Aquaculture 305 (2010) 109–116

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Effects of dietary yeast cell wall β-glucans and MOS on performance, gut health, andsalmon lice resistance in Atlantic salmon (Salmo salar) fed sunflower and soybeanmeal

Ståle Refstie a,b,⁎, Grete Baeverfjord b, Rudi Ripman Seim c, Odd Elvebø c

a Aquaculture Protein Centre (APC), CoE, Norwayb Nofima Marin, N-6600 Sunndalsøra, Norwayc Biorigin Scandinavia AS, N-0253 Oslo, Norway

⁎ Corresponding author. Present address: Sunndal MBox 94, 6601 Sunndalsøra, Norway. Tel: +47 48 12 31

E-mail address: stale.refstie@sunndal.kommune.no (

0044-8486/$ – see front matter © 2010 Elsevier B.V. Aldoi:10.1016/j.aquaculture.2010.04.005

a b s t r a c t

a r t i c l e i n f o

Article history:Received 7 May 2009Received in revised form 22 November 2009Accepted 11 April 2010

Keywords:Feedstuffs–fish meal–soybean meal–sun-flower mealYeast cell wall-β-glucans-MOSFeed intake-growthDigestibility-retentionGut health-salmon lice

This experiment examined and compared the effects of a highly purified immune modulating β-1,3/1,6-glucanproduct (BG) and of a putative receptor blocking,mannan oligosaccharide rich product (MOS) in Atlantic salmonfed extruded diets containing extracted soybean meal (SBM) or a combination of SBM and extracted sunflowermeal (SFM). The BG andMOS productswere derived from the cell walls of baker's yeast. A control dietwas basedon LT-fish meal (FM) and contained no plant protein. Two basic experimental diets were formulated with 32%SBM (FM+S) or with 14% SBM+14% SFM (FM+SS). Following extrusion, four FM+S batches weresupplemented with 500 or 1000 mg BG or 1000 or 2000 mgMOS kg−1, while two FM+SS batches weresupplemented with 1000 mg BG or 2000 mgMOS kg−1. Each diet was fed to three groups of 150 salmon kept insea pens, and effects on feed intake, growth, nutrient utilisation, gut health, sea lice infestation, and overallperformance of the fishwere recorded over a period of 70 days. The initial weight of the fishwas 0.68 kg, and thedifferent feed groups grew to final weights ranging from 1.33 to 1.72 kg. Compared to the control group, fish fedthe dietwith 32% SBMate 18% less, grew30% slower, had 24% poorer feed efficiency ratio (FER), and also sufferedfromserious SBM-induced enteritis, diarrhoea, and reduced capacity to digest lipid. Adding BGorMOS to this diethad no detectable effects. Fish fed the diet with 14% SBM+14% SFM ate as much as the control group, but stillgrew 5% slower, had 7% poorer FER, and suffered from a diarrhoea-like condition and moderate enteritis.Noteworthy, 27% fewer of these fishwere infestedwith salmon licewhen compared to the other groups. AddingBG to this diet further reduced the number of lice-infested fish by 28%. Adding MOS to this diet did not affectappetite or lice infestation, but resulted10%better FER, 8% faster growth (similar to the control group), 11%higherprotein retention, less diarrhoea, and most noteworthy: elimination of the SBM-induced enteritis. This clearlydemonstrates that gut health is an important production parameter for Atlantic salmon.

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© 2010 Elsevier B.V. All rights reserved.

1. Introduction

There is an ongoing effort to define nutritionally acceptable plantprotein sources for Atlantic salmon. Purified plant proteins mayreplace fish meal protein in feed for salmon, provided that the aminoacid profile in the final ration is properly adjusted by amino acidsupplements (Storebakken et al., 1998, 2000a; Refstie et al., 2000,2001; Espe et al., 2006, 2007). Since such refined plant proteinproducts are relatively expensive, the fish feed industry is trying touse cruder and hence cheaper plant proteins in feed formulations.However, crude plant protein products often contain antinutritionalfactors that negatively affect digestion, gut function, growth andoverall performance of the fish (Storebakken et al., 2000b; Franciset al., 2001; Bakke-McKellep and Refstie, 2008). Such products cantherefore be used only at a low dietary inclusion to avoid adverse

effects. In order to allow higher inclusion and lower feed costs, the fishfeed industry is therefore searching for ways to circumvent theadverse effects of the antinutritional factors present in crude plantproteins.

Soybean meals (SBMs) are the most widely used protein source inanimal feeds. These less refined soy products induce inflammation andpathomorphological changes in thedistal intestine of salmonidfish (vanden Ingh et al., 1991, 1996; Rumsey et al., 1994; Baeverfjord andKrogdahl, 1996; Burrells et al., 1999), followed by reduced functionalitysuch as lower activity of enzymes in and nutrient transport across thebrushbordermembraneof this intestinal section (Bakke-McKellepet al.,2000, 2007a; Krogdahl et al., 2003; Refstie et al., 2006a,b; Urán et al.,2008). Dietary SBM also alters the number and diversity of intestinalbacteria in salmonid fish (Heikkinen et al., 2006; Bakke-McKellep et al.,2007a), and may, thus, potentially favour growth of unfavourablebacteria that worsen the inflammation. The hypersensitivity appearsspecific to soy, as similar intolerance reactions are not observed whenfeeding legumes like lupin, field peas, and faba beans, oilseeds likesunflower and rapeseed, wheat and corn gluten, or cereal grains like

Table 1Formulation of the experimental diets.

