Artificial propagation method of Bagarius yarrelli without emasculation and sperm collection and Bagarius yarrelli sperm storage solution

Abstract

The invention provides a Bagarius yarrelli artificial propagation method and Bagarius yarrelli sperm storage liquid without emasculation and sperm collection, and belongs to the technical field of aquaculture. The Bagarius yarrelli sperm storage liquid comprises glucose, sodium chloride, potassium chloride, calcium chloride and water. After the Bagarius yarrelli sperm storage liquid is mixed with Bagarius yarrelli sperms, the Bagarius yarrelli sperms can be temporarily stored for one week, and the problem of low fertilization rate caused by asynchronous development of male and female Bagarius yarrelli parents is solved. According to the method, the male Bagarius yarrelli is subjected to induced spawning by adopting a two-needle method, firstly, luteinizing hormone releasing hormone A < 2 > + domperidone maleate is injected, after 9-11 h, the luteinizing hormone releasing hormone A < 2 > + domperidone maleate + chorionic gonadotropin is injected, and the injection dosage is strictly controlled. By means of the method, artificial breeding, large-scale production and enhancement and release of the Bagarius yarrelli can be achieved under the condition that male fish testis are not cut, and the method has important significance on protection of the Bagarius yarrelli.

Description

Technical Field

[0001] The invention relates to the technical field of aquaculture, and in particular to an artificial breeding method of giant bream without killing males to obtain sperm and a sperm storage liquid of giant bream. Background Art

[0002] The giant yarrelli (Bagarius yarrelli) belongs to the order Siluriformes, family Sioridae, genus Bagarius. Local names: melon fish, tiger fish, mainly distributed in the Lancang River, Nujiang River and Yuanjiang River. It is a national second-class protected animal. In recent years, with the construction and operation of the Lancang River cascade power stations, dam obstruction, shrinking water habitats, and changes in hydrological conditions have posed a great threat to the survival and reproduction of the giant yarrelli. Coupled with the influence of human factors, its wild population has declined significantly. Carrying out research on artificial breeding technology and implementing reproduction and release are important means to protect the wild population resources of the giant yarrelli and the diversity of native fish in the Lancang River. However, the testes of the fish of the family Sioridae are branched, and semen cannot be obtained by direct squeezing. Therefore, when the giant yarrelli is artificially bred, the male fish needs to be dissected to remove the testes and then artificially inseminated. At present, the artificial breeding technology of giant bream is not mature yet. During the breeding process, males need to be killed to extract sperm, which results in low breeding efficiency, large losses of parent fish, and is insufficient to support the large-scale production of giant bream seedlings and the development of protection work such as proliferation and release. For example, the application number "201310375245.8" and the invention name "A method for artificial breeding of giant bream" discloses a method for artificial breeding of giant bream, which is to manually dissect the spermatozoa of male fish, which will cause the death of male fish and large losses of parent fish. In addition, the male and female parents of giant bream develop asynchronously, and semen is not easy to store, resulting in low fertilization rate, hatching rate and survival rate of seedlings. Therefore, it is of great significance to the protection of giant bream to develop a method for artificial breeding of giant bream without killing males to extract sperm and a sperm storage solution for giant bream to realize artificial breeding, large-scale production and proliferation and release of giant bream. Summary of the invention

[0003] The purpose of the present invention is to provide a giant croaker artificial breeding method and a giant croaker sperm storage liquid without killing male fish for sperm collection. The giant croaker artificial breeding method of the present invention can obtain semen without removing the testes of male fish, thereby solving the problem that removing the testes of male fish in the existing giant croaker artificial breeding method may cause the death of male fish and cause large loss of parent fish; at the same time, the giant croaker sperm storage liquid of the present invention can prolong the storage time of giant croaker sperm, thereby solving the problem of low fertilization rate caused by asynchronous development of male and female parent fish.

[0004] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0005] The invention provides a giant trout sperm storage solution, comprising the following components in parts by mass: 48-52 parts of glucose, 8.8-9.2 parts of sodium chloride, 0.2-0.3 parts of potassium chloride, 0.2-0.3 parts of calcium chloride and 900-1100 parts of water.

