JP2014198673A - Preservative for low temperature storage of animal embryo and method of storing animal embryo at low temperature - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a preservative for a low temperature storage of an animal embryo containing a compound having a flavonol structure, a preservation liquid for the low temperature storage of the animal embryo containing the preservative, and a method of storing the animal embryo at low temperature using the compound having the flavonol structure.SOLUTION: There are provided a preservative for a low temperature storage of an animal embryo containing a flavonoid compound represented by the formula (I), where Rto Rare same or different and -H, -OH or an alkoxy group, a preservation liquid for the low temperature storage of the animal embryo containing the preservative, and a method of storing the animal embryo including immersing the animal embryo in a solution containing the flavonoid compound represented by the formula (I) and storing the solution at low temperature.

Description

  The present invention relates to a preservative for cryopreserving animal embryos, and a preservation solution for cryopreserving animal embryos containing the preservative. Furthermore, the present invention relates to a method for preserving animal embryos at low temperatures.

  Cells have different ion compositions inside and outside the cell membrane, and the difference in the distribution of ions having this charge causes a potential difference. Usually, the intracellular potential is negative with respect to the outside of the cell (membrane potential), and this membrane potential exists as a basic principle common to living organisms regardless of animals or plants. The regulation mechanism of membrane potential is indispensable for life support and cell function, and its failure directly leads to life or cell death.

For this reason, there are various ion pumps and ion channels on the inner and outer membranes, and ion balance is constantly adjusted. The most important regulatory mechanism includes a sodium pump (Na ⁺ -K ⁺ ATPase) in animal cells and a proton pump (H ⁺ -ATPase) in plant cells. These ion pumps are membrane proteins that actively transport specific ions using ATP energy. If ATP is depleted for some reason or the ambient temperature deviates from the optimum range, the function of the ion pump will be reduced or stopped.

  Under physiological conditions, three physiological sodium ions are pumped out of the cell each time under physiological conditions. Conversely, two potassium ions are pumped out of the cell into the cell. Therefore, normally, intracellular potassium concentration is high (sodium concentration is low), and extracellular sodium concentration is high (potassium concentration is low). When the temperature of the cell falls below a certain temperature, the function of the sodium pump declines, so that sodium cannot be pumped out of the cell, and the intracellular sodium concentration increases. As the sodium concentration rises, the intracellular osmotic pressure rises, and the influx of water molecules causes the cells to swell and eventually lead to cell rupture (cell damage).

  The above mechanism is considered to be one of the main causes of cell damage when organs for transplantation are cryopreserved at the time of organ transplantation in the medical field. The basic composition of the electrolyte is intracellular low sodium and high potassium. Organ preservation solutions have been developed. Typical examples are Euro Collins (EC) liquid and UW (University of Wisconsin) liquid. Compared to conventional extracellular (high sodium, low potassium) preservation solutions centering on Ringer's solution, these can greatly extend the organ preservation period and are clinically applied as major organ preservation solutions in Japan and overseas. ing. However, these intracellular storage solutions have a risk of reversing and causing cytotoxicity when the storage temperature rises. In addition, these are not applicable to all tissues and organs, and further improvement in performance is expected, including further extension of the storage period.

  The fertilized egg (embryo) transplantation technology for cattle consists of processes such as superovulation treatment with hormonal agents, artificial insemination, embryo recovery and transplantation to the recipient cattle, etc., by using the embryo transfer technology, It becomes possible to efficiently produce excellent breeding cows and bulls with high genetic value. For this reason, research on the cryopreservation of bovine embryos has been conducted around the world, and many rapid freezing methods (vitrification freezing methods) and slow freezing methods have been developed, and a relatively stable conception rate has been obtained. Yes.

  However, conventional techniques for cryopreservation of bovine embryos can only obtain an average conception rate of around 50%, and no major improvement measures for improving the conception rate have been reported for more than 10 years. This fact is thought to mean that the cryopreservation method of mammalian embryos has reached a certain technical limit. In the conventional cryopreservation method, there is a problem that the vitality of the embryo is inevitably impaired due to physical damage in the process of freezing and thawing. In addition, the conventional cryopreservation method uses liquid nitrogen, and therefore has a problem that it cannot be carried on an airplane.

