CN108728401A - The method of IVF of Oocyte in Bovine Embryo Culture and culture medium used - Google Patents

Abstract

The invention relates to a method for culturing bovine oocyte fertilized embryos in vitro and a used culture medium. This basal medium is an aqueous solution containing sodium chloride, sodium dihydrogenphosphate, sodium bicarbonate, sodium acetate, and the like. It also relates to an egg washing liquid and a maturation culture liquid. Wherein the method for culturing bovine in vitro fertilized embryos comprises the following steps: collection and in vitro maturation of oocytes; in vitro fertilization; in vitro culture and preservation of embryos. The method for culturing bovine IVF embryos of the present invention has excellent technical effects as described in the description.

Description

Method for culturing bovine oocyte fertilized embryo in vitro and medium used

technical field

The invention belongs to the technical field of animal reproduction, and relates to a technology for agricultural-animal husbandry and veterinary reproduction, in particular to a method for culturing bovine in vitro fertilized embryos. In addition, the present invention also relates to the use of related culture fluids for the cultivation of bovine in vitro fertilized embryos. Application in in vitro fertilization embryo culture. Further, the present invention also relates to a method for culturing bovine IVF embryos using the relevant culture fluid for culturing bovine IVF embryos. In particular, the method for culturing bovine IVF embryos of the present invention has excellent technical effects. In particular, the present invention relates to a method for in vitro fertilization and embryo culture of live or isolated bovine oocytes.

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. It is 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 by transplanting in vitro fertilized embryos into mothers are called test-tube animals. This technology was successfully developed in the 1950s, and has developed rapidly in the past 20 years. Now it has matured and become an important and conventional animal reproduction biotechnology.

In vitro fertilization technology is of great significance to animal reproductive mechanism research, animal husbandry production, medicine and endangered animal protection. If mice, rats or rabbits are used 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 cheap and efficient means for embryo production, and is of great value in making full use of fine breed resources, shortening the breeding cycle of livestock, and speeding up the speed of breed improvement. In humans, IVF-ET technology is one of the important measures to treat some infertility and overcome sex-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 fine-bred cows and accelerate the process of genetic breeding, it is inevitable to apply efficient new breeding technologies in production practice. Ovum pick UP (OPU) and in vitro fertilization (In Vitro Fertilization, IVF) are new technologies for embryo engineering that developed rapidly in the 1980s. The combination of the two can obtain a large number of embryos with definite genetic pedigrees. thereby shortening the generation interval. At present, these two technologies have become important breeding technologies adopted by farmers in developed animal husbandry countries such as Europe, America and Oceania to expand the herd of fine-bred cows. However, using the conventional bovine embryo culture system (CR1aa and SOF solution), the development rate of bovine in vitro fertilized blastocysts is low, and the quality of embryos is far inferior to that of in vivo embryos, resulting in low pregnancy rates after embryo transfer to recipients, so how to improve Blastocyst development rate and embryo quality have become the focus and research focus of in vitro fertilization embryo production.

As early as 1878, the German Scnenk began to explore the in vitro fertilization technology of mammals using rabbits and guinea pigs as materials. However, it was not until 1951, when Chinese-Americans Zhang Minjue and Austin discovered the phenomenon of sperm capacitation separately, that in vitro fertilization technology made a breakthrough. The bovine in vitro fertilization technique is affected by many aspects such as in vitro maturation of oocytes, in vitro capacitation of sperm, and in vitro culture environment of fertilized eggs.

The in vitro culture of embryos is a key link in 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. After in vitro fertilization, the fertilized egg will undergo a series of important changes during its development to the blastocyst, including the formation of the zygote, the first cleavage, activation of the embryonic genome, compaction, and formation of the blastocyst. During this process, changes in the external environment will lead to changes in gene expression, thereby affecting the normal development and quality of embryos. At present, research on in vitro culture of mammalian early embryos is mainly focused on improving the composition of the culture medium to meet the nutritional needs of embryos at different developmental stages. Based on the Charles Rosenkrans 1 developed by Rosenkrans et al. (CR1) culture fluid and the synthetic oviduct fluid developed by Tervit et al. Synthetic Oviductal Fluid, SOF), two culture systems have been gradually formed after years of continuous improvement. According to Hakan Sagirkaya et al. (Sagirkaya, H., et al., Developmental potential of bovineocytes cultured in different maturation and culture conditions. Anim ReprodSci, 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 Vet Hung, 2010.58 (4): p.465-74) research results show that CR1aa culture medium is used for bovine embryo culture Good effect, can be widely used in bovine embryo culture; Thompson, J.G. et al. ): p.47-55) and Jean M.Feugang et al. ) showed that SOF medium is also a suitable culture system for bovine embryo culture. Zhang Zhiping (Zhang Zhiping, An Zhixing, Zhang Ru, Zhang Yong, Optimization of Bovine Embryo Culture System. Journal of Northwest A&F University, 2006.34) and Sang Guojun et al. (Sang Guojun, Research on bovine oocyte and in vitro embryo culture technology. 2008) research results also show that the optimized CR1aa and SOF culture medium are suitable for bovine embryo culture in vitro, and both have achieved good culture effects. Early mammalian embryonic development is a highly coordinated and precisely regulated process. During evolution, gametocytes gradually formed a series of molecular cascade networks to ensure that the embryonic development cycle proceeded systematically. During development, the balance of reactive oxygen species (Reactive Oxygen Species, ROS) and antioxidants in the embryo plays a decisive role in early embryonic development.

Most biochemical reactions produce ROS, which play an important role inside and outside the cell. 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 can even lead to Inactivation of certain enzymes. Generally speaking, ROS exists in four forms, among which H2O2 has a strong oxidation effect and is the most important factor causing oxidative damage.

A large number of studies have shown that glutathione (GSH) is an antioxidant that exists in a non-protein form and can scavenge a variety of free radicals: superoxide anion radicals, hydroxyl radicals, hydrogen peroxide, hypochlorous acid and lipid oxygen Free radicals, and can maintain intracellular and intracellular redox balance. The levels of GSH and ROS in the internal and external environment of cells 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 on embryodevelopment effect of beta-mercaptoethanol, cysteine and cystine. Theriogenology, 2000.53(3): p .761-71) have 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 cost and low efficiency of in vitro fertilized embryo production due to the low blastocyst rate of in vitro fertilization limit the wide application of this technology in the practice of rapid multiplication 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 solution are mainly used as embryo in vitro culture medium, and on this basis, the development rate of blastocysts has been improved to varying degrees, and the average rate of blastocyst development is 30%-40 %. The quality of blastocysts can be evaluated by the total number of blastocyst cells, the ratio of the number of ICM cells to the total number of cells, and the rate of apoptosis. The total number of blastocyst cells varies according to the stage of the blastocyst, S.Iwasaki et al. .Theriogenology, 1990.33 (3): p.669-75) the average number of bovine early blastocyst total cells obtained is 44, and the ICM cell number/blastocyst total cell number ratio is about 15.8%; Andrew J.Watson etc. (Watson, A.J., et al., Impact of bovine oocyte maturation media on oocyte transcript levels, blastocyst development, cell number, and apoptosis. Biol Reprod, 2000.62(2): p.355-64) statistics bovine blastocyst cell apoptosis rate is about 7.7%-13%.

CN103898046B (Chinese Patent Application No. 201410073635.4) discloses a culture solution specially used for bovine IVF embryos. The formula of the culture solution 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 content of each amino acid 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, 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 the aqueous solution prepared after mixing the following amino acids in proportion, wherein, each 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 L. Bovine in vitro fertilized embryos are placed in the above culture medium for in vitro culture. It is believed that the results are 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 a bovine IVF technology. The application in practice provides an experimental basis, which can greatly accelerate the process of genetic breeding.

Other references:

Chen Dayuan. Fertilization Biology. 2003, Beijing: Science Press;

Fang Junshun. Research on In Vitro Production Technology of Bovine Embryos. "China Excellent Master's Dissertations Full-text Database. Agricultural Science and Technology Series". 2007, (No. 4);

Cao Haiqing. The effect of cumulus cells and culture conditions on the production efficiency of bovine in vitro embryos. "China Excellent Master's Dissertation Full-text Database. Agricultural Science and Technology Series". 2008, (No. 9);

I.H.KIM et al.Effect of exogenous glutathione on the in vitrofertilization of bovine oocytes.Theriogenology.1999,Vol.52(3).

However, the art still expects a method for culturing bovine IVF embryos with improved performance, such as a method for improving the efficiency of bovine IVF embryo cultivation, for example, a method for cultivating bovine IVF embryos with excellent cost advantages.

Contents of the invention

The purpose of the present invention is to provide a method for cultivating bovine IVF embryos with improved performance, especially expecting to improve the production efficiency and embryo quality of bovine IVF embryos. More specifically, in order to achieve the above-mentioned purpose, the present invention needs to provide a related culture fluid specially used for bovine IVF embryos and a method for culturing bovine IVF embryos using the culture fluid. The present inventors have unexpectedly found that the use of the method of the present invention exhibits excellent technical effects, and the present invention has been accomplished by this discovery.

