larvi 2013
ghent university, belgium, 2-5 september 2013
6th fish & shellfish larviculture symposium
The long term effects of DHA supplementationin live feed for first feeding pike perch larvae
on development of neural tissueand behavioral responses
Ivar Lund
Larvi – Ghent2-6 Sept. 2013
Ivar LundErik HøglundPeter V. Skov
Importance of DHA for first feeding pike perch(Sander Lucioperca) larvae– Influence on behavioural responses
Challenges in production of pike perch fry
• Cannibalism• Quality of fry, and survival rate• High stress sensitivity
Causes• Lack of breeding programme• Environmental and nutritional issues
• Optimal nutritional requirements not known(lipids, fatty acids, amino acids, vitamins etc. )
Influence of DHA on stress andneural development in pikeperch larvae
The developing brain may be vulnerable to DHAdeficiency.Neurologic processing,Synapse formation, Neurite outgrowth,Myelination, Neurotransmitter secretionNeurological function.
Low brain DHA levelsduring development can be restored to normal bysubsequent n-3fatty acid supplementation, but will physiologicalfunctions be affected ?
time (min)
0 20 40 60 80 100 120 140
averag
e cum
ulative
mort
ality (
%) at
24 dp
h (19
ppt sa
linity)
0
20
40
60
80
100
OAOA+ 40 DHA
time (min)
0 20 40 60 80 100 120 140
Juve
nile c
umula
tive m
ortal
ity (2
1 ppt
salin
ity)
0
10
20
30
40
50
60
OAOA+ 40 DHA
Selco v OAvideo.mp4
A B C D
com
posi
tion
of e
mul
sion
s (g
kg-1
)
0
200
400
600
800
1000
1200
Refined OODHA oilPhospholipidLecithinE vitamin mix
Experimental set up:
OO: refined olive oil (0.79 kg 18:1 kg oil-1)DHA oil: INCROMEGA, CRODA, (0.51 kg DHA Kg oil-1)Phospholipid oil (PL): fish oil rich in phospholipids (0.44 kg phospholipid kg oil-1)
Larvae:
Physiological implications of early larvae FA nutrition:Short & long term consequences on wellfare, cognitive behaviour;stress
juvenile
Behaviour of larvae (28-30 dph) in presence and absenceof a simulated predator
Fast-escape response (avoidance behaviour) of larvae (28 dph)and juveniles (+120 dph) to a mechanosensory stimulus
Long terms effects on learning ability and stress responsivenessby maze tests and cortisol analyses
Experimental set up:
Experimental set up:
Behaviour of larvae in absence and presence of a simulatedpredator
Fast/ escape (avoidance) test for larvae & juveniles
Learning ability – maze test
Larvae FA composition 27 dph:
Dietary inclusion: A: 890 g OO B: 840 g OO50 g DHA oil
C: 390 g OO500 g DHA oil
D: 390 g OO500 g PL.
