Application of creatine in improving in-vitro blastocyst development rate of sheep oocyte

Abstract

The invention discloses an application of creatine in improving the development rate of an in-vitro blastocyst of a sheep oocyte. The concentration of the creatine is 0.5 mmol / L. The creatine is added into an in-vitro maturation system and an in-vitro embryo culture system. The key metabolite creatine is analyzed and screened based on metabonomics of sheep oocytes, early embryos, follicular fluid and fallopian tube fluid. On the basis, the development regulation effect of the creatine on the oocytes and early embryos is researched, the in-vitro blastocyst development rate of the sheep oocytes can be remarkably increased by adding the creatine into an in-vitro maturation system and an in-vitro embryo culture system of the sheep oocytes, and it is proved that the creatine can serve as an additive of the in-vitro culture system; and the creatine is a key metabolite contained in the development microenvironment and is friendly to oocyte and embryo development.

Description

Technical Field

[0001] The invention relates to a method for improving the in vitro blastocyst development rate of sheep oocytes, and particularly to application of creatine in improving the in vitro blastocyst development rate of sheep oocytes. Background Art

[0002] Although adult sheep have tens of thousands of primordial follicles in their ovaries, only a very small number of oocytes can be fertilized to form embryos during the entire natural breeding process. This indicates that a large number of oocytes have not been fully exploited and utilized, making it difficult to bring into play the germplasm characteristics of excellent ewes. If these oocytes can be used for efficient in vitro embryo production, it will greatly promote the preservation and development and utilization of sheep germplasm resources. However, the in vitro development potential of sheep oocytes is relatively low, and the blastocyst rate is difficult to exceed 50%. Therefore, improving the development potential of sheep oocytes is of great significance for giving full play to the genetic characteristics of excellent ewes and promoting the efficient development of the sheep industry.

[0003] In vitro maturation of oocytes refers to collecting immature cumulus-oocyte complexes from the antral follicles of the ovaries and obtaining mature oocytes through in vitro culture. In vitro embryo production is a core component of modern reproductive technology. In recent years, combined with live egg collection, in vitro embryo production has been rapidly promoted worldwide and has become an important means of genetic improvement and breeding of livestock. However, due to the imperfection of the in vitro culture system, the quality of in vitro produced embryos is much lower than that of in vivo produced embryos.

[0004] The document "Effect of Proanthocyanidin B2 on In Vitro Maturation and Embryonic Development of Sheep Oocytes of Different Ages" (Li Houru, Master's Thesis, Shihezi University, 2023) discloses that by adding different concentrations (0, 2.5, 5, 7.5 μg / mL) of PCB2 (proanthocyanidin B2) to IVC medium (in vitro embryo culture medium), its effects on cleavage rate and blastocyst rate were explored. The in vitro maturation solution used is composed of: 100 mL basic mother solution, 47 mg HEPES (salt), 4 mg sodium pyruvate (salt), 18 mL basic mother solution, 2 mL estrus sheep serum, 20 μL FSH (100,000 IU), 20 μL LH (luteinizing hormone, 100,000 IU), 20 μL E2 (estradiol, 0.0001 g); basic mother solution components: 970 mg M199, 220 mg NaHCO3, 10 mg gentamicin, 100 mL Mill-QH2O, filtered with a 0.22 μm filter. The in vitro culture solution used is composed of: M199 + 25 mmol / L NaHCO3 + 5 mmol / L HEPES + sodium lactate + glutamine (Gln) + sodium pyruvate + essential amino acids + non-essential amino acids. The results showed that the cleavage rate of the 2.5, 5, and 7.5 μg / mL PCB2-added groups (86.32±0.73%, 85.64±1.31%, 87.87±0.82%, P<0.05) was significantly higher than that of the control group (81.52±0.97%), and the blastocyst rate of the 7.5 μg / mL PCB2-added group was significantly higher than that of the control group (64.58±4.16% vs. 46.56±5.19, P<0.05), indicating that the addition of PCB2 can improve the subsequent embryonic development ability. Although the parthenogenetic blastocyst rate was significantly improved (46.56% vs. 64.58%, P<0.05), the nuclear maturation rate of the control group and the treatment group (46.68 vs. 51.51%) was relatively low, and the improvement rate was less than 5%, indicating that the efficiency of proanthocyanidins in improving nuclear maturation under this condition was not high. Moreover, from the perspective of the overall development efficiency of oocytes, the use of proanthocyanidins to improve the development efficiency of sheep oocytes is low. In addition, although its strong antioxidant properties reduce excessive reactive oxygen species (ROS) in cells and improve the parthenogenetic blastocyst development rate of sheep oocytes, it is difficult to guarantee that it has no negative effects on the embryo.

