CN103190366B - Method for cultivating pure aristichthys nobilis red.var groups - Google Patents
Method for cultivating pure aristichthys nobilis red.var groups Download PDFInfo
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- CN103190366B CN103190366B CN201310112737.8A CN201310112737A CN103190366B CN 103190366 B CN103190366 B CN 103190366B CN 201310112737 A CN201310112737 A CN 201310112737A CN 103190366 B CN103190366 B CN 103190366B
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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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Abstract
The invention discloses a method for cultivating pure aristichthys nobilis red.var groups. The method comprises activating roe of aristichthys nobilis red.var with diploid sperms of allotetraploid crucian carps, performing cold shock treatment after activation, and controlling the temperature through flowing water to obtain gynogenesis aristichthys nobilis red.var; feeding feeds containing male hormone after incubation for several days to obtain sex reversal male gynogenesis aristichthys nobilis red.var; and performing selfing with gynogenesis aristichthys nobilis red.var serving as the female parent and with male gynogenesis gynogenesis aristichthys nobilis red.var serving as the male parent to obtain gynogenesis pure aristichthys nobilis red.var groups. Further, the gynogenesis aristichthys nobilis red.var can serve as the female parent, a common male aristichthys nobilis red.var serves as the male parent, and the female parent and the male parent are hybridized to obtain gynogenesis pure aristichthys nobilis red.var groups. According to the method, the roe hatchability and the survival rate can be improved greatly, and the pure aristichthys nobilis red.var groups stable in body color and excellent in performance can be cultivated.
Description
Technical field
The present invention relates to a kind of breeding method of fish colony, particularly relate to the breeding method of a kind of bighead pure lines colony.
Background technology
Xiang Yun bio tech ltd, Hunan has found that body colour sports the individuality of salmon pink in common bighead breeding process, through long-term breeding, obtained a new bighead cultivation strain in 2005---Hunan cloud gold flathead (see Fig. 1) (Aristichthysnobilis red.var).Because possessing, fast growth, high-output stress-resistance, build are beautiful, delicious flavour, fish body colour are the advantages such as salmon pink, in recent years, Hunan cloud gold flathead is subject to the welcome of fishery cultivating family and consumer deeply, carry out rule at present and touch breeding and cultivation, in Sichuan, Chongqing, the tens thousand of mu of the provinces and cities such as Hunan test cultivation, effect reaction is good.
Because Hunan cloud gold flathead is a gene mutation strain, although have passed through long-term directed seed selection, its a lot of character gene is also in heterozygous state, because of inbrde reason, more serious gene decay is found that there is in the breeding, breeding process of Hunan cloud gold flathead, it shows that individual red color is thin out, growth rate is slack-off, resistance reduces, degradation phenomenon (see Fig. 2) under economic characters, this causes adverse effect to the production of Hunan cloud gold flathead and cultivation, therefore necessary by the quality of Biology Breeding technique improvement and stable Hunan cloud gold flathead.
Many scholars utilize artificial induction fish gynogenesis method, have successfully been obtained gynogenesis fishes, have accumulated certain experiences in this fish individual having a merit in screening.But up to the present, gynogenetic report is carried out little about artificial induction bighead, because gynogenesis operating process easily damages fish egg, but also there is sperm deactivation immoderation, the many reasons such as the diplodization of monoploid ovum is dealt with improperly, this causes the incubation rate of Artificial gynogenesis fish and survival rate not high, abductive approach cost is high and process complicated, so far do not set up the report of gynogenesis bighead colony on a large scale, more do not have pertinent literature to propose to adopt artificial induction fish gynogenesis technology to cultivate the relevant report of bighead pure lines colony.
Summary of the invention
The technical problem to be solved in the present invention overcomes the deficiencies in the prior art, provides a kind of and greatly can improve fry hatching rate and survival rate and can cultivate the breeding method of the Hunan cloud gold flathead pure lines colony that body colour is stablized, proterties is excellent.
