JP2000189155A - Preservation of mammalian embryo or ovum and thawing dilution of frozen mammalian embryo or ovum - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for preserving and thawing and diluting mammalian embryos or ova in high survival rate without concern for being infected with virus or bacteria. SOLUTION: This method comprises the following practice: mammalian embryos or ova are stuck with a vitrified liquid in the minimum amount enough to coat them onto the inner surface of a sterilized container for freeze preservation such as frozen straw, frozen vial or frozen tube followed by sealing the resultant container and then bringing it into contact with liquid nitrogen to effect rapid chilling; for thawing the embryos or ova thus frozen, the container preserved by the method described above is withdrawn from the liquid nitrogen, one end of the container is opened, and the resulting container thus opened is directly injected with a diluent at 33-39 deg.C to thaw and dilute the frozen embryos or ova.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to vitrification and preservation of mammalian ova and embryos, and more specifically to a method that can be used for cryopreservation and thawing dilution of ova and embryos at all stages of development in mammals. It is about.

[0002]

2. Description of the Related Art Cryopreservation of mammalian embryos enables the preservation of genetic resources of specific lines and varieties, and is not only an indispensable technique for breeding and raising livestock, but also threatening extinction. There is also a need to maintain zoo and wildlife species. In addition, its importance has been pointed out as a core technology of a fertility treatment program also in the human medical field.

[0003] Studies on the cryopreservation of mammalian embryos have been described by Smith (1.
952, 1953) began using glycerol (Polge et al., 1949), which has been shown to have a cryoprotective effect on sperm, to preserve rabbit embryos for transplantation studies. No major research progress was seen. However, Whittingham et al. (1972) and Wilmut (19
72) independently reported that mouse embryos cryopreserved by slow freezing using dimethyl sulfoxide, which has a molecular structure similar to glycerol, as a cryoprotectant, show high survival after thawing. With this success, research on cryopreservation of mammalian embryos has rapidly become active and has been applied to cryopreservation of eggs and embryos of small experimental animals (rats: Ogawa, 1978; Ogawa et al., 1973; Utsumi and Yubara , 1974a, b; Whittingham, 1975; rabbit: Bank & Ma
urer, 1974; Whittingham & Adams, 1976; Tsunoda &
Sugie, 1977).

[0004] In domestic animals, sheep (Willadsen et al., 197
4; Schwier et al., 1990) and goats (Bilton & Moor,
1976a), and especially in cattle, Wilmut & Rowson (1
973), following the development of technology related to embryo transfer in the context of worldwide commercialization, cryopreservation of embryos for preservation and transport of bovine embryos with excellent genetic characteristics The technology was actively researched and developed as one of the key technologies for embryo transfer (Bilton & Moor, 1976b; Willadsen e
t al., 1976; Leibo & Mazur, 1978; Trounsonet al.,
1978; Willadsen, 1980; Niemann et al., 1982; Renard
et al., 1982; Leibo, 1983, 1984, 1986a, b, 1988;
Renard & Heyman, 1983; Chupin et al., 1984; Massip
& Van Der Zwalmen, 1984; Suzuki et al., 1990; Niema
nn, 1991).

As a result, the cryopreservation technique of embryos has become an important technique widely used not only in research but also in industry. For this reason, cryopreservation of bovine embryos is a practical technique, not only requiring high and stable embryo viability after cryopreservation, but also improving processing cost or convenience (processing time and simplicity). Is required. However, slow freezing requires expensive cooling equipment, requires a long processing time, and exposes embryos to a low temperature region above the freezing point for a certain period of time, so it cannot be used for embryos sensitive to low temperatures or immature or mature eggs thing,
In order to improve the method, it is necessary to repeat an enormous number of trials and errors for setting the optimum dehydration conditions and the like, and disadvantages such as requiring a great deal of labor are problems.

