CN107723272B

CN107723272B - Method for obtaining cattle early embryo trophoblast tissue

Info

Publication number: CN107723272B
Application number: CN201710846496.8A
Authority: CN
Inventors: 陈洪波; 程蕾; 向敏; 胡修忠; 侯永清
Original assignee: Wuhan Polytechnic University
Current assignee: Wuhan Polytechnic University
Priority date: 2017-09-19
Filing date: 2017-09-19
Publication date: 2021-05-18
Anticipated expiration: 2037-09-19
Also published as: CN107723272A

Abstract

The invention belongs to the technical field of biology, and provides a method for obtaining a cattle early embryo trophoblast tissue. The method comprises the following steps: (1) obtaining pregnant cattle: after the cattle are subjected to estrus induction and superovulation treatment, the pregnant cattle are confirmed by combining progesterone detection and fluorescent quantitative PCR detection in one estrus period (as early as 18 days) after artificial insemination. (2) Taking the uterus of the pregnant cow, flushing embryo and obtaining early embryo: picking the uterus and the associated tissue structure of the uterus of a pregnant cow in 18-44 days of pregnancy, and performing embryo washing on the uterine horn of the uterus at the side with good development of an ovarian corpus luteum to obtain an early embryo; (3) morphological observation of early embryonic tissue and isolation of trophoblast tissue: observing the tissue structure of the embryo under a microscope, fixing the pregnant body tissue with complete morphology by 4% paraformaldehyde, performing HE staining, determining the trophoblast tissue and stripping the trophoblast tissue. The method has the advantages of strong accuracy and more economical and effective performance.

Description

Method for obtaining cattle early embryo trophoblast tissue

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a method for obtaining a cattle early embryo trophoblast tissue.

Background

Embryo implantation is a critical link in mammalian reproduction. At the maternal-embryonic interface, trophoblast cells and maternal endometrial cells play a decisive role in pregnancy recognition and embryo implantation, especially trophoblast cells play a leading role in embryo implantation, and the invasion, proliferation and functional regulation of the trophoblast cells are closely related to the occurrence and development of various pregnancy-related diseases. Meanwhile, the trophoblast cell is an important component of a placenta barrier, is the first barrier for maternal blood to enter a fetus, and plays an important role in immune tolerance in the process of identifying and establishing maternal pregnancy. The trophoblast tissue separated by a whole set of technical method at a specific gestational stage is digested into a single cell to be cultured in vitro, the trophoblast cell cultured in vitro is closest to the real in vivo cell biological state, is an ideal in vitro model for researching gestational biological phenomena such as embryo implantation, endometrial changes, trophoblast autocrine control and the like, is also an important cell material and experimental basis for preventing and treating and researching pregnancy-related diseases such as implantation failure, early fetal death and the like, and can provide a theoretical basis for establishing a strategy for improving the conception rate of female animals through manual intervention in the future. In addition, as the trophoblast cell can start maternal pregnancy recognition through paracrine, the success rate of transplantation can be improved by adding the trophoblast cell cultured in vitro during embryo transplantation, and the method has important practical significance for improving the current situation of low success rate of embryo transplantation.

In summary, the acquisition of trophoblast tissue is a key prerequisite for the primary culture of bovine trophoblast cells. Until now, researchers have collected embryonic cotyledons from the uterus of pregnant cows from 45 to 60 days to isolate trophoblast cells, and researchers have collected trophoblast cells from yaks by digestion, isolation and purification from fetal placental tissue from 8 to 10 weeks (56 to 70 days) of gestation. However, the placenta is gradually aged along with the increase of gestational months, and meanwhile, the trophoblast cells are also aged, so that the number of the cells is small, the activity is low, more fibroblasts, interstitial cells and other cell components are easily mixed, and the obtaining of the trophoblast cells with stable property and high purity is difficult to ensure.

Therefore, there is a need to develop a method for precisely obtaining high quality bovine trophoblasts (cells).

Disclosure of Invention

The invention aims to provide a method for obtaining a cattle early embryo trophoblast tissue.

The inventor of the invention finds that 14-19 days after Artificial Insemination (AI) is a maternal pregnancy identification period, embryo implantation starts at 19-20 days, once implantation starts, a fetal chorion extends to a non-pregnant uterine horn, and then a caruncle-placental maternal villus lobule appears according to the development rule of early-stage embryos of dairy cows. Although the trophoblast is in contact with the uterine mucosa only at the mons, blastocyst trophoblast cells invade the uterine mucosal epithelium, making the fetus more closely associated with the mother during the implantation process. Therefore, before the embryo is implanted, the fetus is not in substantial contact with the mother, and the trophoblast cells obtained by separating the obtained embryo have high purity and strong activity and are easy to culture.

