CN111235095A

CN111235095A - Method for improving bovine oocyte in vitro maturation

Info

Publication number: CN111235095A
Application number: CN202010250266.7A
Authority: CN
Inventors: 郭晶; 王小武; 王娜; 张月桥; 郝少强; 赵明礼; 郭春明; 许晓椿
Original assignee: Tianjin Boyu Limu Technology Co ltd
Current assignee: Tianjin Boyu Limu Technology Co ltd
Priority date: 2020-04-01
Filing date: 2020-04-01
Publication date: 2020-06-05
Anticipated expiration: 2040-04-01
Also published as: CN111235095B

Abstract

The invention relates to a method for improving bovine oocyte in vitro maturation, in particular to a method for bovine oocyte in vitro maturation, which comprises the following steps: (1) providing an oocyte; (2) in vitro maturation of oocytes: and transferring the oocyte into a mature culture liquid drop, and carrying out in-vitro mature culture under the culture conditions of 100% humidity, 38.5 ℃ and 5% CO2 for 22-24 h. The maturation medium comprises: ECS, NaHCO3, EGCG, Hepe, estradiol, BFF, luteinizing hormone, cysteine, FSH (follicle stimulating hormone), TCM-199. The method can effectively promote the in vitro maturation of bovine oocytes.

Description

Method for improving bovine oocyte in vitro maturation

Technical Field

The invention belongs to the technical field of biology, and relates to an oocyte in-vitro maturation method, in particular to an improved bovine oocyte in-vitro maturation method. The method can obviously improve the in vitro maturation performance of the bovine oocyte, for example, can obviously improve the cumulus expansion index, the in vitro maturation rate of the oocyte, the cleavage rate and the blastocyst rate.

Background

Oocyte in vitro fertilization refers to the process of fertilizing an oocyte by specially treating the sperm. The completion of in vitro fertilization requires a culture system that facilitates the survival and metabolism of sperm and eggs. In vitro fertilization is the basis for in vitro embryo production. At present, the embryo in vitro production technology is developed rapidly and is mainly applied to the breeding of various domestic animals, foreign breeds, wild animals and endangered animals. Many scientific researchers have a high enthusiasm for in vitro embryo production and have developed large-scale production. Bovine in vitro embryo production is less efficient than other mammals. Among these, in vitro maturation and in vitro fertilization are two major key components of in vitro embryo production.

A large number of high quality mature oocytes are the basis for in vitro fertilization and embryo transfer. The oocyte in vitro maturation technology began in the 30 s of the last century and developed rapidly in the 60 s, and the main study animals were mice and sheep. In 1978 Newcomb et al obtained the first test-tube animal and born bovine oocytes matured in vitro and then fertilized in vivo. In recent years, with the intensive research on mammalian embryo engineering technology and the rapid development of commercial embryo transfer technology, oocyte in vitro maturation technology has become more and more important.

There are many factors that influence oocyte maturation in vitro. E.g. the source of the oocyte, maturation medium and additives, maturation culture conditions, etc.

In terms of the source of oocytes, the species, age and ovarian quality of animals are one of the important factors, and the species of mammals are different, and the performance and quality of maturation are different; the in vitro maturation effect of the cow oocyte is better than that of the horse and the pig. The in vitro maturation and early embryo development of the oocyte of the cattle are better than those of buffalo, which is probably related to the fact that more excellent oocyte of the cattle in the GV stage can be collected in vitro. The age and physical condition, particularly sexual function and ovarian condition, of the same species of animals vary significantly in the number and quality of oocytes collected from each ovary. Ovarian oocytes without a corpus luteum achieved higher maturation rates than ovarian oocytes with a corpus luteum. The egg-taking season is also one of the important factors, and the development potential of the oocyte is related to the season. In the breeding season, the in vitro culture of goat oocytes obtains higher maturation rate than that in the non-breeding season, and the quality and in vitro maturation rate of the oocytes are influenced by over-high temperature and under-low temperature. The egg collection method is also one of important factors, and the egg collection method mainly comprises a suction method and a cutting method, wherein the suction method is more suitable for collecting bovine oocytes and is beneficial to maturation of the oocytes. The cutting method is suitable for individual ovaries with small volume, especially for animals with small volume.

The polyphenol substances are also called tea salubriol and tea tannin, are phenolic compounds containing a plurality of hydroxyl groups, and the content of the polyphenol compounds in dry tea leaves is 20-30 percent, and are generally called Tea Polyphenol (TP). The catechins (C) are one of flavanols, have the largest specific gravity in tea polyphenols, account for 60-80% of the total TP, and have very strong antioxidation. Epigallocatechin gallate (EGCG) is a monomer with the highest content of catechin, and accounts for 50-60% of catechin.

Flavanols are the most abundant active components in tea polyphenols, and two hydroxyl groups and 1-oxygen in the molecular structure of flavanols can make 6-position and 8-position carbon atoms generate strong nucleophilicity, and finally form C-C or C-O skeleton, so that flavanols can generate oxidative polymerization, and the flavanols mainly adopt the following four mechanisms that ① can reduce the content of free iron and iron ions due to EGCG, and are very strong metal ion chelating agents, ② EGCG can react with superoxide anion free radicals and hydroxyl groups to form DNA fragments and damage other intracellular molecules, ③ EGCG can stop the reaction of lipid peroxidation chains, change gene expression and cause DNA fragmentation, and ④ can enhance the activities of various enzymes related to the oxidative reduction reaction, such as ester reductase, glutathione-S-transferase and the like.

EGCG accounts for 50-60% of catechin content and is a high-efficiency antioxidant. EGCG is an ester catechin monomer, has a molecular formula of C22H18O11, and has a relative molecular mass of: 458.38 kDa. EGCG is chemically stable, increasing stability as pH decreases. EGCG can remove free radicals in healthy cells, resist ROS from damaging cells of each organ, and has oxidation resistance higher than that of common vitamin antioxidants. EGCG can prevent cell damage and DNA fragmentation. EGCG is used as a plant-derived antioxidant, has a series of biological activities of preventing heart disease, resisting tumor, resisting mutation, resisting aging and the like, and can prevent and treat obesity by frequently drinking green tea. In medicine, EGCG is also used for the adjuvant treatment of malignant tumors, coronary heart disease, hyperlipidemia, etc.

Due to the special structure, EGCG can react with active oxygen to form a dimer, so that the oxidation-reduction reaction of unsaturated fatty acid and active oxygen can be reduced. Thus, EGCG exhibits higher radical clearance than monophenolic hydroxyl or non-phenolic antioxidants.

