Induced spawning of African Catfish (Clarias gariepinus) using Natural Hormones (HCG & HMG) and Synthetic (Ovaprim)

ABSTRACT

This study was carried out to investigate artificial breeding in female African catfish Clarias gariepinus through injection of synthetic hormones separately and in combination with natural hormones. The study was carried out 1^st time in Muzaffargarh, Punjab, Pakistan. The stock of 1000 fish was imported from Thailand and reared at local government fish hatchery in Muzaffargarh. After identification of male and female fish total 48 (24:24 / Male:Female) equally weighted fishes were selected for this study. All the males were injected with same concentration of ovaprim @ 0.2 ml/kg body weight to get milt. Females were injected with different hormones by dividing them into groups as A, B, C, D, E and F. Group A was control group and received no dose. Other groups received as B, Ovaprim (0.5); C, HCG (0.5); D, HMG (0.5); E, Ovaprim+ HCG (0.3+0.5); F, Ovaprim+ HMG (0.3+0.5). incubation Period (Hours), fertilization Rate (%), survival Rate (%), hatching (%), deformed larvae (%), fecundity (Nos), total egg weight (Grams), spawning success (%) and latency period (Hours) were studied in this experiment. A combination of Ovaprim+ HCG (0.3+0.5) indicates the preeminent results and economically beneficial for induced breeding in Clarias gariepinus

INTRODUCTION

Fish are an important part of the human diet in many countries and are often perceived as having some nutritional advantages over the ‘meat’ of terrestrial live stock (Byelashov et al., 2015; Sargent & Tacon 1999). Fish are important as protein source, are a source of fats and are also valuable source of some vitamins and minerals (Vladau et al., 2008). Fish tissue contain a number of bioactive compounds including peptides, fatty acids, and enzymes that may be beneficial within the context of human health (Jemil et al., 2014).

In the world, the requirement of the fish is increasing day by day due to increase in the worlds population, movement of peoples from villages to cities and growing living standards. (World Bank, 2013). As per the research of world bank the fish consumption will increase ten folds in developed nations and in some other patches of the world, such as Taiwan china, subcontinent, where fish farming flourish, which will result in difference between fish production and demand (World Bank, 2013)

The success of fish culture lies in discovery of fishes that have rapid growth, can adapt to the rapidly fluctuating climatic conditions. Aquaculture is growing very rapidly and providing excellent quality food specially protein to the world. The fastest growth in aquaculture began after 1970 due to improvement in fish breeding technology and with the construction of fish hatcheries. Aquaculture is at the top with an annual average growth rate of 8.8% per year since 1970 as compared to other animal food producing sectors (FAO, 2007). The total production of aquatic food such as fishes, mollusks, crustaceans and other aquatic organisms were 3.9% of total production in 1970 and 27.2% in 2001, 33% in 2004 (Megbowon et al., 2013; Maradun et al., 2018)

Catfishes form an important group and belong to the Order Siluriformes. They are called catfishes because of the presence of thread-like barbels on the snout just like the whiskers of cat (Herrel et al., 2005). They attain a good size ranging from 5 cm to 3 m. Catfishes are resistant in nature and can remain alive out of water for a long period. Some fishes have accessory respiratory organs and have the ability to respire in air and mud(Olson et al., 1995). They are valued as food fishes in many parts of the world and also used for sports and as a ornamental fishes in a glass aquaria. Several catfishes are venomous and can inflict severe wounds with their spines particularly those of pectoral fin (Gjedrem et al., 2012; Ali et al., 2015).

There are about 30 families and more than 2000 species of catfishes in the world. Majority of them are distributed in freshwaters, although a few (family Tachysuridae, Plotosidae, etc.) occur in brackish and marine waters.

Clarias gariepinus is a very important catfish, farmed in Africa, even though more than 100 species found in African waters. Clarias gariepinus is commonly known as African Catfish, Mud catfish and Sharp toothed Catfish. In Africa, the African catfish is indigenous fish of the following countries: Algeria, Angola, Botswana; Cameroon; Central African Republic, The Democratic Republic of Congo, Ethiopia, Egypt, Guinea, Ghana, Israel, Jordan, Kenya, Libya, Malawi; Mozambique, Nigeria, Rwanda, Somalia; South Africa and Sudan (Doadrio., 1994; Huisman and Richter., 1987; Maradun et al., 2018).

The culture of mud cat fish prolonged outside its natural waters and was cultured in Argentina; Bangladesh; Brazil; China; Greece; Bangladesh; India, Indonesia; Iraq; Myanmar; Thailand and Phillippines and Pakistan.

African Catfish Clarias gariepinus belongs to family claridae. Catfish are a sub order of fish grouping several families, all of which are more or less similar in appearance. They are found almost throughout the world. Their bodies may be either long or short but are generally cylindrical in cross section and thick. The skin is generally naked i,e without scales. One to four pair of barbels, which sometimes are very long , are characteristic cat fish, and it is because of this characteristic the fish got the name “Cat fish” (Baras and Jobling, 2002: Kasi et al., 2015).

The cultivation of clarias is very popular in fareast and Africa specially in Nigeria and Egypt. Thialand is also getting high production. Two species are raised: Clarias batrachus and Clarias gariepinus. These fishes are popular for cultivation because they grow rapidly and also because they give high production and are sturdy and resistant. They can withstand transport out of the water over long distances, and can also survive in water which is poor in oxygen and which is not suitable for other species. Clarias can live under all conditions and can be raised in a restricted environment. Generally Clarias gariepinus is omnivorous in nature. (Kumar et al., 2007; Kumar et al., 2017) Their feeding habits are very varied. They eat insects, plankton, worms, crustaceans, toads, frogs, small fishes, even rats, small snakes, decomposing organic matter and also fish meal, rice and rice bran as well as chopped vegetables. They readily accept manufactured diets at all life stages and demonstrate good growth and feed conversion on relatively low-cost diets.

African catfish breeds naturally during the rainy season in flooded river, paddy fields and earthen ponds. Sexes can be recognized at the period of reproduction because the genital aperture is different and the belly of female is swollen and vent is reddish in color. The import of fry from Thailand and Africa of this specie is very expensive and not reliable for culture. To eliminate these problems, induced breeding is thought to be the only solution for fish fry production and supply to fish farmers. Further, the rearing of the wild collected brood stock 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 (Brzuska, E,. 2004). Over the last half century, 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)

Among differnt hormones used in fish induced breeding, salmon gonadotropin releasing hormone (sGnRH) or luteinising hormone releasing hormone (LHRH) analogues in combination with dopamine antagonists proved to be the best agents in fish artificial breeding (Lin and Peter, 1996). Several breeding hormones have effectively been used in various fish species including common carp and cat fishes. Human chorionic gonadotropin (HCG), proved to be very effective in inducing spawning in catfish (Legendre et al., 2000; Adebayo and Fagbenro, 2004). Formation of Ovaprim transformed artificial breeding technique and commonly applied for induced spawning of catfish and in a variety of carp species. Human menopausal gonadotropin (HMG) a innovative hormone was applied for the first time in Pakistan for artificial spawning of Clarias gariepinus with mixture of other hormones (Ahmed, 2018).

