CHAPTER iii

Embryonic and larval development ofOnipok nialabaricus (Val.)

301.00 INTRODUCTION

Studies on biology of fishes have gamed importance in view of

conservation and management of fish genetic resources. Considering

the utilization of vast swampy areas in India for propagation and

culture of air and non-air breathing fishes, it has become imperative to

study and fill up the gaps in the information on the biology of these

fishes. The recognition of early life stages by fish culturists is

important because the requirements of young fish change rapidly with

age. No doubt, fish culture practices the production of fry, rearing and

growth of youngones the primary requisites (Haniffa et al., 1999).

Definitions pertaining to developmental stages have been

described in aquacultural practical (Haylor, 1992; Basheer et al., 2001).

61

In response to calls for a clear definition regarding the length of the

larval period, and enhancement of compatibility of different studies,

developmental definitions of 0, nialabaricus are suggested in the

present study.

The developmental stages of non-air breathing fishes like 0.

hirnaculatus (Sridhar et al., 1998) and air breathings fishes viz., C/ian iia

niarulius (Khan, 1926; Mookerjee, 1945a); Glaiina striatus (Alikunhi

1953) Notopterus notopterus, (Parameswaran and Sinha, 1966); Anahas

testudineus (Moitra et al., 1987; Hughes et al., 1986), Monopterus cuchia

(Singh et al., 1990), Clarias batrachus (Thakur 1978a, 1980; Mookerjee

and Mazumdar, 1950), Heteropneustes fossilis (Thakur et al., 1974; Bhatt,

1968) and Mystus niacropterus (Wang et al., 1992) have been described.

During the embryonic (egg and eleuthero-embryo) stages of

C/arias gariepinus, the yolk material provides the substrates for energy

and growth (Verreth et al., 1993), Haylor (1993) and Haylor and

Oyegunwa (1993) investigated the early developmental stages and the

timing of the onset of air-breathing in young African catfish, C/a rius

gariepinus.

62

A hatchery management in true sense involves breeding of fish,

hatching of eggs and rearing of hatchlings to larval stage. The rearing

of larvae is a very delicate technical work as the larvae are very tender

and easily succumb to adverse changes in physico-chemical conditions

of water and also they easily become prey to predators. The success of

the seed production depends on the efforts and attempts to provide the

most conducive conditions for the survival of the embryos and larvae

in the hatchery (Venugopal, 1995). Therefore, it is of paramount

importance that proper care should be involved at every phase of life

cycle of fish, to ensure high survival and to gain sizable returns in

commercial venture.

Some species accept formulated feed during the last part of the

yolk absorption period, i.e., after the larvae have utilized most of their

yolk reserves they begin to ingest exogenous food, the stage being

defined by Balon (1985) as eleuterembryonic stage and by Kamler

(1992) as the mixed feeding period. However, in many other cultivated

species, initial exogenous feeding of larvae is usually secured by the

supply of live feed organisms mainly Rotifers and Arternia ;iaupli upto a

period of weaning (weaning size or adaptation weight). The weaning

period often concurs with the moment at which the stomach becomes

63

functional with a switch from a digestion exclusively intestinal to a

mainly stomach digestive activity (Dobrowski, 1992).

Asha Landge (1995) while reporting the feeding strategies in

Clarias bat radius suggested that finely sieved zooplankton could be

given to 6 - 8 days old spawn; sieved boiled hen's egg (white) may be

added to the diet for 10-12 days old fry and the yellow of the eggs was

suitable in the diet of advanced fingerlings and fry.

3.2. 0. MATERIALS AND METHODS

3 2 10 Egg collection

After spawning, the eggs (Plate 17) were carefully collected from

the nursery tanks using a beaker and poured into a bucket containing a

small quantity of water (Haniffa et al., 2002a). The number of egg

spawned were determined volumetrically (Laglar, 1956). The

fertilization and hatching rates were determined as the percentage of

normal egg/larvae from the total number of live eggs in each sample.

A few hundred eggs were allowed to hatch and grow along with the

parents in the breeding compartment to observe the parental care

(Haniffa et al., 2001 c).

—

64

3. 2. 2 Embryonic and larval development

Eggs were sampled every hour during the first 24 hours and

every 6 hours for the next three days and then only once a day.

Specimens anaesthetized in a solution of 10% ethanol were used for

observations and measurements were made under a dissecting

microscope with a ocular micrometer (Erica company, Japan). The egg

samples and larvae were fixed in 1% neutral buffered formalin and

different embryonic stages were observed within 4 hours (Fermin,

1991) under Nikon microscope (E400) (Plate 18).

3. 2. 3. Fertilized and unfertilized eggs

The fertilized eggs and fertilized eggs were identified based on

their shape, colour, weight, transparency and adhesiveness.

