Reproductive Cycle, Captive Breeding and Embryonic Development of

Threatened Snakehead Channa gachua (Hamilton, 1822) for Species

Conservation - First Report
JAMES MILTON1, ANANTH KUMAR HANIFFA*,
1

College of Fishery, Key Lab of Agricultural Animal Genetics, Breeding and

Reproduction of Ministry of Education, Huazhong Agricultural University, Wuhan,
Hubei, 430070, PR China
*Centre for Aquaculture Research and Extension (CARE),St.Xaviers Collge
(Autonomous)Palayamkottai- 627002, Tamilnadu, India.
Abstracts:
Over the last few decades wild population of Channa gachua (Hamilton,
1822) has declined due to various reasons and is presently listed under vulnerable
category by CAMP, 1998. The oogenic process of C. gachua shows that it undergoes
distinct morphological changes, which are of cyclic and seasonal nature. six different
developmental stages have been classified under four major categories viz., i)
immature oocyte ii) maturing oocyte iii) mature oocyte and iv) spent. Also, most C.
gachua spawn from December to February. The available data show that all female
C. gachua larger than16 cm standard length were mature. Spawning under captivity
during October, 2011 by injecting three different doses of a natural hormone HCG
(1000, 2000, 3000 IU/Kg body weight). Twelve sets of breeding experiments each
with 2 males (average weight 54g) and one female (average weight of 65g) were
selected from the brood stock and a control set without hormone injection was
maintained for each experiment. Latency period, number of eggs spawned,
fertilization rate, hatching rate and number of hatchlings produced were determined.
No spawning activity was noticed in control set. Fertilization rate varied from 62% 70% and total number of eggs spawned were estimated to be 2740. The fertilized eggs
were spherical in shape (1.16mm 0.05 mm).The eggs hatched out between 24 36h
after fertilization and about 2,672 hatchlings were produced. Newly hatched larvae
measured 4.23mm 0.03 mm length and 0.037g 0.008 g weight and the mean
diameter of yolk sac was 1.1mm 0.08 mm. Percentage survival of hatchlings varied
from 40% - to 56 %. The length of the youngone at flexion stage was 6.2 7.1 mm
from day 8 14. During Post flexion stage the length of the larvae was 7.1 14.0 mm
from day 14 30.
Key words: C. gachua, HCG, Seed production, Ontogeny.
Introduction
1

Study on reproductive activities of fish is of particular importance in

maintenance of healthy populations of fish in the water systems and optimization of
appropriate broodstock management strategies. Different species of fish have various
reproductive strategies from strict gonochorism to simultaneous functional
hermaphroditism. Most species spawn once a year in a demarcated period, but others
spawns spontaneously in a season (Delvin and Nagahama, 2002). Murrels commonly
called snakeheads due to the presence of large scales on their head belong to family
Channidae. They are obligatory air-breathing freshwater fishes possessing accessory
respiratory apparatus and are distributed in African and Asian continents (Li et al.
2006). Snakeheads are important food fishes in South East Asia, and some colorful
species are even common in aquarium trade (Ng and Lim 1990; Musikasinthorn 2000;
Courtenay and Williams 2004).
The Asian genus Channa, presently contains 26 valid species and is widely
distributed in Iran and Southern Asia (Musikasinthorn 2001; Berra 2001;
Musikasinthorn and Taki, 2001; Zhang et al. 2002; Courtenay and Williams, 2004);
most of them have become threatened due to human anthropogenic pressure and one
among them is C.gachua commonly called dwarf snakehead declined drastically
(vulnerable) in India (CAMP 1998; Anjan Kumar Prusty et al. 2007) and endangered
in Asia (Lim and Ng 1990). C. gachua is important as food fish as well as aquarium
fish due to its beautiful colouration (Talwar and Jhingran 1992). Its small size makes
it more appealing as an aquarium species. Ng and Lim (1990) cited individuals being
sold for $30-60 in Singapore, although they listed the species as endangered there.
Freshwater murrels are being seasonal breeders exhibit clear changes in the gonads
during breeding season (Marimuthu et al, 2001 and 2011; Haniffa et al, 2011).
Life history of threatened Channa species is still unknown and the knowledge
on Channa gachua is almost fragmentary. So far, the breeding season of C. gachua is
not confined to a definite period but it varies geographically. The variations in the
reproductive biology of the C. gachua in various geographical regions may be
attributed to the differences in various climatic conditions. Information on
reproductive cycle and histomorphological changes of ovary of C. gachua in captive
conditions is yet to be known. Therefore, the present study was performed to
determine the macroscopical and microscopical changes in ovary of C. gachua during
its annual maturation cycle and attempt was made to breed C.gachua under captivity