According to a first aspect of the present invention, the present invention provides a method for obtaining a bovine early embryo trophoblast tissue, comprising the steps of:

(1) taking and washing embryo of uterus of pregnant cow and obtaining embryo at early stage

Picking the uterus and the associated tissue structure of the uterus of a pregnant cow in 18-44 days of pregnancy, and performing embryo washing on the uterine horn of the uterus at the side with good development of an ovarian corpus luteum to obtain an early embryo;

(2) morphological observation of early embryonic tissue and isolation of trophoblast tissue

Observing the tissue structure of the embryo under a microscope, fixing the pregnant body tissue with complete morphology by 4% paraformaldehyde, performing HE staining, determining the trophoblast tissue and stripping the trophoblast tissue.

Preferably, the uterus and associated tissue structures of 18-19 day-old pregnant cows are harvested for trophoblast tissue to further reduce the cycle time and cost of the method of the invention.

According to the invention, the uterus is preferably slaughtered, which is the most simple, economical and efficient method at present.

The invention aims to obtain embryo trophoblast tissues of 18-44 days, preferably 18-19 days of cow pregnancy, can use naturally estrous cows for cow pregnancy, and can also adopt estrus induction and superovulation technologies to ensure accuracy and high efficiency, preferably, the method comprises the following steps:

(1) oestrus induction and superovulation (hereinafter referred to as superovulation) treatment of cattle

Carrying out superovulation treatment on cattle with a normal oestrus cycle on 8-12 days after oestrus, and then carrying out at least one artificial insemination;

(2) pregnancy diagnosis of cattle

After artificial insemination, it was judged whether the cattle were pregnant.

Specifically, the estrus induction and superovulation treatment of cattle comprises: the continuous 4-day intramuscular injection of the follitropin is carried out once a day in the morning and at night, and specifically comprises the following steps: injecting 68-72 units of Follicle Stimulating Hormone (FSH) in the morning and evening on the first day, 58-62 units in the morning and evening on the second day and 48-52 units in the morning and evening on the fourth day; wherein, on the third day, FSH is injected and at the same time, chlorprostenol (PG) is injected in an amount of 0.4-0.6mg, on the fifth day, gonadotropin releasing hormone (GnRH) 180-; artificial insemination was performed once again in the sixth morning.

According to the present invention, the method for determining whether a cow is a pregnant cow may be a method conventionally used in the art. For example, it may be: collecting peripheral blood of cattle for multiple times within 0-18 days, detecting progesterone content, and judging whether the cattle is pregnant cattle according to progesterone content change.

In order to improve the accuracy of the determination, preferably, the method for determining whether the cattle is a pregnant cattle comprises the following steps:

i. collecting peripheral blood of cattle through tail vein at 0 day, 15 days, 16 days, 17 days and 18 days after artificial insemination, separating serum, and detecting progesterone content in serum;

extracting total RNA of peripheral blood of cattle at 0 day and 18 days, performing reverse transcription to obtain cDNA, and detecting the pregnancy state of the cattle at 18 days after artificial insemination by using a fluorescent quantitative PCR kit; the fluorescent quantitative PCR kit comprises a cattle ISG15 gene, an internal reference gene beta-Actin specific primer pair and a cattle RSAD2 gene specific primer pair, wherein the cattle ISG15 gene primer pair is as follows: SEQ ID NO: 1 and SEQ ID NO: the primer pair of 2, beta-Actin is: SEQ ID NO: 3 and SEQ ID NO: 4, the specific primer pair of the bovine RSAD2 gene is as follows: SEQ ID NO: 5 and SEQ ID NO: 6;

and comprehensively judging whether the cattle is pregnant or not by combining the content change of the progesterone in the serum and the detection result of the fluorescent quantitative PCR kit.

Wherein, the comprehensive judgment basis is as follows: the content of progesterone in serum is lower than 1ng/ml at 0 day after artificial insemination, and the content of progesterone is continuously increased and maintained at a higher level at 15-18 days; meanwhile, the detection result of the kit is positive.

The sample collected by the invention can be serum or plasma, and the detection is not limited by a specific experimental method, namely: other techniques than chemiluminescence can be used, provided that the progesterone (P4) content is accurately obtained.

The fluorescent quantitative PCR kit disclosed in the present invention is the kit disclosed in ZL201410657206.1, which is herein incorporated by reference in its entirety.

