Application of alpha-lipoic acid in enhancing animal embryo in-vitro developmental capacity and method for enhancing animal embryo in-vitro developmental capacity

Abstract

The invention discloses application of alpha-lipoic acid in enhancing animal embryo in-vitro developmental capacity and a method for enhancing animal embryo in-vitro developmental capacity, and belongs to the technical field of sheep embryo in vitro. The application comprises application of alpha-lipoic acid or pharmaceutical salt thereof in enhancing animal in-vitro embryo developmental capacity. The method has the advantages that from the perspective of oxidative stress, the developmental capacity, blastocyst rate and blastocyst quality of the sheep in-vitro embryo can be effectively improved by adding 5 mu M and 10 mu M ALA in the sheep in-vitro embryo culture process.

Description

Technical Field

[0001] The invention relates to the technical field of sheep embryo in vitro, in particular to application of alpha-lipoic acid in enhancing the in vitro development ability of animal embryos and a method for enhancing the in vitro development ability of animal embryos. Background Art

[0002] In vitro embryo production (IVEP) is a comprehensive technology that combines in vitro maturation of oocytes (IVM), in vitro fertilization (IVF), and in vitro embryo culture (IVC). It simulates the environment of the oviduct and uterus of female animals, allowing gametes to be fertilized in vitro to form embryos and undergo certain growth and development in vitro. This technology has been applied on a large scale in production and is an important means of livestock breeding and genetic improvement.

[0003] After in vitro maturation and in vitro fertilization of sheep oocytes, the fertilized eggs continue to develop in vitro for 6 to 8 days into blastocysts. However, only 50 to 60% of the embryos of sheep fertilized eggs cultured in vitro can develop into blastocysts. Among the in vitro embryos that fail to develop into blastocysts, most of their development is arrested at the 8-16 cell stage, which is also the main period of embryonic genome activation. "8-16 cell developmental arrest" is a common event in in vitro cultured embryos of small ruminants, and the occurrence of this event can lead to embryonic death. Inappropriate in vitro embryo culture conditions may also lead to epigenetic changes, placental defects, loss of imprinting, and abnormal offspring. Therefore, the in vitro culture conditions of embryos are very important for in vitro embryo development.

[0004] Embryos often suffer from oxidative stress during in vitro development. The supplementation of antioxidants in embryo culture medium and the use of hypoxic incubators can improve the efficiency of sheep in vitro embryo development and make in vitro embryos have similar low temperature resistance as in vivo embryos. Alpha-lipoic acid (ALA) is synthesized from octanoic acid and sulfur source by lipoic acid synthase. It is a coenzyme in mitochondrial multienzyme complex reactions, responsible for removing excess reactive oxygen, reducing the oxidized form of other antioxidants such as glutathione, and regulating mitochondrial function, which helps produce ATP. Summary of the invention

[0005] The technical problem to be solved by the present invention is to provide a material and method for enhancing the developmental ability and quality of in vitro embryos. The technical problem to be solved is not limited to the described technical subject matter, and those skilled in the art can clearly understand other technical subjects not mentioned in this article through the following description.

[0006] In order to solve the above technical problems, the present invention provides the following technical solutions:

[0007] The invention provides an application of alpha-lipoic acid or a pharmaceutically acceptable salt thereof in enhancing the in vitro embryonic development ability of animals.

[0008] The present invention also provides an application of alpha-lipoic acid or a pharmaceutically acceptable salt thereof in preparing a product for enhancing the in vitro embryonic development ability of an animal.

[0009] In the above application, the enhancing the in vitro embryo development ability of the animal includes increasing the blastocyst rate of the in vitro embryo of the animal.

[0010] In the above application, the product for enhancing the in vitro embryonic development ability of animals may be the following in vitro embryo culture medium.

[0011] In the above application, the embryo in vitro culture medium contains α-lipoic acid or a pharmaceutically acceptable salt thereof.

[0012] The present invention also provides a method for enhancing the in vitro embryonic development ability of an animal, comprising the following steps: culturing a fertilized egg after in vitro fertilization with an in vitro embryonic culture medium, wherein the in vitro embryonic culture medium contains alpha-lipoic acid.

[0013] In the above method, the content of α-lipoic acid or its pharmaceutically acceptable salt in the embryo in vitro culture medium is 5 or 10 μM.

[0014] In the above method, the content of α-lipoic acid or its pharmaceutically acceptable salt in the embryo in vitro culture medium is 5 μM.

