CN109769722B - Method for rapidly producing all-female loaches in large scale without sex hormones - Google Patents
Method for rapidly producing all-female loaches in large scale without sex hormones Download PDFInfo
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Abstract
The invention discloses a method for quickly producing gynoecial loaches on a large scale without sex hormones, which comprises the following steps: (1) artificial propagation and incubation: selecting female loach and male loach with gonads, wherein the parent body surfaces have no trauma, no parasite attachment and no appearance deformity, performing anesthesia before artificial oxytocic, performing artificial insemination after injecting an oxytocic, naturally adhering fertilized eggs on a mesh, and transferring the mesh into a plastic box for artificial incubation; (2) high temperature conductivity reversion: gradually raising the temperature of the culture water by using a heating rod at a speed of one degree per day, continuously treating at high temperature, and then cooling for culture; (3) testing cross and group expanding: breeding the rest male fishes to sexual maturity, dividing the offspring generated by each group of male and female fishes into two parts for independent breeding, rejecting female individuals in the group, and breeding the rest XX type male fishes to sexual maturity; (4) and (5) producing the all-female loaches. The method is easy to implement and simple and convenient to operate, and can be used for quickly producing the all-female loaches with high growth speed without using sex steroid hormones in the whole process.
Description
Technical Field
The invention belongs to the technical field of fish parthenogenesis production or artificial fish sex control, and particularly relates to a method for quickly producing all-female loaches on a large scale without sex hormones.
Background
Loaches (Misgurnus anguillicaudatus) have 39 million tons of yield in the whole country in 2017, the yield is increased by about 6 percent compared with 2016, the loaches are one of breeding varieties with the fastest yield increase of freshwater fishes, and the national yield of the freshwater fishes in 2017 ranks 11 th (2018, China fishery statistics yearbook). The loach yield in China accounts for 99.7% of the worldwide yield, the total world value of 2016 is $ 9.43 billion, and the loach yield value of 2016 ranks 34 th (Fischeries and Aquaculture Software, 2018, FAO) in the world is an important economic fish in the world and China.
Under the same culture condition or experimental condition, loach male and female individuals show obvious growth difference, and the female individuals grow 50-150% faster than the male individuals. The cultivation of the all-female loaches can improve the cultivation benefit and reduce the cultivation cost.
Sex hormones are inevitably used in the process of producing the parthenocarpic fish population on a large scale, and the sex hormones are not only a three-line mating method but also a method for combining and reversing gynogenesis or androgenesis. At present, there are two methods for producing the all-female loaches, one is gynogenesis binding reversion (a method for producing the all-female loaches in a large scale, the patent application number is 201610668830.0), and the other is a method for producing the all-female loaches, wherein the filial generation of the XX type male loaches is reversed once again after hormone induction reversion, the patent application number is 201310546649.9. In both methods, the artificial synthesis of androgen methyltestosterone is used in the production process, the former method needs to use artemia larva soaked by 50mg/kg methyltestosterone as bait to continuously feed loach larva for 30 days, and the latter method uses artemia soaked by 200-400 IU methyltestosterone solution to continuously feed fries which are opened for 4 days for 40-60 days.
Although individuals with sex steroid hormone induced reversion are only used as parents and cannot enter a consumption market, the use of sex hormones (whether direct fish fry soaking, live bait for fish fry soaking or feed mixing feeding) inevitably causes partial sex hormone residues or metabolites to enter aquaculture water bodies or natural water bodies (ponds, lakes, reservoirs and the like) and has negative effects on aquaculture fishes or natural ecosystems. Therefore, an environment-friendly and eco-friendly method for producing parthenocarpic fish is important.
Disclosure of Invention
The invention aims to provide a method for quickly producing the all-female loaches on a large scale without sex hormones, which is easy and convenient to operate, can quickly produce the all-female loaches with high growth speed, does not use sex steroid hormones in the whole process, and is an eco-friendly method for producing the all-female loaches.
