CN107232185B - Vitrification cryopreservation liquid for equine animal oocyte, cryopreservation method and application - Google Patents

Vitrification cryopreservation liquid for equine animal oocyte, cryopreservation method and application Download PDF

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CN107232185B
CN107232185B CN201710639975.2A CN201710639975A CN107232185B CN 107232185 B CN107232185 B CN 107232185B CN 201710639975 A CN201710639975 A CN 201710639975A CN 107232185 B CN107232185 B CN 107232185B
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liquid
vitrification
cryopreservation
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oocytes
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CN107232185A (en
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李莎莎
邹敬清
邹志钢
荣美洁
王浩
李成修
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Yili Derui Junfa Biotechnology Co ltd
Qingdao Derui Junfa Biotechnology Co ltd
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Qingdao Derui Junfa Biotechnology Co ltd
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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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Abstract

The invention discloses an equus animal oocyte vitrification cryopreservation liquid, a cryopreservation method and application, wherein the vitrification cryopreservation liquid comprises a base liquid (BS), a balance liquid (ES) and a vitrification liquid (VS), wherein the vitrification liquid (VS) comprises the following components in percentage by volume: 10-20% Ethylene Glycol (EG), 6-10% dimethyl sulfoxide (DMSO), 6-10% propylene glycol (PROH), 15-25% inactivated fetal bovine serum (NCS), 8-15% carboxylated epsilon-polylysine (COOL-PLL), and the balance HEPES solution; each liter of the glass transition liquid (VS) also comprises 0.3-0.8mol of cane sugar. The invention has the beneficial effects that: the vitrified cryopreservation liquid has low cytotoxicity, strong permeability and obvious vitrified cryopreservation effect, obviously reduces the generation of ice crystals in the freezing and rewarming processes, protects cells from physical damage, improves the survival rate of oocytes, is very suitable for vitrified cryopreservation of oocytes or embryos of equine animals such as horses, donkeys and the like, and is beneficial to popularization and application of artificial insemination and embryo transfer technologies.

Description

Vitrification cryopreservation liquid for equine animal oocyte, cryopreservation method and application
Technical Field
The invention belongs to the technical field of cryopreservation of animal oocytes and embryos, and particularly relates to a vitrification cryopreservation liquid for equine oocytes, a cryopreservation method and application.
Background
The cryopreservation of the oocyte refers to a preservation technology that the collected oocyte is preserved under the condition of ultralow temperature (-196 ℃ liquid nitrogen), so that the metabolic activity of the oocyte is stopped, the oocyte is in a dormant state, and the oocyte can be continuously inseminated and developed into a healthy embryo after the external environment reaches a proper condition. The oocyte cryopreservation technology can provide conditions for establishing an excellent variety oocyte library or a gene library; maintaining various animal varieties, strains and rare or endangered animals lost due to diseases or other reasons; the simple and cheap long-distance transportation can be realized, and the international introduction is convenient; it can replace the transportation of live animals, save a lot of time and money, and reduce the risk of disease transmission during the transportation of live animals.
The first successful cryopreservation of oocytes is the mouse oocytes frozen and matured by Whittingham in 1977, mammals including rabbits, goats, cows, pigs, humans and the like which have been related in recent years, but cryopreservation of oocytes of equine animals is rarely reported. Oocyte refrigerating fluid and a cryopreservation method mainly use embryo cryopreservation fluid and a cryopreservation method for reference, but because the oocyte has the characteristics of special structure, large volume, difficulty in fully dehydrating during freezing and the like, the cryopreservation is always difficult. After the mouse mature oocyte is frozen by the vitrification freezing method and is successfully obtained, the vitrification freezing method is widely used for the cryopreservation research of various oocytes.
The principle of the vitrification cryopreservation is that the vitrification cryoprotectant is a key factor for successfully freezing the cells, and the vitrification cryopreservation adopts the characteristic that the viscosity of the high-concentration cryoprotectant is extremely increased when the high-concentration cryoprotectant is frozen and solidified, so that the liquid state is changed into a structureless vitrification state, the concentration of a damaging solution is reduced, a non-freezing part is increased, the generation of ice crystals and the fluctuation of the volume of oocytes or embryos in the freezing and thawing process are reduced, the damage of osmotic pressure and chilling to the cells is reduced, and the recovery rate of the oocytes or embryos is improved. The existing vitrification cryoprotectant has single component, high cytotoxicity, poor ice crystal generation inhibition effect in the processes of temperature reduction and rewarming, and great damage to cells, which causes relatively low recovery rate of oocytes or embryos.
