CN108753689B - Method for culturing bovine in-vitro fertilized embryo - Google Patents

Method for culturing bovine in-vitro fertilized embryo Download PDF

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CN108753689B
CN108753689B CN201810576762.4A CN201810576762A CN108753689B CN 108753689 B CN108753689 B CN 108753689B CN 201810576762 A CN201810576762 A CN 201810576762A CN 108753689 B CN108753689 B CN 108753689B
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sodium
culture
embryo
hydrochloride
vitro
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王小武
郭晶
王娜
郝少强
赵明礼
马毅
郭春明
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Tianjin Limu Biotechnology Co.,Ltd.
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Abstract

The invention relates to a method for culturing bovine in vitro fertilized embryos. The method comprises the following steps: collecting and in-vitro maturing oocytes; in vitro fertilization; and (5) culturing and preserving embryos in vitro. During in vitro fertilization, mature COCs are cultured in the fertilization culture solution, during embryo in vitro culture, granulosa cells around an embryo are removed cleanly by an egg stripping needle, the embryo is placed into an embryo culture solution for culture, and after the culture is finished, the available embryo is frozen and stored in a freezing solution and liquid nitrogen. The method of the invention presents a number of excellent technical effects as described in the specification.

Description

Method for culturing bovine in-vitro fertilized embryo
Technical Field
The invention belongs to the technical field of animal breeding, relates to a technology for agriculture-animal husbandry and veterinary breeding, in particular to a method for culturing cattle in vitro fertilization embryos, and further relates to application of a culture solution related to the culture of the cattle in vitro fertilization embryos in culture of the cattle in vitro fertilization embryos. Furthermore, the invention also relates to a method for culturing the cattle in vitro fertilization embryos by using the culture solution related to the cattle in vitro fertilization embryo culture. In particular, the method for culturing bovine in vitro fertilized embryos has excellent technical effects.
Background
In Vitro Fertilization (In Vitro Fertilization) or (external Fertilization) refers to a technique In which sperm and eggs of a mammal complete a Fertilization process In an environment artificially controlled In Vitro, abbreviated In the english to IVF. Because it is inseparable from the embryo transfer technique (ET), also referred to as IVF-ET for short. In biology, an animal obtained after an in vitro fertilized embryo is transferred to a mother is called a test-tube animal. The technology is successful in the 50 s of the 20 th century, develops rapidly in the last 20 years, and is mature day by day to become an important and conventional animal breeding biotechnology.
The in vitro fertilization technology has important significance for animal reproductive mechanism research, livestock production, medicine, endangered animal protection and the like. For example, using mouse, rat or rabbit as experimental material, the in vitro fertilization technique can be used to study the gametogenesis, fertilization and early embryonic development mechanism of mammals. In the livestock breed improvement, the in vitro fertilization technology provides a cheap and efficient means for embryo production, and has important values for fully utilizing excellent breed resources, shortening the breeding cycle of livestock, accelerating the breed improvement speed and the like. In humans, IVF-ET technology is one of the important measures to treat certain infertility and to overcome sexual linked diseases. In vitro fertilization is also an indispensable component of modern biotechnology, such as mammalian embryo transplantation, cloning, transgenosis, sex control and the like.
With the development of modern agricultural science and technology, in order to make full use of the breeding potential of elite cows and accelerate the genetic breeding process, it becomes necessary to apply a new efficient breeding technology in production practice. In-vivo egg taking (OPU) and In Vitro Fertilization (IVF) are new embryo engineering technologies that have been developed rapidly In the eighties of the twentieth century, and a large number of embryos with definite genetic pedigrees can be obtained by combining the OPU and the IVF, so that the generation interval is shortened. At present, the two technologies become important breeding technologies adopted by farmers in animal husbandry developed countries such as europe, the united states and oceania for expanding stock cow groups. However, with the conventional bovine embryo culture system (CR1aa and SOF liquid), the blastocyst development rate of bovine in vitro fertilization is low, and the embryo quality is far inferior to that of in vivo embryos, so that the pregnancy rate after embryo transfer recipient is low, and therefore how to improve the blastocyst development rate and the embryo quality becomes the focus of in vitro fertilization embryo production and research.
As early as 1878, German Scnenk began to explore the in vitro fertilization technique of mammals using rabbits and guinea pigs as materials. However, in 1951, the in vitro fertilization technology has not been developed in a breakthrough after sperm capacitation was discovered by Zhang Xuanling and Austin, respectively. The cattle in vitro fertilization technology is influenced by the aspects of oocyte in vitro maturation, sperm in vitro capacitation, fertilized egg in vitro culture environment and the like.
The in vitro culture of the embryo is a key link of the IVF technology, and is also the embodiment and the test of the final effect of the oocyte in vitro maturation and in vitro fertilization technology. After in vitro fertilization, a fertilized egg undergoes a number of important changes during its development into a blastocyst, including zygote formation, first cleavage, activation of the embryonic genome, densification, and blastocyst formation. In the process, the change of the external environment can cause the change of gene expression, thereby influencing the normal development and quality of the embryo. Currently, in vitro culture studies of early mammalian embryos focus mainly on improving the composition of the culture medium to meet the nutritional requirements of the embryos at different developmental stages. Two gradually improved systems were developed based on Charles Rosenkrans 1(CR1) culture Fluid developed by Rosenkrans et al (Rosenkrans, C.F., Jr.and N.L.first, Effect of free amino acids and vitamins on clearance and maintenance of bovine zygotees in vitamins Sci, 1994.72(2): p.434-7) and Synthetic transfusion tube Fluid developed by Tervit et al (Tervit, H.R., D.G.Whittingham, and L.E.Rowson, Saccharomyces focus in vitamins of market and vegetable and vitamin J.reprod.Fertil, 1972.30(3): p.493-7). According to the research results of Hakan Sagirkaya et al (Sagirkaya, H., et al, Development potential of bovine embryos culture in differential formation and culture conditions. animal Reprod Sci,2007.101(3-4): p.225-40) and Somfai et al (Somfai, T., et al, Development of bovine embryos culture in CR1aa and IVD101 medium using differential oxygen delivery and culture system. vector Act Hung,2010.58(4): p.465-74), CR1aa culture solution has a good effect on bovine embryo culture and can be widely applied to bovine embryo culture; studies by Thompson, J.G., et al (Thompson, J.G., et al, effects of inhibitors and undercouplers of oxidative phosphorylation reactions and suspensions of bovine embryos cultured in vitro. J. Reprod. Fertil,2000.118(1): p.47-55) and by J.Feugang et al (Feugang, J.M., O.Camigo-Rodriguez, and E.Memil, Culture systems for bovine embryos Science 2009.121(2-3): p.141-149) show that SOF medium is also a suitable Culture system for bovine embryo Culture. The research results of Zhangzhiping et al (Zhangzhiping, anzhixing, Zhang Rust, Zhangong, optimization of cattle embryo culture system, proceedings of northwest university of agriculture and forestry, 2006.34) and Morgan et al (Jun sang nationality, research on cattle oocyte and in vitro embryo culture technology, 2008) also show that the optimized CR1aa and SOF culture solution are both suitable for cattle embryo culture in vitro and achieve good culture effects. Mammalian early embryonic development is a highly coordinated and precisely regulated process. During evolution, gametocytes gradually form a series of molecular cascade networks to ensure that the embryonic development cycle proceeds systematically. During development, the balance of Reactive Oxygen Species (ROS) and antioxidants in and out of the embryo plays a crucial role in early embryo development.
Most biochemical reactions generate ROS, which play important roles both inside and outside the cell, and some ROS function as 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) report that ROS can cause cellular DNA damage, oxidation of unsaturated fatty acids, oxidation of amino acids in proteins and even inactivation of certain enzymes. In general, ROS exist in four forms, of which H2O2 has a strong oxidizing effect and is the most important factor causing oxidative damage.
Numerous studies have shown that Glutathione (GSH), an antioxidant in a non-protein form, is capable of scavenging a variety of free radicals: superoxide anion free radical, hydroxyl free radical, hydrogen peroxide, hypochlorous acid and lipoxy radical, and can maintain redox balance inside and outside cells. The intracellular and extracellular environment GSH and ROS levels are two important factors influencing the development process of fertilized eggs. As early as 2000, de Matos et al (de Matos, D.G.and C.C.Furnus, The animal of high yield (GSH) level after vitamin in vision formation on embryo development effect of beta-mercaptethanol, cysteine and cysteine, theriogenology,2000.53(3): p.761-71) have increased blastocyst rate by adding beta-mercaptoethanol, cysteine and cystine during in vitro embryo culture.
Although the in vitro fertilization technique can be successfully applied to many mammals, the high production cost and the low efficiency of the in vitro fertilized embryo are caused by the low blastula rate of the in vitro fertilization, so that the wide application of the technique in the rapid propagation practice of the cattle is limited. Therefore, how to reduce the cost and improve the production efficiency and quality of bovine IVF embryos becomes a problem to be solved urgently.
