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Chapter III

.

85

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CHAPTER-III

CAPTIVE BREEDING (HCG AND OVAPRIM) OF

MASTACEMBELUS ARMATUS; THREATENED AND ENDEMIC

SPECIES IN THE CAUVERY RESERVOIR

Introduction

Biodiversity loss is one of the world's most pressing crises and there is global

concern about the status of the biological resources on which so much of human life

depends. The estimated current species extinction rate is between 1,000 and 10,000 times

higher than it would be naturally be (Kumar and Khanna, 2006). Fish forms highest

species diversity among all vertebral groups apart from its economic importance. India is one

of the mega biodiversity hot spots (North East Region and Western Ghat) contributing about

11.72% of global fish diversity mainly from the greater Himalayan range on the

northern plains, long stretches Eastern and Western-ghats. The aquatic environments are

experiencing serious threats to both diversity and ecosystem stability and therefore,

research is being pursued globally to develop systematic conservation planning to protect

freshwater biodiversity (Margules and Pressey, 2000; Saunders et al., 2002; Nel et al.,

2008) and various methods, strategies and priorities have been proposed (Cowx, 1998;

Lakra et al., 2006; Sarkar et al., 2008).

The wild population has steadily declined mainly due to the loss of habitat,

introduction of alien species, diseases, pollution, siltation, poisoning, dynamite, and other

destructive fishing (Katerina et al., 1996). Ecological studies focusing on patterns of

biodiversity are a vital foundation for natural resource management and conservation

(Sarkar et al., 2008). Over the years, the cultural practices have undergone considerable

intensification and with the possibility of obtaining high productivity levels. There has

been a state of flux between different farming practices. Studies on physicochemical

parameters and reproductive fecundity of endangered and commercially important fishes

are required for management and conservation of fish population in natural water bodies.

Hormonal preparations applied in fish aquaculture allow the improvement of

artificial reproduction techniques during the spawning season and off season (Brzuska

and Adamek, 1999; Kucharczyk et al., 2008; Ulikowski, 2004; Cejko et al., 2010).

47

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One of the difficulties in working with a new candidate fish for culture is obtaining

reliable quantities of viable eggs, and it is often necessary to use exogenous hormone

treatment to stimulate ovulation, to achieve this. Successful manipulation of reproductive

processes is dependent on some level of understanding of reproduction in the target

species (Morehead et al., 1998).

Fish propagation is increasingly dependent on artificial reproduction. New hormonal

preparations permit improving techniques for the controlled reproduction of species that

have been propagated successfully for many years. Captive breeding and the release of

captive bred individuals into the wild are among the techniques used for conservation of

rare and endangered fish species (Sarkar et al., 2006). It is also possible to apply these

hormonal preparations in the reproduction of species that, until recently, were not

frequently studied, for example hemophilic cyprinid fishes (Kucharczyk, 2002;

Szabo et al., 2002 and Krejszeff et al., 2008) or species that are under protection

(Philippart 1995; Kaminski et al., 2004). The study of the effectiveness of a given

preparation for wider application in fish reproduction takes into consideration its impact

on quantitative (number of individuals ready to spawn, working and relative fecundity,

ejaculate volume) and qualitative parameters of spawners (post spawning mortality) and

gametes obtained (sperm motility, embryo survival). The facility of administering the

hormonal stimulation is also a consideration.

Hormonal preparations applied in fish aquaculture allow improving artificial

reproduction techniques both during and outside the spawning season (Brzuska and

Adamek, 1999; Kucharczyk et al., 1999; 2000 and 2008, Targonska - Dietrich et al.,

2004;Ulikowski 2004; Targońska et al., 2005 and 2008; Cejko et al., 2008a,b, and 2009).

Also, the economic analysis of hormonal stimulation in wild fishes was done (Kupren et al.,

2008; Turkowski et al., 2008; Hakuc-Błazowska et al., 2009). This allows conducting

restitution programs and studies in the area (Babiak et al., 1998). During the last several

years numerous new hormonal preparations that can be applied in controlled fish

reproduction appeared in the market (Yaron 1995; Yaron et al., 2003; Szabo, 2002;

Brzuska, 2006). Determining their suitability, however, requires many studies aiming at

defining the optimum conditions for conducting the treatment and the type of hormonal

preparation applied as well as its quantity and dose size. In case of cyprinid fish, the

48

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87


analogues of gonado liberine, the effectiveness of which is frequently conditioned by

applying them jointly with a dopamine antagonist. Extensive works have been carried out

on food and feeding habit, cytological, redescription, morphometry and length-weight

relationship of the mastacembelid fishes in India and abroad in last few decades. Of these,

the most notable works were those of Saxena et al., (1979); Serajuddin and Mustafa,

(1994); Serajuddin et al. (1998); Froese and Binohlam, (2000); Serajuddin, (2004);

Serajuddin and Ali, (2005); Ahirrao, (2008); Emmanuel and Melanie, (2009) and Cakmak

and Alp, (2010).

Ovaprim contains 20μg of salmon GnRH and 10mg domperidone per milliliter.

