KR101934969B1 - Composition for low or room temperature preservating of bovine embryos - Google Patents

Abstract

The present invention relates to a composition for preserving bovine embryos at low or room temperature. When the composition for preserving the low-temperature or room temperature of the embryo of the present invention is used, the embryo can be effectively stored at a low temperature and room temperature to improve the survival rate and hatching rate after preservation and increase the pregnancy rate of the surrogate mother. And the defects of fertilized eggs and low resilience. Therefore, the composition of the present invention and the preserving method using the same can be effectively used in the livestock industry and related industries.

Description

TECHNICAL FIELD The present invention relates to a composition for preserving bovine embryos at low or room temperature,

The present invention relates to a composition for preserving bovine embryos at low or room temperature.

Since animal embryos must be preserved until they are implanted in surrogate mothers, a variety of conservation methods are being developed. Currently, most of preservation methods are using cryopreservation method. However, in the cryopreservation method, there is a problem that embryo survival rate and embryo quality are lowered due to physically damaged embryo, and in case of cow embryo, in order to adjust the estrus cycle of the embryo, The state of the melted embryo is significantly damaged compared to before frozen. In addition, a preservation solution and a culture solution are used for embryo transfer. At this time, since the quality of the embryo is not maintained due to the influence of the temperature, the conception rate is greatly reduced. In addition, when frozen embryos are stored, high grade fertilized eggs can be used because they have excellent resilience even after freezing. However, they are economically disadvantageous due to low fusion resilience of low grade embryos.

In order to improve the aforementioned cryopreservation method, various methods and preservation solutions for preserving fertilized eggs have been studied. Among them, embryonic cryopreservation has been developed, but the state of the embryo is sensitive to slight temperature changes, and a method for protecting the embryo in a wider temperature range is required. In addition, problems of synchronization of estrus between surrogate mothers to improve the conception rate and efficiency for application to low quality embryos are becoming problems. Therefore, there is a need for an optimal preservation solution and an improved low temperature preservation method to improve the fertility rate.

Therefore, the inventors of the present invention have been studying a composition and a method capable of efficiently preserving bovine embryos in addition to the cryopreservation method, and have succeeded in preparing a low-temperature or room-temperature stock solution containing BSA, HEPES and TCM 199, The present inventors completed the present invention upon confirming that bovine embryos can be effectively preserved by the selective addition of Thiazovivin, CHIR99021 or Y27632.

Accordingly, an object of the present invention is to provide a composition for preserving bovine embryos at low or room temperature.

It is also an object of the present invention to provide a method for preserving a bovine embryo at low or room temperature.

In order to solve the above-mentioned problems, the present invention relates to a method for producing a bovine serum albumin (BSA), which comprises bovine serum albumin (BSA), HEPES (tetramethylthiouracil) Bos Taurus ) composition for the preservation of embryos at low or room temperature.

Also, the present invention provides a method for preserving a bovine embryo at low or room temperature, comprising mixing the bovine embryo with the composition and preserving the bovine embryo at a low temperature or at a room temperature.

When the composition for preserving the low-temperature or room temperature of the embryo of the present invention is used, the embryo can be effectively stored at a low temperature and room temperature to improve the survival rate and hatching rate after preservation and increase the pregnancy rate of the surrogate mother. And the defects of fertilized eggs and low resilience. Therefore, the composition of the present invention and the preserving method using the same can be effectively used in the livestock industry and related industries.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a schematic diagram showing a method of putting a fertilized egg in a straw into a basic storage solution or a low-matter substance addition storage solution of the present invention.
FIG. 2 shows the results of preserving bovine embryos using a stock solution prepared by adding 1 uM of Ch9 (CHIR99021), 10 uM of Y2 (Y27632) or 2 uM of Thiazovivin to the stock solution of the present invention, A) and the hatched embryo (B). FIG.
Figure 3 shows the group of basal reservoirs of the present invention, 10 uM of Y2 (Y27632) alone additional treatment group; 10 uM of Y2 (Y27632) + 1 uM of Ch9 (CHIR99021) combined treatment group; And the addition of 2 μM of Thiazovivin + 1 μM of Ch9 (CHIR99021), the embryos were preserved and the morphology of the embryos (A) and hatched embryos (B) Fig.

