Yu Shigang
In about 460 BC., Fan Li wrote The Treatise of Pisciculture, the earliest monograph on freshwater fish culture. He summed up the method of common carp culture in ponds in China and, therefore, the first pond fish cultured in China was common carp. Polyculture of black carp, grass carp, silver carp and bighead began during the Tang Dynasty (618–904 AD.).
The high per-unit yield of fish culture in China is due to the many merits of Chinese carps. In general, however, the pond production cycle does not go beyond 3 years; therefore fish gonads do not develop fully. Moreover, in the past it was believed that grass carp, black carp, silver carp, and bighead could not be propagated in ponds; fry were obtained mainly from the Changjiang River and the Pearl River. Collecting the fry in rivers and transporting them to the distant culture ponds required much efforts, increased the cost of production, and resulted in a high fry mortality rate. Furthermore, carp fry became mixed with the fry of wild fish. For these reasons, artificial propagation was seen as a better method of obtaining fry.
In 1958, aquaculture researchers in Guangdong Province obtained fry by injecting both silver carp and bighead brood fish with the hypophysis of common carp. In the same year, researchers in Zhejiang Province succeeded in inducing the spawning of silver carp and bighead by applying human chorionic gonadotropin (HCG). In 1960, the artificial propagation of grass carp was successful. In 1963, black carp fry were obtained by artificial propagation. Within 6 years the artificial propagation of all four cultured carps was successful and had been used for production purposes. Now, most of the fingerlings nurtured in ponds come from artificially propagated fry.
In 1974, synthetic luteinizing hormone-releasing hormone (LRH) was proven to be effective in the induction of spawning. In 1975, the highly effective LRH analogue (LRH-A) was synthesized. This analogue solved the problem of inadequate supply of inducing agents. Its wide application has significantly reduced production costs. At present, many advances are being made in the artificial propagation of Chinese carps.
“Brood fish” refers to the male and female fish used in artificial propagation. Only after a fish reaches sexual maturity can it be induced to spawn; whether the fish is properly reared or not will directly affect the results of induced spawning. he rearing of brood fish therefore is an important step in the artificial propagation of cultured fish.
Collection of Brood Fish
Differentiation between male and female
The male to female ratio of parent fish must be controlled when they are cultured in ponds and induced to spawn. Consequently, it is important to correctly distinguish the male from the female. Sex is usually identified by the characteristics of the pectoral fins. Secondary sexual characteristics of Chinese carps are shown in Table 2.1.
Table 2.1. Secondary sex characteristics of male and female Chinese carp.
| Species | Male | Female |
| Silver carp | There is a row of fine bony atenoid serrations on several of the foremost fin rays of the pectoral fin. They are coarse and thorny and present throughout the life of the fish. | Pectoral fin is smooth. |
| Bighead | Edges of the upper rims of several of the foremost pectoral fin rays are sharp and slant backward. | Pectoral fin is smooth. |
| Grass carp and black carp | The pectoral fin rays are thick and long, extending freely like sharp knives. In the reproduction season, pearl organs appear on the pectoral fins and opercula of mature male fish. They are coarse to the touch. | The pectoral fins are thin and short, spreading out spontaneously like a fan. No pearl organs appear. |
Selection and transportation of parent fish
Parent fish may be captured from rivers, lakes, or reservoirs, or may be reared in ponds from fry, fingerling, or adult. In China, mature grass carp, black carp, silver carp, and bighead are captured from open bodies of water and, for a short time, in ponds as parent fish. This method requires much less time than rearing parent fish from fry.
The most appropriate time to collect parent fish is the autumn or the winter, when the water temperature is around 10°C. At this time, the fish are less active so there is less chance of serious injury to the fish, dissolved oxygen is abundant, and transportation is convenient. After selection, the parent fish are reared in ponds for the coming spawning season. Fish whose gonads do not develop well can be reared as reserve parent fish for the next spawning season. During the capture and selection of the parent fish, the male and female should be taken from different bodies of water or different drainage systems: parents from different bloodlines will improve the vitality of the next generation.
There are many methods for transporting the parent fish. Four of these are described here.
