Freezing carrier device capable of absorbing embryo refrigerating fluid and application thereof

Abstract

The invention relates to a freezing carrier device capable of absorbing embryo freezing fluid and application of the freezing carrier device. The freezing carrier device comprises a freezing pipe body, fluid-absorbing resin particles are adhered to the inner wall of the freezing pipe body through a polyurethane adhesive, tiny gaps exist between the fluid-absorbing resin particles, numerous micro-channels are formed, and a capillary effect is generated on the fluid. The liquid-absorbing resin particles used for absorbing the liquid can be polyacrylamide, sodium polyacrylate or a mixture of the polyacrylamide and the sodium polyacrylate. When the freezing carrier device is used, an embryo and a protective agent are mixed and then put into the freezing pipe body, and due to the liquid absorption performance (the hydrophilic effect of resin and the capillary effect of particle gaps) of the liquid absorption resin particle layer, the liquid absorption resin particle layer can rapidly absorb redundant embryo freezing liquid in embryo freezing liquid, so that the volume of the embryo freezing liquid is reduced, and the embryo freezing effect is improved. The freezing and unfreezing speed is increased, the damage of ice crystals to embryos is reduced, and the survival rate and conception rate of the embryos are increased.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology and specifically relates to a cryo-carrier device capable of absorbing embryo cryo-fluid and its application in absorbing fluid, particularly in animal breeding. The present invention is a cryo-carrier device for embryo cryopreservation that can be widely used in biomedical research and clinical applications, particularly in embryo cryopreservation technology. Background Art

[0002] Embryo freezing is a technology that preserves reproductive cells or embryos under low temperature conditions to prolong their life activity. Embryo freezing technology has a wide range of applications in animal genetic improvement, reproductive medicine, biotechnology and other fields. At present, there are two main methods of embryo freezing technology, one is programmed freezing and the other is vitrification. Programmed freezing refers to a method in which the temperature of the embryo is gradually lowered under a certain program until it reaches the liquid nitrogen temperature (-196°C), and the temperature is gradually increased during thawing. Vitrification refers to a method in which the embryo is rapidly cooled to the liquid nitrogen temperature in a high concentration of protective agent to form a non-crystallized glass state, and the temperature is also rapidly increased during thawing.

[0003] Compared to programmed freezing, vitrification offers higher freezing efficiency and lower cell damage. This is because vitrification prevents the formation of ice crystals inside and outside cells, thereby preserving their structure and function. Vitrification has become the most advanced embryo freezing technology, particularly for sensitive embryo stages like oocytes and blastocysts, where it can achieve near-100% recovery rates and high pregnancy rates.

[0004] However, vitrification also presents some problems and challenges. First, high concentrations of protective agents may adversely affect the biological characteristics of the embryo, such as affecting its metabolism, differentiation, and implantation. Second, vitrification requires high technical skills and specialized equipment and personnel to ensure the speed and accuracy of freezing and thawing. Finally, vitrification is costly, primarily due to the high price of cryotransfer media.

[0005] A cryocarrier is a tool used to freeze and thaw embryos along with a cryosol (protective agent). Its function is to provide a microenvironment suitable for embryo freezing while protecting the embryo from external contamination and damage. Currently, common embryo vitrification cryocarriers on the market, such as open stretched straws (OPS), increase freezing speed by controlling the volume of the embryo and cryosol to a range of 2 to 5 μL. However, a disadvantage of this type of cryocarrier is that the volume of cryosol is still relatively large, resulting in slower freezing and thawing speeds. This causes damage to the embryos from ice crystals during the freezing and thawing process, reducing embryo survival and conception rates. Other types of cryocarriers, such as Cryotop, require manual aspiration of excess liquid, reducing the volume of the embryo and protective agent to approximately 1 μL, thereby achieving ultra-fast freezing and thawing. However, these cryocarriers require high operator skills and are relatively expensive, making large-scale use costly.

[0006] How to conveniently and quickly reduce the volume of the frozen liquid in the freezing carrier is an urgent problem to be solved. Water-absorbing resin is a new functional polymer material commonly used as a personal care product and water-retaining agent, and can be used for water absorption. However, the frozen liquid to be absorbed by the present invention contains not only water as a main component but also an organic solvent (such as glycerol). Glycerol is a polar molecule. Although it can well combine and mix with other polar liquids, the liquid-absorbing resin cannot absorb glycerol.