At present, in a cattle in-vitro fertilization technical system, CR1aa and SOF liquid are mainly used as embryo in-vitro culture solutions, and improvement is carried out on the basis, the blastocyst development rates are improved to different degrees, and the average blastocyst development rate is 30-40%. For blastocyst quality, it can be evaluated by the total number of blastocyst cells, the ratio of ICM cell number/total cell number, and the apoptosis rate. The total number of blastocysts varies depending on the stage of the blastocyst, and the average of the total number of blastocysts obtained from bovine early stage blastocysts obtained from S.Iwasaki et al (Iwasaki, S.and T.Nakahara, Cell number and intention of chromosomal antibodies in bone blast embryos transferred in vitro well formed by culture in vitro or in vivo in vitro culture, 1990.33(3): p.669-75) is 44, and the ratio of the number of ICM cells to the total number of blastocysts is about 15.8%; andrew J.Watson et al (Watson, A.J., et al, Impact of bone marrow regulation media on embryo transitions, blast definition, cell number, and apoptosis. biol Reprod,2000.62(2): p.355-64) count bovine blastocyst cell apoptosis rates at about 7.7% -13%.
CN103898046B (chinese patent application No. 201410073635.4) discloses a culture solution specially used for bovine in vitro fertilization embryos, the formula of the culture solution is: NaCl 109.5mM, KCl 3.1mM, NaHCO 326.2 mM, MgCl 2.6H 2O 0.8.8 mM, KH2PO31.19mM, sodium pyruvate 0.4mM, glucose 1.5mM, calcium hemi-lactobionate 5mM, 10 v/v% fetal bovine serum, L-glutamine 1mM, 2v/v% essential amino acids, 1v/v% nonessential amino acids and glutathione 3mM, formulated in 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 as follows: l-arginine hydrochloride 6.32g/L, L-cystine dihydrochloride 1.564g/L, L-histidine hydrochloride monohydrate 2.1g/L, L-isoleucine 2.625g/L, L-leucine 2.62g/L, L-lysine hydrochloride 3.625g/L, L-methionine 0.755g/L, L-phenylalanine 1.65g/L, L-threonine 2.38g/L, L-tryptophan 0.51g/L, L-tyrosine 1.8g/L and L-valine 2.34 g/L; the non-essential amino acid is an aqueous solution prepared by mixing the following amino acids in proportion, wherein the content of each amino acid is as follows: l-alanine 0.89g/L, L-asparagine monohydrate 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 1.05 g/L. The results of in vitro fertilization of cattle embryos placed in the culture solution are believed to be obviously superior to the control group without GSH, the blastocyst development rate and the embryo quality are improved, the cost of in vitro embryo production is reduced, an experimental basis is provided for the application of cattle IVF technology to practice, and the genetic breeding process can be greatly accelerated.
Other references:
chenda Yuan, fertilization biology 2003, Beijing, science Press;
research on technology for in vitro production of bovine embryos, namely Junshun, full-text database of Chinese Excellent Master academic paper, agricultural science and technology edition, 2007 (stage 4);
influence of caochai, cumulus cells and culture conditions on the efficiency of production of bovine in vitro embryos [ full-text database of the Chinese outstanding Master academic thesis, agricultural science and technology edition ] (stage 9);
I.H.KIM et al.Effect of exogenous glutathione on the in vitro fertilization of bovine oocytes.Theriogenology.1999,Vol.52(3)。
however, there remains a need in the art for methods of culturing bovine in vitro fertilized embryos with improved performance, for example, methods of increasing the efficiency of culturing bovine in vitro fertilized embryos, for example, methods of culturing bovine in vitro fertilized embryos with superior cost advantages.
Disclosure of Invention
The invention aims to provide a method for culturing bovine in vitro fertilized embryos with improved performance, and particularly to expect improvement of the production efficiency and the quality of bovine IVF embryos. More specifically, the present invention provides a culture solution specially used for bovine in vitro fertilization embryos and a method for culturing bovine in vitro fertilization embryos by using the culture solution. The present inventors have surprisingly found that excellent technical effects are exhibited using the method of the present invention, and the present invention has been accomplished by this finding.
To this end, the invention provides, in a first aspect, a method for culturing bovine in vitro fertilized embryos, comprising the steps of:
(1) collection and in vitro maturation of oocytes
Taking slaughterhouse ovaries, placing in a heat-preserving barrel added with double-resistance normal saline, and transporting back to a laboratory within 3h at the temperature of 30-33 ℃; extracting follicle with surface of 2-8mm, collecting precipitate, picking out oocyte COCs (i.e. cumulus-oocyte complex) with at least 3 layers of cumulus cells under a stereoscopic microscope, washing for 2 times in ovum washing solution, and removing excessive impurities;
washing the obtained COCs in oocyte maturation culture solution for 1 time, transferring to new maturation culture solution, and culturing for 22-24h under the conditions of 38.8 deg.C, 5.5-6.5% CO2, and saturated humidity;
(2) in vitro fertilization
Washing mature COCs in a fertilization culture solution for 1 time, transferring the COCs into the fertilization culture solution, and putting the COCs into an incubator for later use;
taking a frozen tubule from liquid nitrogen, and unfreezing in a water bath at 37 ℃; aseptically cutting two ends of the thin tube, injecting semen into 15mL centrifuge tube containing semen preparation culture solution, centrifuging at 328 Xg for 2 times (5 min each time), and discarding supernatant after centrifuging; adding 300 mu L of semen preparation culture solution into the centrifuge tube, re-suspending the sperm precipitate, and taking proper sperm suspension for sperm counting;
adding the calculated volume of sperm suspension into the fertilization culture liquid drop containing the oocyte, and placing the culture disc into an incubator to incubate the sperm and the ovum for 16-20h, wherein the culture conditions are 38.8 ℃, 5.5-6.5% CO2 and saturated humidity;
(3) embryo in vitro culture and preservation
After the in vitro fertilization operation is finished, removing granular cells around the embryo by using an ovum-peeling needle, putting the granules into embryo culture solution for culture, recording the culture condition as the 1 st day of the embryo culture, wherein the culture condition is 38.8 ℃, 6% O2, 88% N2 and saturation humidity, and recording the cleavage rate on the 3 rd day; recording the blastocyst rate on the 7 th day, counting the blastocyst hatching rate (which is the percentage obtained by dividing the number of hatched blastocysts by the number of blastocysts) by the 9 th day, and carrying out quality identification;
washing the available embryo in preservation solution for 3 times, balancing in balancing solution for 10min, transferring into freezing solution, loading into embryo according to 5-stage liquid loading method, marking, cooling to-35 deg.C at 0.5 deg.C/min in program cooling instrument, taking out the tubule of embryo rapidly, and placing into liquid nitrogen for freezing.
The method according to the first aspect of the present invention, wherein in the step (1), the superantibisaline is a physiological saline containing 400IU/mL of penicillin and 400. mu.g/mL of streptomycin.
The method according to the first aspect of the present invention, wherein in the step (1), the egg-washing solution is BY basal medium supplemented with 3mg/mL bovine serum albumin.
The preparation method of the egg-washing solution or other culture solution of the present invention is easily realized BY those skilled in the art, for example, for the egg-washing solution, bovine serum albumin is usually added to the BY basal culture solution of the present invention prepared in advance to reach the corresponding required concentration, and such preparation method is also commonly used BY those skilled in the art.
The method according to the first aspect of the present invention, wherein in step (1), the maturation medium is BY basal medium supplemented with 100mL/L FBS, 10. mu.g/mL FSH, 10. mu.g/mL LH, 1. mu.g/mL E2, 20ng/mL EGF.
In the present invention, EGF-epidermal growth factor, FSH-follicle stimulating hormone, FBS-fetal bovine serum, E2-estradiol, LH-luteinizing hormone.
The method according to the first aspect of the present invention, wherein in step (2), the fertilization medium is an aqueous solution comprising 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 sodium pyruvate, 10. mu.g/ml heparin, 4mg/ml Bovine Serum Albumin (BSA), 100U/ml penicillin, 100. mu.g/ml streptomycin.
The method according to the first aspect of the present invention, wherein in the step (2), the semen preparation medium is an aqueous solution 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 sodium pyruvate, 10. mu.g/ml heparin, 4mg/ml Bovine Serum Albumin (BSA), 10mM caffeine, 100U/ml penicillin, 100. mu.g/ml streptomycin.
