CN107142239B - Method for improving culture efficiency of bovine in vitro fertilization embryos - Google Patents
Method for improving culture efficiency of bovine in vitro fertilization embryos Download PDFInfo
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- CN107142239B CN107142239B CN201710433656.6A CN201710433656A CN107142239B CN 107142239 B CN107142239 B CN 107142239B CN 201710433656 A CN201710433656 A CN 201710433656A CN 107142239 B CN107142239 B CN 107142239B
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- 239000000725 suspension Substances 0.000 description 1
- 238000010998 test method Methods 0.000 description 1
- 238000010257 thawing Methods 0.000 description 1
- 210000001519 tissue Anatomy 0.000 description 1
- 230000007704 transition Effects 0.000 description 1
- 238000002054 transplantation Methods 0.000 description 1
- GPRLSGONYQIRFK-MNYXATJNSA-N triton Chemical compound [3H+] GPRLSGONYQIRFK-MNYXATJNSA-N 0.000 description 1
- 210000002993 trophoblast Anatomy 0.000 description 1
- 150000003722 vitamin derivatives Chemical class 0.000 description 1
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Abstract
The invention relates to a method for improving the culture efficiency of bovine in vitro fertilized embryos. Specifically, the invention relates to a culture solution for bovine in vitro fertilization embryos, which comprises: NaCl, KCl, NaHCO3, MgCl2, KH2PO3, sodium pyruvate, glucose, calcium hemi-lactobionate, fetal bovine serum, L-glutamine, essential amino acids, non-essential amino acids, glutathione and water. Also relates to a method for culturing bovine in vitro fertilized embryos with improved efficiency, comprising the steps of: the bovine in vitro fertilized embryo is placed in bovine in vitro fertilized embryo culture solution, and the embryo in vitro culture is carried out under the conditions of 38.5 ℃, 0.5% CO2 and 100% humidity. The method and the culture solution used in the present invention exhibit excellent technical effects as described in the specification.
Description
Technical Field
The invention belongs to the technical field of animal breeding, relates to a technology for agriculture-animal husbandry and veterinary breeding, particularly relates to a method for improving the culture efficiency of cattle in-vitro fertilization embryos, and more particularly relates to application of a cattle in-vitro fertilization embryo culture solution in the culture of cattle in-vitro fertilization embryos. Furthermore, the invention also relates to a method for culturing the cattle in-vitro fertilization embryos by using the cattle in-vitro fertilization embryo culture solution. In particular, the method for culturing bovine in vitro fertilized embryos has excellent cost advantages.
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 and culture system. acta t 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; the results of 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 Jreprod Fertil,2000.118(1): p.47-55) and by Jean M.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 improvement on embryo development of beta-mercaptoethanol, 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 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, 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 vitrofertilization 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 bovine IVF embryo production efficiency and embryo quality. More specifically, the present invention provides a culture solution specifically for bovine in vitro fertilized embryos and a method for culturing bovine in vitro fertilized embryos using the culture solution to achieve the above object. 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 bovine in vitro fertilization embryo culture solution comprising:
109-110mM NaCl,
2.9-3.1mM of KCl,
26.0-26.5mM NaHCO3,
0.5-1.0mM MgCl 2.6H2O,
1.0-1.3mM KH2PO3,
0.4mM of sodium pyruvate,
1.5mM glucose,
5mM of calcium hemi-lactamate,
2-3v/v% of fetal calf serum,
1mM of L-glutamine,
2v/v% of essential amino acids,
1v/v% of non-essential amino acids,
1-10mM glutathione, and
water as a solvent for the liquid preparation.
A bovine in vitro fertilization embryo culture solution according to any one of the embodiments of the first aspect of the invention, the culture solution comprising:
109mM NaCl,
3.1mM of KCl,
26.2mM NaHCO3,
0.8mM MgCl2 & 6H2O,
1.19mM KH2PO3,
0.4mM of sodium pyruvate,
1.5mM glucose,
5mM of calcium hemi-lactamate,
2.5v/v% of fetal bovine serum,
1mM of L-glutamine,
2v/v% of essential amino acids,
1v/v% of non-essential amino acids,
1-10mM glutathione, and
water as a solvent for the liquid preparation.
