Artificial breeding method of culter oxycephalus in xingkai lake

Abstract

The present application relates to the technical field of aquaculture, and particularly relates to an artificial breeding method of Cottus hangi in Xingkai Lake. The present application uses LRH-A2 and HCG combination oxytocin to reasonably inject female parent fish and male parent fish, especially to twice injection of female parent fish, and strictly controls injection dose of each injection and reasonable control time interval, which not only can efficiently induce labor, but also can reach 95% of the female parent fish induction efficiency, and can obtain good ovum and semen, so that the ovum can be better fertilized, the quality of the fertilized ovum is improved, the hatching rate is improved, the stress of the parent fish is reduced, and the death of the parent fish is reduced. The present application improves the physical fitness of the parent fish through reasonable parent fish cultivation, and combines reasonable artificial induction operation and post-production nursing measures, so that the post-production death of the parent fish is reduced from multiple aspects, and the post-production survival rate of the parent fish is significantly improved, so that the repeated cultivation cost of the parent fish is reduced.

Description

Technical Field

[0001] This invention relates to the field of aquaculture technology, and in particular to an artificial breeding method for the Culter alburnus from Lake Xingkai. Background Technology

[0002] The Culter alburnus Basilewsky, commonly known as the big white fish, is called "Juezuidaozi" along the Songhua River. It is widely distributed in the Pearl River, Yangtze River, and Heilongjiang River basins of my country. The Xingkai Lake Culter alburnus is a naturally independent population of this species. Due to the excellent geographical and aquatic environment of Xingkai Lake, its Culter alburnus has a silvery-white body, tender flesh, and delicious flavor, making it an important and valuable economic fish species. Historically, it was a tribute fish to the capital and a delicacy at high-end banquets, enjoying a high market reputation and significant economic value. Furthermore, the Xingkai Lake Culter alburnus is highly favored by fish farmers due to its rapid growth, strong disease resistance, high harvest rate, and ability to be artificially domesticated. In recent years, among the aquaculture species that Heilongjiang Province has accelerated in its strategy to revitalize and develop cold-water fisheries, the Xingkai Lake Culter alburnus, known as one of China's "Four Famous Freshwater Fish," has been included.

[0003] With the booming popularity of the Culter alburnus from Lake Xingkai, market demand is increasing, while wild Culter alburnus resources are gradually decreasing. Farming Culter alburnus has become one of the main models for the development of ecological circular agriculture. As early as 2011, the Harbin Municipal Fisheries Technology Extension Station successfully conducted artificial breeding and propagation trials of Culter alburnus. However, due to the fish's unique reproductive habits and poor stress resistance, the hatching rate of fry after artificial breeding is low, and the mortality rate of broodstock can reach over 90%. Furthermore, the sexual maturity cycle of Culter alburnus in Northeast China is as long as 5-6 years, resulting in a long broodstock cultivation period, high breeding costs, and low reuse rates, which severely restricts the development of the Culter alburnus farming industry.

[0004] Therefore, it is essential to develop a breeding method for the Culter alburnus in Lake Xingkai with low parent fish mortality and high hatching rate. Summary of the Invention

[0005] Therefore, based on the above background, the present invention provides an artificial breeding method for Culter alburnus in Lake Xingkai, which can effectively solve the problems of low hatching rate and high mortality rate of parent fish caused by the existing artificial breeding process of Culter alburnus in Lake Xingkai, and can better promote the development of the Culter alburnus aquaculture industry in Lake Xingkai.

[0006] The technical solution provided by this invention is as follows: An artificial breeding method for Culter alburnus from Lake Xingkai includes the following steps: S100 parent fish breeding Select healthy, disease-free Culter alburnus from Xingkai Lake that are over 5 years old and released into parent fish rearing ponds. The ratio of female to male fish is 1:(1.2-1.5), and the stocking density is 250-500 kg / mu.

[0007] The pH of the water in the broodstock rearing pond is controlled between 7.5 and 8.5; S200 Artificial Labor Induction Mature gonadal-developing Culter alburnus from Lake Xingkai were selected as breeding broodstock, and oxytocin was injected into the breeding broodstock. The oxytocin is composed of luteinizing hormone-releasing hormone analog LRH-A2 and human chorionic gonadotropin HCG. The female parent fish were injected with two doses of oxytocin, totaling 2-5 μg / kg of luteinizing hormone-releasing hormone analog LRH-A2 and 800-1200 μI / kg of human chorionic gonadotropin (HCG). The first injection was 1 / 20 to 2 / 10 of the full dose of oxytocin, with the water temperature controlled at 21-26℃. The remaining dose was injected after an interval of 8-10 hours. At the same time as the female parent fish receive the second injection, the male parent fish are injected with 3 / 10 to 7 / 10 of the full dose of oxytocin given to the female parent fish. S300 Artificial Insemination The water temperature is controlled at 21-26℃. After the broodstock fish are injected with oxytocin, they are cultured for 7-12 hours. Eggs and semen are collected artificially. The eggs and semen are placed in a dry container with 100,000-220,000 eggs per ml of semen. The mixture is stirred thoroughly to ensure that the eggs and sperm are evenly mixed. Water is then added at a volume of 2-4 times the volume of the egg and semen mixture. The mixture is stirred for 2-5 minutes and then left to stand for 2-3 minutes to complete the artificial insemination. S400 fertilized eggs hatch The fertilized eggs obtained in step S300 are distributed at a rate of 600,000-900,000 eggs / m³. 3 The eggs are placed in water at 22-26℃ for incubation. The dissolved oxygen in the water is ≥6mg / L. The initial water flow rate is controlled at 5m / s. After the eggs have fully absorbed water, the water flow rate is reduced to 2m / s and maintained for 18-22 hours until hatching and breaking the membrane. Once the eggs emerge from the waist and can swim horizontally, the fry can be obtained. S500 Postpartum Care On the day after the collection of eggs and sperm in step S300, which is counted as day 1, gentamicin sulfate at a dose of 5-10 mg / kg is injected into the dorsal fin muscle of the breeding broodstock. Then, the broodstock are cultured in a pond at a temperature of 20-25℃ until day 8-12 postpartum, after which the breeding broodstock are transferred with water.

