CN103404461B - Ussuriensis Artificial gynogenesis method - Google Patents
Ussuriensis Artificial gynogenesis method Download PDFInfo
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- CN103404461B CN103404461B CN201310357534.5A CN201310357534A CN103404461B CN 103404461 B CN103404461 B CN 103404461B CN 201310357534 A CN201310357534 A CN 201310357534A CN 103404461 B CN103404461 B CN 103404461B
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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 discloses ussuriensis Artificial gynogenesis method, comprise the genetic inactivation of sperm, fertilized egg cold shock treatment, incubation step, by adjustment ultraviolet irradiation Distance geometry irradiation time, find out the Best Times of deactivation ussuriensis sperm; Carry out the cold shock of different time length at the start time that after fertilization is different, determine the processing time with most high viability, as the right times length of cold shock, thus filter out the optimum condition being suitable for ussuriensis Artificial gynogenesis.According to the technology path of milter sex reversal → artificial gynogenesis → supermale fish → full milter, the present invention provides technical foundation for cultivation ussuriensis supermale fish and the full milter of unisexuality cultivate, expand cultivation scale, significantly improve output, raiser is significantly increased income, there is wide industrialization breeding prospect.
Description
Technical field
The present invention relates to genetics-breeding in fish technical field, be specifically related to ussuriensis Artificial gynogenesis method.
Background technology
Fish have the various ways such as bisexual reproduction, gynogenesis, hybrid reproduction and produce offspring, continuity population.Gynogenesis (gynogenesis) refers to that ovum must excite the sexual reproduction mode that could produce the individuality only with maternal genetic material through sperm.The feature that the fish of battalion's Natural Gynogenesis are common is: the chromosome number of mature egg does not reduce by half, still identical with somatic chromosome number.After the sperm of both sexes relationship kind enters ovum, only play activation, sperm nucleus is generally in densifie state, protokaryon does not occur, not with female pronucleus, the filial generation produced is similar to female parent, genotype is identical with female parent or be not quite similar, the final female colony forming unisexuality.
Prove that fish gynogenesiss can since artificial induction from Opperman et al, the research of artificial gynogenesis is just very active, be widely used in the research fields such as damaging character, genetic improvement, Sex Differentiation and reproduction control, particularly at sexual control with set up fast and to have unique application in pure lines.The fish that the artificial gynogenesis reported obtains survival offspring have kind more than 30, comprise sturgeon, rainbow trout, carp, crucian carp, grass carp, mandarin fish, Tilapia mossambica etc.But different fish are because of life habit difference, reproduction condition difference, and the gynogenesis technology of a kind of fish can not directly apply to another kind of fish.All need to develop corresponding gynogenesis artificial induction technology and condition for each fish.Artificial gynogenesis by making Human Sperm Chromosome genetic inactivation, this not there is the insemination of hereditary information after, ovum germinates; Double to develop into normal juvenile fish to ovum chromosome again.
Ussuriensis belong to Silurformes (
silurformes) Chang section (
bagridae) plan Chang genus (
pseudobgrus), it is the kind that in Chang section fish, growth rate is the fastest, and maximum individual lengths can reach 1m, gonochorism, abnormal shape, and male is significantly greater than female individuals, adult fish body weight is male be almost female 2 times even more.Its fat content is high, fine and tender taste, and meat flavour is delicious, has very high edibility and economic worth.If the full milter cultivation of unisexuality can be carried out, will greatly improve output and economic benefit.According to the technology path of milter sex reversal → artificial gynogenesis → supermale fish → full milter, Artificial gynogenesis determines an important ring of success or failure by becoming.But the gynogenesis of so far there are no both at home and abroad this fish report.
Summary of the invention
The technical problem to be solved in the present invention is to provide ussuriensis Artificial gynogenesis method, sets up pure lines fast, realizes the complete male unisexuality breeding of ussuriensis, improves output, brings abundant return to raiser.
The present invention is achieved through the following technical solutions: ussuriensis Artificial gynogenesis method, and step comprises:
(1) genetic inactivation of sperm: the seminal fluid getting ussuriensis sexual maturity milter, described seminal fluid and spermatozoa preservative fluid by volume 1:5 dilute, yarn thin,tough silk is set in culture dish with 1.0 ~ 1.8mm thickness uniform spreading after filtering, culture dish is placed in the ultra violet lamp 10 ~ 25min of camera bellows wavelength 2537A, illumination is 66 μ W/cm
2, irradiation distance is 10 ~ 25cm, and shaking speed is 30 ~ 50R/min;
Spermatozoa preservative fluid: containing NaCl 2.5 ~ 4.0g, KCl 1.0 ~ 1.6g, CaCl in every 500ml solution
20.1 ~ 0.2g, MgSO
4.7H
2o 0.1 ~ 0.2g, NaHCO
30.2 ~ 0.4g, preservative 0.1 ~ 0.3g;
(2) fertilized egg cold shock treatment: the sperm got in the ovum of ussuriensis sexual maturity raun and step (1) after genetic inactivation carries out artificial insemination, 5 ~ 10 min after ovum fertilization, transfer to 10 ~ 40min in the cold water of 0 ~ 4 DEG C, make fertilized ovum chromosome diplodization;
(3) hatch: hatched by the hatching apparatus that the fertilized egg after cold shock treatment is placed under normal temperature, keep water to have certain mobility, water oxygen level is sufficient, cultivates fry.
