CN103215220A - Method for producing dzo gender controllable in vitro embryos - Google Patents

Abstract

The invention discloses a method for producing dzo gender controllable in vitro embryos. The method comprises the steps of: removing connective tissues surrounding a Bos grunniens ovary and conducting cleaning, selecting oocytes and washing them; transferring the washed oocytes into a culture dish added with serum, hormone, a follicular fluid and a Hepes buffer solution, and then placing the culture dish into an incubator to perform maturation culture; taking X or Y chromosome-containing sperms from Bos taurus, separating dead sperms from live sperms in semen by a Percoll gradient separation liquid; picking the oocytes that are cultured to maturity from the Hepes buffer solution, washing them with a fertilization fluid, then adding the X or Y chromosome-containing sperms into oocyte-containing fertilization microdroplets, and placing the microdroplets into the incubator to conduct in vitro fertilization culture; after fertilization, carrying out a blastocyst maturation treatment; and at a mature time, recovering the blastocyst, and performing embryo transplantation or storage. By combining the method provided in the invention and other modern embryo production technologies, gender controllable dzo breeding can be carried out, and the number of high quality dzos can be increased rapidly.

Description

A method for producing in vitro embryos with controllable gender

technical field

The invention relates to a method for producing in vitro embryos of Y cattle, in particular to a method for producing in vitro embryos of Y cattle with controllable sex.

Background technique

Pyak is the offspring of common cattle Bos taurus (♂) as the male parent and yak Bos grunniens (♀) as the female parent. , In addition, it has a long service life and a high market price. It is an important supplement to yak production and is very popular among people. In addition, Pyak plays an extremely important role in people's production and life: First, the castrated male Pyak not only has the same adaptability to the plateau climate and environment as yaks, but also has the advantages of being more docile and easy to control. Therefore, in pastoral areas, P cattle are often used as pack cattle, and in semi-agricultural and semi-pastoral areas or agricultural areas, they are used as plow cattle for plowing; second, for milk, compared with yaks, the milk production of female P cattle It will increase exponentially, and the milk production cycle will be long, which can better meet the increasing demand for milk from herdsmen or large and medium cities in the Qinghai-Tibet Plateau. The problem of competing with yak calves for yak milk, on the other hand, can also meet the demand for milk from urban population growth to a greater extent; third, for meat, male P cattle have the characteristics of fast growth and high meat production. Cattle can either graze in pastoral areas, or take advantage of rich forage resources such as crop stalks in semi-agricultural and semi-pastoral areas or agricultural areas to provide people with a large amount of beef.

The use of in vitro fertilization to produce fine bovine embryos has been reported in the literature at home and abroad. However, there is no method for using in vitro fertilization to carry out interspecific hybridization to produce in vitro embryos with controllable sex.

Contents of the invention

The object of the present invention is to provide a method for producing in vitro embryos with controllable gender in order to solve the above problems. This method utilizes the combination of sexually controlled semen, in vitro fertilization and interspecies hybridization, a new animal breeding technology, to produce gendered embryos. Pyak embryos are used to control the sex of Pyak, and at the same time, it also provides technical support for the factory and gendered production of high-quality sex-controlled Pyak embryos.

In order to achieve the above object, the present invention adopts the following technical solutions:

The method for producing the sex-controllable in vitro embryos of the present invention comprises the following steps:

(1) After removing the surrounding connective tissue from the yak ovary, rinse it with sterile saline for 3 to 4 times, extract the oocytes from the surface of the cleaned yak ovary, place the oocytes in the collection solution, and examine them under a stereomicroscope Select oocytes, wash the selected oocytes with the collection solution for 2 to 3 times, and then wash with the maturation medium once;

(2) Transfer the washed oocytes into a petri dish added with serum, hormones, follicular fluid and Hepes buffer, and then put the petri dish into an incubator for maturation culture;

(3) Take fresh ordinary bovine sperm containing X or Y chromosomes separated by flow cytometry or common bovine sperm containing X or Y chromosomes thawed at 37°C, and separate the dead sperm in the semen through Percoll gradient separation medium separated from live sperm;

(4) Pick out the mature oocytes cultured in step (2) from the Hepes buffer, wash them twice with fertilization fluid, and then transfer them into fertilization droplets made with fertilization fluid. After dilution, the sperm will contain X or The sperm of the Y chromosome is added to the fertilization droplet containing the oocyte, and placed in an incubator for in vitro fertilization;

(5) In vitro fertilization and culture until the blastocysts are matured after fertilization;

(6) Recover blastocysts on the 6th, 7th, and 8th days after fertilization when the blastocysts are mature, and if there is a suitable recipient cow, perform embryo transfer; otherwise, cryopreserve the well-developed high-quality sex-controlled embryos , and then stored in a liquid nitrogen tank for future embryo transfer.

