larvi 2013

ghent university, belgium, 2-5 september 2013

6th fish & shellfish larviculture symposium

Reproduction of European eel and larval cultureState of the art

Jonna Tomkiewicz

Ian A.E. Butts, Sune Riis Sørensen, Sebastian N. Politis, Josianne Støttrup,Peter Munk & Maria Krüger-Johnsen, DTU, DenmarkPeter De Schryver & Mathias Bouillart, Ghent University, BelgiumJosé Zambonino-Infante, Geneviève Corraze & Sachi Kaushik, INRA, FranceSylvie Dufour & Anne-Gaëlle Lafont, CNRS, FranceJuan F. Asturiano, ICTA-UPV, SpainElin Kjørsvik, NTNU, NorwayTerje van der Meeren, IMR, NorwayPeter Lauesen, Billund Aquaculture Service, Denmark

Reproduction of European eel andlarval culture - State of the art

Jonna Tomkiewicz, Technical University of Denmark

Larvi 2013

• Eel aquaculture relies on wild caught glass eels• Severe decline in eel stocks renders present aquaculture

production unsustainable• Captive breeding is needed to sustain eel aquaculture

CHALLENGES FOR EEL AQUACULTURE

Continental

Oceanic

Challenges for hatcheryproduction of glass eelsComplex life cycle

Lack of knowledge about wild eelreproduction and early life stages

Hormonal control and inhibition ofmaturation during silvering

PRO-EEL OBJECTIVES

Increase knowledge about eel physiology and hormonalcontrol of reproduction→ Improvement of broodstock nutrition and hormonaltreatments for induction of maturation

Uncover mechanisms of final oocyte maturation,sperm function and fertilisation capability→ Establishment of a stable production of viable eggs andembryos

Access requirements for successful egg and larvaldevelopment and identify suitable larval feeds→ Establishment of rearing technology and first feeding inculture of European eel larvae

PRO-EEL CONCEPT

Neuro-endocrine function andhormonal control mechanisms

Liver function and energyallocation

Male maturation and spermquality

Female maturation, ovulationand egg quality

Development and functionalanatomy of embryos and larvae

Establishment of aknowledgebase

Establishment of aknowledgebase

Broodstock nutrition andselection criteria

Controlled maturation andgamete production

Fertilisation procedures andincubation methods

Larval culture and feeding

ApplicationdevelopmentApplication

development

Protocols foreel reproduction

and larvalculture

Protocols foreel reproduction

and larvalculture

Full scaleexperiments

Link basic research to application and technology development to fillgaps in knowledge and methodology

FULL SCALE EXPERIMENTS

Broodstock selectionand nutrition

Broodstock selectionand nutrition

Gamete production offemales and males

Gamete production offemales and males

Fertilisation and incubation

Larval production andculture

Full scaleexperiments

An integratedapproach

Hormonal treatmentsand responsivenessHormonal treatmentsand responsiveness

Protocols

Broodstock maturation –Wild and farmed: differentdiets and hormonaltreatment

Fertilisation (4-5 males perfemale) and incubation

Hatching and larval rearingimproving graduallytechnology

Comparison of reproductive success of farmedand wild broodstocks

Results

Broodstock responseStripping success

Fertilisation successEmbryonic developmentalsuccess

Larval production successLarval quality and longevity

FULL SCALE EXPERIMENTS

BROODSTOCK RESPONSE

Female response tohormonal treatmentvariesResponse of females asweight change over timeclassified by ovarianhistology at sacrifice