In India trials with Ovaprim have given very encouraging results (Nandeesha et al., 1990

and 1991). Kaula and Rishi, (1986) reported the successful spawning of Mrigal with

Ovaprim. Nandeesha et al., (1990 and 1991) reported very satisfactory results in trials

with Ovaprim. Khan et al., (1992) reported successful spawning of rohu and mrigal with

Ovaprim. However, a major handicap in the use of Ovaprim is its high viscosity, which

causes difficulty in injection. Its high cost is also a prohibitive factor. The high demand for

fish fingerlings in the phenomenal growing aquaculture industry has stimulated the need for

artificial propagation of cultural warm water fishes. Statistics of global fish production shows

that fish farming represents about 15% of the global fish yields and was expected to

exceed 20% by the year 2000 FAO (2000). However, inspite of the break through

reported for its artificial propagation (Richter and Vander Hurk, 1982; Madu et al., 1987;

Madu et al., 1989), the demand for fish seed still outstrips the supply. Richter and Vander

Hurk, (1982) reported that the problem of inadequate supply of fish seed can only be

solved through induced breeding by the application of various inducement materials. Various

types of fishes have been induced to spawn, using various hormonal materials (Nwadukwe,

1993; Eyo, 1992, 1997, 1998 and 2000; Nwuba and Aguigwo, 2002). Some of these

hormonal materials (natural and synthetic) include cHCG. The formulation of a standardized

method to induce milkfish (Chanos chanos ) to spawn has elated investigators for over a

decade (Lam, 1984; Kuo, 1985). Initially, spawning attempts have involved administering

piscine pituitary extracts (salmon or carp), plus human chorionic gonadotropin

(Vanstone et al., 1977; Kuo et al., 1979). More recently, HCG alone has been used to

bring about the final maturation of ova (Tseng and Hsiao, 1979; Lin, 1982, 1984).

49

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The salmon LHRH analogue (i.e., that included in Ovaprim) is effective to induce

maturation in cyprinids. This is important in the view of the fact that mammalian

analogues are available more widely and their price is much more attractive. However,

recent data showed that in many cyprinids, including hemophilic fishes, Ovaprim worked

better than Ovopel (Targonska et al., 2008; Zarski et al., 2011a, b and 2012). The present

study compares the effect of HCG and Ovaprim hormones in induce breeding of M. armatus,

with the following specific objectives : to compare the level of egg productivity, egg

hatchability, deformatities, survival and Starter formulated feed fed fishes were examined

with regard to growth rate, survival and Standard Growth Rate (SGR).

Materials and Methods

Brooder Fish Collection and Acclimatization

The Brooder fish Mastacembalus armatus was collected from Kaveripatti

(Latitude 11 º 32ʹ 26 ″ N Longitude 77 º43ʹ 50″) Cauvery River bank, Tamil Nadu, India

by using special Grass Bait and Nets without disturbance. The fishes were acclimatized to

four artificial cave fitted tanks. M.armatus (125 Males and 125 Females) Brooders were

selected based on the external morphology and ovarian biopsy.

Fish Gonads for Histological Examination

Histology studies is applied currently in many biological phenomena such as fish

reproduction to invent new and effective methods for increasing efficiency of broodstock,

increasing fish production and ultimately increase efficiency and higher fish are predicted.

Determination of the peak period of spawning helps to understand the biological

characteristics and life cycle of a species and also supplies necessary data for the

management and reconstruction population.

M. armatus is a gonochoristic species with no sexual dimorphism between male

and female. Externally, sex could be recognized only during breeding season when

female shows a fully bulged, soft abdomen and red bulging cloacal region. The female

reproductive system consisted of a pair of ovaries, suspended anterio-posteriorly in the

peritoneal cavity and attached to the dorsal air bladder by thin mesenteries. Ovaries joined

together posteriorly, sharing a single oviduct which opened into cloaca through genital pore

50

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along with the openings of gut and ureters. Oviduct very thin and narrow white tube,

which varied in its length and prominent seasonally. The length and girth of both ovaries

were not always equal. Their cephalic end was thicker when compared with the caudal

end. Externally, each ovary was covered by thin peritoneal membrane, beneath the

peritoneal layer lies tunica albugenia. Tunica albugenia becomes thinner as fish reaches

full maturity.

Gonads collected from 5 pairs of mature male and female with ABW of 200-400gms

in early and late reproductive period was used for the present work. Gonads were surgically

removed without damage, collected and fixed for histological examination by light

microscopy.

Histological slide preparation

The histological approach uses a semi quantitative numerical assignment, to rank

reproductive stage. The gonads of male and female were subjected to micro-technique

sequences i.e., fixation, embedding, sectioning and staining. The stained sections were

examined under a compound microscope, and the state of gonadal development was

determined. Fixation follows the method described by Preece (1972).

Reagents

Acetone, Ammonium hydroxide, Ethanol, Ferric ammonium sulphate, Formaldehyde

(37% solution) Glacial acetic acid, Hematoxylin powder, Eosin, Paraffin - Paraplast

tissue embedding media (melting point 56 °C), mounting, Phosphotungstic acid, Sodium

chloride, Sulfuric acid, Tissue Clear III, Tissue Dry, histological grade xylene.

