JP2005261413A - Animal embryo freezing and storing liquid and animal embryo freezing and storing method using the same - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide an animal embryo freezing and storing liquid not containing a protein component derived from an animal, hygienic, highly safe, and capable of easily conducting direct transplantation of a thawed embryo to an embryo receiving animal, without requiring an embryo handling operation after thawing of the frozen embryo, and to provide an animal embyro freezing and storing method using the same. <P>SOLUTION: This animal embryo freezing and storing method comprises a process for injecting the animal embryo freezing and storing liquid into a straw 1, forming two parts of air layers 4 in the straw 1, positioning the animal embryo 2 in the animal embryo freezing and storing liquid 3 located between the two parts of the air layers 4, and sealing both ends of the straw, a process for forming ice in the straw 1 by a slow-freezing procedure, and freezing and storing the animal embryo 2 in the straw 2, and a process for thawing the frozen and stored animal embryo 2, wherein the animal embryo freezing and storing liquid is prepared by adding polyvinyl alcohol (PVA) in an amount of about 1.5 %(w/v) to a basal culture medium. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

  The present invention is a preservation solution for cryopreserving animal embryos, and particularly does not contain animal-derived protein components, and an animal that does not require embryo manipulation after thawing of a frozen embryo and can be directly transplanted to a recipient animal. The present invention relates to an embryo cryopreservation solution and an animal embryo cryopreservation method using the same.

Conventionally, it has been widely practiced that fertilized eggs are collected for breeding mammals, frozen and then thawed and transplanted to other embryos. There are some proven ones.
For example, there is a vitrification preservation method, which can instantly freeze-thaw animal eggs or embryos, such as electron microscope grid method, open-pulled straw method, nylon loop method, microdrop method, etc. . In any of these vitrification preservation methods, a frozen-thawed egg or embryo exhibits a high survival rate, and can be said to be a simple and effective cryopreservation method for animal embryos.

  As this vitrification preservation method, for example, as disclosed in Patent Document 1 as a “vitrification device and method for ovum or embryo”, a cylindrical ultrathin tube portion, and a suction and discharge device following this ultrathin tube portion are loaded. There are disclosed a vitrification tool and a vitrification method using the vitrification tool in which two eggs or embryos cannot coexist in the vertical direction by limiting the dimensions of the ultrathin tube portion including the connecting portion.

Patent Document 2 discloses (1) 10% (v / v) ethylene glycol-added M2 solution as “a liquid composition for vitrification preservation of embryo and method for ultra-low temperature preservation of embryo using the same”, (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 M sucrose and 2% ( w / v) A liquid composition for vitrification preservation of porcine early blastocysts composed of polyethylene glycol-added M2 liquid and an ultra-low temperature preservation method using the composition are disclosed. By rapidly cooling with such a high concentration of cryoprotectant, cells can be preserved without forming ice crystals inside and outside the cells. Furthermore, osmotic shock can be avoided by sequentially transferring to a liquid having a different ethylene glycol concentration.
However, such a vitrification preservation method has a problem that rapid embryo manipulation is necessary to reduce cytotoxicity caused by cryoprotectants such as ethylene glycol or cryoprotectants added at a high concentration. That is, since the osmotic pressure is increased by the high concentration of the cryoprotectant and the cytotoxicity is increased, it is necessary to reduce the time during which the embryo is immersed in the cryoprotectant as much as possible.
Moreover, even in the technique disclosed in Patent Document 2, in order to avoid osmotic shock due to this cryoprotectant, an addition-dilution treatment by a stepwise method for acclimating an embryo to a high-concentration solution stepwise is possible. There was a problem of necessity.
Furthermore, since the above-described embryo manipulation is required, there has been a major problem that the thawed embryo cannot be directly transferred to the recipient animal after the frozen embryo has been thawed.

Therefore, a technique has also been developed for cryopreservation of embryos, in which ethylene glycol or serum is added to a basal medium, frozen, and directly transplanted after thawing.
For example, Patent Document 3 discloses a technique for use in the cryopreservation of pig embryos by adding ethylene glycol, propylene glycol, serum and textlan in the presence of a basal medium as a “direct freezing method of pig embryos”. According to this technique, frozen embryos can be thawed at the time of use and directly transferred to recipient pigs. The technique disclosed in Patent Document 3 is a so-called slow freezing method. In this case, since embryo manipulation is unnecessary, it can be directly transplanted into an animal after thawing the embryo as described above. Therefore, it can be said that it is a more practical cryopreservation method.

