Yak oocyte long-distance transportation in-vitro maturation culture method and culture maturation liquid

Abstract

The invention relates to a Tibetan yak oocyte in-vitro maturation method under a long-distance transportation condition. According to the method, the optimal transportation preservation temperature of the yak ovary is firstly determined to be 30 DEG C under the long-time transportation condition, so that the activity and the quality of the oocyte after long-distance transportation are improved from the first step. Secondly, the problems of transportation distance and transportation time of the yak oocytes are solved, long-distance transportation tools such as high-speed rails and airplanes are fully utilized, a portable constant-temperature incubator is used, in-vitro maturation culture of the yak oocytes on a long-distance transportation road is realized by utilizing a maturation tube, the maturation rate can reach 55.29%, and the survival rate of the yak oocytes is greatly improved. And the influence of long-distance transportation distance and temperature on later embryonic development is greatly reduced. The invention provides a new thought for developing and utilizing livestock germplasm resources in remote places, greatly saves the manufacturing time of laboratory embryos through long-distance transportation and in-vitro maturation, and provides reference for further improving the production efficiency of the yak in-vitro embryos.

Description

Technical Field

[0001] The invention relates to the technical field of cytology, and in particular to a method for long-distance transportation of yak ovaries and oocytes and a maturation and culture system for oocytes during long-distance transportation. Background Art

[0002] Yaks are a unique livestock resource of the Qinghai-Tibet Plateau. They are also known as universal livestock and are known as the "ship of the plateau". Their meat and milk are not only the main food source in the plateau, but their feces and fur are also the main heating and insulation materials in pastoral areas. They are an important economic foundation and pillar industry in high-altitude pastoral areas. In vitro maturation of oocytes, in vitro embryo production and embryo transplantation are important links in the preservation and transmission of high-quality germplasm resources. The use of appropriate methods to preserve and transport yak ovaries and oocytes is particularly important for better collection and utilization of precious livestock resources in the Qinghai-Tibet Plateau.

[0003] Due to the limited number of oocytes collected from living bodies, the oocytes required for embryo engineering experiments are currently collected from the ex vivo ovaries of animals slaughtered in slaughterhouses and sent to the laboratory for processing as soon as possible within 1 to 2 hours. Animal slaughterhouses are generally far away from urban areas and located in remote counties and towns, with inconvenient transportation. Therefore, in actual production, the quality of oocytes collected from the ovaries will be affected by the transportation distance, method and temperature, and the quality of oocytes directly affects the subsequent embryonic development. Previous studies have shown that long ovarian transportation time will affect the quality of oocytes, inhibit protein synthesis and the occurrence of germinal vesicle rupture, and significantly reduce the in vitro maturation rate of oocytes. The in vitro maturation of oocytes is the basis of in vitro fertilization and in vitro embryo development, and its maturation is the key to determining the cleavage rate and subsequent blastocyst development rate. Therefore, the transportation time and storage temperature of the ovaries will affect the in vitro embryonic development. The yak living area is located in the plateau, and transportation is more inconvenient. It takes a longer time to transport ovaries from the slaughterhouse to the laboratory, which greatly limits the research and utilization of yak ovary resources. If a suitable method can be found to preserve and transport yak ovaries, it will be of great significance for better collection and utilization of yak oocytes and improving the quality of subsequent embryonic development.

[0004] At present, the collected ovaries are generally stored in physiological saline containing antibiotics. The transportation and storage temperatures of ovaries of different species are different, and the commonly used range for short-distance transportation is generally 35-38°C. At present, the existing technology believes that for the research on the transportation conditions of yak ovaries, the temperature is generally around 35°C as the best storage temperature. At this temperature, the survival rate of yak oocytes is the highest. However, when transported over long distances at this temperature, the survival rate of yak oocytes is low. In the existing technology, yak ovaries are rarely transported over long distances. Therefore, exploring the research on the transportation of yak ovaries under long-distance transportation conditions is of great significance to improving the subsequent efficiency of yak oocyte maturation.

