Chapter VII

CAPTIVE BREEDING AND SEED PRODUCTION PROTOCOLS OF HORABAGRUS BRACHYSOMA(GUNTHER)

7.1. Introduction

Stocking of natural waters using hatchery bred seeds is an important and in

some cases very successful tool for species conservation. Historically, Horabagrus

brachysoma was locally abundant in the riverine areas in Kerala, but populations

have declined drastically and the species is now restricted to sparse populations in

tributaries of Chalakkudy, Meenachil, Manimala and Pampa. H. brachysoma, the

endemic rarity of the Western Ghats is also an excellent table fish with high market

demand and consumer preference. Their omnivorous feeding habit, high fecundity

and attributed nutraceutical properties also make it a potential species for

commercial aquaculture. The species is however listed as endangered by the IUCN

and as a species of special concern (CAMP, 1998; Shaji et al., 2000;

Gopalakrishnan and Ponniah, 2000). In the context that artificial breeding of

threatened species, for restocking in their natural habitats, is the most widespread

measure adopted for species recovery. Investigations were undertaken for

artificial breeding of this endemic species under farm conditions. The technology

will be of immense use for artificial propagation of this species.

In many countries, catfishes are farmed on commercial scale. The problems

that confront rapid development of aquaculture of such endemic species, despite

their high demand, is the lack of technologies for mass production of seeds.

Although, this fish is known to attain maturity in pond conditions, they do not

spawn naturally in confinement. In this context, taking cue from life history

parameters described in chapter VI, investigations were undertaken to develop and

standardize techniques for captive breeding and artificial propagation of H.

brachysoma.

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7.2. Results

7.2.1 Collection and maintenance of brooders

Long term fishery survey indicated that H. brachysoma, in nature spawns

during monsoons. Hence, fishes were collected from the riverine locations of

Vembanad lake, and maintained in the farm pond as brood stock during the

premonsoon months when the mature spawners were available. A total of 535 fishes

were collected and conditioned to maturity in the pond situations. These pond

reared fishes were utilized for artificial maturation and breeding studies. Since the

size of the broodstock population was small, an effective breeding number (Ne) of

over 500 (FAO/UNDP, 1981) fishes were utilized to avoid problems of inbreeding

and genetic drift. As the fish was found to prefer omnivorous diet comprising

filamentous algae and fish offal, broodstocks were raised in manured ponds with

abundant plankton, supplemented with commercial feed comprising rice bran, fish

meal, ground nut oil cake and commercial feed pellets.

Although under culture systems vitellogenesis in brood fishes was rapidly

induced, similar to that of natural stocks, they never attained final maturation or

ovulation in the pond. The female fish was found to mature even at a small size

13.5cm and male at 12cm in the culture tanks. Spermiation among males were also

rapid and the fishes attained milting stage by late March.

The brood fish ponds were therefore subjected to frequent water exchange

and replenishment, to facilitate re-circulation and aeration, so as to stimulate

gonadal maturation. With frequent water exchange and use of nutritionally

balanced diets, these fishes attained good breeding condition even earlier before

the normal monsoon months. Such fishes were seined from the holding ponds

during the reproductive season and assessed for readiness for induced breeding.

Healthy ones were only selected to ensure the production of healthy progenies.

Brood fishes collected from nature and transported carefully to the laboratory in

fish carriers were also utilized for captive breeding trials.

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7.2.2. Maturation Assessment and Selection of Brooders

In H. brachysoma, the male and female can be identified based on their

external secondary sexual characters (Plate 25).

However, sexual dimorphism was most apparent among the ripe fishes close

to the breeding season. Ripe females have

distended and swollen abdomen and have

bright red genital papillae, eggs extrude

under slight pressure. Males, however have

a streamlined body, look more brighter and

are generally smaller in size. In variance to

most other catfishes, males of H.

brachysoma was found exude milt freely at

slight pressure, close to breeding periods.

7.2.3. Induced Breeding by Hormonal Manipulation

Induced breeding experiments were taken up during May-September

coinciding with the natural spawning of the species in nature. From the life history

parameters, it was evident that the fish attain the highest mean GSI of 6.6 for male

and 16.3 for female during June-July months. The water column in the spawning

tank was maintained between 13-32cm in various trials. Water depth ranging from

21 and 31cm was also found suitable for natural spawning.

Plate 26.Breeding set of H. brachysoma Plate 27.Hormone injection

A total of 368 fishes comprising 222 male and 146 female were utilized for

the induced breeding studies. Out of the 104 trials undertaken, induced ovulation

Plate 25.Sexual dimorphism in H. brachysoma

♀ ♂

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was successfully realised in 70.2 percent trials and hatching was also successful in

50.7 percent of observations. The pond reared broodstock was found to respond

well to artificial spawning as compared to virgin wild spawners collected and

utilized during the same season Results of the breeding experiments are shown in

Table 7.1.

Table 7.1. Captive breeding trials in Horabagrus brachysoma

Table 7.1a Fertilisation Hatching Month

No. of

Trials

Fertili-sation

Hatc-hing >90% 70-

90% <70% >90% 70-90% <70%

March 1 100.0 0.0 0.0 0.0 100.0 0.0 0.0 0.0 May 13 76.9 20.0 60.0 20.0 20.0 50.0 0.0 50.0 June 40 80.0 68.8 78.1 18.8 3.1 31.8 22.7 45.5 July 24 62.5 46.7 73.3 6.7 20.0 14.3 14.3 71.4 August 15 53.3 37.5 75.0 0.0 25.0 33.3 0.0 66.7 September 8 75.0 50.0 66.7 16.7 16.7 0.0 0.0 100.0 October 1 100.0 0.0 0.0 100.0 0.0 0.0 0.0 0.0 December 2 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 Total 104 70.2 50.7 71.2 15.1 13.7 27.0 16.2 56.8 Table 7.1b

Fertilisation Hatching Inducing agents

No. of Trials

Fertili-sation

Hatc-hing >90% 70-

90% <70% >90% 70-90% <70%

Ovaprim 78 74.4 42.3 74.1 17.2 8.6 30.3 18.2 51.5 CPE 19 78.9 21.1 60.0 6.7 33.3 0.0 0.0 0.0

