JPH0729881B2 - Mammalian embryo or ovum embedding medium, embedding medium, and method for producing the same - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION [Field of Industrial Application] The present invention relates to cryopreservation and
The present invention relates to an embedding agent for forming an embedding body such as a capsule and a microcapsule suitable for handling in vitro, an embedding body formed thereby, and a method for producing an embedding body.

[Conventional technology]

For the purpose of improving productivity of mammals, preserving traits, etc., the activity of preserving and utilizing genes has been activated. Particularly in the field of livestock, the utilization of valuable genetic resources has been dramatically combined with the production of monozygotic multiple fetuses, which was developed by SM Willadsen in 1979, using a micromanipulator-based cutting technique for embryos, and cryopreservation technology. Is being expanded to.

Cryopreservation of bovine embryos or ova is generally carried out by immersing them in a liquid containing a cryoprotectant.However, as in the case of cut embryos, the zona pellucida is incomplete before cooling or is broken during cooling. The effect of ice crystals directly reaches the embryo cytoplasm of said embryo, and the conception rate after transplantation is said to decrease. In fact, H. Heyman shows that the survival rate of frozen embryos after freeze-thawing is generally low, 30-40% compared to uncut embryos.
It is also reported to be low (Theriogenology, 23: 63,198
Five). Also, Y. Tsunoda et al. Have a poor survival rate after thawing because the embryos placed in the artificially cut zona pellucida are directly affected by ice crystals, but if the embryos are embedded in agar, It has been confirmed that the survival rate is significantly improved (Theriogeno
logy, 28: 317-322, 1987).

Thus, in cryopreservation of embryos, it goes without saying that the cooling method should be devised, but it is also important to devise how to prevent damage to the zona pellucida in order to improve the survival rate after transplantation. It will be. The zona pellucida is, of course, damaged when the embryo is cut, but it is also damaged during ice crystal growth during handling in vitro or during the freezing process, and also when the frozen embryo is thawed. Ice crystal faults caused by different expansion initiation times between sites are damaged as they pass through the surface of the embryo or zona pellucida. As a technique to prevent such damage of the transparent zone, the above-mentioned Y.Ts
Although there is a method of using agar of unoda et al. as an embedding medium, another technique for wrapping the embryo in the embedding medium is TS Hollingswort.
The method reported by H et al. for making microcapsules using polylysine / alginate (Theriogenology, 29:26.
2,1988), and a method of encapsulating sodium alginate by gelling with calcium ions as reported by G. Adaniya et al. (Biology of Reproductin, 19th Annual Mee.
ting of Society of Biology of Reproduction, July 14
-17, 1986).

However, the above-mentioned conventional techniques have the following problems when agar is used as an embedding medium.

1) The gelation temperature and the survival limit temperature of the embryo are close to each other, and if the temperature is not properly controlled, the embryo will die.

2) Low gel strength and poor practicality.

3) The transparency is low and the state of the embryo or the cut embryo cannot be accurately grasped from the outside.

4) In handling using a microscope, since the refractive index of the agar gel is similar to that of the treatment liquid conforming to the periphery thereof, it is difficult to determine the boundary surface, and workability is poor.

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

5) The gel strength of alginate gel remarkably decreases due to the influence of various salts contained in the reaction solution or the change of pH.

6) In order to improve the gel strength, a large amount of calcium ion, which is a gelling agent, is added to the reaction solution. This method improves the gel strength but increases the osmotic pressure. Reduces the viability of embryos or cut embryos.

7) When the gel containing embryos or eggs is treated with a culture medium containing phosphate ions having a chelating effect such as modified Dulbecco's phosphate buffered saline (hereinafter abbreviated as PBS), the gel formed is dissolved. Will end up.

8) The capsules are often deformed or disintegrated after the steps of freezing and thawing.

Each of the conventional technologies has such problems,
A method that can solve all of these problems has not yet been developed.

