a 1.6c 0.2a 0.0a 0.2a 0.1c 1.5a 1A: Easy DHA Selco 1B: multilamellar liposomes of soya phosphatidylcholine Data represent mean Æ SD (n = 3) Data that not share the same letter among incubation times in the same treatment differ significantly (one-way ANOVA and Tukey’s test, P 0.05) Aquaculture Nutrition 19; 837–844 ª 2013 John Wiley & Sons Ltd Inorganic phosphorous (μg g dw–1) was achieved in detriment of HUFA incorporation, with an obvious dilution effect derived from the incorporation of LOA (18:2n-6) present in liposome soya PC (Table 2) DHA/EPA ratios reflected the enrichment product FA composition and significantly higher values (0.8) were obtained in Treatment 2A compared with liposome-treated nauplii (0.5–0.6) Comparison of liposome-enriched metanauplii (sequential or mixed treatments) showed that Treatment 2B metanauplii contained more PL (Fig 1b), although no differences (one-way ANOVA, P 0.05) in the contents of EPA and DHA were detected (Table 2) (a) c a b b 1A 1B 24 Time (h) Inorganic phosphorous (μg g dw–1) (b) c b a Inorganic phosphorous (μg g dw–1) 2A (c) 2B 2C b ab a The use of lecithins (soya and marine origins) for enriching Artemia metanauplii was explored in Experiment Thus, metanauplii treated with Marine lecithin LC60 (Treatments 3A and 3B) exhibited notable EPA and DHA levels, particularly in Treatment 3A metanauplii with 12.6% (EPA) and 13.1% (DHA) (Table 3) Consequently, DHA/EPA ratio also reached significant values, with the highest (1.0) corresponding to that of Treatment 3A metanauplii Moreover, Treatment 3A metanauplii showed the highest Pi contents, significantly different (one-way ANOVA, P 0.001) compared with the levels of Treatment 3C metanauplii, despite they were treated with soya lecithin (Fig 1c) 3A 3B 3C Figure Inorganic phosphorous contents in the Artemia metanauplii lipid fraction collected from Experiments 1–3 Data represent means and error bars are standard deviations (n = 3) Experiment (panel a) consisted of the following treatments: Treatment 1A, Easy docosahexaenoic (DHA) Selco; Treatment 1B, multilamellar liposomes of soya phosphatidylcholine Experiment (panel b) consisted of the following treatments: Treatment 2A, 4-h enrichment with Easy DHA Selco; Treatment 2B, 2-h enrichment with Easy DHA Selco + 2-h enrichment with soya phosphatidylcholine liposomes; Treatment 2C, 4-h enrichment with a mixture of Easy DHA Selco and soya phosphatidylcholine liposomes Experiment included the following treatments: Treatment 3A, 4-h enrichment with Marine lecithin LC60; Treatment 3B, 4-h enrichment with a mixture of Marine lecithin LC60 and Easy DHA Selco; Treatment 3C, 4-h enrichment with a mixture of soya lecithin and Easy DHA Selco Sampling times that not share the same letter in panel A differ significantly (two-way ANOVA and Tukey’s test, P 0.05) Data that not share the same letter in panels (b and c) differ significantly (one-way ANOVA and Tukey’s test, P 0.05) Aquaculture Nutrition 19; 837–844 ª 2013 John Wiley & Sons Ltd On-grown stages of Artemia (metanauplii) have been regarded as adequate live preys of suitable size for common octopus paralarvae, since eating large-size preys increase the food intake per hunting effort, thus potentially improving growth and survival (Okumura et al 2005; Iglesias et al 2006) Like naupliar stages, however, Artemia metanauplii appear to have suboptimal dietary lipid profiles as live preys for feeding marine organism larvae The present study aimed to develop adequate enrichment protocols for Artemia metanauplii with key lipid nutrients for octopus