[0005] Secondly, the cost of long-distance transportation is high, and the yak germplasm resources are rare and precious. How to maximize the recovery efficiency of yak ovaries and whether yak oocytes can be matured in vitro during transportation are also urgent issues to be solved. Generally, oocytes are first obtained by extracting cumulus-oocyte complexes (COCs) from follicular fluid. At present, the quality assessment of COCs is generally classified according to microscopic morphology: COCs with more than three layers of cumulus cells, uniform cytoplasm and dense structure are classified as grade A; COCs with 1-3 layers of cumulus cells are classified as grade B; naked eggs and COCs with poor cytoplasm morphology are classified as grade C. Research on the maturation rate of yak oocytes generally revolves around the three more mature COCs, while COCs (Ex-COCs) whose cumulus cells have begun to spread under the microscope are usually discarded and rarely studied. If Ex-COCs can also be transported for long-distance in vitro maturation, the recovery efficiency of yak ovaries and the in vitro maturation efficiency of oocytes can be greatly improved.

[0006] If we can solve the above problems, realize the long-term transportation and preservation of yak ovaries, and establish an in vitro maturation system for long-distance transportation of yak oocytes, turn the distance shortcoming into a bridge for scientific research, and realize the nationwide transportation of yak ovaries and oocytes, it will be of great significance to improve the maturation efficiency of yak oocytes, improve scientific research efficiency, save scientific research costs, and better develop and utilize the precious yak germplasm resources of the Qinghai-Tibet Plateau. Summary of the invention

[0007] In order to overcome the problems of related technologies and laboratory distance, the present invention provides a convenient method for preserving yak ovaries during long-distance transportation and a system for in vitro maturation and culture of yak oocytes during long-distance transportation.

[0008] The purpose of the present invention is to improve the recovery rate of oocytes by determining the optimal storage temperature of yak ovaries during long-distance transportation and studying the maturation efficiency of two cumulus ovarian complexes (Cp-COCs; Ex-COCs). At the same time, a portable constant temperature incubator is used to establish an in vitro maturation system for yak oocytes in long-distance transportation tubes, thereby reducing the impact of long-term transportation on the developmental potential of yak in vitro embryos.

[0009] In order to realize the above invention, the present invention adopts the following technical scheme, a method for in vitro maturation of Tibetan yak oocytes during long-distance transportation, characterized in that it comprises the following steps:

[0010] (1) Determine the optimal storage temperature for long-distance transportation of yak ovaries: The in vitro ovaries collected from the slaughterhouse were transported to the laboratory closest to the transportation hub using ovarian preservation fluids with different storage temperatures. Follicular fluid with a diameter of 3-8 mm on the upper surface of the yak ovaries was extracted, diluted with egg-picking fluid, and placed under a microscope to select cumulus-oocyte complexes (COCs) wrapped with more than 2 to 3 layers of GCs. According to the diffusion morphology of GCs on the surface of the oocytes, the collected oocytes were divided into extended oocytes (Ex-COCs) with a loose diffusion shape and compact oocytes (Cp-COCs) with a compact diffusion state. After washing with egg washing fluid and maturation fluid, the recovery efficiency was calculated. The difference in the recovery efficiency of COCs at different transportation and storage temperatures of yak ovaries was obtained, and the effect of different transportation temperatures on the recovery efficiency of COCs was clarified based on the results.

[0011] (2) In vitro maturation of oocytes during long-distance transportation: The Cp-COCs and Ex-COCs obtained in step (1) were washed and grouped into pre-prepared maturation tubes, which were sealed tightly and placed in a portable constant temperature incubator for culture. The incubator temperature was set at 38.5°C.

[0012] (3) Statistical analysis of oocyte maturation efficiency: The long-distance transportation maturation efficiency of the collected Cp-COCs and Ex-COCs in a portable constant temperature incubator was measured, and the in vitro maturation efficiency data of dairy cow oocytes in previous short-distance transportation (within 2 h) were used for comparative analysis. At the same time, high-quality mature oocytes were selected from the groups with high maturation efficiency and no significant difference for parthenogenetic activation (PA) to further verify the effect of the culture system of the present invention on the subsequent embryonic development of yaks.

[0013] Furthermore, in step (1), determining the optimal storage temperature for long-distance transportation of yak ovaries comprises the following steps:

[0014] 1.1 Ovarian collection: The ovaries of yaks that have just been slaughtered and isolated from the slaughterhouse are immediately placed in thermos cups of physiological saline at different temperatures. The different storage and transportation temperatures include: 4°C, room temperature, 20°C, 25°C, 30°C, and 38°C.

