In-vitro fertilization liquid capable of improving fertilization ability of bovine sperms and preparation method thereof

Abstract

The invention provides an in-vitro fertilization liquid capable of improving the fertilization capacity of bovine sperms and a preparation method thereof, and belongs to the technical field of biology. According to the in-vitro fertilization fluid capable of improving the fertilization capacity of the bovine sperms and the preparation method of the in-vitro fertilization fluid, the in-vitro fertilization fluid comprises a sperm treatment fluid and an in-vitro fertilization culture fluid, and the sperm treatment fluid and the in-vitro fertilization culture fluid respectively comprise a storage fluid and a working fluid, in-vitro embryo production and transplantation are carried out by using oocytes obtained from live egg collection and ovaries in a slaughter house, and a relatively high blastocyst rate and a similar transplantation conception rate can be obtained. Meanwhile, reagents involved in the in-vitro fertilization liquid are simple, easy to obtain and low in price, the production cost is reduced while the bovine in-vitro embryo production efficiency is guaranteed, sperm movement parameters can be improved by treating sperms through the in-vitro fertilization liquid, the in-vitro fertilization capacity of the sperms is improved, and the in-vitro fertilization liquid is suitable for popularization and application. And a relatively high blastocyst rate and embryo transplantation conception rate are obtained.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, in particular to an in vitro fertilization solution capable of improving the fertilization ability of bovine sperm and a preparation method thereof. Background Art

[0002] With the widespread application of artificial insemination and in vitro fertilization technology in bovine in vitro embryo related production practice and research, how to further improve the fertilization ability of thawed sperm has become a focus of increasing attention. The ultimate goal of in vitro sperm treatment is to improve sperm motility and enhance sperm fertilization ability. Sperm capacitation, that is, sperm activation treatment process, is a key link in the bovine in vitro embryo production technology system. After the sperm is activated, the harmful components in the semen are removed, the capacitation inhibitory factors attached to the sperm cell membrane are removed, the sperm metabolic function is improved, the sperm survival time in vitro is prolonged, the sperm survival rate in vitro is improved, and the sperm undergoes acrosome reaction, thereby having the ability to fertilize the oocyte.

[0003] Artificial sperm activation methods are divided into physical activation and chemical activation. The commonly used method of physical activation is to use a low-intensity helium-neon laser to treat sperm, thereby stimulating sperm activity. The commonly used method of chemical activation is to use certain special chemical reagents, such as non-selective phosphodiesterase (PDE) inhibitors, biotin, sperm agonists, etc., to incubate with sperm in vitro for a certain period of time, thereby achieving the purpose of improving sperm motility. At present, the most widely used sperm activation factors in artificial sperm activation are non-selective phosphodiesterase inhibitors, mainly including caffeine, pentoxifylline and theophylline.

[0004] Theophylline is 1,3-dimethylxanthine, which is mainly found in tea leaves and coffee beans in nature. It is soluble in water, ethanol, chloroform, alkali hydroxide and ammonia water, and can also be soluble in dilute hydrochloric acid and nitric acid, but is difficult to dissolve in ether. In clinical practice, theophylline is often used to treat respiratory diseases such as chronic obstructive pulmonary disease and asthma. In addition, theophylline is also an important sperm activator. Theophylline can enhance sperm motility. Its mechanism of action is to inhibit the activity of sperm phosphodiesterase (PDE). PDE is a type of intracellular hydrolase responsible for the degradation of cyclic adenosine monophosphate (cAMP). Theophylline inhibits PDE activity, leading to increased intracellular cAMP levels. cAMP is an important second messenger that mediates protein kinase A and downstream signaling pathways to induce axonemal protein phosphorylation, thereby promoting sperm motility.

[0005] Hishinuma M et al. added 2 mmol / L theophylline to the dog epididymal sperm culture medium and successfully induced epididymal sperm to penetrate the egg. Ebner et al. proved that theophylline is a harmless and effective biological agent that can be used to restore immobile sperm in human retrograde ejaculation problems. In 2015, Ferre et al. found that theophylline can increase the in vitro fertilization rate of sperm. In 2018, Gorji et al. found that in the freezing process of freshly ejaculated sperm and testicular puncture sperm, adding 40 mmol / L theophylline to the freezing fluid can reduce the extent of the decline in sperm motility after recovery, suggesting that theophylline has the effect of protecting sperm against freezing damage.

