CN111269876A

CN111269876A - Method for in vitro fertilization and embryo culture by in vivo collection of bovine oocytes

Info

Publication number: CN111269876A
Application number: CN202010082016.7A
Authority: CN
Inventors: 王娜; 王小武; 赵明礼; 郝少强; 郭晶; 张月桥; 郭春明
Original assignee: Tianjin Boyu Limu Technology Co ltd
Current assignee: Tianjin Boyu Limu Technology Co ltd
Priority date: 2020-02-06
Filing date: 2020-02-06
Publication date: 2020-06-12

Abstract

The invention relates to a method for in vitro fertilization and embryo culture by in vivo collection of bovine oocytes. Specifically, in one aspect, the method for culturing bovine in vitro fertilized embryos comprises the following steps: taking follicle fluid from a cow living body egg taking source, picking out a cumulus-oocyte complex at least containing 3 layers of cumulus cells under a stereoscopic microscope, and putting the cumulus-oocyte complex into a transport culture solution to transport to a laboratory within 24 hours; washing COCs in oocyte maturation culture solution for 1 time, transferring to new maturation culture solution, and culturing for 22-24 hr under the conditions of 38.8 deg.C, 5.5-6.5% CO2, and saturated humidity; in vitro fertilization; and (5) culturing and preserving embryos in vitro. The invention also relates to a transport culture solution for the oocyte. The method and the transport culture solution of the present invention exhibit excellent technical effects as described in the specification.

Description

Method for in vitro fertilization and embryo culture by in vivo collection of bovine oocytes

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. In particular, the invention relates to a method for in vivo collective oocytes from cattle for in vitro fertilization and embryo culture.

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 stepwise 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 acids and vitamins on clearance and maintenance of bovine zygoesin vision.J animal 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, Successful focus in vitro of sheet and maintenance a.J. Reprod foil, 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 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 degradation and purification of bovine embryo cultures 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 embryo cultures LiveStokes, 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.

A number of studies have shown that Glutathione (GSH), an antioxidant present in a non-protein form, is capable of scavenging various free radicals, superoxide anion free radicals, hydroxyl free radicals, hydrogen peroxide, hypochlorous acid and lipoxygen free radicals, and of maintaining The redox balance inside and outside The cell, environmental GSH and ROS levels inside and outside The cell are two important factors in The development of fertilized eggs, as early as 2000, de Matos et al (de Matos, D.G. and C.C.Furns), The animal of living high yield one (GSH) level after vitamin in vitro synthesis of beta-mercaptohanol, cysteine and cysteine, theriogenology,2000.53(3): p.761-71) have increased The blastocyst rate by The addition of β -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 S.Iwasaki et al (Iwasaki, S.and T.Nakahara, cell number and intention of chromosomal antibodies in bovine blast transferred in vitro followed by tissue culture in vitro or in vivo in clinical science, 1990.33(3): p.669-75) obtained the average total number of blastocysts in early cattle, and the ratio of the number of ICM cells/total number of blastocysts was 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, 2 v/v% essential amino acids, 1 v/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.

The inventor's prior application CN108728404A (application No.: 201810576806.3) discloses a method for culturing bovine in vitro fertilized embryos, which comprises the following steps: (1) collection of oocytes and in vitro maturation in vitro collection: taking slaughterhouse ovaries, placing in a heat-preserving barrel added with double-resistance normal saline, and transporting back to a laboratory within 3h at 31-33 ℃; 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; collecting living bodies: collecting ovum of cattle in vivo, picking up cumulus-oocyte complex (COCs) at least containing 3 layers of cumulus cells under a stereomicroscope from the obtained follicular fluid, and putting into mature culture solution containing HEPES at 38.8 deg.C for 3h for transporting to laboratory; washing COCs obtained by the in vitro collection or the living body collection 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 ℃, 5.5-6.5% CO2 and saturated humidity; (2) in vitro fertilization, mature COCs are washed in a fertilization culture solution for 1 time, transferred into the fertilization culture solution and placed 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) after the embryo in-vitro culture and preservation and in-vitro fertilization operations are finished, removing granular cells around the embryo by using an egg stripping needle, putting the embryo into an embryo culture solution for culture, recording the culture conditions as 1 day of the embryo culture, wherein the culture conditions are 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.

