JP2005270006A - Agent for preserving sperm by freezing - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To obtain an agent for preserving sperms by freezing, capable of keeping fertilizing capacity of a sperm effective even after thawing. <P>SOLUTION: This agent for preserving the sperms by freezing contains an ice nucleus-activator and a freeze-protecting material of a cell membrane-impermeable type. A kit for preserving the sperms by freezing includes the agent for preserving the sperms. A method for preserving the sperms by freezing involves using the agent for preserving the sperms by freezing or the kit for preserving the sperms by freezing. A container and a method for transporting the sperms preserved by freezing by the method are also provided. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to a cryopreservation agent for cryopreserving sperm, particularly mouse sperm, and a sperm cryopreservation kit containing the cryopreservation agent. The present invention also relates to a method of cryopreserving sperm using the cryopreservation agent or cryopreservation kit, and a container and method for transporting sperm cryopreserved by the method.

  Long-term preservation of embryos and germ cells that have the ability to generate new individuals based on their own genetic information is a vital technology in a wide range of fields including medicine, pharmacy, and biotechnology. is there. This technology is particularly important for breeding livestock and preserving genetic resources such as precious wild animals and genetically modified organisms. Furthermore, when an individual having a desired genetic trait is to be obtained, it is possible to greatly reduce the labor and cost of transactions by transporting embryos and germ cells instead of the individual itself. Over.

  However, living cells generally change over time due to metabolism and denaturation under high temperature conditions including room temperature, and their functions and activities eventually decrease or disappear. In order to suppress such changes in living cells, a cryopreservation method is usually used. This method prevents cell deterioration by suppressing cell metabolism and denaturation at an ultra-low temperature such as −80 ° C. or lower. However, cells contain water inside and are generally used for storage. Therefore, there is a problem that cells are damaged by ice crystals formed inside and outside in the cooling process. Therefore, various cryopreservatives for embryos and germ cells have been developed so far in the field of animal husbandry for the purpose of improving such harmful effects (see, for example, Patent Documents 1 to 3). Such cell storage technology has recently become increasingly important in many fields including basic research on biosystems and treatment of human infertility (see Patent Document 4).

  In the gene-related field, attention has been focused on the next stage of gene function research after the completion of decoding of the human genome. In this field, mice that are highly homologous to humans and whose genome has already been deciphered have become major experimental animals, and currently genetically modified mice such as knockout mice and transgenic mice. Many lines have been created. Traditionally, these strains have been maintained by breeding and breeding multiple mouse individuals, but with the increase in the number of strains, in recent years the lack of breeding space and increased labor costs have become a major issue. (Refer nonpatent literature 1). Therefore, in order to improve such a situation, attempts have been made to efficiently preserve the gene resources of the prepared strains in the form of cryopreserved embryos and sperm.

  When embryos and sperm are compared, the number of cells obtained from one individual is overwhelmingly higher in sperm, and sperm is far more desirable for the above purposes, such as requiring no complicated pretreatment such as superovulation treatment. Is excellent. However, mouse spermatozoa are very vulnerable to freezing, and a normal cryopreservation agent could not obtain a sufficient fertilization rate after thawing.

  In order to overcome this situation, a cryopreservation solution containing raffinose and a protein source has recently been developed (see Non-Patent Document 2 and Patent Document 5), and some results have been obtained for cryopreservation of mouse sperm. However, when this cryopreservation solution is used, the fertilization rate of sperm after freezing and thawing is as high as about 95% in the DBA / 2 line, but about 10% in the C57BL / 6 line and in the BALB / c line. As low as about 20%, sufficient fertility could not be ensured depending on the mouse strain. In addition, since the mouse strain currently most frequently used for basic and applied research as a genetically modified mouse is the C57BL / 6 strain, the above cryopreservation solution was not sufficiently effective for cryopreservation of genetically modified mice. On the other hand, as a method for improving the fertilization rate, there are reports that a method using a zona pellucida ovum and an intracytoplasmic sperm injection method are effective. However, such a method is complicated and requires advanced techniques. For this reason, cryopreservation of embryos is still the mainstream for preserving genetic resources of genetically modified mice, and sperm cryopreservation plays only a secondary role.