Latency period refers to the duration between administration of hormone and spawning, and it is a very important tool to observe any confined breeding researches. Latency period is important parameter in calculating the efficiency of hormones in induced breeding (Hogendoorn and Vismanas, 1980; Legendre and Oteme, 1995). An appropriate mixture of suitable dose of various drugs and stripping time always yield the maximum number of the eggs during artificial breeding, so for the fish fry production of Clarias gariepinus at commercial level, an experiment of its own kind was designed in which variety of hormone sources and /or their analogues were used for artificial ovulation of this important African fish.

Interactions between Central nervous system and environmental signals plays an important role in fish breeding and spawning. In a fish, central nervous system detects the signals from the environment and transmits to the hypothalamus which is a part of forebrain to secrete gonadotropin releasing hormone (GnRH), this releasing hormone stimulates the anterior pituitary gland to release gonadotropins as luteinizing hormone. Then the gonads (Ovaries and testis) are stimulated by this hormone to increase the secretion and release of steroid hormones like progesterone and prostaglandins which in turn helps in the development and maturation of gonads till the ovulation. (sGnRHa) is a synthetic hormone which stimulates the pituitary gland to secrete and release the Follicle stimulating hormone and luteinizing hormone (Ali et al., 2015).

Like major and exotic important carps, artificial breeding of Clarias gariepinus can be another marvelous and beneficial option. Artificial propagation and breeding of this specie can ensure improvement and continuous availability of fish seed to the progressive fish farmers. Now a days the seed of Clarias gariepinus is being imported from Thailand which is time consuming and costly for the fish farmers due to mortality and transportation charges. It will be not wrong if we say that no effort have been made in the artificial breeding of this very important specie. Various inducing hormones are applied for the artificial propagation of different fish species, because spawning of these species is impossible in captivity. Fishes successfully completes the initial stages of oogenesis but refused to spawn in captivity due to lack of inducing factors viz environmental and hormonal factors (Peter et al., 1988; Podhorec and Kouril, 2009).

Human chorionic gonadotropin (HCG) and Ovaprim are widely injected to major carps and Chinese carps as a stimulator for artificial spawning. Each specie is injected intramuscularly at dorsal side near caudal peduncle and lateral line of the fishes with specific quantity of both hormones (Pinillos et al. 2002; Vischer et al. 2003; Hill et al., 2005). Both the hormones are frequently used in fish breeding due to perfect spawning response of fishes and local availability and simplicity in the administration process (Akhtar, 2001). Whereas the fish breeders do not follow the same dose for different fish species due to different environmental conditions (Nandesha et al., 1991). Physical characteristics of the external environment triggers the nervous system which ultimately controls and stimulate the glands for hormonal synthesis and these hormonal changes starts the process of fish breeding which is otherwise impossible under the conditions of captivity (Hochachka and Mommsen., 1995; Luskova., 1997; Svoboda et al., 2000). Therefore in stagnant waters and fish ponds fish requires some hormonal agents to cross this barrier and break the pause in maturation of its eggs. The main purpose of this experiment is to provide important guidelines to the fisheries workers and fish breeders for easily breeding of this valuable exotic specie to save their precious time and money (Huisman and Richter, 1987; Kumar et al., 2017).

Objectives

To induce ovulation by using different hormones in female African Catfish (Clarias gariepinus)
To assess dose dependent ovulation in female African Catfish (Clarias gariepinus) by using different horomnes

REVIEW OF LITERATURE

Hafeez-ur-Rehman et al,.(2015), measured the Effect of various artificial hormones and their equivalents on breeding efficiency of snakehead catfish Channa marulius by means of four different hormonal treatments. Ovaprim (gonadotropin releasing hormone and dopamine antagonist) was injected to males in different percentages with human menopausal gonadotropin (HMG), human chorionic gonadotropin (HCG) & HCG+HMG; while, females were injected mixture of ovaprim with HMG and HCG. After 12, 16, 20 and 24 hours of first dose, ovaprim was injected and blood testosterone, follicle stimulating hormone and luteinizing hormone were analyzed every 4 h for 48 h.

Outcomes shows that mixture of HCG+HMG and ovaprim+HCG were best and reliable inducing hormones for artificial breeding in Channa marulius. Fecundity and egg fertilization rate were highest for HCG+HMG at latency period of 43.20-44.45 h. Body fluid hormonal levels were non-significant (P<0.05) and increased progressively till 28 h of after injection and then reduced slowly. Fish injected with ovaprim+HCG does not spawn at all. Eggs obtained were yellow, spherical, non-adhesive and translucent. After fertilization, first cleavage was observed within 2 h, second was between 3-4 h and after 4-6 h, a shield appeared inside and two-layered structure appeared with an outer epiblast and inner hypoblast. However, no further development was observed as the eggs succumb to fungal infection

Oyekutor and Onisokytu.,(2016). investigated that how the hormones ovaprim and ovulin are effective in the induced spawning of African cat fish Clarias gariepinus, and they successfully calculated their effectiveness. They take eighteen clarias females and six sexually mature males for this experiment purpose. The experiments outcomes shows that the hormone ovulin is significantly more effective (p<0.05) in overall parameters assessed in all the experimented fish specially at 50% concentration as the percentage mean value was respectively 81.00±18.51% and 57.33%±4.15%. The hatching rate shows that the percentage mean value of hatchability in both the hormnes ovulin and ovaprim were measured at the concentration of hundred percent. The fishes that were treated with ovulin were have the highest latency period. It shows that the high dose of both the hormones resulted in low latency time. The females injected with ovulin had more healthy fingerlings and its weight (8.66± 0.53 g). This experiment proved that the ovulin is more effective and cheap than the ovaprim since 0.36 ml of ovulin generated mean weight of 8.66 ± 0.53 g of fingerling at N 180.00.

Oyeleye, et al., (2016), investigated the ability of pituitary gland cells to divide and increase in number in a medium like artificial culture media and its hormones for the artificial spawning of Clarias gariepinus. Three culture media were designed for the culture of these trypsinized hypophysial cells. Culture media were named RPMI 1640, McCoy 5 a and M2 media with the supplementation of 10% fetal bovine serum. The female cat fish of Clarias gariepinus (800±200) that was injected with the hormone of culture media resulted in minimum population repetition time and maximum cell count (RPMI 1640). In vitro fertilization was performed in the female catfish. Accurate and precise percentage of fertilization and hatching were calculated. Sampling of blood of the experimented brood stock were performed before the hormone administration, and after administration of hormone, 3,6,9 and 12 hours respectively. Enzyme linked immunosorbent assay procedure was adopted for analyzing the concentration and difference in blood plasma level of follicle stimulating hormone.

After 12 hours of administration of hormone a significant rise in cell count was observed which was 4.1(106)/ ml after 12 hour of culture. 97.5 g/kg of mature eggs were obtained from female fish that was injected with cultured pituitary cells (CPC), 127.5 g/kg were spawned by the fish injected with fresh pituitary gland (FPPG) and a maximum 157 g/kg was spawned by the fish injected with gonadotropin releasing hormone analogue (Ovaprim of syndel Laboratories Canada) in African cat fishes (800±200 g). The fertilization rate of cultured hypophyseal cells contrasted significantly (P<0.05) lacking significant fluctuations in hatching rate (P>0.05).

The outcomes of the blood GnRH level showed that the maximum concentrations of blood luteinizing hormone and follicle stimulating hormones were touched among 9 &12 hours of spawning in all the brood stock. These 2 hypophyseal based hormones (FSH and LH) showed opposite results while spawning & ovulation of African mud fish(Clarias gariepinus) eggs. Above experiment showed that cultured cells from pituitary can be used for induced and artificial breeding of Clarias gariepinus.

