3. Inland Fish. Sac. India, 23 ( I ) 199 1 : 16-26

E W E LARVAL REARmG OF GW)EU ETS AND SEA G m U LAICE

T. RAJYALAKSHMI, S. M. PILLAI AND P. RAVICIIANDRAN

Central Iwtitute of Brackishwater A yumlture, J t , Leith C a t b Street,

Santhom, Madras-600 028

The results of induced spawning and larval rearing of two species of grey mullets, Mugil cephallcr (Linnaeus) and Lira mmrolepis (Smith) and sea bream, Spa- m datnia (Forskal) at the Chilka lake mouth area are presented in this paper. Both homoplastic and heteroplastic pituitary injections were used for hypophysation. Female mullets in oozing condition were also .used.

A total of seventeen experiments were conducted with M. cephalus, thirteen with L. mamolepis and six with S. datnia. The respective size of brood fishes of M. cephalu, L. mmrulepis andS. datnia ranged from 800 g - 1.5 kg, 620 g - 1.5 kg and 760 g - 1.7 kg and their hatchings survived for 8, 14 and 7 days respectively. The role of environmental factors on broodstock availabiiity, maintenance, larval rearing, water quality management and the problems encountered in field condi- tions are discussed.

The grey mullets, Mugil cephalus (Linnaeus) and Liza macroltpis (Smith) and the sea bream, Sparus dztnia (Forskal) contribute significantly to the commercial fisheries of certain coastal and estuarine areas. The mullets are ideal specie7 for coastal aquaculture. The advantage of mullets for aquaculture have been well documented (Anon, 1983; Pillai et al., 1984; Rajyalakashmi and Chandra, 1987).

In India, experiments on induced breeding of mullets were initiated in 1961 and the first successful breeding of M. cephalus was reported in 1962 (Anon, 1962) followed by another report by Chaudhuri et al., in 1977. The controlled breeding and mass propagation techniques for M. cephalus have been reviewed by Nash and Shehadeh (1980). Induced spawningand larval rearing of L. nzacrolepir of peninsular India have been reported by Sebast'an and Nair (1975), Radhakrishnan et al., (1976), James et al. (1983), Kowtal and Gupta (1986). Natarajan and Patnaik (1972) described the embryonic and larval development of L. macrolepis of the Chilka lake. Studies on similar aspects have not been undertaken so far, in the case of sparids of India. Species belonging to the family Sparidae are of high culture value in the Mediterranean region (Mazzola and Rallo, 1971).

As part of the development of a comprehensive hatchery technology for mullets and sea bream, a series of experiments were conducted du-ing 1983-85 on the induced spawning and larval re%-ing of these species at Chilka lake and the preliminary results obtained are presented in this paper.

BREEDING AND LARVAL REARING OF MULLET AXD BREAM

Materid and methods

Experimental site

Short duration camps lasting 2-3 weeks were set up during October/November-January at the mouth area of Chilka lake. The camp site was 55 km away from Puri town and about 1 km from the lake mouth proper.

Collection and transportation of spawners

The grey mullets undertake seaward migration for spawning from the lake during October- January (Jhingran, 1958) and these spawners were caught in large numbers at the lake mouth area in dragnet (Khadijal). Mullets are also caught from the sea adjacent to the lake mouth. Commercial fishing for sea bream near the lake mouth is done with cast net, and hook & lines. They are also caught in dragnet.

The spawners were transported to the camp site by holding them in hand nets and wadillg through knee deep water along the shore. On reaching the camp sites. the fishes were swiftly transferred into large holding tanks. On many occasions female L. mac~olepis started oozing as soon as they were caught in nets and in such instances the spawners were quickly brought to the camp site and stripped immediately.

Maintenance of spawners

Brood fish were maintained in plastic pools of 60 to 100 I capacity. 50% of the water in the holding tanks was exchanged daily. A portable generator (1 HP) was used to maintain aeration. Aeration was also provided by an air compressor (1 HP).

Larval rearing system

For rearing the larvae, glass jars (12 l), plastic tubs (40 l), drums (70 1) and pools (4.9 m dia.) were used. The larval rearing units were also aerated. Daily 50% of the water was replenished from rearing units with fresh filtered water. A plastic drurc (70 1) was modified into a sand-gravel filter with an outlet at its base. For feeding the larvae, culture of Chlorella was maintained in 0.9 m dia. plastic pools by fertilization. The larvae were fed @ lo3 cells ml-1 from third day onwards.

