CHAPTER II. ARTIFICIAL PROPAGATION OF BLACK CARP, GRASS CARP, SILVER CARP AND BIGHEAD

In about 460 B.C., Fan Li wrote “The Treatise of Pisciculture”, the earliest monograph on fresh water fish culture in the world. He summed up the method of Common carp culture in ponds in China and therefore, the earliest practice of pond fish culture in China is Common carp culture. So far as the most popular polyculture of Black carp, Grass carp, Silver carp and Bighead is concerned, it began in Tang Dynasty (618– 904 A.D.). Why is the per-unit yield of pond culture in China so high? One of the reasons is that these species have many merits; however, the production cycle in ponds did not last more than three years in general; thus the gonads of fish in ponds could not develop well. Moreover, it was believed that Grass carp, Black carp, Silver carp and Bighead could not propagate in ponds; therefore, no one thought of stimulating farm fish in ponds to propagate. The fry source relied on natural resources in the Changjiang River and the pearl River, etc. for a long time.

Collecting the fry in rivers and transporting part of them to the distant places called for much exertion and increased the cost of production. The mortality of fry was high. Furthermore, they were mixed with the fry of wild fish. This is the reason why people have been inclined to get the fry through artificial propagation so far.

In 1958, aquaculture researchers of Guangdong Province first obtained the fry by injecting the hypophysis of Common carp into the brood fish of Silver carp and Bighead cultured in ponds.

In autumn of the same year, researchers of Zhejiang Province also succeeded in induced spawning of Silver carp and Bighead by applying Human Chorionic Gonadotropin (HCG). In 1960, artificial propagation of Grass carp succeeded too. In 1963, the fry of Black carp were obtained by artificial propagation. It was only within 6 years that artificial propagation of all four cultured carps was carried out successfully and has been used for production so far. In recent years, most of the fingerlings nurtured in ponds come from the fry gained through artificial propagation, which plays an important role in developing aquaculture.

In 1974, the experiment of synthetic Luteinizing Release Hormone (LRH) proved its effectiveness. In 1975, the high effective Luteinizing Release Hormoneanalogue (LRH-A) was synthesized. It was widely applied in practice. It solved the problem of inadequate supply of estrualizing agents. The effect of estrualization was raised while the cost of production was reduced. Artificial propagation of Chinese carps is developing more rapidly than ever.

1. Rearing of brood fish

Brood fish here means the male and female fish which are used for artificial propagation. Only after they reach sexual maturity through culture in ponds, can they be induced to spawn; therefore, whether they are reared well or not will directly affect the results of induced spawning. Rearing of brood fish is a very important link in a chain of artificial propagation of cultured fish.

1) Collection of brood fish

(1) Differentiation between the male and the female fish

It is necessary to control the ratio of the male and the female parent fish when they are cultured in ponds and induced to spawn. Consequently, the correct differentiation between two sexes is important. The method of distinguishing the sex of carps is generally the same. The sex is mainly identified in accordance with the characteristics of the pectoral fins. The secondary characteristics of both sexes of Chinese carps are shown in Table 2-1.

Table 2–1 Comparison between the secondary characteristics of both sexes of the Chinese carps

(2) Selection and transportation of parent fish

Parent fish for artificial propagation may be captured from rivers, lakes and reservoirs or reared in ponds from fry, fingerling up to adult. The most common way in China is to catch mature Grass carp, Black carp, Silver carp and Bighead from great water bodies as parent fish and rear them in ponds for a short period of time for inducement. By this way, the time is shorter than that of rearing them from fry to parent fish in ponds.

Seasons of collecting parent fish: The most proper time is in autumn and winter when the water temperature is rather low, about 10°C. At that time, the activeness of fish is rather weak. So there will be no serious injury to fish, no shortage of dissolved oxygen for fish and it is convenient to transport the fish. After selection, the parent fish are reared in ponds for propagation season to come. Fish whose gonads do not develop well can be reared in ponds as reserve parent fish for the next spawning season. During capture and selection of parent fish, choose the male or the female fish from different water bodies or different drainage systems if possible, for different blood relations will improve the vitality of the next generation.

A few kinds of methods of transportation for parent fish are as follows:

1. Transportation with canvas tub or wooden barrel: Generally, it is suitable for short range transportation by truck or by train, etc. In 100 litres of water, put one parent fish of about 10 kg in body weight. The quantity of fish in transportation is determined according to water, temperature, size of fish and time limitation. It is necessary to change water if the water quality deteriorates on the way. It is better to contain each fish in a perforated plastic bag which is about 30 cm longer than the fish itself, bind the openings of the bag and put the fish and the bag into the barrel together. That will prevent the fish from injury.

