Thawing solution for bovine vitrified in vitro embryos and use method thereof

Abstract

The present invention belongs to the field of embryo biotechnology and embryo transplantation technology, and specifically relates to a thawing solution for bovine vitrified frozen in vitro embryos and a method for using the same. The thawing solution comprises the following components: trehalose 340-515 g / L, hyaluronic acid 0.2-0.3 g / L, sodium chloride 6-10 g / L, potassium chloride 0.1-0.3 g / L, magnesium chloride 0.05-0.15 g / L, disodium hydrogen phosphate 0.2-0.35 g / L, potassium dihydrogen phosphate 0.1-0.3 g / L, glucose 0.5-1.5 g / L, sodium pyruvate 0.03-0.04 g / L, calcium chloride 0.05-0.15 g / L, and bovine serum albumin 45-55 g / L. The present invention can significantly shorten the embryo thawing time without compromising the thawing and transplantation effects, improve the efficiency of embryo thawing, and simplify the operation process, which is conducive to the popularization and large-scale application of embryo transplantation technology.

Description

Technical Field

[0001] The invention belongs to the field of embryo biotechnology and embryo transplantation technology, and particularly relates to a thawing solution for bovine vitrified frozen in vitro embryos and a use method thereof. Background Art

[0002] Bovine embryo transfer is a core biotechnology in modern livestock farming, improving the reproductive efficiency of high-quality breeding cattle, accelerating genetic improvement, controlling disease transmission, and enabling international exchange of genetic resources. Long-term and effective embryo preservation is a key prerequisite for the widespread application and commercialization of embryo transfer technology, enabling the separation of embryo production, transportation, and transfer in time and space. Vitrification has become the preferred method for embryo preservation. Its core principle is to utilize a high-concentration cryoprotectant (CPA) solution and extremely rapid cooling rates to prevent the formation of ice crystals in the intracellular and extracellular solution when the temperature drops below the glass transition temperature. Instead, the solution directly transforms into an amorphous, highly viscous, glass-like solid, minimizing the physical damage to cell membranes, organelles, and cytoskeleton caused by ice crystal formation. The successful thawing of vitrified embryos is a crucial step in the technical process, directly determining the ultimate effectiveness of embryo cryopreservation.

[0003] The current traditional vitrification thawing method, a multi-step dilution method, offers advantages in terms of cell viability, functional recovery, and sample adaptability. However, sucrose is often used as an osmotic pressure regulator and protectant. This regulates the solution osmotic pressure, maintains a stable internal environment during cell thawing, and reduces ice crystal formation. However, direct removal of the sucrose during thawing can lead to a sudden drop in extracellular osmotic pressure, a rapid influx of water, and potentially cell swelling or even rupture. Incomplete sucrose removal can also leave residual sucrose. This residual sucrose may competitively inhibit glucose absorption, affecting glycolysis in early embryos. The interaction of sucrose with cell membrane phospholipids may increase membrane rigidity, impairing cytoskeletal reorganization during subsequent embryonic divisions. Furthermore, the traditional vitrification thawing method is time-consuming, complex, and requires high operator skill. Inexperienced operators can lead to poor control precision in solution concentration, embryo residence time, and operating temperature, which in turn affects thawing efficiency and embryo survival, resulting in a lack of stability and reproducibility in vitrified embryo thawing results. Summary of the Invention

[0004] The purpose of the present invention is to provide a thawing solution for bovine vitrified in vitro embryos and a method for using the same, thereby overcoming the shortcomings of the prior art, enabling one-step thawing of bovine vitrified in vitro embryos and obtaining a higher embryo hatching rate and conception rate.

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

[0006] In a first aspect, the present invention provides a thawing solution for bovine vitrified in vitro embryos, comprising the following components:

[0007] Trehalose 340-515 g / L, hyaluronic acid 0.2-0.3 g / L, sodium chloride 6-10 g / L, potassium chloride 0.1-0.3 g / L, magnesium chloride 0.05-0.15 g / L, disodium hydrogen phosphate 0.2-0.35 g / L, potassium dihydrogen phosphate 0.1-0.3 g / L, glucose 0.5-1.5 g / L, sodium pyruvate 0.03-0.04 g / L, calcium chloride 0.05-0.15 g / L, bovine serum albumin 45-55 g / L.