[Problems to be Solved by the Invention]

The object of the present invention is to solve the problems of the prior art, to reliably protect an embryo or an egg from various physical shocks caused by handling in vitro, freezing, thawing, and the like, and to encapsulate under mild conditions. Alternatively, it can be microencapsulated to form a stable and transparent embedding medium for cryopreservation, and can improve the survival rate of an embryo or an egg. It is to provide a manufacturing method.

[Means for Solving the Problems]

As a result of intensive studies on the industrially usable embedding agents, the present inventors have found that capsules or microcapsules made from an alkali metal alginate having a specific composition ratio as raw materials have excellent handling characteristics and freeze resistance. And that it is suitable for the survival of embryos and ova, and after forming capsules or microcapsules, a culture solution that does not contain phosphate ions or calcium ions, such as Ringer's solution, or a very small amount of culture solution containing them. It was found that the transparency can be maintained for a long period of time by washing with a liquid, and the present invention was achieved.

The present invention provides the following mammalian embryo or egg embryo embedding agents, embedding bodies, and methods for producing the same.

(1) A mammalian embryo or ovum is covered with a 0.5 to 5% by weight aqueous solution of an alkali metal alginate, and the liquid covering the embryo or ovum is brought into contact with an aqueous solution containing calcium ions to form a gel. An embedding medium for forming an embedding medium for cryopreservation, comprising D- of an alginic acid moiety.
Embedding of a mammalian embryo or egg containing an alkali metal alginate having a component ratio (M / G ratio) of mannuronic acid (M) and L-guluronic acid (G) of 0.3 to 1.8 Agent.

(2) Covering the periphery of a mammalian embryo or ovum with a 0.5 to 5% by weight aqueous solution of an alkali metal alginate and contacting the liquid covering the embryo or ovum with an aqueous solution containing calcium ions to form a gelled embryo or ovum. An embedding medium for cryopreservation, wherein the alginic acid alkali metal salt has a component ratio (M / G ratio) of D-mannuronic acid (M) and L-guluronic acid (G) in the alginic acid moiety of 0.3 to 1.8. An embedding body of a mammalian embryo or egg, which is characterized in that

(3) D-mannuronic acid (M) and L- of the alginic acid moiety
Covering the periphery of a mammalian embryo or ovum with a 0.5 to 5 wt% aqueous solution of an alkali metal alginate having a component ratio (M / G ratio) with guluronic acid (G) of 0.3 to 1.8, and covering this embryo or ovum The liquid is brought into contact with an aqueous solution containing calcium ions to form a gel, and the formed embryos or embryos for cryopreservation embedding are washed with a low washing rate of phosphate ions. Of the embedded body of the above.

In the present invention, an embryo includes a cut embryo, and an egg includes a fertilized egg and an unfertilized egg before becoming an embryo. The embedding medium includes capsules, microcapsules, and all other materials that embed and protect embryos or eggs.

The embedding medium of the present invention covers the periphery of a mammalian embryo or ovum with an aqueous solution of an alkali metal alginate, gels the liquid covering the embryo or ovum by contacting it with an aqueous solution containing calcium ions, and An embedding agent for forming an embedding medium, comprising a component ratio (M / G ratio) of D-mannuronic acid (M) and L-guluronic acid (G) in an alginic acid moiety.
Containing an alginic acid alkali metal salt of 0.3 to 1.8.

The component ratio (M / G ratio) of D-mannuronic acid (M) and L-guluronic acid (G) constituting alginic acid is closely related to the strength of the calcium alginate gel forming the embedded body of the present invention. Are involved in. This M / G ratio is deeply involved in the survival of embryos and ova, and is also involved in the resistance to physical shock generated during freezing and thawing. That is, the higher the M / G ratio (more M), the more elastic the gel that is formed, and the lower the M / G ratio (more G), the higher the gel strength and the harder the gel. A gel is obtained.

