CN112501114B - Method for improving cattle in-vitro fertilization efficiency - Google Patents

Abstract

The present invention relates to a method for improving the efficiency of bovine in vitro fertilization. On the one hand, it relates to a method for culturing bovine in vitro fertilized embryos, including the following steps: collection and in vitro maturation of oocytes, in vitro fertilization by using fertilization medium and semen to prepare culture medium, and in vitro culturing and preservation of embryos. The mature medium used in this method is the BY basal medium supplemented with 100mL/L FBS, 10μg/mL FSH, 10μg/mL LH, 1μg/mL E2, and 20ng/mL EGF. The fertilization medium and semen used The prepared culture medium contains sodium pyruvate, heparin and bovine serum albumin. The present invention also relates to methods for in vitro fertilization of bovines, as well as to fertilization cultures and semen preparation cultures used in these methods. The method of the present invention using the reagent of the present invention exhibits excellent technical effects such as significantly improving the efficiency of in vitro fertilization.

Description

Method for improving the efficiency of bovine in vitro fertilization

technical field

The invention belongs to the technical field of animal reproduction, and relates to a technology for agriculture-animal husbandry and veterinary reproduction, in particular to a method for bovine in vitro fertilization. In addition, the invention also relates to a related working test solution used in bovine in vitro fertilization. application in fertilization. The bovine in vitro fertilization method of the present invention has excellent technical effects.

Background technique

In Vitro Fertilization (In Vitro Fertilization) or (external fertilization) refers to the technology in which mammalian sperm and eggs complete the fertilization process in an artificially controlled environment outside the body, referred to as IVF in English. Because it is inseparable from embryo transfer technology (ET), it is also referred to as IVF-ET. In biology, animals obtained after in vitro fertilization embryos are transplanted into mothers are called test-tube animals. Successful in the 1950s, this technology has developed rapidly in the last 20 years and has now matured into an important and routine animal reproductive biotechnology.

In vitro fertilization technology is of great significance to the research of animal reproductive mechanism, animal husbandry, medicine and protection of endangered animals. For example, using mice, rats or rabbits as experimental materials, in vitro fertilization technology can be used to study the mechanism of mammalian gametogenesis, fertilization and early embryonic development. In the improvement of livestock breeds, in vitro fertilization technology provides a cheap and efficient means for embryo production, which is of great value in making full use of fine breed resources, shortening the breeding cycle of livestock, and speeding up breed improvement. In humans, IVF-ET technology is one of the important measures to treat certain infertility and overcome sexually linked diseases. In vitro fertilization technology is also an indispensable part of modern biotechnology such as mammalian embryo transfer, cloning, transgenic and sex control.

With the development of modern agricultural science and technology, in order to make full use of the reproductive potential of high-quality cows and accelerate the process of genetic breeding, it is inevitable to apply new efficient breeding techniques in production practice. Ovum pick UP (OPU) and in vitro fertilization (In Vitro Fertilization, IVF) are new embryo engineering technologies developed rapidly in the 1980s. The combination of the two can obtain a large number of embryos with clear genetic pedigrees. This shortens the generation interval. At present, these two technologies have become important breeding technologies adopted by farmers in developed countries of animal husbandry, such as Europe, America and Oceania, in order to expand the herd of fine-bred cows. However, using the conventional bovine embryo culture system (CR1aa and SOF fluid), the blastocyst development rate of bovine in vitro fertilization is low, and the quality of embryos is far less than that of in vivo embryos, resulting in a low pregnancy rate after embryo transfer recipients, so how to improve The blastocyst development rate and embryo quality have become the focus and research focus of in vitro fertilization embryo production.

As early as 1878, German Scnenk began to explore mammalian in vitro fertilization technology using rabbits and guinea pigs. But it was not until 1951, when Chinese-American Zhang Minjue and Austin respectively discovered the phenomenon of sperm capacitation in vitro, that in vitro fertilization technology achieved a breakthrough. Bovine in vitro fertilization technology is affected by the in vitro maturation of oocytes, the in vitro capacitation of sperm, and the in vitro culture environment of fertilized eggs.

The in vitro culture of embryos is a key link of IVF technology, and it is also the embodiment and test of the final effect of in vitro maturation of oocytes and in vitro fertilization technology. Following in vitro fertilization, the fertilized egg will undergo a series of important changes during development towards the blastocyst, including zygote formation, first cleavage, activation of the embryonic genome, densification, and blastocyst formation. During this process, changes in the external environment will lead to changes in gene expression, which will affect the normal development and quality of embryos. At present, the in vitro culture research of early mammalian embryos mainly focuses on improving the composition of the culture medium to meet the nutritional needs of embryos at different developmental stages. Based on Charles Rosenkrans 1 developed by Rosenkrans et al. (Rosenkrans, C.F., Jr. and N.L. First, Effect of free amino acids and vitamins on cleavage and developmental rate of bovinezygotes in vitro. J Anim Sci, 1994.72(2): p.434-7) (CR1) culture fluid and synthetic fallopian fluid developed by Tervit et al. (Tervit, H.R., D.G. Whittingham, and L.E. Rowson, Successful culture in vitro of sheep and cattle ova. J Reprod Fertil, 1972.30(3): p.493-7) (Synthetic Oviductal Fluid, SOF), after years of continuous improvement, two culture systems have been gradually formed. According to Hakan Sagirkaya et al. (Sagirkaya, H., et al., Developmental potential of bovine oocytes cultured in different maturation and culture conditions. Anim Reprod Sci, 2007.101(3-4): p.225-40) and Somfai et al. (Somfai, T. ., et al., Development of bovine embryos cultured in CR1aa and IVD101 media using different oxygen tensions and culture systems. Acta VetHung, 2010.58(4): p.465-74) The research results show that CR1aa culture medium is effective for bovine embryo culture. It has better effect and can be widely used in bovine embryo culture; Thompson, J.G., etc. (Thompson, J.G., et al., Effectof inhibitors and uncouplers of oxidative phosphorylation during compaction and blastulation of bovine embryos cultured in vitro. J Reprod Fertil, 2000.118 ( 1): p.47-55) and Jean M.Feugang et al. (Feugang, J.M., O. Camargo-Rodriguez, and E. Memili, Culture systems for bovine embryos. Livestock Science, 2009.121(2-3): p.141 -149) research results show that SOF medium is also a culture system suitable for bovine embryo culture. Zhang Zhiping (Zhang Zhiping, An Zhixing, Zhang Xi, Zhang Yong, Optimization of bovine embryo culture system. Journal of Northwest A&F University, 2006. 34) and Sang Guojun et al (Sang Guojun, Bovine oocyte and in vitro embryo culture techniques Research. 2008) The results also showed that the optimized CR1aa and SOF mediums were both suitable for the in vitro embryo culture of bovines, and both achieved good culture effects. Early mammalian embryonic development is a highly coordinated and precisely regulated process. In the process of evolution, gamete cells gradually formed a series of molecular cascade networks to ensure the systematic progress of the embryonic development cycle. During development, the balance of internal and external reactive oxygen species (ROS) and antioxidants in the embryo plays a decisive role in early embryonic development.

Most biochemical reactions generate ROS, which play an important role both inside and outside cells. Some ROS play the role of signaling molecules, but most ROS are harmful to the body. Brooker, R.J., et al. (Brooker, R.J., Genetics: analysis and principles (4th ed.). McGraw-Hill Science, 2011) reported that ROS can cause cellular DNA damage, oxidation of unsaturated fatty acids, oxidation of amino acids in proteins and even lead to Inactivation of certain enzymes. Generally speaking, ROS exist in four forms, among which H2O2 has a strong oxidation effect and is the most important factor causing oxidative damage.

Numerous studies have shown that glutathione (GSH) is an antioxidant that exists in non-protein form and can scavenge a variety of free radicals: superoxide anion radicals, hydroxyl radicals, hydrogen peroxide, hypochlorous acid and lipoxygen Free radicals, and can maintain intracellular and extracellular redox balance. The levels of GSH and ROS in the intracellular and extracellular environment are two important factors affecting the development of fertilized eggs. As early as 2000, de Matos et al. (de Matos, D.G. and C.C. Furnus, The importance of having high glutathione(GSH) level after bovine in vitro maturation onembryo development effect of beta-mercaptoethanol, cysteine and cystine. Theriogenology, 2000.53(3): p.761-71) had previously increased blastocyst rate by adding β-mercaptoethanol, cysteine and cystine during in vitro embryo culture.

Although in vitro fertilization technology can be successfully applied to many mammals, the high production cost and low efficiency of in vitro fertilized embryos due to the low blastocyst rate of in vitro fertilization limit the wide application of this technology in the practice of rapid propagation of cattle. Therefore, how to reduce the cost and improve the production efficiency and embryo quality of bovine IVF embryos has become an urgent problem to be solved.

At present, in the technical system of bovine in vitro fertilization, CR1aa and SOF are mainly used as the in vitro culture medium of embryos, and improvements are made on this basis. %. For blastocyst quality, it can be assessed by the total number of blastocyst cells, the ratio of ICM cells/total cells, and the rate of apoptosis. The total number of blastocyst cells varies according to the stage of blastocyst, S. Iwasaki et al. (Iwasaki, S. and T. Nakahara, Cellnumber and incidence of chromosomal anomalies in bovine blastocystsfertilized in vitro followed by culture in vitro or in vivo in rabbitoviducts . Theriogenology, 1990.33(3): p.669-75) The average number of total cells in bovine early blastocysts was 44, and the ratio of ICM cells/total cells in blastocysts was about 15.8%; Andrew J. Watson et al. (Watson, A.J., etal., Impact of bovine oocyte maturation media on oocyte transcript levels, blastocyst development, cell number, and apoptosis. Biol Reprod, 2000.62(2):p.355-64) The statistical bovine blastocyst cell apoptosis rate is about 7.7 %-13%.

CN103898046B (Chinese Patent Application No. 201410073635.4) discloses a culture fluid specially used for bovine in vitro fertilization embryos, the culture fluid formula is: NaCl 109.5mM, KCl 3.1mM, NaHCO3 26.2mM, MgCl2 6H2O 0.8mM, KH2PO3 1.19mM , sodium pyruvate 0.4mM, glucose 1.5mM, calcium galactonate 5mM, 10v/v% fetal bovine serum, L-glutamine 1mM, 2v/v% essential amino acids, 1v/v% non-essential amino acids and glutathione Peptide 3mM, prepared with water; the essential amino acid is an aqueous solution prepared by mixing the following amino acids in proportion, wherein the amino acid content is: L-arginine hydrochloride 6.32g/L, L-cystine dihydrochloride 1.564 g/L, L-histidine hydrochloride monohydrate 2.1g/L, L-isoleucine 2.625g/L, L-leucine 2.62g/L, L-lysine hydrochloride 3.625g/ L, L-methionine 0.755g/L, L-phenylalanine 1.65g/L, L-threonine 2.38g/L, L-tryptophan 0.51g/L, L-tyrosine 1.8g/L and L-valine 2.34g/L; the non-essential amino acid is an aqueous solution prepared by mixing the following amino acids in proportion, wherein the amino acid content is: L-alanine 0.89g/L, L-asparagine- Water 1.5g/L, L-aspartic acid 1.33g/L, L-glutamic acid 1.47g/L, glycine 0.75g/L, L-proline 1.15g/L and L-serine 1.05g/ L. The bovine in vitro fertilized embryos were placed in the above-mentioned culture medium for in vitro culture. It is believed that the results show that it is significantly better than the control group without GSH, which improves the blastocyst development rate and embryo quality, and reduces the cost of producing embryos in vitro. It is the bovine IVF technology. Applied to practice to provide experimental basis, which can greatly accelerate the process of genetic breeding.