[0006] On the other hand, the vitrification preservation method proposed by Luyet (1937) is a method of vitrifying a solution inside and outside cells by rapid cooling and storing the solution at the temperature of liquid nitrogen without using an expensive cooling device. The rapid cooling of embryos was considered to be an ideal practical technique, but its success was not long available (Luyet, 1966; Rapatz et al., 1966; Fah
y et al., 1984; Fahy, 1988). However, by using high concentrations of cryoprotectants, plant cells (Grout & Hensha
Following the success of vitrification in insect cells (James, 1980) and Rall & Fahy (1985), the vitrification of mouse 8-cell embryos and the vitrification of mammalian embryos were succeeded. The possibility was shown. This success led to a rapid activation of vitrification approaches in various animal embryos. Successful preservation of mouse embryos by vitrification using a simpler vitrification solution was reported by Sheffen et al. (1986), and mice (Exp., 1986; Kono and Kakuda) Matsumoto et al., 1987; Ka.
sai et al., 1990), rat (Konoet al., 1988; Is)
chenko et al., 1992; Nakamichi et al., 1993), rabbits (Kobayashi et al., 1990; Smorag et al .; 198)
9, Smorag & Gadja, 1991; Kasaiet al., 1992; Papis et
al., 1993) and livestock sheep (Schwieretal., 1).
990), pig (Yoshino et al., 1993; Kobayashi et a
l., 1994; Dobrinsky & Johnson, 1994), reported successful vitrification preservation.

In cows, Massip et al. (1986)
Applying the method of Sheffen et al. (1986) in mice,
Survival cases after vitrification preservation were obtained in late morula embryos recovered from the living body. However, blastocysts generally used for embryo transfer did not survive, indicating difficulty in vitrification and preservation of blastocysts (Massip et al., 1986; Doji et al., 1990;
Van Der Zwalmen et al., 1989). In recent years, Kuwayama et al. (1992) showed the first success in blastocysts in in vitro fertilization-derived embryos, the value of which has been rapidly increasing not only experimentally but also industrially with the development of in vitro fertilization technology. Following the reporting of cases, similarly successful cases of vitrification preservation have been reported (Tachikawa et al, 1994).

In order to put this technology to practical use in the field of bovine in vitro fertilized embryos, it is necessary to develop a method capable of thawing and diluting cryopreserved embryos at the transplantation site. In the slow freezing method,
A direct transplantation method or a direct dilution method in which a frozen embryo is thawed, undiluted, or diluted in a straw, and then transplanted to a recipient cow has been used (Leibo 1983; Renard & Heymann, 198).
3; Massip & Van Der Zwalmen 1984; Voelkel & Hu, 199
For 2a, b), it is necessary to establish a simple and practical method that enables direct transfer even to vitrified and preserved embryos.

[0009] Kuwayama et al. (1994) disclose in a straw a diluent layered on a vitrification solution, and after thawing, dilute and remove a cryoprotectant from embryos to allow direct transfer to recipients. The dilution method was reported. High fertility and normal litters were obtained in experimental conditions using the same method, and the validity of the experiment was demonstrated by follow-up tests performed by other researchers. Melting, dilution and handling during transplantation are difficult,
There is a need to improve on a simpler approach. In addition, for viable blastocysts and in vivo fertilized embryos, the viability of morula embryos, which are also used for embryo transfer, after vitrification preservation, is not sufficient.
It is necessary to develop an effective vitrification method suitable for these embryos. In addition, especially for pre-fertilized ova, the size of ovum cells has been increasing despite the necessity for the in vitro fertilization system in animals and the dramatic development of human fertility treatment methods. Due to its high sensitivity and low temperature sensitivity, survival after slow cryopreservation has hardly been obtained, and satisfactory results have not been obtained using vitrification storage (Haman
o et al. 1992, Kuchenmeister & Kuwayama, 1996).

[0010] Martino (1996) obtained a higher viability after vitrification and storage by using a grid for an electron microscope and immersing eggs in liquid nitrogen directly together with a minimized vitrification solution. Vajta et al. Also reported that a 0.25 ml plastic straw (Ope
n Pulled Straw (OPS), a small amount of vitrification solution containing embryos was directly exposed to liquid nitrogen and cooled to obtain survival after vitrification storage. However, in both cases, the eggs are brought into direct contact with the liquid nitrogen at the time of cooling, and thus there is a risk of virus infection, which is not suitable for practical use. In addition, the shape of the vitrified sample on the grid is difficult to preserve, as is the case with the OPS method, which is already 0.25 ml worldwide.
It is difficult to use the standardized and unified system for storing and distributing bovine frozen embryos for straws, and it is not possible with these techniques to directly transfer the container used for freezing to the transplantation. Currently, in the in vitro fertilization system of livestock, it is necessary to cryopreserve many eggs of the same donor or the same species at a time in order to increase the efficiency of embryo production and reduce costs.
The method has not been developed yet.