3.2.4. Rearing of post larvae

The early post larvae of 0. malabaricus were reared in plastic

troughs (50 individuals! 25 1 capacity) (Plate 23) by supplying plankton

soup, chironomus larvae, mosquito larvae and earth worms for a total

duration of 21 days. The larvae were grouped into three batches.

Water was changed daily with minimal disturbance to the larvae; they

were fed with live feed daily three times (9h, 13h and 17h) at 10% of

their total body weight. Faeces, unIed and dead larvae if any were

65

siphoned out from each aquarium every morning, before the first

feeding (Haniffa et al., 2001a). Dissolved oxygen (5.9 ± 0.3 mg/ 1), water

temperature (28°C ± 5°C) and pH (6.6 - 7.0) were recorded. After the

experimental period the length and weight of the fish were recorded

and tabulated.

3 2 5 Rearing of fry

Feeding trails were conducted in 200 1 cement tanks (3 m x 1 m

x 1 m) (Plate 7). Water temperature was 28 ± 2°C and a photoperiod of

12 h light and 12 h dark were recorded. In each tank, 50 fry were

stocked and assigned to one of the feeding regimes viz., plankton,

chicken intestine and beef liver.

3.3. 0. RESULTS AND DISCUSSION

A summary of the timing of the important ontogenetic events

and structure is presented in Table 3.1. In the present study, the

developmental stages were divided into embryonic development,

larval development and post-larval development. Similarly

developmental pattern was reported by (Fermin, 1991). The

description of the visible stages of development is as follows.

66

3. 3. 1. Morphological development

3. 3. 1. 0. Fertilized eggs

The fertilized eggs of 0. malabaricus were golden brown, non-

floating, adhesive and found attached to the sand bed of the tank. Un

fertilized eggs also showed the same charactertics but pale white in

colour. The diameter of the fertilized eggs ranged from 1.20 ± 0.04 mm

to 1.22 ± 0.05 mm at a temperature of 26 - 28°C. Descript on the

morphology of fertilized eggs of 0. nialabaricus were more or less

similar to that of reported by Haniffa et al. (2001a).

Fertilized eggs of 0. nzalaharicus were adhesive, demersal and

spherical in form. The yolk sphere contained no oil globule. Due to

the adhesive nature of the egg, debris got adhered to the capsule of the

egg. Often, along with debris some living protozoan such as vorti ce/la

were also found attached to the egg-capsule. The egg-capsule was

transparent and golden brown while the yolk was brown in colour.

The pre-vitelline space was large measuring 0.1 - 0.3 mm in width. The

eggs became translucent as the development proceeded.

3. 3. 1. 10 2-celled stage

First cleavage commenced in about 40 mm. after fertilization

dividing the blastodisc in to two blastomeres.

67

3. 3. 1. 2. 4-celled stage

Meroblastic verticle cleavage occurred that separated the

blastodisc into two equal halves; second cleavage at right angles to the

first cleavage, divided two cells into four equal sized cells; each

blastomere with rounded outer and straight inner margin.

3. 3. 1. 3. 16-celled stage

Further vertical cleavages resulted in the formation of 16 - cells,

smaller in size than the 4-celled stage.

3, 3, 1. 4, 32 celled stage

Horizondal cleavage resulted in 32-cells, forming a layer of

upper and lower blastomeres; cell size was further reduced over the 16-

celled stage.

3. 3. 1. 5. Morula stage

Blastoderm cells increased in number but decreased in size;

tightly packed and invaded areas over the yolk Blastodermal cells

were golden yellow whereas uncovered yolk was blackwish red in

colour.

3. 3. 1. 60 Gastrulation stage

Most of yolk was invaded by blastoderm cells; only a small

portion of uncovered yolk persisted towards one pole c egg,

68

extending slightly sidewards; margins of blastoderms towards this

pole and its side were thickened to form a germ ring, which were the

embryonic rudiments.

3. 3. 1. 7, Differentiation of cephalic and caudal ends

Embryo elongates on the periphery of yolk, distinguishing a

slightly thickened anterior head region and an angular posterior tail

region but without any sign of internal structures or massing of cells.

3, 3. 1. 8 Initiate of optic-vesicle and 16-19 somites

Embryo 'c' shaped with broad and anteriorly blunt head region,

rudiments of optic - vesicles and 16 19 somites; cephalic and caudal

ends further differentiated and became distinguished.

3. 3 1. 9. Appearance of brain, auditary - vesicle rudiments and25-28 somites

Embryo with distinct head and tail region; inner thickening in

the head indicates initiation of brain. Auditory vesicle rudiments

without otoliths, appeared posterior to brain. Embryo with 25 - 28

somites, made occasional twitching movements within the egg arid had

narrow embryonic fin fold surrounding the tail. Kupffer's vesicle

appeared (in one specimen only) as an oval area at the base of caudal

region.

