Artificial insemination method for freshwater ornamental fish swallow fish

Abstract

The invention belongs to the technical field of aquaculture, and discloses an application of triiodothyronine in induced spawning of aquatic products and an artificial insemination method among different varieties of swallow fishes by using triiodothyronine, and the method comprises the following steps: (1) parent fish treatment; (2) hormone feeding; (3) collecting sperms and eggs; (4) artificial insemination; (5) restoring the form; and (6) embryo incubation. According to the method, artificial insemination of the fresh water symphysodon is carried out, the fertilization rate of the symphysodon can reach 90%, and interspecies hybridization can be well carried out. According to the method, an artificial insemination mode of the swallow fish is created, and the embryo fertilization rate of the freshwater symphysodon is increased; the method plays a positive role in the development of a new variety of the freshwater fish; reference is provided for artificial breeding of the water fish; and a good experimental material can be provided for molecular biology.

Description

Technical Field

[0001] The invention belongs to the technical field of aquaculture, in particular to an application of triiodothyronine in aquatic induced labor and an artificial insemination method for scalare (Pterophyllum scalare). Background Art

[0002] Angelfish (Pteroghyllum scalare) is an important ornamental fish in the aquarium market. It is native to the Amazon waters of South America. It belongs to the genus Angelfish of the phylum Chordata, class Osteichthyes, order Perciformes, family Cichlidae. It was officially introduced to Europe in 1911 and was introduced to my country in the late 1950s, becoming the most high-end ornamental fish at that time. Because its dorsal fin and anal fins on both sides have long fins, it looks like a swallow when swimming in the water, so it is also called swallow fish. Angelfish are docile and eat quietly, and are loved by most people.

[0003] There are many types of angelfish. According to their colors, they include white angelfish, black angelfish (black angelfish), gray angelfish, blue angelfish, zebra angelfish, golden-headed angelfish, and tri-color angelfish (juvenile and adult are called panda angelfish). Some merchants also classify them according to the length of their fins into long-fin angelfish, medium-fin angelfish and short-fin angelfish.

[0004] The prices of different species of angelfish vary greatly. For example, the price of a 2.5cm fry of Egyptian angelfish is basically more than ten times that of a tricolor angelfish. The price of a new species of angelfish drops rapidly in the second year. For example, when a 2.5cm fry of a golden angelfish appears in its first year, the price can even reach about 50 times that of the second year. This shows that it is extremely critical and important to have an excellent variety and how to ensure that the variety does not flow out. This not only ensures the input-output ratio of new variety research and development, but also is a test of how to develop new varieties.

[0005] In the field of artificial aquatic breeding, existing technologies have many defects, which limit the efficiency, quality and development of aquatic breeding and urgently need to be improved:

[0006] Limitations in the effect of hormone use: When commonly used hormones in aquaculture, such as human chorionic gonadotropin (HCG), dioestrogen maleate (DOM), and luteinizing hormone-releasing hormone (A2), are used to induce labor in angelfish, even if they are fed at the same dosage as grass carp, the combination of two or three cannot achieve the ideal effect of inducing labor, and cannot effectively induce ovulation in angelfish, affecting the progress and efficiency of artificial breeding. This shows that the existing technology has not fully adapted to the physiological characteristics of angelfish in terms of hormone selection and use, and it is difficult to meet the actual needs of inducing labor.

[0007] Difficulties in hybridization and limited variety breeding: The unique one-male-one-female pairing breeding form of swallowfish during natural reproduction makes hybridization between different varieties extremely difficult. Existing technologies mostly rely on natural reproduction methods and cannot effectively overcome this obstacle, resulting in a low success rate of hybridization between swallowfish varieties. This seriously restricts the cultivation and development of new varieties and cannot meet the market demand for diversified and high-quality swallowfish varieties. Artificial insemination and unified fertilization time can be of great significance for the research and development of new varieties and to ensure that germplasm resources do not flow out. Breeding technology is a prerequisite for controlling new varieties. For example, triploid breeding requires fertilized eggs of the same time.

