CN103250667A - Method for cultivating arctic charr triploid offspring seeds - Google Patents
Method for cultivating arctic charr triploid offspring seeds Download PDFInfo
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- CN103250667A CN103250667A CN2013101955015A CN201310195501A CN103250667A CN 103250667 A CN103250667 A CN 103250667A CN 2013101955015 A CN2013101955015 A CN 2013101955015A CN 201310195501 A CN201310195501 A CN 201310195501A CN 103250667 A CN103250667 A CN 103250667A
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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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- Y02A40/80—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in fisheries management
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Abstract
The invention provides a method for cultivating arctic charr triploid offspring seeds. Oosperms of arctic charrs are placed at 6-8 DEG C, the oosperms are fertilized for 12-14 min, then the oosperms are moved into a 6-DMAP (6-Dimethylaminopurine) solution with concentration being 220-240mg/L and temperature being 9-11 DEG C to be socked for 14-16 min, and the oosperms are moved into a 6-8 DEG C water body to be normally incubated. After about 60 days, fries of the arctic charrs are incubated from the oosperms. During the incubating process, developmental rate of the oosperms is above 61 %, the hatching rate of the fries reaches above 90%, the obtained fries are detected through a flow cytometry, and the triploid rate reaches 79%. According to the method for cultivating the arctic charr triploid offspring seeds, the problems, caused by long-time near relation propagation and shortage of systematic seed selection, of sexual precocity, individual miniaturization, disease breeding and the like in a breeding individual are relieved, and the method has good market value.
Description
Technical field
The present invention relates to the aquatic biological breeding technical field, relate to the preparation method of a kind of arctic charr 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.
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 is that it is 67.80%-70.28% that fertilized egg is sent out a rate when under 26.5 ℃ condition, and the fry triploid rate can reach 50%-65%; When under 28 ℃ of conditions, it is 29.78%-32.75% that fertilized egg is sent out a rate, and the fry triploid rate can reach 80.95%-83.18%; Under 30 ℃ of conditions; can access very high triploid rate; reach more than 90%; but its eye rate is no more than 10% usually; and seed abnormal rate height; 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 induction so far especially.
Summary of the invention
The object of the present invention is to provide the preparation method of a kind of arctic charr triploid seed, to overcome the deficiencies in the prior art.
The preparation method of arctic charr triploid seed provided by the invention may further comprise the steps:
1) makes the fertilization of arctic charr parent population ovum and sperm in vitro;
2) the 6-dimethylaminopurine is handled fertilized egg;
3) fertilized egg is hatched in breeding water.
Wherein, the arctic charr fish-egg of step 1) fertilization temperature is 6-8 ℃.
Step 1) arctic charr parent fish rearing water temperature is 8-10 ℃, and flowing water pond water exchange capacity is 2h/ time.
Described step 1) is taken out arctic charr 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 220-240mg/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 9-11 ℃.Soak time is 14-16min.
With step 2) fertilized egg of immersion treatment moved in the 6-8 ℃ of hatching water body hatching rapidly 55-65 days, obtained arctic charr triploid prelarva.
The present invention also provides the application of 6-dimethylaminopurine (6-DMAP) in preparation arctic charr triploid seed.
Further, adopting concentration is that 220-240mg/L, temperature are 9-11 ℃ 6-dimethylaminopurine solution-treated arctic charr fertilized egg.
Preferably, the preparation method of a kind of arctic charr triploid seed is provided in an embodiment of the present invention, concrete operations are: choose arctic charr raun fish-egg and the milter sperm carries out in vitro fertilization, under 7 ℃ of conditions behind the fish-egg fertilization 12min, it is that 240mg/L, temperature are to soak arctic charr fertilized egg 15min in 10 ℃ the 6-DMAP solution that fertilized egg is moved into concentration rapidly; Move into normal the hatching 60 days in 7 ℃ of water bodys afterwards rapidly, obtain arctic charr 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 arctic charr triploid seed breeding work.