paralarvae, namely PL and HUFA (Navarro & Villanueva 2000) Our results clearly show that it is possible to obtain Artemia metanauplii enriched in PL and HUFA, if appropriate materials and procedures are considered Results of Experiment demonstrate that individual supply of HUFA or PL into Artemia metanauplii is possible Thus, remarkable contents of HUFA, including EPA and DHA, were obtained by treating Artemia metanauplii with a commercial emulsion like Easy DHA Selco Fish oilbased emulsions are widely used as enrichment diets for Table Selected fatty acid content (percentage of total fatty acids) in the Artemia metanauplii total lipid fraction collected during Experiment Treatment 2A 16:0 18:0 18:1n-9 18:1n-7 18:2n-6 18:3n-3 20:4n-6 20:5n-3 22:6n-3 DHA:EPA Ratio Σ% FAME mg of Total lipids g dwÀ1 13.7 6.4 18.7 6.6 8.5 11.6 2.2 7.5 6.0 0.8 Æ Æ Æ Æ Æ Æ Æ Æ Æ Æ 0.5b 0.1b 0.5b 0.2b 0.4a 1.1a 0.1b 0.3b 0.4b 0.0b Treatment 2B 12.4 5.9 15.6 5.8 27.2 11.3 1.7 5.1 2.6 0.5 94.7 Æ 0.2a 18.3 Æ 0.6a Æ Æ Æ Æ Æ Æ Æ Æ Æ Æ 0.0a 0.2a 0.4a 0.2a 0.2c 1.2a 0.1a 0.3a 0.4a 0.1a 95.6 Æ 1.3a 19.6 Æ 1.3a Treatment 2C 12.9 6.2 14.9 6.0 24.9 11.9 1.6 4.8 2.7 0.6 Æ Æ Æ Æ Æ Æ Æ Æ Æ Æ 0.0a 0.2a,b 0.3a 0.2a 1.4b 1.3a 0.0a 0.1a 0.3a 0.0a 94.0 Æ 0.5a 18.6 Æ 0.8a 2A: 4-h enrichment with Easy DHA Selco 2B: 2-h enrichment with Easy DHA Selco + 2-h enrichment with soya phosphatidylcholine liposomes 2C: 4-h enrichment with a mixture of Easy DHA Selco and soya phosphatidylcholine liposomes Data represent mean Æ SD (n = 3) Data in the same row that not share the same letter differ significantly (one-way ANOVA and Tukey’s test, P 0.05) Table Selected fatty acid content (percentage of total fatty acids) in the Artemia metanauplii total lipid fraction collected during Experiment Treatment 3A 16:0 18:0 18:1n-9 18:1n-7 18:2n-6 18:3n-3 20:4n-6 20:5n-3 22:6n-3 DHA:EPA Ratio Σ% FAME mg of Total lipids g dwÀ1 19.4 7.6 11.7 6.1 2.8 11.2 1.8 12.6 13.1 1.0 95.9 17.6 Æ Æ Æ Æ Æ Æ Æ Æ Æ Æ Æ Æ b 0.4 0.1c 0.0a 0.0a 0.1a 0.5a 0.1b 0.4c 0.3c 0.0c 0.2a 0.1b Treatment 3B 14.2 7.0 18.4 6.5 4.9 11.4 1.8 9.4 8.6 0.9 96.6 17.3 Æ Æ Æ Æ Æ Æ Æ Æ Æ Æ Æ Æ a 0.3 0.1a 0.2b 0.2a 0.1b 0.4a 0.0b 0.2b 0.2b 0.0b 0.1b 0.1a Treatment 3C 14.2 7.2 19.6 6.6 9.1 11.2 1.5 7.6 5.8 0.8 96.8 16.5 Æ Æ Æ Æ Æ Æ Æ Æ Æ Æ Æ Æ 0.2b 0.1b 0.2c 0.3a 0.9c 0.5a 0.1a 0.3a 0.3a 0.0a 0.3b 1.0a 3A: 4-h enrichment with marine lecithin LC60 3B: 4-h enrichment with a mixture of marine lecithin LC60 and Easy DHA Selco 3C: 4-h enrichment with a mixture of soya lecithin and Easy DHA Selco Data represent mean Æ SD (n = 3) Data in the same row that not share the same letter differ significantly (one-way ANOVA and Tukey’s test, P 0.05) Artemia nauplii (Han et al 2001), and the herein reported results clearly show that these products can be efficiently utilized as well for metanauplii enrichments The use of emulsion-based products, however, appears inefficient for delivering PL into Artemia metanauplii, consistently with their formulation based on neutral lipids, in particular TAG (Monroig et al 2006) Liposomes, in contrast, are an efficient means for delivering PL into metanauplii, and Artemia enriched with soya PC liposomes clearly exhibited enhanced PL levels compared with the emulsion-enriched metanauplii Rather than emulsion-treated Artemia, a more adequate control treatment reflecting the basal PL levels of Artemia was unfortunately not available, as non-enriched on-grown metanauplii still retained phytoplankton cells, thus altering Artemia natural PL composition (Ruız et al 2008) Similarly, early studies pointed out