A bovine oocyte maturation fluid and its application

Abstract

The present invention provides a bovine oocyte maturation fluid and application, belonging to the field of animal breeding technology. The present invention effectively improves the bovine oocyte maturation efficiency, promotes the proliferation of cumulus cells, improves the embryo development ability, and thus improves the bovine in vitro embryo production efficiency by adding lipoxin to the basic culture medium.

Description

Technical Field

[0001] The invention belongs to the technical field of animal reproduction, and in particular relates to a bovine oocyte maturation fluid and an application thereof. Background Art

[0002] The statements in this section merely provide background information related to the present invention and do not necessarily constitute prior art.

[0003] Livestock embryo engineering technology is an important way to rapidly breed excellent germplasm. In order to improve the efficiency of using donor cows, live egg collection-in vitro embryo production technology is usually used to expand and breed fine breeds. In vitro maturation of bovine oocytes is a key step in producing in vitro embryos, and low oocyte quality is the main factor leading to low embryo production efficiency. Optimizing bovine oocyte in vitro maturation culture medium and improving in vitro maturation methods are effective means to improve oocyte quality and increase the efficiency of in vitro embryo production technology.

[0004] During the in vitro maturation of oocytes, hormones and cytokines secreted by the cumulus cells on the surface play an important role in promoting the maturation of oocytes. However, in vitro culture environments often cause cumulus cells to undergo apoptosis and grow slowly, resulting in impaired cumulus cell function, poor oocyte maturation, and reduced blastocyst acquisition rates and in vitro embryo transfer success rates. Summary of the invention

[0005] In order to overcome the above-mentioned deficiencies of the prior art, the present invention provides a bovine oocyte maturation solution and its application. The main purpose of the present invention is to further improve the in vitro bovine oocyte maturation rate, promote the blastocyst acquisition rate, and improve the bovine in vitro embryo production efficiency.

[0006] To achieve the above objectives, one or more embodiments of the present invention provide the following technical solutions:

[0007] In a first aspect of the present invention, there is provided the use of lipoxin in any one or more of the following:

[0008] 1) Application in promoting oocyte maturation in vitro or in preparing reagents for promoting oocyte maturation;

[0009] 2) Application in improving the blastocyst acquisition rate in vitro or in preparing reagents for improving the blastocyst acquisition rate;

[0010] 3) Use in promoting the proliferation of cumulus cells in vitro or in preparing reagents for promoting the proliferation of cumulus cells;

[0011] 4) Use in in vitro neutralization of inflammatory factors or preparation of anti-inflammatory factor reagents;

[0012] 5) Application in improving the efficiency of bovine in vitro embryo production and in preparing reagents for improving the efficiency of in vitro embryo production;

[0013] The lipoxins include LXA4;

[0014] The oocyte is an oocyte wrapped by more than two layers of cumulus cells;

[0015] The oocyte is derived from bovine;

[0016] The blastocyst is a blastocyst obtained 7 days after fertilization;

[0017] The applications include preparation of cell models or animal models, and preparation of scientific research reagents.

[0018] Lipoxin is an endogenous lipoxygenase-derived eicosanoid regulator that inhibits the release of cellular inflammatory factors and neutralizes cellular inflammatory responses. In previous studies, emphasis was placed on the anti-inflammatory effects of lipoxin in vivo, but it was unknown whether it could effectively inhibit the release of inflammatory factors and neutralize inflammatory factors in vitro, or whether it could be used as a cell culture medium additive to improve the efficiency of in vitro embryo production.

[0019] In a specific embodiment of the present invention, lipoxin is configured into 1 μM, 10 μM, and 100 μM 1000X concentrated solutions. The specific operation method is to add 457.5 μL of ethanol to 250 μL of 283 μM lipoxin to obtain a 100 μM 1000X concentrated solution; take 100 μL of the 100 μM 1000X concentrated solution, add 900 μL of ethanol and mix, to obtain a 10 μM 1000X concentrated solution; take 100 μL of the 100 μM 1000X concentrated solution, add 900 μL of ethanol and mix, to obtain a 1 μM 1000X concentrated solution. After each concentration of 1000X concentrated solution is dispensed into 10 μL per tube, it is stored in a -80°C ultra-low temperature refrigerator.

