Essential Fatty Acid Requirements of Catfish (Clarias batrachus Linn.) for Broodstock Development

I.

INTRODUCTION

I n t e n s i v e aquacul t u r e r e q u i r e s adequate suppl y o f seed
the

whole

year

demonstrated
fecundity
( Pagrus

round.

that

the

and t h e
major),

Recently,
quality

of

some
diets

h a t c h i n g r a t e of

and

rainbow

experiments
could

eggs o f

affect

r e d sea

( Oncorhynchus

trout

Itoh,

Murakami, Tsukashima,

1984a ; Watanabe, Ohhashi,
Watanabe,

Arakawa,

Kitajima,

and

Takeuchi, S a i t o and Nishimura,

Fujita,

1984c;

1984d; Watanabe,

bream

1981;

and F u j i t a ,

I t o h , K i t a j i m a and F u j i t a ,

Kitajima

the

myk i s s )

(Takeuchi, Watanabe, Ogino, S a i t o , Nishimura and Nose,
Watanabe,

have

1984b;

Watanabe,

I t o h , Saito,

Satoh, K i t a j i m a and F u j i t a , 1985).
Hatching

rate of

eggs

produced

by

r e d sea

bream and

rainbow t r o u t f e d l i n o l e n i c a c i d d e f i c i e n t d i e t i s v e r y low
(Watanabe e t a7.

1984a,

1984b,

19844).

M o r p h o l o g i c a l study

o f d e v e l o p i n g embryo showed some cleavage d i s o r d e r a t t h e 1 6
t o 32 c e l l s t a g e , a b l o c k i n g e f f e c t b e f o r e g a s t r u l a t i o n ,
1a t e r

various

a1 t e r a t i ons

in

the

phase

( L e r a y , Nonnote, Roubaud and Leger,

1985).

of

organogenesi s

L i n o l e i c and l i n o l e n i c a c i d s a r e e s s e n t i a l
(EFA) f o r some f r e s h w a t e r f i s h .
i s known as € F A
d i e t containing
w i l l

for

Especial l y ,

rainbow t r o u t .

and

f a t t y acids

1i n o l e n i c a c i d

Rainbow t r o u t f e d upon

l i n o l e n i c acid only, without l i n o l e i c acid,

produce normal eggs and normal

larva.

Yu,

Sinnhuber

and Hendricks (1979) analyzed t h e f a t t y a c i d c o n t e n t o f t h e
eggs

and

was

found t h a t t h e

eggs

contained

arachidonic

acid too.
acid

Presumably rainbow t r o u t s t i l l

for

normal

egg

development,

Yu e t a ? . ( 1 9 7 9 )

amount.

requires l i n o l e i c

eventhough

concluded t h a t

in

a

linoleic

small

acid

in

t h e eggs was e i t h e r f r o m

incompletely extracted d i e t ingre-

d i e n t s such as

c a s e i n and g e l a t i n ,

dextrin,

1 i z e d f r o m t h e body o f
from

Rahn,

embryos
acid,

Sand

and

apparently

whereas

the broodfish.

Schlenk

required

only

(1977)

both

linolenic

acid

or

was mobi-

Another
showed

linolenic
is

experiment

that
and

gourami
linoleic

essential

to

older

fish.
To g a i n t h e maximum growth,

the juvenile

of

catfish

( C 7 a r i a s b a t r a c h u s L i n n . ) r e q u i r e s 1 . 5 % o f 1 i n o l e i c a c i d and

0 . 6 % l i n o l e n i c a c i d (Mokoginta,

1986).

But f o r

broodstock

development o f t h i s s p e c i e s t h e l i n o l e i c and l i n o l e n i c a c i d
r e q u i r e m e n t s have n o t y e t been determined.
S o t h e o b j e c t i v e s o f t h i s experiment a r e :
1.

t o determine t h e

l i n o l e i c and l i n o l e n i c a c i d r e q u i r e -

ments o f b r o o d f i s h , t h a t i s s u i t a b l e t o become an a d u l t
diet,

t o develop gonad m a t u r a t i o n ,

good q u a l i t y
2.

to

study

embryonic
Linn. ) .

the

and p r o d u c t i o n o f

eggs.
essential

development

of

fatty

acid

catfish

metabolism
( C 7arias

during

batrachus

I I.
Although
requirements
lacking,

LITERATURE REVIEW

precise
for

information

gonadal

maturation

on

the

of

the

nutritional
broodstock

i t has been agreed t h a t t h e q u a n t i t y ,

is

qual i t y and

f e e d i n g regiment would a f f e c t f i s h spawning and egg q u a l i t y .
Some experiments showed t h a t t h e q u a l i t y o f
d i e t c o u l d a f f e c t t h e qual i t y o f

t h e broodstock

t h e eggs produced by t h e

broodstock.
A

group o f

r e d sea bream broodstock

f e d -upon t h e

low

p r o t e i n d i e t (33.0%), o r f e d upon t h e d i e t w i t h o u t supplemental phosphorus has a lower number o f eggs compared t o t h e
broodstock fed

upon t h e c o n t r o l

w i t h supplemental

of

diet

phosphorus,

(45.0% p r o t e i n l e v e l

Watanabe et a 7 .

,

1984a).

Growth r a t e and feed e f f i c i e n c y o f t h e rainbow t r o u t broods t o c k f e d upon t h e d i e t c o n t a i n i n g 36.0% p r o t e i n ,

and 18.0%

1i p i d l e v e l was h i g h e r compared t o t h e growth r a t e and feed
efficiency

of

the

broodstock

fed

20.0% l i p i d l e v e l (Watanabe e t a l . ,

upon

28.0% p r o t e i n

1984d).

and

Another e x p e r i -

ment by Oahl green ( 1980) u s i n g guppy ( Poeci 7 i a r e t i c u l a t a )
broudstock

has found o u t t h a t i f t h e

f i s h were f e d upon

t h e d i e t c o n t a i n i n g 47.0% p r o t e i n l e v e l w i l l produce average
gonad0 somatic r a t i o h i g h e r than i f i t was f e d upon a d i e t
c o n t a i n i n g 31.0% and 15.0% p r o t e i n l e v e l .
Takeuchi

et

al.

(1981)

demonstrated

that

the

eggs

produced by rainbow t r o u t r e c e i v i n g a d i e t w i t h o u t supplemental o f t r a c e element were s i g n i f i c a n t l y low i n t h e per-

centage

of

eyed

eggs

as

well

as

the

hatchability.

The

e s s e n t i a l f a t t y a c i d (EFA) d e f i c i e n t

broodstock r e c e i v i n g

d i e t a l s o produced eggs w i t h s i g n i f i c a n t l y

low h a t c h a b i l i t y ,

and most o f t h e hatched l a r v a e showed d e f o r m i t y o f t h e body
as w e l l (Watanabe et a l . ,

1984a, d ) .
t h e d i e t s g i v e n t o t h e brood-

Nutritional quality o f
stock

would

eventhough
1984a).

influence the
on

the

verge

quality
of

of

r e d sea

spawning

bream eggs

(Watanabe

et

al.,

The f a t t y a c i d o f t h e eggs were g r e a t l y a f f e c t e d by

d i e t a r y f a t t y a c i d s u p p l i e d t o t h e broodstock.