  Therefore, if there is a method that can keep the embryos collected from the laying cattle in a non-frozen state while suppressing growth, the embryos that are not damaged by freezing and thawing can be used efficiently for calf production. It becomes possible to do. By using such a method, when the estrus cycle between the recipient cow and the recipient cow is shifted by several days, it is possible to adjust the transplanting time according to the estrous cycle of the recipient cow.

  As a method for dealing with such problems, Non-Patent Document 1 uses a medium 199 containing FBS and HEPES at a certain ratio to maintain bovine embryos at 4 ° C for up to 7 days. Has been reported to be possible. Patent Document 1 reports that, in non-freezing cryopreservation of bovine embryos, a medium supplemented with serum and Nfe11 peptide exhibits a strong life-prolonging effect on bovine embryos in a low-temperature environment.

  Further, techniques for preserving cells and organs are disclosed in the following Patent Documents 2 to 5, for example.

  Patent Document 2 discloses a method for preserving biological material by adding a preservation solution containing one or more polyphenols to the biological material and cooling. In Examples, catechins are disclosed as polyphenols. Only.

  Patent Document 3 discloses a method for refrigerated storage of cells at a temperature at which water does not crystallize, for example, a temperature of about 4 ° C., by adding an enkephalin derivative to the cell culture medium.

  Patent Document 4 discloses a medical polyphenol solution containing polyphenol and 0.0001 to 0.05% by weight of ascorbic acid or a metal salt of ascorbic acid for use as a cell preservative, tissue preservative, or the like. It is disclosed that decomposition is suppressed and generation of hydrogen peroxide is suppressed. In Examples, only epigallocatechin gallate (EGCg) is disclosed as a polyphenol.

  Patent Document 5 discloses a preservative composition containing epigallocatechin gallate as an active ingredient in an amount of 90% by mass or more, and the effect of preserving cells is more constant by purifying epigallocatechin gallate with high purity. It is described that it can be.

  Patent Document 6 discloses a flavonoid glycoside having an ability to promote the supercooling ability of an aqueous solution. By using this supercooling promoting substance, an antifreeze liquid that can be used at about -15 ° C and water. Therefore, it is described that biological materials and the like can be stored in the antifreeze liquid. As a method for using the flavonoid glycoside of the supercooling promoting substance disclosed in Patent Document 6, the following Patent Documents 7 to 9 are also reported.

  Patent Document 7 discloses a beverage that can maintain a supercooled state containing the flavonoid glycoside. Patent Document 8 discloses a cryopreservation solution in which the above flavonoid glycoside is contained in a vitrification solution, which is less toxic than conventional vitrification solutions and increases the viability of cells and the like by storage. It describes what you can do.

  Furthermore, Patent Document 9 discloses that by using an organ preservation solution containing the above flavonoid glycoside, it becomes possible to preserve an animal organ at a temperature of 0 ° C. or lower without freezing. Has been.

JP 2012-176940 A Special Table 2007-519712 JP 2002-335954 A JP 2006-188436 A JP 2003-267801 A International Publication No. 2008/007684 JP 2009-219394 JP 2009-219395 A JP 2009-221128 JP

Scientific Reports 3: 1173 doi: 10.1038 / srep01173

  Non-patent document 1 and Patent documents 1 to 5 do not substantially disclose the use of a compound having a flavonol structure in a solution for preserving cells and organs. Patent Documents 6 and 9 disclose that by using flavonoid glycosides, cells can be stored at 0 ° C. or lower without freezing injury because they can be cooled to a temperature below freezing point without freezing. However, there is no disclosure about the reduction of low temperature damage under normal low temperature conditions. Moreover, there is no such disclosure about a flavonol compound containing no sugar.

  It is a commonly practiced method to easily store cells, organs, living tissues, and the like at low temperatures. However, it is also known that a failure due to the low temperature condition occurs even when the object is not frozen.

