Additive for freezing solution in one-step bovine in vitro embryo process and one-step bovine in vitro embryo freezing method

Abstract

The present invention discloses an additive for freezing liquid in a one-step method for bovine in vitro embryos and a one-step freezing method for bovine in vitro embryos, and belongs to the technical field of animal embryo cryopreservation and embryo transplantation. The additive of the present invention is an aqueous solution comprising sodium chloride, potassium chloride, magnesium chloride, disodium hydrogen phosphate, potassium dihydrogen phosphate, glucose, sodium pyruvate, calcium chloride, bovine serum albumin, sucrose, ethylene glycol and hyaluronic acid; the concentration of sucrose is 4.5 to 5.0 M. Based on the one-step freezing method for bovine in vitro embryos, the present invention increases the sucrose concentration in the conventional one-step freezing liquid and additionally adds hyaluronic acid, thereby increasing the osmotic pressure of the freezing liquid, facilitating the discharge of water inside the embryo, reducing the formation of ice crystals inside the embryo during the freezing process, reducing the damage to the embryo during the freezing process, and improving the survival rate, hatching rate and transplantation pregnancy rate of bovine in vitro embryos after freezing and thawing.

Description

Technical Field

[0001] The invention belongs to the technical field of animal embryo cryopreservation and embryo transplantation, and particularly relates to an additive for freezing liquid in a one-step bovine in vitro embryo process and a one-step bovine in vitro embryo freezing method. Background Art

[0002] The information disclosed in this background technology section is only intended to enhance understanding of the overall background of the invention and should not necessarily be regarded as an admission or any form of suggestion that the information constitutes the prior art already known to those skilled in the art.

[0003] Embryo cryopreservation allows for the use of embryos without time or space constraints, and prevents the loss of animal strains and rare mutants due to disease and other reasons. However, cryopreserved embryos account for less than 50% of bovine embryos transplanted annually, with the majority still transferred fresh. This is due to the sensitivity of in vitro embryos to cryopreservation, resulting in significantly lower survival and pregnancy rates for frozen in vitro embryos than for in vivo embryos. Currently, the use of cryoprotectants, such as dimethyl sulfoxide, glycerol, propylene glycol, methanol, polyethanol, polyvinyl pyrrolidone, glucose, lactose, maltose, and xylose, is a common method for improving embryo survival and pregnancy rates after freezing. However, the effectiveness of cryoprotectants is currently suboptimal, with low 24-hour embryo survival rates, 48-hour hatching rates, and post-transplant pregnancy rates after thawing. Further research is needed to effectively improve the efficiency of bovine in vitro embryo freezing, as well as the survival and hatching rates of frozen and thawed embryos and the post-transplant pregnancy rates. Summary of the Invention

[0004] In order to address the deficiencies of the prior art, the present invention aims to provide an additive for freezing solution in a one-step bovine in vitro embryo process and a one-step bovine in vitro embryo freezing method. The additive for freezing solution in a one-step bovine in vitro embryo process provided by the present invention can be added to embryo freezing solution and obtain an ideal transplantation conception rate.

[0005] In order to achieve the above object, the technical solution of the present invention is:

[0006] The first aspect of the present invention provides an additive for freezing solution in a one-step bovine in vitro embryo process, wherein the additive is an aqueous solution comprising sodium chloride, potassium chloride, magnesium chloride, disodium hydrogen phosphate, potassium dihydrogen phosphate, glucose, sodium pyruvate, calcium chloride, bovine serum albumin, sucrose, ethylene glycol, and hyaluronic acid;

[0007] The concentration of the sucrose is 4.5-5.0M.

[0008] The present invention develops an additive for freezing solution in the one-step freezing method for bovine in vitro embryos based on the one-step freezing method for bovine in vitro embryos. By increasing the sucrose concentration in the conventional one-step freezing solution and additionally adding hyaluronic acid, the osmotic pressure of the freezing solution is increased, which is beneficial to the discharge of water inside the embryo, reduces the formation of ice crystals inside the embryo during the freezing process, reduces the damage to the embryo during the freezing process, and improves the survival rate and hatching rate of bovine in vitro embryos after freezing and thawing, as well as the transplantation pregnancy rate of the thawed embryos.

