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

I .

INTRODUCTION

I n East Java, Indonesia, t h e Asian c a t f i s h ( C l a r i a s #

b a t r a c h u s L . ) 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 becoining an

i m p o r t a n t species f o r aquaculture. F i s h farmers a r e i n t e n s i f y i n g t h e i r e f f o r t s , s i n c e

C l a r i a s

b a t r a c h u s , 1ocally.known as " i k a n l e l e " , i s t h e h i g h e s t p r i z e d f r e s h - water f i s h species i n t h i s r e g i o n . U n f o r t u n a t e l y t h e farmers f a c e a complex o f problems which hamper t h e i r e f f o r t t o expand and i n t e n s i f y t h e c u l t u r e o f i k a n l e l e . One o f t h e i r problems concerns a 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 . T h i s problem can \ b e s o l v e d

by

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

A r t i f i c i a l induced breeding techniques i n Asian c a t f i s h have been r e c e n t l y developed by Zonneveld e t a l . ,

(1988).

A s i n g l e i n t r a m u s c u l a r i n j e c t i o n o f cPS ( c a r p p i t u i t a r y suspension) ( dosage 6 mg/kg body welght ) i n combination w i t h a l a t e n c y t i m e o f 17 hours ( a t 25OC )

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 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 v a r i o u s techniques, i n c l u d i n g s e l e c t l o r i f o r disease r e s i s t a n c e and behavior c h a r a c t e r s ,

I

Chromosomal manipulation and artificial mutations have

long been established a s highly advantageous in plant

breeding. It i s very likely

that they will be j u s t

as \

efficient in fish. T h e fish breeder, unlike that of tarn1

animals, can handle both male and female gametes outslde

the organisms

and has manipulative control o v e r the

developing zygote (Moav, 1976).

Polyploidy h a s been induced in a number of fish

species using a variety of techniques (Purdom, 1972;

Valenti, 1975; Refstie e t al.,

1977; Allen and Stanley,

1979; Gervai

e t al.,

1980a; Wolters e t al.,

1981).

Induction of triplaidy in

f i s h

might potentially

be

useful

for t h e control of overpopulation, f o r increasing the

growth rate in juveniles, and for extending survival and

improving growth in mature fish.

Most of the interest in induced triploidy has been

from a n aquaculture perspective

w i t h t h e

hope that

triploids might grow faster than diploid a s juvenile o r a s

mature fish. T h i s might result from triploidy

p e r

se

o r

a s

an indirect result of sterility o f triploids (Thorgaard,

Juvenile triploid have generally been found t o grow n o

faster than diploids.

Growth of j u v e n i l e triploids was

similar t o t h a t

of

diploids

in stickleback

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

aculeatus) (Swarup, 19591, common c a r p

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

3

punctatus) (Wolters

et

dl.,

1982a). In t h e blue tilapia

,

(Oreochramis aurea)

,

juvenile triploid w e r e found t o

be

larger than diploids {Valenti, 1975). However, in Pacific

s a

1

mon

(Oncurhynchus

kisutch)

(

Utter

e t

a1

.

,

1983)

triploids may grow slower than diploids.

Several s t u d i e s 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

i s used for growth.

Triploid

channel

catfish were

significantly heavier than diploids a t t h e a g e of 8 months

and older (Wolters e t

dl.,

1982a).

In African catfish

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

affected by triploidy. Body composition, however, w a s

strongly affected.

Triploid fish deposited, per gram of

Qrowth, less protein and more fat (Richter e t al., 1987).

Induction o f triploidy of Clarias batrachus

L.

is

possible through t h e manipulation of chromosome. However,

the problems

are

X )

how fish conditon (eggs

and sperm

quality) be s e t up,

2 )

what methods will be used,

3 )

how to

determine t h e triploid fish,

and

4)

what

is the

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

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 m a n i p u l a t i o n o f chromosomes becomes f e a s i b l e d u r i n g t h e n u c l e a r c y c l e s o f c e l l 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 s e t . I n animals, m e i o s i s i n t h e eggs is t t t e

p r i n c i p a l c e l l d i v i s i o n phase where m a n i p u l a t i o n

is

p o s s i b l e , and i n f i s h , and o t h e r animals w i t h e x t e r n a l f e r t i l i z a t i o n , a r t i f i c i a l processes can 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 p e r i o d d u r i n g t h e f o r m a t i o n o f t h e zygote. C o n t r o l o f t h e 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 i n eggs b u t 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 (Purdom, 1983).

I n commercial f i s h , d e v i a t i o n from t h e normal r e p r o d u c t i o n p a t t e r n can be induced e a s i l y ; by r a i s i n g p a r e n t s o f two d i f f e r e n t species ( h y b r i d i z a t i o n ) ; o n l y one p a r e n t (gynogenesis and androgenesis) o r i n c r e a s e o f chromosome numbers ( p o l y p l o i d y ) . These phenomena can be e x p l o i t e d i n p l a n n i n g new schemes o f g e n e t i c improvement f o r animal breeding ( a l r e a d y c l a s s i c a l i n p l a n t ) , such as t h e c r o s s i n g o f i n b r e d l i n e s (produced by gynogenesis) o r t h e use o f p o l y p l o i d s (Purdom, 1983; Chourrout, 1984).

