Spawning
Response time
Under normal conditions, there is a delay from the time of the last injection until the parent fish enter estrus. This period is called the response time.

Response time vary slightly depending on water temperature, the spawning inducing agent, the injection frequency, and the species induced. When the water temperature raise 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 7-8 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 LRHa injection is longer than that of PG injection. In addition, given the same hormone at same dosage during the same season, the response times of various species are slightly different. Generally, silver carp show the shorter response time than black carp.

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 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.

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. There are two methods of artificial insemination for Chinese carps: the dry method and the semi-dry method. In the dry method, about 15min after the beginning of estrus, the brood fish is captured and stripped. The eggs are collected with a basin. 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 1min and a little clean water is added. The mixture is stirred again for 1 min, allow 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 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.

Incubation

Incubation not only involves bringing the eggs through embryonic development to hatching out but also includes all the management work from hatching to stocking ponds. The hatching instrument of black carp and silver carp is raceway or hatch jar.  

The eggs of Chinese carps are semibuoyant. After the eggs have been fertilized and have absorbed water, the egg membrane expends 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.

For normal embryonic development and hatching of Chinese carp the water temperature must range from 17°C  to 30°C. The optimum range is 25-27°C. As water temperature increases, the speed of embryonic development also increases.

1.2.2    Common carp (Cyprinus carpio)

A month before spawning, the female and male parent fish should be reared separately in order to keep the breeders spawning in the meantime.

The initial artificial reproduction age of female and male parent fish should be 3 years and 2 years respectively. The weight of female and male parent fish should be more than 1.5 and 1 kg.

Artificial inducing and natural pond spawning can be used in common carp spawning.

Spawning inducing could improve spawning effectiveness and obtain strong fry groups. Spawning inducing agents are PG, HCG and LRHa. Dosage for female (1kg body weight) is HCG 500-600IU  + LRHa 10-20 µg, or LRHa 10-20 µg + PG 2-4 mg, or PG 4-6 mg, ovaprim 0.5ml, dosage for male is the half of female. Intraperitoneal injection is common.

The female to male ratio of common carp stocked in spawning pond should be 1:1-1:1.5. Water temperature of natural spawning is 16-18°C. The response time from injection to parent fish entering estrus is 20-24 hours. After fertilized eggs stuck to palm fiber, move the palms to fry-rearing pond.

At 20°C, the fertilized eggs of common carp take 101-104 hours to incubate; at 25°C, 49-53 h. Generally speaking, hatching of fertilized eggs need 4-5 days at the water temperature of 18-20°C. The eggs hatching can also be conducted in running water system, such as hatching jar and hatching circulator.

1.2.3    Crucian carp (Carassius carassius)

The artificial propagation of crucian carp is similar to common carp. Crucian carp begins to spawning when the water temperature reaches 18°C, in middle April to May in Changjiang River drainage, a little later than common carp. The optimum spawning temperature is 20-22°C. The initial artificial reproduction age of parent fish is 2 years. The weight of female parent fish should be more than 300g and male 250g. The quantity of male crucian carp is usually less than the female, so the female to male ratio of artificial propagation is 2-3:1. Crucian carp can use artificial induced spawning and natural pond hatching. Spawning inducing agents and dosage refer to common carp.  

1.2.4    Mandarin fish (Siniperca chautsi)

Artificial inducing and natural raceway spawning can be used in mandarin fish.

Spawning inducing agents are PG, HCG and LRHa. Dosage for female (1kg body weight) is HCG 1500IU + LRHa 150 µg, or LRHa 50-100 µg + PG 5-8 mg, or PG 5 mg +HCG 500IU + LRHa 50 µg, or ovaprim 0.5ml, dosage for male is the half of female.

The female to male ratio of mandarin fish stocked in spawning pond should be 1:1-1:1.5. Water temperature of natural spawning is 25-28°C. The response time from injection to parent fish entering estrus is 24-35 hours.

At 25-30°C, the fertilized eggs of mandarin fish take 30-50 hours to incubate. The eggs hatching usually conducts in running water system, such as hatching jar and hatching circulator.

1.2.5    Ricefield eel (Monpterus albus)

Artificial propagation of ricefield eel is similar to black carp and silver carp. But more parent fish is needed because the fecundity of female is small, just 200-600 eggs a fish.