and rear the captive bred youngones at Centre for Aquaculture Research and
Extension (CARE) Aquafarm.
Materials and methods
Ovary Morphology:
C. gachua is ranging in total length from 12-20 cm and weight 15-100 g were
collected from Tamirabarani river (8.44N, 77.44E) fed systems during the months of
December 2008 -February 2009 and were stocked in earthen pond ( 3 x 3 x 1m) of
Center for Aquaculture Research and Extension (CARE) Aquafarm. Fresh chicken
viscera was collected from local market, cleaned and sliced into small pieces and
supplied to the fish ad libitum. For the study of maturation and gonad development,
monthly samples of 4-5 female fish were collected from the pond using drag net
during the period April 2009 to March 2010. By visual morphological observations
like slightly bulged abdomen in female fish were made for selection of the gravid fish,
which could be easily distinguished during the month of the July-December 2009.
After anesthesia, macroscopical appearances of ovary length, ovary weight,
body length and body weight were recorded.

Ganado-somatic index (GSI) was

calculated using the following formula:

GSI

GW
100
TW

Where,
GW = Gonad weight
TW = Body weight
For microscopical studies, ovary samples were immersed in Bouins solution
for 24h before processing for routine paraffin embedding. Sections of 5m thickness
were stained with haematoxylin and eosin and periodic acid Schiff (PAS). The
maturity stages of ovary of C. gachua were determined using the modified gonad
maturity scale developed by Arockiaraj et al, 2004b (Table .1).
Captive Breeding:
The brood fishes were fed with small live tadpoles and semimoist feed
(anchovy, rice bran, soy flour and fish oil) at 4% of body weight upto 4 months. After
4 months of rearing, they were found mature for captive breeding. The female
brooders showed bulged abdomen than males and their vent was reddish in colour;
fully mature females showed freely oozing eggs when pressed gently (Fig. 2). Twelve
sets of breeding experiments each with 2 males (average weight 54g) and one female
3

(average weight of 65g) were selected from the brood stock and a control set without
hormone injection was maintained for each experiment. Three different doses (low
dose - 1000IU, medium dose - 2000IU and high dose - 3000IU) of Human Chorionic
Gonadotropin (HCG) were injected intramuscularly to both males and females. After
HCG injection, each set was released into separate breeding tanks (15'3'2') and
Hydrilla verticilata was introduced into the breeding tanks for hiding purposes
(Haniffa et al. 2004). Spent fishes were removed from the breeding pool, washed in
KMnO4 solution and released back into the stocking pond.
Embryonic Development:
Diameter of eggs was measured using Magnus Pro Software with accuracy
level of 0.01 mm. Hatching rate was estimated on fourth day after spawning. After 14
days, hatchlings were transferred to glass tanks (50L capacity) for further rearing.
Paste of boiled egg yolk and plankton (Rotifers and Cladocerans) were supplied as
feed. Survival of hatchlings was calculated randomly by taking samples from glass
tank upto 15 days at 7 days interval. Data were analysed using statistical software
package SPSS version 11.5. A probability level of 0.05 was applied to account the
statistical difference if any between the means.
Results and Discussion:
The GSI of C. gachua varied significantly among Tamirabarani River
populations. The minimum observed GSI was in April (0.79 2.15%) and May (0.9
1.02%), while the maximum was recorded in December (3.61 1.85%). The rise of
GSI from June to August indicated the preparatory cum active phase or maturing
phase of the fish (Fig. 1) as 1.87%, 1.97% and 2.01% in June, July and August
respectively. The high GSI during September to February clearly indicated that the
vittellogenic period or active cum quiescent period. In December, the index was
suddenly raised from 2.42% to 3.61% as the ovaries mature, and was maximum in
December (3.61%), when the ovaries were in ripe condition (Table 2). This result
indicated that the winter season (December, January and February) is the possible
spawning season of C. Gachua. The highest proportion of spent gonads was
noticed in March, April and May, where the GSI value declined rapidly from 3.31 to
0.79 after spawning. Regarding the ova diameter, the size gradually increase from
March and rapidly reaching the maximum size until December (Fig. 2). Therefore it
was confirmed that the fish spawned once in year with one spawning season during
winter (December-February). Gonadal development and reproductive strategy have
4

