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2 Aquculture (2011) Contents lists vilble t SciVerse ScienceDirect Aquculture journl homepge: Replcement of fishmel by incresing levels of plnt protein blends in diets for Seneglese sole (Sole seneglensis) juveniles E.M. Cbrl,b, M. Bcelr,b, S. Btist,b, M. Cstro-Cunh c, R.O.A. Ozório, L.M.P. Vlente,b, CIMAR/CIIMAR, Centro Interdisciplinr de Investigção Mrinh e Ambientl, Ru dos Brgs, 289, Porto, Portugl b ICBAS, Instituto de Ciêncis Biomédics de Abel Slzr, Universidde do Porto, Lrgo Prof. Abel Slzr, 2, Porto, Portugl c ACC A. Coelho & Cstro, Ld, Prç Luís de Cmões, 15 R/C, Póvo de Vrzim, Portugl rticle info bstrct Article history: Received 27 July 2011 Received in revised form 16 September 2011 Accepted 21 September 2011 Avilble online 29 September 2011 Keywords: Environmentl impct Fish-in:Fish-out Flt fish N nd P budgets Plnt protein Sustinble diets A growth tril ws conducted to evlute the growth potentil nd nutrient utiliztion of Seneglese sole fed diets contining incresing replcement levels of dietry fishmel (FM) by mixtures of plnt protein (PP) sources. Six extruded isonitrogenous nd isolipidic diets (55% crude protein nd 8% crude ft on dry mtter bsis) were fed to juvenile sole (men initil body weight: 8 g±0.06) during 92 dys. A reference diet contining FM s the min protein source (Control) ws compred with five PP bsed-diets with incresing PP levels: 25% (PP25), 35% (PP35), 45% (PP45), 60% (PP60) nd 75% (PP75). PP45, PP60 nd PP75 diets were supplemented with crystlline mino cids to simulte the level present in the Control diet. The environmentl impct of the tested diets ws ssessed through the determintion of N nd P budgets nd clculting pproximte Fish-in:Fish-out (Fi:Fo) rtios. Fish fed PP25 nd PP75 reched finl body weight nd dily growth index (DGI) similr to the Control group (PN0.05). Diet PP45 displyed the lowest DGI (0.8 vs 1.3%), lowest protein efficiency rtio (1.03 vs 1.8) nd highest FCR (1.7 vs 1.01). The dietry tretments with highest percentge of FM replcement (PP60 nd PP75) displyed similr FCR vlues s the Control ( ). Apprent digestibility coefficients (ADCs) of protein vried between 77 nd 85% nd were not significntly ffected by the PP inclusion level. Whole body protein nd energy content did not differ significntly mong tretments, but protein gin ws significntly higher in the Control group compred to PP35, PP45 nd PP60 groups. The results indicte tht Seneglese sole cn effectively cope with plnt protein-bsed diets, but growth rte nd nutrient gin minly depend on the selection of dequte plnt protein blends, rther thn on the plnt protein incorportion level. Sole fed the highest PP level (PP75) showed good growth performnce, efficient dietry nutrient utiliztion nd lower Fi:Fo rtio compred to the Control. The present results clerly show tht incresing FM replcement level cn hve positive environmentl impct s reduces P fecl wste nd the fishmel used per kg of sole produced Elsevier B.V. All rights reserved. 1. Introduction Totl quculture production hs incresed from tonnes in 1995 to tonnes in During the sme period, production of mrine fish species hs grown from tonnes to tonnes. Consequently, the shre of mrine fish species in overll quculture output hs risen from 13% in 1995 to 36% in 2007 (FAO, 2010). Southern Europe quculture industry expnded extensively in the lst few decdes minly due to the culture of few mrine species. The recent decrese in profit mrgins for the min cultured species like gilthed sebrem (Sprus urt), Europen sebss (Dicentrrchus lbrx) nd turbot (Scophthlmus mximus) pointed to the need of finding new cndidte species for mrine culture (Dinis et l., 1999). In this scenrio, Corresponding uthor t: CIIMAR Centro Interdisciplinr de Investigção Mrinh e Ambientl, Ru dos Brgs, 289, Porto, Portugl. Tel.: ; fx: E-mil ddress: lvlente@icbs.up.pt (L.M.P. Vlente). Seneglese sole (Sole seneglensis, Kup 1858) ppers s strong cndidte to diversify the Southern Europe quculture. Since the lte 1990s, the high interest in culture of the species hs led to significnt dvnces in optimizing wening techniques nd lrve feeding (Conceição et l., 2007, 2008; Dinis et l., 1999; Engrol et l., 2009,b, 2010; Moris et l., 2006). Recently informtion on nutritionl needs of this fltfish during juvenile stge hs been ttined (Borges et l., 2009; Conceição et l., 2008; Dis et l., 2004, 2010; Rem et l., 2008; Silv et l., 2009, 2010) nd contributed decisively for tody's progressive estblishment of lrge-scle commercil frming of sole in Portugl nd Spin. Protein is the most importnt nd expensive dietry nutrient of quculture diets. In sole juveniles, protein requirement for good overll growth performnce hs been estimted to be bove 53% crude protein (DM), while the requirement for mximum protein ccretion (N gin) ws met by diet contining 60% crude protein level (Rem et l., 2008). Due to its high nutritionl vlue nd pltbility, fishmel (FM) still remins the mjor dietry protein source comprising 20% to 60% of fish feeds (Tcon, 1995). But supplies of fish mel /$ see front mtter 2011 Elsevier B.V. All rights reserved. doi: /j.quculture

