Efficacy of Different Hormones on Induced Spawning of Channa gachua

Transcription

1 Chapter III Efficacy of Different Hormones on Induced Spawning of Channa gachua Introduction Reproduction in fishes is regulated by external environmental factors that trigger internal mechanisms into action. The final event of the reproductive cycle, the release of eggs (ovulation) and sperm (spermeation) known as spawning, can be induced by either placing the fish in an appropriate environment or by changing the fish s internal regulating factors by hormones or other substances. The single most important drawback of large-scale commercial culture of several fish species is the non - availability of quality seed and disease free of uniform size. These strict requisites are seldom fulfilled where the seed is obtained from the natural water bodies (Zohar and Mylonas, 2001). Furthermore, the brood fish 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 arrest reproductive development at later stage of vitellogenesis. For this reason, hormonal treatment been attempted for stimulation of gamete 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 action of various natural and synthetic hormones along the brain-hypothalamus-pituitary-gonad axis. Under natural conditions 48

2 environmental stimuli are detected and relayed to the brain, resulting in release of hormones and neurotransmitters that regulate ovulation (Yaron 1995, Peter and Yu, 1997). The most important reproductive hormone is gonadotropin-releasing hormone (GnRH) that regulates gonadotropic hormone, GtH (Peter and Yu, 1997). Gonadotropin released 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). Failure of female broodfish to undergo final oocyte maturation (FOM), ovulation and spawning in captivity is a common reproductive dysfunction of cultured fishes (Zohar, 1989). This is due to the absence of the appropriate spawning environment, as well as the stress imposed under captivity. More serious is the failure of cultured females to complete oogenesis, as observed in the greater amberjack (Marino et al., 1995; Micale et al., 1999) and wreck fish Polyprion americanus (Suquet et al., 2001; Papandroulakis et al., 2002), and is exemplified by the complete absence of gametogenesis in the freshwater eel Anguilla sp. (Miura et al., 1991 Ohta et al., 1997). Manipulation of reproductive processes and induction of final oocyte maturation (FOM) and ovulation can be achieved using a variety of hormonal treatments (Zohar and Mylonas, 2001). Induced spawning involves induction of final oocyte maturation i.e. germinal vesicle breakdown and ovulation. One of the main reasons for lack of ovulation and spawning in a number of cultured fishes is failure of the pituitary release gonadotropin (GtH - II), one of the hormones involved in the regulation of reproduction (Lin and Peter, 1996). The major concern of any seed production and hatchery system is to produce the maximum number of quality fingerlings from the available brood stock for aquaculture. This is particularly 49

3 important in the farming of catfish species because their wild seed collection is very much limited due to the absence of seasonal monsoons in India (Vijayakumar et al., 1998). Information on early embryonic and larval development and organogeny is of critical importance in understanding the basic biology of a particular species and their dietary needs and environmental preferences (Koumoundouros et al., 2001, Borcato et al., 2004). Further, studies on embryonic and early larval development are imperative and consequential to the successful rearing of larvae for large scale seed production in aquaculture (Khan and Mollah, 1998, Rahman et al., 2004). Murrels breed naturally during southwest moonsoon and northeast moonsoon in flooded rivers and ponds in India. Since monsoon failure often limits their seed production, Parameswaran and Murugesan (1976) attempted induced breeding by carp pituitary glands. Hypophysation is a simple practical technique but suffers from the disadvantage that often gonadotropic potency of pituitary glands used is unknown and difficult to standardise. Hence alternative sources viz. human chorionic gonadotropin (HCG) (Mollah and Tan, 1983; Zairin et al., 1992; Inyang and Hettiarachchi, 1994) luteinizing hormone releasing hormone analogue (LH-RHa) (Billard et al., 1984; De Leeuw et al., 1985; Fermin, 1992) and Ovaprim (Alok et al., 1993; Francis, 1996; Haniffa et al., 1996) have been attempted in air-breathing fishes. Hormones for induced spawning Hormone-induced spawning techniques influence this sequential mechanism at several levels, by either promoting or inhibiting the process. The primary substances used for hormone-induced spawning are; Human Chorionic Gonadotrophin (HCG) - Infar India Ltd. Calcutta. 50

