CN112914784B - Bovine embryo segmentation method - Google Patents
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Abstract
The invention relates to a method for dividing bovine embryo, which comprises the following steps: collecting oocytes and allowing them to mature in vitro; incubating the sperm suspension with the mature oocyte in a fertilized culture drop to complete in vitro fertilization; culturing the embryo in embryo culture solution after in vitro fertilization operation until morula grows well, and selecting morula under a microscope; manufacturing divided liquid drops; transferring morula into divided liquid drops, placing under an inverted microscope, fixing embryo with a fixing tube, and gradually pressing embryo from top to bottom with a dividing knife to divide embryo into two parts; transferring the half embryo obtained by segmentation into embryo culture solution for culture until morula develops into expanded blastula for embryo transplantation, wherein the segmentation solution comprises dipotassium hydrogen phosphate, potassium dihydrogen phosphate, sucrose, povidone, etc. The method and the used parting liquid have 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 agricultural-livestock veterinary breeding, in particular to a method for dividing bovine in-vitro embryos, and further relates to application of a test solution used for dividing bovine embryos in bovine embryo division. The method for dividing the bovine embryo has excellent technical effects. In particular, the invention relates to a technique for segmenting embryos obtained after in vitro fertilization of bovine oocytes obtained by ex vivo collection.
Background
With the development of modern agriculture technology, in order to make full use of the reproductive potential of improved cows, the genetic breeding process is accelerated, and the application of efficient new reproductive technology in production practice becomes necessary.
Living egg collection (Ovum pick UP, OPU) and in vitro fertilization (In Vitro Fertilization, IVF) are new embryo engineering technologies which are rapidly developed in the eighties of the twentieth century, and a large number of embryos with definite genetic genealogy can be obtained by combining the two technologies, so that the generation interval is shortened. Currently, these two techniques have become important breeding techniques for growing groups of fine breed cows for farmers in developed countries such as europe, america and oceans. In addition, embryo segmentation techniques, which are extended based on in vivo spawning and in vitro fertilization techniques, are powerful tools for accelerating the genetic breeding process.
Embryo segmentation techniques are described in detail in Liu Yonghua (Liu Yonghua, et al, progress in mammalian embryo segmentation techniques research, chinese livestock veterinarian, 2007, 34 (3): 52), the entire contents of which are incorporated herein by reference. Specifically, embryo segmentation refers to taking out a mammalian embryo from a uterus or a fallopian tube by a surgical or non-surgical method, artificially dividing the embryo into 2 or more parts by a mechanical or chemical method, and culturing each of the divided parts in vitro under a proper condition, transplanting the cultured parts back to a recipient uterus or fallopian tube (or directly transplanting the cultured parts) to obtain 2 or more offspring with consistent genotypes and phenotypes 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 and multiple embryos in the same egg, can be used for protecting endangered animals, and can also provide a biopsy method for early embryo genetic disease detection, sex identification and other technologies. The mammalian embryo segmentation technique began with the isolation and culture study of the rat 2-cell embryo blastomeres by Nicholas (1942). Tarkowski (1959) found that half of the blastomeres of the 2-cell mouse embryos had been destroyed were able to develop into blastocysts and mice were developed, after which he studied the development laws of the 1/4, 2/4, 3/4 blastomeres of mice, which showed that: each blastomere of the early embryo has developmental totipotency, namely, can develop into a normal individual, and living animals of the first half embryo source in the world are obtained through embryo segmentation. Mullen et al (1970) isolated 2 blastocysts of a mouse 2-cell embryo, cultured and transplanted to recipients, resulting in a first artificial twins of mammals. Thereafter, animal embryo segmentation techniques have rapidly evolved and reached a climax in the middle and late 80 s of the 20 th century. Willadsen (1979) initially uses a microsurgery method to divide sheep 2-cell embryo and morula into two or four respectively, then uses agar to embed the split balls, cultures for 3-5 d in ovine oviduct, takes out, and then strips and transplants the embryo to a receptor to obtain syngeneic twin and syngeneic tetrad. However, the method has a plurality of intermediate links, is complex to operate and is difficult to apply and popularize in production. In 1981, he removed the zona pellucida of sheep 4-cell stage embryos, separated 4 split spheres, and transplanted to recipients to obtain four lambs in common eggs and three lambs in common eggs. In the same year, willadsen, in cooperation with Ploge, divided 8-cell stage bovine embryos into 4 1/4 embryos and obtained a pair of co-oval twin and single embryo. Willadsen (1980) first combined embryo segmentation with embryo ultralow temperature freezing, and used sheep to cryopreserve half-embryos to obtain 3 live lambs. In 1984 he improved the method to divide more advanced embryos (morula or blastocyst) and the divided embryos could be transferred directly without culturing, thus having great practical value. Gyu-Jin (1998) also utilized oocyte in vitro maturation and in vitro fertilization to obtain bovine embryos, and transferred 12 quadrotola embryos to 6 recipients to obtain calves with 2 quadrotola embryos. This opens up new research and application fields for embryo segmentation technology.
The embryo of mammal can be divided from 2-cell stage to blastula stage and offspring can be produced, and the embryo dividing method is mainly different according to embryo development stage. In the 2-8 cell phase, embryo segmentation (blastomere separation) is mainly mechanical and chemical-assisted mechanical methods. Morula and blastocyst segmentation are the main execution phases of embryo segmentation of mammals, and in this phase, a Willadsen segmentation method, a Williams segmentation method, a TSuzuki segmentation method, a micropipette separation method, an enzyme-softened zona pellucida microscopic glass needle segmentation method, a freehand segmentation method and the like are typical.
Zheng Haiying (Zheng Haiying, et al, influence of different partitioning solutions on the embryo partitioning effect of buffalo, livestock and veterinary, 2009, 41 (7): 32) describes methods for partitioning bovine embryos, however these methods still have certain limitations.
Efficient embryo segmentation from morula is still highly desirable to those skilled in the art.
Disclosure of Invention
The present invention aims to provide a method for dividing bovine embryo, in particular to a method for efficiently dividing embryo from morula.