Reagent preparation

1. Ferric alum mordant: 25g Ferric ammonium sulphate dissolved in 500ml distilled water.

2. Basic ethanol: 26 ml ammonium hydroxide in a solution of 3370 ml 95% ethanol

and 630 ml distilled water.

3. 1% acetic acid: 20 ml glacial acetic acid in 1980ml distilled water.

4. 1% acid acetone: 20 ml glacial acetic acid in 1980ml acetone

5. Groat/Weigert hematoxylin working solution: 245ml distilled water, 5ml sulphuric

acid, 5g ferric ammonium sulphate, 245ml 95% ethanol, and 2.5g hematoxylin powder.

51

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6. Phosphotungstic acid solution: 10g phosphotungstic acid crystals dissolved in

490ml distilled water.

7. Davidson's fixative solution: 1 part glycerine, 1 part glacial acetic acid, 2 parts 37%

formalin, 3 parts 95% ethanol, and 3 parts isotonic sodium chloride (usually 20 – 30%).

Tissue embedding

Individual fish gonad tissue samples were prepared for embedding in paraffin

using an established dehydration protocol. The solutions used for dehydration, clearing, and

infiltration were changed frequently to maintain solution purity. Embedding was done

manually by moving the tissues through the sequence. The paraffin was melted at 60°C.

After the tissues are infiltrated with paraffin, they were transferred to a vacuum infiltrator

set at 60°C and placed under a vacuum for 30min. Tissues were transferred to a holding

tank of melted paraffin and removed singly to stainless steel moulds. The tissues were

oriented with the cross-sectional face down for sectioning, and a plastic mould embedding

ring is placed on top. The ring is filled with paraffin and the mould moved to a cold plate

of the embedding system. As the tissue/paraffin cools and hardens, the paraffin shrinks.

The block was then placed in a freezer until sectioning.

Tissue sectioning

The paraffin blocks were sliced at 10μm sections using microtome. The sections were

placed on the surface of a water bath maintained at 45-50°C and allowed to expand. Once the

sections expand to full size, a microscope slide is held at an angle and slide under one or

more of the tissue sections. The sections are then lifted out of the water onto the slide.

The sections were positioned on the slide in the orientation in which they will be stained and

observed. The slide was allowed to air dry until it can be placed in a slide rack. The slide

rack is placed in a drying oven at 40°C. After drying overnight or longer, the slides are ready

to stain.

Tissue staining

Sections are de-paraffinized and hydrated using Xylene-ethanol series. Following

hydration, slides were stained in hematoxylin and eosin, dehydrated in a series of acetic

acid dips followed by acetone, cleared in xylene and mounted in Permount.

52

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Analysis

Each slide was examined microscopically to determine sex and stage of gonadal

development. Careful examination of early developmental stages was needed to distinguish

males and females.

Hormonal Administration

Induced breeding of M.armatus was done using synthetic hormone Ovaprim

(Olubiyi, 2005). For each concentration 5 male and 5 female fishes (24 groups) were

exposed to separate spawning plastic tanks, with volume of 1m3

and 80 cm height of

water body inside. One third of the water surface of the water in each tank was covered

with water hyacinth and grass rhizome so that the fish could shelter under it. Both male

(1.70ml/kg to 2.10 ml/kg) and female (2.40ml/kg to 2.95 ml/kg) fishes were administrated

with a single dose intra muscularly. The dosage level has been standardized as HCG

(Lam, 1982) at 0.064ml/kg to0.069 ml/kg body weight for females and 0.031ml/kg to

0.036ml/kg body weight for males. Both the hormones were injected at 10 different

concentrations as shown in the Table-1 and 2. The injected fishes were introduced into

the artificial spawning tank at 1:1 ratio 5 pairs in each tank. The spawning rate, hatching

rate, deformities and survival rate were analyzed using standard procedure (Uthayakumar

and Ramasubramanian, 2011).

Challenging Behaviors of M. armatus breeding

Spiny eels are generally shy and ignore tank mates too big to eat. Towards their

own kind, spiny eels run from being hostile at one extreme to relatively peaceful at the

other. As a broad rule, species of Mastacembelus tend to be territorial, whereas species of

M. armatus are much more sociable. Nonetheless, while M. armatus usually get along, in

small groups (twos or threes) there's still the potential of bullying, especially if the tank is

small and lacks hiding places. When kept in groups, though, spiny eels tend to be more

outgoing and more likely to settle down quickly.

Another benefit of keeping spiny eels in groups is the opportunity for breeding.

Only a few spiny eels have spawned in captivity at all, though this is likely more about

them being rarely kept in groups than in any intrinsic difficulty. Identifying the two sexes

53

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92


is the first challenge, and this is impossible with immature fish. One fully grown, females

are obviously more deeper-bodied than the males. The exact spawning trigger is

unknown, but feeding the fish well and performing substantial water changes appear to be

important. Perhaps the abundance of food and the influx of clean water mimic the 'rainy

season' of their natural habitat? Certainly species like Mastacembelus armatus are known

to spawn only during the monsoon (May-September).

Spawning Behaviors of M. armatus

Courtship is a lengthy, elaborate process that lasts several hours. The fish chase one

another and swim around in tight circles before spawning. The sticky eggs are deposited

among the leaves or roots of floating plants such as water hyacinth. Up to a thousand eggs are

produced, about 1.25 mm in diameter, and these hatch after three or four days.