[0006] Bovine in vitro embryo production technology can effectively utilize cow eggs, improve the reproductive efficiency of excellent cows through in vitro fertilization, and accelerate the proliferation of excellent breeds. How to improve sperm fertilization ability has become the core technology that limits the efficiency of bovine in vitro embryo production, and plays a vital role in improving embryo production efficiency. Although domestic experts have added theophylline to the bull sperm capacitation fluid to improve various sperm movement parameters, how to reasonably use theophylline in bovine in vitro embryo production, improve sperm activation processing technology, and improve fertilization rate and in vitro embryo production efficiency still needs further research.

[0007] To achieve this goal, the present invention has developed an in vitro fertilization fluid and a preparation method thereof that can improve the fertilization ability of bovine sperm. The method of the present invention can significantly improve the in vitro fertilization ability of bovine sperm, for example, it can significantly improve sperm motility and fertilization rate, and further improve cleavage rate and blastocyst rate. Summary of the invention

[0008] In order to make up for the above deficiencies, the present invention provides an in vitro fertilization solution and a preparation method thereof that can improve the fertilization ability of bovine sperm and overcome the above technical problems or at least partially solve the above problems.

[0009] The present invention is achieved in that:

[0010] The present invention provides an in vitro fertilization solution capable of improving the fertilization ability of bovine sperm, comprising an in vitro fertilization solution, wherein the in vitro fertilization solution is composed of a sperm treatment solution and an in vitro fertilization culture solution, and is characterized in that;

[0011] The sperm treatment solution and in vitro fertilization culture solution include a storage solution and a working solution respectively.

[0012] In a preferred embodiment, the sperm processing storage solution includes sperm processing storage solution A and sperm processing storage solution B, and the sperm processing storage solution A is an aqueous solution containing 35mM potassium chloride, 700mM sodium chloride, 3mM sodium dihydrogen phosphate, 100mM 4-hydroxyethylpiperazineethanesulfonic acid, 15mM calcium chloride dihydrate, and 5mM magnesium chloride hexahydrate.

[0013] In a preferred embodiment, the sperm treatment fluid storage solution B is an aqueous solution containing 100 mM sodium bicarbonate, and the sperm treatment fluid working solution contains sperm treatment fluid working solution A, sperm treatment fluid working solution B, and sperm treatment fluid working solution C.

[0014] In a preferred embodiment, the sperm treatment fluid working solution A is an aqueous solution containing 10% (v / v) sperm treatment fluid storage solution A, 90% percoll, 25mM sodium bicarbonate, and 25mM sodium lactate, and the sperm treatment fluid working solution B contains an aqueous solution of 10% (v / v) sperm treatment fluid working solution A, 25% sperm treatment fluid storage solution B, 2mM sodium pyruvate, and 15mM sucrose.

[0015] In a preferred embodiment, the sperm processing fluid working solution C is a solution containing 50% (v / v) sperm processing fluid working solution A, 50% sperm processing fluid working solution B, and 100 μl phenol red, and the in vitro fertilization culture medium includes in vitro fertilization culture medium storage solution and in vitro fertilization culture medium working solution.

[0016] In a preferred embodiment, the in vitro fertilization culture medium stock solution includes in vitro fertilization culture medium stock solution A and in vitro fertilization culture medium stock solution B, and the in vitro fertilization culture medium stock solution A is an aqueous solution containing 115mM sodium chloride, 4mM potassium chloride, 3mM calcium chloride dihydrate, 0.6mM magnesium chloride hexahydrate, and 1mM sodium dihydrogen phosphate dihydrate.