The bovine in vitro fertilization embryo culture solution according to any one of the embodiments of the first aspect of the invention, wherein the essential amino acids are aqueous solutions prepared from the following amino acids in proportions and concentrations:
6.32 g/L-arginine hydrochloride,
1.564g/L of L-cystine dihydrochloride,
2.1g/L of L-histidine hydrochloride monohydrate,
2.625g/L of L-isoleucine,
2.62g/L of L-leucine,
3.625g/L of L-lysine hydrochloride,
0.755g/L of L-methionine,
1.65g/L of L-phenylalanine,
2.38g/L of L-threonine,
0.51g/L of L-tryptophan,
L-tyrosine 1.8g/L, and
l-valine 2.34 g/L.
The bovine in vitro fertilization embryo culture fluid according to any embodiment of the first aspect of the invention, wherein the non-essential amino acid is an aqueous solution prepared from the following amino acids in proportion and concentration:
0.89g/L of L-alanine,
1.5g/L of L-asparagine monohydrate,
1.33g/L of L-aspartic acid,
1.47g/L of L-glutamic acid,
0.75g/L of glycine,
L-proline 1.15g/L, and
l-serine 1.05 g/L.
The culture solution for bovine in vitro fertilization embryos according to any one of the embodiments of the first aspect of the invention, wherein the concentration of glutathione in the culture solution is 1-7 mM.
The culture solution for bovine in vitro fertilization embryos according to any one of the embodiments of the first aspect of the invention, wherein the concentration of glutathione in the culture solution is 3-5 mM.
The bovine in vitro fertilization embryo culture solution according to any one of the embodiments of the first aspect of the invention, wherein the concentration of glutathione in the culture solution is 1mM, 3mM, 5mM or 7 mM.
The bovine in vitro fertilization embryo culture solution according to any one of the embodiments of the first aspect of the invention, wherein the concentration of glutathione in the culture solution is 3mM or 5 mM.
The bovine in vitro fertilization embryo culture solution according to any one of the embodiments of the first aspect of the invention, wherein the concentration of glutathione in the culture solution is 3 mM.
The bovine in vitro fertilization embryo culture solution according to any one of the embodiments of the first aspect of the present invention further comprises sodium citrate and maltose. In one embodiment, the concentration of sodium citrate and maltose in the bovine in vitro fertilization embryo culture fluid is 0.03-0.05 w/v% and 0.01-0.03 w/v%, respectively. In one embodiment, the concentration of sodium citrate and maltose in the bovine in vitro fertilization embryo culture fluid is 0.04w/v% and 0.02w/v%, respectively. It has been unexpectedly found that the addition of trace amounts of sodium citrate and maltose to the culture solution of bovine in vitro fertilized embryos helps to improve blastocyst hatchability, and other indicators are not affected. The blastocyst hatching rate is an important evaluation index of the quality of the blastocyst, the blastocyst hatching rate is improved on the premise that other indexes are not influenced, and the method has important significance for the culture work of the bovine in vitro fertilization embryos. In addition, the sodium citrate and maltose are added, so that the concentration of fetal calf serum in the bovine in vitro fertilization embryo culture solution can be greatly reduced, and basically the same culture effect is obtained; since fetal calf serum in the culture solution of bovine in vitro fertilization embryos is extremely expensive relative to other components (including sodium citrate and maltose) in the culture solution, the main cost of the culture solution of bovine in vitro fertilization embryos is that of fetal calf serum, and the reduction of the dosage of fetal calf serum is extremely beneficial to improving the culture efficiency of bovine in vitro fertilization embryos and reducing the production cost.
Further, the second aspect of the present invention provides a method for culturing bovine in vitro fertilized embryos with improved efficiency, the method comprising the steps of:
the bovine in vitro fertilized embryo is placed in bovine in vitro fertilized embryo culture solution, and the embryo in vitro culture is carried out under the conditions of 38.5 ℃, 0.5% CO2 and 100% humidity.