D i p l o i d gynogenetic i n d i v i d u a l s c o u l d be produced i t

o f sperm, induced r e t e n t i o n o f t h e second p o l a r body, o r induced suppression o f t h e f i r s t cleavage ( F i g . 1 ) . D i p l o i d

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 maternal chromosome s e t . I f t h e l a t t e r was achieved by r e t e n t i o n o f second p o l a r body, t h e r e s u l t i n g embryo s t a r t s from two d i f f e r e n t t e r m i n a l p r o d u c t s o f t h e same m e i o s i s and so i s n o t homozygous a t a l l l o c i . I f suppression o f t h e f i r s t cleavage i s used t o d i p l a i d i z e t h e maternal s e t , t h e 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 t h e f u s i o n o f two m i t o t i c p r o d u c t s (Purdom, 1983;

Chourrout, 1984).

A v a r i e t y of r a d i a t i o n t r e a t m e n t s a r e a v a i l a b l e t o i n a c t i v a t e sperm chromosomes. R a d i a t i o n 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 w i t h gamma r a y s u s u a l l y from 'OCo o r &OCs (Purdom, 1969; Nagy e t

d l . , 1978; Chourrout e t d l . , 1980; R e f s t i e e t d l . , 1982), X

M e i o s i s I 1 1 s t m i t o s i s

I

0 .

sperm

i n a c t i v a t i o n

-

9

h a p l o i d

r ' -

gynogenetic

R e t e n t i o n

o f i n a c t i v a t i o n PB II+sperm

@ - 0 - @ - @ 3

h e t e r o z d i p l o i d ygous gynogenetic Suppression

o f 1st cleavage

p-

0-

@

-

@

homoz d i p l o i d ygous g y n o g e n e t ~ c Reten t i o n

of

PB

I 1

t r i p l o i d

I

Suppression o f

1st cleavage

,p

-

Gi

@

-

@

t e t r a p l o i d

F i g u r e 1. Three o p e r a t i o n s used f o r producing

gynogenetic i n d i v i d u a l s , t r i p l o i d s , and t e t r a p l o i d s i n lower v e r t e b r a t e s : sperm i n a c t i v a t i o n , r e t e n t i o n o f second p o l a r body and suppression o f f i r s t cleavage

(Chourrout, 1984).

H t l o p h o r c 11 Anophosr 1 1 0t98nrrol1119 H t ~ o p l ~ o t t ol 2nd polof body 1st ormlon ol

rmnd )o

199 n u ~ l r u r fuctd sptrm 8

( P ~ ~ P ~ O S ! 11) Cold 'hock 899 ~ l o ~ v c l t ~ c o l d

I

sMch

5.

w

'8'

Y O H b l l L

1 R I f'lOIO l r l R A i ; 0 1 0 Oll'LOIO

[image:113.540.46.511.4.749.2]

2. P o l y p l o i d p r o d u c t i o n

I

Induced p o l y p l o i d y r e f e r s t o t h e p r o d u c t i o n o f i n d i v i d u a l s w i t h e x t r a s e t s o f chromosomes. T h i s can be done by t r e a t i n g f e r t i l i z e d eggs w i t h e i t h e r temperature shock, h y d r o s t a t i c pressure o r chemical treatment. I f t h e treatments a r e a p p l i e d s h o r t l y a f t e r f e r t i l i z a t i o n , t r i p l o i d s can be produced due t o r e t e n t i o n o f t h e second p o l a r body o f t h e eggs. I f t h e t r e a t m e n t s a r e s h o r t l y b e f o r e t h e f i r s t cleavage d i v i s i o n , 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 t h a t scheme t r i p l o i d f i s h can be produced by c o l d shocking 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, t r e a t m e n t s o f f e r t i l i z e d eggs have been w i d e l y used t o suppress t h e second m e i o t i c d i v i s i o n

or

second p o l a r body e x t r u s i o n i n f i s h , b o t h c o l d shock (e.g., Purdom, 1969; V a l e n t i , 1975; Nagy e t al.,1978; Chaurrout,

-.

1980; R e f s t i e e t d l . , 1982; R i c h t e r e t al.,1987; Lomen e t

a l . , 1988), and heat shock (Chourrout, 1980; Thorgaard

et

al., 1981#

BenPey and S u t e r l i n ,

1984).

r e l a t e d t o g e n e t i c background and egg m a t u r i t y stage 1 R e f s t i e e t al., 1982). A simple, p r a c t i c a l approach for- i n d u c t i o n o f p o l y p l o i d y by a temperature shock i s c a r r i e d o u t s h o r t l y a f t e r f e r t i l i z a t i o n ( f o r induced t r i p l o i d y ) o r s h o r t l y b e f o r e f i r s t cleavage ( f o r t e t r a p l o i d y ) a t j u s t below l e t h a l temperatures. An advantage i s t h a t a u n i f o r m t r e a t m e n t can be g i v e n t o a1 1 eggs. Temperature treatments a r e inexpensive 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 p o l y p l o i d s prove v a l u a b l e . I n d u c t i o n o f t r i p l o i d y by temperature shocking o f eggs i n some t e l e o s t species

used

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

Table 1. I n d u c t i o n o f t r i p l o l d y by temperature shocking o f eggs i n some t e l e o s t species

( R i c h t e r e t d l . , 1987)

Solmo gairdnerl

Solmo salor 1 0

-

3 2

Cyprinus corpio

I

5 1 2 0 - 2

I I

-

~ e l a t i v c t o controls

IVOLTEItS C I id., 1 9 8 1

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

[image:115.540.48.506.30.743.2]

Hydrostatic pressure ha5 been used t o black second

polar body extrusion o r first mitotic division. Streisinger

*

e t dl.,

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

and Yamazaki

(1983) and Chourrout (1984) with rainbow

trout(Salmo gairdneri)

.

Although application of hydrostatic

pressure treatments requlres

more

specific equipment

[pressure cell and hydraulic press) than temperature shock

,

treatments, t h e method diserves wide investigation because

it may be 1

s

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

shock.

