Bovine embryo cryopreservation method and unfreezing method thereof
By adopting a two-stage balanced and step-type frozen storage mode in the cryopreservation of bovine embryos and using a specific cryopreservation solution, the problem of low frozen survival rate of bovine embryos in the prior art is solved, and high survival rate and excellent breeding are achieved. performance.
Patent Information
- Application Number
- CN202411633919.4
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-11-15
- Publication Date
- 2025-02-25
AI Technical Summary
In the prior art, there are fewer methods for cryopreservation of bovine embryos, and the combination of cryopreservation solution and cryopreservation method is not effective, resulting in a low cryopreservation rate of embryos.
A method of cryopreservation of bovine embryos is adopted, which includes mixing the bovine embryos with the balance solution and balancing them through two stages; then mixing them with the cryopreservation solution, adopting a stepped freezing mode, and combining specific cryopreservation solution selection and use methods.
The frozen survival rate of cattle embryos was improved, and the embryo transfer test was carried out to ensure excellent breeding performance of the recipient cows, calving rate and calving survival rate.
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Abstract
Description
Technical Field
[0001] The present application relates to the technical field of embryo cryopreservation, and in particular to a method for cryopreservation of bovine embryos and a method for thawing the same. Background Art
[0002] Embryo cryopreservation is a biotechnology that freezes early animal embryos using special protective agents and cooling measures so that their metabolism stops or is weakened to a small enough level in liquid nitrogen, but without losing the ability to resume metabolism after warming up, thereby preserving the embryos for a long time.
[0003] Cryopreservation of bovine embryos was successfully achieved by Wilmut in 1973. Bovine embryo freezing methods can be divided into slow cooling method and vitrification freezing method according to different cooling methods. Slow cooling method refers to the embryos treated with cryoprotective solution, using an embryo freezer with a computer-controlled cooling speed, first cooling from room temperature to ice planting temperature -7°C at a rate of -1.0°C / min, keeping for 10 minutes, and then cooling to -30°C at a rate of -0.3°C / min after ice planting, and then directly putting into liquid ammonia for long-term storage. This slow cooling method can prevent ice formation in cells or reduce the formation of ice crystals. Ice planting induces crystallization, which can effectively prevent the damage to embryos caused by supercooling. Vitrification freezing method is to treat embryos with cryoprotective solution and directly put them into liquid nitrogen for long-term storage. The principle is that the viscosity of high-concentration cryoprotective solution increases when cooling, and solidifies when the critical value is reached.
[0004] However, there are relatively few methods for cryopreservation of bovine embryos, and the combination of cryopreservation fluid and cryopreservation methods has a poor cryopreservation effect on embryos, resulting in a low cryopreservation survival rate of embryos. Summary of the invention
[0005] In order to solve the above technical problems, the present application provides a bovine embryo cryopreservation method and a thawing method thereof.
[0006] In a first aspect, the present application provides a method for cryopreservation of bovine embryos, which specifically comprises the following steps: mixing the bovine embryos with a balancing solution, controlling the cooling rate to be ≤2°C / min, and balancing at a temperature of 15-20°C and 8-15°C for 10-20 minutes respectively to obtain a pretreatment solution; The pretreatment liquid is mixed with the cryoprotectant liquid, and the temperature is lowered to -2 to -8°C at a cooling rate of 0.5 to 2°C / min, and the temperature is kept for 20 to 30 minutes; then the temperature is lowered to -20 to -30°C at a cooling rate of 0.2 to 0.8°C / min, and the temperature is kept for 20 to 30 minutes; then the temperature is lowered to -50 to -60°C at a cooling rate of 0.05 to 0.2°C / min, and the temperature is kept for 2 to 5 hours; finally, the temperature is put into liquid nitrogen for long-term storage; The balance solution specifically includes the following components in concentration: 200-260 mg / L of carbon source, 340-390 mg / L of salt, 100-150 mg / L of ethylene glycol, and 80-120 mg / L of stabilizer; the stabilizer is composed of a mixture of triethanolamine and albumin in a weight ratio of 10-15:1-3; The cryoprotectant solution specifically includes components with the following concentrations: 500-560 mg / L of carbon source material, 180-220 mg / L of salt material, 400-450 mg / L of ethylene glycol, 80-100 mg / L of glutathione, 30-50 mg / L of fetal bovine serum, and 100-140 mg / L of antifreeze agent; the antifreeze agent is composed of a mixture of N-acetylcysteine, hydroxyethyl starch, EGF factor, and IL18 in a weight ratio of 80-100:20-30:2-8:10-20.
