CN103039386A - Method for inducing gynogenesis of natural tetraploid loach - Google Patents
Method for inducing gynogenesis of natural tetraploid loach Download PDFInfo
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- CN103039386A CN103039386A CN2012105082526A CN201210508252A CN103039386A CN 103039386 A CN103039386 A CN 103039386A CN 2012105082526 A CN2012105082526 A CN 2012105082526A CN 201210508252 A CN201210508252 A CN 201210508252A CN 103039386 A CN103039386 A CN 103039386A
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- loach
- dliploid
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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
- 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
Abstract
The invention discloses a method for inducing gynogenesis of a natural tetraploid loach. The method sequentially includes the following steps that (1) a natural female tetraploid loach and a natural male diploid loach are subjected to spawning induction, eggs of the female tetraploid loach and semen of the male diploid loach are respectively taken, and the obtained semen is subjected to 100-folded dilution by a Kurokura's solution for usage; (2) the semen of the diploid loach is uniformly spread on a clean culture dish which is coated with 1% of bull serum albumin (BSA) with the thickness of 1mm on the bottom surface, the semen of the diploid loach is subjected to ultraviolet irradiation, and the irradiation dose is 50erg/mm2/s*120s; (3) the eggs obtained at the step (1) are subjected to insemination with the semen of the diploid loach subjected to the ultraviolet irradiation at the step (2), the insemination is performed under conditions away from light, and incubation is performed at the state away from light for 15 minutes; and (4) fertilized egg incubation and breeding are performed. The method for inducing the gynogenesis of the natural tetraploid loach has the advantages that the operation method is simple and rapid, and the process of egg chromosome doubling is not required.
Description
Technical field
The present invention relates to the gynogenetic method of a kind of polyploid fish, especially a kind of method of operating is simple, quick, need not the ovum chromosome doubling and process, can be fast, the gynogenetic method of inducing natural Tetraploid Loach, Misgurnus of High-efficient Production gynogenesis diploid loach.
Background technology
Artificial gynogenesis (artificially induced gynogenesis) is the activation of spermatozoa ovum that makes genetic inactivation by physics or chemical method, sperm does not participate in the formation of synkaryon, and ovum only depends on gynogenesis to form the embryo and develops into the technology of normal individual through chromosome doubling.Up to the present, both at home and abroad successively to zebra fish (
Danio rerio), medaka
(Oryzias latipes), sweetfish
(Plecoglossus altivelis), chum salmon
(Oncorhynchus rhodurus), rainbow trout
(Oncorhynchus mykiss), Tilapia mossambica
(Oreochromis niloticus), African catfish
(Clarias gariepinus),Brown lefteye flounder (
Paralichthys olivaceus), Paralichthys lethostigma (
Paralichthys lethostigma), large yellow Crocker (
Pseudosciaena crocea, existing name
Larimichthys crocea), hump back salmon (
Oncorhynchus gorbuscha) etc. carried out gynogenesis and induced.Artificial gynogenesis is the stronger scientific method of a kind of practicality, at aspects such as producing pure lines, chromosome operation, genetic analysis and sex control potential using value is arranged.
At present, artificial induction fish gynogenesis diploid comprises two committed steps: the genetic inactivation of (1) Human Sperm Chromosome; (2) the chromosomal dliploidization of ovum.The former is mainly used to activate egg development; The latter makes fertilized egg recover to have guaranteed individual normal development since the normal diploid ploidy.The existing chromosomal dliploidization of ovum mainly contains three kinds of physics method, chemical method and biological methods, which kind of method no matter, various processing in the multiploid induction process (such as physical operations or medicament residue) to cell in the biochemical effect of specific protein have certain injury, this injury usually produces certain negative effect to growth and the survival of inducer.
Summary of the invention
The present invention is in order to solve the existing above-mentioned technical problem of prior art, provide a kind of method of operating simple, quick, need not the ovum chromosome doubling and process, can be fast, the gynogenetic method of inducing natural tetraploid fish of High-efficient Production gynogenesis diploid loach.
