SPAWNING PERFORMANCE OF NATIVE THREATENED SPOTTED SNAKEHEAD FISH, CHANNA PUNCTATUS (ACTINOPTERYGII: CHANNIDAE: PERCIFORMES), INDUCED WITH OVATIDE

* Correspondence: Dr. Kasi Marimuthu, Department of Biotechnology, Faculty of Applied Sciences, AIMST University, Batu 3 1/2 Bukit Air Nasi, Jalan Bedong Semeling 08100 Bedong, Kedah Darul Aman, Malaysia, phone: 00604–429 8000, fax: 00604–429 8009, e-mail: aquamuthu2k@yahoo.com INTRODUCTION The single most important drawback of large-scale commercial culture of several fish species is the deficiency of quality seed of uniform size, and free of diseases, parasites, and pests at the time of stocking in culture ponds. These strict requisites are seldom fulfilled where the seed is obtained from the natural water bodies (Zohar and Mylonas 2001). Furthermore, the broodfish that are obtained from the wild and taken to captivity or reared in captive conditions may receive inappropriate environmental cues for reproduction and these can cause reproductive development to be arrested in late vitellogenesis. For this reason, hormonal treatment been attempted for stimulating of gametes maturation and have been successfully used to spawn many commercially important fish species that exhibit arrested reproductive development (Zohar and Mylonas 2001). It is well known that reproductive processes in fishes are controlled by endogenous biological rhythms as well as by environmental cues (Munro 1990). Endogenous SPAWNING PERFORMANCE OF NATIVE THREATENED SPOTTED SNAKEHEAD FISH, CHANNA PUNCTATUS (ACTINOPTERYGII: CHANNIDAE: PERCIFORMES), INDUCED WITH OVATIDE


INTRODUCTION
The single most important drawback of large-scale commercial culture of several fish species is the deficiency of quality seed of uniform size, and free of diseases, parasites, and pests at the time of stocking in culture ponds. These strict requisites are seldom fulfilled where the seed is obtained from the natural water bodies (Zohar and Mylonas 2001). Furthermore, the broodfish that are obtained from the wild and taken to captivity or reared in captive conditions may receive inappropriate environ-mental cues for reproduction and these can cause reproductive development to be arrested in late vitellogenesis. For this reason, hormonal treatment been attempted for stimulating of gametes maturation and have been successfully used to spawn many commercially important fish species that exhibit arrested reproductive development (Zohar and Mylonas 2001).
It is well known that reproductive processes in fishes are controlled by endogenous biological rhythms as well as by environmental cues (Munro 1990). Endogenous control is mediated through actions of various hormones along the brain-hypothalamus-pituitary-gonad axis. Under natural conditions environmental stimuli are detected and relayed to the brain, resulting in a release of hormones and neurotransmitters that regulate ovulation (Yaron 1995, Peter andYu 1997). The most important reproductive hormone is gonadotropin-releasing hormone (GnRH) that regulates gonadotropic hormone, GtH (Peter and Yu 1997). Gonadotropin release in teleost fishes is also influenced by a gonadotropin-inhibiting factor (GRIF) from the hypothalamus. This factor has been identified as dopamine and demonstrated to have inhibitory activity on the release of GtH (Peter et al. 1988). Several commercially available synthetic ovulating agents in ready made form containing GnRHa and dopamine antagonist like Ovaprim, Ovopel, Dagin, and Aquaspawn are becoming very popular nowadays and found to be efficient and successful spawning agent in different fish species (Peter et al. 1988, Cheah and Lee 2000, Das 2004, Brzuska 2001, 2003, 2006. Ovatide, a new ovulating agent has successfully been tested by the Central Institute of Fisheries Education (ICAR), Mumbai including some other parts of India since 1997. Ovatide, a readily injectable spawning agent inducing gonadotropic hormone, consisting of GnRH analogue and dopamine antagonist, is also found to be efficient in induced spawning (Sahoo et al. 2005, Marimuthu et al. 2000, 2007. Recently Ovatide has been used to induce spawning in stinging catfish, Heteropneustes fossilis (cf. Marimuthu et al. 2000); snakehead murrel, Channa striatus (cf. Marimuthu et al. 2007); and in walking catfish, Clarias batrachus (cf. Sahoo et al. 2005).
The spotted snakehead, Channa punctatus (Bloch, 1793), is locally known as spotted murrel and one among the highly priced freshwater food fish species in India. It is found to be distributed throughout the South East Asian countries and has been identified as a potential candidate species for aquaculture in derelict and swampy water as it is a hardy and an air-breathing fish. The fish is well known for its taste, high protein content and low intramuscular spines, high nutritive value, recuperative and medicinal qualities, and is recommended as a diet during convalescence (Haniffa et al. 2004). Over the last 10 years, its wild population has undergone a steady decline due to overexploitation, loss of habitat, introduction of alien species, disease, pollution, siltation, poisoning, dynamite, and destructive fishing. These factors not only destroyed the feeding and breeding grounds but also caused havoc to the biodiversity of this important fishery. As a result, according to IUCN status (Molur and Walker 1998), it has been listed among the 66 low-risk nearthreatened fish species in India. Information on the induction of spawning and artificial propagation of C. punctatus is limited (Parameshwaran and Murugesan 1976). Therefore, the present study was attempted to investigate the efficacy of a synthetic GnRH, with a dopamine antagonist for the induction of ovulation and the initiation of spawning in C. punctatus, and to determine the minimum effective dose of Ovatide that could be used to spawn and produce seed of the candidate fish species under a controlled captive condition.

