JP2002212001A - Liquid composition for vitrifying preservation of embryo and ultra-low temperature preservation thereof using the same - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a liquid composition for vitrifying preservation of embryos so as to establish the practical vitrifying preservation, wherein the composition is capable of preserving porcine early blastocyst covered with a perfect zona pellucida also applicable to disease control thereof, and to provide a simple and highly reproducible ultra-low temperature preservation method for porcine embryos. SOLUTION: This liquid composition for the vitrifying preservation for porcine early blastocyst is characterized by comprising (1) an M2 liquid added with v/v 10% ethylene glycol, (2) an M2 liquid added with v/v 10% ethylene glycol, 0.6 mol sucrose and w/v 1% polyethylene glycol and (3) an M2 liquid added with v/v 40% ethylene glycol, 0.6 mol sucrose and w/v 2% polyethylene glycol. The other objective ultra-low temperature preservation method for porcine embryos comprises using the composition.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to ultra-low temperature preservation of embryos by vitrification preservation method, and more particularly to a liquid composition for vitrification preservation and a method for ultra-low temperature preservation of pig embryos using the same.

[0002]

2. Description of the Related Art Fertilized egg transplantation has been performed as one of the livestock breeding methods. For that purpose, it is necessary to establish a method for preserving embryos (fertilized eggs). In a conventional fertilized egg freezing method, cells are damaged by ice crystals formed inside and outside the cells during freezing, which is one of the causes of decreasing the viability of the embryo after thawing.

[0003] In order to solve this problem, a technique for preserving cells without using ice crystals both inside and outside the cells by using a cryoprotectant at a higher concentration than the conventional freezing medium and rapidly cooling the cells has been proposed. This has made it possible to store embryos at ultra-low temperatures without destroying cells. This technique is a vitrification preservation method. By using the vitrification preservation method, we succeeded in improving the survival rate of in vitro fertilized embryos of cattle, which had been said to be vulnerable to freezing, after ultra-low temperature storage. Furthermore, by using the OPS method or the micro-droplet method, which increases the cooling rate during vitrification, it is becoming possible to store unfertilized eggs of cattle, which had been difficult to store at very low temperatures. However, this vitrification preservation method also has problems. This means that the chemical toxicity of highly concentrated vitrification solutions and the osmotic shock that occurs when adding or removing vitrification solutions can adversely affect embryos. In addition, when the concentration of ethylene glycol, which is usually used in the vitrification solution, is low, although the cytotoxicity is low, there is a problem that the solution freezes without becoming a glassy solid. Therefore, development of a vitrification solution having low toxicity to cells and easy handling is desired.

[0004] On the other hand, with respect to the technique for ultra-low temperature storage of pig embryos,
The reproducible technique has not yet been established, and embryos that can be stored at ultra-low temperature are limited to expanded blastocysts or blastocysts immediately after escape from the zona pellucida. However, by centrifuging the pig early embryo in 1995, the intracellular fat granules accumulate in one place, and after removing it with a micromanipulator, freezing the piglet from the ultra-low-temperature-preserved early embryo. Successfully produced (Nagashima H.,
Kashiwazaki N., Ashman RJ, Grupen CG, Nottle
MB, Cryopreservation of porcine embryos, Nature 1
955; 373. 416). However, this method requires specialized equipment and requires skill in operation of a micromanipulator, and thus has not yet been widely used.

[0005] A report that piglets were produced from ultra-cryogenically preserved embryos of early blastocysts completely wrapped in a zona pellucida using the vitrification preservation method, similarly to the above-described method, was used to produce centrifuged pig embryos. The OPS (Open Pulled Straw) method (Camcro
n RD, Beebe LF, Blacksbaw AW, Higgins A., Not
tle MB, Piglets born from vitrified carly blastc
ysts using a simpletechnique, Aust Vet J., 2000, M
ar .; 78 (3), 195-6). However, also in this method, there is a problem that equipment for centrifugation is required, and
Centrifugation of the embryo is required before vitrification, which is troublesome.

[0006]

SUMMARY OF THE INVENTION Accordingly, an object of the present invention is to provide a liquid composition for vitrification preservation of porcine early blastocysts completely wrapped in a zona pellucida, which can be applied to disease control, for ultra-low temperature preservation. In addition, a simple and highly reproducible method for preserving pig embryos at ultra-low temperature is to be developed. This makes the technique of transplanting pig embryos more practical.