Diet code Fish meal(FM) control

FM+SBM(S) basic

FM+SBM+SFM(SS) basic

Formulaa, g kg−1

LT-fish meal 525.0 242.0 300.0Soybean meal 320.0 135.0Sunflower meal 135.0Wheat gluten 10.0Wheat 188.0 100.5 116.5Fish oil 286.0 305.0 291.0Lysine 1.0 1.0Methionin 1.5 1.5MCPb 1.0 20.0 20.0

a All ingredients were supplied by Skretting AS (Averøy, Norway).b Mono calcium phosphate.

110 S. Refstie et al. / Aquaculture 305 (2010) 109–116

wheat and oat to Atlantic salmon (Storebakken et al., 2000a; Refstieet al., 2006a; Aslaksen et al., 2007).

The causative soy component(s) is found in the alcohol-extractablefraction of soybeans (van den Ingh et al., 1996; Knudsen et al., 2007).Thus SBMs for salmonids are refined by alcohol-washing to produce soyprotein concentrates (Lusas and Riaz, 1995), which do not alter thedistal intestine in these species (van den Ingh et al., 1991, 1996; Rumseyet al., 1994;Refstie et al., 2000, 2001; Escraffe et al., 2007). An alternativeapproach may be the use of immunostimulatory, anti-inflammatory,and/or prebiotic feed additives in diets containing less refined sourcesof soy protein such as full-fat or extracted (de-oiled) SBM.

Live yeasts are present and appear commensal in fish guts(Gatesoupe, 2007), and different yeast preparations have immuno-stimulatory effects in fish (Robertsen; 1999; Sakai, 1999). Phagocyticcells have receptors that recognise and bind highly purifiedmixed linkβ-1,3/1,6-glucans (BG) extracted from baker's yeast cell walls(Jørgensen et al., 1991; Engstad and Robertsen, 1993, 1994; Jørgensenand Robertsen, 1995). Thus, BG is routinely used as a vaccine adjuvant(Jørgensen et al., 1993; Rørstad et al., 1993) and immunostimulatoryfeed ingredients (Robertsen et al., 1990; Burrells et al., 2001), and isalso reported to increase sea lice resistance (Ritchie, 2000) in Atlanticsalmon. BG furthermore has anti-inflammatory effect(s) (Anderssonet al., 2000; Li et al., 2005; Sandvik et al., 2007).

Mannan oligosaccharides (MOS) is another yeast cell wall derivedfeed ingredient working locally in the gut. MOS are known to improvedigestion and gut health in animals by binding to and blocking glyco-protein receptors on pathogens (Newman, 2001; Fernandez et al.,2002). By the samemode of action they may bind and inactivate plantantigens of glycoprotein nature. MOS may furthermore function as aprebiotic, favouring growth of beneficial bacteria in the gut. In linewith this, moderate dietary supplementation (1.5 to 10 gkg−1 diet) ofpurified MOS derived from baker's yeast cell walls have been shownto improve feed efficiency in Atlantic salmon (Grisdale-Helland et al.,2008), growth and immune status in rainbow trout (Oncorhynchusmykiss; Staykov et al., 2007; Yilmaz et al., 2007a), and gutdevelopment in rainbow trout and hybrid tilapia (Oreochromisniloticus×O. aureus; Genc et al., 2007).

Basedon this, the objectivesof thisworkwere 1) to compare growth,nutrient utilisation, and severity of SBM-induced pathomorphologicalchanges in the intestine of Atlantic salmon fed diets containing either amixture of 14% extracted sunflowermeal (SFM) and 14% extracted SBMor 32% SBM, 2) to evaluate and compare effects on performance and guthealth when adding a highly purified immune modulating βBG or aputative receptor blocking, MOS, both extracted from baker's yeast cellwalls, to these basic diets, and 3) to investigate potential salmon licerepelling effects of BG and MOS in the diet.

2. Materials and methods

2.1. Ingredients and diets

Highly purified cell wall mixed link β-1,3/1,6-glucans (BG;MacroGard®) from baker's yeast (Saccharomyces cerevisiae) cell andayeast cellwall fractionwithhighmannanoligosaccharide concentration(MOS;PatoGard™)wereproducedand suppliedbyBiotec ParmaconASA(Tromsø, Norway). This BG typically contains 65% carbohydrate and 6%protein, while the MOS typically contains 51% carbohydrate, of which25% is mannan oligosaccharides, and 30% protein. All other ingredientswere supplied by Skretting AS (Averøy, Norway).

The formulation of the diets is given in Table 1. A standard fishmeal based control diet (FM), a basic high-vegetable diet with 32%extracted and toasted soybean meal (SBM; FM+S), and a basic high-vegetable diet with 14% SBM and 14% extracted sunflowermeal (SFM;FM+SS) were manufactured by high-pressure moist extrusion bySkretting. The particle size was 6 mm, and the diets were dried priorto coating with fish oil.

