CN114507636A - Method for improving animal embryo development efficiency - Google Patents
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Abstract
The invention relates to a method for promoting in vitro development of pre-implantation embryos and improving embryo transfer efficiency, which comprises the step of culturing the embryos in a culture solution containing alpha-ketoglutaric acid or adding the alpha-ketoglutaric acid in the culture process of the embryos. The addition of alpha-ketoglutaric acid in the in vitro embryo culture solution can improve the blastocyst rate, the hatching blastocyst rate, the number of cell masses in the blastocyst and the proportion of the total number of the cells of the blastocyst to the blastocyst rate of in vitro-developed embryos, thereby obviously improving the pregnancy rate of embryo transplantation. The method for promoting the in vitro development of the embryo provided by the invention has the advantages of simple and easy operation process and low cost, and provides an effective method and a new thought for further improving the in vitro culture and development of the mammalian embryo and the development of an assisted reproduction technology.
Description
The invention is the invention patent division with application number 201811488196.8 entitled "a method for promoting embryo in vitro development before implantation and improving embryo transfer efficiency
Technical Field
The invention relates to the field of animal genetic breeding, in particular to a method for promoting in-vitro development of embryos before implantation and improving embryo transfer efficiency.
Background
Embryo transplantation, as a fast-developing animal breeding technology at present, can enable animals with excellent performance to exert the potential and the function in variety improvement and breeding work to the maximum extent, improve the breeding efficiency, accelerate the variety improvement, expand the fine breed animal groups, and have very wide application and development prospects. The in vitro culture of the embryo is a key link of the embryo transplantation technology, and the quality of the in vitro cultured embryo and the pregnancy rate of the transplantation are key factors influencing the embryo transplantation efficiency of animals. How to improve the in vitro development efficiency and quality of embryos and further improve the transplantation efficiency of in vitro cultured embryos is always a research hotspot of scientific research and technicians.
Alpha-ketoglutaric acid (alpha-KG) is an important intermediate product of tricarboxylic acid cycle and other various metabolic pathways in an organism, and has the characteristics of stable structure, difficult degradation, no toxicity, safety and the like. At present, alpha-ketoglutarate has been applied to sports functional health products and sports drink additives to help athletes quickly recover physical fitness and increase muscle vitality. As a precursor substance of glutamine, the alpha-ketoglutaric acid can promote the recovery of wounds of burn patients clinically. The existing research shows that the alpha-ketoglutaric acid can relieve the diarrhea of piglets, promote the weight gain and the skeletal development, and the growth and the development of broiler chickens can be promoted by adding the alpha-ketoglutaric acid into the daily ration. In addition, the alpha-ketoglutaric acid also has the functions of increasing the insulin level in blood plasma, increasing the osteocalcin level under a stress state, improving the negative nitrogen balance and muscle dissimilation of postoperative patients and the like.
Disclosure of Invention
In order to solve the technical problems in the prior art, the invention aims to provide a method for promoting the in vitro development of a pre-implantation embryo and improving the embryo transfer efficiency.
Alpha-ketoglutarate is an important intermediate metabolite of an organism and has important physiological functions in vivo, and the functions of the alpha-ketoglutarate are researched more in the prior art, but no research report of the action relationship between the alpha-ketoglutarate and the embryonic development exists. The inventors have long dedicated research on animal genetic breeding, and in the research related to in vitro embryo culture and development, the inventors have discovered that the addition of alpha-ketoglutarate to the culture solution can promote the embryo development and significantly improve the embryo transfer efficiency.
First, the present invention provides the use of alpha-ketoglutarate for promoting embryo in vitro development or improving embryo transfer efficiency.
In particular, said application is the culturing of said embryo in a culture medium containing alpha-ketoglutarate or the addition of alpha-ketoglutarate during the culturing of said embryo.
The inventors have further found that the effect of alpha-ketoglutarate on embryonic development is particularly important and significant in the initial stage of embryonic development, i.e., prior to implantation of the embryo.
Preferably, the addition is at an in vitro culture stage prior to embryo implantation.
