2006) 259–268www.elsevier.com/locate/aqua-online

Aquaculture 261 (

Effects of soybean meal based diet on growth performance,gut histopathology and intestinal microbiota of juvenile

rainbow trout (Oncorhynchus mykiss)

Jouni Heikkinen a,⁎, Jouni Vielma b, Outi Kemiläinen a, Marja Tiirola c,Päivi Eskelinen b, Tapio Kiuru d, Dina Navia-Paldanius a, Atte von Wright a

a Institute of Applied Biotechnology, University of Kuopio, FIN-70211 Kuopio, Finlandb Finnish Game and Fisheries Research Institute (FGFRI), Jyväskylä Game and Fisheries Research, FIN-40500 Jyväskylä, Finland

c Department of Biological and Environmental Science, University of Jyväskylä, FIN-40351 Jyväskylä, Finlandd Finnish Game and Fisheries Research Institute (FGFRI), Laukaa Fish Farm, FIN-41341 Laukaa, Finland

Received 23 March 2006; received in revised form 7 July 2006; accepted 7 July 2006

Abstract

Effects of soybean meal (SBM) based diet on growth performance, histology of the intestinal epithelium and on the gut microbiotaof juvenile rainbow trout (Oncorhynchus mykiss) were investigated on two trials lasting for 8 weeks (Trial I) and 18 weeks (Trial II).The microbiological characterization was done both with conventional plating techniques, biochemical profiling and lengthheterogeneity analysis of PCR amplified 16S rDNA (LH-PCR).

Typical histological changes were found after 18 weeks of SBM feeding (Trial II). Mean height of simple foldings weresignificantly higher within the group fed with fish meal (FM) based diet. These changes were not, however, reflected in theappearance, behavior and weight gain of the fish, although the feed conversion rate (FCR) was higher, compared to fish fed with thetraditional FM feed.

In both SBM and FM groups the counts of cultivable intestinal bacteria dropped within the first 4 weeks of feeding (Trial I).Subsequently, the bacterial numbers increased in the FM group, but not in the SBM group by the 8th week of the trial. The LH-PCRdata suggested also that diet-related qualitative changes in the microbiota had occurred (Trial II). The main identified bacteria weremainly representatives of the genera Aeromonas, Sphingomonas and Chryseomonas, and among the lactic acid bacteria, Lactococcusand Lactobacillus.

Future research will be directed to the identification and eventual elimination the SBM-associated factors involved in theinflammatory responses and unfavorable FCR, in particular by modifying the intestinal microbiota.© 2006 Elsevier B.V. All rights reserved.

Keywords: Gastrointestinal tract; Bacteria; Fish meal; Soybean meal; Gut histology; Rainbow trout; Feeding experiment

⁎ Corresponding author. Tel.: +35817163373; fax: +35817163752.E-mail address: jouni.heikkinen@uku.fi (J. Heikkinen).

0044-8486/$ - see front matter © 2006 Elsevier B.V. All rights reserved.doi:10.1016/j.aquaculture.2006.07.012

1. Introduction

Soybean meal (SBM) is a valuable protein sourcewith a generally good nutritional balance for aquafeeds.Upper inclusion level for carnivorous fish such assalmonids is restricted to 20–30% due to the presence of

Table 1Composition (g kg−1) of the diets

Ingredient g kg−1 diet

Fish meal Soybean meal

Fish meal1 537 270Soybean meal2 – 450Extruded wheat3 330 105Wheat gluten3 20 20Vitamin–mineral premix4 15 15Monocalcium phosphate – 10L-Lysine – 7DL-Methionine – 3Soy lecithin 10 10Fish oil 88 1101 Icelandic low temperature capelin meal, 70.2% crude protein and13.8% crude lipid.2 Hexane extracted GMO-free soybeans. SBM included hulls andcontained 45.5%crude protein as-fed basis. RaisioGroup, Raisio Finland.3 Raisio Group, Raisio Finland.4 Vitamins added to supply the following (kg−1 diet): retinol acetate,5000 IU; cholecalciferol, 2500 IU; all-race-α-tocopheryl acetate,250 IU; menadione sodium bisulfite, 30 mg; thiamin.HCl, 40 mg;riboflavin, 50 mg; calcium d-pantothenate, 150 mg; biotin, 0.8 mg;folic acid, 15 mg; vitamin B12, 0.05 mg; niacin, 200 mg; pyridoxine.-

HCl, 30 mg; ascorbic acid (Stay C), 300 mg; inositol, 400 mg.Minerals added to supply the following (mg kg−1 diet): magnesium,200; sodium, 500; iron, 20; copper, 3; zinc, 15; manganese, 15;selenium, 0.5; iodine, 1.9; aluminium, 0.03; cobalt, 2.5.

