JPH02142701A - Embedding agent of mammalian embryo or egg, embedded material and production thereof - Google Patents

Abstract

PURPOSE:To obtain an embedding agent forming an embedded material having excellent handling characteristics and freeze resistance under a mild condition, improving survival rate of embryo or egg, comprising an alkali metallic salt of alginic acid having a specific composition ratio. CONSTITUTION:The circumference of mammalian embryo or egg is coated with an aqueous solution (preferably 1-3wt.% concentration) of an alkali metallic salt of alginic acid having a component ratio (ratio of M/G) of D-mannuronic acid (M) and L-gluronic acid (G) of alginic acid part of 0.3-1.8, especially 0.6-1.6, then the solution covering the mammalian embryo or egg is brought into contact with an aqueous solution containing calcium ion and a formed embedded material is washed with a cleaning solution having a small amount of phosphate ion. When an embryo or egg is embedded, a clear zone is protected and when a cut embryo is embedded, a germ cell is protected in such a way that the germ cell is not directly damaged by cryohydrate. Survival rate of embryo, egg and cut embryo and fertility after transplantation are extremely improved.

Description

DETAILED DESCRIPTION OF THE INVENTION [Field of Industrial Application] The present invention relates to cryopreservation of mammalian embryos or eggs and
The present invention relates to an embedding agent for forming embedding bodies such as capsules and microcapsules suitable for handling in vitro, an embedding body formed using the same, and a method for manufacturing the embedding body.

[Conventional technology]

There is an increasing movement toward preserving and utilizing genes for purposes such as improving productivity and preserving traits of mammals. In particular, in the livestock field, in 1979 S. M. Wil
The creation of monozygotic multiple fetuses through embryo cutting technology using a micromanipulator and cryopreservation technology developed by LADSEN, combined with the cryopreservation technology, are rapidly expanding the use of valuable genetic resources.

In general, cow embryos or eggs are cryopreserved by immersing them in a solution containing a cryoprotectant; however, in cases where the zona pellucida is incomplete before cooling, such as in cut embryos, or the zona pellucida is damaged during cooling. It is said that the impact of ice crystals directly reaches the embryonic cytoplasm, resulting in a lower conception rate after transplantation. fact.

H. Heyman said that the survival rate of cut embryos after freezing and thawing is generally low, with a survival rate of 30 to 40% compared to when using uncut embryos.
It has been reported that the percentage decreases (Theriogenol
ogy.

23:63.1985). Also, Y, Tsun
Oda et al. found that cut embryos placed in artificially cut zona pellucida have a low survival rate after thawing because they are directly affected by ice crystals, but if cut embryos are embedded in agar, the survival rate is significantly higher. We have confirmed that it will improve (Tberiogenolo
gy, 28:317-322.1987).

In cryopreservation of embryos, it goes without saying that the cooling method should be devised, but it is also important to devise ways to prevent damage to the zona pellucida in order to increase the survival rate after transplantation. It turns out. Of course, the zona pellucida is damaged when the embryo is cut, but it is also damaged when ice crystals grow during in vitro handling or during the freezing process, and when the frozen embryo is thawed. The ice crystals are damaged as they pass through the surface of the embryo or zona pellucida, which is caused by the difference in the time of onset of expansion between regions. As a technique to prevent such damage to the zona pellucida, there is the method of using agar as an embedding agent by Y. and Tsunoda et al.
A method for making microcapsules using polylysine/alginate reported by S., Holljngsworth et al. (Theriogenology, 29
:262°1988), and G, Ac1aniya
A method of gelling and encapsulating sodium alginate with calcium ions reported by et al. (Biology
of Reproduction.

19th Annual Meeting of 5o
Society of Biology ofReprod
(July 14-17.1986).

However, in the conventional techniques as described above, when agar is used as an embedding agent, there are the following problems.

1) The temperature at which gelation occurs is close to the critical temperature for embryo survival, and incorrect temperature control will lead to death of the embryo.

2) Low gel strength and poor practicality.

3) Low transparency makes it impossible to accurately assess the condition of the embryo or cut embryo from the outside.

4) In handling using a microscope, since the refractive index of the agar gel and the processing liquid located around it are similar, it is difficult to determine the boundary surface, resulting in poor workability.

Furthermore, the conventional embedding method using sodium alginate has the following problems.