More preferably, the addition is between the prokaryotic stage to the blastocyst stage of embryonic development.
As a preferred embodiment of the invention, the addition is at the beginning of the in vitro development of the embryo.
The inventor further finds that the influence of alpha-ketoglutaric acid on embryo development is particularly remarkable when the alpha-ketoglutaric acid is added at a certain concentration.
Specifically, the concentration of the alpha-ketoglutaric acid in the culture solution of the embryo is 0.1-5 mM; preferably 0.1 to 3 mM.
In the present invention, the embryo source is mammalian.
It will be appreciated by those skilled in the art that the process and mechanism of in vitro development of mammalian embryos are very similar, and the method for promoting embryo in vitro development by using alpha-ketoglutarate provided by the invention is also suitable for in vitro embryo culture of small mammals such as mice, medium or large mammals such as cows, pigs and horses.
In the present invention, the embryo is preferably derived from a non-human mammal, and more preferably from any one of mouse, cow, pig, sheep, horse, deer, mink, and fox.
As a preferred embodiment of the present invention, when the embryo is derived from a mouse, the concentration of α -ketoglutarate in the culture solution of the embryo is 0.1 to 2 mM; preferably 0.1 to 1 mM; more preferably 0.1 to 0.3 mM.
When the embryo is of bovine origin, the concentration of alpha-ketoglutaric acid in the culture solution of the embryo is 0.15-1 mM; preferably 0.15 to 0.5 mM.
In another aspect, the present invention also provides a method for promoting the in vitro development of an embryo, wherein the embryo is cultured in a culture solution containing alpha-ketoglutarate, or alpha-ketoglutarate is added during the culture of the embryo.
The inventor proves that the promotion effect of the alpha-ketoglutaric acid on the development of the animal embryo is that the alpha-ketoglutaric acid plays an independent role and does not depend on being matched with a specific culture solution or a specific substance for use through a large number of experiments. Therefore, it will be understood by those skilled in the art that the culture medium used in the method for promoting the development of animal embryo using α -ketoglutaric acid provided by the present invention may be any of the various culture media disclosed in the prior art or any other non-disclosed culture medium suitable for various animal embryos, including but not limited to commonly used culture media such as KSOM, M16, PZM-3, etc.
Preferably, the addition is at an in vitro culture stage prior to embryo implantation.
More preferably, the addition is between the prokaryotic stage to the blastocyst stage of embryonic development.
In a preferred embodiment of the present invention, the addition is performed at the start of in vitro embryo culture.
Specifically, the concentration of the alpha-ketoglutaric acid in the culture solution of the embryo is 0.1-5 mM; preferably 0.1 to 3 mM.
As a preferred embodiment of the present invention, when the embryo is derived from a mouse, the concentration of α -ketoglutarate in the culture solution of the embryo is 0.1 to 2mM, preferably 0.1 to 1 mM; more preferably 0.1 to 0.3 mM.
When the embryo is of bovine origin, the concentration of alpha-ketoglutaric acid in the culture solution of the embryo is 0.15-1 mM; preferably 0.15 to 0.5 mM.
The method for promoting the in vitro development of the embryo comprises the following steps:
(1) collecting the embryo in the prokaryotic stage: obtaining embryo in prokaryotic stage by natural mating or artificial insemination; the embryo source is a mammal;
(2) in vitro culture of embryos: and placing the prokaryotic stage embryo in a culture solution containing alpha-ketoglutaric acid with the concentration of 0.1-3 mM for in vitro culture of the embryo, or adding the alpha-ketoglutaric acid until the final concentration is 0.1-3 mM before the prokaryotic stage embryo is cultured to the blastocyst stage.
In addition, the invention also provides an in-vitro culture solution of the embryo, and the culture solution contains the alpha-ketoglutaric acid.
Preferably, the concentration of the alpha-ketoglutaric acid is 0.1-5 mM.
More preferably, the concentration of the alpha-ketoglutaric acid is 0.1-3 mM.
Preferably, the culture solution further contains one or more of inorganic salts, carbohydrates, essential amino acids and non-essential amino acids.