[0017] In a preferred embodiment, the in vitro fertilization culture medium stock solution B is an aqueous solution containing 40mM sodium bicarbonate, and the in vitro fertilization culture medium working solution is an aqueous solution containing 10% (v / v) in vitro fertilization culture medium stock solution A, 25% (v / v) in vitro fertilization culture medium stock solution B, 1.5mM sodium pyruvate, 15mM sucrose, 1% (v / v) penicillin-streptomycin, 5mM theophylline, and 4mg / mL bovine serum albumin.

[0018] In a preferred embodiment, the purity of the reagents used in the present invention is above the cell culture grade, and the in vitro fertilization culture medium can be used for in vivo oocyte collection and in vitro fertilization of ovarian oocytes.

[0019] In a preferred embodiment, each of the storage solutions can be stored for a long time in an environment of 2-8 degrees after preparation, and each of the working solutions can be stored for 30 days in an environment of 2-8 degrees after preparation. The in vitro fertilization solution used to treat sperm can improve various sperm movement parameters. The in vitro fertilization solution used to perform in vitro fertilization on oocytes can obtain a higher blastocyst rate and a similar embryo transplantation pregnancy rate than commercial fertilization solution.

[0020] A method for preparing in vitro fertilization solution capable of improving the fertilization ability of bovine sperm comprises the following steps:

[0021] (1) Add 1.5 ml of sperm treatment solution A to a 15 ml centrifuge tube, slowly add sperm treatment solution C to it, and place the centrifuge tube in a 37 degree metal bath for insulation;

[0022] (2) Gently add semen to the upper layer of sperm treatment solution C. At this time, the liquid in the centrifuge tube is clearly divided into three layers: the bottom layer is sperm treatment solution A, the middle layer is sperm treatment solution C, and the top layer is semen;

[0023] (3) Centrifuge the tube at 2500 rpm for 15 minutes;

[0024] (4) After centrifugation, remove the lower precipitate and place it in a new 15 ml centrifuge tube. Add 3 ml of in vitro fertilization culture medium and centrifuge at 1500 rpm for 5 minutes. Remove the supernatant after centrifugation.

[0025] (5) Adjust the sperm density by taking an appropriate amount of treated semen and adding it to the oocytes in the fertilization solution to make the final sperm density 5×10 6 Pieces / ml.

[0026] The invention provides an in vitro fertilization solution capable of improving the fertilization ability of bovine sperm and a preparation method thereof, and its beneficial effects include:

[0027] 1. Using live egg collection and oocytes obtained from slaughterhouse ovaries for in vitro embryo production and transplantation can obtain a higher blastocyst rate and a similar transplantation pregnancy rate. At the same time, the reagents involved in the in vitro fertilization solution of the present invention are simple, easy to obtain and low in price, which reduces the production cost while ensuring the efficiency of bovine in vitro embryo production. DETAILED DESCRIPTION

[0028] In order to make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below in combination with the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0029] Example 1

[0030] An in vitro fertilization fluid capable of improving the fertilization ability of bovine sperm comprises in vitro fertilization fluid, wherein the in vitro fertilization fluid comprises a sperm treatment fluid and an in vitro fertilization culture fluid, wherein the sperm treatment fluid and the in vitro fertilization culture fluid respectively comprise a storage fluid and a working fluid.

[0031] 1. Preparation of bovine in vitro fertilization solution;

[0032] (1) Sperm treatment solution stock A: an aqueous solution containing 35 mM potassium chloride, 700 mM sodium chloride, 3 mM sodium dihydrogen phosphate, 100 mM 4-hydroxyethylpiperazineethanesulfonic acid, 15 mM calcium chloride dihydrate, and 5 mM magnesium chloride hexahydrate.

[0033] (2) Sperm treatment solution stock B: an aqueous solution containing 100 mM sodium bicarbonate.

[0034] (3) Sperm processing solution Working solution A: An aqueous solution containing 10% (v / v) sperm storage solution A, 90% percoll, 25 mM sodium bicarbonate, and 25 mM sodium lactate.

[0035] (4) Sperm treatment solution working solution B: an aqueous solution containing 10% (v / v) sperm treatment solution working solution A, 25% sperm treatment solution stock solution B, 2 mM sodium pyruvate, and 15 mM sucrose.

