CN112980778A - Method for culturing and cryopreserving bovine in-vitro fertilization embryos - Google Patents

Method for culturing and cryopreserving bovine in-vitro fertilization embryos Download PDF

Info

Publication number
CN112980778A
CN112980778A CN202110493547.XA CN202110493547A CN112980778A CN 112980778 A CN112980778 A CN 112980778A CN 202110493547 A CN202110493547 A CN 202110493547A CN 112980778 A CN112980778 A CN 112980778A
Authority
CN
China
Prior art keywords
sodium
culture
embryo
vitro
fertilization
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Granted
Application number
CN202110493547.XA
Other languages
Chinese (zh)
Other versions
CN112980778B (en
Inventor
王娜
王小武
韩勇权
郝少强
赵明礼
郭春明
许晓椿
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Tianjin Limu Biotechnology Co ltd
Original Assignee
Tianjin Boyu Limu Technology Co ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Tianjin Boyu Limu Technology Co ltd filed Critical Tianjin Boyu Limu Technology Co ltd
Priority to CN202110493547.XA priority Critical patent/CN112980778B/en
Publication of CN112980778A publication Critical patent/CN112980778A/en
Application granted granted Critical
Publication of CN112980778B publication Critical patent/CN112980778B/en
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0603—Embryonic cells ; Embryoid bodies
    • C12N5/0604—Whole embryos; Culture medium therefor
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N1/00—Preservation of bodies of humans or animals, or parts thereof
    • A01N1/02—Preservation of living parts
    • A01N1/0205—Chemical aspects
    • A01N1/021—Preservation or perfusion media, liquids, solids or gases used in the preservation of cells, tissue, organs or bodily fluids
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N1/00—Preservation of bodies of humans or animals, or parts thereof
    • A01N1/02—Preservation of living parts
    • A01N1/0205—Chemical aspects
    • A01N1/021—Preservation or perfusion media, liquids, solids or gases used in the preservation of cells, tissue, organs or bodily fluids
    • A01N1/0221—Freeze-process protecting agents, i.e. substances protecting cells from effects of the physical process, e.g. cryoprotectants, osmolarity regulators like oncotic agents
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/05—Inorganic components
    • C12N2500/10—Metals; Metal chelators
    • C12N2500/12—Light metals, i.e. alkali, alkaline earth, Be, Al, Mg
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/05—Inorganic components
    • C12N2500/10—Metals; Metal chelators
    • C12N2500/12—Light metals, i.e. alkali, alkaline earth, Be, Al, Mg
    • C12N2500/14—Calcium; Ca chelators; Calcitonin
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/05—Inorganic components
    • C12N2500/10—Metals; Metal chelators
    • C12N2500/12—Light metals, i.e. alkali, alkaline earth, Be, Al, Mg
    • C12N2500/16—Magnesium; Mg chelators
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/30—Organic components
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/30—Organic components
    • C12N2500/32—Amino acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/30—Organic components
    • C12N2500/38—Vitamins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/02—Compounds of the arachidonic acid pathway, e.g. prostaglandins, leukotrienes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/10—Growth factors
    • C12N2501/11—Epidermal growth factor [EGF]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/30—Hormones
    • C12N2501/31—Pituitary sex hormones, e.g. follicle-stimulating hormone [FSH], luteinising hormone [LH]; Chorionic gonadotropins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/90—Polysaccharides
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/999—Small molecules not provided for elsewhere

Landscapes

  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Wood Science & Technology (AREA)
  • Zoology (AREA)
  • Biomedical Technology (AREA)
  • General Health & Medical Sciences (AREA)
  • Reproductive Health (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Gynecology & Obstetrics (AREA)
  • Environmental Sciences (AREA)
  • Dentistry (AREA)
  • Biotechnology (AREA)
  • Organic Chemistry (AREA)
  • Developmental Biology & Embryology (AREA)
  • Genetics & Genomics (AREA)
  • Chemical & Material Sciences (AREA)
  • Microbiology (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • Cell Biology (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

The invention relates to a method for culturing and cryopreserving bovine in-vitro fertilization embryos, which comprises the following steps: washing the mature cumulus-oocyte complex in a fertilization culture solution, transferring the mature cumulus-oocyte complex into the fertilization culture solution, and putting the mature cumulus-oocyte complex into an incubator for later use; taking the frozen semen tubule from the liquid nitrogen, thawing, and taking the semen suspension for counting the semen; adding the sperm suspension into the fertilization culture liquid drop containing the oocyte to incubate the sperm and the ovum, completing the in vitro fertilization operation, removing the granular cells around the embryo by using an ovum-peeling needle, putting the granular cells into an embryo culture solution for culturing, transferring the available embryo into a refrigerating fluid, and putting the refrigerating fluid into liquid nitrogen for freezing and storing; when the embryo needing to be frozen and stored is used, the thin tube is taken out of the liquid nitrogen and is transferred into the thawing solution, and the embryo is put into the small drop of the embryo culture solution for culture, thereby completing the thawing process. The invention also relates to the refrigerating fluid and the thawing fluid used. The method can obviously improve the survival rate of the in vitro fertilized embryo of the cattle after cryopreservation and thawing.