The propor-

t i o n o f a 3 h i g h l y unsaturated f a t t y a c i d s ((#J3 HUFA) was h i g h
i n t h e eggs o f t h e broodstock f e d upon t h e d i e t c o n t a i n i n g a
h i g h l e v e l o f cJ3 HUFA,

and low i n t h e eggs o f t h e EFA-def i-

c i e n t f i s h (Watanabe et a l . ,
composition

in

the

organs

1984b;
like

1985).

liver,

The f a t t y a c i d

heart,

kidney and

b r a i n were a l s o i n f l u e n c e d by f a t t y a c i d composition o f t h e
diet

(Lee,

Andrews,

Roehm,

1971; C a s t e l l ,

and Buckley,

and

Sinnhuber,

composition

1967;

Lee and Sinnhuber,

1980; R e i n i t z and Yu,

s i t i o n of dietary
acid

Yu

1984).

Stickney

and

1972; C a s t l e d i n e
F a t t y a c i d compo-

l i p i d had a g r e a t e r e f f e c t on t h e f a t t y
of

phospholipid

than

on

the

fatty

acid

composition o f n e u t r a l 1i p i d .
The

precise

known y e t .
no

marked

metabolism o f

Watanabe
chemical

et a l .

EFA

i n the

embryo

is

not

(1985) showed t h a t t h e r e were

differences

between

(buoyant eggs)

and t h e abnormal

eggs

r e d sea bream.

I n t h e o t h e r experiment,

(

the

normal

eggs

deposited eggs)
Leray e t a l .

of

(1985)

studied the

incidence o f G 3 - f a t t y

t r o u t r e p r o d u c t i v e processes.

acid deficiency

on t h e

Eggs from broodstock f e d upon

E F A - d e f i c i e n t d i e t , has t h e same f e r t i l i z a t i o n e f f i c i e n c y i n
two days development compared t o t h e eggs from broodstock
f e d uQon a commercial d i e t .

After that,

two peaks o f mor-

t a l i t y , one a t day 8 and t h e o t h e r a t day 22 a f t e r f e r t i l i z a t i o n were observed from t h e eggs produced by t h e d e f i c i e n t
fish.

I n addition,

v i t e l l u s r e s o r p t i o n was s h o r t e r i n t h e

a l e v i n s from t h e EFA-deficient broodstock ( 5 0 days) than t h e
a l e v i n s from t h e broodstock f e d upon a commercial
d i e t ( 6 5 days).
the

control

Morphological study o f t h e development o f

c33 d e f i c i e n t embryo showed some cleavage d i s o r d e r a t 16

t o 3 2 c e l l stage, a b l o c k i n g e f f e c t b e f o r e g a s t r u l a t i o n , and
l a t e r v a r i o u s a l t e r a t i o n i n organogenesis.
that

t h e G=3 f a t t y

acids,

and e s p e c i a l l y

It was concluded
22:6 G3,

play

a

c r u c i a l and s p e c i f i c r o l e i n t h e t r o u t embryo development.
Former

experiments

demonstrated,

that

some

dietary

p h o s p h o l i p i d were necessary f o r good growth and s u r v i v a l o f
the l a r v a l f i s h ,

such as t h e r e d sea bream (Pagrus major),

t h e ayu ( PLecog 7ossus a 7 t i v e 1 i s ) ,

t h e s t a r y f l o u n d e r (Para-

7ychthys o 1ivaccus) and t h e k n i f e jaw

tus) (Teshima,
1987).

Kanazawa,

Horinouchi,

( Op legnathus f a s c i a -

Yamasaki

and H i r a t a ,

Those f i n d i n g s a r e unusual because animal i n general

can synthesize p h o s p h o l i p i d from d i e t a r y o r endogenous f a t t y
acid,

d i g l y c e r i d e s and t r i g l y c e r i d e s .

Teshima e t a?.,

Kanazawa e t a l .

(in

1 9 8 7 ) found t h a t t h e p h o s p h a t i d y l c h o l i n e and

p h o s p h a t i d y l i n o s i t o l c o n t a i n i n g unsaturated f a t t y

a c i d such

as l i n o l e i c a c i d (18:Z U 6 ) ,
ect.

as

fatty

docosahexaenoic a c i d (22:6 G3),

acids moieties

were

effective

i n enhancing

growth and s u r v i v a l r a t e s of t h e l a r v a l ayu.
The
r e 1a t e d
fluid

function
to

the

mosaic

membranes
bilayer

of

membrane

model

of

consists

with

EFA

of

the

f 1u i d i t y

membrane

embryogenesis
and

protein

1980).

might

permeabi 1it y .

structure

phospholipid

globular

b i l a y e r (Capaldi,

in

The

postulate

arranged

penetrating

be

that

in

a

fluid

into

a

lipid

Length and degree o f u n s a t u r a t i o n

o f hydrocarbon s i d e c h a i n c o u l d i n f l u e n c e membrane properties,

lowering

the

melting

temperature

of

i n Morson and C l a n d i n i n ,

( a r e t s c h e r and R a f t ,

complex
1986).

lipid
Criti-

c a l temperature o r t r a n s i ' t i o n 1 i p i d phase ( T t ) and Arrhenius
activation
determine

(Ea)

energy
a

of

the

enzyme

temperature-induced

have

change

in

been

used

the

physical

p r o p e r t i e s o f t h e l i p i d membrane ( I n n i s and C l a n d i n i n ,
McMurchie,

Gibson,

Abeywardena

and

Charnock,

McMurchi, Abeywardena, Charnock and Gibson,
ran

and

Venkataraman

demonstrated
influenced
brane.

that

by

(1977);

the

membrane

the physical

Recently,

Morson and
enzyme

Divaka-

Clandinin

properties o f

their

1981;
1983a;

1983b).

activity

to

(1986)

will

be

h o s t mem-

i t was shown a l s o t h a t t h e r a t i o o f phos-

hat i d y l c h o l ine : phosphatidyl ethanol ami ne i n t h e m i tochond r i a l membrane o f cold-aclimated
function o f
Clandinin,

temperature
1984).

p o i k i l o t h e r m s changes as a

( T h i l l a r t and Brum

i n Robblee and

I t i s n o t known whether these changes i n

l i p i d composition c o u l d a l t e r physicochemical p r o p e r t i e s o f

the

membrane.