The method according to the first aspect of the present invention, wherein in step (3), the embryo culture fluid comprises: 109.5mM sodium chloride, 3.1mM potassium chloride, 26.2mM sodium bicarbonate, 0.8mM magnesium chloride hexahydrate, 1.19mM monopotassium phosphate, 0.4mM sodium pyruvate, 1.5mM glucose, 5mM calcium half-lactobionate, 2.5v/v% Fetal Bovine Serum (FBS), 1mM L-glutamine, 2v/v% essential amino acids, 1v/v% nonessential amino acids, 3mM glutathione, 0.04w/v% sodium citrate, 0.02w/v% maltose in water; the essential amino acid is added by the following amino acids according to the weight proportion: 6.32g of L-arginine hydrochloride, 1.564g of L-cystine dihydrochloride, 2.1g of L-histidine hydrochloride monohydrate, 2.625g of L-isoleucine, 2.62g of L-leucine, 3.625g of L-lysine hydrochloride, 0.755g of L-methionine, 1.65g of L-phenylalanine, 2.38g of L-threonine, 0.51g of L-tryptophan, 1.8g of L-tyrosine and 2.34g of L-valine, wherein the optional amino acids are added in the following weight ratio: 0.89g of L-alanine, 1.5g of L-asparagine monohydrate, 1.33g of L-aspartic acid, 1.47g of L-glutamic acid, 0.75g of glycine, 1.15g of L-proline and 1.05g of L-serine.
The method according to the first aspect of the present invention, wherein in step (1), the BY basal medium is an aqueous solution comprising: 180-220 mg/L calcium chloride, 0.70-0.75 mg/L ferric nitrate nonahydrate, 380-420 mg/L potassium chloride, 90-100 mg/L magnesium sulfate, 6500-7000 mg/L sodium chloride, 130-150 mg/L monosodium phosphate monohydrate, 2000-2500 mg/L sodium bicarbonate, 40-60 mg/L, L-alanine 20-30 mg/L, L-sodium acetate, 60-80 mg/L, L-aspartic acid, 25-35 mg/L, L-cysteine hydrochloride monohydrate, 0.10-0.12 mg/L, L-cystine dihydrochloride, 20-30 mg/L, L-glutamic acid, 40-60 mg/L, L-glycine-histidine hydrochloride monohydrate, 20-25 mg/L, L-hydroxyproline 8-12 mg/L, 15-25 mg/L, L-leucine 50-70 mg/L, L-lysine hydrochloride 60-80 mg/L, L-methionine 10-20 mg/L, L-phenylalanine 20-30 mg/L, L-proline 30-50 mg/L, L-serine 20-30 mg/L, L-threonine 25-35 mg/L, L-tryptophan 8-12 mg/L, L-tyrosine disodium dihydrate 55-60 mg/L, L-valine 20-30 mg/L, ascorbic acid 0.04-0.06 mg/L, alpha-D-tocopherol phosphate 0.008-0.012 mg/L, biotin 0.008-0.012 mg/L, calcitol 0.08-0.12 mg/L, D-calcium pantothenate 0.008-0.012 mg/L, 0.4-0.6 mg/L choline chloride, 0.008-0.012 mg/L folic acid, 0.04-0.06 mg/L inositol, 0.015-0.025 mg/L menadione sodium bisulfite trihydrate, 0.02-0.03 mg/L nicotinic acid, 0.02-0.03 mg/L nicotinamide, 0.04-0.06 mg/L L, p-aminobenzoic acid, 0.04-0.06 mg/L pyridoxine hydrochloride, 0.008-0.012 mg/L riboflavin, 0.008-0.012 mg/L thiamine hydrochloride, 0.1-0.2 mg/L vitamin A acetate, 8-12 mg/L adenine sulfate, 0.15-0.25 mg/L adenine, 0.8-1.2 mg/L disodium adenosine triphosphate, 0.15-0.25 mg/L cholesterol, 2-deoxy-D-0.4-0.6 mg/L glucose, 0.04-0.84 mg/L glutathione, 0.06mg/L glucose, 0.25-0.35 mg/L guanine hydrochloride, 0.3-0.4 mg/L hypoxanthine sodium, 0.4-0.6 mg/L ribose, 0.25-0.35 mg/L thymosin, 804-6 mg/L tween, 0.25-0.35 mg/L uracil, 0.3-0.4 mg/L xanthine sodium and 8-12 mg/L phenol red.
The method according to the first aspect of the present invention, wherein in step (1), the BY basal medium is an aqueous solution comprising: 200mg/L calcium chloride, 0.72mg/L ferric nitrate nonahydrate, 400mg/L potassium chloride, 97.7mg/L magnesium sulfate, 6800mg/L sodium chloride, 140mg/L monobasic sodium phosphate monohydrate, 2200mg/L sodium bicarbonate, 50mg/L, L-sodium acetate, 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 leucine, 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, alpha-D-tocopherol phosphate 0.01mg/L, biotin 0.01mg/L, calciferol 0.1mg/L, D-calcium pantothenate 0.01mg/L, choline chloride 0.5mg/L, folic acid 0.01mg/L, inositol 0.05mg/L, menadione trihydrate sodium bisulfite 0.019mg/L, nicotinic acid 0.025mg/L, Nicotinamide 0.025mg/L, p-aminobenzoic acid 0.05mg/L, pyridoxine hydrochloride 0.05mg/L, riboflavin 0.01mg/L, thiamine hydrochloride 0.01mg/L, vitamin A acetate 0.14mg/L, adenine 10mg/L, adenine 0.2mg/L, disodium adenosine triphosphate 1mg/L, cholesterol 0.2mg/L, 2-deoxy-D-ribose 0.5mg/L, D-glucose 1000mg/L, glutathione 0.05mg/L, guanine hydrochloride 0.3mg/L, sodium hypoxanthine 0.354mg/L, ribose 0.5mg/L, thymosin 0.3mg/L, tween 805mg/L, uracil 0.3mg/L, sodium xanthine 0.34mg/L, phenol red 10 mg/L.
The method according to the first aspect of the present invention, wherein in step (1), the BY basal medium further comprises sodium selenite at a concentration of 0.2-0.3 mg/L, such as 0.25 mg/L; and/or the BY basic culture solution also comprises copper sulfate, and the concentration of the copper sulfate is 0.05-0.1 mg/L (calculated BY anhydrous substance), such as 0.075 mg/L.
In the present invention, for example, in step (3), the preservation solution, equilibration solution, freezing solution and the like for embryos are well known in the art and are readily available from commercial sources, for example, the freezing solution may be FreezeKit sold domestically by Vitroffe, SwedenTM Cleave。
Further, the second aspect of the present invention provides an egg-washing solution, which is BY basal medium supplemented with 3mg/mL bovine serum albumin.
The egg wash according to the second aspect of the present invention, wherein the BY basal medium is an aqueous solution comprising: 180-220 mg/L calcium chloride, 0.70-0.75 mg/L ferric nitrate nonahydrate, 380-420 mg/L potassium chloride, 90-100 mg/L magnesium sulfate, 6500-7000 mg/L sodium chloride, 130-150 mg/L monosodium phosphate monohydrate, 2000-2500 mg/L sodium bicarbonate, 40-60 mg/L, L-alanine 20-30 mg/L, L-sodium acetate, 60-80 mg/L, L-aspartic acid, 25-35 mg/L, L-cysteine hydrochloride monohydrate, 0.10-0.12 mg/L, L-cystine dihydrochloride, 20-30 mg/L, L-glutamic acid, 40-60 mg/L, L-glycine-histidine hydrochloride monohydrate, 20-25 mg/L, L-hydroxyproline 8-12 mg/L, 15-25 mg/L, L-leucine 50-70 mg/L, L-lysine hydrochloride 60-80 mg/L, L-methionine 10-20 mg/L, L-phenylalanine 20-30 mg/L, L-proline 30-50 mg/L, L-serine 20-30 mg/L, L-threonine 25-35 mg/L, L-tryptophan 8-12 mg/L, L-tyrosine disodium dihydrate 55-60 mg/L, L-valine 20-30 mg/L, ascorbic acid 0.04-0.06 mg/L, alpha-D-tocopherol phosphate 0.008-0.012 mg/L, biotin 0.008-0.012 mg/L, calcitol 0.08-0.12 mg/L, D-calcium pantothenate 0.008-0.012 mg/L, 0.4-0.6 mg/L choline chloride, 0.008-0.012 mg/L folic acid, 0.04-0.06 mg/L inositol, 0.015-0.025 mg/L menadione sodium bisulfite trihydrate, 0.02-0.03 mg/L nicotinic acid, 0.02-0.03 mg/L nicotinamide, 0.04-0.06 mg/L L, p-aminobenzoic acid, 0.04-0.06 mg/L pyridoxine hydrochloride, 0.008-0.012 mg/L riboflavin, 0.008-0.012 mg/L thiamine hydrochloride, 0.1-0.2 mg/L vitamin A acetate, 8-12 mg/L adenine sulfate, 0.15-0.25 mg/L adenine, 0.8-1.2 mg/L disodium adenosine triphosphate, 0.15-0.25 mg/L cholesterol, 2-deoxy-D-0.4-0.6 mg/L glucose, 0.04-0.84 mg/L glutathione, 0.06mg/L glucose, 0.25-0.35 mg/L guanine hydrochloride, 0.3-0.4 mg/L hypoxanthine sodium, 0.4-0.6 mg/L ribose, 0.25-0.35 mg/L thymosin, 804-6 mg/L tween, 0.25-0.35 mg/L uracil, 0.3-0.4 mg/L xanthine sodium and 8-12 mg/L phenol red.