[0007] The freezing method is an important factor affecting the activity of bovine embryos after thawing and recovery. An excellent freezing method is the key to ensuring the activity and biological properties of bovine embryos after recovery. The freezing method designed by the present application for bovine embryos is to mix the bovine embryos with a balancing solution, undergo a two-stage balancing process, and then mix them with a cryoprotectant solution. A step-type freezing mode is adopted and combined with a specific cryoprotectant solution selection and use method, so that after the bovine embryos are frozen, the frozen bovine embryos have a higher survival rate after thawing, and the recipient cows that have undergone embryo transplantation tests have excellent breeding performance in terms of conception rate, calving rate, and calf survival rate.
[0008] In the preparation stage before cryopreservation, the balancing solution plays a vital role. By using a balancing solution containing specific concentrations of polysucrose, sodium bicarbonate, ethylene glycol, and stabilizers to balance the bovine embryos, it is beneficial to maintain the osmotic pressure balance inside and outside the bovine embryos and prevent cells from being damaged by osmotic pressure changes. And after balancing, it is beneficial to induce the adaptive evolution of bovine embryos to freezing conditions. Among them, triethanolamine can reduce the concentration of electrolytes in the unfrozen solution inside and outside the cells, thereby protecting the cells from damage by high concentrations of electrolytes; albumin has a large molecular weight and is not easy to pass through the blood vessel wall, so it can keep the osmotic pressure of the bovine embryo stable and prevent excessive penetration of water into the embryonic gap; and triethanolamine combined with albumin also has a certain antioxidant function, which can remove free radicals in the system and reduce the damage of oxidative stress to bovine embryos.
[0009] During cryopreservation, a cryoprotectant containing a carbon source substance, a salt substance, ethylene glycol, glutathione, fetal bovine serum, and an antifreeze agent containing a specific concentration is used to form a cryoprotectant solution, which can maintain the salt ion concentration of the tissue osmotic pressure and maintain the pH stability of the physiological environment, provide a suitable osmotic pressure and pH, maintain the activity of the embryo, and improve the anti-osmotic pressure effect of the embryo. The carbon source can be used as an energy substrate for the embryo, providing energy to maintain the minimum physiological metabolic conditions of the embryo and maintain the long-term preservation of the embryo. Ethylene glycol can penetrate into the cell through the cell membrane before the cell freezing suspension is completely solidified, and produce a certain molar concentration inside and outside the cell, thereby protecting the cell from damage by high concentrations of electrolytes. Glutathione has antioxidant properties, and fetal bovine serum provides certain nutrients. Under the synergistic effect of the above-mentioned specific combination, the biological development process of the bovine embryo is greatly delayed, the cell activity of the bovine embryo is maintained, the cell metabolism is reduced, and cell death or programmed apoptosis is reduced. After the bovine embryo is thawed, the original function of the embryo can be exerted and the development of the embryo can be restored.
[0010] The inventors found through research that the use of a mixture of N-acetylcysteine, hydroxyethyl starch, EGF factor, and IL18 in a weight ratio of 80-100:20-30:2-8:10-20 as an antifreeze agent can promote N-acetylcysteine and hydroxyethyl starch to preferentially combine with water molecules in the solution, reduce the content of free water in the solution, lower the freezing point, reduce the formation of ice crystals, and promote the outflow of water inside the bovine embryo, thereby ultimately reducing the formation of ice crystals inside the bovine embryo; at the same time, due to the large molecular weight of hydroxyethyl starch, the electrolyte concentration in the solution is reduced, thereby reducing solute damage. The EGF factor and IL18 in the antifreeze agent can not only protect the bovine embryo from damage during the freezing process, but also promote the rapid recovery and proliferation of the bovine embryo after thawing; the growth factor can also synergize with other components to improve the effect of cryopreservation. This combination brings better cell survival rate and functional recovery.