[0007]

SUMMARY OF THE INVENTION The present inventor has proposed a liquid composition useful for the vitrification and preservation of pig embryos by referring to the microdroplet method which has succeeded in the vitrification and preservation of unfertilized bovine eggs. Was successfully developed, and a method for ultra-low temperature storage of pig embryos with high reproducibility was established. Then, it was confirmed that piglets could be produced using the preserved pig embryos by applying this method. The present invention has been completed based on such findings.

The present invention according to claim 1 provides (1) 10%
(V / v) M2 liquid with ethylene glycol added, (2) 10
% (V / v) ethylene glycol, 0.3 M sucrose and 1% (w / v) polyethylene glycol added M2 solution, and (3) 40% (v / v) ethylene glycol, 0.6
A liquid composition for vitrification and preservation of porcine early blastocysts, comprising M sucrose and 2% (w / v) polyethylene glycol added M2 solution.

According to a second aspect of the present invention, a swine early blastocyst is immersed in the solution (1) and the solution (2) according to the first aspect using a pipette to equilibrate the solution and the solution (2). A wound is made near the tip of the pipette during the equilibration in step 2, and after the equilibration is completed, the blastocysts sucked into the pipette are transferred to each of the solutions (3) according to claim 1, and then sucked into the pipette. Extruding the containing liquid to form microdroplets of the liquid at the tip of the pipette,
The microdroplets were moved onto the liquid nitrogen water surface to solidify the surface, then immersed in liquid nitrogen together with the pipette, and then the pipette was cut at the damaged portion near the pipette tip and filled with liquid nitrogen. An ultra-low-temperature preservation method for pig early blastocysts, which is contained in a container.

[0010]

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a vitrification-preserving liquid composition for preserving porcine early blastocysts at an ultra-low temperature and a method for preserving porcine early blastocysts using the composition. . The liquid composition for vitrification preservation of the present invention is used when vitrifying and preserving swine early blastocysts, and particularly has the ability to freeze water in cells and suppress the formation of ice, That is, it means a vitrification solution having high vitrification ability and low toxicity to cells.

The liquid composition for preservation of vitrification of the present invention comprises the following three types of liquids. That is, one liquid is 10
% (V / v) ethylene glycol added M2 solution, 2 solutions 1
M2 with 0% (v / v) ethylene glycol, 0.3 M sucrose and 10% (w / v) polyethylene glycol
Solution 3 and Solution 3 consist of M2 solution with 40% (v / v) ethylene glycol, 0.6M sucrose and 2% (w / v) polyethylene glycol. Table 1 shows the composition of each liquid constituting the vitrification storage liquid composition and the preparation method thereof, and Table 2 shows the composition of the M2 liquid.

[0012]

TABLE 1 Composition and preparation method of liquid composition for vitrification preservation EG: ethylene glycol PEG: polyethylene glycol (molecular weight 7300-9
000)

[0013]

Table 2 Composition of M2 liquid (when 2L is prepared) ^* BSA: Bovine serum albumin

In the conventional vitrification preservation method, ethylene glycol is used as one component of the vitrification solution, and its concentration is 6.5 mol / L (JR Dobrinsk).
y, VG Pursel, CR Long, and LA Johnson, Birth
of Piglets After Transferof Embryo Cryopreserved
by Cytoskeletal Stabilization and Vitrification, BI
OLOGY OF REPRODUCTION 62,564-570, 2000) or 8mol
/ L (Kobayashi S., Takei M., Kano M. et al., Pigle
ts produced by transfer of vitrified porcine embry
o after stepwise dillution of cryoprotectants, Cry
obiology, 1998; 36, 20-31). On the contrary,
In the vitrification preservation liquid composition of the present invention, ethylene glycol of three liquids is 7.0 to 7.5 mol / L (about 40 mol / L).
% (V / v)) is appropriate. The sucrose concentration is about 0.6 mol / L, which is equivalent to the concentration of a vitrification solution generally used for cattle. Incidentally, polyethylene glycol having a molecular weight of usually 7300 to 9000 is used.