The present invention relates to the preservation of Bos Taurus embryos at low or room temperature, including bovine serum albumin (BSA), HEPES (4- (2-hydroxyethyl) -1-piperazineethanesulfonic acid) and TCM 199 (Tissue culture medium 199) ≪ / RTI >

In the present invention, "preservation" means that the embryo of a cow is stably maintained over a long period of time under low or room temperature conditions.

In the present invention, the term "room temperature" means the atmospheric temperature of the room, and is generally a temperature that human beings can comfortably enjoy, usually a temperature of about 15 to 20 degrees.

The BSA is contained in an amount of 0.1 to 10% (w / v), preferably 1 to 5% (w / v) based on the final weight of the composition, and the composition is for cold storage at 1 to 5 캜, In order to achieve the purpose of effectively preserving cow embryos at low temperatures, the temperature conditions are not limited thereto.

Thiazovivin, CHIR99021 (CAS 252917-06-9), Y27632 (CAS 129830-38-2), PD184352 (CAS 212631-79-3) and SU5402 (CAS 215543-92-3) The temperature condition is not limited thereto so long as it includes one or more kinds of small molecule substances selected from the group consisting of cow's embryos stored at 4 to 20 degrees but for the purpose of effectively preserving cattle embryos. The small molecule material may preferably be treated with the addition of CHIR99021 or Y27632 alone to the reservoir, or optionally with the addition of thiazovivin, CHIR99021 or Y27632 to the storage solution.

In the present invention, "Thiazovivin" is a Rho-associated kinase (ROCK) inhibitor. It is a small molecule substance that protects the life of human embryonic stem cells and is related to cell survival.

[Chemical Formula 1]

In the present invention, "CHIR99021" is a GSK-3? /? Kinase inhibitor and is named CT99021, CHIR99021, CHIR 99021 and CT-99021.

(2)

In the present invention, "Y27632" is a ROCK (Rho-associated kinase) inhibitor and has an effect of inhibiting stress fibers.

(3)

The small molecule material is contained at a concentration of 1 to 30 uM, preferably, when the small molecule material is singly treated, the concentration of Thiazovivin is 3 to 25 uM, the concentration of CHIR99021 is 1 to 10 uM, To 25 uM in the composition of the present invention. In addition, the concentration of each small molecule substance is not limited in the complex treatment of the small molecule substance, and is not limited as long as the small molecule substance can be added to the composition of the present invention to achieve the purpose of survival and hatching of the cattle embryo.

The cow may be at least one selected from the group consisting of Korean cattle, cow, beef cattle, cross breed, and imported Korean cattle. Preferably, the composition of the present invention is used to protect and preserve the embryo, Can be used without limitation in cattle varieties.

The fertilized egg is an In Vitro Fertilization-Embryo Transfer (IVF), a somatic cell nuclear trasnfer embryo or a parthenogenetic embryo, preferably an in vitro fertilized egg or a low temperature Or embryos to be stored at room temperature.

In the present invention, the term " In Vitro Fertilization-Embryo Transfer (IVF) "means embryos fertilized in the culture medium after the ovaries that have been grown up to the state just before ovulation are collected from the ovaries.

In the present invention, the "somatic cell nuclear transplant embryo" is a fertilized egg produced by somatic cell nuclear transfer technology for mass production of genetically identical cloned animals. The somatic cell that has already been differentiated is transplanted into the enucleated unfertilized egg And reprogramming the nucleus, resulting in a new embryo.

In the present invention, the term " parthenogenetic embryo "means an embryo in which the embryo has grown and developed through chemical and electrical activation signals without sperm modification.

The embryo is a development stage of blastocyst. The blastocyst is an embryo ending in the early development of mammals. For embryo transfer, a blastocyst, which is a fertilized embryo, It is most effective.

The low temperature or the room temperature is a condition of 1 to 30 캜, preferably 4 to 20 캜, and the temperature condition can be selectively set according to the composition, the small molecule material of the present invention, or the combination treatment and storage time. In addition, since the breeding optimum temperature of most livestock is in the range of 10 to 20 ° C, for the purpose of the present invention, the breeding optimum temperature condition is met, and the optimum breeding condition that can not be achieved by the conventional cryopreservation method is provided can do.

The preservation is carried out for 20 to 180 hours, preferably 24 to 168 hours, and the preservation time can be selectively set according to the composition of the present invention, the small molecule material, or the combination treatment and the temperature condition.