To this end, a first aspect of the invention relates to a method of bovine embryo segmentation comprising the steps of:
(1) Collecting oocytes and allowing them to mature in vitro;
(2) Incubating the sperm suspension with the mature oocyte in a fertilized culture drop to complete in vitro fertilization;
(3) Culturing the embryo in embryo culture solution after in vitro fertilization operation until morula grows well, and selecting morula under a microscope;
(4) Manufacturing split liquid drops in a sterile plastic plate by using split liquid; washing the sorted morula with a partitioning solution for 2 times, transferring into PBS solution for pretreatment, transferring into partitioning liquid drops, placing under an inverted microscope, fixing the embryo with a fixing tube, and gradually pressing the embryo from top to bottom with a partitioning knife to divide the embryo into two parts;
(5) Transferring the half embryo into another clean split liquid drop with a straw for 10min, transferring into embryo culture solution for culturing, and culturing to obtain expanded blastocyst for embryo transfer.
The method according to the first aspect of the invention, wherein the cow is selected from the group consisting of: chinese cattle, holstein cattle, siemens cattle, chinese buffalo.
According to the method of the first aspect of the invention, in step (1), the oocyte collection is performed as follows:
and (3) in-vitro collection: placing the slaughterhouse ovary in a heat-preserving barrel added with double antibiotic saline water, 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 the precipitate, picking up oocyte COCs (namely, cumulus-oocyte complexes) wrapped by at least 3 layers of cumulus cells under a stereoscopic microscope, washing the oocysts in the egg washing liquid for 2 times, and removing redundant impurities.
According to the method of the first aspect of the invention, in step (1), oocyte maturation is performed in the following manner: and (3) washing the obtained COCs in oocyte maturation culture solution for 1 time, transferring the COCs into new maturation culture solution, and culturing for 22-24 hours under the culture conditions of 38.8 ℃ and 5.5-6.5% CO2 and saturated humidity to mature the oocytes in vitro.
According to the method of the first aspect of the invention, the in vitro fertilization of step (2) is performed as follows:
washing mature COCs in fertilization culture solution for 1 time, transferring the COCs into fertilization culture solution, and placing the COCs into an incubator for standby;
taking a frozen seminiferous tubule from liquid nitrogen, and thawing in a water bath at 37 ℃; cutting off two ends of the tubule in a sterile operation, injecting semen into a 15mL centrifuge tube containing a semen preparation culture solution, centrifuging 328 Xg for 2 times each for 5min, and discarding the supernatant after centrifuging; adding 300 mu L of semen preparation culture solution into the centrifuge tube, re-suspending sperm sediment, and taking proper sperm suspension for sperm counting;
adding the sperm suspension with calculated volume into fertilized culture liquid drop containing oocyte, and placing a culture dish into an incubator to incubate sperm and egg for 16-20h under the culture conditions of 38.8 ℃ and 5.5-6.5% CO2 and saturated humidity, thus completing in vitro fertilization.
According to the method of the first aspect of the present invention, the step (3) is to perform embryo in vitro culture and sorting morula as follows:
after the in vitro fertilization operation is finished, the granulosa cells around the embryo are removed by an egg stripping needle, and are put into embryo culture solution for culture, at the moment, the culture condition is 38.8 ℃ at the 1 st day of embryo culture, 6% O2, 88% N2 and saturated humidity, and the morula which grows well is selected under a stereo microscope at the 5 th day for embryo segmentation operation.
According to the method of the first aspect of the present invention, the step (4) is to perform the morula segmentation as follows:
in a sterile plastic plate (e.g. 100mm diameter), several split droplets, each 100 μl, are made from the split solution; washing the separated morula with a partitioning solution for 2 times, transferring into PBS for pretreatment for 20min, transferring into partitioning liquid drops, and placing under an inverted microscope; the embryo dividing knife and the glass fixing tube are arranged on the micromanipulator, the embryo is fixed by the fixing 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 invention, step (4) is carried out to divide the development of the embryo as follows:
transferring the half embryo into another clean split liquid drop with a straw for 10min, transferring into embryo culture solution for culturing (38.8deg.C, 6% O2, 88% N2, saturated humidity) for 24 hr, and culturing to give expanded blastocyst for embryo transfer.
According to the method of the first aspect of the invention, the dividing liquid is a sterilized aqueous solution comprising the following components: 9.39g of dipotassium hydrogen phosphate, 3.5g of potassium dihydrogen phosphate, 68g of sucrose, 12g of povidone K30 and adding water for injection to 1000ml; for example, it is prepared using the following conventional method: dissolving the above materials with appropriate amount of water for injection, adding water for injection to 1000ml, filtering, and sterilizing at 115deg.C for 30 min.
According to the method of the first aspect of the invention, the dividing liquid is a sterilized aqueous solution comprising the following components: 9.39g of dipotassium hydrogen phosphate, 3.5g of potassium dihydrogen phosphate, 68g of sucrose, 12g of povidone K30, 1.6g of calcium ketophenylalanine, 5g of glycerol and adding water for injection to 1000ml; for example, it is prepared using the following conventional method: dissolving the above materials with appropriate amount of water for injection, adding water for injection to 1000ml, filtering, and sterilizing at 115deg.C for 30 min.
According to the method of the first aspect of the invention, the HEPES-containing transportation culture solution is formulated as described in the specific embodiment.
According to the method of the first aspect of the invention, the double antibiotic-added normal saline is normal saline containing 400IU/mL penicillin and 400 mug/mL streptomycin.
According to the method of the first aspect of the invention, the egg washing liquid is BY basal culture liquid added with 3mg/mL bovine serum albumin.
According to the method of the first aspect of the invention, the maturation medium is a 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 invention, the maturation medium comprising HEPES is a 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.
According to the method of the first aspect of the invention, the fertilization medium comprises: 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 in water.
According to the method of the first aspect of the invention, the semen preparation culture solution comprises: 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 in aqueous solution.
According to the method of the first aspect of the invention, the embryo culture solution is formulated as described in the detailed description.
According to the method of the first aspect of the invention, the BY basal culture solution is prepared according to the specific embodiment.
According to the method of the first aspect of the invention, the PBS solution is phosphate buffer solution, the pH value of the PBS solution is 6.8-7.2, and the PBS solution is prepared according to the following mode: 9.39g of dipotassium hydrogen phosphate and 3.5g of potassium dihydrogen phosphate are taken, 1000ml of water is added, filtration is carried out, and sterilization is carried out for 30 minutes at 115 ℃.