[0008] Furthermore, in step S100, the feed is provided during the cultivation process, and the nutritional composition (by mass) of the feed is 35-42% crude protein, 8-12% crude fat, 10-15% crude ash, 3-8% crude fiber, 0.5-1.5% total phosphorus, 2-5% lysine, and 10-15% moisture.

[0009] Furthermore, in step S100, 20-35 days before artificial spawning, wild miscellaneous fish or silver carp and bighead carp fry are released into the parent fish rearing pond as bait fish.

[0010] Furthermore, in step S400, before hatching the fertilized eggs, the eggs are soaked in potassium permanganate at a concentration of 8-12 mg / L for 3-10 minutes.

[0011] Furthermore, during the first 1-11 days of postpartum rearing of breeding broodstock in S500, antimicrobial peptides were administered in addition to the feed. The nutritional composition (by weight) of the feed is 35-42% crude protein, 8-12% crude fat, 10-15% crude ash, 3-8% crude fiber, 0.5-1.5% total phosphorus, 2-5% lysine, and 10-15% moisture.

[0012] Furthermore, in S500, before transferring the breeding broodstock with water, the breeding broodstock are soaked in povidone-iodine solution for 3-5 minutes, and after transferring the breeding broodstock, anti-stress vitamin C is sprinkled into the pond after the transfer.

[0013] Furthermore, in S200, the female parent fish were injected with 1 / 10 of the full dose of oxytocin for the first time, and the remaining amount was injected after an interval of 8-10 hours.

[0014] Furthermore, in step S300, the collection time of sperm and eggs is controlled based on the water temperature during cultivation after artificial ovulation. When the water temperature is 21-23℃, after the parent fish that have been injected with oxytocin continue to be cultured for 9-11 hours, eggs and semen are collected artificially. When the water temperature is 24-25℃, after the broodstock fish injected with oxytocin are cultured for another 8-9 hours, eggs and semen are collected artificially.

[0015] The beneficial effects achieved by this invention are as follows: ① This invention utilizes a combination of LRH-A2 and HCG as an oxytocin for the rational injection of both female and male broodstock. In particular, the female broodstock are injected twice, with strict control over the dosage of each injection and the time interval between injections. This not only achieves highly efficient spawning induction, with an efficiency of up to 95% for female broodstock, but also yields good quality eggs and sperm, leading to better fertilization, improved egg quality, increased hatching rate, reduced stress on broodstock, and reduced mortality.

[0016] ② This invention improves the physical condition of broodstock through reasonable parent fish breeding, and reduces postpartum mortality of broodstock from multiple aspects by combining reasonable artificial spawning induction operations and postpartum care measures. The postpartum mortality rate of female fish is as low as 17% or less, and the mortality rate of male fish is as low as 10% or less. The postpartum survival rate of broodstock is significantly improved, which can also reduce the cost of repeated breeding of broodstock. Detailed Implementation

[0017] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0018] This invention provides a method for the artificial breeding of Culter alburnus from Lake Xingkai, comprising the following steps: S100 parent fish breeding Select healthy, disease-free Culter alburnus from Lake Xingkai that are 5 years or older and release them into parent fish rearing ponds (1.5-2m deep, outdoors). The ratio of female to male fish should be 1:(1.2-1.5); the stocking density should be 250-500 kg / mu. The pH of the water in the broodstock pond should be controlled between 7.5 and 8.5; the water quality in the broodstock pond must meet the fishery water quality standard GB11607. In specific applications, one way to implement this step is to use aquatic peptide fertilizer and biological agents to control the pH at 7.5-8.5. The biological agents include carbon sources for bacteria and algae, EM bacteria and brown sugar.

[0019] Of course, the pH of the water in the broodstock pond can also be adjusted using other conventional methods.

[0020] During this breeding process, the feed is provided, and the nutritional composition (by weight) of the feed is 35-42% crude protein, 8-12% crude fat, 10-15% crude ash, 3-8% crude fiber, 0.5-1.5% total phosphorus, 2-5% lysine, and 10-15% moisture.