Technical solution of the present invention is: the gynogenetic method of a kind of inducing natural Tetraploid Loach, Misgurnus is characterized in that carrying out as follows successively:
A. the acquisition of essence, ovum
Get the native female Tetraploid Loach, Misgurnus and male dliploid loach is hastened parturition, get respectively the ovum of female Tetraploid Loach, Misgurnus and the seminal fluid of male dliploid loach, 100 times of gained seminal fluid Kurokura ' s solution solution dilutions are stand-by;
B. the genetic inactivation of sperm
Dliploid loach seminal fluid uniform spreading is located at the bottom surface scribbles in the clean culture dish of 1%BSA that thickness is 1mm, with the seminal fluid of ultraviolet ray irradiation dliploid loach, exposure dose is 50erg/mm2/s * 120s;
C. artificial insemination
Get a step gained ovum and inseminate with the sperm of the dliploid loach of shining through the ultraviolet ray of b step, insemination is carried out under the lucifuge condition, and hatches 15min in the lucifuge state;
D. incubating oosperm and cultivation
Gained fertilized egg is collected in the culture dish that fresh water is housed hatches, 23 ± 1 ℃ of water temperatures are changed water twice every day, change water 1/3 at every turn, and another is cultivated vessel and cultivates wait hatching rear immigration, produces the loach gynogenesis diploid fish fry.
The present invention is with the ovum through the natural Tetraploid Loach, Misgurnus of activation of spermatozoa of the dliploid loach of ultraviolet inactivation, then cultivates the gynogenesis diploid loach.Because natural Tetraploid Loach, Misgurnus is to contain the genomic hereditary tetraploid of quadruplet (4n=100) and is autotetraploid, the male and female loach all can produce normal 2n gamete, so need not double to process by ovum, method of operating is simple, quick, the injury of having avoided loaded down with trivial details artificial treatment operation sequence and physics and chemistry to induce means to cause fertilized egg, having improved incubation rate etc., is to realize practicable approach of fish gynogenesis, has huge using value.
Description of drawings
Fig. 1 is the picture of the prepared gynogenesis diploid loach chromosome (2n=50) of the embodiment of the invention.
Fig. 2 is the picture of control group (2n ♀ * 4n ♂) loach chromosome (3n=75).
Embodiment
Carry out as follows successively:
A. the acquisition of essence, ovum
Choose well-developed female natural tetraploid (4n=100) and male dliploid (2n=50) loach is hastened parturition, 24 ± 1 ℃ of water temperatures, after 10 ~ 12 hours effect time, confirm female natural tetraploid ovulation after, gently press belly to adopt ovum; The same light male dliploid loach belly capillary semen collection of pressing, 100 times of gained seminal fluid Kurokura ' s solution solution dilutions are stand-by; The compound method of spermatozoa preservative fluid Kurokura ' s solution solution is: NaCl 750 mg, CaCl
220 mg, NaHCO
320 mg, KCl 20 mg are dissolved in the 100 ml distilled water;
B. the genetic inactivation of sperm
To be located at diameter be 6cm and scribble in the clean culture dish of 1%BSA that thickness is 1mm with dliploid loach seminal fluid uniform spreading, and with the seminal fluid of ultraviolet ray irradiation dliploid loach, exposure dose is 50erg/mm2/s * 120s;
C. artificial insemination
Get the sperm insemination of the natural Tetraploid Loach, Misgurnus of a step the gained mature egg (dliploid) that produces and the dliploid loach of shining through the ultraviolet ray of b step, insemination is carried out under the lucifuge condition, and hatches 15min in the lucifuge state;
D. incubating oosperm and cultivation
Gained fertilized egg is collected in the culture dish that fresh water is housed hatches, 23 ± 1 ℃ of water temperatures are chosen unfertilized ovum or dead ovum, change water every day twice, change water 1/3 at every turn, cultivate wait hatching the larger cultivation vessel of another volume of rear immigration, produce the loach gynogenesis diploid fish fry.
Embodiment of the invention gained loach ploidy is detected:
Detect by the loach chromosome number of somatic of early stage mixing embryo method of tableting to the embodiment of the invention and control group, the difference of control group and the embodiment of the invention is the fertilized egg non-irradiated with ultraviolet radiation, i.e. the heredity of sperm is inactivation not.
The early stage embryo of mixing method of tableting is: get 30 ~ 50 of the embryos that the embodiment of the invention and control group are grown eye born of the same parents' phase, cut open to put into behind egg membrane and the yolk with tweezers and fill 0.0025% colchicine and process 45min, the hypotonic 20min of 0.8% citric acid, use again the Kano fixer (methyl alcohol: glacial acetic acid=3:1) fix 3 times of precooling, each 15 ~ 20min, last-20 ℃ of freeze overnight.Cold sheet, 10% Giemsa staining, microscopy is taken pictures.