[0008] Problems with fertilized egg processing: In the prior art, there is a lack of specific methods for configuring hatching liquids during the subsequent incubation of fertilized eggs, and there is no effective means to restore the morphology of the eggs, making it difficult to ensure the quality of the fertilized eggs, resulting in a low hatching rate. For example, the prior art does not clearly propose mature measures such as adding embryo culture fluid containing thyroid hormones to restore egg morphology and improve the quality of fertilized eggs, as in the present invention.

[0009] Through searching, the following journals and patent publications related to the patent application of this invention were found:

[0010] 1. A method for improving the artificial insemination rate and hatching rate of pike (CN109329138A), comprising the following steps: (1) strengthening the cultivation of pike parents and waiting for them to mature; (2) when the pike eggs mature, adjusting the water temperature to 15-17°C and injecting gonadotropin into the mother; (3) raising the pike eggs in constant temperature still water at 15-17°C for 20 hours and then repeating the injection; (4) raising the eggs in constant temperature still water at 15-17°C for 14 hours and when the fertilization hole of the eggs is about to open, closely observing the opening process of the fertilization hole and completing artificial insemination within 30-40 minutes after the fertilization hole of the eggs is fully opened; (5) controlling the water temperature at 15-17°C, After 20 hours, the fertilized eggs begin to fission into small fry; (6) after 48 hours, the small fry are moved into an earthen pond for cultivation, and after 148-150 hours, they are fed with starter feed; the dog liver grass extract is added to the constant temperature still water described in steps (3) and (4), and the amount of the dog liver grass extract added is 0.065% of the total volume of the constant temperature still water; the dog liver grass extract is prepared by the following method: the dog liver grass is dried and crushed, and then 10 times the amount of 15% ethanol is added, soaked for 2 hours, then refluxed for extraction for 1 hour, filtered, and the filtrate is reserved, the filter residue is repeatedly extracted twice, filtered, and the filtrate is combined, and the filtrate is concentrated under reduced pressure at 45°C to a relative density of 1.07-1.09, so as to obtain the dog liver grass extract.

[0011] 2. A method for artificial insemination of basa fish for improving fertilization rate (CN117581813A), specifically comprising the following steps: S1, selecting 4-year-old male basa fish and 5-year-old female basa fish and placing them in a suitable water quality environment, and ensuring that the gonads of the selected female basa fish are mature and the reproductive sinus of the male basa fish is prominent; S2, in the period from the end of May to the beginning of June, putting the male basa fish and the female basa fish selected in S1 into an induced labor pond in a ratio of 1:2; S3, selecting DOM, HCG, and LRH-A2 as induced labor drugs, injecting them into female and male fish, and after induced labor, temporarily raising the male basa fish and female basa fish in separate ponds for female fish spawning; S4, after the female fish in S3 have completed spawning, completing artificial insemination of the eggs by dry method.

[0012] 3. A goldfish fertilization medium and a method for artificial insemination and hatching of goldfish (CN118716255A), wherein the specific implementation steps are: selecting female and male parent fish that are breeding by chasing each other, squeezing the abdomen of the fish to discharge fish eggs and semen into the fertilization medium, stirring and mixing for artificial insemination; transferring the fish eggs to a demucin solution to debond the fish eggs; placing the demucined eggs in a hatching bag after simple rinsing; and transferring the fry to a nursery pond for breeding after hatching. The suitable osmotic pressure and the egg white protein-containing environment provided by the fertilization medium of the present invention can significantly prolong the duration of goldfish eggs and sperm vitality and improve the effect of fish egg fertilization rate; the demucin solution is used to debond the fish eggs to prevent the fish eggs from adhering to each other to form a mass or cluster, thereby improving the hatching rate of goldfish, allowing the various heritable ornamental traits of the goldfish to fully develop, and preventing pathogenic microorganisms from being transmitted from the parent generation to the offspring. The selection of the hatching bag provides a good hatching environment for the fish eggs.

[0013] By comparison, the patent application of the present invention is essentially different from the above-mentioned public documents. Summary of the invention

[0014] The purpose of the present invention is to overcome the shortcomings of the prior art and provide an application of triiodothyronine in aquatic induction of labor and an artificial insemination method for swallow fish.

[0015] The technical solution adopted by the present invention to solve its technical problem is:

[0016] Application of triiodothyronine in aquatic labor induction.

