Post on 06-Apr-2018
transcript
Nutrition and reproduction of the pacific oyster Crassostrea gigas : main results of the European project GIGANUGA (Gigas Nutrition and Gametogenesis)
JF. Samain1, P.Sorgeloos4, M.Caers4, C. Van Ryckeghem4, P.Soudant4, O.Garcia5, J.Espinosa5, Y.Marty2, E.Palacios1, M.Mathieu3, C.Berthelin3, C.Quere1, J.R.Le Coz1, C.Seguineau1, J.Moal1
1IFREMER Brest, 2UBO-CNRS Brest, 3University of Caen : France4ARC Gent University : Belgium5University of Santiago de Compostela : Spain
Project FAIR CT.96.1852 (France, Belgium, spain)
5 aspects were studied :
– Cellular reproductive and storage cycles– Biochemical level : compare natural reproductive
cycle and hatchery conditioning at two seasons– Define deficiencies and problems– Evaluate possibilities to supplement hatchery
conditioning standard diet or low cost diet using emulsions or other particules
– Evaluate possibility to solve the question of fall conditioning
GIGANUGA 1:
CELLULAR ASPECTS OF REPRODUCTIVE AND STORAGE TISSUE CYCLES OF CRASSOSTREA GIGAS.
C. Heude Berthelin (1), J. Espinosa (2), O. Garcia(2), G. Hernandez (2),
K. Kellner (1) and M. Mathieu (1)
1 IBBA ,Biologie et Biotechnologies Marines, Université de Caen, 14032 Caen, France2 Facultad de Farmacia, Universidad de Santiago de Compostela, Espana
Course of the oyster gametogenesis
M onth J F M A M J J A S O N DGonad I/II II II II IIIA II/IIIA IIIA IIIB IIID IIID/0 0/I I
Cellular aspects of storage tissue development and mobilisation
Sstage Age A
Stage B Stage C Stage D
Month J F M A M J J A S O N D
Storage B/C C C C D D D D A B B B
Gonad I/II II II II IIIA IIIA IIIA IIIB IIID IIID/0 0/I I
GIGANUGA 2:
Nutrient storage and transfer during the reproductive cycle in nature and in hatchery
of the Pacific Oyster Crassostrea gigas
Soudant P.(1), M. Caers(2), Y.Marty(3), E.Palacios, C.VanRyckeghem(2), C. Heude Berthelin(4), JR Le Coz(1), C.Quere(1),
C.Seguineau(1), J.Moal(1), JF.Samain(1)
1 IFREMER Brest, Physiologie des Invertébrés, BP 70, 29280 Plouzané, France;2 ARC, Laboratory of Aquaculture , University of Gent, Rozier 44, 9000 Gent, Belgium3UMR/CNRS 6521, Université de Bretagne Occidentale, BP 809, 29285 Brest, France
4 IBBA ,Biologie et Biotechnologies Marines, Université de Caen, 14032 Caen, France
1/ Natural cycle : lipidsTotal lipid percentage
Natural cycle
0
5
10
15
20
25
20-Jan 11-Mar 30-Apr 19-Jun 8-Aug 27-Sep 16-Nov 5-Jan 24-Feb
Sampling date
% o
f the
DW
Gonad+mantleDigestive glandMuscle
1/ Natural cycle : sterols 97-98STERYL ESTERS
0
50
100
150
200
250
janv févr mars avr mai juin juil août sept oct nov déc janv févr mars
µg o
f ste
ryl e
ster
s/10
0 m
g of
dry
wei
ght
Glycogen metabolism in vesicular cell during gametogenesis
0
5
10
15
20
25
30
35
40
J F M A M J J A S O N D
Glycogène (% du PS)
0,00
2,00
4,00
6,00
8,00
10,00
12,00
Glucose incorporé (nmol)
1/ Natural cycle : thiamine andriboflavin
RIBOFLAVIN
02468
101214161820
20/01 11/03 30/04 19/06 08/08 27/09 16/11 05/01
µg /
g dr
y w
eigh
t
stade 1 3 4 1stade 2
Gonade + mantledigestive gland
2/ Deficiencies : comparisonNature-Hatchery: Spring period
PUFAs Steryl Esters
COMPARISON NATURE / HATCHERY
5
1 0
1 5
2 0
2 5
3 0
3 5
4 0
4 5
2 0 - a v r 2 0 - m a i 1 9 - ju i n 1 9 - ju i l
% of
ste
ryl e
ster
s in
tota
l ste
rols
Comparison natural / hatchery
0
5
10
15
20
25
20-Apr 10-May 30-May 19-Jun 9-Jul
Sampling date or larvae stage
% o
f the
tota
l fat
ty a
cids
20:5(n-3) in NL20:5(n-3) in PL
•2/ Relation between diet qualityand gonad composition : PUFAs
Information from gonad neutral lipids (fatty acids)
Neutral lipids from female gonads
y = 0.8373x + 1.9856R 2 = 0.8162
05
1015202530
0 10 20 30% AG Algues
% A
G L
NG
onad
s
2/ Relation between diet quality and gonad composition : sterols
STERYL ESTERS
y = 0.27x + 0.57R2 = 0.76
0
0.2
0.4
0.6
0.8