that liposomes can enhance the PL fraction of Artemia naupliar stages (McEvoy et al 1996; Monroig et al 2003, 2006), despite their inherent metabolic activity addressed to conserve the homoeostasis phospholipid fraction (Rainuzzo et al 1994; Coutteau et al 1997) Additionally to the provision of PL, it is noteworthy to mention that liposomes can facilitate the delivery of cholesterol that, while added as membrane stabilizer in our liposome formulations, can also exert beneficial effects for the octopus paralarvae as suggested by Navarro & Villanueva (2000) Further investigations are underway in our laboratories to ascertain the efficiency of liposomes as a tool for the cholesterol enrichment of Artemia The incorporation of HUFA or PL into Artemia metanauplii, which have a better developed filter-feeding apparatus than nauplii (Dhont et al 1991), appears to be a rapid process In contrast to longer (18–24 h) incubations required in the enrichment procedures of newly hatched Artemia nauplii (Monroig et al 2006), enhanced HUFA or PL levels in metanauplii were achieved within only h, with unremarkable increases observed thereafter These results suggest that short-term incubations of h for delivering each lipid ingredient (HUFA or PL) are sufficient and that longer incubations can be therefore avoided to prevent autoxidation of enrichment diets (McEvoy et al 1995; Monroig et al 2007) or undesired metabolic conversions of lipid classes (Rainuzzo et al 1994; McEvoy et al 1996) and fatty acids (Navarro et al 1999) Combined use of emulsions and liposomes was investigated to achieve a simultaneous enhancement of HUFA and PL in metanauplii, and two different approaches were assessed One strategy consisted of a sequential supply of commercial emulsion and a subsequent replacement by soya liposomes (Treatment 2B) The other strategy consisted of a mixture of emulsion and liposomes that was dispensed at the beginning of the incubation (Treatment 2C) Aquaculture Nutrition 19; 837–844 ª 2013 John Wiley & Sons Ltd While the two strategies resulted in an increase in PL fractions compared with emulsion-enriched Artemia, PL content was significantly higher in metanauplii enriched through the sequential emulsion/liposome treatment compared with the mixture emulsion/liposome approach Interestingly, no differences in HUFA contents including EPA and DHA were encountered in metanauplii enriched through the two distinct strategies These results enlighten a novel strategy for simultaneously delivering HUFA and PL into Artemia, and more importantly, simpler and cheaper than the sequential emulsion/liposome treatment proposed by Barr et al (2005) for naupliar stages that involved renewal and larger total amounts of enrichment products Despite liposome-based treatments in Experiment demonstrated their ability to increase the HUFA contents of Artemia metanauplii, the significance of such increases might still be insufficient, if it is compared with natural preys (Navarro & Villanueva 2000; Iglesias et al 2004) or to metanauplii treated exclusively with commercial emulsions Thus, in Experiment 2, the DHA/EPA ratios, regarded as a good indicator of nutritional quality in live preys for marine larvae (Rodrıguez et al 1998; Sui et al 2007), were significantly lower in Treatments 2B (0.5) and 2C (0.6) than in Treatment 2A (0.8) These results indicated a dilution effect caused by the soya FA composition with which the liposomes were formulated This was further supported by the increased levels of LOA (18:2n-6) accounting for over 24% of total FA in