[0006] The present invention also provides a method for artificially breeding giant bream without killing male breams to obtain sperm, comprising the following steps:

[0007] S1. Select sexually mature giant bream parents during the breeding season of giant bream;

[0008] S2. The male parent is injected with luteinizing hormone releasing hormone A2 2.8-3.2 μg / kg + dioconazole maleate 1.8-2.2 mg / kg, and 9-11 hours later, luteinizing hormone releasing hormone A2 5.5-6.5 μg / kg + dioconazole maleate 4.5-5.5 mg / kg + chorionic gonadotropin 900-1100 IU / kg is injected; 5.5-6.5 hours after the injection, the abdomen of the male parent is squeezed to discharge urine, and semen can be squeezed out 11-12 hours after the injection is completed, and the semen is mixed with the giant croaker sperm storage solution and stored to obtain diluted semen;

[0009] S3. Inducing ovulation in female parents to obtain mature eggs;

[0010] S4. Mix the semen diluted in S2 with the mature eggs, add water and stir, then rinse with water to obtain fertilized eggs;

[0011] S5. Incubate the fertilized eggs in water at 26-29°C for 19-24 hours to obtain giant snapper seedlings.

[0012] Preferably, the breeding season of the giant snapper is from May to October.

[0013] Preferably, the sexually mature giant bream parent is a male giant bream weighing more than 3 kg and with a genital process protruding more than 0.2 cm; a female giant bream weighing more than 4 kg, with a red and swollen genital opening, and a swollen, soft and shaken lower abdomen.

[0014] Preferably, the method of inducing labor for female parents with better development is: injecting luteinizing hormone releasing hormone A29-11 μg / kg + dioxigenin maleate 4.5-5.5 mg / kg + chorionic gonadotropin 450-550 IU / kg; the method of inducing labor for female parents with poorer development is: first injecting luteinizing hormone releasing hormone A22.8-3.2 μg / kg, and then injecting LARHA26-8 μg / kg + dioxigenin maleate 4.5-5.5 mg / kg + chorionic gonadotropin 450-550 IU / kg 9-11 hours later.

[0015] Preferably, the better-developed female parent has a round, swollen and soft abdomen in both the upper and lower parts, a red, particularly swollen genital opening, and the eggs dug out with an egg digger are regular round individuals; the poorly developed female parent has a relatively flat upper abdomen, only the lower abdomen is swollen, the genital opening is slightly red, and the eggs dug out with an egg digger are irregular round individuals.

[0016] Preferably, the volume ratio of the semen to the giant trout sperm storage solution is 1:18-22.

[0017] Preferably, the stirring time is 2 to 3 minutes.

[0018] Preferably, mature eggs can be obtained by gently pressing the abdomen of the female parent 10 to 16 hours after induction of labor.

[0019] Preferably, the dissolved oxygen during the incubation period of the fertilized eggs is 8-16 mg / L, and the incubation density is 13,000-17,000 eggs / m 3 .

[0020] By adopting the above technical solution, the present invention has the following beneficial effects:

[0021] (1) The present invention provides a method for obtaining semen of giant bream without killing males. This method can avoid the loss of parent fish caused by killing males for semen collection, and can also improve the quantity and quality of semen obtained. At the same time, the giant bream males that have been induced to spawn can continue to be used for induced spawning more than 20 days later, filling the gap in the current technology for obtaining semen of giant bream males without killing males for semen collection.

[0022] (2) The present invention provides a storage solution for giant catfish semen, which can prolong the storage time of giant catfish semen, firstly avoiding the situation of different development between males and females, and also improving the utilization rate of semen.

[0023] (3) The present invention classifies and induces spawning of female giant bream. By classifying the female fish before spawning, the spawning rate and egg-laying quality can be significantly improved, the fertilization rate and the physical fitness of newly hatched fry can be improved, and the survival rate of seedlings can be significantly increased. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 The present invention is a flowchart of artificial breeding of giant bream.

[0025] Figure 2 These are the mature gonads of the male parent of the giant catfish.

[0026] Figure 3 These are the mature gonads of the female giant snapper.

[0027] Figure 4 A diagram of squeezing out the female parent's gonads and squeezing out the eggs.

[0028] Figure 5This is a fertilized egg of a giant snapper 12 hours after artificial insemination.

[0029] Figure 6 These are giant snapper fry 8 days after hatching.