[0015] 1.2 Long-distance transportation of ovaries: Before the collected ovaries are shipped out of the factory, they are washed 2-3 times with physiological saline at different temperatures and placed in a thermos cup filled with ovarian preservation fluid at the corresponding temperature for transportation, ensuring that the temperature in the cup is constant at the transportation temperature.

[0016] 1.3 Recovery of oocytes: After the ovaries arrive at the egg collection laboratory, they are washed 2-3 times with 30°C saline until no blood is washed out. The washed ovaries are placed in a beaker filled with saline and the beaker is placed on a hot table. Use disposable sterile hand towels to dry the surface of the ovaries, and use a disposable syringe with a specification of 10mL to extract follicular fluid from follicles with a diameter of 3-8mm. Add an appropriate amount of egg washing solution to the extracted follicular fluid and shake it well, then transfer it to a 60mm glass dish. Under a stereomicroscope, pick out COCs with complete cumulus encapsulation and good morphology, wash them 3 times with egg washing solution, wash away impurities, and then wash them twice with oocyte maturation culture medium. According to the degree of diffusion of cumulus cells, the cleaned COCs are sorted into compact and diffuse types under the microscope, and the recovery rate is calculated.

[0017] Furthermore, in step (2), the long-distance transport in vitro maturation of oocytes comprises the following steps:

[0018] 2.1 Preparation of oocyte maturation solution: prepare 5 mL round-bottom glass centrifuge tubes, dispense 4 mL of the prepared maturation solution into the centrifuge tubes, open the tubes and place them in an incubator at 38.5°C, 5% volume CO2 and maximum saturated humidity for 10-15 hours to equilibrate, then seal them and store them at -20°C.

[0019] 2.2 In vitro maturation of oocytes using a portable incubator: Cp-COCs and Ex-COCs sorted under a microscope were transferred into tubes of maturation solution in groups. The tube walls were marked with compact and extended types and the number of cells. After being tightly sealed, they were placed in a portable constant temperature incubator for culture. The incubator temperature was set to 38.5°C.

[0020] Furthermore, the ovarian preservation solution in step (1) is composed of physiological saline plus 5% double antibody; the double antibody refers to 75 μg / mL penicillin and 100 μg / mL streptomycin.

[0021] Furthermore, the ovarian egg washing solution in step (1) is composed of Dulbecco's phosphate buffered saline (DPBS) plus 5% fetal bovine serum.

[0022] Furthermore, in step (2), the maturation fluid includes TCM199 basal culture fluid, fetal bovine serum FBS, follicle stimulating hormone FSH, luteinizing hormone LH, estradiol E2, epidermal growth factor EGF, cysteine, sodium pyruvate and double antibody (penicillin-streptomycin); in the maturation fluid, the volume ratio of TCM199 basal culture fluid to fetal bovine serum FBS is 10:1; the volume ratio of TCM199 basal culture fluid to double antibody is 100:1. In the maturation fluid, the concentration of follicle stimulating hormone FSH is 5-10 μg / mL; the concentration of luteinizing hormone LH is 5-10 μg / mL, the concentration of estradiol E2 is 1 μg / mL, the concentration of epidermal growth factor EGF is 100 ng / mL, the concentration of cysteine ​​is 0.05-0.1 mg / mL, and the concentration of sodium pyruvate is 0.04-0.05 mg / mL.

[0023] Furthermore, in the step (2), the oocyte maturation tube is thawed inverted at room temperature before use, and then temporarily stored in a portable constant temperature incubator at 38.5°C;

[0024] Furthermore, the oocyte maturation tube in step (2) can be transported at -20°C cold chain to any egg collection laboratory.

[0025] Furthermore, in step (2), 80-100 oocytes are placed in each maturation tube. Oocytes have an interaction effect during the maturation process, and this number can achieve the optimal culture effect. Too much or too little will affect the maturation efficiency of oocytes.

[0026] Furthermore, in step (2), the maturation tube needs to be slowly shaken up and down every four hours until the incubator is transported to the laboratory by high-speed rail or airplane;

[0027] Furthermore, in step (3), after the incubator arrives at the laboratory, the two COCs need to continue to mature in the tube for 22-24 hours, and then the maturation efficiency is calculated.

