JPWO2011070973A1 - Tubular for vitrification preservation of animal embryos or eggs - Google Patents

Abstract

Provided is a vitrification storage tubule capable of vitrifying and storing animal embryos and the like with excellent operability and high survival rate. A thin tube for vitrifying and storing an animal embryo or egg consisting of a container main body and a sheath, wherein the container main body comprises an outer handle and an inner handle connected to the outer handle, A vitrification preservative removal material is fixed to the inner handle, and the vitrification preservative removal material is provided with at least one embryo or ovum, and a vitrification preservative solution present on the outer periphery of the embryo or ovum. It is a tubule for vitrification preservation of an animal embryo or egg to be removed.

Description

  The present invention relates to a tube for vitrification preservation of animal embryos or eggs and a kit for transplantation of animal embryos, and more particularly, vitrification preservation of biological specimens such as animal eggs, embryos, tissue cells, and the like, and The present invention relates to an animal embryo vitrification storage tube for transplantation, a melting method after vitrification storage, an animal embryo transfer kit comprising the vitrification tube and a diluent straw, and the like.

  Embryos used in bovine embryo transfer technology are transplanted according to the estrous cycle of the recipient cow, and the produced embryo is placed in a cooling substance such as liquid nitrogen to match the embryo transfer with the estrous cycle. It is preserved in a frozen state, and the embryo is thawed and transplanted in accordance with the estrous cycle. As a freezing method for storing the embryo in a frozen state, there is a so-called vitrification storage method.

  The vitrification preservation method uses the principle that ice crystals are difficult to form even below the freezing point due to the freezing point depression of an aqueous solution containing a large amount of antifreezing agents such as glycerol and ethylene glycol. When this solution is rapidly cooled in liquid nitrogen, it can be solidified without generating ice crystals, and the water becomes a glassy solid without crystallization, which is called vitrification.

  When vitrification preservation method is used, physical damage (freezing damage) can be avoided because there are no ice crystals inside and outside the cell, but there is chemical toxicity due to protective substances (freezing agents) contained in vitrification preservation solution. In order to avoid this, it is necessary to dilute the vitrification stock immediately after thawing. Moreover, bovine embryos are generally filled in thin straws and stored in liquid nitrogen. If the embryos are not removed from the straws after thawing and are injected directly into the uterus of the recipient cow, they can be used in the garden. Can be performed directly.

  As such a straw for embryo transfer that can be vitrified, there is a straw for animal embryo transfer in which a frozen body of a diluted solution and a vitrification preserving solution containing an embryo are brought into contact with each other and enclosed in the straw (Patent Document) 1). One end of the straw for embryo transfer is sealed, and a diluent is laminated on the upper part of the sealing plug, and a vitrification cryopreservation liquid is laminated on the upper part of the diluent. The embryo is injected into the vitrification solution, the other end of the straw is sealed, and cooled in liquid nitrogen to be vitrified. After thawing, the frozen straw is transplanted without removing the molten embryo from the straw. Can be used as.

In addition, the animal embryo vitrification storage straw characterized by laminating a dilution liquid layer having an animal embryo inside, an air layer, a vitrification storage liquid layer, an air layer, and a dilution liquid layer in this order in the straw Yes (Patent Document 2). Can be frozen with a small amount of vitrification preservative, can avoid contact between the vitrification preservative layer and the dilute layer through the air layer, can be easily diluted with the dilute solution after thawing, such as cattle It is said that it is possible to perform embryo transfer in the garden for domestic animals.
Further, a cryopreservation tubule for storing a vitrification preservation solution and a diluent containing an animal biological specimen in a frozen state, and then thawing and transplanting the biological specimen to the animal. The linear support device in which the diluting solution is filled from one end side to the other end side in the thin tube main body and the vitrification preservation solution containing the biological specimen is attached to the tip portion is the same as the linear support device. There is also a tubule for cryopreservation of biological specimens characterized in that the tip side is arranged in the tubule body so that the distal end side is adjacent to the end face of the diluent freezing layer (Patent Document 3). The vitrification preservation solution containing the embryo is placed on the tip of the linear support and frozen, and after thawing, the molten embryo can be diluted in the adjacent diluent from the linear support and transplanted. It can be transplanted with a straw attached to the vessel.

  Moreover, although it is a cryopreservation means for porcine embryos, a jig called a cryotop having a freezing plate having a concavity at the tip is used, and the porcine with the minimum amount of freezing liquid is provided in the concavity of the freezing plate. A method of placing an embryo and vitrifying it by directly immersing it in liquid nitrogen is disclosed (Non-patent Document 1). A 0.1 μl vitrification solution is used for the depression.

  Further, germ cells immersed in the vitrification preservation solution are dropped onto the vitrification preservation solution removing material together with the vitrification preservation solution, and the vitrification solution adhering to the periphery of the germ cells is added to the vitrification preservation solution. Also known is a method for cryopreserving germ cells, characterized in that the germ cells are vitrified and stored by being immersed in liquid nitrogen while being absorbed on the vitrification preservation solution removal material after being absorbed by the removal material. (Patent Document 4). If the germ cells before dipping in liquid nitrogen are present in the vitrification preservation solution, the vitrification preservation solution also solidifies, and when the germ cells are thawed, the solidified vitrification preservation solution also becomes liquid. The phase transition between the liquid phase and the solid phase occurs abruptly, so that a fracture surface occurs due to a difference in expansion rate or contraction rate between the vitrification preservation solution and the germ cell, and the fracture surface passes through the germ cell. Sometimes the germ cells are damaged. For this reason, vitrification preservation is performed without the presence of a vitrification preservation solution.

JP-A-5-176946 JP-A-10-248860 JP 2006-149231 A JP 2005-40073 A

http://www.agri-kanagawa.jp/seika/pdf/4197.pdf#search='Cryotop method frozen storage '

  Cattle embryo transfer is a technique used to produce excellent cows regardless of whether they are cows or not, and is performed by licensed professionals who have acquired certain techniques, such as veterinarians and livestock artificial insemination teachers. . A good bovine embryo is valuable and expensive, and a high conception rate by experts is expected.