been described in many teleost fish species in an effort to understand the time course
and energetic consequences of reproductive effort. Oocyte growth follows a similar
general pattern in most teleosts (Sundarabarathy et al.,2004; Encina and Lorencio,
1997; Stoumboudi etal., 1993; Dixit and Agarwal, 1974). The highest/lowest values
of conditional factor (CF) and GSI were due to the active somatic energy
accumulation/depletion (Encina and Lorencio 1997). Mishra (1991) described C.
gachua mature (stage V) oocytes ranging from 2.1 to 2.6 mm in diameter with the
highest percentage of stage V oocytes in July from specimens collected near
Berhampur, Orissa, India. The highest gonadosomatic index of 3.61 was noticed
during December. Estimated fecundity ranged from 2,539 to 7,194 in 15 mature
specimens ranging from 13.4 to 17.2 cm in length.
The present observation on fecundity revealed that C. gachua is a less fecund
fish when compared to carps, catfish and other Channa species like C. striatus(5000
10,000) and C. punctatus (3000 - 6000) with a maximum of 3450 and a minimum of
925 eggs. Bhuiyan and Rahman (1982) reported fecundity of C. gachua ranging from
487 4482. C. gachua measuring 15.5 cm total length and 53g body weight with
0.52g ovary weight produced 3450 eggs, whereas another fish of the same produced
2980 eggs. Similar kind of variation was also observed in other two fishes with the
total length of 15.0 and 14.7 cm produced 2340 and 2050 eggs respectively. The same
type of variation was also reported by Marimuthu et al., (2006) in spotted snakehead
Channa punctatus and Musa and Bhuiyan (2007) in Mystus bleekeri. The average
fecundity from an individual is 1750 550.12 (average body length 13.07 1.09cm
and average body weight 34.8 3.28g).
Histological features of oocyte developmental stages:
Based on the shape, size, changes in the nuclear and cytoplasmic components
of oocytes, six different developmental stages have been classified under four major
categories viz., i) immature oocyte ii) maturing oocyte iii) mature oocyte and iv)
spent. The process of oogenesis was classified according to oocyte location, size,
staining characters, number of nucleoli, presence of the follicular layer and the
distribution of cytoplasmic inclusions. According to these criteria oogenesis was
found to six different stages of ovary of C. gachua which is reported in Table 1. The
structural alterations were observed in the C. gachua oocytes during the oocyte
development in the histological studies performed. In this study, the oocyte
development of the C. gachua was divided to six stages. In a majority of teleost
5

fishes, the process of oogenesis may be divided five-eight stages (Fishelson et al.,
1996, Nagahama 1983, nal et al., 1999, West 1990, Goke et al.,2003, isa 1996).
Arockaraj et al., (2004b) described the morphological changes in the gonad of
Mystus montanus and histologically divided into five stages. In the present
investigation, C. gachua breeds from December to February. Similar observations
were made in Gobioides rubicundus(otherwise known as Odontamblyopus
rubicundus) by Kader et al., (1988). In the present study six oocytic stages (I to VI
stages) were clearly defined. It can also be inferred from the observed findings that in
Channa gachua, there occurred atleast 3 phases in the annual ovarian activity. Based
on the histological studies on the growing oocyte, cleardistinctions viz; stage I and
Stage II as immature, stage III,IV and V oocyte as maturing and stage VI as mature
which is similar to that of sundararaj and Sehagal (1970) and Daham and Bhatti
(1979).
Captive Breeding and Embryonic Development:
Spawning took place approximately 20 h - 26 h after hormone injection.
Chasing behaviour by male was observed after 14 18 h of injection Mating was
preceded by elaborate courtship. The active male chased the female and frequently
excited its movement. In all sets, the male was more actively involved in courtship. It
was seen hitting the female snout and vent region more frequently. The spawning
activity continued till the release of gametes. At the culmination of courtship, the male
bent its body close to the female and the breeders joined together (Fig. 3, 4) and the
male released its milt and the female its eggs. Eggs were found floating on the surface
from where the male took them inside its mouth. No spawning activity was observed
in the control set. C. gachua injected with 2000IU and 3000IU HCG / kg body weight
showed 2024 h latency period as compared to those injected with 1000IU /kg where
it took 2529 h. complete spawning was observed in medium dose and high dose
whereas partial spawning was noticed in the low dose. The total numbers of eggs
spawned ranged from 1,160 - 2,740 (Table 1). The fertilized eggs were clearly
distinguished from unfertilized ones; the former were larger, pale yellow in colour,
floating and spherical in shape (1.16 mm 0.05 mm) (Fig. 5).
Fertilization rate ranged from 64% - 70% in medium dose and high dose
whereas in the case of low dose the fertilization rate was (55%). The eggs hatched out
between 24h 32 h after fertilization. Changes in color of eggs and other
characteristics were noticed during embryonic development. The pale yellow eggs (0
6

