Artificial induced breeding and triploidy in the asian catfish (Clarias Batrachus L.)

I.

In

#

East

Java,

species

intensifying

fish

farmers

for

their

1ocally.known
water

Indonesia,

aquaculture.

efforts,

species
a

i n

complex

e f f o r t t o expand and

farmers

Clarias

batrachus,

this

region.

of

problems

concerns a

Unfortunately

\be

can

solved

the

which hamper t h e i r

culture of

ikan l e l e .

r e l i a b l e and c o n t r o l l e d

method f o r q u a l i t y and q u a n t i t y f i n g e r l i n g p r o d u c t i o n .
problem

are

i s the highest prized fresh-

i n t e n s i f y the

t h e i r problems

becoining an

Fish

since

as " i k a n l e l e " ,

face

One o f

t h e Asian c a t f i s h ( C l a r i a s

i s p r e s e n t l y on t h e t h r e s h o l d o f

batrachus L . )
important

INTRODUCTION

This

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

by

g e n e t i c improvement.
Artificial
c a t f i s h have

(1988). A
pituitary

induced

breeding

been r e c e n t l y

single

dosage

(

combination w i t h a l a t e n c y

injection

time o f

of

cPS ( c a r p

17 hours

(

at

25OC

)

%.

and s u p p o r t i n g g e n e t i c research I n

a q u a c u l t u r e use5

various

for

resistance

disease

Asian

6 mg/kg body welght ) i n

r e s u l t e d i n h a t c h i n g r a t e s up t o 82.5
S e l e c t i v e breeding

i n

developed by Zonneveld e t a l . ,

intramuscular

suspension)

techniques

techniques,
and

including selectlori

behavior

characters,

I

intraspecific
including

hybridization,

sterile

gynogenesis,

and

interspecific

monosex h y b r i d s ,

p o l y p l o i d y and mutagenesis.

hybridrzation

g e n e t l c breeding,

Chromosomal manipulation and artificial mutations have
long

been

breeding.

established

as

It i s very likely

efficient in

fish. T h e

animals, can

handle both

the

highly

organisms

and

that

advantageous
they

fish breeder,

has

male and

will

in plant

be

j u s t as

unlike that of tarn1

female gametes outslde

manipulative

control

over

the

developing zygote (Moav, 1976).
Polyploidy
species

has

using

a

been

variety

Valenti, 1975; Refstie e t
1979;

Gervai

induced

et

al.,

of

in

the

control

growth rate

of

al.,

1977;

1980a;

Wolters

and for

of fish

(Purdom,

Allen

1972;

and Stanley,

et

al.,

1981).

potentially be useful

overpopulation,

in juveniles,

number

techniques

Induction of triplaidy in f i s h might
for

a

for

increasing the

extending survival and

improving growth in mature fish.
Most
from

an

of

the

interest

aquaculture

triploids might

in induced triploidy has been

perspective

with

the

indirect

that

grow faster than diploid a s juvenile o r a s

mature fish. T h i s might result from triploidy p e r
an

hope

result

se o r a s

of sterility o f triploids (Thorgaard,

Juvenile triploid have generally been found t o grow n o
faster

than

diploids.

similar t o t h a t of

Growth

diploids

aculeatus)

(Swarup,

(Gervai e t

al.,

19591,

1960a1,

and

in

of

j u v e n i l e triploids was

stickleback ( G a s t e r o s t e u s

common c a r p ( C y p r z n u s c a r p z u )
channel

catfish ( I c t a l u r ~ l s

\

3

punctatus)

et

(Wolters

dl.,

1982a).

, (Oreochramis aurea) , juvenile

triploid

larger than
s a 1mon

diploids {Valenti,

(Oncurhynchus

In t h e blue tilapia
were

found

t o be

1975). However, in Pacific

kisutch) ( Utter

et

a1

.,

1983)

triploids may grow slower than diploids.
Several

studies

have

found

that triploids may grow

faster than diploids a t sexual maturity, presumably because
energy that i s channeled t o gonadal development in d i p l o ~ d s
is

used

for

growth.

Triploid

significantly heavier
(Wolters

and older

channel

catfish

were

than diploids a t t h e a g e of 8 months
et

1982a).

dl.,

In

African catfish

(Clarias gariepinus), t h e growth rate w a s not significantly
affected

by

triploidy.

strongly

affected.

Body

composition,

Triploid

fish

however,

deposited, per gram of

Qrowth, less protein and more fat (Richter e t al.,
Induction o f triploidy

of

Clarias

possible through

t h e manipulation

the problems are

X)

how

fish

was

1987).

batrachus

L. is

of chromosome. However,

conditon

(eggs

and sperm

quality) be s e t up, 2 ) what methods will be used, 3 ) how to
determine

the

characteristic
Experiments
problems.

triploid
of

were,

fish,

triploid
therefore,

and
fish

4)

what

(growth

per--1ormed t o

pr:

is

the

formance~.

solve

these

the objectives of the experiments w e r e :

+.t3 develop

a practical method for inducing triploidy,

2. t o identify triploidy a t juvenile fish,
3. t o c o m p a r e t h e growth performance of diploid and

triploid fish
gonads.

during early and

late development of t h e

11. L I T E R A T U R E REVIEW

1. Genotype m a n i p u l a t i o n
The

manipulation

during the

of

chromosomes

nuclear cycles

of c e l l

becomes

feasible

d i v i s i o n and b a s i c a l l y

comprises t h e a d d i t i o n o r s u b t r a c t i o n o f a complete h a p l o i d
o r d i p l o i d set.

I n animals,

principal

cell

possible,

and i n

fertilization,

meiosis

division
fish,

phase

and

artificial

i n

where

other

the

is t t t e

manipulation

animals

processes can

eggs

is

w i t h external

he a p p l i e d e i t h e r

t o t h e gamete b e f o r e f e r t i l i z a t i o n o r t o t h e f e r t i l i z e d egg
a t any

period during

t h e f o r m a t i o n o f t h e zygote.

o f the f i r s t m i t o t i c d i v i s i o n i s a l s o f e a s i b l e
r e p o r t s o f i t so f a r a r e u n s u b s t a n t i a t e d
In

commercial

reproduction pattern
parents o f
parent

can

numbers

and

induced

(Purdom,
from

easily;

androgenesis)

(polyploidy).

e x p l o i t e d i n p l a n n i n g new
f o r animal

be

deviation

i n eggs b u t

the crossing o f inbred

t h r e e techniques

of

normal

by r a i s i n g
o n l y one

increase

of

phenomena can be

g e n e t i c improvement

c l a s s i c a l i n p l a n t ) , such as

l i n e s (produced

t h e use o f p o l y p l o i d s (Purdom,
D i p l o i d gynogenetic

These

schemes

breeding ( a l r e a d y

or

1983).

the

two d i f f e r e n t species ( h y b r i d i z a t i o n ) ;

(gynogenesis

chromosome

fish,

Control

by gynogenesis) o r

1983; Chourrout,

individuals

are available,

viz.