[0007] Therefore, the water-absorbing resin used in embryo freezing cannot only have water absorption, but also needs to have good absorption, adsorption or solubility properties for specific freezing fluids, especially organic solvents.

[0008] To address these issues, the present invention provides a cryo-carrier device based on a liquid-absorbing resin. By coating the inner wall of a cryo-carrier tube with specialized water-absorbing resin particles, forming a porous particle layer structure, the device boasts excellent liquid absorption (including the ability to absorb organic solvents and water), rapid freezing rates, ease of operation, and low cost, effectively improving the quality and success rate of embryo freezing. Summary of the Invention

[0009] The object of the present invention is to provide a freezing carrier device based on a liquid-absorbing resin and its application in animal breeding in view of the problems existing in the prior art. In the freezing carrier device of the present invention, it is necessary to consider the distribution pattern of the liquid-absorbing resin material on the inner wall of the carrier tube. If the liquid-absorbing resin particles adhered are too large or directly piled up, the contact area between the freezing liquid (water and organic solvent) and the liquid-absorbing resin particles will be reduced, thereby affecting the liquid absorption rate (absorption time exceeds 1 minute). At the same time, the adhesive used to adhere the water-absorbing resin particles can be resistant to low temperatures, adapt to the situation of rapid cooling, and have good adhesion properties to both the carrier tube material and the liquid-absorbing resin material.

[0010] The purpose of the present invention can be achieved by the following solutions:

[0011] The present invention provides a freezing carrier device capable of absorbing embryo freezing liquid, characterized in that the freezing carrier device comprises: a freezing tube body, a liquid-absorbing resin particle layer;

[0012] The liquid-absorbing resin particles are adhered to the inner wall of the freezing tube body by an adhesive to form a liquid-absorbing resin particle layer;

[0013] The liquid-absorbing resin particles are one or more of polyacrylamide and sodium polyacrylate particles; the particle size of the liquid-absorbing resin particles does not exceed 70 μm;

[0014] The adhesive is a polyurethane adhesive.

[0015] As an embodiment of the present invention, the particle diameter of the liquid-absorbing resin particles is preferably 20~70 μ m. The average thickness of the resin particle layer is no more than 70 μ m. The present invention processes the liquid-absorbing resin particles through a screen, and a diameter is collected less than 70 μ m, is evenly sprinkled on the pipe inner wall that scribbles adhesive, so the thickness of the granular layer is less than 70 μ m in theory, but considers the possible aggregation situation of the particle, the granular layer local thickness may surpass 70 μ m, so the average thickness is no more than 70 μ m.

[0016] As one embodiment of the present invention, the liquid-absorbing resin particles are one or more of polyacrylamide particles and sodium polyacrylate particles. The liquid-absorbing resin particles are preferably sodium polyacrylate particles. Although the type of water-absorbing resin is various, the liquid-absorbing resin particles selected by the present invention, particularly sodium polyacrylate, possess high water absorbability and also have a liquid-absorbing effect, and can realize that embryo freezing solution (mainly water and an organic solvent, wherein the organic solvent includes glycerol etc.) is rapidly absorbed in a short time. Simultaneously, in the face of the situation of rapid cooling, the liquid-absorbing resin particles used by the present invention have little volume change before and after absorbing water and in the ultra-low temperature conversion process of room temperature, and adhere to the inner wall of the pipe and are not easy to fall off.

[0017] As one embodiment of the present invention, the liquid-absorbing resin particles are closely arranged on the inner wall of the freezing tube body, and the tiny gaps between the particles form numerous micro-channels, which produce capillary action on the liquid.

[0018] As one embodiment of the present invention, 5 μL of embryo freezing solution is dropped on a layer of liquid-absorbing resin particles and can be absorbed within 1 minute.

[0019] The embryo freezing solution includes a base solution and an organic solvent; the base solution includes one or more of Dulbecco's phosphate buffer, calf serum, sodium pyruvate, glucose, and bovine serum albumin; and the organic solvent includes glycerol.