First, there is transportation by canvas tub or wooden barrel. Generally, this method is suitable for short-range transportation by truck or train. There should be 100 L of water for every 10 kg parent fish and the quantity of fish to be transported is determined according to water temperature, fish size, and time limitations. If the water quality deteriorates enroute the water must be changed. It is recommended that each fish be placed in a perforated plastic bag that is about 30 cm longer than the fish itself. The openings of the bag should then be bound and the fish and the bag placed in the barrel together. This will prevent injury to the fish.
Second, parent fish may be transported in plastic bags with oxygenated water. One plastic bag, which is made of a vinyl-film cylinder 30–35 cm in diameter and 50 cm longer than the fish body, can carry one parent fish. The water level must be as high as the fish's body. The fish and the water should occupy one-third of the bag. The openings of the bag must be tightly bound after oxygenation. The bag is then placed in a paper box for transportation by truck. For long-distance transportation, some spare bags, both empty and filled with oxygenated water, should be readily available.
Third is transportation by “perforated boat”. If transportation by water is possible, a perforated boat should be used. Because water can come in and out through the holes in the hull of the boat, more fish can be transported using this method than with the other methods.
Fourth, fish may be transported after tranquilization. Tranquilization reduces both breathing frequency and metabolic rate, leaving the fish in an unconscious state. This greatly reduces the possibility of injuring the fish during transport and reduces the consumption of oxygen. Sodium barbital at a concentration of 13.3 ppm is an effective tranquilizer. With a water temperature of 10°C, fish can be tranquilized for more than 10 h. After being placed in the culture pond, the fish revive in 6–10 min.
Parent Fish Rearing Pond
Site selection
Rearing ponds should be close to a water source to facilitate water management. If possible, rearing ponds should be next to the spawning pond and the incubation pool. The pond should be 3–4 mu (15 mu = 1 ha) with a water depth of 1.5 m. Flat-bottomed ponds simplify catching and management. If the pond is too large and contains too many parent fish, multiple spawning inductions must be performed. If the pond contains several different catches of fish, gonad development may be adversely affected; in fact, gonads have been observed to degrade in such conditions. In the pond for rearing silver carp and bighead, the bottom should be choice loam with some humus (about 10 cm thick). Leakage must be prevented to save manure and keep the pond water fertile. In the pond for rearing grass carp and black carp, no humus is needed at the bottom and the water must be kept clear with a high oxygen content. Some leakage is allowable if it is convenient to irrigate the pond.
Pond clearing
The pond must be cleared without neglect to the rearing of the parent fish. It should be done each year, after artificial propagation. Pond clearing is important in preventing disease and improving water quality. In one year of rearing parent fish, a lot of leftover manure dregs and silt are deposited at the bottom of the pond. If they are not removed, they will dissolve and produce poisonous substances, such as H2S and CH4, which will contaminate the pond water and retard the growth and gonad development of the fish. If the pollution is serious, the fish might suffocate or become diseased. In clearing the pond, the surplus humus is removed after draining away the water. Pond-clearing chemicals can help kill pathogens, and wild fish. Some chemicals (e.g., quicklime) can improve bottom quality. After draining the pond, the dikes, outlet, and inlet can also be easily repaired.
Stocking of Parent Fish
Monoculture and polyculture
Parent fish can be cultured in either monoculture or polyculture. In monoculture, the pond contains only one species. In polyculture, the pond contains one major fish species and several other species to help fully utilize the natural food and maintain water quality. Grass carp and black carp prefer clear, fresh water; however, their excreta can grow large quantities of plankton, making the water fertile. In fertile water, grass carp and black carp reduce or stop feeding, adversely affecting their gonad development. Therefore, grass carp or black carp that are cultured as the major species in one pond should be mixed with some silver carp or bighead. Polyculture cannot only control plankton reproduction and adjust water quality, but also ensures the full utilization of the natural food in pond; thus, the major and minor species are mutually beneficial. However, there are some disadvantages to polyculture as well. For example, catching one species for spawning induction will interfere with the normal life of the other species. Frequent catching might not only result in injury to the fish but also cause the gonads to stop developing or even degenerate. However, measures can be taken to reduce these undesirable effects. For example, before induced spawning the minor and major species could be separated; alternatively, polyculture could be adopted with either reserve parent fish or commercial fish.
Sex ratio
The female to male ratio of the major species stocked in one pond should be the same as that during spawning induction, i.e., 1:1 or 2:3.
