INDUCTION OF OVULATION AND SPAWNING IN M~JGIL CEPHALUS(L) USING HCG AND OVAPRIM - -

Mathew Abraham ,P.Kishore Chandra , P.Shiranee P.%ri'iasam and A.Vasant Kumar Charles

Central Institute o f Brackishwater Aquaculture, 141 ,Marshal 1s Rd., Egmore, Chennai 600 008.

INDUCTION OF OVULATION AND SPAWNING I N MUGIL CEPHALUS ( L ) USING HCG AND OVAPRIM';S--

Mathew Abraham ,P.Kishore Chandra, P.Shiranee H.Kailasam and Vasant Kumar Char les

Cent ra l I n s t i t u t e o f Brackishwater Aquaculture, 141,Marshalls Road,Egrnore,Chennai 600 008.

ABSTRACT

E x p e r i m e n t s on i n d u c e d b r e e d i n g o f g r e y m u l l e t , Mtrgi7 c e p h a l i r s were c a r r i e d o u t u s i n g b o t h c a p t i v e broodstock and b r e e d e r s f rom w i l d catc f - tes . ferna7es w i t h average ova diameter o f 525u and above were se lec ted f o r hormonal i nduc t i on of spawning. I n a1 1 t,he 6 experiments , Human Chorionic; Gonadotropin (HCG) was used as t h e p r i m i n g dose @10,0001 .U . / kg body w e i g h t , Sucessful ovu la t i on and f e r t i l i s a t i o n was observed w i t h a r e s o l v i n g dose o f ovapr im 8 5ml /kg body w e i g h t . L e u t i n i z i n g hormone re l eas ing hormone analogue (LHRH-a) was a l so used a t a dose o f 300-400ug/kg body weight as reso l v i ng dose and was found t o be e f fec t i ve .0vapr im a syn the t i c hormone p repara t ion p r s e n t l y used f o r induced breeding o f f reshwater f i shes , was success fu l l y used f o r t he f i r s t t ime i n brackishwater f i s h Mugi7 c e p h a l u s . F e r t i l i s a t i o n r a t e was 90% when d r y s L r i p p i n g was fo l lowed and 50% i n na tu ra l spawning.

INDUCTION OF OVULATION AND SPAWNING I N - MUGIL CEPHALUS --c-- USING HCG AND OVAPRIM

INTRODUCTION

The endocrine f u n c t i o n o f t he hypothalamus - p i t u i t a r y - g o n a d a x i s m a i n t a i n s t h e r e p r o d u c t i v e

a c t i v i t i e s i n f i s h e s i n t u n e w i t h t h e s e a s o n a l and

environmental cyc les as most of the f i s h e s a re seasonal

breeders.The most common problem encountered i n c a p t i v e

breeding o f f i shes i s the i n h i b i t . i o n o f t he f i n a l s tages

o f gamete deve lopment due t o t h e f a i l u r e o f t h e

p i t u i t a r y t o re lease gonadotropin owing t o t h e s h i f t . i n g

o f f i s h from t h e i r natciral environment t o a hatchery

tank o r pond f o r breeding. T r a d i t i o n a l methods o f

induced spawning a re based on i n j e c t i o n o f gonadotropi n

from d i f f e r e n t sources such as carp p i t u i t a r y e x t r a c t

(CPE) and mammalian gonadotropin i . e . Human Chor ion ic

g o n a d o t r o p i n ( H C G ) ( L i n and P e t e r 1996 ;Lee e t

a7,1996). I n I n d i a f i s h breeding a c t i v i t y dates back t o

a mass sca le carp breeding programme i n t h e l a t e f i f t - i e s

u s i n g bundh b reed ing t e c h n i q u e . I n d u c e d b r e e d i n g i n

I n d i a s t a r t e d w i t h t he use o f p i k u i t a r y e x t r a c t s i n

major carps i n 1 9 5 7 f o l l owed by chinese carps i~ 1 9 6 2

( T r i p a t h i , 1 9 9 6 ) . A t p r e s e n t , s e v e r a l hqrmone

combinations are i n vogue.

