CN112914784A - Method for segmenting bovine embryo - Google Patents
Method for segmenting bovine embryo Download PDFInfo
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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- A61D19/00—Instruments or methods for reproduction or fertilisation
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- 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
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- 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
- C12N5/0608—Germ cells
- C12N5/0609—Oocytes, oogonia
Abstract
The invention relates to a method for segmenting bovine embryos, which comprises the following steps: collecting oocytes and maturing the oocytes in vitro; incubating the sperm suspension with the mature oocyte in a drop of fertilization culture to complete in vitro fertilization; after the in vitro fertilization operation is finished, culturing the embryos in an embryo culture solution until the morula grows well, and selecting the morula under a microscope; making a segmentation liquid drop; moving the morula alba into the cutting liquid drop, placing under an inverted microscope, fixing the embryo by using a fixing tube, and gradually pressing the embryo from top to bottom by using a cutting knife to divide the embryo into two parts; transferring the semi-embryo obtained by segmentation into embryo culture solution, and culturing until the morula develops into expanded blastocyst for embryo transplantation, wherein the segmentation solution contains dipotassium hydrogen phosphate, potassium dihydrogen phosphate, sucrose, polyvidone, etc. The method of the present invention and the partitioning liquid used exhibit excellent technical effects as described in the specification.
Description
Technical Field
The invention belongs to the technical field of animal breeding, relates to a technology for agriculture-animal husbandry and veterinary breeding, in particular to a method for segmenting cattle in vitro embryos, and further relates to application of a test solution for segmenting cattle embryos in cattle embryo segmentation. The method for segmenting the bovine embryo has excellent technical effects. In particular, the invention relates to a technology for segmenting an embryo obtained after in vitro fertilization of a bovine oocyte obtained by in vitro collection.
Background
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. In addition, embryo segmentation technology based on in vivo egg collection and in vitro fertilization technology is a powerful tool for accelerating genetic breeding process.
Embryo segmentation techniques are described in detail in the liu immortality literature (liu immortality, et al, advances in the study of mammalian embryo segmentation techniques, chinese veterinary medicine, 2007, 34(3):52), the entire contents of which are incorporated herein by reference. Specifically, embryo segmentation means that a mammalian embryo is surgically or non-surgically removed from the uterus or oviduct, the embryo is artificially divided into 2 or more parts by mechanical or chemical means, each of the divided parts is cultured in vitro under appropriate conditions and transplanted back into the uterus or oviduct of a recipient (or directly transplanted without culture), and 2 or more offspring having the same genotype and phenotype can be obtained from 1 embryo. The embryo segmentation technology can be used for amplifying the number of available excellent embryos and producing cloned animals with double embryos in the same ovum and multiple embryos in the same ovum, can be used for protecting endangered animals, and can also provide a method for checking living tissues for the technologies of checking genetic diseases of early embryos, identifying sexes and the like. The mammalian embryo segmentation technique began with the isolation and culture study of rat 2-cell embryo blastomeres by Nicholas (1942). Tarkowski (1959) found that half of the blastomeres had been destroyed and 2-cell mouse embryos could develop into blastocysts and mice were generated, after which he investigated the developmental regularity of the blastomeres in mice 1/4, 2/4, 3/4, which revealed that: each blastomere of the early embryo has totipotency of development, namely, the blastomere can be developed into a normal individual, and the first living animal from the world half embryo is obtained by embryo segmentation. Mullen et al (1970) isolated 2 blastomeres of mouse 2-cell embryos, cultured and transplanted into recipients to produce the first mammalian artificial twin. Since then, animal embryo segmentation techniques developed rapidly and reached a climax in the middle and late 80 s of the 20 th century. Willadsen (1979) firstly divides the 2-cell embryo and the morula of a sheep into two or four respectively by a microsurgical method, then embeds the obtained blastomeres with agar, cultures the blastomeres in the oviduct of the sheep for 3-5 days, and detaches and transplants the embryos to a receptor after taking out, thereby obtaining the homozygotic twins and homozygotic quadruplets. But the method has many intermediate links and troublesome operation and is difficult to apply and popularize in production. In 1981, he removed the zona pellucida of sheep embryos at the 4-cell stage, separated 4 blastomeres, and transplanted them to recipients to obtain four lambs on the same egg and three lambs on the same egg. In the same year, Willadsen, in turn, worked with Ploge to divide the 8-cell stage bovine embryos into 4 1/4 embryos and obtain an egg-in-three, a pair of egg-in-two and a single embryo. Willadsen (1980) first combined the embryo segmentation technique with the ultra-low temperature freezing technique of the embryos and used sheep to cryopreserve the half-embryos to obtain 3 live lambs. In 1984 he improved the method, and segmented the more advanced embryos (morula or blastocyst), and the segmented embryos could be directly transplanted without culture, thus having great practical value. Gyu-Jin (1998) uses oocyte in vitro maturation and in vitro fertilization to obtain cattle embryo, and transfers 12 quartets of embryos to 6 recipients to obtain 2 quartets of embryos of calves. This opens up new research and application field for embryo segmentation technique.
Mammalian embryos can be divided from the 2-cell stage to the blastocyst stage and offspring can be generated, and the methods for dividing the embryos mainly vary according to the developmental stages of the embryos. In the 2-8 cell stage, the embryo division (separation of blastomeres) mainly comprises a mechanical method and a chemical auxiliary mechanical method. Morula and blastocyst division is a main execution stage of embryo division in mammals, and typically includes willardsen division, Williams division, TSuzuki division, micropipette division, enzyme-softened zona pellucida microscopic glass needle division, free-hand division, and the like.
Zhenghaiying documents (Zhenghaiying, et al, influence of different split solutions on the splitting effect of buffalo embryos, zootechnics and veterinarians, 2009, 41 (7): 32) describe methods for splitting bovine embryos, however, these methods still have certain limitations.