In order to achieve the purpose, the invention adopts the following technical measures:
a method for rapidly producing hologynic loaches in a large scale without sex hormones comprises the following steps:
(1) artificial propagation and incubation: selecting female loach and male loach with good gonad development, requiring no obvious trauma, no parasite attachment and no appearance deformity on the body surface of the parent, performing mild anesthesia by using MS-222 before artificial oxytocic, performing artificial insemination after injecting an oxytocic, naturally adhering fertilized eggs on a mesh, transferring the mesh into a plastic box for artificial incubation, and controlling the incubation temperature to be 23-28 ℃;
(2) high-temperature (31-32 ℃) conductive reversion: when the larval fish grows to about 8 days old, the water temperature of the culture is gradually increased to 31-32 ℃ by using a heating rod (purchased from the market) at a speed of one degree per day, the culture is continuously treated at a high temperature (31-32 ℃ and the same below) for about 30 days, then the temperature is gradually reduced to 23-28 ℃ for culture until about 4 months old, and female individuals in the larval fish are removed through pectoral fins. The remaining male individuals were bred to sexual maturity. Wherein 70-90% of XX genetic female fish can be reversibly transformed into XX physiological male fish.
(3) Testing cross and group expanding: breeding the rest male fishes (including XX physiological male fishes and XY male fishes) to sexual maturity, pairing the rest male fishes and normal XX female fishes one to one according to the method in the step 1) of the invention, dividing the filial generation generated by each group of male and female fishes into two parts to be bred independently, wherein one part is bred normally to distinguish male and female fishes, the other part is bred to distinguish male and female fishes according to the method in the step 2) of the invention to lead most XX genetic female fishes to be XX physiological male fishes at high temperature, keeping a male parent (namely XX physiological male fishes) with 100% of filial generation female rate under normal breeding condition and the filial generation thereof to be changed into a male fish group by the method in the step 2) of the invention, rejecting a small amount of female individuals in the male fish group, and breeding the rest XX male fishes to sexual maturity;
(4) production of all-female loaches: the XX physiological type male fish obtained in the step 3) of the invention and the normal XX physiological type female fish are mixed according to the male-female ratio of 10: 1, artificial propagation is carried out, or the XX physiological type male fish juvenile fish obtained in the step 3) of the invention is cultured until sexual maturity, and the male fish is mixed with the normal XX physiological type female fish in a male-female ratio of 5: 1, artificial propagation is carried out, and mass production of the all-female loaches is carried out.
The artificial propagation and incubation step in the step 1) is as follows: before artificial induction, 50mg/L MS-222 is used for mild anesthesia, 5ug/kg LHRH-2, 3mg/kg DOM and 1000IU HCG are used for one-time injection, the dosage of male fish is halved, and after the effect time (6-20 hours), 0.6 percent (mass percent, the same below) saline sperm and egg are mixed for wet artificial insemination.
In the step 1), after sperm and eggs are mixed by 0.6 percent (mass percentage) of saline water for wet artificial insemination, the fertilized eggs are naturally adhered to the net piece and are placed in a plastic box for incubation, and the temperature is controllable.
The water temperature of the culture in the step 2) is 31-32 ℃, the high-temperature (31-32 ℃) treatment time is about 30 days, and then the temperature is gradually reduced to the normal temperature for feeding (23-28 ℃).
In the step 3), the filial generation is divided into two parts to be raised, one part is normally raised until male and female parts can be distinguished, and the other part adopts high-temperature (31-32 ℃) induced reversion.
The male-female ratio in the step 4) is 10: 1, fully utilizing the male fish sperm to produce the loaches or expanding the male XX loaches.
Through the technical measures: the invention produces the all-female loach by the most advanced and environment-friendly method in genetics, inducing the sex reversal of the loach through physical stimulation and combining the means of modern genetics. Compared with the existing method for producing the all-female loaches, the method does not use sex steroid hormones in the whole process, and can obtain better environmental benefit, breeding benefit and economic benefit. Meanwhile, the applicant combines the test cross and the population expanding, and compared with the prior art, the production period can be shortened by at least about 3 months.
Compared with the prior art, the invention has the following advantages and effects:
1. in any link of the invention, sex hormone is not used for inducing sexual reversion.
2. Because the gynogenesis or the hormone treatment process is omitted, the potential influence of the damage of the artificial induction gynogenesis process to the DNA of the fertilized eggs or the hormone treatment of the juvenile fish can be reduced, and the health condition of the bred offspring is improved.
3. In the invention, test crossing and population expansion are carried out in the same breeding period, so that the production speed of the all-female loaches can be increased.
4. A breeding system of the all-female loaches can be established in a short time, large-scale production of the all-female loaches is carried out, the loach breeding yield can be improved, and the cost is reduced.