Also, the present invention provides a method for preserving a bovine embryo at low or room temperature, comprising mixing the bovine embryo with the composition and preserving the bovine embryo at a low temperature or at a room temperature.

The low temperature or room temperature is a condition of 1 to 30 캜, preferably 4 to 20 캜. The preservation is carried out for 20 to 180 hours, preferably 24 to 168 hours. The temperature condition and the preservation time can be selectively set depending on the composition or small molecule material of the present invention, alone or in combination.

In one embodiment of the present invention, through the series of results, Ch9 (CHIR99021) or Y2 (Y27632), which is a small molecule substance, is treated in a stock solution containing BSA replacing the conventional FBS and composed of HEPES and TCM199 (From 4 to 20 degrees Celsius) over a wide temperature range when stored at room temperature or in combination. In particular, it was confirmed that the stock solution containing the small molecule material of the present invention was excellent in the preservation effect of Hanwoo embryos even at 20 ° C for 72 hours (day 3). In addition, compared with the conventional cryopreservation method, the pregnancy rate was further increased at low temperature (4 degrees) and at room temperature (20 degrees), and it was confirmed that Hanwoo embryos were effectively protected and quality of embryos was maintained.

It will be apparent to those skilled in the art that various modifications and variations can be made in the present invention without departing from the spirit or scope of the invention as defined by the appended claims. It will be obvious to you.

Experimental Example  1. Preparation of In vitro Embryos for Laboratory Materials and Hanwoo

1-1. Experimental material

(4- (2-hydroxyethyl) -1-piperazineethanesulfonic acid), BSA (bovine serum albumin) and TCM 199 were purchased from ThermoFisher Scientific. In addition, the small molecule substances PD184352 (MEK 1/2 inhibitor) (CAS 212631-79-3), SU5402 (FGF receptor inhibitor) (CAS 215543-92-3), CHIR99021 (GSK3 inhibitor) (CAS 252917-06-9) And Y27632 (Rock inhibitor) (CAS 129830-38-2) were purchased from Selleckchem. IVMD101, IVF100 and IVD 101 cultures without serum were purchased from Functional Peptides Research Institute for the maturation of oocytes, the production of embryos and cultivation. The straw for storage of Hanwoo embryos was purchased from Japan FHK (Fujihira industry co.).

1-2. In vitro fertilization Blastocyst  get

Ovum Pick Up (OPU) is a technique that utilizes an ultrasonic guide to continuously collect eggs from live living Hanwoo. It is a technology that can utilize the genetic ability of a cow together with improvement of conventional hydrogen (semen) Obtained the in vitro fertilization blastocyst using the above technique. Specifically, oocytes were obtained from follicles ≥2 mm in diameter using an ultrasonic diagnostic system (Ibex ^™ Portable Ultrasound, USA). The follicular aspiration was performed using a 18G X 90mm needle (JEIL TECH CO, Korea) and a 17G X 76mm needle (GMMC CO, Korea) using a vacuum pump (Gast, USA) Tubes (BD Falcon, USA) and immediately moved to the laboratory. The inoculated follicular fluid was washed with a cone filter, and only the oocyte was removed and the in vitro maturation step was carried out.

1-3. In vitro maturation of oocytes

The ovaries of the slaughtered cows were collected and inoculated with cumulus-oocyte complex (COC) in follicles 2-5 mm in diameter with an 18G syringe. Using a 0.5 ml (IV) In vitro maturation was induced for 20-22 hours. After 20-22 hours of culture, the cumulus cell-oocyte complex (COC) matured with metaphase II (MII) was immediately donated to in vitro fertilization.

1-4. In vitro fertilization

In vitro fertilization culture medium IVF100 was coated with mineral oil in a 45-μl aliquot of a microtiter plate, and then matured oocytes washed twice with IVF100 were added to each microtiter plate. Spermatozoa for in vitro fertilization were prepared by first adding 4 ml of IVF100 to a 15 ml tube and then freezing the frozen straw at 37 for 1 minute and dispensing the spermatozoa in the tube which was fused to a tube containing IVF100. Then, centrifugation was performed at 600 g for 10 minutes to separate the sperm and the diluent for freezing, and then the supernatant was removed. The remaining sperm pellet was further rinsed once with 4 ml IVF100, and the final concentration was adjusted to 1 × 10 ⁷ / ml, and the cells were injected into the prepared ovarian microspheres for modification for 6 hours. The fertilized oocytes were further cultured via IVMD101 for 20-24 hours. After completion of the addition, it was determined that the sperm around the oocyte and the cumulus cells were removed, and the blastocyst was obtained by incubating for 7 days in the same culture medium after washing three times through the IVD101 medium.