Richard et al., (2016), investigated efficiency of Osteolaemus tetraspis hypophyseal gland hormones on artificial breeding of African cat fish Clarias gariepinus. The experiment was conducted during the months of January and May 2008. The selected site for this experiment was the Nigerian Institute of Oceanography and Marine Research Sipale. 39 brooders of this African cat fish (36 females and 3 males)& 3 alligators also selected for the research work. The average weight of the above said brooders was 800 ± 20 g whereas weight of alligator was 5.0 ± 0.2 kg. 3 replicate treatments were carried out to observe the breeding behavior by applying 3 dissimilar dosages, (0.5 ml, 1.0 ml and 2.0 ml) APG dried in acetones. Spawning behavior was monitored after 11-13 hours of injecting the hormone.

The process of hand stripping was performed for expulsion of eggs from the gonads of female Clarias garirpinus and fertilized with sperms(milt) obtained from male by dissecting and sacrificing the male cat fish of same species and kept for incubation in plastic containers. Hatching takes place after 20-26 hours of fertilization. The temperature of water during the experiment was measured and remain between 25-26°c. Fertilization and hatchability ratio raised (p < 0.05) by increasing the dose of Osteolaemus tetraspis hypophyseal glands hormone. The overall effect of Osteolaemus tetraspis hypophyseal glands hormone on breeding of African cat fish was satisfactory.

Okomoda et al., 2017 conducted an experiment to observe the effect of the preserved and fresh hypophyseal gland extract on the induced breeding performance of the African catfish Clarias gariepinus (Burchell,1822). To determine the survival rate and weight gain performance of the fish fry, test netting was performed in the nursery ponds after every 15 days for the evaluation of effect of this hormone. Twelve males of Clarias gariepinus were sacrificed to remove pituitary gland, 96 percent ethanol was used to preserve six removed Pituitary Glands 24 hour before injection, whereas the second sample was removed by dissecting the male catfish and used in their fresh condition. The females Clarias gariepinus were injected with pituitary gland mixture (1 g ml -1 of saline water) at a rate of 1 ml/kg body weight.

Synthetic hormone Ovaprim of syndel laboratories Canada used as a control and standard and injected to female African cat fish at a rate of 0.5 ml/kg body weight of fish. All the fishes were stripped manually with hand stripping after a same latency period which was 9 h 30 minutes. Eggs were fertilized with milt of same quantity and then incubated to evaluate the efficacy of treatments. Outcomes of the experiment shown that Ovaprim which is a synthetic hormone given best hatching results (64.52%), however, the preserved form of gland extract gave better satisfactory hatching (59.74%) as compared to the freshly extracted pituitary gland mixture (51.39%).

Fry of both groups were fed with artemia cyst whose shell was removed before feeding for 15 days, weight gain and growth of fry of control group was compared with the fry of preserved pituitary extract. In the end the conclusion was clear that the preservation of the hypophyseal gland extract in ethanol for twenty four hour prior to administration into fish had a best effect on induced propagation of African cat fish and can be used in the large scale breeding of Africa cat fish Clarias gariepinus.

Sime., 2017 performed an experiment of artificial breeding on African Mud fish Clarias gariepinus with a synthetic hormone Ovaprim and pituitary extract of African catfish. The brood stock both male and female of Clarias gariepinus were chosen with ages above eight months. Three pieces of pituitary gland extract were perfectly grinded and mixed with 1 ml of water and injected to female broodfish of 1 kg, 1 ml distilled water was mixed with synthetic hormone Ovaprim and injected to female of same weight.

After a constant latency period of 12 hours all the females were hand stripped and the eggs obtained were fertilized with milt of dissected/ sacrificed male. The performance of both the hormones were analyzed by the parameters like total egg weight, fecundity, latency period, incubation time, hatching and survival rate of fry. The females injected with Synthetic hormone Ovaprim yielded eggs with better weight (0.004g/egg) as compared to the egg of fish injected with Pituitary gland (0.003g/egg) . Though the Pituitary gland extract had larger fecundity (100,000±8724) and less number of eggs for the Ovaprim injected Clarias gariepinus which was (75000±5432) however the result of percent hatchability the synthetic hormone Ovaprim exceeded in number(33.73%) value as compared to the fish treated with the Catfish pituitary extract whose value was (29.6%).

Whereas the fish fry weight and survival rate of fish fry was better in fishes treated with Ovaprim whose value was 0.002g and 85% respectively as compared to fishes injected with Pituitary gland extract had value0.001g and 72% of fish fry weight and survival rate respectively. The final weight of fingerling was also better in Ovaprim treated fish (0.09) as compared to the Pituitary gland extract treated fishes (0.066g). finally concluded that the Ovaprim is much more efficient as compared to Pituitary gland for all the parameters of induced breeding except fecundity were Pituitary gland extract given better and higher value.

Abdul Rahman., et al,. (2017), experimented on artificial and induced breeding of Clarias gariepinus by using Synthetic hormones and pituitary extract and studied the growth performance of fish fry at the hatchery unit of the Fisheries Department farm of the Modibbo Adama University of technology Yola, Adamawa State, Nigeria.

Total 12 matured Cat fishes were used for the breeding experiment. Two matured Catfish brooders were dissected for the removal and extraction of the Pituitary gland extract to use in the breeding experiment. Ovaprim which is a synthetic hormone was used as a control (T1) hormone for both the sexes of Clarias gariepinus and injected in pure 100% form. Other treatments were T2, T3, T4, T5, in every group same number of brooders one male and one female were selected and injected with same dose of hormone, T2 (African Cat fish PE, 100%), T3 (Combination of 50% Synthetic + 50% African catfish PE), T4 ( 25% African Catfish PE + 75% Synthetic Ovaprim), T5 (25% Synthetic hormone Ovaprim + 75% African Catfish PE). Highest fecundity was recorded in broodfish of group T2 with (38.37%) minimum was recorded in fishes of the group T4 with (27.88%).

The hatchability and latency periods were recorded among 48 hours and 9.05-12 hours respectively for each female during the experiment. The lowest hatchability (56.30%) was recorded in female African Catfish injected with 100% Ovaprim (T1), highest hatchability (60.99) was recorded in broodfish injected with pure 100% African Catfish pituitary extract (T2). The efficicieny of Combinations of both the hormones were also analyzed 2nd highest hatchability ((59.39%) was observed in fishes injected with 25% Ovaprim and 75% African Catfish extract as compared to % Ovaprim + 50% African Catfish extract. Survival percentage were also recorded and follow the same ratio as hatchability. Highest survival rate was recorded in females of group injected with pure 100% African Catfish Pituitary extract (92.60%) and minimum survival rate was recorded in fishes injected with 100% Ovaprim (63.45%) growth rate of fish fry also shown clear differences in all the different groups treated with different hormones; maximum growth rate was observed in T2 ranging from of 0.029-0.236 and the minimum was recorded in T4 with 0.029-0.200. Some other parameters observed include was the Mean weight gain and the highest recorded in group T2 with 14.01 and minimum was found in group T4 which was recorded 0.171.

highest specific growth rate was observed in group T2 with 14.01 and minimum was observed in group T4 with 16.96. The overall results indicated that Ovaprim and African Catfish Pituitary extract induced ovulation and spawning in Clarias gariepinus (African Catfish), whereas the female African catfish injected with African Catfish Pituitary extract showed higher hatchability and the fish larvae produced from Catfish Pituitary extract injected females had maximum survival rate,