  Here, in the vitrification preservation method described above, it is necessary to quickly immerse the cow embryo after thawing in a diluting solution in order to remove the toxicity caused by the antifreezing agent. That is, the embryo before freezing is generally vitrified after the embryo is equilibrated in advance with a solution having a low ethylene glycol concentration, and the cryoprotectant penetrates the embryo. Immediately after thawing, the bovine embryo is immersed in a diluent, and the cryoprotectant contained in the embryo after thawing is diluted from the embryo to remove the toxicity caused by the cryoprotectant. Patent Documents 1 to 3 are characterized in that the bovine embryo can be thawed in a straw filled with a diluent so that the thawed cow embryo can be directly transplanted.

  However, the straw for embryo transfer described in Patent Document 1 is a method of filling a vitreous preservative solution of 10 to 20 μl and a large amount of vitrification preservative together with a diluent in the straw, and vitrifies the embryo with the vitrification preservative, Freezing and thawing takes time and embryo viability may be reduced.

  Moreover, since the straw for animal embryo vitrification preservation | save of patent document 2 forms a dilution liquid layer and a vitrification preservation liquid layer, introducing an air layer in a straw, manufacture is not easy. In addition, in order to form a vitrification preservation liquid layer formed at a height of 5 to 15 mm in a 0.25 ml plastic straw and freeze the embryo in this vitrification preservation liquid layer, as in Patent Document 1, There are many vitrification preservation solutions, and it takes time for freezing and thawing, and the viability of the embryo may be reduced.

  Furthermore, the cryopreservation tubule described in Patent Document 3 is one in which a vitrification preservation solution containing an embryo is placed on the tip of a linear support and frozen, and the vitrification preservation solution is vitrified in order to minimize the amount of preservation solution. Although there is an advantage that the amount of the preservation solution is small, it is necessary to remove the linear support tool introduced into the cryopreservation capillary after thawing, and the operation is complicated.

  Further, in Non-Patent Document 1, the glass is vitrified using a very small amount of a vitrification preservation solution of 0.1 μl, but even a specialist such as a veterinarian cannot easily control it to 0.1 μl. In practice, however, the vitrification preservation solution amount is more varied, and it is not easy to ensure stable operability such as vitrification preservation of embryos in a large amount of vitrification preservation solution.

  On the other hand, in Patent Document 4, the vitrification preservation is performed without vitrification preserving solution, but the container used for preservation after vitrification is composed of a container main body and a lid, and the animal embryo is contained in the container main body. A vitrification preservative removing material having a diameter of 13 mm is placed. The vitrification preservation solution present on the outer periphery of the animal embryo is removed by the vitrification preservation solution removing material, placed in liquid nitrogen to be vitrified, placed in a container body, and then fitted with a lid portion in liquid nitrogen. Although preserved, the vitrification preservation solution removing material has an advantage that a plurality of animal embryos can be placed, but it is not easy to remove at the time of melting. Moreover, the embryo cannot be directly transferred using the container.

  The vitrification preservation technique is not limited to animal embryos, but is preferably applicable to eggs.

  Furthermore, animal embryos and eggs that have been equilibrated with a vitrification preservation solution are directly put into liquid nitrogen for rapid vitrification preservation, but liquid nitrogen is not aseptic. For this reason, a capillary tube that can preserve vitrified animal embryos and ova not exposed to liquid nitrogen is desired.

  In view of the above situation, animal embryos and ova that have been equilibrated can be maintained at a high survival rate without direct contact with liquid nitrogen, and animal embryos can be melted in a straw. An object of the present invention is to provide a tube for vitrification preservation of animal embryos, which can be directly transplanted in a farm garden.

  The present invention, as a result of a detailed study of the vitrification preservation tubule of animal embryos or eggs, is a tubule composed of a container body portion sheath portion, the container body portion to be inserted into the sheath portion, the outer handle, If it consists of a handle and a vitrification preservative removal material, the vitrification preservative existing on the periphery is absorbed and removed by placing the animal embryo equilibrated with the vitrification preservative on the vitrification preservative removal material. Can be vitrified without direct contact between liquid nitrogen and animal embryos and eggs equilibrated by inserting into the sheath after inserting it into the sheath. It is possible to dilute in a short time by using a dilution straw filled with a diluent and inserting a container main body to which the animal embryos after thawing are attached into the dilution straw. Heading, present invention It was completed.

  That is, the present invention is a tubule for vitrifying and preserving an animal embryo or egg (hereinafter, also referred to as an animal embryo or the like in the present invention) comprising a container body and a sheath. The container main body portion includes an outer handle and an inner handle connected to the outer handle, and a vitrification preservative removing material is fixed to the inner handle, and the vitrification preservative removing material is at least one An object of the present invention is to provide a vitrification-preserving capillary such as an animal embryo, on which the embryo or ovum is placed and from which the vitrification preservation solution present on the outer periphery of the embryo or ovum is removed.

  Further, it is a method of vitrifying and preserving animal embryos and the like with vitreous preserving tubules such as animal embryos, wherein vitrification and preservation that constitutes the vitrification and preserving tubes of animal embryos and the like equilibrated with vitrification preservation solution Place on the liquid removal material, remove the vitrification preservation solution present on the outer periphery of the animal embryo and the like by suction, and then store the container main body constituting the vitrification preservation thin tube in the sheath, liquid nitrogen It is intended to provide a vitrification preservation method for animal embryos, characterized by being placed inside.

  Furthermore, the present invention provides a kit for transplantation of animal embryos, comprising the above-mentioned thin tubes for vitrification preservation of animal embryos and the like, and a dilution straw filled with a diluent.

  According to the thin tube for vitrification preservation of animal embryos and the like of the present invention, it is possible to vitrify without the presence of vitrification preservation solution in the container main body, so there is no need to adjust the vitrification preservation solution amount, and vitrification preservation operation And the stability of the melting operation is excellent.

  According to the vitrification preservation method of the present invention, when animal embryos and the like equilibrated with the vitrification preservation solution are vitrified and preserved, the vitrification preservation solution present on the periphery of the animal embryos and the like is removed in advance, Is cooled in a bare state that does not float in the vitrification preservation solution, rapid vitrification and melting are possible, and a high survival rate of animal embryos and eggs can be ensured.