Chemicals may a l s o be used t o block polar body

extrusion o r mitotic division in fertilized eggs. Refstie

e t dl.

(1977) reported producing mosaic polyploid-diploid

Atlantic salmon [ S a l m o salar) after exposing fertilized

e g g s t o cytochalasin B .

Kanka and R a b i n Thorgaard (1983)

reported observing diplaid-triploid

mosaic, triploid and

tetraploid Tinca tinca after treating fertilized e g g s with

cytochalasin

B.

3.

Viability o f polyploids

T h e 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.

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

t o

increased viability in

interspecific hybrids (Sheerer and Thorgaard, 1983). In

frogs, triploid interspecific hybrids a r e sometimes more

viable than diploid hybrids (Bogart

In

Thorgaard, 1983).

Interspecific triploid hybrids could prove useful in

fish culture because hybrid 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

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. F a i l u r e o f m e i o s i s may p r e v e n t o o c y t e development and t h e a s s o c i a t e d i n c r e a s e i n s i z e o f t h e gonad. T r i p l o i d female rainbow t r o u t a t m a t u r i t y had s m a l l , s t r i n g l i k e gonads w i t h many c e l l s a r r e s t e d a t the pachytene stage o f m e i o s i s (Thorgaard and G a l l , 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 c a t f i s h ( W o l t e r s e t al., 1982b3. Gonadal development was a l s o s u b s t a n t i a l l y i n h i b i t e d i n female common c a r p ( G e r v a i e t a l . , 1980b)

I n e x t e r n a l appearence t h e o v a r i e s o f t r l p l o i d C .

g a r i e p i n u s

and s t e r i l e rainbow t r o u t

( S a l m o

g a i r d n e r i )

resemble t h e undeveloped gonads (Thorgaard and G a l l , 1979; L i n c o l n and S c o t t , 1983; Yamazaki, 1983; R i c h t e r e t a l . ,

1987), b u t t r i p l o i d t e s t e s a r e w e l l developed. Such sexual dimorphism i n gonadal development has p r e v i o u s l y been d e s c r i b e d i n t r i p l o i d f i s h ( Purdom, 1972; Thorgaard and G a l l , 1979; O e r v a i

et

d l . ,

1980a;

W o l t e r s e t a l . , 1982a;

Johnson

et

d l . , 1986) and amphibians (Fankhauster, 1941 and

13

process accur l a t e r i n t h e l i f e o f salmonids (Nakamura, 1982).

1

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 f i r s t m e i o t i c d i v i s i o n i n b o t h t h e o v a r i e s and t e s t e s o f t r i p l o i d animals; female t r i p l o l d rainbow t r o u t a r e t h e o n l y r e p o r t e d e x c e p t i o n ( L i n c o l n and S c o t t ,

1983).

Nothing i s known about t h e m e i o t i c nbechanisrn whereby p o s t m e i o t i c c e l l s a r e produced i n such t r i p l o i d s , b u t i t may be s i m p l y t h r o u g h t h e random s e g r e g a t i o n o f chromosomes. T h i s i s supported by t h e f a c t t h a t 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 i n Thorgaard,

1983).

However, f u n c t i o n a l m e i o t i c mechanisms have evolved i n gynogenetic t r i p l o i d , i n v o l v i n g e i t h e r t h e f o r m a t i o i l o f t r i p o l a r s p i n d l e s

(Cherfas,

1969)

or

t h e e n d o m i t o t i c d u p l i c a t i o n o f chromosomes p r i o r t o normal m e i o t i c d i v i s i o n (Cimino,

5. I d e n t i f i c a t i o n o f t r i p l o i d f i s h

T h e assessment a f t h e 'success o f t r e a t m e n t s designed t o produce d i p l o i d s from o t h e r w i s e h a p l o i d eggs i s easy because o f t h e v e r y g r e a t d i f f e r e n c e s i n appearance between d i p l o i d s and h a p l o i d s which become apparent e a r l y i n embryonic development. No such easy o r definitive assay

1 4 methods have consequently been used. Nuclear

site

is

t h e 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 p l o i d y and f o l l o w s

6

t h e e x t e n s i v e work i n amphibians (Fankhausten i n Purdom, 1983). V a s e t s k i i (1967) measured t h e areas o f n u c l e i o f epidermal c e l l s from sturgeon (Acipencer

s t u r i o )

larvae, Purdom (1969) used c a r t i l a g e c e l l n u c l e i o f rainbow t r o u t and v a r i e t y 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., 1982b).

None o f these methods can be r e a d i l y 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 , b u t t h e e r y t h r o c y t e s t u d i e s do employ a r e a d i l y i d e n t i f i a b l e c e l l t y p e and should be r e l i a b l e . I t i s necessary, however, t o r a i s e f i s h t o 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 DNA c o n t e n t o f n u c l e i (Gervai e t al., 1980b) seems t o be more p r e c i s e 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 o f p l o i d y o f any s o r t b u t i t i s d i f f i c u l t i n f i s h and u s u a l l y r e q u i r e s t h a t f i s h be grown t o f r y o r f i n g e r l i n g stage before assay. Chromosome analyses i n blastomere have been used i n salmonids (Purdom and L i n c o l n , 1973; L i n c o l n and S c o t t , 1984) b u t t h e r e s o l v i n g power o f t h e technique i s n o t g r e a t .

(Platichythys f lesus)

and

between

turbot

(Scapthamus

maximus)

and brill

(Scupthamus

rhombus) a r e distinguishable

I

from parental type shortly after hatching by pigment

patterns, and triploids can 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 of triploidy.