Further, the second aspect of the present invention provides a split solution for bovine embryo split, which is a sterilized aqueous solution comprising: 9.39g of dipotassium hydrogen phosphate, 3.5g of potassium dihydrogen phosphate, 68g of sucrose, 12g of povidone K30 and adding water for injection to 1000ml; for example, it is prepared using the following conventional method: dissolving the above materials with appropriate amount of water for injection, adding water for injection to 1000ml, filtering, and sterilizing at 115deg.C for 30 min.
The partitioning solution according to the second aspect of the present invention is a sterilized aqueous solution comprising: 9.39g of dipotassium hydrogen phosphate, 3.5g of potassium dihydrogen phosphate, 68g of sucrose, 12g of povidone K30, 1.6g of calcium ketophenylalanine, 5g of glycerol and adding water for injection to 1000ml; for example, it is prepared using the following conventional method: dissolving the above materials with appropriate amount of water for injection, adding water for injection to 1000ml, filtering, and sterilizing at 115deg.C for 30 min.
Any technical feature provided in any aspect of the present invention or any embodiment of any aspect is equally applicable to any other embodiment or any other embodiment of any aspect, provided that they do not contradict each other, and of course the corresponding features can be modified appropriately as appropriate when applicable to each other. Various aspects and features of the invention are described further below.
All documents cited herein are incorporated by reference in their entirety and are incorporated by reference herein to the extent they are not inconsistent with this invention. Furthermore, various terms and phrases used herein have a common meaning known to those skilled in the art, and even though they are still intended to be described and explained in greater detail herein, the terms and phrases used herein should not be construed to be inconsistent with the ordinary meaning in the sense of the present invention.
The fetal bovine serum used in the present invention can be easily purchased in standardized commercial forms from the market, for example, australian fetal bovine serum (accession number: 10099141), new Zealand fetal bovine serum (accession number: 10091148), north American fetal bovine serum (accession number: 16000044), mexico fetal bovine serum (accession number: 10437028) and the like from various agents. In the test 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) is a new biological technique for obtaining the same-egg twin or multiple Embryo by dividing the early Embryo into two, four and other equal parts by means of micromanipulation technique or freehand operation method and then transplanting the equal parts to the recipient female animal.
In 1890, heape reported for the first time that rabbit embryo transfer was successful at Cambridge university, UK, and embryo transfer technology has been studied for over 100 years. Embryo transfer has been increasingly studied in the 30 s of the 20 th century, and has been successful in various domestic animals, mainly sheep, goats, pigs, cows and horses. The first international embryo transfer society was held in Colorado, USA, month 1 in 1975, indicating that embryo transfer technology entered a higher stage of development. In 1976, the non-operative transplanting technique of cattle was promoted, and in 1977, commercialization was started, and 13 countries established hundreds of commercial embryo transfer companies. Cattle pregnant after 1978 in north america alone are nearly 1 million, and cattle pregnant by embryo transfer technology in the united states alone are up to 30 ten thousand per year.
The technology in China starts later, rabbit embryo transfer is successful in 1973, sheep in 1974, cow in 1978 and milk goat embryo transfer in 1980 are successful; the rabbit embryo and sheep embryo are successfully transplanted after being kept at low temperature for 1 day and 10 days in 1976-1977; after ultralow temperature preservation of sheep embryo in 1980, transplanting lambing; 3 calves are transplanted after the bovine embryo is frozen for 374 days in 1982; horse embryo transfer was successful in 1982. The success of dividing cattle and sheep before and after 90 s is equal to Guo Zhiqin which is equal to that of obtaining the same-egg double calves after dividing and transplanting frozen embryo of the dairy cow in 1989 and the same-egg lambs after dividing and transplanting fresh embryo of sheep in 1992; in 1989, cow quarter embryo successfully calving.
The bovine embryo segmentation technique is a technique developed on the basis of the bovine embryo transfer technique. The method is to divide an embryo into two or more by microsurgery operation on the embryo, and each blastomere has the potential possibility of developing into a complete individual in early stage of embryo development of the cow, so that the embryo dividing technology can be used for artificially manufacturing double or multiple embryo with the same egg, and the embryo number and calving number can be increased exponentially, thereby expanding the fine breed cow group rapidly and accelerating the development of the cow industry. In addition, the embryo segmentation technology can also obtain the homoegg twin or the volunteer offspring with the same sex and genetics, and provides precious test materials for researches of genetics, physiology, nutrition and the like. The problems of the effective rate of embryo segmentation, the pregnancy rate of embryo transplantation and the like are mainly solved in the implementation of the bovine embryo segmentation technology in China.
The application of embryo segmentation technology can not only increase the number of embryos used for embryo transfer by multiple times. But also can produce offspring with the same genetic properties. The method has special effects on animal husbandry production and experimental research, such as researching influence of external environment and conditions on growth, development, production performance and the like of livestock. The test results obtained by using the same-egg twin or multiple twin with the same genetic property are more definite. Meanwhile, sex can be indirectly controlled by applying embryo segmentation technology, so that the value and the practical effect of embryo transfer are greatly improved.
Embryo segmentation technology has great promotion effect on physiological, nutritional, genetic, embryological and animal breeding research, especially animal genetic research, and can provide valuable materials for research of these subjects. The research of the cryopreservation technology of the mammalian embryo starts in the 50 th century of 20, and the technology is combined with embryo segmentation, so that the number of embryos can be increased, the calving rate can be improved, the embryo transfer is not limited by time and region, the long-distance transportation of the embryos is solved, the high cost of international introduction is reduced, the disease transmission is reduced, and the popularization and application of the embryo transfer technology in production are facilitated; and embryo freezing has important significance in accelerating the breeding improvement process of livestock, establishing a gene library, protecting resources and the like.
The embryo segmentation technique provided by the present invention exhibits 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. Those skilled in the art will appreciate that various changes and modifications can be made to the invention without departing from the spirit and scope thereof. The present invention generally and/or specifically describes the materials used in the test as well as the test methods. Although many materials and methods of operation are known in the art for accomplishing the objectives of the present invention, the present invention will be described in as much detail herein. Unless otherwise indicated, the technical means used in the examples are conventional means well known to those skilled in the art, and all raw materials used are commercially available. In the present invention, unless otherwise specified, references to glutamic acid or sodium glutamate refer to either L-glutamic acid or sodium L-glutamate. In the present invention, if necessary, the test data are expressed as mean ± standard deviation, and analysis of variance is performed using SPSS21.0 software to determine the significance of the difference in results, if necessary. In the present invention, the embryo cutter is modified from a double sided razor unless otherwise specified.