Stocking
Generally, the stocking rate of the major species is about 150 kg/mu; that of the minor species 50 kg/mu; this total gives a stocking rate of about 150–200 kg/mu. Stocking density varies with the condition of the pond, the rearing-management technique etc. The grass carp or black carp rearing pond is supplied mainly with artificial feeds. It is possible to raise the stocking density slightly if there is sufficient food and if 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 polyculture pond with silver carp brooders as the major species, the stocking weights should be as follows: silver carp, 120 kg/mu; bighead, 25 kg/mu; grass carp, 25 kg/mu.
In a pond with bighead brooders as the major species, the stocking rate of bighead should be 100 kg/mu, with 30–40 kg/mu of grass carp. In the bighead brooder rearing pond, silver carp is not a minor species because they would compete with bighead so vigorously for food that the growth and development of the bighead brooders would be hindered. In a pond with grass carp brooders as the major species, the stocking rate of grass carp should be 150 kg/mu, with 40 kg/mu of silver carp and 10 kg/mu of bighead. In a pond with black carp brooder as the major species, the stocking rate should be as follows: black carp, 150 kg/mu; silver carp, 40 kg/mu; bighead, 10 kg/mu. Grass carp is not included in the black carp rearing pond because they would compete with the black carp for fine feed, adversely affecting the growth and development of the black carp.
Rearing Silver Carp and Bighead Brooders
Silver carp and bighead chiefly feed on natural food (plankton). By fertilization, a large quantity of plankton can be quickly produced so that the parent fish have a sufficient food supply. Therefore, fertilization, in accordance with water colour, is crucial to the success of rearing silver carp and bighead brooders. In the silver carp rearing pond, human feces is the principal manure (70 per cent); the remaining 30 per cent is animal manure. In the bighead rearing pond, there is 70 per cent cow manure and human excreta. Cow manure is suitable for the reproduction of zooplankton. A base manure should be applied before stocking; the general amount is 300–400 kg/mu. After stocking, additional manure should be applied at a rate dependent on condition of the pond and seasonal changes. Generally, additional manure is applied in small amounts with the frequency of application depending on the water colour. An average of 700–1000 kg of manure is applied each month.
Commercial feeds such as bean cakes, wheat bran, and rice bran can be supplemented in the winter or before spawning. Required yearly amounts of commercial feed are as follows: bighead, 20 kg; silver carp, 15 kg. The daily ration is 1–2 per cent of body weight.
Culture after spawning
After spawning, the weather gets hot. The brooders need special attention during this period. The weather, water colour, and water quality should be watched closely. Fertilization should be conducted in accordance with water quality. As mentioned earlier, fertilizer should be applied frequently and in small quantities. Fresh water should be added in the same manner to prevent deterioration of the pond water.
Autumn and winter cultivation
Before winter, manure should be heavily applied to make the water fertile. During the winter, small amounts of manure should be applied and supplemented with certain feeds. There is no need to add fresh water during the winter.
Spring cultivation
As spring approaches, the water depth should be controlled at about 1 m to raise the water temperature. This makes it easier for the water to become fertile. The amount of manure applied can be increased gradually. The compost heaped at the corner of the pond can be combined with animal wastes and spread into the pond. Manure spreading is carried out every other day or every other 2 days. Some fine feeds must also be added. Just over 2 weeks before spawning, manure application should stop. In the early spring, fresh water is added one or two times each month; this stimulates the development of the gonads.
Rearing Grass Carp and Black Carp Brooders
Reasonable feeding is the key to the success in rearing grass carp and black carp brooders.
Grass carp
Combining commercial feeds, green fodders and regular changes of fresh water is an effective way of rearing grass carp brooders. Appropriate commercial feeds include barley, wheat, wheat sprout, bean cake, and peanut cake. Appropriate green fodders include English ryegrass (Lolium sp.), clover, lettuce leaves and aquatic and terrestrial plants. At a water temperature of 10°C, grass carp begin feeding and their appetite increases with temperature. At water temperatures over 20°C, grass carp feed well. When the gonads mature, feeding decreases sharply. After spawning, however, the fish again increase their food intake. When the water temperature is over 30°C in July and August, feeding decreases again. When the water temperature reaches 70°C, feeding stops and overwintering begins.