According to the vitrification preservation method of the present invention, by using a vitrification preservation capillary such as the animal embryo, the equilibrated animal embryo or egg can be vitrified and preserved without directly contacting liquid nitrogen. Therefore, it is possible to prevent infections such as bacteria and is excellent in safety.
According to the animal embryo transplantation kit of the present invention, the animal embryo after thawing can be directly transplanted to the target animal.

FIG. 1 is an example of a thin tube for vitrification preservation such as animal embryos of the present invention, and FIG. 1A is a plan view of a container body (10) of a thin tube for vitrification preservation such as animal embryos (1). . Moreover, FIG.1 (b) is a top view of a sheath part (20), and FIG.1 (c) is a plane perspective view at the time of inserting the said container main-body part (10) in a sheath part (20). . FIG. 2 is another embodiment of the vitreous preserving capillary for animal embryos of the present invention, and FIG. 2 (a) is a container main body with a metal net attached to the inner handle (13) as a vitrification preservative removing material. It is a perspective view of (10), FIG.2 (b) is a perspective view of a sheath part (20), FIG.2 (c) is the animal which equip | installed the sheath part (20) with the container main-body part (10). It is a see-through | perspective perspective view of thin tubes (1) for vitrification preservation | save, such as an embryo, FIG.2 (d) is an enlarged view of the vitrification preservation liquid removal material (15) of a container main-body part (10). FIG. 3 shows another embodiment of a vitreous preserving capillary for animal embryos according to the present invention. FIG. 3A shows a vitrification preservative removing material (15) formed by laminating two film-like materials. FIG. 3 (b) is a perspective view of the container main body (10) arranged, and FIG. 3 (b) shows the film-like material, metal net, etc. at the tip of the support handle (15a) connected to the inner handle (13). FIG. 3C is a perspective view of a mode in which the entire length of the vitrification preservative removing material (15) is shortened and is transformed into a U-shape. FIG. 4 is a front view of a dilution straw (40) used in the present invention, FIG. 4 (a) is a diagram for explaining the overall configuration, and FIG. 4 (b) is a diagram showing a sealing plug (47). FIG. 4 (c) shows a mode in which the sealing plug (47) is configured to have a conical tapered shape. FIG. 5 is a process diagram for explaining the vitrification preservation method for animal embryos of the present invention. FIG. 6 is a diagram for explaining a method for thawing and diluting animal embryos and the like preserved by the vitrification preservation method for animal embryos of the present invention.

  1st of this invention is a thin tube for vitrifying preservation | save of the animal embryo etc. which consist of a container main-body part and a sheath part, Comprising: The said container main-body part is an inner handle connected with the outer handle and the said outer handle. A vitrification preservative removing material is fixed to the inner handle, and the vitrification preservative removing material is placed on the outer periphery of the embryo or ovum on which at least one embryo or egg is placed. It is a thin tube for vitrification preservation such as animal embryos for removing vitrification preservation solution.

  It is characterized in that a vitrification preservative removing material is disposed at the tip of the inner handle, and an animal embryo or the like is placed on the vitrification preservative removing material together with the vitrification preservative, and the vitrification preservative is absorbed. Alternatively, the vitrification preservative can be removed by suction from the lower part of the vitrification preservative removal material. In the present invention, “vitrification” means that it is frozen and solidified without generating ice crystals by immersing it in liquid nitrogen or a refrigerant that can be cooled in a shorter time than liquid nitrogen. “Vitrification preservation” means that the target tissue is semipermanently preserved by vitrification of an animal embryo or the like that has been equilibrated with a vitrification preservation solution containing a cryoprotectant. The outer periphery of the egg means the outer periphery of the egg cell. Hereinafter, the present invention will be described in detail.

(1) Animal Embryo and Ovum The tissue to be vitrified and preserved in the vitrification and preservation tube of the present invention is an animal embryo and egg. In general, in the case of vitrification preservation, an “equilibrium treatment” is performed in which an animal embryo or the like is immersed in a vitrification preservation solution before being put into liquid nitrogen or the like. When an animal embryo or the like is immersed in a vitrification preservation solution, the cell membrane of the animal embryo or the like can move moisture more easily than the antifreeze agent, so that intracellular moisture flows into the preservation solution, and instead in the vitrification preservation solution. Thus, the osmotic pressure inside the cells such as animal embryos and the extracellular osmotic pressure is maintained at the same pressure. At this time, cells such as animal embryos can be vitrified and preserved easily by immersion in a high-concentration freezing agent. In the present invention, prior to vitrification preservation, animal embryos and eggs that have been equilibrated in advance with a vitrification preservation solution are used as objects for vitrification preservation, so that the vitrification preservation solution does not exist on the periphery of the object. Vitrification preservation can be performed.

  As animals, cattle, pigs, horses, mice, Xenopus, humans and other animals can be targeted, and embryos of various stages such as morula and blastocyst should be used as embryos. Can do.

(2) Vitreous Preservation Tubular for Animal Embryos, etc. FIG. 1 shows a preferred example of a thin tube for vitrification preservation for animal embryos of the present invention. FIG. 1 (a) is a plan view of a container main body (10) of a vitrification thin tube (1) such as an animal embryo, and is connected to an outer handle (11) and the outer handle (11). It consists of a handle (13) and a vitrification preservative removal material (15) connected to the inner handle (13). Moreover, FIG.1 (b) is a top view of a sheath part (20), and FIG.1 (c) is a plane perspective view at the time of inserting the said container main-body part (10) in a sheath part (20). .

  As the material of the outer handle (11) and the inner handle (13) of the container main body (10), a material having low temperature resistance, liquid nitrogen resistance, stretch resistance and alteration resistance can be suitably used. It can be formed of metal, synthetic resin, or the like. Examples of the metal include iron, copper, and an alloy such as SUS containing iron as a main component, and examples of the synthetic resin include a linear thread-like fiber material made of polyamide fiber. In this invention, it is preferable that it is metal, especially stainless steel, such as SUS, at the point which is excellent in thermal conductivity.