In a specific test of the present invention, the relevant reagents used, unless otherwise specified, are described in detail as follows:
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, menaquinone 0.01mg/L, nicotinamide 0.025mg/L, nicotinic acid 0.025mg/L, p-aminobenzoic acid 0.05mg/L, pyridoxal hydrochloride 0.025mg/L, pyridoxine hydrochloride 0.025mg/L, riboflavin 0.01mg/L, thiamine hydrochloride 0.01mg/L, vitamin A acetate 0.1mg/L, vitamin D2, i.e., calcitol 0.1mg/L, alpha-tocopherol phosphoric acid 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, vitamin A acetate 0.1mg/L, 6800.0mg/L of sodium chloride, 140.0mg/L of sodium dihydrogen phosphate monohydrate, 10.0mg/L of adenine sulfate, 0.2mg/L of 5' -adenosine 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 Tween 80, 0.3mg/L of uracil, 0.3mg/L of sodium xanthine, and 0.01IU/mL of FSH, 0.01IU/mL of LH, 1. Mu.g/mL of E 2 EGF 50ng/mL, IGF 100ng/mL, 10% gentamicin, sodium pyruvate 55 μg/mL, cysteine 1.2mM/L, BSA 3mg/mL, HEPES 10mM/L, and taurine 40mg/L, zinc gluconate 2mg/L。
Adding double antibiotic normal saline: comprises 400IU/mL of penicillin and 400 mug/mL of streptomycin.
Egg washing liquid: BY basal medium with 3mg/mL bovine serum albumin was added.
Maturation culture solution: BY basal medium with 100mL/L FBS, 10. Mu.g/mL FSH, 10. Mu.g/mL LH, 1. Mu.g/mL E2, 20ng/mL EGF was added.
Mature culture broth comprising HEPES: BY basal medium was 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-epidermal growth factor, FSH-follicle stimulating hormone, FBS-fetal bovine serum, E2-estradiol, LH-luteinizing hormone.
A fertilisation broth 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 in water.
A semen preparation culture fluid 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 in aqueous solution.
An embryo culture fluid comprising: 109.5mM sodium chloride, 3.1mM potassium chloride, 26.2mM sodium bicarbonate, 0.8mM magnesium chloride hexahydrate, 1.19mM potassium dihydrogen phosphate, 0.4mM sodium pyruvate, 1.5mM glucose, 5mM calcium galactonate, 10v/v% Fetal Bovine Serum (FBS), 1mM L-glutamine, 2v/v% essential amino acids, 1v/v% optional amino acids, 3mM glutathione, sodium citrate 0.04w/v%, maltose 0.02w/v% aqueous solution; the essential amino acids are added according to the weight ratio: l-arginine hydrochloride 6.32g, L-cystine dihydrochloride 1.564g, L-histidine hydrochloride monohydrate 2.1g, L-isoleucine 2.625g, L-leucine 2.62g, L-lysine hydrochloride 3.625g, L-methionine 0.755g, L-phenylalanine 1.65g, L-threonine 2.38g, L-tryptophan 0.51g, L-tyrosine 1.8g and L-valine 2.34g, wherein the optional amino acids are added according to the following amino acid weight ratio: l-alanine 0.89g, L-asparagine monohydrate 1.5g, L-aspartic acid 1.33g, L-glutamic acid 1.47g, glycine 0.75g, L-proline 1.15g and L-serine 1.05g.
BY basal culture solution is an aqueous solution comprising the following components: 200mg/L of calcium chloride, 0.72mg/L of ferric nitrate nonahydrate, 400mg/L of potassium chloride, 97.7mg/L of magnesium sulfate, 6800mg/L of sodium chloride, 140mg/L of sodium dihydrogen phosphate monohydrate, 2200mg/L of sodium bicarbonate, 50mg/L, L-alanine 25mg/L, L-arginine hydrochloride 70mg/L, L-aspartic acid 30mg/L, L-cysteine hydrochloride monohydrate 0.11mg/L, L-cystine dihydrochloride 26mg/L, L-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, 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, calcitonin 0.1mg/L, D-calcium pantothenate 0.01mg/L, choline chloride 0.5mg/L, folic acid 0.01mg/L, inositol 0.05mg/L, menaquinone sodium bisulfite 0.019/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 sulfate 10mg/L, adenine 0.2mg/L, disodium adenosine triphosphate 1mg/L, cholesterol 0.2mg/L, 2-deoxy-D-ribose 0.5mg/L, D-glucose 1000mg/L, glutathione 0.05mg/L, guanine hydrochloride 0.3mg/L, sodium hypoxanthine 0.354mg/L, ribose 0.5mg/L, thymosin 0.3mg/L, tween 805mg/L, uracil 0.3mg/L, xanthine sodium 0.34mg/L, phenol red 10mg/L, sodium selenite 0.25mg/L, and anhydrous copper sulfate 0.075 mg/L.
The PBS solution is phosphate buffer solution, the pH value of the PBS solution is 6.8-7.2, and the PBS solution is prepared according to the following mode: 9.39g of dipotassium hydrogen phosphate and 3.5g of potassium dihydrogen phosphate are taken, 1000ml of water is added, filtration is carried out, and sterilization is carried out for 30 minutes at 115 ℃.
The remaining reagents were purchased from Sigma-Aldrich as not specifically described.
Example 1: segmentation method of cattle in vitro fertilized embryo (in vitro egg collection)
Test cattle: chinese cattle (Nanyang cattle, service species).