[0005] Chinese patent CN101892194A, the application document discloses a method for improving the in vitro utilization efficiency of sheep oocytes. In order to improve the quality of sheep oocytes, BCB staining is first used for screening, and then the screened oocytes are inhibited and cultured with a PDE3 inhibitor (milrinone). Adding milrinone can effectively inhibit PDE3 activity, maintain a high concentration of intracellular cAMP, thereby blocking the spontaneous meiosis of oocytes, providing time for nuclear and cytoplasmic synchronous maturation, and after resuming mature culture, the in vitro culture blastocyst development rate can be significantly improved. This practice of determining cell activity by using bright cresyl blue staining is risky. Although it can analyze cell activity and screen oocytes of better quality for in vitro embryo production, it still needs to be verified whether it has a negative impact on the development of the embryo. In addition, although milrinone, as an inhibitor, can effectively improve the quality of oocyte maturation, it still needs to be tested whether it has a negative effect.

[0006] There are two constraints in establishing an efficient embryo culture system. First, there is a lack of comprehensive understanding of metabolism and metabolic gene expression during oocyte maturation and embryonic development. Second, traditional metabolic analysis methods require a large number of cell samples and are not applicable to livestock oocytes and early embryos. Therefore, using micro-metabolomics technology to identify key metabolites in oocyte maturation and embryonic development and to improve in vitro culture systems is an urgent need in the field of livestock reproduction. Summary of the invention

[0007] The purpose of the present invention is to provide an application of creatine in improving the in vitro blastocyst development rate of sheep oocytes. Creatine is added to a sheep oocyte in vitro maturation system and an in vitro embryo culture system for the first time. Creatine can significantly improve the in vitro blastocyst development rate of sheep oocytes.

[0008] In order to achieve the above object, the present invention provides the use of creatine in improving the blastocyst development rate of sheep oocytes in vitro, wherein the concentration of the creatine is 0.5 mmol / L.

[0009] Preferably, the creatine is added to an in vitro maturation system and an in vitro embryo culture system.

[0010] Preferably, the creatine is added to an in vitro maturation solution, and the in vitro maturation solution used comprises: M199, 0.5 IU / mL follicle stimulating hormone, 0.5 IU / mL luteinizing hormone, 1 μg / mL estradiol, 50 μg / mL gentamicin, 4 mmol / L sodium pyruvate and 50 μmol / L L-cystine.

[0011] Preferably, the creatine is added to an in vitro embryo culture medium, and the in vitro embryo culture medium used comprises: SOF, 2% essential amino acids, 1% non-essential amino acids, 0.48 mg / mL inositol, 1 mmol / L L-glutamine and 6 mg / mL BSA; wherein the SOF comprises: 6.29 mg / mL NaCl, 0.534 mg / mL KCl, 0.162 mg / mL KH2PO4, 0.6 μL / mL sodium lactate, 0.089 mg / mL MgSO4, 2.1 mg / mL NaHCO3, 0.0357 mg / mL sodium pyruvate and 0.299 mg / mL CaCl2·2H2O.

[0012] Preferably, the application comprises: after the cumulus-oocyte complex is matured in an in vitro maturation solution containing 0.5 mmol / L creatine for 22 to 24 hours, the cumulus cells are removed and the oocytes are retained for standby use; the mature oocytes are transferred to parthenogenetic activation solution A and incubated for 5 minutes, and then washed several times with in vitro embryo culture solution, and then transferred to parthenogenetic activation solution B and incubated in an incubator for 2 to 3 hours; finally, the oocytes are taken out, washed several times with in vitro embryo culture solution, and transferred to in vitro embryo culture solution for culture, wherein the parthenogenetic activation solution A is a calcium-magnesium DPBS buffer containing calcium ionomycin; and the parthenogenetic activation solution B is an in vitro embryo culture solution containing 6-DMAP.

[0013] More preferably, the parthenogenetic activation solution A comprises: DPBS buffer containing calcium and magnesium, 0.5 μmol / L calcium ionomycin, and 3 mg / mL bovine serum albumin; the parthenogenetic activation solution B comprises: in vitro embryo culture medium and 2 mmol / L 6-DMAP.