Experimental Example  2. Survival rate and hatching rate of Hanwoo embryos by storage method

The blastocysts obtained by in vitro culture were washed three times in a basic stock solution containing HEPES, BSA and TCM199 or as a stock solution containing the small molecule substance added to the basic stock solution, and then put into a 0.25 ml plastic straw (FHK, Tokyo, Japan) , Which is shown in Fig. The blastocysts stored in the stock solution were stored under the conditions of temperature and time according to each of the following examples.

In order to verify the survival rate and hatching rate of Hanwoo embryos, Hanwoo embryos stored in the stock solutions of each of the following conditions were taken out, washed three times with IVMD culture medium, and then cultured further. The survival rate was determined 24 h after culture and hatching rate after 48 h. The survival rate was determined by microscopic examination. After 24 hours of incubation, the cells in the blastocyst of embryo were arrested or shrunk to show no further proliferation. The hatchability of the embryos was determined by incubating the zona pellucida at 48 hours after the incubation.

Example  1. Optimization of the concentration of BSA constituting the basic stock solution of the present invention

The survival rate and hatching rate of Hanwoo embryos were measured to confirm the optimal concentration of BSA in the basal stock solution.

Specifically, the basic stock solution of the present invention was prepared by constituting 25 mM HEPES, 1 × TCM199 and 1% or 5% BSA (w / v), and a blastocyst Hanwoo embryo was used as the embryo.

Forty-six embryos were stored in a stock solution containing 1% (w / v) BSA and 42 embryos were preserved in the same manner as in Experimental Example 2 using a stock solution containing 5% (w / v) BSA And the survival rate and hatching rate of each embryo were confirmed. The results are shown in Table 1 below.

Percentage of BSA No. of Blastocysts Viable embryos (%) Hatching embryos (%) One 46 41 (89.1) 38 (82.6) 5 42 38 (90.5) 35 (83.3)

As shown in Table 1, Hanwoo embryos of 1% (w / v) BSA group and 5% (w / v) BSA group showed survival rate and hatching rate of 80% or more. In addition, the survival rate and hatching rate of each of the above-mentioned groups were similar, and finally, 1% (w / v) BSA was selected for the stock solution of the present invention and used for the following series of experiments.

Example  2. Survival rate of Hanwoo embryos according to the basic stock solution of the present invention Hatching rate

As confirmed in Example 1 above, the survival rate and hatching rate of Hanwoo embryos according to storage days were confirmed in order to confirm basic storage level of the basic stock solution of the present invention containing 1% (w / v) BSA.

The basic stock solution of the present invention was prepared by constituting 25 mM HEPES, 1 × TCM199 and 1% BSA (w / v), and a blastocyst Hanwoo embryo was used as the embryo. 50 embryos for 24 hours, 60 embryos for 48 hours, 100 embryos for 72 hours, 40 embryos for 96 hours, 46 embryos for 120 hours, 22 embryos for 168 hours at 4 degrees , And preserved in the same manner as in Experimental Example 2, and the survival rate and hatching rate of each fertilized egg were confirmed. The results are shown in Table 2 below.

Time No. of Blastocysts Viable embryos (%) Hatching embryos (%) 24 50 50 (100.0) 49 (98.0) 48 60 60 (100.0) 56 (93.3) 72 100 93 (93.0) 87 (87.0) 96 40 36 (90.0) 32 (80.0) 120 46 35 (76.1) 22 (47.8) 168 22 10 (45.5) 8 (36.4)

As shown in Table 2, a high survival rate and hatching rate of about 80% or more were observed until 96 hours of storage. In addition, it was confirmed that the embryos could survive and hatch at 120 hours of storage. Thus, it was confirmed that the basic stock solution containing HEPES, TCM199 and BSA of the present invention efficiently stored the Korean embryos.

Example  3. Small molecule  Identification of survival rate and hatching rate of Hanwoo embryos according to the storage solution of the present invention containing the substance and storage temperature of 4 degrees

Single or combined use of a small molecule material (Ch9 (CHIR99021), Y2 (Y27632) or Th (Thiazovivin)) was added to the basic stock solution of the present invention and the survival rate and hatching rate of Hanwoo embryos Respectively.