[0020] A second aspect of the present invention provides an oocyte maturation solution, wherein the oocyte maturation solution is supplemented with 10-100 nM lipoxin in a basic culture medium;

[0021] The lipoxin is LXA4;

[0022] The oocytes are derived from bovine.

[0023] In a specific embodiment of the present invention, the basal culture medium is any one of M199, NCSU-37, NCSU-23, Ham's F10, and α-MEM; preferably M199.

[0024] In a specific embodiment of the present invention, the oocyte maturation solution further comprises fetal bovine serum, FSH, EGF, IGF-1, GlutaMax, and penicillin-streptomycin 100X concentrate;

[0025] Preferably, the oocyte maturation solution further comprises 8-15% by volume of fetal bovine serum, 0.3-0.8 μg / ml FSH, 8-15 ng / ml EGF, 8-15 ng / ml IGF-1, 0.5-1.5% by volume of GlutaMax, and 0.5-1.5% by volume of penicillin-streptomycin 100X concentrate.

[0026] In a specific embodiment of the present invention, the oocyte maturation solution also includes 10% fetal bovine serum, 0.5 μg / ml FSH, 10 ng / ml EGF, 10 ng / ml IGF-1, 1% GlutaMax, and 1% penicillin-streptomycin 100X concentrate.

[0027] The third aspect of the present invention provides the use of the oocyte maturation solution described in the second aspect in any of the following:

[0028] 1) Application in promoting oocyte maturation in vitro or in preparing reagents for promoting oocyte maturation;

[0029] 2) Application in improving the blastocyst acquisition rate in vitro or in preparing reagents for improving the blastocyst acquisition rate;

[0030] 3) Use in promoting the proliferation of cumulus cells in vitro or in preparing reagents for promoting the proliferation of cumulus cells;

[0031] 4) Use in in vitro neutralization of inflammatory factors or preparation of anti-inflammatory factor reagents;

[0032] 5) Application in improving the efficiency of in vitro embryo production in cattle and in the preparation of reagents for improving the efficiency of in vitro embryo production.

[0033] A fourth aspect of the present invention provides a method for promoting bovine oocyte maturation in vitro, wherein the bovine cumulus oocyte complex is placed in the oocyte maturation solution described in the second aspect, and cultured in an incubator at 5% carbon dioxide, saturated humidity, and 38.5° C. for 20-24 hours;

[0034] or,

[0035] The bovine cumulus-oocyte complex is placed in cell culture medium I, cultured in an incubator at 5% carbon dioxide, saturated humidity, and 38.5° C. for 6-18 hours, and then lipoxin is added dropwise and cultured for 2-18 hours under the same conditions. The total culture time is 20-24 hours.

[0036] The oocyte is wrapped by more than two layers of cumulus cells.

[0037] As a preferred solution, 30-50 bovine cumulus-oocyte complexes can be placed in a 50-100 μL oocyte maturation solution droplet or 80-120 bovine cumulus-oocyte complexes can be placed in a well containing 200-300 μL oocyte maturation solution for in vitro maturation culture.

[0038] In a specific embodiment of the present invention, the bovine cumulus-oocyte complex is placed in cell culture medium I, cultured in an incubator at 5% carbon dioxide, saturated humidity, and 38.5°C for 12 hours, and then lipoxin is added dropwise and cultured for another 2-18 hours under the same culture conditions to obtain the product.

[0039] A fifth aspect of the present invention provides a method for promoting cumulus cell proliferation, comprising the following steps:

[0040] 1) Isolating and obtaining cumulus cells from bovine cumulus-oocyte complexes;

[0041] 2) placing the cumulus cells obtained in step 1) in cell culture medium II, and culturing them in an incubator at 5% carbon dioxide, saturated humidity, and 38.5° C. for 6-24 hours;

[0042] Wherein, cell culture medium II contains 10-100 nM lipoxin;

[0043] Preferably, the cell culture medium II uses M199 culture medium as the basic culture medium, supplemented with 8-15% fetal bovine serum, 0.5-1.5% penicillin-streptomycin and 10-100 nM lipoxin;

[0044] Preferably, the amount of lipoxin added is 10 nM or 100 nM.