  The total length from the end of the outer handle (11) of the container body (10) to the end of the vitrification preservative removal material (15) can be appropriately selected depending on the animal species to be used, but generally 20 to 100 mm, More preferably, it is 30-70 mm. If the outer handle (11) is 8 to 40 mm, it is easy to grip. Further, if the inner handle (13) is 5 to 30 mm, the sheath of liquid nitrogen is formed by the close contact between the outer periphery of the inner handle (13) and the inner periphery of the sheath portion (20) after the sheath portion (20) is mounted. Intrusion into the part (20) can be prevented.

  Moreover, if the length of the vitrification preservative removal material (15) is 5 to 30 mm, the vitrification preservative removal material adhering to animal embryos and the like can be easily removed, and the vitrification preservative removal material (15) can be easily inserted into the sheath (20).

  In addition, the cross-sectional shape of the inner handle (13) is the same as the cross-sectional shape of the inner periphery of the sheath part (20), and when the container main body part (10) is housed in the sheath part (20), it is liquid-tight. If it can be secured, it may be a circle or a polygon. The inner handle (13) has a diameter of 0.5 to 3 mm in terms of an equivalent circle.

  The sheath (20) is configured with a uniform diameter over the entire length and is easy to put in and out of the canister because there is no unevenness. In addition, the full length of the sheath part (20) can be inserted into the vitrification preservative removing material (15) and the internal handle (13) of the container body part (10), and the vitreous preservative capillary (1) ) In a canister in the liquid nitrogen storage device, and is preferably 80 to 200 mm.

  In addition, as the material of the sheath part (20), a material having low temperature resistance, liquid nitrogen resistance, stretch resistance and alteration resistance can be suitably used, and it can be formed of metal, synthetic resin or the like. Examples of the metal include iron, copper, and an alloy such as SUS containing iron as a main component, and examples of the synthetic resin include a linear thread-like fiber material made of polyamide fiber. In this invention, it is preferable that it is metal, especially stainless steel, such as SUS, at the point which is excellent in thermal conductivity.

  The “vitrification preservation solution removing material” used for the vitrification preservation tubule (1) of animal embryos and the like of the present invention is a vitrification preservation solution present on the outer periphery of an animal embryo or the like equilibrated with the vitrification preservation solution. Widely include those that can be removed, such as those that absorb and remove the vitrification preservative, and those that can remove the vitrification preservative by aspiration from the lower part of the vitrification preservative removal material. be able to. Therefore, examples of the material include sheet-like materials made of natural products such as paper, synthetic resins, metals, etc., and net-like materials made of these fibrous materials.

  Here, if it explains with a cow embryo as an animal embryo, the magnitude | size of the cytoplasm of an egg is 138-143 micrometers, The outer periphery is coat | covered with the zona pellucida. The bovine embryo is approximately the same size, but when equilibrated with a vitrification preservation solution of bovine embryo, the zona pellucida is the original size because it does not shrink, but the embryo itself shrinks to about 69-71 μm, about half of it. It becomes size. Therefore, it is necessary to use a vitrification preservative removing material having an opening or a pore size capable of holding such contracted animal embryos. In general, it is preferable to use a material having an opening or a hole diameter of 0.001 to 0.8 times, more preferably 0.01 to 0.7 times the diameter of the object. For example, when the vitrification preservative removing material is a metal net, and the embryo diameter is 70 μm, a metal net of 0.7 to 49 μm square mesh is used. Thereby, the vitrification preservative which adheres by suction from the lower part of a metal net | network can be removed, hold | maintaining an animal embryo etc. to the vitrification preservative removal material.

  According to the thin tube for vitrification preservation of the present invention, since the vitrification preservation solution existing on the outer periphery of the animal embryo or the like can be removed, the liquid amount of the vitrification preservation solution when placing an embryo or egg is minimized. There is no need to adjust the amount. That is, commercially available tubules for vitrification store embryos and eggs on the tubule for vitrification storage with a minimum volume of, for example, 0.1 μl of vitrification storage solution. It was difficult. For this reason, the non-uniformity of the liquid volume occurred and contributed to the influence of the embryo survival rate, but this problem is solved. Note that, even if the above-described absorption or suction operation of the vitrification preservation solution present on the outer periphery of the animal embryo or the like is performed, the vitrification preservation solution present inside the cell such as the animal embryo necessary for vitrification preservation is maintained.

  When such a metal net is used as the vitrification preservative removing material, as shown in FIG. 2, the vitrification preservative removal material can be used in a cross-sectional view and deformed into a U shape. FIG. 2 (a) is a perspective view of the container body (10) in which such a metal net is attached to the inner handle (13) as a vitrification preservative removing material, and FIG. 2 (b) is a sheath ( 20 (c) is a perspective view of FIG. 2 (c), and FIG. 2 (c) is a perspective view of a thin tube (1) for vitrification preservation such as an animal embryo in which a container body (10) is attached to a sheath (20). (D) is an enlarged view of the vitrification preservative removal material (15) of the container body (10). By wrapping a flat metal net around a metal round bar or the like and deforming it into a substantially U shape, the retention stability of animal embryos and the like is improved, and even when the vitrification preservative removal material (15) is long, The linearity can be kept high and insertion into the sheath (20) can be easily performed.

  On the other hand, as the vitrification preservative removal material (15), it is 0.001 to 0.8 times the diameter of the vitrification target object in a film-like product made of a natural product such as paper or a synthetic resin, more preferably 0.01. You may use the film-like thing in which the hole diameter (through-hole) of -0.7 times was formed. By placing an animal embryo or the like on the upper part of such a film and sucking it from the lower part, the vitrification preservation solution adhering by suction can be removed in the same manner as described above. As such a film-like material, a polyamide fiber or other film having liquid nitrogen resistance can be suitably used as in the case of the synthetic resin constituting the container main body (10).