1
0 0.2 0.4 0.6 0.8 1 1.2Arc sin % sterols in diet
Arc
sin
% s
tero
ls in
Gon
ad
Brassicasterol24Methylene cholesterolCampesterol
Cholesterol
Giganuga 3: Advances in liposoluble and hydrosolublecompound supplementation for broodstockand larvae of the Pacific oyster Crassostreagigas
C.Seguineau(1), M.Cansell(2), P.Soudant(1), C.Langdon(3), B.PoncePinon(4), M.Val Sanles(4), J.R.Le Coz(1), C.Quere(1), J.Moal(1), M.Caers(5), P.Coutteau(6), P.Sorgeloos(5), J.F.Samain(1)
1 IFREMER Brest, Physiologie des Invertébrés, BP 70, 29280 Plouzané, France;2 ISTAB, Université Bordeaux I, avenue des Facultés, 33405 Talence, France3 Hatfield Marine Science Center, 2030S Marine Science Drive, Newport, OR 973654 Facultad de Farmacia, Universidad de Santiago de Compostela, Espana5 ARC, Laboratory of Aquavculture, University of Gent, Rozier44, 9000Gent, Belgium6 INVE Technologies NV Oeverstraat7-9200 Baasrode, Belgium
EMULSION AND ALGAL SIZE
0
5
10
15
20
25
30
35
40
0 10 20 30
diameter µm
% v
olum
e
microalgal mixture
emulsion
EFFECT OF EMULSION CONCENTRATION ON ALGAL FILTRATION
algae
algae + emulsion
No algal filtration pertubation for emulsion concentration < 1.2 mg/L
emulsion 1.2mg/L
y = -0,0051x + 1,5535
y = -0,004x + 1,5249
1,21,31,31,41,41,51,51,6
0 20 40 60minutes
log
DW
(mg)
EMULSION ASSIMILATION EXPERIMENT ON SPAT
Control
0,1
0719330
2
4
6
µg/in
divi
dual
Days
Stigmasterol
Stigmasterolassimilation wasdose and timedependent
Emulsion: 0%, 3%, 10%, 20%
2/ Emulsion supplementation : absorption efficiency was lowAbsorption rate of sterols
010
20
30
4050
6070
80
90
100
Control 3% 10% 20%Treatments
Inco
rpor
atio
n ra
te (%
)
CholesterolStigmasterol
010
20
30
4050
6070
80
90
100
Control 3% 10% 20%Treatments
Inco
rpor
atio
n ra
te (%
) BrassicasterolCampesterol
From microalgae From emulsion
SPRAY BEADS
Incorporated materialsdissolved in an aqueous phasedry particules
within a triglyceridebead composed of:60% tripalmitin40% fish oil
Riboflavin crystal
Lipid matrix
GIGANUGA 4: EFFECTS OF LIPID SUPPLEMENTATION DURING CONDITIONING OF Crassostrea gigas FED A STANDARD MIXED ALGAL DIET AND A LOW COST ALGAL DIET
M.Caers*1, P.Soudant, E.Palacios, K. Curé*1, P.Sorgeloos1, J.R.Le Coz2, C.Quere2, Y.Marty5, O. Garcia3, J.Espinoza3, C. Heude Berthelin4, E.Danton6, B.Diss6, J.F.Samain2, J.Moal2
3/ Spring : effect of emulsiondoses on biochemistry
– No effect at 2.5, 5.0, 10% emulsion FA/DW algae (Hatchery diet)
– Positive effect at 20 and 40% emulsion and Skeletonema (Low cost diet)
DHA/EPA in Polar Lipids
0
0,2
0,4
0,6
0,8
1
Natural Skeleto Skeleto + 40% Emulsion
Conditioning diet
DHA/
EPA
3/ Spring : biological effectNo biological effect at less than 10%Some compensatory effect at 20 and 40%, but
survival and fecundity were affected depending on rearing systems
Open circulating system
0
10
20
30
40
50
% larvae-D % abnormality
Ske +T-isoSkeSke + 40%
Enclosed recirculating system
0
10
20
30
40
50
60
% larvae-D % abnormality
Ske +T-isoSkeSke + 40%
gigas in fall, a combination of physical and nutritional factors?
◆ J.F. Samain, M. Mathieu, C.Heude Berthelin, C.Cure, J. Espinosa, P. Soudant, E.Palacios, J.R.Le Coz, C.Quéré, J. Moal
Perspectives for a better reproduction process in hatcheries
3/ Hatchery conditioning in spring/ fall :
In spring hatchery conditioning was normalIn fall, hatchery conditioning after spawning
was impossible– No effect of the increase in temperature and
food
Problems in fall conditioning:
Gonad dry weight 1997-98
0
0,5
1
1,5
2
2,5
01 02 03 04 05 06 07 08 09 10 11 12 01 02 03
months
g / o
rgan
NatureFall 97
Conditioning in fall did not allow storage restoration
Problems in fall conditioning:
Fall 97 conditioning
0%
20%
40%
60%
80%
100%
start (sept) end (jan)
mitosis
no gametogenesisfrequ
ency
Histology : no gametogenesis was observed (C stage).
