Effects of DHA-enriched live food on growth, survival and incidence of opercular deformities in milkfish ž Chanos chanos/

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1 Ž. Aquculture Effects of DHA-enriched live food on growth, survivl nd incidence of operculr deformities in milkfish ž Chnos chnos/ R.S.J. Gpsin ), M.N. Dury Aquculture Deprtment, Southest Asin Fisheries DeÕelopment Center SEAFDECr AQD, 5021 Tigun, Iloilo, Philippines Received 28 Ferury 2000; received in revised form 27 June 2000; ccepted 27 June 2000 Astrct The use of commercil enrichers to improve the nutritionl qulity of live food in lrviculture of milkfish ws investigted. Fish were either fed rotifers cultured on Chlorell sp. nd newly htched Artemi nuplii Ž Control, Trt I. or rotifers nd Artemi given DHA enrichment diets Ž DHA-treted, Trt II.. Results showed survivl ws significntly etter Ž P in the DHA-treted fish thn in the untreted fish fter 25-dy culture period. Although growth ws not sttisticlly different Ž P) etween the control nd DHA-treted fish during the htchery phse, extensive rering of the postlrve Ž fry. in nursery ponds for nother 60 dys showed tht DHA-treted fish exhiited significntly etter Ž P growth thn the untreted fish. Operculr deformities in 85-dy old milkfish juveniles were lso significntly lower Ž P in the DHA-treted fish thn the control. Survivl fter nursery culture, however, ws high for oth tretments ut not significntly different Ž P) The lck of vile nd relile method of mss culturing copepods s live food in the htchery mkes the use of off-the-shelf commercil enrichment diets for rotifers nd Artemi prcticl option in the lrvl culture of milkfish. q 2001 Elsevier Science B.V. All rights reserved. Keywords: Milkfish; Essentil ftty cids; Lrvl food; Growth; Survivl; Operculr deformity ) Corresponding uthor. Tel.: q ; fx: q Ž. E-mil ddress: rgpsin@qd.sefdec.org.ph R.S.J. Gpsin r01r$ - see front mtter q 2001 Elsevier Science B.V. All rights reserved. Ž. PII: S

2 50 R.S.J. Gpsin, M.N. DuryrAquculture Introduction The long-chin highly unsturted ftty cids Ž HUFAs., prticulrly eicospentenoic cid Ž EPA, 20:5n y 3. nd docoshexenoic cid Ž DHA, 22:6n y 3., re importnt in the nutrition of young mrine fish Ž Knzw, 1985; Wtne et l., Vrious investigtors hve used DHArEPA rtios s n index of the optiml level required for norml growth nd development in fish lrve ŽKoven et l., 1993; Mourente et l., 1993; Rinuzzo et l., 1994; Reitn et l., 1994; Tocher et l., 1997; Rodriguez et l., This is sed on the proposition tht optimum DHA nd EPA levels re determined not on totl mounts per se Ž s excess cn e hrmful. ut rther on the reltive proportions of these essentil ftty cids Ž EFAs. in the diet ŽWtne nd Kiron, 1994; Rodriguez et l., 1997, Aprt from DHA nd EPA, rchidonic cid Ž ARA, 20:4n y 6. hs lso een recognized s essentil for mrine fish ŽCstell et l., ARA is the min precursor of eicosnoids responsile for osmoregultion, crdiovsculr functions, neurl control nd reproduction ŽMustf nd Srivstv, Srgent et l. Ž 1997, hve suggested tht desirle rtios of 22:6n y 3r20:5n y 3r20:4n y 6 cn e useful in determining optiml requirements in fish lrvl nutrition. Operculr normlities in fish ffect its morphology Ž Koumoundouros et l., nd iologicl performnce Ž Andrdes et l., 1996; Sumgysy et l., Shortened operculum nd distortion of the support crtilge hve een descried to e signs of nutritionl deficiency Ž e.g., vitmin C. in fish cused y impired collgen formtion nd support crtilge formtion Ž Hlver et l., Some reserchers hve theorized tht operculr deformities Ž e.g., milkfish. re cused y mechnicl stress, especilly during egg collection, trnsport or routine htchery opertions ŽToledo et l., 1996; Hilomen-Grci, 1997, Toledo et l. Ž even recommended trnsporting milkfish eggs t C-shped emryonic stge to improve viility. Other cuses such s genetic vritions or fctors, however, cnnot e discounted Ž Srgent, Recently, Gpsin et l. Ž oserved tht the incidence of operculr deformities mong htchery-rered milkfish lrve could e llevited y feeding them live food supplemented with EFAs nd vitmin C. The DHArEPA rtios reported in tht study rnged from for HUFA-enriched rotifers nd Artemi nuplii while vlues for the unenriched live food were very low rnging from This follow-up study ws therefore conducted to determine whether incresing the DHA levels nd the corresponding DHArEPA rtio Ž to vlue of t lest G1.0. in live food orgnisms Ž using commercilly ville enrichers. could further improve milkfish lrve performnce. 2. Mterils nd methods 2.1. Milkfish eggs Ž. Nturl spwned eggs used for tril 1 cme from milkfish Chnos chnos spwners rered t SEAFDECrAQD Tigun Min Sttion in 10=10=10 m concrete tnk