[0020] Regarding the resin selection, the present invention selected a liquid-absorbing resin whose main component is low-crosslinked sodium polyacrylate, which can absorb deionized water hundreds or thousands of times its own weight. Regarding the resin structure design, the present invention grinds the resin and then sieves it to obtain water-absorbing resin particles with a diameter of no more than 70 microns. Compared to unground particles (diameter exceeding 200 microns), the small-diameter liquid-absorbing resin particle layer has a larger contact area with the freezing liquid, which can shorten the time it takes to absorb the embryo freezing liquid.

[0021] In one embodiment of the present invention, the adhesive is a polyurethane adhesive. To address the problem of the smooth inner wall of the cryotube body, which makes it difficult for water-absorbing resin particles to adhere, the present invention first coats the tube wall with a non-toxic polyurethane adhesive, then evenly sprinkles the water-absorbing resin particles onto the adhesive-coated tube wall, thereby forming a layer of liquid-absorbing resin particles on the inner wall.

[0022] In addition to the bonding effect, the reason why the present invention uses polyurethane adhesive is that it has low-temperature resistance. Polyurethane adhesive can withstand the ultra-low temperature of liquid nitrogen (-196°C) without becoming brittle and falling off, nor will it produce a vitrified state at ultra-low temperatures, causing the adhesive to easily break and fall off, thereby adversely affecting the quality of the embryo. The polyurethane adhesive used in the present invention has both good adhesion to the carrier tube material and good ultra-low temperature resistance. Even if the frozen carrier device is put into liquid nitrogen, it will not fall off from the inner wall of the frozen carrier tube. Other typical adhesives include epoxy resin adhesives and silicone adhesives, but epoxy resin adhesives are generally not resistant to liquid nitrogen low temperatures, and silicone adhesives have weaker adhesion to plastics such as frozen carrier tubes than polyurethane adhesives. Therefore, if the adhesive is not resistant to low temperatures and has weak adhesion, it is impossible to form a stable polymer resin particle layer on the surface of the carrier tube.

[0023] At the same time, when the polyurethane adhesive adheres to the particles, it forms numerous micropores with the particles, creating a capillary effect on the liquid. The liquid-absorbing resin particles and micropores absorb not only water but also gases and liquids. Specifically, it absorbs other substances in the cryopreservation solution (such as phosphate buffer, serum, sodium pyruvate, glucose, and protein) in the same proportions as the original cryopreservation solution. Any remaining cryopreservation solution remains unchanged, without affecting embryo preservation.

[0024] Even after the liquid-absorbing resin swells after absorbing liquid, it remains intact due to the excellent adhesion of the polyurethane adhesive and the presence of cryopreservation liquid in the micropores, which creates strong hydrogen bonding between the resin structure and water molecules. The cryo-support carrier is cryogenically resistant and has undergone repeated freeze-thaw tests, with neither the adhesive nor the liquid-absorbing resin particles attached to it falling off. Furthermore, if the particle size of the liquid-absorbing polymer (e.g., exceeding 200 microns) and its distribution on the tube wall (e.g., direct accumulation) are not considered, it is still impossible to absorb the residual embryo cryo-fluid in a short period of time.

[0025] Polyurethane adhesives are adhesives containing carbamate or isocyanate groups in their molecular chains. They are resistant to low and ultra-low temperatures and exhibit high reactivity, forming covalent bonds with many materials containing active hydrogen atoms on their surfaces, such as metals, rubbers, fibers, wood, leather, and plastics. After curing, they contain carbamate and highly polar bonds and groups, easily forming secondary bonds with the substrate. These combined chemical and physical bonding forces result in a high bond strength between the substrates. They possess excellent shear strength and impact resistance, making them suitable for a variety of structural bonding applications and possessing exceptional flexibility.

[0026] As one embodiment of the present invention, the material of the freezing tube body is a material suitable for freezing, specifically including one or more of plastic, glass, metal, and ceramics; the shape of the freezing tube body is a shape suitable for loading embryos, specifically including one of cylindrical, U-shaped, and V-shaped.

[0027] As one embodiment of the present invention, the length of the cryovial body is 2-5 cm and the diameter is 2-5 mm. The length can be adjusted as needed and the diameter can be adjusted according to the size and number of embryos.