Description

Method for culturing and cryopreserving bovine in-vitro fertilization embryos
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 cattle in-vitro fertilization, and further relates to application of relevant working test solution used in cattle in-vitro fertilization, in particular to a method for cattle in-vitro fertilization, such as a method for cryopreserving and unfreezing cattle in-vitro fertilization blastocysts with excellent effects. The method for cattle in-vitro fertilization has excellent technical effects.
Background
In Vitro Fertilization (In Vitro Fertilization) or (external Fertilization) refers to a technique In which sperm and eggs of a mammal complete a Fertilization process In an environment artificially controlled In Vitro, abbreviated In the english to IVF. Because it is inseparable from the embryo transfer technique (ET), also referred to as IVF-ET for short. In biology, an animal obtained after an in vitro fertilized embryo is transferred to a mother is called a test-tube animal. The technology is successful in the 50 s of the 20 th century, develops rapidly in the last 20 years, and is mature day by day to become an important and conventional animal breeding biotechnology.
The in vitro fertilization technology has important significance for animal reproductive mechanism research, livestock production, medicine, endangered animal protection and the like. For example, using mouse, rat or rabbit as experimental material, the in vitro fertilization technique can be used to study the gametogenesis, fertilization and early embryonic development mechanism of mammals. In the livestock breed improvement, the in vitro fertilization technology provides a cheap and efficient means for embryo production, and has important values for fully utilizing excellent breed resources, shortening the breeding cycle of livestock, accelerating the breed improvement speed and the like. In humans, IVF-ET technology is one of the important measures to treat certain infertility and to overcome sexual linked diseases. In vitro fertilization is also an indispensable component of modern biotechnology, such as mammalian embryo transplantation, cloning, transgenosis, sex control and the like.
With the development of modern agricultural science and technology, in order to make full use of the breeding potential of elite cows and accelerate the genetic breeding process, it becomes necessary to apply a new efficient breeding technology in production practice. In-vivo egg taking (OPU) and In Vitro Fertilization (IVF) are new embryo engineering technologies that have been developed rapidly In the eighties of the twentieth century, and a large number of embryos with definite genetic pedigrees can be obtained by combining the OPU and the IVF, so that the generation interval is shortened. At present, the two technologies become important breeding technologies adopted by farmers in animal husbandry developed countries such as europe, the united states and oceania for expanding stock cow groups. However, with the conventional bovine embryo culture system (CR1aa and SOF liquid), the blastocyst development rate of bovine in vitro fertilization is low, and the embryo quality is far inferior to that of in vivo embryos, so that the pregnancy rate after embryo transfer recipient is low, and therefore how to improve the blastocyst development rate and the embryo quality becomes the focus of in vitro fertilization embryo production and research.
As early as 1878, German Scnenk began to explore the in vitro fertilization technique of mammals using rabbits and guinea pigs as materials. However, in 1951, the in vitro fertilization technology has not been developed in a breakthrough after sperm capacitation was discovered by Zhang Xuanling and Austin, respectively. The cattle in vitro fertilization technology is influenced by the aspects of oocyte in vitro maturation, sperm in vitro capacitation, fertilized egg in vitro culture environment and the like.
The in vitro culture of the embryo is a key link of the IVF technology, and is also the embodiment and the test of the final effect of the oocyte in vitro maturation and in vitro fertilization technology. After in vitro fertilization, a fertilized egg undergoes a number of important changes during its development into a blastocyst, including zygote formation, first cleavage, activation of the embryonic genome, densification, and blastocyst formation. In the process, the change of the external environment can cause the change of gene expression, thereby influencing the normal development and quality of the embryo. Currently, in vitro culture studies of early mammalian embryos focus mainly on improving the composition of the culture medium to meet the nutritional requirements of the embryos at different developmental stages. Two gradually improved systems were developed based on Charles Rosenkrans 1(CR1) culture Fluid developed by Rosenkrans et al (Rosenkrans, C.F., Jr. and N.L. First, Effect of free amino acids and vitamins on clearance and maintenance of bovine zygoes in vision, J Anim Sci, 1994.72(2): p.434-7) and Synthetic egg tube Fluid developed by Tervit et al (Tervit, H.R., D.G. Whittingham, and L.E. Rowson, Saccharomyces focus in vision of sheet and vegetable and ova. J. Reprod. Fertil, 1972.30(3): p.493-7). According to the research results of Hakan Sagirkaya et al (Sagirkaya, H., et al, Development potential of bovine embryos culture in differential formation and culture conditions, animal Reprod Sci, 2007.101(3-4): p.225-40) and Somfai et al (Somfai, T., et al, Development of bovine embryos culture in CR1aa and IVD101 medium using differential oxygen delivery and culture systems, 2010.58(4): p.465-74), CR1aa culture solution has a good effect on bovine embryo culture and can be widely applied to bovine embryo culture; studies by Thompson, J.G., et al (Thompson, J.G., et al, effects of inhibitors and undercouplers of oxidative phosphorylation reactions and suspensions of bovine embryos cultured in vitro, J. Reprod. Fertil, 2000.118(1): p.47-55) and by J.Feugang et al (Feugang, J.M., O.Camago-Rodriguez, and E.Memil, Culture systems for bovine embryos Science 2009.121(2-3): p.141-149) show that SOF Culture fluid 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, and unsaturationAnd oxidation of fatty acids, amino acids in proteins may even lead to the inactivation of certain enzymes. In general, ROS exist in four forms, where H is2O2The strong oxidation is the most important factor causing oxidative damage.
Numerous studies have shown that Glutathione (GSH), an antioxidant in a non-protein form, is capable of scavenging a variety of free radicals: superoxide anion free radical, hydroxyl free radical, hydrogen peroxide, hypochlorous acid and lipoxy radical, and can maintain redox balance inside and outside cells. The intracellular and extracellular environment GSH and ROS levels are two important factors influencing the development process of fertilized eggs. As early as 2000, de Matos et al (de Matos, D.G. and C.C. Furnus, The animal of high yield (GSH) level after vitamin in vision formation on embryo development effect of beta-mercaptethanol, cysteine and cysteine, Theriogenology, 2000.53(3): p.761-71) have increased blastocyst rate by adding beta-mercaptoethanol, cysteine and cystine during in vitro embryo culture.
Although the in vitro fertilization technique can be successfully applied to many mammals, the high production cost and the low efficiency of the in vitro fertilized embryo are caused by the low blastula rate of the in vitro fertilization, so that the wide application of the technique in the rapid propagation practice of the cattle is limited. Therefore, how to reduce the cost and improve the production efficiency and quality of bovine IVF embryos becomes a problem to be solved urgently.
At present, in a cattle in-vitro fertilization technical system, CR1aa and SOF liquid are mainly used as embryo in-vitro culture solutions, and improvement is carried out on the basis, the blastocyst development rates are improved to different degrees, and the average blastocyst development rate is 30-40%. For blastocyst quality, it can be evaluated by the total number of blastocyst cells, the ratio of ICM cell number/total cell number, and the apoptosis rate. The total number of blastula cells is different according to the stage of the blastula, and the total number of blastula cells obtained by S. Iwasaki et al (Iwasaki, S. and T.Nakahara, Cell number and intention of chromosomal antibodies in bone blast cultures transformed in vitro followed by culture in vitro or in vivo in nucleic acids, theriogenesis, 1990.33(3): p.669-75) is 44 on average, and the ratio of ICM Cell number/total number of blastula cells is about 15.8%; andrew J. Watson et al (Watson, A.J., et al, Impact of bone marrow regulation media on embryo transcripts, blast severity, 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, NaHCO3 26.2mM、MgCl2·6H2O 0.8mM、KH2PO31.19mM, sodium pyruvate 0.4mM, glucose 1.5mM, calcium hemi-lactobionate 5mM, 10v/v% fetal bovine serum, L-glutamine 1mM, 2v/v% essential amino acids, 1v/v% non-essential amino acids and glutathione 3mM, 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.
