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1 Aquculture 283 (2008) Contents lists ville t ScienceDirect Aquculture journl homepge: Processing soyen mel for iodiesel production; effect of new processing method on growth performnce of rinow trout, Oncorhynchus mykiss Frederic T. Brrows,, T. Gison Gylord, Wendy M. Seley, Michel J. Hs c, Roert L. Stroup d U.S. Deprtment of Agriculture, Agriculturl Reserch Service, Hgermn Fish Culture Experiment Sttion, 3059-F Ntionl Fish Htchery Rod, Hgermn, ID, 83332, USA University of Idho, Hgermn Fish Culture Experiment Sttion, 3059-F Ntionl Fish Htchery Rod, Hgermn, ID, 83332, USA c U.S. Deprtment of Agriculture, Agriculturl Reserch Service, Estern Regionl Reserch Center, 600 Est Mermid Lne., Wyndmoor, PA, 19038, USA d R.L. Stroup Co. LTD., 1835 Fox Run, Troy, OH, USA ARTICLE INFO ABSTRACT Article history: Received 20 Novemer 2007 Received in revised form 28 June 2008 Accepted 29 June 2008 Keywords: Soyen mel processing Biodiesel Trout feed Alternte protein source In situ trnsesterifiction A new method of soyen mel processing hs een developed, which my simplify the process of iodiesel production. This method, in situ trnsesterifiction, elimintes hexne extrction to remove the oil, comining the extrction nd trnsesterifiction steps so s to synthesize iodiesel vi single tretment conducted directly on lipid-ering solid mteril. If the resulting mel is comprle in nutritionl vlue to commercilly ville hexne-extrcted soyen mel (SE-SBM) the new process could ecome widely used in the io-fuel industry. Two levels (17.5 nd 35%) of ech of three types of soyen mel were fed to triplicte lots of 30 (initil wt 22 g) rinow trout for 9 weeks in flow-through 15 C spring wter. The three types of soyen mel included SE-SBM, n experimentlly produced hexne-extrcted SE-SBM (ESE-SBM), nd mel produced using in situ trnsesterifiction (IS-SBM) nd ech ws fed t two levels for totl of 6 diets. Growth of fish fed the diets ws good, verging over 600% gin. There ws no effect of source of soyen mel on weight gin of trout. The fish fed the mel processed y the new method, IS-SBM, gined s much weight s fish fed either of the two control mels, t ech inclusion level. Fish fed the diets contining IS-SBM, however, did hve higher feed intke (2.51% w/d) compred to fish fed the ESE-SBM or SE-SBM, 2.38 nd 2.46% w/d, respectively. Since growth ws equl, feed conversion rtios were higher for fish fed the IS-SBM diets. Protein nd energy retention vlues were lower for fish fed the IS-SBM diets. There ws no effect of soyen source on crcss composition. Apprent digestiility for protein ws lower for the IS-SBM (85.9%) thn for the ESE-SBM (89.3%). Feeding IS-SBM did not decrese weight gin in this study, ut due to the incresed feed intke nd FCR, long term feeding trils should e conducted to further evlute the mel. Pulished y Elsevier B.V. 1. Introduction As the quculture industry serches for sustinle replcements for fish mel, the energy industry lso serches for sustinle replcements for fossil fuels. Both industries re evluting oilseed crops, ut with miniml forethought their uses could e complementry rther thn competitive. For exmple, iodiesel which consists of the simple lkyl esters of ftty cids cn e mde effectively from soyen oil, ut current methods of production re costly. While replcement for fish oil is needed nd soyen oil cn e used in quculture feeds, ffordle high qulity soy protein is currently of greter interest. Mention of trde nmes or commercil products in this rticle is solely for the purpose of providing specific informtion nd does not imply recommendtion or endorsement y the U.S. Deprtment of Agriculture. Corresponding uthor. E-mil ddress: Rick.Brrows@rs.usd.gov (F.T. Brrows). Soyen mel hs een stple protein source for the niml feed industries ut its vlue hs een limited for rinow trout for numer of resons. The most prominent fctor limiting soyen mel inclusion in trout or slmon feeds hs een the occurrence of enteritis (Refstie et l., 2000). The oligoscchride content of soyen mel, primrily stchyose nd rffinose, hs een identified s possile custive gent of soyen enteritis (vn den Ingh et l., 1991, 1996; Bureu et l., 1998). Lending support for this theory is the fct tht soy protein concentrte contins only 3% oligoscchrides