The combina t ion o f a g o n a d o t r o p i n r e l e a s i n g

hormone analogue and a dopami ne an tagon is t f o r - i nduced

spawn ing i n c u l t u r e d f i s h i s t h e h i g h l y e f f e c t i v e

method c a l l e d t he Linpe method (Peter et a7 1988).This

method has r a p i d l y gained acceptance and has been

commercial i s e d b y Syndel Labs , I n c . ,Vancouver ,Canada,

under t he t rade name 0vaprim;whic.h has r e v o l u t i n n i s e d

' t h e freshwater f i s h induced breeding a c t i v i t i e s a l l

over the wor ld ( L i n and Peter,1996; K i m Harker,1992

Samanta,l995). The ovapr im spawning k i t i s s p e c i a l l y

formulated f o r use w i t h salmon'ids, c y p r i n i d s and o the r

freshwater f i shes .

The c u l t u r e o f t h e b r a c k i s h w a t e r f i s h , M u g i 7

c e p h a I i ~ s , cou ld n o t be t a k e n u p i n a l a r g e s c a l e i n

I n d i a because o f t h e l a c k o f f o o l p r o o f h a t c h e r y

technology f o r seed product.ion . Several r e p o r t s on

i n d u c e d b r e e d i n g o f M . c e p h a 7 u s i n c a p t i v i t y a r e

avai l a b l e by us ing d i f f e r e n t combinations o f GPH ,HCG

and LHRHa(Lee et a7, 1987; Tamaru ee ~17,1994; Tamaru et

a7,1992 ; Rajalakshrni et a7,1991). I n t he present s tudy,

a new combination o f HCG/Ovaprim was t e s t e d a long

w i t h t h e o t h e r combina t ions . As o v a p r i m i s l o c a l l y

a v a i l a b l e and i s r e p o r t e d t o be a d v a n t a g e o u s i n

economic terms compared t o CPE o r HCG (Nandeesha et a7

,1991 ) ; t h e study was conducted t o t e s t i t s e f f i c a c y

i n t he induced breeding o f t h e brackishwater f i s h , M u g i 7

cepha 7us.

MATERIALS AND METHODS A t o t a l o f s i x induced breeding experiments were

conducted us ing both w i l d and cap t i ve broodstock f i s h o f

M.cepha7us,. i n Feb. '97 and i n Dec. ' 9 7 r e p e c t i v e l y.

The important and b a s i ~ cr i : ter ia f o l l owed i n

t h e s e l e c t i o n o f f i s h f o r t h e i n d u c e d b r e e d i n g

experiments , were t h a t the females had an average ova

diameter u f 5 2 5 u o r above and t.he males were i n oozing

cond i t i on . The se lec ted f i s h were g iven a d i p t rea tment

i n Ippm a c r i f l a v i n and a I h r b a t h i n IOpprn

Furazol idone t r e a t e d water. Ovar ian b iopsy was always

done using a po lyethy lene canula. A s M.cepha7us i s very

s e n s i t i v e t o handl ing s t r e s s and looses sca les even w i t h

c a r e f u l and m i n i rna l hand1 i ng (Mathew A b r - a h a m s t

a 1 , 1 9 9 R ) , a1 1 t h e o b s e r v a t i o n s made on t h e f i s h were

done under anaesthesia w i t h 20pfzm c h i n a l d i n o r 2 5 O p p m

M S - 2 2 2 . L e n g t h and w e i g h t o f t h e f i s h w e r e a l s u

recorded. The se lec ted f i s h were s h i f t e d t o the hatchery

f o r hormonal t reatment.

A 24hr i n te r va l - two i n j e c t i o n p ro toco l w a s f o l l owed

f o r the hormonal i nduc t i on o f o v u l a t i o n and spawning . I n all t he experiments , H1~mat-1 Char ion ic Gonadotropin