Japanese Patent Laid-Open No. 10-7501 JP 11-228301 A Japanese Patent Laid-Open No. 11-98935 JP 2000-239101 A JP 2001-328901 A JP 2001-247401 A Nature, June 20, 2002, vol. 417, p. 785-786. Theriogenology, 1992, 37, p. 1283-1291

  An object of the present invention is to provide a cryopreservative capable of maintaining the fertility of sperm, particularly mouse sperm, at a high level even after freezing and thawing.

  The inventors of the present invention are diligently studying to achieve the above-described object. The cause of the decrease in fertility of mouse sperm after freezing and thawing is not the decrease in sperm motility, which has been generally accepted, but the sperm head. It was found that this was due to significant damage to the part, especially the acrosome. The acrosome contains enzymes that decompose the zona pellucida of the ovum, and if the enzymes are lost due to damage to the site, sperm cannot enter the ovum. This coincides with the fact that fertilization rate increases when zona pellucida ova are used (URL: http://card.medic.kumamoto-u.ac.jp/card/japanese/sigen/h13/18. html [Search September 9, 2003]).

  As a result of further research based on this knowledge, the use of a cryopreservation agent containing an ice nucleus active substance and a cell membrane non-permeabilized cryoprotectant prevents the sperm head from being damaged by freezing and thawing. In mouse strains that were not, we found that sufficient fertilization rate was obtained using sperm after freeze-thawing, and this study was completed.

That is, the present invention relates to a sperm cryopreservation agent that contains, as active ingredients, an ice nucleus active substance and a cell membrane impermeable cryoprotectant.
The present invention also relates to the cryopreservation agent, wherein the ice nucleus active substance is one or more amino acids.
The present invention further relates to the cryopreservation agent, wherein the cell membrane impermeable cryoprotectant is one or more saccharides.
The present invention also relates to the cryopreservation agent, wherein the amino acid is one or more selected from the group consisting of glycine, alanine, valine, asparagine, isoleucine, glutamine, proline and histidine.

Furthermore, the present invention relates to the cryopreservation agent, wherein the saccharide is selected from the group consisting of raffinose, lactose and trehalose.
The present invention further relates to the cryopreservation agent, wherein the amino acid is selected from the group consisting of alanine, glycine and glutamine, and the saccharide is raffinose.
The present invention also relates to the cryopreservation agent, wherein the content of the ice nucleus active substance is 5 to 200 mM and the content of the cell membrane impermeable cryoprotectant is 1 to 30% (w / v).
The present invention further relates to the cryopreservation agent, wherein the sperm is mouse sperm.
Furthermore, the present invention relates to the cryopreservation agent, wherein the mouse is a C57BL / 6 strain, DBA / 2 strain or BALB / c strain mouse, or a genetically modified mouse having these in the background.

The present invention also relates to a sperm cryopreservation kit comprising the above cryopreservation agent, a petri dish for collecting sperm in the cryopreservation agent, and a straw filled with sperm in the cryopreservation solution.
Furthermore, this invention relates to the cryopreservation method of a sperm including freezing a sperm in said cryopreservation agent.
The present invention also relates to a sperm transport container comprising the above-mentioned cryopreservation agent and sperm frozen therein.
The present invention further relates to a method for transporting sperm using the transport container described above.

In the cryopreservation agent of the present invention, the cell membrane impermeable cryoprotectant protects the cell membrane, and the presence of an ice nucleus active substance makes it easier for ice crystals to form in the cryopreservation agent during the freezing process. It is thought that the cells are dehydrated by surrounding the sperm and acting to pump out the moisture in the cells that have not been frozen to the outside of the cells, and the damage to the cells due to freezing is suppressed.
Conventionally, the formation of ice crystals inside and outside the cells has been the cause of cell damage due to freezing, so in the development of cryopreservatives, emphasis has been placed on the suppression of ice crystal formation. In general, those containing an ice crystal formation inhibitor such as sulfoxide (see Patent Document 6). On the other hand, the cryopreservation agent of the present invention is a completely new one that suppresses the formation of intracellular ice crystals by actively forming ice crystals in the solution by the presence of the ice nucleus active substance, thereby suppressing freezing injury. It is based on the idea.