The egg wash according to the second aspect of the present invention, wherein the BY basal medium is an aqueous solution comprising: 200mg/L calcium chloride, 0.72mg/L ferric nitrate nonahydrate, 400mg/L potassium chloride, 97.7mg/L magnesium sulfate, 6800mg/L sodium chloride, 140mg/L monobasic sodium phosphate monohydrate, 2200mg/L sodium bicarbonate, 50mg/L, L-sodium acetate, 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 leucine, 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, alpha-D-tocopherol phosphate 0.01mg/L, biotin 0.01mg/L, calciferol 0.1mg/L, D-calcium pantothenate 0.01mg/L, choline chloride 0.5mg/L, folic acid 0.01mg/L, inositol 0.05mg/L, menadione trihydrate sodium bisulfite 0.019mg/L, nicotinic acid 0.025mg/L, Nicotinamide 0.025mg/L, p-aminobenzoic acid 0.05mg/L, pyridoxine hydrochloride 0.05mg/L, riboflavin 0.01mg/L, thiamine hydrochloride 0.01mg/L, vitamin A acetate 0.14mg/L, adenine 10mg/L, adenine 0.2mg/L, disodium adenosine triphosphate 1mg/L, cholesterol 0.2mg/L, 2-deoxy-D-ribose 0.5mg/L, D-glucose 1000mg/L, glutathione 0.05mg/L, guanine hydrochloride 0.3mg/L, sodium hypoxanthine 0.354mg/L, ribose 0.5mg/L, thymosin 0.3mg/L, tween 805mg/L, uracil 0.3mg/L, sodium xanthine 0.34mg/L, phenol red 10 mg/L.
The egg washing solution according to the second aspect of the invention, wherein the BY basal medium further comprises sodium selenite with a concentration of 0.2-0.3 mg/L, such as 0.25 mg/L; and/or the BY basic culture solution also comprises copper sulfate, and the concentration of the copper sulfate is 0.05-0.1 mg/L (calculated BY anhydrous substance), such as 0.075 mg/L.
Further, the third aspect of the present invention provides a maturation culture solution comprising BY basal culture solution supplemented with 100mL/L FBS, 10. mu.g/mL FSH, 10. mu.g/mL LH, 1. mu.g/mL E2, and 20ng/mL EGF.
The maturation culture solution according to the third aspect of the present invention, wherein the BY base culture solution is an aqueous solution comprising: 180-220 mg/L calcium chloride, 0.70-0.75 mg/L ferric nitrate nonahydrate, 380-420 mg/L potassium chloride, 90-100 mg/L magnesium sulfate, 6500-7000 mg/L sodium chloride, 130-150 mg/L monosodium phosphate monohydrate, 2000-2500 mg/L sodium bicarbonate, 40-60 mg/L, L-alanine 20-30 mg/L, L-sodium acetate, 60-80 mg/L, L-aspartic acid, 25-35 mg/L, L-cysteine hydrochloride monohydrate, 0.10-0.12 mg/L, L-cystine dihydrochloride, 20-30 mg/L, L-glutamic acid, 40-60 mg/L, L-glycine-histidine hydrochloride monohydrate, 20-25 mg/L, L-hydroxyproline 8-12 mg/L, 15-25 mg/L, L-leucine 50-70 mg/L, L-lysine hydrochloride 60-80 mg/L, L-methionine 10-20 mg/L, L-phenylalanine 20-30 mg/L, L-proline 30-50 mg/L, L-serine 20-30 mg/L, L-threonine 25-35 mg/L, L-tryptophan 8-12 mg/L, L-tyrosine disodium dihydrate 55-60 mg/L, L-valine 20-30 mg/L, ascorbic acid 0.04-0.06 mg/L, alpha-D-tocopherol phosphate 0.008-0.012 mg/L, biotin 0.008-0.012 mg/L, calcitol 0.08-0.12 mg/L, D-calcium pantothenate 0.008-0.012 mg/L, 0.4-0.6 mg/L choline chloride, 0.008-0.012 mg/L folic acid, 0.04-0.06 mg/L inositol, 0.015-0.025 mg/L menadione sodium bisulfite trihydrate, 0.02-0.03 mg/L nicotinic acid, 0.02-0.03 mg/L nicotinamide, 0.04-0.06 mg/L L, p-aminobenzoic acid, 0.04-0.06 mg/L pyridoxine hydrochloride, 0.008-0.012 mg/L riboflavin, 0.008-0.012 mg/L thiamine hydrochloride, 0.1-0.2 mg/L vitamin A acetate, 8-12 mg/L adenine sulfate, 0.15-0.25 mg/L adenine, 0.8-1.2 mg/L disodium adenosine triphosphate, 0.15-0.25 mg/L cholesterol, 2-deoxy-D-0.4-0.6 mg/L glucose, 0.04-0.84 mg/L glutathione, 0.06mg/L glucose, 0.25-0.35 mg/L guanine hydrochloride, 0.3-0.4 mg/L hypoxanthine sodium, 0.4-0.6 mg/L ribose, 0.25-0.35 mg/L thymosin, 804-6 mg/L tween, 0.25-0.35 mg/L uracil, 0.3-0.4 mg/L xanthine sodium and 8-12 mg/L phenol red.
The maturation culture solution according to the third aspect of the present invention, wherein the BY base culture solution is an aqueous solution comprising: 200mg/L calcium chloride, 0.72mg/L ferric nitrate nonahydrate, 400mg/L potassium chloride, 97.7mg/L magnesium sulfate, 6800mg/L sodium chloride, 140mg/L monobasic sodium phosphate monohydrate, 2200mg/L sodium bicarbonate, 50mg/L, L-sodium acetate, 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 leucine, 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, alpha-D-tocopherol phosphate 0.01mg/L, biotin 0.01mg/L, calciferol 0.1mg/L, D-calcium pantothenate 0.01mg/L, choline chloride 0.5mg/L, folic acid 0.01mg/L, inositol 0.05mg/L, menadione trihydrate sodium bisulfite 0.019mg/L, nicotinic acid 0.025mg/L, Nicotinamide 0.025mg/L, p-aminobenzoic acid 0.05mg/L, pyridoxine hydrochloride 0.05mg/L, riboflavin 0.01mg/L, thiamine hydrochloride 0.01mg/L, vitamin A acetate 0.14mg/L, adenine 10mg/L, adenine 0.2mg/L, disodium adenosine triphosphate 1mg/L, cholesterol 0.2mg/L, 2-deoxy-D-ribose 0.5mg/L, D-glucose 1000mg/L, glutathione 0.05mg/L, guanine hydrochloride 0.3mg/L, sodium hypoxanthine 0.354mg/L, ribose 0.5mg/L, thymosin 0.3mg/L, tween 805mg/L, uracil 0.3mg/L, sodium xanthine 0.34mg/L, phenol red 10 mg/L.
The maturation culture solution according to the third aspect of the present invention, wherein the BY basal culture solution further comprises sodium selenite at a concentration of 0.2-0.3 mg/L, such as 0.25 mg/L; and/or the BY basic culture solution also comprises copper sulfate, and the concentration of the copper sulfate is 0.05-0.1 mg/L (calculated BY anhydrous substance), such as 0.075 mg/L.
Any technical feature possessed by any one aspect of the invention or any embodiment of that aspect is equally applicable to any other embodiment or any embodiment of any other aspect, so long as they are not mutually inconsistent, although appropriate modifications to the respective features may be made as necessary when applicable to each other. Various aspects and features of the disclosure are described further below.
All documents cited herein are incorporated by reference in their entirety and to the extent such documents do not conform to the meaning of the present invention, the present invention shall control. Further, the various terms and phrases used herein have the ordinary meaning as is known to those skilled in the art, and even though such terms and phrases are intended to be described or explained in greater detail herein, reference is made to the term and phrase as being inconsistent with the known meaning and meaning as is accorded to such meaning throughout this disclosure.
The fetal bovine serum used in the present invention can be easily obtained in a standardized commercial form from the market, and for example, Australian fetal bovine serum (cat # 10099141) from Gibco, New Zealand fetal bovine serum (cat # 10091148), North American fetal bovine serum (cat # 16000044), Mexico fetal bovine serum (cat # 10437028) and the like are available from various agents, and 500ml of these commercial fetal bovine serum is mostly 8000 yuan or more, and when used in the bovine in vitro fertilization embryo culture solution of the present invention, fetal bovine serum is a major contributor to the cost. In the experiments in the context of the present invention, the fetal bovine serum used was Australian fetal bovine serum from Gibco (cat # 10099141), unless otherwise specified.
