CN107787885B - Pure line cultivation method for artificial induction of body color character of gynogenesis goldfish - Google Patents
Pure line cultivation method for artificial induction of body color character of gynogenesis goldfish Download PDFInfo
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K61/00—Culture of aquatic animals
- A01K61/10—Culture of aquatic animals of fish
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2227/00—Animals characterised by species
- A01K2227/40—Fish
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
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- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
- Y02A40/80—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in fisheries management
- Y02A40/81—Aquaculture, e.g. of fish
Abstract
The invention relates to a pure line cultivation method for artificially inducing the body color character of gynogenesis goldfish. Performing spawning induction and sperm elimination on the grey male crucian carps by adopting a secondary injection method; performing sperm inactivation treatment on the sperm diluent in batches by using a normal-pressure room-temperature plasma breeding instrument; obtaining crucian inactivated sperm liquid and goldfish spawning granules with target body color to complete fertilization; treating fertilized eggs of goldfish by colchicine and cold treatment, diploidizing gynogenesis and naturally hatching; selecting and culturing to obtain the F1 generation goldfish with the target body color character; and (3) selecting female goldfishes of the F1 generation as parent fishes and male crucian carps, and repeating the steps to obtain the pure lines of the goldfishes of the F2 generation with the target body color character. The F2 generation pure line parent of the target body color character obtained by the technical scheme of the invention is used for breeding production, and a new quick way is created for the directional cultivation of the target body color character of the ornamental fish.
Description
Technical Field
The invention belongs to the technical field of artificial breeding induction of goldfishes, and particularly relates to a method for breeding a pure line of a body color character of a gynogenesis goldfish by artificial induction.
Background
Goldfish (Carassius auratus) is the most common consumer variety in the domestic ornamental fish market at present, and the common body colors in the market comprise red, red white, yellow, five flowers and the like, but the number of strains capable of being stably inherited is small. Therefore, the genetic research of the oriented development of the body color of the goldfish is developed, the time for breeding high-quality product color is greatly shortened, the breeding efficiency is improved, a new economic value can be created, and the method has wide practical prospect.
According to the research data: the method is prominent in the body color breeding research of marine fishes and tropical ornamental fishes such as zebra fish, peacock fish, medaka, moonfish and Japanese koi. Related researches are carried out by scholars in China aiming at the body color of fishes, such as body color type genetic researches of Zhang Jiansen and the like; study on the body color and scale coat genetic characteristics of filial generations such as Xuwei; the study on the inheritance and variation of body color of the royal jelly. At present, most of fishes still obtain specific body colors by artificially regulating and controlling the directional development of the body colors of the fishes by the following methods: genetic factor regulation, endocrine factor regulation, nutritional factor regulation, coloring matter regulation, environmental control, etc. In addition, a large number of studies have shown that stable inheritance can only be obtained with the body color of a pure line variety.
How to stably maintain the ornamental body color character of the excellent goldfish becomes an urgent problem to be solved in the genetic breeding of the ornamental goldfish. The gynogenesis method is used for establishing the goldfish genetic pure line, the goldfish pure line with stable ornamental body color characters can be rapidly bred, and a large number of goldfishes with excellent ornamental body color characters are bred to meet the requirements of people, so that the targeted cultivation of the ornamental fish body color characters is realized.
Disclosure of Invention
According to the invention, a normal-pressure room-temperature plasma breeding instrument (ARTP) is adopted to perform crucian sperm inactivation treatment, the crucian sperm inactivation treatment is used for fertilizing with female goldfish eggs with selected body color shapes, a colchicine combined cold treatment mode is adopted to artificially induce the gynogenesis and diploidization of the goldfish, and a goldfish pure line with target body color characters is obtained through identification and screening of the goldfish with the target body color shapes, so that a new rapid way is created for directional cultivation of the target body color characters of the ornamental fishes.
The technical scheme adopted for achieving the purpose of the invention is as follows:
1. sperm inactivation treatment
Adopting male crucian with grey body color, inducing parturition and discharging sperm by adopting a secondary injection method, and preparing a needle for injecting male parent crucian by using 2 mu g of luteinizing hormone releasing hormone analogue (LHRH-A2) for the first time; after 16 hours, the compound ripener of 3-5 mg is injected into each kg of fish for the second injection. Collecting crucian sperm liquid by a conventional method, and diluting by using 40-50 times of Hank's liquid to obtain sperm diluent.
The formula of the compound ripener is that every 10mg of the compound ripener contains 2mg of Diospyrone (DOM), 6mg of carp Pituitary Gland (PG) and 800 international units IU of Human Chorionic Gonadotropin (HCG).