  The cryopreservation agent of the present invention effectively freezes sperm even in mouse strains that have not been able to obtain a sufficient fertilization rate after freezing and thawing, particularly in C57BL / 6 strains and BALB / c strains that are important in studies such as gene analysis. It can be preserved, and a great contribution can be expected in efficient long-term preservation and transportation of genetic resources of genetically modified mice.

Hereinafter, the suitable aspect of the cryopreservation agent of this invention is demonstrated in detail.
The cryopreservation agent of the present invention comprises an ice nucleus active substance and a cell membrane impermeable cryoprotectant as active ingredients.

The ice nucleus active substance used in the cryopreservation agent of the present invention is a substance that forms ice nuclei in a solution at a temperature higher than that in which intracellular water freezes, and does not impair the viability of sperm. Either may be sufficient. Such substances include amino acids, long-chain saturated alcohols (C _n H _{2n + 1} OH, where n is preferably in the range of 10 to 31 and n is preferably odd), and steroids such as cholesterol. Silver iodide, silica and the like are preferable, and amino acids are preferable, and glycine, alanine, valine, asparagine, isoleucine, glutamine, proline and histidine are particularly preferable. Of these, the most preferred ice nuclei active substances are glycine, alanine and glutamine.
The cryopreservation agent of the present invention can contain one or more of the above ice nucleus active substances. The amount of the above ice nucleus active substance in the cryopreservation agent of the present invention is such that the fertilizing ability of sperm can be increased even after freezing and thawing. Although it will not restrict | limit especially if it can keep effectively, When using an amino acid, it may be 5-200 mM, Preferably it is 50-150 mM.
In this specification, fertilizing ability of sperm is effective or sufficient, for example, the sperm is in vitro applied to an unfertilized egg without using a special technique such as zona pellucida incision method or intracytoplasmic sperm injection method. When fertilized, it means that a fertilization rate of 20% or more, preferably 25% or more, more preferably 30% or more, particularly preferably 50% or more is obtained.

The cell membrane impermeable cryoprotectant used for the cryopreservation agent of the present invention is not particularly limited as long as it is a substance that does not permeate the cell membrane and has cryoprotective activity in principle, but for example, sugars, dextran, polyvinylpyrrolidone ( PVP), polyethylene glycol (PEG), Ficoll and the like. A preferred cell membrane non-permeabilized cryoprotectant is a saccharide, with disaccharides or more being particularly preferred. Particularly preferred cell membrane impermeable cryoprotectants are raffinose, lactose and trehalose, and raffinose is most preferred.
The cryopreservation agent of the present invention may contain one or more of the cell membrane impermeable cryoprotectants.
The content of the cell membrane impermeable cryoprotectant in the cryopreservation agent of the present invention is not particularly limited, but is preferably 1 to 30% (w / v), more preferably 10 to 25% (w / v), most preferably Preferably it is 15 to 21% (w / v). A particularly preferred content is 18% (w / v). If the content is less than 1% (w / v), the sperm motility may decrease, and if it exceeds 30% (w / v), the cell membrane non-permeabilized freezing in the cryopreservation solution There is a tendency for the protective substance to precipitate.

The cryopreservation agent of the present invention can further contain a dispersion medium in order to further enhance the cell membrane protecting effect of the cell membrane impermeable cryoprotectant. Examples of such a dispersion medium include skim milk, milk, whey, egg yolk, bovine serum albumin, and serum. In this case, skim milk is preferable because it contains various proteins and minerals such as albumin and globulin, and therefore a composite protective effect can be expected. The dispersion media can be used alone or in combination.
The content of the dispersion medium is preferably 0.1 to 20% (w / v), more preferably 1 to 10% (w / v), and most preferably 2 to 4% (w / v). When the content is 0.1% by weight (w / v), the cell membrane protective effect may be low. When the content is more than 20% (w / v), the osmotic pressure of the cryopreservation agent is increased. Sperm viability tends to decrease.

