CN101669449A - Gynogenesis method of grass carp and application thereof - Google Patents
Gynogenesis method of grass carp and application thereof Download PDFInfo
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Abstract
The invention discloses a gynogenesis method of grass carp and application thereof. The method comprises the following steps: taking the diploid sperms of a properly inactivated allotetraploid cruciancarp as a stimulus to stimulate mature eggs of the grass carp, after activating for 2-3 min or fertilizing for 20-22 min, carrying out the water-cooling shock treatment for 10-12 min at 4-6 DEG C, and hatching and controlling the temperature by to obtain the gynogenesis grass carp restraining the discharge of a second polar body or the first cleavage. The method greatly improves the hatching rateand the survival rate of the gynogenesis diploid grass carp, simplifies the preparation process, and can prepare a gynogenesis grass carp colony with good characters and stable heredity.
Description
Technical field
The present invention relates to cultivation and cultural method and the application thereof of a kind of fish, relate in particular to breeding method and the application thereof of a kind of grass carp.
Background technology
Grass carp (Ctenopharyngodon idellus) is one of important economic freshwater cultured fishes of China, because communicable diseases such as its easy infection hemorrhage, enteritis, gill rot, red skin cause the fingerling survival rate not high.In recent years, many scholars are at this problem of grass carp resistance against diseases difference, by traditional gynogenesis method, adopt the activation grass carp ovums such as carp sperm of deactivation, handle through chromosomal doubling again, have the disease-resistant basis of biology that the grass carp of strong this merit of resistance against diseases has accumulated rich experience aspect individual and tentatively proved the gynogenesis grass carp in screening.But up to the present, because the gynogenesis operating process damages the grass carp ovum easily, and sperm deactivation immoderation, the diplodization of monoploid ovum such as deal with improperly at multiple reason, caused artificial induction's gynogenesis grass carp incubation rate and survival rate not high, abductive approach cost height and process complexity are not set up the report that the gynogenesis grass carp is sheerly colony so far on a large scale.
Summary of the invention
The technical problem to be solved in the present invention is to overcome the deficiencies in the prior art, gynogenesis method of multiple grass carp and the method for using with the grass carp of this gynogenesis method preparation are provided, method of the present invention has not only improved incubation rate and the survival rate of gynogenesis diploid grass carp greatly, and simplified preparation technology, can prepare that proterties is good, the gynogenesis grass carp colony of inheritance stability.
For solving the problems of the technologies described above, the technical scheme that the present invention proposes is the gynogenesis method of a kind of grass carp, this method is to be stimulus with the allotetraploid crucian carp carp diploid sperm after the appropriate deactivation, stimulate the grass carp mature egg, activate back 2～3min and handle 10～12min with 4 ℃～6 ℃ water-cooled shock, through the hatching of flowing water temperature control, prepare and suppress the gynogenesis grass carp that second polar body is discharged then.
In the technique scheme, the original female parent of described allotetraploid crucian carp carp (4n=200) is red crucian carp (Carassius auratusred var.), and original male parent is carp (Cyprinus carpio).Tetraploid crucian carp carp two performances are educated, and can produce diplont ovum and diploid sperm respectively, and both mating have guaranteed that the tetraploid performance is handed down from age to age, and have bred F18 at present, have formed the tetraploid fish new population of stabilization characteristics of genetics.Male tetraploid crucian carp carp produces diploid sperm, and this diploid sperm has been proved to be by the analysis of sox-HMG dna fragmentation and has been the allos sperm, has both contained the hereditary fragment of red crucian carp, contains the hereditary fragment of carp again.We have also carried out long-term sexual hybridization experiment, the result shows: be maternal with the grass carp, allotetraploid crucian carp carp is that the hybrid combination of male parent can not obtain to survive fry, cross hybrid seedling is often in the profile (shown in B figure among Fig. 1) that keeps obviously being different from the grass carp embryo in the growth course, keep features such as spheroidal (normal grass carp seedling yolk sac elongates with embryo development) as embryo's present deformity, idiosome yolk sac in various degree always, grow the phase of ingesting promptly to be eliminated.Therefore, the sperm that produces with allotetraploid crucian carp carp can be used as allos sperm stimulus, induces the grass carp gynogenesis, but can not produce experiment statistics and observation that the survival cross hybrid seedling influences the later stage; In addition, the allotetraploid crucian carp carp activation of spermatozoa after appropriate deactivation but suffer from tangible monoploid syndrome without the gynogenesis grass carp embryo that chromosome doubling is handled can not be grown the phase of ingesting.Therefore, if the fry (seeing A figure among Fig. 1) that grows the phase of ingesting is arranged, can be defined as the gynogenesis diploid grass carp in the test of handling through diplodization.As seen, the technical scheme of the invention described above has improved incubation rate and the survival rate of artificial induction's gynogenesis grass carp greatly, for extensive, high efficiency, prepare gynogenesis grass carp pure lines colony in enormous quantities and opened up new approach.
