Comparative studies on Spawning-inducing using Ovaprim and other hormones. Xu Pao Min Kuanhong Zhu Jian Wang Jianxin Gong Yongseng

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1 Comparative studies on Spawning-inducing using Ovaprim and other hormones Xu Pao Min Kuanhong Zhu Jian Wang Jianxin Gong Yongseng (Freshwater Fisheries Research Center, Chinese Academy of Fishery Sciences, Wuxi, China) Abstract Ovaprim and PG, HCG, LRH were concurrently used to induce final maturation and spawning in freshwater fishes. Trials of five freshwater fish species as mandarin fish, black carp, silver carp, crucian carp and common carp were conducted. The rice field eel is not be able for a trial because the climate was abnormal and a serious flood happened affecting the gonadal in maturation fully. From the data of trials, it is found that the effectiveness of Ovaprim inducing fish spawning is better than the products used before and Ovaprim can also induce final maturation, increase milt production and improve the fecundity greatly. The training is also conducted for marketing potentials. Key words: Ovaprim, PG, HCG, LRHa, spawning inducing, Freshwater Fisheries Research Center (FFRC) cooperated with Syndel International Inc. Canada on the experiment for fish hormone product. According to the program, FFRC introduced the Syndel product Ovaprim in April The objective of project is to induce final maturation and spawning in high economic value species or hard to spawn species with Ovaprim and conduct trials to compare product pituitary gland (PG), human chorionic gonadotropin (HCG), luteinizing (hormone) releasing hormone analogue (LHRHa, LRHa) made in China with Ovaprim. Six freshwater fish species, mandarin fish (Siniperca chautsi), black carp (Mylopharyngodon piceus), silver carp (Hypophthalmichtys molitrix), crucian carp (Carassius carassius), common carp (Cyprinus carpio) and ricefield eel (Monpterus albus) were selected in the trial. Experimental targets in our trial involve response time (time from to spawning), spawning rate, fertilization rate, hatching rate and survival rate, etc. 1 Materials and methods 1.1 Materials Mandarin fish (Siniperca chautsi) Mandarin fish is a famous and precious freshwater fish with high economic value. Ovaprim is used to induce final maturation and spawning and the effectiveness is to be compared with other hormone product Black carp (Mylopharyngodon piceus) The spawning inducing of black carp is difficult and need a lot of labor and time because the injection for female is usually twice or even three times. For two injections, the first one is for promoting maturation, second one is for inducing spawning. Interval between two injections is about 10 hours. The male black carp is given only one injection at the second time of the female. For three injections, the first and second are promoting maturation, the interval is 15days. The third is about 10h after the second for inducing spawning. For the male fish, one injection is 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. A single injection of Ovaprim is most often sufficient to induce spawning. It can save manpower and expense Crucian carp (Carassius carassius) Crucian carp is delicious and in great demand by consumers. It is found that fertilization rate of crucian carp is low, sometimes even below 10%. It affected the production of fry. The objective of using Ovaprim is to induce final maturation, increase milt production and improve the fertilization rate greatly Silver carp (Hypophthalmichtys molitrix) and Common carp (Cyprinus carpio)

2 Silver carp and Common carp are the species with very high production. They are the principle food fish in China. The spawning inducing of silver carp and common carp is relatively easy. The purpose of the trial is to compare Ovaprim with hormone product made in China Ricefield eel (Monpterus albus) Ricefield eel is a nutritious and delicious species, it sells well in the market with high economic value. But the spawning inducing of ricefield eel is not so easy because the sex reversal makes the differentiation between male and female difficult. The parent fish is captured from field or brought from market, it is easy to be injured and the survival rate is low. And the fecundity of female is small, just eggs a fish. It is also difficult to get large quantities of fries. The objective of project is to induce final maturation and spawning with Ovaprim, and obtain fries with large scale. 1.2 Methods Table 1. A brief introduction to the trial Species Mandarin fish Date treated Water temp. Trial site Product used May, 8th. 25 C Fish farm of Changshu city Ovaprim, PG, LRHa Black carp May, 22nd. 25 C Fish farm of Changshu city Ovaprim, PG, LRHa Silver carp May, 20th. 22 C Crucian carp April, 21st. 18 C Fish fry and fingerling farm of Xishan city Experimental fish farm of Wuxi Fisheries Research Institute (FFRC) Ovaprim, HCG, LRHa Ovaprim, HCG, LRHa Common carp April, 16th. 18 C Experimental fish farm of FFRC Ovaprim, HCG, LRHa Ricefield eel June, 22nd 26 C Experimental fish farm of FFRC Ovaprim, LRHa Black carp (Mylopharyngodon piceus) and Silver carp (Hypophthalmichtys molitrix) Only after a fish reaches sexual maturity can it be induced to spawn; whether the fish is properly reached or not will directly affect the results of induced spawning. The rearing of brood fish therefore is an important step in the artificial propagation of cultured fish. Induced Spawning In induced spawning, the mature parent fish are injected with spawning inducing agents. The basic principle behind induced spawning comes from the biological mechanism of natural propagation. Because the external ecological conditions in ponds cannot satisfy the reproductive requirements of the brood fish, the fish are injected with extraneous hormones. Some extraneous hormones (e.g., PG and 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 (LRHa) only stimulate the hypophysis of the parent fish, accelerating the secretion of gonadotrophin and inducing the parent fish to spawn or discharge milt. Choosing the most suitable season to induce spawning is an important step in the artificial propagation. 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 C. 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