  In the present invention, a preservative for cryopreservation of animal embryos by using a flavonol compound having no sugar to reduce these low-temperature damages, a preservation solution for cryopreservation of animal embryos containing the preservative, And a method for preserving animal embryos at low temperatures using flavonol compounds.

  The inventors of the present invention use a specific compound having a flavonol structure that does not have sugar to store a cow embryo at a low temperature from around the freezing point without freezing to a normal refrigeration temperature. It was found that the survival rate and embryo hatching rate were improved, and the effect of protecting against cold injury was obtained. The present invention has been completed based on these findings, and provides the following preservatives, preservatives, and storage methods.

(I) Preservative
(I-1) Formula (I):

A preservative for cryopreservation of animal embryos comprising a flavonoid compound represented by the formula: wherein R ^{1 to} R ⁹ are the same or different and are —H, —OH or an alkoxy group.
(I-2) The preservative according to (I-1), wherein the flavonoid compound is at least one selected from the group consisting of myricetin, quercetin, kaempferol, galangin, isorhamnetin, fisetin and flavonol.
(I-3) The preservative according to (I-1) or (I-2), wherein the animal embryo is a cow embryo.
(I-4) The low temperature is a temperature at which the animal embryo does not completely freeze, that is, a temperature of -5 ° C to 20 ° C, according to any one of (I-1) to (I-3). Preservatives.

(II) Stock solution
(II-1) A preservation solution for cryopreserving animal embryos, comprising the preservative according to any one of (I-1) to (I-4).

(III) Storage method
(III-1) Formula (I):

[In the formula, R ^{1 to} R ⁹ are the same or different and are —H, —OH or an alkoxy group] The animal embryo is immersed in a solution containing the flavonoid compound, and the solution is kept at a low temperature. A method for preserving animal embryos.
(III-2) The method according to (III-1), wherein the flavonoid compound is at least one selected from the group consisting of myricetin, quercetin, kaempferol, galangin, isorhamnetin, fisetin and flavonol.
(III-3) The method according to (III-1) or (III-2), wherein the animal embryo is a cow embryo.
(III-4) The low temperature is a temperature at which the animal embryo does not freeze completely, that is, a temperature of -5 ° C to 20 ° C, according to any one of (III-1) to (III-3) the method of.

  With the preservative and method of the present invention, it is possible to increase the survival rate of animal embryos such as bovine embryos by preserving animal embryos at a low temperature of normal refrigerated storage at -5 ° C to 20 ° C that is not completely frozen. Low temperature damage protection effect is obtained. Therefore, since it becomes possible to preserve the animal embryo at a low temperature suitable for preservation of the animal embryo and under conditions that can suppress the low-temperature injury caused by it, further improvement of the survival rate of the animal embryo is expected. it can. Therefore, the present invention can be expected to be applied in fields such as livestock breeding.

6 is a graph showing the relationship between the amount of compounds (1) to (7) added and the number of viable cells in Test Example 3 (added compound final concentrations of 10 μg / ml, 1 μg / ml, 0.1 μg / ml, 0.01 μg / ml). 6 is a graph showing the relationship between the amount of catechins and gallic acid added and the number of viable cells in Test Example 3 (added compound final concentrations of 100 μg / ml, 10 μg / ml, 1 μg / ml).

  Hereinafter, the present invention will be described in detail.

  The preservative for cryopreservation of the animal embryo of the present invention has the formula (I):

[Wherein R ^{1 to} R ⁹ are the same or different and are —H, —OH, or an alkoxy group], and a flavonoid compound represented by the formula:

  The animal embryo storage method of the present invention is characterized in that the animal embryo is immersed in a solution containing the flavonoid compound and the solution is kept at a low temperature.

R ^{1 to} R ⁴ are preferably the same or different and are —H or —OH.