compred to up to 15% in some soyen mel (Russett, 2002) nd soy protein concentrte cn e used t higher dietry inclusion levels thn soyen mel without reducing growth (Gtlin et l., 2007). A new method for processing soyen mel my ffect the chemicl composition of the mel nd thus ffect either the presence of enteritis, or the level t which enteritis occurs in trout fed the new mel. An dditionl limittion to the utiliztion of soyen mel in rinow trout diets hs een tht the mino cid lnce of soyen mel is slightly low in methionine (NRC, 1993). This limittion cn generlly e overcome y dietry supplementtion with commercilly /$ see front mtter. Pulished y Elsevier B.V. doi: /j.quculture

2 142 F.T. Brrows et l. / Aquculture 283 (2008) Tle 1 Composition of reference diet for determining pprent digestiility coefficients Ingredient g kg 1 (dry weight sis) Menhden fish mel, Specil Select Whet flour Menhden Fish oil 80.0 Vitmin C c 3.0 Choline Cl 50% 5.0 Vitmin premix 6.0 Trce Minerl 1.0 Yttrium oxide d 0.1 Chromic oxide d 10.0 Anlyzed composition g kg 1 (dry weight sis) Crude protein Phosphorus 17.3 Energy (kj/g) c d Omeg Protein Corp., Hmmond, Louisin, USA. Nelson & Sons Inc., Murry, UT, USA. Vitmin C s Rovimix Sty-C 35, DSM Nutritionl Products, Bsel, Switzerlnd. Sigm-Aldrich Compny, St. Louis, Missouri, USA. ville sources of methionine or y utilizing complimentry protein sources. Supplementtion thus cn improve the protein vlue of soyen mel nd remove some of the potentil growth reduction when soy protein concentrtes re utilized s the primry protein ingredient (Medle et l., 1998). These fctors comined with limittions on the use of fish mel nd terrestril niml y-product mels hve further incresed the utiliztion nd therey demnd for soy protein mel in feeds for crnivorous fish. The iodiesel industry hs the potentil to utilize vst mount of soyen oil, if production methods cn e cost effective. Estimted iodiesel production in 2007 is 400 million gllons (chnge to metric), nd the industry is experiencing rpid growth (Ntionl Biodiesel Bord, pers. comm.). New methods re eing developed tht might support cost-effective production nd result in lrge quntities of soyen mel produced y method different from tht trditionlly used (Hs et l., 2004, 2007; Hs nd Scott, 2007). While the new processing technique effectively produces high qulity iodiesel (Hs nd Scott, 2007) it my lter the composition nd nutritionl vlue of the soyen mel in either positive or negtive wy. Therefore, the current tril ws performed to ssess chnges in composition of soyen mel from n experimentl in situ trnsesterifiction process for oil removl compred to solvent extrction methodology, ssess effects of processing technique on pprent nutrient digestiility for rinow trout, nd to mesure production performnce of rinow trout fed incresing levels of the in situ esterified soyen mel. 2. Mterils nd methods 2.1. Soyen mel processing Two soyen mels were experimentlly produced. The preprtion of in situ extrcted soyen mel (ISE-SBM) ws s previously descried (Hs nd Scott, 2007). Flkes were dried in convection oven to less thn 1% moisture, determined grvimetriclly. In typicl rection, 2.25 kg of dried flkes ws then mixed y gentle rolling t room temperture in seled continer with 5.75 L of 0.1 N dry NOH in nhydrous methnol. After 5.5 h the gittion ws stopped nd the liquid removed y filtrtion. The flkes were then wshed twice y suspension for 5 min in 3.2 L of nhydrous methnol, nd recovered y filtrtion. The ulk of the residul lcohol in the flkes ws removed y ir drying in shllow pns. Multiple runs were conducted to ccumulte 30 kg of ISE-SBM. The mel ws then sujected to regimes of het nd stem. Determintion of lipid content y extrction nd high performnce liquid chromtogrphy (Hs et l., 2007), indicted n verge residul mel oil content of 1.1% (rnge: 0.2 to 2.2%). Experimentl hexne-extrcted soyen mel (ESE-SBM) differed from the in situ extrcted soyen mel (ISE-SBM) in tht the flked soyen mel used in preprtion of the in situ extrcted soyen mel ws extrcted with hexne s opposed to sodium hydroxide nd methnol to remove the lipid. Finl lipid levels, determined y exhustive extrction with hexne in Soxhlet extrctor, were less thn 1.0% in ESE-SBM. As with ISE-SBM, the ESE-SBM ws susequently treted with het/moisture