CN112535123A - Method for artificially inducing female siniperca chuatsi sexual reversion - Google Patents
Method for artificially inducing female siniperca chuatsi sexual reversion Download PDFInfo
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; 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
-
- 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
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- 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
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- Life Sciences & Earth Sciences (AREA)
- Environmental Sciences (AREA)
- Marine Sciences & Fisheries (AREA)
- Zoology (AREA)
- Animal Husbandry (AREA)
- Biodiversity & Conservation Biology (AREA)
- Farming Of Fish And Shellfish (AREA)
Abstract
The invention discloses a method for artificially inducing female siniperca chuatsi sexual reversion, which comprises the following steps: (1) selecting mandarin fish parent; (2) artificial induced spawning of siniperca chuatsi; (3) obtaining siniperca scherzeri heterogenous sperms; (4) genetic inactivation of siniperca scherzeri heterologous spermatozoa; (5) obtaining mature eggs of siniperca chuatsi; (6) artificial gynogenesis of siniperca chuatsi to obtain female individuals; (7) artificial induction and transformation of gynogenesis mandarin fish. The method can be used for inducing the artificial female nucleus development of the siniperca chuatsi to obtain the all-female mandarin fish, then inducing the all-female mandarin fish by the artificial androgen Methyltestosterone (MT) to generate the pseudo-male fish with the XX genotype, wherein the sperms of the pseudo-male fish have the same fertilization capability as the ordinary male fish, and a large number of all-female seedlings can be obtained with the ordinary female fish parent to serve as breeding seeds, so that the breeding yield and the commercial value are greatly improved.
Description
Technical Field
The invention belongs to the field of fish genetic breeding, and particularly relates to a method for artificially inducing female siniperca chuatsi sexual reversion.
Background
The siniperca chuatsi is commonly called mandarin fish, belongs to the order Perciformes and the family Serratidae, has the largest body type and the fastest growth speed in the mandarin fish, and is a rare edible fish native to major rivers and lakes in China. The study subjects "siniperca chuatsi" in this project are all "siniperca chuatsi". According to statistics, the annual output of mandarin fish culture in China reaches over 50 million tons, the direct annual output value is about 300 hundred million yuan, and the method plays a very important role in fresh water famous fish culture. However, 90% of mandarin fish seedling breeding units in China do not pay attention to the selective breeding and seed preservation of mandarin fish parent, the close breeding is serious, the outstanding problems of germplasm degradation, slow growth, poor disease resistance, high aberration rate and the like are exposed during the breeding period, and the extreme lack of high-quality improved varieties greatly restricts the development of the mandarin fish breeding industry.
The application of the artificial gynogenesis technology in fish genetic breeding is very wide, and the artificial gynogenesis technology plays an important role in the aspects of quickly establishing a pure line, improving the growth rate, controlling the sex, improving the quality and the like, and the breeding of all-male tilapia, all-male pelteobagrus fulvidraco and other varieties has epoch-making influence on the industrial development. Research data show that the growth of the mandarin fish has hermaphrodite property, the growth of the mandarin fish in Poyang lake and the gonad development are researched by Lida and the like, the growth speed of the female mandarin fish is considered to be faster than that of the male mandarin fish in all ages, the gonad development shows annual change, the annual change of the female maturation coefficient is 0.79-10.07%, the annual change of the male sex is 0.24-3.83%, and the male sex reaches the maximum in 4-5 months. Under the condition of Wangxiangqing and the like, the growth of female and male mandarinfish is not obviously different when the weight is within 170 g; 170g to 800g, the growth rate of the female fish is obviously faster than that of the male fish; the growth of female fish above 800g is slowed down, and the growth of male fish is faster than that of female fish; the size of the male and female mandarin fish is less than 1kg, while the size of the male and female mandarin fish is more than 1 kg.
The hermaphroditic growth of the fish promotes the unisexual culture to become one of effective breeding methods, and the unisexual culture can greatly improve the culture yield and the commercial value. Therefore, the method can be used for cultivating the all-female unisexual variety and has direct production and application values. Researches show that the hereditary decision type of siniperca chuatsi is XX/XY type, the artificial gynogenesis of siniperca chuatsi is induced to obtain female offspring for cultivating all-female variety, the offspring female fish (XX type) is induced by artificial androgen (MT) to generate 'pseudo male fish' with XX genotype, and the sperm of the pseudo male fish has the same fertilization capability as that of common male fish, so that the all-female seedling can be normally bred in large scale as cultivation seedling.