Efficient embryo segmentation from morula remains a desideratum for those skilled in the art.
Disclosure of Invention
The present invention aims to provide a method for segmenting bovine embryos, in particular a method for efficiently segmenting embryos from morulae.
To this end, the invention relates in a first aspect to a method for bovine embryo segmentation comprising the steps of:
(1) collecting oocytes and maturing the oocytes in vitro;
(2) incubating the sperm suspension with the mature oocyte in a drop of fertilization culture to complete in vitro fertilization;
(3) after the in vitro fertilization operation is finished, culturing the embryos in an embryo culture solution until the morula grows well, and selecting the morula under a microscope;
(4) preparing a cutting liquid drop by using a cutting liquid in a sterile plastic plate; washing the sorted morula alba for 2 times by using a segmentation solution, then transferring into a PBS solution for pretreatment, then transferring into segmentation liquid drops, placing under an inverted microscope, fixing the embryos by using a fixing tube, and gradually pressing the embryos from top to bottom by using a segmentation knife to divide the embryos into two parts;
(5) transferring the semi-embryo obtained by division into another clean divided liquid drop by using a suction pipe, treating for 10min, transferring into an embryo culture solution, and culturing until the morula develops into an expanded blastocyst for embryo transfer.
The method according to the first aspect of the present invention, wherein said bovine is selected from the group consisting of: chinese cattle, Holstein cattle, Simmental cattle, and Chinese buffalo.
According to the method of the first aspect of the present invention, in step (1), the collection of oocytes is performed as follows:
collecting in vitro: taking and placing the slaughterhouse ovary in a heat-preserving barrel added with double-resistance normal saline, and transporting the slaughterhouse ovary back to a laboratory within 3 hours at the temperature of 31-33 ℃; and (3) extracting follicles with the surface of 2-8 mm, collecting precipitates, picking up oocyte COCs (namely, cumulus-oocyte complexes) at least containing 3 layers of cumulus cells under a stereoscopic microscope, washing for 2 times in an egg washing liquid, and removing redundant impurities.
According to the method of the first aspect of the present invention, in step (1), the in vitro maturation of the oocyte is carried out in the following manner: and (3) washing the collected COCs in the oocyte maturation culture solution for 1 time, transferring the COCs into a new maturation culture solution, and culturing for 22-24 hours under the conditions of 38.8 ℃, 5.5-6.5% CO2 and saturated humidity so as to mature the oocytes in vitro.
According to the method of the first aspect of the present invention, the in vitro fertilization of step (2) is carried out in the following manner:
washing mature COCs in a fertilization culture solution for 1 time, transferring the COCs into the fertilization culture solution, and putting the COCs into an incubator for later use;
taking a frozen tubule from liquid nitrogen, and unfreezing in a water bath at 37 ℃; aseptically cutting two ends of the thin tube, injecting semen into 15mL centrifuge tube containing semen preparation culture solution, centrifuging at 328 Xg for 2 times (5 min each time), and discarding supernatant after centrifuging; adding 300 mu L of semen preparation culture solution into the centrifuge tube, resuspending the sperm precipitation, and taking appropriate sperm suspension for sperm counting;
adding the calculated volume of sperm suspension into the fertilization culture liquid drop containing the oocyte, putting the culture disc into an incubator, and incubating the sperm and the ovum for 16-20h under the culture conditions of 38.8 ℃, 5.5-6.5% CO2 and saturated humidity, thereby completing the in vitro fertilization.
According to the method of the first aspect of the present invention, step (3) is the in vitro culture and sorting of morula embryos as follows:
after the in vitro fertilization procedure was completed, granulosa cells around the embryos were removed with an ovum-dissecting needle, and cultured in an embryo culture medium under conditions of 38.8 ℃, 6% O2, 88% N2, and saturation humidity on day 5, which was taken as day 1 of embryo culture, and morula with good growth was selected under a stereo microscope and used for embryo segmentation.
According to the method of the first aspect of the present invention, step (4) is carried out by morula segmentation as follows:
making several split droplets, each 100. mu.L, from the split fluid in a sterile plastic plate (e.g., 100mm in diameter); washing the sorted morula alba with a partitioning solution for 2 times, then transferring into PBS solution for pretreatment for 20min, then transferring into a partitioning liquid drop, and placing under an inverted microscope; the embryo dividing knife and the glass fixed tube are arranged on the micromanipulator, the embryo is fixed by the fixed tube, and the embryo is divided into two parts by gradually pressing the embryo from top to bottom by the dividing knife.
According to the method of the first aspect of the present invention, the step (4) is carried out by dividing the development of embryos as follows:
transferring the semi-embryo obtained by division into another clean divided liquid drop by using a suction tube, treating for 10min, transferring into an embryo culture solution, and culturing (38.8 ℃, 6% O2, 88% N2, saturation humidity) for 24h until the morula develops into an expanded blastocyst for embryo transfer.
According to the method of the first aspect of the invention, the partitioning liquid is a sterile aqueous solution comprising: 9.39g of dipotassium hydrogen phosphate, 3.5g of monopotassium phosphate, 68g of cane sugar, 12g of povidone K30 and supplementing injection water to 1000 ml; for example, it is prepared using the following conventional method: dissolving the above materials in appropriate amount of water for injection, adding water for injection to 1000ml, filtering, and sterilizing at 115 deg.C for 30 min.
According to the method of the first aspect of the invention, the partitioning liquid is a sterile aqueous solution comprising: 9.39g of dipotassium hydrogen phosphate, 3.5g of monopotassium phosphate, 68g of cane sugar, 12g of povidone K30, 1.6g of ketophenylalanine calcium, 5g of glycerin and supplementing water for injection to 1000 ml; for example, it is prepared using the following conventional method: dissolving the above materials in appropriate amount of water for injection, adding water for injection to 1000ml, filtering, and sterilizing at 115 deg.C for 30 min.