3 E.M. Cbrl et l. / Aquculture (2011) nd oil re finite, nd the choice of ingredients from vilble plnt sources in qufeed formultions is minly driven by the price of FM nd oil reltive to those lterntive ingredients. Incresing inclusion of plnt proteins in qufeeds is still chllenge for the sector due to insufficient informtion on the biovilbility of essentil nutrients needed to formulte lterntive feeds for mjor frmed species (Hrdy, 2010). However, gret reserch efforts hve been devoted to the evlution of vrious plnt protein (PP) ingredients s sustinble lterntives to FM. Replcement of FM by blend of PP sources in fish feeds is presently mjor trend in quculture (Gtlin et l., 2007; Hrdy, 2010; Tcon nd Metin, 2008). Replcement of FM by PP sources hs been ccomplished in number of species t different levels (50 95%) with minimum mino cids (AA) supplementtion, without jeoprdizing growth performnce, feed utiliztion nd protein ccretion (Burel et l., 2000; Dis et l., 2009; Kushik et l., 2004; Refstie et l., 1998). Seneglese sole cpcity to cope with diets in which the mrine-derived protein ws replced by PP ingredients ws recently demonstrted (Dis et l., 2009; Silv et l., 2009, 2010; Vlente et l., 2011), nd it ws further evidenced tht sole juveniles cn grow eqully well on diets completely devoid of FM provided the dietry AA were blnced (Silv et l., 2009). Reserch hs to focus on the development of diets tht minimize the inclusion of crystlline AA, which re expensive, in order to hve prcticl nd sustinble diets vilble for the feed industry. Additionlly, the development of eco-friendly feeds should lso trget the reduction of outputs such s phosphorus (P) nd nitrogen (N) wste. This cn be ttined through improving nutrient vilbility nd optimizing the digestible protein to energy blnce of the diet (Wtnbe, 2002). The environmentl impct of fish frming is closely ssocited with excessive feed wstge nd sub-optiml nutrient utiliztion. FM contins reltively high levels of P nd this minerl is one of the most importnt environmentl pollutnts. Yng et l. (2010) demonstrte tht reducing dietry FM levels, reducing P excretion, nd incresing P digestibility re criticl pproches to reduce environmentl pollution. In this context, 92 dy growth tril ws undertken with Seneglese sole juveniles to test six isonitrogenous diets contining incresing PP levels. FM ws grdully replced (25, 35, 45, 60 nd 75% PP of totl crude protein) by blend of different PP sources (pes, soyben, whet gluten nd corn gluten). The tril ws conducted to evlute the growth potentil nd nutrient utiliztion of Seneglese sole juveniles fed diets contining incresing PP levels. The environmentl impct of the tested diets ws lso ssessed by determining N nd P budgets nd clculting pproximte Fish-in:Fish-out (Fi:Fo) rtios. 2. Mterils nd methods The current study ws conducted under the supervision of n ccredited expert in lbortory niml science by the Portuguese Veterinry Authority (1005/92, DGV-Portugl, following FELASA ctegory C recommendtions), ccording to the Europen Economic Community niml experimenttion guidelines, Directive of 24 November 1986 (86/609/EEC) Experimentl diets Formultion nd the proximte composition of the experimentl diets re presented in Tble 1 nd the mino cid (IAA) profile in Tble 2. Six extruded isonitrogenous nd isolipidic diets (55% crude protein, nd 8% crude ft on dry mtter bsis) were formulted: reference diet contining FM s the min protein source (Control) nd five PP bsed-diets (blend of soyben mel, pes, corn gluten, nd whet) with incresing PP levels: 25% (PP25), 35% (PP35), 45% (PP45), 60% (PP60) nd 75% (PP75). All diets contined hydrolysed protein concentrte (CPSP) nd PP60 nd PP75 diets hd lso 5% squid mel to improve pltbility (Espe et l., 2006). FO levels were djusted Tble 1 Feed ingredients nd proximte composition of the experimentl diets. Dietry tretments Feed ingredients (%) Fishmel 70 LT Fishmel CPSP Squid mel Soyben mel Soy PC Pes Whet gluten Whet mel Potto concentrte Corn gluten Fish oil Vit b nd Min Premix c Lutvit C Lutvit E Choline chloride Betfin MCP L-Lysine DL-Methionine Proximte composition Dry mtter (DM, %) Ash (% DM) Crude protein (% DM) Crude ft (% DM) Totl phosphorus (% DM) NDF (% DM) Strch (% DM) Gross energy (kj/g DM) MCP monoclcium phosphte, NDF nondigestible fiber. Soluble fish protein hydrolyste (75% crude protein, Sopropêche, Frnce). b Vitmins (mg or IU kg 1 diet): Vitmin A (retinyl cette), IU; vitmin D3 (DL-choleclciferol), 2125 IU; vitmin K3 (mendione sodium bisulfite), 12.5 mg; vitmin B12 (cynocoblmin), mg; vitmin B1 (thimine hydrochloride), 10 mg; vitmin B2 (riboflvin), 25 mg; vitmin B6 (pyridoxine hydrochloride), 12.5 mg; folic cid, 7.5 mg; biotin, mg; inositol, 375 mg; nicotinic cid, 87.5 mg; pntothenic cid, 37.5 mg. c Minerls (g or mg kg 1 diet): Mn (mngnese oxyde), 25 mg; I (potssium iodide), mg; Cu (cupric sulfte), 6.25 mg; Co (coblt sulfte), 0.13 mg; Zn (zinc oxide), 37.5 mg; Se (sodium selenite), 0.31 mg; Fe (iron sulfte), 75 mg. Tble 2 Amino cid composition (g 16 g 1 N) of experimentl diets. Dietry tretments His Arg Thr Vl Met Ile Leu Phe Lys Tyr Cys Asx Glx Ser Gly Al Pro Tu IAA DAA IAA/DAA Trp ws not nlyzed.