Rearing after spawning — After spawning, special care should be taken in rearing. Green fodder should be supplied at 09:00–10:00 every day at a daily rate of 30–40 per cent of body weight. This schedule provides sufficient food, without leftover fodder. Commercial food is supplied at a rate of 100 g per parent fish every afternoon.
Rearing in the autumn and the winter — Grass carp requires less feeding as water temperature declines. Because the supply of green fodder is minimal at this time of year, commercial feeds are used. When the water temperature is below 10°C, feeding is not needed.
Rearing in the spring — At the beginning of spring, half of the pond water should be drained and replaced with fresh water, keeping the water depth at about 1 m. From early March, 50–100 g of wheat sprout or bean cake should be fed to each fish every day. The staple food is green fodder, which must be supplied as early as possible at a rate of 40–50 per cent body weight. Commercial food should be given only as supplement. Shortly before spawning, grass carp sharply reduce their food intake or stop feeding entirely. This indicates that the gonads have reached maturity. Each grass carp brooder needs 500 kg of green fodder and 20 kg of commercial food per annum. An accumulation of leftover feed and grass carp feces will make the grass carp brooder rearing pond overfertile. This must be avoided because it hinders the growth and development of grass carp. During the culture period, fresh water must be added at regular intervals, depending on the season and the fertility of the pond water. In winter, when the water temperature is low, one or two water changes per month is adequate. However, 1 or 2 months before spawning, the water should be changed three or four times with slight flows lasting 3–4 h each and raising the water level by 10 cm. About 2 weeks before spawning, fresh water should be added every day. Clear, fresh water is important in promoting the gonad development of grass carp brooders.
Black carp
Black carp mainly feed on snails and clams supplemented with small amounts of bean cake. The daily feeding rate is about 20 per cent of body weight. Food is spread on the flat bottom of the pond, 2–3 m from the dike and is supplied year-round. Each black carp needs 500 kg of snails and clams and about 15 kg of bean cake per annum. Like grass carp, black carp favour clear, fresh water; therefore, leftover feed must be avoided. Water must be changed at regular intervals although water changes need not be as frequent as those for the grass carp rearing pond.
In induced spawning, the mature parent fish are injected with spawning inducing agents. Without these agents, Chinese carp will not spawn in ponds.
The Fundamental Principle
The natural spawning of Chinese carps in rivers is controlled by certain environmental factors. When these ecological conditions stimulate the external sensory organs, the nerves of these external organs produce impulses that are immediately sent via the central nervous system to the hypothalamus. The hypothalamus then secretes LRH. The pituitary gland reacts to LRH by secreting two gonadotrophic hormones: luteinizing hormone (LH) and follicle-stimulating hormone (FSH). These hormones are transmitted through blood circulation. Stimulated by LH and FSH, the gonads are rapidly developed and mature from stage IV to stage V. After the dissolution of the follicle cell membrane, ovulation occurs and a sex hormone is secreted. The sex hormone acts in coordination with the gonadotrophic hormones, arousing sexual desire in the brood fish. The fish then engage in active sexual activities discharging eggs and milt.
The basic principle behind induced spawning comes from the biological mechanism of natural propagation. Because the external ecological conditions in the fish ponds cannot satisfy the reproductive requirements of the brood fish, the fish are injected with extraneous hormones. Some extraneous hormones (e.g., fish pituitary gland [PG] and human chorionic gonadotrophin [HCG] can take the place of the hormones directly secreted by the pituitary gland of the parent fish and directly stimulate the gonads. Other extraneous hormones (LRH-A) only stimulate the hypophysis of the parent fish, accelerating the secretion of gonadotrophin and inducing the parent fish to spawn or discharge milt.
Spawning Inducing Agents
LRH-A
The secretion activities of the hypophysis are directly controlled by the hypothalamus, which secretes LRH. In China, LRH was refined from the hypothalamus of sheep in 1971. Analysis showed LRH to be peptide of 10 amino acids; pyroglutamic acid, histidine, tryptophan, serine, tyrosine, glutamic acid, leucine, arginine, proline, and glycine amide. Its molecular weight is 1182. Artificially synthesized LRH has a high biological activity for cows, sheep, and humans. When it is used to induce spawning in Chinese carps, however, the dose must be 100 times higher than that for mammals because LRH is easily destroyed by fish protease. In 1975, LRH-A was synthesized. This analogue of LRH consists of nine amino acids (pyroglutamic acid, histidine, trytophan, serine, tyrosine, Dalanine, leucine, arginine, and proline) and acetyl amine. Its molecular weight is 1167. The 6th (glutamic acid) and the 10th (glycine amide) amino acids of LRH are replaced by D-alanine and acetyl amine in LRH-A. The biological activity of LRH-A is about 100 times higher than that of LRH to fish. The LRH-A available on the market is a white powder and is combined with mannite as a filler. It is soluble in water and should be stored in a dry, shady, airtight-environment.