[0028] Furthermore, in step (3), the specific steps of parthenogenetic activation (PA) are as follows: mature yak oocytes are selected and first treated with mSOF solution containing 5 μmol / L ionomycin for 5 minutes, and then cultured in mSOF containing 2 mmol / L 6-DMAP for 4 hours. The activated embryos are washed 3 times with culture medium and then transferred to culture droplets for culture. The cleavage rate is counted after 24 hours of culture, and the cleavage and blastocyst formation rates are counted after 168 hours.

[0029] All raw materials of the present invention can be purchased commercially or prepared according to conventional methods in the art.

[0030] The technical route described in the present invention uses yaks as the research object, but is also applicable to other studies that require long-distance transportation of ovaries, oocytes and embryos.

[0031] The present invention is also applicable to all long-distance transportation modes, including road transportation, high-speed rail, airplanes, etc.

[0032] Compared with the prior art, the present invention has the following advantages:

[0033] 1. The present invention overcomes the technical prejudice that the best storage temperature of yak ovaries during transportation is about 35°C. The present invention creatively finds that storing yak ovaries at about 30°C is more conducive to long-distance transportation of ovaries and improves the survival rate of oocytes. By storing yak ovaries at 30°C for long-distance transportation, the average maturation rate of oocytes (55.18%), the cleavage rate (77.25%) and the blastocyst rate (8.47%) of parthenogenetic embryos are significantly improved.

[0034] 2. It does not affect the recovery efficiency of ovaries after long-distance transportation, which helps to improve the maturation efficiency of oocytes and the development quality of embryos after long-distance transportation;

[0035] 2. A simple egg collection laboratory is set up during long-distance transportation to shorten the ovarian transportation time. The oocytes obtained mature during the next long-distance transportation process, saving the time and cost of embryo production throughout the process, breaking the time and space limitations, and facilitating the protection and utilization of livestock germplasm resources nationwide;

[0036] 4. The yak ovaries were preserved at 30°C for long-distance transportation, and the yak oocyte maturation types were subdivided into compact oocytes (Cp-COCs) and extended oocytes (Ex-COCs). The maturation efficiency was compared respectively. There was no significant difference in the maturation efficiency of the two types of oocytes, which helps to improve the efficiency of ovarian recovery under conditions of high transportation costs.

[0037] 5. The present invention has developed a yak oocyte culture maturation solution, which promotes the maturation culture of yak oocytes. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] The invention will be further described below in conjunction with the accompanying drawings.

[0039] Figure 1 Compact (A) and expanded (B) oocytes selected under the microscope of the present invention, with a magnification of 20 times;

[0040] Figure 2 The compact (A) and expanded (B) oocytes matured for 22-24 hours after long-distance transportation according to the present invention are shown at a magnification of 20 times;

[0041] Figure 3 The portable transport box (Biotherm) suitable for any mode of transportation used in the present invention TM INC-RB1);

[0042] Figure 4 The parthenogenetic embryos are made from oocytes that matured after long-distance transportation in the present invention. A shows the blastocyst development in the 25°C group; B shows the blastocyst development in the 30°C group; C shows the blastocyst development in the 35°C group; the magnification is 100 times. DETAILED DESCRIPTION

[0043] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention. Unless otherwise specified, the technical means used in the examples are conventional means known to those skilled in the art, and all raw materials are commercially available products.

[0044] Experimental materials: Yak ovaries were collected from slaughterhouses in Maqu County and Luqu County, Gannan Tibetan Autonomous Prefecture, Gansu Province.

[0045] A method for in vitro maturation of yak oocytes during long-distance transportation comprises the following steps:

[0046] (1) Ovarian collection: Yak ovaries collected from slaughterhouses need to be placed in a thermos cup, and 1 / 3 of the volume of ovarian preservation fluid at different temperatures is added to the cup in advance. Different storage temperatures include: 4°C, room temperature, 20°C, 25°C, 30°C, and 38°C. Among them, the ovarian preservation fluid is composed of physiological saline plus 5% double antibody.

[0047] The above dual antibodies refer to 75 μg / mL penicillin and 100 μg / mL streptomycin.