Disclosure of Invention
The invention provides an equine animal oocyte vitrification cryopreservation liquid and a preparation method, a cryopreservation method and application thereof, which aim to solve the problems of chemical and physical damage of ice crystals to cells, cytotoxicity of a freezing liquid and the like in the cryopreservation process of equine animal oocytes and embryos and improve the recovery rate of the oocytes or embryos.
In order to achieve the purpose, the invention provides an equine oocyte vitrification cryopreservation liquid, which comprises 3 liquids of a base liquid (BS), a balance liquid (ES) and a vitrification liquid (VS), wherein the vitrification liquid (VS) comprises the following components in percentage by weight: 10-20% Ethylene Glycol (EG), 6-10% dimethyl sulfoxide (DMSO), 6-10% propylene glycol (PROH), 15-25% inactivated Fetal Bovine Serum (FBS), 0.3-0.8mol/L sucrose, 8-15% carboxylated epsilon-polylysine, and the balance of HEPES solution with a concentration of 10 mmol/L.
The content of each component of the equilibrium liquid (ES) is as follows: 5-10% Ethylene Glycol (EG), 2-6% dimethyl sulfoxide (DMSO), 2-6% propylene glycol (PROH), 15-25% inactivated Fetal Bovine Serum (FBS), 0.1-0.3mol/L sucrose, 2-8% carboxylated epsilon-polylysine, and the balance 10mmol/L HEPES solution.
The Base Solution (BS) consists of inactivated fetal calf serum with volume fraction of 15-25% and HEPES solution with concentration of 75-85% and 10 mmol/L; preferably, the base fluid (BS) consists of a volume fraction of 20% inactivated fetal bovine serum and 80% HEPES solution at a concentration of 10 mmol/L.
The molecular formula of the Ethylene Glycol (EG) is C2H6O2(ii) a Molecular formula of the dimethyl sulfoxide (DMSO)Is (CH3)2 SO; the molecular formula of the propylene glycol (PROH) is C3H8O2(ii) a The molecular formula of the sucrose is C12H22O11(ii) a The molecular formula of the carboxylated epsilon-polylysine is C181H362N60O33(ii) a The molecular formula of the HEPES is C8H18N2O4S; the molecular formula of the hydroxyapatite is Ca10(PO4)6(OH)2。
Preferably, the vitrification liquid (VS) further includes hydroxyapatite having a particle size of 40 nm; the glass transition liquid (VS) consists of the following components in percentage by volume: 10-20% Ethylene Glycol (EG), 6-10% dimethyl sulfoxide (DMSO), 6-10% propylene glycol (PROH), 15-25% inactivated Fetal Bovine Serum (FBS), 8-15% carboxylated epsilon-polylysine, and the balance of HEPES solution with a concentration of 10 mmol/L; each liter of the Vitrification Solution (VS) also comprises 0.3 to 0.8mol of sucrose and 1 to 5g of hydroxyapatite with the particle size of 40 nm;
the equilibrium liquid (ES) also comprises hydroxyapatite with the grain diameter of 40 nm; the equilibrium liquid (ES) consists of the following components in percentage by volume: 5-10% Ethylene Glycol (EG), 2-6% dimethyl sulfoxide (DMSO), 2-6% propylene glycol (PROH), 15-25% inactivated Fetal Bovine Serum (FBS), 2-8% carboxylated epsilon-polylysine, and the balance of HEPES solution with a concentration of 10 mmol/L; each liter of the equilibrium liquid (ES) also comprises 0.1-0.3mol/L of sucrose and 1-5g of hydroxyapatite with the particle size of 40 nm.
Preferably, the vitrification liquid (VS) consists of the following components in volume percent: 12-17% Ethylene Glycol (EG), 7-9% dimethyl sulfoxide (DMSO), 7-9% propylene glycol (PROH), 18-21% inactivated Fetal Bovine Serum (FBS), 10-13% carboxylated epsilon-polylysine, and the balance of HEPES solution with a concentration of 10 mmol/L; each liter of the Vitrification Solution (VS) also comprises 0.4 to 0.7mol of sucrose and 2 to 4g of hydroxyapatite with the particle size of 40 nm;
the equilibrium liquid (ES) consists of the following components in percentage by volume: 6-8% Ethylene Glycol (EG), 3-5% dimethyl sulfoxide (DMSO), 3-5% propylene glycol (PROH), 17-22% inactivated Fetal Bovine Serum (FBS), 4-7% carboxylated epsilon-polylysine, and the balance of HEPES solution with a concentration of 10 mmol/L; each liter of the equilibrium liquid (ES) also comprises 0.1-0.3mol/L of sucrose and 2-4g of hydroxyapatite with the particle size of 40 nm.