0.8

0.9

1

1.1

1.2

1.3

1.4

Wei

ght c

hang

e slow responders 1E5CB6CAC6F2BDEFD40E

0.8

0.9

1

1.1

1.2

1.3

1.4no responders 1670

20712341B52DBF04CF13D98A

0.8

0.9

1

1.1

1.2

1.3

1.4

Time (weeks)

fast responders

41D5C0A1B794B79D1198C092C5D7C918C91ECBBF5B152B3ACBDCCC6ACCF7CD76CF11262A

Enhanced farmed broodstocknutrition optimising egg &larvae quality

• Comparing farmed and wild eels

• Formulated diets-10

-5

0

5

-6 -4 -2 0 2 4 6 MuskelNLVilde_L…, X-expl: 44%,15%

Klint

Klint

Klint

KlintKlint

KlintKlint

Klint

KlintKlint

LyksLyksLyksLyksLyksLyksLyks Lyks

LyksLyksNorge

Norge

NorgeNorge

NorgeNorge

Norge

Norge

NorgeNorge

PC1

PC2 Scores

FEMALE BROODSTOCK NUTRITION

0

2

4

6

8

10

ARA EPA DHA DHA/EPA EPA/ARA

Rel

ativ

efa

ttyac

idco

nten

t

Ovary NL

Farmed broodstock on different diets

400163Vit. E ppm

400257Vit. C ppm

3.124.51ARA g/kg

20.517.2DHA g/kg

11.98.1EPA g/kg

24.622.9Fat %

47.447.3Protein %

JD2JD1DAN-EX

163

257

1.08

24.4

19.4

25.7

48.1

Essential FA in different diets

100 100 100 100 100

85

100

90 89

75

23

44

10

4450

15

33

10

33

50

0

25

50

75

100

FarmedStandarddose 4

FarmedFeed 1dose 4

FarmedFeed 2dose 4

Wilddose 4

Wilddose 8

% r

esp

ond

ing

ind

ivid

ual

s

Broodstock type –feed and treatment

Comparison wild and farmed eels

stripped

fertilised

embryos

larvae

FEMALE NUTRITION AND REPRODUCTIVE SUCCESS

0

2

4

6

8

10

12

14

K-high K-low K-high K-low K-high K-low Wild 1 Wild 2

Standard Standard Feed 1 Feed 1 Feed 2 Feed 2 Natural Natural

ARA

EPA

DHA

DHA/EPA

EPA/ARA

Does feed matter – Yes!

Comparing performance of farmed and wild broodstocks

0

20

40

60

80

100

0,5 1,2 1,9

% o

f st

rip

ped

fem

ales

ARA content in feed (%)

d eggs

Feed1

K-Fulton’sconditionfactor

Firstcleavages3h

Cells overyolk-mass14t

Evidentembryo24t

Apparentsegments, tail isfree 40 t

FWRTILISATION AND EMBRYONIC DEVELOPMENT

Embryonic stage duration app. 48 h until hatch

6-7 hours

2-3 hours

DEVELOPMENT OF YOLKSAC LARVAE

10-11 hours500 µm

2 days

5 days

14 days

9 days

1 mm

DEVELOPMENT OF YOLKSAC LARVAE

Amount and use of yolk

Length increase

Measurments of morphological characteristics and generaldevelopmental success

QUALITY OF YOLKSAC LARVAE

Measuring development in general morphology,amount and use of yolk and length increase

QUALITY OF YOLKSAC LARVAE

PRO-EEL Series A and B, Length at age

Age (dph)

0 2 4 6 8 10

Sta

ndar

d le

ngth

(mm

)

2

3

4

5

6

7 5414CO92C91EDD221065 5492B5FE BE82C452C881 D042 D37D D727

PRO-EEL Series A and B , Yolk at length

Standard length (mm)

2 3 4 5 6 7 8

Yolk

are

a (m

m2)

0.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.954145B15C092 C91E DD221065 5492B5FE BE82C452C881 D042 D37D D727

Red=ABlue=B

Red=ABlue=B

PRO-EEL Series A and B, Length at age

Age (dph)

0 2 4 6 8 10

Sta

ndar

d le

ngth

(mm

)

2

3

4

5

6

7 5414CO92C91EDD221065 5492B5FE BE82C452C881 D042 D37D D727

PRO-EEL Series A and B , Yolk at length

Standard length (mm)