JP 2001-252293 A Japanese Patent Laid-Open No. 2002-212001 JP-A-9-122158

  However, in the conventional slow freezing method as shown in Patent Document 3 and the like, protein derived from the animal such as serum having an embryo washing action for direct transplantation is included, which is harmful. There was a problem that there was a risk that the virus would infect embryos, embryo recipient cows and recipient pigs. In addition, there has been a problem that growth abnormalities such as post-transplantation miscarriage and fetal overgrowth frequently occur by culturing bovine embryos in a serum-added medium.

  The present invention has been made to solve the above-described problems of the prior art, and does not contain animal-derived protein components, is hygienic and highly safe, and does not require embryo manipulation after thawing of a frozen embryo. It is an object to provide an animal embryo cryopreservation solution that can be directly transplanted into a recipient animal and an animal embryo cryopreservation method using the same.

In the animal embryo cryopreservation solution according to claim 1 of the present invention, in order to solve the above-mentioned problems, 1.8M ethylene glycol, 0.05M trehalose, 25 mM hepes and approximately 1. 5% (w / v) polyvinyl alcohol is added.
In such an animal embryo cryopreservation solution, ethylene glycol and trehalose act as cryoprotectants, and Hepes acts as a buffer that suppresses changes in the pH of the animal embryo cryopreservation solution. About 1.5% polyvinyl alcohol has the effect of maintaining the skeletal structure of the germ cell as a protein substitute, but has the effect of exerting sufficient surface tension at a concentration of about 1.5%.

In addition, the animal embryo cryopreservation solution described in claim 2 of the present invention is an animal embryo cryopreservation solution whose concentration is the concentration shown in Table 1 of the claims.
The animal embryo cryopreservation solution according to the above configuration has the same action as the animal embryo cryopreservation solution described in claim 1. In addition, the description of ± 10% as the concentration range means that it is not particularly limited only to the numerical value, and at the same time, when a culture solution is prepared when a test or the like is performed. The liquid amount measurement error of about ± 10% occurs, and it is based on the fact that the test result obtains substantially the same effect even in such a range.

In the animal embryo cryopreservation method according to claim 3, the animal embryo cryopreservation solution according to claim 1 or claim 2 is injected into a straw, and two air layers are formed in the straw. Placing the animal embryo in the animal embryo cryopreservation solution between two air layers and sealing both ends of the straw, and freezing the animal embryo inside the straw by inoculating the straw by a slow freezing method And thawing the cryopreserved animal embryo.
In such an animal embryo cryopreservation method, since the animal embryo cryopreservation solution described in claim 1 or 2 is used, the amino acid constituents contained in the basal medium serve as an energy source, and have a detoxification action or pH. In addition to the action of promoting adjustment, ethylene glycol and trehalose act as cryoprotectants, and Hepes acts as a buffer that suppresses changes in the pH of the solution. Furthermore, polyvinyl alcohol has the effect of maintaining the skeleton of the germ cell as a protein substitute. Moreover, since sufficient surface tension is exhibited by a concentration of approximately 1.5%, the shape of the air layer becomes more spherical than in the case of a low concentration such as 0.1%. Therefore, the contact area between the straw and the air layer is reduced, and the heat insulation effect by the air layer is diminished, so that ice crystals formed on the outer surface of the straw at the time of ice planting exceed the air layer region and the region where the embryo exists Also has the effect of reaching.

  The animal embryo cryopreservation solution described in claim 1 and claim 2 of the present invention can exhibit a function as an energy source and a purification action by an amino acid component without containing animal-derived protein. In addition, the animal embryo is frozen by the slow freezing method using a straw by preparing polyvinyl alcohol to a concentration of about 1.5% for the embryo in the straw placed so as to be sandwiched between air layers. In the case of preservation, it is possible to promote the arrival of ice crystals in a cryopreservation solution containing embryos sandwiched between air layers.

  In addition, in the animal embryo cryopreservation method described in claim 3, by using the animal embryo cryopreservation solution described in claim 1 or claim 2, the above-mentioned effects can be exerted to facilitate the animal embryo. In addition, embryos that can be stored hygienically hygienically and have a high survival rate after lysis are provided. Furthermore, since embryo manipulation is not required after thawing, direct transplantation after thawing, which cannot be performed by vitrification, can be performed.