5) The gel strength of the alginate gel is significantly reduced due to the influence of various salts contained in the reaction solution or changes in pl+.

's5) In order to improve the gel strength, a large amount of calcium ions as a gelling agent is added to the reaction solution, but although this method improves the gel strength, the osmotic pressure The survival rate of embryos or cut embryos decreases due to an increase in

7) When a gel containing embryos or eggs is treated with a culture solution containing phosphate ions with a chelating effect, such as modified Dulbecco's phosphate buffered saline (hereinafter abbreviated as PBS), the formed gel will dissolve. It ends up.

8) The capsule often deforms or collapses during the freezing and thawing process.

Each of the conventional technologies has such problems,
At present, no method has been developed that can solve all of these problems.

[Problem to be solved by the invention]

The purpose of the present invention is to solve the problems of the prior art, to reliably protect livers or eggs from various physical shocks caused by in vitro handling, freezing, thawing, etc., and to encapsulate them under mild conditions. Another object of the present invention is to provide an embedding agent for mammalian embryos or eggs, which can form an embedding body through microencapsulation, and which can improve the survival rate of the embryo or egg, an embedding body, and a method for producing the same. .

[Means to solve the problem]

As a result of intensive studies on industrially usable embedding agents, the present inventors found that capsules or microcapsules made from alkali metal alginate having a specific composition ratio have excellent hand-linking properties and freeze resistance. and suitable for the survival of embryos and eggs, and after forming capsules or microcapsules, a culture medium that does not contain phosphate ions or calcium ions, such as Ringer's solution, or contains only a small amount of them. It was discovered that transparency could be maintained for a long period of time by washing with a culture solution, and the present invention was achieved.

The present invention relates to the following mammalian embryo or egg embedding agent, embedding body, and method for producing the same.

(1) Cover a mammalian embryo or egg with an aqueous solution of an alkali metal alginate, and contact the solution covering the embryo or egg with an aqueous solution containing calcium ions to form a gel, forming an embedded body of the embryo or egg. An embedding agent for D-mannuronic acid (M) and 14-
The component ratio (M/G ratio) with guluronic acid (G) is 0.3~
An embedding agent for mammalian embryos or eggs, characterized in that it contains an alginate alkali metal salt having a concentration of 1.8.

(2) An embedded body of an embryo or egg that is obtained by covering a mammalian embryo or egg with an aqueous solution of an alkali metal alginate, and then contacting the solution covering the embryo or egg with an aqueous solution containing calcium ions to form a gel. Therefore, the alginate alkali metal salt is D-mannuronic acid (A) of the alginic acid moiety.
and L-guluronic acid (G) (M/G ratio) is 0.
．． 3 to 1.8.

(3) The component ratio (M/G ratio) of D-mannuronic acid (M) and L guluronic acid (G) in the alginic acid part is 0.3 to 1
．． Embedding of the embryo or egg formed by covering a mammalian embryo or egg with an aqueous solution of an alkali metal salt of alginate, and contacting the solution covering the embryo or egg with an aqueous solution containing calcium ions to form a gel. A method for producing an embedded body of a mammalian embryo or egg, which comprises washing the body with a washing liquid containing little phosphate ions.

In the present invention, embryos include cut embryos, and eggs include fertilized eggs and unfertilized eggs before becoming embryos. In addition, embedding bodies include capsules, microcapsules, and anything that embeds and protects embryos or eggs.

The embedding agent of the present invention covers a mammalian embryo or egg with an aqueous solution of an alginate alkali metal salt, and gels the embryo or egg by contacting the solution covering the embryo or egg with an aqueous solution containing calcium ions. An embedding agent for forming an embedding body, which contains D-mannuronic acid (alginic acid moiety)
Component ratio of M) and L-guluronic acid (G) (M/G ratio)
contains an alginate alkali metal salt having a value of 0.3 to 1.8.

The strength of the calcium alginate gel forming the embedded body of the present invention is determined by the strength of D-mannuronic acid (M), which constitutes alginic acid, and 1. - The component ratio (M/G ratio) of guluronic acid (G) is closely involved. This M/G ratio is deeply involved in the survival of embryos and eggs, and is also involved in resistance to physical shock that occurs during freezing and thawing. In other words, the higher the M/G ratio (the more M there is), the more elastic the formed gel will be, and the lower the M/G ratio (the more G), the higher the gel strength and the harder it will be. A gel is obtained.

