CN110604128A - Beef cattle propagation embryo cryopreservation liquid and freezing method - Google Patents
Beef cattle propagation embryo cryopreservation liquid and freezing method Download PDFInfo
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- CN110604128A CN110604128A CN201910998213.0A CN201910998213A CN110604128A CN 110604128 A CN110604128 A CN 110604128A CN 201910998213 A CN201910998213 A CN 201910998213A CN 110604128 A CN110604128 A CN 110604128A
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N1/00—Preservation of bodies of humans or animals, or parts thereof
- A01N1/02—Preservation of living parts
- A01N1/0205—Chemical aspects
- A01N1/021—Preservation or perfusion media, liquids, solids or gases used in the preservation of cells, tissue, organs or bodily fluids
- A01N1/0221—Freeze-process protecting agents, i.e. substances protecting cells from effects of the physical process, e.g. cryoprotectants, osmolarity regulators like oncotic agents
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N1/00—Preservation of bodies of humans or animals, or parts thereof
- A01N1/02—Preservation of living parts
- A01N1/0205—Chemical aspects
- A01N1/021—Preservation or perfusion media, liquids, solids or gases used in the preservation of cells, tissue, organs or bodily fluids
- A01N1/0226—Physiologically active agents, i.e. substances affecting physiological processes of cells and tissue to be preserved, e.g. anti-oxidants or nutrients
Abstract
The invention belongs to the technical field of embryology, and provides frozen preservative fluid and a freezing method for beef cattle breeding embryos, wherein the frozen preservative fluid comprises the following components in parts by weight: 40-50 parts of HPES buffer solution, 0.1-0.5 part of bovine serum albumin, 2-5 parts of sorbitol, 0.01-0.02 part of glutamine, 10-15 parts of polyethyleneimine and 20-25 parts of sucrose. The freezing method of beef cattle propagation embryos comprises the following steps: the method comprises the steps of firstly balancing a bovine embryo to be frozen in a pre-balancing solution for 5-8 min, then balancing the bovine embryo to be frozen in a frozen preservation solution for beef propagation embryos of any one of claims 1-3 for 30-40 seconds, transferring the bovine embryo to a freezing carrier, and putting the freezing carrier into liquid nitrogen for freezing. Through above-mentioned technical scheme, solved among the prior art cryopreservation liquid to the cryopreservation effect of cell not good, lead to the problem that the freezing survival rate of cell is low.
Description
Technical Field
The invention belongs to the technical field of embryology, and relates to a frozen preservation solution and a freezing method for beef cattle breeding embryos.
Background
The embryo cryopreservation is a biological technology which is used for freezing early embryos of animals by adopting a special protective agent and a cooling measure, so that the metabolism of the early embryos is stopped or weakened to a small enough degree in liquid nitrogen at the temperature of 196 ℃ below zero, and the capacity of recovering the metabolism after the temperature is raised is not lost, thereby being capable of preserving the embryos for a long time. Cryopreservation of bovine embryos was successful by Wilmut in 1973. The bovine embryo freezing method can be divided into a slow cooling method and a vitrification freezing method according to different cooling methods. The slow cooling method is that the embryo treated by freezing protective agent is cooled from room temperature to-7 deg.c at-1.0 deg.c/min by means of embryo freezing instrument with computer controlled cooling speed, maintained for 10min, cooled to-30 deg.c at-0.3 deg.c/min after being frozen, and then thrown into liquid ammonia for long term preservation. This slow cooling process can prevent intracellular freezing or reduce ice crystal formation. The ice planting induces crystallization, and can effectively prevent the damage of supercooling phenomena to embryos. The vitrification freezing method is that embryo is treated with freezing protectant and then thrown into liquid nitrogen for long term preservation. The principle is that a high concentration of cryoprotectant increases in viscosity upon cooling and solidifies when a critical value is reached.