3 R.S.J. Gpsin, M.N. DuryrAquculture wheres eggs for tril 2 cme from roodstock mintined t SEAFDECrAQD Igng Mrine Susttion in 10-m dimeter= 3-m deep se cge. Eggs were incuted in 500-l cpcity circulr, flt-ottomed, fierglss tnks following stndrd procedures Ž Gpsin nd Mrte, 1990; Gpsin et l., LiÕe food enrichment Rotifers Ž Brchionus rotundiformis, S-type. were first cultured in ker s yest ŽRed Str rnd. for 3 dys nd then enriched with DHA Protein Selco ŽINVE Aquculture, Dendermonde, Belgium. on the 4th dy s descried y Lvens et l. Ž with modifiction. Rotifers cultured extensively in Chlorell sp. served s control. Artemi nuplii were htched following stndrd prctice Ž Sorgeloos et l., nd enriched therefter with DHA Selco Ž INVE Aquculture, Dendermonde, Belgium. following the procedures of Leger et l. Ž Newly htched Artemi served s control LrÕl culture Newly htched milkfish lrve were estimted volumetriclly nd stocked in 5-ton circulr, concrete lrvl rering tnks t density of 30 lrverl. Wter mngement nd feeding scheme followed previous protocol outlined in Gpsin et l. Ž The Tle 1 ) Certin ftty cids in the totl lipids Ž % dry weight sis. from Brchionus rotundiformis fed Chlorell sp. Ž A. nd rotifers enriched with commercil diet, DHA Protein Selco Ž B. Ftty cid A B 14:0 0.18" " :0 1.61" " :0 0.60" " :1ny9 1.87" " :2ny6 0.45" " :3ny3 1.01" " :4ny6 0.02" " :1ny9 0.10" " :4ny6 0.26" " :5ny3 0.45" " :4ny6 0.24" " :4ny3 0.15" :5ny6 0.09" :5ny3 0.05" " :6ny3 0.03" "0.01 Sny6 0.97" "0.16 Sny3 1.54" "0.05 DHArEPA 0.07" "0.04 Sny3r Sny6 1.59" "0.06 ) Mens Ž "S.E.M.. within rows not ering the sme letter superscripts re significntly different Ž P

4 52 R.S.J. Gpsin, M.N. DuryrAquculture tretments Ž with three replictes. were s follows: Ž. Trt 1 Ž control. lrve fed Chlorell-cultured rotifers nd newly htched Artemi nuplii nd Ž. Trt 2 lrve fed rotifers nd Artemi nuplii enriched with DHA Protein Selco nd DHA Selco, respectively. Fish lrve Ž n s were smpled for totl length every week from ech replicte tnk. Percent survivl Ž surviving fishrinitil stock = 100. were determined t hrvest Ž dy 25.. Two lrviculture trils were conducted. Hrvested 25-dy old lrve were pcked Žin seprte plstic gs ccording to tretments., trnsported y lnd nd stocked in 27=38 m erthen nursery ponds Žthree. 2 replictes t density of lrverm. Prior to stocking, ponds were fertilized nd prepred following stndrd prctice Ž Lijuco et l., Fish were rered extensively Ž on nturl food present in ponds. for 60 dys fter which the young juveniles or fingerlings Ž n s 100. were rndomly smpled. Live fish were individully exmined for operculr deformities following procedures in Gpsin et l. Ž Of the 100 fingerlings Ž per replicte pond. checked for normlities, hlf of these Ž n s 50. were smpled for totl length mesurements. The rest of the fish stock were seined nd counted to determine survivl Ž done only for tril Lipid extrction nd FAME nlysis Rndom smples of rotifers Ž Chlorell-cultured or DHA Protein Selco-enriched., Artemi nuplii Ž newly htched or DHA Selco-enriched. nd 25-dy old fish lrve Žfed Tle 2 ) Certin ftty cids in the totl lipids Ž % dry weight sis. from newly htched Artemi nuplii Ž A. nd Artemi metnuplii enriched with commercil diet, DHA Selco Ž B. Ftty cid A B 14:0 0.06" " :0 0.31" " :0 0.21" " :1ny9 0.52" " :2ny6 0.13" " :3ny3 0.47" " :4ny6 0.14" " :1ny9 0.10" :4ny6 0.05" " :5ny3 0.03" " :4ny6 0.02" " :4ny3 0.11" :5ny6 0.02" :5ny3 0.08" :6ny3 0.61"0.05 Sny6 0.33" "0.02 Sny3 0.51" "0.09 DHArEPA 1.27"0.04 Sny3r Sny6 1.54" "0.05 ) Mens Ž "S.E.M.. within rows not ering the sme letter superscripts re significntly different Ž P

