CN113774148A

CN113774148A - Method for sex identification and embryo segmentation of early-stage embryo of cow

Info

Publication number: CN113774148A
Application number: CN202111129088.3A
Authority: CN
Inventors: 王小武; 王娜; 陈建美; 郝少强; 赵明礼; 郭春明; 李琳; 许晓椿
Original assignee: Tianjin Boyu Limu Technology Co ltd
Current assignee: Tianjin Boyu Limu Technology Co ltd
Priority date: 2021-09-26
Filing date: 2021-09-26
Publication date: 2021-12-10

Abstract

The invention relates to a method for sex determination and embryo segmentation of early cattle embryos, and further relates to a Phosphate Buffer Solution (PBS) solution for sex determination of early cattle embryos, wherein the pH value of the buffer solution is 6.8-7.2 and the buffer solution 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 method comprises the following steps: fertilizing the oocyte and the sperm in vitro to obtain a morula; cutting the morula alba in the cutting liquid drop by using a cutting knife to obtain embryo tissues as a sex identification sample; preparing a sample by using a sex determination kit by a loop-mediated isothermal amplification method and preparing a Master Mix; after the samples are mixed, the reaction tube is placed in a loop-mediated isothermal amplification real-time turbidity detection system for reaction, and the samples are determined to be male, female and one of three situations which need to be redone indefinitely according to results. The method of the present invention exhibits excellent technical effects as described in the specification.

Description

Method for sex identification and embryo segmentation of early-stage embryo of cow

Technical Field

The invention belongs to the technical field of biology, relates to a sex identification method, in particular to a method for identifying the sex of an early-stage embryo of a cow, and further relates to an embryo segmentation method. In particular to a method for carrying out sex determination and embryo segmentation on early cattle embryos by using a loop-mediated isothermal amplification method.

Background

The animal husbandry is a production department for obtaining animal products such as meat, eggs, milk, wool, cashmere, hide, silk and medicinal materials by utilizing the physiological functions of animals such as livestock and poultry which are domesticated by human beings or wild animals such as deer, musk, fox, mink, otter, quail and the like and converting the plant energy such as pasture, feed and the like into animal energy through artificial propagation and feeding. Is different from self-sufficient livestock breeding, has the characteristics of centralization and scale in animal husbandry, and becomes a supporting industry for ensuring basic livelihood.

In Vitro Fertilization (In Vitro Fertilization) or (external Fertilization) refers to a technique In which sperm and eggs of a mammal complete a Fertilization process In an environment artificially controlled In Vitro, abbreviated In the english to IVF. Because it is inseparable from the embryo transfer technique (ET), also referred to as IVF-ET for short. In biology, an animal obtained after an in vitro fertilized embryo is transferred to a mother is called a test-tube animal. The technology is successful in the 50 s of the 20 th century, develops rapidly in the last 20 years, and is mature day by day to become an important and conventional animal breeding biotechnology.

The in vitro fertilization technology has important significance for animal reproductive mechanism research, livestock production, medicine, endangered animal protection and the like. For example, using mouse, rat or rabbit as experimental material, the in vitro fertilization technique can be used to study the gametogenesis, fertilization and early embryonic development mechanism of mammals. In the livestock breed improvement, the in vitro fertilization technology provides a cheap and efficient means for embryo production, and has important values for fully utilizing excellent breed resources, shortening the breeding cycle of livestock, accelerating the breed improvement speed and the like. In humans, IVF-ET technology is one of the important measures to treat certain infertility and to overcome sexual linked diseases. In vitro fertilization is also an indispensable component of modern biotechnology, such as mammalian embryo transplantation, cloning, transgenosis, sex control and the like.

With the development of modern agricultural science and technology, in order to make full use of the breeding potential of elite cows and accelerate the genetic breeding process, it becomes necessary to apply a new efficient breeding technology in production practice. In-vivo egg taking (OPU) and In Vitro Fertilization (IVF) are new embryo engineering technologies that have been developed rapidly In the eighties of the twentieth century, and a large number of embryos with definite genetic pedigrees can be obtained by combining the OPU and the IVF, so that the generation interval is shortened. At present, the two technologies become important breeding technologies adopted by farmers in animal husbandry developed countries such as europe, the united states and oceania for expanding stock cow groups.

For example, prior application CN108728404A (application No.: 201810576806.3) discloses a method for the culture of bovine in vitro fertilized embryos comprising the steps as described in this document. Prior application CN111254109A (chinese patent application No. 2020100820171) provides a transport culture solution containing glycine, etc. to overcome the problem of transport difficulty, and the method for in vitro fertilization and embryo culture of bovine oocytes using the transport culture solution comprises the following steps: collecting oocytes, maturing in vitro, fertilizing in vitro, culturing embryo in vitro and preserving. The method and the transport culture solution of the invention achieve excellent technical effects. Further, the prior application CN112322579A (chinese patent application No. 2021100106576) relates to a culture solution for cattle in vitro fertilization and a method of improving cattle in vitro fertilization. The method of in vitro fertilization of cattle comprises the steps as described in this document. After the in vitro fertilization operation is finished, embryo in-vitro culture is carried out by carrying out in vitro development on fertilized eggs, and the obtained usable embryo can be frozen and preserved by liquid nitrogen. Temporary cryopreservation is beneficial for increasing animal reproductive efficiency. The above-mentioned CN108728404A, CN111254109A and CN112322579A are incorporated herein by reference in their entirety.

After obtaining a large number of in vitro fertilized embryos, on the one hand, the embryos are usually frozen and then wait for recipient cows with appropriate physiological phases for in vivo embryo reproduction, and on the other hand, sex determination is carried out on the embryos so as to select embryos with appropriate sex for transplantation into maternal cows for reproduction.

The sex selection for embryo transfer by embryo sex identification is a sex control technology in the general sense.