69

3. 3. 2 Observations

3A. 2. 0. Embryonic development

The spawning of 0. nialabaricus is generally completed in abou1:

one hour during which the eggs are ejected in batches at irregular

intervals and as a result the eggs from a given set of spawners do not

develop at the same place. The time of developing stage stated in the

present description may, therefore, in general be considered with the

allowance of about one hour (Fig. 3.1).

3. 3. 2. 1. Cleavage

The first blastomere was formed 35 min after fertilization. The

first meridional cleavage was apparent with in 40 min of fertilization

and produced two blastomeres of equal size (Plate 25). The second

meridional cleavage was at right angles to the first cleavage and

produced four equally sized blastomeres 1 hr - 0.5 min after

fertilization (Plate 26). The third cleavage commenced 1 hr - 22 mm

after fertilization and produced eight blastomeres of equal size. A

horizontal fourth cleavage produced a double layered bias todisc

consisting of 16 blastomeres of equal size, 1 hr - 42 min after

fertilization. As successive cleavages occur the blastomeres decrease in

size and the morula stage is reached 3 hrs - 25 min after fertilization,

following the morula-stage, the crown of the blastoderm starts

70

Fig. 3. 1. Embryonic developmental stages of 0. malabaricus

1. Formation of Pre-vitelline space, 6 min. after fertilization.2. Formation of blastodisc, 35 mm. after fertilization.3. 2 celled stage, 40 mm. after fertilization.4. 4 celled stage, 1 hr-10 min. after fertilization.5. 8 celled stage, I hr-22 mm. after fertilization.6. 16 celled stage, I hr42 mm. after fertilization.7. 32 celled stage, I hr-50 mm. after fertilization.8. 64 celled stage, 2 hr-21 min. after fertilization.9. Morula stage, 3 hr-25 min. after fertilization.10. Post gastrula stage, 8 hr-20 mm. after fertilization11. Formation of embryo, 9.45 hr after fertilization.12. 34 somites stage, formation optic & brain vesicles,

12 brs after fertilization.13. 6-7 somites stage, formation of optic vesicles,

13 hrs after fertilization.14. 16 somites stage, 17.30 hrs after fertilization.15. 25-28 somites stage, formation of eye lens,

specialization of brain, 21 hrs after fertilization.16-12-36 somites stage, 23 hrs after fertilization.17. Hatching of embryo, 23 hrs 45 min. after fertilization.18.Just hatched larvae, 24 hrs after fertilization.

0206,

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Fig. 3.1. Embryonic developmental stages of 0. rnalaharicus

invading the yolk by spreading over the latter in the form of a thin

layer (Plate 27 & 28). In the successive stages of development, the

spread of embryo over the yolk-sphere gradually increases in length.

In Clarias bat radius, the morula stage was attained in 3 hours

after fertilization (Thakur and Das, 1986; Zairin et al., 1992). In Channa

punctatus after about 45 min of fertilization the 16 - celled stage is

reached (Banerji, 1974, Munshi and Hughes, 1991). In Indian major

carps, the morula stage is reached within 2 - 3 1 /2h after fertilization

(Chondar, 1999). In 1-teteropenustes fossilis, the first cleavage begins in

about 30 min after fertilization and the 16 - celled stage develops in

about 70 - 80 min and about 100 mm, the morula stage was attained

(Thakur et al., 1974; Kohil and Goswami, 1987; Alok et al., 1993;

Koteeswaran et al., 2000).

30 3. 2. 2. Gastrulation

Initial expansion of the epiblast was uneven before becoming

more uniform between 8 hr - 20 min after fertilization. When the

germinal ring and embryonic shield is appearing, yolk invasion

progresses considerably. At this stage, the head and eye of the embryo

become distinguishable. After 8 hrs about two third of yolk invasion is

complete (Plate 29).

71

In Mono nterus cuchia, as the time of gastrulation approaches, the

under-rim of the blastodisc thickens to form a marginal ridge or

germinal ring (Sing et al., 1990). According to Thakur et al. (1974), the

head and tail ends of the H. fossilis embryo become distinguishable

after about 10 hours after spawning. Invasion of the yolk by the

blastoderm is gradually completed (Munshi and Hughes, 1991). In

Indian major carps, the yolk plug stage is almost completed within 5

hours alter fertilization (Chondar, 1994). Baneiji (1974), Munshi and

Hughes (1991) reported that in C. punctatus, the blastula stage appears

after 2 - 3 hours and the yolk invasion is completed 9 hours after

fertilization. Thakur (1980), reported that in C. batrachus, the head and

tail ends of Clarias batrachus embryo become distinguistable after 3

hours and the yolk invasion is complete by the end of 10 th hour of

development.

