A pretreatment solution for bovine in vitro embryo programmed freezing and a programmed freezing method

Abstract

The application provides a pretreatment liquid for bovine in-vitro embryo programmed freezing and a programmed freezing method, and the pretreatment liquid comprises an embryo culture liquid and 2-10 mu g / L cytochalasin B and 0.2-1.5 mM 4-phenylbutyric acid. The freezing liquid comprises the following components: 0.05-0.25 mg / mL hyaluronic acid, 2-20 uM astaxanthin, 0.01-0.12 mg / mL L-carnitine, 33.8-34.5 mg / mL sucrose, 0.3-2.0 mg / mL glucose, 5-20 %v / v ethylene glycol, 3-15 mg / mL bovine serum albumin and 25-45 mg / L sodium pyruvate PBS solution. The application comprehensively solves the in-vitro embryo freezing damage repair problem from two aspects of before freezing and during freezing, so that the in-vitro embryo is frozen by using the programmed freezing mode, and ideal thawing recovery rate and pregnancy rate are achieved, the industry problem is solved, and the development and application of the in-vitro embryo production technology are promoted.

Description

Technical Field

[0001] This invention belongs to the field of bovine in vitro embryo production technology, and in particular relates to a pretreatment solution for programmed freezing of bovine in vitro embryos and a programmed freezing method. Background Technology

[0002] In vitro embryo production (IVP) in cattle is one of the core cutting-edge technologies in modern bovine reproduction. Compared with traditional in vivo embryo production and transfer techniques, it has many significant advantages, fully utilizing maternal resources, maximizing the reproductive potential of superior female animals, and shortening breeding intervals while reducing breeding costs. However, compared to in vivo embryos, IVP embryos are highly sensitive to low temperatures and have poor cryopreservation tolerance. Therefore, vitrification is the preferred method for preserving the developmental capacity of IVP embryos after thawing, with 95% of embryos surviving after thawing. Traditional programmed freezing (the freezing method commonly used for in vivo embryos) has a recovery rate of only about 60% for IVP embryos. However, vitrification requires specialized technicians and microscopic equipment for embryo thawing and transfer, posing a significant challenge for cross-farm transfers and cattle farm breeders. Vitrification is not as quick and convenient as programmed freezing with direct water bath thawing and transfer, and it also requires less technical expertise from technicians. Therefore, the freezing problem of IVP embryos has been a major factor limiting the further widespread application of IVP technology and a key focus of research for many years. Summary of the Invention

[0003] In view of this, the present invention aims to overcome the deficiencies in the prior art and proposes a pretreatment solution for programmed cryopreservation of bovine in vitro embryos and a programmed cryopreservation method.

[0004] To achieve the above objectives, the technical solution of the present invention is implemented as follows: In a first aspect, the present invention provides a pretreatment solution for programmed cryopreservation of bovine in vitro embryos, the pretreatment solution comprising embryo culture medium and 2-10 μg / L cytochalasin B and 0.2-1.5 mM 4-phenylbutyric acid.

[0005] More preferably, the concentration of cytochalasin B in the pretreatment solution is 5 μg / L, and the concentration of 4-phenylbutyric acid is 1 mM.

[0006] Preferably, the embryo culture medium comprises the following components: 108-110.5 mM NaCl, 2.8-3.8 mM KCl, 26.2-27.5 mM NaHCO3, 0.5-1.5 mM MgCl2•6H2O, 1.10-1.25 mM KH2PO3, 0.2-1.0 mM sodium pyruvate, 1.0-2.0 mM glucose, 3-10 mM calcium galactobionate, 50-150 μM cysteine, 0.5-2.0 mM L-glutamine, 2.85-3.79 g / L 4-hydroxyethylpiperazine ethanesulfonic acid, 10-30 μL / mL essential amino acids, 5-20 μL / mL non-essential amino acids, 2-10 mg / mL bovine serum albumin, and 0.5-2.0% v / v gentamicin.