As is well known, embryo biotechnology is an effective technical means for breeding and rapid propagation of fine cattle, wherein in vivo ovum collection-in vitro embryo production (OPU-IVP) technology can rapidly obtain a large amount of high-quality embryos under in vitro conditions by collecting excellent individual oocytes in vivo and carrying out In Vitro Maturation (IVM), In Vitro Fertilization (IVF), embryo In Vitro Culture (IVC) and the like. Compared with in vivo embryos, the production efficiency of the method is 4-8 times, the breeding potential of excellent dams can be effectively improved, but under general conditions, a cow oocyte donor cow farm is far away from an embryo production laboratory and needs to be transported for hours, and the transportation time and the transportation conditions are very important for maintaining the activity and the quality of oocytes. The method comprises the following steps of firstly, putting collected oocytes in a pre-maturation solution to inhibit the oocytes from maturing, transferring the oocytes to a maturation solution after reaching a laboratory, and putting the oocytes in an incubator for in vitro maturation culture; secondly, the collected oocytes are put in a mature liquid and transported in a micro incubator, and CO is supplied continuously during the transportation₂However, it is difficult to maintain the supply of carbon dioxide in a transport state. Therefore, there is a need to develop a culture medium for oocytes that can be used during long-distance transportation without affecting their maturation rate, and without the need to supply CO₂And time and cost are saved.

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. In addition, the present inventors have also found that a culture medium for culturing bovine in vitro fertilized embryos, which is a culture medium for transporting oocytes over a long distance, surprisingly shows excellent technical effects using the method of the present invention, and have completed the present invention.

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

a) Collecting living bodies: taking follicular fluid from ovum collection source of living cattle, picking out cumulus-oocyte complexes (COCs) at least containing 3 layers of cumulus cells under a stereomicroscope, putting into transport culture solution containing HEPES, transporting to laboratory at 38.8 deg.C without carbon dioxide supply for 24 hr;

b) washing COCs obtained by the in vitro collection or the living body collection 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 ℃, 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) (b), 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.

According to the method of the first aspect of the present invention, in the step (1) (a), the transport culture solution contains glycine 50.0mg/L, L-alanine 25.0mg/L, L-arginine hydrochloride 70.0mg/L, L-aspartic acid 30.0mg/L, L-cystine dihydrochloride 26.0mg/L, L-glutamic acid 75.0mg/L, L-glutamine 100.0mg/L, L-histidine hydrochloride monohydrate 21.88mg/L, L-hydroxyproline 10.0mg/L, L-isoleucine 40.0mg/L, L-leucine 60.0mg/L, L-lysine hydrochloride 70.0mg/L, L-methionine 15.0mg/L, L-phenylalanine 25.0mg/L, L-proline 40.0mg/L, L-serine 25.0mg/L, L-threonine 30.0mg/L, L-tryptophan 25.0mg/L, L-tyrosine disodium salt dihydrate 58.0 mg/5735.0 mg/28 mg/28.0 mg/L, L mg/serine 25.0mg/L, L mg/threonine 30.0mg/L, L mg/tryptophan, vitamin D, vitamin E, L hydrochloride 0.0.0.0.0.0.0 mg/1000 mg/L5 mg/L, vitamin E hydrochloride0.01IU/mL FSH, 0.01IU/mL LH, 1 ug/mL E₂50ng/mL EGF, 100ng/mL IGF, 10% gentamicin, 55. mu.g/mL sodium pyruvate, 1.2mM/L cysteine, 3mg/mL BSA, 10mM/L HEPES.

The method according to the first aspect of the invention, wherein the concentration of HEPES, 4-hydroxyethylpiperazine ethanesulfonic acid in the maturation medium is 5-15 mmol/L, such as 10 mmol/L. In the present invention, EGF-epidermal growth factor, FSH-follicle stimulating hormone, FBS-fetal bovine serum, E2-estradiol, LH-luteinizing hormone, IGF-insulin-like growth factor, BSA-bovine serum albumin.

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

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

The method according to the first aspect of the present invention, wherein in step (1), the concentration of zinc gluconate in the transport culture broth is 1 to 3mg/L, for example 2 mg/L. It has surprisingly been found that when defined amounts of taurine and zinc gluconate are added simultaneously to the transport medium according to the invention, the desired maturation of the oocytes is achieved during transport even after transport for up to 24 hours in the absence of carbon dioxide.

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, 2.5 v/v% Fetal Bovine Serum (FBS), 1mM L-glutamine, 2 v/v% essential amino acids, 1 v/v% nonessential amino acids, 3mM glutathione, 0.04 w/v% sodium citrate, 0.02 w/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.

