Embryo thawing device and embryo thawing and transfer method using the same

Abstract

To provide an embryo thawing instrument that can realize superior survival rates and operability, when thawing embryos cryopreserved by vitrification, and to provide an embryo thawing transfer technique.SOLUTION: Provided are an embryo-thawing instrument comprising: a thawing part for thawing a cryopreserved embryo with a thawing solution; an insertion part for inserting an embryo into the thawing part; and an ejection part for ejecting the thawed embryo from the thawing part, the maximum inner diameter of the thawing part or the maximum diagonal length of the inner surface of the thawing part being larger than the inner diameter of the insertion part, and the inner diameter of the ejection part being the same as or smaller than the inner diameter of the insertion part; and an embryo thawing transfer technique using the same.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to an embryo thawing instrument that can achieve excellent survival rates and operability when thawing vitrified, cryopreserved embryos, and to an embryo thawing and transfer method using the thawing instrument. [Background technology]

[0002] Cryopreservation of animal embryos allows for the preservation of genetic resources of specific lineages or breeds, and is necessary for the maintenance of endangered animal species. Furthermore, in the livestock industry, the technique of transplanting cryopreserved embryos into the reproductive tract of mammals (embryo transfer) has become widespread with the aim of increasing the production and improving mammalian species. Traditionally, in embryo transfers performed on farms, the embryos are frozen in plastic straws with cryopreservation solution, stored in liquid nitrogen, and at the time of transfer, the embryos in the straws are thawed and loaded into a transfer vessel along with the cryopreservation solution, allowing the embryos to be transferred directly into the uterus (direct transfer method).

[0003] One known method for cryopreserving embryos is the slow freezing method. In this method, embryos are immersed in a cryopreservation solution containing a relatively low concentration of cryoprotectant and cooled to -30°C to -35°C at a relatively slow rate of -0.3 to -0.5°C / min for an extended period of time, resulting in the formation of ice crystals in the cryopreservation solution. During this process, an osmotic pressure difference occurs between the cryopreservation solution and the embryo, where no ice crystals have formed, and the embryo gradually dehydrates. This sufficiently concentrates the cryopreservation solution within and around the embryo, resulting in a vitrified state in which no ice crystals form in the cryopreservation solution within or around the embryo, even when cooled to the temperature of liquid nitrogen (-196°C). By storing vitrified embryos in liquid nitrogen, molecular movement is essentially eliminated, making it possible to preserve them semi-permanently.

[0004] When embryos cryopreserved by slow freezing are transferred into the uterus by the direct method, the one-step straw method, in which the embryos are cryopreserved in a straw, is known (Non-Patent Document 1). When performing transfer using the one-step straw method, similar to artificial insemination, the preserved embryos are thawed in a straw outdoors, such as in a livestock farmer's yard, and the straw is directly loaded into a transfer device for embryo transfer. This method is the most widely used because it does not require special facilities at the transfer site and can be performed with the same ease as artificial insemination. However, the current survival rate of embryos stored using this method is 80% to 90%, and the conception rate is less than 50%. Therefore, a simple method that can achieve high survival and conception rates has been sought.

[0005] Vitrification is also known as a method for cryopreserving embryos (Non-Patent Document 2). This method involves using a vitrification solution containing a high concentration of a cryoprotectant (cryoprotectant) to penetrate the sample cells, thoroughly dehydrating the cells, and then vitrifying the intracellular and extracellular solutions of the sample by rapid cooling in liquid nitrogen, making it possible to freeze the sample with almost no ice crystal formation. For this reason, embryos cryopreserved using vitrification are known to have a higher pregnancy rate and produce healthier offspring than embryos cryopreserved using slow freezing.

[0006] However, because the high concentration of cryoprotectants contained in the vitrification solution is highly toxic to embryos, the frozen embryos must be removed from the straw after thawing and immersed in a diluent. Therefore, in conventional vitrification methods, the cryoprotectant that had permeated the embryos must be removed by immersion in a diluent, and the embryos must then be returned to the straw and loaded into the transfer device. However, this procedure must be performed under a microscope, and from a hygienic standpoint, it is difficult to perform on livestock farms.