In Hepluiestus carbo the yolk plug becomes visible 9 h alter

fertilization, blastopore closure completed the gastrulation phase of

embryonic development 10 - 11 h alter fertilization (Close et al., 2001).

3. 3. 2. 3. Organogenesis

Differentiation of somites occurs with in 12 hours and

appearance of 3 - 4 somites marks the distinction of embryonic

72

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rudiment (Plate 30). In the 6 - 7 somites stage two small otic vesicles

are formed. With the increase in the number of somites (Plate 31), the

embryo gets further elongated till it encircles a little more than 2/3 way

round the yolk mass and appears like V. By 17.30 hrs, the number of

somites increases to 16 and the position of Kupfer's vesicle and a

pulsating mechanism, which is the fore runner of heart are discernible.

After an hour, the Kupfer's vesicle vanishes and the lens in the otic

cups, appear. The eye and eye lens are further developed and

demarcation of brain is also noticed (Plate 32), At this stage the caudal

portion of the embryo becomes free from the yolk mass, which gets

steadily used up. The tip of the tail is round while the fin-fold is

hardly recognizable. Notochord becomes visible in the form of a

tubular structure running longitudinally along with the body of the

embryo which is a little dilated at the head region and slightly tapering

at the tail-end.

The heart could be identified within the pericardial cavity in the

form of a pulsating tube, 21 hrs after ferti1 7 o. Anterior to the yolk

sac the pulsating heart beats about 150 times/ min (Plate 33 & 34). In 23

hrs old embryos, the whole space inside the egg was fully occupied by

the embryo (Plate 35), blood circulation also begins. The caudal region

73

begins to detach from the yolk mass and the round tip of the tail

becomes free. The eyes become heavily pigmented and the mouth

structure develops 24 hrs after fertilization (Plate 36).

In Heteropneustes fossilis the eye lens and heart appear at about 13

hours after fertilization (Thakur et al., 1974). It was observed that the

outline of the embryo becomes defined within 12 hours after

fertilization, as reported earlier in case of Channa niarulius (Khan, 1926;

Mookerjee, 1945 a).

Banerji (1974) reported that in C. punctatus, the heart beat begins

20 hours after fertilization and blood circulation also commences at this

stage. In Anabas testudineus (Munchi and Hughes, 1991), the lashing

movements of the tail help to rupture the egg membrane covering the

head portion. In H. fossilis, the average hatching time was 16 -18 hours

at 26°C (Kohli and Vidyarthi, 1990). The rhythm of the heart was

measured at 100 beats per minute in Mystus macropterus (Wang et al.,

1992) and in C. punctatus 160 times per minute (Munshi and Hughes,

11).

74

30 3 3. Larval Development

30 3 3 O Hatching

The measurements of newly hatched larva is given in Table 3.1;

Fig. 32. The newly hatched larva of 0. nialabaricus showed a

transparent laterally compressed body and was characterised by the

presence of almost round yolk-sac which was about 38% of the total

length of larva and chromatophores were completely absent. Mouth,

alimentary canal and gills however had not differentiated. The streak

of notochord was quite prominent and 19 - 25 somites were distinct

and another 11 - 13 were apparent at the tail region. Tip of the tail was

round and distinct. Newly hatched larvae were not active and

generally remained on their sides at the bottom of the container

(Plate 37),

3. 3. 3. 1. Three hours old larva

The 3 hours old larva was brown in colour about 215 mm in

length. The mouth was not formed and the anal invagination

appeared on the ventral side. The optic vesicles were still

unpigment'cL The embryo displayed a continuous dorso-ventral

unpaired fin. The heart became two - chambered, blood circulation

was seen around the notochord in addition to the brain and yolk.

75

Fig. 3. 2. Larval development of 0. nialabaricus

Li - Just hatched larvae-lateral view.L2 - Pre larvae, 5-9 hrs after hatching.L3 - Pre larvae, 12-20 hrs after hatching.L4 - Pre larvae, 29-36 firs after hatching,L5 - Pre larvae, 3942 hrs after hatching.L6 - Pre larvae, 43-48 hrs after hatching.L6(a) - Pre larvae, 48 hrs after hatching-dorsal view.L6(b) - Pre larvae, 48 hrs after hatching-ventral view.L7 - Post larvae, 3 days after hatching.L8 - Post larvae, 4 days after hatching.L9 - Post larvae, 6 days after hatching.L10 - Post larvae, 8 days after hatching.LII - Post larvae, 10 days after hatching.L12 - Juvenile, 15 days alter hatching.L13 - Fingerling, 30 days after hatching.