It has been shown that Wuyi tea TP can reduce the Acid Value (AV) of oil and fat. The research also shows that the tea polyphenol and the monomer thereof have very strong scavenging effect on ROS, the concentration of the tea polyphenol and the monomer thereof is increased and enhanced within a certain range, even reaching 98 percent, and the effect is obviously higher than that of a vitamin antioxidant. Research has proved that EGCG can eliminate ROS in cells of organisms and enhance the capacity of the organisms to resist oxidative stress. It has also been shown that EGCG is beneficial in maintaining the balance of the body's oxidative system and can reverse the lead-induced peroxidative damage to cells in the hippocampus. In medicine, EGCG gradually plays an important role in the treatment of chronic poisoning by heavy metals.

In addition, EGCG can inhibit signal transmission required by cancer cell survival, interact with other antioxidants to reduce the activity of certain carcinogenic substances, and reduce the damage of harmful free radicals to the body. EGCG was tested by Japan scholars in 1987 to have the effect of inhibiting the binding of the skin mucosa potential carcinogen Tetradecanoyl Phorbol Acetate (TPA) and its receptor (i.e. phorbol ester receptor modulation), and the principle of the test is to use [3H ] labeled TPA, which utilizes the characteristic that TPA can be specifically bound with TPA receptor on cell membrane. A two-stage chemical carcinogenic experiment is subsequently carried out to prove that the EGCG can inhibit skin tumors.

During in vitro maturation, in vitro manipulation exposes the gametes to more oxygen, and oxygen free radicals in the gametes themselves and the environment inhibit maturation of the oocytes. Meanwhile, oocytes are often in an unbalanced state of the antioxidant system during in vitro culture. Therefore, it is important to add exogenous antioxidants to the oocyte in vitro maturation medium. In some researches, green tea polyphenol is added in-vitro maturation researches of bovine oocytes to research the influence of the green tea polyphenol on maturation and fertilization of the oocytes. The test results showed that the highest GSH content (p <0.05) was observed in MII oocytes when the amount of TP was 15. mu. mol/L. The results show that the addition of 15. mu. mol/L of green tea polyphenols during in vitro culture increases the rate of oocyte development to MII stage and blastocyst development rate. Research shows that EGCG has certain protection effect on oocyte damage caused by maternal heat stress and promotes the embryonic development of mice. In mouse experiments, researchers find that the addition of 20 mu mol/L of EGCG in a maturation solution promotes the maturation of oocytes and the development of later embryos. It is shown by the research that the addition of 15 mu mol/L EGCG in the in vitro maturation liquid promotes the in vitro of goat oocytes and reduces the oxidation rate of glutathione in the oocytes.

Active oxygen is a by-product of sperm oxidation reactions, normally, the oxidant and antioxidant in the sperm are in equilibrium. When the intracellular ROS level is higher than physiological dosage, the ROS can corrode polyunsaturated fatty acid on the surface of a sperm membrane, and oxidative damage is caused to the sperm. EGCG has strong ability to scavenge oxygen free radicals, and can protect cells from damage and prevent DNA fragmentation. In recent years, EGCG has been intensively studied on mammalian sperm. Research shows that the addition amount of EGCG is 10 mug/mL in the process of pig IVF, the effect is optimal, and the generation of hydrogen peroxide can be effectively reduced.

The oocyte in vitro embryo production technology can accelerate the breeding rate of cattle and the propagation of fine varieties by effectively utilizing the bovine oocytes. The in vitro maturation quality of the ovum and the sperm fertilization capability are used as the core technology of in vitro embryo production, and play an important role in the high-efficiency production of embryos. However, oxidative stress is one of the major factors that restrict the quality of maturation of ova and the capacity of sperm fertilization. Although literature such as Li Ming's laboratories (southern Agreement, 2016, 47 (3): 478-. However, there is still a need for improvement in the use of EGCG.

Disclosure of Invention

It is an object of the present invention to provide a method for improving the in vitro maturation of bovine oocytes, which method is expected to achieve one or more technical effects. It has been surprisingly found that the process conditions of the invention exhibit surprising technical effects, and the invention has been completed on the basis of such findings.

To this end, in a first aspect the present invention provides a method for improving the in vitro maturation of bovine oocytes, the method comprising the steps of:

(1) providing an oocyte;

(2) in vitro maturation of oocytes: and transferring the oocyte into a mature culture liquid drop, and carrying out in-vitro mature culture under the culture conditions of 100% humidity, 38.5 ℃ and 5% CO2 for 22-24 h.

According to the method of any one of the embodiments of the first aspect of the present invention, the oocyte is collected by a live bovine oocyte collection method comprising the steps of:

i. superovulation treatment before egg taking

Screening cattle with follicles of 2-8 mm in diameter on two sides and the number of the follicles being more than or equal to 20 as oocyte donor cattle, wherein the cattle age is 2-5 years old; performing superovulation by injecting FSH preparation at equal dose of 4 times every two days, wherein the interval of each injection is 12h, and the injection dose is 0.5 mu g/kg of bovine body weight calculated by FSH;

ii. Living egg taking

In vivo egg collection was performed 48 hours after the last injection of the FSH preparation and was performed as follows:

(ii1) preoperative preparation: debugging equipment to working state (the pressure of an egg sucking vacuum pump is 50-90mmHg), injecting about 5ml of egg sucking liquid into a collecting pipe, placing in a temperature balancer, and adjusting the temperature of the temperature balancer to 39 ℃; connecting the needle tail of the ovum taking needle and one interface on the collecting tube plug by a transparent plastic catheter, and connecting the other interface of the collecting tube plug with a vacuum pump; standing stably, removing rectum feces, sterilizing vulva, performing caudal gap epidural space nerve trunk conduction blocking anesthesia with 2-4ml of 20% lidocaine, and operating after the tail root is soft;

(ii2) Observation of ovary and ovarian follicle: an operator holds the handle of the B-ultrasonic probe with one hand, inserts the B-ultrasonic probe into the vaginal fornix gently, extends the other hand into the rectum, holds the ovary from the rectum, places the ovary on the probe, moves the ovary, observes the structure and the size of the ovary through a B-ultrasonic screen, distinguishes follicles and corpus luteum, and records the number and the size of the follicles in detail; on the B-ultrasonic screen, the ovary has a weak echo structure and has clear outline; the follicle presents a plurality of anechoic areas in the ovary area, has clear outline and can carry out ovum collection;

