Early weaning in meagre Argyrosomus regius: Effects on growth, survival, digestion and skeletal deformities

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1 Received: 5 Decemer 216 Revised: 27 Jnury 217 Accepted: 1 Mrch 217 DOI: /re ORIGINAL ARTICL rly wening in megre Argyrosomus regius: ffects on growth, survivl, digestion nd skeletl deformities Cindy Cmpoverde 1 Covdong Rodriguez 2 Jose Perez 2 nric Gisert 1 Alici stevez 1 1 Centro de Sn Crlos de l Rpit, IRTA, Sn Crlos de l Rpit, Trrgon, Spin 2 Deprtmento de Biologı Animl, dfologı y Geologı, Universidd de L Lgun, Sn Cristol de L Lgun, Spin Correpondence Alici stévez, IRTA, Centro de Sn Crlos de l Rpit, Sn Crlos de l Rpit, Trrgon, Spin. mil: lici.estevez@irt.es unding informtion Seventh rmework Progrmme, Grnt/ Awrd Numer: KBB single stge, GA Astrct Megre Argyrosomus regius is considered new species for the diversifiction of finfish quculture in the Mediterrnen. Severl ottlenecks hve een identified y producers, nd mong them, the necessity to estlish erly wening protocols to reduce production costs. In this study, two experiments were crried out with megre lrve from 2 to 35 dys post htch (dph) using different wening strtegies, including the erly introduction of rtificil diets nd the reduction of Artemi metnuplii to hlf of the norml mounts. A high frequency of cnnilism nd high vriility in growth rte nd survivl were otined in one of the trils nd severl chnges were introduced (reduction of light intensity, higher frequency of food distriution) in the second tril to increse the survivl rte. In oth trils, wening strted efore the complete morphologicl nd functionl development of the stomch; thus, pncretic enzymes, minly trypsin nd lipse tended to e more ctive in erly wened lrve compred to the control groups. rly wening delyed the development of the stomch formtion nd secretion of cid proteses, which my explin the lower growth rtes oserved in our study. The effect of wening on skeletl development ws lso studied nd in this sense the results otined showed no mjor influence of the erly wening on the incidence of skeletl deformities. Wening of megre lrve cn e performed s erly s 12 dph, ut importnt spects such s voiding cnnilism nd co-feeding live prey nd rtificil diets for t lest 5 dys were recommended. KYWORDS cnnilism, digestive enzymes, growth, megre, skeletl deformities, wening 1 INTRODUCTION Lrvl rering of megre Argyrosomus regius is usully crried out following protocol sed in uropen se ss (Dicentrrchus lrx) nd gilthed se rem (Sprus urt) lrvl rering (stevez, Trevi~no & Gisert, 27; Roo, Hernndez-Cruz, Borrero, ernndez- Plcios & Schuchrdt, 27; Roo, Hernndez-Cruz, Borrero, Schuchrdt & ernndez-plcios, 21), including the use of enriched live feeds (rotifers, Brchionus sp. nd Artemi sp. metnuplii). However, lrviculture prctices sed on live preys represent high cost compred to inert diets, oth in terms of production nd lour costs. Different studies hve reveled tht these protocols need to e dpted to the iologicl demnds of this species, s megre lrve re quite sensitive to stress produced y high light intensity (>5 lux t wter surfce), long photoperiods or high densities of live prey (Roo et l., 21; Suzer, Kmci, Con, irt & Sk, 213; Vlles & stevez, 213). Although the precise nutritionl requirements for megre hve not een completely estlished, lrve show very good growth performnce nd survivl rtes using commercilly ville products for live prey enrichment nd feeds Aquculture Reserch. 217;1 11. wileyonlinelirry.com/journl/re 217 John Wiley & Sons Ltd 1

2 2 CAMPOVRD T AL. for wening (Vlles & stevez, 215). Megre producers do not consider lrvl rering to e mjor ottleneck for megre culture (Lzo, Holt, uvel, Suquet & Quemener, 21), nd only cnnilism nd vrile size distriution in juveniles re considered the min concern in the intensive production of megre fry, s they reduce production yield nd increse the production costs. Therefore, dvncing the erly wening of lrve from its dependence on Artemi onto dry feed is priority nd the mjor focus of the current lrvl reserch on megre. In this sense, etter knowledge of lrvl digestive physiology under new feeding protocol my contriute to the optimiztion of diets (Zmonino-Infnte et l., 28) nd my help to understnd functions nd limittions in the processing cpcity of the digestive system, nd consequently the delivery of nutrients to the rpidly