Detailed Description
The present invention will be further described by the following examples, however, the scope of the present invention is not limited to the following examples. It will be understood by those skilled in the art that various changes and modifications may be made to the invention without departing from the spirit and scope of the invention. The present invention has been described generally and/or specifically with respect to materials used in testing and testing methods. Although many materials and methods of operation are known in the art for the purpose of carrying out the invention, the invention is nevertheless described herein in as detail as possible. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art, and the raw materials used are commercially available products.
Example 1: culture method of bovine in vitro fertilized embryo
Reagent
In the specific test of the present invention, the reagents used are described in detail below, unless otherwise specified:
adding double-resistant normal saline: contains normal saline solution of penicillin 400IU/mL and streptomycin 400 mug/mL.
Washing the egg: BY basal medium supplemented with 3mg/mL bovine serum albumin.
Mature culture solution: BY basal medium supplemented with 100mL/L FBS, 10. mu.g/mL FSH, 10. mu.g/mL LH, 1. mu.g/mL E2, 20ng/mL EGF. Wherein EGF is epidermal growth factor, FSH is follicle stimulating hormone, FBS is fetal bovine serum, E2 is estradiol, and LH is luteinizing hormone.
Fertilization culture solution: an aqueous solution comprising 112.0mM sodium chloride, 4.02mM potassium chloride, 2.25mM calcium chloride dihydrate, 0.52mM magnesium chloride hexahydrate, 0.83mM monopotassium phosphate, 37.0mM sodium bicarbonate, 1.25mM sodium pyruvate, 10. mu.g/ml heparin, 4mg/ml Bovine Serum Albumin (BSA), 100U/ml penicillin, 100. mu.g/ml streptomycin.
Preparing a culture solution from semen: an aqueous solution comprising 112.0mM sodium chloride, 4.02mM potassium chloride, 2.25mM calcium chloride dihydrate, 0.52mM magnesium chloride hexahydrate, 0.83mM monopotassium phosphate, 37.0mM sodium bicarbonate, 1.25mM sodium pyruvate, 10. mu.g/ml heparin, 4mg/ml Bovine Serum Albumin (BSA), 10mM caffeine, 100U/ml penicillin, 100. mu.g/ml streptomycin.
Embryo culture solution: comprises the following steps: 109.5mM sodium chloride, 3.1mM potassium chloride, 26.2mM sodium bicarbonate, 0.8mM magnesium chloride hexahydrate, 1.19mM monopotassium phosphate, 0.4mM sodium pyruvate, 1.5mM glucose, 5mM calcium half-lactobionate, 2.5v/v% Fetal Bovine Serum (FBS), 1mM L-glutamine, 2v/v% essential amino acids, 1v/v% nonessential amino acids, 3mM glutathione, 0.04w/v% sodium citrate, 0.02w/v% maltose in water; the essential amino acid is added by the following amino acids according to the weight proportion: 6.32g of L-arginine hydrochloride, 1.564g of L-cystine dihydrochloride, 2.1g of L-histidine hydrochloride monohydrate, 2.625g of L-isoleucine, 2.62g of L-leucine, 3.625g of L-lysine hydrochloride, 0.755g of L-methionine, 1.65g of L-phenylalanine, 2.38g of L-threonine, 0.51g of L-tryptophan, 1.8g of L-tyrosine and 2.34g of L-valine, wherein the optional amino acids are added in the following weight ratio: 0.89g of L-alanine, 1.5g of L-asparagine monohydrate, 1.33g of L-aspartic acid, 1.47g of L-glutamic acid, 0.75g of glycine, 1.15g of L-proline and 1.05g of L-serine.
The BY basal medium is an aqueous solution containing the following components: 200mg/L calcium chloride, 0.72mg/L ferric nitrate nonahydrate, 400mg/L potassium chloride, 97.7mg/L magnesium sulfate, 6800mg/L sodium chloride, 140mg/L monobasic sodium phosphate monohydrate, 2200mg/L sodium bicarbonate, 50mg/L, L-sodium acetate, 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 leucine, 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, alpha-D-tocopherol phosphate 0.01mg/L, biotin 0.01mg/L, calciferol 0.1mg/L, D-calcium pantothenate 0.01mg/L, choline chloride 0.5mg/L, folic acid 0.01mg/L, inositol 0.05mg/L, menadione trihydrate sodium bisulfite 0.019mg/L, nicotinic acid 0.025mg/L, Nicotinamide 0.025mg/L, p-aminobenzoic acid 0.05mg/L, pyridoxine hydrochloride 0.05mg/L, riboflavin 0.01mg/L, thiamine hydrochloride 0.01mg/L, vitamin A acetate 0.14mg/L, adenine 10mg/L, adenine 0.2mg/L, disodium adenosine triphosphate 1mg/L, cholesterol 0.2mg/L, 2-deoxy-D-ribose 0.5mg/L, D-glucose 1000mg/L, glutathione 0.05mg/L, guanine hydrochloride 0.3mg/L, sodium hypoxanthine 0.354mg/L, ribose 0.5mg/L, thymosin 0.3mg/L, tween 805mg/L, uracil 0.3mg/L, sodium xanthine 0.34mg/L, phenol red 10 mg/L.
II, cattle in-vitro fertilization and embryo culture:
step (1), collection and in vitro maturation of oocytes
Taking slaughterhouse ovaries, placing in a heat-preserving barrel added with double-resistance normal saline, and transporting back to a laboratory within 3h at the temperature of 30-33 ℃; extracting follicle with surface of 2-8mm, collecting precipitate, picking out oocyte COCs (i.e. cumulus-oocyte complex) with at least 3 layers of cumulus cells under a stereoscopic microscope, washing for 2 times in ovum washing solution, and removing excessive impurities;
washing the obtained COCs in oocyte maturation culture solution for 1 time, transferring to new maturation culture solution, and culturing for 22-24h (actual operation for 24 hr) under the conditions of 38.8 deg.C, 5.5-6.5% CO2, and saturated humidity;
step (2), in vitro fertilization
Washing mature COCs in a fertilization culture solution for 1 time, transferring the COCs into the fertilization culture solution, and putting the COCs into an incubator for later use;
taking a frozen tubule from liquid nitrogen, and unfreezing in a water bath at 37 ℃; aseptically cutting two ends of the thin tube, injecting semen into 15mL centrifuge tube containing semen preparation culture solution, centrifuging at 328 Xg for 2 times (5 min each time), and discarding supernatant after centrifuging; adding 300 mu L of semen preparation culture solution into the centrifuge tube, re-suspending the sperm precipitate, and taking proper sperm suspension for sperm counting;
adding the calculated volume of sperm suspension into the fertilization culture liquid drop containing the oocyte, and placing the culture disc into an incubator to incubate the sperm and the ovum for 16-20h (actual operation is 18 h), wherein the culture conditions are 38.8 ℃, 5.5-6.5% CO2 and saturated humidity;
step (3), embryo in vitro culture and preservation
After the in vitro fertilization operation is finished, removing granular cells around the embryo by using an ovum-peeling needle, putting the granules into embryo culture solution for culture, recording the culture condition as the 1 st day of the embryo culture, wherein the culture condition is 38.8 ℃, 6% O2, 88% N2 and saturation humidity, and recording the cleavage rate on the 3 rd day; recording the blastocyst rate on the 7 th day, counting the blastocyst hatching rate (which is the percentage obtained by dividing the number of hatched blastocysts by the number of blastocysts) by the 9 th day, and carrying out quality identification;
washing the available embryo in preservation solution for 3 times, balancing in balancing solution for 10min, transferring into freezing solution, loading into embryo according to 5-stage liquid loading method, marking, cooling to-35 deg.C at 0.5 deg.C/min in program cooling instrument, taking out the tubule of embryo rapidly, and placing into liquid nitrogen for freezing.
Method for differential staining of embryo
1. Blastocysts from day 7 of in vitro culture were selected and fixed with 2% paraformaldehyde for 20 min.
2. The cells were washed twice with phosphate buffered saline (PBS-BSA) containing 0.5% BSA, and placed in a permeabilizing solution (50. mu.l Triton, 5. mu.l Tween 80 and 9.945ml PBS) at room temperature for 30 min.
3. CDX2 protein was able to bind to primary antibodies by treatment with 2M hydrochloric acid at room temperature for 20min, followed by treatment with 100mM Tris-HCl at room temperature for 10 min.
4. Washed three times with PBS-BSA, the blastocysts were placed in blocking solution (1ml goat serum, 5. mu.l Tween 80 and 8.995ml PBS), blocked for 1h at room temperature, and then blocked overnight in a refrigerator at 4 ℃.
5. Discarding the blocking solution, diluting CDX2 primary antibody with the blocking solution at a ratio of 1:200, incubating at room temperature for 2h, discarding the primary antibody dilution, and washing with PBS-BSA for 5min for 3 times.
Caspase-3 primary antibody (available from Cell Signaling Technology) was diluted 1:200 with blocking solution, incubated at room temperature for 2h, the primary antibody dilution was discarded, and washed 3 times with PBS-BSA for 5min each.