[0007] More preferably, the embryo culture medium comprises the following components: 109.5 mM NaCl, 3.1 mM KCl, 26.2 mM NaHCO3, 0.8 mM MgCl2•6H2O, 1.19 mM KH2PO3, 0.4 mM sodium pyruvate, 1.5 mM glucose, 5 mM calcium galactobionate, 100 μM cysteine, 1 mM L-glutamine, 3.58 g / L 4-hydroxyethylpiperazine ethanesulfonic acid, 20 μL / mL essential amino acids, 10 μL / mL non-essential amino acids, 6 mg / mL bovine serum albumin, and 1% v / v gentamicin.

[0008] Secondly, the present invention also provides a method for programmed cryopreservation of bovine embryos in vitro, the method comprising the following steps: After placing the bovine in vitro embryos into the pre-treatment solution that has been balanced, they are placed in an incubator and cultured for 15-30 minutes. Then, the bovine in vitro embryos are washed in a cleaning solution and placed in a programmed freezing solution to balance for 5-10 minutes. Finally, they are placed in a wheat tube for programmed freezing. After the programmed freezing is completed, the wheat tubes are removed and stored in liquid nitrogen.

[0009] Preferably, the programmed cryogenic fluid comprises the following components: 0.05-0.20 mg / mL hyaluronic acid, 2-15 μM astaxanthin, 0.01-0.10 mg / mL L-carnitine, 32.5-35.5 mg / mL sucrose, 0.2-1.5 mg / mL glucose, 5-15% v / v ethylene glycol, 3-12 mg / mL bovine serum albumin, and 25-45 mg / L sodium pyruvate in PBS solution.

[0010] Preferably, the programmed cryosol comprises the following components: 0.15 mg / mL hyaluronic acid, 10 μM astaxanthin, 0.05 mg / mL L-carnitine, 34.2 mg / mL sucrose, 1 mg / mL glucose, 10% v / v ethylene glycol, 6 mg / mL bovine serum albumin, and 36 mg / L sodium pyruvate in PBS solution.

[0011] Compared with the prior art, the present invention has the following advantages: The pretreatment solution of this invention contains cytochalasin B, a cytoskeleton stabilizer (capable of protecting the cytoskeleton, enhancing plasma membrane toughness, stabilizing the spindle, and targeting and regulating the microfilament cytoskeleton to resist physical and biochemical damage caused by freezing), and 4-phenylbutyric acid, an endoplasmic reticulum stress inhibitor (the endoplasmic reticulum is an important organelle for maintaining cellular homeostasis, responsible for protein synthesis and folding, lipid metabolism, and intracellular calcium ion regulation. When the function of the endoplasmic reticulum is impaired, it triggers an endoplasmic reticulum stress response, which can disrupt intracellular homeostasis and cause apoptosis). The combination of these two substances, pretreatment with a culture medium containing cytochalasin B and 4-phenylbutyric acid for 0.5 h before in vitro programmed embryo freezing, can stabilize the embryonic cytoskeleton and maintain normal cell spatial structure. On the other hand, it can inhibit endoplasmic reticulum stress caused by freezing, maintain normal intracellular ion distribution, and maintain homeostasis. By maintaining cell morphology from both spatial structure and ion distribution perspectives, it protects cells from freezing damage and significantly improves overall survival and development after cryopreservation.

[0012] The programmed cryoprotectant used in this invention contains additional small amounts of hyaluronic acid (a natural mucopolysaccharide, a major component of the extracellular matrix, which reduces the proportion of free water during freezing, decreases ice crystal nucleation sites, and its high viscosity hinders ice crystal diffusion, preventing mechanical damage to embryonic cell membranes and organelles from large ice crystals; it can also interact with receptors on the cell surface, activating a series of signaling pathways and promoting cell repair), astaxanthin (a natural and potent antioxidant that reduces oxidative stress, lipid peroxidation, and lysosomal activity, while increasing mitochondrial activity and inhibiting cryopreservation-induced apoptosis), and L-carnitine (an amino acid-like substance that promotes the conversion of fat into energy, also has antioxidant properties, improves embryonic mitochondrial function, and effectively repairs cryopreservation damage to embryos). These additions further protect embryonic cells during freezing and enhance the embryo's self-repair and developmental capabilities after thawing. Ethylene glycol is used as a cryoprotectant (which does not require additional removal during thawing; glycerol and other types of cryoprotectants require gradual removal from the cells after thawing, a cumbersome process).