[0014] More preferably, 0.5 mmol / L of creatine is added to the in vitro maturation solution; then the cumulus-oocyte complex is transferred into the in vitro maturation solution and cultured in a CO2 incubator at 5% CO2, 95% air and a temperature of 38.5°C for 22 to 24 hours; then, the cumulus-oocyte complex is transferred into an egg-picking solution containing 0.1% hyaluronidase, the cumulus cells are removed, and the oocytes are retained for later use.

[0015] Preferably, the oocyte is transferred to parthenogenetic activation solution A and incubated in a CO2 incubator with 5% CO2, 95% air and a temperature of 38.5°C for 5 minutes, then washed several times with in vitro embryo culture solution, and then transferred to parthenogenetic activation solution B and incubated in a CO2 incubator with 5% CO2, 95% air and a temperature of 38.5°C for 2 to 3 hours; finally, the oocyte is taken out, washed several times with in vitro embryo culture solution, and transferred to in vitro embryo culture solution for culture.

[0016] The second object of the present invention is to provide an in vitro maturation solution for improving the in vitro blastocyst development rate of sheep oocytes, the in vitro maturation solution comprising: M199, 0.5 IU / mL follicle stimulating hormone, 0.5 IU / mL luteinizing hormone, 1 μg / mL estradiol, 50 μg / mL gentamicin, 4 mmol / L sodium pyruvate and 50 μmol / L L-cystine, creatine is added to the in vitro maturation solution, and the concentration of creatine in the in vitro maturation solution is 0.5 mmol / L.

[0017] The third object of the present invention is to provide an in vitro embryo culture medium for improving the in vitro blastocyst development rate of sheep oocytes, characterized in that the in vitro embryo culture medium comprises: SOF, 2% essential amino acids, 1% non-essential amino acids, 0.48 mg / mL inositol, 1 mmol / L L-glutamine and 6 mg / mL BSA; wherein the SOF comprises: 6.29 mg / mL NaCl, 0.534 mg / mL KCl, 0.162 mg / mL KH2PO4, 0.6 μL / mL sodium lactate, 0.089 mg / mL MgSO4, 2.1 mg / mL NaHCO3, 0.0357 mg / mL sodium pyruvate and 0.299 mg / mL CaCl2·2H2O; creatine is added to the in vitro embryo culture medium, and the concentration of creatine in the in vitro embryo culture medium is 0.5 mmol / L.

[0018] The application of creatine in improving the blastocyst development rate of sheep oocytes in vitro has the following advantages:

[0019] The present invention is based on the key metabolite creatine screened out by metabolomics analysis of sheep oocytes, early embryos, follicular fluid and fallopian tube fluid. On this basis, the present invention studies the developmental regulatory effect of creatine on oocytes and early embryos. Adding creatine to the sheep oocyte in vitro maturation system and in vitro embryo culture system can significantly improve the in vitro blastocyst development rate of sheep oocytes, which proves that creatine can be used as an additive to the in vitro culture system to promote the production of sheep in vitro embryos, and creatine is a key metabolite contained in the developmental microenvironment and is friendly to the development of oocytes and embryos. DETAILED DESCRIPTION

[0020] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0021] Note: The experimental methods in the following experimental examples, unless otherwise specified, are all conventional methods, and are performed according to the techniques or conditions described in the literature in this field or according to the product instructions. The materials, reagents, etc. used in the following experimental examples, unless otherwise specified, can all be obtained from commercial channels.

[0022] In the present invention, all features defined in the form of numerical ranges or percentage ranges, such as values, quantities, contents and concentrations, are only for brevity and convenience. Accordingly, the description of numerical ranges or percentage ranges should be considered to have included and specifically disclosed all possible secondary ranges and individual values ​​within the range (including integers and fractions).

[0023] The features mentioned in the present invention can be combined arbitrarily, as long as there is no contradiction in the combination of these features, all possible combinations should be considered as the scope of this specification. Each feature disclosed in the specification can be replaced by any alternative feature that can provide the same, equal or similar purpose. Therefore, unless otherwise specified, the disclosed features are only general examples of equal or similar features.

[0024] The present invention aims at the problem of low in vitro development potential of sheep oocytes. Based on metabolomics and cell culture technology, the key metabolic regulator creatine in the process of oocyte maturation and embryonic development is excavated, and the effect of creatine added to the in vitro culture system on sheep oocytes is studied. It is found that creatine can promote the in vitro blastocyst development rate of sheep oocytes, and creatine is a key metabolite contained in the developmental microenvironment, which is friendly to oocyte and embryonic development. The present invention adds creatine to the oocyte in vitro culture system, which improves the efficiency of in vitro embryo production of sheep oocytes.