In both the slow cooling method and the vitrification freezing method, a cryoprotectant must be added. The cryoprotectant may be combined with water to avoid freezing or to reduce the degree and rate of freezing, or to form only small ice crystals, while at the same time the cryoprotectant may reduce solute damage caused by elevated salt concentrations during freezing. The cryoprotectants are divided into two categories according to whether they can penetrate into cells, wherein- -the category is permeability protectant, and most of the compounds are low molecular compounds, can penetrate through cell membranes, can be frozen quickly at high molecular concentration, and can protect cells especially in slow freezing. The protective agent mainly comprises glycerol, dimethyl sulfoxide, propylene glycol, ethylene glycol, glucose and the like; another class is the non-osmotic protectants, mainly sucrose, polyvinylpyrrolidone, etc. The main characteristic of its action is to protect cells more effectively in faster freezing and thawing speeds. At present, more than two cryoprotectants are generally combined to form the cryopreservation liquid. However, the conventional cryopreservation solution has a poor effect of cryopreservation cells, resulting in a low cell cryopreservation survival rate. Therefore, there is a need for improvement of cryopreservation solutions to increase the freezing survival rate of cells.
Disclosure of Invention
The invention provides a beef cattle propagation embryo cryopreservation liquid and a freezing method, and solves the problem that in the prior art, the cryopreservation liquid has poor effect on cryopreservation of cells, so that the freezing survival rate of the cells is low.
The technical scheme of the invention is realized as follows:
the cryopreservation liquid for the beef cattle propagation embryos comprises the following components in parts by weight:
40-50 parts of HPES buffer solution, 0.1-0.5 part of bovine serum albumin, 2-5 parts of sorbitol, 0.01-0.02 part of glutamine, 10-15 parts of polyethyleneimine and 20-25 parts of sucrose.
As a further technical scheme, the paint comprises the following components in parts by weight:
45 parts of HPES buffer solution, 0.3 part of bovine serum albumin, 3 parts of sorbitol, 0.015 part of glutamine, 12 parts of polyethyleneimine and 23 parts of sucrose.
As a further technical scheme, the molecular weight of the polyethyleneimine is 70000.
A freezing method of beef cattle propagation embryos comprises the following steps: the method comprises the steps of firstly balancing a bovine embryo to be frozen in a pre-balancing solution for 5-8 min, then balancing the bovine embryo to be frozen in a frozen preservation solution for beef propagation embryos of any one of claims 1-3 for 30-40 seconds, transferring the bovine embryo to a freezing carrier, and putting the freezing carrier into liquid nitrogen for freezing.
As a further technical scheme, the method comprises the step of preheating the cryopreservation liquid to 37-40 ℃ before placing the bovine embryos which are balanced in the pre-balancing liquid for 5-8 min into the cryopreservation liquid.
As a further technical scheme, the freezing carrier is OPS.
As a further technical scheme, the pre-equilibrium liquid comprises the following components in parts by weight:
45 parts of HPES buffer solution, 0.4 part of bovine serum albumin, 0.03 part of glutamine and 15 parts of polyethyleneimine.
As a further technical scheme, the bovine embryo is a blastocyst, a 2-cell embryo, a 4-cell embryo or an 8-cell embryo.
The working principle and the beneficial effects of the invention are as follows:
1. according to the invention, the cryopreservation liquid and the freezing method remarkably improve the cryopreservation effect of the embryo, so that the survival rate of the frozen embryo after thawing is greatly improved, and the freezing survival rate of the bovine embryo is improved to more than 90%, such as: the freezing survival rate of the bovine blastocyst is improved to 95.1%, the freezing survival rate of the bovine 2-cell embryo is improved to 91.1%, the freezing survival rate of the bovine 4-cell embryo is improved to 91.6%, and the freezing survival rate of the bovine 8-cell embryo is improved to 93.8%, so that the problem of low freezing survival rate of cells caused by poor freezing preservation effect of the freezing preservation solution on the cells in the prior art is effectively solved.
