Biochemical aspects of ovarian maturation in Liza parsia (Hamilton-Buchanan).

BIO CH EM ICAL ASPECTS OF OVARIAN MATURATION IN

(HAMILTON-BUCHANAN)

DISSERTATION SUBMITTED BY Shri MUTHUKARUPPAN S.

IN PARTIAL FULFILMENT FOR THE DEGREE OF MASTER OF SCIENCE (MARICULTURE)

OF THE

COCHIN UNIVERSITY OF SCIENCE AND TECHNOLOGY

> 35 - T 5 L

L . :, r ^ lU T

OCTOBER 1987

tS

^ ^ \

POST-GRADUATE EDUCATION AND RESEARCH PROGRAMME

I

IN MARICULTURE

\

/ CENTRAL MARINE FISHERIES RESEARCH INSTITUTE

^3 COCHIN - 682 031

record o f the work carried out by Shrl. M U TH U K A R U PPA N , S.

under my supervision and that no part th ereof has been presented before fo r any other degree.

Countersigned by:

Dr.(Mrs.) S.SIVAKAMI, SCIENTIST S-2,

C E N TR A L M ARINE FISHERIES RESEARCH INSTITUTE,

COCHIN - 682 031,

C E N TR A L MARINE FISHERIES RESEARCH INSTITUTE,

COCHIN - 682 031.

C O N T E N T S

PAGE NO.

PR E F A C E 1

IN TR O D U C TIO N 5

M A TE R IA LS A N D METHODS 15

RESULTS

DISCUSSION 45-

SU M M A R Y 60

REFERENCES 63

organisms frcin water •» a process analogous to agriculture where animals and p la n ts are c u ltiv a te d from land. The f i e l d o f aquaculture i s an emerging bioin du stry based upon the c u lt u r e and husbandry o f a l l u s e fu l aquatic organisins*

I t i s a s ig n ific a n t d ^ a r t u r e fro n the t r a d itio n a l "search and cap tu re" f i s h e r i e s and may in c re asin g ly d isp lace the

/

hunting and catching of w ild stock . Of la t e there has been a g l d s a l upsurge f o r aquaculture. Increasing world popula- t io n , the d e cisio n

by developing u n u t ilis e d or p a r t i a l l y u t il is e d water resources^ and d e p le tio n o f natvural stock due to excessive e a ^ lo it a t io n have been some o f the reasons f o r the need to produce more f i s h tiirough aquaculture.

fb r aquaculture^ seed i s the b a s ic need and i s usually c o lle c t e d from the w ild . However^ sin ce the success of aq­

u a c u ltu re i s dependent on the d e sire d , as w e ll as uniform s iz e o f tiie stock and because the nature i s not ^ l e to meet the demand of the aquacu I t u r ls t s f o r seed, the method o f pro*

cu rin g seed by induced breeding i s resorted to . In order to

proceed w ith induced breed in g, the aquaculturlsts have to be

f u l l y aware o f the spa»rning season as w e ll as the n u tritio n al

s ta t e o£ the b re e a e c s . Further, i t I s indispens Ib le to have a good Icn w led ge o f th e b io lo g y , li'y s io lo g y and biochemistry o f the fis h e s th at a r e t o be rea re d . In order th a t the fis h may be placed in the most s u ita b le enviraruaental ccndltlons

and be s u p p lie d w ith ap p rop riate fe e d and energy. This in tu rn can e f f i c i e n t l y enhance th e growth o f the stock with the r e s u lt a n t In c re a se i n the o v e r a ll f i s h production*

E a r l i e r works o f s c ie n t is t s which were re s tric te d only t o the b io lo g y has taken a tu rn in the re c e n t years with b i o l o g is t s te n d in g t o u se a more biochemical approach and biochem ists envinclng more in te re s t in fis h cihemistry. Hence the o v e r a l l view i s now more biochem ical and indeed more so­

p h is tic a te d than b e fo r e . ‘Hie fa c t that more s c ie n t is t s have s ta rte d u s in g f i s h fOT^ biochem ical stu dies, which used to be c o ifln e d to sm all mammals i s evident from the remark o f N alin s and Sargent (1974) i **We sense the presence o f g re a te r number o f biochem ists and b io p h y s ic is t s showing an in te re s t in marine

^ gan lsm sth an e v e r b e f o r e " .

A qu atic organisms when con^ared with t e r r e s t i a l animals have an e n t ir e ly d i f f e r e n t way o f l i f e causing fis h e s to evolve

a nxamber o f structxares and system unique to themselves, 5he

three prim ary c o n s titu e n ts

l i p i d which fu rn is h a f i s h with energy fo r v ario u s purposes

due t o phenonenon lilce maturation, m igraticn, e t c ., which most f i s h e s undergo as a p a rt o f t h e ir normal liv in g , have a profcund e f f e c t on the composition o f the body, that fis h chemistary beccmes e s s e n t ia l and re q u ire s a s p e c ia l approach.

Wittj a s h i f t in p r i o r i t y away fra n “hunted" food f i s h t o c u ltu re d f i s h e s p e c ia lly u s in g hypophysation techniques, i t becomes an e s s e n t i a l p r e r e q u is it e to know the n u tritio n a l s ta t e o f th e f i s h b e fo r e , a f t e r and at the time o f spawning*

"Hie o ld Ycarkshire fa rm e r’ s ad vice to the young man seeking a l i f e p a rtn e r (H e r r io t t , 1974), "Have a good look at the mother f i r s t , la d " t o g e t an id e a of how tiie daughter would develop, can be a p t ly quoted h e re to emphasise the importance o f studying the n u t r it io n a l s t a t e of the mother f is h at the time o f spawning.