The kit relates to a method for detecting early pregnancy of a cow based on a fluorescent quantitative PCR technology, which comprises the following steps:

(1) blood sample collection

Collecting 1.5-2ml of peripheral blood of the cow after oestrus and before artificial insemination at 0d and after artificial insemination at 18d, performing anticoagulation, and refrigerating at low temperature;

(2) extraction of RNA from specimen

The total RNA extraction in the sample is completed by using a blood total RNA extraction kit within 2h after the sample is collected, and then the concentration of the RNA sample is analyzed by using a NanoDrop2000 ultramicro spectrophotometerAnd purity, OD_260/280Detecting the integrity of RNA by 1.2% agarose gel electrophoresis at the same time, subpackaging samples subjected to quality inspection, and storing at-80 ℃ for later use;

(3) reverse transcription reaction

Reverse transcription is carried out by adopting a kit, the using amount of each reaction RNA is 500ng, the volume required by each reaction is calculated according to the concentration of the RNA, and the reverse transcription is carried out according to the kit instruction;

(4) fluorescent quantitative PCR detection of relative expression of ISG15 and RSAD2 genes in cow peripheral blood

1) Design and synthesis of target gene and reference gene primers

The primer pair of the cattle ISG15 gene is shown as SEQ ID NO: 1 and SEQ ID NO: 2; the primer pair of the reference gene beta-Actin is shown as SEQ ID NO: 3 and SEQ ID NO: 4; the primer pair of the bovine RSAD2 gene is shown as SEQ ID NO: 5 and SEQ ID NO: 6;

2) fluorescent quantitative PCR reaction system

The bovine ISG15, RSAD2 gene and reference gene beta-Actin fluorescent quantitative PCR amplification systems are all as follows:

green 10. mu.l, upstream and downstream primers 0.5. mu.l each (10. mu. mol/L), cDNA template 0.3. mu.l, ddH₂O8.7. mu.l, 20. mu.l in total;

3) fluorescent quantitative PCR reaction condition

ISG15 Gene reaction conditions: pre-denaturation at 95 ℃ for 60s, denaturation at 95 ℃ for 15s, and annealing at 62 ℃ for 15 s; extension at 72 ℃ for 30s for 40 cycles;

RSAD2 Gene reaction conditions: pre-denaturation at 95 ℃ for 60s, denaturation at 95 ℃ for 15s, and annealing at 58 ℃ for 15 s; extension at 72 ℃ for 30s for 40 cycles;

the reaction condition of the beta-Actin gene is as follows: pre-denaturation at 95 ℃ for 60s, denaturation at 95 ℃ for 15s, and annealing at 60-62 ℃ for 15 s; extension at 72 ℃ for 30s for 40 cycles;

(5) statistical analysis

1) Analysis of ISG15 and RSAD2 Gene expression levels

Detection of target genes by relative quantificationExpression amount of (3): calculating the threshold difference (delta Ct) between the target gene and the reference gene in the sample to be detected

Expressing the relative expression quantity of target gene mRNA in the sample, correcting the relative expression quantity of each gene of 0d after artificial insemination of the cow to be 1, and multiplying the expression quantity when the expression quantity of 18d after artificial insemination is 0 d;

2) early pregnancy diagnosis of cow by using expression level of ISG15 and RSAD2 genes

After artificial insemination, the expression level of ISG15 in the peripheral blood of 18d cows was X1, the expression level of RSAD2 was X2, and X1 and X2 were substituted into the formula P ═ 1/[1+ e ]^{-(-6.53+2.830X1+0.866X2)}]If the P value is more than 0.680, the pregnancy is determined, namely the positive is stated in the invention.

According to the invention, the expression of the interferon stimulating gene in the peripheral blood of the cow after artificial insemination is detected by a fluorescent quantitative PCR method, and the detection method is not limited by specific reagents and different dye types marked by the detected gene probe.

According to the invention, the uterus of a pregnant cow is obtained intact, said uterus comprising the cervix, the corpus uteri and the uterine horn, said connective tissue structure comprising the reproductive tract.

According to the invention, the step of blanking preferably comprises: before embryo flushing, placing a sterile PBS solution in a 37 ℃ water bath for preheating in advance, and expanding the aperture of a No. 18 2-path type embryo flushing tube by more than half through manual shearing; during embryo washing, a cervix dilatation rod is firstly used for moderate dredging, then an embryo washing catheter is inserted, preheated PBS is poured for washing for many times, the recovered embryo washing liquid is kept stand in a liquid collecting cup, and the deposition part is transferred into a sterile glass culture dish.