[0011]

SUMMARY OF THE INVENTION The present invention has been made in view of the above problems, and has as its object to minimize the amount of vitrification without directly contacting a mammalian embryo or egg with liquid nitrogen. An object of the present invention is to provide a preservation method which can obtain a high survival rate without the risk of infection by viruses or bacteria by cryopreservation with a liquid.

Another object of the present invention is to provide a method for rapidly thawing and diluting an embryo or egg frozen and stored with a minimum amount of vitrification liquid while maintaining a high survival rate.

Another object of the present invention is to provide a method for thawing and diluting embryos or ova frozen and stored with a minimum amount of vitrification solution so that they can be directly transplanted quickly and while maintaining a high survival rate.

[0014]

In order to achieve the above object, the method for preserving a mammalian embryo or ovum according to the present invention employs a sterilized frozen straw, a frozen vial or a frozen tube for a mammalian embryo or ovum. Paste the inner surface of the cryopreservation container with a minimum amount of vitrification liquid sufficient to enclose the embryo or egg, seal the cryopreservation container, and contact the container with liquid nitrogen for rapid It is cooled down.

[0015] As a result, the embryo or ovum is frozen in the sealed cryopreservation container without directly contacting the liquid nitrogen outside thereof, so that the embryo or ovum may be infected with a virus or a bacterium. There is no. In addition, since the minimum amount of vitrification liquid is used, the vitrification liquid can be quickly frozen and the survival rate of embryos or eggs can be maintained at a high level.

[0016] Preferably, in the above method, the amount of the vitrification solution is 0.5 μl or less per embryo or egg. Since the amount of the vitrification liquid is sufficient to envelop the embryo or ovum and is extremely small, the vitrification liquid can be quickly frozen and the viability of the embryo or ovum can be maintained high.

Preferably, in the above method, the diluent layer and the diluent droplets are separated and stored in a cryopreservation container with the embryo or egg interposed therebetween. This allows the frozen embryos or ova to be directly transplanted to the recipient after thawing and dilution.

Preferably, in the above method, a low-toxic vitrification solution obtained by adding about 30% ethylene glycol and 1M sucrose to a buffer solution is used as the vitrification solution for the embryo. Thereby, the survival rate of the embryo can be further increased.

Preferably, in the above method, a vitrification solution obtained by adding about 40% ethylene glycol and 1M sucrose to a buffer solution is used as the vitrification solution for the eggs. Thereby, the survival rate of the ovum can be further increased.

[0020] Preferably, in the method for preserving embryos, before exposing the embryos to the vitrification solution, the embryos are stored for about 1 hour.
Equilibration in 5% ethylene glycol solution or about 10% glycerol solution for 5-10 minutes. Thereby, the survival rate of the embryo can be increased as compared with the case where the embryo is directly exposed to the vitrification solution.

Preferably, in the method for preserving an egg, the egg is equilibrated with an approximately 20% ethylene glycol solution for 5 to 10 minutes before exposing the egg to the vitrification solution. Thereby, the survival rate of the ovum can be increased as compared with the case where the ovum is directly exposed to the vitrification liquid.

In the method for thawing and diluting a frozen embryo or egg according to the present invention, the container for cryopreservation stored by the above method is taken out of the liquid nitrogen, and one end of the container is opened.
A method of directly injecting a diluent at 33 ° C. to 39 ° C. into the container and thawing and diluting the freezing of the embryo or egg. As a result, the thawed dilution of the frozen embryo or egg can be rapidly performed, and the survival rate of the embryo or egg can be increased.

Preferably, the container for cryopreservation stored by the above-described method for direct transplantation is taken out of the liquid nitrogen and immersed in warm water at 33 ° C. to 39 ° C. to freeze the embryo or ovum after thawing. Gently mixing the embryo and the diluent. This allows direct transfer of the embryo or egg.

[0024]

DESCRIPTION OF THE PREFERRED EMBODIMENTS The method of the present invention will be described below in detail. The animal species of eggs and embryos used for cryopreservation in the present invention is not specified. That is, the present invention can be applied to livestock and experimental animals represented by cattle, pigs, sheep, and the like, as well as to all mammals including humans.