Next, a method for preserving porcine early blastocysts at a very low temperature using the above-mentioned liquid composition for vitrification preservation will be described. (1) Collection of embryos When embryonic immature pigs are used as embryo pigs, pregnant horse serum gonadotropin (hereinafter may be abbreviated as PMSG).
Is administered, and 72 hours later, 500 IU of human chorionic gonadotropin (hereinafter sometimes abbreviated as hCG) is administered. Artificial insemination (hereinafter sometimes abbreviated as AI) is performed 24 hours to 48 hours after the hCG administration. Then, the day after the hCG administration day is set as day 0, embryos are collected surgically or after sacrifice on day 5, and the uterine horns are perfused with M2 solution, and the embryos are collected from the perfusate.

On the other hand, when sexually mature pigs are used as embryo pigs,
Prostaglandin F2α (hereinafter sometimes abbreviated as PGF2α) was administered in an appropriate amount 20 to 40 days after AI, and 24 hours after that, PGF2 was again administered.
Administer α. At the time of the second administration of PGF2α, PMSG
At the same time as 1500 IU. 72 hours later, h
CG is administered at 500 IU. 24 after administration of hCG
48 hours later, AI was performed. Embryos were collected surgically or after sacrifice on day 5, with the day following the day of hCG administration as day 0, and peritoneal horns were perfused with M2 solution and perfused. Collect the embryo from the solution.

(2) Culture of embryos until vitrification storage The embryos collected by the above (1) are placed in an incubator kept at 39 ° C. under 5% CO ₂ conditions in NCSU.
23 liquids (Petters RM, Reed ML, Additionof taurin
e or hypotaurine to culture medium improves develo
pment of one-and two-cell pig embryos in vitro, Th
Culture until vitrification preservation is carried out by a microdrop culture method using eriogenology, 1991; 35,253 (Abstract)). The culture time is usually within one hour. Note that NCSU
Table 3 shows the composition of the 23 liquids.

[0018]

Table 3 Composition of NCSU23 solution (at the time of making 100 mL) ^* BSA: Bovine serum albumin

The microdrop culture method was performed in the following manner. That is, a microdroplet of a culture solution of 30-100 μL is prepared in a Petri dish, and the Petri dish is allowed to stand in a carbon dioxide gas incubator.
After gas phase equilibration, the embryos are introduced into microdroplets. Next, the petri dish containing the embryo is cultured in a carbon dioxide gas incubator.

(3) Preparation of vitrification solution In the first hole of a four-hole petri dish, about 1 mL of the above-mentioned liquid composition for vitrification preservation liquid is placed in the first hole, and about 2 m in the second hole.
L Inject about 1 mL of the third solution into the third and fourth holes. Each liquid is kept at 35-39 ° C, preferably at 38 ° C.

(4) Equilibration with Vitrification Solution One solution obtained by injecting 1 to 10, preferably 5 pig embryos obtained by culturing according to the above (2) into the petri dish of the above (3) using a pipette or the like. And then equilibrate for 5 minutes, then further transfer to 2 solutions and equilibrate for 5 minutes. Here, the pipette or the like is preferably a Pasteur pipette, but any pipette or the like that has a thin tip and is easy to form microdroplets, such as a thin and stretched straw-shaped object or a micropipette tip. During equilibration with the two solutions, a place (D in FIG. 1) about 1 cm from the tip of the pipette or the like used when moving the embryo is lightly scratched with an ampule cutter or the like. Pre-scratching is preferable because it facilitates the work of cutting at this location during vitrification and storage. FIG. 1 shows one embodiment of the vitrification preservation method according to the present invention,
(I) shows the state before immersion in liquid nitrogen, and (II) shows the state after immersion in liquid nitrogen. On the other hand, liquid nitrogen is put in a container such as a mortar and prepared for vitrification and storage.

After the completion of the equilibration with the two solutions, the embryos are transferred to the third well containing the third solution, and further immediately to the third well in the fourth well. Subsequently, after the embryo is inhaled with a pipette or the like, the embryo is moved to a container containing liquid nitrogen, and a microdrop containing an embryo is placed on the tip of the pipette or the like at a height of about 5 cm from the upper surface of the liquid nitrogen (about 5 μL).