The method according to any embodiment of the second aspect of the present invention, wherein the in vitro culture of embryos comprises the steps of:
(1) after the sperms and the eggs are incubated for 8h, taking out the oocytes, digesting and treating for 3min by using 1mg/ml hyaluronidase, and then repeatedly blowing and beating by using a pipette until cumulus cells are basically shed;
(2) using TCM199 containing 10% FBS to terminate the digestion of hyaluronidase, washing suspected fertilized eggs in a pre-embryo culture solution added with bovine serum albumin (usually also abbreviated as BSA) for 3 times by using a mouth suction tube, and then putting the suspected fertilized eggs into a pre-embryo culture drop for culture under the culture conditions of 38.5 ℃, 0.5% CO2 and 100% humidity;
(3) after 48 hours, transferring the embryos cracked to 4-8 cells into a late-stage embryo culture solution (namely the bovine in vitro fertilization embryo culture solution) drop for culture (the culture condition is 38.5 ℃, 0.5% CO2 and 100% humidity), and replacing the late-stage embryo culture solution after 48 hours and continuing to culture (38.5 ℃, 5% CO2 gas and 95% humidity);
(4) counting the day of fertilization by 0 day, counting blastocyst rate by 7 days, and counting blastocyst rate (which is the percentage of the number of hatched blastocysts divided by the number of blastocysts) by 9 days.
According to the method of any one of the embodiments of the second aspect of the invention, the culture solution of bovine in vitro fertilized embryo comprises:
109-110mM NaCl,
2.9-3.1mM of KCl,
26.0-26.5mM NaHCO3,
0.5-1.0mM MgCl 2.6H2O,
1.0-1.3mM KH2PO3,
0.4mM of sodium pyruvate,
1.5mM glucose,
5mM of calcium hemi-lactamate,
2-3v/v% of fetal calf serum,
1mM of L-glutamine,
2v/v% of essential amino acids,
1v/v% of non-essential amino acids,
1-10mM glutathione, and
water as a solvent for the liquid preparation.
According to the method of any one of the embodiments of the second aspect of the invention, the culture solution of bovine in vitro fertilized embryo comprises:
109mM NaCl,
3.1mM of KCl,
26.2mM NaHCO3,
0.8mM MgCl2 & 6H2O,
1.19mM KH2PO3,
0.4mM of sodium pyruvate,
1.5mM glucose,
5mM of calcium hemi-lactamate,
2.5v/v% of fetal bovine serum,
1mM of L-glutamine,
2v/v% of essential amino acids,
1v/v% of non-essential amino acids,
1-10mM glutathione, and
water as a solvent for the liquid preparation.
According to the method of any one of the embodiments of the second aspect of the invention, the essential amino acids in the culture solution of the bovine in vitro fertilized embryo are aqueous solutions prepared by the following amino acids in proportion and concentration:
6.32 g/L-arginine hydrochloride,
1.564g/L of L-cystine dihydrochloride,
2.1g/L of L-histidine hydrochloride monohydrate,
2.625g/L of L-isoleucine,
2.62g/L of L-leucine,
3.625g/L of L-lysine hydrochloride,
0.755g/L of L-methionine,
1.65g/L of L-phenylalanine,
2.38g/L of L-threonine,
0.51g/L of L-tryptophan,
L-tyrosine 1.8g/L, and
l-valine 2.34 g/L.
According to the method of any one of the embodiments of the second aspect of the invention, the non-essential amino acids in the culture solution of bovine in vitro fertilized embryos are aqueous solutions prepared from the following amino acids in proportions and concentrations:
0.89g/L of L-alanine,
1.5g/L of L-asparagine monohydrate,
1.33g/L of L-aspartic acid,
1.47g/L of L-glutamic acid,
0.75g/L of glycine,
L-proline 1.15g/L, and
l-serine 1.05 g/L.
The method according to any one of the embodiments of the second aspect of the present invention, wherein the concentration of glutathione in the culture solution is 1 to 7 mM.
The method according to any one of the embodiments of the second aspect of the present invention, wherein the concentration of glutathione in the culture solution is 3 to 5 mM.
The method according to any one of the embodiments of the second aspect of the present invention, wherein the concentration of glutathione in the culture liquid is 1mM, 3mM, 5mM or 7 mM.
The method according to any one of the embodiments of the second aspect of the present invention, wherein the concentration of glutathione in the culture solution is 3mM or 5 mM.
The method according to any one of the embodiments of the second aspect of the present invention, wherein the concentration of glutathione in the culture solution is 3 mM.