Availability of oogonia ?Annual cycle
0,0
5,0
10,0
15,0
20,0
25,0 T°C
Photophase
Mitosis
J F M A M J J A S O N D
E1 E2 E2 E2 E3A1 E3A1 E3A2 E3A3 C C E0 E1
Hypothese : all oogonia weremobilized for the main spawning during summer.
Are temperature and low photoperiod necessary for reinitiation of the sexualcycle?
Protocol :Preconditioning : 4 weeks at 9°C,
decreasing photoperiod. Algal mixture.Then, conditioning at 19°C and high
photoperiod. Algal mixture +/- emulsion
0
5
1 0
1 5
2 0
2 5
2 4 /0 7 /9 8 1 2 /0 9 /9 8 0 1 /1 1 /9 8 2 1 /1 2 /9 8 0 9 /0 2 /9 9
tem
pera
ture
C o n t r o l
c o n t r o l w ith o u tp r e c o n d it io n in g
Preconditioning conditioning
1/ Preconditioning on gametogenesis0
5
10
15
20
25
24/07/98 12/09/98 01/11/98 21/12/98 09/02/99
tem
pera
ture
Control
control w ithoutpreconditioning
In fall, a decrease of temperature and photoperiod allowed the initiation of a new gametogenesis cycle
Gametogenesis
0
20
40
60
80
100
120
no preconditioning with preconditioning
mitosisno gametogenesis
1/ Preconditioning on storage
No storage tissue restorationNo gain in DW, lipid, and steryl esters.
0
5
10
15
20
25
24/07/98 12/09/98 01/11/98 21/12/98 09/02/99
tem
pera
ture
Control
control w ithoutpreconditioning
Storage tissue development
0,020,040,060,080,0
100,0120,0
no medium
nopreconditioning
w ithpreconditioning
Storage composition
02468
10121416
gonad DW % Lipid/DW % steryl ester
3/ Fall : Effect of supplementation on gonad biochemistry and weight
Emulsion increased vitellogenesis (biochemical criteria)However, weak gonad development
Gonad composition
05
10152025
lipid/DW % EPA inPL
% sterylester
Algae5% emulsion10% emulsion
Gonad development
00,5
11,5
22,5
gonad DW
Algae5% emulsion10% emulsion
spring reference
3/Fall : effect of supplementationon gametogenesis
0
20
40
60
80
100
indifferenciated differenciated
no precondAlgae5% EM3010% EM30
emulsion accelerated vitellogenesis rate
Temperature and photoperiod
FoodNo storage-?
High
No Mitosis-?
No gametogenesis
Temperature and photoperiod No storage Food
1/Low
Mitosis+?
Oogonia
Mitosis
Oogonia
1/Low 2/High
+?
+?
Temperature and photoperiod
2/FoodSmall storage
Quantity :vesicular cell
number
Fecundity?+?
Oocytes
Fecundity?+?
Mitosis
Oogonia
Low High
+?
+?
Temperature and photoperiod Food
Small storageQuantity :vesicular cell
number
Oocytes
Quality :EPA, DHA,
AA, Vitamines...Emulsion
Vitellogenesisrate?
Conclusions...
The reproductive cycle of C.gigas is well characterized in Marennes Oleron on cellular and biochemical aspects.
Gonial mitosis should be re-initiated at fall by external factors as temperature-photoperiod. Regulations are understudy.
But storage control is not yet documented
...Conclusions ...Deficiencies in essential nutriments can be
evidenced comparing natural and hatchery processes : PUFAs, Sterols, Vitamins
Promising results were obtained using emulsions :» At spring, deficiencies of monospecific algal
diet can be compensated, but problems of doses and nutriments should be solved
» At fall, emulsion stimulates vitellogenesisrate, but control of storage is not yet solved
...Conclusions :Other particules as spraybeads or liposomes
encapsulating hydrosoluble compounds are understudy (vitamins).
These techniques are promising for a better understanding of essential nutriments for reproduction of bivalves all over the year, and using a low cost algal diet.
But other non nutritional factors affecting storage, gonial mitosis should be investigated and needmore cooperative researches.
Contributions :
» Experiments : IFREMER Brest and La Tremblade» Histology : J.Espinosa, O.Garcia, Univ. Santiago de
Compostella, Espagne, M.Mathieu IBBA Caen» Biochemistry of essential nutriments : IFREMER Brest,
Y.Marty, UMR CNRS 6521 UBO Brest, P.Sorgeloos, P.Coutteau ARC Gent, Belgique
» Glycogen metabolism : M.Mathieu et al. Univ.Caen» Supplementation technics : P.Sorgeloos, P.Coutteau ARC
Gent, Belgique, M.Cansell, Univ.Bordeaux, France, C.Langdon, Oregon Univ. USA
» Hatcheries : SATMAR, Guernesey Sea Farm