4 Ovaprim - Syndel Laboratoreis, Glaxo Ltd. Canada Lutenizing Hormone Releasing Hormone analogue (LHRHa) - Sigma Aldrich, Bangalore. The appropriate hormone preparation should be selected on the basis of the species to be spawned and the availability of the hormones. Many factors which have impact on ability of induced spawning, include: 1) Condition of the fish, 2) Stage of sexual maturity, 3) Size of the fish, 4) Previous spawning history, 5) Water temperature, 6) Season of the year and 7) Dosage of hormone. Human Chorionic Gonadotropin Hormone (HCG) Treatment HCG is measured not by weight but biological activity called International Units (IU). It is usually available in sterile vials containing 5,000 or 10,000 IU. The unopened vial of hormone should be stored in a refrigerator at 35-45ºF (2-7 C). HCG is mixed with bacteriostatic water, usually supplied with the hormone. The hormone solution should either be used immediately or divided into small volumes and kept in a freezer; otherwise potency maybe reduced. HCG can be obtained from the urine of the pregnant woman. Hence it is very cheap and easily available. HCG has been used successfully to induce ovulation and spawning in a number of fishes (Zairin et al., 1992, and Basu et al., 2000). Eyo (1997) reported crude HCG collected from (9 th to 14 th day) urine of early pregnant women for induced spawning in C.gariepinus. It was highly effective in vivo induction for spawning with a dose of 5 mg HCG / 100g. Oocyte maturation and ovulation were induced by HCG in Heteropneustes longifilis and 100% ovulation was obtained after a single intramuscular injection (Legendre, 1986). 51

5 Luteinizing Hormone - Releasing Hormones analogue (LH-RHa) Injection of native form of luteinizing hormone releasing hormone is not very effective in elevating the plasma concentration of gonadotropin (Crim et al., 1988), it is the changes in the amino acids that make LHRHa more resistant to degradation by tissue enzymes and the binding to the pituitary better than the natural form of LHRH. Over 2000 analogues have been synthesized (Struthers et al., 1985) and some of these have been tested for induced spawning of various fishes. Being a non - peptide, LHRH alalogues are received great attention as a potential agent for controlled reproduction in fishes. In majority of studies LHRH and its analogues have been used for induction of egg and sperm production at the final stage of gamete maturation (Crim et al., 1983), Billard et al., 1983). Crim et al., (1983) reported that intraperitoneal implantation of pellets containing LRHAa that give a long-term release of a superactive analogue caused a prolonged increase in plasma GtH levels, resulting in acceleration of the time of ovulation of landlocked Atlantic salmon (Salmo salar) by several weeds. The combination of PIM and LHRHa was very effective in inducing ovulation in goldfish (Change and Peter, 1983), Asian catfish (Manickam and Joy, 1989), African catfish (De Leeuw et al., 1985) and Indian major carps (Sahu and Biswal, 1988). Some recent studies on induced ovulation by LRHAa and LRHAa + PIM are summarized in Table. Ovaprim This product contains an analogue of salmon gonadotropin releasing hormone (sgnrh) and domperidone, an effective dopamine antagonist (Harvey and Carolsfeld, 1993). sgnrh analogue (Glu His Trp Ser Tyr D Arg Trp Leu Pro NH CH2 CH3] has amino acid change in the sixth and tenth position of the native form [Glu His 52

6 Trp Ser Tty Gly Trp Leu Pro Gly NH 2 ]. Ovaprim acts on pituitary leading to secretion of endogenous gonadotropin. Endogenous gonadotropin appears to significantly enhance the secretion of the right type of steroids in abundant quantity enabling complete maturity of ova for spawning. sgnrha proved to be more potent than LHRHa and domperidone was found to be more potent than pimozide in common carp (Lin et al., 1988). An additional advantage of dompridone is that it does not cross the blood brain barrier in fish (Omeljaniuk et al., 1987), there by reducing to probability of detrimental side effects in treated 53