7. CDX 2-specific secondary antibody (purchased from Sigma) was diluted 1:200 with blocking solution under exclusion of light and left at room temperature for 1h without light. The secondary antibody dilutions were discarded in the dark and washed 3 times with PBS for 5min each.
8. Caspase-3 specific secondary antibodies (purchased from Life Technologies) were diluted 1:200 with blocking solution under dark conditions and left at room temperature for 1h under dark conditions. The secondary antibody dilutions were discarded in the dark and washed 3 times with PBS for 5min each.
9. Cell nuclei were stained with 10. mu.g/mL Hochest 33342 dye, allowed to act at room temperature for 5min, observed under a fluorescent microscope and photographed.
10. The experiment is repeated three times, 10 blasts are randomly selected each time, and the blast quality is evaluated by calculating the apoptosis rate and the number of ICM cells/total number of cells.
The data statistical method comprises the following steps: experimental data were analyzed using the ANOVA program in statistical software SAS V8 and the Duncan's multiple-range test method judged significant differences between treatments, which were considered significant when p < 0.05.
In the present invention, cleavage rate is fertilization cleavage number/fertilization egg number. In the present invention, blastocyst rate is blastocyst number/cleavage embryo number.
Fourth, In Vitro Maturation (IVM) Effect of oocytes-maturation Rate
In step (1) of this example 1, after the in vitro maturation culture, the oocytes were observed under an inverted microscope, and the oocytes were judged to have matured if they had released the first polar body, had retained the viscous matrix secreted from the cumulus cells, had significantly expanded cell layers, and had spread radially around the ovum as the center, and the number of matured oocytes was recorded, and the maturation rate was calculated.
Five results
In this example 1, a test was conducted on a Chinese cattle (Nanyang cattle, working 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, blastocyst hatchability reached 72.3% on day 9. The maturation rate of the oocyte in vitro maturation reaches 75.6 percent.
In a supplementary test, with reference to the method of example 1 above, three types of cattle, holstein cattle (dairy cattle breed), simmental cattle (beef cattle breed), and chinese buffalo (service breed), were tested, and the results were: the cleavage rate is within 84-89%, the morula rate is within 62-65%, the blastocyst rate is within 50-53%, the apoptosis rate is within 4-7%, and the blastocyst hatching rate on day 9 is within 71-75%; the in vitro maturation rate of oocytes of three holstein cattle, Simmental cattle and Chinese buffalo cattle is 69.3%, 65.6% and 57.8% respectively.
Example 2: culture method of bovine in vitro fertilized embryo
The main difference between this example 2 and the above example 1 is that 0.25mg/L sodium selenite and 0.075mg/L anhydrous copper sulfate are additionally added into BY basal medium.
Reagent
In the specific test of the present invention, the reagents used are described in detail below, unless otherwise specified:
adding double-resistant normal saline: contains normal saline solution of penicillin 400IU/mL and streptomycin 400 mug/mL.
Washing the egg: BY basal medium supplemented with 3mg/mL bovine serum albumin.
Mature culture solution: BY basal medium supplemented with 100mL/L FBS, 10. mu.g/mL FSH, 10. mu.g/mL LH, 1. mu.g/mL E2, 20ng/mL EGF. Wherein EGF is epidermal growth factor, FSH is follicle stimulating hormone, FBS is fetal bovine serum, E2 is estradiol, and LH is luteinizing hormone.
Fertilization culture solution: an aqueous solution comprising 112.0mM sodium chloride, 4.02mM potassium chloride, 2.25mM calcium chloride dihydrate, 0.52mM magnesium chloride hexahydrate, 0.83mM monopotassium phosphate, 37.0mM sodium bicarbonate, 1.25mM sodium pyruvate, 10. mu.g/ml heparin, 4mg/ml Bovine Serum Albumin (BSA), 100U/ml penicillin, 100. mu.g/ml streptomycin.
Preparing a culture solution from semen: an aqueous solution comprising 112.0mM sodium chloride, 4.02mM potassium chloride, 2.25mM calcium chloride dihydrate, 0.52mM magnesium chloride hexahydrate, 0.83mM monopotassium phosphate, 37.0mM sodium bicarbonate, 1.25mM sodium pyruvate, 10. mu.g/ml heparin, 4mg/ml Bovine Serum Albumin (BSA), 10mM caffeine, 100U/ml penicillin, 100. mu.g/ml streptomycin.
Embryo culture solution: comprises the following steps: 109.5mM sodium chloride, 3.1mM potassium chloride, 26.2mM sodium bicarbonate, 0.8mM magnesium chloride hexahydrate, 1.19mM monopotassium phosphate, 0.4mM sodium pyruvate, 1.5mM glucose, 5mM calcium half-lactobionate, 10 v/v% Fetal Bovine Serum (FBS), 1mM L-glutamine, 2v/v% essential amino acids, 1v/v% nonessential amino acids, 3mM glutathione, sodium citrate 0.04w/v%, maltose 0.02w/v% aqueous solution; the essential amino acid is added by the following amino acids according to the weight proportion: 6.32g of L-arginine hydrochloride, 1.564g of L-cystine dihydrochloride, 2.1g of L-histidine hydrochloride monohydrate, 2.625g of L-isoleucine, 2.62g of L-leucine, 3.625g of L-lysine hydrochloride, 0.755g of L-methionine, 1.65g of L-phenylalanine, 2.38g of L-threonine, 0.51g of L-tryptophan, 1.8g of L-tyrosine and 2.34g of L-valine, wherein the optional amino acids are added in the following weight ratio: 0.89g of L-alanine, 1.5g of L-asparagine monohydrate, 1.33g of L-aspartic acid, 1.47g of L-glutamic acid, 0.75g of glycine, 1.15g of L-proline and 1.05g of L-serine.
The BY basal medium is an aqueous solution containing the following components: 200mg/L calcium chloride, 0.72mg/L ferric nitrate nonahydrate, 400mg/L potassium chloride, 97.7mg/L magnesium sulfate, 6800mg/L sodium chloride, 140mg/L monobasic sodium phosphate monohydrate, 2200mg/L sodium bicarbonate, 50mg/L, L-sodium acetate, 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 leucine, 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, alpha-D-tocopherol phosphate 0.01mg/L, biotin 0.01mg/L, calciferol 0.1mg/L, D-calcium pantothenate 0.01mg/L, choline chloride 0.5mg/L, folic acid 0.01mg/L, inositol 0.05mg/L, menadione trihydrate sodium bisulfite 0.019mg/L, nicotinic acid 0.025mg/L, Nicotinamide 0.025mg/L, p-aminobenzoic acid 0.05mg/L, pyridoxine hydrochloride 0.05mg/L, riboflavin 0.01mg/L, thiamine hydrochloride 0.01mg/L, vitamin A acetate 0.14mg/L, adenine 10mg/L, adenine 0.2mg/L, disodium adenosine triphosphate 1mg/L, cholesterol 0.2mg/L, 2-deoxy-D-ribose 0.5mg/L, 1000mg/L of D-glucose, 0.05mg/L of glutathione, 0.3mg/L of guanine hydrochloride, 0.354mg/L of hypoxanthine sodium, 0.5mg/L of ribose, 0.3mg/L of thymosin, 805mg/L of tween, 0.3mg/L of uracil, 0.34mg/L of xanthine sodium, 10mg/L of phenol red, 0.25mg/L of sodium selenite and 0.075mg/L of anhydrous copper sulfate.
II, cattle in-vitro fertilization and embryo culture:
step (1), collection and in vitro maturation of oocytes
Taking slaughterhouse ovaries, placing in a heat-preserving barrel added with double-resistance normal saline, and transporting back to a laboratory within 3h at the temperature of 30-33 ℃; extracting follicle with surface of 2-8mm, collecting precipitate, picking out oocyte COCs (i.e. cumulus-oocyte complex) with at least 3 layers of cumulus cells under a stereoscopic microscope, washing for 2 times in ovum washing solution, and removing excessive impurities;
washing the obtained COCs in oocyte maturation culture solution for 1 time, transferring to new maturation culture solution, and culturing for 22-24h (actual operation for 24 hr) under the conditions of 38.8 deg.C, 5.5-6.5% CO2, and saturated humidity;
step (2), in vitro fertilization
Washing mature COCs in a fertilization culture solution for 1 time, transferring the COCs into the fertilization culture solution, and putting the COCs into an incubator for later use;
taking a frozen tubule from liquid nitrogen, and unfreezing in a water bath at 37 ℃; aseptically cutting two ends of the thin tube, injecting semen into 15mL centrifuge tube containing semen preparation culture solution, centrifuging at 328 Xg for 2 times (5 min each time), and discarding supernatant after centrifuging; adding 300 mu L of semen preparation culture solution into the centrifuge tube, re-suspending the sperm precipitate, and taking proper sperm suspension for sperm counting;
adding the calculated volume of sperm suspension into the fertilization culture liquid drop containing the oocyte, and placing the culture disc into an incubator to incubate the sperm and the ovum for 16-20h (actual operation is 18 h), wherein the culture conditions are 38.8 ℃, 5.5-6.5% CO2 and saturated humidity;
step (3), embryo in vitro culture and preservation
After the in vitro fertilization operation is finished, removing granular cells around the embryo by using an ovum-peeling needle, putting the granules into embryo culture solution for culture, recording the culture condition as the 1 st day of the embryo culture, wherein the culture condition is 38.8 ℃, 6% O2, 88% N2 and saturation humidity, and recording the cleavage rate on the 3 rd day; recording the blastocyst rate on the 7 th day, counting the blastocyst hatching rate (which is the percentage obtained by dividing the number of hatched blastocysts by the number of blastocysts) by the 9 th day, and carrying out quality identification;
washing the available embryo in preservation solution for 3 times, balancing in balancing solution for 10min, transferring into freezing solution, loading into embryo according to 5-stage liquid loading method, marking, cooling to-35 deg.C at 0.5 deg.C/min in program cooling instrument, taking out the tubule of embryo rapidly, and placing into liquid nitrogen for freezing.
