A method for cryopreservation of mongolica oocytes

Abstract

The invention belongs to the field of biotechnology, and discloses a method for cryopreservation of oocytes of Mongolian croaker, comprising the steps of: (1) dispersing Mongolian croaker oocytes in equilibrium solution one and equilibrium solution two, respectively, for 8-12 minutes; (2) transferring to a cryoprotectant, subpackaging into a freezing container, and inserting liquid nitrogen for preservation. This method uses a variety of solvents and an antioxidant, melatonin, to prepare a freezing preservation solution, mixes the cryoprotectant with a diluent containing KCl, and forms equilibrium solution one and equilibrium solution two for the freezing preservation process of oocytes. This method can reduce toxic damage, oxidative damage, and mechanical damage to cells; simultaneously, using a straw as a freezing preservation container reduces the cost and operational difficulty of freezing oocytes, improves the freezing preservation efficiency of oocytes, achieves long-term preservation of oocytes, and is suitable for the production needs of fish germplasm preservation.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and in particular relates to the field of biological material / tissue cryopreservation, and in particular to an oocyte cryopreservation method. Background Art

[0002] Cryopreservation is the technique of preserving biological gametes at ultra-low temperatures for long periods of time. It can reduce genetic drift, maintain active reproductive populations, and provide a solution for protecting germplasm resources. Due to the presence of maternal inheritance, the maternal genome is crucial in germplasm conservation, and cryopreservation of oocytes will improve species conservation efficiency. Currently, commonly used cryopreservation methods include slow freezing and vitrification. Fish oocytes are difficult to cryopreserve due to their large size, poor membrane permeability, high yolk content, and sensitivity to antifreeze toxicity. Lujic used a vitrification solution of 3 mol / L LDMSO and 3 mol / L PG to cryopreserve ovarian tissue from juvenile sea trout, achieving an oocyte survival rate of up to 40%. Cao Junguo also found that adding 10-9 mol / L melatonin to mink GV stage oocytes during vitrification and in vitro maturation improved oocyte cryopreservation efficiency. Fish are the most diverse group of vertebrates. The morphological structure of oocytes of different fish varies greatly, and their sensitivity to low temperature and toxicity also varies. However, the cryopreservation technology of Mongolian carp oocytes has not been studied yet.

[0003] The paper "Cryopreservation of zebrafish (Danio rerio) oocytes by vitrification" published by David M Rawson et al. in 2010 studied the diluent, cryoprotectant, and storage container for zebrafish oocyte cryopreservation, and summarized a better method for zebrafish oocyte cryopreservation: the diluent is a solution containing KCl, the specific formula of which has not been disclosed; the cryoprotectant formula is 3M dimethyl sulfoxide (DMSO), 2M methanol (MeOH), and 2M propylene glycol (PG); and the storage container uses CryoLogic's CVA65 freezing hook and 0.25mL straw. The specific operation process is as follows: (1) Collect stage III zebrafish oocytes and place them in diluent; (2) Equilibrate in ES1 (1M DMSO, 1M MeOH, 0.75M PG) for 15 minutes, then transfer to ES2 (2M DMSO, 2M MeOH, 1.5M PG) for 10 minutes, and finally transfer to antifreeze for 5 minutes; (3) Transfer to CVA65 cryohoo hooks and quickly insert into liquid nitrogen for storage; (4) After 10 minutes, remove and thaw in a 26±1℃ water bath, transfer to ES2 or 0.5M sucrose, and then equilibrate to diluent through four steps; (5) Use trypan blue to detect survival rate. The survival rate of oocytes stored using this method using CVA65 cryohoo hooks reached 68.5±7.2% 30 minutes after thawing. In this study, the survival rate of oocytes stored using the same formula and process but in 0.25mL straws was 48.3±4.6%.