For this reason, the first aspect of the present invention provides a kind of basal culture solution that can be called BY basal culture solution, which is an aqueous solution comprising the following components: calcium chloride 180-220 mg/L, iron 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 monohydrate 0.10～0.12mg/L, L-cystine dihydrochloride 20～30mg/L, L-glutamic acid 70～80mg/L, glycine 40～60mg/L, L-group Amino acid hydrochloride 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 sodium bisulfite trihydrate 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, 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 0.25～0.35mg/L, sodium hypoxanthine 0.3～0.4mg/L, ribose 0.4～0.6mg/L, thymosin 0.2 5~0.35mg/L, Tween 804~6mg/L, uracil 0.25~0.35mg/L, xanthine sodium 0.3~0.4mg/L, phenol red 8~12mg/L.

The basal culture solution according to the first aspect of the present invention 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 Esters 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 L, menaquinone 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, nuclear Flavin 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 basal culture solution of the first aspect of the present invention, it also includes sodium selenite at a concentration of 0.2-0.3 mg/L, such as 0.25 mg/L; and/or, it also includes copper sulfate at a concentration of The anhydrous content is 0.05-0.1 mg/L, such as 0.075 mg/L.

Furthermore, the second aspect of the present invention provides an egg washing liquid, which is BY basic culture liquid supplemented with 3 mg/mL bovine serum albumin.

According to the egg washing solution of the second aspect of the present invention, wherein the BY basic culture solution is an aqueous solution comprising the following components: 180-220 mg/L of calcium chloride, 0.70-0.75 mg/L of ferric nitrate nonahydrate, and 380 mg/L of potassium chloride ～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 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 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-threo Amino acid 25～35mg/L, L-tryptophan 8～12mg/L, L-tyrosine disodium dihydrate 55～60mg/L, L-valine 20～30mg/L, ascorbic acid 0.04～0.06 mg/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, chlorine Choline 0.4～0.6mg/L, folic acid 0.008～0.012mg/L, inositol 0.04～0.06mg/L, menaquinone sodium bisulfite trihydrate 0.015～0.025mg/L, niacin 0.02～0.03mg/L 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, 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 0.25～0.35mg/L, Hypoxanthine Sodium 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, xanthine sodium 0.3~0.4mg/L, phenol red 8~12mg/L.

According to the egg washing solution of the second aspect of the present invention, wherein the BY basic culture solution is an aqueous solution comprising the following components: calcium chloride 200mg/L, ferric nitrate nonahydrate 0.72mg/L, potassium chloride 400mg/L, sulfuric acid Magnesium 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-glutamine Glycine 75mg/L, Glycine 50mg/L, L-Histidine Hydrochloride Monohydrate 21.88mg/L, L-Hydroxyproline 10mg/L, L-Isoleucine 20mg/L, L-Leucine Amino acid 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, Menaquinone Sodium Bisulfite Trihydrate 0.019mg/L, Nicotinic Acid 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, Hypoxanthin Purine 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 egg washing solution according to the second aspect of the present invention, wherein the BY base culture solution also includes sodium selenite, the concentration of which is 0.2-0.3 mg/L, such as 0.25 mg/L; and/or, the BY base The culture solution also includes copper sulfate, the concentration of which is 0.05-0.1 mg/L in terms of anhydrous matter, such as 0.075 mg/L.

Further, the third aspect of the present invention provides a mature culture medium, which is BY basal culture medium supplemented with 100mL/L FBS, 10μg/mL FSH, 10μg/mL LH, 1μg/mL E2, and 20ng/mL EGF.

According to the mature culture solution of the third aspect of the present invention, wherein the BY basic culture solution is an aqueous solution comprising the following components: 180-220 mg/L of calcium chloride, 0.70-0.75 mg/L of ferric nitrate nonahydrate, and 380 mg/L of potassium chloride ～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 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 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-threo Amino acid 25～35mg/L, L-tryptophan 8～12mg/L, L-tyrosine disodium dihydrate 55～60mg/L, L-valine 20～30mg/L, ascorbic acid 0.04～0.06 mg/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, chlorine Choline 0.4～0.6mg/L, folic acid 0.008～0.012mg/L, inositol 0.04～0.06mg/L, menaquinone sodium bisulfite trihydrate 0.015～0.025mg/L, niacin 0.02～0.03mg/L 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, 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 0.25～0.35mg/L, Hypoxanthine Sodium 0.3～0.4mg/L, Ribose 0.4～0.6mg/L, Thymosin 0.25～0.35 mg/L, Tween 804~6mg/L, uracil 0.25~0.35mg/L, xanthine sodium 0.3~0.4mg/L, phenol red 8~12mg/L.

According to the mature culture solution of the third aspect of the present invention, wherein the BY basic culture solution is an aqueous solution comprising the following components: calcium chloride 200mg/L, ferric nitrate nonahydrate 0.72mg/L, potassium chloride 400mg/L, sulfuric acid Magnesium 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-glutamine Glycine 75mg/L, Glycine 50mg/L, L-Histidine Hydrochloride Monohydrate 21.88mg/L, L-Hydroxyproline 10mg/L, L-Isoleucine 20mg/L, L-Leucine Amino acid 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, Menaquinone Sodium Bisulfite Trihydrate 0.019mg/L, Nicotinic Acid 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, Hypoxanthin Purine 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 mature culture solution of the third aspect of the present invention, wherein the BY base culture solution also includes sodium selenite at a concentration of 0.2-0.3 mg/L, such as 0.25 mg/L; and/or, the BY base The culture solution also includes copper sulfate, the concentration of which is 0.05-0.1 mg/L in terms of anhydrous matter, such as 0.075 mg/L.

Further, the fourth aspect of the present invention provides a mature culture medium, which is added with 10-20mmol/L (for example, 15mmol/L) 4-hydroxyethylpiperazineethanesulfonic acid, 100mL/L FBS, 10μg/mL BY basal culture medium of FSH, 10 μg/mL LH, 1 μg/mL E2, 20 ng/mL EGF.

According to the mature culture solution of the fourth aspect of the present invention, wherein the BY basic culture solution is an aqueous solution comprising the following components: 180-220 mg/L of calcium chloride, 0.70-0.75 mg/L of ferric nitrate nonahydrate, and 380 mg/L of potassium chloride ～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 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 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-threo Amino acid 25～35mg/L, L-tryptophan 8～12mg/L, L-tyrosine disodium dihydrate 55～60mg/L, L-valine 20～30mg/L, ascorbic acid 0.04～0.06 mg/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, chlorine Choline 0.4～0.6mg/L, folic acid 0.008～0.012mg/L, inositol 0.04～0.06mg/L, menaquinone sodium bisulfite trihydrate 0.015～0.025mg/L, niacin 0.02～0.03mg/L 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, 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 0.25～0.35mg/L, Hypoxanthine Sodium 0.3～0.4mg/L, Ribose 0.4～0.6mg/L, Thymosin 0.25～0.35 mg/L, Tween 804~6mg/L, uracil 0.25~0.35mg/L, xanthine sodium 0.3~0.4mg/L, phenol red 8~12mg/L.

According to the mature culture solution of the fourth aspect of the present invention, wherein the BY basic culture solution is an aqueous solution comprising the following components: calcium chloride 200mg/L, ferric nitrate nonahydrate 0.72mg/L, potassium chloride 400mg/L, sulfuric acid Magnesium 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-glutamine Glycine 75mg/L, Glycine 50mg/L, L-Histidine Hydrochloride Monohydrate 21.88mg/L, L-Hydroxyproline 10mg/L, L-Isoleucine 20mg/L, L-Leucine Amino acid 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, Menaquinone Sodium Bisulfite Trihydrate 0.019mg/L, Nicotinic Acid 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, Hypoxanthin Purine 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 mature culture solution of the fourth aspect of the present invention, wherein the BY basal culture solution further includes sodium selenite at a concentration of 0.2-0.3 mg/L, such as 0.25 mg/L; and/or, the BY basal The culture solution also includes copper sulfate, the concentration of which is 0.05-0.1 mg/L in terms of anhydrous matter, such as 0.075 mg/L.

