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This reoort not to be cited without prior reference to the council·

International Council for theExploration of the Sea

C.M. 1991/F:2

REPORT OF THE WORKING GROUP ON MASS REARING OF

JUVENILE MARINE FISH TO THE MARICULTURE COMMITTEE OF ICES

Gent, Belgium 31 August· 2 September, 1991.

This document is areport of a Working Group of the International Council for theExploration of the Sea and does not necessarily represent the views of the Council.Therefore, it should not be quoted without consuItation with the General Secretary•

•General SecretaryICES, Palregade 2-4DK-1261 Copenhagen KDenmark.

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APPENDIX L List of participants and addresses.

APPENDIX II. Bibliography on nutrition and histology.

APPENDIX 111. LC.E.S. Study on (n-3) HUFA Requirementsin Marine Fish Larvae (draft).

1. PARTICIPATION

The \Vorking Group convened its fourth meeting at Gent, BeIgium on August 31 - 2September, 1991. Members attending the meeting were:

BeIgium: P. Sorgeloos. Denmark: J. G. Stottrup. Canada: J.A. Brown, G. Goff, K.Waiwood. Faroe IsIands: L Fjallstein. France: B. Chatain. Germany: G. Quantz.Norway: D. DanieIssen, I. Holmefjord, I. Huse, E. Kjorsvik, I. Lein, A. Mangor-Jensen,K. Naas, Y. Olsen, G. Rosenlund. Portugal: P. Pausao-Ferreria. Spain: J. Iglesias, I.Martinez, G. Minkoff, J.B. Peleteiro. Sweden: P.-G. Larsson. UK: B.R. HoweII.

During 1990, several members attended by invitation the World Aquaculture Societyannual meeting in Halifax, Canada, where a Larviculture Task Force within the WAS

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b)

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chaired by Patrick Sorgelaos (Belgium) and David Bengtson (USA) was formed. Several\VAS/LTF members also attended part of this meeting. Thus, apart from the ICESmembers, the following were present:

Belgium: T. BasteeIs, Ph. Dhert, A. Komis, P. Lavens, Ph. Leger, I. Roelants, G. VanStappen, L. Verdonck, F. Volckaert. Denmark: N.E. Poulsen. France: S. Kaushik.Germany: H. Segner. Greece: G.M. Robbins, E. Sweetman, J. Sweetman. Israel: Y.Barr, L. Samuel, A. Tandler, O. Zmora. Japan: K.. Muroga, M. Tanaka. Mexico: A.Abreu Gobrois. Netherlands: J. Verreth. Norway: G. Adoff, A. Folkvord. Scotland: J.Dye, J.R. Sargent. Singapore: T.J. Lam, C.L. Lim, J. \Valford. S.Africa: T. Hecht.Sweden: J. Pickova. Taiwan: H.-Y. Chien. Thailand: D. Fegan, P. Menasveta, S.Piyatiratitivorakul, J.-F. Rees. USA: H. Ako, D. Bengtson, J.G. Holt, S. Kraul, J.A.Tellock.

See Appendix I for addresses.

I. Huse, Norway, (chairman) and J.G. Stottrup, Denmark, kindly served as rapporteursfor the meeting.

2. TERMS OF REFERENCE

The ICES \Vorking Group on Mass Rearing ofJuvenile Marine Fish met to workaccording to the follmving terms of reference (ICES C.M. 1990jF:65):

a) prepare areport describing standardized procedures and conditions forexperimental fry production of turbot and sea bass as model species,including criteria in addition to growth and survival, for the evaluation of'the quality of eggs, larvae and juveniles;

describ'e nutritional requirements of marine fish, primarily for fatty acidsand amino acids and collect information on the function of individualcompounds in the organisms;

c) advice on alternative strategies to the use of antibiotics in the control ofmicroflora in culture systems.

3. AGENDA

During the meeting, three plenary sessions and one concurrent group session wereheldwith the following subjects covered:

A) Egg, larval and juvenile quality (E. Kjorsvik, Norway)

B) Nutrition (P. Sorgeloos, Belgium and J. Verreth, Holland)J. Sargent: Lipid metabolismS. Kaushik: Protein metabolism

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C) Standardized procedures and conditions for experimental fry productionof model species. (8. Chatain, France and J. G. Stottrup, Denmark)

D) Hygiene strategies (G. Minkoff, Spain)

In the first two plenary sessions each topic was introduced by 1-2 short presentations,followed by discussion. The nutrition session also included two invited speakers on themain topics; lipid and protein metabolism.

Each subject was discussed in more detail in smaller groups during the concurrentsessions.

The recommendations were discussed and revised during the final plenary session.

4. EGG, LARVAL AND JUVENILE QUALI1Y

Convener: Elin KjorsvikRapporteurs: Bari Howell, Kenneth \Vaiwood

Considerable variability in the performance of eggs and larvae is observed during themass rearing of marine fish. Much of this variability may be attributable to egg qualitydefined as the potential oJ the ef3 at he completioll oJJertilizatioll to produce viable Jry. Itwill consequently embrace any effects associated with the quality of sperm, a subject thathas been studied little in marine fish but one that can not be ignored.

There is an urgent need among practitioners to identify characteristics of eggs and larvaethat reflect their quality as weil as to improve our understanding of the mechanismsinvolved in generating the observed variability. The. \Vorking Group consequentlyrecognized two important aspects for future work:

The requirement for making simple, rapid predictions of subsequentperformance of eggs and larvae.

II The development of strategies for the in~estigation of factors andmechanisms that influence egg quality.