[0008] The present invention introduces both trehalose and hyaluronic acid (HA) into the embryo thawing solution. Trehalose primarily exerts its intracellular protective effect by maintaining osmotic balance and protein stability. Hyaluronic acid, on the other hand, provides extracellular protection by building a physical barrier and maintaining cell membrane stability. The high viscosity of hyaluronic acid helps slow the permeation rate of trehalose, preventing drastic changes in osmotic pressure and thereby enhancing cellular tolerance. Furthermore, as an adjuvant, hyaluronic acid promotes the formation of a more stable glassy matrix with trehalose, thereby enhancing the effectiveness of embryo vitrification. The addition of hyaluronic acid also improves embryo adhesion to a certain extent, thereby increasing embryo implantation rates. The synergistic effect of trehalose and hyaluronic acid improves the survival rate of thawed embryos and enhances their developmental potential.

[0009] In some other embodiments, the thawing solution comprises the following components: trehalose 420-430 g / L, hyaluronic acid 0.2-0.25 g / L, sodium chloride 7-9 g / L, potassium chloride 0.15-0.25 g / L, magnesium chloride 0.08-0.12 g / L, disodium hydrogen phosphate 0.25-0.3 g / L, potassium dihydrogen phosphate 0.15-0.25 g / L, glucose 0.8-1.2 g / L, sodium pyruvate 0.035-0.04 g / L, calcium chloride 0.08-0.12 g / L, and bovine serum albumin 48-52 g / L.

[0010] Thawing solution within this ratio range can significantly shorten the time required for embryo thawing without affecting the embryo thawing and transplantation effects, thereby greatly improving the thawing efficiency.

[0011] For example, the components of the thawing solution are: trehalose 420 g / L, 425 g / L, 430 g / L, hyaluronic acid 0.2 g / L, 0.25 g / L, sodium chloride 7 g / L, 8 g / L, 9 g / L, potassium chloride 0.15 g / L, 0.2 g / L, 0.25 g / L, magnesium chloride 0.08 g / L, 0.09 g / L, 0.1 g / L, 0.11 g / L, 0.12 g / L, disodium hydrogen phosphate 0.25 g / L, 0.28 g / L, 0.3 g / L, potassium dihydrogen phosphate 0.15 g / L, 0.2 g / L, 0.25 g / L, glucose 0.8 g / L, 1.0 g / L, 1.2 g / L, sodium pyruvate 0.035 g / L, 0.036 g / L. g / L, 0.038 g / L, 0.04 g / L, calcium chloride is 0.08g / L, 0.09g / L, 0.10g / L, 0.11g / L, 0.12g / L, and bovine serum albumin is 48g / L, 49g / L, 50g / L, 51g / L, 52 g / L.

[0012] In some other embodiments, the thawing solution comprises the following components: 427.87 g / L trehalose, 0.25 g / L hyaluronic acid, 8 g / L sodium chloride, 0.2 g / L potassium chloride, 0.1 g / L magnesium chloride, 0.29 g / L disodium hydrogen phosphate, 0.2 g / L potassium dihydrogen phosphate, 1.0 g / L glucose, 0.036 g / L sodium pyruvate, 0.1 g / L calcium chloride, and 50 g / L bovine serum albumin. This thawing solution can rapidly thaw bovine vitrified in vitro embryos and achieve a high frozen embryo transfer pregnancy rate after transplantation.

[0013] In some other embodiments, the thawing solution of bovine vitrified in vitro embryos is stored at 2-8° C. for more than 30 days. The thawing solution has good stability and is easy to promote and apply.

[0014] In a second aspect, the present invention provides a method for using the thawing solution of bovine vitrified in vitro embryos according to the first aspect, comprising the following steps:

[0015] After the vitrified bovine in vitro embryos were taken out from liquid nitrogen, they were transferred into preheated thawing solution and allowed to stand;

[0016] The embryos after resting are transferred into embryo culture medium and cultured or transplanted after washing.

[0017] This thawing method enables one-step thawing of bovine vitrified in vitro embryos, achieving high embryo hatching and conception rates. While maintaining the thawing and transfer outcomes, it significantly shortens the time required for embryo thawing, thereby greatly improving embryo thawing efficiency.

[0018] In some other embodiments, the preheating temperature of the thawing solution is 35-37°C and the standing time is 0.5-1.5 minutes. In some other embodiments, the preheating temperature of the thawing solution is 37°C and the standing time is 1.0 minute. The thawing effect is best at this temperature.

[0019] When the embryo is vitrified, both inside and outside the cells are filled with high concentrations of cryoprotectants (such as ethylene glycol EG, dimethyl sulfoxide DMSO, etc.). These protectants form a glassy solid with the water in the cells to prevent the formation of ice crystals. When thawing, it is necessary to quickly rewarm (such as in a 37°C water bath) to instantly convert the glassy state into a liquid state to avoid recrystallization. Traditional vitrified embryo thawing adopts the method of gradually reducing the osmotic pressure of the thawing solution and gradually removing the cryoprotectant in the cells to achieve the purpose of embryo thawing. The content of cryoprotectant in existing vitrified embryos is significantly lower than that of traditional programmed frozen embryos. Therefore, the removal of cryoprotectant in cells does not require a long time. The inventors have found that the thawing solution provided by the present invention can complete the removal of most of the cryoprotectant in 1 minute and avoids the multi-step dilution method for thawing vitrified embryos. The embryos are placed in an environment with unsuitable temperature, osmotic pressure, etc. for a long time, resulting in the risk of reduced developmental potential of the embryos after thawing.