According to the method of the first aspect of the invention, the transport medium comprising HEPES is formulated as described in the detailed description.
According to the method of the first aspect of the invention, the saline plus diabrotic salt is a saline containing penicillin 400IU/mL and streptomycin 400. mu.g/mL.
According to the method of the first aspect of the present invention, the egg-washing solution is BY basal medium supplemented with 3mg/mL bovine serum albumin.
According to the method of the first aspect of the present invention, the maturation medium is BY basal medium supplemented with 100mL/L FBS, 10. mu.g/mL FSH, 10. mu.g/mL LH, 1. mu.g/mL E2, 20ng/mL EGF.
According to the method of the first aspect of the present invention, the maturation culture solution containing HEPES is BY basal culture solution supplemented with 15 mmol/L HEPES, 100mL/L FBS, 10. mu.g/mL FSH, 10. mu.g/mL LH, 1. mu.g/mL E2, 20ng/mL EGF.
According to the method of the first aspect of the invention, the fertilization medium comprises: an aqueous solution of 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.
According to the method of the first aspect of the invention, the semen preparation medium comprises: an aqueous solution of 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.
The method according to the first aspect of the invention, the embryo culture fluid, the formulation of which is as described in the detailed description.
According to the method of the first aspect of the present invention, the BY basal medium is formulated as described in the detailed description.
According to the method of the first aspect of the present invention, the PBS, i.e., the phosphate buffer, has a pH of 6.8 to 7.2 and is prepared 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.
Further, the second aspect of the present invention provides a parting solution for parting bovine embryos, which is a sterilized aqueous solution comprising the following components: 9.39g of dipotassium hydrogen phosphate, 3.5g of monopotassium phosphate, 68g of cane sugar, 12g of povidone K30 and supplementing injection water to 1000 ml; for example, it is prepared using the following conventional method: dissolving the above materials in appropriate amount of water for injection, adding water for injection to 1000ml, filtering, and sterilizing at 115 deg.C for 30 min.
A partitioning fluid according to a second aspect of the present invention is a sterilized aqueous solution comprising: 9.39g of dipotassium hydrogen phosphate, 3.5g of monopotassium phosphate, 68g of cane sugar, 12g of povidone K30, 1.6g of ketophenylalanine calcium, 5g of glycerin and supplementing water for injection to 1000 ml; for example, it is prepared using the following conventional method: dissolving the above materials in appropriate amount of water for injection, adding water for injection to 1000ml, filtering, and sterilizing at 115 deg.C for 30 min.
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.
Embryo segmentation (Embryo bissection) is a new biological technology for obtaining monozygotic twins or multipaths by segmenting early embryos into two, four, etc. multiple equal parts by means of micromanipulation technology or free-hand operation method and then transplanting the multiple parts to recipient dams.
Embryo transfer technology has been studied for over 100 years since Heape in 1890 first reported success in rabbit embryo transfer at Cambridge university, UK. Since the 30 s of the 20 th century, embryo transplantation has been studied more and more, and has been successful for various livestock, mainly including sheep, goats, pigs, cattle and horses. The first international society for embryo transfer was held in the united states of kororado in 1975, indicating that embryo transfer technology is at a more advanced stage. In 1976, the non-surgical transplantation technique of cattle was popularized, and in 1977, the technique was commercially applied, and 13 countries established hundreds of commercial embryo transplantation companies. In north america alone, nearly 1 million cows are pregnant after 1978 transplantation, and up to 30 million cows are pregnant by embryo transfer techniques in the united states alone each year.
The technology starts late in China, rabbit embryo transplantation is successful in 1973, sheep in 1974, cows in 1978 and dairy goats in 1980 are successful in embryo transplantation; the rabbit embryo and the sheep embryo are kept at low temperature for 1 day and 10 days in 1976-1977, and then are successfully transplanted; after ultralow temperature preservation of sheep embryos in 1980, the sheep embryos are transplanted to lambs; transferring 3 calving embryos after 374 days of freezing of bovine embryos in 1982; in 1982, horse embryo transfer was successful. The cattle and sheep are successfully divided before and after the 90 s, Guo Shiqin is equal to that the cattle are obtained in 1989 and are subjected to frozen embryo division and transplantation to produce double calves with the same eggs, and the sheep are subjected to fresh embryo division and four embryo division and transplantation in 1992 to produce lambs with the same eggs; in 1989, four embryos of cattle were successfully calved.
The bovine embryo segmentation technology is developed on the basis of the bovine embryo transplantation technology. The method is to divide an embryo into two or more embryos by microsurgical operation of the embryo, and because each blastomere has the potential possibility of developing into a complete individual at the early development stage of the cow embryo, double or multiple embryos in the same egg can be artificially manufactured by the embryo dividing technology, the number of embryos and calving can be multiplied, thereby rapidly expanding the fine breed cow group and accelerating the development of the cow industry. In addition, twins or multiple offspring of the same ovum with completely same sex and heredity can be obtained by embryo segmentation technology, thereby providing valuable test materials for the researches of genetics, physiology, nutriology and the like. The important point to be solved when the cattle embryo segmentation technology is implemented in China is the efficiency of embryo segmentation, the pregnancy rate of embryo transplantation and the like.
The application of the embryo segmentation technology can not only increase the number of embryos used for embryo transplantation by times. But also progeny with identical genetic properties can be generated. The method has special effects on animal husbandry production and experimental research, such as research on influences of external environment and conditions on growth, development and production performance of livestock. The obtained experimental result is more exact by applying the twin or multi-twin test with the same genetic property. Meanwhile, sex can be indirectly controlled by applying the embryo segmentation technology, so that the value and the practical effect of embryo transplantation are greatly improved.