[0030] Figure 7 These are giant snapper fry 60 days after hatching. DETAILED DESCRIPTION

[0031] The invention provides a giant trout sperm storage solution, comprising the following components in parts by mass: 48-52 parts of glucose, 8.8-9.2 parts of sodium chloride, 0.2-0.3 parts of potassium chloride, 0.2-0.3 parts of calcium chloride and 900-1100 parts of water.

[0032] In the giant slug sperm storage solution of the present invention, the mass fraction of glucose is 48 to 52 parts, more preferably 49 to 51 parts, and more preferably 50 parts;

[0033] The mass fraction of the sodium chloride is 8.8 to 9.2 parts, more preferably 8.9 to 9.1 parts, and more preferably 9.04 parts;

[0034] The mass fraction of potassium chloride is 0.2 to 0.3 parts, more preferably 0.22 to 0.28 parts, and more preferably 0.24 parts;

[0035] The mass fraction of calcium chloride is 0.2 to 0.3 parts, more preferably 0.23 to 0.27 parts, and more preferably 0.26 parts;

[0036] The mass fraction of the water is 900 to 1100 parts, more preferably 950 to 1050 parts, and even more preferably 1000 parts.

[0037] The present invention also provides a method for artificially breeding giant bream without killing male breams to obtain sperm, comprising the following steps:

[0038] S1. Select sexually mature giant bream parents during the breeding season of giant bream;

[0039] S2. The male parent is injected with luteinizing hormone releasing hormone A2 2.8-3.2 μg / kg + dioconazole maleate 1.8-2.2 mg / kg, and 9-11 hours later, luteinizing hormone releasing hormone A2 5.5-6.5 μg / kg + dioconazole maleate 4.5-5.5 mg / kg + chorionic gonadotropin 900-1100 IU / kg is injected; 5.5-6.5 hours after the injection, the abdomen of the male parent is squeezed to discharge urine, and semen can be squeezed out 11-12 hours after the injection is completed, and the semen is mixed with the giant croaker sperm storage solution and stored to obtain diluted semen;

[0040] S3. Inducing ovulation in female parents to obtain mature eggs;

[0041] S4. Mix the semen diluted in S2 with the mature eggs, add water and stir, then rinse with water to obtain fertilized eggs;

[0042] S5. Incubate the fertilized eggs in water at 26-29°C for 19-24 hours to obtain giant snapper seedlings.

[0043] The method first selects sexually mature parents during the breeding season of giant bream, which is from May to October every year. The sexually mature male parents are preferably male giant breams with a body weight of more than 3 kg and a reproductive process protruding more than 0.2 cm. The sexually mature female parents are preferably female giant breams with a body weight of more than 4 kg, a red and swollen reproductive aperture, and a swollen, soft and shaken lower abdomen.

[0044] The present invention induces labor in male giant bream parents to obtain semen, and the obtained semen is mixed with the giant bream sperm storage solution to obtain diluted semen. Specifically, the male parent is subjected to a two-injection injection method for inducing labor, the first injection is injected with luteinizing hormone releasing hormone A2 2.8-3.2 μg / kg + dioconazole maleate 1.8-2.2 mg / kg, the injection dose of the luteinizing hormone releasing hormone is further preferably 2.9-3.1 μg / kg, more preferably 3.0 μg / kg; the injection dose of dioconazole maleate is further preferably 1.9-2.1 mg / kg, more preferably 2 mg / kg; 9-11 hours later, preferably 10 hours later, the second injection is injected, the second injection is injected with luteinizing hormone releasing hormone A2 5.5-6.5 μg / kg + dioconazole maleate 4.5-5.5 mg / kg + chorionic gonadotropin 900-1100 IU / kg; the injection dose of the luteinizing hormone releasing hormone A2 is further preferably 5.8-6.2 μg / kg, more preferably 6 μg / kg; the injection dose of dioconazole maleate is further preferably 4.8-5.2 mg / kg, more preferably 5 mg / kg; the injection amount of chorionic gonadotropin is further preferably 950-1050 IU / kg, more preferably 1000 IU / kg. 5.5-6.5 hours after injection, more preferably, 6 hours, squeeze the abdomen of the male parent to discharge urine, squeeze and discharge urine every 1 hour, and squeeze out semen 11-12 hours after the injection is completed. The squeezed semen is mixed with the giant slug sperm storage solution and stored to obtain a sperm diluent. The volume ratio of the semen to the giant croaker sperm storage solution is 1:18 to 22, and more preferably 1:19 to 21. The sperm diluent is preferably stored in a brown bottle, and the storage temperature is preferably 0 to 4°C, more preferably 1 to 3°C, and more preferably 2°C.

