2006) 392–406www.elsevier.com/locate/aqua-online

Aquaculture 261 (

Capacity for digestive hydrolysis and amino acid absorption inAtlantic salmon (Salmo salar) fed diets with soybean meal

or inulin with or without addition of antibiotics

Ståle Refstie a,b,⁎, Anne Marie Bakke-McKellep a,c, Michael H. Penn a,c,Anne Sundby a,c, Karl D. Shearer d, Åshild Krogdahl a,c

a Aquaculture Protein Centre (APC), CoE, Norwayb AKVAFORSK (Institute of Aquaculture Research AS), N-6600 Sunndalsøra, Norway

c Norwegian School of Veterinary Science, Department of Basic Sciences and Aquatic Medicine, P.O.Box 8146 Dep, N-0033 Oslo, Norwayd Northwest Fisheries Science Center, NOAA/NMFS, Seattle, WA, USA

Received 26 April 2006; received in revised form 4 August 2006; accepted 8 August 2006

Abstract

This experiment was done to study the effects of dietary soybean meal (SBM) and inulin (a prebiotic) on the capacity fordigestive hydrolysis and amino acid absorption by Atlantic salmon, and how a dietary supplement of the broad-spectrum antibioticoxytetracycline (OTC) modulated these responses. A control diet (FM) was made from fish meal, fish oil and extruded wheat. Twosimilar diets were made with 250 g soybean meal (SBM) or 75 g inulin kg−1. Each diet was made with or without a supplement of3 g OTC kg−1. All six diets contained yttrium oxide for estimation of apparent nutrient absorption. Each diet was fed to two groupsof 172 g salmon kept in 1 m2 tanks with 9 °C saltwater for 3 weeks. Intestinal organs were then sampled and weighed.Gastrointestinal tracts (GIT) were sectioned for analyses of brush border alkaline phosphatase (ALP) and leucine aminopeptidase(LAP) activities. Tissue from the distal intestine (DI) was also fixed for histological examination. Digesta from the differentsections were freeze dried for estimation of trypsin and amylase activities, and of apparent absorption of amino acids (AA),nitrogen (N), and sulphur (S). About 85% of the trypsin activity, 70% of the amylase activity, 85% of the ALP activity, and 82% ofthe LAP activity were found in the proximal (PI) and mid (MI) intestine of fish with functional DI, and the absorption of AA, N,and S was quantitatively completed in the MI. Dietary OTC resulted in lower relative liver weight, but apart from increased ALPand LAP activities in DI when feeding OTC in combination with inulin, OTC did not modify the responses to dietary SBM orinulin. Dietary SBM resulted in lower relative liver weight, and induced pathomorphological changes in the DI mucosa, thus lowerthe ALP and LAP activities in the DI. SBM also stimulated absorption of AA, N, and S in the PI, but at the same time increased theactivities of trypsin and amylase in the DI, indicating reduced re-absorption and increased faecal losses of these endogenousenzymes. Dietary inulin did not damage the DI, and stimulated intestinal growth and higher relative mass of the GIT. Inulin withoutOTC did not affect the hydrolytic and absorptive capacity of the salmon GIT.Crown Copyright © 2006 Published by Elsevier B.V. All rights reserved.

Keywords: Fish feed; Prebiotic; Oxytetracycline; Sulphuric amino acid; Trypsin; Amylase; Alkaline phosphatase; Leucine aminopeptidase

⁎ Corresponding author. Aquaculture Protein Centre, N-6600 Sunndalsøra, Norway. Tel.: +47 71 69 53 22; fax: +47 71 69 53 01.E-mail address: stale.refstie@akvaforsk.no (S. Refstie).

0044-8486/$ - see front matter. Crown Copyright © 2006 Published by Elsevier B.V. All rights reserved.doi:10.1016/j.aquaculture.2006.08.005

393S. Refstie et al. / Aquaculture 261 (2006) 392–406

1. Introduction

Due to steady supply, constant composition, lowprotein price, and high content of available protein withwell balanced amino acid profile, full fat and extractedsoybean meals are potentially very good protein ingre-dients for fish. However, soybean meals contain severalpotent antinutritional factors that disturb the digestiveprocess in carnivorous fishes (Storebakken et al., 2000).Among these, still unidentified heat stabile and alcoholsoluble soy component(s) cause pathomorphologicalchanges in the distal intestine of salmonids (Ingh et al.,1991, 1996; Rumsey et al., 1994; Burrells et al., 1999).Such changes were first noted by Ingh and Krogdahl(1990), and were described in detail by Baeverfjord andKrogdahl (1996). In short they are characterised byshortening of the primary and secondary mucosal foldswith a widening of the central stroma (lamina propria)and submucosa, shortened microvilli of the brush bordermembrane and increased formation of microvillarvesicles, and a dramatic decrease or even absence of thenormal supranuclear absorptive vacuoles in the enter-ocytes. The lamina propria is widened with a profoundinfiltration of a mixed population of inflammatory cells.

These morphological changes reduce the mass of thedistal intestine in salmon (Nordrum et al., 2000). Thedigestive process is also altered. The activity of brushborder membrane bound (Krogdahl et al., 1995; Bakke-McKellep et al., 2000a; Krogdahl et al., 2003) andcytosolic (Bakke-McKellep et al., 2000a) digestiveenzymes in the distal enterocytes is reduced, and thecarrier-mediated transport of amino acids and glucose islowered while the permeability of distal intestinalepithelium for nutrient transport is increased (Nordrumet al., 2000). The absorption of macromolecules by thedistal intestine is also decreased (Bakke-McKellep,1999), apparently causing reduced re-absorption ofendogenous digestive secretions, as indicated bydramatically increased activity of trypsin in the distalintestine (Dabrowski et al., 1989; Krogdahl et al., 2003).

Due to infiltration of inflammatory cells in theintestinal mucosa and rapid regression of the conditionfollowing withdrawal of soybean meal from the diet, thepathomorphological changes have been classified asnon-infectious, sub-acute soybean meal-induced enter-itis (Baeverfjord and Krogdahl, 1996), suggesting anetiology involving immunological mechanisms. In-creased number of proliferating cells lining the villousfolds of the distal intestine of soybean meal fed salmon(Sanden et al., 2005) suggests disturbed functionality ofenterocytes due to alterations in enterocyte turnover anddegree of maturation.

It is possible that the intestinal microbiota is involved inthe development of soybean meal-induced enteritis. As interrestrial animals, the intestinal microbiota in fish isaffected by diet (Cahill, 1990; Ringø et al., 1995; Ringøand Olsen, 1999), and the gastrointestinal tract appears asa major route of infection in fish (Ringø et al., 2004;Birkbeck and Ringø, 2005). In this context, physicaldamage to the mucosa by indigestible plant structuralcomponentsmay also play a role. Supporting this, Olsen etal. (2001) noted a destructive effect of high dietary levels(150 g kg−1) of inulin on the enterocytes in the distalintestine of the salmonid Arctic charr (Salvelinus alpinus),possibly caused by inulin absorption and accumulation.