The embryo segmentation technology has great promotion effect on the research of physiology, nutrition, genetics, embryology and animal breeding, especially on the research of animal genetics, and can provide valuable materials for the research of the subjects. The research of the cryopreservation technology of the mammalian embryo starts in the 50 th of the 20 th century, and the cryopreservation technology is combined with embryo segmentation, so that the number of the embryos can be increased, the calving rate can be improved, the embryo transplantation is not limited by time and regions, the remote transportation of the embryos is solved, the high cost of international introduction is reduced, the disease transmission is reduced, and the popularization and the application of the embryo transplantation technology in production are facilitated; and the embryo freezing has important significance in accelerating the breeding improvement process of livestock, establishing a gene bank, protecting resources and the like.
The embryo segmentation techniques provided by the present invention exhibit excellent technical effects as described herein.
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. In the present invention, if necessary, test data are expressed as mean ± standard deviation, and variance analysis is performed using SPSS21.0 software to determine the significance of the difference in the results thereof, if necessary, unless otherwise specified. In the present invention, the embryo-dividing knife is modified from a double-sided razor, unless otherwise specified.
In the specific test of the present invention, the reagents used are described in detail below, unless otherwise specified:
a transport medium comprising HEPES, comprising: glycine 50.0mg/L, L-alanine 25.0mg/L, L-arginine hydrochloride 70.0mg/L, L-aspartic acid 30.0mg/L, L-cystine dihydrochloride 26.0mg/L, L-glutamic acid 75.0mg/L, L-glutamine 100.0mg/L, L-histidine hydrochloride monohydrate 21.88mg/L, L-hydroxyproline 10.0mg/L, L-isoleucine 40.0mg/L, L-leucine 60.0mg/L, L-lysine hydrochloride 70.0mg/L, L-methionine 15.0mg/L, L-phenylalanine 25.0mg/L, L-proline 40.0mg/L, L-serine 25.0mg/L, L-threonine 30.0mg/L, L-tryptophan 10.0mg/L, L-tyrosine disodium salt dihydrate 58.0mg/L, L-valine 25.0mg/L, ascorbic acid 0.05mg/L, biotin 0.01mg/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, no-vitamin ACalcium chloride hydrate 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, monosodium phosphate monohydrate 140.0mg/L, adenine sulfate 10.0mg/L, adenosine 5' -phosphate 0.2mg/L, adenosine triphosphate 1.0mg/L, cholesterol 0.2mg/L, glucose 1000.0mg/L, deoxyribose 0.5mg/L, reduced glutathione 0.05mg/L, guanine 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 20.0mg/L, uracil 0.3mg/L, sodium xanthine 0.3mg/L, and FSH 0.01 mL, LH of 0.01IU/mL, E of 1. mu.g/mL250ng/mL of EGF, 100ng/mL of IGF, 10% gentamicin, 55 mug/mL of sodium pyruvate, 1.2mM/L of cysteine, 3mg/mL of BSA, 10mM/L of HEPES, 40mg/L of taurine and 2mg/L of zinc gluconate.
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 15 mmol/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.
A fertilization medium comprising: an aqueous solution of 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.
A semen preparation medium comprising: an aqueous solution of 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.
An 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, 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.
BY basal medium, which is an aqueous solution comprising the following components: 200mg/L calcium chloride, 0.72mg/L ferric nitrate nonahydrate, 400mg/L potassium chloride, 97.7mg/L magnesium sulfate, 6800mg/L sodium chloride, 140mg/L monobasic sodium phosphate monohydrate, 2200mg/L sodium bicarbonate, 50mg/L, L-sodium acetate, 25mg/L, L-arginine hydrochloride, 70mg/L, L-aspartic acid, 30mg/L, L-cysteine hydrochloride monohydrate, 0.11mg/L, L-cystine dihydrochloride, 26mg/L, L-glutamic acid, 75mg/L glycine, 50mg/L, L-histidine hydrochloride monohydrate, 21.88mg/L, L-hydroxyproline, 10mg/L, L-isoleucine, 20mg/L, L-leucine, 60mg/L leucine, L-lysine hydrochloride 70mg/L, L-methionine 15mg/L, L-phenylalanine 25mg/L, L-proline 40mg/L, L-serine 25mg/L, L-threonine 30mg/L, L-tryptophan 10mg/L, L-tyrosine disodium dihydrate 57.66mg/L, L-valine 25mg/L, ascorbic acid 0.05mg/L, alpha-D-tocopherol phosphate 0.01mg/L, biotin 0.01mg/L, calciferol 0.1mg/L, D-calcium pantothenate 0.01mg/L, choline chloride 0.5mg/L, folic acid 0.01mg/L, inositol 0.05mg/L, menadione trihydrate sodium bisulfite 0.019mg/L, nicotinic acid 0.025mg/L, Nicotinamide 0.025mg/L, p-aminobenzoic acid 0.05mg/L, pyridoxine hydrochloride 0.05mg/L, riboflavin 0.01mg/L, thiamine hydrochloride 0.01mg/L, vitamin A acetate 0.14mg/L, adenine 10mg/L, adenine 0.2mg/L, disodium adenosine triphosphate 1mg/L, cholesterol 0.2mg/L, 2-deoxy-D-ribose 0.5mg/L, 1000mg/L of D-glucose, 0.05mg/L of glutathione, 0.3mg/L of guanine hydrochloride, 0.354mg/L of hypoxanthine sodium, 0.5mg/L of ribose, 0.3mg/L of thymosin, 805mg/L of tween, 0.3mg/L of uracil, 0.34mg/L of xanthine sodium, 10mg/L of phenol red, 0.25mg/L of sodium selenite and 0.075mg/L of anhydrous copper sulfate.
The PBS (phosphate buffer solution) has a pH value of 6.8-7.2 and is prepared in the following way: 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 remaining reagents were purchased from Sigma-Aldrich as not specified.
Example 1: method for segmenting bovine in vitro fertilization embryo (in vitro egg collection)
Cattle for testing: chinese cattle (south-yang cattle, working variety).