[0045] The present invention induces labor according to the development of the female parent. If the development is good, the following method is used to induce labor: injecting luteinizing hormone releasing hormone A2 9-11 μg / kg + dioxigenin maleate 4.5-5.5 mg / kg + chorionic gonadotropin 450-550 IU / kg; the injection dose of the luteinizing hormone releasing hormone A2 is preferably 9.5-10.5 μg / kg, more preferably 10 μg / kg; the injection dose of dioxigenin maleate is preferably 4.8-5.2 mg / kg, more preferably 5 mg / kg; the injection dose of the chorionic gonadotropin A2 is preferably 480-520 IU / kg, more preferably 500 IU / kg. The following method is used to induce labor for poorly developed female parents: first, inject luteinizing hormone releasing hormone A2 2.8-3.2 μg / kg, preferably 2.9-3.1 μg / kg, and more preferably 3 μg / kg; 9-11 hours later, preferably 10 hours later, inject LARH A2 6-8 μg / kg + dioconazole maleate 4.5-5.5 mg / kg + chorionic gonadotropin 450-550 IU / kg; the injection dose of LARHA2 is more preferably 6.5-7.5 μg / kg, and more preferably 7 μg / kg; the injection dose of dioconazole maleate is more preferably 4.8-5.3 mg / kg, and more preferably 5 mg / kg; the injection dose of chorionic gonadotropin is more preferably 480-430 IU / kg, and more preferably 500 IU / kg. At 10-16 hours after the injection of labor induction, gently press the abdomen to obtain mature eggs.

[0046] The well-developed female giant catfish parent described in the present invention has a round, swollen and soft abdomen in both the upper and lower parts, a red genital opening that is particularly swollen, and eggs dug out with an egg digger are regular round individuals; the poorly developed female giant catfish parent has a relatively flat upper abdomen, only the lower abdomen is swollen, the genital opening is slightly red, and eggs dug out with an egg digger are irregular round individuals.

[0047] After obtaining the giant croaker eggs, the present invention mixes the diluted semen and mature eggs in a dry and clean basin, and then adds water. The volume of the water added is 2 to 3 times the sum of the volumes of the diluted semen and the mature eggs, more preferably 2.2 to 2.8 times, and more preferably 2.5 times. Then continue stirring for 2 to 3 minutes, more preferably 2.5 minutes, and then rinse with clean water several times, and incubate after counting. The water temperature during incubation is 26 to 29°C, more preferably 27 to 28°C, and more preferably 27.5°C. The water temperature fluctuates less than 2°C during incubation. The dissolved oxygen content is 8 to 16 mg / L, more preferably 10 to 14 mg / L, and more preferably 13 mg / L; the density of the incubation is 13,000 to 17,000 grains / m 3 , more preferably 14,000 to 16,000 particles / m 3, more preferably 15,000 particles / m 3 .

[0048] After 19 to 24 hours, giant bream seedlings are obtained; the hatching is preferably carried out under micro-flow conditions in an incubator. The present invention has no special restrictions on the flow rate of the water flow, and the conventional flow rate for hatching giant bream fertilized eggs in the field can be used. The water quality meets the requirements of the fishery water quality standard GB11607.

[0049] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0050] Example 1

[0051] On June 30, 2024, select 2 male giant breams weighing more than 3kg and with a genital protrusion of more than 0.2cm; select 3 female giant breams weighing more than 4kg, with a red and swollen genital opening, and a swollen, soft and shaken lower abdomen (two of them have round, swollen and soft abdomens, red genital openings, and particularly swollen genital openings. The eggs dug out with an egg digger are regular round; the other one has a relatively flat upper abdomen, only a swollen lower abdomen, a slightly red genital opening, and an irregular round egg dug out with an egg digger);