For solving the problems of the technologies described above, the technical scheme that the present invention proposes is the breeding method of a kind of Hunan cloud gold flathead pure lines colony, comprises the following steps:
(1) Artificial gynogenesis: using the diploid sperm of allotetraploid hybrids as stimulus, this diploid sperm after appropriate deactivation is made to activate the mature egg of Hunan cloud gold flathead, under water temperature is 23 DEG C ~ 26 DEG C conditions, 2min ~ 4min water cold shock treatment 10min ~ 13min of 4 DEG C ~ 6 DEG C after activating, then through the hatching of flowing water temperature control, the gynogenesis Hunan cloud gold flathead suppressing second polar body to be discharged is prepared;
(2) male gynogenesis fishes is obtained: after the gynogenesis Hunan cloud gold flathead obtained after step (1) hatches a couple of days, throw something and feed containing the feed of androgenic hormone, after meeting the requirement in cycle of throwing something and feeding, feed to sexual maturity with the normal diet not containing androgenic hormone, the male gynogenesis Hunan cloud gold flathead of acquired reversion;
(3) selfing: feed to sexual maturity to the gynogenesis Hunan cloud gold flathead obtained in step (1), then using this sexually matured gynogenesis Hunan cloud gold flathead as female parent, the male gynogenesis Hunan cloud gold flathead obtained in step (2) is as male parent, make this female parent and male parent carry out selfing, and then obtain gynogenetic Hunan cloud gold flathead pure lines colony.
As a total technical conceive, the present invention also provides the breeding method of a kind of Hunan cloud gold flathead pure lines colony, comprises the following steps:
(1) Artificial gynogenesis: using the diploid sperm of allotetraploid hybrids as stimulus, this diploid sperm after appropriate deactivation is made to stimulate the mature egg of Hunan cloud gold flathead, under water temperature is 23 DEG C ~ 26 DEG C conditions, 21min ~ 24min water cold shock treatment 10min ~ 13min of 4 DEG C ~ 6 DEG C after activating, then through the hatching of flowing water temperature control, the gynogenesis Hunan cloud gold flathead suppressing First cleavage is prepared;
(2) male gynogenesis fishes is obtained: after the gynogenesis Hunan cloud gold flathead obtained after step (1) hatches a couple of days, throw something and feed containing the feed of androgenic hormone, after meeting the requirement in cycle of throwing something and feeding, feed to sexual maturity with the normal diet not containing androgenic hormone, the male gynogenesis Hunan cloud gold flathead of acquired reversion;
(3) selfing: feed to sexual maturity to the gynogenesis Hunan cloud gold flathead obtained in step (1), then using this sexually matured gynogenesis Hunan cloud gold flathead as female parent, the male gynogenesis Hunan cloud gold flathead obtained in step (2) is as male parent, make this female parent and male parent carry out selfing, and then obtain gynogenetic Hunan cloud gold flathead pure lines colony.
Compare two kinds of above-mentioned technical schemes, its difference is mainly the time upon activation makes an adjustment, and two kinds of gynogenesis modes all can obtain gynogenesis Hunan cloud gold flathead.
The breeding method of above-mentioned Hunan cloud gold flathead pure lines colony, in described step (2), androgenic hormone preferably uses 17 Alpha-Methyl stosterones, and the 17 Alpha-Methyl stosterones preferably containing 40 μ g ~ 60 μ g in every gram of feed.
The breeding method of above-mentioned Hunan cloud gold flathead pure lines colony, in described step (2), the number of days of gynogenesis Hunan cloud gold flathead hatching is preferably 4 ~ 6 days, and the cycle of throwing something and feeding of feed is preferably 80 ~ 100 days, and the number of times of throwing something and feeding of every day is preferably 2 times.
As a total technical conceive, the present invention also provides the breeding method of the third Hunan cloud gold flathead pure lines colony, comprises the following steps:
(1) Artificial gynogenesis: using the diploid sperm of allotetraploid hybrids as stimulus, this diploid sperm after appropriate deactivation is made to activate the mature egg of Hunan cloud gold flathead, under water temperature is 23 DEG C ~ 26 DEG C conditions, 2min ~ 4min water cold shock treatment 10min ~ 13min of 4 DEG C ~ 6 DEG C after activating, then through the hatching of flowing water temperature control, the gynogenesis Hunan cloud gold flathead suppressing second polar body to be discharged is prepared;
(2) hybridize: feed to sexual maturity to the gynogenesis Hunan cloud gold flathead obtained in step (1), then using this sexually matured gynogenesis Hunan cloud gold flathead as female parent, using common male Hunan cloud gold flathead as male parent, this female parent and male parent are hybridized, and then obtains gynogenetic Hunan cloud gold flathead pure lines colony.