15 h) became light yellowish (15 h- 20 h) and finally turned yellowish black (21h 32h). Average hatching rate ranged between 60% 73%. Increase in dosage of
hormone produced elevated values in hatching rate. For instance maximum hatching
rate of 73% was recorded as a function of high dose 3000IU when compared to 60%
in the case of low dose 1000IU. Correlation attempted for statistical difference
showed a positive relationship (P<0.01) between hormone dose versus latency period,
number of eggs spawned, fertilization rate and hatching rate.
In the present study, a single intramuscular injection of HCG resulted in
successful spawning of Channa gachua. The results showed that complete spawning
of Channa gachua occurred when injected with 2000IU and 3000 IU/kg and the dose
of the hormone significantly affected the percentage of fertilization, egg output,
hatching rate and survival of hatchlings (Table 1). Higher latency period as a function
of 1000IU /kg indicated the difference in the mode of action of the hormone. Haniffa
and Sridhar (2002) observed a latency period of 28 34hr for C.punctatus and
fertilization of eggs varied from 75% 80%. Marimuthu et al (2007) reported a
latency period of 23h 27hr and fertilization rate of 60 -70% for C.striatus injected
with ovatide. In the present study high dose (3000IU) showed better results in terms
of fertilization (70%) and hatching (73%) rates (Fig.1). Haniffa and Sridhar (2002)
reported fertilization rate of 75.5% for C. punctatus injected with HCG and according
to Hossain et al (2008) the same was 58.83% as a function of PG hormone. In the
present study hatching took place between 24h - 32 h after fertilization. Marimuthu
and Haniffa (2007) reported incubation period 23h -24h for C.striatus eggs.
The embryonic development of C. gachua started when the egg was fertilized
by sperm and ended when the embryo attained the generalized organ systems (Table 2
and 3). Newly hatched larvae showed large oval yolksac (Fig. 6). The length of the
yolksac on day 1 was 1.1 mm and the yolk was rapidly absorbed from day 1 to 3
(1.1mm 0.12mm). At this stage the head and tail ends of embryo became
distinguishable. The mouth was open at the end of the yolksac, while the eyes became
pigmented. Yolk moved dorsally above abdominal cavity beside upper base of the
pectoral fin and was completely absorbed by day 3.
Just before 1-2 h hatching, the embryo of C.gachua showed twisting
movements inside the egg. The fertilized egg diameter of C.gachua ranged from 1.04
mm- 1.16 mm, which is more or less similar to C.striatus (1.4-1.6 mm) as observed
by Parameshwaran and Murugesan (1976b). According to Parameswaran and Kamal
7