Further, the fifth aspect of the present invention provides a method for culturing bovine in vitro fertilized embryos, which includes the following steps:

(1) Oocyte collection and in vitro maturation

In vitro collection: Take the ovary from the slaughterhouse and put it in an insulated barrel with double-antibiotic saline, and transport it back to the laboratory within 4 hours under the condition of 30.5-33°C; extract the follicles with a surface size of 2-8mm, collect the precipitate, and examine it under a stereo microscope Next, pick out oocyte COCs (that is, cumulus-oocyte complexes) that contain at least three layers of cumulus cells, and wash them twice in egg washing liquid to remove excess impurities;

Live collection: live bovine eggs are collected, and the resulting follicular fluid is picked out under a stereomicroscope to pick out cumulus-oocyte complexes (COCs) that contain at least 3 layers of cumulus cells, and put them into the mature culture medium containing HEPES Temperature 38.8 ℃, transported back to the laboratory within 3 hours;

Wash the COCs collected above in vitro or in vivo in the oocyte maturation culture medium once, and then transfer to a new maturation culture medium for 22-24 hours. The culture conditions are 38.8°C, 5.5-6.5% CO2, and saturated humidity. ;

(2) In vitro fertilization

Wash the mature COCs once in the fertilization medium, then transfer them to the fertilization medium, put them in the incubator, and set aside;

Take a frozen fine tube from liquid nitrogen and thaw it in a water bath at 37°C; cut both ends of the thin tube aseptically, inject the semen into a 15mL centrifuge tube filled with the semen preparation culture medium, and centrifuge twice at 328×g, each time After centrifuging for 5 minutes, discard the supernatant; add 300 μL of semen preparation culture medium to the above centrifuge tube, resuspend the sperm pellet, and take an appropriate sperm suspension for sperm counting;

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

(3) In vitro culture and storage of embryos

After the in vitro fertilization operation, remove the granulosa cells around the embryo with the egg stripping needle, and put them into the embryo culture medium for culture. At this time, it is recorded as the first day of embryo culture. The culture conditions are 38.8°C, 6% O2, 88% N2, saturated humidity, record the cleavage rate on the 3rd day; record the blastocyst rate on the 7th day, and count the blastocyst hatching rate (it 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 usable embryos in the preservation solution for 3 times, equilibrate in the balance solution for 10 minutes, transfer to the freezing solution, load the embryos according to the 5-stage liquid filling method, mark them well, 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 straw was quickly taken out and placed in liquid nitrogen for cryopreservation.

According to the method of the fifth aspect of the present invention, wherein in step (1), the double-antibiotic-added normal saline is normal saline containing 400 IU/mL of penicillin and 400 μg/mL of streptomycin.

According to the method of the fifth aspect of the present invention, wherein in step (1), the egg washing solution is BY basal culture solution added with 3 mg/mL bovine serum albumin.

The preparation method of egg washing liquid or other culture fluid of the present invention is easily realized by those skilled in the art, for example, for egg washing liquid, it is usually to add bovine serum albumin to The corresponding required concentration, this preparation method is also commonly used by those skilled in the art.

According to the method of the fifth aspect of the present invention, wherein in step (1), the mature culture solution is the BY base added with 100mL/LFBS, 10μg/mL FSH, 10μg/mL LH, 1μg/mL E2, 20ng/mL EGF culture medium.

The method according to the fifth aspect of the present invention, wherein the concentration of HEPES, namely 4-hydroxyethylpiperazineethanesulfonic acid, in the maturation culture solution is 10-20 mmol/L, for example 15 mmol/L. In the present invention, EGF—epidermal growth factor, FSH—follicle stimulating hormone, FBS—fetal bovine serum, E2—estradiol, LH—luteinizing hormone.

According to the method of the fifth aspect of the present invention, wherein in step (2), the fertilization culture liquid is calcium chloride dihydrate comprising 112.0mM sodium chloride, 4.02mM potassium chloride, 2.25mM, 0.52mM 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 in water.

According to the method of the fifth aspect of the present invention, wherein in step (2), the seminal fluid preparation culture fluid is calcium chloride dihydrate containing 112.0mM sodium chloride, 4.02mM potassium chloride, 2.25mM, 0.52mM hexahydrate Magnesium chloride, 0.83mM 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 chain Aqueous solution of mycin.

According to the method of the fifth aspect of the present invention, wherein in step (3), the embryo culture solution 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 galactose, 2.5v/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/v%, maltose 0.02w/v% aqueous solution; the essential amino acids are the following amino acids added in proportion 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 in proportion 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.

According to the method of the fifth aspect of the present invention, wherein in step (1), the BY basal culture solution is an aqueous solution comprising the following components: 180-220 mg/L of calcium chloride, 0.70-0.75 mg/L of ferric nitrate nonahydrate, 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 Salt 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, menaquinone sodium bisulfite trihydrate 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 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, hypoxanthine sodium 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, xanthine sodium 0.3~0.4mg/L, phenol red 8~12mg/L.

According to the method of the fifth aspect of the present invention, wherein in step (1), the BY basic 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, menaquinone 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 L, Sodium Hypoxanthine 0.354mg/L, Ribose 0.5mg/L, Thymosin 0.3mg/L, Tween 805mg/L, Uracil 0.3mg/L, Sodium Xanthine 0.34mg/L, Phenol Red 10mg/L .

According to the method of the fifth aspect of the present invention, wherein in step (1), the BY basal culture solution further includes sodium selenite at a concentration of 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 in terms of anhydrous matter, such as 0.075 mg/L.

In the present invention, for example, in step (3), the preservation solution, balance solution, freezing solution, etc. used by the embryo are well known in the art and can be easily obtained from commercially available channels. For example, the freezing solution can be obtained from Vitrolife, Sweden FreezeKit ^TM Cleave sold domestically.

Any aspect of the present invention or any technical feature of any implementation of this any aspect is also applicable to any other implementation or any implementation of any other aspect, as long as they do not contradict each other, of course When applicable, the corresponding features can be appropriately modified if necessary. Various aspects and features of the present invention are further described below.

All the documents cited in the present invention are incorporated herein by reference in their entirety, and if the meaning expressed in 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 common meanings known to those skilled in the art. Even so, the present invention still hopes to make a more detailed description and explanation of these terms and phrases here. The terms and phrases mentioned are as follows: 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 commodity form, for example, Australian fetal bovine serum (article number: 10099141), New Zealand fetal bovine serum (article number: 10091148) can be purchased from various agents. ), North American fetal bovine serum (article number: 16000044), Mexican fetal bovine serum (article number: 10437028), etc., the price of these commercial fetal bovine serum is mostly more than 8000 yuan for 500ml, when used in the bovine in vitro fertilization embryo culture medium of the present invention Fetal bovine serum is a major contributor to costs. In the tests in the context of the present invention, unless otherwise specified, the fetal bovine serum used was Australian fetal bovine serum from Gibco (Product No.: 10099141).

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. Those skilled in the art can understand that various changes and modifications can be made in the present invention without departing from the spirit and scope of the present invention. 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 manipulation which are employed for the purposes of the invention are well known in the art, the invention has been described here 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 products.

Embodiment 1: the culture method of bovine in vitro fertilization embryo (no selenium in vitro)

1. Reagents

In the specific test of the present invention, if not otherwise specified, the relevant reagents used are described in detail as follows:

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

Egg washing solution: BY basal culture solution supplemented with 3mg/mL bovine serum albumin.

Maturation 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/mL EGF.

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

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

Fertilization medium: containing 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, 1.25mM pyruvate Aqueous solution of sodium, 10 μg/ml heparin, 4 mg/ml bovine serum albumin (BSA), 100 U/ml penicillin, 100 μg/ml streptomycin.

Semen preparation medium: containing 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, 1.25mM acetone An aqueous solution of sodium nitrite, 10 μg/ml heparin, 4 mg/ml bovine serum albumin (BSA), 10 mM caffeine, 100 U/ml penicillin, and 100 μg/ml streptomycin.

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 carbonate, 2.5v/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 /v%, maltose 0.02w/v% aqueous solution; the essential amino acids are the following amino acids added in proportion by weight: L-arginine hydrochloride 6.32g, L-cystine dihydrochloride 1.564g, L-hydrochloride group Amino 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, described non-essential amino acid is that following amino acid is added by weight ratio: L-alanine 0.89 g of acid, 1.5 g of L-asparagine monohydrate, 1.33 g of L-aspartic acid, 1.47 g of L-glutamic acid, 0.75 g of glycine, 1.15 g of L-proline, and 1.05 g of L-serine.

BY basic culture solution 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 Sodium dihydrogen phosphate water 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, menaquinone trihydrate Sodium bisulfate 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, hypoxanthine sodium 0.354mg/L, ribose 0.5mg/L, thymus Su 0.3mg/L, Tween 805mg/L, uracil 0.3mg/L, xanthine sodium 0.34mg/L, phenol red 10mg/L.

2. Cattle in vitro fertilization and embryo culture:

Step (1), collection and in vitro maturation of oocytes

Take the ovaries from the slaughterhouse and put them in an insulated bucket with double-antibiotic saline, and transport them back to the laboratory within 4 hours at 30.5-33°C; extract follicles with a surface size of 2-8mm, collect the precipitate, and pick out under a stereo microscope at least Oocyte COCs (i.e., cumulus-oocyte complex) wrapped by three layers of cumulus cells are washed twice in egg washing liquid to remove excess impurities;

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

Step (2), in vitro fertilization

Wash the mature COCs once in the fertilization medium, then transfer them to the fertilization medium, put them in the incubator, and set aside;

Take a frozen fine tube from liquid nitrogen and thaw it in a water bath at 37°C; cut both ends of the thin tube aseptically, inject the semen into a 15mL centrifuge tube filled with the semen preparation culture medium, and centrifuge twice at 328×g, each time After centrifuging for 5 minutes, discard the supernatant; add 300 μL of semen preparation culture medium to the above centrifuge tube, resuspend the sperm pellet, and take an appropriate sperm suspension for sperm counting;

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

Step (3), in vitro culture and preservation of embryos

After the in vitro fertilization operation, remove the granulosa cells around the embryo with the egg stripping needle, and put them into the embryo culture medium for culture. At this time, it is recorded as the first day of embryo culture. The culture conditions are 38.8°C, 6% O2, 88% N2, saturated humidity, record the cleavage rate on the 3rd day; record the blastocyst rate on the 7th day, and count the blastocyst hatching rate (it 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 usable embryos in the preservation solution for 3 times, equilibrate in the balance solution for 10 minutes, transfer to the freezing solution, load the embryos according to the 5-stage liquid filling method, mark them well, 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 straw was quickly taken out and placed in liquid nitrogen for cryopreservation.