2. Transportation in oxygenated plastic water bags: generally, one plastic bag which is made of vinyle-film cylinder 30–35 cm in diameter and 50 cm longer than the fish body can contain one parent fish after being filled with some water. The water level is as high as fish body. Fish and water occupy ⅓ of the bag capacity. Bind tightly the openings of the bag after oxygenation, put it in a paper box for transportation by truck. Long distance transportation needs some spare bags filled with oxygen as well as some empty bags for fear that there should be some urgent needs.

3. Transportation with a “perforated boat”: If there is a water way, it is more convenient to use a perforated boat. As the water can come in and out through the holes of the hull of the boat, the density of fish can be higher than the other.

4. Transportation after tranquillization: Fish tranquilized with drugs are in a state of unconsciousness. They can not swim or jump. The breath frequency of fish decreases and the metabolic rate reduces. This can avoid injuring fish body and reducing consumption of oxygen as well. For example, put a fish in sodium barbital solution at a concentration of 13.3ppm, within a short period of time, the fish will be in a coma with its abdomen up and its breath slow. The fish now can not swim. When water temperature is at 10°C, fish can be traquilized for more than 10 hours. Being put back in ponds for 5–10 minutes, the fish is able to revive.

2) Parent fish rearing pond

(1) Site selection for parent fish rearing pond

The location of rearing ponds should be near the water source so as to facilitate drainage and irrigation. If possible, rearing ponds should be next to the spawning pond and the incubation pool. The proper size of the pond is 3–4 mu and the water depth, 1.5 m. The flat bottom of the pond is easy for catching and management. If the area of the pond is too large and there are too many parent fish in it, it is impossible to estrualize at one time. Several hauls in one pond can affect the gonad development of parent fish and even cause the fish gonads to degrade. In the pond for rearing Silver carp and Bighead, the bottom should be choice loam with some humus of about 10 cm thick. No leakage is allowed so as to save manure and keep pond water fertile. In the pond for rearing Grass carp and Black carp, no humus is needed in the bottom and no fertile water as well in order to keep water clear with high oxygen content. There is no problem with a little leakage if it's convenient to irrigate the pond.

(2) Pond clearing

Pond clearing has to be done without neglect in rearing parent fish. It should be carried out at the end of artificial propagation each year. Pond clearing is of great significance in preventing diseases and improving water quality. In one year of rearing parent fish, the pond deposited much leftover, manure dregs and silt at its bottom. If they are not cleared, they will dissolve and produce poisonous substances, such as H₂S, CH₄, etc. which will contaminate the pond water and retard the growth and the gonad development of fish. If the pollution is serious, the fish might suffer from suffocation and also, these rotten organic substances will easily cause fish diseases. When pond clearing is ocnducted, the surplus humus can be dug out after draining the water away from the pond. Drugs for pond clearing can help to kill pathogens, bacteria and wildfish. Some drugs for instance, quicklime, can help improve bottom quality. At the same time, pond dikes, the outlet, the inlet, etc. can be easily repaired after draining the water away.

3) Stocking of parent fish

(1) Monoculture and polyculture

parent fish can be cultured in either monoculture or polyculture. Polyculture means to stock one kind of fish in one pond as the major species and to mix with several other species for full utilization of natural food and adjustment of water quality. For example, Grass carp and Black carp would like clear and fresh water but their excreta can proliferate large quantity of plankton and then, water will become fertile soon, causing Grass carp and Black carp to lose their appetite or even to have no appetite. This will affect the gonad development. Therefore, Grass carp or Black carp which are cultured as major species in one pond should be mixed with some Silver carp or Bighead in it. Polyculture not only can control reproduction of plankton and adjust water quality, but also can fully utilize natural food in the pond; thus the major and minor species can benefit each other; however, there exists certain demerits in polyculture too. For example, catching one species for estrualization will interfere the normal life of the other; frequent catching not only might injure the fish body, but also cause the gonads to stop developing, even to retrograde. But some measures could be taken to reduce bad effects to some extent. Before estrualizing period, separate the minor species from the major species or adopt polyculture with either reserve parent fish or commercial fish to avoid all these demerits.

(2) Sex ratio

Sex ratio of the major species stocked in one pond should be the same as that of the fish for estrualizing, that is, 1 : 1 or 1 : 1.5 for the male and the female fish, it is convenient for grouping when estrualizing.

(3) The amount of stocking

Generally, the stocking weight of the major species is 150kg/mu with 50 kg of minor species. The total amount is about 150–200 kg. Stocking density varies with the conditions of the pond and the techniques of rearing management, etc. The Grass carp or Black carp rearing pond mainly is supplied with artificial feeds, It is possible that the stocking density can be raised a little if the food is sufficient and the pond is easy to drain and irrigate. The stocking density of Bighead should be lower, because zooplankton grow more slowly than phytoplankton. In a pond with Silver carp brooders as the major species, the stocking weight of Silver carp is 120kg/mu, accompanied with 25 kg of Bighead and 25 kg of Grass carp.