[0009] Specifically, hyaluronic acid is a glycosaminoglycan composed of disaccharide units of D-glucuronic acid and N-acetylglucosamine. It is naturally present in the extracellular matrix and intercellular matrix of mammalian tissues and is one of the most abundant macromolecules in the reproductive tract of female animals. The addition of hyaluronic acid to the one-step freezing solution of bovine in vitro embryos, combined with the presence of high concentrations of sucrose, can promote the discharge of excess water in the embryo during the freezing process, reduce the formation of ice crystals, and improve the survival rate and hatching rate of embryos after freezing and thawing. After thawing, the one-step frozen bovine in vitro embryos are directly transplanted, and the freezing solution serves as the transplant solution. The hyaluronic acid in the transplant solution can indirectly promote angiogenesis and promote cell-to-cell and cell-to-matrix adhesion, thereby facilitating embryo implantation. At the same time, the high viscosity of hyaluronic acid can prevent the embryo from being expelled from the uterine cavity, thereby improving the transplant pregnancy rate.

[0010] In some embodiments of the present invention, the concentrations of the components in the bovine in vitro embryo one-step freezing solution additive are: 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, 1-5 g / L bovine serum albumin, 4.5-5.0 M sucrose, 1.0-2.0 M ethylene glycol, and 3-8 mg / mL hyaluronic acid.

[0011] In some embodiments of the present invention, the concentrations of the components in the bovine in vitro embryo one-step freezing solution additive are: 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, 4.6 M sucrose, 1.5 M ethylene glycol, and 5 mg / mL hyaluronic acid.

[0012] In some embodiments of the present invention, the purity of the sodium chloride, potassium chloride, magnesium chloride, disodium hydrogen phosphate, potassium dihydrogen phosphate, glucose, sodium pyruvate, calcium chloride, bovine serum albumin, sucrose, ethylene glycol, and hyaluronic acid are all cell culture grade or above, for example, products from Sigma-Aldrich.

[0013] A second aspect of the present invention provides a one-step freezing method for bovine in vitro embryos, comprising the following steps:

[0014] (1) preparing an additive for the freezing solution in the above-mentioned bovine in vitro embryo one-step method;

[0015] (2) mixing the additive with the embryo freezing solution to obtain a freezing solution containing the additive;

[0016] (3) mixing the freezing solution containing the additive with the embryo freezing solution to obtain a balanced solution;

[0017] (4) transferring the embryos to be frozen into the equilibrium droplet, allowing it to stand, and then transferring the embryos to be frozen from the equilibrium droplet into a freezing solution containing an additive;

[0018] (5) Place the embryos to be frozen into straws and control the temperature for freezing.

[0019] In some embodiments of the present invention, the embryo freezing solution is a homemade embryo freezing solution or a commercial embryo freezing solution.

[0020] Preferably, the homemade embryo freezing solution 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, 1-5 g / L bovine serum albumin, 0.1-0.5 M sucrose and 1.0-2.0 M ethylene glycol.

[0021] Further preferably, the homemade embryo freezing solution 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.

[0022] In some embodiments of the present invention, the embryo freezing solution and the one-step freezing solution additive are mixed evenly in a volume ratio of 5-10:1-3 to form a freezing solution containing the additive; the volume ratio is preferably 8:2.

[0023] In some embodiments of the present invention, the embryo preservation solution and the additive-containing freezing solution are mixed uniformly in a volume ratio of 1-2:1-2 to form equilibrium droplets; the volume ratio is preferably 1:1. At a volume ratio of 1:1, the osmotic pressure of the equilibrium solution is between that of the embryo preservation solution and the additive-containing freezing solution. During embryo processing, the embryo is exposed to an environment with steadily increasing osmotic pressure. Specifically, the following equation is used: preservation solution (osmotic pressure equal to that of the embryo) - equilibrium solution (osmotic pressure intermediate between that of the preservation solution and the freezing solution) - freezing solution (highest osmotic pressure).

[0024] In some embodiments of the present invention, the embryos to be frozen are placed in the equilibration droplet and allowed to rest for 1-2 minutes, preferably 1 minute. During this resting period, the equilibration droplet rapidly permeates the embryo with the protective agent and dehydrates the embryo, allowing the embryo to reach equilibrium in the freezing agent.