The invention has the beneficial effects that:
(1) the invention discovers for the first time that the addition of alpha-ketoglutaric acid in-vitro embryo culture solution can obviously improve the blastocyst rate, the hatching blastocyst rate, the number of cell mass cells in the blastocyst and the proportion of the cell mass cells in the blastocyst to the total number of the blastocyst cells of animals such as mice, cattle and the like, thereby obviously improving the pregnancy rate of embryo transplantation (improved by 150%), the farrowing rate (improved by 60%) and the birth weight of young children (improved by 47%).
(2) In the method for promoting the in vitro development of the embryo, the used alpha-KG has no toxic effect on animals and is safe to use; the solvent has excellent dissolving performance, is easy to dissolve in water and organic solute, and does not need other solvent media (such as dimethyl sulfoxide, propylene glycol and the like) to help promote the dissolution; and the physical property is stable, the film is not easy to decompose when exposed to light, and the film is not easy to degrade in a solution, so that the operation in the practical use process is very easy.
(3) In the method for promoting the in vitro development of the embryo, the alpha-KG can be directly added into the embryo culture solution, the whole process of culture is carried out, the solution change is not needed in the middle, and the adverse effect of the solution change on the embryo development is avoided.
(4) The method for promoting the in vitro development of the embryo provided by the invention has the advantages of obvious effect on promoting the development of the embryo, simple and easy operation process and low cost, and provides an effective method and a new thought for further improving the culture and development of the in vitro embryo of mammals such as mice, livestock and the like and the development of an assisted reproduction technology.
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FIG. 1 is a graph showing the effect of α -KG on blastocyst gene expression in example 2; wherein, Control represents a Control group without adding alpha-KG, and alpha KG represents an experimental group with adding alpha-KG.
FIG. 2 is a graph showing the effect of α -KG on the rate of apoptosis of blastocysts in example 2; wherein, Control represents a Control group without adding alpha-KG, and alpha-KG represents an experimental group with adding alpha-KG.
FIG. 3 Effect of α -KG on mitochondrial function of blastocytes in example 4, wherein a is ATP level; b is the mitochondrial copy number; c is mitochondrial membrane potential; control represents the Control group without addition of α -KG, and α -KG represents the experimental group with addition of α -KG.
FIG. 4 is a graph of the effect of α -KG on the methylation level of blastocyst cells in example 5, wherein a is the picture of the fluorescent staining of 5-hmC in the blastocyst; b is content analysis of 5-hmC In a Control group (Control, without adding alpha-KG), an alpha-KG treated experimental group (alpha-KG) and an In vivo embryo (In vivo); c is expression level analysis (FPKM) of Tet1 gene; d is expression level analysis (FPKM) of Tet2 gene; e is expression level analysis (FPKM) of Tet3 gene; f is the relative expression level of Tet1 and Tet2 genes of Control (Control) and alpha-KG treated experimental (alpha-KG) embryos.
Detailed Description
Preferred embodiments of the present invention will be described in detail with reference to the following examples. It is to be understood that the following examples are given for illustrative purposes only and are not intended to limit the scope of the present invention. Various modifications and alterations of this invention will become apparent to those skilled in the art without departing from the spirit and scope of this invention.
The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.
Materials, reagents and the like used in the following examples are commercially available unless otherwise specified. Statistical analysis of all experimental data was performed using SPSS software.
Example 1: collection of prokaryotic embryos
After ICR female mice of 6-7 weeks of age are adaptively raised for 1-2 weeks, PMSG (10IU) is injected into the abdominal cavity, and hCG (10IU) is injected at intervals of 48h for superovulation. Female hCG-injected mice were immediately caged with sexually mature syngeneic male mice. Vaginal plugs were examined in the next morning (approximately after hCG16h injection) and female spiked mice were sacrificed by cervical dislocation 24h after hCG injection. The oviduct is flushed with pre-warmed M2 operating fluid to collect morphologically normal prokaryotic embryos. The operation was carried out at room temperature of 25 ℃ on a thermostatic table at 37 ℃.