(5) Precooling The microdroplets formed in the above (4) are brought close to the upper surface of liquid nitrogen and held for 2 to 5 seconds, preferably about 3 seconds, to solidify the surface of the microdroplets. By this pre-cooling, the surface of the microdroplets solidifies, but the embryos in the microdroplets are not frozen. If the pre-cooling time is too short, the cooling rate will be too fast, which may have a large impact on the embryos in the microdroplets. On the other hand, if the pre-cooling time is too long, the cooling temperature of the embryo will be slowed down, and the embryo will be exposed to a low temperature region for a long time, which may cause a low-temperature injury.

(6) Vitrification storage After confirming that the surface of the microdroplets has solidified by pre-cooling, the microdroplets are immersed in liquid nitrogen together with a pipette.
Thereafter, a portion of about 1 cm from the tip of the pipette is gripped with tweezers, and the pipette is cut at the previously scratched portion (D in FIG. 1). The work from transferring the embryo to the third solution in the fourth hole and immersing it in liquid nitrogen is preferably performed within 30 seconds. The reason for this is that prolonged exposure of embryos to highly vitrified solutions has a negative effect on embryo viability. The embryo vitrified by the above method is placed in a predetermined container, for example, a microtube or the like having a hole in the lid, and stored in liquid nitrogen for an arbitrary period. That is, since the vitrified liquid at the tip of the pipette is small and fragile, it is stored in a microtube with tweezers together with the pipette. Note that a hole is formed in the lid so that liquid nitrogen always exists in the microtube.

The embryo preserved at an ultra-low temperature by the above vitrification preservation method can be melted by an ordinary method, if necessary, and after a predetermined process, can be subjected to embryo transfer. Hereinafter, an example of a method for melting vitrified preserved embryos will be described. Since a diluted solution is used for melting the embryo, the solution is first prepared. The diluting solution is used to dilute and remove the liquid composition used for thawing the vitrified preserved embryo and vitrifying and storing.

(7) Preparation of Dilute Solution The dilute solution is composed of three types of liquids, ie, 4 to 7 liquids. The fourth solution was NCSU23 solution to which 5% (v / v) ethylene glycol and 0.6 M sucrose were added.
NCSU23 solution to which% (v / v) ethylene glycol and 0.3 M sucrose were added. In addition, 6 liquids are 0.3M
NCSU23 solution to which sucrose was added. Solution 7 was NC
Consists of only SU23 solution. Table 4 shows the composition of each solution constituting the diluted solution and the preparation method thereof.

[0027]

Table 4 Composition of diluted solution and preparation method EG: ethylene glycol

Into a Petri dish having a diameter of 35 mm, 4 liquids, which are components of the above-mentioned diluted solution, are put so as to be about 3 mL.
In addition, about 1 mL of 4 solution was placed in the first hole of a 4-hole petri dish,
Approximately 1 mL of 5th liquid in the 3rd hole Approximately 1m of 6th liquid in the 3rd hole
L, inject about 7 mL of the 7 solution into the 4th hole. Each liquid is kept at 35-39 ° C, preferably at 38 ° C.

(8) Method of Melting Vitrified Preserved Embryo and Dilution of Vitrification Solution First, the contents of the microtube containing the vitrified preserved embryo are transferred to a container containing liquid nitrogen. Next, the vitrified embryo is pinched with tweezers, and the vitrified embryo is put into a 35 mm-diameter dish containing 4 liquids, which are the components of the diluted solution prepared in the above (7), and the microdroplets are melted. Let it. Immediately after the microdrops are thawed, the embryo is placed in a 4-well petri dish.
Transfer to solution 4 in the second well and dilute for 5 minutes. Subsequently, the mixture was diluted with 5 liquids and 6 liquids for 5 minutes each.
Dilute for 5 minutes with 7 solutions consisting of U23 solution only. Thereby, the vitrification-preserved embryo is completely melted, and the vitrification-preservation liquid composition is completely removed. Embryos thawed in this way have a high survival rate and are used for embryo transfer. Two of the embryos collected from one litter were thawed and cultured, and the remaining embryos were vitrified and stored for transplantation.
By subjecting the individual to an in vitro culture test, the viability of the embryos collected from the same abdomen after thawing can be determined.

[0030]

The present invention will be described below in detail with reference to examples, but the present invention is not limited to these examples. Example 1 In this example, the effectiveness of pre-cooling before vitrification was examined. As a control, a fresh 5-day-old embryo obtained by a microdrop culture method using NCSU23 solution supplemented with 10% FBS (fetal calf serum) was used.