As a total technical conceive, the present invention also provides the breeding method of the 4th kind of Hunan cloud gold flathead pure lines colony, comprises the following steps:
(1) Artificial gynogenesis: using the diploid sperm of allotetraploid hybrids as stimulus, this diploid sperm after appropriate deactivation is made to stimulate the mature egg of Hunan cloud gold flathead, 21min ~ 24min water cold shock treatment 10min ~ 13min of 4 DEG C ~ 6 DEG C after activating, then through the hatching of flowing water temperature control, the gynogenesis Hunan cloud gold flathead suppressing First cleavage is prepared;
(2) hybridize: feed to sexual maturity to the gynogenesis Hunan cloud gold flathead obtained in step (1), then using this sexually matured gynogenesis Hunan cloud gold flathead as female parent, using common male Hunan cloud gold flathead as male parent, this female parent and male parent are hybridized, and then obtains gynogenetic Hunan cloud gold flathead pure lines colony.
In four kinds of above-mentioned breeding methods, its (1) step is all cultivate gynogenesis Hunan cloud gold flathead based on Artificial gynogenesis technology, wherein have employed the diploid sperm of allotetraploid hybrids (4n=200) especially as stimulus.The original female parent of allotetraploid hybrids is red crucian carp (Carassius auratus red var.), original male parent is carp (Cyprinus carpio), these allotetraploid hybrids both sexes of selecting can be educated, and female and male difference can produce diplont ovum and diploid sperm, both ensure that being handed down from age to age of this allotetraploid hybrids performance at mating, breed F21 at present, define the tetraploid fish new population of stabilization characteristics of genetics.The diploid sperm that this male allotetraploid hybrids produces is proved to be as Heterologous Sperm by the analysis of sox-HMG DNA fragmentation, and it had both contained the DNA fragmentation of red crucian carp, the DNA fragmentation again containing carp.We are tested by long-term sexual hybridization, its experimental result shows: with female Hunan cloud gold flathead for female parent, with male allotetraploid hybrids for male parent, its hybrid combination can not obtain and survive fry, cross hybrid seedling often keeps the profile being obviously different from Hunan cloud gold flathead embryo in growth course, as embryo present deformity to some extent, idiosome yolk sac keeps the features such as spheroidal always (normal Hunan cloud gold flathead seedling yolk sac elongates with embryo development), grow the phase of ingesting namely to be eliminated.Therefore, can as Heterologous Sperm stimulus using the diploid sperm that allotetraploid hybrids produces, to induce Hunan cloud gold flathead to carry out gynogenesis, but the cross hybrid seedling of survival can not be produced and affect experiment statistics and the observation in later stage.In addition, find after the experiment that we are long-term, allotetraploid hybrids activation of spermatozoa after appropriate deactivation but without the gynogenesis Hunan cloud gold flathead of chromosome doubling process, its embryo suffers from obvious monoploid syndrome, can not grow the phase of ingesting (see Fig. 3).Therefore, if there is the fry grown to the phase of ingesting in the test through diplodization process, can be judged to be it is gynogenetic dliploid Hunan cloud gold bighead seedling (see Fig. 4).Visible, the technical scheme of the invention described above substantially increases incubation rate and the survival rate of Artificial gynogenesis Hunan cloud gold flathead, for high efficiency, prepares gynogenesis Hunan cloud gold flathead pure lines colony in enormous quantities and provides basic prerequisite.
