CN102246708A - Triploid induction method of cynoglossus semilaevis - Google Patents

Abstract

The invention discloses a triploid induction method of cynoglossus semilaevis, belonging to the technical field of marine organisms. The method mainly comprises the following steps of: selecting mature cynoglossus semilaevis spawner and milter; respectively collecting mature ova and sperm; artificially fertilizing; carrying out cold shock treatment for 15-55 minutes with sea water at 2-10 DEG C after fertilizing for 2-10 minutes; and then, hatching in sea water at 20-25 DEG C, thereby finishing the triploid induction of a cold shock method. The method disclosed by the invention has the advantages of simple process, low cost and strong operability and greatly improves the inductivity of triploid cynoglossus semilaevis. The experimental statistics show that the artificial inductivity of the obtained triploid cynoglossus semilaevis can reach more than 80%, and the maximum artificial inductivity of the triploid cynoglossus semilaevis can reach 88%. Thus, the invention has favorable economic benefit and social benefit.

Description

The triploid abductive approach of Cynoglossus semilaevis

Technical field

The present invention relates to the cultural method of a kind of fish, particularly relate to a kind of production method of triploid Cynoglossus semilaevis, this technology belongs to the marine biotechnology field.

Background technology

Cynoglossus semilaevis delicious flavour, mouthfeel are smooth, are the breed varieties that China's mariculture industry is developed in recent years, have very high economic worth.But Cynoglossus semilaevis male and female growth differences is big, and is female obviously faster than male.Male growth rate is slow, reaches marketable fish size through the cultivations in 2 years also difficulty, and the culturist is everlasting and milter is eliminated gradually after half a year, and only stays female fish culture to commercial specification.Therefore, actual survival rate is equivalent to the ratio of raun.In the fry that artificial breeding obtained, male ratio sometimes can be nearly to 70% generally more than 60%.Mantissa more than 50% just is eliminated after culturing more than half a year, and aquaculture cost is significantly improved, and culture benefit descends significantly, has seriously influenced culturist's enthusiasm.

For addressing these problems, researchers have carried out many good tries, concentrate on mainly that female unisexualityization is induced and the optimization aspect of breeding environment, but do not have obvious effects so far.But from the trend of domestic and international aquatic animal research, the triploid technology is a kind of method of potentialization.The triploid aquatic livestock is owing to have sterility, and promptly sexual gland can not be reached maturity or developmental deformity, and irreproducible offspring, does not therefore have the energy loss of sexual maturity or proliferation, has avoided the growth in breeding stage to stop or high mortality.At present, the triploid aquatic livestock has all shown good effect at aspects such as control excessive multiplication, quickening growth rate.

Therefore according to the characteristic of Cynoglossus semilaevis, develop a kind of abductive approach, obtain the triploid Cynoglossus semilaevis, avoid male growth rate shortcoming slowly, to improve the growth rate of Cynoglossus semilaevis, improving its economic benefit and social benefit is the common expectation of culturing the dealer.

Summary of the invention

The present invention aims to provide the triploid abductive approach of a kind of Cynoglossus semilaevis.

Step of the present invention is:

1) chooses the Cynoglossus semilaevis milter and the raun of gonad maturity, respectively collecting semen and mature egg;

2) seminal fluid is evenly mixed with the ovum grain, room temperature leaves standstill 2min, adds seawater and carries out artificial insemination;

3) triploid obtains: the fertilized egg of after fertilization 2-10min is transferred in the 0-10 ℃ of seawater bath carries out cold shock 15-55min, suppress the formation and the discharge thereof of second polar body, chromosome set is doubled.

4), remove shock and ovum is moved in the normal temperature seawater hatch, according to common semi-smooth tongue sole offspring breed production method management, cultivate Cynoglossus semilaevis triploid fry.

In step 2) in, the seawater water temperature is 20-25 ℃.

In step 3), the time of initial cold shock is after fertilization 2-8min in the seawater; The cold shock duration is 15-55min; The cold shock temperature is 3-8 ℃.

In step 4), the temperature of seawater hatching is 20-25 ℃.

More preferably,

In the step 3), the time of initial cold shock is after fertilization 6-8min in the seawater; The cold shock duration is 20-40min, and the cold shock temperature is 4-6 ℃;

In the step 4), the temperature of seawater hatching is 22 ℃.