It is well known that sex control techniques are of great importance in animal husbandry, as described in the Liuyang literature [ Liuyang, et al, research advances in animal embryo sex determination techniques, livestock and poultry industry, 2007, (03): 4 to 5 are described in detail. Firstly, the livestock sex control technology is a bioengineering technology which can obviously improve the breeding rate of livestock, control the sex ratio of offspring can increase the seed selection intensity, accelerate the breeding process, and has very important significance for breeding, production and prevention and treatment of genetic diseases of livestock. Secondly, the technology of sex control of livestock is also a significant breeding technology in animal husbandry, since many important economic traits in animal husbandry are related to sex, for example: meat, egg, milk, wool, antler and the like, and by controlling the sex ratio of offspring, the maximum economic benefit of production traits (such as lactation) limited by sex and production traits (such as production speed, meat quality and the like) influenced by sex can be allowed to be exerted. With the rapid development of molecular biology, cytogenetics, immunohistology and other disciplines, and the advent of modern high-precision instruments, research on sex control has been greatly developed. Currently, sex control technology is mainly performed in 2 stages of pre-fertilization sex control and post-fertilization sex control. The most desirable approach for pre-fertilization sex control is to take X and Y sperm separation and then purposefully perform fertilization. The sex control after fertilization is realized by the sex identification of the embryo before implantation. But from the development of current X, Y sperm cell separation technology. Although X, Y sperm cells have been successfully isolated using a flow cytometer. But the cost is high, the separation efficiency is low, and the popularization in practical application is difficult. The embryo sex identification has been greatly developed and is already in practical use.

The development of sex control technology comes from the gene regulation theory of sex determination, and sex control is divided into two processes of sex determination and sex differentiation. Animal sex determination refers to the process by which the undifferentiated gonads of early embryos of animals differentiate into testes or ovaries. The key to determining sex is the Y chromosome, the presence of which determines the male and female respectively. The male is determined by the short arm and 1 micro-fragment of the Y chromosome, called sex-determining region on the Y chromosome, which is the major gene that initiates testis formation. In recent years, it has been discovered that genes such as SRY, DMRT1, SOX9, SF-1, WT1, LIM1, LHX9 and DAX-1 play an important role in determining sex and differentiating sexual organs of animals [ Zhanghua, et al, research progress on sex determination mechanism and sex identification, Chinese veterinary medicine, 2004, 7 (31): 23 to 25 ]. In this process, expression of SRY is primarily involved in male embryos, which initiates testicular formation and, in turn, secondary sex characteristics in males. Sexual differentiation refers primarily to the formation of committed gonadal-regulated reproductive structures. This process is mainly regulated by gonadal hormones. For males, the gonadal hormones mainly include MIS and testosterone.

With the development of molecular genetics, developmental biology and related disciplines, people's understanding of sex differentiation has increased to the molecular level, and livestock embryo sex identification methods have also been developed to the molecular level. At present, a plurality of methods for identifying embryo sex are developed, mainly including cytogenetics method, H-Y antigen method, male specificity DNA probe method, PCR method, loop-mediated isothermal amplification method and the like.

PCR sexing techniques are currently known to be quite sophisticated. Herr, et al 1990, was the first successful PCR method for sexing bovine and ovine embryos. The PCR method has the advantages of strong specificity, high sensitivity, rapidness, accuracy, small influence on embryos and the like, so that the PCR method occupies an increasingly important position in livestock embryo sex identification. The essence is the detection technique of Y-chromosome specific fragments or sex-determining genes on the Y-chromosome. The embryo with the target band amplified by PCR is the male embryo, otherwise, the embryo is the female embryo. The accuracy of the PCR method depends, in the first place, on the quality of the nucleic acid extraction. However, since the method of manually extracting nucleic acid has many disadvantages in handling a large amount of samples, such as high cost and long time consumption, it is difficult to completely remove the inhibitory factors present in the cells and outside the cells, and it easily causes contamination of the samples, thereby affecting the accuracy of PCR and increasing the probability of false negative results [ B. Rittich et a1, PCR identification of Salmonella cells in food and stool samples after immunological separation, Bioseparation, 2001(9): 379-384 ]. Therefore, in order to expand the application scale of PCR, it is necessary to use a high-throughput fully-automatic PCR workstation which can automatically perform DNA extraction and PCR. The main processes of PCR for embryo sexing generally include: (1) obtaining an embryo: frozen embryos, fresh embryos or embryos cultured by in vitro fertilization can be used for sex determination; (2) designing a primer: designing a primer sequence according to the base sequence of a specific gene or DNA fragment on the Y-chromosome; (3) embryo cell sampling and processing: sucking 6-8 cells from the embryo by using a microscope needle, boiling and performing PCR amplification; (4) and (3) PCR: the total reaction volume and reaction conditions vary depending on the kind of PCR used; (5) electrophoretic analysis: 8-L2 mu L PCR amplification product is electrophoresed on agarose gel, and whether specific amplification is brought or not is observed under ultraviolet light to judge the embryo sex. The two-temperature gradient multiplex PCR method of Wangman et al is a stable, reliable and quick method for identifying embryo sex [ Wangman et al, two-temperature gradient multiplex PCR technology for identifying early embryo sex of cattle Hereditas (Beijing), 2006, 3(28): 334-338 ]. The method can shorten the PCR amplification time to 38min, and the obtained PCR product is small, the difference between two bands is large, and the PCR product can be obviously distinguished in a short time during electrophoresis detection. Due to smaller product fragments and shortened amplification time, compared with the traditional PCR detection method, the detection time is greatly shortened, and the whole sex identification time can be shortened to 57 min. Can basically meet the requirements of rapidness and high efficiency required by embryo sex determination practice.

