CN107723272A - A kind of preparation method of cattle early embryo Trophoblast tissue - Google Patents
A kind of preparation method of cattle early embryo Trophoblast tissue Download PDFInfo
- Publication number
- CN107723272A CN107723272A CN201710846496.8A CN201710846496A CN107723272A CN 107723272 A CN107723272 A CN 107723272A CN 201710846496 A CN201710846496 A CN 201710846496A CN 107723272 A CN107723272 A CN 107723272A
- Authority
- CN
- China
- Prior art keywords
- embryo
- days
- pregnant
- day
- tissue
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0603—Embryonic cells ; Embryoid bodies
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61D—VETERINARY INSTRUMENTS, IMPLEMENTS, TOOLS, OR METHODS
- A61D19/00—Instruments or methods for reproduction or fertilisation
- A61D19/02—Instruments or methods for reproduction or fertilisation for artificial insemination
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2509/00—Methods for the dissociation of cells, e.g. specific use of enzymes
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/158—Expression markers
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/166—Oligonucleotides used as internal standards, controls or normalisation probes
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Organic Chemistry (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- General Health & Medical Sciences (AREA)
- Biomedical Technology (AREA)
- Analytical Chemistry (AREA)
- Genetics & Genomics (AREA)
- Biotechnology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Veterinary Medicine (AREA)
- Reproductive Health (AREA)
- General Engineering & Computer Science (AREA)
- Microbiology (AREA)
- Biochemistry (AREA)
- Animal Behavior & Ethology (AREA)
- Molecular Biology (AREA)
- Immunology (AREA)
- Biophysics (AREA)
- Physics & Mathematics (AREA)
- Developmental Biology & Embryology (AREA)
- Gynecology & Obstetrics (AREA)
- Public Health (AREA)
- Cell Biology (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention belongs to biological technical field, there is provided a kind of preparation method of cattle early embryo Trophoblast tissue.This method comprises the following steps：(1) acquisition of pregnant ox：After implementing heat induction and superfecundation processing to ox, (early to 18 days) makes a definite diagnosis pregnant ox with reference to progesterone detection and fluorescence quantitative PCR detection within artificial insemination the latter feelings phase.(2) acquisition of embryo and body early embryo is won, rushed to pregnant cattle uterus：The uterus of the gestation pregnant ox of 18 44 days and its related institutional framework are won, the cornua uteri in the uterus for the side that physically well developed to corpus luteum carries out rushing embryo, obtains body early embryo；(3) morphologic observation of body early embryo tissue and the separation of Trophoblast tissue：The institutional framework of embryo is observed under the microscope, after the complete conceptus tissue of form is fixed with 4% paraformaldehyde, is carried out HE dyeing, is determined Trophoblast tissue and peel off Trophoblast tissue.The method accuracy of the present invention is strong, more economical effective.
Description
Technical field
The invention belongs to biological technical field, more particularly, to a kind of acquisition side of cattle early embryo Trophoblast tissue
Method.
Background technology
Embryonic limb bud cell is the key link of Mammalian Reproduction.On parent-embryo interface, trophocyte and parent
Endometrial cells play decisive role, especially trophocyte during gestation identification and Embryonic limb bud cell, are planted in embryo
Played a leading role during entering, it is attacked and the not normal and generation of a variety of pregnancy related disorders of propagation and function point analysis, hair
Exhibition has substantial connection.It is that maternal blood enters the first of fetus meanwhile trophocyte is the important component of placental barrier
Road barrier, identified for maternal gestational and played important function with the immune tolerance during establishing.Utilize technological package side
Method digestion process after specific stages of gestation isolates Trophoblast tissue is that individual cells can be to carry out in vitro culture, this body
The trophocyte of outer culture closest to real cell biology state in vivo, be research Embryonic limb bud cell, endometrium change,
The preferable external model of the Aschheim-Zondek phenomenon such as trophocyte's autocrine regulation, and carry out implantation failure, early stage fetus
The important cells material and experiment basis of the pregnancy related disorders such as death prevention and treatment research, can be that formulation in the future is artificial dry
The pre- strategy for improving dam conception rate is provided fundamental basis.Further, since trophocyte can be pregnant by paracrine startup parent
It is pregnent identification, transplanting success rate can be improved by adding the trophocyte of in vitro culture during embryo transfer, for improving embryo
Transplanting low success rate of present situation has important practice significance.