[0028] The present invention also provides a method for preparing the frozen carrier device, comprising the following steps:

[0029] An adhesive is applied to the inner wall of the freezing tube body, and then liquid-absorbing resin particles are evenly sprinkled on the inner wall coated with the adhesive. After the adhesive is solidified, a liquid-absorbing resin particle layer is formed on the inner wall with tiny gaps between the particles, thereby obtaining the freezing carrier device.

[0030] As one embodiment of the present invention, after the adhesive is cured, the loosely bonded liquid absorbing resin particles are blown away to form a liquid absorbing resin particle layer.

[0031] As one embodiment of the present invention, the frozen carrier device is sterilized by ultraviolet irradiation before use.

[0032] The present invention also provides an application of the freezing carrier device in animal breeding, specifically in embryo cryopreservation.

[0033] The present invention also provides a method for using the frozen carrier device, comprising the following steps:

[0034] After mixing the embryos with vitrification solution (i.e., protective agent), use a pipette to transfer them onto the layer of absorbent resin particles. Once the excess vitrification solution has been absorbed by the absorbent resin particles, immediately place the cryovial into liquid nitrogen for freezing. After freezing, store the cryovial in a liquid nitrogen tank. When thawing, quickly remove the cryovial and place it in preheated thawing solution. Use a pipette to remove the embryos and vitrification solution from the cryovial before subsequent culture or transplantation.

[0035] The cryotransport device of the present invention works as follows: embryos are mixed with a protective agent and placed into a cryovial. Due to the liquid-absorbing properties of the liquid-absorbing resin granules (capillary action and the resin's hydrophilicity), they rapidly absorb excess embryo vitrification solution from the embryo cryosol. This reduces the volume of embryo cryosol, speeds freezing and thawing, reduces ice crystal damage to the embryo, and improves embryo survival and conception rates. Furthermore, the material and shape of the cryovial protect the embryos from external contamination and damage, facilitating embryo loading and transfer.

[0036] The cryo-carrier device of the present invention can provide embryo protection: the liquid-absorbing resin particles coating the inner wall of the freezing device can quickly absorb excess cryo-fluid, significantly reducing the risk of ice crystal formation during the freezing process. Furthermore, the polyurethane adhesive used in this device is resistant to ultra-low temperatures and remains firmly attached to the tube wall even under liquid nitrogen freezing conditions, ensuring that the polymer resin layer remains stable and does not fall off throughout the freezing and thawing process. The design of the freezing device further reduces the possibility of embryos being exposed to the external environment, lowering the risk of contamination and physical damage, and improving embryo survival and recovery rates, making it suitable for widespread application in efficient embryo cryopreservation and breeding.

[0037] Compared with the prior art, the present invention has the following beneficial effects:

[0038] (1) It has good liquid absorption performance and can quickly absorb excess embryo freezing liquid, thereby reducing the volume of embryo freezing liquid, increasing the speed of freezing and thawing, reducing the damage of ice crystals to embryos, and improving the survival rate and conception rate of embryos.

[0039] (2) The operation is simple and does not require professional personnel to absorb excess freezing fluid, which greatly improves the convenience of embryo freezing and significantly shortens the operation time of the embryo freezing process.

[0040] (3) Low cost. Since the materials and manufacturing processes of the liquid-absorbing resin particles and the freezing tube body are relatively simple and cheap, the cost of a single freezing carrier device of the present invention is less than 1 yuan, and the cost of use is much lower than that of imported freezing carrier devices. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Other features, objects and advantages of the present invention will become more apparent upon reading the detailed description of non-limiting embodiments with reference to the following drawings:

[0042] Figure 1 It is a schematic structural diagram of the freezing carrier device of the present invention;

[0043] Figure 2 This is a schematic diagram of the use process of the refrigeration device. DETAILED DESCRIPTION

[0044] The present invention is described in detail below with reference to the accompanying drawings and specific embodiments. The following examples are implemented under the premise of the technical solution of the present invention, provide detailed implementation methods and specific operating procedures, and will help those skilled in the art to further understand the present invention. It should be pointed out that the scope of protection of the present invention is not limited to the following embodiments, and a number of adjustments and improvements made under the premise of the concept of the present invention all fall within the scope of protection of the present invention.