T h e labor intensive chrontosomal preparation has limited

volume of data (Thorqaard e t al.,

1981; 1982),

while

concern about

reliability h a s

arisen regarding data

generated by more rapid procedures (Lemoine and Smith,

1980;

Benfey e t al.,

19843.

Johnson et

dl.

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

counter w i t h channelizer and t h e

ICP-22

flow cytometer.

Both equipment allowed rapid identification of diploid and

triploid.

However

differences

in

accuracy

between

instruments w a s revealed in comparison of data from the

s a m e individuals.

Ploidy measurement on c o h o salmon w a s clear for the

t w o instruments in

8 5

o f

100

individuals

( 2 2

triploids,

63

diploids). T e n of t h e remaining individuals had skewed

histagram and t h u s indeterminate from

coulter counter

analyses, but were definitely triploids based o n f l o w

cytometery.

T h e five additional individuals produced n o

histogr-ams in t h e coulter counter and were determined

Ly

Both the coulter counter with channelizer and

ICP-22

flow cytometer are able to rapidly differentiate triploid

blood samples, however the flow cytometer is more accurate.

The flow cytometer measures ploidy by

fluorescent staining

of nuclear

DNA

and the coulter counter by measuring

erythrocyte cellular volume.

The differences of accuracy

could be attributed to the fragility of cellular shape and

volume a s contrasted to the maintenance of integrity of

nuclei

under conditions

of shear, altered osmotic

environments, and storage conditions.

Analysis by flow

cytometry i s therefore resilient to cellular disruptions

that d o not affect

DNA

fluorescence.

The use of flow cytometry is very accurate, it is

reasonable to assume that the fish with

a

high

DNA

content

were triploid, since both aneuploids (Gervai et dl.,

198th;

Lincoln, 1981a) and tetraploids (Thorgaard et al., 1981;

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

The

identification of triploid fish can be based

solely on the measurement of the major axis of either the

cell or the nucleus (Thorgaard and Gall, 1979; Chourrout

and Quillet, 1982;

Wolters et al.,

1982a; Beck and

Biggers, 1983; Richter et

dl.,

1987).

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.

1 7

c o u l t e r counter s i z i n g o f e r y t h r o c y t e s appears t o

be

t h e most s u i t e d 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 . I n #

t h e absence o f a c o u l t e r counter c h a n n e l i z e r o r a f l o w cytometer, t h e measurement o f 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 r e l i a b l e 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 . However, t h i s technique i s more time consuming and, hence, l i m i t s t h e number o f f i s h t h a t can be screened i n a p a r t i c u l a r study

6. 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 t h e assumptian t h a t they would be s t e r i l e and consequently might a v o i d o v e r p o p u l a t i o n problems, and p o s s i b l y grow f a s t e r o r s u r v i v e longer 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 l e a d t o d i s r u p t i o n o f meiosis and e i t h e r a f a i l u r e o f gonad development o r p r o d u c t i o n o f aneuploid gametes. The f a i l u r e o f gonad development might, i n t u r n , prevent t h e appearance o f u n d e s i r a b l e s i d e e f f e c t o f sexual maturation, such as poor meat q u a l i t y , slower growth, and h i g h m o r t a l i t y . I t appears t h a t t r i p l o i d f i s h a r e indeed f u n c t i o n a l l y s t e r i l e , secondary sexual c h a r a c t e r s a r e n o t always suppressed.

18 t h e f a c t t h a t 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, 1984). T h i s has been shown t o be associated w i t h 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 asymmetry (Leary e t al.,

1985).

Bingham i n Crosby (1986) proposed t h a t increased h e t e r o z y g o u s i t y was a primary advantage o f u s i n g p o l y p l o i d i e s i n p l a n t breeding programs. Heterozygousity might be maximized i n induced t r i p l o i d s by u s i n g h y b r i d s between two s t r a i n s as t h e female parent and crossxng t o male o f a t h i r d s t r a i n .

Purdom (1972) induced t r i p l o i d p l a i c e , flounder, and h y b r i d s by c o l d shocks ((3 t o 5OC f o r 2 t o 4 hours) a p p l i e d t o newly f e r t i l i z e d eggs. T r i p l o i d h y b r i d s s u r v i v e a t a s i g n i f i c a n t l y lower r a t e than

athar

hybrids. I n

larval

pigment p a t t e r n , number o f vertebrae, and metamorphosis c h a r a c t e r i s t i c s , t h e t r i p l o i d s d i s p l a y a d d i t i v e i n h e r i t a n c e i n v o l v i n g a l l t h r e e s e t s o f chromosomes. Concerning t h e growth r a t 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

may r e s u l t i n an excessive growth r a t e .

s t e r i l e , probably because abnormal spermatogenesis was t a k i n g place, b u t gonad s i r e appeared t o 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, b u t t h e mean ovary weight was l e s s than 13 %

o f t h e d i p l o i d c o n t r o l . H i s t o l o g i c a l examinations revealed t h a t o v a r i e s o f t r i p l o i d h y b r i d s were more abnormal than those o f d i p l o i d hybrids. The oocytes appeared t o undergo degeneration, and o v u l a t i o n was n o t observed.

T r i p l o i d f i s h do n o t appear t o be s t r i k i n g l y m o r p h o l o g i c a l l y d i f f e r e n t from d i p l o i d s (Thorgaard and G a l l , 1979; Gervai e t dl., 1980b). Swarup (1959) found t h a t t r i p l o i d s t i c k l e b a c k (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 t h a t t h i s may lead

*

t o extended growth and/or s u r v i v a l i n mature f i s h . Data on t h e performance o f s t e r i l e t r i p l o i d s a r e s t i l l accumulating. The r e s u l t o f t r i p l o i d 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)

.