The method of the invention is suitable for cows, beef or buffalos, preferably cows.

In the invention, the tissue morphology observation is a tissue morphology observation method of a paraffin tissue section, and is a conventional laboratory technology.

According to a preferred embodiment of the present invention, the technical solution is as follows, and the flow is as shown in fig. 1:

firstly, oestrus induction and superovulation treatment of cows

Observing the oestrus cycle of the cow, carrying out superovulation treatment on the cow with a normal oestrus cycle on the 9 th day after oestrus, and carrying out intramuscular injection of Follicle Stimulating Hormone (FSH) for 4 consecutive days, wherein the superovulation treatment is carried out once in the morning and at night every day, and specifically comprises the following steps: 70 units of FSH are injected in the morning and evening on the first day, 60 units are injected in the morning and evening on the second day and the third day, and 50 units are injected in the morning and the evening on the fourth day; wherein, on the third day, 0.5mg of chlorprostenol (PG) is injected while FSH is injected, on the fifth day, 200 mu g of gonadotropin releasing hormone (GnRH) is injected, and artificial insemination is performed once after 2 hours; artificial insemination was performed once again in the sixth morning.

② pregnancy diagnosis of cow

i. Collecting peripheral blood of cow via tail vein at 0, 15, 16, 17 and 18 days after artificial insemination, separating serum, and detecting progesterone (P) in serum by chemiluminescence method₄) And (4) preliminarily judging the early embryonic development state of the dairy cow according to the content change rule.

Extracting total RNA from peripheral blood at 0 and 18 days, and reverse transcribing into cDNA. The pregnancy state of the cow at 18 days after artificial insemination is detected by using a fluorescent quantitative PCR kit and a detection method (a patent granted by the inventor before: ZL201410657206.1) for early pregnancy of the cow.

Binding of progesterone (P) in serum₄) The content change and the detection result of the kit carry out comprehensive diagnosis on whether the cow is pregnant or not. The judgment basis is as follows: the content of progesterone in serum is lower than 1ng/ml at 0 day after artificial insemination, and the content of progesterone is continuously increased and maintained at a higher level at 15-18 days; meanwhile, when the detection result of the kit is positive, the detected individual is judged as a pregnant cow, otherwise, the detected individual is judged as a non-pregnant cow.

③ slaughtering and embryo washing of pregnant cow and obtaining of early embryo

The pregnant cow is delivered to a slaughter house for slaughtering 18-19 days after artificial insemination, the uterus (including cervix, uterus body and uterine horn) and associated tissue structure (reproductive tract) of the cow are completely picked up within 30 minutes after slaughtering, and the cow uterus and the associated tissue structure are placed on ice in time and brought back to a laboratory within 1 hour. In a laboratory, embryo washing is carried out on uterine horn on one side with good development of an ovary corpus luteum, before embryo washing, a sterile PBS solution is placed in a 37 ℃ water bath kettle to be preheated in advance, and the aperture of a No. 18 2-path type embryo washing tube is enlarged by more than half through manual shearing. When embryo washing, firstly, a cervix dilating rod is used for moderate dredging, then an embryo washing catheter is inserted, 50-70ml of PBS is filled for washing each time, the continuous washing is carried out for 6-7 times, the recovered embryo washing liquid is kept stand in a liquid collecting cup for 10-15 minutes, and the precipitate is transferred into a sterile glass culture dish with the diameter of 9 cm.

Morphological observation of early embryo tissue and isolation of trophoblast tissue

And (3) placing the glass culture dish under a stereo microscope to observe the tissue structure of the embryo, fixing the pregnant body tissue with complete shape by 4% paraformaldehyde, and performing HE staining. The trophoblast tissue was stripped using a sterile pipette tip and washed 3 times with PBS solution for primary culture of the trophoblast cells in cows.

