INDUCED SPAWNING OF INDIAN MAJOR CARPS
THROUGH SINGLE APPLICATION OF OVAPRIM-C

Mudnakudu C. Nandeesha, Kondapalli Gopal Rao (*1), Rama Naik Jayanna (*2), Nick C. Parker (*3), Tharayil J. Varghese, Perar Keshavanath and Handady P. C. Shetty

College of Fisheries, University of Agricultural Sciences, Mangalore-575002, India. (*1) Department of Fishery Sciences, Andhra Pradesh Agricultural University, Kakinada - 533007, India. (*2) Bhadra Fish Seed Farm, Government of Karnataka, B. R. Project - 571115, India. (*3) Texas Cooperative Fish and Wildlife Research Unit, Department of Range and Wildlife Sciences, P. O. Box 4169, Texas Technology University, Lubbock, TX 79409, USA.

Abstract

A new drug, Ovaprim-C, manufactured by Syndel Laboratories, Canada, was tested for its induced breeding efficacy in three species of Indian major carps, viz. catla (Catla catla), rohu (Labeo rohita) and mrigal (Cirrhinus mrigala). The drug, prepared based on the 'Linpe' method of breeding carps in China, contains an analogue of salmon gonadotropin releasing hormone and the dopamine antagonist, domperidone. Breeding response of the three carps was evaluated vis-a-vis that obtained in the controls, injected split doses of carp pituitary extract or a single dose of carp pituitary extract or a single dose of Ovaprim solvent. All the three species could be bred with a single intramuscular injection of Ovaprim at 0.5 ml/kg body weight. Mrigal responded positively to even lower dosages of 0.3 and 0.4 ml/kg, but 0.4 ml/kg was found to be the minimum dosage required for rohu. In all these trials, males were injected with carp pituitary extract at 3-4 mg/kg, 6 hr after injecting Ovaprim to females. In another trial, wherein female and male rohu were injected simultaneously with Ovaprim at 0.4 and 0.15 ml/kg, respectively, the spawning response was excellent. As against this, the control fish receiving pituitary extract in a single dose failed to respond. The percentage of fertilization in most cases ranged from 70 to 99%. The results of this investigation clearly indicate the suitability of Ovaprim-C for inducing breeding of the Indian major carps.

Introduction

The carp culture industry is rapidly expanding in India. But shortage of major carp seed is one of the major constraints in the development of this industry. To mitigate this problem, efforts are ongoing to establish hatcheries in places where the environment is favorable for carp breeding. Though the technology of hypophysation is fairly well standardized and is being employed widely, availability, quality and potency of pituitary glands have become undependable. As a result, spawning failure is very common in several farms.

With the objective of finding out an effective substitute for pituitary glands, the present investigation was undertaken wherein a new drug, Ovaprim-C, was used for the first time for induced breeding of three Indian major carps, namely, catla (Catla catla), rohu (Labeo rohita), and mrigal (Cirrhina mrisala).

Material and Methods

The experiments were conducted in fish farms in three southern states of India, viz. Karnataka, Andhra Pradesh and Tamil Nadu. Mature male and female carp were selected based on external secondary sexual characters (Jhingran and Pullin 1984). In the case of rohu and mrigal, varying doses of Ovaprim (0.2-0.5 ml/kg) were tried, while in catla only one dose (0.5 ml/kg) was tried due partly to insufficient number of brood fish. Females of all the three species were injected intramuscularly with Ovaprim in a single dose, while males were given carp pituitary extract at 3-4 mg/kg, 6 hr after injecting females. The males were released with the injected females only after hormone administration, which was adjusted to fall between 17 and 20 hr. Control fish were injected carp pituitary extract in two doses for females (4 and 8 mg/kg) at an interval of 6 hr and a single dose for males (3-4 mg/kg) at the time of second injection to females. On a few occasions, fish treated with a single dose of Ovaprim solvent (0.5 ml/kg for females and 0.15 ml/kg for males) were used as control. In some of the trials carried out in July with robe, both female and male fish were injected Ovaprim simultaneously at 0.4 and 0.15 ml/kg, respectively. However, in August, higher dosages (0.6 and 1.2 ml/kg for female and 0.2 ml/kg for male) were also tried, since it has been reported that the carp pituitary dose required for induced breeding is higher when the water temperature goes beyond 30 C (Rao and Gnaneshwar 1988). For the August trials, female fish receiving either split dose or a single dose (total of 18 mg/kg) and males getting only a single dose (6 mg/kg) of pituitary extract served as control. The Ovaprim used in our study had 20/ug of an analogue of salmon gonadotropin releasing hormone (sGnRH) (D-Arg6, Trp7, Leu8, Pro9 NEt)-LH-RH and 10 mg domperidone, a dopamine antagonist, per ml.