The gynogenesis Xiang Yunjin flathead colony that each breeding method above-mentioned prepares is female, on the basis obtaining gynogenesis Hunan cloud gold flathead, for obtaining the gynogenesis Hunan cloud gold flathead that both sexes can be educated, the gynogenesis Hunan cloud gold flathead that above-mentioned the first of the present invention and the second breeding method start obtaining in step (2) hatches, feed, containing androgenic hormone material (MT) in the feed of feeding, after lasting till the number of days required by feeding cycle, throw something and feed not containing the normal diet of androgenic hormone to sexual maturity, can be succeeded male gynogenesis Hunan cloud gold flathead (see Fig. 5) of sex reversal like this.And then gynogenesis Hunan cloud gold flathead (female) to obtain with above-mentioned gynogenesis method and gynogenesis Hunan cloud gold flathead (male) by sex reversal are carried out selfing and can be obtained Hunan cloud gold flathead and be sheerly colony, thus enable the proterties such as the body colour of gynogenesis Hunan cloud gold flathead stably hereditary.In addition, because the stimulus in step (1) is crucian carp carp hybridization diploid sperm, its " different essence " benefit produced also helps that screening high-quality is efficient, the gynogenesis Hunan cloud gold flathead of strong stress resistance, reverses the strain degradation phenomena that existing Hunan cloud gold flathead causes because of inbreeding.
Improve as another kind, on the basis obtaining gynogenesis Hunan cloud gold flathead, above-mentioned the third and the 4th kind of breeding method then eliminate the incubation step of sex reversal, directly using gynogenesis Hunan cloud gold flathead (female) that above-mentioned steps (1) prepares as maternal, using common Hunan cloud gold flathead (male) as male parent, make this female parent and male parent carry out hybridizing (gynogenesis Hunan cloud gold flathead ♀ × common Hunan cloud gold flathead ♂), can obtain by this cross method the gynogenesis Hunan cloud gold flathead offspring F that body colour is stablized, fast growth, resistance against diseases are strong equally
1.And the operating procedure of this kind of method is more easy, reproductive efficiency is higher.
In the breeding method of above-mentioned various Hunan cloud gold flathead pure lines colony, the concrete operations of described appropriate deactivation are preferably: first use Hank ' s liquid to dilute the seminal fluid of allotetraploid hybrids, again by ultraviolet inactivation, deactivation degree weakens with sperm tail swing during microscopy and the sperm of 80% ~ 90% still has good travelling vigor for standard.The diploid sperm that allotetraploid hybrids produces comprises the two cover chromosome sets of red crucian carp and carp, the sperm that head obviously produces than common diploid carp is large, in UV irradiation inactivation process, under microscopic field, more easily judge the deactivation degree of illuminated sperm, generally can aforesaid microscopy sperm tail swing compared with slightly to weaken before deactivation and the sperm of 80% ~ 90% still has good travelling vigor is standard.Visible, allotetraploid hybrids sperm had a kind ofly not only possessed genetic screening mark, but also is easy to control sperm appropriateness deactivation and is applicable to the excellent stimulus of Hunan cloud gold flathead gynogenesis operation.In the process of appropriate deactivation, preferably use power to be the uviol lamp (two) of 15W during described ultraviolet inactivation, the distance of described uviol lamp and described seminal fluid is preferably 10cm ~ 12cm, and the irradiation time of described uviol lamp is preferably 20min ~ 35min.Carry out deactivation by the mode of above-mentioned Ultraviolet radiation process, the time is shorter compared with carp, more easily grasps when batch process seminal fluid.Activate Hunan cloud gold flathead ovum with the allotetraploid hybrids diploid sperm after this processing mode deactivation, do not produce hybridization fry, " different essence " benefit is more obvious, and inducing effect is very good.
In the breeding method of above-mentioned various Hunan cloud gold flathead pure lines colony, temperature during described flowing water temperature control hatching is 23 DEG C ~ 26 DEG C.
Compared with prior art, the invention has the advantages that:
First, the present invention substantially increases incubation rate and the survival rate of gynogenesis diploid Hunan cloud gold flathead;
The second, the sperm selected in breeding method of the present invention is allotetraploid hybrids diploid sperm, has better " different essence " benefit;
3rd, the time required for the process of allotetraploid hybrids diploid sperm Ultraviolet radiation is short compared with carp sperm, is convenient to carry out batch process to a large amount of sperm;
In addition, differentiate in the present invention that the method for gynogenesis diploid Hunan cloud gold flathead is very simple, distant hybridization body and monoploid can be different from gynogenesis diploid Hunan cloud gold flathead in shape, and all can not survive to the phase of ingesting, and ensure that living individuals is gynogenesis diploid Hunan cloud gold flathead.