5 R.S.J. Gpsin, M.N. DuryrAquculture unenriched or DHA-enriched live food. were periodiclly tken nd plced in codedplstic vils. For reference nd comprison, newly spwned milkfish eggs Žfrom roodstock mintined t SEAFDECrAQD Igng Mrine Susttion. nd wild milkfish fry Žpproximtely 3-week old postlrve seined y fry collectors long Tigun costl wters. were lso collected. All smples were kept in deep freeze Ž y708c. until nlyzed for totl lipids nd ftty cid methyl esters Ž FAME.. Totl lipids were extrcted from triplicted pooled smples Žexcept for the eggs nd wild milkfish fry which hd only one pooled replicte smple. fter homogeniztion in chloroformrmethnol Ž 2:1, vrv. with 0.05% utylted hydroxytoluene Ž BHT. following the method of Folch et l. Ž Extrcted lipid frctions were sponified with 0.5 N KOH in methnol nd ftty cids were esterified in 14% oron triflouride methnol complex Ž Metclfe et l., FAMEs were resuspended in isooctne, flushed with nitrogen in glss vils nd stored in deep freeze Ž y708c. efore injection into the chromtogrph. Ftty cid compositions were nlyzed using Shimdzu gs liquid chromtogrph Ž GC-4PTF, Jpn. equipped with flme ioniztion detector Žinitil temperture: 1508C for 5 min, progrm rte: 108Crmin, finl temperture: 2008C., using 30 m=0.32 mm = 0.2 m film thickness SPB-PUFA cpillry column Ž Supelco, USA.. The chromtogrph ws linked to Shimdzu integrtorrrecorder ŽChromtopc C-R7A Plus, Tle 3 ) Certin ftty cids in the totl lipids Ž % dry weight sis. from whole ody tissues of 25-dy old milkfish lrve fed Chlorell-cultured rotifersrnewly htched Artemi sp. Ž A., lrve fed DHA Protein Selco-enriched rotifersrdha Selco-enriched Artemi sp. Ž B., wild milkfish fry Ž C. nd newly spwned milkfish eggs Ž D. Ftty cid A B C D 14:0 0.03" " :0 0.70" " :0 0.37" " :1ny9 0.40" " :2ny6 0.04" " :3ny3 0.19" " :4ny6 0.01" " :1ny9 0.14" " :4ny6 0.13" " :5ny3 0.30" " :4ny6 0.03" " :4ny3 0.14" " :5ny6 0.07" " :5ny3 0.06" " :6ny3 0.16" " Sny6 0.29" " Sny3 0.85" " DHArEPA 0.53" " Sny3r Sny6 2.93" " ) Mens Ž "S.E.M.. within rows not ering the sme letter superscripts re significntly different Ž P

6 54 R.S.J. Gpsin, M.N. DuryrAquculture Fig. 1. Growth of 25-dy-old milkfish fed Chlorell-cultured rotifers nd newly htched Artemi nuplii ŽTrt 1, control. nd DHA-enriched rotifers nd Artemi metnuplii Ž Trt 2, DHA-treted.. Ech dt point represents men totl length"s.e.m. Ž ns3, fish per replicte smple..

7 R.S.J. Gpsin, M.N. DuryrAquculture Jpn.. Individul ftty cids were identified y compring the retention times with commercilly ville stndrds Ž Sigm-Aldrich, USA Sttisticl nlysis Totl length, percent survivl nd deformities Žlog- or rcsine-trnsformed where pproprite., individul ftty cid levels, totl ny3, totl ny6, DHArEPA nd totl n y 3rn y 6 rtios etween the two tretments were sujected to Student s t-test nlysis Ž P s using the Sttisticl Anlysis System Ž SAS. softwre progrm ŽSAS Institute, for PCs. 3. Results Tle 1 presents the ftty cid profile of rotifers cultured in Chlorell sp. nd those enriched with DHA-Protein Selco. The individul ftty cids, including the EFAs linoleic cid Ž 18:2ny6., linolenic cid Ž 18:3ny3., ARA Ž 20:4ny6., EPA Ž 20:5ny3. nd DHA Ž 22:6ny3., s well s the totl ny6 nd ny3 were significntly higher Ž P in the DHA-enriched rotifers compred with the unenriched rotifers. The Fig. 2. Percent survivl Ž men"s.e.m.. of 25-dy-old milkfish fed Chlorell-cultured rotifers nd newly htched Artemi nuplii Ž Trt 1, control. nd DHA-enriched rotifers nd Artemi metnuplii ŽTrt 2, DHA-treted..

