CN117264878A - Culture solution added with echinacoside for improving porcine oocyte aging and application thereof - Google Patents

Abstract

The invention discloses a culture solution for improving aging of porcine oocytes by adding echinacoside and application thereof, and the culture solution comprises a basal culture solution, D-galactose and echinacoside, wherein the basal culture solution is based on an M199 culture solution which does not contain HEPES and has the volume fraction of 82.9%, 10% of porcine follicular fluid and 5% of fetal bovine serum are added, and meanwhile, 1% of L cysteine with the volume fraction of 6.9mg/mL, 0.1% of epidermal growth factor with the volume fraction of 10 mug/mL, 1% of gonadotropin with the volume fraction of 10IU/mL and chorionic gonadotrophin with the volume fraction of 10IU/mL are added, the addition of D-galactose is 9mg/mL, and the addition of the echinacoside is 39.337 mug/mL or 78.673 mug/mL. According to the invention, a damage model is built by adding 9 mg/mLD-galactose into a basic culture solution, and 78.673 mug/mL of echinacoside is added on the basis, so that the maturation rate of oocytes can be remarkably improved.

Description

A culture medium that adds echinaceaside to improve the aging of porcine oocytes and its application

Technical field

The invention relates to an in vitro maturation culture medium for pig oocytes, specifically a culture medium for adding echinacea to improve the aging of pig oocytes and its application, and belongs to the application field of livestock reproduction technology.

Background technique

Porcine oocyte in vitro maturation culture (IVM), as one of the ways to produce pig embryos, is an important link in livestock reproduction technology. The quality of in vitro mature oocytes affects methods including in vitro fertilization, parthenogenetic activation and embryo cloning. Multiple technical links. Since the existing in vitro culture system cannot completely restore the in vivo microenvironment, oocytes matured in vitro have significantly different maturation rates, maturation quality and developmental potential than oocytes matured in vivo. Therefore, a more stable oocyte is established. An in vitro culture system with a higher mother cell maturation rate is urgent.

At present, it is not clear what core factors are added during in vitro maturation that are beneficial to oocyte development. It is generally believed that factors that contribute to oocyte maturation include hormones, growth factors, cytokines and other components.

Therefore, providing an in vitro maturation culture medium for porcine oocytes and its application is an urgent problem that those skilled in the art need to solve.

D-galactose is an aldohexose, a reducing sugar that occurs naturally in the body and many foods. At high levels, it can be converted into aldose and hydrogen peroxide under the catalysis of galactose oxidase. , thereby producing reactive oxygen species (ROS). This may subsequently lead to oxidative stress, inflammation, mitochondrial dysfunction, and apoptosis. It has become the most popular drug for establishing aging models due to its convenience, small side effects, and high survival rate.

Echinacea is a natural phenylethanoid glycoside and the main active ingredient in Cistanche deserticola. Studies have shown that echinacea plays a role in neuroprotection, anti-tumor, anti-aging, myocardial remodeling, antioxidant, anti-inflammation, and anti-apoptosis. has a beneficial effect. Currently, there are no studies on the combined addition of echinaceaside during the in vitro maturation of porcine oocytes. It is of great significance to explore whether the addition of echinaceaside can significantly improve the in vitro maturation efficiency of porcine oocytes, thereby improving the quality of porcine oocytes.

Contents of the invention

The purpose of the present invention is to solve the above problems and provide a culture medium that adds echinacea to improve the aging of porcine oocytes and its application.

The present invention achieves the above object through the following technical solution: a culture medium that adds echinaceaside to improve the aging of porcine oocytes, including a basic culture medium, D-galactose (Sigma, G5388) and echinaside (MCE, HY-N0020), the basic culture medium is based on 82.9% volume fraction M199 culture medium (Sigma, M4530) without HEPES, with 10% volume fraction of porcine follicular fluid and 5% volume fraction of fetal bovine serum added (Cellmax, SA311.01), while supplementing 1% volume fraction of 6.9 mg/mL L-cysteine (Sigma, C7880), 0.1% volume fraction of 10 μg/mL epidermal growth factor (Sigma, E4127), 1 % volume fraction of a mixture of 10 IU/mL gonadotropin and 10 IU/mL chorionic gonadotropin (Ningbo Sansheng), the added amount of D-galactose is 9 mg/mL, the added amount of echinaceaside The amount is 39.337μg/mL or 78.673μg/mL,

The preparation method of the culture medium for adding echinaceaside to improve the aging of porcine oocytes is specifically:

SA. Add 9 mg/mL D-galactose to the basic culture medium to establish an in vitro culture medium model of porcine oocyte aging;

SB. Dissolve 5 mg echinaceaside in 127.108 μL of DMSO to obtain a 50mM concentrated stock solution, and then use the aging in vitro culture medium of porcine oocytes prepared in SA to dissolve the 50mM echinaceaside concentrated stock solution respectively. Dilute to 78.673μg/mL, 39.337μg/mL;

SC. Add 39.337 μg/mL or 78.673 μg/mL echinacea to the aging in vitro culture medium model of porcine oocytes prepared in SA, and finally obtain a culture in which echinacea is added to improve the aging of porcine oocytes. liquid.

Preferably, the amount of echinaceaside added to the culture medium for improving the aging of porcine oocytes is 78.673 μg/mL.

Preferably, the molecular formula of echinaceaside is C ₃₅ H ₄₅ O ₂₀ , its molecular weight is 786.73, and its structural formula is as follows:

Preferably, the preparation process of the porcine follicular fluid is:

Take fresh ovaries from pigs and place them in a 38.5°C physiological saline thermos. Use an 18G needle syringe to extract the follicular fluid from the follicles on the ovarian surface. Collect the extracted follicular fluid in a 15mL centrifuge tube and place it in a 38.5°C incubator for 30 minutes. , collect the upper layer of follicular fluid and centrifuge it at 3000 rpm for 30 minutes. The lower layer contains cumulus granulosa cell oocyte complexes (COCs). Filter the centrifuged supernatant with a 0.22 μm filter. The filtered porcine follicular fluid is aliquoted and stored at 20°C. save.

Preferably, follicles with a diameter of 2 to 8 mm are selected during the extraction of follicular fluid.

The invention also discloses the application of a culture medium that adds echinaceaside to improve the aging of pig oocytes, specifically the application of echinaceaside in improving the maturity rate and development quality of damaged pig oocytes, including the use of echinaceaside in the aging of pig oocytes. Study on the expansion degree of mother cell cumulus, first pole efflux in porcine oocyte MII stage, and spindle and chromosome morphology after porcine oocyte maturity. The experimental design is as follows:

Control group: in vitro maturation culture medium without D-galactose and echinacea.

Based on the HEPES-free 82.9% volume fraction M199 culture medium, 10% volume fraction of porcine follicular fluid and 5% volume fraction of fetal bovine serum were added, and at the same time, 1% volume fraction of L-half at 6.9 mg/mL was added. A mixture of cystine, 0.1% epidermal growth factor with a volume fraction of 10 μg/mL, 1% gonadotropin with a volume fraction of 10 IU/mL, and chorionic gonadotropin with a volume fraction of 10 IU/mL;

D-galactose control group: in vitro maturation culture medium containing 9mg/mL D-galactose

Based on the HEPES-free 82.9% volume fraction M199 culture medium, 10% volume fraction of porcine follicular fluid and 5% volume fraction of fetal bovine serum were added, and at the same time, 1% volume fraction of L-half at 6.9 mg/mL was added. Cystine, 0.1% volume fraction 10 μg/mL epidermal growth factor, 1% volume fraction 10IU/mL gonadotropin and 10IU/mL mixture of chorionic gonadotropin, 9mg/mL D-galactose;

Echinaceaside group 1: in vitro maturation culture medium containing 39.337μg/mL echinaceaside

Based on the HEPES-free 82.9% volume fraction M199 culture medium, 10% volume fraction of porcine follicular fluid and 5% volume fraction of fetal bovine serum were added, and at the same time, 1% volume fraction of L-half at 6.9 mg/mL was added. Cystine, 0.1% volume fraction 10 μg/mL epidermal growth factor, 1% volume fraction 10 IU/mL gonadotropin and 10 IU/mL mixture of chorionic gonadotropin, 9 mg/mL D-galactose, 39.337 μg /mL of echinaceaside;

Echinaceaside group 2: in vitro maturation culture medium containing 78.673μg/mL echinaceaside

Based on the HEPES-free 82.9% volume fraction M199 culture medium, 10% volume fraction of porcine follicular fluid and 5% volume fraction of fetal bovine serum were added, and at the same time, 1% volume fraction of L-half at 6.9 mg/mL was added. Cystine, 0.1% volume fraction 10 μg/mL epidermal growth factor, 1% volume fraction 10 IU/mL gonadotropin and 10 IU/mL mixture of chorionic gonadotropin, 9 mg/mL D-galactose, 78.673 μg /mL of echinaceaside.