Prior to vacuumcoatingwith oil, four batches of thebasic FM+Sdietswere pre-coatedwith 500 (FM+S+500BG) or 1000 (FM+S+1000BG)mg BG or 1000 (FM+S+1000MOS) or 2000 (FM+S+2000MOS)mgMOS kg−1 diet. Likewise, two batches of the basic FM+SS diet waspre-coated with 1000 mg of BG (FM+SS+1000BG) or 2000 mg of MOS(FM+SS+2000MOS) kg−1 diet. This gave a series of nine experimentaldiets. More levels of BG and MOS were tested in the FM+S diet than inthe FM+SS diet because this diet would give a higher SBM challenge inthe salmon.

2.2. Fish, rearing conditions, and sampling

This experiment was conducted in accordance with laws andregulations that control experiments and procedures in live animals inNorway, as overseen by the Norwegian Animal Research Authority.The experiment was done at Nofima Marin's model sea farm atEkkilsøy in Norway.

Atlantic salmon (Salmo salar) were fed the experimental diets for atotal of 69 to 71 feeding days. Prior to the experiment, the fish were fedcommercial diets (Skretting AS, Stavanger, Norway). The experimentwas initiated in week 25 and terminated in week 36 of 2006. The watertemperature varied from 12.3 to 17.4 °C during the course of theexperiment, averaging 15.3 °C.

At the start of the experiment, 27 groups of salmon (679±2 g (penmean±std), 150 fish per group) were randomly distributed to5×5×5 m3 sea pens. Each diet was then allocated to three groups offish in a triplicate randomised experimental design. The fish were fedcontinuously by electrically driven feeders, and uneaten feed wascollected from underneath the pens and pumped up into wire meshstrainers as described by Einen et al. (1999). The feeding rate wasplanned to be 15% in excess, andwas adjusted according to the recordedoverfeeding every 3 days as described by Helland et al. (1996).

The fishwereweighed in bulk at the start of the experiment and onfeeding day 70. At the final weighing a sufficient number of fish fromall groups except those fed the FM+S+500BG and FM+S+1000BGdiets were also anesthetised with tricaine methanesulfonate (MS 222,Argent Chemical Laboratories Inc., Redmont, Wa, USA) and stripped asdescribed by Austreng (1978) to collect faeces for digestibilityestimation. The faecal samples were pooled per pen and immediatelyfrozen at −20 °C.

Before the final weighing 20 fish per penwere weighed individuallyand sampled for counting of salmon and sea lice to evaluate degree oflice infestation.Out of these,fivefishper penwere euthanisedby a sharpcranial blow for sampling of tissue from the distal intestine, defined asthe region characterised by the transverse luminal folds and increasedintestinal diameter to the anus. From each fish, 5 mm tissue sampleswere cut (a transverse cut relative to the length of the tract) from thecentral area of the distal intestine. These samples were placed and

111S. Refstie et al. / Aquaculture 305 (2010) 109–116

stored in phosphate-buffered formalin (4%, pH 7.2) for histologicalexamination.

Before distributing fish to the experimental sea pens, 15 fish weresampled from the holding pen. These fish were euthanised in waterwith a lethal concentration of MS 222, weighed individually, andfrozen immediately at −20 °C as three pooled samples of five fish.After feeding day 70, the fish were fasted for 2 days before thisprocedure was repeated, sampling five fish per pen. These pooledsamples were ground frozen and homogenised for analyses ofchemical composition.

2.3. Calculations

Crude protein (CP) was calculated as N×6.25. Feed intake (FI) wasestimated by subtracting uneaten feed from fed feed on a dry-matterbasis. Recovery of uneaten feedwas estimated as described by Hellandet al. (1996), and the recorded uneaten feed was corrected for dry-matter losses during feeding and collection. Feed efficiency ratio (FER)was calculated as: G×F−1, where G is the weight gain and F is theconsumption of dry matter from the feed. Thermal-unit growthcoefficient (TGC) was calculated according to Iwama and Tautz(1981), modified by Cho (1992), as: TGC=(W1

1/3−W01/3)×(ΣD°)−1,

where W0 and W1 are the initial and final weights (pen means),respectively, and ΣD° is the thermal sum (feeding days×averagetemperature, °C). Apparent digestibility was estimated by the indirectmethod, as described by Maynard and Loosli (1969), using yttriumoxide (Y2O3) as an inert marker (Austreng et al., 2000).

2.4. Chemical analyses

Homogenised fish were freeze-dried (Hetosicc Freeze drier CD 13-2HETO, Birkerød, Denmark) and analysed for dry matter (105 °C toconstant weight), ash (combusted at 550 °C to constant weight), andnitrogen (Kjeltec Auto Analyser, Tecator, Höganäs, Sweden). Faeceswere freeze-dried prior to analyses. Diets, and freeze-dried faeces wereanalysed for dry matter, ash, nitrogen, lipid (pre-extraction withdiethylether and hydrolysis with 4 M HCl prior to diethyletherextraction (Stoldt, 1952) in a Soxtec (Tecator) hydrolysing (HT-6) andextraction (1047) apparatus), starch (determined as glucose afterhydrolysis by α-amylase and amylo-glucosidase, followed by glucosedetermination by the “GODPOD method” (Megazyme, Bray, Ireland)),and yttrium (at Jordforsk, Ås, Norway, by inductivity coupled plasma(ICP) mass-spectroscopy, as previously described by Refstie et al.(1997)). Diets were furthermore analysed for gross energy (Parr 1271Bomb calorimeter, Parr, Moline, IL, USA).

