esearch VINANIE PUBLISING Department of Biotechnology, Faculty of Applied Sciences, AIMST University Bedong, Kedah Darul Aman, Malaysia 2

Transcription

1 Journal of Environment & B iotechnology esearch VINANIE PUBLISING R Induced ovulation and spawning of African catfish Clarias gariepinus (Bloch) using ovaprim Kasi Marimuthu, 1, * Nirmell Satthiyasilan, 1 M. Aminur Rahman, 2 A. Arshad, 2 M. Gokul Raj, 3 Jesu Arockiaraj 4, * 1 Department of Biotechnology, Faculty of Applied Sciences, AIMST University Bedong, Kedah Darul Aman, Malaysia 2 Laboratory of Marine Biotechnology, Institute of Bioscience, Universiti Putra Malaysia, UPM Serdang, Selangor, Malaysia 3 Department of Civil Engineering, Sree Sastha Institute of Engineering and Technology Chembarambakkam, Chennai , Tamil Nadu, India 4 Division of Fisheries Biotechnology & Molecular Biology, Department of Biotechnology, Faculty of Science and Humanities, SRM University, Kattankulathur , Chennai, Tamil Nadu, India ORIGINAL RESEARCH ARTICLE A BSTRACT Experiments were conducted to study the effects of different doses of ovaprim in stimulating ovulation and spawning response of African catfish, Clarias gariepinus. Nine matured female and 9 male fishes weighing from 1.46 to 2.80 kg were randomly selected for three different doses of hormonal injection. Both male and female fishes from all the three replicate groups (3 females and 3 males per replicate) were administered a single dose of 0.4, 0.5 and 0.6 ml of ovaprim/kg body weight (BW), respectively. After 12 h of post-injection, the females were stripped individually into dry and pre weighed plastic containers to record the stripped egg weight per female. The breeding performance was assessed on the basis of total egg mass and stripping response. To assess the egg quality of hormone induced fishes, the fertilization rate and hatching rates were examined among the three doses administered. Complete ovulation was observed in all the three doses of hormonal injection. The results indicated that the total weight of stripped eggs were significantly highest (P < 0.05) at the individuals received 0.4 ml ovaprim/kg BW compared to those injected with other higher doses. Highest fertilization (97.88%) and hatching (93.66%) were also recorded in the individuals received 0.4 ml ovaprim/kg BW. However, breeding performance and egg quality were not statistically significant among the three hormone doses tested in this study. Hence, it was evidenced that ovaprim, the synthetic gonadotropin-releasing hormone with a dopamine antagonist at a dose of 0.4 ml/kg BW could be used as an appropriate spawning dose for C. gariepinus. KEYWORDS African catfish; Clarias gariepinus; hormonal treatment; induced breeding; seed production 1. INTRODUCTION African catfish, Clarias gariepinus is one among the highly demanded freshwater food fish and cultivar species in Malaysia and other Asian and African countries due to its higher resistance to diseases, ability to tolerate a wide range of environmental parameters and high stocking culture conditions, relatively fast Corresponding authors: K. Marimuthu (KM); J. Arockiaraj (JA) Tel: (KM); (JA) Fax: (KM); (JA) E. mail: aquamuthu2k@yahoo.com (KM); jesuaraj@gmail.com (JA) growth rate and good quality meat (Hogendoorn, 1980; Huisman and Richter, 1987; Haylor, 1991; Goos and Richter, 1996). It is one of the popular native fish species of African countries and has been introduced and commercially cultured in several countries in Europe (Netherlands, Germany and Belgium) and Asian countries (Indonesia, Thailand and Malaysia) and South America (Brazil). C. gariepinus inhabits Recieved: Revised: Accepted: Available online: Journal of Environment and Biotechnology Research, Vol. 1, No: 1, Pages 2-9,