Preferably, in the research experiment on the degree of cumulus expansion of porcine oocytes and the excretion of the first pole of porcine oocytes in the MII stage, a control group, a D-galactose control group, an echinaceaside group 1, and an echinacea group are set. Echinaceaside group 2, conducted three independent repeated experiments, each repeated testing of 60 MII stage oocytes, cultured for 42-48h; in the research experiment on the spindle and chromosome morphology of porcine oocytes after maturation, echinaceaside was eliminated Group 1, retain the control group, D-galactose control group and echinacea glycoside group 2. Each group of experiments is set up with three independent repetitions. Each repetition detects 15-20 MI stage oocytes and culture them for 24-28 hours. The results Difference statistics were analyzed using GraphPad Prism statistical software.

The beneficial effects of the present invention are: compared with the prior art, the present invention provides a culture medium that adds echinacea to improve the aging of porcine oocytes and its application. By adding 9 mg/mLD-half to the basic culture medium, Establishing a lactose injury model, and adding 78.673 μg/mL echinaceaside at the same time can significantly improve the maturation rate of oocytes by nearly 20%, and the normal proportion of spindles by nearly 26%. This program It can effectively obtain a large number of high-quality oocytes, provide support for technologies such as in vitro fertilization, parthenogenetic activation, and nuclear transfer to prepare cloned embryos, and play an important role in the propagation of fine livestock, construction of animal models, and production of transgenic animals.

Description of the drawings

Figure 1 shows the degree of cumulus expansion of porcine oocytes with or without echinaceaside added to the culture medium during the in vitro maturation of porcine oocytes of the present invention: the control group, the D-galactose control group, and the echinaceaside in that order. Group 1 (39.337 μg/mL), echinaceaside group 2 (78.673 μg/mL); the scale bar of the original picture is 200 μm; the scale bar of the enlarged picture is 50 μm.

Figure 2 shows the efflux of the first pole in the MII stage of porcine oocytes in the culture medium with or without the addition of echinaceaside during the in vitro maturation process of porcine oocytes of the present invention;

Among them, A is the control group, D-galactose control group, echinaceaside group 1 (39.337 μg/mL echinaceaside is added to the basal culture medium), and echinaceaside group 2 (78.673 μg is added to the basal culture medium). /mL Echinaceaside) The first polar efflux of porcine oocytes in the MI stage. The arrow points to the first polar body. The scale bar of the original image is 200 μm; the scale bar of the enlarged image is 50 μm.

B is the control group, D-galactose control group, echinaceaside group 1 (39.337 μg/mL echinaceaside is added to the basal culture medium), and echinacoside group 2 (78.673 μg/mL is added to the basal culture medium). Echinaceaside) Percentage of first pole efflux of porcine oocytes at MI stage.

Figure 3 shows the spindle and chromosome morphology of mature porcine oocytes with or without echinaceaside added to the culture medium during the in vitro maturation process of porcine oocytes of the present invention;

Among them, A is the spindle morphology of MI stage porcine oocytes in the control group, D-galactose control group, and echinaceaside group 2 (78.673 μg/mL echinaceaside was added to the basal culture medium), using a confocal microscope Photographed; scale bar is 20 μm;

B is the percentage of normal spindle morphology of MI stage porcine oocytes in the control group, D-galactose control group, and echinaceaside group 2 (78.673 μg/mL echinaceaside was added to the basal culture medium);

Detailed ways

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only some of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts fall within the scope of protection of the present invention.

The reagents used in the present invention are as follows:

D-galactose: purchased from Sigma, model number: G5388;

Echinaceaside: purchased from MCE, model: HY-N0020;

M199 culture medium: purchased from Sigma, model: M4530;

5% volume fraction of fetal bovine serum: purchased from Cellmax, model: SA311.01;

L-cysteine: purchased from Sigma, model: C7880;

Epidermal growth factor: purchased from Sigma, model number: E4127;

Mixed solution of gonadotropin and chorionic gonadotropin: purchased from Ningbo Sansheng;

Hyaluronidase solution: purchased from Sigma, model: H3506;

anti-α-tubulin-FITC: purchased from Sigma, model: F2168);

Fixative: purchased from Sigma, model number: 818715;

Permeabilizing fluid: purchased from Solebao, model number: 9002-93-1;

Blocking solution: purchased from Sigma, model: A1933;

washing buffer: purchased from Biyuntian, model P0106;

Hoechst 33342: purchased from Thermo Fisher, model number 2189158.