According to the first aspect of the present invention, in the step (1), the BY-based culture solution includes 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-arginine hydrochloride, 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, 70-80 mg/L glycine, 20-60 mg/L, L-histidine hydrochloride monohydrate, 20-25 mg/6-hydroxyproline, 8mg/L, L-15 mg/0.008-0.8 mg/L methionine hydrochloride, 0.06-0.06 mg/L choline chloride, 0.06-20 mg/L choline chloride, 0.06-12 mg/L choline chloride monohydrate, 0.06-60 mg/L, 0.06-12 mg/L choline chloride monohydrate, 0.8-200 mg/L, 0.8-200 mg/L choline acetate monohydrate, 0.8 mg/L, 0.8-200 mg/L methionine hydrochloride monohydrate, 0.8-200 mg/L methionine hydrochloride, 0.8-5 mg/L0.8-200 mg/L, 0.8-5 mg/L, 0.8-.

According to the method of the first aspect of the present invention, in the step (1), the BY base medium is an aqueous solution containing 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 sodium dihydrogen phosphate monohydrate, 2200mg/L sodium bicarbonate, 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-75 mg/L sodium acetate, 50mg/L, L-glycine hydrochloride monohydrate, 21.88mg/L, L-hydroxyproline 10 mg/84-20 mg/L, L-leucine 60mg/L, L-lysine hydrochloride 70mg/L, L-methionine 15mg/L, L-phenylalanine 25 mg/5-proline 40mg/L, L mg/40 mg/24 mg/L serine folate, 0.5mg/L folate hydrochloride, 0.05mg/L adenine, 0.05mg/L sodium folate hydrochloride monohydrate, 0.27 mg/L, 0.5mg/L adenine, 0.8-arginine hydrochloride monohydrate, 27, 0.8-L, 0.8-cysteine hydrochloride monohydrate, 26 mg/L-cystine hydrochloride monohydrate, 26 mg/L-cystine hydrochloride monohydrate, 26 mg/L-26 mg/L, 26 mg/L-cystine hydrochloride monohydrate, 26 mg/L-26 mg/L, 0.8-histidine hydrochloride, 0.8-26 mg/L, 0.8-histidine hydrochloride monohydrate, 26mg/L, 0.8-histidine hydrochloride, 26mg/L, 0.8-histidine hydrochloride, 26mg/L, 3-histidine hydrochloride, 0.8-arginine hydrochloride, 0.8-L, 3-L, 0.8-arginine hydrochloride, 0.8-L, 3-L, 0.8-L, 3mg/L, 3-L, 3mg/L, 0.8-L, 0.

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, Sweden^TMClean (the invention is not specifically illustrated, and the freezing fluid used in the example is Freezekit^TMClean, the embryo preservation solution is Whittingham modified Du's phosphate buffer containing 20% bovine serum and 500IU/ml penicillin G potassium, and the equilibrium solution is ES solution from Kitazato Biopharma, Japan).

Further, the second aspect of the present invention provides a transport culture solution for oocyte.

The transport culture solution according to the second aspect of the present invention comprises: glycine 50.0mg/L, L-alanine 25.0mg/L, L-arginine hydrochloride 70.0mg/L, L-aspartic acid 30.0mg/L, L-cystine dihydrochloride 26.0mg/L, L-glutamic acid 75.0mg/L, L-glutamine 100.0mg/L, L-histidine hydrochloride monohydrate 21.88mg/L, L-hydroxyproline 10.0mg/L, L-isoleucine 40.0mg/L, L-leucine 60.0mg/L, L-lysine hydrochloride 70.0mg/L, L-methionine 15.0mg/L, L-phenylalanine 25.0mg/L, L-proline 40.0mg/L, L-serine 25.0mg/L, L-threonine 30.0mg/L, L-tryptophan 10.0mg/L, L-tyrosine disodium salt dihydrate58.0mg/L, L-valine 25.0mg/L, ascorbic acid 0.05mg/L, biotin 0.01mg/L, choline chloride 0.5mg/L, D-calcium pantothenate 0.01mg/L, folic acid 0.01mg/L, menadione 0.01mg/L, nicotinamide 0.025mg/L, nicotinic acid 0.025mg/L, p-aminobenzoic acid 0.05mg/L, pyridoxal hydrochloride 0.025mg/L, pyridoxine hydrochloride 0.025mg/L, riboflavin 0.01mg/L, thiamine hydrochloride 0.01mg/L, vitamin A acetate 0.1mg/L, vitamin D2, i.e., calcitol 0.1mg/L, α -tocopherol sodium phosphate 0.01mg/L, inositol 0.05mg/L, anhydrous calcium chloride 200.0mg/L, ferric nitrate nonahydrate 0.7mg/L, anhydrous 97.67mg/L, calcium triphosphate 0.354mg/L, sodium adenine chloride 0.01mg/L, adenine sodium chloride 0.80 mg/L, glutathione hydrochloride 0.20 mg/L, adenine sodium chloride 0.80 mg/L, adenine sodium chloride/L, glutathione L, adenine sodium chloride/L, sodium chloride 0.1mg/L, adenine sodium chloride 0.20 mg/L, adenine sodium chloride/L, sodium chloride 0.80 mg/L, sodium chloride L/L, sodium chloride L/L, sodium chloride L/L, sodium chloride L/2.1.80 mg/L, sodium chloride L/L₂50ng/mL EGF, 100ng/mL IGF, 10% gentamicin, 55. mu.g/mL sodium pyruvate, 1.2mM/L cysteine, 3mg/mL BSA, 10mM/L HEPES.

