CN111493064A - Bovine cloned embryo refrigerating fluid, thawing fluid, kit and bovine cloned embryo freezing and thawing method - Google Patents
Bovine cloned embryo refrigerating fluid, thawing fluid, kit and bovine cloned embryo freezing and thawing method Download PDFInfo
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Abstract
The invention provides a bovine cloned embryo refrigerating fluid, a thawing fluid, a kit and a method for freezing and thawing bovine cloned embryos, belonging to the in vitro embryo freezing technology, wherein the freezing method comprises the following steps: placing the cloned bovine embryos in a basic solution, and balancing the embryos for 5-10 min at 24-25 ℃; the embryos are sequentially placed in a first vitrification refrigerating fluid to be balanced for 4-6 min at 24-25 ℃, a second vitrification refrigerating fluid to be balanced for 4-6 min at 24-25 ℃, a third vitrification refrigerating fluid to be balanced for 35-45 s at 24-25 ℃, and finally the embryos are placed in liquid nitrogen to be frozen. And (3) unfreezing the frozen bovine cloned embryo in a first unfreezing liquid at 37-39 ℃ for 50-70 s, then placing the frozen bovine cloned embryo in a second unfreezing liquid to balance the embryo at 24-25 ℃ for 4-6 min, and finally placing the frozen bovine cloned embryo in a base liquid to balance the embryo at 24-25 ℃ for 4-6 min. By using the method, the conception rate of the cloned cow embryo reaches up to 25 percent.
Description
Technical Field
The invention belongs to an in-vitro embryo freezing technology, and particularly relates to a bovine cloned embryo freezing solution, a thawing solution, a kit and a bovine cloned embryo freezing and thawing method.
Background
Cryopreservation of human and animal embryos has been an essential part of the field of artificial reproduction. The development of cryopreservation techniques in the past two decades has also reflected the degree of development of artificial reproduction techniques, and modern cryopreservation techniques have been used to cryopreserve sperm, eggs and embryos at different times. In particular, the Vitrification freezing technique (vitrifying), not only has gained wide acceptance in the medical field, but also gradually replaces the traditional slow freezing method, and becomes the best choice for freezing technique. Compared with the traditional slow freezing method, the vitrification freezing process ensures that water crystallization is not generated in the cell body, thereby avoiding the damage to the embryo.
In vitro fertilized embryos and cloned embryos are of inferior quality and weak freezing resistance compared to in vivo embryos. The low quality of the embryo leads to further reduction of the embryo revival rate and conception rate after freezing and thawing. The traditional slow freezing method can not effectively freeze and preserve the embryo, thereby greatly limiting the application of in vitro fertilization and cloning technology in livestock breeding. The present inventors of the bovine embryo vitrification freezing technology, the scheme of G Vajta, is as follows: refrigerating fluid: 15% glycerol + 15% DMSO; 38 ℃; thawing the liquid: 1M sucrose, 38 ℃; compared with the traditional slow freezing method, the method improves the freezing effect and the conception rate of the embryo in vivo, but the application effect on cloning the embryo is not ideal, and the conception rate is only 5 percent generally.
Disclosure of Invention
In view of the above, the present invention aims to provide a bovine cloned embryo freezing solution, a thawing solution, a kit and a bovine cloned embryo freezing and thawing method with high conception rate.
In order to achieve the above purpose, the invention provides the following technical scheme:
the invention provides bovine cloned embryo refrigerating fluid which comprises base fluid, first vitrification refrigerating fluid, second vitrification refrigerating fluid and third vitrification refrigerating fluid which independently exist;
the basic solution takes Du's phosphate buffer solution as a solvent and comprises calf serum with the volume fraction of 18-22%, bovine serum albumin with the volume fraction of 0.2-0.4%, sodium pyruvate with the volume fraction of 0.25-0.35 mmol/L and glucose with the volume fraction of 3.0-3.5 mmol/L;
the first vitrification refrigerating fluid comprises 88-92% of base fluid by volume fraction and 8-12% of glycerin by volume fraction;
the second vitrification refrigerating fluid comprises 68-72% of base fluid by volume fraction, 18-22% of glycerin by volume fraction and 8-12% of glycol by volume fraction;
the third vitrification refrigerating fluid comprises base fluid with volume fraction of 48% -52%, glycerol with volume fraction of 24% -26% and glycol with volume fraction of 24% -26%.