(1988) the length of newly hatched murrel hatchling was; 3.88 mm 4.47 mm for
Channa marulius 2.81 mm 3.22 mm for C.striatus and 2.49 mm 2.7 mm for
C.punctatus. In C.gachua newly hatched larvae showed a length of 2.5 mm 2.8mm.
Preflexion larvae showed a large head with a conspicuous occipital
melanophore and terminal mouth. The head, oil globule and the yolksac together
appeared as a bulb like structure. Melanophores were scattered on the yolk and a few
were present on the unpaired fin. The length of the youngone at flexion stage was
6.2mm 7.1 mm on day 8 14 (Fig. 7). Caudal fin began to separate; faint
pigmentation of eyes was noticed; alimentary tract was distinct and pectoral fin bud
appeared. During Post flexion stage the length of the larvae was 7.1mm 14.0 mm
from day 14 30. The development of pectoral and pelvic fins was completed on day
26 (11.3mm), whereas caudal fin completed its development on day 16 (7.2mm). The
number of rays in pectoral, caudal and pelvic fins were 14 rays, 13 15 rays and 5 6
rays respectively (Fig. 8).
In the present study pectoral fin buds and mouth cleft appeared 48 h after
hatching. Larvae commenced feeding at 5.4 mm length (48 hr), and its first feeding
took place 12 hr after the mouth opening. Verreth et al (1992) reported that
morphological and functional development of stomach was not completed at the onset
of exogenous feeding in C. gariepinus.In the present study alimentary canal was
observed 48 hr after hatching but not fully developed physiologically. Marimuthu and
Haniffa (2007) reported that the yolksac of C.striatus was fully resorbed 48 h after
hatching. Yolk sac of C. gachua was fully reabsorbed by the third day, when the
larvae measured 5.0 mm -5.5 mm in length. Aerial breathing of C. gachua larvae was
observed on 15th day after hatching. Similarly in other air breathing fishes the air
breathing habit was observed in C.striatus from 10th day onwards after hatching
(Marimuthu and Hanifffa 2004) and after 14th day in C. marulius (Parameshwaran
and Murugesan (1976b). The movement of larvae and capture of prey improved when
the larvae measured 6.3 mm on the 10th day onwards. This is likely due to the
development of caudal fin rays and the emergence of rays on the dorsal fin. Brown
(1986) opined that improvement in capture of prey could be due to the maturation of
the visual system as well as improvement in locomotive ability.
Our results clearly demonstrated The gonadosomatic indices of females varied
significantly between different months for Tamirabarani River populations. The GSI
value declined rapidly from 3.31 to 0.79 after spawning. Therefore it was confirmed
8

that the fish spawned once in a year with spawning peak during December to
February. However, in the present study, six oocytic stages (I to VI stages) were
clearly defined. It can also be inferred from the observed findings that in Channa
gachua there occurred atleast 3 phases in the annual ovarian activity. The possibility
of using natural hormone Human Chorionic Gonadotropin for effective induced
spawning and seed production of dwarf snakehead, C. gachua at experimental level.
In conclusion it is recommended that the seed of C. gachua could be produced in
captivity through scientific management. The success of breeding experiments will
be of immense help in conservation programmes of this threatened murrel species.
The successful protocols for captive breeding are likely to pave way towards
commercialization of the technology. The present dosage of 3000IU / Kg BW of HCG
showed encouraging results for induced spawning in terms of spawning, rate of
fertilization and rate of hatching and may be used as a standard dose in future
breeding of C. gachua.
Acknowledgement:
This research work was funded by CSIR Emeritus Scientist grant (No.21
(0670)/07/EMR-II). We are grateful to Dr. Alphone Manickam, Principal, St.Xaviers
College, Tamilnadu, India for providing necessary facilities.

References:
Anjan Kumar Prusty B., Rachna Chandra Azeez PA., Sharma LL. 2007. New
Additions to the ichthyofauna of Keoladeo national park, A world heritage site in
India. Zoos Print Journal 22 (10) pp.2848 2852.
Anon. 1998. Report of the workshop "Conservation Assessment and Management
Plan

(CAMP) for freshwater fishes of India 1997" organized by Zoo Outreach

Organization (ZOO) and National Bureau of Fish Genetic Resources (NBFGR),

Lucknow, held at NBFGR in September 1997. Zoo Outreach Organization,
Coimbatore, India, pp. 156.
Arockia Raj AJ., Haniffa MA., Seetharaman S., Singh SP. 2003.