[0020] It can be seen that the thawing solution and thawing method of the present invention can control the thawing time to 1 minute, remove most of the CPA, and the remaining very small amount of CPA in the cells that has not been removed has limited impact on the embryo's ability to continue development.

[0021] Beneficial effects of the present invention:

[0022] (1) The thawing solution for bovine vitrified in vitro embryos provided by the present invention has a gradient osmotic pressure (trehalose, salt, sugar) and buffering capacity (phosphate) to prevent osmotic shock and pH fluctuations through fine concentration ratio and functional complementation; BSA binds toxins, and HA and BSA provide physical buffering to reduce mechanical damage. Ions (Na⁺, K⁺, Mg⁺) 2+ , Ca 2+ ) maintains membrane potential and function, while the buffer stabilizes pH. Glucose and sodium pyruvate provide immediate energy. This synergistic effect ensures a smooth transition of embryos after the intense freeze-thaw process, maximizing their vitality and laying a solid foundation for subsequent in vitro culture or transplantation.

[0023] (2) The present invention optimizes the traditional "multi-step dilution" method in the vitrified embryo thawing process into a "one-step thawing" method. This method can significantly shorten the time required for embryo thawing without affecting the embryo thawing and transplantation results, thereby greatly improving the thawing efficiency. At the same time, it reduces the instability of the results caused by the multi-step operation, facilitates the standardization of the operation process, and is conducive to the popularization and large-scale application of embryo transplantation technology. DETAILED DESCRIPTION

[0024] Those skilled in the art will appreciate that the following examples are intended to illustrate the present invention only and should not be construed as limiting the scope of the invention. Unless otherwise specified, the examples were performed under conventional conditions or manufacturer's recommended conditions. Components used without manufacturer's indication are commercially available conventional products.

[0025] The current traditional vitrification thawing method is the "multi-step dilution method". Its basic principle is to remove the cryocarrier containing the embryo from liquid nitrogen and quickly place it in a solution containing high-concentration sucrose. The embryo stays in the high-concentration sucrose solution for a period of time to complete the elution of the cryoprotectant (CPA) solution and the initial recovery of the cell osmotic pressure. After that, the embryo is moved into sucrose solutions of decreasing concentrations in sequence to further remove CPA and prevent water from penetrating into the cells too quickly, causing osmotic shock damage.

[0026] However, the traditional vitrified embryo thawing method has the following problems, such as the thawing process is time-consuming, the operation process is complicated, and the embryo thawing effect is highly volatile. The embryos must stay in sucrose solutions of different concentrations in sequence, resulting in thawing of one embryo usually taking more than 10 minutes, which significantly increases the overall operation time. Especially when a large number of embryos need to be processed for transplantation, the bottleneck seriously affects the thawing efficiency; although the "multi-step dilution" method can gently remove CPA, it needs to stay in a variety of liquids of different concentrations, and different steps need to be performed at different operating temperatures, which increases the risk of operational errors and the exposure time of embryos in non-ideal environments. In addition, the "multi-step dilution" method has high requirements for the operator, and requires strict control of solution concentration, embryo residence time, operating temperature, etc. Otherwise, it will affect the thawing efficiency and embryo survival rate, making the thawing results of vitrified embryos lack stability and repeatability.

[0027] The present invention addresses the above technical issues and provides a method for thawing vitrified embryos that is rapid, simple, stable, and easy to operate. The technical solutions adopted are as follows:

[0028] An embodiment of the present invention provides a thawing solution for bovine vitrified in vitro embryos, comprising the following components: 340-515 g / L of trehalose, 0.2-0.3 g / L of hyaluronic acid, 6-10 g / L of sodium chloride, 0.1-0.3 g / L of potassium chloride, 0.05-0.15 g / L of magnesium chloride, 0.2-0.35 g / L of disodium hydrogen phosphate, 0.1-0.3 g / L of potassium dihydrogen phosphate, 0.5-1.5 g / L of glucose, 0.03-0.04 g / L of sodium pyruvate, 0.05-0.15 g / L of calcium chloride, and 45-55 g / L of bovine serum albumin.