The prior application CN108728404A (application number: 201810576806.3) discloses a method for culturing bovine in vitro fertilized embryos, which includes the following steps: (1) collection of oocytes and in vitro maturation in vitro collection: taking slaughterhouse ovaries and placing them in Place the eggs in an incubator with double-antibody saline, and transport them back to the laboratory within 3 hours at 31-33 °C; extract the follicles with a surface of 2-8 mm, collect the precipitate, and pick out the cumulus cells containing at least 3 layers under a stereo microscope. The oocyte COCs (i.e., cumulus-oocyte complex) were washed twice in the egg wash to remove excess impurities; live collection: live eggs were collected from cattle, and the obtained follicular fluid was examined under a stereo microscope. Pick up cumulus-oocyte complexes (COCs) with at least 3 layers of cumulus cells, put them in a mature medium containing HEPES at 38.8°C, and transport them back to the laboratory within 3 hours; collect the above in vitro or in vivo collections. The obtained COCs were washed once in the oocyte maturation medium, and then transferred to a new maturation medium for 22-24 hours. The culture conditions were 38.8 °C, 5.5-6.5% CO2, and saturated humidity; (2) In vitro fertilization will mature The COCs were washed once in the fertilization medium, then transferred to the fertilization medium, placed in the incubator for use; take a frozen fine tube from liquid nitrogen and thawed in a 37°C water bath; cut the fine tube aseptically At both ends, inject the semen into the 15mL centrifuge tube containing the semen preparation medium, centrifuge twice at 328 × g for 5 min each time, and discard the supernatant after centrifugation; add 300μL of the semen preparation medium to the above centrifuge tube, and resuspend the sperm pellet. , take the appropriate sperm suspension for sperm count; add the calculated volume of sperm suspension to the fertilization culture droplet containing oocytes, put the culture plate into the incubator, and incubate the sperm and eggs for 16-20h. The culture conditions are 38.8°C, 5.5-6.5% CO2, and saturated humidity; (3) embryos are cultured and preserved in vitro. After the in vitro fertilization operation is completed, the granulosa cells around the embryos are removed with an egg stripper and placed in the embryo culture medium for culture. This time was recorded as the first day of embryo culture. The culture conditions were 38.8°C, 6% O2, 88% N2, and saturated humidity. The cleavage rate was recorded on the third day; the blastocyst rate was recorded on the seventh day, and the cysts were counted on the ninth day. Embryo hatching rate (it is the percentage obtained by dividing the number of hatched blastocysts by the number of blastocysts), and carry out quality identification; wash the available embryos in the preservation solution 3 times, equilibrate in the equilibration solution for 10 minutes, and transfer them into the freezing solution. The embryos were loaded by the liquid filling method, marked, and then placed in a programmed cooling device to -35 °C at a speed of 0.5 °C/min, and the embryo straw was quickly taken out and placed in liquid nitrogen for cryopreservation.

As we all know, embryo biotechnology is an effective technical means for the cultivation and rapid propagation of high-quality cattle. Among them, in vivo egg collection-in vitro embryo production (OPU-IVP) technology collects excellent individual oocytes in vivo, and undergoes in vitro maturation (IVM) and in vitro fertilization. (IVF), in vitro embryo culture (IVC), etc., a large number of high-quality embryos can be quickly obtained under in vitro conditions. Compared with in vivo embryos, its production efficiency is as high as 4~8 times, which can effectively improve the reproductive potential of excellent female animals. However, under normal conditions, the cattle oocyte donor cattle farm is far away from the embryo production laboratory, requiring several hours of transportation. Shipping time and shipping conditions are critical for maintaining oocyte viability and quality. There are two traditional solutions. One is that the collected oocytes are first placed in the pre-maturation solution to inhibit the maturation of the oocytes, and after arriving in the laboratory, they are transferred to the maturation solution and placed in an incubator for in vitro maturation culture; The collected oocytes are placed in the maturation medium and transported in a micro-incubator, during which CO ₂ is continuously supplied, but it is difficult to maintain the supply of carbon dioxide in the transport state.

The prior application CN111254109A (Chinese Patent Application No. 2020100820171) provides a transport medium containing glycine, alanine, arginine hydrochloride, aspartic acid, cystine Acid dihydrochloride, glutamic acid, L-glutamine, histidine, threonine, tryptophan, tyrosine, valine, ascorbic acid, biotin, choline chloride, calcium pantothenate, folic acid , menadione, etc., the method for in vitro fertilization and embryo culture of bovine oocytes using the transport medium includes the following steps: oocyte collection and in vitro maturation, in vitro fertilization, in vitro culture and preservation of embryos. The inventive method and the transporting medium have achieved excellent technical effects. The aforementioned CN108728404A and CN111254109A are incorporated herein by reference in their entirety.

However, the efficiency of in vitro fertilization of bovine frozen semen during in vitro fertilization with oocytes is still limited. Therefore, it is necessary to develop a method to improve the efficiency of in vitro fertilization of bovine frozen semen during in vitro fertilization with oocytes.

SUMMARY OF THE INVENTION

The object of the present invention is to provide a method for improving the efficiency of in vitro fertilization of bovine frozen semen during in vitro fertilization with oocytes. More specifically, the present invention provides a kind of semen preparation culture fluid and fertilization culture fluid specially used for freezing sperm in bovine in vitro fertilization in order to achieve the above-mentioned purpose, and use these semen preparation culture fluid and fertilization culture fluid to pre-treat frozen sperm to improve its fertilization culture. Efficiency during subsequent in vitro fertilization. The inventors of the present invention have unexpectedly found that the method of the present invention and the related working test solutions exhibit excellent technical effects, and the present invention has been completed accordingly.

To this end, a first aspect of the present invention provides a method for culturing bovine in vitro fertilization embryos, comprising the steps of:

(1) Collection and in vitro maturation of oocytes

a) In vitro collection: take bovine ovaries from slaughterhouses, extract follicles with a surface of 2-8 mm, collect sediment, and pick out oocyte COCs (ie, cumulus- oocyte complex), placed in a transport medium containing HEPES at 38.8°C, without carbon dioxide supply, and transported back to the laboratory within 24 hours; or

a) In vivo collection: take follicular fluid from bovine live egg collection, pick out cumulus-oocyte complexes (COCs) containing at least 3 layers of cumulus cells under a stereo microscope, and put them into a transport culture containing HEPES 38.8℃ in the liquid, no carbon dioxide supply, transported back to the laboratory within 24h;

b) The COCs collected above in vitro or in vivo were washed once in the oocyte maturation medium, and then transferred to a new mature medium for 22-24 hours under the conditions of 38.8°C, 5.5-6.5% CO2, saturated humidity;

(2) In vitro fertilization

The mature COCs were washed once in the fertilization medium, then transferred to the fertilization medium, and placed in an incubator for use;

Take a frozen fine tube from liquid nitrogen and thaw it in a water bath at 37°C; cut both ends of the fine tube aseptically, pour the semen into a 15 mL centrifuge tube containing the culture medium for semen preparation, and centrifuge at 328 × g twice, each time After centrifugation for 5 min, the supernatant was discarded; 300 µL of semen preparation medium was added to the above centrifuge tube, the sperm pellet was resuspended, and an appropriate sperm suspension was taken for sperm count;

Add the calculated volume of sperm suspension to the fertilization culture droplet containing the oocytes, and put the culture plate into the incubator to incubate the sperm and eggs for 16-20 hours. The culture conditions are 38.8°C, 5.5-6.5% CO2 , saturated humidity;

(3) Embryos are cultured and preserved in vitro

After the in vitro fertilization operation is completed, the granulosa cells around the embryos are removed with an egg stripper and cultured in the embryo culture medium. This time is recorded as the first day of embryo culture. N2, saturated humidity, record the cleavage rate on the 3rd day; record the blastocyst rate on the 7th day, count the blastocyst hatching rate (which is the percentage obtained by dividing the number of hatched blastocysts by the number of blastocysts) on the 9th day, and carry out quality identification ;

Wash the available embryos in the preservation solution for 3 times, equilibrate in the equilibration solution for 10 minutes, and transfer them into the freezing solution. Load the embryos according to the 5-stage filling method, make a mark, and then put them into the programmed cooling device at a temperature of 0.5 °C/min. After the speed was reduced to -35°C, the embryo tube was quickly taken out and placed in liquid nitrogen for cryopreservation.

The method according to the first aspect of the present invention, wherein in step (1) (b), the mature culture medium is added with 100mL/L FBS, 10μg/mL FSH, 10μg/mL LH, 1μg/mL E2, 20ng/mL mL of EGF in BY basal medium.

The method according to the first aspect of the present invention, wherein in step (1) (a), the transport medium comprises: glycine 50.0 mg/L, L-alanine 25.0 mg/L, L-arginine salt Acid 70.0mg/L, L-aspartic acid 30.0mg/L, L-cystine dihydrochloride 26.0mg/L, L-glutamic acid 75.0mg/L, L-glutamine 100.0mg/ L, L-histidine hydrochloride monohydrate 21.88mg/L, L-hydroxyproline 10.0mg/L, L-isoleucine 40.0mg/L, L-leucine 60.0mg/L, L-Lysine hydrochloride 70.0mg/L, L-methionine 15.0mg/L, L-phenylalanine 25.0mg/L, L-proline 40.0mg/L, L-serine 25.0mg /L, L-threonine 30.0mg/L, L-tryptophan 10.0mg/L, L-tyrosine disodium salt dihydrate 58.0mg/L, L-valine 25.0mg/L, ascorbic acid 0.05mg/L, Biotin 0.01mg/L, Choline Chloride 0.5mg/L, D-Calcium Pantothenate 0.01mg/L, Folic Acid 0.01mg/L, Menaquinone 0.01mg/L, Niacinamide 0.025mg/L , niacin 0.025mg/L, p-aminobenzoic acid 0.05mg/L, pyridoxal hydrochloride 0.025mg/L, pyridoxine hydrochloride 0.025mg/L, riboflavin 0.01mg/L, thiamine hydrochloride 0.01mg/ L, vitamin A acetate 0.1mg/L, vitamin D2 (calcidol 0.1mg/L, α-tocopherol sodium phosphate 0.01mg/L, inositol 0.05mg/L, anhydrous calcium chloride 200.0mg/L , ferric nitrate nonahydrate 0.7mg/L, anhydrous magnesium sulfate 97.67mg/L, potassium chloride 400.0mg/L, sodium chloride 6800.0mg/L, sodium dihydrogen phosphate monohydrate 140.0mg/L, sodium sulfate Purine 10.0mg/L, Adenosine 5'-Phosphate 0.2mg/L, Adenosine Triphosphate 1.0mg/L, Cholesterol 0.2mg/L, Glucose 1000.0mg/L, Deoxyribose 0.5mg/L, Reduced Glutathione 0.05mg /L, Guanine Hydrochloride 0.3mg/L, Sodium Hypoxanthine 0.354mg/L, Phenol Red 20.0mg/L, Ribose 0.5mg/L, Sodium Acetate 50.0mg/L, Thymine 0.3mg/L, Tween 80 20.0mg/L, uracil 0.3mg/L, sodium xanthine 0.3mg/L, and 0.01IU/mL FSH, 0.01IU/mL LH, 1μg/mL _E2 , 50ng/mL EGF, 100ng IGF/mL, 10% gentamicin, 55 µg/mL sodium pyruvate, 1.2 mM/L cysteine, 3 mg/mL BSA, 10 mM/L HEPES.

According to the method of the first aspect of the present invention, the concentration of HEPES, ie, 4-hydroxyethylpiperazineethanesulfonic acid, in the mature culture medium is 5 to 15 mmol/L, for example, 10 mmol/L. In the present invention, EGF--epidermal growth factor, FSH--follicle-stimulating hormone, FBS--fetal bovine serum, E2--estradiol, LH--luteinizing hormone, IGF--insulin-like growth factor, BSA - bovine serum albumin.

According to the method of the first aspect of the present invention, in step (1), the transport culture solution further comprises taurine and zinc gluconate.

The method according to the first aspect of the present invention, wherein in step (1), the concentration of taurine in the transport culture solution is 30-50 mg/L, such as 40 mg/L or 45 mg/L.

According to the method of the first aspect of the present invention, in step (1), the concentration of zinc gluconate in the transport culture solution is 1-3 mg/L, for example 2 mg/L.

According to the method according to the first aspect of the present invention, wherein in step (2), the fertilization medium contains 112.0 mM sodium chloride, 4.02 mM potassium chloride, 2.25 mM calcium chloride dihydrate, 0.52 mM magnesium chloride hexahydrate , 0.83mM potassium dihydrogen phosphate, 37.0mM sodium bicarbonate, 1.25mM sodium pyruvate, 10μg/ml heparin, 4mg/ml bovine serum albumin (BSA), 100U/ml penicillin, 100μg/ml streptomycin, 10umoL/ A solution of L alpha-lipoic acid, 25 μg/ml folic acid, 2.5 mg/ml glutamic acid in water.

According to the method of the first aspect of the present invention, wherein in step (2), the semen preparation culture medium is composed of 112.0 mM sodium chloride, 4.02 mM potassium chloride, 2.25 mM calcium chloride dihydrate, 0.52 mM hexahydrate Magnesium chloride, 0.83 mM potassium dihydrogen phosphate, 37.0 mM sodium bicarbonate, 1.25 mM sodium pyruvate, 10 μg/ml heparin, 4 mg/ml bovine serum albumin (BSA), 10 mM caffeine, 100 U/ml penicillin, 100 μg/ml chain Mycin, 10umoL/L α-lipoic acid, 25 μg/ml folic acid, 2.5 mg/ml glutamic acid in water.

