WO2024085220A1 - Straw tube, straw, and cryopreservation method of semen or fertilized egg - Google Patents

Abstract

[Problem] To provide a means that enables easy and highly sure ice seeding in cryopreservation of sperms/fertilized eggs while minimizing exposure of a fertilized/inseminated animal to an ice nucleus material and concerns about safety and toxicity resulted therefrom.　[Solution] Provided are: a straw tube A in which a semen- or fertilized egg-containing solution is housed inside a hollow part 2, wherein a plug part 1 formed on one end A1 side comprises three layers including an outer plug 11, a hygroscopic coagulating material powder layer 12 and an inner plug 13 arranged in this order from the A1 side toward the hollow part 2 side, and an ice nucleus material is held by a fiber member forming the inner plug 13; and others. In this straw tube A, the ice nucleus material is held by the fiber member forming the inner plug 13 so that ice seeding can be automatically performed during the sperm/fertilized egg freezing procedure.

Description

Straw tube, straw, and method for freezing and storing sperm or fertilized eggs

The present invention relates to a straw tube in which a solution containing sperm or fertilized eggs is stored in the hollow portion, in which the plug portion formed on one end is composed of three layers from that end side to the hollow portion side: an outer plug, a layer of moisture-absorbing coagulant powder, and an inner plug, in that order, and in which an ice nucleating substance is held in the fibers forming the inner plug, a sperm-encapsulated straw or a fertilized egg-encapsulated straw in which sperm or fertilized eggs are encapsulated in the straw tube, and a method for freezing and storing sperm or fertilized eggs.

Artificial insemination and embryo transfer are extremely important techniques for promoting livestock improvement and making advanced use of superior breeding stock, and are widely used worldwide in the livestock industry, including cattle.

Artificial insemination is a technique in which semen is artificially injected into the female reproductive organs to fertilize and impregnate her instead of natural mating. For example, the most common method for artificial insemination of cattle and other animals is to use a straw tube. In this method, diluted semen is first sealed in a straw tube, frozen by quick freezing, and stored in liquid nitrogen. Then, at the time of artificial insemination, the frozen semen in the straw tube is thawed, the straw tube is loaded into an injector, and the tip of the injector is advanced into the female's cervical canal, etc., and the semen is injected. For example, Patent Document 1 describes a livestock semen ampoule with an inner stopper, powdered glue, and a stopper inserted in that order into one end of the tube as an example of an artificial insemination straw tube.

Furthermore, fertilized egg transfer (embryo transfer) is a technique in which a fertilized egg (embryo) is transferred into the reproductive tract of a female individual to induce pregnancy. Straw tubes are also widely used to store the fertilized eggs. For example, in the case of in vivo fertilized egg transfer, which is widely used in cattle and other animals, first, a female donor is superovulated and artificially inseminated, after which fertilized eggs (morulae, blastocysts) are collected from the female and sealed one by one in a straw tube together with a cryoprotectant, and then frozen and stored using a slow freezing method, vitrification method, ultra-rapid vitrification method, or other method. Then, at the time of fertilized egg transfer, the frozen embryo in the straw tube is thawed, and the embryo transfer straw containing the thawed embryo is loaded into a transfer device, and the tip of the transfer device is advanced into the uterine horn of the recipient female, and the embryo is transferred. As an example of a straw tube for embryo transfer, Patent Document 2 describes a straw for containing embryos that is filled in sequence from the opening side of one end with a fiber layer, a gelatin layer, and a synthetic fiber layer.

When cryopreserving for artificial insemination or embryo transfer, cooling a solution containing sperm or fertilized eggs results in a supercooled state (a state in which ice crystals do not form even when the temperature drops below the temperature at which ice crystals form). If ice crystals form after the solution has become excessively supercooled, the ice crystals will grow larger, causing intracellular freezing and damaging the cells. Therefore, it is preferable to perform an ice planting operation to prevent the solution from becoming excessively supercooled during cooling. Here, ice planting refers to planting ice nuclei that can grow at that temperature in a supercooled solution to induce ice crystal formation. In normal ice planting operations, ice nuclei are planted at a temperature slightly lower than that near the freezing point of the solution to induce ice crystal formation. The ice planting operation can induce ice crystal formation in the extracellular fluid before the supercooling state becomes excessive, thereby suppressing intracellular freezing (cell damage).

Currently, in artificial insemination of cattle, sperm viability after thawing can be maintained at a practical level without the ice planting procedure, so the ice planting procedure is rarely performed when cryopreserving semen. However, it has been shown that freezing cattle semen using the ice planting procedure improves sperm viability after thawing. In addition, for example, in artificial insemination of pigs, there is a large variation in sperm's freeze resistance and conception rate and number of offspring after artificial insemination, and it is believed that the main cause of this is a decrease in sperm viability after freezing and thawing. It has also been shown that freezing pig semen using the ice planting procedure improves sperm viability after thawing.

