Bovine oocyte cryoprotectant and cryopreservation method

Abstract

The invention relates to the technical field of animal husbandry, and particularly discloses a bovine oocyte cryoprotectant and a cryopreservation method. The bovine oocyte cryoprotectant disclosed by the invention is prepared from the following components: 510 to 595 mg / L of excipient, 220 to 280 mg / L of antioxidant, 20 to 30 mg / L of antibiotic, 120 to 180 mg / L of salt substance, 320 to 380 mg / L of ethanol, 65 to 85 mg / L of glycerol and 55 to 68 mg / L of fetal bovine serum, the excipient is prepared by mixing polysaccharose, collagen and leifrog according to the weight ratio of 10: (2-6): (0.5-1.5); the antibiotic is formed by mixing kanamycin and nalidixic acid in a weight ratio of (1-3): (1-3). The cryoprotectant is used for cryoprotection of bovine oocytes, the integrity and biological activity of the cells can be effectively protected, and the fertilization ability and subsequent development ability of the oocytes can be protected.

Description

Technical Field

[0001] The present application relates to the technical field of animal husbandry and biology, and in particular to a bovine oocyte cryoprotectant and a cryopreservation method. Background Art

[0002] With the development of animal husbandry biotechnology, the demand for cryopreservation of bovine oocytes is increasing. At present, although a variety of protective agents and methods are used for cell cryopreservation, the particularity of bovine oocytes still makes them face the problems of cell damage and low survival rate during cryopreservation.

[0003] Traditional cryopreservation methods often cause bovine oocytes to be damaged by ice crystal formation during freezing and thawing. Ice crystals formed inside or between cells may destroy the cell structure, causing membrane damage, cytoskeleton disorder, etc., resulting in impaired cell function, which seriously affects the subsequent development and fertilization ability of oocytes. Therefore, it is urgent to develop a bovine oocyte cryoprotectant and cryopreservation method. Summary of the invention

[0004] In order to solve the above technical problems, the present application provides a bovine oocyte cryoprotectant and a cryopreservation method.

[0005] In a first aspect, the present application provides a bovine oocyte cryoprotectant, specifically comprising the following components in concentrations: excipient 510-595 mg / L, antioxidant 220-280 mg / L, antibiotic 20-30 mg / L, salt substance 120-180 mg / L, ethanol 320-380 mg / L, glycerol 65-85 mg / L, fetal bovine serum 55-68 mg / L; The excipient is composed of polysucrose, collagen, and caerulein in a weight ratio of 10:2-6:0.5-1.5; the specification of the collagen is: type II, derived from bovine nasal membrane, and has a molecular weight of 3000-4000; The antibiotic is composed of kanamycin and nalidixic acid mixed in a weight ratio of 1-3:1-3.

[0006] The inventors of the present application have discovered that the use of excipients, antibiotics and specific weight portions of antioxidants, salt substances, ethanol, glycerol and fetal bovine serum as raw material components of bovine oocyte cryoprotectants can form a unique protective film on the surface of bovine oocytes under harsh environmental conditions such as freezing, dehydration and dehydration, effectively protecting the bovine oocytes from denaturation and inactivation, protecting the integrity and biological activity of the bovine oocytes during the freeze-drying process, and protecting the fertilization ability and subsequent development ability of the oocytes.

[0007] The present application utilizes polysucrose, collagen, and caerulein as excipients in the dehydrated product of bovine oocytes. The hydroxyl group in the polysucrose structure can replace the hydrogen bond between protein and water to provide a stable effect; the filamentous collagen fibers in type II collagen interweave with elastin and polysaccharide proteins to form a mesh structure, which produces a certain mechanical strength to avoid the bovine oocytes from being damaged during the dehydration process during freezing; caerulein polypeptide substances are an excellent compatible solute, which can promote polysucrose sugar substances to quickly penetrate bovine oocytes after combining with polysucrose and collagen, improve the permeability of cell membrane to water, reduce the freezing point, and enable the water in the cell to penetrate the cell before freezing, and delay the thawing process, reduce the ice crystals in the cell, thereby reducing the damage of the thawing process to the bovine oocytes. That is, excipients can be used to stabilize bovine oocytes and their activity, especially in the dehydration process, which can play a good cell protection role, thereby reducing cell damage and keeping the cells active.