(1) Collection of Embryos The immature pigs were given 1500 IU of PMSG, and
One hour later, hCG was administered at 500 IU. hCG administration 2
After 4 and 48 hours, artificial insemination was performed. hC
Embryos were collected by surgical means or after sacrifice on day 5, with day 0 as the day after G administration, and perfused with M2 solution in both horns, and embryos were collected from the perfusate.

(2) Culture of embryos until vitrification preservation The collected embryos were cultured by a microdrop culture method using NCSU23 solution in an incubator maintained at 39 ° C. under 5% CO ₂ conditions.

(3) Preparation of vitrification solution In the first hole of a 4-hole petri dish, about 1 mL of one liquid of the vitrification preservation liquid composition, and about 1 m of the second liquid in the second hole.
L, about 1 mL each of the third solution was injected into the third and fourth holes. Each solution used was kept at 38 ° C.

(4) Equilibration with Vitrification Solution A predetermined number of embryos cultured up to the initial blastocyst in the above (2) is transferred to the first solution in the first hole with a Pasteur pipette and equilibrated for 5 minutes. Transferred to the second solution in the second well and equilibrated for 5 minutes. During equilibration with the two solutions, a place about 1 cm from the tip of the Pasteur pipette used to transfer the embryo (FIG. 1)
D) in the middle was lightly scratched with an ampoule cutter. In addition,
Separately, liquid nitrogen was put in a mortar to prepare for vitrification storage.

After the completion of the equilibration in the second solution, the embryo was transferred to the third well containing the third solution, and immediately transferred to the third well in the fourth well.
Transferred to liquid. Next, suck the embryo with a Pasteur pipette,
Move to a mortar containing liquid nitrogen, and place a microdrop (approximately 5 μL) containing embryos at a height of about 5 cm from the upper surface of liquid nitrogen.
Was formed at the tip of a Pasteur pipette. When the preliminary cooling according to the present invention was not performed, the liquid containing the embryo was directly dropped into liquid nitrogen from a Pasteur pipette and frozen as in the case of the conventional micro droplet method.

(5) Pre-cooling The microdroplets formed in the above (4) were brought close to the upper surface of liquid nitrogen and held for 3 seconds to solidify the surface of the microdroplets.

(6) Vitrification Storage Method Next, after confirming the freezing of the surface of the microdroplets, the microdroplets were immersed in liquid nitrogen together with a Pasteur pipette. Thereafter, a portion about 1 cm from the tip of the Pasteur pipette was grasped with tweezers, and the Pasteur pipette was cut at a previously scratched portion (D in FIG. 1). The work from transferring the embryos to the three liquids and immersing them in liquid nitrogen was completed within 30 seconds. The vitrified embryo was placed in a microtube having a hole in the lid and stored in liquid nitrogen to obtain a vitrified preserved embryo.

(7) Preparation of Dilute Solution Into a Petri dish having a diameter of 35 mm, apply about 4
The preparation for melting the vitrified stored embryos was started. Also, in the first hole of the 4-hole petri dish,
Approximately 1 mL of diluted solution 4 and approximately 1 m of solution 5 in the second hole
L, about 1 mL of the 6 solution was placed in the third hole, and about 1 mL of the 7 solution was placed in the fourth hole. Each solution used was kept at 38 ° C.

(8) Melting of Vitrified Preserved Embryo and Dilution of Liquid Composition for Vitrification Transfer the contents of the microtube containing the vitrified preserved embryo into a container containing liquid nitrogen, and pinch the vitrified preserved embryo with tweezers. The vitrified preserved embryos were placed in a 35 mm diameter petri dish containing four diluted solutions. After the droplets melt,
Immediately transfer the embryos to the first 4 liquids of a 4-well petri dish and dilute for 5 minutes, and then use the second 5 liquids and the third 6 liquids for 5 minutes each.
And finally diluted with 7 liquid NCSU23 for 5 minutes. Thereby, the vitrification-preserved embryo was completely melted, and the liquid composition used for vitrification-preservation was removed.