Furthermore, in this invention, the member which has the characteristic which itself absorbs vitrification preservative can be used as said vitrification preservative removal material. If there is a property to absorb the vitrification preservative, the vitrification preservative without performing the suction operation when the embryo or egg equilibrated with the vitrification preservative is placed on the vitrification preservative removal material. Can be removed from the periphery of the embryo or egg. The absorption properties of the vitrification solution, water absorption 1 to 10 [mu] m / cm ^2, more preferably, is sufficient 3 to 7 [mu] m / cm ^2.

  The thickness of the film-like material used as the vitrification preservative removing material (15) should be thick enough to maintain linearity so that it can be easily inserted into the sheath (20). There is no particular limitation. Further, such a film-like object may be attached to the inner handle (13) after being deformed into a U shape in a sectional view as shown in FIG. 2 (d). In addition, in FIG.2 (d), although the front-end | tip of the vitrification preservative removal material (15) is cut off perpendicularly | vertically, you may cut a front-end | tip into a shovel shape. By thinning the tip, the insertion into the sheath (20) can be performed more stably.

  Further, the vitrification preservative removal material (15) may be a composite of two or more. For example, in the case of a composite comprising the above-described film-like material and a metal net, the film-like material can be laminated on the upper surface of the metal net. In this case, the opening and the hole diameter of the wire mesh are not limited to the above ranges, and a wire having a larger opening and a hole diameter may be used. Further, two or more of the above film-like materials can be laminated and used as a vitrification preservative removing material. Under the present circumstances, the size of the hole diameter (through-hole) and water absorption of the film-like material to be used may be the same or different. In addition, as long as at least the contact surface of an animal embryo or the like has the above-described opening and pore size, the lower layer is not limited to this, and a material having a larger opening and pore size can be used. The perspective view of the container main-body part (10) by which the vitrification preservative removal material (15) formed by laminating | stacking two film-like materials is arrange | positioned at Fig.3 (a) is shown.

  As described above, in the present invention, since the vitrification preservative removing material (15) is 5 to 30 mm in length, for example, 10 or more animal embryos can be placed. In the case of direct transplantation, one is sufficient, but when only preservation is performed, there is an advantage that a plurality of animal embryos can be vitrified and preserved by a single operation.

  Furthermore, as shown in FIG. 3 (b), the vitrification preservative removing material (15) includes the film-like material, the metal net, and the like at the tip of the support handle (15a) connected to the inner handle (13). The vitrification preservative removing material (15) may be provided. By interposing the support handle (15a), even when a flexible material is used as the vitrification preservative removal material (15), it can be easily inserted into the sheath portion (20). The total length of the vitrification preservative removing material (15) includes the length of the support handle (15a) when the support handle (15a) is provided.

  Moreover, as shown in FIG.3 (c), shortening the full length of the vitrification preservative removal material (15), and transforming it into a U shape in a cross-sectional view, it is set as the vitrification preservative removal material (15). it can. Since the film-like material constituting the vitrification preservative removal material (15) is a thin material, even when it is not easy to maintain linearity, it is possible to easily ensure linearity by shortening the overall length.

  The thin tube for vitrification preservation of animal embryos and the like of the present invention is composed of the container body and the sheath described above, and as shown in the vitrification preservation method described later, vitrification existing on the outer periphery of the equilibrated animal embryo etc. After removing the preservative solution by absorption or suction removal, the container main body can be inserted into a sheath and put into liquid nitrogen for vitrification. It is preferable that the vitrification preservative removing material of the container main body has a shape that can be quickly inserted into the sheath. According to the above configuration, the container can be easily inserted into the sheath, and the sheath before being put into liquid nitrogen. A vitrification preservative removing material can be accommodated in the part to prevent contamination by liquid nitrogen. For this reason, the sterilization process of liquid nitrogen etc. can also be avoided.

  As described above, if the outer handle, inner handle, and sheath of the container main body are made of metal, it is excellent in thermal conductivity, so that the animal placed on the vitrification preservative removing material housed in the sheath is used. It is possible to vitrify and store embryos and the like quickly. Moreover, since it is excellent in mechanical strength, it can be reused.

(3) Kit for transplantation of animal embryo The kit for transplantation of animal embryo of the present invention comprises a capillary tube for vitrification preservation (1) such as the animal embryo and a straw for dilution (40) filled with a diluent. Consists of. Vitrification preservation is intended for animal embryos and ova, but since animal ovum is not expected to be transplanted directly, it was designated as an “animal embryo transfer kit”.

  As shown in FIG. 4 (a), the dilution straw (40) is provided with a cotton plug (41) at its end and filled with a diluent (43). An air layer (45) is formed on the other end, and a sealing plug (47) is attached to the other end. In addition, the cotton plug (41) includes two cotton plugs (41a) via an adhesive layer (41b).

  The air layer (45) in the dilution straw (40) is formed to avoid breakage of the straw (40) due to expansion of the diluent when stored in liquid nitrogen. The total length of the dilution straw (40) is such a length that can be easily charged and recovered into liquid nitrogen and charged into and recovered from the melt, and is preferably 80 to 200 mm.

  Further, the sealing stopper (47) may be a covering stopper as shown in FIG. 4B, and is particularly preferable from the viewpoint of aseptic operation. Furthermore, as shown in FIG.4 (c), the sealing stopper (47) may be comprised so that it may become a conical taper. If it is conical, the container main body part (10) of the thin tube for vitrification preservation (1) such as the animal embryo of the present invention can be easily inserted. The composition of the diluent can be appropriately selected according to the type of vitrification preservative. Generally, the antifreezing agent ethylene glycol and glycerol are removed from the vitrification preservative and the sucrose concentration is reduced. Those having different compositions can be preferably used.

  In the present invention, after melting an animal embryo that has been vitrified and preserved by a vitreous preservation capillary (1) such as an animal embryo, the container main body from the sheath (20) of the vitrification preservation tube (1) such as an animal embryo. The part (10) is extracted, the sealing plug (47) of the dilution straw (40) is removed, the container body (10) is inserted therefrom, and the animal embryo that adheres to the vitrification preservative removal material (15) Is diluted in the diluent. Even if the height of the air layer (45) is 10 mm, the animal embryo can be dropped into the diluent (43) as long as the vitrification preservative removal material (15) is 30 mm.