The oxytocin mentioned in the present invention is different from the sex hormones mentioned above for sex reversal.
Drawings
FIG. 1 is a flow chart of a method for producing all-female loaches (populations) without using sex hormones.
FIG. 2 is a technical explanatory diagram of a method for producing a population of all-female loaches without using sex hormones.
In the figure, XX and XY respectively represent hereditary female fish and hereditary male fish, symbols 'male' and 'female' respectively represent physiological female fish and physiological male fish, F0, F1, F2 and F3 respectively represent breeding basic population, first generation, second generation and third generation.
Detailed Description
Example 1:
the present invention will be further described with reference to the following specific examples and accompanying drawings so that those skilled in the art can better understand the invention and practice it, but the examples are not intended to limit the invention.
The present invention will be described in further detail with reference to fig. 1 and 2, and a method for rapidly mass-producing gynoecial loaches without sex hormones, comprising the steps of:
(1) artificial propagation and incubation: selecting female loach and male loach with good gonad development, requiring no obvious trauma, no parasite attachment and no appearance deformity on the body surface of the parents, carrying out mild anesthesia by using 50mg/L MS-222 before artificial oxytocic, carrying out one-time injection by using three oxytocics of 5ug/kg LHRH-2, 3mg/kg DOM and 1000IU HCG, halving the dosage of the male loach till the effect time (6-20 hours), mixing the sperm and the ovum by using 0.6% saline water for wet artificial insemination, naturally adhering the fertilized ovum on a mesh, transferring the fertilized ovum to a plastic box for artificial incubation, and controlling the incubation temperature to be between 23 and 28 ℃;
(2) high temperature induced reversal: when the larval fish grows to 8 days old, the water temperature of the culture is gradually increased to 31 or 32 ℃ by using a heating rod (purchased from the market) at a speed of one degree per day, the high-temperature treatment is continuously carried out for 30 days, siphoning and water changing are carried out every day, the total water changing amount does not exceed 1/4 of the volume of the culture water, then the temperature is gradually reduced to 23 or 26 or 28 ℃, the larval fish is transferred to a cement pond for continuous culture until the larval fish is about 4 months old, and female individuals in the larval fish are removed through pectoral fins. The remaining males are bred to sexual maturity and are subjected to prenatal intensive cultivation before the breeding season. Wherein 70% to 90% of XX hereditary female fish can be reversibly converted into XX physiological male fish.
(3) Testing cross and group expanding: breeding the rest male fishes (including XX physiological male fishes and XY male fishes) to sexual maturity, pairing the rest male fishes with normal XX female fishes one by one according to the method in the step 1), dividing filial generations generated by each group of male and female fishes into two parts to be bred independently, wherein one part is bred normally to be capable of distinguishing males and females, the other part is bred to be most of XX genetic female fishes at high temperature into XX physiological male fishes according to the method in the step 2), keeping male parents (namely the XX physiological male fishes) with 100% of filial generation female rate under normal breeding conditions and the filial generations of the male fishes are subjected to sex reversion to be male fish populations by the method in the step 2), removing a small number of female individuals in the populations, breeding the rest male fishes to sexual maturity, and carrying out prenatal intensive breeding before the breeding season;
(4) production of all-female loaches: comparing the XX physiological type male fish obtained in the step 3) with the normal XX physiological type female fish in a male-female ratio of 10: 1, artificial propagation is carried out, or the XX physiological type male fish juvenile fish obtained in the step 3) is cultured until sexual maturity, and the male fish is mixed with the normal XX physiological type male fish in a male-female ratio of 5: 1, artificial propagation is carried out, and mass production of the all-female loaches is carried out.
Thus, a process for rapidly producing the all-female loaches in a large scale without using sex steroid hormones and a breeding system for continuously producing the all-female loaches are formed.
The invention produces the all-female loach by the most advanced and environment-friendly method in genetics, inducing the sex reversal of the loach through physical stimulation and combining the means of modern genetics. Sex steroid hormone is not used in the whole process, so that the damage of artificially induced gynogenesis to fertilized egg DNA or potential damage to offspring caused by hormone treatment can be avoided, and germplasm degeneration possibly caused by the damage can be avoided. Meanwhile, the applicant combines the test cross and the population expanding, and compared with the prior art, the production period can be shortened by at least about 3 months.