1984).

c o u l d be produced i t
genetic i n a c t i v a t i o n

o f sperm,

induced r e t e n t i o n

o f the

second p o l a r body,

induced suppression o f t h e f i r s t cleavage

(Fig.1).

or

Diploid

1

gynogenesis r e q u i r e s

t h e combination o f sperm i n a c t i v a t i o n

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

set.

I f the

achieved by r e t e n t i o n o f second p o l a r body,

resulting

embryo

starts

t h e same

products o f
a l l loci.

maternal chromosome

from

m e i o s i s and

I f suppression o f t h e

diplaidize

the

two

maternal

set,

different

terminal

so i s n o t homozygous a t

f i r s t cleavage
the

the

i s used t o

c o l l e c t e d embryos a r e

considered t o be homozygous a t a l l l o c i because they r e s u l t
from

the

Chourrout,

fusion

two

m i t o t i c p r o d u c t s (Purdom,

sperm

radiation

treatments

chromosomes.

are

Radiation

available t o

treatments t h a t

have been used s u c c e s s f u l l y i n c l u d e i r r a d i a t i o n
rays u s u a l l y
dl.,

1978;

from

O
' Co

Chourrout e t

or

dl.,

I r i ,

&OCs

(Purdom,

1980; R e f s t i e e t

1969; Nagy e t
dl.,

176;

l i g h t (Stanley,

1981; Chourrout and Q u i l l e t , 1982;

1988).

1990), and

w i t h gamma

r a y s (Stanley,

al.,

1983;

1984).

A v a r i e t y of

inactivate

of

1982), X

u l t r a v i o l e t (UV)
Komen e t

Meiosis I 1
I

-

sperm
inactivation

1st mitosis

0 .

9

r'-

Retention
o
i nf aPB
c t i vII+sperm
ation

@-0-@-@3

Suppression
o f 1st cleavage

Retent i o n
of PB I 1
Suppression o f
1st cleavage

haploid
gynogenetic

hetero
dzi p
ygous
loid
gynogenetic

p0-

@- @

,p
I

- Gi

homoz
dip
ygous
loid
gynogenet~c

triploid

@- @

tetraploid

used f o r
producing
F i g u r e 1. Three o p e r a t i o n s
gynogenetic
individuals,
triploids,
and
tetraploids i n
lower
vertebrates
: sperm
inactivation,
retention of
second
polar
first
cleavage
body and suppression o f
(Chourrout, 1984).

H t l o p h o r c 11

Anophosr 11

0t98nrrol1119
2nd polof body

H t ~ o p l ~ o tolt
1st ormlon ol

rmnd )o

199 n u ~ l r u r
( P ~ ~ P ~ 11)
OS!

fuctd sptrm

Cold 'hock

8

I

899 ~ l o ~ v c l t ~ cold sMch

5.

w
1 R I f'lOIO

F i g u r e 2.

Schematic r e p r e s e n t a t i o n
(Purdom, 1983).

lrlRAi;010

'8'

YOHbllL
Oll'LOIO

of polyploids

2.

Polyploid production

I

Induced

individuals

polyploidy
with

refers

extra

sets

done by t r e a t i n g f e r t i l i z e d
shock,

hydrostatic

treatments

are

be produced

p o l a r body o f

the

the

first

with

of

If

e i t h e r temperature

after

due t o

cleavage

production

chemical treatment.

shortly

t r i p l o i d s can

before

eggs

applied

the

o f chromosomes. T h i s can be

pressure o r

eggs.

to

I f the

fertilization,

r e t e n t i o n o f t h e second

the

treatments

division,

are s h o r t l y

t e t r a p l o i d s can be

produced.
I n t h e method o f

temperature shocking,

Purdom (1983)

presented t h e scheme f o r p o l y p l o i d s p r o d u c t i o n (Fig. 2). I n
produced by c o l d shocking

t h a t scheme t r i p l o i d f i s h can be
f e r t i l i z e d eggs

a t t h e metaphase I 1 stage,

and t e t r a p l o i d s

by c o l d shocking t h e f e r t i l i z e d eggs a t t h e metaphase stage
d u r i n g t h e f i r s t cleavage d i v i s i o n i n embryos.
Temperature,
w i d e l y used t o

treatments

suppress

of

the

f e r t i l i z e d eggs have been

second

second p o l a r body e x t r u s i o n i n f i s h ,
Purdom,

1969;

1980; R e f s t i e

Valenti,
et

dl.,

1975;

Nagy e t

al.,1978;

al.,

1981# BenPey and S u t e r l i n ,
duration,

(Chourrout,

1980;

(e.g.,

Chaurrout,

1982; R i c h t e r e t al.,1987;

1988), and heat shock

timing,

d i v i s i o n or

b o t h c o l d shock

-.

al.,

The

meiotic

Lomen e t

Thorgaard et

1984).

and

temperature o f t r e a t m e n t

must be determined f o r each species.

There a r e i n d i c a t i o n s

that

shock

differences

of

temperatures

susceptibility

related

to

genetic

1Refstie e t

al.,

background

A simple,

1982).

i n d u c t i o n o f p o l y p l o i d y by
out shortly

below l e t h a l

cleavage

temperatures.

stage

p r a c t i c a l approach forshock i s c a r r i e d

( f o r induced t r i p l o i d y ) o r
(for

An

t r e a t m e n t can be g i v e n t o a1 1
a r e inexpensive

egg m a t u r i t y

a temperature

after fertilization

s h o r t l y before f i r s t

and

tetraploidy)

a t just

advantage i s t h a t a u n i f o r m
eggs.

Temperature treatments

t o apply and m i g h t be s u c c e s s f u l l y adapted

f o r m a s s p r o d u c t i o n by f i s h farms o r management agencies i f
polyploids

prove

valuable.

temperature shocking o f eggs i n

Induction

of

triploidy

some t e l e o s t

by

species used

i n f i s h c u l t u r e i s shown i n Table 1.

Table

1.

Induction o f
triploldy
shocking o f
eggs
i n some
1987)
( R i c h t e r e t dl.,

Solmo gairdnerl
Solmo salor
Cyprinus corpio

10

I

5

-

by
temperature
t e l e o s t species

32

1 2 0 - 2

IVOLTEItS
1981
I

~ e l a t i v c t o controls

I

-

CI

id.,

I t I C I I I ' E R ut al.,
1986

Hydrostatic

pressure

ha5

been

used t o black second

polar body extrusion o r first mitotic division. Streisinger

*

et
and

dl.,

( 1 9 8 1 ) worked wlth zebra fish CBrachydanio reria),

Yamazaki

(1983)

and

Chourrout

(1984)

with rainbow

trout(Salmo gairdneri)

. Although

application of hydrostatic

pressure

requlres

more

treatments

[pressure cell

may

be

equipment

and hydraulic press)
than temperature shock
,

treatments, t h e method diserves
it

specific

1

s

damaging

wide investigation because

t o t h e e m b r y o than temperature

shock.
Chemicals
extrusion o r
et

dl.

may

also

used

mitotic division

(1977) reported

Atlantic

be

salmon

polar

body

producing mosaic polyploid-diploid

[Salmo

observing

block

in fertilized eggs. Refstie

salar)

e g g s t o cytochalasin B . Kanka and
reported

to

after exposing fertilized
Rab in

diplaid-triploid

Thorgaard (1983)

mosaic, triploid and

tetraploid Tinca tinca after treating fertilized

e g g s with

cytochalasin B.