(HCG) was used as t h e p r im ing dose 89000-10,000 I U / ' k g

body weight . I n t h r e e exper i rnen ts ,Ovapr im 133-5ml/kg

body weight was used as t he r e s o l v i n g dose and i n t h e

o ther t h ree experiments LHRHa @ 300ug/kg body weight was

ilsed as t.he reso l v i ng dose . A 1 l t h e i n j e c t i o n s were

administered under anaesthesia. Speci a1 care was t,aken

w h i l e i n j e c t i n g O v a p r i m , a s t h e i n c i d e n c e o f

subcutaneous swe l l i ng and sp i 1 l over f rom t h e i n j e c t i o n

s i t e was observed i n the course o f t he s tudy. Ovaprim, a

combinat ion-o f salmon gonadotropin re l eas ing hormone and

domperidone i s t h i c k i n consistency because of i t s non-

t o x i c organic so lven t i . e . propylene - g l y c o l base. To

overcome the problems i n admin is te r ing Ovaprim , a long

20 gauge s t a i n l e s s s t e e l needle was used t o g i v e a deep

intra-muscu7ar i n j e c t i n n a t a s low r e l e a s i n g pace. When

ever t h e t o t a l vol i~rne o f Ovaprim t o be a d m i n i s t e r e d was

more than 4m1, t h e dose was s p l i t i n t o two equa l volumes

and g i ven a t two d i f f e r e n t s i t e s , one a f t e r ano the r .

RCC r e ~ t a n g i l l a r t a n k s o f 10 t u n c a p 8 c i t .y

f i l l e d w i t h sand f i t s r e d sea water o f 2 6 - 2 8 p p t s a l i n i t y

were used as spawning tanks . The sea water was e a r l i e r

t r e a t e d w i t h c a l c i ~ ~ m h y p o c h l c - ] r i t e @ I O p p r n a n d

d e c h l o r i n a t e d by v igo rc> i~s a e r a t i o n . Female and males

were re leased i n t o t.hese spawning tanka i n t h e r;lt.in

1 : 2 , a f t e r t h e p r i m i n g dose. F i shes were observed f o r

i nc rease i n b e l l y s i z e a f t e r 2 4 h r s , when t h e r e s o l v i n g

dose was a l s o g i ven .

RESULTS D e t a i 1 s o f t h e e x p e r i m e n t s c r ~ f i d i i c t e d o n

M . c : e p h a Ills a r e shown i n T a b l e 1 . Two o f t h e s e

exper iments were conducted i n Feb. '97 w i t h w i l d b reeders

and t h e o t h e r f o u r were done i n Dec. '97 u s i n g c a p t i v e

b r o o d s t o c k . I n t h e second and t h i r d e x p e r i m e n t , t h e

f i s h e s d i e d p r o b a b l y due t o s t r e s s . I n t h e f i f t h

exper iment , no spawning w a s observed even a f t e r 7 2 h r s

and hence t h e f i s h was r e l e a s e d back t o t h e b roods tock

t a n k .

I n t h e f i r s t experiment. , a f t e r 24h rs o f t h e p r i m i n g

i n j e c t i o n o f HCG, ova d iameter i nc reased t o 5 4 0 - 5 R O u and

t h e r e s o l v i ng dose o f ovapr i rn w a s admi n i s t e r e d . Hormone

t r e a t m e n t was n o t g i v e n t o males. W i t h i n 12hrs a f t e r

a d m i n i s t e r i n g t h e r e s o l v i n g d o s e , a r e m a r k a b l e

i n c r e a s e i n b e l l y s i z e was o b s e r v e d and c o m p l e t e

ovu la t i on was observed a f t e r 22hrs. Eggs were sLrdpped

and co ' l l ec ted i n a c l e a n b a s i n . The eggs were f r e e

f lowing and had a brown t i n g e . The c o l l e c t e d eggs were

f e r i l i s e d w i t h m i l t c o l l e c t e d from two ooz ing males . A f t e r 2-3 minutes, c lean water o f the same s a l i n i t y w a s

added and mixed f o r 2 m inu tes . L a t e r t h e eggs were

d i s t r i b u t e d i n d i f f e r e n t FRP tanks of assor ted capac i t y

o f 5001-20001 w i t h aerat ion 8 140e~gs/7 f o r hatch ing.

The t o t a l number o f eggs released were I d l akhs and the

f e r t i l i z a t i o n r a t e was 90%. F i r s t hatch ing was no t i c~ed

a f t e r 3 0 h r s o f f e r t i l i s a t i o n and t.he h a t c h i n g wes

complete w i t h i n 2hrs. The hatch ing r a t e was est imated t o

be 42%.