2 3 4 5 6 7 8

Yolk

are

a (m

m2)

0.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.954145B15C092 C91E DD221065 5492B5FE BE82C452C881 D042 D37D D727

Red=ABlue=B

Red=ABlue=B

Differences in larval quality between farmed andwild broodstock

Age (dph)

0 2 4 6 8 10 12

Sta

ndar

d le

ngth

(mm

)

2

3

4

5

6

7

8

Series ASeries BSeries CSeries D

Differences in larvalquality

Farmed:Series: A and C

Wild:Series B and D

QUALITY OF YOLKSAC LARVAE

Sperm to egg ratio

Fert

iliza

tion

succ

ess

(%)

0

20

40

60

80

a

ab ab

bccd cd

cdcd

d d dd

1.0e+67.5e+55.0e+52.5e+51.0e+57.5e+45.0e+42.5e+41.0e+45.0e+32.5e+31.3e+3

Time post-stripping (min)

Fert

iliza

tion

succ

ess

(%)

0

20

40

60

80

100

120

0 5 10 15 20 40 6030

a

ab

abc

bc bc

bc

bc

c

Standardisation offertilisation proceduresImportance of sperm to eggratio and time post-stripping

Egg fertilisation capacity inrelation to time post-stripping

Optiomal sperm # and timepost-stripping–25,000 sperm per egg–Eggs fertilized within 10min

STANDARDISATION OF FERTILISATION PROCEDURES

Butts et al., Larvi 2013, poster

EGG AND LARVAL REARING TECHNIQUES

Establishment of biotechnicalconditions for egg incubation andlarval rearing

• Water quality management• Recirculation, flow-through• Mesocosmos etc.

Transport and export

• Transport of eggs by flight fromDenmark to Norway

• Successful parallel experiments

Incubation:Test of eggdisinfectiontreatments

Treatments:

Filtered autoclavedSeawater

Glutar aldehyde

Hydrogen peroxide

low 2 g L-1

high 6 g L-1

EGG AND LARVAL REARING TECHNIQUES

Disinfection agentFASW GLUT S.HYP 50 S.HYP 100 H2O2 2g H2O2 6g

Larv

al h

atch

(%)

0

20

40

60

80

100

Hours after fertilization50 100 150 200 250

Larv

al s

urvi

val (

%)

0

20

40

60

80

100

FASWGLUTH2O2 2gH2O2 6g

WATER MANAGEMENT AND MICROBIAL CONTROL

Larval survival in relation to disinfection treatment

Amount and use of yolk

Length increase

Measurments of morphological characteristics and generaldevelopmental success

QUALITY OF YOLKSAC LARVAE

Digestivehistology

Europeaneel 14 DPH

12 DPH

6 DPH 12 DPH

• Large yolk syncytium at 6 DPH,• Relatively immature liver/pancreas at 12 DPH

LARVAL DIGESTIVE SYSTEM

12 DPH

Enzyme activity (biochemical study)

Enzyme gene expression

LARVAL DIGESTIVE SYSTEM

Digestive enzymes in eellarvae day 3-14

Very high activities in lipase-likeenzymes after hatching Similar development in expression

of the enzymes’ correspondingcoding genes• lipase, amylase, trypsin and

aminopeptidase N

Data suggest that yolksac eel larvaehave an elevated nutritionalrequirement for lipids

Important for development of newstrategies for feeding eel larvae?

M.

Bouillart

et al.,Lariv

2013LARVAL FEEDING STAGE AND FEED?

European eel14 DPH

What can they eat?

Analysis of larval headmorphology

Study of larval behaviour

European eel larvae 14-15 DPH

CONCLUSION

Enhanced broodstock feeds for high quality eggsand larvae comparable to wild broodstocks

Optimal fertilization protocols Stable production of viable eggs and larvae Culture of yolksac larvae Insight in to larval nutritional requirements

Successful results of PRO-EEL

Future goals Identification of suitable feed, feeding larval culture and

ongrowing