4 76 E.M. Cbrl et l. / Aquculture (2011) ccordingly to keep the diets isolipidic nd to supply essentil ftty cid. Diets PP45, PP60 nd PP75 were supplemented with L-lysine, wheres PP75 lso included DL-methionine to simulte the level present in the Control diet. Prior to extrusion (twin-screw extruder, 2.0-mm pellet size, SPAROS, Portugl) ll ingredients were grounded with hmmer mill (0.8-mm sieve) nd mixed. The digestibility tril ws crried out with the sme diets, supplemented with 1% chromic oxide (Cr 2 O 3 ) s n inert mrker. The diets were dried t 45 C for 12 h nd stored t 4 C until use Growth tril The feeding tril ws crried out with htchery-borned (Coelho & Cstro Ld., Portugl) Seneglese sole (S. seneglensis) juveniles. Prior to the experiment, fish were held in qurntine t the experimentl fcilities of CIIMAR for 4 weeks, to dpt for the new rering conditions. For ech tretment, triplicte groups of 16 homogeneous fish (men initil body weight: 8 g±0.06) were distributed to 18 fiber glss tnks of 50 L (50 35 cm=0.175 m 2, initil fish density: 0.75 kg/m 2 )inclosed recircultion sewter system. Ech tnk ws supplied with filtered nd heted (19±1 C) sltwter (30±1 ), t flow rte of 2 L min 1.Dissolved O 2 (N90% sturtion in wter), ph nd nitrogenous compounds were monitored dily during the entire tril nd mintined t levels within limits recommended for mrine fish species. Fish were kept under constnt photoperiod (12 h light:12 h drkness). The tril lsted 12 weeks nd fish were fed using temporized utomtic feeders to pprent stiety over 24 h. Uneten feed ws dily collected nd used to djust dily rtion when necessry s previously described by Borges et l. (2009) Smpling procedures All fish were individully weighed nd mesured t the beginning nd t the end of the tril fter 24 h fsting period. Dt on totl feed consumption nd bulk weight of ech tnk were recorded every four weeks. Prior to finl smpling, fish were fsted for 24 h nd killed by shrp blow on the hed. Ten fish from the initil stock nd four fish from ech tnk t the end of the feeding tril were pooled nd stored t 20 C for subsequent whole-body composition nlysis. The liver nd viscer weight ws recorded nd the heptosomtic index nd viscerosomtic index were clculted. At the end of the growth tril, the remining groups of fish were used to determine the pprent digestibility coefficients (ADCs) of experimentl diets by the indirect method, using 1% chromic oxide s dietry inert trcer (Mynrd et l., 1979). Eight to ten hours fter the lst mel, s required for digest to rech the posterior intestine (Dis et l., 2010), fish were scrificed by shrp blow on the hed nd dissected. The posterior intestine (from the ileocecl vlve to the nus) ws identified nd its contents were crefully collected. Feces smples from ech tnk were pooled nd frozen t 20 C prior to nlysis Chemicl nlyses All chemicl nlyses were crried in duplicte out ccording to AOAC (2006). Frozen whole fish from ech tnk were minced without thwing, using met mincer, pooled nd moisture content ws determined. Fish were subsequently freeze-dried before further nlysis. Feed nd whole body smples were nlyzed for dry mtter (105 C for 24 h), sh (550 C for 6 h), crude protein (N 6.25, Leco N nlyser, Model FP-528, Leco Corportion, St. Joseph, USA), crude lipid (petroleum ether extrction, Soxtherm, Gerhrdt, Germny), totl P (molybdte-blue/scorbic cid method t 820 nm fter minerliztion nd cid digestion) ccording to AFNOR (1992) nd gross energy (dibtic bomb clorimeter, Werke C2000, IKA, Germny). Fecl smples were scrce being just nlyzed for dry mtter, crude protein, totl P nd chromic oxide ccording to Bolin et l. (1952) fter perchloric cid digestion. Totl mino cids (AA) were determined in experimentl diets fter hydrolysis (6 M HCl t 106 C over 24 h in N-flushed glss vils) nd pre-column derivtiztion with phenyl isothiocynte (PITC; Pierce). Anlyses were done by high-performnce liquid chromtogrphy (HPLC) in Wters Reversed-Phse AA Anlysis System, using the PicoTg method (Wters, Bedford, USA), nd norleucine s n internl stndrd, following the procedures described by Cohen et l. (1989). Tryptophn ws not determined, since it is prtilly destroyed by cid hydrolysis. Asprgine is converted to sprtte nd glutmine to glutmte during cid hydrolysis, so the reported vlues for these mino cids (Asx nd Glx) represent the sum of the respective mide nd cid. The resultnt peks were nlyzed with BREEZE softwre (Wters, USA) Clcultions Feed