Disclosure of Invention
The invention aims to solve the problems of germplasm degeneration, slow growth, poor disease resistance, high aberration rate and the like in the mandarin fish seedling breeding and breeding period in the prior art, and provides a method for artificially inducing the sex reversal of female siniperca chuatsi.
The invention realizes the aim through the following technical scheme, and provides a method for artificially inducing female siniperca chuatsi sexual reversion, which comprises the following steps:
s1: and (3) breeding of siniperca chuatsi parents: the siniperca chuatsi female fish is derived from female fish bred by 4 generations from a Yangtze river wild population, and the siniperca scherzeri male fish is derived from siniperca chuatsi from Jiangxi Cudrania tricuspidata forest lake wild;
s2: artificial induced spawning of siniperca chuatsi: injecting by one needle, wherein the injection dosage is 4-6 mug/kg of luteinizing hormone releasing hormone analogue and 800IU/kg-1200IU/kg of chorionic gonadotropin, diluting by 0.7% physiological saline, the injection dosage of parent fish is 0.5-1.0 mL/kg, and the dosage of male fish is halved compared with that of female fish;
s3: obtaining a siniperca scherzeri heterogenous sperm: after injecting sex oxytocin into mature siniperca scherzeri male fish for 12 hours, obtaining semen by adopting a manual semen squeezing mode;
s4: siniperca scherzeriGenetic inactivation of source sperm: extruding the siniperca scherzeri semen according to the proportion of 1: 4 is diluted by Hanks liquid and then put into a culture dish with the diameter of 10cm, the mixture is evenly shaken on an ice box and then put on a horizontal shaking table together with the ice box, the mixture and the shaking table are put into an artificial induction gynogenesis device, two 15W UV irradiation tubes are used for full irradiation for 25 minutes, and the irradiation intensity is 80mJ/cm2The irradiation distance is 17cm, after irradiation, the inactivated semen is siphoned into a plurality of Ep tubes with the volume of 5mL, and is stored at the low temperature of 0-4 ℃, and the storage life is 24 h;
s5: obtaining mature eggs of siniperca chuatsi: after the female fish estruses, manually extruding the ovum into a dry vessel;
s6: fertilization and incubation of artificial gynogenesis mandarin fish: shaking up genetically inactivated siniperca scherzeri heterologous sperms and mature mandarin fish eggs, mixing and fertilizing for 30s, transferring into fresh water for cold shock treatment after fertilization is completed, controlling the water temperature to be 4 ℃ and the cold shock induction treatment time to be 20-25min to obtain diploid eggs, transferring into a square hatching tank for hatching, controlling the water temperature to be 25 ℃ according to a common mandarin fish seedling cultivation method to obtain mandarin fish gynogenesis diploid fish seedlings with tilted mouth, and cultivating the fish seedlings to 2.5cm after seedling emergence for transformation;
s7: artificially inducing gynogenesis mandarin fish transformation, which mainly comprises the following steps:
(1) preparation of 17 α -Methyltestosterone (MT) solution: calculating the total dosage of the required MT, weighing the MT in a brown glass bottle, adding analytically pure alcohol for dissolution, and adding a mother solution of (i): 100mg/L, fully oscillating and dissolving, and then storing in a refrigerator at 4 ℃;
(2) soaking the fairy shrimp with methyltestosterone solution (2LH 2O: 20mL mother solution (first)) for 0.5 h;
(3) fishing out bait dace starving for 24h to 50L of water according to 5% of the weight of the artificial gynogenesis mandarin fish, and feeding the dace containing MT fairy shrimp in the step (2) for 0.5 h;
(4) feeding the Siniperca micrantha kurz fed with the fairy shrimp in the net cage for 2 times each time, wherein the sex cycle of artificial gynogenesis mandarin fish is 30 days;
(5)1 piece of 5m3The net cage can transform 300 fish to breed the fishKilling fish in 5 months, taking out essence, fertilizing and incubating with mandarin fish ovum to obtain full-female fry.
In the above method for artificially inducing sex reversal of a female siniperca chuatsi, the formula of Hanks diluent in step S3 is as follows: KCL 0.4g, NaCL 8.00g, NaHCO30.35g, KH2PO40.06g, Na2HPO4 & 7H2O 0.09.09 g, Na2HPO4 & 12H2O 0.10.10 g, MgSO4 & 7H2O 0.10.10 g, MgCL2 & 6H2O 0.10g, CaCl20.14g and Glucose 1.00 g.