[0011] Preferably, the bovine embryo cryopreservation method specifically comprises the following steps in sequence: The bovine embryos are mixed with the balancing solution, the cooling rate is controlled to be 0.5-1°C / min, and the mixture is balanced at 16-19°C and 10-12°C for 13-18 minutes respectively to obtain a pretreatment solution; The pretreatment liquid is mixed with the cryoprotectant liquid, and the temperature is reduced to -6 to -4°C at a cooling rate of 0.7 to 1.5°C / min, and the temperature is kept in equilibrium for 22 to 27 minutes; then the temperature is reduced to -23 to -28°C at a cooling rate of 0.3 to 0.6°C / min, and the temperature is kept in equilibrium for 22 to 27 minutes; then the temperature is reduced to -53 to -57°C at a cooling rate of 0.08 to 0.15°C / min, and the temperature is kept in equilibrium for 3 to 4 hours; finally, the temperature is put into liquid nitrogen for long-term storage.
[0012] Preferably, the balance solution specifically comprises the following components in concentrations: 220-240 mg / L carbon source, 350-370 mg / L salt, 120-130 mg / L ethylene glycol, 90-100 mg / L stabilizer; the solvent is HEPES buffer with a pH of 6.6-7.4.
[0013] Preferably, in the balancing solution, the stabilizer is composed of a mixture of triethanolamine and albumin in a weight ratio of 11 to 14:1.5 to 2.5.
[0014] Preferably, the cryoprotectant solution specifically comprises components in the following concentrations: 520-540 mg / L carbon source, 190-210 mg / L salt, 420-430 mg / L ethylene glycol, 85-95 mg / L glutathione, 35-45 mg / L fetal bovine serum, and 110-130 mg / L antifreeze; the solvent is HEPES buffer with a pH of 6.6-7.4.
[0015] Preferably, in the cryoprotectant solution, the antifreeze agent is composed of a mixture of N-acetylcysteine, hydroxyethyl starch, EGF factor, and IL18 in a weight ratio of 85-95:23-28:3-6:12-17.
[0016] Preferably, the carbon source material is selected from one or more of polysucrose, trehalose, dextran and maltose.
[0017] Preferably, the salt substance is selected from one or more of sodium chloride, potassium chloride, sodium octanoate, sodium bicarbonate, and sodium N-acetyltryptophanate.
[0018] In a second aspect, the present application provides a method for thawing bovine embryos preserved by the above-mentioned cryopreservation method, which specifically comprises the following steps in sequence: When thawing, take the culture bottle containing frozen bovine embryos out of liquid nitrogen, let it stay in the air for 3 to 5 seconds, then quickly put it into a 36 to 39°C water bath, shake it gently for 90 to 120 seconds, add thawing solution into the clean bench, shake the culture bottle gently, balance it for 5 to 8 minutes, and then put it into the clean bench; The thawing solution is composed of the following components in concentrations: 3.2-3.6 g / L sucrose, 0.45-0.60 g / L arginine, and 0.04-0.08 g / L tetracycline.
[0019] Preferably, the thawing solution is composed of components with the following concentrations: 3.3-3.5 g / L sucrose, 0.50-0.55 g / L arginine, 0.05-0.07 g / L tetracycline; the solvent is HEPES buffer, and the pH is 6.6-7.4.
[0020] The thawing solution provided in the present application can not only help the frozen bovine embryos to thaw quickly, but also provide necessary nutrients and antioxidant and antibacterial effects, help the bovine embryos restore normal physiological functions, reduce bovine embryo damage, and protect the bovine embryos from oxidative stress damage during the thawing process.
[0021] In summary, the technical solution of this application has the following effects: The cryopreservation method for bovine embryos designed in the present application comprises mixing the bovine embryos with a balancing solution, undergoing a two-stage balancing process, and then mixing with a cryoprotectant solution, adopting a step-type freezing mode and combining a specific cryoprotectant solution selection and use method, so that the bovine embryos have a higher survival rate after freezing and thawing, and the recipient cows that have undergone embryo transplantation experiments have excellent breeding performance in terms of conception rate, calving rate, and calf survival rate. DETAILED DESCRIPTION
[0022] 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.