Aiming at the defects of the prior art that a cow is pregnant for more than 45 days to collect trophoblast tissues, the invention firstly applies a plurality of reproductive hormones in a combined manner to induce oestrus and superovulation treatment on the cow, slaughters the pregnant cow through pregnancy diagnosis, picks up the uterus, washes the embryo, combines cell morphology identification to obtain the cow embryo trophoblast tissues, and provides materials for efficient primary culture of trophoblast cells. Therefore, the method of the present invention aims to obtain more accurately, efficiently and economically trophoblast tissue from 18-19 day old embryos after artificial insemination of cows, around this purpose: firstly, reproductive hormones FSH, PG and GnRH are jointly applied, an optimal using dose and an optimal combination strategy are established, and accurate estrus induction and superovulation are carried out on cows; innovatively integrating a molecular detection technology, and establishing an accurate ultra-early cow pregnancy diagnosis method in a matching way; at the moment, the embryo vitality is high, the trophoblast cells are in the early stage of differentiation, and the trophoblast cells are easier to culture in the later stage. ② focusing on embryo trophoblast tissues in the period of maternal pregnancy recognition (18-19 days after mating). At the moment, the embryo is not in close contact with the mother body, the purity of the trophoblast tissue is high, and few impurity cells are mixed when the trophoblast tissue is used for culturing trophoblast cells; the regulation of the pore size of the punched embryo tube and the identification of tissue morphology are combined, so that the experimental steps of later cell culture can be greatly simplified, and high-quality trophoblast tissues can be effectively obtained; and improving the economy of the separation of the cattle trophoblast tissues. The method has the advantages of large number of embryos obtained by superovulation, accurate judgment of maternal pregnancy, improvement of test efficiency and saving of test cost.

Specifically, the advantages and positive effects of the invention include:

the accuracy is high.

One of the key technologies of the invention is the induction of estrus and superovulation of the dairy cattle, and the key of obtaining more embryos and trophoblast tissues is the step. The induced estrus expression obtained by jointly applying FSH, PG and GnRH and combining the optimal dosage of the optimal combination better conforms to the actual reproductive physiological state of the follicle development of the dairy cow, and the external estrus expression is ensured to be accurate and consistent with the actual follicle development.

The second key technology is the early pregnancy diagnosis of the cow after artificial insemination, and the embryo in the early development stage can be obtained only from the pregnant cow obtained by the early pregnancy diagnosis and identification. Different from the traditional ultrasonic diagnosis or artificial rectum examination, the two methods depend on more clinical work experience of inspectors and have high misdiagnosis rate on one hand, and cannot perform ultra-early pregnancy diagnosis at all on the other hand due to the technical limitation of the two methods. The method achieves further optimization and improvement: the method integrates the level detection of progesterone (P4) in peripheral blood and the detection of the expression of interferon stimulating genes in the peripheral blood after artificial insemination of cows by a fluorescence quantitative PCR method, can ensure that more accurate pregnancy diagnosis is carried out on cow pregnancy 18 days after insemination, realizes 100% pregnancy diagnosis positive rate in one estrus cycle (21 days after insemination) after artificial insemination, and is difficult to realize by other pregnancy diagnosis technologies.

② more effective.

The invention compares two oestrus induction and superovulation schemes and determines the optimal method: the first is the combined use of PMSG, PG and GnRH, and the second is the combined use of FSH, PG and GnRH. Although both of the two schemes have obvious oestrus expression, the dose of PMSG in the first scheme is difficult to control, when the dose of PMSG is too large, a test cow may oestrous in advance, and the conception rate is poor although typical oestrus expression such as cross-crawl and flow mucus exists. The method finally established by the invention has more obvious effects on the aspects of estrus induction and superovulation.

The cow which is successfully pregnant for 18-19 days is adopted, and because the cow is in the period of identifying the pregnancy of the mother to the fetus, the cow and the fetus are not closely physically connected, compared with the cow pregnant for 45 days or more, the embryo trophoblast tissue in the period is relatively independent and is easy to separate; more importantly, the trophoblast tissue has high activity without excessive interference of other tissue type impurity cells, and effective acquisition of target tissues can be ensured by combining HE staining and embryo section of early embryo and trophoblast tissue morphology microscopic examination, so that the yield and purity of viable cells can be ensured when the trophoblast tissue is used for primary culture.

In addition, in the aspect of obtaining embryo and trophoblast tissues, which is the third key technology of the invention, the test of the invention compares two methods of directly flushing embryos and cutting off uterine tissues to pick up embryos, and respective optimal strategies under different experimental purposes are established. Although embryo tissue is not broken by embryo washing (integrity of embryo body tissue is guaranteed) after embryo picking after cutting the uterus, because the embryo ectotrophoblast tissue is adhered to the uterine wall, the embryo ectotrophoblast tissue is very thin and is difficult to distinguish by naked eyes, so that the yield is reduced. However, the technical contrast test of the method shows that when directly punching embryos, the pore size of the embryo punching tube can be ensured to be proper through manual shearing, and the introduction and the leading-out processes of the embryo punching liquid are ensured to be soft and slow during operation, so that the damage to the embryos can be reduced, the integrity of the embryos is ensured, and the yield of trophoblast tissues is also ensured.

And the cost is saved.