[0052] First, induce labor in the male parent, and perform two injections of induced labor on the male parent. The first injection is luteinizing hormone releasing hormone A2 (LARHA2) 3μg / kg + dioxigenin maleate (DOM) 2mg / kg. After 10 hours, luteinizing hormone releasing hormone LARHA2 6μg / kg, dioxigenin maleate (DOM) 5mg / kg, and human chorionic gonadotropin (HCG) 1000IU / kg are injected. Starting from the 6th hour after the injection, the abdomen of the male parent is squeezed every 1 hour to discharge urine. After 12 hours after the injection, the semen is squeezed out, and the semen and giant bream sperm storage solution are diluted in a volume ratio of 1:20 and stored in a brown bottle in a refrigerator at 4°C. The formula of giant bream sperm storage solution is 50g glucose, 9.04g sodium chloride, 0.24g potassium chloride, and 0.26g calcium chloride dissolved in 1000mL water;

[0053] On July 2, 2024, the female parents were induced to give birth. First, the female parents with poor development were injected with LRH-A23μg / kg. Ten hours later, the female fish were injected with LARHA27μg / kg, DOM 5mg / kg, and HCG 500IU / kg again. At the same time, the female parents with better development were injected with LARHA210μg / kg, DOM 5mg / kg, and HCG 500IU / kg at one time.

[0054] After 10 hours, the female parents were checked, and artificial insemination was performed on the female fish that could ovulate successfully, that is, the female fish eggs were squeezed into a dry and clean basin, the diluted semen was mixed with mature eggs, and 2 times of water was added to activate the sperm, and the eggs were stirred continuously for 3 minutes with feathers. After rinsing several times, the fertilized eggs were counted and the female fish that could not ovulate successfully were put back into the water and checked every 1 hour. Finally, 2 female fish were successfully induced to spawn, with an induced spawning rate of 66.67%, and a total of about 40,000 eggs were laid.

[0055] The eggs are placed in a hatching ring with micro-flow water for incubation, so that the fertilized eggs roll in the hatching ring without sinking to the bottom. The hatching density is about 15,000 eggs / m 3 ; Oxygen was continuously introduced, and the dissolved oxygen in the water was greater than 7mg / L during the entire incubation period, the water temperature was 26-29℃, and the water quality met the requirements of the fishery water quality standard GB11607. After about 10 hours, the neurula entered, and the fertilization rate was about 90%. After 24-28 hours, the hatching rate was 80%, and 28,800 giant bream seedlings were obtained.

[0056] Example 2

[0057] On July 2, 2024, two female giant snappers weighing more than 4 kg, with red and swollen genital openings and swollen, soft and shaky lower abdomens were selected (one of them had a round, swollen and soft abdomen, a red and particularly swollen genital opening, and eggs dug out with an egg digger were regular round; the other one had a relatively flat upper abdomen, only a swollen lower abdomen, a slightly red genital opening, and eggs dug out with an egg digger were irregular round);

[0058] On July 2, 2024, the female parents were induced to give birth. First, the female parents with poor development were injected with LRH-A23μg / kg. Ten hours later, the female fish were injected with LARHA27μg / kg, DOM 5mg / kg, and HCG 500IU / kg again. At the same time, the female parents with better development were injected with LARHA210μg / kg, DOM 5mg / kg, and HCG 500IU / kg at one time.

[0059] After 10 hours, the female parents were checked, and artificial insemination was performed on the female fish that could ovulate smoothly, that is, the female fish eggs were squeezed into a dry and clean basin, the semen diluent temporarily stored in Example 1 was mixed with mature eggs, 2 times of water was added to activate the sperm, and the mixture was stirred continuously for 3 minutes with a feather, and the fertilized eggs were counted after being rinsed several times; the female fish that could not ovulate smoothly were put back into the water and checked every 1 hour. Finally, 2 female fish were successfully induced to spawn, with an induced spawning rate of 100%, and a total of about 40,000 eggs were laid.

[0060] The eggs are placed in a hatching ring with micro-flow water for incubation, so that the fertilized eggs roll in the hatching ring without sinking to the bottom. The hatching density is about 15,000 eggs / m 3; Oxygen was continuously introduced, and the dissolved oxygen in the water was greater than 7mg / L during the entire incubation period, the water temperature was 26-29℃, and the water quality met the requirements of the fishery water quality standard GB11607. After about 10 hours, the neurula entered, and the fertilization rate was about 75%. After 24-28 hours, the hatching rate was 80%, and 24,000 giant bream seedlings were obtained.