The method according to the first aspect of the present invention, wherein in step (3), the embryo culture medium comprises: 109.5mM sodium chloride, 3.1mM potassium chloride, 26.2mM sodium bicarbonate, 0.8mM magnesium chloride hexahydrate, 1.19mM phosphoric acid Potassium dihydrogen, 0.4mM sodium pyruvate, 1.5mM glucose, 5mM calcium galactonate, 2.5v/v% fetal bovine serum (FBS), L-glutamine 1mM, 2v/v% essential amino acids, 1v/v% An aqueous solution of non-essential amino acids, 3mM glutathione, 0.04w/v% sodium citrate, and 0.02w/v% maltose; the essential amino acids are the following amino acids added by weight: L-arginine hydrochloride 6.32g , L-cystine dihydrochloride 1.564g, L-histidine hydrochloride monohydrate 2.1g, L-isoleucine 2.625g, L-leucine 2.62g, L-lysine hydrochloride 3.625g, L-methionine 0.755g, L-phenylalanine 1.65g, L-threonine 2.38g, L-tryptophan 0.51g, L-tyrosine 1.8g and L-valine 2.34g, The non-essential amino acids are the following amino acids added by weight: L-alanine 0.89g, L-asparagine monohydrate 1.5g, L-aspartic acid 1.33g, L-glutamic acid 1.47g, glycine 0.75g, L-proline 1.15g and L-serine 1.05g.

The method according to the first aspect of the present invention, wherein in step (1), the BY basal culture solution is an aqueous solution comprising the following components: calcium chloride 180-220 mg/L, ferric nitrate nonahydrate 0.70-0.75 mg/L, Potassium chloride 380~420mg/L, magnesium sulfate 90~100mg/L, sodium chloride 6500~7000mg/L, sodium dihydrogen phosphate monohydrate 130~150mg/L, sodium bicarbonate 2000~2500mg/L, sodium acetate 40 ~60mg/L, L-alanine 20~30mg/L, L-arginine hydrochloride 60~80mg/L, L-aspartic acid 25~35mg/L, L-cysteine hydrochloride Salt monohydrate 0.10~0.12mg/L, L-cystine dihydrochloride 20~30mg/L, L-glutamic acid 70~80mg/L, glycine 40~60mg/L, L-histidine Acid monohydrate 20~25mg/L, L-hydroxyproline 8~12mg/L, L-isoleucine 15~25mg/L, L-leucine 50~70mg/L, L-lysine Hydrochloride 60~80mg/L, L-methionine 10~20mg/L, L-phenylalanine 20~30mg/L, L-proline 30~50mg/L, L-serine 20~30mg/L , L-threonine 25~35mg/L, L-tryptophan 8~12mg/L, L-tyrosine disodium dihydrate 55~60mg/L, L-valine 20~30mg/L, Ascorbic acid 0.04~0.06mg/L, α-D-tocopheryl phosphate 0.008~0.012mg/L, biotin 0.008~0.012mg/L, calcidol 0.08~0.12mg/L, D-calcium pantothenate 0.008~0.012mg /L, choline chloride 0.4~0.6mg/L, folic acid 0.008~0.012mg/L, inositol 0.04~0.06mg/L, menadione trihydrate sodium bisulfite 0.015~0.025mg/L, niacin 0.02 ~0.03mg/L, nicotinamide 0.02~0.03mg/L, p-aminobenzoic acid 0.04~0.06mg/L, pyridoxine hydrochloride 0.04~0.06mg/L, riboflavin 0.008~0.012mg/L, sulfur hydrochloride Amine 0.008~0.012mg/L, vitamin A acetate 0.1~0.2mg/L, adenine sulfate 8~12mg/L, adenine 0.15~0.25mg/L, adenosine triphosphate disodium 0.8~1.2mg/ L, cholesterol 0.15~0.25mg/L, 2-deoxy-D-ribose 0.4~0.6mg/L, D-glucose 800~1200mg/L, glutathione 0.04~0.06mg/L, guanine hydrochloride 0.25~0.35 mg/L, sodium hypoxanthine 0.3~0.4mg/L, ribose 0.4~0.6mg/L, thymosin 0.25~ 0.35mg/L, Tween 804~6mg/L, uracil 0.25~0.35mg/L, sodium xanthine 0.3~0.4mg/L, phenol red 8~12mg/L.

The method according to the first aspect of the present invention, wherein in step (1), the BY basal culture solution is an aqueous solution comprising the following components: calcium chloride 200mg/L, ferric nitrate nonahydrate 0.72mg/L, potassium chloride 400mg /L, magnesium sulfate 97.7mg/L, sodium chloride 6800mg/L, sodium dihydrogen phosphate monohydrate 140mg/L, sodium bicarbonate 2200mg/L, sodium acetate 50mg/L, L-alanine 25mg/L, L -Arginine hydrochloride 70mg/L, L-aspartic acid 30mg/L, L-cysteine hydrochloride monohydrate 0.11mg/L, L-cystine dihydrochloride 26mg/L , L-glutamic acid 75mg/L, glycine 50mg/L, L-histidine hydrochloride monohydrate 21.88mg/L, L-hydroxyproline 10mg/L, L-isoleucine 20mg/L , L-leucine 60mg/L, L-lysine hydrochloride 70mg/L, L-methionine 15mg/L, L-phenylalanine 25mg/L, L-proline 40mg/L, L- Serine 25mg/L, L-Threonine 30mg/L, L-Tryptophan 10mg/L, L-Tyrosine Disodium Dihydrate 57.66mg/L, L-Valine 25mg/L, Ascorbic Acid 0.05mg /L, α-D-tocopheryl phosphate 0.01mg/L, biotin 0.01mg/L, calcidol 0.1mg/L, D-calcium pantothenate 0.01mg/L, choline chloride 0.5mg/L, folic acid 0.01mg/L, inositol 0.05mg/L, menadione trihydrate sodium bisulfite 0.019mg/L, niacin 0.025mg/L, nicotinamide 0.025mg/L, p-aminobenzoic acid 0.05mg/L, Pyridoxine Hydrochloride 0.05mg/L, Riboflavin 0.01mg/L, Thiamine Hydrochloride 0.01mg/L, Vitamin A Acetate 0.14mg/L, Adenine Sulfate 10mg/L, Adenine 0.2mg/L, Adenosine triphosphate disodium 1mg/L, cholesterol 0.2mg/L, 2-deoxy-D-ribose 0.5mg/L, D-glucose 1000mg/L, glutathione 0.05mg/L, guanine hydrochloride 0.3mg/ L, Hypoxanthine Sodium 0.354mg/L, Ribose 0.5mg/L, Thymosin 0.3mg/L, Tween 805mg/L, Uracil 0.3mg/L, Xanthine Sodium 0.34mg/L, Phenol Red 10mg/L .

According to the method of the first aspect of the present invention, wherein in step (1), the BY basal culture solution further includes sodium selenite, and its concentration is 0.2-0.3 mg/L, such as 0.25 mg/L; and/or, The BY basal culture solution also includes copper sulfate, the concentration of which is 0.05-0.1 mg/L calculated as anhydrous, for example, 0.075 mg/L.

In the present invention, for example, in step (3), the preservation solution, equilibration solution, freezing solution, etc. used for embryos are well known in the art and can be easily obtained from commercial sources. For example, the freezing solution can be obtained from Vitrolife Company, Sweden. FreezeKit ^TM Cleave sold in China (if not specified in the present invention, the freezing solution used in the example is FreezeKit ^TM Cleave, the embryo preservation solution is Whittingham's modified Dulbecco's phosphate buffer containing 20% bovine serum and 500 IU/ml penicillin G potassium, balanced The liquid is ES liquid of Kitazato Biopharma Company of Japan).

Further, the second aspect of the present invention provides an oocyte transport medium.

The transport medium according to the second aspect of the present invention comprises: glycine 50.0 mg/L, L-alanine 25.0 mg/L, L-arginine hydrochloride 70.0 mg/L, L-aspartic acid 30.0 mg/L mg/L, L-cystine dihydrochloride 26.0mg/L, L-glutamic acid 75.0mg/L, L-glutamine 100.0mg/L, L-histidine hydrochloride monohydrate 21.88 mg/L, L-Hydroxyproline 10.0mg/L, L-Isoleucine 40.0mg/L, L-Leucine 60.0mg/L, L-Lysine Hydrochloride 70.0mg/L, L -Methionine 15.0mg/L, L-phenylalanine 25.0mg/L, L-proline 40.0mg/L, L-serine 25.0mg/L, L-threonine 30.0mg/L, L -Tryptophan 10.0mg/L, L-Tyrosine Disodium Salt Dihydrate 58.0mg/L, L-Valine 25.0mg/L, Ascorbic Acid 0.05mg/L, Biotin 0.01mg/L, Chloride Choline 0.5mg/L, D-Calcium Pantothenate 0.01mg/L, Folic Acid 0.01mg/L, Menaquinone 0.01mg/L, Niacinamide 0.025mg/L, Niacin 0.025mg/L, Para-Aminobenzoic Acid 0.05mg /L, pyridoxal hydrochloride 0.025mg/L, pyridoxine hydrochloride 0.025mg/L, riboflavin 0.01mg/L, thiamine hydrochloride 0.01mg/L, vitamin A acetate 0.1mg/L, vitamin D2 Calcidol 0.1mg/L, α-tocopherol sodium phosphate 0.01mg/L, inositol 0.05mg/L, anhydrous calcium chloride 200.0mg/L, iron nitrate nonahydrate 0.7mg/L, anhydrous sulfuric acid Magnesium 97.67mg/L, Potassium Chloride 400.0mg/L, Sodium Chloride 6800.0mg/L, Sodium Dihydrogen Phosphate Monohydrate 140.0mg/L, Adenine Sulfate 10.0mg/L, 5'-Adenosine Phosphate 0.2mg /L, Adenosine Triphosphate 1.0mg/L, Cholesterol 0.2mg/L, Glucose 1000.0mg/L, Deoxyribose 0.5mg/L, Reduced Glutathione 0.05mg/L, Guanine Hydrochloride 0.3mg/L, Hypoxanthine Sodium 0.354mg/L, phenol red 20.0mg/L, ribose 0.5mg/L, sodium acetate 50.0mg/L, thymine 0.3mg/L, Tween 80 20.0mg/L, uracil 0.3mg/L, yellow Sodium purine 0.3mg/L, and 0.01IU/mL FSH, 0.01IU/mL LH, 1μg/mL E ₂ , 50ng/mL EGF, 100ng/mL IGF, 10% gentamicin, 55μg/mL mL sodium pyruvate, 1.2 mM/L cysteine, 3 mg/mL BSA, 10 mM/L HEPES.

The transport medium according to the second aspect of the present invention further comprises taurine and zinc gluconate.

According to the transport medium of the second aspect of the present invention, the concentration of taurine is 30-50 mg/L, for example 40 mg/L or 45 mg/L.

According to the transport medium of the second aspect of the present invention, the concentration of zinc gluconate is 1-3 mg/L, for example 2 mg/L.

Further, a third aspect of the present invention provides a method for bovine in vitro fertilization, comprising the steps of:

The mature cumulus-oocyte complexes (COCs) were washed once in the fertilization medium, then transferred to the fertilization medium, and placed in an incubator for use;

Take a frozen fine tube from liquid nitrogen and thaw it in a water bath at 37°C; cut both ends of the fine tube aseptically, pour the semen into a 15 mL centrifuge tube containing the culture medium for semen preparation, and centrifuge at 328 × g twice, each time After centrifugation for 5 min, the supernatant was discarded; 300 µL of semen preparation medium was added to the above centrifuge tube, the sperm pellet was resuspended, and an appropriate sperm suspension was taken for sperm count;

Add the calculated volume of sperm suspension to the fertilization culture droplet containing the oocytes, and put the culture plate into the incubator to incubate the sperm and eggs for 16-20 hours. The culture conditions are 38.8°C, 5.5-6.5% CO2 , saturated humidity, complete in vitro fertilization operation, ready for in vitro culture of embryos.

The method according to the third aspect of the present invention, wherein the fertilization medium comprises 112.0 mM sodium chloride, 4.02 mM potassium chloride, 2.25 mM calcium chloride dihydrate, 0.52 mM magnesium chloride hexahydrate, 0.83 mM dihydrogen phosphate Potassium, 37.0mM sodium bicarbonate, 1.25mM sodium pyruvate, 10μg/ml heparin, 4mg/ml bovine serum albumin (BSA), 100U/ml penicillin, 100μg/ml streptomycin, 10umoL/L alpha-lipoic acid, 25 μg/ml folic acid, 2.5 mg/ml glutamic acid in water.

The method according to the third aspect of the present invention, wherein the semen preparation medium comprises 112.0 mM sodium chloride, 4.02 mM potassium chloride, 2.25 mM calcium chloride dihydrate, 0.52 mM magnesium chloride hexahydrate, 0.83 mM phosphate dihydrate Potassium hydrogen, 37.0mM sodium bicarbonate, 1.25mM sodium pyruvate, 10μg/ml heparin, 4mg/ml bovine serum albumin (BSA), 10mM caffeine, 100U/ml penicillin, 100μg/ml streptomycin, 10umoL/L Alpha-lipoic acid, 25 μg/ml folic acid, 2.5 mg/ml glutamic acid in water.