[0025] The application of creatine provided by the present invention in improving the in vitro blastocyst development rate of sheep oocytes is described in detail below through experimental examples.

[0026] The reagents used in the experiments are as follows:

[0027] (1) Egg collection solution: M199 (containing HEPES, purchased from Gibco, product number 31100035) + 0.42 g / L NaHCO3 + 0.01 g / L heparin sodium + 0.05 g / L penicillin + 0.065 g / L streptomycin + 1% FBS (fetal bovine serum);

[0028] (2) In vitro maturation solution: M199 + 0.5 IU / mL FSH (follicle stimulating hormone) + 0.5 IU / mL LH (luteinizing hormone) + 1 μg / mL E2 (estradiol) + 50 μg / mL gentamicin + 4 mmol / L sodium pyruvate + 50 μmol / L L-cystine;

[0029] (3) Parthenogenetic activation solution: Solution A: DPBS (Dublin's phosphate buffer containing calcium and magnesium, purchased from Gibco, product number 14040133) + 0.5 μmol / L Ionomycin (calcium ionomycin) + 3 mg / mL BSA (bovine serum albumin); Solution B: in vitro embryo culture medium + 2 mmol / L 6-DMAP (N,N-dimethylaminopurine); (4) In vitro embryo culture medium: SOF + 2% EAA (essential amino acids, purchased from Sigma, product number M5550) + 1% NEAA (non-essential amino acids, purchased from Sigma, product number M7145) + 0.48 mg / mL Myo-inositol + 1 mmol / L L-glutamine + 6 mg / mL BSA;

[0030] (5) Sheep in vitro fertilization fluid: SOF + 1% EAA (essential amino acid, purchased from Sigma, product number M5550) + 1% NEAA (non-essential amino acid, purchased from Sigma, product number M7145) + 1 mmol / L L-glutamine + 0.01 g / L heparin sodium + 8 mg / mL BSA + 2% fetal bovine serum;

[0031] (6) Floating solution: SOF solution + 5.208 g / L HEPEs + 0.1% L-glutamine + 0.01 g / L heparin sodium + 2% fetal bovine serum;

[0032] (7) SOF formula: 6.29 mg / mL NaCl + 0.534 mg / mL KCl + 0.162 mg / mL KH2PO4 + 0.6 μL / mL sodium lactate + 0.089 mg / mL MgSO4 + 2.1 mg / mL NaHCO3 + 0.0357 mg / mL sodium pyruvate + 0.299 mg / mL CaCl2·2H2O.

[0033] Experimental Example 1: Metabolomics analysis of creatine, a key metabolic regulator in oocyte maturation and embryonic development

[0034] (1) Ovaries of sheep were obtained from the slaughtered sheep on the same day, placed in 30°C saline containing 200 IU / mL penicillin and 200 IU / mL streptomycin, and transported to the laboratory within 2 to 4 hours;

[0035] (2) After the ovaries were washed three times with saline containing antibiotics, they were transferred to a 100 mm culture dish containing oocyte collection fluid. The ovarian follicles with a diameter of 2 to 5 mm on the surface were cut with a No. 23 surgical blade. The cumulus-oocyte complexes (COCs) were selected under a microscope and washed 3 to 5 times with in vitro maturation fluid. Some COCs were transferred to oocyte collection fluid containing 0.1% hyaluronidase to remove the peripheral granulosa cells (cumulus cells) of the oocytes. The obtained germinal vesicle stage (GV stage) oocytes were transferred to saline and washed three times. According to the requirements of metabolomics sampling, they were collected and stored in a -80 degree refrigerator.

[0036] (3) Another part of COCs was transferred to in vitro maturation solution and cultured in a CO2 incubator at 5% CO2, 95% air and 38.5°C for 22 to 24 hours; then, the COCs were transferred to egg-picking solution containing 0.1% hyaluronidase, the granulosa cells were removed, and a part of mature (MⅡ stage) oocytes were selected and washed three times in physiological saline. According to the requirements of metabolomics sampling, they were collected and stored in a -80 degree refrigerator;