Preferably, the basic solution takes Du's phosphate buffer solution as a solvent, and comprises calf serum with the volume fraction of 19-21%, bovine serum albumin with the volume fraction of 0.25-0.35%, sodium pyruvate with the volume fraction of 0.28-0.32 mmol/L and glucose with the volume fraction of 3.1-3.4 mmol/L.
Preferably, the first vitrification refrigerating fluid comprises 89-91% of base fluid by volume fraction and 9-11% of glycerol by volume fraction.
Preferably, the second vitrification refrigerating fluid comprises 69-71% of base fluid by volume fraction, 19-21% of glycerol by volume fraction and 9-11% of glycol by volume fraction.
Preferably, the third vitrification freezing fluid includes 50% by volume of the base fluid, 25% by volume of glycerin and 25% by volume of ethylene glycol.
The invention provides a method for freezing bovine cloned embryos by using the freezing liquid, which comprises the following steps:
placing the cloned bovine embryos in the basic solution, and balancing the embryos for 5-10 min at 24-25 ℃; and (3) sequentially placing the embryos in the first vitrification refrigerating fluid for balancing the embryos for 4-6 min at 24-25 ℃, placing the embryos in the second vitrification refrigerating fluid for balancing the embryos for 4-6 min at 24-25 ℃, placing the embryos in the third vitrification refrigerating fluid for balancing the embryos for 35-45 s at 24-25 ℃, and finally placing the embryos in liquid nitrogen for freezing.
Preferably, the bovine cloned embryos are frozen in liquid nitrogen after being packed in an OPS tube.
The invention provides a bovine cloned embryo thawing solution, which comprises a first thawing solution and a second thawing solution; the first unfreezing liquid comprises 88-92% of the base liquid and 8-12% of galactose by volume fraction; the second unfreezing liquid comprises 93-97% of the base liquid and 3-7% of galactose by volume fraction.
The invention provides a method for unfreezing cloned bovine embryos, which comprises the following steps: placing the frozen bovine cloned embryos in a first thawing solution to balance the embryos for 50-70 s at 37-39 ℃, then placing the bovine cloned embryos in a second thawing solution to balance the embryos for 4-6 min at 24-25 ℃, and finally placing the bovine cloned embryos in a base solution to balance the embryos for 4-6 min at 24-25 ℃.
The invention also provides a bovine cloned embryo freezing kit, which comprises the bovine cloned embryo freezing solution and the bovine cloned embryo unfreezing solution.
The invention has the beneficial effects that: the bovine cloned embryo refrigerating fluid provided by the invention accelerates the cell dehydration process by improving the concentration of an antifreeze in the refrigerating fluid; the full cell dehydration prevents the generation of ice crystals in the freezing process, thereby greatly reducing the damage to the embryo; can improve the recovery rate and the survival rate of the cloned bovine embryos.
The bovine cloned embryo freezing method provided by the invention balances the embryo at the room temperature of 24-25 ℃ instead of the 38 ℃ in the traditional system, thereby reducing the toxicity of the antifreeze to the embryo; the thawing solution of the bovine cloned embryo freezing method provided by the invention adopts galactose, so that the toxicity to the embryo is greatly lower than that of the traditional sucrose; the bovine cloned embryo refrigerating fluid and the method provided by the invention can generate a conception rate of 25-35%.
Detailed Description
The invention provides a bovine cloned embryo refrigerating fluid which comprises a base fluid, a first vitrification refrigerating fluid, a second vitrification refrigerating fluid and a third vitrification refrigerating fluid which are independent, wherein the base fluid takes a Duchen phosphate buffer solution as a solvent and comprises calf serum with the volume fraction of 18-22%, bovine serum albumin with the volume fraction of 0.2-0.4%, sodium pyruvate with the volume fraction of 0.25-0.35 mmol/L and glucose with the volume fraction of 3.0-3.5 mmol/L, the first vitrification refrigerating fluid comprises a base fluid with the volume fraction of 88-92% and glycerol with the volume fraction of 8-12%, the second vitrification refrigerating fluid comprises a base fluid with the volume fraction of 68-72%, glycerol with the volume fraction of 18-22% and ethylene glycol with the volume fraction of 8-12%, and the third vitrification refrigerating fluid comprises a base fluid with the volume fraction of 48-52%, glycerol with the volume fraction of 24-26% and ethylene glycol with the volume fraction of 24-26%.