(ii3) aspiration: moving the ovary to enable the follicle to be punctured to be positioned on an ovum taking line of a B-ultrasonic display screen, fully absorbing ovum absorbing liquid in an ovum absorbing needle, inserting the ovum absorbing needle into a puncture needle fixing cylinder at the back of the probe, puncturing the vaginal wall and the follicle wall by the needle head, and simultaneously controlling a vacuum pump by using a foot switch to absorb the follicle content into a collecting pipe; after repeatedly puncturing a plurality of follicles, timely drawing out the puncture needle, sucking a proper amount of ovum-sucking liquid from the needle head to flush the needle tube so as to prevent the ovum-sucking liquid from being blocked by the coagulated blood, and collecting the flushing liquid into the collecting tube together to obtain the follicle liquid;

iii detection of oocytes

Immediately diluting the collected follicular fluid with an egg washing solution, pouring into an embryo filter, repeatedly washing for 3-4 times, finally pouring the liquid and precipitate in the filter into a culture dish, examining the number of collected oocytes (number of collected ova) under a dissecting mirror, and counting the number of oocytes (number of available ova) as available oocytes having the following properties: the cytoplasm of the oocyte is uniform, at least two layers of cumulus cells, at least more than 5 layers of granular cells and densely arranged around the oocyte are wrapped by the COCs.

The method according to any one of the embodiments of the first aspect of the invention, wherein the FSH preparation used in the live bovine egg collection method consists of: follicle stimulating hormone (35 μ g/ml), estriol (FSH: estriol: 100: 15), poloxamer 188 (concentration 0.4 mg/ml).

The method according to any one of the embodiments of the first aspect of the present invention, wherein the donor cattle is yellow cattle in china (south yang).

The method according to any one of the embodiments of the first aspect of the invention, wherein the composition of the maturation medium is: 10% ECS (oestrus calf serum), 26.2mmol/L NaHCO3, 10. mu. mol/L EGCG, 5mmol/L Hepe, 1. mu.g/mL E2 (estradiol), 3% BFF (bovine follicular fluid), 10. mu.g/mL LH (luteinizing hormone), 100. mu. mol/L cysteine, 10. mu.g/mL FSH (follicle stimulating hormone), TCM-199 to 100%.

The method according to any embodiment of the first aspect of the present invention, wherein the estrus bovine serum (ESC) is prepared according to a method comprising the steps of: selecting an individual in an oestrous state for jugular vein blood collection, and collecting along the tube wall by using a 50mL sterilized disposable centrifuge tube with an inclination angle of 30-45 degrees; standing at normal temperature in a blood sampling place, returning the blood sample to a laboratory after the blood sample is observed to be coagulated, avoiding sunlight irradiation, standing at room temperature for 4-6 h, observing layering, and separating out partial serum from the upper layer to avoid shaking; centrifuging after 12h and collecting serum; inactivating the serum in 56 deg.C water bath for 30min, and packaging with disposable 15mL centrifuge tube according to dosage; one small tube is unfrozen every time, so that repeated freezing and thawing is avoided, and the quality of serum is reduced.

The method according to any embodiment of the first aspect of the present invention, wherein the Bovine Follicular Fluid (BFF) is prepared according to a method comprising the steps of: taking slaughtered cow ovaries, shearing other tissues around the ovaries by using surgical scissors, cleaning the ovaries once by using alcohol, cleaning the ovaries for 2-3 times by using normal saline, and extracting follicular fluid; centrifuging the follicular fluid for tens of seconds, sucking the upper clear follicular fluid, filtering, sterilizing, and packaging at-80 deg.C; the proper amount of unfreezing is used every time, and repeated freeze thawing is avoided.

The method according to any one of the embodiments of the first aspect of the present invention, further comprising the step (3) of:

(3) in vitro fertilization of oocytes: putting the F liquid into an incubator for balancing 4-6 hours in advance; after the frozen semen is processed by an upstream method, sucking 1mL of liquid at the upper layer, and centrifuging at normal temperature; discarding 800-900 mu L of supernatant immediately after centrifugation, putting the supernatant back into an incubator for incubation for 5-10 min, and gently mixing semen by using a pipette before use; placing 15-20 oocytes subjected to in vitro maturation culture in each fertilization liquid drop, and adding 12-15 mu L of semen to enable the density of the semen in each fertilization liquid drop to reach (1.0-1.5) x10⁶And (4) dropping the fertilization liquid into an incubator with 39 ℃, 5% CO2 and 100% humidity for incubation for 20-22 h to perform in vitro fertilization culture.

According to the method of any one of the embodiments of the first aspect of the invention, the composition of the F fluid, i.e. the receptor fluid, is: tyrode's solution (modified), 1mg/mL glucose, 20. mu.g/mL heparin sodium, 0.6% BSA (bovine serum albumin).

The method according to any one of the embodiments of the first aspect of the present invention, further comprising the step (4) of:

(4) in vitro culture of fertilized eggs: after fertilization is carried out for 20-22 h, the sperms are washed by fertilization liquid to remove other liquid attached in the process of transferring zygote; washing the fertilized eggs with the solution C, and then transferring the fertilized eggs into a granular cell monolayer for culture; the culture conditions were 5% CO2, 100% humidity and 38.5 ℃ for 48 hours.

According to the method of any one of the embodiments of the first aspect of the present invention, the composition of the solution C, i.e. the embryo culture solution, is: 54% Tyrode's solution (modified), 10% FBS (fetal bovine serum) and 36% TCM-199 solution, and are pre-balanced in an incubator for 1-2 h before use.

According to the method of any one of the embodiments of the first aspect of the invention, the granulosa cell monolayer is cultured as follows: centrifuging and washing the blown granular cells on the oocytes by using an embryo culture solution; in each dish, 20. mu.L of microdroplets of granular cells were placed, covered with mineral oil, and the impurities were removed with a glass needle and placed in an incubator for culture.

The method according to any one of the embodiments of the first aspect of the invention, wherein the maturation medium further comprises sodium caprylate and polyvinyl alcohol. For example, the composition also comprises 20mg/L of sodium caprylate and 5mg/L of polyvinyl alcohol.