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

The transport culture solution according to the second aspect of the present invention, wherein the concentration of taurine is 30 to 50mg/L, for example 40mg/L or 45 mg/L.

The transport culture solution according to the second aspect of the present invention, wherein the concentration of zinc gluconate is 1 to 3mg/L, for example 2 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), and Mexico fetal bovine serum (cat # 10437028) can be obtained from various agents. 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 cattle in vitro fertilization embryo (in vitro collection)

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.

Maturation medium containing HEPES: BY basal medium supplemented with 15mmol/L HEPES, 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, 2 v/v% essential amino acids, 1 v/v% nonessential amino acids, 3mM glutathione, sodium citrate 0.04 w/v%, maltose 0.02 w/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.

BY basal medium is aqueous solution containing 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 20 mg/42-leucine 60mg/L, L-lysine hydrochloride 70mg/L, L-methionine 15mg/L, L-phenylalanine 25 mg/5-proline 40mg/L, L-serine 25mg/L, L mg/30 mg/24 mg/tryptophan hydrochloride 70mg/L, L mg/460.45 mg/methionine 0mg/L, L mg/phenylalanine 25 mg/5 mg/proline 40mg/L, L mg/serine 25mg/L, L mg/24 mg/L-lysine hydrochloride 0.45 mg/L, choline chloride monohydrate 0.45/L, calcium adenine dinucleotide phosphate monohydrate 0.05mg/L, calcium sulfate monohydrate 0.05mg/L, calcium chloride monohydrate 0.05mg/L, calcium adenine disodium adenine dinucleotide sulfate monohydrate 0.05mg/L, calcium chloride monohydrate 0.8 mg/L, calcium chloride monohydrate 0.05mg/L, calcium adenine dinucleotide phosphate monohydrate 0.8 mg/L, vitamin D0.8-cysteine hydrochloride monohydrate 0.8-arginine hydrochloride monohydrate 0.8-cysteine hydrochloride 0.8-arginine hydrochloride, vitamin D0.8-cysteine hydrochloride 0.8-arginine hydrochloride 0.8-cysteine hydrochloride, vitamin D0.8-cysteine hydrochloride 0.8-arginine hydrochloride, vitamin D0.8-cysteine hydrochloride monohydrate 0.8-arginine hydrochloride, vitamin D0.8-arginine hydrochloride 0.8-arginine hydrochloride, vitamin D.8-arginine hydrochloride monohydrate 0.8-arginine hydrochloride 0.8-cysteine hydrochloride 0..

II, cattle in-vitro fertilization and embryo culture:

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

Placing the obtained cow ovary in a heat-insulating barrel containing double-resistant normal saline, carrying out transportation to the laboratory within 3h at 31-33 ℃ and 5.5-6.5% CO2 and saturated humidity; 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

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

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 in the dark. 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' smultiple-range test method judged the significance of 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, after the in vitro maturation culture, the oocyte is observed under an inverted microscope, and when the oocyte has the first polar body released, viscous matrix secreted from the cumulus cells is maintained, and the cell layer is significantly expanded, and the cell spreads around the ovum in a substantially radial manner, the oocyte is judged to be mature, the number of mature oocytes is recorded, and the maturation rate is calculated.

Five results

In this example, a test was conducted on a cattle of China (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 the oocyte in vitro maturation reaches 88.3 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 84-90%, 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 maturation rates of the oocytes of three holstein cattle, Simmental cattle and Chinese buffalo in vitro maturation are 84.5%, 79.5% and 73.2%, respectively.

Example 2: culture method of cow in vitro fertilization embryo (Living body collection)

Reagent

Washing the egg: BY basal medium supplemented with 3mg/mL bovine serum albumin.