[0025] In some embodiments of the present invention, the embryos to be frozen are loaded into straws in a five-stage manner according to the order of embryo preservation fluid, air bubbles, embryo preservation fluid, air bubbles, freezing fluid containing embryos and additives, air bubbles, embryo preservation fluid, air bubbles, and embryo preservation fluid, wherein the length of each liquid column is the same, and the embryos are loaded into the straws.

[0026] The embryo preservation solution can be a self-prepared embryo preservation solution or a commercial embryo preservation solution. The self-prepared embryo preservation solution is an aqueous solution containing 8g / L sodium chloride, 0.2g / L potassium chloride, 0.1g / L magnesium chloride, 2.9g / L disodium hydrogen phosphate, 0.2g / L potassium dihydrogen phosphate, 1g / L glucose, 0.036g / L sodium pyruvate, 0.1g / L calcium chloride, and 4g / L bovine serum albumin. The brand of commercial embryo preservation solution is ICPbio, and its commercial name is EMCARE. TM Commercial freezing solution is used in conjunction with commercial holding solution, and homemade embryo freezing solution is used in conjunction with homemade embryo holding solution.

[0027] The present invention has carried out special design to the embryo tube mode, embryo preservation solution and the freezing liquid containing additives containing embryo are loaded into straws in order, due to the special tube mode in the freezing process, the embryo can realize the dilution of high concentration sucrose in the straw inside the straw without changing the straw contents after thawing. Finally, the freezing liquid in the straw after thawing is diluted as embryo transplantation liquid, and the hyaluronic acid wherein contained can serve as a bonding agent and protective agent in the subsequent transplantation process, which helps embryo implantation. Therefore, the bovine in vitro embryo one-step freezing method provided by the present invention is utilized, the bovine in vitro embryo is frozen, the embryo damage in the freezing process can be reduced, the survival rate and the hatching rate of the embryo after thawing are improved, and a more ideal transplantation conception rate can be obtained.

[0028] In some embodiments of the present invention, the temperature-controlled freezing is specifically as follows: the straw containing the embryo to be frozen is frozen at -5°C to -10°C, preferably -6°C, and then cooled to -30°C to -40°C, preferably -35°C, at a cooling rate of 0.5°C / min, and then the straw containing the embryo to be frozen is put into liquid nitrogen.

[0029] The present invention uses conventional freezing methods to cryopreserve in vitro embryos for the following reasons: 1. Conventionally cryopreserved embryos can be directly transplanted after thawing, reducing production requirements and significantly saving the time and cost of embryo transplantation; 2. This method does not require high technical skills from the operator. However, compared with unfrozen embryos, the hatching rate and survival rate of conventionally frozen embryos are significantly lower. The present invention uses additives and a special tube packaging method to freeze bovine in vitro embryos, which can reduce embryo damage during the freezing process, improve the survival rate and hatching rate of thawed embryos, and achieve a relatively ideal transplantation and conception rate.

[0030] In some embodiments of the present invention, bovine in vitro embryos are thawed and transplanted into recipient cows on estrus day 7 within 5 minutes. Thirty days after embryo transplantation, the recipient cows can be tested for pregnancy using ultrasound to calculate the embryo transfer pregnancy rate.

[0031] In some embodiments of the present invention, bovine in vitro embryos can be thawed and then cultured, specifically by transferring the thawed embryos from the straws into embryo culture medium, washing with the embryo culture medium, placing them into new embryo culture medium, and continuing to culture them for 48 hours in an environment of 38.5°C, 5-7% carbon dioxide, 5-7% nitrogen (volume percentage), and saturated humidity.

[0032] In some embodiments of the present invention, thawing involves removing the cryopreserved embryos from liquid nitrogen, exposing them to air for 10 seconds, and then placing them in 30°C warm water for 30 seconds. After 30 seconds, the straw is shaken to evenly mix the liquid columns within the straw. When using the method provided by the present invention to freeze bovine in vitro embryos in one step, the thawed embryos can be directly transplanted, bypassing the steps of thawing and removing the cryoprotectant.