Hirayama in 2004 reported a Rapid Loop-mediated Isothermal Amplification (LAMP) [ Hirayama H, Kageyama et al, Rapid sequencing of bone transplantation activities using Loop-mediated Isothermal Amplification, 2004, 62(5): 887-896 ], and Loopamp company subsequently introduced its corresponding embryo sex determination kit and nephelometer. The method uses 4 different primers to carry out amplification reaction on a male specificity nucleic acid series and a male and female common nucleic acid series in the bovine embryonic cells, and identifies the sex of the bovine embryonic cells by detecting whether the amplification reaction exists or not. The presence or absence of the amplification reaction is determined by detecting the turbidity of a white precipitate formed by the by-product magnesium pyrophosphate obtained during the reaction. The method has the following advantages: (1) rapidly: the time from the embryo flushing to the identification is only 2-3 h, so that the in-vitro culture time of the embryo is greatly shortened; (2) and (3) precision: the kit has high specificity, male specificity and a male-female common primer are adopted, so that the error cutting can be eliminated to the maximum extent, but the kit has extremely sensitive reaction to DNA pollution, and a false positive result can occur once a very small amount of exogenous DNA is polluted [ Huanghe, Lixin, application of the loop-mediated isothermal amplification method for identifying the sex of the embryo in production, herbivorous livestock, 2004, (8): 42-43 ]; (3) the method is simple and convenient: the whole process of amplification, detection and judgment can be completed only by one Loopamp end-point turbidity meter without electrophoresis.

As is well known, the loop-mediated isothermal amplification method for determining the sex of a mammalian embryo has high sensitivity, is easy to be polluted, has strict requirements on the operation process, and can cause misjudgment only by mixing a very small amount of gene or other test product amplificates except a target gene.

However, as described above, embryos obtained by in vitro fertilization usually need to be obtained from embryo culture fluid, or the embryos undergo cryopreservation, and animal-derived substances, such as FBS, i.e., fetal bovine serum, are typically contained in the embryo culture fluid, the cryopreservation fluid (e.g., containing PBS + FBS + ethylene glycol + sucrose) used, and the embryo thawing fluid (e.g., containing PBS + FBS + sucrose) used before embryo sexing, which may seriously affect the accuracy of the loop-mediated isothermal amplification method. It has been found that the most common problems of the loop-mediated isothermal amplification method, which may be caused by such embryo culture solution, freezing solution and thawing solution, are that the result of the common male and female primer reaction is negative and needs to be re-detected.

Therefore, overcoming the adverse effect of animal-derived substances in embryo culture solution, embryo freezing solution and thawing solution on the loop-mediated isothermal amplification method for sex determination of early-stage bovine embryos remains an important problem for the technicians in the field.

Disclosure of Invention

The present invention is intended to provide a method for sex determination and embryo segmentation of early bovine embryos, and more particularly, to a method for sex determination and embryo segmentation of early bovine embryos using loop-mediated isothermal amplification (LAMP), such as morula sex determination and embryo segmentation of bovine embryos using loop-mediated isothermal amplification, which is expected to exhibit desirable technical effects. It has been surprisingly found that the method of the invention, as described herein, exhibits excellent technical results after treatment with a specific PBS solution prior to embryo dissection. The present invention has been completed based on such findings.

To this end, the present invention relates in a first aspect to a method for sexing and segmenting early bovine embryos, comprising the steps of:

(1) performing in vitro fertilization on the oocyte and the sperm, and culturing the fertilized ovum to obtain a morula;

(2) washing the sorted morula with a partitioning solution, then transferring into a PBS solution for pretreatment for 20min, then transferring into a partitioning liquid drop, placing under an inverted microscope, cutting (for example, at least 2 parts, for example, 2 parts) of embryo tissues containing 10-12 cells by using a partitioning knife as a sample for sex identification, dividing the rest part into two to obtain 2 semi-embryos, treating the semi-embryos in the partitioning liquid drop, then transferring into an embryo culture solution for culture until the morula develops into an expanded blastocyst for embryo transplantation; (3) providing a loop-mediated isothermal amplification real-time turbidity detection system and a loop-mediated isothermal amplification method sex determination kit, wherein the kit consists of a Master Mix I for male reaction, a Master Mix II for male-female common reaction, BstDNA polyrrerras, Extraction Solution and Control DNA (positive Control);

(4) sample preparation

Embryo sample: placing the cut embryo sample containing 6 μ l of the cutting Solution into a sterilized centrifuge tube, adding 12 μ l of DNA extract Solution to suspend, standing at room temperature for more than 5min, mixing, and centrifuging to obtain sample Solution;

negative control samples: placing 6 μ l of the split Solution into a sterilized centrifuge tube, adding 12 μ l of DNA extract Solution to suspend, standing at room temperature for more than 5min, mixing, and centrifuging to obtain negative control sample Solution;

positive control sample: placing 6 μ l of Control DNA into a sterilized centrifuge tube, adding 12 μ l of DNA extract Solution, standing at room temperature for more than 5min, mixing, and centrifuging to obtain positive Control sample Solution;

(5) prepare Master Mix

The preparation is carried out under the ice-bath condition,

master mix for male reactions (i.e. reaction mix I): putting 60 mul of Master Mix I into a sterilized centrifuge tube, adding 3 mul of BstDNA polyrrerras, uniformly mixing, and centrifuging to obtain the product;

master mix (reaction mixture II) was used for the hermaphroditic reaction: putting 60 mul of Master Mix II into a sterilized centrifuge tube, adding 3 mul of BstDNA polyrrerras, mixing uniformly, and centrifuging to obtain the product;

(6) sample mixing

Each sample is used for 2 reaction centrifuge tubes;

taking 6 reaction centrifuge tubes, respectively adding 20 mul of reaction mixed solution I into tubes No. 1, No. 2 and No. 3, and respectively adding 20 mul of reaction mixed solution II into tubes No. 4, No. 5 and No. 6;

taking 5 mul of each of the negative sample, the embryo sample and the positive Control (Control DNA) to be respectively added into No. 1, No. 2 and No. 3 reaction tubes, taking 5 mul of each of the negative sample, the embryo sample and the positive Control (Control DNA) to be respectively added into No. 4, No. 5 and No. 6 reaction tubes, fully mixing and centrifuging;

(7) loop-mediated isothermal amplification reaction, detection and result judgment

Placing 6 reaction centrifuge tubes in a loop-mediated isothermal amplification real-time turbidity detection system for amplification for 40min at 63 ℃, recording results indicated by "+" and "-" in each tube after the reaction is finished, and performing male and female judgment according to the following table:

under the premise that the results of the negative control and the positive control are both correct, the samples are judged to be male, female and uncertain to need to redo three types of conditions according to the results of the samples in the two reaction mixed liquids.