The alginic acid alkali metal salt used in the embedding agent of the present invention has an M/G ratio of the constituent alginic acid in the range of 0.3 to 1.8, particularly in the range of 0.6 to 1.6. It has excellent freeze resistance, does not form wrinkles due to cracking or shrinkage even after freezing and thawing processes, and does not cause much damage to the cytoplasm of embryos or eggs. Those with an M/G ratio of more than 1.8 have low gel strength and are likely to break when handled, while those with an M/G ratio of less than 0.3 have high gel strength but are brittle and difficult to freeze. , alginic acid alkali metal salts made of alginic acid having such M/G ratios are not suitable for the purpose of the present invention because they crack when subjected to the melting process. Furthermore, when alginate alkali metal salts are used without specifying the M/G ratio, variations in the strength and freeze resistance of embedding bodies such as capsules and microcapsules are observed, and furthermore, Since the reproducibility is not sufficient, it is necessary to carry out 4-7σ fractional distillation for industrial use.

The alginate alkali metal salt used in the embedding agent of the present invention is
Examples include sodium salts and potassium salts of alginic acid having the M/G ratio as described above. These alginate alkali metal salts are used in aqueous solution, and the concentration is between 0.5 and 5.
Weight percent, preferably about 1 to 3 weight percent.

The gelling agent for gelling these alginate alkali metal salts is an aqueous solution containing calcium ions, and calcium salts such as calcium chloride, calcium lactate, and calcium acetate are used, but among these, calcium chloride is particularly inexpensive and easy to use. .

In addition to alginate alkali metal salts, it is a natural water-soluble crab polymer that has gelling ability and can form a capsule-like protective body.
The following items are industrially produced: For grains and tubers, starch and konnyaku; for seaweed extracts, agar, carrageenan, and furcellan; for fruit extracts, pectin; for legumes, guar gum, tamarind, and locust hinka 11; for sap-derived products, gum arabic, 1 herakan 1 - Strength 11; Examples of microorganism-derived substances include dextran, pullulan, curdlan, and xanthan gum; and animal-derived substances such as gelatin.

However, although starch can gel and temporarily create a capsule state, this state does not last long and is extremely degraded and becomes brittle upon freezing, so it is not suitable for the purpose of the present invention. Konjac does not gel at around 60°C unless an alkali such as calcium hydroxide is added, and the temperature exceeds the survival limit of embryos or eggs, so it cannot be used. Agar, Farcellan, and Kartran have gelation temperatures close to the survival limit temperature of liver or eggs, and they require skill to handle, and all have low gel strength and are easily damaged during operation. There is. There are three types of carrageenan: kappa, lambda-carrageenan, and iota-carrageenan, but the gels of kappa-carrageenan and iota-carrageenan have low strength and are not suitable for practical use, and lambda-carrageenan does not gel. Pectin, both high methoxyl pectin and low methoxyl pectin, is
Tamarind does not gel without the sugar content, and similarly, tamarind requires the addition of monosaccharides or brown sugars in order to gel, so it is not suitable for the purpose of the present invention.

Both guar gum and locust bean gum are pi-dependent, so the system must be moved to an acidic or alkaline region when gelling.
The survival rate of embryos and eggs decreases.

Gum arabic and gum tragacanth have low gelling ability;
Unable to maintain a certain shape. Although it is known that dextran and pullulan can be gelled using a low-molecular cross-linking agent such as epichlorohydrin, it is not appropriate to use such an irritating cross-linking agent. Xanthan gum does not gel by itself, but when used in combination with locust bean gum, a gel with elasticity and strong water retention can be created. It is necessary to heat both gummy aqueous solutions to a temperature exceeding 80°C. Gelatin capsules are used as carriers for pharmaceuticals, but when used in the application of the present invention, the capsule coating begins to dissolve into water during the melting process, and livers or eggs may jump out into the water. Gelatin is not suitable when trying to preserve If or eggs with as little damage as possible.

As mentioned above, there are many substances that form capsules like alginate alkali metal salts, but at present, only industrially produced water-soluble polymer substances that can serve as protectors for embryos or eggs exist. In this case, alginate alkali metal salts are most suitable.