In a pond with Bighead brooders as the major species, the stocking weight of Bighead is 100 kg/mu, accompanied with 30–40 kg of Grass carp. In Bighead brooder rearing pond, we do not stock Silver carp, because they will compete with Bighead so vigourously for food that the growth of Bighead will be hindered. In a pond with Grass carp brooders as the major species, the stocking weight is 150 kg/mu, accompanied with 40 kg of Silver carp and 10 kg of Bighead. In a pond with Black carp brooders as the major species, the stocking weight is 150 kg/mu, accompanied with 40 kg of Silver carp and 10 kg of Bighead. In Black carp rearing pond, we do not stock Grass carp because they compete with Black carp for fine food, affecting the growth of Black carp.

4) Rearing Silver carp and Bighead brooders

Silver carp and Bighead chiefly feed on natural food, plankton. By means of fertilization, a large quantity of plankton will be produced so that the parent fish may have sufficient food supply. Therefore, fertilization in accordance with the water colour is the key to the success of rearing Silver carp and Bighead brooders. In Silver carp rearing pond, human faeces are used as principal manure which generally occupies 70% with 30% animal manure. By virtue of quick effectiveness, large amount of phytoplankton will come into bloom while in Bighead rearing pond, the general amount is 70% with 30% human excreta. Cow manure is suitable for reproduction of zooplankton. Base manure should be applied before stocking of brooders. The general amount is 300–400 kg/mu. After stocking, additional manure should be applied in accordance with the actual conditions of the pond and seasonal changes. The principle of applying additional manure is “In less amount but with more frequencies and by observing water colour.” Usually, an average of 700–1000 kg of manure is applied each month. Some commercial feeds such as bean cake, wheat bran, rice bran could be supplemented in winter or before spawning. The amount of commercial food for each individual Bighead is 20 kg each year; Silver carp, 15 kg. Daily quantity is 1–2% of fish body weight.

Culture after spawning:

After spawning, the weather is getting hot. The brooders need convalescence. They can not stand shortage of oxygen; therefore, mortality from suffocation often happens. They should be looked after by special persons. Special attention should be paid to weather, water colour and water quality. Fertilization should be conducted in accordance with water quality. It should be done frequently, sparsely, with small quantities. At the same time, input fresh water at short intervals so as to prevent the water from deterioration.

Autumn and winter cultivation:

Before winter comes, manures should be heavily applied in order to make the water fertile. During winter time, small amounts of manure are applied and supplemented with certain feeds. There is no need to add fresh water in winter.

Spring cultivation:

When spring is coming, pond water depth should be controlled at about one meter in order to raise water temperature and that is easier for the water to get fertile. The amount of manure applied can be increased gradually. The compost heaped at the corner of a pond combines with animal wastes, which are spread into the pond. manure spreading is carried out every other day or every other 2 days. Some fine feeds are needed. In half a month before spawning, no manure is needed. In early spring, fresh water is poured 1–2 times each month, which stimulates the development of the gonads.

5) Rearing Grass carp and Black carp brooders

Reasonable feeding is the key to the success of rearing Grass carp and Black carp brooders.

Rearing Grass carp brooders:

Combination of commercial feeds and green grass and regular fresh water filling are the effective methods of rearing Grass carp brooders. kinds of commercial food are barley, wheat, wheat sprout, bean cake, peanut cake, etc. Kinds of green grass are English rye grass (Lolium sp.), clover, lettuce leaves, aquatic and terrestrial plants, etc. When water temperature rises up to 10°C, Grass carp start feeding. Their appetite is stimulated by the rise of temperature. When water temperature is over 20°C, the fish show good appetite; however, upon the maturity of the goands, fish will lose appetite obviously. But it is improving shortly after spawning. When the water temperature is over 30°C in July-August, they will lose their appetite again. When the water temperature comes down to some degree in late autumn, they lose their appetite too. When it is down to about 7°C, they get into over-wintering period and stop eating.

Rearing after spawning:

After spawning, special care should be taken at once in rearing because water temperature is quite suitable at this time. Grass carp have the best appetite. Green fodder is supplied at 9-10 o'clock a.m. each day (equivalent to 30–40% of body weight. The principle is to give them sufficient food, but never let fodder to be left over. 100 grams of commercial food is supplied for each parent fish in the afternoon everyday.

Rearing in autumn and winter:

Grass carp will lose their appetite when the temperature goes down. Besides, sources of grass shrink day by day. It is time to feed them with commercial food. When the temperature is below 10°C, feeding is not needed.

Rearing in spring:

At the beginning of spring, drain half of the pond water away and add some fresh water so as to keep the water depth at about 1 meter. From early March, 50–100 g of wheat sprout or bean cake should be fed to each individual fish. The staple food is green fodder, which must be supplied as early as possible. The supplemental food is commercial food, but it should not be the sole food, because overfatness will have some bad effects on spawning. Green fodder is provided by about 40–50% of body weight. Shortly before spawning, Grass carp obviously reduce food intake or stop feeding. It shows that the gonads have been fully mature. They are ready for spawning.