The employing said method is induced, and can access higher inductivity.

Further scheme of the present invention is: induce obtain the triploid Cynoglossus semilaevis after, utilize technology such as chromosome karyotype analysis, flow cytometer to measure the chromosome multiple of above-mentioned fish body, differentiate, filter out the triploid Cynoglossus semilaevis and cultivate.

Beneficial effect of the present invention is:

1. the present invention induces by utilizing low temperature shock method, and, suppress second polar body release, thereby improve the inductivity of triploid Cynoglossus semilaevis greatly by the inducing sustained time of control, test statistics factually, the artificial induction of the triploid Cynoglossus semilaevis that is obtained leads and can reach more than 80%.

2. technology of the present invention is simple, and cost is low, and is workable, has avoided chemical method to induce the pollution that brings.

3. advantages such as the triploid Cynoglossus semilaevis that adopts the method for the invention to cultivate has fast growth, big, the breed survival rate height of individual specification, and resistance against diseases is strong have good economic benefit and social benefit.

In the Cynoglossus semilaevis triploid induction, wherein most preferred inductive condition is,

Choose the Cynoglossus semilaevis raun and the milter of gonad maturity, gather mature egg and seminal fluid respectively, artificial insemination, after fertilization 8min handles 30min with 4 ℃ seawater bath cold shock, suppress second polar body and discharge, in 22 ℃ seawater, hatch then, finish the triploid induction of cold shock method.

Adopt this condition to carry out the triploid induction of Cynoglossus semilaevis, experimental result shows, fertilization rate=34.1%, and incubation rate=82.45%, triploid rate reaches 88%.During the triploid Cynoglossus semilaevis is induced, the low further raising that has influenced triploid induction rate of incubation rate, even under experiment condition of the present invention, general incubation rate also only about 50%, yet induce under this condition, do not influence its incubation rate.

Description of drawings

Fig. 1 Cynoglossus semilaevis diploid fish fry cell DNA fluorescence distribution histogram

Fig. 2 Cynoglossus semilaevis triploid fry cell DNA fluorescence distribution histogram

Embodiment

Following experimental example and embodiment will the present invention is described further.

Experimental example 1

The triploid ploidy of Cynoglossus semilaevis is identified

Adopt the German Partec Cystain DNA of company 1 step dye liquor one-step method dyeing, identify ploidy by the cells were tested by flow cytometry cell DNA content.

1, preparation of histocyte suspension and mensuration

Employing is randomly drawed mode 60 age in days Cynoglossus semilaevis triploids is identified, at every turn with 4 tail dliploids in contrast.

(1) the about 1mm of live body tail fin of each tail fish is got in histocyte extraction and DNA fluorescent staining ³, put into the 1.5mL microcentrifugal tube, add Cystain DNA 1 step dye liquor 0.5ml, shred tail fin and organize to naked eyes and cannot see block, grind gently again to equal pulpous state; Cell filtration net filtration with 300 orders Germany Partec company produces adds CystainDNA 1 step dye liquor 0.5ml, further dyeing again in filtrate.

(2) cells were tested by flow cytometry PARTEC is that German Partec Gmbh company produces, model PA, and this machine excitation source is a negative ion laser.Before the measurement, adjust cell concentration about 1 * 106/ml, keep measuring speed during measurement at 200～300/s with sheath fluid.Selecting the normal diploid cell suspension is control sample.

(3) detect the preceding adjustment flow cytometer of control mensuration and make pipeline unimpeded, nozzle cleaning, instrument is in optimum state, carries out FLOWCHECK and proofreaies and correct, and the coefficient of variation (CV) of each passage is stabilized in below 3%, and dna content is detected automatically by instrument.Keep cell separation purity by the control flow velocity.

2, detect principle and method for expressing

With the dliploid cellular control unit is reference calculation dliploid and triploid frequency, and cell DNA content is with DNA index (DNAindex, DI) expression.

DI=processed group cell G0/1 peak channel/cellular control unit G0/1 peak channel average

With DI=1.0 ± 0.1 is the diplontic criterion of DNA.If DI=1.5 ± 0.1 then is a triploid.

DNA to triploid processed group and dliploid control group analyzes (table 1), and the result shows the G0/1 peak channel significant difference of the two cell DNA.

Control group that flow cytometer provides and triploid processed group cell DNA content fluorescence distribution histogram are seen Fig. 1 and Fig. 2.