Embodiment of the invention photo is as shown in Figure 1: the result shows that androgenesis loach chromosome is dliploid (2n=50).
The control group photo is as shown in Figure 2: the result shows that chromosome is triploid (3n=75).
Claims (1)
1. gynogenetic method of inducing natural Tetraploid Loach, Misgurnus is characterized in that carrying out as follows successively:
A. the acquisition of essence, ovum
Get the native female Tetraploid Loach, Misgurnus and male dliploid loach is hastened parturition, get respectively the ovum of female Tetraploid Loach, Misgurnus and the seminal fluid of male dliploid loach, 100 times of gained seminal fluid Kurokura ' s solution solution dilutions are stand-by;
B. the genetic inactivation of sperm
Dliploid loach seminal fluid uniform spreading is located at the bottom surface scribbles in the clean culture dish of 1%BSA that thickness is 1mm, with the seminal fluid of ultraviolet ray irradiation dliploid loach, exposure dose is 50erg/mm2/s * 120s;
C. artificial insemination
Get a step gained ovum and inseminate with the sperm of the dliploid loach of shining through the ultraviolet ray of b step, insemination is carried out under the lucifuge condition, and hatches 15min in the lucifuge state;
D. incubating oosperm and cultivation
Gained fertilized egg is collected in the culture dish that fresh water is housed hatches, 23 ± 1 ℃ of water temperatures are changed water twice every day, change water 1/3 at every turn, and another is cultivated vessel and cultivates wait hatching rear immigration, produces the loach gynogenesis diploid fish fry.
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| CN2012105082526A CN103039386A (en) | 2012-12-03 | 2012-12-03 | Method for inducing gynogenesis of natural tetraploid loach |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103651195A (en) * | 2013-11-22 | 2014-03-26 | 大连海洋大学 | Cold shock induction method for paramisgurnus dabryanus androgenesis haploid |
| CN103875569A (en) * | 2014-03-24 | 2014-06-25 | 华中农业大学 | Method for obtaining high-proportion male loaches through interspecies cross |
| CN105994077A (en) * | 2016-06-30 | 2016-10-12 | 湖南文理学院 | Efficient breeding method for spiral shell male tetraploid pure line |
| CN106376501A (en) * | 2016-10-24 | 2017-02-08 | 大连海洋大学 | Method for producing loach tetraploid |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN1685806A (en) * | 2005-04-29 | 2005-10-26 | 中国科学院南海海洋研究所 | Induction method of flat porgy gynogenesis diploid |
| CN1709047A (en) * | 2005-06-10 | 2005-12-21 | 湖南师范大学 | Fish gynogenesis method |
| CN102007879A (en) * | 2010-11-16 | 2011-04-13 | 湖南师范大学 | Method for cultivating gynogenetic carps |
-
2012
- 2012-12-03 CN CN2012105082526A patent/CN103039386A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1685806A (en) * | 2005-04-29 | 2005-10-26 | 中国科学院南海海洋研究所 | Induction method of flat porgy gynogenesis diploid |
| CN1709047A (en) * | 2005-06-10 | 2005-12-21 | 湖南师范大学 | Fish gynogenesis method |
| CN102007879A (en) * | 2010-11-16 | 2011-04-13 | 湖南师范大学 | Method for cultivating gynogenetic carps |
Non-Patent Citations (3)
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| 尹洪滨: "应用天然四倍体泥鳅生产多倍体和能生存的雌核鱼", 《水产学杂志》 * |
| 李雅娟: "《水产动物遗传育种学实验指导》", 31 July 2012, 中国农业科学技术出版社 * |
| 饶浩 等: "《无公害泥鳅养殖技术》", 30 November 2009 * |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103651195A (en) * | 2013-11-22 | 2014-03-26 | 大连海洋大学 | Cold shock induction method for paramisgurnus dabryanus androgenesis haploid |
| CN103875569A (en) * | 2014-03-24 | 2014-06-25 | 华中农业大学 | Method for obtaining high-proportion male loaches through interspecies cross |
| CN103875569B (en) * | 2014-03-24 | 2016-01-20 | 华中农业大学 | A kind of method being obtained male loach at high proportion by interspecific cross |
| CN105994077A (en) * | 2016-06-30 | 2016-10-12 | 湖南文理学院 | Efficient breeding method for spiral shell male tetraploid pure line |
| CN106376501A (en) * | 2016-10-24 | 2017-02-08 | 大连海洋大学 | Method for producing loach tetraploid |
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Application publication date: 20130417 |