A one-step freezing solution for bovine in vitro embryos and a one-step freezing method for bovine in vitro embryos

Abstract

The application discloses a one-step freezing solution for bovine in-vitro embryos and a one-step freezing method for bovine in-vitro embryos and belongs to the technical field of biotechnology.The method comprises the following steps: bovine oocyte in-vitro maturation; bovine in-vitro fertilization embryo production; after culturing the bovine in-vitro fertilization embryo for 6-8 days, the embryo is moved into freezing solution A, and after standing, the shriveled blastocyst is moved from the freezing solution A into freezing solution B, the embryo sinking to the bottom of the freezing solution B is moved into freezing solution C, is loaded into a macaroni tube, and is subjected to programmed freezing; after the freezing program is completed, the macaroni tube is put into liquid nitrogen. The bovine in-vitro embryos are frozen by using the three kinds of freezing solutions, on the premise that the conventional freezing method has low technical requirements for the operator and can be directly transplanted after thawing, the conventional freezing method is overcome, the survival rate, the hatching rate and the transplantation pregnancy rate of the embryos after thawing are low, the convenience of use and the effective integration of the high transplantation pregnancy rate are realized.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a one-step cryopreservation solution for bovine in vitro embryos and a one-step cryopreservation method for bovine in vitro embryos. Background Technology

[0002] The information disclosed in this background section is intended only to enhance understanding of the overall background of the invention and is not necessarily to be construed as an admission or in any way implying that such information constitutes prior art known to those skilled in the art.

[0003] Embryo cryopreservation breaks down the limitations of time and space, facilitating the exchange of germplasm resources. However, less than 50% of bovine embryos transferred annually are cryopreserved, with the majority still used for fresh embryo transfer. Of the nearly 80% of in vitro embryos produced, almost 60% are used for fresh embryo transfer. Conversely, although the number of in vivo embryos produced is smaller, 60% are used for frozen embryo transfer.

[0004] Compared to in vivo embryos, in vitro embryos have a higher vacuolar content and less intercellular density, making them more sensitive to cryopreservation. The survival rate and pregnancy rate of frozen in vitro embryos are significantly lower than those of in vivo embryos. This greatly limits the application of bovine in vitro embryos.

[0005] There are two common methods for cryopreservation of bovine embryos: conventional freezing and vitrification, each with its own advantages and disadvantages. Conventionally cryopreserved embryos can be directly transferred after thawing, requiring less technical expertise from the operator. However, the survival and hatching rates of conventionally cryopreserved embryos are significantly lower than those of vitrified embryos, and the pregnancy rate after transfer is also lower. Conversely, vitrified embryos have a high pregnancy rate, but the thawing process requires specialized technicians and equipment. Therefore, to further promote the application of bovine in vitro embryos in production, it is necessary to develop an embryo cryopreservation method and related reagents that balance convenience and pregnancy rate. Summary of the Invention

[0006] To address the shortcomings of existing technologies, the purpose of this invention is to provide a one-step cryopreservation solution and a one-step freezing method for bovine in vitro embryos. By using this invention to freeze bovine in vitro embryos, the thawed embryos can be directly transplanted, and a high implantation success rate can be achieved.

[0007] To achieve the above objectives, the technical solution of the present invention is as follows:

[0008] In a first aspect, the present invention provides a one-step cryopreservation solution for bovine in vitro embryos, comprising cryopreservation solution A, cryopreservation solution B, and cryopreservation solution C;

[0009] The cryogenic solution A is an aqueous solution containing 5–10 g / L sodium chloride, 0.1–0.5 g / L potassium chloride, 0.1–0.3 g / L magnesium chloride, 2.5–3.0 g / L disodium hydrogen phosphate, 0.1–0.5 g / L potassium dihydrogen phosphate, 0.8–1.2 g / L glucose, 0.03–0.04 g / L sodium pyruvate, 0.1–0.3 g / L calcium chloride, 0.1–0.3 g / L bovine serum albumin, and 1–1.5 M sucrose.