The fry (elvers) become free swimming another three to four days later, at which

point they need tiny foods such as radiolarians, Cyclops nauplii, and hard-boiled egg

yolk. A particular problem with newly hatched spiny eels is a certain susceptibility to

opportunistic fungal infections. Regular water changes are very important, and the use of

a safe antifungal agent like Pimafix might also be worthwhile.

Statistical analysis

All the statistical analysis was performed using SPSS 13.0 for windows.

The significance of variation in the Ovaprim and HCG administration effect of Egg

productivity, Egg hatchability, Deformatities, Survival Rate of was M. armatus analyzed

by One-way ANOVA (DMRT- Duncan Multiple Range Test).

Results

Hormonal Administration

In the present study, the successful captive breeding of M. armatus with the

administration of Ovaprim and HCG was observed. The different dose of the hormone

significantly altered the percentage of fertilization, number of egg laid, hatching rate,

deformatities and Survival Rate (Table 1 and 2). Induced breeding was done using

synthetic hormone Ovaprim. For each concentration 5 male and 5 female fishes (12 groups)

were exposed in separate spawning tanks. Both male Ovaprim (1.70 to 2.30 ml/kg)

54

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93


TA

BL

E-1

. RE

SUL

TS O

F C

APT

IVE

BR

EE

DIN

G U

SIN

G D

IFFE

RE

NT

DO

SES

OF

OV

APR

IM

**S

ignif

ican

t at

0.0

5 l

evel

; *S

ignif

ican

t at

0.0

1 l

evel

, ns-

Not

signif

ican

t. M

ean i

n a

colu

mn f

oll

ow

ed b

y a

dif

fere

nt

lett

ers

are

S

ignif

ican

tly (

P<

0.0

5).

No.

T

rial

s

M.a

rmatu

s H

orm

one(

ml/k

g)

Ave

rage

Wei

ght(g

) N

umbe

r of

E

gg

Rel

ease

d

(n)

Hat

chin

g R

ate

(%)

Def

orm

ities

(%

) Su

rviv

al

(%)

Mal

e

(n

) Fe

mal

e (n

) M

ale

Fem

ale

Mal

e Fe

mal

e

1

5

5

1.7

0

2.4

0

295.7

±2.3

0c*

280.2

±1.7

0e*

387.2

±0.9

5i*

48.0

±1.8

2f*

*

23.7

±0.9

5f*

26.5

±1.9

1g*

2

5

5

1.7

5

2.4

5

305.7

±0.9

6ab

* 2

35.7

±1.2

5j*

396.5

±2.3

8h

*

46.2

±2.5

0f*

*

24.5

±0.5

7f*

34.5

±1.2

9f*

3

5

5

1.8

0

2.5

0

267.5

±1.2

9e*

267.0

±1.2

7f*

403.5

±1.2

9g*

57.2

±2.5

0e*

26.2

±1.7

0e*

46.5

±1.2

9e*

4

5

5

1.8

5

2.5

5

256.5

±1.2

9f*

233.9

±1.4

6j*

*

425.7

±0.9

5f*

72.2

±2.2

1d

*

16.0

±0.8

1g*

55.5

±2.3

8d

*

5

5

5

1.9

0

2.6

0

227.5

±3.5

1g*

254.0

±1.4

1h

*

457.2

±1.5

0e*

74.0

±0.8

1c*

*

12.5

±1.2

9h

*

56.2

±1.8

9d

*

6

5

5

2.0

0

2.6

5

256.5

±1.2

9f*

344.2

±0.9

5c*

458.7

±0.9

5e*

66.5

±1.2

9e*

09.0

±0.8

1i*

74.2

±0.9

5c*

7

5

5

2.0

5

2.7

0

267.7

±1.2

5e*

257.0

±2.9

4g*

486.2

±1.7

0d

*

87.7

±0.9

5b

*

7.6

±0.4

7j*

78.0

±1.8

2b

*

8

5

5

2.1

0

2.7

5

226.0

±1.8

2h

*

337.7

±0.9

5d

*

562.7

±2.0

6a*

95.0

±3.5

5a*

03.5

±0.5

7k

*

93.0

±2.9

8a*

9

5

5

2.1

5

2.8

0

288.0

±0.8

1d

*

397.2

±2.2

1a*

536.2

±2.2

1c*

58.2

±1.7

0e*

42.7

±0.5

0c*

44.2

±0.5

0e*

10

5

5

2.2

0

2.8

5

307.2

±0.9

5a*

215.7

±0.9

5k

*

544.2

±1.2

5b

*

08.2

±0.9

5h

*

36.5

±0.5

7d

*

35.2

±1.7

0f*

11

5

5

2.2

5

2.9

0

288.5

±1.2

9d

*

244.1

±2.3

5i*

396.5

±1.7

3h

*

11.7

±0.9

5g*

45.0

±1.4

1b

*

12.2

±1.2

5h

*

12

5

5

2.3

0

2.9

5

297.7

±1.2

5c*

348.5

±1.0

0b

*

394.0

±0.8

1h

*

08.7

±0.5

0h

*

62.5

±1.2

9a*

03.0

±0.8

1i*

.