MATERIALS AND METHODS
Broodfish, weighing from 50 to 90 g, were maintained in earthen ponds (3 × 3 × 1 m) at the Centre for Aquaculture Research and Extension (CARE), Palayamkottai, Tamilnadu, India. The fish were fed cleaned chicken viscera ad libitum daily for their normal growth and gonadal development. Mature male fish was identified by a slightly pointed genital papilla, and mature females by a swollen abdomen and a reddish, swollen vent (Haniffa et al. 1996). In addition, maturity of the female was confirmed by slightly pressing along the ventral side of the fish for oozing of eggs. A sample of 10-20 eggs from each female was collected by hand stripping and immersed in a solution containing 70% acetic acid and 30% ethanol for clarification of the cytoplasm. About 3 min subsequent to immersion, the position of the oocyte nuclei was determined. Migration of the nucleus from the centre of eggs to its periphery indicates the readiness of fish for breeding by hormonal stimulation. Only those females containing the highest percentage of mature oocytes having germinal vesicle in the centre or initial stage of migration were selected for the hormone treatment (Billard et al. 1984).
In total, 12 matured females and 24 male fish weighing from 50 to 90 g were randomly selected for three hormonal treatment groups and one control group. One day before the experiment, the fish were selected and transferred to 1500-L concrete tanks (3 × 1 × 1 m) filled with de-chlorinated water to the depth of 50 cm. Each breeding set consisted of two males and one female (Haniffa et al. 1996). The selected fish were injected intramuscularly either with saline (0.9% NaCl, control group) or with Ovatide at 0.2, 0.4, or 0.6 mL · kg -1 body weight (BW), respectively. For each dose, three breeding trials were made to find out the differences in response by the fish and to observe the variation in latency period, the rate of fertilization, and percentage of hatching in each treatment groups.
The hormone-treated broodfish were introduced into the cemented breeding tank (3 × 1 × 1 m). Aquatic macrophytes such as Eichhornia crassipes and Hydrilla verticillata were introduced into the breeding tank for performing their breeding activities under hiding condition. After spawning, eggs were collected from the breeding tank, and the number of eggs spawned (spawning fecundity) and rate of fertilization was calculated. Dead eggs were removed from the egg batches by siphoning. Two hours post-spawning, a total of 500 fertilized eggs from each breeding set were collected and incubated in glass aquaria to determine the incubation period and hatching rate. The water quality parameters recorded during the study were as follows, temperature, 29 ± 1°C; dissolved oxygen, 5.8-6.5 mg · L -1 ; and pH 7.5-8.1.
The fertilization and hatching rate were calculated as follows: • Fertilization rate [%] = number of fertilized eggs/total number of eggs counted × 100; • Hatching rate (%) = number of eggs hatched/total number of eggs in a batch × 100.
Statistical analysis. The data obtained for mean number of spawning fecundity, fertilization rate, latency period, and hatching rate from each hormone dose were analyzed using one-way analysis of variance (ANOVA) to find significant difference among the hormone doses and each treatment mean were analysed by Duncan's multiple range tests (P = 0.05) using SPSS package Version.11.

RESULTS
The spawning performances of C. punctatus induced at different Ovatide doses are presented in Table 1. No spawning behaviour and performance was observed in the control groups. The hormone-induced fish showed breeding behaviour 3-4 h after injection irrespective of dosages. Each female paired with a single male. At all times, the more active and aggressive male paired with the female while the other one was found to be passive and idle in the corner of the breeding tank. Mating was preceded by elaborate courtship. Spawning rituals commenced after 8-12 h of the hormone injection and continued until the releasing of gametes. Spawning was noticed within 25-30.5 h after the hormone administration. Minimum latency period was observed for the Ovatide dose of 0.4 mL · kg -1 BW but no significant difference (P > 0.05) was recognized between the low and high hormone doses of 0.2 and 0.6 mL · kg -1 BW.
The spawning fecundity (per g female) in fish that were administered with 0.2, 0.4, and 0.6 mL · kg -1 BW of fish were 20 ± 6, 102 ± 7, and 39 ± 4, respectively. Significantly highest (P < 0.05) spawning fecundity was obtained in fish administered with a dose of 0.4 mL · kg -1 BW, than those with 0.2 mL · kg -1 , and 0.6 mL · kg -1 BW doses of Ovatide, respectively. The highest fertilization rate (90.6 ± 1.52%) and hatching rate (91.33 ± 2.51%) observed in the medium dose of 0.4 mL · kg -1 BW and were significantly higher than the fish injected with 0.2 mL · kg -1 and 0.6 mL · kg -1 BW (Table 1). Hatching rates were not statistically significant between the doses of 0.4 mL · kg -1 and 0.6 mL · kg -1 BW administered groups. The incubation period ranged from 24 to 28 h at the water temperatures of 29 ± 1.5ºC. In the present study, no size differences of larvae were observed, despite the hatching performances varied among the hormone doses tested.