Dosage — For female silver carp and bighead, the injection dose is 10 microgram/kg body weight. Two consecutive injections are more effective: 1–2 microgram/ kg for the first injection and, 8–10 h later, 8–9 microgram/kg for the second injection. If LRH-A is used in combination with HCG, the dose of LRH-A is 2 microgram/kg for the first injection and, 8–12 h later, 8 microgram/kg combined with a dose of 200 IU/kg of HCG for the second injection. This treatment stimulates the ova to mature or promotes the polarization of nucleus and the rate of successful induction is high and constant. For the male fish, the dose should be reduced by half, and only one injection should be given (when the female gets the second injection).
Grass carp are more sensitive and responsive to LRH-A. Both males and females need only one injection: females 10 microgram/kg; males, 5 microgram/kg.
For black carp, LRH-A is used in combination with PG. The dose of LRH-A is 15 microgram/kg and the dose of PG is 1 mg/kg. Three injections are given. First, a 5 microgram/kg dose of LRH-A; second, 15 days after, another 5 microgram/kg dose of LRH-A; and third, 12 h later, a 10 microgram/kg dose of LRH-A and a 1 mg/kg dose of PG. For the male fish, doses should be reduced by half. Just one injection should be given at the same time the female receives the last injection. If maturity of the male fish is not achieved, i.e., the sperm cannot be squeezed out, a double injection method may be adopted. The dose of the first injection for the male is the same as that for the female and is given at the same time (15 days in advance). The second injection of the male is synchronized with the third injection of the female.
Common carp pituitary gland (PG)
The pituitary gland, or the hypophysis, lies beneath the diencephalon, connecting with the hypothalamus. When the fish's brain is lifted upside down, the PG is easily separated from the hypothalamus. It is buried in the sphenoid. The hypophysis excretes two gonadotrophic hormones: LH, which stimulates ovulation, and FSH, which promotes the development and maturity of the eggs and stimulates the development and maturity of the follicles.
Collection — Hypophysis can be collected from mature male or female common carp weighing more than 0.5 kg. It can also be collected from dead, unspoiled fish. The hypophysis of a common carp that has never spawned is the best. To remove the hypophysis, first, cut off the head of the fish, set the head on its cut surface with the snout facing upward, and use a knife to cut from the nostrils to the upper edge of the eyes and remove the front parietal bone. Turn the whole brain upside down with a pair of forceps and the hypophysis can be seen. Carefully remove the tissues around it with a pair of forceps and the hypophysis can be removed.
Preservation — If fresh hypophysis is used, it should be ground before use or dehydrated and defatted with pure acetone or absolute alcohol (use a volume about 15–20 times that of the hypophysis). The hypophysis can be preserved for future use after the acetone or alcohol has been renewed twice (immersed for 1–2 h each time). There are methods of preservation. First, the dehydrated hypophysis is dried on a piece of filter paper for 15–20 min and then stored in a tightly sealed, labeled, small, brown bottle. Second, the hypophysis is left in the second immersion liquid and stored in a tightly sealed, labeled, small, brown bottle. Both methods give good results. Generally, the hypophysis is still effective after 2 years of preservation.
Dosage — The estrus-inducing effect of PG on Chinese carps is immediately evident. For females, a dose of 4 mg dry weight/kg body weight should be used; for males, the dose should be reduced by half. After dehydration with pure acetone, the hypophysis of a 0.5 kg common carp weighs about 1 mg.
Human chorionic gonadotropin (HCG)
Human chorionic gonadotropin is a polypeptide hormone. Its molecular weight is 36,000. In physiological function, it is similar to LH and FSH. It promotes ovulation, gonad development, and sex-hormone secretion. The hormone is refined from the urine of women who are 2–4 months pregnant. It is secreted from the chorionic membrane of the placenta and is more effective on silver carp and bighead than on grass carp. At present, the ready-made material available on the market in China is “veterinary gonadotropin.” It is a white powder that is soluble in water and must be stored in a cool, shady, dry, airtight environment.