[0021] 20-35 days before artificial spawning, wild fish or silver carp and bighead carp fry are introduced into the broodstock rearing pond as feed fish to provide animal protein and comprehensive nutrition for the development of the broodstock's gonads. The feeding amount is 1-2% of the broodstock's body weight, and the fish should be about 5cm in size.

[0022] S200 Artificial Labor Induction Selected breeding broodstock from Lake Xingkai with gonads developed to stage IV, and transferred them to an indoor pond, where they were injected with oxytocin; When gonadal development reaches stage IV, it means that gonadal development is about to mature. The criteria for judging that gonadal development is about to mature are: male parent fish have tubercles on their body surface, feel rough to the touch, and a small amount of white semen will be discharged when the abdomen is gently pressed. The semen disperses immediately upon entering the water. Microscopic examination shows that the sperm have strong motility and few abnormalities. Female parent fish have a full abdomen, obvious ovarian outline, soft and swollen ovaries, slightly red genital opening, and a small amount of eggs will flow out when the anus is squeezed. After soaking in a clear solution (acetic acid: alcohol = 3:1), the egg nucleus has been deviated to the animal pole when examined under a dissecting microscope. At this time, spawning can be induced.

[0023] The oxytocin is composed of luteinizing hormone-releasing hormone analog LRH-A2 and human chorionic gonadotropin HCG. The female parent fish were injected with two doses of oxytocin, totaling 2-5 μg / kg of luteinizing hormone-releasing hormone analog LRH-A2 and 800-1200 μI / kg of human chorionic gonadotropin (HCG). The first injection was 1 / 20 to 2 / 10 of the full dose of oxytocin, with the water temperature controlled at 21-25℃. The remaining dose was injected 8-10 hours later. Simultaneously with the second injection of oxytocin to the female broodstock, the male broodstock are injected with 3 / 10 to 7 / 10 of the full dose of oxytocin given to the female broodstock. Preferably, the female broodstock are first injected with 1 / 10 of the full dose of oxytocin, and after an interval of 8-10 hours with the water temperature controlled at 21-25°C, the remaining dose is injected. Simultaneously with the second injection of oxytocin to the female broodstock, the male broodstock are injected with 1 / 2 of the full dose of oxytocin given to the female broodstock. S300 Artificial Insemination The water temperature is controlled at 21-25℃. After the broodstock fish are injected with oxytocin and cultured for another 8-12 hours, eggs and semen are collected artificially. The eggs and semen are placed in a dry container with 100,000-220,000 eggs per ml of semen. After stirring thoroughly to ensure that the eggs and sperm are evenly mixed, water is added at a volume of 2-4 times the volume of the egg and semen mixture. After stirring for another 2-5 minutes and letting it stand for 2-3 minutes, artificial insemination can be completed. In this step, the parent fish are examined according to temperature and effect time to collect semen and eggs for artificial insemination. Specifically, when the water temperature is 21-23℃, the parent fish that have been injected with oxytocin are cultured for another 9-11 hours before the eggs and semen are collected artificially. When the water temperature is 24-26℃, after the broodstock fish injected with oxytocin are cultured for another 7-9 hours, eggs and semen are collected artificially.

[0024] S400 fertilized eggs hatch The fertilized eggs obtained in step S300 are distributed at a rate of 600,000-900,000 eggs / m³.3 The eggs are placed in water at 22-26℃ for incubation. The dissolved oxygen in the water is ≥6mg / L. The initial water flow rate is controlled at 5m / s. After the eggs have fully absorbed water, the water flow rate is reduced to 2m / s and maintained for 18-22 hours until hatching and breaking the membrane. Once the eggs emerge from the waist and can swim horizontally, the fry can be obtained. In this step, when incubating the fertilized eggs, they are soaked in potassium permanganate at a concentration of 8-12 mg / L for 3-10 minutes, and then the water flow rate is controlled to continue incubation.

[0025] S500 Postpartum Care On the day after collecting eggs and sperm in step S300, designated as postpartum day 1, gentamicin sulfate at a dose of 5-10 mg / kg is injected into the dorsal fin muscles of the breeding broodstock. After 8-12 days of cultivation in an indoor pond at 20-25℃, the breeding broodstock are transferred to an outdoor pond while still in water. Specifically, the breeding broodstock are transferred from the indoor pond to an outdoor pond. After introducing water from the outdoor pond into the indoor pond, continue the rearing process for 3-5 days. Once the water temperature in the indoor pond matches that in the outdoor pond, transfer the breeding broodstock to the outdoor pond while still submerged. In this step, the water quality of both the indoor and outdoor ponds must meet the Fisheries Water Quality Standard GB11607.

[0026] Specifically, before transferring the breeding broodstock from the indoor pond to the outdoor pond while still in water, soak the breeding broodstock in a povidone-iodine solution for 3-5 minutes, and after transferring the breeding broodstock to the outdoor pond, sprinkle anti-stress vitamin C into the outdoor pond.

[0027] In this step, during the first 1-11 days of postpartum rearing of the breeding broodstock, in addition to the regular feed, antimicrobial peptide feed will be added. The feed nutrient composition (by weight) is 35-42% crude protein, 8-12% crude fat, 10-15% crude ash, 3-8% crude fiber, 0.5-1.5% total phosphorus, 2-5% lysine, and 10-15% moisture.