regimes nd residul levels of trypsin inhiitor ctivity determined Soyen mel digestiility To determine the digestile nutrients in the two sources of experimentlly produced soyen mels, the methods of Cho et l. (1982) nd Bureu et l. (1999) were used to estimte pprent digestiility coefficients (ADCs). Yttrium oxide served s the inert mker. A complete reference diet meeting or exceeding ll known nutritionl requirements for rinow trout (NRC, 1993) (Tle 1) ws lended with the test ingredients (ISE-SBM or ESE-SBM) in 70:30 rtio (dry weight sis) to form test diets. All diets were produced y cold-extrusion (Advnced Hydrolyzing Systems, Inc., Astori, OR, USA) with 3-mm die nd dried to 10% moisture. Rinow trout, Oncorhynchus mykiss, Housecreek strin, were otined from the College of Southern Idho (Twin Flls, ID) with 50, 250-g fish per 140-L fierglss tnk. Wter temperture ws mintined t 14.5 C throughout the feeding tril using flow-through spring wter. Lighting ws mintined on 14:10-h diurnl cycle. Ech diet ws fed to three tnks of fish. Ech diet ws rndomly ssigned to tnk of fish nd fed to pprent stition twice dily for 7 dys prior to fecl collection. Fecl smples were otined in one collection y mnul stripping h post-feeding. Mnul stripping of fish ws ccomplished y netting nd nesthetizing ll fish in the tnk, followed y gently drying nd then pplying pressure to the lower dominl region to express fecl mtter into plstic weighing pn. Cre ws tken to exclude urinry excretions from the collection. Fecl smples for given tnk were dried overnight t 50 C nd stored t 20 C until chemicl nlyses were performed. Apprent digestiility coefficients of ech nutrient in the test diet nd ingredients were clculted ccording to the following equtions (Kleier, 1961; Forster, 1999): ADCN diet ¼ fkYt in diet knutrient in fecesg= fkyt in feces knutrient in dietg ADCN ingredient ¼ fð þ ÞADCN t ðþadcn r where, ADCN ingredient ADCN t ADCN r p g 1 pprent digestiility coefficient of the nutrient in the test ingredient pprent digestiility coefficients of the nutrient in the test diets pprent digestiility coefficients of the nutrient in the reference diet (1 p) nutrient content of the reference diet p nutrient content of the test ingredient proportion of test ingredient in the test diet 2.3. Soyen mel utiliztion A three y two fctoril design ws used with soyen mel source nd level of soyen mel in the diet s the min effects. Three sources of soyen mel included commercilly produced, hexne-extrcted soyen mel (SE-SBM), in situ extrcted soyen mel (ISE-SBM) nd experimentl hexne-extrcted soyen mel (ESE-SBM). The sme

3 F.T. Brrows et l. / Aquculture 283 (2008) Tle 2 Ingredient composition (g/kg) of experimentl diets Soy 17.5 Soy 35 Ingredient Fish mel Blood mel Soyen mel c Corn gluten mel Whet gluten Whet flour Fish oil Di-clcium phosphte Methionine Vitmin premix d Choline Cl Ascoric cid Trce minerl premix e Nutrient composition Crude protein, g/kg f Lipid, g/kg f Ash, g/kg f Energy, kj/g Peruvin nchovy, 70% protein. Spry dried, 70% protein, Interntionl Ingredients, Firfield, NJ. c Soyen mel ws either commercilly solvent extrcted soyen mel (SE-SBM), experimentlly solvent extrcted soyen mel (ESE-SBM) or in situ esterified soyen mel (ISE-SBM). d Contriuted per kilogrm of diet: vitmin A (s retinol plmitte), 10,000 IU; vitmin D 3, 720 IU; vitmin E (s DL-%-tocopheryl-cette), 530 IU; nicin, 330 mg; clcium pntothente, 160 mg; rioflvin, 80 mg; thimin mononitrte, 50 mg; pyridoxine hydrochloride, 45 mg; mendione sodium isulfte, 25 mg; folcin, 13 mg; iotin, 1 mg; vitmin B 12, 30 mg. e Contriuted in mg/kg of diet: zinc, 37; mngnese, 10; iodine, 5; copper, 3. f Wet weight sis. source of soyens ws used to produce oth the ISE-SBM nd ESE- SBM. Ech of the three mel sources ws included in the diet t either 17.5 or 35% of the diet (Tle 2), nd chosen to e ove nd elow the generlly regrded threshold of soyen mel inclusion (20%) tht cuses intestinl enteritis. The diets were formulted to contin 18% fish mel nd other ingredients common in commercil trout feeds, to meet or exceed known nutrient requirements for trout (NRC, 1993) nd to e iso-nitrogenous nd iso-lipidic, with 40% crude protein nd 12% crude ft. Prior to mixing the diets, ll ingredients were ground using n irswept pulverizer (Jcosen 