The breeding tests were conducted by pairing each female with two males in a cloth hapa measuring 2.5 x 1.2 x 1 m, fixed in ponds provided with moderate circulation of water. All the trials in Tamil Hadu and a few trials in Andhra Pradesh with rohu were conducted in Chinese hatcheries of 4.2 m diameter and 1.2 m height. These hatcheries are not the breeding pools, but the hatching pools which are commonly used for breeding by keeping the fish in the outer chamber. Fish were examined for the breeding response usually 10 hr after the first injection and subsequently at hourly intervals. The percentage of fertilization was calculated using samples of eggs developed for a minimum period of 4-6 hr. Before releasing the spawned females, they were examined for the extent of spawning. Whenever a large number of eggs oozed out freely by applying slight pressure on the abdomen, they were classified as partially spawned. In cases where no natural spawning had taken place, if the eggs were oozing out with slight pressure on the abdomen, they were classified as ovulated.

In most of the experiments, eggs from the same species were grouped together and hatched in the Chinese hatcheries. However, in Karnataka, catla eggs were hatched in traditional cloth hapas (2 x 1 x 1 m).

Results

The general weather that prevailed during the month of July in [arnataka and Andhra Pradesh was conducive for breeding of carps. Though the temperature was relatively higher in Andhra Pradesh (27.529C) as compared with [arnataka (2324.5.C), there were light showers in both the states on almost all days during the experiments. However, temperatures recorded during the experiments conducted in Andhra Pradesh in August (31-32.5-C) and in Tamil Nadu in September (30-30.5.C) were not very favorable for breeding of carps, especially because of little rainfall.

Results of the experiments on mrigal are presented in Table 1. A total of 21 females were treated with different doses of Ovaprim, nine were administered a uniform dosage of 12 mg/kg carp pituitary extract, and three were given Ovaprim solvent, the latter two being control groups. Of the seven females treated with Ovaprim at 0.5 ml/kg, five spawned, while one got plugged and in another, spawning was partial. All the six females injected wit 0.4 ml/kg spawned naturally. Among the 4 females treated with 0.3 ml/kg, three spawned naturally and the fourth one showed only ovulation. Out of the four females injected with 0.2 ml/kg, only one spawned, while none of those treated with Ovaprim solvent responded. Except for one fish which spawned partially and another that did not spawn, all the others treated with pituitary extract bred naturally. The percentage of fertilization was usually high in Ovaprim treated fish (74-99:) as compared with pituitary extract injected fish (11-97%).

All the seven females of catla treated with Ovaprim at 0.5 ml/kg responded well, though the spawning was partial in one fish and fertilization rate varied from 60 to 87Z (Table 1). The two females injected with Ovaprim solvent did not respond. Since in one instance most of the eggs went out of the breeding hapa through a hole, the number of eggs released could not be ascertained.

Table 2 summarizes the results of the experiments with rohu. All the three females treated with carp pituitary spawned, though spawning was partial in one instance with low fertilization percentage (43%). Out of the 16 females administered with 0.5 ml/kg Ovaprim, 15 females spawned, whereas one female did not respond. However, spawning was partial in two females and fertilization was poor (3 and 31%). All the 29 females treated with 0.4 ml/kg responded positively. At 0.3 ml/kg, no female spawned naturally, but all ovulated. Out of the three females treated with 0.25 ml/kg, none spawned but ovulation was recorded in one. Both the groups treated with 0.2 ml/kg Ovaprim and Ovaprim solvent did not respond.

The results of experiments pertaining to single and simultaneous administration of Ovaprim to both sexes of rohu are given in Table 3. Out of the 19 sets treated at 0.4 ml/kg for female and 0.15 ml/kg for male, 14 sets responded positively with high fertilization (76 - 94%). Even in the experiments conducted during August, fish injected with 0.4 ml/kg also spawned like those injected with 0.6 and 1.2 ml/kg, the latter doses were tried to confirm the need for higher doses of inducing agents at higher temperatures. None of the fish treated with pituitary extract (18 mg/kg for female and 6 mg/kg for male) in a single dose responded. However, those fish given pituitary in two doses responded positively (Table 3). The number of eggs obtained, spawning rate and fertilization were poor in most of the trials conducted during August.

Discussion

Certain drugs and different analogies of LH-RH are being tested for breeding fishes with varying degrees of success (Harvey and Hoar 1979). However, it was only when the dopamine inhibitory activity in the synthesis of gonadotropin was demonstrated (Peter et al. 1986) that the reasons behind the spawning failures became clear. Investigations have now clearly shown the potentiated action of analogies when they are combined with dopamine antagonists like pimozide or domperidone (Billard et al. 1983, Chang and Peter 1983; Chang et al. 1984: Sokolowska et al. 1984; Lin et al. 1986; Lin et al. 1988; Peter et al. 1988). Based on the extensive research on Chinese carps, Peter et al. (1988) defined a new method of breeding fishes called the 'Linpe' method in which LH-RH analogue is combined with a dopamine antagonist. Ovaprim_C was developed essentially based on this principle.