growing lrvl tissues under n erlier wening protocol (Rønnestd et l., 213). Thus, estlishing n dequte feeding protocol dpted to the digestive cpcities nd nutritionl needs during erly development while lso ddressing options to reduce cnnilism nd size dispersion re of primry importnce to improve survivl nd growth in megre. Skeletl deformities in cultured fish re mjor fctor tht reduces production, suppresses growth nd increses economic loss, s well s leds to high mortlity rtes. Most skeletl normlities pper during the lrvl nd juvenile stges where severl fctors cn interfere with the norml development of lrve. xisting literture clerly suggests tht unfvourle iotic conditions, inpproprite nutrition nd genetic fctors re the most possile custive fctors of skeletl nomlies in rered fish (Boglione et l., 213). In this regrd, nutritionl imlnces re known to ply key role in morphogenesis nd skeletogenesis t erly stges (Boglione et l., 213; Person Le Ruyet, Alexndre, Theud & Mugnier, 1993). Thus, the efficiency of erly wening prctices nd its effects on skeletl development in megre lrve needs lso to e considered. Thus, the ojectives of the present study were (1) estlish wening protocol for megre to reduce production losses due to cnnilism, (2) study the chnges in digestive enzyme ctivity when live preys re replced y dry feed nd (3) descrie possile skeletl deformities derived from erly wening onto rtificil diets. 2 MATRIAL AND MTHODS 2.1 Lrvl rering nd experimentl design ertilized eggs of megre were otined from wild roodstock mintined in 4, l circulr tnks connected to recircultion units (IRTAMr â, IRTA, Spin) t IRTA Centre of Sn Crles de l Rpit under controlled conditions nd fter hormonl induction (Duncn et l., 212). loting eggs were incuted in 35 L cylindricl PVC continers provided with ir-lift systems nd high ertion supply. On dy 2 post htching (dph), lrve were stocked into 1 L tnks t density of 1 lrve/l nd cultured from 2 to 35 dph on different dietry tretments. The 1 L tnks were connected to IRTAmr â units with 5% dily wter renewl. Temperture ( C), slinity ( g/l), dissolved oxygen (7.9.3 mg/l) nd ph (7.9.2) were checked dily, wheres nitrites (<.25 mg/l) nd mmoni (<.7 mg/l) were mesured once per week (Hch Colorimeter DR/89, USA). Light intensity ws regulted with mnul potentiometer connected to ech fluorescent lmp (Philips LPS1) nd mesured t the wter surfce in the middle of the tnk with luxometer (Lutron LX-11 LUX MTR) nd mintined t 5 lux t wter surfce, wheres the light regime ws 12-hr light: 12-hr drk. Lrve were fed enriched rotifers (Brchionus sp) from 2 dph until 14 dph t density of 1 rotifers/ml nd Artemi metnuplii (Sep Art Artemi, Inve, Belgium) from 9 dph strting with.5 metnuplii/ml, incresing the density up to 6 metnuplii/ml t 2 dph nd decresing the density down to 1.5 nuplii/ml t 25 dph, nd keeping tht density until the end of the tril. Both live preys were enriched using Red Pepper TM (Bernqu, Belgium) for 12 hr t 28 C in the cse of rotifers nd 6 h t 25 C in the cse of Artemi. Lrve were fed two doses of live prey (morning nd evening) every dy, wheres dry feed (Gemm Micro, Skretting, Norwy) ws dministered y hnd every morning t 9: hours nd using utomtic feeders every hour, from 9: to 2: hours. The mount of feed ws djusted to rech the level of pprent feeding stition. very dy, the ottom of the tnk ws siphoned to remove ded fish, uneten food nd feces. Two experiments were crried out with megre lrve. In Tril 1, the following experimentl protocols were tested in triplicte: Group A: wening on dry feed strted from 2 dph nd it ws completed t 3 dph (control group) following the stndrd protocol descried ove; Group B: wening strted from 2 dph nd it ws completed t 3 dph (the sme s in Group A ut using hlf the mount of Artemi metnuplii); Group C: wening strted from 15 dph nd it ended t 25 dph using lso hlf the mount of Artemi metnuplii; nd Group D: wening strted t 12 dph nd it ws completed t 23 dph using hlf the mount of Artemi metnuplii. Due to the high incidence of cnnilism (so-clled coevl cnnilism; olkvord, 1997) mong similr-ged individuls oserved in Tril 1, severl chnges in the rering protocol were introduced in Tril 2. Thus, light intensity ws reduced from 5 lux to 15 2 lux from 13 dph onwrds, s well s the numer of doses/mels of Artemi (given t 1:, 13:, 16: nd 18: hours) nd the rtificil diet tht were incresed in order to