Example 2: effect of alpha-ketoglutarate on embryonic development
Preparing 60 mu L culture drops of embryo culture solution in a 35mm culture dish, wherein the embryo culture solution is KSOM culture solution and KSOM culture solution respectively added with alpha-ketoglutaric acid with final concentration of 100 mu M, 150 mu M, 200 mu M and 300 mu M, the KSOM culture solution without alpha-ketoglutaric acid is used as a control group, and the group added with 100 mu M, 150 mu M, 200 mu M and 300 mu M alpha-ketoglutaric acid is used as an experimental group; placing into an incubator (37 ℃, 5% CO)2) Pre-equilibration is performed. 20-30 prokaryote embryos collected according to example 1 were placed in each culture drop and cultured in an incubator (5 groups in total for the experimental group and the control group, and 1326 prokaryote embryos were cultured in total for each group as shown in Table 1).
And culturing the prokaryotic embryos in an incubator for 24h, and counting the cleavage rate. And (4) counting the blastocyst rate and the hatching blastocyst rate after culturing in the incubator for 3.5-4 days.
The results are shown in table 1, the cleavage rate of the embryo in the prokaryotic stage obtained by adding alpha-ketoglutaric acid for culture is not significantly different from that of the control group, but the blastocyst rate and the hatching blastocyst rate are both significantly improved compared with that of the control group. Wherein, the blastocyst rates of the prokaryotic embryos cultured by adding 100, 150, 200 and 300 mu M alpha-ketoglutaric acid are 62.64 +/-5.19%, 69.90 +/-2.51%, 64.56 +/-5.06% and 61.33 +/-8.46% respectively, which are all obviously higher than that of a control group (53.82 +/-4.58%) (p is less than 0.05); the hatching blastocyst rates of the prokaryotic embryos cultured by adding 100, 150, 200 and 300 mu M alpha-ketoglutaric acid are 27.60 +/-2.67%, 30.60 +/-1.56%, 32.22 +/-3.33% and 35.17 +/-1.42% respectively, which are obviously higher than 19.82 +/-3.14% of the control group.
Table 1: effect of alpha-KG addition on blastocyst Rate and blastocyst hatching Rate
| Group of | Hatching in number (pieces) | Cleavage Rate (%) | Percentage of blastocyst (%) | Hatching blastocyst Rate (%) |
| Mean±SEM | Mean±SEM | Mean±SEM | ||
| Control | 302 | 95.36±2.09 | 53.82±4.58a | 19.82±3.14a |
| 100μM | 302 | 95.36±1.52 | 62.64±5.19b | 27.60±2.67ab |
| 150μM | 285 | 95.80±1.31 | 69.90±2.51b | 30.60±1.56bc |
| 200μM | 256 | 96.33±1.30 | 64.56±5.06b | 32.22±3.33bc |
| 300μM | 181 | 95.33±1.48 | 61.33±8.46b | 35.17±1.42c |
Note: the table shows significant differences (P <0.05) when the different lowercase letters are placed on the shoulder, and no significant difference (P >0.05) when the same lowercase letters are placed on the shoulder.
The blastocyst rate is a key index for measuring the development efficiency of the embryo, and the results show that the addition of alpha-ketoglutaric acid with the final concentration of 100-300 mu M has a promoting effect on the in vitro development of the embryo, wherein the promoting effect is the best when 150 mu M is added, so that the subsequent experiments are carried out by selecting the addition amount of the alpha-ketoglutaric acid with the final concentration of 150 mu M.
The blastocysts were collected and transferred to 4% paraformaldehyde, fixed overnight at 4 ℃, punched with Triton, BSA blocked, incubated with primary antibody (Oct4 antibody), washed, incubated with secondary antibody, washed, DAPI stained, and photographed under fluorescent microscope. Oct4 fluorescence positive cells were labeled as inner cell mass and DAPI fluorescence labeled total number of embryonic cells. As a result, as shown in Table 2, the cell number of the Inner Cell Mass (ICM) and the ratio of the ICM to the total cell number of the embryo to which 150. mu.M of α -ketoglutarate was added were 25.50. + -. 3.46 and 38.25. + -. 5.85%, respectively, which were significantly higher than those of the control group, i.e., 16.87. + -. 1.87 and 27.61. + -. 2.96% (p < 0.05).