[0029] The hyaluronic acid (HA) added to the thawing solution of this invention is a glycosaminoglycan composed of disaccharide units of D-glucuronic acid and N-acetylglucosamine. It occurs naturally in the extracellular matrix and intercellular matrix of mammalian tissues and is one of the most abundant macromolecular substances in the female reproductive tract. HA's high viscoelastic properties help stabilize the thawing solution's microenvironment, buffering physical shock and maintaining the stability of cell membranes and cytoskeleton, thereby reducing potential damage to embryonic cell membranes during temperature fluctuations and osmotic pressure adjustments. Furthermore, HA can regulate oxidative stress and apoptosis pathways by binding to hyaluronic acid receptors, thereby improving the survival rate of post-thaw embryos.

[0030] Trehalose is a non-reducing disaccharide composed of two glucose molecules. As a typical stress metabolite, trehalose can form a unique protective film on the cell surface under extreme conditions such as high temperature, low temperature, high osmotic pressure, and dehydration, effectively protecting the structure of biological molecules from damage and maintaining the vital activities and biological characteristics of living organisms. Compared with sucrose used in traditional vitrification thawing fluids, trehalose shows multiple advantages in the frozen embryo thawing process: (1) higher stability and stronger resistance to hydrolysis; (2) long-term effectiveness, maintaining osmotic pressure balance; (3) antioxidant properties, which are beneficial for protecting mitochondrial function; (4) more gentle osmotic pressure regulation, reducing cell swelling; (5) good biocompatibility and high safety; (6) transmembrane permeability differences, continuously forming an osmotic gradient.

[0031] The purity of the reagents used in the present invention is above the cell culture grade, such as products from Sigma-Baldric. The thawing solution needs to be stored at low temperature after preparation, and can be stored at 2-8 degrees for 30 days.

[0032] Another embodiment of the present invention provides a method for using a thawing solution for bovine vitrified in vitro embryos, comprising the following steps:

[0033] After the vitrified bovine in vitro embryos were taken out from liquid nitrogen, they were transferred into preheated thawing solution and allowed to stand;

[0034] The embryos after resting are transferred into embryo culture medium and cultured or transplanted after washing.

[0035] The preheating temperature of the thawing solution is 35-37°C, and the standing time is 0.5-1.5 minutes.

[0036] More specifically, the method for using a thawing solution for vitrified bovine in vitro embryos involves freezing bovine in vitro embryos using a commercial embryo vitrification solution. The specific steps are as follows: the embryos are allowed to rest in a frozen equilibrium droplet for 1 minute; then, they are transferred to a cryostat droplet and allowed to rest for 45 seconds; within 10 seconds, the embryos are removed from the cryostat droplet, loaded onto an embryo cryocarrier, and plunged into liquid nitrogen. The entire process is performed at room temperature. The embryo cryocarrier is a straw filled with five sections in the order of embryo preservation solution, air bubble, embryo preservation solution, air bubble, cryostat containing the embryo, air bubble, embryo preservation solution, air bubble, and embryo preservation solution. Each section of liquid column has the same length. The straw is sealed with a straw stopper and allowed to rest at room temperature for 5-10 minutes. The embryos are then placed in a programmable thermostat pre-chilled to -6°C. After ice is applied, the temperature is lowered to -35°C at a rate of 0.5°C / minute. After the freezing program is completed, the straw is plunged into liquid nitrogen.

[0037] Use a one-step rapid thawing method: remove the embryo freezing carrier from liquid nitrogen, quickly immerse it in embryo rapid thawing solution preheated to 37°C, and let it stand for 1 minute; suck the embryo out of the thawing solution and place it in a droplet made of embryo culture solution. After washing 3 times, the embryo is transferred to the corresponding embryo culture solution for culture or directly transplanted.

[0038] Thawed embryo transfer: After thawing, the embryos are washed three times with embryo preservation solution, then placed in five-stage tubes. The tubes are sealed with straw stoppers and then transferred into recipient cows on day 7 of estrus. Thirty days after embryo transfer, the recipient cows are tested for pregnancy using ultrasound, and the pregnancy rate is calculated.

[0039] The thawing solution for bovine vitrified in vitro embryos of the present invention can realize one-step rapid thawing of bovine vitrified in vitro embryos, and can obtain a higher recovery rate and embryo hatching rate.

[0040] The technical solution of the present invention is further described below in conjunction with specific embodiments:

[0041] Example 1

[0042] Thawing solution for bovine vitrified in vitro embryos and use method thereof

[0043] (1) Thawing solution for bovine vitrified in vitro embryos, comprising the following components: trehalose 427.87 g / L, hyaluronic acid 0.25 g / L, sodium chloride 8 g / L, potassium chloride 0.2 g / L, magnesium chloride 0.1 g / L, disodium hydrogen phosphate 0.29 g / L, potassium dihydrogen phosphate 0.2 g / L, glucose 1.0 g / L, sodium pyruvate 0.036 g / L, calcium chloride 0.1 g / L, and bovine serum albumin 50 g / L.