It needs to be noted, Artificial gynogenesis technology is the basis of breeding method of the present invention, moderate control sperm deactivation and reduce the impaired artificial gynogenesis method of ovum quality, the production efficiency of Artificial gynogenesis Hunan cloud gold flathead can be significantly improved, but it is applied in the present invention and the more important thing is to solve the inbreeding problem of gynogenesis Hunan cloud gold flathead in large-scale promotion application, solve the gene decline of Hunan cloud gold flathead, deterioration of variety problem, to ensure Hunan this excellent strain ground genetic stability of cloud gold flathead and extensive foundation better, this and conventional gynogenesis technology are only used to obtain the hybrid vigour of allogynogensis or exist significantly distinguish to obtain other novel hybridization fish strains.Male gynogenesis Hunan cloud gold flathead is obtained by artificial sex reversal technology in breeding method of the present invention; make itself and the selfing of gynogenesis diploid Hunan cloud gold flathead; and then obtain body colour genetic stability and there is the gynogenesis Hunan cloud gold flathead pure lines colony of merit, ensure that the large-scale production of gynogenesis Hunan cloud gold flathead.Breeding method of the present invention provides solution, simultaneously also for related biological theoretical research provides important colony's gene pool for setting up gynogenesis Hunan cloud gold flathead pure lines colony on a large scale.Breeding method of the present invention has major application and is worth and wide popularizing application prospect in the breeding of gynogenesis Hunan cloud gold flathead excellent seed, sexual control and complete female breeding.
Accompanying drawing explanation
Fig. 1 is the photo in kind (body colour presents salmon pink) of common Hunan cloud gold flathead female parent.
Fig. 2 is the photo in kind (body colour is thin out by salmon pink) of common Hunan cloud gold flathead self-bred progeny.
Fig. 3 is through activation of spermatozoa but without the gynogenesis Hunan cloud gold bighead seedling outline drawing of chromosome doubling process.
Fig. 4 is through activation of spermatozoa and through the gynogenesis Hunan cloud gold bighead seedling outline drawing of chromosome doubling process.
Fig. 5 is the outward appearance photo of the golden flathead of I male gynogenesis Hunan cloud in age that breeding method of the present invention obtains.
Fig. 6 is the outward appearance photo of the golden flathead of I female gynogenesis Hunan cloud in age that breeding method of the present invention obtains.
Fig. 7 is the gynogenesis Hunan cloud gold flathead chromosome map that the suppression second polar body obtained in the embodiment of the present invention is discharged.
Fig. 8 is the gynogenesis Hunan cloud gold flathead chromosome map of the suppression First cleavage obtained in the embodiment of the present invention.
Embodiment
Below in conjunction with Figure of description and concrete preferred embodiment, the invention will be further described, but protection domain not thereby limiting the invention.
Embodiment:
A breeding method for Hunan of the present invention cloud gold flathead pure lines colony, specifically comprises the following steps:
1. sperm deactivation: extrude and reach sexually matured allotetraploid hybrids (allotetraploid hybrids fish is purchased from Xiang Yun bio tech ltd, Hunan, its breeding method can see CN1263704A Chinese patent literature) seminal fluid is in culture dish, with the seminal fluid of Hank ' s liquid dilution allotetraploid hybrids, the volume ratio of Hank ' s liquid and seminal fluid is 3: 1, seminal fluid after dilution distributes with thin layer in culture dish, the culture dish containing seminal fluid is placed on the shaking table being lined with ice bag and carries out centrifugal treating, rotating speed is 45r/min, and be placed in two power and be respectively treatment with irradiation under the uviol lamp of 15W, the distance of uviol lamp and seminal fluid is 11cm, irradiation time is 30min, during microscopy, sperm tail swings and to weaken and the sperm of about 85% still has good travelling vigor, now seminal fluid lucifuge good for deactivation is placed in 4 DEG C of refrigerators to preserve.