[0061] Comparative Example 1

[0062] On June 10, 2024, two male giant snappers weighing more than 3kg and with a genital protrusion of more than 0.2cm were selected; three female giant snappers weighing more than 4kg, with red and swollen genital openings, and enlarged, soft and shaken lower abdomens were selected (one of them had a round, enlarged and soft abdomen, a red and particularly swollen genital opening, and the eggs dug out with an oviduct were regular round; the other two had relatively flat upper abdomens, only swollen lower abdomens, slightly red genital openings, and irregular round eggs dug out with an oviduct); female fish were uniformly injected with two-injection injections to induce labor. The first injection was used to inject luteinizing hormone-releasing hormone analogues (LRH-A2) 5μg / kg into male fish; 12h later, male fish were injected with LRH-A2 12μg / kg + dioxin maleate (DOM) 8mg / kg + human chorionic gonadotropin (HCG) 500IU / kg, and female fish were injected with LRH-A2 10μg / kg + DOM 5mg / kg+HCG 500IU / kg.

[0063] After 10 hours, the female parents were checked, and artificial insemination was performed on the female fish that could ovulate successfully, that is, the female fish eggs were squeezed into a dry and clean basin, the male fish testis was dissected, cut into pieces and ground, and diluted with 0.9% saline and mixed evenly with mature eggs, and 2 times of water was added to activate the sperm, and it was stirred continuously for 3 minutes with feathers, rinsed several times and counted to obtain fertilized eggs; the female fish that could not ovulate successfully were put back into the water and checked every 1 hour. Finally, one female fish was successfully induced to spawn, with an induced spawning rate of 33.33%, and a total of about 30,000 eggs were laid.

[0064] The eggs are placed in a hatching ring with micro-flow water for incubation, so that the fertilized eggs roll in the hatching ring without sinking to the bottom. The hatching density is about 15,000 eggs / m 3 ; Oxygen was continuously introduced, and the dissolved oxygen in the water was greater than 7mg / L during the entire incubation period, the water temperature was 26-29℃, and the water quality met the requirements of the fishery water quality standard GB11607. After about 10 hours, the neurula entered, and the fertilization rate was about 20%. After 24-28 hours, the hatching rate was 80%, and 4,800 giant bream seedlings were obtained.

[0065] Comparative Example 2

[0066] On June 18, 2024, select 2 male giant bream weighing more than 3kg and with a genital protrusion of more than 0.2cm; select 3 female giant bream weighing more than 4kg, with a red and swollen genital opening, and a swollen, soft and shaky lower abdomen (two of them had round, swollen and soft abdomens, red genital openings, and particularly swollen genital openings. The eggs dug out with an egg digger were regular round. The other one had a relatively flat upper abdomen, only a swollen lower abdomen, a slightly red genital opening, and irregular round eggs dug out with an egg digger).

[0067] First, the male parent was induced to spawn. The male fish was first injected with luteinizing hormone-releasing hormone analog (LRH-A2) 5μg / kg. After 12 hours, the male fish was injected with LRH-A2 12μg / kg + dioxin maleate (DOM) 8mg / kg + human chorionic gonadotropin (HCG) 500IU / kg. To induce spawning of female parents, the poorly developed female parents were first injected with LRH-A2 3μg / kg. After 10 hours, the female fish were injected with LARHA 27μg / kg, DOM 5mg / kg, and HCG 500IU / kg again. At the same time, the well-developed female parents were injected with LARHA 210μg / kg, DOM 5mg / kg, and HCG 500IU / kg at one time.

[0068] After 10 hours, the female parents were checked, and artificial insemination was performed on the female fish that could ovulate successfully, that is, the female fish eggs were squeezed into a dry and clean basin, the male fish testis was dissected, cut into pieces and ground, and diluted with 0.9% saline and mixed evenly with mature eggs, and 2 times of water was added to activate the sperm, and it was stirred continuously for 2 minutes with feathers, rinsed several times and counted to obtain fertilized eggs; the female fish that could not ovulate successfully were put back into the water and checked every 1 hour. Finally, 2 female fish were successfully induced to spawn, with an induced spawning rate of 66.67%, and a total of about 45,000 eggs were laid.

[0069] The eggs are placed in a hatching ring with micro-flow water for incubation, so that the fertilized eggs roll in the hatching ring without sinking to the bottom. The hatching density is about 15,000 eggs / m 3 ; Oxygen was continuously introduced, and the dissolved oxygen in the water was greater than 7mg / L during the entire incubation period, the water temperature was 26-29℃, and the water quality met the requirements of the fishery water quality standard GB11607. After about 10 hours, the neurula entered, and the fertilization rate was about 25%. After 24-28 hours, the hatching rate was 80%, and 9,000 giant bream seedlings were obtained.