The alginic acid alkali metal salt used in the embedding medium of the present invention has a M / G ratio of its constituent alginic acid in the range of 0.3 to 1.8, and among them, those in the range of 0.6 to 1.6 are excellent in freezing resistance and are frozen. Even after the melting step, there is no formation of wrinkles due to cracking, shrinkage, etc., and there is little damage to the cytoplasm of the embryo or egg. If the M / G ratio exceeds 1.8, the gel strength is low, and there is a high rate of breakage during handling, and if the M / G ratio is less than 0.3, the gel strength is high, but it is rather fragile and requires a freeze / thaw process. As it goes through cracks,
Alkali metal alginates composed of alginic acid having an M / G ratio such as these are not suitable for the purpose of the present invention. Further, when the alkali metal alginate is used without specifying the M / G ratio, there are variations in the strength and freeze resistance of the embedded bodies such as capsules and microcapsules. Since the viability will not be reproducible, it is necessary to pay sufficient attention in industrial use.

Alginic acid alkali metal salt used in the embedding medium of the present invention,
Examples thereof include sodium salt and potassium salt of alginic acid having the above M / G ratio. These alkali metal alginates are used in an aqueous solution and their concentration is 0.5 to 5% by weight, preferably 1 to 3% by weight.

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

In addition to the alkali metal alginate, natural water-soluble polymers that have gelling ability and can form a capsule-shaped protective body and are industrially produced include the following. . Starch and konjak in cereals and potatoes; agar, carrageenan, and furcellan in seaweed extracts; pectin in fruit extracts; guar gum, tamarind, in beans
Locust bean gum; sap-derived gum arabic and tragacanth gum; microbial-derived dextran, pullulan, curdlan, xanthan gum; and animal-derived gelatin.

However, although starch can be gelled to temporarily form a capsule state, the state is not long-lasting and is extremely deteriorated and becomes brittle due to freezing, which 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 it cannot act because it exceeds the survival limit temperature of the embryo or egg.
Agar, furcellan and curdlan have a drawback that the gelation temperature is close to the survival limit temperature of the embryo or egg, it requires skill to handle and the gel strength is low in any case, and it is easily damaged during operation. There is. There are three types of carrageenan, copper, lambda and iota carrageenan, but the gels of copper carrageenan and iota carrageenan have low strength and cannot be used practically, and lambda carrageenan does not gel. Both high methoxyl pectin and low methoxyl pectin do not gel without a certain amount of sugar, and similarly tamarind requires gelling of monosaccharides and disaccharides in order to gel. Is not suitable. Since both guar gum and locust bean gum are pH-dependent, it is necessary to transfer the system to an acidic region or an alkaline region when gelling, so that the survival rate of embryos and ova is reduced. Because gum arabic and gum tragacanth have low gelling ability, they cannot maintain a certain shape. Dextran and pullulan are known to gel with small molecule crosslinkers such as epichlorohydrin, but it is not appropriate to use such irritating crosslinkers. 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 made, but in order to form this gel, the survival temperature of the embryo or egg is exceeded. Both gum aqueous solutions need to be heated at a temperature of 80 ° C. Gelatin capsules are used as a carrier for pharmaceuticals, but when used for the purpose of the present invention, the capsule coating may start to dissolve in water during the melting step and the embryo or egg may jump out into hot water. Gelatin is not suitable when trying to preserve embryos or eggs with as little damage as possible.

As described above, there are many substances that form capsules like alginic acid alkali metal salts, but those that can be a protector of embryos or ova are currently among the industrially produced water-soluble polymer substances. Then, the alkali metal alginate is most suitable.

The embedding medium of the present invention covers the periphery of a mammalian embryo or ovum with an aqueous solution of an alkali metal alginate having an M / G ratio as described above, and the liquid covering the embryo or ovum is contacted with an aqueous solution containing calcium ions. It was made to gel. The embedding medium is preferably in the form of capsules or microcapsules.

Capsule- or microcapsule-forming calcium alginate gel forming an embedding body has only the advantage that an arbitrary number of embryos or eggs can be inserted into one capsule or microcapsule and treated as a unit. However, the shape is suitable for protecting the zona pellucida of the inserted embryo, and when using the embryo or ovum stored in the cut zona pellucida, position it so that the embryo or ovum does not protrude outside the zona pellucida. Suitable for fixing.