In summary, the acquisition of Trophoblast tissue is the key precondition of ox trophocyte's original cuiture.So far, have
Researcher separates trophocyte by collecting the pregnancy ox uterus embryo collection cotyledon of 45-60 days, and some scholars are in yak
With the fetal placenta setup action material of pregnant 8-10 week old (56-70 days) in ox, grown by digesting, separating and purify to obtain
Support confluent monolayer cells.But placenta gradual aging with the increase in pregnant month, while trophocyte's also aging therewith, cause
Cell quantity is few, vigor is low, and is easily mixed with more fibroblast, interstitial cell and other cell components, it is difficult to ensures
The trophocyte that energy acquired character is stable and purity is high, so does the complexity that can not only increase the screening of succeeding target cell purification
Property, influence operating efficiency, the vigor and/or motility rate of trophocyte can be also had a strong impact on because of cumbersome additional manipulation processes.
Therefore, a kind of method for precisely obtaining high quality ox trophoderm (cell) of exploitation is needed badly.
The content of the invention
It is an object of the invention to provide a kind of preparation method of cattle early embryo Trophoblast tissue.
It was found by the inventors of the present invention that according to the law of development of milk cattle early embryo, artificial insemination (AI) 14-19 days is afterwards
Maternal gestational identifies period, and Embryonic limb bud cell starts within 19-20 days, once implantation starts, fetus chorion stretches to unpregnancy cornua uteri, after
And caruncula-cotyledons occurs.Although during attached plant, trophoderm only contacts in uterus Fu Chu with uterine mucosa,
But blastocyst trophocyte can invade uterine mucosa epithelium so that contacting for fetus and parent is more close.Therefore, in embryo
Before implantation, substantial contact not yet occurs for fetus and parent, now obtain the isolated trophocyte's purity of embryo it is high,
It is energetic, and easily culture.
According to the first aspect of the invention, the present invention provides a kind of preparation method of cattle early embryo Trophoblast tissue, its
It is characterised by, this method comprises the following steps：
(1) acquisition of embryo and body early embryo is won, rushed to pregnant cattle uterus
The uterus of the pregnant ox of pregnant 18-44 days and its related institutional framework are won, physically well develop side to corpus luteum
The cornua uteri in uterus rush embryo, obtain body early embryo；
(2) morphologic observation of body early embryo tissue and the separation of Trophoblast tissue
The institutional framework of embryo is observed under the microscope, after the complete conceptus tissue of form is fixed with 4% paraformaldehyde,
HE dyeing is carried out, Trophoblast tissue is determined and peels off Trophoblast tissue.
Preferably, the uterus and its related institutional framework that can win the pregnant ox of 18-19 days obtain Trophoblast tissue, to enter
One step reduces the cycle and cost of the inventive method.
, according to the invention it is preferred to win uterus by the way of butchering, under present circumstances, this method be it is most simple and direct, economical,
Efficiently.
It is contemplated that the cow gestation embryonic feeder layer tissue of preferred 18-19 days 18-44 days is obtained, it is pregnant for cow
It is pregnent, using the ox of spontaneous estrus, can also uses estrus induction accurate and efficient to ensure with super ovulation techniques, it is preferable that
The step includes：
(1) the heat induction of ox and superfecundation (hereinafter referred to as super row) processing
The superfecundation processing is proceeded by for 8-12 days after heat to oestrous cycle normal ox, then advanced to few
Artificial insemination；
(2) cyesiognosis of ox
After artificial insemination, judge whether ox is pregnant ox.
Specifically, the heat induction of ox includes with superfecundation processing：Continuous 4 days intramuscular injection follicle-stimulating hormone, it is early daily
Evening is each once, is specially：Sooner or later follicle-stimulating hormone (FSH) 68-72 units, second day and the 3rd day morning and evening each note are respectively injected within first day
58-62 units are penetrated, respectively inject 48-52 units sooner or later within the 4th day；Wherein, chlorine forefront is injected while the 3rd day injects FSH at night
Alcohol (PG) 0.4-0.6mg, the 5th day descendant for injecting gonadotropin-releasing hormone (GRH) (GnRH) 180-220 μ g, 2-3 hours at night
Work is inseminated once；6th day morning carried out artificial insemination once again.