The vitrification liquid (VS) also comprises hydroxyapatite with the grain diameter of 40 nm; the glass transition liquid (VS) consists of the following components in percentage by volume: 10-20% Ethylene Glycol (EG), 6-10% dimethyl sulfoxide (DMSO), 6-10% propylene glycol (PROH), 15-25% inactivated Fetal Bovine Serum (FBS), 8-15% carboxylated epsilon-polylysine, and the balance of HEPES solution with a concentration of 10 mmol/L; each liter of the Vitrification Solution (VS) also comprises 0.3 to 0.8mol of sucrose and 1 to 5g of hydroxyapatite with the particle size of 40 nm;
the equilibrium liquid (ES) also comprises hydroxyapatite with the grain diameter of 40 nm; the equilibrium liquid (ES) consists of the following components in percentage by volume: 5-10% Ethylene Glycol (EG), 2-6% dimethyl sulfoxide (DMSO), 2-6% propylene glycol (PROH), 15-25% inactivated Fetal Bovine Serum (FBS), 2-8% carboxylated epsilon-polylysine, and the balance of HEPES solution with a concentration of 10 mmol/L; each liter of the equilibrium liquid (ES) also comprises 0.1-0.3mol/L of sucrose and 1-5g of hydroxyapatite with the particle size of 40 nm.
Preferably, 0.3-0.7g of vitamin D is also added into each liter of the glass melting liquid; each liter of the balance liquid is also added with 0.2 to 0.6g of vitamin D; furthermore, the vitamin D added in the vitrification liquid and the equilibrium liquid is vitamin D2.
The preparation methods of the vitrification liquid (VS), the equilibrium liquid (ES) and the base liquid (BS) are as follows:
the preparation method of the glass transition metal (VS) comprises the following steps: adding the components into the HEPES solution according to the volume fraction ratio, dissolving and shaking uniformly, and filtering and sterilizing to obtain the Vitrification Solution (VS).
The preparation method of the equilibrium liquid (ES) comprises the following steps: adding the components into the HEPES solution according to the volume fraction ratio, dissolving and shaking uniformly, and filtering and sterilizing to obtain the Equilibrium Solution (ES).
The preparation method of the base liquid (BS) comprises the following steps: and adding the inactivated Fetal Bovine Serum (FBS) into the HEPES solution according to the volume fraction ratio to obtain the Base Solution (BS).
In order to better achieve the above object, the present invention further provides a vitrification cryopreservation method for equine oocyte, the preservation method comprises:
(1) putting the oocyte or embryo to be frozen into a Basic Solution (BS) and washing for 1-2 times;
(2) transferring the washed oocyte or embryo to an equilibration fluid (ES) for 3-7 minutes; preferably, the washed oocyte or embryo is transferred to an equilibration fluid (ES) for 5 minutes;
(3) rapidly transferring the balanced oocyte or embryo to a Vitrification Solution (VS), rapidly transferring the vitrification solution containing the oocyte or embryo to a closed elongated straw (CPS tube), and putting liquid nitrogen for preservation at the temperature of-196 ℃, wherein the whole process is completed within 1 minute.
Wherein the base liquid (BS) consists of the following components in percentage by volume: 15-25% inactivated fetal calf serum and 75-85% HEPES solution with concentration of 10 mmol/L; preferably, the base fluid (BS) consists of a volume fraction of 20% inactivated fetal bovine serum and 80% HEPES solution at a concentration of 10 mmol/L.
The equilibrium liquid (ES) consists of the following components in percentage by volume: 5-10% Ethylene Glycol (EG), 2-6% dimethyl sulfoxide (DMSO), 2-6% propylene glycol (PROH), 15-25% inactivated Fetal Bovine Serum (FBS), 2-8% carboxylated epsilon-polylysine, and the balance of HEPES solution with a concentration of 10 mmol/L; each liter of the equilibrium liquid (ES) also comprises 0.1-0.3mol/L of sucrose;
the glass transition liquid (VS) consists of the following components in percentage by volume: the glass transition liquid (VS) consists of the following components in percentage by volume: 10-20% Ethylene Glycol (EG), 6-10% dimethyl sulfoxide (DMSO), 6-10% propylene glycol (PROH), 15-25% inactivated Fetal Bovine Serum (FBS), 8-15% carboxylated epsilon-polylysine, and the balance of HEPES solution with a concentration of 10 mmol/L; and each liter of the Vitrification Solution (VS) also comprises 0.3-0.8mol of sucrose.
