Capacity for digestive hydrolysis and amino acid absorption in Atlantic salmon (Salmo salar) fed diets with soybean meal or inulin with or without addition of antibiotics

Aquaculture 261 (2006) 392 – 406 www.elsevier.com/locate/aqua-online

Capacity for digestive hydrolysis and amino acid absorption in Atlantic 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, Norway AKVAFORSK (Institute of Aquaculture Research AS), N-6600 Sunndalsøra, Norway Norwegian School of Veterinary Science, Department of Basic Sciences and Aquatic Medicine, P.O.Box 8146 Dep, N-0033 Oslo, Norway d Northwest Fisheries Science Center, NOAA/NMFS, Seattle, WA, USA b

c

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 for digestive hydrolysis and amino acid absorption by Atlantic salmon, and how a dietary supplement of the broad-spectrum antibiotic oxytetracycline (OTC) modulated these responses. A control diet (FM) was made from fish meal, fish oil and extruded wheat. Two similar diets were made with 250 g soybean meal (SBM) or 75 g inulin kg− 1. Each diet was made with or without a supplement of 3 g OTC kg− 1. All six diets contained yttrium oxide for estimation of apparent nutrient absorption. Each diet was fed to two groups of 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 different sections 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% of the 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 ALP and LAP activities in DI when feeding OTC in combination with inulin, OTC did not modify the responses to dietary SBM or inulin. Dietary SBM resulted in lower relative liver weight, and induced pathomorphological changes in the DI mucosa, thus lower the 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 the activities of trypsin and amylase in the DI, indicating reduced re-absorption and increased faecal losses of these endogenous enzymes. Dietary inulin did not damage the DI, and stimulated intestinal growth and higher relative mass of the GIT. Inulin without OTC 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: [email protected] (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

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1. Introduction Due to steady supply, constant composition, low protein price, and high content of available protein with well balanced amino acid profile, full fat and extracted soybean meals are potentially very good protein ingredients for fish. However, soybean meals contain several potent antinutritional factors that disturb the digestive process in carnivorous fishes (Storebakken et al., 2000). Among these, still unidentified heat stabile and alcohol soluble soy component(s) cause pathomorphological changes 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 and Krogdahl (1996). In short they are characterised by shortening of the primary and secondary mucosal folds with a widening of the central stroma (lamina propria) and submucosa, shortened microvilli of the brush border membrane and increased formation of microvillar vesicles, and a dramatic decrease or even absence of the normal supranuclear absorptive vacuoles in the enterocytes. The lamina propria is widened with a profound infiltration of a mixed population of inflammatory cells. These morphological changes reduce the mass of the distal intestine in salmon (Nordrum et al., 2000). The digestive process is also altered. The activity of brush border membrane bound (Krogdahl et al., 1995; BakkeMcKellep et al., 2000a; Krogdahl et al., 2003) and cytosolic (Bakke-McKellep et al., 2000a) digestive enzymes in the distal enterocytes is reduced, and the carrier-mediated transport of amino acids and glucose is lowered while the permeability of distal intestinal epithelium for nutrient transport is increased (Nordrum et al., 2000). The absorption of macromolecules by the distal intestine is also decreased (Bakke-McKellep, 1999), apparently causing reduced re-absorption of endogenous digestive secretions, as indicated by dramatically increased activity of trypsin in the distal intestine (Dabrowski et al., 1989; Krogdahl et al., 2003). Due to infiltration of inflammatory cells in the intestinal mucosa and rapid regression of the condition following withdrawal of soybean meal from the diet, the pathomorphological changes have been classified as non-infectious, sub-acute soybean meal-induced enteritis (Baeverfjord and Krogdahl, 1996), suggesting an etiology involving immunological mechanisms. Increased number of proliferating cells lining the villous folds of the distal intestine of soybean meal fed salmon (Sanden et al., 2005) suggests disturbed functionality of enterocytes due to alterations in enterocyte turnover and degree of maturation.

393

It is possible that the intestinal microbiota is involved in the development of soybean meal-induced enteritis. As in terrestrial animals, the intestinal microbiota in fish is affected by diet (Cahill, 1990; Ringø et al., 1995; Ringø and Olsen, 1999), and the gastrointestinal tract appears as a major route of infection in fish (Ringø et al., 2004; Birkbeck and Ringø, 2005). In this context, physical damage to the mucosa by indigestible plant structural components may also play a role. Supporting this, Olsen et al. (2001) noted a destructive effect of high dietary levels (150 g kg− 1) of inulin on the enterocytes in the distal intestine of the salmonid Arctic charr (Salvelinus alpinus), possibly caused by inulin absorption and accumulation. Inulin is a large oligosaccharide naturally occurring in many plants, and is produced commercially from the chicory (Cichorium intybus) root. It is a fructosan consisting of fructose monomers linked in linear chains of varying length by β(2–1) bonds, and with terminal glucose moieties (Roberfroid et al., 1998; Pool-Zobel et al., 2002). Inulin cannot be hydrolysed by pancreatic or brush border digestive enzymes in the intestine of monogastric animals (Pool-Zobel et al., 2002), but is fermented in the large intestine or colon (Roberfroid, 2002; Flickinger et al., 2003), where it enhances the relative populations of bifidobacteria and other lactic acid producing bacteria (Pool-Zobel et al., 2002). It is therefore considered a prebiotic, which are defined as non-digestible feed ingredients that benefit the host by selectively stimulating beneficial bacterial species already resident in the intestine (Gibson and Roberfroid, 1995; Grittenden and Playne, 1996). As such, moderate dietary levels (20 g kg− 1) of inulin have been applied attempting to establish a stable and healthy intestinal microbiota in larvae of the carnivorous marine turbot (Psetta maxima; Mahious et al., 2006). Based on this, the objectives of this work were 1) to evaluate effects of dietary soybean meal on the capacity for digestive hydrolysis and absorption of amino acids in Atlantic salmon, 2) to compare the digestive effects of dietary soybean meal with those produced by dietary inulin, and 3) to evaluate how a dietary supplement of the broadspectrum antibiotic oxytetracycline (OTC) modulates the digestive responses in salmon to soybean meal and inulin. 2. Materials and methods 2.1. Ingredients and diets The low-temperature dried (LT) fishmeal (FM; Norse LT-94, Vedde Herring Oil Factory, Egersund, Norway) was made from 80% blue whiting, 10% herring, and 10% processing offal from herring and mackerel. This LT-FM