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 ovary and placing in double-resistance normal salineIn a warm barrel, transporting the mixture back to a laboratory within 3 hours at the temperature of 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 in vitro collection or in vivo collection in oocyte maturation culture solution for 1 time, transferring to new maturation culture solution, and culturing for 22-24 hr at 38.8 deg.C and 5.5-6.5% CO2Saturated 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 drop of fertilization culture liquid containing oocyte, placing the culture disc into an incubator, and incubating sperm and ovum for 16-20h under the culture conditions of 38.8 deg.C and 5.5-6.5% CO2Saturated humidity; (3) after the embryo in-vitro culture and preservation and in-vitro fertilization are finished, the granular cells around the embryo are removed by an ovum-peeling needle and are put into the embryo culture solution for culture, which is marked as the 1 st day of the embryo culture, the culture conditions are 38.8 ℃ and 6 percent O2、88% N2Saturation humidity, recording the cleavage rate on day 3; 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, and placing into solutionFreezing and storing in nitrogen.
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.
Prior application CN111254109A (chinese patent application No. 2020100820171) provides a transport medium containing glycine, alanine, arginine hydrochloride, aspartic acid, cystine dihydrochloride, glutamic acid, L-glutamine, histidine, threonine, tryptophan, tyrosine, valine, ascorbic acid, biotin, choline chloride, calcium pantothenate, folic acid, menadione, etc. to overcome the above-mentioned transport difficulty problem, and the method for in vitro fertilization and embryo culture of bovine oocytes using the transport medium comprises the following steps: collecting oocytes, maturing in vitro, fertilizing in vitro, culturing embryo in vitro and preserving. The method and the transport culture solution of the invention achieve excellent technical effects.
Further, the prior application CN112322579A (chinese patent application No. 2021100106576) relates to a culture solution for cattle in vitro fertilization and a method of improving cattle in vitro fertilization. The method for cattle in vitro fertilization comprises the following steps: washing the mature cumulus-oocyte complex in a fertilization culture solution, transferring the mature cumulus-oocyte complex into the fertilization culture solution, and putting the mature cumulus-oocyte complex into an incubator for later use; taking the frozen semen tubule from liquid nitrogen, thawing in water bath at 37 deg.C to inject semen into a centrifuge tube containing semen preparation culture solution, centrifuging, and discarding supernatant; adding the semen preparation culture solution into the centrifugal tube, re-suspending the sperm precipitate, and taking a proper sperm suspension for sperm counting; adding the calculated volume of sperm suspension into the fertilization culture liquid drop containing the oocyte, and putting the culture disc into an incubator to incubate the sperm and the ovum so as to finish the in vitro fertilization operation. After the in vitro fertilization operation is finished, embryo in-vitro culture is carried out by carrying out in vitro development on fertilized eggs, and the obtained usable embryo can be frozen and preserved by liquid nitrogen. Temporary cryopreservation is beneficial for increasing animal reproductive efficiency.
The above-mentioned CN108728404A, CN111254109A and CN112322579A are incorporated herein by reference in their entirety.
However, in the case of embryos obtained after in vitro development and culture of fertilized eggs, the survival rate of the embryos may be reduced after undergoing poor freezing and thawing processes. Therefore, there is a need to develop a method for improving the survival rate of the embryo obtained after the in vitro development and culture of bovine fertilized egg after freezing and thawing.
Disclosure of Invention
The invention aims to provide a method for improving the efficiency of in vitro fertilization of frozen bovine semen in the process of in vitro fertilization with oocytes, in particular to a method for improving the survival rate of embryos obtained after in vitro development and culture of bovine fertilized eggs after freezing and thawing. More particularly, the present invention provides a freezing solution and a thawing solution after freezing, which are specially used for the embryo obtained after the in vitro development and culture of the bovine fertilized egg, so as to improve the survival rate of the embryo after freezing and thawing. The present inventors have surprisingly found that the method of the present invention and the related working solutions exhibit excellent technical effects, and thus the present invention has been completed.
To this end, the invention provides in a first aspect a method for culturing and cryopreserving bovine in vitro fertilized embryos, which comprises the following steps:
(1) collection and in vitro maturation of oocytes
a) Collecting in vitro: taking cow ovaries obtained from slaughterhouses, extracting follicle with 2-8mm on the surface, collecting precipitate, picking out oocyte COCs (i.e. cumulus-oocyte complex) at least containing 3 layers of cumulus cell packages under a stereomicroscope, putting the oocyte COCs into transport culture solution containing HEPES, transporting to a laboratory at 38.8 ℃ without carbon dioxide supply within 24 h;
b) washing COCs obtained by in vitro collection or in vivo collection in oocyte maturation culture solution for 1 time, transferring to new maturation culture solution, and culturing for 22-24 hr at 38.8 deg.C and 5.5-6.5% CO2Saturated humidity;
(2) in vitro fertilization
Washing mature COCs in a fertilization culture solution for 1 time, transferring the COCs into the fertilization culture solution, and putting the COCs into an incubator for later use;
taking a frozen tubule from liquid nitrogen, and unfreezing in a water bath at 37 ℃; aseptically cutting two ends of the thin tube, injecting semen into 15mL centrifuge tube containing semen preparation culture solution, centrifuging at 328 Xg for 2 times (5 min each time), and discarding supernatant after centrifuging; adding 300 mu L of semen preparation culture solution into the centrifuge tube, resuspending the sperm precipitation, and taking appropriate sperm suspension for sperm counting;
adding the calculated volume of sperm suspension into the drop of fertilization culture liquid containing oocyte, placing the culture disc into an incubator, and incubating sperm and ovum for 16-20h under the culture conditions of 38.8 deg.C and 5.5-6.5% CO2Saturated humidity;
(3) embryo in vitro culture, preservation and thawing
After the in vitro fertilization operation is finished, the granulosa cells around the embryo are removed by an ovum-removing needle and are put into the embryo culture solution for culture, which is marked as the 1 st day of the embryo culture, the culture conditions are 38.8 ℃ and 6 percent O2、88% N2Saturation humidity, recording the cleavage rate on day 3; 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, placing into programmed cooling instrument, cooling to-35 deg.C at 0.5 deg.C/min, taking out the tubule rapidly, and placing into liquid nitrogen for freezing;
when the embryo needing to be frozen and stored is used, taking out the thin tube from the liquid nitrogen, staying in the air for 5s, putting into a water bath at 37 ℃, cutting off the thin tube until the ice crystals are completely melted, quickly transferring the embryo into the thawing solution, keeping at room temperature for 5min, then putting the embryo into the small drop of the embryo culture solution for culture, and finishing the thawing process.
The method according to the first aspect of the present invention, wherein in the step (3), the composition of the freezing fluid is: PBS +20% FBS +1.8mol/L ethylene glycol +0.25mol/L sucrose +0.2% dextran 40+5mmol/L acetylcysteine.
The method according to the first aspect of the present invention, wherein in the step (3), the composition of the thawing solution is: PBS +20% FBS +0.25mol/L sucrose +0.2% dextran 40+5mmol/L acetylcysteine. It has been surprisingly found that the survival rate of embryos subjected to freezing and thawing can be significantly improved by adding appropriate amounts of dextran 40 and acetylcysteine to both the freezing and thawing solutions.
In the present invention, when the compositions of the refrigerating fluid or the thawing fluid are expressed, they are prepared by adding FBS and other materials to PBS as a base fluid to reach corresponding concentrations. For example, when the composition of the refrigerating fluid is: PBS +20% FBS +1.8mol/L ethylene glycol +0.25mol/L sucrose +0.2% dextran 40+5mmol/L acetylcysteine, it means to PBS as a base solution to which adding FBS to the concentration of 20%, adding ethylene glycol to 1.8mol/L, adding sucrose to the concentration of 0.25mol/L, adding dextran 40 to the concentration of 0.2%, adding acetylcysteine to the concentration of 5mmol/L preparation. The present invention relates to other fluids, such as culture fluids, whose composition is formulated in a similar manner to that described above for the freezing fluid.
In the context of the invention, the PBS, i.e. the phosphate buffer, has a pH value of 7.0 and is formulated as follows, unless otherwise specified: adding water into dipotassium hydrogen phosphate 9.39g and potassium dihydrogen phosphate 3.5g to make 1000ml, filtering, and sterilizing at 115 deg.C for 30 min.
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.