Natea.,et al.,2017 evaluated the breeding behavior of African catfish Clarias gariepinus using various piscine and artificially synthetic hormones. Three different treatments were designed and each treatment contained three gravid females, all the females were injected with the hormone intramuscularly. Different parameters like spawning, fecundity, fertilization, incubation hours, survival and hatchability rate were studied and collected. The group of gravid females injected with African catfish pituitary extract showed maximum number of eggs (fecundity) (9731.6 eggs/g) followed by the group of gravid females that was injected intramuscularly with pituitary extract of Gulfam fish or Common carp (Cyprinus carpio) and yielded (5813 eggs/g).

whereas the females of Clarias gariepinus of different groups did not show significant difference (p=0.073) in spawning fecundity/g body weight/ female. Gravid females injected with pituitary extract of African catfish shown maximum mean hatchability rate (73.3%) followed by the group that was injected with common carp pituitary extract (63.5%). The experiment concluded that the use of African catfish pituitary extract for induce breeding of Clarias gariepinus is more efficient as compared to the pituitary extract of the Common carp (Cyprinus carpio) and synthetic hormones.

Mullera.,et al., 2018 used the in vitro method of fertilization in African catfish Clarias gariepinus and successfully induced the process of spawning in fish. In practice normally different hormones are administered in fishes intramuscularly in a very precise and calculated manner. Intraperitoneally hormones are injected with a special syringe and a needle to induce spawning and release of sperms by the male fish. We experimented a unique and new method to fertilize the eggs inside the ovary of the fish by placing sperms and hormone together with a special catheter and syringe, and concluded that a mixture of hormone and sperms injected inside the ovary of fish can induce breeding and successful ovulation in Clarias gariepinus and collection of developing embryos in different experimental methods applied to fish. The outcomes of the experiment shown that injected sperms and carp pituitary extract are stored and remain alive in the ovarian lobes for up to 10 hours and retain the metabolism and biological activity and the eggs inside the ovary can be fertilized by the injected spermatozoa, eggs after ovulation from the ovary and released from fish body cavity. Seminal fluid also played a role as a vehicle for sperms and ovulation inducing hormone via ovarian lavage.

Maradun., et al., 2018, investigated the effect of various sex ratio (male-female ratio) on the breeding performance of African catfish Clarias gariepinus by conducting an experiment on induced breeding. National institute for Freshwater Fisheries Research, New Bussa provided the broodstock for the experiment. The experiment was performed at the Fish hatchery of Happy Island Garden, Sokoto. Completely randomized design was used for the experiment with treatment combinations as follows; (Treatment A) 1:1 male-female ratio, (Treatment B) 1:2 male-female ratio, (Treatment C) 1:3 male:female ratio. The female African catfish Clarias gariepinus were administered with the recommended dosage of Ovulin 0.5 ml/kg body weight of fish whereas males were given half dose.

Maximum fertilization rate (71.6%) was recorded in treatment A and hatchability rate (84.54%) and survival rate (73.17%) followed by Treatment B with fertilization rate (68.69%), and had the minimum value for hatchability (80.58%) whereas survival rate (72.13%). The minimum value of fertilization (64.25%) was existed in Treatment C but followed treatment A in terms of hatchability (84.54%) whereas survival rate (72.13%). Breeding performance in different groups indicated no significant difference (p>0.05) among the treatments. This experiment indicated that good quality of eggs and larvae can be obtained from Clarias gariepinus at different broodstock ratios even the small sized 600g female African catfish breed well with Ovulin.

DiMaggio.,et al., 2014 performed an experiment on Pigfish, Orthopristis chrysoptera, and he investigated that this fish readily undergo the vitellogenesis when kept in captivity, but the spawning, and ovulation becomes random due to the delay in final oocyte maturation (FOM). The major purpose of this experiment were to investigate the efficacy of different doses of human chorionic gonadotropin (HCG) and Ovaprim (sGnRHa) [salmon gonadotropin-releasing hormone analog] + domperidone) on spawning, ovulation and final oocyte maturation in Orthopristis chrysoptera. Different doses of ovaprim used were 0.25, 0.50, 1.00, and 2.00 ml/kg, that was administered at the dorsal musculature near lateral line of the female pigfish,, Orthopristis chrysoptera. Male Orthopristis chrysoptera the Pigfish was administered half the dose of hormone as to female Orthopristis chrysoptera were injected.

Different doses for human chorionic gonadotropin were assessed and injected to female Pigfish Orthopristis chrysoptera were 500, 1000, 2000, and 4000 IU/kg, but the male Pigfish Orthopristis chrysoptera was injected half the dose of female fish to make sure the process of spermiation. Spawning started after 72 hour of injection of hormone and fertilized and then the hatching percentage and survival to first feeding was assessed. Photographs were taken at each step of ovulation and larvae and morphology was determined. Experiment indicated that low doses of synthetic hormone Ovaprim (0.25 and 0.50 ml/kg) had better results as compared to the higher doses. Whereas the better quality of eggs and larvae indicated that the 0.50 ml/kg dose as the best choice for breeding of Orthopristis chrysoptera. Out of four different doses of human chorionic gonadotropin hormone injected to female pigfish Orthopristis chrysoptera responded unreliable in this experiment, therefore no dose can be recommended for induced breeding of Orthopristis chrysoptera.

Yisa., et al. (2014) investigated that matured African catfish Clarias gariepinus whose eggs range in size from 550-800 g total body weight were disinfected for 60 seconds with formalin at a concentration of 0.5 ml and 1ml formalin to find out its effect and benefit on hatchability, growth performance and survival of fish fry. Three replicates of each treatment were used. The selected site for research was Toxicology center at fish farm, Federal university of Technology, (FUT), Bosso Campus, Minna. No significant difference was found in mean fecundity (P<0.05) between 1ml, and 0.5 ml (64.43a and 54.59b) and (62.48a and 42.59b) respectively. Total mortality was recorded and was 15, 13, and 20 for 0.5 ml, 1 ml and control respectively when the bred maintained for eight weeks. Significant difference was recorded in Mean Total Body weight (TBW) that was (P<0.05) between 1ml (3.27a) and 0.5 ml (0.82b). Better results were recorded for fertilization, hatchability and growth efficiency in treatment No. 3. This experiment indicates that formalin at a concentration of 1 ml for a treatment period of 60 seconds is suitable to disinfect the eggs. This kind of disinfection is very beneficial in terms of survival of fish fry of Clarias gariepinus and hence recommended before incubation of eggs.

Yisa., et al., (2013) used matured and sexually ripe African catfish Clarias gariepinus with size between 400-650g average total body weight and conducted an experiment. They performed their experiment at Federal University of Technology, (F.U.T) Minna, Bosso campus used indoor hatchery complex. The broodstock was obtained from a private fish farm on payment and carried to the research site in a perforated 50 liter jerry can.

The male and female broodstock was provided an optimum temperature and water quality for two weeks and fed with 30% crude protein feed to sexually mature the fish. During the induced breeding experiment an incision was made in the abdominal region of the male African catfish through (AIM) Abdominal incision method to take milt from the testis of the male catfish. Volume of milt in various males differ significantly and maximum milt extracted from testis to be (0.86±0.006a). The spent males was reused after 45 days and can be used for a maximum of 6 times in a year.