[0008] At the same time, the applicant of this application also found that the antibiotics were mixed with kanamycin and tetracycline in a weight ratio of 1.2:2.5, and added to the protective agent in a small concentration of 20-30 mg / L, which can be used as a protective agent carrier or auxiliary component, which can protect cells during freezing and play the role of antibiotics after thawing, prevent microbial contamination that may be introduced during freezing or in the environment after thawing, and inhibit microbial changes in the solution, thereby achieving the purpose of protecting active components, thereby reducing cell damage and keeping the cells active. During the experiment, the applicant found that the use of other types of antibiotics as raw material components in the cryoprotectant resulted in a lower cell survival rate, or a lower cleavage rate and blastocyst rate after fertilization; this shows that the type of antibiotics has a greater impact on the freezing of bovine oocytes. Only by reasonably selecting kanamycin and tetracycline mixed with a weight ratio of 1.2:2.5 and using them in combination with other raw materials can the purpose of protecting bovine oocytes be achieved.

[0009] In addition, the antioxidant substances in the cryoprotectant can be used to prevent the oxidation and deterioration of bovine oocytes during the freezing and dehydration process and storage process, effectively remove free radicals in the system, and inhibit the oxidative damage of bovine oocytes. Salt substances can adjust the pH changes of the solution during the freezing and dehydration of bovine oocytes, thereby protecting the functions of the active components. Ethanol and glycerol, as fillers and cryoprotectants in the cell freezing process, will crystallize during the freezing process, thereby providing a support structure for the active components and will not react with other active components; at the same time, they can work together with excipients to increase the permeability of the cell membrane to water, lower the freezing point, and delay the thawing process, reduce ice crystals in the cells, and reduce the damage to bovine oocytes during the thawing process.

[0010] Therefore, the cryoprotectant provided in the present application has an unexpected protective effect on the dehydration and drying of bovine oocytes. The obtained dehydrated and dried bovine oocyte preparation has high safety, strong stability, and high recovery rate. The effect of cryodehydration on bovine oocytes is extremely small, which is conducive to the long-term storage and transportation of bovine oocytes.

[0011] The concentration of each component in the bovine oocyte cryoprotectant is directly related to the protection of the bovine oocyte during the dehydration and drying process. When the concentration is too low, the protective effect is weak; and when the concentration reaches a certain level, the ability to stabilize the cells reaches its limit. Too high a concentration will in turn cause the cells to denature during the dehydration and drying process, resulting in cell function loss. Therefore, this application needs to reasonably control the concentration of each raw material component within the above range.

[0012] Preferably, the bovine oocyte cryoprotectant specifically includes components in the following concentrations: excipient 530-560 mg / L, antioxidant 240-260 mg / L, antibiotic 23-28 mg / L, salt substance 140-160 mg / L, ethanol 340-360 mg / L, glycerol 70-80 mg / L, and fetal bovine serum 60-64 mg / L.

[0013] Preferably, the excipient is composed of a mixture of polysucrose, collagen and caerulein in a weight ratio of 10:3-5:0.7-1.3.

[0014] Furthermore, the excipient is composed of a mixture of polysucrose, collagen and caerulein in a weight ratio of 10:3.5-4.5:0.9-1.1.

[0015] In some specific embodiments, in the excipient, the weight ratio of polysucrose, collagen and caerulein can be 10:2-3.5:1.5, 10:3-4.5:1.3-1.5, 10:3.5-4.5:1.1-1.3, 10:4-4.5:1, 10:4.5:0.9-1.3, 10:5:0.7-1.1, 10:6:0.5-1.3.

[0016] In a specific embodiment, in the excipient, the weight ratio of polysucrose, collagen and caerulein can also be 10:2:1.5, 10:3:1.3, 10:3.5:1.1, 10:4:1, 10:4.5:0.9, 10:5:0.7, 10:6:0.5.

[0017] Through experimental analysis, it can be known that the application of the present invention using the mixed excipient of polysucrose, collagen and caerulein in the above weight ratio can further improve the application effect of the bovine oocyte cryoprotectant.

[0018] Preferably, among the antibiotics, the amount of kanamycin used is less than the amount of nalidixic acid used.

[0019] Preferably, the antibiotic is composed of a mixture of kanamycin and nalidixic acid in a weight ratio of 1-1.5:2-3.

[0020] In some specific embodiments, in the antibiotic, the weight ratio of kanamycin to nalidixic acid can be 1-1.2:1, 1-1.2:2, 1-1.2:2.5, 1-1.2:3, 1.2-1.5:1, 1.2-1.5:2, 1.2-1.5:2.5, 1.2-1.5:3, 1.5:1-2, 1.5:2, 1.5-2:2.5, 1.5:2-3, 2:1-2, 2:2.5-3, 2:2-3, 3:1-2, 3:2-3, 2:2.5-3.