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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 in mink. The soybean meal (SBM) was dehulled, extracted, and toasted, and was declared to have a trypsin inhibitor activity (TIA) of 4.5 mg trypsin inhibited per g protein. The inulin powder (Frutanimal ND, Suker Unie, Dinteloord, the Netherlands) was extracted from chicory root, and was declared to contain ≥ 85% inulin with chain lengths ranging from 2 to 60 and averaging 8 to 11 fructose moieties. Six diets with a pellet size of 4 mm were produced on a laboratory cold pellet press at AKVAFORSK. The diets were formulated to contain three different combinations of raw materials: 1) 100% of crude protein (CP) from LT-FM; 2) LT-FM and 24% of CP from SBM, and 3) 100% of CP from LT-FM, but added 7.5% inulin. Each of these three diets was produced with or without a supplement of 3 g oxytetracycline hydrochloride (OTC; Norsk medisinaldepot, Oslo, Norway) kg− 1. All diets were formulated to contain 555 g crude protein, 190 g lipid, and from 41 to 114 g starch (DM basis), and to be iso-energetic on a gross energy basis. The calculated concentration of non-starch polysaccharides was similar in the SBM and inulin diets. The diets were supplemented with D,L-methionine to contain similar amounts of (calculated) methionine. All diets contained 100 mg yttrium oxide kg− 1 dry mix as an inert marker to permit apparent absorption measurements. 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 with laws and regulations that control experiments and procedures in live animals in Norway, as overseen by the Norwegian Animal Research Authority. The experiment was done at AKVAFORSK (Sunndalsøra, Norway), where seawater adapted Atlantic salmon (Salmo salar) were fed the experimental diets for a total of 21 days. Fourteen days prior to the experiment, 12 groups of salmon (172 g, 37 fish/group) were randomly distributed from a holding tank to fibreglass tanks (1 × 1 × 0.6 m, water depth 40–50 cm) supplied with seawater. The fish were continued on a commercial diet (Skretting AS, Stavanger, Norway) until day 15, when the experimental diets were randomly allocated to 2 groups of fish each. The fish were then fed the experimental diets for 21 feeding days. The fish were fed continuously (24 hr d− 1) by electrically driven disc feeders, aiming for 15% overfeeding based on expected

Table 1 Formulation of the diets Diet code

FM

FM + OTC

SBM

SBM + OTC

Formulation, g kg − 1 LT-fish meala 696 696 535 535 Soybean mealb 250 250 Fish oil 120 120 132 132 Extruded wheat 164 161 61 58 Inulinc Oxytetracyclined 3 3 e DL-methionine 2 2 Premixf 19.9 19.9 19.9 19.9 Yttrium oxideg 0.1 0.1 0.1 0.1

Inulin Inulin + OTC

710

710

119 76 75

119 73 75 3

19.9 0.1

19.9 0.1

a

Norse LT-94 (Vedde Herring Oil Factory, Langevåg, Norway). Extracted soybean meal (Hamlet, Horsens, Denmark). c Frutanimal ND (Suiker Unie, Dinteloord, The Netherlands). d Oxytetracycline hydrochlorid (Norsk medisinaldepot, Oslo, Norway). e D,L-methionine (Degussa, Hanau, Germany). f Vitamin and mineral premix (FeedTech, Ås, Norway). g Sigma (St. Louis, Mo, USA). b

feed intake. The water temperature during the experimental period ranged 8 to 10 °C, and the O2 saturation of the outlet water was above 80%. At feeding day 21, 21 fish randomly selected from each tank were euthanised in water with a lethal concentration of tricaine methanesulfonate (MS 222, Argent Chemical Laboratories Inc., Redmont, Wa, USA), weighed individually, and the gastrointestinal tracts (GITs) were dissected out. Five fish per tank were sampled for analysis of alkaline phosphatase (ALP) and leucine aminopeptidase (LAP) activities. These GITs were sectioned into stomach (ST); pyloric intestine (PI), defined as the intestine from the most proximal to the most distal pyloric caeca; mid intestine (MI), defined as the intestine between the most distal pyloric caeca and the appearance of transverse luminal folds and increase in intestinal diameter, and; distal intestine (DI), defined as the region characterised by the transverse luminal folds and increased intestinal diameter to the anus. Surrounding adipose and connective tissues were carefully removed, the sections cut open and rinsed (with the exception of the pyloric caeca) before frozen in liquid nitrogen and stored at − 80 °C. Liver was also sampled from this fish and weighed individually. Blood and intact intestines were furthermore sampled from 10 fish per tank. Blood was collected from the caudal vein into vacutainers containing anticoagulant (EDTA) and protease inhibitor (Pefabloc® SC, Sigma no. 76307, Sigma Chemical Co., St. Louis, MO, USA). Samples were kept on ice until centrifugation at

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Table 2 Composition of the diets Diet code