The method according to the third aspect of the invention, wherein the cumulus-oocyte complex is harvested and matured in vitro by:

a) In vitro collection: take bovine ovaries from slaughterhouses, extract follicles with a surface of 2-8 mm, collect sediment, and pick out oocyte COCs (ie, cumulus- oocyte complex), placed in a transport medium containing HEPES at 38.8°C, without carbon dioxide supply, and transported back to the laboratory within 24 hours; or

a) In vivo collection: take follicular fluid from bovine live egg collection, pick out cumulus-oocyte complexes (COCs) containing at least 3 layers of cumulus cells under a stereo microscope, and put them into a transport culture containing HEPES 38.8℃ in the liquid, no carbon dioxide supply, transported back to the laboratory within 24h;

b) The COCs collected above in vitro or in vivo were washed once in the oocyte maturation medium, and then transferred to a new mature medium for 22-24 hours under the conditions of 38.8°C, 5.5-6.5% CO2, Saturate the humidity to obtain mature cumulus-oocyte complexes.

The method according to the third aspect of the present invention has the features described in the first or second aspect of the present invention.

Further, the fourth aspect of the present invention provides a fertilization medium for bovine in vitro fertilization, which comprises 112.0 mM sodium chloride, 4.02 mM potassium chloride, 2.25 mM calcium chloride dihydrate, 0.52 mM magnesium chloride hexahydrate , 0.83mM potassium dihydrogen phosphate, 37.0mM sodium bicarbonate, 1.25mM sodium pyruvate, 10μg/ml heparin, 4mg/ml bovine serum albumin (BSA), 100U/ml penicillin, 100μg/ml streptomycin, 10umoL/ A solution of L alpha-lipoic acid, 25 μg/ml folic acid, 2.5 mg/ml glutamic acid in water.

Further, the fifth aspect of the present invention provides a seminal fluid preparation for bovine in vitro fertilization, which comprises 112.0 mM sodium chloride, 4.02 mM potassium chloride, 2.25 mM calcium chloride dihydrate, 0.52 mM hexahydrate Magnesium chloride, 0.83 mM potassium dihydrogen phosphate, 37.0 mM sodium bicarbonate, 1.25 mM sodium pyruvate, 10 μg/ml heparin, 4 mg/ml bovine serum albumin (BSA), 10 mM caffeine, 100 U/ml penicillin, 100 μg/ml chain Mycin, 10umoL/L α-lipoic acid, 25 μg/ml folic acid, 2.5 mg/ml glutamic acid in water.

Any technical feature possessed by any aspect of the present invention or any embodiment of any aspect of the present invention is also applicable to any other embodiment or any embodiment of any other aspect, as long as they do not contradict each other. When applicable, appropriate modifications can be made to the corresponding features if necessary. Various aspects and features of the present invention are further described below.

All documents cited in the present invention, their entire contents are incorporated herein by reference, and if the meaning expressed by these documents is inconsistent with the present invention, the expression of the present invention shall prevail. In addition, various terms and phrases used in the present invention have ordinary meanings known to those skilled in the art. Even so, the present invention still hopes to make more detailed descriptions and explanations for these terms and phrases. The terms and phrases mentioned are such as If there is any inconsistency with the known meaning, the meaning expressed in the present invention shall prevail.

The fetal bovine serum used in the present invention can be easily purchased from the market in its standardized commercial form, for example, Australian fetal bovine serum (article number: 10099141) and New Zealand fetal bovine serum (article number: 10091148 of Gibco Company can be purchased from various agents ), North American Fetal Bovine Serum (Cat. No. 16000044), Mexican Fetal Bovine Serum (Cat. No. 10437028), etc. In the experiments in the context of the present invention, the fetal bovine serum used was Australian fetal bovine serum (Cat. No. 10099141) from Gibco, unless otherwise specified.

Detailed ways

The present invention can 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 can be made in the present invention without departing from the spirit and scope of the inventions. The present invention provides general and/or specific descriptions of the materials and test methods used in the tests. While many of the materials and methods of operation used for the purposes of the present invention are known in the art, the present invention is described in as much 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 all commercially available commodities. In the present invention, unless otherwise specified, when referring to glutamic acid or sodium glutamate, it refers to L-glutamic acid or sodium L-glutamate.

Example 1: Cultivation method of bovine in vitro fertilized embryos (collected in vitro)

In this example, in step (1), the collected oocytes were placed in a transport medium containing HEPES at 38.8° C., without carbon dioxide supply, and transported back to the laboratory within 24 hours.

1. Reagents

In the specific test of the present invention, unless otherwise stated, the relevant reagents used are described in detail as follows:

The transport medium containing HEPES used in this example contains: glycine 50.0 mg/L, L-alanine 25.0 mg/L, L-arginine hydrochloride 70.0 mg/L, L-aspartic acid 30.0mg/L, L-cystine dihydrochloride 26.0mg/L, L-glutamic acid 75.0mg/L, L-glutamine 100.0mg/L, L-histidine hydrochloride monohydrate 21.88mg/L, L-hydroxyproline 10.0mg/L, L-isoleucine 40.0mg/L, L-leucine 60.0mg/L, L-lysine hydrochloride 70.0mg/L, L-methionine 15.0mg/L, L-phenylalanine 25.0mg/L, L-proline 40.0mg/L, L-serine 25.0mg/L, L-threonine 30.0mg/L, L-tryptophan 10.0mg/L, L-tyrosine disodium salt dihydrate 58.0mg/L, L-valine 25.0mg/L, ascorbic acid 0.05mg/L, biotin 0.01mg/L, chlorine Choline 0.5mg/L, calcium D-pantothenate 0.01mg/L, folic acid 0.01mg/L, menadione 0.01mg/L, niacinamide 0.025mg/L, niacin 0.025mg/L, p-aminobenzoic acid 0.05 mg/L, pyridoxal hydrochloride 0.025mg/L, pyridoxine hydrochloride 0.025mg/L, riboflavin 0.01mg/L, thiamine hydrochloride 0.01mg/L, vitamin A acetate 0.1mg/L, vitamin D2 Namely calcidol 0.1mg/L, α-tocopherol sodium phosphate 0.01mg/L, inositol 0.05mg/L, anhydrous calcium chloride 200.0mg/L, iron nitrate nonahydrate 0.7mg/L, anhydrous Magnesium sulfate 97.67mg/L, potassium chloride 400.0mg/L, sodium chloride 6800.0mg/L, sodium dihydrogen phosphate monohydrate 140.0mg/L, adenine sulfate 10.0mg/L, 5'-adenosine phosphate 0.2 mg/L, Adenosine Triphosphate 1.0mg/L, Cholesterol 0.2mg/L, Glucose 1000.0mg/L, Deoxyribose 0.5mg/L, Reduced Glutathione 0.05mg/L, Guanine Hydrochloride 0.3mg/L, Hypoxanthone Purine sodium 0.354mg/L, phenol red 20.0mg/L, ribose 0.5mg/L, sodium acetate 50.0mg/L, thymine 0.3mg/L, Tween 80 20.0mg/L, uracil 0.3mg/L, Xanthine sodium 0.3mg/L, and 0.01IU/mL FSH, 0.01IU/mL LH, 1μg/mL E ₂ , 50ng/mL EGF, 100ng/mL IGF, 10% gentamicin, 55μg /mL sodium pyruvate, 1.2mM/L cysteine, 3mg/mL BSA, 10mM/L HEPES, and taurine 40mg/L, grape Zinc gluconate 2mg/L.

Add double antibody physiological saline: physiological saline containing penicillin 400IU/mL and streptomycin 400μg/mL.

Egg wash: BY basal medium supplemented with 3 mg/mL bovine serum albumin.

Mature medium: BY basal medium supplemented with 100 mL/L FBS, 10 μg/mL FSH, 10 μg/mL LH, 1 μg/mL E2, and 20 ng/mLEGF.

Mature medium containing HEPES: BY basal medium supplemented with 15 mmol/L HEPES, 100 mL/L FBS, 10 μg/mL FSH, 10 μg/mL LH, 1 μg/mL E2, 20 ng/mL EGF.

Among them, EGF - epidermal growth factor, FSH - follicle stimulating hormone, FBS - fetal bovine serum, E2 - estradiol, LH - luteinizing hormone.

Fertilization medium: contains 112.0 mM sodium chloride, 4.02 mM potassium chloride, 2.25 mM calcium chloride dihydrate, 0.52 mM magnesium chloride hexahydrate, 0.83 mM potassium dihydrogen phosphate, 37.0 mM sodium bicarbonate, 1.25 mM pyruvate Sodium, 10 μg/ml heparin, 4 mg/ml bovine serum albumin (BSA), 100 U/ml penicillin, 100 μg/ml streptomycin in water.

Semen preparation medium: contains 112.0 mM sodium chloride, 4.02 mM potassium chloride, 2.25 mM calcium chloride dihydrate, 0.52 mM magnesium chloride hexahydrate, 0.83 mM potassium dihydrogen phosphate, 37.0 mM sodium bicarbonate, 1.25 mM acetone Sodium, 10 μg/ml heparin, 4 mg/ml bovine serum albumin (BSA), 10 mM caffeine, 100 U/ml penicillin, 100 μg/ml streptomycin in water.

Embryo culture medium: contains: 109.5mM sodium chloride, 3.1mM potassium chloride, 26.2mM sodium bicarbonate, 0.8mM magnesium chloride hexahydrate, 1.19mM potassium dihydrogen phosphate, 0.4mM sodium pyruvate, 1.5mM glucose, 5mM galactose Calcium acid, 10v/v% fetal bovine serum (FBS), L-glutamine 1mM, 2v/v% essential amino acids, 1v/v% non-essential amino acids, 3mM glutathione, sodium citrate 0.04w/ The aqueous solution of v% and maltose 0.02w/v%; the essential amino acids are the following amino acids added by weight: L-arginine hydrochloride 6.32g, L-cystine dihydrochloride 1.564g, L-histidine hydrochloride Acid monohydrate 2.1g, L-isoleucine 2.625g, L-leucine 2.62g, L-lysine hydrochloride 3.625g, L-methionine 0.755g, L-phenylalanine 1.65g, L-threonine 2.38g, L-tryptophan 0.51g, L-tyrosine 1.8g and L-valine 2.34g, the non-essential amino acids are the following amino acids added by weight: L-alanine 0.89 g, L-asparagine monohydrate 1.5 g, L-aspartic acid 1.33 g, L-glutamic acid 1.47 g, glycine 0.75 g, L-proline 1.15 g, and L-serine 1.05 g.

BY basal culture medium is an aqueous solution containing the following components: calcium chloride 200mg/L, ferric nitrate nonahydrate 0.72mg/L, potassium chloride 400mg/L, magnesium sulfate 97.7mg/L, sodium chloride 6800mg/L, a Water sodium dihydrogen phosphate 140mg/L, sodium bicarbonate 2200mg/L, sodium acetate 50mg/L, L-alanine 25mg/L, L-arginine hydrochloride 70mg/L, L-aspartic acid 30mg /L, L-cysteine hydrochloride monohydrate 0.11mg/L, L-cystine dihydrochloride 26mg/L, L-glutamic acid 75mg/L, glycine 50mg/L, L-group Amino acid hydrochloride monohydrate 21.88mg/L, L-hydroxyproline 10mg/L, L-isoleucine 20mg/L, L-leucine 60mg/L, L-lysine hydrochloride 70mg/L, L-methionine 15mg/L, L-phenylalanine 25mg/L, L-proline 40mg/L, L-serine 25mg/L, L-threonine 30mg/L, L-tryptophan Acid 10mg/L, L-Tyrosine Disodium Dihydrate 57.66mg/L, L-Valine 25mg/L, Ascorbic Acid 0.05mg/L, α-D-Tocopheryl Phosphate 0.01mg/L, Biotin 0.01mg/L, calcidol 0.1mg/L, D-calcium pantothenate 0.01mg/L, choline chloride 0.5mg/L, folic acid 0.01mg/L, inositol 0.05mg/L, menadione trihydrate Sodium hydrogen sulfate 0.019mg/L, niacin 0.025mg/L, nicotinamide 0.025mg/L, p-aminobenzoic acid 0.05mg/L, pyridoxine hydrochloride 0.05mg/L, riboflavin 0.01mg/L, hydrochloric acid Thiamine 0.01mg/L, vitamin A acetate 0.14mg/L, adenine sulfate 10mg/L, adenine 0.2mg/L, adenosine triphosphate disodium 1mg/L, cholesterol 0.2mg/L, 2- Deoxy-D-ribose 0.5mg/L, D-glucose 1000mg/L, glutathione 0.05mg/L, guanine hydrochloride 0.3mg/L, sodium hypoxanthine 0.354mg/L, ribose 0.5mg/L, thymus Sulphur 0.3mg/L, Tween 805mg/L, uracil 0.3mg/L, sodium xanthine 0.34mg/L, phenol red 10mg/L, 0.25mg/L sodium selenite and 0.075mg/L anhydrous copper sulfate .