Further, the vitrification liquid (VS) also comprises hydroxyapatite with the particle size of 40 nm; the glass transition liquid (VS) consists of the following components in percentage by volume: 10-20% Ethylene Glycol (EG), 6-10% dimethyl sulfoxide (DMSO), 6-10% propylene glycol (PROH), 15-25% inactivated Fetal Bovine Serum (FBS), 8-15% carboxylated epsilon-polylysine, and the balance of HEPES solution with a concentration of 10 mmol/L; each liter of the Vitrification Solution (VS) also comprises 0.3 to 0.8mol of sucrose and 1 to 5g of hydroxyapatite with the particle size of 40 nm; preferably, the vitrification liquid (VS) consists of the following components in volume percent: 12-17% Ethylene Glycol (EG), 7-9% dimethyl sulfoxide (DMSO), 7-9% propylene glycol (PROH), 18-21% inactivated Fetal Bovine Serum (FBS), 10-13% carboxylated epsilon-polylysine, and the balance of HEPES solution with a concentration of 10 mmol/L; each liter of the Vitrification Solution (VS) also comprises 0.4 to 0.7mol of sucrose and 2 to 4g of hydroxyapatite with the particle size of 40 nm;
the equilibrium liquid (ES) also comprises hydroxyapatite with the grain diameter of 40 nm; the equilibrium liquid (ES) consists of the following components in percentage by volume: 5-10% Ethylene Glycol (EG), 2-6% dimethyl sulfoxide (DMSO), 2-6% propylene glycol (PROH), 15-25% inactivated Fetal Bovine Serum (FBS), 2-8% carboxylated epsilon-polylysine, and the balance of HEPES solution with a concentration of 10 mmol/L; each liter of the equilibrium liquid (ES) also comprises 0.1 to 0.3mol of sucrose and 1 to 5g of hydroxyapatite with the particle size of 40 nm; preferably, the equilibrium liquid (ES) consists of the following constituents in volume percent: 6-8% Ethylene Glycol (EG), 3-5% dimethyl sulfoxide (DMSO), 3-5% propylene glycol (PROH), 17-22% inactivated Fetal Bovine Serum (FBS), 4-7% carboxylated epsilon-polylysine, and the balance of HEPES solution with a concentration of 10 mmol/L; each liter of the equilibrium liquid (ES) also comprises 0.1 to 0.3mol of sucrose and 2 to 4g of hydroxyapatite with the particle size of 40 nm.
Preferably, 0.3-0.7g of vitamin D is also added into each liter of the glass melting liquid; each liter of the balance liquid is also added with 0.2 to 0.6g of vitamin D; furthermore, the vitamin D added in the vitrification liquid and the equilibrium liquid is vitamin D2.
The preparation methods of the vitrification liquid (VS), the equilibrium liquid (ES) and the base liquid (BS) are as follows:
the preparation method of the glass transition metal (VS) comprises the following steps: adding the components into the HEPES solution according to the volume fraction ratio, dissolving and shaking uniformly, and filtering and sterilizing to obtain the Vitrification Solution (VS).
The preparation method of the equilibrium liquid (ES) comprises the following steps: adding the components into the HEPES solution according to the volume fraction ratio, dissolving and shaking uniformly, and filtering and sterilizing to obtain the Equilibrium Solution (ES).
The preparation method of the base liquid (BS) comprises the following steps: and adding the inactivated Fetal Bovine Serum (FBS) into the HEPES solution according to the volume fraction ratio to obtain the Base Solution (BS).
The invention also provides an application of the vitrification cryopreservation liquid for equine animal oocytes, and the vitrification cryopreservation liquid is suitable for cryopreservation of oocytes and embryos of all equine animals such as horses, donkeys and the like; furthermore, the vitrified cryopreservation solution is not limited to cryopreservation of oocytes and embryos of all equine animals such as horses, donkeys and the like.
The invention has the beneficial effects that: the vitrified cryopreservation liquid has low cytotoxicity, strong permeability and obvious vitrified cryopreservation effect, obviously reduces the generation of ice crystals in the freezing and rewarming processes, protects cells from physical damage, improves the survival rate of oocytes, is very suitable for vitrified cryopreservation of oocytes or embryos of equine animals such as horses, donkeys and the like, and is beneficial to popularization and application of artificial insemination and embryo transfer technologies.