Based on above-mentioned identical technical conceive, the present invention also provides another kind of technical scheme, it also is the gynogenesis method of a kind of grass carp, this method is to be stimulus with the allotetraploid crucian carp carp diploid sperm after the appropriate deactivation, stimulate the grass carp mature egg, after fertilization 20～22min handles 10～12min with 4 ℃～6 ℃ water-cooled shocks, and then through the hatching of flowing water temperature control, prepares the gynogenesis grass carp that suppresses the spilting of an egg for the first time.Compare with first kind of above-mentioned technical scheme, the technical program mainly is to have done adjustment on the time of being fertilized after the activation, can obtain a kind of gynogenesis grass carp equally.
In each above-mentioned technical scheme, the concrete operations of described deactivation are preferably: the seminal fluid of using earlier Hank ' s liquid dilution allotetraploid crucian carp carp, again by ultraviolet inactivation, the deactivation degree during with microscopy the sperm tail swing weaken and 80%～90% sperm still to have the good vigor of moving about be standard.The diploid sperm that allotetraploid crucian carp carp produces comprises red crucian carp with carp two cover chromosome groups, head is obviously big than the sperm that common diploid carp produces, in the ultraviolet irradiation inactivation process, the deactivation degree of the illuminated sperm of easier judgement under microscopic field, generally can be aforesaid the swing of microscopy sperm tail than have slightly before the deactivation weaken and 80%～90% sperm still to have the good vigor of moving about be standard.As seen, allotetraploid crucian carp carp sperm is a kind of good stimulus that is applicable to the operation of grass carp gynogenesis that had not only possessed the genetic screening mark but also be easy to control the deactivation of sperm appropriateness.What preferably use during described ultraviolet inactivation is the uviol lamp of 15W, and the distance of described uviol lamp and described seminal fluid is preferably 10～12cm, and the irradiation time of described uviol lamp is preferably 20～30min.Carry out deactivation with the mode that above-mentioned ultraviolet irradiation is handled, the time is shorter than carp, easier grasp when the batch process seminal fluid.Activate the grass carp ovum with the allotetraploid crucian carp carp diploid sperm after this processing mode deactivation, do not produce the hybridization fry, " different essence " benefit is more obvious, induces effect very good.
In each above-mentioned technical scheme, the temperature when described flowing water temperature control is hatched is preferably 21 ℃～26 ℃.
The gynogenesis grass carp colony of method for preparing all is female, because stimulus is crucian carp carp hybridization diploid sperm, " different essence " benefit of its generation helps screening high-quality and efficient gynogenesis disease-resistant grass carp.Further improvement as above-mentioned each technical scheme, the gynogenesis grass carp pure lines of gynogenesis grass carp that can prepare with above-mentioned each gynogenesis method and sex reversal are carried out selfing, thereby make the merit heredity stably of described gynogenesis grass carp.
As the further improvement of above-mentioned each technical scheme, the invention allows for more than one state gynogenesis grass carp that each gynogenesis method prepares as maternal, with common grass carp as male parent (
) method of hybridizing.Can obtain fast growth, gynogenesis grass carp Offspring F1 that resistance against diseases is strong by this cross method.
Compared with prior art, advantage of the present invention mainly is: at first, improved incubation rate and the survival rate of gynogenesis diploid grass carp greatly; Secondly, employed sperm is an allotetraploid crucian carp carp diploid sperm in the inventive method, has better " different essence " benefit; Once more, it is short than the carp sperm that allotetraploid crucian carp carp diploid sperm is handled the needed time with ultraviolet irradiation, is convenient to a large amount of sperms are carried out batch process; At last, differentiate that the method for gynogenesis diploid is very simple, distant hybridization body and monoploid can be different from gynogenesis diploid in shape, and all can not survive to the phase of ingesting, and have guaranteed that the survival individuality is gynogenesis diploid.