The alkoxy group is preferably an alkoxy group having 1 to 6 carbon atoms, more preferably an alkoxy group having 1 to 3 carbon atoms, and particularly preferably —OCH ₃ . The alkyl part of the alkoxy group may be linear or branched. Examples of the alkoxy group having 1 to 6 carbon atoms include methoxy, ethoxy, propoxy, isopropoxy, butoxy, isobutoxy, tert-butoxy, pentyloxy, isopentyloxy, hexyloxy and the like.

  Examples of the flavonoid compound represented by the formula (I) include myricetin, quercetin, kaempferol, galangin, isorhamnetin, fisetin, and flavonol. Is mentioned.

  The above flavonoid compounds can be chemically synthesized by known methods, and are contained in living organisms such as plants, and therefore can be obtained by extraction from these by known methods. Moreover, it is also possible to obtain with a commercial item.

  In the present invention, the low temperature is a temperature at which the animal embryo does not completely freeze, that is, −5 ° C. or higher, preferably −3 ° C. or higher, more preferably higher than 0 ° C. and 20 ° C. or lower, preferably 15 ° C. or lower. More preferably, the temperature is 10 ° C or lower.

  As used herein, low temperature injury means cell damage caused by low temperature, and the low temperature injury protection effect means the effect of protecting animal embryos from the low temperature injury. Therefore, taking this meaning into consideration, the preservative of the present invention can also be referred to as a low-temperature damage protective agent.

  The animal embryo used in the present invention is not limited as long as the effects of the present invention can be obtained, but it is preferably a mammal (human, monkey, cow, pig, horse, dog). , Cats, rabbits, mice, rats, etc.), more preferably bovine embryos.

  The bovine embryo can be appropriately prepared by a known method, and any bovine embryo produced by either in vitro fertilization or in vivo fertilization can be used. Examples of the method for producing a bovine embryo include the following methods. The follicular ovum collected from the ovary of the cow is cultured in vitro and cocultured with the sperm of the cow (measles), and then the morphologically from the mulberry stage to the blastocyst stage is used as the cow embryo.

  In addition to the flavonoid compound (I), the preservative of the present invention may contain known additives as appropriate.

  The preservation solution for cryopreservation of animal embryos of the present invention is characterized by containing the above preservative. In the present invention, when the animal embryo is stored at a low temperature, the flavonoid compound (I) is usually used as a solution. The solvent for dissolving the flavonoid compound (I) is not particularly limited. ), Buffer (PBS, Tris buffer, Hepes buffer, MOPS buffer, PIPES buffer, etc.), cell culture solution (Medium199, RPMI1640, DMEM, etc.), organ preservation solution (EC solution, UW solution, etc.), modena Liquid and the like. When cattle embryos are cryopreserved, the solvent preferably contains serum (preferably bovine serum) and cell culture medium (preferably Medium 199), and more preferably contains Hepes buffer. Examples of bovine serum include fetal bovine serum, calf serum, and adult bovine serum, but fetal bovine serum is preferable.

  The concentration of the flavonoid compound (I) in the preservation solution of the present invention is usually 0.001 to 1000 μg / ml, preferably 0.01 to 100 μg / ml, more preferably 0.1 to 10 μg / ml. In the preservation solution of the present invention, other conventional components such as buffers, antibiotics, antibacterial agents, antioxidants, serum, saccharides, lipids, vitamins, proteins, peptides, amino acids, pH indicators, chelating agents, An osmotic pressure regulator and the like can also be included.

  In the present invention, animal embryos can be preserved by immersing the animal embryos in a solution containing the flavonoid compound (I). In this case, the solution may be cooled to a low temperature before immersing the animal embryo, or may be cooled to a low temperature after immersing the animal embryo. Once the solution containing the animal embryo is once cooled to a low temperature, it is kept at a low temperature. However, it is not always necessary to maintain a constant temperature, and the temperature may be outside the low temperature range for a short time.

  The container for immersing the animal embryo in the solution containing the flavonoid compound is not particularly limited as long as it does not show toxicity to the animal embryo. For example, a semen freezing straw, a cryotube, a sampling tube, and a fertilized egg transplant Examples include straws, cell culture plates, cell culture dishes, and various vials.