8 56 R.S.J. Gpsin, M.N. DuryrAquculture DHArEPA rtio in DHA-enriched rotifers Ž ws likewise significntly higher Ž P thn those cultured in Chlorell sp. Ž Totl ny3rny6 rtio, however, ws not sttisticlly significnt etween the control nd DHA-enriched rotifers. The ftty cid composition of the newly htched nd DHA-enriched Artemi re shown in Tle 2. EPA ws present in low mounts while no DHA ws detected in the newly htched Artemi nuplii confirming erlier reports ŽLeger et l., 1987; Tuncer nd Hrrell, This my perhps explin why Artemi is n inferior live food for mrine fish lrve unless enriched with HUFA-rich mrine fish oils. The DHA-enriched Artemi contined reltively sustntil mounts of DHA Ž 0.61%. nd EPA Ž 0.48%. with rtio of 1.28 compred with the control. The rest of the EFAs, s well s 16:0, 18:4n y 6 nd 22:4n y 6, were significntly elevted Ž P in the DHA-enriched Artemi compred with the newly htched Artemi nuplii. In terms of totl ny3rny6 rtio, vlue for the DHA-enriched Artemi Ž ws significntly higher Ž P thn the newly htched Artemi Ž The whole-ody tissue ftty cid pttern of 25-dy old milkfish lrve fed DHA-enriched rotifers nd Artemi nd fish fed unenriched live food Ž control. is presented in Tle 3. Generlly, the DHA-treted fish hve incresed ny3 nd ny6 PUFA levels compred with the untreted fish reflecting the ftty cid compositions of their diet Žsee Tles 1 nd 2.. As expected, the DHA Ž 0.60%. nd EPA Ž 0.39%. levels in the ody tissue of the fish fed DHA-enriched live food were significntly higher Ž P thn Fig. 3. Percent incidence Ž men"s.e.m.. of operculr deformity Ž minly cleft rnchiostegl memrne. mong 85-dy-old milkfish fed Chlorell-cultured rotifers nd newly htched Artemi nuplii Ž Trt 1, control. nd DHA-enriched rotifers nd Artemi metnuplii Ž Trt 2, DHA-treted..

9 R.S.J. Gpsin, M.N. DuryrAquculture Fig. 4. Men totl length Ž "S.E.M.. of 85-dy-old milkfish fed Chlorell-cultured rotifers nd newly htched Artemi nuplii Ž Trt 1, control. nd DHA-enriched rotifers nd Artemi metnuplii Ž Trt 2, DHA-treted.. Fig. 5. Percent survivl Ž men"s.e.m.. of 85-dy-old milkfish fed Chlorell-cultured rotifers nd newly htched Artemi nuplii Ž Trt 1, control. nd DHA-enriched rotifers nd Artemi metnuplii ŽTrt 2, DHA-treted. for tril 2 only.

10 58 R.S.J. Gpsin, M.N. DuryrAquculture those of the untreted fish Ž 0.16% nd 0.30% for DHA nd EPA, respectively.. ARA seemed to e conserved t certin levels, i.e., 0.13% in the control, 0.16% in the DHA-treted fish nd 0.12% in the wild fry. The DHArEPA rtio ws significntly higher Ž P for the DHA-treted fish Ž thn the control Ž EPA nd DHA levels in the tissue of wild milkfish postlrve or fry were 0.42% nd 0.96%, respectively while newly htched milkfish eggs hd 0.29% EPA, 1.99% DHA nd 0.36% ARA Ž Tle 3.. Growth, in terms of totl length, ws not significntly different Ž P) etween the DHA-treted nd the control fish during the 25-dy culture period Ž Fig. 1.. Survivl of 25-dy old milkfish lrve, however, ws significntly higher Ž P mong the fish fed DHA-enriched live food Ž %. compred with those given unenriched diet Ž %, Fig. 2. in contrst to the previous findings Ž Gpsin et l., After 60-dy extensive culture in erthen nursery ponds, incidence of deformity ws significntly lower Ž P in milkfish fingerlings fed DHA-enriched live food Ž %. thn those given unenriched diet Ž %, Fig. 3.. Totl length ws oserved to e significntly etter Ž P in 85-dy old milkfish juveniles fed DHA-enriched rotifers nd Artemi Ž during the htchery phse. compred with the control Ž Fig. 4.. Although men percent survivl ws slightly higher in the DHA-treted fish Ž rnge: %. thn the untreted fish Ž rnge: %., vlues were not sttisticlly different Ž Fig Discussion Live food enriched with the EFAs DHA nd EPA improved lrvl performnce in striped ss nd plmetto ss Ž Tuncer nd Hrrell, 1992., cod Ž Tkeuchi et l., 1994., red se rem Ž Furuit et l., 1996., yellowtil Ž Furuit et l., 1996., striped jck Ž Tkeuchi et l., nd the summer flounder Ž Bker et l., In the present investigtion, no significnt difference ws oserved in growth of milkfish lrve fed DHA-enriched live food Ž high DHArEPA rtio. compred with those fed unenriched Ž control. diet Ž low DHArEPA rtio. fter 25-dy lrvl culture. However, extensive rering of milkfish in nursery ponds showed tht DHA-treted fish exhiited significntly etter growth thn the control confirming our erlier results ŽGpsin et l., Mourente et l. Ž reported the est growth rte in gilthed se rem lrve fed HUFA-enriched rotifers with high DHArEPA rtio. In milkfish, the effects of DHA-enriched live food on growth my not e redily discerned over short period Ž htchery phse. ut rther fter extended rering Ž nursery stge.. Gpsin et l. Ž oserved no significnt difference in survivl of 25-dy old milkfish lrve fed HUFA-enriched live food or those fed unenriched diet. The DHArEPA rtios reported in the previous study were less thn 1.0 Žtht is nd for HUFA-enriched rotifers nd Artemi, respectively.. In the present study, however, survivl ws significntly etter Ž consistent for the two trils. in milkfish fed DHA-enriched live food compred with the fish fed unenriched diet Ž despite egg tches coming from different spwners.. Noteworthy were the DHArEPA