I. IDENTIFICATION OF QUALITY CRITERIA

Ia~

The following parameters have been or may prove 10 be useful indicators of egg qualityin several marine species:

- fertilization rate- hatching rate- buoyancy

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- c1eavage pattern (morphology)- mortality rates (at different stages)- relative developmental rates- turgor pressure (egg hardness)- chorion appearance / fertilization process- oil globule distribution- opacity- 'stress' tests (eg. mechanical shock)

Of these, it was considered that fertilization and hatching rates, buoyancy and c1eavagepatterns offered the greatest potential for routine assessment and that thesemeasurements should be included in an assessments of quality. Nevertheless, it wasrecognized that, because egg quality may vary in response to a variety of factors, theremay not be a universal indicator of egg quality and that different parameters may needto be used according to species and procedures used.

• Ib Larvae

It is important that larvae quality is assessed at a defined developmental stage. the onsetof exogenous feeding would be an appropriate stage and is the last stage at which larvalquality can realistically be attributed to maternal influence. This should not, however,preclude eventual studies of effects on later developmental stages.

The following parameters were identified as being worthy of further evaluation:

- deformities- pigmentation- survival- growth- synchrony in development- development of stages with time- yolk absorption rate- disease resistance- 'stress tests' (eg. salinity, air exposure)- 'performance tests' (eg. behavior, response to stimuli)

11 DEVELOPMENT OF RESEARCH STRATEGIES

IIa Factors affecting egg quality

The following broodstock-related factors were identified as potential determinants of eggquality:

- time within the spawning season (batch spawners)- fish age- nutrition- stripping/handling/fertilization- over-ripening

- induced ovulation /spawning (hormones)- environmental conditions (eg. temperature, salinity, light)- water quality (eg. 02' NH3)

IIb Mechanisms and manifestations of egg Quality

The investigation of the following factors may provide an insight into the mechanismsinvolved in the deterinination of egg quality:

- energy charge (ATP/ ADP)- time dependent changes in, for example, hormones (eg. thyroxine), enzymes

and nutrients- cell cycles (eg. regulation of cell cleavage)- chromosome aberrations/karyotyping of chromosomes

It was stressed that the value of 'snap-shot' analyses were often limited and that more •effort should be devoted to studies of dynamic processes during development.

IIc Research priorities

The following priority areas for future research were identified:

1. Standardization of methods and criteria.An effort should be made to link the empirical routine criteria to the results ofmore advanced scientific investigations in order to more fully understand theunderlying mechanisms involved in the determination of egg quality.

2. Development of methods for determining the timing of ovulation and the post­ovulatory age of eggs. Such methods may be related to:- rates of drinking / salt excretion- ultrasonie observation- ovulatory cycles- refinement of methods for natural spawning

These studies would be facilitated by an understanding of the hormonal controlmechanisms which could even lead to the development of 'fish-ovulation test kits'.

3. Sperm quality assessment which should also include effects of storage methods.

RECOMMENDATIONS

The following recommendations were proposed and accepted by the Working Group:

1. A standardized protocol for assessment of egg and larval quality should bedevised.

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2. The design and distribution of a proforma data sheet to record eggproduetion methods, the eharaeteristies of eggs at fertilization and theirsubsequent performance.

3. . Research should be encouraged in the following areas:

a) broodstoek management proceduresb) reproduetion meehanisms of the fishc) ovulation/over-ripening mechanismsd) time dependent compositional changes during the egg and

larval stages.

5. NUTRITION

Convener: Patrick Sorgeloos & Johan VerrethRapporterus: Patrick Sorgeloos & Dave Bengtson

There is an urgent need to develop an appropriate protocol for nutritional experimentsin fish larviculture. In that respect the following proposal was worked out:

al) A standard live diet should be used consisting of:

ICES-reference Artemia (smaIl nauplii with high content of n-3HUFAs) as starter feed (to be seleeted and made available by theArtemia Reference Center, Belgium), followed by

Great Salt Lake (Utah, USA) Artemia enriehed by a standardprotocol using:- ICES-standard emulsion as external standard- loeal enriehment proeedure as internal standard

a2) Regarding the green-water teehnique and 'Brachiorius culturing, eaehlaboratory should use the procedure they feel appropriate for the specieseultured. Applied procedures should be weIl documented

a3) A protoeol should be developed so that nutritional tests ean be performedunder standardized, optimal conditions, including at least:

removal of aIl remaining food at least onee a dayidentification of feeding levels

a4) Experimental conditions need to be optimize in order to realize maximumgrowth for use as referenee eonditions in later dose-response studies

aS) Experimental results should be evaluated in relation to the maximumgrowth potential and in relation to a low growth under known diet­deficient conditions

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a6) The following parameters should be considered for evaluating theexperimental results:

survivalsize of the fish, e.g. length, dry weight and any other mass-relatedparameterstress resistancesize variationtime and size until certain developmental stage, such as gastric pHchange, metamorphosis, pigmentation, organ developmentRNA/DNA ratio

a7) The need is expressed that a literature search shoul be conducted on thetechnology, formulation and use of standard reference inert diets forlarvae. Dr. J. \Valford has accepted to take up this responsibility.