Experimental fishes weighing 25g to 430g were subjected to induced

ovulation by hormonal manipulation. They were injected with Carp Pituitary

Extract(CPE) @50-60mg kg-1 body weight or synthetic hormones

(Ovaprim/Ovatide) @1ml per kg body weight for artificial maturation. Final

maturation was induced by administration of hormones during late evening hrs, 4.00

and 5.00 pm, so that the fish spawned during the night hours. Breeding set

comprising male and female in both 1:1 by weight or 2:1 by number was found to

function as effective breeding partners (Plate 26). As the fish has the habit of

hiding in dark corners, PVC pipes of 30-40cm were placed inside the breeding tank

as hideouts and the shelter substrates provided were utilised effectively by the

spawners. The results of the breeding trials are summarised in Table 7.3. Oocyte

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maturation and ovulation were induced with administration of hormones in single

dose, and the males received half the dose, at the same time, as that for females.

Since H. brachysoma is very hardy, the survival rate of brooders, even subjected to

stripping was very high (98 percent). Spawning was successful in fishes weighing

an average of 90-100g among males and 150g in females. The percentage success of

ovulation, with different inducing agents is given in Table 7.2.

Table 7.2. Response of Horabagrus brachysoma to Ovaprim and Carp pituitary

Extract (CPE)

Fertilisation success (%)

Hatching success (%)

Not Responded (%)

Type of Breeding (%)

Month

Trial No.

Ovaprim CPE Ovaprim CPE Ovaprim CPE Natural StrippedMarch 1 0.0 100.0 0.0 0.0 0.0 0.0 0.0 100.0 May 13 76.9 0.0 20.0 0.0 23.1 0.0 30.8 46.2 June 39 82.1 0.0 68.8 0.0 10.3 7.7 71.8 10.3 July 22 45.5 9.1 60.0 50.0 31.8 0.0 54.5 13.6 August 11 18.2 54.5 50.0 33.3 27.3 0.0 9.1 63.6 September 8 37.5 75.0 66.7 16.7 25.0 0.0 25.0 50.0 October 1 100.0 0.0 0.0 0.0 0.0 0.0 0.0 100.0 December 2 0.0 0.0 0.0 0.0 0.0 50.0 0.0 0.0 Total 97 59.8 15.5 56.9 26.7 20.6 4.1 48.5 26.8

Table 7.3. Breeding trials in Horabagrus brachysoma

Inducing Agents Ovaprim CPE Ovatide Total number of trials 78 19 7 Fertilisation success 58 15 0 Hatching success 33 4 0 Fishes not responded 20 4 7 Fertilisation % 74.36 78.95 -- Hatching % 56.90 26.67 --

Out of the total 146 female fish subjected to hormonal manipulation, 37.7

percent ovulated spontaneously and 5.5 percent spawned partially. Fishes that

underwent final maturation but failed to breed naturally (32.2 percent) were

subjected to stripping, on expiry of latency period. Over 24 percent of fishes failed

to respond to hormonal administration. Out of 109 females subjected to Ovaprim

administration, 82.6 percent ovulated and hatching was also successful in 33

percent of the trials. Out of 26 females subjected to final oocyte maturation using

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crude CPE @50-60 mg kg-1, only 42.3 percent ovulated naturally and 50 percent

had to be subjected to stripping. The inducing agent, ovatide was used in 7 trials

@1 ml kg-1 body weight, but none of the Ovatide administered fishes completed

final maturation and spawning (Table 7.4). Egg viability assessed in terms of

hatching rate at ambient conditions was perceptibly higher when administered with

Ovaprim than with CPE. When administered with Ovaprim, hatching success was

56.9 percent, whereas when CPE was utilized, the percentage success was observed

to be only 26.67 percent. Evaluation of the quality of eggs indicated that egg

quality was comparatively high with Ovaprim (74 percent) followed by CPE(60

percent). The mean size of the ovulated eggs ranged from 1.4 to 1.6mm. Ovulation

time was assessed precisely in 58 cases, treated with Ovaprim and 15 cases with

CPE. There was enormous variability in the latency response among treated

females, even at similar temperature regime.

Table 7.4. Performance of females during captive breeding

Table 7.4.a

Month Total Number of females

Complete spawning

(%)

Partial spawning

(%)

Stripping (%)

Not responded

(%) March 3 0 0 100 0 May 18 27.8 5.6 44.4 22.2 June 55 61.8 5.5 14.5 18.2 July 26 46.2 7.7 19.2 26.9 August 25 8 0 52 40 September 13 15.4 15.4 46.2 23.1 October 4 0 0 100 0 December 2 0 0 0 100 Total 146 37.7 5.5 32.2 24.7

Table 7.4.b

Inducing agents used

Total Number of

females

Complete spawning

(%)

Partial spawning

(%)

Stripping (%)

Not responded

(%) Ovaprim 109 44 7.3 31.2 17.4

CPE 26 26.9 0 50 23.1

Ovatide 11 0 0 0 100

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Latency period was observed to range from 8 to 15hr in Ovaprim treatment

and 8 to 12 hr with CPE. Latency response however did not demonstrate any

significant effect on oocyte diameter or egg quality. Obviously, there was no

apparent variation in incubation time with reference to inducing agents. It ranged

from 22 to 29 hr with Ovaprim and 22.30 to 24 hr with CPE. Testicular maturation

in response to hormonal manipulation indicated that males spermiate freely with

hormonal injection. Hormonal stimulation was found to enhance sperm motility

significantly. Sperm motility in induced males was observed 35 to 40 seconds and

the average sperm count was 16.4 + 4.02 x 109 per ml. The quality of egg obtained

in natural spawning was perceptibly superior as compared to stripped ova.