conversion rtio (FCR) ws clculted s: dry feed intke (g) wet weight gin 1 (g), nd the dily growth index (DGI, 100) s: 100 [(W 1 ) 1/3 (W 0 ) 1/3 ] (dys) 1,whereW 0 nd W 1 re the initil nd the finl fish men weights in grms. The protein efficiency rtio (PER) ws clculted s weight gin (g)/protein ingested (g) 1.The dily nutrient gin, (g kg 1 ABW dy 1 ) ws clculted s: (finl body nutrient content initil body nutrient content) ABW 1 dys 1, where ABW ws clculted s: (W 1 +W 0 )/2. The heptosomtic index (HSI, 100) ws clculted s: 100 liver weight (g) whole body weight (g) 1 nd the viscerosomtic index (VSI, 100) s 100 viscer weight (g) whole body weight (g) 1. The pprent digestibility coefficients (ADCs) of the dietry nutrients nd energy for the reference nd test diets were clculted ccording to Mynrd et l. (1979) s follows: ADC ð% Þ ¼ dietry Cr 2 O 3 level feces nutrient or energy level : feces Cr 2 O 3 level dietry nutrient or energy level ADC of dry mtter ws clculted ccording to the following formul: ADC ð% Þ ¼ dietry Cr 2O 3 level : feces Cr 2 O 3 level Dily N nd P blnce in sole fed the experimentl diets ws clculted s follows: N Gin (g ABW 1 kg dy 1 ): finl crcss N content initil crcss N content; fecl N losses (g ABW 1 kg dy 1 ): crude N intke (100 ADC N); nd metbolic N losses (g ABW 1 kg dy 1 ): digestible N intke N gin; P gin (g ABW 1 kg dy 1 ): finl crcss Pcontent initil crcss P content; fecl P losses (g ABW 1 kg dy 1 ): crude P intke (100 ADC P). The ssessment of the quntity of live fish from cpture fisheries required for ech unit of frmed fish produced, Fi:Fo rtios (kg feed fish used/kg sole produced), were clculted ccording to Tcon nd Metin (2008) from the known dietry FM nd FO contents of the experimentl diets, feed conversion rtios of sole fed ech diet, nd ssuming yield from feed fish of 22.5% for FM nd 5% for FO, s follows: kg FM in the diet FCR 100/ 22.5 nd kg FO in the diet FCR 100/ 5, respectively (Fig. 3) Sttisticl nlysis Sttisticl nlyses followed methods outlined by Zr (1999). Dt re expressed s men±stndrd devition. All dt were tested for homogeneity of vrinces by Levene's tests, nd then submitted to one-wy ANOVA with the IBM SPSS STATISTICS, 17.0 pckge (IBM Corportion, New York, USA, 2011). When these tests showed

5 E.M. Cbrl et l. / Aquculture (2011) significnce, individul mens were compred using Tukey's post hoc test. Significnt differences were considered when P Results Dt on pprent digestibility coefficient (ADC) of dry mtter, crude protein nd phosphorus re reported in Tble 3. ADC of dry mtter vried from 44 to 60%, in PP45 nd Control, wheres protein ADC rnged between 77 nd 85% for PP25 nd PP75, but did not differ significntly mong tretments. Phosphorus vilbility ws low in ll diets (23 46%), nd given the vribility within tretments no significnt differences were registered mong them. After the 12-week tril, with the exception of PP45, ll groups of fish more thn tripled their initil body weight. Finl body weight nd dily growth index (DGI) were significntly higher (Pb0.05) in fish fed the FM bsed diet (Control) thn in those fed with PP35, PP45 nd PP60 (Tble 4). Sole fed with PP75 grew t similr rte s ll other groups, including fish fed the Control diet. Feed conversion rtio (FCR) rnged between 1.01 in the Control, nd 1.73 in fish fed PP45 (Pb0.05). Conversely, protein efficiency rtio (PER) rnged from 1.03 (PP45) to 1.77 (Control) nd ws lowest in PP45 (Pb0.05). Nutrient intke ws not significntly ffected (Pb0.05) by the experimentl tretments (Tble 4), with the exception of P intke tht ws significntly higher in fish fed with Control, PP25 nd PP45 diets thn in other tretments. Heptosomtic nd viscerosomtic indexes were not ffected by dietry tretments (Tble 5). Whole body composition, expressed in terms of wet weight, ws not significntly ffected (Pb0.05) by the increment of PP levels, with the exception of P content of fish fed PP35 tht ws significntly lower thn those fed the Control diet (Pb0.05). Nutrient nd energy retention nd gin were highest in fish fed the Control diet, but did not differ significntly from those fed PP25 or PP75. Dily phosphorous nd nitrogen blnce ws determined for ll tretments nd results re presented in Figs. 1 nd 2. Fecl P losses were lowest (Pb0.05) in fish fed with PP60 nd PP75, wheres P gin ws highest in fish fed the Control. Fecl N losses were significntly lower in fish fed with PP60 nd PP75 diets thn in ll the other tretments. Moreover, groups fed with the Control diet showed the lowest metbolic N losses. The nitrogen retention efficiency (% digestible intke) vried significntly mong tretments, being highest in the Control (43%) nd lowest in the PP45 fed fish (27%). In wht concerns Fi:Fo rtios (Fig. 3), dt show tht reducing dietry FM inclusion clerly reduces the mount of FM used to produce 1 kg of sole, but increses the FO input. 