The method according to any one of the embodiments of the first aspect of the invention, wherein the composition of the maturation medium is: 20mg/L sodium caprylate, 5mg/L polyvinyl alcohol, 10% ECS (oestrus calf serum), 26.2mmol/L NaHCO3, 10. mu. mol/L EGCG, 5mmol/L Hepe, 1. mu.g/mL E2 (estradiol), 3% BFF (bovine follicular fluid), 10. mu.g/mL LH (luteinizing hormone), 100. mu. mol/L cysteine, 10. mu.g/mL FSH (follicle stimulating hormone), TCM-199 to 100%.

The method according to any one of the embodiments of the first aspect of the present invention, comprising the steps of:

(1) providing an oocyte, which is obtained by taking an egg from a living bovine body, wherein the egg taking from the living bovine body comprises the following steps:

i. superovulation treatment before egg taking

ii. Living egg taking

iii detection of oocytes

Immediately diluting the collected follicular fluid with an egg washing solution, pouring into an embryo filter, repeatedly washing for 3-4 times, finally pouring the liquid and precipitate in the filter into a culture dish, examining the number of collected oocytes (number of collected ova) under a dissecting mirror, and counting the number of oocytes (number of available ova) as available oocytes having the following properties: the cytoplasm of the oocyte is uniform, at least two layers of cumulus cells, at least more than 5 layers of granular cells and the COCs are wrapped by the oocyte and are densely arranged around the oocyte;

(2) in vitro maturation of oocytes: transferring the oocyte into a mature culture liquid drop, performing in-vitro maturation culture under the culture conditions of 100% humidity, 38.5 ℃ and 5% CO2 for 22-24 h; (for example, the maturation medium is composed of 10% ECS (estrus bovine serum), 26.2mmol/L NaHCO3, 10. mu. mol/L EGCG, 5mmol/L Hepe, 1. mu.g/mL E2 (estradiol), 3% BFF (bovine follicular fluid), 10. mu.g/mL LH (luteinizing hormone), 100. mu. mol/L cysteine, 10. mu.g/mL FSH (follicle stimulating hormone), TCM-199 added to 100%; for example, the maturation medium optionally further comprises 20mg/L sodium caprylate, 5mg/L polyvinyl alcohol);

(3) in vitro fertilization of oocytes: putting the F liquid into an incubator for balancing 4-6 hours in advance(ii) a After the frozen semen is processed by an upstream method, sucking 1mL of liquid at the upper layer, and centrifuging at normal temperature; discarding 800-900 mu L of supernatant immediately after centrifugation, putting the supernatant back into an incubator for incubation for 5-10 min, and gently mixing semen by using a pipette before use; placing 15-20 oocytes subjected to in vitro maturation culture in each fertilization liquid drop, and adding 12-15 mu L of semen to enable the density of the semen in each fertilization liquid drop to reach (1.0-1.5) x10⁶Dropping the fertilized liquid into an incubator with 39 ℃, 5% CO2 and 100% humidity for incubation for 20-22 h to perform in vitro fertilization culture;

Further, the second aspect of the present invention provides a maturation culture solution, which comprises: 10% ECS (oestrus calf serum), 26.2mmol/L NaHCO3, 10. mu. mol/L EGCG, 5mmol/L Hepe, 1. mu.g/mL E2 (estradiol), 3% BFF (bovine follicular fluid), 10. mu.g/mL LH (luteinizing hormone), 100. mu. mol/L cysteine, 10. mu.g/mL FSH (follicle stimulating hormone), TCM-199 to 100%.

The maturation culture solution according to any one of the embodiments of the second aspect of the present invention, further comprising sodium caprylate and polyvinyl alcohol. For example, the composition also comprises 20mg/L of sodium caprylate and 5mg/L of polyvinyl alcohol.

The maturation medium according to any one of the embodiments of the second aspect of the present invention, comprising: 20mg/L sodium caprylate, 5mg/L polyvinyl alcohol, 10% ECS (oestrus calf serum), 26.2mmol/L NaHCO3, 10. mu. mol/L EGCG, 5mmol/L Hepe, 1. mu.g/mL E2 (estradiol), 3% BFF (bovine follicular fluid), 10. mu.g/mL LH (luteinizing hormone), 100. mu. mol/L cysteine, 10. mu.g/mL FSH (follicle stimulating hormone), TCM-199 to 100%.

According to any aspect and/or any embodiment of the present invention, there is further provided any one of the features and/or aspects as described in the examples.

Any technical feature possessed by any one aspect of the invention or any embodiment of that aspect is equally applicable to any other embodiment or any embodiment of any other aspect, so long as they are not mutually inconsistent, although appropriate modifications to the respective features may be made as necessary when applicable to each other. Various aspects and features of the disclosure are described further below.

All documents cited herein are incorporated by reference in their entirety and to the extent such documents do not conform to the meaning of the present invention, the present invention shall control. Further, the various terms and phrases used herein have the ordinary meaning as is known to those skilled in the art, and even though such terms and phrases are intended to be described or explained in greater detail herein, reference is made to the term and phrase as being inconsistent with the known meaning and meaning as is accorded to such meaning throughout this disclosure.

The present inventors have surprisingly found that the improvement of the maturation medium can significantly improve the biological properties of oocytes, such as the cumulus expansion performance during oocyte maturation, the first polar body expulsion performance of oocytes, the maturation and maturation quality of oocytes, the fertilization ability of oocytes, the nuclear maturation and early embryo development ability of oocytes, which could not be expected at all in the prior art.

Detailed Description

The present invention will be further described by the following examples, however, the scope of the present invention is not limited to the following examples. It will be understood by those skilled in the art that various changes and modifications may be made to the invention without departing from the spirit and scope of the invention. The present invention has been described generally and/or specifically with respect to materials used in testing and testing methods. Although many materials and methods of operation are known in the art for the purpose of carrying out the invention, the invention is nevertheless described herein in as detail as possible. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art, and the raw materials used are commercially available products. For example, FSH and LH for use in the present invention are readily available from Bevack Biotech, Beijing.