Appropriateness control sperm deactivation that the present invention proposes and the impaired artificial gynogenesis method of minimizing ovum quality, can significantly improve artificial induction's gynogenesis grass carp production efficiency, solved the parent population bottleneck problem of gynogenesis disease-resistant grass carp in large-scale promotion application, for setting up gynogenesis grass carp pure lines colony on a large scale, also providing important colony's gene pool simultaneously for the research of associated biomolecule scientific principle opinion.Method of the present invention has major application and is worth and broad prospect for its application in the good seed breeding of gynogenesis grass carp, sex controlling and complete female breeding.
Description of drawings
Fig. 1 is gynogenesis grass carp seedling and the contrast of cross hybrid seedling outline drawing; Wherein A is a gynogenesis grass carp seedling outline drawing, and B is the cross hybrid seedling outline drawing;
Fig. 2 is the photo of the gynogenesis grass carp for preparing in the embodiment of the invention; Wherein A figure is the gynogenesis grass carp of the inhibition second polar body discharge at 4 monthly ages, and B figure is the gynogenesis grass carp of the inhibition spilting of an egg for the first time at 4 monthly ages;
The gynogenesis grass carp chromosome map of Fig. 3 for preparing in the embodiment of the invention; Wherein A figure suppresses the gynogenesis grass carp chromosome (2n=48) that second polar body is discharged, and arrow is depicted as minute chromosome (microchromosome) among the figure, and B figure is the gynogenesis grass carp chromosome (2n=48) that suppresses the spilting of an egg for the first time.
Embodiment
Embodiment:
Be described in further detail the gynogenesis method of grass carp of the present invention below in conjunction with accompanying drawing, specifically implement according to the following steps:
1, sperm deactivation: extrude and reach sexually matured allotetraploid crucian carp carp seminal fluid in culture dish, use Hank ' s liquid dilution seminal fluid, the volume ratio of Hank ' s liquid and seminal fluid is 4: 1, the seminal fluid of dilution distributes with thin layer in culture dish, the culture dish of containing seminal fluid is placed on carries out centrifugal treating on the shaking table that is lined with ice bag, rotating speed is 40～50r/min, and place two power to be respectively treatment with irradiation under the uviol lamp of 15W, the distance of uviol lamp and seminal fluid is 10～12cm, irradiation time is 20～30min, sperm tail swing weakens and 80%～90% sperm still has the good vigor that moves about during microscopy, and this moment, the seminal fluid lucifuge that deactivation is good placed 4 ℃ of refrigerators to preserve.
The prescription of Hank ' s liquid is as follows: KCL 0.40g, NaCl 8.00g, NaHCO
30.35g, KH
2PO
40.06g, Na
2HPO
47H
2O 0.09g, Na
2HPO
412H
2O 0.10g, MgSO
47H
2O 0.10g, MgCl
26H
2O 0.10g, CaCl
20.14g and glucose (Glucose) 1.00g; Adding distil water is assigned to 1L.
2, ovum activates: the seminal fluid of handling through ultraviolet irradiation in the step 1 is mixed with the grass carp mature egg, and with clean goose feather with its abundant stirring and evenly mixing, the interior adding of 1min clear water, and begin the calculating activationary time of being fertilized, the ovum activity ratio can reach more than 90%.
3, chromosome doubling is handled: present embodiment adopts the cold shock method inhibition second polar body discharge and the inhibition spilting of an egg for the first time to make and activates ovum chromosome recovery diploidy.By thermograde and processing time comparative trial (see Table 1 and table 2) are set, obtained best PROCESS FOR TREATMENT parameter, that is: activate back 2～3min, handle 10～12min with 4 ℃～6 ℃ water-cooled shocks, discharge to suppress second polar body, the activation ovum is crossed the primitive gut rate and can be reached more than 60%, and gynogenesis grass carp emergence rate can reach more than 40%; Perhaps, after fertilization 20～22min, with 4 ℃～6 ℃ water treatment 10～12min, to suppress the spilting of an egg for the first time, the activation ovum is crossed the primitive gut rate and can be reached more than 50%, and gynogenesis grass carp emergence rate can reach 10%.
Table 1: different cold shock temperature are induced the influence of effect to the grass carp gynogenesis
Table 2: the different cold shock processing times are induced the influence of effect to the grass carp gynogenesis
Table is annotated: go up in table 1 and the table 2, cross ovum number * 100% of primitive gut rate=the mistakes embryo number of gastrul stage/always; The field run plant number of the survival rate=phase of ingesting/total ovum number * 100%.