Step (1), collection and in vitro maturation of oocytes
Collecting in vitro: taking and placing the slaughterhouse ovary in a heat-preserving barrel added with double-resistance normal saline, and transporting the slaughterhouse ovary back to a laboratory within 3 hours at the temperature of 31-33 ℃; extracting follicles with the surface of 2-8 mm, collecting precipitates, picking up oocyte COCs (namely, cumulus oophorus-oocyte complexes) at least containing 3 layers of cumulus oophorus cell packages under a stereoscopic microscope, washing for 2 times in an egg washing liquid, and removing redundant impurities;
washing the COCs obtained by the collection in an oocyte maturation culture solution for 1 time, transferring the COCs into a new maturation culture solution, and culturing for 22-24 hours under the conditions of 38.8 ℃, 5.5-6.5% CO2 and saturated humidity so as to mature the oocyte in vitro;
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 sperm suspension with the calculated volume into the fertilization culture liquid drop containing the oocyte, putting the culture disc into an incubator, and incubating the sperm and the ovum for 16-20h (actual operation for 18 h), wherein the culture conditions are 38.8 ℃, 5.5-6.5% CO2 and saturated humidity, and the in vitro fertilization is completed;
step (3), culturing embryo in vitro and sorting morula
After the in vitro fertilization procedure was completed, granulosa cells around the embryos were removed with an ovum-dissecting needle, and cultured in an embryo culture medium under conditions of 38.8 ℃, 6% O2, 88% N2, and saturation humidity on day 5, which was taken as day 1 of embryo culture, and morula with good growth was selected under a stereo microscope and used for embryo segmentation.
Step (4), morula segmentation
Preparing a plurality of cutting liquid drops with each 100 mu L in a sterile plastic plate with the diameter of 100mm by using the cutting liquid; washing the sorted morula with a partitioning solution for 2 times, then transferring into PBS solution for pretreatment for 20min, then transferring into a partitioning drop, and placing under an inverted microscope (200 times, Olympus corporation); the embryo dividing knife and the glass fixed tube are arranged on the micromanipulator, the embryo is fixed by the fixed tube, and the embryo is divided into two parts by gradually pressing the embryo from top to bottom by the dividing knife.
Step (5) development of divided embryos
Transferring the semi-embryo obtained by segmentation into another clean segmented liquid drop by using a suction pipe for treatment for 10min, then transferring the semi-embryo into an embryo culture solution for culture (38.8 ℃, 6% O2, 88% N2 and saturation humidity) for 24h, checking the recovery number of the semi-embryo (namely the segmentation success number for calculating the segmentation success rate) during 5-6 h, and checking whether the morula develops to an expanded blastocyst (if the morula successfully develops to the expanded blastocyst, the development success can be used for embryo transplantation, thereby calculating the blastocyst development rate).
The partitioning solution used in this example was a sterile aqueous solution containing the following components: 9.39g of dipotassium hydrogen phosphate, 3.5g of monopotassium phosphate, 68g of cane sugar, 12g of povidone K30 and supplementing injection water to 1000 ml; the preparation method is a conventional preparation method: dissolving the above materials in appropriate amount of water for injection, adding water for injection to 1000ml, filtering, and sterilizing at 115 deg.C for 30 min.
Example 2: method for segmenting bovine in vitro fertilization embryo (in vitro egg collection)
Cattle for testing: holstein cattle (dairy cattle breed).
Step (1), collection and in vitro maturation of oocytes
Collecting in vitro: taking and placing the slaughterhouse ovary in a heat-preserving barrel added with double-resistance normal saline, and transporting the slaughterhouse ovary back to a laboratory within 3 hours at the temperature of 31-33 ℃; extracting follicles with the surface of 2-8 mm, collecting precipitates, picking up oocyte COCs (namely, cumulus oophorus-oocyte complexes) at least containing 3 layers of cumulus oophorus cell packages under a stereoscopic microscope, washing for 2 times in an egg washing liquid, and removing redundant impurities;
washing the COCs obtained by the collection in an oocyte maturation culture solution for 1 time, transferring the COCs into a new maturation culture solution, and culturing for 22-24 hours under the conditions of 38.8 ℃, 5.5-6.5% CO2 and saturated humidity so as to mature the oocyte in vitro;
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 sperm suspension with the calculated volume into the fertilization culture liquid drop containing the oocyte, putting the culture disc into an incubator, and incubating the sperm and the ovum for 16-20h (actual operation for 18 h), wherein the culture conditions are 38.8 ℃, 5.5-6.5% CO2 and saturated humidity, and the in vitro fertilization is completed;
step (3), culturing embryo in vitro and sorting morula
After the in vitro fertilization procedure was completed, granulosa cells around the embryos were removed with an ovum-dissecting needle, and cultured in an embryo culture medium under conditions of 38.8 ℃, 6% O2, 88% N2, and saturation humidity on day 5, which was taken as day 1 of embryo culture, and morula with good growth was selected under a stereo microscope and used for embryo segmentation.
Step (4), morula segmentation
Preparing a plurality of cutting liquid drops with each 100 mu L in a sterile plastic plate with the diameter of 100mm by using the cutting liquid; washing the sorted morula with a partitioning solution for 2 times, then transferring into PBS solution for pretreatment for 20min, then transferring into a partitioning drop, and placing under an inverted microscope (200 times, Olympus corporation); the embryo dividing knife and the glass fixed tube are arranged on the micromanipulator, the embryo is fixed by the fixed tube, and the embryo is divided into two parts by gradually pressing the embryo from top to bottom by the dividing knife.
