CN106106280A - A kind of improve crucian carp, the method for Cyprinus carpio male tetraploid incubation rate - Google Patents
A kind of improve crucian carp, the method for Cyprinus carpio male tetraploid incubation rate Download PDFInfo
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- CN106106280A CN106106280A CN201610496553.XA CN201610496553A CN106106280A CN 106106280 A CN106106280 A CN 106106280A CN 201610496553 A CN201610496553 A CN 201610496553A CN 106106280 A CN106106280 A CN 106106280A
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- 238000000034 method Methods 0.000 title claims abstract description 43
- 208000035199 Tetraploidy Diseases 0.000 title claims abstract description 31
- 241000252233 Cyprinus carpio Species 0.000 title claims abstract description 23
- 241001609213 Carassius carassius Species 0.000 title claims abstract description 17
- 238000011534 incubation Methods 0.000 title claims abstract description 17
- 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
- 238000002347 injection Methods 0.000 claims abstract description 31
- 239000007924 injection Substances 0.000 claims abstract description 31
- 239000000243 solution Substances 0.000 claims abstract description 30
- 229960001603 tamoxifen Drugs 0.000 claims abstract description 30
- 239000003112 inhibitor Substances 0.000 claims abstract description 19
- 229940011871 estrogen Drugs 0.000 claims abstract description 18
- 239000000262 estrogen Substances 0.000 claims abstract description 18
- 230000002706 hydrostatic effect Effects 0.000 claims abstract description 16
- 238000003825 pressing Methods 0.000 claims abstract description 13
- 230000035939 shock Effects 0.000 claims abstract description 13
- 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 13
- 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
- 108010062540 Chorionic Gonadotropin Proteins 0.000 claims description 4
- 102000011022 Chorionic Gonadotropin Human genes 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
- 229940015047 chorionic gonadotropin Drugs 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
- 238000009472 formulation Methods 0.000 claims description 4
- 229940088597 hormone Drugs 0.000 claims description 4
- 239000005556 hormone Substances 0.000 claims description 4
- 239000000203 mixture Substances 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
- 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
- 241000251468 Actinopterygii Species 0.000 abstract description 10
- 208000026487 Triploidy Diseases 0.000 abstract description 5
- 238000009396 hybridization Methods 0.000 abstract description 3
- 239000007787 solid Substances 0.000 abstract description 2
- 241000252229 Carassius auratus Species 0.000 description 5
- 241000209094 Oryza Species 0.000 description 3
- 238000006243 chemical reaction Methods 0.000 description 2
- 210000000349 chromosome Anatomy 0.000 description 2
- 239000012153 distilled water Substances 0.000 description 2
- 235000013372 meat Nutrition 0.000 description 2
- 230000001629 suppression Effects 0.000 description 2
- 241000252211 Carassius Species 0.000 description 1
- 241000246211 Carassius auratus red var. Species 0.000 description 1
- 241000511340 Carassius cuvieri Species 0.000 description 1
- 241001677838 Cyprinus carpio nudus Species 0.000 description 1
- 230000004543 DNA replication Effects 0.000 description 1
- 208000035240 Disease Resistance Diseases 0.000 description 1
- 108010000912 Egg Proteins Proteins 0.000 description 1
- 102000002322 Egg Proteins Human genes 0.000 description 1
- 238000009395 breeding Methods 0.000 description 1
- 238000003776 cleavage reaction Methods 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 230000009977 dual effect Effects 0.000 description 1
- 230000009931 harmful effect Effects 0.000 description 1
- 230000013011 mating Effects 0.000 description 1
- 230000011278 mitosis Effects 0.000 description 1
- 210000004681 ovum Anatomy 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 230000007017 scission Effects 0.000 description 1
- 238000002791 soaking Methods 0.000 description 1
- 230000004083 survival effect Effects 0.000 description 1
Classifications
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- 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
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/13—Amines
- A61K31/135—Amines having aromatic rings, e.g. ketamine, nortriptyline
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/13—Amines
- A61K31/135—Amines having aromatic rings, e.g. ketamine, nortriptyline
- A61K31/138—Aryloxyalkylamines, e.g. propranolol, tamoxifen, phenoxybenzamine
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/56—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids
- A61K31/565—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids not substituted in position 17 beta by a carbon atom, e.g. estrane, estradiol
- A61K31/568—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids not substituted in position 17 beta by a carbon atom, e.g. estrane, estradiol substituted in positions 10 and 13 by a chain having at least one carbon atom, e.g. androstanes, e.g. testosterone
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- A61K38/22—Hormones
- A61K38/24—Follicle-stimulating hormone [FSH]; Chorionic gonadotropins, e.g. HCG; Luteinising hormone [LH]; Thyroid-stimulating hormone [TSH]
-
- 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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- Medicinal Chemistry (AREA)
- Pharmacology & Pharmacy (AREA)
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- Animal Behavior & Ethology (AREA)
- General Health & Medical Sciences (AREA)
- Public Health (AREA)
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- Endocrinology (AREA)
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- Gastroenterology & Hepatology (AREA)
- Engineering & Computer Science (AREA)
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Abstract
The invention discloses and a kind of improve crucian carp, the method for Cyprinus carpio male tetraploid 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, the acquisition of tetraploid pure lines.Pass through the method, crucian carp, the Cyprinus carpio male tetraploid pure lines of 100% can be obtained, be effectively increased tetraploid incubation rate and emergence rate, for comparing hybridization, the male tetraploid fish system that purity is higher, offspring is more stable can be obtained, established solid foundation for sterile triploid crucian carp, the incubation of Cyprinus carpio.