Dosage — For silver carp and bighead, the recommended dose is 4–5 mg/kg (800–1000 IU/kg).
Injection
Preparation of injection
To prepare the PG suspension fluid, first calculate the required amount of PG based on the total body weight of the parent fish. Second, place the PG into a mortar (if the PG is preserved in liquid, dry it on a filter paper for 15 min) and grind thoroughly; add enough normal saline to suspend the ground PG. The volume of injections should be controlled at 2–3 mL for each parent fish.
When HCG and LRH-A are injected, the required dose must be combined with normal saline at a concentration of 0.7 per cent. When the powder dissolves, the solution is ready to be injected. It should be prepared right before use so as to prevent the suspension fluid from becoming ineffective.
Method of injection
In traperitoneal injection is common. During injection, a brood fish is placed in a cloth bag, lying laterally in the water. The upper half of the fish is held above the surface. At the inner side of the basal part of the pectoral fin where it is scaleless, the syringe needle is inserted toward the head at an angle of 45° to the body's longitudinal axis and to a depth of about 1.5 cm. The fluid is then slowly injected.
Frequency and time of injection
There are two frequencies of injection: single and double. For the single injection, the predetermined dose is completely injected into the fish. For the double injection, the predetermined dose is divided into two separate dosages and injected twice. The amount of the first injection is usually 10–20 per cent of the total dose; 90-80 per cent is in the second injection. Milters always receive only one injection. The injection is given to the milter when the spawner receives the second injection. Both the single-injection and double-injection techniques give satisfactory results. The time of injection depends upon water temperature and working conditions. The brooders are usually controlled to spawn around midnight of at dawn. If the single-injection method is adopted, the injection is done in the afternoon or at dusk to allow the brooders to spawn at dawn. If the double-injection method is adopted, the first injection is given in the morning. The interval between the two injections is 8–12 h. If the water temperature is high, it is better for the brooders to spawn at midnight; fertilization rate and the hatching rate are enhanced.
Season for Spawning Induction
Choosing the most suitable season to induce spawning is an important step in the artificial propagation of Chinese carps. The optimum time depends on the weather and the gonad development of fish. The initial propagation period in Guangdong Province is early May; that in Jiangsu and Zhejiang provinces is from the middle of May to the middle of June. The optimum temperature range is 22–28°C. The sequence for spawning induction is as follows: grass carp, silver carp and big-head, black carp.
Selection of Parent Fish
A female fish with a bulging abdomen and a swollen, soft, elastic genital opening is the usual choice. When mature female silver carp and bighead are held out of the water, their ribs can be faintly seen. If the tail is raised, the contour of the ovary is seen to move forward. If the abdomen of a female silver carp or bighead brooder is exceedingly expanded and less elastic, the fish is overmature and should not be selected for spawning induction. For the mature male fish, if the abdomen is pressed gently, milt will be exuded.
For grass carp, feeding should be stopped 2 days before selection for induction. If a mature female grass carp is placed belly up and the contour of the ovary is seen to move downward on each side of the abdomen, the middle of the abdomen is indented, and the abdomen feels soft, the fish can be selected for induction.
Selection can also be based on the degree of maturity of the eggs. Collect a small amount of eggs from the genital opening and fix them in a solution of 85 mL of 95 per cent alcohol, 10 mL of 40 per cent formaldehyde, and 5 mL of glacial acetic acid for 2 or 3 min. The cytoplasm and yolk will then be transparent and the nucleus opaque. The nucleus, if observed in the centre of the egg, indicates that the egg has not yet matured, and the fish is not ready for induction. If the nucleus is eccentric, the egg is mature and the fish is ready for spawning. If the nucleus is vague or cannot be seen, the egg is overmature or degraded and the fish should not be induced.
Response Time
Under normal conditions, there is a delay from the time of the last injection until the parent fish enters estrus. This period is called the response time.