As a preference, in the step (1), the yak ovaries are taken from the slaughterhouse, and the yak ovaries are recovered from the slaughterhouse and placed in sterile saline at 30°C, brought back to the laboratory as soon as possible with a thermos bottle, and injected with a No. 8 syringe , extract oocytes from follicles with a diameter of 2-8mm on the surface of the ovary, and select oocytes with more than three layers of dense granulosa cells, bright appearance and uniform cytoplasm under a stereo microscope; the collection solution is buffered with Hepes, The oocyte collection solution of TCM199 was added with 5mmol/L NaHCO ₃ and 3% OCS (estrous calf serum).

In the step (2), the maturation culture medium used in the maturation culture is Hepes-buffered TCM199+5%OCS+0.1μg/mLFSH (follicle-stimulating hormone)+3%BFF (bovine follicle fluid). The culture solution was 1 mL, the culture time of the mature culture was 21 to 24 hours, and the conditions in the incubator were 39° C., 5% CO ₂ air and saturated humidity.

In the step (3), the method of separating the dead sperm and the live sperm in the semen is as follows: put the semen into a centrifuge tube with a gradient of 2mL90%:2mL45%Percoll, centrifuge at 700Xg for 20 minutes, discard the supernatant, and add 1mL of the fertilized fluid Centrifuge at 400×g for 5 minutes, remove the supernatant suspension, and leave about 50 μL of semen; the common cattle are Holstein cattle, Simmental cattle, Charolais cattle, Jersey cattle, Hereford cattle, and Western yellow hybrid cattle ( The offspring of Simmental cattle and Tibetan yellow cattle) or Dutch yellow hybrid cattle (the offspring of Holstein cattle and Tibetan yellow cattle crossbreeds).

In the step (4), the fertilization droplet is 20 μL, and 16 to 18 oocytes are placed in each drop. After the sperm motility meets the requirements under a microscope, the sperm is added to the fertilization droplet to make the final concentration up to 2X106/mL; incubate in an incubator at 39°C and 5% CO ₂ saturated humidity for 6-8 hours.

In the step (5), the blastocyst maturation treatment method is as follows: after fertilization, use a pipette to repeatedly blow the assumed fertilized egg in the fertilized droplet, remove part of the cumulus cells, and then remove the assumed fertilized egg from the fertilized droplet. Removed from the medium, washed twice with in vitro culture medium and transferred to a co-culture system containing cattle granulosa cells for cultivation, the culture medium was Hepes-buffered TCM199+10% NCS (newborn calf serum)+0.11mg/mL sodium pyruvate, Add 25 μL of culture solution per drop, put 50 fertilized eggs, and culture in vitro for 6-8 days under the saturated humidity conditions of 39°C, 5% CO ₂ , 5% O ₂ , and 90% N ₂ , and replace them every two days during the culture period. Liquid once, replace 1/2 liquid each time.

The beneficial effects of the present invention are:

Combining the present invention with other modern embryo production techniques can carry out the breeding of sex-controlled P cattle, and rapidly increase the number of high-quality P cattle; since the current accuracy of separating sperm containing X and Y chromosomes is more than 90%, using the method containing More than 90% of the offspring of embryos fertilized with X-chromosomal sperm are cows, and more than 90% of the embryos fertilized with Y-chromosome-containing sperm are bulls, so gendering can be done according to needs. Production.