I Z C b

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

is d e s i r a b l e . T r i p l o i d

grass

c a r p

( C t e m p h a r y n ~ o d o n 1

i d e l l w )

a r e

b e i n g

a d o p t e d i n a q u a t i c

*

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 , 1 9 8 3 1 ,

a n d t r i p l o i d s may

b e

d e s i r a b l e

f o r

s p e c i e s

w h e r e

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

a s s o c i a t e d s t u n t i n g o c c u r

( T h o r g a a r d ,

1986).

A n o t h e r

a p p l i c a t i o n

o f

i n d u c e d

t r i p l o i d y

lies i n t h e f a c t t h a t

t r i p l o i d

h y b r i d s

are t y p i c a l l y much

more

v i a b l e t h a n

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 ,

1983).

T h i s m a k e

i t

p o s s i b l e

t o combine

d e s i r a b l e c h a r a c t e r s f r o m

t w o

s p e c i e s i n

a

s t e r i l e h y b r i d .

T k

m o s t

s u c c e s s f u l a p p l i c a t i o n

o f

p o l y p l o i d

so

f a r

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

A

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

method h a v e p r o v e n e f f e c t i v e , i n a n i m a l s , s u c h

a s f i s h and

m o l l u s c s , w i t h 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

c a n d i d a t e s

( A l l e n a n d

S t a n l e y ,

1981). T r i p l o i d h y b r i d s

b e t w e e n 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

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 o f Wageningen A g r i c u l t u r e U n i v e r s i t y , t h e Netherlands

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. I d e n t i f i c a t i o n o f t r i p l o i d f i s h .

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.

P a r e n t a l f i s h , husbandry o f f r y and experimental f i s h

#

Larvae o f t h e Asian c a t f i s h

( C l a r i a s b a t r a c h u s )

were c o l l e c t e d from a f i s h pond i n Kabupaten B l i t a r , East Java,

f i s h served as p a r e n t a l f i s h . The experiment, s t a r t e d when t h e f i r s t g e n e r a t i o n had reached an age o f

14

months and e

w e i g h t o f

200

-

600 g. 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 .

(1988).

F r y produced f o r experiments 2 and

J

were r a i s e d i n 250 1 g l a s s f l o w through tanks, p u t a t i v e d i p l o i d

( D

group) 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 . They were f e d n a u p l i i Artemia s a l i n a f o r t h e f i r s t two weeks, f o l l o w e d by a commercial t r o u t d i e t f e d from two weeks onwards a t a r a t i o n o f 16.8 g.kg-O-o.d-z, recommended by Hoogendorn

(1981)

as t h e o p t i m a l f e e d i n g r a t i o n f o r 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 .

I n t h e w e i g h t range o f 1 t o 20 g t h e i n c i d e n c e o f " r u p t u r e d i n t e n t i n e syndroms" (RIS) has o f t e n been r e p o r t e d f o r

C l a r i a s

g a r i e p i n u s (Boon e t al.,

1987).

J u v e n i l e f i s h seemed t o be l e s s s u s c e p t i b l e t o RIS when f e d a t a low l e v e l . F o r 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 a q u a r i a w i t h 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

1 ) w i t h f l o w through water system, and one s e t of a q u a r i a

( 2 0

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.

23 2 l / m i n u t e f o r each aquarium (volume 140 1). The dissolved 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 was k e p t near

4

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

o u t f l o w i n g water. C o n c e n t r a t i o n o f NH4* and

NOn-

never exceeded v a l u e s o f 2 and

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 ( c a r p p i t u i t a r y e x t r a c t ) manufactured by Crescent Research Chemicals, V i r g i n i a 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 i n j e c t i o n .

1.3. Equipment

The equipmept f o r analyses o f p r o t e i n ( k j e l t e c ) , f a t ( s o x l e t t ) , and energy (bomb c a l o r i m e t e r ) were used f o r d e t e r m i n i n g body composition. The c e n t r i f u g e , p l a s t i c tubes

(2.5

c c ) , s y r i n g e and needle were used f o r blood sample

*

a n a l y s i s . The f l o w cytometer was used f o r r e d blood c e l l s (RBC) o r DNA measurement. 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).

Measurements

:

Forward scatter

CFSCI;

Side scatter LSSC3;

Fluorescence

1 CFL11;

F l u o r e s ~ e n c e

2

CFt21.

'Set-up

:

laser

:

488 nm

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,

FL2

was

amplified linear.

A 1 1

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

and staining of blood cells were filtered with a 0.2 mm

bacterial filter before usage.

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

2.1.2.

Cold shocking eggs

4

About 200 eggs per sample were f e r t i l i z e d w i t h m i l t and incubated a t 27OC i n p l a s t i c c i r c u l a r chambers

(diameter

10

cm), 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 t o water o f S°C. T h i s was done a t v a r i o u s times a f t e r f e r t i l i z a t i o n . The d u r a t i o n o f t h e shock was c o n s t a n t ( R i c h t e r e t al., 1987

1 .

A f t e r t h e c o l d shock treatment, t h e eggs were t r a n s f e r r e d again t o water o f 27OC. The e f f e c t s o f t h e treatment were measured by undeveloped eggs

(U.D),

hatching r a t e o f deformed l a r v a e

(H.D.)

and hatching r a t e o f normal l a r v a e ( H . N . ) . These were expressed as percentages o f number o f eggs incubated.

2.1.3.

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

The e f f e c t i v e n e s s o f cold-shocking c.q. t h e suppression of t h e second 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

2.1.4.