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anti-nutrients, which eventually leads to reduced growthand feed utilization. Most anti-nutrients can be reducedby physical, chemical or biochemical treatments, such asinactivation of trypsin inhibitor by heat, reduction ofcarbohydrate content by methanol extraction or hydro-lysis of phytic acid by phytase. Due to economic reasons,conventional, heat-treated SBM remains the major soyproduct in salmonid feeds (Storebakken et al., 2000).

SBM causes pathological changes in the morphologyand function of salmonid distal intestinal epithelium.Typical morphological changes include shortening ofmucosal foldings, abnormal vacuolization, loss ofmucosal integrity, widening of central stroma andinflammatory cell infiltration in lamina propria (Vanden Ingh et al., 1991; Baeverfjord and Krogdahl, 1996;Burrels et al., 1999), and they appear both in therainbow trout (Oncorhynchus mykiss) and the Atlanticsalmon (Salmo salar) (Refstie et al., 2000). Immuno-histochemical study by Bakke-McKellep et al. (2000)demonstrated the inflammatory nature of distal intestineepithelial reaction to SBM by increased number ofmacrophages, neutrophilic granulocytes and IgM.Functional changes associated with SBM-inducedenteritis include increased susceptibility to bacterialfish diseases (Krogdahl et al., 2000) and digestivedisorders such as osmotic diarrhea and reduced nutrientabsorption (Storebakken et al., 2000).

The gastrointestinal tract (GIT) can be considered as amicrobiological ecosystem, the complexity of which mostevidently depends on theGITanatomy.Most of the studieson intestinal microbiota have been focused on warm-bloodied animals. With mammals the bacterial densities,especially in colon can reach up to 1012 cfu/g intestinalcontent (Berg, 1996; Drasar and Hill, 1974). Intestinalbacteria typically represent obligately or facultativelyanaerobic nutritionally fastidious species and genera withoptimal growth temperatures close to the body tempera-ture. The physiological role of mammalian intestinalmicrobiota is manifold, including colonization resistanceagainst pathogens, metabolic conversions of xenobioticsand endogenous compounds, production of short-chainfatty acids and interactions with host immune system(Lhoste et al., 2003; Cummings and Macfarlane, 1997).

The intestinal microbiota of fish is still fairly poorlyknown. The physiology of the fish gut differs in manyrespects from that of the homeothermal animals (Bud-dington et al., 1997), and this can be expected to affect thebacterial numbers and the species' composition. Therelevant findings of the published studies on fish intestinalmicrobiota can be summarized as follows: the totalcultivable bacterial numbers are seldom higher than106 cfu/g (Ringo, 1993; Ringo and Olsen, 1999), the

many typical genera of homeothermal animals such asBifidobacteria, Bacteroides, Eubacterium (Isolauri et al.,2004), are either absent or only occasionally present.Lactic acid bacteria (LAB) are relatively common, buttheir numbers are low (Ringo et al., 2000). Typical fishspecific species and genera include, among others Pseu-domonas, Aeromonas and Vibrio (Sugita et al., 1996;Ringo and Olsen, 1999; Huber et al., 2004). Thesignificance of the intestinal microbial community to thehealth andwell-being of fish is poorly known, and also theknowledge of the dietary effects on the composition ofmicrobiota is limited.

The aim of the study was to follow the intestinalresponse of juvenile rainbow trout (O. mykiss) fed with apractical type SBM-based diet. The emphasis was on thecondition of the intestinal epithelium and intestinalbacterial community under different dietary regimes.The relevance of the observed changes to the furtherdevelopment of aqua-feeds will be discussed.