[0045] A sixth aspect of the present invention provides a method for improving the production efficiency of bovine in vitro embryos, the method comprising:

[0046] 1) Obtaining bovine cumulus-oocyte complex;

[0047] 2) using the method described in the fourth aspect to culture the bovine cumulus-oocyte complex obtained in step (1) to obtain a mature bovine oocyte;

[0048] 3) placing the mature bovine oocyte obtained in step 2) in an in vitro fertilization culture medium, and mixing and culturing with sperm for 10-16 hours to obtain zygote embryos;

[0049] 4) Using hyaluronidase to remove the cumulus cells in the zygotic embryos of step 3), and transferring the embryos after the cumulus cells are removed to an embryo in vitro culture medium for culture for 7-8 days.

[0050] One or more of the above technical solutions have the following beneficial effects:

[0051] In the prior art, the main ways to optimize bovine oocyte in vitro maturation are to achieve the purpose of anti-oxidation, growth promotion, etc. by adding exogenous cytokines or hormones, or by adding serum-free culture medium, etc. In the present invention, it is found for the first time that lipoxin can effectively promote bovine oocyte maturation.

[0052] The present invention finds that the addition of lipoxin can effectively improve the efficiency of bovine oocyte maturation, promote the proliferation of cumulus cells, and reduce the release of inflammatory factors of cumulus cells, thereby improving the embryo development ability and the efficiency of bovine in vitro embryo production.

[0053] The present invention also determines a method for further improving the bovine oocyte maturation rate and blastocyst acquisition rate. Through research, it is found that adding a certain amount of lipoxin after culturing the bovine cumulus-oocyte complex in vitro for 12 hours is more conducive to improving the bovine in vitro embryo production efficiency.

[0054] Advantages of additional aspects of the present invention will be given in part in the following description, and in part will become obvious from the following description, or will be learned through practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0055] The accompanying drawings in the specification, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0056] Figure 1 This is the effect of adding different concentrations of lipoxins on the proliferation ability of cumulus cells on the surface of oocytes according to Example 11 of the present invention. ab represents significant difference P<0.05. DETAILED DESCRIPTION

[0057] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods in the following examples without specifying specific conditions are usually carried out under conventional conditions or under conditions recommended by the manufacturer.

[0058] It should be noted that the details of the present invention are known to those skilled in the art. The experimental methods in the following examples that do not specify specific conditions are usually carried out under conventional conditions or according to the conditions recommended by the manufacturer. The experimental steps that are not recorded in detail refer to "Animal Cell Culture - Basic Techniques and Special Applications Guide" ((British) RI Freshney (RI Freshney), translated by Zhang Jingbo et al., 7th edition), online databases, etc.

[0059] In a specific embodiment of the present invention, cell culture medium I refers to M199 as a basic culture medium, with the addition of fetal bovine serum, follicle-stimulating hormone FSH, insulin-like growth factor EGF, insulin-like growth factor IGF-1, Glutamax, and penicillin-streptomycin 100X concentrated solution; more specifically, taking 100 mL as an example, 10 mL of fetal bovine serum, 50 μg FSH, 1 μg EGF, 1 μg IGF-1, 1 mL Glutamax, and 1 mL penicillin-streptomycin 100X concentrated solution are added to 88 mL of basic culture medium M199.

[0060] In a specific embodiment of the present invention, the cell culture medium II refers to M199 culture medium containing 10% by volume of fetal bovine serum and 1% by volume of penicillin-streptomycin, supplemented with 10-100 nM lipoxin.