The instruments and equipment used in this invention are as follows:

Stereo microscope: purchased from Olympus, model SZX7;

Confocal microscope: purchased from Olympus, model FV3000.

Example 1 Adding echinaceaside to culture medium to improve aging of porcine oocytes and its preparation method

A culture medium that adds echinaceaside to improve the aging of porcine oocytes, including a basic culture medium, D-galactose and echinaceaside. The base culture medium is an 82.9% volume fraction M199 culture medium that does not contain HEPES. As a base, add 10% volume fraction of porcine follicular fluid and 5% volume fraction of fetal bovine serum. At the same time, add 1% volume fraction of L-cysteine at 6.9 mg/mL and 0.1% volume fraction of epidermis at 10 μg/mL. A mixed solution of growth factors, 1% volume fraction of gonadotropins and 10 IU/mL of chorionic gonadotropin, the added amount of D-galactose is 9 mg/mL, and the added amount of echinaceaside is 39.337μg/mL or 78.673μg/mL.

The preparation method of the culture medium for adding echinaceaside to improve the aging of porcine oocytes is specifically:

SA. Add 9 mg/mL D-galactose to the basic culture medium to establish an in vitro culture medium model of porcine oocyte aging;

SB. Dissolve 5 mg echinaceaside in 127.108 μL of DMSO to obtain a 50mM concentrated stock solution, and then use the aging in vitro culture medium of porcine oocytes prepared in SA to dissolve the 50mM echinaceaside concentrated stock solution respectively. Dilute to 78.673μg/mL, 39.337μg/mL;

SC. Add 39.337 μg/mL or 78.673 μg/mL echinacea to the aging in vitro culture medium model of porcine oocytes prepared in SA, and finally obtain a culture in which echinacea is added to improve the aging of porcine oocytes. liquid.

In the above embodiments, the molecular formula of echinaceaside is C ₃₅ H ₄₅ O ₂₀ , its molecular weight is 786.73, and its structural formula is as follows:

In the above embodiment, the preparation process of porcine follicular fluid is specifically as follows:

Take fresh ovaries from pigs and place them in a 38.5°C physiological saline thermos. Use an 18G needle syringe to extract the follicular fluid from the follicles on the ovarian surface. During the process of extracting follicular fluid, select follicles with a diameter of 2 to 8 mm. Collect the extracted follicular fluid in a 15 mL centrifuge. tube and placed in a 38.5°C incubator for 30 minutes. The upper layer of follicular fluid was collected and centrifuged at 3000 rpm for 30 minutes. The lower layer was cumulus granulosa cell oocyte complexes (COCs). The centrifuged supernatant was filtered with a 0.22 μm filter. , aliquot the filtered porcine follicular fluid and store it at 20°C.

Example 2 Application of adding echinacea to culture medium to improve aging of porcine oocytes

2.1 Experimental design

Control group: in vitro maturation culture medium without D-galactose and echinacea.

Based on the HEPES-free 82.9% volume fraction M199 culture medium, 10% volume fraction of porcine follicular fluid and 5% volume fraction of fetal bovine serum were added, and at the same time, 1% volume fraction of L-half at 6.9 mg/mL was added. A mixture of cystine, 0.1% epidermal growth factor with a volume fraction of 10 μg/mL, 1% gonadotropin with a volume fraction of 10 IU/mL, and chorionic gonadotropin with a volume fraction of 10 IU/mL;

D-galactose control group: in vitro maturation culture medium containing 9mg/mL D-galactose

Based on the HEPES-free 82.9% volume fraction M199 culture medium, 10% volume fraction of porcine follicular fluid and 5% volume fraction of fetal bovine serum were added, and at the same time, 1% volume fraction of L-half at 6.9 mg/mL was added. Cystine, 0.1% volume fraction 10 μg/mL epidermal growth factor, 1% volume fraction 10IU/mL gonadotropin and 10IU/mL mixture of chorionic gonadotropin, 9mg/mL D-galactose;

Echinaceaside group 1: in vitro maturation culture medium containing 39.337μg/mL echinaceaside