Induced breeding procedure

Only healthy fishes were selected for experimcntation. The males were taken for stripping where free flow of milt occurred with slight pressure on the abdomen. The readiness of females for spawning was determined following the method of Shehadeh et aZ. (1973 a). The female fishes were given either homoplastic (mullet or sea bream pituitary) or heteroplastic (freshwater carp pituitary) pituitary injections to induce ovulation. The male fishes were not given any pituitary injection. The dosage of the hormone varied according to the condition of the fish and is expressed as mg/kg weight of fish or number of glands per fish.

Dry method of stripping was foiiowed (Chaudhuri et aE., 1977) to artificially fertilize the eggs when the females failed to spawn.

Wesdts

Table4 : Det& of Induced Breeding ExpePinnents of iMugil cef lhdu -. - - - - - - - - --

S i e Tirne of the injections and. dosage kg-"dght of the fishes bm/k8;) received homoplastic pituitary injections Remarks

Stripped after 11 h of the second injection. No fertilization of the egg.

Stripped after 13 h of the second injection. No fertilization.

Stripped after 12 h of the second injection. No fertilization of the eggs.

0600 h on 27-1 1-83 & 14 mg.

The fish died 7 h after the third injection.

Stripped after 10 h of the second injection. The eggs were ferti- lized but failed to develop further.

Stripped after 12 h of the thud injection. The eggs were ferti- Iized but there was no further development.

Stripped after 14 h of the second injection. The eggs were ferti- lized but development stopped at 4 cell stage.

The fish died after the third injection.

Stripped after 20 h of the second injection. Development stopped at gastrula stage.

BREEDING AND LARVAL REARING OF MULLETS AND BREAM 19

550/1.13 1700 h on 7-12-83 0800 h on 9-12-83 - Stripped after 30 h of the second 8; 16 mg & 28 mg injection. Development arrested

at gastda stage.

43011.0 0945 h on 15-12-83 . - - Stripped after 4 h. The eggs were Br 20 mg not fertilized.

510il.4 1230 h on 2412-83 1830 h on 2412-83 - Stripped after 9.30 h of the second & 9 mg & 18 mg injection. Development stopped

at 2-cell stages.

43210.8 2000 h on 7-12-84 0700 h on 8-12-84 2000 h on 9-12-84 The fish died aftcr the rhird & 10 mg & !5mg & 8 mg injection.

188/1.4 2000 h on 8-12-84 2000 h on 9-12-84 - Stripped after 10 11 of the sewn3 in- &4mg & 12 mg iection. No fertibation of the cgys.

Experiments with hormone administration

Sixteen experiments were conducted during 1983-84 breeding season. The size of the females ranged from 420-525 mm in length and 1 .O-1.5 kg in weight. The total length and weight of the males ranged between 300 and 362 mm, and 250 and 400 g respectively. The female fish received only hom~plastic pituitary injections. The abdomen of the recipient spawners distended considerably after treatmect and the genital pore became reddish and dilated. The details of the experiments are given in Table-I.

Out of the sixteen experiments, fertilized eggs were obtained in six cases only. Of these, embryonic development was observed only in four cases. However, there was no hatching of the eggs. The effective dose of the hormone which inducedovulation varied from 5 to 28 mg kg-% body weight. The ovulation period generally ranged from 10 to 30 h after the last injection at a water temperature of 21 to 27'C. The percentage of fertilization was in the range of 50-80.

Natural spawning

In November, 1983 one female M. cephalus (525 mml1.5 kg) kept in the holding tank to re- cover from the stress of capture and transportation, started spawning in the tank itself before hormone administration. By the time the fish was taken out of the tank, it had almost completed spawning. Upm stripping, the fish released 1000 eggs and they were fertilized with the milt of males. Normal development was observed. The rate of fertilization was 80%.

Larval development

The larval development of M. cephalus was similar to the observations made by Chaudhwi et al. (1977). The fertilized eggs were 0.6-0.8 mm in diameter. The incubation period was 44 h

20 T. RAJUAURSHMI ef al.

at a water temperature 21-25OC and salinity 30-32 ppt. The hatching rate was 80%. Only 800 hatchlings were produced. The larvae developed mouth on the third day and it became functional on the fourth day. The hatchlings were fed with Chlorella @ lo3 cells mi-' from the third day. Heavy mortality of the hatchlings were observed daily. The hatchlings survived up to 8 days.

Liza macrole@is

Experiments with hormone administration

During 1983-85, five experiments were undertaken with hormone administration. In four trials, homoplastic pituitary injections were used and in the fifth one heteroplastic (carp) pituitary injections were given. Only females were injected. Males collected were all in oozing stage. The size of the females ranged between 424 and 500 nm in length, and 0.660 and 1.5 kg in weight. The males were in the size range of 260-373 mm in length, and 180-600 g in weight. The details of the experiments are shown in Table-2. In one experiment, only hatchlings were obtained.