[0007] Therefore, efforts have been made to develop methods for diluting the cryoprotectant in embryos frozen by vitrification in a straw and then directly transplanting them. One such method is the "shaking method" (Patent Document 1), in which a vitrification solution and a diluting solution are placed in a straw with an air gap between them, and the two layers are mixed by shaking the straw during thawing, thereby diluting the cryoprotectant from the embryo. However, vitrification solutions containing high concentrations of cryoprotectant are highly viscous, making their mixing with the diluting solution unstable. Furthermore, methods using straws cannot completely prevent various damages (such as ice crystal formation damage and fracture damage) associated with freezing and thawing, and these remain issues that have prevented this method from becoming widespread.

[0008] In recent years, ultra-rapid vitrification methods have been developed, in which embryos are frozen more rapidly by directly contacting a film carrying an embryo together with a minute amount of vitrification solution (Patent Documents 2 to 4) or a special loop (Patent Document 5, embryo placement site) with liquid nitrogen. This ultra-rapid vitrification method allows the embryos to pass through the temperature range in which ice crystals are likely to form during the freezing and thawing process in an extremely short time, reduces the concentration of cryoprotectants, and prevents damage to ice crystal formation or fracture, resulting in a very high survival rate. Clinical application of this method has been progressing in the field of assisted reproductive technology in humans in recent years, and it is expected that it will also be used in livestock.

[0009] However, these ultra-rapid vitrification methods require the use of minute amounts of vitrification fluid and special cryopreservation equipment for handling the embryos. Therefore, in order to transfer the embryos, the frozen embryos must be thawed in a petri dish or other container, the cryoprotectant diluted, and then placed in a transfer straw and loaded into a transfer device. Furthermore, these procedures must be performed under a microscope and in a hygienic environment, which is extremely cumbersome and difficult to perform on livestock farms' backyards. Therefore, efforts have been made to develop technologies that allow embryos cryopreserved by ultra-rapid vitrification to be thawed in a straw or special equipment, the cryoprotectant diluted, and then loaded directly into a transfer device for direct transfer. Techniques that enable direct transfer and suitable equipment for such techniques have been proposed. Three examples are presented below, each of which is explained.

[0010] The aforementioned Patent Document 5 discloses a transfer straw and its use for cryopreserving vitrified mammalian embryos in a container, thawing and diluting them by heating, and then transplanting them. A vitrification device with an embryo placement section is attached to the straw, allowing embryos on the embryo placement section to be vitrified and preserved using an ultra-rapid vitrification method. The melting procedure, on the other hand, involves immersing the transfer straw in hot water, causing the air layer to expand, moving the diluent layer toward the embryo placement section, thereby immersing the frozen embryo in the diluent layer and thawing it. However, because this method involves immersing the straw in lukewarm water, the melting rate is slower than in methods where the embryo is directly immersed in the melting solution, which may result in a lower survival rate.

[0011] Patent Document 6 discloses a mammalian embryo storage device and a mammalian embryo transfer straw, and methods for using the same. The storage device has an embryo placement area, and by placing an embryo on the embryo placement area together with a small amount of vitrification liquid, cryopreservation by ultra-rapid vitrification is possible. The thawing procedure involves immersing the straw, which has been stored with the storage device attached, in warm water, which raises concerns about survival rates, similar to the method disclosed in Patent Document 5.

[0012] Patent Document 7 discloses an instrument and method for thawing vitrified germ cells. The thawing procedure in this method also involves immersing the thawing instrument, into which the cell-mounting instrument is inserted, into warm water. This raises concerns about the results, similar to the methods disclosed in Patent Documents 5 and 6.

[0013] The thawing methods for frozen embryos described in the above-mentioned Patent Documents 5 to 7 involve thawing by immersing a straw in warm water, which does not achieve a sufficient thawing rate. Furthermore, the cryopreservation devices used in these thawing methods require a cryopreservation device with a shape that matches the immersion of frozen embryos or cells in a melting or dilution solution contained in a straw. Therefore, commercially available cryopreservation devices suitable for ultra-rapid vitrification cannot be used. Therefore, there is a need for a thawing and transfer method for embryos preserved by ultra-rapid vitrification that not only has a high survival rate but also offers excellent operability, as well as a thawing device that enables this method. [Prior art documents] [Patent documents]