3. Viability o f polyploids

The

successful

inductions

of triploidy in many fish

s p e c i e s [ s e e T a b l e 1) support t h e belief that triploid fish
have good viability.
Most s t u d i e s

of induced triploid f i s h have found that

they have normal viability.
and common

Tilapia a u r e a

(Valenti, 1973)

c a r p (Cyprinus carpia) triploid (Gervai e t al.,

1980a) apparently survive as

well a s

diploids. Studies in

rainbow trout (Thorgaard et al.,

1982) suggest that induced

triploid in t h i s species may be

somewhat less

viable than

diploids.
Triploidy

may

lead

interspecific hybrids
frogs,

triploid

to

(Sheerer

interspecific

increased
and

viability

in

1983).

Thorgaard,

In

hybrids a r e sometimes more

viable than diploid hybrids (Bogart In Thorgaard, 1983).
Interspecific triploid
fish culture

because hybrid

hybrids could

prove useful in

vigor and desirable atributes

of both s p e c i e s might be combined

in a

relatively healthy

sterile hybrid (Allen and Stanley, 1981).

4. Gonad development
Triploid
development

males
than

may

triploid

generally
females,

show

more

probably

gonad
because

triploid d o e s n o k interfere with t h e many mitotic divisions
involved in bringing t h e testis
gonad

of

to

spontaneous-triploid

considerable development, similar t o
developing m a l e s

diploids

at

mature

rainbow

size. T h e

trout

t h e normal

showed

testis An

(Thorgaard and Gall, 1979). T h e testes of

triploid channel catfish were
of

its

age

slightly smaller

than those

8 months and, unlike diploid testes,

histologically evinced n o sperm production (Welters e t

dl.,

T r i p l o i d y a p p a r e n t l y i n h i b i t s gonadal developifrent more
i n females than i n

males.

o o c y t e development

and t h e

t h e gonad.
small,

Failure

pachytene stage

rainbow t r o u t

gonads w i t h

of

may p r e v e n t

associated increase i n s i z e o f

T r i p l o i d female

stringlike

o f meiosis

meiosis

a t m a t u r i t y had

many c e l l s
(Thorgaard

a r r e s t e d a t the

and

Gall,

1979).

D i p l o i d s have o v a r i e s about f o u r t i m e s as l a r g e a t m a t u r i t y
as t r i p l o i d i n channel

Gonadal

development

catfish
was

also

(Wolters

gariepinus

and

appearence
sterile

1987),

Scott,

i n

described i n

O e r v a i et

Kawamura,

1983;

has

( Purdom,

1980a;

Such sexual

previously

1972;

1951

I n Thorgaard,

been

Thorgaard and

Wolters e t al.,

1986) and amphibians (Fankhauster,

dl.,

1979;

Richter e t al.,

developed.

development

dl.,

trlploid C.

(Thorgaard and G a l l ,

Yamazaki,

triploid fish

1979;

Johnson et

gonadal

of

(Salmo g a i r d n e r i )

trout

but t r i p l o i d testes are well

dimorphism

Gall,

ovaries

rainbow

1983;

1982b3.

1980b)

the

resemble t h e undeveloped gonads
L i n c o l n and

al.,

substantially inhibited i n

female common c a r p ( G e r v a i e t a l . ,

I n external

et

1982a;
1941 and

1983) and i t i s l i k e l y due t o

t e s t e s a b e i n g a t much

larger size

c e l l s enter

a t which p o i n t t r i p l o i d gametogenesis

i s

presumably

begins
and

meiosis,

when

i n

secondary

disrupted.

oogonia

the

testis

than o v a r i e s

Meiosis

in

the

when t h e i r

t e l e a s t ovary

a r e transformed i n t o p r i m a r y oocytes
when

spermatocytes

primary
(Nagahama,

spermatocytes
19831,

the

becorne
lattrr-

13
process accur l a t e r i n
1

the

life

of

salmonids (Nakamura,

1982).

It i s apparent t h a t a s m a l l number o f c e l l s succeed i n
passing through t h e
ovaries

and

first

testes

of

meiotic

t r i p l o i d animals;

rainbow t r o u t a r e t h e o n l y r e p o r t e d
Scott,

1983).

division

it

may

female t r i p l o l d

Nothing i s known about t h e m e i o t i c nbechanisrn

be

chromosomes.

both the

e x c e p t i o n ( L i n c o l n and

whereby p o s t m e i o t i c c e l l s a r e produced
but

in

simply

This

i n such t r i p l o i d s ,

t h r o u g h t h e random s e g r e g a t i o n o f

i s supported

by t h e

fact that t r i p l o i d

amphibians g e n e r a l l y produce a n e u p l o i d gametes (Fankhauster
and

Humprey

meiotic

Thorgaard,

mechanisms

involving

either

(Cherfas,

1969)

chromosomes

5.

i n

prior

have

1983).

functional

evolved i n gynogenetic t r i p l o i d ,

the

formatioil

or

the

to

However,

normal

of

tripolar

endomitotic
meiotic

spindles

duplication

of

(Cimino,

division

Identification of triploid fish

T h e assessment

a f t h e 'success o f

t o produce d i p l o i d s from
because

of

the

very

otherwise
great

between d i p l o i d s and h a p l o i d s
i n embryonic

development.

t r e a t m e n t s designed

haploid

differences
which become

eggs
in

i s easy

appearance

apparent e a r l y

No such easy o r definitive assay

i s possible f o r detection o f palyploids,

and a

variety

tit

14

methods have
6

consequently been

used.

most w i d e l y used c r i t e r i o n t o e s t a b l i s h
the

extensive

epidermal

(1967) measured

cells

from

the

areas

of

nuclei of

sturgeon (Acipencer s t u r i o ) larvae,

Purdom (1969) used c a r t i l a g e
and v a r i e t y

p l o i d y and f o l l o w s

i n amphibians (Fankhausten i n Purdom,

work

1983). V a s e t s k i i

site is t h e

Nuclear

c e l l nuclei

o f rainbow t r o u t

o f authors have used n u c l e i o f r e d blood c e l l s

( M o l t e r s e t al.,
None o f

1982b).

these

methods

can

be

readily

accepted as

d e f i n i t i v e because o f v a r i a t i o n i n c e l l o r n u c l e a r s i z e f o r
reasons o t h e r than p l o i d y ,
employ

a

reliable.

readily

but the

identifiable

I t i s necessary,

e r y t h r o c y t e s t u d i e s do

cell

however,

type
to

and should be

raise

fish

to a

reasonable s i z e b e f o r e blood can be c o l l e c t e d .
Measurement o f
1980b) seems t o

DNA c o n t e n t

be

more

o f n u c l e i (Gervai e t al.,

precise

than

s i z e measurement.