7 Materials and Methods Experimental Fish Brood fishes of C. gachua were collected from river Godavari, Tamirabarani and Brahmaputra during April 2008 February 2009 and transported to CARE Aquafarm. The brood fishes were stocked in the stocking pond and after one month they were reared in cement tanks (area 6.8x4.4ft, average depth 3ft). The length ranged from 10.6 to 21.1 cm with a weight range of 35 65g (Plate 3a - c&d). The brood fishes were maintained in a monoculture system and fed with small live tadpoles, liver, rice bran and oil cake (2:1) at 4% of the body weight upto 4 months. After 4 months of rearing the fishes were found mature enough for captive breeding. The induced breeding experiments were conducted between July and December Mature healthy males and females were selected by external morphological characteristics. We have observed that in maximum cases females are bigger in length than male. After dissection we observed that both male and female gonads are spongy in appearance and situated on the left side of the abdomen. The shape of the gonad is sac like structure in which the eggs are embedded like fimbrae. In male there is a point like protrusion, white in colour and the margin of dorsal fins are orange coloured at the time of maturity (Plate 3a - e). The female brooder shows bulged abdomen, vent is red in colour and dorsal fins are orange in colour at the time of maturity. Fully mature female shows freely oozing of eggs (Plate 3a - f). The eggs collected by hand stripping, were immersed in 70% acetic acid and 30% alcohol. After about three minutes, the position of the nuclei was observed. Migration of nucleus from center of egg to the periphery region indicated the readiness of fish for breeding. The fish 54

8 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, 1984). A day before experiment, the required fishes were selected and transferred to glass tanks of 50l capacity filled with dechlorinated water (Plate 3a - a). Each breeding set consisted of two males and one female (Jhingran and Pullin, 1985; Haniffa et al., 1996). Different types of hormones (Human Chorionic Gonadotropin, Ovaprim and LHRHa) were used for induced spawning. For each hormone three doses were used and for each dose, three breeding trials were made to find out the response of the fish and to observe the variation in latency period, the rate of fertilization and percentage of hatching. The details of the doses of hormones used in the experiments are given in Table 1. Injections were administered intramuscularly in the dorso lateral region of the body (Plate 3a - h). The injections were given during late afternoon or early evening (17-19 h). Immediately after administering the hormones, the breeding sets were released into the spawning tanks provided with Hydrilla verticillata for hiding purposes. The weed was useful for holding the floating eggs together without dispersion. The water quality parameters recorded during the study were as follows: water temperature (30 ± C), ph 7.5 ± 0.23, and dissolved oxygen (6.2 ± 2.3 ppm). 55

9 Table 3.0: Hormonal Application for Induced Breeding of Channa gachua S.No. Hormone Dosage 1. Human Chorionic Gonadotropin (HCG) 2. Ovaprim 3. LHRHa Low dose 1000 IU Medium dose 2000 IU High dose 3000 IU Low dose 0.3 ml Medium dose 0.4 ml High dose 0.5 ml Low Dose 40µg Medium Dose 50 µg Heavy Dose 60 µg In the early hours of the day, the breeders were closely observed for their response to hormone administration. After spawning, eggs were collected from each breeding tank using a beaker and immediately total eggs were enumerated and percentage of fertilization was assessed. Thereafter the fishes were removed and checked individually for the extent of spawning by giving a gentle pressure to the belly. If large number of eggs oozed out freely on applying pressures, they were classified as partially spawned. The hatchability was determined as the percentage of normal larvae from the total number of fertilized eggs in each sample (Haniffa et al., 2000; Marimuthu et al., 2000). Remaining eggs were allowed to hatch out and grow along with the parents in the breeding tanks to observe parental care. 56

