CN103238547B - Method for preparing golden trout and brook trout hybridization triploid fries - Google Patents
Method for preparing golden trout and brook trout hybridization triploid fries Download PDFInfo
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- CN103238547B CN103238547B CN201310195318.5A CN201310195318A CN103238547B CN 103238547 B CN103238547 B CN 103238547B CN 201310195318 A CN201310195318 A CN 201310195318A CN 103238547 B CN103238547 B CN 103238547B
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Abstract
The invention provides a method for preparing golden trout and brook trout hybridization triploid fries. Fertilized eggs are obtained by hybridizing a golden trout and a brook trout, soaked in 6-DMAP (dimethylaminopurine) solution, and then hatched and cultivated to generate the hybridization triploid fries. The eyed rate of the fertilized eggs is higher than 78% during hatching, the hatching rate of the fries reaches more than 90%, and the triploid rate of obtained larva fishes reaches 95% as the larva fishes are tested by a flow cytometry. By the method, the golden trout and brook trout hybridization triploid fries can be produced in a large-scaled manner, so that the growth speed and the disease resistance of trouts are improved, economic benefits of farmers are improved, and fine market values are achieved.
Description
Technical field
The present invention relates to aquatic biological breeding technical field, relate to the preparation method of a kind of golden trout and brook trout hybridization triploid seed particularly.
Background technology
Salmon trout is one of fish of mainly cultivating at present of the world, due to the quality of himself and the particular/special requirement to living environment, determines that its is green, the character of environmental protection nutritive food, therefore quite by the favor of consumer.Salmon trout is the main at present cold aqueous cultured fishes of China, along with developing rapidly of China's salmon trout cultivation, fundamental research relatively lags behind, under the degeneration of salmon trout parent population kind matter causes fry quality degeneration productivity effect, degradation problem is given prominence to gradually, thus constrains the sustainable development of salmon trout aquaculture.Utilize modern biotechnology, cultivate growth rapidly, individual large, meat is good, resistance against diseases is strong seedling, the continuing of China's salmon trout aquaculture, stable, the only way of developing in a healthy way will be become.
Hybridization sharply can shake the conservative of heredity, makes the inheritance of hybrid be imbued with motility, has larger plasticity, thus can improve rapidly and significantly the vitality of hybrid, thus obtains hybrid vigour; Hybrid can enrich genetic structure, and by hybridization by kinds different for two or more hereditary basis or the independent assortment of genes of planting above individuality, occur new hereditary form, people can select excellent individuality then, cultivate into new varieties.
Compared with common dliploid fish, the advantages such as triploid fish have fast growth, survival rate is high, premunition is strong, meat is good, safety and Health: 1. triploid fish growth rate 30-40% faster than dliploid, be conducive to producing Large scale individual, effectively can improve population yield; ②Gui section triploid fish are sterile, avoid gonad development stage and spawning season and decline and appearance features change because of the flesh quality that energy trasfer and nuptial coloration occur and cause, avoid its biological death breeding period, extend the culture-cycle; 3. 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, belonging to the mankind can the healthy food of safe edible, compared with transgenic technology more people accepted; 5. triploid fish is Fresh & Tender in Texture, edible part is high, the bodily form stablize attractive in appearance, commodity value is high.Because triploid fish have above-mentioned advantage, therefore by triploid technology of preparing obtain growth fast, adaptability and the strong high-quality seed of premunition have huge potentiality.
Since the eighties in last century, artificial induction's work of triploid fish makes significant progress.The research prepared triploid seed at present mainly concentrates on the triploid preparation of purebred rainbow trout, and its method is heat shock or hydrostatic pressing method.The technology that existing heat shock procedures prepares purebred Triploid Rainbow Trout fish depends on accurate temperature; temperature does not slightly convert; seed abnormal rate can be caused high; make way to cultivate unstable; all the time large-scale production cannot be formed; its reason is that the triploid rate that existing purebred production triploid seed technology of preparing maybe cannot reach production requirement maybe cannot reach the weed survival rate that can be used for producing, and cannot accomplish scale production always.Therefore, in the urgent need to a kind of technology maturation, effect stability and can the triploid fry technology of preparing of large-scale production, that improves fertilized egg sends out eye rate and seed triploid rate.