[0017] A use of triiodothyronine as an oxytocin in aquatic labor induction.

[0018] Application of triiodothyronine in inducing spawning in swallow fish.

[0019] Application of triiodothyronine as an oxytocin in inducing spawning in swallow fish.

[0020] A hormone composition for artificial insemination of swallow fish using triiodothyronine, the hormone composition comprising: human chorionic gonadotropin HCG, diodonone maleate DOM, luteinizing hormone releasing hormone A2, and triiodothyronine T3.

[0021] Furthermore, the dosage of the hormone composition when used is: 200 IU / kg chorionic gonadotropin HCG of swallow fish + 1 mg / kg domylone maleate of swallow fish + 10 μg / kg luteinizing hormone releasing hormone A2 of swallow fish + 0.2 mg / kg triiodothyronine T3 of swallow fish.

[0022] The method of artificial insemination between different species of swallow fish using triiodothyronine comprises the following steps:

[0023] (1) Broodstock treatment;

[0024] (2) hormone feeding;

[0025] (3) Sperm and egg collection;

[0026] (4) Artificial insemination;

[0027] (5) Morphological restoration;

[0028] (6) Embryo hatching.

[0029] Furthermore, the specific steps are as follows:

[0030] (1) Water conditions for broodstock breeding of swallowtail: water temperature: 28°C; pH: 6.0-6.5;

[0031] (2) Broodstock requirements: Female fish should be seven to eight months old, and male fish should be more than one year old;

[0032] (3) Broodstock pairing: Broodstock are paired after purchase. The breeding period is determined by cleaning the spawning tank and checking the time when the genitals bulge. Broodstock feed is used to feed the broodstock for 15 days.

[0033] Among them, the broodstock feed is: 0.3g / pair of bloodworms from 07:00 to 09:00 every day; 0.3g / pair of imitation bloodworms from 14:00 to 16:00; 0.3g / pair of Artemia at 20:00; after 15 days,

[0034] (4) Feeding broodstock hormones: After 15 days of broodstock feed feeding, on the 16th day, the female fish were fed with specific hormones, including 200 IU / kg of human chorionic gonadotropin HCG + 1 mg / kg of dioconazole maleate DOM + 10 μg / kg of luteinizing hormone-releasing hormone A2 + 0.2 mg / kg of triiodothyronine T3; the male fish were fed the same dosage; only one feeding was required;

[0035] The method of hormone feeding is as follows: injecting hormone into thawed red worms, and then feeding the injected red worms;

[0036] When feeding hormones, the first feeding is bloodworms injected with HCG and A2. After the reproductive pores of the broodstock are prominent, the second feeding is bloodworms injected with T3+DOM to promote sperm, follicle development and ovulation.

[0037] (5) Collecting eggs from female fish: After feeding the broodstock hormones, take a clean culture dish and add pure water to the dish. After ovulation and ejaculation 2 hours after the second feeding, the female fish is taken out and wrapped with a wet towel. Then, gently press the genitals on the abdomen to scatter the eggs in the water of the culture dish.

[0038] (6) Extracting sperm from male fish: First, anesthetize the male fish with eugenol at a concentration of 60-90 mg / L; select male fish of different species or paired fish; align the male fish's genital opening with the culture dish and press its genital opening. Liquid will flow out and the sperm will be sucked out with a syringe. The sperm motility will be examined under a microscope. If it is qualified, the sperm will be mixed with the egg;

[0039] (7) After the eggs are fertilized, embryo culture medium containing thyroid hormone is added to the culture dish to restore the egg morphology and improve the quality and hatching rate of the fertilized eggs;

[0040] The formula of the embryo culture medium containing thyroid hormone is as follows: TCM-199 basic solution, supplemented with 10% volume fraction of fetal bovine serum, 25mM HEPES, 0.55mg / ml sodium pyruvate, 100IU / ml penicillin-streptomycin and 8μg / L levothyroxine L-T4;

[0041] (8) Egg incubation method: Pour the prepared low-concentration potassium permanganate solution into a sterilized glass dish, place the fertilized eggs after recovery into the low-concentration potassium permanganate solution in the glass dish and let it stand for 10 to 15 seconds, then use a sterilized plastic straw to suck out all the eggs, put them into a sterilized container, pour them into the sterilized aquaculture water, add 8000 units / L of gentamicin, and ventilate for incubation;

[0042] The aquaculture water body is configured as follows: the water is composed of 70% volume of aerated tap water with TDS=200 and pH8.2 + 30% volume of pure water with TDS=5 and pH8.2, and 0.1 ml / L phosphoric acid is added.

