Induced spawning and hatching rate of Grass Carp (Ctenopharyngodon idella) by using a single intramuscular injection of ovaprim at Demar fish hatchery Dhamtari (C.G), India.

Mukesh Raghav¹, Dr. M.S.Chari¹ and Shudhansu Shekhar Mishra²

¹Department of Fisheries, College of Agriculture, I.G.K.V, Raipur, Chhattisgarh, India

²Central Institute of freshwater Aquaculture, Bhubhneswar, Orissa, India

ABSTRACT:

The study was carried out at Demar fish hatchery, Dhamtari (Chhattisgarh), India. Intramuscular injection of ovaprim was studied on the number of eggs/kg, fertilization rate and hatching percentage during May to July 2010 on Grass carp (Ctenopharyngodon idella). Fishes were spawned successfully by a single dose of injection of ovaprim (luteinizing hormone releasing hormone analogue) with 0.5 ml/kg for female and 0.2 ml/kg for male. Ova and milt were stripped simultaneously and mixture was stirred for 20 to 30 s during which fertilization occurred. Hatching occurred within 16 to 20 h after fertilization. Experiment was conducted in circular spawning tank with 2 m diameter. Regression analysis was applied to assess the body weight dependence of absolute fecundity (total ripe eggs in the ovary) and relative fecundity (number of eggs/kg). It was observed that body weight has positive influence on absolute fecundity (r = 0.930). Equations were developed to describe these relationships. If it is impossible to determine the absolute and relative fecundity, then these parameters can be determined from the body weight. The equations of these parameters are highly significant (P < 0.002) and can be used to estimate the absolute and relative fecundity with a fair amount of accuracy.

KEYWORDS: Fish, induced spawning, fecundity, ovaprim-C, Ctenopharyngodon idella.

1. INTRODUCTION:

Grass carp (Ctenopharyngodon idella) was first introduced in India from Hongkong for the first time in 1959 at Cuttack research station. The purpose of its introduction, in addition to culture, biological control of aquatic weed in natural waterways, rivers and man-made lakes. Grass carp (C. idella) matures at the age of 2 years and above; generally, males mature little earlier than female in cultivable carps (Rath, 1993). Spawning is the release of sexual products (ova in the case of female and milt in the case of male) to the exterior of the body (Basaran et al., 2008). Induced breeding is a method in which exogenous hormones are injected into the body of mature parent fish for induction of breeding (Heggberget, 1996). During the past two decade, pituitary extract was used for induced breeding. The ever increasing cost of donor pituitary and cumbersome process obliged expert to test alternative hormones such as HCG (human chorionic gonadotropin), LHRH (leutinizing hormone releasing hormone) and ovaprim (Haniffa and Sridhar, 2002). Ovaprim is a product that contains salmon gonadotropin releasing hormone analogue (sGnRH;D-Arg6,Pro9,Net) at a concentration of 20 ug/ml and dompridone, a dopamine antagonist at 10 mg/ml (Hill et al., 2009). Dopamine antagonists are used for ceasation of dopamine activity which acts as an inhibitory factor for the synthesis of gonadotropin (Naeem et al., 2005).

Ovaprim is used to induce ovulation and spermiation in fishes mostly by intramuscular or intraperitonial route. The aim of this study was to investigate the induction of spawning, fertilization rate and hatching rate of the Grass carp (C. idella) in culture conditions at Sanjay Gandhi Fish Hatchery, Demar, Chhattisgarh, India.

MATERIALS AND METHODS:

The experiment was conducted at Sanjay Gandhi Fish Hatchery, Demar, Chattisgarh, India, during the month of May to July 2010 with the aim of accessing the relative fecundity, that is, number of eggs/kg of body weight, by artificial spawning in Grass carp (C.idella). Eighty two specimens were used for this investigation, their weight ranged from 2.8 to 6.5 kg; they were free of all diseases and sexually ripened. Tests were conducted in circular spawning tank 2 m in diameter and 1 m in depth with optimum water quality variables and fish was acclimatized for one day before the commencement of experiment. Ovaprim was injected in a single dose because trials by Nandeesha et al. (1990), has found the effectiveness of a single and simultaneous injection. Good quality and well matured male and female brood fish are prerequisite for successful artificial propagation (Muir and Robert, 1985), so, most suitable size of spawners taken was 4 to 6 kg, to avoid difficulty in handling large size and requirement of large doses of expensive hormones. Brood fishes were transferred into cemented holding tanks of hatchery and nesthetized with 100 to 200 ppm 2-phenoxy ethanol in 1000 L one ton capacity fiberglass tank half filled with tap water. Sex ratio of one female to two males was used in induced spawning for achievement of best results.

