CN105670988A - Mammal embryo flushing solution as well as preparation method and application thereof - Google Patents
Mammal embryo flushing solution as well as preparation method and application thereof Download PDFInfo
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- CN105670988A CN105670988A CN201610073489.4A CN201610073489A CN105670988A CN 105670988 A CN105670988 A CN 105670988A CN 201610073489 A CN201610073489 A CN 201610073489A CN 105670988 A CN105670988 A CN 105670988A
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- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
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- A61D—VETERINARY INSTRUMENTS, IMPLEMENTS, TOOLS, OR METHODS
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- C12N2500/05—Inorganic components
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- C12N2500/12—Light metals, i.e. alkali, alkaline earth, Be, Al, Mg
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- C12N2500/10—Metals; Metal chelators
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- C12N2500/05—Inorganic components
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- C12N2500/16—Magnesium; Mg chelators
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Abstract
The invention provides a mammalian embryo flushing solution and a preparation method thereof. The embryo flushing solution comprises a solution I, a solution II and neonatal bovine serum, wherein the solution I contains sodium chloride and at least one of potassium chloride, calcium chloride and magnesium chloride; the solution II contains disodium hydrogen phosphate and/or disodium hydrogen phosphate hydrate and fructose, and preferably, the solution II contains monopotassium phosphate. The preparation method of the mammalian embryo flushing solution comprises steps as follows: the solution I and the solution II are prepared with water respectively, the solution I and the solution II are proportionally mixed after high-pressure sterilization is performed for 15-20 min at the temperature of 120 DEG C, an appropriate amount of the neonatal bovine serum is added to a mixed solution, and the mammalian embryo flushing solution is obtained. The invention further provides an application of the embryo flushing solution in the non-surgical embryo transfer technology for mammals. The mammalian embryo flushing solution is simple to prepare, the osmotic pressure and the pH range from 7.1 to 7.4 are closer to those of a physiological environment in the uterus of a donor, the storage time of an embryo in vitro can be prolonged, and large-scale production of embryos of cattle and sheep and popularization and application of the embryo transfer technology are facilitated.
Description
Technical field
The present invention relates to animal embryo implantation technique field, specifically, relate to a kind of mammal egg liquid and compound method thereof and application.
Background technology
Superovulation and embryo transfer technology (MOET) extensive use in cattle, sheep improvement and breeding. In MOET process, the height of embryo's (ovum) organic efficiency directly affects the carrying out that subsequent embryo is transplanted. The recovery of embryo's (ovum) is typically in carrying out after super row's donor is bred 3～8 days, and embryo's (ovum) recovery method adopted is divided into modus operandi and Nonoperative method. Embryo's recovery method of cattle mainly adopts Nonoperative method, its specific operation process is to insert side donor cornua uteri with the metal-cored oviduct that rushes, to airbag aeration, fixed punch oviduct, extract out metal-cored, by the egg liquid prepared, the embryo being positioned at cornua uteri is developed. At present, domestic conventional TCM-199, Hank ' the import synthesis buffer such as s liquid uses as cattle embryo's egg liquid, and expensive due to it, synthesis condition requires the problems such as strict to have impact on the promotion and application of cattle super ovulation techniques.
Summary of the invention
It is an object of the invention to provide a kind of mammal egg liquid and compound method thereof.
It is a further object of the present invention to provide the application in mammal Nonoperative method embryo transfer technology of the above-mentioned egg liquid.
In order to realize the object of the invention, a kind of mammal egg liquid of the present invention, described egg liquid includes I liquid, II liquid and calf serum;
Described I liquid includes at least one in sodium chloride and potassium chloride, calcium chloride and magnesium chloride; Described II liquid includes disodium hydrogen phosphate and/or its hydrate and fructose, optionally includes potassium dihydrogen phosphate.
Specifically, described I liquid includes sodium chloride 5.0-10.0g/L, potassium chloride 0-0.5g/L, calcium chloride 0-0.5g/L and magnesium chloride 0-0.5g/L. Preferably, described I liquid includes sodium chloride 7.0-9.0g/L, potassium chloride 0.1-0.3g/L, calcium chloride 0.1-0.3g/L and magnesium chloride 0.1-0.3g/L. It is highly preferred that described I liquid includes sodium chloride 8.0g/L, potassium chloride 0.2g/L, calcium chloride 0.1g/L and magnesium chloride 0.1g/L.
Described II liquid includes disodium hydrogen phosphate 1.0-2.0g/L or seven hypophosphite monohydrate disodium hydrogen 1.0-3.0g/L or disodium hydrogen phosphate dodecahydrate 1.0-3.0g/L, potassium dihydrogen phosphate 0-0.5g/L and D-Fructose 1.0-3.0g/L. Preferably, described II liquid includes disodium hydrogen phosphate 1.0-1.2g/L or seven hypophosphite monohydrate disodium hydrogen 2.0-2.2g/L or disodium hydrogen phosphate dodecahydrate 2.6-3.0g/L, potassium dihydrogen phosphate 0.1-0.3g/L and D-Fructose 1.0-1.5g/L. It is highly preferred that described II liquid includes disodium hydrogen phosphate dodecahydrate 2.9g/L, potassium dihydrogen phosphate 0.2g/L and D-Fructose 1.0g/L.