  Such a straw for dilution (40) can be prepared, for example, by filling a commercially available cotton plug straw with a diluent and inserting a sealing plug. The capacity of the dilution straw (40) can be appropriately selected according to the type of animal embryos preserved by vitrification. For example, in the case of bovine embryos, it is preferable to use 0.25 ml. it can.

  In the animal embryo transplantation kit of the present invention, the length of the vitrification preservative removal material (15) in the vitrification storage tubule (1) such as animal embryo is diluted from the end of the dilution straw (40). Since it is related to the length to the liquid (43), the length of the vitrification preservative removal material (15) is set so that the vitrification preservative removal material (15) can be easily inserted into the sheath (20). For example, when it is prepared as short as 12 mm, the length from the end on the side of the sealing plug (47) of the dilution straw (40) to the diluent (43) is the length of the vitrification preservative removal material (15). The container main body (10) may be inserted into the dilution straw (40) after being cut short correspondingly.

  According to the kit of the present invention, when the animal embryo or the like is thawed, the thin tube (1) for vitrification preservation of the animal embryo or the like and the straw for dilution (40) are immersed in a melt at a predetermined temperature, Since the container body (10) can be easily guided to the dilution liquid (45) of the dilution straw (40), it can be surely diluted with the dilution liquid (45) and maintains high viability. It is possible to improve the conception rate while maintaining simplicity. In the present invention, liquid nitrogen is described as the cooling substance, but other substances may be used, and the present invention is not limited to this.

  In the dilution straw (40) used in the present invention, animal embryos and the like that have been thawed in the diluent (43) are sedimented and diluted by gravity, and the diluent (43) is removed from the cotton plug (41) side at the time of transplantation. It can be transplanted directly by extruding.

(4) Vitrification preservation method The vitrification preservation method of the present invention is a method of vitrifying and preserving animal embryos and the like in the above-mentioned vitreous preservation tubules such as animal embryos, and animal embryos equilibrated with a vitrification preservation solution Is placed on the vitrification preservative removing material constituting the vitrification preserving thin tube, and the vitrification preserving solution present on the outer periphery of the animal embryo or the like is removed by suction, and then the vitrification preserving thin tube is removed. It is a vitrification preservation method of animal embryo characterized by storing a container main part which constitutes in a sheath, and placing it in liquid nitrogen. The case of the vitrification preservation | save of a cow embryo is demonstrated using FIG.

(I) Equilibrium treatment The recovered bovine embryo is stored in a development medium. The development medium is selected at a suitable temperature depending on the type of embryo, and for example, 39 ° C. for bovine embryos.

  The embryo is then immersed in a vitrification base solution at a temperature of 15 ° C. Such a vitrification base solution can be appropriately selected from those used in the conventional vitrification preservation method. The infiltration time is about 10 minutes.

  Next, it is immersed for about 5 minutes in a vitrification pretreatment liquid at a temperature of 15 ° C. The vitrification pretreatment liquid is a vitrification base liquid containing an antifreezing agent such as ethylene glycol or glycerol.

  Then, it is immersed in a vitrification preservation solution at a temperature of 4 ° C. for a maximum of 25 seconds, and then the bovine embryo is again immersed in a fresh vitrification preservation solution at a temperature of 4 ° C. for a maximum of 25 seconds. As a result, the vitrification preservation solution is immersed in the bovine embryo, and the concentration thereof becomes equilibrium with the vitrification preservation solution. The vitrification preservation solution is a solution obtained by increasing the ethylene glycol concentration and the glycerol concentration of the above-mentioned vitrification pretreatment solution and adding sucrose.

(Ii) Vitrification preservation The filter paper is placed on the support, and the vitrification preservation solution removing material (15) of the container body (10) constituting the thin tube for vitrification preservation (1) such as the animal embryo of the present invention (15) Is fixed and the bovine embryo equilibrated with the vitrification preservation solution is placed on the vitrification preservation solution removing material (15). The vitrification preservation solution present on the outer periphery of the cow embryo is removed by suction from the lower part of the support. In addition, when the vitrification preservative removal material (15) has the water-absorbing property of the vitrification preservative, the embryo obtained by equilibrating the vitrification preservative with the vitrification preservative just by placing without removing by suction. And can be removed from the outer periphery of the egg.

  After removing the vitrification preservative, the container body (10) is inserted into the sheath (20). The inner handle (13) of the container body (10) and the inner periphery of the sheath (20) are fitted in a liquid-tight manner and charged into liquid nitrogen. Thus, even when aseptically treated liquid nitrogen is not used, liquid nitrogen and animal embryos and the like are not in direct contact with each other, and contamination with liquid nitrogen can be prevented and cow embryos can be vitrified and preserved. It should be noted that it takes 10 seconds or less for the vitrification preservative removing material (15) to be vitrified by sucking and removing the vitrification preservative from the vitrification preservative removal material (15).

(Iii) Thawing and dilution method In the present invention, as shown in FIG. 6 (d), a straw for dilution (40) previously stored in liquid nitrogen is used as a melting solution prior to melting of a vitrified cow embryo. Immerse.
Next, the vitrification storage tubule (1) such as an animal embryo stored in liquid nitrogen shown in FIG. 6 (a) is kept upright at a temperature of 20 to 39 ° C. as shown in FIG. 6 (b). Immerse in the melt and warm the embryo to a temperature of 4 ° C.

  Next, the container body part (10) of the vitrification preservation thin tube (1) such as animal embryo is extracted from the sheath part (20), and the diluted cow embryo is attached to the dilution liquid of the dilution straw (40). Soak. The bovine embryo adhering to the vitrification preservative removing material (15) of the container main body (10) falls by its own weight in the diluted solution. Hold in dilution for about 5-30 minutes to remove antifreeze.

  When the vitreous preserving tube (1) for animal embryos or the like of the present invention is made of metal, the melting time is extremely fast due to high thermal conductivity, and the vitreous preserving tube for animal embryos after heating ( 1) Since it can be handled safely with bare hands, it is easy to insert and attach the vitrification preservative removal material (15) of the container body (10) to the diluting solution straw (40). The rate can be kept high. It should be noted that the time from warming to 4 ° C. until the vitrification preservative removing material (15) holding the embryo is submerged in the diluent (45) of the diluent straw (40) is within 10 seconds.