The

surface

( z e r o a t n e u t r a l pH)
dylethanolamine,
Therefore,

of

phosphatidylchol i n e

i s d i f f e r e n t than t h a t o f phosphati-

which has a n e g a t i v e charge a t n e u t r a l pH.

i t was concievable t h a t a change i n t h e r a t i o o f

phosphatidylcholine
membrane s u r f a c e
(Robbiee

charge

and

:

phosphatidylethanolamine

potential

Clandinin,

and

their

1984).

membrane

Change

in

may

alter

properties

the

physical

p r o p e r t i e s o f b i o l o g i c a l membrane have been shown t o i n f l u ence t h e

behavior o f

c e r t a i n membrane-associated

enzymes,

and t h i s might a l t e r p h y s i o l o g i c a l processes.
F i s h eggs c o n t a i n most e s s e n t i a l enzymes o f g l y c o l y s i s ,
t r i c a r b o x y l i c acid cycle,
Yurovi t s k y

and r e s p i r a t i o n c h a i n ( M i lman and

i n Neyfakh and Abramova,

1974).

A1 t e r a t i o n o f

some o f these a c t i v i t i e s might a f f e c t o t h e r c e l l metabolism.
Inhibition of

all

metabolic processes as

i n d i c a t e d by t h e

decrease o f r e s p i r a t i o n may cause t h e i n a b i 1 i t y o f c e l l s to
aggregate ( F l o r k i n and S t o t z , 1967).
Other
re1a.ted

to

function
the

of

EFA

synthesis

c y c l i c derivatives.

i n t h e embryogenesis might

of

oxygenated

cyclic

be

and non-

Precursors o f oxygenated hormone-like

compounds belong t o t h e 0 3 and c36 s e r i e s .

These compounds

might a c t as a modulator o r mediator i n v a r i o u s p h y s i o l o g i cal

events,

especially

in

the

processes

of

the

cellular

r e c o g n i t i o n o c c u r r i n g d u r i n g embryonic development (Leray e t

a l . , 1985).
Some warmwater f i s h r e q u i r e s
nic

and

linoleic

acids

i n their

various levels o f l i n o l e diets.

Cyprinus

carpio

-

requires 1.0% linoleic (18:2 9 6 ) and 1

%

linolenic (18:3d3)

acid (Takeuchi and Watanabe, 1977); Tilapia z i 7 i i requires
linoleic acid more than linolenic acid (Kanazawa, Teshima,
Sakamoto and Awal, 1980); Anguilla japonica requires 0.5%
linolenic acid 0 . 5 % linoleic acid (Takeuchi, Arai, Watanabe
and Shimma, 1980).

Bandyopadhay and Dutta (1982) demon-

strated that catfish (C7arias batrachus Linn. ) had no capability

to bi'osynthesize linolenic (18:3 c . 4 )

and linoleic

acid (18:2 06). Catfish (Clarias batrachus Linn.) juvenile
requires

1.5%

their maximum

linoleic acid
growth

and 0.6%

linolenic acid

(Mokoginta, 1986).

for

Requirements of

1 inolenic and 1 inoleic acids for catfish (Clarias batrachus

Linn.) broodstock development has not yet been determined.

111.

The
Complex

experiment
of

the

MATERIALS AND METHODS

was

Faculty

conducted
of

at

the

Fisheries,

Research

Institut

Bogor ( I P B ) , Bogor, f r o m March t o December 1990.
anal y s i s

was

Technology

conducted

and

Human

at

the

Nutrition,

laboratory
Faculty

Pertanian
Fatty acid

of
of

Pond

Departement
Agricultural

E n g i n e e r i n g and Technology, IPB.
3.1
3.1.1

Materials
Experimental f i s h
F i v e months o l d f i s h ,

and 9 0 . 0 - 110.0 g/male,

weighing 91.7

-

122.3

g/female,

were randomly stocked i n 15 t a n k s .

S i x females and t h r e e males were p l a c e d i n each t a n k .
3.1.2

Experimental d i e t s
F i v e experimental

d i e t s were used i n t h i s experiment.

T a b l e 1 shows t h e composition o f t h e d i e t s .
d i e n t s were mixed t o g e t h e r .

A t first,

mixed w i t h v i t a m i n mix i n a bowl.

dextrin,

mix,

and

was

Then,

c a s e i n and l i p i d .

followed

other

by

was c o l d ,

ingredient,

I t was s t a r t e d

CMC,

a-cellulose,

I n another bowl, 400 m l

kg d i e t ) o f b o i l e d water was added t o g e l a t i n
gelatin

the ingre-

c h o l i n e c h l o r i d e was

one by one, was added t o t h e former m i x t u r e .
from. mineral

A l l

.

(for

After

1

the

i t was added t o t h e o t h e r i n g r e d i e n t , and

was mixed t o g e t h e r f o r f i v e minutes.
were s t o r e d i n a f r e e z e r a t -12'~.

The e x p e r i m e n t a l d i e t s

Futher, sample

of each experimental diet was analyzed

for proximate composition.

Crude protein content of diets

was analyzed by using Kjeldahl method;
Table 1 .

Composition

of the

l i p i d . by

and crude

experimental diets

*

Component
(s/lOOs)

Casei n
35.0
Gelatin
9.0
Dextrin
35.0
CMC
3.0
a-cellulose
5.0
Mineral mixa
5.0
1.5
Vitamin mix b
Choline chloride
0.5
Beef tallow
6.0
Corn oil
0.0
Linseed oil
0.0
Cuttle fish liver oil 0 . 0
(g/100g dry

1 u6**

-----

basic)

0.2

35.0

35.0

35.0

35.0

9.0

9.0

9.0

9.0

35.0

35.0

35.0

35.0

3.0

3.0

3.0

3.0

5.0

5.0

5.0

5.0

5.0

5.0

5.0

5.0

1.5

1.5

1.5

1.5

0.5

0.5

0.5

0.5

2.0

2.0

2.0

0.0

0.0

4.0

3.0

0.0

4.0

0.0

1 .O

0.0

0.0

0.0

0.0

6.0

0.6

2.0

1.5

0.2

-

(a) See Appendix 1 for the composition of mineral mix.
( b ) See Appendix 2 for the composition of vitamin mix.
* Takeuchi, personal communication; Mokoginta, 1 9 8 6 .
** 1 d 6 contain 1 6 : 3 w6, 18:2 U S , 20:2 t36, 20:3 d6
20:4 d6, 22:5 W6
*** 1 d 3 contain 18:3 a,18:4 w3, 20:4 W3, 20:5 W3
2 2 : 5 W3, 22:6 W3

e t h e r e x t r a c t i o n method.

F a t t y a c i d composition o f t h e d i e t

was analyzed u s i n g gas l i q u i d chromatography.
Table 2 shows t h e proximate composition o f t h e e x p e r i mental d i e t s ,
diets.

and t h e W6 and d 3 - f a t t y

a c i d contents o f t h e

The composition o f f a t t y a c i d s i n t h e d i e t s can be

seen i n Appendix 3.
Table 2.