And (3) performing sperm inactivation treatment on the sperm diluent by utilizing a normal-pressure room-temperature plasma (ARTP) in batches, and combining multiple batches to obtain the crucian inactivated sperm liquid with the total amount of more than 300 mu L.
After the sperm inactivation treatment, 5 mul of 0.6M mannitol and 10 mul of Hank's solution are added into every 20 mul of inactivated sperm solution, and the mixture is stored at low temperature of 2-4 ℃ in a dark place for later use.
The sperm inactivation treatment is carried out under the conditions of 20 mu L of sperm diluent sample loading amount, 125W of treatment output power and 10L min of gas flow rate － 1 The irradiation distance is 2 mm, and the treatment time is 20-60 s.
2. Fertilization of
Pouring 0.5L-1L of fish normal saline into a clean 2L ceramic basin, placing a fish nest which is sterilized on the bottom surface of the ceramic basin, selecting 2-4-year-old female goldfishes different from the body color spawning period of the crucian, injecting carp pituitary gland 3-4 mg per kg of the weight of the female goldfishes for artificial spawning induction, then completely squeezing female goldfish eggs into the fish normal saline by hands, simultaneously pouring 200 mu L of crucian inactivated sperm liquid at low temperature around the squeezed goldfish eggs, and gently stirring the eggs in water by using feathers to ensure that the sperm eggs are fertilized in the water and adhered to the fish nest.
The formula of the fish normal saline is as follows: naCl 15 g, KCl 0.3g, caCl 2 0.6 g, 10 μ L of 0.6M Adenosine Triphosphate (ATP), and 20g of purified water, and adjusting pH of the fish physiological saline to 7.0.
3. Gynogenesis diploidization and hatching
The gynogenesis diploidization of the fertilized eggs of the goldfish is treated by adopting a colchicine combined cold treatment mode. Pouring 1 liter of colchicine aqueous solution with the concentration of 40-6 Oppm into an incubation cylinder, cooling to the temperature of-1-3 ℃, and then soaking fertilized goldfish eggs in the colchicine aqueous solution for 20-30 minutes. After soaking, the eggs are transferred to a conventional hatching pool with the water temperature of 24 ℃ for gynogenesis diploidization and then natural hatching.
4. Screening of target body color character F1-substituted goldfish
The goldfish eggs doubled after gynogenesis are hatched, developed and grown in a hatching pond, and the body color is changed after 50 to 100 days. Because the body color of the female goldfish fry is recessive relative to the gray body color of the male crucian fry, the body color of the hybridized next generation (F1 generation) is gray, only the actual goldfish individual which realizes diploidization of gynogenesis is, and the body color is different from the gray body color of the crucian fry. Therefore, the individuals with the grey color discarded are selected in the hatching pond, and the F1 generation goldfish individuals with the original target body color character and female nucleus development are reserved.
5. F1-substituted goldfish for establishing target body color character
Transferring the F1-substituted goldfish fry with the target body color and female nucleus developed into a seedling raising pool, removing individual fries which present some characters controlled by the expression harmful recessive gene in the growth and development process after hatching and abnormal or damaged fries generated due to poor parent characters in the growth and development process, retaining the goldfish fry which selects the target body color and female nucleus developed healthily, and continuously culturing to 2-4 years to obtain the F1-substituted goldfish individual with the target body color and character.
6. Cultivation of pure line of target body color F2-substituted goldfish
Selecting 2-4-age F1-generation female goldfish with target body color property as parent fish, and adopting crucian with body color of grey for male parent fish. And (3) repeating the steps 1-5 in the technical scheme to obtain the healthy F2-substituted goldfish pure line with the target body color character with high inheritance rate. The F2 generation pure line parent of the goldfish with the target body color character, which is obtained by adopting the technical scheme of the invention, is used for breeding production, the ornamental body color character of the goldfish is stably kept, and a large number of goldfishes with the excellent ornamental body color character are bred to meet the requirements of people.
The luteinizing hormone releasing hormone analogue (LHRH-A2), diosdone (DOM), carp Pituitary Gland (PG), IU Human Chorionic Gonadotropin (HCG), mannitol and colchicine used in the invention are all purchased from the market. The normal-pressure room-temperature plasma breeding instrument is purchased from Qingdao Yijin testing technology Co.