T h is work has b e » i put forward to serve the purpose in a m u lle t o f h l ^ p o t e n tia l namely L iza p a r s la .

I exp ress my deep sense o f g ra titu d e t o D r.(M rs .) S, Sivakam i, S c i e n t is t , C entral Marine F is h e rie s Research I n s t i t u t e f o r h e r In v a lu a b le guidance and encouragement throughout tlie p e rio d o f th is study. I a ls o express my g r a t it u d e t o D ir e c t o r , C e n tra l Marine F is h e rie s R ese at^

I n s t it u t e f o r g r a t i n g a l l f a c i l i t i e s fo r the study«

I v/ould lik e to thank S h rl. Narayana Rirup and D r, Peer Kohaironed, S c ie n t is t s , C entral Marine F ish e rie s Research

In s t it u t e f o r t h e ir v a lu a b le suggestions and h elp during the study, I a ls o thank S h ri Nandakumar f o r the h e lp rendered d u rin g the study, “l^anks are due to D r, Sankaranarayanan, S c ie n t is t , R egional Centre o f N a tio n a l In s titu te of Oceano- grphy. Cochin f o r having helped me in carrying out the ash content a n a ly s is . My h e a r t ie s t thanks are due to Shri Gopala- krishnan. Senior Research Fellow f o r h is constant help through-

cut the p e rio d o f t h i s study. A l l my frie n d s, e s p e c ia lly S a lly , S heela, D a lje e t , S a ji , Ramraj and Dinesh have helped me on v a rio u s occasion s during the course o f study, Dipak Narendra C h au d h ari's in flu e n c e had bearing during this study.

My s in c e re thanks are due to a l l o f them, I would lik e to p lace on re c o rd , my thanks to the Indian Council of Agricul­

t u ra l -■Research fo r gra n tin g me the Jrmior Research Fellowship,

I N T R O D U C T I O N

The grey m u lle t sp ecies o f the fa m ily M ugllidae are re p r e s e n t a t iv e o f a t r u ly In te rn a tio n a l group o f f i s h . Ihey c o n s t itu t e 0.l..0.3?i o f tiie t o t a l marine landings o f In d ia and the p rodu ction from K erala back w aters forms about

o f

i t s t o t a l annual landings (Jhingran, 1983) • F isheries o f m u lle t are s u f f i c i e n t l y important th at i t has stim ulated c o n s id e ra b le r e s e a r ^ ,

Tliere are ^ o u t 281 ncminate sp ecie s in the family

M u g ilid a e , o f which Thomson (1964) had recognised 70 as v a l i d . In d ia n waters^ Day (1878) i d e n t if ie d 26 sp ecies o f which 3 v e r e re p o rte d to e n te r freshw ater and another

Of the m u lle ts , M uqil cephalus i s the most abundant and w idely d i s t r i b u t e d sp e c ie s, and has re c e iv e d maximum attenticn from S c i e n t i s t s . L iz a p a r s i a (Hamiltcn-Buchanan) i s o f considerable Importance in In d ia , second only t o M, cephalus. According to Jh in gran (19 8 2 ), L . p a r s ia . one o f the common grey mullets in the Cochin estu ary , co n stitu te s a t h r iv in g fis h e r y in e stu aries and backw aters o f In d ia* I t i s a l s o abundant in West Bengal, M adras, V i^akapatnam , Palk Bay, G u lf o f Mannar and Andaman

I s la n d s . Cutside In d ia , th is sp e c ie s i s r e s t r ic t e d to the

I n d b -P a c i f i c re g io n where i t is f a i r l y d is trib u te d along the

c o a st o f P ^ l s t a n , S r i Lanka, Hong Kong, A u s tra lia , Indonesia, e t c .

■The p o p u la rit y o f raullets as c u ltu re fis h e s is no

a c c id e n t. I>lke most fis h e s , they are h ig h ly rated food f i s h e s . L, p a rs l a i s c la s se d as one o f the ric h e s t sources o f Vitamin A among the ccinmon Indian food s t u f f s by Ghosh and Guha(1934).

The p r o t e in content o f the whole m u llet i s about 20%, Jacqout (1961) c l a s s i f i e s them among the s e m i-fa tty fis h e s with a f a t content o f around l,S -5 *0 9 i. ihey a r e a ls o proclaimed fo r t h e ir extreme t o le ra n c e to s a l i n i t y and t«n p e ra tu re . Last, but not the l e a s t , m u llets occupy the low est p o sitio n in food chain, e n a b lin g transform ation o f n u trien ts a v a ila b le in the environ­

ment in t o f i s h f le s h a t the minimum expense o f energy.

In view o f the s ig n ific a n t ch aracters o f mullets as c u lt u r e f i s h e s , f i s h c u lt u r ls t s and fis h e r y s c ie n tis ts are e v in c in g more In t e r e s t in d evelopin g th is group in to a f u l l -

fle d g e d sou rce o f c u ltu re , not m erely by the present status, b u t a l s o by the promise o f even g re a t e r sig n ific a n c e in

f u t u r e .

The p r in c ip a l areas o f m u llet c u ltu re in India ^ e

lo c a te d In West Bengal and K e ra la , A ppreciable advancements

have been made in r e c a i t past in resea rc h leading to the

p o t a i t i a l f o r m u lle t c u ltu re w ith ^^peen is la n d o f Cochin, a f i s h i n g hamlet, talcing

le a d * I t i s cu ltu red in s ig n i­

f i c a n t l e v e l s in a r e ^ o f Tamil nadu a ls o .