2 a wide range of water bodies like swamps, lakes and rivers. It is a hardy fish and is able to thrive in harsh environmental conditions such as muddy, turbid and oxygen depleted water bodies with the help of accessory air-breathing organ which allows them to breathe oxygen from the atmospheric air. Generally, C. gariepinus is omnivorous in nature and usually feeds on insects, plankton, snails and plant matters in the natural water bodies (Bruton, 1979; Uys, 1989). However, this species is highly cannibalistic when substantial differences occur in size (Baras and Jobling, 2002). African catfish breeds naturally during the rainy season in flooded rivers, inundated paddy fields and earthen ponds. The seed collection of this species from the wild is unreliable, time consuming and uneconomical for large-scale culture of this fish. To overcome these problems, induced spawning is thought to be the only alternative method for seed production and supply. Further, the rearing of the wild collected broodstock in captive conditions may not receive appropriate environmental cues for gonad maturation and spawning and it can cause reproductive development to be arrested in late vitellogenesis stage. Hence, matured females are induced to spawn by hormonal manipulation (Zohar and Mylonas, 2001). Over the last few decades, hormonal administration techniques have been used to induce final oocyte maturation and spawning in fishes which allowed the reproduction in controlled conditions (Marimuthu et al., 2007 & 2009; Marimuthu and Haniffa 2010). Moreover, induced breeding techniques have significantly contributed a lot to the expansion and diversification of the aquaculture industry (Zohar and Mylonas, 2001). The injection of different spawning agents in fish is adopted for successful ovulation and collection of eggs. Traditional methods of induced spawning in fish are based on the injection of GtH-II from different sources, including extract of carp pituitary gland, partially purified fish GtH-II and mammalian GtH, especially human chorionic gonadotropin (HCG) (Lam, 1982; Donaldson and Hunter, 1983; Peter et al., 1988; Zairin et al., 1992; Goswami and Sharma, 1997). The GnRHa and domperidone are the most popular compounds for induction of ovulation and spermiation in various fish species. The introduction of GnRH analogues has been proven to be efficient in inducing maturation and spawning in many fish species (Tamaru et al., 1988; Thomas and Boyde, 1988; Zohar, 1988; Slater et al., 1995; Berlinsky et al., 1996; Larsson et al., 1997; Mylonas et al., 1997 & 1998). Similarly, an antidopaminergic drug, pimozide has also been found to be highly effective for stimulating the spawning process of fishes mainly in cyprinids and catfishes (Billard et al., 1984; Tan-Fermin, 1997). These hormones are used to stimulate the secretion of endogenous gonadotropin (Zohar, 1989 & 2001). Lin and Peter (1996) reported that among the several inducing agents used in fish breeding, salmon gonadotropin releasing hormone (sgnrh) or luteinising hormone releasing hormone (LHRH) analogues in combination with dopamine antagonists were identified as effective agent in fish breeding and seed production. Several practical problems have been reported using exogenous hormones and endogenous hormones, such as weighing of such low quantity, preparation of these analogues and storage of this hormone in prepared solutions. Due to these difficulties, fish breeders and farmers are unwilling to use these hormonal preparations under farm conditions. Commercially available synthetic ovulating agents in ready-made form which contained GnRHa and dopamine antagonist such as ovaprim, ovatide, ovopel, dagin and aquaspawn are becoming very popular nowadays and found to be efficient and successful spawning agents in different fish species (Nandeesha et al., 1990; Peter et al., 1988; Brzuska, 1998; Cheah and Lee, 2000; Das, 2004). Recently, successful spawning through a synthetic analogue of GnRH has been reported in several air breathing fish species including Clarias batrachus (Basu et al., 2000; Sahoo et al., 2008), Heteropneustes fossilis (Alok et al., 1993 