In the above method for artificially inducing sex reversal of female siniperca chuatsi, the incubation method of fairy shrimp used in step S7 includes:
(1) unfreezing: before hatching, refrigerating and thawing for 24 hours, and soaking for 2 hours in fresh water;
(2) hatching density: adding 2g of fairy shrimp eggs into each liter of water, wherein the weight of the fairy shrimp when the fairy shrimp are hatched every time is 20 g;
(3) hatching salinity: the most suitable incubation salinity is 15 per mill;
(4) incubation temperature: controlling the temperature to be 26-28 ℃, and keeping the temperature constant;
(5) illumination: the illumination above 2000lux is kept for 24 hours;
(6) pH value: controlling pH at 8.2-8.3, and adding 0.2-0.5 g sodium bicarbonate per liter water if pH is low;
(7) air inflation, namely continuously inflating air without dead angles in the hatching process;
(8) hatching conditions: keeping the requirement of the incubation water body, and after incubation for 30-36 hours, turning off the heating equipment, ventilation and illumination;
(9) collecting: standing for 10-20 minutes, sinking the fairy shrimp larva to the bottom of an incubation barrel, and collecting the larva from the bottom.
In the step S7, the size of the net cage for artificially inducing female siniperca chuatsi sexual reversion is 5 mx 1m, 40 meshes, 3 inflatable stones are uniformly placed in each net cage to ensure sufficient oxygen supply, and the net cage is suspended in a cement pond to keep micro-flowing water and is cleaned once every 2 days.
Generally, compared with the prior art, the technical scheme of the invention can achieve the following beneficial effects: the invention relates to a method for artificially inducing sex reversal of female siniperca chuatsi, which is characterized in that the siniperca chuatsi is subjected to artificial gynogenesis induction, the obtained gynogenesis mandarin fish (all-female fish) is induced by artificial androgen to generate 'pseudo-male fish' with XX genotype, and the sperms of the pseudo-male fish have the same fertilization capability of common male fish, so that the all-female seedlings can be normally bred in large scale to serve as breeding seeds, the breeding yield is greatly improved, the breeding period is shortened, the breeding risk is reduced, and huge commercial value is generated.
Drawings
The following detailed description of embodiments of the invention is provided in conjunction with the appended drawings, in which:
FIG. 1 is a diagram of a real object of a pseudo-male fish according to the present invention;
FIG. 2 is a histological section of gynogenesis mandarin fish oogonial cells according to the invention;
FIG. 3 is a histological section of spermatogonium of a "pseudo-male fish" according to the present invention;
FIG. 4 is a histological section of gynogenesis mandarin fish egg cells according to the invention;
FIG. 5 is a histological section of "pseudo-male" sperm cells of the present invention.
Detailed Description
The technical solutions of the present invention will be described clearly and completely with reference to the following detailed description, but those skilled in the art will understand that the following described examples are some, not all, of the examples of the present invention, and are only used for illustrating the present invention, and should not be construed as limiting the scope of the present invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.
A method for artificially inducing female siniperca chuatsi sexual reversion, which comprises the following steps:
s1: and (3) breeding of siniperca chuatsi parents: the siniperca chuatsi female fish is derived from female fish bred by 4 generations from a Yangtze river wild population, and the siniperca scherzeri male fish is derived from siniperca chuatsi from Jiangxi Cudrania tricuspidata forest lake wild;
s2: artificial induced spawning of siniperca chuatsi: injecting by one needle, wherein the injection dosage is 4-6 mug/kg of luteinizing hormone releasing hormone analogue and 800IU/kg-1200IU/kg of chorionic gonadotropin, diluting by 0.7% physiological saline, the injection dosage of parent fish is 0.5-1.0 mL/kg, and the dosage of male fish is halved compared with that of female fish;
s3: obtaining a siniperca scherzeri heterogenous sperm: after injecting sex oxytocin into mature siniperca scherzeri male fish for 12 hours, obtaining semen by adopting a manual semen squeezing mode;
s4: genetic inactivation of siniperca scherzeri heterologous spermatozoa: extruding the siniperca scherzeri semen according to the proportion of 1: 4 is diluted by Hanks liquid and then put into a culture dish with the diameter of 10cm, the mixture is evenly shaken on an ice box and then put on a horizontal shaking table together with the ice box, the mixture and the shaking table are put into an artificial induction gynogenesis device, two 15W UV irradiation tubes are used for full irradiation for 25 minutes, and the irradiation intensity is 80mJ/cm2The irradiation distance is 17cm, after irradiation, the inactivated semen is siphoned into a plurality of Ep tubes with the volume of 5mL, and is stored at the low temperature of 0-4 ℃, and the storage life is 24 h;
