Application of L-aspartic acid in preparation of goat oocyte in-vitro maturation culture solution

Abstract

The invention discloses an application of L-aspartic acid in preparation of a culture solution for in-vitro maturation of goat oocytes. The goat oocyte in-vitro maturation culture solution is composed of a certain amount of L-aspartic acid and a basic culture solution. It is verified that the goat oocyte culture fluid L-Asp added with the L-Asp can reduce the ROS level of goat oocytes, the mitochondrial membrane potential activity is improved, and the maturation rate of the oocytes is remarkably increased. The L-Asp provided by the invention can also be used for preparing a pharmaceutical preparation for promoting in-vitro maturation of goat oocytes. The goat oocyte in-vitro culture solution containing the L-Asp and the application of the goat oocyte in-vitro culture solution have important significance on improvement of growth and development of goat in-vitro embryos.

Description

Technical Field

[0001] The invention belongs to the field of oocyte culture, and in particular relates to application of L-aspartic acid in preparing goat oocyte in vitro maturation culture medium. Background Art

[0002] L-Aspartic acid (L-Asp) is an organic compound with the chemical formula C4H7NO4. It is a white crystal or crystalline powder with a slightly sour taste. The amino acid is an essential component of the culture medium used to culture human fertilized eggs to the blastocyst stage in vitro. The use of such culture medium can significantly increase the proportion of embryos that implant after transfer to the uterus and reduce the number of embryos that need to be transferred to achieve pregnancy.

[0003] Studies have found that specific amino acids in pigs [1] ,mouse [2] ,goat [3] Wagyu [4] For example, follicular aspartate concentrations have been shown to be directly correlated with improved oocyte morphology, increased metaphase II (MII) oocytes, and fertilization rates in humans. [5] Bahrami et al. reported that a specific culture medium containing glutamine, proline, and isoleucine could be used for bovine oocyte maturation and successful fertilization. [6] Cysteine, alone or in combination with glutamate and glycine, increases glutathione levels in oocytes and cumulus cells, thereby promoting development. Glutamine has been shown to promote oocyte nuclear maturation and induce cumulus expansion in the presence of glucose or lactate. [7] Oocyte and embryo quality is related to amino acid metabolism [8] Studies have shown that poorly developed oocytes exhibit higher levels of glutamine consumption, higher levels of alanine synthesis, and lower levels of total amino acid consumption [9] The above studies show that amino acids play an important role in improving the reproductive performance of female livestock, but there is no systematic report on the research of L-Asp in promoting the maturation of goat oocytes.

[0004] The research results of the present invention show that L-Asp can reduce the ROS level of goat oocytes, increase mitochondrial activity, and improve the maturation rate of oocytes. Therefore, the present invention provides a new culture medium composed of L-Asp and goat oocyte culture medium, which is used to improve the maturation rate of goat oocytes. In order to deeply and systematically study the mechanism of the influence of L-Asp on the maturation of goat oocytes, the team of the present invention intends to comprehensively utilize the methods of cell biology, molecular biology, proteomics and bioinformatics on the basis of existing work to carry out research on the molecular mechanism of L-Asp on improving the maturation rate of goat oocytes, in order to provide a theoretical basis for the development of culture medium for improving the maturation rate of goat oocytes. Summary of the invention

[0005] The purpose of the present invention is to provide an application of L-aspartic acid in preparing a goat oocyte in vitro maturation culture medium. The prepared goat oocyte in vitro maturation culture medium can improve the in vitro maturation rate of goat oocytes, that is, the application of L-aspartic acid in promoting the in vitro maturation of goat oocytes can improve the quality of goat oocytes.

[0006] In order to achieve the above-mentioned object of the invention, the present invention is implemented by the following technical solutions:

[0007] The invention discloses an application of L-aspartic acid in preparing a goat oocyte in vitro maturation culture medium. The goat oocyte in vitro maturation culture medium consists of an L-aspartic acid solution and a basic culture medium.

[0008] Furthermore, the L-aspartic acid solution is obtained by heating L-aspartic acid powder to 55° C. with goat embryo water (Sigma, W1503) to fully dissolve it.

[0009] Furthermore, the basic culture medium is prepared from TCM-199 containing 20% ​​estrus goat serum, 0.5 IU / ml FSH, 0.5 IU / ml LH, 1 μg / ml estradiol, 1 mM sodium pyruvate, 0.05 mM L-cysteine, 1% ITS, 20 ng / ml EGF and 25 μg / ml gentamicin.