7.2.3.1. Viability of ova

Viability of the eggs was assessed with reference to hatching rate and

hatchling survival. The eggs collected at ovulation were generally of good quality

as compared to stripped eggs produced from females after the expiry of latency

period (Table 7.5). Egg viability was found to deteriorate rapidly when stripping

time was considerably delayed than the normal ovulation time. The hatching rate

obtained in mediated stripping is given in Table 7.5. Nevertheless, hatching

percentage was not seriously affected for eggs stripped within one hour of the

ovulation time. Although, in few exceptional cases high hatching rates were

obtained, even when the stripping was conducted in two hours, assessment of

viability of ova retained in the ovarian cavity after ovulation time indicated that the

process of over ripening occurs rapidly and overall quality of ova begin to

deteriorate fast within 1-2 hr. Hatching rates of such eggs were also perceptibly

decreased. Apparently, the decline in egg quality due to over ripening had

significant bearing on egg viability than the fertilizing ability of the sperm. Such a

temporal change in quality of ova was perceptible also with reference to ovulating

agents, viz., Ovaprim and CPE. Notwithstanding above, the egg quality was found

to be better lasting in treatment with Ovaprim.

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7.2.3.2. Stripping/Artificial fertilisation

Hormonal manipulation followed by stripping (Plate 29) resulted in

successful hatching only in 19 percent of the trials and hatching rate was found to

vary widely (10-95 percent). Highest rate of hatching was obtained when stripping

was preceded by administration of Ovaprim. Dry stripping, within 1-2 hrs of the

latency period was observed efficous when natural spawning was not successful.

Unlike carps, H. brachysoma was found to be hardy and the stripped fishes was

found to regain most rapidly. The same males have been observed to recoup

milting condition during the same season. There was no perceptible variation in

egg yield in natural spawning and stripping. Eggs expelled in natural spawning

varied from 20229 to 145000 with a mean of 89756 while in individual stripping

the fecundity fluctuated between 21388 to 140952, mean being 88697. The

relative weight of the stripped eggs ranged from 13.3 percent to 22.7 percent of the

fish biomass. Highest G.S.I. of female H. brachysoma at prime spawning state was

29.7. Egg released through stripping almost approximated this potential fecundity

value. It is therefore reasonable to suggest that administration of Ovaprim

followed by stripping brings about a near complete ovulation.

Table 7. 5. Spawning Fecundity in Horabagrus brachysoma

Natural Breeding (N = 12)

Stripped breeding (N = 12) Sl.

No. Range Mean + SD Range Mean + SD

1 Fish Body weight (Female) (g) 110 -500 248.33 +

114.88 150-320 209.17 + 52.99

2 Number of eggs/ fish 4248 -72500

22345.33 +18177.85

3422-36855

19071.42 + 10892.72

3 Eggs/kg body weight 20229 -145000

89755.92 + 49765.88

21388-140952

88697.25 + 39645.33

4 Latency period (h) 8-14 12.42 + 1.79 13-18.15 16.31 + 1.56 5 Fertilisation rate (%) 3.0 -98.4 74.97 + 33.93 75-100 95.23 + 7.33 6 Hatching rate (%) 0-98 35.15 + 37.00 0-79.2 7.43 + 22.78 7 Water Temperature (0C) 23-27 24.48 + 1.24 24-28.4 25.71 + 1.47 8 Water pH 6.5-8.5 7.05-0.65 6-8.5 7.26 + 0.97

7.2.4. Breeding behaviour

Catfishes have evolved a series of ecological and behavioural adaptations

and reproductive strategies that make them one of the most exciting group of fishes.

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Like vast majority of catfishes, H. brachysoma is active at night and spawning

occurs in high intensities during night hours. Spawning crescendo in H. brachysoma

is preceded by vibrant mating and courting behaviour (Plate 28). The pre-spawning,

spawning and post-spawning habits are characteristics. Prior to spawning, the

induced fish disappear from the spawning tank to dark corners. The pre-spawning

female rest quietly in the tank and could be taken hiding in dark corners and hide

outs. The males swim back and forth and occasionally remain motionless. During

the pre-mating time, the fish that take shelter at the bottom, occasionally come to the

surface and slowly returns back to the tank corners. Among the males, the primary

male slowly begin to move on either side of the female. Approximately in 3-4 hrs

after injection, the males appear to become more aggressive and go around

chasing the female, nosing and beating the abdomen, move forward and pound at

the mate. In the end, the male succeed in enticing the female to participate in

nuptial embrace. The pair occasionally moves into the hideouts, the PVC pipe

refuges, provided in the tank bottom. Close to mating, the fish become profoundly

excited, become restless and exhibit vigorous fast movements. The pair swim

together up close to the surface back and forth. The male follows the female

closely, swim underneath her and occasionally rub his snout on her vent and incite

her. He also butt her vent, swim behind her in fast movements, some times

encircling each other and move restlessly up and down. As the females become

excited, she in quick movements attract the attention of the nearby males. The

male in a tightening grip with spines locked assume an enfolding posture and

terminally enter in to active courting. In an obvious jerk, the excited females

quiver and the peristaltic body contractions result in aggressive spawning in 8-14

hrs. During spawning climax, the fish exhibit arching and waving of the tail and

discharge eggs in spurts. Simultaneously with the expulsion of eggs, the males

slightly quivers, curve around with rapid contraction of the abdomen and ejaculate

spontaneously releasing a puff of milky milt that make the water almost cloudy.

Female completes her egg expulsion in few minutes. The fish exhibit a violent

quivering during egg laying and sometimes exhibit a vibrating and trembling

behaviour. Spawning occurs at water temperatures 23 to 28.40C. During spawning,

the fish has been observed to make a creaking noise. This acoustic orchestration is

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apparently facilitated by the rapid contraction and relaxation of the swim bladder

and muscles on the lateral walls of the swim bladder. H. brachysoma is a

broadcast spawner and exhibit no parental care. A single female is often

characteristically serviced by more than one male and the eggs are broadcast over

the bottom

After release of eggs and sperm, the pair swim side by side and at times enter

into pseudo-spawning. As the pair completes spawning, the fatigued female

appears to loose interest and move away, while the male continue to entice the

female for repeat spawning. Male repeatedly try to continue persuasion of female

for mating and female often drive them off. Some males also exhibit false bouts

and pseudo mating in between actual spawning bouts. Towards the end of the

spawning activity, the females become completely exhausted and can be observed to

move with her collapsed belly. Deposited eggs are free and benthic. The mating

pair always keep away from the released eggs. Probably this help to protect the

eggs from damages. Captive breeding protocols of H. brachysoma by hormonal

manipulation is summarized in Table 7.6.

