CN106135093A - A kind of method improving Mylopharyngodon piceus triploid incubation rate - Google Patents
A kind of method improving Mylopharyngodon piceus triploid incubation rate Download PDFInfo
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- CN106135093A CN106135093A CN201610497870.3A CN201610497870A CN106135093A CN 106135093 A CN106135093 A CN 106135093A CN 201610497870 A CN201610497870 A CN 201610497870A CN 106135093 A CN106135093 A CN 106135093A
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- 238000000034 method Methods 0.000 title claims abstract description 43
- 208000026487 Triploidy Diseases 0.000 title claims abstract description 27
- 241001275898 Mylopharyngodon piceus Species 0.000 title claims abstract description 23
- 238000011534 incubation Methods 0.000 title claims abstract description 15
- NKANXQFJJICGDU-QPLCGJKRSA-N Tamoxifen Chemical compound C=1C=CC=CC=1C(/CC)=C(C=1C=CC(OCCN(C)C)=CC=1)/C1=CC=CC=C1 NKANXQFJJICGDU-QPLCGJKRSA-N 0.000 claims abstract description 60
- 239000000243 solution Substances 0.000 claims abstract description 35
- 238000002347 injection Methods 0.000 claims abstract description 31
- 239000007924 injection Substances 0.000 claims abstract description 31
- 229960001603 tamoxifen Drugs 0.000 claims abstract description 30
- 239000003112 inhibitor Substances 0.000 claims abstract description 24
- 239000000262 estrogen Substances 0.000 claims abstract description 18
- 229940011871 estrogen Drugs 0.000 claims abstract description 18
- 208000035199 Tetraploidy Diseases 0.000 claims abstract description 16
- 230000002706 hydrostatic effect Effects 0.000 claims abstract description 16
- 230000035939 shock Effects 0.000 claims abstract description 14
- 238000003825 pressing Methods 0.000 claims abstract description 12
- 238000002360 preparation method Methods 0.000 claims abstract description 11
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 16
- MUMGGOZAMZWBJJ-DYKIIFRCSA-N Testostosterone Chemical compound O=C1CC[C@]2(C)[C@H]3CC[C@](C)([C@H](CC4)O)[C@@H]4[C@@H]3CCC2=C1 MUMGGOZAMZWBJJ-DYKIIFRCSA-N 0.000 claims description 16
- 210000004602 germ cell Anatomy 0.000 claims description 14
- 239000010413 mother solution Substances 0.000 claims description 12
- 230000008774 maternal effect Effects 0.000 claims description 8
- 229960003604 testosterone Drugs 0.000 claims description 8
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 8
- 239000003205 fragrance Substances 0.000 claims description 6
- ZAMOUSCENKQFHK-UHFFFAOYSA-N Chlorine atom Chemical compound [Cl] ZAMOUSCENKQFHK-UHFFFAOYSA-N 0.000 claims description 4
- 235000007164 Oryza sativa Nutrition 0.000 claims description 4
- 229910052801 chlorine Inorganic materials 0.000 claims description 4
- 239000000460 chlorine Substances 0.000 claims description 4
- GKIRPKYJQBWNGO-OCEACIFDSA-N clomifene Chemical compound C1=CC(OCCN(CC)CC)=CC=C1C(\C=1C=CC=CC=1)=C(\Cl)C1=CC=CC=C1 GKIRPKYJQBWNGO-OCEACIFDSA-N 0.000 claims description 4
- 229960003608 clomifene Drugs 0.000 claims description 4
- 230000008775 paternal effect Effects 0.000 claims description 4
- 230000000149 penetrating effect Effects 0.000 claims description 4
- 230000002085 persistent effect Effects 0.000 claims description 4
- 239000000843 powder Substances 0.000 claims description 4
- 235000009566 rice Nutrition 0.000 claims description 4
- 206010062767 Hypophysitis Diseases 0.000 claims description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 2
- 239000003795 chemical substances by application Substances 0.000 claims description 2
- 239000012895 dilution Substances 0.000 claims description 2
- 238000010790 dilution Methods 0.000 claims description 2
- 238000004821 distillation Methods 0.000 claims description 2
- 239000007788 liquid Substances 0.000 claims description 2
- 230000032696 parturition Effects 0.000 claims description 2
- 240000007594 Oryza sativa Species 0.000 claims 1
- 238000009395 breeding Methods 0.000 abstract description 3
- 230000001488 breeding effect Effects 0.000 abstract description 2
- 230000007423 decrease Effects 0.000 abstract description 2
- 239000008239 natural water Substances 0.000 abstract description 2
- 241000251468 Actinopterygii Species 0.000 description 7
- 241000209094 Oryza Species 0.000 description 3