[0043] Furthermore, in step (4), the time after the genital pore of the broodstock protrudes is 16 hours after the first feeding.

[0044] The advantages and positive effects achieved by the present invention are:

[0045] 1. The present invention performs artificial insemination of freshwater angelfish, so that the fertilization rate can reach 90% and can perform good inter-variety hybridization. The present invention creates an artificial insemination model for swallowfish, improves the embryo fertilization rate of freshwater angelfish; and has a positive effect on the development of new varieties of freshwater angelfish; provides a reference for the artificial breeding of water angelfish; and can also provide good experimental materials for molecular biology experiments.

[0046] 2. The present invention has created an artificial insemination method for freshwater angelfish, and can achieve hybridization between different varieties of freshwater angelfish instead of natural reproduction, effectively improving the fertilization rate of swallowfish embryos and the success rate of cross-species hybridization, and providing a good application technology for the development of new varieties.

[0047] 3. The present invention has a positive effect on the development of new varieties of freshwater angelfish. The present invention can provide a reference for the artificial breeding of freshwater angelfish. The present invention can improve the embryo fertilization rate of freshwater angelfish.

[0048] 4. The method of the present invention can effectively obtain a large number of eggs with uniform fertilization time, and can provide good experimental materials for molecular biology experiments.

[0049] 5. The method of the present invention can shorten the spawning period of freshwater angelfish and improve the physical fitness of parent fish.

[0050] 6. The present invention overcomes the problem that the existing freshwater angelfish can only be bred through natural breeding in an artificial breeding environment. There are many varieties of angelfish, but due to the differences between different varieties and their unique pairing breeding form (one female and one male pairing), hybridization between varieties is difficult. The present invention realizes artificial insemination of freshwater angelfish. This method performs artificial insemination on freshwater angelfish, so that its fertilization rate can reach 90% and hybridization between varieties can be achieved.

[0051] 7. The present invention is the first to use thyroid hormones in aquatic induced labor. The present invention uses triiodothyronine (T3) as an oxytocin hormone in conjunction with human chorionic gonadotropin (HCG), dithyroxine maleate (DOM), and luteinizing hormone-releasing hormone (A2). Triiodothyronine (T3) is a key addition, that is, the induced labor effect cannot be achieved without T3. The present invention also adds levothyroxine sodium (L-T4) as an additive to the cell morphology recovery solution to restore the cell morphology. BRIEF DESCRIPTION OF THE DRAWINGS

[0052] Figure 1 The photos are of the continuous development of the fertilized eggs of swallow fish obtained by artificial insemination in the present invention: a is the two-cleft stage, b is the eight-cleft stage, c is the 32-cleft stage, d and e are the multicellular stage, and f is the blastocyst stage;

[0053] Figure 2Figures A, B and C are embryo diagrams of the present invention; wherein, a, b and c are embryo diagrams without adding culture medium, and it can be seen that the embryos are abnormal in morphology, and are irregular oval in shape and cannot be artificially inseminated; Figures A, B and C are embryo diagrams after adding culture medium, and it can be seen that after adding morphological recovery culture medium, the eggs quickly become full and recover to regular oval shape, and can be artificially inseminated. DETAILED DESCRIPTION

[0054] The present invention will be further described below in conjunction with the embodiments. The following embodiments are descriptive rather than restrictive, and the protection scope of the present invention cannot be limited by the following embodiments.

[0055] The various experimental operations involved in the specific embodiments are all routine techniques in the art. For parts not specially annotated in this document, ordinary technicians in the art can implement them by referring to various commonly used reference books, scientific and technological literature or related instructions, manuals, etc. before the filing date of this invention.

[0056] Application of triiodothyronine in aquatic labor induction.