Table 7.6. Captive Breeding protocols of Horabagrus brachysoma

Ovaprim* CPE** Sl. No.

Range Mean + SD Range Mean + SD

1 Fish Body weight (Male)(g) 200-534 372.57+ 76.64 230-255 242.50 + 17.68

2 Fish body weight(Female) (g) 140 - 390 223.29+ 64.05 280-320 300.00 +28.28

3 Hormone dose (ml) 0.14- 0.39 0.22+ 0.06 0.27-0.33 0.30 +0.04 4 Latency period (h) 8.0-16.0 13.19+ 1.83 8.0-14.0 11.00 +4.24 5 Incubation time (h) 23.0-29.0 25.36+ 1.74 22.0-25.0 23.50 +2.12

6 Fertilisation rate( %) 80.0-100.0 97.05+ 5.17 50.0-98.0 74.00 +33.94

7 Hatching rate (%) 23.0-100 73.09+ 30.56 6.0-10.0 8.00 +2.83 8 Water Temperature (0C) 20.0-27.0 24.84+ 1.99 25.0 25.0 +0.00 9 Water pH 6.5-7.8 6.87+ 5.17 7.0-8.5 7.75 +1.06

* Ovaprim @1ml /kg **Carp Pituitary Extract @50-60mg/kg

7.2.5. Embryonic Development

The fertilized eggs of H. brachysoma is golden yellow in colour, heavily

yolked, glossy, translucent, and spherical with an average size of 1.6mm

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diameter. Unlike most other catfishes, the eggs of H. brachysoma are less

adhesive and are free and demersal. The eggs are easily collected and transferred

for incubation in hatching tanks with continuous oxygenation. Egg incubation and

hatching is better performed in shallow water conditions. Egg development,

monitored simultaneously and the embryonic developments were captured using a

Magnus Imaging System connected to a computer monitor (Plate 30). The

observation reveals that hatching of eggs are successfully accomplished in 22-29

hrs under a temperature regime of 24-27oC. Hatching rate above 95 percent was

achieved at water pH 6.5 to 7.

Ova diameter in H. brachysoma was found to be highest during

June(1.63mm). The embryonic stages from fertilized eggs to hatching are

summarised in Table 7.7. The fertilized egg is telolecethal and cleavage is

meroblastic. The blastoderm formed, is seen restricted to animal pole formed at the

point of entrance of the sperm at the level of the micropyle, leaving large yolk at

the vegetal pole. First cleavage is meridional and incomplete. The second division

is at perpendicular to the first and the third division results in the formation of 8

cells. The 4th cleavage results in the formation of sixteen celled stage at one and

half hour, and 32 celled stage at about two hours. A clear blastocoel begins to

appear at about 3.30 hours and the blastula at this stage appears as a cap of cells

over the yolk. By 4 hrs, the embryo starts to roll on in the cytoplasm. At about 6

hrs, the early gastrula stage is reached, and an embryonic shield appears.

Gradually, epibolic germ layers spreads to the equator of the spherical yolk surface

and at 7½ hrs, the germ ring invades 3/4th of the yolk surface and at 8 hrs, the

neural plate is formed and gradually almost 5/6th of the yolk surface become

invaded. As the blastopore gets closed, yolk plug projects and the head rudiment

is seen lifted up. By 9 hrs, the optic rudiment appears and gradually by 11hrs it

becomes differentiated in to a vesicle. At this stage, the head and tail get

differentiated and the myotomes also become clearly visible. At 12th hr the tail

bud appear and the embryo appear very much elongated and is seen encircled over

the yolk, reaching nearly three fourth of its circumference. At 14 hrs, caudal fin

fold rudiment draws out from the yolk and the head region become more and more

differentiated. Yolk sac stretches and assumes a characteristic beaked appearance.

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Tail buds projects out, from the beak like yolk mass distally, at around 15.30hrs.

Paired somites also become distinct at this point of time. At 18-19 hrs, optic

vesicle become conspicuous and the head region gets separated; the caudal fin

fold become very much elongated and the embryo appear ‘C’ shaped encircling

the yolk. At this period, the muscular somites are seen twitching at intervals. At

20 hrs, the heart and otic capsule become conspicuous and embryonic

movement become rapid. At 21 hrs, tail become free and the embryo encircles

almost 90 percent of the yolk mass. Gradually, the heart pulsation becomes more

rhythmic. The embryo begin to roll within the egg case. Egg membrane also

appears soft and wrinkled. As the development advances, the embryo appear more

and more elongated and the tail appears to overlap the head. The myotomes and

the auditory vesicle become prominent and the embryo at this stage begin to twitch

and roll within the cytoplasm. The twitching becomes fast and rapid (30-32 per

min) and gradually the pulsation of heart become regular at 100-102 beats per

minute.

Table 7.7. Embryonic Development in Horabagrus brachysoma

Time after fertilisation

Egg Developmental stages

1.00 Blastodisc appears 1.30 16 celled stage 2.00 32 celled stage 3.00 Early morula 4.00 Blastulation; blastodisc formation 6.00 Gastrulation; Early gastrula 7.30 Late gastrula 8.00 Neurula stage 8.30 Closure of blastopore. Yolk plug projects, head rudiment lift. 9.00 Optic rudiment appears

11.00 Formation of head and tail ; myotomes visible 14.00 Tail region detach from the yolk 18.00 Movement of the embryo 20.00 Development of eyes, heart and auditory vesicles advances 21.00 Heart beat starts 24.00 Hatching

Close to hatching, the twitching and lashing of the embryo inside the egg

capsule becomes rapid. The egg shell breaks up and the tail emerges out first,

followed by the head. Hatching occurs at around 24 hrs. Egg hatching is

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protracted and the incubation time was fluctuated between 22-29 hours post

fertilisation, at temperature 24-270C. Hatching rate above 95 percent was achieved

at water pH 6.5 to 7. Hatching time was invariably linked to water temperature and

at warmer temperatures (250C), hatching time was considerably reduced (22hrs).