[0010] The cryogenic solution B is an aqueous solution containing 5–10 g / L sodium chloride, 0.1–0.5 g / L potassium chloride, 0.1–0.3 g / L magnesium chloride, 2.5–3.0 g / L disodium hydrogen phosphate, 0.1–0.5 g / L potassium dihydrogen phosphate, 0.8–1.2 g / L glucose, 0.03–0.04 g / L sodium pyruvate, 0.1–0.3 g / L calcium chloride, 3–5 g / L bovine serum albumin, 0.1–0.3 M sucrose, and 0.5–1 M anhydrous ethanol.

[0011] The cryogenic solution C is an aqueous solution containing 5–10 g / L sodium chloride, 0.1–0.5 g / L potassium chloride, 0.1–0.3 g / L magnesium chloride, 2.5–3.0 g / L disodium hydrogen phosphate, 0.1–0.5 g / L potassium dihydrogen phosphate, 0.8–1.2 g / L glucose, 0.03–0.04 g / L sodium pyruvate, 0.1–0.3 g / L calcium chloride, 3–5 g / L bovine serum albumin, 0.1–0.3 M sucrose, and 1.3–1.7 M anhydrous ethanol.

[0012] All reagents used in this invention are of cell culture grade or higher purity, such as products from Sigma-Aldrich.

[0013] In some embodiments of the present invention, the cryosol A is an aqueous solution containing 8 g / L sodium chloride, 0.2 g / L potassium chloride, 0.1 g / L magnesium chloride, 2.9 g / L disodium hydrogen phosphate, 0.2 g / L potassium dihydrogen phosphate, 1 g / L glucose, 0.036 g / L sodium pyruvate, 0.1 g / L calcium chloride, 0.1 g / L bovine serum albumin and 1 M sucrose.

[0014] In some embodiments of the present invention, the cryosol B is an aqueous solution containing 8 g / L sodium chloride, 0.2 g / L potassium chloride, 0.1 g / L magnesium chloride, 2.9 g / L disodium hydrogen phosphate, 0.2 g / L potassium dihydrogen phosphate, 1 g / L glucose, 0.036 g / L sodium pyruvate, 0.1 g / L calcium chloride, 4 g / L bovine serum albumin, 0.1 M sucrose, and 0.75 M ethylene glycol.

[0015] In some embodiments of the present invention, the cryosol C is an aqueous solution containing 8 g / L sodium chloride, 0.2 g / L potassium chloride, 0.1 g / L magnesium chloride, 2.9 g / L disodium hydrogen phosphate, 0.2 g / L potassium dihydrogen phosphate, 1 g / L glucose, 0.036 g / L sodium pyruvate, 0.1 g / L calcium chloride, 4 g / L bovine serum albumin, 0.1 M sucrose, and 1.5 M ethylene glycol.

[0016] A second aspect of the present invention provides a one-step in vitro embryo freezing method for bovine embryos, using the one-step in vitro embryo freezing solution described in the first aspect, comprising the following steps:

[0017] (1) In vitro maturation of bovine oocytes;

[0018] (2) Production of bovine in vitro fertilized embryos;

[0019] (3) One-step freezing of in vitro embryos, the steps are as follows:

[0020] After culturing bovine in vitro fertilized embryos for 6-8 days, they are transferred to cryogenic solution A and left to stand.

[0021] After standing, the shrunken blastocyst is transferred from cryosol A to cryosol B and allowed to settle naturally.

[0022] The embryos that had sunk to the bottom of cryogenic solution B were transferred into cryogenic solution C and loaded into straws;

[0023] The wheat tubes containing the embryos were cryopreserved using a programmed freezing process. Once the freezing process was complete, the wheat tubes were placed in liquid nitrogen.

[0024] This invention adds an artificial shrinkage of the blastocyst cavity before embryo freezing, building upon conventional freezing methods. By culturing bovine in vitro fertilized embryos for 6-8 days and then placing them in freezing solution A, the blastocyst cavity is shrunk, draining the fluid from the cavity and reducing osmotic damage and ice crystal formation during cryopreservation. Unlike mechanical methods (injection needle aspiration, thin tube blowing) and laser methods (laser perforation), this invention uses a physical method, utilizing the osmotic pressure difference between the inside and outside of the embryo to automatically drain the fluid from the blastocyst cavity, achieving artificial shrinkage. This successfully avoids the drawbacks of mechanical artificial shrinkage (causing significant embryo damage) and laser artificial shrinkage (requiring specialized equipment).