. bl) The procedure for quantitative (n-3) HUFA analysis as prepared by Ph.Leger has been improved since the Palavas \VG-meeting, will be furtheramended to exclude the use of carcinogens (benzene) and will besubmitted for final evaluation to J.Sargent. The \VG recommends that thismethod should be proposed for adoption as a standard ICES-procedure for(n-3) HUFA analysis.

b2) Regarding the (n-3) HUFA requirement study proposed· a the \VGmeeting in Vigo (1989) experimental results are available for sea bass, seabream, turbot, striped bass, red drum, and summer flounder. it appearsthat best results are obtained with the medium (n-3) HUFA emulsion.sometimes better results were obtained with high (n-3) HUFA emulsion,although analytical data show that enrichment levels obtained withmedium· may equal those obtained with high, which indicated thatenrichment success might have been different. it is therefore essential thealong with the biological tests, analysis for the enriched prey is taken intoconsideration. The \VG recommends the the experimental results shouldbe compiled and proposed in a final report (draft as Appendix 11).

b3) Since it appears from the previous study that much variability exists in (n­3) HUFA enrichment success with Brachionus and Artemis, even whenusing the same enrichment emulsions, and the present standardprocedures, it is important to esiimate present variability in (n-3) Hufaenrichment procedures. In this respect and in order to be able to adviceoptimal and standard enrichment procedures, an enrichmentintercalibration exercise should be performed using an ICES-referenceemulsion (made available by the Artemia Reference Center, Belgium) andGreat Salt Lake (Utah, USA) Artemia. Protocols for experimental and

. analytical procedures will be prepared by J. Sargent and P. Sorgeloos.

c) A bibliography on nutrition and histology has been compiled by D.A.Bengtson. .

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RECOMMENDATIONS

The following recommendations were proposed and accepted by the \Vorking Group:

1. A protocol should be developed so that nutritional tests can be performedunder standardized, optimal conditions.

2. It is recommended that a reference diet should be included in nutritionalstudies and this should consist of:

ICES-reference Artemia as starter feed (to be selected und madeavailable by the Artemia Reference Center, Belgium), followed by

Great Salt Lake (Utah, USA) Artemia enriched by a standardprotocol (to be developed), using:-ICES-standard emulsion as external standard-local enrichment procedures as internal standard

3.

4.

5.

6.

Results should be evaluated in terms of both maximal growth and somelow-growth reference

Experimental results should be evaluated following well-defined criteria

To standardize (n-3) HUFA enrichment procedures, an enrichmentintercalibration exercise should be performed.

A literature search should be conducted on the technology, formulationund use of standard reference inert diets for larvae (J. Walford).

6. STANDARDIZED PROCEDURES FOR EXPERIMENTAL FRY PRODUCTION

• Conveners & rapporteurs: Beatrice Chatain & Josianne G. St0ttrup

It was decided that the standardized system should serve as a reference protocol ratherthan a rigid experimental procedure. That is, the protocol should ascribe to each physicalparameter a certain value and quality control within which effects of variation arenegligible. \Vhere actual experiments deviate from this protocol, it would then serve asa reference protocol.

The protocol could contain certain general laboratory procedures in sufficient detail toenable an operator to carry them out. It should be intended for experience biologists andlaboratory staff, familiar with these types of experiments.

The following protocol was proposed and accepted by the Working Group for thestandardization of procedures for experimental fry production of turbot and sea bass:

PROTOCOL

1. Introductory information

- water quality; description of water treatment and analytical procedures- egg and larval quality; how to assess the egg and larval quality

2. Method

- definitions- principles of rearing methods- replicates and controls- experimental design- description of experimental parameters- experimental data requirements- description of experimental procedures- conditions for judging the validity of the experiment

3. Report

- information to be incIuded in the report

B. Chatain, France provided information on sea bass rearing techniques as a basis fordiscussion and l.G. Stottrup, Denmark that on turbot. Due to the complexity of thesubject and the short time available, many important details were not discussed, nor wasthere time to work out a comprehensive draft of the protocol at the meeting. The tworeports on sea bass and turbot will therefore be compiled by each of the two convenersand sent to the \VG members for further comments before the final draft can besubmitted. At the earliest this may be for the ICES Statutory Meeting, 1992.

It was proposed that the final draft be submitted as an ICES Cooperative ResearchReport.

RECOMMENDATIONS

The following recommendations were proposed and accepted by the Working Group:

1. Research priorities should incIude:

a) the elucidation of the role of algae in rearing systems.

b) an assessment of the effects of the quality and quantity of light onlarval performance (including effects of tank.color/reflectivity)

c) the development of an empirical feeding model to provide a basisfor hatchery feeding strategies

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2. Composition, source and shelf-life of ingredients and storage conditionswhen commercial products are used in nutritional studies, should beprovided by the manufacturers.

3. Research reports should incIude starvation (100%) times in cIear waterunde~ the same experimental conditions as control groups.

7. HYGIENE STRATEGIES

Convener: Gidon MinkoffRapporteur: 0ivind Bergh

RECOMMENDATIONS

The following recommendations were proposed and accepted by the \Vorking Group:

A. Marine finfish hatcheries are recommended to take the followingmeasures:

1. Disinfect eggs, employing methods adapted to the species in question

2. Divide the hatchery into production zones, physically separated byinfection barriers. Movement of material should be carried outdown the production line. Duplicate facilities are advisable.

3. Periodic shut-down of the plant should be carried out in order todisinfect the facilities.

4. As the live food is seen as a major vector of contaminants, it isadvisable to use methods such as rinsing to reduce the associatedbacterial abundance.

5.For monitoring bacterial growth, florescence microscopy isrecommended if possible, otherwise marine agar and TCBS.

B. There is a need for research on:

6a. Bacterial populations associated with different life stages underdifferent conditions

6b. Interactions between larvae and associated bacteria including bothpathogens and probiotics, taking into account the nutritionalcondition of the larvae, as well as their physical rearing conditions.

6c. Procedures to improve system stability with regards to bacterialpopulations

6d. The development of the immune system in early life stages.

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8. NEXT MEETING

Tbe \Vorking Group on Mass Rearing of Juvenile Marine Fish recommends that thegroup should continue its work and meet in Bergen on June 25-26, 1993 with IngvarHuse as Chairman.