2.5. Histological examination

Formalin-fixed distal intestinal tissue was routinely dehydrated inethanol, equilibrated in xylene and embedded in paraffin according to

Table 2Composition of the experimental diets.

Basic diet FM FM+S FM+SS FM+S

Added dose, mg kg−1 BG 500

Dry matter, g 937.3 934.0 941.0 933.1In dry matter, kg−1

Crude proteina, g 410.8 373.1 362.2 372.2Lipid, g 359.2 350.8 369.4 359.0Starch, g 107.3 57.8 60.6 62.2NSP+OSb, g 37.4 151.1 134.8 138.9Ash, g 85.3 67.2 72.9 67.7Energy, MJ 24.3 24.2 24.7 24.4

a CP; N×6.25.b Non-starch polysaccharides+oligosaccharides; calculated by difference.

standard histological techniques. Sections of approximately 5 μmwere cut and stained with haematoxylin and eosin before histologicalexamination under a Nikon Optiphot light microscope.

Pathomorphological changes associated with feeding of SBM wereidentified according to the criteria given by Baeverfjord and Krogdahl(1996). The severity of these changes was evaluated by a semiquantitative scoring system (Urán score) according to the criteriagiven by Knudsen et al. (2007). By this system the following fourintestinal responses to dietary SBM are evaluated separately: (1)presence and size of supranuclear vacuoles (2) degree of widening ofthe lamina propria of simple folds (3) amount of connective tissuebetween base folds and stratum compactum, and (4) degree ofthickening of the mucosal folds. Each response is ranked according toa scale ranging from 1 to 5, where Urán score 1 represents normal andundamaged intestines, 2 represents slight but noticeable pathomor-phological changes, 3 represents fully developed enteritis, 4 repre-sents serious enteritis, and 5 represents severe and potentially lethalenteritis. A pooled Urán score for each intestine was calculated as themean of these four separate scores. Illustrative pictures are given inFig. 2.

The histological evaluation was blind, i.e., identification of pen anddiet was done after the evaluation. Selected sections were photo-graphed with a Coolpix 990 digital camera connected to themicroscope.

2.6. Statistical analyses

The results were analysed by the General Linear Model procedurein the SAS computer software (SAS, 1985). Mean results per pen weresubjected to one-way analysis of variance (ANOVA) with diet as theindependent variable. Prior to analysis, the percent-wise frequency ofsea lice-infested fish was arcsine transformed, and the number of liceper infested fish and number of lice per 100 fish were ln transformed.Significant differences were indicated by Duncan's multiple rangetest. The level of significance was P≤0.05, and the results arepresented as mean±s.e.m. (standard error of the mean).

3. Results

3.1. Diet composition

The compositions of the diets are given in Table 2. Whenformulating the diets, the intention was to have similar crude proteinand lipid content in all diets. However, due to feed technologicalproblems when manufacturing the fish meal (FM) control diet, thestarch content in this diet had to be reduced, and this was achieved byreplacing part of the wheat by fish meal. Consequently, the FM dietcontained more protein than the other diets. The other diets wereuniform with respect to protein and lipid contents.

FM+SS

MOS 1000 BG 1000 MOS 2000 BG 1000 MOS 2000

930.3 932.7 930.8 940.8 940.7

371.9 376.3 371.4 362.7 351.7343.6 355.9 350.4 370.5 365.157.7 64.0 58.3 60.2 68.9

162.6 134.3 152.7 133.2 133.064.2 69.5 67.3 73.4 81.324.2 24.1 24.0 24.6

112 S. Refstie et al. / Aquaculture 305 (2010) 109–116

3.2. Feed intake, growth, and feed efficiency

When comparing the feed intake in groups fed the basic diets, thetotal intake was 18% lower in the groups fed the diet with 32% SBM(FM+S) than in the groups fed the FM control diet and the diet with14% SFM+14% SBM (FM+SS; Table 3). As shown by Fig. 1, this was aconsistent difference that lasted throughout the experiment. Further-more, the feed intake dropped noticeably 3 days after introducing thefish to all diets containing 32% SBM, but then rose steadily until thebeginning of the third experimental week. As opposed to this, the feedintake in the groups fed the FM and FM+SS diets increased steadily inthis period. Supplementation of BG or MOS to the FM+S and FM+SSdiets did not alter the feed intake.

When compared to the groups fed the FM diet, growth rateestimated both as SGR and TGC (×1000) was 30% and 5% lower ingroups fed the FM+S and FM+SS diets, respectively (Table 3). Thisin turn resulted in respective 22% and 5% lower final body weights.Both growth rate and final body weight differed significantly betweenthe FM+S and FM+SS groups. Supplementing the FM+S diet withBG or MOS did not alter the growth rates. The same was seen whensupplementing the FM+SS diet with 1000 mg BG kg−1diet. Whensupplementing this diet with 2000 mg MOS kg−1, however, the fishgrew 8% faster and at a similar rate as when feeding the FM diet.