11 R.S.J. Gpsin, M.N. DuryrAquculture rtios of more thn 1.0 Žtht is 1.49 nd 1.26 for the DHA-enriched rotifers nd Artemi, respectively; Tles 1 nd 2.. Results suggest tht incresing the DHArEPA rtio Ž to vlue of t lest G1.0. in the live food further improved survivl of the htchery-rered milkfish lrve. For mrine fish lrve, Tucker Ž recommended dietry ny3 HUFA content of 2 4%, including t lest 1.0% EPA nd 1.0% DHA. Srgent s Ž estimte of 2:1 rtio for DHA nd EPA Žsed on the vlues found in most fish eggs., however, is more resonle. While the optiml DHA nd EPA levels required y milkfish lrve hve not yet een determined, dietry DHA level of out % Ž dry weight sis. nd DHArEPA rtio of t lest G 1.0 in the live food would e pproprite for norml growth nd good survivl in intensive lrviculture system. In fltfish, Estevez Ž reported higher pigmenttion success in the Jpnese flounder fed diets with DHArEPA rtio of 1.0. Similrly, Rinuzzo et l. Ž found positive correltion etween pigmenttion success nd the DHArEPA rtio in the totl nd polr lipid frction of the turot lrve. Bker et l. Ž lso noted direct reltion etween norml pigmenttion nd levels of DHA in the diet of summer flounder lrve where the DHArEPA rtios in the enriched live food rnged from These reports underscore the importnce of DHA nd EPA Žin pproprite proportions. in survivl, growth nd development of vrious fltfish species. In milkfish, the need for high DHArEPA rtio Ž proly t lest G1.0. in the lrvl diet is vlidted y the fct tht the DHArEPA rtios in the newly spwned milkfish eggs Ž 6.8. nd whole-ody tissue of wild milkfish fry Ž 2.3. were lso high Ž Tle 3.. Aundnce of DHA in the tissue of the wild fry further suggests tht the zooplnkton diet Že.g., copepods. of milkfish lrve in the wild is reltively superior in terms of nutritionl qulity nd quntity compred with the live food given in the htchery Ždespite eing fortified with HUFA-rich mrine fish oils or commercil enrichers.. Mrine copepods Ž nd their nupliircopepodites. re considered excellent live food for fish lrve not only of their smll size ut more importntly, ecuse of their high nutritive vlue. Evjemo nd Olsen Ž reported the predominnt ftty cids in the mrine copepods Temor longicornis nd Eurytemor sp. were DHA, EPA nd plmitic cid Ž 16:0. with DHArEPA rtios rnging from High mounts of DHA were detected in the ftty cid composition of the copepods Oithon sp., Pseudodiptomus spp. nd Acrti tsuensis with DHArEPA vlues of 1.28, 1.37 nd 2.64, respectively ŽToledo et l., With the exception of plmitic cid Ž 16:0., the predominnce of DHA nd EPA in the tissues of the wild mikfish fry Ž this study. nd the copepods ŽEvjemo nd Olsen, 1997; Toledo et l., indicte the preference nd importnce of these EFAs in the diet of milkfish lrve Ž nd perhps in other mrine fish species s well.. The ftty cid pttern of the predtor lrve generlly reflects tht of its prey. Thus, it is importnt in enrichment tht the ftty cid profile of live food produced in the htchery emultes or pproximtes the ftty cid ptterns of the nturl food orgnisms in the wild through the use of pproprite commercil enrichers nd proper enrichment procedures. Incidence of operculr deformity ws significntly lower in the DHA-treted fish compred with the untreted fish despite the eggs coming from different roodstock confirmed our previous results Ž Gpsin et l., Similr normlities hve lso een reported in slmonids Ž Bruno, 1990., Europen se ss Ž Douls et l., 1991., tilpi Ž Tve nd Hndwerker, 1994., nd gilthed se rem Ž Andrdes et l., 1996.