[0014] [Patent Document 1] Japanese Patent Application Publication No. 10-277067 [Patent Document 2] Japanese Patent Application Laid-Open No. 2002-315573 [Patent Document 3] Japanese Patent Application Laid-Open No. 2006-271395 [Patent Document 4] International Publication No. 2015 / 064380 Brochure [Patent Document 5] Japanese Patent Application Publication No. 2019-170962 [Patent Document 6] Japanese Patent Application Laid-Open No. 2014-184056 [Patent Document 7] Japanese Patent Application Publication No. 2018-7662 [Non-patent literature]

[0015] [Non-Patent Document 1] Suzuki Tatsuyuki et al., "Transplantation of Bovine Frozen Embryos by the One-Step Straw Method," Journal of Animal Theriogenology, Vol. 29 (1983) No. 3, pp. 162-163, Published by the Japanese Society of Reproduction Biology [Non-patent document 2] Masashige Kuwayama, "Cryopreservation of Livestock, Especially Cow's Oocytes and Embryos," Journal of Cryogenic Biotechnology, Vol. 46 (2000) No. 1, pp. 26-29, Published by the Society of Cryogenic Biotechnology Summary of the Invention [Problem to be solved by the invention]

[0016] An object of the present invention is to provide an embryo thawing device that can achieve a high survival rate and ease of use when thawing embryos that have been cryopreserved by vitrification, preferably by ultra-rapid vitrification, and to provide an embryo thawing and transfer method that can achieve a high survival rate and ease of use.

[0017] As a result of extensive research into solving the above problems, the present inventors have found that the above problems can be solved by the following embryo thawing device and embryo thawing and transfer method. [Means for solving the problem]

[0018] (1) An embryo thawing device comprising a melting section for melting a cryopreserved embryo with a melting solution, an insertion section for inserting the embryo into the melting section, and a discharge section for discharging the thawed embryo from the melting section, wherein the maximum inner diameter of the melting section or the maximum diagonal length of the inner surface of the melting section is larger than the inner diameter of the insertion section, and the inner diameter of the discharge section is the same as or smaller than the inner diameter of the insertion section. (2) A method for thawing and transferring embryos, comprising at least the steps of: a filling step of filling a melting liquid into the melting section of the embryo thawing device described in (1) above; an insertion step of inserting a cryopreserved embryo into the melting section from the insertion section of the thawing device; a melting step of melting the embryo in the melting liquid; and a transfer step of sending a gas or liquid from the insertion section into the melting section, discharging the thawed embryo from the discharge section, and transferring it into the mother's body (excluding humans). [Effects of the Invention]

[0019] According to the present invention, an embryo thawing device that can achieve a high survival rate and ease of use when thawing embryos that have been cryopreserved by vitrification, preferably by ultra-rapid vitrification, can be provided. Furthermore, according to the present invention, an embryo thawing and transfer method that can achieve a high survival rate and ease of use can be provided. [Brief explanation of the drawings]

[0020] [Figure 1] 1 is a schematic cross-sectional view showing an example of a melting apparatus of the present invention. [Figure 2] FIG. 2 is a schematic cross-sectional view showing another example of the melting apparatus of the present invention. [Figure 3] FIG. 10 is a schematic cross-sectional view showing yet another example of the melting apparatus of the present invention. [Figure 4] 10A and 10B are perspective views showing examples of shapes of transplanter connecting devices. [Figure 5] 1 is a perspective view showing an example of the shape of the discharge portion of the melting device of the present invention. FIG. [Figure 6] 6 is a schematic cross-sectional view of the implanter connecting device shown in FIG. 4 and the discharge section shown in FIG. 5 when mated. DETAILED DESCRIPTION OF THE INVENTION

[0021] The present invention will be described in detail below.

[0022] The embryo thawing instrument of the present invention (hereinafter also referred to as the thawing instrument of the present invention) and the embryo thawing and transfer method using the thawing instrument (hereinafter also referred to as the thawing and transfer method of the present invention) are suitable for use in thawing and transfer procedures of vitrified, cryopreserved embryos of mammalian species.