[0023] Polysucrose (CAS No.: 26873-85-8, product No.: F5415) was purchased from Sigma; albumin (CAS No.: 9048-46-8) was purchased from Weiyi Peptide (Guangdong) Biotechnology Co., Ltd.; fetal bovine serum (Product No.: M85271) was purchased from Shanghai Myrrel Biochemical Technology Co., Ltd.; EGF factor (Product No.: WKQ-0015798) was purchased from Sichuan Weikeqi Biotechnology Co., Ltd., IL18 (Product No.: 10119-HNCE) was purchased from Beijing Yiqiao Shenzhou Technology Co., Ltd.; FGF21 (Product No.: xy-db-1097) was purchased from Shanghai Xuanya Biotechnology Co., Ltd.; tetracycline (CAS No.: 60-54-8, Product No.: T580568) was purchased from Shanghai Aladdin Chemical Reagent Co., Ltd.; the remaining reagents and raw materials can be obtained commercially.
[0024] Preparation Example This preparation example provides a bovine embryo and a preparation method thereof.
[0025] The specific steps of this preparation example are as follows.
[0026] Cows over 2 months postpartum (local yellow cattle, 550kg±10kg per head, 3 years old) were selected as donor cows, with obvious breed characteristics, good body shape and appearance, stable genetic performance, clear pedigree, healthy and disease-free, normal reproductive function, no history of abortion, normal estrus cycle; rectal examination showed that the uterus and ovaries were normally developed and there were no reproductive system diseases. During the experiment, the cows were allowed to eat and drink freely, and the cowshed environment was cleaned and disinfected every day according to conventional methods.
[0027] After the first intramuscular injection of 4 ml of PG, the donor cow was injected with 4 ml of PG again after 10 days. Superovulation treatment began on the 14th day after the second intramuscular injection of PG. FSH was injected with a decreasing method for 4 consecutive days with an interval of 12 hours in the morning and evening. The injection volume for 4 days was 70 international units / 70 international units, 60 international units / 60 international units, 50 international units / 50 international units, and 40 international units / 40 international units, respectively, with a total of 440 international units. On the third day of FSH injection, PG was injected at the same time. On the second day after PG injection (the day of injection was day 0), insemination was performed once in the morning and evening, and on the third day, insemination was performed once again depending on the estrus status of the cow. For cows in estrus and ovulation, artificial insemination was performed in a timely manner according to the technical operating procedures of the "Technical Regulations for Artificial Insemination of Cattle".
[0028] On the 7th day after estrus mating (the day of estrus is day 0), embryo flushing is performed according to the conventional method, the donor cow is restrained according to the requirements of human insemination, and 5-10 ml of 2% procaine is used for epidural anesthesia of the coccyx.
[0029] The recovered egg flushing liquid was directly injected into the filter cup, and the remaining 35 ml of the recovered liquid in the filter cup was poured into the plate, and the filter cup was rinsed with the egg flushing liquid for 2-3 times, and the liquid was flushed into the plate, and then the embryo was inspected under a stereo microscope. After quality identification, the bovine embryo blastocyst was obtained, with complete embryo morphology, clear spherical outline, uniform blastomere size, compact structure, moderate color tone and transparency, and no attached cells and vacuoles. Example
[0030] Examples 1-5 Examples 1-5 each provide a method for cryopreservation of bovine embryos.
[0031] The bovine embryo blastocysts in the above examples are derived from the preparation example.
[0032] The difference between the above embodiments is that the amounts of the components in the balancing solution and the cryoprotectant solution are different, as shown in Table 1.
[0033] Preparation of equilibrium solution: According to Table 1, according to the 1L specification of cryoprotectant solution, weigh the corresponding weights of carbon source material polysucrose, salt material sodium bicarbonate, ethylene glycol, and stabilizer (composed of a mixture of triethanolamine and albumin with a weight ratio of 13:2); 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 equilibrium solution.