[0057] A use of triiodothyronine as an oxytocin in aquatic labor induction.

[0058] Application of triiodothyronine in inducing spawning in swallow fish.

[0059] Application of triiodothyronine as an oxytocin in inducing spawning in swallow fish.

[0060] A hormone composition for artificial insemination of swallow fish using triiodothyronine, the hormone composition comprising: human chorionic gonadotropin HCG, diodonone maleate DOM, luteinizing hormone releasing hormone A2, and triiodothyronine T3.

[0061] Preferably, the dosage of the hormone composition when used is: 200 IU / kg chorionic gonadotropin HCG of swallow fish + 1 mg / kg domylone maleate of swallow fish + 10 μg / kg luteinizing hormone releasing hormone A2 of swallow fish + 0.2 mg / kg triiodothyronine T3 of swallow fish.

[0062] The method of artificial insemination between different species of swallow fish using triiodothyronine comprises the following steps:

[0063] (1) Broodstock treatment;

[0064] (2) hormone feeding;

[0065] (3) Sperm and egg collection;

[0066] (4) Artificial insemination;

[0067] (5) Morphological restoration;

[0068] (6) Embryo hatching.

[0069] Preferably, the specific steps are as follows:

[0070] (1) Water conditions for broodstock breeding of swallowtail: water temperature: 28°C; pH: 6.0-6.5;

[0071] (2) Broodstock requirements: Female fish should be seven to eight months old, and male fish should be more than one year old;

[0072] (3) Broodstock pairing: Broodstock are paired after purchase. The breeding period is determined by cleaning the spawning tank and checking the time when the genitals bulge. Broodstock feed is used to feed the broodstock for 15 days.

[0073] Among them, the broodstock feed is: 0.3g / pair of bloodworms from 07:00 to 09:00 every day; 0.3g / pair of imitation bloodworms from 14:00 to 16:00; 0.3g / pair of Artemia at 20:00; after 15 days,

[0074] (4) Feeding broodstock hormones: After 15 days of broodstock feed feeding, on the 16th day, the female fish were fed with specific hormones, including 200 IU / kg of human chorionic gonadotropin HCG + 1 mg / kg of dioconazole maleate DOM + 10 μg / kg of luteinizing hormone-releasing hormone A2 + 0.2 mg / kg of triiodothyronine T3; the male fish were fed the same dosage; only one feeding was required;

[0075] The method of hormone feeding is as follows: injecting hormone into thawed red worms, and then feeding the injected red worms;

[0076] When feeding hormones, the first feeding is bloodworms injected with HCG and A2. After the reproductive pores of the broodstock are prominent, the second feeding is bloodworms injected with T3+DOM to promote sperm, follicle development and ovulation.

[0077] (5) Collecting eggs from female fish: After feeding the broodstock hormones, take a clean culture dish and add pure water to the dish. After ovulation and ejaculation 2 hours after the second feeding, the female fish is taken out and wrapped with a wet towel. Then, gently press the genitals on the abdomen to scatter the eggs in the water of the culture dish.

[0078] (6) Extracting sperm from male fish: First, anesthetize the male fish with eugenol at a concentration of 60-90 mg / L; select male fish of different species or paired fish; align the male fish's genital opening with the culture dish and press its genital opening. Liquid will flow out and the sperm will be sucked out with a syringe. The sperm motility will be examined under a microscope. If it is qualified, the sperm will be mixed with the egg;

[0079] (7) After the eggs are fertilized, embryo culture medium containing thyroid hormone is added to the culture dish to restore the egg morphology and improve the quality and hatching rate of the fertilized eggs;

[0080] The formula of the embryo culture medium containing thyroid hormone is as follows: TCM-199 basic solution, supplemented with 10% volume fraction of fetal bovine serum, 25mM HEPES, 0.55mg / ml sodium pyruvate, 100IU / ml penicillin-streptomycin and 8μg / L levothyroxine L-T4;

[0081] (8) Egg incubation method: Pour the prepared low-concentration potassium permanganate solution into a sterilized glass dish, place the fertilized eggs after recovery into the low-concentration potassium permanganate solution in the glass dish and let it stand for 10 to 15 seconds, then use a sterilized plastic straw to suck out all the eggs, put them into a sterilized container, pour them into the sterilized aquaculture water, add 8000 units / L of gentamicin, and ventilate for incubation;

[0082] The aquaculture water body is configured as follows: the water is composed of 70% volume of aerated tap water with TDS=200 and pH8.2 + 30% volume of pure water with TDS=5 and pH8.2, and 0.1 ml / L phosphoric acid is added.