18H, Minnepolis, MN). Dry ingredients were mixed in horizontl mixer nd portion ( 1/3) of the dded oil ws mixed into the dry ingredients long with the lecithin. The msh ws then extruded through 3.0-mm die of Buhler twin-screw cooking extruder (DNDL-44, Buhler AG, Uzwil, Switzerlnd). Brrel temperture verged 130 C in sections 2 6, die pressure ws 360 psi nd the feed hd rrel residence time of pproximtely 18 s. The diets were dried in pulse ed drier (Buhler AG, Uzwil, Switzerlnd) with ir dischrge temperture remining elow 104 C to finl moisture content less thn 8%. After the diets were dried, they were top-coted with the remining oil (8%) using vcuum coter (A.J. Mixing Interntionl, Ontrio, CA) nd stored t room temperture ( C). Groups of 35 rinow trout (House Creek strin, College of Southern Idho), with n verge initil weight of 22 g, were rndomly plced in ech of 18, 150-L fierglss tnks. Ech tnk ws supplied with 8 L/min of untreted, constnt temperture (14.5 C), grvity-fed spring wter t the Hgermn Fish Culture Experiment Sttion, University of Idho. There were three tnks of fish per diet nd fish were fed three times per dy, 6 dys per week to pprent stition for period of 64 dys. A 14-h photoperiod, controlled y timers nd fluorescent lights ws provided. The experimentl protocol ws pproved y the University of Idho's Animl Cre nd Use Committee Chemicl nlyses Feed nd fecl smples were dried, nd nlyzed using AOAC (1995) methods for proximte composition, with the exception of protein nd lipid. Crude protein (N 6.25) ws determined in ingredients, diets nd feces y the Dums method (AOAC, 1995) on Leco TruSpec N nitrogen determintor (LECO Corportion, St. Joseph, MI, USA). Crude ft ws nlyzed using Soxhlet extrction pprtus (Soxtec System HT, Foss Tector AB, Hogns, Sweden) with methylene chloride s the extrcting solvent, nd sh y incinertion t 550 C in muffle furnce. Totl energy ws determined y isoperiol om clorimetry (Prr1281, Prr Instrument Compny Inc., Moline, IL, USA). Yttrium nlyses were conducted t the University of Idho Anlyticl Sciences Lortory, Moscow, ID, using n Optim 3200 rdil inductively-coupled plsm tomic emission spectrometer (Perkin-Elmer Corp., Norwlk, CT). Amino cids were nlyzed y AAA Lortories (Mercer Islnd, WA) on Beckmn 6300 AA nlyzer (Beckmn Instruments, Inc., Fullerton, CA). The three sources of soyen mel were nlyzed for the following constituents y the Archer Dniels Midlnd Compny, Specilty Feeds Division (Dectur, IL): trypsin inhiitor ctivity (ISO 14902) (AOCS, 1983); stchyose, rffinose, nd oligosccrides concentrtions were determined using HPLC nd clcium column; group A sponins were seprted nd quntified using methods descried y Shiriw et l. (1991); group B sponins were seprted nd quntified using methods descried y Shiriw et l. (1991), Hu et l. (2002),ndDlluge et l. (2003) Performnce indices The concentrtions of moisture, crude protein (N 6.25), energy nd phosphorus in the feed nd fish t the eginning nd end of the study were determined s descried ove. The mount of protein nd totl energy fed during the study ws used to clculte pprent protein nd energy retention during the 64-dy study. Indices re expressed on per-fish sis for ech dietry tretment group. Performnce indices were clculted using the following formule: Feed conversion rtioðfcrþ ¼ feed intkeðdry weightþ=ody weight ginðwet weightþ Heptosomtic indexðhsiþ ¼ liver mssðgþt100=fish mssðgþ Apprent protein retentionðprekþ ¼ ðprotein gin in fishðgþ=protein intke in feedðgþþ 100 Apprent energy retentionðerekþ ¼ ðenergy gin in fishðclþ=energy intke in feedðclþþ100: Feed intke expressed s percent of ody weight per dy ws clculted s percentge of the verge of the initil nd finl weights per fish from ech tnk Histology Fish fed ech of the experimentl diets were smpled t the end of the tril for histologicl nlyses. Five fish from ech of the replicte tnks were euthnized nd smples of kidney, liver, nd pyloric nd rectl intestines were preserved in Dvidson's solution for 48 h. Tissues were then trnsferred to 65% lcohol until processed y stndrd histologicl procedures (Sheehn nd Hrpchek, 1983) Sttisticl nlyses Fish performnce, nutrient retention, nutrient digestiility, nd crcss composition dt were nlyzed using the generl liner models procedure of the Sttisticl Anlysis System (SAS, 1988).