Table 2: influence on blastocyst cell number and inner cell mass number after addition of alpha-KG
Note: the table shows significant differences (P <0.05) when the different lowercase letters are placed on the shoulder, and no significant difference (P >0.05) when the same lowercase letters are placed on the shoulder.
ICM is the main source of stem cells, therefore, qRT-PCR method is used to detect the expression of genes related to embryo pluripotency in control group and experiment group added with alpha-KG. As shown in FIG. 1, the expression levels of the embryo pluripotency-associated genes Oct4, Sox2 and Nanog were not significantly different between the control group and the α -ketoglutarate group (p >0.05), but the expression level of the pluripotency-associated gene in the α -KG-treated group showed a significant upward trend (FIG. 1).
Apoptosis is one of the important factors affecting embryo quality, and therefore, the apoptosis rate is measured. TUNEL staining showed that the apoptosis rate of 150 μ M α -KG treated blastocysts was 6.26% ± 0.66%, similar to the control group (7.42% ± 1.02%), indicating that α -KG did not cause embryonic apoptosis (fig. 2).
Example 3: effect of alpha-ketoglutarate on pregnancy and farrowing efficiency of transferred embryos
To examine the quality of α -KG treated embryos, 150 μ M α -KG treated embryos of example 2 were transplanted into mice and analyzed for pregnancy and litter after embryo transplantation. Blastocysts cultured for 3.5 days in the experimental group to which α -KG (150 μ M) was added and the control group (to which α -KG was not added) were transplanted into recipient mice, the mice were normally bred, and the pregnancy and litter status of the mice were examined. The results show that the pregnancy rate, the labor rate and the birth weight of the alpha-KG (150 mu M) experimental group are respectively 62.5%, 25.0% and 1.46g, which are obviously higher than those of the control group (25.0%, 15.6% and 0.99g), the pregnancy rate of the alpha-KG (150 mu M) experimental group is improved by 150%, the birth rate is improved by 60% and the birth weight is improved by 47%.
Table 3: post-transplantation pregnancy and farrowing conditions of alpha-KG cultured embryos
| Group of | Pregnancy Rate (%) | Percentage of farrowing (%) | Newborn body weight (g) |
| Control | 25.0％(2/8)b | 15.6％(15/96)b | 0.99±0.05b |
| 150μM | 62.5％(5/8)a | 25.0％(24/96)a | 1.46±0.07a |
Note: the table is marked with different lowercase letters in the shoulder indicates significant difference (P <0.05), and marked with no or the same lowercase letters indicates no significant difference (P > 0.05).
Example 4: effect of alpha-ketoglutarate on mitochondrial function
To analyze the mitochondrial function of embryos, the most direct indicators reflecting mitochondrial function were detected separately: ATP, mitochondrial membrane potential and mitochondrial copy number. As shown in FIG. 3, the 150. mu.M alpha-KG treatment significantly decreased the ATP level of the embryos compared to the control group (0.12. + -. 0.02 for the experimental group, 0.29. + -. 0.03 for the control group; p <0.05) (a in FIG. 3), and significantly increased the mitochondrial membrane potential (3.93. + -. 0.60 for the experimental group, 1.34. + -. 0.20 for the control group; p <0.05) (c in FIG. 3). There was no significant difference in mitochondrial copy number between the experimental and control groups (487.7. + -. 137.3 for the experimental group, 531.9. + -. 116.2 for the control group; p >0.05) (FIG. 3 b).