2. According to the invention, the cryopreservation liquid is composed of HPES buffer solution, bovine serum albumin, sorbitol, glutamine, polyethyleneimine and sucrose, each component has low cytotoxicity, and small damage to cells in the freezing process, and the components are compatible with each other, so that the freezing survival rate of embryos is remarkably improved. The small molecule sorbitol and glutamine can penetrate into cells to generate a certain concentration inside and outside the cells, and the cells are protected from being damaged by high-concentration electrolytes. During the process of freezing and unfreezing the cells, glutamine can also provide energy for the cells, thereby further improving the freezing survival rate of the cells. The polyethyleneimine with high molecular weight is selected to be matched with sucrose, so that the polyethyleneimine can be combined with water molecules in solution in preference to reduce the content of free water in the solution, so that the freezing point is lowered, the formation of ice crystals is reduced, and meanwhile, the concentration of electrolyte in the solution can be reduced, so that the cells are further protected from being damaged by high-concentration electrolyte, therefore, the freezing survival rate of the cells is greatly improved, and the polyethyleneimine with high molecular weight is suitable for popularization and use.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example 1
Bovine blastocyst freezing test
1. Liquid preparation:
pre-balancing liquid: 45 parts of HPES buffer solution, 0.4 part of bovine serum albumin, 0.03 part of glutamine and 15 parts of polyethyleneimine, wherein the preparation method of the pre-equilibrium solution is to stir and mix the components uniformly at room temperature to obtain the pre-equilibrium solution.
Freezing and preserving fluid: 45 parts of HPES buffer solution, 0.3 part of bovine serum albumin, 3 parts of sorbitol, 0.015 part of glutamine, 12 parts of polyethyleneimine and 23 parts of sucrose, and the preparation method of the frozen preservation solution comprises the following steps: adding polyethyleneimine into HPES buffer solution, dissolving, adding bovine serum albumin, standing at 4 deg.C for dissolving, adding sucrose, sorbitol and glutamine, standing at 4 deg.C for dissolving to obtain frozen storage solution; before use, the cryopreservation solution is preheated to 38 ℃.
Wherein the molecular weight of the polyethyleneimine is 70000.
Thawing solution 1: sucrose was added at 1mg/mL based on HPES buffer.
Thawing solution 2: sucrose was added at 2mg/mL based on HPES buffer.
Thawing solution 3: sucrose (2 mg/mL) and bovine serum albumin (3 mg/mL) were added to the HPES buffer solution.
Embryo culture solution: KSOM culture medium, commercially available.
2. Embryo freezing: the bovine blastocysts to be frozen are firstly balanced in a pre-balancing solution for 5min, then are put into a preheated freezing preservation solution for balancing for 30 seconds, then are transferred into OPS, and are put into liquid nitrogen for freezing.
3. And (3) unfreezing the embryo: respectively placing the thawing solution 1, the thawing solution 2 and the thawing solution 3 in an incubator at 37 ℃ for balancing for 15min, then placing on a constant temperature table, taking out OPS from liquid nitrogen, directly immersing the part containing the embryo into the thawing solution 1, balancing for 1min, transferring the embryo into the thawing solution 2, balancing for 5min, transferring the embryo into the thawing solution 3, balancing for 5min, transferring the embryo into an embryo culture solution, continuously culturing in the incubator, and observing the result after 16 h.
And (3) freezing 16 batches of blastocysts with good growth states simultaneously according to the method, and setting 4-6 parallel tests in each batch. And observing the preserved result.
Meanwhile, a conventional freezing preservation solution is arranged for carrying out a freezing comparison test, the freezing and unfreezing processes are the same as those of the invention, and the formula of the conventional pre-equilibrium solution used in a comparison group is as follows: 45 parts of HPES buffer solution, 0.4 part of bovine serum albumin, 10 parts of ethylene glycol and 25 parts of sucrose; the formula of the conventional cryopreservation solution used in the control group was: 45 parts of HPES buffer solution, 0.3 part of bovine serum albumin, 18 parts of ethylene glycol and 23 parts of sucrose.
4. And (3) test results:
the survival rates of the cryopreserved 16 batches of blastocysts were tested as shown in table 1:
TABLE 1 cryopreservation effect of inventive and conventional freezing fluids on bovine blastocysts
As can be seen from the data in Table 1, the cryopreservation solution of the invention has higher cryopreservation effect on bovine blastocysts than the conventional cryopreservation solution, and the bovine blastocysts have higher survival rate after cryopreservation and more stable performance.