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, Chinese buffalo, Japan and cattle.

The method according to the first aspect of the present invention, wherein the step (1) is carried out as follows:

(11) collecting oocytes and maturing the oocytes in vitro;

(12) incubating the sperm suspension with the mature oocyte in a drop of fertilization culture to complete in vitro fertilization;

(13) after the in vitro fertilization procedure is completed, the embryos are cultured in an embryo culture solution until the morula grows well, and the morula is selected under a microscope.

According to the method of the first aspect of the present invention, in step (11), the collection of oocytes is performed as follows:

collecting living bodies: collecting ovum of cattle in vivo, picking up cumulus-oocyte complex (COCs) at least containing 3 layers of cumulus cells under a stereomicroscope, placing into mature culture medium containing HEPES, and transporting to laboratory at 38.8 deg.C for 3 hr.

According to the method of the first aspect of the present invention, in step (11), 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 invention, the step (12) 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.

According to the method of the first aspect of the present invention, step (13) is the in vitro culture and sorting of morula for embryos as follows:

after the in vitro fertilization operation is finished, the granular cells around the embryo are removed by an ovum-peeling needle, and are put into an embryo culture solution for culture, which is marked as the 1 st day of the embryo culture, the culture conditions are 38.8 ℃, 6% O2, 88% N2 and saturation humidity, and the mulberry embryo with good growth is selected under a stereo microscope on the 5 th day for the operation of embryo segmentation and/or sex identification.

The method according to the first aspect of the present invention, wherein the step (2) is divided as follows: in a sterile plate (for example, a sterile plastic plate with a diameter of 100 mm), making a plurality of split droplets of 100. mu.L each with the split solution; 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 cutter and the glass fixed tube are arranged on a micromanipulator, the embryo is fixed by the fixed tube, and the embryo tissue containing 10-12 cells is cut by the cutter to be used as a sample for sex identification.

The method according to the first aspect of the present invention, wherein the embryo is subjected to further culture and development of the embryo as a production embryo after division for sex determination and division of the remaining part into two.

The method according to the first aspect of the present invention, wherein the embryo is divided into two halves to obtain 2 semi-embryos after being divided for sex determination, the semi-embryos obtained by dividing are transferred into another clean divided droplet by a pipette and treated for 10min, then transferred into an embryo culture solution and cultured for 24h, the number of recovered semi-embryos is checked for 5-6 h, and whether morula develops into expanded blastocysts for embryo transfer is checked for 23-24 h.

The method according to the first aspect of the present invention, wherein the partitioning liquid is a sterilized aqueous solution comprising: 9.39g dipotassium hydrogen phosphate, 3.5g potassium dihydrogen phosphate, 68g sucrose, 12g povidone K30, and supplementing water for injection to 1000 ml. For example, the preparation method is as follows: 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 method according to the first aspect of the present invention, wherein in step (4), the sterilized centrifuge tube used for sample preparation is a sterilized 0.5ml centrifuge tube.

According to the method of the first aspect of the present invention, in the step (4), the DNA extraction solution is added during sample preparation, and then the sample is placed at room temperature for 5-6 min.

The method according to the first aspect of the present invention, wherein in step (5), the sterilized centrifuge tube used is a sterilized 1.5ml centrifuge tube.

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 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.

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 to 1000ml of 9.39g of dipotassium hydrogen phosphate, 3.5g of monopotassium phosphate, 20-30 g (for example, 25g) of fructose and 1.0-1.5 g (for example, 1.2g) of sodium caprylate, filtering, and sterilizing at 115 ℃ for 30 minutes.

According to the method of the first aspect of the invention, the sex determination Kit by the loop-mediated isothermal Amplification method is Loopamp DNA Amplification Kit.

According to the method of the first aspect of the invention, the loop-mediated isothermal amplification real-time turbidity detection system is a Loopamp LA-500 isothermal amplification real-time turbidity detection system.

Further, the invention provides a phosphate buffer solution which is PBS (phosphate buffer solution) for the sex determination of early bovine embryos, wherein the pH value of the phosphate buffer solution is 6.8-7.2 and the phosphate buffer solution 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 PBS solution of the second aspect of the invention has a pH of 6.8-7.2 and is prepared as follows: adding water to 1000ml of 9.39g of dipotassium hydrogen phosphate, 3.5g of monopotassium phosphate, 20-30 g (for example, 25g) of fructose and 1.0-1.5 g (for example, 1.2g) of sodium caprylate, filtering, and sterilizing at 115 ℃ for 30 minutes.

The PBS solution according to the second aspect of the present invention is used for pre-treating morulae in step (2) according to any embodiment of the first aspect of the present invention.

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.

The relevant principle of the loop-mediated isothermal amplification method for identifying the gender of the early-stage bovine embryo has been reported in detail. For example, in the Mingming literature (Mingming et al, research on the effect of the loop-mediated isothermal amplification method for identifying the sex of early-stage cow embryos, news of livestock ecology, 26(1):20), it is described that loop-mediated isothermal amplification is a novel DNA amplification method, which is characterized by simplicity, rapidness and strong specificity and has the possibility of replacing the PCR method. The method mainly utilizes 4 different specific primers to identify 6 specific regions of a target gene, and can carry out amplification reaction under isothermal condition. The amplification of the gene and the detection of the product can be finished in one step, the amplification efficiency is high, and the amplification can be carried out by 109-1010 times in 15-60 min; the specificity is high, and the detection of all target gene sequences can be distinguished only by the existence of amplification products. The presence or absence of amplification reaction is determined by detecting the turbidity of a white precipitate formed by the by-product magnesium pyrophosphate in the reaction process using a dedicated "Loopamp endpoint turbidity detector".

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. For example, the applicant's prior application CN2021105115124 invention details the method of embryo segmentation, the entire contents of which are incorporated herein by reference. It has been surprisingly found that minor modifications to the embryo segmentation method of the applicant's above-mentioned prior application can be effectively used for embryo sexing, and that the method of the present invention can be used for both sexing and embryo segmentation on the same embryo, with accuracy and reliability.