The following terms of reference were suggested by tbe group for tbe Working Groupmeeting in 1993. The group should meet to work towards the establishment of:

a) a protocol for standardized monitoring of egg and larval quality

b) an inter-Iaboratory investigation of egg and larval quality

c) a protocol for hygiene procedures in rearing systems

d) a protocol for standard nutrition research taking into account dataavailable on the performance of the proposed standard live diet andexperimental procedure as weIl as the results of the (n-3) HUFAenricbment intercalibration exercise.

Furthermore, the group should meet to:

e) compile information on standard inert reference diets.

APPENDIX I

List of participants fromICES countries

Belgium

Bosteels, ThomasLaboratory of Aquaculture &Artemia Reference CenterUniversity of GhentRozier 449000 Gent

Dhert, Ph.Laboratory of Aquaculture &Artemia Reference CenterUniversity of GhentRozier 44

.000 Gent

Komis, AntoniosLaboratory of Aquaculture& Artemia Reference CenterUniversity of GhentRozier 449000 Gent

Lavens, PatrickLaboratory of Aquaculture &Artemia Reference CenterUniversity of GhentRozier 449000 Gent

Leger, Philippee'utemia Systems NVISA

\Viedauwkaai 799000 Gent

Roelants, 1.Laboratory of Ecology and AquacultureCatholic University of LeuvenNaamsestraat 59B-3000 Leuven

Sorgeloos, PatrickLaboratory of Aquaculture &Artemia Reference CenterUniversity of GhentRozier 44, 9000 Gent

Van Stappen, GLaboratory of Aquaculture& Artemia Reference CenterUniversity of GhentRozier 449000 Gent

Verdonck, LindaLaboratory of MicrobiologyUniversity of GhentKL Ledeganckstraat 359000 Gent

Volckaert, F.Laboratory of Ecologyand AquacultureCatholic Universityof LeuvenNaamsestraat 593000 Leuven

Denmark

Poulsen, Niels EgDanish Institute of Fisheriesand Marine ResearchNorth Sea CenterP.D. Box 1019850 Hirtshals

Stcttrup, JosianneDanish Inst. for Fisheriesand Marine ResearchNorth Sea CenterP.D.Box 1019850 Hirtshals

Canada

Brown, Joseph ADcean Science CenterMemorial Universityof NewfoundlandSt JohnsNew FoundlandAlL 557

Goff, GregFisheries Resouree Development Ltd2021 Brunswiek St., Suite 317Halifa.xNova Scotia B3K 2Y5

\Vaiwood, KennethBiological StationSt. AndrewsNew BrunswickEOG 2XO Canada

Faroe Islands

Fjallstein, IngvardFiskirannsoknarstovanP.O. Box 3051NoatunFr-lOO Torshavn

France

Chatain, B.Gie Recherche AquacoleStation Experimentale d'AquacultureIFREMERroute de Maguelone34250 Palavas-Ies-Flots

Kaushik, SLaboratorie de Nutritiondes PoissonsStation d'Hydrobiologie INRA64310 St. Pee-sur-Nivelle

Person-Le Ruyet, JCentre de BrestIFREMERB.P.7029280 Plouzane

Germany

Quantz, G.Institut für Hydrobiologie undFishereiwissenschaftOlbersweg 24W-2000 Hamburg 50

Segner, HelmuthZoologie IIUniversität KarlsruheKaiserstrasse 127500 Karlsruhe

Netherlands

Verreth, JohanDepartment of Fish Cultureand Fisheries\Vageningen Agricultural UniversityP.O.Box 3386700 AH WageningenThe Netherlands

Norway

Adoff, GretheSea Farm AS5419 Fitiar

Danielssen, DidrikInstitute of Marine ResearchFlödeigen Marine Research Station4817 His

Folkvord, ArildFr. Stangsv. 35032 Minde

Holmefjord, IvarInstitute of Aquaeulture ResearchAKVAFORSK6600 Sunndals~ra

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Huse, IngvarAustevoll Aquaculture Research StationInstitute of Marine Research5392 Stcrebo

Kjcrsvik, ElinDept. of ZoologyUniversity of Trondheim7055 Dragvoll-Trondheim

Lein, IngridInstitute of Aquaculture Resarch.

~VAFORSK6600 Sunndalscra

Mangor-Jensen, AndersResearch Station AustevollInstitute of Marine Research5392 Storebc

Naas, KjelAustevoll Aquaculture Research StationInstitute of Marine Research5392 Storebc

Olsen, YngvarCentre of AquacultureSINTEF

.034 Trondheim

Rosenlund, GretheBP Nutrition AquacultureResearch CentreP.O.Box 5324001 Stavanger

Portugal

Pousao-Ferreira, PedroInstituto National deInvestigacao das PescasAv.5 de Outubro S/N87000lh

Spain

Iglesias, JoseInstituto Espanol de OceanografiaApdo 155236280 Vigo

Martinez, 1.Instituto Espacol deOceanografiaLaboratorio de SantanderApdo 24039080 Santander

Minkoff, G.Tinamenor S.A. PesuesCantabria

Peleteiro, Jose BInstituto Espanol de OceanografiaApdo 152236280 VigoPontevedra

Sweden

Larsson, Per-OlovInstitute of Marine ResearchP.O. Box 445300 Lysekil

Pickova, JanaInstitute of Marine ResearchP.O. Box 445900 Lysekil

United Kingdom

Bromage, NiallInstitute of AquacultureUniversity of StirlingStirlingFK94LAScotland

Dye, JohnFish Industry AuthorityMarine Farmaing UnitArdtoe, AcharacleArgyll, PH36 4LD

Howell, B. R.Fisheries LaboratoryMAFFBenarth RoadConwyG\,,,ynedd LL32 8UB\Vales

United States of Amerika

Ako. HarryDept of Environmental BiochemistryUniversity of Hawaii1800 East West RdHonoluluHI96822

Bengtson, DavidDept. of ZooIogyUniversity of Rhode IslandKingston, RI 02881

Holt, G JoanMarine Sciences InstituteThe University of Texas at AustinP.O.Box 1267Port AransasTX 78373

Tellock, Jeff A.Provesta Corporation93·D Philips Research CenterBartlesvilleOK 74004

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Appendix 11

3IBLICGnAPHY OU NUTRITION Alm HISTOLOGYD.A. 3engtson

(Draft No. 1)

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Al-Maghazachi, s.~. 1983. Histological and histochemical studiescn gut develcpment and physiclogy of larval turbot, Sccpht~al~us

~axi0us (L.l. Ph.n. Dissertation, Liverpoc1 P01ytechnic, 23i pp.