4 144 F.T. Brrows et l. / Aquculture 283 (2008) Tle 3 Chemicl composition of soyen mels SE-SBM ESE-SBM IS-SBM Anti-nutrients Glycinine, mg/kg 25,000 10,000 25,000 Bet con-glycinine, mg/kg 30,000 9,000 30,000 Lectins, mg/kg Dry solids, g/kg Stchyose, g/kg Rffinose, g/kg Oligoscchrides, g/kg TIA, mg/g Sponin, type B B, g/kg Sponin, type B (type I), g/kg Sponin, type B (type I), g/kg Sponin, type B Bc (type II, III), g/kg Sponin, type B Bc (type II, III), g/kg Type B sponins, g/kg Type DDMP sponins, g/kg Totl type B nd DDMP sponins, g/kg Nutrients Protein, % Energy, cl/g NA Moisture, % Ash, % Arginine Histidine Isoleucine Leucine Lysine Methionine Phenyllnine Threonine Tyrosine Vline Leucine Lysine Methionine Phenyllnine Threonine Tyrosine Vline Wet weight sis. Expressed s percentge of the protein. Differences in tretments mens were seprted using the Tukey's multiple rnge test. Any vlue expressed s percentge ws rcsine trnsformed prior to nlysis (Sokl nd Rohlf, 1981). Dt were lso nlyzed s 3 y 2 fctoril tretment design to evlute min effects rther thn just tretment mens. When significnt interction of the min effects ws detected, tretment mens were compred using Tukey's multiple rnge test within dietry inclusion levels. 3. Results 3.1. Chemicl composition of the soyen mels Concentrtion of mjor nutrients in the soyen mels ws similr, ut protein content of ESE-SBM (49.8%) nd ISE-SBM (49.9%) ws slightly higher thn of the commercil source (SE-SBM, 48.3%) on wet weight sis nd much higher on dry mtter sis (Tle 3). The experimentlly produced mels hd higher moisture content (vg. 11.4%) ut did not mold efore incorportion into the experimentl diets nd were then thoroughly dried. The protein content of the SE- SBM diets ws 42.4% compred to 41.4% for the ESE-SBM diets nd 41.7% for the ISE-SBM diets. Amino cid concentrtions of the three soyen mels were lso similr (Tle 3). Trypsin inhiitor ctivity, glycinine, et con-glycinine, lectins, vrious crohydrtes nd sponins were determined for ech of the mels (Tle 1). The concentrtions of ech of these sustnces, which re commonly referred to s nti-nutrients, were found in the ISE-SBM to e pproximtely equivlent to the levels found in either the SE-SBM or the ESE-SBM (Tle 3) Apprent digestiility nd vilility coefficients The pprent digestiility coefficients (ADC) for orgnic mtter, dry mtter, energy nd phosphorus were ll similr for the ESE-SBM nd ISE-SBM (Tle 4). The ADC for protein ws significntly higher for the ESE-SBM (87.3%) thn for the IS-SBM (85.9%). Significnt differences were detected for pprent mino cid vilility coefficients (AAC) etween the soyen mels processed y the different methods (Tle 4). The AAC for four essentil mino cids, rginine, isoleucine, leucine, nd threonine were higher for the ESE-SBM thn for ISE-SBM. The opposite trend ws oserved for nonessentil mino cids. The AAC for sprtic cid, serine nd proline ws higher in the ISE-SBM thn in ESE-SBM (Tle 4) Fish performnce nd nutrient retention There were effects of dietry soyen mel source nd level on growth, FCR nd feed intke (Tle 5). The level of soyen mel in the diet significntly ffected growth; trout fed the diets with 35% soyen mel hd greter growth (126.5 g) thn the fish fed the 17.5% soyen mel diets (119.1 g). There ws trend for n effect of source of soyen mel on weight gin (P=0.09). Trout fed diets contining SE-SBM hd n verge gin of g compred to g fish fed diets with ESE-SBM, or g for trout fed diets with ISE-SBM (Tle 6). FCR for fish fed ll diets rnged from 0.86 to 0.98 g feed/g gin (Tle 5). There ws n effect of oth level nd source of soyen mel on FCR. The fish fed the diets contining 17.5% soyen mel hd significntly etter FCR thn fish fed the 35% soyen mel diets. The fish fed the IS-SBM diets hd the highest FCR of 0.93 compred to 0.90 for fish fed the SE-SBM or 0.87 for fish fed the ESE-SBM diets (Tle 6). A significnt interction of level nd source on FCR lso ws oserved. At 17.5% SBM, the FCR for fish fed the IS diets ws not different from fish fed either SE or ESE-SBM. When diets contined 35% soyen mel, fish fed the ISE diets hd significntly higher FCR thn fish fed either SE or ESE (Tle 5). Source nd level of soyen mel in the diet lso hd n ffect on feed intke (Tle 6). Trout fed diets contining ISE-SBM consumed more feed (2.51% w/d) thn fish fed the diet with SE-SBM (2.46% w/d) or the diets with ESE-SBM (2.38% w/d). Incresing the inclusion level of soyen mel from 17.5 to 35% resulted in n increse in feed intke, from 2.39 to 2.51% w/d. There ws no significnt interction of these two effects. Neither source nor inclusion level of soyen mel hd n