The feed efficiency ratio (FER) was significantly highest (i.e., thebest conversion of feed to body gain) when feeding the FM diet, 24%lower when feeding the FM+S diet, and 7% lower when feeding theFM+SS diet (Table 3). FER differed significantly between the FM+Sand FM+SS groups. Supplementing the FM+S diet with BG andMOS actually resulted in lower FER when adding 500 mg BG or2000 mg MOS kg−1 diet, while there was no effect when supple-menting 1000 mg BG or MOS kg−1 diet. In line with this, supple-menting the FM+SS diet with 1000 mg BG kg−1 did not affectFER. However, supplementing this diet with 2000 mg MOS kg−1 dietresulted in a significant 10% increased FER.

The mortality in the experiment was low, and was not affected bydiet (Table 3).

3.3. Diarrhoea, nutrient digestibility, and retention

Feeding the FM+S and FM+SS diets generally resulted in lowerdry-matter content (i.e., more water) in the faeces than when feedingthe FM diet, indicating a diarrhoea-like condition (Table 4). Whencomparing the FM+SS diets, this diarrhoeic condition was signifi-cantly improved when adding 2000 mg kg−1 of MOS, and it alsotended to improve when adding 1000 mg kg−1 of BG. No such effectof BG and MOS was seen when added to the FM+S basic diet.

The apparent digestibility of nitrogen (i.e., crude protein) was notaffected by diet (Table 4). Likewise, the apparent digestibility of starchwas similar when feeding the FM+S and FM+SS diets, but was

Table 3Body weight (BW), dry feed intake (% of initial BW), specific growth rate (SGR), thermal-undry feed eaten), and mortality when feeding the experimental diets for 70 days (n=3).

Diet Initial BW, g Final BW, g SGR, % d−1

FM 679 1716a 1.32a

FM+S 680 1346c 0.97cd

FM+SS 679 1632b 1.26b

FM+S+500BG 679 1327c 0.95cd

FM+S+1000MOS 679 1354c 0.99c

FM+S+1000BG 679 1327c 0.95cd

FM+S+2000MOS 679 1299c 0.93d

FM+SS+1000BG 678 1649b 1.26b

FM+SS+2000MOS 679 1707a 1.32a

ANOVAP 0.99 b0.0001 b0.0001ffiffiffiffiffiffiffiffiffiffi

MSEp

3 31 0.03

Different superscripts abcde within column indicates significant differences as indicated by D

reduced when feeding the FM control diet in response to the muchhigher starch level in this diet. The lipid digestibility was, however,significant and 9 to 20% lower when feeding the FM+S diets. This wasnot seen when feeding the FM+SS diets. Addition of BG or MOS didnot affect the apparent digestibility of nutrients.

When comparing the basic diets, the retention of nitrogen(i.e., crude protein) was intermediate when feeding the FM diet, 8%higher when feeding the FM+SS diet, but 13% lower when feedingthe FM+S diet (Table 4). When comparing the FM+SS diets, thenitrogen retention was significantly improved and 11% higher whenadding 2000 mg kg−1 of MOS to the diet. No such effect of BG wasseen, nor of adding MOS to the FM+S basic diet.

3.4. Soybean meal-induced enteritis

All fish fed the FM control diet had normal and undamaged distalintestines (Table 5 and Fig. 2). In contrast, all fish fed the FM+S dietswith 32% SBM displayed typical inflammation and severe pathomor-phological changes in the distal intestine, in accordance with thecriteria for SBM-induced enteritis established by Baeverfjord andKrogdahl (1996), and graded by the Urán-score (Knudsen et al.,2007). Fish fed the FM+SS diet, which contained only 14% SBM,displayed noticeable but moderate distal enteritis, and the severity ofthe pathomorphological changes varied more among individuals. Thisis illustrated by higher coefficient of variation for the Urán-scores (CV;standard deviation as a percentage of the group mean), whichaveraged 44% in fish groups fed the FM+SS diet, but only 11% and 14%in the groups fed the FM control and FM+S diets, respectively.

BG andMOS did not alter the severity of the SBM-induced enteritisat any level of supplementation when feeding the FM+S diets. Whenadded to the FM+SS diet, however, the supplement of2000 mg MOS kg−1 diet eliminated the pathomorphological changes,giving a 24% lower pooled Urán-score that was similar to whenfeeding the FM control diet (Table 5).