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

* _{The m i l t stock was d i l l u t e d 1 : 1 O w i t h}

0.4

_%

NaCi

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 . Samples o f

10

m l were spread on a l a r g e watch g l a s s ( i n o r d e r t o o b t a i n a t h i n l a y e r o f spermatozoa) and placed on a p e t r i d i s h f i l l e d w i t h i c e . The m i l t was mechanically s t i r r e d d u r i n g i r r a d i a t i o n ( Komen e t al., 1988

1 .

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

(%I

was e s t i m a t e d immediately a f t e r t h e treatment. The d i s t a n c e between t h e lamp and t h e sperm sample was 25 cm. Samples o f

200

eggs were mixed w i t h 100 u l o f i r r a d i a t e d sperm ( c o n t r o l on sperm i r r a d i a t i o n ) o r w i t h u n t r e a t e d 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

( % I

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

2.1.5.

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

Experiment

1

(

23-10-1987)

I

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 b o t h o f s t r i p p e d m i l t and o f m i l t #

o b t a i n e d from t h e t e s t i s was determined by

e g g

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

Experiment 2 ( 11-11-1987

T h i s experiment was c a r r i e d o u t t o determine t h e b e s t s t r i p p i n g l a t e n c y t i m e f o r

C l a r i a s

batrachus k e p t under "Wageningen hatchery c o n d i t i o n s " . Twenty females were hypophysized and d i v i d e d i n 5 subgroups o f 4 females, which

were

s t r i p p e d a t l a t e n c y

times

o f 13, 15, 17, 19, and 21 hours, r e s p e c t i v e l y . I n c u b a t i o n o f f e r t i l i z e d eggs was

I

c a r r i e d o u t a t 29OC and s u r v i v a l r a t e s were c a l c u l a t e d a f t e r w a r d s .

*

Experiment 3 ( 19-11-1987, 24-11-1987, 7-12-1987, A1-12- 1987 )

The t h i r d experiment was designed t o determine t h e

/

28 eggs were estimated. The p l o i d y was n o t determined i n t h i s experiment. T h i s experiment was repeated t h r e e times, because o f unexpected t e c h n i c a l problems, which c o u l d have a f f e c t e d t h e r e s u l t s .

Experiment 4 ( 30-11-1987)

T h i s experiment was c a r r i e d o u t t o determine t h e e f f e c t o f i r r a d i a t i o n 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. Four males o f

C l a r i a s

batrachus were s a c r i f i c e d and t h e t e s t e s were grinded. The 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, 10, 20,

30,

40, 5 0 , and 60 minutes. Sperm 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.

Experiment 5, 6, 7 and 8 (22-12-1987,7-1-1988, 20-4-1988, 4-5-1988)

.

embryonic development. The e f f e c t o f t h e l a t t e r was compared w i t h 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 w i t h i r r a d i a t e d 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 t h e f i f t h experiment, eggs were cold-shocked a t 3, 5, and 8 minutes a f t e r f e r t i l i z a t i o n . They were f e r t i l i z e d e i t h e r w i t h u n t r e a t e d sperm o r w i t h i r r a d i a t e d 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, eggs were c o l d shocked a t 3, 5,

and

8 minutes a f t e r f e r t i l i z a t i o n . D u r a t i o n o f sperm i r r a d i a t i o n was 5, 10, 20, o r 30 minutes.

In

t h e seventh experiment, eggs were c o l d shocked a t 3 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 5, 10, 15, 20, 25, and 30 minutes.

I n t h e e i g h t experiment, eggs were c o l d shocked a t 1, 2, 3, 4, 5, and 8 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 r a t e o f eggs, 48 h r and 72 h r a f t e r f e r t i l i z a t i o n ware used

as

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 t h e hatching r a t e o f eggs, i n c l u d i n g deformed and h a p l o i d larvae. *

Hatching rate

=

((nl)/(nl+ud+dl))SlOO

nl

=

normal larvae

0

ud

=

undeveloped eggs

dl

=

deformed larvae

(Richter et dl.,

1985)

Statistical analyses

The data were tested for normality using W-values and

homogeneity of variance using Bartlett's test (Sokal and

Rohlf, 1981).

The data were normalized by arcsine square

root transformation and subsequently, difference between

groups were tested with students t-test (Sokal and Rohlf,

1981). Calculation of

W

values, t-student test, and Anova

was performed using An Interactive Statistical Analysis

Program for Microcomputers by NH Analytical Software (Nimis

and Heisey, 1982).

2.2.

Identification of triploid

and

diploid fish

2.2.1.

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 previous experiment.

Five Clarias

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 t h e mixed sperm and incubated i n water o f 27OC t o produce t h e normal ( d i p l o i d ) f i s h . T r i p l o i d f i s h 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. F i s h were r a i s e d i n 250 1 g l a s s f l o w t h r o u g h tanks a t (25

t

1 ) O C and were fed n a u p l i i A r t e m i a

salina

f o r t h e f i r s t two weeks, and a f t e r t h i s p e r i o d a commercial t r o u t d i e t a t a r a t i o n o f

16.8 g.kg-o-6.d-a (Henken e t a l , 1987; Hoogendorn,

1981).

