CN103229742A - Preparation method of oncorhynchus mykiss walbaum and oncorhynchus mykiss hybrid triploid offspring seed - Google Patents
Preparation method of oncorhynchus mykiss walbaum and oncorhynchus mykiss hybrid triploid offspring seed Download PDFInfo
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Abstract
The invention provides a preparation method of an oncorhynchus mykiss walbaum and oncorhynchus mykiss hybrid triploid offspring seed. The preparation method comprises the steps of: carrying out in vitro fertilization on oncorhynchus mykiss walbaum female parent fish ova and oncorhynchus mykiss male parent fish sperms, transferring into a 170-190 mg/L 6-DMAP (6-dimethylaminopyridine) solution with the temperature of 12-14 DEG C after fertilized ova is fertilized for 12-14minutes, soaking for 14-16minutes, then transferring in a water body with the temperature of 6-8 DEG C and then hatching; hatching the fertilized ovum to obtain hybrid triploid larva fishes after about 50 days; and detecting the larva fishes by using a flow cytometry, wherein the eyed rate of the fertilized ova in a hatching period is above 71 percent, the hatching rate of the seeds reaches above 90 percent and the triploid rate reaches above 80 percent. By using the preparation method of the oncorhynchus mykiss walbaum and oncorhynchus mykiss hybrid triploid offspring seed, the problems of sexual precocity, miniaturization, disease propagation and the like of individuals in a trout cultivating process are solved, the advantages of hybridization and triploid are obtained, and a good market value is achieved.
Description
Technical field
The present invention relates to the aquatic biological breeding technical field, relate to the preparation method of a kind of golden trout and rainbow trout (Salmo gairdneri) hybridization triploid seed particularly.
Background technology
The salmon trout is the world one of main fish of culturing at present, because the quality of himself and to the specific (special) requirements of living environment, has determined the character of its green, environmental protection nutritive food, therefore quite is subjected to consumer's favor.The salmon trout is China's main cold water cultured fishes at present, along with developing rapidly that China salmon trout is cultured, fundamental research relatively lags behind, salmon trout parent population germplasm is degenerated and to be caused under the seed quality degradation productivity effect degradation problem outstanding gradually, thereby has restricted the sustainable development of salmon trout aquaculture.Utilize modern biotechnology, cultivate growth rapidly, individual big, meat is good, resistance against diseases is strong seedling, will become the continuing of China's salmon trout aquaculture, stable, the only way of developing in a healthy way.
Hybridization can be rapid the conservative that shakes heredity, make the inheritance of hybrid be imbued with motility, have bigger plasticity, thereby can be rapidly and improve the vitality of hybrid significantly, thereby obtain hybrid vigour; Hybrid can be enriched genetic structure, with two kind or above individual independent assortment of genes of kind that above hereditary basis is different, occurs new hereditary form by hybridization, and people can select good individuality then, cultivate into new varieties.
Compare with common dliploid fish, advantages such as the triploid fish have fast growth, survival rate height, premunition is strong, meat good, safety and Health: 1. triploid fish growth rate is than the fast 30-40% of dliploid, be conducive to produce large-scale individuality, can effectively improve population yield; 2. salmon section triploid fish are sterile, avoided the flesh quality that gonad development stage and spawning season shift because of energy and nuptial coloration occurs causing to descend and the appearance features variation, avoided its biological death breeding period, prolong the culture-cycle; 3. the triploid fish do not have potential threat to environment, and the gene that triploid can reduce fish mixes, and effectively control the excessive multiplication of natural water area Mesichthyes, avoid the degeneration of breed variety, the diversity of protection species; 4. do not have alien gene, belong to the healthy food that the mankind can eat safely, compared with transgenic technology more people accept; 5. triploid fish is Fresh ﹠ Tender in Texture, edible part is high, the bodily form stablize attractive in appearance, commodity value is high.Because the triploid fish have above-mentioned advantage, therefore by the triploid technology of preparing obtain growth fast, adaptability and the strong high-quality seed of premunition have great potential.
Since the eighties in last century, the artificial induction of triploid fish work makes significant progress.At present the research of triploid seed preparation is mainly concentrated on the triploid preparation of purebred rainbow trout, its method is heat shock method or hydrostatic pressing method.The technology that existing heat shock method prepares purebred triploid rainbow trout depends on precise dose; temperature is not conversion slightly; can cause seed abnormal rate height; make and cultivate the effect instability; all the time can't form large-scale production; it is former because the triploid rate that existing purebred production triploid seed technology of preparing maybe can't reach production requirement maybe can't reach the weed survival rate that can be used for producing, and can't accomplish scale production always.Therefore, press for the triploid fry technology of preparing of a kind of technology maturation, effect stability and energy large-scale production, improve eye rate and a seed triploid rate of fertilized egg.