The method according to the first aspect of the present invention, wherein in the step (1) (a), the transport culture solution 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 dihydrate 58.0mg/L, L-valine 25.0mg/L, ascorbic acid 0.05mg/L, biotin 0.01mg/L, choline chloride 0.5mg/L, D-calcium pantothenate 0.01mg/L, folic acid 0.01mg/L, 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 (calciferol 0.1 mg/L), alpha-tocopherol phosphate sodium salt 0.01mg/L, inositol 0.05mg/L, anhydrous calcium chloride 200.0mg/L, 0.7mg/L of ferric nitrate nonahydrate, 97.67mg/L of anhydrous magnesium sulfate, 400.0mg/L of potassium chloride, 6800.0mg/L of sodium chloride, 140.0mg/L of sodium dihydrogen phosphate monohydrate, 10.0mg/L of adenine sulfate, 0.2mg/L of adenosine 5' -phosphate, 1.0mg/L of adenosine triphosphate, 0.2mg/L of cholesterol, 1000.0mg/L of glucose, 0.5mg/L of deoxyribose, 0.05mg/L of reduced glutathione, 0.3mg/L of guanine hydrochloride, 0.354mg/L of sodium hypoxanthine, 20.0mg/L of phenol red, 0.5mg/L of ribose, 50.0mg/L of sodium acetate, 0.3mg/L of thymine, 20.0mg/L of 80, 0.3mg/L of uracil, 0.3mg/L of sodium xanthine, 0.3mg/L of FSH, 0.01IU/mL of LH, E2 of 1. mu.g/mL, EGF of 50ng/mL, IGF of 100ng/mL, gentamicin of 10%, sodium pyruvate of 55. mu.g/mL, cysteine of 1.2mM/L, BSA of 3mg/mL, HEPES of 10 mM/L.
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.
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, 10umoL/L alpha-lipoic acid, 25. mu.g/ml folic acid, 3mg/ml sodium glutamate.
The method according to the first aspect of the present invention, wherein in step (2), the semen preparation culture solution 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), 10mM caffeine, 100U/ml penicillin, 100. mu.g/ml streptomycin, 10. mu.L/L alpha. -lipoic acid, 25. mu.g/ml folic acid, 3mg/ml sodium glutamate.
The method according to the first aspect of the present invention, wherein in step (3), the embryo culture fluid comprises: 109.5mM sodium chloride, 3.1mM potassium chloride, 26.2mM sodium bicarbonate, 0.8mM magnesium chloride hexahydrate, 1.19mM monopotassium phosphate, 0.4mM sodium pyruvate, 1.5mM glucose, 5mM calcium half-lactobionate, 2.5v/v% Fetal Bovine Serum (FBS), 1mM L-glutamine, 2v/v% essential amino acids, 1v/v% nonessential amino acids, 3mM glutathione, 0.04w/v% sodium citrate, 0.02w/v% maltose in water; the essential amino acid is added by the following amino acids according to the weight proportion: 6.32g of L-arginine hydrochloride, 1.564g of L-cystine dihydrochloride, 2.1g of L-histidine hydrochloride monohydrate, 2.625g of L-isoleucine, 2.62g of L-leucine, 3.625g of L-lysine hydrochloride, 0.755g of L-methionine, 1.65g of L-phenylalanine, 2.38g of L-threonine, 0.51g of L-tryptophan, 1.8g of L-tyrosine and 2.34g of L-valine, wherein the optional amino acids are added in the following weight ratio: 0.89g of L-alanine, 1.5g of L-asparagine monohydrate, 1.33g of L-aspartic acid, 1.47g of L-glutamic acid, 0.75g of glycine, 1.15g of L-proline and 1.05g of L-serine.
The method according to the first aspect of the present invention, wherein in step (1), the BY basal medium is an aqueous solution comprising: 180-220 mg/L calcium chloride, 0.70-0.75 mg/L ferric nitrate nonahydrate, 380-420 mg/L potassium chloride, 90-100 mg/L magnesium sulfate, 6500-7000 mg/L sodium chloride, 130-150 mg/L monosodium phosphate monohydrate, 2000-2500 mg/L sodium bicarbonate, 40-60 mg/L, L-alanine 20-30 mg/L, L-sodium acetate, 60-80 mg/L, L-aspartic acid, 25-35 mg/L, L-cysteine hydrochloride monohydrate, 0.10-0.12 mg/L, L-cystine dihydrochloride, 20-30 mg/L, L-glutamic acid, 40-60 mg/L, L-glycine-histidine hydrochloride monohydrate, 20-25 mg/L, L-hydroxyproline 8-12 mg/L, 15-25 mg/L, L-leucine 50-70 mg/L, L-lysine hydrochloride 60-80 mg/L, L-methionine 10-20 mg/L, L-phenylalanine 20-30 mg/L, L-proline 30-50 mg/L, L-serine 20-30 mg/L, L-threonine 25-35 mg/L, L-tryptophan 8-12 mg/L, L-tyrosine disodium dihydrate 55-60 mg/L, L-valine 20-30 mg/L, ascorbic acid 0.04-0.06 mg/L, alpha-D-tocopherol phosphate 0.008-0.012 mg/L, biotin 0.008-0.012 mg/L, calcitol 0.08-0.12 mg/L, D-calcium pantothenate 0.008-0.012 mg/L, 0.4-0.6 mg/L choline chloride, 0.008-0.012 mg/L folic acid, 0.04-0.06 mg/L inositol, 0.015-0.025 mg/L menadione sodium bisulfite trihydrate, 0.02-0.03 mg/L nicotinic acid, 0.02-0.03 mg/L nicotinamide, 0.04-0.06 mg/L L, p-aminobenzoic acid, 0.04-0.06 mg/L pyridoxine hydrochloride, 0.008-0.012 mg/L riboflavin, 0.008-0.012 mg/L thiamine hydrochloride, 0.1-0.2 mg/L vitamin A acetate, 8-12 mg/L adenine sulfate, 0.15-0.25 mg/L adenine, 0.8-1.2 mg/L disodium adenosine triphosphate, 0.15-0.25 mg/L cholesterol, 2-deoxy-D-0.4-0.6 mg/L glucose, 0.04-0.84 mg/L glutathione, 0.06mg/L glucose, 0.25-0.35 mg/L guanine hydrochloride, 0.3-0.4 mg/L hypoxanthine sodium, 0.4-0.6 mg/L ribose, 0.25-0.35 mg/L thymosin, 4-6 mg/L tween 80, 0.25-0.35 mg/L uracil, 0.3-0.4 mg/L xanthine sodium and 8-12 mg/L phenol red.
The method according to the first aspect of the present invention, wherein in step (1), the BY basal medium is an aqueous solution comprising: 200mg/L calcium chloride, 0.72mg/L ferric nitrate nonahydrate, 400mg/L potassium chloride, 97.7mg/L magnesium sulfate, 6800mg/L sodium chloride, 140mg/L monobasic sodium phosphate monohydrate, 2200mg/L sodium bicarbonate, 50mg/L, L-sodium acetate, 25mg/L, L-arginine hydrochloride, 70mg/L, L-aspartic acid, 30mg/L, L-cysteine hydrochloride monohydrate, 0.11mg/L, L-cystine dihydrochloride, 26mg/L, L-glutamic acid, 75mg/L glycine, 50mg/L, L-histidine hydrochloride monohydrate, 21.88mg/L, L-hydroxyproline, 10mg/L, L-isoleucine, 20mg/L, L-leucine, 60mg/L leucine, L-lysine hydrochloride 70mg/L, L-methionine 15mg/L, L-phenylalanine 25mg/L, L-proline 40mg/L, L-serine 25mg/L, L-threonine 30mg/L, L-tryptophan 10mg/L, L-tyrosine disodium dihydrate 57.66mg/L, L-valine 25mg/L, ascorbic acid 0.05mg/L, alpha-D-tocopherol phosphate 0.01mg/L, biotin 0.01mg/L, calciferol 0.1mg/L, D-calcium pantothenate 0.01mg/L, choline chloride 0.5mg/L, folic acid 0.01mg/L, inositol 0.05mg/L, menadione trihydrate sodium bisulfite 0.019mg/L, nicotinic acid 0.025mg/L, Nicotinamide 0.025mg/L, p-aminobenzoic acid 0.05mg/L, pyridoxine hydrochloride 0.05mg/L, riboflavin 0.01mg/L, thiamine hydrochloride 0.01mg/L, vitamin A acetate 0.14mg/L, adenine 10mg/L, adenine 0.2mg/L, disodium adenosine triphosphate 1mg/L, cholesterol 0.2mg/L, 2-deoxy-D-ribose 0.5mg/L, D-glucose 1000mg/L, glutathione 0.05mg/L, guanine hydrochloride 0.3mg/L, sodium hypoxanthine 0.354mg/L, ribose 0.5mg/L, thymosin 0.3mg/L, Tween 80 5mg/L, uracil 0.3mg/L, sodium xanthine 0.34mg/L, phenol red 10 mg/L.
The method according to the first aspect of the present invention, wherein in step (1), the BY basal medium further comprises sodium selenite at a concentration of 0.2-0.3 mg/L, such as 0.25 mg/L; and/or the BY basic culture solution also comprises copper sulfate, and the concentration of the copper sulfate is 0.05-0.1 mg/L (calculated BY anhydrous substance), such as 0.075 mg/L.
In the present invention, for example, in step (3), the preservation solution, equilibration solution and the like for embryos are well known in the art and can be readily obtained from commercial sources, for example, the embryo preservation solution is Whittingham modified Duchen phosphate buffer containing 20% bovine serum and 500IU/ml penicillin G potassium, and the equilibration solution is ES solution from Kitazato Biopharma, Japan). In addition, the refrigerating fluid provided by the invention is obviously superior to some commercial refrigerating fluids in cost, for example, the comprehensive cost of the refrigerating fluid provided by the invention is about the comprehensive cost of the refrigerating fluid which can be FreezekitTMThe cost of clean refrigerating fluid (sold by vitrolite corporation, sweden) is about 1/10.
Further, the second aspect of the present invention provides a transport culture solution for oocyte, which is described in the first aspect of the present invention.
Further, the third aspect of the present invention provides a method for bovine in vitro fertilization, comprising the steps of:
washing mature cumulus-oocyte complexes (COCs) in a fertilization culture solution for 1 time, transferring the mature cumulus-oocyte complexes into the fertilization culture solution, and putting the mature cumulus-oocyte complexes (COCs) into an incubator for later use;
taking a frozen tubule from liquid nitrogen, and unfreezing in a water bath at 37 ℃; aseptically cutting two ends of the thin tube, injecting semen into 15mL centrifuge tube containing semen preparation culture solution, centrifuging at 328 Xg for 2 times (5 min each time), and discarding supernatant after centrifuging; adding 300 mu L of semen preparation culture solution into the centrifuge tube, resuspending the sperm precipitation, and taking appropriate sperm suspension for sperm counting;
adding the calculated volume of sperm suspension into the drop of fertilization culture liquid containing oocyte, placing the culture disc into an incubator, and incubating sperm and ovum for 16-20h under the culture conditions of 38.8 deg.C and 5.5-6.5% CO2And saturating the humidity to finish the in vitro fertilization operation for the in vitro culture of the embryo.
The method according to the third aspect of the invention, wherein 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 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, 10umoL/L alpha-lipoic acid, 25. mu.g/ml folic acid, 3mg/ml sodium glutamate.
The method according to the third aspect of the invention, wherein the semen preparation 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 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, 10umoL/L alpha-lipoic acid, 25. mu.g/ml folic acid, 3mg/ml sodium glutamate.
The method according to the third aspect of the present invention, wherein the cumulus-oocyte complex is collected and matured in vitro by:
a) collecting in vitro: taking cow ovaries obtained from slaughterhouses, extracting follicle with 2-8mm on the surface, collecting precipitate, picking out oocyte COCs (i.e. cumulus-oocyte complex) at least containing 3 layers of cumulus cell packages under a stereomicroscope, putting the oocyte COCs into transport culture solution containing HEPES, transporting to a laboratory at 38.8 ℃ without carbon dioxide supply within 24 h;