[0010] Furthermore, the final concentration of the L-aspartic acid solution in the goat oocyte in vitro maturation culture medium is 0.5-2.5 mM.

[0011] Furthermore, the final concentration of the L-aspartic acid solution in the goat oocyte in vitro maturation culture medium is 2.0 mM.

[0012] The use of L-aspartic acid in the preparation of a goat oocyte in vitro maturation culture medium according to the present invention comprises the following steps:

[0013] S1, heating L-aspartic acid powder with embryo water at 55°C to fully dissolve it to obtain L-aspartic acid solution for later use;

[0014] S2, taking the L-aspartic acid solution obtained in the above step, adding it to the basal culture medium and shaking it well to form a goat oocyte in vitro maturation culture medium with a final L-Asp concentration of 2.0 mM;

[0015] S3, adding the goat oocyte in vitro maturation culture medium obtained in the above step S2 into a four-well plate with a pipette, covering it with paraffin oil and placing it in a 38.5°C, 5% CO2, 100% humidity incubator for preheating for a certain period of time, placing the oocytes of the intact and dense cumulus mass in a preheated four-well plate with goat oocyte in vitro maturation culture medium added and placing it in a 38.5°C, 5% CO2, 100% humidity incubator for in vitro maturation culture;

[0016] S4. Use a pipette to aspirate the oocytes after 22 hours of culture and place them in a 3.5 cm culture dish. Add 1 ml of 1% hyaluronidase and blow repeatedly to remove the granulosa cells around the oocytes. Under a stereomicroscope, pick out the mature oocytes that have expelled the first polar body (the first polar body is located between the zona pellucida and the oocyte membrane), put the mature oocytes back into the maturation fluid, and count the maturation of the oocytes.

[0017] The invention discloses an application of the L-aspartic acid in the preparation of a pharmaceutical preparation for promoting in vitro maturation of goat oocytes.

[0018] Preferably, the pharmaceutical preparation comprises 2.0 mM L-aspartic acid.

[0019] In a further aspect, the pharmaceutical preparation includes but is not limited to: injection, capsule, granule, tablet.

[0020] Beneficial effects of the present invention:

[0021] The goat oocyte in vitro maturation culture medium added with 2.0 mM L-aspartic acid has an in vitro maturation rate of 76.66%±4.07% for goat oocytes, which is 17.09% higher than that of the blank control group (59.57%±4.16%). The ROS level of goat oocytes is reduced, the mitochondrial activity is increased, and the in vitro maturation rate of goat oocytes is significantly improved. Moreover, the expansion of most cumulus cells is also significantly improved. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 :The effect of adding different concentrations of L-Asp to goat in vitro maturation medium on the in vitro maturation rate of oocytes. (A represents the maturation rate statistics of the control group and different concentrations of L-Asp (0.5, 1.0, 1.5, 2, 2.5mM), different lowercase letters indicate significant differences (P<0.05), and the same letters indicate no significant differences (P>0.05); B represents the representative images of cumulus cell expansion in the control group and the 2.0mM L-Asp treatment group.)

[0023] Figure 2:The effect of adding 2.0mM L-Asp to goat in vitro maturation medium on ROS in oocytes. (A represents the fluorescence images of ROS in oocytes of the control group and the experimental group. The ROS level is expressed as the average fluorescence intensity value, which is calculated from the fluorescence intensity values ​​of multiple oocytes. Each treatment was repeated 3 times, with about 15 oocytes each time. The data were analyzed by independent sample t-test; B shows the quantitative statistical graph of ROS fluorescence values. The "*" at the top of the bar graph indicates a statistically significant difference (P<0.05).)

[0024] Figure 3 :The effect of adding 2.0mM L-Asp to goat in vitro maturation medium on the mitochondrial membrane potential of oocytes (A represents the fluorescence images of mitochondrial membrane potential in oocytes of the control group and the experimental group; B represents the quantitative statistical graph showing the fluorescence value of mitochondrial membrane potential, and the "**" at the top of the bar graph indicates a statistically significant difference (P<0.01).) DETAILED DESCRIPTION

[0025] The present invention is further described in detail below in conjunction with the examples and drawings, but the embodiments of the present invention are not limited thereto. Unless otherwise defined, all scientific and technical terms used in the present invention have the same meanings as those commonly understood by those skilled in the art to which the present invention relates. In order to have a clearer understanding of the technical features, purposes and beneficial effects of the present invention, the technical solution of the present invention is now described in detail below, but it should not be construed as a limitation on the scope of implementation of the present invention.