Example 5: effect of alpha-ketoglutarate on early embryonic development epigenetics
alpha-KG and Fe (II) are cofactors that activate the Tet family of enzymes, which are important epigenetic modifying enzymes capable of converting 5-methylcytosine (5-mC) to 5-hydroxymethylcytosine (5-hmC). Thus, the 5hmc level and the expression level of the Tet gene of the blastocysts in vitro and in vivo were examined to analyze the effect of alpha-KG treatment on the level of methylation of the embryos. The 5-hmC ratio Mean Fluorescence Intensity (MFI) level (1814.21 + -338.63) of the in vitro-developing embryos of the experimental group with added alpha-KG (150. mu.M) was significantly higher than that of the control group without added alpha-KG (184.19 + -61.13; p <0.05), but significantly lower than that of the in vivo-developing embryos group (4250.78 + -738.64; p <0.05) (a and b of FIG. 4). The relative expression levels of the Tet1 and Tet2 genes of the α -KG-added experimental group embryos were slightly improved compared with the control group, but the expression levels of Tet1, Tet2 and Tet3 were not significantly different (c, d, e and f of fig. 4). The results indicate that alpha-KG treatment was able to reduce the DNA methylation level of embryos, but did not significantly alter Tet gene expression.
Example 6: effect of alpha-ketoglutarate on bovine in vitro fertilization embryo development
Obtaining bovine ovary from slaughterhouse, extracting follicular fluid, sorting out cumulus oocyte complex under stereoscope, adding bovine embryo in vitro maturation culture solution, and culturing at 38.5 deg.C with 5% CO in saturated humidity2Culturing in vitro maturation for 44h in incubator, removing cumulus cells with ovum suction needle, performing in vitro fertilization for 10h, washing supposedly fertilized oocyte with early stage embryo development liquid for 3 times, transferring to early stage embryo development liquid drop containing 0.15mM alpha-KG and pre-balancing for 2h, each drop contains 20-25 oocytes, and 5% CO at 38.5 deg.C and saturated humidity2After the culture box is developed for 48 hours, the culture box is transferred into the later-stage development liquid of the cattle embryo, the liquid is changed half and half every 48 hours, and the group without adding alpha-KG in the early-stage development liquid of the cattle embryo is taken as a control group. During the period, the cleavage rate and blastocyst development rate of the embryo (day 8 of embryo development) are counted, and the number of cells of the blastocyst is counted by DAPI fluorescent staining.
The formula of the bovine embryo in-vitro maturation culture solution is as follows: m199 culture medium was supplemented with 10% FBS (fetal bovine serum), 10. mu.g/mL FSH (follicle stimulating hormone), 1. mu.g/mL LH (luteinizing hormone), 1. mu.g/mL E2 (estradiol), 100. mu.g/M penicillin, 100. mu.g/mL streptomycin.
The formula of the cattle early embryo development liquid is as follows: CR1aa +3 mg/mLBSA. Wherein, the formula of CR1aa is as follows (100 mL): 0.67g NaCl, 0.023g KCl, 0.22g NaHCO30.002g sodium lactate, 100 μ L phenol red; the medicine is added into 90mL of ultrapure water for full dissolution, 0.055g of half calcium lactobionate is added, the pH is adjusted to 7.4, and the volume is determined to be 100 mL. On the day of use, 3mg of fatty acid-free BSA, 20. mu.L of essential amino acids (50 Xconcentrated), 10. mu.L of non-essential amino acids (100 Xconcentrated) and 10. mu.L of L-Glutamine (100 Xconcentrated) were added per 1mL of CR1aa, sterilized by filtration through a 0.22 μm filter and stored at 4 ℃.
The formula of the bovine embryo later-stage development liquid is as follows: CR1aa + 6% FBS; wherein the formula of CR1aa is as follows (100 mL): 0.67g NaCl, 0.023g KCl, 0.22g NaHCO30.002g of sodium lactate and 100. mu.L of phenol red.
The results show that similar to the effect of alpha-ketoglutaric acid on promoting the in vitro development of mouse embryos in example 2, the addition of alpha-ketoglutaric acid to the culture solution of bovine in vitro fertilized embryos can also significantly improve the blastocyst rate and hatching blastocyst rate of the development of bovine embryos, and the ratio of the number of inner cell masses to the number of blastocyst cells.