6-dimethylaminopurine (N6-Dimethyladenine, 6-DMAP) in inducing, multiple polyploid shellfish obtained good result, but because the salmon trout is higher to the growing environment water quality requirement, to the chemical substance sensitivity, there is not the 6-dimethylaminopurine to be applied to the successful report of salmon trout triploid or hybridization triploid induction so far especially.
Summary of the invention
The object of the present invention is to provide the preparation method of a kind of golden trout and rainbow trout (Salmo gairdneri) hybridization triploid seed, to overcome the deficiencies in the prior art.
The preparation method of golden trout provided by the invention and rainbow trout (Salmo gairdneri) hybridization triploid seed may further comprise the steps:
1) makes golden trout parent population ovum and the exomixis of rainbow trout (Salmo gairdneri) parent population sperm;
2) the 6-dimethylaminopurine is handled fertilized egg;
3) fertilized egg is hatched in breeding water.
Wherein, step 1) fertilization temperature is 6-8 ℃.
It is 10-13 ℃ that step 1) gold trout parent population, rainbow trout (Salmo gairdneri) parent population are cultivated water temperature, and flowing water pond water exchange capacity is 2h/ time.
Described step 1) is taken out rainbow trout (Salmo gairdneri) parent population fish-egg and seminal fluid respectively, and seminal fluid uses the hatching water to clean, and method is that the volume ratio of seminal fluid and hatching water is 1-1.5:2500-3000, outwells water behind the stirring 1-1.5min, washes 3-5 time repeatedly with quadrat method; Mantissa's ratio according to male parent population and female parent population 1:2-4 is carried out the fertilization of sperm and ovum.
Preparation method's of the present invention step 2) in, fertilized egg moves into fertilized egg immersion treatment in 6-dimethylaminopurine (6-DMAP) solution of 170-190mg/L behind fertilization 12-14min.
Preferably, fertilized egg moves into immersion treatment in the 6-dimethylaminopurine solution behind fertilization 12min.
The temperature of 6-dimethylaminopurine solution is 12-14 ℃.Soak time is 14-16min.
With step 2) fertilized egg of immersion treatment moved in the 6-8 ℃ of hatching water body hatching rapidly 48-60 days, obtained hybridizing the triploid prelarva.
The present invention also provides the application of 6-dimethylaminopurine (6-DMAP) in the golden trout of preparation and rainbow trout (Salmo gairdneri) hybridization triploid seed.
Further, employing concentration is that 170-190g/L, temperature are 12-14 ℃ 6-dimethylaminopurine solution-treated gold trout and rainbow trout (Salmo gairdneri) fertilized egg, and the time is 14-16min.
The invention provides the preparation method of a kind of golden trout and rainbow trout (Salmo gairdneri) triploid seed, concrete operations are: choose the female parent population fish-egg of golden trout and rainbow trout (Salmo gairdneri) milter parent population sperm carries out in vitro fertilization, under 7 ℃ of conditions behind the fish-egg fertilization 12min, it is that 180mg/L, temperature are to soak rainbow trout (Salmo gairdneri) fertilized egg 15min in 13 ℃ the 6-DMAP solution that fertilized egg is moved into concentration rapidly; Move into normal the hatching 50 days in 7 ℃ of water bodys afterwards rapidly, obtain golden trout and rainbow trout (Salmo gairdneri) hybridization triploid fry.
The used water body of preparation method of the present invention is the hatching water that meets national water quality standard for fishery regulation.
The invention provides the application of above-mentioned preparation method in rainbow trout (Salmo gairdneri) triploid seed breeding work.
The present invention is surprised to find that and passes through finite concentration; 6-dimethylaminopurine solution-treated gold trout and the rainbow trout (Salmo gairdneri) fertilized egg of temperature; can suppress the discharge of fertilized egg second polar body; on this basis; the present invention gropes experiment condition repeatedly through a large amount of experiments; scale preparation gold trout and rainbow trout (Salmo gairdneri) hybridization triploid seed have been realized; the fertilized egg of using this method is sent out a rate and is reached more than 71%; the seed incubation rate reaches more than 90%; the seed triploid rate reaches more than 80%; this inventive method fertilized egg is sent out a rate and is produced close with normal salmon trout; triploid rate has reached the production demand; realized the large-scale production of hybridization triploid seed; the golden trout that obtains and rainbow trout (Salmo gairdneri) hybrid seedlings have hybridization and triploid advantage; obviously being better than existing dliploid rainbow trout aspect growth rate and the resistance against diseases, have favorable economic benefit and market value.