  By using the solution containing the flavonoid compound (I) of the present invention, the survival rate of the animal embryo can be significantly increased by preserving the animal embryo at a low temperature, and a low-temperature injury protection effect can be obtained. Since the present invention can suppress a low-temperature injury caused by low temperature suitable for preservation of animal embryos, it is possible to preserve animal embryos in an appropriate state.

  The preservation solution of the present invention having the above characteristics is expected to be applied as a preservation solution for fertilized embryos in the field of livestock breeding.

  Animal embryos preserved at a non-freezing low temperature with the preservation solution of the present invention are not subject to physical damage during freezing and thawing in the cryopreservation method, so that an improvement in conception rate can be expected. Furthermore, although the conventional cryopreservation method using liquid nitrogen could not be transported by airplane, according to the present invention, high speed transportation by airplane is possible.

  Examples are given below to illustrate the present invention in more detail. However, the present invention is not limited to these examples.

Test example 1
For the compounds of the present invention, the protective effect against cold injury of cells was evaluated by the following method.

RPMI-1640 (SIGMA, No.R8758) containing 10% fetal calf serum (Thermo, No.SH3D396.03) in a 5% carbon dioxide, 37 ° C. incubator (Sanyo Electric Co., MCO-17AIC) (10% FBS-RPMI) Human promyelocytic leukemia cell line HL-60 cultured in culture medium was collected, centrifuged at 4 ° C, 1,000 rpm, 5 minutes (TOMY, EIX-135), and the supernatant was It was removed and resuspended in physiological saline to 2 × 10 ⁶ cells / ml. 0.25 ml of this cell suspension and 0.25 ml of the test compound were mixed in a 2 ml sterilized microtube. The test compound was dissolved in 100 mg / ml with dimethyl sulfoxide (Nacalai Tesque, No. 13407-45) (DMSO) before the test, and then in physiological saline (Otsuka Pharmaceutical Co., Ltd. Was diluted 500 times and prepared as a 0.2 mg / ml physiological saline solution.

The mixture of the cell suspension and the test compound was allowed to stand for about 24 hours in a cooling vessel (No. SC-DF25, manufactured by TWINBIRD) set at 4 ° C. Thereafter, 2.5 ml of 10% FBS-RPMI culture solution was added and centrifuged at 4 ° C. and 1,000 rpm for 5 minutes. After removing the supernatant, it is suspended in 2.5 ml of 10% FBS-RPMI culture solution, added to a 96-well microplate (IWAKI, No. 3860-096) at 0.1 ml / well, and 5% carbon dioxide gas at 37 ° C. The cells were cultured for about 24 hours in an incubator. Subsequently, 10 μl / well of WST-8 solution (Nacalai Tesque, Cell Count Reagent SF, No. 07553-44) was added, and after further culturing in an incubator for 3 hours, the absorbance was measured at a wavelength of 450 nm. (WAKO, SPECTRA MAX250). From this absorbance value, the number of viable cells was calculated based on the standard line of cell number and absorbance prepared beforehand (viable cell number = 56375 × absorbance, R ² = 0.9995).

  As a result, as shown in Table 1, in the case of no addition, cells were allowed to stand at 4 ° C for 24 hours (cold injury), resulting in the number of viable cells from the initial number of cells from 20,000 cells / well to 500 cells / well. Decreased to. On the other hand, the effect of protecting against cold injury was observed by adding the compounds (1) to (7) to the cells at a concentration of 100 μg / ml during low-temperature storage. The number of viable cells at this time was 21.0 to 50.6 times higher than that of the physiological saline alone without addition of the compound.

Test example 2
For test compound (3) (final concentration: 100 μg / ml, 10 μg / ml, 1 μg / ml or 0.1 μg / ml), the temperature during low-temperature storage was set to −5 ° C., 0 ° C., 4 ° C. or 10 ° C. Except for this, the same test method as in Test Example 1 was followed. As a result, as shown in Table 2, the low temperature failure protection effect was confirmed at all storage temperatures.