Based on the HEPES-free 82.9% volume fraction M199 culture medium, 10% volume fraction of porcine follicular fluid and 5% volume fraction of fetal bovine serum were added, and at the same time, 1% volume fraction of L-half at 6.9 mg/mL was added. Cystine, 0.1% volume fraction 10 μg/mL epidermal growth factor, 1% volume fraction 10 IU/mL gonadotropin and 10 IU/mL mixture of chorionic gonadotropin, 9 mg/mL D-galactose, 39.337 μg /mL of echinaceaside;

Echinaceaside group 2: in vitro maturation culture medium containing 78.673μg/mL echinaceaside

Based on the HEPES-free 82.9% volume fraction M199 culture medium, 10% volume fraction of porcine follicular fluid and 5% volume fraction of fetal bovine serum were added, and at the same time, 1% volume fraction of L-half at 6.9 mg/mL was added. Cystine, 0.1% volume fraction 10 μg/mL epidermal growth factor, 1% volume fraction 10 IU/mL gonadotropin and 10 IU/mL mixture of chorionic gonadotropin, 9 mg/mL D-galactose, 78.673 μg /mL of echinaceaside.

2.2 Research on the degree of cumulus expansion of porcine oocytes and the efflux of the first pole of porcine oocytes during the MII stage

2.2.1 Preliminary treatment of pig oocytes

Take fresh ovaries from pigs and place them in a 38.5°C physiological saline thermos. Use an 18G needle syringe to extract the follicular fluid from the follicles on the ovarian surface. During the process of extracting follicular fluid, select follicles with a diameter of 2 to 8 mm. Collect the extracted follicular fluid in a 15 mL centrifuge. tube and place it in a 38.5°C incubator for 30 minutes. After standing, the upper layer is follicular fluid and the lower layer is cumulus granulosa cell oocyte complex (COCs). The upper layer of follicular fluid is discarded, leaving the lower layer of COCs. Add and precipitate Use an equal volume of DPBS as the porcine oocyte operating fluid, mix by inversion, pour into a 60mm petri dish, select under a stereomicroscope, use a 500μm glass needle to select COCs with three or more layers of cumulus cells. Wash three times in in vitro maturation culture medium containing echinaceaside and D-galactose, and then move to the control or echinaceaside group for maturation culture.

2.2.2 In vitro maturation culture of porcine oocytes

Control group: Add in vitro maturation culture medium without echinaceaside and D-galactose to a 4-well plate, add 500 μL of culture medium to each well, cover with 300 μL of paraffin oil, and incubate at 38.5°C, 5% CO ₂ , saturated Preheat in a humid incubator for more than 5 hours, place the selected and washed COCs in mature wells, 60 COCs per well, and culture for 42-48 hours.

D-galactose control group: Add the in vitro maturation culture medium containing 9 mg/mL D-galactose to a 4-well plate, add 500 μL of culture medium to each well, cover with 300 μL of paraffin oil, and incubate at 38.5°C, 5% CO ₂ , Preheat in an incubator with saturated humidity for more than 5 hours. Place the selected and washed COCs in mature wells, 60 COCs per well, and culture for 42-48 hours.

Echinacea group 1: Add the in vitro maturation culture medium containing 9 mg/mL D-galactose and 39.337 μg/mL echinacea to a 4-well plate, add 500 μL of culture medium to each well, and cover with 300 μL of paraffin oil. Preheat for more than 5 hours in an incubator at 38.5°C, 5% CO ₂ and saturated humidity. Place the selected and washed COCs in mature wells, 60 pieces per well, and culture them for 42-48 hours.

Echinacea group 2: Add the in vitro maturation culture medium containing 9 mg/mL D-galactose and 78.673 μg/mL echinacea to a 4-well plate, add 500 μL of culture medium to each well, and cover with 300 μL of paraffin oil. Preheat in an incubator at 38.5°C, 5% CO ₂ and saturated humidity for more than 5 hours. Place the selected and washed COCs in mature wells, 60 pieces per well, and culture them for 42-48 hours.

2.2.3 MII porcine oocyte collection

Use TCM-199 as the solvent to prepare a hyaluronidase solution with a concentration of 1 mg/mL. Use a glass needle with a diameter of 200 μm to absorb the mature COCs and put them into 200 μL of the 1 mg/mL hyaluronidase solution, and use a 100 μL pipette Gently blow with the gun until the oocytes with sufficient cumulus granules can be seen under the stereomicroscope. Use a 200 μm glass needle to transfer the oocytes to the TCM-199 solution and wash them three times. Each wash must be performed in clean TCM-199. , and then use a 200 μm glass needle under a stereomicroscope to select the oocytes that discharge the first polar body, which are the mature MII stage oocytes.