As the cryopreservation container used for the cryopreservation, any container can be used as long as it can withstand storage in liquid nitrogen and can be hermetically sealed, such as a fertilized egg transplanting straw, a semen freezing straw, and various cryopreservation tubes and vials. Is possible.

The vitrification liquid has any of the compositions reported so far, that is, glycerol, propylene glycol,
Dimethyl sulfoside (DMSO), ethylene glycol,
A vitrified solution containing butanediol and sucrose, trehalose, percoll, polyethylene glycol, polyvinylpyrrolidone, bovine serum albumin, ficoll and the like as a cell membrane impermeant cryoprotectant can be used. In the embryo
The highest viability is obtained with a low-toxic vitrification solution in which 30% ethylene glycol and 1M sucrose are added to the buffer, and a vitrification solution in which eggs are added with 40% ethylene glycol and 1M sucrose in the buffer.

Before exposing the eggs and embryos to vitrification liquid,
Preliminary equilibrium treatment in which a low concentration of cell membrane permeable cryoprotectant is penetrated into cells to reduce physical damage due to osmotic pressure difference of embryos when injected into vitrification solution, and to further reduce cells during cooling and heating By preventing the formation of inner ice crystals, it is possible to obtain higher viability after storage.
In the present invention, preferably, the ovum is placed in a 20% ethylene glycol solution, and the embryo is placed in 15% ethylene glycol or
Equilibrate with 10% glycerol solution for 5 to 10 minutes. The work is performed at room temperature for blastocysts, and at a temperature of 30 to 33 ° C. for embryos or eggs at the earlier development stage. The eggs and embryos that have completed equilibration are introduced into the vitrification liquid by means of a hot and thin tip pasteur pipette with a fine and long working. Since the main purpose here is to replace the extracellular solution, the sample is moved in the vitrification solution and sufficiently washed. At this time, strong cell shrinkage occurs due to an extreme osmotic pressure difference between the equilibration solution and the vitrification solution, but the viability is not affected by exposure to the vitrification solution within 90 seconds. After washing the eggs and embryos with the vitrification solution, the same amount of vitrification solution is taken out with the same Pasteur pipette, the tip of the pipette is inserted into a straw, and the eggs and embryos are attached (attached) to the inner wall.

Here, the minimum amount of vitrification liquid means the minimum amount of vitrification liquid capable of securely and safely enclosing an egg or an embryo. That is, although the size of each embryo or egg is different, in the present invention, by encapsulating them with the smallest possible amount of vitrification liquid, it is ensured and safe.
The vitrification liquid should not exceed 0.5 μl (microliter) at most. Exceeding this amount may reduce the rate of freezing of the vitrification liquid and reduce the viability of the embryo or egg.

In the present invention, when the eggs or embryos are stored frozen for direct transplantation, it is preferable to perform dilution in a straw at the time of thawing. In this case, 6 to 7 cm (in the case of a 0.25 ml straw, , 150-175
A column of about 10 μl of diluted solution (buffer containing 10% chicken egg yolk and 0.5 M sucrose) is created by suction, and a vitrification solution containing eggs and embryos is attached to the column (tip side). . Further, a microdroplet of the diluent is prepared before this, and then the straw tip is sealed by heat or ultrasonic waves, immediately put into liquid nitrogen, rapidly cooled, and stored. When using a large freezing container other than a straw, such as a freezing vial or a freezing tube, cool the container to ultra-low temperature in liquid nitrogen in advance and use the minimum amount of vitrification liquid containing embryos and eggs on the inner wall. It is discharged as if sprayed, fixed to the inner wall, and cooled.

For thawing in storage of vitrification liquid without using a straw for direct transplantation, remove the frozen container from liquid nitrogen, open one end of the container, and immediately at 37 or 38 ° C.
A high heating rate can be obtained by injecting the diluent into a cryocontainer or, in the case of a straw, inhaling and thawing. In this method, melting and dilution are performed simultaneously,
After standing for 1 minute, it can be washed and immediately used as a molten egg or embryo for experiments and the like.

In the case of storage of a vitrified liquid using a straw for direct transplantation, thawing is performed by immersing the straw in warm water at 33 ° C. to 39 ° C., preferably at 37 or 38 ° C. to thaw the freezing of the embryo or egg. To further increase the melting rate, the straw is shaken to promote mixing of the diluent. Next, the tip of the straw is cut, set in a transplanter, and transplanted into a recipient.