Example 1: ovum collection of living cow

The method of the inventor group (CN110251660A, Chinese patent application No. 2019106516223, Y19032) is referred to for live cow egg taking, and the specific steps are as follows:

1. superovulation treatment before egg taking

2. living egg taking

(21) preoperative preparation: debugging equipment to working state (the pressure of an egg sucking vacuum pump is 50-90mmHg), injecting about 5ml of egg sucking liquid into a collecting pipe, placing in a temperature balancer, and adjusting the temperature of the temperature balancer to 39 ℃; connecting the needle tail of the ovum taking needle and one interface on the collecting tube plug by a transparent plastic catheter, and connecting the other interface of the collecting tube plug with a vacuum pump; standing stably, removing rectum feces, sterilizing vulva, performing caudal gap epidural space nerve trunk conduction blocking anesthesia with 2-4ml of 20% lidocaine, and operating after the tail root is soft;

(22) observation of ovaries and follicles: an operator holds the handle of the B-ultrasonic probe with one hand, inserts the B-ultrasonic probe into the vaginal fornix gently, extends the other hand into the rectum, holds the ovary from the rectum, places the ovary on the probe, moves the ovary, observes the structure and the size of the ovary through a B-ultrasonic screen, distinguishes follicles and corpus luteum, and records the number and the size of the follicles in detail; on the B-ultrasonic screen, the ovary has a weak echo structure and has clear outline; the follicle presents a plurality of anechoic areas in the ovary area, has clear outline and can carry out ovum collection;

(23) taking eggs: moving the ovary to enable the follicle to be punctured to be positioned on an ovum taking line of a B-ultrasonic display screen, fully absorbing ovum absorbing liquid in an ovum absorbing needle, inserting the ovum absorbing needle into a puncture needle fixing cylinder at the back of the probe, puncturing the vaginal wall and the follicle wall by the needle head, and simultaneously controlling a vacuum pump by using a foot switch to absorb the follicle content into a collecting pipe; after repeatedly puncturing a plurality of follicles, timely drawing out the puncture needle, sucking a proper amount of ovum-sucking liquid from the needle head to flush the needle tube so as to prevent the ovum-sucking liquid from being blocked by the coagulated blood, and collecting the flushing liquid into the collecting tube together to obtain the follicle liquid;

3. detection of oocytes

Immediately diluting the collected follicular fluid with an egg washing solution, pouring into an embryo filter, repeatedly washing for 3-4 times, finally pouring the liquid and precipitate in the filter into a culture dish, examining the number of collected oocytes (number of collected ova) under a dissecting mirror, and counting the number of oocytes (number of available ova) as available oocytes having the following properties: the cytoplasm of the oocyte is uniform, at least two layers of cumulus cells, at least more than 5 layers of granular cells and densely arranged around the oocyte are wrapped by the COCs. The number of eggs collected from the head of each animal and the number of usable eggs per head were calculated based on the number of test animals.

FSH preparation: follicle stimulating hormone (35 μ g/ml), estriol (FSH: estriol: 100: 15), poloxamer 188 (concentration 0.4 mg/ml).

Donor cattle: chinese cattle (south Yang).

Example 2: in vitro maturation of bovine oocytes

EGCG (10 mu mol/L) is added into in-vitro maturation liquid, the in-vitro maturation of bovine oocytes is inspected by referring to a Leminling literature method, the methodological performance of the invention is inspected by indexes such as maturation rate, cumulus expansion and the like, and the developmental capacity of the matured oocytes is inspected by using an in-vitro fertilization method and a parthenogenetic activation method so as to improve the in-vitro culture efficiency and the developmental quality of bovine embryos. Unless otherwise specified, cattle involved in the procedure were Chinese (south Yang) cattle.

1. Material

(1) Frozen semen (southern yang yellow cattle, straw) was self-stored by the company. TCM-199 was purchased from GIBCO, Dulbecco's PBS was purchased from GIBCO, Fetal Bovine Serum (FBS) was purchased from GIBCO, Neonatal Bovine Serum (NBS) was purchased from GIBCO, epigallocatechin gallate (EGCG) was purchased from Dougui and HPLC pure 99.6%; the remaining non-listed biochemicals/reagents were purchased from Sigma-Aldrich.

(2) Ovary preservation solution: adding penicillin, streptomycin, Mg2+, K +, Ca2+ physiological saline.

(3) Egg wash (liquid H): the main components are TCM-199, 3% NBS, 5mmol/L NaHCO3 and 20mmol/L Hepes are added, and the mixture is put into a water bath kettle before use.

(4) Maturation medium (M liquid): the main components of the base liquid are TCM-199, and 10% ECS (estrus bovine serum), 26.2mmol/L NaHCO3, 10 mu mol/L EGCG and 5mmol/L Hepe are added; e2 (estradiol) at 1. mu.g/mL, 3% BFF (bovine follicular fluid), LH (luteinizing hormone) at 10. mu.g/mL, cysteine at 100. mu. mol/L and FSH (follicle stimulating hormone) at 10. mu.g/mL were added to the basal fluid the morning of the experiment. Briefly, the composition of the maturation medium (M fluid) was: 10% ECS (oestrus calf serum), 26.2mmol/L NaHCO3, 10. mu. mol/L EGCG, 5mmol/L Hepe, 1. mu.g/mL E2 (estradiol), 3% BFF (bovine follicular fluid), 10. mu.g/mL LH (luteinizing hormone), 100. mu. mol/L cysteine, 10. mu.g/mL FSH (follicle stimulating hormone), TCM-199 to 100%.

(5) Receptor fluid (fluid F): the components are Tyrode's solution (modified), 1mg/mL glucose, 20. mu.g/mL heparin sodium, and 0.6% BSA (bovine serum albumin).

(6) Embryo culture solution (solution C): the components are 54% Tyrode's solution (modified), 10% FBS (fetal bovine serum) and 36% TCM-199 solution, and are pre-balanced in an incubator for 1-2 h before use.

The water source for preparing the culture solution is Milli-Q water, the culture solution is distilled by sub-boiling water to prepare ultrapure water for use, and streptomycin and penicillin are added into all the culture solutions, but the concentrations are different and are respectively as follows: 100mg/L and 60 mg/L. The pH value of the prepared liquid needs to be measured within 10 minutes, the pH values of the liquid in the H liquid, the M liquid and the C liquid are all 7.2-7.4, and the pH value of the liquid F is slightly higher and is 7.5-7.8. The working solution needs to be filtered and sterilized because the embryo culture needs a sterile environment, and a microporous filter membrane with the pore diameter of 0.22 mu m is used for filtering, sealing with sealing glue, and storing in a refrigerator at 4 ℃ after marking. All the glass bottles and centrifuge tubes containing the solution are subjected to aseptic processing.