provide enough food to lrve. This strtegy ws chosen ccording to Smith nd Rey (1991) who reported tht cnnilism is enhnced y low food vilility, high fish densities, size disprity nd lck of refuges. Thus, in Tril 2, only two tretments were tested (five replictes ech): Group : wening strted from 2 dph nd it ws completed t 3 dph (control group); nd Group : wening strted t 12 dph nd it ended t 23 dph. In oth trils, 1 lrve from ech tnk were rndomly collected to mesure growth (stndrd length, SL nd dry weight, DW) every week. ish were previously nesthetized with tricine methnosulphonte (MS-222, Sigm-Aldrich, Spin) nd SL mesured under stereomicroscope Nikon SMZ8 (Nikon, Tokyo, Jpn) equipped with digitl cmer Olympus DP7 (Olympus, Hmurg, Germny)

3 CAMPOVRD T AL. 3 nd n imge nlyser (AnlySIS, SIS Gmh, Hmurg, Germny). The sme lrve were used to estimte wet nd dry weight, plcing them, previously wshed with distilled wter, on preweighted coverslips, dried in n oven t 6 C for 24 hr, nd weighted in microlnce Mettler MX5 (Mettler Toledo, Brcelon, Spin). Specific growth rte (SGR) ws clculted using the following formul: SGR (%dy 1 ) = Ln DW f Ln DW i /t f t i ; where DW f nd DW i were the finl nd initil lrvl dry weight nt t f t i the dys from the initil until the end of the smpling periods. The coefficient of vrition (CV, %) of weight ws clculted ccording to the formul CV = stndrd devition/men 9 1. Lrve were rndomly collected when the wening strted nd the feed ws chnged from live to inert diets tht ws t 12 dph (Group D), 15 dph (Group C), 2 dph (Groups A nd B) nd t 24 dph in Tril 1 nd t the end of Tril 2 (35 dph), to nlyse the ctivity of digestive enzymes. In this cse, smpled lrve were scrificed with overdose of MS-222, rinsed in distilled wter nd conserved t 8 C until nlysis. At the end of the tril, survivl ws evluted y counting the survivors t the end of the experiment nd clculted ccording to Buckley et l. (1984) tht consider the numer of smpled individuls during the experiment. 2.2 Determintion of digestive enzyme ctivities Lrve in oth Trils 1 nd 2 (5 15 individuls depending on ge nd size) were collected for enzyme nlyses t 12, 15, 2 nd 24 dph in Tril 1 nd t 35 dph in Tril 2. or quntifying the ctivity of pncretic nd gstric enzymes (totl lkline proteses, - mylse, lipse nd pepsin), smples were homogenized (Ultr-Turrx T25 sic, IKA -Werke, Germny) in 5 volumes (v/w) of ice-cold Milli-Q wter, centrifuged t 3,3 g for 3 min t 4 C, the superntnt removed for enzyme quntifiction nd kept t 8 C until further nlysis. or the quntifiction of the intestinl rush order (BB) enzyme, lkline phosphtse smples were homogenized in cold 5 mm mnnitol, 2 mm Tris-HCl uffer (ph 7.) nd intestinl BB memrnes purified ccording to Crne et l. (1979). nzyme ctivities of pncretic, gstric nd intestinl enzymes were conducted s descried in Gisert, Gimenez, ernndez, Kotzmnis nd stevez (29) nd Solovyev et l. (216). In ddition, spectrophotometric nlyses were performed s recommended y Solovyev nd Gisert (216) in order to prevent smple deteriortion due to their short- nd long-term storge. In rief, trypsin (.C ) ctivity ws ssyed t 25 C using BAPNA (N--enzoyl- DL-rginine p-nitronilide) s sustrte. One unit of trypsin per ml (U) ws defined s 1 lmol BAPNA hydrolysed per min per ml of enzyme extrct t 47 nm (Holm, Hnssen, Krogdhl & lorholmen, 1988). Alph-mylse (.C ) ctivity ws determined ccording to Metis nd Bieth (1968), using.3% solule strch s sustrte, nd its ctivity (U) ws defined s the mg of strch hydrolysed during 3 min nd ml of tissue homogente t 37 C t 58 nm. Bile slt-ctivted lipse (BALT,.C ) ctivity ws ssyed for 3 min t 3 C using p-nitrophenyl myristte s sustrte. The rection ws stopped with mixture of cetone: n-heptne (5:2), the extrct centrifuged for 2 min t 6,8 g nd 4 C nd the sornce of the superntnt red t 45 nm. Bile slt-ctivted lipse ctivity (U/ml) ws defined s the nmol of sustrte hydrolysed per min per ml of enzyme extrct (Iijim, Tnk & Ot, 1998). Regrding intestinl enzymes, lkline phosphtse (.C ) ws quntified t 37 C using 4-nitrophenyl phosphte (PNPP) s sustrte. One unit (U) ws defined s 1 lg BT relesed per min per ml of rush order homogente t 47 nm (Bessey, Lowry & Brock, 1946). inlly, pepsin ctivity (U) ws defined s the lmol of tyrosine lierted per min t 37 C per ml of tissue homogente t 28 nm (Worthington Biochemicl Corportion, 1991). All enzymtic ctivities were expressed s specific ctivity defined s milliunits per milligrm of protein (mu/mg protein). Solule protein of crude enzyme extrcts ws quntified y mens of the Brdford s method (Brdford, 1976) using ovine serum lumin s stndrd. All the ssys were mde in triplicte from ech pool of lrve nd sornce red using spectrophotometer (Tecnᵀᴹ Infinite M2, Mnnedorf, Switzerlnd). 