  The form of the cryopreservation agent of the present invention is not particularly limited, but a solution is preferable. As the solvent, water is preferable. In the case of an aqueous solution, it is preferable to use distilled water, distilled water for injection, distilled water for cell culture, distilled water for embryo manipulation, etc. as a solvent.

  The cryopreservation agent of the present invention can be prepared by dissolving components containing an ice nucleus active substance and a cell membrane impermeable cryopreservation substance, for example, in water. The cryopreservation agent can be further sterilized, for example, by filtration sterilization, injected into a container (eg, ampoule, vial, etc.), and sealed. In addition, the ice nucleus active substance and the cell membrane non-permeabilized cryopreservation substance may be stored together or separately in a container (eg, ampoule, vial, etc.). At that time, at least one of them may be an aqueous solution, or both may be an aqueous solution. Further, as a kit configuration described below, only water is filled in a container, for example, an ampoule, and stored, and the aqueous solution may be used at the time of use. .

  Sperm cryopreserved by the cryopreservation agent of the present invention may be from any animal, but mouse sperm is preferred. Among these, sperm of C57BL / 6 strain, BALB / c strain or DBA / 2 strain, or genetically modified mice having these strains in the background is particularly preferable.

The sperm cryopreservation kit of the present invention includes the sperm cryopreservation agent, a petri dish for collecting sperm in the cryopreservation agent, and a straw filling the sperm in the cryopreservation agent.
The straw functions as a cryopreservation container for filling sperm suspended in a sperm cryopreservation agent and cryopreserving it. The inner diameter and length of the straw are not particularly limited, but those having an inner diameter of 0.5 mm to 3 mm are preferred for reasons of uniformity in temperature during sperm freezing.

The petri dish is used as a container for preparing a sperm suspension in a cryopreservation agent. A plurality of wells are provided on the upper surface of the petri dish used in the sperm cryopreservation kit of the present invention. The number of wells is not particularly limited, but it is desirable that the number of wells is plural in consideration of washing with a cryopreservative in order to remove blood and fat on the extracted epididymis tail, and preferably 4 to It is about 96. In addition, the well needs to have a size that allows easy cleaning and shredding of the epididymis tail. Therefore, the number of wells is particularly preferably about 4 to 12.
The straw and petri dish can be packaged after sterilization.
The sperm cryopreservation kit of the present invention can appropriately include an outer box, an instruction manual, a straw case and the like as necessary.

Next, a procedure for cryopreserving sperm using the sperm cryopreservation kit of the present invention will be described using a mouse as an example. However, this does not limit the present invention.
First, the epididymal tail of an adult male mouse (8 weeks of age or older) is transferred into a cryopreservation agent in a well on the petri dish. Suspend in a preparation to make a sperm suspension.

  Next, after attaching a straw to a syringe and filling a culture solution such as an HTF medium, take a sperm suspension in a petri dish lid or the like and sandwich an air phase between the filled culture solution and sperm suspension. Fill the straw. An air phase is provided at both ends of the straw and sealed with a sealer. Next, a straw filled with sperm is placed in a freezing float, and left for 5 to 15 minutes on the surface of liquid nitrogen in the liquid nitrogen storage device. Then, the float is immersed in liquid nitrogen, and the sperm is stored frozen.

