CN106135089A - The male tetraploid of Siniperca chuatsi is sheerly efficient breeding method - Google Patents
The male tetraploid of Siniperca chuatsi is sheerly efficient breeding method Download PDFInfo
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- CN106135089A CN106135089A CN201610495640.3A CN201610495640A CN106135089A CN 106135089 A CN106135089 A CN 106135089A CN 201610495640 A CN201610495640 A CN 201610495640A CN 106135089 A CN106135089 A CN 106135089A
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- solution
- tamoxifen
- injection
- siniperca chuatsi
- tetraploid
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- 208000035199 Tetraploidy Diseases 0.000 title claims abstract description 26
- 241000264847 Siniperca chuatsi Species 0.000 title claims abstract description 23
- 238000009395 breeding Methods 0.000 title claims abstract description 9
- 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 56
- 238000000034 method Methods 0.000 claims abstract description 33
- 239000000243 solution Substances 0.000 claims abstract description 32
- 238000002347 injection Methods 0.000 claims abstract description 30
- 239000007924 injection Substances 0.000 claims abstract description 30
- 229960001603 tamoxifen Drugs 0.000 claims abstract description 28
- 239000003112 inhibitor Substances 0.000 claims abstract description 21
- 229940011871 estrogen Drugs 0.000 claims abstract description 16
- 239000000262 estrogen Substances 0.000 claims abstract description 16
- 230000002706 hydrostatic effect Effects 0.000 claims abstract description 16
- 238000003825 pressing Methods 0.000 claims abstract description 14
- 230000035939 shock Effects 0.000 claims abstract description 14
- 238000002360 preparation method Methods 0.000 claims abstract description 11
- 238000011534 incubation Methods 0.000 claims abstract description 9
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 16
- 210000004602 germ cell Anatomy 0.000 claims description 13
- 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 12
- 239000010413 mother solution Substances 0.000 claims description 12
- 230000008774 maternal effect Effects 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
- 229960003604 testosterone Drugs 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
- 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
- 230000000149 penetrating effect Effects 0.000 claims description 3
- 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
- 150000002576 ketones Chemical class 0.000 claims description 2
- 230000032696 parturition Effects 0.000 claims description 2
- 210000001550 testis Anatomy 0.000 claims description 2
- 240000007594 Oryza sativa Species 0.000 claims 1
- 241000251468 Actinopterygii Species 0.000 abstract description 8
- 208000026487 Triploidy Diseases 0.000 abstract description 4
- 238000009396 hybridization Methods 0.000 abstract description 3
- 239000007787 solid Substances 0.000 abstract description 2
- 235000013372 meat Nutrition 0.000 description 4
- 241000209094 Oryza Species 0.000 description 3
- 241000404975 Synchiropus splendidus Species 0.000 description 3
- 241001580679 Siniperca knerii Species 0.000 description 2
- 210000000349 chromosome Anatomy 0.000 description 2
- 239000012153 distilled water Substances 0.000 description 2
- 239000003652 hormone inhibitor Substances 0.000 description 2
- 230000001629 suppression Effects 0.000 description 2
- 101100279436 Caenorhabditis elegans egg-2 gene Proteins 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
- 206010020852 Hypertonia Diseases 0.000 description 1
- 208000020584 Polyploidy Diseases 0.000 description 1
- 230000009087 cell motility Effects 0.000 description 1
- 238000006243 chemical reaction 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
- 239000000796 flavoring agent Substances 0.000 description 1
- 235000019634 flavors Nutrition 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
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
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Environmental Sciences (AREA)
- Marine Sciences & Fisheries (AREA)
- Zoology (AREA)
- Animal Husbandry (AREA)
- Biodiversity & Conservation Biology (AREA)
- Acyclic And Carbocyclic Compounds In Medicinal Compositions (AREA)
Abstract
The invention discloses a kind of male tetraploid of Siniperca chuatsi and be sheerly efficient breeding method, 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, obtain Siniperca chuatsi male tetraploid pure lines.Pass through the method, the Siniperca chuatsi male tetraploid pure lines of 100% can be obtained, be effectively increased tetraploid incubation rate and emergence rate, for comparing hybridization, can obtain the male tetraploid fish system that purity is higher, offspring is more stable, solid foundation has been established in the incubation for sterile triploid Siniperca chuatsi.