- 210000000349 chromosome Anatomy 0.000 description 2
- 238000003776 cleavage reaction Methods 0.000 description 2
- 239000012153 distilled water Substances 0.000 description 2
- 238000005304 joining Methods 0.000 description 2
- 230000007017 scission Effects 0.000 description 2
- 230000001629 suppression Effects 0.000 description 2
- 241000252210 Cyprinidae Species 0.000 description 1
- 230000004543 DNA replication Effects 0.000 description 1
- 208000035240 Disease Resistance Diseases 0.000 description 1
- 206010020852 Hypertonia Diseases 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 230000009977 dual effect Effects 0.000 description 1
- 239000013505 freshwater Substances 0.000 description 1
- 238000009396 hybridization Methods 0.000 description 1
- 230000013011 mating Effects 0.000 description 1
- 235000013372 meat Nutrition 0.000 description 1
- 230000011278 mitosis Effects 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 238000002791 soaking Methods 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 238000005728 strengthening Methods 0.000 description 1
- 230000004083 survival effect Effects 0.000 description 1
Classifications
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K61/00—Culture of aquatic animals
-
- 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
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
- Y02A40/80—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in fisheries management
- Y02A40/81—Aquaculture, e.g. of fish
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- Life Sciences & Earth Sciences (AREA)
- Environmental Sciences (AREA)
- Marine Sciences & Fisheries (AREA)
- Zoology (AREA)
- Animal Husbandry (AREA)
- Biodiversity & Conservation Biology (AREA)
- Medicines Containing Plant Substances (AREA)
Abstract
The invention discloses a kind of method improving Mylopharyngodon piceus triploid incubation rate, comprise the steps: the outfit of tamoxifen inhibitor, the preparation of tamoxifen injection, injection treatment female parent, cold shock+hydrostatic pressing process, the preparation of estrogen inhibitors solution, estrogen inhibitors solution soak at room temperature, triploid acquisition: take male tetraploid as male parent, take common diploid as female parent, use the mode backcrossed, it is thus achieved that triploid Mylopharyngodon piceus is.By the method, can obtain the Mylopharyngodon piceus triploid pure lines of 100%, improve triploid incubation rate and emergence rate, decrease the trouble of breeding and seed selection, more importantly matter all to Natural Water product and ecological environment are pollution-free.
Description
Technical field
The present invention relates to the triploid mating system of Mylopharyngodon piceus, be specifically related to a kind of side improving Mylopharyngodon piceus triploid incubation rate
Method.
Background technology
Mylopharyngodon piceus is one of four large Chinese carps of CHINESE FRESHWATER cultivation.The sterile feature of triploid Mylopharyngodon piceus so that it is the speed of growth,
The aspects such as resistance, disease resistance and meat show significantly more advantage.In prior art, sterile triploid Mylopharyngodon piceus is logical
Cross tetraploid fish to obtain with liploid fish interspecies crossing, but to obtain the triploid fish of more than 98%, then be accomplished by setting up heredity
Stable, the tetraploid fish system of very high purity.
In genetics-breeding in fish, cold heat is generally utilized to suffer a shock, or the first time by method suppression germ cell such as hybridization
Mitosis or directly chromosome nonhomologous double, and prepare homology or allotrtraploid fish, and wherein cold shock is usually and hydrostatic
Pressure process together, it is difficult to obtain higher degree tetraploid system, although and the mode of heat shock is easily operated, equally, it is difficult to obtain
Obtain the tetraploid system that purity is high；Although and the method purity height hybridized, but success rate is low, the cycle is long.Additionally, these methods above-mentioned
The tetraploid fish of preparation, some can be educated, some can not be educated again, also likely to be present gene simultaneously and mixes phenomenon (allotetraploid),
Easily cause bio-hazard.