I n t h e 4 t h and 6 t h expe r imen ts t h e f ema les w e r e

administered HCQ 8 10,000I.U.ikg and 9000I.U./kg as the

pr iming dose and were re leased i n t o the spawning tanks

along w i t h males i n the r a t i o o f 1 : 2 respect ive7y. A f t e r

24hrs, an inc rease o f 100-1509 i n t h e w e i g h t a f t h e

females was observed. Resolv ing dose o f LHRHa @ 300ug/kg

was administered t o t he females. M a l e s were a l so g iven a

hormonal treatment o f ovaprim 81ml/kg body weight along

w i t h the reso l v i ng dose f o r the females. I n t he 4 t h

experiment na tu ra l spawning and f e r t i 1 s a t i o n occur-ed

a f t e r 24hrs o f a d m i n i s t e r i n g t h e r e s o l v i n g dose. The

t o t a l number o f eggs r e l e a s e d was f o u n d t o be 1 7 . 5

l akhs w i t h a f e r t i l i s a t i o n ra t .e o f 50%. Ha- t ch ing w a s

observed between 30-32hrs a f t e r f e r t i l i z a t i o n . However,

the hatching percentage was v e r y low at 10%. I n t h e 6 t h

exper iment though natcr ra l spawning occrdred a f t e r 19hrs

a f t e r t h e r e s o l v i n g dose t h e r e was no f e r t i l i z a t i o n .

I n a l l t h e cases , t h e o v u l a t e d eggs w e r e

t r a n s l u c e n t a n d n o n - a d h e s i v e . The d i a m e t e r o f % h e

o v u l a t e d e g g s r a n g e d beLween 750-76011. The new1 y

h a t c h e d l a r v a e were t r a n s p a r e n t a n d h a d n a v e r a g e

l e n g t h o f 2.29rnm.

DISCCISSION

Hormone combi n a t i ons t h a t . iir-e h e i ng pr-esent.1 y 14sed f o r

M. cepha 7 ~ s induced k ~ r eed ing ar-e CPH/CPH, CPH,/LHRHa,

LHRHa/CPH, LHRHa/LHRHa, HCG/LHRHa a n d LHRHa/HCG ( L e e

eta 7 , 1 9 8 8 ) . I n t h e present. sttrdy , a new c;c)mhinal:.inri c 3 f

HCG/ovapri m was success fu l 1 y c~serl i n i ndtrc i ng o v t l l a t i o n

and spawning a l o n g w i t h t h e o the r - c o m b i n a t i o n c l f

HCG/LHRHa. The d o s e o f o v a p r i m i n t h e scrccess f t r l

exper iment was Eiml/kg body weight. o f !?.he f i s h , wh l c - : h 1s

10 t i m e s more t h a n t.he ~ rsc ra l d o s e o f 0 . 5 m l / k g b o d y

w e i g h t as suggestr.e[d by t h e m a n u f a ~ t u r - e r s f o r L h e

induced b reed ing (Kim Har-ker, 1 9 9 2 ; Sanintlta, 1 9 9 5 ) . The

dosage r-equi r e d to b r i n g about. s p n w n i n y i n r n l ~ l l e t w i t.h

HZG o r CPH, a r e bet.weetn 10 Lo FjT, t.irnes repc>rF.ecj f o r t.l-ie

o t h e r f i s h e s ( D o n a l d s o n a n d H ~ n t . e r - , 1 9 8 3 ; L e e et.

a7, 1 9 8 6 ) . T h i s was fnur id t.o be t i t i 1;he c a s e o f

o v a p r i m a l s o i n i h e p r e s e n t s t . ~ . ~ r ~ y a s t . i ~ e S I J ~ : R S S ~ I J ~

i n d u c t i o n o f spawning was ach ieved w i t h 10 t.itries h i g h e r

dose than t h a t p r e s c r i b e d f o r ot.her f i s h e s . T h i s h i g h e r

dose i n mu l le t . may be due to t h e p o s i t.inn a t whic:h t.hese

hormones act. on t h e hypothalamus-y i t r ~ i t a r y - g o n ~ t l a x i s

which s1~ggest.s t h a t t h e b l o c k t.cz spawnit>g i n mcr l lel. m a y

res ide i n t he b r a i n (Lee e t a7,1987). The lowes t total

dose o f LHRHa observed t o b r i n g a b o u t spawn ing was

302ug/kg body weight (Lee et a7,1957). I n t he p resen t

study a l so a dose o f 300ug/kg body weight o f LHRHa was

found t o be e f f e c t i v e i n b r i n g i n g about spawning.