Brood fishes were weighed and dosage of ovaprim solution was calculated according to Nandeesha (1991): Quantity to be injected (ml) = weight of brood fish (kg) x dosage of ovaprim Male brooders were injected 0.2 ml/kg and females 0.5 ml/kg by intramuscular route into the orsolateral region of the fish in a single dose (Haniffa and Sridhar, 2002) by using hypodermic syringe after cleaning the area with cotton swab soaked in alcohol. Ripeness can be accessed by several indicators example, in female, the abdomen was round, soft and genital opening was swollen, protruding and pinkish red, anus was also swollen and reddish. In male, secondary sexual characteristics were evident (Metwally et al., 2008). Required amount of ovaprim-C was withdrawn from the bottle by keeping the needle upward and air was removed from syringe. During injection, brood fish was placed in cloth bag, lying laterally in water and upper half of fish was held above the water surface. At the inner side of basal part of the pectoral fin where it was scaleless, needle was inserted gently towards the head at an angle of 45° to the body's longitudinal axis to a depth of about 1.5 cm and injected slowly.

Immediately after administering the hormones, the breeding sets (two males and one female) were released into cemented tanks (capacity 500 L) containing dechlorinated tap water, flowing at the rate of 12 L/min (Haniffa and Sridhar, 2002). Fish were observed for behavioral signs after 8.30 h of dose administration. Estrus is restlessness of female and its abdomen and tail become extremely constricted, which lasts for 30 to 60 min after which fish were netted out for stripping. Sex product was stripped from the spawners by gentle massage and pressure on the abdomen (Jamroz et al., 2008) into a dry plastic bowl. Following the semi dry fertilization method by Chaudhary et al., (1984), milt was mixed with the eggs using a bird feather for two minutes, this will decrease the distance from the sperm to the micropyle of the egg. Eggs were washed with water for 10 min; they absorb water and attain the size of 1 to 1.4 mm in diameter. The number of eggs released was calculated following the gravimetric method (Haniffa and Sridhar, 2002) in which 1 g of egg sample was weighed three times and mean value was multiplied with the total weight of egg sample:

No. of eggs kg-1 = total no. of eggs / total weight of fish

After 3 to 4 h of fertilization, division of cell was so regular that the size and shape of blastomeres was equal in fertilized egg, and distinguishable from unfertilized eggs. So, the fertilized eggs were calculated according to Muir and Robert (1985):

Fertilization rate (%) = no. of fertilized eggs / total no. of eggs x 100.

Table 1. Effect of ovaprim on spawning of Grass carp (C. idella).

Parameter	Ovaprim treatment
No. of females treated .	32
Total weight of females	111.7 kg
Total no. of eggs	11’711’968
Total no. of fertilized eggs	9’326’240
Total no. of hatchling	7’162’552
Overall fertilization percentage	79.63%
Overall hatching percentage	76.80%
Average no. of eggs/Kg	104852
Average no. of fertilized eggs/kg	83494
Average no. of hatching/kg	64123

Hatching occur after 16 to 20 h at water temperature of 20.0 to 24.5°C and hatchling were kept in circular spawning tanks with bolting cloth for three days until yolk was absorbed, and percentage hatchling calculated by:

Percentage of hatchling = total no. of hatchlings / total no. of fertilized eggs x 100

RESULTS:

Fish given single injection of ovaprim were successfully induced to spawn during the month of May, 2010. 82 females were injected with ovaprim, ovulation of fish in these treatments were 100%, total number of obtained eggs were 104852 kg-1, while overall fertilization and hatching rate was 79.63 and 76.80 respectively (Table1). Absolute and relative fecundity was found to be related to body weight in Grass carp (C. idella). Regression analysis was applied to assess the body weight dependence of these variables. Body weight had a positive influence on absolute fecundity, while there was no influence on relative fecundity (Table 3) with increasing body weight. Each of these relationships was statistically significant (P < 0.002) and well described by a linear equation:

Y = a + bX

Where, ‘a’ and ‘b’ are constant; X is the body weight and Y is the dependent variable. When total values of absolute fecundity and relative fecundity of Grass carp (C. idella) were transformed into log-log scale, a linear relationship of the following form was obtained showing a high degree of correlation (Table 4):

Log Y = a + b log X

Statistical analysis, including regression analysis and calculation of correlation was carried out by using a computer package EXCEL following Zar (1996).