2. Material and methods

2.1. Experimental design and diets

Two diets were prepared using fish meal or SBM asthe major protein feedstuff (Table 1). SBM was made of

Table 2Progress of mean weight of juvenile rainbow trout in two differentfeeding trials

Feed Trial I Trial II

0 weeks 4 weeks 8 weeks 0 weeks 18 weeks

Fish meal 20.7±1.1 53.5±2.9 110.4±5.8 8.7±0.1 264.5±10.7Soybean

meal21.1±1.4 51.6±3.2 105.4±8.0 8.6±0.1 260.2±18.2

n=5, mean±SEM.

Fig. 1. Intestinal epithelium of juvenile rainbow trout fed with fishmeal based diet (H&E, 200×). Absorptive vacuoles (av) fill the apicalpart of mucosal cells and nuclei (n) are located in regular rows.

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hexane extracted GMO-free soybeans. SBM includedhulls and contained 45.5% crude protein as-fed basis.Fish meal was Icelandic low temperature capelin mealcontaining 70.2% crude protein and 13.8% crude lipid.Diets were made isonitrogenous and isolipidic on grossbasis by adjusting the levels of conventional wheat meal(12.6% crude protein) and capelin oil. SBM-based dietwas fortified with lysine, methionine and phosphate toavoid apparent deficiency during the trial. Dry feedingredients were mixed in a batch mixer, conditioned byadditional 20% moisture, and manufactured by a twin-screw laboratory extruder at 120 °C to 2 and 3.5 mmpellets. Fish oil was coated onto dried pellets. Diets werestored at 4 °C until feeding.

2.2. Fish, feeding, and sampling

Two feeding trials were run with juvenile rainbowtrout allocated into round fibre-glass tanks to providefive replicate tanks in both trials. Trial I lasted for8 weeks, during which cultivable microbes and growthwere measured at 0, 4 and 8 weeks. At the beginning ofthe trial, there were 25 fish per tank (0.8 m2). Trial IIcontinued for 18 weeks during which samples for PCR-based microbial community analysis were taken at 4 and18 weeks and samples for histology at 18 weeks. In TrialII, groups of 55 fish per tank were transferred to larger1.5 m2, round fibre-glass after 4 weeks to raise them inreasonable densities for the longer period. Fish were feddaily with belt feeders during 1 am–8 am at 70% level ofcommercially used feeding table ratios. This wascomplemented by hand-feeding to full satiation withoutundue feed wastage on weekdays. Fish were weighed inbulk and counted for mean body weight at 0, 4 and8 weeks in Trial I, and at 0 and 18 weeks in Trial II. Inaddition, fish were biweekly weighed to correct auto-matic feeding ratios. Approximately 70% of the dailyfeeding was covered by the belt feeders and theremaining 30% by hand-feeding. Each tank was suppliedwith aerated, temperature-controlled fresh water in aflow-through system.Water temperature was maintained

constant at 15.0 °C with the help of a computer con-trolled valves, and 24:0, light:dark illumination wasprovided throughout the trial. Oxygen saturation waskept above 70% by adjusting water inflows.

Samples for distal intestine bacterial analysis werecollected at 0, 4, 8 weeks in Trial I and at 4 and 18 weeksin Trial II. During the day of sampling, fish werenormally fed until randomly selected, anesthetized withtricaine and killed with a sharp blow to the head. Thelateral side of the fish was carefully wiped with ethanoland the abdominal cavity was opened with sterileinstruments. Proximally first half of the distal intestinewas ligated with sterile thread and removed to petridishes. Five fish per tank were sampled, and petri disheswere inserted into anaerobic bags (Anaerocult P, Merck,Darmstadt, Germany) at room temperature. Afteranaerobic conditions had developed, bags were insertedon ice and transported to the laboratory for cultivation.In Trial II, the contents of the ligated intestine wereemptied into sterile eppendorf tubes without anaerobicconditions and frozen.

The study followed protocols approved by theFGFRI Animal Care Committee, Helsinki Finland.