A n a l y s i s o f chromosome complements i s t h e most d i r e c t assay
of ploidy

o f any

usually requires

s o r t but
that f i s h

stage before assay.

it

i s difficult

be grown

t o f r y or fingerling

Chromosome analyses i n

been used

i n salmonids

and S c o t t ,

1984) b u t t h e r e s o l v i n g

i n f i s h and

blastomere have

(Purdom and L i n c o l n ,
power o f

1973; L i n c o l n
t h e technique

i s n o t great.

A

genetic

produced

from

between

plaice

assay
hybrid

i s

possible

embryos.

(Pleurunectes

In

for

triploids

flatfish,

platessa)

and

when

the hybrid

flounder

(Platichythys

and

f lesus)

between

turbot

(Scapthamus

maximus) and brill (Scupthamus rhombus) a r e distinguishable
I

from

parental

type

patterns, and

shortly

triploids can

after

hatching

by

pigment

be recognised a s intermediate

between t h e hybrid and the maternal type (Purdom, 1972).
P r o b l e m s have arisen in t h e measurement
The

labor

volume

of

concern

chrontosomal

intensive
data

(Thorqaard

about

generated by

reliability
more

rapid

1980; Benfey e t al.,
Johnson

et

has

1982), while

1981;

arisen

regarding

(Lemoine

data

and Smith,

(1984) compares t h e u s e of coulters

and

the

flow cytometer.

ICP-22

allowed rapid identification of diploid and

However

instruments

al.,

procedures

counter w i t h channelizer

triploid.

preparation has limited

19843.

dl.

Both equipment

et

of triploidy.

was

differences

revealed

in

in

accuracy

between

comparison of data from the

s a m e individuals.
Ploidy measurement
t w o instruments

on c o h o

salmon w a s

clear for the

in 85 o f 100 individuals ( 2 2 triploids, 63

diploids).

Ten

of

the

remaining

histagram

and

thus

indeterminate

analyses,

but

were

definitely

cytometery.

The

histogr-ams in

t h e coulter

individuals
from

had skewed

coulter counter

triploids

based

on f l o w

five additional individuals produced n o
counter and

were determined Ly

flow cytometry t o be two triploid and three diploids.

Both the

with channelizer and ICP-22

coulter counter

flow cytometer are able

to rapidly

differentiate triploid

blood samples, however the flow cytometer is more accurate.
The flow cytometer measures ploidy by
of

DNA

nuclear

and

the

coulter

counter

The

differences

erythrocyte cellular volume.
could be

under

environments,
cytometry

by

measuring
of accuracy

attributed to the fragility of cellular shape and

volume a s contrasted to
nuclei

fluorescent staining

is

the

maintenance

conditions

and

storage

therefore

of

shear,

conditions.

of

integrity of

altered

osmotic

Analysis

by flow

resilient to cellular disruptions

that d o not affect DNA fluorescence.
The use of flow
reasonable to

cytometry

is

very

accurate,

assume that the fish with a high DNA content

were triploid, since both aneuploids (Gervai et
Lincoln,

it is

1981a)

and

dl.,

tetraploids (Thorgaard et al.,

198th;
1981;

Allen, 1983; Chourrout, 1984) are generally non viable.
The

solely on
cell or
and

identification

of

the measurement
the nucleus

Quillet,

Biggers, 1983;

1982;

triploid

can

be based

of the major axis of either the

(Thorgaard and
Wolters

Richter et

fish

dl.,

et

Gall, 1979; Chourrout
al.,

1987).

1982a;

Beck and

The calculation of

cell surface area or nuclear volume, which necessitates the
measurement of

the

second

axis,

does

not

increase t h e

probability of identifying triploids correctly.
Of

the

three

techniques

employed

in the research,

17
c o u l t e r counter
most s u i t e d

sizing of

e r y t h r o c y t e s appears

t o be t h e

f o r t h e r o u t i n e screening o f t r i p l o i d f i s h .

In

#

t h e absence o f a
cytometer,

the

coulter

counter

measurement o f

channelizer

or

e r y t h r o c y t e c e l l o r nucleus

major a x i s from t h e blood smears can be used as
a l t e r n a t i v e method f o r i d e n t i f y i n g t r i p l o i d s .
technique i s more time
number o f

6.

f i s h that

a flow

consuming

and,

a reliable

However,

this

l i m i t s the

hence,

can be screened i n a p a r t i c u l a r study

4spects and c h a r a c t e r i s t i c s o f t r i p l o i d s

The i n t e r e s t o f producing t r i p l o i d f i s h has been based
on

the

assumptian

consequently

might

p o s s i b l y grow

that

they

avoid

faster o r

would

be

overpopulation

s u r v i v e longer

sterile

problems,

and

and

than normal f i s h .

T r i p l o i d s a r e expected t o be s t e r i l e because t h e odd number
o f thrbm~some $ e t s w i l l
either a failure
aneuploid gametes.
i n turn,
of

are

gonad

development

maturation,

such

and h i g h m o r t a l i t y .

indeed

or

production o f

The f a i l u r e o f gonad development might,

prevent t h e appearance o f

sexual

growth,

of

l e a d t o d i s r u p t i o n o f meiosis and

functionally

undesirable s i d e e f f e c t

as poor meat q u a l i t y ,

I t appears
sterile,

slower

that t r i p l o i d f i s h
secondary

sexual

c h a r a c t e r s a r e n o t always suppressed.

A p o s s i b l e a p p l i c a t i o n o f induced t r i p l o i d

may lxe In

18

the

fact

that

t r i p l o i d s have h i g h e r heterozygousity than

d i p l o i d s ( A l l e n d o r f and Leary,
to

be

associated

with

in

asymmetry

Crosby

(1986)

heterozygousity

was

polyploidies

plant

might be

i n

a

has been shown

(Leary

et

proposed

primary

s t r a i n s as

increased

advantage

induced t r i p l o i d s
t h e female

1985).

al.,

that

breeding programs.

maximized i n

between two

This

h i g h e r developmental s t a b i l i t y as

measured by f l u c t u a t i n g
Bingham

1984).

of

using

Heterozygousity
by u s i n g h y b r i d s

parent and crossxng t o

male o f a t h i r d s t r a i n .
Purdom (1972) induced
h y b r i d s by

triploid

plaice,

flounder,

and

c o l d shocks ((3 t o 5OC f o r 2 t o 4 hours) a p p l i e d
Triploid

t o newly f e r t i l i z e d eggs.
significantly

lower

pigment p a t t e r n ,
characteristics,

rate

number

athar

than

of

hybrids

survive

hybrids.

vertebrae,

In

at a

larval

and metamorphosis

the t r i p l o i d s display a d d i t i v e inheritance

involving a l l three

sets

of

chromosomes.

Concerning t h e

t h e r e was some i n d i c a t i o n t h a t t r i p l o i d per se

growth r a t e ,

may r e s u l t i n an excessive growth r a t e .
Such experiments
(1981b).
hybrids

He
to

identified
19721,

maturity
by

direct

measurements.
triploid

reared

larval

were

triploid

till

hybrids,

carried

male

three

melanophore

chromosome
Males

also

Triplaid

plaice

years.

by L i n c o l n

and

and
i n

hybrids

nuclear
both

wake

flounder

Triploids

distribution

count,

predominated

out

were

(Purdom,
volume

diploid

shown

to

and
be

-

sterile,

probably

t a k i n g place,

because

b u t gonad

abnormal

sire

spermatogenesis

appeared

to

was

be unaffected.