4, flowing water temperature control hatching: will put into hatching pail through the ovum that gynogenesis is handled and carry out the hatching of flowing water temperature control, water velocity can float in bucket with ovum, at the bottom of neither can sinking to barrel, can not broken through egg membrane for spending by current again.Incubation temperature is controlled in 21 ℃～26 ℃ scopes.
5, hatching and culture: hatch and can descend cement pond behind the sterilization training water to carry out early stage to the fry of fish glue inflation phase to raise, the bimester after go to the Tu Chi raising.
Gynogenesis method by above-mentioned grass carp has successfully obtained gynogenesis grass carp colony 2000 tails (seeing the A figure among Fig. 2) of discharging from the inhibition second polar body of same grass carp female parent and gynogenesis grass carp colony 500 tails (seeing the B figure among Fig. 2) that suppress the spilting of an egg for the first time.With the direct method of tableting of nephrocyte to one age the juvenile fish chromosome number detect.The method of the direct method of tableting of nephrocyte is: after cultivating experiment fish 1～3d under 18 ℃～22 ℃ water temperatures, to experiment fish injection PHA1～3 time, each dosage is 2～8 μ g/ gram body weight, be 12～24h blanking time, at the dissection preceding 2～6h that draws materials, injection colchicine, dosage are 2～4 μ g/ gram body weight.Take out nephridial tissue, under physiological saline, shred nephridial tissue, the hypotonic processing of 0.075mol/L KCl, hypotonic 40～60min under 20 ℃ of temperature; Glacial acetic acid with 1: 3: the mixed liquor of methyl alcohol is nephrocyte 1～3 time fixedly; On freezing slide, drip sheet; The dyeing of Giemsa dye liquor.The result shows: suppressing the gynogenesis grass carp (seeing the A figure among Fig. 3) of second polar body discharge and gynogenesis grass carp (seeing the B figure among Fig. 3) chromosome number of the inhibition spilting of an egg for the first time all is 2n=48.And found 1～3 minute chromosome (shown in the arrow of A figure among Fig. 3) in the division mutually of gynogenesis grass carp chromosome, this minute chromosome may cause " different essence " benefit.It is female that different development stage sexual gland paraffin section result in enormous quantities shows that gynogenesis grass carp that suppresses the second polar body discharge and the gynogenesis grass carp that suppresses the spilting of an egg for the first time are, and is complete female colony.Paraffin section method is: the sexual gland Bouin ' s that got 4～8 tail gynogenesis grass carps in every month at random is liquid-solid fixed, paraffin embedding, and slice thickness is 6 μ m, hematoxylin eosin staining, the neutral gum mounting, the Olympus microscopically is observed, and takes pictures.
Carry out selfing with the gynogenesis grass carp of above-mentioned present embodiment method preparation and the gynogenesis grass carp pure lines of sex reversal, because father and mother's gene pure degree originally is all quite high, be not easy to take place proterties and separate, so the merit that gynogenesis produces can be given the offspring by both selfing genetic stabilities.
As maternal and common male grass carp mating, obtained large batch of with the gynogenesis grass carp of above-mentioned present embodiment method preparation
Hybridization fish offspring F
1, the contrast culture experiment showed in 2 years: support the grass carp kind and do not use under the situation of disease-resistant vaccine F main
1Remove few the infection bacterial gill exception takes place, grass carp bacillary enteritis and the viral hemorrhagic disease big to popular harm do not infect substantially, and growth rate is faster 10%～15% than common grass carp, and that common grass carp disease takes place is serious, and survival rate is low, yields poorly.Different provinces large tracts of land contrast culture experiment statistics sees Table 3.
Table 3: contrast culture experiment data statistics
Therefore, adopt present embodiment method seed selection gynogenesis grass carp, can obviously improve gynogenesis and induce efficient, the gynogenesis grass carp colony that obtains from same grass carp parent in enormous quantities, the gynogenesis grass carp colony of this genetic background unanimity provides valuable research material for several important biomolecule knowledge topic researchs such as relevant Sex determination are machine-processed and evolve.In addition, the gynogenesis grass carp by seed selection of the present invention can be used as high-quality female parent, large-scale production
F
1, this F
1More common grass carp aspect growth rate and the resistance against diseases remarkable advantages is arranged, overcome to a certain extent one, two age grass carp resistance against diseases difference problem, on producing, significantly improved the economic benefit of culturing grass carp.Gynogenesis grass carp resistance against diseases strengthens, not only handle with gynogenesis cause gene pure, to get rid of deleterious gene relevant, also relevant with mixing of allotetraploid crucian carp carp diploid sperm correlated inheritance fragment.