Detailed Description
In order to clearly illustrate the technical features of the present solution, the present solution is explained below by way of specific embodiments.
EXAMPLE 1 refrigerating fluid
The embodiment of the invention provides an equus animal oocyte vitrification cryopreservation liquid, which comprises 3 liquids of basic liquid (BS), equilibrium liquid (ES) and vitrification liquid (VS); wherein the content of the first and second substances,
the Base Solution (BS) consists of inactivated fetal calf serum with the volume fraction of 20% and HEPES solution with the volume fraction of 80% and the concentration of 10 mmol/L; the preparation method comprises the following steps: in a sterile environment, inactivated Fetal Bovine Serum (FBS) is added into HEPES solution according to volume percentage, and dissolved and shaken uniformly to prepare 10ml of Base Solution (BS).
The equilibrium liquid (ES) consists of the following components in percentage by volume: 8% Ethylene Glycol (EG), 4% dimethyl sulfoxide (DMSO), 4% propylene glycol (PROH), 20% inactivated Fetal Bovine Serum (FBS), 5% carboxylated epsilon-polylysine, and the remainder as a 10mmol/L HEPES solution; each liter of the equilibrium liquid (ES) also comprises 0.25mol of sucrose; the preparation method comprises the following steps: adding the components into HEPES solution according to volume fraction ratio, dissolving, shaking, filtering, sterilizing to obtain 100ml of Equilibrium Solution (ES), and placing in a refrigerator at 4 ℃ for later use.
The glass transition metal (VS) consists of the following components in percentage by volume: 16% Ethylene Glycol (EG), 8% dimethyl sulfoxide (DMSO), 8% propylene glycol (PROH), 20% inactivated Fetal Bovine Serum (FBS), 10% carboxylated epsilon-polylysine, and the remainder as a 10mmol/L HEPES solution; 0.5mol of sucrose per liter of vitrification liquid (VS); the configuration method comprises the following steps: adding the components into HEPES solution according to the volume fraction ratio, dissolving and shaking uniformly, filtering and sterilizing to prepare 10ml of vitrified liquid (VS), and placing the vitrified liquid in a refrigerator at 4 ℃ for later use.
The vitrification cryopreservation liquid of the embodiment is used for vitrification cryopreservation of the oocyte of the equine animal.
EXAMPLE 2 refrigerant two
The embodiment of the invention provides an equus animal oocyte vitrification cryopreservation liquid, which comprises 3 liquids of basic liquid (BS), equilibrium liquid (ES) and vitrification liquid (VS); wherein the content of the first and second substances,
the Base Solution (BS) consists of inactivated fetal calf serum with the volume fraction of 20% and HEPES solution with the volume fraction of 80% and the concentration of 10 mmol/L; the preparation method comprises the following steps: in a sterile environment, inactivated Fetal Bovine Serum (FBS) is added into HEPES solution according to volume percentage, and dissolved and shaken uniformly to prepare 10ml of Base Solution (BS).
The equilibrium liquid (ES) consists of the following components in percentage by volume: 8% Ethylene Glycol (EG), 4% dimethyl sulfoxide (DMSO), 4% propylene glycol (PROH), 20% inactivated Fetal Bovine Serum (FBS), 5% carboxylated epsilon-polylysine, and the remainder as a 10mmol/L HEPES solution; each liter of the equilibrium liquid (ES) also comprises 0.25mol of sucrose and 3g of hydroxyapatite with the particle size of 40 nm; the preparation method comprises the following steps: adding the components into HEPES solution according to volume fraction ratio, dissolving, shaking, filtering, sterilizing to obtain 100ml of Equilibrium Solution (ES), and placing in a refrigerator at 4 ℃ for later use.
The glass transition metal (VS) consists of the following components in percentage by volume: 16% Ethylene Glycol (EG), 8% dimethyl sulfoxide (DMSO), 8% propylene glycol (PROH), 20% inactivated Fetal Bovine Serum (FBS), 10% carboxylated epsilon-polylysine, and the remainder as a 10mmol/L HEPES solution; each liter of the Vitrification Solution (VS) also comprises 0.5mol of sucrose and 4g of hydroxyapatite with the grain diameter of 40 nm; the configuration method comprises the following steps: adding the components into HEPES solution according to the volume fraction ratio, dissolving and shaking uniformly, filtering and sterilizing to prepare 10ml of vitrified liquid (VS), and placing the vitrified liquid in a refrigerator at 4 ℃ for later use.