Female t r i p l o i d h y b r i d s contained o v a r i e s which were normal
i n appearance,
o f the

b u t t h e mean ovary weight was l e s s than 13 %

d i p l o i d control.
of triploid

t h a t ovaries
those o f

more abnormal than

The oocytes appeared t o undergo

and o v u l a t i o n was n o t observed.

Triploid

fish

morphologically

do

not

different

1979; Gervai e t

triploid

h y b r i d s were

d i p l o i d hybrids.

degeneration,

Gall,

H i s t o l o g i c a l examinations revealed

dl.,

stickleback

appear
from

to

diploids

be

strikingly

(Thorgaard

and

1980b). Swarup (1959) found t h a t

(Gasterosteus

aculeatus) had s h o r t e r

t r u n k s and longer t a i l s than t h e d i p l o i d c o n t r o l s .

However

t r i p l o i d i n t e r s p e c i f i c h y b r i d s may be r e a d i l y d i s t i n g u i s h e d
from d i p l o i d h y b r i d s i n

some cases

because o f d i f f e r e n c e s

i n gene dosage from t h e parent species.

I

7. Application
The primary

i n t e r e s t i n induced t r i p l o i d f i s h l i e s i n

t h e i r s t e r i l i t y and i n t h e p o s s i b i l i t y

that t h i s

may lead
*

t o extended
the

growth and/or s u r v i v a l i n mature f i s h .

performance

accumulating.

The

of

sterile

result of

triploids

triploid

are

Data on
still

may m a i n t a i n t h e i r

growth much b e t t e r than d i p l o i d s a r e shown by Wolter e t al.
( 1982b)

.

Sterility i s

a l s o advantageous i n s i t u a t i o n where t h e

IZCb

c o n t r o l o f r e p r o d u c t i o n is

*

desirable. Triploid

(Ctempharyn~odon i d e l l w )
1

are

being

weed c o n t r o l p r o g r a m s ( T h o r g a a r d ,
be

desirable

associated

species

stunting

application
triploid

for

occur

of

induced

hybrids

are

adopted i n a q u a t i c

19831,
where

a n d t r i p l o i d s may
o v e r p o p u l a t i o n 1 and

(Thorgaard,

typically

1986).

lies

triploidy

grass c a r p

much

Another

in the fact that

more

viable

than

d i p l o i d h y b r i d s ( A l l e n a n d S t a n l e y , 1981; C h e v a s s u s et a l . ,
1983; S h e e r e r a n d T h o r g a a r d ,

to

combine

desirable

1983). T h i s

characters

from

make it possible

two

species i n a

sterile h y b r i d .
T k m o s t successful application
h a s been t h e i n d u c t i o n o f t r i p l o i d s .
method h a v e p r o v e n e f f e c t i v e ,

polyploid

so f a r

A v a r i e t y of i n d u c t i o n

i n animals, such as

f i s h and

a r r e s t e d m e i o s i s p r i o r t o f e r t i l i z a t i o n are

molluscs, with
candidates

of

(Allen

and

Stanley,

1981).

Triploid hybrids

between p l a i c e a n d f l o u n d e r are o b s e r v e d t o b e m o r e s t e r i l e
v

t h a n t h e i r d i p l o i d c o u n t e r p a r t s (Purdom, 1 9 7 2

; Thorgaard,

111. MATERIALS AND METHODS

*

The experiments were conducted a t t h e hatchery o f F i s h
C u l t u r e and F i s h e r i e s Department
University,

the

Netherlands

o f Wageningen A g r i c u l t u r e

fram October 1987 t o September

1988 i n t h e f o l l o w i n g phases.

1. I n d u c t i o n o f r e p r o d u c t i o n and t r i p l o i d y o f C l a r i a s

batrachus L . c o v e r i n g :
1) I n d u c t i o n o f spermatogenesis,

2 ) Determination o f s t r i p p i n g l a t e n c y time,
3 ) c o l d shocking eggs,

4) sperm i r r a d i a t i o n ,
5 ) up t o 8 ) gynogenesis.

2.

Identification of t r i p l o i d fish.

3.

Growth performance o f t r i p l o i d and d i p l o i d f i s h .

1. M a t e r i a l s
1.1.

Parental f i s h ,

husbandry o f f r y and experimental f i s h
#

Larvae o f t h e Asian
c o l l e c t e d from
Indonesia.

c a t f i s h (Clarias

b a t r a c h u s ) were

a f i s h pond i n Kabupaten B l i t a r ,

They were r a i s e d t o m a t u r i t y a t t h e

East Java,
hatchery o f

t h e Department o f F i s h C u l t u r e and F i s h e r i e s o f A g r i c u l t u r e
U n i v e r s i t y i n Wageninqen.

The

first

generation

o f these

f i s h served
e

as p a r e n t a l f i s h .

t h e f i r s t g e n e r a t i o n had

200

weight o f

-

600 g.

The experiment,

reached an

s t a r t e d when

14 months and

age o f

The techniques used f o r a r t i f i c i a l

induced breeding a r e d e s c r i b e d by Zonneveld e t a l .
F r y produced f o r experiments

250 1

g l a s s f l o w through tanks,

2 and

J were

raised i n

putative d i p l o i d

and t r i p l o i d ( T group) f i s h were k e p t s e p a r a t e l y .
fed

nauplii

salina

Artemia

f o l l o w e d by a commercial
onwards a t

a ration

Hoogendorn

(1981)

for

trout

o f 16.8

as

the

the

diet

(D group)
They were

f i r s t two weeks,

fed

from

g.kg-O-o.d-z,
optimal

(1988).

two weeks

recommended by

feeding

ration

for

commercial p r o d u c t i o n o f C l a r i a s g a r i e p i n w .
In

the

weight

range

" r u p t u r e d i n t e n t i n e syndroms"
f o r Clarias

1 t o 20 g t h e i n c i d e n c e o f

of

g a r i e p i n u s (Boon

(RIS) has o f t e n been r e p o r t e d
e t al.,

seemed t o be l e s s s u s c e p t i b l e t o
level.

For

RIS

1987). J u v e n i l e f i s h
when

fed

at

a low

t h i s reason f e e d i n g r a t i o n s were lowered i n t h e

mentioned weight range.
One s e t o f

aquaria

with

water

r e c i r c u l a t i n g system

were used f o r eggs i n c u b a t i o n and c o l d shocking treatments.
F o r l a r v a e r e a r i n g and growing were used one s e t o f a q u a r i a

( 4 aquaria,

volume 400

and one s e t of

1 ) w i t h f l o w through water system,

a q u a r i a (20 aquarla,

volume 80 1 ) w i t h water

r e c i r c u l a t i n g system were used f o r f e e d i n g experiment.
During t h e

f e e d i n g experiments

m a i n t a i n e d a t (25

0.5j°C.

water temperature was

The f l o w r a t e ranged from

1 to

-

23

2 l/minute

f o r each aquarium (volume 140 1).

oxygen c o n c e n t r a t i o n o f t h e

i n f l o w i n g water

The dissolved
was k e p t near

4

saturation

and

was

o u t f l o w i n g water.
exceeded

values

always

above 40 % s a t u r a t i o n f o r tire

Concentration

2

of

and

of

and

NH4*

NOn- never

1 mg/l r e s p e c t i v e l y ,

w h i l e pH

v a l u e s ranged from 7 t o 7.5.