[0021] In a specific embodiment, in the antibiotic, the weight ratio of kanamycin to nalidixic acid can also be 1:1, 1:2, 1:2.5, 1:3, 1.2:1, 1.2:2, 1.2:2.5, 1.2:3, 1.5:1, 1.5:2, 1.5:2.5, 1.5:3, 2:1, 2:2.5, 2:3, 3:1, 3:2, 2:2.5.

[0022] Through experimental analysis, it can be known that the present application uses kanamycin and nalidixic acid in the above weight ratio as antibiotics to further improve the application effect of bovine oocyte cryoprotectant.

[0023] Preferably, the antioxidant is selected from any one or more of vitamin E and glutathione.

[0024] Preferably, the salt substance is selected from one or more of sodium chloride, potassium chloride, sodium N-acetyltryptophanate, sodium octanoate, and sodium bicarbonate.

[0025] Preferably, in the bovine oocyte cryoprotectant, the solvent is HEPES buffer with a pH of 6.6-7.4.

[0026] The cryoprotectant provided in the present application uses HEPES buffer as a buffer system, which can effectively stabilize the pH of the environment in which bovine oocytes are located during the freezing process. During the drying and storage process, the pH of the bovine oocyte dried product can be adjusted to the most stable region of the active substance, thereby protecting the integrity and biological activity of the bovine oocytes.

[0027] In a second aspect, the present application provides a method for cryopreservation of bovine oocytes, using the above-mentioned bovine oocyte cryoprotectant for cryopreservation.

[0028] Preferably, the bovine oocyte cryopreservation method specifically comprises the following steps: transferring the bovine oocyte into a treatment solution for equilibration for 30-60 seconds, transferring the bovine oocyte into the cryoprotectant for equilibration for 25-45 seconds, and then freezing and preserving the bovine oocyte with liquid nitrogen; the pretreatment solution is prepared from EG, DMSO, FBS, and DPBS in a volume ratio of 1:1:2:6.

[0029] In summary, the technical solution of this application has the following effects: The present application utilizes excipients, antibiotics and specific weight portions of antioxidants, salt substances, ethanol, glycerol, and fetal bovine serum as raw material components of a bovine oocyte cryoprotectant, which can effectively protect the bovine oocytes from denaturation and inactivation under freezing, dehydration, and drying conditions, can protect the integrity and biological activity of the bovine oocytes during the freeze-drying process, and can protect the fertilization ability and subsequent development ability of the oocytes.

[0030] The highly efficient bovine oocyte cryoprotectant and cryopreservation method developed in this application can maintain its activity and stability during long-term storage and transportation, meet the needs of subsequent research or practical applications, and contribute to the protection and utilization of cattle species. DETAILED DESCRIPTION

[0031] Polysucrose (CAS No.: 26873-85-8, Product No.: F5415) was purchased from Sigma; trehalose (CAS No.: 99-20-7, Product No.: ST8950) was purchased from Solebao Biotechnology Co., Ltd.; collagen and caerulein were purchased from Shanghai Yuanye Biotechnology Co., Ltd.; kanamycin (CAS No.: 8063-07-8, Product No.: K331597), nalidixic acid (CAS No.: 389-08-2, Product No.: N104920), gentamicin (CAS No.: 1403-66-3, Product No.: G302822), and tetracycline (CAS No.: 60-54-8, Product No.: T580568) were purchased from Shanghai Aladdin Chemical Reagent Co., Ltd.; fetal bovine serum (Product No.: M85271) was purchased from Shanghai Myrel Biochemical Technology Co., Ltd.; the remaining reagents and raw materials can be obtained commercially.

[0032] The present application is further described in detail below in conjunction with examples, comparative examples and performance testing experiments. These examples should not be construed as limiting the scope of protection claimed in the present application. Example

[0033] Examples 1-5 Examples 1-5 each provide a bovine oocyte cryoprotectant.

[0034] The difference between the above embodiments is that the dosage of each component in the cryoprotectant is different, as shown in Table 1.

[0035] The preparation method of the cryoprotectant in the above embodiment is: Excipient: A mixture of polysucrose, collagen (type II, derived from bovine nasal membrane, molecular weight 3000-4000), and caerulein in a weight ratio of 10:4:1.