[0033] The beneficial effects of the present invention are:

[0034] The present invention develops an additive for freezing solution in the one-step freezing method for bovine in vitro embryos based on the one-step freezing method for bovine in vitro embryos. By increasing the sucrose concentration in the conventional one-step freezing solution and additionally adding hyaluronic acid, the osmotic pressure of the freezing solution is increased, which is beneficial to the discharge of water inside the embryo, reduces the formation of ice crystals inside the embryo during the freezing process, reduces the damage to the embryo during the freezing process, and improves the survival rate and hatching rate of bovine in vitro embryos after freezing and thawing, as well as the transplantation pregnancy rate of the thawed embryos.

[0035] By the additive of freezing solution in the cattle in vitro embryo one-step method involved in the present invention, add in cattle in vitro embryo one-step freezing solution in proportion, and according to method provided by the present invention, cattle in vitro embryo is carried out one-step freezing, thawing rear embryo can skip the steps such as step-by-step thawing, removing cryoprotectant and realize direct transplantation.In freezing process, due to the use of additive, the freezing solution contains higher concentration of sucrose, and added hyaluronic acid, increased the osmotic pressure of freezing solution, is conducive to the discharge of embryo internal moisture, reduced the embryo internal ice crystal formation in freezing process, reduced the damage of freezing process to embryo.Meanwhile, due to special pipe-loading mode in freezing process, embryo after thawing can, when not changing straw contents, realize the dilution of high concentration sucrose in straw inside.Finally, the freezing solution in the wheat straw after thawing is diluted as embryo transplantation liquid, and the hyaluronic acid wherein contained can serve as bonding agent and protective agent in subsequent transplantation process, helps embryo implantation. Therefore, by using the additive for freezing liquid in a one-step bovine in vitro embryo method and the one-step bovine in vitro embryo freezing method provided by the present invention, bovine in vitro embryos are frozen, which can reduce embryo damage during the freezing process, improve the survival rate and hatching rate of embryos after thawing, and obtain a more ideal transplantation conception rate. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] The accompanying drawings, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0037] Figure 1 This is a flow chart of a one-step freezing method for bovine in vitro embryos according to the present invention;

[0038] Figure 2 A schematic diagram of tube loading during the one-step freezing of bovine in vitro embryos using the present invention;

[0039] Figure 3 Schematic diagram of tube loading during the conventional one-step freezing process of bovine in vitro embryos. DETAILED DESCRIPTION

[0040] In order to enable those skilled in the art to more clearly 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.

[0041] Glossary

[0042] One-step freezing: One-step freezing is for transplantation, which is commonly known as programmed freezing. Embryos frozen in this way can be thawed in one step and then directly transplanted.

[0043] The purity of the reagents used in the following examples is above the cell culture grade, such as products from Sigma-Aldrich.

[0044] Example 1

[0045] A one-step freezing method for bovine in vitro embryos, comprising freezing and transplanting bovine in vitro embryos using a homemade embryo freezing solution containing additives, the specific contents of which are as follows:

[0046] (1) Preparation of one-step freezing fluid additive

[0047] The one-step freezing solution additive 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, 4.6 M sucrose, 1.5 M ethylene glycol, and 5 mg / mL hyaluronic acid.

[0048] Accurately weigh each component and add it into water and mix well to obtain the one-step freezing liquid additive.

[0049] The purity of all reagents used was above cell culture grade.

[0050] (2) Preparation of homemade embryo freezing solution

[0051] Homemade embryo freezing solution 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.

[0052] Accurately weigh each component and add it to water and mix well to obtain the homemade embryo freezing solution.

[0053] (3) Configuration of the refrigerant containing additives

[0054] The homemade embryo freezing solution prepared in step (2) and the one-step freezing solution additive prepared in step (1) are uniformly mixed in a volume ratio of 8:2 to form a freezing solution containing the additive.

[0055] (4) Embryo freezing

[0056] ① Prepare 500 μL equilibrium droplets by mixing the self-prepared embryo preservation solution and the freezing solution containing additives in a 1:1 ratio;

[0057] The self-prepared embryo preservation solution is an aqueous solution containing 8g / L sodium chloride, 0.2g / L potassium chloride, 0.1g / L magnesium chloride, 2.9g / L disodium hydrogen phosphate, 0.2g / L potassium dihydrogen phosphate, 1g / L glucose, 0.036g / L sodium pyruvate, 0.1g / L calcium chloride, and 4g / L bovine serum albumin.