Embodiment
Following examples further specify content of the present invention, but should not be construed as limitation of the present invention.Without departing from the spirit and substance of the case in the present invention, modification or replacement to the inventive method, step or condition are done all belong to scope of the present invention.
If do not specialize the conventional means that used technological means is well known to those skilled in the art among the embodiment.Golden trout parent population of the present invention and rainbow trout (Salmo gairdneri) parent population are that the eyed eggs cultivation that Beijing Fisheries Research Institute is bought by Northern Europe agate bio tech ltd forms.6-DMAP is available from U.S. SIGMA company.
Embodiment 1 gold medal trout and rainbow trout (Salmo gairdneri) hybridization triploid seed preparation method (1)
The employing current stimulate, control illumination, and the parent population of throwing something and feeding is strengthened means such as manufactured feed, and golden trout parent population and rainbow trout (Salmo gairdneri) parent population are reached maturity synchronously.Gold trout, rainbow trout (Salmo gairdneri) parent fish rearing water temperature are 10 ℃, and flowing water pond water exchange capacity is 2h/ time.
Fish-egg adopts the dry method fertilization.Adopt extrusion that fish-egg is clamp-oned in the clean porcelain basin fully-developed gold trout raun earlier, per 4 tail rauns are 1 group, the seminal fluid of the male rainbow trout (Salmo gairdneri) of 2 tails is clamp-oned in the basin, 8 ℃ of water temperatures stir immediately again, adding seminal fluid is the hatching water of 1:2500 with the volume ratio of hatching water, stir gently, behind the 1min water is poured out 3 times repeatedly, seminal fluid is cleaned up, and placement etc. are pending.
Behind the fish-egg fertilization 12min, it is that 180mg/L, temperature are immersion treatment in 12 ℃ the 6-DMAP solution that fertilized egg is moved into concentration rapidly, soak 15min after, move into normally hatching in 7 ℃ of hatching water bodys rapidly.
Through hatching fry in 50 days, hybridizing triploid zygote during this time, to send out a rate be 71%, the seed incubation rate is 92%, and it is 83% golden trout and rainbow trout (Salmo gairdneri) hybridization triploid seed that its seed detects to triploid rate through flow cytometer, and stage at eyed eggs to 4 monthly age triploid survival rate is 85% of a stage.Hybridize triploid little satellite analysis by golden trout and the rainbow trout (Salmo gairdneri) that golden trout, rainbow trout (Salmo gairdneri) and present embodiment are made, the hybridization triploid that the presentation of results present embodiment makes is golden trout and rainbow trout (Salmo gairdneri) hybridization triploid.
Embodiment 2 gold medal trouts and rainbow trout (Salmo gairdneri) hybridization triploid seed preparation method (2)
The employing current stimulate, control illumination, and the parent population of throwing something and feeding is strengthened means such as manufactured feed, and golden trout and rainbow trout (Salmo gairdneri) parent population are reached maturity synchronously.Gold trout, rainbow trout (Salmo gairdneri) parent fish rearing water temperature are 13 ℃, and flowing water pond water exchange capacity is 2h/ time.
Fish-egg adopts the dry method fertilization.Adopt extrusion that fish-egg is clamp-oned in the clean porcelain basin fully-developed gold trout raun earlier, per 4 tail rauns are 1 group, the seminal fluid of the male rainbow trout (Salmo gairdneri) of 2 tails is clamp-oned in the basin, 8 ℃ of water temperatures stir immediately again, adding seminal fluid is the hatching water of 1:3000 with the volume ratio of hatching water, stir gently, behind the 1.5min water is poured out 3 times repeatedly, seminal fluid is cleaned up, and placement etc. are pending.
Behind the fish-egg fertilization 13min, it is that 170mg/L, temperature are immersion treatment in 13 ℃ the 6-DMAP solution that fertilized egg is moved into concentration rapidly, soak 14min after, move into normally hatching in 8 ℃ of hatching water bodys rapidly.
Through hatching fry in 52 days, during the hybridization triploid zygote to send out a rate be 72%, the seed incubation rate is 91%, its seed through flow cytometer detect for triploid rate be that 82% golden trout and rainbow trout (Salmo gairdneri) hybridize the triploid seed.Stage at eyed eggs to 4 monthly age triploid survival rate is for sending out 86% of the eye stage.Adopt little satellite analytical method of reporting in the prior art, the result shows that the hybridization triploid that present embodiment makes is golden trout and rainbow trout (Salmo gairdneri) hybridization triploid.