[0015] In the above method, the content of α-lipoic acid or its pharmaceutically acceptable salt in the embryo in vitro culture medium is 10 μM.

[0016] The method further comprises the following steps: ① collecting cumulus oocyte complexes (COCs) from fresh eggs of adult sheep by using egg suction fluid. ② transferring COCs into preheated equilibrium in vitro maturation (IVM) solution, and culturing for 24 to 26 hours at 38.5° C., 5% CO2, 95% air, and saturated humidity to obtain mature oocytes. ③ removing the fluffy cumulus cells from the outer layer of mature oocytes and transferring them into preheated equilibrium in vitro fertilization (IVF) solution to obtain IVF solution containing oocytes, adding sperm to the IVF solution containing oocytes, and culturing for 12 to 16 hours at 38.5° C., 5% CO2, 95% air, and saturated humidity to obtain eggs after in vitro fertilization, i.e., fertilized eggs. ④ remove the sperm and cumulus cells attached to the surface of the fertilized egg, transfer it into a preheated and balanced embryo in vitro culture (IVC) solution to obtain a four-well plate with embryos, and then culture the four-well plate in an incubator at 38.5°C, a mixed gas environment of 88% N2, 5% CO2 and 7% O2.

[0017] The composition of each 1L of the IVM solution is as follows: TCM199 9.5g, NaHCO3 2.1g, glutamine 0.1g, sodium pyruvate 0.03g, 10μg / mL FSH, 10μg / mL LH, 1μg / mL E2, 10% fetal bovine serum, 1% penicillin-streptomycin, and the rest is ultrapure water.

[0018] The IVF solution is composed of 0.6542 g NaCl, 0.0555 g KCl, 0.0168 g KH2PO4, 0.0189 g MgSO4·7H2O, 0.0036 g sodium pyruvate, 0.0374 g CaCl2·2H2O, 0.0128 mL sodium lactate, 2% estrus sheep serum and 1% penicillin-streptomycin, and the rest is ultrapure water.

[0019] The composition of each 1L of the embryo in vitro culture medium (IVC medium) is as follows: NaCl 6.542g, KCl 0.555g, KH2PO40.168g, MgSO4·7H2O 0.189g, sodium pyruvate 0.036g, CaCl2·2H2O 0.374g, 60% sodium lactate 0.128mL, 2% essential amino acids, 1% non-essential amino acids, 8mg / mL BSA, 1% penicillin-streptomycin, 5 or 10μM α-lipoic acid (ALA), and the rest is ultrapure water.

[0020] The present invention also provides an in vitro embryo culture medium, which is composed of an in vitro embryo culture solution and a substance A, wherein the substance A is alpha-lipoic acid or a pharmaceutically acceptable salt thereof.

[0021] In the above in vitro embryo culture medium, the content of α-lipoic acid or its pharmaceutically acceptable salt in the embryo in vitro culture medium is 5 or 10 μM.

[0022] The in vitro embryo may be a mammalian in vitro embryo.

[0023] In one embodiment of the present invention, the embryo in vitro culture medium is composed of: NaCl 6.542g, KCl0.555g, KH2PO4 0.168g, MgSO4·7H2O 0.189g, sodium pyruvate 0.036g, CaCl2·2H2O 0.374g, 60% sodium lactate 0.128mL, 2% essential amino acids, 1% non-essential amino acids, 8mg / mL BSA, 1% penicillin-streptomycin, 5 or 10μM α-lipoic acid (ALA), and the rest is ultrapure water.

[0024] The advantage of the present invention is that, from the perspective of oxidative stress, by adding 5 μM and 10 μM ALA during the sheep in vitro embryo culture process, the development ability, blastocyst rate and blastocyst quality of the sheep in vitro embryo can be effectively improved. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 The effect of adding different concentrations of ALA to IVC medium on blastocyst development. The figure shows the development of sheep in vitro embryos on the 7th day of IVC in culture medium with ALA concentrations of 0 (control), 2.5, 5, and 10 μM. Scale bar: 100 μm.

[0026] Figure 2 The effect of adding different concentrations of ALA to IVC fluid on the number of blastocyst cells. (A) Representative images of DAPI-stained blastocyst nuclei in the control group and the groups treated with different concentrations of ALA, scale: 100μm; (B) Statistical results of the number of sheep blastocyst cells in vitro in the four groups of ALA concentrations; ns indicates no significant difference, P>0.05, where the vertical axis Total Cell Number represents the total number of cells in the blastocyst.