The above method is applicable to the control group, D-galactose control group, echinaceaside group 1 and echinaceaside group 2.

Maturation rate = number of oocytes expelling the first polar body (MII stage)/total number of oocytes.

Table 1 MII stage oocyte maturation rate in different experimental groups

The results are shown in Figures 1 and 2. Compared with the D-galactose control group, when 78.673 μg/mL echinacea was added to the culture medium, the maturation rate was significantly increased, and the cumulus cells expanded more fully, and the eggs In the MII stage of the mother cell, the first pole is excreted more obviously.

The test results showed that adding 78.673 μg/mL echinaceaside to the basic culture medium had a better effect. In subsequent tests, the content of echinaside in group 2 was used to add echinaside.

2.3 Study on the spindle and chromosome morphology of mature porcine oocytes

2.3.1 Experimental design

Control group: in vitro maturation culture medium without D-galactose and echinacea.

Based on the HEPES-free 82.9% volume fraction M199 culture medium, 10% volume fraction of porcine follicular fluid and 5% volume fraction of fetal bovine serum were added, and at the same time, 1% volume fraction of L-half at 6.9 mg/mL was added. A mixture of cystine, 0.1% epidermal growth factor with a volume fraction of 10 μg/mL, 1% gonadotropin with a volume fraction of 10 IU/mL, and chorionic gonadotropin with a volume fraction of 10 IU/mL;

D-galactose control group: in vitro maturation culture medium containing 9mg/mL D-galactose

Based on the HEPES-free 82.9% volume fraction M199 culture medium, 10% volume fraction of porcine follicular fluid and 5% volume fraction of fetal bovine serum were added, and at the same time, 1% volume fraction of L-half at 6.9 mg/mL was added. Cystine, 0.1% volume fraction 10 μg/mL epidermal growth factor, 1% volume fraction 10IU/mL gonadotropin and 10IU/mL mixture of chorionic gonadotropin, 9mg/mL D-galactose;

Echinaceaside group 2: in vitro maturation culture medium containing 78.673μg/mL echinaceaside

Based on the HEPES-free 82.9% volume fraction M199 culture medium, 10% volume fraction of porcine follicular fluid and 5% volume fraction of fetal bovine serum were added, and at the same time, 1% volume fraction of L-half at 6.9 mg/mL was added. Cystine, 0.1% volume fraction 10 μg/mL epidermal growth factor, 1% volume fraction 10 IU/mL gonadotropin and 10 IU/mL mixture of chorionic gonadotropin, 9 mg/mL D-galactose, 78.673 μg /mL of echinaceaside.

The above control group, D-galactose control group and echinacea glycoside group 2 were all set up in three repeated groups, and 15 to 20 MI stage oocytes were repeatedly detected in each group. The difference statistics of the results were analyzed using GraphPad Prism statistical software.

2.3.2 In vitro maturation culture of porcine oocytes

Refer to the above embodiment 2.2.2.

2.3.3 MI stage oocyte collection

Use TCM-199 as the solvent to prepare a hyaluronidase solution with a concentration of 1 mg/mL. Use a glass needle with a diameter of 200 μm to absorb the COCs cultured for 24-28 h and put them into 200 μL of the 1 mg/mL hyaluronidase solution, and use a 100 μL pipette. Gently blow with the liquid gun until the oocytes with fully stripped cumulus granules can be seen under the microscope. Use a glass needle to transfer the oocytes to the TCM-199 solution and wash them three times. Each wash must be carried out in clean TCM-199. The resulting cells are MI stage oocytes.

The above method is applicable to the control group, D-galactose control group and echinacea glycoside group 2.

2.3.4 Detection of spindle and chromosome morphology of porcine oocytes

Anti-α-tubulin-FITC was used to detect the spindle and chromosome morphology of mature porcine oocytes. MI stage oocytes obtained from the control group, D-galactose control group and echinacea group 2 were fixed in The solution was left to stand at room temperature for 30 minutes, washed three times with washing solution, and then transferred to permeabilization solution and left to stand at room temperature for 8 hours. Afterwards, they were moved to the blocking solution and left to stand at room temperature for 1 hour. The MI stage oocytes in the control group, D-galactose control group and echinacea group 2 after standing at room temperature were placed in a container containing anti-α-tubulin-FITC ( 1:800) solution, incubate overnight at 4°C in a humidified box, then wash the MI stage oocytes three times with washing buffer, no less than 3 minutes each time, use Hoechst 33342 to stain the nuclei, and use a confocal microscope to Observe below. The results are shown in Figure 3.