6-dimethylaminopurine (N6-Dimethyladenine, 6-DMAP) in multiple polyploid shellfish induction, achieve good result, but because salmon trout is higher to growing environment water quality requirement, responsive to chemical substance especially, there is not 6-dimethylaminopurine to be applied to the reported success of salmon trout triploid or hybridization triploid induction so far.
Summary of the invention
The object of the present invention is to provide the preparation method of a kind of golden trout and brook trout hybridization triploid seed, to overcome the deficiencies in the prior art.
The preparation method of golden trout provided by the invention and brook trout hybridization triploid seed, comprises the following steps:
1) golden trout parent population ovum and the exomixis of brook trout parent population sperm is made;
2) 6-dimethylaminopurine process fertilized egg;
3) fertilized egg is hatched in breeding water.
Wherein, step 1) insemination temperature is 6-11 DEG C.
It is 10-13 DEG C that step 1) gold trout parent population cultivates water temperature, and flowing water pond water exchange capacity is 2h/ time, and brook trout parent fish rearing water temperature is 8-10 DEG C, and flowing water pond water exchange capacity is 4h/ time.
Brook trout parent population fish-egg and seminal fluid take out by described step 1) respectively, and seminal fluid Spawning water cleans, and method is the volume ratio of seminal fluid and Spawning water is 1-1.5:2500-3000, and outwell water after stirring 1-1.5min, same method rinses 3-5 time repeatedly; The fertilization of sperm and ovum is carried out according to mantissa's ratio of male parent population and female parent population 1:2-4.
The step 2 of preparation method of the present invention) in, fertilized egg, after fertilization 12-14min, is moved into immersion treatment in 6-dimethylaminopurine (6-DMAP) solution of 170-190mg/L by fertilized egg.
Preferably, fertilized egg, after fertilization 12min, moves into immersion treatment in 6-dimethylaminopurine solution.The temperature of 6-dimethylaminopurine solution is 12-14 DEG C.Soak time is 14-16min.
By step 2) fertilized egg of immersion treatment moves into rapidly in 6-11 DEG C of hatching water body and hatches 48-55 days, obtains golden trout and brook trout hybridization triploid prelarva.
Present invention also offers the application of 6-dimethylaminopurine (6-DMAP) in the golden trout of preparation and brook trout hybridization triploid seed.
Further, 6-dimethylaminopurine solution-treated gold trout and the brook trout fertilized egg that concentration is 170-190mg/L, temperature is 12-14 DEG C is adopted.
The invention provides the preparation method of a kind of golden trout and brook trout triploid seed, concrete operations are: choose golden trout female parent population fish-egg and brook trout milter parent population sperm carries out in vitro fertilization, under 7 DEG C of conditions after fish-egg fertilization 12min, fertilized egg is moved into rapidly concentration is 180mg/L, temperature is soak brook trout fertilized egg 15min in the 6-DMAP solution of 12 DEG C; To move into rapidly in 7 DEG C of water bodys normal incubation afterwards 50 days, obtain golden trout and brook trout hybridization triploid fry.
Preparation method of the present invention water body used is the Spawning water meeting national water quality standard for fishery and specify.
The invention provides the application of above-mentioned preparation method in brook trout triploid seed breeding work.
The present invention is surprised to find that and passes through finite concentration, the 6-dimethylaminopurine solution-treated gold trout of temperature and brook trout fertilized egg, the discharge of fertilized egg second polar body can be suppressed, on this basis, the present invention gropes experiment condition repeatedly through great many of experiments, achieve scale and prepare golden trout and brook trout hybridization triploid seed, the fertilized egg of application the method is sent out a rate and is reached 78%, hatching rate reaches more than 90%, seed triploid rate reaches 95%, this inventive method fertilized egg sends out a rate and normal salmon trout produces close, triploid rate reaches Production requirement, achieve the large-scale production of hybridization triploid seed, the golden trout obtained and brook trout hybrid seedlings have hybridizes and triploid advantage, existing dliploid rainbow trout is obviously better than in growth rate and resistance against diseases, there is good economic benefit and market value.