[0083] Preferably, the time after the genital pore of the broodstock protrudes in step (4) is 16 hours after the first feeding.

[0084] Specifically, the relevant preparation and detection are as follows:

[0085] A method for improving the artificial insemination efficiency of swallowfish (Pterophyllum scalare), comprising the following steps:

[0086] The broodstock of swallowfish are placed in aerated tap water and fed to reduce stress response.

[0087] The three commonly used aquatic hormones HCG, DOM, and A2 were combined in pairs or in combination according to the dosage of grass carp and fed, but it was found that the effect of inducing labor could not be achieved. Therefore, T3 (triiodothyronine) was added. After adding it, it was found that although the egg morphology was not full, labor could be induced.

[0088] The eggs were collected from the females within half an hour of injection, while the males were anesthetized and the semen was gently aspirated using a needle.

[0089] The collected semen is inseminated with eggs at the same time to control the fertilization time and improve the fertilization rate.

[0090] After the eggs are fertilized, embryo culture medium containing thyroid hormone is added to restore the egg morphology and improve the quality and hatching rate of the fertilized eggs (the fertilization rate can exceed 90%).

[0091] Through optimized hormone formula and insemination technology, batch artificial insemination of angelfish can be achieved, improving efficiency and success rate.

[0092] The more specific steps are as follows:

[0093] A method for artificial insemination breeding of freshwater angelfish, comprising the following steps:

[0094] Broodstock culture water conditions: water temperature: 28℃; pH: 6.0-6.5;

[0095] Broodstock requirements: female fish should be seven to eight months old; male fish should be over one year old.

[0096] The composition of broodstock feed, its daily feeding time and dosage are as follows: 08:30 bloodworm: 1 grain / pair; 14:00 compound feed imitation bloodworm: 0.3g / pair; 20:00 Artemia: 0.3g / 1 pair.

[0097] Broodstock pairing: 6-7 broodstock are initially determined to be male or female for pairing. The broodstock with a bulge on the head is a male, and the external genitalia are thin and pointed. The female fish has no bulge on the head, and the external genitalia are tubular. The color is lighter than that of the male fish. Put one female fish and one male fish into the broodstock tank prepared in advance. Feed them with broodstock feed for 15 days;

[0098] Configuration of the breeding fish tank: 30cm*30cm*35cm fish tank, add a water fairy cotton, add a spawning tank.

[0099] Preparation of aquaculture water: The water is prepared by adding 70% volume of aerated tap water with a pH of 8.2 (TDS: 200) + 30% volume of pure water with a pH of 8.2 (TDS: 5) and 0.1 ml / L phosphoric acid.

[0100] Hormone feeding: After 15 days of feeding the broodstock, on the 16th day, specific hormones are fed to the broodstock. A certain amount of hormones is injected into the thawed bloodworms, and then the injected bloodworms are fed. When feeding hormones, the bloodworms injected with HCG and A2 are fed first. After the reproductive pores of the broodstock are protruding (about 16 hours), the bloodworms injected with T3+DOM are fed to promote sperm, follicle development and ovulation.

[0101] Specifically, when feeding broodstock hormones, female fish are fed with specific hormones, including 200 IU / kg of human chorionic gonadotropin HCG + 1 mg / kg of dioconazole maleate DOM + 10 μg / kg of luteinizing hormone releasing hormone A2 + 0.2 mg / kg of triiodothyronine T3; male fish are fed with the same dosage; and only one feeding is required;

[0102] The method of hormone feeding is as follows: injecting hormone into thawed red worms, and then feeding the injected red worms;

[0103] When feeding hormones, the first feeding is bloodworms injected with HCG and A2. After the reproductive pores of the broodstock are protruding, the second feeding is bloodworms injected with T3+DOM to promote sperm, follicle development and ovulation.