Hereinafter, the best mode for carrying out the animal embryo cryopreservation solution and the animal embryo cryopreservation method using the same according to the present invention will be described with reference to FIG.
FIG. 1 is a conceptual diagram showing a state in which a bovine embryo is enclosed in a straw for cryopreserving a bovine embryo using the animal embryo cryopreservation solution according to the present embodiment. In FIG. 1, a cryopreservation solution 3 is enclosed in a straw 1, an air layer 4 having a length of about 3 to 5 mm is provided on both sides, and an embryo 2 is placed between the air layers 4. Further, both ends of the straw 1 are sealed with a seal 5 so that the embryo 2 and the cryopreservation solution 3 do not leak. The straw 1 is made of plastic and has a capacity of about 0.25 ml. Although one of the seals 5 needs to be completely sealed, it is desirable that the other is not a complete seal but is gently sealed with a cotton plug or the like. This is to make it possible to absorb changes in the volume of the animal embryo cryopreservation solution at the time of freezing, and at the same time to facilitate removal of the embryo upon thawing. Therefore, the term “sealing” as used in the present application is a concept including a case where sealing is performed moderately to a certain extent as in the above-described cotton plug from a state where there is no leakage.
Thus, the straw 1 encapsulating the embryo 2 is immersed in an ethanol bath of a program freezer set at 0 ° C. Then, for example, it is cooled at a rate of 1 ° C. per minute, and after reaching −7 ° C., it is planted and kept at the same temperature for about 10 minutes. Thereafter, it is cooled to about −0.3 ° C. to −30 ° C. per minute, immersed in liquid nitrogen, and stored. The term “planting ice” refers to attaching seed ice, but if such seed ice is not applied, ice crystals will not grow, so that ice is planted when cooling proceeds to some degree, such as −7 ° C.
After freezing, in use, it is thawed in warm water in a temperature range of 30 ° C. to 37 ° C. and directly transferred to a recipient embryo.
Since the straw 1 shown in FIG. 1 is actually quite thin, it is shown enlarged in the radial direction in the figure, so the ratio between the longitudinal direction and the radial direction is not necessarily different from the actual one.

According to the animal embryo cryopreservation method according to the present embodiment, the cryopreservation solution does not need to be removed at the time of thawing and can be directly transplanted.
The animal embryo cryopreservation solution according to the present embodiment will be described in detail. The animal embryo cryopreservation solution contains three types of amino acid components, alanine, glycine and taurine, as a basal medium. This amino acid component serves as an energy source and promotes detoxification and pH adjustment.
More specifically, alanine has a chelating effect that neutralizes toxic ammonium ions. Glycine is a protein that exists in the fallopian tube or uterus, but this glycine also has the function of adjusting the osmotic pressure of embryonic tissue and the function of improving the metabolic capacity of the embryo. Taurine is also a protein originally present in the follicle and fallopian tube, but it has been found that it acts as a chelating agent or an antioxidant that neutralizes the cytotoxin, and uses this action.

In the animal embryo cryopreservation solution according to the present embodiment, 1.8 M ethylene glycol, 0.05 M trehalose and 25 mM hepes are added to the basal medium as described above. Both ethylene glycol and trehalose are added as cryoprotectants, and are added to protect embryos that are cryopreserved. Hepes is a buffer and suppresses changes in the pH of the animal embryo cryopreservation solution according to the present embodiment.
Further, PVA having a concentration of about 1.5% (w / v) is added. Although this PVA is added as a protein substitute, it has the effect of maintaining the skeleton of the germ cell with high viscosity. However, in the slow freezing method, although it is necessary to add a cryoprotectant or a cryoprotectant, it is not necessary to freeze rapidly and it is prepared with only a low concentration component. A low concentration such as% is desirable.
However, at such a low concentration, the above-mentioned ice crystals do not grow to the region where the embryo 2 is enclosed beyond the air layer 4, and thus the embryo 2 cannot be frozen. Therefore, in the animal embryo cryopreservation solution according to the present embodiment, the concentration is increased to 1.5%. Since the test was conducted on the appropriateness of this concentration, the result will be described later.
Gentamicin is an antibiotic. In addition, myo-inositol is also a component contained in serum and is a component that promotes embryo development, and itself and its metabolites transmit intracellular substances, promote mitosis, and enter the cell during fertilization. It has the effect of adjusting osmotic pressure together with calcium release and glycine. In addition, sorbitol promotes metabolism and supports embryo development, and citric acid has an action of chelating metal ions and the like in addition to stimulating fatty acid synthesis. This action is thought to be involved in embryo development by maintaining cell-cell connections and promoting embryo compaction and blastocoel formation.
Apo-transferrin has an antioxidant effect. BME (EAA) is an abbreviation for Basel Medium Eagles (Essential Amino Acids Solution), and MEM (NEAA) is a Modified Eagles Medium (Non-essential Amino Acid Solution) , A MEM non-essential amino acid solution, which is a commercially available amino acid solution. These amino acid solutions promote embryo development when added to a medium.
Moreover, in the animal embryo cryopreservation method in the present embodiment, ice is necessary to gradually cool while using the program freezer in this way, and there is a trouble such as taking time for cooling, Alternatively, although there are disadvantages such as exposing the embryo 2 to a low temperature for a long period of time, there is no need to avoid osmotic shock by the stepwise method, etc., and since the straw is used, the operator directly touches the embryo 2 or the cryopreservation solution 3 There is no hygiene. Furthermore, since liquid nitrogen used at the time of freezing may be contaminated by microorganisms or the like, the fact that liquid nitrogen itself does not come into contact with an embryo or animal embryo cryopreservation solution is also highly effective in terms of hygiene.