[0026] Example 1 Preparation of goat oocyte in vitro maturation culture medium

[0027] Step 1: Take 0.3328 g of L-Asp powder and heat it to 55° C. with 50 mL of embryo water (Sigma, W1503) to fully dissolve it to obtain a L-Asp solution with a concentration of 50 mM.

[0028] Step 2: Take 5.0 μL of the L-Asp solution in the above step, add it to 500 μL of goat oocyte in vitro maturation culture medium and shake well to obtain a goat oocyte in vitro maturation culture medium with a final L-Asp solution concentration of 0.5 mM.

[0029] The basic culture medium is prepared from TCM-199 containing 20% ​​estrus goat serum, 0.5 IU / ml FSH, 0.5 IU / ml LH, 1 μg / ml estradiol, 1 mM sodium pyruvate, 0.05 mM L-cysteine, 1% ITS, 20 ng / ml EGF and 25 μg / ml gentamicin.

[0030] Example 2 Preparation of goat oocyte in vitro maturation culture medium

[0031] Step 1: Take 0.3328 g of L-Asp powder and heat it to 55° C. with 50 mL of embryo water (Sigma, W1503) to fully dissolve it to obtain a L-Asp solution with a concentration of 50 mM.

[0032] Step 2: Take 10.0 μL of the L-Asp solution in the above step, add it to 500 μL of goat oocyte in vitro maturation culture medium and shake well to obtain a goat oocyte in vitro maturation culture medium with a final concentration of 1.0 mM L-Asp solution.

[0033] The basic culture medium is prepared from TCM-199 containing 20% ​​estrus goat serum, 0.5 IU / ml FSH, 0.5 IU / ml LH, 1 μg / ml estradiol, 1 mM sodium pyruvate, 0.05 mM L-cysteine, 1% ITS, 20 ng / ml EGF and 25 μg / ml gentamicin.

[0034] Example 3 Preparation of goat oocyte in vitro maturation culture medium

[0035] Step 1: Take 0.3328 g of L-Asp powder and heat it to 55° C. with 50 mL of embryo water (Sigma, W1503) to fully dissolve it to obtain a L-Asp solution with a concentration of 50 mM.

[0036] Step 2: Take 15.0 μL of the L-Asp solution in the above step, add it to 500 μL of goat oocyte in vitro maturation culture medium and shake well to obtain a goat oocyte in vitro maturation culture medium with a final L-Asp solution concentration of 1.5 mM.

[0037] The basic culture medium is prepared from TCM-199 containing 20% ​​estrus goat serum, 0.5 IU / ml FSH, 0.5 IU / ml LH, 1 μg / ml estradiol, 1 mM sodium pyruvate, 0.05 mM L-cysteine, 1% ITS, 20 ng / ml EGF and 25 μg / ml gentamicin.

[0038] Example 4 Preparation of goat oocyte in vitro maturation culture medium

[0039] Step 1: Take 0.3993 g of L-Asp powder and heat it to 55° C. with 100 mL of embryo water (Sigma, W1503) to fully dissolve it to obtain a 30 mM L-Asp solution.

[0040] Step 2: Take 20.0 μL of the L-Asp solution in the above step, add it to 500 μL of goat oocyte in vitro maturation culture medium and shake well to obtain a goat oocyte in vitro maturation culture medium with a final concentration of 2.0 mM L-Asp solution.

[0041] The basic culture medium is prepared from TCM-199 containing 20% ​​estrus goat serum, 0.5 IU / ml FSH, 0.5 IU / ml LH, 1 μg / ml estradiol, 1 mM sodium pyruvate, 0.05 mM L-cysteine, 1% ITS, 20 ng / ml EGF and 25 μg / ml gentamicin.

[0042] Example 5 Preparation of goat oocyte in vitro maturation culture medium

[0043] Step 1: Take 0.3993 g of L-Asp powder and heat it to 55° C. with 100 mL of embryo water (Sigma, W1503) to fully dissolve it to obtain a 30 mM L-Asp solution.