[0025] In some embodiments of the present invention, the bovine in vitro fertilization embryo culture conditions are as follows: the outer granulosa cells of the fertilized oocyte are removed, and the embryo is cultured in a fetal culture medium. The culture environment is 35-40°C, 5-10% carbon dioxide, 5-10% nitrogen, and saturated humidity, by volume percentage; preferably, the culture environment is 38.5°C, 5-7% carbon dioxide, 5-7% nitrogen, and saturated humidity.

[0026] In some embodiments of the present invention, the settling time is 1 to 2 minutes, preferably 1 minute.

[0027] In some embodiments of the present invention, the shrunken blastocyst is transferred from cryosol A to the surface of a droplet of cryosol B with a volume of 400-600 μL, and allowed to settle naturally.

[0028] In some embodiments of the present invention, the embryos are loaded into wheat tubes in the following order: cryosol C, bubbles, cryosol C, bubbles, cryosol C containing embryos, bubbles, cryosol C, bubbles, and cryosol C, with each liquid column being of the same length.

[0029] Preferably, after the straw is filled, it is sealed and placed at an angle with the cotton plug facing down for 8 to 12 minutes to allow it to balance.

[0030] In some embodiments of the present invention, a one-step in vitro embryo freezing method for bovine embryos includes the following steps:

[0031] (1) In vitro maturation of bovine oocytes

[0032] Oocytes were collected from bovine ovaries or the ovaries of live cows. The oocyte collection fluid was recovered. Oocytes containing three or more layers of granulosa cells and with uniform cytoplasm were selected for maturation culture. The culture environment was 38.5℃, 5-7% carbon dioxide, 5-7% nitrogen, and saturated humidity. The culture time was 20-24 hours.

[0033] (2) Production of bovine in vitro fertilized embryos

[0034] After maturation, oocytes are transferred into fertilization fluid. Sperm are treated with a sperm processing solution to adjust sperm density. The treated semen is then added back into the fertilization fluid to achieve a final sperm density of 1–5 × 10⁻⁶. 6 Cells / mL were incubated at 38.5℃ in a saturated humidity environment with 5-7% carbon dioxide for 16-20 hours.

[0035] In some embodiments of the present invention, when collecting oocytes from bovine ovaries, the bovine ovaries may come from slaughterhouses. After being rinsed clean with 0.9% physiological saline, the bovine ovaries obtained from slaughterhouses are transported to the laboratory at a transport temperature of 30-35°C. The time from the collection of the first ovary to its arrival at the laboratory does not exceed 6 hours.

[0036] After the ovaries are transported to the laboratory, they are first rinsed with 0.9% saline solution at 30-35℃. After rinsing, follicles are extracted from the ovaries using a sterile syringe with a 12-gauge needle.

[0037] In some embodiments of the present invention, embryos frozen using the one-step in vitro embryo freezing method described in the invention can be further cultured or directly transferred after thawing.

[0038] The method for thawing the frozen embryos may be to remove the frozen embryos from liquid nitrogen, expose them to air for 10 seconds, immerse them in warm water at 30°C, and leave them for 30 seconds.

[0039] The beneficial effects of this invention are as follows:

[0040] This invention adds an artificial shrinkage of the blastocyst cavity before embryo freezing, building upon conventional freezing methods. This shrinkage process drains the fluid from the blastocyst cavity, reducing osmotic damage and ice crystal formation during cryopreservation. Unlike mechanical methods (injection needle aspiration, thin tube blowing) and laser methods (laser perforation), this invention uses a physical method, leveraging the osmotic pressure difference between the inside and outside of the embryo to automatically drain the fluid from the blastocyst cavity, achieving artificial shrinkage. This successfully avoids the drawbacks of mechanical artificial shrinkage (causing significant embryo damage) and laser artificial shrinkage (requiring specialized equipment).

[0041] The cryosol involved in this invention is used to freeze bovine embryos in vitro. After thawing, the embryos can be directly transferred, and a high implantation success rate can be achieved. While taking into account the fact that conventional freezing methods do not require high operator skills and can be directly transferred after thawing, this invention overcomes the shortcomings of conventional freezing methods, such as low embryo survival and hatching rates after thawing and low implantation pregnancy rates, achieving a combination of ease of use and high implantation success rate. Attached Figure Description

[0042] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0043] Figure 1 This is a flowchart illustrating the use of the bovine in vitro embryo one-step cryopreservation solution of the present invention.

