IS IT POSSIBLE TO INFLUENCE METABOLIC PROCESS OF EUROPEAN SEA BASS JUVENILES BY A PROCESS OF EUROPEAN SEA BASS JUVENILES BY A

NUTRITIONAL CONDITIONING DURING LARVAL STAGE ?

• M Vagner• M. Vagner• J.L. Zambonino Infante • J.H. Robin • J. Person-Le Ruyet

larvi 20095th fish & shellfish larviculture symposium

ghent university, belgium7 - 10 september 2009

INTRODUCTION

N dNatural fish stocks Nowadays

Carnivorous marine fish are mainly fed with fish meals and oils

Natural fish stocks

meals and oils

BUT fish aquaculture and natural fish stocks

Substitution of fish lipid sources with vegetable lipid sources

• Not adapted to the requirement in essential fatty acids (HUFA):

Disadvantages of vegetable products:

Not adapted to the requirement in essential fatty acids (HUFA):

rich in C18 fatty acids (i.e. HUFA precursors) BUT poor in HUFA (EPA, DHA)

• Low capacity for marine fish to convert C18 FA into HUFA, in particular because p y , pof the low expression of the Delta-6 desaturase (D6D)

*

EPAAA EPAAA

DHADHA

Hi h i i d i fi h hi h

INTRODUCTION

High interest in producing fish, which

could better incorporate vegetable p g

products.

BUT HOW ?

Could it be possible using the concept of p g p

metabolic programming ?

INTRODUCTION

Adaptive process at a cellular, molecular and biochemical levels occuring during young stages, and then maintained in further developmental stages during young stages, and then maintained in further developmental stages

and potentially transmissible to the offspring (Lucas, 1998)

I it ibl i b t i t h i l gi l f j il b l l Is it possible in sea bass to orient physiological process of juveniles by a larval

conditioning to dietary vegetable products, characterized by a low HUFA

content?

1. Experimental design

I- Nutritional conditioning 1

HH22HH16LH22LH16d-5-45

Mouth openning

stag

e

LH Low HUFA EPA+DHA= 0.8% DM HH High HUFA EPA+DHA=2.2% DM

16°C 22°C

2 diet

2 T°C

C1

Larv

al s

1 commercial IP

90 days

ex-LH22ex-LH16 ex-HH16 ex-HH22

ex LH22LH16 ex-HH16 ex-HH221 HUFA-d-151-21160 days

R1

diet

nile

sta

ge EPA+DHA= 2.7% DM 19°C

EPA+DHA= 0.5% DM

ex-LH22ex-LH16 ex-HH16 ex HH22

19°C

1 HUFArestricted

diet

R1

Juve

n

2. Growth performances

I- Nutritional conditioning 1 s

tage

100

120gh

t (m

g)

No effect of diet on T***d-45

C1Larv

al

40

60

80

vidu

al m

ean

wei

g

the survival ratediet ***

0

20

LH16 HH16 LH22 HH22

Indi

v

exHH2218

nile

sta

ge

R1

100% survival rate in all groups

exLH16exHH16

exLH22

14

18

an w

eigh

t (g)

Juve

n R1No significant effect of

nutritional conditionning10

Indi

vidu

al m

ea

J 211

6-10 0 10 20 30 40 50 60

time (days)

5. Lipid metabolism in larvae

I- Nutritional conditioning 1

D6D mRNA level at d-45 D6D Desaturation product at d-45 :

18:3n-6 in PL

0 4R l ti i i

***

***

0.3

0.418:3n-6 in % FAME***

6

8 Relative expression in relation to an

housekeeping gene and to a referential condition

HH22

0 1

0.2

NS

***

2

4

HH22

0

0.1

LH16 HH16 LH22 HH220

2

LH16 HH16 LH22

Stimulation of desaturation pathways for HUFA synthesis, without any effect of temperature