(7) Preparation of estrus bovine serum (ESC): the blood collection variety is female cattle, an individual in an oestrous state is selected for jugular vein blood collection, and a 50mL sterilized disposable centrifuge tube is used for collecting blood along the tube wall in an inclined angle of 30-45 degrees. Standing at the local normal temperature of blood sampling, sending back to the laboratory after observing the blood sample coagulation, avoiding the sunshine to shine, standing for 4 ~ 6h at the room temperature, can observing the layering, going up the chromatography and appearing partial serum, avoid rocking. After 12h, centrifugation and serum collection. The serum was inactivated in a 56 ℃ water bath for 30min and dispensed into disposable 15mL centrifuge tubes at each dose. One small tube is unfrozen every time, so that repeated freezing and thawing is avoided, and the quality of serum is reduced.

(8) Preparation of Bovine Follicular Fluid (BFF):

taking slaughtered cow ovaries, shearing other tissues around the ovaries by using surgical scissors, cleaning the ovaries once by using alcohol, cleaning the ovaries for 2-3 times by using normal saline, and extracting follicular fluid; and then quickly centrifuging the follicular fluid for tens of seconds, sucking the upper clear follicular fluid, filtering and sterilizing, and subpackaging according to the dosage for each time, and freezing and storing the follicular fluid in a refrigerator at-80 ℃. The proper amount of unfreezing is used every time, and repeated freeze thawing is avoided.

2. Test method

(1) Culture of granular cell monolayer: washing the blown-down granulosa cells from the oocytes obtained in example 1 by centrifugation using an embryo culture solution; in each dish, 20. mu.L of microdroplets of granular cells were placed, covered with mineral oil, and the impurities were removed with a glass needle and placed in an incubator for culture.

(2) In vitro maturation of oocytes: oocytes obtained by collecting ova from the living bodies in example 1 were transferred to a drop of mature culture medium (55. mu.L/drop) and subjected to in vitro maturation culture under conditions of 100% humidity, 38.5 ℃ and 5% CO2 for 22-24 hours.

(3) Assessment of cumulus expansion and determination of oocyte maturation: evaluation of Cumulus Expansion and calculation of Cumulus Expansion Index (CEI) were reported in Fagbohun and Down [ Fagbohun C F, Down SM. distribution of the motor effect-Cumulus cell complex: deposition by molecules [ J ]. Biology of reproduction,1990,42(3):413-423 ]. Cumulus expansion is divided into 5 grades: level 0, no expansion of cumulus, and sticking the oocyte to the bottom of the culture dish; grade 1, only the outmost 1-2 layers of cumulus cells are expanded; grade 2, the cumulus cells on the outer layer radially expand, and the whole COCs are observed to be fluffy; grade 3, the radiation crown part is not expanded, and the rest are expanded; grade 4, total cumulus cell expansion. The CEI is [ (0 grade oocyte number × 0) + (1 grade oocyte number × 1) + (2 grade oocyte number × 2) + (3 grade oocyte number × 3) + (4 grade oocyte number × 4) ]/total oocyte number. The criterion for oocyte maturation is first polar body ejection. Bovine oocytes matured in vitro were gently blasted with a pipette to remove all cumulus cells and the first polar body was observed under a stereomicroscope.

(4) In vitro fertilization: and (4) balancing the F liquid in an incubator 4-6 hours in advance. The frozen semen is processed by an upstream method (namely, a frozen semen tubule is scratched left and right in water at 38.5 ℃, the flowing of the semen in the frozen semen tubule is observed, after sterilization, a section of the tubule without a cotton plug is cut off, then one end with the cotton plug is cut off, the semen flows into the bottom of a centrifuge tube along the wall of the tube, the semen is added to the bottom of an F liquid to avoid generating bubbles, the frozen semen is incubated in an incubator by inclining at an angle of 45 degrees, and after 30-60 min, the high-activity semen can flow up to the upper layer liquid), the upper layer of 1mL of liquid is absorbed, and the centrifugation is; discarding 800-900 mu L of supernatant immediately after centrifugation, putting the supernatant back into an incubator for incubation for 5-10 min, and gently mixing semen by using a pipette before use; placing 15-20 oocytes subjected to in vitro maturation culture in each fertilization liquid drop, and adding 12-15 mu L of semen to enable the density of the semen in each fertilization liquid drop to reach (1.0-1.5) x10⁶And (4) dropping the fertilization liquid into an incubator with 39 ℃, 5% CO2 and 100% humidity for incubation for 20-22 h to perform in vitro fertilization culture.

(5) Parthenogenetic activation: and after the oocyte is matured in vitro for 22-24 hours, slightly blowing and beating by using a gun head to remove most of cumulus cells. The activation conditions are: 5 μmol/L ionomycin (Ion) for 5min, 2 mmol/L6-dimethylaminopurine (6-DMAP) for 4 h. Washing with solution C for 2 times, transferring into particle monolayer cells, and culturing. The culture conditions were 39 ℃, 5% CO2 and 100% humidity.

(6) In vitro culture of fertilized eggs: after fertilization is carried out for 20-22 h, the sperms are washed by fertilization liquid to remove other liquid attached in the process of transferring zygote; washing fertilized eggs or activated oocytes for 3 times by using the solution C, and then transferring the fertilized eggs or activated oocytes into a granular cell monolayer for culture; the culture conditions were 5% CO2, 100% humidity and 38.5 ℃.

(7) Evaluation of early embryo development potential: the putative fertilized egg or activated oocyte is transferred into a granular cell monolayer and cultured to the 2d (the day of IVF or parthenogenetic activation is marked as 0d, namely 48h), and the division condition of the fertilized egg or parthenogenetic embryo is checked. The division rate is the number of divided ova/total number of fertilized oocytes or total number of parthenogenetically activated oocytes. Blastocyst rate is the number of blastocysts per total number of oocytes fertilized or parthenogenetically activated.

The experimental data were analyzed for one-way anova using SPSS Statistics 17 software. The representation mode is as follows: mean ± standard error (Mean ± SE).

3. Test results

(1) And after the oocytes are matured and cultured in vitro for 22-24 h, observing and recording the cumulus expansion grade of each oocyte, and calculating the cumulus expansion index. The experiment was repeated 5 times, and the total number of eggs counted was 217, and the results were: cumulus expansion index CEI (% ± s.e.) 2.43 ± 0.06. The result shows that the cumulus expansion performance is good in the oocyte maturation process.

(2) After the oocyte is matured and cultured in vitro for 22-24 h, the mark of the GV stage development to the MII stage is first polar body discharge, and the discharge rate of the first polar body is taken as the standard for judging the maturation of the oocyte. The experiment was repeated 5 times, and the results (total number of eggs counted 233): maturation rate (% ± s.e.) 58.52 ± 1.73. The results show that the first polar body of the oocyte is well expelled.