liposome-treated metanauplii To increase the PL contents of Artemia metanauplii while preserving their HUFA, a marine lecithin, a coarse material containing HUFA-rich PL, was examined in Experiment The metanauplii treated exclusively with marine lecithin (Treatment 3A) exhibited a DHA/EPA ratio of More importantly, the individual HUFA content was notably high, with impressive percentages of EPA and DHA (~13% each) These results revealed a remarkable efficiency for the HUFA enrichment in Artemia metanauplii Thus, if the efficiency is estimated in an approximate way as [DHA incorporated in metanauplii/DHA present in the enrichment diet] 100, it can be easily deduced that the marine lecithin enrichment is highly efficient with values of 40% incorporation, whereas much lower efficiency (11%) was observed for metanauplii enriched with a prototype DHArich emulsion containing 70% of total FA that produced DHA contents of 8% of total FA in nauplii (Viciano et al 2011) In addition to HUFA delivery, the marine lecithin was also efficient in the provision of PL into metanauplii, Aquaculture Nutrition 19; 837–844 ª 2013 John Wiley & Sons Ltd showing the highest PL content among treatments in Experiment 3, even higher than PL levels of metanauplii enriched with soya lecithin (Treatment 3C) Overall our results demonstrate that Marine lecithin LC60 is a promising novel enrichment diet capable to enhance the HUFA and PL contents of Artemia metanauplii Moreover, the marine lecithin can also be efficient when used for enrichment of other live preys employed in marine larviculture, including rotifers or Artemia naupliar stages In conclusion, this study demonstrates that it is possible to increase simultaneously the contents of PL and essential HUFA of Artemia metanauplii treated with both fish oilbased emulsions and liposomes, as well as with marine origin lecithins, resulting in a hypothetically more equilibrated living food for octopus paralarvae Further investigations are required to elucidate the physiological impacts that metanauplii enriched here presented have for octopus paralarvae This study was funded by the Ministerio de Ciencia e Innovaci on (Spanish Government) under Project OCTOPHYS (AGL-2010-22120-CO3-02), by the Generalitat Valenciana under Project PROMETEO (2010/006) and by JACUMAR under Project NUTRIPULPO DG was recipient of a Spanish fellowship from CSIC-Bancaja OM was supported by a Marie Curie Reintegration Grant within the 7th European Community Framework Programme (PERG08GA-2010-276916, LONGFA) and a Juan de la Cierva postdoctoral contract from Ministerio de Ciencia e Innovaci on Bangham, A.D., Standish, M.M & Watkins, J.C (1965) Diffusion of univalent ions across the lamellae of swollen phospholipids J Mol Biol., 13, 238–252 Barclay, W & Zeller, S (1996) Nutritional Enhancement of n-3 and n-6 fatty acids in rotifers and Artemia nauplii by feeding spray-dried Schizochytrium sp J World Aquacult Soc., 27, 314– 322 Barr, Y., Terjesen, B.F & Helland, S (2005) Intensive short-term enrichment – An efficient method for enhancing phospholipids and free amino acids in live feed In: Larvi 2005 – Fish & Shellfish Larviculture Symposium (Hendry, C.I., Van Stappen, G., Willie, M & Sorgeloos, P eds.), pp 15–18 European Aquaculture Society, Special Publication No 36, Oostende, Belgium Christie, W.W (1982) Lipid Analysis Pergamon Pres, Oxford Copeman, L.A., Parrish, C.C., Brown, J.A & Harel, M (2002) Effects of docosahexaenoic, eicosapentaenoic, and arachidonic acids on the early growth, survival, lipid composition and pigmentation