5. Lipid metabolism in larvae

I- Nutritional conditioning 1

******0.8

1.2

1.6

2

AA % FAME PL

10

0

0.4

LH16 HH16 LH22 HH22

EPA % FAME PL

******

4

6

8

HUFA deficiency

0

2

LH16 HH16 LH22 HH22

30DHA % FAME PL

******

5

10

15

20

25DHA % FAME PL

0

5

LH16 HH16 LH22 HH22LH16 HH16 LH22 HH22

6. Lipid metabolism in juveniles

I- Nutritional conditioning 1

D6 mRNA level – 1st part of the challenge PL final composition in DHA

*24% FAME

***6

Relative expression in

*22

% FAME

***3

4

5 relation to an house-keeping gene

20NS

NS

1

2

3

• Significant effect of larval conditionning

18ex-LH16 ex-HH16 ex-LH22 ex-HH22

0ex-LH16 ex-HH16 ex-LH22 ex-HH22

• Better capacity of ex-LH groups to Significant effect of larval conditionning

• BUT transient (30 days)

Better capacity of ex LH groups to adapt to a low-HUFA diet

• BUT with a low amplitude

• NS for EPA

7. Conclusions

I- Nutritional conditioning 1

POSSIBLE to influence fatty acid desaturation pathways for HUFA synthesis in

juveniles, using a nutritional conditioning during larval stage, without any effect

of temperature on the capacity of FA desaturation (- 30% of HUFA requirement)

BUT this modulation seems to be limited

Is it possible to obtain a response with a larger magnitude?

2. Experimental design

II. Nutritional conditioning 2

LH1 HH1d-5-45= C1

Mouth opening

sta

ge

C2XLH1 XH1

19°C

LH1 = 0.7% HH1 = 1.7%

30 days

Larv

al C24 diets XH1 =3.7%XLH1= 0.5%

30 days≠ 90 (C1)

PI

ge

1 commercial

diet EPA+DHA= 2.7% DM 19°C

XLH2 XH2LH2 HH2

d-83- 118≠d151-211 en

ile s

tag

R2

diet

1 more HUFA XLH2 XH2LH2 HH2

EPA DHA 2.7% DM

(R1)

35 daysJuve R2restricted

diet EPA+DHA= 0.3% DM 19°C

3. Growth performances

II. Nutritional conditioning 2ge a a

b b

***

No effect of diet on 50

60

ght (

mg)

Larv

al s

tag

C2

the survival rate

30

40

n in

divi

dual

wei

g

Idem C110

20

XLH1 LH1 HH1 XH1

Mea

nile

sta

ge

R2

100% of survival rate in all groups3.5

4.5

ivid

ual w

eigh

t (g

)