[0004] The shortcomings of David M Rawson's zebrafish oocyte cryopreservation method are as follows: (1) due to the large differences in characteristics of fish oocytes between species, it is often not feasible for economic fish; (2) the CVA65 cryohoo used by CryoLogic can only preserve one oocyte at a time, but fish are oviparous organisms and lay a large number of eggs each time. Cryopreservation of fish oocytes often requires a large number of storage, which is not feasible in production practice; containers such as cryohooks and cryorings for preserving single germ cells have a very small storage volume and much stronger thermal conductivity than straws, cryovials, etc., so the expensive and cumbersome cryohooks cannot meet the needs; (3) since vitrification requires the solution to be kept in a glassy state between liquid and solid, it is more sensitive to changes in the storage environment. During long-term storage, crystallization is likely to gradually occur, resulting in a decrease in survival rate or even failure of preservation. Therefore, this method can only cryopreserve oocytes for 10 minutes, and long-term cryopreservation is not achieved, which has a great impact on the survival rate of oocytes.

[0005] Therefore, it is necessary to improve the existing method and provide a method and reagent that can improve the cryopreservation effect, so that fish oocytes can be cryopreserved for a long time and the survival rate can be improved when using straws as containers, while being easy to use. Summary of the Invention

[0006] Aiming at the problem that the prior art lacks a cryopreservation technology for oocytes of Mongol brucei, the present invention provides a cryopreservation method for oocytes of Mongol brucei.

[0007] A method for cryopreservation of mongolica oocytes comprises the following steps:

[0008] (1) The dispersed oocytes of Mongoloid brookii were equilibrated in equilibration solution 1 and equilibration solution 2 for 8-12 minutes respectively;

[0009] (2) Transfer to cryoprotectant, dispense into freezing containers, and store in liquid nitrogen;

[0010] The first balance solution contains 50-60 mM potassium chloride, 50-60 mM potassium acetate, 0.5-1.5 mM magnesium chloride, 5-15 mM 4-hydroxyethylpiperazineethanesulfonic acid potassium salt, 0.5-1.5 M dimethyl sulfoxide, 0.5-1.0 M methanol, 0.5-1.0 M propylene glycol and 0.1-0.5 mM melatonin;

[0011] The second equilibrium solution contains 50-60 mM potassium chloride, 50-60 mM potassium acetate, 0.5-1.5 mM magnesium chloride, 5-15 mM 4-hydroxyethylpiperazineethanesulfonic acid potassium salt, 1.5-2.5 M dimethyl sulfoxide, 1.0-2.0 M methanol, 1.0-2.0 M propylene glycol, and 0.5-1.0 mM melatonin;

[0012] The cryoprotectant contains 50-60 mM potassium chloride, 50-60 mM potassium acetate, 0.5-1.5 mM magnesium chloride, 5-15 mM 4-hydroxyethylpiperazineethanesulfonic acid potassium salt, 2.5-3.5 M dimethyl sulfoxide, 1.5-2.5 M methanol, 1.5-2.5 M propylene glycol and 0.5-1.5 mM melatonin;

[0013] Preferably, the balance solution 1 contains 53-57 mM potassium chloride, 53-57 mM potassium acetate, 0.8-1.2 mM magnesium chloride, 8-12 mM 4-hydroxyethylpiperazineethanesulfonic acid potassium salt, 0.9-1.1 M dimethyl sulfoxide, 0.66-0.67 M methanol, 0.66-0.67 M propylene glycol, and 0.33 mM-0.34 mM melatonin;

[0014] Preferably, the second equilibrium solution contains 53-57 mM potassium chloride, 53-57 mM potassium acetate, 0.8-1.2 mM magnesium chloride, 8-12 mM 4-hydroxyethylpiperazineethanesulfonic acid potassium salt, 1.9-2.1 M dimethyl sulfoxide, 1.33-1.34 M methanol, 1.33-1.34 M propylene glycol, and 0.66-0.67 mM melatonin;

[0015] More preferably, the cryoprotectant comprises 53-57 mM potassium chloride, 53-57 mM potassium acetate, 0.8-1.2 mM magnesium chloride, 8-12 mM 4-hydroxyethylpiperazineethanesulfonic acid potassium salt, 3 M dimethyl sulfoxide, 2 M methanol, 2 M propylene glycol, and 1 mM melatonin. More preferably, the balanced solution ES2 comprises 55 mM potassium chloride, 55 mM potassium acetate, 1 mM magnesium chloride, 10 mM 4-hydroxyethylpiperazineethanesulfonic acid potassium salt, 1.9-2.1 M dimethyl sulfoxide, 1.33-1.34 M methanol, 1.33-1.34 M propylene glycol, and 0.66-0.67 mM melatonin.