[0027] Figure 3 Effects of adding different concentrations of ALA to IVC fluid on apoptosis of blastocysts. (A) Representative images of TUNEL staining of blastocysts in the control group and groups treated with different concentrations of ALA. DAPI staining (blue fluorescence) locates the nucleus, and green fluorescence is the apoptosis signal. Scale bar: 100μm; (B) Relative quantitative analysis of apoptosis signals; the results are expressed as Mean+SEM, ns indicates no significant difference, P>0.05, where the vertical axis Fluorescence Ratio of TUNEL represents the fluorescence ratio; (C) RT-qPCR detection of the expression levels of apoptosis genes CASPASE3, p53 and BAX / BCL-2 in sheep blastocysts in the control group and ALA treatment group. GAPDH was used as an internal reference, and the results were expressed as Mean+SEM; ns indicates no significant difference, P>0.05; * indicates significant difference, P<0.05; n=3, where the vertical axis Expression Level represents the expression level.

[0028] Figure 4 Comparison of proliferation and antioxidant gene expression levels in blastocysts between the control group and the ALA-treated group. RT-qPCR was used to detect the expression levels of (A) proliferation genes (PCNA, CYCLIND1, CDK2, and C-MYC) and antioxidant genes (SOD1, SOD2, CAT, and GPX2) in sheep blastocysts in the control group and the ALA-treated group. GAPDH was used as an internal reference, and the results were expressed as Mean+SEM; ns indicates no significant difference, P>0.05; * indicates significant difference, P<0.05; n=3. In Figures A and B, the ordinate ExpressionLevel represents the expression level. DETAILED DESCRIPTION

[0029] The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way.

[0030] The experimental methods in the following examples, unless otherwise specified, are all conventional methods, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial channels.

[0031] The following examples used SPSS Statistics26 statistical software to process the data, and the experimental results were expressed as mean ± standard deviation. The t-test was used for the test. P < 0.05 (*) indicated a significant difference, P < 0.01 (**) indicated a very significant difference, and P < 0.001 (***) indicated a very significant difference.

[0032] The commonly used sheep in vitro embryo culture medium is IVC liquid. The composition of each 1L embryo in vitro culture medium (IVC liquid) is as follows: NaCl 6.542g, KCl 0.555g, KH2PO4 0.168g, MgSO4·7H2O 0.189g, sodium pyruvate 0.036g, CaCl2·2H2O 0.374g, sodium lactate 0.128mL, 2% essential amino acids (Sigma, B6766), 1% non-essential amino acids (Sigma, M7145), 8mg / mL BSA and 1% penicillin-streptomycin (purchased from Gibco, item number 15140122), and the rest is ultrapure water.

[0033] The sheep in vitro embryo culture medium used in the present application is a culture medium for enhancing the in vitro embryonic development ability of animals, which is a liquid culture medium obtained by adding α-lipoic acid (ALA) (Sigma, T1395) to the aforementioned IV C solution. The content of ALA in the culture medium for enhancing the in vitro embryonic development ability of animals is 5 μM.

[0034] Alpha-lipoic acid (ALA) is an organic sulfur component produced by plants and animals. It is synthesized from octanoic acid and a sulfur source through the action of lipoic acid synthetase. It is an important cofactor in the mitochondrial multienzyme reaction complex, responsible for scavenging ROS, reducing other antioxidants such as oxidized forms of glutathione, and regulating mitochondrial function. ALA is often used to treat neurodegenerative diseases by inhibiting the formation of reactive oxygen species and neuronal damage and regulating protein levels. The CAS number of alpha-lipoic acid in the following examples is 1077-28-7, and it is a product of Sigma with a product number of T1395. Reference website: https: / / www.chemicalbook.com / ProductChemicalPropertiesCB5180699.htm.

[0035] Example 1

[0036] 1. Oocyte Collection

[0037] Egg suction solution: It is composed of 0.1-1% penicillin-streptomycin, 10 IU heparin and phosphate buffered saline (PBS).

[0038] Egg aspiration fluid: The egg aspiration fluid contains Medium 199 9.5g / L (purchased from Gibco, product number 31100035), NaHCO3 0.4g / L, HEPES 10.412g / L, heparin sodium 50mg / L, 50mL / L fetal bovine serum (purchased from Hyclone, product number SH3008) and 10mL / L penicillin-streptomycin (purchased from Gibco, product number 15140122).

[0039] Fetal bovine serum was purchased from Hyclone Company, catalog number SH30088.