12 60 R.S.J. Gpsin, M.N. DuryrAquculture nd seemed to e ssocited with intensive culture. While it ws oserved tht high dietry DHArEPA rtio decresed incidence of operculr normlities in milkfish lrve, it is not cler why or how these EFAs Ž in pproprite proportions. function to llevite such deformities. Further study is therefore required to understnd such syndrome. ARA is n importnt constituent of the food chin Ž Nichols et l., The presence of ARA in moderte mounts, s well s other n y 6 PUFAs which re generlly of plnt origin, in the tissue of wild milkfish fry Ž Tle 3. suggests tht the fish t such developmentl stge lso ingest microlge, prt from zooplnkton, s food. The present dt confirmed Bnno s Ž nd Kinoshit s Ž oservtions tht wild milkfish fry in shore wters feed minly on copepods nd ditoms. As fish grows from one developmentl stge to the other, there is corresponding ontogenetic shift in its nutrient requirements Ž Drowski, With the yolk fully resored nd lcking functionl stomch, first feeding milkfish lrve must rely on stedy exogenous supply of HUFA-rich diet of zooplnkton from the nturl environment. As milkfish lrve metmorphoses into fry Ž postlrve., it ecomes n omnivore ŽBnno, 1980; Kinoshit, As the fry susequently ecomes young juvenile Žor fingerling., the crdic stomch develops cid- nd pepsin-secreting glnds while mucosl folds cquire secondry rnching Ž Ferrris et l., At this stge, the morphology of the digestive system of juvenile Ž nd lso of the dult. milkfish indictes tht it hs ecome n herivore with generlist tendencies Ž Kinoshit, Algl mt Žlocl term ll., nturl food se of milkfish in ponds, is complex community of unicellulr lge, ditoms nd other orgnisms Ž Rnl, nd reportedly constitutes in prt 2.0% 18:2ny6, 4.0% 18:3ny3 ut no detectle mounts of 20:4ny6, 20:5ny3 or 22:5ny3 Ž Gorricet, The presence of ARA, EPA nd DHA in the liver, despite their sence in ll, suggests tht young milkfish juveniles hve the cpcity to convert dietry ny3 nd ny6 PUFAs into long-chin HUFAs ŽBenitez nd Gorricet, nd lter confirmed y Butist nd de l Cruz Ž nd Borlongn Ž This proly explins the high percent survivl of 85-dy old milkfish juveniles ŽDHA-treted or not. s the fish t this stge Ž nd perhps erlier t metmorphosis. re lredy le to utilize the ville nturl food. This informtion hs importnt implictions on the feeding mngement in the culture of milkfish for optimum growth nd survivl. Acknowledgements The uthors thnk I. Borlongn for her constructive comments on the erlier drft nd to the two nonymous referees for the criticl review on the finl version of the mnuscript. We gretly pprecite R. Snres for the sttisticl dvice, I. Borlongn for guidnce in the use of the gs chromtogrph nd nlyses of the chromtogrms nd T. Hute, Jr. for providing the nursery ponds. We re grteful to F. Pudder, Jr. nd B. Polohn for the ssistnce in the lrvl culture nd lipid extrctionrfame nlyses, respectively. We cknowledged the finncil support of SEAFDECrAQD under the study code Nr-12-F90T.

13 R.S.J. Gpsin, M.N. DuryrAquculture References Andrdes, J.A., Becerr, J., Skeletl deformities in lrvl, juvenile nd dult stges of cultured se rem Ž Sprus urt L... Aquculture 141, Bker, E.P., Alves, D., Bengtson, D.A., Effects of rotifer nd Artemi ftty cid enrichment on survivl, growth nd pigmenttion of summer flounder Prlichthys denttus lrve. J. World Aqucult. Soc. 29, Bnno, J.E., The food nd feeding hits of milkfish fry, Chnos chnos Ž Forsskl. collected from two hitts long the cost of Hmtik, Antique. MSc Thesis, University of the Philippines-Visys, Iloilo, Philippines, 77 pp. Butist, M.N., de l Cruz, M.C., Linoleic Ž v y6. nd linolenic Ž v y3. cids in the diet of fingerling milkfish Ž Chnos chnos Forsskl.. Aquculture 71, Benitez, L.V., Gorricet, I.R., Lipid composition of milkfish grown in ponds y trditionl quculture. In: Cho, C.Y., Cowey, C.B., Wtne, T. Ž Eds.., Finfish Nutrition in Asi: Methodologicl Approches to Reserch nd Development. Interntionl Development Centre Ž IDRC., Ottw, Cnd, pp Borlongn, I.G., The essentil ftty cid requirement of milkfish Ž Chnos chnos Forsskl.. Fish Physiol. Biochem. 9, Bruno, D.W., Miscellneous externl normlities of frmed slmonids. Aqucult. Inform. Serv. 11, 1 6. Cstell, J.D., Bell, J.G., Tocher, D.R., Srgent, J.R., Effects of purified diets contining different comintions of rchidonic nd docoshexenoic cid on the survivl, growth nd ftty cid composition of juvenile turot Ž Scophthlmus mximus.. Aquculture 128, Drowski, K., Ontogenetic spects of nutritionl requirements in fish. Comp. Biochem. Physiol. 85A, Douls, C., Economou, A.N., Bntvs, I., Osteologicl normlities in lortory rered se-ss Ž Dicentrrchus lrx. fingerlings. Aquculture 97, Estevez, A., Effects of lipids nd vitmin A on pigmenttion success of fltfish. PhD Thesis, University of Kgoshim, Jpn, 149 pp. Evjemo, J.O., Olsen, Y., Lipid nd ftty cid content in cultivted live feed orgnisms compred to mrine copepods. Hydroiologi 358, Ferrris, R.P., Tn, J.D., de l Cruz, M.C., Development of the digestive trct of milkfish Chnos chnos Ž Forsskl.: histology nd histochemistry. Aquculture 61, Folch, J., Lees, M., Stnley, G.H., A simple method for the isoltion nd purifiction of totl lipids from niml tissues. J. Biol. Chem. 226, Furuit, H., Tkeuchi, T., Toyot, M., Wtne, T., EPA nd DHA requirements in erly juvenile red se rem using HUFA enriched Artemi nuplii. Fish. Sci. 62, Furuit, H., Tkeuchi, T., Wtne, T., Fujimoto, H., Sekiy, S., Imizumi, K., Requirements of lrvl yellowtil for eicospentenoic cid, docoshexenoic cid nd highly unsturted ftty cid. Fish. Sci. 62, Gpsin, R.S.J., Mrte, C.L., Milkfish htchery opertions, Aquculture Extension Mnul No. 17. SEAFDECrAQD, Tigun, Iloilo, Philippines, 24 pp. Gpsin, R.S.J., Bomeo, R., Lvens, P., Sorgeloos, P., Nelis, H., Enrichment of live food with essentil ftty cids nd vitmin C: effects on milkfish Ž Chnos chnos. lrvl performnce. Aquculture 162, Gorricet, I.R., Studies on the digestive lipses nd lipid composition of milkfish, Chnos chnos Ž Forsskl.. MSc Thesis, University of the Philippines-Dilimn, Quezon City, Philippines, 56 pp. Hlver, J.E., Smith, R.R., Tolert, B.M., Bker, E.M., Utiliztion of scoric cid in fish. Ann. N.Y. Acd. Sci. 258, Hilomen-Grci, G.V., Morphologicl normlities in htchery-red milkfish Ž Chnos chnos Forsskl. fry nd juveniles. Aquculture 152, Hilomen-Grci, G.V., Sensitivity of fertilized milkfish Ž Chnos chnos Forsskl. eggs to mechnicl shock nd simulted trnsport. Aquculture 159, Knzw, A., Essentil ftty cid nd lipid requirements of fish. In: Cowey, C.B., Mckie, A.M., Bell, J.G. Ž Eds.., Nutrition nd Feeding in Fish. Acdemic Press, London, pp