[0028] The following examples and comparative examples of artificial breeding of Culter alburnus from Xingkai Lake were all conducted at the Aquatic Experiment and Demonstration Base of Harbin Agricultural Technology Extension Station.

[0029] Unless otherwise specified, the experimental methods used in the following examples and comparative examples are conventional methods; unless otherwise specified, the experimental materials used in the following examples and comparative examples are all purchased from commercial channels.

[0030] The peptide fertilizers used in the following examples and comparative examples were purchased from Wuhan Keyang Biotechnology Co., Ltd. as type B peptide fertilizer. The carbon source for bacteria and algae was purchased from Hailianke's Junzaowang. The antimicrobial peptides were purchased from Wuhan Jixin Yibang Biotechnology Co., Ltd., and the CAS number of the active ingredient is 136212-91-4.

[0031] Example 1: Artificial breeding of Culter alburnus in Lake Xingkai, including the following steps: S100 parent fish breeding Five-year-old or older, disease-free and uninjured Culter alburnus from Xingkai Lake were selected and placed in a parent fish rearing pond. The ratio of female to male fish was 1:1.5. The water depth of the parent fish rearing pond was 2.0 m, and the stocking density was 250 kg / mu. The pH of the water in the broodstock rearing pond is controlled between 7.5 and 8.5 by adding peptide fertilizer and biological agents as needed.

[0032] Feed according to water temperature (feed less when water temperature is low, feed more when water temperature is high). Specifically, when the water temperature is 15-20℃, the feed amount should be... Feed the fish at 1-2% of their body weight when stocked in the pond; 2-3% at 20-25℃; and 3-5% at 25-30℃. The feed is pelleted feed, with the following nutritional composition (by weight): crude protein 40%, crude fat 10%, crude ash 12%, crude fiber 5%, total phosphorus 1%, lysine 2.2%, and moisture 12%. (Purchased from Hongwang Feed Co., Ltd., a compound feed for Culter alburnus). Continue feeding wild fish or silver carp fry as bait fish daily until 30 days before spawning.

[0033] S200 Artificial Labor Induction The breeding broodstock of Culter alburnus from Lake Xingkai, whose gonads have developed to stage IV, were selected and injected with oxytocin. The oxytocin consisted of luteinizing hormone-releasing hormone analog LRH-A2 and human chorionic gonadotropin (HCG). The criteria for judging whether gonadal development has reached stage IV or below are as follows: Male parent fish have tubercles on their bodies, feel rough to the touch, and a small amount of white semen will leak out when the abdomen is gently pressed. The semen disperses immediately upon entering the water. Microscopic examination shows that the sperm are highly motile and have few deformities. Female parent fish have full abdomens, clear outlines of ovaries, and are soft and swollen. The genital opening is slightly red, and a small number of eggs will flow out when the anus is squeezed. After soaking in a clear solution (acetic acid: alcohol = 3:1), the egg nucleus has been deviated to the animal pole when examined under a dissecting microscope, and spawning can be induced.

[0034] In this embodiment, based on the above criteria, 10 female fish and 15 male fish were selected from the parent fish rearing pond for induced spawning.

[0035] The female broodstock were injected with two doses of oxytocin, totaling 3 ug / kg of luteinizing hormone-releasing hormone analogue LRH-A2 and 1000 UI / kg of human chorionic gonadotropin (HCG). The first injection was 1 / 10 of the full dose (i.e., the female broodstock were injected with 0.3 ug / kg of LRH-A2 and 100 UI / kg of HCG), with the water temperature controlled at 24℃. Ten hours later, the remaining dose was injected (i.e., the female broodstock were injected with 2.7 ug / kg of LRH-A2 and 900 UI / kg of HCG). At the same time as the female parent fish receive the second injection, the male parent fish are injected with half the full dose of oxytocin given to the female parent fish (i.e., the male parent fish are injected with a single dose of luteinizing hormone-releasing hormone analog LRH-A2 1.5ug / kg and human chorionic gonadotropin HCG 500UI / kg).

[0036] S300 Artificial Insemination The water temperature was kept at 24℃. After the broodstock fish injected with oxytocin were cultured for another 8 hours, eggs and sperm were collected artificially. The sperm collected from the males could be added to 0.7% fish physiological saline and kept in an environment of 10℃, or collected immediately for artificial insemination.

[0037] In this embodiment, after collecting semen immediately, the eggs and semen are placed in a dry container at a ratio of 200,000 eggs per ml of semen. After stirring thoroughly to ensure that the sperm and eggs are evenly mixed, water is added at a ratio of 2-4 times the volume of the egg and semen mixture. Stirring is continued for 2-5 minutes, and then the mixture is allowed to stand for 2-3 minutes before artificial insemination can be completed. S400 fertilized eggs hatch The fertilized eggs obtained in step S300 were soaked in potassium permanganate at a concentration of 10 mg / L for 5 minutes, followed by treatment at a concentration of 800,000 eggs / m³. 3 The eggs are placed in water at 22-24℃ for incubation. The dissolved oxygen in the water is ≥6mg / L. The initial water flow rate is controlled at 5m / s. After the eggs have fully absorbed water, the water flow rate is reduced to 2m / s and maintained for 18-22 hours until hatching and breaking the membrane. Once the eggs emerge from the waist and can swim horizontally, the fry can be obtained. S500 Postpartum Care On the day after collecting eggs and sperm in step S300, which is counted as the first day after spawning, gentamicin sulfate at 10 mg / kg is injected into the dorsal fin muscle of the breeding broodstock. On the 11th day of cultivation in an indoor pond at a temperature of 20-25°C, water from the outdoor pond is introduced into the indoor pond. After continuing cultivation for 3-5 days, the water in the indoor pond is heated to the same temperature as the water in the outdoor pond, and the breeding broodstock are transferred to the outdoor pond with the water still in it.