b) washing COCs obtained by in vitro collection or in vivo collection in oocyte maturation culture solution for 1 time, transferring to new maturation culture solution, and culturing for 22-24 hr at 38.8 deg.C and 5.5-6.5% CO2And saturation humidity to obtain mature cumulus-oocyte complexes.
A method according to the third aspect of the present invention having the features of the first or second aspect of the present invention.
Further, the fourth aspect of the present invention provides a fertilization culture solution for in vitro fertilization of cattle, which 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, 10. mu.l/L alpha-lipoic acid, 25. mu.g/ml folic acid, 3mg/ml sodium glutamate.
Further, the fifth aspect of the present invention provides a semen preparation culture solution for fertilization in cattle in vitro, which 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), 10mM caffeine, 100U/ml penicillin, 100. mu.g/ml streptomycin, 10umoL/L alpha-lipoic acid, 25. mu.g/ml folic acid, 3mg/ml sodium glutamate.
Further, the sixth aspect of the invention provides a freezing solution for cryopreservation and thawing of bovine in vitro fertilization blastocysts, which comprises the following components: PBS +20% FBS +1.8mol/L ethylene glycol +0.25mol/L sucrose +0.2% dextran 40+5mmol/L acetylcysteine.
Further, the seventh aspect of the present invention provides a thawing solution for cryopreservation and thawing of bovine in vitro fertilization blastocysts, which comprises the following components: PBS +20% FBS +0.25mol/L sucrose +0.2% dextran 40+5mmol/L acetylcysteine.
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. In the present invention, unless otherwise specified, all references to glutamic acid or sodium glutamate refer to L-glutamic acid or sodium L-glutamate.
Example 1: culture method of cattle in vitro fertilization embryo (in vitro collection)
This example in step (1), the collected oocytes were placed in a transport medium containing HEPES at 38.8 ℃ and transported back to the laboratory without carbon dioxide supply for 24 h.
Reagent
In the specific test of the present invention, the reagents used are described in detail below, unless otherwise specified:
the transport medium used in this example, which contained HEPES, contained: 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-cerealAminamide 100.0mg/L, L-histidine hydrochloride monohydrate 21.88mg/L, L-hydroxyproline 10.0mg/L, L-isoleucine 40.0mg/L, L-leucine 60.0mg/L, L-lysine hydrochloride 70.0mg/L, L-methionine 15.0mg/L, L-phenylalanine 25.0mg/L, L-proline 40.0mg/L, L-serine 25.0mg/L, L-threonine 30.0mg/L, L-tryptophan 10.0mg/L, L-tyrosine disodium salt dihydrate 58.0mg/L, L-valine 25.0mg/L, ascorbic acid 0.05mg/L, biotin 0.01mg/L, choline chloride 0.5mg/L, D-calcium pantothenate 0.01mg/L, Folic acid 0.01mg/L, menadione 0.01mg/L, nicotinamide 0.025mg/L, nicotinic acid 0.025mg/L, para aminobenzoic acid 0.05mg/L, pyridoxal hydrochloride 0.025mg/L, pyridoxine hydrochloride 0.025mg/L, riboflavin 0.01mg/L, thiamine hydrochloride 0.01mg/L, vitamin A acetate 0.1mg/L, vitamin D2I.e., calciferol 0.1mg/L, alpha-tocopherol phosphate sodium salt 0.01mg/L, inositol 0.05mg/L, anhydrous calcium chloride 200.0mg/L, ferric nitrate nonahydrate 0.7mg/L, anhydrous magnesium sulfate 97.67mg/L, potassium chloride 400.0mg/L, sodium chloride 6800.0mg/L, sodium dihydrogen phosphate monohydrate 140.0mg/L, adenine sulfate 10.0mg/L, 5' -adenosine phosphate 0.2mg/L, adenosine triphosphate 1.0mg/L, cholesterol 0.2mg/L, glucose 1000.0mg/L, deoxyribose 0.5mg/L, glutathione reduced form 0.05mg/L, guanine hydrochloride 0.3mg/L, sodium hypoxanthine 0.354mg/L, phenol red 20.0mg/L, ribose 0.5mg/L, sodium acetate 50.0mg/L, thymine 0.3mg/L, Tween 80 is 20.0mg/L, uracil 0.3mg/L, xanthine sodium 0.3mg/L, and 0.01IU/mL FSH, 0.01IU/mL LH, 1 μ g/mL E2, 50ng/mL EGF, 100ng/mL IGF, 10% gentamicin, 55 μ g/mL sodium pyruvate, 1.2mM/L cysteine, 3mg/mL BSA, 10mM/L HEPES, and taurine 40mg/L, zinc gluconate 2 mg/L.
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, 10v/v% Fetal Bovine Serum (FBS), 1mM L-glutamine, 2v/v% essential amino acids, 1v/v% nonessential amino acids, 3mM glutathione, sodium citrate 0.04w/v%, maltose 0.02w/v% aqueous solution; the essential amino acid is added by the following amino acids according to the weight proportion: 6.32g of L-arginine hydrochloride, 1.564g of L-cystine dihydrochloride, 2.1g of L-histidine hydrochloride monohydrate, 2.625g of L-isoleucine, 2.62g of L-leucine, 3.625g of L-lysine hydrochloride, 0.755g of L-methionine, 1.65g of L-phenylalanine, 2.38g of L-threonine, 0.51g of L-tryptophan, 1.8g of L-tyrosine and 2.34g of L-valine, wherein the optional amino acids are added in the following weight ratio: 0.89g of L-alanine, 1.5g of L-asparagine monohydrate, 1.33g of L-aspartic acid, 1.47g of L-glutamic acid, 0.75g of glycine, 1.15g of L-proline and 1.05g of L-serine.
The BY basal medium is an aqueous solution containing the following components: 200mg/L calcium chloride, 0.72mg/L ferric nitrate nonahydrate, 400mg/L potassium chloride, 97.7mg/L magnesium sulfate, 6800mg/L sodium chloride, 140mg/L monobasic sodium phosphate monohydrate, 2200mg/L sodium bicarbonate, 50mg/L, L-sodium acetate, 25mg/L, L-arginine hydrochloride, 70mg/L, L-aspartic acid, 30mg/L, L-cysteine hydrochloride monohydrate, 0.11mg/L, L-cystine dihydrochloride, 26mg/L, L-glutamic acid, 75mg/L glycine, 50mg/L, L-histidine hydrochloride monohydrate, 21.88mg/L, L-hydroxyproline, 10mg/L, L-isoleucine, 20mg/L, L-leucine, 60mg/L leucine, L-lysine hydrochloride 70mg/L, L-methionine 15mg/L, L-phenylalanine 25mg/L, L-proline 40mg/L, L-serine 25mg/L, L-threonine 30mg/L, L-tryptophan 10mg/L, L-tyrosine disodium dihydrate 57.66mg/L, L-valine 25mg/L, ascorbic acid 0.05mg/L, alpha-D-tocopherol phosphate 0.01mg/L, biotin 0.01mg/L, calciferol 0.1mg/L, D-calcium pantothenate 0.01mg/L, choline chloride 0.5mg/L, folic acid 0.01mg/L, inositol 0.05mg/L, menadione trihydrate sodium bisulfite 0.019mg/L, nicotinic acid 0.025mg/L, Nicotinamide 0.025mg/L, p-aminobenzoic acid 0.05mg/L, pyridoxine hydrochloride 0.05mg/L, riboflavin 0.01mg/L, thiamine hydrochloride 0.01mg/L, vitamin A acetate 0.14mg/L, adenine 10mg/L, adenine 0.2mg/L, disodium adenosine triphosphate 1mg/L, cholesterol 0.2mg/L, 2-deoxy-D-ribose 0.5mg/L, 1000mg/L of D-glucose, 0.05mg/L of glutathione, 0.3mg/L of guanine hydrochloride, 0.354mg/L of hypoxanthine sodium, 0.5mg/L of ribose, 0.3mg/L of thymosin, 5mg/L of Tween 80, 0.3mg/L of uracil, 0.34mg/L of xanthine sodium, 10mg/L of phenol red, 0.25mg/L of sodium selenite and 0.075mg/L of anhydrous copper sulfate.
The refrigerating fluid comprises the following components: PBS +20% FBS +1.8mol/L ethylene glycol +0.25mol/L sucrose +0.2% dextran 40+5mmol/L acetylcysteine.
The composition of the thawing solution is as follows: PBS +20% FBS +0.25mol/L sucrose +0.2% dextran 40+5mmol/L acetylcysteine.
PBS, i.e. phosphate buffer, has a pH of 7.0 and is formulated as follows: adding water into dipotassium hydrogen phosphate 9.39g and potassium dihydrogen phosphate 3.5g to make 1000ml, filtering, and sterilizing at 115 deg.C for 30 min.
II, cattle in-vitro fertilization and embryo culture:
step (1), collection and in vitro maturation of oocytes
(a) Taking cow ovaries obtained from slaughterhouses, extracting follicles with the surface of 2-8mm, collecting precipitates, picking up oocyte COCs (namely, cumulus-oocyte complex) at least containing 3 layers of cumulus cell packages under a stereomicroscope, putting the oocycs into a transport culture solution containing HEPES, transporting the oocycs to a laboratory at 38.8 ℃ without carbon dioxide supply within 24 hours (in the example, transporting the oocycs to the laboratory for the next step treatment within 15 hours);
(b) washing the obtained COCs in oocyte maturation culture solution for 1 time, transferring to new maturation culture solution, and culturing for 22-24h (24 hr for practical operation) at 38.8 deg.C and 5.5-6.5% CO2Saturated humidity;
step (2), in vitro fertilization
Washing mature COCs in a fertilization culture solution for 1 time, transferring the COCs into the fertilization culture solution, and putting the COCs into an incubator for later use;
taking a frozen tubule from liquid nitrogen, and unfreezing in a water bath at 37 ℃; aseptically cutting two ends of the thin tube, injecting semen into 15mL centrifuge tube containing semen preparation culture solution, centrifuging at 328 Xg for 2 times (5 min each time), and discarding supernatant after centrifuging; adding 300 mu L of semen preparation culture solution into the centrifuge tube, resuspending the sperm precipitation, and taking appropriate sperm suspension for sperm counting;
adding the calculated volume of sperm suspension into the drop of fertilization culture liquid containing oocyte, placing the culture disc into an incubator, and incubating the sperm and the ovum for 16-20h (actual operation for 18 h) under the culture conditions of 38.8 ℃ and 5.5-6.5% CO2Saturated humidity;
step (3), embryo in vitro culture, preservation and thawing
After the in vitro fertilization operation is finished, the granulosa cells around the embryo are removed by an ovum-removing needle and are put into the embryo culture solution for culture, which is marked as the 1 st day of the embryo culture, the culture conditions are 38.8 ℃ and 6 percent O2、88% N2Saturation humidity, recording the cleavage rate on day 3; 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, placing into programmed cooling instrument, cooling to-35 deg.C at 0.5 deg.C/min, taking out the tubule rapidly, and placing into liquid nitrogen for freezing;