The embedding body of the present invention covers a mammalian embryo or egg with an aqueous solution of an alkali metal alginate having the M/G ratio as described above, and contacts the solution covering the embryo or egg with an aqueous solution containing calcium ions. It is made into a gel. The embedding body is preferably capsule-shaped or microcapsule-shaped.

When the calcium alginate gel that forms the embedding body is made into a capsule or microcapsule, the only advantage is that an arbitrary number of embryos or eggs can be inserted into one capsule or microcapsule and treated as a unit. The shape is suitable for protecting the inserted embryo's zona pellucida, and when using embryos or eggs stored in the cut zona pellucida, the shape is suitable to prevent the embryo or egg from protruding outside the zona pellucida. suitable for fixing.

The method for manufacturing an embedded body of the present invention includes covering an embryo or an egg with an aqueous solution of an alginate alkali metal salt having the above M/G ratio, and bringing the solution covering the embryo or the egg into contact with an aqueous solution containing calcium ions to form a gel. , the formed embedding body is washed with a washing solution containing few phosphate ions.

When forming a capsule or microcapsule as an embedding body, droplets of an aqueous alginate salt aqueous solution covering an embryo or egg can be dropped into an aqueous solution surrounding calcium ions to form a gel. When forming a cylindrical embedding body, the aqueous alginate salt aqueous solution covering the embryo or egg is extruded into a string shape and gelled in an aqueous solution containing calcium ions.

Hereinafter, the manufacturing method of the present invention will be explained with reference to embodiments of the drawings. FIG. 1 is a schematic diagram showing a method for manufacturing an embedding body in which an embryo or an egg is embedded in an alginate gel capsule or microcapsule, and FIG. 2 is a cross-sectional IMI of the obtained embedding body. In the figure, 1 is an embedding body, in which an embryo or egg 3 is embedded in alginate gel 2. The embryo or egg 3 has a structure in which a cytoplasm 4 is surrounded by a perivitelline cavity 5 and a zona pellucida 6. 7 is alginate alkali metal salt aqueous solution,
8 is an aqueous solution containing calcium ions, and 9 is a reaction vessel.

The method for producing the embedding body 1 is to transfer the embryo or egg 3 into an alginate alkali metal salt aqueous solution 7, such as 2% sodium alginate Ringer's solution, and suck it into a syringe barrel, and then use a 20 gauge syringe with the tip dropped. From the injection needle 10, the liquid level of the aqueous solution 8 containing calcium ions, such as a 100mM calcium chloride aqueous solution, is increased drop by drop as droplets 11]Oc
It is dropped into the same aqueous solution 8 from a height of m. When the alginate alkali metal salt aqueous solution 7 comes into contact with the aqueous solution 8 containing calcium ions, it instantly transforms into an alginate gel 2 made of calcium alginate at room temperature. Encased in alginate gel 2, the embryo or egg 3 is placed in an aqueous solution 8 containing calcium ions for about 30 minutes.

FIGS. 3 and 4 are schematic diagrams showing a method for manufacturing an embedding body in which an embryo or an egg is embedded in a cylinder of alginate gel, and FIG. 5 is a cross-sectional view of the obtained embedding body. The method for manufacturing the embedding body 1 is to transfer the embryo or egg 3 into an alkali metal alginate aqueous solution 7 such as 2% sodium alginate Ringer's solution, and then transfer the embryo or egg 3 to an alkali metal alginate aqueous solution 7 such as 2% sodium alginate Ringer's solution.
Aspirate together with the above solution 7 into a Pasteur pipette 12 with an inner diameter of ~3 times. At this time, the embryos or eggs 3 are aspirated in a string. The pipette 12 is placed in a glass Petri dish (
Filter sterilization in a container 9 such as 90 mm x 15 mm)1. Transfer the alkali metal alginate containing the embryo or egg 3 to an aqueous solution 8 containing calcium ions, such as a 100mM distilled calcium chloride aqueous solution, and while gently rotating the Petri dish (rotating to the left if using a pipette with your right hand), gently remove the alkali metal alginate containing the embryo or egg 3. The aqueous salt solution 7 is extruded from the pipette 12 in a cylindrical shape. When the alginate alkali metal salt aqueous solution 7 comes into contact with the calcium ion-containing aqueous solution 8, it instantly changes to the alginate gel 2 at room temperature. Place the string-like alginate gel 2 around the embryo or egg 3 using a surgical scalpel 13.
The embedded body 1 is formed by cutting into strips about 1 mm long under a stereoscopic microscope using a blade. Encased in alginate gel 2, the embryo or egg 3 is placed in an aqueous solution 8 containing calcium ions for about 30 minutes.