1.2.

Hormone

The

cPE

Crescent

(carp

Research

pituitary

Chemicals,

extract)
Virginia

manufactured

by

USA were used f o r

i n d u c t i o n o f eggs o v u l a t i o n i n t h e a r t i f i c i a l r e p r o d u c t i o n .
The cPE

powder was

suspended i n 0.9

% NaCl (cPS) p r i o r t o

injection.

1.3.

Equipment

The equipmept f o r analyses
(soxlett),

and

energy

(bomb

d e t e r m i n i n g body composition.

(2.5

cc),

syringe

and

o f protein
calorimeter)

The c e n t r i f u g e ,

(kjeltec),

fat

were used f o r
p l a s t i c tubes

needle were used f o r blood sample
*

analysis.

The f l o w cytometer was used

(RBC) o r

DNA measurement.

f o r red

blood c e l l s

The s p e c i f i c a t i o n o f t h e machine

Machine t y p e : Fluorescence Associated C e l l - S o r t e r
(FACStar).
Laser t y p e
: brgon-ion l a s e r ; o u t p u t = 5 watts.
Manufacturer : Becton Dickinson.

Measurements : Forward scatter CFSCI; Side scatter LSSC3;
Fluorescence 1 CFL11; F l u o r e s ~ e n c e 2 CFt21.
: laser : 488 nm

'Set-up

filter: long pass 585 [default).
The signal

from the

size measurements

( F S C and S S C ) were

linearly amplified. F L I was amplified logarithmic,

A 1 1 solutions used for washing, f i x a t ~ a n

amplified linear.
and staining

FL2 was

of blood

bacterial filter

cells were

before usage.

filtered with

a 0.2 mm

Only FSC and FL2 were used

to measure cells size and amount of DNA respectively.

2. Methods
2.1.

Induction of reproduction and triploidy

2.1.1.

Artificial reproduction
Three to four days prior to hypophysation the parental

fish were

sexed, macroecapically,

and transferred

to 70 1

aquaria. The water temperature was held

constant at

0.5)OC.

mg

Males

were

suspension t cPS

)

injected

with

4

in order to stimulate

(25 +_

carp pituitary

and estimate milt

production. Females of C l a r i a s batrachus received a similar
dosage of cPS in order
time

far

ovulation.

to

determine

Viability

determined by estimating the
eggs.

of

hatching

the

optimal latency

sperm
rate

and

eggs

was

of fertilized

The means of duplos were calculated per fist).

2.1.2.
4

and

Cold shocking eggs
About 200

eggs per

incubated

at

(diameter 10

sample were

27OC

cm),

in

f e r t i l i z e d with m i l t

plastic

circular

chambers

which were provided w i t h a gauze bottom.

Cold shocking was c a r r i e d o u t by t r a n s f e r r i n g t h e eggs from
water o f

27*C

times a f t e r

Richter e t

treatment,

t h e eggs

The

and

hatching

The

treatment

hatching

t h e shock was

1. A f t e r

transferred

rate

was done a t v a r i o u s

duration o f

1987

the

(U.D),

were expressed as

2.1.3.

of

This

S°C.

al.,

were

effects

undeveloped eggs

(H.D.)

water o f

fertilization.

constant (

27OC.

to

rate

t h e c o l d shock

again
were
of

to

water o f

measured

by

deformed l a r v a e

o f normal l a r v a e (H.N.).

These

percentages o f number o f eggs incubated.

Assessment o f t r i p l o i d y

The

effectiveness

suppression of

t h e second

of

cold-shocking

c.q.

the

m e i o t i c d i v i s i o n o f t h e egg was

determined by f e r t i l i z i n g u n t r e a t e d eggs

( c o n t r o l o f sperm

i r r a d i a t i o n ) and eggs (gynogenetic c o n t r o l ) w i t h i r r a d i a t e d
sperm.

I n t h e f i r s t case h a p l o i d u n v i a b l e embryos should be

obtained,

i n d i c a t i n g t h a t t h e i r r a d i a t e d sperm was,

genetically inactive.
diploid

embryos

indeed,

I n t h e second case gynogenetic viable

should

be

expected,

which

r e t e n t i o n o f t h e second p o l a r body had occurred.

showed t h a t

-

2.1.4.
*

I r r a d i a t i o n o f sperm

dilluted 1

The m i l t stock was

: 1O

s o l u t i o n t o p r e v e n t sperm a c t i v a t i o n .
spread on a l a r g e watch g l a s s
layer
with

of

spermatozoa)

ice.

estimated

d i s t a n c e between

1988 1 .

a thin

stirred

during

The m o t i l i t y o f sperm

after

the

treatment.

The

and t h e sperm sample was 25 cm.

were mixed

c o n t r o l on

t o obtain

mechanically

al.,

t h e lamp

NaCi

placed on a p e t r i d i s h f i l l e d

immediately

Samples o f 200 eggs
sperm (

was

Komen e t

irradiation (

(%I
was

m i l t

The

%

Samples o f 10 m l were

( i n order

and

w i t h 0.4

w i t h 100

sperm i r r a d i a t i o n

u l of irradiated

) o r w i t h untreated

sperm ( c o n t r o l on eggs q u a l i t y I. The h a t c h i n g r a t e o f t h e
f e r t i l i z e d eggs

2.1.5.

(%I

was c a l c u l a t e d .

Experimental designs and s t a t i s t i c a l methods

Experiment 1 ( 23-10-1987)
I

M i l t

production

"Wageningen
experiment

hatchery
was

of

Clarias

conditions"

designed

to

batrachus

i s

very

kept

under

moderate.

The

s t i m u l a t e m i l t production i n

t h i s f i s h species. F i v e a d u l t males were i n j e c t e d w i t h 4 mg
cPS/kg body weight every 2 days d u r i n g a 12 day p e r i o d ,

5

adult

males

physiological

received

salt

corresponding

solution.

s a c r i f i c e d afterwards.

The

The t e s t i s

volumes

of

and
a

f i s h were s t r r p p e d and
somatic index

( T S I J and

-

t h e s e m i n a l i s v e s i c u l a somatic index ( S V S I ) were determined
#

and t h e sperm q u a l i t y
obtained

from

both

the

m i l t and

of stripped

testis

was

determined

of m i l t
by

egg

fertilization.

Experiment 2 ( 11-11-1987

T h i s experiment was c a r r i e d o u t t o
stripping

latency

"Wageningen

time

hatchery

determine t h e b e s t

C l a r i a s batrachus k e p t under

for

conditions".