II, cattle in-vitro fertilization and embryo culture:

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

Taking follicular fluid from ovum collection source of living cattle, picking out cumulus-oocyte complex (COCs) at least containing 3 layers of cumulus cells under a stereomicroscope, placing into mature culture solution containing HEPES, and transporting to laboratory at 38.8 deg.C, 5.5-6.5% CO2, saturation humidity, within 3 h;

step (2), in vitro fertilization

step (3), embryo in vitro culture and preservation

Method for differential staining of embryo

Fourth, In Vitro Maturation (IVM) Effect of oocytes-maturation Rate

Five results

In this example, a test was conducted on a cattle of China (Nanyang cattle, working 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, blastocyst hatchability reached 74.4% on day 9. The maturation rate of the oocyte in vitro maturation reaches 89.6 percent.

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 the range of 85-90%, the morula rate is within the range of 62-65%, the blastocyst rate is within the range of 51-54%, the apoptosis rate is within the range of 4-6%, and the blastocyst hatching rate on day 9 is within the range of 71-75%; the in vitro maturation rate of oocytes of three holstein cattle, Simmental cattle and Chinese buffalo cattle is 85.2%, 79.1% and 74.32% respectively.

Example 12: culture method of cow in vitro fertilization embryo (Living body collection)

The main difference between this example and example 2 above is that in step (1) the harvested oocytes were placed in transport medium containing HEPES at 38.8 ℃ without carbon dioxide supply and transported back to the laboratory within 24 h.

Reagent

the transport medium used in this example contained 50.0mg/L, L mg-alanine 25.0mg/L, L-arginine hydrochloride 70.0mg/L, L-aspartic acid 30.0mg/L, L-cystine dihydrochloride 26.0mg/L, L-glutamic acid 75.0mg/L, L-glutamine 100.0mg/L, L-histidine hydrochloride monohydrate 21.88mg/L, L-hydroxyproline 10.0mg/L, L-isoleucine 40.0mg/L, L-leucine 60.0mg/L, L-lysine hydrochloride 70.0mg/L, L-methionine 15.0mg/L, L-histidine 25.0mg/L, L-proline 40.0mg/L, L-serine 25.0mg/L, L-threonine 30.0mg/L, L-tryptophan 10.0mg/L, L-tyrosine dihydrate 58.0mg/L, L-valine 25.0mg/L, ascorbic acid 0 mg/vitamin E0 mg/phenylalanine hydrochloride 0.0 mg/240 mg/L, calcium chloride 0.0.0 mg/1000 mg/folate 0 mg/folate 0.0 mg/240 mg/folate 0.0.0.0 mg/240 mg/L, vitamin E5-arginine hydrochloride monohydrate, 0.0.0.0.0.0.0 mg/240 mg/L0.0.0.0 mg/240 mg/vitamin D, 0 mg/1.8-arginine hydrochloride, 0.0.0.0.0 mg/1.0 mg/8-arginine hydrochloride, 0.0.0.0.0.0 mg/1, 0.0.0 mg/1.0 mg/8-arginine hydrochloride, 0.0.0.0 mg/1.0.0 mg/1.0 mg/1, 0.0 mg/1, 0.8-arginine hydrochloride, 0.0.0.0.0.0 mg/1, 0.0 mg/1, 0.0.0 mg/1.0.0 mg/1, 0.0 mg/8-arginine hydrochloride, 0.0 mg/1, 0 mg/1, 0.8-arginine hydrochloride, 0.0.0.0 mg/1, 0.0.0 mg/1, 0.0.0.0 mg, 0 mg/1, 0.0.0 mg, 0 mg/1, 0 mg/1, 0.0.0.0.0 mg/1, 0 mg/1, 0.8, 0 mg/1, 0mg, 0.8, 5-arginine, 0.8, 1, 0mg, 1, 0, 0.8, 5, 1, 5, 0, 1₂50ng/mL EGF, 100ng/mL IGF, 10% gentamicin, 55. mu.g/mL sodium pyruvate, 1.2mM/L cysteine, 3mg/mL BSA, 10mM/L HEPES, as well as 45mg/L taurine and 2mg/L zinc gluconate.

Washing the egg: BY basal medium supplemented with 3mg/mL bovine serum albumin.

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

II, cattle in-vitro fertilization and embryo culture:

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

(a) Taking follicular fluid from ovum of living cattle, picking out cumulus-oocyte complexes (COCs) at least containing 3 layers of cumulus cell packages under a stereomicroscope, putting the cumulus-oocyte complexes into a transportation culture solution containing HEPES, transporting the cumulus-oocyte complexes to a laboratory at 38.8 ℃ without carbon dioxide supply within 24h (in the example, transporting the cumulus-oocyte complexes to the laboratory for the next step in 15 h);

(b) 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

step (3), embryo in vitro culture and preservation

Method for differential staining of embryo

Fourth, In Vitro Maturation (IVM) Effect of oocytes-maturation Rate

Five results

In this example, a test was conducted on a cattle of China (Nanyang cattle, working breed). As a result, the cleavage rate was 87.1%, the morula rate was 65.3%, the blastocyst rate was 49.7%, and the apoptosis rate was 5.6%; in addition, blastocyst hatchability reached 73.2% on day 9. The maturation rate of the oocyte in vitro maturation reaches 88.2 percent.