[0016] In a more preferred embodiment of the present invention, the balanced solution ES1 contains 55mM potassium chloride, 55mM potassium acetate, 1mM magnesium chloride, 10mM 4-hydroxyethylpiperazineethanesulfonic acid potassium salt, 1M dimethyl sulfoxide, 0.66-0.67M methanol, 0.66-0.67M propylene glycol, and 0.33-0.34mM melatonin; the balanced solution ES2 contains 55mM potassium chloride, 55mM potassium acetate, 1mM magnesium chloride, 10mM 4-hydroxyethylpiperazineethanesulfonic acid potassium salt, 2M dimethyl sulfoxide, 1.33-1.34M methanol, 1.33-1.34M propylene glycol, 0.66-0.67mM melatonin; the cryoprotectant contains 55mM potassium chloride, 55mM potassium acetate, 1mM magnesium chloride, 10mM 4-hydroxyethylpiperazineethanesulfonic acid potassium salt, 3M dimethyl sulfoxide, 2M methanol, 2M propylene glycol and 1mM melatonin.

[0017] Preferably, the freezing container used in the freezing preservation method is a straw, and each straw can store 5-10 Mongolian brook oocytes.

[0018] The present invention also provides a mongolica oocyte cryopreservation reagent comprising at least one of a diluent and a cryoprotectant (CPA).

[0019] The cryoprotectant contains 50-60 mM potassium chloride, 50-60 mM potassium acetate, 0.5-1.5 mM magnesium chloride, 5-15 mM 4-hydroxyethylpiperazineethanesulfonic acid potassium salt, 2.5-3.5 M dimethyl sulfoxide, 1.5-2.5 M methanol, 1.5-2.5 M propylene glycol, and 0.5-1.5 mM melatonin.

[0020] The diluent contains 50-60 mM potassium chloride, 50-60 mM potassium acetate, 0.5-1.5 mM magnesium chloride, and 5-15 mM 4-hydroxyethylpiperazineethanesulfonic acid potassium salt.

[0021] Preferably, the cryoprotectant solution (CPA) contains 55 mM potassium chloride, 55 mM potassium acetate, 1 mM magnesium chloride, 10 mM 4-hydroxyethylpiperazineethanesulfonic acid potassium salt, 3 M dimethyl sulfoxide, 2 M methanol, 2 M propylene glycol, and 1 mM melatonin.

[0022] Preferably, the diluent contains 55 mM potassium chloride, 55 mM potassium acetate, 1 mM magnesium chloride, and 10 mM 4-hydroxyethylpiperazineethanesulfonic acid potassium salt.

[0023] When in use, the diluent and cryoprotectant can be mixed to prepare equilibrium solution 1 and equilibrium solution 2 respectively.

[0024] The diluent and the cryoprotectant CPA are mixed in a volume ratio of 1.8-2.4:1 to prepare the equilibrium solution one; preferably, the diluent and the cryoprotectant CPA are mixed in a volume ratio of 2:1 to prepare the equilibrium solution one.

[0025] The diluent and the cryoprotectant CPA are mixed in a volume ratio of 1:2 to prepare the equilibrated solution II; preferably, the diluent and the cryoprotectant CPA are mixed in a volume ratio of 1:2 to prepare the equilibrated solution II.

[0026] The method for thawing the Mongolian carp oocytes after cryopreservation by the above method includes the following steps: the frozen Mongolian carp oocytes are allowed to stand at room temperature for 8-12 seconds, thawed at 38-42°C, transferred to a 0.4-0.6 mol / L sucrose solution, equilibrated at room temperature for 8-12 minutes, then transferred to a 0.15-0.35 mol / L sucrose solution, equilibrated at room temperature for 8-12 minutes, and finally transferred to a diluent.