The method for producing an embedded body of the present invention covers the periphery of an embryo or an egg with an aqueous solution of an alkali metal alginate having the M / G ratio,
The liquid covering the embryo or egg is brought into contact with an aqueous solution containing calcium ions to gel, and the formed embedding medium is washed with a washing solution containing a small amount of phosphate ions.

When forming a capsule or a microcapsule as an embedding medium, a droplet of the aqueous solution of alkali metal alginate covering the embryo or egg can be dropped into the aqueous solution encapsulating calcium ions to cause gelation. When forming a cylindrical embedding medium, an aqueous solution of alkali metal alginate covering the embryo or egg is extruded in the form of a string and gelled in an aqueous solution containing calcium ions.

Hereinafter, the manufacturing method of the present invention will be described with reference to the embodiments of the drawings. FIG. 1 is a schematic diagram showing a method for producing an embedding medium in which an embryo or an ovum is embedded in an alkyne acid gel capsule or microcapsule, and FIG. 2 is a sectional view of the obtained embedding medium. In the figure, reference numeral 1 denotes an embedding body in which an embryo or an egg 3 is embedded in an alginic acid gel 2. The embryo or egg 3 is manufactured by surrounding the cytoplasm 4 with the perivitelline cavity 5 and the zona pellucida 6. 7 is an aqueous solution of alkali metal alginate,
Reference numeral 8 is an aqueous solution containing calcium ions, and 9 is a reaction vessel.

The embedding body 1 is manufactured by transferring the embryo or egg 3 into an aqueous solution 7 of an alkali metal alginate such as 2% by weight sodium alginate-Ringer solution, sucking it into a syringe, and then dropping the tip. From the injection needle 10, each droplet 11 is dropped into the aqueous solution 8 from a height of about 10 cm of the aqueous solution 8 containing calcium ions such as 100 mM calcium chloride aqueous solution. When the aqueous solution of alkali metal alginate 7 comes into contact with the aqueous solution 8 containing calcium ions,
It instantly changes to alginate gel 2 composed of calcium alginate at room temperature. The embryo or egg 3 wrapped in the alginate gel 2 is placed in an aqueous solution 8 containing calcium ions for about 30 minutes.

3 and 4 are schematic views showing a method for producing an embedding medium in which an embryo or an ovum is embedded in a cylinder of alginate gel, and FIG. 5 is a sectional view of the obtained embedding medium. Embedded body 1
In the method for producing, the embryo or egg 3 is transferred into an aqueous solution of alkali metal alginate 7 such as a 2 wt% sodium alginate / Ringer solution, and then the tip has an inner diameter of about 2-3 times the diameter of the embryo or egg 3. Aspirate with solution 7 above into a Pasteur pipette 12. At this time, the embryo or the egg 3 is sucked in a pearl shape. Place the pipette 12 in a glass Petri dish (90
mm × 15mm) 100mM which has been sterilized by filtration in a container 9
The solution is transferred to an aqueous solution 8 containing calcium ions, such as a distilled aqueous solution of calcium chloride, and gently rotated while the Petri dish is rotated (when the right hand is a pipette, rotated left) while gently calcining the aqueous solution of alkali metal alginate 7 containing the embryo or egg 3. Is extruded from the pipette 12 in a cinder shape. When the aqueous solution 7 of alkali metal alginic acid salt comes into contact with the aqueous solution 8 containing calcium ions, the alginic acid gel 2 is instantly formed at room temperature.
Changes to. Embed egg-shaped alginate gel 2 or embryo 3
With the blade of a surgical scalpel 13 as the center, the embedded body 1 is formed by cutting into a strip shape of about 1 mm length under a stereoscopic microscope. When the embryo or egg 3 is wrapped in alginate gel 2,
It is placed in an aqueous solution 8 containing calcium ions for 30 minutes.