Embodiment
Following examples further illustrate 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, the amendment do the inventive method, step or condition or replacement, all belong to scope of the present invention.
If do not specialize, the conventional means that technological means used in embodiment is well known to those skilled in the art.Golden trout parent population of the present invention and brook trout parent population are that the eyed eggs cultivation that Beijing Fisheries Research Institute is bought by agate bio tech ltd, Northern Europe forms.6-DMAP purchased from American SIGMA company.
Embodiment 1 gold medal trout and brook trout hybridization triploid seed preparation method (1)
Employing current stimulate, controlled light, means such as parent population of throwing something and feeding strengthening manufactured feed etc., and golden trout parent population and brook trout parent population are synchronously reached maturity.Gold trout parent fish rearing water temperature is 10 DEG C, and flowing water pond water exchange capacity is 2h/ time, and brook trout parent fish rearing water temperature is 10 DEG C, and flowing water pond water exchange capacity is 4h/ time.
Fish-egg adopts dry method fertilization.First extrusion is adopted to clamp-on in clean porcelain basin by fish-egg fully-developed gold trout raun, every 4 tail rauns are 1 group, clamp-on in basin by the seminal fluid of the male brook trout of 2 tail again, water temperature 10 DEG C, stirs immediately, the volume ratio adding seminal fluid and Spawning water is the Spawning water of 1:2500, stir gently, after 1min, water is poured out, 3 times repeatedly, cleaned up by seminal fluid, placement etc. are pending.
After fish-egg fertilization 12min, fertilized egg is moved into rapidly concentration is 180mg/L, temperature is immersion treatment in the 6-DMAP solution of 12 DEG C, after soaking 15min, move into rapidly normal incubation in 7 DEG C of hatching water bodys.
Hatched fry through 50 days, period hybridization triploid fertilized egg to send out a rate be 82%, hatching rate is 93%, its seed through flow cytomery to be triploid rate be 95% golden trout and brook trout hybridization triploid seed.Stage at eyed eggs to 4 monthly age triploid survival rate is send out the eye stage 76%.By micro-satellite analysis of the golden trout that obtains golden trout, brook trout and the present embodiment and brook trout hybridization triploid, result illustrates that the obtained hybridization triploid of the present embodiment is golden trout and brook trout hybridization triploid.
Embodiment 2 gold medal trout and brook trout hybridization triploid seed preparation method (2)
Employing current stimulate, controlled light, means such as parent population of throwing something and feeding strengthening manufactured feed etc., and golden trout and brook trout parent population are synchronously reached maturity.Gold trout parent fish rearing water temperature is 13 DEG C, and flowing water pond water exchange capacity is 2h/ time, and brook trout parent fish rearing water temperature is 8 DEG C, and flowing water pond water exchange capacity is 4h/ time.
Fish-egg adopts dry method fertilization.First extrusion is adopted to clamp-on in clean porcelain basin by fish-egg fully-developed gold trout raun, every 4 tail rauns are 1 group, clamp-on in basin by the seminal fluid of the male brook trout of 2 tail again, water temperature 8 DEG C, stirs immediately, the volume ratio adding seminal fluid and Spawning water is the Spawning water of 1:3000, stir gently, after 1.5min, water is poured out, 3 times repeatedly, cleaned up by seminal fluid, placement etc. are pending.
After fish-egg fertilization 13min, fertilized egg is moved into rapidly concentration is 170mg/L, temperature is immersion treatment in the 6-DMAP solution of 13 DEG C, after soaking 16min, move into rapidly normal incubation in 10 DEG C of hatching water bodys.