Summary of the invention
The deficiency existed for above-mentioned prior art, it is an object of the invention to provide the raising Mylopharyngodon piceus three that a kind of method is stable
The method of times body incubation rate.
For reaching above-mentioned purpose, technical scheme is as follows:
A kind of method improving Mylopharyngodon piceus triploid incubation rate, comprises the steps:
(1) tamoxifen inhibitor is equipped with: take the tamoxifen powder that purity is more than 95%, and the ethanol solution of addition 90% is carried out
Dissolving, make mother solution, wherein tamoxifen content is about 60～90mg/ml, stand-by；
(2) preparation of tamoxifen injection: take above-mentioned mother solution and be added directly in the odinagogue solution prepared, its ratio
Join for 1ml tamoxifen mother solution in the odinagogue solution prepared of 240～400ml, make injection stand-by；Hasten parturition
Agent solution is to be prepared through 0.9% normal saline dilution by hypophysis cerebri, and the mesencephalohypophysial dosage of odinagogue solution is 0.1-0.2mg/
g；
(3) injection treatment is maternal: before maternal artificial spawning, routinely induced spawning method female parent is carried out manual injection he not
Former times sweet smell injection, wherein the consumption of tamoxifen injection is about 40～160 μ g/kg, i.e. every kg female parent injection tamoxifen note
Penetrate liquid 40-160 μ g；
(4) cold shock+hydrostatic pressing processes: when a spilting of an egg, uses the method that cold shock combines with hydrostatic pressing, through note
The female parent penetrating process puts into, with the germ cell obtained without the paternal hybrid of any process, the cold water that temperature is-1～1.0 DEG C
In, hydrostatic pressing pressure is 250～340Kg/cm2, the persistent period is 3～5min；
(5) preparation of estrogen inhibitors solution: clomifene is dissolved in the ethanol of 80%～90% together with testosterone, then adds distillation
Water is configured to estrogen inhibitors solution, and the concentration that wherein estrogen inhibitors Chlorine in Solution rice is fragrant is 60～180mg/l, testosterone
Concentration be 10～50mg/l；
(6) taking the germ cell after step (4) processes and put in the estrogen inhibitors solution as prepared by step (5), room temperature soaks
Bubble 10～30s, then takes out and is put in normal incubation in couveuse；Thus obtain Mylopharyngodon piceus male tetraploid pure lines；
(7) triploid acquisition: take the male tetraploid of step (6) as male parent, takes common diploid as female parent, uses back
The mode handed over, it is thus achieved that the triploid Mylopharyngodon piceus system of 100%.
Advantages of the present invention: 1, inject tamoxifen in the present invention in female parent, play suppression DNA replication dna and promote male
While dual function, the germ cell First cleavage time is put off 3-5min, and improves all germ cell First cleavages
Synchronization degree (up to 99%), for chromosome doubling and improve its tetraploid rate provide the time ensure and established solid base
Plinth；2, use cold shock and relatively low hydrostatic pressing (less than conventional 350kg/cm in spilting of an egg of germ cell2), and shorter
The process time (less than 5min), not only effectively reduce because of cold shock overlong time, the hypertonia destructiveness to germ cell,
Improve the survival rate of germ cell, and reduce operation easier and pressurization cost；3, estrogen inhibitors solution soaking, again
Strengthening and the male conversion ratio of guarantee；4, backcross with original diploid female with the male tetraploid of pure lines, be effectively ensured three
The purity of times body.Thereby through the method, the Mylopharyngodon piceus triploid pure lines of 100% can be obtained, improve triploid incubation rate and go out
Seedling rate, decreases the trouble of breeding and seed selection, and more importantly matter all to Natural Water product and ecological environment are pollution-free.
Detailed description of the invention
In conjunction with specific embodiment, the invention will be further described.