[0037] (4) The mature oocytes obtained in step (3) without granulosa cells were transferred into sheep in vitro fertilization (IVF) solution and washed three times, and then transferred into IVF droplets at a rate of 15 to 20 sperm per drop for later use; the thawed frozen semen was added to the bottom of the test tube containing the supernatant, and the sperm suspension was taken from the upper layer and centrifuged at 800 rpm for 5 minutes, and the supernatant was discarded. The density of the remaining sperm pellet was calculated at 2 to 4 × 10 6 The density of oocytes / mL was added into IVF droplets containing mature oocytes;

[0038] (5) taking out the cells fertilized 20 to 22 hours after step (4), washing them 3 to 4 times in an in vitro embryo culture medium, transferring them to an in vitro culture medium and culturing them in a CO2 incubator for 24 to 26 hours, removing the oocytes that have not cleaved, and continuing to culture the remaining cleaved cells until the sixth or seventh day, during which the medium is replaced by half every 48 hours; collecting 2-cell embryos, 8-cell embryos and expanded blastocysts at 28 hours, 48 ​​hours and the sixth day after fertilization, respectively, and storing the collected samples in a -80 degree refrigerator according to the requirements of metabolomics sampling;

[0039] (6) Metabolomics samples were analyzed and the key metabolite was determined to be creatine.

[0040] (7) To determine the concentration of creatine added, the concentration of creatine in the fallopian tube fluid was referenced, and the final concentration of creatine in the in vitro culture system was selected as 0.5 mmol / L.

[0041] Experimental Example 2 Application of Creatine in Improving the Blastocyst Development Rate of Sheep Oocytes in Vitro

[0042] (1) The cumulus-oocyte complex is cultured in an in vitro maturation solution (containing 0.5 mmol / L creatine) in a CO2 incubator at 5% CO2, 95% air, and a temperature of 38.5°C for 22 to 24 hours; after 22 to 24 hours of maturation, the COCs are transferred to an oocyte-collecting solution containing 0.1% hyaluronidase, the granulosa cells are removed, and the oocytes are retained for standby use;

[0043] (2) Using the parthenogenetic activation method to develop the embryo in vitro: The oocytes in step (7) were transferred to parthenogenetic activation solution A (calcium-magnesium DPBS containing 0.5 μmol / L calcium ionomycin), incubated in a CO2 incubator at 5% CO2, 95% air, and a temperature of 38.5°C for 5 minutes, then washed 3 times with in vitro embryo culture solution, and transferred to parthenogenetic activation solution B (in vitro embryo culture solution containing 2 mmol / L 6-DMAP), and incubated in a CO2 incubator at 5% CO2, 95% air, and a temperature of 38.5°C for 2 to 3 hours. Finally, the oocytes were taken out, washed 3 times with in vitro embryo culture solution, and transferred to in vitro embryo culture solution for culture. The day when parthenogenetic activation began was recorded as day 0, and the cleavage rate and blastocyst rate were counted on the second day and the seventh day, respectively. The results are shown in Table 1.

[0044] Table 1 Creatine promotes the parthenogenetic blastocyst development rate of sheep oocytes

[0045]

[0046] Note: The control group refers to the group without the addition of creatine to the in vitro maturation medium and in vitro embryo culture medium; the treatment group refers to the group with the addition of creatine to the in vitro maturation medium and in vitro embryo culture medium; "COCs(n)" here refers to the number of COCs planned for subsequent parthenogenetic activation after 22-24 hours of maturation; "PA(n)" refers to the number of oocytes used for parthenogenetic activation after the granulosa cells in COCs are digested; the cleavage rate is the ratio of the number of cleaved cells to the number of parthenogenetically activated oocytes; the blastocyst rate is the ratio of the number of blastocysts to the number of cleavage cells.

[0047] In summary, the present invention applies creatine to in vitro culture of sheep oocytes and frozen-thawed oocytes, has a high embryo blastocyst rate and high safety, and can be extended to in vitro fertilization embryo development.

[0048] Although the content of the present invention has been described in detail through the above preferred embodiments, it should be appreciated that the above description should not be considered as a limitation of the present invention. After reading the above content, it will be apparent to those skilled in the art that various modifications and substitutions of the present invention will occur. Therefore, the protection scope of the present invention should be limited by the appended claims.

Claims

1. The application of creatine in improving the blastocyst development rate of sheep oocytes in vitro, characterized in that: The concentration of creatine is 0.5 mmol / L.

2. The use according to claim 1, characterized in that: The creatine is added to an in vitro maturation system and an in vitro embryo culture system.