[0104] Female fish egg collection: After feeding the broodstock hormone, take a clean culture dish and add pure water to the dish. Ovulation and ejaculation will occur 2 hours after the second feeding. Take out the female fish and wrap it with a wet towel, then gently press its abdominal genitals to scatter the eggs in the water of the culture dish.

[0105] Sperm collection from male fish: After anesthetizing the male fish with eugenol (60-90 mg / L), squeeze the genital opening to allow fluid to flow out, collect the semen, and after microscopic examination to determine if the semen is viable, mix it with the eggs for fertilization.

[0106] After the eggs are fertilized, embryo culture medium containing thyroid hormone is added to restore the egg morphology and improve the quality and hatching rate of the fertilized eggs.

[0107] The formula of thyroid hormone embryo culture medium: TCM-199 basic solution, supplemented with 10% volume fraction of fetal bovine serum, 25mM HEPES, 0.55mg / ml sodium pyruvate, penicillin-streptomycin (100IU / ml) and levothyroxine L-T4 (8μg / L);

[0108] Incubation method: Observe the stage of the cells under a stereoscope and take photos to record; pour the prepared low-concentration potassium permanganate solution into a sterilized glass dish, put the fertilized eggs that have recovered their morphology into the low-concentration potassium permanganate solution in the glass dish and let it stand for 10 to 15 seconds, then use a sterilized plastic straw to suck out all the eggs, put them into a sterilized container, pour them into the disinfected breeding water, add 8000 units / L of gentamicin for ventilation and incubation.

[0109] Batch processing: Through optimized hormone formula and insemination technology, batch artificial insemination of swallow fish is achieved to improve efficiency and success rate. The hatching rate of fertilized eggs in vitro operation of the present invention is about 90%.

[0110] Record the results:

[0111] Figure 1 The following are photos of the continuous development of the fertilized eggs of swallow fish obtained by artificial insemination in the present invention: a is the two-cleft stage, b is the eight-cleft stage, c is the 32-cleft stage, d and e are the multi-cell stage, and f is the blastocyst stage. It can be seen that the swallow fish embryos obtained by artificial insemination have clear morphology, artificial insemination can more accurately combine sperm and eggs, avoid the interference of environmental factors such as water flow factors in natural fertilization, and the fertilization water body does not contain debris and enemies. The fertilization rate of artificially inseminated swallow fish embryos is 14% higher than the natural fertilization rate on average. By controlling the time of fertilization, the breeding process can be carried out in the most suitable season, the breeding success rate is improved, and the planning and continuity of aquaculture production are guaranteed.

[0112] Figure 2 Figures a, b, and c are embryos without culture medium added in the present invention. It can be seen that the embryos are abnormally morphologically irregular and cannot be artificially inseminated. Figures A, B, and C are embryos after culture medium is added in the present invention. It can be seen that after adding morphological recovery culture medium, the eggs quickly become full and restored to regular oval shapes for artificial insemination. The present invention creatively uses cell morphological recovery fluid to make unfull and unfertilizable eggs fertilizable.

[0113] 1. The hatching rate of fertilized eggs in vitro is about 90%.

[0114] 2. The use of the invention can prevent the fertilized eggs from being infected by aquatic microorganisms during the hatching process, thereby preventing the fertilized eggs from becoming bad eggs.

[0115] 3. The in vitro culture technology of the present invention is simple to operate and does not have high requirements on the conditions of the surrounding environment.

[0116] Although the embodiments of the present invention are disclosed for illustrative purposes, those skilled in the art will appreciate that various substitutions, changes and modifications are possible without departing from the spirit and scope of the present invention and the appended claims. Therefore, the scope of the present invention is not limited to the contents disclosed in the embodiments.

Claims

1. An application of triiodothyronine in inducing labor in aquaculture.

2. A method for using triiodothyronine as an oxytocin in aquatic labor induction.

3. An application of triiodothyronine in inducing spawning in swallow fish.

4. A method for using triiodothyronine as an oxytocin in inducing spawning in swallow fish.

5. A hormone composition for artificial insemination of swallow fish using triiodothyronine, characterized in that: The hormone composition comprises: human chorionic gonadotropin HCG, diodothyronine maleate DOM, luteinizing hormone releasing hormone A2 and triiodothyronine T3.