FM

FM + OTC

SBM

SBM + OTC

Inulin

Inulin + OTC

925.7

927.7

923.0

922.6

918.3

924.9

542.1 445.3 209.6 89.9 125.6 0.1 22.6

539.8 450.0 217.7 85.0 120.6 0.1 22.9

544.9 454.7 208.0 32.1 112.8 0.1 23.0

542.6 457.5 207.0 29.3 111.6 0.1 23.1

545.7 450.4 193.8 36.2 127.1 0.1 22.4

534.2 444.4 196.8 39.2 126.7 0.1 22.6

6.0 2.0 4.3 7.9 7.7 2.9 3.9 4.2 1.2 5.0

6.1 2.1 4.4 8.1 7.8 2.9 4.0 4.3 1.3 5.1

6.4 2.2 4.4 7.9 7.4 2.7 4.2 4.1 1.3 5.1

6.3 2.2 4.5 8.0 7.4 2.7 4.2 4.0 1.4 5.4

6.2 2.0 4.4 8.0 7.9 2.9 3.8 4.3 1.3 4.8

6.2 2.0 4.5 8.0 7.9 2.9 3.9 4.4 1.3 4.9

6.3 9.8 0.9 15.0 6.3 4.1 4.4 3.6

6.3 10.0 1.0 15.2 6.4 4.2 4.4 3.6

5.8 10.2 1.1 15.5 5.9 4.8 4.5 3.7

6.3 10.2 1.2 15.8 5.9 4.5 4.5 3.7

6.5 10.1 0.9 14.7 6.5 3.7 4.4 3.6

6.6 10.2 1.0 14.9 6.5 3.7 4.5 3.6

−1

Composition, g kg Dry matter (DM), g kg− 1 In DM Crude proteina (CP), g kg− 1 Amino acid proteinb, g kg− 1 Lipid, g kg− 1 Starch, g kg− 1 Ash, g kg− 1 Yttrium oxide, g kg− 1 Energy, MJ kg− 1 In CP, % (g 16− 1 g N) Essential amino acidsc Arginine Histidine Isoleucine Leucine Lysine Methionine Phenylalanine Threonine Tryptophan Valine Non essential amino acidsc Alanine Aspartate + asparagine Cysteine Glutamate + Glutamine Glycine Proline Serine Tyrosine a

N × 6.25. Expressed as the sum of peptide-bound (dehydrated) amino acids. c Expressed as free amino acids. b

3000 rpm for 10 min. Plasma samples were aliquoted into three separate Eppendorf tubes, frozen in liquid nitrogen and stored at − 80 °C until analysis. The intact intestines were sampled for estimation of apparent amino acid absorption and activities of trypsin and amylase, and were wrapped in aluminium foil, frozen in liquid nitrogen and stored at − 40 °C. Frozen intestinal contents were sampled from the same GIT sections as described above after careful thawing of the intestinal wall, and the contents were pooled per tank for analysis. For this sampling the PI was further subdivided into proximal, PI1, and distal, PI2, portions, and the DI into proximal, DI1, and distal, DI2, portions. From the last six fish sampled per tank, a 5 mm tissue sample was cut (a transverse cut relative to the length of the tract) from the central area of DI. These samples were 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 to standard methodology by the Central Laboratory at The Norwegian School of Veterinary Science. Intestinal contents from PI1, PI2, MI, DI1 and DI2 were freeze dried (Hetosicc Freeze drier CD 13-2 HETO, Birkerød, Denmark) prior to analyses. Diets and intestinal contents were analysed for amino acids (Biochrom 30 Amino Acid Analyser, Biochrom, Cambridge, UK, after hydrolysis according to EC Commission Directive 98/ 64/EC (1999)), nitrogen and sulphur (Automatic Elemental Analyser Flash EA 1112, reading the results with Eager 300 software, Thermo Electron Corporation, Waltham, MA, USA), and yttrium (by inductivity coupled plasma (ICP) mass-spectroscopy, as previously described by Refstie et al. (1997)). Protein in

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homogenates of intestinal tissues were analysed using BioRad Protein Assay (BioRad Laboratories, Munich, Germany). Diets were also analysed for dry matter (105 °C to constant weight), ash (combusted at 550 °C to constant weight), nitrogen (Kjeltec Auto Analyser, Tecator, Höganäs, Sweden), lipid (pre-extraction with diethylether and hydrolysis with 4 M HCl prior to petroleum ether extraction (Stoldt, 1952) in a Soxtec (Tecator) hydrolysing (HT-6) and extraction (1047) apparatus), starch (determined as glucose after hydrolysis by á-amylase and amylo-glucosidase, followed by glucose determination by the “GODPOD method” (Megazyme, Bray, Ireland)), and gross energy (Parr 1271 Bomb calorimeter, Parr, Moline, IL, USA). 2.4. Enzyme assays Trypsin and amylase activities were determined colourometrically in freeze dried intestinal contents from PI1, PI2, MI, DI1 and DI2. Trypsin activity was determined colorimetrically as described by Kakade et al. (1973) using the substrate benzoyl-arginine-p-nitroanilide (BAPNA; Sigma no. B-4875, Sigma Chemical Co., St. Louis, MO, USA) and a curve generated from a standardised bovine trypsin solution. For amylase measurements, the samples were suspended in aqua dest. to a final concentration of 0.1 mg ml− 1, vortexed for 1 min, then centrifuged at 9300 ×g for 4 min at 4 °C. Amylase activity was measured in the supernatant immediately following centrifugation by hydrolysis of benzylidene blocked p-nitrophenyl maltoheptaoside (pNPG7) using a Randox amylase assay kit (AY892, Randox Laboratories Ltd., Crumlin, UK). Absorbance was 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 U mg− 1 dry intestinal contents (relative activity) and as U in each intestinal section (total activity). Activities of brush-border membrane bound ALP and LAP were determined in homogenates of intestinal tissue from PI, MI, and DI. ALP was also analysed in stomach (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 serine protease inhibitor. Aliquots of homogenates were frozen in liquid N and stored at − 80 °C prior to analysis. The ALP and LAP activities were determined colorimetrically as previously described by Krogdahl et al. (2003). Incubations were performed at 37 °C. Enzyme activities are 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 the fish. 2.5. Histological examination Formalin fixed DI tissue was routinely dehydrated in ethanol, equilibrated in xylene and embedded in paraffin according to standard histological techniques. Sections of approximately 5 μm were cut and stained with haematoxylin and eosin before examination under a light microscope. Intestinal morphology was evaluated according to the following criteria: (1) widening and shortening of the intestinal folds (2) loss of the supranuclear vacuolisation in the absorptive cells (enterocytes) in the intestinal epithelium; (3) widening of the central lamina propria within the intestinal folds, with increased amounts of connective tissue and (4) infiltration of a mixed leukocyte population in the lamina propria and submucosa. These are the characteristics of the condition previously described as SBMinduced enteritis in Atlantic salmon (Baeverfjord and Krogdahl, 1996). 2.6. Calculations Crude protein (CP) was calculated as N × 6.25. Protein was estimated after hydrolysing the protein for amino acid analysis as the sum of dehydrated amino acids (as when peptide-bound). Apparent cumulative absorption of amino acid protein (Σ amino acids), cysteine, nitrogen, and sulphur in different intestinal sections was estimated by the indirect method, as described by Maynard and Loosli (1969), using Y2O3 as an inert marker (Austreng et al., 2000). Absorption of cysteine was estimated separately because of the high content of this sulphuric amino acid in endogenous digestive enzymes. 2.7. Statistical analyses The results were analysed by the General Linear Model procedure in the SAS computer software (SAS, 1985). Mean results per tank were subjected to two-way analysis of variance (ANOVA) with interaction, with oxytetracycline (OTC; with or without) and Diet (FM, SBM, or inulin) as the independent variables. The results from the ANOVA are presented with pffiffiffiffiffiffiffiffiffi ffi the square root of the mean square error ( MSE ) indicating variation, and as the proportion of total variation explained by each of the factors and their interaction, calculated as the marginal contribution of the mean