2.3. Histological studies

Samples for histological analysis were collected at 0and 18 weeks, two fish per tank. Proximally 0.5 cm ofdistal intestine were cut with sterile scalpel, opened by alengthwise slit, rinsed carefully with physiological salinesolution to remove all intestine contents and fixed withphosphate-buffered 4% formalin solution. Histologicalsamples were dehydrated and embedded in paraffin andcut to approximately 5 μm sections according to routine

Fig. 2. Intestinal epithelium of juvenile rainbow trout fed with soybeanmeal based diet (H&E, 200×). Decreased and irregularly arrangedabsorptive vacuoles (av), goblet cells (g) and slightly widened stroma (s).

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methods of the regional laboratory of Finnish FoodSafety Authority Evira in Kuopio. Three glass slides ofsections from both fish of each tank were made. Sectionswere stained with H and E, examined by lightmicroscopy and digitally photographed with ZeissAxioskop-equipment. Heights of simple mucosal fold-ings were measured with an ocular micrometer.

2.4. Bacterial cultivations

Intestinal contents from fish held in the same tankwere pooled and 1 g of intestinal content was suspendedinto 9 ml sterile peptone saline water (0.1% peptone,0.9% NaCl). The suspension was serially diluted into10−6, and 100 μl of the solution was spread in duplicateinto agar plates. Bacteria associated with gut werequantified as colony forming units (CFU), using threedifferent types of agar. The total numbers of cultivable

Fig. 3. Total aerobic bacteria levels (log cfu/g intestinal content) in thehindgut of juvenile rainbow trout fed test diets in Trial I (n=5). Valuesare presented as the means±SD.

aerobic bacteria were estimated with plate count agar(LAB M, Bury, England). Lactic acid bacteria wereestimated with MRS-agar (LAB M, Bury, England) andbacteria with lipolytic activity were estimated with platecount agar fortified with 1% Tween 40. Anaerobicbacteria were quantified with plate count agar (LAB M,Bury, England) using anaerobic jars (Merck KGaA,Darmstadt, Germany) and Anaerocult A (Merck KGaA,Darmstadt, Germany) gas generating kits. Plates wereincubated at +15 °C for 21 days. Bacteria levels areinterpreted as geometric means with standard deviation.The morphologically dominant colonies were picked upand purified at random and blindly without takingaccount of the dietary groups.

2.5. Characterization of cultivable aerobic or aerotolerantmicroflora

The tentative characterization of the cultivablebacteria was done by checking the cell morphology,gram staining and biochemical characteristics of theisolates. The aerobic Gram negative rod shaped bacteriagrown on plate count agar, MRS-agar or on test platesfor lipolysis were tested using the API 20NE strips(Biomerieux, Marcy l'Etoile, France) according to theinstruction of the manufacturer, while Gram positive rodor coccoid shaped bacteria were tested with API 50CHand cocci shaped bacteria with API 50 CH, API Staphand API 20 Strep.

2.6. PCR-based microflora community analyses

The bacterial community composition of fish intes-tine samples was analysed using length heterogeneityanalysis of PCR amplified 16S rDNA (LH-PCR) (Suzukiet al., 1998). Bacterial cells were recovered from the

Fig. 4. Aerotolerant lactic acid bacteria levels (log cfu/g intestinalcontent) in the hindgut of juvenile rainbow trout fed test diets in Trial I(n=5). Values are presented as the means±SD.

Fig. 5. Total anaerobic bacteria levels (log cfu/g intestinal content) inthe hindgut of juvenile rainbow trout fed test diets in Trial I (n=5).Values are presented as the means±SD.

Table 3Number of different microbial isolates representing identified bacterialgenera by biochemical characterization in Trial I after 0, 4 and 8 weeksfeeding with test feeds

Time of feeding Fish meal Soybean meal

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distal intestine samples by a differential centrifugationtechnique (Apajalahti et al., 1998; Holben et al., 2002),since direct lysis methods did not yield PCR-amplifiableDNA. Briefly, approximately 100 mg of intestinalsample (pooled from five fish pooled) was suspendedin 1.2 ml of sodium phosphate buffer (50 mM, pH 8)amended with 0.1% Tween 20. The sample was shakenfor 20 min at 100 rpm/min and centrifuged at 200 ×g for15 min. The supernatant was collected and kept on ice,and the pellet was again suspended in wash buffer,shaken, and centrifuged at low speed for 4 more times.Collected supernatants from each sample were pooledand the bacteria were collected by centrifugation at30000 ×g for 20 min. The bacterial pellet was suspendedin 180 μl of wash buffer and 20 μl of 10% SDS. Glassbeads (400 mg, diameter 0.1 mm) and phenol:chlor-ophorm:isoamylalcohol (25:24:1) solution (400 μl) wasadded, and the sample was homogenized at 1600 rpm for6 min using a bead-beating instrument (Braun Mikro-UDismembrator, B. Braun Biotech International, Mes-slungen, Germany). After centrifugation of the sample at