I n M. cepha Jus, most o f t he sucess fu l spawnings

by hormonal t r ea tmen ts occ i l r ed when t h e i n i t i a l egg

diameter was 600u and above (Lee et a7, 1 9 8 7 ; Gr-eel i g et.

~ 7 , 1 9 8 7 ; Tamaru e t a ? , 1991 1 . However, i n t h e p r e s e n t

s tudy , sucess fu l o v u l a t i o n and spawn ing ~ o c ~ I d be

observed i n 3 f i s h e s w i t h i n i t i a l average egg diameter

o f 525u,536u and 61du respec t i ve l y . Th is f i n d i n g l e d t o

the conclusion t h a t mu1 l e t cou ld be s ~ ~ c e s s f u l 1 y induced

t o spawn even when the i n i t i a l egg diameter i s as low

as 525u. However, the e f f e c t o f i n i t i a l egg diameter on

t he hatching percentages and l a r v a l development has t o

he f u r t h e r inves t iga ted . The t ime o f o v u l a t i o n and

spawning i n the present study ranged between 43-4Rhrs.

The r e p o r t s f rom o t h e r wo rke rs r e v e a l e d a range o f

ovu la t i on per iods betwen 32 hrs(Yashouv,l969) and 4 S h r s

(L ing, 1970). I n the present experiments, t h e incuba t ion

per iod ranged between 30-32hrs. The incuba t ion per-iod

r e p o r t e d e l s e w h e r e i n M. cepha 7c1s was 32-52.30h1-s

( C h a u d h a r i e t a7 ,1977 ) and 4 4 h r s ( R a j a l a k s h m i et.

a7,1991).

The f e r t i l i s a t i o n r a t e was f o u n d t o be 50% when

n a t u r a l spawning was observed and was 90% when d r y

s t r i p p i n g and a r t i f i c i a l f e r t i l i z a t i o n was done .

However, e a r l i e r r e p o r t s d i d n o t show any d i f f e r e n c e i n

t h e f e r t i 1 i s a t i o n r a t e s between d r y s t r i p p i n g qnd

na tu ra l spawning(Lee et a7,1987).

ACKNOWLEDGEMENT 6. P.M.. RRV,

The au tho rs a r e t h a n k f i ~ 1 t o D i r e c t o r , C I B A , f o r h i s A

encouragement dur ing t he course o f t h i s work.

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7

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Table 1 .Detai I s o f induced breed 'ng experiments i n Mugi 7 cepha lus.

Expt. Per iod Source Female I n i t i a l P r im ing Resol- Remarks No. o f length/ avg. ova dose v i ng

breeders weight diameter (HCG) dose (mm/g) (u) ( T . I J . / ~ ~ )

1 Feb.'97 Wi ld 380/675 5 2 5 10,000 Fiml/kg Ovulated, Ovaprim s t r i p p i n g

done. 90% fer t . - i l i s a t i n r i , iZ%hatch ing.

2. Feb.'97 Wild 415/900 540 9000 3ml/kg Fish Ovaprim died,

B e l l y s i z e i nc reased.

3. Dec.'97 Capt ive 490/?725 544 10,000 5ml/kg Nn brood- Ovaprim o w l a t i o n . s tock F i s h d ied .

4. Dec. '97 Capt ive 480/1500 535 10,060 300ug/kg Na tu ra l brood- LHRHa spawrii ng. s tock 50%

f e r t i l - i s a t i o n . 10% ha t ch i ng

5 . Dec. '97 Capt ive 550/1600 545 10,000 300ug/kg Nn brood- LHRHa spawning. s tock

6. Dec. '97 Capt ive 460/1300 614 9,000 .300ug/kg Nat .ura l brood- LHRHa spawning. s tock No

f e r t i l - i s a l i o n .