The vitrification cryopreservation liquid of the embodiment is used for vitrification cryopreservation of the oocyte of the equine animal.
EXAMPLE 3 refrigerant fluid III
The embodiment of the invention provides an equus animal oocyte vitrification cryopreservation liquid, which comprises 3 liquids of basic liquid (BS), equilibrium liquid (ES) and vitrification liquid (VS); wherein the content of the first and second substances,
the Base Solution (BS) consists of inactivated fetal calf serum with the volume fraction of 20% and HEPES solution with the volume fraction of 80% and the concentration of 10 mmol/L; the preparation method comprises the following steps: in a sterile environment, inactivated Fetal Bovine Serum (FBS) is added into HEPES solution according to volume percentage, and dissolved and shaken uniformly to prepare 10ml of Base Solution (BS).
The equilibrium liquid (ES) consists of the following components in percentage by volume: 8% Ethylene Glycol (EG), 4% dimethyl sulfoxide (DMSO), 4% propylene glycol (PROH), 20% inactivated Fetal Bovine Serum (FBS), 5% carboxylated epsilon-polylysine, and the remainder as a 10mmol/L HEPES solution; each liter of the equilibrium liquid (ES) also comprises 0.25mol of sucrose and 0.3g of vitamin D2; the preparation method comprises the following steps: adding the components into HEPES solution according to volume fraction ratio, dissolving, shaking, filtering, sterilizing to obtain 100ml of Equilibrium Solution (ES), and placing in a refrigerator at 4 ℃ for later use.
The glass transition metal (VS) consists of the following components in percentage by volume: 16% Ethylene Glycol (EG), 8% dimethyl sulfoxide (DMSO), 8% propylene glycol (PROH), 20% inactivated Fetal Bovine Serum (FBS), 10% carboxylated epsilon-polylysine, and the remainder as a 10mmol/L HEPES solution; each liter of the glass transition liquid (VS) also comprises 0.5mol of sucrose and 0.5g of vitamin D2; the configuration method comprises the following steps: adding the components into HEPES solution according to the volume fraction ratio, dissolving and shaking uniformly, filtering and sterilizing to prepare 10ml of vitrified liquid (VS), and placing the vitrified liquid in a refrigerator at 4 ℃ for later use.
The vitrification cryopreservation liquid of the embodiment is used for vitrification cryopreservation of the oocyte of the equine animal.
EXAMPLE 4 refrigerant four
The embodiment of the invention provides an equus animal oocyte vitrification cryopreservation liquid, which comprises 3 liquids of basic liquid (BS), equilibrium liquid (ES) and vitrification liquid (VS); wherein the content of the first and second substances,
the Base Solution (BS) consists of inactivated fetal calf serum with the volume fraction of 20% and HEPES solution with the volume fraction of 80% and the concentration of 10 mmol/L; the preparation method comprises the following steps: in a sterile environment, inactivated Fetal Bovine Serum (FBS) is added into HEPES solution according to volume percentage, and dissolved and shaken uniformly to prepare 10ml of Base Solution (BS).
The equilibrium liquid (ES) consists of the following components in percentage by volume: 8% Ethylene Glycol (EG), 4% dimethyl sulfoxide (DMSO), 4% propylene glycol (PROH), 20% inactivated Fetal Bovine Serum (FBS), 5% carboxylated epsilon-polylysine, and the remainder as a 10mmol/L HEPES solution; each liter of the equilibrium liquid (ES) also comprises 0.25mol of cane sugar, 2g of hydroxyapatite with the particle size of 40nm and 0.4g of vitamin D2; the preparation method comprises the following steps: adding the components into HEPES solution according to volume fraction ratio, dissolving, shaking, filtering, sterilizing to obtain 100ml of Equilibrium Solution (ES), and placing in a refrigerator at 4 ℃ for later use.
The glass transition metal (VS) consists of the following components in percentage by volume: 16% Ethylene Glycol (EG), 8% dimethyl sulfoxide (DMSO), 8% propylene glycol (PROH), 20% inactivated Fetal Bovine Serum (FBS), 10% carboxylated epsilon-polylysine, and the remainder as a 10mmol/L HEPES solution; each liter of the Vitrification Solution (VS) also comprises 0.5mol of sucrose, 3g of hydroxyapatite with the particle size of 40nm and 0.6g of vitamin D2; the configuration method comprises the following steps: adding the components into HEPES solution according to the volume fraction ratio, dissolving and shaking uniformly, filtering and sterilizing to prepare 10ml of vitrified liquid (VS), and placing the vitrified liquid in a refrigerator at 4 ℃ for later use.