[0061] In a specific embodiment of the present invention, the formula of in vitro fertilization culture medium is: taking 100 mL as an example, 0.63 g NaCl, 0.053 g KCl, 0.016 g KH2PO4, 0.038 g MgSO4·7H2O, 0.026 g CaCl2·2H2O, 0.21 g NaHCO3, 0.004 g sodium pyruvate, 0.08 mg sodium lactate, 0.003 g glutamine, 0.02 g caffeine, 0.005 g sodium heparin, 4 g bovine serum albumin, and 1.2 mg gentamicin are added to 100 mL deionized water.

[0062] In a specific embodiment of the present invention, the embryo in vitro culture medium is purchased from Sigma, with the product number MR-061-D.

[0063] In a specific embodiment of the present invention, the zygote stage embryo refers to the embryo obtained after sperm and mature egg cells are co-cultured for 10-16 hours.

[0064] In a specific embodiment of the present invention, the cattle does not refer to buffalo. The cattle in the present invention is common cattle, more specifically, Bos taurus.

[0065] In the specific implementation of the present invention, the specific information of the products used is as follows:

[0066] Lipoxin, manufacturer: MCE; item number: HY-113509.

[0067] Fetal bovine serum, manufacturer: ThermoFisher, catalog number: 1009915.

[0068] FSH, manufacturer: Vetoquinol, trademark: Folltropin.

[0069] EGF, manufacturer: ThermoFisher, product number: PHG0311.

[0070] IGF-1, manufacturer: ThermoFisher, product number: 100-11-100UG.

[0071] GlutaMax, manufacturer: ThermoFisher, product number: 35050061.

[0072] Penicillin-Streptomycin 100X concentrate, manufacturer: ThermoFisher, product number: 15140122.

[0073] M199, manufacturer: ThermoFisher, product number: 11150059.

[0074] Hyaluronidase, manufacturer: Sigma-Aldrich; product number: H3506.

[0075] In the following examples, the materials and reagents used were obtained from commercial sources unless otherwise specified.

[0076] The present invention will be further described in detail below in conjunction with specific embodiments. It should be noted that the specific embodiments are intended to explain the present invention rather than to limit it.

[0077] Embodiment 1

[0078] This embodiment discloses an oocyte maturation solution. Taking 100 mL as an example, 10 mL of fetal bovine serum, 50 μg FSH, 1 μg EGF, 1 μg IGF-1, 1 mL Glutamax, and 1 mL of penicillin-streptomycin 100X concentrate are added to 88 mL of basic culture medium M199, and 10 nM lipoxin is added and mixed.

[0079] Embodiment 2

[0080] This embodiment discloses an oocyte maturation fluid. Taking 100 mL as an example, 10 mL of fetal bovine serum, 50 μg of FSH, 1 μg of EGF, 1 μg of IGF-1, 1 mL of Glutamax, and 1 mL of penicillin-streptomycin 100X concentrate are added to 88 mL of basal culture fluid M199, and 100 nM of lipoxin is added and mixed.

[0081] Embodiment 3

[0082] This embodiment discloses a method for promoting bovine oocyte maturation in vitro. Specifically, 500 mL of 28°C physiological saline containing 1% volume fraction of penicillin-streptomycin 100X concentrate is added to a thermos cup, and bovine ovaries are collected from a slaughterhouse. The ovaries are transported back to the laboratory within 4 hours, and follicles with a diameter of 2-6 mm on the ovaries are aspirated with a 10 mL syringe. Pick up oocytes (cumulus-oocyte complexes) wrapped with more than 2 layers of cumulus cells, wash them 3 times in DPBS containing 15% fetal bovine serum, and then put them into the oocyte maturation culture medium of Example 1 that has been pre-equilibrated in a carbon dioxide incubator for more than 2 hours. If the droplet culture method is used, 30-50 cumulus-oocyte complexes are placed in 80 μL droplets; if the 4-well plate culture method is used, 80-120 cumulus-oocyte complexes are placed in 250 μL culture medium. In vitro maturation culture is carried out for 20-24 hours in an incubator with 5% carbon dioxide, saturated humidity, and 38.5°C. After the end, 2 mg / mL hyaluronidase was used to remove the cumulus cells on the surface of the oocytes, and the proportion of mature oocytes was counted by polar body exclusion under a stereomicroscope. The results of bovine oocyte maturation rate are shown in Table 1. The results showed that the addition of 10 nM lipoxin can effectively promote the in vitro maturation of bovine oocytes.