[0038] This step involves transferring the breeding broodstock to the outdoor pond while still submerged in water, using a solution of 0.5 g / m³ for the breeding broodstock. 3 Soak for 3-5 minutes, and after transferring the breeding broodstock to the outdoor pond, spray the outdoor pond with anti-stress vitamin C at a rate of 500g / acre.

[0039] During the first 1-10 days of postpartum rearing in indoor tanks, antimicrobial peptides were added to the feed provided. The feed is the same as that used for raising the parent fish.

[0040] To administer antimicrobial peptides, dissolve them in water, spray the solution onto the surface of the pelleted feed, and then let it air dry in a cool place. Add 0.5g-1g of antimicrobial peptides per kg of pelleted feed.

[0041] Comparative Example 1: In this comparative example, 10 female fish and 15 male fish from Example 1 that had been bred as parent fish and met the requirements for induced spawning were artificially induced to spawn. The female broodstock were injected with a single dose of oxytocin consisting of 3 ug / kg of luteinizing hormone-releasing hormone analog LRH-A2 and 1000 IU / kg of human chorionic gonadotropin (HCG). Simultaneously, the male broodstock were injected with half the full dose of oxytocin given to the female broodstock (i.e., the male broodstock were injected with a single dose of 1.5 ug / kg of luteinizing hormone-releasing hormone analog LRH-A2 and 500 IU / kg of human chorionic gonadotropin HCG).

[0042] Other operations are the same as in Example 1.

[0043] Comparative Example 2: In this comparative example, 10 female fish and 15 male fish from Example 1, which had been bred as parent fish and met the requirements for induced spawning, were artificially induced to spawn. The female broodstock were injected with two doses of oxytocin, totaling 3 ug / kg of luteinizing hormone-releasing hormone analogue LRH-A2 and 1000 UI / kg of human chorionic gonadotropin (HCG). The first injection was 1 / 20 of the full dose of oxytocin (i.e., the female broodstock were injected with 0.15 ug / kg of luteinizing hormone-releasing hormone analogue LRH-A2 and 50 UI / kg of HCG). The water temperature was controlled at 24℃. After a 10-hour interval, the remaining dose was injected (i.e., the female broodstock were injected with 2.85 ug / kg of luteinizing hormone-releasing hormone analogue LRH-A2 and 950 UI / kg of HCG)). At the same time as the female parent fish receive the second injection, the male parent fish are injected with half the full dose of oxytocin given to the female parent fish (i.e., the male parent fish are injected with a single dose of luteinizing hormone-releasing hormone analog LRH-A2 1.5ug / kg and human chorionic gonadotropin HCG 500UI / kg).

[0044] Other operations are the same as in Example 1.

[0045] Comparative Example 3: In this comparative example, 10 female fish and 15 male fish from Example 1, which had been bred as parent fish and met the requirements for induced spawning, were artificially induced to spawn. The female broodstock were injected with two doses of oxytocin, totaling 3ug / kg of luteinizing hormone-releasing hormone analogue LRH-A2 and 1000UI / kg of human chorionic gonadotropin (HCG). The first injection was half the full dose of oxytocin (i.e., 1.5ug / kg of LRH-A2 and 500UI / kg of HCG). The water temperature was controlled at 24℃. Ten hours later, the remaining dose was injected (i.e., the second injection of 1.5ug / kg of LRH-A2 and 500UI / kg of HCG). At the same time as the female parent fish receive the second injection, the male parent fish are injected with half the full dose of oxytocin given to the female parent fish (i.e., the male parent fish are injected with a single dose of luteinizing hormone-releasing hormone analog LRH-A2 1.5ug / kg and human chorionic gonadotropin HCG 500UI / kg).

[0046] Other operations are the same as in Example 1.

[0047] Comparative Example 4: In this comparative example, 10 female fish and 15 male fish from Example 1 that had been bred as parent fish and met the requirements for induced spawning were artificially induced to spawn.

[0048] Because a single overdose of human chorionic gonadotropin (hCG) can cause blindness or dystocia in broodstock, we attempted to induce spawning by administering hCG in multiple injections. In this comparative study, female broodstock were injected with two doses of hCG totaling 3 ug / kg of luteinizing hormone-releasing hormone analogue LRH-A2 and 1000 IU / kg of hCG. The first injection was hCG 100 IU / kg, with the water temperature controlled at 24°C. Ten hours later, the second injection was hCG 3.0 ug / kg and hCG 900 IU / kg.