[0044] Figure 2 This is an image of the embryo before freezing in Example 1 of the present invention;

[0045] Figure 3 This is an image of an embryo after treatment with cryosol 1 in Example 1 of the present invention. Detailed Implementation

[0046] To enable those skilled in the art to better understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below with reference to specific embodiments.

[0047] Glossary

[0048] One-step freezing: One-step freezing, also known as programmed freezing, refers to the process of thawing in which frozen embryos can be directly transferred after a single thawing step.

[0049] The reagents used in the following examples are all of cell culture grade or higher purity, such as products from Sigma-Aldrich.

[0050] Example 1

[0051] 1. One-step preparation of refrigerant

[0052] Cryosol A: An aqueous solution containing 8 g / L sodium chloride, 0.2 g / L potassium chloride, 0.1 g / L magnesium chloride, 2.9 g / L disodium hydrogen phosphate, 0.2 g / L potassium dihydrogen phosphate, 1 g / L glucose, 0.036 g / L sodium pyruvate, 0.1 g / L calcium chloride, 0.1 g / L bovine serum albumin, and 1 M sucrose.

[0053] Cryosol B: An aqueous solution containing 8 g / L sodium chloride, 0.2 g / L potassium chloride, 0.1 g / L magnesium chloride, 2.9 g / L disodium hydrogen phosphate, 0.2 g / L potassium dihydrogen phosphate, 1 g / L glucose, 0.036 g / L sodium pyruvate, 0.1 g / L calcium chloride, 4 g / L bovine serum albumin, 0.1 M sucrose, and 0.75 M ethylene glycol.

[0054] Cryosol C: An aqueous solution containing 8 g / L sodium chloride, 0.2 g / L potassium chloride, 0.1 g / L magnesium chloride, 2.9 g / L disodium hydrogen phosphate, 0.2 g / L potassium dihydrogen phosphate, 1 g / L glucose, 0.036 g / L sodium pyruvate, 0.1 g / L calcium chloride, 4 g / L bovine serum albumin, 0.1 M sucrose, and 1.5 M ethylene glycol.

[0055] All reagents used in this invention are of cell culture grade or higher purity, such as products from Sigma-Aldrich.

[0056] 2. Live egg retrieval - in vitro embryo production

[0057] (1) Live oocyte retrieval procedure:

[0058] Instrument preparation: Prepare a live oocyte retrieval instrument, a negative pressure pump, and a metal bath; connect the oocyte retrieval needle, oocyte retrieval tube, and negative pressure pump.

[0059] Donor cow preparation: The donor cow is restrained using a restraint frame, feces are cleared from the rectum, and the follicles and corpus luteum in the donor cow's ovaries are examined. After confirming that the cow is suitable for oocyte collection, it is anesthetized with lidocaine hydrochloride at the tailbone area via epidural anesthesia.

[0060] Live oocyte retrieval: Using a live oocyte retrieval instrument and a negative pressure pump, oocytes are retrieved from the cow's ovary under ultrasound guidance, and the oocyte collection fluid is also collected.

[0061] (2) In vitro embryo production

[0062] Oocyte maturation: Oocytes are collected from the oocyte collection fluid, and those containing three or more layers of granulosa cells and with uniform cytoplasm are selected for maturation culture. The culture environment is 38.5℃, 5-7% carbon dioxide, and saturated humidity, and the culture time is 20-24 hours.

[0063] The maturation solution used for oocyte maturation is the maturation solution disclosed in patent application number 2023111645589, which is based on commercially available TCM199 and contains 0.01 IU / mL follicle-stimulating hormone, 0.01 IU / mL luteinizing hormone, 1 μg / mL estrogen, and 0.1 g / L bovine serum albumin.