Because the test cattle has high cost and long test period, the improvement of the working efficiency, the saving of the operation time and the reduction of unnecessary animal loss are fundamental ways for ensuring the reduction of the test cost. The invention has the advantages of more compact test process, less time consumption and greatly reduced test economic loss risk. The comprehensive pregnancy diagnosis scheme can be used for timely searching the non-pregnant cows (unsuccessful pregnant cows) after mating in a oestrus cycle (within 21 days after mating) after artificial insemination, so that the compounding can be carried out at the first time according to the method disclosed by the invention, the slaughtering waste of test cows caused by inaccurate diagnosis can be avoided, and the trial economic cost is increased by at least 1.2 ten thousand RMB by mistakenly slaughtering one non-pregnant cow according to the current cow market. In addition, compared with the prior art which utilizes pregnant cows of 45 days of pregnancy and even 8-10 weeks of gestation, the invention can better realize the test purpose in 18-19 days of pregnancy, thereby shortening the test period by 27-52 days; the test cattle are generally low-yield cattle with normal reproductive capacity, the milk yield is about 5.5 kg/day, the milk price is 4.2 yuan/kg, and the income of milk production per day is 23.1 yuan; and the actual feeding cost (43.3 yuan/day) of one adult cow per day is calculated at present: average feed amount is 30 yuan/day, labor cost is about 8 yuan/day, equipment cost is 5 yuan/day, and veterinary drug cost is 0.3 yuan/day. Therefore, for one test cow, the invention can save the feeding management cost by 20.2 yuan/day, and the feeding management cost can be saved by 545.4-1050.4 yuan/day in the whole test period.

Additional features and advantages of the invention will be set forth in the detailed description which follows.

Drawings

The above and other objects, features and advantages of the present invention will become more apparent by describing in more detail exemplary embodiments thereof with reference to the attached drawings.

FIG. 1 is a flow chart of a method according to a preferred embodiment of the present invention.

FIG. 2 shows the results of fluorescent quantitative detection of early pregnancy of test cattle; a, 1208-1 amplification curve chart; 1127 amplification graph; 1208-2 amplification curve chart; 1089 amplification plot.

FIG. 3 is a graph of HE staining of embryonic tissue from a cow at day 19 of gestation.

Detailed Description

Preferred embodiments of the present invention will be described in more detail below. While the following describes preferred embodiments of the present invention, it should be understood that the present invention may be embodied in various forms and should not be limited by the embodiments set forth herein.

(1) Test animals:

the 3 test cows are from a standard cow demonstration breeding base in Wuhan city, and have healthy body conditions, low milk production performance and normal reproductive capacity.

(2) Main reagents and instruments:

follitropin (FSH), chlorprostinol (PG), gonadotropin releasing hormone for injection (GnRH), and Pregnant Mare Serum Gonadotropin (PMSG) were purchased from sansheng pharmaceutical ltd, ningbo. The embryo-flushing catheter is purchased from Takara science and technology development center in Beijing, Tanzhou, the RNA extraction kit is purchased from Tiangen Biotechnology (Beijing) Inc., the reverse transcription kit is purchased from ThermoFisher Scientific, the premix solution for real-time quantitative PCR amplification is purchased from Toyobo (Shanghai) Biotechnology Inc., and other PCR-related reagents are purchased from Takara. The main instrument platform is: maglumi 800 chemiluminescence apparatus (Snibe), BioRad CFX ConnectTM real-time fluorescence quantitative PCR apparatus.

(3) The test method comprises the following steps:

firstly, oestrus induction and superovulation treatment

Scheme 1: the first day, the afternoon, 2000IU of Pregnant Mare Serum Gonadotropin (PMSG), the third day, the afternoon, 0.5mg of chlorprostenol (PG), the fifth day, the afternoon, 200 mu g of gonadotropin releasing hormone (GnRH), artificial insemination is performed once after 2h, and artificial insemination is performed again in the morning of the next day.

Scheme 2: superovulation treatment was started on the ninth day of the oestrus cycle of the test cows, and four consecutive days of intramuscular injection of Follicle Stimulating Hormone (FSH) were carried out once a day, morning and evening. FSH 70 units were injected in the morning and evening on the first day, 60 units were injected in the morning and evening on the second and third days, and 50 units were injected in the morning and evening on the fourth day. Wherein, another injection of 0.5mg of treprostinil (PG) is performed on the third night, and 200 μ g of gonadotropin releasing hormone (GnRH) is performed on the fifth night. Artificial insemination was performed once each in the fifth evening and the sixth morning.