[0040] Penicillin-streptomycin was purchased from Gibco, catalog number 15140122.

[0041] Fresh ovaries of freshly slaughtered adult sheep were collected from the slaughterhouse, stored in 37°C saline, and transported back to the laboratory within 2 hours. The ovaries were washed 3 times with 75% alcohol, and then washed 3 times with 37°C saline, and the washed ovaries were soaked in 37°C saline. 2mL of egg suction fluid was drawn with a 10mL syringe, and then the contents of the follicles on the surface of the ovaries were sucked. The contents were poured into a 90mm diameter culture dish, and the cumulus-oocyte complexes (COCs) were picked out with an oviduct under a stereoscope with a constant temperature stage.

[0042] 2. Oocyte maturation in vitro

[0043] Egg washing solution: The egg washing solution contains Medium 199 9.5 g / L (purchased from Gibco, product number 31100035), NaHCO3 0.4 g / L, HEPES 10.412 g / L, 50 mL / L fetal bovine serum and 10 mL / L penicillin-streptomycin.

[0044] The oocyte in vitro maturation medium (IVM medium) contains Medium 199 9.5 g / L (Gibco, 31100035), NaHCO3 2.1 g / L, glutamine 0.1 g / L, sodium pyruvate 0.03 g / L, 0.15 IU FSH (follicle stimulating hormone, CAS number: 9002-68-0), 0.15 IU (luteinizing hormone, CAS number: 39341-83-8), 1 μg / mL E2 (estradiol), 100 mL / L fetal bovine serum (purchased from Hyclone, product number SH3008) and 10 mL / L penicillin-streptomycin (purchased from Gibco, product number 15140122).

[0045] The COCs were washed twice in the egg washing solution, and then washed twice with the in vitro maturation (IVM) culture medium (hereinafter referred to as IVM solution), and finally transferred to the pre-equilibrated IVM solution (600 μL IVM solution in a four-well plate was covered with 300 μL paraffin oil), and 30 to 50 COCs were placed in each well. The culture conditions for in vitro oocyte maturation were 38.5°C, 5% CO2, 95% air, saturated humidity, and the maturation time was 24 to 26 hours to obtain mature oocytes.

[0046] 3. In vitro fertilization of oocytes

[0047] After taking the sperm from the liquid nitrogen, place it in a 39℃ water bath for about 10 seconds, and take it out and place it in a clean bench when it thaws and changes color. Use 75% alcohol to clean the outer wall of the sperm and wipe it dry.

[0048] The composition of each 100 mL of in vitro fertilization fluid (IVF fluid) is as follows: NaCl 0.6542 g, KCl 0.0555 g, KH2PO4 0.0168 g, MgSO4·7H2O 0.0189 g, sodium pyruvate 0.0036 g, CaCl2·2H2O 0.0374 g, sodium lactate 0.0128 mL, 2% estrus sheep serum and 1% penicillin-streptomycin, and the rest is ultrapure water.

[0049] Estrus sheep serum: Collect blood from the vein of estrus ewes, let it stand at 4℃ overnight, centrifuge at 3000rmp for 30min, collect serum, and freeze it for future use.

[0050] The specific steps of in vitro fertilization are as follows:

[0051] (1) Aspirate the thawed semen and slowly add it to the bottom of a glass test tube containing 600 μL IVF solution. Transfer the test tube steadily into the incubator to avoid shaking. Incubate the sperm upstream in the incubator for 25 minutes and take about 300 μL of the supernatant, which is the capacitated semen.

[0052] (2) The mature oocytes were picked out, digested with 0.2% hyaluronidase and the outer fluffy cumulus cells were removed, leaving only 2-3 layers. The oocytes were washed 3 times with pre-equilibrated IVF solution and transferred to pre-heated and equilibrated IVF solution to obtain IVF solution containing oocytes.

[0053] (3) Add about 100 μL of capacitated semen to the IVF solution containing oocytes, record fertilization time as 0 hours, and culture at 38.5°C, 5% CO2, 95% air, and saturated humidity for 12 to 16 hours to obtain eggs after in vitro fertilization, i.e., fertilized eggs.

[0054] 4. Embryo in vitro culture

[0055] The fertilized eggs were picked out, and the sperm and cumulus cells attached to the surface of the eggs were gently blown and suctioned to remove them. After being washed three times with embryo in vitro culture medium (IVC solution), they were finally transferred to the preheated and balanced embryo in vitro culture medium (IVC solution) (cultured in four-well plates, 400 μL IVC solution in each well was covered with 300 μL wax oil), and a four-well plate with embryos was obtained. The plates were cultured at 38.5°C in a mixed gas environment of 88% N2, 5% CO2 and 7% O2.