4. Discussion Over recent yers, substntil reserch effort hs been conducted towrds the replcement of fishmel (FM) by sustinble lterntive sources, such s plnt ingredients (PP). The suitbility of this replcement in terms of growth performnce is highly vrible mong fish species nd experimentl conditions. Avilble knowledge shows tht sensible blend of different plnt protein sources is needed to blnce the indispensble mino cid (IAA) profile, nd hence minimizing the required mino cid (AA) supplementtion (Dis et l., 2009; Kushik et l., 2004; Schulz et l., 2007; Silv et l., 2009). In the present study, t the end of the growth tril men body weight tripled in most groups nd displyed DGI vlues between 0.8 nd 1.3. Borges et l. (2009) reported similr growth vlues for Seneglese sole juveniles fed FM-bsed diets, nd Silv et l. (2009) observed even superior growth performnce with PP-bsed diets when supplemented with crystlline AA. DGI differences mong studies re probbly relted to the genetic origin nd juvenile qulity, since sole broodstock is still wild. The present results show tht incresing replcement of FM by blend of PP sources in the diets hd significnt impct on the growth performnce of sole. Sole fed with PP75 grew t similr rte s ll other groups, including fish fed the Control diet. Both PP60 nd PP75 diets included pondered blend of plnt ingredients tht required miniml IAA supplementtion. These dt suggest gret potentil for plnt ingredients to replce fishmel s dietry protein source for sole, provided the mino cids re blnced, s observed by Silv et l. (2009, 2010). An lmost totl replcement of fishmel by PP ws successfully ccomplished in Europen se bss (Kushik et l., 2004) nd in Atlntic slmon (Slmo slr L.) (Espe et l., 2006) without compromising growth or protein utiliztion. High levels of FM replcement (with mino cids supplementtion) led to growth reduction in turbot (Regost et l., 1999), Jpnese flounder (Prlichthys olivceus) (Kikuchi, 1999), rinbow trout (Oncorhynchus mykiss) (Gomes et l., 1995), Europen sebss (Dis et l., 2005) nd Atlntic cod (Gdhus morhu L.) (Hnsen et l., 2007). In most of these studies, poor growth rtes of fish fed plnt protein bsed diets were ssocited with poor feed intke, though in Seneglese sole the incresing replcement of FM by blend of PP ingredients ffected neither nutrients nor energy intke. The reltively high nutrient intke mong ll tretments (13 16 g/kg verge body weight per dy) ws probbly induced by the incorportion of hydrolysed protein concentrte (CPSP) nd squid mel tht improved dietry pltbility, s previously suggested in other fish species (Espe et l., 2006). An increse in feed conversion rtio (FCR) nd reduction in protein utiliztion seem to be mjor concern when FM is replced by PP (Opstvedt et l., 2003; Refstie et l., 1998; Robin et l., 1995). The poorest FCR vlues were registered when PP35 nd PP45 diets were used (1.4 nd 1.7, respectively). Interestingly, the dietry tretments with highest percentge of FM replcement (PP60 nd PP75) displyed similr FCR vlues s the Control ( ), nd within the rnge of vlues normlly observed in growth trils with sole juveniles (Borges et l., 2009; Rem et l., 2008; Rued-Jsso et l., 2004; Silv et l., 2009). A similr trend ws registered for PER, with PP60 nd PP75 diets presenting similr vlues s the Control ( ), tht were lso comprble to previous vlues registered with sole juveniles (Borges et l., 2009; Rem et l., 2008; Silv et l., 2009). Fish fed PP75 diet demonstrted nutrient retention (% intke) nd gin similr to tht of the Control fed fish. These results reflect high Tble 3 Apprent digestibility coefficients (ADCs, %) of nutrients of experimentl diets. Dietry tretments ADC (%) Dry mtter 59.59± ± ± ± ± ±9.64 Protein 79.20± ± ± ± ± ±1.36 Phosphorus 30.72± ± ± ± ± ±4.32 N retention efficiency (% digestible intke) 43.62± ±2.88 b 32.47±1.46 bc ±2.35 c 36.57±2.55 b 34.70±1.95 bc Vlues represent men nd stndrd devition. In ech line, different superscript letters indicte significnt differences between tretments (Pb0.05).