Proximate composition o f t h e experimental d i e t s ,
t h e G;6 and d 3 - f a t t y a c i d c o n t e n t o f t h e d i e t .

P r o x i mate

Treatments/diets

composition
A

B

c

D

E

47.47

48.29

46.51

47.72

47.58

Crude l i p i d

5.83

5.89

5.96

5.85

5.81

Ash

5.02

5.69

5.16

6.08

6.15

Crude f i b e r

3.19

4.86

5.10

4.79

5.83

1 u6

0.21

0.67

2.24

1.85

0.26

i: W 3

0.03

2.09

0.07

0.56

1.68

0.00

0.00

0.00

0.00

1.45

(g/100 g d r y
basic d i e t )

Crude p r o t e i n

E a3

3.1.3

HUFA

Experimental t a n k s
The f i s h were placed i n

x 80 x 100 cm.
320

liters.

r e c t a n g u l a r concrete tanks,

80

The volume o f water i n each tank was about
Each tank was p r o v i d e d w i t h a

filter

system

(see ~ p p e n d i x4 ) .
range o f 25Oc

-

The water temperature was r e g u l a t e d a t a

27Oc by using a thermostat.

quar ria, 60 x 50 x 40 cm, were used f o r egg incubation.

F e r t i l i z e d eggs were spread on g l a s s surface 20 x 10 cm, and
t h e n they were p l a c e d i n aquaria.
water was we1 1 aerated,

Ouring incubation,

and 1.0 Qpm o f methylene b l u e was

added %o t h e water t o g r o t e c t the eggs from fungus.
water temperature was 2 6 . 5 ' ~

3.2.1

the

-

The

2 8 . ~ ~ ~ .

Feeding and spawning of the b r o o d f i s h
The f i s h

were t r a i n e d t o feed

and acclimated t o t h e

l a b o r a t o r y c o n d i t i o n and f e d w i t h c o n t r o l d i e t f o r t w o weeks
~riot
r.o t h e beginning of the experiment.
iment, t h e f i s h were f e d experimental

Ouring t h e exper-

diets,

a t satiation,

once a day, a t 5.00 o ' c l o c k i n the aflternoon.
t h e water i n t h e tank was changed 100% volume,
t w o days.

once

once i n

The tank were scrubbed and cleaned thoroughly

a month.

Ouring cleaning,

the

fish

were

observed

w h e C h s r t h e y ware mature b y sexing them macroscopically

Afeer s i x t h month o f experiment,
grow a t a l l , and some died.
the f i s h was too high.
2.9

.

several f i s h d i d n o t

Presumably t h e s t o c k i n g r a t e o f

Oissolved oxygen was a t a range from

to 5.6 ppm, and the t o t a l ammonia concentration ranged

from 0.13

t o 1 .19 ppm.

Some o f t h e f i s h were then remved

i n order t o save, and l e f t t o 4 females per tank.

A l l males

used f o r t h e t r e a t m e n t s were p l a c e d i n d i f f e r e n t tanks.
The mature females were removed t o a f i b e r g l a s s t a n k
b e f o r e t h e f i s h were i n j e c t e d w i t h c a r p p i t u i t a r y suspension
i n order t o stimulate ovulation.
ias

batrachus

Linn.)

r e c e i v e d one

donor : body w e i g h t of
suspension f o r

at

recepient
injection.

one a t 4:00

injections,
other

every

12:OO o ' c l o c k

t h e morning,

Females o f c a t f i s h ( C l a r -

=

dosage

(body w e i g h t

1 : 1 ) of

r e c e i v e d two

i n the afternoon,

a t night.

A t

t h e f i s h were s t r i p p e d .

7:00/8:00

and t h e

o'clock

in

The eggs produced by

t h e females were f e r t i l i z e d w i t h sperms o f
a n o t h e r pond.

carp p i t u i t a r y

The females

o'clock

of

t h e males f r o m

I n o r d e r t o g e t v i a b l e spermatozoa, t h e males

were i n j e c t e d w i t h one dosage o f c a r p

p i t u i t a r y suspension,

and were s a c r i f i c e d f o r t h a t purpose.
About 20% of u n f e r t i l i z e d eggs were c o l l e c t e d f o r p o l a r
and

nonpolar

l i p i d fraction

composition analysis.

analysis,

and t h e

fatty

acid

The r e s t o f eggs were f e r t i l i z e d by

spermatozoa, and t h a n wqre spread on g l a s s s u r f a c e ( 2 0 x 10
cm) which was p l a c e d i n a f i b e r g l a s s t a n k c o n t a i n i n g w a t e r .
Futhermore,

three

or

four

pieces

of

those

glasses

were

removed f r o m f i b e r g l a s s t a n k and were p l a c e d i n an aquarium
c o n t a i n i n g water f o r eggs i n c u b a t i o n .

The number o f eggs on

t h o s e g l a s s e s and t h e l a r v a e produced were counted and were
used t o determine t h e h a t c h i n g r a t e o f eggs.
fertilized

eggs

were

divided

i n c u b a t e d i n another a q u a r i a .

into

three

The r e s t o f

batches and were

One b a t c h o f eggs were c o l -

l e c t e d one

hour a f t e r f e r t i l i z a t i o n ;

another

batch eleven

hours a f t e r f e r t i l i z a t i o n ;

and t h e r e s t were c o l l e c t e d a f t e r

t h e eggs hatched ( l a r v a e ) .

Those samples o f eggs and l a r v a e

were used f o r

nonpolar l i p ~ df r a c -

l i p i d c o n t e n t , p o l a r and

t i o n a n a l y s i s , and f a t t y a c i d

composition a n a l y s i s .

The female f i s h were weighed b e f o r e and a f t e r spawning,
so

the

weight

calculated.

of

eggs

produced

Futhermore,

the

by

each

number o f

female

eggs

could

from

be

0.5

g

( w i t h t w o r e p l i c a t e s ) sample were counted i n o r d e r t o e v a l u a t e d i f f e r e n c e s o f egg s i z e between t r e a t m e n t s .
Seven

lots of

histological

abnormal

evaluation

(see

l a r v a e were
Appendix

5

collected
for

for

procedure

of

h i s t o l o g i c a l preparation).
I n o r d e r t o e v a l u a t e t h e v i a b i 1i t y of

t h e spermatozoa,

t h e eggs produced by one female i n t r e a t m e n t D were d i v i d e d
into five

batches,

f r o m t r e a t m e n t s A,
Furthermore,

and were
B, C,

by m i l t

fertilized

of

males

D and E.

eggs produced by one female f r o m

treat-

ment E were d e v i d e d i n t o t h r e e batches,

and were f e r t i l i z e d

by m i l t o f males f r o m t r e a t m e n t s A,

and E.

after

f e r t i l i z a t i ~ n ~ t h eeggs

incubation

aquariua.