[0034] Preparation of cryoprotectant solution: According to a 1L cryoprotectant solution meter, weigh the corresponding weights of carbon source material polysucrose, salt material sodium bicarbonate, ethylene glycol, glutathione, fetal bovine serum, and antifreeze (composed of a mixture of N-acetylcysteine, hydroxyethyl starch, EGF factor, and IL18 in a weight ratio of 90:25:5:15); 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 then obtain cryoprotectant solution.
[0035] The specific steps of the bovine embryo cryopreservation method are as follows: 2 bovine embryos are mixed with 10 g of balancing solution, the cooling rate is controlled by a program freezer at 1°C / min, and the pretreatment solution is obtained at 17°C for 45 seconds; The pretreatment solution was mixed with 100 g of cryoprotectant solution and allowed to equilibrate at 12°C for 160 seconds. Finally, the embryos were placed on a freezing rod (OPS tube) and immersed in liquid nitrogen for long-term storage.
[0036] Table 1 Amount of each component in the balance solution and cryoprotectant solution in Examples 1-5 and Comparative Examples 1-8 Embodiment 6-7 Examples 6-7 each provide a method for cryopreservation of bovine embryos.
[0037] The difference between the above embodiment and embodiment 1 is that the type of stabilizer in the balancing solution is different, as shown below.
[0038] In Example 6: the stabilizer is composed of a mixture of triethanolamine and albumin in a weight ratio of 10:3.
[0039] In Example 7: the stabilizer is composed of a mixture of triethanolamine and albumin in a weight ratio of 15:1.
[0040] The other process parameters in the above embodiment are the same as those in embodiment 1.
[0041] Embodiment 8-11 Examples 8-11 each provide a method for cryopreservation of bovine embryos.
[0042] The difference between the above embodiment and embodiment 1 is that the types of antifreeze agents in the cryoprotectant solution are different, as shown below.
[0043] In Example 8, the antifreeze agent is composed of a mixture of N-acetylcysteine, hydroxyethyl starch, EGF factor, and IL18 in a weight ratio of 80:30:2:20.
[0044] In Example 9, the antifreeze agent is composed of a mixture of N-acetylcysteine, hydroxyethyl starch, EGF factor, and IL18 in a weight ratio of 100:20:8:10.
[0045] In Example 10, the antifreeze agent is composed of a mixture of N-acetylcysteine, hydroxyethyl starch, EGF factor, and IL18 in a weight ratio of 85:23:6:17.
[0046] In Example 11, the antifreeze agent is composed of a mixture of N-acetylcysteine, hydroxyethyl starch, EGF factor, and IL18 in a weight ratio of 95:28:3:12.
[0047] The other process parameters in the above embodiment are the same as those in embodiment 1.
[0048] Examples 12-15 Examples 12-15 each provide a method for cryopreservation of bovine embryos.
[0049] The differences between the above embodiment and embodiment 1 are specifically as follows.
[0050] In Example 12: 2 bovine embryos were mixed with 10 g of equilibration solution, the cooling rate was controlled to 2°C / min by a program freezer, and the mixture was equilibrated at 20°C for 30 seconds to obtain a pretreatment solution; the pretreatment solution was mixed with 100 g of cryoprotectant solution, and the mixture was equilibrated at 12°C for 160 seconds; finally, the embryos were placed on a freezing carrier (OPS tube), immersed in liquid nitrogen, and stored for a long time.
[0051] In Example 13: 2 bovine embryos were mixed with 10 g of equilibration solution, the cooling rate was controlled to be 1°C / min by a program freezer, and the mixture was equilibrated at 15°C for 60 s to obtain a pretreatment solution; the pretreatment solution was mixed with 100 g of cryoprotectant solution, and the mixture was equilibrated at 12°C for 160 s; finally, the embryos were placed on a freezing carrier (OPS tube), immersed in liquid nitrogen, and stored for a long time.
[0052] In Example 14: 2 bovine embryos were mixed with 10 g of equilibration solution, the cooling rate was controlled to be 1°C / min by a program freezer, and the mixture was equilibrated at 17°C for 45 seconds to obtain a pretreatment solution; the pretreatment solution was mixed with 100 g of cryoprotectant solution, and the mixture was equilibrated at 15°C for 210 seconds; finally, the embryos were placed on a freezing carrier (OPS tube), immersed in liquid nitrogen, and stored for a long time.