[0048] (2) Long-term transportation of ovaries: Due to the long transportation time, the collected ovaries need to be pre-treated before leaving the factory for transportation. That is, they need to be washed 2-3 times with physiological saline at the corresponding storage temperature until there is no blood, and then placed in a thermos cup filled with ovarian preservation solution at different temperatures for transportation to ensure that the temperature in the cup is constant at the corresponding storage temperature.

[0049] (3) Recovery of oocytes: After the ovaries arrive at the laboratory, the connective tissue, fat and attached fallopian tubes on the surface of the ovaries are cut off. They are then washed with 30°C saline for 2-3 times until there is no blood. The washed ovaries are placed in a beaker filled with 30°C saline and placed on a hot plate. The surface of the ovaries is wiped dry with disposable sterile hand towels, and follicular fluid from follicles with a diameter of 3-8 mm is extracted using a 10 mL disposable syringe. The extracted follicular fluid is added with an appropriate amount of egg washing solution and shaken well, then transferred to a 60 mm glass dish. COCs with intact cumulus encapsulation and good morphology are picked out under a stereomicroscope, washed three times with egg washing solution, and after washing away impurities, they are washed twice with oocyte maturation culture medium. According to the degree of diffusion of cumulus cells, the washed COCs are sorted under the microscope into compact (Cp-COCs) and extended (Ex-COCs), and the recovery rate is calculated.

[0050] The egg washing solution is composed of 5% fetal bovine serum plus Dulbecco's phosphate buffered saline (DPBS).

[0051] (4) In vitro maturation of oocytes: The pre-balanced thawed maturation tubes are placed in a portable constant temperature incubator (temperature 38.5°C) for temporary storage, and the compact (Cp-COCs) and extended (Ex-COCs) oocytes cleaned in step (3) are transferred to the preheated maturation tubes, and the maturation tubes are placed in a portable constant temperature incubator (temperature 38.5°C) for transportation and maturation. The portable constant temperature incubator is selected from Biotherm TM INC-RB1 portable incubator; wherein the composition of the egg washing solution is 5% fetal bovine serum plus Dulbecco's phosphate buffered saline (DPBS); wherein the preparation of oocyte maturation solution refers to the preparation of maturation solution in the laboratory, which is divided into 4mL to 5mL round-bottom glass tubes, and the centrifuge tubes are opened and placed in an incubator at 38.5℃, 5% volume CO2 and maximum saturated humidity for 10-15h, and then sealed and stored at -20℃. The frozen maturation tubes are transported to the egg collection laboratory at -20℃ cold chain, thawed at room temperature before use, and then preheated in a 38.5℃ constant temperature portable incubator.

[0052] The maturation fluid includes TCM199 basic culture fluid, fetal bovine serum FBS, follicle stimulating hormone FSH, luteinizing hormone LH, estradiol E2, epidermal growth factor EGF, cysteine, sodium pyruvate and double antibody (penicillin-streptomycin); in the maturation fluid, the volume ratio of TCM199 basic culture fluid to fetal bovine serum FBS is 10:1; the volume ratio of TCM199 basic culture fluid to double antibody is 100:1. In the maturation fluid, the concentration of follicle stimulating hormone FSH is 5-10μg / mL; the concentration of luteinizing hormone LH is 5-10μg / mL, the concentration of estradiol E2 is 1μg / mL, the concentration of epidermal growth factor EGF is 100ng / mL, the concentration of cysteine ​​is 0.05-0.1mg / mL, and the concentration of sodium pyruvate is 0.04-0.05mg / mL; 80 to 100 oocytes are placed in each maturation tube.

[0053] (5) Statistics of MⅡ oocyte maturation efficiency

[0054] After the oocytes arrive at the destination laboratory after long-distance transportation, the two COCs need to continue to mature in the tube for 22-24 hours, and then the maturation efficiency is counted. The matured oocytes are mixed with 1mg / mL hyaluronidase and repeatedly blown with a 100μL pipette for 2-5 minutes to remove the cumulus cells. Under a stereomicroscope, oocytes with intact yolk membrane, clear perivitelline space, uniform cytoplasm, and expelling the first polar body are selected as mature oocytes for counting.