The

C,

were

number

removed
of

Seven h o u r s
from

unfertilized

the
and

f e r t i l i z e d eggs were c a l c u l a t e d .

3.2.2 Chemical analysis
The crude

l i p i d of

sample was

extracted

by

using

i n Takeuchi, 1988), and

F o l c h , Lee and Stanley method (1957,
then nonpolar
polar

l i p i d (neutral

l i p i d (phospholipid)

column (Sep-Pak C a r t r i d g e ;

l i p i d ) was separated from t h e
by u s i n g s i l i c i c

fraction

Water Associates,

USA;

acid

Takeuchi,

1988).
Further,

t h e f a t t y a c i d composition o f

nonpolar and

p o l a r 1 i p i d f r a c t i o n were determined by u s i n g gas chromatography
3.2.2.1

L i p i d extraction

Sample o f eggs,

l a r v a e o r body t i s s u e ( A g) was placed

i n a waring blender.
20 x

ml

A

sample

was

Chloroform/methanol

2

:

vol./vol.)

was added i n t o t h e waring blender,and
homogenized

for

5

minutes.

The

then t h e

homogeni zed

sample was f i l t e r e d , and t h e lower l a y e r s o l u t i o n was placed
i n an erlenmeyer (250 m l ) .

The upper l a y e r was discarded.

The lower l a y e r s o l u t i o n i n t h a t erlenmeyer was t r a n s f e r e d
MgC12 0.2 x (20

i n t o a separatory funnel (500 m l ) .
0.03

M was added i n t o t h a t separatory

vigorously f o r

1 minute.

That funnel

funnel,

x

A)

ml

then shaken

was . f i l l e d w i t h N2

gas, and i t s stopper was replaced again

.

n i g h t a t room temperature.

l a y e r was f i l t e r e d ,

The

lower

I t was k e p t over-

and was placed i n a p r e v i o u s l y weighed round bottom f l a s k ( a
g),

the

flask

upper

was

evaporator,

l a y e r was

evaporated
and

then

to

the

discarded.
dryness
flask

The s o l u t i o n
using

was

a

weighed

rotary
again

i n the
vacuum

(b

g).

T o t a l weight o f l i p i d can be c a l c u l a t e d by s u b t r a c t i n g b-a
( D g).

Crude l i p i d
3.2.2.2

=

D I A x 100%

P o l a r and nonpolar l i p i d f r a c t i o n a n a l y s i s

About 100.0 mg of crude l i p i d was d i s s o l v e d i n 1.0 m l
chloroform.
tridge.

That- sample was

i n j e c t e d a t t h e top o f

m l o f chloroform

Another s y r i n g e c o n t a i n i n g 20.0

was connected i n t o t h e top of c a r t r i d g e ,

car-

and then t h e c h l o -

r o f o r m was pushed down Shrough t h e c a r t r i d g e w i t h t h e f l o w
r a b

25 ml/minute.

Nonpolar l i ~ i df r a c t i o n was c o l l e c t e d

i n a p r e v i o u s l y weighed round bottom f l a s k ( a g).

Then,

ch1oroform/methano1

used to

elute

she

(49:1,

remaining

Monoglycerides

vol./vol.)

m i x t u r e was

monog1ycerides

in

the

a

cartridge.

was a l s o added to t h e nonpolar l i p i d frac-

tion.

F u r t h e r , 30.0 m l methanol was used t o e l u t e t h e

w l a r lipid,

and t h a t p o l a r l i p i d f r a c t i o n was c o l l e c t e d i n

another p r e v i o u s l y wei.ghed round bottom f l a s k ( x g ) .

Both

p o l a r and nonpolar l i g i d s o l u t i o n i n t h e f l a s k were evapor a t e d fo

dryness u s i n g a r o t a r y vacuum evaporator.

,the f l a s k was weighed again.
c o n t a i n i n g nonpolar

Then

I f t h e weight o f f i r s t f l a s k

l i p i d was .b g,

and t h e

second

flask

p o l a r l i p i d was y g, t h a n t h e weight o f nonpolar l i p i d can
be c a l c u l a t e d by s u b r a c t i n g b-a g (C

s u b t r a c t i n g y-x g

(Z g).

g),

and p o l a r l i p i d b y

Nonpolar l i p i d f r a c t i o n

C

=

x 100%.
(C + Z )

z

--

Polar l i p i d f r a c t i o n

(C +

x 100%.

Z)

3.2.2.3 F a t t y a c i d a n a l y s i s
M e t h y l e s t e r i f i c a t i o n o f f a t t y a c i d i n p o l a r and nonpol a r l i p i d has t o be done b e f o r e those sample can be i n j e c t e d
t o t h e gas chromatography.
a. M e t h y l

esterification

method,

of

Supelco

Inc.

1975)

About 5.0
Then 0 . 4

(modification

-

-

15.0 mg

l i p i d was p l a c e d i n a small v i a l .

0.8 m l 8F3 methanol

(14.5%, b / v )

was added i n t o

t h e v i a l , and t h e cap o f v i a l was r e p l a c e d again.

The v i a l

was h e a t e d a t 6 0 O ~f o r 60 minutes by p l a c i n g i t i n a h e a t e r
b l o c k which was p l a c e d on a s m a l l h o t p l a t e .
0.8

ml

s a t u r a t e d NaCl was added i n t o t h e v i a l ,

the

vial.

0.4

-

and i t was

F i n a l l y 0.4 m l petroleum e t h e r was added

shaken v i g o r o u s l y .
into

Futher,

The

upper

layer

s o l u t i o n c o u n t a i n methyl

e s t e r o f f a t t y acid.

b. F a t t y a c i d a n a l y s i s
F a t t y a c i d c o m p o s i t i o n analyses were
Shimadzu Gas Chromatography GC-SAM,
pac C-RGA.

done by

using

w i t h Shimadzu Chromato-

S t a t i o n a r y phase used i n t h i s a n a l y s i s was SP-

2330 on 100/120

chromosorb,

temperature 27!j0c,

68% cyanoprophyl,

from Supelco.

USA.

wi%h maximum

Standard f a t t y a c i d s

were

from

was 260°c,

Sigma Chemical

Comp.

i n j e c t o r temperature was 230°c,

p e r a t u r e was 150-21 OOC ( 5 O ~ / mni u t e )
About 0.5
to

t h e GC.

would

D e t e c t o r temperature

USA.

t h e column tem-

.

yl s t a n d a r d f a t t y a c i d s o l u t i o n was i n j e c t e d

The chromatogram o f

be used f o r

sample f a t t y

this

standard

fatty

a c i d determination.

acid
About

0.5 p1 methyl e s t e r o f sample f a t t y a c i d was i n j e c t e d t o GC.
I t had t h e same temperature c o n d i t i o n as above.

matogram was compared t o t h e s t a n d a r d f a t t y
gram.