6. The hormone composition according to claim 5, characterized in that: The dosage of the hormone composition when used is: 200 IU / kg chorionic gonadotropin HCG of swallow fish + 1 mg / kg domylone maleate of swallow fish + 10 μg / kg luteinizing hormone releasing hormone A2 of swallow fish + 0.2 mg / kg triiodothyronine T3 of swallow fish.

7. A method for artificial insemination of different species of swallow fish using triiodothyronine, characterized in that: The steps include: (1) Broodstock treatment; (2) hormone feeding; (3) Sperm and egg collection; (4) Artificial insemination; (5) Morphological restoration; (6) Embryo hatching.

8. The method according to claim 7, characterized in that: The specific steps are as follows: (1) Water conditions for swallowfish broodstock culture: Water temperature: 28℃; pH: 6.0-6.5; (2) Broodstock requirements: Female fish should be seven to eight months old, and male fish should be more than one year old; (3) Broodstock pairing: Broodstock are paired after purchase. The breeding period is determined by cleaning the spawning tank and checking the time when the genitals bulge. Broodstock feed is used to feed the broodstock for 15 days. Among them, the broodstock feed is: 0.3g / pair of bloodworms from 07:00 to 09:00 every day; 0.3g / pair of imitation bloodworms from 14:00 to 16:00; 0.3g / pair of Artemia at 20:00; after 15 days, (4) Feeding broodstock hormones: After 15 days of broodstock feed feeding, on the 16th day, the female fish were fed with specific hormones, including 200 IU / kg of human chorionic gonadotropin HCG + 1 mg / kg of dioconazole maleate DOM + 10 μg / kg of luteinizing hormone-releasing hormone A2 + 0.2 mg / kg of triiodothyronine T3; the male fish were fed the same dosage; only one feeding was required; The method of hormone feeding is as follows: injecting hormone into thawed red worms, and then feeding the injected red worms; When feeding hormones, the first feeding is bloodworms injected with HCG and A2. After the reproductive pores of the broodstock are prominent, the second feeding is bloodworms injected with T3+DOM to promote sperm, follicle development and ovulation. (5) Collecting eggs from female fish: After feeding the broodstock hormones, take a clean culture dish and add pure water to the dish. After ovulation and ejaculation 2 hours after the second feeding, the female fish is taken out and wrapped with a wet towel. Then, gently press the genitals on the abdomen to scatter the eggs in the water of the culture dish. (6) Extracting sperm from male fish: First, anesthetize the male fish with eugenol at a concentration of 60-90 mg / L; select male fish of different species or paired fish; align the male fish's genital opening with the culture dish and press its genital opening. Liquid will flow out and the sperm will be sucked out with a syringe. The sperm motility will be examined under a microscope. If it is qualified, the sperm will be mixed with the egg; (7) After the eggs are fertilized, embryo culture medium containing thyroid hormone is added to the culture dish to restore the egg morphology and improve the quality and hatching rate of the fertilized eggs; The formula of the embryo culture medium containing thyroid hormone is as follows: TCM-199 basic solution, supplemented with 10% volume fraction of fetal bovine serum, 25mM HEPES, 0.55mg / ml sodium pyruvate, 100IU / ml penicillin-streptomycin and 8μg / L levothyroxine L-T4; (8) Egg incubation method: Pour the prepared low-concentration potassium permanganate solution into a sterilized glass dish, place the fertilized eggs after recovery into the low-concentration potassium permanganate solution in the glass dish and let it stand for 10 to 15 seconds, then use a sterilized plastic straw to suck out all the eggs, put them into a sterilized container, pour them into the sterilized aquaculture water, add 8000 units / L of gentamicin, and ventilate for incubation; The aquaculture water body is configured as follows: the water is composed of 70% volume of aerated tap water with TDS=200 and pH8.2 + 30% volume of pure water with TDS=5 and pH8.2, and 0.1 ml / L phosphoric acid is added.

9. The method according to claim 8, characterized in that: The time after the genital pore of the broodstock in step (4) becomes prominent is 16 hours after the first feeding.

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