[0013] In summary, this invention addresses the problem of in vitro embryo freezing damage repair from both pre-freezing and freezing processes, enabling programmed freezing of in vitro embryos. This significantly improves post-thawing recovery, achieving a recovery rate of 82% or higher and a pregnancy rate of 48% or higher after implantation. It solves industry pain points and promotes the wider development and application of in vitro embryo production technology. Attached Figure Description

[0014] Figure 1 A schematic diagram of programmed cryopreservation of bovine in vitro embryos.

[0015] Explanation of reference numerals in the attached figures: A: Programmed cryoprotectant; B: Embryo; C: Air; D: Cotton plug. Detailed Implementation

[0016] The embodiments of the present invention are described in detail below. The embodiments described below are exemplary and are only used to explain the present invention, and should not be construed as limiting the present invention.

[0017] In this document, unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains.

[0018] In this document, when values ​​are described as ranges, it should be understood that such disclosure includes disclosure of all possible subranges within that range, as well as the specific numerical values ​​falling within that range, regardless of whether the specific numerical value or specific subrange is explicitly specified.

[0019] In this article, the terms "multiple" or "more than" are used unless otherwise specified, referring to a quantity greater than or equal to 2. For example, "one or more" means one or more types.

[0020] In this document, the terms "preferred" and "more preferred" are used only to describe implementation methods or embodiments with better effects, and should be understood as not constituting a limitation on the scope of protection of this invention.

[0021] In this document, terms such as "further" are used for descriptive purposes to indicate differences in content, but should not be construed as limiting the scope of protection of this invention.

[0022] In this article, the term "and / or" describes an association between objects, indicating that three relationships can exist. For example, A and / or B means: A or B, or A and B.

[0023] In this document, the term "about" means + / - 10% of a specified value, preferably + / - 5%, and more preferably + / - 1%.

[0024] In this article, the terms “include,” “including,” “have,” “contain,” etc., are all open-ended terms, meaning that they include but are not limited to.

[0025] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar to or equivalent to those described herein may be used in the implementation or testing of this invention.

[0026] In a first aspect, the present invention also provides a pretreatment solution for programmed cryopreservation of bovine in vitro embryos. The pretreatment solution comprises embryo culture medium and 2-10 μg / L cytochalasin B and 0.2-1.5 mM 4-phenylbutyric acid.

[0027] In one embodiment of this application, the concentration of cytochalasin B can be 2 μg / L, 3 μg / L, 4 μg / L, 5 μg / L, 6 μg / L, 7 μg / L, 8 μg / L, 9 μg / L, or 10 μg / L.

[0028] In one embodiment of this application, the concentration of 4-phenylbutyric acid can be 0.2 mM, 0.3 mM, 0.4 mM, 0.5 mM, 0.6 mM, 0.7 mM, 0.8 mM, 1.0 mM, 1.2 mM, 1.3 mM, 1.4 mM, or 1.5 mM.

[0029] More preferably, the concentration of cytochalasin B in the pretreatment solution is 5 μg / L.

[0030] More preferably, the concentration of the 4-phenylbutyric acid is 1 mM.

[0031] Preferably, the embryo culture medium comprises the following components: 108-110.5 mM NaCl, 2.8-3.8 mM KCl, 26.6-27.5 mM NaHCO3, 0.5-1.5 mM MgCl2•6H2O, 1.10-1.25 mM KH2PO3, 0.2-1.0 mM sodium pyruvate, 1.0-2.0 mM glucose, 3-10 mM calcium galactobionate, 50-150 μM cysteine, 0.5-2.0 mM L-glutamine, 2.85-3.79 g / L 4-hydroxyethylpiperazine ethanesulfonic acid, 10-30 μL / mL essential amino acids, 5-20 μL / mL non-essential amino acids, 2-10 mg / mL bovine serum albumin, and 0.5-2.0% v / v gentamicin.