In the invention, the base solution takes Du's phosphate buffer solution as a solvent, preferably comprises 19-21% by volume of calf serum, 0.25-0.35% by volume of bovine serum albumin, 0.28-0.32 mmol/L of sodium pyruvate and 3.1-3.4 mmol/L of glucose, more preferably comprises 20% by volume of calf serum, 0.3% by volume of bovine serum albumin, 0.3 mmol/L of sodium pyruvate and 3.3 mmol/L of glucose.
In the invention, the first vitrified refrigerating fluid preferably comprises 89-91% of base fluid by volume fraction and 9-11% of glycerol by volume fraction, and more preferably comprises 90% of base fluid by volume fraction and 10% of glycerol by volume fraction.
In the invention, the second vitrification refrigerating fluid preferably comprises 69-71% of base fluid by volume fraction, 19-21% of glycerol by volume fraction and 9-11% of glycol by volume fraction, and more preferably comprises 70% of base fluid by volume fraction, 20% of glycerol by volume fraction and 10% of glycol by volume fraction.
In the present invention, the third vitrification refrigerating fluid preferably includes 50% by volume of the base fluid, 25% by volume of glycerin and 25% by volume of ethylene glycol.
In a preferred embodiment of the present invention, the base solution uses Du's phosphate buffer solution as a solvent, and comprises 20% by volume of calf serum, 0.3% by volume of bovine serum albumin, 0.3 mmol/L of sodium pyruvate and 3.3 mmol/L of glucose, the first vitrification freezing solution comprises 90% by volume of base solution and 10% by volume of glycerol, the second vitrification freezing solution comprises 70% by volume of base solution, 20% by volume of glycerol and 10% by volume of ethylene glycol, and the third vitrification freezing solution comprises 50% by volume of base solution, 25% by volume of glycerol and 25% by volume of ethylene glycol.
The invention provides a method for freezing bovine cloned embryos by using the freezing liquid, which comprises the following steps:
placing the cloned bovine embryos in the basic solution, and balancing the embryos for 5-10 min at 24-25 ℃; and (3) sequentially placing the embryos in the first vitrification refrigerating fluid for balancing the embryos for 4-6 min at 24-25 ℃, placing the embryos in the second vitrification refrigerating fluid for balancing the embryos for 4-6 min at 24-25 ℃, placing the embryos in the third vitrification refrigerating fluid for balancing the embryos for 35-45 s at 24-25 ℃, and finally placing the embryos in liquid nitrogen for freezing.
In the invention, preferably, a bovine cloned embryo is placed in the base liquid to balance the embryo at 24.5 ℃ for 5min, then sequentially placed in the first vitrified refrigerating liquid to balance the embryo at 24.5 ℃ for 5min, placed in the second vitrified refrigerating liquid to balance the embryo at 24.5 ℃ for 5min, placed in the third vitrified refrigerating liquid to balance the embryo at 24.5 ℃ for 40s, and finally placed in liquid nitrogen for freezing.
The invention also provides a bovine cloned embryo thawing solution, which comprises a first thawing solution and a second thawing solution; the first unfreezing liquid comprises 88-92% of the base liquid and 8-12% of galactose by volume fraction; preferably comprising 90% by volume of said base liquid and 10% galactose; the second thawing solution comprises 93-97% of the base solution and 3-7% of galactose by volume fraction, and preferably comprises 95% of the base solution and 5% of galactose by volume fraction.
The invention also provides a method for unfreezing the cloned bovine embryo, which comprises the following steps: placing the frozen bovine cloned embryos in a first thawing solution to balance the embryos for 50-70 s at 37-39 ℃, then placing the bovine cloned embryos in a second thawing solution to balance the embryos for 4-6 min at 24-25 ℃, and finally placing the bovine cloned embryos in a basic solution to balance the embryos for 4-6 min at 24-25 ℃; preferably, the frozen cloned bovine embryo is placed in the first thawing solution to equilibrate the embryo at 38 ℃ for 60s, then placed in the second thawing solution to equilibrate the embryo at 24.5 ℃ for 5min, and finally placed in the base solution to equilibrate the embryo at 24.5 ℃ for 5 min.