when the embryo needing to be frozen and stored is used, taking out the thin tube from the liquid nitrogen, staying in the air for 5s, putting into a water bath at 37 ℃, cutting off the thin tube until the ice crystals are completely melted, quickly transferring the embryo into the thawing solution, keeping at room temperature for 5min, then putting the embryo into the small drop of the embryo culture solution for culture, and finishing the thawing process.
The embryos are placed into the embryo culture solution droplets for culture, and the survival rate after thawing is counted for 36 hours.
At the time of the above statistics of survival rate, the survival criterion was the re-emergence of blastocoel cavities. In the present experiments, the survival rate of the cryopreservation test was examined, and all of them were cryopreserved in liquid nitrogen for 30 days, unless otherwise stated.
The blastocysts used in the following procedures of this example were not freeze-thawed.
Method for differential staining of embryo
1. Blastocysts from day 7 of in vitro culture were selected and fixed with 2% paraformaldehyde for 20 min.
2. The cells were washed twice with phosphate buffered saline (PBS-BSA) containing 0.5% BSA, and placed in a permeabilizing solution (50. mu.l Triton, 5. mu.l Tween 80 and 9.945ml PBS) at room temperature for 30 min.
3. CDX2 protein was able to bind to primary antibodies by treatment with 2M hydrochloric acid at room temperature for 20min, followed by treatment with 100mM Tris-HCl at room temperature for 10 min.
4. Washed three times with PBS-BSA, the blastocysts were placed in blocking solution (1ml goat serum, 5. mu.l Tween 80 and 8.995ml PBS), blocked for 1h at room temperature, and then blocked overnight in a refrigerator at 4 ℃.
5. Discarding the blocking solution, diluting CDX2 primary antibody with the blocking solution at a ratio of 1:200, incubating at room temperature for 2h, discarding the primary antibody dilution, and washing with PBS-BSA for 5min for 3 times.
Caspase-3 primary antibody (available from Cell Signaling Technology) was diluted 1:200 with blocking solution, incubated at room temperature for 2h, the primary antibody dilution was discarded, and washed 3 times with PBS-BSA for 5min each.
7. CDX 2-specific secondary antibody (purchased from Sigma) was diluted 1:200 with blocking solution under exclusion of light and left at room temperature for 1h without light. The secondary antibody dilutions were discarded in the dark and washed 3 times with PBS for 5min each.
8. Caspase-3 specific secondary antibodies (purchased from Life Technologies) were diluted 1:200 with blocking solution under dark conditions and left at room temperature for 1h under dark conditions. The secondary antibody dilutions were discarded in the dark and washed 3 times with PBS for 5min each.
9. Cell nuclei were stained with 10. mu.g/mL Hochest 33342 dye, allowed to act at room temperature for 5min, observed under a fluorescent microscope and photographed.
10. The experiment is repeated three times, 10 blasts are randomly selected each time, and the blast quality is evaluated by calculating the apoptosis rate and the number of ICM cells/total number of cells.
The data statistical method comprises the following steps: experimental data were analyzed using the ANOVA program in statistical software SAS V8 and the Duncan's multiple-range test method judged significant differences between treatments, which were considered significant when p < 0.05.
In the present invention, cleavage rate is fertilization cleavage number/fertilization egg number. In the present invention, blastocyst rate is blastocyst number/cleavage embryo number.
Fourth, In Vitro Maturation (IVM) Effect of oocytes-maturation Rate
In step (1) of this example, 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 86.4%, the morula rate was 66.3%, the blastocyst rate was 51.6%, and the apoptosis rate was 5.3%; in addition, blastocyst hatchability reached 75.1% on day 9. The maturation rate of the oocyte in vitro maturation reaches 87.5 percent.
Example 1A: culture method of cattle in vitro fertilization embryo (in vitro collection)
With reference to the method of example 1, three types of cattle, a holstein cattle (dairy cattle breed), a simmental cattle (beef cattle breed), and a chinese buffalo (service breed), were tested, and the results were as follows: the cleavage rate is within the range of 85-90%, the morula rate is within the range of 62-67%, the blastocyst rate is within the range of 50-55%, the apoptosis rate is within the range of 4-6%, and the blastocyst hatching rate on day 9 is within the range of 72-77%; the maturation rates of oocytes in vitro maturation of three holstein cattle, Simmental cattle and Chinese buffalo cattle are 83.3%, 80.6% and 75.6% respectively.
In the experiments of example 1 and example 1A on four cattle, when embryo in vitro culture, preservation and thawing treatment were performed in step (3), freezing-thawing treatment was performed using a freezing solution and a thawing solution containing dextran 40 and acetylcysteine, and then the survival rate after thawing was counted for 36 hours, and as a result: the survival rate of the 36h four bovine blastocysts is within the range of 54-60%, for example, the survival rate of the blastocysts of Chinese cattle is 57.8%.
The test was performed on four cattle with reference to example 1 and example 1A, except that no dextran 40 was added to the freezing and thawing solutions, and the survival rate after thawing was counted for 36h in the same way, and the results were: the survival rate of the four bovine blastocysts is within 22-25% after 36h, for example, the survival rate of the blastocysts of Chinese cattle is 24.1%.
The test was performed on four cattle with reference to example 1 and example 1A, except that acetylcysteine was not added to the freezing and thawing solutions, and the survival rate after thawing was counted for 36 hours in the same manner as follows: the survival rate of the four bovine blastocysts is within the range of 24-30% in 36h, for example, the survival rate of the blastocysts of Chinese cattle is 27.7%.
The test was performed on four cattle with reference to example 1 and example 1A, except that neither dextran 40 nor acetylcysteine was added to the freezing and thawing solutions, and the survival rate after thawing was counted for 36h as follows: the survival rate of the four bovine blastocysts is 18-24% in 36h, for example, the survival rate of the blastocysts of Chinese cattle is 21.2%.
Example 1B: culture method of cattle in vitro fertilization embryo (in vitro collection)
Referring to the method of example 1, experiments were conducted on four types of cattle, i.e., cattle of Chinese yellow cattle (south-yang cattle, working breed), Holstein cattle (dairy cattle breed), Simmental cattle (beef breed), and buffalo cattle (working breed), but the formulas of the fertilization culture solution and the semen preparation culture solution were modified, respectively, and the rest of the test solutions and the operations were unchanged. The formula of the fertilization culture solution is changed into: 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 μ g/ml heparin, 4mg/ml Bovine Serum Albumin (BSA), 100U/ml penicillin, 100 μ g/ml streptomycin, 10umoL/L alpha-lipoic acid, 25 μ g/ml folic acid, 3mg/ml sodium glutamate; the formula of the semen preparation culture solution is changed into that: 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, 10umoL/L alpha-lipoic acid, 25. mu.g/ml folic acid, 3mg/ml sodium glutamate. As a result: the cleavage rate of each of the four cattle is 88-91%, the morula rate is 64-67%, the blastocyst rate is 77-82%, the apoptosis rate is 3-5%, and the blastocyst hatching rate on day 9 is 74-78%, for example, the cleavage rate of Chinese cattle is 89.7%, the morula rate is 66.1%, the blastocyst rate is 79.4%, the apoptosis rate is 3.8%, and the blastocyst hatching rate on day 9 is 75.3%; the maturation rates of oocytes of four Chinese cattle, namely cattle of Huang Bai Sten, cattle of Ximenta and cattle of Buffalo, in vitro maturation are 85.3%, 86.4%, 81.8% and 75.3%, respectively.
In the experiment of example 1B for four cattle, in the embryo in vitro culture, preservation and thawing treatment in step (3), freeze-thaw treatment was performed using a freezing solution and a thawing solution containing dextran 40 and acetylcysteine, and then the survival rate after thawing was counted at 36 hours. As a result: the survival rate of the 36h four bovine blastocysts is within the range of 56-61%, for example, the survival rate of the blastocysts of Chinese cattle is 59.4%.
The test was performed on four cattle with reference to example 1B, except that no dextran 40 was added to the freezing and thawing solution, and the survival rate after thawing was counted for 36h, as follows: the survival rate of the 36h four bovine blastocysts is within the range of 23-26%, for example, the survival rate of the blastocysts of Chinese cattle is 25.1%.
The test was performed on four cattle with reference to example 1B, except that acetylcysteine was not added to the freezing and thawing solutions, and the survival rate after thawing was counted for 36 hours by the same method, and the results were as follows: the survival rate of the four bovine blastocysts is within the range of 24-31% in 36h, for example, the survival rate of the blastocysts of Chinese cattle is 26.8%.
The test was performed on four cattle with reference to example 1B, except that neither dextran 40 nor acetylcysteine was added to the freezing and thawing solutions, and the survival rate after thawing was counted for 36h as follows: the survival rate of the four bovine blastocysts is within the range of 17-25% in 36h, for example, the survival rate of the blastocysts for Chinese cattle is 22.3%.
Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.