On the other hand, at the site of embryo transfer for cattle and other animals, the ice planting procedure is actually widely carried out by keeping the straw tube at a temperature close to but lower than the freezing point, cooling tweezers with liquid nitrogen, and pinching the outside of the straw tube with the tweezers. While this procedure is relatively simple to perform, it is inefficient because it is a manual operation, and the ice planting can be unreliable.

In the cryopreservation in artificial insemination and embryo transfer, a freezing operation using silver iodide as a nucleating material has been investigated. Here, the nucleating material is a material that functions as an ice nucleus for ice crystal formation. For example, Non-Patent Document 1 reports that in cryopreservation of boar semen using a straw tube, sperm viability and conception rate after thawing are improved by sealing one fixed silver iodide particle with a diameter of 3 mm in the straw tube together with the semen and freezing it. In addition, for example, Patent Document 3 describes a technology for freezing and preserving embryos by sealing a storage solution containing an embryo and fixed silver iodide in a straw tube, and Patent Document 4 describes a straw tube in which the plug part on one end of the straw tube is composed of three layers, an inner plug, a powder layer, and an outer plug, in that order from the hollow part side, and an ice nucleating material is mixed into the powder layer.
Jpn. Jpn. Published No. 48-7582 Japanese Utility Model Application Publication No. 8-862 Patent Publication No. 3-1013 JP 2016-67807 A "Effect of boar semen processing method on sperm viability and fertility after freezing and thawing", Nakajima Kiyoshi and Doi Tomoko, Toyama Prefectural Livestock Research Station Research Report No. 12, p9-14 (1995).

As mentioned above, in artificial insemination and embryo transfer, the planting procedure during cryopreservation is important for improving the survival of sperm and embryos after thawing. However, to perform the planting procedure reliably, special procedures and processes for planting the ice are required separately, making the operation complicated.

On the other hand, since artificial insemination and embryo transfer involve the injection and implantation of sperm and fertilized eggs deep inside the female reproductive tract, it is preferable that the dilution solution is as free of other substances as possible, taking into consideration the safety of the inseminated or egg-recipient animals and concerns about contamination of these animals. In contrast, for example, in the case of the technology described in Patent Document 4, the ice nucleation material is merely mixed into the powder layer and not retained therein, and it has been found that the ice nucleation material may adhere to the inner wall surface of the straw tube when the semen or fertilized eggs are injected or transferred. As a result, the ice nucleation material may contaminate the injector or transfer device, and through this, the ice nucleation material may be mixed into the semen or fertilized eggs during artificial insemination or embryo transfer, and there is a problem that it is not possible to completely eliminate concerns about the exposure of the fertilized or egg-recipient animals to the ice nucleation material and the resulting safety and toxicity.

The present invention aims to provide a means for freezing and preserving sperm and fertilized eggs by easily and reliably planting ice, while minimizing exposure of fertilized or egg-recipient animals to ice nucleating materials and the associated safety and toxicity concerns.

The present invention provides a straw tube in which a solution containing sperm or fertilized eggs is stored in a hollow portion, the plug portion formed on one end of the straw tube is composed of three layers, in order from the end side to the hollow portion side: an outer plug, a layer of moisture-absorbing coagulant powder, and an inner plug, and ice nucleating material is held in the fiber member that forms the inner plug.

In this straw tube, a layer of moisture-absorbing coagulant powder is sandwiched between an outer plug and an inner plug at one end that forms the plug portion. For example, when a solution containing sperm or fertilized eggs is injected into the straw tube and the solution is stored in the hollow portion, the solution penetrates into the moisture-absorbing coagulant powder layer, and the moisture-absorbing coagulant powder absorbs moisture and coagulates into a gel-like substance. Therefore, from before freezing to after thawing, the plug side of the straw tube is mostly sealed, preventing leakage or loss of solution from that side.

In addition, in this straw tube, an ice nucleation material is held in the fiber member that forms the inner plug. Therefore, for example, when cryopreserving semen or fertilized eggs, if a solution containing semen or fertilized eggs is injected into the straw tube and the solution is stored in the hollow portion, the solution containing semen or fertilized eggs will infiltrate the inner plug and come into contact with the ice nucleation material in the inner plug. Then, the ice nucleation material functions as ice nuclei during the freezing procedure, inducing the formation of ice crystals.

In the present invention, an ice nucleating substance is preliminarily contained in the plug of the straw tube, and this ice nucleating substance functions as an ice nucleus during freezing, so when performing the sperm/fertilized egg freezing procedure, the worker can automatically plant the ice using only the conventional freezing procedure without having to perform any additional complicated procedures.