  In the above, bovine embryos were explained under the handling conditions, but when applied to human and other animal embryos and ova, change to conditions suitable for these embryos and ova, and vitrify, preserve, and dilute. Can do.

  In the present invention, vitrification preservation is performed in a state where the vitrification preservation solution does not exist on the outer periphery of the animal embryo or egg, so vitrification and melting can be performed rapidly, and dilution can also be performed rapidly accordingly. .

  EXAMPLES Next, although an Example is given and this invention is demonstrated concretely, these Examples do not restrict | limit this invention at all.

(Example 1)
A membrane filter for dry filtration sterilization (Nippon Millipore Corporation; pore size 5.0-8.0 μm) cut to a width of 2 mm and a length of 20 mm was used as a vitrification preservative removal material (15), and is shown in FIG. Vitreous preservation tubules (1) such as animal embryos shown were prepared. The outer handle, the inner handle, and the sheath (20) of the container body (10) were made of SUS. The outer handle (11) is 30 mm long, the inner handle (13) is 19 mm, the membrane filter protrudes from the inner handle by 17 mm, and the vitreous preserving tubule (such as an animal embryo equipped with a sheath (20)) ( The total length of 1) was 134 mm, and the outer diameter was 1.2 mm.

  On the other hand, a commercially available cotton plug straw for freezing storage having a length of 132 mm and an internal volume of 0.25 ml is filled with a diluent having the composition shown in Table 1 below, and a sealing plug is attached to the other end portion in liquid nitrogen. Stored and used as dilution straw.

(Example 2)
A vitreous preservative removing material (15) was prepared using a SUS metal net knitted with 30 μm thick SUS metal with an opening of 38 μm, and the vitreous preservative thin tube (1) such as animal embryo shown in FIG. 2 was prepared. The outer handle, inner handle, and sheath (20) of the container body (10) were also made of SUS. The outer handle (11) has a length of 30 mm, the inner handle (13) has a length of 19 mm, the vitrification preservative removal material (15) has a length of 19 mm, and is vitrified such as an animal embryo equipped with a sheath (20). The total length of the storage capillary (1) was 134 mm, and the outer diameter was 1.2 mm. The vitrification preservative removing material (15) was formed by winding the SUS metal net having a width of 2 mm around the outer periphery of a 22G needle to form a U-shaped recess.

Example 3
(1) The bovine blastocyst was immersed in a vitrification base solution having a temperature of 15 ° C. for 10 minutes. Table 2 shows the composition of the vitrification base solution.
(2) Next, it was immersed in a pre-vitrification liquid at a temperature of 15 ° C. for about 5 minutes. Table 3 shows the composition of the vitrification pretreatment liquid.

  (3) Next, it was immersed in a vitrification preservation solution at a temperature of 4 ° C. for 25 seconds, and then a cow embryo was again immersed in a fresh vitrification preservation solution at a temperature of 4 ° C. for 25 seconds. Table 4 shows the composition of the vitrification stock solution.

  (4) Next, a filter paper is placed on the support, and the vitrification preserving liquid removing material of the container main body constituting the vitrification preserving thin tube produced in Example 1 is fixed thereon and equilibrated with the vitrification preserving liquid. The treated bovine blastocyst is placed on a vitrification preservative removal material, and the vitrification preservative present on the outer periphery of the bovine blastocyst is removed by aspiration from the lower part of the support, and immediately The said container main-body part was inserted in the sheath part, and it prepared in liquid nitrogen. The time from when the vitrification preservative solution on the vitrification preservative removal material was removed by suction, rapidly cooled in liquid nitrogen and vitrified was within 10 seconds.

(5) On the other hand, the dilution straw prepared in Example 1 was immersed in a melt at a temperature of 39 ° C.
(6) Next, vitrification tubules such as animal embryos preserved in liquid nitrogen in (4) above are immersed for 7 seconds in a molten solution at a temperature of 39 ° C. while standing upright, so that bovine blastocysts are heated to a temperature of 4 Warmed to ° C.

(7) Next, the container main body part of the vitrification preservation thin tube was extracted from the sheath part, and immersed in the diluting solution of the diluting straw for 5 minutes while the molten bovine blastocyst was adhered.
(8) Next, the bovine blastocyst was cultured for 96 hours.
The above process was similarly performed on 12 bovine blastocysts, and the survival rate after dilution in a straw was evaluated. The results are shown in Table 5.

Example 4
(1) Instead of a membrane filter for dry filtration sterilization, a hard filter paper (manufactured by Toyo Filter Paper, trade name “No. 4A”, water absorption 5.3 μm / cm ² ) cut to a width of 2 mm and a length of 20 mm is made of glass. A thin tube for vitrification preservation was prepared in the same manner as in Example 1 except that the vitrification preservation solution removing material (15) was used.

  (2) In the same manner as in Example 3, bovine blastocysts were immersed in a vitrification base solution at a temperature of 15 ° C. for 10 minutes, and immersed in a pre-vitrification solution at a temperature of 15 ° C. for about 5 minutes, and then preserved by vitrification. It was immersed in the solution for 25 seconds, and again the cow embryo was immersed in a fresh vitrification preservation solution at a temperature of 4 ° C. for 25 seconds.

  (3) The bovine blastocyst equilibrated with this vitrification preservative, together with about 0.5 μl of vitrification preservative, above the vitrification preservative removal material of the vitrification preservative prepared in (1) above Placed on. Absorption removal of the vitrification preservation solution existing on the outer periphery of the blastocyst was confirmed visually. Immediately, the container body was inserted into the sheath and charged into liquid nitrogen. The time from placing the bovine blastocyst on the vitrification preservative removal material to rapid cooling in liquid nitrogen to vitrification was within 10 seconds.

(4) On the other hand, the dilution straw prepared in Example 1 was immersed in a melt at a temperature of 39 ° C.
(5) Next, vitrification tubules such as animal embryos preserved in liquid nitrogen in (3) above are immersed in a molten solution at a temperature of 39 ° C. for 7 seconds while being kept upright, so that the bovine blastocysts have a temperature of 4 Warmed to ° C.