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

As the cleaning liquid, one having a phosphate ion of 100 mg / or less, preferably 50 mg / or less is suitable. It is preferable that the calcium ion content is low, and calcium ion content is 0.8% by weight.
Below, preferably 0.4% by weight or less is suitable.
As such a washing solution, Ringer's solution or a culture solution having a low content of phosphate ions and calcium ions can be used. It is also possible to prepare an aqueous solution containing calcium ion for reacting with the alkali metal alginate or the alkali metal alginate with such a Ringer's solution or a culture solution having a low content of phosphate ions and calcium ions.

Conventionally, calcium alginate capsules or microcapsules in which embryos or eggs are inserted were often treated in a culture medium such as PBS. As the gel begins to dissolve under the influence of phosphate ions and white crystals of calcium phosphate appear on the surface almost at the same time, the strength of the capsule or microcapsule decreases, and it becomes difficult to observe the inside. come. Thus, it is not appropriate to use a type of culture solution such as PBS containing a large amount of phosphate ions and calcium ions.

The embedded body thus obtained is subjected to in vitro handling and stored by freezing.

Forming an embedding body of a mammalian embryo or egg into a capsule or microcapsule of the calcium alginate gel formed as described above facilitates handling in in vitro handling and cryopreservation,
When embedding an embryo or an egg, the zona pellucida is protected, and when embedding a cut embryo, the embryo cytoplasm is protected from damage by direct ice crystals. Etc., and the conception rate after transplantation is greatly improved. Also, after encapsulation or microencapsulation, instead of treating in a culture solution containing a large amount of phosphate ions or calcium ions such as PBS, do not include these ions,
Alternatively, by treating in a culture medium containing an extremely small amount, for example, Ringer's solution or cell culture medium GIT solution, the transparency of the capsules or microcapsules can be maintained, the inside can be easily observed, and the workability is remarkably improved.

〔The invention's effect〕

According to the present invention, since an embedding medium for cryopreservation is formed using a solution of a specific concentration of an alkali metal alginate having a specific M / G ratio as an embedding medium, the embedding medium under mild conditions is used. It is possible to form an embedding medium which is excellent in strength and elasticity, and is stable and transparent against pH change and the influence of salts, which allows the embryo or egg to be handled or frozen in vitro, It is possible to improve the survival rate of the embryo or egg by protecting it from physical impacts such as thawing.

〔Example〕

 Examples of the present invention will be described below.

Example 1 An embedding body consisting of the capsule shown in FIG. 2 was formed by the method shown in FIG. For the test, a 2% by weight aqueous solution of each sodium alginate shown in Table 1 was prepared, and this aqueous solution was sucked up with a syringe, dropped into a 100 mM calcium chloride aqueous solution and reacted for 30 minutes to form a capsule with a diameter of 1.0 to 1.5 mm. It was 30 capsules of 40 wt% glycerin solution 3 ml
After transferring into a test tube with a stopper, immersed in liquid nitrogen for exactly 3 minutes, put it in a constant temperature water bath at 37 ° C to melt it,
The number of breakage and changes in appearance were observed.

Table 1 shows the results of the freeze resistance test of capsules using sodium alginate having different M / G ratios.

Example 2 A case where Ringer's solution was used for the treatment of calcium alginate gel and a case where PBS was used were compared. That is, 2 g of sodium alginate (M / G ratio of 1.2) was put into Ringer's solution or PBS, and after thorough thawing, 1
The mixture was dropped into a 00 mM calcium chloride aqueous solution and reacted for 30 minutes to form a capsule. The capsule was placed in a glass Petri dish containing Ringer's solution or PBS and observed with a stereomicroscope. The results are shown in Table 2.

Example 3 The embedded body of FIG. 5 was manufactured by the method of FIGS. 3 and 4.