3.5. Lice infestation

The degree of sea lice (Caligus elongatus) infestationwasmoderate,and was not affected by dietary raw materials nor BG or MOSsupplementation (Table 6). The infestation with salmon lice(Lepeophteirus salmonis) was more pronounced, and was significantlylower when feeding the FM+SS diets than when feeding the FM andFM+S diets, with 27% fewer fish infested and 42% fewer lice perinfested fish. The salmon lice infestation was not affected by dietaryMOS supplementation. However, when comparing the FM+SS diets,the frequency of salmon lice-infested fishwas significantly reduced by28% when supplementing the FM+SS diet with 1000 mg BG kg−1.The salmon lice repelling effect of supplemented BG was not seenwhen feeding the FM+S diets.

it growth coefficient (TGC×1000), feed efficiency ratio (FER; unit BW increase per unit

TGC×1000 Feed intake, % IBW FER Mortality, %

2.95a 133.5a 1.14a 0.222.09cd 109.9bc 0.89c 0.442.79b 132.6a 1.05b 1.112.04cd 116.9b 0.82e 0.002.12c 113.7bc 0.87cd 0.222.04cd 106.7c 0.89c 0.221.98d 107.3c 0.85d 0.222.81b 133.1a 1.07b 0.672.95a 130.3a 1.16a 0.44

b0.0001 b0.0001 b0.0001 0.680.07 4.2 0.02 0.68

uncan's Multiple Range Test (Pb0.05).

Fig. 1. Daily dry feed intake in sea pens with 150 fish fed the experimental diets (n=3).

113S. Refstie et al. / Aquaculture 305 (2010) 109–116

4. Discussion

The major finding of this experiment was that dietary supple-mentation with a baker's yeast cell wall fraction rich in MOS to a dietwith moderate (14%) inclusion of solvent-extracted SBM eliminatedSBM-induced enteritis in the distal intestine of Atlantic salmon, andalso improved the diarrhoeic condition associated with feeding SBM.This coincided with more efficient utilisation of nutrients for growth,and, thus, faster growth. Furthermore, dietary SFM prevented salmonlice, and there was a co-action between SFM and supplemented highlypurified BG from baker's yeast cell walls in this respect.

The salmon fed thefishmeal (FM)diet and the dietswith 14% SFM+14% SBM(FM+SS) grew from11 to 14% faster than tabled growth ratesobtained with Atlantic salmon of comparable size at similar tempera-tures (Austreng et al., 1987). Considering the high water temperatureduring theexperiment (averaging15.3 °C), this illustrates that thesefishwere eating and performing well. Growth was, however, noticeablyslowed in all groups fed the diets with 32% SBM (FM+S), in line withprevious resultswhen feeding dietswithmore than 20% SBM to Atlanticsalmon (Olli et al., 1994, 1995; Refstie et al., 1998; 2000; 2005).

The observed low faecal dry-matter content (indicating diarrhoea)and low lipid digestibility when feeding the FM+S diets was in linewith previous results (Refstie et al., 2000, 2001, 2005). The apparentdigestibility estimates were, however, unexpectedly low. The reasons

Table 4Apparent digestibility of nutrients and retention of nitrogen by the fish after feeding theexperimental diets for 70 days (n=3).

Diet Faecal drymatter, %

Apparent digestibility (%) of Retention ofnitrogen (%)

Nitrogen Lipid Starch

FM 12.2a 71.0 84.5ab 27.5b 46.5c

FM+S 8.8c 75.1 77.3bc 49.8a 40.4d

FM+SS 8.7c 75.6 87.7a 46.1a 50.1b

FM+S+1000BG 8.5c 73.3 70.0c 51.0a 39.8d

FM+S+2000MOS 8.8c 72.8 71.0c 47.3a 38.9d

FM+SS+1000BG 9.5bc 77.5 88.2a 48.6a 47.8bc

FM+SS+2000MOS 10.7b 77.1 89.7a 42.6a 55.5a

ANOVAP b0.0001 0.27 0.0008 0.02 b0.0001ffiffiffiffiffiffiffiffiffiffi

MSEp

0.7 3.4 5.2 7.2 1.7

Different superscripts abcd within column indicates significant differences as indicatedby Duncan's Multiple Range Test (Pb0.05).

for this are unclear. It may in part be caused by leaching of inertdigestibility marker (yttrium oxide) from the diets, as yttrium oxideand yeast products were coated on the feed particles post-extrusion.Hillestad et al. (1999) showed that the leaching of yttrium oxide wastenfold higher than the leaching of dry matter from extruded feedparticles when this digestibility marker was added by coating.However, in the present experiment both yttrium oxide and yeastproducts were coated on the feed particles before vacuum coatingwith oil, so that all supplements were partly pressed into the feedparticles before being covered by a layer of hydrophobic fat. Second,the feed particles were eatenwithin amatter of seconds after enteringthe water, not sinking more than a few meters. Thus, one should notexpect significant leaching of these components from the diets.

The depressed apparent digestibility of lipid when feeding the FM+Sdietswas still in linewith previous results (Refstie et al., 2000, 2001, 2005,2006a). The high apparent lipid digestibility when feeding the FM+SSdiets was, however, unexpected. Supporting this result, Aslaksen et al.(2007) found that dietary sunflower, lupin, and rapeseed meal increasedthedigestibility of dietary lipid inAtlantic salmon, and similar effectswereindicated for different lupin meals by Refstie et al. (2006a). Thus, thepresent results may indicate that certain plant meals including SFMcounteractsnegative effects of SBMon lipiddigestion and/or absorption inAtlantic salmon.

Table 5Severity of SBM-induced enteritis as ranked by Urán score after feeding theexperimental diets for 70 days (n=3).