According to the method of any one of the embodiments of the second aspect of the present invention, the culture solution of the bovine in vitro fertilized embryo further comprises sodium citrate and maltose. In one embodiment, the concentration of sodium citrate and maltose in the bovine in vitro fertilization embryo culture fluid is 0.03-0.05 w/v% and 0.01-0.03 w/v%, respectively. In one embodiment, the concentration of sodium citrate and maltose in the bovine in vitro fertilization embryo culture fluid is 0.04w/v% and 0.02w/v%, respectively.
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.
The invention has the following advantages: the invention can obviously improve the developmental capacity of in vitro embryos by adding Glutathione (Glutathione, GSH) with certain concentration into the culture solution. Meanwhile, the blastocyst is differentially stained, Inner Cell Masses (ICMs), trophoblast cells and apoptotic cells are distinguished, the total Cell number is compared, the proportion of the ICM Cell number to the total Cell number and the apoptosis rate are calculated, and the result shows that a test group added with GSH is superior to a control group, so that 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 the cattle IVF technology to practice, and the genetic breeding process can be greatly accelerated. In addition, the invention has surprisingly found that the use of the bovine in vitro fertilization embryo culture solution with the formula can obviously improve the blastocyst hatching rate without influencing other indexes, the blastocyst hatching rate is an important evaluation index of the quality of the blastocyst, the blastocyst hatching rate is improved on the premise that other indexes are not influenced, and the invention has important significance for the culture work of the bovine in vitro fertilization embryo. In addition, the invention also discovers that the concentration of fetal calf serum in the bovine in vitro fertilization embryo culture solution can be greatly reduced under the condition of adding the sodium citrate and the maltose, and the basically same culture effect is obtained; since fetal calf serum in the culture solution of bovine in vitro fertilization embryos is extremely expensive relative to other components (including sodium citrate and maltose) in the culture solution, the main cost of the culture solution of bovine in vitro fertilization embryos is that of fetal calf serum, and the reduction of the dosage of fetal calf serum is extremely beneficial to improving the culture efficiency of bovine in vitro fertilization embryos and reducing the production cost.
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
Mature culture solution:contains 0.01IU/ml FSH, 10IU/ml LH, 1 μ g/ml estradiol, 100ng/mlIGF (1), EGF (50 ng/ml), penicillin (100U/ml), streptomycin (100 mu g/ml) and TCM-199 culture medium of 10% fetal bovine serum.
Washing semen:an aqueous solution containing 112.0mM NaCl, 4.02mM KCl, 2.25mM CaCl 2.2H 2O, 0.52mM MgCl 2.6H 2O, 0.83mM KH2PO3, 37.0mM NaHCO3, 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.
Receiving semen:an aqueous solution containing 112.0mM NaCl, 4.02mM KCl, 2.25mM CaCl 2.2H 2O, 0.52mM MgCl 2.6H 2O, 0.83mM KH2PO3, 37.0mM NaHCO3, 1.25mM sodium pyruvate, 10. mu.g/ml heparin, 4mg/ml BSA, 100U/ml penicillin, 100. mu.g/ml streptomycin.
Embryo prophase culture solution:containing 109.5mM NaCl, 3.1mM KCl, 26.2mM NaHCO3, 0.8mM MgCl 2.6H2O, 1.19mM KH2PO3, 0.4mM sodium pyruvate, 1.5mM glucose, 5mM calcium hemi-lactobionate, 6mg/ml BSA, 1mM L-glutamine, 2v/v% essential amino acids, 1v/v% optionalAn aqueous solution of an amino acid.
And (3) culture solution at the later stage of embryo:an aqueous solution containing 109.5mM NaCl, 3.1mM KCl, 26.2mM NaHCO3, 0.8mM MgCl 2.6H2O, 1.19mM KH2PO3, 0.4mM sodium pyruvate, 1.5mM glucose, 5mM calcium hemi-lactamate, 10 v/v% Fetal Bovine Serum (FBS), 1mM L-glutamine, 2v/v% essential amino acids, 1v/v% non-essential amino acids.
The essential amino acid is an aqueous solution prepared from the following amino acids according to proportion/concentration, 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 from the following amino acids according to proportion/concentration, 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.