(1) Embedding of embryo 100 ml of Ringer's solution containing 2 g of sodium alginate (M / G ratio 1.2, duck algin 150-M, manufactured by Kibun Food Chemifa Co., Ltd.) was autoclaved at 121 ° C. for 20 minutes. Sterilization was performed to obtain a test sodium alginate solution. Rabbit morula was transferred into this sodium alginate Ringer's solution and then aspirated with this solution in a Pasteur pipette whose tip had an inner diameter approximately 2-3 times that of the embryo.
At this time, the embryo was sucked in a pearl shape. The pipette was placed in a glass Petri dish (90 x 15 mm) and was sterile filtered.
The solution was transferred into a 0 mM calcium chloride distilled water solution, and the sodium alginate solution containing the embryo was gently extruded from the pipette while gently rotating the petri dish (counterclockwise when the right hand was a pipette), as shown in FIG. Then, as shown in FIG. 4, the cord-shaped alginate gel was cut into strips of about 1 mm length with a blade of a surgical knife under a stereomicroscope, centering on the embryo. In a state wrapped in alginate gel,
The embryo was immersed in an aqueous calcium chloride solution for about 30 minutes.

(2) Cryopreservation test of rabbit mulberry seeds embedded in alginate gel The rabbit morula seeds embedded in alginate gel were removed from the calcium chloride aqueous solution by the method described above, and the cell culture medium GIT Wash with liquid (trademark, manufactured by Wako Pure Chemical Industries, Ltd.),
Freezing medium (11% dimethyl sulfoxide-GIT solution)
It was stored in the same solution until the equilibrium with and was started. 30 at room temperature
While the rabbit mulberry seeds embedded in alginate gel were equilibrated with the freezing medium for a minute, the gel-embedded embryos were sucked into a 0.25 ml plastic straw as a freezing container. For cooling, a program freezer manufactured by Osaka Oxygen Industry Co., Ltd. was used, and after cooling from room temperature to -30 ° C at a speed of 1 ° C per minute,
Quenched into liquid nitrogen. The sample temperature was kept at -7 ° C for 10 minutes on the way, but no forced ice crystal formation-inducing treatment (ice planting) from outside was performed. Instead, Japanese Patent Laid-Open No. 61-255655
As shown in Japanese Patent Application No. 62-265125 and Japanese Patent Application No. 62-265125, induction of ice crystal formation was automatically carried out by immersing silver iodide immobilized in an alginate gel in a freezing medium together with an embryo. Upon thawing, the straw stored in liquid nitrogen was immersed in warm water at 37 ° C., waiting for the ice crystals to completely disappear, and then quickly returning to room temperature. After thawing, the gel containing the embryo was recovered from the straw, transferred directly into the GIT solution and washed several times to remove the antifreezing agent (dimethyl sulfoxide) from the embryo. After that, the normality of the embryo cytoplasm and the degree of breakage of the zona pellucida and mucin layer were observed, and the gel was placed in a glass Petri dish and the surrounding gel was removed with a 27-gauge sterile injection needle. The embryo from which the gel was removed was subjected to a transplantation test and a culture test in a pseudopregnant rabbit. Only embryos with normal cytoplasm, zona pellucida and mucin layer were provided for transplantation. Rabbits from which embryos were transplanted as a litter were injected with 50 IU of hCG (human placental gonadotropin, manufactured by Sankyo Organs Co., Ltd.) into the ear vein 60 hours before transplantation to induce pseudopregnancy. Under general anesthesia, the lateral part of the incision was incised to expose the ovary and fallopian tube to the outside of the body, and the transplanted embryo was transplanted into the fallopian tube via the fallopian tube together with a minimal amount of GIT. On the 14th day after the transplantation, a midline incision was made on the lower abdomen to count the number of implantations and the number of surviving fetuses. Thereafter, the abdomen was closed and the delivery of normal children was observed. The in vitro culture was carried out in a GIT solution under liquid paraffin oil at 37 ° C. in a gas phase of 5% by volume CO ₂ and 95% by volume air.

On the other hand, an embryo that was frozen, thawed, transplanted or cultured by the same method as above without being embedded in alginate gel was used as a control.