[0056] The cleavage rate was calculated at 24h and 48h after IVF, and the blastocyst rate was observed and calculated on days 7 to 9.

[0057] 5. Detection of blastocyst apoptosis level

[0058] The fluorescence intensity of TUNEL staining and the relative expression of apoptosis-related genes CASPASE3, p53 and BAX / BCL-2 were detected to determine the level of blastocyst apoptosis in the control group and the ALA-treated group.

[0059] TUNEL staining: After the blastocyst sample fixed with 4% paraformaldehyde was washed once with PBS, a PBS solution containing 0.5% Triton X-100 was added thereto and incubated at room temperature for 5 minutes to obtain the infiltrated blastocyst. Subsequently, the infiltrated blastocyst was washed twice with PBS solution, 30 μL of TUNEL detection solution was transferred, and incubated at 37°C in the dark for 60 minutes to obtain the TUNEL-stained blastocyst. After the TUNEL-stained blastocyst was washed three times with PBS solution, it was incubated with 5 μg / mL DAPI for 30 seconds, and then observed and photographed using an inverted fluorescence microscope at an excitation wavelength of 488 nm. The fluorescence intensity of the fluorescence image was analyzed using image J software, and statistical analysis was performed using SPSS.

[0060] Detection of relative gene expression: Real-time fluorescence quantitative PCR was used to detect the relative expression of apoptosis-related genes CASPASE3, p53 and BAX / BCL-2 in the blastocysts of the control group and the 5μM ALA-treated group. The blastocysts obtained by in vitro culture were washed with PBS and collected in cell cryopreservation tubes containing 10μL RNase-free H2O and 0.3μL RNase inhibitors. 2 to 3 blastocysts were added to each tube and quickly transferred to dry ice for storage. The Single Cell Sequence Specific Amplification Kit (Nanjing Novogene Biotechnology Co., Ltd.) was used for trace cell transcriptome amplification, and then the TransStart Tip Green qPCR Super Mix (+Dye II) (Beijing Quanshijin Biotechnology Co., Ltd.) kit was used for fluorescence quantitative PCR detection. The results were based on GAPDH as a reference and 2 -ΔΔCT The relative expression of genes was analyzed by SPSS.

[0061] 6. Detection of proliferation gene and antioxidant gene expression in blastocysts

[0062] Real-time fluorescence quantitative PCR was used to detect the relative expression of proliferation genes (PCNA, CYCLIND1, CDK2 and C-MYC2) and antioxidant genes (SOD1, SOD2, CAT and GPX2) in blastocysts of the control group and the 5 μM ALA-treated group.

[0063] Detection of relative gene expression: The blastocysts obtained by in vitro culture were washed with PBS and collected in cell cryopreservation tubes containing 10μL RNase-free H2O and 0.3μL RNase inhibitor. 2 to 3 blastocysts were added to each tube and quickly transferred to dry ice for storage. The Single Cell Sequence Specific Amplification Kit (Nanjing Novogene Biotech Co., Ltd.) was used for trace cell transcriptome amplification, and then the TransStart Tip Green qPCR Super Mix (+Dye II) (Beijing Quanshijin Biotech Co., Ltd.) kit was used for fluorescence quantitative PCR detection. The results were compared with GAPDH as a reference and 2 -ΔΔCT The relative expression of genes was analyzed by SPSS.

[0064] 7. Results

[0065] The effects of adding different concentrations of ALA to IVC on the in vitro development of blastocysts are shown in Figure 1 The cleavage rate and blastocyst rate are shown in Table 1. Compared with the control group, the in vitro culture medium supplemented with 5μM and 10μM ALA significantly increased the blastocyst rate (P<0.05), and there was no significant difference between the two. The in vitro culture medium supplemented with 2.5μM ALA had no significant effect on the blastocyst rate of in vitro embryos.

[0066] The effect of adding different concentrations of ALA to IVC on the number of blastocyst cells is shown in Figure 2 The blastocysts obtained from IVC fluids with different concentrations of ALA were stained with DAPI, with 15 to 20 blastocysts in each group. The cell nuclei were counted and the number of blastocyst cells was statistically analyzed. Figure 2 As shown, the number of blastocyst cells in the 2.5μM and 5μM groups was slightly higher than that in the control group and the 10μM group, but the difference was not significant (P>0.05).