7.2.6. Larval and Post larval Development

7.2.6.1. Newly hatched larva

Immediately after hatching, the heavily yolked hatchling appear rolling on

with their attached spherical yolk sac on the ventral side. At this stage the

hatchlings appear slender and transparent except for the opaque and creamy yolk

mass and are seen congregating in the tank bottom. The hatchlings gradually

straightens with flashing tail movements. Anal fin fold become extending from the

postero-dorsal end to the ventral region. The mean length of the hatchling at this

stage is 4.0 mm.

7. 2.6.2. Hatchling – 2 day old

In about 24 hrs, after hatching, the larva attains a mean length of 5mm.

Mouth cleft become clearly distinguishable; upper and lower jaws are also

prominent. Two pairs of barbels also become visible. The intestine is visible through

the transparent body. Eyes appear pigmented and the auditory vesicles are more

distinct. The pulsating, two chambered heart and the streaming larval blood

circulation to the caudal fin are perceptible ventrally at this stage.

7. 2.6.3. Hatchling – 3 day old

At 48 hours, the larva appear slender and elongated. The yolk is very much

reduced, though not completely exhausted. The hatchlings become segregated and

they gradually begins to swim up towards the water surface. Melanophores

become conspicuous on the body. Pelvic fin become visible and the stomach is

visible through the transparent body, as a pouch like sac. The fin fold appear

continuous extending dorsally from behind the head, up to the postero-ventral

margin of the yolk.

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7. 2.6.4. Hatchling – 4 day old

By day 4, the yolk is completely used up and the hatchling attains a reliable

landmark of larval period and begin to depend on exogenous feeding. Head

appears broad and well differentiated. The characteristic black blotches appear on

the shoulder. Barbels become elongated, the maxillary pair is perceptibly longer

than the others. Pectoral fin rays become well differentiated with their

characteristic spines. Opercular fold is strong and gills become more distinct at

this point of time. Distribution of melanophores is conspicuous and the body

appear highly pigmented. The larva which is 6mm at this stage, rapidly begins

to grow and attain a size of 9 mm by the end of the first week.

7. 2.6.5. Nursing of hatchlings

After the embryogenic phase, H. brachysoma begins to subsist on

exogenous feeding, from 4th day onwards. During this stage, the digestive and

urinogenital tracts are visible externally and the fry grow to an average size of 1cm

in a week (Fig.7.1) The spawn at this stage can be transferred for rearing in large

open nursery ‘hapas’ (5m x4m x1m) fixed in nursery ponds. During this period, the

larvae is fed on powdered yolk of boiled eggs, spread on the water surface by

squeezing it along with water. The larvae is gradually weaned to powdered

compounded feed concentrate, in addition to the live plankton. Fry nursing of the

seeds were carried out in floating hapa system in fertilized ponds and in cemented

nurseries. In the nursery, the hatchlings attain a size of 3.5cm in 30-45 days and the

survival varies from 2 to 65 percent (Average 41 percent). The seeds attain a size of

6.5 cm/3.08g and 5.8cm/ 2.17g within three months in hapa rearing and tank

nurseries respectively (Fig.7.2 and 7.3). The rate of growth was appreciably high in

hapa nurseries maintained in fertilized ponds, or manured nurseries, and fed on

wet feed comprising groundnut oil cake and rice bran mixture in combination with

commercial feed pellets @5 percent of biomass.

Based on the multiple regression method, the inter relationships of various

breeding parameters viz., fish weight, latency period, incubation period, water

temperature and water pH with fertilization percentage and hatching percentage

were analysed. Among the different parameters studied, incubation period and

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water temperature were observed to indicate significant correlation with fertilization

percentage, the relation between incubation period and fertilization rate was

negative (coefficient 1.869619 and R square 0.820294). The relation between water

temperature and fertilization rate was positive (coefficient 2.208603 and R square

value 0.820294). As regards relationship of hatching rate with other parameters, the

significance could not be proved statistically.

7.3. Discussion.

Although, Horabagrus brachysoma is a valued candidate species as food

fish and ornamental fish, their artificial propagation is largely impeded by lack of

technologies for captive breeding. The species do not breed spontaneously in

captivity. Their utilization in ornamental fish trade is largely dependent on

capture from the wild, where the fish has become largely endangered. The technique

of induced breeding developed and standardized assumes significance in this

context. Despite its high economic importance, published data on its biology and

culture are scarce.

7.3.1. Induced maturation by hormonal manipulation

In the present study, it is demonstrated that H. brachysoma can be brought

to final maturation and ovulation by the administration of a single dose of Ovaprim

@1ml kg-1 body weight or fish pituitary extract @50-60 mg kg-1 body weight, the

former being more effective. Although the technique of induced breeding,

involving the process of final gonadal maturation, ovulation/spermiation and

spawning, is being adopted increasingly since it was first developed (Chaudhuri and

Alikunhi,1957), the use of second generation techniques involving effective

substitutes such as mammalian hormones, HCG, steroids and LHRH and its

analogues have come to stay. The valuable report of Breton et al. (1972) on an in

vitro study on the possibility of using LHRH for stimulation of gonadotropin

release from fish pituitary, led to a number of valuable investigations on these

lines. The observations that LHRH-a combined with pimozide or domperidone

induced high rate of spawning in Indian Major Carps (Nandeesha et al., 1990),

stimulated its extensive use in induced breeding of a variety of fish species. Trials

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conducted with Ovaprim the synthetic formulation has indicated that

H. brachysoma can also be induced to maturity and spawning similar to carps with

a single dose of Ovaprim.