and 1994; Marimuthu et al., 2000), murrel (Haniffa et al., 2003 & 2004; Marimuthu et al., 2001a & 2001b, Marimuthu et al., 2007 & 2009; Marimuthu and Haniffa 2010). However, the information on the induction of spawning and artificial reproduction using synthetic hormones in C. gariepinus is limited. Therefore, the present study was conducted to investigate the efficacy of a synthetic GnRH, with a dopamine antagonist (ovaprim) for the induction of ovulation and spawning performance in C. gariepinus; also to determine the minimum effective dose of ovaprim for induced spawning and seed production of the candidate fish species under a controlled condition. 2. MATERIALS AND METHODS 2.1. Broodstock collection, maintenance and selection Brood fishes (live body weight ranged between 1.5 and 3 kg) were obtained from a local fish farm at Sungai Petani, Kedah Darul Aman, Malaysia. The collected 3

3 K. Marimuthu, Journal of Environment and Biotechnology Research, Vol. 1, No.1, Pages 2-9, 2015 fishes were transported to the Aquaculture Research Laboratory, AIMST University, Kedah Darul Aman, Malaysia and maintained in circular cement tanks. They were regularly fed with a commercial pellet feed containing 30% crude protein at 5% of their estimated body weight for two weeks. Nine matured females and 9 males were randomly selected and divided into three treatment groups, each group comprised of three males and three females. The matured male and female fishes were identified based on their external morphological features. Matured male fish was identified by a prominent slightly pointed genital papilla, and mature Figure 1. Identification of female (left side) and male fish (right side) Figure 2. Intramuscular injection of hormone ovaprim Figure 3. Collection of eggs Figure 4. Egg mass of catfish Figure 5. Macerate testis and collection of sperm Figure 6. Mixing sperm suspension and eggs for fertilization 4

4 females by a swollen abdomen and a reddish round vent (Figure 1). In addition, maturity of the female was confirmed by gentle pressing on the ventral side of the fish for oozing eggs. The eggs were collected by handstripping, and immersed in a solution consists of 70% acetic acid and 30% absolute alcohol for clarification of the cytoplasm. After about three minutes, the position of the oocyte nuclei was determined under microscope. Migration of the nucleus from the center of eggs to the periphery region indicates the readiness of fish for breeding and is the best moment for hormonal stimulation as observed in other fish species. Only those females showing the highest percentage of mature oocytes having germinal vesicle in the center or initial stage of migration were selected for the hormonal treatment (Billard et al., 1984) Hormone administration, gamete collection, fertilization and hatching The selected fishes were randomly assigned to three treatment groups and were injected intramuscularly with 0.4, 0.5 or 0.6 ml ovaprim/kg BW (Figure 2). For each dose, three breeding trials were made to find out the spawning response of the fish and to observe the variation in incubation period, rate of fertilization and percentage of hatching. The hormone-administered males and females were released separately into the circular cement tanks. After 12 h, the females were stripped individually and eggs were collected into dry and pre-weighed plastic containers to record the weight of stripped eggs (Figures 3 and 4). The testes were removed from male fishes, incised and squeezed to get concentrated sperm (Figure 5). The sperms were diluted with 5 ml of physiological saline to prepare a sperm suspension. The eggs were thoroughly mixed with sperm suspension and left for 5 minutes (Figure 6). After 3-4 consecutive washing with water, they were transferred into glass aquaria (40 L) provided with continuous aeration. The breeding performance was assessed on the basis of total egg mass and stripping response. The water quality