(9) In vitro culture test of embryos after thawing The embryos thawed and diluted in the above (8) were subjected to 5% CO ₂
In an incubator kept at 39 ° C under conditions,
The cells were cultured by a microdrop culture method using NCSU23 solution supplemented with 10% FBS (fetal calf serum). The state of development of the embryo 24 hours and 48 hours after the start of the culture was examined. The results are shown in Table 5. For the blastocysts that escaped the zona pellucida, the ratio to the number of embryos was calculated as the zona pellucida escape rate.

[0041]

[Table 5] Table 5

As is clear from the table, the results of the in vitro culture test of 23 embryos that had been pre-cooled according to the present invention were as follows:
After 24 hours, 73.9%, or 17 embryos, grew into expanded blastocysts, and after 48 hours, all of the embryos that had grown into expanded blastocysts after 24 hours were transformed into escaped blastocysts. Was growing. The clear zone escape rate was 60.8%.

On the other hand, embryos frozen directly by dripping into liquid nitrogen as in the conventional method without pre-cooling were subjected to an in vitro culture test after thawing.
Even after the time, the survival rate was low, and a significant difference was observed as compared with the case of pre-cooling according to the present invention.

Example 2 In this example, an embryo transfer test was performed on the melted embryo that had been precooled according to the present invention in Example 1. The thawed embryos were washed three times with the M2 solution, and then surgically placed in the uterine horn of the embryonic pigs on the third day of estrus together with five 5-day-old embryos collected from another pig on the day of transplantation. Transplanted.
In addition, M2 solution was used as a transplant solution.

As a result, the fertilized pigs conceived, and three males and three females
The piglets were delivered. As a result of DNA determination of all piglets, it was found that one male was a litter derived from a vitrified preserved embryo. From this, the pig early embryo stored at ultra-low temperature using the microdrop vitrification storage method of the present invention has a high survival rate and incidence even after storage, and furthermore, the offspring derived from the vitrification storage embryo It was clear that he had the ability to produce.

[0046]

Industrial Applicability According to the present invention, there is provided a liquid composition for preservation of porcine early blastocysts, and by using the same, it is possible to efficiently preserve porcine early blastocysts by ultra-low temperature preservation by a microdrop vitrification preservation method. Can be implemented. Moreover, the method of the present invention is simple and, after thawing, the embryo has a higher survival rate after thawing than that obtained by ultra-low temperature storage according to the conventional method.
Therefore, embryos vitrified and stored by the method of the present invention are:
After thawing, it can be used for embryo transfer at a practical level.

[Brief description of the drawings]

FIG. 1 shows one embodiment of a vitrification preservation method according to the present invention. (I) shows a state before immersion in liquid nitrogen, and (II)
Indicates the state after immersion in liquid nitrogen.

[Explanation of symbols]

 A liquid nitrogen B microdrop containing embryo C pipette D damaged part

 ────────────────────────────────────────────────── ─── Continued on the front page (72) Inventor Misae Suzuki Odakura Hara 1 Odakura Hara, Nishigo-mura, Nishishirakawa-gun, Fukushima F-term (Reference) 4H011 BA01 BB03 BB08 BB19 CA01 CB08 CD02 CD06 DH03

Claims (2)

[Claims] 1. An M2 solution containing (1) 10% (v / v) ethylene glycol, (2) a solution containing 10% (v / v) ethylene glycol, 0.3M sucrose and 1% (w / v) polyethylene glycol. M2 solution and (3) 40% (v / v)
Ethylene glycol, 0.6M sucrose and 2% (w /
v) A liquid composition for preserving vitrification of early porcine blastocysts, comprising a polyethylene glycol-added M2 solution. 2. The equilibrium by immersing the porcine blastocysts in the solution (1) or (2) according to claim 1 using a pipette, and the pipette during equilibration with the solution (2). After the equilibration is completed, the liquid is transferred to each of the liquids (3) according to claim 1, and then the liquid containing the blastocysts sucked into the pipette is pushed out to remove the pipette. A microdroplet of the liquid is formed at the tip, and the microdroplet is moved onto a liquid nitrogen water surface to solidify the surface, then immersed in liquid nitrogen together with the pipette, and then the pipette is cut at a damaged portion near the pipette tip. An ultra-low temperature preservation method for pig early blastocysts, which comprises cutting and storing the cut cells in a container filled with liquid nitrogen.