Second, bovine in vitro fertilization and embryo culture:

Step (1), oocyte collection and in vitro maturation

(a) Take bovine ovaries from slaughterhouses, extract follicles with a surface of 2-8 mm, collect the precipitate, and pick out oocyte COCs (i.e., cumulus-oocytes) containing at least 3 layers of cumulus cells under a stereomicroscope complex), put it in the transport medium containing HEPES at 38.8°C, without carbon dioxide supply, and transported back to the laboratory within 24 hours (in this case, it was transported back to the laboratory within 15 hours for the next step processing);

(b) The obtained COCs were washed once in the oocyte maturation medium, and then transferred to a new mature medium for 22-24 hours (actual operation 24 hours). The culture conditions were 38.8°C, 5.5-6.5% CO2, saturated humidity;

Step (2), in vitro fertilization

The mature COCs were washed once in the fertilization medium, then transferred to the fertilization medium, and placed in an incubator for use;

Take a frozen fine tube from liquid nitrogen and thaw it in a water bath at 37°C; cut both ends of the fine tube aseptically, pour the semen into a 15 mL centrifuge tube containing the culture medium for semen preparation, and centrifuge at 328 × g twice, each time After centrifugation for 5 min, the supernatant was discarded; 300 µL of semen preparation medium was added to the above centrifuge tube, the sperm pellet was resuspended, and an appropriate sperm suspension was taken for sperm count;

Add the calculated volume of sperm suspension to the fertilization culture droplet containing oocytes, and put the culture plate into the incubator to incubate the sperm and eggs for 16-20 hours (actual operation 18 hours), and the culture condition is 38.8 ℃ , 5.5-6.5% CO2, saturated humidity;

Step (3), embryo culture and preservation in vitro

After the in vitro fertilization operation is completed, the granulosa cells around the embryos are removed with an egg stripper and cultured in the embryo culture medium. This time is recorded as the first day of embryo culture. N2, saturated humidity, record the cleavage rate on the 3rd day; record the blastocyst rate on the 7th day, count the blastocyst hatching rate (which is the percentage obtained by dividing the number of hatched blastocysts by the number of blastocysts) on the 9th day, and carry out quality identification ;

Wash the available embryos in the preservation solution for 3 times, equilibrate in the equilibration solution for 10 minutes, and transfer them into the freezing solution. Load the embryos according to the 5-stage filling method, make a mark, and then put them into the programmed cooling device at a temperature of 0.5 °C/min. After the speed was reduced to -35°C, the embryo tube was quickly taken out and placed in liquid nitrogen for cryopreservation.

Third, the method of differential staining of embryos

1. The blastocysts on the 7th day of in vitro culture were selected and fixed with 2% paraformaldehyde for 20 min.

2. Wash twice with phosphate buffered saline (PBS-BSA) containing 0.5% BSA, put into permeabilization solution (50 μl Triton, 5 μl Tween 80 and 9.945 ml PBS), and leave at room temperature for 30 min.

3. Treat with 2M hydrochloric acid for 20 min at room temperature, and then treat with 100 mM Tris-HCl for 10 min at room temperature, so that the CDX2 protein can bind to the primary antibody.

4. The blastocysts were washed three times with PBS-BSA, and the blastocysts were placed in blocking solution (1 ml of goat serum, 5 μl of Tween 80 and 8.995 ml of PBS), blocked at room temperature for 1 h, and then transferred to a 4°C refrigerator to block overnight.

5. The blocking solution was discarded, the CDX2 primary antibody was diluted 1:200 with the blocking solution, incubated at room temperature for 2 h, the primary antibody dilution was discarded, and washed three times with PBS-BSA, 5 min each time.

6. Caspase-3 primary antibody (purchased from Cell Signaling Technology) was diluted 1:200 with blocking solution, incubated at room temperature for 2 h, discarded the primary antibody dilution, and washed three times with PBS-BSA for 5 min each time.

7. The CDX2-specific secondary antibody (purchased from Sigma) was diluted 1:200 with blocking solution under dark conditions, and placed in the dark for 1 h at room temperature. The secondary antibody diluent was discarded in the dark and washed three times with PBS for 5 min each time.

8. The caspase-3-specific secondary antibody (purchased from LifeTechnologies) was diluted 1:200 with blocking solution under dark conditions, and placed in the dark for 1 h at room temperature. The secondary antibody diluent was discarded in the dark and washed three times with PBS for 5 min each time.

9. Add 10 μg/mL of Hochest 33342 dye solution to stain the nucleus, let it act for 5 min at room temperature, observe and take pictures under a fluorescence microscope.

10. The experiment was repeated three times, and 10 blastocysts were randomly selected each time, and the apoptotic rate and the number of ICM cells/total cells were calculated to evaluate the blastocyst quality.

Data Statistical Methods: The experimental data were analyzed by the ANOVA program in the statistical software SAS V8. The Duncan's multiple-range test method was used to determine the significance of the difference between treatments. When p<0.05, the difference was considered significant.

In the present invention, the cleavage rate=the number of fertilized cleavage/the number of fertilized eggs. In the present invention, blastocyst rate=number of blastocysts/number of cleavage embryos.

4. In vitro maturation (IVM) effect of oocytes - maturation rate

In step (1) of the present embodiment, after in vitro maturation and culture, observed under an inverted microscope, the oocyte has a first polar body released, a viscous matrix secreted between cumulus cells is maintained, and the cell layer is significantly swelled , the cells are centered on the ovum, and those that spread radially to the surrounding are judged to be mature, the number of mature oocytes is recorded, and the maturation rate is calculated.

5. Results

In this example, the Chinese yellow cattle (Nanyang cattle, a draught breed) were tested. The results showed that the cleavage rate was 86.4%, the morula rate was 66.3%, the blastocyst rate was 51.6%, and the apoptotic rate was 5.3%. In addition, the blastocyst hatching rate was 75.1% on the ninth day. The maturation rate of oocytes matured in vitro was 87.5%.

Example 1A: Cultivation method of bovine in vitro fertilized embryos (collected in vitro)

With reference to the method of Example 1, tests were carried out for three kinds of cattle of Holstein (dairy cattle variety), Simmental cattle (beef cattle variety), Chinese water buffalo (dramatic variety), and the result: the cleavage rate was all in the range of 85 to 90%. The morula rate was in the range of 62~67%, the blastocyst rate was in the range of 50~55%, the apoptosis rate was in the range of 4~6%, and the hatching rate of blastocyst on the 9th day was in the range of 72~50%. Within the range of 77%; the maturation rates of in vitro maturation of oocytes from Holstein, Simmental, and Chinese buffalo were 83.3%, 80.6%, and 75.6%, respectively.

Example 1B: Cultivation method of bovine in vitro fertilized embryos (collected in vitro)

With reference to the method of Example 1, tests were carried out for four kinds of cattle of Chinese yellow cattle (Nanyang cattle, draught type), Holstein cattle (dairy cattle species), Simmental cattle (beef cattle species), Chinese buffalo (draft cattle species), However, the recipes of fertilization medium and semen preparation medium were modified respectively, and the other test solutions and operations remained unchanged. The fertilization medium formula was changed to: 112.0mM sodium chloride, 4.02mM potassium chloride, 2.25mM calcium chloride dihydrate, 0.52mM magnesium chloride hexahydrate, 0.83mM potassium dihydrogen phosphate, 37.0mM sodium bicarbonate, 1.25 mM sodium pyruvate, 10μg/ml heparin, 4mg/ml bovine serum albumin (BSA), 100U/ml penicillin, 100μg/ml streptomycin, 10umoL/L α-lipoic acid, 25μg/ml folic acid, 2.5mg/ml Aqueous solution of glutamic acid; semen preparation medium formula changed to: containing 112.0 mM sodium chloride, 4.02 mM potassium chloride, 2.25 mM calcium chloride dihydrate, 0.52 mM magnesium chloride hexahydrate, 0.83 mM potassium dihydrogen phosphate, 37.0mM sodium bicarbonate, 1.25mM sodium pyruvate, 10μg/ml heparin, 4mg/ml bovine serum albumin (BSA), 10mM caffeine, 100U/ml penicillin, 100μg/ml streptomycin, 10umoL/L α-sulfur Caprylic acid, 25 μg/ml folic acid, 2.5 mg/ml glutamic acid in water. Results: The cleavage rates of the four types of cattle were in the range of 87-91%, the morula rates were in the range of 64-68%, the blastocyst rates were in the range of 78-82%, and the apoptosis rates were all in the range of 3-6%. The hatching rate of blastocysts on day 9 was in the range of 75-78%. For example, the cleavage rate of Chinese cattle was 90.2%, the morula rate was 66.4%, the blastocyst rate was 80.7%, and the apoptosis rate was 3.3%. %, the blastocyst hatching rate on the 9th day was 76.4%; the in vitro maturation rates of oocytes from Chinese cattle, Holstein cattle, Simmental cattle, and Chinese buffalo were 86.8%, 85.1%, 82.2%, and 76.4%, respectively. %.

Example 1C: Cultivation method of bovine in vitro fertilized embryos (collected in vitro)

Carry out with reference to Example 1B, the only difference is that no folic acid is added in the fertilization medium and the semen preparation medium. Results: The cleavage rates of the four types of cattle were in the range of 84-90%, the morula rates were in the range of 61-65%, the blastocyst rates were in the range of 52-56%, and the apoptosis rates were all in the range of 3-5%. The hatching rate of blastocysts on day 9 was in the range of 73-76%. For example, the cleavage rate of Chinese cattle was 88.4%, the morula rate was 64.3%, the blastocyst rate was 53.4%, and the apoptosis rate was 3.8. %, the blastocyst hatching rate on the 9th day was 74.1%; the in vitro maturation rates of oocytes from Chinese cattle, Holstein cattle, Simmental cattle, and Chinese buffalo were 85.2%, 86.7%, 80.5%, and 77.2%, respectively. %.

Example 1D: Cultivation method of bovine in vitro fertilized embryos (collected in vitro)

Carry out with reference to Example 1B, the only difference is that glutamic acid is not added in the fertilization medium and the semen preparation medium. Results: The cleavage rates of the four types of cattle were in the range of 86-90%, the morula rates were in the range of 62-65%, the blastocyst rates were in the range of 53-57%, and the apoptosis rates were all in the range of 4-6%. The hatching rate of blastocysts on day 9 was in the range of 72-76%. For example, the cleavage rate of Chinese cattle was 87.2%, the morula rate was 62.8%, the blastocyst rate was 55.7%, and the apoptosis rate was 5.1%. %, the blastocyst hatching rate on the 9th day was 74.7%; the in vitro maturation rates of oocytes from Chinese cattle, Holstein cattle, Simmental cattle, and Chinese buffalo were 86.4%, 83.6%, 84.7%, and 76.7%, respectively. %.

Example 1E: Cultivation method of bovine in vitro fertilized embryos (collected in vitro)

Carry out with reference to Example 1B, the only difference is that neither folic acid nor glutamic acid is added to the fertilization medium and the semen preparation medium. Results: The cleavage rates of the four types of cattle were in the range of 86-92%, the morula rates were in the range of 61-65%, the blastocyst rates were in the range of 51-55%, and the apoptosis rates were all in the range of 4-7%. The hatching rate of blastocysts on day 9 was in the range of 71-76%. For example, the cleavage rate of Chinese cattle was 87.6%, the morula rate was 64.1%, the blastocyst rate was 53.4%, and the apoptosis rate was 5.5%. %, the blastocyst hatching rate on the 9th day was 73.1%; the in vitro maturation rates of oocytes from Chinese cattle, Holstein cattle, Simmental cattle, and Chinese buffalo were 85.2%, 85.7%, 80.8%, and 74.5%, respectively. %.

Example 2: Cultivation method of bovine in vitro fertilized embryos (living collection)

In this example, in step (1), the collected oocytes were placed in a transport medium containing HEPES at 38.8° C., without carbon dioxide supply, and transported back to the laboratory within 24 hours.