Embodiment 3 gold medal trouts and rainbow trout (Salmo gairdneri) hybridization triploid seed preparation method (3)
The employing current stimulate, control illumination, and the parent population of throwing something and feeding is strengthened means such as manufactured feed, and golden trout and rainbow trout (Salmo gairdneri) parent population are reached maturity synchronously.Gold trout, rainbow trout (Salmo gairdneri) parent fish rearing water temperature are 11 ℃, and flowing water pond water exchange capacity is 2h/ time.
Fish-egg adopts the dry method fertilization.Adopt extrusion that fish-egg is clamp-oned in the clean porcelain basin fully-developed gold trout raun earlier, per 4 tail rauns are 1 group, the seminal fluid of the male rainbow trout (Salmo gairdneri) of 2 tails is clamp-oned in the basin, 8 ℃ of water temperatures stir immediately again, adding seminal fluid is the hatching water of 1.5:3000 with the volume ratio of hatching water, stir gently, behind the 1min water is poured out 3 times repeatedly, seminal fluid is cleaned up, and placement etc. are pending.
Behind the fish-egg fertilization 14min, it is that 190mg/L, temperature are immersion treatment in 14 ℃ the 6-DMAP solution that fertilized egg is moved into concentration rapidly, soak 15min after, move into normally hatching in 7.5 ℃ of hatching water bodys rapidly.
Through hatching fry in 55 days, during the hybridization triploid zygote to send out a rate be 75%, the seed incubation rate is 93%, its seed through flow cytometer detect for triploid rate be that 81% golden trout and rainbow trout (Salmo gairdneri) hybridize the triploid seed.Stage at eyed eggs to 4 monthly age triploid survival rate is for sending out 84% of the eye stage.Adopt little satellite analytical method of reporting in the prior art, the result shows that the hybridization triploid that present embodiment makes is golden trout and rainbow trout (Salmo gairdneri) hybridization triploid.
Embodiment 4 gold medal trouts and rainbow trout (Salmo gairdneri) hybridization triploid seed preparation method (4)
The employing current stimulate, control illumination, and the parent population of throwing something and feeding is strengthened means such as manufactured feed, and golden trout and rainbow trout (Salmo gairdneri) parent population are reached maturity synchronously.Gold trout, rainbow trout (Salmo gairdneri) parent fish rearing water temperature are 12 ℃, and flowing water pond water exchange capacity is 2h/ time.
Fish-egg adopts the dry method fertilization.Adopt extrusion that fish-egg is clamp-oned in the clean porcelain basin fully-developed gold trout raun earlier, per 4 tail rauns are 1 group, the seminal fluid of the male rainbow trout (Salmo gairdneri) of 2 tails is clamp-oned in the basin, 8 ℃ of water temperatures stir immediately again, adding seminal fluid is the hatching water of 1.5:2500 with the volume ratio of hatching water, stir gently, behind the 1min water is poured out 5 times repeatedly, seminal fluid is cleaned up, and placement etc. are pending.
Behind the fish-egg fertilization 12.5min, it is that 185mg/L, temperature are immersion treatment in 12 ℃ the 6-DMAP solution that fertilized egg is moved into concentration rapidly, soak 16min after, move into normally hatching in 7.5 ℃ of hatching water bodys rapidly.
Through hatching fry in 58 days, during the hybridization triploid zygote to send out a rate be 74%, the seed incubation rate is 91%, its seed through flow cytometer detect for triploid rate be that 80% golden trout and rainbow trout (Salmo gairdneri) hybridize the triploid seed.Stage at eyed eggs to 4 monthly age triploid survival rate is for sending out 89% of the eye stage.Adopt little satellite analytical method of prior art report, the result shows that the hybridization triploid that present embodiment makes is golden trout and rainbow trout (Salmo gairdneri) hybridization triploid.
Embodiment 5 variable concentrations 6-DMAP solution are to the influence of hybridization triploid seed incubation rate and hybridization triploid rate
Prepare concentration and be 120,150,180,210mg/l, temperature is 12 ℃ 6-DMAP solution for later use.It is 12min in 7 ℃ the water body that gold trout ♀ * rainbow trout (Salmo gairdneri) ♂ after fertilization fertilized egg is placed on temperature, the 6-DMAP solution of putting into concentration behind the 12min immediately respectively and be 120mg/l, 180mg/l soaks, time is 15min, stirring fertilized egg frequently during this time, so that fertilized egg is able to even immersion, other operations are carried out with reference to the method for embodiment 1.The hybridization triploid rate situation of two kinds of concentration 6-DMAP solution-treated fertilized eggs the results are shown in Table 1 in the record hatching process.The result shows: concentration be the 6-DMAP solution solution-treated fertilized egg of 180mg/l the hybridization triploid rate, send out the eye rate apparently higher than 120mg/l concentration group.Illustrate that the 6-DMAP solution solution-treated fertilized egg of selecting 180mg/l for use can obtain hatching effect preferably.