Sperm cryopreserved as described above can be thawed at the time of use and used for in vitro fertilization with unfertilized eggs.
An example of thawing frozen sperm is as follows.
First, a frozen frozen straw is taken out of liquid nitrogen and immersed in warm water. Thereafter, the straw is pulled up from the warm water, and only the sperm suspension is transferred to a pre-culture medium prepared in advance (mainly HTF medium, but may be TYH medium, CZB medium, etc.), and pre-culture is performed. Sperm suspension after pre-culture can be used for in vitro fertilization

Sperm cryopreserved with the cryopreservation solution of the present invention can be stored in a transport container and transported.
As the transport container, any container that can stably hold frozen sperm at −120 ° C. or lower, preferably −196 ° C. or lower for 3 days or longer can be used. Examples of such containers include dry sippers, but are not limited thereto. When transporting, for example, the straw containing the sperm frozen as described above is stored directly or in a straw case, and then stored in the transport container. The transportation means is not particularly limited, but it is preferable that the transportation container is kept in a low temperature atmosphere during transportation.

  EXAMPLES Hereinafter, although this invention is demonstrated further more concretely based on an Example, this invention is not limited to a following example.

Example 1 (Preparation of sperm cryopreservation solution)
10 ml of distilled water was added to 0.0891 g of alanine and mixed by stirring to prepare a 100 mM alanine solution. The solution was added to a container containing 1.80 g of raffinose and 0.30 g of skim milk, mixed with stirring, and dissolved in a 60 ° C. hot water bath. The lysate was centrifuged and the supernatant was collected. Thus, the sperm cryopreservation agent of the present invention containing 100 mM alanine, raffinose 18% (w / v) and skim milk 3% (w / v) was obtained.

Example 2 ( Preparation of sperm cryopreservation kit)
The cryopreservative prepared in Example 1 was sterilized by filtration, poured into an ampule, and sealed. Then, γ-sterilized 0.25 ml plastic kusu-type sperm freezing straws having an inner diameter of 1.2 mm and 4-well dishes were individually packaged. These ampoules, straws and petri dishes were combined to obtain the sperm cryopreservation kit of the present invention.

Example 3 (Cryopreservation of sperm)
Using the sperm cryopreservation kit obtained in Example 2, sperm was cryopreserved as shown below.
From the sex-matured 8-40 week old male C57BL / 6 strain mice, the epididymis of the left and right testes was taken out immediately after euthanasia. In each well on the petri dish, 100 μl of a cryopreservation agent is placed in advance, and the two epididymis tails are lightly shaken in 100 μl of the cryopreservation agent in one of the wells. Unnecessary blood and the like were removed by moving to. This washing operation is repeated three times, and the epididymis tail is transferred to the fourth well, then it is cut into about 5 places with an ophthalmic scissor or a Weckel scissors and left as it is for 3 minutes in the cryopreservation agent. Sperm were collected. Thereafter, the tissue piece was removed, and the sperm was sufficiently diffused in the cryopreservation agent to obtain a sperm suspension.
Next, after attaching a straw to a 1 ml syringe and filling about 100 μl of HTF medium, take 12 to 15 μl of sperm suspension in a petri dish lid and sandwich a 10 mm air phase between the filled culture medium. Then, the straw was filled, and a 10 mm air phase was provided at both ends of the straw and sealed with a sealer. Next, the straw was heated at 37 ° C. for 1 minute so that the sperm was uniformly distributed in the straw.

  A sperm freezing float in which an outer cylinder of a commercially available disposable 50 ml syringe is filled with a polystyrene foam piece having a thickness of 2 to 3 cm toward the tip is fixed to a sperm freezing float fixed to one end of an acrylic stick with a sperm. Was placed with the end filled with In this state, the float was floated on liquid nitrogen and allowed to stand for 10 minutes. This lowers the sperm temperature to about -120 ° C at a cooling rate of about -20 ° C / min. The sperm freezing float was then introduced into liquid nitrogen and the straw was exposed in the liquid nitrogen liquid phase. The straw was stored in liquid nitrogen for 2 days to 6 months.

Example 4 (Transportation of frozen sperm)
Mouse sperm frozen using the cryopreservation kit of Example 2 was transported by the method as described below.
The straw containing the frozen spermatozoa was stored frozen in a state stored in a straw case. The frozen sperm was transferred to a dry sipper filled with liquid nitrogen in advance in a straw case so that the frozen sperm would not melt in the air phase. The dry shipper was transported by courier. The time required for this transportation was 2 to 3 days. The frozen sperm was taken out from the transported dry sipper and subjected to in vitro fertilization.