94


TA

BL

E -2

. RE

SUL

TS O

F C

APT

IVE

BR

EE

DIN

G U

SIN

G D

IFFE

RE

NT

DO

SES

OF

HC

G

**S

ignif

ican

t at

0.0

5%

lev

el;

*S

ignif

ican

t at

1.0

0%

lev

el, ns-

Not

signif

ican

t. M

ean i

n a

colu

mn f

oll

ow

ed b

y a

dif

fere

nt

lett

ers

are

S

ignif

ican

tly (

P<

0.0

5).

No.

Tr

ials

M.a

rma

tus

Hor

mon

e(m

l/kg)

A

vera

ge W

eigh

t(g)

Num

ber

of

Egg

Rel

ease

d

(n)

Hat

chin

g R

ate

(%)

Def

orm

ities

(%

) Su

rviv

al (%

) M

ale

(n)

Fem

ale

(n)

Mal

e Fe

mal

e M

ale

Fem

ale

1

5

5

0.0

31

0.0

64

276.2

±2.9

8e*

224.2

±0.9

5i*

314.2

±1.5

9f*

24.5

±1.2

9h

*

35.0

±0.8

1a*

0.0

±0.0

i*

2

5

5

0.0

32

0.0

65

267.7

±0.9

5f*

217.4

±1.5

0j*

317.0

±1.4

1f*

27.5

±1.2

9g*

28.5

±1.2

9b

*

0.0

±0.0

i*

3

5

5

0.0

33

0.0

66

236.0

±1.4

1i*

291.0

±1.4

1f*

417.0

±0.8

1d

*

38.0

±0.8

1d

*

13.0

±0

.81

d*

27.0

±0.8

1g*

4

5

5

0.0

34

0.0

67

257.5

±1.7

3g*

238.5

±1.2

9h

*

452.0

±1.4

1c*

31.7

±1.7

0f*

11.7

±0.5

0e*

30.0

±1.4

1f*

5

5

5

0.0

35

0.0

68

266.2

±0.9

5f*

237.5

±1.2

9h

*

457.5

±1.2

9c*

32.5

±0.5

7e*

08.2

±0.5

0f*

31.5

±0.5

7e*

6

5

5

0.0

36

0.0

69

297.2

±0.9

5c*

298.7

±1.6

7c*

*

343.5

±2.3

8e*

47.0

±081

c*

01.0

±0.0

5ij

*

32.0

±0.8

1e*

7

5

5

0.0

37

0.0

70

302.5

±1.7

3c*

294.7

±1.7

0e*

477.2

±1.5

0b

*

55.5

±0.5

7b

*

1.2

5±

0.0

5i*

55.7

±0.9

5b

*

8

5

5

0.0

38

0.0

71

333.2

±2.7

5a*

313.5

±1.2

9b

*

492.0

±0.8

1a*

*

69.5

±1.2

9a*

0.0

±0.0

j*

65.7

±0.5

0a*

9

5

5

0.0

39

0.0

72

313.7

±1.2

5b

*

297.2

±1.7

0d

*

496.7

±3.3

0a*

*

21.7

±0.5

0i*

04.1

±1.1

5h

*

43.7

±0.9

5d

*

10

5

5

0.0

40

0.0

73

297.5

±0.5

7c*

326.5

±1.2

9a*

419.7

±3.7

7d

*

11.0

±0.5

0j*

06.2

±0.0

g*

45.7

±0.9

5c*

11

5

5

0.0

41

0.0

74

244.0

±3.3

6h

* 298.2

±0.9

5d

*

347.0

±2.1

6e*

0.0

±0.0

k*

13.5

±1.2

9d

*

12.7

±0.9

5 h

*

12

5

5

0.0

42

0.0

75

295.2

±3.5

9c*

276.2

±1.2

5g*

268.7

±1.2

5g*

0.0

±0.0

k*

23.5

±1.2

9c*

0.0

±0.0

i*

.

95


HCG (0.031 to 0.042 ml/kg) and female Ovaprim (2.40 to 2.95 ml/kg) HCG (0.064 to

0.075 ml/kg) fishes were injected with a single dose intra muscularly. Both the hormones

were injected at 12 different concentrations as shown in the Table.1 and 2. The injected

fishes were introduced into the artificial spawning tank at 1:1 ratio.

Courtship Behavior

Spawning response varied from 8-12 hours. Courtship begins with the male

chasing the female and swimming in a tight circle. Later, the pair encircles each other

around the water hyacinth for spawning. The eggs stick to the roots of the water hyacinth.

The male releases sperm as the eggs are laid. The eggs are round, light orange in color,

adhesive in nature and about 1.30 mm in diameter.

Effect of Hormones

Total 60 females and male brooders were treated with Ovaprim; the effective dose

was found to be 2.75 ml/kg and 2.10 ml/kg body weight respectively, were induced

spawning occurred after 10-12 hrs of injection. The number of eggs released was maximum

(562.7±2.06), Minimum 387.2±0.95 found and percentage of hatchling (95.0±3.55 %) was

observed when treated with Ovaprim. The total deformities 62.5±1.29 % and survival

rate high in 93.0±2.98 lowest 03.5±0.57%; 03.0±0.81% was observed in Ovaprim

administrated groups respectively.