10 Results Spawning behavior and Parental Care In the present study, the hormone induced fishes showed breeding behaviour after 6 h of administration irrespective of the ovulating agents used. Each female paired with single male. At all times, the more active and aggressive male paired with the female and the other male was found passive and idle in the corner of the breeding tank. Mating was proceeded by elaborate courtship. Chasing behavior commenced from h after the hormone injection. The active male chased the female frequently indicated the excited movement of the paired breeders. In all the spawning attempts, the male was more actively involved in the courtship and mating. It was seen hitting the female snout and vent region more frequently. The spawning actively continued till the releasing of gametes. During spawning, the female fish bent its body close to the male and breeders joined together at the opposite direction (Plate 3b a & b). This is followed by the male releasing its milt, the female releasing the eggs and after that external fertilization occurred. The eggs were laid in a clear area harbored by weeds (Plate 3b - c). The fertilized eggs usually floated and adhered to each other forming an egg mass, 4-5 cm in diameter while fertilized eggs lost their adherent ability and were scattered throughout the tank. Subsequently after fertilization the scattered eggs in the breeding tank were pooled in the vicinity of weeds by the moving activity of the male parent. The female parent was seen in the vicinity of the egg mass in the breeding tank whereas both the parents took care of eggs and hatchlings. While guarding the egg mass, male parent remained quiet curving 57

11 around eggs or intermittently swam in a slow circle fanning the eggs with its pectoral fins. One of the interesting characters of parental care in C. gachua is that they are mouth brooders (Plate 3a - d). To protect the youngones from predators, they took almost all hatchlings in their mouth. Induced Spawning using natural and synthetic hormones In the present study, three different types of hormones (HCG, LHRHa and Ovaprim) were used for induced spawning (Table 3.0). In the low dose of HCG (1000IU/Kg), latency period varied from hr with an average of 26.0 ± 0.46 h. Each female spawned resulting in an an average of 1196 ± eggs with a fertilization rate of 57.4 ± 1.83% and hatching rate of 61.6 ± 1.29%. In the medium dose of HCG (2000IU/Kg), the average latency period was 23.0 ± 0.12 h. Each female spawned an average of 1978 ± eggs with a fertilization rate of 62.1 ± 1.55 % and hatching rate of 66.3 ± 1.14 %. In the high dose (3000IU/Kg) of HCG, latency period of 21.3 ± 0.18 h was recorded (Table 3.1). Each female spawned an average of 2471 ± eggs. The average fertilization rate and hatching rate were 70.3 ± 1.67% and 72.3 ± 1.96 % at high dose respectively. There was complete spawning due to medium and high dose of hormonal injection whereas partial spawning was noticed with regard to lower doses. Some eggs were retained in the ovary itself. Statistical analysis indicated that there was no statistical difference with regard to latency period. However, the number of eggs spawned due to medium dose of HCG was significantly different (P<0.05) from those of low and high dose and also a significant difference was noticed between the low and high dose. With regard to fertilization rate, there was significant difference (P>0.05) noticed between medium versus high dose and high versus low dose. A significant difference in the hatching rate was found between low and high doses and also 58

12 medium and high doses but no significant difference was noticed between low and high doses. Results of induced spawning by administering LHRHa are summarized in Table 3.1 In low dose of this hormone, the average latency period was found to be 25.3 ± h and average fertilization rate was 54.7 ± 0.64 %. In all the three trials conducted with low dose (40µg / Kg), partial spawning was observed. Each female released an average of 877 ± eggs with a hatching rate of 58.3 ± 1.79 %. In the medium dose of LHRHa, (50µg / Kg) the average latency period was 23.0 ± 0.81 h. Each female spawned an average of 1650 ± eggs. A fertilization rate of 65.2 ± 3.17 % and hatching rate of 68.3± 1.47% were observed in the medium dose of LHRHa. In the high dose (60µg / Kg), an average latency period of 20.8 ± 1.12 h was observed. Each female spawned 1080 ± eggs and fertilization rate was 60.6 ± 1.07 % at the high dose of LHRHa. Statistical analysis indicated that, there was no significant difference (P>0.05) between the doses with regard to latency period. However, a significant difference (P<0.05) was observed between the three doses with regard to number of eggs spawned. Statistical inference indicated that the medium dose significantly differed from the rest of the doses. The fertilization rate as a function of the medium dose of LHRHa was significantly different (P<0.05) from those of low and high doses. A significant difference in the hatching rate was found between the medium and high doses and the medium and low doses. However, no significant difference (P>0.05) was observed between the low and high doses of LHRHa. Results of induced spawning of C.gachua using ovaprim are presented in the Table 3.1 Three different doses of (0.3, 0.4 and 0.5ml / Kg body weight) ovaprim were tested. The low dose of ovaprim (0.3ml / Kg body weight) injected fish did not respond. In the medium 59