Fig. 6. Lipolytic bacterial levels (log cfu/g intestinal content) in thehindgut of juvenile rainbow trout fed test diets in Trial I (n=5). Valuesare presented as the means±SD.

13000 rpm for 10 min the DNA was purified from theupper phase by using isopropanol precipitation. Finally,the DNAwas suspended in 50 μl of sterile water.

LH-PCR amplification was performed as describedearlier (Tiirola et al., 2003) using universal bacterialprimers 27f (Weisburg et al., 1991) and PRUN518(Muyzer et al., 1993) amplifying the first third of the16S rRNA gene (nucleotides 8–534, Escherichia colinumbering). The fragment lengths of the PCR productwere analyzed using LI-COR automated sequencer (LI-COR BioTech, Lincoln, NE) and Quantity One software(Bio-Rad Laboratories, Hercules, CA) as previouslydescribed (Tiirola et al., 2003). Band intensity data wasthen expressed as a proportion of the total intensity of allbands comprising a particular community profile.

2.7. Statistical analyses

Growth rate and feed efficiency data were analyzedby t-test using tank (n=5) as the unit of observation.Data on the heights of simple mucosal foldings wasanalyzed by two sample t-test (SYSTAT Software Inc.,Richmond, CA) using individual fish (n=10) as the unitof observation. Differences at pb0.05 were consideredsignificant. Data matrixes of the proportional bandintensities in LH-PCR band size classes were analyzedby two-way ANOVA (p=0.05) using program SYSTAT(SPSS Inc., Richmond, CA). Jackard similarity coeffi-cients were calculated and a UPGMA dendrogram waschained using group average method using program PC-

(weeks)0 4 8 Number of

isolates0 4 8 Number of

isolates

Aeromonas 7 3 10 2 2 4Bacillus 0 1 2 3Brevundimonas 1 1 1 1Brochotrix 1 1 1 1Burkholderia 1 1 0Chryseomonas 2 2 2 6 8Lactobacillus 4 1 2 7 2 2Lactococcus 1 2 3 2 3 1 6Micrococcus 0 1 2 3Sphingobacterium 1 1 2 1 1Sphingomonas 3 8 11 1 1Staphylococcus 1 1 2 2Stenotrophomonas 0 1 1Unidentified 3 8 1 12 5 5 0 10

Total number of isolates was 94.

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ORD 4.0 for Windows (MjM Software, GlenedenBeach, Oregon). SPSS 12.0.1 for Windows (SPSS Inc.,Richmand, CA) was used to conduct principal compo-nent analysis (PCA) ordination from the covariancematrix of the relative LH-PCR band intensity data.

3. Results

3.1. Growth rate and feed conversion

Meanweights of juvenile rainbow trout fed with SBMand FM based diets are represented in Table 2. Theobserved growth rate was fairly good with appr. 80%growth rate in comparison to commercial operations atsimilar fish size and water temperature. Contrary tohypothesis replacement of FM with SBM did notdecrease the growth of juvenile rainbow trout. Therewas statistically significant difference in feed conversionratio (FCR) between test meals. Fish fed with SBM-based diet had higher FCR than fish fed with FM baseddiet (FCR 0–8 weeks 0.75 vs. 0.79; p=0.038).

3.2. Histological analysis

Typical histological changes in the distal intestinewere apparent after 18 weeks of SBM diet feeding.Absorptive vacuoles of epithelial cells of simplemucosal foldings were round-shaped and irregularlyarranged. Also goblet cells and totally non-vacuolatedareas were present. Inflammatory cells were seen tosome extent. Mean height of simple foldings was0.75 mm (SD 0.102) in SBM diet and 1.08 mm (SD0.120) in FM diet groups. Height differences werehighly significant (t=7.079, df=16.0, pb0.001). Typ-

Fig. 7. Proportions of the main fragment size classes (shown in basepairs) infeeding with fish meal and soybean meal diet.

ical effects of test diets on intestinal epithelium areshown in Figs. 1 and 2.