[0044] Step 2: Take 25.0 μL of the L-Asp solution in the above step, add it to 500 μL of goat oocyte in vitro maturation culture medium and shake well to obtain a goat oocyte in vitro maturation culture medium with a final L-Asp solution concentration of 2.5 mM.

[0045] Example 6 Application of L-Asp in Promoting in Vitro Maturation of Goat Oocytes

[0046] S1, take 0.3328 g of L-Asp powder, heat it to 55°C with 50 mL of embryo water (Sigma, W1503) to fully dissolve it, and obtain an L-Asp solution with a concentration of 50 mM.

[0047] S2, taking 5.0 μL of the L-Asp solution in the above step, adding it to 500 μL of goat oocyte in vitro maturation culture medium and shaking well to obtain a goat oocyte in vitro maturation culture medium with a final L-Asp solution concentration of 0.5 mM.

[0048] The basic culture medium is prepared from TCM-199 containing 20% ​​estrus goat serum, 0.5 IU / ml FSH, 0.5 IU / ml LH, 1 μg / ml estradiol, 1 mM sodium pyruvate, 0.05 mM L-cysteine, 1% ITS, 20 ng / ml EGF and 25 μg / ml gentamicin.

[0049] S3, adding the goat oocyte in vitro maturation culture medium obtained in the above step S2 into a four-well plate with a pipette, covering it with paraffin oil and placing it in a 38.5°C, 5% CO2, 100% humidity incubator for preheating for a certain period of time, placing the oocytes of the intact and dense cumulus mass in a preheated four-well plate with goat oocyte in vitro maturation culture medium added and placing it in a 38.5°C, 5% CO2, 100% humidity incubator for in vitro maturation culture;

[0050] S4. Use a pipette to aspirate the oocytes after 22 hours of culture and place them in a 3.5 cm culture dish. Add 1 ml of 1% hyaluronidase and blow repeatedly to remove the granulosa cells around the oocytes. Under a stereomicroscope, pick out the mature oocytes that have expelled the first polar body (the first polar body is located between the zona pellucida and the oocyte membrane), put the mature oocytes back into the maturation fluid, and count the maturation of the oocytes.

[0051] Example 7 Application of L-aspartic acid in the preparation of a pharmaceutical preparation for promoting in vitro maturation of goat oocytes

[0052] L-Asp powder is added to conventional pharmaceutical excipients, and prepared into granules, tablets, capsules, injections and other pharmaceutical preparations according to conventional pharmaceutical preparation processes, and added to the basic culture medium of goat oocyte in vitro maturation culture medium. The culture medium is preheated using a four-well plate as a dish, and the complete and dense oocytes are placed in an incubator at 38.5°C, 5% CO2, and 100% humidity for in vitro maturation culture.

[0053] Test example:

[0054] In order to verify the effectiveness of the present invention, the invention team conducted a series of studies, as follows:

[0055] 1 Materials

[0056] 1.1 Animals

[0057] After collecting goat ovaries at the slaughterhouse, place them in a thermos bottle containing normal saline (500ml 9% normal saline + 1mL penicillin-streptomycin (10,000IU / mL)) supplemented with penicillin-streptomycin and transport them back to the laboratory within 1 hour. Rinse the collected ovaries three times with preheated normal saline to remove impurities and blood, and use scissors to remove fat masses, connective tissues and fallopian tubes on the surface of the ovaries, and wash the trimmed ovaries again with normal saline. Place the treated ovaries in a beaker of normal saline with 1mL penicillin-streptomycin (10,000IU / mL) and place it on a 37°C constant temperature table for use. Then use a 10mL syringe (with a No. 12 needle) to aspirate the contents of the follicles with a diameter of 2-5mm on the surface of the ovaries.

[0058] 1.2 Drugs and reagents

[0059] L-Asp was purchased from Sigma-Aldrich (Shanghai) Trading Co., Ltd.; ROS detection kit was purchased from Nanjing Jiancheng Bioengineering Institute, and mitochondrial membrane potential detection kit was purchased from Shanghai Bio-Tech Biotechnology Co., Ltd. 。

[0060] 2 Test methods

[0061] 2.1 Experimental Methods and Steps

[0062] Preparation of basal culture medium: TCM-199 prepared with 20% estrus goat serum, 0.5 IU / ml FSH, 0.5 IU / ml LH, 1 μg / ml estradiol, 1 mM sodium pyruvate, 0.05 mM L-cysteine, 1% ITS, 20 ng / ml EGF and 25 μg / ml gentamicin.