The embedded body 1 formed as described above is taken out of the aqueous solution 8 containing calcium ions and washed with a washing liquid.

The cleaning solution should contain 100mg/Q or less of phosphate ions,
Preferably, the amount is 50 mg/Q or less. It is preferable that the amount of calcium ions is small, and 0.8% by weight or less, preferably 0.4% by weight or less of calcium ions is suitable. As such a washing solution, Ringer's solution or a culture solution with a low content of phosphate ions or calcium ions can be used. It is also possible to prepare an aqueous solution containing calcium ions for reaction with the alginate alkali metal salt or alginate alkali metal salt using such Ringer's solution or a culture medium with a low content of phosphate ions or calcium ions.

Conventionally, calcium alginate capsules or microcapsules into which embryos or eggs have been inserted have often been treated in a culture medium such as PBS.

As the gel begins to dissolve under the influence of phosphate ions, white crystals of calcium phosphate begin to form on the surface almost simultaneously, which reduces the strength of the capsule or microcapsule and also reduces the internal $1'! becomes difficult.

Thus, it is not appropriate to use a type of culture solution such as PBS that contains large amounts of phosphate ions and calcium ions.

The embedding body obtained as described above is subjected to in vitro handling and stored by freezing.

When the calcium alginate gel formed as described above is made into a capsule or microcapsule form and used to embed mammalian embryos or eggs, it becomes easy to handle in vitro and cryopreservation, and it also facilitates handling in vitro and cryopreservation. When embedding eggs, the zona pellucida is protected, and when embedding cut embryos, the liver cytoplasm is protected from being directly damaged by crystals, so that the survival of the embryos, eggs, cut embryos, etc. rates, and post-transplant conception rates are significantly improved.

Also, after encapsulation or microencapsulation [”B
Instead of processing in a culture medium containing a large amount of phosphate ions or calcium ions such as S, processing in a culture medium that does not contain these ions or contains very little of these ions, such as Ringer's solution or cell culture medium GIT solution. By doing so, the transparency of the capsule or microcapsule can be maintained, the inside can be easily observed, and the workability is significantly improved.

〔Effect of the invention〕

According to the present invention, the embedding body is formed at a specific M/G ratio using the alkali metal salt of diuroarhenic acid as the embedding agent, so that the embedding body can be formed under mild conditions and the strength It is possible to form an embedding body that has excellent elasticity and is stable against the effects of p++ changes and salts. It can protect against physical shock and improve the survival rate of embryos or eggs.

〔Example〕

Examples of the present invention will be described below.

Example 1 By the method shown in FIG. 1, an embedded body consisting of a capsule shown in FIG. 2 was formed. In the test, a 2% by weight aqueous solution of each sodium alginate shown in Table 1 was prepared, this aqueous solution was sucked up with a syringe, and dropped into a 100mM calcium chloride aqueous solution and reacted for 30 minutes.
A capsule was formed. 30 of these capsules were placed in a test tube with a stopper, containing 3 mΩ of 40% glycerin solution, and immersed in liquid nitrogen for exactly 73 minutes.
They were placed in a thermostatic water bath and thawed, and the number of breakages and changes in appearance were observed.

Table 1 shows the results of freeze resistance tests of capsules using monohelium alkynates with different H7G ratios.

Table 1 Examples of freezing resistance of alginate Kel Compared to comparative example M/1.2 0.6 1.6 0.1 0.
2 1.9 Chimura number 30 10 30 30
: Only) : 30 Number of responses ; (0 old 110
3 (1:10 30 broken numbers 3'+4
7 5 9 Notes --- Wrinkles (Note) Wrinkles were observed on the surface of the cocapsule.