(6) Next, the container body part of the vitrification preservation thin tube was extracted from the sheath part, and immersed in a dilute solution at 25 ° C. for 30 minutes with the melted bovine blastocyst attached.
(7) Thereafter, the bovine blastocyst was cultured for 96 hours.

  (8) The above steps were performed in the same manner for 25 bovine blastocysts, and the survival rate after dilution in the straw was evaluated. The results are shown in Table 6.

(Comparative Example 1)
(1) Example 1 except that a non-water-absorbing plastic film (water absorption 0 μm / cm ² ) cut to a width of 1 mm and a length of 20 mm was used as a freezing plate in place of the membrane filter for dry filtration sterilization. In the same manner, a comparative vitrification storage tube was prepared.

  (2) In the same manner as in Example 3, bovine blastocysts were immersed in a vitrification base solution at a temperature of 15 ° C. for 10 minutes, and immersed in a pre-vitrification solution at a temperature of 15 ° C. for about 5 minutes, and then preserved by vitrification. It was immersed in the solution for 25 seconds, and again the cow embryo was immersed in a fresh vitrification preservation solution at a temperature of 4 ° C. for 25 seconds.

  (3) Next, the bovine blastocyst equilibrated with the vitrification preservation solution is placed on the freezing plate tip of the comparative vitrification preservation capillary prepared in (1) above with about 0.3 μl of the vitrification preservation solution. Placed. The vitrification stock solution present on the outer periphery of the blastocyst remained on the frozen plate. Subsequently, the frozen plate of the comparative vitrification preservation thin tube was put into liquid nitrogen, and then inserted into the sheath of the comparative vitrification preservation thin tube.

(4) On the other hand, the dilution straw prepared in Example 1 was immersed in a melt at a temperature of 39 ° C.
(5) Next, vitrification preservation tubules such as animal embryos preserved in liquid nitrogen in the above (4) are immersed for 7 seconds in the melting solution at a temperature of 39 ° C. while standing upright, and the bovine blastocyst is heated to a temperature. Warmed to 4 ° C.

(6) Next, the container main body part of the comparative vitrification preservation thin tube was extracted from the sheath part, and immersed in a dilution solution of a 39 ° C. dilution straw for 5 minutes with the melted bovine blastocyst attached.
(7) Thereafter, the bovine blastocyst was cultured for 96 hours.

  (8) The above process was performed in the same manner for three bovine blastocysts, and the survival rate after dilution in the straw was evaluated. The results are shown in Table 7.

(result)
(1) As shown in Example 3, the thin tube for vitrification preservation of the present invention is an embryo of an animal equilibrated with vitrification preservation solution by performing a suction operation from the lower part of the vitrification preservation solution removing material to be disposed. The vitrification preservation solution present on the periphery of the ovum and ovum can be removed, so that the number of morphologically viable embryos in bovine blastocysts and the ratio of viable embryos to morphologically viable embryos at the end of culture can be improved. It was.

  (2) As shown in Example 4, when the vitrification preservative removing material disposed in the vitrification preservation thin tube has a high water absorption, 0.5 μl of glass together with the equilibrated bovine blastocyst Even when the vitrification preservation solution is placed on the vitrification preservation solution removing material, the vitrification preservation solution present on the outer periphery of the embryo or egg can be removed without performing the suction operation, and the operation is simple. It was.

  (3) In Comparative Example 1, only 0.3 μl of vitrification preservation solution smaller than Example 4 was placed together with equilibrated bovine blastocysts, but the number of morphologically viable embryos of bovine blastocysts As a result, the ratio of the number of viable embryos to the morphological viable embryos at the end of the culture was low. This means that by removing the vitrification preservation solution present around the embryos and ova of animals equilibrated with the vitrification preservation solution, the number of morphological surviving embryos of bovine blastocysts and morphological survival at the end of the culture This shows that the ratio of the number of viable embryos to the embryos can be improved.

  (4) Comparative Example 1 was that a bovine blastocyst equilibrated with a vitrification preservation solution was directly put into liquid nitrogen and frozen, and 0.3 μl of a vitrification preservation solution was left in the bovine blastocyst. It differs from Example 3 and Example 4 in the point which freezes as it is. Since the vitrification preservation capillaries of Examples 3 and 4 are inserted into the liquid nitrogen after inserting the container main body portion into the sheath portion, they are frozen without leaving the vitrification preservation solution on the outer periphery of the embryo or egg. Can be made. Further, since it is not necessary to directly introduce embryos and eggs into liquid nitrogen, it is not necessary to consider contamination of embryos and eggs with liquid nitrogen.

  The cryopreservation tubule of the present invention can be widely used for transplantation into animals while maintaining the high viability of biological specimens, and is particularly effective when used for transplantation of humans and bovine embryos.

1 ... Various tubules for vitrification of animal embryos,
10: Container body,
20 ... sheath,
40 ... Dilution straw

Claims (3)

A capillary tube for vitrifying and preserving an animal embryo or egg consisting of a container body and a sheath,
The container body portion is composed of an outer handle and an inner handle connected to the outer handle, and a vitrification preservative removing material is fixed to the inner handle,
The vitrification preservative removal material is a vitrification of an animal embryo or ovum on which at least one embryo or egg is placed and which removes the vitrification preservative present on the periphery of the embryo or ovum Storage tube. A method for vitrifying and preserving an animal embryo or ovum in a thin tube for vitrification preservation of an animal embryo or ovum according to claim 1,
An animal embryo or egg equilibrated with a vitrification preservation solution is placed on the vitrification preservation solution removing material constituting the thin tube for vitrification preservation,
Aspirate the vitrification preservation solution present on the periphery of the animal embryo or egg,
Subsequently, the vitrification preservation method of the animal embryo characterized by storing the container main-body part which comprises the said thin tube for vitrification preservation | save in a sheath part, and mounting in liquid nitrogen.   A kit for transplanting an animal embryo comprising the capillary tube for vitrification of an animal embryo or egg according to claim 1 and a dilution straw filled with a diluent.

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