~Accelbaum, S., H. Seqner and V. Storch. 1986. Sleceren microscocic.. s~~dy cn :~e influence cf different nutritienal conditions on liver

and white -:runk muscles of whitefish (Ccreqenus lavaretus) larvae.:co1. An=. 217:34-64.

3ell, M.V., ~.=. Hendersen, 3.J.S. Pirie and ~.R. Sargent. 1985.E::fects :; f di etarj' polyunsa~urated :a t ty aci d deii ci enci es enmor-:a1ity, grcw~h and gill str~cture in the turbot, Scophthal~us

~ax~~us. ~. Fish Biol. 25:131-191.

31axter, =.H.S., D. ~anielssen, E. Moksness and V. Oiestad. 1983.Descripticn cf the early development of the halibut Hippoglossushi;;oqlqssus and attempts tc rear the larvae past first feeding.Mar. 3iol. i4:99-10i.

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ttc:;nnes, R. and K. 3encalima. 1sa4. u1trastructur€ ds l'intestin du:ouo Dicentrarchus labrax L. au cours du developpement larvaire.3uli. Soc. Zool. Fr. 109:19-33.

Cousin, J. C. 3. and F. Baudin-Laurencin. 1985. Morphogenese del'appareil digestif et de la vessie gazeuse du turbot, Scophthalrnusmaximus L. Aquaculture 4i:30S-319.

Cousin, J.C.B., G. Balouet and F. Baudin-Laurencin. 1986.Alterations histolocricrues observees chez des larves de turbot(Scophthalmus maximus L.) en elevage intensif. Aquaculture 52:173­189.

Cousin, J.C.B., F. Baudin-Laurencin and J. Gabaudan. 1987. Ontogenyof enzymatic activities in fed and fasting turbot, Scophthalmusmaximus L. J. Fish Biol. 30:15-33.

Dabrowski, K. 1989. Formulation of a bioenergetic model forcoregonine early life history. Trans. Am. Fish. Soc. 118:138-150.

Eckmann, R. 1985. Histopathological alterations in the intestineof whitefish (Coregonus sp.) larvae reared on zooplankton from LakeConstance. Dis. aquat. Org. 1:11-17. , .

Eckmann, R. 1987. pathological changes in the midgut epithelium ofgrayling, Thymallus thymallus L., larvae reared on different kindsof food, and the relation to mortality and growth. J. Fish Dis.10:91-99.

Ehrlich, K.F., J.H.S. Blaxter and R. Pemberton~ 1976. Morphologicaland histological changes during the growth and starvation ofherring and plaice larvae. Mar. Biol. 35:105-118.

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smelt larvae. Bull. Jap. Soc. Sci. Fish. 33:1116-1119.

Iwai, T. 1968a. The comparative study of the digestive tract ofteleost larvae V. Fat absorption in the gut epi thelium ofgoldfish larvae. Bull. Jap. Soc. Sci. Fish. 34:973-978.

Iwai, T. 1968b. Fine structure and absorption patterns ofintestinal epithelial cells. in rainbow trout alevins. Z.Zellforsch. mikroskop. Anat. 91:366-379.

Iwai, T. 1969. Fine structure of gut epithelium cells of larval andjuvenile carp during absorption of fat and protein. Arch. Histol.Jap. 30:183-199.

Iwai, T.digestiveposterior48 .

and X1. Tanaka. 1968a. The comparative study' of thetract of teleost larvae - III. Epithelial cells in thegut of halfbeak larvae. Bull. Jap. Soc. Sci. Fish. 34:44-

• Iwai, T. and M. Tanaka. 1968b. The comparative study of thedigestive tract of teleost larvae - IV. Absorption of fat by thegut of halfbeak larvae. Bull. Jap. Soc. Sci. Fish. 34:871-875.

Kjorsvik, E., T. van der Meeren, H. Kryvi, J. Arnfinnson and P.G.Kvenseth. 1991. Early deve10pment of the digestive tract of cod1arvae, Gadus morhua L., during start-feeding and starvation. J.Fish Biol. 38:1-15.

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Mitchell, L·~G., J.G. Nickum and M.T. Long. 1986. Histochemicallocalization of some digestive enzymes in larval walleyes. Prog.Fish Cult. 48:279-281.

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and functionalthe round goby,of development.

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•

Segner, H., R. Rosch, H. Schmidt and K.J. von'Poeppinghausen. 1989. ..Digestive enzymes in larval Coregonus lavaretus L. J. Fish Biol.35:249-263.

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Stroband, H.W.J. and F.H. van der Veen~

protein absorption during transport of foodgrasscarp (Ctenopharyngodon idella Val.).156.

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•

Tanaka, H. 1972b. Studies on the structure and function of thedigestive system in teleost 1arvae - V. Epithelial changes in theposterior gut and protein absorption. Jap. J. Ichthyol. 19:172- ..180.