The chro-

a c i d chromato-

Each peak o f f a t t y a c i d from t h e sample chromatogram

which

has

the

same

retention

time

as

that

from

standard

f a t t y a c i d chromatogram was c o n s i d e r e d t o be t h e same f a t t y
a c i d as t h a t s t a n d a r d f a t t y a c i d .
3.2.3

Experimental d e s i g n and s t a t i s t i c a l methods

T h i s experiment used t h e c o m p l e t e l y randomized d e s i g n ,
w i t h f i v e treatments
ment.

The

weight,

weight

and t h r e e

of

egg

t h e s i z e o f eggs,

replicates

produced

for

compared

each t r e a t to

the hatching r a t e o f

the

body

eggs,

per-

centage o f abnormal l a r v a e and f e e d c o n v e r s i o n were s u b j e c t ed t o one-way
test

v a r i a n c e and Tuckey's

t o determine s i g n i f i c a n t

(Steel
were

a n a l y s i s of

and T o r r i e

,

1980).

n o t subjected t o

procedure

d i f f e r e n c e s among t r e a t m e n t s
Percentage o f

statistical

analysis,

f e r t i 1i z e d eggs
because t h e r e

were n o t enough samples o f eggs.
F o l l o w i n g parameters were c a l c u l a t e d f r o m each t r e a t -

ment:
a.

The weight o f egg

produced compared t o t h e body weight

I t was c a l c u l a t e d as:

t h e weight o f egg

produced
x

100%

t h e 4ody weight

.

Hatching r a t e :
Hatching r a t e was c a l c u l a t e d as:

the number o f hatching eggs
x 100 %
t h e number o f incubated eggs
c.

Percentage o f abnormal l a r v a e
Percentage o f abnormal l a r v a e was c a l c u l a t e d as:

t h e number of abnormal l a r v a e
x 100 %
t h e number o f t o t a l l a r v a e
d.

V i a b i 1it y o f t h e spermatozoa
V i a b i l i t y o f the spermatozoa was c a l c u l a t e d as:
t h e number o f f e r t i l i z e d eggs
x 100 %

t h e number o f incubated eggs

e.

Feed conversion
Feed conversion was c a l c u l a t e d as:
t o t a l feed consumed by f i s h ( d r y b a s i c d i e t ) ( g )
f i n a l body w e i g h t - i n i t i a l
weight ( g )

body weight+dead f i s h body

Feed conversion was evaluated from s i x months o f feeding
t r i a l s , b e f o r e t h e males were separated from females.

3.

Water quality analysis

Alkalinity,

pH,

d i s s o l v e d oxygen,

temperature,

t o t a l ammonia o f t h e w a t e r where f i s h l i v e d
Samples
later.
quai i t y .

were

taken

at

the

b e g i n n i n g and

and

were a n a l y s e d .

then

s i x t h month

These were r e c o g n i z e d as t h e i n d i c a t o r s o f t h e w a t e r

IV.
4.1

RESULTS

The percentage o f egg weight/body
eggs, t h e hatching
&normal

weight,

the size o f

r a t e o f eggs and t h e percentage o f

larvae

N a t u r a l l y , c a t f i s h ( C t a r i a s batrachus Linn.) w i l l spawn

at t h e age o f 10
s p a w n e d at t h e
groblems

-

11 months, but i n t h i s experiment,

age o f

arosed

14

t o the

-

fish

15

months.

Presumably some

i n adjusting

syswm d u r i n g t h i s experiment.

However,

fish

to

the

rearing

a t t h a t age,

all

female f i s h were maturle, and they could then be induced t o

spawn.
The body weight o f f i s h before and a f t e r spawning, and

the weight
f a b l e 3.

of

eggs produced by

each female are

shown i n

It was found t h a t one female f i s h from treatment A

ptuduced non v i a b l e eggs, which was seen t h a t the colour o f
.eggs were opague, and a f t e r

of

,

those eggs d i d n o t

Besides, three f i s h from treatment B and two

haeh at all.
fish

incubation

treatment

C

were

macroscopically

determined

as

famale at t h e beginning of t h i s experiment, but unfortunatel y they turned o u t t o be males l a t e r on (Table 3 ) .
The weight
individuals,

o f egg produced by each f i s h varied between

and bigger

eggs compared to smaller

f i s h d i d n o t always produce more
ones.

For example, one f i s h from

treatment A weighing 260.0 g produced 7 . 0 g eggs (about 2 . 7 %
o f t h e body weight),

b u t another f i s h from the same t r e a t -

ment weighing 1 4 6 . 0 g, produced 1 0 . 0 g eggs (about 6.8

X of

t h e body weight).

Treatments/
'replications

1
2

1
2
3

Diet C

Body
weight o f
fish
before
spawn ing
(9)

I

0

Diet 8

was

The
broodfish
spawni ng, and
each female.

Table 3.

Diet A

This

1

2
3

also

found

i n the other four

body weight before and
after
the weight
o f eggs produced by

8ody
weight o f
fish
after
spawn ing
fs)

II

Weight
o f eggs
(9)

I11

Total
weight
compared
t o body
weight

(%I
I V

Table 3. Continued
Treatments/
r e* p l i c a t i o n s

0

1
:

a*

***

Body
weight o f
fish
before
spawning
(9
I

Body
weight of
fish
after
spawni ng
(9)

Weight
o f eggs
(9)

I1

Total
weight
compared
t o body
weight

I11

(%I
IV

non v i a b l e eggs
t h e f i s h i s male, eventhough macroscopically determined
as female
a l l eggs were used f o r t e s t o f the v i a b i l i t y o f
wermatozoa

treatments as we1 1.

Statistical ly,

ences o f t o t a l egg w e i g h t / t o t a l
ments (?

>

0.05)

t h e r e were no d i f f e r -

body weight between t r e a t -

(see Appendix 6 f o r s t a t i s t i c a l a n a l y s i s ) .

It was found a l s o t h a t the s i z e o f eggs produced by t h e
b r o o d f i s h f e d on €FA-deficient d i e t ( d i e t A) was t h e same as
those produced by the broodf i s h f e d
a c i d d i e t s ( d i e t 0 , C , 0 , and E )
f o r s t a t i s t i c a l analysis).The

on h i g h d 6 and cJ3-fatty

(P > 0.05,

see Appendix

number o f eggs per 0.5

from each treatment i s presented i n

Table 4.

7

g egg

From t h i s r e s u l t i t was shown t h a t t h e d 6 and w 3 - f a t t y
acid level

i n t h e d i e t d i d n o t a f f e c t t h e weight

of

egg

produced by those broodfish, b u t i t a f f e c t e d t h e q u a l i t y o f
eggs.

This was shown by the hatching r a t e o f eggs.

Table 4.

The number o f eggs per 0.5
ment

Rep1i c a t i o n

g egg from each t r e a t -

.