2.3 Anlysis of skeletl deformities To evlute the impct of different wening strtegies on the incidence of skeletl deformities in megre, 2 erly juveniles ged 35 dph per tnk were rndomly smpled t the end of Tril 2. ish were preserved in 1% uffered formlin nd stored until doule stining. Animls were stined with Alcin lue 8X nd lizrin red (Sigm-Aldrich, Brcelon, Spin) to detect crtilginous nd ony tissues s descried in Dris, Ln Chow Wing, Chu, Zmonino- Infnte nd Mzuris (21). Once stined, fish were individully exmined under dissection microscope y two independent oservers. The incidence of skeletl normlities ws determined in the crnium, verterl column nd cudl fin complex. Specil ttention ws given to verterl deformities, which were divided in two ctegories: severe, which included the fusion nd compression of djcent verterl odies, deformtion of verterl odies nd chnges in the nterior posterior lignment of vertere (kyphosis nd lordosis), nd light, including deformed heml spines nd neurl spines, nd chnges in the osteologicl orgniztion of the cudl fin complex. The nomenclture of skeletl elements ws conducted ccording to the description of megre skeletogenesis (Crdeir et l., 212). 2.4 Sttisticl nlyses Dt were expressed s men stndrd devition (SD) except for skeletl nomlies tht were expressed in men stndrd error of the men (SM) nd tested y Student s t test (Tril 2, with only two tretments) or one-wy ANOVA (Tril 1, with four tretments). When significnt difference ws found etween tretments, Tukey s test ws performed for multiple rnge comprisons with the level of significnt difference set t p <.5. All the dt were tested for normlity, homogeneity nd independence to stisfy the

4 4 CAMPOVRD T AL. ssumptions of ANOVA, wheres dt expressed s percentge were previously rcsine-trnsformed. 3 RSULTS 3.1 Growth nd survivl The results in terms of lrvl growth in SL nd DW from Tril 1 re shown in igure 1. Growth in SL nd DW of megre ged 35 dph from Groups C, wened t 15 dph with hlf the mount of Artemi metnuplii, nd A, control, ws higher thn in the rest of the other groups (p <.5), wheres fish from Group B showed the lowest vlues in SL nd DW mong groups (dt not shown). Specific growth rtes of the different groups were 25.74% dy 1 for Group A nd 21.86%, 24.79% nd 23.39% for Groups B, C nd D respectively. Survivl rtes were significntly ffected y the wening strtegy (p <.5), eing higher in fish from the Group B (2.8.6%), wheres megre from Groups A, C nd D showed similr nd lower vlues (1.7.1%, 1.2.3% nd 1.8.3% respectively). Lrge differences in size were detected mong experimentl groups t the end of the Tril 1, s indicted y the vlues of the SL (mm) Dry weight (mg) A B C D Dry weght (mg) Age (dph) Age (dph) Age (dph) IGUR 1 Growth in stndrd length (SL) (mm, men SD) nd dry weight (DW) (mg, men SD) of megre (Argyrosomus regius) lrve from the different groups t different smpling times in Tril 1. Different letters show significnt differences (ANOVA, p <.1) c c c A B C D coefficient of vrition (CV) for SL (igure 2). In prticulr, fish showing the lrgest size dispersion vlues were those from Group D ( %), wheres fish from Groups A, B nd C showed similr CV vlues ( %, % nd % respectively). Differences in DW mong smll, medium nd lrge lrve re lso shown in Tle 1, with lrve weighing from 1.28 to mg of dry weight depending on the group, such wide rnge of sizes might hve enhnced lrvl cnnilism. The results in lrvl growth in SL nd DW from Tril 2 re shown in igure 3. Growth performnce nd survivl rtes were higher in megre from the Group, wened t 2 dph nd fed using the stndrd protocol, thn those otined in Group (lrve wened t 12 dph) (p <.5). Specific growth rtes of lrve etween 1 nd 3 dph were 18.95%/dy for the control group nd 17.6%/dy for erly wened lrve, vlues tht were lmost 1.5 times lower thn those oserved in Tril 1. In this cse, the size etween the groups ws similr, with CV vlue of.12 nd.13 for groups nd respectively (igure 3). Survivl rte from Group (4.9.7%) ws lso higher thn in the erly wened Group (3.9.5%), similrly to the results otined in Tril 1. Mesures dopted in Tril 2 to reduce cnnilistic ehviour (reduction of light intensity nd higher feeding frequency) hd positive results lowering the size differences mong the lrve nd improving the survivl rte t the end of the tril. 3.2 Activity of digestive enzymes In oth trils, digestive enzyme ctivities nlysed in lrve t the end of live prey feeding period were the sme for ll the tretments (dt not shown). The ctivity of the pncretic nd intestinl digestive enzymes ws ssessed in 24 dph lrve in the cse of Tril 1 nd 35 dph lrve for Tril 2; the results re shown in igure 4. In Tril 1, trypsin ctivity ws higher in megre from Group C in comprison with lrve from Groups A nd B, wheres fish from Group D showed intermedite vlues in trypsin ctivity (p <.5). Similrly, lipse ctivity ws higher in lrve from Group B in comprison with the rest of the tretments (p <.5), wheres no sttisticlly significnt differences were found with regrd to -mylse ctivity etween groups (p >.5). The ctivity of the rush order enzyme, lkline phosphtse, ws highest in fish from Group A, wheres the lowest vlues were oserved in megre from Group D (p <.5), nd Groups B nd C showed intermedite vlues. Similrly to Tril 1, there were no differences neither in -mylse ctivity nor in trypsin regrdless of the wening strtegy used in Tril 2 (p >.5), wheres lipse ctivity ws higher in erly wened lrve (Group, p <.5). The ctivity of lkline phosphtse ws higher in lrve from the control Group thn in Group (p <.5). Regrding the ctivity of cid proteses, pepsin ws only nlysed in lrve from Tril 2; in prticulr, erly wened lrve (12 dph) showed significntly lower ctivity of this cid protese ( U mg protein 1 ) compred with lrve wened t 2 dph ( U mg protein 1 )(p <.5).

5 CAMPOVRD T AL. 5 () () (d) (c) requency (%) Group A (24 dph) ± 2.86 mm CV = 23.2% >24 requency (%) Size (mm) Group B (24 dph) 9.33 ± 2.32 mm CV = 24.9% >24 6 Group C (24 dph) 6 Group D (24 dph) ± 3.19 mm 1.84 ± 4.57 mm CV = 24.8% CV = 42.2% requency (%) >24 requency (%) 4 2 Size (mm) >24 IGUR 2 Photogrphs nd frequency digrms showing the differences in the size (SL) of the lrve (24 dph) of the experiment 1 in the groups (left), (centre, up), c (centre, down) nd d (right), due to the high incidence of cnnilism. CV, coefficient of vrition TABL 1 Differences in dry weight (mg, verge SD) mong smll, medium nd lrge lrve in the four feeding groups (A, B, C nd D) Group Smll Medium Lrge A B C D Skeletl deformities In Tril 2, erly wening of the lrve did not hve ny effect on the incidence of totl skeletl deformities in erly juveniles of megre (p >.5) with n verge frequency of deformed fish rnging from 17.6% to 21.% nor in the numer of verterl odies (98.7% specimens with 25 vertere nd 1.3% with 24). In ddition, no differences were found in the incidence of light nd severe skeletl deformities mong groups (p <.5), eing the light one the most

6 6 CAMPOVRD T AL. () 16 1 Growth in length (mm) Dry weight Student's t test 8 P < Student's t test P < Age (dph) Age (dph) SL (mm) () 8 6 Group (3 dph) ± 1.3 mm CV =.12 DW (mg) 8 6 Group (3 dph) ± 1.13 mm CV =.13 requency (%) 4 requency (%) < >16 < >16 Size (mm) IGUR 3 () Growth in stndrd length (SL) (mm, mensd) nd dry weight (DW) (mg, men SD) of Tril 2 lrve, wened t 2 dph (group ) nd 12 dph (group ). Letters indicte significnt differences (Student s t test, p <.1). () requency digrms showing the differences in the size (SL) of the lrve (3 dph) in the groups (left) nd (right) of Tril 2. CV, coefficient of vrition common mong exmined fish. In ny of the groups exmined, crnil deformities were oserved, wheres most of skeletl normlities were detected in the verterl column nd cudl fin complex, prticulrly fusions etween 2 to 24 heml vertere, deformtion of epurls 1 to 2 nd in the lst two heml vertere efore the urostile (igure 5). No significnt differences in the frequency of skeletl normlities ffecting the verterl column (preheml nd heml regions) were oserved, lthough when exmining ech type of verterl deformity, the incidence of verterl fusion in the heml region ws different etween the two groups of Tril 2. igure 5 shows some exmples of the skeletl nomlies detected in oth groups. Thus, most of the nomlies oserved cn e considered mild nomlies such s lordosis nd scoliosis (.4% for oth groups). usion of heml vertertes ws oserved in.4 to 1.6% of the exmined lrve in Group nd only in.4 to.8% in the lrve from Group. Skeletl structures of the cudl fin complex were lmost not ffected showing some defects in ossifiction ssocited with the underdevelopment or sence of epurls (Group = % nd Group = %). The incidence of severe deformities (lordosis, kyphosis, scoliosis, deformed verterl centr) ws similr in oth dietry tretments ( % for group nd % for group ), s well s in the cse of light deformities (heml spines nd neurl spines nd modified epurl) % for Group lrve nd % for Group (igure 6). 