[0058] ② Move the embryos to be frozen into the equilibrium droplet and let it stand for 1 minute;

[0059] ③ Transfer the embryos to be frozen from the equilibrium droplet into the freezing solution containing additives;

[0060] ④ Place the embryos into 0.25 mL straws in the following order: embryo preservation solution, air bubble, embryo preservation solution, air bubble, cryosol containing embryos and additives, air bubble, embryo preservation solution, air bubble, and embryo preservation solution. Each liquid column should be the same length.

[0061] ⑤ Seal the straw with a straw stopper and let it sit at room temperature for 5-10 minutes;

[0062] ⑥ Place the embryo in a programmable temperature controller pre-cooled to -6°C. After planting on ice, cool it down to -35°C at a rate of 0.5°C / min.

[0063] ⑦After the freezing process is completed, put the straw into liquid nitrogen;

[0064] (5) Embryo thawing

[0065] Embryo thawing: Take out the frozen embryos from liquid nitrogen, expose them to air for 10 seconds, and put them into 30℃ warm water for 30 seconds; after 30 seconds, shake the straw to mix the liquid columns in the straw evenly.

[0066] Embryo culture: Thawed embryos were transferred from the straws to embryo culture medium. After washing with the embryo culture medium, the embryos were placed in fresh embryo culture medium and cultured for 48 hours at 38.5°C in an environment with 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. The specific results are shown in Table 1.

[0067] (6) Embryo transfer

[0068] The frozen embryos were thawed in one step. The thawing steps were as follows: the frozen embryos were taken out of liquid nitrogen, exposed to air for 10 seconds, and placed in 30°C warm water for 30 seconds; after 30 seconds, the straws were shaken to mix the liquid columns in the straws evenly.

[0069] Thawed embryos were transferred within 5 minutes into recipient cows on day 7 of estrus (the day of estrus is considered day 0, and day 7 is calculated from this). Thirty days after embryo transfer, recipient cows were tested for pregnancy using ultrasound, and the embryo transfer pregnancy rate was calculated. Specific results are shown in Table 2.

[0070] In order to verify the effect of the present invention, comparative examples are set for comparison, as follows:

[0071] Comparative Example 1

[0072] Bovine in vitro embryos were frozen and transplanted using the same method as in Example 1. However, in Comparative Example 1, only the homemade embryo freezing solution was used to freeze the embryos, without the additives provided by the present invention. The embryo packaging method and the remaining embryo freezing, embryo thawing, and embryo transplant procedures were similar to those in Example 1.

[0073] Comparative Example 2

[0074] Bovine in vitro embryos were frozen and transplanted using the same method as in Example 1. However, in Comparative Example 2, embryos were frozen using only a homemade embryo freezing solution, without the additives provided by the present invention. The embryos were loaded into 0.25 mL straws using a five-stage loading process: embryo freezing solution, air bubbles, embryo freezing solution, air bubbles, freezing solution containing embryos, air bubbles, embryo freezing solution, air bubbles, and embryo freezing solution. Each liquid column had the same length, and the embryos were then loaded into 0.25 mL straws.

[0075] Example 2

[0076] The bovine in vitro embryos were frozen and transplanted in the manner described in Example 1, except that a commercial freezing solution containing additives was used to freeze and transplant the bovine in vitro embryos.

[0077] (1) Preparation of one-step freezing liquid additive: same as in Example 1.

[0078] (2) Preparation of embryo freezing solution containing additives

[0079] The commercial freezing solution and the one-step freezing solution additive prepared in step (1) were mixed evenly in a volume ratio of 8:2 to form an embryo freezing solution containing the additive. The commercial freezing solution brand is ICPbio and its commercial name is EMCARE TM Ethylene Glycol 1.5M

[0080] The purity of the reagents used is above the cell culture grade, such as products from Sigma-Aldrich.

[0081] (3) Embryo freezing: Refer to Example 1, the details are as follows

[0082] ① Prepare 500 μL equilibrium droplets of commercial embryo preservation solution and freezing solution containing additives at a ratio of 1:1; the brand of commercial embryo preservation solution is ICPbio, and its commercial name is EMCARE. TM HoldingSolution.