Maximum percentage survival existed (75.20%) which never significantly (P>0.05) differed, and the fingerlings fed for 12 weeks and SD obtaine was (1.789) and ±SEM (0.516). Eggs were fertilized by the milt of dissected catfish. African catfishes have greater fecundity as compared to the small Clarias gariepinus and significantly differed (P<0.05) from each other. The male African catfish survived after surgery and healing within 14 Days of surgery. The most suitable and effective incision length 3.40 cm was followed during surgery of male fish. These males of Clarias gariepinus can be used again and again for induced breeding through abdominal incision or for genetic studies without killing the male brooders although adequate nutrition should be given to these males for early healing and immunity as well as development of gonads.

Olumuji, O.K., and Mustapha M.K., (2012), investigated on induced breeding of African catfish Clarias gariepinus using diluted synthetic hormone Ovaprim. In West Africa Clarias gariepinus is a very important farmed fish whose artificial spawning is performed by very costly synthetic hormone Ovaprim. The purpose of this experiment is to minimize the expenditure of farmer by diluting the costly hormone by normal saline in artificial breeding of Clarias gariepinus. 5 various doses of normal saline diluted ovaprim at 0%, 25%, 75%, and 100% were injected to fish to assess the breeding performance of African catfish Clarias garirpinus, whereas pure Ovaprim used as the control.

Fishes of all treatments were injected with recommended dose of Ovaprim which is 0.5 ml/kg body weight of the fish. Fishes were divided into treatments A, B, C, D and E. stripping was done by hand pressing of the belly of female fish, mean weight of the ovulated eggs were 18.45 g, 17.50 g and 17.25 g in treatments A, B and C respectively with no significant difference (p<0.05) in the values. The fishes of the treatments D and E did not spawn, there was no significant difference (P<0.05) in the value of percentage fertilization in the groups A, B and C respectively. The fertilization rate of ovulated eggs were 88.70%, 87.50% and 77.38% in the groups A, B, and C respectively.

Treatment A showing significant difference (p<0.05) as compared to treatment B and C. hatchability percentage of the ovulated eggs in the treatments A, B and C were 56.58%, 54.07% and 57.75% respectively. No significant difference (P<0.05) existed between the groups. Whereas the survival of fry were recorded 40.27%, 40.87% and 42.52% in treatment A, B and C. No significant difference (P<0.05) recorded in the groups. Overall the experiment shown that the 50% normal saline diluted group is the most cost effective group. Experiment indicated that 50% normal saline diluted ovaprim can induce spawning in Clarais gariepinus with maximum hatching rate and survival of fish larvae.

Achionye., and Obaroh., 2012, conducted artificial breeding of African catfish Clarias gariepinus using various doses of synthetic hormone Ovaprim to assess the dose that yield maximum number of eggs. Various doses of Ovaprim 0.5 ml/kg, 1.0ml/kg and 1.5ml/kg were administered to female Clarias gariepinus. While the females African catfishes of control groups were injected with 0.0ml/kg of Ovaprim. The eggs were fertilized by the milt of dissected and sacrificed male of Clarias gariepinus. The fertilized eggs of all the different groups were placed in separate netted fabrics in water containers inside the laboratory.

The outcomes of the experiment indicated that higher dosage of Ovaprim increases the maturation of eggs in fish ovary, so maximum number of eggs are spawned by the fish injected with 1.5ml/kg of Ovaprim. It minimizes the latency time in female fish. Lowest number of eggs were collected from the fish that were injected with 0.5ml/kg of Ovaprim and the fish never survived due to stress while stripping of eggs. No mortality was observed in the group of fishes that were injected with 1.0ml/kg of Ovaprim and eggs released by the fish by gently pressing the belly. Therefore the the dose 1.0ml/kg of the fish body weight is recommended for the induced breeding of Clarias gariepinus. Maximum dose which is 1.5ml/kg can be injected if the farmer has the area for the rearing of the maximum number of fish seed.

Kipper., et al 2013, performed an experiment in order to help fish farmers to identify the eggs, larvae and juveniles of Clarias gariepinus, they investigated the developmental stages of Clarias gariepinus due to its importance in aquaculture and its farming worldwide. All the experimental stages of this fish were obtained by induced spawning. After breeding of Clarias gariepinus samples were taken at different stages and with varied periodicity as per ontogenic development. 6 juveniles, 146 fish fry (larvae) and 12 eggs were analyzed during the experiment.

The density of the eggs varies and the eggs of Clarias gariepinus are spherical, having a double membranes. At the beginning the fertilized eggs have pigmented spot which becomes clearer and darker during further development. Clarias gariepinus have 4 pairs of barbels, dorsal and anal fins are elongated, and lacks adipose fin. Caudal fin develops first then dorsal, anal, pectoral and pelvic respectively. Metamorphosis occur at the flexion stage of development. This specie is ideal for aquaculture because its reproductive organs develops earlier than other catfishes. This characteristic makes it invasive specie for natural environments and dangerous for the extinction of native species.

Haniffa., and Sridhar., 2002, used human chorionic gonadotropin (HCG) and Synthetic hormone Ovaprim for the induced breeding of the spotted murrel (Channa punctatus) and catfish (Heteropneustes fossilis). The matured broodstock was injected with varied single doses of the hormones at the dorsal musculature near caudal peduncle. No of eggs (Fecundity) in H. fossilis was 6692 ± 790 for ovaprim and 82922 ± 5432 for HCG. No of eggs (Fecundity) in C. punctatus was 3273 ± 75 for ovaprim and 1253 ± 126 for HCG. Channa punctatus spawned successfully with 0.3 and 0.5 ml/kg body weight of Ovaprim whereas human chorionic gonadotropin hormones optimum dose was 2000 and 3000 IU/kg body weight of fish. Whereas the catfish (Heteropneustes fossilis) spawned successfully at the dosage of 0.3, 0.5 and 0.7ml/kg for Ovaprim and 1000, 2000, and 3000 IU/kg body mass for Human Chorionic Gonadotropin.

Hafeez-ur-Rehman, et al., 2015 investigated on the efficiency of different doses of synthetic hormone Ovaprim on the spawning behavior of Snakehead catfish Channa punctatus. Before starting the experiment the fish were prepared for induced breeding and fed on pelleted feed containing 40% protein at 5% of the body weight of broodstock. After four months of feeding the females breeders were weighed 1150-1350 g and males were 1100-1340 g. pairing was done randomly. Two males and two females were selected for each pair.

The groups were named as treatment 1, 2, 3 and the control group.(two replicate tanks). Ovaprim was administered in two doses after the interval of 24 hour intramuscularly near the caudal peduncle dorsally just above the lateral line. Blood samples were obtained from the experimented fishes after the interval of 2 hours for the estimation of hormonal changes like testosterone, FSH, and LH concentrations. All the fishes did not responded and never spawned at all even after the duration of forty eight hours.

Each female Channa punctatus were dissected for the removal of ovaries, eggs were counted and fecundity of the female brooders were estimated that was 871.1 ±75.37, 812.3 ±104.20, 901.0 ±95.57 and 941.0 ±39.80 in Control group, treatment 1 , Treatment 2, and Treatment 3 respectively. The value of diameter (Mean) of egg recorded was 1.50 ±0.03 to 1.64±0.01mm. The minimum mean egg width (1.49±0.02 mm) was recordrd in control circular breeding tank (0.4 ml Ovaprim/kg) whereas the maximum (1.55±0.01 mm) was recorded in Treatment 1 (0.5ml Ovaprim/kg). The maximum bllod testosterone concentration was recorded in treatment no. 3 that was (2.05±0.11 ng ml-1) after the duration of twenty four hours after 1st dose of Ovaprim. The level of follicle stimulating hormone (FSH) was maximum (0.88±0.02 ng ml-1) in the same group. The experiment proved that the dosages of synthetic hormone Ovaprim are insufficient for the artificial breeding of this fish.