Table 1 variable concentrations 6-DMAP solution is to the influence of hybridization triploid seed
| Concentration for the treatment of mg/l | The hatching water temperature (℃) | Send out eye rate (%) | Incubation rate | Triploid rate (%) |
| 120 | 7 | 65 | 91 | 43 |
| 150 | 7.5 | 63 | 89 | 74 |
| 180 | 7 | 61 | 92 | 81 |
| 210 | 7.2 | 60 | 90 | 82 |
Embodiment 6 6-DMAP solution (180mg/L, 12 ℃) are handled the fertilized egg different time to the influence of hybridization triploid seed incubation rate and hybridization triploid rate
Cultivate golden trout and hardhead trout hybridization triploid seed with reference to the method for embodiment 1, concentration is that 180mg/L, temperature are the asynchronism(-nization) of 12 ℃ 6-DMAP solution-treated fertilized egg, sees Table 2, and other breeding conditions are with embodiment 1.Hatched 50 days, and observed and record 6-DMAP solution-treated fertilized egg different time, the influence to golden trout and hardhead trout hybridization triploid seed the results are shown in Table 2.Presentation of results, fertilized egg triploid rate raise gradually along with the prolongation of soak time, do not have to embody the phenomenon of a rate of setting out and the reduction of seed incubation rate in 15min, and the seed incubation rate slightly reduces when soaking 20min, but the seed triploid rate is not promoted.
Table 2 different disposal time 6-DMAP solution soaks fertilized egg to the influence of triploid seed
| Duration (min) | The hatching water temperature (℃) | Send out eye rate (%) | Incubation rate | Triploid rate (%) |
| 5 | 7 | 62 | 89 | 43 |
| 10 | 7 | 65 | 88 | 68 |
| 15 | 7 | 61 | 91 | 81 |
| 20 | 7 | 60 | 85 | 82 |
Claims (10)
1. the preparation method of a golden trout and rainbow trout (Salmo gairdneri) hybridization triploid seed is characterized in that, may further comprise the steps:
1) makes golden trout parent population ovum and the exomixis of rainbow trout (Salmo gairdneri) parent population sperm;
2) the 6-dimethylaminopurine is handled fertilized egg;
3) fertilized egg is hatched in breeding water.
2. preparation method as claimed in claim 1 is characterized in that, the fertilization temperature of step 1) is 6-8 ℃.
3. preparation method as claimed in claim 1 is characterized in that, it is 10-13 ℃ that step 1) gold trout parent population, rainbow trout (Salmo gairdneri) parent population are cultivated water temperature, and flowing water pond water exchange capacity is 2h/ time.
4. preparation method as claimed in claim 1, it is characterized in that, described step 1) is taken out the seminal fluid of the female parent population fish-egg of golden trout and the male parent population of rainbow trout (Salmo gairdneri) respectively, seminal fluid cleans with the hatching water, method is that seminal fluid is 1-1.5:2500-3000 with the volume ratio of hatching water, outwell water after stirring 1-1.5min, wash 3-5 time repeatedly with quadrat method; Mantissa's ratio according to male parent population and female parent population 1:2-4 is carried out the fertilization of sperm and ovum.
5. preparation method as claimed in claim 1 is characterized in that step 2) in, fertilized egg moves into fertilized egg immersion treatment in the 6-dimethylaminopurine solution of 170-190mg/L behind fertilization 12-14min.
6. preparation method as claimed in claim 5 is characterized in that, the temperature of 6-dimethylaminopurine solution is 12-14 ℃.
7. preparation method as claimed in claim 5 is characterized in that, soak time is 14-16min.
8. preparation method as claimed in claim 1 is characterized in that, with step 2) fertilized egg of immersion treatment moved in the 6-8 ℃ of hatching water body hatching rapidly 48-60 days.
9.6-the application of dimethylaminopurine in the golden trout of preparation and rainbow trout (Salmo gairdneri) hybridization triploid seed.
10. application as claimed in claim 9 is characterized in that, is that 170-190mg/L, temperature are 12-14 ℃ 6-dimethylaminopurine solution immersion treatment gold trout and rainbow trout (Salmo gairdneri) fertilized egg with concentration.
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| CN107581111A (en) * | 2017-11-08 | 2018-01-16 | 重庆市同盟农业开发有限公司 | A kind of Triploid Rainbow Trout fish seeding technique method |
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