13 dose of ovaprim (0.4 ml/ Kg body weight), the average latency period was 24.7 ± 0.37 h. Each female spawned an average of 863 ± eggs. A fertilization rate of 55.2± 0.92% and hatching rate of 58 ± 2.37 % were observed as a function of medium dose. In the high dose (0.5ml / Kg body weight) of Ovaprim an average latency period of 23.2 ± 0.89% was recorded. Each female spawned an average of 1319 ± eggs. In the high dose of ovaprim, the fertilization rate and hatching rate were 64.1 ± 4.16% and 61.3 ± 2.39% respectively. 60

14 Discussion Breeding behaviour or courtship behaviour is a very important act in fish breeding. It varies from the simple swimming of the breeders along the side of each other to the elaborate act of nest building and intense male competition inherent in group spawning. Spawning depends not only on gametogenesis but also on behaviors such as pre spawning migration, habitat selection, mate selection and courtship. In Channa gachua the breeding behaviour was noticed in 10 13hr after the hormonal injection and continued till spawning upto 23 24hrs. These results are similar to those reported previously viz: Channa punctatus (Haniffa et al., 2004), Ananbas testudineus (Johannessen et al., 1993), Clarias batrachus (Moitra et al., 1979), Heteropneutes fossilis (Thahur, 1976) and Hopilas malabaricus (Prado et al., 2006). The unpaired male remained passive in a corner of the breeding tank as observed for A.testudineus (Moitra et al., 1979) and C. batracus (Thakur, 1976). Breder and Rosen (1966) stated that C. gachua spawning in India occurs with the female swimming upside-down under the male, with eggs being released and fertilized in groups of every minute or two. Females in Indonesia and Malaysia, however, are reported to produce from 20 to 200 eggs per spawning, with the male orally brooding and developing eggs and fry (Lee and Ng, 1991). Egg guarding is the most common form of parental care (Clutton Brock, 1991), and in a majority of species only one parent involves in parental care. Among the teleost families male care is much more common than female care with 61% against 39% respectively; Bipaerntal care occurs in less than 25% of the families (Gross & Shine, 1981) as reported by Haniffa et al (2004) in induced bred spotted murrel C.punctatus. In the present study very 61

15 strong parental care behavior was observed in the breeders induced by different hormones. According to Alikuni (1957), the murrel breeds in natural conditions and both the parents were involved in parental care of eggs upto fry stage. But in our study the induced bred dwarf murrel C. gachua bred in tank which is the first of its kind. Although C. gachua showed biparental care it was also observed that males show much care towards their youngones. Moreover when the eggs were removed and incubated without parental care, they were affected by fungal infection resulting in poor hatching. The dwarf snakehead C.gachua is widely distributed in Asia, declined drastically (VU) in India (CAMP, 1998, Anjan Kumar Prusty et al., 2007) and endangered in Asia (Lim & Ng, 1990). C. gachua is well known in the aquarium trade (Deharadrai et al., 1973, Talwar and Jhingran, 1992). The dwarf snakehead does not spawn spontaneously when held in captivity. The success of reproduction largely depends upon the complex interactions taking place along the hypothalamus pituitary gonads axis (Hassin, 1991). The hormonal approach is presently more feasible for the induction and synchronization of ovulation and spawning in commercial hatcheries as reported by Zohar (1989). Various hormones were used for inducing maturation and spawning in a variety of teleosts with considerable success (see table 1). The dosage of the different hormones viz; HCG, LHRHa and Ovaprim were selected based on previous reports for induced spawning in murrels and catfishes and found to be in the range of 1000IU 3000IU/kg body weight for HCG, 40µg - 60 µg for LHRHa and 0.1ml 0.5ml for Ovaprim (Haniffa et al., 1996, 2000 and 2002, Francis 1996, Mollah and Tan 1983, Zarrin et al., 1992 and Zonneveld, 1988). In the present study three hormones were used to assess their impact on latency period, number of eggs spawned, fertilization rate, 62