During use, I liquid and II liquid are pressed volume ratio (preferred volume ratio 1:1) mixing of 1:2-2:1, in mixed liquor, then adds the calf serum of 10-15v/v%, obtain described mammal egg liquid.
The present invention also provides for the compound method of described egg liquid, with deionized water or distilled water, prepares I liquid and II liquid respectively according to each constituent content, 120 DEG C of autoclavings are after 15-20 minute, I liquid and II liquid are mixed in proportion, in mixed liquor, then add calf serum, to obtain final product.
The present invention further provides the application in mammal Nonoperative method embryo transfer technology of the described egg liquid.
Mammal of the present invention includes but not limited to cattle, sheep, horse, mice. Preferably, described mammal is cattle.
Mammal egg liquid provided by the invention; preparation is simple; osmotic pressure and pH scope (pH7.1-7.4) are closer to the intrauterine physiological environment of donor, it is possible to extend embryo's holding time in vitro, be conducive to the popularization and application of cattle, sheep embryo's large-scale production and embryo transfer technology.
Detailed description of the invention
Following example are used for illustrating the present invention, but are not limited to the scope of the present invention. If not specializing, the conventional means that technological means used in embodiment is well known to those skilled in the art, raw materials used it is commercial goods.
Embodiment 1 mammal egg liquid and preparation thereof
The preparation of egg liquid I liquid: use electronic balance to weigh sodium chloride, potassium chloride, calcium chloride and magnesium chloride, deionized water or distilled water in table 1 ratio and fully dissolve, 120 DEG C of autoclavings 15-20 minute, room temperature stands and preserves.
The preparation of egg liquid II liquid: use electronic balance to weigh disodium hydrogen phosphate dodecahydrate, potassium dihydrogen phosphate and D-Fructose, deionized water or distilled water in table 2 ratio and fully dissolve, 120 DEG C of autoclavings 15-20 minute, room temperature stands and preserves.
The preparation of egg liquid: I liquid and II liquid are sufficiently mixed in table 3 ratio, then adds the calf serum of mixed liquor 10-15v/v%, obtains mammal egg liquid.
Table 1 egg liquid I formula of liquid
Table 2 egg liquid II formula of liquid
Table 3 egg liquid formula
The impact on cow embryo production and embryo transfer of the embodiment 2 different egg liquid
1, donor cattle: selecting 180,18-20 monthly age young cow is donor.
2, donor cattle Superovluation: super mining conventional method, i.e. injection follicle stimulating hormone (FSH) in continuous 4 days, dosage tapered dosage. Particularly as follows: donor cattle estrus cycle any heeling-in CIDR (the 0th day), the 5-8 days morning, late each intramuscular injection FSH, within the 7th day, injection FSH distinguishes intramuscular injection prostaglandin (PG) simultaneously, removes CIDR evening simultaneously. It is 7.8-8.4mg that FSH injects accumulated dose.
3, artificial insemination: donor oestrus latter 12 hours first time artificial insemination, interval after 12 hours second time artificial insemination. Using 1 freezing straw semen, insemination site is body of uterus every time.
4, Embryo Collection: adopt non-operative treatment to reclaim embryo in after donor cows breeding the 7th day. Every side cornua uteri rinses 6～7 times repeatedly, egg liquid used is the egg liquid A～G of preparation in embodiment 1, equus egg liquid disclosed in CN102864122A and TCM-199 (osmotic pressure of several egg liquids compares in Table 4 with pH value), egg liquid makes consumption be 250-400mL.
5, embryo transfer: select 30 pieces of available embryos, be randomly divided into 2 groups, often 15 pieces of embryos of group, are saved in above-mentioned egg liquid respectively, and room temperature preservation carried out embryo transfer after 2 hours.
Result shows, compared to equus egg liquid and TCM-199, use the egg liquid prepared of the present invention to reclaim and embryo transfer for the embryo of cattle, it is thus achieved that available embryo number and Recipient embryo transfer conception rate higher, improve the efficiency (table 5 and table 6) of cattle superovulation.
The osmotic pressure of several egg liquid of table 4 compares with pH value
Impact that cow embryo is produced by the different egg liquid of table 5 (unit: head, piece)
The preservation of the different egg liquid of table 6 impact on embryo transfer
The Embryo Production of the black Angus beef of embodiment 3
1, donor cattle: select 34, the 18-22 monthly age, young black Angus beef was donor.
2, donor cattle FSH injects dosage is 10.0mg, and egg liquid used is the egg liquid A of preparation in embodiment 1, and concrete operations are with the description in experimental example 2.
It is shown that use the egg liquid A black Angus beef of super row to be obtained in that available embryo 8.3 pieces (table 7).
The black Angus beef Embryo Production result of table 7 (unit: head, piece)
Result above shows, utilizes the egg liquid that the present invention prepares can improve cattle superovulation efficiency and embryo transfer conception rate. Egg liquid preparation method is simple, with low cost, is conducive to embryo's short-term preservation in vitro, it is possible to meets the large-scale production needs of cattle, sheep embryo, has promoted the popularization and application of embryo transfer technology.