[0032] In one embodiment of this application, the concentration of NaCl can be 108.0 mM, 108.5 mM, 109 mM, 109.2 mM, 109.5 mM, 110.0 mM, or 110.5 mM.

[0033] In one embodiment of this application, the concentration of KCl can be 2.8 mM, 3.0 mM, 3.1 mM, 3.4 mM, 3.5 mM, or 3.8 mM.

[0034] In one embodiment of this application, the concentration of NaHCO3 can be 26.2 mM, 26.6 mM, 26.8 mM, 27.0 mM, 27.2 mM, or 27.5 mM.

[0035] In one embodiment of this application, the concentration of MgCl2•6H2O can be 0.5mM, 0.6mM, 0.8mM, 1.0mM, 1.2mM, or 1.5mM.

[0036] In one embodiment of this application, the concentration of KH2PO3 can be 1.10 mM, 1.15 mM, 1.19 mM, 1.20 mM, or 1.25 mM.

[0037] In one embodiment of this application, the concentration of sodium pyruvate can be 0.2 mM, 0.4 mM, 0.5 mM, 0.6 mM, 0.7 mM, 0.8 mM, or 1.0 mM.

[0038] In one embodiment of this application, the concentration of glucose can be 1.0 mM, 1.2 mM, 1.3 mM, 1.4 mM, 1.5 mM, 1.6 mM, 1.8 mM, or 2.0 mM.

[0039] In one embodiment of this application, the concentration of calcium galactobionate can be 3mM, 5mM, 6mM, 7mM, 8mM, or 10mM.

[0040] In one embodiment of this application, the concentration of cysteine ​​can be 50 μM, 60 μM, 80 μM, 100 μM, 120 μM, 140 μM, or 150 μM.

[0041] In one embodiment of this application, the concentration of L-glutamine can be 0.5 mM, 0.6 mM, 0.8 mM, 1.0 mM, 1.2 mM, 1.5 mM, 1.8 mM, or 2.0 mM.

[0042] In one embodiment of this application, the concentration of 4-hydroxyethylpiperazine ethanesulfonic acid can be 2.85 g / L, 3.00 g / L, 3.20 g / L, 3.26 g / L, 3.50 g / L, 3.58 g / L, 3.60 g / L, 3.62 g / L, 3.65 g / L, 3.70 g / L, or 3.79 g / L.

[0043] In one embodiment of this application, the concentration of essential amino acids can be 10 μL / mL, 12 μL / mL, 16 μL / mL, 18 μL / mL, 20 μL / mL, 22 μL / mL, 24 μL / mL, 25 μL / mL, 26 μL / mL, 27 μL / mL, 28 μL / mL, or 30 μL / mL.

[0044] In one embodiment of this application, the concentration of non-essential amino acids can be 5 μL / mL, 6 μL / mL, 8 μL / mL, 10 μL / mL, 12 μL / mL, 14 μL / mL, 15 μL / mL, 16 μL / mL, 17 μL / mL, 18 μL / mL, or 20 μL / mL.

[0045] In one embodiment of this application, the concentration of bovine serum albumin can be 2 mg / mL, 3 mg / mL, 4 mg / mL, 5 mg / mL, 6 mg / mL, 8 mg / mL, or 10 mg / mL.

[0046] In one embodiment of this application, the volume fraction (v / v) of gentamicin can be 0.5%, 0.6%, 0.8%, 1.0%, 1.2%, 1.5%, 1.6%, 1.8%, or 2%.