parameters recorded during the study were as follows: temperature, C; dissolved oxygen, mg/l and ph The percentage of fertilization and hatching rates were calculated as follows: Fertilization (%) = number of fertilized eggs/total number of eggs counted x 100 (1) Hatching (%) = number of eggs hatched/ total number of eggs in a batch x 100 (2) Data analysis The data for total egg mass, fertilization rate, incubation period and hatching rate were analyzed using one way analysis of variance (ANOVA) followed by Duncan s New Multiple Range Tests at the significant level, P = All the statistical analyses were performed using the computerized statistical package, SPSS (ver. 13.0). 3. RESULTS AND DISCUSSION Complete ovulation and spermiation was observed in all the three hormone dosages tested in the study. No significant size variations of the male and female fishes were used for breeding among the three hormonal treatments. The results obtained from the induced spawning experiments are summarized in Table 1. The results indicated that the total weight of stripped eggs were significantly highest (P < 0.05) in females received 0.4 ml hormonal dose than those females administered with higher doses. Significantly highest percentages (P < 0.05) of fertilization rate (97.88%) and hatching rate (93.66%) were also recorded in the females received 0.4 ml ovaprim than those received higher doses. However, both fertilization rate and hatching rates were not statistically significant among the females received 0.5 and 0.6 ml/kg BW. Significantly longer (P < 0.05) incubation period was observed in the individuals administered with 0.4 ml ovaprim than those administered with 0.5 and 0.6 ml hormone, the latter two doses did not statistically significant among them. In this study, a single intramuscular injection of synthetic hormone, ovaprim resulted in successful spawning of C. gariepinus. Successful spawning using ovaprim and its analogues have also been reported in several fish species viz., carps (Nandeesha et al., 1990), stinging catfish H. fossilis (Vijayakumar et al., 1998), Ompok bimaculatus (Sridhar et al., 1998), murrel (Haniffa et al., 2003 and 2004; Francis et al., 2000), catfish Neosilurus ater (Cheah and Lee, 2000) and C. batrachus (Sahoo et al., 2008). Table 2 summarizes the results of induced spawning and success rate of different fish species using ovaprim. It has been reported that the ability to induce oocyte maturation and ovulation depend on the dose, species and maturation stage of the oocytes (Donaldson and Hunter, 1983; Yaron, 1995; Peter et al., 1988). In the present study, significantly highest (P < 0.05) amount of egg mass was found in C. gariepinus when injected with 0.4 ml/kg BW of ovaprim. Further,

5 Table 1. Induced spawning of African catfish, C. gariepinus using synthetic hormone ovaprim Hormone dose ml/kg BW Female fish weight (kg) Male fish weight (kg) Total egg mass (g) Fertilization rate (%) Incubation period (h) Hatching (%) ± 0.73 a 2.53 ± 0.46 a 300 ± 90.0 b ± 0.38 a 27.6 ± 1.2 a ± 2.96 a ± 0.15 a 2.80 ± 0.10 a 167 ± 61.0 a ± 5.59 b 23.5 ± 1.0 b ± 7.47 b ± 0.46 a 2.46 ± 0.15 a 163 ± 60.0 a ± 3.67 a 24.0 ± 0.9 b ± 2.51 b Mean values in the each column with the different superscript letter are significantly different (P < 0.05) Table 2. Summary of induced breeding in different fish species using ovaprim Species Ovaprim (ml/ kg bw) Latency period (h) Spawning success complete spawning was also observed in all the three doses tested. Complete spawning has also been reported using ovaprim in carps (Nandeesha et al., 1990), Puntius japonicus (Azad and Shimray, 1991), N. ater (Cheah and Lee, 2000), C. batrachus (Basu et al., 2000) and using ovatide in H. fossilis (Marimuthu et al., 2000) and Ompok pabo (Mukherjee and Das, 2001). No significant differences were noticed in the hatching rates between the doses, 0.5 and 0.6 ml/kg BW tested. This observation suggests that inducing ovulation