Second, collection and in vitro maturation of oocytes
1. Bovine ovaries were collected from slaughterhouses, placed in physiological saline at 37 ℃ and sent to the laboratory within 3 hours.
2. The ovaries were washed 3-5 times with warm saline containing penicillin and streptomycin.
3. Follicular fluid was withdrawn from 5-8 mm follicles using a peristaltic vacuum pump with an 18 gauge needle.
4. Oocytes-cumulus cell complexes (COCs) were selected under a stereomicroscope, washed twice in an egg wash and once in mature medium.
5. Putting the washed COCs into a four-hole plate containing mature culture solution and covered with mineral oil, culturing 50 COCs in each hole, and culturing for 22-24 h.
Third, in vitro fertilization
1. The COCs were transferred from the maturation medium into 50. mu.l fertilisation drops, 15 COCs per drop.
Thawing semen in water bath at 2.38 deg.C, and centrifuging 5ml of semen for 5min under 500 g; discarding the supernatant, adding 5ml of semen, centrifuging for 5min, discarding the supernatant, and adjusting the semen concentration to 2 × 107One per ml.
3. 50 μ l of seminal fluid was mixed with the fertilization drop containing COCs to form a 100 μ l drop.
4.38.5 deg.C, 5% CO2 gas, and 95% humidity for 8 h.
Fourthly, embryo in vitro culture
1. After the seminal egg is incubated for 8h, the oocyte is taken out, digested and treated for 3min by using 1mg/ml hyaluronidase, and then blown and beaten repeatedly by using a pipette until the cumulus cell is basically shed.
2. Digestion with hyaluronidase was terminated using TCM199 containing 10% FBS, and suspected fertilized eggs were washed 3 times in pre-embryo culture medium supplemented with bovine serum albumin using a mouth pipette and then placed in pre-embryo culture drops for culture (38.5 ℃, 5% CO2 gas, 95% humidity).
After 3.48h, the embryos which have been lysed to 4-8 cells are transferred to the late-embryo culture drops for culture (38.5 ℃, 5% CO2 gas, 95% humidity), and the late-embryo culture solution is replaced after 48h and the culture is continued (38.5 ℃, 5% CO2 gas, 95% humidity).
4. Counting the day of fertilization by 0 day, counting blastocyst rate by 7 days, and counting blastocyst rate (which is the percentage of the number of hatched blastocysts divided by the number of blastocysts) by 9 days.
Fifthly, screening the optimum addition concentration of GSH (glutathione) in the culture solution of the later period of the embryo
1. GSH was formulated at 0.5M as stock solution.
2. The test group prepared as the late embryo culture solution was added to the late embryo culture solution using GSH stock solution in a gradient of 1mM, 3mM, 5mM, and 7mM final concentrations, and the non-added group was used as a control.
3. And counting the rate of eggs cracked and 8 cells, the rate of morulae and blastula of each group on 48h, 5 th and 7 th days of culture respectively.
Sixthly, 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.
Seventh, data statistics
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.
Eighthly, the addition of GSH in the culture solution of the embryo at the later stage can obviously improve the development rate of the bovine in-vitro fertilization blastocyst
GSH was selected at 0, 1, 3, 5, and 7mM concentrations for screening, and the cleavage rate (i.e., cleavage rate ═ fertilized cleavage number/fertilized egg number), 4-8 cell rate, morula rate, and blastocyst rate (i.e., blastocyst rate ═ blastocyst number/cracked embryo number) were counted.
As a result:
the difference between the cleavage rate and the 4-8 cell rate of five groups with the GSH addition concentration of 0, 1, 3, 5 and 7mM in the embryo later-stage culture solution is not significant (p is more than 0.05), for example, the cleavage rate of five groups is within the range of 0.80-0.88, and the 4-8 cell rate of five groups is within the range of 0.67-0.79;
for the morula development rate (i.e. morula rate, morula rate which is the morula number/cleavage embryo number), the GSH addition groups of 3mM and 5mM are both significantly higher than the control group and the experimental groups of 5mM and 7mM (p <0.05), but the difference between the two groups of 3mM and 5mM is not significant (p >0.05), for example, the morula rate of the control group and the three groups of 5mM and 7mM is in the range of 0.46-0.49, while the morula rate of the two groups of 3mM and 5mM is in the range of 0.61-0.63;
for blastocyst rate, the 1mM, 3mM and 5mM GSH addition groups are all significantly higher than the control group (p <0.05), while the 7mM GSH addition group has no significant difference from the control group (p >0.05), for example, the blastocyst rate of the control group and 7mM group is 0.32 and 0.36, the blastocyst rate of the 1mM group is 0.42, and the blastocyst rate of the 3mM and 5mM groups is in the range of 0.46-0.51; these results are substantially consistent with CN103898046A table 1 results. It can be seen that the addition of GSH to the late-stage culture solution of the embryo can improve the in vitro developmental rate of the bovine IVF embryo.