1. Reagents

In the specific test of the present invention, unless otherwise stated, the relevant reagents used are described in detail as follows:

The transport medium containing HEPES used in this example contains: glycine 50.0 mg/L, L-alanine 25.0 mg/L, L-arginine hydrochloride 70.0 mg/L, L-aspartic acid 30.0mg/L, L-cystine dihydrochloride 26.0mg/L, L-glutamic acid 75.0mg/L, L-glutamine 100.0mg/L, L-histidine hydrochloride monohydrate 21.88mg/L, L-hydroxyproline 10.0mg/L, L-isoleucine 40.0mg/L, L-leucine 60.0mg/L, L-lysine hydrochloride 70.0mg/L, L-methionine 15.0mg/L, L-phenylalanine 25.0mg/L, L-proline 40.0mg/L, L-serine 25.0mg/L, L-threonine 30.0mg/L, L-tryptophan 10.0mg/L, L-tyrosine disodium salt dihydrate 58.0mg/L, L-valine 25.0mg/L, ascorbic acid 0.05mg/L, biotin 0.01mg/L, chlorine Choline 0.5mg/L, calcium D-pantothenate 0.01mg/L, folic acid 0.01mg/L, menadione 0.01mg/L, niacinamide 0.025mg/L, niacin 0.025mg/L, p-aminobenzoic acid 0.05 mg/L, pyridoxal hydrochloride 0.025mg/L, pyridoxine hydrochloride 0.025mg/L, riboflavin 0.01mg/L, thiamine hydrochloride 0.01mg/L, vitamin A acetate 0.1mg/L, vitamin D2 Namely calcidol 0.1mg/L, α-tocopherol sodium phosphate 0.01mg/L, inositol 0.05mg/L, anhydrous calcium chloride 200.0mg/L, iron nitrate nonahydrate 0.7mg/L, anhydrous Magnesium sulfate 97.67mg/L, potassium chloride 400.0mg/L, sodium chloride 6800.0mg/L, sodium dihydrogen phosphate monohydrate 140.0mg/L, adenine sulfate 10.0mg/L, 5'-adenosine phosphate 0.2 mg/L, Adenosine Triphosphate 1.0mg/L, Cholesterol 0.2mg/L, Glucose 1000.0mg/L, Deoxyribose 0.5mg/L, Reduced Glutathione 0.05mg/L, Guanine Hydrochloride 0.3mg/L, Hypoxanthone Purine sodium 0.354mg/L, phenol red 20.0mg/L, ribose 0.5mg/L, sodium acetate 50.0mg/L, thymine 0.3mg/L, Tween 80 20.0mg/L, uracil 0.3mg/L, Xanthine sodium 0.3mg/L, and 0.01IU/mL FSH, 0.01IU/mL LH, 1μg/mL E ₂ , 50ng/mL EGF, 100ng/mL IGF, 10% gentamicin, 55μg /mL sodium pyruvate, 1.2mM/L cysteine, 3mg/mL BSA, 10mM/L HEPES, and taurine 45mg/L, grape Zinc gluconate 2mg/L.

Add double antibody physiological saline: physiological saline containing penicillin 400IU/mL and streptomycin 400μg/mL.

Egg wash: BY basal medium supplemented with 3 mg/mL bovine serum albumin.

Mature medium: BY basal medium supplemented with 100 mL/L FBS, 10 μg/mL FSH, 10 μg/mL LH, 1 μg/mL E2, and 20 ng/mLEGF.

Mature medium containing HEPES: BY basal medium supplemented with 15 mmol/L HEPES, 100 mL/L FBS, 10 μg/mL FSH, 10 μg/mL LH, 1 μg/mL E2, 20 ng/mL EGF.

Among them, EGF - epidermal growth factor, FSH - follicle stimulating hormone, FBS - fetal bovine serum, E2 - estradiol, LH - luteinizing hormone.

Fertilization medium: contains 112.0 mM sodium chloride, 4.02 mM potassium chloride, 2.25 mM calcium chloride dihydrate, 0.52 mM magnesium chloride hexahydrate, 0.83 mM potassium dihydrogen phosphate, 37.0 mM sodium bicarbonate, 1.25 mM pyruvate Sodium, 10 μg/ml heparin, 4 mg/ml bovine serum albumin (BSA), 100 U/ml penicillin, 100 μg/ml streptomycin in water.

Semen preparation medium: contains 112.0 mM sodium chloride, 4.02 mM potassium chloride, 2.25 mM calcium chloride dihydrate, 0.52 mM magnesium chloride hexahydrate, 0.83 mM potassium dihydrogen phosphate, 37.0 mM sodium bicarbonate, 1.25 mM acetone Sodium, 10 μg/ml heparin, 4 mg/ml bovine serum albumin (BSA), 10 mM caffeine, 100 U/ml penicillin, 100 μg/ml streptomycin in water.

Embryo culture medium: contains: 109.5mM sodium chloride, 3.1mM potassium chloride, 26.2mM sodium bicarbonate, 0.8mM magnesium chloride hexahydrate, 1.19mM potassium dihydrogen phosphate, 0.4mM sodium pyruvate, 1.5mM glucose, 5mM galactose Calcium acid, 10v/v% fetal bovine serum (FBS), L-glutamine 1mM, 2v/v% essential amino acids, 1v/v% non-essential amino acids, 3mM glutathione, sodium citrate 0.04w/ The aqueous solution of v% and maltose 0.02w/v%; the essential amino acids are the following amino acids added by weight: L-arginine hydrochloride 6.32g, L-cystine dihydrochloride 1.564g, L-histidine hydrochloride Acid monohydrate 2.1g, L-isoleucine 2.625g, L-leucine 2.62g, L-lysine hydrochloride 3.625g, L-methionine 0.755g, L-phenylalanine 1.65g, L-threonine 2.38g, L-tryptophan 0.51g, L-tyrosine 1.8g and L-valine 2.34g, the non-essential amino acids are the following amino acids added by weight: L-alanine 0.89 g, L-asparagine monohydrate 1.5 g, L-aspartic acid 1.33 g, L-glutamic acid 1.47 g, glycine 0.75 g, L-proline 1.15 g, and L-serine 1.05 g.

BY basal culture medium is an aqueous solution containing the following components: calcium chloride 200mg/L, ferric nitrate nonahydrate 0.72mg/L, potassium chloride 400mg/L, magnesium sulfate 97.7mg/L, sodium chloride 6800mg/L, a Water sodium dihydrogen phosphate 140mg/L, sodium bicarbonate 2200mg/L, sodium acetate 50mg/L, L-alanine 25mg/L, L-arginine hydrochloride 70mg/L, L-aspartic acid 30mg /L, L-cysteine hydrochloride monohydrate 0.11mg/L, L-cystine dihydrochloride 26mg/L, L-glutamic acid 75mg/L, glycine 50mg/L, L-group Amino acid hydrochloride monohydrate 21.88mg/L, L-hydroxyproline 10mg/L, L-isoleucine 20mg/L, L-leucine 60mg/L, L-lysine hydrochloride 70mg/L, L-methionine 15mg/L, L-phenylalanine 25mg/L, L-proline 40mg/L, L-serine 25mg/L, L-threonine 30mg/L, L-tryptophan Acid 10mg/L, L-Tyrosine Disodium Dihydrate 57.66mg/L, L-Valine 25mg/L, Ascorbic Acid 0.05mg/L, α-D-Tocopheryl Phosphate 0.01mg/L, Biotin 0.01mg/L, calcidol 0.1mg/L, D-calcium pantothenate 0.01mg/L, choline chloride 0.5mg/L, folic acid 0.01mg/L, inositol 0.05mg/L, menadione trihydrate Sodium hydrogen sulfate 0.019mg/L, niacin 0.025mg/L, nicotinamide 0.025mg/L, p-aminobenzoic acid 0.05mg/L, pyridoxine hydrochloride 0.05mg/L, riboflavin 0.01mg/L, hydrochloric acid Thiamine 0.01mg/L, vitamin A acetate 0.14mg/L, adenine sulfate 10mg/L, adenine 0.2mg/L, adenosine triphosphate disodium 1mg/L, cholesterol 0.2mg/L, 2- Deoxy-D-ribose 0.5mg/L, D-glucose 1000mg/L, glutathione 0.05mg/L, guanine hydrochloride 0.3mg/L, sodium hypoxanthine 0.354mg/L, ribose 0.5mg/L, thymus Sulphur 0.3mg/L, Tween 805mg/L, uracil 0.3mg/L, sodium xanthine 0.34mg/L, phenol red 10mg/L, 0.25mg/L sodium selenite and 0.075mg/L anhydrous copper sulfate .

Second, bovine in vitro fertilization and embryo culture:

Step (1), oocyte collection and in vitro maturation

(a) Take follicular fluid from bovine live egg collection, pick out cumulus-oocyte complexes (COCs) containing at least 3 layers of cumulus cells under a stereo microscope, and put them in a transport medium containing HEPES 38.8 ℃, no carbon dioxide supply, transported back to the laboratory within 24 hours (in this case, it was transported back to the laboratory within 15 hours for the next step processing);

(b) The obtained COCs were washed once in the oocyte maturation medium, and then transferred to a new mature medium for 22-24 hours (actual operation 24 hours). The culture conditions were 38.8°C, 5.5-6.5% CO2, saturated humidity;

Step (2), in vitro fertilization

The mature COCs were washed once in the fertilization medium, then transferred to the fertilization medium, and placed in an incubator for use;

Take a frozen fine tube from liquid nitrogen and thaw it in a water bath at 37°C; cut both ends of the fine tube aseptically, pour the semen into a 15 mL centrifuge tube containing the culture medium for semen preparation, and centrifuge at 328 × g twice, each time After centrifugation for 5 min, the supernatant was discarded; 300 µL of semen preparation medium was added to the above centrifuge tube, the sperm pellet was resuspended, and an appropriate sperm suspension was taken for sperm count;

Add the calculated volume of sperm suspension to the fertilization culture droplet containing oocytes, and put the culture plate into the incubator to incubate the sperm and eggs for 16-20 hours (actual operation 18 hours), and the culture condition is 38.8 ℃ , 5.5-6.5% CO2, saturated humidity;

Step (3), embryo culture and preservation in vitro

After the in vitro fertilization operation is completed, the granulosa cells around the embryos are removed with an egg stripper and cultured in the embryo culture medium. This time is recorded as the first day of embryo culture. N2, saturated humidity, record the cleavage rate on the 3rd day; record the blastocyst rate on the 7th day, count the blastocyst hatching rate (which is the percentage obtained by dividing the number of hatched blastocysts by the number of blastocysts) on the 9th day, and carry out quality identification ;

Wash the available embryos in the preservation solution for 3 times, equilibrate in the equilibration solution for 10 minutes, and transfer them into the freezing solution. Load the embryos according to the 5-stage filling method, make a mark, and then put them into the programmed cooling device at a temperature of 0.5 °C/min. After the speed was reduced to -35°C, the embryo tube was quickly taken out and placed in liquid nitrogen for cryopreservation.

Third, the method of differential staining of embryos

1. The blastocysts on the 7th day of in vitro culture were selected and fixed with 2% paraformaldehyde for 20 min.

2. Wash twice with phosphate buffered saline (PBS-BSA) containing 0.5% BSA, put into permeabilization solution (50 μl Triton, 5 μl Tween 80 and 9.945 ml PBS), and leave at room temperature for 30 min.

3. Treat with 2M hydrochloric acid for 20 min at room temperature, and then treat with 100 mM Tris-HCl for 10 min at room temperature, so that the CDX2 protein can bind to the primary antibody.

4. The blastocysts were washed three times with PBS-BSA, and the blastocysts were placed in blocking solution (1 ml of goat serum, 5 μl of Tween 80 and 8.995 ml of PBS), blocked at room temperature for 1 h, and then transferred to a 4°C refrigerator to block overnight.

5. The blocking solution was discarded, the CDX2 primary antibody was diluted 1:200 with the blocking solution, incubated at room temperature for 2 h, the primary antibody dilution was discarded, and washed three times with PBS-BSA, 5 min each time.

6. Caspase-3 primary antibody (purchased from Cell Signaling Technology) was diluted 1:200 with blocking solution, incubated at room temperature for 2 h, discarded the primary antibody dilution, and washed three times with PBS-BSA for 5 min each time.

7. The CDX2-specific secondary antibody (purchased from Sigma) was diluted 1:200 with blocking solution under dark conditions, and placed in the dark for 1 h at room temperature. The secondary antibody diluent was discarded in the dark and washed three times with PBS for 5 min each time.

8. The caspase-3-specific secondary antibody (purchased from LifeTechnologies) was diluted 1:200 with blocking solution under dark conditions, and placed in the dark for 1 h at room temperature. The secondary antibody diluent was discarded in the dark and washed three times with PBS for 5 min each time.

9. Add 10 μg/mL of Hochest 33342 dye solution to stain the nucleus, let it act for 5 min at room temperature, observe and take pictures under a fluorescence microscope.

10. The experiment was repeated three times, and 10 blastocysts were randomly selected each time, and the apoptotic rate and the number of ICM cells/total cells were calculated to evaluate the blastocyst quality.

Data Statistical Methods: The experimental data were analyzed by the ANOVA program in the statistical software SAS V8. The Duncan's multiple-range test method was used to determine the significance of the difference between treatments. When p<0.05, the difference was considered significant.