3.3. Microbiological analysis of cultivable microflora

In Trial I, levels of total bacteria (both aerobic andanaerobic) decreased during first 4 weeks with both fishfeeds (Figs. 3, 4, 5 and 6). Mean levels after 4 weeksfeeding were as low as 2.7 log CFU/g intestinal content.During the next 4 weeks levels of aerobic bacteriaincreased with the FM based diet, but remained still lowwith the SBM-based diet. The numbers of total lacticacid bacteria and lipolytic bacteria followed closely thepattern of the total aerobic/anaerobic bacterial counts.According to the API-results the dominant generaamong aerobic species were Aeromonas, Sphingomo-nas and Chryseomonas. Among the lactic acid bacteriathe genera Lactococcus and Lactobacillus were repre-sented (Table 3).

3.4. PCR-based analysis of microbiota in Trial II

The microbial communities of fish intestines wereanalyzed after 4 and 18 weeks of FM and SBM diet. Thelengths of the amplified LH-PCR fragments from theintestinal samples varied between 465 and 551 bp.Altogether 17 different fragment sizes were detected, 11of which contributed to over 95% of the amplified DNAof all of the samples, showing relatively low bacterialdiversity. Significant effects were not seen in the averagenumber of LH-PCR band length classes between thedifferent diet groups during the first sampling, but amarginally significant (p=0.09) differencewas seen in thesamples taken after 18 weeks of different diet (in average

the LH-PCR analysis of fish intestinal samples after 4 and 18 weeks of

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5.2 LH-PCR bands in the fish diet group and 7.8 LH-PCRbands in the SBM-based diet group), indicating that theSBM increased the number of new detectable groups inthe LH-PCR diversity analysis.

The most common LH-PCR lengths were 466, 499,505 and 520 bp long (Fig. 7.), and bacteria belonging tothose LH-PCR groups were prevalent in all samples.Some new groups of bacteria emerged in the intestinalcommunities of SBM fed fish that were totally absentfrom the LH-PCR profiles from FM fed fish. Significant(pb0.05) differences existed due to diet in the relative

Fig. 8. A andB.Clustering of themicrobial communities of fish intestinal samplescomponent analysis of 16S rRNAgene community fingerprints. Tank number (1–using the Jackard similarity coefficient and PCAwas based on the covariance m

intensities of separate LH-PCR bands 473, 505, 520,522 and 551 bp. However, the effect of time was alsosignificant in all those peak lengths (except 473 bp) aswell as in bands 465 and 466 bp. Other detected peaksizes varied between the samples of each treatmentgroups, showing differences between the samplespooled from separate tanks. This is especially seen inthe UPGMA cluster analysis (Fig. 8A), in which thetreatments did not form separate clusters but time sortedthe samples better, which some exceptions. Theprincipal component analysis (Fig. 8B) showed clearly

in the feeding experiment using (A)UPGMAdendrogramand (B) principal10) is also shown in the UPGMA tree. UPGMAclusteringwas constructedatrix.

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the effect of time and the phenomenon that the sampleordinations were more scattered after 4 weeks diet thanafter 18 weeks diet. The analysis showed also an effectof meal, although the separation was on the y-axis,which did not have as much explanation power as the x-axis. The two components shown in the Fig. 8Bexplained 83% of the variance in the diversity analysis.

4. Discussion

SBM-associated histological changes (supranuclearvacuolization of the absorptive cells in the distalintestine epithelium and shortening of heights ofmucosal foldings, presence of inflammatory cells) seenin this study confirm previous findings (Van den Ingh etal., 1991; Baeverfjord and Krogdahl, 1996; Burrels etal., 1999). Despite these pathological findings, thegrowth of fish was not affected, although SBM dietresulted in higher feed conversion ratio than FM diet.Measuring the heights of mucosal foldings is prone tovarious sources of error, for instance position of tissue ina section and shape of a folding. In spite of these,prominent differences in heights were evident, as alsoseen in studies of Baeverfjord and Krogdahl (1996) andRefstie et al. (2005). However, the appearance andbehavior of the fish in the SBM groups were normal,and — with the exception of the higher FCR — theirgrowth parameters were similar to those observed in fishfed with the traditional diet. This implies that animalwelfare was not compromised in the trial. The severityof intestinal inflammation is largely dependent onprocessing method and level of soy as well as on thefish species (Storebakken et al., 2000).