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

square of the parameter (type I sum of squares) as a percentage of the corrected total sum of squares. Significant differences among treatments were indicated by least-squares means comparison. The level of significance was chosen at P ≤ 0.05, and the results are presented as group means. 3. Results No fish died during the 21 days experimental feeding period. When terminating the experiment there were no effects of treatment on final body weight and length, which ranged from 232–260 g among the feeding groups. 3.1. Plasma chemistry No clear effects of dietary supplementation of oxytetracycline (OTC) on the plasma concentration of free fatty acids, glucose, cholesterol, or triacylglycerides (Table 3) were detected. There was, however, a tendency for higher plasma concentration of free fatty acids when feeding 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 and gastrointestinal sections sampled for measurements of brush border membrane bound alkaline phosphatase (ALP) and leucine aminopeptidase (LAP) are given in Table 3 Concentration of glucouse, cholesterol, triglyderides (TG), and free fatty acids (FFA) in plasma (mean, n = 2) Glucose, mM

Cholesterol, mM

TG, mM FFA, mM

OTC Without 5.43 8.56 2.26 0.36y With 5.65 8.35 2.34 0.45x Diet FM 5.63 9.24 2.32 0.42 SBM 5.59 8.12 2.34 0.38 Inulin 5.40 8.00 2.25 0.43 Two-way pffiffiffiffiffiffiffiffiffiffi ANOVA: MSE 0.34 0.74 0.50 0.07 Proportion (type I SS) of variation (corrected total SS) explained (%) by effect of OTC 11.0 1.7 1.1 34.8⁎ Diet 9.3 46.1 1.1 8.6 OTC × Diet 28.0 11.1 12.4 10.5 Different superscripts indicate a statistical tendency (P ≤ 0.1). ⁎ ≤ 0.1. pPffiffiffiffiffiffiffiffiffi ffi MSE is the square root of the mean square error.

x,y

397

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

Stomach Intestinal section PI

MI

Total GIT DI

OTC Without 10.1a 5.7 17.9 1.9 4.3 29.8 5.9 16.9 1.9 4.3 29.0 With 9.5b Diet FM 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: pffiffiffiffiffiffiffiffiffiffi MSE 0.3 0.2 1.4 0.1 0.2 1.5 Proportion (type I SS) of variation (corrected total SS) explained (%) by effect of OTC 14.3⁎ 18.0 7.6 4.8 0.0 2.9 Diet 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 Different superscripts indicate a statistical difference (P ≤ 0.05). Different superscripts indicate a statistical tendency (P ≤ 0.1). ⁎ ≤ 0.05; ⁎⁎P ≤ 0.01; ⁎⁎⁎P ≤ 0.1; ⁎⁎⁎⁎P ≤ 0.001. pPffiffiffiffiffiffiffiffiffi ffi MSE is the square root of the mean square error.

a,b,c x,y

Table 4. The relative liver weight was higher in fish fed diets without antibiotic supplementation than in fish fed OTC, but dietary OTC did not affect the relative weight of any gastrointestinal section. With regard to effects of raw material use in the diets (Diet), the relative weight of the liver was similar in fish fed the FM control and inulin diets, but lower in fish fed the SBM diets. There was no effect of Diet on the relative weight of the stomach (ST). The relative weight of the pylorus intestine (PI) was higher in fish fed inulin than in fish fed FM, with intermediate weights in fish fed SBM. A similar tendency (P b 0.1) was seen for relative weight of the mid intestine (MI). The relative weight of the distal intestine (DI), however, was lower in fish fed SBM than in fish fed FM and inulin. This caused a higher relative weight of the total gastrointestinal tract (GIT) in fish fed inulin than in fish fed FM and SBM, but similar weight of the total GIT in fish fed FM and SBM. 3.3. Intestinal morphology As judged by light microscopy, there were no apparent effects of dietary OTC on the morphology of the DI. In accordance with the descriptions of Baeverfjord and Krogdahl (1996), all examined fish fed FM (12/12) and all but one examined fish fed inulin (11/12) showed normal morphology of the DI, characterised by the presence of well-differentiated enterocytes with many absorptive vacuoles. In contrast, all fish fed SBM showed moderate (8/12) to severe (4/12)

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Table 5 Trypsin 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 of the distal intestine (DI1 and DI2), and the total intestinal tract (IT; mean, n = 2) Relative trypsin activity, U mg− 1 PI1

1

P12

MI

DI1

Total trypsin activity, U section− 1 DI2

P121

PI1

OTC Without 118.6 93.2 108.3 64.2 37.8 66874 51219 With 145.4 123.3 113.9 59.3 37.5 87045 68890 Diet FM 159.9 153.5 135.4 60.3 16.6c 84926 71883 SBM 130.2 85.3 86.8 68.9 72.2a 87043 48610 Inulin 105.8 113.6 111.0 56.0 24.2b 58910 70373 Two-way pffiffiffiffiffiffiffiffiffiffi ANOVA MSE 31.9 67.3 49.5 14.6 4.2 24244 21460 Proportion (type I SS) of variation (corrected total SS) explained (%) by effect of OTC 14.1 11.3 0.5 2.1 0.0 18.0 9.5 Diet 38.6 34.9 24.2 9.7 97.4⁎⁎⁎ 29.0 13.4 OTC × Diet 7.2 0.3 52.4 1.2 1.1