[0053] In Example 15: 2 bovine embryos were mixed with 10 g of equilibration solution, the cooling rate was controlled to be 1°C / min by a program freezer, and the mixture was equilibrated at 17°C for 45 seconds to obtain a pretreatment solution; the pretreatment solution was mixed with 100 g of cryoprotectant solution, and the mixture was equilibrated at 8°C for 120 seconds; finally, the embryos were placed on a freezing carrier (OPS tube), immersed in liquid nitrogen, and stored for a long time.
[0054] The other process parameters in the above embodiment are the same as those in embodiment 1.
[0055] Comparative Example Comparative Examples 1-8 Comparative Examples 1-8 respectively provide a method for cryopreservation of bovine embryos.
[0056] The difference between the comparative example and Example 1 is that the amounts of the components in the balancing solution and the cryoprotectant solution are different, as shown in Table 1.
[0057] The other process parameters in the above comparative example are the same as those in Example 1.
[0058] Comparative Examples 9-10 Comparative Examples 9-10 respectively provide a method for cryopreservation of bovine embryos.
[0059] The differences between the above comparative example and Example 1 are specifically as follows.
[0060] In Comparative Example 9: the stabilizer in the equilibrium solution is composed of a mixture of formamide and albumin in a weight ratio of 13:2.
[0061] In Comparative Example 10: the antifreeze agent in the cryoprotectant solution is a mixture of N-acetylcysteine, hydroxyethyl starch, EGF factor, and FGF21 factor in a weight ratio of 90:25:5:15.
[0062] The other process parameters in the above comparative example are the same as those in Example 1.
[0063] Comparative Examples 11-12 Comparative Examples 11-12 respectively provide a method for cryopreservation of bovine embryos.
[0064] The differences between the above comparative example and Example 1 are specifically as follows.
[0065] Comparative Example 11: The specific steps of the bovine embryo cryopreservation method are as follows: 0.1 g of bovine embryo liquid is mixed with 100 g of cryoprotectant liquid, the cooling rate is controlled to 1°C / min, and the liquid is equilibrated at 17°C and 11°C for 15 min respectively to obtain a pretreatment liquid; the temperature is reduced to -5°C at a cooling rate of 1°C / min, and the temperature is kept for 25 min; the temperature is then reduced to -25°C at a cooling rate of 0.5°C / min, and the temperature is kept for 25 min; the temperature is then reduced to -55°C at a cooling rate of 0.1°C / min, and the temperature is kept for 3.5 h; and finally, the liquid nitrogen is put into the liquid nitrogen for long-term storage.
[0066] Comparative Example 12: The specific steps of the bovine embryo cryopreservation method are as follows: 0.1g of bovine embryo liquid is mixed with 10g of balancing liquid, the cooling rate is controlled to 1℃ / min, and the temperature is balanced at 25℃ and 15℃ for 15min respectively to obtain a pretreatment liquid; 10g of the pretreatment liquid is mixed with 100g of cryoprotectant liquid, and the temperature is reduced to 0℃ at a cooling rate of 3℃ / min, and the temperature is kept balanced for 25min; then the temperature is reduced to -15℃ at a cooling rate of 1.5℃ / min, and the temperature is kept balanced for 25min; then the temperature is reduced to -45℃ at a cooling rate of 0.5℃ / min, and the temperature is kept balanced for 3.5h; finally, liquid nitrogen is put into the liquid nitrogen for long-term storage.
[0067] The other process parameters in the above comparative example are the same as those in Example 1.
[0068] Performance testing Test Example 1: Survival rate of bovine embryos Preparation of thawing solution: According to a 1L cryoprotectant solution meter, weigh 3.4g sucrose, 0.52g arginine, and 0.06g tetracycline 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 then obtain the thawing solution.