s5: obtaining mature eggs of siniperca chuatsi: after the female fish estruses, manually extruding the ovum into a dry vessel;
s6: fertilization and incubation of artificial gynogenesis mandarin fish: shaking up genetically inactivated siniperca scherzeri heterologous sperms and mature mandarin fish eggs, mixing and fertilizing for 30s, transferring into fresh water for cold shock treatment after fertilization is completed, controlling the water temperature to be 4 ℃ and the cold shock induction treatment time to be 20-25min to obtain diploid eggs, transferring into a square hatching tank for hatching, controlling the water temperature to be 25 ℃ according to a common mandarin fish seedling cultivation method to obtain mandarin fish gynogenesis diploid fish seedlings with tilted mouth, and cultivating the fish seedlings to 2.5cm after seedling emergence for transformation;
s7: inducing artificial gynogenesis mandarin fish transformation, which mainly comprises the following steps:
(1) preparation of 17 α -Methyltestosterone (MT) solution: calculating the total dosage of the required MT, weighing the MT in a brown glass bottle, adding analytically pure alcohol for dissolution, and adding a mother solution of (i): 100mg/L, fully oscillating and dissolving, and then storing in a refrigerator at 4 ℃;
(2) soaking the fairy shrimp with methyltestosterone solution (2LH 2O: 20mL mother solution (first)) for 0.5 h;
(3) fishing out bait dace starving for 24h to 50L of water according to 5% of the weight of the artificial gynogenesis mandarin fish, and feeding the dace containing MT fairy shrimp in the step (2) for 0.5 h;
(4) feeding mandarin fish with artificial gynogenesis in a net cage for 2 times in each time, wherein the mandarin fish is fed with the dace of fairy shrimp in step (3), the sex cycle of artificial gynogenesis mandarin fish is 30 days, and the mandarin fish is a pseudomale fish with artificial gynogenesis mandarin fish sex as shown in figure 1.
(5)1 piece of 5m3300 fish of the net cage convertible pseudo-male fish are cultivated until 5 months in the next year, and the fish is killed, the sperm is taken out, and the fish is fertilized with mandarin fish eggs to be incubated, so that the all-female fry is obtained.
The shape difference between the sex glands of the transformed pseudo male fish and the sex glands of the common male mandarin fish is large, the sex glands of the common male fish are in a bidirectional long-belt shape, the left side and the right side are symmetrical, and the appearance is regular; the gonad of the pseudomale fish is irregular in shape, wherein one side of the pseudomale fish is atrophied in development, and the other side of the pseudomale fish is enlarged and irregular, as shown in fig. 2 to fig. 5, the pseudomale fish is a section diagram of the spermary tissue of the gynogenesis mandarin fish ovary and the pseudomale fish.
The computer aided sperm analysis system (CASA) is utilized to analyze and obtain that the 'pseudo-male fish' has the sperm concentration with the same order of magnitude as the mandarin fish with siniperca chuatsi, the quantity of the moving sperms is basically the same as that of the conventional male mandarin fish, the path movement speed of the sperms of the 'pseudo-male fish' is consistent with that of the conventional male fish, the 'pseudo-male fish' has the same vitality as the conventional male fish, the comparison of the sperm capacity of the 'pseudo-male fish' and the sperm capacity of the sperm in 1mL of the semen of the conventional male fish is shown in the table 1, and the comparison of the sperm movement parameter detection is.
TABLE 1 comparison of sperm capacities in 1mL semen of Mandarin fish and pseudo-male fish
TABLE 2 comparison table of sperm movement parameters of mandarin fish and pseudo-male fish
In table 2:
path Velocity (VAP): the motion speed of the sperm head along the space average track;
linear movement Velocity (VSL): also called forward motion speed, i.e. the speed of the linear movement distance of the sperm head;
track speed (VCL): i.e. the average movement speed of the sperm head along its actual walking curve;
linearity (LIN): also called linearity, is the linear resolution of the sperm motion curve, namely VSL/VCL;
whip frequency (BCF): also known as the wobble frequency, i.e. the frequency at which the sperm head crosses its average path.
It will be understood by those skilled in the art that the foregoing is only a preferred embodiment of the present invention, and is not intended to limit the invention, and that any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the scope of the present invention.