Step (5) development of divided embryos
Transferring the semi-embryo obtained by segmentation into another clean segmented liquid drop by using a suction pipe for treatment for 10min, then transferring the semi-embryo into an embryo culture solution for culture (38.8 ℃, 6% O2, 88% N2 and saturation humidity) for 24h, checking the recovery number of the semi-embryo (namely the segmentation success number for calculating the segmentation success rate) during 5-6 h, and checking whether the morula develops to an expanded blastocyst (if the morula successfully develops to the expanded blastocyst, the development success can be used for embryo transplantation, thereby calculating the blastocyst development rate).
The partitioning solution used in this example was a sterile aqueous solution containing the following components: 9.39g of dipotassium hydrogen phosphate, 3.5g of monopotassium phosphate, 68g of cane sugar, 12g of povidone K30 and supplementing injection water to 1000 ml; the preparation method is a conventional preparation method: dissolving the above materials in appropriate amount of water for injection, adding water for injection to 1000ml, filtering, and sterilizing at 115 deg.C for 30 min.
Example 3: method for segmenting bovine in vitro fertilization embryo (in vitro egg collection)
Cattle for testing: simmental cattle (beef variety).
Step (1), collection and in vitro maturation of oocytes
Collecting in vitro: taking and placing the slaughterhouse ovary in a heat-preserving barrel added with double-resistance normal saline, and transporting the slaughterhouse ovary back to a laboratory within 3 hours at the temperature of 31-33 ℃; extracting follicles with the surface of 2-8 mm, collecting precipitates, picking up oocyte COCs (namely, cumulus oophorus-oocyte complexes) at least containing 3 layers of cumulus oophorus cell packages under a stereoscopic microscope, washing for 2 times in an egg washing liquid, and removing redundant impurities;
washing the COCs obtained by the collection in an oocyte maturation culture solution for 1 time, transferring the COCs into a new maturation culture solution, and culturing for 22-24 hours under the conditions of 38.8 ℃, 5.5-6.5% CO2 and saturated humidity so as to mature the oocyte in vitro;
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 sperm suspension with the calculated volume into the fertilization culture liquid drop containing the oocyte, putting the culture disc into an incubator, and incubating the sperm and the ovum for 16-20h (actual operation for 18 h), wherein the culture conditions are 38.8 ℃, 5.5-6.5% CO2 and saturated humidity, and the in vitro fertilization is completed;
step (3), culturing embryo in vitro and sorting morula
After the in vitro fertilization procedure was completed, granulosa cells around the embryos were removed with an ovum-dissecting needle, and cultured in an embryo culture medium under conditions of 38.8 ℃, 6% O2, 88% N2, and saturation humidity on day 5, which was taken as day 1 of embryo culture, and morula with good growth was selected under a stereo microscope and used for embryo segmentation.
Step (4), morula segmentation
Preparing a plurality of cutting liquid drops with each 100 mu L in a sterile plastic plate with the diameter of 100mm by using the cutting liquid; washing the sorted morula with a partitioning solution for 2 times, then transferring into PBS solution for pretreatment for 20min, then transferring into a partitioning drop, and placing under an inverted microscope (200 times, Olympus corporation); the embryo dividing knife and the glass fixed tube are arranged on the micromanipulator, the embryo is fixed by the fixed tube, and the embryo is divided into two parts by gradually pressing the embryo from top to bottom by the dividing knife.
Step (5) development of divided embryos
Transferring the semi-embryo obtained by segmentation into another clean segmented liquid drop by using a suction pipe for treatment for 10min, then transferring the semi-embryo into an embryo culture solution for culture (38.8 ℃, 6% O2, 88% N2 and saturation humidity) for 24h, checking the recovery number of the semi-embryo (namely the segmentation success number for calculating the segmentation success rate) during 5-6 h, and checking whether the morula develops to an expanded blastocyst (if the morula successfully develops to the expanded blastocyst, the development success can be used for embryo transplantation, thereby calculating the blastocyst development rate).
The partitioning solution used in this example was a sterile aqueous solution containing the following components: 9.39g of dipotassium hydrogen phosphate, 3.5g of monopotassium phosphate, 68g of cane sugar, 12g of povidone K30 and supplementing injection water to 1000 ml; the preparation method is a conventional preparation method: dissolving the above materials in appropriate amount of water for injection, adding water for injection to 1000ml, filtering, and sterilizing at 115 deg.C for 30 min.
Example 4: method for segmenting bovine in vitro fertilization embryo (in vitro egg collection)
Cattle for testing: chinese buffalo (used breed).
Step (1), collection and in vitro maturation of oocytes
Collecting in vitro: taking and placing the slaughterhouse ovary in a heat-preserving barrel added with double-resistance normal saline, and transporting the slaughterhouse ovary back to a laboratory within 3 hours at the temperature of 31-33 ℃; extracting follicles with the surface of 2-8 mm, collecting precipitates, picking up oocyte COCs (namely, cumulus oophorus-oocyte complexes) at least containing 3 layers of cumulus oophorus cell packages under a stereoscopic microscope, washing for 2 times in an egg washing liquid, and removing redundant impurities;
washing the COCs obtained by the collection in an oocyte maturation culture solution for 1 time, transferring the COCs into a new maturation culture solution, and culturing for 22-24 hours under the conditions of 38.8 ℃, 5.5-6.5% CO2 and saturated humidity so as to mature the oocyte in vitro;
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 sperm suspension with the calculated volume into the fertilization culture liquid drop containing the oocyte, putting the culture disc into an incubator, and incubating the sperm and the ovum for 16-20h (actual operation for 18 h), wherein the culture conditions are 38.8 ℃, 5.5-6.5% CO2 and saturated humidity, and the in vitro fertilization is completed;
step (3), culturing embryo in vitro and sorting morula
After the in vitro fertilization procedure was completed, granulosa cells around the embryos were removed with an ovum-dissecting needle, and cultured in an embryo culture medium under conditions of 38.8 ℃, 6% O2, 88% N2, and saturation humidity on day 5, which was taken as day 1 of embryo culture, and morula with good growth was selected under a stereo microscope and used for embryo segmentation.