As can be seen from Figure 3, the normal spindle rate in the control group was 70.07±2.754%; the normal spindle rate in the D-galactose control group was 30.60±7.002%; the normal spindle rate in the echinacea group 2 was 57.13±7.150 %, compared with the D-galactose control group, when 78.673 μg/mL echinaceaside was added to the porcine oocyte basal culture medium, the spindle normal rate significantly increased, the spindle morphology was significantly improved, and the spindle normal rate The increase was about 26%, while the degree of chromosome arrangement disorder was reduced.

In summary, the present invention provides a culture medium that adds echinaceaside to improve the aging of porcine oocytes and its application. The damage model is established by adding 9 mg/mL D-galactose to the basic culture medium, and on this basis The addition of 78.673 μg/mL echinaceaside can significantly increase the maturation rate of oocytes by nearly 20%, and the normal proportion of spindles by nearly 26%. This program can effectively obtain a large number of high-quality oocytes. , provides support for technologies such as in vitro fertilization, parthenogenetic activation, and nuclear transfer to prepare cloned embryos, and plays an important role in the expansion of breeding livestock, construction of animal models, and production of transgenic animals.

It is obvious to those skilled in the art that the present invention is not limited to the details of the above-described exemplary embodiments, and that the present invention can be implemented in other specific forms without departing from the spirit or essential characteristics of the present invention. Therefore, the embodiments should be regarded as illustrative and non-restrictive from any point of view, and the scope of the present invention is defined by the appended claims rather than the above description, and it is therefore intended that all claims falling within the claims All changes within the meaning and scope of equivalent elements are included in the present invention. Any reference signs in the claims shall not be construed as limiting the claim in question.

In addition, it should be understood that although this specification is described in terms of implementations, not each implementation only contains an independent technical solution. This description of the specification is only for the sake of clarity, and those skilled in the art should take the specification as a whole. , the technical solutions in each embodiment can also be appropriately combined to form other implementations that can be understood by those skilled in the art.

Claims (9)

1. A culture solution for improving aging of porcine oocytes by adding echinacoside, which is characterized by comprising a basal culture solution, D-galactose and echinacoside, wherein the basal culture solution is based on an M199 culture solution which does not contain HEPES and has a volume fraction of 82.9%, 10% of porcine follicular fluid and 5% of fetal bovine serum are added, and a mixture of L cysteine with a volume fraction of 6.9mg/mL, epidermal growth factor with a volume fraction of 10 μg/mL of 0.1% and gonadotropin with a volume fraction of 10IU/mL and chorionic gonadotropin with a volume fraction of 10IU/mL is supplemented, the addition amount of D-galactose is 9mg/mL, and the addition amount of echinacoside is 39.337 μg/mL or 78.673 μg/mL.

The preparation method of the culture solution for improving the aging of the porcine oocyte by adding echinacoside comprises the following steps:

SA, adding 9mg/mL of D-galactose into the basic culture solution, and establishing an aging in-vitro culture solution model of the porcine oocyte;

SB, dissolving 5mg of echinacoside in 127.108 mu L of DMSO to obtain 50mM concentrated stock solution, and respectively diluting the 50mM concentrated stock solution to 78.673 mu g/mL and 39.337 mu g/mL by using the aging in vitro culture solution of the porcine oocyte prepared in SA;

and SC, adding 39.337 mug/mL or 78.673 mug/mL echinacoside into the aging in vitro culture solution model of the pig oocyte prepared in SA, and finally obtaining the culture solution for improving the aging of the pig oocyte by adding the echinacoside.

2. The culture solution for improving aging of porcine oocytes by adding echinacoside according to claim 1, wherein the culture solution is characterized in that: the addition amount of the echinacoside in the culture solution for improving the aging of the porcine oocyte is 78.673 mug/mL.

3. A culture broth for improving aging of porcine oocytes by adding echinacoside according to claim 1 or claim 2, wherein: the molecular formula of the echinacoside is C ₃₅ H ₄₅ O _20， The molecular weight is 786.73, and the structural formula is as follows:

4. the culture solution for improving aging of porcine oocytes by adding echinacoside according to claim 1, wherein the culture solution is characterized in that: the preparation method of the pig follicular fluid specifically comprises the following steps:

collecting fresh ovary of pig, placing in 38.5deg.C normal saline thermos bottle, extracting follicular fluid of ovarian surface follicle with 18G needle syringe, collecting the extracted follicular fluid in 15mL centrifuge tube, standing in 38.5deg.C incubator for 30min, collecting upper follicular fluid, centrifuging at 3000rpm for 30min, collecting lower layer to obtain cumulus granulosa cell oocyte complex, filtering the centrifuged supernatant with 0.22 μm filter, packaging the filtered follicular fluid, and storing at 20deg.C.