Total IT1

MI

DI1

126074 151960

44994 50840

22607 2728

310923 387285

146431 123340 147293

45773 53464 44516

10355b 46574a 17909b

352521 359030 346318

35847

19963

6868

71830

17.6 12.9 2.1

DI2

2.9 5.3 24.6

2.0 89.1⁎⁎⁎ 0.2

32.5 2.9

Different superscripts indicate a significant statistical difference (P ≤ 0.05). ⁎⁎⁎P ≤ 0.001. pffiffiffiffiffiffiffiffiffiffi MSE is the square root of the mean square error. 1 ANOVA calculated without interaction due to insufficient material for analysis from 3 experimental units and, thus, insufficient replication. a,b,c

morphological changes in the DI consistent with SBM induced enteritis. These changes included variable degrees of inflammatory cell infiltration of the lamina propria, reduced vacuolisation of the enterocytes, and shortening of the villi. 3.4. Enzymology No effects of dietary OTC were found on the activities of trypsin and amylase in the intestinal

contents (Tables 5 and 6), nor on the activities of ALP and LAP in the brush border membrane of any gastrointestinal section (Table 7). No effects of Diet were detected on the trypsin activity (Table 5) in PI, MI, the first half of DI (DI1), or the 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 fish fed SBM than in fish fed FM and inulin. The relative trypsin activity in DI2 was intermediate in fish fed

Table 6 Amylase 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 of the distal intestine (DI1 and DI2), and the total intestinal tract (IT; mean, n = 2) Relative amylase activity, U mg− 1 PI1

P12

MI

DI1

Total amylase activity, U section− 1 DI2

OTC Without 2.99 2.91 3.17 2.27 1.73 With 2.56 2.62 2.71 2.39 1.53 Diet FM 2.63 2.65 2.57 1.89y 1.17b SBM 3.00 2.86 2.95 2.66x 2.05a xy Inulin 2.69 2.77 3.31 2.45 1.66ab Two-way ANOVA pffiffiffiffiffiffiffiffiffiffi MSE 0.62 0.46 0.48 0.42 0.38 Proportion (type I SS) of variation (corrected total SS) explained (%) by OTC 14.9 11.9 17.4 1.7 4.7 Diet 8.2 4.3 29.8 52.2⁎ 58.6⁎⁎ OTC × Diet 16.7 24.7 15.2 2.5 3.9

PI1

P12

1735 1541

1370 1363

3946 3788

1521 2083

1055 1102

9626 9877

1419 2015 1480

1069 1598 1431

2970y 4216x 4415x

1357 2099 1949

731y 1320x 1185x

7545y 11248x 10460x

347

803

515

312

1917

643 effect of 2.7 20.9 16.1

Different superscripts indicate a significant statistical difference (P ≤ 0.05). Different superscripts indicate a statistical tendency (P ≤ 0.10). ⁎ ≤ 0.10; ⁎⁎P ≤ 0.05. pPffiffiffiffiffiffiffiffiffi ffi MSE is the square root of the mean square error.

a,b

x,y

0.0 25.7 42.6

MI

DI1

0.6 42.7⁎ 23.0

23.6 30.7 6.1

DI2

0.5 55.9⁎ 0.7

Total IT

0.3 46.8⁎ 19.1

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

399

Table 7 Alkaline phosphatase (ALP) activity in the stomach (ST), pylorus intestine (PI), mid intestine (MI), distal intestine (DI), and the total gastrointestinal tract (GIT; mean, n = 2) ALP activity, μmol h− 1 g− 1 tissue

ALP activity, μmol h− 1 mg− 1 protein

ALP activity, μmol h− 1 in whole tissue kg− 1 BW

ST

ST

ST

PI

MI

DI

PI

MI

DI

OTC Without 0.08 0.18 0.15 0.10 0.67 1.75 2.04 With 0.07 0.17 0.12 0.10 0.67 1.75 2.04 Diet FM 0.07 0.19 0.13 0.12a 0.69 1.96 2.08 SBM 0.08 0.16 0.13 0.07b 0.67 1.62 1.90 0.65 1.68 2.13 Inulin 0.07 0.17 0.15 0.11a Two-way p ffiffiffiffiffiffiffiffiffiffi ANOVA MSE 0.01 0.02 0.05 0.01 0.05 0.34 0.27 Proportion (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 Diet 13.3 31.1 7.4 66.5⁎⁎ 19.1 22.9 14.5 OTC × Diet 13.8 26.1 7.5 22.4⁎ 0.4 17.8 30.5

PI

MI

DI

Total GIT

1.35 1.59

0.42 0.43

3.08 2.92

0.28 0.23

0.42 0.44

4.20 4.02

1.47ab 1.13b 1.81a

0.41 0.44 0.42

3.04 2.89 3.07

0.22 0.24 0.29

0.49a 0.24b 0.57a

4.16 3.82 4.35

0.23

0.03

0.50

0.10

0.07

0.49

9.6 51.1⁎ 20.6

2.7 11.5 36.5

3.3 2.8 32.5

10.8 13.9 6.1

0.4 74.9** 15.2

2.9 16.5 39.5

Different superscripts indicate a significant statistical difference (P ≤ 0.05). ⁎ ≤ 0.05; ⁎⁎P ≤ 0.01. pPffiffiffiffiffiffiffiffiffi ffi MSE is the square root of the mean square error.

a,b

and, thus, in the whole intestinal tract of fish fed SBM and inulin than in fish fed FM. The activity of ALP in ST, PI, and MI did not differ among fish fed the different diets (Table 7). Likewise, the LAP activity in PI and MI was similar in all Diet groups (Table 8). In DI the activities of both ALP and LAP were lower in fish fed SBM than in fish fed FM and inulin when related to g DI tissue or kg BW. However, as the contribution of DI to the total enzyme activity in the gastrointestinal tissue was low, the total ALP and

inulin, while the total trypsin activity in DI2 was similar in fish fed FM and inulin, although the numerical ranking was similar to that of the relative activity. No effect of Diet was found on the relative amylase activity (mU mg− 1 DM) in contents from PI and MI (Table 6). In DI2, however, the relative amylase activity was highest in fish fed SBM, lowest in fish fed FM, and intermediate in fish fed inulin. A similar tendency (P b 0.1) was seen in DI1. There was likewise a tendency (P b 0.1) for higher total amylase activity (U) in MI, DI2,