Step (4), morula segmentation
Preparing a plurality of cutting liquid drops with each 100 mu L in a sterile plastic plate with the diameter of 100mm by using the cutting liquid; washing the sorted morula with a partitioning solution for 2 times, then transferring into PBS solution for pretreatment for 20min, then transferring into a partitioning drop, and placing under an inverted microscope (200 times, Olympus corporation); the embryo dividing knife and the glass fixed tube are arranged on the micromanipulator, the embryo is fixed by the fixed tube, and the embryo is divided into two parts by gradually pressing the embryo from top to bottom by the dividing knife.
Step (5) development of divided embryos
Transferring the semi-embryo obtained by segmentation into another clean segmented liquid drop by using a suction pipe for treatment for 10min, then transferring the semi-embryo into an embryo culture solution for culture (38.8 ℃, 6% O2, 88% N2 and saturation humidity) for 24h, checking the recovery number of the semi-embryo (namely the segmentation success number for calculating the segmentation success rate) during 5-6 h, and checking whether the morula develops to an expanded blastocyst (if the morula successfully develops to the expanded blastocyst, the development success can be used for embryo transplantation, thereby calculating the blastocyst development rate).
The partitioning solution used in this example was a sterile aqueous solution containing the following components: 9.39g of dipotassium hydrogen phosphate, 3.5g of monopotassium phosphate, 68g of cane sugar, 12g of povidone K30 and supplementing injection water to 1000 ml; the preparation method is a conventional preparation method: dissolving the above materials in appropriate amount of water for injection, adding water for injection to 1000ml, filtering, and sterilizing at 115 deg.C for 30 min.
In the present invention, when the effect of the embryo segmentation method is examined, as not specifically described, 179 to 196 embryos are segmented in each example, and the segmentation success rate (%) and the blastocyst development rate (%) after the segmentation are examined and calculated; the results of examples 1 to 4 are as follows: the success rate (%, mean) of the segmentation in examples 1 to 4 were: 58.7%, 56.2%, 49.2%, 60.6%; the blastocyst development rates (%, mean) of examples 1 to 4 were: 53.4%, 52.1%, 43.2%, 54.8%.
Example 5: method for segmenting bovine in vitro fertilized embryo
In this example, the procedures and conditions of examples 1 to 4 were referred to, except that 1.6g of ketophenylalanine calcium and 5g of glycerin were further added to 1000ml of the split solution. It is also prepared by conventional methods, for example: dissolving the materials in appropriate amount of water for injection, adding water for injection to 1000ml, filtering, and sterilizing at 115 deg.C for 30 min; the pH value of the cutting fluid is 6.8-7.2. The success rate (%) of division and the percentage (%) of blastocyst development after division were examined and calculated as above, and the results were as follows: in this example 5, the success rates (%, mean) of the segmentation performed with the additive component segmentation solution in reference to examples 1-4 were: 78.2%, 70.6%, 66.4%, 78.9%; in this example 5, the blastocyst development rates (%, mean) of the experiments performed with the supplemented composition-partitioning solution with reference to examples 1 to 4 were: 54.1%, 50.3%, 47.0%, 55.6%.
Example 6: method for segmenting bovine in vitro fertilized embryo
In this example, the procedures and conditions of examples 1 to 4 were referred to, except that 1.6g of ketophenylalanine calcium was further added to each 1000ml of the split solution. It is also prepared by conventional methods, for example: dissolving the above materials in appropriate amount of water for injection, adding water for injection to 1000ml, filtering, and sterilizing at 115 deg.C for 30 min. The success rate (%) of division and the percentage (%) of blastocyst development after division were examined and calculated as above, and the results were as follows: in this example 6, the success rates (%, mean) of the segmentation performed with the additive component segmentation solution in reference to examples 1-4 were: 61.2%, 55.7%, 43.8%, 61.4%; in this example 6, the blastocyst development rates (%, mean) of the experiments performed with the supplemented composition-partitioning solution with reference to examples 1 to 4 were: 50.8%, 49.6%, 45.2%, 53.7%.
Example 7: method for segmenting bovine in vitro fertilized embryo
In this example, the operations and conditions of examples 1 to 4 were referred to, except that 5g of glycerin was further added to each 1000ml of the above-mentioned split liquid. It is also prepared by conventional methods, for example: dissolving the above materials in appropriate amount of water for injection, adding water for injection to 1000ml, filtering, and sterilizing at 115 deg.C for 30 min. The success rate (%) of division and the percentage (%) of blastocyst development after division were examined and calculated as above, and the results were as follows: in this example 7, the success rates (%, mean) of the segmentation performed with the additive component segmentation solution in reference to examples 1-4 were: 59.6%, 55.7%, 45.8%, 62.6%; in this example 7, the blastocyst development rates (%, mean) of the experiments performed with the supplemented composition-partitioning solution with reference to examples 1 to 4 were: 53.4%, 54.1%, 45.8% and 53.2%.
According to the results of the embodiments 6 to 7, 1 to 4 and 5 of the present invention, it was unexpectedly found that the success rate of segmentation can be significantly improved by supplementing two substances in the segmentation solution, and the embryo segmentation technology can be generally improved even though the blastocyst development rate is not improved; however, when one substance is supplemented alone or both substances are not supplemented, the segmentation success rate cannot be improved at all. In addition, it was unexpectedly found that although the literature teaches that it is not desirable to add calcium ions to the partitioning liquid, example 5 of the present invention does not cause a success rate of partitioning and a deterioration in the success rate of partitioning after adding a substance containing calcium ions to the partitioning liquid, which has not been taught at all in the prior art.