Step (1), oocyte collection and in vitro maturation
And (3) in-vitro collection: placing the slaughterhouse ovary in a heat-preserving barrel added with double antibiotic saline water, 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 the precipitate, picking up oocyte COCs (namely, cumulus-oocyte complexes) wrapped by at least 3 layers of cumulus cells under a stereoscopic microscope, washing the oocyte COCs in a cumulus-oocyte complex for 2 times in a liquid for removing redundant impurities;
washing the COCs obtained by the collection in oocyte maturation culture solution for 1 time, transferring the COCs into new maturation culture solution, and culturing for 22-24 hours under the culture conditions of 38.8 ℃ and 5.5-6.5% CO2 and saturated humidity to mature oocytes in vitro;
step (2), in vitro fertilization
Washing mature COCs in fertilization culture solution for 1 time, transferring the COCs into fertilization culture solution, and placing the COCs into an incubator for standby;
Taking a frozen seminiferous tubule from liquid nitrogen, and thawing in a water bath at 37 ℃; cutting off two ends of the tubule in a sterile operation, injecting semen into a 15mL centrifuge tube containing a semen preparation culture solution, centrifuging 328 Xg for 2 times each for 5min, and discarding the supernatant after centrifuging; adding 300 mu L of semen preparation culture solution into the centrifuge tube, re-suspending sperm sediment, and taking proper sperm suspension for sperm counting;
adding the sperm suspension with calculated volume into fertilization culture liquid drop containing oocyte, and placing a culture dish into an incubator to incubate sperm and egg for 16-20h (18 hours of practical operation), wherein the culture condition is 38.8 ℃ and 5.5-6.5% CO2 and saturated humidity, thus completing in vitro fertilization;
step (3), embryo in vitro culture and sorting morula
After the in vitro fertilization operation is finished, the granulosa cells around the embryo are removed by an egg stripping needle, and are put into embryo culture solution for culture, at the moment, the culture condition is 38.8 ℃ at the 1 st day of embryo culture, 6% O2, 88% N2 and saturated humidity, and the morula which grows well is selected under a stereo microscope at the 5 th day for embryo segmentation operation.
Step (4), morula segmentation
In a sterile plastic plate with the diameter of 100mm, a plurality of divided droplets are manufactured by using a dividing liquid, and each 100 mu L of the divided droplets are prepared; washing the separated morula with the partitioning solution for 2 times, transferring into PBS solution for pretreatment for 20min, transferring into the partitioning solution, and placing under an inverted microscope (200 times, olympus company); the embryo dividing knife and the glass fixing tube are arranged on the micromanipulator, the embryo is fixed by the fixing 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 the split embryo
Transferring the half embryo obtained by segmentation into another clean segmentation liquid drop by using a suction tube, treating for 10min, transferring into embryo culture solution for culturing (38.8 ℃, 6% O2, 88% N2 and saturated humidity) for 24h, checking the half embryo recovery number (namely the segmentation success number for calculating the segmentation success rate) during 5-6 h, and checking whether the morula is developed to an expanded blastula (if the morula is successfully developed to the expanded blastula, the development success can be used for embryo transplantation, and thus calculating the blastula development rate) during 23-24 h.
The partitioning solution used in this example was a sterilized aqueous solution comprising the following components: 9.39g of dipotassium hydrogen phosphate, 3.5g of potassium dihydrogen phosphate, 68g of sucrose, 12g of povidone K30 and adding water for injection to 1000ml; the preparation method comprises the following steps of: dissolving the above materials with appropriate amount of water for injection, adding water for injection to 1000ml, filtering, and sterilizing at 115deg.C for 30 min.
Example 2: segmentation method of cattle in vitro fertilized embryo (in vitro egg collection)
Test cattle: holstein cow (dairy variety).
Step (1), oocyte collection and in vitro maturation
And (3) in-vitro collection: placing the slaughterhouse ovary in a heat-preserving barrel added with double antibiotic saline water, 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 the precipitate, picking up oocyte COCs (namely, cumulus-oocyte complexes) wrapped by at least 3 layers of cumulus cells under a stereoscopic microscope, washing the oocyte COCs in a cumulus-oocyte complex for 2 times in a liquid for removing redundant impurities;
Washing the COCs obtained by the collection in oocyte maturation culture solution for 1 time, transferring the COCs into new maturation culture solution, and culturing for 22-24 hours under the culture conditions of 38.8 ℃ and 5.5-6.5% CO2 and saturated humidity to mature oocytes in vitro;
step (2), in vitro fertilization
Washing mature COCs in fertilization culture solution for 1 time, transferring the COCs into fertilization culture solution, and placing the COCs into an incubator for standby;
taking a frozen seminiferous tubule from liquid nitrogen, and thawing in a water bath at 37 ℃; cutting off two ends of the tubule in a sterile operation, injecting semen into a 15mL centrifuge tube containing a semen preparation culture solution, centrifuging 328 Xg for 2 times each for 5min, and discarding the supernatant after centrifuging; adding 300 mu L of semen preparation culture solution into the centrifuge tube, re-suspending sperm sediment, and taking proper sperm suspension for sperm counting;
adding the sperm suspension with calculated volume into fertilization culture liquid drop containing oocyte, and placing a culture dish into an incubator to incubate sperm and egg for 16-20h (18 hours of practical operation), wherein the culture condition is 38.8 ℃ and 5.5-6.5% CO2 and saturated humidity, thus completing in vitro fertilization;
step (3), embryo in vitro culture and sorting morula
After the in vitro fertilization operation is finished, the granulosa cells around the embryo are removed by an egg stripping needle, and are put into embryo culture solution for culture, at the moment, the culture condition is 38.8 ℃ at the 1 st day of embryo culture, 6% O2, 88% N2 and saturated humidity, and the morula which grows well is selected under a stereo microscope at the 5 th day for embryo segmentation operation.
Step (4), morula segmentation
In a sterile plastic plate with the diameter of 100mm, a plurality of divided droplets are manufactured by using a dividing liquid, and each 100 mu L of the divided droplets are prepared; washing the separated morula with the partitioning solution for 2 times, transferring into PBS solution for pretreatment for 20min, transferring into the partitioning solution, and placing under an inverted microscope (200 times, olympus company); the embryo dividing knife and the glass fixing tube are arranged on the micromanipulator, the embryo is fixed by the fixing 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 the split embryo
Transferring the half embryo obtained by segmentation into another clean segmentation liquid drop by using a suction tube, treating for 10min, transferring into embryo culture solution for culturing (38.8 ℃, 6% O2, 88% N2 and saturated humidity) for 24h, checking the half embryo recovery number (namely the segmentation success number for calculating the segmentation success rate) during 5-6 h, and checking whether the morula is developed to an expanded blastula (if the morula is successfully developed to the expanded blastula, the development success can be used for embryo transplantation, and thus calculating the blastula development rate) during 23-24 h.