(3) After the oocyte is fertilized in vitro, the assumed fertilized ovum is transplanted into a granular cell monolayer for culturing, the cleavage rate and the blastocyst development rate are counted, 5 times of experiments are repeated, and the result is obtained (the counted total number of fertilized ovum is 197): cleavage (% ± s.e.) 57.26 ± 2.84, blastocyst development (% ± s.e.) 25.37 ± 2.03. The result shows that the oocyte is well matured, the maturation quality of the oocyte is good, and the fertilization capability of the oocyte is good.

(4) Parthenogenetic activation treatment is carried out on bovine oocytes in the MII stage, parthenogenetic activation embryos are transferred into granular cell monolayers for culture after parthenogenetic activation of the oocytes, the cleavage rate and the blastocyst development rate are counted, 5 times of experiments are repeated, and the result is shown (the counted total activation number of the ova is 213): the cleavage rate (% ± s.e.) is 65.38 ± 3.87, and the blastocyst development rate (% ± s.e.) is 30.41 ± 1.87. The result shows that the oocyte has good nuclear maturation and good early embryo development capability.

Example 3: in vitro maturation of bovine oocytes

Reference is made to examples 1 and 2, except that the composition of the maturation medium (M liquid) used in example 2 is: 20mg/L sodium caprylate, 5mg/L polyvinyl alcohol, 10% ECS (oestrus calf serum), 26.2mmol/L NaHCO3, 10. mu. mol/L EGCG, 5mmol/L Hepe, 1. mu.g/mL E2 (estradiol), 3% BFF (bovine follicular fluid), 10. mu.g/mL LH (luteinizing hormone), 100. mu. mol/L cysteine, 10. mu.g/mL FSH (follicle stimulating hormone), TCM-199 to 100%.

The test procedure of reference example 2 resulted in the following:

(1) after the oocytes are matured and cultured in vitro for 22-24 h, the total number of eggs counted is 221, and the cumulus expansion index CEI (% + -S.E.) is 2.81 +/-0.09.

(2) After the oocytes are matured and cultured in vitro for 22-24 h, the total number of counted eggs is 235, and the maturation rate (% + -S.E.) is 69.33 +/-2.13.

(3) After in vitro fertilization of oocytes, the total number of fertilized ova was 214, and the cleavage rate (% ± s.e.) was 71.32 ± 3.45 ″, and the blastocyst development rate (% ± s.e.) was 31.22 ± 2.83 ″.

(4) Parthenogenetic activation treatment was performed on MII-stage bovine oocytes, and the total number of statistical egg activations was 204, cleavage rate (% ± s.e.) 73.44 ± 5.12 ·, and blastocyst development rate (% ± s.e.) 39.24 ± 2.26 ·.

Note: in this example, it is shown that there is a significant difference from the corresponding index data in the section "3, test result" of example 2, and p is < 0.05.

Example 4: in vitro maturation of bovine oocytes

Reference was made to example 3, except that sodium octanoate was not used, and the results were as follows:

(1) after the oocytes are matured and cultured in vitro for 22-24 h, the total number of the counted ova is 187, and the cumulus expansion index CEI (% + -S.E.) is 2.47 +/-0.05.

(2) After the oocytes are matured and cultured in vitro for 22-24 h, the total number of the counted ova is 216, and the maturation rate (% + -S.E.) is 58.17 +/-1.65.

(3) After the oocytes are fertilized in vitro, the total number of fertilized ova is 176, the cleavage rate (% + -S.E.) is 57.83 + -2.57, and the blastocyst development rate (% + -S.E.) is 24.86 + -2.11.

(4) Parthenogenetic activation treatment is carried out on bovine oocytes in the MII stage, the total number of counted ovum activation is 223, the cleavage rate (% + -S.E.) is 65.02 +/-3.44, and the blastocyst development rate (% + -S.E.) is 29.86 +/-1.92.

Example 5: in vitro maturation of bovine oocytes

Reference was made to example 3, except that polyvinyl alcohol was not used, and the results were as follows:

(1) after the oocytes are matured and cultured in vitro for 22-24 h, the total number of the counted ova is 194, and the cumulus expansion index CEI (% + -S.E.) is 2.51 +/-0.08.

(2) After the oocytes are matured and cultured in vitro for 22-24 h, the total number of the counted ova is 217, and the maturation rate (% + -S.E.) is 57.34 + -1.52.

(3) After the oocytes are fertilized in vitro, the total number of fertilized ova is 202, the cleavage rate (% + -S.E.) is 58.11 + -2.43, and the blastocyst development rate (% + -S.E.) is 25.84 + -1.84.

(4) Parthenogenetic activation treatment is carried out on bovine oocytes in the MII stage, the total number of counted ovum activation is 183, the cleavage rate (% + -S.E.) is 64.84 +/-4.02, and the blastocyst development rate (% + -S.E.) is 31.36 +/-1.58.

According to the difference between the results of examples 3-5 and example 2, it can be understood that the addition of sodium caprylate and polyvinyl alcohol to the maturation culture solution can significantly improve the biological properties of the oocyte, such as the cumulus expansion performance during the maturation of the oocyte, the first polar body expulsion performance of the oocyte, the maturation and maturation quality of the oocyte, the fertilization ability of the oocyte, the nuclear maturation and early embryo development ability of the oocyte, and the like, which is not expected at all in the prior art.

Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications and improvements can be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.

Claims

1. A method for in vitro maturation of bovine oocytes, the method comprising the steps of:

(1) providing an oocyte;

2. The method according to claim 1, wherein said oocyte is collected by a live bovine oocyte collection method comprising the steps of:

i. superovulation treatment before egg taking

ii. Living egg taking

iii detection of oocytes

3. A method according to claim 1, characterized in that:

the FSH preparation used in the method for taking the eggs from the living cattle comprises the following components: follicle stimulating hormone (35 μ g/ml), estriol (FSH: estriol: 100: 15), poloxamer 188 (concentration 0.4 mg/ml);

the donor cattle is Chinese yellow cattle (south Yang);

the composition of the maturation medium was: 10% ECS (estrus calf serum), 26.2mmol/L NaHCO3, 10. mu. mol/L EGCG, 5mmol/L Hepe, 1. mu.g/mL E2 (estradiol), 3% BFF (bovine follicular fluid), 10. mu.g/mL LH (luteinizing hormone), 100. mu. mol/L cysteine, 10. mu.g/mL FSH (follicle stimulating hormone), TCM-199 to 100%; and/or

The F liquid, namely the receptor fluid, comprises the following components: tyrode's solution (modified), 1mg/mL glucose, 20. mu.g/mL heparin sodium, 0.6% BSA (bovine serum albumin).