Each of Grass carp brooders needs 500 kg of green fodder and 20kg of commercial food per annum. In Grass carp brooder rearing pond, huge amount of feeds, leftover and Grass carp faeces easily make the pond water overfertile which causes the fish to lose appetite. During the culturing period, water filling must be conducted at regular intervals according to the change of seasons and the fertility of water in order to keep water clear and fresh. The water temperature is lower in winter, filling times can be reduced to once or twice in a month; but 3–4 times in 1–2 months before spawning with slight flow lasting 3-4 hours each time and with the water level rising 10 cm. Half a month before spawning, it is better to add fresh water day in and day out to keep water clear and fresh. This is one of the important measures in promoting the development of the gonads of Grass carp parent fish.

Rearing Black carp parent fish

Black carp are mainly fed with snails and clams and fresh water mussel flesh. Daily amount of feeding is about 20% of body weight, supplemented with small amount of bean cake. Food is spread into the flat bottom of the pond, one meter from the surface, 2-3 meters from the dike. Food is supplied in all seasons. It is better to feed sufficient food, but without any leftover. The remaining dregs must be removed out in time. Each of Black carp needs 500 kg of snails and clams and about 15 kg of bean cake per annum. Black carp favour clear and fresh water; therefore, water filling at regular intervals are needed, but the times of water filling could be less than that in Grass carp rearing pond.

2. Induced Spawning

Induced spawning also means estrualizing. That is to say, the mature parent fish are injected with estrualizing agents to reach estrus and spawning. The gonads of Chinese carps can mature after rearing, but they can not spawn naturally in ponds. So long as they are injected with estrualizaing agents, they are able to spawn.

1) The fundamental principle of induced spawning

The natural spawning of Chinese carps in rivers is controlled by certain factors of external environment. That is to say, when certain ecological condition stimulates the external sensing organs, the nerves of these external sensing organs produce impulses which are immediately sent to the nerve center to make the hypothalamus /ill/ secrete lutenizing release hormone(LRH). When the pituitary gland has been excited by LRH, it answers or reacts by secreting gonadotrophic hormone--luteinizing hormone(LH) and follicle stimulating hormone (FSH). These hormones are transmitted by body fluids through blood circulation. When the hormones reach the gonads, the gonads in turn become stimulated and begin rapidly to develop and mature from stage IV to stage V. After the dissolution of the membrane of the follicle cell ovulation begins, and a sex hormone is secreted simultaneously. The sex hormone acts in coordination with the gonadotrophic hormones, and this arouses sexual desire in the brood fish. Thus the fish engage in active sexual activities--laying eggs and exuding milt.

The basic principle of the artificial estrualization rests on the general biological principle of the natural propagation of fish. Because the ecological conditions in fish ponds can not satisfy the propagation requirements of the brood fish, the fish are injected with extraneous harmaones. Some extraneous hormaones (PG or HCG) can take the place of the hormones secreted by the pituitary gland of the parent fish and could directly stimulates the gonads. Other extraneous hormones (LRH-A) only stimulates the hypophysis of parent fish to accerate secretion-activities of gonadotrophin which will induce the parent fish to spawn or discharge milt.

2) Estrualizing agents

(1) Kinds of estrualizing agents

At present, three kinds of estrualizing agents are mainly employed in practice: luteinizing release hormone-analogue (LRH-A), the pituitary gland of fish (PG) and human chorionic gonadotrophin (HCG).

(i) LRH-A:

As mentioned above, the secretion activities of hypophysis of fish are directly controlled by the hypothalamus which secretes luteinizing release hormone. In China, it was refined out from the hyphthalamus of sheep in 1971. Through analysis it is found that that it is a polypeptide which is constructed of 10 amino acids. They are Pyroglutamic acid, Histisine, Tryptophan, Serine, Tyrosine, Glutamic acid, leucine, Arginine, Proline and Glycine amide. Molecular weight is 1182. Artificial LRH synthesized in terms of that molecular structure has very high biological activity. for cow, sheep and human. When it is used in estrualizing carps, the dose must be 100 times higher than that for mammals because LRH is easy to be destroyed by the protease in fish body. In 1975, China synthesized LRH-A, a nine peptide hormone which is constructed of 9 amino acids. They are Pyroglutamic acid, Histidine, Tryptophan, Serine, Tyrosine, D-Alanine, Leucine, Arginine, Proline and acetyl amine. Its molecular weight is 1167. The sixth glutamic acid and the tenth glycine amide are replaced by D-Alanine and acetyl amine. The biological activeness of LRH-A is about 100 times higher than LRH to fish. It is a high effective estrualizing agent. LRH-A is a white powder. The LRH-A available in the market is often added with mannite as a filler, that is 10 times as much as its weight. It easily dissolves in water. So it should be kept in dry and shady place under airtight condition.