[0044] (2) A rapid one-step thawing method for bovine vitrified in vitro embryos, comprising the following steps:

[0045] Remove the embryo cryocarrier from liquid nitrogen and quickly immerse it in embryo rapid thawing solution preheated to 37°C and let it stand for 1 minute;

[0046] The embryos are sucked out of the thawing solution, placed in the corresponding embryo culture medium, washed three times, and then placed in the embryo culture medium for further culture or directly transplanted.

[0047] The present invention does not impose any particular limitations on the embryo freezing method or the thawed embryo culture or transplantation method; commonly used culture or transplantation methods in the art may be employed. The embryo transplantation solution comprises the following components: 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, and 4 g / L bovine serum albumin.

[0048] Example 2

[0049] A thawing solution for bovine vitrified in vitro embryos and a method for using the same, which differs from Example 1 in that the concentration of trehalose is 171.15 g / L. Specifically, the thawing solution for bovine vitrified in vitro embryos comprises the following components: 171.15 g / L trehalose, 0.25 g / L hyaluronic acid, 8 g / L sodium chloride, 0.2 g / L potassium chloride, 0.1 g / L magnesium chloride, 0.29 g / L disodium hydrogen phosphate, 0.2 g / L potassium dihydrogen phosphate, 1.0 g / L glucose, 0.036 g / L sodium pyruvate, 0.1 g / L calcium chloride, and 50 g / L bovine serum albumin.

[0050] The method of use is the same as that in Example 1.

[0051] Example 3

[0052] A thawing solution for bovine vitrified in vitro embryos and a method for use thereof, which differs from Example 1 in that the concentration of trehalose is 513.45 g / L. Specifically, the thawing solution for bovine vitrified in vitro embryos comprises the following components: 513.45 g / L trehalose, 0.25 g / L hyaluronic acid, 8 g / L sodium chloride, 0.2 g / L potassium chloride, 0.1 g / L magnesium chloride, 0.29 g / L disodium hydrogen phosphate, 0.2 g / L potassium dihydrogen phosphate, 1.0 g / L glucose, 0.036 g / L sodium pyruvate, 0.1 g / L calcium chloride, and 50 g / L bovine serum albumin.

[0053] The method of use is the same as that in Example 1.

[0054] Comparative Example 1

[0055] A thawing solution for bovine vitrified in vitro embryos and a method for using the same, which differs from Example 1 in that the addition of hyaluronic acid is omitted.

[0056] Thawing solution for bovine vitrified in vitro embryos contains the following components: trehalose 427.87 g / L, sodium chloride 8 g / L, potassium chloride 0.2 g / L, magnesium chloride 0.1 g / L, disodium hydrogen phosphate 0.29 g / L, potassium dihydrogen phosphate 0.2 g / L, glucose 1.0 g / L, sodium pyruvate 0.036 g / L, calcium chloride 0.1 g / L, and bovine serum albumin 50 g / L.

[0057] The rapid one-step thawing method for bovine vitrified in vitro embryos is the same as that in Example 1.

[0058] Comparative Example 2

[0059] A thawing solution for bovine vitrified in vitro embryos and a method for using the same, which differs from Example 1 in that the addition of trehalose is omitted.

[0060] Thawing solution for bovine vitrified in vitro embryos contains the following components: hyaluronic acid 0.25 g / L, sodium chloride 8 g / L, potassium chloride 0.2 g / L, magnesium chloride 0.1 g / L, disodium hydrogen phosphate 0.29 g / L, potassium dihydrogen phosphate 0.2 g / L, glucose 1.0 g / L, sodium pyruvate 0.036 g / L, calcium chloride 0.1 g / L, and bovine serum albumin 50 g / L.

[0061] The rapid one-step thawing method for bovine vitrified in vitro embryos is the same as that in Example 1.

[0062] Comparative Example 3

[0063] A thawing solution for bovine vitrified in vitro embryos and a method for use thereof, which differs from Example 1 in that the concentration of trehalose is 68.46 g / L and the concentration of hyaluronic acid is 0.25 g / L. Specifically, the thawing solution for bovine vitrified in vitro embryos comprises the following components: 68.46 g / L trehalose, 0.2 g / L hyaluronic acid, 8 g / L sodium chloride, 0.2 g / L potassium chloride, 0.1 g / L magnesium chloride, 0.29 g / L disodium hydrogen phosphate, 0.2 g / L potassium dihydrogen phosphate, 1.0 g / L glucose, 0.036 g / L sodium pyruvate, 0.1 g / L calcium chloride, and 50 g / L bovine serum albumin.