Experimental Example 1 (Changes in fertility of frozen-thawed sperm depending on the concentration of ice nucleating substance)
In order to examine the effect of the cryopreservation agent of the present invention on sperm fertilization ability, in vitro fertilization was performed using sperm freeze-thawed in cryopreservation agents containing various concentrations of ice nucleation substances.

a) Freezing and thawing of sperm Six kinds of cryopreservatives 1 to 6 were prepared in the same manner as in Example 1 so that the alanine concentrations were 5, 10, 50, 100, 150 and 200 mM, respectively. For comparison with a conventional cryopreservation agent, a cryopreservation agent A prepared in the same manner as in Example 1 except that alanine was not included was also prepared (see Patent Document 6).
Sperm were cryopreserved as described in Example 3 using cryopreservatives 1-6 and A, respectively. After 5 days, frozen mouse sperm was removed from liquid nitrogen, allowed to stand at room temperature (25 ° C.) for 5 seconds, and then heated in a 37 ° C. water bath for 10 minutes. Thereafter, sperm was introduced into 90 μl of HTF medium prepared in advance, and the sperm was precultured by allowing it to stand in a CO ₂ incubator controlled at 37 ° C. and 5% CO ₂ for 1 hour. After pre-culture, motor spermatozoa were selected and collected while avoiding dead spermatozoa and immobilized spermatozoa collected in the center.

b) In vitro fertilization Mouse eggs subjected to in vitro fertilization were collected from 8-week-old C57BL / 6 female mice according to a standard method. That is, the mouse ovum was superovulated by the superovulation induction method using PMSG and hCG, and the ovum cumulus complex for one female obtained by collecting the ovulated cumulus complex from the enormous part of the mouse oviduct, It was introduced into 90 μl of HTF medium prepared in advance. For freeze-thawed sperm obtained from one sperm straw, five egg cumulus complexes (each present in 90 μl of HTF medium) were prepared.
10 μl of the solution containing the sperm obtained in the above a) was introduced into a medium containing one egg ovule complex, respectively, and then spermized.

c) Determination of in vitro fertilization rate 10 hours after in vitro fertilization, fertilized eggs and unfertilized eggs were collected and fixed on a slide glass with glutaraldehyde to prepare a fixed specimen. Fertilization was determined based on whether or not there were two pronuclei, and two were used as fertilized eggs, and none were used as unfertilized eggs and counted. The number obtained was substituted into the following formula to calculate the in vitro fertilization rate.
In vitro fertilization rate (%) = {number of fertilized eggs / (number of fertilized eggs + number of unfertilized eggs)} × 100

表１により、アラニンを従来の凍結保存剤に加えることで、マウス精子の凍結保存率が飛躍的に向上することがわかる。またその効果はアラニン濃度に依存的であり、至適濃度である１００ｍＭでの体外受精率は、アラニンを含まない従来の凍結保存剤を用いた場合の約５倍に達した。 d) Results Table 1 shows the in vitro fertilization rates obtained in c).

Table 1 shows that adding alanine to a conventional cryopreservation agent dramatically improves the cryopreservation rate of mouse sperm. In addition, the effect is dependent on the alanine concentration, and the in vitro fertilization rate at 100 mM, which is the optimum concentration, has reached about 5 times that when a conventional cryopreservative containing no alanine is used.

表２により、氷核活性物質としてアラニン以外のアミノ酸を用いても、凍結融解精子の体外受精率が顕著に改善されることが分かる。また、生体内に存在するアミノ酸以外の修飾アミノ酸についても、同様な結果を得ている。例えば、氷核形成物質として修飾アミノ酸の一種であるベタイン１００ｍＭを用いること以外は実施例１と同様にして凍結融解精子の受精能を評価したところ、体外受精率は２４.９±１６.８％であった。 Experimental Example 2 (Change in fertilizing ability of frozen and thawed sperm depending on the type of ice nucleus active substance)
As an ice nucleus active substance, glycine 100 mM, valine 100 mM, isoleucine 100 mM, asparagine 100 mM, glutamine 100 mM, aspartic acid 100 mM, histidine 100 mM, methionine 100 mM, threonine 100 mM, or proline 100 mM are used instead of alanine 100 mM. In the same manner as in Example 1, the fertilizing ability of freeze-thawed sperm was evaluated. Table 2 shows the fertilization rate obtained.