In addition, in the present invention, when the sperm/fertilized eggs freezing procedure is performed, the ice nucleation material actually comes into contact with the solution containing the semen or fertilized eggs, so the ice nucleation material effectively functions as an ice nucleus during the freezing procedure. In other words, in the present invention, ice nuclei are planted not by the manual operation of the worker freezing the sperm/fertilized eggs, but by pre-positioning the ice nucleation material in the straw tube at a position where it will come into contact with the solution containing the semen or fertilized eggs, so ice crystals can be formed with high precision and certainty.

In this way, when freezing and storing sperm and fertilized eggs, freezing can be done easily and with high reliability, and ice crystals can be formed without excessive supercooling, so the procedure for freezing and storing sperm or fertilized eggs can be carried out efficiently, and cell damage during freezing can be suppressed, improving the survival of sperm or fertilized eggs after thawing. This also improves the conception rate during artificial insemination and embryo transfer, and in the case of artificial insemination, the number of sperm to be enclosed in each straw tube can be reduced, allowing for efficient use of the collected sperm.

In addition, in the present invention, because the ice nucleation material is held in the fiber member that forms the inner plug, there is no concern that the ice nucleation material will adhere to the inner wall surface of the straw tube when injecting or transferring sperm or fertilized eggs, and there is no possibility that the ice nucleation material will contaminate the injector or transfer device, or that it will be mixed into the sperm or fertilized eggs during artificial insemination or fertilized egg transfer. This makes it possible to minimize the risk of ice nucleation material being mixed into solutions containing sperm or fertilized eggs, and further to eliminate concerns about the safety and toxicity of individuals undergoing artificial insemination or fertilized egg transfer due to exposure to ice nucleation material.

Another advantage of the present invention is that it eliminates the need for procedures to separate or recover ice nucleation material during artificial insemination or embryo transfer.

The present invention allows for easy and highly reliable freezing of sperm and fertilized eggs during cryopreservation, without the need for any additional complicated procedures. In addition, in principle, there is no risk of exposure of the recipient animal to ice nucleating materials, and therefore no safety or toxicity concerns.

<About the straw tube according to the present invention>
The present invention broadly encompasses straw tubes in which a solution containing sperm or fertilized eggs is contained in a hollow portion, in which a plug portion formed on one end side is composed of three layers, in order from the end side to the hollow portion side, an outer plug, a moisture-absorbing solidifying material powder layer, and an inner plug, and an ice nucleating material is held in the fiber member forming the inner plug. An example of this is described below using Figure 1. Note that the present invention is not narrowly limited to this embodiment.

FIG. 1 is a schematic diagram of a longitudinal cross section showing an example of a straw tube according to the present invention.

The straw tube A in Figure 1 is formed of a long, thin cylindrical member that is open at one end A1 and the other end A2. Inside the tube, there is a plug portion 1 formed at one end A1, a hollow portion 2 that contains a solution containing sperm or a fertilized egg, and a stopper 3 formed at the other end A2. The plug portion 1 is made up of three layers, from the end A1 side to the hollow portion 2 side: an outer plug 11, a layer of moisture-absorbing coagulant powder 12, and an inner plug 13.

The straw tube A is formed of a hollow, elongated cylindrical member with both ends A1 and A2 open. Any straw tube used in artificial insemination or embryo transfer can be widely used for the straw tube A. For example, in artificial insemination or embryo transfer of cattle, a colorless or colored transparent straw tube made of synthetic resin with a capacity of 0.25 mL or 0.5 mL, an inner diameter of 1.6 to 3.0 mm, and a length of 133 mm is commonly used, and can be used in the present invention.

The straw tube A can be made of any material, and is not particularly limited; for example, those made by extruding synthetic resin are disposable, highly productive, and easy to use, and are widely used.

When the straw tube is made of, for example, glycol-modified polyethylene terephthalate resin, among various synthetic resins, the tube is unlikely to discolor or deteriorate even when subjected to freezing and thawing processes or electron beam sterilization processes, so there is an advantage that the presence or absence of ice crystal formation, the quality of the sperm and embryo, etc. can be clearly and visually confirmed from outside the tube while the sperm or fertilized egg is still contained within the straw tube.

The plug portion 1 is formed near one end A1 of the straw tube A, and has a structure that almost completely blocks the hollow portion 2 of the straw tube A at the one end A1 side. It is composed of three layers: an outer plug 11, a moisture-absorbing solidifying material powder layer 12, and an inner plug 13.

The plug portion 1 can be formed, for example, by packing the outer plug 11, the moisture-absorbing solidifying material powder layer 12, and the inner plug 13 in sequence from the other end A2 side to a position near one end A1 of the straw tube.

The outer plug 11 is the layer located on the outermost side (one end A1 side) of the plug portion 1, with the other end A2 side facing the moisture-absorbing solidifying material powder layer 12 and the side surface facing the inner wall surface of the straw tube.

A wide variety of known fibrous materials can be used for the outer plug 11, and there are no particular limitations. For example, fibrous materials made of fibrous materials such as absorbent cotton, glass fiber, cotton thread, semi-synthetic fibers such as cellulose and acetate, and synthetic fibers such as polyester, polyamide, polyacrylic, and polyolefin can be used.