DISCUSSION:

In the present study, a single intramuscular injection of synthetic hormone, ovaprim resulted in successful spawning of Grass carp (C. idella). The results of the hormonal stimulation in the current work are similar to the effectiveness and usefulness by using ovaprim (Jamroz et al., 2008). But speed and gentleness during fish capture and handling are of utmost important (Basaran et al., 2008). Certain hormones are used individually like different analogues of LH-RH, without pituitary gland, which results in failure of spawning and clearly indicates that dopamine blocks the action of LH-RH on the secretion of gonadotropin (Naeem et al., 2005a). However, use of dopamine antagonists like pimozide or doperidon, potentiate the action of LH-RH, resulting in successful spawning (Chang et al., 1983). Major breakthrough in the history of aquaculture happened when extensive research on Chinese carp (Peter et al., 1988) and a new Linpe method was introduced in which LH-RH analogue is combined with a dopamine antagonist. Then Canada introduced the ovaprim containing the analogue of salmon gonadotropin releasing hormone (D-Arg6, Pro9, Net) and dopamine antagonist, and studies conducted in India (Nandeesha et al., 1990) and Pakistan (Khan et al., 1992; Naeem et al., 2005a, b, c) revealed the superiority of ovaprim in induced spawning. Dose of ovaprim used in Grass carp (C. idella) in the present experiment is 0.5 ml/kg, while experiments conducted by Nandeesha et al. (1990) and Peter et al. (1986) reported the dose rate of 0.7 ml/kg. So, the present study reveals that ovaprim use is more economical in commercial carp seed production, as it saves a considerable amount of time and avoids the excessive handling of brood fish. Dosage of ovaprim for carps at different locations is given in Table 5. Grass carp (C. idella) preferably spawn during mid April to June (Table 2). Fertilization rate is 79.63% and hatchling percentage is 76.80% (Table 1) at the favorable temperature of 26 to 30°C as shown in Table 2.

Table 2. Spawning response of female Grass Carp (C. idella)

Month	Temperature Of Air/water (⁰C)	No. Of Females	Total Weight Of Females	Dose of Ovaprim (ml/kg)	No. Of Eggs (Lac)	Fertilization Rate (Lac)	No. Of Hatchlings (Lac)
May	34/31	4	12.5	0.5	13.106	10.436	8.015
May	32/30	4	14.175	0.5	14.863	11.835	9.089
June	34/32	5	15.625	0.5	16.383	13.046	10.019
June	32/30	5	14.30	0.5	14.993	11.938	9.169
June	30/27	5	20.40	0.5	21.389	17.030	13.079
June	31/27	5	16.350	0.5	17.146	13.635	10.485
July	28/26	4	18.350	0.5	19.240	15.321	11.7665

Table 3. Statistical parameters of body weight versus total no. of eggs and no. of eggs/kg of Grass Carp (C. idella)

Relationship

S.E.(b)

Wet bodyweight (x)

Total No. of eggs (y)

0.900***

-19654

66220

7133

Wet bodyweight (x)

No. of eggs/kg (y)

0.243^n.s

53746

1628.5

1447

r=correlation coefficient, a=intercept, b=slope, S.E.= standard error; *****,

Table 4. Statistical parameters of log body weight versus log total no. of eggs and no. of eggs/kg of Grass Carp (C. idella)

Relationship

S.E.(b)

Wet bodyweight (x)

Total No. of eggs (y)

0.924***

4.6630

1.1778

0.105

Wet bodyweight (x)

No. of eggs/kg (y)

0.347 ^n.s

4.6630

0.1778

0.105

r=correlation coefficient , a=intercept, b=slope, S.E.= standard error; *****,

Generally, the number of eggs spawned by Grass carp (C. idella) in the present study was lower than previous report (Ling et al., 1980; Chaudhary et al., 1984; Armando et al., 1989). Statistical parameters reveals that body weight had a positive influence on absolute fecundity (Table 3), while there was no influence on relative fecundity (Table 4). This may be due to low nutritional status of the brood fish as brood stock depended completely on the natural productivity of the reservoir and were not given artificial feed throughout the rearing period. Further studies are needed to determine minimum effective dose of ovaprim that could be used to spawn a brood fish under captive condition.

CONCLUSION:

The present study shows the advantages of ovaprim over commercial pituitary (example reduced handling of brood fish due to the single dose, which not only decrease the post spawning mortality, but also increase spawning response, adverse effects on the health and growth), and are very easy to use by unskilled farmers.

ACKNOWLEDGEMENTS:

The authors would like to thank the Director of Fisheries, Chhattisgarh, India for technical support, provision of fish and accessing their facilities for experiments.

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Received on 19.01.2012

Modified on 06.02.2012

Accepted on 12.03.2012

Research J. Science and Tech. 4(1): Jan.-Feb. 2012: 28-31