In HCG administrated fishes, the number of eggs released was highest in

496.7±3.30 (male 0.039and female0.072ml/kg), less in 268.7±1.25 (male 0.042 and

female 0.075ml/kg). The percentage of hatchling rate was maximum in 69.5±1.29%,

minimum 0.0±0.0% was observed in (male 0.042 and female 0.075 mg/kg). The total

deformities and survival rate was high in (65.7±0.50%) lowest (0.0±0.0%) was observed

in HCG administrated groups.

Histology

Immature Ovary

Ovaries were small and transparent. They were thread-like and attached to the

vertebral column. Oocytes were not visible to the naked eye. The mean diameter of

oocytes was 0.100 mm. small, round and transparent oocytes with a central nucleus was

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observed in histological sections of ovaries. Nucleoli were found in few oocytes. No lipid

droplets were found. These cells had basophilic cytoplasm and an acidophilic nucleus.

The ratio of nucleus to cytoplasm volume was high. The thickness of the gonads was

slightly higher than the immature ovary. The majority of the oocytes present in these

gonads was small and comprised of oogonia and primary oocytes. Some oocytes were in

the chromatin nucleolar stage. The biggest oocytes present were in the early peri-nucleolus

stage. The size of the oocytes never surpassed 200 µm; however, the majority was in the

range of 75 to 150 µm. The oogonia were basophilic in nature and stained darkly. The

nucleus was quite big, while the cytoplasm was in the form of a narrow rim around the

nucleus. The stroma of the ovary was well developed, while the tunica albugenia was thick.

Matured Ovary

During this stage, the ovary is filled mainly with previtellogenic and vitellogenic

oocytes in different stages of yolk deposition. Most of the vacuoles in this stage were

connected to each other and formed spaces between the yolk granules in the cytoplasm.

The deposition of yolk granules that contain lipoprotein appeared at the marginal regions

of the maturing oocytes and then spread until the entire central cytoplasm of the oocytes.

Nucleoli migrated towards the center of the nucleus away from the nuclear membrane

which loses its circularity and stiffness being a winding weak membrane in the way of

disintegration completely. During the migration of nucleus it began to liberate it’s

substances into the cytoplasm. The average of oocytes diameters in this stage was about

390µm. in M.armatus.

Matured Ova

During this stage, the ovary was filled mainly with previtellogenic and vitellogenic

oocytes in different stages of yolk deposition. Most of the vacuoles in this stage were

connected to each other and formed spaces between the yolk granules in the cytoplasm.

Cortical alveoli began to be liquefied, with a concomitant increase in size. As a result of

this, the yolk homogeneity started in the periphery of the oocytes. The nucleus starts to

migrate to the animal pole. With continuous growth the oocyte membrane became well

developed with average diameter of 200µm in the present study, zona radiata was formed

of two different layers, an internal spongy thick layer followed by a very thin external

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non-cellular homogeneous layer. A thick layer of well-developed epithelial follicle with

very prominent large cubic cells surrounds these two layers externally. The external layer

of connective tissues as a thin layer was detected.

Development of Male Gonads

At different stages of male gonad cells, spermatocytes, spermatids and spermatozoa

were found in testicular tissue using histology in the early reproductive and late reproductive

stage.

Immature Stage

Testis was thread-like, thin and whitish gray. Spermatogonia and primary

spermatocytes were the dominant cells of this stage. Spermatogonia had a light cytoplasm

and a large nucleus. Some secondary spermatocytes having basophilic cytoplasm were

also observed. The gonads, with active spermatogenesis were at the initial phase which

showed the presence of cysts containing type B spermatogonia (SG B). A cyst is formed as a

result of spermatogonia being surrounded by the Sertoli cell processes which form a tight

sheath. The youngest cysts contain at first two and later on four cells (Plate -1; Fig. 1&2).

Mature Stage

The mature spermatozoa were released from gonads during spawning, but some

spermatozoa were always left in the testis. They were observed in the gonads for a long

period, in some cases until the beginning of a new spermatogenetic cycle. The lobules shrink

and the Sertoli cells retract their processes, for which reason the lobule boundary wall

becomes higher. The highest lobule wall epithelium (51.0 µm) was recorded in M. armatus

(Plate -1; Fig. 3 -5).

In November to April the testis has spermatocytes and spermatids. The testicular

gland had numerous large lipid vacuoles in the luminal cell regions. The spermatids were

released from the seminiferous tubules into the ducts of the testicular gland. In May to

October its maximum stage of development was observed, and in the testis and ducts of

the testicular gland the spermatid number was comparatively greater.