Claims (4)
1. A method for artificially inducing female siniperca chuatsi sexual reversion is characterized by comprising the following steps:
s1: and (3) breeding of siniperca chuatsi parents: the mandarin fish parent is originated from Changjiang river wild population and is bred for 4 generations, and the male mandarin fish parent is originated from Jiangxi Cudrania tricuspidata forest and wild siniperca scherzeri;
s2: artificial induced spawning of siniperca chuatsi: injecting by one needle, wherein the injection dosage is 4-6 mug/kg of luteinizing hormone releasing hormone analogue and 800IU/kg-1200IU/kg of chorionic gonadotropin, diluting by 0.7% of physiological saline, the injection dosage of parent fish is 0.5-1.0 mL/kg, and the dosage of male fish is half of that of female fish;
s3: obtaining a siniperca scherzeri heterogenous sperm: injecting sex hormone into mature male siniperca scherzeri for 12 hours, and manually extruding sperm to obtain semen;
s4: inheritance of siniperca scherzeri heterogenous spermInactivation: extruding the siniperca scherzeri semen according to the proportion of 1: 4 is diluted by Hanks liquid and then put into a culture dish with the diameter of 10cm, the mixture is evenly shaken on an ice box and then put on a horizontal shaking table together with the ice box, the mixture and the shaking table are put into an artificial induction gynogenesis device, two 15W UV irradiation tubes are used for full irradiation for 25 minutes, and the irradiation intensity is 80mJ/cm2The irradiation distance is 17cm, after irradiation, the inactivated semen is siphoned into a plurality of Ep tubes with the volume of 5mL, and is stored at the low temperature of 0-4 ℃, and the storage life is 24 h;
s5: obtaining mature eggs of siniperca chuatsi: after the female fish estruses, manually extruding the ovum into a dry vessel;
s6: fertilization and incubation of artificial gynogenesis mandarin fish: shaking up genetically inactivated siniperca scherzeri heterologous sperms and mature mandarin fish eggs, mixing and fertilizing for 30s, transferring into fresh water for cold shock treatment after fertilization is completed, controlling the water temperature to be 4 ℃ and the cold shock induction treatment time to be 20-25min to obtain diploid eggs, transferring into a square hatching tank for hatching, controlling the water temperature to be 25 ℃ according to a common mandarin fish seedling cultivation method to obtain mandarin fish gynogenesis diploid fish seedlings with tilted mouth, and cultivating the fish seedlings to 2.5cm after seedling emergence for transformation;
s7: inducing artificial gynogenesis mandarin fish transformation, which mainly comprises the following steps:
(1) preparation of 17 α -Methyltestosterone (MT) solution: calculating the total dosage of the required MT, weighing the MT in a brown glass bottle, adding analytically pure alcohol for dissolution, and adding a mother solution of (i): 100mg/L, fully oscillating and dissolving, and then storing in a refrigerator at 4 ℃;
(2) soaking the fairy shrimp with methyltestosterone solution (2LH 2O: 20mL mother solution (first)) for 0.5 h;
(3) fishing out bait dace starving for 24h to 50L of water according to 5% of the weight of the artificial gynogenesis mandarin fish, and feeding the dace containing MT fairy shrimp in the step (2) for 0.5 h;
(4) feeding the Siniperca micrantha kurz fed with the fairy shrimp in the net cage for 2 times each time, wherein the sex cycle of artificial gynogenesis mandarin fish is 30 days;
(5)1 piece of 5m3The net cage can transform 300 fish to breed the fishKilling fish in 5 months, taking out essence, fertilizing and incubating with mandarin fish ovum to obtain full-female fry.
2. The method for artificially inducing female siniperca chuatsi sexual reversal of claim 1, wherein the Hanks diluent of step S3 is formulated as follows: KCL 0.4g, NaCL 8.00g, NaHCO30.35g, KH2PO40.06g, Na2HPO4.7H2O 0.09g, Na2HPO4.12H2O 0.10g, MgSO4.7H2O 0.10g, MgCL2.6H2O 0.10g, CaCL20.14g and Glucore 1.00 g.