The embryo sexing 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, anhydrous calcium chloride 200.0mg/L, 0.7mg/L of ferric nitrate nonahydrate, 97.67mg/L of anhydrous magnesium sulfate, 400.0mg/L of potassium chloride, 6800.0mg/L of sodium chloride, 140.0mg/L of sodium dihydrogen phosphate monohydrate, 10.0mg/L of adenine sulfate, 0.2mg/L of adenosine 5' -phosphate, 1.0mg/L of adenosine triphosphate, 0.2mg/L of cholesterol, 1000.0mg/L of glucose, 0.5mg/L of deoxyribose, 0.05mg/L of reduced glutathione, 0.3mg/L of guanine hydrochloride, 0.354mg/L of sodium hypoxanthine, 20.0mg/L of phenol red, 0.5mg/L of ribose, 50.0mg/L of sodium acetate, 0.3mg/L of thymine, 20.0mg/L of 80, 0.3mg/L of uracil, 0.3mg/L of sodium xanthine, 0.3mg/L of FSH, 0.01IU/mL of LH, 1 mug/mL of E₂50ng/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.

A partitioning solution which 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 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.

A sex determination Kit by a loop-mediated isothermal Amplification method (Loopamp DNA Amplification Kit, Japan research, which consists of Master Mix I for male reaction, Master Mix II for male and female common reaction, BstDNA polyrrerras, Extraction Solution and Control DNA (positive Control)).

A loop-mediated isothermal amplification real-time turbidity detection system (Loopamp LA-500 isothermal amplification real-time turbidity detection system, EIKEN CHEMICAL CO., LTD).

The remaining reagents were purchased from Sigma-Aldrich as not specified.

Example 1: method for segmenting bovine in vitro fertilization embryo (in vivo egg taking)

Cattle for testing: chinese cattle (south-yang cattle, working variety).

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

Collecting living bodies: collecting ovum of cattle in vivo, picking up cumulus-oocyte complex (COCs) at least containing 3 layers of cumulus cells under a stereomicroscope from the obtained follicular fluid, and putting into mature culture solution containing HEPES at 38.8 deg.C for 3h for transporting to laboratory;

washing 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

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

Step (4), morula segmentation and development of segmented embryos

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); mounting an embryo cutter and a glass fixed tube on a micromanipulator, fixing the embryo by using the fixed tube, and cutting an embryo tissue containing 10-12 cells by using the cutter to be used as a sample for sex determination [ according to the method, 6-8 parts of sample can be cut for each morula to be used for methodology investigation of sex determination; however, when the sex-identified embryo needs to be obtained or the embryo needs to be cut, two embryo tissues containing 10-12 cells can be cut from one embryo according to the method for sex identification, and the rest part is divided into two parts to be continuously cultured and developed, so that the embryo for production is obtained.

Development of the split embryos: after two morula division samples are used for sex determination, the remaining part is divided into two parts to obtain 2 semi-embryos, the semi-embryos obtained by division are moved into another clean division liquid drop by a straw to be treated for 10min, then the semi-embryos are moved into an embryo culture solution to be cultured (38.8 ℃, 6% O2, 88% N2 and saturation humidity) for 24h, the number of semi-embryo recovery is checked during 5-6 h (namely the division success number is used for calculating the division success rate), and whether the morula develops to an expansion blastocyst is checked during 23-24 h (if the morula develops to the expansion blastocyst successfully, the development success rate can be used for embryo transplantation, so that the blastocyst development rate is calculated). 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, potassium dihydrogen phosphate 3.5g, fructose 25g and sodium caprylate 1.2g to make 1000ml, filtering, and sterilizing at 115 deg.C for 30 min.

Step (5), sample preparation and Master Mix preparation

Embryo sample: placing the cut embryo sample into a sterilized 0.5ml centrifuge tube, keeping the liquid volume of the cutting liquid about 6 mul when transferring the embryo sample, adding 12 mul of DNA extract Solution to suspend the embryo sample, standing at room temperature for more than 5min (5-6 min during specific operation), mixing uniformly, and centrifuging to obtain a sample Solution for later use.

Negative control samples: placing 6 μ l of the partition liquid into a sterilized 0.5ml centrifuge tube, adding 12 μ l of DNA extract Solution to suspend, standing at room temperature for more than 5min (5-6 min for specific operation), mixing, and centrifuging to obtain negative control sample Solution;

positive control sample: placing 6 μ l of Control DNA into a sterilized 0.5ml centrifuge tube, adding 12 μ l of DNA extract Solution, suspending, standing at room temperature for more than 5min (5-6 min for specific operation), mixing, and centrifuging to obtain positive Control sample Solution;

the preparation is carried out under the ice-bath condition,

master mix for male reactions (i.e. reaction mix I): placing 60 μ l Master Mix I in a sterilized 1.5ml centrifuge tube, adding 3 μ l BstDNA polyrrerras, mixing, and centrifuging to obtain the final product;

master mix (reaction mixture II) was used for the hermaphroditic reaction: 60 mul of Master Mix II is put into a sterilized 1.5ml centrifuge tube, 3 mul of BstDNA polyrrerrase is added, mixed evenly and centrifuged, thus obtaining the product.

Step (6), mixing the samples

2 reaction centrifuge tubes (for male reaction and female reaction) are used for each sample;

taking 6 reaction centrifuge tubes, respectively adding 20 μ l of reaction mixed solution I into the first 3 reaction tubes (tubes 1, 2 and 3) and respectively adding 20 μ l of reaction mixed solution II into the last 3 reaction tubes (tubes 4, 5 and 6);

adding 5 μ l of each of the negative sample, the embryo sample and the positive Control (Control DNA) into No. 1, No. 2 and No. 3 reaction tubes, adding 5 μ l of each of the negative sample, the embryo sample and the positive Control (Control DNA) into No. 4, No. 5 and No. 6 reaction tubes, mixing thoroughly and centrifuging.