Example 2
Bovine 2-cell embryo freezing assay
1. Liquid preparation:
pre-balancing liquid: 45 parts of HPES buffer solution, 0.4 part of bovine serum albumin, 0.03 part of glutamine and 15 parts of polyethyleneimine, wherein the preparation method of the pre-equilibrium solution is to stir and mix the components uniformly at room temperature to obtain the pre-equilibrium solution.
Freezing and preserving fluid: 40 parts of HPES buffer solution, 0.1 part of bovine serum albumin, 2 parts of sorbitol, 0.01 part of glutamine, 10 parts of polyethyleneimine and 20 parts of sucrose, and the preparation method of the frozen preservation solution comprises the following steps: adding polyethyleneimine into HPES buffer solution, dissolving, adding bovine serum albumin, standing at 4 deg.C for dissolving, adding sucrose, sorbitol and glutamine, standing at 4 deg.C for dissolving to obtain frozen storage solution; before use, the cryopreservation solution is preheated to 37 ℃.
Wherein the molecular weight of the polyethyleneimine is 70000.
Thawing solution 1: sucrose was added at 1mg/mL based on HPES buffer.
Thawing solution 2: sucrose was added at 2mg/mL based on HPES buffer.
Thawing solution 3: sucrose (2 mg/mL) and bovine serum albumin (3 mg/mL) were added to the HPES buffer solution.
Embryo culture solution: KSOM culture medium, commercially available.
2. Embryo freezing: the bovine 2-cell embryo to be frozen is firstly balanced in a pre-balancing solution for 5min, then is put into a preheated freezing preservation solution to be balanced for 30 seconds, then is transferred into OPS, and is put into liquid nitrogen for freezing.
3. And (3) unfreezing the embryo: respectively placing the thawing solution 1, the thawing solution 2 and the thawing solution 3 in an incubator at 37 ℃ for balancing for 15min, then placing on a constant temperature table, taking out OPS from liquid nitrogen, directly immersing the part containing the embryo into the thawing solution 1, balancing for 1min, transferring the embryo into the thawing solution 2, balancing for 5min, transferring the embryo into the thawing solution 3, balancing for 5min, transferring the embryo into an embryo culture solution, continuously culturing in the incubator, and observing the result after 16 h.
15 batches of 2-cell embryos with good growth state are frozen simultaneously according to the method, and 10-20 parallel tests are set for each batch. And observing the preserved result.
Meanwhile, a conventional freezing preservation solution is arranged for carrying out a freezing comparison test, the freezing and unfreezing processes are the same as those of the invention, and the formula of the conventional pre-equilibrium solution used in a comparison group is as follows: 45 parts of HPES buffer solution, 0.4 part of bovine serum albumin, 10 parts of ethylene glycol and 25 parts of sucrose; the formula of the conventional cryopreservation solution used in the control group was: 45 parts of HPES buffer solution, 0.3 part of bovine serum albumin, 18 parts of ethylene glycol and 23 parts of sucrose.
4. And (3) test results:
frozen 15 batches of 2-cell embryos were tested for viability as shown in table 2:
TABLE 2 cryopreservation Effect of the freezing fluid of the present invention and the conventional freezing fluid on bovine 2-cell embryos
As can be seen from the data in Table 2, the cryopreservation liquid of the present invention has higher cryopreservation effect on bovine 2-cell embryos than the conventional cryopreservation liquid, and the bovine 2-cell embryos have higher survival rate after cryopreservation and more stable performance.
Example 3
Bovine 4-cell embryo freezing assay
1. Liquid preparation:
pre-balancing liquid: 45 parts of HPES buffer solution, 0.4 part of bovine serum albumin, 0.03 part of glutamine and 15 parts of polyethyleneimine, wherein the preparation method of the pre-equilibrium solution is to stir and mix the components uniformly at room temperature to obtain the pre-equilibrium solution.
Freezing and preserving fluid: 50 parts of HPES buffer solution, 0.5 part of bovine serum albumin, 5 parts of sorbitol, 0.02 part of glutamine, 15 parts of polyethyleneimine and 25 parts of sucrose, and the preparation method of the frozen preservation solution comprises the following steps: adding polyethyleneimine into HPES buffer solution, dissolving, adding bovine serum albumin, standing at 4 deg.C for dissolving, adding sucrose, sorbitol and glutamine, standing at 4 deg.C for dissolving to obtain frozen storage solution; before use, the cryopreservation solution is preheated to 40 ℃.