Detailed ways

Below in conjunction with specific embodiment the present invention is described in further detail:

Yak ovaries were collected from the slaughterhouse, placed in sterile saline at 30°C, and brought back to the laboratory as soon as possible with a thermos bottle. Wash the ovaries with sterile normal saline for 3 to 4 times, and extract oocytes from follicles with a diameter of 2 to 8 mm on the surface of the ovary with a No. 8 syringe. Under a stereo microscope, select oocytes with more than three layers of dense granulosa cells, bright appearance, and uniform cytoplasm, and then use Hepes buffered oocytes with TCM199 supplemented with 5mmol/L NaHCO3 and 3% OCS (estrous bovine serum) The collected solution was washed 2 to 3 times, and then washed once with Hepes-buffered TCM-199+5%OCS+0.1μg/mL FSH (follicle-stimulating hormone)+3%BFF (bovine follicle fluid) maturation culture medium. Transfer the washed oocytes into a petri dish added with serum, hormone, follicular fluid and Hepes buffer, and then put the petri dish into an incubator for maturation culture. The maturation solution of each petri dish is 1mL, put Oocytes were cultured for 21-24 hours at 39°C, 5% CO2 air and saturated humidity.

Take fresh common bovine sperm containing X or Y chromosomes separated by flow cytometry or common bovine sperm containing X or Y chromosomes thawed at 37°C, and separate the dead sperm and live sperm in the semen by Percoll gradient separation medium Separate, the method is: put the semen into a centrifuge tube with 2mL90%:2mL45%Percoll gradient, centrifuge at 700Xg for 20 minutes, discard the supernatant, add 1mL of fertilized fluid and centrifuge at 400Xg for 5 minutes, remove the supernatant suspension, leaving about 50μL semen. The common cattle are Holstein cattle, Simmental cattle, Charolais cattle, Jersey cattle, Hereford cattle, Xihuang hybrid cattle (the offspring of Simmental cattle and Tibetan yellow cattle) or Dutch yellow hybrid cattle (Dutch cattle) The offspring of Stan cattle and Tibetan yellow cattle).

Pick out the cultured mature oocytes from the Hepes buffer, wash them twice with fertilization solution, and then transfer them into fertilization microdrops made of fertilization fluid. Mother cells, after the sperm motility is checked under a microscope to meet the requirements, the sperm is added to the fertilization microdroplet to make the final concentration reach 2X106/mL, after the sperm is diluted, the sperm containing X or Y chromosome is added to the fertilization microdroplet containing the oocyte In vitro fertilization was carried out in an incubator at 39°C and a saturated humidity of 5% CO ₂ for 6-8 hours.

After fertilization, the putative fertilized eggs were blown and blown repeatedly with a straw in the fertilized droplet to remove part of the cumulus cells, and then the putative fertilized eggs were removed from the fertilized droplet, washed twice with in vitro culture medium and transferred to a co-culture containing cattle granulosa cells. Cultivate in a culture system, the culture medium is Hepes-buffered TCM199 + 10% NCS (newborn calf serum) + 0.11 mg/mL sodium pyruvate, add 25 μL of culture medium per drop, put 50 fertilized eggs, and store at 39 °C, Under the saturated humidity conditions of 5% CO ₂ , 5% O ₂ , and 90% N ₂ , culture in vitro for 6-8 days, and change the medium every two days during the culture period, and replace 1/2 of the solution each time.

On the 6th, 7th, and 8th days after fertilization, when the blastocysts are mature, the blastocysts are recovered, and if there is a suitable recipient cow, embryo transfer is performed; otherwise, the well-developed high-quality sex-controlled embryos are cryopreserved, and then placed in Store frozen in a liquid nitrogen tank at -196°C, and then transplant when the conditions of the cow are mature.

Claims (6)

1. produce pien niu controllability complicated variant embryo's method outward for one kind, it is characterized in that: may further comprise the steps:

(1) with behind the reticular tissue around the removal of yak ovary, with stroke-physiological saline solution flushing 3～4 times, from the yak ovary surface extraction ovocyte that cleans up, ovocyte is placed collection liquid, under stereoscopic microscope, choose ovocyte, ovocyte after choosing with collecting liquid washing 2～3 times, is washed 1 time with maturation culture solution again;

(2) ovocyte after will washing changes in the culture dish that is added with serum, hormone, liquor folliculi and Hepes damping fluid, culture dish is put into incubator again and is carried out maturation and cultivate;

(3) get by sperm that contains X or Y chromosome of the isolating fresh common ox of flow cytometer or the sperm that contains X or Y chromosome of 37 ℃ of common oxen of thawing, the Necrospermia in the seminal fluid and the sperm of living are separated by Percoll gradient separations liquid;