In a supplementary test (i.e., supplementary test 12A), tests were carried out for three kinds of cattle, holstein cattle (dairy cattle breed), simmental cattle (beef cattle breed), and chinese buffalo (service breed), respectively, with reference to the method of example 12 above, and the results were: the cleavage rate is within the range of 86-89%, the morula rate is within the range of 63-66%, the blastocyst rate is within the range of 50-54%, the apoptosis rate is within the range of 4-6%, and the blastocyst hatching rate on day 9 is within the range of 70-75%; the maturation rates of the oocytes of three holstein cattle, Simmental cattle and Chinese buffalo in vitro maturation are 84.6%, 79.5% and 76.1%, respectively.

In a supplementary experiment (i.e. supplementary experiment 12B), reference was made to the method of example 12 above, but in step (1) the oocyte COCs were transported in transport medium for 12 and 24 hours respectively to the laboratory for further processing, 2 transport times results: the cleavage rate is within the range of 85-90%, the morula rate is within the range of 59-67%, the blastocyst rate is within the range of 50-58%, the apoptosis rate is within the range of 4-6%, and the blastocyst hatching rate on day 9 is within the range of 70-77%; the maturation rates of oocytes matured in vitro for the two transport times of 12 and 24 hours were 86.8% and 84.0%, respectively, which indicates that the transport medium used in step (1) can achieve a relatively high in vitro maturation rate over a relatively long (wide) time range of 12 and 24 hours.

In a supplementary test (i.e. supplementary test 12C), reference was made to the method of example 12 above, but in step (1) the transport medium was not supplemented with taurine and the oocytes COCs were transported in the transport medium for 12, 15 and 24 hours respectively back to the laboratory for further processing; under the test condition, the maturation rate of the oocyte in vitro maturation is obviously different from that of the supplement test 11B, and the maturation rates of the three transportation times are respectively 52.1%, 43.2% and 35.3%.

In a supplementary test (i.e., supplementary test 12D), the method of example 12 above was referenced, but in step (1) the transport medium was not supplemented with zinc gluconate, and the oocyte COCs were transported in the transport medium for 12, 15, and 24 hours, respectively, back to the laboratory for further processing; under the test condition, the maturation rate of the oocyte in vitro maturation is obviously different from that of the supplement test 11B, and the maturation rates of the three transportation times are respectively 48.2%, 41.7% and 32.5%.

In a supplementary test (i.e., supplementary test 12E), the method of example 12 above was referenced, but in step (1) neither taurine nor zinc gluconate was added to the transport medium, and the oocytes COCs were transported in the transport medium for 12, 15, and 24 hours, respectively, back to the laboratory for further processing; under the test condition, the maturation rate of the oocyte in vitro maturation is obviously different from that of the supplement test 12B, and the maturation rates of the three transportation times are 46.4%, 37.0% and 28.7% respectively.

In a supplemental test (i.e., supplemental test 12F), reference was made to the test conditions for 24 hour transport in supplemental test 12B, supplemental test 12C, supplemental test 12D, and supplemental test 12E, respectively, above, except that a 5.5% carbon dioxide supply was provided during transport; under the test conditions, the maturation rates of the oocytes in vitro are 87.7%, 70.8%, 75.4% and 73.2%, respectively.

The results show that the maturation rate of the oocytes obtained in vitro maturation in the step (1) of the method of the invention under different conditions is obviously different: the high maturation rate can be maintained even when the transport culture solution to which both taurine and zinc gluconate are added is transported for 24 hours under the condition of no carbon dioxide supply, while the maturation rate is considerably low when the culture solution to which both taurine and zinc gluconate are not added is transported under the condition of no carbon dioxide supply. This indicates that the simultaneous addition of the two reagents to the transport medium is very beneficial for the in vitro maturation of oocytes during transport, especially for environments where carbon dioxide is not available during transport.

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

1. The method for culturing the bovine in vitro fertilized embryo comprises the following steps:

(1) collection and in vitro maturation of oocytes

(2) in vitro fertilization

(3) embryo in vitro culture and preservation

2. The method according to claim 1, wherein in step (1) (b), the maturation medium is BY basal medium supplemented with 100mL/LFBS, 10 μ g/mL FSH, 10 μ g/mL LH, 1 μ g/mL E2, 20ng/mL EGF.