16 hatching rate and survival rate of fry. Spawning was observed in all the attempts irrespective of doses for HCG and LHRHa; but in case of ovaprim injected fish spawning was observed as a function of medium and doses, where positive response only observed only for high doses. In HCG and LHRHa spawning was complete in the medium and high doses, but partial spawning was observed in the low dose. Similar report was observed in C.striatus showing complete spawning for medium and high doses of HCG, LHRHa and Ovaprim, whereas low dose of Ovaprim injected fish did not respond (Haniffa et al., 2000). In the present study among the different hormones used, fish administered with HCG performed better than those injected with LHRHa and Ovaprim. Among the hormonal doses, HCG was tested in three doses of (1000IU, 2000IU and 3000IU /Kg body weight of fish). Lowest latency period of 21.3 ± 0.18hr and highest fertilization rate of 70.3 ± 1.67 were recorded in C. gachua injected with 2000IU/Kg body weight of HCG. The latency period reported in the literature are 28 34h for C. punctatus (Haniffa and Sridhar, 2002), 22 25hr for H.fossilis (Kohil and Goswami, 1987), 17 22h for Pangasius bocourti (Philippe Cacot et al., 2002) 14 23h for C.batrachus (Sahoo et al., 2008). C.gachua given a low dosage of HCG showed partial spawning. Incubation period was more or less same among different doses. Zaki et al (2007) reported the usage of HCG at a cumulative dose of IU/Kg body weight of HCG for successful breeding in Sparus aurata. Sahoo et al (2005 & 2007) reported that the survival rate of hatchlings was higher (60 72%) for HCG injected C. batrachus. Salami et al (1994) also reported a better performance due to HCG administration than pituitary extracts in C. garipinus. 63

17 Ovaprim is the commercial name of hormonal preparation with 20µg of SGnRHa and 10mg of domperidone in one ml of solution and widely used for induced breeding and seed production of commercially important fish species. Among the doses used for C. gachua, complete spawning was observed in medium and high doses (0.4 and 0.5ml/Kg body weight); whereas for the low dose (0.2ml / Kg) there was a negative response. Sarkar (2006) reported that the complete ovulation in Chitala chitala was achieved when ovaprim was administered at a level of 1 and 1.5 ml/kg body weight of female. Nandeesa et al., (1990 b) reported positive response of mrigal due to ovaprim at a dose of 0.3ml/Kg indicating a high potency of this drug in induced spawning. Induced spawning of murrel C. striatus with various inducing agents was reported by Francis (2000) and Haniffa et al (2000). They reported that among the different hormones used, Ovaprim showed better performance in terms of higher fertilization rate and lower latency period. Latency period as a function of ovaprim induction in the present study was 23 24h. The latency period in the range of 11 23h was observed in induced spawning of C. batrachus (11 23h), (Sahoo et al., 2007) and also Australian eel tailed Catfish Neosilurus ater (Cheah and Lee, 2000). The results of the breeding experiments using ovaprim showed that the mean rate of spawning, percentage of fertilization and percentage of ovulation being 1091, 55 and 64.1% respectively. The mean percentage of survivorship of hatchling was 61% after 10 days. Till date, there was no report regarding the breeding behaviour of C. gachua under captivity. Ovaprim has been found to be effective in inducing ovulation in Ompok bimaculatus (Sridhar et.al., 1998), O. malabaricus ((Vijayakumar, 2002) and Cirrhinus reba (Sarkar et al., 2004). Nandeesha et al (1990 b) reported the positive response in mrigal with ovaprim at a dose of 0.3 ml Kg-1 indicating the high potency of this drug in induced 64