The invention also provides a bovine cloned embryo freezing kit, which comprises the bovine cloned embryo freezing solution and the bovine cloned embryo unfreezing solution.
The technical solutions provided by the present invention are described in detail below with reference to examples, but they should not be construed as limiting the scope of the present invention.
Example 1
The refrigerating fluid comprises the following components:
basic solution (MH) is composed of calf serum with volume fraction of 20%, sodium pyruvate with volume fraction of 0.3 mmol/L + glucose with volume fraction of 3.3 mmol/L%, bovine serum albumin with volume fraction of 0.3%, and Du's phosphate buffer with balance.
First vitrified cryo fluid (VS 1): 90% volume fraction base liquid + 10% volume fraction glycerin
Second vitrified cryo fluid (VS 2): 70 volume percent of base fluid, 20 volume percent of glycerin and 10 volume percent of glycol
Third vitrified cryo fluid (VS 3): 50% by volume base fluid + 25% by volume glycerol + 25% by volume ethylene glycol
Freezing procedure:
balancing the embryos for 5min at 24-25 ℃ of the base solution;
balancing the embryos for 5min at 24-25 ℃ by using first vitrification refrigerating fluid;
balancing the embryos for 5min by using a second vitrification refrigerating fluid at the temperature of 24-25 ℃;
balancing the embryos for 40s at 24-25 ℃ by using third vitrification refrigerating fluid;
loading into OPS tubule, and directly freezing with liquid nitrogen.
Example 2
The refrigerating fluid comprises the following components:
basic solution (MH) is calf serum with volume fraction of 18%, sodium pyruvate of 0.25 mmol/L + glucose of 3.0 mmol/L%, bovine serum albumin with volume fraction of 0.2%, and Du's phosphate buffer solution in balance.
First vitrified cryo fluid (VS 1): volume fraction of 92% base fluid + volume fraction of 8% glycerol
Second vitrified cryo fluid (VS 2): 72 volume percent base fluid, 19 volume percent glycerin, and 9 volume percent ethylene glycol
Third vitrified cryo fluid (VS 3): 52 volume fraction base fluid + 24 volume fraction glycerin + 24 volume fraction ethylene glycol
Freezing procedure:
balancing the embryos for 5min at 24-25 ℃ of the base solution;
balancing the embryos for 5min at 24-25 ℃ by using first vitrification refrigerating fluid;
balancing the embryos for 5min by using a second vitrification refrigerating fluid at the temperature of 24-25 ℃;
balancing the embryos for 40s at 24-25 ℃ by using third vitrification refrigerating fluid;
loading into OPS tubule, and directly freezing with liquid nitrogen.
Example 3
The refrigerating fluid comprises the following components:
base liquid (MH) is calf serum with volume fraction of 22%, sodium pyruvate of 0.35 mmol/L + glucose of 3.5 mmol/L%, bovine serum albumin with volume fraction of 0.4%, Du's phosphate buffer with the balance.
First vitrified cryo fluid (VS 1): 88% by volume base fluid + 12% by volume glycerol
Second vitrified cryo fluid (VS 2): 68% by volume of base fluid, 21% by volume of glycerol and 11% by volume of ethylene glycol
Third vitrified cryo fluid (VS 3): 48 volume percent base fluid, 26 volume percent glycerin, and 26 volume percent ethylene glycol
Freezing procedure:
balancing the embryos for 5min at 24-25 ℃ of the base solution;
balancing the embryos for 5min at 24-25 ℃ by using first vitrification refrigerating fluid;
balancing the embryos for 5min by using a second vitrification refrigerating fluid at the temperature of 24-25 ℃;
balancing the embryos for 40s at 24-25 ℃ by using third vitrification refrigerating fluid;
loading into OPS tubule, and directly freezing with liquid nitrogen.
Example 4
The composition of the thawing solution is as follows:
first thawing solution (WS 1): 90% by volume of base fluid + 10% by volume of galactose
Second thawing solution (WS 2): volume fraction 95% base fluid + volume fraction 5% galactose thawing procedure:
the first thawing solution balances the embryos for 1min at 38 ℃, the second thawing solution balances the embryos for 5min at 24-25 ℃, and the base solution balances the embryos for 5min at 24-25 ℃.