(3) The results are shown in Table 3, and the number of normal embryos after thawing, that is, the proportion of embryos in which not only the cytoplasm but also the zona pellucida and the mucin layer were not damaged was observed in the test group embedded in the alginate gel. The ratio was higher than that of the test group without embedded treatment (P <0.
025). Since the zona pellucida is essential for the implantation of rabbit embryos in the uterus, and having an unbroken mucin layer is also necessary for improving the conception rate,
This result means that the transplant availability of rabbit embryos after freezing and thawing was improved (P <0.005).

In order to confirm how the embedding treatment with alginate gel has an effect on the development of embryos, the embryos embedded in alginate gel, frozen and thawed were transferred to the oviducts of pseudopregnant rabbits. The viability and its in vitro culture system were examined.
As a result, no significant difference was found between the viability of each embryo that was desired to be embryo-embedded with alginate gel, and it was confirmed that the embedding treatment in alginate gel had no adverse effect on embryo development.

Table 4 shows the effect of the embedding process with alginate gel on the fertility of frozen and thawed rabbit embryos. Embedding rabbit embryos in alginate gel ensures a high rate of transplantable embryos after thawing. This makes it possible (P <0.025) to improve the ratio of embryos to be conceived with respect to the embryos subjected to freezing.

From these results, the viability of the embryos or cut embryos embedded in alginate gel obtained by the method of the present invention, frozen,
It can be seen that the survival rate and workability after thawing are significantly improved as compared with the conventional method.

[Brief description of drawings]

1, 3 and 4 are schematic views showing the manufacturing method,
2 and 5 are cross-sectional views of the manufactured embedding bodies. In each figure, the same reference numerals indicate the same or corresponding parts, 1 is an embedding body, 2 is an alginic acid gel, 3 is an embryo or egg, 7 is an aqueous solution of an alkali metal alginate, and 8 is an aqueous solution containing calcium ions.

 ─────────────────────────────────────────────────── --- Continuation of the front page (72) Inventor Kenichi Hamoto 5-31-30 Minamioizumi, Nerima-ku, Tokyo (72) Inventor Masatsugu Ito 2-19-3-505 (72) Horifune, Kita-ku, Tokyo Inventor Yahachiro Hori 2-19-201 Nakahara-cho, Kawagoe City, Saitama Prefecture (56) Reference JP-A 63-177791 (JP, A) JP-A 63-160584 (JP, A) Biological of Reproduction, 19th Annual Meeting of Society of Biology of Reproduction, July 14-17 (1986).

Claims (3)

[Claims] 1. A 0.5 to 5% by weight aqueous solution of an alkali metal alginate covers the periphery of a mammalian embryo or egg, and the liquid covering the embryo or egg is brought into contact with an aqueous solution containing calcium ions to gel the embryo or An embedding agent for forming an embedding medium for cryopreservation of an egg, wherein the component ratio (M / G ratio) of D-mannuronic acid (M) and L-guluronic acid (G) in the alginic acid moiety is An embedding agent for a mammalian embryo or egg, which comprises an alkali metal alginate of 0.3 to 1.8. 2. An embryo formed by gelling a mammal embryo or egg covered with a 0.5 to 5% by weight aqueous solution of an alkali metal alginate and contacting the liquid covering the embryo or egg with an aqueous solution containing calcium ions. An embedding medium for cryopreservation of an egg, wherein the alginic acid alkali metal salt has a component ratio (M / G ratio) of D-mannuronic acid (M) and L-guluronic acid (G) in the alginic acid moiety of 0.3 to. A mammalian embryo or ovum embedding characterized in that it is 1.8. 3. D-mannuronic acid (M) in the alginic acid moiety.
And the component ratio (M / G ratio) of L-guluronic acid (G) is 0.3-
Embryo formed by covering the periphery of a mammalian embryo or ovum with a 0.5 to 5 wt% aqueous solution of an alkali metal alginate, which is 1.8, and gelling the liquid covering the embryo or ovum by contacting it with an aqueous solution containing calcium ions Alternatively, a method for producing a mammalian embryo or an ovum embedding, which comprises washing the ovum cryopreservation embedding with a washing solution containing a small amount of phosphate ions.