Table-2. Dee& of Induced Breeding Experiments of Liza rnu4ro&@is

Size Time of the injections andl dosage kg-" i-Ilrlk) fish Remark

42810.66 1130 h on 12-12-83 1000 h on 13-12-83 Stripped after 7 h of the second injection. Ob- & 30 mg (MP) & 30 mg (MP) served development upto 2-cell stage.

42910.78 1430 h on 12-12-83 - After 2.30 h of the injection the fish started & 20 mg (MP) oozing. Development progreued up to gastrula

stage.

45710.8 1015 h on 16-12-83 - Stripped after 11 h of the injection. Normal dwe- & 25 mg (MP) lopment noticed. 4 lakh hatchlings obtained.

Incubation period 31 h. The hatchlings survived 14 days.

42410.7 1530 h on 22-12-83 - Stxippcd after 1830 h of the injection. Develop- & 22 mg IMP) ment observed up to 4-cell stage.

5001 1.5 1700 h on 6-1-85 0700 h on 7-1-85 Stripped after 28 h of the second injection. The & 14 mg (MP) & 14 mg (CP) eggs were fertilized but no further development

MP ZI Mullet pituitary; CP = Carp pituitary

The period of ovulation from the last injection varied from 2.30 to 28 h at a water tempera- ture of 20-32OC and saliinty of 28-38 ppt. The effective dose of the hormone ranged from 14 to 30 mg kg-' body weight. The percentage o f fertilization ranged from 20 to 70.

BREEDING AND LARVAL REARING OF XIULLETS AND BREAM 2 1

Natural spawning

L, rnncrolepis were caught in drag nets within the outer channel area near the mouth. More than 90% of the females thus caught were in oozing condition. A total of eight breeding trials were conducted during 1983-85 with these fish. The size of the females ranged between 391 and 530 mm in length, and 0.4 arid 1.4 kg in weight. Out of the eight trials, hatchlings were produ- ced in two cases only. The details are furnished in Table-3.

Table 3. Details of Breeding Experherats wi& natural spawners of Lira macrolcpis

Size (mike) Date Remarks

40610.62 2-1 1-83 Collected in oozing condition. Stripped. The eggs were fertilized but no further development.

4-12-83 Collected in oozing condition. The eggs were fertilized. Develop- ment stopped at gastmla stage.

44010.8 12-12-83 Stripped the oozing fish. The eggs were fertilized, but no further development.

47211.4 10-12-84 Collected in oozing stage and stripped. The fertilized eggs stopped development at gastrula stage.

53011.4 14-12-84 The fish was in oozing condition and partially spawned, at rhe time of collection. Stripped and fertilized the eggs. Normal develop- ment was observed and obtained 20,000 hatchlings. The larvae swived 2 days only.

7-1-85 The fish was partially spawned in oozing stage at the time of collection. Stripped and fertilized the eggs. Normal development was noticed. Produced 1.32 lakh hatchlings. The larvae died on the 3rd day.

Larval development

The larval development of L. macrale$is observed in the present study was similar to that re- ported by James et al. (1983). Of the five trials conducted with hormone administration, develop- ment of eggs was observed in four cases. However, only in one fish, the development was com- plete and hatchlings obtained. The size of the fertilized eggs varied from 0.8 to 0.9 mm. The incu- bation period was 31 11 at 22-30°C and the hatching rate was 66.6%. A total of 4 lakh hatchlings were obtained from this trial. They survived for 14 days.

In the trials with natural spawners, hatchlings were obtained in two cases. The size of the eggs varied from 0.8 to 0.9 mm in this case also. I n the first trial, the incubation period was 28 h at 24-30°C. The hatching rate was 50%. A total of 20,000 hatchlings were obtained which sur-

vived upto 2 days. In the second :rid, the incubation period was 27 11 at 24-28'6. The hatching rate was 88% and a tota! of 1.32 iak'n hatch!irgs were obtained. They survived for 3 days only.

In ail, a ~ o t a i of 5.52 iakh hatchlings were produced from three successful trials. The larvae were reared in plastic drums, basins and pools under aeration. The mouth formation was corn- plete in the larvae on the fourth day and they were fed with Chlorella @ 103 cells mYs.

Experiments with hormone administration

Females of S. dat:lin were given both homoplastic and heteroplastic (carp) pituitary injections. In 1983, five experiments were conducted. The length and weight of the females ranged between 341 and 453 mm and 0.760 and 1.7 kg respectively. The size of males ranged between 273 and 405 mm (0.38 and 1.23 kg). Of the five experiments, hatchlings were obtained from one trial only. The details are given in Table-4.