[0049] At the same time as the female parent fish receive the second injection, the male parent fish are injected with half the full dose of oxytocin given to the female parent fish (i.e., the male parent fish are injected with a single dose of luteinizing hormone-releasing hormone analog LRH-A2 1.5ug / kg and human chorionic gonadotropin HCG 500UI / kg).

[0050] Comparative Example 5: In this comparative example, 10 female fish and 15 male fish from Example 1 that had been bred as parent fish and met the requirements for induced spawning were artificially induced to spawn.

[0051] In this comparative experiment, female broodstock were injected with a total of 3ug / kg of luteinizing hormone-releasing hormone analogue LRH-A2 in two injections. The first injection was 0.3ug / kg of luteinizing hormone-releasing hormone analogue LRH-A2, with the water temperature controlled at 24℃. After a 10-hour interval, the second injection was 2.7ug / kg of luteinizing hormone-releasing hormone analogue LRH-A2.

[0052] At the same time as the female parent fish receive the second injection, the male parent fish are injected with half the full dose of oxytocin given to the female parent fish (i.e., the male parent fish are injected with a single dose of luteinizing hormone-releasing hormone analog LRH-A2 1.5ug / kg).

[0053] Comparative Example 6: In this comparative example, 10 female fish and 15 male fish from Example 1, which had been bred as parent fish and met the requirements for induced spawning, were artificially induced to spawn. The female broodstock were injected with a total dose of 1000 IU / kg of human chorionic gonadotropin (HCG) in two separate injections. The first injection was 1 / 10 of the full dose of oxytocin (i.e., the female broodstock was injected with 100 IU / kg of HCG for the first time), with the water temperature controlled at 24°C. After a 10-hour interval, the remaining dose was injected (i.e., the female broodstock was injected with 900 IU / kg of HCG for the second time). At the same time as the female parent fish receive the second injection, the male parent fish are injected with half the full dose of oxytocin given to the female parent fish (i.e., the male parent fish are injected with human chorionic gonadotropin (HCG) 500 IU / kg in a single injection).

[0054] Comparative Example 7: In this comparative example, 10 female fish and 15 male fish from Example 1, which had been bred as parent fish and met the requirements for induced spawning, were artificially induced to spawn. In this comparative control, the female broodstock were injected with two doses of oxytocin totaling 6 ug / kg of luteinizing hormone-releasing hormone analogue LRH-A2 and 1000 UI / kg of human chorionic gonadotropin (HCG). The first injection was 1 / 10 of the full dose of oxytocin (i.e., the female broodstock were injected with 0.6 ug / kg of luteinizing hormone-releasing hormone analogue LRH-A2 and 100 UI / kg of HCG), with the water temperature controlled at 24℃. After a 10-hour interval, the remaining dose was injected (i.e., the female broodstock were injected with 5.4 ug / kg of luteinizing hormone-releasing hormone analogue LRH-A2 and 900 UI / kg of HCG)). At the same time as the female parent fish receive the second injection, the male parent fish are injected with half the full dose of oxytocin given to the female parent fish (i.e., the male parent fish are injected with a single dose of luteinizing hormone-releasing hormone analog LRH-A2 3ug / kg and human chorionic gonadotropin HCG 500UI / kg).

[0055] Other operations are the same as in Example 1.

[0056] Comparative Example 8: In this comparative example, 10 female fish and 15 male fish from Example 1 that had been bred as parent fish and met the requirements for induced spawning were artificially induced to spawn.

[0057] In this comparative control, female broodstock were injected with two doses of oxytocin, totaling 3ug / kg of luteinizing hormone-releasing hormone analog LRH-A2 and 3mg / kg of dioxin. The first injection was 1 / 10 of the full dose of oxytocin (i.e., the female broodstock were injected with 0.3ug / kg of luteinizing hormone-releasing hormone analog LRH-A2 and 0.3mg / kg of dioxin), with the water temperature controlled at 24℃. After a 10-hour interval, the remaining dose was injected (i.e., the female broodstock were injected with 2.7ug / kg of luteinizing hormone-releasing hormone analog LRH-A2 and 2.7mg / kg of dioxin for the second time). At the same time as the female parent fish receive the second injection, the male parent fish are injected with half the full dose of oxytocin given to the female parent fish (i.e., the male parent fish are injected with a single dose of luteinizing hormone-releasing hormone analog LRH-A2 1.5ug / kg and dioxin 1.5mg / kg).

[0058] Comparative Example 9: In this comparative example, 10 female fish and 15 male fish from Example 1 that had been bred as parent fish and met the requirements for induced spawning were artificially induced to spawn.

[0059] The female broodstock were injected with two doses of oxytocin, totaling 3 ug / kg of luteinizing hormone-releasing hormone analogue LRH-A2 and 1000 UI / kg of human chorionic gonadotropin (HCG). The first injection was 1 / 10 of the full dose of oxytocin (i.e., the female broodstock were injected with 0.3 ug / kg of luteinizing hormone-releasing hormone analogue LRH-A2 and 100 UI / kg of HCG). The water temperature was controlled at 24℃. Five hours later, the remaining dose was injected (i.e., the female broodstock were injected with 2.7 ug / kg of luteinizing hormone-releasing hormone analogue LRH-A2 and 900 UI / kg of HCG)). At the same time as the female parent fish receive the second injection, the male parent fish are injected with half the full dose of oxytocin given to the female parent fish (i.e., the male parent fish are injected with a single dose of luteinizing hormone-releasing hormone analog LRH-A2 1.5ug / kg and human chorionic gonadotropin HCG 500UI / kg).