6 78 E.M. Cbrl et l. / Aquculture (2011) Tble 4 Growth performnce nd feed intke of Seneglese sole fed the dietry tretments with incresing plnt protein levels. Dietry tretments Growth Initil body weight (g) 7.98± ± ± ± ± ±0.22 Finl body weight (g) ± ±3.62 b 24.23±0.06 bc ±0.02 c 24.86±3.95 bc 25.77±1.55 bc Dily growth index (DGI) 1.30 ± ±0.15 b 0.98 ±0.06 bc 0.75 ±0.07 c 0.99 ±0.18 bc 1.04 ±0.08 bc Feed conversion rtio (FCR) 1.01 ±0.10 c 1.20 ±0.04 bc 1.37 ±0.10 b 1.73 ± ±0.07 bc 1.19 ±0.06 bc Protein efficiency rtio (PER) 1.77 ± ±0.05 b 1.30 ±0.09 bc 1.03 ±0.10 c 1.53 ±0.09 b 1.50 ±0.07 b Intke (g or kj/kg ABW/dy) Dry mtter 13.36± ± ± ± ± ±1.33 Protein 7.51 ± ± ± ± ± ±0.74 Lipids 0.94 ± ± ± ± ± ±0.10 Phosphorus 0.29 ± ± ±0.01 b 0.30 ± ±0.03 b 0.21 ±0.02 b Energy ± ± ± ± ± ±28.44 ABW verge body weight. Vlues represent men nd stndrd devition. In ech line, different superscript letters indicte significnt differences between tretments (Pb0.05). qulity of the plnt protein blend used in those diets nd the cpcity of sole to cope with them. In generl, PP45 diet induced the poorest nutrient utiliztion nd gin, though whole body composition nd heptosomtic index of the fish were not ffected by PP inclusion level, confirming previous dt on juveniles (Silv et l., 2009). Differences in digestibility coefficients mong feed ingredients do occur frequently nd re usully n effect of vritions in the rw mterils due to their origin nd processing conditions. Differences cn lso occur mong fish species nd due to procedures for fecl collection or the clcultion of pprent digestibility coefficients (ADCs) (Tibbetts et l., 2006). Dt on ingredient nd nutrient digestibility for fltfish, prticulrly for sole is quite scrce. Most of the previous studies in Seneglese sole juveniles filed in determining nutrients' ADC vlues due to the low cohesion of the feces nd the morphology of the intestine, tht prevent the use of trditionl methodologies for fecl collection like decnttion or striping methods (Borges et l., 2009). Present dt regrding the ADC of dry mtter ws generlly low (44 60%) in comprison with other species (Burel et l., 2000b; Tibbetts et l., 2006), nd ws not significntly ffected by the tested diets. Digestibility of dry mtter decreses with the inclusion of indigestible subtrcts nd cn contribute for the high suspended solids often observed in recirculted wter systems with sole (personl observtion). Apprent digestibility coefficients (ADCs) of protein vried between 77 nd 85% nd were not significntly ffected by the PP inclusion level. Such vlues re within the rnge of those previously registered in turbot (Dy nd González, 2000; Regost et l., 1999), Atlntic hlibut (Berge et l., 1999), Atlntic slmon (Mundheim et l., 2004; Øverlnd et l., 2009), nd Atlntic cod (Førde-Skjærvik et l., 2006), confirming the cpcity of this species to digest protein from vegetble ingredients. Dis et l. (2010) determined the ADC coefficients of prcticl feed ingredients (fish mel, solvent-extrcted soyben mel, corn gluten nd whet mel) dissecting the intestine of severl lrge sized sole. They registered high protein ADC for fishmel nd corn gluten (87 91%), but moderte vlues for whet mel (59%). Although dry mtter ADC vlues did not vry significntly mong dietry tretments, PP35 nd PP45 resulted in the poorest ADC vlues (44 45%). One possible explntion could be the highest inclusion of whet mel (23%) in those diets. Similr dietry whet inclusion levels resulted in good growth rtes in previous experiment with sole juveniles, but without ny concomitnt FM replcement (Borges et l., 2009). In the present study, PP60 nd 75 groups displyed the uppermost protein ADC vlues (84 85%) tht could explin Tble 5 Whole body composition nd nutrient utiliztion of Seneglese sole fed the dietry tretments with incresing plnt protein levels. Dietry tretments VSI 2.48± ± ± ± ± ±0.23 HSI 1.12± ± ± ± ± ±0.13 Whole body composition (% WW) Dry mtter 24.10± ± ± ± ± ±0.30 Ash 2.02± ± ± ± ± ±0.10 Protein 18.19± ± ± ± ± ±0.49 Lipids 4.92± ± ± ± ± ±0.43 Phosphorous 0.29± ±0.02 b 0.23±0.02 b 0.28 ±0.02 b 0.27±0.01 b 0.26 ±0.02 b Energy (kj) 6.00± ± ± ± ± ±0.10 Gin (g or kj/abw kg/dy) Dry mtter 3.42± ±0.32 b 2.90±0.05 bc 2.44 ±0.12 c 3.03±0.17 b 3.06 ±0.13 b Protein 2.57± ±0.23 b 2.18±0.03 bc 1.87 ±0.08 c 2.20±0.17 bc 2.25 ±0.11 b Lipids 0.79± ± ± ± ± ±0.07 Energy 87.08± ± ± ±5.53 b 77.68± ±3.93 Retention (% intke) Dry mtter 25.83± ±0.97 b 19.28±0.92 bc 15.55±1.40 c 23.86±2.10 b 22.46±1.53 b Protein 34.44± ±2.17 b 25.62±1.15 bc 21.06±1.84 c 30.76±2.14 b 29.51±1.66 b Lipids 84.17± ±2.11 b 69.95±2.00 b 55.89±5.54 b 87.02± ±11.02 b Energy (kj) 32.79± ±0.93 b 24.14±1.16 bc 18.94±2.21 c 28.39±3.33 b 28.07±1.93 b ABW verge body weight. Vlues represent men nd stndrd devition. In ech line, different superscript letters indicte significnt differences between tretments (Pb0.05).