[0023] The embryos to be thawed and transferred using the thawing instrument and thawing and transfer method of the present invention are not particularly limited as long as they have been cryopreserved by a known method such as slow freezing, vitrification, or ultra-rapid vitrification. However, from the viewpoint of embryo survival rate and conception rate, it is preferable that the embryos be cryopreserved by ultra-rapid vitrification.

[0024] The melting device of the present invention comprises a melting section for melting cryopreserved embryos with a melting liquid, an insertion section for inserting embryos into the melting section, and a discharge section for discharging the melted embryos from the melting section. From the viewpoint of workability, each of the insertion section and discharge section is preferably hollow and cylindrical. The inner diameter of the cavity in each section may or may not be constant, but it is preferred that the inner diameter of the cavity in the insertion section and discharge section be constant. When the melting section is cylindrical, its cross-sectional shape (the cross-sectional shape of the dashed-dotted line in FIG. 1 , specifically, the internal cross-sectional shape in the direction perpendicular to the direction of melting liquid flow (x direction in FIG. 1 )) is circular. Other examples of the cross-sectional shape include polygons such as squares and hexagons, but from the viewpoint of workability, a circular cross-sectional shape is preferred.

[0025] The materials used for the insertion section, melting section, discharge section, and other sections described above, which are filled with or pass through the melting liquid, are not particularly limited as long as they can retain the melting liquid without permeating or leaking. Suitable materials include, for example, various resins such as ABS resin, acrylic resin, polypropylene resin, polystyrene resin, and polyethylene resin; various metals such as iron and aluminum; and even glass and rubber. Resins are particularly preferred because they have excellent impact resistance and water resistance, as well as a small weight and high transparency, allowing for good visibility when filling the container with melting liquid and inserting frozen embryos from the insertion section into the melting section. Each section of the melting instrument of the present invention may be made of a single material or multiple materials.

[0026] The insertion part of the thawing instrument of the present invention has an appropriate inner diameter and length, which allows for easy operation when filling with thawing liquid, inserting and thawing the cryopreservation jig that cryopreserves embryos, and fitting the ejection instrument described below. The outer diameter of the insertion part is preferably 5.0 to 9.0 mm, and the inner diameter is preferably 3.0 to 7.0 mm. This allows for easy insertion of the aforementioned film or loop carrying an embryo together with a very small amount of vitrification liquid (the width of the film or loop is preferably 0.5 to 2.5 mm, from the viewpoints of easy operation when placing the embryo and limiting the amount of vitrification liquid placed together with the embryo) into the thawing part, and also allows for easy attachment of a liquid delivery means (e.g., an extrusion syringe) used when transplanting an embryo into the uterus, described below. The length of the insertion part (length in the liquid transfer direction) is not particularly limited as long as it does not interfere with the insertion of the cryopreservation jig or the connection and discharge operations of the liquid transfer means, but is preferably 3.0 to 7.0 mm.

[0027] The outer diameter of the melting part of the melting device of the present invention, or the maximum diagonal length of the outer surface of the melting part in a direction perpendicular to the direction of melting liquid flow (hereinafter referred to as the maximum diagonal length of the outer surface of the melting part), is preferably 8.0 to 15.0 mm. Furthermore, the maximum inner diameter of the melting part, or the maximum diagonal length of the inner surface of the melting part in a direction perpendicular to the direction of melting liquid flow (hereinafter referred to as the maximum diagonal length of the inner surface of the melting part), is preferably 6.0 to 9.0 mm. This allows frozen cells or tissues to be immersed in the melting liquid in the melting part with good workability. Furthermore, the length of the melting part (internal length in the direction of liquid flow) is preferably 15 to 35 mm. In the present invention, as described below, when cells or tissues are discharged from the thawing device, preferably 0.3 to 2.0 ml of thawing liquid is used. In this case, it is preferable that the maximum inner diameter of the thawing section or the maximum diagonal length of the inner surface of the thawing section is 6.0 to 9.0 mm, since this allows the cells or tissues to be moved into the transplanter at a sufficient speed.