Twenty

females

hypophysized and d i v i d e d i n 5 subgroups o f 4 females,

were s t r i p p e d a t l a t e n c y times o f

13, 15,

hours,

of

respectively.

Incubation

17,

which
and 21

19,

fertilized

were

eggs was

I

carried out a t

29OC

and

survival

rates

were c a l c u l a t e d

afterwards.

Experiment

3

*
(

19-11-1987,

24-11-1987,

7-12-1987,

A1-12-

1987 )

The t h i r d experiment

was

designed

to

determine t h e
/

best time

interval for

c o l d shocking

o f eggs i n o r d e r t o

o b t a i n t h e r e t e n t i o n o f t h e second

p o l a r body

al.,

a single

1987).

Eggs were

exposed t o

c o n s t a n t d u r a t i o n ( 20 minutes
with

one

minute

fertilization.

intervals

)

The

up

till

c o l d shock o f

shocks
10

(Richter e t

were g i v e n

minutes

The f e r t i l i z a t i o n r a t e and h a t c h i n g

after
rate of

28

eggs were

estimated.

experiment.
because o f

This

The p l o i d y was n o t determined i n t h i s

experiment

was

repeated

unexpected t e c h n i c a l problems,

three

times,

which c o u l d have

affected the results.

Experiment 4 ( 30-11-1987)

This
effect

experiment

of

was

irradiation

sacrificed

out

Four

and

the

males
testes

i r r a d i a t i o n d u r a t i o n s o f sperm were 5,
and

60

minutes.

to

determine t h e

d u r a t i o n on g e n e t i c a l i n a c t i v a t i o n

and m o r t a l i t y o f sperm.
were

carried

Sperm

C l a r i a s batrachus

of

were
10, 20,

grinded.

30, 40,

The

50,

m a t i l i t y and t h e hatching r a t e o f

f e r t i l i z e d eggs were used as parameters i n t h i s experiment.

6,

Experiment 5,

Experiments
reproduce

the

five

up

effects

t h i r d experiment,
by

and 8 (22-12-1987,7-1-1988,

using

of

to

eight

was c a r r i e d

and t o check t h e

p o l a r body
to

assess

the

and t h e

adverse

to

c o l d shocked

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

The f i r s t f e r t i l i z a t i o n

determine whether

was r e t a i n e d

designed

obtained i n the

ploidy i n

gynogenetic c o n t r o l s .

out t o

were

cold-shocking

w i t h i r r a d i a t e d o r u n t r e a t e d sperm.

done

20-4-1988,

.

4-5-1988)

eggs

7

and when t h e second

second f e r t i l i z a t i o n was

effect

o f c o l d shocking on

-

embryonic

development.

compared w i t h

The

effect

of

the

latter

was

hatching percentages o f non t r e a t e d eggs and

I

sperm.

Untreated eggs were a l s o f e r t i l i z e d

with irradiated

sperm t o check t h e sperm c a p a c i t y t o f e r t i l i z e d eggs.
I n the

5,

f i f t h experiment,

eggs were cold-shocked

and 8 minutes a f t e r f e r t i l i z a t i o n .

either

with

untreated

sperm

or

a t 3,

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

with

irradiated

sperm

( i r r a d i a t i o n d u r a t i o n 30 and 35 minutes).
I n t h e s i x t h experiment,

5,

and

8

minutes

i r r a d i a t i o n was

5,

after fertilization.
10,

20,

a t 3,

eggs were c o l d shocked

D u r a t i o n o f sperm

o r 30 minutes.

In t h e seventh experiment, eggs were c o l d shocked a t 3
minutes
10,

after

15, 20,

25,

and 30 minutes.

I n t h e e i g h t experiment,
2,

3,

4,

5,

and sperm i r r a d i a t e d f o r 5,

fertilization,

and 8

a t 1,

eggs were c o l d shocked

minutes a f t e r f e r t i l i z a t i o n ,

and sperm

i r r a d i a t e d f o r 20 minutes.
Hatching

rate

f e r t i l i z a t i o n ware

of

eggs,

used as

48

hr

and

of

eggs,

including

Hatching r a t e o f 72
larvae,

excluding

hr

i s

after

v a r i a b l e i n these experiments.

Hatching r a t e o f 48 h r a f t e r f e r t i l i z a t i o n i s
rate

hr

72

deformed
the

deformed

experiments used hatching r a t e ,

and

hatching
and

t h e hatching

haploid
rate

haploid

larvae.

o f normai

larvae.

as one o f t h e v a r i a b l e s

All

*

Hatching rate = ((nl)/(nl+ud+dl))SlOO
nl = normal larvae

0

ud = undeveloped eggs
dl = deformed larvae
(Richter et

1985)

dl.,

Statistical analyses
The data were tested for normality
homogeneity

of

Rohlf, 1981).
root

variance
The data

transformation

using Bartlett's test (Sokal and
were normalized

and

was performed

of W

using

An

by arcsine square

subsequently, difference between

groups were tested with students
1981). Calculation

using W-values and

t-test (Sokal

and Rohlf,

values, t-student test, and Anova
Interactive

Statistical Analysis

Program for Microcomputers by NH Analytical Software (Nimis
and Heisey, 1982).

2.2.
2.2.1.

Identification of triploid and diploid fish
Mass production of diploid and triploid fish

.
The

cold-shocked

fish,

putative

triploid

(T)

and

untreated fish

or diploid (D) were mass produced using the

best procedure

from

batrachus

females

previous
were

weight 1 6 hours prior
sacrificed

to

obtain

experiment.

injected

to stripping,
milt.

with

4

Five Clarias
mg cPS/kg body

and three

Testes

were

males were

grinded

and

suspended w i t h p h y s i o l o g i c a l NaCl 0.9

.

%

A l l

eggs were

mixed and placed i n 5 t r a y s .
One p a r t

o f them

were f e r t i l i z e d

w i t h 1 p a r t o f the

mixed sperm and incubated i n water

o f 27OC

normal

fish

(diploid)

fish.

Triploid

t o produce t h e

were

produced by

f e r t i l i z i n g t h e remaining eggs w i t h sperm and c o l d shocking
them from

27%

t o 5% a t 3 minutes a f t e r f e r t i l i z a t i o n f o r

20 minutes (based on t h e experiment 3 and 8 ) .
Eggs hatched a t 4/3/88.
glass flowthrough

tanks a t (25

A r t e m i a salina f o r t h e
period a
16.8

first

in

250 1

weeks,

and

after this

1987; Hoogendorn,

1981).

Identification of t r i p l o i d f i s h

vasculature o f

m l

,40 randomly

and T and s t o r e d i n i c e .
solution

used

were

drawn

A volume

o f 0.15

sample

for

washing,

o f b o t h group

D

f i x a t i o n and s t a i n i n g o f

mm

bacterial f i l t e r .

m l o f a 6 % N a - c i t r a t e s o l u t i o n was added

were

coagulation.

suspended

c o n t a i n i n g 10 % h a - c i t r a t e
200 G

t h e caudal

Sampled f i s h were 140 days o f age.

t o every blood sample t o prevent
every

from

selected f i s h

blood c e l l s were f i l t e r e d w i t h a 0.20

of

raised

1 ) O C and were fed n a u p l i i

two

(Henken e t a l ,

Blood samples o f 1.5

A l l

t

were

commercial t r o u t d i e t a t a r a t i o n o f

g.kg-o-6.d-a

2.2.2.