Ndimele., and , Owodeinde, 2012, Carried out an experiment to measured the relative and comparative growth, reproductive efficiency and consumption of various nutrients in the hybrid of Clarias gariepinus namely “heteroclarias” and in the wild Clarias gariepinus. Six females of Clarias gariepinus were taken for the experiment, out of six females two were injected with ovaprim, two with male pituitary of Clarias gariepinus whereas two were injected with pituitary gland extract of female C. gariepinus.

Three males of Heterobranchus bidorsalis and three of Clarias gariepinus were taken. The two females that were injected with artificial hormone Ovaprim were fertilized separately with male of Heterobranchus bidorsalis and Clarias gariepinus. Eggs of Other two female groups that were injected with pituitary gland extract of male and female Clarias gariepinus were also fertilized with the same pattern. The outcomes proved that there was a significant difference (P<0.05) exist between various groups in terms of all the parameters observed like growth, reproductive success and utilization of nutrient uptake. The maximum %age of fertilization (87.45±8.08g) protein uptake (16.40±4.0) feed intake (26.45±8.0) FCR (1.20±0.15) and protein efficiency ratio (1.75±1.19) were calculated in wild line of African catfish Clarias gareipinus injected with the synthetic hormone. Reproductive success were also recorded and found maximum in the wild type C.gareipinus. the above experiment therefore proved that the larvae of Clarias gariepinus produced by injecting artificial hormone Ovaprim showed good qualities as compared to the larvae resulted by injecting with pituitary gland extract of male and female Clarias gariepinus.

MATERIALS AND METHODS

Study area

The experiments were conducted at Government Fish Hatchery and Farms Ghazanfargarh near Khangarh district Muzzafargarh, Punjab, Pakistan during the months of June and July 2018. The fish farms and hatchery complex were constructed by the Fisheries department government of the Punjab, Pakistan during the year 2014-2018. The purpose of this fish farm and hatchery is development of fish farms at saline and brackish waters by producing fish seed of salt tolerant species like tilapia, pangasius, African catfish etc. 48 specimens of Clarias gariepinus were used for induced breeding experiment, brood fishes weighed about 2.4 to 2.6 kg. These experiments were performed in circular spawning tanks with 2 m in a diameter with optimum water quality parameters. Ovaprim, human chorionic gonadotropin and human menopausal hormones were injected in a single dose. These experiments were done to produce fish fry of African catfish for the very first time in Pakistan for the farmers of Pakistan and Punjab Fisheries department.

Procurement of Fishes.

Broodstock of Clarias gariepinus were imported by the Punjab Fisheries department in march 2017 from Asian Institute of Technology (AIT) Thailand, and raised for one year at the Government fish farms and hatchery Ghazanfargarh, district, Muzzafargarh. Before stocking in broodstock pond, the fishes were disinfected with 0.5 % salt bath (5 g NaCl / 1 litre water) as recommended by (Tonguthai et al., 1993). Bathing was done by dipping the fish into the solution for fifteen minutes. Salt bath is effective against ectoparasites, bacteria and fungi and also reduces stress (Tonguthai et al., 1993).

Thailand is major producer of catfish’s specially African catfish Clarias gariepinus. The brooders were fed by artificial pelleted feed manufactured by AMG aquatic feed company Lahore, containing 30% crude protein at the rate of 3% of body weight up to January 2018. Last five months from February to May 2018 the brood fishes were fed with feed containing 40% crude protein at a rate of 5% of their body weight to prepare the fishes for breeding experiment. 1000 broodfishes were stocked in a one hectare fish pond. Water quality parameters of the brood stock fish pond were maintained by regular addition of fresh water and by use of aerator for dissolved oxygen. pH of the broodfish pond was maintained by adding lime in the pond fortnightly.

Selection and handling of brood fishes.

Well matured Brood fishes with homogenous size of Clarias gariepnus of good quality were selected from the bulk stock because healthy and well matured brood fishes are the two conditions for successful induced spawning of fishes (Muir and Robert.,1985). Therefore, the ideal size of the broodfishes is from 2.5 to 3.5 kg, bigger fishes are difficult to control and handle during the induced spawning experiment and the expenditure also increased due to requirement of high doses of Ovaprim, human chorionic gonadotropin and human menopausal hormone.

At the start of breeding season mature brooders (2543.25±10.39 g) to (2545.75± 7.08 g) were selected for induce spawning using the method of Ayinla et al. (1989). Sexual distinction can be made by observing the external portion of the primary sexual organs or genitals, on the belly or ventral side of a fish. Two openings are present, the anus and the genital opening. The opening nearest to the head is the anus while that nearest to the tail is the genital opening. In the male of Clarias gariepinus genital opening has an appearance of a tiny raised nipple known as the genital papilla.

In the female the genital opening is rounded and flat, prior to spawning the genital opening of the female Clarias gariepinus may be swollen and reddish in colour with folds of skin, appearing on the each side of the urinary and genital openings.

At the day of experiment brooders were captured by using drag net from the brood stock pond early morning. All the brooders were physically checked for examination of maturation according to the method of (Blythe et al., 1994). Mature females of Clarias gariepinus were netted out for experiment by the examination of belly and vent by applying gentle pressure on the belly by finger. Few eggs were released on gently pressing the belly with finger. Mature gravid females were picked out based on bulging, well distended soft and swollen belly, reddish vent. Females of homogenous size with light green colored eggs were selected. Matured males were also netted out on the basis of their reddish pointed genital papillae.

The belly of the male brood fishes were gently pressed and those males which have reddish genital papilla and comparatively soft belly were selected for the induced breeding experiment. These selected brood fishes were then transferred to the fish hatchery complex and kept in the cemented holding tanks. The sex ratios of four males to four females were used to get the best results during the induced breeding experiment.

Conditioning and shifting of brood stock

The selected brood stock of C. gariepinus were shifted to the fish hatchery complex and kept for 12 hours in concrete holding tanks for conditioning and acclimatization before the experiment of induced breeding. Both males and females broodfishes were kept for conditioning and acclimatized in separate concrete holding tank of two meter diameter.

Experimental Desgin

Hatchery raised 24 months old brooders of African catfish Clarias gariepinus of homogenous size were selected. These brooders were selected on the basis of morphological characteristics, as described earlier by Ayinla et al., (1989). The experimental procedure was laid in a completely randomized design (CRD). 24 females and 24 males of Clarias gariepinus with body weight ranges between 2430 g to 2475g were selected for the induced breeding experiment. Fishes were divided in 6 treatment groups. Four females and four males were considered for each group. The six treatments used in the experiment were T1 (control group) = normal saline, T2 (standard) Ovaprim 0.5, T3 (HCG), T4 (HMG), T5 (Ovaprim+HCG), T6 (Ovaprim+HMG). Four females and four males were randomly selected for each group and kept in a separate indoor tank.

Hormone injection.