Inulin is a large oligosaccharide naturally occurringin many plants, and is produced commercially from thechicory (Cichorium intybus) root. It is a fructosanconsisting of fructose monomers linked in linear chainsof varying length by β(2–1) bonds, and with terminalglucose moieties (Roberfroid et al., 1998; Pool-Zobel etal., 2002). Inulin cannot be hydrolysed by pancreatic orbrush border digestive enzymes in the intestine ofmonogastric animals (Pool-Zobel et al., 2002), but isfermented in the large intestine or colon (Roberfroid,2002; Flickinger et al., 2003), where it enhances therelative populations of bifidobacteria and other lacticacid producing bacteria (Pool-Zobel et al., 2002). It istherefore considered a prebiotic, which are defined asnon-digestible feed ingredients that benefit the host byselectively stimulating beneficial bacterial speciesalready resident in the intestine (Gibson and Roberfroid,1995; Grittenden and Playne, 1996). As such, moderatedietary levels (20 g kg−1) of inulin have been appliedattempting to establish a stable and healthy intestinalmicrobiota in larvae of the carnivorous marine turbot(Psetta maxima; Mahious et al., 2006).

Based on this, the objectives of this work were 1) toevaluate effects of dietary soybean meal on the capacity fordigestive hydrolysis and absorption of amino acids inAtlantic salmon, 2) to compare the digestive effects ofdietary soybeanmealwith those produced by dietary inulin,and 3) to evaluate how a dietary supplement of the broad-spectrum antibiotic oxytetracycline (OTC) modulates thedigestive responses in salmon to soybean meal and inulin.

2. Materials and methods

2.1. Ingredients and diets

The low-temperature dried (LT) fishmeal (FM; NorseLT-94, Vedde Herring Oil Factory, Egersund, Norway)wasmade from 80%blue whiting, 10% herring, and 10%processing offal from herring and mackerel. This LT-FM

Table 1Formulation of the diets

Diet code FM FM+OTC

SBM SBM+OTC

Inulin Inulin+OTC

Formulation,g kg −1

LT-fish meala 696 696 535 535 710 710Soybean mealb 250 250Fish oil 120 120 132 132 119 119Extruded wheat 164 161 61 58 76 73Inulinc 75 75Oxytetracyclined 3 3 3DL-methioninee 2 2Premixf 19.9 19.9 19.9 19.9 19.9 19.9Yttrium oxideg 0.1 0.1 0.1 0.1 0.1 0.1aNorse LT-94 (Vedde Herring Oil Factory, Langevåg, Norway).bExtracted soybean meal (Hamlet, Horsens, Denmark).cFrutanimal ND (Suiker Unie, Dinteloord, The Netherlands).dOxytetracycline hydrochlorid (Norsk medisinaldepot, Oslo, Norway).eD,L-methionine (Degussa, Hanau, Germany).fVitamin and mineral premix (FeedTech, Ås, Norway).gSigma (St. Louis, Mo, USA).

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met the following specifications: 1.5 g kg−1 volatile N,0.51 g kg−1 cadaverine, 0.05 g kg−1 histamine, 23.1%water soluble protein, and 92.2% nitrogen digestibility inmink. The soybean meal (SBM) was dehulled, extracted,and toasted, and was declared to have a trypsin inhibitoractivity (TIA) of 4.5 mg trypsin inhibited per g protein.The inulin powder (Frutanimal ND, Suker Unie,Dinteloord, the Netherlands) was extracted from chicoryroot, and was declared to contain ≥85% inulin withchain lengths ranging from 2 to 60 and averaging 8 to 11fructose moieties.

Six diets with a pellet size of 4 mm were produced ona laboratory cold pellet press at AKVAFORSK. Thediets were formulated to contain three differentcombinations of raw materials: 1) 100% of crudeprotein (CP) from LT-FM; 2) LT-FM and 24% of CPfrom SBM, and 3) 100% of CP from LT-FM, but added7.5% inulin. Each of these three diets was produced withor without a supplement of 3 g oxytetracyclinehydrochloride (OTC; Norsk medisinaldepot, Oslo,Norway) kg−1. All diets were formulated to contain555 g crude protein, 190 g lipid, and from 41 to 114 gstarch (DM basis), and to be iso-energetic on a grossenergy basis. The calculated concentration of non-starchpolysaccharides was similar in the SBM and inulin diets.The diets were supplemented with D,L-methionine tocontain similar amounts of (calculated) methionine. Alldiets contained 100 mg yttrium oxide kg−1 dry mix asan inert marker to permit apparent absorption measure-ments. The formulation of the diets is given in Table 1,and the composition is given in Table 2.

2.2. Fish, rearing conditions and sampling

This experiment was conducted in accordance withlaws and regulations that control experiments andprocedures in live animals in Norway, as overseen bythe Norwegian Animal Research Authority. The exper-iment was done at AKVAFORSK (Sunndalsøra, Nor-way), where seawater adapted Atlantic salmon (Salmosalar) were fed the experimental diets for a total of21 days. Fourteen days prior to the experiment, 12groups of salmon (172 g, 37 fish/group) were randomlydistributed from a holding tank to fibreglass tanks(1×1×0.6 m, water depth 40–50 cm) supplied withseawater. The fish were continued on a commercial diet(Skretting AS, Stavanger, Norway) until day 15, whenthe experimental diets were randomly allocated to 2groups of fish each. The fish were then fed theexperimental diets for 21 feeding days. The fish werefed continuously (24 hr d−1) by electrically driven discfeeders, aiming for 15% overfeeding based on expected

feed intake. The water temperature during the experi-mental period ranged 8 to 10 °C, and the O2 saturation ofthe outlet water was above 80%.

At feeding day 21, 21 fish randomly selected fromeach tank were euthanised in water with a lethalconcentration of tricaine methanesulfonate (MS 222,Argent Chemical Laboratories Inc., Redmont, Wa,USA), weighed individually, and the gastrointestinaltracts (GITs) were dissected out. Five fish per tank weresampled for analysis of alkaline phosphatase (ALP) andleucine aminopeptidase (LAP) activities. These GITswere sectioned into stomach (ST); pyloric intestine (PI),defined as the intestine from the most proximal to themost distal pyloric caeca; mid intestine (MI), defined asthe intestine between the most distal pyloric caeca andthe appearance of transverse luminal folds and increasein intestinal diameter, and; distal intestine (DI), definedas the region characterised by the transverse luminalfolds and increased intestinal diameter to the anus.Surrounding adipose and connective tissues werecarefully removed, the sections cut open and rinsed(with the exception of the pyloric caeca) before frozen inliquid nitrogen and stored at −80 °C. Liver was alsosampled from this fish and weighed individually.