Hatched fry through 52 days, period hybridization triploid fertilized egg to send out a rate be 78%, hatching rate is 89%, and its seed is golden trout and the brook trout hybridization triploid seed of 93% through flow cytomery triploid rate.Stage at eyed eggs to 4 monthly age triploid survival rate is send out the eye stage 81%.Adopt the micro-satellite analytical method reported in prior art, the hybridization triploid that result shows the present embodiment obtained is golden trout and brook trout hybridization triploid.
Embodiment 3 gold medal trout and brook trout hybridization triploid seed preparation method (3)
Employing current stimulate, controlled light, means such as parent population of throwing something and feeding strengthening manufactured feed etc., and golden trout and brook trout parent population are synchronously reached maturity.Gold trout parent fish rearing water temperature is 11 DEG C, and flowing water pond water exchange capacity is 2h/ time, and brook trout parent fish rearing water temperature is 9 DEG C, and flowing water pond water exchange capacity is 4h/ time.
Fish-egg adopts dry method fertilization.First extrusion is adopted to clamp-on in clean porcelain basin by fish-egg fully-developed gold trout raun, every 4 tail rauns are 1 group, clamp-on in basin by the seminal fluid of the male brook trout of 2 tail again, water temperature 11 DEG C, stirs immediately, the volume ratio adding seminal fluid and Spawning water is the Spawning water of 1.5:3000, stir gently, after 1min, water is poured out, 3 times repeatedly, cleaned up by seminal fluid, placement etc. are pending.
After fish-egg fertilization 14min, fertilized egg is moved into rapidly concentration is 190mg/L, temperature is immersion treatment in the 6-DMAP solution of 12 DEG C, after soaking 15min, move into rapidly normal incubation in 8 DEG C of hatching water bodys.
Hatched fry through 48 days, period hybridization triploid fertilized egg to send out a rate be 79%, hatching rate is 90%, and its seed is golden trout and the brook trout hybridization triploid seed of 94% through flow cytomery triploid rate.Stage at eyed eggs to 4 monthly age triploid survival rate is send out the eye stage 77%.Adopt the micro-satellite analytical method reported in prior art, the hybridization triploid that result shows the present embodiment obtained is golden trout and brook trout hybridization triploid.
Embodiment 4 gold medal trout and brook trout hybridization triploid seed preparation method (4)
Employing current stimulate, controlled light, means such as parent population of throwing something and feeding strengthening manufactured feed etc., and golden trout and brook trout parent population are synchronously reached maturity.Gold trout parent fish rearing water temperature is 12 DEG C, and flowing water pond water exchange capacity is 2h/ time, and brook trout parent fish rearing water temperature is 8 DEG C, and flowing water pond water exchange capacity is 4h/ time.
Fish-egg adopts dry method fertilization.First extrusion is adopted to clamp-on in clean porcelain basin by fish-egg fully-developed gold trout raun, every 4 tail rauns are 1 group, clamp-on in basin by the seminal fluid of the male brook trout of 2 tail again, water temperature 7.5 DEG C, stirs immediately, the volume ratio adding seminal fluid and Spawning water is the Spawning water of 1.5:2500, stir gently, after 1min, water is poured out, 5 times repeatedly, cleaned up by seminal fluid, placement etc. are pending.
After fish-egg fertilization 12.5min, fertilized egg is moved into rapidly concentration is 180mg/L, temperature is immersion treatment in the 6-DMAP solution of 14 DEG C, after soaking 14min, move into rapidly normal incubation in 7.5 DEG C of hatching water bodys.
Hatched fry through 55 days, period hybridization triploid fertilized egg to send out a rate be 96%, hatching rate is 92%, and its seed is golden trout and the brook trout hybridization triploid seed of 94% through flow cytomery triploid rate.Stage at eyed eggs to 4 monthly age triploid survival rate is send out the eye stage 77%.Adopt the micro-satellite analytical method reported in prior art, the hybridization triploid that result shows the present embodiment obtained is golden trout and brook trout hybridization triploid.