[0027] More preferably, the oocytes of Mongol brook croaker are allowed to stand at room temperature for 9-11 seconds, thawed at 39-41° C., transferred to 0.45-0.55 mol / L and 0.2-0.3 mol / L sucrose solutions in sequence, and equilibrated at room temperature for 9-11 minutes.

[0028] In a more preferred embodiment of the present invention, the oocytes of Mongol croaker are kept at room temperature for 10 seconds, thawed at 40°C, and transferred to 0.5 mol / L and 0.25 mol / L sucrose solutions in sequence, and equilibrated at room temperature for 10 minutes each.

[0029] The beneficial effects of the present invention are:

[0030] The present invention provides a method for cryopreservation of oocytes from Mongolian croaker. This method utilizes a cryopreservation solution containing a mixture of multiple solvents and the antioxidant melatonin. While achieving the high organic solvent concentration required for vitrification, it also reduces the toxicity of a single organic solvent, minimizing both toxic damage to cells and oxidative damage, a major hazard during cryopreservation. A three-step balancing process is employed during both cryopreservation and thawing, minimizing mechanical damage to oocytes during transfer between hypertonic and hypotonic solutions and improving cryopreservation effectiveness. This method enables long-term oocyte preservation, with a survival rate of 63.08±2.50% after 7 days of storage and 53.23±3.07% after 30 days of storage, making the invention applicable in production practice.

[0031] The invention provides a new mongolica oocyte cryopreservation reagent, which is convenient for preparing a balance solution, can be used for cryopreservation and subsequent thawing of mongolica oocytes, and effectively improves the survival rate of cryopreserved oocytes.

[0032] The present invention can use wheat straws as cryopreservation containers, and each wheat straw can store 5-10 Mongolian croaker oocytes. The cost is low and the operation is convenient, so the invention is suitable for the production needs of fish germplasm preservation. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 A picture of trypan blue staining of Mongolian croaker oocytes after cryopreservation for 7 days DETAILED DESCRIPTION

[0034] The embodiments of the present invention and comparative examples are described in detail below with reference to the accompanying drawings:

[0035] Example 1

[0036] The Mongolian croaker oocyte cryopreservation technology of the present invention was used to preserve the oocytes and detect their survival, as follows:

[0037] Step 1) Preparation of diluent, the solvent is ddH2O, the formula is as shown in Table 1:

[0038] Table 1: Component ratio of diluent

[0039]

[0040] Step 2): Preparation of cryoprotectant (CPA). The solvent is the diluent prepared in step 1). There is no special requirement for the order of adding solutes. The formula is shown in Table 2:

[0041] Table 2 Composition ratio of cryoprotectant (CPA)

[0042]

[0043] Step 3): Preparation of equilibrium solutions ES1 and ES2: The CPA prepared in step 2) was mixed with the diluent prepared in step 1 at a ratio of 1:2 to obtain equilibrium solution ES1 with a concentration of one-third CPA; the CPA prepared in step 2) was mixed with the diluent prepared in step 1 at a ratio of 2:1 to obtain equilibrium solution ES2 with a concentration of two-thirds CPA.

[0044] Step 4): During the breeding season of Mongolian brook, female adult Mongolian brook are collected, the ovaries are removed by dissection, and the stage III oocytes are separated into individual oocytes in the diluent prepared in step 1.

[0045] Step 5): The Mongolian carp oocytes were transferred to equilibration solution ES1, equilibrated for 10 minutes, then transferred to equilibration solution ES2 for 10 minutes, and finally transferred to cryoprotectant CPA.

[0046] Step 6): The oocytes in the CPA were divided into 0.25 mL straws, with 5-10 oocytes per straw, and then quickly inserted into liquid nitrogen for storage.