[0036] (5) Sperm treatment solution C: a solution containing 50% (v / v) sperm treatment solution A, 50% sperm treatment solution B, and 100 μl of phenol red.

[0037] (6) In vitro fertilization culture medium stock solution A: an aqueous solution containing 115 mM sodium chloride, 4 mM potassium chloride, 3 mM calcium chloride dihydrate, 0.6 mM magnesium chloride hexahydrate, and 1 mM sodium dihydrogen phosphate dihydrate.

[0038] (7) In vitro fertilization culture medium stock solution B: an aqueous solution containing 40 mM sodium bicarbonate.

[0039] (8) In vitro fertilization culture medium working solution: an aqueous solution containing 10% (v / v) in vitro fertilization culture medium stock solution A, 25% (v / v) in vitro fertilization culture medium stock solution B, 1.5 mM sodium pyruvate, 15 mM sucrose, 1% (v / v) penicillin-streptomycin, 5 mM theophylline, and 4 mg / mL bovine serum albumin.

[0040] The purity of the reagents used in the present invention is above the cell culture grade, such as products of Sigma-Aldrich. After each storage solution is prepared, it can be stored for a long time in an environment of 2-8 degrees, and after each working solution is prepared, it can be stored for 30 days in an environment of 2-8 degrees.

[0041] 2. Live egg collection;

[0042] Instrument preparation: prepare the live egg collection instrument, vacuum pump and metal bath, connect the egg collection needle, egg collection tube and vacuum pump. Adjust the vacuum pump pressure to 80mmHg, put 5-10ml preheated PBS buffer in the oocyte collection tube, and place the collection tube in a 37 degree metal bath.

[0043] Donor cattle preparation: The donor cattle are restrained using a restraining frame, the feces in the rectum are cleared, and the vulva is disinfected. The status of the ovarian follicles and corpus luteum on the donor cattle is checked by B-ultrasound. After confirming that the cattle can be used for egg collection, 2 ml of lidocaine hydrochloride is used to perform epidural anesthesia at the coccyx.

[0044] Live egg collection: Using a live egg collection instrument and a negative pressure pump, ultrasound guidance is used to recover oocytes from the cow's ovaries, and the egg collection fluid is recovered into an oocyte collection tube.

[0045] 3. In vitro maturation of oocytes;

[0046] After filtering the oocyte recovery solution in the collection tube through the egg picking cup, the oocytes containing more than three layers of granulosa cells and uniform cytoplasm are selected for maturation culture. The culture environment is 38.5 degrees, 5-7% carbon dioxide saturated humidity environment, and the culture time is 20-24 hours.

[0047] 4. In vitro fertilization, a method for preparing in vitro fertilization solution that can improve the fertilization ability of bovine sperm, comprising the following steps.

[0048] ① Add 1.5 ml of sperm treatment solution A to a 15 ml centrifuge tube, slowly add sperm treatment solution C on it, and place the centrifuge tube in a 37 degree metal bath for insulation; ② Take the frozen semen from liquid nitrogen and put it into a 38 degree water bath for thawing; ③ Gently add the thawed semen to the upper layer of sperm treatment solution C. At this time, the liquid in the centrifuge tube is clearly divided into three layers: the bottom layer is sperm treatment solution A, the middle layer is sperm treatment solution C, and the top layer is semen; ④ After the centrifugation, centrifuge the centrifuge tube at 2500rpm for 15 minutes; ⑤ After the centrifugation, remove the lower layer of sediment into a new 15 ml centrifuge tube, add 3 ml of in vitro fertilization culture medium working solution to it, centrifuge at 1500rpm for 5 minutes, and remove the supernatant after the centrifugation; ⑥ Adjust the sperm density, take an appropriate amount of treated semen and add it to the oocytes in the fertilization solution, so that the final sperm density is 5×10 6 ⑦ Sperm and oocytes were cultured in a saturated humidity environment of 38.5 degrees and 5-7% carbon dioxide for 16-20 hours.