Description
Technical field
The present invention relates to the tetraploid mating system of crucian carp, Cyprinus carpio, be specifically related to a kind of raising crucian carp, Cyprinus carpio male tetraploid incubation rate
Method.
Background technology
Carassius auratus: refer to diploid Carassius auratus (including Carassius auratusvar, red crucian carp, white crucian carp and black crucian carp etc.)；Cyprinus carpio: be commonly called as Cyprinus carpio cripple, Cyprinus carpio, bag
Include mirror Cyprinus carpio, red Cyprinus carpio, naked carp etc..Its fast growth, delicious meat.And Artificial Triploid crucian carp (the natural triploid of Carassius auratus is to educate
) and the sterile feature of Cyprinus carpio so that it is show significantly more at aspects such as the speed of growth, resistance, disease resistance and meats
Advantage.In prior art, sterile triploid fish is to be obtained with liploid fish interspecies crossing by tetraploid fish, but to obtain 98%
Above tetraploid fish, then be accomplished by setting up the tetraploid fish system of inheritance stability, 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 stable raising crucian carp of a kind of method, Cyprinus carpio
The method of male tetraploid incubation rate.
For reaching above-mentioned purpose, technical scheme is as follows:
A kind of improve crucian carp, the method for Cyprinus carpio male tetraploid incubation rate, comprise 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 240～450ml, make injection stand-by；Hasten parturition
Agent solution is to be prepared with 0.9% normal saline dilution by chorionic-gonadotropin hormone, chorionic-gonadotropin hormone in odinagogue solution
Dosage be 5-10 IU/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 60～120 μ g/kg, i.e. every kg female parent injection tamoxifen note
Penetrate liquid 60-120 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 80～200mg/l, testosterone
Concentration be 20～80mg/l；
(6) acquisition of tetraploid pure lines: take the estrogen put into as prepared by step (5) of germ cell after step (4) processes and press down
In formulation soln, soak at room temperature 10～30s, then take out and be put in normal incubation in couveuse；Thus it is male to obtain 100% crucian carp, Cyprinus carpio
Tetraploid is sheerly.
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, put off the time (3-5min) of germ cell First cleavage, improve all germ cell the first deutovums
The synchronization extent (up to 99%) split, for operating the guarantee of next step offer time and having established the basis of chromosome doubling；2, it is being subject to
An essence spilting of an egg of ovum uses cold shock and relatively low hydrostatic pressing (less than conventional 350kg/cm2), and the shorter process time
(less than 5min), not only effectively reduces the harmful effect to germ cell, improves the survival rate of germ cell, and strengthen four further
The conversion ratio of times body；3, estrogen inhibitors solution soaking, strengthens again and ensures male conversion ratio.Thereby through this side
Method, can obtain crucian carp, the Cyprinus carpio male tetraploid pure lines of 100%, be effectively increased tetraploid incubation rate and emergence rate, compare hybridization
For, the male tetraploid fish system that purity is higher, offspring is more stable can be obtained, establish for sterile triploid crucian carp, the incubation of Cyprinus carpio
Determine solid foundation.
Detailed description of the invention
In conjunction with specific embodiment, the invention will be further described.
Embodiment one
The method improving Carassius auratus male tetraploid 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 310Kg/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 160mg/l, and the concentration of testosterone is
60mg/l；
(6) acquisition of tetraploid pure lines: take the estrogen put into as prepared by step (5) of germ cell after step (4) processes and press down
In formulation soln, soak at room temperature 30s, then take out and be put in normal incubation in couveuse；Thus obtain male four times of 100% Carassius auratus
Body is sheerly.
Embodiment two
The method improving Cyprinus carpio male tetraploid 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 450ml, make injection, in injection, he is not
The former times concentration of sweet smell is 0.2mg/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 120 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 250Kg/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 200mg/l, and the concentration of testosterone is
80mg/l；
(6) acquisition of tetraploid pure lines: take the estrogen put into as prepared by step (5) of germ cell after step (4) processes and press down
In formulation soln, soak at room temperature 10s, then take out and be put in normal incubation in couveuse；Thus it is pure to obtain the male tetraploid of Cyprinus carpio
System.
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: crucian carp, the male tetraploid of Cyprinus carpio are bred as result statistics
Note: the above results is soak at room temperature 10S under conditions of the concentration of tamoxifen is 0.2mg/ml in injection, hydrostatic pressing
Process 3 minutes acquired results.
Claims (1)
1. improve crucian carp, a method for Cyprinus carpio male tetraploid incubation rate, comprise 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 240～450ml, make injection stand-by；Hasten parturition
Agent solution is to be prepared with 0.9% normal saline dilution by chorionic-gonadotropin hormone, chorionic-gonadotropin hormone in odinagogue solution
Dosage be 5-10 IU/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 60～120 μ g/kg, i.e. every kg female parent injection tamoxifen note
Penetrate liquid 60-120 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 80～200mg/l, testosterone
Concentration be 20～80mg/l；
(6) acquisition of tetraploid pure lines: take the estrogen put into as prepared by step (5) of germ cell after step (4) processes and press down
In formulation soln, soak at room temperature 10～30s, then take out and be put in normal incubation in couveuse；Thus it is male to obtain 100% crucian carp, Cyprinus carpio
Tetraploid is sheerly.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107494358A (en) * | 2017-09-28 | 2017-12-22 | 中国科学院南海海洋研究所 | A kind of preparation method of Hong Kong oyster tetraploid children shellfish |
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| CN107494358A (en) * | 2017-09-28 | 2017-12-22 | 中国科学院南海海洋研究所 | A kind of preparation method of Hong Kong oyster tetraploid children shellfish |
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