It is described to judge whether ox is that the method for pregnant ox use the conventional method of this area according to the present invention.Such as can
Think：In 0-18 days, ox peripheral blood is gathered several times, detects progesterone content, and whether ox is judged according to progesterone content change
For pregnant ox.
To improve the precision judged, it is preferable that described to judge whether ox is that the method for pregnant ox includes：
I. gather ox peripheral blood through tail vein within 0 day, 15 days, 16 days, 17 days and 18 days after artificial insemination, separate serum,
Detect the progesterone content in serum；
Ii. 0 day and 18 longicorn peripheral blood total serum IgEs are extracted, and reverse transcription is examined into cDNA using PCR kit for fluorescence quantitative
Pregnant state when 18 days after survey ox artificial insemination；The PCR kit for fluorescence quantitative includes ox ISG15 genes, reference gene
β-Actin specific primers pair and ox RSAD2 gene-specific primers pair, wherein, the primer pair of the ox ISG15 genes is：
SEQ ID NO：1 and SEQ ID NO：2, β-Actin primer pair is：SEQ ID NO：3 and SEQ ID NO：4, ox RSAD2 bases
Because specific primer is to being：SEQ ID NO：5 and SEQ ID NO：6；
With reference to progesterone content change in serum, to ox, whether gestation integrates with PCR kit for fluorescence quantitative testing result
Judge.
Wherein, the foundation of comprehensive descision is：Progesterone content is less than 1ng/ml in 0 day serum after artificial insemination, and 15-18 days pregnant
Ketone content continues to increase and maintains higher level；Meanwhile kit testing result is the positive.
The sample that the present invention is gathered can be that serum can also be blood plasma, while detection is not limited by specific experiment method
System, i.e.,：The other technologies in addition to chemoluminescence method can be used, as long as progesterone (P4) content can be obtained accurately.
Heretofore described PCR kit for fluorescence quantitative be ZL201410657206.1 disclosed in kit, the patent
It is all incorporated herein by reference herein.
The kit is related to the milk cow early pregnancy detection method based on fluorescent quantitative PCR technique, comprises the following steps：
(1) collection of blood preparation
Gather after cow oestrus the peripheral blood 1.5-2ml of 18d after 0d and artificial insemination before artificial insemination, anti-freezing, low temperature cold
Hide；
(2) sample RNA extraction
Total RNAs extraction in sample is completed with blood total RNA extraction reagent box in 2h after collection of specimens, is utilized afterwards
NanoDrop2000 ultramicrospectrophotometers analyze RNA concentration of specimens and purity, OD260/280=1.9-2.1, is used simultaneously
1.2% agarose gel electrophoresis detects RNA integralities, after the sample packing of quality inspection, is saved backup in -80 DEG C；
(3) reverse transcription reaction
Reverse transcription is carried out using kit, and each RNA dosages of reacting are 500ng, are calculated every time according to RNA concentration
Volume needed for reaction, is carried out according to kit specification；
(4) relative expression quantity of fluorescence quantitative PCR detection ISG15 and RSAD2 genes in milk cow peripheral blood
1) design and synthesis of target gene and reference gene primer
The primer pair of ox ISG15 genes is such as SEQ ID NO：1 and SEQ ID NO：Sequence shown in 2；Reference gene β-
Actin primer pair is such as SEQ ID NO：3 and SEQ ID NO：Sequence shown in 4；Ox RSAD2 gene primers are to for such as SEQ
ID NO：5 and SEQ ID NO：Sequence shown in 6；
2) quantitative fluorescent PCR reaction system
Ox ISG15, RSAD2 gene and reference gene β-Actin fluorescent quantitative PCR systems are：
The μ l of Green 10, each 0.5 μ l of upstream and downstream primer (10 μm of ol/L), cDNA templates 0.3 μ l, ddH2The μ l of O 8.7, system totally 20 μ l；
3) quantitative fluorescent PCR reaction condition
ISG15 Gene response conditions：95 DEG C of pre-degenerations 60s, 95 DEG C of denaturation 15s, 62 DEG C of annealing 15s；72 DEG C of extension 30s,