Wherein the molecular weight of the polyethyleneimine is 70000.
Thawing solution 1: sucrose was added at 1mg/mL based on HPES buffer.
Thawing solution 2: sucrose was added at 2mg/mL based on HPES buffer.
Thawing solution 3: sucrose (2 mg/mL) and bovine serum albumin (3 mg/mL) were added to the HPES buffer solution.
Embryo culture solution: KSOM culture medium, commercially available.
2. Embryo freezing: the bovine 4-cell embryo to be frozen is firstly balanced in a pre-balancing solution for 5min, then is put into a preheated freezing preservation solution to be balanced for 30 seconds, then is transferred into OPS, and is put into liquid nitrogen for freezing.
3. And (3) unfreezing the embryo: respectively placing the thawing solution 1, the thawing solution 2 and the thawing solution 3 in an incubator at 37 ℃ for balancing for 15min, then placing on a constant temperature table, taking out OPS from liquid nitrogen, directly immersing the part containing the embryo into the thawing solution 1, balancing for 1min, transferring the embryo into the thawing solution 2, balancing for 5min, transferring the embryo into the thawing solution 3, balancing for 5min, transferring the embryo into an embryo culture solution, continuously culturing in the incubator, and observing the result after 16 h.
15 batches of 4-cell embryos with good growth state are frozen simultaneously according to the method, and 4-6 parallel tests are set for each batch. And observing the preserved result.
Meanwhile, a conventional freezing preservation solution is arranged for carrying out a freezing comparison test, the freezing and unfreezing processes are the same as those of the invention, and the formula of the conventional pre-equilibrium solution used in a comparison group is as follows: 45 parts of HPES buffer solution, 0.4 part of bovine serum albumin, 10 parts of ethylene glycol and 25 parts of sucrose; the formula of the conventional cryopreservation solution used in the control group was: 45 parts of HPES buffer solution, 0.3 part of bovine serum albumin, 18 parts of ethylene glycol and 23 parts of sucrose.
4. And (3) test results:
frozen 15 batches of 4-cell embryos were tested for viability as shown in table 3:
TABLE 3 cryopreservation Effect of the freezing fluid of the present invention and the conventional freezing fluid on bovine 4-cell embryos
As can be seen from the data in Table 3, the cryopreservation solution of the present invention has higher cryopreservation effect on bovine 4-cell embryos than the conventional cryopreservation solution and has more stable performance.
Example 4
Bovine 8-cell embryo freezing assay
1. Liquid preparation:
pre-balancing liquid: 45 parts of HPES buffer solution, 0.4 part of bovine serum albumin, 0.03 part of glutamine and 15 parts of polyethyleneimine, wherein the preparation method of the pre-equilibrium solution is to stir and mix the components uniformly at room temperature to obtain the pre-equilibrium solution.
Freezing and preserving fluid: 45 parts of HPES buffer solution, 0.3 part of bovine serum albumin, 3 parts of sorbitol, 0.015 part of glutamine, 12 parts of polyethyleneimine and 23 parts of sucrose, and the preparation method of the frozen preservation solution comprises the following steps: adding polyethyleneimine into HPES buffer solution, dissolving, adding bovine serum albumin, standing at 4 deg.C for dissolving, adding sucrose, sorbitol and glutamine, standing at 4 deg.C for dissolving to obtain frozen storage solution; before use, the cryopreservation solution is preheated to 37.5 ℃.
Thawing solution 1: sucrose was added at 1mg/mL based on HPES buffer.
Thawing solution 2: sucrose was added at 2mg/mL based on HPES buffer.
Thawing solution 3: sucrose (2 mg/mL) and bovine serum albumin (3 mg/mL) were added to the HPES buffer solution.
Embryo culture solution: KSOM culture medium, commercially available.