7 E.M. Cbrl et l. / Aquculture (2011) g/ ABW Kg/ dy 0,30 0,25 0,20 0,15 0,10 0,05 0,00 b bc cd cd d b b b c bc bc bc Ctrl PP25 PP35 PP45 PP60 PP75 Metbolic P losses Fecl P losses P Gin g/ ABW Kg/ dy 1,60 1,40 1,20 1,00 0,80 0,60 0,40 0,20 0,00 b b b b b b b b bc c bc b Ctrl PP25 PP35 PP45 PP60 PP75 Metbolic N losses Fecl N losses N Gin Fig. 1. Dily phosphorous (P) blnce in Sole seneglensis fed the vrious experimentl diets. Brs represent men nd stndrd devition. Different superscript letters indicte significnt differences between tretments (pb0.05). Fig. 2. Dily nitrogen (N) blnce in Sole seneglensis fed the vrious experimentl diets. Brs represent men nd stndrd devition. Different superscript letters indicte significnt differences between tretments (pb0.05). the good growth performnce registered in these groups. These results might be n effect of the dietry inclusion of pe nd soy protein concentrte ingredients with proven performnce benefits in severl fish species (Dvis et l., 2002; Hjen et l., 1993; Øverlnd et l., 2009; Tibbetts et l., 2006). In the future, the evlution of ADC vlues of different feedstuffs, including pe nd soy protein concentrte used in the formultion of PP60 nd PP75 diets, is wrrnted to ssure blnced mixtures of ingredients ble to sustin good growth performnce nd nutrient utiliztion. Comprtive crcss nlysis combined with dt on the digestibility of the different diets, llowed the clcultion of the P nd N budgets (Figs. 1 nd 2). Dily fecl N losses nd N gin re reltively smll frctions in comprison to metbolic N losses. A slightly increse in metbolic N losses nd decline in N gin were observed when the dietry inclusion level of PP incresed up to 45%. However, in PP60 nd PP75 groups concomitnt decrese in the fecl nd metbolic N losses tended to increse N gin in comprison to PP45. According to Fournier et l. (2004), these results might be due to the fct tht some of the tested plnt ingredients might not be eqully digested nd/or my depend on the mount nd nture of the nutrient present in the selected plnt ingredients. The tested diets were isonitrogenous nd with similr IAA/DAA rtios, resulting in similr protein ADC vlues. Nevertheless, the PP blend used in PP75 resulted in the highest protein ADC tht could explin n N gin similr to the Control fed fish. Generlly, n increse in mmoni N excretion nd fecl N losses is coupled with decrese in protein efficiency nd such pttern hs been reported in severl studies with FM substitution by PP or niml protein sources (Ergün et l., 2008,b; Robin et l., 1995; Turker et l., 2005; Yng et l., 2010; Yigit et l., 2006). But in se brem, Dis et l. (2009) reported similr metbolic N losses nd dily N gin in fish fed with diets with incresing substitution of FM. When using plnt ingredients s principl dietry protein sources in fish feeds, specil ttention to P biovilbility is needed since those ingredients usully present low P level, nd mostly in the phytte form which is unvilble to most fish species (Lll, 1991; NRC, 1993). Hence, in this study, PP60 nd PP75 diets were supplemented with n inorgnic P form (monoclcium phosphte). The registered ADC vlues for P were generlly low (23 46%), but within the rnge of those reported for rinbow trout (Médle et l., 1998), Europen se bss (Dis et l., 2005) nd Atlntic slmon (Storebkken et l., 1998). Incresing levels of dietry PP did not seem to significntly ffect P pprent digestibility coefficient, though high vrition ws registered within groups. The P digestibility of PP45 ws the lowest, resulting in significnt reduction of the digestible P intke in those fish (0.05 g/kg ABW/d) compred to the ones fed PP25, PP75 nd Control diets ( g/kg ABW/d). These results seem to suggest dietry deficiency of digestible P in PP45 (0.3% vs % in other diets) tht could prtly explin the poorest growth performnce nd feed efficiency of those fish. Previous studies showed tht soluble P excretion ws significntly reduced by the use of plnt-rich diets (Dis et l., 2009; Kushik et l., 2004; Vielm et l., 2000). Similrly, in our study, fecl P losses were lower in groups fed with PP60 nd PP75 diets thn in those fed the Control, reflecting the P dietry profile, once P ADC did not vry significntly mong diets. Yng et l. (2010) hve lredy suggested tht dietry P levels re clerly ssocited with FM levels in the diets nd the totl P losses re grdully reduced when more FM is replced. The present results clerly show tht incresing FM replcement level cn hve positive environmentl impct s reduces P fecl wste. The soluble metbolic P losses followed the sme trend up to PP60, but diet PP75 showed the highest dissolved P wste, suggesting concentrtion of vilble P bove dietry requirement level for sole. The vilbility of FM nd FO is n importnt fctor limiting further expnsion of fish frming industry. The impct of FM replcement ws determined by clculting pproximte Fish-in:Fish-out (Fi:Fo) rtios ccording to Tcon nd Metin (2008). The present results demonstrted tht reducing dietry FM inclusion clerly reduced the mount of FM used to produce 1 kg of sole, but incresed the FO input. Moreover, higher FCR were reflected in incresed Fi:Fo rtio for FO use s previously suggested in other studies (Nylor et l., 2009; Prtoomyot et l., 2010). Prtoomyot et l. (2010) showed tht reducing FM inclusion in slmon diets reduced the feed fish required for fishmel usge, but lso evidenced the higher impct of dietry FO on the sustinbility issue thn the utiliztion of FM. Seneglese sole hs lower dietry lipid tolernce thn Atlntic slmon tht is reflected in its lower feed fish for oil usge. The use of vegetl oil sources in diets for Seneglese sole should lso be considered in the ner future to reduce fish oil input, but ssuring essentil ftty cids nd without compromising the finl qulity of the fish. In conclusion, these results indicte tht Seneglese sole cn effectively cope with plnt protein-bsed diets, but growth rtes nd nutrient gin minly depend on the selection of dequte PP blends, rther thn on the PP incorportion level. In fct, sole fed the highest Kg feed fish used/kg sole produced 3,0 2,5 2,0 1,5 1,0 0, ,0 CTRL PP25 PP35 PP45 PP60 PP75 Feed fish for fishmel usge, kg kg-1 Feed fish for oil usge, kg kg-1 Fig. 3. Amount (kg) of fish feed used per kg of sole produced. Dt were clculted from the known dietry fishmel nd fish oil contents of the experimentl feeds (Control, PP25, PP35, PP45, PP60 nd PP75), feed conversion rtios of sole fed ech feed, nd ssuming yield from feed fish of 22.5% for fishmel nd 5% for fish oil (Tcon nd Metin, 2008).