The formula 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. Oocyte Activation: mix in step 1 through the mature egg of the seminal fluid of Ultraviolet radiation process with Hunan cloud gold flathead, and with clean goose feather by its abundant stirring and evenly mixing, add clear water (water temperature is 23 DEG C ~ 26 DEG C) in 1min, and start to calculate activationary time, Oocyte Activation rate can reach more than 92%.
3. chromosome doubling process: the present embodiment adopts cold shock method to suppress second polar body to be discharged and suppresses First cleavage to make activation ovum chromosome recover diploidy.By set temperature gradient and processing time comparative trial (see table 1 and table 2), obtain best PROCESS FOR TREATMENT parameter, that is: 3min after activating, with 5 DEG C of water cold shock treatment 12min, discharge to suppress second polar body, activation ovum is crossed primitive gut rate and can be reached more than 62%, and gynogenesis Hunan cloud gold flathead emergence rate can reach more than 31%; Or 24min after activating, with 5 DEG C of water treatment 13min, to suppress First cleavage, activation ovum is crossed primitive gut rate and can be reached more than 48%, and gynogenesis Hunan cloud gold flathead emergence rate can reach 8.0%.
Table 1: different shock temperature is on the impact of Hunan cloud gold flathead gynogenesis inducing effect
Table 2: the different cold shock treatment time is on the impact of Hunan cloud gold flathead gynogenesis inducing effect
Table note: in upper table 1 and table 2, crosses embryo number/always ovum number × 100% of primitive gut rate=mistakes gastrul stage; Field run plant number/total ovum number × 100% of survival rate=phase of ingesting.
4. flowing water temperature control hatching: the ovum through gynogenesis process is put into hatching pail and carries out the hatching of flowing water temperature control, water velocity can float with ovum in bucket, at the bottom of both can not having sunk to barrel, can not be broken through egg membrane for degree by current again.Incubation temperature controls within the scope of 23 DEG C ~ 26 DEG C.
5. artificial sex reversal: gynogenesis Hunan cloud Jin Yong hatches latter 4 ~ 6 days, lower cement pond clearing after sterilization training water, with the forage feed containing 17 Alpha-Methyl stosterones, concentration is 40 ~ 60 μ g MT/g feeds, every day regularly throws something and feeds twice sooner or later, cycle of throwing something and feeding continues 80 ~ 100 days, goes to Tu Chi afterwards and raises, feed with normal diet.
Suppression second polar body gynogenesis Xiang Yunjin flathead colony 1120 tail of discharging and gynogenesis Xiang Yunjin flathead colony 360 tail (see Fig. 6) suppressing First cleavage from same Hunan cloud gold flathead female parent is have successfully been obtained by the gynogenesis method of above-mentioned Hunan cloud gold flathead.With nephrocyte direct preparation to one age juvenile fish chromosome number detect.The method of nephrocyte direct preparation is: under 18 DEG C ~ 22 DEG C water temperatures after cultural aim fish 1d ~ 3d, to experiment fish injection PHA1 ~ 3 time, each dosage is 2 ~ 8 μ g/ gram body weight, interval time is 12h ~ 24h, 2h ~ 6h before dissection is drawn materials, injection colchicine, dosage is 2 ~ 4 μ g/ gram body weight.Take out nephridial tissue, under physiological saline, shred nephridial tissue, 0.075mol/L KCl Hypotonic treatment, hypotonic 40min ~ 60min at 20 DEG C of temperature; Glacial acetic acid with 1: 3: the mixed liquor of methyl alcohol fixes nephrocyte 1 ~ 3 time; Freezing slide drips sheet; Giemsa dye liquor dyes.Result shows: gynogenesis Hunan cloud gold flathead (see Fig. 7) suppressing second polar body to be discharged and gynogenesis Hunan cloud gold flathead (see Fig. 8) chromosome number suppressing First cleavage are 2n=48.Different development stage sexual gland paraffin section result in enormous quantities shows: the gynogenesis Hunan cloud gold flathead suppressing second polar body to be discharged and the gynogenesis Hunan cloud gold flathead suppressing First cleavage are female, are Quan Ci colony.Paraffin section method is: the sexual gland Bouin ' s monthly getting 4 ~ 8 tail gynogenesis Hunan cloud gold flatheads is at random liquid-solid fixed, and paraffin embedding, slice thickness is 6 μm, hematoxylin eosin staining, neutral gum mounting, and Olympus basis of microscopic observation, takes pictures.