with ovaprim at higher doses did not have any adverse effect on egg viability. However, fish responds differently, when induced with GnRH or their analogue(s) in terms of elevation in the gonadotropin levels (Peter et al., 1985; Breton et al., 1990), steroid 6 Fertilization rate (%) Hatching rate (%) References Acanthopagrus latus Leu and Chou (1996) Aristichthys nobilis Complete Afzal et al. (2008) Catla catla Complete Nandeesha et al. (1990) Channa striatus Francis et al. (2000) Channa punctatus Haniffa et al. (2003) Cirrhinus mrigala Complete Nandeesha et al. (1990) Clarias batrachus Complete Mohapatra et al. (2000) Clarias batrachus Complete Basu et al. (2000) Clarias batrachus Complete - - Sahoo et al. (2008) Ctenopharyngodon idella Complete Nandeesha et al. (1990) Heteropneustes fossilis Complete - - Alok et al. (1993) Heteropneustes fossilis Complete Francis (1996) Heteropneustes fossilis Vijayakumar et al. (1998) Hypopthalmichthys molitrix Peter et al. (1988) Hypopthalmichthys molitrix Complete 53 - Nandeesha et al. (1990) Labeo rohita Complete Nandeesha et al. (1990) Neosilurus ater Complete Cheah and Lee (2000) Ompok bimaculatus Sridhar et al. (1998) Puntius japonicus Complete Azad and Shimray (1991) Tor puttitora Pandey et al. (1998) Channa striatus complete Marimuthu et al. (2001b) - not mentioned levels (Weil et al., 1980; Van Der Kraak et al., 1987), ovulation (Ramos, 1986; Peter et al., 1987; Fermin, 1991), latency period (De Leeuw et al., 1985; Glubokov et al., 1986; Kaul and Rishi, 1986) and spawning (Lee et al., 1986; Nandeesha et al., 1990). The variations in response can be attributed to the fish species, amino acid sequence and purity of the GnRH analogue (Peter et al., 1985 & 1987), dose of the GnRH and based on weak or strong dopaminergic inhibitory mechanism (Fermin, 1991). In general, the response of fish to ovaprim was found to be better, considering the spawning success, egg mass, percentages of fertilization and hatching. Further, the synthetic hormones like ovatide, ovapel and ovaprim are known to act at the pituitary level

6 leading to the secretion of the fish s own endogenous gonadotropins, while in the case of hypophysation technique and administration of HCG, exogenous gonadotropins 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 appropriate quantities, enabling complete maturation of ova. 4. CONCLUSIONS From the present findings, it is evidenced that the synthetic gonadotropin-releasing hormone with a dopamine antagonist at the low dose of 0.4 ml/kg BW could be used as an appropriate spawning agent for successful breeding and seed production of C. gariepinus. The results emerged from the present study would immensely be helpful for quality seed production in African catfish. Further studies are required to examine the development and growth performances of larvae and fry produced by the induced breeding techniques. REFERENCES K. Marimuthu, Journal of Environment and Biotechnology Research, Vol. 1, No.1, Pages 2-9, 2015 Afzal, M., Rab, A., Akhtar, N., Khan, M.F., Khan, S.U. and Qayyum, M. (2008) Induced spawning of bighead carp, Aristichthys nobilis (Richardson), by using different hormones/hormonal analogues. Pakistan Journal of Zoology, 40, Alok, D., Krishnan, T., Salunke, D., Gupta, H., Talwar, G.P. and Garg, L.C. (1994) Precocious ovarian recrudescence and induced off season spawning of catfish Heteropneustes fossilis (Bloch) by D-Lys6 salmon gonadotropin releasing hormone analog (SGnRHa). Journal of Fish Biology, 45, Alok, D., Krishnan, T., Talwar, G.P. and Garg, L.C. (1993) Induced spawning of catfish Heteropneustes fossilis (Bloch) using D-Lys6 salmon gonadotropin releasing hormone analog. Aquaculture, 115, Azad, I.S. and Shimray, D.K. (1991) First success in induced breeding of Indian and exotic carps in Manipur using ovaprim-c. Fishing Chimes, 10, Baras, E. and Jobling, M. (2002) Dynamics of intracohort cannibalism in cultured fish. Aquaculture