Blood and intact intestines were furthermore sampledfrom 10 fish per tank. Blood was collected from thecaudal vein into vacutainers containing anticoagulant(EDTA) and protease inhibitor (Pefabloc® SC, Sigmano. 76307, Sigma Chemical Co., St. Louis, MO, USA).Samples were kept on ice until centrifugation at

Table 2Composition of the diets

Diet code FM FM+OTC SBM SBM+OTC Inulin Inulin+OTC

Composition, g kg−1

Dry matter (DM), g kg−1 925.7 927.7 923.0 922.6 918.3 924.9In DMCrude proteina (CP), g kg−1 542.1 539.8 544.9 542.6 545.7 534.2Amino acid proteinb, g kg−1 445.3 450.0 454.7 457.5 450.4 444.4Lipid, g kg−1 209.6 217.7 208.0 207.0 193.8 196.8Starch, g kg−1 89.9 85.0 32.1 29.3 36.2 39.2Ash, g kg−1 125.6 120.6 112.8 111.6 127.1 126.7Yttrium oxide, g kg−1 0.1 0.1 0.1 0.1 0.1 0.1Energy, MJ kg−1 22.6 22.9 23.0 23.1 22.4 22.6In CP, % (g 16−1 g N)

Essential amino acidsc

Arginine 6.0 6.1 6.4 6.3 6.2 6.2Histidine 2.0 2.1 2.2 2.2 2.0 2.0Isoleucine 4.3 4.4 4.4 4.5 4.4 4.5Leucine 7.9 8.1 7.9 8.0 8.0 8.0Lysine 7.7 7.8 7.4 7.4 7.9 7.9Methionine 2.9 2.9 2.7 2.7 2.9 2.9Phenylalanine 3.9 4.0 4.2 4.2 3.8 3.9Threonine 4.2 4.3 4.1 4.0 4.3 4.4Tryptophan 1.2 1.3 1.3 1.4 1.3 1.3Valine 5.0 5.1 5.1 5.4 4.8 4.9

Non essential amino acidsc

Alanine 6.3 6.3 5.8 6.3 6.5 6.6Aspartate+asparagine 9.8 10.0 10.2 10.2 10.1 10.2Cysteine 0.9 1.0 1.1 1.2 0.9 1.0Glutamate+Glutamine 15.0 15.2 15.5 15.8 14.7 14.9Glycine 6.3 6.4 5.9 5.9 6.5 6.5Proline 4.1 4.2 4.8 4.5 3.7 3.7Serine 4.4 4.4 4.5 4.5 4.4 4.5Tyrosine 3.6 3.6 3.7 3.7 3.6 3.6

aN×6.25.bExpressed as the sum of peptide-bound (dehydrated) amino acids.cExpressed as free amino acids.

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3000 rpm for 10 min. Plasma samples were aliquotedinto three separate Eppendorf tubes, frozen in liquidnitrogen and stored at −80 °C until analysis. The intactintestines were sampled for estimation of apparentamino acid absorption and activities of trypsin andamylase, and were wrapped in aluminium foil, frozen inliquid nitrogen and stored at −40 °C. Frozen intestinalcontents were sampled from the same GIT sections asdescribed above after careful thawing of the intestinalwall, and the contents were pooled per tank for analysis.For this sampling the PI was further subdivided intoproximal, PI1, and distal, PI2, portions, and the DI intoproximal, DI1, and distal, DI2, portions.

From the last six fish sampled per tank, a 5 mm tissuesample was cut (a transverse cut relative to the length ofthe tract) from the central area of DI. These sampleswere placed and stored in phosphate-buffered formalin(4%, pH 7.2) for histological examination.

2.3. Chemical analyses

Plasma was analysed for glucose, cholesterol,triacylglycerides, and free fatty acids according tostandard methodology by the Central Laboratory atThe Norwegian School of Veterinary Science. Intestinalcontents from PI1, PI2, MI, DI1 and DI2 were freezedried (Hetosicc Freeze drier CD 13-2 HETO, Birkerød,Denmark) prior to analyses. Diets and intestinal contentswere analysed for amino acids (Biochrom 30 AminoAcid Analyser, Biochrom, Cambridge, UK, afterhydrolysis according to EC Commission Directive 98/64/EC (1999)), nitrogen and sulphur (AutomaticElemental Analyser Flash EA 1112, reading the resultswith Eager 300 software, Thermo Electron Corporation,Waltham, MA, USA), and yttrium (by inductivitycoupled plasma (ICP) mass-spectroscopy, as previouslydescribed by Refstie et al. (1997)). Protein in

396 S. Refstie et al. / Aquaculture 261 (2006) 392–406

homogenates of intestinal tissues were analysed usingBioRad Protein Assay (BioRad Laboratories, Munich,Germany). Diets were also analysed for dry matter(105 °C to constant weight), ash (combusted at 550 °Cto constant weight), nitrogen (Kjeltec Auto Analyser,Tecator, Höganäs, Sweden), lipid (pre-extraction withdiethylether and hydrolysis with 4 M HCl prior topetroleum ether extraction (Stoldt, 1952) in a Soxtec(Tecator) hydrolysing (HT-6) and extraction (1047)apparatus), starch (determined as glucose after hydro-lysis by á-amylase and amylo-glucosidase, followed byglucose determination by the “GODPOD method”(Megazyme, Bray, Ireland)), and gross energy (Parr1271 Bomb calorimeter, Parr, Moline, IL, USA).

2.4. Enzyme assays

Trypsin and amylase activities were determinedcolourometrically in freeze dried intestinal contentsfrom PI1, PI2, MI, DI1 and DI2. Trypsin activity wasdetermined colorimetrically as described by Kakade et al.(1973) using the substrate benzoyl-arginine-p-nitroani-lide (BAPNA; Sigma no. B-4875, Sigma Chemical Co.,St. Louis, MO, USA) and a curve generated from astandardised bovine trypsin solution. For amylasemeasurements, the samples were suspended in aquadest. to a final concentration of 0.1mgml−1, vortexed for1 min, then centrifuged at 9300 ×g for 4 min at 4 °C.Amylase activity was measured in the supernatantimmediately following centrifugation by hydrolysis ofbenzylidene blocked p-nitrophenyl maltoheptaoside(pNPG7) using a Randox amylase assay kit (AY892,Randox Laboratories Ltd., Crumlin, UK). Absorbancewas measured with a Heλios α UV spectrophotometer(Thermo Spectronic, Cambridge, UK) in 4 cycles,1.5 min between subsequent readings for each sample.Trypsin and amylase activities were expressed both as Umg−1 dry intestinal contents (relative activity) and as U ineach intestinal section (total activity).

Activities of brush-border membrane bound ALP andLAP were determined in homogenates of intestinaltissue from PI, MI, and DI. ALP was also analysed instomach (ST) homogenates. The tissues were thawed,weighed and homogenized (1:20) in ice-cold 2 mM Tris/50 mM mannitol, pH 7.1, containing phenyl–methyl–sulphonyl fluoride (Sigma no. P-7626) as serineprotease inhibitor. Aliquots of homogenates were frozenin liquid N and stored at −80 °C prior to analysis. TheALP and LAP activities were determined colorimetri-cally as previously described by Krogdahl et al. (2003).Incubations were performed at 37 °C. Enzyme activitiesare expressed as mmol (ALP) or μmol (LAP) substrate

hydrolysed h−1 and related to g tissue, mg protein(specific activity), and whole tissue and kg BW of thefish.