(ii) Common carp pituitary gland

The pituitary gland or the hypophysis lies beneath the diencephalon, connecting with the hypothalamus. When the fish's brain is lifted upside down, it is easy to separate it from the hypothalamus. It is buried in the sphenoid. The hypophysis secretes the gonadotropin. There are two kinds of gonadotrophins, i.e. luteinizing hormone (LH) which stimulates ovulation function and follicle stimulating hormone (FSH) which promotes the development and maturity of eggs and stimulates, the development and maturity of follicle.

A. Method of collection and preservation of the hypophysis:

Hypophysis could be collected from both sexes of the matured Common carp of more than 0.5 kg by weight. It can also be collected from the dead but unspoiled fish. The quality of the hypophysis of the Common carp which have never spawned is the best.

Collection: First, cut off the head of a fish, set the head on its cut surface with the snout facing upward, use a knife to cut from the nostrils to the upper edge of the eyes to remove the front-parietal bone, and then turn the whole brain upside down with a pair of forceps, and the hypophysis can be seen. Tear off the membrane around it with a pair of forceps carefully and the hypophysis can be taken out.

B. Preservation : If fresh hypophysis is used, it should be ground before used. It can also be dehydrated and defatted by pure acetone or absolute alcohol (the volume is about 15–20 times that of the hypophysis). It can be preserved for future use after the acetone or alcohol is renewed twice (immersed 1–2 hours each time). Two methods may be adopted for preservation. One is to dry the dehydrated hypophsis on a piece of filter paper for 15– 20 minutes, then put it into a small brown bottle and seal it tightly. A label of species and the collection date is stuck on the bottle. The other is to preserve the hypophysis in the second immersion liquid and seal it tightly in a small brown bottle. Both methods mentioned above bring forth good results. Generally, the hypophysis still remains effective in two years' preservation.

(iii) Human chorionic gonadotropin:

The human chorionic gonadotropin (HCG) is an protein hormone. Its molecular weight is 36000. It is similar to LH and FSH of mammal in physiological function. It promotes the ovulation, the development of the gonads and the secretion of sex hormone.

HCG is refined from the urine of women who is pregnant for 2–4 months. It is a hormone which is secreted from the chorionic membrane of placenta. The HCG is more effective to silver carp and Bighead than to Grass carp. At present, the ready made material available in the market in our country is “the veterinary gonadotropin”. It is in the form of white power and easily dissolves in water. It must be preserved under airtight condition in cool, shady and dry place.

(2) Dose

(i) LRH-A: This agent has obvious estrualizing effect on all species of Chinese carps.

For Silver carp and Bighead, the injection dose is 10 μg per kg in body weight. (omitted thereafter) (lg= 1000μg). The effect of two injections is better, 1–2 μg/kg for the first injection and 8–9 μg/kg for the second injection after an 8–10 hours' interval. If LRH-A is used in combination with HCG, the dose of LRH-A is 2 μg/kg for the first injection and after an 8–12 hours' interval, the dose of LRH-A, 8μg/kg and the dose of HCG, 200 IU/kg for the second injection. This can mature the ova or promote the polarization of cells. The estrualizing rate is high and steady.

For the male fish, the dose should be reduced by half. It receives only one injection when the female gets the second injection. Grass carp is more sensitive to LRH-A with better effects. They need only one injection. The dose of LRH-A is 5–10μg/kg. For the male fish, the dose should be reduced by half.

For Black carp, LRH-A is used in combination with PG. The dose of LRH-A is 15μg/kg and the dose of PG is lmg/kg. They are given in three injections: LRH-A, 5μg/kg for the first injection; 15 days later, the same amount for the second injection and after 12 hours, LRH-A, 10μg/kg and PG, 1mg/kg for the last injection. For the male fish, the dose should be reduced by half. Just one injection is given at the same time as the female receives the last injection. If the maturity of the male fish is not desirable, that is, the sperm can not be squeezed out, the double injection may be adopted. The dose of the first injection for the male is the same as the one for the female at the same time i.e. 15 days in advance. The second injection of the male is synchronized with the third injection of the female.

(ii) PG of Common carp

The estrualizing effect of this agent is obvious when applied to any species of Chinese carps. The dose to be used is 4mg (dry weight) per kg of body weight. For the male fish, the dose should be reduced by half. For comparison, the PG of Common carp of about 0.5 kg by weight is only about 1mg after dehydration by pure acetone.

(3) HCG: For both Silver carp and Bighead, the dose 4–5mg/kg or 800– 1000IU/kg.

3) Injection

(1) Preparation of injection suspension fluid

The preparation of PG suspension fluid: according to total body weight of parent fish, calculate the required number of PG. First, put the PG (if the PG is preserved in liquid, dry it on the filter paper for 15 minutes) into a mortar and grind them thoroughly, then add normal saline to make suspension fluid. The dose of injection suspension is controlled in 2–3 mg for each parent fish. When HCG and LRH-A are used, the dose required must mix with the normal saline at a concentration of 0.7 %.

when the powder dissolves, it is ready for use. It should be prepared impromptu so as to prevent the suspension fluid from becoming ineffective.