6. selfing: the gynogenesis Hunan cloud gold flathead obtained in the present embodiment step 4 is fed to sexual maturity, then using this sexually matured gynogenesis Hunan cloud gold flathead as female parent, the male gynogenesis Hunan cloud gold flathead obtained in the present embodiment step 5 is as male parent, this female parent and male parent is made to carry out selfing, because the gene homozygosity of father, female parent is all quite high, trait segregation is less likely to occur, therefore, gynogenesis produce merit by both selfing genetic stabilities to offspring; And then obtain gynogenetic Hunan cloud gold flathead pure lines colony.
7. hybridize: the gynogenesis Hunan cloud gold flathead obtained in the present embodiment step 4 is fed to sexual maturity, then using this sexually matured gynogenesis Hunan cloud gold flathead as female parent, using common male Hunan cloud gold flathead as male parent, make this female parent and male parent carry out hybridizing (gynogenesis Hunan cloud gold flathead ♀ × common Hunan cloud gold flathead ♂), and then gynogenetic Hunan cloud gold flathead pure lines colony (i.e. hybridization fish offspring F can be obtained in enormous quantities
1).The contrast culture experiment of 2 years shows: support Hunan cloud gold flathead kind and when not using anti-disease vaccines when main, hybridization fish offspring F
1except bacterial gill exception occurs few infection, pop endangers large bacillary enteritis and viral hemorrhagic disease does not infect substantially, growth rate flathead fast 10% ~ 15% golden than common Hunan cloud.Different provinces large area contrast culture experiment statistics is in table 3.
Table 3: contrast culture experiment data statistics
Therefore, adopt the present embodiment method seed selection gynogenesis Hunan cloud gold flathead, gynogenesis induced efficiency can be significantly improved, acquisition in enormous quantities is from the gynogenesis Hunan cloud gold flathead pure lines colony of same Hunan cloud gold flathead parent, and the gynogenesis Hunan cloud gold flathead pure lines colony that this genetic background is consistent is that several important biomolecule Study on Problems such as relevant Sex Determination Mechanism and evolution provides valuable research material.In addition, can be used as high-quality female parent by the gynogenesis Hunan cloud gold flathead of seed selection of the present invention, large-scale production (gynogenesis Hunan cloud gold flathead ♀ × common Hunan cloud gold flathead ♂) F
1, this F
1in body colour, growth rate and resistance against diseases etc., more common Hunan cloud gold flathead has obvious advantage, gynogenesis Hunan cloud gold flathead resistance against diseases strengthens, not only cause gene pure with gynogenesis process, to get rid of deleterious gene relevant, also relevant with mixing of allotetraploid hybrids diploid sperm correlated inheritance fragment, this overcomes the problem of Hunan cloud gold flathead body colour degeneration in I, II age, anti-adversity ability difference to a certain extent, significantly improves the economic benefit of cultivation Hunan cloud gold flathead on producing.
Claims (6)
1. a breeding method for Hunan cloud gold flathead pure lines colony, comprises the following steps:
(1) Artificial gynogenesis: using the diploid sperm of allotetraploid hybrids as stimulus, this diploid sperm after appropriate deactivation is made to activate the mature egg of Hunan cloud gold flathead, under water temperature is 23 DEG C ~ 26 DEG C conditions, 2min ~ 4min water cold shock treatment 10min ~ 13min of 4 DEG C ~ 6 DEG C after activating, then through the hatching of flowing water temperature control, the gynogenesis Hunan cloud gold flathead suppressing second polar body to be discharged is prepared;
(2) male gynogenesis fishes is obtained: the gynogenesis Hunan cloud gold flathead hatching obtained after step (1) is after 4 ~ 6 days, throw something and feed containing the feed of androgenic hormone, the cycle of throwing something and feeding of feed is 80 ~ 100 days, after meeting the requirement in cycle of throwing something and feeding, feed to sexual maturity with the normal diet not containing androgenic hormone, the male gynogenesis Hunan cloud gold flathead of acquired reversion; Described androgenic hormone selects 17 Alpha-Methyl stosterones, and the 17 Alpha-Methyl stosterones containing 40 μ g ~ 60 μ g in every gram of feed;
(3) selfing: feed to sexual maturity to the gynogenesis Hunan cloud gold flathead obtained in step (1), then using this sexually matured gynogenesis Hunan cloud gold flathead as female parent, the male gynogenesis Hunan cloud gold flathead obtained in step (2) is as male parent, make this female parent and male parent carry out selfing, and then obtain gynogenetic Hunan cloud gold flathead pure lines colony.