[0045] The frozen carrier device of the present invention comprises the following steps:

[0046] 1. Design and preparation of a cryo-carrier device based on an absorbent resin. The design and preparation process of this device have been carefully optimized to ensure that it can absorb the maximum amount of embryo cryo-carrier in the shortest time.

[0047] 2. This freezing device was used to cryopreserve bovine embryos. Rigorous experimental validation has proven its effectiveness and reliability in embryo cryopreservation.

[0048] 3. Thaw and incubate the frozen embryos to assess their recovery and hatch rates. This step is the final verification of the performance of the cryo-carrier device. The performance of the device can be intuitively seen through the data of recovery and hatch rates.

[0049] Example 1

[0050] Preparation of frozen carriers, such as Figure 1 As shown:

[0051] Use scissors to cut off 3 / 4 of the front 2 cm of a 0.5 mL frozen fine tube to form a U-shaped notch. Apply a polyurethane adhesive (cyanoacrylate: Yi Shang Xiang YS-1996) to the inner tube wall at the inner surface of the notch. Liquid-absorbing resin particles (sodium polyacrylate particles) are then evenly sprinkled on the adhesive-coated inner tube wall. After the adhesive solidifies, loose particles are blown away, forming a layer of liquid-absorbing resin particles on the inner tube wall.

[0052] The inner surface of the incision is coated with a liquid-absorbing resin material with an average thickness of approximately 70 μm. This is sterilized by UV irradiation before use. The liquid-absorbing resin material should not exceed 70 μm in diameter and should absorb 5 μL of embryo cryosol within 1 minute. The resulting cryocarrier device is 2 cm long and 3 mm wide.

[0053] Example 2

[0054] The preparation of the frozen carrier is basically the same as that in Example 1, except that the liquid-absorbing resin particles are polyacrylamide particles.

[0055] Comparative Example 1

[0056] The preparation of the cryotransfer medium was essentially the same as in Example 1, except that the liquid-absorbing resin particles were not adhered to the inner tube wall but instead accumulated directly within the tube. This method has significant application drawbacks: 1) resin powder easily falls off during operation; 2) the contact area between the embryo-containing cryotransfer medium and the accumulated particles is small, resulting in slow and incomplete absorption of the cryotransfer medium (over a minute).

[0057] Comparative Example 2

[0058] The preparation of the freezing carrier is basically the same as that of Example 1, except that the diameter of the liquid-absorbing resin particles is greater than 200 μm, the contact area with the freezing liquid is greatly reduced, and the freezing liquid absorption time exceeds 1 minute.

[0059] Comparative Example 3

[0060] The preparation of the frozen carrier is basically the same as in Example 1, except that the adhesive is an epoxy resin adhesive (3M TM Scotch-Weld TM Epoxy resin adhesive 460). Epoxy resin adhesive is not resistant to liquid nitrogen low temperatures, and some resin particles will fall off after freezing.

[0061] Comparative Example 4

[0062] The preparation of the frozen carrier was essentially the same as in Example 1, except that the adhesive was silicone adhesive (Weili Intrinsic Silicone Sealant E50). Silicone adhesive has weak adhesion to the frozen fine tubes and sodium polyacrylate particles. When blowing away the loosely adhered liquid-absorbing resin particles, some particles on the tube inner wall were also blown away.

[0063] Example 3

[0064] Cryopreservation of bovine embryos

[0065] 1. In vitro ovarian egg collection

[0066] The ovaries of the slaughtered cows were cut and separated, placed in a thermos cup containing physiological saline and double-antibody liquid preheated to 35°C, and transported back to the laboratory within 6 hours.

[0067] 1.1 Ovarian Cleansing

[0068] Pour out the ovaries from the thermos cup, measure the temperature, and wash them three times with 35.5℃ saline solution supplemented with penicillin, streptomycin, and gentamicin until the saline solution is clear. Place them in a 35.5℃ water bath to maintain a constant temperature.

[0069] 1.2 Oocyte collection by aspiration

[0070] Use a 5mL disposable syringe with OCM egg collection fluid to aspirate follicles with a diameter of 2-8mm.