Response times vary slightly depending on water temperature, the spawning inducing agent, the injection frequency, and the species induced. When the water temperature rises 1°C, the response time decreases 1–2 h. The double-injection method has a shorter response time than the single-injection method. For example, when the water temperature is 24–25°C and a silver carp is induced with a single PG injection, the response time is 12–14 h.; with the double-injection method, the response time is 7 or 8 h (counted from the second injection). The response time to PG injection is 1 or 2 h shorter than that to HCG injection; the response time to LRH-A injection is longer than that of PG injection. In addition, given the same hormone at the same dosage during the same season, the response times of various species are slightly different. Generally, grass carp show the shortest response times, followed by silver carp, and bighead and black carp showing the longest response time.
Natural Spawning and Fertilization
After injection, the parent fish are placed in the spawning pond to spawn or exude milt and complete the fertilization process. This is called natural spawning and fertilization.
Spawning pond
After the injection of the spawning-inducing agent, the parent fish need a pond with suitable ecological conditions in which they can perform estrus and spawn. The pond should also allow the convenient collection of fertilized eggs. The spawning pond is usually made of bricks and trowelled with cement. There are two common varieties: circular (Fig. 2.1) and elliptical (Fig. 2.2).
Circular spawning pond — The inlet of the pond is set at a 40° angle tangent to the pond wall. The bottom is concave and an outlet is usually installed in the centre of the bottom. The outlet passes, through an underground pipe, either directly to the incubators or to an egg collection chamber beside the spawning pond. A small cage is set in the chamber to collect the eggs carried by water from the spawning pond.
Elliptical spawning pond — The inlet and the outlet are situated on the same straight line. The water is 1 m deep. The bottom of the pond slopes downward to the outlet. There is an adjacent egg-collecting pond with an egg-collecting cage that is connected to the outlet of the spawning pond.
Fig. 2.1. Circular spawning pond (Unit = cm)
Fig. 2.2. Elliptical spawning pond. (Unit = cm)
Estrus and spawning
After injection and if the fish react normally to the spawning inducing agent, they will begin chasing each other excitedly. This phenomenon is called estrus. The chasing produces irregular ripples on the water surface. As the chasing continues, the fish may sometimes break the surface of the water. At the climax of estrus, the male hits the female's abdomen with its head. The female then lies on its side either underwater or on the surface; its abdomen and tail become extremely constricted and eggs are discharged. At the same time, the male nestles up to the female's abdomen and discharges milt. Sometimes, the male and the female are entangled and swing their pectoral fins as they spawn or discharge milt.
Methods of collecting fertilized eggs
There are two methods of collecting fertilized eggs. One method involves the addition of slowly flowing water to the spawning pond during estrus. This helps to bring the gametes together and raise the fertilization rate. After spawning, the blood fish are removed and the eggs are collected by flushing the pool. The eggs then flow into the egg-collecting cage, where they are collected and counted. Another method involves pouring water into the pond after spawning and flushing the fertilized eggs, which sink to the bottom, into the egg-collecting cage. This is called still-water spawning.
Methods of counting eggs
A volumetric method can be used to calculate the number of eggs. First, the capacity of a container is measured. The eggs in 10 or 20 mL are then counted and the total number of eggs in the container is calculated. On average, after absorption of water, there are 10,000 grass carp and silver carp eggs or 8,000 bighead eggs per litre.
Artificial Insemination
When the brood fish are in estrus and begin to spawn, they are immediately captured. Eggs and milt are collected to bring the mature egg and sperm together. This is called artificial insemination.
The eggs must be at the proper stage of maturity so that after fertilization they will develop normally. At a water temperature of about 28°C, after ovulation Chinese carp eggs keep fertile for 1–2 h until spawning. The time of egg and milt collection is dependent on the spawning-inducing agent used, species, water temperature, etc. These factors are key to the success of artificial insemination. For example, when the water temperature is 23°C, silver carp eggs develop from early maturation to proper maturation in 40 min. They become overmature 1.5–2 h after reaching proper maturity. Only properly matured eggs are able to develop normally; immature or overmature eggs develop abnormally.
After stripping, eggs remain fertile for 10–20 min if free from water; however, in freshwater the eggs lose their fertility in 1 min. In a 0.7 per cent saline solution, eggs remain fertile for 10 min.
The sperm of Chinese carps is active only after it is released into the water. In fresh water, sperm survive for 1 min. The sperm is most fertile for the first 20–30 s in fresh water. Sperm in normal saline can survive for 2–3 min.