effect on HSI or fillet yield (Tles 5 nd 6). There were effects of soyen mel source nd level on oth PRE nd ERE. Fish fed diets with ESE-SBM hd PRE vlues of 43.9%, compred to 41.8 nd 40.7% for fish fed SE-SBM nd ISE-SBM, respectively. Incresing dietry inclusion level from 17.5 to 35% resulted in n increse in PRE from 41.3 to 43.0% (Tle 6). This sme increse in inclusion level, however, resulted in decrese of ERE from 42.3% for fish fed diets with 17.5 to 40.9% for the fish fed diets with 35% SBM. Source of soyen mel did not ffect ERE, ut significnt interctions of the min effects were oserved. When diets contining 17.5% SBM were fed, there ws no difference in ERE mong the three sources. When diets contining 35% SBM were fed, fish fed the ISE-SBM diets hd significntly lower ERE (38.3%) compred to 42.5 nd 41.8% for fish fed the SE-SBM nd ESE-SBM diets, respectively (Tle 5) Body composition Soyen mel source did not ffect ody composition, ut level of soyen mel in the diet hd n effect on ody protein, lipid nd energy content. The ody protein content incresed significntly when the level of soyen mel incresed from 17.5 to 35%. Body lipid

5 F.T. Brrows et l. / Aquculture 283 (2008) Tle 4 The effect of source of soyen mel on the pprent digestiility coefficients nd mino cid vilility coefficients ADC or AAC (%) ESE-SBM +SD IS-SBM +SD R-squre PNF Orgnic mtter Dry mtter Protein Energy Phosphorus Arginine Histidine Isoleucine Leucine Lysine Methionine Phenyllnine Threonine Tyrosine Vline Tryptophn Glycine Asprtic cid Serine Alnine Glutmic cid Proline nd energy content decresed with incresing level of soyen mel in the diet (Tle 5). Soyen mel source nd level hd no effect on ody sh or minerl (C, P, Mg, K, N, S) content (Tle 5) Histology There ws no difference in incidence or severity of the soyen melinduced enteritis mong fish fed the different sources of soyen mel within inclusion level. Although soyen enteritis ws oserved in fish irregrdless of dietry tretment, the severity ws much greter for trout fed diets contining 35% soyen mel. Lrge colonies of grm positive cteri were oserved in the intestine of fish fed diets contining 35% soyen mel. Some cteri ws oserved in the intestines of trout fed the 17.5% diets, ut incidence nd numers were less thn tht found in fish fed the 35% soyen mel diets. 4. Discussion Sustinility is ecoming incresingly importnt in oth the quculture nd energy industries. The serch for lterntive protein sources to fish mel, nd the serch for lterntives to fossil fuels hve oth led in the sme direction focusing on oilseeds. A new method of soyen mel processing hs een developed, which could provide enefit to oth industries nd my simplify the process of iodiesel production. If the resulting mel is comprle in nutritionl vlue to commercilly ville hexne-extrcted soyen mel (SE-SBM) the new process could ecome widely used in the io-fuel industry. The results of this study indicte tht the new processing methods did not significntly lter the proximte composition or the nti-nutrient content of the resultnt soyen mels. Further, ecuse the vlue of feed ingredient cnnot e judged y chemicl composition lone, in vivo comprison of the mels in growth nd digestiility trils were conducted nd results indicte no effect of typeofsoyenmelonweightginoftrout.weightginoftroutfedthe experimentl diets ws good during the 9-week study rnging from 520 to 570% increse over initil ody weight. In different studies t this lortory using fish of equivlent size, similr growth hs een oserved. In 9 weeks trout fed diet contining 15% soyen mel gined 450% of initil weight (Gylord et l., 2006). Gins of 450% were oserved when trout were fed diets contining pproximtely 50% soyen mel for 10 weeks (Brrows et l., 2007). In longer study, trout gined over 600% of initil weight fter 12 weeks (Stone et l., 2005). The TIA (define) levels (Tle 1) oserved in the ESE-SBM (1.8) were well elow the mximum suggested levels for slmon y Olli et l. (1994). Levels of TIA comprle to these were not found to ffect growth or nutrient digestiility in rinow trout (Olli nd Krogdhl, 1994). The TIA level of IS-SBM of 5.3% my hve hd n ffect on growth s suggested y significntly lower ADC for protein (Tle 4). An ADC for protein of 87.3% ws oserved for trout fed the diets with ESE-SBM compred to 85.9% for fish fed the IS-SBM diets. Romrheim et l. (2006) fed diets contining 246 g/kg of soyen mel with TIA level of 0.6, however, nd lso oserved decrese in weight gin of Atlntic slmon. Other fctors in ddition to TIA my e ffecting growth performnce. The trout fed the soyen mel produced y in situ trnsesterifiction