The present invention is surprised to find that and passes through finite concentration; the 6-dimethylaminopurine solution-treated arctic charr fertilized egg of temperature; can suppress the discharge of arctic charr fertilized egg second polar body; on this basis; the present invention gropes experiment condition repeatedly through a large amount of experiments; realized scale preparation arctic charr triploid seed; the fertilized egg of using this method is sent out a rate and is reached 61%; the seed incubation rate reaches more than 90%; the seed triploid rate reaches 79%; 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 triploid seed, the arctic charr seed that obtains has triploid advantage.
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.Arctic charr parent population of the present invention is that the eyed eggs cultivation that Beijing Fisheries Research Institute is bought by Northern Europe agate bio tech ltd forms.The 6-DMAP that uses in the embodiment of the invention is available from U.S. SIGMA company.
Embodiment 1 arctic charr 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 the arctic charr parent population is reached maturity.Arctic charr parent fish rearing water temperature is 8 ℃, 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 arctic charr raun earlier, per 4 tail rauns are 1 group, the seminal fluid of the male arctic charr of 2 tails is clamp-oned in the basin, water temperature is 8 ℃, stirs 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 arctic charr fish-egg fertilization 12min, it is that 240mg/L, temperature are immersion treatment in 10 ℃ 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 60 days, during triploid zygote to send out a rate be 61%, the seed incubation rate is 92%, it is 79% arctic charr triploid seed that its seed detects to triploid rate through flow cytometer.Stage at eyed eggs to 4 monthly age triploid survival rate is for sending out 78% of the eye stage.
Embodiment 2 arctic charr triploid seed preparation methods (2)
The employing current stimulate, control illumination, and the parent population of throwing something and feeding is strengthened means such as manufactured feed, and the arctic charr parent population is reached maturity.Arctic charr parent fish rearing water temperature is 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 arctic charr raun earlier, per 4 tail rauns are 1 group, the seminal fluid of the male arctic charr of 2 tails is clamp-oned in the basin, water temperature is 7 ℃, stirs 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 arctic charr fish-egg fertilization 13min, it is that 220mg/L, temperature are immersion treatment in 10 ℃ 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 62 days, during triploid zygote to send out a rate be 65%, the seed incubation rate is 90%, it is 77% arctic charr triploid seed that its seed detects to triploid rate through flow cytometer.Stage at eyed eggs to 4 monthly age triploid survival rate is for sending out 80% of the eye stage.
Embodiment 3 arctic charr triploid seed preparation methods (3)
The employing current stimulate, control illumination, and the parent population of throwing something and feeding is strengthened means such as manufactured feed, and the arctic charr parent population is reached maturity.Arctic charr parent fish rearing water temperature is 9 ℃, 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 arctic charr raun earlier, per 4 tail rauns are 1 group, the seminal fluid of the male arctic charr of 2 tails is clamp-oned in the basin, water temperature is 6 ℃, stirs 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 arctic charr fish-egg fertilization 14min, it is that 240mg/L, temperature are immersion treatment in 9 ℃ 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 triploid zygote to send out a rate be 63%, the seed incubation rate is 91%, it is 81% arctic charr triploid seed that its seed detects to triploid rate through flow cytometer.Stage at eyed eggs to 4 monthly age triploid survival rate is for sending out 79% of the eye stage.
Embodiment 4 arctic charr triploid seed preparation methods (4)
The employing current stimulate, control illumination, and the parent population of throwing something and feeding is strengthened means such as manufactured feed, and the arctic charr parent population is reached maturity.Arctic charr parent fish rearing water temperature is 8 ℃, 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 arctic charr raun earlier, per 4 tail rauns are 1 group, the seminal fluid of the male arctic charr of 2 tails is clamp-oned in the basin, water temperature is 8 ℃, stirs 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 arctic charr fish-egg fertilization 12.5min, it is that 220mg/L, temperature are immersion treatment in 11 ℃ 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 61 days, during triploid zygote to send out a rate be 62%, the seed incubation rate is 91%, it is 77% arctic charr triploid seed that its seed detects to triploid rate through flow cytometer.Stage at eyed eggs to 4 monthly age triploid survival rate is for sending out 85% of the eye stage.