3. The method according to claim 1, wherein in step (1) (a), said transport medium comprises: glycine 50.0mg/L, L-alanine 25.0mg/L, L-arginine hydrochloride 70.0mg/L, L-aspartic acid 30.0mg/L, L-cystine dihydrochloride 26.0mg/L, L-glutamic acid 75.0mg/L, L-glutamine 100.0mg/L, L-histidine hydrochloride monohydrate 21.88mg/L, L-hydroxyproline 10.0mg/L, L-isoleucine 40.0mg/L, L-leucine 60.0mg/L, L-lysine hydrochloride 70.0mg/L, L-methionine 15.0mg/L, L-phenylalanine 25.0mg/L, L-proline 40.0mg/L, L-serine 25.0mg/L, L-threonine 30.0mg/L, L-Tryptophan 10.0mg/L, L-disodium tyrosine58.0mg/L, L mg/valine 25.0mg/L of salt dihydrate, 0.05mg/L of ascorbic acid, 0.01mg/L of biotin, 0.5mg/L, D-calcium pantothenate 0.01mg/L of choline chloride, 0.01mg/L of folic acid, 0.01mg/L of menadione, 0.025mg/L of nicotinamide, 0.025mg/L of nicotinic acid, 0.05mg/L of p-aminobenzoic acid, 0.025mg/L of pyridoxal hydrochloride, 0.025mg/L of pyridoxine hydrochloride, 0.01mg/L of riboflavin, 0.01mg/L of thiamine hydrochloride, 0.1mg/L of vitamin A acetate, 0.1mg/L of vitamin D2 i.e. calcitol, 0.1mg/L of α -tocopherol phosphate sodium salt, 0.01mg/L of inositol, 0.05mg/L of inositol, 200.0mg/L of anhydrous calcium chloride, 0.7mg/L of nonaferrihydrate, 0.354mg/L of anhydrous magnesium sulfate, 0.57-L of guanine, 0.01mg/L of adenine chloride, 0.80 mg/L of adenine chloride, 0.20 mg/L of adenine sodium adenine, 0.10 mg/L of glutathione, 0.20 mg/L of adenine chloride, 0.80 mg/L of adenine chloride, 0.7mg/L of glutathione, 0.10L of adenine sodium acetate, 0.7mg/L of adenine chloride, 0.7mg/L of glutathione, 0.7mg/L of potassium acetate, 0.7mg/L of adenine sodium acetate, 0.1, 0.7mg/L of adenine sodium acetate, 0.1mg/L of adenine₂50ng/mL EGF, 100ng/mL IGF, 10% gentamicin, 55. mu.g/mL sodium pyruvate, 1.2mM/L cysteine, 3mg/mL BSA, 10mM/L HEPES.

4. The method according to claim 1, wherein the concentration of HEPES, 4-hydroxyethylpiperazine ethanesulfonic acid, in the maturation culture solution is 5-15 mmol/L, such as 10 mmol/L; or, in the step (1), the transport culture solution also comprises taurine and zinc gluconate; or in the step (1), the concentration of taurine in the transport culture solution is 30-50 mg/L, such as 40mg/L or 45 mg/L; alternatively, in step (1), the concentration of zinc gluconate in the transport culture broth is 1-3 mg/L, for example 2 mg/L.

5. 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 μ g/ml heparin, 4mg/ml Bovine Serum Albumin (BSA), 100U/ml penicillin, 100 μ g/ml streptomycin; or

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, and 100. mu.g/ml streptomycin.

6. 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, 2.5 v/v% Fetal Bovine Serum (FBS), 1mM L-glutamine, 2 v/v% essential amino acids, 1 v/v% nonessential amino acids, 3mM glutathione, 0.04 w/v% sodium citrate, 0.02 w/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.

7. The method according to claim 1, wherein in step (1), the BY-based culture solution comprises 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 sodium chloride, 130-150 mg/L monosodium phosphate monohydrate, 2000-2500 mg sodium bicarbonate, 40-60 mg/L, L-alanine, 20-30 mg/L, L-arginine hydrochloride, 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, 70-80 mg/L glycine, 20-60 mg/L, L-histidine hydrochloride monohydrate, 20-25 mg/6-hydroxyproline, 8-12 mg/L, L-15 mg/0.008-0.8 mg/8 mg/L methionine hydrochloride, 0.06-0.008-0.8 mg/L choline chloride, 0.06-200 mg/L choline chloride, 0.06-150 mg/L, 0.8 mg/L, 0.06-150 mg/L choline chloride, 0-150 mg/L choline chloride monohydrate, 0.8-150 mg/L, 0-150 mg/L, 0.8-150 mg/L sodium glutamate monohydrate, 0.8-150 mg/L, 0.8-200 mg/L2-200 mg/L methionine hydrochloride monohydrate, 0.8-200 mg/L, 0.8-200 mg/L methionine hydrochloride monohydrate, 0.8-5 mg/L, 0.8-0.0.0.8-0.8-0.0.8-0.8-0.0.0.0.0.8-0.0.8-0.8.