Step (7), loop-mediated isothermal amplification reaction, detection and result judgment

sex determination was performed using the method of example 1, and 107 samples obtained by dividing 15 morulae were measured, and the results were: the results of the negative control group and the positive control group are both correct, 50 samples cut from 7 embryos are all male results, 57 samples cut from the rest 8 embryos are all female results, and two uncertain and redo results with reaction mixture II as "-" do not occur.

Example 2: method for segmenting bovine in vitro fertilization embryo (in vivo egg taking)

Cattle for testing: holstein cattle (dairy cattle breed).

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

step (2), in vitro fertilization

step (3), culturing embryo in vitro and sorting morula

Step (4), morula segmentation and development of segmented embryos

Development of the split embryos: after two morula division samples are used for sex determination, the remaining part is divided into two parts to obtain 2 semi-embryos, the semi-embryos obtained by division are moved into another clean division liquid drop by a straw to be treated for 10min, then the semi-embryos are moved into an embryo culture solution to be cultured (38.8 ℃, 6% O2, 88% N2 and saturation humidity) for 24h, the number of semi-embryo recovery is checked during 5-6 h (namely the division success number is used for calculating the division success rate), and whether the morula develops to an expansion blastocyst is checked during 23-24 h (if the morula develops to the expansion blastocyst successfully, the development success rate can be used for embryo transplantation, so that the blastocyst development rate is calculated).

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, potassium dihydrogen phosphate 3.5g, fructose 25g and sodium caprylate 1.2g to make 1000ml, filtering, and sterilizing at 115 deg.C for 30 min. Step (5), sample preparation and Master Mix preparation

the preparation is carried out under the ice-bath condition,

Step (6), mixing the samples

6 reaction centrifuge tubes were placed in a loop-mediated isothermal amplification real-time turbidity detection system for amplification at 63 ℃ for 40min, and after the reaction was completed, the results indicated by "+" and "-" in each tube were recorded, and male and female determinations were performed according to the criteria shown in the table of example 1.

Sex determination was performed using the method of example 2, and a total of 139 samples obtained by dividing 20 morulae were measured, with the results: the results of the negative control group and the positive control group are both correct, 71 samples cut from 10 embryos are all male results, 68 samples cut from the rest 10 embryos are all female results, and two uncertain and redo results with reaction mixture II as "-" do not occur.

Example 3: method for segmenting bovine in vitro fertilization embryo (in vivo egg taking)

Cattle for testing: simmental cattle (beef variety).

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

step (2), in vitro fertilization

step (3), culturing embryo in vitro and sorting morula

Step (4), morula segmentation and development of segmented embryos

the preparation is carried out under the ice-bath condition,

Step (6), mixing the samples

Sex determination was performed using the method of example 3, and 121 samples obtained by dividing 17 morulae were measured, and the results were: the results of the negative control group and the positive control group are both correct, 65 samples cut from 9 embryos are all male results, 56 samples cut from the rest 8 embryos are all female results, and two uncertain and redo results with reaction mixture II as "-" do not occur.

Example 4: method for segmenting bovine in vitro fertilization embryo (in vivo egg taking)

Cattle for testing: chinese buffalo (used breed).

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

step (2), in vitro fertilization

step (3), culturing embryo in vitro and sorting morula

Step (4), morula segmentation and development of segmented embryos

the preparation is carried out under the ice-bath condition,

Step (6), mixing the samples

Sex determination was performed using the method of example 4, and 125 samples obtained by dividing 18 morulae were measured, and the results were: the results of the negative control group and the positive control group are both correct, 56 samples cut from 8 embryos are all male results, 69 samples cut from the rest 10 embryos are all female results, and two uncertain and redo results with reaction mixture II as "-" do not occur.

Example 5: method for segmenting bovine in vitro fertilization embryo (in vivo egg taking)

Cattle for testing: japan and cattle (beef breeds).

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

step (2), in vitro fertilization

step (3), culturing embryo in vitro and sorting morula

Step (4), morula segmentation and development of segmented embryos

the preparation is carried out under the ice-bath condition,

Step (6), mixing the samples

Sex determination was performed using the method of example 5, and 142 samples obtained by dividing 20 morulae were measured, and the results were: the results of the negative control group and the positive control group are both correct, 78 samples cut from 11 embryos are all male results, 64 samples cut from the rest 9 embryos are all female results, and two uncertain and redo results with reaction mixture II as "-" do not occur.

Example 6: development of divided embryos

Using the method of examples 1-5, continuing to the morula segmentation of step (4), after cutting 2 samples from each morula for sex identification, dividing the rest into two halves to obtain two half-embryos, and continuing to culture and develop the embryos; transferring the semi-embryo into another clean split liquid drop with a pipette for 10min, and culturing in embryo culture solution (38.8 deg.C, 6% O2, 88% N)₂Saturation humidity) for 24h, checking the number of semi-embryo recovery (i.e. the number of successful segmentation, used for calculating the success rate of segmentation) in the period of 5-6 h, checking whether the morula grows to the expansion blastula in the period of 23-24 h (if the success rate occurs)When the expanded blastocyst is developed, the successful development can be used for embryo transplantation, and the blastocyst development rate is calculated).

In this example, the effect of the embryo sex determination and embryo segmentation method was examined; 31, 29, 27, 32 and 34 morulae were examined by the methods of examples 1 to 5, and the identified sexes were (male/female) 15/16, 15/14, 13/14, 16/16 and 16/18, respectively, and the results of the negative control group and the positive control group were both correct, and neither of the two "uncertain" and "redo" results, in which the reaction mixture II was "-", occurred; continuously culturing and developing embryos of the semi-embryos obtained by segmenting the morula subjected to sex identification, and inspecting and calculating the segmentation success rate (%) and the blastocyst development rate (%) after segmentation; the results of examples 1 to 5 are as follows: the success rate (%, mean) of the segmentation in examples 1 to 5 were: 57.3%, 61.2%, 51.6%, 60.3%, 57.7%; the blastocyst development rates (%, mean) of examples 1 to 5 were: 51.4%, 50.6%, 49.2%, 54.2%, 53.3%. In other comparative experiments, referring to the methods of examples 1 to 5, the morula was divided into two halves directly without sex determination sampling, and development of the divided embryos was performed by the same method, and the division success rate (%) and blastocyst development rate (%) of each of the 16 to 19 morula division cultures were 54 to 62% and 49 to 54%. These results show that the semi-embryos obtained by continuously dividing morula subjected to sex determination, division and sampling in the above embodiments 1-5 of the present invention into two have good division success rate and blastocyst development rate, which indicates that the sex determination method of the present invention does not have adverse effects on embryos.