[0060] Comparative Example 10: In this comparative example, 10 female fish and 15 male fish from Example 1 that had been bred as parent fish and met the requirements for induced spawning were artificially induced to spawn.

[0061] The female broodstock were injected with two doses of oxytocin, totaling 3ug / kg of luteinizing hormone-releasing hormone analogue LRH-A2 and 1000UI / kg of human chorionic gonadotropin (HCG). The first injection was 1 / 10 of the full dose of oxytocin (i.e., the female broodstock were injected with 0.3ug / kg of luteinizing hormone-releasing hormone analogue LRH-A2 and 100UI / kg of HCG). The water temperature was controlled at 24℃. After a 15-hour interval, the remaining dose was injected (i.e., the female broodstock were injected with 2.7ug / kg of luteinizing hormone-releasing hormone analogue LRH-A2 and 900UI / kg of HCG)). At the same time as the female parent fish receive the second injection, the male parent fish are injected with half the full dose of oxytocin given to the female parent fish (i.e., the male parent fish are injected with a single dose of luteinizing hormone-releasing hormone analog LRH-A2 1.5ug / kg and human chorionic gonadotropin HCG 500UI / kg).

[0062] The induced spawning rate (%), fertilization rate (%), hatching rate (%), and mortality rate (%) of the female parent fish in Example 1, Comparative Examples 1 to 9 were statistically analyzed, and the results are shown in Table 1.

[0063] Table 1: Statistical analysis of artificial breeding results of the examples and their comparative examples

[0064] The induced spawning rate is calculated as follows: (Number of spawning female broodstock * 100%) / (Total number of induced spawning female broodstock). Fertilization rate = Number of fertilized eggs * 100% / Total number of eggs; Hatching rate = Number of newly hatched fry * 100% / Number of fertilized eggs; Mortality rate of parent fish = Number of dead parent fish * 100% / Number of fish induced to spawn As can be seen from the table above, the choice of oxytocin type and its injection method are very important, as they not only affect the production and quality of eggs and sperm, but also the stress of the parent fish. In Example 1, a combination of LRH-A2 and HCG was used, administered in two injections. The first injection was 1 / 10 of the dose to induce maturation, and the remaining dose was injected 10 hours later (at a water temperature of 24°C). Simultaneously with the second injection into the female broodstock, half of the full dose of oxytocin was injected into the male broodstock. This method effectively promotes the maturation and ovulation of female broodstock eggs, ensuring a large number of mature eggs are available for fertilization. This results in the highest induced spawning rate, and the quality of the obtained eggs and sperm is excellent, making fertilization easier and obtaining higher quality fertilized eggs. This leads to the highest fertilization and hatching rates, while minimizing adverse effects and stress on the broodstock, thus reducing mortality.

[0065] Compared to Example 1, Comparative Example 1 involved a single injection of oxytocin into the female broodstock. This single high-dose injection caused significant stress and damage to the broodstock, leading to increased mortality. Furthermore, the single injection affected egg production and quality, resulting in decreased fertilization and hatching rates. While Comparative Examples 2 and 3 also involved two separate injections into the female broodstock, both excessively high and low initial doses significantly impacted egg quality (too low an initial dose resulted in immature eggs, while too high a dose led to premature rupture or aging), consequently reducing fertilization and hatching rates. This increased stress on the female broodstock also contributed to higher mortality rates. Comparative Examples 4 and 5 involved injection of a single hormone. Comparative Example 4 lacked the luteinizing hormone release signal of LRH-A2, resulting in reduced efficiency of HCG alone. In Comparative Example 5, LRH-A2 alone required a high dose and had a weak effect, failing to effectively trigger ovulation. This led to ovulation failure in most broodstock fish, resulting in the lowest induced spawning rate. Even after fertilization, most of the released eggs failed to develop into embryos, leading to the lowest hatching rate. The difficulty or failure of ovulation in the broodstock increased stress, affecting mortality. Comparative Example 7 involved injection of a high dose of LRH-A2, which increased stimulation in both female and male broodstock, significantly increasing mortality and noticeably affecting normal egg development and ovulation, thus impacting induced spawning, egg fertilization, and hatching of fertilized eggs. Comparative Example 8 used dioxin to mention HGG, indicating that the synergistic effect of DOM and LRH-A2 is weaker than that of HCG and LRH-A2, affecting the spawning efficiency and the uniformity of egg maturation, thus affecting fertilization and hatching. Furthermore, the combined effect of DOM and LRH-A2 on the parent fish is greater than the combined effect of HCG and LRH-A2. Comparative Examples 9 and 10, both at 24℃, showed the impact of excessively long or short intervals between the two injections on the breeding results. These results indicate that both excessively long and short intervals are detrimental to catalysis and egg quality improvement, and exacerbate stress responses in the female parent fish.