Claims (10)

1. The method for culturing and cryopreserving the bovine in vitro fertilized embryos comprises the following steps:
washing the mature cumulus-oocyte complex in a fertilization culture solution for 1 time, transferring the mature cumulus-oocyte complex into the fertilization culture solution, and putting the mature cumulus-oocyte complex into an incubator for later use;
taking a frozen tubule from liquid nitrogen, and unfreezing in a water bath at 37 ℃; aseptically cutting two ends of the thin tube, injecting semen into 15mL centrifuge tube containing semen preparation culture solution, centrifuging at 328 Xg for 2 times (5 min each time), and discarding supernatant after centrifuging; adding 300 mu L of semen preparation culture solution into the centrifuge tube, resuspending the sperm precipitation, and taking appropriate sperm suspension for sperm counting;
adding the calculated volume of sperm suspension into the drop of the fertilization culture liquid containing the oocyte, and putting the culture disc into an incubatorIncubating the sperm and egg for 16-20 hr at 38.8 deg.C and 5.5-6.5% CO2And saturation humidity, and finishing the in vitro fertilization operation;
after the in vitro fertilization operation is finished, the granulosa cells around the embryo are removed by an ovum-removing needle and are put into the embryo culture solution for culture, which is marked as the 1 st day of the embryo culture, the culture conditions are 38.8 ℃ and 6 percent O2、88% N2Saturation humidity, recording the cleavage rate on day 3; recording the blastocyst rate on the 7 th day, counting the blastocyst hatchability by the 9 th day, and performing quality identification;
washing the available embryo in preservation solution for 3 times, balancing in balancing solution for 10min, transferring into freezing solution, loading into embryo according to 5-stage liquid loading method, marking, placing into programmed cooling instrument, cooling to-35 deg.C at 0.5 deg.C/min, taking out the tubule rapidly, and placing into liquid nitrogen for freezing;
when the embryo needing to be frozen and stored is used, taking out the thin tube from the liquid nitrogen, staying in the air for 5s, putting into a water bath at 37 ℃, cutting off the thin tube until all ice crystals are melted, quickly transferring the embryo into a thawing solution, keeping at room temperature for 5min, then putting the embryo into a small drop of an embryo culture solution for culture, and finishing the thawing process;
wherein,
the fertilization 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, 100U/ml penicillin, 100. mu.g/ml streptomycin, 10umoL/L alpha-lipoic acid, 25. mu.g/ml folic acid, 3mg/ml sodium glutamate,
the semen preparation culture solution 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 monopotassium phosphate, 37.0mM sodium bicarbonate, 1.25mM sodium pyruvate, 10. mu.g/ml heparin, 4mg/ml bovine serum albumin, 10mM caffeine, 100U/ml penicillin, 100. mu.g/ml streptomycin, 10umoL/L alpha-lipoic acid, 25. mu.g/ml folic acid, 3mg/ml sodium glutamate,
the refrigerating fluid comprises the following components: PBS +20% FBS +1.8mol/L ethylene glycol +0.25mol/L sucrose +0.2% dextran 40+5mmol/L acetylcysteine,
the thawing solution comprises the following components: PBS +20% FBS +0.25mol/L sucrose +0.2% dextran 40+5mmol/L acetylcysteine.
2. The method according to claim 1, wherein the cumulus-oocyte complexes are collected and matured in vitro by:
a) collecting in vitro: taking cow ovaries obtained in a slaughterhouse, extracting follicle with the surface being 2-8mm, collecting precipitates, picking out oocyte COCs (oocytes complexes) containing at least 3 layers of cumulus cells under a stereomicroscope, namely cumulus-oocyte complexes, putting the oocytes COCs into a transport culture solution containing HEPES (high efficiency particulate matter), transporting the oocytes COCs to a laboratory at 38.8 ℃ without carbon dioxide supply within 24 h;
b) washing COCs obtained by in vitro collection in oocyte maturation culture solution for 1 time, transferring to new maturation culture solution, and culturing for 22-24 hr at 38.8 deg.C and 5.5-6.5% CO2And saturation humidity to obtain mature cumulus-oocyte complexes.
3. The method according to claim 2, wherein the maturation medium is BY basal medium supplemented with 100mL/L FBS, 10 μ g/mL FSH, 10 μ g/mL LH, 1 μ g/mL E2, 20ng/mL EGF.
4. The method of claim 2, wherein the 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-tyrosine disodium salt dihydrate 58.0mg/L, L-valine 25.0mg/L, ascorbic acid 0.05mg/L, biotin 0.01mg/L, choline chloride 0.5mg/L, D-calcium pantothenate 0.01mg/L, folic acid 0.01mg/L, 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 (calciferol 0.1 mg/L), alpha-tocopherol phosphate sodium salt 0.01mg/L, inositol 0.05mg/L, anhydrous calcium chloride 200.0mg/L, 0.7mg/L of ferric nitrate nonahydrate, 97.67mg/L of anhydrous magnesium sulfate, 400.0mg/L of potassium chloride, 6800.0mg/L of sodium chloride, 140.0mg/L of sodium dihydrogen phosphate monohydrate, 10.0mg/L of adenine sulfate, 0.2mg/L of adenosine 5' -phosphate, 1.0mg/L of adenosine triphosphate, 0.2mg/L of cholesterol, 1000.0mg/L of glucose, 0.5mg/L of deoxyribose, 0.05mg/L of reduced glutathione, 0.3mg/L of guanine hydrochloride, 0.354mg/L of sodium hypoxanthine, 20.0mg/L of phenol red, 0.5mg/L of ribose, 50.0mg/L of sodium acetate, 0.3mg/L of thymine, 20.0mg/L of 80, 0.3mg/L of uracil, 0.3mg/L of sodium xanthine, 0.3mg/L of FSH, 0.01IU/mL of LH, E2 of 1. mu.g/mL, EGF of 50ng/mL, IGF of 100ng/mL, gentamicin of 10%, sodium pyruvate of 55. mu.g/mL, cysteine of 1.2mM/L, BSA of 3mg/mL, HEPES of 10 mM/L.
5. The method according to claim 4, wherein the transport culture solution further comprises taurine and zinc gluconate, wherein the concentration of taurine is 30-50 mg/L, and the concentration of zinc gluconate is 1-3 mg/L.
6. The method according to claim 1, said embryo culture fluid comprising: 109.5mM sodium chloride, 3.1mM potassium chloride, 26.2mM sodium bicarbonate, 0.8mM magnesium chloride hexahydrate, 1.19mM monopotassium phosphate, 0.4mM sodium pyruvate, 1.5mM glucose, 5mM calcium half-lactobionate, 2.5v/v% fetal bovine serum, 1mM L-glutamine, 2v/v% essential amino acids, 1v/v% nonessential amino acids, 3mM glutathione, sodium citrate 0.04w/v%, maltose 0.02w/v% aqueous solution; the essential amino acid is added by the following amino acids according to the weight proportion: 6.32g of L-arginine hydrochloride, 1.564g of L-cystine dihydrochloride, 2.1g of L-histidine hydrochloride monohydrate, 2.625g of L-isoleucine, 2.62g of L-leucine, 3.625g of L-lysine hydrochloride, 0.755g of L-methionine, 1.65g of L-phenylalanine, 2.38g of L-threonine, 0.51g of L-tryptophan, 1.8g of L-tyrosine and 2.34g of L-valine, wherein the optional amino acids are added in the following weight ratio: 0.89g of L-alanine, 1.5g of L-asparagine monohydrate, 1.33g of L-aspartic acid, 1.47g of L-glutamic acid, 0.75g of glycine, 1.15g of L-proline and 1.05g of L-serine.
7. The method according to claim 3, wherein the BY basal medium is an aqueous solution comprising: 180-220 mg/L calcium chloride, 0.70-0.75 mg/L ferric nitrate nonahydrate, 380-420 mg/L potassium chloride, 90-100 mg/L magnesium sulfate, 6500-7000 mg/L sodium chloride, 130-150 mg/L monosodium phosphate monohydrate, 2000-2500 mg/L sodium bicarbonate, 40-60 mg/L, L-alanine 20-30 mg/L, L-sodium acetate, 60-80 mg/L, L-aspartic acid, 25-35 mg/L, L-cysteine hydrochloride monohydrate, 0.10-0.12 mg/L, L-cystine dihydrochloride, 20-30 mg/L, L-glutamic acid, 40-60 mg/L, L-glycine-histidine hydrochloride monohydrate, 20-25 mg/L, L-hydroxyproline 8-12 mg/L, 15-25 mg/L, L-leucine 50-70 mg/L, L-lysine hydrochloride 60-80 mg/L, L-methionine 10-20 mg/L, L-phenylalanine 20-30 mg/L, L-proline 30-50 mg/L, L-serine 20-30 mg/L, L-threonine 25-35 mg/L, L-tryptophan 8-12 mg/L, L-tyrosine disodium dihydrate 55-60 mg/L, L-valine 20-30 mg/L, ascorbic acid 0.04-0.06 mg/L, alpha-D-tocopherol phosphate 0.008-0.012 mg/L, biotin 0.008-0.012 mg/L, calcitol 0.08-0.12 mg/L, D-calcium pantothenate 0.008-0.012 mg/L, 0.4-0.6 mg/L choline chloride, 0.008-0.012 mg/L folic acid, 0.04-0.06 mg/L inositol, 0.015-0.025 mg/L menadione sodium bisulfite trihydrate, 0.02-0.03 mg/L nicotinic acid, 0.02-0.03 mg/L nicotinamide, 0.04-0.06 mg/L L, p-aminobenzoic acid, 0.04-0.06 mg/L pyridoxine hydrochloride, 0.008-0.012 mg/L riboflavin, 0.008-0.012 mg/L thiamine hydrochloride, 0.1-0.2 mg/L vitamin A acetate, 8-12 mg/L adenine sulfate, 0.15-0.25 mg/L adenine, 0.8-1.2 mg/L disodium adenosine triphosphate, 0.15-0.25 mg/L cholesterol, 2-deoxy-D-0.4-0.6 mg/L glucose, 0.04-0.84 mg/L glutathione, 0.06mg/L glucose, 0.25-0.35 mg/L guanine hydrochloride, 0.3-0.4 mg/L hypoxanthine sodium, 0.4-0.6 mg/L ribose, 0.25-0.35 mg/L thymosin, 4-6 mg/L tween 80, 0.25-0.35 mg/L uracil, 0.3-0.4 mg/L xanthine sodium, 8-12 mg/L phenol red, 0.2-0.3 mg/L sodium selenite and 0.05-0.1 mg/L copper sulfate.
8. A fertilization culture and semen preparation culture for use in the method for the culture and cryopreservation of bovine in vitro fertilized embryos according to claim 1, wherein:
the fertilization 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, 100U/ml penicillin, 100. mu.g/ml streptomycin, 10umoL/L alpha-lipoic acid, 25. mu.g/ml folic acid, 3mg/ml sodium glutamate,
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 monopotassium phosphate, 37.0mM sodium bicarbonate, 1.25mM sodium pyruvate, 10. mu.g/ml heparin, 4mg/ml bovine serum albumin, 10mM caffeine, 100U/ml penicillin, 100. mu.g/ml streptomycin, 10umoL/L alpha-lipoic acid, 25. mu.g/ml folic acid, 3mg/ml sodium glutamate.
9. The refrigerating fluid for culturing and cryopreserving the bovine in vitro fertilization embryos comprises the following components: PBS +20% FBS +1.8mol/L ethylene glycol +0.25mol/L sucrose +0.2% dextran 40+5mmol/L acetylcysteine.
10. The thawing solution for the culture and cryopreservation of bovine in vitro fertilization embryos comprises the following components: PBS +20% FBS +0.25mol/L sucrose +0.2% dextran 40+5mmol/L acetylcysteine.
CN202110493547.XA 2021-05-07 2021-05-07 Method for culturing and cryopreserving bovine in-vitro fertilization embryos Active CN112980778B (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN202110493547.XA CN112980778B (en) 2021-05-07 2021-05-07 Method for culturing and cryopreserving bovine in-vitro fertilization embryos