Example 2 A comparison was made between the use of Rinkel's solution and the use of li II S in the treatment of calcium alginate gel. Sunao] also contains 2g of sodium alkinate (M/6
Put the ratio 1, 2) into Rinkel's solution or LLBS,
After dissolving for 1 minute, the mixture was added dropwise to a 100 mM calcium chloride aqueous solution using a syringe, and reacted for 30 minutes to form capsules. This capsule is made of glass containing Ringer's solution or PBS. It was placed in a rim dish and observed under a stereomicroscope. The results are shown in Table 2.

Table 2 Comparison of Treatment Solutions Observation Results Workability Example 33 The embedding body shown in FIG. 5 was produced by the method shown in FIGS. 3 and 4.

(1) Embedding of embryos 100 mQ of Ringer's solution containing 2 g of sodium alginate (M/G ratio 1.2, Duck Algin + 50-M, Kibun Food Chemifa Co., Ltd., trademark) was heated at 121 °C for 2 hours.
Autoclave sterilization was performed for 0 minutes to obtain a sample sodium alginate solution. After the rabbit morula embryo was transferred into this sodium alginate Ringer's solution, the solution was aspirated into a Pasteur pipette whose tip had an inner diameter approximately 2 to 3 times the diameter of the embryo. At this time, the embryos were aspirated in a string.

Place the pipette in a glass plate (90X l5).
+Iu) of filter sterilized 100mM 4
+,・, Transfer to the distilled aqueous solution of Calcilla 11 and gently rotate the plate (with your right hand or counterclockwise if using a pipette)
As shown in FIG. 3, the 1-lium alginate solution containing the liver was gently pushed out of the pipette. Then, as shown in Figure 4, a string-like alginate gel was placed around the embryo and examined under a stereomicroscope using the blade of a scalpel.
It was cut into strips of mm length. The embryos were immersed in an aqueous calcium chloride solution for approximately 30 minutes while encased in alginate gel.

(2) Cryopreservation test of rabbit morula embryos embedded in alginate gel The rabbit morula embryos embedded in alginate gel as described above were removed from the calcium chloride aqueous solution and used for cell culture. Wash with medium GIT solution (manufactured by Wako Pure Chemical Industries, Ltd., trademark), freeze medium solution (11% dimethyl sulfoxide-〇I
It was stored in the same solution until equilibration with T solution) was started. While the rabbit morula embedded in alginate Kel was equilibrated with the freezing medium for 30 minutes at room temperature,
The embryos embedded in the gel were aspirated into a 1 ton plastic draw (25 m 12 volumes). For cooling, Osaka Sanso Kogyo Co., Ltd.
Using a Procla 11 freezer made by
After cooling to 1° C. per minute, the mixture was rapidly cooled into liquid nitrogen.

During the test, the sample temperature was maintained at -7°C for 10 minutes, but no external forced ice crystal formation induction treatment w (plants) was performed.

Instead, silver iodide immobilized in alginate gel is immersed in freezing medium along with the embryos, as shown in JP-A No. 61-255,655 and Japanese Patent Application No. 62-265,125. Crystal formation induction was performed automatically. For thawing, the straws stored in liquid nitrogen were immersed in warm water at 37°C, and after waiting for the ice crystals to completely disappear, the straws were quickly returned to room temperature. After thawing, the gel containing the embryos was collected from the straw and transferred directly into GITI and washed several times to remove the cryoprotectant (dimethyl sulfoxide) from the embryos. Thereafter, the normality of the embryonic cytoplasm and the degree of damage to the zona pellucida and mucin layer were observed, and the embryos were placed in a glass Petri dish and the surrounding gel was removed using a sterile injection end of a 27 gauge. The embryos from which the gel was removed were subjected to transplantation tests into pseudopregnant rabbits and culture tests. Only embryos with normal cytoplasm, zona pellucida, and mucin layer were used for transplantation. 50 IU of hCG (human placental gonadotropin, manufactured by Sankyo Kinki Co., Ltd.) was injected into the ear vein of the rabbit to induce pseudopregnancy 60 hours before the transplantation. Under general anesthesia, the ovaries and fallopian tubes are removed from the body through an incision in the flank, and the transferred embryos are transferred to a very small amount of GT.
It was transplanted into the fallopian tube via the oviduct tube together with T. 14 days after transplantation
On the following day, midline incisions were made in the upper and lower abdomen to count the number of implantations and the number of viable fetuses. Afterwards, the abdomen was opened and the delivery of a normal baby was observed. In vitro culture was carried out in GIT fluid under liquid paraffin oil for 3.
The experiment was carried out at 7°C in a gas phase of 5% CO2 by volume and 95% air by volume.