By v ir t u e o f the wider range o f toleran ce of mullets t o s a l i n i t y , att^ n p ts are made t o a c clim a tise m ullets in fre s h w a te r as w e l l . Hie f i r s t ac c lim a tisatio n studies of L , p a r s i a i n Benged was due to MooMierjee ^ a l « (1946), w h ile farm in g o f v a rio u s species of m u llets in freshwater b o d ie s i s re p o rte d by Ganapathi ^ (1 9 5 0 ), Mullets are g e n e r a lly cu ltu red alon g with oth er fis h e s liJce Stroplus

s u r a t e n s is and Chan os chanos in southern In d ia (Bardach e t a l , 1972) and w ith Indian major carps and e x o tic carps (P ak rasi e£ a l . , 1975).

T i l l r e c e n t ly seed f o r m u llet c u ltu re was obtained from w i ld depending on t i d e s . Since i t has i t s lim itation s lik e

in ad equ ate a v a i l a b i l i t y o f q u a lit y seeds and a ls o because o f the d i f f i c u l t y in segg rega tin g the d e sire d sized s t 0 ^{(dc f c r}

c u lt u r e , i t has beccwie e s s e n t ia l to adept hypophysation

tech n iq u es f o r seed production in good q uantity. Moreover

a c c o rd in g t o » i o e t aj,. (1974b), gonads o f m ullets in

8

confinement f a i l t o a tta in m aturity without hormone stim ula- t io n . An ij^ o r t a n t break through in t h is lin e was a ^ ie v e d in 1964 b y Yh-An-Tang o f Taiwan F is h e rie s Research In stitu te in 1964 (quoted

Bardach a l . 1972)* This was followed by attem pts o f Yashouv (1969), Kuo ^ g i * Cl974b) and others.

In In d ia , picmeer works o f th is kind were c a rrie d out in the C e n tral In land F is h e rie s ^ s e a r c h I n s t it u t e Barrackpore and

a ls o b y S eb astian and N a ir (1974) and J ^ e s ^ ^ (1983).

For s u c c e s s fu l hypophysation and a r t i f i c i a l propagation o f c u lt i v a b l e f i s h e s , i t i s h ig h ly e s s ^ t i a l t o have a thorcugh knowledge o f th e reprodu ctive ph ysiology, b io lo g y , and b i o - chem istry o£ the b re e d e rs . A proper understanding of the b i o l o g i c a l s t r a t e g i e s , p h y s io lo g ic a l r e q u is it io n s and b io - c h e n ic a l ccm position o f parent f i s h keeps us informed about

t h e i r s u i t a b i l i t y t o fa c e the n a tu ral p^encmenon of procreation.

S tu d ie s o f biochem ical composition o f eggs can help us to a s s e s s the q u a lit y o f the eggs and hence the condition of the em erging young ones.

The rep rod u ctive b io lo g y of v ario u s species of m ullets

in In d ian w aters has been reported by Jacob and Krishnamurthl

(1 9 4 2 ), P i l l a y (1954), S a r o jln i (1957; 1958), luther (1962),

Rangaswami (1972), Sunny (1975), Red<ty (1977), Das (1978),

N a ta r a ja n and Reddy (1980), Sulochanamma (1981), IQirup and

Samuel (1 9 8 3 ), Surendra Babu aad Neelakantan (1983).

N e v e rth e le ss, co n tribu tio n s o f the various aspects of breed­

ing b i o l o g y of L , p a r s ia belong only to S a r o jin i (1957), Kurup Samuel (1983) and Surendra Batai and Neelal^antan

(1 9 8 3 ).

M aturation r e f e r s to c y c lic moirphological changes

whlcSi a fem ale and male gonad undergo to a t t a in f u l l m aturity.

Attainment of f u l l m aturity almost always marTcs a change in the g ro ’.rth p attern r e s u lt in g from the 'rep rod u ctive drain*

due to the d iv e rs io n of m aterial meant fo r san atic growth to the gonads. P a rris h and S a v ille (quoted by li e s , 1974) m aintain th a t in h e rrin g stocks, th ere i s a marked reduction

In growth r a t e at the a is e t o f m aturity r e f le c t in g the u t i l i ­ zation o f the growth m aterial f o r gonad maturation. lie s

(1974) d is c u s s in g the ta c tic s and stra te g y of grcwth in fis h e s , s ta t e s th a t gonadal maturation i s mainly dependent on the

n u t r i t i o n a l 's t o r e * accumulated d u rin g the major feeding and growing p e r io d .

There i s a co n sid erable body o f lit e r a t u r e on the v a r ia t io n s in body composition in r e la t io n to the ecophyslo- l o g i c a l changes li k e attainment of sexual m aturity and

spawning m igration o f f i s h e s . Atwater (1888) had pointed out the body composition as an in d ic a to r o f n u tritiv e value o f f i s h e s . Observations on the d e p le tiv e e f f e c t s of matu­

r a t io n and spawning are made by Sekharan (1949), Bachanlal

10

(1963), Appa Hao (1967), Chaturvedi e t (1976), Pandey e t a l . (1 9 7 6 ), Slvalcami (1981), Shreni (1983), S elvaraj

(1984), and Sivakami ^ (1 9 8 6 ). S ig n ific a n t v ariatio n s in biochem ical composition of d i f f e r e n t tissu e s of fis h

d u rin g prespawning and postspavming periods have been obser­

ved w ith s p e c ia l refere n c e to c h o le s t e r o l, glycogen, water, p ro te in and t o t a l f a t contents ( I d l e r and B ltners, 1958;

Robertson, e t a l , , 1961? S id d iq u i, 1966? and Josh i, 1977), D e sp ite the voluminous li t e r a t u r e that have accumu­

la te d on th e proximate ccmposition o f various groups of fis h e s , c o n trib u tio n s c « s im ila r aspects in fis h e s of the fa m ily M u gilid a e are v e ry few . P ro te in , f a t , water and ash contents a r e e s ta b lis h e d in c e r^ a lu s by Saby (1934) Jewett and D avies (19 38) and in L . ramada and L* s a lie n s by Saby

(1 9 3 4 ). V a r ia t io n s in body ccm position of m ullets caught in the Swartlcops estuary of South A f r ic a have been discussed by K arias and Erasmus (1977).