Table-4. Details of Indaeed Breedhe E x n e b e n t s of Sfiarrts datnia

Size Time off the injection a n d dosage kg-! body of the iish (mm1kg)

1st 2nd 3rd Remark

43011.5 1000 h on 30-11-83 1600 h on 30-11-83 1600 h on 1-12-83 Stripped 16h after the third & 16 mg (CP) & 32 mg (CP) & 64 mg (CP) injection. Fertilization md nor-

mal development were obsuved. Produced 2000 hatchlings and the larvae survivcd 5 days.

36311.12 1800 h on 1 -12-83 1715 h on 16-12-83 - No response. The fish died. & I gland (SP) & I gland (SP)

34110.76 1700 h on 22-12-83 1830 11 on 23-12-83 - No response. The fish died after & 52 mg (CP) & 52 mg (CP) the second injection.

36810.85 1700 h on 22-12-83 I830 h on 23-12-83 0800 h on 26-12-83 No response. The fish died. & 58 mg (CP) & 24 mg (CP) & 24 mg (CP)

34810.84 1800 h on 2412-83 - - By 12 h after the injection, the & 19 mg (CP) fish started oozing. Stripped but

no development of the eggs.

CP = Carp pituitary

At a water temperature of 20-2fi0C and salinity 30-38 ppt, the ovulation time varied from 12-24 h. The effective dose of the hormone ranged from 16 to 64 mg kg-' body weight. The rate of fertilization was 520%.

Natural spawning

One female S. datnia in the size of 453 mm11.7 kg was collected in .Tanuary 1984 from the

BREEDING AND LARVAL REARING OF MULLETS AND BREAM 23

outer channel near the mouth. The fish was stripped. Only partial spa~ning was noticed. The eggs were fertilized and normal development observed. The fertilization rate was 50% and 40,000 hatchlings were produced.

Larval development

On two occasions complete larval development was observed and resulted in production of hatchlings. In the first case the fish was given heteroplastic pituitary extract injections. At 24- 26°C temperature and 30 ppt salinity, the incubation period was 24 h. The fertilized eggs were in size range of 0.4-0.6 mm. The hatching rate was 2% and 2,000 hatchlings were obtained. The larvae survived for 5 days. In the second case, larvae were produced by using a natural spawner. The size of the fertilized eggs varied from 0.4 to 0.6 mm in diameter. The incubation period was shorter, 20 h at a lower temperature of 20 OC. The hatching rate was higher, 20% and produced 40,000 hatchlings. They survived for 7 days.

A total of 42,000 larvae were produced during 1983-84 from the two experiments. As in mullets, the larvae of S. datnia developed mouth on the fourth day and started feeding. The lanrae were fed with Chlorella @ 103 cells/mI. Heavy mortality of the larvae were noticed daily especially during early morning hours.

Discussion

The effective dose of the pituitary extract to precipitate ovulation varied in mulIets and sea bream. Homoplastic and heteroplastic pituitary extract injections alone or in combination with other synthetic hormones were used by various workers to induce ovulation in mullets, Further, the number of injections required for hypophysation also varied. According to Nash and Shehadeh (1980) the efficiency of any dose is related to the sexual maturity of both the donor and recipient, the gonadotropin potency of the injected preparation and the physiological state of the fish. For precipitating ovulation of M. cephalus, a threshold dosage of 3 mullet pituitary glands and 40 rabbit units of Synahorin (Tang, 1964), 2.5 to 6.0 pituitary glands along with 10-16 rabbit units of Syna- horin and 0 to 3,000 mg vitamin E (Liao et al., 1971), 20 I.U/100 g body weight of HCG (Shehadeh et al., 1973 b) and 2-8 mullet pituitary glands per fish (Chaudhuri et al., 1977) were successfully tried. In the present studies, the effective dosage varied from 9-28 mg kg1 body weight of mullet pituitary glands.

The threshold dose to induce ovulation in L. macrolepis was found to be 7 homopIastic pituitary glands (Sebastjan and Nair, 1975), 4-20 mg kg-' body weight of mullet pituitary glands (Radha- krishnan et al., 1976),600-1200 mg kg-' body weight of heteroplastic glands and 1,10,000 to 3,40,000 I.Ukg-'body weight of HCG(James et al., 1983) and 1-6 homoplastic glands(Kowta1 and Gupta, 1986). The effective dose of the hormone varied from 14-30 mg k g ' weight of pituitary glands in the present experiments. In the case of S. datnia, the effective dose varied from 16-64 mg kg-' body weight of pituitary glands.