[0028] The inner diameter of the discharge part of the thawing device of the present invention is not particularly limited as long as it is the same as or smaller than the inner diameter of the insertion part, but is preferably 0.25 to 3 mm. Furthermore, in the thawing device of the present invention, the maximum inner diameter of the melting part or the maximum diagonal length of the inner surface of the melting part is larger than the inner diameter of the insertion part, and the inner diameter of the discharge part is the same as or smaller than the inner diameter of the insertion part. This improves the operability when inserting frozen embryos into the melting part and immersing them in the melting liquid filled in the melting part, and the reliability when extruding thawed embryos into a transplanter connected to the discharge part, thereby enabling the realization of particularly excellent survival rates and operability.

[0029] The melting device of the present invention is characterized in that the inner diameter of the discharge part of the melting device is the same as or smaller than the inner diameter of the insertion part, and preferably also satisfies the following formula (1). This makes it possible to obtain a melting device that is particularly excellent in operability and reliability as described above. In formula (1), D1 represents the inner diameter of the insertion part, and D2 represents the inner diameter of the discharge part. 1.0mm<(D1-D2)≦3.0mm (1)

[0030] From the viewpoint of operability, the total length of the melting device of the present invention, which includes the insertion section, melting section, and discharge section described below (length in the liquid transfer direction), is preferably 30 to 60 mm.

[0031] The outer diameter of the discharge part of the thawing device of the present invention is not particularly limited as long as it can be connected to a transplanter, but is preferably 1 to 5 mm. Furthermore, the length of the discharge part (length in the direction of fluid transfer) is preferably 3 to 20 mm. Because the discharge part is used to discharge embryos, appropriate adjustment of its outer diameter, inner diameter, length, etc. ensures reliable connection to the transplanter or to the transplanter-connecting device described below, which is expected to improve operability and stability and the embryo discharge rate.

[0032] The inlet of the insertion part and the outlet of the discharge part of the melting device of the present invention can be provided with a lid or cap that fits the shape of the inlet and the outlet.

[0033] The embryo thawing device of the present invention will be described in detail below with reference to the drawings: Figure 1 is a schematic cross-sectional view showing an example of the thawing device of the present invention. The melting device 1 shown in Fig. 1 comprises an insertion section 2, a melting section 3, and a discharge section 4. From the viewpoint of operability, the shape of the melting section of the melting device of the present invention is preferably such that the inner diameter D1 of the insertion section 2 is larger than the inner diameter D2 of the discharge section 4, as in the melting device 1 shown in Fig. 1, but may also be such that the inner diameter D1 of the insertion section 2 and the inner diameter D2 of the discharge section 4 are equal, as in the melting device 1 shown in Fig. 2.

[0034] The shape of the melting portion of the melting device of the present invention is not particularly limited as long as its maximum inner diameter or the maximum diagonal length of the inner surface of the melting portion is greater than the inner diameter of the insertion portion, and it may be cylindrical like the melting device 1 shown in Figure 1 (the inner diameter is constant throughout the melting portion), or may be a tube-like shape like the melting device 1 shown in Figure 3 (the inner diameter varies throughout the melting portion).

[0035] Figure 3 is a cross-sectional view showing another example of the melting apparatus of the present invention. The cylindrical shape of the melting section in Figure 3 has a shape in which the inner diameter of the melting section 3 continuously increases from the outlet of the insertion section 2 toward the discharge section on the insertion section side, and a shape in which the inner diameter of the melting section 3 continuously decreases toward the discharge section 4 on the discharge section side. The cylindrical shape shown in Figure 3 is preferred because it provides a melting apparatus that is excellent at discharging thawed embryos.

[0036] The smaller the angle α (the spread angle of the melting section on the insertion section side) and angle β (the constriction angle of the melting section on the discharge section side) in the melting device 1 in Figure 3, the smoother the connection between the various sections (angle α between the insertion section 2 and the melting section 3, and angle β between the melting section 3 and the discharge section 4). For example, when angles α and β are each 90°, the melting device of the present invention is as shown in Figures 1 and 2. In the transfer operation using the melting device of the present invention, it is most important that the thawed embryos are smoothly discharged from the discharge section, so it is preferable that angle β is relatively small. Specifically, from the perspective of ensuring reliable discharge of the thawed embryos, an angle that satisfies the following formula (2) is preferred. Furthermore, it is preferable that angle α be an angle that satisfies the following formula (3). 5°≦β≦10° (2) 30°≦α≦90° (3)