Juve

n R2 No significant effect of larval conditionning

Idem R11.5

2.5

Mea

n in

d

Idem R10.5

0 5 10 15 20 25 30 35

4. Lipid metabolism in larvae

II. Nutritional conditioning 2

0 6%18:3n-6 in % FAME

D6D mRNA level at d-45 D6D desaturation product at d-45 in PL

6 Relative expression in relation to an house keeping gene

a

a0.4%

0.5%

0.6%***

a

4

5 ***

house-keeping gene

b0.2%

0.3%

cc

b

2

3

c0.0%

0.1%

XLH LH HH XH0

1

XLH1 LH1 HH1 XH1

Stimulation of desaturation pathways for HUFA synthesis

Idem C1Idem C1

4. Lipid metabolism in larvaeII. Nutritional conditioning 2

d3.5%

d

c

b1.5%

2.0%

2.5%

3.0%

***

AA (% FAME in PL)Idem C1

d10%

12%

b

a

0.0%

0.5%

1.0%

XLH1 LH1 HH1 XH1

EPA (% FAME in PL)c

ba4%

6%

8%

10%

***EPA (% FAME in PL)

HUFA deficiency

0%

2%

XLH1 LH1 HH1 XH1d

30%

35%

40%

***DHA (% FAME in PL)

c

b

a10%

15%

20%

25%

30% ***

0%

5%

XLH1 LH1 HH1 XH1XLH1 LH1 HH1 XH1

5. Lipid metabolism in juveniles

II. Nutritional conditioning 2

D6D mRNA level PL final composition in DHA

NS18

% FAMEa

6

7XLH2 LH2 HH2 XH2

Relative expression in relation to an house-keeping gene

16

18

aa

ab a

b b4

5

6Diet ***

14

a

aa

bb

bc

cab

b

c1

2

3 IDEM for EPA

• Significant effect of larval

12ex-XLH ex-LH ex-HH ex-XH

bc c

0

1

J-83 J-90 J-107 J-118

• Non significant effect of larval conditioning

≠ R1

• D6D stimulation NON transient

gconditionning

≠ R1 ≠ R1≠ R1

Summary

Although no beneficial effect on growth performances and no clear higher HUFA content in PL, POSSIBLE to influence FA desaturation pathways for HUFA synthesis for a better incorporation of vegetable products, using a nutritional

Possible to use the concept of metabolic programming in sea bass

y p g p , gconditioning during larval stage (-60% of the HUFA requirement)

Possible to use the concept of metabolic programming in sea bass

BUT is it a:Long lasting adaptation?Long-lasting adaptation?

Transient acclimatation of juveniles to their nutritional environment ?

Need to better investigate mechanisms involved in desaturation pathways in response to a low HUFA dietary content in larvae and in juveniles

PPAR

InhibitionStimulation of desaturation pathways for HUFA synthesis

0.8 1.7%0.5 0.7XLH1 LH1 LH HH1 XH1

3.7%HH2.2

D6D+ + D6D - D6DPPAR+ PPAR- PPAR-

Implementation of metabolic programming using a nutritional conditioning during larval stage

InhibitionStimulation of desaturation pathways for HUFA synthesis

D6D+ + D6D - D6DPPAR+ PPAR- PPAR-

Possible to influence the FA desaturation pathways for HUFA synthesis for a better incorporation of vegetable products

BUT

NO transient stimulation Transient stimulation

0,3 0,5HUFA

requirement at 0,7%

R2 R1

Additional factors: HUFA n-3 from the juvenile environment

DHA content in PL

NO transient stimulation Transient stimulationDHA content in PL

Control of desaturation mechanisms previously gained

Control of the fish lipid composition

IV- Perspectives

Was the metabolic programming really present ?

Transmission to Intermediate Transmission to the offspring ?period: Stop

or Non stop

?Challenge extension:

what will happen during

?adulthood ?

IF YES

Application to other carnivorous species with high commercial value in aquaculture

Thanks to the financially support: Ifremer, INRA, RAQ, Larvi.

Th k h h i l M M L G ll A Sé è H

y pp , , Q,

Thanks to the technical support: M.M Le Gall, A. Sévère, H. Le Delliou, P. Quazuguel, N. Le Bayon, J. Moriceau, C.

Huelvan & E. Desbruyères.

Thanks for your attention

y

Thanks for your attention

larvi 20095th fish & shellfish larviculture symposium

ghent university, belgium7 - 10 september 2009

Récapitulatif

XLH1 LH1 LH HH1HH XH10 5% 0 7% 0 8% 1 7% 2 2% 3 7%

LARVES

0,5% 0,7% 0,8% 1,7% 2,2% 3,7%

Adaptation à un régime carencé en HUFA n-3

Stimulation des mécanismes de désaturation

Niveau ARNm D6 continue Niveau ARNm D6 transitoire

R2 R10,3 0,5

JUVENILES

Niveau ARNm D6 continue Niveau ARNm D6 transitoire

D6 non fonctionnelle ?D6 fonctionnelle ?