[0083] ② Move the embryos to be frozen into the equilibrium droplet and let it stand for 1 minute;

[0084] ③ Move the embryos to be frozen from the equilibrium droplet into the freezing solution containing additives;

[0085] ④ Place the embryos into 0.25 mL straws in the following order: embryo preservation solution, air bubble, embryo preservation solution, air bubble, cryosol containing embryos and additives, air bubble, embryo preservation solution, air bubble, and embryo preservation solution. Each liquid column should be the same length.

[0086] ⑤ Seal the straw with a straw stopper and let it sit at room temperature for 5-10 minutes;

[0087] ⑥ Place the embryo in a programmable thermostat pre-cooled to -6°C. After planting on ice, cool it down to -35°C at a rate of 0.5°C / min.

[0088] ⑦After the freezing process is completed, put the straw into liquid nitrogen;

[0089] (4) Embryo thawing is the same as in Example 1

[0090] (5) Embryo transplantation is the same as in Example 1

[0091] In order to verify the effect of the present invention, comparative examples are set for comparison, as follows:

[0092] Comparative Example 3

[0093] Bovine in vitro embryos were frozen and transplanted using the same procedures as in Example 2. However, in Comparative Example 3, only commercial freezing fluid was used to freeze the embryos, without the additives provided by the present invention. The embryo packaging method and the remaining embryo freezing, embryo thawing, and embryo transplant procedures were similar to those in Example 2.

[0094] Comparative Example 4

[0095] Bovine in vitro embryos were frozen and transplanted using the same method as in Example 2. However, in Comparative Example 4, only a commercial freezing solution was used to freeze the embryos, without the additives provided by the present invention. The embryos were loaded into 0.25 mL straws using a five-stage loading process: embryo freezing solution, air bubbles, embryo freezing solution, air bubbles, freezing solution containing embryos, air bubbles, embryo freezing solution, air bubbles, and embryo freezing solution. Each liquid column had the same length, and the embryos were then loaded into 0.25 mL straws.

[0096] In order to verify the effectiveness of the methods described in Examples 1 and 2 and Comparative Examples 1-4, the number of test embryos, the number of embryos that survived within 24 hours, the number of embryos that hatched within 48 hours, the 24-hour survival rate (%), the 48-hour hatching rate (%), and the number of embryos transplanted, the number of pregnancies, and the conception rate after embryo transplantation in each example were tested. The test results are shown in Tables 1 and 2.

[0097] Table 1

[0098]

[0099]

[0100] Table 2

[0101]

[0102] Comparative Example 5-Comparative Example 13

[0103] In order to explore the optimal mixing ratio of embryo freezing solution and one-step freezing solution additive, comparative examples 5 to 13 were set up, which differ from Example 1 in that:

[0104] The homemade embryo freezing solution prepared in step (2) and the one-step freezing solution additive prepared in step (1) were mixed evenly according to the volume ratio in Table 3 or Table 5.

[0105] Table 3

[0106]

[0107]

[0108] Table 4

[0109]

[0110] Table 5

[0111]

[0112]

[0113] Table 6

[0114]

[0115] Description of indicators in the table:

[0116] An additive for a one-step freezing solution for bovine in vitro embryos according to the present invention was added to a homemade embryo freezing solution and a commercial embryo freezing solution, and the bovine in vitro embryos were frozen in a one-step manner according to the embryo tube loading method provided by the present invention. The results were compared with freezing according to the freezing method provided by the present invention when the homemade embryo freezing solution and the commercial embryo freezing solution were not added (i.e., Example 1, Comparative Example 1, Example 2, and Comparative Example 3). The results showed that the embryo survival rate 24 hours after thawing, the hatching rate 48 hours after thawing, and the embryo transplantation conception rate were all improved.