[0067] The effect of adding different concentrations of ALA to IVC solution on apoptosis of blastocysts is shown in Figure 3 The blastocysts obtained from IVC fluid with different concentrations of ALA were stained with TUNEL, with 15 to 20 blastocysts in each group. The fluorescence signals were detected and statistically analyzed. Figure 3 As shown in the figure, the TUNEL fluorescence intensity in the 2.5μM, 5μM and 10μM ALA treatment groups was lower than that in the control group, with no statistical difference (P>0.05). In the blastocysts of the 5μM ALA treatment group, the relative expression ratio of BAX / BCL2 genes was significantly lower than that of the control group (P<0.05).

[0068] The effects of adding 5 μM ALA to IVC medium on cell proliferation and antioxidant gene expression in blastocysts are shown in Figure 4 The relative expression of proliferation genes (PCNA, CYCLIND1, CDK2, and C-MYC2) and antioxidant genes (SOD1, SOD2, CAT, and GPX2) in the blastocysts treated with 5 μM ALA was detected by real-time fluorescence quantitative PCR, and statistical analysis was performed. Figure 4 As shown, the expression levels of PCNA and CYCLIND1 in the sheep in vitro blastocysts in the ALA treatment group were not significantly different from those in the control group (P>0.05), but the expression levels of CDK2 and c-MYC were significantly increased compared with the control group (P<0.05). The expression levels of SOD1 and CAT in the sheep in vitro blastocysts in the ALA treatment group were not significantly different from those in the control group (P>0.05), but the expression levels of SOD2 and GPX2 were significantly increased compared with the control group (P<0.05).

[0069] Table 1 Effect of adding ALA to in vitro culture medium on the blastocyst rate of sheep in vitro embryos

[0070]

[0071] Cleavage rate = number of cleavage embryos / number of oocytes, blastocyst rate = number of blastocysts / number of cleavages, and the experiment was repeated four times.

[0072] Different superscript letters (a, b) in the same column indicate significant differences (P<0.05), and the same superscript letters in the same column indicate insignificant differences (P≥0.05).

[0073] It can be seen that the above method can significantly improve the blastocyst development ability of sheep in vitro embryos, reduce the apoptosis level of blastocyst cells, and promote cell proliferation and the expression of antioxidant genes.

[0074] The present invention has been described in detail above. For those skilled in the art, without departing from the purpose and scope of the present invention, and without the need to carry out unnecessary experimental conditions, the present invention can be implemented in a wide range under equivalent parameters, concentrations and conditions. Although the present invention provides specific embodiments, it should be understood that the present invention can be further improved. In a word, according to the principles of the present invention, the application is intended to include any changes, uses or improvements to the present invention, including departure from the disclosed scope in the application, and changes made with conventional techniques known in the art.

Claims

1. Application of α-lipoic acid or its pharmaceutically acceptable salt in enhancing the in vitro embryonic development ability of animals.

2. Application of α-lipoic acid or its pharmaceutically acceptable salt in the preparation of products for enhancing the in vitro embryonic development ability of animals.

3. The use according to claim 1 or 2, characterized in that: The enhancing the in vitro embryo development ability of the animal includes increasing the blastocyst rate of the in vitro embryo of the animal.

4. A method for enhancing the in vitro embryonic development ability of an animal, characterized in that: The method comprises the following steps: culturing the fertilized egg after in vitro fertilization with an embryo in vitro culture medium, wherein the embryo in vitro culture medium contains alpha-lipoic acid.

5. The method according to claim 4, characterized in that The content of the α-lipoic acid or its pharmaceutically acceptable salt in the embryo in vitro culture medium is 5 or 10 μM.

6. The method according to claim 4, characterized in that The content of the α-lipoic acid or its pharmaceutically acceptable salt in the embryo in vitro culture medium is 5 μM.

7. The method according to claim 4, characterized in that The content of the α-lipoic acid or its pharmaceutically acceptable salt in the embryo in vitro culture medium is 10 μM.

8. An in vitro embryo culture medium, comprising an in vitro embryo culture medium and a substance A, wherein the substance A is α-lipoic acid or a pharmaceutically acceptable salt thereof.

9. The in vitro embryo culture medium according to claim 8, characterized in that The content of the α-lipoic acid or its pharmaceutically acceptable salt in the embryo in vitro culture medium is 5 or 10 μM.

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