2.5. Histological examination

Formalin fixed DI tissue was routinely dehydrated inethanol, equilibrated in xylene and embedded in paraffinaccording to standard histological techniques. Sectionsof approximately 5 μm were cut and stained withhaematoxylin and eosin before examination under alight microscope. Intestinal morphology was evaluatedaccording to the following criteria: (1) widening andshortening of the intestinal folds (2) loss of thesupranuclear vacuolisation in the absorptive cells(enterocytes) in the intestinal epithelium; (3) wideningof the central lamina propria within the intestinal folds,with increased amounts of connective tissue and (4)infiltration of a mixed leukocyte population in thelamina propria and submucosa. These are the character-istics of the condition previously described as SBM-induced enteritis in Atlantic salmon (Baeverfjord andKrogdahl, 1996).

2.6. Calculations

Crude protein (CP) was calculated as N×6.25.Protein was estimated after hydrolysing the protein foramino acid analysis as the sum of dehydrated aminoacids (as when peptide-bound). Apparent cumulativeabsorption of amino acid protein (Σ amino acids),cysteine, nitrogen, and sulphur in different intestinalsections was estimated by the indirect method, asdescribed by Maynard and Loosli (1969), using Y2O3 asan inert marker (Austreng et al., 2000). Absorption ofcysteine was estimated separately because of the highcontent of this sulphuric amino acid in endogenousdigestive enzymes.

2.7. Statistical analyses

The results were analysed by the General LinearModel procedure in the SAS computer software (SAS,1985). Mean results per tank were subjected to two-wayanalysis of variance (ANOVA) with interaction, withoxytetracycline (OTC; with or without) and Diet (FM,SBM, or inulin) as the independent variables. Theresults from the ANOVA are presented with the squareroot of the mean square error (

ffiffiffiffiffiffiffiffiffiffi

MSEp

) indicatingvariation, and as the proportion of total variationexplained by each of the factors and their interaction,calculated as the marginal contribution of the mean

Table 4Relative weights (g kg−1 BW) of the liver and different sections of thegastrointestinal tract (GIT; mean, n=2)

Liver Stomach Intestinal section TotalGIT

PI MI DI

OTCWithout 10.1a 5.7 17.9 1.9 4.3 29.8With 9.5b 5.9 16.9 1.9 4.3 29.0

DietFM 10.1a 5.6 15.7b 1.8y 4.3b 27.3b

SBM 9.0b 5.8 17.6ab 1.9xy 3.5c 28.7b

Inulin 10.4a 5.9 18.9a 2.0x 5.2a 32.0a

Two-way ANOVA:ffiffiffiffiffiffiffiffiffiffi

MSEp

0.3 0.2 1.4 0.1 0.2 1.5Proportion (type I SS) of variation (corrected total SS) explained (%)

by effect ofOTC 14.3⁎ 18.0 7.6 4.8 0.0 2.9Diet 66.3⁎⁎ 22.0 56.9⁎ 47.7⁎⁎⁎ 92.6⁎⁎⁎⁎ 69.1⁎

OTC×Diet 9.6 24.6 4.4 16.6 3.6 7.5

a,b,cDifferent superscripts indicate a statistical difference (P≤0.05).x,yDifferent superscripts indicate a statistical tendency (P≤0.1).⁎P≤0.05; ⁎⁎P≤0.01; ⁎⁎⁎P≤0.1; ⁎⁎⁎⁎P≤0.001.ffiffiffiffiffiffiffiffiffiffi

MSEp

is the square root of the mean square error.

397S. Refstie et al. / Aquaculture 261 (2006) 392–406

square of the parameter (type I sum of squares) as apercentage of the corrected total sum of squares.Significant differences among treatments were indicatedby least-squares means comparison. The level ofsignificance was chosen at P≤0.05, and the resultsare presented as group means.

3. Results

No fish died during the 21 days experimental feedingperiod. When terminating the experiment there were noeffects of treatment on final body weight and length,which ranged from 232–260 g among the feedinggroups.

3.1. Plasma chemistry

No clear effects of dietary supplementation ofoxytetracycline (OTC) on the plasma concentration offree fatty acids, glucose, cholesterol, or triacylglycerides(Table 3) were detected. There was, however, a tendencyfor higher plasma concentration of free fatty acids whenfeeding the OTC diets. The raw material use in the diets(Diet) did not affect the measured plasma chemistry.

3.2. Relative organ weights

Relative weights (g kg−1 BW) of the liver andgastrointestinal sections sampled for measurements ofbrush border membrane bound alkaline phosphatase(ALP) and leucine aminopeptidase (LAP) are given in

Table 3Concentration of glucouse, cholesterol, triglyderides (TG), and freefatty acids (FFA) in plasma (mean, n=2)

Glucose,mM

Cholesterol,mM

TG, mM FFA,mM

OTCWithout 5.43 8.56 2.26 0.36y

With 5.65 8.35 2.34 0.45x

DietFM 5.63 9.24 2.32 0.42SBM 5.59 8.12 2.34 0.38Inulin 5.40 8.00 2.25 0.43

Two-way ANOVA:ffiffiffiffiffiffiffiffiffiffi

MSEp

0.34 0.74 0.50 0.07Proportion (type I SS) of variation (corrected total SS) explained (%)

by effect ofOTC 11.0 1.7 1.1 34.8⁎

Diet 9.3 46.1 1.1 8.6OTC×Diet 28.0 11.1 12.4 10.5

x,yDifferent superscripts indicate a statistical tendency (P≤0.1).⁎P≤0.1.ffiffiffiffiffiffiffiffiffiffi

MSEp

is the square root of the mean square error.

Table 4. The relative liver weight was higher in fish feddiets without antibiotic supplementation than in fish fedOTC, but dietary OTC did not affect the relative weightof any gastrointestinal section. With regard to effects ofraw material use in the diets (Diet), the relative weight ofthe liver was similar in fish fed the FM control andinulin diets, but lower in fish fed the SBM diets. Therewas no effect of Diet on the relative weight of thestomach (ST). The relative weight of the pylorusintestine (PI) was higher in fish fed inulin than in fishfed FM, with intermediate weights in fish fed SBM. Asimilar tendency (Pb0.1) was seen for relative weight ofthe mid intestine (MI). The relative weight of the distalintestine (DI), however, was lower in fish fed SBM thanin fish fed FM and inulin. This caused a higher relativeweight of the total gastrointestinal tract (GIT) in fish fedinulin than in fish fed FM and SBM, but similar weightof the total GIT in fish fed FM and SBM.