I n s p it e of the fa c t th at high q u a lit y m ullet seed production depends on the understanding of proximate compo-

i s i t i o n o f the b ree d ers, co n trib u tio n s r e la t in g body ccmpo­

s it i o n w ith th at o f spawning h a b its o f m ullets are not much

accom plished. The only r e s u lt s obtained in th is lin e are

those o f Kuo in M. cephalus(quoted by Nash and Shehadeh, 1980)

and Gopalakrishnan (P erso n a l coirmtanication).

Because o f the physical and b io lo g ic a l hazards in the environment f i s h u n lik e other in v e rte b ra te s, have a high

re p ro d u c tiv e p o t e n t ia l where they produce s u ffic ie n t quan- t i r i e s o f eggs so as to enable s u c c e ss fu l recruitment*

A cco rdin g to Oven (1961), the mass o f g e n ita l products

c o n s t it u t e upto 30% o f the body and even much raore - a process c a l l i n g f o r in t e n s iv e energy. The f i r s t source o f energy i s su ggested to b e body f a t (G ie s s e , 1966) with t h e ir m obilization

becoming prominant a t the time o f maturation, even in non- m lgratory fis h e s *

Eggs a r e found to use p ro te in s te a d ily f o r energy

purposes d u rin g developrtent* S in ce newly spawned eggs cannot absorb much o f th e nutrim oit d i r e c t ly from the water, i t

sto re s adequate q u a n t it ie s o f e s s e n t ia l sulcstaoces fo r the growth* Sorvachev and Shatunovkll (1968) noted that as the co n cen tratio n s o f ‘ fr e e * amino acid s r i s e in the developing gonad, th e re i s a f a l l in corresponding amino acid s in the l i v e r , w h ile tc a n slo c a tio n of muscle proteins f o r gonad deve­

lopment has been reported by Masurekar and P a l (1979) in C y p rin u s c a r p lo *

Though glycogen le v e ls in the muscle, l i v e r and gonad a r e co m p aratively low (Needham, 1931), i t s r o le in m obilizat­

io n o f energy during maturation and spawning may not be

c o n s id e re d n e g li g ib l e * i^ccumulation o f glycogen and glucose

12

In the ovary has been reported (Greene, 1926; Chang and I d l e r , 1960 and Venugopalan, 1962) w hile Kuo (unpublished d a ta ) found a d i s t i n c t decrease o f glycogen le v e ls in the ovary b e fo r e spawning in M* eeohalus.

C h o le ste ro l i s an important precursor o f steroid hormone and i s a co n stitu e n t o f c e l l w a lls . Serum ^ o l e - s t e r o l le v e ls i s minimal at the time o f g re a te s t sexual conversion t o se x hormones. Shreni and J a f f r i (1974) have suggested ttiat the egg c h o le s te ro l r e f le c t s the parental

food in t a k e . C h o le s t e ro l le v e ls in gonads in r e la t io n with mat\xrity i s a ls o stu d ie d by Singh and Singh (1979), Sen and Bhattacharya (19 8 1 ), N a u ria l and Singh (1985) and (Dlwan and

K rishnan (1 9 6 6 ).

C arotenoids a re considercjd t o be the ultim ate source o f vitam in A and a re found to be derived from the d ie t .

C aroten oid s a re w id e ly d is trib u te d in the sk in and serves f o r cam ou flagin g. These terpene compounds are also deposted in the ovary d u rin g m aturation and are attrib u te d with the fu n c tio n s o f im p airin g colouration and v i a b i l i t y to the e g g s . While Matsuno ^ a l . (1985) have reported the change in the c o n p o sitio n o f carotenoids during maturation of

m arine f i s h e g g s, th e re i s evidence o f accumulation of

c a ro ten o id s in th e muscle, l i v e r and skin and further

m o b ilis a t io n in to gonads during maturation in Salmonids

(Steven , 1948, 1949; K ith ara , 1983). However, there has been l i t t l e e v a lu a tio n on the relatlcai between egg v ia b i­

l i t y and i t s pigm entation (C raik , 1985).

Ohe r o l e o f t r a c e e l u e n t s in f i s h are in man^

cases com pletely unknown (Berman and V itin , 1968). These authors fcwnd th at requirem ents o f tra c e elements d i f f e r at d i f f e r e n t sta g e s o f m aturation. However, since mineral ccm posltion o f gonad i s very low in comparison with the content In the body as a whole, the increase in gonad m in erals s i g n i f i e s e x c lu s iv e ly the growth o f f i s h proper.

Fu rth er, as Shul*man (1972) points out, there i s a close r e la t io n s h ip between th e trend and in ten sity o f protein and m ineral metabolism in fis h e s .

In v e s t ig a t io n s on the biochemical ccmpoeition of

f i s h en ables us to u n v e il the p o te n tia l storehouse of various n u trie n ts and to t r a c e the pathways through which they are m o b ilise d fo r the b i o l o g i c a l needs of the f i s h . Further, the le v e l s o f these substances could indicate the "q u a lity ” o f the eggs and ex a ct stage o f maturation of the fis h

(tiove, 1970) and so •Uiere aj^ e a rs to be p o ten tia l in

continuous e f f o r t in t h is f i e l d .