[0066] The present invention and its embodiments have been described above. This description is not restrictive, and the embodiments shown are only one of the embodiments of the present invention. The actual structure is not limited to this. In conclusion, if those skilled in the art are inspired by this description and design similar structures and embodiments without departing from the spirit of the present invention, they should all fall within the protection scope of the present invention.

Claims

1. A method for the artificial breeding of Culter alburnus from Lake Xingkai, characterized in that, It includes the following steps: S100 parent fish breeding Five-year-old or older, disease-free and uninjured Culter alburnus from Xingkai Lake were selected and placed in a parent fish rearing pond for cultivation. The ratio of female to male fish was 1:(1.2-1.5). The pH of the water in the broodstock rearing pond is controlled between 7.5 and 8.5; S200 Artificial Labor Induction Mature gonadal-developing Culter alburnus from Lake Xingkai were selected as breeding broodstock, and oxytocin was injected into the breeding broodstock. The oxytocin is composed of luteinizing hormone-releasing hormone analog LRH-A2 and human chorionic gonadotropin HCG. The female parent fish were injected with two doses of oxytocin, totaling 2-5 μg / kg of luteinizing hormone-releasing hormone analog LRH-A2 and 800-1200 μI / kg of human chorionic gonadotropin (HCG). The first injection was 1 / 20 to 2 / 10 of the full dose of oxytocin, with the water temperature controlled at 21-26℃. The remaining dose was injected 8-10 hours later. At the same time as the female parent fish receive the second injection, the male parent fish are injected with 3 / 10 to 7 / 10 of the full dose of oxytocin given to the female parent fish. S300 Artificial Insemination The water temperature is controlled at 21-26℃. After the broodstock fish are injected with oxytocin, they are cultured for 7-12 hours. Eggs and semen are collected artificially. The eggs and semen are placed in a dry container with 100,000-220,000 eggs per ml of semen. The mixture is stirred thoroughly to ensure that the eggs and sperm are evenly mixed. Water is then added at a volume of 2-4 times the volume of the egg and semen mixture. The mixture is stirred for 2-5 minutes and then left to stand for 2-3 minutes to complete the artificial insemination. S400 fertilized eggs hatch The fertilized eggs obtained in step S300 are distributed at a rate of 600,000-900,000 eggs / m³. 3 The eggs are placed in water at 22-26℃ for incubation. The dissolved oxygen in the water is ≥6mg / L. The initial water flow rate is controlled at 3-5m / s. After the eggs have fully absorbed water, the water flow rate is reduced to 1-2m / s and maintained for 18-22 hours until hatching and breaking the membrane. Once the eggs emerge from the waist and can swim horizontally, the fry can be obtained. S500 Postpartum Care On the day after collecting eggs and sperm in step S300, which is counted as postpartum day 1, gentamicin sulfate at a dose of 5-10 mg / kg is injected into the dorsal fin muscle of the breeding broodstock. Then, the broodstock are cultured in a pond at a temperature of 20-25℃ until 8-12 days postpartum, after which the breeding broodstock are transferred with water.

2. The method for artificially breeding Culter alburnus in Lake Xingkai according to claim 1, characterized in that, In step S100, the feed is provided during the cultivation process. The nutritional composition (by mass) of the feed is 35-42% crude protein, 8-12% crude fat, 10-15% crude ash, 3-8% crude fiber, 0.5-1.5% total phosphorus, 2-5% lysine, and 10-15% moisture.

3. The method for artificially breeding Culter alburnus in Lake Xingkai according to claim 1, characterized in that, In step S100, 20-35 days before artificial spawning, wild miscellaneous fish or silver carp and bighead carp fry are released into the parent fish rearing pond as bait fish.

4. The method for artificial breeding of Culter alburnus in Lake Xingkai according to claim 1, characterized in that, In step S400, before hatching the fertilized eggs, they are soaked in potassium permanganate at a concentration of 8-12 mg / L for 3-10 minutes.

5. The method for artificially breeding Culter alburnus in Lake Xingkai according to claim 1, characterized in that, During the first 1-11 days of postpartum rearing of breeding broodstock in S500, antimicrobial peptides were added to the feed. The nutritional composition (by weight) of the feed is 35-42% crude protein, 8-12% crude fat, 10-15% crude ash, 3-8% crude fiber, 0.5-1.5% total phosphorus, 2-5% lysine, and 10-15% moisture.

6. The method for artificially breeding Culter alburnus in Lake Xingkai according to claim 1, characterized in that, In S200, the female parent fish were injected with 1 / 10 of the full dose of oxytocin for the first time, and the remaining amount was injected after an interval of 8-10 hours.

7. The method for artificially breeding Culter alburnus in Lake Xingkai according to claim 1, characterized in that, Step S300 controls the collection time of sperm and eggs based on the water temperature during cultivation after artificial ovulation: When the water temperature is 21-23℃, after the parent fish that have been injected with oxytocin are cultured for another 9-11 hours, eggs and semen are collected artificially. When the water temperature is 24-26℃, after the broodstock fish injected with oxytocin are cultured for another 8-9 hours, eggs and semen are collected artificially.

Images