Table 1 shows the comparison of the current all-female loach production method and the key technology in the method of the present invention
The above-mentioned embodiments are merely preferred embodiments for fully illustrating the present invention, and the scope of the present invention is not limited thereto. The equivalent substitution or change made by the technical personnel in the technical field on the basis of the invention is all within the protection scope of the invention. The protection scope of the invention is subject to the claims.
Claims (2)
1. A method for rapidly producing hologynic loaches in a large scale without sex hormones comprises the following steps:
(1) artificial propagation and incubation: selecting female loach and male loach with good gonad development, wherein the parent body surfaces have no trauma, no parasite attachment and no appearance deformity, anesthetizing by 50mg/L MS-222 before artificial oxytocic, injecting by 5ug/kg LHRH-2, 3mg/kg DOM and 1000IU HCG for one time, halving the male fish dosage, mixing with 0.6 mass percent saline solution eggs after 6-20 hours of effect, carrying out wet artificial insemination, naturally adhering fertilized eggs on a mesh, transferring into a plastic box for artificial incubation, and controlling the incubation temperature to be 23-28 ℃;
(2) high temperature conductivity reversion: after the larval fish grow to 8 days old, gradually raising the culture water temperature to 31-32 ℃ at a speed of one degree per day by using a heating rod, continuously performing high-temperature treatment for 30 days, then gradually cooling to 23-28 ℃ for culture until the larval fish is 4 months old, removing female individuals through pectoral fins, culturing the remaining male individuals until sexual maturity, wherein 70-90% of XX genetic female fishes are reversely converted into XX physiological male fishes;
(3) testing cross and group expanding: breeding the rest male fishes to sexual maturity, pairing the rest male fishes with the normal XX female fishes one to one according to the method in the step (1), dividing the filial generation of each group of male and female fishes into two parts for independent breeding, wherein one part is bred normally to distinguish the male and female fishes, the other part leads most of the XX genetic female fishes to be the XX physiological male fishes according to the method in the step (2) at high temperature, keeping the male parents with 100% of the female rate of the filial generations under the normal breeding condition and the filial generations thereof to be the male fish population through the method in the step (2), removing the female individuals in the population, and breeding the rest XX type male fishes to sexual maturity;
(4) production of all-female loaches: and (3) comparing the XX physiological type male fish obtained in the step (3) with the normal XX physiological type female fish in a male-female ratio of 10: 1, artificial propagation is carried out, or the XX physiological type male fish juvenile fish obtained in the step (3) is cultured until sexual maturity, and the male fish is bred to the normal XX physiological type male fish juvenile fish in a male-female ratio of 5: 1, carrying out artificial propagation and producing the all-female loaches.
2. The method for rapidly mass-producing the all-female loach without using sex hormones as claimed in claim 1, wherein: in the step (4), the male-female ratio reaches 10: 1, producing loaches or expanding the male loaches XX by using male fish semen.
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| CN101120658A (en) * | 2006-08-08 | 2008-02-13 | 白宝海 | All female method for salmon and bulltrout |
| CN103548740A (en) * | 2013-11-07 | 2014-02-05 | 武汉百瑞生物技术有限公司 | Method for producing gynoecial loaches |
| CN106259089A (en) * | 2016-08-15 | 2017-01-04 | 安徽省农业科学院水产研究所 | A kind of method of large-scale production gynoecial loaches |
| KR20190001381A (en) * | 2017-06-27 | 2019-01-04 | 순천향대학교 산학협력단 | A method inducting triploid of albino Misgurnus anguillicaudatus |
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Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101120658A (en) * | 2006-08-08 | 2008-02-13 | 白宝海 | All female method for salmon and bulltrout |
| CN103548740A (en) * | 2013-11-07 | 2014-02-05 | 武汉百瑞生物技术有限公司 | Method for producing gynoecial loaches |
| CN106259089A (en) * | 2016-08-15 | 2017-01-04 | 安徽省农业科学院水产研究所 | A kind of method of large-scale production gynoecial loaches |
| KR20190001381A (en) * | 2017-06-27 | 2019-01-04 | 순천향대학교 산학협력단 | A method inducting triploid of albino Misgurnus anguillicaudatus |
Non-Patent Citations (2)
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| 温度对泥鳅和大鳞副泥鳅性腺分化的影响和CYP19a 基因的克隆与时空表达;南平等;《中国水产科学》;20050731;第12卷(第4期);第407-413页 * |
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