In the procedure for storing semen or fertilized eggs in the straw tube A, the solution may be stored in the straw tube A by sucking at one end A1 of the straw tube A (see symbol X1) while supplying a solution containing semen or fertilized eggs from the other end A2 (see symbol X2). Therefore, it is preferable that the outer plug 11 is made of a material that allows air to pass between the inside and outside of the tube A even after the plug portion 1 is formed.

Furthermore, in the procedure of artificial insemination or embryo transfer, when a solution containing semen or fertilized eggs is to be expelled from straw tube A, a push rod may be inserted from one end A1 and pushed toward the other end A2 (see symbol X3) to expel the contents from tube A (see symbol X4). Therefore, it is preferable that outer plug 11 is not adhered to the inner wall of straw tube A but is installed in a state in which it can slide freely.

The outer plug 11 is positioned, for example, so that its end A1 is located 3.0 to 5.0 mm from the end A1. The length of the outer plug 11 (longitudinal length, symbol X5) is not particularly limited, and may be set to the same length as a normal straw tube (for example, about 6 mm).

The moisture-absorbing solidifying material powder layer 12 is a layer sandwiched between the outer plug 11 and the inner plug 13, which keep the powder in place at a fixed position near one end A1 of the straw tube.

The moisture-absorbing solidifying material powder layer 12 must contain at least moisture-absorbing solidifying material powder, and may also contain powders of other substances depending on the purpose and application.

The length of the powder layer 12 (longitudinal length, symbol X6) is preferably 1.0 to 5.0 mm, and more preferably 1.2 to 4.0 mm. For example, by shortening the length of the powder layer 12, when sperm or fertilized eggs are sucked into the straw tube A from one end A1 (see symbol X1) during the procedure of storing the sperm or fertilized eggs in the straw tube A, air circulation inside and outside the straw tube A can be ensured, and a significant increase in suction pressure can be prevented.

The moisture-absorbing solidifying powder in the powder layer 12 is a material that can be solidified and solidified by absorbing moisture, and can maintain that state for a long period of time. For example, powder materials such as sodium alginate, polyvinyl alcohol, and gelatin powder can be used as the moisture-absorbing solidifying powder.

By containing moisture-absorbing coagulant powder in the plug portion 1, when a solution containing sperm or fertilized eggs is placed in the straw tube A and the solution penetrates the inner plug 13 and reaches the moisture-absorbing coagulant powder layer 12, the moisture causes the moisture-absorbing coagulant powder to coagulate and solidify, and one end A1 of the straw tube A is automatically sealed. This makes it possible to prevent the solution from leaking from one end A1 of the straw tube A.

For example, in the procedure of artificial insemination or embryo transfer, when a solution containing semen or fertilized eggs is forced out of straw tube A, a push rod is inserted from one end A1 and pushed toward the other end A2 to force the contents out of tube A, so it is preferable that the powder in moisture-absorbing solidifying material powder layer 12 is a material that does not easily adhere to the inner wall of tube A, and from that perspective, it is most preferable that the moisture-absorbing solidifying material powder is sodium alginate.

The inner plug 13 is the layer located closest to the hollow section 2 inside the plug section 1, with one end A1 facing the moisture-absorbing coagulant powder layer 12, the other end A2 facing the hollow section 2, and the side facing the inner wall surface of the straw tube.

The inner plug 13 has the function of substantially isolating the solution containing the sperm or fertilized eggs contained in the hollow portion 2 within the straw tube A, and together with the outer plug 11, it has the function of sandwiching the moisture-absorbing solidifying powder layer 12 to retain the powder, and at the same time, it has the function of allowing the solution to permeate and the moisture to reach the moisture-absorbing solidifying powder layer 12.

The inner plug 1 may be any material that can be infiltrated with a solution containing sperm or fertilized eggs, and in principle, the same fiber material as the outer plug 11 described above can be widely used, and fiber materials made of known fiber materials can be widely used, and there is no particular limitation. For example, from the viewpoint of ease of carrying ice nucleation material, it is preferable that the inner plug 1 is made of polyester fiber or nylon fiber, and furthermore, it is more preferable that the inner plug 1 is made of nylon fiber, in terms of ease of carrying an appropriate amount. The fiber structure of these materials can also be widely used, and there is no particular limitation, but, for example, from the viewpoint of liquid permeability, it is preferable that the fiber is a multifilament made of long fibers, and furthermore, from the viewpoint of ease of carrying ice nucleation material and liquid permeability, it is more preferable that the fiber is a woolly processed multifilament.

The inner plug 13, like the outer plug 11, is preferably made of a material that allows air to flow between the inside and outside of the tube A even after the plug portion 1 is formed, and is preferably installed in a freely slidable state without being glued to the inner wall of the tube A.