[0069] The culture bottles (120 days of liquid nitrogen storage time) containing frozen bovine embryos in the embodiment and the comparative example (80 embryos in each test group) were taken out from the liquid nitrogen, left in the air for 3-5 seconds, and then quickly placed in a 36-39° C. water bath, gently shaken for 100 seconds, thawing solution was added in the clean bench, the culture bottles were gently shaken, balanced for 6 minutes, and then placed in the clean bench; The thawed bovine embryos were observed in the culture dish to see whether their morphology was normal (based on membrane integrity and cell gloss) and judged to be alive. The experiment was repeated 3 times, the average number of survivors was recorded (rounded to an integer), and the survival rate was calculated (survival rate = number of surviving bovine embryos after thawing / number of bovine embryos before freezing × 100%).
[0070] Test results: as shown in Table 2.
[0071] Test Example 2: Evaluation of Embryo Transfer Performance of Bovine Embryos Evaluation of embryo transfer performance of bovine embryos: Cows more than 2 months after giving birth were used as recipient cows. The recipient cows were empty-breasted, multiparous cows that had not been in estrus, and the rest of their shapes were the same as the donor cows. The recipient cows were synchronized and embryo transfer was performed on the 8th day of estrus; each experimental group had 50 recipient cows, and 50 thawed surviving bovine embryos in each group of the test case were taken for embryo transfer.
[0072] Pregnancy judgment and conception rate calculation: blood was collected from the anterior vena cava on the 50th day after embryo transfer. The blood was collected in a centrifuge tube and centrifuged at 3500r / min and 4°C for 15min. The supernatant was taken to test the serum progesterone level, the number of cows that successfully conceived was recorded, and the conception rate was calculated; where conception rate = number of conceptions / 50×100%.
[0073] Group management: pregnant cows are raised separately in groups until they give birth, and their calving rate and calf survival rate are calculated; among which, calving rate = number of calves born / 50×100%, calf survival rate = number of calves alive / number of calves born×100%.
[0074] Test results: as shown in Table 2.
[0075] Table 2 Performance test results of frozen bovine embryos in Examples and Comparative Examples Combined with the test results in Table 2, it can be seen that in Comparative Example 1, the concentration of carbon source substances in the balanced solution is higher and the concentration of carbon source substances in the cryopreservation solution is lower, no stabilizer is added to the balanced solution in Comparative Example 4, no stabilizer is added to the balanced solution in Comparative Example 6, and no antifreeze is added to the cryopreservation solution, the stabilizer concentration in the balanced solution in Comparative Example 7 is lower and the antifreeze concentration in the cryopreservation solution is higher, the components of the antifreeze in the cryopreservation solution in Comparative Example 10 do not match, the cryopreservation method in Comparative Example 11 does not use balanced solution balance, and the step cooling temperature and cooling rate of the cryopreservation method in Comparative Example 12 do not match, and the survival rate of the frozen bovine embryos after thawing in the above comparative examples is less than 75%, and the survival rate is low.
[0076] In Comparative Example 2, the concentration of salt substances in the balancing solution is low, and the concentration of salt substances in the cryopreservation solution is high. In Comparative Example 3, the concentration of salt substances in ethylene glycol is high, and the concentration of ethylene glycol substances in the cryopreservation solution is low. In Comparative Example 5, no antifreeze is added to the cryopreservation solution. In Comparative Example 8, the concentration of stabilizer in the balancing solution is high, and the concentration of antifreeze in the cryopreservation solution is low. In Comparative Example 9, the components of the stabilizer in the balancing solution do not match. In the above comparative examples, the frozen bovine embryos were thawed for embryo transplantation experiments, and the breeding performance of the recipient cows in terms of conception rate, calving rate, and calf survival rate were poor.
[0077] In contrast, the frozen bovine embryos obtained using the cryopreservation method provided in the embodiments of the present application have a higher survival rate after thawing, and after embryo transplantation experiments, the recipient cows have excellent breeding performance in terms of conception rate, calving rate, and calf survival rate.