3. Method of Differential Staining of Embryos

1. Select blastocysts on day 7 of in vitro culture and fix them with 2% paraformaldehyde for 20 minutes.

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 stand at room temperature for 30 min.

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

4. Wash three times with PBS-BSA, put the blastocyst into blocking solution (1ml goat serum, 5μl Tween 80 and 8.995ml PBS), seal at room temperature for 1h, and then transfer to 4°C refrigerator to seal overnight.

5. Discard the blocking solution, dilute the CDX2 primary antibody with blocking solution at 1:200, incubate at room temperature for 2 hours, discard the primary antibody dilution, and wash with PBS-BSA 3 times, 5 minutes each time.

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

7. Dilute the CDX2-specific secondary antibody (purchased from Sigma) at a ratio of 1:200 with blocking solution in the dark, and place it in the dark at room temperature for 1 hour. The secondary antibody dilution was discarded under dark conditions, and washed 3 times with PBS, 5 min each time.

8. Dilute the caspase-3-specific secondary antibody (purchased from Life Technologies) at a ratio of 1:200 with blocking solution under dark conditions, and place in the dark at room temperature for 1 hour. The secondary antibody dilution was discarded under dark conditions, and washed 3 times with PBS, 5 min each time.

9. Add 10 μg/mL Hochest 33342 staining solution to stain cell nuclei, react at room temperature for 5 minutes, observe and take pictures under a fluorescent microscope.

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

Data statistics method: The experimental data was analyzed using the ANOVA program in the statistical software SAS V8, and 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, cleavage rate=number of fertilized cleavage/number of fertilized eggs. In the present invention, the blastocyst rate=the number of blastocysts/the number of cleavage embryos.

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

In the step (1) of this embodiment, after in vitro maturation and culture, observe under an inverted microscope, the oocyte has the first polar body released, the viscous matrix secreted between the cumulus cells is maintained, and the cell layer is significantly enlarged. , The cells are centered on the ovum, and the cells that spread radially to the surroundings are judged to be mature, record the number of mature oocytes, and calculate the maturity rate.

5. Results

In this example, a test is carried out on Chinese yellow cattle (Nanyang cattle, a service breed). As a result, the cleavage rate was 88.7%, the morula rate was 63.7%, the blastocyst rate was 51.2%, and the apoptosis rate was 5.1%. In addition, the blastocyst hatching rate reached 72.3% on the 9th day. The maturation rate of in vitro maturation of oocytes reached 75.6%.

In a supplementary test, with reference to the method of Example 1, tests were carried out on Holstein cattle (dairy breed), Simmental cattle (beef breed), and Chinese buffalo (service breed), and the results: cleavage The rate of morula was in the range of 84-89%, the rate of morula was in the range of 62-65%, the rate of blastocyst was in the range of 50-53%, the rate of apoptosis was in the range of 4-7%. The hatching rates of embryos were all in the range of 71-75%. The maturation rates of in vitro maturation of oocytes from Holstein, Simmental and Chinese buffalo were 69.3%, 65.6% and 57.8% respectively.

Embodiment 2: the culture method of bovine in vitro fertilization embryo (living body without selenium)

1. Reagents

In the specific test of the present invention, if not otherwise specified, the relevant reagents used are described in detail as follows:

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

Egg washing solution: BY basal culture solution supplemented with 3mg/mL bovine serum albumin.

Maturation 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/mL EGF.

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

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

Fertilization medium: containing 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, 1.25mM pyruvate Aqueous solution of sodium, 10 μg/ml heparin, 4 mg/ml bovine serum albumin (BSA), 100 U/ml penicillin, 100 μg/ml streptomycin.

Semen preparation medium: containing 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, 1.25mM acetone An aqueous solution of sodium nitrite, 10 μg/ml heparin, 4 mg/ml bovine serum albumin (BSA), 10 mM caffeine, 100 U/ml penicillin, and 100 μg/ml streptomycin.

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 carbonate, 2.5v/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 /v%, maltose 0.02w/v% aqueous solution; the essential amino acids are the following amino acids added in proportion by weight: L-arginine hydrochloride 6.32g, L-cystine dihydrochloride 1.564g, L-hydrochloride group Amino 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, described non-essential amino acid is that following amino acid is added by weight ratio: L-alanine 0.89 g of acid, 1.5 g of L-asparagine monohydrate, 1.33 g of L-aspartic acid, 1.47 g of L-glutamic acid, 0.75 g of glycine, 1.15 g of L-proline, and 1.05 g of L-serine.

BY basic culture solution 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 Sodium dihydrogen phosphate water 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, menaquinone trihydrate Sodium bisulfate 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, hypoxanthine sodium 0.354mg/L, ribose 0.5mg/L, thymus Su 0.3mg/L, Tween 805mg/L, uracil 0.3mg/L, xanthine sodium 0.34mg/L, phenol red 10mg/L.

2. Cattle in vitro fertilization and embryo culture:

Step (1), collection of oocytes and in vitro maturation

Live bovine eggs were collected, and the obtained follicular fluid was picked out under a stereo microscope to find cumulus-oocyte complexes (COCs) that contained at least 3 layers of cumulus cells, and put them into the mature culture medium containing HEPES at 38.8°C 、Transport back to the laboratory within 3 hours;

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

Step (2), in vitro fertilization

Wash the mature COCs once in the fertilization medium, then transfer them to the fertilization medium, put them in the incubator, and set aside;

Take a frozen fine tube from liquid nitrogen and thaw it in a water bath at 37°C; cut both ends of the thin tube aseptically, inject the semen into a 15mL centrifuge tube filled with the semen preparation culture medium, and centrifuge twice at 328×g, each time After centrifuging for 5 minutes, discard the supernatant; add 300 μL of semen preparation culture medium to the above centrifuge tube, resuspend the sperm pellet, and take an appropriate sperm suspension for sperm counting;

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

Step (3), in vitro culture and preservation of embryos

After the in vitro fertilization operation, remove the granulosa cells around the embryo with the egg stripping needle, and put them into the embryo culture medium for culture. At this time, it is recorded as the first day of embryo culture. The culture conditions are 38.8°C, 6% O2, 88% N2, saturated humidity, record the cleavage rate on the 3rd day; record the blastocyst rate on the 7th day, and count the blastocyst hatching rate (it 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 usable embryos in the preservation solution for 3 times, equilibrate in the balance solution for 10 minutes, transfer to the freezing solution, load the embryos according to the 5-stage liquid filling method, mark them well, 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 straw was quickly taken out and placed in liquid nitrogen for cryopreservation.

3. Method of Differential Staining of Embryos

1. Select blastocysts on day 7 of in vitro culture and fix them with 2% paraformaldehyde for 20 minutes.

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 stand at room temperature for 30 min.

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

4. Wash three times with PBS-BSA, put the blastocyst into blocking solution (1ml goat serum, 5μl Tween 80 and 8.995ml PBS), seal at room temperature for 1h, and then transfer to 4°C refrigerator to seal overnight.

5. Discard the blocking solution, dilute the CDX2 primary antibody with blocking solution at 1:200, incubate at room temperature for 2 hours, discard the primary antibody dilution, and wash with PBS-BSA 3 times, 5 minutes each time.

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

7. Dilute the CDX2-specific secondary antibody (purchased from Sigma) at a ratio of 1:200 with blocking solution in the dark, and place it in the dark at room temperature for 1 hour. The secondary antibody dilution was discarded under dark conditions, and washed 3 times with PBS, 5 min each time.

8. Dilute the caspase-3-specific secondary antibody (purchased from Life Technologies) at a ratio of 1:200 with blocking solution under dark conditions, and place in the dark at room temperature for 1 hour. The secondary antibody dilution was discarded under dark conditions, and washed 3 times with PBS, 5 min each time.

9. Add 10 μg/mL Hochest 33342 staining solution to stain cell nuclei, react at room temperature for 5 minutes, observe and take pictures under a fluorescent microscope.

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

Data statistics method: The experimental data was analyzed using the ANOVA program in the statistical software SAS V8, and 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, cleavage rate=number of fertilized cleavage/number of fertilized eggs. In the present invention, the blastocyst rate=the number of blastocysts/the number of cleavage embryos.

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

In the step (1) of this embodiment, after in vitro maturation and culture, observe under an inverted microscope, the oocyte has the first polar body released, the viscous matrix secreted between the cumulus cells is maintained, and the cell layer is significantly enlarged. , The cells are centered on the ovum, and the cells that spread radially to the surroundings are judged to be mature, record the number of mature oocytes, and calculate the maturity rate.