[0036] Antibiotics: A mixture of kanamycin and nalidixic acid in a weight ratio of 1.2:2.5.

[0037] According to a 1L cryoprotectant meter, weigh the corresponding weights of excipients, antioxidant vitamin E, antibiotics, salt substances sodium bicarbonate, ethanol, glycerol, and fetal bovine serum respectively; then fully dissolve them in 950mL HEPES buffer, stir to mix and dissolve, adjust the pH to 7.2, add HEPES buffer to make up to 1000mL, and obtain the bovine oocyte cryoprotectant.

[0038] Table 1 Amount of each component in the cryoprotectant in Examples 1-5 and Comparative Examples 1-2 Embodiment 6-9 Examples 6-9 each provide a bovine oocyte cryoprotectant.

[0039] The difference between the above embodiment and embodiment 1 is that the types of excipients are different, as shown below.

[0040] In Example 6: Excipient: A mixture of polysucrose, collagen (type II, derived from bovine nasal membrane, molecular weight 3000-4000), and caerulein in a weight ratio of 10:2:1.5.

[0041] In Example 7: Excipient: A mixture of polysucrose, collagen (type II, derived from bovine nasal membrane, molecular weight 3000-4000), and caerulein in a weight ratio of 10:6:0.5.

[0042] In Example 8: Excipient: A mixture of polysucrose, collagen (type II, derived from bovine nasal membrane, molecular weight 3000-4000), and caerulein in a weight ratio of 10:3:1.3.

[0043] In Example 9: Excipient: A mixture of polysucrose, collagen (type II, derived from bovine nasal membrane, molecular weight 3000-4000), and caerulein in a weight ratio of 10:5:0.7.

[0044] The other raw materials and their amounts in the above examples and the preparation method of the bovine oocyte cryoprotectant are the same as those in Example 1.

[0045] Examples 10-13 Examples 10-13 each provide a bovine oocyte cryoprotectant.

[0046] The difference between the above embodiment and embodiment 1 is that the types of antibiotics are different, as shown below.

[0047] In Example 10: Antibiotics: a mixture of kanamycin and nalidixic acid in a weight ratio of 1:3.

[0048] In Example 11: Antibiotics: a mixture of kanamycin and nalidixic acid in a weight ratio of 1.5:2.

[0049] In Example 12: Antibiotics: a mixture of kanamycin and nalidixic acid in a weight ratio of 2.5:1.2.

[0050] In Example 13: Antibiotics: a mixture of kanamycin and nalidixic acid in a weight ratio of 1:1.

[0051] The other raw materials and their amounts in the above examples and the preparation method of the bovine oocyte cryoprotectant are the same as those in Example 1.

[0052] Comparative Example Comparative Example 1-2 Comparative Examples 1-2 respectively provide a bovine oocyte cryoprotectant.

[0053] The difference between the comparative example and Example 1 is that the dosage of each component in the cryoprotectant is different, as shown in Table 1.

[0054] The types and amounts of the raw materials and the preparation method of the bovine oocyte cryoprotectant in the above comparative example are the same as those in Example 1.

[0055] Comparative Examples 3-7 Comparative Examples 3-7 each provide a bovine oocyte cryoprotectant.

[0056] The differences between the above comparative example and Example 1 are specifically as follows.

[0057] In comparative example 3: the excipient is composed of a mixture of polysucrose, collagen (type I, derived from bovine heel, molecular weight 3000-4000), and caerulein in a weight ratio of 10:4:1.

[0058] In Comparative Example 4: the excipient is composed of a mixture of trehalose, collagen (type II, derived from bovine nasal membrane, molecular weight 3000-4000), and caerulein in a weight ratio of 10:4:1.

[0059] In Comparative Example 5: the excipient is composed of a mixture of polysucrose and collagen (type II, derived from bovine nasal membrane, molecular weight 3000-4000) in a weight ratio of 10:4.

[0060] In Comparative Example 6, the antibiotic is composed of a mixture of gentamicin and nalidixic acid in a weight ratio of 1.2:2.5.

[0061] In Comparative Example 7, the antibiotics are composed of a mixture of kanamycin and tetracycline in a weight ratio of 1.2:2.5.

[0062] The types and amounts of the raw materials and the preparation method of the bovine oocyte cryoprotectant in the above comparative example are the same as those in Example 1.