The length of the inner plug 13 (longitudinal length, symbol X7) is preferably 2.0 to 7.0 mm, more preferably 2.0 to 6.0 mm, and most preferably 2.0 to 5.5 mm.

For example, if the length of the inner plug 13 is made shorter than the normal length (e.g., the length of the outer plug 11), the function of allowing the solution containing semen or fertilized eggs to reach the moisture-absorbing coagulant powder layer 12 can be maintained, which has the advantage that when performing the sperm/fertilized eggs freezing procedure, the operator can automatically plant the ice with a high degree of certainty without having to change the conventional freezing procedure.

In the present invention, the ice nucleating material is held in the fiber member that forms the inner plug 13.

The ice nucleation substance is a substance that functions as ice nuclei for the formation of ice crystals. By containing the ice nucleation substance in the plug portion 1, when the sperm or fertilized eggs are cryopreserved, the solution containing the sperm or fertilized eggs also comes into contact with the ice nucleation substance held in the inner plug 13, and the ice nucleation substance functions as ice nuclei during the freezing procedure, inducing the formation of ice crystals. Therefore, when performing the freezing procedure for sperm/fertilized eggs, the worker can automatically plant the ice with a high degree of certainty using only the conventional freezing procedure, without having to perform any additional complicated procedures.

In the present invention, any known ice nucleation material can be used, and there is no particular limitation, but for example, it is most preferable that the ice nucleation material is silver iodide.

Silver iodide is a substance that effectively functions as an ice nucleus for the formation of ice crystals, so by using silver iodide as an ice nucleus material, it is possible to freeze sperm and fertilized eggs with a high degree of certainty without having to carry out separate, complicated procedures.

The silver iodide is not particularly limited as long as it can be held by the fibrous material that forms the inner plug 13, and any known silver iodide can be used.

For example, silver iodide can be retained on a fiber material by immersing the fiber material in an iodine aqueous solution, then immersing it in a silver nitrate aqueous solution, rinsing with water and drying. In addition, for example, by using a radiation graft polymerization method, synthetic fibers such as nylon 6 can be irradiated with gamma rays, then immersed in a glycidyl methacrylate (GMA)/methanol solution to graft polymerize GMA, the GMA-grafted fiber can be immersed in a triethylenediamine aqueous solution of about pH 9.5 to which hydrochloric acid has been added to introduce triethylenediamine, the fiber can be converted to an iodide ion form by contacting it with a potassium iodide aqueous solution, and then the fiber can be contacted with a silver nitrate aqueous solution to fix silver iodide to the fiber.

The amount of silver iodide held in the inner plug 13 is not particularly limited as long as it is an amount that induces ice crystal formation, but if the amount of silver iodide is too large, there is a possibility that the silver iodide may physically fall off. Therefore, for example, it is preferable that 0.05 to 400 mg of silver iodide is held in the inner plug 13, more preferably 0.05 to 100 mg, and most preferably 0.1 to 25 mg.

In addition, in order to prevent the silver iodide held in the inner plug 13 from physically falling off as much as possible, each fiber member may be subjected to a predetermined coating treatment. The means for coating the fiber members may be any known method and is not particularly limited.

Hollow section 2 is a hollow section formed near the center of straw tube A, between plug section 1 and plug 3, and is the section that contains a solution containing semen or fertilized eggs.

For example, when freezing, cryopreserving, or thawing sperm, the sperm prepared with a known diluent is inserted into the straw tube and contained in the hollow section 2. Also, when freezing, cryopreserving, or thawing fertilized eggs, the fertilized eggs prepared with a known freeze-resistant or preservative solution are inserted into the straw tube and contained in the hollow section 2. The fertilized eggs according to the present invention broadly include those to be used for fertilized egg transplantation, that is, eggs that have cleaved and differentiated from fertilized eggs to morulae, blastocysts, etc.

When storing a solution containing sperm or fertilized eggs, the entire area of the hollow section 2 does not need to be filled with the solution, and one or more air areas (air bubble areas) or areas containing other solutions may be formed as appropriate depending on the purpose and use.

　Any known procedure can be used to store the solution containing semen or fertilized eggs. For example, the solution containing semen or fertilized eggs can be sucked into one end A1 of the straw tube A (see symbol X1) and injected into the tube A from the other end A2 (see symbol X2), to store the solution containing semen or fertilized eggs in the hollow portion 2.

The stopper 3 is the sealing portion on the other end A2 side of the straw tube A after the solution containing sperm or fertilized eggs is placed in the hollow portion 2, i.e., on one of the two ends of the straw tube A, opposite the A1 side where the stopper portion 1 is formed.

The plug portion 1 formed on one end A1 of the straw tube A and the plug 3 formed on the other end A2 substantially seal the solution containing the sperm or fertilized eggs contained in the hollow portion 2, preventing leakage.