Research, 33, Basu, D., Rana, G.C., Mondal, B.K., Sengupta, K.K. and Dhar, P.K. (2000) Studies on the comparative efficiency of ovaprim, HCG and piscine pituitary gland in induced breeding of Clarias batrachus (Linn). Fishing Chimes, 19, Berlinsky, D.L., King, V.W., Smith, T.I.J., Hamilton, R.D. and Holloway, S.C.R. (1996) Induced ovulation of southern flounder Paralichthys lethostigma using gonadotropin releasing hormone analogue implants. Journal of World Aquaculture Society, 27, Billard, R., Reinaud, P., Hollebecq, M.G. and Breton, B. (1984) Advancement and synchronization of spawning in Salmo gairdneri and Salmo trutta following administration of LHRHacombined or not with pimozide. Aquaculture, 43, Breton, B., Weil, C., Sambroni, E. and Zohar, Y. (1990) Effect of acute versus sustained administration of GnRHa on GtH release and ovulation in the rainbow trout, Oncorhynchus mykiss. Aquaculture, 91, Bruton, M.N. (1979) The food and feeding behaviour of Clarias gariepinus (Pisces; Clariidae) in Lake Sibaya, South Africa, with emphasis on its role as a predator of cichlids. Transactions of the Zoological Society of London, 35, Brzuska, E. (1998) Ovopel as ovulation stimulator in African cat fish (Clarias gariepinus Burchell 1822) females. Scientific Papers of Polish Academy of Science. Selected Problems, 5, Cheah, M.S.H. and Lee, C.L. (2000) Induced ovulation of the Australian eel tailed catfish Neosilurus ater (Perugia) with ovaprim. Asian Fisheries Science, 13, Das, S.K. (2004) Evaluation of a new spawning agent, ovopel in induced breeding of Indian carps. Asian Fisheries Science, 17, De Leeuw, R., Goose, H.J.T., Richter, C.J.J. and Eding, E.H. (1985) Pimozide- LHRH-induced breeding of the African catfish, Clarias gariepinus (Burchell). Aquaculture, 44, Donaldson, E.M. and Hunter G.A. (1983) Induced final maturation, ovulation and spermiation in cultured fish. In: Hoar, W.S., Randall, D.J. and Donaldson, E.M. (Eds.), Fish Physiology. Vol. IX, Part B: Reproduction. Academic Press: Orlando, FL, pp Fermin, C.A. (1991) LHRHa and domperiodone-induced oocyte maturation and ovulation in bighead carp, Aristichthys nobilis (Richardson). Aquaculture, 93, Francis, T. (1996) Studies on the effect of pituitary hormones and feeds on the reproduction of Heteropneustes fossilis (Bloch). Ph.D Thesis, Tamilnadu Veterinary and Animal Sciences University, Madras, India. Francis, T., Ramanathan, N., Athithan, S. and Cheryl, H.F. (2000) Induced breeding of murrel, Channa striatus using various inducing agents. Fishing Chimes, 19, Glubokov, A.I., Motloch, N.N. and Sedova, M.A. (1991) Effect of a synthetic LHRH analogue and dopamine antagonists on the maturation of bream, Abramis brama L. Aquaculture, 95, Goos, H.J.T. and Richter, C.J.J. (1996) Internal and external factors controlling reproduction in the African catfish, Clarias gariepinus. In: Legendre, M., Proteau, J. P. (Eds.), The Biology and Culture of Catfishes. Aquatic Living Resources, 9, Goswami, U.G. and Sharma, N.N. (1997) Pituitary dose optimization for induced ovulation, in vitro fertilization and production of normal fry of Clarias batrachus (Linn). Asian Fisheries Science, 10, Habibi, H.R., Marchant, T.A., Nahorniak, C.S., Van Deroo, H., Peter, R.E., Rivier, J.E. and Vale, W.W. (1989) Functional relationship between receptor binding and biological activity for analogs of mammalian and salmon gonadotropin-releasing hormones in the pituitary of goldfish (Carassius auratus). Biology of Reproduction, 40, Haniffa, M.A., Marimuthu, K., Nagarajan, M., Arockiaraj, J. and Kumar, D. (2004) Breeding behaviour and parental care of the induced bred spotted murrel Channa punctatus under captivity. Current Science, 86, Haniffa, M.A., Nagarajan, M., Marimuthu, K. and Arockiaraj, J. (2003) Embryonic and larval development of spotted murrel Channa punctatus (Bloch). Indian Journal of Fisheries, 50, Haylor, G.S. (1991). Controlled hatchery production of Clarias gariepinus (Burchell 1882): growth and survival of fry at high stocking density. Aquaculture and Fisheries Management, 22,