[0037] The shape of the discharge part of the thawing device of the present invention may be changed from a cylindrical shape to ensure a stronger connection with the transplanter used and more reliable embryo transfer. For example, the animal embryo transfer catheter Mo No. 4 (hereinafter referred to as Mo No. 4), a transplanter manufactured by Misawa Medical Industries Co., Ltd., has a transplanter connection part 5 for connecting the thawing device, which has an internal shape hollowed out by two cylinders of different diameters, as shown in Figure 4. When connecting the thawing device of the present invention to a transplant catheter of this shape, by changing the shape of the discharge part to the shape shown in Figure 5 (specifically, a shape in which two cylinders with the same inner diameter but different outer diameters are connected so that the internal spaces are continuous), the Mo No. 4 connection part 5 and the deformed discharge part 6 of the thawing device fit together without any gaps, as shown in Figure 6, which is expected to improve the stability of the connection and the reliability of the transfer.

[0038] The internal volume of the melting section of the melting device of the present invention is preferably 0.3 to 2.0 ml, more preferably 0.35 to 1.5 ml, from the viewpoint of the melting rate of the cryopreserved embryos and avoiding excessive amounts of melting liquid delivered to the animal's body along with the embryos. If the internal volume of the melting section is too small, the melting rate of the cryopreserved embryos will be slow, which may cause damage to the embryos. Furthermore, if the internal volume of the melting section is large, more melting liquid will be required when transferring the embryos to the animal's body, which may affect the intrauterine environment of the animal to be transplanted.

[0039] The thawing instrument of the present invention can be referred to as an instrument for thawing a cryopreserved embryo, an instrument for thawing a cryopreserved embryo, an instrument for thawing a cryopreserved embryo, an instrument for thawing a cryopreserved embryo, an instrument for thawing a cryopreserved embryo, an instrument for thawing a cryopreserved embryo, or the like.

[0040] The thawing solution used to thaw embryos using the thawing device of the present invention can be any solution commonly used for thawing cells such as embryos, oocytes, etc. For example, a thawing solution containing a physiological solution such as phosphate-buffered saline containing 1 M sucrose to adjust the osmotic pressure can be used.

[0041] The melting apparatus filled with the melting solution along with the embryos is preferably operated to a temperature of 39±2°C, with a more preferred temperature range being 39±1°C.

[0042] The ejection instrument used to eject the thawed embryos (the ejection instrument connected to the insertion part 2) should be an ejection instrument that can be connected to the embryo transplanter, as this is convenient and eliminates the need to prepare multiple ejection instruments when transplanting, and is not particularly limited as long as it is capable of pushing out the embryos in the thawing instrument and the transplanter and ejecting them from the tip of the transplanter.However, it is preferable that the ejection instrument be a syringe with an internal volume of approximately 1.0 to 2.0 ml so that no embryos are left behind in the thawing instrument and the transplanter and ejected.

[0043] It is preferable to fill the syringe used as the ejection device with 0.3 to 1.0 ml of thawing solution, so that the embryos can be ejected from the thawing device and the transfer device by pumping the thawing solution into the syringe during the transfer operation, allowing for smooth transfer.

[0044] Furthermore, in the transplantation method using the thawing device of the present invention, the following exemplary operations can also be carried out: The outlet of the discharge part is sealed with a lid or cap, and frozen embryos are thawed using the thawing device with the melting part filled with melting liquid, and after sealing the inlet of the insertion part with the lid or cap, the seal on the outlet of the discharge part is removed and the discharge part of the thawing device is connected to the connection part of a transplanter (e.g., one equipped with a connection part for connecting the thawing device and a tube reaching from the vagina to the uterus of the recipient animal). The seal on the inlet of the insertion part of the thawing device connected to the transplanter is removed, and a discharge device filled with gas or liquid in an amount sufficient to discharge the melting liquid containing the embryos from the thawing device is attached to the inlet of the insertion part of the thawing device, and the melting liquid containing the embryos from the thawing device is transferred to the transplanter using the discharge device. After the transfer of the thawing liquid is completed, the thawing device is removed from the implanter, and a discharge device filled with a volume of gas or liquid sufficient to discharge the thawing liquid containing the embryos from the implanter is attached to the connection part of the implanter. After the implanter is set in the mother's body, the discharge device is used to discharge the thawing liquid containing the embryos from the implanter. This method is preferable because it is easy to operate and can be performed without causing significant stress to the mother, since the embryos are transferred into the implanter before it is set in the mother's body.