Early

development of threatened freshwater catfish Mystus montanus (Jerdon). Acta

Zoologica Taiwanica. 14(1)pp. 1-10.
Berra TM. 200.1 Freshwater fish distribution. Academic press. New york. New york.
Xxxviii +. Pp. 604.
Bruton MN. 1979. The breeding and early development of Clarias gariepinus
(Pisces: Clariidae) in lake sibaya South Africa, with a review of breeding in species of
the subgenus Clarias (Clarias). Trans Zoo. Soc., Lond. 35: pp. 1-45.
CAMP. 1998. Report of the workshop on Conservation Assessment and Management
Plan (CAMP) for fresh water fishes of India, zoo out reach organization and NBFGR,
Lucknow, India. Pp.156.
Courtenay WR, Williams JD (2004) Snakeheads (Pisces: Channidae), A biological
synopsis and risk assessment. U.S. Department of the Interior, U.S. Geological.
Haniffa MA. 2010. Indian Snakeheads. Fishin Chimes.30 (1) pp.34-36.
Haniffa MA., Sridhar S. 2002. Induced Spawning of spotted murrel (Channa
punctatus) and catfish (Heteropneustes fossilis) using human chorionic gonadotropin
and synthetic hormone (ovaprim). Vet. Arhiv., 72 (1) pp.51-56.
Hossain MK., Latifa GA., Rahman MM. 2008. Observations On Induced Breeding
Of Snakehead Murrel, Channa striatus (BLOCH, 1793). Int. J. Sustain. Crop Prod.
3(5) pp. 65-68.
Lee PG., Ng PKL. 1994. The systematic and ecology of snakeheads (Pisces:
Channidae) in Peninsular Malaysia and Singapore. Hydrobiologia, v. 285, pp. 59-74.
10

Li Xia., Prachya Musikasinthorn., Yoshinori Kumazawa. 2006. Molecular

phylogenetic analyses of snakeheads Perciformes:Channidae) using mitochondrial
DNA sequences. Ichthyol Res 53: 148159.
Lim KKP., Ng PKL. 1990. The freshwater fishes of Singapore. Singapore Science
Centre, p160.
Marimuthu K., Haniffa MA. 2007. Embryonic and Larval Development of the
Striped Snakehead Channa striatus. Taiwania 52(1): 84-92
Marimuthu K., Kumar D., Haniffa MA. 2007. Induced

spawning of striped

snakehead, Channa striatus, using Ovatide. Journal of Applied Aquaculture 19 (4):

95103.
Musikasinthorn P. 2000. Channa aurantimaculata, a new channid fish from Assam
(Brahmaputra River basin), India, with designation of a neotype for C. amphibeus
(McClelland, 1845). Ichthyol Res 47:2737.
Musikasinthorn P., Taki Y. 2001. Channa siamensis (Gnther, 1861), a junior
synonym of Channa lucius (Cuvier in Cuvier and Valenciennes, 1831). Ichthyol Res
48:319324.
Ng PKL.,

Lim KKP. 1990. Snakeheads (Pisces: Channidae): Natural, history,

biology and economic importance: Essays in Zoology, Papers Commemorating the

40th Anniversary of the Department of Zoology, National University of Singapore, p.
127-152.
Ogunji JO., Rahe RE. 1999. Larval development of the African catfish
Heterobranchus longifilis VAL, 1840 (Teleostei; Claridae) and its larval behavior.
Journal of Aquaculture in the Tropics 14, 11-25.
Parameshwaran S., Kamal MY. 1988. Synoposis of biological data on the giant
murrel, (Channa marulius), striped murrel (Channa striatus) and the spotted murel
(Channa punctatus). Central Inland Capture Fisheries Research Institute Bulletin 53,
77-94.
Parameswaran S., Murugesan VK. 1976b. Observations on the hypophysation of
murrels (Ophiocephalidae). Hydrobiologia 50 (1): 8187.
Puvaneswari1 S., Marimuthu K., Karuppasamy R., Haniffa MA. 2009. Early
embryonic and larval development of Indian catfish, Heteropneustes fossilis.
EurAsia J BioSci 3, 84-96.

11

Talwar PK., Jhingran AG. 1992. Inland fishes of India and adjacent countries. Vol.
I and II Oxford and IBH Publishing company, New Delhi, India, 1158p.
Verreth J., Torreele E., Sparzier E., Slurszen A. 1993. The development of a
functional digestive system in Clarias gariepinus (Burchell). J. of the World Aqua.
Soc. 23: 286-998.
Zhang FC., Li Z., Wang

H., Wu. 2006. Modeling impacts of management

alternatives on soil carbon storage of farmland in Northwest China. Biogeosciences,

3,451466.

12