14

These f a c t s and the lack o f p r < ^ r Information on the b io c h e m ic al composition o f mullets In r e la t io n w ith m a tu rity has prompted t o take up the present work.

The b ioch em ical ccm position o f three tissues o f L , p a rs ia nafnely m uscle, l i v e r , and gonad are studied. Seven

param eters v i z « m o istu re , p ro te in , f a t , glycogen,

c h o le s t e r o l, c a ro te n o id , and ash are estimated in

r e la t io n t o the m aturation o f the gonad.

Note the Chinese d ip nets in Operation

s i r e s OF SPECIM EN C O L L E C T I O N A T T H E

COCHIN BARMOUTM

COLI£CTION OF SPSCU^NS

Ihe f i s h san ^les vece c o lle c te d bimonthly during the p e rio d froD February t o SeptenOser^ 1987 from the barmouth area o f Cochin estuaey; They

craght

the Chinese d i ^ e t s operated d u rin g the hours o f high tid e every day.

The specimens c o lle c t e d had a length range o f 115 to 236 nvn*

The f l s h a f t e r c o lle c tio n were carried in Icebox to the la b o ra to ry where they were washed thoroughly* Ihey were measured fo r t h e ir t o t a l lengths as w e ll as stand 2 trd lengths*

T heir body w eights a l s o were taken accurately u s in g a sen sitiv e b alan ce, The specimens were then cut cpen t o I d ^ t i f y t^e sex.

Males were d iscarded from the c o lle c tio n and fem ales alone were used fo r fu rth e r a n a ly se s. M aturity stages o f females were determined based on the macroscopic appearance o f the ovary

lik e shape and s iz e in r e la t io n to body cavity (Bowers/ 1954), co lo u r (Graham, 1924), extent o f yolk formation and mlcroscqpic s tru c tu re such as ova diameter m e a su r^ e its (C la rk , 1934), The gonads were assigned t o f i v e various reproductive stages v iz . Stage I ( immature}, s ta g e

s p e n t), s ta g e I I I (r ip e n in g ). Stage XV (r ip e ) and Stage V

(Sp en t) fo llo w in g the method suggested by Qazlm (1973) and

adopted by Kurtip.and Samuel (1983)•

16

Tissues o f muscle^ l i v e r and gonad were removed from the sam ples. Muscle tis s u e was taken frcia

f i r s t d o r s a l f i n , c a r e b ein g taken not t o include any s k e le t a l p a r t s . Weights o f th e whole l i v e r and gonad were a ls o noted t o the n earest mg. Enough portion s o f a l l the tissues were used f o r m oisture content an a ly sis and a p a rt was frozen t i l l

an aly ses were c a r r ie d out.

Simultaneous w ith each san p lln g o f the f is h , water tem­

p e ra tu re , s a l i n i t y and d isso lved oxygen o f the s it e of collectiCTi were determined. S a lin it y was estimated using M^u: t i t r a t i o n and ojjygen by W in k le r's lodcm etric method (S trick lan d and Parsons, 1968).

1 , BIOCHEMICAL ANALYSES

i ) Estim ation o f m oisture content:

Ihe sample prepared as above were washed in d i s t i l l e d w ater and the excess moisture removed u sin g a b lo ttin g

Ihe t is s u e s were weighed accu rately and la t e r dried In an a i r oven a t 70*C t o constant weight, ITie moisture ccntent was c a lc u la t e d as the d iffe re n c e between the wet weight and dry w eight o f the t is s u e which r.epresents the lo s s at 70*C and t h i s i s expressed as percentage o f wet weight.

Ihe samples d rie d in th is manner were po«dered in a mort:<ir, tra r;s fe rre d to la b e lle d polythajv'i bail's and stored in d s s s i c i t o r fo r c a rry in g out an a ly sis o f ash content an3 l i p i d

n t.

i l ) Sstim ation o£ t o t a l p ro teln t

For the estim ation o f t o t a l protein, the method o f Lowry e t a l . (1951)

fo llo w e d ,

25 rog o f the wet tis s u e was weighed accurately and a f t e r adding 2 ml o f d e p ro te in is in g agent (5% T rich lo ro a c e t ic a c id ), was homogenised thoroughly. When the p ro tein was ccfn- p le t e ly p r e c ip ita t e d , the saitple was subjected to c e n trifu g a tio n at 3000 rpm f o r 5 minutes. The supernatant was ranoved and used fo r glycogen a n a ly s is . To the precipitate^ 4 ml o f 1 N NaCH was added fo r d is s o lv in g the p ro te in . Frcin the re s u lta n t soluticm , 0.5 ml was p ip e tte d oat and was made upto 1 ml w ith

1 N NaCH, To t h is , 5 ml Of a33caline mix (50 ml o f 2X Na^CO^

in 0.1 N NaOH + 1 ml o f

prepared a fre s h ) was added and kept at rocm temperature f o r 10

m inutes. A ft e r 10 minutes, 0.5 ml o f Polin c io c a lte a u 's reagent (d ilu t e d t o 5056 with d i s t i l l e d water) was added,

A standard solu tion was prepared u sin g bovine serum albumin c r y s t a ls at a concentration o f 25 mg/lOO ml and a liq u o te s in the range 2 5 -2 5 0 ^g was prepared and the same procedure as fo r unknown Has fo llo w e d ,

A blank was prepared w ith 1 ml 1 N NaCH fo llo w in g the

same procedure as above.