3.3. Intestinal morphology

As judged by light microscopy, there were noapparent effects of dietary OTC on the morphology ofthe DI. In accordance with the descriptions ofBaeverfjord and Krogdahl (1996), all examined fishfed FM (12/12) and all but one examined fish fed inulin(11/12) showed normal morphology of the DI, char-acterised by the presence of well-differentiated enter-ocytes with many absorptive vacuoles. In contrast, allfish fed SBM showed moderate (8/12) to severe (4/12)

Table 5Trypsin activity in contents from the first and second halves of the pyloric intestine (PI1 and PI2), the mid intestine (MI), the first and second halves ofthe distal intestine (DI1 and DI2), and the total intestinal tract (IT; mean, n=2)

Relative trypsin activity, U mg−1 Total trypsin activity, U section−1

PI1 P121 MI DI1 DI2 PI1 P121 MI DI1 DI2 Total IT1

OTCWithout 118.6 93.2 108.3 64.2 37.8 66874 51219 126074 44994 22607 310923With 145.4 123.3 113.9 59.3 37.5 87045 68890 151960 50840 2728 387285

DietFM 159.9 153.5 135.4 60.3 16.6c 84926 71883 146431 45773 10355b 352521SBM 130.2 85.3 86.8 68.9 72.2a 87043 48610 123340 53464 46574a 359030Inulin 105.8 113.6 111.0 56.0 24.2b 58910 70373 147293 44516 17909b 346318

Two-way ANOVAffiffiffiffiffiffiffiffiffiffi

MSEp

31.9 67.3 49.5 14.6 4.2 24244 21460 35847 19963 6868 71830Proportion (type I SS) of variation (corrected total SS) explained (%) by effect ofOTC 14.1 11.3 0.5 2.1 0.0 18.0 9.5 17.6 2.9 2.0 32.5Diet 38.6 34.9 24.2 9.7 97.4⁎⁎⁎ 29.0 13.4 12.9 5.3 89.1⁎⁎⁎ 2.9OTC×Diet 7.2 0.3 52.4 1.2 1.1 2.1 24.6 0.2

a,b,cDifferent superscripts indicate a significant statistical difference (P≤0.05).⁎⁎⁎P≤0.001.ffiffiffiffiffiffiffiffiffiffi

MSEp

is the square root of the mean square error.1ANOVA calculated without interaction due to insufficient material for analysis from 3 experimental units and, thus, insufficient replication.

398 S. Refstie et al. / Aquaculture 261 (2006) 392–406

morphological changes in the DI consistent with SBMinduced enteritis. These changes included variabledegrees of inflammatory cell infiltration of the laminapropria, reduced vacuolisation of the enterocytes, andshortening of the villi.

3.4. Enzymology

No effects of dietary OTC were found on theactivities of trypsin and amylase in the intestinal

Table 6Amylase activity in contents from the first and second halves of the pyloric inof the distal intestine (DI1 and DI2), and the total intestinal tract (IT; mean,

Relative amylase activity, U mg−1 To

PI1 P12 MI DI1 DI2 PI

OTCWithout 2.99 2.91 3.17 2.27 1.73 17With 2.56 2.62 2.71 2.39 1.53 15

DietFM 2.63 2.65 2.57 1.89y 1.17b 14SBM 3.00 2.86 2.95 2.66x 2.05a 20Inulin 2.69 2.77 3.31 2.45xy 1.66ab 14

Two-way ANOVAffiffiffiffiffiffiffiffiffiffi

MSEp

0.62 0.46 0.48 0.42 0.38 6Proportion (type I SS) of variation (corrected total SS) explained (%) by effOTC 14.9 11.9 17.4 1.7 4.7Diet 8.2 4.3 29.8 52.2⁎ 58.6⁎⁎

OTC×Diet 16.7 24.7 15.2 2.5 3.9a,bDifferent superscripts indicate a significant statistical difference (P≤0.05)x,yDifferent superscripts indicate a statistical tendency (P≤0.10).⁎P≤0.10; ⁎⁎P≤0.05.ffiffiffiffiffiffiffiffiffiffi

MSEp

is the square root of the mean square error.

contents (Tables 5 and 6), nor on the activities of ALPand LAP in the brush border membrane of anygastrointestinal section (Table 7).

No effects of Diet were detected on the trypsinactivity (Table 5) in PI, MI, the first half of DI (DI1), orthe total intestinal tract. In the second half of DI (DI2),however, both the relative (U mg−1 DM) and the total(U) trypsin activities were significantly higher in fishfed SBM than in fish fed FM and inulin. The relativetrypsin activity in DI2 was intermediate in fish fed

testine (PI1 and PI2), the mid intestine (MI), the first and second halvesn=2)

tal amylase activity, U section−1

1 P12 MI DI1 DI2 Total IT

35 1370 3946 1521 1055 962641 1363 3788 2083 1102 9877

19 1069 2970y 1357 731y 7545y

15 1598 4216x 2099 1320x 11248x

80 1431 4415x 1949 1185x 10460x

43 347 803 515 312 1917ect of2.7 0.0 0.6 23.6 0.5 0.320.9 25.7 42.7⁎ 30.7 55.9⁎ 46.8⁎

16.1 42.6 23.0 6.1 0.7 19.1

.

Table 7Alkaline phosphatase (ALP) activity in the stomach (ST), pylorus intestine (PI), mid intestine (MI), distal intestine (DI), and the total gastrointestinaltract (GIT; mean, n=2)

ALP activity, μmol h−1 g−1 tissue ALP activity, μmol h−1 mg−1

proteinALP activity, μmol h−1 in whole tissue kg−1

BW

ST PI MI DI ST PI MI DI ST PI MI DI Total GIT

OTCWithout 0.08 0.18 0.15 0.10 0.67 1.75 2.04 1.35 0.42 3.08 0.28 0.42 4.20With 0.07 0.17 0.12 0.10 0.67 1.75 2.04 1.59 0.43 2.92 0.23 0.44 4.02

DietFM 0.07 0.19 0.13 0.12a 0.69 1.96 2.08 1.47ab 0.41 3.04 0.22 0.49a 4.16SBM 0.08 0.16 0.13 0.07b 0.67 1.62 1.90 1.13b 0.44 2.89 0.24 0.24b 3.82Inulin 0.07 0.17 0.15 0.11a 0.65 1.68 2.13 1.81a 0.42 3.07 0.29 0.57a 4.35

Two-way ANOVAffiffiffiffiffiffiffiffiffiffi

MSEp

0.01 0.02 0.05 0.01 0.05 0.34 0.27 0.23 0.03 0.50 0.10 0.07 0.49Proportion (type I SS) of variation (corrected total SS) explained (%) by effect of

OTC 2.2 1.1 10.9 1.5 0.7 0.0 0.0 9.6 2.7 3.3 10.8 0.4 2.9Diet 13.3 31.1 7.4 66.5⁎⁎ 19.1 22.9 14.5 51.1⁎ 11.5 2.8 13.9 74.9** 16.5OTC×Diet 13.8 26.1 7.5 22.4⁎ 0.4 17.8 30.5 20.6 36.5 32.5 6.1 15.2 39.5

a,bDifferent superscripts indicate a significant statistical difference (P≤0.05).⁎P≤0.05; ⁎⁎P≤0.01.ffiffiffiffiffiffiffiffiffiffi

MSEp

is the square root of the mean square error.