(2) The method of injection

The coelomic injection is commonly adopted. During injection, a brood fish is placed in a cloth clamp. Let the fish lie laterally in water. Hold the upper half of the fish above the surface. Wipe off the water at the inner side of the basal part of the pectoral fins. Point the syringe needle towards the head at an angle of 45^o to the body's longitudinal axis, insert the needle through to a depth of about 1.5 cm, inject the fluid slowly.

(3) Frequency and time of injection

Frequency of injection: The frequency is of two kinds, i.e. the single injection and double injection. For the single injection, the predetermined dose is completely injected into the fish body. For the double injection, the predetermined dose is divided into two and injected twice. The amount of the first injection is usually 10–20% of the total dose, the rest is for the second injection. For milters, only the single injection is adopted. The injection is given to the milter when the spawner receives the second injection.

Both the single injection and the double injection adopted at present give satisfactory results. In respect of the double injection, especially to those less matured parent fish, it can accelerate the eggs to reach maturity.

Time of injection: The time of injection depends upon water temperature and convenience of working condition. The brooders are usually controlled to spawn in midnight or dawn. If the single injection is adopted, the injection is carried out in the afternoon or at dusk in order to allow the brooders to spawn at dawn. If double injection is adopted, the first injection is carried out in the morning. The interval between two injections is 8–12 hours. If water temperature is high, it's better to control the brooders to spawn at midnight. It can enhance the fertilization rate and the hatching rate.

4) Season of estrualization

To choose the most suitable season to estrualize is one of the key links in a chain of artificial propagation of Chinese carps.

The optimum period is dependent on the weather and the gonad development of fish. The initial propagation period in Guangdong Province is early May, while that in Jiangsu and Zhejiang Provinces is from the middle of May to the middle of June. The optimum temperature is 22–28°C. The sequence for estrualization is Grass carp the first, Silver carp and Bighead the second, Black carp the last.

5) Selection of parent fish

Usually we choose the female fish with its abdomen bulging out, especially the part around genital opening being swollen, soft and elastic. If Silver carp and Bighead are held out of water, ribs are faintly seen on the abdomen. If the tail is raised, the contour of the ovary is moving forward. As for the male fish, if the abdomen is slightly pressed, the milt will flow out and spread in water at once. These parent fish are matured ones.

If the abdomen of the female Silver carp or Bighead brooders is exceedingly expanded, less elastic, this is the phenomenon of over maturation of parent fish, which can not be chosen for estrualization.

Feeding should be stopped two days before choosing Grass carp. If the female Grass carp is placed belly up, the contour of the ovary is seen downwards on both sides of the abdomen. If the middle of abdomen is in shape of dented shrinkage, and the abdomen portion feels soft, it can be chosen.

It can also be done by an egg collecting spoon to determine the maturity degree of the eggs. Collect a small amount of eggs from the cloacal opening, fix it in the tranparent liquid (85ml of a solution at a concentration of 95 % alcohol, 10ml of formalin at a concentration of 40 % formaldehyde, 5ml of frozen acetic are mixed together) for 2–3 minutes. If the transparency of cytoplasm and yolk is in the center, it's an unmatured egg, which will have a little effect for estrualizing. If the nucleus is eccentric, it is a matured one, ready for spawning.

If the egg nucleus is vague or is not seen, it is an over-matureed one or a degraded one, which will have no effect for estrualizing.

3. Spawning

1) Under normal conditons it will take a while after the last injection that parent fish appear to be in a state of estrus. This period of time is called the response time.

The response time varies slightly depending on water temperature, kinds of estrualizing agents, the frequency of injection and species of parent fish. When water temperature rises 1°C, the response time is shorter by 1–2 hours. In case of the double injection, the response time counted from the second injection is shorter than that of single injection. For example, when water temperature is 24–25°C, using PG to Silver carp for the single injection, the response time is 12–14 hours, whereas for the double injection, (counted from the second injection), it is 7–8 hours. The response time also varies with different hormones used. The response time to PG injection is shorter by one or two hours than that to HCG injection, while the response time for LRH-A injection is longer than that of PG injection. In addition, given the same hormone and the same dosage in the same season, the response time of various species are similar with some slight differences. Generally, Grass carp have the shortest response time of the three, Bighead a little longer, and the response time of Silver carp is the longest.

2) Natural spawning & fertilization:

After having been injected with estrualizing agent, the parent fish are stocked into the spawning pond to spawn or exude milt to complete fertilization process. This is called natural spawning and fertilization.

(1) Spawning pond:

Spawning pond is also called estrualizing pond. After having been injected with estrualizing agents, the parent fish need a pond with suitable ecological conditions in which they can perform estrus and spawning. It is also convenient to collect fertilized eggs.