[0071] 1.3 Collecting eggs

[0072] Oocyte-cumulus cell complexes (COCs) that meet the culture conditions were selected under a stereomicroscope, and the COCs were washed twice in egg washing solution and once in maturation culture medium.

[0073] 2. In vitro maturation (IVM)

[0074] Oocyte maturation culture: Place the washed COCs into a four-well plate containing maturation culture medium and covered with mineral oil, culturing 30-50 COCs per well for 24 hours.

[0075] 3. In vitro fertilization (IVF)

[0076] 3.1 Semen storage conditions

[0077] The liquid nitrogen tanks used to store sperm comply with the requirements of GB / T 5458. The quality of frozen semen complies with the requirements of GB 4143.

[0078] 3.2 Fertilization process

[0079] Washing eggs: Wash the COCs three times in preheated IVF fluid and transfer them into 50 μL fertilization drops, with 15 COCs placed in each drop.

[0080] 3.2.1 Thawing of semen: Take out a sperm from the liquid nitrogen tank and let it stay in the air for 5 seconds. Then put it into 37℃ hot water to thaw for 45 seconds. Use gauze to wipe off the water stains on the outer wall of the sperm and wipe both ends with alcohol.

[0081] 3.2.2 Sperm Washing: Cut the ends of the thawed sperm and slowly pour into 3 mL of semen washing solution. Centrifuge at 300G for 5 minutes and discard the supernatant. Add another 3 mL of semen washing solution and centrifuge for 5 minutes. Remove the supernatant with a pipette, retaining 100 μL of the solution and add an equal volume of IVF solution.

[0082] 3.2.3 Sperm and egg incubation: Sperm density was measured using a sperm density meter and the sperm concentration was adjusted to approximately 2×106 10 μL of semen was added to the fertilized drop and the mixture was returned to the CO2 incubator for 20 h.

[0083] 4. In vitro culture (IVC)

[0084] 4.1 De-cumulus cell removal: 20 hours after fertilization, the fertilized eggs were removed from the fertilization solution and placed in a 1.5 mL centrifuge tube containing OCM. The tubes were shaken for 2 minutes until the cumulus cells were basically detached. The fertilized eggs with uniform cytoplasm and clear zona pellucida were selected, washed with 3 drops of development culture medium, placed in preheated development culture medium drops, and cultured in a three-gas incubator.

[0085] 4.2 Change medium and calculate fertilization rate: After 72 hours of in vitro culture, select and discard unfertilized eggs and 2-cell embryos, replace 1 / 3 drop of developmental culture medium, continue to cultivate embryos with more than 4 cells, and calculate the fertilization rate.

[0086] 4.3 Calculation of blastocyst rate: Observe the fertilized eggs on the 7th day after fertilization and calculate the blastocyst rate.

[0087] Table 1 Developmental culture solution formula

[0088]

[0089]

[0090] 5. Embryo Freezing (Vitrification)

[0091] 5.1 Embryo freezing medium: MH, V1, V2, V3.

[0092] Table 2 Embryo freezing solution formula

[0093]

[0094] Equilibrate the embryos in the order of base solution - vitrification solution 1 - vitrification solution 2 - vitrification solution 3.

[0095] 5.2 Freezing procedure:

[0096] a) Basal solution: Equilibrate the embryos for 5 minutes at room temperature (24-25°C).

[0097] b) Vitrification Solution 1: Equilibrate embryos for 5 minutes at room temperature (24-25°C).

[0098] c) Vitrification Solution 2: Equilibrate embryos for 5 minutes at room temperature (24-25°C).

[0099] d) Vitrification Solution 3: Equilibrate embryos for 45 seconds at room temperature (24-25°C).

[0100] e) If Figure 2 As shown, 3 drops of 5 μL of vitrification solution containing embryos are placed on the liquid-absorbing resin particle layer. After standing for about 40 seconds, the excess freezing solution is absorbed by the liquid-absorbing resin particle layer, and then the freezing carrier is immersed in liquid nitrogen.

[0101] 5.3 Embryo freezing storage: The liquid nitrogen tanks used to store embryos must comply with the requirements of GB / T 5458.