Example 7: embryo sex identification

Sex determination was performed by referring to the methods of examples 1 to 5, respectively, except that fructose was not added to the PBS solution used in the operation. In reference example 1, a total of 103 samples obtained by dividing 15 morulae were measured, and the results were: the results of the negative control group and the positive control group were correct, 16 samples showed uncertain and redo results with reaction mixture I of "+" and reaction mixture II of "-", and the remaining 87 samples were male or female. In reference example 2, a total of 92 samples obtained by dividing 13 morulae were measured, and the results were: the results of the negative control group and the positive control group were correct, 18 samples showed uncertain and redo results with reaction mixture I of "+" and reaction mixture II of "-", and the remaining 74 samples were male or female. In reference example 3, a total of 91 samples obtained by dividing 13 morulae were measured, and the results were: the results of the negative control group and the positive control group were correct, 14 samples showed the uncertain and redo results with the reaction mixture I being "+" and the reaction mixture II being "-", and the remaining 77 samples were male or female. In reference example 4, a total of 113 samples obtained by dividing 16 morulae were measured, and the results were: the results of the negative control group and the positive control group were correct, 19 samples showed the uncertain and redo results with the reaction mixture I being "+" and the reaction mixture II being "-", and the remaining 94 samples were male or female. In reference example 5, a total of 101 samples obtained by dividing 14 morulae were measured, and the results were: the results of the negative control group and the positive control group were correct, 20 samples showed the results of uncertain and redo with the reaction mixture I of "+" and the reaction mixture II of "-", and the remaining 81 samples were male or female. As can be seen from the above results, if fructose is not added to the PBS solution, there is a probability of about 16 to 20% of the uncertain and redo results that the reaction mixture I is "+" and the reaction mixture II is "-", but there are no uncertain and redo results that the reaction mixture I is "-" and the reaction mixture II is "-".

Example 8: embryo sex identification

Sex determination was performed by referring to the methods of examples 1 to 5, respectively, except that no sodium caprylate was added to the PBS solution used in the procedure. In reference example 1, a total of 114 samples obtained by dividing 16 morulae were measured, and the results were: the results of the negative control group and the positive control group were correct, 25 samples showed the uncertain and redo results that the reaction mixture I was "+" and the reaction mixture II was "-", and the remaining 89 samples were male or female. In reference example 2, a total of 97 samples obtained by dividing 14 morulae were measured, and the results were: the results of the negative control group and the positive control group were correct, 23 samples showed the results of uncertain and redo with reaction mixture I of "+" and reaction mixture II of "-", and the remaining 74 samples were male or female. In reference example 3, a total of 90 samples obtained by dividing 13 morulae were measured, and the results were: the results of the negative control group and the positive control group were correct, 18 samples showed the uncertain and redo results that the reaction mixture I was "+" and the reaction mixture II was "-", and the remaining 72 samples were male or female. In reference example 4, 99 samples obtained by dividing 14 morulae were measured, and the results were: the results of the negative control group and the positive control group were correct, 22 samples showed uncertain and redo results with the reaction mixture I being "+" and the reaction mixture II being "-", and the remaining 77 samples were male or female. In reference example 5, a total of 97 samples obtained by dividing 14 morulae were measured, and the results were: the results of the negative control group and the positive control group were correct, 22 samples showed the uncertain and redo results that the reaction mixture I was "+" and the reaction mixture II was "-", and the remaining 75 samples were male or female. As can be seen from the above results, if sodium caprylate is not added to the PBS solution, there is a probability of about 20 to 24% of the uncertain and redo results that the reaction mixture I is "+" and the reaction mixture II is "-", but there are no uncertain and redo results that the reaction mixture I is "-" and the reaction mixture II is "-".

Example 9: embryo sex identification

Sex determination was performed by referring to the methods of examples 1 to 5, except that neither fructose nor sodium caprylate was added to the PBS solution used in the procedure. In reference example 1, a total of 106 samples obtained by dividing 15 morulae were measured, and the results were: the results of the negative control group and the positive control group were correct, 23 samples showed the results of uncertain and redo with the reaction mixture I of "+" and the reaction mixture II of "-", and the remaining 61 samples were male or female. In reference example 2, a total of 97 samples obtained by dividing 14 morulae were measured, and the results were: the results of the negative control group and the positive control group were correct, 23 samples showed the uncertain and redo results that the reaction mixture I was "+" and the reaction mixture II was "-", and the remaining 76 samples were male or female. In reference example 3, a total of 113 samples obtained by dividing 16 morulae were measured, and the results were: the results of the negative control group and the positive control group were correct, 25 samples showed uncertain and redo results with reaction mixture I of "+" and reaction mixture II of "-", and the remaining 68 samples were male or female. In reference example 4, 93 samples obtained by dividing 13 morulae were measured, and the results were: the results of the negative control group and the positive control group were correct, 21 samples showed uncertain and redo results with the reaction mixture I being "+" and the reaction mixture II being "-", and the remaining 77 samples were male or female. In reference example 5, a total of 111 samples obtained by dividing 16 morulae were measured, and the results were: the results of the negative control group and the positive control group were correct, 24 samples showed the uncertain and redo results that the reaction mixture I was "+" and the reaction mixture II was "-", and the remaining 83 samples were male or female. From the above results, it can be seen that there is a probability of about 21-24% of uncertain and redo results in which the reaction mixture I is "+" and the reaction mixture II is "-" in the PBS solution, but there are no uncertain and redo results in which the reaction mixture I is "-" and the reaction mixture II is "-".