Low levels (less than 3.9 log cfu/g intestinal content)of cultivable bacteria were identified from the hindgut ofrainbow trout. All bacteria levels decreased during thefirst 4 weeks and started to recover in FM-fed fish duringthe next 4 weeks. This recovery was not observed in fishfed with SBM diet during the first 8 weeks of the trial.

The lipolytic bacteria which were analyzed in orderto detect possible microbiological adaptations to SBMcontaining diet did not show any feed related quantita-tive differences between the experimental groups. Lowincubation temperatures were applied in the cultivationof lactic acid bacteria in order to detect species favoringcommon temperature conditions of coldwater fishfarming and to avoid possible errors caused by applyingconditions suitable for bacteria from homeothermalanimals (Ringo and Gatesoupe, 1998).

The tentative taxonomic characterization of thecultivable aerobic or aerotolerant microflora did notreveal any observable differences between the two

feeding regimes regarding the identified genera amongthe intestinal isolates. Sphingomonas appeared to bemore common among the bacteria isolated from the fishmeal fed group, but this might be just an accidentalfinding, and would require additional studies forconfirmation. In both FM and SBM groups thedominant aerobic genera among the isolates wererepresentatives of Aeromonas, Sphingomonas andChryseomonas, while among the lactic acid bacteriathe common genera were Lactococcus and Lactobacil-lus. However, the results are based on the data obtainedwith API-strips, and these can be considered reliableonly at the genus level. The species designations shouldbe performed using more advanced techniques (such as16S rDNA sequencing) in order to detect eventualdifferences in species' composition.

Although quantitatively the effects of feeding on themajor cultivable bacterial groups appear marginal, the16S rDNA length variation demonstrated by PCR clearlysuggests changes also in species' composition. In thefuture more thorough studies on the actual composition ofboth the cultivable and non-cultivable microflora shouldbe undertaken in order to find out the nature of thesechanges and to what extent they reflect the adaptation ofthe fish and fish microbiota to variable diets.

The LH-PCR analysis is a robust method to visualisethe overall diversity of bacterial community, givingpossibility to screen relatively many samples at the sametime. The analysis showed low numbers of dominatingLH-PCR fragments suggesting relatively narrow bacte-rial diversity in the intestines in both dietary groups.Both the meal type and especially the time had an effecton the proportional abundances of different LH-PCRsize classes, showing an effect in the bacterialcomposition, which is an interesting result. Appearanceof distinct new bands that were unique for SBM fed fishand indication of generally higher number of bands inthe LH-PCR of samples from SBM fed fish may reflectdifferent nutritional conditions for the bacteria allowingdifferent groups to establish in the intestine. Anotherpossible explanation for new LH-PCR bands seen onlyin the intestines of SBM fed fish could be the reductionof bacterial numbers in the dominating bacterial groupsenabling the proportion of other bacteria to rise abovethe detection limit. When looking at the bacterialdiversity as a whole, principal component analysisindicated some correlation between the bacterialdiversity profiles, time and diet, although no explicitseparation of time or meal groups could be seen in theUPGMA dendrogram. Generally, differences betweenthe intestinal bacterial diversity between parallel tankswere high, as it is seen from the standard errors,

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UPGMA tree topology and scattering of the PCAordination, although the samples were pooled fromseveral (five) fish individuals. It is possible that therandom drift leads to differentiation of intestinalmicrobiota in different tanks, but we do not, at present,have data from separate individuals to check this. Themicrobiota of fish intestines may be affected by the otherfish in the same tank since the fish eat from the samewater to which they defecate.