[0063] Performance testing The bovine oocytes were cryopreserved for 180 days using the cryoprotectants prepared in the examples and comparative examples, and then the performance of the bovine oocytes was evaluated. The specific experimental operation steps are as follows: (1) Obtaining bovine oocytes: routine collection and collection of bovine oocytes; 3-year-old cattle, ovaries were taken and washed 2-3 times with saline solution containing antibiotics to remove adherent blood stains, and excess surrounding tissue was cut off with scissors; oocytes (number of follicles ≥ 20) in follicles with a diameter of 2-8 mm were extracted from the surface, and complete and dense cumulus-oocyte complexes (COCs) were selected and placed in IVM fluid for maturation for 22-24 hours. After IVM, granulosa cells were removed from COCs, and oocytes with the first polar body and uniform cytoplasm were selected for subsequent research.

[0064] (2) Pretreatment: Adjust the room temperature to 25±1℃ to allow the test equipment and reagents to be fully balanced. The test was performed on a 37-39℃ constant temperature bench. The bovine oocytes were transferred into the treatment solution (the treatment solution consisted of EG, DMSO, FBS, DPBS (-Ca 2+ Mg 2+ ) were prepared in a volume ratio of 1:1:2:6) and then equilibrated for 45 seconds, and then transferred into the cryoprotectant prepared in the example and the comparative example and equilibrated for 30 seconds, and then transferred into OPS tubes and directly put into liquid nitrogen for cryopreservation for 180 days; 120 bovine oocytes were placed in each group of OPS tubes.

[0065] (3) Thawing: Before thawing, prepare a 37°C operating table and a room temperature operating table, and place 1 ml of 0.25M sucrose solution and 1 ml of 0.15M sucrose solution in a 37°C CO2-free incubator for preheating. After the OPS tube is taken out of liquid nitrogen, the oocytes are placed in a culture dish containing 0.25M sucrose solution for 60 seconds, and then moved into 0.15M sucrose thawing solution for 5 minutes. The oocytes are washed twice with IVM solution. Those with normal morphology after thawing (based on membrane integrity and cell gloss) are judged as alive. Repeat the experiment 3 times, record the average number of survival (rounded), and calculate the survival rate (survival rate = average number of survival / 120×100%), as shown in Table 2.

[0066] (4) Fertilization: The frozen semen in liquid nitrogen was thawed in a 38°C water bath, moved to a clean bench, and the semen was added to a 15 mL centrifuge tube. The tube was centrifuged with sperm washing solution (1500 r / min, 5 min / time), and the sperm was washed twice. The supernatant was discarded, and finally the fertilization solution was added to make the sperm density become 1×10 6 / mL, put it in a CO2 incubator, and incubate it for 18 hours at 5% CO2, 38°C, and 100% humidity. Then move the thawed oocytes to the fertilization drop, 20 to 30 live oocytes / drop, and put it in a CO2 incubator for fertilization for 7 hours. After the fertilized eggs were cultured for 7 h, they were placed in culture medium (composed of 54% Tyrode's solution, 10% FBS and 36% TCM-199 solution, pre-balanced in an incubator for 1.5 h before use) for washing, surrounding sperms were removed, and the eggs were then cultured in culture medium. After 48 h, the cleavage rate (number of embryos from the fertilized eggs / total number of fertilized eggs × 100%; as shown in Table 2) was recorded, and the cleaved embryos were transferred to culture medium (composed of 54% Tyrode's solution, 10% FBS and 36% TCM-199 solution, pre-balanced in an incubator for 1.5 h before use) for culture. The solution was changed every 48 h thereafter, and the blastocyst rate on the 7th day was counted (as shown in Table 2).

[0067] Test results: as shown in Table 2.

[0068] Table 2 Application performance test results of bovine oocyte cryoprotectants in Examples and Comparative Examples Combined with the test results of Table 2, the present application uses excipients, antibiotics and antioxidants, salt substances, ethanol, glycerol, and fetal bovine serum in specific weight portions to prepare bovine oocyte cryoprotectants, which are used for the cryoprotection of bovine oocytes. The damage of thawed bovine oocytes is small, the survival rate is high, and the cleavage rate and blastocyst rate of bovine oocytes after fertilization are high. The results show that the use of the cryoprotectant of the present application can protect the integrity and biological activity of bovine oocytes in the freeze-drying process, and can protect the fertilization ability and subsequent development ability of oocytes.