18

A l l the tes-t tubes were l e f t at room temperature foe 30 minutes and readings were taken at 660 nin u sin g a spectrin photometer•

A standard grajAi was p lo tte d and anount o f protein s in the san^le ccmpared and ca lcu la ted frCRi the graph*

l i i ) Estim ation o f glycogens

Glycogen was estimated by the method suggested by V ile s e t a l - (1949) u sin g anthrcme reagent (0.2^ ^ t h r o n e in concen­

tra te d H 2 ^S 0 ^{^ ) ,} The su lp h u ric acid in anthrone reagent hydro­

ly s e s glycogen in to glucose and then dd^ydrates in to fu r fu r a l which g iv e s a coloured ccntplex with anthrone*

The supernatant obtained fra n the sample f o r protein estim ation was used f o r the a n a ly sis*

0,5 ml o f the supernatant was taken and made upto 1 ml with d i s t i l l e d w a te r. To t h is , 4 ml of anthrone reagent was added and kept in a water bath f c r 10«15 minutes and then cooled t o room tem perature in dark* the o p tic a l density was determined at 620 nm u sin g a spectrophotometer. The readings were compared w ith a brank prepared using 1 ml d i s t i l l e d

w ater and 4 ml anthrone reagent*

A standard was prepared using O .glu cose (concentration

10 mg/lOO m l) and a llq u o te s o f the range lOyug to lO O ^ g were

used t o p lo t a standard graph* Hie value obtained was

d iv id e d by 1,11, a conversion fa c t o r o f glucose to glycogen.

I

) E stim ation o f l i p i d s s

The d r ie d t is s u e powdered and stored in the d esslca tor was used f o r the eatiniation o f l i p i d ccntent. % e method followed was th a t

B llg h and Dyer (1959), using chlorofom wnethanol

m ixture in the r a t i o 2*1 v/v. To the weighed amount (100 mg) 3 ml o f ch o lo ro fo rm ^ eth a n o l mixture was added, mixed thoroughly and kept overnigh t at rocm temperature in dark. At the end of the p e rio d , another a d d itio n o f 2 ml o f chlorofcarm and 2 ml of w ater was made. The r e s u lt in g so lu tio n was subjected to

c e n t r ifu g a t io n , when g e n e ra lly 3 la y e rs were seen v iz , a c le a r low er la y e r o f chloroform ccntaining a l l li p i d s , an upper coloured aqueous la y e r o f methanol with a l l w ater soluble m a t e ria l and a thick pasty In te rfa c e ,

The methanol la y e r was discarded and the lower layer was c a r e f u ll y c o lle c t e d fr e e o f interphase by sucking with a f i n e c a p i l l a r y or by f i l t r a t i o n through g la s s w ool. The o rg a n ic la y e r taken In a preweighed beaker and c a r e fu lly evaporated in an a i r over) a t 60®C in order t o obtain the r e s i d u a l f a t . The beaker w ith the content was weighed a ft e r

c o o lin g t o room tem perature. The d lf f e r a i c e in weight gave

th e w eight o f l i p i d s . I t s percentage was c a lc u la te d on the

20

w eight Of d ry tis s u e taken and f i n a l l y converted to wet tis s u e b a s i s ,

v ) B stim ation o f t o t a l c h o le s te ro l:

Ih e method fo llo w e d fo r the estiniation of ch olesterol was th a t o f Henly (1957), a m odification of Hestrin*s(1949) method*

25 mg o f wet t is s u e was taken and was sxihjected to hcmogenisation* To t h is , 10 ml o f 0.05% PeCl^.SHjO prepared in p u r i f i e d a c e tic acid was added and l e f t fo r p recip itatio n o f p r o t e in . When the p re c ip ita tic m was ccm plet^ the tube was c e n t r ifu g e d at 3000 rpm f o r 5 minutes. 5 ml o f fiie supernatant was taken in a fre s h t e s t tube t o which 3 ml o f conc.H^SO^ was added. The tube was k ^ t a t rocm temperature for 20 minutes a f t e r thorough m ixing. The co lo u r developed was read at 560 nm u sin g a spectrophotoneter ag ain st a blank prepared using 5 ml FeC1^.6H20 g l a c i a l ac e tic acid and 3 ml concentrated

The standard so lu tio n used had a concentntion of 100 mg pure c h o le s t e r o l in 100 ml o f g l a c i a l ac e tic acJd d ilu ted In the r a t i o 1 :2 5 . The c h o le s te r o l content was calculated usin g the fo r m u la :

O .D *of unknown 0.2 - __

O io le s t e r o l content -

standard ^ i l l s *

v i ) S stlm a tio n o f ceurotenolds:

OlscTi's (1979) m^jthod was follow ed to estim ate the t o t a l carotOTOid content in v ario u s tiss u e s * In th is method chloroform s t a b i li s e d w ith 0,7596 absolute ethanol was used t o e x tra c t the carotenolds*

Ih e samples o f tis s u e s qu ick ly removed from the f i s h were p la c e d in g la s s v i a l s closed with t e f f lo n stopper and

sto re d in deep fr e e z e r u n t i l a n a ly sis * The samples were analysed w ith in a week of storage*

1 mg o f tiss u e prepared as above was weighed quickly and p la ce d in a 10 ml screw cap clean g la s s v i a l * To th is 2*5 gm o f anhydrous sodium sulphate was added and th is sample g e n tly mashed with a g la s s rod against the sid e o f the v i a l u n t i l i t i s reasonably w e ll mixed w ith sodium sulphate, 5 ml

o f chloroform was added and the v i a l was sealed and placed a t 0*C overnigh t (8-24 h o u rs ). When chloroform formed a c le a r la y e r o f 1-2 cm weight above the caked residue, the o p tic a l d e n s ity was read at 380 nm, 450

all<yiotes o f chloroform d ilu t e d to a colume o f 3 ml with ethanol* A blank prepared in a s im ila r manner was used fo r can p arison. The wavelength at which maximum absorption

obtained was used f o r c a lc u la tio n *

2 2

The t o t a l carotenoid content was calcu lated asyag carotenoid/gm t is s u e ,

C aroten oid content » y>3orptlon a t 450 nm X _dllution factcar 0.25 X sample weight

t h is case, d ilu t io n fa c t o r = 50, 0«25 i s ex tin ctio n c o e f f i c i e n t .

v i i ) B s tim a tim of ash content!