3. The use according to claim 2, characterized in that: The creatine is added to an in vitro maturation solution, and the in vitro maturation solution used contains: M199, 0.5 IU / mL follicle stimulating hormone, 0.5 IU / mL luteinizing hormone, 1 μg / mL estradiol, 50 μg / mL gentamicin, 4 mmol / L sodium pyruvate and 50 μmol / L L-cystine.

4. The use according to claim 2, characterized in that: The creatine is added to an in vitro embryo culture medium, and the in vitro embryo culture medium used comprises: SOF, 2% essential amino acids, 1% non-essential amino acids, 0.48 mg / mL inositol, 1 mmol / L L-glutamine and 6 mg / mL BSA; The SOF contains: 6.29 mg / mL NaCl, 0.534 mg / mL KCl, 0.162 mg / mL KH2PO4, 0.6 μL / mL sodium lactate, 0.089 mg / mL MgSO4, 2.1 mg / mL NaHCO3, 0.0357 mg / mL sodium pyruvate and 0.299 mg / mL CaCl2·2H2O.

5. The use according to any one of claims 1 to 4, characterized in that: The app contains: After the cumulus-oocyte complex matured in an in vitro maturation solution containing 0.5 mmol / L creatine for 22 to 24 hours, the cumulus cells were removed and the oocytes were retained for later use; The oocytes are transferred to parthenogenetic activation solution A and incubated for 5 minutes, then washed several times with in vitro embryo culture solution, transferred to parthenogenetic activation solution B, and incubated in an incubator for 2 to 3 hours; finally, the oocytes are taken out, washed several times with in vitro embryo culture solution, and transferred to in vitro embryo culture solution for culture; Wherein, the parthenogenetic activation solution A is a calcium-magnesium-containing DPBS buffer solution containing calcium ionomycin; The parthenogenetic activation solution B is an in vitro embryo culture solution containing 6-DMAP.

6. The use according to claim 5, characterized in that: The parthenogenetic activation solution A comprises: DPBS buffer containing calcium and magnesium, 0.5 μmol / L calcium ionomycin, and 3 mg / mL bovine serum albumin; The parthenogenetic activation solution B contains: in vitro embryo culture solution and 2mmol / L 6-DMAP.

7. The use according to claim 5, characterized in that: Add 0.5mmol / L creatine to the in vitro maturation solution; then transfer the cumulus-oocyte complex into the in vitro maturation solution and culture it in a CO2 incubator with 5% CO2, 95% air and a temperature of 38.5°C for 22 to 24 hours; then, transfer the cumulus-oocyte complex into an egg-picking solution containing 0.1% hyaluronidase, remove the cumulus cells, and retain the oocytes for later use.

8. The use according to claim 5, characterized in that: The oocytes are transferred to parthenogenetic activation solution A and incubated in a CO2 incubator at 5% CO2, 95% air, and a temperature of 38.5°C for 5 minutes, and then washed several times with in vitro embryo culture solution, and then transferred to parthenogenetic activation solution B and incubated in a CO2 incubator at 5% CO2, 95% air, and a temperature of 38.5°C for 2 to 3 hours; finally, the oocytes are taken out, washed several times with in vitro embryo culture solution, and transferred to in vitro embryo culture solution for culture.

9. An in vitro maturation solution for improving the in vitro blastocyst development rate of sheep oocytes, characterized in that: The in vitro maturation solution comprises: M199, 0.5 IU / mL follicle stimulating hormone, 0.5 IU / mL luteinizing hormone, 1 μg / mL estradiol, 50 μg / mL gentamicin, 4 mmol / L sodium pyruvate and 50 μmol / L L-cystine. Creatine is added to the in vitro maturation solution, and the concentration of creatine in the in vitro maturation solution is 0.5 mmol / L.

10. An in vitro embryo culture medium for improving the in vitro blastocyst development rate of sheep oocytes, characterized in that: The in vitro embryo culture medium comprises: SOF, 2% essential amino acids, 1% non-essential amino acids, 0.48 mg / mL inositol, 1 mmol / L L-glutamine and 6 mg / mL BSA; Wherein, the SOF comprises: 6.29 mg / mL NaCl, 0.534 mg / mL KCl, 0.162 mg / mL KH2PO4, 0.6 μL / mL sodium lactate, 0.089 mg / mL MgSO4, 2.1 mg / mL NaHCO3, 0.0357 mg / mL sodium pyruvate and 0.299 mg / mL CaCl2·2H2O; Creatine is added to the in vitro embryo culture medium, and the concentration of creatine in the in vitro embryo culture medium is 0.5 mmol / L.

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