Larvae FA comp (%total) (27 dph):
16:018:0SFA16:1 n-718:1 n-9MUFA18:3 n-620:4 n-6n-6 PUFA18:3 n-320:5 n-322:6 n-3n-3 PUFADHA/EPAn-3 /n-6
16.9±0.48.3±0.326.61.6±0.134.1±1.4d
38.6d
0.4±0.00.9±0.1a
7.918.6±0.32.6±0.2a
3.5±0.6a
26.1a
1.43.3
17.0±0.38.6±0.327.21.5±0.131.9±1.5c
36.4c
0.4±0.01.0±0.1a
7.518.9±0.53.3±0.3a
4.6±0.1b
28.1b
1.43.7
15.9±1.08.9±0.326.61.3±0.124.0±1.1a
29.6a
0.3±0.01.5±0.1b
6.719.2±0.75.4±0.3b
10.3±1.3c
36.2d
1.9b
5.4
15.9±0.47.8±0.325.52.1±0.428.1±1.0b
34.1b
0.3±0.01.0±0.1a
6.417.2±0.36.4±0.4c
8.5±1.4c
33.3c
1.35.2
Larvae growth until 27 Dph
dietary code
A: OO B: +5% DHA C: +50 % DHA D: +50 % PL
larva
e weig
ht (m
g dw
-1 )
0
1
2
3
4
Time at edge
A B C D
%
0
20
40
60
80
100
120
Restpredator stressor
Time spent in lower half
A B C D
%
0
20
40
60
80
100
Routine swimming speed
Diet
A B C D
BL
sec-1
0,0
0,5
1,0
1,5
2,0
2,5
3,0
Time in rheotaxis
A B C D
%
0
20
40
60
80
100
a a
bb
significant interactionbetween Diet andStress. (P = 0,029)
C
b
a a
Stress vs no stressP = 0,020
Larvae behaviour in absence and presence of a simulated predator
c
b
aa
A B C D
Esc
ape
late
ncy
(ms)
0
10
20
30
40
A B C D
Pea
k ve
loci
ty (b
l/s)
0
10
20
30
40
50
A B C D
Dis
tanc
e co
vere
d (b
l)
0,0
0,5
1,0
1,5
2,0
2,5
A B C D
Pea
k ac
cele
ratio
n (m
s-2
)
0
20
40
60
80
100
Larvae (avoidance) escape response
Annova, P=0.1192 Annova, P=0.399
Annova, P=0.656 Annova, P=0.001
bb
ab
a
dietary code
A B C D
Dis
tanc
e co
vere
d (b
l in
80 m
s)
0,0
0,1
0,2
0,3
0,4
dietary code
A B C D
Esc
ape
late
ncy
(ms)
0
5
10
15
20
25
30 Annova, P=0.093
dietary code
A B C D
Peak
vel
ocity
(bl s
-1)
0
2
4
6
8
10
12
14
dietary code
A B C D
peak
acc
eler
atio
n m
s-2
0
200
400
600
800
1000
1200
1400Annova, P=0.107
Annova, P=0.106
Annova, P=0.04
Juvenile (avoidance) escape response
a
b bb
Tim
eun
tilfir
stm
ovem
ent
(sec
)
Learning session
0
500
1000
1500
1 2 3 4 5 60
500
1000
1500
1 2 3 4 5 6To
tal t
ime
inm
aze
(sec
)
Emulsion: A: OOo C+D (50% DHA + PL)
Learning session
Two way repeated ANOVALearning session:P<0.001Fatty acid:P<0.20Fatty acid x Trail:0.55
Two way repeated ANOVALearninng session:P<0.001Fatty acid:P<0.05Fatty acid x Trail:0.56
Learning ability and stress responsiveness in maze tests
Emulsion: A: OOo C+D (50% DHA + PL)
Significance: (P=0.25).
0
5
10
15
20
25
30
35
40
45
Code A+B (OO; OO+5 % DH Code C+D (50%DHA & PL)
Cor
tiso
lcon
ten
tn
g/g
tiss
ue
Cortisol levels in juveniles after test in maze
Conclusions
Dietary DHA influences on larvae tissue composition and affectslarvae stress responsiveness and behavior during rest or to an acute stressor.
No apparent influence on growth or visual acuity.
Peak accelaration for larvae and juveniles were significantly decreased by lowdietary DHA concentrations.
- n should be increased in such studies as individual variation is high.
Juvenile learning ability in a maze was similar between groups, but ”inititalfreezing time” higher in groups fed low levels of DHA as larvae.
- How is stress and behavior linked to neurophysiology ?- a cross disciplinary approach including brain signalingsystems as well as behavior and metabolic physiology.
- future studies
- From an applied point of view, gene expression orprotein levels of BDNF (i.e neutrophic factor associatedwith learning) may be utilized as biological markers forstress coping ability and mental robustness in fish.
Aknowledgements
The study was made possible by an internal grant from DTU Aqua
Thanks to:
Aquapri for providing larvaeUniversity of Roskilde, Enspac for GC/MS analyses