<Experimental Examples of the Invention> Experiment 1 Vitrification and preservation of bovine blastocysts by the minimum volume method: <Materials and methods> In the experiment, the expanded blastocysts of Day 7 (insemination day = Day 0) produced according to a conventional method Vesicles were used. The embryo is equilibrated (25
% Ethylene glycol, 3 mg / ml polyvinylpyrrolidone; PVP MW40000, PBS supplemented for 10 minutes, and then poured into a vitrification solution (PBS supplemented with 25% ethylene glycol, 20% sucrose, 20% PVP). After injection of the vitrification solution, experiment group 1: embryos were introduced into a 25 μl vitrification liquid column in a straw in the same manner as before (see FIG. 1).
Embryos were affixed to the inside of the straw with a minimum volume (0.1 μl) of vitrification solution (see FIG. 2). In Experiments 1 and 2, after the straw tip was heat-sealed, the embryos were cooled by cooling and stored in liquid nitrogen. The embryos frozen in the experimental section 1 were taken out of liquid nitrogen, kept in air at room temperature for 10 seconds, thawed in warm water at 37 ° C for 10 seconds, thawed, and diluted (10% chicken egg yolk). , 0.5 M sucrose and PBS containing 20% calf serum) to dilute and remove the cryoprotectant. In Experimental Section 2, the embryo was directly immersed in the diluent by injecting the heated diluent into the straw, and rapidly melted in one step (see FIG. 3).
In the experimental section 3, the embryo frozen in the same manner as the experimental section 2 was thawed by the method in the experimental section 1.

The embryos after diluting and removing the cryoprotectant were washed twice with a washing medium (TCM 199 containing 10% calf serum), and co-cultured with a cumulus cell monolayer at 38.5 ° C., 5% CO 2 in the same medium. _2. Performed for 24 hours under the condition of 95% air to determine the viability.

<Results and Discussion>

[Table 1] As is clear from the results of the above experiment, in the method of the present invention according to Experimental Section 2, the survival rate of Experimental Section 1 according to the conventional method (56.
7%), the survival rate is significantly improved.

From the above results, the viability of embryos vitrified and stored by the minimum volume method and rapidly heated was significantly higher than that of the conventional method, and the viability of embryos after vitrification was improved by the same method. It became clear that it would be. It was also found that it is preferable to heat the embryos frozen by the minimum volume method rapidly in one step.

Experiment 2 <Materials and Methods> Temperature changes during cooling and heating under the conditions of Experiment 1 were recorded, and the cooling and heating rates were calculated. <Results> In the two experimental sections, the cooling and heating rates were about -9600 ° C / min and about 43800 ° C / min, respectively. was gotten.

Experiment 3 <Materials and Methods> In order to directly transfer the embryo vitrified and stored by the minimum volume cooling method, the embryo was immersed in warm water with the straw closed at a high rate by a simple method in which the straw was sealed. We examined whether it could be thawed and diluted.

A bovine blastocyst produced outside the body according to a conventional method was subjected to an experiment. Aspirate 150 μl of the diluent layer into a straw, create a minimum volume of vitrification liquid containing embryos, and further dilute droplets immediately before that, heat-enclose, and directly pour into liquid nitrogen for rapid cooling did. Thaw the straw directly at 37 ° C
In warm water, and shaken for 5 seconds. Subsequently, each liquid in the straw was mixed by holding the heat sealing portion of the straw and shaking lightly to dilute the cryoprotectant. After washing, embryos were cultured for 24 hours in the same manner as in Experiment 1 to determine viability. The vitrification liquid used was PBS to which 30% EG, 1M sucrose and 20% CS were added.

As a control for the dilution method, embryos were vitrified and stored by the method of the two sections in Experiment 1 in which a high survival rate was obtained, and the survival rates were compared. That is, after the embryo is similarly vitrified and stored,
The 37 ° C. diluent was sucked into the straw and mixed with the vitrification liquid to perform melting and dilution.

Further, the conventional in-straw dilution method (Kuwayama
et al. 1994), vitrified embryos, thawed, diluted,
After culturing, the utility of the method was compared and evaluated. That is, 25
After melting the embryos vitrified and stored in the μl column in two steps by treating them in air for 10 seconds and in warm water for 10 seconds, hold the straw vertically with the cotton plug up for 30 seconds,
The dilution in the straw was performed by mixing the vitrification liquid and the diluting liquid. After washing, the embryos were cultured for 24 hours as in Experiment 1, and the viability was determined.