Treatments/diets

a

A

c

D

and

G6-fatty

acid level

contained 0.21% * - f a t t y

a c i d and

E

Mean

T h e broodfish,

i n their

diet

0.03% d3-fatty

fed on low *3

( d i e t A,
acid)

produced eggs w i t h

(see Table 5 and Appendix 8 and 9 ) .

low

The hatching r a t e o f

eggs improved by a d d i t i o n o f G36 and (33-fatty
diet,

acid

i n the

as was shown by treatments D and E ( d i e t D contained

1 -85%iw.6-fatty a c i d and 0.56%

diet E
acid

hatchabi 1 ity

contained 0.26% a 6 - f a t t y

level).

was t o o
contained

high,

o f a3

--fatty

C

acid

contained

acid i n t o the d i e t

and 2.09%
2.24%

a3-fatty

eggs decreased again,

as was shown by treatments B

0.67% W6-fatty
diet

or

a c i d l e v e l , whereas

a c i d and 1.68%

The hatching r a t e o f

when t h e a d d i t i o n

whereas

Q3-fatty

and C ( d i e t B

u3 f a t t y

cJ6-fatty a c i d

acid,

Table 5.

The h a t c h i n g r a t e o f eggs produced by t h e broodf i s h r e c e i v i n g experimental d i e t f o r n i n e months.

Rep1 i c a t i o n

Mean

Treatments/diets
C
D

A

B

46.0

74.0

74.4

E

82.3

80.7

and 0.07% d 3 - f a t t y a c i d ) .

It was i n t e r e s t i n g t o n o t e t h a t t h e b r o o d f i s h f e d on

*

d i e t E which contained very
h i g h c 3 -HUFA
G33,

low

f a t t y a c i d (0.26%) and

(1.68%), and most o f i t were 20:5t;3

and 22;6

c o u l d produce t h e same h a t c h i n g r a t e o f eggs as those

b r o o d f i s h f e d on d i e t D, which contained 1.85Xc36-fatty

acid

and 0.56% U 3 - f a t t y acid, most o f i t was 18:3 C3.
The

€FA-deficient

eggs

i n treatment A

produced

low

h a t c h a b i l i t y and h i g h percentage o f abnormal l a r v a e as w e l l .
Table 6 and Appendix

10

show t h e

percentage

o f 'abnormal

l a r v a from each treatment. The percentage o f abnormal l a r v a
~ r o d u c e dby t h e b r o o d f i s h which r e c e i v e d lowG)6 and w 3 - f a t t y
a c i d ( d i e t A) was h i g h e r than by those which r e c e i v e d h i g h
~ 2 6and 4>3-fatty

a c i d d i e t s ( d i e t s B,

C,

0 , and E,

P3 and

1 HAF-eggs,

11 HAF-eggs,

6,

and

7,

8,

9,

and

Appendices

14,

15,

16,

17,

f a t t y acid.

18).

Fishcouldnotsynthesizec36andGl3-

I n general, f i s h would synthesize h i g h W9-fatty

a c i d if t h e f i s h r e c e i v e d lowW6 and W 3 - f a t t y a c i d
diet.

So,

i n their

Show f a t t y a c i d s would be t h e focus o f

fatty

acid e v a l u a t i o n d u r i n g embryogenesis.
From F i g u r e 5,

low G;6 and d 3 - f a t t y

eggs were a f f e c t e d by t h e low
b r w d f i s h diet.

a c i d l e v e l s o f UF-

and w 3 - f a t t y

6.8

a c i d s i n the*

t h e d 9 - f a t t y a c i d l e v e l o f t h e UF-eggs was

h i g h , e s p e c i a l l y i n t h e NP l i p i d f r a c t i o n .

The

unsaturated

f a t t y a c i d l e v e l o f eggs changed s i n c e t h e f i r s t hour a f t e r

K t was i n t e r e s t i n g t o note, thatc36,W3 and

Fertilization.
W9-fatty

acid

level

of

HAF-eggs,

1

11

HAF-eggs,

I t seemed

l a r v a s were h i g h e r than t h a t from t h e UF-eggs.

&hat a c e r t a i n

level

of d 6 , 0 3

and &.%-fatty

and t h e

acids

were

needed for embryogenes is .
F i g u r e 6 shows t h e fatty a c i d d i s t r i b u t i o n o f UF-eggs,
1 HAF-eggs,

1 1 HAF-eggs,

and t h e

l a r v a e from treatment 8

X T a t t y a c i d d i s t r i b u t i o n of UF-eggs p o l a r 1i p i d was missing
by

aecibent).

1% was found

t h a t t h e W6 and d 3 - f a t t y
by t h e c26 and

l e v e l o f UF-eggs was a160 affec-d

acid l e v e l

acid

d3-fatty

in d i e t 8, the d 3 - f a t t y

o f -the b r o o d f i s h d i e t .

a c i d l e v e l was h i g h e r than t h a t o f the w6-fatty

acid.

This

~ r o f i l s
of b o t h F a t t y a c i d s i n t h e d i e t c o u l d be found a l s o

i n t h e W-eggs.

furthermore

,

lower l e v e l o f (36 and d 3 -

f a t t y a c i d was found i n 1 HAF-eggs,
b o t h eggs,

o r i n 1 1 HAF-eggs o r i n

b u t t h e l e v e l of G6 and 3 3 - f a t t y

a c i d increased

uc

1-nrr

tc -nbr

E m w p p o e o s ~ sphosm
40

Figure 5 .

'Of

1

e,

Total
G)3 and 0 - f a t t y acids d i s t r i b u t i o n o f
p o l a r ( a ) and nonpolar l i p i d ( b ) during embryogenesis o f treatment A , ( d 6 - f a t t y acid 0.21% and
4 3 - f a t t y acid 0.03%).

Figure 6.

ToCal06.
a n d ~ 9 - f a t t y acids d i s t r i b u t i o n o f
p o l a r ( a ) and nonpolar 1i p i d ( b ) during embryogenesis of treatment 8 ( Wb-fatty a c i d 0.67% and
W3-fatty a c i d 2 . 0 9 % ) .

again i n t h e larvae.

On t h e o t h e r hand,

level

increased i n 1 HAF-eegs,

again

i n 11 HAF-eggs

and

the d 9 - f a t t y

acid

b u t a f t e r t h a t i t decreased
F i n a l l y t h e a 6 and a3

larvae.

- f a t t y a c i d l e v e l o f l a r v a e was t h e same as t h a t o f UF-eggs,
-

b u t t h e a 9 - f a t t y a c i d l e v e l was h i g h e r t h a t t h a t o f UF-eggs.
D i e t C contained very
low u 3 - f a t t y a c i d (0.07%).
U3-fatty
and

high d6-fatty

acid (2.24%)

and

T h i s h i g h d 6 - f a t t y a c i d and low

a c i d l e v e l i n t h e b r o o d f i s h d i e t would a f f e c t t h e
03-fatty

acid

level

of

c o u l d be seen from F i g u r e 7.
high d 6 - f a t t y

acid,

Further,

the U 6 - f a t t y

HAF-eggs,

11 HAF-eggs,

t h e UF-eggs.