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Fig. 3.2. Larval development of 0. ,naluharicus

2. Six hours - old larva

The 6 hours old larva was about 3.15 mm and barbels weve

absent. Buccal invagination was apparent. There was no si 8 n of

development of opercies. The yolk - sac was round but as the larva

grew the yolk - sac became oval and gradually got elongated with the

growing body of the larva.

3. 3. 3. 3. Twel hour old larva

The 12 hours old larva measured about 3.40 mm in length; dark

brown chromatophores were present on eyes. Auditory capsules

moved closer to the eye. Length of yolk - sac was reduced and mouth

cleft was clear.

Larva at this stage was still rested on its lateral side showing no

sign of any obvious movement. When disturbed it just moved around

for a while and again went to rest. Larva showed sticking tendency

and avoided the lighted zones of the rearing container by congregating

at the corners or at a place where they can conceal themselves. To

confirm whether they really prefer to remain concealed, same stone

chips were placed in the plastic droughs and it was found that most of

the larvae took shelter at the bases of these stone chips. This shows

76

that hiding tendency in 0. inalabaricus developed in its early larval

stage itself.

13. 3. 4. 24 hours - old larva

The 24 hours old larva measured about 3.70 mm in length. The

embryo hatched by rupturing the egg capsule near the head region, at

hatching and 38 somites could be counted.

The first pair of barbels were 0.5 mm long; the otic capsule was

enlarged. Upper and lower jaws were formed; lower jaw showed

movements occasionally. Alimentary canal was in the form of a

straight tube, running from the yolk-sac to the vent. The anal opening

was still closed and situated below the 10th myomere. The operculum

was formed covering the branchial plate. Notochord was deflected

upward at the tail end and fins had not differentiated. The eyes were

darkly pigmented. Movement of the larva was restricted only to the

bottom, but most of the time it rested on its lateral sides and confined

to the darker zones of the rearing container.

3. 3. 3. 5 36 hours - old larva

The 36 hours old larva was about 4.00 mm in length and post

anal length was 1.8 mm. The pectoral fin was a round membranous

rap and was being actively used for free movement. Mouth was

77

e - ,- - .- - - - - --S F - .- - - •5_ 5; _ F - I - - - -___5 -_-.•_-.- - --.5--- - -5-, - __5_ 5-- _#_5___ .-_-. - S

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formed as a terminal opening; the lower jaw was less developed

vent Wa, .c:r (Plate 38).

3. 3. 3. 6. 2 days old larva

The two days old larva was about 4.4 mm in length and post

anal length was 1.8 mm. The mouth was formed but remained closed.

The pro larva displayed a continous dorso-ventral unpaired fin. The

eye was oval in shape with the choroids fissure. Circulation of the

blood could be observed in the gills,-heart and tail. The second pair of

barbles appeared. The two pairs of barbels were 0.4 and 0.7 mm in

length respectively. Upper and lower jaws were completely formed.

Lower jaw showed the presence of conical teeth. Operculum and gills

were formed and became functional. Heart beat rate was 120

times/minute at a temperature of 29.2°C; movement of the larvae was

still restricted to bottom only. Eye, were 0.22 mm in diameter and

darkly pigmented. Altogether 37-46 somites could be counted. Yolk-

sac was differentiated having a dirty brown colouration (Plate 39).

3. 3. 3. 7, 3 days old larva

The three days old larva was about 4.50 mm in length and post

anal length was 2.3 mm. Barbels became longer. Arterial and venous

flow of blood through the vessels in barbels was clearly observed

78

under the microscope. Barbels were smooth on one side while on the

other there was a row of elevated points, which were actually the

precursors of nerve ends. The mouth was perforated, with a opening

ventral to the head. Larva still restricted its movement to the bottom

layer only. Further shrinkage in the yolk-sac and the resultant space,

got gradually filled up by the developing stomach and the intestine

(Plate 40).

3.3.4. Post - larval development

3. 3. 4. 0. Four days old larva

The four days old larva was about 4.70 mm in length with a post

anal length of 2.4 mm. The head was pigmented. On the body,

melanophores appeared on the dorsal surface of the yolk-sac. The

mouth was formed and the teeth on the jaw, bones were translucent.

Fin fold remained continuous and the differentiation of dorsal and

caudal fins became apparent. Pectoral fin-fold was more conspicuous.

Yolk exhausted by the end of the 4 th day of development and larva

begin feeding on the food available in ambient water even before the

completion of the yolk absorption (Plate 41).

79

3. 3. 4. 1. Six days old post - larva

The six days old post-larva was about 5.60 mm in length.

