Diapositiva 1 - UGent 4 - Wednesday... · Benítez-Santana et al., 2007 Sargent et al., 1995...

larvi 2013

ghent university, belgium, 2-5 september 2013

6th fish & shellfish larviculture symposium

Oxidative stress in sea bass(Dicentrarchus labrax) larvae

interaction of high dietary DHA contentsand several antioxidant nutrients

Monica Betancor

Oxidative stress in sea bass (Dicentrarchus labrax)larvae fed on high DHA microdiets. Involvement of

several antioxidant nutrients

Mónica B Betancor*Mª José CaballeroMarisol IzquierdoUniversity of Las Palmas de GC,

Canary Islands, Spain*Author present address

Carbohydrates<2%

Protein15-20%

Lipids1-20%

Carriers

PLs

Energy

Hormones

EPA20:5n-3

ARA20:4n-6

DHA22:6n-3

Growth & stressresistance

Watanabe & Kiron, 1994

Membrane functionIzquierdo & Koven, 2010

Selectively retainedRainuzzo et al., 1993

LC-PUFA

Fernández-Palacios et al., 2011

Decrease in DHA 10 dphWatanabe, 1993

Species Larvae DHArequirements (%)

Larvae DHA content(% total fatty acid)

Authors (Requirements)

Sparus aurata >3 30.1± 0.4 Izquierdo, 2005

Scophtalmusmaximus

3.2 27.0 ± 0.3 Le Milinaire, 1984

Seriola dumerilii 4 - Izquierdo, 2005

Pagrus pagrus 3.4 26.9 Hernández et al., 1999

Dentex dentex 4 27.6 Mourente et al., 1999

Mourente et al., 2003;Benítez-Santana et al., 2007

Sargent et al., 1995 Izquierdo, 2005

Laurel et al., 2010

ROS

ROS

ROS

Increased susceptibility to

Nagaoka et al., 1990

What is the effect of high DHA dietarylevels on sea bass larvae?

Experimental conditions:

- 170 L grey cylinder fibre glass tanks

-Photoperiod 12:12

-Sea bass larvae

-Temperature 19.5-20°C

-Oxygen 5-8 g L-1

-Salinity 34 g L-1

-Manually cleaned-Water flow 1.0-1.5 L min-1

Diets and Experimental Design

DHA (g/100g)

α-TO

H(m

g/10

0g)

1 3 5

150

300

1/150

1/300 3/300 5/300

3/150 5/150

-Five experiments-Two, three or five weeks

Betancor et al., 2011. Aquacult NutrBetancor et al., 2011. Aquacult Nutr

12,5

13,0

13,5

14,0

14,5

15,0

1/150 3/150 5/150

mm

Dietary DHA/vitamin E

d

bc

cd

Total length

High DHA

Reduced growth and survival

75

80

85

90

95

100

1/150 3/150 5/150

%


c

bc

ab

Final survival

Muscular dystrophyLovell et al., 1984; Gatlin et al., 1986;Frischknecth et al., 1994; Bowater &

Burren, 2007; Lebold et al., 2013

Fibre swelling Eosinophilic and pale cytoplasm

Muscular debris+InfiltrateFibre breakage Monocyte infiltrate

Granular cytoplasm

2,00E+03

4,00E+03

6,00E+03

8,00E+03

1,00E+04

1,20E+04

14 26 35

IGF-

I mRN

A co

py n

º/ng

tota

l RN

A

1/150 5/150

b

a

b

a

IGF-I

Regenerating fibresRowlerson et al., 1997Regenerating fibresRowlerson et al., 1997Regenerating fibresRowlerson et al., 1997

Satellite cell pool controlGrounds ,1999; Seale & Rudnicki, 2000



Muscle regeneration processMuscle regeneration processMuscle regeneration process