4. A method according to claim 1, characterized in that:

estrus bovine serum (ESC) was prepared according to a method comprising the steps of: selecting an individual in an oestrous state for jugular vein blood collection, and collecting along the tube wall by using a 50mL sterilized disposable centrifuge tube with an inclination angle of 30-45 degrees; standing at normal temperature in a blood sampling place, returning the blood sample to a laboratory after the blood sample is observed to be coagulated, avoiding sunlight irradiation, standing at room temperature for 4-6 h, observing layering, and separating out partial serum from the upper layer to avoid shaking; centrifuging after 12h and collecting serum; inactivating the serum in 56 deg.C water bath for 30min, and packaging with disposable 15mL centrifuge tube according to dosage; a small tube is unfrozen every time, so that repeated freeze thawing is avoided, and the quality of serum is reduced;

the Bovine Follicular Fluid (BFF) is prepared by the following steps: taking slaughtered cow ovaries, shearing other tissues around the ovaries by using surgical scissors, cleaning the ovaries once by using alcohol, cleaning the ovaries for 2-3 times by using normal saline, and extracting follicular fluid; centrifuging the follicular fluid for tens of seconds, sucking the upper clear follicular fluid, filtering, sterilizing, and packaging at-80 deg.C; the proper amount of unfreezing is used every time, and repeated freeze thawing is avoided.

5. The method according to claim 1, further comprising the step (3) of:

(3) in vitro fertilization of oocytes: putting the F liquid into an incubator for balancing 4-6 hours in advance; after the frozen semen is processed by an upstream method, sucking 1mL of liquid at the upper layer, and centrifuging at normal temperature; immediately after centrifugation, discardClearing about 800-900 mu L, putting the mixture back into an incubator for incubation for 5-10 min, and gently mixing semen by using a pipette before use; placing 15-20 oocytes subjected to in vitro maturation culture in each fertilization liquid drop, and adding 12-15 mu L of semen to enable the density of the semen in each fertilization liquid drop to reach (1.0-1.5) x10⁶And (4) dropping the fertilization liquid into an incubator with 39 ℃, 5% CO2 and 100% humidity for incubation for 20-22 h to perform in vitro fertilization culture.

6. The method according to claim 1, further comprising the step (4) of:

(4) in vitro culture of fertilized eggs: after fertilization is carried out for 20-22 h, the sperms are washed by fertilization liquid to remove other liquid attached in the process of transferring zygote; washing the fertilized eggs with the solution C, and then transferring the fertilized eggs into a granular cell monolayer for culture; the culture conditions are 5% CO2, 100% humidity and 38.5 ℃, and the culture time is 48 hours; for example

The C solution, namely the embryo culture solution, comprises the following components: 54% Tyrode's solution (modified), 10% FBS (fetal bovine serum) and 36% TCM-199 solution, and are pre-balanced in an incubator for 1-2 h before use.

7. The method according to claim 1,

the granulosa cell monolayer was cultured as follows: centrifuging and washing the blown granular cells on the oocytes by using an embryo culture solution; making 8 particle cell microdroplets of 20 mu L in each culture dish, covering with mineral oil, removing impurities by using a glass fine needle, and placing in an incubator for culture;

the maturation culture solution also comprises sodium caprylate and polyvinyl alcohol; for example, the composition also comprises 20mg/L of sodium caprylate and 5mg/L of polyvinyl alcohol; and/or

The mature culture solution comprises the following components: 20mg/L sodium caprylate, 5mg/L polyvinyl alcohol, 10% ECS (oestrus calf serum), 26.2mmol/L NaHCO3, 10. mu. mol/L EGCG, 5mmol/L Hepe, 1. mu.g/mL E2 (estradiol), 3% BFF (bovine follicular fluid), 10. mu.g/mL LH (luteinizing hormone), 100. mu. mol/L cysteine, 10. mu.g/mL FSH (follicle stimulating hormone), TCM-199 to 100%.

8. The method according to claim 1, comprising the steps of:

i. superovulation treatment before egg taking

ii. Living egg taking

iii detection of oocytes

(3) in vitro fertilization of oocytes: putting the F liquid into an incubator for balancing 4-6 hours in advance; after the frozen semen is processed by an upstream method, sucking 1mL of liquid at the upper layer, and centrifuging at normal temperature; discarding 800-900 mu L of supernatant immediately after centrifugation, putting the supernatant back into an incubator for incubation for 5-10 min, and gently mixing semen by using a pipette before use; placing 15-20 oocytes subjected to in vitro maturation culture in each fertilization liquid drop, and adding 12-15 mu L of semen to enable the density of the semen in each fertilization liquid drop to reach (1.0-1.5) x10⁶Dropping the fertilized liquid into an incubator with 39 ℃, 5% CO2 and 100% humidity for incubation for 20-22 h to perform in vitro fertilization culture;

9. A maturation culture fluid comprising: 10% ECS (oestrus calf serum), 26.2mmol/L NaHCO3, 10. mu. mol/L EGCG, 5mmol/L Hepe, 1. mu.g/mL E2 (estradiol), 3% BFF (bovine follicular fluid), 10. mu.g/mL LH (luteinizing hormone), 100. mu. mol/L cysteine, 10. mu.g/mL FSH (follicle stimulating hormone), TCM-199 to 100%.

10. The maturation culture according to claim 9, further comprising sodium caprylate and polyvinyl alcohol; for example, the sodium caprylate/L and the polyvinyl alcohol/L also comprise 20mg/L and 5 mg/L; for example, it consists of: 20mg/L sodium caprylate, 5mg/L polyvinyl alcohol, 10% ECS (oestrus calf serum), 26.2mmol/L NaHCO3, 10. mu. mol/L EGCG, 5mmol/L Hepe, 1. mu.g/mL E2 (estradiol), 3% BFF (bovine follicular fluid), 10. mu.g/mL LH (luteinizing hormone), 100. mu. mol/L cysteine, 10. mu.g/mL FSH (follicle stimulating hormone), TCM-199 to 100%.