A known cjuantity of the oven d rie d sample was ig n ite d in s i l i c a c ru c ib le at 600*C in a m uffle furnace t i l l the organic m atter was burnt out le a v in g no carbon re sid u e . !Hid content was weighed and d iffe r e n c e gave the ash content

2.

The l i v e r index was ca lcu la ted in specimens at d if f e r e n t m atu rity s t a g ^ u sin g the formula

Hhe average o f the l i v e r index values at each m aturity s ta g e s w ere c a lc u la te d .

3,

-

(G S I )>

The gonadosomatic index was determined in each m aturity

s ta g e in v a rio u s in d iv id u a ls w ith a view to ascertaining the

c o n d itio n o f the gonad in each sta g e ; The GSl was determined

u sin g th e form ulai

weiaht o f oonad ______

“ ^ Igh t o f fis h ' ^

T^ie average o f a l l the GSI and the range was d e te r­

mined in each m aturity sta g e ,

4 . MICROSCOPIC BXAMINATICW OF OVARIES S

Aroind 500 ova were measured frcm 3 sair^les

at each m atu rity stage u sin g an o c u la r micrometer in order to determ ine the ova diameter fre<^ency in various stages (C lark ,

1934; Prabhu, 1955), No s p e c if ic are a was chosen to take the eggs as according t o Shehadeh e t ^ <1973b), n n lle t oocytes develop in synchroi^ and so samples taken frcm any area in the ovary would b e rep re se n tativ e o f the a i t i r e ovary',

1 micrcmeter d iv is io n was found to be equivalent t o 1 3 , 1 ^ , Ova diam eter frequency polygons were drawn a fte r

grou p in g the ova in to SOyu c la s s in t e r v a ls , 5 , STATISTICAL ANAIifSIS!

A l l the biochemical analyses except ash content a n a ly s is and carotenoid a n a ly sis were done atle a st 5 times and hence a l l the v alu es were pooled up and standard d e v ia tio n was c a lc u la t e d .

A n a ly s is o f varian ce was c a rrie d out to ascertain the p o s s ib le d iffe r e n c e in the contents of v a ria is parameters in the th re e t is s u e s namely muscle« l i v e r and gcmad at

le v e l o f s ig n ific a n c e .

24

R E S U L T S

d e s c r i p t i o n o f s p e c ie s

LIZA PARSIA (HAMILTCW-BUCHANAN) ( H a t e 2) SYNCNYM : MDGIL PARSIA HAMII/TCM-BUCHANAN 1822 VEraJAOJIAR NAME * GOID-SPOT MULIET

D IV ; I 8 A I I I , 8 -9 . P 14? C 14 L l a t 31-36 T r, 11-12 DISTINCTIVE CHARACTERISTICS!

Body s le n d e r, head moderately wide, d o r s a lly flatten e d . Head le n gth 23-26% o f standard len gth ; fa t ty (ad ip ose) tissu e

covers most o f the i r i s p o s t e rio r ly and p a rt o f i t a n te rio rly ; l i p s t h in , low er l i p w ith a high symphysial kncb; hind end o f upper jaw re a < ^ in g v e r t i c a l between p o s te rio r n o s t r i l and

a n t e r io r rim o f eye; mid gape above h o rla o n ta l thrqigh centre o f the p u p i l ; teeth - l ^ i a l « 2 rcws o f short teeth in upper l i p , lo w er l i p t o o t h le s s ; p r e o r b lt a l bone w ide, f i l l i n g the space between l i p and eye, notched a n t e r io r ly ; Pins t F ir s t d o r s a l f i n o r ig in n earer t o snout t i p tiian t o caudal f i n base - above 11th s c a le and second above 22nd s c a le ; pectoral a x i l l a r y s c a l e absent? p e c to ra l f i n 76-79% o f head length;

a n a l f i n w ith 3 sp in e s and 8-9 s o f t ra y s ; second d o rsa l and

an a l f i n s d e n s e ly s c a le d . S cales in l a t e r a l s e rie s 31-36*

I I (V

P la t e 3: Various m aturity stages o f female L . p a rsia

I , limiature, I I « Maturing, I I I . Mature,

I V . Ripe and V. Spent.

go ld en sp o t on upper operculum/ base of second d o rs a l, anal and c a u d a l f i n s y e llo w is h , other f i n s o ff-w h it e with dusky m argin s.

S iz e * Maximum s i z e o f species recorded i s 33 am, though comtonly a v a i l ^ l e s iz e range i s 15 - 16 cm.

DISTRIBUTION

A ccomon sp e c ie s i n In d ia d is trib u te d alon g the west c o a s t, sou th o f Bcmbay and a l l alon g the e a st coast« This s p e c ie s i s abundant in the c o a s ta l w aters, estueuries, lagoons,

and i n t e r t i d a l r i v e r s o f K era la. I t i s a ls o recognised in Andaman is la n d s . Outside In d ia , d is t r ib u t io n i s r e s tric te d t o I n d o » P a c ific area, where i t occurs in the se a and b r a ^ i s h w a te r in Indonesia, P h ilip p in e s and Thailand, Hongkong,

A u s t r a li a , Ceylon and K arachi.