Description
Technical field
The present invention relates to the polyploid mating system of Siniperca chuatsi, be specifically related to the Siniperca chuatsi male tetraploid pure lines efficiently side of incubation
Method.
Background technology
Siniperca chuatsi, is divided into again Mandarin fish, Siniperca kneri, speckle mandarin fish etc..Health is flat-sided, and back is swelled, and health is thicker, tip, its meat
Matter is delicate, and the few meat of thorn is many, and meat is flap, delicious flavour, actually the good merchantable brand in fish.The sterile feature of triploid Siniperca chuatsi so that it is
The aspects such as the speed of growth, resistance, disease resistance and meat show significantly more advantage.In prior art, sterile three times
Body Siniperca chuatsi is to be obtained with diploid Siniperca chuatsi interspecies crossing by tetraploid Siniperca chuatsi, but to obtain the triploid fish of more than 98%, then
It is 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 that a kind of method is stable, population purity is high
The male tetraploid of Siniperca chuatsi reaching 100% is sheerly efficient breeding method.
For reaching above-mentioned purpose, technical scheme is as follows:
The male tetraploid of Siniperca chuatsi is sheerly efficient breeding method, 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 80～120mg/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 obtain by being prepared with 0.9% normal saline dilution by hypophysis cerebri, and the mesencephalohypophysial dosage of odinagogue solution is
0.01-0.05mg/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 100～280 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 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 150～300mg/l, testis
The concentration of ketone is 50～140mg/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 100% Siniperca chuatsi male tetraploid pure lines.
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
Use cold shock and relatively low hydrostatic pressing (less than conventional 350kg/cm during an essence spilting of an egg of ovum2), and during shorter process
Between (less than 5min), effectively reduce cold shock overlong time, the hypertonia destructiveness to germ cell, improve the one-tenth of germ cell
Motility rate；3, estrogen inhibitors solution soaking, strengthens again and ensures male conversion ratio.Thereby through the method, can obtain
The Siniperca chuatsi male tetraploid pure lines of 100%, are effectively increased tetraploid incubation rate and emergence rate, for comparing hybridization, can obtain
The male tetraploid fish system that purity is higher, offspring is more stable, solid foundation has been established in the incubation for sterile triploid Siniperca chuatsi.
Detailed description of the invention
In conjunction with specific embodiment, the invention will be further described.
Embodiment one
The male tetraploid of Mandarin fish is sheerly efficient breeding method, 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 120mg/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 0.5mg/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 220 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) estrogen inhibitors preparation: clomifene is dissolved in the ethanol of 90% together with testosterone, then adds distilled water and be configured to female
Hormone inhibitors solution, the concentration that wherein estrogen inhibitors Chlorine in Solution rice is fragrant is 250mg/l, and the concentration of testosterone is 110mg/
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 30s, then takes out and is put in normal incubation in couveuse；Thus obtain 100% Siniperca chuatsi male tetraploid pure lines.
Embodiment two
The male tetraploid of Siniperca kneri fish is sheerly efficient breeding method, 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 120mg/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 0.2mg/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 280 μ g/kg；
(4) cold shock+hydrostatic pressing processes: before a spilting of an egg 2～5min, uses the method that cold shock combines with hydrostatic pressing,
It is-1 DEG C cold that the germ cell that female parent through injection treatment and paternal hybrid without any process are obtained puts into temperature
In water, hydrostatic pressing pressure is 280Kg/cm2, the persistent period is 3min；
(5) estrogen inhibitors preparation: clomifene is dissolved in the ethanol of 90% together with testosterone, then adds distilled water and be configured to female
Hormone inhibitors solution, the concentration that wherein estrogen inhibitors Chlorine in Solution rice is fragrant is 300mg/l, and the concentration of testosterone is 140mg/
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% Siniperca chuatsi male tetraploid pure lines.