A spawning pond is usually made of bricks with covered with concrete. There are two common ponds, i.e. circular and elliptical pond.
Circular spawning pond: (fig. 2--1)

The inlet of the pond is set at a 40^o angle tangent with the pond wall. The rim of the bottom is higher than the center. An outlet is usually installed in the center of the bottom which serves as a passage for collection of eggs. The pipe which is hidden underground passes to the egg-collecting pond. There is an egg-collecting cage in that pond to collect eggs, which can be transferred to incubation ponds or the pipe can directly go to incubation ponds for hatching.

Elliptical spawning pond:

The inlet and the outlet are on the same straight line. Water is 1 meter deep. The bottom of the pond inclines to the outlet. Outside, there is an egg collecting pond in which there is an egg collecting cage that is connected with the outlet of the spawning pond. (see Fig 2--2)

2) Estrus and spawning:

After they have received the estrualization treatment, and if they react normally to the hormones used, brood fish will generally begin chasing each other excitedly. This phenomenon is called estrus. At the beginning, some irregular ripples appear on the water surface. They are caused by the parent fish beginning to chase each other underwater. If the chasing continues, sometimes, emerging on the surface of the water or forming sprays, it is regarded as a normal estrus. At the climax of estrus, the male hits female's abdomen with its head. The female lies on its side on the surface of water (or underwater). Its abdomen and tail become intensely constricted, and then, eggs flow out. At the same time, the male nestles closely up to the female's abdomen, discharging milt. Sometimes, the male and the female are entangled together and swing their pectoral fins to spawn or discharge milt.

3) Methods of collecting fertilized eggs:

There are two methods of collecting fertilized eggs. One is to fill the pond with water one to two hours before the fish begin to estrualize. (This has a stimulating function to the parent fish). During estrus, slowly-flowing water is better because it can bring the gametes together so as to raise the fertilization rate. At the end of spawning, take the brood fish out, then flush the eggs with a great current in order to collect eggs. Eggs consecutively flow into the egg collecting cage by flushing. Eggs are collected and counted, then put into hatching boxes to hatch. Another method is to pour water into the pond at the end of spawning and to flush the fertilized eggs which sink down to the bottom to flow into egg collecting cage in great force. This is so called still-water spawning.

The effect of these two methods are related to the ecological factors of artificial estrualizing. There are no identical views at present. Some people believe that under artificial propagation conditions, the extraneous hormones have produced the decisive effects on egg maturation and ovulation and water flowing is of no importance. Others consider that the ecological conditions are still necessary and that the physiological activities of endocrine system keep going and their requirements for ecological conditions still exist although the estrualizing agent can make the fish spawn; therefore, water-flowing stimulation will raise egg-laying rate and fertilization rate.

Method of counting eggs:

Volumetric method can be adopted to calculate eggs. First of all, the capacity of a container is measured, take out 10 or 20ml of eggs and count the number of eggs and then, calculate the total number according to the capacity of the container and the times of ladling. The concentration of eggs in each ladling must be uniform for accuracy. There are about 1000 eggs in 100ml of eggs of Grass carp and Silver carp after absorbing water, and 800 eggs for Bighead, i.e. eggs of Chinese carps after absorbing water are about 8000--10000 eggs per litre.

3. Artificial insemination:

When the brood fish are in estrus and begin to spawn, they are captured immediately. Egg-collection and milt collection are done so as to bring the matured .

The eggs must be proper mature so that after fertilization, they may develop normally. Under the water temperature of about 28°C, the normal fertilization time of Chinese carps eggs (from separating from follicles to being released) is only 1--2 hours; therefore, when we perform artificial insemination, the time of egg-collection and milt-collection is dependent on various estrualizing agents, different species and water temperature, etc. This is the key to the success of artificial insemination. The experiments show when temperature is at 23°C, eggs of silver carp from early maturation to proper maturation needs 40 minutes, from proper maturation to slight overmaturation lasts 1.5--2 hours. Only proper-matured eggs are able to develop normally, immatured or over-matured eggs develop abnormally because of their low fertilization rate.

Most of the eggs from ovulation will not immediately lose their fertilization ability in 10 minutes when they stay in ooplasm and more than half of them may maintain this ability for over 20 minutes, however they will soon lose their fertilization ability in one minute if they come into fresh water. Eggs in saline solution at the concentration of 0.7% can survive for 10 minutes.

The sperm of Chinese carps in semen or in spermary are not active. After they come into water, they start moving. The sperm in fresh water can survive for one minute. The time when the sperm has higher fertilization ability is only 20--30 seconds in fresh water. The sperm in normal saline can survive for 2--3 minutes.

Methods of artificial insemination of Chinese carps:

Two methods are usually adopted, i.e. dry method and semi-dry method.