Pectoral fin was differentiated in the form of a flap just behind the

operculum and the larva began to show side wise movement. Streaks

denoting rudimentary rays started appearing in caudal fin. Yolk was

completely absorbed and the larva began to wander in search of food.

If the food was not supplied abundantly in the ambient water at this

stage, the larvae tend to congregate into minor groups and majority

were found at the bottom, while a few occupied different water levels

upto the surface. Post larvae in their early period fed best on minute

zooplanktonic organisms such as ciliates, brachionus, the nauplii of

copepods, small cladocera etc. The presence of cyclops in the ambient

water even in small number may cause serious damage to the growing

post-larvae. Rearing at this stage, there fore, should be done with

utmost care (Plate 42).

3. 3. 4. 2. Eight days old post - larva

The eight day old post - larva was about 6.40 mm in length. The

intestine was convoluted ventrally to the stomach. The larva swam

freely and fed on finely chopped chironomus larvae cooked egg, blood

meal and beef liver. The yolk - sac was reduced and contained the

residues of yolk materials (Plate 43).

80

3. 3. 4. 3. Ten days old post - larva

The ten days old post larva was about 7.10 mm in length. Dorsal

fin became free with six branched rays. Spine and rays were

developed in pectoral fin and pelvic fin had not differentiated. Anal

fin was still continuous with larval fin fold. Caudal fin, remained to be

separated from the larval fin - fold, with eight branched rays, all

placed, beneath the upward deflection of the notochord (Plate 44).

Larva at this stage was an active swimmer and moved into every

nook and corner of the container in search of food. It also undertook

vertical trips to the surface of the water by wriggling hard through the

column. While doing so it broke the surface of the water by its mouth

and then passively started sinking to the bottom keeping the body

vertical. Often before reaching the bottom it again wriggled up and

broke the surface to sink again passively. When observed closely, it

was found that the post-larva not only came upto the surface and

broke the surface of the water but also took in mouthful of air and then

sank. The phenomenon of aerial respiration began from 8 th day of

development.

81

3. 3. 4. 4. 12 days old post - larva

The twelve days old post larva was about 8.90 mm in length.

The colour bands were more distinct. The caudal fin rays were five in

number and hypurals were indicated as basal thickenings (Plate 45).

3. 3. 4. 5. 15 days old post - larva

The fifteen days old post-larva was about 11.20 mm in length.

The body surface with exception of the ventral position of the yolk was

pigmented. The yolk material was found exclusively in the ventral

portion of the yolk sac and appeared solely as a yellow spot.

Pigmentation appeared in the dorsal and caudal fins (Plate 46).

The dorsal fin was differentiated into 5 - 6 branched rays as in

adult. Pectoral spine became stout. Ventral fin showed six indistinct

branched rays and there were 63 - 69 anal fin rays. The anal and

caudal fins were still continuous with the embryonic fin - fold. The

embryonic fin - folds were yet to disappear. Vertebral segmentation of

tha notochord took place with distinct neural and haemal spines at the

caudal region. Pigmentation was more pronounced through out the

head and the body. The larvae were capable of ingesting more food.

The traisition from larval to juvenile period was gradually completed.

82

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/ kJ •)I .J .J.EJI-

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• I

?/ThTta.. At-

/ I• / I I• '

3. 3. 4. 6. 30 days - old fingerlings

The thirty days old fingerlings were about 19 - 21 mm in length

(Plate 47). All fins were partially pigmented. The pectoral spine

became serrated. The barbels became completely, pigmented and the

fin folds disappeared. The schooling behaviour of 0. nialabaricus broke

up, during this stage.

chaudhuri et al. (1978) and Khan (1972) have reported yellowish

- brown colour while during observation he was observed to be of pale

or greenish yellow colour commences in the colouration of eggs.

Similar observation have also been reported in the case of H. fossilis by

Thakur et al. (1974). The disappearance of yolk - sac was observed in

Clarius lazera on 4 th day (Panjonghua and Zhengwenbiao, 1987) and

Clarias batrachus (Asha landge, 1995) on 3rd day.

In Channa marulius, the tip of the notochord turns upwards on 9th

day and on the 13th day the caudal fin gets separated from the dorsal

and anal fins (Khan, 1926). In Mystus macropterus, flexion of notochord,

which starts 2 days after hatching completes development within four

days after hatching (Wang et al, 1992). Eggs of T. putitora are orange in

colour and range between 2.2 mm and 3.2 mm (Pandey et al., 1998).

The air - breathing habits develop within 12 - 13 days in A,ials

83

testudineus (Munshi and Hughes, 1991). Heteropenustes fossilis (Kohli

and Vidyarthi, 1990) and Clarias spp (Dehadrai, 1972) start air

breathing around 5 11, to 8th day after hatching.