[0083] Embodiment 4

[0084] This embodiment discloses a method for promoting bovine oocyte maturation in vitro. The difference from the third embodiment is that the oocyte maturation solution shown in the second embodiment is used for culture. The results of the bovine oocyte maturation rate are shown in Table 1. The results show that the addition of 100nM lipoxin can effectively promote the in vitro maturation of bovine oocytes.

[0085] Table 1 Effects of different lipoxin addition concentrations on bovine oocyte maturation efficiency.

[0086] Group Total number of oocytes tested Maturity rate (%) 0 388 <![CDATA[68.3±1.8 a ]]> 1nM 376 <![CDATA[69.0±2.2 a ]]> 10nM 411 <![CDATA[78.1±2.7 b ]]> 100nM 364 <![CDATA[75.2±1.7 b ]]>

[0087] Note: Numbers with different letters in the same column indicate significant differences (P<0.05).

[0088] Comparative Example 1

[0089] This comparative example provides a method for promoting bovine oocyte maturation in vitro, which is different from Example 3 in that the oocyte maturation fluid used to culture the cumulus-oocyte complex in this comparative example does not contain lipoxin. The results of in vitro maturation rate of oocytes are shown in Table 1.

[0090] Comparative Example 2

[0091] This comparative example provides a method for promoting bovine oocyte maturation in vitro, which is different from Example 3 in that the oocyte maturation solution used to culture the cumulus-oocyte complex in this comparative example contains only 1 nM lipoxin. The results of in vitro maturation rate of oocytes are shown in Table 1.

[0092] Embodiment 5

[0093] This embodiment discloses a method for promoting bovine oocyte maturation in vitro. Specifically, 500 mL of 28°C physiological saline containing 1% volume fraction of penicillin-streptomycin 100X concentrate is added to a thermos cup, and bovine ovaries are collected from a slaughterhouse. The ovaries are transported back to the laboratory within 4 hours, and follicles with a diameter of 2-6 mm on the ovaries are aspirated with a 10 mL syringe. Pick up oocytes (cumulus-oocyte complexes) wrapped with more than 2 layers of cumulus cells, wash them 3 times in DPBS containing 15% fetal bovine serum, and then put them into cell culture medium I that has been pre-equilibrated in a carbon dioxide incubator for more than 2 hours. If the droplet culture method is used, 30-50 cumulus-oocyte complexes are placed in 80 μL droplets; if the 4-well plate culture method is used, 80-120 cumulus-oocyte complexes are placed in 250 μL culture medium. After 6 hours of in vitro maturation culture in an incubator with 5% carbon dioxide, saturated humidity and 38.5°C, 10 nM lipoxin was added to the system and cultured for 24 hours. After the end, 2 mg / mL hyaluronidase was used to remove the cumulus cells on the surface of the oocytes, and the proportion of mature oocytes was counted by polar body exclusion under a stereo microscope. The results of bovine oocyte maturation rate are shown in Table 2, and the in vitro maturation rate of oocytes reached 70.1%.

[0094] Embodiment 6

[0095] This embodiment discloses a method for promoting bovine oocyte maturation in vitro. The difference from the fifth embodiment is that the lipoxin in this embodiment is added after the cumulus oocyte complex is cultured in cell culture medium I for 12 hours. The results of the bovine oocyte maturation rate are shown in Table 2. It can be seen that the addition of 10 nM lipoxin during the in vitro maturation culture for 12 hours can promote the in vitro maturation of bovine oocytes.

[0096] Embodiment 7

[0097] This example discloses a method for promoting bovine oocyte maturation in vitro. The difference from Example 5 is that in this example, lipoxin is added after the cumulus-oocyte complex is cultured in cell culture medium I for 18 hours. The results of bovine oocyte maturation rate are shown in Table 2.