The positive response of mrigal to Ovaprim in the present investigation even at a dose of 0.3 ml/kg indicated the higher potency of this drug in inducing spawning Haul and Rishi (1986) could spawn mrigal with 10 m g of LH-RHa (des-Gly LH-RH (1-9) ethylamide) or 10 mg of pimozide. Peter et al. (1985; 1987) reported sGnRH to be 17 times more potent than LH-RHa, when it is combined with low doses of pimozide. Silver carp (Hypophthalmichthys molitrix) and loach (Paramisgurnus debryanus) have been bred with sGnRH at 10/m g/kg and l ug/kg, respectively when combined with domperidone at 10 mg/kg, as compared with the higher dose of LH-RHa (50 m g/kg) required even when it is injected with antagonist domperidone or pimozide (Lin et al. 1988). The partial spawning observed in a few sets of mrigal appears to be due to the poor state of maturity of the fish.

Based on the results obtained with mrigal, it can be presumed that dopamine activity is higher in rohu, since it has responded to only higher doses of Ovaprim. Our earlier trials with rohu have indicated a higher requirement of LH-RHA (12 m g/kg) for successful spawning (M. C. Nandeesha, pers. comm.). Peter et al. (1986) have reviewed dopamine activity in various fish species and indicated that it may vary considerably between species. The positive response of both male and female rohu to a single, simultaneous injection of Ovaprim is very significant from the point of view commercial carp seed production, as it saves a considerable amount of time and avoids excessive handling of brood fish. The dosage for males was arbitrarily fixed at 0.15 ml/kg, since major carp males respond well to 1/3 the pituitary dose required by females (812 mg/kg). It is necessary to inject carp females with pituitary extract in split doses, since single dose is ineffective in inducing effective spawning. In the present study also, fish injected with pituitary in a single dose failed to respond, while those given a split dose spawned successfully. Though the actual reason for this difference in response to single injection and double injection is not clearly known, Peter et al. (1986) ascribed it to the self potentiating action of the releasing hormone to some drugs when given in two doses. As could be seen from Table 3, in the month of July spawning was complete and fertilization high when both male and female rohu were injected simultaneously with Ovaprim. Peter et al, (1988) obseved successful spawning of Chinese carps and loach through a single injection of sGnRH combined with dopamine antagonist. Spawning success and fertilization were relatively poor in the trials conducted in August which could be attributed to initiation of resorption of ovaries coupled with high temperature. Though no strong evidence can be provided for the higher requirement of hormone with increasing temperature, Rao and Gnaneshwar (1988) have observed a linear relationship between dosage and temperature. Farmers generally use high dose of pituitary extract towards the end of the breeding season (August-September) when the temperature is generally high.

Breeding of catla is relatively difficult as compared with that of rohu and mrigal. Our preliminary trials with LHRHa on catla yielded positive results at 10 - 14 m g/kg (M. C. Nandeesha, Pers. comm.). However, the response to LN-RNa was not uniform. The positive response of all the catla to Ovaprim in the present study could be mainly due to the combined effect of sGnRN and the dopamine antagonist. Due to unavailability of broodfish, lower doses of Ovaprim could not be tested on catla. It is likely that the dose required would be less than 0.5 ml/kg even in this species.

The spawning response time varied between 10-14 hr. Fish injected with pituitary extract spawned earlier than Ovaprim-treated fishes. This difference in spawning time could be because Ovaprim was given in a single injection unlike the pituitary extract, which was given in two doses, the latter is known to reduce spawning time considerably (Harvey and Hoar 1979).

The results of the present investigation clearly demonstrate the possibility of using Ovaprim-C for breeding carps. Though the cost of Ovaprim at present is almost double the cost of pituitary, it has the advantage of known potency and assured breeding response. Spawning failure due to poor quality of glands and varying potency has become too common and when the loss incurred is considered, the heigher cost of Ovaprim is offset by the lower risk of spawning failure. A survey conducted in 1980-82 among 550 fish seed producers in India has shown that only 152 of them use the hypophysation technique for breeding carps which has been attributed to the complexity of the technique (Dehadrai 1984). Ovaprim-C being in ready-to-use form will simplify the breeding operation and hence can be easily adopted in carp seed production. Nonetheless, further trials are necessary to standardize the dosage for adoption on a commercial scale. Work in this direction is in progress.