8. The method according to claim 1, wherein in step (1), the BY basal medium is an aqueous solution containing 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 sodium dihydrogen 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 mg/L glutamic acid, 50mg/L, L-histidine hydrochloride monohydrate 21.88mg/L, L-hydroxyproline 10 mg/84-20 mg/L, L-leucine 60mg/L, L-lysine hydrochloride 70mg/L, L-methionine 15mg/L, L-phenylalanine 25 mg/5-proline 40mg/L, L mg-20 mg/L leucine 60mg/L, L-serine hydrochloride, 0.5 mg/L5 mg/26 mg/L methionine hydrochloride, 0.5mg/L methionine hydrochloride, 0.05mg/L adenine, 0.05mg/L sodium folate hydrochloride, 0.05mg/L adenine, 0.05mg/L sodium folate hydrochloride monohydrate, 0.05mg/L, 0.8-arginine hydrochloride monohydrate, 0.8-L, 0.8-cysteine hydrochloride monohydrate, 0.27-L-cysteine hydrochloride monohydrate, 0.27-L-cysteine hydrochloride monohydrate, 0.27-L-histidine hydrochloride, 0.27-L-histidine hydrochloride, 0.8-L-histidine hydrochloride, 0.27-L-0.8-L-.

9. 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, 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.

10. A transport medium for oocytes comprising: glycine 50.0mg/L, L-alanine 25.0mg/L, L-arginine hydrochloride 70.0mg/L, L-aspartic acid 30.0mg/L, L-cystine dihydrochloride 26.0mg/L, L-glutamic acid 75.0mg/L, L-glutamine 100.0mg/L, L-histidine hydrochloride monohydrate 21.88mg/L, L-hydroxyproline 10.0mg/L, L-isoleucine 40.0mg/L, L-leucine 60.0mg/L, L-lysine hydrochloride 70.0mg/L, L-methionine 15.0mg/L, L-phenylalanine 25.0mg/L, L-proline 40.0mg/L, L-serine 25.0mg/L, L-threonine 30.0mg/L, L-tryptophan 10.0mg/L, L-tyrosine disodium salt dihydrate 58.0mg/L, L-valine 25.0mg/L, ascorbic acid 0.05mg/L, biotin 0.01 mg/based onL, choline chloride 0.5mg/L, D-calcium pantothenate 0.01mg/L, folic acid 0.01mg/L, menadione 0.01mg/L, nicotinamide 0.025mg/L, nicotinic acid 0.025mg/L, p-aminobenzoic acid 0.05mg/L, pyridoxal hydrochloride 0.025mg/L, pyridoxine hydrochloride 0.025mg/L, riboflavin 0.01mg/L, thiamine hydrochloride 0.01mg/L, vitamin A acetate 0.1mg/L, vitamin D2 i.e. calcitol 0.1mg/L, α -tocopherol sodium phosphate 0.01mg/L, inositol 0.05mg/L, anhydrous calcium chloride 200.0mg/L, ferric nitrate nonahydrate 0.7mg/L, anhydrous magnesium sulfate 97.67mg/L, potassium chloride 400.0mg/L, sodium chloride 6800.0mg/L, sodium dihydrogen phosphate monohydrate 140.0mg/L, adenine triphosphate 0.10 mg/L, adenine triphosphate 0.7mg/L, anhydrous magnesium sulfate 97.67mg/L, calcium acetate 0.80 mg/L, adenine L, 3mg/L, 3.80 mg/L, 3mg/L, 3.80 mg/L, 3.80L, 3.5 mg/L, 3.5L, 3.80L, 3.5 mg/L, 3.0.0.5 mg/L, 3.5 mg/L, 3.5 mg/L, 3.5L₂50ng/mL EGF, 100ng/mL IGF, 10% gentamicin, 55. mu.g/mL sodium pyruvate, 1.2mM/L cysteine, 3mg/mL BSA, 10mM/L HEPES; for example, taurine and zinc gluconate are also included therein; for example, the concentration of taurine is 30-50 mg/L, such as 40mg/L or 45 mg/L; for example, the concentration of zinc gluconate is 1 to 3mg/L, for example 2 mg/L.