Although induced breeding using pituitary extract has contributed to the

rapid development of carp culture in India and several drugs, hormones and

homoeopathic preparations have been tried (Tripathi and Khan, 1990) to replace

pituitary gland because of the cumbersome process of its preparation, storage and

potency. The use of synthetic hormone Ovaprim has been demonstrated to be an

effective substitute as evidenced by better spawning success in case of Indian

major carp, Catla (Dhawan and Kaur, 2004) comparative efficiency of different

synthetic hormones have also established the effectiveness of Ovaprim. In the

present study also, fishes treated with Ovaprim performed well with high

fertilization(74 percent) and hatching rate(57 percent). Several authors have

indicated the superiority of Ovaprim in induced breeding of a variety of teleost

species. Among a series of inducting agents such as fish pituitary extract, HCG,

Ovaprim and Ovatide, tried for induce ovulation, Ovaprim injected individuals

showed a better performance with regard to fertilization and survival in case of

catfish, O. malabaricus (Haniffa et al., 2001). Similarly, Francis(1996) obtained

higher percentage of fertilisation (84 percent) using Ovaprim as compared to other

hormonal preparations such as Carp Pituitary Extract, HCG, mixture of HCG and

Pituitary extract, LHRH in induced breeding H. fossilis. Sreedhar and Haniffa

(1999), successfully bred H. fossilis using Ovaprim @0.5ml/kg body weight and

attained 95 percent fertilization and 90 percent hatching. Sahu et al.(2000) and

Mahapatra et al.( 2000) also reported performance of Ovaprim on par with other

hormonal mutations in successfully inducing spawning in Asian catfish, Clarias

batrachus. Bhowmik et al.(2001) also reported successful spawning of catfish

M. vittatus using Ovaprim, similar to the present observations. Negative responses

observed with Ovatide in the limited number of trials calls for further verification

using diverse dose patterns.

Although a number of factors have been implicated to influence natural

spawning, gonadal hydration (Sinha et al., 1974) as a factor responsible for

stimulated spawning, appear to hold good also in case of these catfishes. The

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failure of the non-spawner in pond condition appear to be due to reduced

accumulation of gonadotropin in its pituitary gland and thus additional amount of

exogenous hormone appear to induce spawning by enhancing ion/ water

transport to the gonad resulting in final maturation among catfishes too.

7.3.2. Hormonal analogues

Captive breeding of catfishes have been accomplished by using diverse

hormonal regimes by various workers. Clemens & Sneed (1971) and Hogendoorn

and Vismans (1980) reported breeding of C. gariepinus using Carp pituitary extract

@4mg/kg body weight. Mollah and Tan(1983) reported breeding of

C. macrocephalus using frog pituitary @4-6mg. Legendre (1986) performed

captive breeding of Heterobranchus longifilis using HCG @2.5 IU and a

fertilization rate of upto 75 percent was reported to be achieved. Zairin et al.

(1992) successfully bred C. batrachus, the tropical walking catfish using HCG

@0.80 IU. Fagnerno et al.(1992) accomplished breeding of C. isheriensis and

Mystus tengara using pituitary extract from non piscean sources @4mg/kg.

Legendre et al. (2000) succeeded in oocyte maturation and ovulation in Pangasius

hypophthalmus with two successive applications of Ovaprim @1ml and they

reported a relative fecundity of 3.17 lakh for this species per kg body weight.

Haniffa et al.(2001) reported captive breeding of Ompok malabaricus using

pituitary extract, HCG, Ovatide and Ovaprim and better performance was reported

with Ovaprim @ 0.7ml/kg. Szyper et al. (2001) gave details of induced maturation

and ovulation in the Chinese catfish Clarias fuscus using HCG @4 IU per gram of

body weight.

7.3.3. Stripping in catfishes

Among catfishes, males are generally not free milting and males in most

silurids are considered not amenable to stripping eg. Clarias batrachus,

Heteropneustes fossilis (Pandian and Koteeswaran, 1998). Therefore, in Clarias, the

males are sacrificed for collecting the milt since it is not possible to extract milt

through stripping (Rao et al., 1994). H. brachysoma appears to be an exception, as

mature males could be freely stripped. Pangasius sutchi is another catfish observed

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to be amenable for stripping. Chattopadhyay et al. (2002) has also reported induced

spawning of Pangasius sutchi by hormonal manipulation and stripping. Legendre et

al. (2000) also reported stripping method for breeding of Pangasius hypophthalmus.

Needless to state that, as observed in the study, fertilization of natural spawning

through hormonal manipulation is the most promising approach, as this is less

stressful for the broodstock than stripping.

The eggs expelled in stripping was observed to be almost double than that of

natural spawning in H. brachysoma. Stripping or artificial insemination is generally

adopted when the spawners fail to respond naturally under optimum conditions. As

the individual ova become discharged from the follicles inside the ovary wall, the

loosened eggs are collected through the genital aperture, with gentle pressure.

Pandian et al. (2001) observed that stripping is easily achieved in species where

the reproductive tract is straight, while in others where it is curved at the posterior

end, stripping may not be successful. Asynchrony in maturity or males becoming

mature much earlier than female was characteristic to H. brachysoma. Recent

reports of the standardisation of cryopreservation technique of milt of

H. brachysoma (Gopalakrishnan et al., 2006) opens up possibilities not only on

maintaining the viability of sperm but also of utilization of cryopreserved milt for

germplasm conservation. This is especially important in the context of the observed

asynchrony in final maturation in this species.

7.3.4. Latency period and ova viability

The duration for which ova remain viable in the ovarian cavity after

ovulation is apparently critical for successful stripping. The moment at which the

first ova could attain maturity or the ovulation time or the latency period in case of

H. brachysoma was observed to be 12-14 h. Stripping, beyond that critical period

was observed to result in poor fertilization and hatching, due to short viability of

ova. Campet(1997) observed that egg quality decrease significantly after ovulation

in Asian catfish Pangasius hypophthalmus and the viability is lost in three hours.

Legendre et al., (1996) reported that duration of ova survival is relatively longer

upto 2-4hr in Heterobranchus longiphilis and 10-12hr in C. monocephalus. The

correct timing of ovulation and ova collection is particularly crucial for egg

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development and successful hatching. In case of H. brachysoma, induced females

were found to ovulate in 12-14 hours and therefore the fish may be inspected at this

time after injection for readiness for stripping and stripping in less than 2-3hours

was found to achieve better success.