[0047] After cryopreservation of Mongoloid brook oocytes for 7 and 30 days using the above method, they were thawed and revived in vitro according to the following method:

[0048] a) Remove the straw containing Mongol brook oyster oocytes from liquid nitrogen, let it stand in air for 10 seconds, and then quickly thaw it in a 40°C water bath.

[0049] b) The thawed oocytes were transferred to a 0.5 mol / L sucrose solution, equilibrated at room temperature for 10 minutes, then transferred to a 0.25 mol / L sucrose solution, equilibrated at room temperature for 10 minutes, and finally transferred to the diluents listed in Table 1.

[0050] Thawed Mongolian croaker oocytes can be tested for viability using 0.4% trypan blue solution. The method is as follows: transfer the thawed Mongolian croaker oocytes to a clean culture dish, aspirate the diluent, add 0.4% trypan blue solution, incubate at room temperature for 2 minutes, aspirate the trypan blue solution, and observe the oocyte morphology. Cells with a transparent interior and no staining are considered viable, and the viability is calculated.

[0051] The oocytes of Mongolian croaker were thawed after being cryopreserved for 7 days, and the results of trypan blue staining were as follows: Figure 1 In the red box, the oocyte is transparent and the trypan blue has not penetrated into the cell, indicating a viable state. In the blue box, the oocyte is dark blue and the trypan blue has penetrated into the cell, indicating a dead state.

[0052] The survival rates of Mongoloid brook oocytes frozen for 7 and 30 days were shown in Tables 3 and 4 after thawing and resuscitation in vitro.

[0053] Table 3 Survival rate of thawed oocytes of Mongoloid croaker after cryopreservation for 7 days in Example 1

[0054]

[0055] Table 4 Survival rate of thawed oocytes of Mongoloid brook croaker after cryopreservation for 30 days in Example 1

[0056]

[0057] Comparative Example 1

[0058] The cryoprotectant (CPA) of this comparative example uses the diluent (KCl buffer) prepared in step 1 as the solvent. There is no special requirement for the order of adding the solutes. Formulas a and b are shown in Table 5 and Table 6, respectively.

[0059] Table 5 Comparative Example 1 using cryoprotectant (CPA) formula a

[0060]

[0061] Table 6 Formula b of cryoprotectant (CPA) in Comparative Example 1

[0062]

[0063]

[0064] Except for the formula of cryoprotectant, the rest is the same as Example 1.

[0065] In this comparative example, we successfully cryopreserved zebrafish oocytes using the method for cryopreservation of mongolica oocytes of the present invention, and the zebrafish oocytes were cryopreserved for 7 days and revived in vitro after thawing. The survival rates are shown in Tables 7 and 8 below:

[0066] Table 7 Cryoprotectant formula a thaw survival rate results after 7 days of cryopreservation

[0067]

[0068] Table 8 Cryoprotectant formula b cryopreservation after 7 days of thawing survival rate results

[0069]

Claims

1. A method for cryopreservation of mongolica oocytes, characterized in that: The steps include: (1) The dispersed oocytes of Mongoloid brook oyster were equilibrated in equilibration solution 1 and equilibration solution 2 for 8-12 minutes respectively; (2) Transfer to cryoprotectant, dispense into cryopreservation containers, and store in liquid nitrogen; (3) Frozen Mongolian croaker oocytes were placed at room temperature for 8-12 seconds, thawed at 38-42°C, transferred to 0.4-0.6 mol / L sucrose solution, equilibrated at room temperature for 8-12 minutes, then transferred to 0.15-0.35 mol / L sucrose solution, equilibrated at room temperature for 8-12 minutes, and finally transferred to diluent. The first balance solution contains 50-60 mM potassium chloride, 50-60 mM potassium acetate, 0.5-1.5 mM magnesium chloride, 5-15 mM 4-hydroxyethylpiperazineethanesulfonic acid potassium salt, 0.5-1.5 M dimethyl sulfoxide, 0.5-1.0 M methanol, 0.5-1.0 M propylene glycol and 0.1-0.5 mM melatonin; The second equilibrium solution contains 50-60 mM potassium chloride, 50-60 mM potassium acetate, 0.5-1.5 mM magnesium chloride, 5-15 mM 4-hydroxyethylpiperazineethanesulfonic acid potassium salt, 1.5-2.5 M dimethyl sulfoxide, 1.0-2.0 M methanol, 1.0-2.0 M propylene glycol, and 0.5-1.0 mM melatonin; The cryoprotectant contains 50-60 mM potassium chloride, 50-60 mM potassium acetate, 0.5-1.5 mM magnesium chloride, 5-15 mM 4-hydroxyethylpiperazineethanesulfonic acid potassium salt, 2.5-3.5 M dimethyl sulfoxide, 1.5-2.5 M methanol, 1.5-2.5 M propylene glycol and 0.5-1.5 mM melatonin; The diluent contains 50-60 mM potassium chloride, 50-60 mM potassium acetate, 0.5-1.5 mM magnesium chloride, and 5-15 mM 4-hydroxyethylpiperazineethanesulfonic acid potassium salt.