[0117] An additive for a one-step freezing solution for bovine in vitro embryos according to the present invention was added to a homemade embryo freezing solution and a commercial embryo freezing solution, and the bovine in vitro embryos were frozen in a one-step manner according to the method provided by the present invention. Compared with freezing the bovine in vitro embryos according to a conventional embryo tube loading method without adding the homemade embryo freezing solution or the commercial embryo freezing solution of the present invention (i.e., Example 1 was compared with Comparative Example 2, and Example 2 was compared with Comparative Example 4). The results showed that the embryo survival rate 24 hours after thawing, the hatching rate 48 hours after thawing, and the embryo transplantation conception rate were significantly improved.

[0118] Using the self-prepared embryo freezing solution and commercial embryo freezing solution without adding the present invention, bovine in vitro embryos were frozen in one step according to the embryo tube loading method provided by the present invention (i.e., comparing Comparative Example 1 with Comparative Example 2, and Comparative Example 3 with Comparative Example 4). The results showed that the embryo survival rate 24 hours after thawing, the hatching rate 48 hours after thawing, and the embryo transfer conception rate were all improved.

[0119] The foregoing description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Those skilled in the art will readily appreciate that various modifications and variations of the present invention are possible. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention are intended to be within the scope of protection of the present invention.

Claims

1. A one-step freezing method for bovine in vitro embryos, characterized in that: The steps include: (1) Preparation of additives; (2) mixing the additive with the embryo freezing solution to obtain a freezing solution containing the additive; (3) mixing the freezing solution containing the additive with the embryo preservation solution to obtain equilibrium droplets; (4) transferring the embryos to be frozen into the equilibrium droplet, allowing it to stand, and then transferring the embryos to be frozen from the equilibrium droplet into a freezing solution containing an additive; (5) Place the embryos to be frozen into straws and freeze them under controlled temperature; The concentrations of the components in the additive are as follows: 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, 4.6 M sucrose, 1.5 M ethylene glycol, and 5 mg / mL hyaluronic acid; The embryo freezing solution is a homemade embryo freezing solution or a commercial embryo freezing solution; The homemade embryo freezing solution 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; The embryo freezing solution and the one-step freezing solution additive were mixed evenly in a volume ratio of 8:2 to form a freezing solution containing the additive; The embryo preservation solution and the freezing solution containing additives are mixed evenly in a volume ratio of 1:1 to form equilibrium droplets. The osmotic pressure of the equilibrium droplets is between that of the embryo preservation solution and the freezing solution containing additives. When the embryos are processed, the osmotic pressure in the environment in which the embryos are located is steadily increasing. The embryos to be frozen are loaded into the straws in a five-stage manner in the order of embryo preservation solution, air bubbles, embryo preservation solution, air bubbles, freezing solution containing embryos and additives, air bubbles, embryo preservation solution, air bubbles, and embryo preservation solution, wherein the length of each liquid column is the same, and the embryos are loaded into the straws.

2. The one-step freezing method for bovine in vitro embryos according to claim 1, wherein: The purity of the sodium chloride, potassium chloride, magnesium chloride, disodium hydrogen phosphate, potassium dihydrogen phosphate, glucose, sodium pyruvate, calcium chloride, bovine serum albumin, sucrose, ethylene glycol and hyaluronic acid is cell culture grade or above.

3. The one-step freezing method for bovine in vitro embryos according to claim 1, wherein: The temperature-controlled freezing is specifically as follows: the straw containing the embryo to be frozen is placed in ice at -5°C to -10°C, then cooled to -30°C to -40°C at a cooling rate of 0.5°C / min, and then the straw containing the embryo to be frozen is placed in liquid nitrogen.

4. The one-step freezing method for bovine in vitro embryos according to claim 1, wherein: The temperature-controlled freezing is specifically as follows: the straw containing the embryos to be frozen is placed in ice at -6°C, then cooled to -35°C at a rate of 0.5°C / min, and then the straw containing the embryos to be frozen is placed in liquid nitrogen.

5. The one-step freezing method for bovine in vitro embryos according to claim 1, wherein: The bovine in vitro embryos were thawed and transplanted into recipient cows on the 7th day of estrus within 5 minutes.

6. The one-step freezing method for bovine in vitro embryos according to claim 5, wherein: The thawing step is as follows: taking out the cryopreserved embryos from liquid nitrogen, exposing them to air for 10 seconds, and then putting them into 30°C warm water for 30 seconds; after 30 seconds, shaking the straw to evenly mix the liquid columns in the straw.

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