[0064] The method of use is the same as that in Example 1.

[0065] Comparative Example 4

[0066] A thawing solution for bovine vitrified in vitro embryos and a method for use thereof, which differs from Example 1 in that the concentration of trehalose is 684.59 g / L and the concentration of hyaluronic acid is 0.3 g / L. Specifically, the thawing solution for bovine vitrified in vitro embryos comprises the following components: 684.59 g / L trehalose, 0.3 g / L hyaluronic acid, 8 g / L sodium chloride, 0.2 g / L potassium chloride, 0.1 g / L magnesium chloride, 0.29 g / L disodium hydrogen phosphate, 0.2 g / L potassium dihydrogen phosphate, 1.0 g / L glucose, 0.036 g / L sodium pyruvate, 0.1 g / L calcium chloride, and 50 g / L bovine serum albumin.

[0067] The method of use is the same as that in Example 1.

[0068] Comparative Example 5

[0069] A thawing solution for bovine vitrified in vitro embryos and a method for using the same, which differs from Example 1 in that the concentration of sucrose is 427.87 g / L. Specifically, the thawing solution for bovine vitrified in vitro embryos comprises the following components: 427.87 g / L sucrose, 0.25 g / L hyaluronic acid, 8 g / L sodium chloride, 0.2 g / L potassium chloride, 0.1 g / L magnesium chloride, 0.29 g / L disodium hydrogen phosphate, 0.2 g / L potassium dihydrogen phosphate, 1.0 g / L glucose, 0.036 g / L sodium pyruvate, 0.1 g / L calcium chloride, and 50 g / L bovine serum albumin.

[0070] The method of use is the same as that in Example 1.

[0071] Comparative Example 6

[0072] A thawing solution for bovine vitrified in vitro embryos and a method for use thereof, which differs from Example 1 in that the concentration of trehalose is 342.30 g / L and the addition of hyaluronic acid is omitted. Specifically, the thawing solution for bovine vitrified in vitro embryos comprises the following components: 342.30 g / L trehalose, 8 g / L sodium chloride, 0.2 g / L potassium chloride, 0.1 g / L magnesium chloride, 0.29 g / L disodium hydrogen phosphate, 0.2 g / L potassium dihydrogen phosphate, 1.0 g / L glucose, 0.036 g / L sodium pyruvate, 0.1 g / L calcium chloride, and 50 g / L bovine serum albumin.

[0073] The method of use is the same as that in Example 1.

[0074] Comparative Example 7

[0075] A thawing solution for bovine vitrified in vitro embryos and a method for use thereof, differing from Example 1 in that the concentration of sucrose is 342.30 g / L and the addition of hyaluronic acid is omitted. Specifically, the thawing solution for bovine vitrified in vitro embryos comprises the following components: 342.30 g / L sucrose, 8 g / L sodium chloride, 0.2 g / L potassium chloride, 0.1 g / L magnesium chloride, 0.29 g / L disodium hydrogen phosphate, 0.2 g / L potassium dihydrogen phosphate, 1.0 g / L glucose, 0.036 g / L sodium pyruvate, 0.1 g / L calcium chloride, and 50 g / L bovine serum albumin.

[0076] The method of use is the same as that in Example 1.

[0077] Comparative Example 8

[0078] Traditional "multi-step dilution" thawing solution and its use method

[0079] 1. Traditional thawing fluids include thawing fluids , thawing solution , thawing solution and thawing solution Among them, the thawing liquid , thawing solution , thawing solution and thawing solution The only difference is the concentration of trehalose. The concentration of trehalose in the solution is 342.30 g / L. The concentration of trehalose in the solution is 171.15 g / L. The concentration of trehalose in the thaw solution was 85.57 g / L. The concentration of trehalose in the solution is 0 g / L. The details are as follows:

[0080] thawing solution The mixture contains 342.30 g of trehalose, 8 g / L of sodium chloride, 0.2 g / L of potassium chloride, 0.1 g / L of magnesium chloride, 2.9 g / L of disodium hydrogen phosphate, 0.2 g / L of potassium dihydrogen phosphate, 1 g / L of glucose, 0.036 g / L of sodium pyruvate, 0.1 g / L of calcium chloride, and 50 g / L of bovine serum albumin.

[0081] thawing solution The solution contains 171.15 g / L trehalose, 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, and 50 g / L bovine serum albumin.

[0082] thawing solution The solution contains 85.57 g / L trehalose, 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, and 50 g / L bovine serum albumin.

[0083] thawing solution The solution contains 0 g / L trehalose, 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, and 50 g / L bovine serum albumin.