[0055] The statistical results of long-distance transportation of ovaries and oocytes using this method are as follows:

[0056] Table 1: Effects of different transport temperatures on yak ovary preservation and oocyte maturation efficiency

[0057]

[0058] Note: Different letters in the same column (a, b, c, d) indicate significant differences (P<0.05); the data format in the table is mean ± standard deviation;

[0059] The results in Table 1 show that when the yak ovary is stored at 30℃, the long-distance transportation effect is the best. The maturation efficiency of compact oocytes (Cp-COCs) and extended oocytes (Ex-COCs) are 55.29% and 56.10%, respectively. The maturation rates of the two COCs are not much different, but they are significantly higher than the maturation efficiency of COCs transported under other storage and transportation temperature conditions. In addition, referring to the laboratory's previous short-distance transportation of Holstein cows for 1 to 2 hours, the ovaries were stored at 30℃, and the temperature range of arrival in the laboratory was 27 to 30℃. The maturation efficiency of Cp-COCs was 48.78% (4436 / 8927), and the maturation efficiency of Ex-COCs (435 / 1060) was 45.29%, and the difference in maturation rate was not significant.

[0060]

Conclusion

[0061] (6) Determination of the development of reconstructed embryos from mature oocytes after long-distance transportation

[0062] To further evaluate the effect of long-distance ovarian transportation on yak oocytes and the quality of yak oocytes matured in the transport box, a group with a maturation rate of more than 30% and no significant difference was selected, and a part of the high-quality mature oocytes obtained in (5) were parthenogenetically activated (PA) to determine whether the mature oocytes after long-distance transportation can support the early development of PA. The mature oocytes were first treated with mSOF solution containing 5μmol / L ionomycin for 5min, and then cultured in mSOF containing 2mmol / L 6-DMAP for 4h. The activated embryos were washed 3 times with culture medium and then transferred to culture microdroplets for culture. The cleavage rate was counted after 24h of culture. The blastocyst formation rate was counted after 168h of culture.

[0063] Table 2 Developmental efficiency of PA embryos under different transport temperature conditions

[0064]

[0065] The results in Table 2 show that when the yak ovary is stored at 30°C, the cleavage rate (77.25%) and blastocyst rate (8.47%) are the highest. When the yak ovary is stored at 25°C, the oocyte maturation rate (48.86±0.11) is significantly different from that at 38°C (30.95±0.05), but the subsequent embryo development results are not ideal. This shows that the influence of ovarian storage temperature on subsequent embryo development is crucial, and the oocyte maturation rate does not directly represent the degree of cleavage and blastocyst rate of later embryo development.

[0066]

Conclusion

[0067] In summary, under long-distance transportation conditions (6 to 8 hours), the optimal storage temperature of yak ovaries is 30°C, which can significantly improve the recovery efficiency, maturation rate, and cleavage rate and blastocyst rate of yak oocytes in late embryonic development.

[0068] Obviously, the above embodiments are merely examples for the purpose of clear explanation, and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived therefrom are still within the scope of protection of the invention.

Claims

1. A method for in vitro maturation of yak oocytes during long-distance transportation, characterized in that The steps include: (1) Ovarian collection: The ovaries of freshly slaughtered yaks collected from the slaughterhouse were immediately placed in a thermos cup filled with 30°C saline and washed 2-3 times with 30°C saline. (2) Long-distance transportation of ovaries: The cleaned ovaries are transported to a laboratory near a large transportation hub at 30°C, and the temperature in the cup must be kept constant at 30°C; (3) Oocyte recovery: In the laboratory, follicular fluid was extracted from follicles with a diameter of 3-8 mm on the upper surface of yak ovaries. Under a stereomicroscope, diffuse and compact cumulus-oocyte complexes (COCs) were selected and sorted according to the degree of diffusion of cumulus cells. (4) In vitro maturation of oocytes during long-distance transportation: The cumulus ovarian-oocyte complex (COCs) obtained in step (3) is washed with an egg washing solution and a maturation solution, respectively, and then placed in a maturation tube containing a maturation solution prepared in advance; the maturation tube is tightly sealed and placed in a portable constant temperature incubator for culture, and the cumulus ovarian-oocyte complex (COCs) is cultured and matured in the maturation tube; the incubator temperature is set to 38.5°C, and the incubator is shaken up and down every four hours until the incubator is delivered to the destination.