of yellowtail flounder (Limanda ferruginea): a live food enrichment experiment Aquaculture, 210, 285–304 Coutteau, P., Geurden, I., Camara, M.R., Bergot, P & Sorgeloos, P (1997) Review on the dietary effects of phospholipids in fish and crustacean larviculture Aquaculture, 155, 149–164 Dhont, J., Lavens, P & Sorgeloos, P (1991) Development of a lipid-enrichment technique for Artemia juveniles produced in an intensive system for use in marine larvicultura In: Larvi 2005 – Fish & Shellfish Larviculture Symposium (Lavens, P., Sorgeloos, P., Jaspers, E & Ollevier, F., eds.), pp 51–55 European Aquaculture Society, Special Publication No 15, Ghent, Belgium Folch, J., Lees, N & Sloane-Stanley, G.H (1957) A simple method for the isolation and purification of total lipids from animal tissues J Biol Chem., 226, 497–509 Han, K., Geurden, I & Sorgeloos, P (2000) Enrichment strategies for Artemia using emulsions providing different levels of nÀ3 highly unsaturated fatty acids Aquaculture, 183, 335–347 Han, K., Geurden, I & Sorgeloos, P (2001) Fatty acid changes in enriched and subsequently starved Artemia franciscana nauplii enriched with different essential fatty acids Aquaculture, 199, 93 –105 Iglesias, J., Otero, J.J., Moxica, C., Fuentes, L & Sanchez, F.J (2004) The completed life cycle of the octopus (Octopus vulgaris, Cuvier) under culture conditions: paralarval rearing using Artemia and zoeae, and first data on juvenile growth up to eight months of age Aquacult Int., 12, 481–487 Iglesias, J., Fuentes, L., Sanchez, J., Otero, J.J., Moxica, C & Lago, M.J (2006) First feeding of Octopus vulgaris Cuvier, 1797 paralarvae using Artemia: effect of prey size, prey density and feeding frequency Aquaculture, 261, 817–822 Iglesias, J., S anchez, F.J., Bersano, J.G.F et al (2007) Rearing of Octopus vulgaris paralarvae: present status, bottlenecks and trends Aquaculture, 266, 1–15 Leger, P., Bengtson, D.A., Simpson, K.L & Sorgeloos, P (1986) The use and nutritional value of Artemia as a food source Oceanogr Mar Biol Annu Rev., 24, 521–623 McEvoy, L.A., Navarro, J.C., Bell, J.G & Sargent, J.R (1995) Autoxidation of oil emulsions during the Artemia enrichment process Aquaculture, 134, 101–112 McEvoy, L.A., Navarro, J.C., Hontoria, F., Amat, F & Sargent, J.R (1996) Two novel Artemia enrichment diets containing polar lipid Aquaculture, 144, 339–352  Navarro, J., Amat, I., Gonzalez, P., Amat, F & Monroig, O., Hontoria, F (2003) Enrichment of Artemia nauplii in PUFA, phospholipids, and water-soluble nutrients using liposomes Aquacult Int., 11, 151–161  Navarro, J.C., Amat, F., Gonzalez, P., Bermejo, A Monroig, O., & Hontoria, F (2006) Enrichment of Artemia nauplii in essential fatty acids with different types of liposomes and their use in the rearing of gilthead sea bream (Sparus aurata) larvae Aquaculture, 251, 491–508  Navarro, J.C., Amat, F., Gonzalez, P & Hontoria, Monroig, O., F (2007) Oxidative stability and changes in the particle size of liposomes used in the Artemia enrichment Aquaculture, 266, 200 –210 Navarro, J.C & Villanueva, R (2000) Lipid and fatty acid composition of early stages of cephalopods: an approach to their lipid requirements Aquaculture, 183, 161–177 Navarro, J.C & Villanueva, R (2003) The fatty acid composition of Octopus vulgaris paralarvae reared with live and inert food: deviation from their natural fatty acid profile Aquaculture, 219, 613–631 Navarro, J.C., Amat, F & Sargent, J.R (1993) The lipids of the cysts of freshwater-and marine-type Artemia Aquaculture, 109, 327–336 Navarro, J.C., Henderson, R.J., McEvoy, L.A., Bell, M.V & Amat, F (1999) Lipid conversions during enrichment of Artemia Aquaculture, 174, 155–166 Okumura, S., Kurihara, A., Iwamoto, A & Takeuchi, T (2005) Improved survival and growth in Octopus vulgaris paralarvae by feeding large type Artemia and Pacific sandeel, Ammodytes personatus: improved survival and growth of common octopus paralarvae Aquaculture, 244, 147–157 Rainuzzo, J.R., Reitan, K.I., Jørgensen, L & Olsen, Y (1994) Lipid composition in turbot larvae fed live feed cultured by emulsions of different lipid classes Comp Biochem Physiol A, 107, 699–710 Rodrıguez, C., Perez, J.A., Badı́a, P., Izquierdo, M.S., FernandezPalacios, H & Hernandez, A.L (1998) The nÀ3 highly unsaturated fatty acids requirements of gilthead seabream (Sparus aurata L.) larvae when using an appropriate DHA/EPA ratio in the diet Aquaculture, 169, 9–23 Ruız, O., Amat, F & Navarro, J.C (2008) A comparative study of the fatty acid profile of Artemia franciscana and A persimilis cultured at mesocosm scale J Exp Mar Biol Ecol., 354, 9–16 Sargent, J.R., McEvoy, L.A & Bell, J.G (1997) Requirements, presentation and sources of polyunsaturated fatty acids in marine fish larval feeds Aquaculture, 155, 117–127 Sorgeloos, P., Dhert, P & Candreva, P (2001) Use of the brine shrimp, Artemia spp in marine fish larviculture Aquaculture, 200, 147–159 Sui, L., Wille, M., Cheng, Y & Sorgeloos, P (2007) The effect of dietary n-3 HUFA levels and DHA/EPA ratios on growth, survival and osmotic stress tolerance of Chinese mitten crab Eriocheir sinensis larvae Aquaculture, 273, 139–150 Tocher, D.R., Bendiksen, E. A., Campbell, P.J & Bell, J.G (2008) The role of phospholipids in nutrition and metabolism of teleost fish Aquaculture, 280, 21–34 Vaz-Pires, P., Seixas, P & Barbosa, A (2004) Aquaculture potential of the common octopus (Octopus vulgaris Cuvier, 1797): a review Aquaculture, 238, 221–238 Viciano, E., Iglesias, J., Lago, M.J., Sanchez, F.J., Otero, J.J & Navarro, J.C (2011) Fatty acid composition of polar and neutral lipid fractions of Octopus vulgaris Cuvier, 1797 paralarvae reared with enriched on-grown Artemia Aquacult Res., 42, 704– 709 Villanueva, R & Bustamante, P (2006) Composition in essential and non-essential elements of early stages of cephalopods and dietary effects on the elemental profiles of Octopus vulgaris paralarvae Aquaculture, 261, 225–240 Villanueva, R & Norman, M.D (2008) Biology of the planktonic stages of benthic octopuses Oceanogr Mar Biol., 46, 105–202 Villanueva, R., Riba, J., Ruız-Capillas, C., Gonzalez, A.V & Baeta, M (2004) Amino acid composition of early stages of cephalopods and effects of amino acid dietary treatments on Octopus vulgaris paralarvae Aquaculture, 242, 455–478 Villanueva, R., Escudero, J.M., Deulofeu, R., Bozzano, A & Casoliva, C (2009) Vitamin A and E content in early stages of cephalopods and their dietary effects in Octopus vulgaris paralarvae Aquaculture, 286, 277–282 Zhou, X & Arthur, G (1992) Improved procedures for the determination of lipid phosphorus by malachite green J Lipid Res., 33, 1233–1236 Aquaculture Nutrition 19; 837–844 ª 2013 John Wiley & Sons Ltd [...]