Fish

on

0.2

(TbS-Na) and

Two d r o p l e t s

m l i c e c o l d TBS
centrifuged

at

f o r 10 minutes and 4OC. The supernatant was decanted

and packed c e l l s

were

resuspended

in

TbS-Na.

The above

washing procedure was repeated t h r e e times.
d e c a n t a t i o n c e l l s were

suspended

#

formalin

and

samples were

stored

overnight

containing

1 %

The n e x t morning

4%.

t h r e e times

again washed

washing packed

TBS

in
at

A f t e r the t h i r d

w i t h TBS-Na.

After

suspended i n 1 m l o f a s o l u t i o n

c e l l s were

c o n t a i n i n g 5 % propidium i o d i d e ( a DNA s p e c i f i c f l u o r e s c e n t
dye)

and

1

%

and vortesed f o r 1 minute.

Na-citrate

sample was s y r i n g e d through
clumping.

Sample

temperature.
times

and

were

a

26

stored

gauge
for

needle

two

hours

The

t o avoid
at

room

A f t e r s t a i n i n g samples were again washed t h r e e
resuspended

i n

volume

a

c y t o m e t r i c a n a l y s i s was performed

of

within

3

m l TBS.

two

Flow

hours a f t e r

t h e l a s t washing.

DNA f l u o r e s c e n c e

(FL) and

p r o p o r t i o n a l t o t h e amount
using a
20,000

FACStar f l o w

MPL )

.

2.2.3.

cytometer.

was kecorded.

[mean peak l o c a t i o n
i s

o f DNA

sufficiently

present,

was measured

Fluorescence o f 10,000

c e l l s were measured from every

value ( W )

it

c e l l s s i z e (FSC), which i s

fish,

and

to

t h e modal

The corresponding channel number

[MPLI) was chosen as assay
linear

unit,

( t r i p l o i d M P L = 1.5

since

1 diploid

-

Statistics

Far each p l o i d y group mean MPL
were c a l c u l a t e d .

and standard d e v i a t r o n

Successively ' r i g h t - o n e - s i d e d '

and ' l e f t - o n e - s i d e d '

( t r i p l o i d s ) 0.05

(diploids)

c r i t i c a l l e v e l s were

calculated,

u s i n g t h e f o l l o w i n g formula o f A.

where XI
X
t,-a
s

= c r i t i c a l level
= a r i t h m a t i c mean peak l o c a t i o n
= t - v a l u e a t n-1 degrees o f freedom

= standard d e v i a t i o n .
= c r i t i c a l level a t 0-05

OC

2.3.

Growth performance o f t r i p l o i d and d i p l o i d f i s h

The

feeding

experiment

was

done

i n two steps.

f i r s t experiment was done when t h e f i s h a t 109 days
(the

gonad

of

normal

fish

experiment was conducted
second

was

done

when

normal f i s h was mature),
August t o

from
the

started
July
fish

to

o f age

develop).

August

This

1988.

The

a t 178 days of age ( t h e

T h i s experiment was conducted from

October 1988. Each experiment r e q u i r e d two weeks

o f a d a p t a t i o n and 6 weeks o f feeding.
t h e same

to

The

population o f

fish,

Both experiments used

which were produced i n March

1988 (mass p r o d u c t i o n ) .

2.3.1.

Experimental desiqns

Both

experiments

(diploid,CDll

and

of

W i t h i n group D and T,
resulting
feeding

in

8

-

contained
cold-treated
four

treatment

groups

untreated

(triploid,LTI)

feeding r a t i o n s
combinations

levels). The mentianed

of

level is

fish.

were employed,

( 2 p l o i d y and

4

t h e feeding level

resulting
#

in

the

best

feed conversion ratio for C l a r l a s

gariepinus (Hoogendorn, 1 9 8 1 ) .

w a s carried

o u t in

Each

treatment combination

duplicate. D ~ s t r i b u t i o n o f t h e various

treatment combinations

over

the

experimental

aquaria

15

given in Fig. 3. Treatments w e r e not placed a t random s i n c e
t h e laboratory condition was sufficiently homogeneous.

I';'-"-"..."

.

.
r-.---.----.

r-.----.--

-

r--

--.--

,--.-

-

-.....-....-.... .---.-p-..-.--..-..

..
...............
"-., r."---"--l
1

II
II
II
II
, Ii
iI
II
II
5
11
7
11
8
11
3
11
4
11
6
11
1
11
2 11
II
II
II
II
II
II
iI
II
l l C D , l l II CT,1111 CD,2111 CT,2111 CD,3111 CT,3111 CD,4111 CT,4111
II
II
II
II
II
II
II
II
II
.--....-.-.................-.-..
........., I......-.--L..-...-.-...-..
.-....-..
L
...................
...........I.L--r
. ....-.-.
. .--..-.....-...-,.- 4 L
,"."...*l.--""-"..l
.-.--....-.
r.-"".-.." ..-..
,.".- -.-.- -.. ..---..-..--.--,
I1
II
II
II
II
11
II
II
II
II
11 9
11 10 11 11 11 12 11 13 11 14 11 19 II 16 II
1I
II
II
11
II
II
II
II
11
I I C T r l l II CD,4111CT,41
l l C D , l I llCT,21 llCD,23 llCT,31 llCD,31 ii
II
II
II
II
11
II
II
II
II
Ii
Ii
II

<

4 L

"-(

1 L

P.."""'."

L.&rL:----.."-...--czL-;lz
L:2::zzz::-;f,C=:;zL.

-2

1

:z;;2:yI2.
::

=;;

I'

--.-J

I
'
L
T
z
;
:
z

J-L.

L.

I

,JJ;::;L:z;-LTI';:L

I

C'

!,k;:,;:

I::yL.-

Ploidy : D = diploid ,(normal)
T = triploid (putative)
1 2 1
= numbers of aquaria.
CD.11,
CD,2I,.CD,41
= diploid
fish with feeding ration 1,
2,.4 o f optimum feeding ration for C l a r i a s gariepinus.
CT,13,

ration

~ ~ ~ 2L 1
T . .,41
.
= putative triploid fish
1,2. .4
optimum
feeding
ration

with feeding
for Clarias

gariepinus.

F i g u r e 3.

Schematic representation of t h e
experimental unit used and t h e distribution
of t h e treatment combinations.

E x p e r i m e n t a l procedure

2.3.2.
*

A t feeding

D

both

and

separately
fish

experiment 1,

T

over

each.

group.
8

Fish

environment

for

t h e number

g

two

weeks.

that

Q,

to

adapt

s t a r t of

distributeo

to

new

i

I n t h ~ sp e r i o d they were f e d
when

t h e experiment s t a r t e d ,

i n aquarium was reduced from 45 t o 35.
an age

time.

o f 123

days and

a weight o f

Ten randomly sampled f i s h o f b o t h

group D and T