5. Results

In this example, a test is carried out on Chinese yellow cattle (Nanyang cattle, a service breed). As a result, the cleavage rate was 87.4%, the morula rate was 64.2%, the blastocyst rate was 51.8%, and the apoptosis rate was 4.8%. In addition, the blastocyst hatching rate reached 71.1% on the 9th day. The maturation rate of in vitro maturation of oocytes was 76.3%.

In a supplementary test, with reference to the method in Example 2 above, tests were carried out on Holstein cattle (dairy breed), Simmental cattle (beef breed), and Chinese buffalo (service breed), and the results: The cleavage rates were all in the range of 83-89%, the morula rates were all in the range of 61-65%, the blastocyst rates were all in the range of 50-54%, and the apoptosis rates were all in the range of 4-6%. The hatching rates of blastocysts were all in the range of 72-75%. The maturation rates of oocytes matured in vitro from Holstein, Simmental and Chinese buffalo were 68.8%, 66.3% and 58.3% respectively.

Embodiment 3: the culture method of bovine in vitro fertilization embryo (isolated selenium copper)

The main difference between this example and the above example 1 is that 0.25 mg/L sodium selenite and 0.075 mg/L anhydrous copper sulfate are additionally added to the BY basal culture solution.

1. Reagents

In the specific test of the present invention, if not otherwise specified, the relevant reagents used are described in detail as follows:

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

Egg washing solution: BY basal culture solution supplemented with 3mg/mL bovine serum albumin.

Maturation 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/mL EGF.

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

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

Fertilization medium: containing 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, 1.25mM pyruvate Aqueous solution of sodium, 10 μg/ml heparin, 4 mg/ml bovine serum albumin (BSA), 100 U/ml penicillin, 100 μg/ml streptomycin.

Semen preparation medium: containing 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, 1.25mM acetone An aqueous solution of sodium nitrite, 10 μg/ml heparin, 4 mg/ml bovine serum albumin (BSA), 10 mM caffeine, 100 U/ml penicillin, and 100 μg/ml streptomycin.

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 carbonate, 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/ v%, maltose 0.02w/v% aqueous solution; the essential amino acid is the following amino acids added in proportion by weight: L-arginine hydrochloride 6.32g, L-cystine dihydrochloride 1.564g, L-histamine 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, described non-essential amino acids are the following amino acids added in proportion 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.

BY basic culture solution 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 Sodium dihydrogen phosphate water 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, menaquinone trihydrate Sodium bisulfate 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, hypoxanthine sodium 0.354mg/L, ribose 0.5mg/L, thymus Sodium 0.3mg/L, Tween 805mg/L, Uracil 0.3mg/L, Sodium Xanthine 0.34mg/L, Phenol Red 10mg/L, Sodium Selenite 0.25mg/L and Copper Sulfate Anhydrous 0.075mg/L .

2. Cattle in vitro fertilization and embryo culture:

Step (1), collection and in vitro maturation of oocytes

Take the ovaries from the slaughterhouse and put them in an insulated bucket with double-antibiotic saline, and transport them back to the laboratory within 4 hours at 30.5-33°C; extract follicles with a surface size of 2-8mm, collect the precipitate, and pick out under a stereo microscope at least Oocyte COCs (i.e., cumulus-oocyte complex) wrapped by three layers of cumulus cells are washed twice in egg washing liquid to remove excess impurities;

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

Step (2), in vitro fertilization

Wash the mature COCs once in the fertilization medium, then transfer them to the fertilization medium, put them in the incubator, and set aside;

Take a frozen fine tube from liquid nitrogen and thaw it in a water bath at 37°C; cut both ends of the thin tube aseptically, inject the semen into a 15mL centrifuge tube filled with the semen preparation culture medium, and centrifuge twice at 328×g, each time After centrifuging for 5 minutes, discard the supernatant; add 300 μL of semen preparation culture medium to the above centrifuge tube, resuspend the sperm pellet, and take an appropriate sperm suspension for sperm counting;

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

Step (3), in vitro culture and preservation of embryos

After the in vitro fertilization operation, remove the granulosa cells around the embryo with the egg stripping needle, and put them into the embryo culture medium for culture. At this time, it is recorded as the first day of embryo culture. The culture conditions are 38.8°C, 6% O2, 88% N2, saturated humidity, record the cleavage rate on the 3rd day; record the blastocyst rate on the 7th day, and count the blastocyst hatching rate (it 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 usable embryos in the preservation solution for 3 times, equilibrate in the balance solution for 10 minutes, transfer to the freezing solution, load the embryos according to the 5-stage liquid filling method, mark them well, 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 straw was quickly taken out and placed in liquid nitrogen for cryopreservation.

3. Method of Differential Staining of Embryos

1. Select blastocysts on day 7 of in vitro culture and fix them with 2% paraformaldehyde for 20 minutes.

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 stand at room temperature for 30 min.

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

4. Wash three times with PBS-BSA, put the blastocyst into blocking solution (1ml goat serum, 5μl Tween 80 and 8.995ml PBS), seal at room temperature for 1h, and then transfer to 4°C refrigerator to seal overnight.

5. Discard the blocking solution, dilute the CDX2 primary antibody with blocking solution at 1:200, incubate at room temperature for 2 hours, discard the primary antibody dilution, and wash with PBS-BSA 3 times, 5 minutes each time.

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

7. Dilute the CDX2-specific secondary antibody (purchased from Sigma) at a ratio of 1:200 with blocking solution in the dark, and place it in the dark at room temperature for 1 hour. The secondary antibody dilution was discarded under dark conditions, and washed 3 times with PBS, 5 min each time.

8. Dilute the caspase-3-specific secondary antibody (purchased from Life Technologies) at a ratio of 1:200 with blocking solution under dark conditions, and place in the dark at room temperature for 1 hour. The secondary antibody dilution was discarded under dark conditions, and washed 3 times with PBS, 5 min each time.

9. Add 10 μg/mL Hochest 33342 staining solution to stain cell nuclei, react at room temperature for 5 minutes, observe and take pictures under a fluorescent microscope.

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

Data statistics method: The experimental data was analyzed using the ANOVA program in the statistical software SAS V8, and 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, cleavage rate=number of fertilized cleavage/number of fertilized eggs. In the present invention, the blastocyst rate=the number of blastocysts/the number of cleavage embryos.

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

In the step (1) of this embodiment, after in vitro maturation and culture, observe under an inverted microscope, the oocyte has the first polar body released, the viscous matrix secreted between the cumulus cells is maintained, and the cell layer is significantly enlarged. , The cells are centered on the ovum, and the cells that spread radially to the surroundings are judged to be mature, record the number of mature oocytes, and calculate the maturity rate.

5. Results

In this example, a test is carried out on Chinese yellow cattle (Nanyang cattle, a service breed). As a result, the cleavage rate was 88.2%, the morula rate was 64.2%, the blastocyst rate was 50.8%, and the apoptosis rate was 5.6%. In addition, the blastocyst hatching rate reached 73.4% on the 9th day. The maturation rate of the in vitro maturation of oocytes reached 88.3%, an increase of about 13 percentage points relative to the maturation rate of the method in Example 1.

In a supplementary test, with reference to the method in Example 3 above, tests were carried out on Holstein cattle (dairy breed), Simmental cattle (beef breed), and Chinese buffalo (service breed), and the results: The cleavage rates were all in the range of 84-90%, the morula rates were all in the range of 61-65%, the blastocyst rates were all in the range of 50-54%, and the apoptosis rates were all in the range of 3-6%. The hatching rates of blastocysts were all in the range of 71-76%; the maturation rates of in vitro maturation of oocytes from Holstein, Simmental, and Chinese buffalo were 84.5%, 79.5%, and 73.2%, respectively. The maturity rates of the methods in Example 1 all increased by about 14 to 15 percentage points. The above-mentioned results, although the method of Example 1 of the present invention can obtain completely satisfactory results, however, it is unexpectedly found that when trace selenide and copper sulfate are added to the BY basal culture solution of the present invention, it can significantly increase the oocyte in vitro. The maturation rate of maturation, which is very meaningful, especially there is no technical teaching in any prior art that the above-mentioned addition of selenide and copper sulfate can significantly increase the maturation rate of in vitro maturation of oocytes.