Test example 3
Test Example 1 except that each tested compound shown in (1) to (7) in Table 1 was added at 10 μg / ml, 1 μg / ml, 0.1 μg / ml, or 0.01 μg / ml (final concentration) during storage at low temperature. A similar test was conducted. In addition, at low temperature storage, catechins (catechin, epicatechin, epicatechin gallate, epigallocatechin, epigallocatechin gallate) and gallic acid (100 μg / ml, 10 μg / ml, or 1 μg / ml) The same test as in Test Example 1 was conducted except that (final concentration) was added.

  The results are shown in FIGS. From these results, it can be seen that catechins and gallic acid cannot obtain a low-temperature damage protection effect at 1 μg / ml or less, whereas the compounds of the present invention can provide a low-temperature damage protection effect even at 1 μg / ml. I understand.

  In addition, as shown in Table 3 below, polyphenols are generally known to have antioxidant ability, but a compound having high antioxidant ability does not necessarily have a high low-temperature damage protection effect. Absent. Since there is no clear correlation between the antioxidant ability and the low temperature injury protection effect, it can be seen that the low temperature injury protection effect of the compound of the present invention is not simply due to the antioxidant ability of polyphenols.

Test example 4
Solvents to which the compounds to be added during low-temperature storage are added from physiological saline, phosphate buffered saline (PBS, SIGMA No. D 8537), EC solution (composition K ₂ HPO ₄ ; 7.4 g / L, KH ₂ PO ₄ 2.05 g / L, KCl; 1.12 g / L, NaHCO ₃ ; 0.84 g / L, glucose; 35 g / L), UW solution (Biaspan; manufactured by Astellas Pharma Inc.), RPMI1640 culture solution (RPMI), or 10% FBS -The same test as in Test Example 1 was performed by changing to the RPMI culture solution. As a result, as shown in Table 4, the low temperature damage protection effect was confirmed in all the solvents used. That is, the solvent of the compound of the present invention is not limited to physiological saline, and it has been confirmed that it exhibits a low-temperature damage protection effect even in widely used solvents such as physiological buffers, organ preservation solutions, and cell culture solutions. It was done.

Test Example 5 (Cryogenic preservation of bovine embryo 1)
Measurement of the protective effect of the compound of the present invention against cold injury of in vitro fertilized embryos was evaluated by the following method.

(Preparation of preservation solution)
The test compound was added to Medium 199 medium (GIBCO) containing 20% (V / V) fetal bovine serum and 25 mM Hepes (DOJINDO) to a final concentration of 1 μg / ml.

(In vitro fertilization and embryo selection)
Follicular ova were aspirated from bovine ovaries and cultured in vitro in medium 199 medium containing 5% (V / V) fetal bovine serum under conditions of 5% CO ₂ and 38.5 ° C. for 20 hours. On the other hand, freeze-thawed bovine sperm was suspended in sperm IVF-100 (Functional Peptide Institute) and centrifuged at 2000 rpm for 10 minutes for sperm washing. Sperm adjusted to a final sperm concentration of 5 million sperm / ml and the above eggs were co-cultured in a 100 μl spot, and the sperm was performed for 6 hours. On day 1, after cumulation, cumulus cells attached to fertilized eggs are removed by pipetting up to about 1 to 2 layers and cultured in vitro in CR1aa medium containing 2% (V / V) fetal bovine serum. On the fifth day, the medium was replaced with 5% (V / V) fetal bovine serum-containing USU medium. On the 7th day, in accordance with the index of the International Fertilized Egg Transfer Study Group, fertilized embryos of grade 1 to 2 that were morphologically normally developed up to the blastocyst stage were selected and subjected to the following cryopreservation test.

(Low temperature storage test)
A low-temperature storage test was conducted by the following method.