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I

•

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de Bar,

•

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•

Appendix III

r.c.~.s. study cn (n-3)HUFA Requirements i~ Mari~e Fish Larvae.

_..-- ----::-l~.L..:=''::' :fCER

Ar~E~~~ Sys~e~s ~.~., :~iedau~kaai 79, 3-9000 Ghen~,Eelgiun.

cf ~ings~=~, ,RI,

?ATRICK SORGELOOS

:l.S ..;.

:~is ~a;er s~~~arizes t~e results ebtained in the fraI:lework cf as~~dy lau~ched a~d c=c==i~a~ed =1 I.C.E.S. (International council:or t~e Explcra~ion of the Seas). This study ai=s at establishingrequiremen~ levels cf t~e essen~ial fatty acids (n-3)HUFA for theearly life s~ages cf ~ari~e aquaculture organiscs. Sofar results~a'\·e =een ::-s;:==-:ed f:r -:he fc llc·... ing species: the qiant prawn"-c--;..--r;..···- --c:-.:::";"-,.._''': sea;"'ass -·C0,., ......-_....... ··s 7a;.. ... "''' seab-ea'".0.':: .::_ •• __ .... ••_=_;: __ , _ -: .-"__ ~."""'_.,;:_'-'.• "'" ,_ "''''-i.•• ' ........S~~::-::.s .::'::':=::~, -:.~r~ct S==;::"Ca~.'7:~s .::a.~~::us, st=:.ped bass !·Iorc::esaxa~~2~s,and red drum S=~ae~=~s ccella~us.

:uri~; i~s =eeti~q in Vigo (19S8~Spain) the I.C.E.S. ~orki~g

=r::u~ :::1 I ~a~2.·: :"i:e Sta::es of :·!arine Fish Lar':ae I reccr:rr::ended toi ::"e--"'- ~~~':'~~:.. -\..e c~'-li"'--~"e and the auan-~"'a·t~"e· (n-3)HT '1'":'A••• - '- '= •• .1 -... ..._0 __ a.i...._V ... ~_'- ~__ 1..ti.

require::,.er'.~5 in =ar ine lar':ae ... I. The ter::l. (n-3) HUFA refers to~eng c~ain ~ighly unsa~~ra~ed fatty aC1Cs, ~ore particularlyeic::sa~en~aenci:: acid (E?A er 20:5n-3) and dcccsahexaenoic acid(CRA er 22:5n-3). These fatty acids have an inportant strueturalrele i:1 ~he =uild up of =ic~ernbranes and they act on a nu~ber cf=icchenicals called 'eiccsanoids' such as leukotrienes and~restaglandins. These ~clecules act as hor::l.ones involved inregulati:1q several vital =cdy functicns such as the immune response~echanis~ and inflarnmatcry prccesses. The essentiality of thesefatty acids f::~ young ~a~ine a~inals has been docurnented quietextensively the last years. Several publications evidence thequalitative dietary irnportance of these fatty acids fcr the larvaeof several species of marine orqanisms. Establishing quantitativerequirenent levels fcr cc~nercially inportant species would be very=eneficial fcr i~prcving and opti::l.izing their larviculture. This ishc~ever a ce~plex issue as quantitative dietary requirement levels::l.ay dif:er fr::::l. species to species and may furthernore be affectedcy.larval age, larval quality, environmental ccnditions, etc.?rovided these ccnditicns are deterrnined and standardized, stillthen establishing dietary requirement levels of f.ex. (n-3)HUFA isvery difficul~ as tcday no reference ccrnpound larval diets -wit~

(n-3)HUFA as the only variable- i5 available. Even when such die~

~ould ce available it might no~ ce suitable as the firs~ feedingstages of nost ~arine fish larvae da not accept cornpound feeds as

I'I

a sole diet. For these reasons i t was suggested that for thepresent I.C.E.S. study the commonly used feeding regime based onthe use of the live food organisms Brachionus plicatilis andArtemia nauplii would be applied in a first step. The fatty acidprofile of the rotifer Brachionus plicatilis and the nauplii of thebrine shrimp P~temia, especially their content of the (n-3)HUFA,canbe modified by applying the technique of enrichment (Leger etal.1985, 1986; Watanabe et ale 1983). This way the content of (n­3)HUFA can significantly be increased from a few milligrams pergram dry weight ~o 50 milligrams and more (Leger et ale 1987).These high values are obtained by feeding the live food organisms(n-3)HUFA rich emulsions.

Materials and Methods

For the present study ~hree emulsions have been formulated for theenrichment of the live food organisms. The emulsions had adifferent ccntent of (n-3)HUFA : low, medium and high (see TableI) .Detailed instructions for rotifer and Artemia enrichment wereprovided by I.C.E.S. ~o the 14 participants of ~he study:

1. Artenia enrichment

- incubate ~he daily required amount of Artemia cysts (Great SaltLake strain, 2 g per li~er sea\·:ater) in natural or artificialsea\Vater:

- salini~y = 35· ppt- tenperature = 28°C +j- lOC- pH = 3 a 9 throughout hatching; eventually add sodiumbicarbonate- light = > 2000 lux- aeration = fairly strong

•

- harvest and rinse the .~te~ia nauplii after 24 h incubation andtransfer them to the enrichment vessel filled with filteredseawater at a density of 100 individuals per ml. tt- mix (with kitchenblender) 0.3 g enrichment emulsion (per literenrichment medium) for 30 seconds in a small volume of water andadd to the enrichment vessel.

- maintain a temperature of 28°C +j- 1°C.

- ensure high oxygen levels (min. 4 ppm D. 0.) by applying anairstone aeration.