In a supplementary test, with reference to this embodiment 3, sodium selenite is not added (but only the corresponding amount of copper sulfate is supplemented) or copper sulfate is not added (and only the corresponding amount of sodium selenite is supplemented) in the BY basal medium ), in the four kinds of cattle species, the four parameters of cleavage rate, morula rate, blastocyst rate, and apoptosis rate were basically consistent with the results of Example 3 in both cases, and the cleavage rates of the four kinds of cattle species were all In the range of 84-89%, the rate of morula is in the range of 63-65%, the rate of blastocyst is in the range of 52-55%, the rate of apoptosis is in the range of 3-6%, and the blastocyst hatches on the 9th day The rates are all in the range of 70 to 75%. For example, the cleavage rates for Chinese yellow cattle are 87.7% and 88.5% respectively; but the maturation rate of oocyte maturation in vitro is not increased compared with the results of Example 1. In both cases The in vitro maturation rates of oocytes from Chinese Yellow Cattle, Holstein Cattle, Simmental Cattle, and Chinese Buffalo are 73-75%, 67-70%, 65-66%, and 55-57%, respectively. This shows that the addition of selenide and copper sulfate in the BY basal medium will not affect the four parameters of cleavage rate, morula rate, blastocyst rate, and apoptosis rate, but it will be effective only when selenide and copper sulfate are added at the same time. Improves the maturation rate of oocyte maturation in vitro.

Embodiment 4: the culture method of bovine in vitro fertilization embryo (living body selenium copper)

The main difference between this example and the above example 2 is that 0.25 mg/L sodium selenite and 0.075 mg/L anhydrous copper sulfate are additionally added to the BY basal culture solution.

1. Reagents

In the specific test of the present invention, if not otherwise specified, the relevant reagents used are described in detail as follows:

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

Egg washing solution: BY basal culture solution supplemented with 3mg/mL bovine serum albumin.

Maturation 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/mL EGF.

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

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

Fertilization medium: containing 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, 1.25mM pyruvate Aqueous solution of sodium, 10 μg/ml heparin, 4 mg/ml bovine serum albumin (BSA), 100 U/ml penicillin, 100 μg/ml streptomycin.

Semen preparation medium: containing 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, 1.25mM acetone An aqueous solution of sodium nitrite, 10 μg/ml heparin, 4 mg/ml bovine serum albumin (BSA), 10 mM caffeine, 100 U/ml penicillin, and 100 μg/ml streptomycin.

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 carbonate, 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/ v%, maltose 0.02w/v% aqueous solution; the essential amino acid is the following amino acids added in proportion by weight: L-arginine hydrochloride 6.32g, L-cystine dihydrochloride 1.564g, L-histamine 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, described non-essential amino acids are the following amino acids added in proportion 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.

BY basic culture solution 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 Sodium dihydrogen phosphate water 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, menaquinone trihydrate Sodium bisulfate 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, hypoxanthine sodium 0.354mg/L, ribose 0.5mg/L, thymus Sodium 0.3mg/L, Tween 805mg/L, Uracil 0.3mg/L, Sodium Xanthine 0.34mg/L, Phenol Red 10mg/L, Sodium Selenite 0.25mg/L and Copper Sulfate Anhydrous 0.075mg/L .

2. Cattle in vitro fertilization and embryo culture:

Step (1), collection and in vitro maturation of oocytes

Live bovine eggs were collected, and the obtained follicular fluid was picked out under a stereo microscope to find cumulus-oocyte complexes (COCs) that contained at least 3 layers of cumulus cells, and put them into the mature culture medium containing HEPES at 38.8°C 、Transport back to the laboratory within 3 hours;

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

Step (2), in vitro fertilization

Wash the mature COCs once in the fertilization medium, then transfer them to the fertilization medium, put them in the incubator, and set aside;

Take a frozen fine tube from liquid nitrogen and thaw it in a water bath at 37°C; cut both ends of the thin tube aseptically, inject the semen into a 15mL centrifuge tube filled with the semen preparation culture medium, and centrifuge twice at 328×g, each time After centrifuging for 5 minutes, discard the supernatant; add 300 μL of semen preparation culture medium to the above centrifuge tube, resuspend the sperm pellet, and take an appropriate sperm suspension for sperm counting;

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

Step (3), in vitro culture and preservation of embryos

After the in vitro fertilization operation, remove the granulosa cells around the embryo with the egg stripping needle, and put them into the embryo culture medium for culture. At this time, it is recorded as the first day of embryo culture. The culture conditions are 38.8°C, 6% O2, 88% N2, saturated humidity, record the cleavage rate on the 3rd day; record the blastocyst rate on the 7th day, and count the blastocyst hatching rate (it 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 usable embryos in the preservation solution for 3 times, equilibrate in the balance solution for 10 minutes, transfer to the freezing solution, load the embryos according to the 5-stage liquid filling method, mark them well, 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 straw was quickly taken out and placed in liquid nitrogen for cryopreservation.

3. Method of Differential Staining of Embryos

1. Select blastocysts on day 7 of in vitro culture and fix them with 2% paraformaldehyde for 20 minutes.

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 stand at room temperature for 30 min.

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

4. Wash three times with PBS-BSA, put the blastocyst into blocking solution (1ml goat serum, 5μl Tween 80 and 8.995ml PBS), seal at room temperature for 1h, and then transfer to 4°C refrigerator to seal overnight.

5. Discard the blocking solution, dilute the CDX2 primary antibody with blocking solution at 1:200, incubate at room temperature for 2 hours, discard the primary antibody dilution, and wash with PBS-BSA 3 times, 5 minutes each time.

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

7. Dilute the CDX2-specific secondary antibody (purchased from Sigma) at a ratio of 1:200 with blocking solution in the dark, and place it in the dark at room temperature for 1 hour. The secondary antibody dilution was discarded under dark conditions, and washed 3 times with PBS, 5 min each time.

8. Dilute the caspase-3-specific secondary antibody (purchased from Life Technologies) at a ratio of 1:200 with blocking solution under dark conditions, and place in the dark at room temperature for 1 hour. The secondary antibody dilution was discarded under dark conditions, and washed 3 times with PBS, 5 min each time.

9. Add 10 μg/mL Hochest 33342 staining solution to stain cell nuclei, react at room temperature for 5 minutes, observe and take pictures under a fluorescent microscope.

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

Data statistics method: The experimental data was analyzed using the ANOVA program in the statistical software SAS V8, and 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, cleavage rate=number of fertilized cleavage/number of fertilized eggs. In the present invention, the blastocyst rate=the number of blastocysts/the number of cleavage embryos.

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

In the step (1) of this embodiment, after in vitro maturation and culture, observe under an inverted microscope, the oocyte has the first polar body released, the viscous matrix secreted between the cumulus cells is maintained, and the cell layer is significantly enlarged. , The cells are centered on the ovum, and the cells that spread radially to the surroundings are judged to be mature, record the number of mature oocytes, and calculate the maturity rate.

5. Results

In this example, a test is carried out on Chinese yellow cattle (Nanyang cattle, a service breed). As a result, the cleavage rate was 88.4%, the morula rate was 64.7%, the blastocyst rate was 50.3%, and the apoptosis rate was 5.1%. In addition, the blastocyst hatching rate reached 74.4% on the 9th day. The maturation rate of the in vitro maturation of oocytes reaches 89.6%, which is about 13 percentage points higher than the maturation rate of the method in Example 2.

In a supplementary test, with reference to the method in Example 4 above, the tests were carried out on Holstein cattle (dairy breed), Simmental cattle (beef breed), and Chinese buffalo (service breed), and the results: The cleavage rates were all in the range of 85-90%, the morula rates were all in the range of 62-65%, the blastocyst rates were all in the range of 51-54%, and the apoptosis rates were all in the range of 4-6%. The hatching rates of blastocysts were all in the range of 71-75%; the maturation rates of oocytes in vitro maturation of Holstein, Simmental, and Chinese buffalo were 85.2%, 79.1%, and 74.32%, respectively. The maturation rates of the methods in Example 2 both increased by about 14 to 15 percentage points. The above-mentioned results, although the method of Example 2 of the present invention can obtain completely satisfactory results, however, it is unexpectedly found that when traces of selenide and copper sulfate are added to the BY basal culture solution of the present invention, it can significantly increase the oocyte in vitro The maturation rate of maturation, which is very meaningful, especially there is no technical teaching in any prior art that the above-mentioned addition of selenide and copper sulfate can significantly increase the maturation rate of in vitro maturation of oocytes.

In a supplementary test, with reference to this embodiment 4, in BY basal medium, do not add sodium selenite (but only supplement the corresponding amount of copper sulfate) or do not add copper sulfate (but only supplement the corresponding amount of sodium selenite ), in the four kinds of cattle species, the four parameters of cleavage rate, morula rate, blastocyst rate, and apoptosis rate were basically consistent with the results of Example 4 in both cases, and the cleavage rates of the four kinds of cattle species were all In the range of 84-89%, the rate of morula is in the range of 63-66%, the rate of blastocyst is in the range of 53-55%, the rate of apoptosis is in the range of 3-5%, and the blastocyst hatches on the 9th day The rates are all in the range of 70 to 74%. For example, the cleavage rates for Chinese yellow cattle are 88.4% and 88.1% respectively; but the maturation rate of oocyte maturation in vitro is not increased compared with the results of Example 2. In both cases The in vitro maturation rates of oocytes from Chinese Yellow Cattle, Holstein Cattle, Simmental Cattle, and Chinese Buffalo are 72-74%, 67-71%, 65-67%, and 55-58%, respectively. This shows that the addition of selenide and copper sulfate in the BY basal medium will not affect the four parameters of cleavage rate, morula rate, blastocyst rate, and apoptosis rate, but it will be effective only when selenide and copper sulfate are added at the same time. Improves the maturation rate of oocyte maturation in vitro.