4 DISCUSSION Wening, the trnsfer from live food to n rtificil diet, is successful with most mrine fish with completely developed digestive trct (Person Le Ruyet et l., 1993). In the current study, wening ws crried out with commercil diet (Gemm Micro, Skretting, Norwy) using grdul trnsfer of live prey to this rtificil diet over minimum of five dys, lthough in some other mrine species like uropen se ss, there is n rupt replcement (Person-Le Ruyet, 199). Durn et l. (29) using wening protocol for megre, similr to the one used in Tril 2, otined similr results in growth performnce nd survivl rtes thn those otined in the present study. Thus, erly wening cn e crried out with megre lrve if severl mesures to reduce cnnilism re in plce. In nturl environments, cnnilism is regrded s n lterntive feeding strtegy, more likely to e dopted y lrve nd erly juveniles which re crnivorous, when resources ecome limiting (Hecht & Pienr, 1993) or when the popultion is too crowded (Bitt &

7 CAMPOVRD T AL. 7 Tril Amylse P= Lipse P=.3. A B C D A B C D.6.5 Trypsin P = Alkline phosphtse P < c c.1.1. A B C D. A B C D Tril Amylse P= Lipse P P<.1 < Trypsin Alkline phosphtse P =.2P=.2.. IGUR 4 Results of digestive enzyme ctivity (men SD) mesured in the lrve from Trils 1 nd 2 t the end of the experiments. Letters indicte significnt differences (ANOVA, dt from Tril 1 nd Student s t test, dt from Tril 2, p <.5) Meshk, 2). It is mjor prolem in the culture of mny mrine fish lrve ecuse, eing size-selective form of predtion, which hs consequences on oth the undnce nd size structure of the popultion. Size heterogeneity is the primry cuse of cnnilism in lrvl fish (Ktvic, Jug-Dujkovic & Glmuzin, 1989), lthough other fctors such s food vilility, lrvl density, feeding frequency, light intensity, wter clrity nd shelter hve een lso identified (see review y Hecht & Pienr, 1993). In prticulr,

8 8 CAMPOVRD T AL. IGUR 5 Different typologies of skeletl deformities (%) found in 37 dph megre lrve in Tril 2 (, control group;, erly wened lrve), considering the numer of norml skeletl elements per fish (men nd SM in rckets). HV, heml vertere; P, epurl IGUR 6 Skeletl deformities (in %, men nd SM in rckets) in megre from the control group () nd erly wened (), considering the degree of the normlities mong tretments Kestemont et l. (23) considered size heterogeneity s the min cuse for this ehviour, s the smllest fish re consumed y the lrgest ones, nd considered the period of wening in crnivorous species s one of the most importnt during lrvl rering ecuse the trnsition from live to dry diet is generlly size dependent nd fish tht re slightly lrger thn others my gin definite dvntge while hving ccess to more energetic diets. Depending on species ehviour nd morphology, the resulting size dvntge my result in more intense nd frequent gonistic interctions in the context of dominnce hierrchies, or in cnnilism. Cnnilism occurs in other cultured mrine fish such s cod Gdus morhu (Puvnendrn, Lurel & Brown, 28), yellowtil Seriol quinquerdit (Skkur & Tsukmoto, 1996) or dusky ko A. jponicus (Timmer & Mgelln, 211), nd uthors ttriuted the ggressions to the high size vrition within the cohorts with frequencies of cnnilistic ehviour incresing s the size differences etween prey nd predtor incresed. Density-dependent cnnilism hs lso een oserved (Otterlei, olkvord & Moller, 1994), eing very rre when fish re cultured in extensive pond systems or mesocosm (N. Ppndroulkis, pers. com.). In the present study, high cnnilistic ehviour in Tril 1 resulted in high reduction in survivl rtes nd high dispersion in fish size especilly from 16 dph onwrds when ggressions, fin npping nd ttcks from cnnil lrve were more frequent, which ws completely unsuitle for commercil rering purposes. Susequently, severl mesures were dopted in Tril 2 to void this ehviour, such s incresing