5. The culture solution for improving aging of porcine oocytes by adding echinacoside according to claim 4, wherein the culture solution is characterized in that: and selecting the follicles with the diameter of 2-8 mm in the process of extracting the follicular fluid of the follicles on the surface of the ovary.

6. The use of a culture broth for improving aging of porcine oocytes by adding echinacoside according to claim 1.

7. The use of a culture broth for improving aging of porcine oocytes with echinacoside as claimed in claim 6, wherein: in particular to application of echinacoside in improving the maturation rate and the development quality of injured pig oocytes.

8. The use of a culture broth for improving aging of porcine oocytes with echinacoside as claimed in claim 7, wherein: comprises the research of the expansion degree of a porcine oocyte cumulus, the first polar body exogenesis of the porcine oocyte in the MII period and the spindle and chromosome morphology of the mature porcine oocyte, and the experimental design is that,

control group: in vitro maturation culture solution without D-galactose and echinacoside

Based on the M199 culture solution without HEPES, 10% of pig follicular fluid and 5% of fetal bovine serum are added, and a mixture of 1% of L cysteine with 6.9mg/mL of L cysteine, 0.1% of epidermal growth factor with 10 mug/mL of epidermal growth factor, 1% of gonadotropin with 10IU/mL of chorionic gonadotrophin is added in a supplementary manner;

d-galactose control group: in vitro maturation culture solution containing 9mg/mL D-galactose

Based on the M199 culture solution without HEPES, which is 82.9% in volume fraction, 10% in volume fraction of pig follicular fluid and 5% in volume fraction of fetal bovine serum are added, and 1% in volume fraction of L cysteine, 0.1% in volume fraction of 10 mug/mL of epidermal growth factor, 1% in volume fraction of a mixed solution of gonadotropin with chorionic gonadotrophin with a volume fraction of 10IU/mL and 9mg/mL of D-galactose are added in a supplementary manner;

echinacoside group 1: in vitro maturation culture solution containing 39.337 mug/mL echinacoside

Based on the M199 culture solution without HEPES, 10% of pig follicular fluid and 5% of fetal bovine serum are added, and 1% of L cysteine with the volume fraction of 6.9mg/mL, 0.1% of epidermal growth factor with the volume fraction of 10 mu g/mL, a mixed solution of gonadotropin with the volume fraction of 1% of 10IU/mL and chorionic gonadotrophin with the volume fraction of 10IU/mL, 9mg/mL of D-galactose and 39.337 mu g/mL of echinacoside are added in a supplementary manner;

echinacoside group 2: in vitro maturation culture solution containing 78.673 mug/mL echinacoside

Based on the M199 culture solution without HEPES with 82.9% volume fraction, 10% volume fraction of pig follicular fluid and 5% volume fraction of fetal bovine serum were added, while 1% volume fraction of L-cysteine with 6.9mg/mL, 0.1% volume fraction of epidermal growth factor with 10. Mu.g/mL, 1% volume fraction of a mixture of gonadotropin with 10IU/mL and chorionic gonadotropin with 10IU/mL, 9mg/mL of D-galactose, 78.673. Mu.g/mL of echinacoside were supplemented.

9. The use of a culture broth for improving aging of porcine oocytes with echinacoside as claimed in claim 8, wherein: in the research test of the expansion degree of the porcine oocyte cumulus and the first polar in vitro row of the porcine oocyte MII period, setting a control group, a D-galactose control group, a echinacoside group 1 and a echinacoside group 2, carrying out three independent repeated tests, repeatedly detecting 60 MII-period oocytes each time, and culturing for 42-48 hours; in the study test of spindle and chromosome morphology after pig oocyte maturation, echinacoside group 1 is canceled, a reserved control group, a D-galactose control group and Echinacoside group 2 are reserved, three independent repetitions are arranged in each group of test, 15-20 MI-phase oocytes are detected each time, the culture is carried out for 24-28 hours, and the result difference statistics are analyzed by using GraphPad Prism statistical software.