Method for differential staining of embryo
1. Blastocysts from day 7 of in vitro culture were selected and fixed with 2% paraformaldehyde for 20 min.
2. The cells were washed twice with phosphate buffered saline (PBS-BSA) containing 0.5% BSA, and placed in a permeabilizing solution (50. mu.l Triton, 5. mu.l Tween 80 and 9.945ml PBS) at room temperature for 30 min.
3. CDX2 protein was able to bind to primary antibodies by treatment with 2M hydrochloric acid at room temperature for 20min, followed by treatment with 100mM Tris-HCl at room temperature for 10 min.
4. Washed three times with PBS-BSA, the blastocysts were placed in blocking solution (1ml goat serum, 5. mu.l Tween 80 and 8.995ml PBS), blocked for 1h at room temperature, and then blocked overnight in a refrigerator at 4 ℃.
5. Discarding the blocking solution, diluting CDX2 primary antibody with the blocking solution at a ratio of 1:200, incubating at room temperature for 2h, discarding the primary antibody dilution, and washing with PBS-BSA for 5min for 3 times.
Caspase-3 primary antibody (available from Cell Signaling Technology) was diluted 1:200 with blocking solution, incubated at room temperature for 2h, the primary antibody dilution was discarded, and washed 3 times with PBS-BSA for 5min each.
7. CDX 2-specific secondary antibody (purchased from Sigma) was diluted 1:200 with blocking solution under exclusion of light and left at room temperature for 1h without light. The secondary antibody dilutions were discarded in the dark and washed 3 times with PBS for 5min each.
8. Caspase-3 specific secondary antibodies (purchased from Life Technologies) were diluted 1:200 with blocking solution under dark conditions and left at room temperature for 1h under dark conditions. The secondary antibody dilutions were discarded in the dark and washed 3 times with PBS for 5min each.
9. Cell nuclei were stained with 10. mu.g/mL Hochest 33342 dye, allowed to act at room temperature for 5min, observed under a fluorescent microscope and photographed.
10. The experiment is repeated three times, 10 blasts are randomly selected each time, and the blast quality is evaluated by calculating the apoptosis rate and the number of ICM cells/total number of cells.
The data statistical method comprises the following steps: experimental data were analyzed using the ANOVA program in statistical software SAS V8 and the Duncan's multiple-range test method judged significant differences between treatments, which were considered significant when p < 0.05.
In the present invention, cleavage rate is fertilization cleavage number/fertilization egg number. In the present invention, blastocyst rate is blastocyst number/cleavage embryo number.
Fourth, In Vitro Maturation (IVM) Effect of oocytes-maturation Rate
In step (1) of this example 2, after the in vitro maturation culture, the oocytes were observed under an inverted microscope, and the oocytes were judged to have matured if they had released the first polar body, had retained the viscous matrix secreted by the cumulus cells, had significantly expanded cell layers, and had spread radially around the ovum as the center, and the number of matured oocytes was recorded, and the maturation rate was calculated.
Five results
In this example 2, a test was conducted on a Chinese cattle (Nanyang cattle, working 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, blastocyst hatchability reached 73.4% on day 9. The maturation rate of oocytes matured in vitro reached 88.3%, which was increased by about 13 percentage points relative to the maturation rate of the method of example 1.
In a supplementary test, with reference to the method of example 2 above, three types of cattle, holstein cattle (dairy cattle breed), simmental cattle (beef cattle breed), and chinese buffalo (service breed), were tested, and the results were: the cleavage rate is within 84-90%, the morula rate is within 61-65%, the blastocyst rate is within 50-54%, the apoptosis rate is within 3-6%, and the blastocyst hatching rate on day 9 is within 71-76%; the in vitro maturation rates of oocytes of three holstein cattle, Simmental cattle and Chinese buffalo cattle are 84.5%, 79.5% and 73.2%, respectively, and are respectively increased by about 14-15 percentage points relative to the maturation rate of the method in the embodiment 1. As a result of the above, although the method of example 1 according to the present invention can achieve completely satisfactory results, it was unexpectedly found that the maturation rate of in vitro maturation of oocytes can be significantly increased when trace amounts of selenide and copper sulfate are added to the BY basal medium according to the present invention, which is very significant, particularly without any technical teaching that the addition of selenide and copper sulfate as described above can significantly increase the maturation rate of in vitro maturation of oocytes.
In a supplementation test, with reference to example 2, in BY basal medium without sodium selenite (but supplemented with a corresponding amount of copper sulfate) or without copper sulfate (but supplemented with a corresponding amount of sodium selenite),
in four kinds of cattle, the four parameters of the cleavage rate, the morula rate, the blastocyst rate and the apoptosis rate are basically consistent with the results of example 2 under two conditions, the cleavage rate for the four kinds of cattle is within 84-89%, the morula rate is within 63-65%, the blastocyst rate is within 52-55%, the apoptosis rate is within 3-6%, and the blastocyst hatching rate for the 9 th day is within 70-75%, for example, the cleavage rate for Chinese cattle is 87.7% and 88.5%, respectively; however, the in vitro maturation rate of oocytes was not increased as compared with the results of example 1, and in both cases, the in vitro maturation rates of oocytes of Chinese cattle, Holstein cattle, Simmental cattle and Chinese buffalo were 73-75%, 67-70%, 65-66% and 55-57%, respectively. This shows that whether selenide and copper sulfate are added in the BY basal medium or not does not affect the four parameters of the cleavage rate, the morula rate, the blastocyst rate and the apoptosis rate, but the maturation rate of the oocyte in vitro maturation can be effectively improved only when the selenide and the copper sulfate are added at the same time.
Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.