[0064] In vitro fertilization (IVF): ① After maturation, oocytes are transferred from the maturation solution to the fertilization solution. ② Sperm processing solution is added to the thawed semen (3 mL per sample), centrifuged at 400g for 5 minutes, and the supernatant is discarded. An appropriate amount of the processed semen is added to the fertilization solution containing oocytes to achieve a final sperm density of 1–5 × 10⁻⁶. 6 Sperm / mL. Sperm and oocytes were cultured at 38.5℃ in a saturated humidity environment with 5-7% (volume percentage) carbon dioxide for 16-20 hours.

[0065] The sperm treatment solution mentioned above is the sperm treatment solution disclosed in patent application number 2023111645589, which is an aqueous solution containing 112mM sodium chloride, 4.02mM potassium chloride, 2.25mM calcium chloride dihydrate, 0.83mM sodium dihydrogen phosphate monohydrate, 0.52mM magnesium chloride hexahydrate, 37mM sodium bicarbonate, 1.25mM sodium pyruvate, 10μg / mL sodium heparin, 10mM caffeine, and 4mg / mL bovine serum albumin.

[0066] The aforementioned fertilization fluid is the fertilization fluid disclosed in patent application number 2023111645589, which is an aqueous solution containing 112mM sodium chloride, 4.02mM potassium chloride, 2.25mM calcium chloride dihydrate, 0.83mM sodium dihydrogen phosphate monohydrate, 0.52mM magnesium chloride hexahydrate, 37mM sodium bicarbonate, 1.25mM sodium pyruvate, 10μg / mL heparin sodium, and 4mg / mL bovine serum albumin.

[0067] In vitro embryo culture: After fertilization, the outer granulosa cells of the oocytes are removed and the cells are placed in an embryo culture medium for culture. The culture environment is 38.5℃, 5-7% carbon dioxide, 5-7% nitrogen, and saturated humidity.

[0068] The aforementioned embryo culture medium is the embryo culture medium disclosed in patent application number 2023111645589, which is an aqueous solution containing 109.5mM sodium chloride, 3.1mM potassium chloride, 26.2mM sodium bicarbonate, 0.8mM magnesium chloride hexahydrate, 1.19mM potassium dihydrogen phosphate, 0.4mM sodium pyruvate, 1.5mM glucose, 5mM calcium galactobionate, 6mg / mL bovine serum albumin, 20μL / mL essential amino acids, 10μL / mL non-essential amino acids, and 0.15mg / mL glutamine.

[0069] 3. One-step freezing of in vitro embryos

[0070] Artificial blastocyst collapse: The blastocysts cultured to day 6-8 are transferred from the embryo culture medium into cryogenic solution A and left to stand for 1 minute to remove the water from the blastocyst cavity, causing the blastocyst cavity to collapse.

[0071] Pre-equilibration: (1) Use cryosol B to make microdroplets with a volume of 500 μL; (2) Transfer the shrunken blastocyst from cryosol A to the surface of the droplets made of cryosol B and let it settle naturally.

[0072] Equilibrium: (1) Transfer the embryos that have sunk to the bottom of cryosol B into cryosol C; (2) In the order of cryosol C, air bubbles, cryosol C, air bubbles, cryosol C containing embryos, air bubbles, cryosol C, air bubbles, cryosol C, the embryos are placed into 0.25 mL straws, with each segment of the liquid column being the same length; (3) After sealing the straws with straw stoppers, place them at an angle with the cotton plug end (one end of the straw is the sealed end, and the other end is the cotton plug end) downwards for 10 minutes to equilibrate.

[0073] Freezing: (1) After equilibration, the wheat tube containing the embryo is placed in a temperature-controlled system and the freezing program is started; (2) After the freezing program is completed, the wheat tube is placed in liquid nitrogen. The freezing program is as follows: the packaged wheat tube is placed in a temperature-controlled system pre-cooled to -6℃, equilibrated at -6℃ for 5 minutes, then ice is applied, and after ice application, it is left to stand for 5 minutes. Then, the temperature is lowered to -32℃ at a rate of 0.5℃ / min. After the temperature drops to -32℃, the wheat tube is removed from the temperature-controlled system and placed in liquid nitrogen.

[0074] 4. Embryo thawing and culture

[0075] Embryo thawing: Remove the frozen embryos from liquid nitrogen, expose them to air for 10 seconds, and then immerse them in warm water at 30°C for 30 seconds.