Diet Supranuclearvacuolisation

Laminapropria

Connectivetissue

Mucosalfolds

PooledUrán score

FM 1e 1.0c 1.3b 1.1d 1.1c

FM+S 3.6ab 3.6a 3.7a 3.9a 3.7a

FM+SS 2.0c 2.1b 2.0b 2.1b 2.1b

FM+S+500BG 3.9a 3.3a 3.5a 3.7a 3.6a

FM+S+1000MOS 3.5ab 3.7a 3.5a 3.9a 3.7a

FM+S+1000BG 3.3b 3.4a 3.3a 3.7a 3.4a

FM+S+2000MOS 3.7ab 3.1a 3.3a 3.4a 3.4a

FM+SS+1000BG 1.6cd 1.6bc 1.5b 1.7bc 1.6bc

FM+SS+2000MOS 1.2de 1.1c 1.4b 1.2cd 1.2c

ANOVAP b0.0001 b0.0001 b0.0001 b0.0001 b0.0001ffiffiffiffiffiffiffiffiffiffi

MSEp

0.2 0.4 0.5 0.3 0.3

Different superscripts abcde within column indicates significant differences as indicatedby Duncan's Multiple Range Test (Pb0.05).

Fig. 2. Urán score related to morphology of the distal intestine: Score 1 (A), 3 (B), 4 (C), and 5 (D).

114 S. Refstie et al. / Aquaculture 305 (2010) 109–116

The retention of consumed (mainly protein-bound) nitrogen inAtlantic salmon routinely exceeds 50% when feeding well-balanceddiets (Nordrum et al., 2000; Refstie et al., 2000, 2004; Grisdale-Helland et al., 2008). Presently this was observed in fish fed the dietswith 14% SFM+14% SBM. The lower nitrogen retention when feedingthe FM diet was probably due to the high protein content in this diet,resulting in more protein catabolism for energy and/or conversion ofdietary amino acids to carbohydrate and lipid and, thus, less efficientutilisation of the protein for muscle growth (Grisdale-Hellandand Helland, 1997). Reduced retention of nitrogen when feeding theFM+S diets was in line with the low feed intake and subsequent lowFERwith this diet, as a relatively larger proportion of the nutrients andenergy then must have been used for metabolic maintenancefunctions.

Table 6% of the fish infested by salmon lice (Lepeophteirus salmonis) and sea lice (Caligus elongatus),the experimental diets for 70 days (n=3).

Diet Salmon lice

Infested fish, % Lice per infested fish Lice per

FM 77.3ab 2.54a 197ab

FM+S 77.3ab 2.42ab 191ab

FM+SS 60.6c 1.48d 89cd

FM+S+500BG 72.7abc 1.86bcd 136bc

FM+S+1000MOS 77.3ab 2.24abc 173ab

FM+S+1000BG 84.8a 2.43ab 208a

FM+S+2000MOS 84.8a 2.25abc 191ab

FM+SS+1000BG 43.9d 1.75cd 77d

FM+SS+2000MOS 68.2bc 1.70d 115cd

ANOVA on transformed dataP 0.0004 0.004 0.0001ffiffiffiffiffiffiffiffiffiffi

MSEp

0.1 0.10 0.22

Different superscripts abcd within column indicates significant differences among transform

The pathomorphological changes in the distal intestine of Atlanticsalmon fed the FM+S diets is in line with the well describedhypersensitivity to full-fat and extracted SBMs in Atlantic salmon (vanden Ingh et al., 1991, 1996; Baeverfjord and Krogdahl, 1996; Krogdahlet al., 2003; Bakke-McKellep et al., 2007a, 2007b; Urán et al., 2008).

The noticeable drop in feed intake at the third day of feeding in fishfed FM+S diets indicates that these fish suffered appetite reductionas response to developing SBM-induced enteritis and digestivedisturbances. This is in line with Baeverfjord and Krogdahl (1996),who observed the first signs of pathomorphological changes in thedistal intestine of Atlantic salmon after 2 days on a diet with 33% SBM.However, as no other feed was offered, the appetite of the present fishgradually increased during the following 2 weeks, and then stabilised,indicating that the fish adapted to the enteritis in the distal intestine.

number of lice per infested fish, and estimated number of lice per 100 fish after feeding

Sea lice

100 fish Infested fish, % Lice per infested fish Lice per 100 fish

36.4 1.06 3922.7 1.12 2625.8 1.25 3213.6 1.13 1719.7 1.07 2124.2 1.00 2421.2 1.11 2619.7 1.30 2416.7 1.18 20

0.30 0.60 0.520.12 0.08 0.53

ed data as indicated by Duncan's Multiple Range Test (Pb0.05).

115S. Refstie et al. / Aquaculture 305 (2010) 109–116

The high individual variation in severity of SBM-induced pathomor-phological changes when feeding the FM+SS diets is in accordancewith Krogdahl et al. (2003), who showed that severity of SBM-inducedenteritis in salmon depends on dietary SBM inclusion. As in the presentstudy, dietary SBM inclusion above 25% gave consistent and seriousenteritis, while inclusion of 15% resulted in more variation as a pro-portion of individuals displayed only moderate pathomorphologicalchanges. This result furthermore supports Aslaksen et al. (2007) in thatAtlantic salmon is not hypersensitive to sunflower.