The ovulation period was reported to be 32 h (Ya~houv, 1969) and 48 h (Ling, 1970) kter the first injection in M. cephalus. According to Chaudhuri et al. (1977) the time taken by M, cephalus for ovulation was 10:30 to 20.00 h after the first injection at a water temperature 16-27.5'C.

James et al. (1983) reported that the time taken for ovulation varied between 33.30 h to 85.20 h for L. mamolepis at a water temperature of27-33.2'6. Kowtal and Gupta (1986) reported that the ovulation time ranged from 06.30-09.30 h in L. mcrolepis. In the present studies, the ovulation period varied from 12.30-44.00 h for M. cephulus at a water temperature of 21-27'C, 02.30-36.00 h for E. mrolep i s at a water temperature of 20-32OC and 12-46 h for S. datnia at a water temperature of 20-26OC. As stated by James et al. (1983) these variations may be due to environmental con&- tions.

The diameter of the fertilized eggs of Ad. cephalus varied between 0.93 and 0.95 mm (Ling, 1970). The average size of the fertilized eggs for this species was given as 0.48 mrn in one experi- ment in 1961 and as 0.8 mm in the subsequent experiments by Chaudhuri et al. (1977). Natarajan and Patnaik (1972), Sebastian and Nair (1975) and James et al. (1983) reported that the size of the fertilized eggs of L. nucrolepis varied from 0.677 to 0.732 mrn, 0.665 mm and 0.74 to 0.78 mm, respectively. In the present studies the size of the fertilized eggs of M. cephalus, L. m o l e p i s and S. datnia varied from 0.6 to 0.8 mm, 0.8 to 0.9 mm and 0.4 to 0.6 mm, respectively.

The fertilized eggs of both the mullets were buoyant and floating at the surface initially and as development progressed they slowly settled down in still waters. The same phenomenon was observed by Chaudhuri et d. (1977) in M. cephdus. To avoid sinking, aeration was provided and the eggs were thus kept in suspension. Sebastian and Nair (1975) and James e t al. (1983) have reported that the fertilized eggs of L. macrolepis floated at the surface of the rearing units. The fertilized eggs of S. datnia also floated at the surface as in case of mullets.

According to Chaudhuri et al. (1977), the incubation period in M. ce th lus ranged from 32 ' to 52.30 h a t a temperature of 19.5-28OC. An incubation period of 23 h was reported by Sebastian and Nair (1975) and 18 h by James et al. (1983) at a temperature of 27 to 30.6'6 for L. macrolepis. In the present studies, incubation period was 44 h at 21-25OC for M, cephalus. For L. macrolepis the hatching time varied from 27 to 31 h at 22-30°C. In S. datnia, the hatching time ranged from 20 to 24 h at 20-26OC.

The larval phase is the critical period in all the above species. Very high mortality of mullet larvae was reported on the 3rd to 4th day when the larvae start feeding (Kuo et al., 1973; Chau- dhuri et al., 1977, Nash and Shehadeh, 1980; James et al., 1983). In 5'. dafnia also, heavy mortality of the larvae was observed on the 4th day when mouth became functional.

Apart from feeding, sudden drop in temperature coupled with low dissolved oxygen at late night and early morning adversely affected the survival of the larvae. Mass mortality of larvae were recorded during certain days when cold wind was observed. At Chilka lake mouth area, the camp was set up on the sea beach and wide fluctuations in temperature was felt during Novem- ber-January when the experiments were conducted. In addition to the low temperatures, unseasonal and delayed monsoon rains lashed the coastal districts of Orissa upto November in 1985 and 1986 and resulted in heavy discharge of flood waters to the sea. This, in turn, affected the sali- nity regime of the near-shore waters and the outer channel of the Chilka lake. The salinity declined to 16-20 ppt in 1985-1986 breeding season. Consequently, the mullet fishery became very poor during this period resulting in sparse availability of spawners. Another problem experienced, especially with S. dotnicr was to get ripe fish of both sexes at the same time. Cryopreservation of

BREEDING L4RVAL REARING OF MUWaETS AND BRErlh4 25

sperm might have to be undertaken for this spcc'rq (Kurokura and Sakamura, 1985,. However, these studies haw jadicated the possibility of hatcl~t,-y ;>roductio~ ef T::-J~ of the above species.

The authors extend their grateful thaakr trr Dr. B. rilagars:va:ri, Dira:tc)r, Ccntrai ins- titute of Brackishwater Aquaculture, Madras for his critical readin-g of tllo rnacus. ript a rd sugges- tions for it4 improvement.

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