2. A method for long-term in vitro maturation of yak oocytes according to claim 1, characterized in that: In the above step (2), during transportation: the thermos cup needs to be placed in a foam insulation box and wrapped with simple insulation material. After 3 to 4 hours of long-distance transportation, the physiological saline solution should be replaced to ensure that the temperature in the cup is kept constant at 30°C.

3. A method for long-term transport in vitro maturation of yak oocytes according to claim 1, characterized in that: The egg washing solution in step (4) is Dulbecco's phosphate buffered saline (DPBS) containing 5% fetal bovine serum.

4. A method for long-term transport in vitro maturation of yak oocytes according to claim 1, characterized in that: The maturation fluid includes TCM199 basic culture fluid, fetal bovine serum FBS, follicle stimulating hormone FSH, luteinizing hormone LH, estradiol E2, epidermal growth factor EGF, cysteine, sodium pyruvate and double antibody (penicillin-streptomycin); in the maturation fluid, the volume ratio of TCM199 basic culture fluid to fetal bovine serum FBS is 10:1; the volume ratio of TCM199 basic culture fluid to double antibody is 100:

1. In the maturation fluid, the concentration of follicle stimulating hormone FSH is 5-10 μg / mL; the concentration of luteinizing hormone LH is 5-10 μg / mL, the concentration of estradiol E2 is 1 μg / mL, the concentration of epidermal growth factor EGF is 100 ng / mL, the concentration of cysteine ​​is 0.05-0.1 mg / mL, and the concentration of sodium pyruvate is 0.04-0.05 mg / mL.

5. A method for long-term transport in vitro maturation of yak oocytes according to claim 1, characterized in that: The maturation tube in step (4) is a 5 mL round-bottom glass centrifuge tube, and the volume of the built-in maturation liquid is 4 mL.

6. The method for in vitro maturation of yak oocytes during long-distance transportation according to claim 1, characterized in that: The method for preparing the mature tube prepared in advance in step (4) is as follows: S1. Before collecting ovaries, prepare yak oocyte maturation solution according to the composition of maturation solution; S2. The prepared mature solution is dispensed into centrifuge tubes with the mouth of the centrifuge tubes open; S3. Place the packaged mature tubes in an incubator at 38.5°C, 5% volume CO2 and maximum saturated humidity for 10-15 hours, seal them and store them at -20°C; S4. Transport the frozen maturation tubes at -20℃ cold chain to the egg collection laboratory. Thaw them upside down at room temperature before use, and then preheat them in a portable constant temperature incubator at 38.5℃.

7. A method for long-term in vitro maturation of yak oocytes according to claim 1, characterized in that: In the step (4), 80-100 oocytes are placed in each maturation tube.

8. The method for long-term in vitro maturation of yak oocytes according to any one of claims 1 to 7, characterized in that: The method is applicable to any mammalian ovary and oocyte that are stored at an optimal temperature and need to be transported over long distances.

9. The method for long-term in vitro maturation of yak oocytes according to any one of claims 1 to 7, characterized in that: The method is applicable to in vitro maturation culture of oocytes in any long-distance transportation mode such as automobile, train, high-speed rail, airplane, etc.

10. A yak oocyte culture maturation solution, the maturation solution comprising TCM199 basic culture solution, fetal bovine serum (FBS), follicle stimulating hormone (FSH), luteinizing hormone (LH), estradiol (E2), epidermal growth factor (EGF), cysteine, sodium pyruvate and double antibody (penicillin-streptomycin); in the maturation solution, the volume ratio of TCM199 basic culture solution to fetal bovine serum (FBS) is 10:1; the volume ratio of TCM199 basic culture solution to double antibody is 100:

1. In the maturation solution, the concentration of follicle stimulating hormone (FSH) is 5-10 μg / mL; the concentration of luteinizing hormone (LH) is 5-10 μg / mL, the concentration of estradiol (E2) is 1 μg / mL, the concentration of epidermal growth factor (EGF) is 100 ng / mL, the concentration of cysteine ​​is 0.05-0.1 mg / mL, and the concentration of sodium pyruvate is 0.04-0.05 mg / mL.

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