On the other hand, embryos that were frozen, thawed, transplanted, or cultured in the same manner as above without being embedded in alginate gel were used as controls.

(3) Results The results are shown in Table 3, and the number of correct livers after thawing, that is, the percentage of embryos in which not only the cytoplasm but also the zona pellucida and the 13-thin layer were not damaged, was determined by the number of embryos in the test group embedded in alginate gel. showed a higher proportion than the test group without embedding treatment (P
<0.025). The presence of a zona pellucida is essential for implantation in the liver or crest of a rabbit, and the presence of an intact mucin layer is also considered necessary to improve conception rates. This result means that the transplant utilization rate of rabbit liver after freezing and thawing was improved (P<0.0O5
),.

What is the viability of embryos embedded in alginate gel, frozen, and thawed when transplanted into the fallopian tubes of pseudopregnant offspring, and their in vitro culture in cows? f-・l'l: +V+heta 1, as a result, the respective viability of Il, f that was not embedded in alginate gel, and the right, 1, no difference was observed 1) Re, embedding in alginate gel. It was confirmed that there were no adverse effects after burial treatment or embryonic development.
(Table 4 shows the effect of sterilization on fertility, but by embedding fetuses in alginate gel, it is possible to preserve a high percentage of transplanted i+J livers after thawing.
<0.025), the proportion of fertile livers to embryos subjected to freezing is significantly lower.

Yes 97/+06 (92%) 4/+06 (4%
) 5/106 (5%) None 29/51 (57
%) 10151 (20%) 12/5] (24
%) In order to confirm the effect of embedding with alginate gel on embryo development,
! 12*, 5041 pressure: 35*P(0,025) From these results, the survival rate of the embryo or cut layer embedded in the alginate gel obtained by the method of the present invention θζ, the survival rate after freezing and thawing, and It can be seen that the workability is significantly improved compared to the conventional method.

[Brief explanation of the drawing]

Figures 1, 3 and 4 are schematic diagrams showing the manufacturing method;
FIGS. 2 and 5 are cross-sectional views of the manufactured embedding bodies. In each figure, the same reference numerals represent the same or corresponding parts; ] is the embedded body; 2 is the alginate gel; 3 is the liver or egg; 7 is the alginate alkali metal salt aqueous solution; 8 is the aqueous solution containing calcium ions. . Attorney Sei Yanagihara! -1 Procedural Amendment 7゜Contents of the Amendment March 24, 1999 "Alkal" in line 15, page 17 of the specification is corrected to "alkali". Table 3 on page 27 is corrected as follows. Patent Application No. 296596 (1988) 2 Title of Invention Embedding agent for mammalian embryo or egg, embedding body and method for producing the same 97/106 (92%) 4/106 (4%) 5/106 ( 5%) 3. Relationship with the person making the amendment

Claims (3)

[Claims] (1) Cover a mammalian embryo or egg with an aqueous solution of an alkali metal alginate, and contact the solution covering the embryo or egg with an aqueous solution containing calcium ions to form a gel, forming an embedded body of the embryo or egg. An embedding agent for the purpose of
．． 8. An embedding agent for mammalian embryos or eggs, characterized by containing an alginate alkali metal salt of No. 8. (2) An embedded body of an embryo or egg that is obtained by covering a mammalian embryo or egg with an aqueous solution of an alkali metal alginate, and then contacting the solution covering the embryo or egg with an aqueous solution containing calcium ions to form a gel. Therefore, the alginic acid alkali metal salt is D-mannuronic acid (M) of the alginic acid moiety.
and L-guluronic acid (G) (M/G ratio) is 0.
．． 3 to 1.8. (3) D-mannuronic acid (M) and L- of the alginic acid moiety
The component ratio (M/G ratio) with guluronic acid (G) is 0.3~
A mammalian embryo or egg is covered with an aqueous solution of alginate alkali metal salt (1.8), and the solution covering the embryo or egg is brought into contact with an aqueous solution containing calcium ions to form a gel. 1. A method for producing an embedded body of a mammalian embryo or egg, which comprises washing the embedded body with a washing liquid containing little phosphate ions.