Comparative example 1
The refrigerating fluid comprises the following components:
base liquid (MH): volume fraction 80% TCM199+ volume fraction 20% calf serum
First vitrified cryo fluid (VS 1): 85% by volume of base fluid, 7.5% by volume of ethylene glycol and 7.5% by volume of dimethyl sulfoxide;
second vitrified cryo fluid (VS 2): 70 volume percent of base fluid, 15 volume percent of ethylene glycol, 15 volume percent of dimethyl sulfoxide and 0.5 mole of cane sugar;
freezing procedure:
balancing embryo in first vitrification refrigerating fluid at 37 deg.C for 3 min;
second vitrification refrigerating fluid equilibrates embryo at 37 deg.C for 30s
Loading into OPS tubule, and directly freezing with liquid nitrogen.
The composition of the thawing solution is as follows:
first thawing solution (WS 1): base liquid +0.5 mol sucrose
Second thawing solution (WS 2): base liquid +0.25 mol sucrose
And (3) unfreezing procedure:
the first thawing solution balances embryos for 1min at 37 ℃, the second thawing solution balances embryos for 5min at 37 ℃, and the base solution balances embryos for 5min at 37 ℃.
Example 5
Freezing, unfreezing and embryo transferring the bovine cloned embryos according to the refrigerating fluid composition and the freezing and unfreezing methods of the bovine cloned embryos in the embodiments 1 and 4, and counting the conception rate;
the bovine cloned embryos were frozen, thawed and embryo-transferred according to the method of comparative example 1, and the conception rate was counted.
The results are shown in Table 1.
TABLE 1 bovine cloned embryo freezing and transplantation results
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Claims (10)
1. The bovine cloned embryo refrigerating fluid is characterized by comprising a base fluid, a first vitrification refrigerating fluid, a second vitrification refrigerating fluid and a third vitrification refrigerating fluid which are independently present;
the basic solution takes Du's phosphate buffer solution as a solvent and comprises calf serum with the volume fraction of 18-22%, bovine serum albumin with the volume fraction of 0.2-0.4%, sodium pyruvate with the volume fraction of 0.25-0.35 mmol/L and glucose with the volume fraction of 3.0-3.5 mmol/L;
the first vitrification refrigerating fluid comprises 88-92% of base fluid by volume fraction and 8-12% of glycerin by volume fraction;
the second vitrification refrigerating fluid comprises 68-72% of base fluid by volume fraction, 18-22% of glycerin by volume fraction and 8-12% of glycol by volume fraction;
the third vitrification refrigerating fluid comprises base fluid with volume fraction of 48% -52%, glycerol with volume fraction of 24% -26% and glycol with volume fraction of 24% -26%.
2. The bovine cloned embryo freezing solution as claimed in claim 1, wherein the base solution uses Du's phosphate buffer as solvent, and comprises calf serum with volume fraction of 19-21%, bovine serum albumin with volume fraction of 0.25-0.35%, sodium pyruvate with volume fraction of 0.28-0.32 mmol/L and glucose with volume fraction of 3.1-3.4 mmol/L.
3. The bovine cloned embryo freezing fluid according to claim 1, wherein the first vitrified freezing fluid comprises 89-91% by volume of base fluid and 9-11% by volume of glycerol.
4. The bovine cloned embryo freezing fluid according to claim 1, wherein the second vitrified freezing fluid comprises 69-71% volume fraction of base fluid, 19-21% volume fraction of glycerol and 9-11% volume fraction of ethylene glycol.
5. The bovine cloned embryo freezing fluid according to claim 1, wherein the third vitrification freezing fluid comprises 50% volume fraction of base fluid, 25% volume fraction of glycerin and 25% volume fraction of ethylene glycol.
6. A method for freezing bovine cloned embryos by using the freezing fluid as claimed in any one of claims 1 to 5, comprising the following steps of:
placing the cloned bovine embryos in the basic solution, and balancing the embryos for 5-10 min at 24-25 ℃; then, the embryos are sequentially placed in the first vitrification refrigerating fluid to be balanced for 4-6 min at 24-25 ℃, placed in the second vitrification refrigerating fluid to be balanced for 4-6 min at 24-25 ℃, placed in the third vitrification refrigerating fluid to be balanced for 35-45 s at 24-25 ℃, and finally placed in liquid nitrogen for freezing.
7. The method for freezing bovine cloned embryos of claim 6, wherein the bovine cloned embryos are frozen in liquid nitrogen after being packed in an OPS tube.