The method of sealing the other end A2 (i.e., the method of forming the plug 3) can be a wide variety of known methods and is not particularly limited. For example, after a solution containing sperm or a fertilized egg is placed in the hollow portion 2, the other end A2 may be plugged with wax or the above-mentioned moisture-absorbing solidifying material, or may be sealed by thermocompression or the like.

<About the semen-encapsulated straw according to the present invention>
The present invention broadly encompasses a semen-filled straw in which a solution containing semen is contained within the hollow portion 2 of the above-mentioned straw tube A and the other end is sealed, as well as an artificial insemination method that includes a procedure for using the semen-filled straw.

For example, a semen-filled straw can be produced by a method including at least the steps of: (1) inserting the components of the outer plug 11, the moisture-absorbing solidifying powder layer 12, and the inner plug 13, in that order, into one end A1 of the straw tube A to form the plug portion 1; (2) sucking in the one end A1 of the straw tube A (see symbol X1) and injecting a solution containing semen into the tube A from the other end A2 (see symbol X2), thereby injecting and storing the solution containing semen into the hollow portion 2 of the straw tube; and (3) sealing the other end A2.

The artificial insemination method according to the present invention includes, for example, (4) a step of freezing and storing the semen-filled straw, (5) a step of thawing the semen-filled straw and loading it into an artificial insemination syringe, and (6) a step of injecting the semen using the syringe.

By using the semen-encapsulated straw of the present invention, the procedure for freezing and storing semen can be performed in the same manner as the conventional method. And because the semen-encapsulated straw of the present invention contains an ice nucleating substance in the plug portion 1, ice can be planted automatically and ice crystals can be formed with a high degree of certainty without changing the conventional freezing procedure.

By using the semen-encapsulated straw of the present invention, the semen thawing procedure can be performed in the same manner as the conventional method. With the semen-encapsulated straw of the present invention, ice crystals are formed with high reliability through an automatic ice planting operation, and cell damage during freezing is suppressed, resulting in high sperm viability after thawing.

After the straw containing the semen has melted, the other end A2 near the stopper 3 is cut off with a straw cutter or similar (see symbol X8), and the straw tube A is loaded into an artificial insemination syringe.

Then, by manually pushing the tip of the syringe into the female's cervical canal, etc., the syringe is operated to insert the pusher rod into the straw tube A from one end A1 side (see symbol X3), and by pushing the contents out to the other end A2 side of the straw tube A, the semen escapes from the straw tube (see symbol X4) and is injected into the female's cervical canal, etc.

In the present invention, ice crystals are formed with high reliability through automatic ice planting operations, and sperm survival after thawing is high, so the number of sperm to be enclosed in each straw tube can be reduced, allowing for efficient use of collected sperm and improving the conception rate through artificial insemination.

<Straw containing fertilized eggs according to the present invention>
The present invention broadly encompasses a fertilized egg-enclosing straw in which a solution containing a fertilized egg is contained within the hollow portion 2 of the above-mentioned straw tube A and the other end is sealed, as well as a fertilized egg transfer method that includes a procedure for using the fertilized egg-enclosing straw.

For example, a straw containing a fertilized egg can be produced by a method including at least the steps of: (1) inserting the components of an outer plug 11, a moisture-absorbing solidifying powder layer 12, and an inner plug 13, in that order, into one end A1 of a straw tube A to form a plug portion 1; (2) sucking in the one end A1 of the straw tube A (see symbol X1) and injecting a solution containing the fertilized egg into the tube A from the other end A2 (see symbol X2), thereby containing the solution containing the fertilized egg in the hollow portion 2 of the straw tube; and (3) sealing the other end A2.

The fertilized egg transfer method according to the present invention includes, for example, (4) a step of freezing and storing the straw containing the fertilized egg, (5) a step of thawing the straw containing the fertilized egg and loading it into an injector for fertilized egg transfer, and (6) a step of transferring the fertilized egg using the injector.

By using the straw enclosing a fertilized egg according to the present invention, the procedure for freezing and storing the fertilized egg can be performed in the same manner as the conventional method. Furthermore, because the straw enclosing a fertilized egg according to the present invention contains an ice nucleating substance in the plug portion 1, ice can be planted automatically and ice crystals can be formed with a high degree of certainty without changing the conventional freezing procedure.

By using the straw containing fertilized eggs according to the present invention, the procedure for thawing fertilized eggs can be performed in the same manner as conventional methods. With the straw containing fertilized eggs according to the present invention, ice crystals are formed with high reliability through automatic ice planting operations, and cell damage during freezing is suppressed, resulting in high survival rates of fertilized eggs after thawing.

After the straw containing the fertilized egg is thawed, the area near the stopper 3 on the other end A2 side is cut off with a straw cutter or similar (see symbol X8), and the straw tube containing the fertilized egg is loaded directly or after the freeze-resistant solution removal procedure into an injector for embryo transfer.