Ninth, the quality of cow in-vitro fertilized embryo can be improved by adding GSH into the culture solution of the later embryo stage
By counting the total cell number, ICM cell number/total cell number, and apoptosis rate (i.e., apoptosis rate ═ apoptotic cell number/total cell number), blastocyst mass obtained from different GSH addition concentration groups was comparatively analyzed. The results are as follows:
for total cell number, the four GSH concentration added groups of 1mM, 3mM, 5mM, 7mM were all significantly higher than the control group (p <0.05), and the 3mM, 5mM, and 7mM groups were all significantly higher than the 1mM group (p <0.05), e.g., the 0mM group total cell number was 42.4, the 1mM group total cell number was 63.6, and the 3mM, 5mM, and 7mM groups total cell number was in the range of 82-89;
for the ratio of ICM cell number/total cell number, the 1mM addition group and the control group have no significant difference (p >0.05), the 3mM, 5mM and 7mM groups have no significant difference (p <0.05), but the difference between the three groups is no significant (p >0.05), for example, the ICM cell number/total cell number ratio of the 1mM addition group and the control group are all in the range of 0.41-0.48, and the ICM cell number/total cell number ratio of the 3mM, 5mM and 7mM groups are all in the range of 0.41-0.48 and 0.27-0.34;
for the apoptosis rate, the difference between the four addition groups and the control group is not significant (p >0.05), and the apoptosis rate of the 1mM, 3mM, 7mM GSH addition group and the control group is significantly lower than that of the 5mM addition group (p < 0.05); these results are substantially consistent with CN103898046A table 2 results. It can be seen that the addition of GSH contributes to the improvement of the quality of the bovine in vitro fertilized embryos.
Tenth, the optimal addition concentration of GSH added into the culture solution at the later stage of the embryo is 3mM
In the four experimental groups, the morula rate and blastocyst rate obtained by the 3mM and 5mM GSH addition groups are significantly higher than those of the control group and other experimental groups (p < 0.05); however, the morula rate of the 3mM GSH-added group was not significantly different from that of the 5mM GSH-added group (p >0.05), but the blastocyst rate was significantly higher than that of the 5mM GSH-added group (p <0.05), and it was found that the bovine IVF embryo development rate was the highest in the 3mM GSH-added group, and therefore the GSH-added group had the optimal concentration of 3 mM.
The optimal addition concentration is screened out in the embodiment, the production efficiency of the blastula is greatly improved, and the blastula rate is improved from 30% to 50%; the evaluation of embryo quality also shows that the quality of the blastocyst added with the product is better than that of a control group.
Example 2: culture method of bovine in vitro fertilized embryo
Referring to example 1, except that the composition of the post-embryo culture fluid was changed to:
109mM NaCl,
3.1mM of KCl,
26.0mM NaHCO3,
0.5mM MgCl2 & 6H2O,
1.3mM KH2PO3,
0.4mM of sodium pyruvate,
1.5mM glucose,
5mM of calcium hemi-lactamate,
10 v/v% of fetal bovine serum,
1mM of L-glutamine,
2v/v% of essential amino acids,
1v/v% of non-essential amino acids,
3mM glutathione, and
water as a solvent for the liquid preparation.
The test was conducted in accordance with example 1, and the results were substantially the same as those of the test conducted in example 1 using the glutathione group at 3mM, i.e., the morula ratio was 0.633, the blastocyst ratio was 0.508, and the apoptosis ratio was 0.050.