[0076] Embryo culture: After thawing, the embryos were transferred from the straw to the embryo culture medium. After washing three times with the embryo culture medium, they were placed in a new embryo culture medium and cultured for 48 hours in an environment of 38.5℃, 5-7% carbon dioxide, 5-7% nitrogen, and saturated humidity. The embryo survival rate after 24 hours and the embryo hatching rate after 48 hours were calculated and recorded. See Table 1 for details.

[0077] The aforementioned embryo culture medium is also the embryo culture medium disclosed in patent application number 2023111645589, which is an aqueous solution containing 109.5mM sodium chloride, 3.1mM potassium chloride, 26.2mM sodium bicarbonate, 0.8mM magnesium chloride hexahydrate, 1.19mM potassium dihydrogen phosphate, 0.4mM sodium pyruvate, 1.5mM glucose, 5mM calcium galactobionate, 6mg / mL bovine serum albumin, 20μL / mL essential amino acids, 10μL / mL non-essential amino acids, and 0.15mg / mL glutamine.

[0078] Example 2

[0079] The in vitro embryos obtained from live oocyte retrieval were frozen according to the method in Example 1. The difference was that the frozen embryos were not further cultured after thawing, but were directly transferred, including the following steps:

[0080] (1) Live oocyte retrieval and in vitro embryo production were performed according to the method in Example 1;

[0081] (2) The embryos were frozen and thawed according to the method in Example 1;

[0082] (3) The thawed embryos were directly transferred into the recipient cow on the 7th day after estrus;

[0083] (4) Thirty days after embryo transfer, the recipient cow was examined by ultrasound to determine the embryo transfer conception rate. See Table 2 for details.

[0084] Example 3

[0085] 1. The one-step method for preparing the cryogenic fluid is as described in Example 1.

[0086] 2. In vitro embryo production: The oocytes used are derived from ovaries in slaughterhouses, not obtained through live oocyte retrieval. The specific method of obtaining them is as follows:

[0087] (1) Oocyte collection

[0088] After rinsing the bovine ovaries obtained from the slaughterhouse with 0.9% saline solution, they are transported to the laboratory at a temperature of 30-35℃. The time from the collection of the first ovary to its arrival at the laboratory should not exceed 6 hours.

[0089] After the ovaries are transported to the laboratory, they are first rinsed with 0.9% saline solution at 30-35℃. After rinsing, follicles are extracted from the ovaries using a sterile syringe with a 12-gauge needle to obtain oocyte extraction fluid.

[0090] (2) In vitro embryo production

[0091] Oocyte maturation: Oocytes were collected from the oocyte extraction fluid, and those containing three or more layers of granulosa cells and with uniform cytoplasm were selected for maturation culture. The culture environment was 38.5℃, 5-7% carbon dioxide, and saturated humidity, for 20-24 hours. The maturation solution was the same as that used in Example 1.

[0092] In vitro fertilization (IVF): ① After maturation, oocytes are transferred from the maturation fluid to the fertilization fluid. ② Sperm are treated with a sperm treatment solution to adjust sperm density. An appropriate amount of the treated semen is then added to the fertilization fluid containing oocytes, resulting in a final sperm density of 1–5 × 10⁻⁶. 6 Sperm / mL. Sperm and oocytes were cultured at 38.5°C and 5-7% CO2 in a saturated humidity environment for 16-20 hours. The fertilization solution and sperm treatment solution were the same as those in Example 1.

[0093] In vitro embryo culture: After fertilization, the outer granulosa cells of the oocytes were removed, and the cells were placed in embryo culture medium for culture. The culture environment was 38.5°C, 5-7% carbon dioxide, 5-7% nitrogen, and saturated humidity. The embryo culture medium was the same as that used in Example 1.

[0094] 3. In vitro embryo freezing in one step: Refer to Example 1

[0095] 4. Embryo thawing and culture: Refer to Example 1, and the specific results are shown in Table 1.

[0096] To verify the effectiveness of the present invention, a comparative analysis was conducted, as follows:

[0097] Comparative Example 1

[0098] In vitro embryos were produced using oocytes obtained from live oocyte retrieval, following the method described in Example 1. Commercially available embryo freezing solution was used for embryo freezing and thawing. The entire process of embryo freezing and thawing was consistent with that in Example 1. The embryo survival rate after 24 hours of embryo culture and the embryo hatching rate after 48 hours were statistically analyzed, as detailed in Table 1.