[0098] Comparative Example 3

[0099] The difference between this comparative example and Example 5 is that in this example, lipoxin is added after the cumulus-oocyte complex is cultured for 0 hours in cell culture medium I. The results of bovine oocyte maturation rate are shown in Table 2.

[0100] Table 2 Effects of different lipoxin addition times on bovine oocyte maturation efficiency.

[0101] Group Total number of oocytes tested Maturity rate (%) 0h 294 <![CDATA[75.7±2.4 a ]]> 6h 295 <![CDATA[74.6±3.1 a ]]> 12h 309 <![CDATA[85.8±3.2 b ]]> 18h 276 <![CDATA[73.3±2.2 a ]]>

[0102] Note: Numbers with different letters in the same column indicate significant differences (P<0.05).

[0103] Embodiment 8

[0104] This embodiment provides a method for culturing bovine embryos in vitro.

[0105] The oocytes were matured in vitro with reference to the methods of Example 3, Example 4, Comparative Example 1 and Comparative Example 2, and then transferred to in vitro fertilization culture medium. Two conventional bovine frozen sperm were thawed, and dead sperm and cryoprotectant were removed by Percoll separation. Sperm was added to the in vitro fertilization solution containing oocytes, and the sperm was diluted to a final concentration of 1 million per milliliter using the in vitro fertilization solution, and cultured in an incubator at 5% carbon dioxide, saturated humidity, and 38.5°C for 10-16 hours.

[0106] 2mg / mL hyaluronidase was used to remove the cumulus cells on the surface of the zygotic embryos, and then the zygotic embryos were transferred to the embryo in vitro culture medium for 7 days. In vitro fertilization was day 0, and the embryo cleavage efficiency was counted under a stereo microscope on day 3, and the blastocyst development efficiency was counted under a stereo microscope at the end of in vitro culture. The effect of adding different concentrations of lipoxin during oocyte maturation on subsequent embryo development is shown in Table 3. Both 10nM and 100nM lipoxin can promote the blastocyst development efficiency of subsequent embryos.

[0107] Table 3 Effects of different lipoxin addition concentrations on the subsequent embryo blastocyst development efficiency.

[0108]

[0109] Note: Numbers with different letters in the same column indicate significant differences (P<0.05).

[0110] Embodiment 9

[0111] Refer to the methods of Example 5, Example 6, Example 7, and Comparative Example 3 to perform in vitro maturation of oocytes, and transfer them to in vitro fertilization culture medium after completion. Thaw two conventional bovine frozen sperm, remove dead sperm and cryoprotectant by Percoll separation. Add sperm to in vitro fertilization solution containing oocytes, dilute sperm to a final concentration of 1 million per milliliter using in vitro fertilization solution, and culture in an incubator at 5% carbon dioxide, saturated humidity, and 38.5°C for 10-16 hours.

[0112] 2mg / mL hyaluronidase was used to remove the cumulus cells on the surface of the zygotic embryos, and then the zygotic embryos were transferred to the embryo in vitro culture medium for 7 days. In vitro fertilization was day 0, and the embryo cleavage efficiency was counted under a stereo microscope on day 3, and the blastocyst development efficiency was counted under a stereo microscope at the end of in vitro culture. The effects of different addition times of lipoxin on subsequent embryo development are shown in Table 4. The results show that adding 10nM lipoxin at 12h of in vitro maturation culture can promote the blastocyst development efficiency of subsequent embryos.

[0113] Table 4 Effects of different lipoxin addition times on subsequent embryo blastocyst development efficiency.

[0114] Group Total number of embryos tested Cleavage rate (%) Blastocyst rate (%) 0h 211 <![CDATA[75.2±2.0 a ]]> <![CDATA[26.2±2.3 a ]]> 6h 197 <![CDATA[74.1±2.5 a ]]> <![CDATA[32.1±2.1 b ]]> 12h 201 <![CDATA[75.5±1.9 a ]]> <![CDATA[36.6±2.5 b ]]> 18h 188 <![CDATA[76.7±2.2 a ]]> <![CDATA[24.3±3.2 a ]]>

[0115] Note: Numbers with different letters in the same column indicate significant differences (P<0.05).