Different fish are because of life habit difference, reproduction condition difference, and the gynogenesis technology of a kind of fish can not directly apply to another kind of fish.All need to develop corresponding gynogenesis artificial induction technology and condition for each fish.Major technique of the present invention is created part and is: by adjustment ultraviolet irradiation Distance geometry irradiation time, find out the Best Times of deactivation ussuriensis sperm; Start times different after insemination carries out the cold shock of different time length, determines the processing time with most high viability, as the right times length of cold shock.Thus filter out the optimum condition being suitable for ussuriensis Artificial gynogenesis, for the cultivation of supermale fish lays the foundation.
The seminal fluid of ussuriensis sexual maturity milter and spermatozoa preservative fluid by volume 1:5 dilute: make sperm can keep the longer time-to-live after in vitro, and in process subsequently, activity is suppressed and can not be dead; When being convenient to follow-up genetic material deactivation, sperm is in a kind of good mobility simultaneously, prevents from irradiating uneven.
The genetic inactivation of sperm adopts Ultraviolet radiation, avoids using chemical substance to produce toxic action to sperm; Sperm still has the ability stimulating egg cell development after genetic material deactivation; Irradiation apparatus is simple.
Cold shock method process: avoid using chemical substance as the injury to ovum during the recovery ploidies such as colchicine, decrease environmental pollution simultaneously; Cold shock method inhibits the discharge of second polar body, makes genetic material recover diploidy, ensures the normal development of embryo; The genetic material system egg cell of filial generation doubles to form, and the gene pure in most site, can set up pure lines fast.
The further improvement project of the present invention is, be provided with shaking table in camera bellows in described step (1), culture vessel is placed on the ice face of shaking table, and shaking speed is 30 ~ 50R/min.It is even that shaking table rotates the ultraviolet radiation that sperm is subject to.
The present invention further improvement project is, in described step (2), artificial insemination adopts semi-dry process artificial insemination.Semi-dry process artificial insemination is ripe technology, is easy to grasp.
The present invention further improvement project is, in described step (2) after artificial insemination, is layed on nylon wire by fertilized egg uniform monolayers.Ussuriensis is viscid egg, is easy to adhere on nylon wire, and fertilized egg is placed on nylon wire and is convenient to follow-up cold shock operation.
The present invention compared with prior art, has following obvious advantage:
The present invention establishes ussuriensis Artificial gynogenesis technology, according to the technology path of milter sex reversal → artificial gynogenesis → supermale fish → full milter, the acquisition of supermale fish is the prerequisite of full milter cultivation, take milter sperm and the female fish egg of milter sex reversal, utilize ussuriensis Artificial gynogenesis technology of the present invention can obtain supermale fish, after supermale piscinity maturation with former be that common raun hybridizes, namely offspring can be entirely male.Ussuriensis male is significantly greater than female individuals, adult fish body weight is male be almost female 2 times even more, its fat content is high, fine and tender taste, and meat flavour is delicious.Current adult fish main exit Japan, Korea S, rare because measuring little valency height domestic market, the present invention provides technical foundation for realizing the full milter cultivation of ussuriensis unisexuality, full milter cultivation expands cultivation scale, significantly improves output, not only domestic market can be opened up, make it to climb up compatriots' dining table, raiser also can be made significantly to increase income, there is wide industrialization breeding prospect.
Accompanying drawing explanation
Fig. 1 is embodiment of the present invention operational flowchart.
Embodiment
Be described in further detail ussuriensis Artificial gynogenesis method of the present invention below in conjunction with accompanying drawing 1, specifically implement according to the following steps:
1. parent population is hastened parturition: select physique healthy and strong, and skin, without the ussuriensis milter of damaged, the full softness of belly and raun, carries out artificial sexual maturity and hastens parturition.
2. gametocide fish gets spermary: by milter dissect after get spermary, interior the scissors of mortar shreds, grinding homogenate, with seminal fluid than spermatozoa preservative fluid by volume 1 ﹕ 5 dilute, yarn thin,tough silk filtration after be set in diameter 90mm culture dish with 1.0 ~ 1.8mm thickness uniform spreading;
Spermatozoa preservative fluid is formulated as follows: containing NaCl 2.5 ~ 4.0g, KCl 1.0 ~ 1.6g, CaCl in every 500ml solution
20.1 ~ 0.2g, MgSO
4.7H
2o 0.1 ~ 0.2g, NaHCO
30.2 ~ 0.4g, preservative 0.1 ~ 0.3g.