7 Hogendoorn, H. (1980). Controlled propagation of the African catfish, Clarias lazera (CandV), 111. Feeding and growth of fry. Aquaculture, 21, Huisman, E.A. and Richter, C.J.J. (1987) Reproduction, growth, health control and aquaculture potential of the African catfish Clarias gariepinus (Burchell, 1822). Aquaculture, 63, Kaul, M. and Rishi, K.K. (1986) Induced spawning of the Indian major carp, Cirrhina mrigala (Ham.), with LH-RH analogue or pimozide. Aquaculture, 54, Lam, T.J. (1982) Application of endocrinology to fish culture. Canadian Journal of Fisheries and Aquatic Sciences, 39, Larsson, D.G.J., Mylonas, C.C., Zohar, Y. and Crim, L.W. (1997) Gonadotropin releasing hormone analogue (GnRH-A) induces multiple ovulations of high-quality eggs in a cold-water, batch-spawning teleost, the yellowtail flounder (Pleuronectes ferrugineus). Canadian Journal of Fisheries and Aquatic Sciences, 54, Lee, C.S., Tamaru, C.S., Kelley, C.D. and Banno, J.E. (1986). Induced spawning of milkfish, Chanos chanos by a single injection of LHRH analogue. Aquaculture, 58, Leu, M.Y. and Chou, Y.H. (1996) Induced spawning and larval rearing of captive yellowfish porgy, Acanthopagrus latus (Houttunyn). Aquaculture, 143, Lin, H.R. and Peter, R.E. (1996) Hormones and spawning in fish. Asian Fisheries Science, 9, Marimuthu, K. and Haniffa, M.A. (2010) Induced spawning of native threatened spotted snakehead fish, Channa punctatus with Ovaprim. Asian Fisheries Science, 23, Marimuthu, K., Haniffa, M. A. and Rahman, M.A. (2009) Spawning performance of native threatened spotted snakehead fish, Channa punctatus induced with ovatide. Acta Ichthyologica Piscatoria, 39, 1 5. Marimuthu, K., Haniffa, M.A., Arockiaraj, A.J. and Muruganandam, M. (2001a) Spawning and parental behaviour in the induced bred murrels. Indian Journal of Fisheries, 48, Marimuthu, K., Haniffa, M.A., Muruganandam, M. and Arockiaraj A.J. (2001b) Low cost murrel seed production technique for fish farmers. Naga (ICLARM), 24, Marimuthu, K., Kumar D. and Haniffa. M. A. (2007) Induced spawning and seed production of striped snakehead Channa striatus using ovatide. Journal of Applied Aquaculture, 19, Marimuthu, K., Muruganandam, M. and Haniffa, M.A. (2000) Induced spawning of the Indian catfish Heteropneustes fossilis (Singhi) using a synthetic hormone, ovatide. Fishing Chimes, 19, Mohapatra, B.K., Sengupta, K.K., De, U.K., Rana, G.C. Datta, A., Basu, A. and Saha, S. (2000) Controlled breeding and larval rearing of Clarias batrachus (Linn) for mass scale propagation. Fishing Chimes, 19, Mukherjee, M. and Das, S. (2001) Artificial propagation of a silurid fish pabda Ompok pabo (Hamilton). Fishing Chimes, 21, Mylonas, C.C., Gissis, A., Magnus, Y. and Zohar, Y. (1997) Hormonal changes in male white bass (Morone chrysops) and evaluation of milt quality after treatment with a sustained-release GnRH delivery system. Aquaculture, 153, Mylonas, C.C., Woods, L.C., Thomas, P. and Zohar, Y. (1998) Endocrine profiles of female striped bass (Morone saxatilis) in captivity, during postvitellogenesis and induction of final oocyte maturation via controlled-release GnRH-delivery systems. General and Comparative Endocrinology, 110, Nandeesha, M.C., Rao, K.G., Jayanna, R., Parker, N.C., Varghese, T.J. and Keshavanath, P. (1990) Induced spawning of Indian major carps through single application of ovaprim. In: Hirano, R., Hanyu, M. (Eds.), The Second Asian Fisheries Forum, Asian Fisheries Society, Manila, Philippines, pp Pandey, A.K., Patiyal, R.S., Upadhyay, J.C., Tyagi, M. and Mahanta, P.C. (1998) Induced spawning of the endangered golden mahseer, Tor putitora, with ovaprim at the state fish farm near Dehradun. Indian Journal of Fisheries, 45, Peter, R.E., Lin, H.R. and Van Der Kraak G. (1988) Induced ovulation and spawning in cultured fresh water fish in China: advanced in