Table 2 shows that the in vitro fertilization rate of freeze-thawed sperm is significantly improved even when an amino acid other than alanine is used as the ice nucleus active substance. Similar results have been obtained for modified amino acids other than amino acids present in the living body. For example, when fertilizing ability of a freeze-thawed sperm was evaluated in the same manner as in Example 1 except that 100 mM betaine, which is a modified amino acid, was used as an ice nucleation substance, the in vitro fertilization rate was 24.9 ± 16.8%. Met.

Experimental Example 3 (Change in fertilizing ability of frozen and thawed sperm by mouse strain)
In order to evaluate the effectiveness of the cryopreservation agent of the present invention in strains other than the C57BL / 6 strain, the mice are BALB / c strain or DBA / 2 strain, and only the cryopreservation agent 4 was used as the cryopreservation agent. In the same manner as in Experimental Example 1, fertilizing ability of freeze-thawed sperm was evaluated. As a result, the in vitro fertilization rate was 55.4 ± 24.2% (n = 5) in the BALB / c line and 88.9 ± 10.4% in the DBA / 2 line (n = 5). On the other hand, the in vitro fertilization rate by freeze-thawed sperm was the same as that described above except that the cryopreservation agent A described in Experimental Example 1 was used as the cryopreservation agent. 21.5% (n = 5) and 82.2 ± 10.7% in the DBA / 2 line (n = 5). This indicates that the use of the cryopreservation agent of the present invention improved fertility in all strains used in the experiment as compared to the case of using a conventional cryopreservation agent. This shows that the present invention can be applied to any strain of mice including the C57BL / 6 strain and the BALB / c strain, which have been difficult in the past.

Claims (13)

  A sperm cryopreservation agent comprising an ice nucleus active substance and a cell membrane impermeable cryoprotectant as active ingredients.   The cryopreservation agent according to claim 1, wherein the ice nucleus active substance is one or more amino acids.   The cryopreservation agent according to claim 1 or 2, wherein the cell membrane impermeable cryoprotectant is one or more saccharides.   The cryopreservation agent according to claim 2 or 3, wherein the amino acid is one or more selected from the group consisting of glycine, alanine, valine, asparagine, isoleucine, glutamine, proline and histidine.   The cryopreservation agent according to claim 3 or 4, wherein the saccharide is one or more selected from the group consisting of raffinose, lactose and trehalose.   The cryopreservation agent according to any one of claims 3 to 5, wherein the amino acid is selected from the group consisting of alanine, glutamine and glycine, and the saccharide is raffinose.   The cryopreservation agent according to any one of claims 1 to 6, wherein the content of the ice nucleus active substance is 5 to 200 mM, and the content of the cell membrane impermeable cryoprotectant is 1 to 30% (w / v). .   The cryopreservation agent according to any one of claims 1 to 7, wherein the sperm is mouse sperm.   The cryopreservation agent according to claim 8, wherein the mouse is a C57BL / 6 strain, BALB / c strain or DBA / 2 strain mouse, or a genetically modified mouse having these strains in the background.   A sperm cryopreservation kit comprising the cryopreservation agent according to claim 1, a petri dish for collecting sperm in the cryopreservation agent, and a straw filled with sperm in the cryopreservation agent.   A method for cryopreserving sperm, comprising freezing sperm in the cryopreservation agent according to claim 1.   A sperm transport container comprising the cryopreservative according to any one of claims 1 to 9 and sperm frozen therein.   A method for transporting sperm using the transport container according to claim 12.