[0084] 2. Thawing of bovine in vitro embryos using the traditional "multi-step dilution" method includes the following steps:

[0085] Remove the embryo freezing carrier from liquid nitrogen and quickly immerse it in embryo thawing solution preheated to 37°C Let it sit for 1 minute;

[0086] From the thawed liquid Pick up the embryos and transfer them to the thawing solution at room temperature. Bottom, let stand for 3 minutes;

[0087] From the thawed liquid Pick up the embryos and transfer them to the thawing solution at room temperature. Bottom, let stand for 5 minutes;

[0088] From the thawed liquid Pick up the embryos and transfer them to the thawing solution at room temperature. Surface, let stand for 5 minutes;

[0089] After thawing, the embryos are transferred into the corresponding embryo culture medium for culture or directly transplanted.

[0090] The present invention imposes no particular limitations on the culture or transplantation methods for thawed embryos, and commonly used culture or transplantation methods in the art may be employed. The embryo transplantation solution comprises the following components: 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, and 4 g / L bovine serum albumin.

[0091] After thawing, the embryos were washed three times with embryo preservation solution, then placed in five-stage tubes. The tubes were sealed with straw stoppers and transplanted into recipient cows on day 7 of estrus. Thirty days after embryo transfer, the recipient cows were tested for pregnancy using ultrasound, and the pregnancy rate was calculated.

[0092] Comparative Example 9

[0093] Traditional "multi-step dilution" thawing solution and its use method

[0094] The difference from Example 1 is that sucrose is used to replace the trehalose in Example 1. The thawing solution includes the thawing solution , thawing solution , thawing solution and thawing solution Among them, the thawing liquid , thawing solution , thawing solution and thawing solution The only difference is the concentration of sucrose. The concentration of sucrose in the solution is 342.30 g / L. The concentration of sucrose in the solution is 171.15 g / L. The concentration of sucrose in the thaw solution was 85.57 g / L. The concentration of sucrose in the solution is 0 g / L. The details are as follows:

[0095] thawing solution The solution contains 342.30 g / L sucrose, 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, and 50 g / L bovine serum albumin.

[0096] thawing solution The solution contains 171.15 g / L sucrose, 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, and 50 g / L bovine serum albumin.

[0097] thawing solution The solution contains 85.57 g / L sucrose, 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, and 50 g / L bovine serum albumin.

[0098] thawing solution The solution contains 0 g / L sucrose, 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, and 50 g / L bovine serum albumin.

[0099] The thawing method of bovine vitrified in vitro embryos is the same as that in Example 9.

[0100] After thawing, the embryos of Examples 1-3 and Comparative Examples 1-9 were washed three times with embryo culture medium and then continued to be cultured in embryo culture medium. The percentage of embryos whose cysts expanded again 24 hours after thawing and the percentage of embryos that hatched 48 hours after thawing (as shown in Table 1) were counted. The number of transplanted fetuses, the number of pregnancies detected initially, and the conception rate (as shown in Table 2) after embryo transplantation were also tested.

[0101] Table 1 Number and proportion of hatched embryos at different thawing times

[0102]

[0103] Table 2 Number of embryos transferred, number of pregnancies detected initially, and conception rate after embryo transfer

[0104]

[0105] As shown in Tables 1 and 2, when Example 1, in which both trehalose and hyaluronic acid were added to the rapid one-step thawing solution, showed higher rates of cystic cavity re-expansion 24 hours after thawing, higher hatching rates 48 hours after thawing, and higher embryo transfer pregnancy rates than when only trehalose was added (Comparative Example 1) or only hyaluronic acid was added (Comparative Example 2). The combined use of trehalose and hyaluronic acid enhanced the effectiveness of the rapid one-step embryo thawing method.

[0106] Compared with Example 1, in Comparative Examples 3 and 4, too high or too low trehalose concentrations would affect the developmental potential of embryos after thawing and the embryo transfer pregnancy rate.

[0107] Comparing Comparative Example 5 with Example 1, Example 1 achieved a better thawing effect by replacing sucrose with trehalose. The reasons are as follows: Although trehalose and sucrose have the same molecular weight, trehalose's unique α,α-1,1-glycosidic bond structure gives it greater stability, allowing it to more efficiently penetrate cell membranes and distribute evenly inside and outside the cell, alleviating osmotic stress during the thawing process. Furthermore, trehalose can directly stabilize the cell membrane structure by replacing water molecules by binding to the phospholipid bilayer. Trehalose can also inhibit the freeze-induced mitochondrial apoptosis pathway. These membrane-stabilizing and anti-apoptotic properties are not possessed by sucrose.

[0108] Compared with Example 1, Comparative Examples 6 and 7 both adopted a rapid one-step thawing method with trehalose and hyaluronic acid as the main ingredients, but the concentrations of trehalose and hyaluronic acid in the thawing solution were different. When the trehalose concentration in the thawing solution reached 427.87 g / L and the hyaluronic acid concentration reached 0.25 g / L, the thawing effect was optimal.