Example 3: culture method of bovine in vitro fertilized embryo
Referring to example 1, except that the composition of the post-embryo culture fluid was changed to:
110mM NaCl,
2.9mM of KCl,
26.5mM NaHCO3,
1.0mM MgCl2 & 6H2O,
KH2PO3 of 1.0mM,
0.4mM of sodium pyruvate,
1.5mM glucose,
5mM of calcium hemi-lactamate,
10 v/v% of fetal bovine serum,
1mM of L-glutamine,
2v/v% of essential amino acids,
1v/v% of non-essential amino acids,
3mM glutathione, and
water as a solvent for the liquid preparation.
The test was conducted in accordance with example 1, and the results were substantially the same as those of the glutathione group at 3mM in example 1, i.e., the morula ratio was 0.636, the blastocyst ratio was 0.521, and the apoptosis ratio was 0.050.
Example 4: culture method of bovine in vitro fertilized embryo
Referring to example 1, except that the composition of the post-embryo culture fluid was changed to:
109mM NaCl,
3.1mM of KCl,
26.0mM NaHCO3,
0.5mM MgCl2 & 6H2O,
1.3mM KH2PO3,
0.4mM of sodium pyruvate,
1.5mM glucose,
5mM of calcium hemi-lactamate,
10 v/v% of fetal bovine serum,
1mM of L-glutamine,
2v/v% of essential amino acids,
1v/v% of non-essential amino acids,
3mM of glutathione,
0.04w/v% of sodium citrate,
Maltose 0.02w/v%, and
water as a solvent for the liquid preparation.
The results show that, in all the indexes examined in example 1, in example 4, the same criteria as those in example 1 were applied to the glutathione group at 3mM, for example, the morula ratio was 0.634, the blastocyst ratio was 0.518, and the apoptosis ratio was 0.049 in example 4; in addition, the blastocyst hatchability was counted on day 9, and the results showed that the blastocyst hatchability of this example reached 71.6%; in addition, the blastocyst hatchability was found to be in the range of 36 to 39% in all of the experiments of examples 1 to 3 using 3mM glutathione.
In a supplementary experiment, referring to example 4 above, except that the sodium citrate concentration was changed to 0.03w/v% or 0.05w/v% (but maltose was still 0.02 w/v%), the criteria in both experiments were essentially the same as in example 4 above, e.g., blastocyst hatchability ranged from 70.2 to 71.4%. In a supplementary experiment, with reference to example 4 above, except that the maltose concentration was changed to 0.01 w/v% or 0.03w/v% (but sodium citrate was still 0.04 w/v%), the criteria in both experiments were essentially the same as in example 4 above, e.g. blastocyst hatchability was in the range of 71.6-72.6%; the result shows that the hatching rate of the blastula can be effectively improved after a proper amount of sodium citrate and maltose are supplemented in the culture solution at the later stage of the blastula. In a supplementary experiment, referring to example 4 above, except that no sodium citrate was added (but maltose was still 0.02 w/v%), or only that no maltose was added (but sodium citrate was still 0.04 w/v%), the indexes except for blastocyst hatchability in both experiments were substantially the same as in example 4 above, but the blastocyst hatchability was significantly low and ranged from 38-40% (p <0.05, with significant difference); this result indicates that addition of either sodium citrate or maltose alone is not effective in increasing blastocyst hatchability.
Example 5: culture method of bovine in vitro fertilized embryo
Referring to example 1, except that the composition of the post-embryo culture fluid was changed to:
109mM NaCl,
3.1mM of KCl,
26.0mM NaHCO3,
0.5mM MgCl2 & 6H2O,
1.3mM KH2PO3,
0.4mM of sodium pyruvate,
1.5mM glucose,
5mM of calcium hemi-lactamate,
2.5v/v% of fetal bovine serum,
1mM of L-glutamine,
2v/v% of essential amino acids,
1v/v% of non-essential amino acids,
3mM of glutathione,
0.04w/v% of sodium citrate,
Maltose 0.02w/v%, and
water as a solvent for the liquid preparation.