[0047] More preferably, the pretreatment solution comprises the following components: 109.5 mM NaCl, 3.1 mM KCl, 26.2 mM NaHCO3, 0.8 mM MgCl2•6H2O, 1.19 mM KH2PO3, 0.4 mM sodium pyruvate, 1.5 mM glucose, 5 mM calcium galactobionate, 100 μM cysteine, 1 mM L-glutamine, 3.58 g / L 4-hydroxyethylpiperazine ethanesulfonic acid, 20 μL / mL essential amino acids, 10 μL / mL non-essential amino acids, 6 mg / mL bovine serum albumin, and 1% v / v gentamicin.

[0048] Secondly, the present invention also provides a method for programmed cryopreservation of bovine embryos in vitro, the method comprising the following steps: After placing the bovine in vitro embryos in a balanced pretreatment solution, they were placed in an incubator and cultured for 15-30 minutes. Then, the bovine in vitro embryos were washed in a cleaning solution and placed in the above-mentioned programmed freezing solution for equilibration for 5-10 minutes. Finally, they were placed in wheat tubes for programmed freezing. After the programmed freezing was completed, the wheat tubes were removed and stored in liquid nitrogen.

[0049] Preferably, the programmed cryogenic fluid comprises the following components: 0.05-0.20 mg / mL hyaluronic acid, 2-15 μM astaxanthin, 0.01-0.10 mg / mL L-carnitine, 32.5-35.5 mg / mL sucrose, 0.2-1.5 mg / mL glucose, 5-15% v / v ethylene glycol, 3-12 mg / mL bovine serum albumin, and 25-45 mg / L sodium pyruvate in PBS solution.

[0050] In one embodiment of this application, the concentration of hyaluronic acid can be 0.05 mg / mL, 0.10 mg / mL, 0.15 mg / mL, or 0.20 mg / mL.

[0051] In one embodiment of this application, the concentration of astaxanthin can be 5 μM, 6 μM, 8 μM, 10 μM, 12 μM, 14 μM, or 15 μM.

[0052] In one embodiment of this application, the concentration of L-carnitine may be 0.02 mg / mL, 0.04 mg / mL, 0.05 mg / mL, 0.06 mg / mL, 0.08 mg / mL, or 0.10 mg / mL.

[0053] In one embodiment of this application, the concentration of sucrose can be 32.5 mg / mL, 32.8 mg / mL, 33.0 mg / mL, 33.5 mg / mL, 33.8 mg / mL, 33.9 mg / mL, 34.0 mg / mL, 34.1 mg / mL, 34.2 mg / mL, 34.3 mg / mL, 34.4 mg / mL, 34.5 mg / mL, 34.8 mg / mL, 35.0 mg / mL, or 35.5 mg / mL.

[0054] In one embodiment of this application, the concentration of glucose can be 0.2 mg / mL, 0.3 mg / mL, 0.4 mg / mL, 0.5 mg / mL, 0.6 mg / mL, 0.8 mg / mL, 1.0 mg / mL, 1.2 mg / mL, 1.4 mg / mL, or 1.5 mg / mL.

[0055] In one embodiment of this application, the volume fraction (v / v) of ethylene glycol can be 5%, 6%, 8%, 10%, 12%, or 15%.

[0056] In one embodiment of this application, the concentration of bovine serum albumin can be 3 mg / mL, 5 mg / mL, 6 mg / mL, 8 mg / mL, 10 mg / mL, or 12 mg / mL.

[0057] In one embodiment of this application, the concentration of the sodium pyruvate PBS solution can be 25 mg / L, 26 mg / L, 28 mg / L, 30 mg / L, 32 mg / L, 34 mg / L, 35 mg / L, 36 mg / L, 38 mg / L, 40 mg / L, 42 mg / L, or 45 mg / L.

[0058] Preferably, the programmed cryosol comprises the following components: 0.15 mg / mL hyaluronic acid, 10 μM astaxanthin, 0.05 mg / mL L-carnitine, 34.2 mg / mL sucrose, 1 mg / mL glucose, 10% v / v ethylene glycol, 6 mg / mL bovine serum albumin, and 36 mg / L sodium pyruvate in PBS solution.