CLASSIFICATIOT OF MATURITg STAGES (P la t e 3)

F iv e m aturity stages were id e n t ifie d based on the siz e shape and c o lo u r o f the ovary and the m icroscopic stru ctu re o f the ova, as suggested by Qazim (1973) and adopted by l^ r u p and Samuel (19 8 3 ).

S ta g e I (In m atu re): Ovary occupied le s s than one-fourth o f

th e body c a v ity , p in k ish coloured, translucent and J e lly lik e

in appearance. The eggs very sm all and i r r e g u la r without any

y o lk form ation s t a r t e d . Ova diameter ranged frcm 26yu to

196 .5 yu.

26

S-^aqe, ,11 (M aturing T lr g ln and Recovering sp en t): ovary i s almost h a i f th at o f the bocly c a v ity , s lig h t ly yellcwlsh|&ggs a re w h ite t o p a le y e llo w w ith traces o f yolk, granular and s t i l l n o t y e t f u l l y rounded, Diatneter o f ov^a ranged frcm

t o 288y a .

S tage H I (R ipen ing o r M ature): Gonad occupies three^foarth o r even more o f the body cav ity , ova beccming yellow ish aw3 opaque w ith d e p o sitio n o f y olk m aterial* Eggs not f u l l y rounded, w ith

the egg diam eter ran ging from 2 6 ^ to 5 0 0 ^ ,

Stage IV (R ip e )* The e n tire body c a v ity is f i l l e d with the ovary, b u t fo r the space occupied by the v isc era* Ovary deeply y e llo w is h and has co n sp ic w s blood v e sse ls* Seme r ip e ova are v i s i b l e to the e x t e r io r at the vent region* Yolk is found as a homogenous mass intersp ersed w ith vacuoles and f i l l i n g the

i n t e r i o r o f the oocytes* Hie ova diameter ranges from 26^u t o 694^

S tage V (S p e n t): Ovary occupies approximately h a lf of the body c a v l ^ ; dark p in k ish , appearing f l a c c i d , translucent and

shrunken. Ova diam eter ranges from

PROXIMATE COMPOSITION ANALYSES 1 , MtrSCIS (T ab le F ig . l )

1* M o istu re * M oisture content was in the range 76*30 to 60.239^,

From 78.02% In stage I# the value decreased to 76*3(K in Stage I I I

t h e r e a f t e r showing an increase t o 80*239( in Stage V*

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F l g . l * V a ria tio n s In b ioch em ical ccm positlon

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“ I V

M A T U R I T Y S T A G E S

28

l i « P r o t e in : P ro te in c o n t «it was the high est in stage I I I b e in g 18«00%, llie concentration showed a d e c lin in g trend In the su cceed in g stages reaching a lowest le v e l o f 15,7296 in sta g e V , ih e v a lu e s were almost constant in the f i r s t two

stages b e in g 17.30X and l7,15?i in sta ge I and I I re sp e c tiv e ly , i l l . lijp i d t L ip id content was the minimuin in stage I I (0«87?6) and the maxiinum in s ta g e I I (2«54%)« Beyond stage III^ the

l i p i d l e v e l was found decreasin g with a value o f 1,92% in s ta g e V , In s ta g e I , the lip id content was

GlvcoQent On the whole, glycogen content was ccmparatively le s s In the muscle w ith a range o f

The minimum co n c en tra tio n was obtained at stage I I I and the maximum

a t s ta g e I I , Fran s ta g e I I I onwards, an ascending trend was o bviou s,

V ,

O i o l e s t e r o l

C h o le ste ro l showed a pattern s im ila r to that o f glycogen w ith th e values in creasin g from sta ge I I I (0,14%) t o sta ge V (0,18?4)« In stage I , the c h o le ste ro l content was

0.1396 which in c re ased to 0.16% in stage I I , The range was D.13%-0,18%.

v i , A sh t Ash content w ith a range 1,03% to 1,94% was the

maximum in sta ge IV and minimum in stage V , Generally, the

m ineral l e v e l showed an a lte rn a tiv e in c re a sin g and decreasing

p a tte rn *

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LIVER

M A T U R I T Y S T A G E S

v l l , Ggro-tenotd (T a b le 4/ F ig , 4 ) : Carotenoid content was a t I t s minimum in s ta g e V b ein g l ,8 0 ^ g / g w hile the maximum was n o tic e d

I I (7,72%^^^/g), The le v e ls showed a steady d e c re a se f r o n s ta g e I I t o stage V. In stage I the valu e was 6 * 2 0 ^ g / g ,

2. LIVER (T a b le l ; F ig ,2 )

i . M oi3t*irgt In l i v e r , moist*-ra content ranged frcm 68*06%

at s ta g e I I I ■ ’■o

ihe le v e l shewed an a l t e r ­ n a tiv e in c r e a s in g and

pattern .

P rotg

'ihe

concentration o f p ro te in was found in s ta g e V (19,55'i) and a

in stage I I (14,86% ), But f o r a d e c rs s 3 s in sta g e IV <15,12%), the value in d ic ated an in c re a s in g p a tte rn from stage 21 to stage V*

le v e ls v.5re in the range 4,76% t o 5*77%

in d ic a t in g oq-.iable decre.-'-ie

5,77% in stage I to 4,76%

in s ta g e IV . In s ta g e V the le v e l showed recovery reaching a l e v e l o f 5*5C%«

I v * G lv c o ig rt: G lyco gen showed i t s highest le v e l in liv e r# o f a l l t is s u e s * Th ^ "aximum ancxint was found in stage IV (3*75%) w h ile the mini-TnLi was in stage V (0,37%), Prcra a le v e l o f 2,12%

In s ta g e I , i t d acrsased to 0,79% in stage I I and th e re a fte r

continued t o in c r a .is e ste< 3 ilv upto s ta g e IV*