18 spawning.comparative efficacy of pituitary gland and ovaprim as inducing agent was studied in Clarias batrachus by Basu et al., (2000). They opined that ovaprim yielded better results with higher percentage of fertilization and hatching. Induced breeding of C. striatus by various inducing agents was reported by Haniffa et al (2000). They reported that among the different hormones used, ovaprim showed better performance in terms of higher fertilization rate (93%) and lower latency period (21 h.). However, percentage of ferlilization achieved in the present experiment was 64% and percentage of hatching was 61.3%. A similar spawning time (10-14 h) was reported by Nandeesha et al., (1990b) when Labeo rohita was induced bred with ovaprim. Among the hormones studied by Nandeesha et al., (1993), the highest percentage of fertilization (93%) was observed in ovaprim induced fish. The hormonal dose of ovaprim recommended for carp is 0.3 ml to 0.4 ml Kg -1 (Nandeesha et al., 1991). Nandeesha et al., (1990b) also reported that Labeo rohita responded to higher dose of ovaprim and presumed that this was because dopamine activity is higher in rohu. Based on the present study ovaprim dose of 0.5ml/Kg body weight can be recommended. Haniffa et al., (1996) have applied different dosages ( ml /Kg body weight) of ovaprim for induced spawning of catfishes and murrels. In Heteropneustus fossilis the dosage of ovaprim given ml/Kg body weight and the number of eggs spawned increased with increasing dosage upto 0.7ml/Kg (Haniffa and Sridhar, 2000). LHRHa has been successfully used to induce final maturation and synchronize ovulation in many commercially cultured fish (Donaldson and Hunter 1983, Park et al.,, 1997b). The average latency period of C. gachua observed in the present study was 23.7 ± 0.67h. Froud Bosak (2010) recorded a latency period of ± 0.2h for Barbus sharpeyi when injected with LHRHa + CPE. The latency period was longer than those of LHRHa + 65

19 MET treated Cyprinus carpio (14 16hr) (Drori et al., 1994) and Ovatide treated Clarias batrachus (17hr) (Sahoo et al., 2005). In contrast, a latency period of 28 30h was observed for LHRHa administered Common carp (Muhammed et al., 2001). Seyed Abdolsaheb (2010) reported a latency period of 27hr for Barbus xanthopterus injected that LHRHa. In the present study, among the doses oh LHRHa, better result was obtained in the medium dose (50µg / Kg body weight) with regard to number of eggs spawned, fertilization rate and hatching rate. Kumarasini and Seneviratue (1988) conducted experiments on induced breeding of Cyprinus carpio using LHRHa and 100% fertilization rate was recorded. Sahoo et al., (2006) reported that C.batrachus injected with 20µg of SGnRHa + 10mg domperidone / Kg of body weight and Puffer fish with 30µg/ Kg of LHRHa (Josefa et al., 1997). Nazari et al., (2010) observed that the fertilization rate of 72% and hatching rate of 86.30% was observed when fish injected with LHRH-a (10µg / Kg of body weight). Considering the overall performance of the hormones with regard to number of eggs spawned, latency period, fertilization rate and hatching rate, the present investigation confirms that HCG was found to be the most potent ovulating agent in C.gachua. The efficacy of the different hormones at various dosage can then be summarized as HCG > LHRHa> Ovaprim. Thus it is obvious that HCG at a dosage of 3000IU / BW can be recommended for higher and better results in breeding attempt of C.gachua. Hence in the view of conservation of the threatened murrel C.gachua, HCG can be suggested for successful breeding attempts. 66