3 filter paper for 15 min) and grind thoroughly; add enough normal saline to suspend PG. The volume of injections should be controlled at 2-3 ml for each parent fish. When HCG and LRHa are injected, the required dose must be combined with normal saline at a concentration of 0.7%. When the powder dissolves, the solution is ready to be injected. It should be prepared right before use so as to prevent the loss of efficacy. Method and frequency of injection Intraperitoneal injection is common. 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% of the total dose; 90-80% 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. The interval between the two injections is 8-12 hours. Both the single-injection and double-injection techniques give satisfactory results. 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 C and a silver carp is induced with a single PG injection, the response time is 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. 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 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.

4 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 C. As water temperature increases, the speed of embryonic development also increases 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 IU + LRHa µg, or LRHa 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 C. The response time from injection to parent fish entering estrus is 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 hours to incubate; at 25 C, h. Generally speaking, hatching of fertilized eggs need 4-5 days at the water temperature of C. The eggs hatching can also be conducted in running water system, such as hatching jar and hatching circulator 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 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 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 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 C. The response time from injection to parent fish entering estrus is hours. At C, the fertilized eggs of mandarin fish take hours to incubate. The eggs hatching usually conducts in running water system, such as hatching jar and hatching circulator 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 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. HCG LRHa PG

5 Weight (g) Dose (IU) Weight (g) Dose (µg) Weight (g) Female Male *Ovaprim: 0.5ml/kg for female, 0.25ml/kg for male Dose (mg) The female to male ratio of ricefield eel stocked should be 1:1-1:3. Water temperature of natural spawning is C. The response time from injection to parent fish entering estrus is hours. Artificial insemination is common in the artificial spawning inducing. At 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 Spawning Inducing Agents 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 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 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 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 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 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 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 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 2-4

6 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 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 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. Ovaprim ml/kg 0.25 ml/kg 80% 78% The control PG 2.5mg + LRHa 50µg/kg PG 1.25mg + LRHa 25µg/kg Fer.%: fertilization rate. Hat.%: hatching rate. 76% 75% 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. Ovaprim )0.5ml/fish 2)5ml/fish The control )LRHa 2µg/kg 2)PG 2mg + LRHa 20µg/kg Table 4. Result of black carp. 5 ml/fish 90% 70% PG 1mg + LRHa 10 µg/kg 85% 65%

7 2.3 Silver carp Table 5. Result of silver carp. Ovaprim ml/kg 0.25 ml/kg 85% 90% The control HCG 200IU +LRHa 28µg/kg HCG 100IU+ LRHa 14µg/kg 80% 85% 2.4 Crucian carp Ovaprim can induce final maturation, increase milt production and improve the fecundity greatly. Table 6. Result of crucian carp. Ovaprim ml/kg 0.25 ml/kg 40% 60% The control HCG IU +LRHa 50µg/kg 2.5 Common carp Table 7. Result of common carp. HCG IU +LRHa 25µg/kg 10% 60% Ovaprim ml/kg 0.25 ml/kg 90% 75% The control HCG 400IU +LRHa 20µg/kg 2.6 Ricefield eel HCG 200IU+ LRHa 10µg/kg Table. 8 The dosage of hormone treated in ricefield eel Female Male Ovaprim ml/kg 0.25 ml/kg The control LRHa 15µg/fish LRHa 20µg/fish 80% 70% 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.