(4) sort out from the Hepes damping fluid cultivating sophisticated ovocyte in the step (2), change over to after 2 times with in the fertilization droplet that made by seminal fluid with washed by seminal fluid, the sperm that will contain X or Y chromosome after the sperm dilution joins and contains in the fertility of oocytes droplet, puts into incubator and carries out cultivation in vitro fertilization;

(5) after fertilization that is cultured in vitro fertilization carries out the ripe processing of blastaea;

(6) reclaim blastaea at after fertilization the 6th, the 7th with when treating on the 8th day that blastaea was ripe,, then carry out embryo transfer if any suitable recipient cattle; Handle otherwise well-developed quality control embryo is carried out freezing preservation, place liquid nitrogen container to store then, be provided with the back embryo transfer and use.

2. the outer embryo's of production pien niu controllability complicated variant according to claim 1 method, it is characterized in that: in the described step (1), the yak ovary is taken from the slaughterhouse, the slaughterhouse is reclaimed the yak ovary and is placed in 30 ℃ of stroke-physiological saline solution, take back the laboratory as early as possible with vacuum flask, with the syringe that is equipped with No. 8, from the ovary surface diameter is the ovarian follicle of 2～8mm, extract ovocyte, under stereoscopic microscope, chosen more than three layers the dense granule cell and profile is bright, the uniform ovocyte of tenuigenin; Described collection liquid is with the Hepes buffered, is added with 5mmol/LNaHCO ₃, the 3%OCS(bovine serum of oestrusing) the oocytes collection liquid of TCM199.

3. the outer embryo's of production pien niu controllability complicated variant according to claim 1 method, it is characterized in that: in the described step (2), the described ripe maturation culture solution of cultivating employing is Hepes buffered TCM199+5%OCS+0.1 μ g/mLFSH (follitropin)+3%BFF(ox liquor folliculi), the maturation culture solution of every culture dish is 1mL, the described ripe incubation time of cultivating is 21～24 hours, and the condition in the incubator is 39 ℃, 5%CO ₂Air and saturated humidity.

4. the outer embryo's of production pien niu controllability complicated variant according to claim 1 method, it is characterized in that: in the described step (3), the method of separating the Necrospermia in the seminal fluid and the sperm of living is: the centrifuge tube of seminal fluid being put into the 2mL90%:2mL45%Percoll gradient, centrifugal 20 minutes of 700Xg, abandon supernatant, add 1mL and be subjected to seminal fluid 400Xg centrifugal 5 minutes, remove the upper strata suspension, stay the seminal fluid of about 50 μ L; Described common ox is He Sitanniu, Simmental, Xia Luolainiu, beautiful coral ox, hereford cow, western yellow cross-bred ox (Simmental and the offspring who hides ox hybridization) or the yellow cross-bred ox of lotus (He Sitanniu and the offspring who hides ox hybridization).

5. the outer embryo's of production pien niu controllability complicated variant according to claim 1 method, it is characterized in that: in the described step (4), described fertilization droplet is 20 μ L, put into 16～18 pieces of ovocytes for every, after the test under microscope motility of sperm meets the requirements, sperm is added in the fertilization droplet, makes its ultimate density reach 2X106/mL; At 39 ℃, 5%CO ₂The incubator of saturated humidity in cultivate 6～8h.

6. produce the outer embryo's of pien niu controllability complicated variant method according to claim 1 or 5, it is characterized in that: in the described step (5), the ripe method of handling of described blastaea is: after fertilization, in the fertilization droplet, blow and beat the zygote of supposition repeatedly with suction pipe, remove the part cumulus cell, again the zygote of supposition from dripping, is shifted out in fertilization, change in the co-culture system that contains the ox granulosa cell with vitro culture liquid washed twice and to cultivate, nutrient solution is that the pyruvic acid of Hepes buffered TCM199+10%NCS (new-born calf serum)+0.11mg/mL is received, whenever be added dropwise to nutrient solution 25 μ L, put into 50 pieces of zygotes, at 39 ℃, 5%CO ₂, 5%O ₂, 90%N ₂The saturated humidity condition under external cultivation 6～8 days, changed liquid once in per two days between incubation period, change 1/2 liquid at every turn.