[0063] Methods: 0.3328g L-Asp powder was fully dissolved in 50mL embryo water heated at 55℃, and the concentration was 50mM. 5.0, 10.0, 15.0, 20.0, and 25.0μL L-Asp were added to 500μL basal culture medium and shaken well (the final concentrations were 0.5mM, 1mM, 1.5mM, 2.0mM, and 2.5mM, respectively). The culture medium was preheated in a four-well plate, and the oocytes with complete and dense cumulus were placed in an incubator at 38.5℃, 5% CO2, and 100% humidity for in vitro maturation culture.

[0064] 0.3328 g of L-Asp powder was fully dissolved in 50 mL of embryo water (Sigma, W1503) at 55°C to obtain a 50 mM L-Asp solution; 5.0, 10.0, 15.0, 20.0, and 25.0 μL of L-Asp were added to 500 μL of basal culture medium and shaken to obtain goat oocyte in vitro maturation culture medium with a final L-Asp concentration of 0.5 mM, 1 mM, 1.5 mM, 2.0 mM, and 2.5 mM, respectively; the culture medium was added to a four-well plate with a pipette and covered with paraffin oil and placed in a 38.5°C, 5% CO2, 100% humidity incubator for a certain period of time, and the oocytes of the intact and dense cumulus mass were placed in the preheated four-well plate with goat oocyte in vitro maturation culture medium added. The oocytes were placed in a well plate and placed in an incubator at 38.5°C, 5% CO2, and 100% humidity for in vitro maturation culture; the oocytes after 22 hours of culture were aspirated with a pipette and placed in a 3.5 cm culture dish and 1 ml of 1% hyaluronidase was added and repeatedly blown for several times to remove the granulosa cells around the oocytes, and the mature oocytes that expelled the first polar body (the first polar body was located between the zona pellucida and the oocyte membrane) were picked out under a stereomicroscope, the mature oocytes were put back into the maturation solution, and the maturation of the oocytes was counted.

[0065] 2.2 Analysis methods

[0066] (1) Oocyte maturation rate

[0067] After in vitro culture for about 22 hours, the oocytes cultured in vitro were aspirated and added to a culture dish containing 0.1% hyaluronidase, and the granulosa cells around the oocytes were separated by blowing and suction with a pipette for about 3 minutes. All the digested oocytes were aspirated with a pipette, added to the pre-prepared operating liquid dish and washed 3 times, and the field of view and pupil distance of the microscope were adjusted for observation, and the oocytes that had discharged the first polar body were selected (the oocytes that discharged the first polar body were regarded as mature oocytes), and the selected mature oocytes were placed in 200 μL operating liquid droplets, covered with paraffin oil, and the experiment was continued.

[0068] (2) Detection of ROS levels in oocytes

[0069] The ROS level of oocytes was measured using a reactive oxygen species assay kit (E004-1-1, njjcbio, China), and the operation and detection were carried out strictly in accordance with the instructions of each kit. The specific operation is as follows: 2.0mM L-Asp treatment group and control group, 5-8 mature oocytes were selected from each group and pipetted into DPBS-0.1% PVA solution for washing, and then the oocytes were transferred into DCFH-DA staining drops (the dye concentration was 10μM) and incubated in a light-proof incubator at 37℃ for 30min. Then wash with DPBS-0.1% PVA solution 3 times, 5min each time, and observe and photograph under an inverted fluorescence microscope. The fluorescence value of the oocytes was processed by Image-J software, and the mean fluorescence intensity Mean=IntDen / Area was calculated. (The mean fluorescence intensity values ​​of the two groups were subjected to an independent sample T test, and the mean fluorescence intensity of the control group was reset to 1 to draw a statistical graph.)

[0070] (3) Detection of mitochondrial membrane potential of oocytes

[0071] 8-15 mature oocytes were selected from each experimental group and control group, and transferred to DPBS-0.1% PVA solution for washing three times to remove the maturation culture medium. Subsequently, the oocytes were transferred to TMRE staining solution (10 μM concentration) and incubated at 37°C in the dark for 30 minutes. After the incubation, the oocytes were washed 3 times with DPBS-0.1% PVA solution for 5 minutes each time to remove the TMRE dye. The samples were placed under a confocal microscope, the fluorescent dye was excited with a specific wavelength, and the same parameters were set to take pictures. The fluorescence images of the oocytes were processed by Image-J software, and the mean fluorescence intensity (Mean=IntDen / Area) was calculated. An independent sample T test was performed on the mean fluorescence intensity values ​​of the two groups, and the mean fluorescence intensity of the control group was reset to 1 to draw a statistical graph.