5,00E+06

1,00E+07

1,50E+07

2,00E+07

2,50E+07

3,00E+07

3,50E+07

4,00E+07

14 26 35MyH

C m

RNA

copy

nº/

ng to

tal R

NA

1/150 5/150

b

a

b

a

MyHC

↑ LESIONS

Betancor et al., 2013. CBPABetancor et al., 2013. CBPA

α-actin

5,00E+07

6,00E+07

7,00E+07

8,00E+07

9,00E+07

1,00E+08

1,10E+08

1,20E+08

14 19 24 29 34

1/150 5/150

1,40E+05

1,60E+05

1,80E+05

2,00E+05

2,20E+05

2,40E+05

2,60E+05

2,80E+05

3,00E+05

14 19 24 29 34

1/150 5/150

ba

Capn1

↑ ROS

Membrane lesion

↑ Permeability

↑ Ca2+ ↑ Na+↑ H2O

Cellularoedema

Mitochondriaoedema

MPT

Cytochrome c

ApoptosisNecrosis

↑ Calpains

↑ Proteolysis

Cytoskeleton lesion

SR oedema

Several α-actin isoformsWatabe, 2001

Betancor et al., 2013. CBPABetancor et al., 2013. CBPA

THIN

SEC

TIO

NS

*

*

***

VacuolesDilatation SR

*

Hydropic degenerationCotran et al., 2004

Failure of ion pumps

Na+, Ca2+ and water influx

What is the effect of high DHA dietarylevels on sea bass larvae?

1. High DHA (5%) microdiets lead to reduced growth and survival andfavoured the appearance of muscular lesions in sea bass larvae

2. One of the first alterations takes place at the cell membrane,probably due to the direct attack of ROS to phospholipids, causingalteration of its permeability and leading to cell and organellesswelling. Massive influx of calcium occurs and a consequentactivation of µ-calpains causes protein lysis

3. A compensatory muscle response was found, as shown by theincrease in insulin-like growth factors and myosin heavy chain geneexpression, as well as the abundant activated satellite cells

Is there an in vivo oxidation?? Could it beprevented with increased levels of vitamin E?

0

5

10

15

20

25

30

35

1/150 1/300 3/150 3/300 5/150

%

Dietary DHA/vitaminE

Incidence of muscular lesions

ab

b

ab

ab

a

Vitamin E increase can protect seabass larvae from increased DHAcontents

Betancor et al., 2011. Aquacult NutrBetancor et al., 2011. Aquacult Nutr

12,0

12,5

13,0

13,5

14,0

14,5

15,0

15,5

16,0

1/150 1/300 3/150 3/300 5/150

Tota

llen

gth

mm


a

d

bcb

cd

Total length

0

5

10

15

20

25

30

1/150 5/150 5/300

%

TBARS content Incidence of muscular lesions at 35 dph

0

100

200

300

400

500

600

700

Initial 1/150 5/150 5/300

nMM

DA/t

issu

es

Betancor et al., 2012. J Fish DisBetancor et al., 2012. J Fish Dis

High DHA+High VitE↓

Increased oxidation

b

a a

aa

Cell ExteriorCell Exterior

Cell InteriorCell InteriorO2

H2O

H2O

+OH-Fenton

Fe+2

Fe+3

GSSG

2GSH

O2-· H2O2

O2-·

OH·+

CATSOD

GPXGR

= ROS

= AOE

α-TOH α-TOHα-TOH α-TOH

α-TOH α-TOH α-TOH α-TOH

Which other nutrients could protectsea bass larvae when high levels of DHA

are used in their diets?

Active sites of the GPX

Felton et al., 1996

Sparing effect between α-TOH and Se

Lin & Shiau, 2009

Synergistic action between α-TOH and Se

Poston et al., 1976; Bell & Cowey, 1985

Involved in thyroid hormone and insulinfunction, maintenance of fertility andregulation of cell growth

Lall, 2002; Kohlmeier, 2003

0

500

1000

1500

2000

2500

3000

Initial 1/150 5/300 5/300+Se

nMol

/gdr

ym

ass

TBARS

a

cb

0

500

1000

1500

2000

2500

3000

3500

Diet Larvae

µg/g

dry

mas

s

Initial1/1505/3005/300+Se

Vitamin E content

AntioxidantAntioxidant protection

No dose dependant effectKiron et al., 2004

↓ Tissue vitamin EPuangkaew et al., 2005

Betancor et al., 2012. Br J NutrBetancor et al., 2012. Br J Nutr

0

10

20

30

40

50

60

70

1/150 5/300 5/300+Se

%


**

*

* *

*

ab

a

b

**

↓ Se accumulation in muscleMonteiro et al., 2009; Elia et al., 2011

0

1

2

3

4

5

6

7

Diet Larvae

µg/m

gdr

ym

ass

Initial 1/150 5/300 5/300+Se

Selenium content

a

cb

35.88%

83.46%

100%

Antioxidant

↓ Tissue concentration

Low retention rate – GPX??