[0102] 6. Embryo Thawing

[0103] 6.1 Embryo thawing medium: W1, W2, MH.

[0104] Table 3 Embryo thawing solution formula

[0105]

[0106] Equilibrate the embryos in the order of Thawing Solution 1, Thawing Solution 2, and Basal Solution.

[0107] 6.2 Thawing procedure:

[0108] a) Thawing solution 1: Equilibrate embryos for 1 minute at 38°C.

[0109] b) Thawing solution 2: Equilibrate embryos for 5 minutes at room temperature (24-25°C).

[0110] c) Basal medium: Equilibrate the embryos for 5 minutes at room temperature (24-25°C).

[0111] 7. Embryo Development: Thawed blastocysts were placed in M16 culture medium and cultured for 48 hours in an incubator at 37°C and 5% CO2. After 24 hours and 48 hours, the recovery rate and blastocyst hatching rate were recorded and counted (Table 4).

[0112] Recovery rate = number of surviving blastocysts / number of thawed blastocysts × 100%;

[0113] Blastocyst rate = number of blastocysts / total number of thawed embryos × 100%;

[0114] Blastocyst hatching rate = number of hatched blastocysts / total number of blastocysts × 100%.

[0115] Table 4

[0116] Experimental group Number of bovine embryos Number of recoveries Recovery rate Number of hatches hatching rate Control (imported frozen carrier) 20 18 90％ 16 80％ Frozen carriers 9 7 78％ 4 44％

[0117] Experimental results show that the frozen carrier device of the present invention has a bovine embryo recovery rate of approximately 80% and a hatching rate of approximately 45%. Although the embryo hatching and recovery rates of the present invention still need to be improved compared to the commercial frozen carrier device Cryotop, due to its advantages such as ease of use and low price, the present invention has broad application prospects.

[0118] The above describes the specific embodiments of the present invention. It should be understood that the present invention is not limited to the above specific embodiments, and those skilled in the art may make various variations or modifications within the scope of the claims, which do not affect the essence of the present invention.

Claims

1. A freezing carrier device capable of absorbing embryo freezing solution, characterized in that: The freezing carrier device comprises: a freezing tube body, a liquid-absorbing resin particle layer; The liquid-absorbing resin particles are adhered to the inner wall of the freezing tube body by an adhesive to form a liquid-absorbing resin particle layer; The liquid-absorbing resin particles are one or more of liquid-absorbing polyacrylamide and sodium polyacrylate particles; the particle size of the liquid-absorbing resin particles does not exceed 70 μm; The adhesive for liquid absorption is a polyurethane adhesive.

2. The frozen carrier device according to claim 1, characterized in that Drop 5 μL of embryo freezing solution on the absorbent resin particle layer and absorb the solution, which can be absorbed within 1 minute.

3. The frozen carrier device according to claim 1, characterized in that The material of the freezing tube body includes one or more of plastic, glass, metal, and ceramic; And / or, the shape of the freezing tube body includes one of cylindrical, U-shaped and V-shaped.

4. The frozen carrier device according to claim 1, characterized in that The length of the freezing tube body is 2-5 cm and the diameter is 2-5 mm.

5. A method for preparing a frozen carrier device according to claim 1, characterized in that: The steps include: An adhesive is applied to the inner wall of the freezing tube body, and then liquid-absorbing resin particles are evenly sprinkled on the inner wall coated with the adhesive. After the adhesive is solidified, a liquid-absorbing resin particle layer is formed on the inner wall with tiny gaps between the particles, thereby obtaining the freezing carrier device.

6. Use of the frozen carrier device according to claim 1 in animal breeding.

7. A method for using the freezing carrier device according to claim 1 in embryo cryopreservation, characterized in that: The steps include: After mixing the embryos and vitrification solution, transfer them to the layer of absorbent resin particles with a pipette. After the excess freezing solution is absorbed by the absorbent resin particles, immediately place the cryovial body into liquid nitrogen for freezing. After freezing, store the cryovial body in a liquid nitrogen tank. When thawing, quickly take out the cryovial body and place it in preheated thawing solution. Use a pipette to suck out the embryos and protective agent from the cryovial body, and then carry out subsequent culture or transplantation.

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