Detailed Description
Example 1
1. Sperm inactivation treatment
The male fish adopts crucian with grey body color, adopts a secondary injection method to induce spawning and discharge sperm, and uses 2 mu g luteinizing hormone releasing hormone analogue (LHRH-A2) for the first time to prepare a needle for injecting the male parent fish; after 16 hours, a second injection of 5mg of the compounded ripener per kg of fish was carried out. Collecting crucian sperm liquid by a conventional method, and diluting by using 50 times of Hank's liquid to obtain sperm diluent.
The formula of the compound ripener is that each 10mg of the compound ripener contains 2mg of Diutanone (DOM), 6mg of carp Pituitary Gland (PG) and 800 international unit IU of Human Chorionic Gonadotropin (HCG).
Repeatedly carrying out sperm inactivation treatment on the sperm diluent in batches by utilizing an Atmospheric Room Temperature Plasma (ARTP) breeding instrument, and combining the sperm diluent to obtain crucian inactivated sperm liquid with the total amount of more than 300 mu L; after treatment, 5. Mu.L of 0.6M mannitol and 10. Mu.L of Hank's solution are added into each 20. Mu.L of the inactivated sperm solution, and the mixture is stored at low temperature of 4 ℃ in a dark place for later use.
The sperm inactivation treatment is carried out under the conditions of 20 mu L of sperm diluent sample loading amount, 125W of treatment output power and 10L min of gas flow rate － 1 The irradiation distance was 2 mm, and the treatment time was 50s.
2. Fertilization of
Selecting 4-year-old female goldfish with golden body color, and injecting carp pituitary gland 3mg per kg body weight of the female goldfish for artificial spawning induction. Pouring 1L of fish physiological saline into a clean 2L ceramic basin, placing a fish nest which is sterilized on the bottom surface of the ceramic basin, then squeezing female goldfish eggs into the fish physiological saline by hands, pouring 200 mu L of low-temperature preserved crucian inactivated sperm liquid around the squeezed goldfish eggs, and slightly stirring the mixture in water by using feathers to ensure that sperm eggs are fertilized in the water and are adhered to the fish nest.
The formula of the fish normal saline is as follows: naCl 15 g, KCl 0.3g, caCl 2 0.6 g, 10 mul of 0.6M Adenosine Triphosphate (ATP), 20g of purified water, and adjusting the pH of the fish physiological saline to 7.0.
3. Gynogenesis diploidization and incubation
Adopting colchicine and cold treatment to treat gynogenesis and diploidization of goldfish oosperm. Pouring 1 liter of colchicine aqueous solution with the concentration of 6Oppm into an incubation cylinder, cooling to the temperature of 3 ℃, and soaking fertilized goldfish eggs in the colchicine aqueous solution for 20 minutes. After soaking, the eggs are transferred to a conventional hatching pond with the water temperature of 24 ℃ for gynogenesis diploidization and then natural hatching is carried out.
4. Screening of target gilding body color character F1-substituted goldfish
The goldfish eggs doubled after gynogenesis are hatched, developed and grown in a hatching pond, and the body color is changed after 80 days. Because the body color of female goldfish fry is recessive character relative to the grey body color of male crucian fry, the body color of the first filial generation (F1 generation) after hybridization is grey, only the goldfish individual which realizes the diploidization of gynogenesis is really has the body color of gold color. Therefore, individuals discarded in a gray color were selected in the hatching pond, and the remaining female-nucleated goldfish individuals with the original objective chromaticness were F1 generation.
5. Establishing F1 generation goldfish with target golden body color character
Transferring the F1 generation goldfish fry with the target golden color female nucleus development into a seedling raising pool, continuously removing the goldfish fry with the non-target color female nucleus development in the growth and development process, namely removing individual fish fries which show some characters of expressing harmful recessive gene control in the growth and development process after hatching, and some individual fish fries which generate abnormal types or damage fish fries due to poor parent characters, keeping the healthy and developed goldfish fry with the target golden color female nucleus development, and continuously culturing to 4 years to obtain the F1 generation goldfish with the target golden color character.
6. Cultivation of pure breed of target golden body color F2-substituted goldfish
Selecting 4-year-old female F1 generation goldfish with golden body color as parent fish, and selecting the male crucian with body color of grey crucian. Repeating the steps 1-5 in the technical scheme to obtain the healthy F2-substituted goldfish pure line with the target golden body color character with high inheritance rate.
The F2 generation pure line parent of the golden fish with the target gilding body color character obtained by the embodiment is used for breeding production, the ornamental gilding body color character of the excellent goldfish is stably kept, and a large amount of goldfishes with the excellent ornamental gilding body color character are bred.