The DNA of table 1 triploid processed group and dliploid control group is (means standard deviation) relatively

Embodiment 1

Choose the Cynoglossus semilaevis raun and the milter of gonad maturity, gather mature egg and seminal fluid respectively, artificial insemination, after fertilization 2-10min handles 18min with 3 ℃ seawater cold shock, hatches in 22 ℃ seawater then.Experimental result shows, fertilization rate=38.4%, and incubation rate=53.27%, triploid rate is more than 80%.

Embodiment 2

Choose the Cynoglossus semilaevis raun and the milter of gonad maturity, gather mature egg and seminal fluid respectively, artificial insemination, after fertilization 4min handles 25min with 8 ℃ seawater bath cold shock, hatches in 22 ℃ seawater then, finishes the triploid induction of cold shock method.Experimental result shows, fertilization rate=39.7%, and incubation rate=54.85%, triploid rate reaches 85%.

Embodiment 3

Choose the Cynoglossus semilaevis raun and the milter of gonad maturity, gather mature egg and seminal fluid respectively, artificial insemination, after fertilization 8min handles 30min with 4 ℃ seawater bath cold shock, hatches in 22 ℃ seawater then, finishes the triploid induction of cold shock method.Experimental result shows, fertilization rate=34.1%, and incubation rate=82.45%, triploid rate reaches 88%.

Claims (9)

1. the triploid abductive approach of Cynoglossus semilaevis is characterized in that comprising the steps:

1) chooses the Cynoglossus semilaevis milter and the raun of gonad maturity, respectively collecting semen and mature egg;

2) seminal fluid is evenly mixed with the ovum grain, stir the back room temperature and leave standstill 2min, add seawater and carry out artificial insemination;

3) triploid obtains: the fertilized egg of after fertilization 0-10min is transferred to carries out cold shock in the 2-10 ℃ of seawater bath and handle 15-55min, chromosome set is doubled.

4) the releasing cold shock moves to ovum in the seawater and hatches, and according to common semi-smooth tongue sole offspring breed production method management, cultivates Cynoglossus semilaevis triploid fry.

2. Cynoglossus semilaevis triploid induction method as claimed in claim 1 is characterized in that:

Step 2) in, the seawater water temperature is 20-25 ℃.

3. Cynoglossus semilaevis triploid induction method as claimed in claim 1 is characterized in that:

The time of initial cold shock is after fertilization 2-8min in the seawater; The cold shock duration is 15-55min; The cold shock temperature is 3-8 ℃.

4. Cynoglossus semilaevis triploid induction method as claimed in claim 1 is characterized in that:

In the step 4), the temperature of seawater hatching is 20-25 ℃.

5. Cynoglossus semilaevis triploid induction method as claimed in claim 1 is characterized in that:

The time of initial cold shock is after fertilization 6-8min in the seawater; The cold shock duration is 20-40min, and the cold shock temperature is 4-6 ℃;

6. Cynoglossus semilaevis triploid induction method as claimed in claim 1 is characterized in that:

In the step 4), the temperature of seawater hatching is 22 ℃.

7. Cynoglossus semilaevis triploid induction method as claimed in claim 1 is characterized in that:

Choose the Cynoglossus semilaevis raun and the milter of gonad maturity, gather mature egg and seminal fluid respectively, artificial insemination, after fertilization 2-10min handles 18min with 3 ℃ seawater cold shock, suppresses second polar body and discharges, and hatches in 22 ℃ seawater then.

8. Cynoglossus semilaevis triploid induction method as claimed in claim 1 is characterized in that:

Choose the Cynoglossus semilaevis raun and the milter of gonad maturity, gather mature egg and seminal fluid respectively, artificial insemination, after fertilization 4min handles 25min with 8 ℃ seawater bath cold shock, suppress second polar body and discharge, in 22 ℃ seawater, hatch then, finish the triploid induction of cold shock method.

9. Cynoglossus semilaevis triploid induction method as claimed in claim 1 is characterized in that:

Choose the Cynoglossus semilaevis raun and the milter of gonad maturity, gather mature egg and seminal fluid respectively, artificial insemination, after fertilization 8min handles 30min with 4 ℃ seawater bath cold shock, suppress second polar body and discharge, in 22 ℃ seawater, hatch then, finish the triploid induction of cold shock method.

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