- no illumination is required.

a second rationenrichment medium)

of 0.3 g enrichment emulsion ( per literis prepared and added after 16 h enrichment

(between 12 and 18 h ).- after a total enrichnent period of 24 h the metanauplii areharvested and thoroughly rinsed; now they are ready for feeding tothe larvae.

2. Rotifer enrichnent

- harvest and rinse cultured rotifers on an immersed sieve ( 45 a60 /-Ln).

- gently transfer the rinsed rotifers into filtered pre-aeratedseawater of 25 ppt salinity at a density of 500 rotifers per ml.

- maintain a temperature of 27°C +/- 1°C.

naintain a slight aeration as to keep all rotifers weIlsuspended; avoid s~rcng aera~ion in order to minimize clogging.

- the enrichment media are prepared as above for Artemia; insteadof t~o times 0.3 g/l for .;rtemia enrichnent two times 0.1 g/l areused for rotifer enrichment; a first ration is added when therotifers are being transferred to the enrichnent tank (tOh ) (startof enrichnent) and ~he second ration is added after three hours(t3h ) incubation.

- after a total enrich~en~ per iod of six hours (t6h) , gently harvestand rinse the enriched ro~ifers on an immersed sieve; avoid strongturbulence; now the enriched rotifers are ready for feeding.

By applying the above ins~ructions the content of (n-3) HUFA inrotifers and Artemia is altered depending on the type of emulsionused: rotifers and PItemia enriched with the low, medium and high(n-3)HUFA enulsion contain respectively low, medium and high levelsof (n-3)HUFA ( example for .;rtemia see Figure 1 and 2).

Results

The detailed results obtained by the different participatinginstitutes are being published elsewhere as individual papers. Asummary of the first results are being presented hereunder.

1. Dicentrarchus labrax

Two different laboratories ran the requirement study onseabass (Dicentrarchus labrax) larvae.One study (Corneillie et al., Zoological Institute, University ofLeuven, Leuven, Belgiurn)was conducted on first feeding larvae whichduring aperiod of 40 days were offered rotifers and subsequentlyArtemia both enriched with the three emulsions containing

..-------- ---- - - -

increasing levels of (n-3)HUFA.A clearirnpact of the fatty acid composition of the feeds on larvalsurvival was noted: survival after 60 days.culturing was 3.3 % +/­0.1 % in the low (n-3)HUFAgroup , 12.7 % +/- 0.6. % in the medium(n-3)HUFA group and 13.0 % +J- 0.1 % in the high (n-3)HUFA group.A massive mortality ~as noted between day 30 and day 60 in thelarvae fed the low (n-3)HUFA enriched rotifers and Artemia. Duringthis period many larvae in this treatment exhibited the symptoms ofthe so-called '·~:hirling disease I. Ho larvae from the medium andhigh (n-3)HUFA treat~ents ~ere affected. Growth in terms ofindividual dry weight was also affected by the diet compositionthough significant differences appeared only from the third weekonwards (see Fig.4). The same was observed for growth in terms ofindividual length. Besides survival and growth effects on themorphological develcp17.ent of the larvae were also studied. Norelation was fcund between the diet composition and the occurrenceof skeletal deformities, shortening of the opercula nor the degreeof swimrnbladder inflation. A significant interaction was detectedhowever between the (n-3)HUFA content of the diet and thedeveloproent of the gall bladder: low dietary (n-3) HUFA levelsappeared to induce hypertrophy of the gall bladder.

The seccnd study ( Hartinez and Alcazar, Oceanographic Institute,Har Henor, Murcia, Spain) was carried out with 35 d oldDicentrarc.l1us labrax larvae ~vhich were fed the enriched Artemiapreparations during 29 days. During this stage of larvaldeveloproent noticeable effects of dietary fatty acid content onsurvival are only observed between the low (n-3)HUFA treatment andboth others (see Fig.5). Effects on growth were not significant.

Neither of the two studies carried.out fatty acid analysis on theenriched live feed used in the experiment. Hence absoluterequireroents seabass larvae for n-3HUFA may not be drawn from theabove studies. Taking into consideration the values as illustratedin Figures 1 and 2 cne could conclude that estimated requirementswould be about ... to ... ng n-3HUFA per gram dry feed offered~

2.Sparus aurata

Sofar resu1ts with seabream larvae are limited to thoseobtained by Martinez and Alcazar (cfr above). Results relative tothe treatments are very cornparable to those obtained by the sameauthors for seabass D. labrax (see above). Figure 6 shows again amassive mortality in the low (n-3)HUFA treatment and a relativelyhigh survival in both other treatments.Koven et al. carried out a two weeks experiment with ••. days oldseabream larvae fed Artemia nauplii enriched with soybean oil (non-3HUFA) supplemented with increasing levels of SUPER SELCO(Artemia Systems S.A., Ghent Belgium; SUPER SELCO contains anequivalent concentration of n-3HUFA as the I.C.E.S. High emolsion).The supplenentation of n-3HUFA in the Artemia increased survival inthe bream larvae significantly though a levelling off could benoted at .... ~g/g (see Fig .... ). On the contrary however, growth

continued to i~prove along with the level of n-3HUFA in the dietindicating that for growth requirements are above ... mg n-3HUFA pergram dry feed offered (see Figure .... ).