In 0. hirnaculatus, after three days hatching the mouth is

completely formed and the larvae begin to ingest exogenous feed

consisting of cooked egg yolk from 4-day post hatching besides

utilizing their endogenous yolk (Sridhar et al., 1998). Nirmal Thakur,

(1980) reported that the number of pre-anal myomeres is 17 in

C. batrachus and ii in the case of H. fossilis.

In 15 days old larva of Anabas testudineus the scales are

completely formed over the body (Munshi and Hughes, 1991). Thakur

(1980) and Asha Landge (1995) reported aerial respiration in C/arias

bat radius 10 or 11 days after hatching.

3. 4. 0. Rearing of post larvae

With regard to larval rearing of catfishes, both extensive and

intensive procedures have been adopted successfully (Janssen, 1987,

Polling et al., 1988, Hansen et al., 1990). Fish larvae consume

zooplankton and sometimes, phytoplankton and many species switch

to larger prey and leave planktivory as they grow, whereas others feed

84

during their entire lives on plankton. Some are obligate for

planktivorous feeding exclusively on plankton whereas others are

facultative planktivorous feeding on plankton as well as other food

materials.

The early larvae subsist exclusively on zooplankton

(Parameswaran, 1975). Adeyemo et al. (1994), Galbusera (1997),

Legendre et al. (1992) and Ragnon et al. (1998) reported higher

mortality for those fed with the commercial diet than the live food diet

in African catfish Clarias gariepinus larvae. At the onset of exogenous

feeding, the 0. malabaricus larval digestive system after sufficient

development was adapted to ensure efficient utilization of live food,

but not dry food. Liao et al. (1971) mentioned, the quality, size, density

and mobility of the food as the important factors for developing larval

rearing techniques. Kenduchen and Legendre (1994) observed that

three-days of old Heterobranchus ion gifilis ingested prey larger than 500

gm such as Arternia nauplii and Moina. Munshi and Hughes (1991)

reported that Heteropneusl-us fossilis larvae feed mainly on

microcustaceans and Anabas testudineus and Notopterus notopterus are

zooplankton feeders.

85

After hatching stage corresponds to the moment at which larvae

can be switched from feed to formulated diet without significantly

effecting growth and/or survival (Kestemont, 1995). However in

Clarias gariepinus, zooplankton alone cannot fully support the growth

and survival of fry, hence supplementary feeds are necessary for

growth and survival (Okoye et al., 1991).

Hartman (1983) indicated that larvae of two cyprinids, in

comparison with other fish species, have a rapidly increasing threshold

for the size of planktonic organisms eaten i.e., from 100 to 300 gm

during early growth. In the present investigation better highest

growth rate was achieved in 0. rnalabaricus larvae fed on mosquito

larvae followed by those fed on chironomous larvae, plankton soup

and earth warm (Table 3.2; Fig. 3.3 & 3.4).

3.5. 0. Rearing of fry

During the experimental period fry fed on chicken intestine

showed the highest growth rate and survival rate (Table 3.3; Fig. 3.5).

The fry ranged from, 1.8 ± 0.01 ml in length and 0.320 gm in weight.

During this stage the dorsal and anal fins were almost separated from

the caudal fin and ventral fin buds were almost formed. Rudimentary

rays were formed in the fin region. When compared to those fed with

86

beef liver and plankton. The poor growth and survival could be due to

the inadequacy of diet for the digestive specificity of the animal during

larval stage (Cahu and Zambonino, 1995). Probably because of the

change in the pattern of the digestive system, the feeding live food

organisms in 0. nialaharicus was mostly practised for a few days only

and were soon replaced by fresh diets. Varghese et al. (1973) reported

that the zooplankton may not be utilized by Clarias batrachus fry. Test

individuals fed with chicken intestine yielded higher growth (6.4263)

and survival (72%) followed by beef liver (5.3647) and (66%) from 30

days of stocking, the fry reached the fingerlings stage (length 5.0 ± 1.5 -

5.5 ± 1.5 cm; weight 1.4 ± 0.1 - 1.6 ± 0.5 g). Then the fingerlings were

transferred from the rearing tank to stocking ponds (Plate 48).

Table 3.1. Ontogenetic events in the early development of0. malabaricus

Age (hour) Ontogenic eventsCleavage5 min after fertilization Formation of previtalline space

35min Formation of blastodisc

40min 2 blastomeres

1 h - 0.5 mm

4 blastomeres

I h-22min 8 blastomeres

I h-42min 16 blastomeres

I h-50 min 32 blastorneres

2h-21 mm

64 blastomeres

3 h - 25 mm

Morula stage.