It has been noticed that in most of the fish species, the eggs and sperm

remain active and viable for quite sometime, even after the fish is dead (Nair,1999).

This will enable stripping as much fish as possible. Stripping technique has been

used effectively in many of the freshwater species such as carps, mrigal, catla, rohu

etc in which stripping is done after hypophysation and the embryos are reared in

hapas and the fry are used as seeds in grow out systems (Alikunhi,1956; Jhingran,

1969; Khan and Jhingran,1975).

Evaluation of viability of ova collected by stripping after elapse of the

latency period beyond 2 hours was observed to result in poor hatching in H.

brachysoma. This indicates the short viability of ova in case of H. brachysoma.

However, over ripening of eggs was also observed to result in problems such as

plugging of the vent in females (Tripathi and Khan,1990). The process of over

ripening is attributed to morphological, histological, physiological and

biochemical changes of the eggs(Nomura et al., 1974; Craik and Harvey,1984) and

are characterized by an increase in translucency and aggregation of cytoplasm

and oil droplets, reduced fertilisability and hatchability (Rowland,1988). Although

by removal of the knot from the vent, eggs could be freely stripped, such eggs have

been observed to loose their condition.

Several authors have reported variability in latency period among a variety

of fish species. Ohta et al (1996) observed that artificial fetilisation must be

carried out immediately after the latency period in order to obtain good quality

fertilized eggs. Sperm motility is considered to be a good parameter that reflect

sperm viability and fertilization ability (Vladic and Jarvi, 1997). Ohta et al (1997)

observed that sperm motility increased sharply a day after injection and it gradually

decreases 2-3days after injection in Anguilla japonica.

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7.3.5. Eggs expelled in spawning bout

The realized egg yield in case of H. brachysoma is extremely high as

compared to most catfishes; which is comparable to that reported for major carps

(Jhingran and Pullin, 1988; Venugopal,1990; Singh et al, 1990; Nandeesha et

al,1990). Szyper et al (2001) reported that in the Chinese catfish C. fuscus, the egg

number is estimated to be 3000 –20000 per female depending on size. Legendre et

al (2000) obtained a high variability in egg number ie, from 33000 to 317000 ova

kg-1 in induced breeding of P. hypophthalmus using Ovaprim. Thakur and Das

(1985) reported a realized egg yield of 4000 to 5000 for C. batrachus, weighing

about 150 to 200g. Chaudhary and Singh (1984), stated that fishes which do not

exhibit parental care produce large number of eggs possibly to compensate for the

loss of eggs and young ones as a result of unfavourable environmental conditions

and large scale predation. H brachysoma was found to exhibit a character similar to

polyandry which facilitated mass breeding of the fish in circular hatcheries. Bruch

and Binkowski (2002) stated that the characteristic polyandrous behaviour that

apparently maximizes opportunities for mating numerous individuals enhance the

genetic diversity of offsprings.

The pond reared broodstock was found to respond well to artificial

spawning than those collected from the wild during the breeding season. This was

evident from the superior quality of eggs and high survival. This can only be

attributed to the superior rearing conditions of the broodstock under captive

conditions. Nutritional status of the brood stock, husbandry conditions, particularly

water quality under which the brood fish are raised, genetics of the brood stock etc

are the primary factors suggested as possible determinants of egg quality (Bromage,

1995).

7.3.6. Courting and Spawning

H. brachysoma has been observed to exhibit a splendid courting behaviour.

Spawning was preceded and accompanied by characteristic breeding behaviour

during which, fish enter into a widespread chasing activity. This behaviour is

apparently of value in timing the stripping. In natural spawning, the arching of body

and muscular contraction is of immense value in complete discharge of the gonadal

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elements. Pandian et al.(2001) observed that juxtaposition and wriggling of the

body of male and female in H. fossilis, facilitated by contraction and relaxation of

successive myotomes, recall the act of stripping of gametes.

A relatively protracted spawning season was characteristic to pond raised

H. brachysoma, and the pond reared broodstock was observed to retain breeding

condition for a long period from May to September. This is probably due to a

stable environment available for the fish in the pond systems as compared to

riverine situations. However, highest breeding success and ova viability are

observed during June-July, coinciding with their natural breeding season. Catfishes

that spawn at a particular time of the year in rivers with strongly marked seasonal

flow regimes have been found to spawn over a much more protracted season in

environments with stable flow regimes (Van Den Hurk et al., 1985; Reynold,1974 ).

As in several catfishes, binding and locking of spines has been

characteristic also in H. brachysoma. This behaviour is considered to be a

protective adaptation as it increases the effective size of the fish making it more

difficult to be preyed by the predators (Kumrich, 1969; Forbes, 1989). Locking

occurs when the spine forms at right angles with the long axis of the fish.

H. brachysoma caught by netting was found to exhibit pectoral binding and spine

locking. Fine and Ladich (2003), indicated that spine locking is a formidable

defensive weapon among catfishes. These authors therefore concluded that spines

also function as a sound generating organ producing creeking stridulation during

courtship. The pectoral spines apparently functioned as acoustic signaling and

communication device.

7.3.7. Egg size and survival

Oocyte diameter and overall quality of ova was perceptibly superior in

pond reared stocks of H. brachysoma. Bascinar(2004) observed that egg size is

one of the important determinant of egg and larval quality. Eggs of catfishes have

been demonstrated to show wide range of size variations. And usually, it has been

described to vary from 1.5mm and 20 mm (Adriaens and Vandewalle, 2003). The

mean size of the egg was however, higher for early maturing females, and those

that were induced to mature during the early part of the breeding season as

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compared to late spawners and such eggs were also more viable. Gall (1975) also

showed that egg size was positively correlated with the survival of egg and fry

and also with growth rate. Bascinar (2004) observed that larger the egg, larger is

the larvae and larger yolk sac and this has positive effects on later development.