399S. Refstie et al. / Aquaculture 261 (2006) 392–406

inulin, while the total trypsin activity in DI2 was similarin fish fed FM and inulin, although the numericalranking was similar to that of the relative activity.

No effect of Diet was found on the relative amylaseactivity (mU mg−1 DM) in contents from PI and MI(Table 6). In DI2, however, the relative amylase activitywas highest in fish fed SBM, lowest in fish fed FM, andintermediate in fish fed inulin. A similar tendency(Pb0.1) was seen in DI1. There was likewise a tendency(Pb0.1) for higher total amylase activity (U) in MI, DI2,

Table 8Leucine aminopeptidase (LAP) activity in the stomach (ST), pylorus intestine(IT; mean, n=2)

LAP activity, mmol h−1 g−1

tissueLAP activity, μmoprotein

PI MI DI PI MI

OTCWithout 16.8 7.9 9.3 411.7 261.2With 16.0 7.7 10.1 398.2 257.7

DietFM 14.6 7.9 12.5a 366.2 275.1SBM 17.6 7.2 3.8b 436.8 235.6Inulin 17.0 8.3 12.7a 412.0 267.6

Two-way ANOVAffiffiffiffiffiffiffiffiffiffi

MSEp

2.6 1.1 1.2 64.7 53.5Proportion (type I SS) of variation (corrected total SS) explained (%) by eff

OTC 2.9 0.7 1.0 1.4 0.7Diet 30.1 5.4 89.6⁎⁎⁎ 26.0 11.1OTC×Diet 6.4 53.2 5.7 9.0 28.5

a,b,cDifferent superscripts indicate a significant statistical difference (P≤0.05⁎P≤0.05; ⁎⁎P≤0.01; ⁎⁎⁎P≤0.001.ffiffiffiffiffiffiffiffiffiffi

MSEp

is the square root of the mean square error.

and, thus, in the whole intestinal tract of fish fed SBMand inulin than in fish fed FM.

The activity of ALP in ST, PI, and MI did not differamong fish fed the different diets (Table 7). Likewise,the LAP activity in PI and MI was similar in all Dietgroups (Table 8). In DI the activities of both ALP andLAP were lower in fish fed SBM than in fish fed FM andinulin when related to g DI tissue or kg BW. However,as the contribution of DI to the total enzyme activity inthe gastrointestinal tissue was low, the total ALP and

(PI), mid intestine (MI), distal intestine (DI) and the total intestinal tract

l h−1 mg−1 LAP activity, mmol h−1 in whole tissue kg−1

BW

DI PI MI DI Total IT

350.4 288.4 14.2 42.0 344.5404.4 272.1 13.9 45.4 331.4

417.2b 226.3 13.6 53.2a 293.1176.0c 312.8 13.5 13.2b 339.5539.1a 301.5 15.1 64.7a 381.2

49.3 52.8 1.7 8.1 57.9ect of

3.6 2.0 0.3 0.5 1.286.1⁎⁎⁎ 45.0 9.0 87.1⁎⁎⁎ 35.25.5 10.4 64.9* 6.5 18.2

).

Fig. 1. Cumulative apparent absorption of amino acid protein (Σ AA), cysteine, nitrogen, and sulphur through successive sections of the intestinaltract of fish fed the FM, SBM or inulin diets. Different superscripts ab indicate a significant statistical difference (P≤0.05) and xy a statisticaltendency (P≤0.10) as indicated by least squares means comparison.

400 S. Refstie et al. / Aquaculture 261 (2006) 392–406

Table 9Two-way ANOVA for cumulative apparent absorption of amino acidprotein (Σ AA), cysteine, nitrogen and sulphur through successivesections of the intestinal tract

Absorbed Intestinalsection

ffiffiffiffiffiffiffiffiffiffi

MSEp

Proportion (type I SS) ofvariation (corrected total SS)explained (%) by effect of

OTC Diet OTC×Diet

Σ AA PI1 16.2 8.7 8.6 13.5PI2 8.2 5.1 42.2* 30.5MI 2.0 5.8 25.0 13.0DI1 5.3 16.4 0.4 14.8DI2 6.3 3.5 13.7 7.0

Cysteine PI1 31.5 13.8 17.8 7.1PI2 31.1 3.3 48.7⁎⁎ 14.2MI 4.9 10.2 45.8⁎⁎ 6.0DI1 13.2 23.2 5.5 10.3DI2 14.7 7.7 5.8 11.4

Nitrogen PI1 18.7 1.1 7.1 14.7PI2 17.3 0.0 45.4⁎⁎ 22.6MI 2.8 3.3 34.7 15.2DI1 8.4 17.9 2.5 17.2DI21 11.5 16.6 4.8

Sulphur PI1 39.1 1.8 6.5 20.0PI2 32.0 0.2 41.5⁎⁎ 32.0MI 6.4 0.1 35.1 10.1DI1 15.6 19.2 8.8 23.0DI21 17.2 0.2 10.4

⁎P≤0.05; ⁎⁎P≤0.1.ffiffiffiffiffiffiffiffiffiffi

MSEp

is the square root of the mean square error.1ANOVA calculated without interaction due to insufficient material foranalysis from 2 (nitrogen) or 1 (sulphur) experimental units and, thus,insufficient replication.

401S. Refstie et al. / Aquaculture 261 (2006) 392–406

LAP activities in the whole intestinal tract related to BWwere not affected by Diet.

When related to mg protein, the activities of both ALPand LAP in DI tissue were highest in fish fed inulin,lowest in fish fed SBM, and intermediate in fish fed FM.There was also a general tendency (Pb0.1) in the two-way ANOVA for a statistical interaction between theindependent variables OTC and Diet on ALP and LAPactivities in the DI. As indicated by least square meanscomparison, this was caused by generally higher activityin fish fed inulin than in fish fed FM when the diets weresupplemented with OTC, while the activity was similarin fish fed these diets without OTC (data not shown). ForALP the activity was also similar in fish fed FM andSBM with OTC, but lower in fish fed SBM than in fishfed FM without OTC (data not shown).

3.5. Apparent amino acid absorption

The cumulative apparent absorption of amino acidprotein (Σ individual amino acids) and nitrogen in

successive intestinal sections increased gradually fromPI1 to MI, but was unchanged from MI to DI2 (Fig. 1).A similar absorption pattern was seen for the sulphuricamino acid cysteine and for sulphur, but for thesecomponents the apparent absorption was negative in PI1and P2 when feeding all diets. The only exception fromthis was a positive but still very low (9%) apparentabsorption of cysteine in PI2 when feeding the SBMdiets.

Dietary OTC did not affect the apparent absorption ofamino acids, nitrogen, or sulphur significantly (Table 9and Fig. 1). The apparent amino acid absorption in PI2was, however, higher when feeding the SBM diets thanwhen feeding FM and inulin diets. There were alsotendencies (Pb0.1) for higher apparent absorption ofcysteine, nitrogen, and sulphur in PI2 when feeding theSBM diets than when feeding the FM diets, and theapparent absorption of cysteine also tended (Pb0.1) tobe higher in MI when feeding the SBM diets.