Embodiment one
The method improving Mylopharyngodon piceus triploid incubation rate, comprises the steps:
(1) tamoxifen inhibitor is equipped with: take the tamoxifen powder that purity is more than 95%, and the ethanol solution of addition 90% is carried out
Dissolving, make mother solution, wherein tamoxifen content is about 90mg/ml, stand-by；
(2) preparation of tamoxifen injection: take above-mentioned mother solution and be added directly in the odinagogue solution prepared, its ratio
Joining for 1ml tamoxifen mother solution in the odinagogue solution prepared of 240ml, make injection, in injection, he is not
The former times concentration of sweet smell is about 0.375mg/ml, stand-by；
(3) injection treatment is maternal: before maternal artificial spawning, routinely induced spawning method female parent is carried out manual injection he not
Former times sweet smell injection, wherein the consumption of tamoxifen injection is about 80 μ g/kg；
(4) cold shock+hydrostatic pressing processes: when a spilting of an egg, uses the method that cold shock combines with hydrostatic pressing, through note
The female parent penetrating process is put in the cold water that temperature is 0 DEG C with the germ cell obtained without the paternal hybrid of any process, hydrostatic
Pressure pressure is 280Kg/cm2, the persistent period is 5min；
(5) preparation of estrogen inhibitors solution: clomifene is dissolved in the ethanol of 90% together with testosterone, then add distilled water and join
Making estrogen inhibitors solution, the concentration that wherein estrogen inhibitors Chlorine in Solution rice is fragrant is 60mg/l, and the concentration of testosterone is
10mg/l；
(6) male tetraploid pure lines acquisition: take step (4) process after germ cell put into as prepared by step (5) female swash
In element inhibitor solution, soak at room temperature 30s, then take out and be put in normal incubation in couveuse；Thus obtain male four times of Mylopharyngodon piceus
Body is sheerly；
(7) triploid acquisition: take the male tetraploid of step (6) as male parent, takes common diploid as female parent, uses back
The mode handed over, it is thus achieved that 100% triploid.
Embodiment two
The method improving Mylopharyngodon piceus triploid incubation rate, comprises the steps:
(1) tamoxifen inhibitor is equipped with: take the tamoxifen powder that purity is more than 95%, and the ethanol solution of addition 90% is carried out
Dissolving, make mother solution, wherein tamoxifen content is about 60mg/ml, stand-by；
(2) preparation of tamoxifen injection: take above-mentioned mother solution and be added directly in the odinagogue solution prepared, its ratio
Joining for 1ml tamoxifen mother solution in the odinagogue solution prepared of 400ml, make injection, in injection, he is not
The former times concentration of sweet smell is 0.15mg/ml, stand-by；
(3) injection treatment is maternal: before maternal artificial spawning, routinely induced spawning method female parent is carried out manual injection he not
Former times sweet smell injection, wherein the consumption of tamoxifen injection is about 160 μ g/kg；
(4) cold shock+hydrostatic pressing processes: when a spilting of an egg, uses the method that cold shock combines with hydrostatic pressing, through note
The female parent penetrating process is put in the cold water that temperature is-1 DEG C with the germ cell obtained without the paternal hybrid of any process, hydrostatic
Pressure pressure is 280Kg/cm2, the persistent period is 3min；
(5) preparation of estrogen inhibitors solution: clomifene is dissolved in the ethanol of 90% together with testosterone, then add distilled water and join
Making estrogen inhibitors solution, the concentration that wherein estrogen inhibitors Chlorine in Solution rice is fragrant is 180mg/l, and the concentration of testosterone is
50mg/l；
(6) taking the germ cell after step (4) processes and put in the estrogen inhibitors solution as prepared by step (5), room temperature soaks
Bubble 10s, then takes out and is put in normal incubation in couveuse；Thus obtain 100% Mylopharyngodon piceus male tetraploid pure lines；
(7) triploid acquisition: take the male tetraploid of step (6) as male parent, takes common diploid as female parent, uses back
The mode handed over, it is thus achieved that 100% triploid.
Above-mentioned two embodiment is only the two of the preferred version of the present invention, can not limit the scope of the invention, and appoints
Meaning is equal to the present invention or similar technological means is considered as protection scope of the present invention.Hereinafter enclose part processing mode of the present invention
And result statistics.