Embodiment 5: the culture method of bovine in vitro fertilization embryo (no selenium in vitro)

With reference to the above example 1 and the corresponding supplementary test method in this example 1, selenium and copper are not added in the BY basal culture solution, the only difference is that the BY basal culture solution used is replaced by an aqueous solution of the following formula:

Calcium chloride 180mg/L, ferric nitrate nonahydrate 0.75mg/L, potassium chloride 380mg/L, magnesium sulfate 100mg/L, sodium chloride 6500mg/L, sodium dihydrogen phosphate monohydrate 150mg/L, sodium bicarbonate 2000mg /L, sodium acetate 60mg/L, L-alanine 20mg/L, L-arginine hydrochloride 80mg/L, L-aspartic acid 25mg/L, L-cysteine hydrochloride Hydrate 0.12mg/L, L-cystine dihydrochloride 20mg/L, L-glutamic acid 80mg/L, glycine 40mg/L, L-histidine hydrochloride monohydrate 25mg/L, L -Hydroxyproline 8mg/L, L-isoleucine 25mg/L, L-leucine 50mg/L, L-lysine hydrochloride 80mg/L, L-methionine 10mg/L, L-benzene Alanine 30mg/L, L-proline 30mg/L, L-serine 30mg/L, L-threonine 25mg/L, L-tryptophan 12mg/L, L-tyrosine disodium dihydrate 55mg/L, L-valine 30mg/L, ascorbic acid 0.04mg/L, α-D-tocopheryl phosphate 0.012mg/L, biotin 0.008mg/L, calcidol 0.12mg/L, D- Calcium pantothenate 0.008mg/L, choline chloride 0.6mg/L, folic acid 0.008mg/L, inositol 0.06mg/L, menaquinone sodium bisulfite trihydrate 0.015mg/L, niacin 0.03mg/L, Niacinamide 0.02mg/L, p-aminobenzoic acid 0.06mg/L, pyridoxine hydrochloride 0.04mg/L, riboflavin 0.012mg/L, thiamine hydrochloride 0.008mg/L, vitamin A acetate 0.2mg /L, adenine sulfate 8mg/L, adenine 0.25mg/L, disodium adenosine triphosphate 0.8mg/L, cholesterol 0.25mg/L, 2-deoxy-D-ribose 0.4mg/L, D-glucose 1200mg /L, Glutathione 0.04mg/L, Guanine Hydrochloride 0.35mg/L, Hypoxanthine Sodium 0.3mg/L, Ribose 0.6mg/L, Thymosin 0.25mg/L, Tween 806mg/L, Uracil 0.25mg/L, xanthine sodium 0.4mg/L, phenol red 8mg/L.

Result: the result of each test parameter is basically the same as the result of the corresponding supplementary test in the above embodiment 1 and this embodiment 1, for example, for Chinese yellow cattle, the cleavage rate is 88.3%, the morula rate is 64.2%, and the blastocyst rate is 88.3%. The rate of apoptosis was 51.6%, the rate of apoptosis was 5.3%, the hatching rate of blastocyst reached 71.7% on the 9th day, and the maturation rate of in vitro maturation of oocytes reached 74.5%; for Holstein, Simmental and Chinese buffalo The maturation rates of in vitro maturation of oocytes were 68.8%, 65.9%, and 57.3%, respectively.

Embodiment 6: the culture method of bovine in vitro fertilization embryo (living body without selenium)

With reference to the method of the above example 2 and the corresponding supplementary test in this example 2, selenium and copper are not added in the BY basal culture solution, the only difference is that the used BY basal culture solution is replaced by an aqueous solution of the following formula:

Calcium chloride 220mg/L, ferric nitrate nonahydrate 0.70mg/L, potassium chloride 420mg/L, magnesium sulfate 90mg/L, sodium chloride 7000mg/L, sodium dihydrogen phosphate monohydrate 130mg/L, sodium bicarbonate 2500mg /L, sodium acetate 40mg/L, L-alanine 30mg/L, L-arginine hydrochloride 60mg/L, L-aspartic acid 35mg/L, L-cysteine hydrochloride Hydrate 0.10mg/L, L-cystine dihydrochloride 30mg/L, L-glutamic acid 70mg/L, glycine 60mg/L, L-histidine hydrochloride monohydrate 20mg/L, L -Hydroxyproline 12mg/L, L-isoleucine 15mg/L, L-leucine 70mg/L, L-lysine hydrochloride 60mg/L, L-methionine 20mg/L, L-benzene Alanine 20mg/L, L-proline 50mg/L, L-serine 20mg/L, L-threonine 35mg/L, L-tryptophan 8mg/L, L-tyrosine disodium dihydrate 60mg/L, L-valine 20mg/L, ascorbic acid 0.06mg/L, α-D-tocopheryl phosphate 0.008mg/L, biotin 0.012mg/L, calcidol 0.08mg/L, D- Calcium pantothenate 0.012mg/L, choline chloride 0.4mg/L, folic acid 0.012mg/L, inositol 0.04mg/L, menaquinone sodium bisulfite trihydrate 0.025mg/L, niacin 0.02mg/L, Niacinamide 0.03mg/L, p-aminobenzoic acid 0.04mg/L, pyridoxine hydrochloride 0.06mg/L, riboflavin 0.008mg/L, thiamine hydrochloride 0.012mg/L, vitamin A acetate 0.1mg /L, adenine sulfate 12mg/L, adenine 0.15mg/L, disodium adenosine triphosphate 1.2mg/L, cholesterol 0.15mg/L, 2-deoxy-D-ribose 0.6mg/L, D-glucose 800mg /L, Glutathione 0.06mg/L, Guanine Hydrochloride 0.25mg/L, Hypoxanthine Sodium 0.4mg/L, Ribose 0.4mg/L, Thymosin 0.35mg/L, Tween 804mg/L, Uracil 0.35mg/L, xanthine sodium 0.3mg/L, phenol red 12mg/L.

Result: the result of each test parameter is basically the same as the result of the corresponding supplementary test in the above embodiment 2 and this embodiment 2, for example, for Chinese yellow cattle, the cleavage rate is 87.9%, the morula rate is 64.6%, and the blastocyst rate is 87.9%. The rate of apoptosis was 50.9%, the rate of apoptosis was 5.1%, the hatching rate of blastocysts was 72.2% on the 9th day, and the maturation rate of oocytes in vitro was 75.3%. The maturation rates of in vitro maturation of oocytes were 69.5%, 65.3%, and 57.8%, respectively.

Embodiment 7: the culture method of bovine in vitro fertilization embryo (isolated selenium copper)

With reference to the method of the corresponding supplementary test in the above embodiment 3 and this embodiment 3, selenium and copper are added in the BY basal culture solution, the only difference is that the used BY basal culture solution is replaced by an aqueous solution of the following formula:

Calcium chloride 180mg/L, ferric nitrate nonahydrate 0.75mg/L, potassium chloride 380mg/L, magnesium sulfate 100mg/L, sodium chloride 6500mg/L, sodium dihydrogen phosphate monohydrate 150mg/L, sodium bicarbonate 2000mg /L, sodium acetate 60mg/L, L-alanine 20mg/L, L-arginine hydrochloride 80mg/L, L-aspartic acid 25mg/L, L-cysteine hydrochloride Hydrate 0.12mg/L, L-cystine dihydrochloride 20mg/L, L-glutamic acid 80mg/L, glycine 40mg/L, L-histidine hydrochloride monohydrate 25mg/L, L -Hydroxyproline 8mg/L, L-isoleucine 25mg/L, L-leucine 50mg/L, L-lysine hydrochloride 80mg/L, L-methionine 10mg/L, L-benzene Alanine 30mg/L, L-proline 30mg/L, L-serine 30mg/L, L-threonine 25mg/L, L-tryptophan 12mg/L, L-tyrosine disodium dihydrate 55mg/L, L-valine 30mg/L, ascorbic acid 0.04mg/L, α-D-tocopheryl phosphate 0.012mg/L, biotin 0.008mg/L, calcidol 0.12mg/L, D- Calcium pantothenate 0.008mg/L, choline chloride 0.6mg/L, folic acid 0.008mg/L, inositol 0.06mg/L, menaquinone sodium bisulfite trihydrate 0.015mg/L, niacin 0.03mg/L, Niacinamide 0.02mg/L, p-aminobenzoic acid 0.06mg/L, pyridoxine hydrochloride 0.04mg/L, riboflavin 0.012mg/L, thiamine hydrochloride 0.008mg/L, vitamin A acetate 0.2mg /L, adenine sulfate 8mg/L, adenine 0.25mg/L, disodium adenosine triphosphate 0.8mg/L, cholesterol 0.25mg/L, 2-deoxy-D-ribose 0.4mg/L, D-glucose 1200mg /L, Glutathione 0.04mg/L, Guanine Hydrochloride 0.35mg/L, Hypoxanthine Sodium 0.3mg/L, Ribose 0.6mg/L, Thymosin 0.25mg/L, Tween 806mg/L, Uracil 0.25mg/L, sodium xanthine 0.4mg/L, phenol red 8mg/L, sodium selenite 0.3mg/L, copper sulfate (calculated as anhydrous substance) 0.05mg/L.