[0072] 3 Results

[0073] 3.1 Effect of adding different concentrations of L-Asp to goat in vitro maturation medium on the in vitro maturation rate of oocytes

[0074] Results Figure 1 .

[0075] Depend on Figure 1 A shows that the average maturation rate of the blank control group without addition is 59.57% ± 4.16%; the average maturation rate of the group adding 0.5mM L-Asp is 65.66% ± 5.12%, the average maturation rate of the group adding 1mM L-Asp is 58.98% ± 9.02%, the average maturation rate of the group adding 1.5mM L-Asp is 58.92% ± 8.87%, the average maturation rate of the group adding 2mM L-Asp is 76.66% ± 4.07%, and the average maturation rate of the group adding 2.5mM L-Asp is 61.81% ± 7.87%. Compared with the blank control group, the maturation rate of the group adding 2.0mM L-Asp increased by 17.09%, promoting oocyte maturation, Figure 1 B shows that compared with the control group, most of the cumulus cells expanded significantly better. Based on the above considerations, the subsequent experiment selected 2.0mM L-Asp treatment as the experimental group to continue the research.

[0076] 3.2 Effect of adding 2.0 mM L-Asp to goat in vitro maturation medium on ROS in oocytes Figure 2 .

[0077] like Figure 2 As shown in A and B, the fluorescence intensity of oocytes in the L-Asp treatment group was lower than that in the control group. Compared with the control group, the intracellular ROS content in the 2.0 mM L-Asp treatment group was significantly reduced (P<0.05), thereby significantly reducing the oxidative stress suffered by oocytes.

[0078] Depend on Figure 2 It can be seen that adding 2.0 mM L-Asp to the goat in vitro maturation culture medium can significantly reduce the ROS level in oocytes.

[0079] 3.3 Effect of adding 2.0 mM L-Asp to goat in vitro maturation medium on oocyte mitochondrial membrane potential Figure 3 .

[0080] like Figure 3 As shown in A and B, TMRE aggregates in mitochondria and emits bright orange-red fluorescence, which is significantly higher in the experimental group than in the control group. Therefore, the addition of 2.0 mM L-Asp can significantly enhance the mitochondrial membrane potential.

[0081] Depend on Figure 3 It can be seen that adding 2.0 mM L-Asp to the goat in vitro maturation culture medium can significantly increase the mitochondrial membrane potential of oocytes.

[0082] Although the present invention has been described in detail above by general explanation and specific implementation methods, it is obvious to those skilled in the art that some modifications or improvements can be made on the basis of the present invention. Therefore, these modifications or improvements made on the basis of not departing from the spirit of the present invention all belong to the scope of protection claimed by the present invention.

[0083] References:

[0084] [1]Ka HH,Sawai K,Wang WH,Im KS,Niwa K.Amino acids in maturationmediumand presence of cumulus cells at fertilization promote male pronuclearformation inporcine oocytes matured and penetrated in vitro[J].Biol Reprod,1997,57(6):1478-1483.

[0085] [2]Gardner DK, Lane M.Amino acids and ammonium regulate mouseembryodevelopment in culture[J].Biol Reprod,1993,48(2):377-385.

[0086] [3]Gardner DK, Lane M, Spitzer A, Batt PA. Enhanced rates of cleavage and development for sheep zygotes cultured to the blastocyst stage in vitro in the absence of serum and somatic cells: amino acids, vitamins, and culturingembryos in groups stimulate development [J]. Biol Reprod, 1994, 50(2): 390-400.

[0087] [4]Liu Z,Foote RH.Effects of amino acids on the development of in-vitro matured / in-vitro fertilization bovine embryos in a simple protein-freemedium[J].Hum Reprod,1995,10(11):2985-2991.

[0089] [5]D'Aniello G,Grieco N,Di Filippo MA,et al.Reproductive implicationof D-asparticacid in human pre-ovulatory follicular fluid[J].Hum Reprod,2007,22(12):3178-3183.