Table 8 Leucine aminopeptidase (LAP) activity in the stomach (ST), pylorus intestine (PI), mid intestine (MI), distal intestine (DI) and the total intestinal tract (IT; mean, n = 2) LAP activity, mmol h− 1 g− 1 tissue

LAP activity, μmol h− 1 mg− 1 protein

LAP activity, mmol h− 1 in whole tissue kg− 1 BW

PI

PI

PI

MI

DI

Total IT

288.4 272.1

14.2 13.9

42.0 45.4

344.5 331.4

226.3 312.8 301.5

13.6 13.5 15.1

53.2a 13.2b 64.7a

293.1 339.5 381.2

52.8

1.7

8.1

57.9

2.0 45.0 10.4

0.3 9.0 64.9*

MI

DI

MI

DI

OTC Without 16.8 7.9 9.3 411.7 261.2 350.4 With 16.0 7.7 10.1 398.2 257.7 404.4 Diet FM 14.6 7.9 12.5a 366.2 275.1 417.2b b SBM 17.6 7.2 3.8 436.8 235.6 176.0c Inulin 17.0 8.3 12.7a 412.0 267.6 539.1a Two-way pffiffiffiffiffiffiffiffiffiffi ANOVA MSE 2.6 1.1 1.2 64.7 53.5 49.3 Proportion (type I SS) of variation (corrected total SS) explained (%) by effect of OTC 2.9 0.7 1.0 1.4 0.7 3.6 Diet 30.1 5.4 89.6⁎⁎⁎ 26.0 11.1 86.1⁎⁎⁎ OTC × Diet 6.4 53.2 5.7 9.0 28.5 5.5 Different superscripts indicate a significant statistical difference (P ≤ 0.05). ⁎ ≤ 0.05; ⁎⁎P ≤ 0.01; ⁎⁎⁎P ≤ 0.001. pPffiffiffiffiffiffiffiffiffi ffi MSE is the square root of the mean square error.

a,b,c

0.5 87.1⁎⁎⁎ 6.5

1.2 35.2 18.2

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

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

S. Refstie et al. / Aquaculture 261 (2006) 392–406 Table 9 Two-way ANOVA for cumulative apparent absorption of amino acid protein (Σ AA), cysteine, nitrogen and sulphur through successive sections of the intestinal tract pffiffiffiffiffiffiffiffiffiffi Absorbed Intestinal MSE Proportion (type I SS) of section variation (corrected total SS) explained (%) by effect of Σ AA

Cysteine

Nitrogen

Sulphur

PI1 PI2 MI DI1 DI2 PI1 PI2 MI DI1 DI2 PI1 PI2 MI DI1 DI21 PI1 PI2 MI DI1 DI21

16.2 8.2 2.0 5.3 6.3 31.5 31.1 4.9 13.2 14.7 18.7 17.3 2.8 8.4 11.5 39.1 32.0 6.4 15.6 17.2

OTC

Diet

OTC × Diet

8.7 5.1 5.8 16.4 3.5 13.8 3.3 10.2 23.2 7.7 1.1 0.0 3.3 17.9 16.6 1.8 0.2 0.1 19.2 0.2

8.6 42.2* 25.0 0.4 13.7 17.8 48.7⁎⁎ 45.8⁎⁎ 5.5 5.8 7.1 45.4⁎⁎ 34.7 2.5 4.8 6.5 41.5⁎⁎ 35.1 8.8 10.4

13.5 30.5 13.0 14.8 7.0 7.1 14.2 6.0 10.3 11.4 14.7 22.6 15.2 17.2 20.0 32.0 10.1 23.0

⁎ ≤ 0.05; ⁎⁎P ≤ 0.1. pPffiffiffiffiffiffiffiffiffi ffi MSE is the square root of the mean square error. 1 ANOVA calculated without interaction due to insufficient material for analysis from 2 (nitrogen) or 1 (sulphur) experimental units and, thus, insufficient replication.

LAP activities in the whole intestinal tract related to BW were not affected by Diet. When related to mg protein, the activities of both ALP and 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 (P b 0.1) in the twoway ANOVA for a statistical interaction between the independent variables OTC and Diet on ALP and LAP activities in the DI. As indicated by least square means comparison, this was caused by generally higher activity in fish fed inulin than in fish fed FM when the diets were supplemented with OTC, while the activity was similar in fish fed these diets without OTC (data not shown). For ALP the activity was also similar in fish fed FM and SBM with OTC, but lower in fish fed SBM than in fish fed FM without OTC (data not shown). 3.5. Apparent amino acid absorption The cumulative apparent absorption of amino acid protein (Σ individual amino acids) and nitrogen in

401

successive intestinal sections increased gradually from PI1 to MI, but was unchanged from MI to DI2 (Fig. 1). A similar absorption pattern was seen for the sulphuric amino acid cysteine and for sulphur, but for these components the apparent absorption was negative in PI1 and P2 when feeding all diets. The only exception from this was a positive but still very low (9%) apparent absorption of cysteine in PI2 when feeding the SBM diets. Dietary OTC did not affect the apparent absorption of amino acids, nitrogen, or sulphur significantly (Table 9 and Fig. 1). The apparent amino acid absorption in PI2 was, however, higher when feeding the SBM diets than when feeding FM and inulin diets. There were also tendencies (P b 0.1) for higher apparent absorption of cysteine, nitrogen, and sulphur in PI2 when feeding the SBM diets than when feeding the FM diets, and the apparent absorption of cysteine also tended (P b 0.1) to be higher in MI when feeding the SBM diets. There was a tendency (P b 0.1) for a statistical interaction in the two-way ANOVA between the independent variables OTC and Diet on apparent amino acid absorption in PI2. This was caused by higher absorption when feeding the inulin diet with OTC (55%) than when feeding it without OTC (31%). 4. Discussion The main findings of this experiment were that dietary oxytetracycline (OTC) did not alter the capacity for digestive hydrolysis and absorption of amino acids, nitrogen, and sulphur in Atlantic salmon. Apart from increasing the activity of brush border enzymes in the distal intestine when feeding inulin, OTC did not modify the digestive responses to dietary soybean meal (SBM) and inulin. Dietary SBM caused pathomorphological changes that reduced the hydrolytic efficiency in the distal intestine, and also resulted in increased trypsin and amylase activity in the distal intestine that indicated reduced re-absorption and increased faecal losses of these endogenous enzymes. However, SBM at the same time stimulated more efficient apparent absorption of amino acids, nitrogen and sulphur in the proximal intestine, regardless of OTC supplementation. Dietary inulin caused no apparent intestinal damage, but stimulated intestinal growth resulting in increased relative mass of the gastrointestinal tract. During the 2 weeks period of adaptation to the experimental facilities and subsequent 3 weeks of feeding the experimental diets, the fish grew at specific growth rates (SGRs) ranging from 0.84–1.07, averaging 0.95. This was slightly slower than the expected SGR of