UF-eggs

very

low

acid level o f

as

it

a l s o contained very

W3-fatty
nonpolar

acid

level.

l i p i d from 1

and l a r v a e were h i g h e r than t h a t from

But t h e a 6 - f a t t y

f r o m 1 HAF-eggs,
G)3 and d 9 - f a t t y

and

t h e eggs produced,

11 HAF-eggs,

acid level o f

polar

lipid

and l a r v a e were constant.

The

a c i d l e v e l o f p o l a r and nonpolar l i p i d a l s o

changed s i n c e t h e f i r s t hour a f t e r f e r t i l i z a t i o n as w e l l .
Although d i e t C contained h i g h l e v e l o f
eggs produced by

t h e b r o o d f i s h which

d6-fatty

acid,

the

received t h i s d i e t ,

s t i l l contained h i g h l e v e l o f d 9 - f a t t y a c i d .
Figure 8 s k w s the f a t t y
eggs,

acid distribution

1 HAF-eggs and 11 HAF-eggs,

the broodfish receiving d i e t D.
and

o f t h e UF-

and t h e l a r v a e produced by
I t was shown,

t h a t the 0 6

d 3 - f a t t y a c i d l e v e l o f t h e UF-eggs were a l s o

affected

F i g u r e 7.

Total a 6 , c6 and c 9 - f a t t y acids d i s t r i b u t i o n o f
p o l a r ( a ) and nonpolar l i p i d ( b ) during embryogenesis o f treatment C (We-fatty acid 2 . 2 4 % , and
0 3 - f a t t y acid 0.07%).

Figure 8.

Total U 6 ,
and @ - f a t t y acids d i s t r i b u t i o n od
p o l a r ( a ) and nonpolar l i p i d ("b) during embryogenesis o f treatment 0 ( d 6 - f a t t y acid 1 .BSX and
U - f a t t y acid 0 . 5 6 % ) .

by t h e (dl a n d Q 6 - f a t t y a c i d l e v e l i n t h e b r o o d f i s h d i e t , t h e

same symptom as i n treatments A,
D.have h i g h h . 6 - f a t t y a c i d l e v e l ,
a c i d l e v e l than d i e t D.

w6-fatty

b u t d i e t C has much h i g h e r
i t c o u l d be seen from

So,

t h a t the fJ6-fatty

F i w r e 7 and 8,

Both d i e t s C and*

8 , and C.

a c i d l e v e l o f UF-e~gs i n

treatment C was s l s o h i g h e r t h a n t h a t f r o m treatment 0.
t h e o t h e r hand,

d i e t C contained lower ( 3 3 - f a t t y

Qn

acid level

than d i e t D, 'and t h i s s l s o a f f e c t e d t h e a 3 f a t t y a c i d l e v e l
o f W-eggs from treatments C and D.

acid l e v e l

The d 9 - f a t t y

o f eggs from treatment O was lower than treatments
indicating that

the

fish

r e c e i v e d enough a3

A and C,

an& 0 6 - f a t t y

a c i d f r o m d i e t D.

En treatment D, i t was shown t h a t t h e f a t t y a c i d compor

of

sition

eggs

fertilization,
fatty

acid

also

changed

level

of

non p o l a r

eggs,

first

B,

l i p i d from

but a f t e r that,

hour

and C.

after
The w6

1 HAF-eggs

the d 6 - f a t t y

was
acid

and t h e l a r v a were almost t h e same as

11 HAF-ews

from UF-eggs.

the

t h e same as treatments A ,

h i g h e r than from UF-eggs,
l e v e l of

since

I n polar 1 i p i d , 0 6 - f a t t y

a c i d l e v e l o f 1 HAf-

11 HAF-e9gs and l a r v a were h i g h e r than t h a t from t h e

UF-eggs.

Further

the @-fatty
HAF-eggs,

,

the d3-fatty

a c i d l e v e l o f p o l a r lipid.,

a c i d l e v e l o f nonpolar and

11 HAF-eggs,

t h a t from t h e UF-eggs.

p o l a r l i p i d from 1

and from l a r v a were a l s o h i g h e r t h a n
The cd3-fatty

a c i d l e v e l o f nonpolar

l i p i d from 1 HAF-eggs and 11 HAF-eggs and l a r v a e was

almost

the

same as from UF-eggs.
D i e t E contained low c 6 - f a t t y

-fatty

acid

Appendix 3).

level,

acid level,

which most o f

The & - f a t t y

and high a3

them were d3-HUFA

(see

a c i d l e v e l o f UF-eggs from t r e a t -

ment € was lower than t h a t from treatments A,

and C,

i t was

t h e same as i n treaement 0 .

The f a t t y a c i d composition also changed since the f i r s t
hour a f a r f e r t i l i z a t i o n .

The G6 andc23-fatty a c i d l e v e l o f

nonpolar l i p i d and the a 9 - f a t t y
l i p i d f r o m 1 HAF-eggs,

11 HAF-eggs,

higher than from t h e UF-eggs.
fatty

a c i d and c U - f a t t y

HAF-eggs,

11 HAF-eggs,

acid o f p o l a r and nonpolar
and from larvae were

On the other hand, the 06l i p i d from 1

acid l e v e l o f p o l a r

and from the larvae was lower than

From t h e UF-eggs.
From these

all

fatty

acid

distribution

analysis

of

p o l a r and nonpolar 1i p i d o f eggs and larvae,

i t seemed t h a t

the f a M y a c i d composition changed since the

early

genesis.

Besides

f a t 4 y acid
HAF-eggs,
3%

that,

i n general, &%-fatty

and U S - f a t t y acid

l e v e l from

embryo-

acid,

U3-

1 HAF-eggs,

1t

and from larva were higher than t h a t from UF-egss.

seemed t h a t the balance o f

.those f a t t y acide,

espe-

c i a l l y i n t h e p o l a r l i p i d of eggs and larvae was important.

The f a t t y = i d

dJstri-bution of

1i v e r , and muscle were
s

a l s o a f f e c t e d by the f a t t y acid l e v e l o f the broodfish d i e t .
Table
muscle

7

shows

the f a t t y

acid d i s t r i b u t i o n

from treatments A and C.

of

liver

The l i v e r and muscle

and
from

Figure 9.

Total (36, 0 3 and - - f a t t y
acids d i s t r i b u t i o n o f
p o l a r ( a ) and nonpolar l i p i d (b) during embryogenesis o f treatment I (--fatty
acid 0.26% and
--fatty
acid 1.68%).

Table 7.

The f a t t y acid d i s t r i b u t i o n o f l i v e r and muscle o f the f i s h fed d i e t A and
d i e t C (area %).

Fatty

Liver
Diet C

Diet A
Acids

Nonpolar

2