<Results> Even in the case of using this method, that is, a simple one-step method in which an embryo is vitrified and stored in a minimum volume and then a straw is directly poured from liquid nitrogen into warm water to melt the embryo,
A high viability similar to the dilution method of Experiment 1 was obtained (97.7%, 42 /
43 vs 97.6%, 41/42), it became clear that the minimum volume freezing method can be used for dilution in straw.

The survival rate by the conventional in-sloteau dilution method was 92.3% (24/26), which was not significant but lower than that of the present method. Furthermore, a comparison of practicality shows that the melting method requires a two-stage heating method in the conventional method, and in particular, the straw is left in the air for 10 seconds in the first melting step, but this temperature (room temperature) ) Is difficult to control, and this is a problem in cold climates where Holstein is bred a lot, or conversely in summer when the temperature is high. However, in this method where straw in liquid nitrogen is directly immersed in hot water and melted, It is considered to be a more practical method because no serious problems occur.

[0043]

EFFECTS OF THE INVENTION According to the present invention, eggs and embryos of any animal species at any stage of development can be vitrified and stored in a sterile environment by a simple method. It can be used at a practical level in the medical field. The most industrially used bovine embryos can be thawed and diluted by simple operations at the site of embryo transfer, and are expected to be used in the same manner as artificial insemination, which has already become widespread. It can greatly contribute to usage.

[Brief description of the drawings]

FIG. 1 is an explanatory view showing a method of freezing an experimental section 1 in the order of steps.

FIG. 2 is an explanatory view showing a method of freezing an experimental section 2 in the order of steps.

FIG. 3 is an explanatory view showing a melting method of an experimental section 3 in the order of steps.

Claims (9)

[Claims] 1. A minimum amount of vitrification liquid sufficient to cover the inner surface of a cryopreservation container such as a frozen straw, a frozen vial or a cryotube obtained by sterilizing a mammalian embryo or ovum. A method for preserving a mammalian embryo or ovum, comprising sealing the container for cryopreservation, contacting the container with liquid nitrogen and rapidly cooling the container. 2. The method for preserving a mammalian embryo or egg according to claim 1, wherein the amount of the vitrification liquid is 0.5 μl or less per embryo or egg. 3. The mammalian embryo according to claim 1 or 2, wherein the diluent layer and the diluted droplets are separated and housed in the cryopreservation container with the embryo or egg interposed therebetween. How to store eggs. 4. The method according to claim 4, wherein the vitrified liquid of the embryo is about 30%.
The method for preserving a mammalian embryo or egg according to any one of claims 1 to 3, wherein a low-toxic vitrification liquid obtained by adding ethylene glycol and 1M sucrose to a buffer is used. 5. The method according to claim 5, wherein the vitrification liquid of the eggs is about 40%.
2. A vitrification solution obtained by adding 1% sucrose and 1% sucrose to a buffer solution.
4. The method for preserving a mammalian embryo or egg according to any one of items 3 to 3. 6. Prior to exposing said embryo to said vitrification liquid, said embryo is treated with about 15% ethylene glycol solution or about 1%.
The method for preserving a mammalian embryo or egg according to claim 4, wherein equilibration treatment is performed for 5 to 10 minutes with a 0% glycerol solution. 7. The ovum is exposed to an about 20% ethylene glycol solution for 5 to 1 days prior to exposing the ovum to the vitrification solution.
The method for preserving a mammalian embryo or egg according to claim 5, wherein the method is equilibrated for 0 minutes. 8. The container for cryopreservation stored by the method of claim 1 or 2 is taken out of the liquid nitrogen, one end of the container is opened, and a diluent at 33 ° C. to 39 ° C. is placed in the container. Wherein the frozen embryo or egg is thawed and thawed to freeze the embryo or egg. 9. The method according to claim 3, wherein the container for cryopreservation is taken out of the liquid nitrogen, and the container is immersed in warm water of 33 ° C. to 39 ° C. to freeze the embryo or egg, A method of thawing and diluting a frozen embryo or ovum, which comprises lightly mixing a shaking embryo and a diluent.