[0045] The thawed embryo transfer technique of the present invention can be rephrased as a thawed transfer technique for vitrified cryopreserved embryos, a thawed transfer method for vitrified cryopreserved embryos, a thawed transfer protocol for vitrified cryopreserved embryos, a thawed transfer protocol for vitrified cryopreserved embryos, a thawed transfer technique for vitrified cryopreserved embryos, or a thawed transfer method for vitrified cryopreserved embryos. [Example]

[0046] The present invention will be described in more detail below with reference to examples, but the present invention is not limited to the following examples.

[0047] (Example) An example melting device having a cylindrical melting section, as shown in Figure 3, was fabricated by resin cutting using polycarbonate. The inner diameters of the insertion section 2 and discharge section 4 of the melting device were constant, while the angle α of the melting section 3 was 34°, gradually increasing from the boundary with the insertion section. When the inner diameter of the melting section 3 reached its maximum inner diameter, it gradually decreased toward the discharge section 4 (angle β was 8°), smoothly connecting to the discharge section 4. The melting device 1 had a maximum outer diameter (outer diameter of the melting section 3) of 10 mm and a length of 52 mm. The insertion section 2 had a length of 5 mm, an outer diameter of 6 mm, and an inner diameter (D1) of 4 mm. The melting section 3 had a length of 32 mm and an inner diameter of 8 mm, while the discharge section 4 had a length of 15 mm, an outer diameter of 4 mm, and an inner diameter (D2) of 2 mm. Also, D1-D2 is 2 mm.

[0048] (Experimental conditions) 1. Media used and composition 1) Base solution: 20mM HEPES buffer modified TCM199 + 20% fetal bovine serum 2) Equilibration solution: Base solution + 0.25 M sucrose + 7.5% ethylene glycol + 7.5% DMSO 3) Vitrification solution: base solution + 0.5 M sucrose + 15% ethylene glycol + 15% DMSO 4) Melting solution: Base solution + 0.3M sucrose 5) Thawed embryo culture medium: 25mM HEPES buffered TCM199 + 5% fetal bovine serum 2. Temperature during equilibration and vitrification: Room temperature (20-25°C) 3. Temperature during thawing and dilution of cryoprotectant: 39°C 4. Temperature and atmosphere for thawed embryo culture: 39°C and 5% CO2, 5% O2, 90% N2 5. Vitrification storage device used: Diamour-op (manufactured by Mitsubishi Paper Mills, Ltd.)

[0049] (Experimental Method) 1. Embryo Vitrification and Cryopreservation 1) One or two bovine embryos (developmental stage: blastocyst stage) were immersed in the equilibration solution and kept there for 10 minutes. 2) Next, the bovine embryos were transferred into the vitrification solution and held there for 30 seconds while repeatedly pipetting. 3) Immediately afterwards, the bovine embryo was placed on the absorbent body of the vitrification storage device Diamour-op (hereinafter referred to as the device), and after confirming that the excess vitrification liquid around the embryo had been absorbed, the device was immersed in liquid nitrogen for vitrification. 4) The device body was placed in an outer tube and stored in liquid nitrogen for approximately one month. 2. Preparation of melting equipment for vitrified embryo melting 1) The melting device obtained as described above was filled with 0.35 ml of melting liquid in advance. 2) The melting device filled with melting liquid (the inlet of the insertion part and the outlet of the discharge part were sealed with caps) was placed upright (with the insertion part facing upward) on a support stand (test tube stand) and kept at 39°C for several tens of minutes. 3. Thawing of Vitrified Embryos 1) The device body and outer cylinder were separated in liquid nitrogen in preparation for the melting operation. 2) After removing the cap from the insertion part of the thawing device, the tip of the device was quickly removed from the liquid nitrogen and inserted into the insertion part of the thawing device, and the bovine embryo was immersed in the thawing solution. The device was held in place for about 10 seconds, and then gently moved up and down for about 5 seconds before being removed from the thawing device. 3) Next, a 1 ml syringe with an air gap of approximately 0.4 ml (to ensure that the 0.35 ml of melting liquid containing the bovine embryos inside the melting device was expelled from the melting device) was attached to the insertion part, and the melting device was left standing on the support stand for 10 minutes (the time when the tip of the device was immersed in the melting liquid was counted as 0 minutes) to melt the vitrified embryos and dilute the cryoprotectant with the melting liquid. 4) After the cryoprotectant dilution treatment was completed, the cap on the outlet at the bottom of the thawing device was opened, the syringe inner tube was pushed out, and air pressure was applied to eject the embryos together with the thawing solution into a dish, where they were then collected. 5) The collected embryos were washed several times with thawed embryo culture medium, then transferred to 0.030 ml microdroplets of the same medium and cultured for 72 hours.