Then, by manually pushing the tip of the syringe into the female's uterine horn, etc., the syringe is operated to insert the pusher rod into the straw tube A from one end A1 (see symbol X3), and the contents are pushed out to the other end A2 of the straw tube A, causing the fertilized egg to escape from the straw tube (see symbol X4) and be transplanted into the female's uterus, etc.

In the present invention, ice crystals are formed with high reliability through automatic ice planting operations, and the survival rate of fertilized eggs after thawing is high, which also improves the conception rate after embryo transfer.

<Method for freezing and preserving sperm or fertilized eggs according to the present invention>
The present invention broadly encompasses a method for cryopreserving semen or fertilized eggs, which includes the steps of placing a solution containing semen or fertilized eggs in the hollow portion 2 of the above-mentioned straw tube A and sealing the other end A2, and freezing the semen or fertilized eggs placed in the straw tube A.

As described above, the straw tube A of the present invention contains an ice nucleating substance in the plug portion 1, so ice can be planted automatically and ice crystals can be formed with high certainty without changing the conventional freezing procedure.

For example, by filling the hollow portion 2 of the straw tube A of the present invention with a solution containing sperm or fertilized eggs, sealing the other end A2, and then freezing the sperm or fertilized eggs contained in the straw tube A, cell damage during freezing can be suppressed and the survival rate of the sperm or fertilized eggs after thawing can be increased.

In Example 1, a straw tube according to the present invention was prototyped.

The fiber bundles made of synthetic fiber (nylon 6) with a diameter of 1.55 to 1.60 mm were used as the fiber material for the outer plug. In addition, sodium alginate (product name "Kimica Algin I-3", viscosity of 300 to 400 mPa·S in a 1% aqueous solution at 20°C, manufactured by Kimica Co., Ltd.) was prepared as the material for the moisture-absorbing and coagulating powder layer.

The member used for the inner plug was prepared as follows. The same fiber member used for the outer plug was first immersed in an iodine solution for 1 hour, washed with water, and then immersed in a silver nitrate aqueous solution for 4 hours.
After thorough drying, the fiber material was used as the inner plug with silver iodide attached. The weight of the fiber material before silver iodide attachment was 0.613 mg per mm, and the amount of silver iodide attached was 1.357 mg per mm of the fiber material, and the total amount of silver iodide attached to the inner plug was about 5.4 mg.

The fiber material used for the outer plug was cut to 6.0 mm in the longitudinal direction and inserted from the other end to one end of a straw tube (total length 133 ± 0.5 mm, outer diameter 1.95 ± 0.05 mm, inner diameter 1.61 ± 0.05 mm) made of glycol-modified polyethylene terephthalate resin until the tip reached a position 4.5 mm from one end of the straw tube. Next, 4 mg of sodium alginate was packed from the other end to one end until it reached the other end of the outer plug. The longitudinal length of the moisture-absorbing coagulant powder layer was 3.0 mm. Next, the silver iodide-attached fiber material used for the inner plug was cut to 3.5 to 4.0 mm in the longitudinal direction and inserted from the other end to one end until it reached the other end of the moisture-absorbing coagulant powder layer, so that the moisture-absorbing coagulant powder layer (sodium alginate) was sandwiched between the outer plug and the inner plug.

By following these steps, a straw tube with a total length of approximately 133 mm was created, with the following components formed in the hollow space, starting 4.5 mm from one end: an outer plug, a layer of moisture-absorbing coagulant powder, an inner plug, and a hollow section (the section that holds the solution containing semen or fertilized eggs).

In Example 2, a liquid was sealed in the straw tube prototyped in Example 1, and an experiment was conducted to see whether ice crystals would form when the liquid was frozen.

In the hollow part of the straw tube prototyped in Example 1, a mixture of saline and ethylene glycol (volume ratio 9:1) was introduced from one end (the side with the outer plug) by suction from the other end (the side opposite the outer plug, etc.), and the other end was sealed with a sealer.

The liquid tank was cooled to -7°C using a programmed freezer bath (solvent: methanol), and the straw tube was immersed horizontally in the tank to observe whether ice crystals formed within the hollow space. As a result, ice crystals were confirmed to have formed within 2 minutes of being placed in the freezer bath.

In Example 3, the effect of silver iodide on fertilized eggs was investigated.

In the presence of gel droplets of 2% silver iodide-containing 2% sodium alginate solution, bovine in vitro fertilized eggs were cultured to investigate whether the in vitro fertilized eggs differentiated and grew to the stage of emerged blastocysts.

As a result, the rate of escaped blastocyst formation in the control group without the addition of silver iodide was 42.4% (n=33), whereas the rate of escaped blastocyst formation in the control group with the addition of silver iodide was 62.8% (n=43), showing no significant difference. These results suggest that sealing fertilized eggs in the straw tubes of the present invention has no adverse effect on the development of the fertilized eggs, i.e., the safety of using silver iodide is guaranteed without any problems.