Example 2
1. Sperm inactivation treatment
The male fish adopts crucian with grey body color, adopts a secondary injection method to induce spawning and discharge sperm, and prepares a needle for injecting male parent fish by using 2 microgram of luteinizing hormone releasing hormone analogue (LHRH-A2) for the first time; after 16 hours, the second injection is carried out according to the injection of 3mg of the compound ripener per kg of fish. Collecting crucian sperm liquid by a conventional method, and diluting by using 40 times of Hank's liquid to obtain sperm diluent.
The formula of the compound ripener is that each 10mg of the compound ripener contains 2mg of Diutanone (DOM), 6mg of carp Pituitary Gland (PG) and 800 international unit IU of Human Chorionic Gonadotropin (HCG).
Repeatedly inactivating the sperm diluent in batches by using an atmospheric room temperature plasma breeding instrument (ARTP), and adding 5 muL of 0.6M mannitol and 10 muL of Hank's solution into every 20 muL of the inactivated sperm solution after treatment, and storing at low temperature of-1 ℃ in a dark place for later use. The total amount of the combined deactivated sperm liquid of the crucian carps is more than 300 mu L
The sperm inactivation treatment is carried out under the conditions that the sample loading amount of the sperm diluent is 20 mu L, the treatment output power is 125W, and the gas flow rate is 10 L.min － 1 The irradiation distance was 2 mm, and the treatment time was 30 s.
2. Fertilization of
Selecting red female goldfish of 2-year old in oviposition period, and injecting carp pituitary gland 4mg per kg body weight of the female goldfish for artificial spawning induction. Pouring 1L of fish physiological saline into a clean 2L ceramic basin, placing a fish nest which is sterilized on the bottom surface of the ceramic basin, then squeezing female goldfish eggs into the fish physiological saline by hands, pouring 200 mu L of low-temperature preserved crucian inactivated sperm liquid around the squeezed goldfish eggs, and slightly stirring the mixture in water by using feathers to ensure that sperm eggs are fertilized in the water and are adhered to the fish nest.
The formula of the fish normal saline is as follows: naCl 15 g, KCl 0.3g, caCl 2 0.6 g, 10 μ L of 0.6M Adenosine Triphosphate (ATP), and 20g of purified water, and adjusting pH of the fish physiological saline to 7.0.
3. Gynogenesis diploidization and hatching
Adopting colchicine and cold treatment to treat gynogenesis and diploidization of goldfish oosperm. Pouring 1 liter colchicine aqueous solution with the concentration of 40ppm into an incubation cylinder, cooling to the temperature of minus 1 ℃, and soaking fertilized goldfish eggs in the colchicine aqueous solution for 30 minutes. After soaking, the eggs are transferred to a conventional hatching pool with the water temperature of 24 ℃ for gynogenesis diploidization and then natural hatching.
4. Screening of target red body color character F1-substituted goldfish
The goldfish eggs which are diploidized through gynogenesis are hatched, developed and grown in an incubation pool, and the body color is changed after 60 days. Because the body color of the female goldfish fry is recessive relative to the gray body color of the male crucian fry, the body color of the first filial generation (F1 generation) after hybridization is gray, only the actual goldfish individual which realizes diploidization of gynogenesis is, and the body color is red body color different from the gray body color of the crucian carp. Therefore, individuals with green-gray color discarded are selected from the hatching pond, and the gynogenesis goldfish individuals with the original target red body color character are kept as F1 generation.
5. F1-substituted goldfish with target red body color character
Transferring the F1 generation goldfish with the target red body color gynogenesis into a seedling pool, removing individual fish fries which present the characters of expressing harmful recessive gene control in the growth and development process after hatching, and some abnormal or damaged fish fries caused by poor parent characters in the growth and development process, reserving the goldfish which selects the healthy and developed target red body color gynogenesis, and continuously culturing to 2 years to obtain the F1 generation goldfish with the target red body color character.
6. Cultivation of pure line of target red body color F2-substituted goldfish
Selecting 2-year-old F1-generation female goldfish with target red body color character as parent fish, and selecting crucian with body color of grey. And (3) repeating the steps 1-5 in the technical scheme to obtain the healthy F2-substituted goldfish pure line with the target red body color character with high inheritance rate.
The pure-line parent of the F2 generation goldfish with the target red body color character obtained by the embodiment is used for breeding production, the ornamental red body color character of the excellent goldfish is stably maintained, and a large number of goldfishes with the excellent ornamental red body color character are bred.