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Plate 1.The histological sections of the gonads of M.armatus

Figure 1. Immature Male Gonad Figure 2. Mature Male Gonad with

Figure 3. Immature female gonad Figure 4. Matured Female Gonad

Figure 5. Developed Egg with Yolk Figure 6. Matured Ova of M.armatus

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99


Plate 2. Collection, Maintaining and Hormonal Administration of M.armatus

Figure 1.Capturing M.armatus using net, Figure 2. Captured live M.armatus collected and by a Native Fisherman Transported to the Banks in Parisal

Figure 3. Brooders Maintaining Tanks Figure 4. Hormonal administration of M. armatus

Fig 5. An Experimental Tank Fig 6. The Rhizome of the grass with eggs

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Developmental stages

The freshly spawned eggs of M. armatus were light orange in colour. They had a

smooth translucent vitelline envelope surrounded by a mucous layer which allows them

stick to the plant substrate and to one another at the time of spawning. The micropyle is

cone-shaped and located at the egg’s animal pole. Most of the egg volume was occupied

by numerous bright, tightly packed yolk globules. Polarization of the cytoplasm towards

the animal pole becomes evident within 60 min after fertilization, giving rise to a prominent

dome-shaped cytoplasmic layer known as the blastodisc. Cleavage is typically meroblastic,

resulting in a blastula with a blastodermal cellular region above the yolk mass (Plate – 3;

Fig. 1 -8).

The subsequent gastrulation process is characterized by several morphogenetic

movements that result in a rearrangement of the blastoderm relative to the yolk. Towards

the end of the first day of development, about 75% of the yolk mass has been covered by

the blastoderm and the elongated embryo’s shape is discerned along an anterior-posterior

axis. The “yolk plug” is the portion of uncovered yolk protruding in the neighbourhood

of the vegetal pole.

Organogenesis initiates soon thereafter; the optic vesicles being one of the first

organ primordial germ cells that become distinct. After 26 hrs, paired somites start

developing sequentially in an antero posterior direction, on both sides of the notochord.

Otoliths become visible within the otic vesicles. Before hatching, the embryo shows

conspicuous muscular contractions. The beating heart was placed beneath the head and

blood flow is readily observed. Brain regions become distinct and the lenses were evident

within the eyes. Rudiments of the adhesive glands were observed at the midbrain-

hindbrain boundary. Larval hatching occurred 58 hours after the fertilization. The mouth

was not yet formed, and the gut was narrow and straight. No fins are differentiated but a

primordial fin fold was already developed dorso-ventrally in the sagittal plane.

Melanophores were scattered over the oblong yolk-sac and extend along the junction of

the body with the ventral fin fold.

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Plate 3. The Sequential Developmental Stages of a Fertilized Egg

Figure 1. Fertilized Egg Figure 2. Two Cell Stage

Figure 3. Eight Cell Stage Figure 4. Sixteen Cell Stage

Figure 5. 32 Cell Stage Figures 6. Gastrulla stage

Figure 7. Morulla stage Figure 8. Tail Free Stage

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Plate 4

Figure 1. The Hatchlings with the Yolk Sac

Figure 2. The 3 Days Fries Hatchlings Reduced Yolk Sac

Figure 3. The M. armatus elvers (larvae)

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During the fifth day of development, the mouth opens and mobile jaws can be

observed. The yolk-sac has become smaller. Branchial arches were differentiated and

soon thereafter gill filaments started to develop. These larvae do not swim freely yet, but

they rapidly swing their tails while still attached to the substrate by their adhesive glands.

The free-swimming stage was reached four days after hatching, during the eighth day

of development. M. armatus larvae were large elongated with a snake-like body without

pelvic fins. It’s anal and dorsal fins are elongated and are connected to the caudal fin. Zig zag

brown coloured patches were obvious on its body. Melanophores were more heavily

distributed over the head and trunk. A few hours after the onset of free swimming; these

larvae start to feed from the environment, though the yolk is not yet totally consumed

(Plate - 4; Fig. 1-3).

Discussion

The M. armatus is a nocturnal socially associated fish, with a main spawning

period during spring and a secondary period during the fall according to studies based on

captive breeders (Anguis and Canavate, 2005; Garcia-Lopez et al., 2007). Sexual

differences are unknown and it is almost impossible to identify the sexes, though a

mature female may be fuller bodied. At a particular stage of maturity of the fish there is a

close relationship between the gonad weight and the body weight which helps in determining

the breeding period of the fish. Both male and female fishes mature simultaneously.

The gonad weight influences the relative condition, while the visceral weight does not

exert any significant effect on it. The development of the female gonads greatly affect the

curve of relative condition, more than the development of male gonads, though the curve

for male fish also shows a steady rise and fall in the seasonal variations of the relative

condition. Seasonal fluctuations in relative condition correspond to its spawning season.

In females, the tight association observed in males between the winter and the

maximum values of many of the measured variables was not evident. However, many

measured variables exhibited a tendency to increase their values with time, peaking in the

spring, and thus in agreement with the fact that ovarian development reaches its maximum

between the end of the September and the beginning of May, similar results discussed in

Senegalesesole (Anguis and Canavate, 2005; Garcia-Lopez et al., 2006a,b, 2007).