2. Embryo freezing: the bovine 8-cell embryo to be frozen is firstly balanced in a pre-balancing solution for 5min, then is put into a preheated freezing preservation solution to be balanced for 30 seconds, then is transferred into OPS, and is put into liquid nitrogen for freezing.
3. And (3) unfreezing the embryo: respectively placing the thawing solution 1, the thawing solution 2 and the thawing solution 3 in an incubator at 37 ℃ for balancing for 15min, then placing on a constant temperature table, taking out OPS from liquid nitrogen, directly immersing the part containing the embryo into the thawing solution 1, balancing for 1min, transferring the embryo into the thawing solution 2, balancing for 5min, transferring the embryo into the thawing solution 3, balancing for 5min, transferring the embryo into an embryo culture solution, continuously culturing in the incubator, and observing the result after 16 h.
15 batches of 8-cell embryos with good growth state are frozen simultaneously according to the method, and 10-20 parallel tests are set for each batch. And observing the preserved result.
Meanwhile, a conventional freezing preservation solution is arranged for carrying out a freezing comparison test, the freezing and unfreezing processes are the same as those of the invention, and the formula of the conventional pre-equilibrium solution used in a comparison group is as follows: 45 parts of HPES buffer solution, 0.4 part of bovine serum albumin, 10 parts of ethylene glycol and 25 parts of sucrose; the formula of the conventional cryopreservation solution used in the control group was: 45 parts of HPES buffer solution, 0.3 part of bovine serum albumin, 18 parts of ethylene glycol and 23 parts of sucrose.
4. And (3) test results:
frozen 15 batches of 8-cell embryos were tested for viability as shown in table 1:
TABLE 4 cryopreservation Effect of the freezing fluid of the present invention and the conventional freezing fluid on bovine 8-cell embryos
As can be seen from the data in Table 4, the cryopreservation solution of the present invention has higher cryopreservation effect on bovine 8-cell embryos than the conventional cryopreservation solution and has more stable performance.
The present invention is not limited to the above preferred embodiments, and any modifications, equivalent substitutions, improvements, etc. within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (8)
1. The cryopreservation liquid for the beef cattle propagation embryos is characterized by comprising the following components in parts by weight:
40-50 parts of HPES buffer solution, 0.1-0.5 part of bovine serum albumin, 2-5 parts of sorbitol, 0.01-0.02 part of glutamine, 10-15 parts of polyethyleneimine and 20-25 parts of sucrose.
2. The cryopreservation liquid for beef cattle propagation embryos according to claim 1, which is characterized by comprising the following components in parts by weight:
45 parts of HPES buffer solution, 0.3 part of bovine serum albumin, 3 parts of sorbitol, 0.015 part of glutamine, 12 parts of polyethyleneimine and 23 parts of sucrose.
3. The cryopreservation liquid for beef cattle propagation embryos according to claim 1 or 2, wherein the molecular weight of the polyethyleneimine is 70000.
4. A freezing method for beef cattle breeding embryos is characterized by comprising the following steps: the method comprises the steps of firstly balancing a bovine embryo to be frozen in a pre-balancing solution for 5-8 min, then balancing the bovine embryo to be frozen in a frozen preservation solution for beef propagation embryos of any one of claims 1-3 for 30-40 seconds, transferring the bovine embryo to a freezing carrier, and putting the freezing carrier into liquid nitrogen for freezing.
5. The freezing method of beef cattle propagation embryos according to claim 4, wherein the frozen preservation solution is preheated to 37-40 ℃ before the beef cattle embryos which are equilibrated in the pre-equilibration solution for 5-8 min are placed in the frozen preservation solution.
6. The method of claim 4, wherein the freezing medium is OPS.
7. The method for freezing beef cattle propagation embryos according to claim 4, wherein the pre-equilibrium liquid consists of the following components in parts by weight:
45 parts of HPES buffer solution, 0.4 part of bovine serum albumin, 0.03 part of glutamine and 15 parts of polyethyleneimine.
8. The method for freezing a beef cattle propagated embryo according to any one of claims 4 to 7, wherein the beef cattle embryo is a blastocyst, a 2-cell embryo, a 4-cell embryo or an 8-cell embryo.
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