the feeding frequency nd keeping the lrve in low light (15 2 lux) when food ws unville or in short supply. The modifiction of feeding prctices nd the use of low light intensity efore feeding in the morning incresed the survivl nd reduced the size differences mong the lrve of Tril 2 y reducing lrvl cnnilism. Previous lrvl rering studies crried out with megre (Ppdkis, Kentouri, Divnch & Mylons, 213; Roo et l., 21; Suzer et l., 213) reported higher survivl rtes (round 5% t 4 dph) nd lower growth rtes (SGR = 1.55%/dy, Suzer et l., 213) thn those otined in the present study. Present results in terms of growth performnce nd survivl were similr to those recently reported y Solovyev et l. (216), lthough the former uthors hd n initil rering density of 2 lrve/l. Cnnilism ws oserved y these uthors from 2 dph, ut it ws not considered s n importnt issue nd other fctors relted to lrvl stress nd hyperinfltion of the swim ldder were considered s the min cuses of mortlity. Pstor et l. (213) otined similr results in growth rte nd high incidence of cnnilism from 15 dph using lrvl rering density of 5 lrve/l; lthough the uthors did not provide survivl results, they considered size differences nd lrvl density the min cuse for this ehviour. Similrly, in the lrvl rering protocols (Bllgh, ielder & Pnkhurst, 21; ielder & Hesmn, 211) developed for the mullowy (A. jponicus), wening strts when the lrve re 1.5 mm length (pprox dph), chieving higher survivl rtes (4%) lthough using low lrvl densities (2 lrve/l). The wening success of ny finfish lrve from live feeds onto formulted diet is prtly dependnt on the composition of the diet nd the ility of lrve to digest it. Thus, stomch development nd the production of gstric digestive enzymes (cid digestion) re generlly regrded s the key indictors for the trnsition from live feeds to microdiets (Chu & Zmonino Infnte, 21; Rønnestd et l., 213; Wtne & Kiron, 1994). In the present study, wening ws strted efore the complete morphologicl nd functionl development of the stomch (Solovyev et l., 216); thus, protein digestion t erly wening stges ws minly sed on lkline proteolytic enzymes produced y the pncres s it hs een reported for uropen se ss (Chu & Zmonino Infnte, 21). Under current experimentl conditions, pncretic enzymes tended to e more ctive in the erly wened lrve, with significntly higher lipse ctivity compred to the control groups (Groups A nd ). Among different pncretic digestive enzymes, proteolytic enzymes (lkline proteses) re

9 CAMPOVRD T AL. 9 regrded s eing prticulrly significnt in the erly life stges of fish ecuse of the sence of functionl stomch with its cid protese, pepsin (Rønnestd et l., 213), s it hs een recently demonstrted in megre y mens of histologicl nd iochemicl procedures (Solovyev et l., 216). Lipse plys n ctive role in lipid digestion, especilly in the rekdown of tricylglycerol to dicylglycerol nd then to monocylglycerol (Zmonino Infnte & Chu, 21). In mny fish species, including megre, lipse is ctive during resorption of the oil gloule nd the complete trnsition to exogenous feeding, eing relevnt for the digestion of high levels of tricylglycerols present in the enriched live prey, s it ws shown y Solovyev et l. (216). On the contrry, the cpcity to digest proteins y mens of cid digestion (pepsin ctivity) ws significntly lower in the erly wened lrve in Tril 2, lso coinciding with the significntly lower growth rte chieved y this group (see results section nd igures 4 nd 5). On the other hnd, pepsin ctivity ecomes pprent concurrently with formtion of functionl stomch. According to different uthors, the stomch nd gstric ctivity ecomes functionl in megre etween 15 dph ( mm in SL; Suzer et l., 213), 2 dph (6.6 mm in SL; Ppdkis et l., 213) nd even t 31 dph ( mm SL; Solovyev et l., 216), which confirmed tht the functionl development of the digestive system in this species is well-conserved process tht generlly occurs within rnge of ody size (notochord flexion) regrdless of lrvl ge nd rering conditions (Solovyev et l., 216). The results of the present study indicted tht 12 dph (wening ges used in oth trils) ws proly it too erly for wening, especilly hving into ccount the reported differences in lrvl growth t the end of the wening period. The wek ility of erly wened lrve for cid proteolytic clevge of proteins from microdiets could e one of the resons for the lower lrvl growth