2.2.2. I d e n t i f i c a t i o n o f t r i p l o i d f i s h

Blood samples o f 1.5 m l were drawn from t h e caudal v a s c u l a t u r e o f , 4 0 randomly s e l e c t e d f i s h o f b o t h group

D

washing procedure was repeated t h r e e times. A f t e r t h e t h i r d d e c a n t a t i o n c e l l s were suspended i n TBS c o n t a i n i n g 1 % #

f o r m a l i n and s t o r e d o v e r n i g h t a t 4%. The n e x t morning samples were again washed t h r e e times w i t h TBS-Na. A f t e r washing packed c e l l s were suspended i n 1 m l o f a s o l u t i o n 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 % N a - c i t r a t e and vortesed f o r 1 minute. The sample was s y r i n g e d through a 26 gauge needle t o a v o i d clumping. Sample were s t o r e d f o r two hours a t room temperature. A f t e r s t a i n i n g samples were again washed t h r e e t i m e s and resuspended i n a volume o f 3 m l TBS. Flow c y t o m e t r i c a n a l y s i s was performed w i t h i n two 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 c e l l s s i z e

(FSC),

which i s p r o p o r t i o n a l t o t h e amount o f DNA present, was measured u s i n g

a

FACStar f l o w cytometer. Fluorescence o f 10,000 t o 20,000 c e l l s were measured from every f i s h , and t h e modal v a l u e ( W ) was kecorded. The corresponding channel number [mean peak l o c a t i o n

[MPLI)

was chosen as assay u n i t , s i n c e i t i s s u f f i c i e n t l y l i n e a r ( t r i p l o i d M P L

=

1.5

1

d i p l o i d

MPL

)

.

-

2.2.3. S t a t i s t i c s

c a l c u l a t e d , u s i n g t h e f o l l o w i n g formula o f A.

where XI

=

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

X

=

a r i t h m a t i c mean peak l o c a t i o n t,-a

=

t - v a l u e a t n-1 degrees o f freedom s

=

standard d e v i a t i o n .

OC = c r i t i c a l l e v e l a t 0 - 0 5

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. The f i r s t experiment was done when t h e f i s h a t 109 days o f

age

( t h e gonad o f normal f i s h s t a r t e d t o develop). T h i s experiment was conducted from J u l y t o August 1988. The second was done when t h e f i s h a t 178 days of age ( t h e normal f i s h was mature), T h i s experiment was conducted from August t o 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. Both experiments used t h e same p o p u l a t i o n o f f i s h , 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 contained groups o f u n t r e a t e d ( d i p l o i d , C D l l and o f c o l d - t r e a t e d ( t r i p l o i d , L T I ) f i s h . W i t h i n group D and T, f o u r feeding r a t i o n s were employed, r e s u l t i n g i n 8 treatment combinations

( 2

p l o i d y and 4

resulting in t h e best feed conversion ratio for

C l a r l a s gariepinus

(Hoogendorn,

1 9 8 1 ) .

Each treatment combination

#

w a s carried o u t in duplicate. D ~ s t r i b u t i o n

o f

t h e various

treatment combinations over t h e experimental aquaria

1 5

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

I i

I I

,

Ii

i

I

I I

I

I

I I

I

I

I I

Ii

1

11

2

11

3

11

4

11

5

11

6

11

7

11

8

1 1

I

I

I

I

I

I

I I

I

I

I I

I

I

i I

I

I l l C D , l l

II

CT,1111 CD,2111 CT,2111 CD,3111 CT,3111 CD,4111 CT,4111 [image:140.540.44.504.12.763.2]

II

<

I I

I I

I

I

I I

I I

I

I

I I

I

I

...

...

I

_.L--r _.

...-..-.

4 L .--..-...-...-,. 4 L .--....-.-..- 1 L ...-.-.. 1 I...-.--- ..-.. -2 L ..-...-.-...-.. --.-J L. ..--- ..-...-.... I L I

. . . . . . . . . .

.

.

-

"-( ,"."...*l.--""-"..l P.."""'." .-.--....-.

...,

r.-"".-.." ,.".- -.-.-

-

... ..-..--.--, C' I 1

I

I

I I

I

I

I

I

11

I

I

I I

I

I I I

11

9

11

10

11

11

11

12

11

13

11

14

11

19

II 16 II

1 I

I I

I I

11 I

I

I I

I

I

I I

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

I

I

I

I

I

I

I

I

11

I I

I

I I I I I L.&rL-czL-;-z :----.."-...--- l L :2::zzz::-;- f,C= .L:;z;;: :z;;2:yI:: 2.

;=;;

I' J-L. IL'Tzz;zz;:z ,JJ;::; L:z;-LTI';:L !,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, ~ ~ ~ 2 1 . .

L T .

,41

=

putative triploid fish with feeding

ration

1,2. . 4

optimum

feeding

ration

for Clarias

gariepinus.

F i g u r e 3.

Schematic representation of t h e

2.3.2.

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

*

A t f e e d i n g experiment 1, 360 f i s h were s e l e c t e d from b o t h D and T group. Both samples were d i s t r i b u t e o s e p a r a t e l y o v e r 8 a q u a r i a , r e s u l t i n g i n

16

groups o f 45 f i s h each. F i s h were a l l o w e d t o adapt t o i n e w

environment f o r two weeks. I n t h ~ s p e r i o d they were f e d 12.6 g.kg-O-Q.d-L. A t day Q , when t h e experiment s t a r t e d , t h e number o f f i s h i n aquarium was reduced from 45 t o 35. The f i s h had reached an age o f 123 days and a weight o f 50.1 g a t t h a t time. Ten randomly sampled f i s h o f b o t h group D and T were used f o r assessment o f body c o m p o s i t i o n a t t h e s t a r t o f t h e experiment. Ten f i s h o f each sex and p l o i d y were sampled f o r d i s s e c t i o n .

fit f e e d i n g experiment

2

each group c o n t a i n e d 40 f i s h a t t h e s t a r t o f t h e a d a p t a t i o n p e r i o d , which was lowered t o 25 a t day 0. F i s h had reached an age o f 192 days and a w e i g h t o f 114.1,g a t t h a t time. Other procedures used a r e t h e same as i n experiment I.