8 80 E.M. Cbrl et l. / Aquculture (2011) PP level (PP75) showed good growth performnce, efficient dietry nutrient utiliztion nd lower Fi:Fo rtio compred to the Control. Further studies on ingredients digestibility re wrrnted to help selecting the most dequte plnt protein combintion for this species. Moreover, the long term impct of high PP incorportion levels is needed to determine its prcticl vibility. Acknowledgments This work ws crried out by Project PROAMBIENTE for enhncing SMEs orgniztionl nd mngement structure to enter foreign mrkets. PROAMBIENTE hs the finncil support of Qudro de Referênci Estrtégico Ncionl QREN nd Progrm Opercionl Regionl do Norte ON2 supported by the Europen fund for regionl development FEDER. E.M. Cbrl nd M. Bcelr were supported by grnt from PROAMBIENTE. References AFNOR, Vindes, préprtions de vinde et produits à bse de vinde - Détermintion de l teneur en phosphore totl. NF V AOAC, Officil Methods of Anlysis of AOAC Interntionl. 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9 E.M. Cbrl et l. / Aquculture (2011) Silv,J.M.G.,Espe,M.,Conceição,L.E.C.,Dis,J.,Vlente,L.M.P.,2009.Seneglesesolejuveniles (Sole seneglensis Kup, 1858) grow eqully well on diets devoid of fish mel provided the dietry mino cids re blnced. Aquculture 296, Silv, J.M.G., Espe, M., Conceição, L.E.C., Dis, J., Costs, B., Vlente, L.M.P., Feed intke nd growth performnce of Seneglese sole (Sole seneglensis Kup, 1858) fed diets with prtil replcement of fish mel with plnt proteins. Aquculture Reserch 41, e20 e30. Storebkken, T., Sherer, K.D., Roem, A.J., Avilbility of protein, phosphorus nd other elements in fish mel, soy-protein concentrte nd phytse-treted soyprotein-concentrte-bsed diets to Atlntic slmon, Slmo slr. Aquculture 161, Tcon, A.G.J., Feed ingredients for crnivorous fish species: lterntives to fishmel nd other fishery resources. In: Reinertsen, H., Hlnd, H. (Eds.), Sustinble Fish Frming, pp Tcon, A.G.J., Metin, M., Globl overview on the use of fish mel nd fish oil in industrilly compounded qufeeds: trends nd future prospects. Aquculture 285, Tibbetts, S.M., Milley, J.E., Lll, S.P., Apprent protein nd energy digestibility of common nd lterntive feed ingredients by Atlntic cod, Gdus morhu (Linneus, 1758). Aquculture 261, Turker, A., Yigit, M., Ergun, S., Krli, B., Erteken, A., Potentil of poultry byproduct mel s substitute for fishmel in diets for Blck Se turbot Scophthlmus meoticus: growth nd nutrient utiliztion in winter. Isreli Journl of Aquculture- Bmidgeh 57, Vlente, L.M.P., Linres, F., Villnuev, J.L.R., Silv, J.M.G., Espe, M., Escórcio, C., Pires, M.A., Svedr, M.J., Borges, P., Medle, F., Alvárez-Blázquez, B., Peleteiro, J.B., Dietry protein source or energy levels hve no mjor impct on growth performnce, nutrient utilistion or flesh ftty cids composition of mrket-sized Seneglese sole. Aquculture 318, Vielm, J., Mäkinen, T., Ekholm, P., Koskel, J., Influence of dietry soy nd phytse levels on performnce nd body composition of lrge rinbow trout (Oncorhynchus mykiss) nd lgl vilbility of phosphorus lod. Aquculture 183, Wtnbe, T., Strtegies for further development of qutic feeds. Fisheries Science 68, Yng, Y.-H., Wng, Y.-Y., Lu, Y., Li, Q.-Z., Effect of replcing fish mel with soyben mel on growth, feed utiliztion nd nitrogen nd phosphorus excretion on rinbow trout (Oncorhynchus mykiss). Aquculture Interntionl Yigit, M., Erdem, M., Koshio, S., Ergün, S., Türker, A., Krli, B., Substituting fish mel with poultry by-product mel in diets for blck se turbot Psett meotic. Aquculture Nutrition 12, Zr, J.H., Biosttisticl Anlysis. Prentice Hll, London.