In Example 4, bull semen was sealed in the straw tube prepared in Example 1 and freeze-thawed.

A solution containing bovine semen was injected and contained in the straw tube prepared in Example 1 by suction from one end, and the other end was sealed to prepare a straw containing semen. Next, the semen was frozen by a quick freezing method using liquid nitrogen and stored in liquid nitrogen. The frozen and stored semen-containing straw was then immersed in lukewarm water at 35-40°C to thaw, opened, and the semen was removed, and the sperm vitality, axolome normal rate, and mitochondrial activity rate were evaluated using a sperm motility analyzer. As a result, there was no significant difference in the sperm properties when the straw tube according to the present invention was used compared to when semen was sealed in a commercially available straw tube (control group). This result demonstrated that the straw tube according to the present invention can also be used for the cryopreservation of semen for artificial insemination.

In Example 5, we attempted to transfer bovine embryos using bovine fertilized eggs that had been frozen and stored in the straw tubes prepared in Example 1.

First, straws for enclosing fertilized eggs were prepared. Bovine fertilized eggs were placed in the straw tubes prepared in Example 1 and immersed in a liquid bath at -7°C to confirm the formation of ice crystals from the inner plug and the complete freezing of the liquid layer containing the embryos. The straws were then slowly cooled to -30°C in a programmed freezer. The cryopreservation solution consisted of Dulbecco's PBS (Gibco14040-133) supplemented with 1 mg/mL glucose, 36 μg sodium pyruvate, 0.4% albumin, penicillin-streptomycin, 1.5 M (8.3%) ethylene glycol, 0.1 M sucrose, and 20% calf serum.

Next, embryo transfer was performed. The straw containing the fertilized egg was removed from the liquid nitrogen and placed in warm water to melt. The other end was then cut off and loaded into an injector for embryo transfer. The fertilized egg was then transferred into the uterus of a recipient cow on the seventh day after estrus.

As a result, conception of the recipient cow was confirmed. This result demonstrated that the straw tube according to the present invention can be applied to embryo transfer.

As mentioned above, in artificial insemination, fertilized egg transfer, and the like, the ice planting operation during cryopreservation is important for improving the survival of sperm and fertilized eggs after thawing. In contrast, the straw tube of the present invention has an added function of automatically planting ice in the procedure for cryopreservation of sperm and fertilized eggs. Moreover, with this straw tube, there are no concerns about exposure of the recipient animal to ice nucleating substances and the resulting safety and toxicity. Therefore, the present invention is useful for cryopreservation of sperm, fertilized eggs, and the like, in that it allows for simple and highly reliable ice planting, and there are almost no concerns about exposure of the recipient animal to ice nucleating substances and the resulting safety and toxicity.

In particular, in the present invention, ice nuclei are planted by placing an ice nucleating material in advance at a position in the straw tube where it will come into contact with the solution containing semen or fertilized eggs, which suppresses cell damage during freezing and improves the survival of sperm or fertilized eggs after thawing, and further improves the conception rate during artificial insemination and fertilized egg transfer. Therefore, it may be useful in freezing procedures for artificial insemination and fertilized egg transfer in non-human mammals such as cows, pigs, and horses, and also in freezing procedures for artificial insemination and fertilized egg transfer in mammals including humans.

In addition, in recent years, the conception rate in artificial insemination of cattle has been declining, which has become a problem. In response to this, the present invention can suppress cell damage during freezing and improve the survival of sperm after thawing, making it effective in improving the conception rate in artificial insemination of cattle.

1 is a schematic diagram of a longitudinal cross section showing an example of a straw tube according to the present invention. FIG.

1 Plug part 11 Outer plug 12 Moisture-absorbing coagulant powder layer 13 Inner plug 2 Hollow part 3 Plug A Straw tube

Claims (7)

1. A straw tube in which a solution containing semen or a fertilized egg is contained in a hollow portion,
   The plug portion formed on one end side is composed of three layers, in order from the end side to the hollow portion side, an outer plug, a moisture absorbing solidifying material powder layer, and an inner plug,
   A straw tube in which an ice nucleating material is held in the fiber member forming the inner plug.
2. The straw tube of claim 1, wherein the ice nucleating material is silver iodide.
3. The straw tube according to claim 2, in which 0.05 to 400 mg of silver iodide is held in the inner plug.
4. The straw tube according to claim 3, wherein the straw tube is made of glycol-modified polyethylene terephthalate resin.
5. A semen-filled straw in which a solution containing semen is contained in the hollow portion of the straw tube described in claim 1 and the other end is sealed.
6. A straw containing a fertilized egg, the hollow portion of which is filled with a solution containing a fertilized egg, and the other end of the straw tube according to claim 1 is sealed.
7. A step of placing a solution containing sperm or a fertilized egg in the hollow portion of the straw tube according to claim 1 and sealing the other end side;
   and a step of freezing the semen or fertilized egg contained in the straw tube.
   
   
   

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