Specifically, in order to confirm the survival rate and the hatching rate of the embryo-transferred Hanwoo embryos when each of the small molecule materials was separately treated in the basic storage solution of Example 2, Ch9 of each 1, 3 and 6 μM concentration was added to the basic stock solution of Example 2 (CHIR99021); A single treatment group supplemented with Y2 (Y27632) at concentrations of 5, 10 and 20 [mu] M, respectively; Or Th (Thiazovivin) at a concentration of 2, 10 and 20 μM, respectively, and the basic storage solution in which the small molecule material of Example 2 was not added was also tested.

In addition, in order to confirm the survival rate and hatching rate of the embryo-transferred Hanwoo embryos when the small molecule materials were additionally used in the storage solution of Example 2, 10 μM of Y2 (Y27632) + 1 uM Ch9 (CHIR99021) ; And 2 uM of Thiazovivin + 1 uM of Ch9 (CHIR99021).

In each treatment group, the embryo-transferred embryo-transferred embryos were preserved in a 4-degree environment for 96 hours in the same manner as in Experimental Example 2, and survival and hatching rates were confirmed. The results are shown in Table 3 and FIG.

Concentration
(μM) No. of Blastocysts Viable embryos (%) Hatching embryos (%) Single 저장 액체 - 14 11 (76.8) 11 (78.6) Ch9 One 24 22 (91.7) 21 (87.5) 3 24 17 (70.8) 14 (58.3) 6 16 10 (62.5) 10 (62.5) Y 5 24 19 (79.2) 18 (75.0) 10 23 21 (91.3) 18 (78.3) 20 24 19 (79.2) 18 (75.0) T 2 24 20 (83.3) 17 (70.8) 10 24 19 (79.2) 16 (66.7) 20 24 19 (79.2) 16 (66.7) Combination Ch9 + Y 1 and 10 39 25 (64.1) 22 (56.4) Ch9 + T 1 and 2 39 13 (33.4) 10 (25.6)

As shown in Table 3, the survival rate and hatching rate of the embryo were found to be higher in the group treated with the small molecule besides the basic stock solution. Particularly, in the basic stock solution of the present invention, 1 μM of Ch9 (CHIR99021), 10 μM of Y2 (Y27632) or 2 μM Th (Thiazovivin) were added to the fertilized eggs, the survival and hatching rates of Hanwoo embryos were the highest. Therefore, it was confirmed that, in addition to the above optimal concentrations, each of the small molecule materials was added to the basic stock solution of the present invention, and thus, it was effective for low temperature preservation of Hanwoo embryos.

In addition, it was confirmed that the survival rate and hatching rate of Hanwoo embryos were higher than that of other combination treatment groups when 10 μM Y2 (Y27632) + 1 uM Ch9 (CHIR99021) small molecule substance was added to the basic stock solution of the present invention.

Further, as shown in FIG. 2, when the small molecule materials of the present invention are each treated at an optimal concentration, the outer surface of the incubated embryos is smooth and less debris, and the small molecule materials are additionally contained in the storage solution of the present invention, , And effectively protected the embryos.

Example  4. Small molecule  The survival rate and hatching rate of Hanwoo embryos according to the storage solution of the present invention and the storage temperature of 10 degrees

The survival rate and hatching rate of Hanwoo embryos according to the storage temperature of 10 degrees were confirmed by adding a small molecule substance (Ch9 (CHIR99021), Y2 (Y27632) or Th (Thiazovivin)) to the basic stock solution of the present invention singly or in combination.

Specifically, the optimum concentration of 1 μM Ch9 (CHIR99021), 10 μM Y2 (Y27632), and 2 μM Th (Thiazovivin) was confirmed in Example 3, a single treatment group supplemented with Ch9 (CHIR99021) of μM, a single treatment group supplemented with 10 μM Y2 (Y27632), and a single treatment group supplemented with 2 μM Th (Thiazovivin); 10 μM of Y2 (Y27632) + 1 μM of Ch9 (CHIR99021); And 2 [mu] M of Thiazovivin + 1 [mu] M of Ch9 (CHIR99021) were added to the embryo-treated Hanwoo embryos. In addition, experiments were carried out with the basic stock solution in which the small molecule material of Example 2 was not added.