Acknowledgements

We gratefully acknowledge the help of the Managing Director, Rarnataka Inland Fisheries Development Corporation, Bangalore, Rarnataka; Shri Radhakrishna Raju, Venkateshwara Fish Hatchery, Venkatapuram, Andhra Pradesh; and Shri Ch. Srikanth, C.H.R. Farm, Padappai, Madras, Tamil Nadu for providing facilities to conduct the trials. Part of the sample used in this study was provided by Syndel Laboratories, Canada which is also gratefully acknowledged. Thanks are also due to Dr. R.E. Peter of Canada for introducing us to this exciting drug.

References

 

Hirano, R. and I. Hanyu, editors. 1990. The Second Asian Fisheries

Forum. 991 p. Asian Fisheries Society, Manila, Philippines.

 

Table 1.---Spawning response of female mrigal and catle to Ovaprim-C in 1988 in Karnataka and Andhra Pradesh.

Treatment Spawning Rate
Species Place Month Water Temp
(0 C)
Pituitarya
(mg/kg)
Ovaprimb solvent (ml/kg) Ovaprimb
(ml/kg)
Number of fish Total weight of fish (kg) Ovulation rate (%) Partial Complete Number of eggs obtained (lakhs) Fertilization rate
Mrigal Karnataka July 23-24, 5 12 9 9.40 100 11.11 77.78 8.28 11-97
0.5 7 6.20 100 14.29 71.43 5.28 74-98
0.4 6 5.10 100 100 5.18 75-97
0.3 4 3.55 100 75.00 2.34 89-99
0.2 4 3.95 25 25.00 44
0.5 3 2.75
Catls Karnataka July 23, 5-24.5 0.5 3 10.00 100 33.33 66.67 9.57 60-87
Andhra Pradesh July 28.0 0.5 4 6.50 100 100 6.75c 76-87
0.5 2 3.10

a. Applied in two doses at 4 mg and 8 mg.kg at an interval of 6 hr.
b. Males were injected with pituitary extract 6 hr after injecting the females with Ovaprim-C in single dose.
c. Eggs were collected from only three fishes.

 

Table 2.---Spawning response of female rohu to Ovaprim-C in three different states in India during 1988.

Treatment Spawning Rate
Place Month Water Temp
(0 C)
Pituitarya
(mg/kg)
Ovaprimb solvent (ml/kg) Ovaprimb
(ml/kg)
Number of fish Total weight of fish (kg) Ovulation rate (%) Partial Complete Number of eggs obtained (lakhs) Fertilization rate
Karnataka July 24.0-24.5 12 3 3.70 100 33.33 66.67 6.47 43-88
0.50 9 10.40 100 22.22 77.78 12.59 3-89
0.5 4 5.10
Andhra Pradesh July 28.0-29.5 0.50 2 1.55 50 50 3.29 98
0.40 5 5.30 100 100 18.24 79-98
0.30 3 4.00 100
0.25 3 3.60 33.33
0.20 2 1.90
0.5 3 3.00
Tamilc Nadu Sept 30.0-30.5 0.50 5 6.00 100 100 10.00 95
0.40 24 25.60 100 100 42.50 95-98

a. Applied in two doses at 4 mg and 8 mg.kg at an interval of 6 hr.
b. Males were injected with pituitary extract 6 hr after injecting the females with Ovaprim-C in single dose.
c. Bred in Chinese hatchery in groups of 2-10 fish at a time.

 

Table 3.---Spawning response of rohu to Ovaprim-C when males and females are given a single dose simultaneously during 1988 in Andhra Pradesh.

    Weight of fish (kg) Ovaprim (ml/kg) Pituitary (mg/kg)   Spawning rate (%)    
Month Water Temperature (0 C) Female Male Female Male Female Male Ovulation Rate (%) Partial Complete Number of eggs obtained (lakhs) Fertilization (%)
July 27-28 8.55 16.5 0.4 0.15     100   100 27.74 82-94
    (5)a (10)                  
 
Augustb 31-32.5 7 16 1.2 0.3     100   100 21.4 75
    (6) (14)                  
 
    10.9 21 0.6 0.2     100 30 70 15.34 59
    (10) (20)                  
 
    14 30.8 0.4 0.15     100 35.71 64.29 19.94 76
    (14) (26)                  
 
    9.5 21     18c 6c 100        
    (6) (12)                  
 
    24.5 51     18P 6P 100 40 60 30.5 50
    (20) (32)                  

a. Figures in parentheses indicate the number of fish.
b. Breeding operations in August were carried out in groups using Chinese hatcheries.
c. Pituitary extract-treated control.  Both males and females were injected simultaneously with a single dose.
P. Pituitary extract was applied in two doses to females at 6 mg and 12 mg.kg at an interval of 6 hr.  Males were injected once at 6 mg/kg at the time of second injection of females.