[0090] [6]Deleuze S,Goudet G.Cysteamine supplementation of in vitromaturation media:areview[J].Reprod Domest Anim,2010,45(6):e476-482.

[0091] [7]Rezaei N,Chian R.Effects of essential and non-essential aminoacids on in-vitromaturation,fertilization and development of immature bovineoocytes[J].2005.

[0092] [8]Sinclair K,Lunn L,Kwong W,Wonnacott K,Linforth R,Craigon J.Aminoacid andfatty acid composition of follicular fluid as predictors of in-vitroembryodevelopment[J].Reproductive biomedicine online,2008,16(6):859-868.

[0093] [9]Hemmings KE,Leese HJ,Picton HM.Amino acid turnover by bovineoocytesprovides an index of oocyte developmental competence in vitro[J].Biology ofreproduction,2012,86(5):165,161-112.

Claims

1. An application of L-aspartic acid in preparing a goat oocyte in vitro maturation culture medium.

2. The use of L-aspartic acid in preparing a goat oocyte in vitro maturation culture medium as claimed in claim 1, It is characterized in that The goat oocyte in vitro maturation culture medium consists of an L-aspartic acid solution and a basic culture medium.

3. The use of L-aspartic acid in preparing a goat oocyte in vitro maturation culture medium as claimed in claim 2, It is characterized in that The L-aspartic acid solution is obtained by heating L-aspartic acid powder with embryo water to 55° C. to fully dissolve it.

4. The use of L-aspartic acid in preparing a goat oocyte in vitro maturation culture medium as claimed in claim 2, It is characterized in that The basic culture medium is prepared from TCM-199 containing 20% ​​estrus goat serum, 0.5 IU / ml FSH, 0.5 IU / ml LH, 1 μg / ml estradiol, 1 mM sodium pyruvate, 0.05 mM L-cysteine, 1% ITS, 20 ng / ml EGF and 25 μg / ml gentamicin.

5. The use of L-aspartic acid in preparing a goat oocyte in vitro maturation culture medium as claimed in claim 2, It is characterized in that The final concentration of the L-aspartic acid solution in the goat oocyte in vitro maturation culture medium is 0.5-2.5 mM.

6. The use of L-aspartic acid in preparing a goat oocyte in vitro maturation culture medium as claimed in claim 5, It is characterized in that The final concentration of the L-aspartic acid solution in the goat oocyte in vitro maturation culture medium is 2.0 mM.

7. The use according to any one of claims 1 to 6, It is characterized in that After the following steps: S1, heating L-aspartic acid powder with embryo water at 55°C to fully dissolve it to obtain L-aspartic acid solution for later use; S2, taking the L-aspartic acid solution obtained in the above step S1, adding it to the basal culture medium and shaking it well to form a goat oocyte in vitro maturation culture medium with a final L-Asp concentration of 2.0 mM; S3, the goat oocyte in vitro maturation culture medium obtained in step S2 above was added to a four-well plate using a pipette and covered with paraffin oil and placed in a 38.5°C, 5% CO 2 After preheating the incubator with 100% humidity for a certain period of time, the oocytes of the intact and dense cumulus mass were placed in a preheated four-well plate supplemented with goat oocyte in vitro maturation culture medium and placed in a 38.5℃, 5% CO 2 , 100% humidity incubator for in vitro maturation culture; S4. Use a pipette to aspirate the oocytes after 22 hours of culture and place them in a 3.5 cm culture dish. Add 1 ml of 1% hyaluronidase and blow repeatedly to remove the granulosa cells around the oocytes. Pick out the mature oocytes that have expelled the first polar body under a stereomicroscope, put the mature oocytes back into the maturation medium, and count the maturation of the oocytes.

8. Use of the L-aspartic acid as claimed in claim 1 in the preparation of a pharmaceutical preparation for promoting in vitro maturation of goat oocytes.

9. Use of L-aspartic acid as claimed in claim 8 in the preparation of a pharmaceutical preparation for promoting in vitro maturation of goat oocytes, It is characterized in that The pharmaceutical preparation contains 2.0 mM L-aspartic acid.

10. Use of L-aspartic acid as claimed in any one of claims 8 or 9 in the preparation of a pharmaceutical preparation for promoting in vitro maturation of goat oocytes, It is characterized in that The pharmaceutical preparations include but are not limited to injections, capsules, granules, and tablets.

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