[0059] The present invention will be described in detail below with reference to the embodiments.

[0060] To verify the effects of the pretreatment solution and programmed cryosol of the present invention on the cryopreservation and thawing of bovine in vitro embryos, an in vitro fertilization-embryo freezing and transfer experiment was conducted, including the following steps: I. In vitro collection of bovine embryos 1. Bovine oocyte collection and maturation culture Bovine ovaries were taken from the slaughterhouse and transported to the laboratory within 2 hours. After washing the ovaries with physiological saline, follicles with a diameter of 2-8 mm on the surface were extracted using a 10 mL syringe. Cumulus-oocyte complexes (COCs) encapsulating cumulus cells in 3 or more layers were picked out under a stereomicroscope. After washing away excess impurities, the ovaries were placed in maturation medium that had been equilibrated for at least 2 hours and then cultured in a CO2 incubator (38.8℃, 5.5% CO2, saturated humidity) for 22-24 hours.

[0061] 2. In vitro fertilization After washing twice with fertilization fluid, matured cocci cultured in vitro were transferred to fertilization fluid pre-equilibrated for at least 2 hours and placed in a CO2 incubator. Frozen semen was removed from the liquid nitrogen tank and thawed in sterile water at 37°C. The frozen semen tubes were cut open, allowing the semen to flow into 15 mL centrifuge tubes containing the washed semen fluid. The tubes were centrifuged twice (328×g), 5 min each time. The supernatant was discarded, and the precipitate at the bottom of the centrifuge tube was retained. Finally, 300 µL of fertilization fluid was added to resuspend the sperm precipitate. The sperm concentration was calculated using a sperm counter, and the calculated amount of semen was added to the fertilization culture medium to achieve a final fertilization concentration of 1.0–2.0 × 10⁻⁶. 6The cells were collected at a density of 10 cells / mL and then placed in a CO2 incubator (38.8℃, 5.5% CO2, saturated humidity) for 16-18 h.

[0062] 3. Early in vitro embryo culture After incubating the fertilized eggs in an incubator for 16-18 hours, they were washed in embryo culture medium and gently pipetted repeatedly to remove cumulus cells and sperm surrounding the fertilized eggs. They were then cultured in pre-equilibrated embryo culture medium (38.8℃, 5.5% CO2, 6% O2, saturated humidity) for at least 2 hours. No fresh culture medium replacement was required during the culture process. The day of in vitro fertilization was recorded as day 0. Blastocysts were collected on the afternoon of day 6 and on day 7 for subsequent embryo freezing.

[0063] II. Live Collection of Bovine Embryos 1. Bovine oocyte collection and maturation culture Superovulation was performed on Wagyu cattle using the FSH decremental injection method. Under ultrasound guidance, follicles with a diameter of 2-8 mm were punctured from the bovine ovaries. After follicular fluid was collected, cumulus cells encapsulated in 3 or more layers of COCs were picked out under a microscope and placed in 500 µL of maturation solution containing hepes buffer. The mixture was then placed in a 1.5 mL centrifuge tube and placed in an incubator (38.5℃). The tube was transported back to the in vitro embryo production laboratory for culture within 2 hours.

[0064] The methods for in vitro fertilization and early in vitro embryo culture are the same as those for in vitro collection.

[0065] III. Bovine in vitro embryo cryopreservation Pre-conditioning with a pretreatment solution containing cytochalasin B and 4-phenylbutyric acid (components and concentrations of the pretreatment solution in the examples and control examples are shown in Table 1) for 0.5 h in an incubator, followed by heating to a programmed cryoprotectant at 38 °C (components and concentrations of the programmed cryoprotectant in the examples and control examples are shown in Table 2). Simultaneously, a programmed cooling device was prepared and pre-cooled to -6 °C. The collected bovine in vitro embryos were placed in the pre-conditioning solution containing cytochalasin B and 4-phenylbutyric acid, cultured in an incubator for 15-30 min, then cleaned in a washing solution, and finally equilibrated in the programmed cryoprotectant for 5 min. The embryos were then subjected to a 5-stage method (…). Figure 1 Fill 0.25 mL wheat tubes with the filling method, seal the tubes, and place them in a programmed freezing apparatus with the cotton plug end facing down. Start the program and perform programmed freezing. After 5 minutes, pause the program, slightly lift the wheat tubes, and apply ice to the upper part containing the embryo segment. After the ice application is completed, resume the program and continue cooling at a rate of 0.5℃ / min until the temperature reaches -35℃. After the program is completed, remove the wheat tubes and quickly immerse them in liquid nitrogen for preservation.