3. sperm genetic substance deactivation: utilize the destruction of ultraviolet to DNA to make sperm deactivation.In camera bellows, install the uviol lamp of 2 18 watts of wavelength 2537, illumination is 66 μ W/cm2.Put by culture dish on the ice face of the shaking table in camera bellows, shaking speed 30 ~ 50R/min, irradiation distance 10 ~ 25cm, irradiation time is respectively 10 ~ 25minn, slowly moves about for deactivation standard with sperm after deactivation.
4. squeeze and get ovum: at the end of sperm deactivation is fast, gets the good raun of maturity, squeeze and get ovum in basin, cover with wet cloth and prevent drying.
5. artificial insemination: adopt semi-dry process artificial insemination, fertilized egg uniform monolayers is layed on ready nylon wire.
6. fertilized egg cold shock treatment: by above-mentioned with after nylon wire 5 ~ 10 min of fertilized egg, insert in the cold water of 0 ~ 4 DEG C, cold shock time setting 10 ~ 40min, suppresses the discharge of second polar body to make ovum chromosome doubling recover ploidy, makes fertilized ovum chromosome diplodization.
7. hatch: nylon wire taking-up is placed in the hatching apparatus under normal temperature hatches, and keep water to have certain mobility, water oxygen level is sufficient, cultivates fry.
In concrete 1-11 embodiment, the experimental data of ussuriensis Artificial gynogenesis method and fertilization rate, incubation rate, opening phase survival rate are in table one:
Table one
Claims (4)
1. ussuriensis Artificial gynogenesis method, is characterized in that step comprises:
(1) genetic inactivation of sperm: the seminal fluid getting ussuriensis sexual maturity milter, described seminal fluid and spermatozoa preservative fluid by volume 1:5 dilute, yarn thin,tough silk is located in culture dish with 1.0 ~ 1.8mm thickness uniform spreading after filtering, culture dish is placed in the ultra violet lamp 20min of camera bellows wavelength 2537, illumination is 66 μ W/cm
2, irradiation distance is 15cm;
Spermatozoa preservative fluid: containing NaCl 2.5 ~ 4.0g, KCl 1.0 ~ 1.6g, CaCl in every 500ml solution
20.1 ~ 0.2g, MgSO
4.7H
2o 0.1 ~ 0.2g, NaHCO
30.2 ~ 0.4g, preservative 0.1 ~ 0.3g;
(2) fertilized egg cold shock treatment: the sperm got in the ovum of ussuriensis sexual maturity raun and step (1) after genetic inactivation carries out artificial insemination, and 8 min after ovum fertilization, transfer to 30min in the cold water of 0 ~ 4 DEG C, make fertilized ovum chromosome diplodization;
(3) hatch: hatched by the hatching apparatus that the fertilized egg after cold shock treatment is placed under normal temperature, keep water to have certain mobility, water oxygen level is sufficient, cultivates fry.
2. ussuriensis Artificial gynogenesis method according to claim 1, is characterized in that: be provided with shaking table in camera bellows in described step (1), culture vessel is placed on the ice face of shaking table, and shaking speed is 30 ~ 50R/min.
3. ussuriensis Artificial gynogenesis method according to claim 1, is characterized in that: in described step (2), artificial insemination adopts semi-dry process artificial insemination.
4. ussuriensis Artificial gynogenesis method according to claim 1, is characterized in that: in described step (2) after artificial insemination, be layed on nylon wire by fertilized egg uniform monolayers.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN106614116A (en) * | 2016-09-30 | 2017-05-10 | 中国长江三峡集团公司中华鲟研究所 | Method for inducing gynogenesis of Chinese sturgeons through homologous sperms |
Families Citing this family (5)
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| CN107996468A (en) * | 2017-12-20 | 2018-05-08 | 淮阴师范学院 | A kind of ussuriensis Genetic male reverses physiology female fish to educate screen method |
| CN109452263A (en) * | 2018-11-30 | 2019-03-12 | 河南省水产科学研究院 | A kind of quasi- Chang spermatozoa preservative fluid and preparation method thereof |
| CN110250052B (en) * | 2019-07-04 | 2022-04-26 | 淮阴师范学院 | Breeding method for pseudobagrus ussuriensis hypoxia-resistant family |
| CN114058713B (en) * | 2021-11-30 | 2023-09-01 | 淮阴师范学院 | Method for obtaining Y chromosome sequence of Pseudobagrus ussuriensis |
| CN115024251A (en) * | 2022-07-04 | 2022-09-09 | 淮阴师范学院 | Rapid acquisition method for full-male Pseudobagrus ussuriensis |
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| CN106614116B (en) * | 2016-09-30 | 2020-04-14 | 中国长江三峡集团公司中华鲟研究所 | Method for inducing gynogenesis of Acipenser sinensis by homologous sperms |
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