[0069] Through the test results of Examples 1, 6-9 and Comparative Examples 3-5, it can be seen that in Comparative Example 3, the excipient composed of type I collagen from bovine calf with a molecular weight of 3000-4000, polysucrose and caerulein is mixed, and the survival rate of bovine oocytes is low; in Comparative Example 4, the excipient composed of trehalose, collagen and caerulein is mixed, and the survival rate, cleavage rate and blastocyst rate of bovine oocytes after fertilization are low; in Comparative Example 4, the excipient composed of polysucrose and caerulein is mixed, and the survival rate, cleavage rate and blastocyst rate of bovine oocytes after fertilization are low. In contrast, the embodiment of the present application uses a mixture of polysucrose, collagen and caerulein with a weight ratio of 10:2-6:0.5-1.5 to form an excipient, which can ensure the survival rate of bovine oocytes, fertilization ability and subsequent development ability.

[0070] Through the test results of Examples 1, 10-13 and Comparative Examples 6-7, it can be seen that in Comparative Example 6, penicillin and nalidixic acid were mixed at a weight ratio of 1.2:2.5 to form antibiotics, and the survival rate of bovine oocytes and the cleavage rate after fertilization were low; in Comparative Example 7, kanamycin and tetracycline were mixed at a weight ratio of 1.2:2.5 to form antibiotics, and the survival rate of bovine oocytes, the cleavage rate after fertilization and the blastocyst rate were low. In contrast, the embodiment of the present application uses kanamycin and nalidixic acid mixed at a weight ratio of 1-3:1-3 to form antibiotics, which can ensure the survival rate of bovine oocytes, fertilization ability and subsequent development ability.

[0071] Although the present invention has been described in detail above with general descriptions and specific embodiments, it is obvious to those skilled in the art that some modifications or improvements may be made thereto based on the present invention. Therefore, these modifications or improvements made without departing from the spirit of the present invention all fall within the scope of protection claimed by the present invention.

Claims

1. A bovine oocyte cryoprotectant, characterized in that: Specifically, the components include the following concentrations: excipient 510-595 mg / L, antioxidant 220-280 mg / L, antibiotic 20-30 mg / L, salt substance 120-180 mg / L, ethanol 320-380 mg / L, glycerol 65-85 mg / L, fetal bovine serum 55-68 mg / L; The excipient is composed of polysucrose, collagen, and caerulein in a weight ratio of 10:2-6:0.5-1.5; the specification of the collagen is: type II, derived from bovine nasal membrane, and has a molecular weight of 3000-4000; The antibiotic is composed of kanamycin and nalidixic acid mixed in a weight ratio of 1-3:1-3.

2. The bovine oocyte cryoprotectant according to claim 1, characterized in that Specifically, it includes components with the following concentrations: excipient 530-560 mg / L, antioxidant 240-260 mg / L, antibiotic 23-28 mg / L, salt substance 140-160 mg / L, ethanol 340-360 mg / L, glycerol 70-80 mg / L, and fetal bovine serum 60-64 mg / L.

3. The bovine oocyte cryoprotectant according to claim 1, characterized in that The excipient is composed of polysucrose, collagen and caerulein in a weight ratio of 10:3-5:0.7-1.

3.

4. The bovine oocyte cryoprotectant according to claim 1, characterized in that Among the antibiotics, the amount of kanamycin used is less than that of nalidixic acid used.

5. The bovine oocyte cryoprotectant according to claim 4, characterized in that The antibiotic is composed of kanamycin and nalidixic acid mixed in a weight ratio of 1-1.5:2-3.

6. The bovine oocyte cryoprotectant according to claim 1, characterized in that The antioxidant is selected from any one or more of vitamin E and glutathione.

7. The bovine oocyte cryoprotectant according to claim 1, characterized in that: The salt substance is selected from one or more of sodium chloride, potassium chloride, sodium N-acetyltryptophanate, sodium octanoate, and sodium bicarbonate.

8. The bovine oocyte cryoprotectant according to claim 1, characterized in that: In the bovine oocyte cryoprotectant, the solvent is HEPES buffer with a pH of 6.6-7.

4.

9. A method for cryopreservation of bovine oocytes, characterized in that: The bovine oocyte cryoprotectant according to any one of claims 1 to 8 is used for cryopreservation.

10. The method for cryopreservation of bovine oocytes according to claim 9, characterized in that: The specific steps include: The bovine oocytes are transferred into the treatment solution for 30-60 seconds of equilibrium, then transferred into the cryoprotectant for 25-45 seconds of equilibrium, and then cryopreserved in liquid nitrogen; the pretreatment solution is prepared from EG, DMSO, FBS, and DPBS in a volume ratio of 1:1:2:6.

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