Result: the result of each test parameter is basically the same as the result of the corresponding supplementary test in the above embodiment 3 and this embodiment 3, for example, for Chinese yellow cattle, the cleavage rate is 87.4%, the morula rate is 64.9%, and the blastocyst rate is 87.4%. The rate of apoptosis was 51.6%, the rate of apoptosis was 5.4%, the hatching rate of blastocysts reached 72.5% on the 9th day, and the maturation rate of in vitro maturation of oocytes reached 89.1%; for Holstein, Simmental and Chinese buffalo The maturation rates of in vitro maturation of oocytes were 85.6%, 80.2%, and 73.6%, respectively.

Embodiment 8: the culture method of bovine in vitro fertilization embryo (living body selenium copper)

With reference to the method of the above Example 4 and the corresponding supplementary test in this Example 4, selenium and copper are added in the BY basal culture solution, the only difference is that the BY basal culture solution used is changed to the aqueous solution of the following formula:

Calcium chloride 220mg/L, ferric nitrate nonahydrate 0.70mg/L, potassium chloride 420mg/L, magnesium sulfate 90mg/L, sodium chloride 7000mg/L, sodium dihydrogen phosphate monohydrate 130mg/L, sodium bicarbonate 2500mg /L, sodium acetate 40mg/L, L-alanine 30mg/L, L-arginine hydrochloride 60mg/L, L-aspartic acid 35mg/L, L-cysteine hydrochloride Hydrate 0.10mg/L, L-cystine dihydrochloride 30mg/L, L-glutamic acid 70mg/L, glycine 60mg/L, L-histidine hydrochloride monohydrate 20mg/L, L -Hydroxyproline 12mg/L, L-isoleucine 15mg/L, L-leucine 70mg/L, L-lysine hydrochloride 60mg/L, L-methionine 20mg/L, L-benzene Alanine 20mg/L, L-proline 50mg/L, L-serine 20mg/L, L-threonine 35mg/L, L-tryptophan 8mg/L, L-tyrosine disodium dihydrate 60mg/L, L-valine 20mg/L, ascorbic acid 0.06mg/L, α-D-tocopheryl phosphate 0.008mg/L, biotin 0.012mg/L, calcidol 0.08mg/L, D- Calcium pantothenate 0.012mg/L, choline chloride 0.4mg/L, folic acid 0.012mg/L, inositol 0.04mg/L, menaquinone sodium bisulfite trihydrate 0.025mg/L, niacin 0.02mg/L, Niacinamide 0.03mg/L, p-aminobenzoic acid 0.04mg/L, pyridoxine hydrochloride 0.06mg/L, riboflavin 0.008mg/L, thiamine hydrochloride 0.012mg/L, vitamin A acetate 0.1mg /L, adenine sulfate 12mg/L, adenine 0.15mg/L, disodium adenosine triphosphate 1.2mg/L, cholesterol 0.15mg/L, 2-deoxy-D-ribose 0.6mg/L, D-glucose 800mg /L, Glutathione 0.06mg/L, Guanine Hydrochloride 0.25mg/L, Hypoxanthine Sodium 0.4mg/L, Ribose 0.4mg/L, Thymosin 0.35mg/L, Tween 804mg/L, Uracil 0.35mg/L, sodium xanthine 0.3mg/L, phenol red 12mg/L, sodium selenite 0.2mg/L, copper sulfate (calculated as anhydrous substance) 0.1mg/L.

Result: the result of each test parameter is basically the same as the result of the corresponding supplementary test in the above embodiment 4 and this embodiment 4. For example, for Chinese yellow cattle, the cleavage rate is 87.8%, the morula rate is 65.5%, and the blastocyst rate is 87.8%. The rate of apoptosis was 51.2%, the rate of apoptosis was 4.4%, the hatching rate of blastocyst reached 73.8% on the 9th day, and the maturation rate of in vitro maturation of oocytes reached 90.2%; for Holstein, Simmental and Chinese buffalo The maturation rates of in vitro maturation of oocytes were 84.7%, 80.5%, and 73.8%, respectively.

Although the present invention has been described in detail with general descriptions and specific embodiments above, 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, the modifications or improvements made on the basis of not departing from the spirit of the present invention all belong to the protection scope of the present invention.

Claims (10)

1. A basic culture solution, which is an aqueous solution comprising the following components: calcium chloride 180～220mg/L, ferric nitrate nonahydrate 0.70～0.75mg/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 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 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-fertility Phenol 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, menaquinone sodium bisulfite trihydrate 0.015～0.025mg/L, niacin 0.02～0.03mg/L, nicotinamide 0.02～0.03mg/L 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 acetic acid Ester 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, hypoxanthine sodium 0.3～0.4mg/L L, Ribose 0.4～0.6mg/L, Thymosin 0.25～0.35mg/L, Tween 804～6mg/L , Uracil 0.25~0.35mg/L, xanthine sodium 0.3~0.4mg/L, phenol red 8~12mg/L. 2. The basal culture fluid according to claim 1, which 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 Esters 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 L, menaquinone 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, nuclear Flavin 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. 3. The basal culture solution according to claim 1, wherein also comprising sodium selenite, its concentration is 0.2～0.3mg/L, such as 0.25mg/L; And/or, described wherein also comprising copper sulfate, its concentration is The anhydrous content is 0.05-0.1 mg/L, such as 0.075 mg/L. 4. An egg washing solution, which is a basic culture solution added with 3 mg/mL bovine serum albumin. 5. The egg-washing liquid according to claim 4, wherein said basal culture liquid is as described in any one of claims 1-4. 6. A mature culture solution, which is a basal culture solution added with 100 mL/L FBS, 10 μg/mL FSH, 10 μg/mL LH, 1 μg/mL E2, and 20 ng/mL EGF. 7. The maturation culture solution according to claim 6, wherein said basal culture solution is as described in any one of claims 1-4. 8. A mature culture solution, which is added with 10～20mmol/L (such as 15mmol/L) 4-hydroxyethylpiperazineethanesulfonic acid, 100mL/L FBS, 10μg/mL FSH, 10μg/mL LH, 1μg /mL E2, 20ng/mL EGF basal culture medium. 9. The maturation culture solution according to claim 8, wherein said basal culture solution is as described in any one of claims 1-4. 10. A method for culturing bovine IVF embryos, comprising the steps of: (1) Oocyte collection and in vitro maturation In vitro collection: Take the ovary from the slaughterhouse and put it in an insulated barrel with double-antibiotic saline, and transport it back to the laboratory within 4 hours under the condition of 30.5-33°C; extract the follicles with a surface size of 2-8mm, collect the precipitate, and examine it under a stereo microscope Next, pick out oocyte COCs (that is, cumulus-oocyte complexes) that contain at least three layers of cumulus cells, and wash them twice in egg washing liquid to remove excess impurities; Live collection: live bovine eggs are collected, and the resulting follicular fluid is picked out under a stereomicroscope to pick out cumulus-oocyte complexes (COCs) that contain at least 3 layers of cumulus cells, and put them into the mature culture medium containing HEPES Temperature 38.8 ℃, transported back to the laboratory within 3 hours; Wash the COCs collected above in vitro or in vivo in the oocyte maturation culture medium once, and then transfer to a new maturation culture medium for 22-24 hours. The culture conditions are 38.8°C, 5.5-6.5% CO2, and saturated humidity. ; (2) In vitro fertilization Wash the mature COCs once in the fertilization medium, then transfer them to the fertilization medium, put them in the incubator, and set aside; Take a frozen fine tube from liquid nitrogen and thaw it in a water bath at 37°C; cut both ends of the thin tube aseptically, inject the semen into a 15mL centrifuge tube filled with the semen preparation culture medium, and centrifuge twice at 328×g, each time After centrifuging for 5 minutes, discard the supernatant; add 300 μL of semen preparation culture medium to the above centrifuge tube, resuspend the sperm pellet, and take an appropriate sperm suspension for sperm counting; Add the calculated volume of sperm suspension into the fertilization culture drop filled with oocytes, put the culture plate into the incubator, and incubate the sperm and eggs for 16-20 hours. The culture conditions are 38.8°C, 5.5-6.5% CO2 , saturated humidity; (3) In vitro culture and storage of embryos After the in vitro fertilization operation, remove the granulosa cells around the embryo with the egg stripping needle, and put them into the embryo culture medium for culture. At this time, it is recorded as the first day of embryo culture. The culture conditions are 38.8°C, 6% O2, 88% N2, saturated humidity, record the cleavage rate on the 3rd day; record the blastocyst rate on the 7th day, and count the blastocyst hatching rate (it 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 usable embryos in the preservation solution for 3 times, equilibrate in the balance solution for 10 minutes, transfer to the freezing solution, load the embryos according to the 5-stage liquid filling method, mark them well, 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 straw was quickly taken out and placed in liquid nitrogen for cryopreservation.