In the present invention, the cleavage rate=the number of fertilized cleavage/the number of fertilized eggs. In the present invention, blastocyst rate=number of blastocysts/number of cleavage embryos.

4. In vitro maturation (IVM) effect of oocytes - maturation rate

In step (1) of the present embodiment, after in vitro maturation and culture, observed under an inverted microscope, the oocyte has a first polar body released, a viscous matrix secreted between cumulus cells is maintained, and the cell layer is significantly swelled , the cells are centered on the ovum, and those that spread radially to the surrounding are judged to be mature, the number of mature oocytes is recorded, and the maturation rate is calculated.

5. Results

In this example, the Chinese yellow cattle (Nanyang cattle, a draught breed) were tested. The results showed that the cleavage rate was 87.1%, the morula rate was 65.3%, the blastocyst rate was 49.7%, and the apoptotic rate was 5.6%; in addition, the blastocyst hatching rate reached 73.2% on the ninth day. The maturation rate of oocytes matured in vitro was 88.2%.

Example 2A: Cultivation method of bovine in vitro fertilized embryos (living collection)

With reference to the method of Example 2, tests were carried out for three kinds of cattle of Holstein (dairy cattle variety), Simmental cattle (beef cattle variety), Chinese water buffalo (dramatic variety), and the result: the cleavage rate was all at 86-89% The morula rate was in the range of 63-66%, the blastocyst rate was in the range of 50-54%, the apoptosis rate was in the range of 4-6%, and the hatching rate of the blastocyst on the 9th day was in the range of 70-54%. Within the range of 75%; the in vitro maturation rates of oocytes from three types of cattle, Holstein, Simmental, and Chinese buffalo, were 84.6%, 79.5%, and 76.1%, respectively.

Example 2B: Cultivation method of bovine in vitro fertilized embryos (living collection)

With reference to the method of Example 2, tests were carried out for four kinds of cattle of Chinese yellow cattle (Nanyang cattle, draught type), Holstein cattle (dairy cattle species), Simmental cattle (beef cattle species), Chinese water buffalo (draught cattle species), However, the recipes of fertilization medium and semen preparation medium were modified respectively, and the other test solutions and operations remained unchanged. The fertilization medium formula was changed to: 112.0mM sodium chloride, 4.02mM potassium chloride, 2.25mM calcium chloride dihydrate, 0.52mM magnesium chloride hexahydrate, 0.83mM potassium dihydrogen phosphate, 37.0mM sodium bicarbonate, 1.25 mM sodium pyruvate, 10μg/ml heparin, 4mg/ml bovine serum albumin (BSA), 100U/ml penicillin, 100μg/ml streptomycin, 10umoL/L α-lipoic acid, 25μg/ml folic acid, 2.5mg/ml Aqueous solution of glutamic acid; semen preparation medium formula changed to: containing 112.0 mM sodium chloride, 4.02 mM potassium chloride, 2.25 mM calcium chloride dihydrate, 0.52 mM magnesium chloride hexahydrate, 0.83 mM potassium dihydrogen phosphate, 37.0mM sodium bicarbonate, 1.25mM sodium pyruvate, 10μg/ml heparin, 4mg/ml bovine serum albumin (BSA), 10mM caffeine, 100U/ml penicillin, 100μg/ml streptomycin, 10umoL/L α-sulfur Caprylic acid, 25 μg/ml folic acid, 2.5 mg/ml glutamic acid in water. Results: The cleavage rates of the four types of cattle were in the range of 86-91%, the morula rates were in the range of 65-68%, the blastocyst rates were in the range of 78-81%, and the apoptosis rates were all in the range of 3-6%. The hatching rate of blastocysts on day 9 was in the range of 74-78%. For example, the cleavage rate of Chinese cattle was 89.4%, the morula rate was 66.1%, the blastocyst rate was 79.4%, and the apoptosis rate was 4.5%. %, the blastocyst hatching rate on the 9th day was 77.2%; the in vitro maturation rates of oocytes from Chinese cattle, Holstein cattle, Simmental cattle, and Chinese buffalo were 87.3%, 86.2%, 84.3%, and 75.7%, respectively. %.

Example 2C: Cultivation method of bovine in vitro fertilized embryos (living collection)

Carry out with reference to Example 2B, the only difference is that no folic acid is added in the fertilization medium and the semen preparation medium. Results: The cleavage rates of the four types of cattle were in the range of 85-90%, the morula rates were in the range of 62-66%, the blastocyst rates were in the range of 53-57%, and the apoptotic rates were all in the range of 3-6%. The hatching rate of blastocysts on day 9 was in the range of 72-76%. For example, the cleavage rate of Chinese cattle was 87.6%, the morula rate was 63.7%, the blastocyst rate was 55.8%, and the apoptosis rate was 4.2%. %, the blastocyst hatching rate on the 9th day was 73.5%; the in vitro maturation rates of oocytes from Chinese cattle, Holstein cattle, Simmental cattle, and Chinese buffalo were 84.6%, 82.8%, 83.2%, and 78.3%, respectively. %.

Example 2D: Cultivation method of bovine in vitro fertilized embryos (living collection)

Carry out with reference to Example 2B, the only difference is that glutamic acid is not added in the fertilization medium and the seminal fluid preparation medium. Results: The cleavage rates of the four types of cattle were in the range of 85-90%, the morula rates were in the range of 61-65%, the blastocyst rates were in the range of 53-58%, and the apoptosis rates were all in the range of 3-6%. The hatching rate of blastocysts on day 9 was in the range of 71-76%. For example, the cleavage rate of Chinese cattle was 88.4%, the morula rate was 62.4%, the blastocyst rate was 57.1%, and the apoptosis rate was 4.6%. %, the hatching rate of blastocyst on day 9 was 72.6%; the in vitro maturation rates of oocytes from Chinese cattle, Holstein cattle, Simmental cattle and Chinese buffalo were 84.8%, 84.3%, 85.3% and 77.2%, respectively. %.

Example 2E: Cultivation method of bovine in vitro fertilized embryos (living collection)

Carry out with reference to Example 2B, the only difference is that neither folic acid nor glutamic acid is added to the fertilization medium and the semen preparation medium. Results: The cleavage rates of the four types of cattle were in the range of 86-91%, the morula rates were in the range of 60-65%, the blastocyst rates were in the range of 52-55%, and the apoptosis rates were all in the range of 4-6%. The hatching rate of blastocysts on day 9 was in the range of 72-76%. For example, the cleavage rate of Chinese cattle was 88.3%, the morula rate was 62.7%, the blastocyst rate was 53.1%, and the apoptosis rate was 5.1%. %, the blastocyst hatching rate on the 9th day was 74.3%; the in vitro maturation rates of oocytes from Chinese cattle, Holstein cattle, Simmental cattle and Chinese buffalo were 84.3%, 86.5%, 83.7% and 76.2%, respectively. %.

It has been reported in the literature that oxidative stress caused by reactive oxygen species (ROS) is one of the main causes of apoptosis or permanent developmental arrest in animal germ cells and early embryos. One of the ways to damage cells and improve the ability of early embryonic development. As an antioxidant, alpha-lipoic acid is believed to exert antioxidant effects by directly scavenging free radicals, chelating metal ions, and promoting the formation of other antioxidants. In germ cells and early embryos, lipoic acid can improve germ cell quality and early embryonic development by reducing ROS levels, promoting antioxidant enzyme activity and mitochondrial activity. The present inventors attempted to add α-lipoic acid to the fertilization medium and the semen preparation medium in order to increase the efficiency of bovine in vitro fertilization. However, as shown in the above test results, in the fertilization medium and In the semen preparation medium, the blastocyst rate can be significantly improved only when both folic acid and glutamate are supplemented at the same time, which further indicates that the use of the above fertilization medium and semen preparation medium to pre-treat frozen sperm can improve the rate of sperm in the follow-up. efficiency during in vitro fertilization.

Although the present invention has been described in detail above with general description and specific embodiments, it is obvious to those skilled in the art that some modifications or improvements can be made on the basis of the present invention. Therefore, these modifications or improvements made without departing from the spirit of the present invention fall within the scope of the claimed protection of the present invention.

Claims (9)