The synthetic hormone used in this experiment was Ovaprim which is the product of Syndell Laboratories, Canada and is being used for induced breeding of many species of Carps, Catfishes as well as for ornamental fishes Since 1990. Ovaprim is a liquid hormone contains sGnRH(D-Arg6, Pro9,) and domperidone. Ovaprim contains salmon gonadotropin releasing hormone (20 µg) and 10 mg of a dopamine antagonist domperidone (Brzuska and Adamek, 1999). The standard dose recommended by the syndel laboratories which is manufacturer of this valuable hormone is 0.5 ml/ kg body weight of the female fish. Whereas the recommended dose is not suitable for different species and it varies species to species and also differ with respect to location and climate of different countries. The human chorionic gonadotropin (HCG) available in Pakistan with the brand name Ferti-C of RG Pharmaceutical (Pvt.) Ltd. were purchased. The HCG was mixed with solvent provided by the company with hormone in pack. The experiment were carried out in six indoor concrete circular tanks having water holding capacity of 1500 liters and four holding tanks with water holding capacity of 1600 liters measuring 1.5m×2 meter×1meter.

Males and females of the African catfish Clarias gariepinus of each group were placed in separate circular tanks. Each circular tank randomly received four females, and four males. The brooders kept in tank No. 1 were served as control (Group A) and injected with 0.5ml/kg body weight with physiological normal saline solution of NaCl. The brood fishes kept in tank No. 2 served as standard (Group B) and were injected with recommended dose of Ovaprim which is 0.5ml/kg of body weight of the female fish. The fishes of the group C were kept in tank No.3 and injected with 0.5ml/kg of the human chorionic gonadotropin. While the fishes of the group D were placed in tank No.4 and received 0.5ml/kg of the human menopausal gonadotropin.

A combination of Ovaprim and human chorionic gonadotropin (0.3ml Ovaprim+0.5 HCG) per kg were injected to the fishes of group E kept in tank No. 5, while the females of group F were kept in tank No.6 and received a combination of Ovaprim and human menopausal gonadotropin(0.3+0.5ml) per kg body weight of the fish.

Whereas all the males were administered with a uniform dose 0.2 ml/kg body weight of the same hormone Ovaprim. All the experimental fishes were shifted to their respective circular or holding tank after the administration of the hormone. All the fishes received a single dose of the hormone. The injection were administered at the dorsolateral side of the fishes above lateral line near the caudal peduncle.

It was make ensured that syringe 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 fluid was injected slowly. All the equipment’s were cleaned before the experiment. The needle of the syringe was sterilized with cotton swab soaked in ethanol before the administration of hormone. Trapped air in syringe was removed by keeping the syringe in upward direction and by gently squeezing the syringe. All the precautionary measures were followed during experiment and hand nets were used to capture the fish from indoor tanks and the kept in cloth bag and then weighed.

Towels were used to hold the fish during administration of hormone to avoid the slipping of the fish. All the tanks continuously received aerated fresh water of the turbine installed at the fish hatchery. During the course of the experiment temperature was recorded 28-28.5, dissolved oxygen 5.74-5.78 mg/l, pH 8.87-8.9, salinity 0.82 ppt, Electrical Conductivity 1854 us/cm and Total dissolved solids 1080 mg/l. After eight hours of administration of the hormone the females were observed to check their spawning behavior. The females showed restlessness after 9 hours which is the sign of spawning behavior. This activity remained for 30 minutes then the females were netted out for hand stripping.

Stripping

Females were checked after 6 hours of injection for spawning and continued after each hour till the start of stripping. During the course of stripping the belly of the females African catfishes was pressed gently towards the caudal fin, then the eggs were released from the genital pore like beads and these eggs were collected in a plastic bowl. Four females were randomly selected for each combination. All the females of each treatment were hand stripped and eggs were kept in a separate pre weighed plastic bowl to record the breeding performance. The female African catfish that spawned a stream of clear and transparent green brown eggs was considered as completely ovulated. The weight of stripped eggs of all four females were pooled. The mean was taken as average weight of stripped eggs for a particular combination. The number of eggs stripped was estimated by subtracting the weight of eggs from the weight of female brooder that spawned and multiplying the difference by 680 (one gram=680 eggs), (Viveen et al., 1985). The number of eggs spawned by the each female was again counted by volumetric method. One gram eggs were taken out and counted (triplicate counting was made) and the average of triplicate count was taken as the number of eggs in one gram. The eggs of each female were weighed and multiplied with the average number of eggs already counted in each gram of eggs. Fecundity of each female was determined using the same volumetric method.

Milt extraction and Fertilization of eggs

During stripping of the female Clarias gariepinus the male African catfish were captured by using hand nets, killed and dissected for removal of testes. Milt was obtained by using sharp blade and scissors to cut open the testes. The milt was mixed in a small bowl containing 0.9 % saline solution. The stripped eggs were fertilized by using semi dry fertilization method was poured on eggs and were mixed by using soft bird feather for 3-4 minutes to ensure maximum fertilization. Then the fertilized eggs were washed with water for 8-10 minutes, so they gain water and attain the size of 1.2-1.4mm in diameter. Fertilization rate was investigated by taking a sample of 100 eggs from the stripped eggs and examined by using the binocular microscope. Whitish unfertilized eggs were taken out by careful examination after 10 hours of fertilization, placed in separate petri dish and counted (Ayinla, 1988)

Fertilization % = × 100

Incubation and Hatching of fertilized eggs

The fertilized eggs of each combination were separately incubated in indoor tanks on submerged nylon mesh (kakabans). A net having very minute mesh size were spread in each incubation tank to avoid the flow of eggs with water of outlet pipe. Tanks were disinfected with fungicide (malachite green, 0.02%) to minimize the chances of infectious diseases of eggs. The continuous flow of aerated water was maintained in incubation tanks to ensure the maximum hatchability. Continuous supply of aerated water was ensured by using overhead reservoir and aeration chamber installed at the government hatchery ghazanfargarh.

Water was sprayed continuously over the fertilized eggs in incubation tanks by using perforated plastic pipe at a rate of 14 liters water per minute. The level of water in each tank was maintained by using stand pipe. The sticky eggs then left for 20-24 hour for incubation at a temperature of 28.1°c-28.5°c.

(Lambert, 2008)

Survival rate:

Survival rate of hatchlings were also determined by using the method of (Adebayo, 2006) and (Lambert, 2008)

Percentage of deformed larvae:

A samples of hundred larvae from each group were taken and counted to determine the percentage of deformity. Deformed larvae had curved tail and shortened body. The percentage of deformed larvae and normal larvae was observed as suggested by (De Leeuw et al., 1985).

Statistical Analysis:

One way analysis of variance (ANOVA) and Tuckey test was applied by using SPSS version 20 to determine the significant differences (p<0.05) in reproductive performance parameters.

RESULTS

4.1 Physicochemical Parameters:

During the course of breeding experiments of Clarias gariepinus water quality parameters were observed regularly by using different digital meters and the parameters remained in optimum range set suitable for African catfish Clarias gariepinus.