② diagnosis of pregnancy

Collecting the peripheral blood of the cow via tail vein at 0 day, 7 days, 15 days, 16 days, 17 days and 18 days after the hybridization, separating the serum,the progesterone content in serum was determined by chemiluminescence. Total RNA was extracted from peripheral blood at 0 day and 18 days and reverse-transcribed into cDNA, according to the procedures described in the commercial kit. The pregnancy state of the cow is detected by utilizing a fluorescent quantitative PCR kit and a detection method (a patent granted by the inventor before: ZL201410657206.1) which are independently developed in the early stage of the cow pregnancy: detecting the expression quantity of ISG15 and RSAD2 genes in the peripheral blood of the cow at 0 day and 18 days after artificial insemination, correcting the relative expression quantity of each gene at 0 day after artificial insemination of the cow to be 1, wherein the relative expression quantity of the genes is as follows: the expression level at 18 days after artificial insemination was expressed in terms of fold relative to the expression level at 0 day. If the expression level of ISG15 in the peripheral blood of the cow 18 days after artificial insemination is X1 and the expression level of RSAD2 is X2, X1 and X2 are respectively substituted into the formula P ═ 1/[1+ e ]^{-(-6.53+2.830X1+0.866X2)}]If the P value is greater than 0.680, the result is positive (i.e., pregnancy). And (4) comprehensively testing the progesterone content change in the cow serum to judge whether the cow is pregnant.

Collecting and identifying trophoblast tissue

The pregnant cow is transported to a Wufeng slaughterhouse in Wuhan city for slaughtering 19 days after artificial insemination, the uterus (including cervix, uterus body and uterine horn) and the uterus with the tissue structure (reproductive tract) of the cow are completely picked up within 30 minutes after slaughtering and are timely placed on ice, and the collected sample is brought back to the laboratory within 1 hour. In the laboratory, embryo-flushing catheters are inserted into one side of the uterine horn with well-developed ovarian corpus luteum, 70ml of PBS solution (PBS volume is specifically adjusted according to the size of the uterus, preheating is carried out in advance at 37 ℃) is used for flushing each time for 6-7 times, and the collected pregnant body is observed under a stereoscopic microscope and trophoblast tissues are separated. Pregnane tissues were fixed with 4% paraformaldehyde and HE stained for further histological morphology.

(4) Test results

Test the oestrus effect of cattle

Experimental calves 1208 (calves numbered, the same below) and 1127 were estrualized and superovulation treated according to protocol 1 described above. 1208 on the morning of the third day, the disease appears as crawling symptom, and the species is bred on the morning of the fourth day according to rectal examination and follicular development. The hormone dosage for inducing estrus of the test cattle 1127 is further adjusted according to the estrus performance of the test cattle 1208, the dosage of PMSG is halved, after all the estrus drugs are injected, namely, on the sixth day of the test, the 1127 cows begin to have creeping symptoms, and the cows are bred at night.

Because the test cow 1208 returns estrus (fails to be pregnant) on the 19 th day after mating, after entering the next estrus (19 days away from the last mating), the test cow 1208 and another test cow 1089 is subjected to induced estrus and superovulation treatment according to the scheme 2. Two cattle were observed with significant symptoms of estrus (crawling and mucus flow) in the morning on the fifth day of the trial, bred 2 hours after injection of GnRH and bred again the next day.

② test the content of progesterone in bovine serum

TABLE 1 test of progesterone content (ng/ml) in cattle after mating

As can be seen from Table 1, the progesterone content of the test cattle 1208-1 sharply decreases to less than 1ng/ml (which is a sign of estrus return) 17 days and 18 days after the first induced estrus and artificial insemination. The progesterone content in the serum of the experimental cattle 1127 is highest 15 days after artificial insemination, and the progesterone content is continuously reduced 15-18 days, but is still maintained at a higher level, which is different from 1208-1 that returning estrus occurs after mating. By adopting the scheme 2, the content of the progesterone in the serum of the experimental cattle 1208-2 after the second induction estrus and artificial insemination is in an ascending trend and is far higher than that of the 1127 dairy cattle. The expression pattern of progesterone in the serum of test bovine 1089 was similar to that of 1208-2.

③ fluorescent quantitative detection result of early pregnancy of cow

As shown in fig. 2, the results of the fluorescent quantitative PCR assay of early pregnancy of cows indicated that 1208-1 was negative (P ═ 0.005), 1127 was positive (P ═ 1.00), 1208-2(P ═ 1.00) was positive, and 1089(P ═ 0.99) was positive. Since the test is to induce oestrus cattle, the accuracy of pregnancy diagnosis by detecting interferon stimulated gene expression by using a fluorescence quantitative PCR technology may be affected, so the scheme further combines the progesterone detection result to comprehensively judge the cows 1208-2 and 1089 as pregnant cows and 1127 as suspected pregnant cows.