The partitioning solution used in this example was a sterilized aqueous solution comprising the following components: 9.39g of dipotassium hydrogen phosphate, 3.5g of potassium dihydrogen phosphate, 68g of sucrose, 12g of povidone K30 and adding water for injection to 1000ml; the preparation method comprises the following steps of: dissolving the above materials with appropriate amount of water for injection, adding water for injection to 1000ml, filtering, and sterilizing at 115deg.C for 30 min.
Example 3: segmentation method of cattle in vitro fertilized embryo (in vitro egg collection)
Test cattle: siemens cattle (beef breeds).
Step (1), oocyte collection and in vitro maturation
And (3) in-vitro collection: placing the slaughterhouse ovary in a heat-preserving barrel added with double antibiotic saline water, 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 the precipitate, picking up oocyte COCs (namely, cumulus-oocyte complexes) wrapped by at least 3 layers of cumulus cells under a stereoscopic microscope, washing the oocyte COCs in a cumulus-oocyte complex for 2 times in a liquid for removing redundant impurities;
washing the COCs obtained by the collection in oocyte maturation culture solution for 1 time, transferring the COCs into new maturation culture solution, and culturing for 22-24 hours under the culture conditions of 38.8 ℃ and 5.5-6.5% CO2 and saturated humidity to mature oocytes in vitro;
step (2), in vitro fertilization
Washing mature COCs in fertilization culture solution for 1 time, transferring the COCs into fertilization culture solution, and placing the COCs into an incubator for standby;
taking a frozen seminiferous tubule from liquid nitrogen, and thawing in a water bath at 37 ℃; cutting off two ends of the tubule in a sterile operation, injecting semen into a 15mL centrifuge tube containing a semen preparation culture solution, centrifuging 328 Xg for 2 times each for 5min, and discarding the supernatant after centrifuging; adding 300 mu L of semen preparation culture solution into the centrifuge tube, re-suspending sperm sediment, and taking proper sperm suspension for sperm counting;
Adding the sperm suspension with calculated volume into fertilization culture liquid drop containing oocyte, and placing a culture dish into an incubator to incubate sperm and egg for 16-20h (18 hours of practical operation), wherein the culture condition is 38.8 ℃ and 5.5-6.5% CO2 and saturated humidity, thus completing in vitro fertilization;
step (3), embryo in vitro culture and sorting morula
After the in vitro fertilization operation is finished, the granulosa cells around the embryo are removed by an egg stripping needle, and are put into embryo culture solution for culture, at the moment, the culture condition is 38.8 ℃ at the 1 st day of embryo culture, 6% O2, 88% N2 and saturated humidity, and the morula which grows well is selected under a stereo microscope at the 5 th day for embryo segmentation operation.
Step (4), morula segmentation
In a sterile plastic plate with the diameter of 100mm, a plurality of divided droplets are manufactured by using a dividing liquid, and each 100 mu L of the divided droplets are prepared; washing the separated morula with the partitioning solution for 2 times, transferring into PBS solution for pretreatment for 20min, transferring into the partitioning solution, and placing under an inverted microscope (200 times, olympus company); the embryo dividing knife and the glass fixing tube are arranged on the micromanipulator, the embryo is fixed by the fixing 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 the split embryo
Transferring the half embryo obtained by segmentation into another clean segmentation liquid drop by using a suction tube, treating for 10min, transferring into embryo culture solution for culturing (38.8 ℃, 6% O2, 88% N2 and saturated humidity) for 24h, checking the half embryo recovery number (namely the segmentation success number for calculating the segmentation success rate) during 5-6 h, and checking whether the morula is developed to an expanded blastula (if the morula is successfully developed to the expanded blastula, the development success can be used for embryo transplantation, and thus calculating the blastula development rate) during 23-24 h.
The partitioning solution used in this example was a sterilized aqueous solution comprising the following components: 9.39g of dipotassium hydrogen phosphate, 3.5g of potassium dihydrogen phosphate, 68g of sucrose, 12g of povidone K30 and adding water for injection to 1000ml; the preparation method comprises the following steps of: dissolving the above materials with appropriate amount of water for injection, adding water for injection to 1000ml, filtering, and sterilizing at 115deg.C for 30 min.
Example 4: segmentation method of cattle in vitro fertilized embryo (in vitro egg collection)
Test cattle: chinese buffalo (a kind of service supplies).
Step (1), oocyte collection and in vitro maturation
And (3) in-vitro collection: placing the slaughterhouse ovary in a heat-preserving barrel added with double antibiotic saline water, 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 the precipitate, picking up oocyte COCs (namely, cumulus-oocyte complexes) wrapped by at least 3 layers of cumulus cells under a stereoscopic microscope, washing the oocyte COCs in a cumulus-oocyte complex for 2 times in a liquid for removing redundant impurities;
Washing the COCs obtained by the collection in oocyte maturation culture solution for 1 time, transferring the COCs into new maturation culture solution, and culturing for 22-24 hours under the culture conditions of 38.8 ℃ and 5.5-6.5% CO2 and saturated humidity to mature oocytes in vitro;
step (2), in vitro fertilization
Washing mature COCs in fertilization culture solution for 1 time, transferring the COCs into fertilization culture solution, and placing the COCs into an incubator for standby;
taking a frozen seminiferous tubule from liquid nitrogen, and thawing in a water bath at 37 ℃; cutting off two ends of the tubule in a sterile operation, injecting semen into a 15mL centrifuge tube containing a semen preparation culture solution, centrifuging 328 Xg for 2 times each for 5min, and discarding the supernatant after centrifuging; adding 300 mu L of semen preparation culture solution into the centrifuge tube, re-suspending sperm sediment, and taking proper sperm suspension for sperm counting;
adding the sperm suspension with calculated volume into fertilization culture liquid drop containing oocyte, and placing a culture dish into an incubator to incubate sperm and egg for 16-20h (18 hours of practical operation), wherein the culture condition is 38.8 ℃ and 5.5-6.5% CO2 and saturated humidity, thus completing in vitro fertilization;
step (3), embryo in vitro culture and sorting morula
After the in vitro fertilization operation is finished, the granulosa cells around the embryo are removed by an egg stripping needle, and are put into embryo culture solution for culture, at the moment, the culture condition is 38.8 ℃ at the 1 st day of embryo culture, 6% O2, 88% N2 and saturated humidity, and the morula which grows well is selected under a stereo microscope at the 5 th day for embryo segmentation operation.