[0070] Comparative Example 3

[0071] On June 23, 2024, select 2 male giant breams weighing more than 3kg and with a genital protrusion of more than 0.2cm; select 3 female giant breams weighing more than 4kg, with a red and swollen genital opening, and a swollen, soft and shaky lower abdomen (2 of them had round, swollen and soft abdomens, red genital openings, and particularly swollen genital openings. The eggs dug out with an egg digger were regular round; the other one had a relatively flat upper abdomen, only a swollen lower abdomen, a slightly red genital opening, and an irregular round egg dug out with an egg digger);

[0072] First, induce labor in the male parent with two injections. The first injection is luteinizing hormone releasing hormone A2 (LARHA2) 3μg / kg + dioxigenin maleate (DOM) 2mg / kg. 10 hours later, luteinizing hormone releasing hormone LARHA2 6μg / kg, dioxigenin maleate (DOM) 5mg / kg, and human chorionic gonadotropin (HCG) 1000IU / kg are injected. Starting from the 6th hour after the injection, the male parent's abdomen is squeezed every 1 hour to discharge urine. Semen can be squeezed out 11 hours after the injection.

[0073] The male parents were induced to spawn while the female parents were induced to spawn. First, the female parents with poor development were injected with LRH-A23μg / kg. After 10 hours, the female fish were injected with LARHA27μg / kg, DOM 5mg / kg, and HCG 500IU / kg again. At the same time, the female parents with good development were injected with LARHA210μg / kg, DOM 5mg / kg, and HCG 500IU / kg at one time.

[0074] After 10 hours, the female parents were checked, and artificial insemination was performed on the female fish that could ovulate successfully, that is, the female fish eggs were squeezed into a dry and clean basin, and the male fish semen was squeezed onto the mature eggs, mixed evenly, and 3 times water was added to activate the sperm, and the mixture was stirred continuously for 3 minutes with a feather, and the fertilized eggs were counted after being rinsed several times; the female fish that could not ovulate successfully were put back into the water and checked every 1 hour. Finally, 2 female fish were successfully induced to spawn, with an induced spawning rate of 66.67%, and a total of about 50,000 eggs were laid.

[0075] The eggs are placed in a hatching ring with micro-flow water for incubation, so that the fertilized eggs roll in the hatching ring without sinking to the bottom. The hatching density is about 15,000 eggs / m 3 ; Oxygen was continuously introduced, and the dissolved oxygen in the water was greater than 7mg / L during the entire incubation period, the water temperature was 26-29℃, and the water quality met the requirements of the fishery water quality standard GB11607. After about 10 hours, the neurula entered, and the fertilization rate was about 70%. After 24-28 hours, the hatching rate was 80%, and 28,000 giant bream seedlings were obtained.

[0076] Table 1 Statistical results of induced spawning rate, fertilization rate, hatching rate and seedling survival rate of different groups of giant bream

[0077]

[0078] As can be seen from Table 1, Comparative Example 1 adopts a conventional method of inducing labor, killing males and taking sperm, and does not use the technologies of the present invention such as not needing to kill males to take sperm, temporary storage of semen, and classification of female fish for inducing labor. The inducing labor rate and fertilization rate are both lower than those in the embodiment; Compared with Comparative Example 1, Comparative Example 2 classifies female fish for inducing labor, and the inducing labor rate is significantly improved; Comparative Example 3 uses the technology of not needing to kill males to take sperm and the technology of classification of female fish for inducing labor, and the fertilization rate and inducing labor rate are significantly improved; and Example 1 uses the technology introduced in the present invention to carry out artificial breeding, and the inducing labor rate and fertilization rate are both the highest. At the same time, the semen temporarily stored in Example 1 is also used for the artificial breeding work of Example 2. The semen is still active after being temporarily stored for 3 days, not only completing fertilization, but also the fertilization rate is higher than that of Comparative Examples 1 to 3. This is because, the giant catfish sperm storage solution of the present invention temporarily stores giant catfish sperm for a short period of time without affecting sperm motility; too high sperm concentration will cause multiple sperm penetration into the eggs, which will reduce the fertilized eggs. Temporary storage dilutes the sperm concentration to a certain extent and increases the fertilization rate; but water cannot dilute and preserve sperm. Sperm will be activated by water and will die within 1 minute.