R1 trop proche du besoin (0,7% EPA+DHA)Synthèse d’HUFA-

dé i é

teneur en DHA Faible en DHA

dérivées

XLH1 LH1 LH HH1HH XH10 5% 0 7% 0 8% 1 7% 2 2% 3 7%

LARVES

0,5% 0,7% 0,8% 1,7% 2,2% 3,7%

+ +D6D

+D6D

-D6D

PPAR

PPAR-

PPAR-

D6D D6DPPAR PPAR

Stimulation des mécanismes de désaturation

Adaptation à un régime carencé en HUFA n-3

R2 R1R2 R10,3 0,5

HUFA = AGLPI (n-9, n-6 et n-3) = Acides gras insaturés à 20-22 atomes de

carbone et au moins 4 doubles liaisons = Acides gras essentiels

C:X n-Y

g

Constituants principaux des PL de la bi-couche membranaire

O

OH -Acide docosahéxaénoique 22:6n-3 (DHA)

Chez les poissons, majoritairement les HUFA n-3:

1

2

3

45

6

-Acide écosapentaénoique 20:5n-3 (EPA)

Acide arachidonique 20:4n-6 (AA)

6

• Croissance et survie larvaire

• Développement de la vision et du cerveau

• Résistance au stress et aux maladies

• Qualité de la chair, …

PPARs

III- Hypothesis concerning the regulation of lipid metabolism in sea bass

XLH1 LH1 LH HH1HH XH10.5% 0.7% 0.8% 1.7% 2.2% 3.7%

stag

e

5 D6D

+ +

D6D

-

D6D

Larv

al s

d-4

PPAR+

PPAR-

PPAR-

tage

of

nge

++ -

uven

ile s

tTh

e en

d he

cha

lle

D6DD6D

+

D6D

-

J t

PPAR+

PPAR-

PPAR

Étude des PPAR

III- Hypothèses de régulation du métabolisme lipidique du bar

P i P lif t A ti t d R t é t lé iPeroxisome Proliferator-Activated Receptor, récepteurs nucléaires

Trois isoformes: α, β et γ

Décrits chez les mammifères et le bar

Facteur de transcription de la delta 6 chez les mammifères

Dans le contexte de programmation métabolique, les PPAR sont-ils impliqués dans la stimulation de la transcription du gène de la D6D chez le bar?

III- Hypothèses de régulation du métabolisme lipidique du bar

PPAR

D6D+

PPAR

D6D+

Mise en place d’une mémorisation au niveau moléculaire des pmécanismes de désaturation des AG

IV- Conclusions & Perspectives

1. General conclusion

1 Nutritional environment

2

POSSIBLE to influence the fatty acid desaturation pathways for

Nutritional conditionning acid desaturation pathways for

HUFA synthesis for a better incorporation of vegetable meals

conditionning during larval

stagePreviously gained

mechanisms

Fish lipid composition3

0 5 < HUFA di < 0 7

PPAR+

D6D transcription

0.5 < HUFA dietary content < 0.7

0,80,5 0,7 1,7Teneur en EPA+DHA du régime (%MS)

AAA

Inhibition

2,2 3,7

Stimulation des mécanismes de désaturation

-

HUFAHUFA HUFA

HUFA

HUFAHUFA

-

HUFAHUFA HUFA

HUFA

HUFAHUFA

HUFAHUFA HUFA

HUFA

HUFAHUFA

HUFAHUFA

+

HUFAHUFA

HUFAHUFA

HUFA

+

e

Gène de la delta 6

PPAR? ?

de la

rvai

re

-++

Stad

Traduction de l’ARNm en protéine

-+ +Transcription du gène de la Δ6-D

Synthèse d’AG précurseurs des HUFA-+ +

Mise en place d’une programmation métabolique par le conditionnement larvaire

Présence de cette programmation chez les juvéniles

Facteurs additionnels: teneur en HUFA n-3 de l’environnement nutritionnel

Teneur en EPA+DHA du régime (%MS)R2 = 0 3% R1 = 0 5% 0 7g ( )R2 = 0,3% R1 = 0,5% 0.7

Contrôle des mécanismes de désaturation préalablement acquis

e ju

véni

le +

Ni ARN D6 t it i

Transcription du gène de la D6D

-Début du challenge Fin du challenge+

Stad

e

D6 non fonctionnelle ?

D6 fonctionnelle ?

Niveau ARNm D6 continue Niveau ARNm D6 transitoire

+ -

dérivées

-Synthèse d’HUFA

+ -dérivées

teneur en DHA Faible en DHA

+ -