LH-PCR does not reveal, what bacterial species areabundant, but it can exclude the existence of specificgroups, if their biomarkers do not match those seen in theanalysis. In the molecular analysis of intestinal micro-biota of fish, the dominant bacterial species in wild andfarmed salmon have been Acinetobacter jejunii andMycoplasma phylotype (Holben et al., 2002). Thefragments sequenced in that study did not cover theLH-PCR region used in our study, but the sequencesfrom some closest organisms from the EMBL (EuropeanMolecular Biology Laboratory) database had LH-PCRlengths of 522 bp (Acinetobacter jejunii, accessionnumber z93438) and 521 bp (both for Mycoplasmaiowae, m24293, and Mycoplasma muris, m23939). Inthe same study (Holben et al., 2002) the relative propor-tion of Lactobacillus sequences was less than 4%. TheLH-PCR lengths of the Lactobacillus genus (115 se-quences analysed) in the EMBL database, varies be-tween 534 and 568 bp. The samples of this study wereanalyzed to reveal the existence of lactic acid bacteria,and the analysis showed that several Lactococcus lactisstrains could be isolated (our unpublished results). TheLH-PCR length of Lactococcus lactis (9 strainsanalyzed) simulated from the database varied 534 and535 bp. Since all these species have biomarkers that arealso seen or really close to band sizes in our LH-PCRanalysis, we cannot exclude their prevalence withoutfurther studies.

In this study, cultivation and molecular approacheswere used to detect differences between microbialcommunities of fish fed with different diets. Differenceswere found due to sampling time and meal type. It wouldbe an interesting issue to continue the study by cloningand sequencing of the 16S rRNA genes of the bacterialphylotypes only detected in the fish fed with the SBM-based diet. However, as seen from the clusteringanalysis, differences in the fish intestinal microbialcommunities of different tanks with the same diet werehigh, demonstrating the difficulty to make general-izations of the microbial diversity of intestinal samples.

The indication of dietary response in the intestinalbacterial community to SBM seen in this study suggeststhat it might be possible to isolate bacteria capable of

colonizing fish intestine and helping the host to adjust todietary changes. This requires, naturally, thoroughcharacterization of the normal and abnormal fishmicrobiota and subsequent selection of most suitablecandidate strains for further testing.

In homeothermal animals the intestinal microbiotaclosely interacts with the host, and the composition of theintestinal community can be beneficially affected byeither applying probiotics, or live bacterial preparations,or prebiotics, which are selective substrates for bacterialgroups considered as health promoting (Nousiainen et al.,2004). Probiotics have mainly been used to combat acuteintestinal disorders. In fish the aim has been to control fishpathogens (Nikoskelainen et al., 2001; Spanggaard et al.,2001). The possibility to use bacterial preparations toadapt fish to new types of nutrients would represent anovel probiotic application.

Despite pathological changes in gut histology, growthin fish fed with soybean meal based diets supplementedwith phosphorus and amino acids was not compromised.When feed intake was not restricted, fish were able tocompensate slight nutritional deficiencies by increasedfeed intake, which was also demonstrated in the higherFCR by SBM. Good growth of fish reflects physiologicalplasticity to adapt to a evolutionarily non-familiar feed,although also the domestication of rainbow troutmay playa role in the development of tolerance to soybean meal.

5. Conclusions

Inclusion of SBM (45% of FM replaced) wasassociated with increased FCR and with histopatholog-ical changes which were similar to those reported in theliterature. Slight quantitative differences in the develop-ment of cultivable intestinal microflora and apparentlyalso quantitative changes as indicated by the 16S rDNAfragment length polymorphism were also observed.However the diet-associated microbiological differencesor the inflammatory responses did not affect animalperformance in trials lasting up to 18 weeks. Furtherstudies should clarify, whether the inflammatory changesin the gut epithelium can be eliminated by optimizednutrient supplementation, removal of anti-nutrients, orby introducing changes in the fish intestinal microbiota.

Acknowledgements

The authors want to acknowledge the skilful assistanceof the laboratory personnel in the Institute of AppliedBiotechnology and Kuopio regional laboratory of FinnishFood Safety Authority. Financial support for theseexperiments was provided by the National Technology

268 J. Heikkinen et al. / Aquaculture 261 (2006) 259–268

Agency of Finland (TEKES), Kemira Grow-HowOy andSavon Taimen Oy. The data was presented as a poster atthe 11th International Symposium on Nutrition andFeeding in Fish, Phuket, Thailand, 2nd–7th May 2004.

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