[0099] Comparative Example 2

[0100] Following the method of Example 2, oocytes obtained from live oocyte retrieval were used to produce in vitro embryos. Commercially available embryo freezing solution was used for embryo freezing, thawing, and transfer. The entire process of embryo freezing, thawing, and transfer was consistent with Example 2. The embryo transfer conception rate was statistically analyzed, as shown in Table 2.

[0101] Comparative Example 3

[0102] Following the method of Example 3, oocytes obtained from ovaries in slaughterhouses were used to produce in vitro embryos. Commercially available embryo freezing solution was used for embryo freezing and thawing. The embryo survival rate after 24 hours of thawing and the embryo hatching rate after 48 hours were statistically analyzed, as shown in Table 1.

[0103] Table 1

[0104]

[0105] Table 2

[0106]

[0107] Explanation of indicators in the table:

[0108] Using the one-step in vitro embryo freezing solution of the present invention, which improves the freezing effect, in vitro embryos obtained by in vitro fertilization of oocytes collected from live eggs and ovaries in slaughterhouses are frozen. Compared with embryos frozen using commercially available embryo freezing solution, the resulting embryos have significantly improved 24-hour survival rate and 48-hour hatching rate after thawing, and the embryo transfer pregnancy rate is also improved.

[0109] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A one-step cryopreservation solution for bovine in vitro embryos, characterized in that, Including coolant A, coolant B, and coolant C; The cryogenic solution A is an aqueous solution containing 5-10 g / L sodium chloride, 0.1-0.5 g / L potassium chloride, 0.1-0.3 g / L magnesium chloride, 2.5-3.0 g / L disodium hydrogen phosphate, 0.1-0.5 g / L potassium dihydrogen phosphate, 0.8-1.2 g / L glucose, 0.03-0.04 g / L sodium pyruvate, 0.1-0.3 g / L calcium chloride, 0.1-0.3 g / L bovine serum albumin, and 1-1.5 M sucrose. The cryogenic solution B is an aqueous solution containing 5-10 g / L sodium chloride, 0.1-0.5 g / L potassium chloride, 0.1-0.3 g / L magnesium chloride, 2.5-3.0 g / L disodium hydrogen phosphate, 0.1-0.5 g / L potassium dihydrogen phosphate, 0.8-1.2 g / L glucose, 0.03-0.04 g / L sodium pyruvate, 0.1-0.3 g / L calcium chloride, 3-5 g / L bovine serum albumin, 0.1-0.3 M sucrose, and 0.5-0.75 M anhydrous ethanol. The cryogenic solution C is an aqueous solution containing 5-10 g / L sodium chloride, 0.1-0.5 g / L potassium chloride, 0.1-0.3 g / L magnesium chloride, 2.5-3.0 g / L disodium hydrogen phosphate, 0.1-0.5 g / L potassium dihydrogen phosphate, 0.8-1.2 g / L glucose, 0.03-0.04 g / L sodium pyruvate, 0.1-0.3 g / L calcium chloride, 3-5 g / L bovine serum albumin, 0.1-0.3 M sucrose, and 1.3-1.7 M anhydrous ethanol.

2. The bovine in vitro embryo one-step cryopreservation solution as described in claim 1, characterized in that, The cryogenic solution A is an aqueous solution containing 8 g / L sodium chloride, 0.2 g / L potassium chloride, 0.1 g / L magnesium chloride, 2.9 g / L disodium hydrogen phosphate, 0.2 g / L potassium dihydrogen phosphate, 1 g / L glucose, 0.036 g / L sodium pyruvate, 0.1 g / L calcium chloride, 0.1 g / L bovine serum albumin, and 1 M sucrose.

3. The bovine in vitro embryo one-step cryopreservation solution as described in claim 1, characterized in that, The cryogenic solution B is an aqueous solution containing 8 g / L sodium chloride, 0.2 g / L potassium chloride, 0.1 g / L magnesium chloride, 2.9 g / L disodium hydrogen phosphate, 0.2 g / L potassium dihydrogen phosphate, 1 g / L glucose, 0.036 g / L sodium pyruvate, 0.1 g / L calcium chloride, 4 g / L bovine serum albumin, 0.1 M sucrose, and 0.75 M ethylene glycol.