8. The bovine cloned embryo thawing solution is characterized by comprising a first thawing solution and a second thawing solution;
the first thawing solution comprises 88 to 92 volume percent of the base solution as described in claim 1 and 8 to 12 volume percent of galactose;
the second thawing solution comprises 93 to 97 volume percent of the base solution as described in claim 1 and 3 to 7 volume percent of galactose.
9. A thawing method of bovine cloned embryos comprises the following steps: placing the frozen bovine cloned embryos in a first thawing solution to balance the embryos for 50-70 s at 37-39 ℃, then placing the bovine cloned embryos in a second thawing solution to balance the embryos for 4-6 min at 24-25 ℃, and finally placing the bovine cloned embryos in a base solution to balance the embryos for 4-6 min at 24-25 ℃.
10. A bovine cloned embryo freezing kit comprising the bovine cloned embryo freezing solution of claim 1 and the bovine cloned embryo thawing solution of claim 8.
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| CN114617121A (en) * | 2022-04-06 | 2022-06-14 | 清华大学 | Freezing optimization system of non-human embryonic cells and application |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR20020034773A (en) * | 2000-11-03 | 2002-05-09 | 임진호 | Method for embryo storage by vitrificaiton, Straw for embryo storage including the embryo vitrified according to the method and Method for thawing the straw by 1-step dilution |
| KR20020075478A (en) * | 2001-03-24 | 2002-10-05 | (주)마리아 바이오텍 | Method for cryopreserving eggs by minimum volume cooling manner, method for producing elite cow from the eggs cryopreserved by the method and the straw therefor |
| CN102648708A (en) * | 2011-02-25 | 2012-08-29 | 深圳华大方舟生物技术有限公司 | Freezing liquid for embryo or cells and application thereof |
| CN104488853A (en) * | 2015-01-12 | 2015-04-08 | 中国农业大学 | Bovine embryo vitrification freezing tube swinging thawing and direct transplanting method |
| CN109688812A (en) * | 2016-08-22 | 2019-04-26 | 博傲沃德株式会社 | The freezen protective composition and freezing and storing method of ox reproduction cell |
-
2020
- 2020-06-17 CN CN202010554671.8A patent/CN111493064A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR20020034773A (en) * | 2000-11-03 | 2002-05-09 | 임진호 | Method for embryo storage by vitrificaiton, Straw for embryo storage including the embryo vitrified according to the method and Method for thawing the straw by 1-step dilution |
| KR20020075478A (en) * | 2001-03-24 | 2002-10-05 | (주)마리아 바이오텍 | Method for cryopreserving eggs by minimum volume cooling manner, method for producing elite cow from the eggs cryopreserved by the method and the straw therefor |
| CN102648708A (en) * | 2011-02-25 | 2012-08-29 | 深圳华大方舟生物技术有限公司 | Freezing liquid for embryo or cells and application thereof |
| CN104488853A (en) * | 2015-01-12 | 2015-04-08 | 中国农业大学 | Bovine embryo vitrification freezing tube swinging thawing and direct transplanting method |
| CN109688812A (en) * | 2016-08-22 | 2019-04-26 | 博傲沃德株式会社 | The freezen protective composition and freezing and storing method of ox reproduction cell |
Non-Patent Citations (7)
| Title |
|---|
| 冯建忠: "《牛羊胚胎移植实用技术》", 31 January 2005, 中国农业出版社 * |
| 李武 等: ""绵羊胚胎移植研究进展"", 《黑龙江畜牧兽医》 * |
| 温集成 等: "《奶牛繁殖疾病及现代繁殖技术》", 31 January 2015 * |
| 王士勇等: ""利用冷冻的GV期卵母细胞制备延边黄牛核移植胚胎的研究"", 《江西农业大学学报》 * |
| 王珂等: ""奶牛一步细管法冷冻胚胎移植试验"", 《甘肃农业大学学报》 * |
| 管元七郎: "《哺乳动物发育工程试验方法》", 28 February 1992, 南京大学出版社 * |
| 肖定汉: "《奶牛饲养与疾病防治》", 31 January 2001, 中国农业出版社 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114617121A (en) * | 2022-04-06 | 2022-06-14 | 清华大学 | Freezing optimization system of non-human embryonic cells and application |
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