Claims (7)
1, the gynogenesis method of a kind of grass carp, it is characterized in that: this method is to be stimulus with the allotetraploid crucian carp carp diploid sperm after the appropriate deactivation, stimulate the grass carp mature egg, activate back 2～3min, water-cooled shock with 4 ℃～6 ℃ is handled 10～12min, through the hatching of flowing water temperature control, prepare and suppress the gynogenesis grass carp that second polar body is discharged then.
2, the gynogenesis method of a kind of grass carp, it is characterized in that: this method is to be stimulus with the allotetraploid crucian carp carp diploid sperm after the appropriate deactivation, stimulate the grass carp mature egg, after fertilization 20～22min, water-cooled shock with 4 ℃～6 ℃ is handled 10～12min, through the hatching of flowing water temperature control, prepare the gynogenesis grass carp that suppresses the spilting of an egg for the first time then.
3, gynogenesis method according to claim 1 and 2, it is characterized in that: the concrete operations of described deactivation are: the seminal fluid of using earlier Hank ' s liquid dilution allotetraploid crucian carp carp, again by ultraviolet inactivation, the deactivation degree during with microscopy the sperm tail swing weaken and 80%～90% sperm still to have the good vigor of moving about be standard.
4, gynogenesis method according to claim 3 is characterized in that: what use during described ultraviolet inactivation is the uviol lamp of 15W, and the distance of described uviol lamp and described seminal fluid is 10～12cm, and the irradiation time of described uviol lamp is 20～30min.
5, gynogenesis method according to claim 1 and 2 is characterized in that, the temperature when described flowing water temperature control is hatched is 21 ℃～26 ℃.
6, the gynogenesis grass carp pure lines of a kind of gynogenesis grass carp for preparing with each gynogenesis method in the claim 1～5 and sex reversal are carried out selfing, make the proterties of described gynogenesis grass carp carry out the method for genetic stability.
7, a kind of gynogenesis grass carp for preparing with each gynogenesis method in the claim 1～5 is as method maternal, that hybridize as male parent with common grass carp.
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101965814A (en) * | 2010-10-27 | 2011-02-09 | 昆明云锦观赏鱼研究开发有限公司 | Artificial intensive incubation method for cyprinus carpio koi fries |
| CN103190366A (en) * | 2013-04-02 | 2013-07-10 | 湖南湘云生物科技有限公司 | Method for cultivating pure aristichthys nobilis red.var groups |
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| CN107318719A (en) * | 2017-08-16 | 2017-11-07 | 湖南师范大学 | The method of fancy carp sperm induction grass carp gynogenesis and the application of gynogenetic grass carp |
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| CN101965814B (en) * | 2010-10-27 | 2012-07-04 | 昆明云锦观赏鱼研究开发有限公司 | Artificial intensive incubation method for cyprinus carpio koi fries |
| CN103190366A (en) * | 2013-04-02 | 2013-07-10 | 湖南湘云生物科技有限公司 | Method for cultivating pure aristichthys nobilis red.var groups |
| CN103734037A (en) * | 2013-11-15 | 2014-04-23 | 中山大学 | Inducing method for mandarin fish gynogenesis diploid and application thereof |
| CN105746388A (en) * | 2015-01-05 | 2016-07-13 | 湖南海博水产种业科技有限公司 | Double-screening and double-inducing technology system for building disease-resistant grass carp pure line |
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| CN107318719B (en) * | 2017-08-16 | 2020-05-01 | 湖南师范大学 | Method for inducing gynogenesis of grass carp by aid of koi sperms and application of gynogenesis grass carp |
| CN108887207A (en) * | 2018-07-05 | 2018-11-27 | 湘阴县南湖洲镇七姑塘现代农业专业合作社 | A kind of aquaculture method of energy-saving and emission-reducing |
| CN115428752A (en) * | 2022-10-12 | 2022-12-06 | 华中农业大学 | Method for producing all-female grass carp in large scale and application |
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