[0049] 5. Embryo culture in vitro;

[0050] After fertilization, the granulosa cells on the outer layer of the fertilized egg were removed with a Pasteur pipette with an inner diameter of 130-160 μm, washed with commercial embryo manipulation fluid, and then placed in commercial embryo culture fluid for culture. The culture environment was 38.5 degrees, 5-7% carbon dioxide, 5-7% nitrogen, and saturated humidity.

[0051] The embryonic development during the culture process was statistically analyzed and the specific results are shown in Table 1.

[0052] 6. Embryo freezing and transplantation;

[0053] (1) Programmed freezing and thawing

[0054] The cultured embryos are programmed to freeze using commercial embryo preservation solution and embryo programmed freezing solution. The specific steps are as follows: after the embryos are washed 3-5 times with commercial embryo preservation solution, they are transferred into commercial embryo programmed freezing solution and filled into tubes in five steps in the order of freezing solution, bubbles, freezing solution, bubbles, freezing solution containing embryos, bubbles, freezing solution, bubbles, and freezing solution; after sealing with straw stoppers, they are placed into a programmed temperature controller and the freezing program is started; after the freezing program is completed, the straws are immersed in liquid nitrogen.

[0055] Thawing: Take the tube out of liquid nitrogen, expose it to air for 10 seconds, and put it into 30-degree warm water for 30 seconds.

[0056] (2) Vitrification and thawing;

[0057] The cultured embryos were frozen using commercial embryo vitrification solution. The specific steps were as follows: the embryos were allowed to stand in the frozen equilibrium droplet for 5 minutes; then the embryos were moved into the freezing droplet and allowed to stand for 45 seconds; within 10 seconds, the embryos were removed from the freezing droplet, loaded onto an embryo freezing carrier, and immersed in liquid nitrogen.

[0058] Thawing: Take out the embryo cryocarrier from liquid nitrogen, quickly immerse it in embryo thawing solution, and shake the cryocarrier; after the embryo falls off the cryocarrier, continue to stay in the thawing solution for 5 minutes; take the culture out of the thawing solution and transfer it into the embryo preservation solution.

[0059] (3) Embryo transfer;

[0060] Fresh embryo transfer: The blastocysts and expanded blastocysts cultured to the 6th to 8th day were placed in five-stage tubes in the order of embryo preservation solution, air bubble, embryo preservation solution, air bubble, embryo preservation solution containing embryo, air bubble, embryo preservation solution, air bubble, and embryo preservation solution. After sealing with straw plugs, they were transplanted into recipient cows on the 7th day of estrus. On the 30th day after embryo transfer, the recipient cows were examined for pregnancy using B-ultrasound, and the transplantation conception rate was calculated. The specific results are shown in Table 2.

[0061] Programmed frozen embryo transfer: After thawing, the embryos were transferred to the recipient cows on the 7th day of estrus within 15 minutes. On the 30th day after embryo transfer, the recipient cows were tested for pregnancy using B-ultrasound and the transfer pregnancy rate was calculated. The specific results are shown in Table 3.

[0062] Vitrified embryo transfer: After thawing, the embryos were washed three times with embryo preservation solution, then placed in five-section tubes, sealed with straw plugs, and transplanted into recipient cows on the 7th day of estrus. On the 30th day after embryo transfer, B-ultrasound was used to check the pregnancy of the recipient cows, and the transplantation pregnancy rate was calculated. The specific results are shown in Table 4.

[0063] Example 2

[0064] 1. The preparation of bovine in vitro fertilization solution is as described in Example 1.

[0065] 2. Oocyte acquisition: The oocytes used are from slaughterhouse ovaries, not from live oocytes. The specific method is: the bovine ovaries obtained from the slaughterhouse are transported to the laboratory at a transportation temperature of 20-30 degrees and then rinsed with sterilized 0.9% saline at about 30 degrees. After rinsing, the follicles on the ovaries are extracted with a sterile syringe with a 12-gauge needle, and the follicular fluid is recovered into the oocyte collection tube.

[0066] 3. The in vitro maturation of oocytes, in vitro fertilization, in vitro embryo culture, and blastocyst rate statistics refer to Example 1.

[0067] Example 3

[0068] 1. The preparation of bovine in vitro fertilization solution is as described in Example 1.