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Atlantic salmon of similar size when grown at similar temperature (Austreng et al., 1987). However, the growth period was short, and appetite in Atlantic salmon 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 extruded salmon diet to the unfamiliar cold pelleted experimental diets in the middle of the period, appetite and, thus, growth were considered within the normal range. Due to low water stability of the diets, the uneaten feed could not be collected from the water to calculate accurate feed intake (Helland et al., 1996). However, as the ration was similar in all groups, and as there were no differences in growth among the feeding groups, the feed intake can be assumed to have been similar in all groups. Dietary SBM contains components that bind bile acids in the intestine (Storebakken et al., 2000), thereby potentially increasing the faecal steroid and lipid loss. In SBM-fed fish this is indicated by lowered plasma cholesterol (Kaushik et al., 1995; Refstie et al., 1999), changes in cholesterol metabolising hepatic enzymes (Martin et al., 2003), and increased cholesterol requirement (Twibell and Wilson, 2004). The lacking effects of dietary SBM on plasma cholesterol and lipids in the present salmon indicate that cholesterol metabolism changes gradually over time in response to soy, and that a three-week feeding period is too short to induce noticeable effects. The cause(s) of the reduced relative liver weight (g kg− 1 BW) when feeding OTC or SBM is not apparent from this study. However, therapeutic administration of antibiotics may cause liver degenerations in mammals (Lown, 1998), and this may also have been the case in the OTC-fed salmon. Dietary SBM on the other hand is reported to cause histological changes and lipid accumulation in the liver of the salmonid rainbow trout (Oncorhynchus mykiss; Ostaszewska et al., 2005). Profiling of liver proteins from SBM-fed rainbow trout furthermore indicates shifts toward hepatic catabolic pathways, increased or inefficient protein turnover, down-regulation of structural protein expression, heightened immune response, and altered levels of stress proteins (Martin et al., 2003). The increased relative weight of all intestinal sections when feeding inulin, and the similar numerical trend for relative weight of the proximal intestine when feeding SBM, were probably caused by high content of indigestible material in these diets. This is a well known effect in livestock, where hypertrophy in gastrointestinal organs is stimulated by workload when feeding high fibre and low-energy diets due to greater filling 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 have altered the intestinal microbiota in the salmon. Nucleotides, which have prebiotic properties (Uauy, 1994), have been shown to accelerate growth in the intestinal mucosa of Atlantic salmon (Burrells et al., 2001). Prebiotic effect(s) of inulin may, thus, also have played a role in stimulating intestinal growth in the inulin-fed salmon. Reduced mass of the distal intestine in salmon fed SBM was in response to the SBM-induced pathomorphological changes in this intestinal section, as previously shown by Nordrum et al. (2000). The increased trypsin activity in the last part of the distal intestine was in keeping with Krogdahl et al. (2003), who found the faecal trypsin concentration in Atlantic salmon to increase in response to the dietary level of soybean meal. The present lack of significant differences in trypsin and amylase activity in the pyloric and mid intestine was to some extent caused by high variation within feeding groups and, thus, low statistical power. Still, when feeding the fish meal (FM) control diets about 85% of the total trypsin activity was found in these proximal intestinal sections. It was a little lower (about 80%) when feeding the inulin diets, but dramatically lower (about 65%) when feeding the SBM diets, and this was caused by high trypsin activity in the last half of the distal intestine. A similar trend was seen for amylase, where about 72% of the total activity was found in the pyloric and mid intestine when feeding the FM diets, but only 65 to 70% when feeding the SBM and inulin diets. Elevated faecal trypsin was also found in salmonids fed 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 fed salmonids is caused by trypsin inhibitors in the intestinal contents, and potentially worsened by pancreatic growth and hypersecretion of pancreatic enzymes. However, at the given SBM inclusion the trypsin inhibitor activity (TIA) in the present SBM diets should by calculation be only 0.5 mg trypsin inhibited g− 1 meal. This is lower than 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 fed salmon appeared to be a loss in the ability to reabsorb endogenous digestive secretions due to the pathomorphological changes in the distal intestine. This is supported by the concomitant increase in faecal amylase when feeding the SBM diets. The results also indicate that dietary inulin increases the faecal loss of pancreatic