The vitrification cryopreservation liquid of the embodiment is used for vitrification cryopreservation of the oocyte of the equine animal.
EXAMPLE 5 freezing method
The embodiment of the invention provides a vitrification cryopreservation method for equine oocyte, which comprises the following steps:
(1) putting the oocyte or embryo to be frozen into a Basic Solution (BS) and washing for 1-2 times;
(2) transferring the washed oocyte or embryo to an equilibration fluid (ES) for 5 minutes;
(3) the equilibrated oocytes or embryos are rapidly transferred to vitrification liquid (VS), and then the vitrification liquid containing the oocytes or embryos is rapidly transferred to a closed elongated straw (CPS tube) and put into liquid nitrogen (-196 ℃) for preservation, and the whole process is completed within 1 minute.
The base liquid (BS), the equilibrium liquid (ES) and the vitrification liquid (VS) may be any one of embodiments 1 to 4.
Comparison experiment one:
an experiment was performed using 100 oocytes obtained by superovulation of mares of this company, wherein 100 oocytes were all normal in morphology and free from significant vacuoles, debris, and the like, and the 100 oocytes were randomly divided into 5 groups of 20 oocytes, and cryopreserved according to the method of example 5 using the vitrified cryopreservation solution of example 1 (example 1 group), example 2 (example 2 group), example 3 (example 3 group), and example 4 (example 4 group), and the conventional vitrified cryopreservation solution (conventional control group), respectively.
Wherein, the components of the traditional vitrification refrigerating fluid are as follows: the base fluid comprises 20% volume fraction inactivated fetal bovine serum and 80% PBS fluid; the equilibrium liquid comprises 80% of base liquid, 10% of Ethylene Glycol (EG) and 10% of dimethyl sulfoxide (DMSO) by volume fraction; the glass melt contained a volume fraction of 60% base solution, 20% Ethylene Glycol (EG), 20% dimethyl sulfoxide (DMSO), and 0.65mol/L sucrose.
Freezing for 4 weeks, thawing, and observing the zona pellucida integrity rate and oocyte survival rate (oocyte survival standard: no obvious cytoplasm overflow and shrinkage, intact cell membrane, clear peri-oocyte space, normal oocyte size) and oocyte fertilization rate (oocyte fertilization standard: two pronuclei are seen in oocyte cytoplasm 18-20 hours after ICSI). The results are shown in Table 1.
TABLE 1 comparison table of zona pellucida completeness, oocyte survival rate and fertilization rate
Group of Using egg number (one) Transparent tape integer (piece) Zona pellucida integrity (%) Survival number (one) Survival rate (%) Fertilization number (one) Fertilization Rate (%)
Conventional control group 20 16 80 15 75 15 75
EXAMPLE 1 group 20 18 90 17 85 16 80
EXAMPLE 2 group 20 19 95 18 90 17 85
EXAMPLE 3 group 20 18 90 17 85 17 85
EXAMPLE 4 group 20 20 100 19 95 18 90
As can be seen from the data in Table 1, the groups of example 1, example 2, example 3 and example 4 were higher than the conventional control group in terms of the zona pellucida integrity rate, the oocyte survival rate and the oocyte fertilization rate, compared to the conventional control group.
Comparative experiment two:
an experiment was performed using 100 oocytes obtained by superovulation of mares of this company, wherein 100 oocytes were all normal in morphology and free from significant vacuoles, debris, and the like, and the 100 oocytes were randomly divided into 5 groups of 20 oocytes, and cryopreserved according to the method of example 5 using the vitrified cryopreservation solution of example 1 (example 1 group), example 2 (example 2 group), example 3 (example 3 group), and example 4 (example 4 group), and the conventional vitrified cryopreservation solution (conventional control group), respectively.
Wherein, the components of the traditional vitrification refrigerating fluid are as follows: the base fluid comprises 20% volume fraction inactivated fetal bovine serum and 80% PBS fluid; the equilibrium liquid comprises 80% of base liquid, 10% of Ethylene Glycol (EG) and 10% of dimethyl sulfoxide (DMSO) by volume fraction; the glass melt contained a volume fraction of 60% base solution, 20% Ethylene Glycol (EG), 20% dimethyl sulfoxide (DMSO), and 0.65mol/L sucrose.