2. a breeding method for Hunan cloud gold flathead pure lines colony, comprises the following steps:
(1) Artificial gynogenesis: using the diploid sperm of allotetraploid hybrids as stimulus, this diploid sperm after appropriate deactivation is made to stimulate the mature egg of Hunan cloud gold flathead, under water temperature is 23 DEG C ~ 26 DEG C conditions, 21min ~ 24min water cold shock treatment 10min ~ 13min of 4 DEG C ~ 6 DEG C after activating, then through the hatching of flowing water temperature control, the gynogenesis Hunan cloud gold flathead suppressing First cleavage is prepared;
(2) male gynogenesis fishes is obtained: the gynogenesis Hunan cloud gold flathead hatching obtained after step (1) is after 4 ~ 6 days, throw something and feed containing the feed of androgenic hormone, the cycle of throwing something and feeding of feed is 80 ~ 100 days, after meeting the requirement in cycle of throwing something and feeding, feed to sexual maturity with the normal diet not containing androgenic hormone, the male gynogenesis Hunan cloud gold flathead of acquired reversion; Described androgenic hormone selects 17 Alpha-Methyl stosterones, and the 17 Alpha-Methyl stosterones containing 40 μ g ~ 60 μ g in every gram of feed;
(3) selfing: feed to sexual maturity to the gynogenesis Hunan cloud gold flathead obtained in step (1), then using this sexually matured gynogenesis Hunan cloud gold flathead as female parent, the male gynogenesis Hunan cloud gold flathead obtained in step (2) is as male parent, make this female parent and male parent carry out selfing, and then obtain gynogenetic Hunan cloud gold flathead pure lines colony.
3. a breeding method for Hunan cloud gold flathead pure lines colony, comprises the following steps:
(1) Artificial gynogenesis: using the diploid sperm of allotetraploid hybrids as stimulus, this diploid sperm after appropriate deactivation is made to activate the mature egg of Hunan cloud gold flathead, under water temperature is 23 DEG C ~ 26 DEG C conditions, 2min ~ 4min water cold shock treatment 10min ~ 13min of 4 DEG C ~ 6 DEG C after activating, then through the hatching of flowing water temperature control, the gynogenesis Hunan cloud gold flathead suppressing second polar body to be discharged is prepared;
(2) hybridize: feed to sexual maturity to the gynogenesis Hunan cloud gold flathead obtained in step (1), then using this sexually matured gynogenesis Hunan cloud gold flathead as female parent, using common male Hunan cloud gold flathead as male parent, this female parent and male parent are hybridized, and then obtains gynogenetic Hunan cloud gold flathead pure lines colony.
4. the breeding method of the Hunan cloud gold flathead pure lines colony according to any one of claims 1 to 3, it is characterized in that, the concrete operations of described appropriate deactivation are: first use Hank ' s liquid to dilute the seminal fluid of allotetraploid hybrids, again by ultraviolet inactivation, deactivation degree weakens with sperm tail swing during microscopy and the sperm of 80% ~ 90% still has good travelling vigor for standard.
5. the breeding method of Hunan according to claim 4 cloud gold flathead pure lines colony, it is characterized in that, use power to be the uviol lamp of 15W during described ultraviolet inactivation, the distance of described uviol lamp and seminal fluid is 10cm ~ 12cm, and the irradiation time of described uviol lamp is 20min ~ 35min.
6. the breeding method of the Hunan cloud gold flathead pure lines colony according to any one of claims 1 to 3, it is characterized in that, temperature during described flowing water temperature control hatching is 23 DEG C ~ 26 DEG C.
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