The preservation solution was sucked into a straw (1/4 cc clear straw, SPI) having a diameter of 400 μm (capacity: 0.25 ml), and then the preservation solution containing a cow embryo was sucked with an air layer interposed therebetween. Further, only the stock solution was sucked again with an air layer in between, and both ends of the straw were sealed and stored in a refrigerator (4 ± 1 ° C.). After cryopreservation for 120 hours (5 days), the bovine embryos washed by immersing 3 times in Dulbecco's phosphate buffer (D-PBS, GIBCO) containing 5% (V / V) fetal bovine serum were washed with 5% CO ₂ The cells were cultured in CR1aa medium containing 5% (V / V) fetal calf serum for 48 hours under the condition of 38.5 ° C. Morphological observation was carried out under a stereomicroscope, and it was judged that the embryo was alive when the cleavage progress by culture was confirmed. For viable embryos, it was confirmed whether escape (hatching) from the transparent body was observed.

(Test results)
Table 5 below shows the survival rate and hatching rate of bovine embryo after low-temperature storage at 4 ° C. for 120 hours (5 days).

  As shown in Table 5, in the medium 199 medium containing 20% (V / V) fetal calf serum, the non-added group had a survival rate of 44.0% and an embryonic hatching rate of 20.0%. On the other hand, in the compound (3) treatment group, the survival rate was 76.0% and the hatching rate was 40%, confirming the low-temperature injury protection effect of the compound of the present invention on bovine embryos.

Test Example 6 (Cryogenic preservation of bovine embryo 2)
The measurement of the protective effect of the compound of the present invention against low temperature damage of in vivo fertilized embryos was evaluated by the following method.

(Preparation of preservation solution)
The test compound was added to Medium 199 medium (GIBCO) containing 20% (V / V) fetal bovine serum and 25 mM Hepes (DOJINDO) to a final concentration of 1 μg / ml.

(Artificial fertilization and fertilized embryo selection)
During the luteal phase of the estrous cycle, follicle-stimulating hormone was administered gradually for 3-4 days, and PGF2α was administered on the final day to induce estrus. After estrus observation, artificial insemination was performed with frozen semen. Seven-day-old embryos (estrus day 0) were collected non-surgically and cultured in USU medium containing 5% (V / V) fetal bovine serum for 1 day. According to the index of the International Fertilized Egg Transfer Study Group, fertilized embryos of grades 1 to 2 that were morphologically normally developed up to the blastocyst stage were selected and subjected to the following cryopreservation test.

(Low temperature storage test)
The survival rate and the number of embryos hatched were evaluated by the same test method and evaluation method as in vitro fertilized embryos, except that the low-temperature storage period was changed to 120 hours (5 days) and changed to 240 hours (10 days).

(Test results)
Table 6 below shows the survival rate and embryo hatching rate of bovine embryos after low-temperature storage at 4 ° C for 240 hours (10 days).

As shown in Table 6, in the medium 199 medium containing 20% (V / V) fetal bovine serum, the non-added group had a survival rate of 10.0% and an embryonic hatching rate of 0%. On the other hand, in the compound (3) -treated group, the survival rate was 27.6% and the hatching rate was 10.3%, confirming the low-temperature injury protection effect of the compound of the present invention on bovine embryos.

Claims (7)

Formula (I):

A preservative for cryopreservation of animal embryos comprising a flavonoid compound represented by the formula: wherein R ^{1 to} R ⁹ are the same or different and are —H, —OH or an alkoxy group.   The preservative according to claim 1, wherein the flavonoid compound is at least one selected from the group consisting of myricetin, quercetin, kaempferol, galangin, isorhamnetin, fisetin and flavonol.   The preservative according to claim 1 or 2, wherein the animal embryo is a cow embryo.   The preservation | save liquid for the cryopreservation of the animal embryo containing the preservative as described in any one of Claims 1-3. Formula (I):

[In the formula, R ^{1 to} R ⁹ are the same or different and are —H, —OH or an alkoxy group] The animal embryo is immersed in a solution containing the flavonoid compound, and the solution is kept at a low temperature. A method for preserving animal embryos.   The method according to claim 5, wherein the flavonoid compound is at least one selected from the group consisting of myricetin, quercetin, kaempferol, galangin, isorhamnetin, fisetin and flavonol.   The method according to claim 5 or 6, wherein the animal embryo is a cow embryo.

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