[0109] Comparative Examples 8 and 9, compared with Example 1, both used a traditional multi-step dilution method to thaw the embryos. The thawing solution used in Comparative Example 8 was a trehalose thawing solution, while the thawing solution used in Comparative Example 9 was a sucrose thawing solution. The results showed that regardless of whether the main component of the thawing solution was trehalose or sucrose, the traditional multi-step dilution method was inferior to the rapid one-step thawing method for thawing vitrified embryos. The step-by-step dilution thawing method using sucrose thawing solution was the worst. Furthermore, when comparing Comparative Examples 8 and 9, after step-by-step thawing of bovine vitrified embryos, the embryos thawed in Comparative Example 8 using a thawing solution with trehalose as the main component, and the embryos thawed in Comparative Example 9 using a traditional sucrose-based thawing solution, showed a trend toward higher hatching rates and embryo transfer conception rates 48 hours after thawing than the embryos thawed in the sucrose group.

[0110] As can be seen, compared with sucrose used in traditional vitrification thawing solutions, trehalose exhibits multiple advantages during the thawing process of frozen embryos. This is because trehalose's unique α,α-1,1-glycosidic bond structure gives it greater stability, allowing it to more efficiently penetrate cell membranes and distribute evenly inside and outside the cell, thereby alleviating osmotic stress during the thawing process. Furthermore, trehalose can directly stabilize the cell membrane structure by replacing water molecules by binding to the phospholipid bilayer. Trehalose can also inhibit the freezing-induced mitochondrial apoptosis pathway. These membrane-stabilizing and anti-apoptotic properties are not possessed by sucrose.

[0111] The above are merely preferred embodiments of the present invention and are 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 shall be included within the scope of protection of the present invention.

Claims

1. A thawing solution for bovine vitrified in vitro embryos, characterized in that: Contains the following components: trehalose 340-515g / L, hyaluronic acid 0.2-0.3 g / L, sodium chloride 6-10g / L, potassium chloride 0.1-0.3g / L, magnesium chloride 0.05-0.15g / L, disodium hydrogen phosphate 0.2-0.35 g / L, potassium dihydrogen phosphate 0.1-0.3g / L, glucose 0.5-1.5 g / L, sodium pyruvate 0.03-0.04g / L, calcium chloride 0.05-0.15g / L, bovine serum albumin 45-55 g / L.

2. The thawing solution for bovine vitrified in vitro embryos according to claim 1, characterized in that Contains the following components: trehalose 420-430 g / L, hyaluronic acid 0.2-0.25 g / L, sodium chloride 7-9 g / L, potassium chloride 0.15-0.25 g / L, magnesium chloride 0.08-0.12 g / L, disodium hydrogen phosphate 0.25-0.3 g / L, potassium dihydrogen phosphate 0.15-0.25 g / L, glucose 0.8-1.2 g / L, sodium pyruvate 0.035-0.04 g / L, calcium chloride 0.08-0.12 g / L, bovine serum albumin 48-52 g / L.

3. The thawing solution for bovine vitrified in vitro embryos according to claim 1, characterized in that Contains the following components: trehalose 427.87 g / L, hyaluronic acid 0.25 g / L, sodium chloride 8 g / L, potassium chloride 0.2 g / L, magnesium chloride 0.1 g / L, disodium hydrogen phosphate 0.29 g / L, potassium dihydrogen phosphate 0.2 g / L, glucose 1.0 g / L, sodium pyruvate 0.036 g / L, calcium chloride 0.1 g / L, bovine serum albumin 50 g / L.

4. The thawing solution for bovine vitrified in vitro embryos according to claim 1, characterized in that The thawing solution of the bovine vitrified in vitro embryos is stored at 2-8° C. for more than 30 days.

5. A method for using the thawing solution for bovine vitrified in vitro embryos according to any one of claims 1 to 4, characterized in that: The following steps are involved: After the vitrified bovine in vitro embryos were taken out from liquid nitrogen, they were transferred into preheated thawing solution and allowed to stand; The embryos after resting are transferred into embryo culture medium and cultured or transplanted after washing; The methods of use described are not intended for the diagnosis or treatment of disease.

6. The method for using the thawing solution for bovine vitrified in vitro embryos according to claim 5, characterized in that: The preheating temperature of the thawing solution is 35-37℃, and the standing time is 0.5-1.5min.

7. The method for using the thawing solution for bovine vitrified in vitro embryos according to claim 6, characterized in that: The preheating temperature of the thawing solution was 37°C and the standing time was 1.0 min.

Images