Although the above embodiments only provide examples of α -KG for in vitro embryo development of mice and cows, the process and mechanism of in vitro development of mammalian embryos are quite similar, and the mechanism of α -KG for promoting embryo in vitro development and assisted reproduction is also applicable to in vitro embryo culture and assisted reproduction of large mammals such as livestock.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the technical principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Claims (10)
1. The application of alpha-ketoglutaric acid in promoting the in vitro development of bovine embryos or improving the efficiency of bovine embryo transfer.
2. Use according to claim 1, characterized in that bovine embryos of prokaryotic stage are cultured in a culture medium containing α -ketoglutarate; or, alpha-ketoglutaric acid is added to the culture solution in the in vitro culture stage before bovine embryo implantation, preferably in the prokaryotic stage or between the prokaryotic stage and the blastocyst stage of bovine embryo development.
3. Use according to claim 2, wherein the concentration of α -ketoglutarate in the culture broth is 0.15 to 1mM, preferably 0.15 to 0.5mM, more preferably 0.15 to 0.2 mM.
4. A method for promoting the in vitro development of bovine embryos is characterized in that bovine embryos in a prokaryotic stage are cultured in a culture solution containing alpha-ketoglutaric acid; or, alpha-ketoglutaric acid is added to the culture solution in the in vitro culture stage before bovine embryo implantation, preferably in the prokaryotic stage or between the prokaryotic stage and the blastocyst stage of bovine embryo development.
5. The method according to claim 4, wherein the concentration of said α -ketoglutarate in said culture broth is 0.15 to 1mM, preferably 0.15 to 0.5mM, more preferably 0.15 to 0.2 mM.
6. Method according to claim 4 or 5, characterized in that it comprises the following steps: taking a cumulus oocyte complex of a cow, carrying out in-vitro maturation culture, then removing cumulus cells, and carrying out in-vitro fertilization; placing the in vitro fertilized oocyte in early-stage bovine embryo development liquid for culture, and then transferring the oocyte to late-stage bovine embryo development liquid for culture;
the early-stage bovine embryo development liquid comprises CR1aa culture liquid, 2-4 mg/mL bovine serum albumin and 0.15-1 mM, preferably 0.15-0.5 mM, more preferably 0.15-0.2 mM alpha-ketoglutaric acid; the bovine embryo late-stage development liquid comprises CR1aa culture liquid and 5-7% fetal bovine serum.
7. The method according to claim 6, wherein the oocytes after in vitro fertilization are placed in a bovine early embryo development solution for culturing for 45-50 h, and then transferred to a bovine late embryo development solution for culturing;
and/or placing the in vitro fertilized oocyte in the liquid drop of the early-stage bovine embryo development liquid, wherein each liquid drop contains 20-25 cells.
8. The method of claim 6, wherein the pH of the pre-bovine embryonic development solution is 7.4, and further comprising calcium hemi-lactobionate;
preferably, fatty acid-free BSA, essential amino acids, non-essential amino acids and L-glutamine are added to the bovine pre-embryonic development liquid on the day of use;
preferably, every 100mL of the CR1aa culture solution comprises the following components: 0.67g NaCl, 0.023g KCl，0.22g NaHCO30.002g of sodium lactate and 100. mu.L of phenol red.
9. An in vitro culture solution of bovine embryos, which is characterized by containing alpha-ketoglutaric acid, wherein the concentration of the alpha-ketoglutaric acid is preferably 0.15-1 mM, more preferably 0.15-0.5 mM, and even more preferably 0.15-0.2 mM.
10. The in vitro culture solution of claim 9, comprising CR1aa culture solution, 2-4 mg/mL bovine serum albumin, and 0.15-1 mM, preferably 0.15-0.5 mM, more preferably 0.15-0.2 mM of α -ketoglutarate;
preferably, the pH value of the in vitro culture solution is 7.4, and calcium hemilactobionate is also added;
preferably, on the day of use, fatty acid-free BSA, essential amino acids, non-essential amino acids and L-glutamine are added to the in vitro culture;
preferably, every 100mL of the CR1aa culture solution comprises the following components: 0.67g NaCl, 0.023g KCl, 0.22g NaHCO30.002g of sodium lactate and 100. mu.L of phenol red.
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