[0069] 2. Treat the thawed semen according to the method of Example 1 and use minitube's The automated sperm analysis system measured the sperm motility parameters. The specific results are shown in Table 5.

[0070] Example 4

[0071] 1. The preparation of bovine in vitro fertilization solution is as described in Example 1.

[0072] 2. Referring to the method of Example 2, the oocytes from the ovary of the slaughterhouse were subjected to in vitro fertilization using sperm treated with the in vitro fertilization solution provided by the present invention. The oocytes were taken 6 hours after the addition of sperm, and the granulosa cells of the outer layer were removed cleanly with a Pasteur pipette with an inner diameter of 130-160 μm. The cover glass was fixed with 90% vaseline + 10% paraffin, and a fertilized egg specimen was prepared under a stereoscope. The oocytes were fixed with a mixed solution of acetic acid and anhydrous ethanol in a volume ratio of 3:1 for more than 10 hours, stained with orcein red dye at room temperature for 1 hour, and the excess dye was removed with a decolorizing solution (glycerol: glacial acetic acid: water is 1:1:3), and observed with a phase contrast microscope after sealing. Whether the oocyte was fertilized and the number of fertilized sperm were determined according to the formation of pronuclei after sperm penetrated the egg, the expansion of the sperm head and the presence of the sperm tail. The specific results are shown in Table 6.

[0073] In order to verify the effect of the present invention, a comparative example is set for comparison, as follows:

[0074] Comparative Example 1

[0075] Refer to the method of Example 1; in vitro embryos are produced using oocytes obtained from live egg collection, except that the in vitro fertilization fluid in the in vitro embryo production reagent used is replaced by commercial fertilization fluid.

[0076] Comparative Example 2

[0077] Refer to the method of Example 2; in vitro embryos are produced using oocytes obtained from slaughterhouse ovaries, except that the in vitro fertilization fluid in the in vitro embryo production reagent used is replaced by commercial fertilization fluid.

[0078] Comparative Example 3

[0079] The sperm motility parameters were measured by referring to the method of Example 2, except that the in vitro fertilization fluid used was replaced by commercial fertilization fluid.

[0080] Comparative Example 4

[0081] Refer to the method of Example 4; the ovum 6 hours after fertilization was naked, stained and fertilized.

[0082] Table 1

[0083]

[0084] Table 2

[0085]

[0086] Table 3

[0087]

[0088] Table 4

[0089]

[0090] Table 5

[0091]

[0092] Table 6

[0093]

[0094] Description of indicators in the table:

[0095] By using the in vitro fertilization fluid provided by the present invention, using live egg collection and oocytes obtained from slaughterhouse ovaries for in vitro fertilization, producing in vitro embryos and transplanting them, a blastocyst rate and transplantation pregnancy rate similar to those of conventional commercial in vitro fertilization fluid can be obtained. At the same time, using the present invention to treat sperm can significantly improve the overall sperm motility and fertilization rate.

Claims

1. An in vitro fertilization solution capable of improving the fertilization ability of bovine sperm, comprising an in vitro fertilization solution, wherein the in vitro fertilization solution is composed of a sperm treatment solution and an in vitro fertilization culture solution, characterized in that ； The sperm treatment solution and in vitro fertilization culture solution include a storage solution and a working solution respectively.

2. The in vitro fertilization solution capable of improving the fertilization ability of bovine sperm according to claim 1, characterized in that: The sperm treatment storage solution includes sperm treatment storage solution A and sperm treatment storage solution B. The sperm treatment storage solution A is an aqueous solution containing 35mM potassium chloride, 700mM sodium chloride, 3mM sodium dihydrogen phosphate, 100mM 4-hydroxyethylpiperazineethanesulfonic acid, 15mM calcium chloride dihydrate, and 5mM magnesium chloride hexahydrate.

3. The in vitro fertilization solution capable of improving the fertilization ability of bovine sperm according to claim 1, characterized in that: The sperm treatment fluid storage solution B is an aqueous solution containing 100 mM sodium bicarbonate, and the sperm treatment fluid working solution contains sperm treatment fluid working solution A, sperm treatment fluid working solution B, and sperm treatment fluid working solution C.