In order to confirm the preservation effect of Hanwoo embryos according to the temperature condition, Hanwoo embryos in the blastocyst condition were stored in each of the above-mentioned additional treatment groups for 10 hours and 96 hours. At this time, the process of storing the embryos and confirming the survival and hatching rate was carried out in the same manner as in Experimental Example 2. The results are shown in Table 4.

No. of Blastocysts Viable embryos (%) Hatching embryos (%) 저장 액체 30 18 (60.0) 11 (36.7) Ch9 34 22 (64.7) 15 (44.1) Y 34 27 (79.4) * 24 (70.6) * T 33 15 (45.5) 11 (33.3) Ch9 + Y 39 25 (64.1) 22 (56.4) * Ch9 + T 39 13 (33.3) 10 (25.6)

As shown in Table 4, it was confirmed that the group treated with 1 μM of Ch9 (CHIR99021) or 10 μM of Y2 (Y27632) alone in the basic stock solution of the present invention was most effective for 10 ° C low temperature treatment of Hanwoo embryos.

Particularly, when 10 μM of Y2 (Y27632) was treated singly with the basic stock solution of the present invention and 10 μM of Y2 (Y27632) + 1 μM of Ch9 (CHIR99021) small molecule material was added, In addition, the survival and hatching rate of Hanwoo embryos were significantly higher than those of the combined treatment groups.

Therefore, the basic storage liquid processing group of the present invention; It was confirmed that Hanwoo fertilized eggs were effectively preserved even at 10 degrees in the single treatment group or the combined treatment group in which the small molecule materials were added to the basic stock solution.

Example  5. Small molecule  Determination of survival and hatching rate of Hanwoo embryos according to the storage solution of the present invention containing the substance and storage temperature of 20 degrees

The survival and hatching rate of Hanwoo embryos according to the storage temperature of 20 degrees was confirmed by adding a small molecule material (Ch9 (CHIR99021), Y2 (Y27632) or Th (Thiazovivin)

5-1. Determination of survival and hatching rate of Hanwoo embryos according to the storage solution of the present invention and storage temperature of 20 degrees

Specifically, in order to confirm the preservation effect of the basic stock solution of the present invention at a storage temperature of 20 ° C, the embryos were stored for 24 to 96 hours in the basic stock solution of Example 2. The results are shown in Table 5.

Time No. of Blastocysts Viable embryos (%) Hatching embryos (%) 24 13 13 (100) 13 (100) 48 14 9 (64.3) 8 (57.1) 72 13 1 (7.7) * 0 (0) * 96 10 0 (0) * 0 (0) *

As shown in Table 5, when the storage solution of the present invention was used under the condition of 20 degrees, it was confirmed that the survival rate and hatching rate of the Hanwoo storage column were maintained at 60% or more for 48 hours. However, it was confirmed that survival rate and hatching rate were decreased when more than 72 hours had elapsed.

5-2. Small molecule  Single treatment with added material; Or combined treatment and incubation rate and hatching rate of Hanwoo embryos according to 20 ℃ storage temperature

The limit of the basic stock solution of the present invention after 72 hours under the condition of 20 ° C was confirmed and the survival rate and the survival rate of the Korean embryo at 20 ° C and 72 hrs condition, The hatching rate was confirmed.

Specifically, a single treatment group in which 10 μM of Y2 (Y27632) was added to the basic stock solution of Example 2; 10 μM of Y2 (Y27632) + 1 μM of Ch9 (CHIR99021); And 2 μM of Thiazovivin + 1 μM of Ch9 (CHIR99021). In addition, experiments were carried out with the basic stock solution in which the small molecule material of Example 2 was not added. In each group, Hanwoo embryos in blastocyst condition were treated at 20 ° C for 72 hours and preserved in the same manner as Experimental Example 2, and the survival rate and hatching rate of each embryo were confirmed. The results are shown in Table 6 and Fig.

No. of Blastocysts Viable embryos (%) Hatching embryos (%) 저장 액체 30 2 (6.7) 0 (0.0) Y2 30 12 (40.0) * 9 (30.0) * Ch9 + Y 30 15 (50.0) * 11 (36.7) * Ch9 + T 30 8 (26.7) 5 (16.7)

As shown in Table 6, it was confirmed that the survival rate and hatching rate were significantly increased compared to the basal storage solution group when 10 μM Y2 (Y27632) was added to the basic stock solution of the present invention alone. It was also confirmed that the survival rate and hatching rate were significantly increased compared to the basal storage solution when 10 μM of Y2 (Y27632) + 1 μM of Ch9 (CHIR99021) was added.