Artificial inducing and natural pond spawning can be used in common carp spawning.

Spawning inducing agents are PG, HCG or LRHA. For dosage, there is an example in the table. The dosage in the table is for one injection separately.

Table 2. The spawning inducing dose of ricefield eel.

*Ovaprim: 0.5ml/kg for female, 0.25ml/kg for male.

The female to male ratio of ricefield eel stocked should be 1:1-1:3. Water temperature of natural spawning is 25-28°C. The response time from injection to parent fish entering estrus is 40-75 hours. Artificial insemination is common in the artificial spawning inducing.

At 25-28°C, the fertilized eggs of ricefield eel take 5-7 days to incubate. The eggs hatching can be conducted in glass jar, small cage, etc.

1.3.  Spawning Inducing Agents

1.3.1    LRHa

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 aminoacids; pyroglutamic acid, histidine, tryptophan, serine, tyrosine, glutamic acid, leucine, arginine, groline, and glycine amide. Its molecular weight is 1182. Artificial synthesized LRH has a high biological activity for cows, sheep, and humans. When it is used to induce spawning in fish, however, the dose must be 100 times higher than that for mammals because LRH is easily destroyed by fish protease. In 1975, LRHa was synthesized. This analogue of LRH consist of nine amino acids (pyroglutamic acid, histidine, trytophan, serine, tyrosine, D-alanine, 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 LRHa. The biological activity of LRHa is about 100 times higher than that of LRH to fish. The LRHa 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.

LRHa can be used for one injection. Two consecutive injections are more effective. If LRHa is used in combination with HCG or PG, it stimulates the ova to mature or promotes the polarization of nucleus and the rate of successful induction is high and constant.

1.3.2    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. The estrus-inducing effect of PG on Chinese carps is immediately evident.

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 hypophsis can be removed.

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.

1.3.3    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. 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.

1.3.4    Ovaprim

Ovaprim contains Syndel's patented analogue of salmon GnRH (SGnRHa) and domperidone, a dopamine inhibitor, in a sterile liquid for easy injection. SGnRHa works with the natural spawning system of the fish. When injected, it causes the release of natural hormones in the fish brain and gonads to trigger normal spawning and spermiation. SgnRHa is more potent and consistent than any other available spawning product. Domperidone blocks the negative effects of dopamine that normally inhibits hormone release. In hard-to-spawn fish, domperidone is essential to support the effectiveness of SgnRHa. Domperidone is safer and more effective than other available Dopamine inhibitors. Ovaprim must be used during, or at the start of the spawning season. Accurate records of spawning dates and performance of the fish population are required. A single injection of Ovaprim is most often sufficient to induce spawning.  

2          Results

Ovaprim is a liquid product different with HCG, LRHa and PG, it needn't to be dissolved and prepared. It is used for injection directly and easily. The dose for injection is treated accurately.

The effectiveness of Ovaprim inducing fish spawning is better than the control products. It is found that the response time of Ovaprim is one hour longer than the control. The results of the five species are given below, the detailed data could be seen in the clinical field study reporting forms drafted afterwards.

2.1       Mandarin fish

Table 3. Result of mandarin fish.

Fer.%: fertilization rate.  Hat.%: hatching rate.

2.2       Black carp

The injection for female black carp is usually twice, the first one for promoting maturation, second one for inducing spawning. The dosage of the first time usually is one-tenth of the second. Interval between two injections is about 10 hours. The male black carp is given only one injection at the second time of the female.

Table 4. Result of black carp.

2.3    Silver carp

Table 5. Result of silver carp.

2.4       Crucian carp

Ovaprim can induce final maturation, increase milt production and improve the fecundity greatly.

Table 6. Result of crucian carp.

2.5       Common carp

Table 7. Result of common carp.

2.6       Ricefield eel

Table. 8 The dosage of hormone treated in ricefield eel

The climate in Wuxi this year is abnormal. A big flood occurred and the temperature is lower than usual. Low temperature and continuous cloudy and rainy day affected the maturity of parent fish and survival rate. The spawning inducing of ricefield eel wasn't successful and the parent fish didn't spawn normally. We plan to do the trial again next year and then we will get some effective data then.