4. The in vitro fertilization solution capable of improving the fertilization ability of bovine sperm according to claim 1, characterized in that: The sperm treatment fluid working solution A is an aqueous solution containing 10% (v / v) sperm storage solution A, 90% percoll, 25mM sodium bicarbonate, and 25mM sodium lactate, and the sperm treatment fluid working solution B contains an aqueous solution of 10% (v / v) sperm treatment fluid working solution A, 25% sperm treatment fluid storage solution B, 2mM sodium pyruvate, and 15mM sucrose.

5. The in vitro fertilization solution capable of improving the fertilization ability of bovine sperm according to claim 1, characterized in that: The sperm treatment fluid working solution C is a solution containing 50% (v / v) sperm treatment fluid working solution A, 50% sperm treatment fluid working solution B, and 100 μl of phenol red, and the in vitro fertilization culture medium includes in vitro fertilization culture medium storage solution and in vitro fertilization culture medium working solution.

6. The in vitro fertilization solution capable of improving the fertilization ability of bovine sperm according to claim 1, characterized in that: The in vitro fertilization culture medium stock solution includes in vitro fertilization culture medium stock solution A and in vitro fertilization culture medium stock solution B. The in vitro fertilization culture medium stock solution A is an aqueous solution containing 115mM sodium chloride, 4mM potassium chloride, 3mM calcium chloride dihydrate, 0.6mM magnesium chloride hexahydrate, and 1mM sodium dihydrogen phosphate dihydrate.

7. The in vitro fertilization solution capable of improving the fertilization ability of bovine sperm according to claim 1, characterized in that: The in vitro fertilization culture medium stock solution B is an aqueous solution containing 40mM sodium bicarbonate, and the in vitro fertilization culture medium working solution is an aqueous solution containing 10% (v / v) in vitro fertilization culture medium stock solution A, 25% (v / v) in vitro fertilization culture medium stock solution B, 1.5mM sodium pyruvate, 15mM sucrose, 1% (v / v) penicillin-streptomycin, 5mM theophylline, and 4mg / mL bovine serum albumin.

8. The in vitro fertilization solution capable of improving the fertilization ability of bovine sperm according to claim 1, characterized in that: The purity of the reagents used in the present invention is above the cell culture level, and the in vitro fertilization culture medium can be used for in vivo egg collection and in vitro fertilization of ovarian oocytes.

9. The in vitro fertilization solution capable of improving the fertilization ability of bovine sperm according to claim 1, characterized in that: After preparation, each storage solution can be stored for a long time in an environment of 2-8 degrees, and each working solution can be stored for 30 days in an environment of 2-8 degrees. The in vitro fertilization solution can improve various sperm movement parameters by treating sperm. The in vitro fertilization solution can obtain a higher blastocyst rate and a similar embryo transplantation pregnancy rate than commercial fertilization solution by in vitro fertilization of oocytes.

10. A method for preparing an in vitro semen solution capable of improving the fertilization ability of bovine sperm, applicable to the in vitro semen solution capable of improving the fertilization ability of bovine sperm as claimed in any one of claims 1 to 9, characterized in that: The following steps are involved: (1) Add 1.5 ml of sperm treatment solution A to a 15 ml centrifuge tube, slowly add sperm treatment solution C to it, and place the centrifuge tube in a 37 degree metal bath for insulation; (2) Gently add semen to the upper layer of sperm treatment solution C. At this time, the liquid in the centrifuge tube is clearly divided into three layers: the bottom layer is sperm treatment solution A, the middle layer is sperm treatment solution C, and the top layer is semen; (3) Centrifuge the tube at 2500 rpm for 15 minutes; (4) After centrifugation, remove the lower precipitate and place it in a new 15 ml centrifuge tube. Add 3 ml of in vitro fertilization culture medium and centrifuge at 1500 rpm for 5 minutes. Remove the supernatant after centrifugation. (5) Adjust the sperm density by taking an appropriate amount of treated semen and adding it to the oocytes in the fertilization solution to make the final sperm density 5×10 6 Pieces / ml.

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