Claims (4)
1. A method for artificially inducing the pure line cultivation of the body color character of gynogenesis goldfish is characterized in that:
1) Sperm inactivation treatment
The method comprises the following steps of (1) adopting male crucian carps with the body color of grey, and injecting male crucian carps with a preparation needle of 2 mu g of luteinizing hormone releasing hormone analogue for the first time; after 16 hours, injecting 3-5 mg of the compound ripener for the second time according to the weight of each kg of crucian, collecting sperm liquid of the crucian, and diluting by using 40-50 times of Hank's liquid to obtain sperm diluent;
performing sperm inactivation treatment on the sperm diluent in batches by using a normal-pressure room-temperature plasma breeding instrument, and combining the sperm diluent to obtain more than 300 mu L of crucian inactivated sperm liquid; after the sperm inactivation treatment, adding 5 mu L of 0.6M mannitol and 10 mu L of LHank's solution into every 20 mu L of inactivated sperm solution, and storing at the low temperature of-2-4 ℃ in a dark place for later use;
2) Fertilization of
Pouring 0.5L-1L of fish normal saline into a clean 2L ceramic basin, placing a fish nest on the bottom surface of the ceramic basin, then selecting 2-4-year-old female goldfishes different from the body color of the crucian in the spawning period, wherein the 2-4-year-old female goldfishes different from the body color of the crucian are the target body color of offspring screening, injecting carp pituitary gland 3-4 mg per kg of the weight of the female goldfishes for artificial spawning induction, then completely extruding female goldfish eggs into the fish normal saline by hands, simultaneously pouring 200 mu L of inactivated sperm liquid of the crucian at low temperature around the extruded goldfish eggs, and slightly stirring the sperm liquid in water by feathers to ensure that the sperm eggs are fertilized in the water and adhered to the fish nest;
3) Gynogenesis diploidization and hatching
Pouring 1 liter of colchicine aqueous solution with the concentration of 40-60 ppm into an incubation cylinder, cooling to the temperature of-1-3 ℃, soaking fertilized goldfish eggs in the colchicine aqueous solution at the temperature of-1-3 ℃ for 20-30 minutes, transferring the soaked goldfish eggs to an incubation pool with the water temperature of 24 ℃ for gynogenesis and diploidization, and then naturally incubating;
4) Screening of target body color character F1-substituted goldfish
Selecting an individual from which the grey is discarded in an incubation pool, and reserving an F1-substituted goldfish individual with the original target body color character and female nucleus development;
5) F1-substituted goldfish for establishing target body color character
Transferring the F1-substituted goldfish fry with the target body color and female nucleus development into a seedling raising pool, continuously removing individual fries which show some characters controlled by expression harmful recessive genes in the growth and development process after hatching and deformed or damaged fries caused by poor parent characters in the growth and development process, retaining the goldfish fry with the target body color and female nucleus development, and continuously culturing to 2-4 years to obtain an F1-substituted goldfish individual with the target body color and characters;
6) Cultivation of pure line of target body color F2-substituted goldfish
Selecting the F1 generation female goldfish with the target body color character as a parent fish, adopting the crucian with the body color of the grey, and repeating the steps 1) -5) to obtain the F2 generation pure goldfish line with the target body color character.
2. The method for breeding the pure line of the body color character of the gynogenesis goldfish by artificial induction according to claim 1, wherein the formula of the compound ripener is that each 10mg of the compound ripener contains 2mg of diutanone, 6mg of carp pituitary gland and 800 international units IU of human chorionic gonadotropin.
3. The method according to claim 1, wherein the sperm-inactivating treatment is carried out under conditions of a sperm dilution loading of 20. Mu.L, a treatment output of 125W, and a gas flow rate of 10L/min － 1 The irradiation distance is 2 mm, and the treatment time is 20-60 s.
4. The method for breeding the pure line of the gynogenesis goldfish with the body color character through the artificial induction according to claim 1, wherein the formula of the fish normal saline is as follows: naCl 15 g, KCl 0.3g, caCl 2 0.6g、10 μ L of 0.6M adenosine triphosphate, purified water 20g, pH 7.0.
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| CN109258527A (en) * | 2018-11-30 | 2019-01-25 | 河南省水产科学研究院 | A kind of plasma irradiating breeding method of the Yellow River carp |
| CN109258526A (en) * | 2018-11-30 | 2019-01-25 | 河南省水产科学研究院 | A kind of plasma irradiation breeding method of crucian |
| CN109744170A (en) * | 2019-03-13 | 2019-05-14 | 沈阳华泰渔业有限公司 | A kind of producing method for seed being sheerly the paradise fish of China |
| CN112680447B (en) * | 2021-01-22 | 2023-10-20 | 中国科学院水生生物研究所 | Goldfish gene editing technology and method for creating new variety of goldfish by using same |
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