... malformations in Atlantic salmon (Salmo salar L.) Aquaculture, 2 95, 6–14 Ytteborg, E., Baeverfjord, G., Torgersen, J., Hjelde, K & Takle, H (2010) Molecular pathology of vertebral deformities in hyperthermic Atlantic salmon (Salmo salar) BMC Physiol., 10, 12 Aquaculture Nutrition 19; 651–664 ª 2013 John Wiley & Sons Ltd Aquaculture Nutrition 2013 19; 665–676 doi: 10.1111/anu.12014 ... related to experimental treatment There was a dose–response of ROL in liver and muscle to dietary VA inclusion level (Table 3) Liver ROL at Aquaculture Nutrition 19; 651–664 ª 2013 John Wiley & Sons Ltd Aquaculture Nutrition 19; 651–664 ª 2013 John Wiley & Sons Ltd n.a n.a y = À23x + 494 n.a n.a y = À0.1x + 0.9 n.a n.a n.a y = À0.1x2 + 0.1x + 1.15 y = À0.07x2 + 0.06x + 1.21 y = À0.03x2.. .Aquaculture Nutrition 2013 19; 651–664 doi: 10.1111/anu. 12013 1 1 1 1 2 3 2 National Institute of Nutrition and Seafood Research, Bergen, Norway; Department of Biology, University of Bergen, Bergen, Norway; 3 Matre Research Station, Institute of Marine Research (IMR), Matredal, Norway The vitamin A (VA) concentration in salmon aquaculture feeds is varying and... abdominal lesions in Atlantic salmon, Salmo salar L J Fish Dis., 34, 531–546 Aquaculture Nutrition 19; 651–664 ª 2013 John Wiley & Sons Ltd Grisdale-Helland, B., Helland, S.J & A˚sga˚rd, T (1991) Problems associated with the present use of menadione sodium bisulfite and vitamin A in diets for Atlantic salmon Aquaculture, 92, 351–358 Gundersen, T.E., Bastani, N.E & Blomhoff, R (2007) Quantitative... biological responses in juvenile sunshine bass (Morone chrysops x M saxatilis) fed graded levels of vitamin A Aquaculture, 2 35, 645–658 Hernandez, L.H.H., Teshima, S.-I., Ishikawa, M & Koshio, S (2004) Effects of dietary vitamin A on juvenile Red Sea Bream Chrysophrys major J World Aquac Soc., 35, 436–444 Hilton, J.W (1983) Hypervitaminosis A in rainbow trout (Salmo gairdneri): toxicity signs and maximum... Retinoic acid and Cyp26b1 are critical regulators of osteogenesis in the axial skeleton Development, 1 35, 3765–3774 Suzuki, T., Srivastava, A.S & Kurokawa, T (2000) Experimental induction of jaw, gill and pectoral fin malformations in Japanese flounder, Paralichthys olivaceus, larvae Aquaculture, 1 85, 175– 187 Takeuchi, T., Dedi, J., Ebisawa, C., Watanabe, T., Seikai, T., Hosoya, K & Nakazoe, J.-I (1995)... sole (Solea senegalensis) skeletogenesis and larval quality Aquaculture, 2 95, 250–265 Fjelldal, P., Lock, E., Grotmol, S., Totland, G., Nordgarden, U., Flik, G & Hansen, T (2006) Impact of smolt production strategy on vertebral growth and mineralisation during smoltification and the early seawater phase in Atlantic salmon (Salmo salar, L.) Aquaculture, 261, 715–728 Fjelldal, P., Hansen, T.J & Berg,... of concern in salmon aquaculture due to the varying and sometimes high concentrations of VA in fish meal and fish oil commonly included in formulated diets Although acute toxicity resulting in lethal effects is probably not relevant in practical fish farming, sublethal adverse effects as a consequence of long-term exposure to high concentrations may be of importance in salmon aquaculture In the present... toxic effects of VA in fish Several studies have been performed on marine larvae exposed to VA resulting in vertebral compression and malformed jaw and fin bone structures (Dedi et al 19 95, 1997; Takeuchi et al 19 95, 1998; Martinez et al 2007), particularly in flatfish (Fernandez & Gisbert 2011) Similarly, first-feeding fry of Atlantic salmon fed chronic high concentrations of ROL showed abnormal vertebral... al 2002) Although the underlying mechanism of VA toxicity through disruption of expression of central genes involved in early life stage bone formation has been described Aquaculture Nutrition 19; 651–664 ª 2013 John Wiley & Sons Ltd (Laue et al 2008; Spoorendonk et al 2008; Fernandez et al 2011), our understanding of the mechanism at which VA interferes with bone growth in later life stages