Step (4), morula segmentation
In a sterile plastic plate with the diameter of 100mm, a plurality of divided droplets are manufactured by using a dividing liquid, and each 100 mu L of the divided droplets are prepared; washing the separated morula with the partitioning solution for 2 times, transferring into PBS solution for pretreatment for 20min, transferring into the partitioning solution, and placing under an inverted microscope (200 times, olympus company); the embryo dividing knife and the glass fixing tube are arranged on the micromanipulator, the embryo is fixed by the fixing 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 the split embryo
Transferring the half embryo obtained by segmentation into another clean segmentation liquid drop by using a suction tube, treating for 10min, transferring into embryo culture solution for culturing (38.8 ℃, 6% O2, 88% N2 and saturated humidity) for 24h, checking the half embryo recovery number (namely the segmentation success number for calculating the segmentation success rate) during 5-6 h, and checking whether the morula is developed to an expanded blastula (if the morula is successfully developed to the expanded blastula, the development success can be used for embryo transplantation, and thus calculating the blastula development rate) during 23-24 h.
The partitioning solution used in this example was a sterilized aqueous solution comprising the following components: 9.39g of dipotassium hydrogen phosphate, 3.5g of potassium dihydrogen phosphate, 68g of sucrose, 12g of povidone K30 and adding water for injection to 1000ml; the preparation method comprises the following steps of: dissolving the above materials with appropriate amount of water for injection, adding water for injection to 1000ml, filtering, and sterilizing at 115deg.C for 30 min.
In the present invention, when the effect of embryo segmentation method is examined, if not specifically described, 179 to 196 embryos are segmented in each example, and the success rate (%) of segmentation and the development rate (%) of blastocysts developed after segmentation are examined and calculated; the results of examples 1 to 4 are as follows: the success rates (%, means) of the segmentation of examples 1 to 4 are respectively: 58.7%, 56.2%, 49.2%, 60.6%; the blastocyst developmental rates (%), the average values of examples 1 to 4 were: 53.4%, 52.1%, 43.2%, 54.8%.
Example 5: segmentation method of bovine in-vitro fertilized embryo
In this example, the operations and conditions of examples 1 to 4 were referred to, respectively, except that 1.6g of calcium ketophenylalanine and 5g of glycerin were added to 1000ml of the above-mentioned divided solution. It is also prepared by conventional methods, for example: dissolving the above materials with appropriate amount of water for injection, adding water for injection to 1000ml, filtering, and sterilizing at 115deg.C for 30 min; the pH value of the segmentation liquid is 6.8-7.2. The success rate (%) of division and the development rate (%) of blastocysts developed after division of this example were examined and calculated as described above, and the results were as follows: in this example 5, the success rates (%), the average values of the division of the test by referring to examples 1 to 4, but using the supplemental ingredient division liquid were respectively: 78.2%, 70.6%, 66.4%, 78.9%; in this example 5, the blastula development rates (%), the average values) of the blastula according to examples 1 to 4, but tested with the supplemental ingredient split solution were respectively: 54.1%, 50.3%, 47.0%, 55.6%.
Example 6: segmentation method of bovine in-vitro fertilized embryo
In this example, the operations and conditions of examples 1 to 4 were referred to, respectively, except that 1.6g of calcium ketophenylalanine was added to 1000ml of the above-mentioned divided solution. It is also prepared by conventional methods, for example: dissolving the above materials with appropriate amount of water for injection, adding water for injection to 1000ml, filtering, and sterilizing at 115deg.C for 30 min. The success rate (%) of division and the development rate (%) of blastocysts developed after division of this example were examined and calculated as described above, and the results were as follows: in this example 6, the success rates (%), the average values of the division of the test by referring to examples 1 to 4, but using the supplemental ingredient division liquid were respectively: 61.2%, 55.7%, 43.8%, 61.4%; in this example 6, the blastula development rates (%), the average values) of the blastula according to examples 1 to 4, but tested with the supplemental ingredient split solution were respectively: 50.8%, 49.6%, 45.2%, 53.7%.
Example 7: segmentation method of bovine in-vitro fertilized embryo
In this example, the operations and conditions of examples 1 to 4 were referred to, respectively, except that 5g of glycerin was added to 1000ml of the above-mentioned divided solution. It is also prepared by conventional methods, for example: dissolving the above materials with appropriate amount of water for injection, adding water for injection to 1000ml, filtering, and sterilizing at 115deg.C for 30 min. The success rate (%) of division and the development rate (%) of blastocysts developed after division of this example were examined and calculated as described above, and the results were as follows: in this example 7, the success rates (%), the average values of the division of the test by referring to examples 1 to 4, but using the supplemental ingredient division liquid were respectively: 59.6%, 55.7%, 45.8%, 62.6%; in this example 7, the blastula development rates (%), the mean values of the blastula development rates were respectively: 53.4%, 54.1%, 45.8%, 53.2%.
According to the comparison of the results of the embodiments 6-7, the embodiments 1-4 and the embodiment 5, the unexpected finding that the success rate of segmentation can be obviously improved after two substances are added in the segmentation liquid at the same time, and the embryo segmentation technology effect can be improved as a whole even though the embryo development rate is not improved; however, if one or both of the substances are not supplemented, the success rate of segmentation cannot be improved at all. In addition, it was unexpectedly found that, although the literature teaches that calcium ions are not preferable to be added to the segmentation liquid, the embodiment 5 of the present invention does not cause degradation of the segmentation success rate and the segmentation success rate after adding the calcium ion-containing substance to the segmentation 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, especially when fine breed reproduction is carried out on large farm animals, the embryo number and the number of artificial homozygotic double embryo and multiple embryo can be increased by using an embryo segmentation mode, and more fine breeds can be obtained after transplantation; the method can increase embryo number and calving rate, and has important significance in accelerating livestock breeding improvement process, establishing gene library, protecting resources and the like.
The embodiments described herein are only for illustrating the composition and the effect of the present invention and are not limited to the scope of the present invention, so that those skilled in the art can make similar modifications without departing from the structure of the present invention, and they are all included in the scope of the patent of the present invention. These should also be construed as protecting the invention, which does not affect the effect of the practice of the invention and the utility of the patent.