3. The method for artificially inducing sex reversal in female siniperca chuatsi according to claim 1, wherein the fairy shrimp hatching method used in step S7 comprises:
(1) unfreezing: before hatching, refrigerating and thawing for 24 hours, and soaking for 2 hours in fresh water;
(2) hatching density: adding 2g of fairy shrimp eggs into each liter of water, wherein the weight of the fairy shrimp when the fairy shrimp are hatched every time is 20 g;
(3) hatching salinity: the most suitable incubation salinity is 15 per mill;
(4) incubation temperature: controlling the temperature at 26-28 ℃ and keeping the temperature constant;
(5) illumination: the illumination above 2000lux is kept for 24 hours;
(6) pH value: controlling pH at 8.2-8.3, and adding 0.2-0.5 g sodium bicarbonate per liter water if pH is low;
(7) air inflation, namely continuously inflating air without dead angles in the hatching process;
(8) hatching conditions: keeping the requirement of the incubation water body, and after incubation for 30-36 hours, turning off the heating equipment, ventilation and illumination;
(9) collecting: standing for 10-20min, depositing the fairy shrimp larva to the bottom of the hatching barrel, and collecting the larva from the bottom.
4. The method of claim 1, wherein the cage used in the step S7 for artificially inducing female siniperca chuatsi sexual reversal has a size of 5m x 1m and 40 meshes, three air-filled stones are uniformly placed in each cage to ensure sufficient oxygen supply, the cages are suspended in a cement pond to keep micro-flowing water, and the cages are cleaned every two days.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115349496A (en) * | 2022-08-19 | 2022-11-18 | 湖南师范大学 | Method for inducing gynogenesis of micropterus salmoides by siniperca chuatsi sperms |
| CN115500286A (en) * | 2022-09-19 | 2022-12-23 | 中山大学 | Method for obtaining pseudo-female siniperca chuatsi through artificial induction |
| CN116784264A (en) * | 2023-08-18 | 2023-09-22 | 深圳精渔科技有限公司 | Induction method of total-female salmon strain |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103070120A (en) * | 2013-01-29 | 2013-05-01 | 水利部中国科学院水工程生态研究所 | Method for establishing full-female pelteobagrus fulvidraco breeding population |
| CN103734037A (en) * | 2013-11-15 | 2014-04-23 | 中山大学 | Inducing method for mandarin fish gynogenesis diploid and application thereof |
| CN106818552A (en) * | 2017-01-11 | 2017-06-13 | 武汉市农业科学技术研究院水产科学研究所 | A kind of method that Heterologous Sperm induces Mandarin fish artificial gynogenesis |
-
2020
- 2020-11-23 CN CN202011318669.7A patent/CN112535123A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103070120A (en) * | 2013-01-29 | 2013-05-01 | 水利部中国科学院水工程生态研究所 | Method for establishing full-female pelteobagrus fulvidraco breeding population |
| CN103734037A (en) * | 2013-11-15 | 2014-04-23 | 中山大学 | Inducing method for mandarin fish gynogenesis diploid and application thereof |
| CN106818552A (en) * | 2017-01-11 | 2017-06-13 | 武汉市农业科学技术研究院水产科学研究所 | A kind of method that Heterologous Sperm induces Mandarin fish artificial gynogenesis |
Non-Patent Citations (6)
| Title |
|---|
| SHUANG LIU 等: "Production of neo-male mandarin fish Siniperca chuatsi by masculinization Production of neo-male mandarin fish Siniperca chuatsi by masculinization with orally administered 17α-methyltestosterone", 《AQUACULTURE》 * |
| 中国水产科学研究院科技情报研究所 主编: "《八十年代国外渔业先进技术与趋势》", 31 December 1988, 海洋出版社 * |
| 朱定贵: "《塘角鱼黄鳝养殖实用新技术》", 30 September 2018, 广西科学技术出版社 * |
| 楼允东 主编: "《鱼类育种学》", 31 August 2001, 中国农业出版社 * |
| 高晓霞: "全雌翘嘴鳜"鼎鳜壹号"面世 长速快20%可错峰上市", 《海洋与渔业》 * |
| 黄权 等: "《淡水养殖技术》", 31 December 2005, 中国农业出版社 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115349496A (en) * | 2022-08-19 | 2022-11-18 | 湖南师范大学 | Method for inducing gynogenesis of micropterus salmoides by siniperca chuatsi sperms |
| CN115349496B (en) * | 2022-08-19 | 2024-01-16 | 湖南师范大学 | Method for inducing gynogenesis of micropterus salmoides by siniperca chuatsi sperms |
| CN115500286A (en) * | 2022-09-19 | 2022-12-23 | 中山大学 | Method for obtaining pseudo-female siniperca chuatsi through artificial induction |
| CN116784264A (en) * | 2023-08-18 | 2023-09-22 | 深圳精渔科技有限公司 | Induction method of total-female salmon strain |
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