2,00E+05

2,50E+05

3,00E+05

3,50E+05

4,00E+05

4,50E+05

5,00E+05

5,50E+05

6,00E+05

6,50E+05

14 26 35

GPX

mRN

A co

py n

º/ng

tota

l RN

A

Days post hatch

1/150 5/300 5/300+Se

ab

a

b

a

b

GPX

Antioxidant defence(Burk et al., 1997)

17 selenocysteine residues(Tubajeva et al., 2000)

Largely used as an antioxidant

Saturated at normal nutritionalintakes


RO RO+Se DHA DHA+Se

DHA (% total fatty acids) 0.7 0.2 19.0 18.5

Se (ppm) 1.7 7.0 1.3 6.3

Two ages7 months 20 months

2 months trial

Glutathione peroxidases

Thyroid Hormone Deiodinases

Thioredoxin reductases

Selenoproteins

Selenocysteine biosynthesis

GPX3 GPX4b

ID2

TR1

SEPP1 SEPW 15 KDa

SPS2 SEC43

0

0,1

0,2

0,3

0,4

0,5

0,6

0,7

RO RO+Se DHA DHA+Se

mg/

kg

Selenium content

Old Young

a a

a

b

b

bb

c

0

500

1000

1500

2000

2500

3000

3500

RO RO+Se DHA DHA+Se

nMol

MDA

/g li

pid

TBARS

Old Young

a

b

b

b

Young fish more sensitive to oxidative stress

Old Young

Thyr

oid

Horm

one

Deio

dina

ses

0

0,2

0,4

0,6

0,8

1

1,2

1,4

1,6

1,8

2

RO RO+Se DHA DHA+Se

Rela

tive

expr

essi

on

TR1

Old Young

*

*

aaab

b

0

0,2

0,4

0,6

0,8

1

1,2

1,4

1,6

RO RO+Se DHA DHA+Se

Rela

tive

expr

essi

on

DI2

Old Young

*

a

bab

b

Thio

redo

xin

redu

ctas

es Hierarchichal regulation of selenoprotein expression(Schomburg and Schweizer, 2009)

Reduced mRNA copy no.(Benner et al., 2010)

Sele

nopr

otei

ns

Old Young

0

0,2

0,4

0,6

0,8

1

1,2

RO RO+Se DHA DHA+Se

Sep W

Old Young

a a

b b

0

0,2

0,4

0,6

0,8

1

1,2

1,4

RO RO+Se DHA DHA+Se

SPS2

Old Young

Sele

noph

osph

ate

synt

heta

se aab ab

b

Low levels→WMDSelenide+ATP

↓SPS2Selenophosphate

Protects low density LP

Levine, 1986

Recycling of α-TOH

Hamre et al., 1997; Sealey & Gatlin, 2002

Sparing effect between α-TOH and AA

Shiau & Shu, 2002; Yildirim-Aksoy et al., 2008

0

500

1000

1500

2000

2500

3000nM

ol/g

dry

mas

sTBARS

0

500

1000

1500

2000

2500

3000

3500

Diet Larvae

µg/g

dry

mas

sInitial 1/150 5/300 5/300+AA

c

a

b

ab ab

Vitamin E content

Vitamin E sparing/recyclingSealey & Gatlin, 2002; Shiau & Shu, 2002

α-tocopherol depletionHamre & Lie, 1995; Kolkovski et al., 2000

NADP NADPH

O

O·

O

OH

Asc-H

NADHNAD

Asc· Asc-H+DHA

2 Asc·

GSSGGSH

Water phase

Lipid phase

Phospholipidwith PUFAs



Tocopheroxylradical

α-tocopherol

Tappel, 1962Adapted from Hamre, 2011

0

10

20

30

40

50

60

70

1/150 5/300 5/300+AA

%


b

a

b

SR

*

*

*

Inclusion of both organic selenium (5 mg kg-1) and ascorbic acid(180 mg 100 g-1 ) to diets with a 5% of DHA and 300 mg 100 g-1 ofvitamin E controlled in vivo lipid peroxidation and decreased theincidence of muscular lesions when compared to theunsupplemented diets

Which other nutrients could protect seabass larvae when high levels of DHA are

used in their diets?In zebrafish muscle TR1, ID2, SEPW and SPS2 expression is affectedby the addition of 7 ppm of Se when high DHA diets are used,indicating that these could be good biomarkers of the oxidativestatus in teleost fish

Thanks for your attention

Mónica B BetancorMª José CaballeroMarisol IzquierdoUniversity of Las Palmas de GC,

Canary Islands, Spain

Diapositiva 1 - UGent 4 - Wednesday... · Benítez-Santana et al., 2007 Sargent et al., 1995...

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