Attached: Mylopharyngodon piceus triploid is bred as result statistics
Note: the above results is soak at room temperature 10S under conditions of the concentration of tamoxifen is 0.15mg/ml in injection, hydrostatic
Pressure processes 3 minutes acquired results.
Claims (1)
1. the method improving Mylopharyngodon piceus triploid incubation rate, comprises the steps:
(1) tamoxifen inhibitor is equipped with: take the tamoxifen powder that purity is more than 95%, and the ethanol solution of addition 90% is carried out
Dissolving, make mother solution, wherein tamoxifen content is about 60～90mg/ml, stand-by；
(2) preparation of tamoxifen injection: take above-mentioned mother solution and be added directly in the odinagogue solution prepared, its ratio
Join for 1ml tamoxifen mother solution in the odinagogue solution prepared of 240～400ml, make injection stand-by；Hasten parturition
Agent solution is to be prepared through 0.9% normal saline dilution by hypophysis cerebri, and the mesencephalohypophysial dosage of odinagogue solution is 0.1-0.2mg/
g；
(3) injection treatment is maternal: before maternal artificial spawning, routinely induced spawning method female parent is carried out manual injection he not
Former times sweet smell injection, wherein the consumption of tamoxifen injection is about 40～160 μ g/kg, i.e. every kg female parent injection tamoxifen note
Penetrate liquid 40-160 μ g；
(4) cold shock+hydrostatic pressing processes: when a spilting of an egg, uses the method that cold shock combines with hydrostatic pressing, through note
The female parent penetrating process puts into, with the germ cell obtained without the paternal hybrid of any process, the cold water that temperature is-1～1.0 DEG C
In, hydrostatic pressing pressure is 250～340Kg/cm2, the persistent period is 3～5min；
(5) preparation of estrogen inhibitors solution: clomifene is dissolved in the ethanol of 80%～90% together with testosterone, then adds distillation
Water is configured to estrogen inhibitors solution, and the concentration that wherein estrogen inhibitors Chlorine in Solution rice is fragrant is 60～180mg/l, testosterone
Concentration be 10～50mg/l；
(6) taking the germ cell after step (4) processes and put in the estrogen inhibitors solution as prepared by step (5), room temperature soaks
Bubble 10～30s, then takes out and is put in normal incubation in couveuse；Thus obtain Mylopharyngodon piceus male tetraploid pure lines；
(7) triploid acquisition: take the male tetraploid of step (6) as male parent, takes common diploid as female parent, uses back
The mode handed over, it is thus achieved that the triploid Mylopharyngodon piceus system of 100%.
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| Application Number | Priority Date | Filing Date | Title |
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| CN201610497870.3A CN106135093A (en) | 2016-06-30 | 2016-06-30 | A kind of method improving Mylopharyngodon piceus triploid incubation rate |
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| CN201610497870.3A CN106135093A (en) | 2016-06-30 | 2016-06-30 | A kind of method improving Mylopharyngodon piceus triploid incubation rate |
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| CN201610497870.3A Pending CN106135093A (en) | 2016-06-30 | 2016-06-30 | A kind of method improving Mylopharyngodon piceus triploid incubation rate |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107027662A (en) * | 2017-03-24 | 2017-08-11 | 覃忠 | A kind of bar house oil fish hybridization triploid artificial propagation techniques |
| CN107711615A (en) * | 2017-10-31 | 2018-02-23 | 金华职业技术学院 | A kind of method that sterile fish is obtained using immersion fish-egg |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107027662A (en) * | 2017-03-24 | 2017-08-11 | 覃忠 | A kind of bar house oil fish hybridization triploid artificial propagation techniques |
| CN107711615A (en) * | 2017-10-31 | 2018-02-23 | 金华职业技术学院 | A kind of method that sterile fish is obtained using immersion fish-egg |
| CN107711615B (en) * | 2017-10-31 | 2020-08-11 | 金华职业技术学院 | Method for obtaining sterile fish by soaking fish eggs |
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Application publication date: 20161123 |