Claims (10)
1. A method of bovine embryo segmentation comprising the steps of:
(1) Collecting oocytes and allowing them to mature in vitro;
(2) Incubating the sperm suspension with the mature oocyte in a fertilized culture drop to complete in vitro fertilization;
(3) Culturing the embryo in embryo culture solution after in vitro fertilization operation until morula grows well, and selecting morula under a microscope;
(4) Manufacturing split liquid drops in a sterile plastic plate by using split liquid; washing the sorted morula with a partitioning solution for 2 times, transferring into PBS solution for pretreatment, transferring into partitioning liquid drops, placing under an inverted microscope, fixing the embryo with a fixing tube, and gradually pressing the embryo from top to bottom with a partitioning knife to divide the embryo into two parts;
(5) Transferring the half embryo into another clean divided droplet with a straw for 10min, transferring into embryo culture solution for culturing until morula develops into expanded blastocyst for embryo transfer,
Wherein the method comprises the steps of
The step (4) is to divide morula as follows: in a sterile plastic plate, a plurality of divided droplets, each 100 μl, are made from the dividing liquid; washing the separated morula with a partitioning solution for 2 times, transferring into PBS for pretreatment for 20min, transferring into partitioning liquid drops, and placing under an inverted microscope; mounting an embryo dividing knife and a glass fixing tube on a micromanipulator, fixing the embryo by the fixing tube, and gradually pressing the embryo from top to bottom by the dividing knife to divide the embryo into two parts;
the parting liquid is a sterilized water solution containing the following components: 9.39g of dipotassium hydrogen phosphate, 3.5g of potassium dihydrogen phosphate, 68g of sucrose, 12g of povidone K30, 1.6g of calcium ketophenylalanine, 5g of glycerol and adding water for injection to 1000ml.
2. The method of claim 1 wherein said bovine is selected from the group consisting of: chinese cattle, holstein cattle, siemens cattle, chinese buffalo.
3. The method according to claim 1, wherein in step (1), the oocyte is collected as follows: placing the slaughterhouse ovary in a heat-preserving barrel added with double antibiotic saline water, and transporting the slaughterhouse ovary back to a laboratory within 3 hours at the temperature of 31-33 ℃; collecting and precipitating follicles with the surface of 2-8 mm, picking up oocyte COCs (cumulus-oocyte complex) wrapped by at least 3 layers of cumulus cells under a stereoscopic microscope, washing for 2 times in egg washing liquid, and removing redundant impurities.
4. The method according to claim 1, wherein in step (1), the oocyte maturation is performed in vitro as follows: collecting COCs, washing in oocyte maturation culture solution for 1 time, transferring to new maturation culture solution, and culturing for 22-24 h under the conditions of 38.8deg.C and 5.5-6.5% CO 2 Saturated humidity, and maturing oocytes in vitro.
5. The method according to claim 1, wherein the in vitro fertilization of step (2) is performed as follows:
washing mature COCs in fertilization culture solution for 1 time, transferring the COCs into fertilization culture solution, and placing the COCs into an incubator for standby;
taking a frozen seminiferous tubule from liquid nitrogen, and thawing in a water bath at 37 ℃; cutting off two ends of the tubule in a sterile operation, injecting semen into a 15mL centrifuge tube containing a semen preparation culture solution, centrifuging 328 Xg for 2 times each for 5min, and discarding the supernatant after centrifuging; adding 300 mu L of semen preparation culture solution into the centrifuge tube, re-suspending sperm sediment, and taking proper sperm suspension for sperm counting;
adding the calculated volume of sperm suspension to fertilize the oocytePlacing the culture dish into incubator, incubating sperm and egg for 16-20 hr under the conditions of 38.8deg.C and 5.5-6.5% CO 2 Saturated humidity, and completing in vitro fertilization.
6. The method according to claim 1, wherein the step (3) is performed by in vitro embryo culture and morula sorting as follows:
after the in vitro fertilization operation is completed, removing the granulosa cells around the embryo with an egg-peeling needle, and culturing in embryo culture solution at 38.8deg.C under 6% O condition, which is 1 day of embryo culture 2 、88% N 2 Saturated humidity, and on day 5, morula well-grown were selected under a stereo microscope for embryo segmentation.
7. The method according to claim 1, wherein step (4) is performed to divide the development of the embryo as follows: transferring the half embryo into another clean divided droplet with a pipette for 10min, transferring into embryo culture solution, and culturing for 24 hr at 38deg.C under 6% O 2 、88% N 2 Saturated humidity, until morula develop into expanded blastocysts that can be used for embryo transfer.
8. The method of claim 1, wherein the partitioning solution is prepared by: dissolving the above materials with appropriate amount of water for injection, adding water for injection to 1000ml, filtering, and sterilizing at 115deg.C for 30 min.
9. The method according to claim 1, 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 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.
10. The method according to claim 1, wherein the embryo culture broth is an aqueous solution comprising 109.5mM sodium chloride, 3.1mM potassium chloride, 26.2mM sodium bicarbonate, 0.8mM magnesium chloride hexahydrate, 1.19mM potassium dihydrogen phosphate, 0.4mM sodium pyruvate, 1.5mM glucose, 5mM calcium galactonate, 10v/v% fetal bovine serum, 1mM L-glutamine, 2v/v% essential amino acids, 1v/v% optional amino acids, 3mM glutathione, sodium citrate 0.04w/v%, maltose 0.02 w/v%; the essential amino acids are added according to the weight ratio: l-arginine hydrochloride 6.32g, L-cystine dihydrochloride 1.564g, L-histidine hydrochloride monohydrate 2.1g, L-isoleucine 2.625g, L-leucine 2.62g, L-lysine hydrochloride 3.625g, L-methionine 0.755g, L-phenylalanine 1.65g, L-threonine 2.38g, L-tryptophan 0.51g, L-tyrosine 1.8g and L-valine 2.34g, wherein the optional amino acids are added according to the following amino acid weight ratio: l-alanine 0.89g, L-asparagine monohydrate 1.5g, L-aspartic acid 1.33g, L-glutamic acid 1.47g, glycine 0.75g, L-proline 1.15g and L-serine 1.05g.
Priority Applications (1)
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