4.1.1 Temperature.

Temperature was measured regularly after the interval of one hour during the breeding experiment. The value of temperature was observed 28-28.5°c in all the experimental tanks. The temperature was controlled by regular supply of fresh underground water of turbine through a overhead reservoir and aeration chamber installed at hatchery complex. Mean temperature of various tanks is given in the table No.4.7

4.1.2 Dissolved Oxygen (DO)

The concentration of dissolved oxygen was measured with the digital DO meter and fall in range between 5.72-5.80 ppm. During the induced breeding experiment no significance difference exist between the various groups and tanks. Concentration of dissolved oxygen fluctuated slightly but fall in optimum range of African catfish. Aeration chamber was used to mix oxygen in underground water. Continuous supply of fresh underground water through aeration chamber reduced the risk of oxygen depletion in indoor breeding tanks. Concentration of dissolved oxygen is given in the table no. 4.8

4.1.3 pH

Digital pH meter was used to measure the exact pH of various indoor breeding tanks and was observed in range between8.80-8.90 as shown in table No.4.9

4.1.4 Electrical Conductivity (µs/cm)

Mean Electrical Conductivity of the water measured at research site varies from 1842.34±0.4 to 1854.25±0.76 µs/cm during the breeding experiment as shown in table No. 4.10

4.1.5 Salinity (ppt)

The mean salinity of water of different indoor tanks measured during experiment was 0.81±0.01 ppt to 0.84±0.01 ppt as shown in table No. 4.11

4.1.6 Total Dissolved Solids (ppm)

Mean Total dissolved solids of water measured at research site was range in value between 1080.65±0.81 to 1082.41±0.68 as given in table No. 4.12

4.2 Ovulation of the catfish

4.2.1 Body weight of the brood fishes.

The body weight of brood stock is very important with regards to its gonads weight and fecundity. The gonads weight increased as the weight of fish increases. The mean body weight of female brood stock range from 2543.25±10.39 to 2545.75±3.56 as shown in table No.4.13.

4.2.2 Latency period (Hours)

The latency period refers to the duration between administration of hormone until the ovulation. The latency period is very important factor in estimating the breeding and spawning success of various hormones. The minimum mean latency period 9.27±0.11h was shown by the fishes of the group E while the maximum mean latency period 15.15±0.14h was observed in group C as shown in table No. 4.14.

4.2.3 Spawning Success (%)

The maximum mean spawning success percentage (98.25±0.85) was observed in group E while the minimum mean spawning success percentage (41.00±0.7) was observed in group C as shown in table No. 4.15.

4.2.4 Total Egg weight (Grams)

The highest mean total egg weight (460.00±10.60) was observed in group E treated with a combination of Ovaprim+ HCG while the minimum mean total egg weight (128.25±4.62) was observed by the group C and no spawning takes place in group A and D as shown in table No. 4.16.

4.2.5 Fecundity

The highest mean fecundity/ Ovulated eggs (322000±7424.62) was observed in group E treated with Ovaprim+HCG while the minimum mean fecundity (89775±3237.89) was recorded in group C. Significant difference (P>0.05) exist in group E as shown in table No. 4.17.

4.2.6 Fertilization rate (%)

The highest mean fertilization rate (83.00±0.54) was observed in group E injected with a combination of Ovaprim + HCG, while the lowest mean fertilization rate 69.50±0.54) was observed in group C injected with only HCG as shown in table No.4.18.

4.2.7 Incubation period (Hours)

There was no significant difference (P>0.05) between the groups in incubation period because of constant temperature in all the tanks. The mean incubation period range from 23.67±0.01 to 23.90±0.03 hours as given in table. No.4.19.

4.2.9 Hatching Percentage.

The maximum mean hatching percentage (85.75±0.14) was observed in group E while the lowest mean hatching rate (66.12±0.31) was observed in group C as shown in table No. 4.20.

4.2.8 Survival rate of fry (%)

The highest mean survival rate of fry (82.62±0.37) was observed in group E injected with the hormones Ovaprim+ HCG while the lowest mean survival rate of fry (74.62± was observed in group c injected with HCG as given in table No. 4.21.

4.2.10 Deformed larvae (%)

Highest percentage of mean deformed larvae (4.00±0.02) was observed in group C injected with HCG while the lowest percentage of deformed larvae (1.00±0.02) was observed in group E as shown in table No.4.22.

Table No 4.1: Behavioral and physiological changes in Clarias gariepinus in control group.

Parameters	Time Duration
Parameters	3 Hrs	6 Hrs	9 Hrs	12 < Hrs
Hyperness	–	–	–	–
Jumping	–	–	–	–
Random Movement	–	–	–	–
Egg release	–	–	–	–
Operculum movement	–	–	–	–
Bottom belly rubbing	–	–	–	–

(-) No change (+) Mild (++) Moderate (+++) Severe (*) Very Severe

Table No 4.2: Behavioral and physiological changes in Clarias gariepinus after post treatment of Ovaprim during latency period.

Parameters	Time Duration
Parameters	3 Hrs	6 Hrs	9 Hrs	12 < Hrs
Hyperness	–	–	–	++
Jumping	–	–	–	++
Random Movement	–	–	–	++
Egg release	–	–	–	++
Operculum movement	–	–	+	++
Bottom belly rubbing	–	–	–	++

(-) No change (+) Mild (++) Moderate (+++) Severe (*) Very Severe

No 4.3: Behavioral and physiological changes in Clarias gariepinus after post treatment of HCG during latency period.


	3 Hrs	6 Hrs	9 Hrs	12 Hrs	15 < Hrs
Hyperness	–	–	–	+	++
Jumping	–	–	–	+	++
Random Movement	–	–	–	+	++
Egg release	–	–	–	—	++
Operculum movement	–	–	+	+	++
Bottom belly rubbing	–	–	–	+	+

(-) No change (+) Mild (++) Moderate (+++) Severe (*) Very Severe

Table No 4.4: Behavioral and physiological changes in Clarias gariepinus after post treatment of HMG during latency period.

Parameters	Time Duration
Parameters	3 Hrs	6 Hrs	9 Hrs	12 < Hrs
Hyperness	–	–	–	–
Jumping	–	–	–	–
Random Movement	–	–	–	–
Egg release	–	–	–	–
Operculum movement	–	–	–	–
Bottom belly rubbing	–	–	–	–

(-) No change (+) Mild (++) Moderate (+++) Severe (*) Very Severe

Table No 4.5: Behavioral and physiological changes in Clarias gariepinus after post treatment of Ovaprim + HCG during latency period.

Parameters	Time Duration
Parameters	3 Hrs	6 Hrs	9 Hrs	12 < Hrs
Hyperness	–	–	++	++
Jumping	–	–	++	++
Random Movement	–	–	++	++
Egg release	–	–	++	+++
Operculum movement	–	–	++	++
Bottom belly rubbing	–	–	++	++

(-) No change (+) Mild (++) Moderate (+++) Severe (*) Very Severe

Table No 4.6: Behavioral and physiological changes in Clarias gariepinus after post treatment of Ovaprim + HMG during latency period.

Parameters	Time Duration
Parameters	3 Hrs	6 Hrs	9 Hrs	12 < Hrs
Hyperness	–	–	–	++
Jumping	–	–	–	++
Random Movement	–	–	–	++
Egg release	–	–	–	++
Operculum movement	–	–	+	++
Bottom belly rubbing	–	–	–	++

(-) No change (+) Mild (++) Moderate (+++) Severe (*) Very Severe

Table No 4.7: Temperature of water during breeding of Clarias gariepinus

Groups	Dose (ml/kg)	Temperature
A	0.00	28.05±0.05a
B	Ovaprim (0.5)	28.10±0.03a
C	HCG (0.5)	28.06±0.06a
D	HMG (0.5)	28.03±0.04a
E	Ovaprim+ HCG (0.3+0.5)	28.04±0.05a
F	Ovaprim+ HMG (0.3+0.5)	28.06±0.03a