In other experiments, referring to the methods of examples 1 to 5, except that the amount of fructose in PBS was adjusted to be in the range of 20 to 30g/1000ml, each of 81 to 114 divided samples was subjected to sex determination test, and the results were as follows: the results of the negative control group and the positive control group are both correct, all samples show male or female results, and two uncertain and redo results with the reaction mixed liquor II as negative do not appear. It is shown that fructose can be used in a wide range in the methods of examples 1 to 5 of the present invention.

In other experiments, with reference to the methods of examples 1 to 5, except that the amount of sodium caprylate in PBS was adjusted to be in the range of 1.0 to 1.5g/1000ml, 86 to 114 divided samples were subjected to sex determination tests per test, and the results were: the results of the negative control group and the positive control group are both correct, all samples show male or female results, and two uncertain and redo results with the reaction mixed liquor II as negative do not appear. It is shown that the sodium caprylate in the methods of examples 1-5 of the present invention can be used in a wide range.

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

1. The method for sex determination and embryo segmentation of early cattle embryos comprises the following steps:

(4) sample preparation

(5) prepare Master Mix

The preparation is carried out under the ice-bath condition,

(6) sample mixing

Each sample is used for 2 reaction centrifuge tubes;

2. The method of claim 1, wherein:

the cattle are selected from: chinese cattle, Holstein cattle, Simmental cattle, Chinese buffalo, Japan and cattle;

the step (1) is carried out as follows: (11) collecting oocytes and maturing the oocytes in vitro; (12) incubating the sperm suspension with the mature oocyte in a drop of fertilization culture to complete in vitro fertilization; (13) 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;

in the step (11), the collection of oocytes is carried out in the following manner: collecting living bodies: collecting ovum of cattle in vivo, picking up cumulus-oocyte complex (COCs) at least containing 3 layers of cumulus cells under a stereomicroscope from the obtained follicular fluid, and putting into mature culture solution containing HEPES at 38.8 deg.C for 3h for transporting to laboratory;

in step (11), the oocyte in vitro maturation is performed as follows: washing the collected COCs 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 to ensure that the oocyte is matured in vitro;

the in vitro fertilization of step (12) is carried out in the following manner:

3. The method according to claim 1, step (13) is that of culturing and sorting morula in vitro of embryos in the following manner:

4. The method of claim 1, wherein step (2) is segmented as follows: in a sterile plate (for example, a sterile plastic plate with a diameter of 100 mm), making a plurality of split droplets of 100. mu.L each with the split solution; 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 cutter and the glass fixed tube are arranged on a micromanipulator, the embryo is fixed by the fixed tube, and the embryo tissue containing 10-12 cells is cut by the cutter to be used as a sample for sex identification.

5. The method of claim 1, wherein:

after the embryo is divided for sex identification, the rest part is divided into two parts for continuous culture and development of the embryo to be used as the embryo for production;

after the embryo is divided for sex identification, dividing the rest part into two parts to obtain 2 semi-embryos, transferring the semi-embryos obtained by division into another clean divided liquid drop by using a suction pipe for treatment for 10min, then transferring the semi-embryos into an embryo culture solution for culture for 24h, checking the number of recovered semi-embryos during 5-6 h, and checking whether morula develops into an expansion blastula for embryo transplantation during 23-24 h;

the cutting fluid is a sterilized 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;

in the step (4), the sterilized centrifugal tube used for sample preparation is a sterilized 0.5ml centrifugal tube;

in the step (4), DNA extracting solution is added during sample preparation and then placed for 5-6 min at room temperature;

in the step (5), the sterilized centrifuge tube is a sterilized 1.5ml centrifuge tube.

6. The method of claim 1, wherein the partitioning fluid 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 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 transport medium comprising HEPES, formulated as described in the detailed description;

the double-resistant normal saline is normal saline containing 400IU/mL of penicillin and 400 mu g/mL of streptomycin;

the egg washing liquid is BY basal culture solution added with 3mg/mL bovine serum albumin;

the maturation culture solution is BY basal culture solution added with 100mL/L FBS, 10 μ g/mL FSH, 10 μ g/mL LH, 1 μ g/mL E2 and 20ng/mL EGF;

the mature culture solution containing HEPES is BY basal culture solution added with 15 mmol/L HEPES, 100mL/L FBS, 10 mu g/mL FSH, 10 mu g/mL LH, 1 mu g/mL E2 and 20ng/mL EGF;

the fertilization culture solution, 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;

the semen preparation culture solution 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 embryo culture solution is prepared according to the specific embodiment;

the BY basal culture solution has the formula as described in the specific embodiment.

7. The method according to claim 1, wherein the PBS is phosphate buffer solution, has a pH of 6.8-7.2 and is prepared as follows: adding water into 9.39g of dipotassium hydrogen phosphate and 3.5g of monopotassium phosphate to make the volume of the mixture reach 1000ml, filtering the mixture, and sterilizing the mixture for 30 minutes at 115 ℃; or

The PBS solution is phosphate buffer solution, the pH value of which is 6.8-7.2 and is prepared according to the following method: adding water to 1000ml of 9.39g of dipotassium hydrogen phosphate, 3.5g of monopotassium phosphate, 20-30 g (for example, 25g) of fructose and 1.0-1.5 g (for example, 1.2g) of sodium caprylate, filtering, and sterilizing at 115 ℃ for 30 minutes.

8. The method according to claim 1, wherein the sex determination Kit by the loop-mediated isothermal Amplification method is Loopamp DNA Amplification Kit; or, the loop-mediated isothermal amplification real-time turbidity detection system is a Loopamp LA-500 isothermal amplification real-time turbidity detection system.

9. A PBS (phosphate buffer solution) for sex determination of early bovine embryos 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.

10. The PBS solution of claim 9, which has a pH of 6.8 to 7.2 and is prepared as follows: adding water into 9.39g of dipotassium hydrogen phosphate, 3.5g of monopotassium phosphate, 20-30 g (such as 25g) of fructose and 1.0-1.5 g (such as 1.2g) of sodium caprylate to make the mixture 1000ml, filtering, and sterilizing at 115 ℃ for 30 minutes; for example, it is used for the pre-treatment of morula in step (2) according to any of the embodiments of the first aspect of the present invention.