[0079] As can be seen from the above embodiments, the present invention provides a method for artificially propagating giant bream without killing males to obtain sperm and a sperm storage solution for giant bream. The present invention can obtain semen without removing the testes of male fish, and the sperm storage solution for giant bream of the present invention can be temporarily stored for one week after being stored with semen, solving the problem of low fertilization rate caused by asynchronous development of male and female bream parents. The present invention can realize artificial propagation, large-scale production and proliferation and release of giant bream without removing the testes of male fish, which is of great significance to the protection of giant bream.

[0080] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A giant croaker sperm storage solution, characterized in that: The invention comprises the following components in parts by weight: 48-52 parts of glucose, 8.8-9.2 parts of sodium chloride, 0.2-0.3 parts of potassium chloride, 0.2-0.3 parts of calcium chloride and 900-1100 parts of water.

2. A method for artificial breeding of giant bream without killing males to obtain sperm, characterized in that: The following steps are involved: S1. Select sexually mature giant bream parents during the breeding season of giant bream; S2. The male parent is injected with luteinizing hormone releasing hormone A2 2.8-3.2 μg / kg + dioconazole maleate 1.8-2.2 mg / kg, and 9-11 hours later, luteinizing hormone releasing hormone A2 5.5-6.5 μg / kg + dioconazole maleate 4.5-5.5 mg / kg + chorionic gonadotropin 900-1100 IU / kg is injected; 5.5-6.5 hours after the injection, the abdomen of the male parent is squeezed to discharge urine, and semen can be squeezed out 11-12 hours after the injection is completed, and the semen is mixed with the giant croaker sperm storage solution described in claim 1 and stored to obtain diluted semen; S3. Inducing ovulation in female parents to obtain mature eggs; S4. Mix the semen diluted in S2 with the mature eggs, add water and stir, then rinse with water to obtain fertilized eggs; S5. Incubate the fertilized eggs in water at 26-29°C for 19-24 hours to obtain giant snapper seedlings.

3. The artificial breeding method of giant bream according to claim 2, characterized in that: The breeding season of the giant bream is from May to October.

4. The artificial propagation method of giant bream according to claim 3, characterized in that: The sexually mature giant bream parent is a male giant bream weighing more than 3kg and with a reproductive process protruding more than 0.2cm; a female giant bream weighing more than 4kg, with a red and swollen reproductive opening, and a swollen, soft and shaken lower abdomen.

5. The artificial propagation method of giant bream according to claim 4, characterized in that: The method of inducing labor for female parents with better development is: inject luteinizing hormone releasing hormone A29~11μg / kg + dioxifen maleate 4.5~5.5mg / kg + chorionic gonadotropin 450~550IU / kg; the method of inducing labor for female parents with poorer development is: first inject luteinizing hormone releasing hormone A22.8~3.2μg / kg, and then inject LARH A26~8μg / kg + dioxifen maleate 4.5~5.5mg / kg + chorionic gonadotropin 450~550IU / kg 9~11h later.

6. The artificial breeding method of giant bream according to claim 5, characterized in that: The female parent with better development has a round, swollen and soft abdomen both above and below, a red and particularly swollen genital opening, and the eggs dug out with an egg digger are regular round individuals; the female parent with poor development has a relatively flat upper abdomen, only the lower abdomen is swollen, the genital opening is slightly red, and the eggs dug out with an egg digger are irregular round individuals.

7. The artificial propagation method of giant bream according to claim 1, characterized in that: The volume ratio of the semen to the giant trout sperm storage solution is 1:18-22.

8. The artificial propagation method of giant bream according to claim 7, characterized in that: The stirring time is 2 to 3 minutes.

9. The method for artificial propagation of giant bream according to claim 8, characterized in that: For the female parent, 10 to 16 hours after inducing ovulation, gently press the abdomen to obtain mature eggs.

10. The method for artificial propagation of giant bream according to claim 9, characterized in that: During the incubation period, the dissolved oxygen level of fertilized eggs is 8-16 mg / L, and the incubation density is 13,000-17,000 eggs / m 3 .

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