[0078] 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 method for cryopreservation of bovine embryos, characterized in that: Specifically, the following steps are performed in sequence: The bovine embryos are mixed with the balancing solution, the cooling rate is controlled to be ≤2°C / min, and the mixture is balanced at a temperature of 15-20°C for 30-60s to obtain a pretreatment solution; The pretreatment solution is mixed with the cryoprotectant solution, and allowed to stand at 8-15°C for 120-210 seconds; and finally, the mixture is put into liquid nitrogen for long-term storage; The balance solution specifically includes the following components in concentration: 200-260 mg / L of carbon source, 340-390 mg / L of salt, 100-150 mg / L of ethylene glycol, and 80-120 mg / L of stabilizer; the stabilizer is composed of a mixture of triethanolamine and albumin in a weight ratio of 10-15:1-3; The cryoprotectant specifically comprises components of the following concentrations: 500-560 mg / L of carbon source, 180-220 mg / L of salt, 400-450 mg / L of ethylene glycol, 80-100 mg / L of glutathione, 30-50 mg / L of fetal bovine serum, and 100-140 mg / L of antifreeze; the antifreeze is composed of a mixture of N-acetylcysteine, hydroxyethyl starch, EGF factor, and IL18 in a weight ratio of 80-100:20-30:2-8:10-20.
2. The method for freezing and preserving bovine embryos according to claim 1, wherein: Specifically, the following steps are performed in sequence: The bovine embryos are mixed with the balancing solution, the cooling rate is controlled to be 0.5-1°C / min, and the mixture is balanced at a temperature of 16-19°C for 30-60s to obtain a pretreatment solution; The pretreatment solution is mixed with the cryoprotectant solution, and allowed to stand at 10-12° C. for 150-180 seconds for equilibrium; and finally, the mixture is put into liquid nitrogen for long-term storage.
3. The method for freezing and preserving bovine embryos according to claim 1, wherein: The balance solution specifically includes the following components in concentration: 220-240 mg / L of carbon source, 350-370 mg / L of salt, 120-130 mg / L of ethylene glycol, and 90-100 mg / L of stabilizer; the solvent is HEPES buffer solution with a pH of 6.6-7.
4.
4. The method for freezing and preserving bovine embryos according to claim 1, wherein: In the balance solution, the stabilizer is composed of a mixture of triethanolamine and albumin in a weight ratio of 11-14:1.5-2.
5.
5. The method for freezing and preserving bovine embryos according to claim 1, wherein: The cryoprotectant solution specifically includes the following components in concentrations: 520-540 mg / L of carbon source, 190-210 mg / L of salt, 420-430 mg / L of ethylene glycol, 85-95 mg / L of glutathione, 35-45 mg / L of fetal bovine serum, and 110-130 mg / L of antifreeze; the solvent is HEPES buffer solution with a pH of 6.6-7.
4.
6. The method for cryopreservation of bovine embryos according to claim 1, characterized in that: In the cryoprotectant solution, the antifreeze agent is composed of a mixture of N-acetylcysteine, hydroxyethyl starch, EGF factor, and IL18 in a weight ratio of 85-95:23-28:3-6:12-17.
7. The method for cryopreservation of bovine embryos according to claim 1, characterized in that: The carbon source material is selected from one or more of polysucrose, trehalose, dextran and maltose.
8. The method for cryopreservation of bovine embryos according to claim 1, characterized in that: The salt substance is selected from one or more of sodium chloride, potassium chloride, sodium octanoate, sodium bicarbonate, and sodium N-acetyltryptophanate.
9. A method for thawing bovine embryos preserved by the cryopreservation method according to any one of claims 1 to 8, characterized in that: Specifically, the following steps are performed in sequence: When thawing, take the culture bottle containing frozen bovine embryos out of liquid nitrogen, let it stay in the air for 3-5 seconds, then quickly put it into a 36-39℃ water bath, shake it gently for 90-120 seconds, add thawing solution into the clean bench, shake the culture bottle gently, balance it for 5-8 minutes, and then put it into the clean bench; The thawing solution is composed of components with the following concentrations: 3.2-3.6 g / L sucrose, 0.45-0.60 g / L arginine, and 0.04-0.08 g / L tetracycline.
10. The method for thawing bovine embryos according to claim 9, characterized in that: The thawing solution is composed of the following components: 3.3-3.5 g / L sucrose, 0.50-0.55 g / L arginine, and 0.05-0.07 g / L tetracycline; the solvent is HEPES buffer solution, and the pH value is 6.6-7.4.