application of GnRH analogue and dopamine antagonists. Aquaculture, 74, Peter, R.E., Nahorniak, C.S., Sokolowska, M., Chang, J.P., Rivier, J.E., Vale, W.W., King, J.A. and Millar, R.P. (1985) Structure activity relationship of mammalian, chicken and salmon gonadotropin releasing hormone in vivo in goldfish. General and Comparative Endocrinology, 58, Peter, R.E., Sokolowska, M., Nahorniak, C.S., Rivier, J.E. and Vale, W.W. (1987) Comparison of [D-Arg6 Trp7 Leu8 Pro9] net sgnrh-a, and [DAla6, Pro9 net.] LHRH-A, in combination with pimozide in stimulating gonadotropin release and ovulation in the goldfish, Carassius auratus. Canadian Journal of Zoology, 65, Ramos, J. (1986) Luteinizing hormone releasing hormone analogue (LH-RHa) induced precocious ovulation in common sole (Solea solea L.). Aquaculture, 54, Sahoo, S.K., Giri, S.S. and Chandra S. (2008) Induced spawning of Clarias batrachus (Linn.): Effect of ovaprim doses and latency periods on the weight of stripped eggs and ovary. Asian Fisheries Science, 21, Slater, C.H., Schreck, C.B. and Amend, D.F. (1995) GnRHa injection accelerates final maturation and ovulation/spermiation of sockeye salmon (Oncorhynchus nekra) in both fresh and salt water. Aquaculture, 130, Sridhar, S., Vijayakumar, C. and Haniffa, M.A. (1998) Induced spawning and establishment of a captive population for an endangered fish, Ompok bimaculatus in India. Current Science, 75, Tamaru, C.S., Lee, C.S., Kelley, C.D., Banno, J.E., Ha, P.Y., Aida, K. and Hanyu, I. (1988) Characterizing the stage of maturity most receptive to an acute LHRH-analogue therapy for inducing milk fish (Chanos chanos) to spawn. Aquaculture, 74, Tan-Fermin, J.D., Pagador, R.R. and Chavez, R.C. (1997) LHRHa and pimozide- induced spawning of Asian catfish Clarias macrocephalus (Gunther) at different times during an annual reproductive cycle. Aquaculture, 148, Thomas, P. and Boyde, M. (1988) Induced spawning of spotted sea trout, red drum and orangemouth corvine (family: Scianidae) with luteinizing hormone-releasing hormone analog injection. Contributions in Marine Science, 30, Uys, W. (1989) Aspects of the nutritional physiology and dietary requircmcnts of juvenile and adult sharptooth catfish, Clurias gariepinus (Pisces: Clariidae). PhD Thesis, Rhodes University, South Africa. Van Der Kraak, G., Donaldson, E.M., Dye, H.M., Hunter, G.A., Rivier, J.E. and Vale, W.W. (1987) Effects of mammalian and salmon gonadotropin releasing hormone and analogues on plasma gonadotropin levels and ovulation in coho salmon Oncorhynchus kisutch. Canadian Journal of Fisheries and Aquatic Sciences, 44,

8 Vijayakumar, C., Sridhar, S. and Haniffa, M.A. (1998) Low cost breeding and hatching techniques of the catfish (Heteropneustes fossilis) for small-scale farmers. Naga (ICLARM) 21, Weil, C., Fostier, A., Horvath, L., Marlot, S. and Berscenyi, M. (1980) Profiles of plasma gonadotropin and 17 beta-estradiol in the common carp, Cyprinus carpio L., as related to spawning induced by hypophysation or LH-RH treatment. Reproduction, Nutrition, and Development, 20, Yaron, Z. (1995). Endocrine control of gametogenesis and spawning induction in carp. Aquaculture, 129, Zairin, M., Furukawa, K. and Aida, K. (1992) Induction of ovulation by HCG injection in the tropical walking catfish Clarias batrachus reared under C. Bulletin of the Japanese Society of Scientific Fisheries, 59, Zohar, Y. (1988) Gonadotropin releasing hormone in spawning induction in teleosts: basic and applied considerations. In: Zohar, Y. and Breton, B. (Eds.), Reproduction in fish: basic and applied aspects in endocrinology and genetics, INRA Press, Paris, pp Zohar, Y. (1989) Fish reproduction, its physiology and artificial manipulation. In: Shilo, M.C. and Sargi, S.H. (Eds.), Fish culture in warm water systems, problems and trends, CRC Press, pp Zohar, Y. and Mylonas, C.C. (2001) Endocrine manipulation of spawning induction in cultured fish from hormone to gene. Aquaculture, 197,