The results show that, in all the indexes examined in example 1, in example 5, the same criteria as those in example 1 were applied to the 3mM glutathione group, and for example, the morula ratio was 0.628, the blastocyst ratio was 0.522, and the apoptosis ratio was 0.048 in example 5; in addition, the blastocyst hatchability was counted on day 9, and the results showed that the blastocyst hatchability of this example reached 70.2%; in addition, the blastocyst hatchability was found to be in the range of 36 to 39% in all of the experiments of examples 1 to 3 using 3mM glutathione.
In a supplementary experiment, referring to example 5 above, except that the sodium citrate concentration was changed to 0.03w/v% or 0.05w/v% (but maltose was still 0.02 w/v%), the criteria in both experiments were essentially the same as in example 5 above, e.g., blastocyst hatchability ranged from 69.7 to 71.8%. In a supplementary experiment, with reference to example 5 above, except that the maltose concentration was changed to 0.01 w/v% or 0.03w/v% (but sodium citrate was still 0.04 w/v%), the criteria in both experiments were essentially the same as in example 5 above, e.g. blastocyst hatchability was in the range of 71.8-72.4%; the result shows that the hatching rate of the blastula can be effectively improved after a proper amount of sodium citrate and maltose are supplemented in the culture solution at the later stage of the blastula. In a supplementary experiment, referring to example 5 above, except that no sodium citrate was added (but maltose was still 0.02 w/v%), or only that no maltose was added (but sodium citrate was still 0.04 w/v%), the indexes except for blastocyst hatchability in both experiments were substantially the same as those in example 5 above, but the blastocyst hatchability was significantly small and ranged from 36 to 39% (p <0.05, with significant difference); this result indicates that addition of either sodium citrate or maltose alone is not effective in increasing blastocyst hatchability. In a supplementary test, with reference to example 5 above, except that sodium citrate and maltose were not added, the results of the test were substantially the same as those of example 5 above except that the blastocyst hatchability was significantly low, 31.3% (p <0.05, with significant difference); this result indicates that blastocyst hatchability cannot be effectively increased without adding both sodium citrate and maltose in the case of lowering the fetal bovine serum concentration to 2.5 v/v%. In a supplementary experiment, referring to example 5 above, except that the concentration of fetal bovine serum was changed to 2v/v% or 3v/v%, the indexes in both experiments were substantially the same as those in example 5 above, e.g., the blastocyst hatchability was within 70.3-71.7%; the result shows that the blastocyst hatching rate can be effectively improved even if the fetal bovine serum concentration is reduced to the range of 2-3v/v% under the condition that a proper amount of sodium citrate and maltose are additionally added into the embryo late-stage culture solution, at the moment, the dosage of expensive reagent of fetal bovine serum is 20-30% of the dosage of the embodiment 1, and the cost of the added sodium citrate and maltose is very low.
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 (5)
1. A bovine in vitro fertilization embryo culture solution, the culture solution comprising: 109-30.5-1.0mM MgCl2·6H2O, 1.0-1.3mM KH2PO30.4mM sodium pyruvate, 1.5mM glucose, 5mM calcium half-lactobionate, 2-3v/v% fetal bovine serum, 1mM L-glutamine, 2v/v% essential amino acids, 1v/v% nonessential amino acids, 1-10mM glutathione, sodium citrate, maltose, and water as a formulating solvent; wherein:
the concentrations of sodium citrate and maltose are 0.03 ~ 0.05.05 w/v% and 0.01 ~ 0.03.03 w/v%, respectively;
the essential amino acid is an aqueous solution prepared from the following amino acids in proportion and concentration: 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 from the following amino acids in proportion and concentration: 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.
2. The bovine in vitro fertilization embryo culture fluid according to claim 1, which comprises: 109mM NaCl, 3.1mM KCl, 26.2mM NaHCO30.8mM MgCl2·6H2O, 1.19mM KH2PO30.4mM 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, 1-10mM glutathione, and water as a formulating solvent.
3. The bovine in vitro fertilization embryo culture fluid of claim 1 wherein the concentration of glutathione in the culture fluid is 1 ~ 7 mM.
4. The bovine in vitro fertilization embryo culture fluid of claim 1 wherein the concentration of glutathione in the culture fluid is 3 ~ 5 mM.
5. The bovine in vitro fertilization embryo culture fluid of claim 1 wherein the concentrations of sodium citrate and maltose are 0.04w/v% and 0.02w/v%, respectively.
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