14 62 R.S.J. Gpsin, M.N. DuryrAquculture Kinoshit, I., Feeding hit nd development of the digestive system in juvenile milkfish, Chnos chnos Ž Forsskl.. MSc Thesis, Ngski University, Jpn, 42 pp. Koven, W.M., Tndler, A., Skln, D., Kissil, G.W., The ssocition of eicospentenoic nd docoshexenoic cids in the min phospholipids of different-ge Sprus urt lrve with growth. Aquculture 116, Koumoundouros, G., Orn, G., Divnch, P., Stefnkis, S., Kentouri, M., The operculr complex deformity in intensive gilthed se rem Ž Sprus urt L.. lrviculture. Moment of pprition nd description. Aquculture 149, Lvens, P., Dhert, Ph., Merchie, G., Stel, M., Sorgeloos, P., A stndrd procedure for the mss production on n rtificil diet of rotifers with high nutritionl qulity for mrine fish lrve. In: Chou, L.M., Munro, A.D., Lm, T.J., Chen, T.W., Cheong, L.K.K., Ding, J.K., Hooi, K.K., Khoo, H.W., Phng, V.P.E., Shim, K.F., Tn, C.H. Ž Eds.., Proceedings of the Third Asin Fisheries Forum Nutrition, Octoer 1992, Singpore. pp Leger, Ph., Nessens-Foucquert, P., Sorgeloos, E., Interntionl study on Artemi XXXV. Techniques to mnipulte the ftty cid profile in Artemi nuplii nd the effects on its nutritionl effectiveness for the mrine crustcen Mysidopsis hi Ž M... In: Sorgeloos, P., Bengtson, D.A., Decleir, W., Jspers, E. Ž Eds.., Artemi Reserch nd Its Applictions, vol. 3. Universl Press, Wetteren, Belgium, pp Lijuco, M.M., Jurio, J.V., Blio, D.D., Grinom, Quinitio, G., Milkfish culture nd mngement, Extension Mnul No. 4. Southest Asin Fisheries Development Center Aquculture Deprtment, Tigun, Iloilo, Philippines. Metclfe, L.D., Schmitz, A.A., Pelk, J.R., The rpid preprtion of ftty cid methyl esters from lipids for gs chromtogrphic nlysis. Anl. Chem. 38, Mourente, G., Rodriguez, A., Tocher, D.R., Srgent, J.R., Effects of dietry docoshexenoic cid Ž DHA; 22:6ny3. on lipid nd ftty cid compositions nd growth in gilthed se rem ŽSprus urt L.. lrve during first feeding. Aquculture 112, Mustf, T., Srivstv, K.C., Prostglndins Ž eicosnoids. nd their role in ectothermic orgnisms. Adv. Comp. Environ. Physiol. 5, Nichols, P.D., Klumpp, D.W., Johns, R.B., Lipid components of the segrsses Posidoni ustrlis nd Heterozoster tsmnic s indictors of cron source. Phytochem. 21, Rnl, H.R., The culture of ll, the nturl food of milkfish or ngos, Chnos chnos Ž Forsskl. fry nd fingerling under cultivtion. Phil. Fish. J. 35, Rinuzzo, J.R., Reitn, K.I., Jorgensen, L., Olsen, Y., Lipid composition in turot lrve fed live feed cultured y emulsions of different lipid clsses. Comp. Biochem. Physiol. 107A, Reitn, K.I., Rinuzzo, J.R., Olsen, Y., Influence of lipid composition of live feed on growth, survivl nd pigmenttion of turot lrve. Aqucult. Int. 2, Rodriguez, C., Perez, J.A., Diz, M., Izquierdo, M.S., Fernndez-Plcios, H., Lorenzo, H., Influence of the EPArDHA rtio in rotifers on gilthed serem Ž Sprus urt. lrvl development. Aquculture 150, Rodriguez, C., Perez, J.A., Bdi, P., Izquierdo, M.S., Fernndez-Plcios, H., Lorenzo-Hernndez, A., The ny3 highly unsturted ftty cids requirements of gilthed serem Ž Sprus urt L.. lrve when using n pproprite DHArEPA rtio in the diet. Aquculture 169, Srgent, J.R., Origins nd functions of egg lipids: nutritionl implictions. In: Bromge, N.R., Roerts, R.J. Ž Eds.., Broodstock Mngement nd Egg nd Lrvl Qulity. Blckwell, Oxford, pp Srgent, J.R., McEnvoy, L.A., Bell, J.G., Requirements, presenttion nd sources of polyunsturted ftty cids in mrine fish lrvl feeds. Aquculture 155, Srgent, J., Bell, J.G., McEnvoy, L.A., Tocher, D.R., Estevez, A., Recent developments in the essentil ftty cid nutrition of fish. Aquculture 177, SAS Institute, SASrSTAT TM Guide for Personl Computers. 6th edn. SAS Institute, Cry, NC, 1028 pp. Sorgeloos, P., Lvens, P., Leger, Ph., Tckert, W., Versichele, D., Mnul for the Culture nd Use of Brine Shrimp Artemi in Aquculture. Fculty of Agriculture, University of Gent, Belgium, 319 pp. Sumgysy, N.S., Hilomen-Grci, G.V., Grci, L.M.B., Growth nd production of deformed nd nondeformed htchery-red milkfish Ž Chnos chnos. in rckishwter ponds. Isr. J. Aqucult.-Bmidgeh 51,