Freezing for 1 year, taking out, thawing, and observing the zona pellucida integrity rate, oocyte survival rate (oocyte survival standard: no obvious cytoplasm overflow and shrinkage, intact cell membrane, clear perivitelline space, normal oocyte size) and oocyte fertilization rate (oocyte fertilization standard: two pronuclei are seen in oocyte cytoplasm 18-20 hours after ICSI). The results are shown in Table 2.
TABLE 2 comparison table of zona pellucida completeness, oocyte survival rate and fertilization rate
Group of Using egg number (one) Transparent tape integer (piece) Zona pellucida integrity (%) Survival number (one) Survival rate (%) Fertilization number (one) Fertilization Rate (%)
Conventional control group 20 15 75 14 70 14 70
EXAMPLE 1 group 20 17 85 17 85 16 80
EXAMPLE 2 group 20 18 90 18 90 17 85
EXAMPLE 3 group 20 18 90 17 85 16 80
EXAMPLE 4 group 20 19 95 19 95 18 90
As can be seen from the data in Table 2, the groups of example 1, example 2, example 3 and example 4 were higher than the conventional control group in terms of the zona pellucida integrity rate, the oocyte survival rate and the oocyte fertilization rate, compared to the conventional control group.
In conclusion, the oocyte vitrification cryopreservation effect of the scheme of the invention is obviously superior to that of the traditional vitrification cryopreservation liquid, is very suitable for vitrification cryopreservation of oocyte and embryo of equine animals such as horse, donkey and the like, and is beneficial to popularization and application of artificial insemination and embryo transplantation technology.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.

Claims (2)

1. A vitrification cryopreservation liquid for equine animal oocyte, which comprises 3 liquids of a basic liquid (BS), a balance liquid (ES) and a vitrification liquid (VS), and is characterized in that the basic liquid (BS) consists of inactivated fetal calf serum with the volume fraction of 20% and HEPES solution with the volume fraction of 80% and the concentration of 10 mmol/L; the preparation method comprises the following steps: under an aseptic environment, taking inactivated Fetal Bovine Serum (FBS) according to volume percentage, adding the inactivated Fetal Bovine Serum (FBS) into a HEPES solution, and dissolving and shaking up to prepare 10ml of Base Solution (BS); the equilibrium liquid (ES) consists of the following components in percentage by volume: 8% Ethylene Glycol (EG), 4% dimethyl sulfoxide (DMSO), 4% propylene glycol (PROH), 20% inactivated Fetal Bovine Serum (FBS), 5% carboxylated epsilon-polylysine, and the remainder as a 10mmol/L HEPES solution; each liter of the equilibrium liquid (ES) also comprises 0.25mol of cane sugar, 2g of hydroxyapatite with the particle size of 40nm and 0.4g of vitamin D2; the preparation method comprises the following steps: adding the components into HEPES solution according to volume fraction ratio, dissolving, shaking, filtering, sterilizing to obtain 100ml of Equilibrium Solution (ES), and placing in a refrigerator at 4 ℃ for later use; the Vitrification Solution (VS) consists of the following components by volume percentage, 16 percent of Ethylene Glycol (EG), 8 percent of dimethyl sulfoxide (DMSO), 8 percent of propylene glycol (PROH), 20 percent of inactivated Fetal Bovine Serum (FBS), 10 percent of carboxylation epsilon-polylysine and the rest of HEPES solution with the concentration of 10 mmol/L; each liter of the Vitrification Solution (VS) also comprises 0.5mol of sucrose, 3g of hydroxyapatite with the grain diameter of 40m and 0.6g of vitamin D2; the configuration method comprises the following steps: adding the components into HEPES solution according to volume fraction ratio, dissolving and shaking uniformly, filtering and sterilizing to prepare 10ml of Vitrification Solution (VS), and placing the vitrification solution in a refrigerator at 4 ℃ for later use;
the application method of the vitrification cryopreservation liquid comprises the steps of (1) placing oocytes or embryos to be frozen in a base liquid (BS) to be washed for 1-2 times, (2) transferring the washed oocytes or embryos into a balancing liquid (ES) to be balanced for 3-7 minutes, (3) rapidly transferring the balanced oocytes or embryos into a vitrification liquid (VS), rapidly transferring the vitrification liquid containing the oocytes or embryos into a closed elongated straw (CPS) and putting liquid nitrogen into the closed elongated straw to preserve the vitrification liquid at the temperature of-196 ℃, wherein the whole process is completed within 1 minute.
2. The cryopreservation solution for equine animal oocyte vitrification according to claim 1, characterized in that the cryopreservation solution is suitable for cryopreservation of all equine animal oocytes and embryos.
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