[0066] Table 1 Table 2 IV. Thawing of Bovine In Vitro Embryos Frozen bovine embryos were removed from the liquid nitrogen tank, air-bathed for 5-10 seconds, and then immersed in 30-33°C warm water for 30-50 seconds. The embryos were then wiped dry with a sterile cloth to remove any remaining moisture from the straw. The sealed end of the straw was cut open, and the open end was placed in the washing solution. The cotton plug end of the straw was then cut off, allowing the liquid and embryo inside the straw to flow into the washing solution. The embryos were located under a stereomicroscope and transferred to a balanced embryo culture medium for washing 2-3 times to remove the cryoprotectant. Finally, they were placed in oil-coated embryo culture medium droplets and cultured in a CO2 incubator (38.8°C, 5.5% CO2, 6% O2, saturated humidity). Embryo thawing status was assessed after 24 hours, and embryo hatching status was assessed after 48 hours.

[0067] V. Bovine in vitro embryo transfer After thawing, the sealed end of the straw is cut open, and the embryo transfer gun is inserted. A hard plastic outer tube and a soft plastic outer tube are then fitted onto the transfer gun. Recipient cows with satisfactory corpus luteum are anesthetized, their rectum is cleared of feces, and their vulva is disinfected and wiped clean. The transfer gun is inserted through the vulva to the appropriate position at the anterior end of the uterine horn. The transfer gun's core is slowly pushed forward to push the embryo to the transfer site, and the transfer gun is then slowly withdrawn. The tip of the transfer gun is observed for any bleeding points, and these are recorded.

[0068] VI. Pregnancy check-up in cattle Thirty-five days after embryo transfer, a pregnancy check is performed on the cow. The bovine ultrasound machine is turned on and adjusted. Wearing disposable long-arm gloves, the cow is positioned in a natural standing position with its head secured in a neck harness. The hand and ultrasound probe are gently inserted into the cow's rectum. During the scan, the probe is slowly moved, using the bladder as a guide to locate the uterus. Once the bladder is located, the probe continues forward to find the cervix and then moves upward to the uterine body. One uterine horn is then located and scanned from side to side. The right uterine horn, right ovary, left uterine horn, and left ovary are scanned sequentially. The images on the ultrasound screen are observed, paying attention to the condition of the uterus, ovaries, and fetus. If a fetus cannot be detected or the image is uncertain, the probe can be slowly moved repeatedly, carefully observing from top to bottom and bottom to top. If the uterine horn is empty, the probe should be moved to examine the other uterine horn. If both sides are confirmed to be empty, the cow is considered non-pregnant. Conversely, if a fetal image is observed, the cow is considered pregnant, and conception status can be recorded.

[0069] Experimental results: In vitro collection of bovine embryos was conducted only to observe thawing and hatching, without transplantation. In live collection of bovine embryos, a portion of the embryos were randomly selected for thawing and recovery, while the majority were directly transplanted to assess the conception rate. Therefore, Examples 1-4 used in vitro collected embryos to screen for the optimal components, which were then compared with Control Examples 1-7. Finally, live collected embryos were used to verify the results of Examples 1, Control Examples 3, 6, and 7, and the conception rate was assessed. The results are shown in Table 3.