There are two methods of artificial insemination for Chinese carps: the dry method and the semi-dry method. In the dry method, about 15 min after the beginning of estrus, the brood fish is captured and stripped. The eggs are collected with a basin (each basin should not contain more than 500,000 eggs). The semen is either directly squeezed onto the eggs or transferred with a pipette and dropped onto the eggs. The mixture is then stirred gently by hand or with a feather for about 1 min and a little clean water is added. The mixture is stirred again for 1 min, allowed to stand for 1 min, and the dirty water is then removed. After the eggs have been washed 3 times in this way, they are transferred to an incubator.
In the semi-dry method, the semen is diluted with a little normal saline and transferred onto the eggs by pipette. Otherwise, this technique is as the dry method.
During artificial insemination, number of eggs can be estimated by measuring the total weight of eggs stripped. Usually, before absorbing water, there are 700–750 grass carp and silver carp eggs and 650–700 bighead eggs per gram.
Incubation not only involves bringing the egg through embryonic development to hatching out but also includes all the management work from hatching to stocking ponds.
The eggs of Chinese carps are semibuoyant. After the eggs have been fertilized and have absorbed water, the egg membrane expands to about 5–6 mm in diameter. In stagnant water, the eggs sink; in running water, they float. In rivers, fertilized eggs will float and hatch. The embryonic development is closely dependent upon environmental conditions: e.g., oxygen supply, water temperature, and water current.
Water Temperature and Dissolved Oxygen
For normal embryonic development and hatching of Chinese carp the water temperature must range from 17 to 30°C. The optimum range is 25–27°C. As water temperature increases, the speed of embryonic development also increases (Table 2.2).
The dissolved oxygen content of the water should not be below 4–5 mg/L; below 2 mg/L, the embryo will develop abnormally.
Table 2.2. Relationship between hatching time and water temperature of silver carp
| Water temperature (°C) | 18 | 20 | 22 | 24 | 25 | 26 | 27 | 28 | 28.5 | 30 |
| Hatching time (hours from fertilization to hatching out) | 61 | 50 | 33 | 31 | 24 | 21 | 19 | 18 | 17 | 16 |
Hatching Instruments and Operational Management
Hatching jar
Hatching jars (Fig. 2.3) are made of tinplate or plastic. Hatching vats are generally reformed earthenware vats (Fig. 2.4). The recommended volume of each container is 250 L and the density of eggs is recommended to be 1 egg/mL. The hood of the hatching jar (and hatching vats) is made of 50 mesh/in nylon netting. The water flows in at the bottom and out over the upper rim through the hood. The flow of water must be regulated so that the fertilized eggs will float to the surface. Debris and egg shell must be periodically brushed off the hood to allow the water to flow freely throughout hatching and prevent the newly hatched fry from escaping with the over flowing water.
Fig. 2.3. Hatching jar. (Unit = cm)
Fig. 2.4. A reformed earthern ware vat which can be used as hatching instrument for small scale fish hatchery.
Fig.2.5. Hatching circulator: (unit = cm)
Hatching circulator
The hatching circulator (Fig. 2.5) made of cement and bricks, consists of ring-shaped tank. There are two kinds of hatching raceway: single and double. The circular course is 1 m wide and 0.9 m deep, and the pond is 4 m in diameter. It can hold 8 m3 of water and 8 × 106 eggs (106 eggs/m3). The water exchange rate is about 260 L/min. No “dead corners” and an adequate flowing speed are essential qualities of a good circulator. The operational management is same as for hatching jars.
Calculating Fertilization Rate, Hatching Rate and Fry Survival Rate
When the eggs have developed to the middle gastrula stage (6–8 h after fertilization), 100 eggs should be randomly collected with a scoop net and placed in a white dish. Turbin eggs, white eggs, empty eggs, rotten eggs etc., should then be selected and removed. The fertilization rate can then be calculated:
Hatching rate can also be calculated:
In practice, however, it is difficult to obtain accurate values from which to calculate the hatching rate. Therefore, it is more common to calculate the survival rate:
As soon as the air bladders of the fry have been filled with air, the yolk sacs have essentially disappeared, and the fry begin to take food actively (about 4–5 days after hatching); they may now be transferred to nursery pond.