15 R.S.J. Gpsin, M.N. DuryrAquculture Tkeuchi, T., Zheng, K., Yosed, K., Hirokw, J., Wtne, T., Nutritive vlue of DHA-enriched rotifer for lrvl cod. Nippon Suisn Gkkishi 60, Tkeuchi, T., Msud, R., Ishizki, Y., Wtne, T., Knemtsu, M., Imizumi, K., Tsukmoto, K., Determintion of the requirement of lrvl striped jck for eicospentenoic cid nd docoshexenoic cid using enriched Artemi nuplii. Fish. Sci. 62, Tve, D., Hndwerker, T.S., Semi-operculum: non-heritle irth defect in Tilpi nilotic. J. World Aqucult. Soc. 25, Tocher, D.R., Mourente, G., Srgent, J.R., The use of silges prepred from fish neurl tissues s enrichers for rotifers Ž Brchionus plictilis. nd Artemi in the nutrition of lrvl mrine fish. Aquculture 148, Toledo, J.D., Doi, J.D., Dury, M., Viility of milkfish eggs nd lrve fter simulted nd ctul trnsport. In: McKinly, D., Eldridge, M. Ž Eds.., The Fish Egg: Its Biology nd Culture Symposium Proceedings, July. Sn Frncisco Stte University, Sn Frncisco, USA, pp Toledo, J.D., Golez, M.S., Doi, M., Ohno, A., Use of copepod nuplii during erly feeding stge of grouper Epinephelus coioides. Fish. Sci. 65, Tucker, J.W., Feeding intensively-cultured mrine fish lrve. In: Alln, G.L., Dll, W. Ž Eds.., Proceedings of the Aquculture Nutrition Workshop, Slmnder By, April NSW Fisheries, Brckish Wter Fish Culture Reserch Sttion, Slmnder By, Austrli, pp Tuncer, H., Hrrell, R.M., Essentil ftty cid nutrition of lrvl striped ss Ž Morone sxtilis. nd plmetto ss Ž M. sxtilis= M. chrysops.. Aquculture 101, Wtne, T., Izquierdo, M.S., Tkeuchi, T., Stoh, S., Kitjim, C., Comprison etween eicospentenoic cid nd docoshexenoic cid in terms of essentil ftty cid deficiency in lrvl red serem. Nippon Suisn Gkkishi 55, Wtne, T., Kiron, Y., Prospects in lrvl fish dietetics. Aquculture 124,