Further, as shown in Fig. 3, a group of the basic storage solution, a group of single treatment added with 10 [mu] M Y2 (Y27632); 10 μM of Y2 (Y27632) + 1 μM of Ch9 (CHIR99021); And 2 μM of Thiazovivin + 1 μM of Ch9 (CHIR99021). The results showed that the number of incubated embryos in the single or combination treatment group It is confirmed that the surface is smooth and the debris is small and effectively protects the cells.

Example  6. Confirmation of the pregnancy rate of the bovine embryos preserved using the stock solution of the present invention

The pregnancy rate of the preserved bovine embryos was confirmed using the basic stock solution of the present invention.

Specifically, the embryos of cattle stored at room temperature conditions of 20 ° C of the present invention were transplanted into surrogate mothers to confirm the pregnancy rate. For this, the most effective combination of 10 μM Y2 (Y27632) + 1 μM Ch9 (CHIR99021) was selected and stored at 20 ° C for 3 days before transplantation. In order to compare with the conventional cryopreservation method, the pregnancy rate was confirmed by comparing free embryos stored in liquid nitrogen and fresh embryos without storage. The results are shown in Table 7.

Small molecules No. of Blastocysts Pregnant cows (%) Frozen 120 43 (35.8) Fresh 256 136 (53.1) * Storage medium 86 42 (48.8) *

As shown in Table 7, it was confirmed that the pregnancy rate was 53.1% when the low-temperature preserved (four-degree) bovine embryo of the present invention was used, and the pregnancy rate difference was about 18% or more with the conventional cryopreservation method. Further, it was confirmed that the pregnancy rate of the present invention at room temperature (20 degrees) was 48.9%, and it was confirmed that a difference of about 13% or more was observed compared to the conventional cryopreservation method.

Through the above-mentioned series of results, it was found that when treated with Ch9 (CHIR99021) or Y2 (Y27632), which is a small molecule substance, or a combination of HEPES and TCM 199, BSA containing BSA replacing conventional FBS, Range (from 4 to 20 degrees Celsius) Hanwoo embryos can be stored without freezing. In particular, it was confirmed that the stock solution containing the small molecule material of the present invention was excellent in the preservation effect of Hanwoo embryos even at 20 ° C for 72 hours (day 3). In addition, compared with the conventional cryopreservation method, the pregnancy rate was further increased at low temperature (4 degrees) and at room temperature (20 degrees), and it was confirmed that Hanwoo embryos were effectively protected and quality of embryos was maintained.

Claims (13)

BSA (bovine serum albumin), HEPES ((4- (2-hydroxyethyl) -1-piperazineethanesulfonic acid)) and TCM199 (Tissue culture medium 199)
One or more small molecules selected from the group consisting of 1 to 2 uM of CHIR99021 (CAS 252917-06-9), 7 to 25 uM of Y27632 (CAS 129830-38-2) and 1 to 5 uM of thiazovivin A composition for preserving a bovine embryo at a low temperature or a room temperature. The method according to claim 1,
Wherein the BSA is contained in an amount of 0.1 to 10% (w / v) based on the final weight of the composition. The method according to claim 1,
Wherein the composition is for low-temperature storage at 1 to 5 占 폚. delete delete The method according to claim 1,
The composition for preserving a bovine embryo at a low temperature or at a room temperature, wherein the bovine is at least one selected from the group consisting of Korean beef cattle, cow, beef cattle, The method according to claim 1,
Wherein the fertilized egg is an In Vitro Fertilization-Embryo Transfer (IVF), a somatic cell nuclear trasnfer embryo or a parthenogenetic embryo. The method according to claim 1,
Wherein the embryo is a development stage of a blastocyst. The method according to claim 1,
Wherein the low temperature or room temperature is 1 to 30 占 폚. The method according to claim 1,
Wherein the preservation is carried out for 20 to 180 hours. A method for preserving a bovine embryo at low or room temperature by mixing bovine fertilized eggs with the composition of claim 1 and keeping the embryos at low or room temperature. 12. The method of claim 11,
Wherein the low temperature or the room temperature is a temperature of 1 to 30 占 폚. 12. The method of claim 11,
Wherein the preservation is carried out for 20 to 180 hours.

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