The embryo segmentation technology provided by the invention plays an important role in animal husbandry production, and particularly can be used for increasing the number of embryos and the number of artificial monozygotic twins and polyembryons in an embryo segmentation mode when fine breed breeding is carried out on large farm animals, so that more fine breeds can be obtained after transplantation; the method can increase the number of embryos and improve the calving rate, and has important significance in accelerating the breeding improvement process of livestock, establishing a gene bank, protecting resources and the like.
The embodiments are only for illustrating the composition and efficacy of the invention, and not for limiting the scope of the invention, therefore, it will be apparent to those skilled in the art that similar modifications can be made without departing from the structure of the invention, and all such modifications are within the scope of the invention. These should also be construed as the scope of the present invention, and they should not be construed as affecting the effectiveness of the practice of the present invention or the applicability of the patent.
Claims (10)
1. A method of bovine embryo segmentation comprising the steps of:
(1) collecting oocytes and maturing the oocytes in vitro;
(2) incubating the sperm suspension with the mature oocyte in a drop of fertilization culture to complete in vitro fertilization;
(3) after the in vitro fertilization operation is finished, culturing the embryos in an embryo culture solution until the morula grows well, and selecting the morula under a microscope;
(4) preparing a cutting liquid drop by using a cutting liquid in a sterile plastic plate; washing the sorted morula alba for 2 times by using a segmentation solution, then transferring into a PBS solution for pretreatment, then transferring into segmentation liquid drops, placing under an inverted microscope, fixing the embryos by using a fixing tube, and gradually pressing the embryos from top to bottom by using a segmentation knife to divide the embryos into two parts;
(5) transferring the semi-embryo obtained by division into another clean divided liquid drop with a suction tube, treating for 10min, transferring into embryo culture solution, culturing until the morula develops into expanded blastocyst for embryo transfer,
wherein
The morula was divided in the following manner in step (4): in a sterile plastic plate, a plurality of cutting liquid drops are made of cutting liquid, and each cutting liquid drop is 100 mu L; washing the sorted morula alba with a partitioning solution for 2 times, then transferring into PBS solution for pretreatment for 20min, then transferring into a partitioning liquid drop, and placing under an inverted microscope; mounting an embryo cutter and a glass fixing tube on a micromanipulator, fixing the embryo by using the fixing tube, and gradually pressing the embryo from top to bottom by using the cutter to divide the embryo into two parts;
the cutting fluid is a sterilized aqueous solution containing the following components: 9.39g dipotassium hydrogen phosphate, 3.5g potassium dihydrogen phosphate, 68g sucrose, 12g povidone K30, 1.6g calcium ketophenylalanine, 5g glycerin and supplementing water for injection to 1000 ml.
2. The method according to claim 1, wherein said bovine is selected from the group consisting of: chinese cattle, Holstein cattle, Simmental cattle, and Chinese buffalo.
3. The method according to claim 1, wherein in step (1) the oocyte is collected by: taking and placing the slaughterhouse ovary in a heat-preserving barrel added with double-resistance normal saline, and transporting the slaughterhouse ovary back to a laboratory within 3 hours at the temperature of 31-33 ℃; and (3) extracting follicles with the surface of 2-8 mm, collecting precipitates, picking out oocyte COCs (oocytes CoCs) at least containing 3 layers of cumulus cells under a stereoscopic microscope, namely cumulus-oocyte complexes, washing for 2 times in an egg washing liquid, and removing redundant impurities.
4. The method according to claim 1, wherein in step (1) the oocyte maturation in vitro is carried out in the following manner: and (3) washing the collected COCs in the oocyte maturation culture solution for 1 time, transferring the COCs into a new maturation culture solution, and culturing for 22-24 hours under the conditions of 38.8 ℃, 5.5-6.5% CO2 and saturated humidity so as to mature the oocytes in vitro.
5. The method according to claim 1, wherein the step (2) of in vitro fertilization is carried out in the following manner:
washing mature COCs in a fertilization culture solution for 1 time, transferring the COCs into the fertilization culture solution, and putting the COCs into an incubator for later use;
taking a frozen tubule from liquid nitrogen, and unfreezing in a water bath at 37 ℃; aseptically cutting two ends of the thin tube, injecting semen into 15mL centrifuge tube containing semen preparation culture solution, centrifuging at 328 Xg for 2 times (5 min each time), and discarding supernatant after centrifuging; adding 300 mu L of semen preparation culture solution into the centrifuge tube, resuspending the sperm precipitation, and taking appropriate sperm suspension for sperm counting;
adding the calculated volume of sperm suspension into the fertilization culture liquid drop containing the oocyte, putting the culture disc into an incubator, and incubating the sperm and the ovum for 16-20h under the culture conditions of 38.8 ℃, 5.5-6.5% CO2 and saturated humidity, thereby completing the in vitro fertilization.
6. The method according to claim 1, step (3) is that of culturing and sorting morula in vitro of embryos in the following manner:
after the in vitro fertilization procedure was completed, granulosa cells around the embryos were removed with an ovum-dissecting needle, and cultured in an embryo culture medium under conditions of 38.8 ℃, 6% O2, 88% N2, and saturation humidity on day 5, which was taken as day 1 of embryo culture, and morula with good growth was selected under a stereo microscope and used for embryo segmentation.
7. The method according to claim 1, wherein the step (4) is carried out by dividing the development of the embryo as follows: transferring the semi-embryo obtained by division into another clean divided liquid drop by using a suction pipe for treatment for 10min, and then transferring into an embryo culture solution for culture for 24h under the culture conditions of 38.8 ℃, 6% O2, 88% N2 and saturated humidity until the morula develops into an expanded blastocyst for embryo transfer.
8. The method of claim 1, wherein the partitioning fluid is prepared using the following method: dissolving the above materials in appropriate amount of water for injection, adding water for injection to 1000ml, filtering, and sterilizing at 115 deg.C for 30 min.
9. The method according to claim 1, said fertilization medium comprising: an aqueous solution of 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.
10. 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, 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.
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