DISCUSSION
In the present study, a single intramuscular injection of synthetic hormone, Ovatide resulted in successful spawning of Channa punctatus. Table 2 summarizes the results of induced spawning and the success rate of different fish species using Ovatide. To the best of our knowledge, this is the first successful attempt to use Ovatide as a stimulating agent for induced spawning of this commercially-important native threatened fish species in a controlled captive condition. Successful spawning using Ovatide and its analogues has also been reported in several fish species viz., stinging catfish, Heteropneustes fossilis (cf. Marimuthu et al. 2000); snakehead murrel,  Channa striatus (cf. Marimuthu et al. 2007); narrowfront tandan, Neosilurus ater (cf. Cheah and Lee 2000); and in several cyprinid species, like common carp, Cyprinus carpio; silver carp, Hypophthalmichthys molitrix; and tench, Tinca tinca (cf. Horváth et al. 1997).
The latency period of C. punctatus ranged from 25 to 31 h at 29 ± 1.5ºC in the three doses tested. The latency period was longer than those reported in H. fossilis administered with Ovatide (Marimuthu et al. 2000) but it was similar to those in C. striatus using Ovaprim (Haniffa et al. 1996) and in C. striatus using Ovatide (Marimuthu et al. 2007). In contrast, short latency periods using Ovatide ranged between 7 and 10.5 h in different carp species have been reported ( Table 2). The latency period is related to water temperature and often decreases with an increase in temperature (Clemens and Sneed 1962).
In the present experiment, complete spawning was observed at the Ovatide doses of 0.4 mL · kg -1 and 0.6 mL · kg -1 BW whereas the dose of 0.2 mL · kg -1 BW induced partial spawning. Complete spawning has been reported using Ovatide in carps (Thakur and Reddy 1997), in pabo catfish, (Mukherjee and Das 2001), and stinging catfish (Marimuthu et al. 2000). The highest hatching rate was observed in the dose of 0.4 mL · kg -1 BW. However, the overall hatching rates of Ovatide-treated fish were high compared to those reported in the same fish species using pituitary extract (Parameshwaran and Murugesan 1976). Similar results were also observed in other air breathing fishes, C. striatus and H. fossilis (cf. Marimuthu et al. 2000Marimuthu et al. , 2007. In general, the response of fish to Ovatide was found to be better, considering the spawning success, number of released eggs, and, percentages of fertilization and hatching. Further, the synthetic hormones like Ovaprim and Ovatide are known to act at the pituitary level leading to the secretion of fish's own endogenous gonadotropins, while in the case of hypophysation technique and administration of HCG exogenous gonadotropins, they are directly delivered into the body (Habibi et al. 1989, Zairin et al. 1992, Goswami and Sharma 1997. Endogenous gonadotropins appear to significantly enhance the secretion of the right type of steroids in abundant quantity enabling complete maturity of ova for spawning. The results of the present investigation demonstrate the possibility of using the synthetic hormone, Ovatide at a dose of 0.4 mL · kg -1 BW for induced spawning and will be appropriate for mass seed production of Channa punctatus. Low and higher doses are reported to affect the egg quality, lead to partial spawning or reduced fertilization and hatching rate. The new ovulating agent, Ovatide is less expensive, easy to store, simple to use, and has lower viscosity compared to Ovaprim and other hormones used for fish breeding in the aquaculture sector. If Ovatide is made locally available at a competitive low price, it could be used as the first alternate to the high-priced Ovaprim for successful breeding of C. punctatus including other commercial fish species by the hatchery operators and seed producers in their aquaculture farms. On the other hand, this study represents the first successful attempt to propagate C. punctatus using synthetic Ovatide as a stimulating agent. Therefore, the findings emerge from the present study would immensely be helpful for quality seed production in snakehead and other threatened freshwater fishes as well as for their conservation and rehabilitation. Further studies are required to examine the development and growth performances of larvae and fry produced by propagation with Ovatide.