Although, above the present invention is described in detail with a general description of the specific embodiments, but on basis of the present invention, it is possible to it is made some modifications or improvements, and this will be apparent to those skilled in the art. Therefore, these modifications or improvements without departing from theon the basis of the spirit of the present invention, belong to the scope of protection of present invention.
Claims (10)
1. a mammal egg liquid, it is characterised in that described egg liquid includes I liquid, II liquid and calf serum;
Described I liquid includes at least one in sodium chloride and potassium chloride, calcium chloride and magnesium chloride; Described II liquid includes disodium hydrogen phosphate and/or its hydrate and fructose, optionally includes potassium dihydrogen phosphate.
2. egg liquid according to claim 1, it is characterised in that described I liquid includes sodium chloride 5.0-10.0g/L, potassium chloride 0-0.5g/L, calcium chloride 0-0.5g/L and magnesium chloride 0-0.5g/L.
3. egg liquid according to claim 2, it is characterised in that described I liquid includes sodium chloride 7.0-9.0g/L, potassium chloride 0.1-0.3g/L, calcium chloride 0.1-0.3g/L and magnesium chloride 0.1-0.3g/L.
4. egg liquid according to claim 3, it is characterised in that described I liquid includes sodium chloride 8.0g/L, potassium chloride 0.2g/L, calcium chloride 0.1g/L and magnesium chloride 0.1g/L.
5. egg liquid according to claim 1, it is characterized in that, described II liquid includes disodium hydrogen phosphate 1.0-2.0g/L or seven hypophosphite monohydrate disodium hydrogen 1.0-3.0g/L or disodium hydrogen phosphate dodecahydrate 1.0-3.0g/L, potassium dihydrogen phosphate 0-0.5g/L and D-Fructose 1.0-3.0g/L.
6. egg liquid according to claim 5, it is characterized in that, described II liquid includes disodium hydrogen phosphate 1.0-1.2g/L or seven hypophosphite monohydrate disodium hydrogen 2.0-2.2g/L or disodium hydrogen phosphate dodecahydrate 2.6-3.0g/L, potassium dihydrogen phosphate 0.1-0.3g/L and D-Fructose 1.0-1.5g/L.
7. egg liquid according to claim 6, it is characterised in that described II liquid includes disodium hydrogen phosphate dodecahydrate 2.9g/L, potassium dihydrogen phosphate 0.2g/L and D-Fructose 1.0g/L.
8. the egg liquid according to any one of claim 1-7, it is characterised in that during use, I liquid and II liquid are pressed the volume ratio mixing of 1:2-2:1, then in mixed liquor, the calf serum of 10-15v/v% is added, it is preferable that I liquid and II liquid press the volume ratio mixing of 1:1.
9. the compound method of egg liquid according to any one of claim 1-7, it is characterized in that, with deionized water or distilled water, I liquid and II liquid is prepared respectively according to each constituent content, 120 DEG C of autoclavings are after 15-20 minute, I liquid and II liquid are mixed in proportion, in mixed liquor, then add calf serum, to obtain final product.
10. egg liquid application in mammal Nonoperative method embryo transfer technology according to any one of claim 1-7, described mammal includes cattle, sheep, horse, mice, it is preferable that cattle.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106119188A (en) * | 2016-06-27 | 2016-11-16 | 河南农业大学 | One takes embryo buffer and application thereof |
| CN108642001A (en) * | 2018-05-08 | 2018-10-12 | 中国农业科学院北京畜牧兽医研究所 | A method of it improving obstinacy control and freezes essence ability in vitro fertilization |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101880690A (en) * | 2010-06-12 | 2010-11-10 | 中国科学院西北高原生物研究所 | Construction and in vitro culture method of Tibetan antelope-goat interspecific cloned embryo |
| CN104042355A (en) * | 2014-06-23 | 2014-09-17 | 南通惠然生物科技有限公司 | Method for unconventionally breeding improved cows through embryo biotechnology |
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2016
- 2016-02-02 CN CN201610073489.4A patent/CN105670988B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101880690A (en) * | 2010-06-12 | 2010-11-10 | 中国科学院西北高原生物研究所 | Construction and in vitro culture method of Tibetan antelope-goat interspecific cloned embryo |
| CN104042355A (en) * | 2014-06-23 | 2014-09-17 | 南通惠然生物科技有限公司 | Method for unconventionally breeding improved cows through embryo biotechnology |
Non-Patent Citations (1)
| Title |
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| 夏天等: "猪胚胎移植研究初报", 《畜牧兽医杂志》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106119188A (en) * | 2016-06-27 | 2016-11-16 | 河南农业大学 | One takes embryo buffer and application thereof |
| CN106119188B (en) * | 2016-06-27 | 2019-10-11 | 河南农业大学 | One kind taking embryo buffer and its application |
| CN108642001A (en) * | 2018-05-08 | 2018-10-12 | 中国农业科学院北京畜牧兽医研究所 | A method of it improving obstinacy control and freezes essence ability in vitro fertilization |
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