4. The bovine in vitro embryo one-step cryopreservation solution as described in claim 1, characterized in that, The cryogenic solution C is an aqueous solution containing 8 g / L sodium chloride, 0.2 g / L potassium chloride, 0.1 g / L magnesium chloride, 2.9 g / L disodium hydrogen phosphate, 0.2 g / L potassium dihydrogen phosphate, 1 g / L glucose, 0.036 g / L sodium pyruvate, 0.1 g / L calcium chloride, 4 g / L bovine serum albumin, 0.1 M sucrose, and 1.5 M ethylene glycol.

5. A one-step in vitro freezing method for bovine embryos, characterized in that, The bovine in vitro embryo one-step cryopreservation solution according to any one of claims 1-4 includes the following steps: (1) In vitro maturation of bovine oocytes; (2) Production of bovine in vitro fertilized embryos; (3) One-step freezing of in vitro embryos, the steps are as follows: After culturing bovine in vitro fertilized embryos for 6-8 days, they are transferred to cryogenic solution A and left to stand. After standing, the shrunken blastocyst is transferred from cryosol A to cryosol B and allowed to settle naturally. The embryos that had sunk to the bottom of cryogenic solution B were transferred into cryogenic solution C and loaded into straws; The wheat tubes containing the embryos were cryopreserved using a programmed freezing process. Once the freezing process was complete, the wheat tubes were placed in liquid nitrogen.

6. The one-step in vitro freezing method for bovine embryos as described in claim 5, characterized in that, The conditions for culturing bovine in vitro fertilized embryos are as follows: after fertilization, the outer granulosa cells of the oocytes are removed and the embryos are placed in a fetal culture medium for culturing. The culture environment is 35-40℃, 5-10% carbon dioxide, 5-10% nitrogen, saturated humidity, and volume percentage.

7. The one-step in vitro freezing method for bovine embryos as described in claim 6, characterized in that, The culture environment is 38.5℃, 5-7% carbon dioxide, 5-7% nitrogen, and saturated humidity, by volume percentage.

8. The one-step in vitro freezing method for bovine embryos as described in claim 5, characterized in that, The settling time is 1-2 minutes.

9. The one-step in vitro freezing method for bovine embryos as described in claim 8, characterized in that, The settling time is 1 minute.

10. The one-step in vitro freezing method for bovine embryos as described in claim 5, characterized in that, The shrunken blastocyst was transferred from cryosol A to the surface of a 400-600 μL droplet made of cryosol B, and allowed to settle naturally.

11. The one-step in vitro freezing method for bovine embryos as described in claim 5, characterized in that, The embryos are loaded into straws in the following order: cryosol C, air bubbles, cryosol C, air bubbles, cryosol C containing embryos, air bubbles, cryosol C, air bubbles, and cryosol C. Each segment of the liquid column is the same length.

12. The one-step in vitro freezing method for bovine embryos as described in claim 11, characterized in that, After the straw is filled, seal it and place it at an angle with the cotton plug facing down for 8-12 minutes to allow it to balance.

13. The one-step in vitro freezing method for bovine embryos as described in claim 5, characterized in that, Includes the following steps: (1) In vitro maturation of bovine oocytes Oocytes were collected from bovine ovaries or the ovaries of live cows. The oocyte collection fluid was recovered. Oocytes containing three or more layers of granulosa cells and with uniform cytoplasm were selected for maturation culture. The culture environment was 38.5℃, 5-7% carbon dioxide, 5-7% nitrogen, and saturated humidity. The culture time was 20-24 h. (2) Production of bovine in vitro fertilized embryos After maturation, oocytes are transferred into fertilization fluid. Sperm are treated with a sperm processing solution to adjust sperm density. The treated semen is then added back into the fertilization fluid to achieve a final sperm density of 1–5 × 10⁻⁶. 6 Cells / mL were incubated at 38.5℃ in a saturated humidity environment with 5-7% carbon dioxide for 16-20 h.

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