87

Formation of embryo

4 h - 18 min Blastula stage

8 h - 20 min Post gastrula stage; germ ring movestowards the vegetal pole, swallows upcompletely

9 hr. 45 min Formation of embryo; the head, Brainvesicles and tail ends of the embryocan be identified.

12.00 hrs 3 - 4 somites can be counted; three-brain vesicle are formed; the opticcups are also formed.

13.00 hrs 6 - 7 somites and two small oticvesicles are formed; the notochord, isin the process of formation; Thefuture heart appears.

17 hrs -30 min 19 sornites are formed; a tubularnotochord with a slight dilationtowards the head runs along the body;the tail is free of the yolk mass; the eyelens is being formed; Kupfer's vesicleand fin folds are formed,

21 hrs 25 - 28 somites are formed; the eyelens and brain formation is completed;melanophore pigments are visibleover the nerve cord; trunk and caudalregions. Anterior to yolk - sac thepulsating heart beat about 150time/mm.

22 hrs

29 - 32 somites are formed,

23hrs 34 - 36 somites are formed; theembryo makes twitching movementsand lashes its tail against the eggcapsule,

88

24 hrs The embryo hatches by rupturing theegg capsule near the head region;while hatching 38 somites can hecounted; larva just hatched.

3.5 mm long; the eyes are darklypigmented; the head of the larvae liesabove the yolk - sac; the olfactory pithad formed at the side of the olfactoryplacode; the brain and ventricles areclearly visible; the heart beats above120 times/minute.

Larval Development

Pre larval stageLarva just hatched5.00 - 9.00 hrs

12.00 - 20.00 hrs 4.00 mm; the brain differentiation iscompleted; the first pair of barbies are05 mm long, the otic capsule isenlarged; the heart lies in the distinctpericardial cavity.

29.00 - 36.00 hrs The operculum is formed and coversthe branchial plate; later two pairs ofgill arches take shape.

39.00 - 42.00 hrs The pectoral fin fold behind the gillbecomes apparent; the digestive canal,which was straight, begins todifferentiate.

43.00 - 48.00 hrs 4.40 mm long; the mouth is formedbut still not open; the Pro larvadisplays a continous dorso - ventralunpaired fin; the second pair ofbarbels appear; 37 - 46 myomeres canbe counted; the eye is oval in shapewith the choroids fissure; thecirculation of the blood could beobserved in the gills, heart and tail;the two pairs of barbles are 0.4 and 0.7mm in length respectively

89

Post larval development

3rd day 4.50 mm long; the mouth is perforatedwith an opening ventral to the head;the alimentary canal is nowdifferentiated into foregut, midgutand hindgut; the anus is alsoperforated; even though the larva stillhas a heavy yolk sac, they are capableof limited suspended swimming.

4th day 4.70 mm long; melanophores appearon the dorsal surface of the yolk sac;the head is pigmented; the mouth iscompletely formed and the teeth onthe jaw bones are translucent

6th day 5.60 mm long; the operculumcompletely covers the gills.

81h day 6.40 mm long; the larva displays acontinuous dorsal - ventral unpairedmel4nophorous fin.

101h day 7.10 mm long; the dorsal fin buddevelops and the anal fin is separatedfrom the caudal fin by a small notchnear the tail; the larva is light yellowin colour; the yolk sac is reduced andcontains the residues of yolkmaterials.

15 -30 day 11.20 - 21.00 mm long; the continuousdorsal - ventral fin divides into dorsal,caudal and anal fins; the yolkmaterials are found exclusively in theventral portion of the yolk sac andappear solely as a yellow spot;pigmentation appears in the dorsaland caudal fins.

90

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Table 3.3. Survival and growth of 0. nialabaricus fry fed different typesof food.

Initial mean length (mm)

Initial mean weight (gm)

Experiment duration (days)

No. of fry stock

Final length (cm)

Final weight (gm)

Specific growth rate (SGR %)*

Weight gain (%)

Survival (%)***

Plankton

1.8 ± 0.01

0.320

30

50

5.0 ± 1.5

1.4 ± 0.1

4.9196

461.22

64

ChickenIntestine

1.8 ± 0.01

0.320

30

50

5.3 ± 1.01

2.2±1.1

6.4263

169.19

72

Beef liver

1.8 ± 0.01

0.320

30

50

5.5 ± 1.5

1.6 ± 0.5

5.3647

141.24

66

Log final body weight - log initial body weight*Specific growth rate (0/o/d5 = -------------------------------------------------------------- x 100

Experimental days

Final weight - Initial weight**Weight gain (%) = --------------------------------------- x 100

Initial weight

Initial number of fish***Survival rate (%) = ------------------------------- x 100

Final number of fishs

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