[0116] Embodiment 10

[0117] The development of follicles can generally be divided into four stages: primordial follicles, primary follicles, secondary follicles and mature follicles. Primary follicles and secondary follicles are collectively referred to as growing follicles. In the earliest stage of follicular development, the oocyte enlarges and granulosa cells form around the oocyte. When the amount of oocyte cytoplasm increases, follicular development is mainly the proliferation and differentiation of granulosa cells, which will drive the formation of the antrum of the initial follicle, that is, the formation of antral follicles. Then, under the action of follicle-stimulating hormone, the antral follicle develops to the pre-ovulatory stage. As the follicular cavity is formed, granulosa cells gradually differentiate into two different types: wall granulosa cells, which are distributed around the follicular cavity; cumulus cells, which directly surround the egg. Studies have found that cumulus cells transfer some small molecules to the egg, participate in the maturation of the oocyte cytoplasm and nucleus, and also improve the developmental potential of oocytes in vitro, that is, cumulus cells participate in the maturation of oocytes, which is crucial for the normal maturation of oocytes.

[0118] In order to verify the factors affecting the maturation of bovine oocytes in this application,

[0119] The above-mentioned implementation two adopts the method in embodiment three to collect the immature cultured cumulus cell-oocyte complex. By the method of removing cumulus cells in embodiments three, five, eight, and nine, cumulus cells are separated from oocytes using hyaluronidase. According to the planting density of 10,000 cells per well, cumulus cells are placed in 96-well plates for culture. The culture fluid is an M199 culture fluid containing 10% volume percentage of fetal bovine serum and 1% volume fraction of penicillin-streptomycin. According to the addition of lipoxin at a final concentration of 0, 1, 10, 100 nM in the culture fluid, the cells are grouped and processed. Each treatment group is set to process two time points of 0h and 48h, and each treatment group and each time point are set to 6 repetitions to detect the effects of different concentrations of treatment and treatment time on cell number proliferation.

[0120] The cell number detection method before and after culture was CCK-8 method, using CCK-8 kit (Cat. No. GK10001) produced by GLPbio. The specific detection method was referred to the kit instructions. The relative cell viability was represented by calculating the absorbance ratio of the same cell sample at 0h and 48h of culture, which was used as the cell proliferation ability index.

[0121] The results are as follows Figure 1 As shown, adding 10 nM lipoxin to the bovine oocyte maturation medium and culturing for 48 hours can enhance the proliferation ability of cumulus cells on the surface of the oocytes, that is, the cell culture medium II provided by the present invention can effectively promote the proliferation of bovine cumulus cells.

[0122] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention is described in detail with reference to the aforementioned embodiments, those skilled in the art can still modify the technical solutions described in the aforementioned embodiments or replace some of the technical features therein by equivalents. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. Application of lipoxin in any one or more of the following: 1) Application in promoting oocyte maturation in vitro or in preparing reagents for promoting oocyte maturation; 2) Application in improving the blastocyst acquisition rate in vitro or in preparing reagents for improving the blastocyst acquisition rate; 3) Application in promoting the proliferation of cumulus cells in vitro or in preparing reagents for promoting the proliferation of cumulus cells; 4) Application in improving the efficiency of bovine in vitro embryo production and in preparing reagents for improving the efficiency of in vitro embryo production; The lipoxin includes LXA4; the concentration of the lipoxin is 10nM or 100nM; The oocyte is an oocyte wrapped by more than two layers of cumulus cells; The blastocyst, cumulus cell and oocyte are derived from cattle; The blastocyst is obtained 7 days after fertilization.

2. An oocyte maturation fluid, characterized in that: The oocyte maturation solution is added with 10nM or 100nM lipoxin in the basic culture medium; The lipoxin is LXA4; The oocyte is derived from bovine; The basic culture medium is any one of M199, NCSU-37, NCSU-23, Ham's F10, and α-MEM.