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN202110493547.XA CN112980778B (en) 2021-05-07 2021-05-07 Method for culturing and cryopreserving bovine in-vitro fertilization embryos

Publications (2)

Publication Number Publication Date
CN112980778A true CN112980778A (en) 2021-06-18
CN112980778B CN112980778B (en) 2021-07-30

Family

ID=76337196

Family Applications (1)

Application Number Title Priority Date Filing Date
CN202110493547.XA Active CN112980778B (en) 2021-05-07 2021-05-07 Method for culturing and cryopreserving bovine in-vitro fertilization embryos

Country Status (1)

Country Link
CN (1) CN112980778B (en)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113875747A (en) * 2021-11-15 2022-01-04 天津博裕力牧科技有限公司 Embryo vitrification freezing method and freezing liquid used by same
CN114540283A (en) * 2022-01-27 2022-05-27 中国农业科学院北京畜牧兽医研究所 High-efficiency vitrification freezing method for bovine in-vitro embryo production

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH09122158A (en) * 1995-10-30 1997-05-13 Kachiku Jiyuseiran Ishiyoku Gijutsu Kenkyu Kumiai Direct freezing method of swine embryo
US6586247B1 (en) * 1999-01-05 2003-07-01 Nipro Corporation Medium for cultivating animal embryo
CN112322579A (en) * 2021-01-06 2021-02-05 天津博裕力牧科技有限公司 Culture solution for cattle in-vitro fertilization and method for improving cattle in-vitro fertilization
CN112501114A (en) * 2021-01-06 2021-03-16 天津博裕力牧科技有限公司 Method for improving cattle in-vitro fertilization efficiency

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH09122158A (en) * 1995-10-30 1997-05-13 Kachiku Jiyuseiran Ishiyoku Gijutsu Kenkyu Kumiai Direct freezing method of swine embryo
US6586247B1 (en) * 1999-01-05 2003-07-01 Nipro Corporation Medium for cultivating animal embryo
CN112322579A (en) * 2021-01-06 2021-02-05 天津博裕力牧科技有限公司 Culture solution for cattle in-vitro fertilization and method for improving cattle in-vitro fertilization
CN112501114A (en) * 2021-01-06 2021-03-16 天津博裕力牧科技有限公司 Method for improving cattle in-vitro fertilization efficiency

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
L PÉREZ 等: "N-acetyl-L-cysteine pre-treatment protects cryopreserved bovine spermatozoa from reactive oxygen species without compromising the in vitro developmental potential of intracytoplasmic sperm injection embryos", 《ANDROLOGIA》 *

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113875747A (en) * 2021-11-15 2022-01-04 天津博裕力牧科技有限公司 Embryo vitrification freezing method and freezing liquid used by same
CN113875747B (en) * 2021-11-15 2022-06-03 天津力牧生物科技有限公司 Embryo vitrification freezing method and freezing liquid used by same
CN114540283A (en) * 2022-01-27 2022-05-27 中国农业科学院北京畜牧兽医研究所 High-efficiency vitrification freezing method for bovine in-vitro embryo production
CN114540283B (en) * 2022-01-27 2023-10-20 中国农业科学院北京畜牧兽医研究所 Efficient vitrification freezing method for bovine in-vitro embryo production

Also Published As

Publication number Publication date
CN112980778B (en) 2021-07-30

Similar Documents

Publication Publication Date Title
CN111254109B (en) Method for in vitro fertilization and embryo culture of bovine oocytes and transport culture solution
CN112501114B (en) Method for improving cattle in-vitro fertilization efficiency
CN113201484B (en) Method for improving cryopreservation and thawing of bovine in vitro fertilization blastocysts
CN111269876A (en) Method for in vitro fertilization and embryo culture by in vivo collection of bovine oocytes
CN107142239B (en) Method for improving culture efficiency of bovine in vitro fertilization embryos
CN107034173B (en) Method for culturing bovine in vitro fertilized embryo and culture solution used in method
CN112980778B (en) Method for culturing and cryopreserving bovine in-vitro fertilization embryos
Ahumada et al. Effect of supplementation of different growth factors in embryo culture medium with a small number of bovine embryos on in vitro embryo development and quality
CN112322579B (en) Culture solution for cattle in-vitro fertilization and method for improving cattle in-vitro fertilization
Balasubramanian et al. Effect of cysteamine supplementation of in vitro matured bovine oocytes on chilling sensitivity and development of embryos
CN108728404B (en) Method for culturing in vitro fertilization embryo of bovine living body or in vitro collected oocyte
CN108728401B (en) Method for culturing bovine oocyte in vitro fertilization embryo and used culture medium
CN108753689B (en) Method for culturing bovine in-vitro fertilized embryo
CN110066763A (en) Promote the method for ox embryo in vitro culture development of fertilized ova
Agung et al. Effect of maturation culture period of oocytes on the sex ratio of in vitro fertilized bovine embryos
CN113151160B (en) Method for improving cattle in-vitro fertilization efficiency and used freezing medium and thawing medium
CN110066764A (en) Promote the method for ox embryo in vitro culture oocyte in vitro maturation
Jochems et al. Effect of two ‘progressively motile sperm–oocyte’ratios on porcine in vitro fertilization and embryo development
Songsasen et al. Effects of β-mercaptoethanol on formation of pronuclei and developmental competence of swamp buffalo oocytes
CN112914784B (en) Bovine embryo segmentation method
EP2740790B1 (en) Composition for embryo culture
JP6796486B2 (en) Embryo culture method and medium
Bowles et al. Attempts to improve the yield of bovine blastocysts by incorporating insulin, selenium and transferrin in the in vitro system
Yang et al. Motility, fertilizability and subsequent embryonic development of frozen-thawed spermatozoa derived from epididymis in Hanwoo
CN113151155A (en) Cutting liquid for bovine embryo segmentation and bovine embryo segmentation method

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination
GR01 Patent grant
GR01 Patent grant
CP01 Change in the name or title of a patent holder

Address after: 300457 building a, Alexandria, No.3 Haitong street, Binhai New Area Development Zone, Tianjin Boya company

Patentee after: Tianjin Limu Biotechnology Co.,Ltd.

Address before: 300457 building a, Alexandria, No.3 Haitong street, Binhai New Area Development Zone, Tianjin Boya company

Patentee before: TIANJIN BOYU LIMU TECHNOLOGY CO.,LTD.

CP01 Change in the name or title of a patent holder