chieved. In similr study with shi drum (Umrin cirros), Ppdkis et l. (29) lso oserved lower growth in erly wened lrve nd detected short period of strvtion during the dpttion to the rtificil feed, without ny influence on the timing of the ppernce of the vrious components of the digestive system, lthough the differentition or mturtion of some orgns might e delyed when inpproprite feeding protocols or diets re evluted t erly life stges of development (Gisert, Ortiz-Delgdo & Srsquete, 28). Severl studies hve shown tht nutrients re responsile for the ppernce of skeletl deformities when their levels in the diet re inpproprite or unlnced (Afonso et l., 2; Boglione et l., 213; Chu, Zmonino Infnte & Tkeuchi, 23; Lll & Lewis- McCre, 27). During erly lrvl development s well s during wening, chnge to n inpproprite diet, or in the hydrodynmic conditions of the rering tnk, might cuse prolems in the skeleton. In the present study, mlformtion rte of the lrve ws not ffected y the feeding regime, which my indicte tht the wening protocol used, co-feeding for severl dys enriched Artemi metnuplii with commercil microdiets, cn supply dequte nutrients for lrve without compromising the hrmonious development of the skeleton, without ffecting lrvl qulity. As similr typology of deformities ws oserved in oth tretments (lrve wened t 2 dph or t 12 dph), we cnnot discrd other fctors such s the rering conditions (Sfkinkis, Koumoundouros, Divnch & Kentouri, 24), genetic ckground (Afonso et l., 2) or roodstock nutrition (Boglione et l., 213; Chu et l., 23) s the min custive gents for such skeletl disorders. 5 CONCLUSIONS Bsed on these results, megre lrve cn e wened from live feed to rtificil diets t s erly s 12 dph, ut other importnt spects for production success including lrvl performnce nd survivl should e considered. Specil cre should e tken to void cnnilistic ehviour in the rering tnks, either y reducing the light intensity t the wter surfce nd incresing lrvl feeding rte nd dily doses. rly wening did not ffect the incidence of skeletl deformities in megre, which is of specil relevnce in terms of ssuring fry qulity for further on-growing purposes. ACKNOWLDGMNTS This project hs received funding from the uropen Union s Seventh rmework Progrmme for reserch, technologicl development nd demonstrtion (KBB single stge, GA 63121, DIVRSIY). Authors re grteful to S. Mols, M. Sstre nd O. Bellot for their technicl ssistnce in fish rering nd iochemicl nlyses respectively. C. Cmpoverde thnks the Ntionl Council for duction, Science, Technology nd Innovtion (Senescyt) of cudor for her predoctorl fellowship. C. Rodriguez elongs to the Institute of Biomedicl Technologies (ITB) of the Cnry Islnds. RRNCS Afonso J. M., Montero D., Roin L., Astorg N., Izquierdo M. S., & Gines R. (2). 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11 CAMPOVRD T AL. 11 Suzer C., Kmci H. O., Con D., irt K., & Sk S. (213). unctionl chnges in digestive enzyme ctivities of megre (Argyrosomus regius, Asso, 281) during erly ontogeny. ish Physiology nd Biochemistry, 39, Timmer R., & Mgelln K. (211). The effects of light intensity nd color on ggressive interctions in the dusky ko, Argyrosomus jponicus. The Isreli Journl of Aquculture Bmidgeh, 63, 1 9. Vlles R., & stevez A. (213). Light conditions for lrvl rering of megre (Argyrosomus regius). Aquculture, , Vlles R., & stevez A. (215). ffect of different enrichment products rich in docoshexenoic cid on growth nd survivl of megre, Argyrosomus regius (Asso, 181). Journl of the World Aquculture Society, 42, Wtne T., & Kiron V. (1994). Prospects in lrvl fish dietetics. Aquculture, 124, Worthington Biochemicl Corportion (1991) Worthington enzyme mnul: nzymes, enzyme regents, relted iochemicl. reehold, N.J: Worthington Biochemicl Corp. Zmonino Infnte J., & Chu C. (21). Sustitution of live food y formulted diets in mrine fish lrve. Aquculture, 2, Zmonino-Infnte J. L., Gisert., Srsquete C., Nvrro I., Gutierrez J., & Chu C. (28). Ontogeny nd physiology of the digestive system of mrine fish lrve. In J.. P. Cyrino, D. Nureu & B. G. Kpoor (ds.), eeding nd digestive function of fishes (pp ). New Delhi: Oxford & IBH Pulishing Co. Pvt. Ltd. How to cite this rticle: Cmpoverde C, Rodriguez C, Perez J, Gisert, stevez A. rly wening in megre Argyrosomus regius: ffects on growth, survivl, digestion nd skeletl deformities. Aquc Res. 217;: /re.13342