3.Macrobrachium rosenberqii

Two studies have ceen perforr:H~d evaluating the effects offeeding newly hatched M. ~csenbergii larvae a sole diet of Artemianauplii enriched ,vith the r.C.E.S. enulsions. A first study byBuzzi et al. (1989) carried out at the Sterling UniversityTropical Pra~.;n Uni t- indicated that best results in terms ofsurvival, grc·,·;th and r::etanorphosis rate were achieved when theprawn larvae ~ere fed the highest n-3HUFA containing diet (High­emulsion) . The results were significantly better than when feedingthe Hedium enriched nauplii. Lowest culture performance wasobserved in the treatr::ent receiving Low n-3HUFA Artemia (seeFigures .... ) . Fatty acid profiles of the enriched Artemia naupliiare given in Table .... Fron these results the authors concludedthat for ~ee~ing the n-3HCFA requirements of M. rosenbergii larvaethe diet offered shculd ccntain at least .... mg n-3HUFA per gramdry "veight.In a second study Devresse et al. (1990) performed the sameexperinent at the Arter::ia Reference center (University of Ghent,Belgiun). Their results indicated that best culture performance (interns of grc·..;th, survival, vitality and metamorphsis rate) wasachieved ~vhen offering .::'.rtemia nauplii enriched with the Mediumemulsion. No further ir::provenent ,,,as noticed when nauplii wereoffered enriched with the High n-3HUFA emulsion. From the fattyacid analysis (see Table ... ) the authors conclude that therequirenents cf ~. ~cse~~e~gii larvae should be in between 5.2 and37.5 ng per gran dry die~ cffered - probably toward the higher end.In this sense these results agree with those obtained by Buzzi etal.(1989). This further correlates with the results obtained bySandifer and Joseph (1976) \-Iho improved the performance of apostlarval diet by increasing the n-3HUFA content to 19 mg/g.Considering n-6 fatty acids Devresse et al.(1990) could not confirma require~ent in H. rosenbergii larvae as was identified inpostlarvae by Reigh and Stickney (1989).Romdhane et al.(in preparation) carried out an experiment feeding.newly hatched M. rosenbergii larvae during an increasingly shorter(delayed) period \-Jith Arte:nia enriched with SELCO (Artemia systemsS.A., Ghent, Belgium). n-3HUFA levels of SELCO enriched naupliicorrespond to those obtained when using the Medium I.C.E.S.emulsion. From their results (see Figure ... ) the authors concludethat for optimal results (in terms of growth, survival, stressresistance and netanorphosis rate) prawn larvae should be fedn-3HUFA enriched Artemia nauplii preferrably from first feedingonwards.

4.Morone saxatilis

One study on larval striped bass (Morone saxatilis) has been

conducted by Lemm and Lemarie (1991, in press). Four days post­hatch Tennessee striped bass larvae were cultured during 14 days ona diet consisting of freshly hatched or enriched (I.C.E.S.emulsions) Artemia nauplii.The authors report on the fatty acidprofiles of the emulsions, the Artemia, and the fish larvae (valuesof n-3HUFA in Artemia are represented in Table •... ).

Fatty LOH HUFA 11EDIl;1·1 HUFA' HIGH HUFA unenracid

enul I Art. enul I J:...rt. emul Art.

20:5n-3 0.0 I 2.6 9.7 I 8.2 25.0 12.5 3.8

22:6n-31 0.0 10.0 7.8 I 3.1 36.9 9.4 0.0

~n3HUFj\ I0.0 I 2.6' I 19.1 111. 3 I 66.5 22.8 3.8

As can be noticed frem Table ... n-3HUFA levels in Artemia reflectthose of the enrichnent enulsions; they are however inferior tothose presented in Table .... Total lipid levels as reported by theauthors decrease during enrichrnent (about 20 %) which wouldindicate that the enrichment procedure was not optimal. eultureresults are sunnarized in Table ...

.~temia length survival swimmbladdtreatment er

inflation

Unenriched I 9.3 ..../- 1.0 I 5 (29)

LOH HUFA I 9.8 .:-/- 0.9 I 23 26

NED. HUFA 10.3 +/- 0.8 I 64 33

HIGH . HUFA I 10.2 .... /- 0.8 48 32-Growth and surv~val "ere s~gn~t~cantly ~mproved ~n the fish larvae

fed the n-3HUFA enriched .~temia. A n-3HUFA content of 11.3 % oftotal fatty acids in Artemia appears to fulfill the requirements offirst feeding striped bass larvae. Increasing the level to 22.8 %did not further irnprove the performance. Swimmbladder inflation wasnot significantly affected by the dietary n-3HUFA content.

6.Sciaenops ocellatus

Craigh, Holt and Arnold (in press) report on experimentsassessing the effects of feeding enriched rotifers ,using thel.e.E.S. emulsions, on the growth and fatty acid composition of reddrum (Sciaenops ocellatus) larvae. Fish were cultured during tendays and no Artemia was fed. The contents of n-3HUFA in theenriched rotifers are given in Table ....

iIIIrIIII

III

!IIt

II1,

I

Fatty CONTROL LOH MEDIUH HIGHacid n-3HUFA n-3HUFA n-3HUFA

20:5n-3 1.5 8.6 10.7

22:6n-3 0.0 6.7 12.5

~n-3HUFA 1.5 15.3 23.2

Reference List

List of 'I'ables

!Jist of Figures

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Grm.,rth rates i~'ere best ln the larvae fed the n-3HUFA enrichedrotifers (medium and high n-3HUFA emulsions) i they were however notsignificantly different from the control treatment fed rotifersgrown on a mixture of algae, fish oil and yeast. Poorest resultswere recorded in the fish fed the low n-3HUFA enriched rotifers.The authors ccnclude that 22:6n-3 significantly affect growth inred drum larvae. XXXXXXXXXXX--control rotifers ---XXXXXXTen day old red drum larvae exhibit a fairly constant fatty acidprofile independant of the diet offered. An extended trial coveringthe subsequent culturing phase including Artemia would allow toverify the evolution of the fatty acid profile in red drum larvaebeyond the rotifer stage. I·!ore pronounced differences betweentreatments could eventually show up when reserve fatty acid pools

~WOUld get depleted.

~ Ccnclusions(

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