Embodiment 5 variable concentrations 6-DMAP solution are to the influence of triploid seed incubation rate and triploid rate
The 6-DMAP solution of each concentration in the preparation following table 1, temperature be 10 ℃ stand-by.Behind the arctic charr fish-egg fertilization 12min, in the basin that fertilized egg pouring into leaked, treat that fertilized egg does not have water to flow out the back and moves into rapidly in the 6-DMAP solution of following variable concentrations behind the immersion treatment 15min, move into rapidly in 7 ℃ of hatching water bodys and normally hatch that other breeding conditions are referring to embodiment 1.Hatched 60 days, after observation and the record variable concentrations 6-DMAP solution-treated fertilized egg, the influence to arctic charr fish triploid seed the results are shown in Table 1.
Table 1 variable concentrations 6-DMAP solution-treated fertilized egg is to the influence of triploid seed
Embodiment 66-DMAP solution-treated fertilized egg different time is to the influence of triploid seed
Cultivate arctic charr fish triploid seed with reference to the method for embodiment 1, wherein concentration is that 240mg/L, temperature are 10 ℃ 6-DMAP solution-treated arctic charr fish fertilized egg asynchronism(-nization), sees Table 2, and other breeding conditions are with embodiment 1.Hatched 60 days, and observed and record 6-DMAP solution-treated fertilized egg different time, the influence to arctic charr fish triploid seed the results are shown in Table 2.The result shows, fertilized egg is at 5-15min in the processing time, fertilized egg send out an eye rate, the seed incubation rate does not have significant change, but along with the growth in processing time, when 15min, the seed triploid rate significantly increases.
Table 2 different disposal time 6-DMAP solution soaks fertilized egg to the influence of triploid seed
Claims (10)
1. the preparation method of an arctic charr triploid seed is characterized in that, may further comprise the steps:
1) makes the fertilization of arctic charr parent population ovum and sperm in vitro;
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 arctic charr fish-egg fertilization temperature of step 1) is 6-8 ℃.
3. preparation method as claimed in claim 1 is characterized in that, step 1) arctic charr parent fish rearing water temperature is 8-10 ℃, 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 arctic charr parent population fish-egg and seminal fluid 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 220-240mg/L behind fertilization 12-14min.
6. preparation method as claimed in claim 5 is characterized in that, the temperature of 6-dimethylaminopurine solution is 9-11 ℃.
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 55-65 days.
9.6-the application of dimethylaminopurine in preparation arctic charr triploid seed.
10. application as claimed in claim 9 is characterized in that, is that 220-240mg/L, temperature are 9-11 ℃ 6-dimethylaminopurine solution-treated arctic charr fertilized egg with concentration.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109874708A (en) * | 2019-04-09 | 2019-06-14 | 北京市水生野生动植物救护中心 | A kind of method for culturing seedlings of land envelope type cherry salmon triploid seed |
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| CN102696515A (en) * | 2012-06-01 | 2012-10-03 | 北京市水产科学研究所 | Preparation method of game fish triploid fries |
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- 2013-05-23 CN CN201310195501.5A patent/CN103250667B/en not_active Expired - Fee Related
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN1177437A (en) * | 1997-09-09 | 1998-04-01 | 中国科学院海洋研究所 | Method for breeding young of prawns in three times size of general ones |
| CN1385060A (en) * | 2001-05-16 | 2002-12-18 | 中国科学院海洋研究所 | Inductest for triploid of Eriocheir sinensia |
| CN101606501A (en) * | 2009-07-22 | 2009-12-23 | 北京顺通虹鳟鱼养殖中心 | Technical method of triploid seed production of rainbow trout |
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Non-Patent Citations (1)
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| 龙强等: "虹鳟鱼三倍体的育种技术", 《北京水产》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109874708A (en) * | 2019-04-09 | 2019-06-14 | 北京市水生野生动植物救护中心 | A kind of method for culturing seedlings of land envelope type cherry salmon triploid seed |
| CN109874708B (en) * | 2019-04-09 | 2022-03-15 | 北京市水生野生动植物救护中心 | Seedling raising method for land-sealed triploid fry of salmon |
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