2. The method for cryopreservation of oocytes of Mongol croaker according to claim 1, wherein The balance solution 1 contains 53-57 mM potassium chloride, 53-57 mM potassium acetate, 0.8-1.2 mM magnesium chloride, 8-12 mM 4-hydroxyethylpiperazineethanesulfonic acid potassium salt, 1 M dimethyl sulfoxide, 0.66-0.67 M methanol, 0.66-0.67 M propylene glycol, and 0.33 mM-0.34 mM melatonin; The second equilibrium solution contains 53-57 mM potassium chloride, 53-57 mM potassium acetate, 0.8-1.2 mM magnesium chloride, 8-12 mM 4-hydroxyethylpiperazineethanesulfonic acid potassium salt, 2 M dimethyl sulfoxide, 1.33-1.34 M methanol, 1.33-1.34 M propylene glycol, and 0.66-0.67 mM melatonin; The cryoprotectant contains 53-57 mM potassium chloride, 53-57 mM potassium acetate, 0.8-1.2 mM magnesium chloride, 8-12 mM 4-hydroxyethylpiperazineethanesulfonic acid potassium salt, 3 M dimethyl sulfoxide, 2 M methanol, 2 M propylene glycol and 1 mM melatonin.

3. The method for cryopreservation of mongolica oocytes according to claim 1, wherein The freezing container is a straw.

4. The method for cryopreservation of mongolica oocytes according to claim 1, wherein In step (1), the oocytes were equilibrated in equilibration solution 1 and equilibration solution 2 for 10 min respectively.

5. A reagent for cryopreservation of oocytes of Mongol croaker, characterized in that: including at least one of a diluent and a cryoprotectant; The cryoprotectant contains 50-60 mM potassium chloride, 50-60 mM potassium acetate, 0.5-1.5 mM magnesium chloride, 5-15 mM 4-hydroxyethylpiperazineethanesulfonic acid potassium salt, 2.5-3.5 M dimethyl sulfoxide, 1.5-2.5 M methanol, 1.5-2.5 M propylene glycol and 0.5-1.5 mM melatonin; The diluent contains 50-60 mM potassium chloride, 50-60 mM potassium acetate, 0.5-1.5 mM magnesium chloride, and 5-15 mM 4-hydroxyethylpiperazineethanesulfonic acid potassium salt.

6. The cryopreservation reagent for mongolica oocytes according to claim 5, characterized in that The cryoprotectant contains 55 mM potassium chloride, 55 mM potassium acetate, 1 mM magnesium chloride, 10 mM 4-hydroxyethylpiperazineethanesulfonic acid potassium salt, 3 M dimethyl sulfoxide, 2 M methanol, 2 M propylene glycol and 1 mM melatonin.

7. The cryopreservation reagent for mongolica oocytes according to claim 5, characterized in that The diluent contains 55 mM potassium chloride, 55 mM potassium acetate, 1 mM magnesium chloride, and 10 mM 4-hydroxyethylpiperazineethanesulfonic acid potassium salt.

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