At 32% inclusionof SBM in thediet (FM+S), thedietary concentrationof soy component(s) inducing pathomorphological changes in the distalintestine was apparently too high to be inactivated by dietary BG orMOS, at least at the tested dietary inclusion levels. However, adding2000 mgMOS kg−1 diet prevented SBM hypersensitivity in Atlanticsalmonwhen using only 14% SBM in the diet (FM+SS). In line with this,MOS supplementation also improved the diarrhoeic condition of fish fed14% SBM.

The modes of action of MOS to prevent SBM-induced enteritisremain speculation, meriting further investigation. However, MOS areknown to improve digestion and gut health in animals by binding toand blocking glycoprotein receptors on pathogens to make them passthrough the gut instead of colonising and invading the host (Newman,2001; Fernandez et al., 2002). MOS may also function as a prebiotic,favouring growth of beneficial bacteria in the gut (such as Bifido-bacteria and Lactobacilli). Adding to this, the presentMOS product wascrude, containing only 25% MOS. Thus, this product may also havecontained yeast cell wall constituents with intact and functionalreceptors that may have bound and inactivated potentially allergenicglycoprotein(s) found in SBM.

The FM+S diets with and without 2000 mg MOS kg−1 weresimilar except from the MOS supplement, which was coated onto thediet after extrusion. This strongly indicates that the improvements infeed conversion efficiency, growth, and nitrogen retention, despitesimilar feed intake, when feeding the MOS-supplemented diet weredue to the improved gut health. In line with this, similar dietarysupplementation with MOS has been shown to improve growth andimmune status in rainbow trout (Staykov et al., 2007; Yilmaz et al.,2007a), gut development in rainbow trout and hybrid tilapia (Genc etal., 2007; Yilmaz et al., 2007), and feed efficiency in Atlantic salmon(Grisdale-Helland et al., 2008). To our best knowledge it is the firsttime a negative effect of SBM-induced enteritis on growth and feedutilisation has been clearly isolated, not being influenced by differentraw material use and, thus, differing protein quality, antinutrientprofiles, and digestible nutrient contents in the compared diets. Itproves that gut health is an important production parameter in theAtlantic salmon industry.

It remains unclear whether MOS has a more beneficial effect thanBG on the intestinal integrity in SBM-fed salmon, or if this was causedby the twofold higher dietary inclusion of MOS than of BG. It is also aquestion if combined use of MOS and BG will give a better preventiveeffect, combining the beneficial effects of MOS and BG on gut health.

Salmon and sea lice were counted on a sub-sample of 20 fishnetted from each pen, which was 13.3% of the whole salmon grouptested. This may have created a potential sampling bias. However, thenine feed groups were randomly distributed among the 27 test pens,and the feed groupswere sampled in randomorder. As the salmon liceinfestation clearly differed statistically among treatments, the licecounts on sampled fish appear representative for the differenttreatments.

Dietary extracted SFM clearly lowered the salmon lice infestation.The reason for this remains unclear, and the existence of a componentin sunflower preventing salmon lice cannot be ruled out. As a salmonlice preventing effect of dietary BG was only observed when addingBG to the FM+SS diet, the results furthermore indicate an interactionand co-action between SFM and BG in this respect. Alternatively, thefish fed the FM+S diets may have responded differently due to the

severe SBM-induced enteritis these fish were suffering. A salmonlouse preventing effect of BG in Atlantic salmon is in accordance withRitchie (2000), who hypothesised that this was due to stimulation ofthe non-specific immune system of the fish. This immunostimulatoryeffect of BG in salmonid fish has been shown repeatedly (Robertsenet al., 1990; Jørgensen et al., 1991; Engstad et al., 1992; Engstad andRobertsen, 1993, 1994; Jørgensen et al., 1993; Rørstad et al., 1993;Jørgensen and Robertsen, 1995).

To conclude, feeding a diet with 32% SBM to Atlantic salmon keptin sea pens resulted in typical SBM-associated digestive disturbances,SBM-induced enteritis, and poorer appetite and performance of thefish. Feeding a diet with 14% SFM+14% SBM did not alter appetite,induced a diarrhoea-like condition and moderate enteritis, resulted inslightly poorer performance, but also had a significant repelling effecton salmon lice. BG and MOS supplementation did not modify theeffects of the diet with 32% SBM. BG did, however, strengthen thesalmon lice preventing effect of the dietary SFM. When MOS wassupplemented to the diet with 14% SFM+14% SBM the SBM-inducedenteritis was eliminated and the diarrhoea-like condition improved.This did not affect appetite, but was followed by bettered feedutilisation and faster growth, showing that gut health is an importantproduction parameter for Atlantic salmon.

Acknowledgements

The authors want to acknowledge the skilful technical assistanceof Sissel Nergaard, Bjarne Saltkjelvik, and Kirsti Hjelde at NofimaMarin. We are also grateful to Skretting for supplying feed ingredientsand manufacturing feeds. Financial support for the study wasprovided by a research contract between Nofima Marin and BiotecPharmacon.

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