Embodiment 5 variable concentrations 6-DMAP solution is on the impact of hybridization triploid hatching rate and hybridization triploid rate
Prepare concentration be 90,120,150,180,210mg/l temperature is the 6-DMAP solution for later use of 12 DEG C.It is 14min in the water body of 9.8 DEG C that gold trout ♀ × small stream red some ♂ after fertilization fertilized egg is placed on temperature, put into the above-mentioned 6-DMAP solution prepared after 14min immediately respectively to soak, time is 15min, period stirring fertilized egg frequently, to make fertilized egg be able to even immersion, after 15min, move into normal incubation 52 days in 7 DEG C of hatching water bodys.
Table 1 variable concentrations 6-DMAP solution-treated fertilized egg is on the impact of triploid seed
Embodiment 66-DMAP solution (180mg/L, 12 DEG C) process fertilized egg different time is on the impact of hybridization triploid hatching rate and hybridization triploid rate
Cultivate golden trout and brook trout fish hybridization triploid seed with reference to the method for embodiment 1, concentration is 180mg/L, temperature is that the golden trout of 6-DMAP solution-treated of 12 DEG C is different from the processing time of brook trout fish fertilized egg, in table 2, other breeding conditions are with embodiment 1.Hatch 50 days, observe and record 6-DMAP solution-treated fertilized egg different time, on the impact of golden trout and brook trout fish hybridization triploid seed, the results are shown in Table 2.Result explanation, 6-DMAP solution is in process 5-20min time range, the eye rate of sending out of fertilized egg does not have significant change, but seed triploid rate significantly increases along with the growth in processing time in 5-15min, when being when treated 20min, hatchability of fertile eggs decreases, although amplitude is little, and from seed triploid rate not a larger increase, therefore think that processing time 15min is suitable.
The table 2 6-DMAP solution different disposal time is on the impact of triploid seed
Claims (4)
1. a preparation method for golden trout and brook trout hybridization triploid seed, is characterized in that, comprise the following steps:
1) golden trout parent population ovum and the exomixis of brook trout parent population sperm is made;
2) fertilized egg fertilization 12-14min after, with concentration be 170-190mg/L, temperature be 12-14 DEG C 6-dimethylaminopurine solution soak 14-16min;
3) by step 2) fertilized egg of immersion treatment moves into rapidly in 6-11 DEG C of hatching water body and hatches 48-55 days.
2. preparation method as claimed in claim 1, is characterized in that, step 1) temperature in vitro fertilization be 6-11 DEG C.
3. preparation method as claimed in claim 1, is characterized in that, step 1) to cultivate water temperature be 10-13 DEG C to golden trout parent population, flowing water pond water exchange capacity is 2h/ time; Brook trout parent fish rearing water temperature is 8-10 DEG C, and flowing water pond water exchange capacity is 4h/ time.
4. preparation method as claimed in claim 1, it is characterized in that, described step 1) respectively the seminal fluid of female for golden trout parent population fish-egg and the male parent population of brook trout is taken out, seminal fluid Spawning water cleans, method is the volume ratio of seminal fluid and Spawning water is 1-1.5:2500-3000, outwell water after stirring 1-1.5min, same method rinses 3-5 time repeatedly; The fertilization of sperm and ovum is carried out according to mantissa's ratio of male parent population and female parent population 1:2-4.
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| CN106417256B (en) * | 2016-10-12 | 2020-01-31 | 中国水产科学研究院黑龙江水产研究所 | method for improving sperm utilization rate of rainbow trout pseudo-male fish |
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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 |
| 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 CN201310195318.5A patent/CN103238547B/en not_active Expired - Fee Related
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
| 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 |
Non-Patent Citations (1)
| Title |
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| 虹鳟鱼三倍体的育种技术;龙强等;《北京水产》;20041025(第5期);第36页第1.2.2节 * |
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