The egg size of H. brachysoma (1.6mm) has been however larger as compared to

most other catfishes such as M. cavassius (Rahman et al., 2004) and O. pabda

(Kohinoor et al., 1997). In catfishes such as H. fossilis, the mean egg size was

indicated to vary from 1.28+ 0.03 mm (Sreedhar and Haniffa, 1999) while in

C. batrachus it has been reported to be 1.00 to 1.2mm (Rao et al., 1994; Thakur

and Das, 1985) and >1.5mm in C. fuscus ( Szyper et al., 2001).

7.3.8. Embryonic development and hatching

Although embryonic development of teleost fishes have been studied

extensively by several authors(Chakrabarty and Murty,1972; Nair,1999; Kurup,

2001; Liang et al, 2003; Bascinar, 2004), little attention has been paid to the

embryology of siluriform fishes(Legendre and Teugels,1991; Watanabe, 1994;

Cardoso et al., 1995; Rahman et al., 2004). A detailed study on the embryonic and

larval development of Magur, C. batrachus was made by Thakur (1980). In

general, the embryonic and larval developmental period consisted of embryonic,

larval and juvenile stages.

Among catfishes, many strategies have been evolved for attachment of eggs

to some kind of substrates and differentiation of zona radiata play a crucial role in

survival of catfishes (Adriaens and Vandewalle,2003). In some clarids the

chorionic membrane possess specialized adhesive organs while in ictalurids

adhesive discs are found to serve this purpose. However, unlike other catfish

species, eggs of H. brachysoma was only partly adhesive and this may be linked to

their natural occurrence in downstream habitats in river systems.

Generally, in catfishes equatorial cleavage appeared to start only at 32

celled stage and subsequent cleavages results in the formation of a multicelled

morula at three hours. After initial cleavages, the morula enters the blastula stage

wherein the central cells form the blastodisc, while as the marginal cells get merged

with the yolk and form yolk syncytial layer (Kimmel et al., 1995). Epiboly initiates

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when both the blastodisc and yolk syncytial layer start thinning and expanding, and

the blastoderm gradually engulfs the yolk sac (Cardoso et al., 1995). Engulfment

of the yolk cell continues into the gastrulation period during which embryogenesis

starts. Germ ring, formed as a thickening of the blastoderm margin involutes and

differentiate into two layers, the superficial epiblast and the deeper hypoblast. The

hypoblast gives rise to notochord and the mesoderm where as the epiblast

differentiates into ectoderm. Close to hatching, the egg shell weakens consequent to

secretion of larval enzymes and as the movements become vigorous it ruptures.

In H. brachysoma egg hatching occur in around 24hrs post fertilization at

24-270C. Amongst catfishes, the hatching time vary substantially and it has been

reported to range from 24 h post fertilization in C. gariepinus to 75 days in Ariidae

(Legendre and Teugels,1991; Tilney and Hecht, 1993; Kamler et al.,1994).

However, the development of embryo and the variability of hatching time in

fertilized egg of most of the fish is generally influenced by the temperature of water

(Jhingran,1983; Rahman,1975). Evidently, the size of the yolk largely determines

the duration of the free living and pre larval periods of fish species (Kamler et

al.,1994). In M. cavassius with an average diameter of 0.5mm, eggs were

observed to hatch out in 19 hrs after fertilization at a temperature range of 27.5 to

29.50C (Rahman et al., 2004). While in other catfishes like H. fossilis and

C. batrachus the hatching time was observed to be 18hrs, 21-24h respectively at

water temperatures of 25 to 290C (Thakur and Das,1985). In Ompok pabda

hatching was reported to occur in 25.30 hr at 25.3 to 28.7 0C (Kohinoor et al.,

1997). It appears that fishes in torrential rivers have a faster development than still

waters (Arratia, 1983).

7.3.9. Larval rearing and fry nursing

The newly hatched larvae of H. brachysoma has an average length of

4.0mm. The hatchling size also has been found to vary widely among catfishes.

Many factors including species, strain, temperature variations, egg size,

fertilization rate and incubation procedures affect the duration of development,

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survival and initial size and growth of larvae or alevins (Dumas et al., 1995; Jonsson

and Svavarsson,2000). The larval phase begins with hatching and lasts until

metamorphosis (Helfman et al., 1997) and is regarded as the fundamental stage of

early life history. However, the innate behaviour of newly hatched larvae differs

widely among species. The length of the newly hatched larvae was 2.59 to

2.62mm in M. cavassius (Rahman et al., 2004).

The feed management of the newly hatched larvae that begin to depend on

exogenous feeding is considered a major bottleneck in larval rearing in fishes. The

finding that larval H. brachysoma accept and relish the commercial feed, at very

early stage as is evident from the high survival in fry nursing is of great value. The

nutritional requirement of the larvae at this stage should match the composition of

yolk that caters the pre feeding fish. The gape of the mouth opening of the

hatchling apparently determines the size of the food particle accepted by the

larvae (Joseph, 2001). The time at which external food is first given to the larvae

influences their subsequent growth and survival (Kamler, 1992). And probably this

mixed feeding behavior of the early life history stages characteristic to

H. brachysoma appear to favour smooth weaning of the early larvae to artificial

diets.

Captive breeding and Conservation Management

Despite resolute utilization of captive breeding in species recovery for a

variety of species in recent years, the limitations of this approach also cannot be

overlooked. There is an apprehension that the technique has often been invoked

prematurely with out a careful field evaluation of costs and benefits. Merely

demonstrating that a population of a particular species is declining or has fallen

below what may be a minimum viable size does not constitute enough reasons to

justify captive breeding as a recovery measure. This is principally due to inexorable

genetic and phenotypic changes that might occur in captive environments. It cannot

also be an alternative to habitat and ecosystem protection and should therefore be

invoked most judiciously in the absence of other comprehensive efforts to maintain

or restore populations in wild habitats. Nevertheless, captive breeding operated

under carefully defined conditions of disease prevention and genetic management

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can play a crucial role in recovery of species for which effective alternatives are

unavailable. All these highlight the dire need to develop simultaneously valid

programs biodiversity conservation through concerted public education, and in situ

conservation efforts.