I n t h e evening o f day I t h e e x p e r i m e n t a l f e e d i n g r a t i o n s were i n t r o d u c e d f o r t h e f i r s t t i m e u s i n g conveyar- b e l t t y p e feeders. The f i s h were f e d c o n t i n o u s l y o v e r n i g h t . . Mean f r e s h body weight and m o r t a l i t y o f e v e r y aquarium was determined b i w e e k l y and r a t i o n s were a d j u s t e d a c c o r d i n g l y . Feeding stopped a t l e a s t 12 hours b e f o r e weighing.

A t t h e end o f t h e experiment [day 43) f i s h were weighed and counted and sex r a t i o s were determined. A t

l e a s t s i x f i s h from every aquarium were sampled f o r body c o m p o s i t i o n a n a l y s i s .

*

A t d i s s e c t i o n body weight, weight o f ovary o r t e s t e s and seminal v e s i c l e , m e s e n t e r i a l f a t weight and g u t t e d weight (body weight minus weight o f a l l i n t e s t i n e s ) were determined. Together w i t h t h e samples taken a t t h e s t a r t and a feed samples t h e

16

f i n a l samples were analyzed f o r d r y m a t t e r , crude p r o t e i n ( N K j e l d a h l Y 6 - 2 5 ] , crude f a t

( h e x a n e - e x t r a c t i o n ) and g r a s s energy.

i

2.3.3.

S t a t i s t i c s

The f o l l o w i n g v a r i a b l e s were c a l c u l a t e d f o r each o f t h e 16 f i s h groups :

V a r i a b l e s on g r o w t h and body c o m p o s i t i o n :

-

M e t a b o l i c g r o w t h r a t e = MGR

=

Cg-kg-o-=,d-a) MGR

=

GR / pWgO-e

GR

=

(We

-

Wo)/d

BWg

=

exp ( t l n W e + I n Wo)/2) GR = growth

rate

=

Cgl

BWg

=

geometric mean body w e i g h t

=

L k g l Wo and Wt = w e i g h t a t day

0

and day t

=

Cg] d = t i m e (day)

-

Feed c o n v e r s i o n r a t i o CFCRI:

FCR = F /

G

F = amount o f feed g i v e n

=

Cgl

G = f r e s h body weight g a i n ( W e

-

W O ) = Cgl

-

P r o t e i n g a i n = PG

=

[g-kg-o-e-d-ll

PG = RP /

1000

/

BWgO-*

Po and Pt = p r o t e i n i n t h e f i s h a t day O and day t C X I

.

- F a t g a i n

= F G = Cg.kg-o-e.d-*3

I

F G = R F / 1000 / bWgO-P

RF

=

r e t a i n e d f a t = (Wt*Ft)

-

( k * F o )

=

C Q ~

-

Energy g a i n

=

E G

=

CkJ.g-13

E G

=

E t

-

E O

E t and EO

=

energy a t day t and day 0

=

C i ~ J . g - ~ l .

- P r o t e i n e f f i c i e n c y r a t i o (PER)

= Cg.g-il

PER

=

G / GP

G

=

weight g a i n = ( W t

-

WO) = Cgl

GP

=

gross p r o t e i n

=

p r o t e i n provided = Cg3

-

Apparent n e t p r o t e i n u t i l i z a t i o n (app.NPU) =

C % l

NPUa= (RP / GP)

t

100 %

= C % l

RP

=

r e t a i n e d p r o t e i n = Cgl

GP = gross p r o t e i n = p r o t e i n provided

=

Cg1

- E f f i c i e n c y o f energy g a i n

= EEG

E E G

=

E G / GE = C % l

EG = amount o f energy g a i n = CkJ.9-=I

GE

=

gross energy i n food

=

u n i t o f energy provided Ck3.g-.+I

-

F a t - p r o t e i n r a t i o

=

FPH

FPR

=

percentage o f f a t d i v i d e d by percentage o f p r o t e i n ( f r e s h w,eight b a s i s ) .

-

F a t percentage, p r o t e i n percentage and energy i n d r y m a t t e r and/or f r e s h m a t e r i a l s .

D i s s e c t i o n v a r i a b l e s

-

Ovarian weight

-

M e s e n t e r i a l f a t weight

-

W e i g h t

a f t e r g u t t i n g as t h e f i s h weight a f t e r - r e m o v a l n t t h e i n t e s t i n e

-

Gonado somatic index

(GSI

C % l )

as gonad weight S 109

38

-

Fat index (FSI) a 5 mesenterial fat weight

$

1 0 0 divided

by fresh body weight.

-

Coddition factor a s (Weight/Lengthf)*lOO

-

Yield after gutting

=

((Weight after gutting)S100)

divided by fresh body weight.

*

Dissection data

were tested for normality using

Kolmogorov-Snirnov

( W

values) and for homogeneity using

Bartlett's test. When necessary data were normalized

using

an arcsin square root transformation, N o apriory tests were

performed on data concerning growth and body composition

since there were only two replicates for every treatment

combination. Further, data were analyzed by analysis

o f

variance. Both body composition and dissection data were

initially analyzed with the following

6NOVA

model:

where

:

=

value of 'the

j

Seeding level group and i

ploidy

=

overall mean

=

effect of ploidy ( i

=

2,

3

for diploids and

triploids respectively)

=

effect of feeding ration

I j

=

1,2.,3,4)

8

Fj

1

=

two-way interaction between ploidy

and feeding ration

I V . RESULTS

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 *

1.1.

I n d u c t i o n o f spermatogenesis

The r e s u l t s o f t h e i n d u c t i o n o f spermatogenesis experiment a r e presented i n Table 2.

Table 2. Means

2

SD o f t e s t i s somatic index ( T S I ) , s e m i n a l i s v e s i