Collection of trophoblast tissue

i. Pregnancy diagnosis and slaughter of test cattle

And carrying out comprehensive pregnancy diagnosis by using gene detection and progesterone content. After the pregnancy is determined, a short electric shock is carried out for 5 seconds in a slaughterhouse by using 220V voltage, blood is discharged after the unconsciousness and the land is fallen, and the abdominal cavity is cut open to collect a target sample, namely the complete uterus and the affiliated tissues thereof.

Collection and observation of early embryonic tissue of cows

Obtaining early embryonic tissue of experimental cattle 1208

1208, the milk cow adopts a mode of introducing an embryo-flushing tube and flushing the uterus with PBS solution to obtain trophoblast tissue of early embryo of the milk cow. The 1089 cow adopts a method of directly dissecting the uterus on one side, and picking out embryo tissues by using an ophthalmic forceps through visual observation, wherein the embryo tissues obtained by the method have a complete morphological structure, but relatively few trophoblast tissues.

Cow embryo tissue morphological structure

Having described embodiments of the present invention, the foregoing description is intended to be exemplary, not exhaustive, and not limited to the embodiments disclosed. Many modifications and variations will be apparent to those of ordinary skill in the art without departing from the scope and spirit of the described embodiments.

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Claims

1. A method for obtaining early embryo trophoblast tissue from cattle, comprising the steps of:

Picking the uterus and the associated tissue structure of the uterus of a slaughtered pregnant cow with 18-44 days of gestation, and performing embryo washing on the uterine horn of the side with good development of an ovary corpus luteum to obtain an early embryo;

the connective tissue structure comprises the reproductive tract;

Observing the tissue structure of the embryo under a microscope, fixing the pregnant body tissue with complete shape by 4% paraformaldehyde, performing HE staining, determining the trophoblast tissue and stripping the trophoblast tissue;

wherein the method further comprises the step of obtaining a pregnant cow, the step comprising:

(a) estrus induction and superovulation treatment of cattle

(b) pregnancy diagnosis of cattle

After artificial insemination, judging whether the cattle is pregnant cattle;

wherein, the estrus induction and superovulation treatment of the cattle comprises the following steps: the continuous 4-day intramuscular injection of the follitropin is carried out once a day in the morning and at night, and specifically comprises the following steps: injecting 68-72 units of follicle-stimulating hormone respectively in the morning and evening on the first day, 58-62 units respectively in the morning and evening on the second day and the third day, and 48-52 units respectively in the morning and evening on the fourth day; wherein, the FSH is injected in the evening of the third day, the chlorprostenol is injected in the range of 0.4-0.6mg, the gonadotropin releasing hormone 180-220 mu g is injected in the evening of the fifth day, and the artificial insemination is performed once after 2-3 hours; artificial insemination is carried out once again in the sixth morning;

wherein the method for judging whether the cattle is pregnant cattle comprises the following steps:

menses at 0, 15, 16, 17 and 18 days after artificial inseminationCollecting peripheral blood of the cattle through tail veins, separating serum, and detecting the progesterone content in the serum;

comprehensively judging whether the cattle is pregnant or not by combining the content change of progesterone in serum and the detection result of the fluorescent quantitative PCR kit, wherein the comprehensive judgment is based on the following steps: the content of progesterone in serum is lower than 1ng/ml at 0 day after artificial insemination, and the content of progesterone is continuously increased and maintained at a higher level at 15-18 days; meanwhile, the detection result of the kit is positive.

2. The method for obtaining bovine early embryo trophoblast tissue according to claim 1, wherein the uterus of said pregnant bovine comprises cervix, corpus uteri and uterine horn.

3. The method for obtaining bovine early embryo trophoblast tissue of claim 1, wherein the step of flushing comprises: before embryo flushing, placing a sterile PBS solution in a 37 ℃ water bath for preheating in advance, and expanding the aperture of a No. 18 2-path type embryo flushing tube by more than half through manual shearing; during embryo washing, a cervix dilatation rod is firstly used for moderate dredging, then an embryo washing catheter is inserted, preheated PBS is poured for washing for many times, the recovered embryo washing liquid is kept stand in a liquid collecting cup, and the deposition part is transferred into a sterile glass culture dish.

4. The method for obtaining the bovine early embryo trophoblast tissue of claim 1, wherein the bovine is a cow, a beef cow or a buffalo.