[0050] (Experimental results) The above-described embryo vitrification cryopreservation and vitrified embryo thawing experiments were repeated four times using four thawing devices of the present invention. Four devices were used in the vitrification and thawing experiments, and six vitrified embryos were tested. That is, two single embryos and two double embryos were placed on each device, for a total of six embryos. The embryo recovery results after thawing and the culture results of the thawed embryos were as follows. (1) Embryo recovery results After thawing, all six test embryos were recovered, resulting in a recovery rate of 100%. (2) Thawed embryo culture results The survival rate (expansion of the blastocoel) after 24 hours of culture was 100% (6 / 6), and the hatching rate after 72 hours of culture was 83.3% (5 / 6). These results were comparable to the survival rate of 100% (51 / 51) and the hatching rate of 82.2% (42 / 51) in the comparative example described below.

[0051] (Comparative Example) The experimental conditions and methods up to "1. Embryo vitrification and cryopreservation" were carried out in exactly the same manner, except for the thawing of the vitrified embryos, which was carried out as follows in the comparative example. 1) The device body and outer cylinder were separated in liquid nitrogen, and a dish filled with 1.0 ml of melting liquid was placed under a microscope in preparation for the melting operation. 2) The tip of the device was quickly removed from liquid nitrogen and immersed in a melting solution to thaw the bovine embryo. The detachment of the bovine embryo from the device was confirmed under a microscope, and the cryoprotectant was diluted at the same time. 3) After the cryoprotectant dilution treatment, the embryos were collected, washed several times with thawed embryo culture medium, and then transferred to 0.030 ml microdroplets of the same medium and cultured for 72 hours.

[0052] This comparative example simulates a commonly used method for thawing and transferring vitrified-frozen bovine embryos using the ultra-rapid vitrification method. The method of this comparative example requires the use of a microscope for the thawing procedure, making it difficult to perform on a livestock farm's premises. However, because the ultra-rapid vitrification method is used as the freezing method, the results are good. Experiments using the thawing device and thawing and transfer method of the present invention demonstrated results comparable to those of the comparative example. Therefore, the thawing device and thawing and transfer method of the present invention are believed to be capable of achieving both thawing and transfer of frozen embryos on a livestock farm's premises and good results. [Industrial Applicability]

[0053] The present invention can be used mainly in backyard thawing and transfer procedures for embryo transfer and artificial insemination of livestock and animals such as cattle using the ultra-rapid vitrification method. [Explanation of symbols]

[0054] 1. Melting equipment 2 Insertion section 3 Melting section 4 Discharge section 5. Transplanter connection 6. Deformed discharge section

Claims

1. An embryo thawing device comprising: a melting section for melting a cryopreserved embryo with a melting liquid; an insertion section for inserting an embryo into the melting section; and a discharge section for discharging the thawed embryo from the melting section, wherein the maximum inner diameter of the melting section or the maximum diagonal length of the inner surface of the melting section is larger than the inner diameter of the insertion section, and the inner diameter of the discharge section is the same as or smaller than the inner diameter of the insertion section.

2. 1. A method for thawing and transferring embryos, comprising at least the steps of: a filling step of filling a melting liquid into the melting section of the embryo thawing device described in claim 1; an insertion step of inserting a cryopreserved embryo into the melting section from the insertion section of the thawing device; a melting step of thawing the embryo in the melting liquid; and a transplantation step of sending a gas or liquid from the insertion section into the melting section, discharging the thawed embryo from the discharge section, and transplanting it into a mother's body (excluding a human).

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