1. A method for culturing bovine in vitro fertilization embryos, comprising the steps of: (1) Collection and in vitro maturation of oocytes a) Live collection: Take follicular fluid from bovine live egg collection, pick out cumulus-oocyte complexes (COCs) containing at least 3 layers of cumulus cells under a stereo microscope, and put them in a transport medium containing HEPES 38.8℃, no carbon dioxide supply, transported back to the laboratory within 24h; b) The COCs collected from the above living body were washed once in the oocyte maturation medium, and then transferred to a new mature medium for 22-24 hours, and the culture conditions were 38.8°C, 5.5-6.5% CO2, and saturated humidity; (2) In vitro fertilization The mature COCs were washed once in the fertilization medium, then transferred to the fertilization medium, and placed in an incubator for use; Take a frozen fine tube from liquid nitrogen and thaw it in a water bath at 37°C; cut both ends of the fine tube aseptically, pour the semen into a 15 mL centrifuge tube containing the culture medium for semen preparation, and centrifuge at 328 × g twice, each time After centrifugation for 5 min, the supernatant was discarded; 300 µL of semen preparation medium was added to the above centrifuge tube, the sperm pellet was resuspended, and an appropriate sperm suspension was taken for sperm count; Add the calculated volume of sperm suspension to the fertilization culture droplet containing the oocytes, and put the culture plate into the incubator to incubate the sperm and eggs for 16-20 hours. The culture conditions are 38.8°C, 5.5-6.5% CO2 , saturated humidity; (3) Embryos are cultured and preserved in vitro After the in vitro fertilization operation is completed, the granulosa cells around the embryos are removed with an egg stripper and cultured in the embryo culture medium. This time is recorded as the first day of embryo culture. N2, saturated humidity, record the cleavage rate on the 3rd day; record the blastocyst rate on the 7th day, count the blastocyst hatching rate on the 9th day, and conduct quality identification; Wash the available embryos in the preservation solution for 3 times, equilibrate in the equilibration solution for 10 minutes, and transfer them into the freezing solution. Load the embryos according to the 5-stage filling method, make a mark, and then put them into the programmed cooling device at a temperature of 0.5 °C/min. After the speed dropped to -35°C, the embryo tube was quickly taken out and placed in liquid nitrogen for cryopreservation; in: The fertilization medium was 112.0 mM sodium chloride, 4.02 mM potassium chloride, 2.25 mM calcium chloride dihydrate, 0.52 mM magnesium chloride hexahydrate, 0.83 mM potassium dihydrogen phosphate, 37.0 mM sodium bicarbonate, 1.25 mM acetone Sodium, 10μg/ml heparin, 4mg/ml bovine serum albumin, 100U/ml penicillin, 100μg/ml streptomycin, 10umoL/L α-lipoic acid, 25μg/ml folic acid, 2.5mg/ml glutamic acid in water , The semen preparation medium was 112.0 mM sodium chloride, 4.02 mM potassium chloride, 2.25 mM calcium chloride dihydrate, 0.52 mM magnesium chloride hexahydrate, 0.83 mM potassium dihydrogen phosphate, 37.0 mM sodium bicarbonate, 1.25 mM acetone Sodium, 10μg/ml heparin, 4mg/ml bovine serum albumin, 10mM caffeine, 100U/ml penicillin, 100μg/ml streptomycin, 10umoL/L α-lipoic acid, 25μg/ml folic acid, 2.5mg/ml glutathione Aqueous acid solution, The mature culture medium is a BY basal medium added with 100 mL/L fetal bovine serum, 10 μg/mL follicle stimulating hormone, 10 μg/mL luteinizing hormone, 1 μg/mL estradiol and 20 ng/mL epidermal growth factor, The BY basal culture solution is an aqueous solution comprising the following components: calcium chloride 180-220 mg/L, ferric nitrate nonahydrate 0.70-0.75 mg/L, potassium chloride 380-420 mg/L, magnesium sulfate 90-100 mg/L , sodium chloride 6500~7000mg/L, sodium dihydrogen phosphate monohydrate 130~150mg/L, sodium bicarbonate 2000~2500mg/L, sodium acetate 40~60mg/L, L-alanine 20~30mg/L, L-arginine hydrochloride 60~80mg/L, L-aspartic acid 25~35mg/L, L-cysteine hydrochloride monohydrate 0.10~0.12mg/L, L-cystine Dihydrochloride 20~30mg/L, L-glutamic acid 70~80mg/L, Glycine 40~60mg/L, L-Histidine hydrochloride monohydrate 20~25mg/L, L-Hydroxyproline Acid 8~12mg/L, L-isoleucine 15~25mg/L, L-leucine 50~70mg/L, L-lysine hydrochloride 60~80mg/L, L-methionine 10~20mg /L, L-phenylalanine 20~30mg/L, L-proline 30~50mg/L, L-serine 20~30mg/L, L-threonine 25~35mg/L, L-tryptophan Acid 8~12mg/L, L-Tyrosine Disodium Dihydrate 55~60mg/L, L-Valine 20~30mg/L, Ascorbic Acid 0.04~0.06mg/L, α-D-Tocopheryl Phosphate 0.008~0.012mg/L, biotin 0.008~0.012mg/L, calcidol 0.08~0.12mg/L, D-calcium pantothenate 0.008~0.012mg/L, choline chloride 0.4~0.6mg/L, folic acid 0.008 ~0.012mg/L, Inositol 0.04~0.06mg/L, Menadione trihydrate sodium bisulfite 0.015~0.025mg/L, Niacin 0.02~0.03mg/L, Niacinamide 0.02~0.03mg/L, p -Aminobenzoic acid 0.04~0.06mg/L, pyridoxine hydrochloride 0.04~0.06mg/L, riboflavin 0.008~0.012mg/L, thiamine hydrochloride 0.008~0.012mg/L, vitamin A acetate 0.1~ 0.2mg/L, Adenine Sulfate 8~12mg/L, Adenine 0.15~0.25mg/L, Disodium Adenosine Triphosphate 0.8~1.2mg/L, Cholesterol 0.15~0.25mg/L, 2-Deoxy-D- Ribose 0.4~0.6mg/L, D-glucose 800~1200mg/L, Glutathione 0.04~0.06mg/L, Guanine hydrochloride 0.25~0.35mg/L, Sodium hypoxanthine 0.3~0.4mg/L, Ribose 0.4~0.6mg/L, thymosin 0.25~0.35mg/L, Tween 804~6mg/L, urine Pyrimidine 0.25~0.35mg/L, sodium xanthine 0.3~0.4mg/L, phenol red 8~12mg/L. 2. according to the method of claim 1, in (a) of step (1), comprise in the described transport culture liquid: glycine 50.0mg/L, L-alanine 25.0mg/L, L-arginine hydrochloride Salt 70.0mg/L, L-aspartic acid 30.0mg/L, L-cystine dihydrochloride 26.0mg/L, L-glutamic acid 75.0mg/L, L-glutamine 100.0mg/L , L-histidine hydrochloride monohydrate 21.88mg/L, L-hydroxyproline 10.0mg/L, L-isoleucine 40.0mg/L, L-leucine 60.0mg/L, L -Lysine hydrochloride 70.0mg/L, L-methionine 15.0mg/L, L-phenylalanine 25.0mg/L, L-proline 40.0mg/L, L-serine 25.0mg/ L, L-threonine 30.0mg/L, L-tryptophan 10.0mg/L, L-tyrosine disodium salt dihydrate 58.0mg/L, L-valine 25.0mg/L, ascorbic acid 0.05 mg/L, Biotin 0.01mg/L, Choline Chloride 0.5mg/L, D-Calcium Pantothenate 0.01mg/L, Folic Acid 0.01mg/L, Menaquinone 0.01mg/L, Niacinamide 0.025mg/L, Niacin 0.025mg/L, p-aminobenzoic acid 0.05mg/L, pyridoxal hydrochloride 0.025mg/L, pyridoxine hydrochloride 0.025mg/L, riboflavin 0.01mg/L, thiamine hydrochloride 0.01mg/L , Vitamin A acetate 0.1mg/L, Vitamin D2 (calcidol 0.1mg/L, α-tocopherol sodium phosphate 0.01mg/L, Inositol 0.05mg/L, Anhydrous calcium chloride 200.0mg/L, Ferric nitrate nonahydrate 0.7mg/L, anhydrous magnesium sulfate 97.67mg/L, potassium chloride 400.0mg/L, sodium chloride 6800.0mg/L, sodium dihydrogen phosphate monohydrate 140.0mg/L, adenine sulfate 10.0mg/L, 5'-adenosine phosphate 0.2mg/L, adenosine triphosphate 1.0mg/L, cholesterol 0.2mg/L, glucose 1000.0mg/L, deoxyribose 0.5mg/L, reduced glutathione 0.05mg/ L, Guanine Hydrochloride 0.3mg/L, Sodium Hypoxanthine 0.354mg/L, Phenol Red 20.0mg/L, Ribose 0.5mg/L, Sodium Acetate 50.0mg/L, Thymine 0.3mg/L, Tween 80 20.0mg/L, uracil 0.3mg/L, sodium xanthine 0.3mg/L, and 0.01IU/mL FSH, 0.01IU/mL LH, 1μg/mL _E2 , 50ng/mL EGF, 100ng/mL mL of IGF, 10% gentamicin, 55 µg/mL sodium pyruvate, 1.2 mM/L cysteine, 3 mg/mL BSA, 10 mM/L HEPES. 3. according to the method of claim 2, in step (1), also comprise the taurine of 30～50mg/L and the zinc gluconate of 1～3mg/L in the described transport culture liquid. 4. The method according to claim 1, in step (3), the embryo culture fluid comprises: 109.5mM sodium chloride, 3.1mM potassium chloride, 26.2mM sodium bicarbonate, 0.8mM magnesium chloride hexahydrate, 1.19mM diphosphate Potassium hydrogen, 0.4mM sodium pyruvate, 1.5mM glucose, 5mM calcium galactonate, 2.5v/v% fetal bovine serum, L-glutamine 1mM, 2v/v% essential amino acids, 1v/v% non-essential amino acids, The aqueous solution of 3mM glutathione, sodium citrate 0.04w/v%, maltose 0.02w/v%; the essential amino acids are the following amino acids added by weight: L-arginine hydrochloride 6.32g, L-cyst Amino acid dihydrochloride 1.564g, L-histidine hydrochloride monohydrate 2.1g, L-isoleucine 2.625g, L-leucine 2.62g, L-lysine hydrochloride 3.625g, L -Methionine 0.755g, L-phenylalanine 1.65g, L-threonine 2.38g, L-tryptophan 0.51g, L-tyrosine 1.8g and L-valine 2.34g, said optional Amino acids are the following amino acids added by weight: L-alanine 0.89g, L-asparagine monohydrate 1.5g, L-aspartic acid 1.33g, L-glutamic acid 1.47g, glycine 0.75g, L -Proline 1.15g and L-serine 1.05g. 5. method according to claim 1, described BY basal culture liquid is the aqueous solution that comprises following components: calcium chloride 200mg/L, ferric nitrate nonahydrate 0.72mg/L, potassium chloride 400mg/L, magnesium sulfate 97.7mg /L, sodium chloride 6800mg/L, sodium dihydrogen phosphate monohydrate 140mg/L, sodium bicarbonate 2200mg/L, sodium acetate 50mg/L, L-alanine 25mg/L, L-arginine hydrochloride 70mg/L, L-aspartic acid 30mg/L, L-cysteine hydrochloride monohydrate 0.11mg/L, L-cystine dihydrochloride 26mg/L, L-glutamic acid 75mg /L, Glycine 50mg/L, L-Histidine Hydrochloride Monohydrate 21.88mg/L, L-Hydroxyproline 10mg/L, L-Isoleucine 20mg/L, L-Leucine 60mg /L, L-lysine hydrochloride 70mg/L, L-methionine 15mg/L, L-phenylalanine 25mg/L, L-proline 40mg/L, L-serine 25mg/L, L- Threonine 30mg/L, L-tryptophan 10mg/L, L-tyrosine disodium dihydrate 57.66mg/L, L-valine 25mg/L, ascorbic acid 0.05mg/L, α-D- Tocopheryl Phosphate 0.01mg/L, Biotin 0.01mg/L, Calciitol 0.1mg/L, D-Calcium Pantothenate 0.01mg/L, Choline Chloride 0.5mg/L, Folic Acid 0.01mg/L, Inositol 0.05mg/L, menadione trihydrate sodium bisulfite 0.019mg/L, niacin 0.025mg/L, niacinamide 0.025mg/L, p-aminobenzoic acid 0.05mg/L, pyridoxine hydrochloride 0.05mg/ L, riboflavin 0.01mg/L, thiamine hydrochloride 0.01mg/L, vitamin A acetate 0.14mg/L, adenine sulfate 10mg/L, adenine 0.2mg/L, adenosine triphosphate disodium 1mg /L, cholesterol 0.2mg/L, 2-deoxy-D-ribose 0.5mg/L, D-glucose 1000mg/L, glutathione 0.05mg/L, guanine hydrochloride 0.3mg/L, hypoxanthine sodium 0.354 mg/L, Ribose 0.5mg/L, Thymosin 0.3mg/L, Tween 805mg/L, Uracil 0.3mg/L, Xanthine Sodium 0.34mg/L, Phenol Red 10mg/L, Sodium Selenite 0.2~ 0.3mg/L, the concentration of copper sulfate is 0.05~0.1mg/L calculated as anhydrous. 6. A method for bovine in vitro fertilization, comprising the steps of: The mature cumulus-oocyte complex was washed once in the fertilization medium, then transferred to the fertilization medium, and placed in an incubator for use; Take a frozen fine tube from liquid nitrogen and thaw it in a water bath at 37°C; cut both ends of the fine tube aseptically, pour the semen into a 15 mL centrifuge tube containing the culture medium for semen preparation, and centrifuge at 328 × g twice, each time After centrifugation for 5 min, the supernatant was discarded; 300 µL of semen preparation medium was added to the above centrifuge tube, the sperm pellet was resuspended, and an appropriate sperm suspension was taken for sperm count; Add the calculated volume of sperm suspension to the fertilization culture droplet containing the oocytes, and put the culture plate into the incubator to incubate the sperm and eggs for 16-20 hours. The culture conditions are 38.8°C, 5.5-6.5% CO2 , saturated humidity, complete in vitro fertilization operation, ready for embryo culture in vitro, in, The fertilization medium was 112.0 mM sodium chloride, 4.02 mM potassium chloride, 2.25 mM calcium chloride dihydrate, 0.52 mM magnesium chloride hexahydrate, 0.83 mM potassium dihydrogen phosphate, 37.0 mM sodium bicarbonate, 1.25 mM acetone Sodium, 10μg/ml heparin, 4mg/ml bovine serum albumin, 100U/ml penicillin, 100μg/ml streptomycin, 10umoL/L α-lipoic acid, 25μg/ml folic acid, 2.5mg/ml glutamic acid in water , The semen preparation medium was 112.0 mM sodium chloride, 4.02 mM potassium chloride, 2.25 mM calcium chloride dihydrate, 0.52 mM magnesium chloride hexahydrate, 0.83 mM potassium dihydrogen phosphate, 37.0 mM sodium bicarbonate, 1.25 mM acetone Sodium, 10μg/ml heparin, 4mg/ml bovine serum albumin, 10mM caffeine, 100U/ml penicillin, 100μg/ml streptomycin, 10umoL/L α-lipoic acid, 25μg/ml folic acid, 2.5mg/ml glutathione Aqueous acid solution. 7. The method according to claim 6, wherein the cumulus-oocyte complex is harvested and matured in vitro by: a) Live collection: Take follicular fluid from bovine live egg collection, pick out cumulus-oocyte complexes (COCs) containing at least 3 layers of cumulus cells under a stereo microscope, and put them in a transport medium containing HEPES 38.8℃, no carbon dioxide supply, transported back to the laboratory within 24h; b) Wash the COCs obtained from the above living body in the oocyte maturation medium once, and then transfer it to a new mature medium for 22-24 hours under the conditions of 38.8°C, 5.5-6.5% CO2, and saturated humidity to obtain Mature cumulus-oocyte complex. 8. Fertilization broth for bovine in vitro fertilization, which is 112.0 mM sodium chloride, 4.02 mM potassium chloride, 2.25 mM calcium chloride dihydrate, 0.52 mM magnesium chloride hexahydrate, 0.83 mM potassium dihydrogen phosphate, 37.0 mM Sodium bicarbonate, 1.25mM sodium pyruvate, 10μg/ml heparin, 4mg/ml bovine serum albumin, 100U/ml penicillin, 100μg/ml streptomycin, 10umoL/L α-lipoic acid, 25μg/ml folic acid, 2.5mg /ml of glutamic acid in water. 9. Semen preparation broth for bovine in vitro fertilization, which is 112.0 mM sodium chloride, 4.02 mM potassium chloride, 2.25 mM calcium chloride dihydrate, 0.52 mM magnesium chloride hexahydrate, 0.83 mM potassium dihydrogen phosphate, 37.0 mM sodium bicarbonate, 1.25mM sodium pyruvate, 10μg/ml heparin, 4mg/ml bovine serum albumin, 10mM caffeine, 100U/ml penicillin, 100μg/ml streptomycin, 10umoL/L α-lipoic acid, 25μg/ml ml folic acid, 2.5 mg/ml glutamic acid in water.