Dry method: About 15 minutes after the beginning of estrus, capture out brood fish and gently squeeze the brooder's belly to let the eggs flow into a basin (each basin should not contain more than 500000 eggs), and then, either directly squeeze the semen on to the eggs or suck the semen with a pipette and drop it on the eggs. Stir the mixture gently with a hand or a featner for about 1 minute, then add a little clean water and stir again for one minute, leave the mixture along for one minute and finally pour out the dirty water. After the eggs have been washed in this way for 3 times, they may be removed to a hatcher for incubation. sometimes, it is according to the response time that the brooder fish should be captured to be examined for fear that we miss the chance because some of the brood fish have no obvious estrus.

Semi-dry method: With a pipette, suck out the semen and dilute it with a little normal saline and then pour it onto the eggs. Therefore, proceed in the same way as mentioned above in dry method.

There are also two methods of counting eggs for artificial insemination:

Weight method: Weight the eggs before absorbing water, count the number per unit weight and then the total egg quantity can be calculated. (generally 700--750 eggs per gramme for Silver carp and Grass carp, 650--700 eggs for Bighead) or it can be counted out according to the difference between the weight of a brooder before and after spawning.

Volumetric method: Measure the volume of eggs before absorbing water, multiply it by the egg numbers per unit volume and then the total egg quantity can be calculated. (650--700 eggs per ml for Silver carp and Grass carp, and 600--650 eggs for Bighead).

4. Incubation

Incubation means the whole course of fertilized eggs from embryonic development to hatching of fry. But in production the implied meaning of the word is broader. It includes all the management work from hatching to stocking.

The eggs of Chinese carps are semi-buoyant, After the eggs have been fertilized and have absorbed water, the egg membrane expands at once up to about 5--6mm in diameter. They sink to the bottom in stagnant water but will float with a little running water. In rivers and streams, fertilized eggs will float and hatch. As the result of a long period of adaptation, the fish population have formed their unique demands for the environmental conditions of incubation: adequate oxygen, appropriate water temperature and certain current.

1) Hatching temperature and dissolved oxygen

The range of water temperature for the embryonic development of Chinese carps is 17°C--30°C, with the proper range of 22°C--28°C, and the optimum 25°C--27°C; if the temperature is lower than 17°C or higher than 30°C, it will cause stoppage or abnormality of embryonic development.

Hatching occurs with a certain temperature range. The relation between the speed of embryonic development and water temperature are in positive correlation, that is, if the temperature is high, the development is quick. Take the embryo development of Silver carp as an example, when the water temperature is 18°C, the incubating process takes 61 hours; when the water temperature is 28°C, only 18 hours are needed. The relation between the hatching time and water temperature is as follows (see table 2--1).

Table 2--1 Relation between hatching time and water temperature of Silver carp

Dissolved oxygen content in pond water should not be lower than 4--5mg/l. Below 2mg O₂/L, the embryo can not develop normally.

Running-water type hatching jars and circulators are all designed in accordance with the characteristics of Chinese carps eggs.

2) Hatching instruments and operational management

(1) Hatching jar (earthen ware vats):

Hatching jar (Fig 2--3) is made of tinplate or plastics. Hatching vats are generally earthenware vats reformed (Fig 2--4). The proper volume of a container is 250kl. Every kl of water may admit 1000 eggs. The hood of hatching jars (vats) is made of nilon net with 50 meshes. The water flows in at the bottom of hatching jar (vats), and over-flows out from the upper rim of the jar through the net hood.

The operational management of the hatching jar (vat) is firstly to regualte the flowing speed of water so as to get the fertilized eggs to float to the surface and Secondly, to brush the dirty matter and egg shell off the hood in order to get the water to flow freely and to prevent the newly-hatched fry from escaping with over-flowing water. During the whole hatching process, it must allow free flow of water.

(2) Hatching circulator: Hatching circulator (Fig. 2-5) is ring-shaped lanes of cement and brick. There are two kinds of circulators, i.e. single and double. The circular course is 1 meter wide, 0.9 meter deep and the pond is 4 meters in diameter. It may hold 8 m³ of water and admit 8 million pieces of eggs. (Every cubic meter of water may admit 1 millon pieces of eggs). The water must be exchanged every half an hour. The management work of circulator is similar to that of hatching jar.

3) Calculating fertilization rate, hatching rate and fry producing rate:

When fish eggs have developed to the middle gastrula stage (after 6-8 hours of fertilization), collect about 100 eggs with small net at random, put them into a white dish, and give up the eggs such as turbid eggs, white eggs, empty eggs and rotten eggs with naked eyes. Calculate the fertilized eggs by percentage.

Method of calculating hatching rate:

But in production, it is trouble-some to obtain the accurate figures of the actual hatching rate, therefore, it is usual to calculate the fry producing rate in production.

As soon as the air bladders of fry have been filled with air, the yolk sacs have basically disappeared and the fry can take food actively (about 4–5 days after hatching), they may be transferred for pond stocking.

Fig 2--3	Fig 2--4

Fig 2--5