  Here, with respect to the animal embryo cryopreservation solution according to the present embodiment, a test for culturing a freeze-thawed embryo for 3 days was performed using the PVA concentration as a parameter. Table 2 shows animal embryo cryopreservation solutions used in the test. However, BSA indicated as * 1 is an animal embryo cryopreservation solution prepared as containing a protein derived from an animal conventionally used as a control group, and PVA indicated as * 2 relates to the present embodiment. Animal embryo cryopreservation solution. In the test, the concentration of PVA is not only 1.5% but also 3% and 6%. In Table 1, from the left column, the name of the reagent, the weight of the reagent per 100 ml, mM represents the millimolar amount, and FW (Formula Weight> represents the formula weight (molecular weight) of each reagent, provided that in the mM column. The quantity indicated with the unit is the quantity by the unit.

The test was performed by adding 1.8 M ethylene glycol, 0.05 M trehalose, 25 mM hepes and gentamicin as shown in Table 2 to the basal medium, and further adding BSA or PVA.
In the test method, bovine blastocyst stage embryos (7-8 day embryos) produced in vitro using an animal embryo cryopreservation solution were frozen, and freeze-thawed embryos were treated with 10% FBS (Fetal Bovine Serum) and MEM NEAA. Viability was compared by culturing for 3 days in an αMEM solution supplemented with.
Table 3 shows the survival rate of the embryos observed as a result of the test conducted using the animal embryo cryopreservation solution shown in Table 2.

Table 3 summarizes the number of blastocysts, the number of escape stages and the number of escaped blastocysts, and the number of escaped blastocysts for each of 20 or more treated embryos.
The results obtained with the animal embryo cryopreservation solution containing 1.5% PVA according to the present embodiment are almost equivalent to the results obtained with the conventional animal embryo cryopreservation solution containing BSA added to the test as a control group. It became clear.
However, as the concentration of PVA is increased to 3% and 6%, the decrease from the number of blastocysts becomes remarkable, particularly in the escape phase and the number of escaped blastocysts. From the result of this test, when the concentration of PVA is 3% or more, the survival rate is worse than before, but at a concentration of about 1.5%, it is sufficiently high without using animal-derived protein. It has been demonstrated that it is possible to maintain survival.
In addition, a preliminary test was conducted before this test. In this case, an animal embryo cryopreservation solution containing 0.3% to 1.0% PVA was prepared, but ice was frozen at the time of freezing. There was a problem that the crystal did not cross the air layer in the straw, and therefore could not be cryopreserved.

From the above results, it is possible to directly transfer thawed embryos to recipient animals without using a vitrification preservation method that requires embryo manipulation as an animal embryo cryopreservation solution. The animal embryo cryopreservation solution according to the present embodiment is suitable as an animal embryo cryopreservation solution for realizing an easy and safe animal embryo cryopreservation method without using an animal-derived protein of concern. It is understood.
Moreover, according to the method in which the animal embryo cryopreservation solution and the embryo are enclosed in a straw and gradually cooled and frozen using a slow freezing method, the freeze-thawed embryo can be directly transferred from the straw to the recipient animal. Therefore, it is possible to realize a safe and hygienic animal embryo cryopreservation method with less burden on workers and high survival rate.

  The animal embryo cryopreservation solution and the animal embryo cryopreservation method according to the present invention can be used for the preservation of embryos and transplantation of embryos in the breeding of livestock and the like, and can be widely used in the field of biotechnology such as the creation of clones. It is.

  It is a conceptual diagram which shows the state which has enclosed the bovine embryo in the straw for cryopreserving a bovine embryo using the animal embryo cryopreservation liquid which concerns on this Embodiment.

Explanation of symbols

  1 ... Straw 2 ... Embryo 3 ... Cryopreservation solution 4 ... Air layer 5 ... Seal

Claims (3)

  An animal embryo cryopreservation solution characterized by adding 1.8 M ethylene glycol, 0.05 M trehalose, 25 mM hepes, and approximately 1.5% (w / v) polyvinyl alcohol (PVA) to a basal medium. An animal embryo cryopreservation solution characterized in that the contained component has a concentration shown in Table 1.

  The animal embryo cryopreservation solution according to claim 1 or 2 is injected into a straw, two air layers are formed in the straw, and the animal embryo cryopreservation solution between the two air layers is formed. A step of placing an animal embryo to seal both ends of the straw, a step of inoculating the straw by slow freezing and freezing the animal embryo inside the straw, and thawing the cryopreserved animal embryo An animal embryo cryopreservation method comprising the steps of:

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