There was a tendency (Pb0.1) for a statistical inter-action in the two-way ANOVA between the independentvariables OTC and Diet on apparent amino acidabsorption in PI2. This was caused by higher absorptionwhen feeding the inulin diet with OTC (55%) than whenfeeding it without OTC (31%).

4. Discussion

The main findings of this experiment were thatdietary oxytetracycline (OTC) did not alter the capacityfor digestive hydrolysis and absorption of amino acids,nitrogen, and sulphur in Atlantic salmon. Apart fromincreasing the activity of brush border enzymes in thedistal intestine when feeding inulin, OTC did not modifythe digestive responses to dietary soybean meal (SBM)and inulin. Dietary SBM caused pathomorphologicalchanges that reduced the hydrolytic efficiency in thedistal intestine, and also resulted in increased trypsin andamylase activity in the distal intestine that indicatedreduced re-absorption and increased faecal losses ofthese endogenous enzymes. However, SBM at the sametime stimulated more efficient apparent absorption ofamino acids, nitrogen and sulphur in the proximalintestine, regardless of OTC supplementation. Dietaryinulin caused no apparent intestinal damage, butstimulated intestinal growth resulting in increasedrelative mass of the gastrointestinal tract.

During the 2 weeks period of adaptation to theexperimental facilities and subsequent 3 weeks offeeding the experimental diets, the fish grew at specificgrowth rates (SGRs) ranging from 0.84–1.07, averaging0.95. This was slightly slower than the expected SGR of

402 S. Refstie et al. / Aquaculture 261 (2006) 392–406

Atlantic salmon of similar size when grown at similartemperature (Austreng et al., 1987). However, thegrowth period was short, and appetite in Atlanticsalmon is low during the first days after handling(Refstie and Tiekstra, 2003; Refstie et al., 1998, 2004).As the diet was furthermore changed from an extrudedsalmon diet to the unfamiliar cold pelleted experimentaldiets in the middle of the period, appetite and, thus,growth were considered within the normal range. Due tolow water stability of the diets, the uneaten feed couldnot be collected from the water to calculate accurate feedintake (Helland et al., 1996). However, as the ration wassimilar in all groups, and as there were no differences ingrowth among the feeding groups, the feed intake can beassumed to have been similar in all groups.

Dietary SBM contains components that bind bileacids in the intestine (Storebakken et al., 2000), therebypotentially increasing the faecal steroid and lipid loss. InSBM-fed fish this is indicated by lowered plasmacholesterol (Kaushik et al., 1995; Refstie et al., 1999),changes in cholesterol metabolising hepatic enzymes(Martin et al., 2003), and increased cholesterol require-ment (Twibell and Wilson, 2004). The lacking effects ofdietary SBM on plasma cholesterol and lipids in thepresent salmon indicate that cholesterol metabolismchanges gradually over time in response to soy, and thata three-week feeding period is too short to inducenoticeable effects.

The cause(s) of the reduced relative liver weight(g kg−1 BW) when feeding OTC or SBM is not apparentfrom this study. However, therapeutic administration ofantibiotics may cause liver degenerations in mammals(Lown, 1998), and this may also have been the case inthe OTC-fed salmon. Dietary SBM on the other hand isreported to cause histological changes and lipidaccumulation in the liver of the salmonid rainbowtrout (Oncorhynchus mykiss; Ostaszewska et al., 2005).Profiling of liver proteins from SBM-fed rainbow troutfurthermore indicates shifts toward hepatic catabolicpathways, increased or inefficient protein turnover,down-regulation of structural protein expression,heightened immune response, and altered levels ofstress proteins (Martin et al., 2003).

The increased relative weight of all intestinal sectionswhen feeding inulin, and the similar numerical trend forrelative weight of the proximal intestine when feedingSBM, were probably caused by high content ofindigestible material in these diets. This is a wellknown effect in livestock, where hypertrophy ingastrointestinal organs is stimulated by workload whenfeeding high fibre and low-energy diets due to greaterfilling and increased peristaltic activity (Koong et al.,

1985; Sainz and Bentley, 1997; McLeod and Baldwin,2000).

As a prebiotic (Pool-Zobel et al., 2002; Roberfroid,2002; Flickinger et al., 2003), dietary inulin may havealtered the intestinal microbiota in the salmon. Nucleo-tides, which have prebiotic properties (Uauy, 1994),have been shown to accelerate growth in the intestinalmucosa of Atlantic salmon (Burrells et al., 2001).Prebiotic effect(s) of inulin may, thus, also have played arole in stimulating intestinal growth in the inulin-fedsalmon. Reduced mass of the distal intestine in salmonfed SBM was in response to the SBM-inducedpathomorphological changes in this intestinal section,as previously shown by Nordrum et al. (2000).

The increased trypsin activity in the last part of thedistal intestine was in keeping with Krogdahl et al.(2003), who found the faecal trypsin concentration inAtlantic salmon to increase in response to the dietarylevel of soybean meal. The present lack of significantdifferences in trypsin and amylase activity in the pyloricand mid intestine was to some extent caused by highvariation within feeding groups and, thus, low statisticalpower. Still, when feeding the fish meal (FM) controldiets about 85% of the total trypsin activity was found inthese proximal intestinal sections. It was a little lower(about 80%) when feeding the inulin diets, butdramatically lower (about 65%) when feeding theSBM diets, and this was caused by high trypsin activityin the last half of the distal intestine. A similar trend wasseen for amylase, where about 72% of the total activitywas found in the pyloric and mid intestine when feedingthe FM diets, but only 65 to 70% when feeding the SBMand inulin diets.

Elevated faecal trypsin was also found in salmonidsfed purified soy trypsin inhibitors (Krogdahl et al.,1994; Olli et al., 1994). Thus, Krogdahl et al. (2003)suggested that faecal trypsin loss in soybean meal fedsalmonids is caused by trypsin inhibitors in the intestinalcontents, and potentially worsened by pancreatic growthand hypersecretion of pancreatic enzymes. However, atthe given SBM inclusion the trypsin inhibitor activity(TIA) in the present SBM diets should by calculation beonly 0.5 mg trypsin inhibited g−1 meal. This is lowerthan the TIA tolerance in salmonids (Krogdahl et al.,1994; Olli et al., 1994) by an order of magnitude. Thus,the major cause for faecal loss of trypsin in the SBM fedsalmon appeared to be a loss in the ability to reabsorbendogenous digestive secretions due to the pathomor-phological changes in the distal intestine. This issupported by the concomitant increase in faecal amylasewhen feeding the SBM diets. The results also indicatethat dietary inulin increases the faecal loss of pancreatic

403S. Refstie et al. / Aquaculture 261 (2006) 392–406

enzymes in salmon slightly, but the reasons for thisremain unclear.