[0070] Table 3 As shown in Table 3, when this invention is applied to bovine in vitro embryo production (ex vivo collection), the recovery rate is above 82% and the hatching rate is above 61%. When this invention is applied to bovine in vitro embryo production (live embryo collection), some sample results show that the recovery rate is above 91% and the implantation conception rate is above 48%, both of which are significantly improved compared to the control group.

[0071] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A pretreatment solution for programmed cryopreservation of bovine in vitro embryos, characterized in that: The pretreatment solution comprises the following components: The pretreatment solution includes embryo culture medium and 2-10 μg / L cytochalasin B and 0.2-1.5 mM 4-phenylbutyric acid.

2. The pretreatment solution for programmed cryopreservation of bovine in vitro embryos according to claim 1, characterized in that: The concentration of cytochalasin B in the pretreatment solution is 5 μg / L, and the concentration of 4-phenylbutyric acid is 1 mM.

3. The pretreatment solution for programmed cryopreservation of bovine in vitro embryos according to claim 1, characterized in that: The embryo culture medium comprises the following components: 108-110.5 mM NaCl, 2.8-3.8 mM KCl, 26.2-27.5 mM NaHCO3, 0.5-1.5 mM MgCl2•6H2O, 1.10-1.25 mM KH2PO3, 0.2-1.0 mM sodium pyruvate, 1.0-2.0 mM glucose, 3-10 mM calcium galactobionate, 50-150 μM cysteine, 0.5-2.0 mM L-glutamine, 2.85-3.79 g / L 4-hydroxyethylpiperazine ethanesulfonic acid, 10-30 μL / mL essential amino acids, 5-20 μL / mL non-essential amino acids, 2-10 mg / mL bovine serum albumin, and 0.5-2.0% v / v gentamicin.

4. The pretreatment solution for programmed cryopreservation of bovine in vitro embryos according to claim 1, characterized in that: The embryo culture medium comprises the following components: 109.5 mM NaCl, 3.1 mM KCl, 26.2 mM NaHCO3, 0.8 mM MgCl2•6H2O, 1.19 mM KH2PO3, 0.4 mM sodium pyruvate, 1.5 mM glucose, 5 mM calcium galactobionate, 100 μM cysteine, 1 mM L-glutamine, 3.58 g / L 4-hydroxyethylpiperazine ethanesulfonic acid, 20 μL / mL essential amino acids, 10 μL / mL non-essential amino acids, 6 mg / mL bovine serum albumin, and 1% v / v gentamicin.

5. A method for programmed cryopreservation of bovine embryos in vitro, characterized in that: The method includes the following steps: After placing the bovine in vitro embryos into the pretreatment solution described in any one of claims 1-4, which has been balanced, the embryos are placed in an incubator and cultured for 15-30 minutes. Then, the bovine in vitro embryos are washed in a washing solution, and the washed bovine in vitro embryos are placed in a programmed freezing solution for equilibration for 5-10 minutes. Finally, the embryos are placed in a wheat tube for programmed freezing. After the programmed freezing is completed, the wheat tubes are removed and stored in liquid nitrogen.

6. The method for programmed cryopreservation of bovine in vitro embryos according to claim 5, characterized in that: The programmed cryosol comprises the following components: 0.05-0.20 mg / mL hyaluronic acid, 2-15 μM astaxanthin, 0.01-0.10 mg / mL L-carnitine, 32.5-35.5 mg / mL sucrose, 0.2-1.5 mg / mL glucose, 5-15% v / v ethylene glycol, 3-12 mg / mL bovine serum albumin, and 25-45 mg / L sodium pyruvate in PBS solution.

7. The method for programmed cryopreservation of bovine embryos in vitro according to claim 5, characterized in that: The programmed cryosol comprises the following components: 0.15 mg / mL hyaluronic acid, 10 μM astaxanthin, 0.05 mg / mL L-carnitine, 34.2 mg / mL sucrose, 1 mg / mL glucose, 10% v / v ethylene glycol, 6 mg / mL bovine serum albumin, and 36 mg / L sodium pyruvate in PBS solution.

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