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

enzymes in salmon slightly, but the reasons for this remain unclear. The amylase activity in mid and distal intestinal contents appeared to be inversely related to the wheat and, thus, starch level in the diets. Atlantic salmon has limited capacity to digest starch (Krogdahl et al., 2005). In consequence the amylase activity in fish fed the FM diets may have been reduced by adsorption of amylase to 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% of the respective total activities of the brush border membrane bound enzymes alkaline phosphatase (ALP) and leucine aminopeptidase (LAP) were found in the pyloric and mid intestine. The reduced ALP and LAP activities in the distal intestine of salmon fed the SBM diets were in keeping with previous findings (Krogdahl et al., 1995, 2003; Bakke-McKellep et al., 2000a). Quantitatively these differences were, however, much too small to noticeably affect the overall ALP and LAP activities in the intestinal tissue. Increased number of proliferating cells lining the villous folds of the distal intestine in salmon fed soybean meal (Sanden et al., 2005) suggests that reduced activity of brush border membrane bound enzymes in distal intestine is caused by alterations in enterocyte turnover and, thus, degree of maturation and functionality of the enterocytes. In the light of the present results, one may speculate if elevated trypsin concentration in the distal intestine of soybean meal fed salmon is damaging the enterocytes in the distal mucosa. Elevated ALP and LAP specific activities (mg− 1 protein) in the distal intestine of inulin fed fish furthermore indicate that dietary inulin stimulated synthesis and incorporation of hydrolases into the distal brush border membrane. As this effect was only apparent when feeding inulin in combination with OTC, it may have been in response to prebiotic stimulation of beneficial bacteria populations at a concomitant general reduction of the intestinal microbiota. Thus, and since dietary inulin stimulated intestinal growth, the prebiotic effect of inulin in Atlantic salmon merits further investigation. Unlike mammals, amino acid transporters are present along the entire length of the intestine of fish (Buddington et al., 1997), although the absorptive efficiency is highest in the proximal intestine (Buddington and Diamond, 1987; Bakke-McKellep et al., 2000b; Nordrum et al., 2000). Thus, the high proteolytic capacity (trypsin and LAP activities) in the proximal intestine of the present salmon was mirrored by efficient apparent amino acid absorption, which at least quanti-

403

tatively appeared to be completed in the mid intestine, in keeping with previous results (Austreng, 1978; Dabrowski and Dabrowska, 1981, Krogdahl et al., 1999). The consistent negative apparent absorption of cysteine and sulphur and the lower apparent absorption of nitrogen than of amino acids in the pyloric intestine illustrate the impact of exocrine secretion of cysteinerich components like pancreatic enzymes and glutathione, and of nitrogen and sulphur rich non-protein components like bile acids (taurocholate) into the lumen of this intestinal section. Low apparent absorption of cysteine in the pyloric intestine of salmonids has also been shown previously (Dabrowski and Dabrowska, 1981; Krogdahl et al., 1999), although not as low as to be estimated as negative absorption. Incomplete reabsorption of endogenous digestive secretions may in part account for the low overall apparent absorption of cysteine and sulphur. Structural disulphide cross-linking between cysteine sulphydryl groups in proteins may also reduce the general availability of cysteine (Opstvedt et al., 1984), although this appears to depend on the accessibility of the disulphide bonds for cleavage in the intestine (Aslaksen et al., 2006). The fast apparent absorption of amino acids, nitrogen and sulphur in salmon fed the SBM diets indicates stimulation of active amino acid transport in the proximal intestine, possibly as a feed-back response to reduced active transport in the distal intestine (Nordrum et al., 2000). However, despite the present lack of dietary effects on proximal trypsin activity, effects of SBM on secretion of other pancreatic proteases cannot be ruled out. Haard et al. (1996) also found differences in the catalytic properties of trypsin, as seen from reduced trypsin inhibitor sensitivity, and changes in the balance of digestive proteases, as seen from increases in total enzyme concentration, in the pyloric intestine of Coho salmon (Oncorhynchus kisutch) fed soybean meal. Thus, rapid proximal acid amino absorption when feeding the present SBM diets may also have resulted from more efficient protein hydrolysis due to pancreatic secretion of different trypsin isozymes and/or increased secretion of other proteases. Still, dietary SBM did not affect the total apparent absorption of amino acids measured in the last half of the DI. This is in line with previous experiments where Atlantic salmon were fed diets containing 20 to 30% SBM, showing only slight (Refstie et al., 1998, 2001, 2005; Storebakken et al., 1998) or no (Storebakken et al., 1998; Refstie et al., 2000, 2001) negative effect of SBM on the apparent faecal protein digestibility. Thus, the true availability for salmon of amino acids in properly processed SBM appears as high as or higher

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than that of LT-FM protein. However, as the endogenous amino acid losses are higher when feeding SBM, the net effect on apparent amino acid absorption may be negative, in particular for cysteine. To conclude, ≥ 80% of the activities of trypsin, ALP and LAP and about 70% of the amylase activity were found in the proximal and mid intestine of Atlantic salmon with functional distal intestine. The apparent absorption of amino acids, nitrogen, and sulphur was also quantitatively completed in the mid intestine. Feeding diets supplemented with oxytetracycline (OTC) resulted in reduced relative liver weight. Apart from increased activity of brush border enzymes in the distal intestine when feeding OTC in combination with inulin, OTC did not modify how dietary soybean meal or inulin affected gut morphology, hydrolysis of protein and starch, and absorption of amino acids, nitrogen and sulphur. Dietary soybean meal resulted in reduced relative liver weight, pathomorphological changes and lowered hydrolytic efficiency in the mucosa of the distal intestine, and apparently reduced re-absorption and, thus, increased faecal losses of endogenous digestive enzymes. However, soybean meal also stimulated absorption of amino acids, nitrogen, and sulphur in the proximal intestine, resulting in a neutral net effect on the total apparent amino acid absorption. Dietary inulin did not damage the distal enterocytes, but stimulated growth in intestinal tissues that resulted in increased relative mass of the gastrointestinal tract. Apart from the positive effect of inulin fed in combination with OTC on the hydrolytic capacity of the distal intestine, dietary inulin did not affect the hydrolytic and absorptive capacity of salmon. Acknowledgements The authors wish to acknowledge the skilful technical assistance of Ellen Koren Hage, Gunn Østby, and Kristin Vekterud at the Norwegian School of Veterinary Science. We are grateful to Suiker Unie for supplying inulin. Financial support for the study was provided by the Research Council of Norway (grant # 145949/120) and the Norwegian School of Veterinary Science via the CoE Aquaculture Protein Centre (APC). References Aslaksen, M.A., Romarheim, O.H., Storebakken, T., Skrede, A., 2006. Evaluation of content and digestibility of disulfide bonds and free thiols in unextruded and extruded diets containing fish meal and soybean protein sources. Anim. Feed Sci. Technol. 128, 320–330. Austreng, E., 1978. Digestibility determination in fish using chromic oxide marking and analysis of contents from different segments of the gastrointestinal tract. Aquaculture 13, 265–272.

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