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http://en.wikipedia.org/wiki/Snake

http://en.wikipedia.org/wiki/Pelvic

http://en.wikipedia.org/wiki/Anal_fin

http://en.wikipedia.org/wiki/Dorsal_fin

http://en.wikipedia.org/wiki/Caudal_fin

According to literature pertaining to the general structure and function of teleost

gonads, the development of gonads is usually similar on both sides of the fish. However,

asymmetry in gonad size has been observed sometimes in some species, usually at or

near the time of breeding (Reddy et al., 1990). As in most teleosts, the gonads in male

and female albacore are paired, elongate organs, located in the dorsal portion of the body

cavity. They are suspended by the mesentery which contains a fat body closely associated

with the gonad between it and the dorsal body wall.

In the present study we have observed the successful captive breeding of

M.armatus with the administration of Ovaprim and HCG. The different dose of the

hormone significantly altered the percentage of fertilization, number of egg laid, hatching

rate and deformatities. This is an important component of captive-breeding programs as

conservation management must entail preservation of a species’ behavioral patterns as

well as its genetic diversity (Shepherdson, 1994). Ensuring that the captive environment

allows for the appropriate expression of behavior is also a welfare issue and zoo

managers need to ensure that animals humanely adapt to their artificial environment

(Price, 1984). The synchronization of ovulation after the stimulation is a very important

aspect. Brzuska (2003) reported that the deterioration of the egg quality with increasing

doses of gonadotropin (Nepal et al., 1998 and Brzuska, 2003).

A six-stage microscopical ovarian maturity scale was elaborated on the basis of

oocyte development phases, the post-ovulatory follicle and the processes of atresia

described above. The first five stages corresponded with those described macroscopically

for different species of Channichthyidae (Cielniaszek and Parkes, 1989; Kock and

Kellermann, 1991). Stage 6 includes ovaries in oocyte resorption processes, which

macroscopically resemble a maturity phase but microscopically conform to a regression

stage. The main histological features of the development stages are described in Plate-1.

Oocytes in early secondary vitellogenesis (Figure-4), are not present in stages of

advanced maturity and total maturity. This indicates that no immature elements are

incorporated into the cohort of yolky oocytes that will be evacuated during the current

spawning season. This was previously observed by Everson et al. (1991) and Kock and

Kellermann (1991), who suggest that Pre-vitellogenic elements and oocytes in primary

growth conform to the reserve 'stock' for the following spawning season.

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The testis in most teleosts consists of compact paired structures lying in the

abdominal cavity and composed of mass elongated, branched tubular structure with thin

fibrous walls which lack a permanent lining, seminiferous epithelium and because of this

reason, they are generally referred to as lobules, crystal or canals (Lofts,1968). On the

basis of distribution of spermatogonia and spermatogenetic pattern, two kinds of

testicular structures namely, tubular and lobular types have been identified (Redding and

Patino, 1993 and Dabhade et al., 2010). The Leydig cells in the present study also

became activated within 24 hrs synthetic hormones treatments. Same results were found

by Miura et al., 1991a,b.

The percentage of fertilization of hatching eggs obtained in the present trial

ranged 95.0% (2.75 ml/kg Ovaprim) and 69.5%(0.0710 ml/kg HCG.) Similar findings

were reported in rainbow trout O.mykiss Billard and Marcel, 1980. Ovaprim (Rowland,

1983) and LHRH have been successfully induce ovulation in a number of teleosts

(Tyler et al., 1990, Habibi et al., 1989). Certain drugs such as LHRH and Human

chorionic gonadotropin (HCG) have been induced in spawning fishes with variation in

the percentage of breeding (Crim et al., 1987). It also confirms suitability of Ovaprim in

fish reproduction stimulation (Thomas, 1994; Harvey, 1979). In India, most of the

breeders have preferred Ovaprim, as a survey showed that only 10 to 15 % of fish

breeders use extract due to the complexity of the technique

Kucharczyk et al., 2007 Ovaprim (at a dose of 0.5 m/kg body weight) induce

breed of 70% spotted murrel (Channa punctatus) and fertilization (Kujawa, 2007)

In C. striatus fertilization rate was 95 - 98% with Ovaprim as the hormonal material

(Dehadrai, 1984). According to Table 1 and 2, the results of number of eggs released,

deformities and survival rate had significant difference (P<0.05) whereas the hatching

rate was not much significant (P>0.05) between the female broods injected with Ovaprim

and with HCG dosages.

These results are in agreement with the study conducted by (Haniffa and Sridhar,

2002), in terms of fertilization and hatching rates, between the fish treated with LHRH

100 or 200 mg and the control fish which ovulated spontaneously (Haniffa et al., 1998).

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Breton et al., (1990) recorded a considerable number of dead larvae in the last

phase of incubation of eggs obtained from Silurus glanis females treated with LHRH.

The results of previous studies shows that in general the female fishes with high quantity

of eggs are obtained in synthetic ovulation stimulators compared to the treatment with

stimulators of natural origin. The eggs stripped injected with 1-1.5 ml Ovaprim/kg and

stripped during 14-17 hr post-injection is regarded as the good quality due to high

fertilization and hatching (Morehead et al., 1998).

High hatching rate and survival rate was successfully obtained in M. armatus

when stimulated with appropriate (Male 2.10, Female 2.75 ml/kg) and (Male, 0.038

Female 0.071ml/kg) dose of Ovaprim and HCG. Cauvery River eel can be successfully

bred in captive conditions and its population can be reclaimed with immediate effects.

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