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: the male tetraploid of Siniperca chuatsi is 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. the male tetraploid of Siniperca chuatsi is sheerly efficient breeding method, 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 80～120mg/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 obtain by being prepared with 0.9% normal saline dilution by hypophysis cerebri, and the mesencephalohypophysial dosage of odinagogue solution is
0.01-0.05mg/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 100～280 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 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 150～300mg/l, testis
The concentration of ketone is 50～140mg/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 100% Siniperca chuatsi male tetraploid pure lines.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610495640.3A CN106135089A (en) | 2016-06-30 | 2016-06-30 | The male tetraploid of Siniperca chuatsi is sheerly efficient breeding method |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610495640.3A CN106135089A (en) | 2016-06-30 | 2016-06-30 | The male tetraploid of Siniperca chuatsi is sheerly efficient breeding method |
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| CN106135089A true CN106135089A (en) | 2016-11-23 |
Family
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|---|---|---|---|
| CN201610495640.3A Pending CN106135089A (en) | 2016-06-30 | 2016-06-30 | The male tetraploid of Siniperca chuatsi is sheerly efficient breeding method |
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Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102388838A (en) * | 2011-09-13 | 2012-03-28 | 上海博琛生物科技有限公司 | Method and device for improving male hatching rate of oviparity |
| CN102988986A (en) * | 2012-11-02 | 2013-03-27 | 浙江万里学院 | Male trait inducer and induction method for pelodiscus sinensis in embryonic periods |
| CN103243068A (en) * | 2013-04-27 | 2013-08-14 | 上海交通大学 | Method for rapidly evaluating composition effect of estrogen and antiestrogen |
| CN105230533A (en) * | 2015-08-04 | 2016-01-13 | 中国水产科学研究院北戴河中心实验站 | Induction and detection method for androgenesis dihaploid of Paralichthys olivaceus |
| CN105409873A (en) * | 2015-11-13 | 2016-03-23 | 丽水学院 | Method for controlling hoplobatrachus tigerinus sex ratio and application of method |
-
2016
- 2016-06-30 CN CN201610495640.3A patent/CN106135089A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102388838A (en) * | 2011-09-13 | 2012-03-28 | 上海博琛生物科技有限公司 | Method and device for improving male hatching rate of oviparity |
| CN102988986A (en) * | 2012-11-02 | 2013-03-27 | 浙江万里学院 | Male trait inducer and induction method for pelodiscus sinensis in embryonic periods |
| CN103243068A (en) * | 2013-04-27 | 2013-08-14 | 上海交通大学 | Method for rapidly evaluating composition effect of estrogen and antiestrogen |
| CN105230533A (en) * | 2015-08-04 | 2016-01-13 | 中国水产科学研究院北戴河中心实验站 | Induction and detection method for androgenesis dihaploid of Paralichthys olivaceus |
| CN105409873A (en) * | 2015-11-13 | 2016-03-23 | 丽水学院 | Method for controlling hoplobatrachus tigerinus sex ratio and application of method |
Non-Patent Citations (5)
| Title |
|---|
| A. D. WORTHINGTON1等: "The induced spawning of roach, Rutilus rutilus (L.), with the antioestrogens clomiphene and tamoxifen", 《JOURNAL OF FISH BIOLOGY》 * |
| 张覃林: "《抗肿瘤药物的药理与临床应用》", 30 April 1999, 河南医科大学出版社 * |
| 李晓诗等: "他莫昔芬的药理作用机制及其对卵巢功能的影响", 《中国普外基础与临床杂志》 * |
| 桂建芳等: "鱼类染色体组操作的研究Ⅱ静水压处理和静水压与冷休克结合处理诱导水晶彩鲫四倍体", 《水生生物学报》 * |
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