hd higher feed conversion rtio nd feed intke. Compred to fish fed the ESE-SBM, FCR ws out 7% higher for fish fed the diet with IS-SBM. This is slight difference, ut would e significnt when extrpolted over production cycle. Incresed supplementtion of the IS-SBM with mino cids my hve improved performnce of fish fed the mel produced y in situ trnsesterifiction. While growth, in generl, in this experiment ws high, nd n effect of soyen mel inclusion level ws expected, trout fed the diets contining 35% soyen mel grew unexpectedly fster thn trout fed diets with 17.5% soyen mel. An inclusion level of 20% soyen mel is generlly considered to e the threshold for rinow trout, ove which reduction in growth will e oserved. The opposite ws oserved in this study. Anlysis of the essentil mino cid content of the diets my explin the oserved results. The fish mel content of the experimentl diets ws kept low t 18.2%, to reflect diets formulted during fish mel shortges. Arginine, threonine, lysine nd methionine incresed s dietry soyen mel incresed from 17.5 to 35% of diet. Arginine content of the diets with 17.5% soyen mel ws 6.97% of protein, s compred to 8.02% for the 35% SBM diets. Becuse of the high level of plnt products in the diets, oth diets contined more rginine thn reported s required for juvenile rinow trout (NRC, 1993). Reported vlues re 3.3% (Kushik, 1979), 3.6% (Wlton et l., 1986), 4.0% (Kim et l., 1983), 4.7% (Cho et l., 1989), nd 5.9% (Ketol, 1983). Threonine levels of 4.5 nd 4.8% of the diet were found in the 17.5 nd 35% SBM diets, respectively, nd re gin considerly higher thn 3.0% tht hs een reported to e required y chum slmon (Akiym et l.,1985). It is thus unlikely tht vritions in rginine nd threonine levels etween the diets with 17.5 nd 35% soyen mel were the cuse for differences in growth oserved for fish fed the diets. Lysine nd methionine levels showed similr pttern to rginine nd threonine levels. Diets with 17.5% SBM verged 5.2% lysine nd 2.5% methionine compred to 6.1% lysine nd 3.0% methionine for diets with 35% SBM. While lysine levels in diets with 17.5% SBM were much higher thn the reported requirement of 3.7% y Kim nd Kyes (1982) nd 4.2% y Wlton et l. (1984), nd lower thn 6.1% y Ketol (1983). Methionine requirements less thn found in diets with 17.5% SBM hve een reported to e 2.2% (Wlton et l., 1984), 1.4% (Kim et l., 1992), nd 1.5% (Cowey et l., 1992). The methionine content of 2.5% in the diet with 17.5% SBM should hve een more thn dequte. However, Rumsey et l. (1983) nd Kim et l. (1992) reported requirements of 3.0 nd 2.9%, respectively. The methionine levels of the 17.5% SBM diets were the sme s predicted y the formultion, ut the 35% SBM diets were pproximtely 0.20% of the diet higher thn expected. Since ll three diets contining 35% SBM hd similr methionine levels, the error ws proly not mixing error ut more likely due to n inccurte vlue for one or more ingredients during formultion. The rpid growth of trout in this study my hve lso led to detection of growth differences mong fish fed diets with smll differences in mino cid content. All of the reported requirements listed ove were determined y feeding either purified or chemiclly defined diets. The diets in the present study included prcticl ingredients nd synthetic methionine, nd with rpidly growing strin of trout, the ccepted methionine requirement (NRC, 1993) my not e dequte. In ddition to growth,

6 146 F.T. Brrows et l. / Aquculture 283 (2008) Tle 5 Effect of diet on growth performnce, nutrient retention nd ody composition of rinow trout 1 Soy mel level, % 17.5% 35% SEM P Soy mel source SE ESE IS SE ESE IS Growth performnce Gin, g/f FCR 0.92,m 0.86 c,n 0.87 c,mn 0.88 c,y 0.88 c,y 0.98,x Feed intke c 2.35 cd 2.31 d 2.42 cd 2.41 cd HSI, % Fillet yield, % Nutrient retention PRE, % ,m 42.4,m 41.7,m 43.7,x 45.5,x 39.7,y ERE, % ,m 43.2,m 42.5,m 42.5,x 41.8,x 38.3,y Body composition Protein Lipid Ash Moisture Energy, kj/g C (µg/g) 14,166 16,000 16,666 15,666 16,000 10, P (µg/g) 15,166 15,833 16,666 16,500 16,166 15, Mg (µg/g) K (µg/g) 12,000 12,333 12,333 12,833 12,666 12, N (µg/g) S (µg/g) Mens (n=3, pooled stndrd error of the men) in the sme row with the sme superscript re not significntly different (PN0.05), nd superscripts of,, nd c represent comprison of ll 6 diets. When significnt interction is detected, superscripts of m, n, nd o represent comprison of sources within the 17% level, nd superscripts of x, y, nd z represent comprison of sources within the 35% level. 2 % ody weight per dy. 3 Protein retention efficiency. 4 Energy retention efficiency. 