3. The oocyte maturation solution according to claim 2, wherein The basic culture medium is M199.

4. The oocyte maturation solution according to claim 2, wherein The oocyte maturation solution also includes fetal bovine serum, FSH, EGF, IGF-1, GlutaMax, and penicillin-streptomycin 100X concentrated solution.

5. The oocyte maturation solution according to claim 4, characterized in that The oocyte maturation solution also includes 8-15% fetal bovine serum, 0.3-0.8 μg / ml FSH, 8-15 ng / ml EGF, 8-15 ng / ml IGF-1, 0.5-1.5% GlutaMax, and 0.5-1.5% penicillin-streptomycin 100X concentrate.

6. The oocyte maturation solution according to claim 5, characterized in that The oocyte maturation solution also includes 10% fetal bovine serum, 0.5 μg / ml FSH, 10 ng / ml EGF, 10 ng / ml IGF-1, 1% GlutaMax, and 1% penicillin-streptomycin 100X concentrate.

7. Use of the oocyte maturation solution according to any one of claims 2 to 6 in any of the following: 1) Application in promoting oocyte maturation in vitro or in preparing reagents for promoting oocyte maturation; 2) Application in improving the blastocyst acquisition rate in vitro or in preparing reagents for improving the blastocyst acquisition rate; 3) Application in promoting the proliferation of cumulus cells in vitro or in preparing reagents for promoting the proliferation of cumulus cells; 4) Application in improving the efficiency of bovine in vitro embryo production and in preparing reagents for improving the efficiency of in vitro embryo production; The blastocyst, cumulus cell and oocyte are derived from bovine.

8. A method for promoting bovine oocyte maturation in vitro, characterized in that: The bovine cumulus oocyte complex is placed in the oocyte maturation solution according to any one of claims 2 to 6, and cultured in an incubator at 5% carbon dioxide, saturated humidity, and 38.5° C. for 20 to 24 hours to obtain; or, The bovine cumulus-oocyte complex was placed in cell culture medium I, cultured in an incubator at 5% carbon dioxide, saturated humidity, and 38.5° C. for 12 hours, and then lipoxin was added dropwise and cultured for 8-12 hours under the same culture conditions. The total culture time was 20-24 hours. The oocyte is wrapped by more than two layers of cumulus cells; The cell culture medium I refers to a medium based on M199, supplemented with fetal bovine serum, follicle-stimulating hormone FSH, insulin-like growth factor EGF, insulin-like growth factor IGF-1, Glutamax, and penicillin-streptomycin 100X concentrate.

9. A method for promoting cumulus cell proliferation, characterized in that: The following steps are involved: 1) Isolation and acquisition of cumulus cells from bovine cumulus-oocyte complexes; 2) placing the cumulus cells obtained in step 1) in cell culture medium II and culturing them in an incubator at 5% carbon dioxide, saturated humidity, and 38.5° C. for 48 hours; The cell culture medium II contained 10 nM or 100 nM lipoxin.

10. The method for promoting cumulus cell proliferation according to claim 9, characterized in that: The cell culture medium II is based on M199 culture medium, supplemented with 8-15% fetal bovine serum, 0.5-1.5% penicillin-streptomycin and 10 nM or 100 nM lipoxin.

11. A method for improving the production efficiency of buffalo in vitro embryos, characterized in that: The method comprises: 1) Obtaining bovine cumulus-oocyte complexes; 2) using the method of claim 8 to culture the bovine cumulus-oocyte complex obtained in step (1) to obtain mature bovine oocytes; 3) placing the mature bovine oocytes obtained in step 2) in an in vitro fertilization culture medium, and mixing and culturing with sperm for 10-16 hours to obtain zygote embryos; 4) Using hyaluronidase to remove the cumulus cells in the zygotic embryos of step 3), the embryos after the cumulus cells are removed are transferred to an embryo in vitro culture medium and cultured for 7-8 days.

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