The amylase activity in mid and distal intestinalcontents appeared to be inversely related to the wheatand, thus, starch level in the diets. Atlantic salmon haslimited capacity to digest starch (Krogdahl et al., 2005).In consequence the amylase activity in fish fed the FMdiets may have been reduced by adsorption of amylaseto starch molecules (Spannhof and Plantikow, 1983)and/or binding of amylase to wheat amylase inhibitors(Sturmbauer and Hoffer, 1985; Franco et al., 2002).

When feeding the FM and inulin diets, 86 and 82% ofthe respective total activities of the brush bordermembrane bound enzymes alkaline phosphatase (ALP)and leucine aminopeptidase (LAP) were found in thepyloric and mid intestine. The reduced ALP and LAPactivities in the distal intestine of salmon fed the SBMdiets were in keeping with previous findings (Krogdahlet al., 1995, 2003; Bakke-McKellep et al., 2000a).Quantitatively these differences were, however, muchtoo small to noticeably affect the overall ALP and LAPactivities in the intestinal tissue. Increased number ofproliferating cells lining the villous folds of the distalintestine in salmon fed soybean meal (Sanden et al.,2005) suggests that reduced activity of brush bordermembrane bound enzymes in distal intestine is causedby alterations in enterocyte turnover and, thus, degree ofmaturation and functionality of the enterocytes. In thelight of the present results, one may speculate if elevatedtrypsin concentration in the distal intestine of soybeanmeal fed salmon is damaging the enterocytes in thedistal mucosa.

Elevated ALP and LAP specific activities (mg−1 pro-tein) in the distal intestine of inulin fed fish furthermoreindicate that dietary inulin stimulated synthesis andincorporation of hydrolases into the distal brush bordermembrane. As this effect was only apparent when feedinginulin in combination with OTC, it may have been inresponse to prebiotic stimulation of beneficial bacteriapopulations at a concomitant general reduction of theintestinal microbiota. Thus, and since dietary inulinstimulated intestinal growth, the prebiotic effect of inulinin Atlantic salmon merits further investigation.

Unlike mammals, amino acid transporters are presentalong the entire length of the intestine of fish(Buddington et al., 1997), although the absorptiveefficiency is highest in the proximal intestine (Budding-ton and Diamond, 1987; Bakke-McKellep et al., 2000b;Nordrum et al., 2000). Thus, the high proteolyticcapacity (trypsin and LAP activities) in the proximalintestine of the present salmon was mirrored by efficientapparent amino acid absorption, which at least quanti-

tatively appeared to be completed in the mid intestine, inkeeping with previous results (Austreng, 1978; Dab-rowski and Dabrowska, 1981, Krogdahl et al., 1999).The consistent negative apparent absorption of cysteineand sulphur and the lower apparent absorption ofnitrogen than of amino acids in the pyloric intestineillustrate the impact of exocrine secretion of cysteine-rich components like pancreatic enzymes and glutathi-one, and of nitrogen and sulphur rich non-proteincomponents like bile acids (taurocholate) into the lumenof this intestinal section. Low apparent absorption ofcysteine in the pyloric intestine of salmonids has alsobeen shown previously (Dabrowski and Dabrowska,1981; Krogdahl et al., 1999), although not as low as tobe estimated as negative absorption. Incomplete re-absorption of endogenous digestive secretions may inpart account for the low overall apparent absorption ofcysteine and sulphur. Structural disulphide cross-linkingbetween cysteine sulphydryl groups in proteins mayalso reduce the general availability of cysteine (Opstvedtet al., 1984), although this appears to depend on theaccessibility of the disulphide bonds for cleavage in theintestine (Aslaksen et al., 2006).

The fast apparent absorption of amino acids, nitrogenand sulphur in salmon fed the SBM diets indicatesstimulation of active amino acid transport in theproximal intestine, possibly as a feed-back response toreduced active transport in the distal intestine (Nordrumet al., 2000). However, despite the present lack ofdietary effects on proximal trypsin activity, effects ofSBM on secretion of other pancreatic proteases cannotbe ruled out. Haard et al. (1996) also found differencesin the catalytic properties of trypsin, as seen fromreduced trypsin inhibitor sensitivity, and changes in thebalance of digestive proteases, as seen from increases intotal enzyme concentration, in the pyloric intestine ofCoho salmon (Oncorhynchus kisutch) fed soybean meal.Thus, rapid proximal acid amino absorption whenfeeding the present SBM diets may also have resultedfrom more efficient protein hydrolysis due to pancreaticsecretion of different trypsin isozymes and/or increasedsecretion of other proteases.

Still, dietary SBM did not affect the total apparentabsorption of amino acids measured in the last half ofthe DI. This is in line with previous experiments whereAtlantic salmon were fed diets containing 20 to 30%SBM, showing only slight (Refstie et al., 1998, 2001,2005; Storebakken et al., 1998) or no (Storebakkenet al., 1998; Refstie et al., 2000, 2001) negative effect ofSBM on the apparent faecal protein digestibility. Thus,the true availability for salmon of amino acids inproperly processed SBM appears as high as or higher

404 S. Refstie et al. / Aquaculture 261 (2006) 392–406

than that of LT-FM protein. However, as the endogenousamino acid losses are higher when feeding SBM, the neteffect on apparent amino acid absorption may benegative, in particular for cysteine.

To conclude, ≥80% of the activities of trypsin, ALPand LAP and about 70% of the amylase activity werefound in the proximal and mid intestine of Atlanticsalmon with functional distal intestine. The apparentabsorption of amino acids, nitrogen, and sulphur was alsoquantitatively completed in the mid intestine. Feedingdiets supplemented with oxytetracycline (OTC) resultedin reduced relative liver weight. Apart from increasedactivity of brush border enzymes in the distal intestinewhen feeding OTC in combination with inulin, OTC didnot modify how dietary soybean meal or inulin affectedgut morphology, hydrolysis of protein and starch, andabsorption of amino acids, nitrogen and sulphur. Dietarysoybean meal resulted in reduced relative liver weight,pathomorphological changes and lowered hydrolyticefficiency in the mucosa of the distal intestine, andapparently reduced re-absorption and, thus, increasedfaecal losses of endogenous digestive enzymes. Howev-er, soybean meal also stimulated absorption of aminoacids, nitrogen, and sulphur in the proximal intestine,resulting in a neutral net effect on the total apparent aminoacid absorption. Dietary inulin did not damage the distalenterocytes, but stimulated growth in intestinal tissuesthat resulted in increased relative mass of the gastroin-testinal tract. Apart from the positive effect of inulin fedin combination with OTC on the hydrolytic capacity ofthe distal intestine, dietary inulin did not affect thehydrolytic and absorptive capacity of salmon.

Acknowledgements

The authors wish to acknowledge the skilfultechnical assistance of Ellen Koren Hage, Gunn Østby,and Kristin Vekterud at the Norwegian School ofVeterinary Science. We are grateful to Suiker Unie forsupplying inulin. Financial support for the study wasprovided by the Research Council of Norway (grant #145949/120) and the Norwegian School of VeterinaryScience via the CoE Aquaculture Protein Centre (APC).

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