5 % Wet weight sis. other performnce dt lso support possile mrginl mino cid deficiency cusing the reduced growth for trout fed the 17.5% soyen mel diets. Feed intke ws significntly greter for fish fed the 35% SBM diets s compred to trout fed the 17.5% SBM diets. Medle et l. (1998) oserved decrese in oth feed intke nd growth of rinow trout due to methionine deficiency in diet contining high levels of soy protein concentrte. Body protein content ws significntly higher (16.3%) nd ody lipid content ws lower (10.8%) for trout fed the 35% SBM diets compred to 15.3% ody protein nd 11.8% ody ft for trout fed the 17.5% diets. Cheng et l. (2003) found similr results when feeding reduced fish mel diets to rinow trout. Feeding diets with incresed plnt proteins nd decresed methionine levels resulted in decrese in ody protein concentrtion. Crystlline methionine ws dded to the diets in the present study, nd this form of the mino cid my not e used s efficiently s methionine from intct protein s ws oserved for chnnel ctfish with crystlline lysine (Zrte nd Lovell, 1997). The dt from the current study suggest tht with prcticl ingredients, nd only 18% fish mel nd 0.24% synthetic methionine, totl dietry level of 1.0% methionine nd 0.50% cystine my e indequte for mximum growth. Despite the differences in feed intke nd ody composition, growth for fish fed the 17.5% diets ws good, suggesting just mrginl deficiency. The sttisticl strength of the fctoril nlyses is demonstrted with these dt, since the effect of inclusion level ws not pprent using the multiple rnge test, ut ws significnt following fctoril nlysis. Histologicl results from the current study re consistent with those reported y Beverfjord nd Krogdhl (1996) nd Refstie et l. (2000) with regrds to intestinl enteritis. Severity of the enteritis incresed with incresing levels of dietry soyen mel, regrdless of the source or type of soyen mel processing. In situ trnsesterifiction did not seem to llevite or worsen this cellulr response. The presence of Grm positive cteri in the trout intestine in the present study is lso Tle 6 The effect of soyen mel processing method nd diet inclusion rte on feed consumption nd growth efficiency of rinow trout Source of mel Level, % ProilityNF vlue R- squre SE ESE IS Model Source Level S L CV Growth performnce Gin, g/f FCR Feed intke, %w/d HSI, % Fillet yield, % Nutrient retention PRE, % c ERE, % d Body composition Protein % Lipid, % Energy, cl/g c d Mens (n=3, pooled stndrd error of the men) in the sme column with the sme superscript re not significntly different (PN0.05). Source y level interction. Protein retention efficiency. Energy retention efficiency.

7 F.T. Brrows et l. / Aquculture 283 (2008) consistent with feeding soyen mel to slmon. Bkke-McKellep et l. (2007) reported chnges in the cteril popultion due to inclusion of soyen mel in the diet. There were higher totl numers of cteri nd more diversity in cteril popultions with fish fed the higher level of soyen mel. In the present study, or in the study of Bkke-McKellep et l. (2007), direct involvement of the cteri in cusing the enteritis could not e determined, ut should e evluted further. 5. Conclusions This study indictes tht soyen mel produced through in situ trnsesterifiction cn e suitle sustitute for trditionl hexneextrcted soyen mel, ut ttention must e given to mino cid lnce. Acknowledgements This reserch ws funded in s prt of the USDA/Agriculturl Reserch Service, Trout-Grins Project D. We thnk Mike Csten nd Lorrie VnTssel for fish cre, Kren Scott for conducting the in situ trnsesterifiction on soyens, nd Lucs Porter for ssistnce in feed processing. We lso thnk John Less nd ADM Corportion for soyen mel nlyses. References Akiym, T., Ari, S., Muri, T., Nose, T., Threonine, histidine nd lysine requirements of chum slmon fry. Bull. Jpn. Soc. Sci. Fish. 51, AOAC, Officil Methods of Anlysis. Assocition of Officil Anlyticl Chemists, Inc., Arlington, Virgini, USA. AOCS (Americn Oil Chemists' Society), In: Wlker, R.C. (Ed.), Officil methods nd tenttive prctices of the Americn Oil Chemists. AOCS, Chmpign, IL. 840 pp. Beverfjord, G., Krogdhl, A., Development nd regression of soyen mel induced enteritis in Atlntic slmon distl intestine. A comprison with intestines of fsted fish. J. Fish Dis. 19, Bkke-McKellep, A.M., Penn, M.H., Sls, P.M., Reftsie, S., Sperstd, S., Lndsverk, T., Ringo, E., Krogdhl, A., Effects of dietry soyen mel, inulin nd oxytetrcycline on intestinl microiot nd epithelil stress, poptosis nd prolifertion in the teleost Atlntic slmon (Slmo slr L.). Brit. J. 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