Claims (10)

1. A method for culturing bovine in vitro fertilization embryos comprises the following steps:
(1) collection and in vitro maturation of oocytes
Taking slaughterhouse ovaries, placing in a heat-preserving barrel added with double-resistance normal saline, and transporting back to a laboratory within 3h at the temperature of 30-33 ℃; extracting follicle with surface of 2-8mm, collecting precipitate, picking out oocyte COCs (oocyte-oocyte complex) containing at least 3 layers of cumulus cells under a stereoscopic microscope, washing for 2 times in ovum washing liquid, and removing excessive impurities;
washing the obtained COCs in oocyte maturation culture solution for 1 time, transferring to new maturation culture solution, and culturing for 22-24h under the conditions of 38.8 deg.C, 5.5-6.5% CO2, and saturated humidity;
(2) in vitro fertilization
Washing mature COCs in a fertilization culture solution for 1 time, transferring the COCs into the fertilization culture solution, and putting the COCs into an incubator for later use;
taking a frozen tubule from liquid nitrogen, and unfreezing in a water bath at 37 ℃; aseptically cutting two ends of the thin tube, injecting semen into 15mL centrifuge tube containing semen preparation culture solution, centrifuging at 328 Xg for 2 times (5 min each time), and discarding supernatant after centrifuging; adding 300 mu L of semen preparation culture solution into the centrifuge tube, resuspending the sperm precipitation, and taking appropriate sperm suspension for sperm counting;
adding the calculated volume of sperm suspension into the fertilization culture liquid drop containing the oocyte, and placing the culture disc into an incubator to incubate the sperm and the ovum for 16-20h, wherein the culture conditions are 38.8 ℃, 5.5-6.5% CO2 and saturated humidity;
(3) embryo in vitro culture and preservation
After the in vitro fertilization operation is finished, removing granular cells around the embryo by using an ovum-peeling needle, putting the granules into embryo culture solution for culture, recording the culture condition as the 1 st day of the embryo culture, wherein the culture condition is 38.8 ℃, 6% O2, 88% N2 and saturation humidity, and recording the cleavage rate on the 3 rd day; recording the blastocyst rate on the 7 th day, counting the blastocyst hatchability by the 9 th day, and performing quality identification;
washing the available embryo in preservation solution for 3 times, balancing in balancing solution for 10min, transferring into freezing solution, loading into embryo according to 5-stage liquid loading method, marking, cooling to-35 deg.C at 0.5 deg.C/min in program cooling instrument, taking out the tubule of embryo rapidly, placing into liquid nitrogen for freezing and preserving,
wherein the content of the first and second substances,
in the step (1), the egg washing solution is BY basal culture solution added with 3mg/mL bovine serum albumin;
in the step (1), the maturation culture solution is BY basal culture solution added with 100mL/L FBS, 10 μ g/mL FSH, 10 μ g/mL LH, 1 μ g/mL E2 and 20ng/mL EGF;
the BY basal culture solution is an aqueous solution containing the following components: 180-220 mg/L calcium chloride, 0.70-0.75 mg/L ferric nitrate nonahydrate, 380-420 mg/L potassium chloride, 90-100 mg/L magnesium sulfate, 6500-7000 mg/L sodium chloride, 130-150 mg/L monosodium phosphate monohydrate, 2000-2500 mg/L sodium bicarbonate, 40-60 mg/L, L-alanine 20-30 mg/L, L-sodium acetate, 60-80 mg/L, L-aspartic acid, 25-35 mg/L, L-cysteine hydrochloride monohydrate, 0.10-0.12 mg/L, L-cystine dihydrochloride, 20-30 mg/L, L-glutamic acid, 40-60 mg/L, L-glycine-histidine hydrochloride monohydrate, 20-25 mg/L, L-hydroxyproline 8-12 mg/L, 15-25 mg/L, L-leucine 50-70 mg/L, L-lysine hydrochloride 60-80 mg/L, L-methionine 10-20 mg/L, L-phenylalanine 20-30 mg/L, L-proline 30-50 mg/L, L-serine 20-30 mg/L, L-threonine 25-35 mg/L, L-tryptophan 8-12 mg/L, L-tyrosine disodium dihydrate 55-60 mg/L, L-valine 20-30 mg/L, ascorbic acid 0.04-0.06 mg/L, alpha-D-tocopherol phosphate 0.008-0.012 mg/L, biotin 0.008-0.012 mg/L, calcitol 0.08-0.12 mg/L, D-calcium pantothenate 0.008-0.012 mg/L, 0.4-0.6 mg/L choline chloride, 0.008-0.012 mg/L folic acid, 0.04-0.06 mg/L inositol, 0.015-0.025 mg/L menadione sodium bisulfite trihydrate, 0.02-0.03 mg/L nicotinic acid, 0.02-0.03 mg/L nicotinamide, 0.04-0.06 mg/L L, p-aminobenzoic acid, 0.04-0.06 mg/L pyridoxine hydrochloride, 0.008-0.012 mg/L riboflavin, 0.008-0.012 mg/L thiamine hydrochloride, 0.1-0.2 mg/L vitamin A acetate, 8-12 mg/L adenine sulfate, 0.15-0.25 mg/L adenine, 0.8-1.2 mg/L disodium adenosine triphosphate, 0.15-0.25 mg/L cholesterol, 2-deoxy-D-0.4-0.6 mg/L glucose, 0.04-0.84 mg/L glutathione, 0.06mg/L glucose, 0.25-0.35 mg/L guanine hydrochloride, 0.3-0.4 mg/L hypoxanthine sodium, 0.4-0.6 mg/L ribose, 0.25-0.35 mg/L thymosin, 4-6 mg/L Tween 80, 0.25-0.35 mg/L uracil, 0.3-0.4 mg/L xanthine sodium and 8-12 mg/L phenol red.
2. The method according to claim 1, wherein in step (1), the saline added with double antibody is a saline containing penicillin 400IU/mL and streptomycin 400 μ g/mL.
3. The method according to claim 1, wherein in step (2), the fertilization medium is an aqueous solution comprising 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 sodium pyruvate, 10. mu.g/ml heparin, 4mg/ml bovine serum albumin, 100U/ml penicillin, 100. mu.g/ml streptomycin.
4. The method according to claim 1, wherein in step (2), the semen preparation medium is an aqueous solution 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 sodium pyruvate, 10. mu.g/ml heparin, 4mg/ml bovine serum albumin, 10mM caffeine, 100U/ml penicillin, 100. mu.g/ml streptomycin.
5. The method according to claim 1, wherein in step (3), the embryo culture fluid comprises: 109.5mM sodium chloride, 3.1mM potassium chloride, 26.2mM sodium bicarbonate, 0.8mM magnesium chloride hexahydrate, 1.19mM monopotassium phosphate, 0.4mM sodium pyruvate, 1.5mM glucose, 5mM calcium half-lactobionate, 2.5v/v% fetal bovine serum, 1mM L-glutamine, 2v/v% essential amino acids, 1v/v% nonessential amino acids, 3mM glutathione, sodium citrate 0.04w/v%, maltose 0.02w/v% aqueous solution; the essential amino acid is added by the following amino acids according to the weight proportion: 6.32g of L-arginine hydrochloride, 1.564g of L-cystine dihydrochloride, 2.1g of L-histidine hydrochloride monohydrate, 2.625g of L-isoleucine, 2.62g of L-leucine, 3.625g of L-lysine hydrochloride, 0.755g of L-methionine, 1.65g of L-phenylalanine, 2.38g of L-threonine, 0.51g of L-tryptophan, 1.8g of L-tyrosine and 2.34g of L-valine, wherein the optional amino acids are added in the following weight ratio: 0.89g of L-alanine, 1.5g of L-asparagine monohydrate, 1.33g of L-aspartic acid, 1.47g of L-glutamic acid, 0.75g of glycine, 1.15g of L-proline and 1.05g of L-serine.
6. The method according to claim 1, wherein in step (1), the BY basal medium is an aqueous solution comprising: 200mg/L calcium chloride, 0.72mg/L ferric nitrate nonahydrate, 400mg/L potassium chloride, 97.7mg/L magnesium sulfate, 6800mg/L sodium chloride, 140mg/L monobasic sodium phosphate monohydrate, 2200mg/L sodium bicarbonate, 50mg/L, L-sodium acetate, 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 leucine, 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, alpha-D-tocopherol phosphate 0.01mg/L, biotin 0.01mg/L, calciferol 0.1mg/L, D-calcium pantothenate 0.01mg/L, choline chloride 0.5mg/L, folic acid 0.01mg/L, inositol 0.05mg/L, menadione trihydrate sodium bisulfite 0.019mg/L, nicotinic acid 0.025mg/L, Nicotinamide 0.025mg/L, p-aminobenzoic acid 0.05mg/L, pyridoxine hydrochloride 0.05mg/L, riboflavin 0.01mg/L, thiamine hydrochloride 0.01mg/L, vitamin A acetate 0.14mg/L, adenine 10mg/L, adenine 0.2mg/L, disodium adenosine triphosphate 1mg/L, cholesterol 0.2mg/L, 2-deoxy-D-ribose 0.5mg/L, D-glucose 1000mg/L, glutathione 0.05mg/L, guanine hydrochloride 0.3mg/L, sodium hypoxanthine 0.354mg/L, ribose 0.5mg/L, thymosin 0.3mg/L, Tween 5mg/L Tween 80, uracil 0.3mg/L, sodium xanthine 0.34mg/L, phenol red 10 mg/L.
7. The method according to claim 1, wherein in step (1), the BY basal medium further comprises sodium selenite at a concentration of 0.2-0.3 mg/L.
8. The method according to claim 1, wherein in step (1), the BY basal medium further comprises sodium selenite at a concentration of 0.25 mg/L.
9. The method according to claim 1, wherein the BY basal medium further comprises copper sulfate at a concentration of 0.05-0.1 mg/L on an anhydrous basis.
10. The method according to claim 1, wherein the BY basal medium further comprises copper sulfate at a concentration of 0.075mg/L as an anhydride.
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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103898046A (en) * 2014-02-28 2014-07-02 中国农业科学院北京畜牧兽医研究所 Cow in-vitro fertilization embryo culture fluid and culture method thereof
CN107034173A (en) * 2017-06-09 2017-08-11 英科博雅基因科技（天津）有限公司 The method of ox IVF Embryos culture and the nutrient solution used
CN107142239A (en) * 2017-06-09 2017-09-08 英科博雅基因科技（天津）有限公司 The method for improving ox IVF Embryos culture efficiency

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103898046A (en) * 2014-02-28 2014-07-02 中国农业科学院北京畜牧兽医研究所 Cow in-vitro fertilization embryo culture fluid and culture method thereof
CN107034173A (en) * 2017-06-09 2017-08-11 英科博雅基因科技（天津）有限公司 The method of ox IVF Embryos culture and the nutrient solution used
CN107142239A (en) * 2017-06-09 2017-09-08 英科博雅基因科技（天津）有限公司 The method for improving ox IVF Embryos culture efficiency

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
Protective effects of antioxidants on linoleic acid–treated bovine oocytes during maturation and subsequent embryo development;Wael A. Khalil等;《Theriogenology》;20131231;第80卷;161-168 *

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