Totally 40 circulations；
RSAD2 Gene response conditions：95 DEG C of pre-degenerations 60s, 95 DEG C of denaturation 15s, 58 DEG C of annealing 15s；72 DEG C of extension 30s,
Totally 40 circulations；
β-Actin Gene response conditions：95 DEG C of pre-degenerations 60s, 95 DEG C of denaturation 15s, 60-62 DEG C of annealing 15s；72 DEG C of extensions
30s, totally 40 circulations；
(5) statistical analysis
1) analysis of ISG15 and RSAD2 gene expression amounts
Using the expression quantity of the method testing goal gene of relative quantification：Calculate target gene and internal reference in detection sample
The threshold difference (△ Ct) of gene, withTarget gene mRNA relative expression quantities in sample are represented, after milk cow artificial insemination
The relative expression quantity calibration of each genes of 0d is 1, the multiple of expression quantity when 18d expression quantity is 0d after artificial insemination；
2) milk cow diagnosis of early gestation is carried out using the expression quantity of ox ISG15 and RSAD2 gene
ISG15 expression quantity is X1 in 18d milk cows peripheral blood after artificial insemination, and RSAD2 expression quantity is X2, by X1 and X2
Bring formula P=1/ [1+e into-(-6.53+2.830X1+0.866X2)] in, be gestation if P values are more than 0.680, i.e., it is of the present invention
The positive.
According to the present invention, on interferon-stimulated gene in peripheral blood after fluorescence quantitative PCR method detection cow artificial insemination
Expression, detection method do not limited by specific reagent and detected Probe labelling different dyes type.
According to the present invention, the uterus of pregnant ox is completely obtained, the uterus of the pregnant ox includes cervix, corpus uteri and son
Palace angle, the related institutional framework include genital tract.
According to the present invention, described the step of rushing embryo, preferably includes：Proembryo is rushed, sterile PBS solution is placed in 37 DEG C of water-baths
Preheat in advance, and by manually shear by No. 18 2 tunnel formulas rush embryonic tube borehole enlargement more than half；When rushing embryo, uterus is used first
Neck dilator bar carries out appropriate dredging, is inserted into and rushes embryo conduit, pours into the more flushings of PBS of preheating, and the embryo washing water of recovery is collecting
Stood in liquid cup, sediment fraction is transferred in sterile glass culture dish.
The method of the present invention is applied to milk cow, beef cattle or buffalo, preferably milk cow.
In the present invention, the tissue morphology is viewed as the tissue morphology observational technique of paraffin tissue sections, is that laboratory is normal
Rule technology.
According to a kind of preferred embodiment of the present invention, technical scheme is as follows, and flow is as shown in Figure 1：
1. the heat induction of milk cow is handled with superfecundation
The oestrous cycle of milk cow is observed, super several rows were proceeded by the 9th day of oestrous cycle normal milk cow after heat
Ovum processing, continuous 4 days intramuscular injection follicle-stimulating hormone (FSH), once in the morning and once at night, it is specially：Sooner or later FSH is respectively injected within first day
70 units, 60 units, 50 units of the 4th day morning and evening each injection are respectively injected within second day and the 3rd day sooner or later；Wherein, the 3rd day evening
Cloprostenol (PG) 0.5mg is injected while injecting FSH, injects gonadotropin-releasing hormone (GRH) (GnRH) 200 μ at night within the 5th day
G, artificial insemination afterwards in 2 hours is once；6th day morning carried out artificial insemination once again.
2. the cyesiognosis of milk cow
I. gather milk cow peripheral blood through tail vein within 0 day, 15 days, 16 days, 17 days and 18 days after artificial insemination, separate blood
Clearly, the progesterone (P in serum is detected with chemoluminescence method4) content, tentatively judge milk cattle early embryo according to its changes of contents rule
Developmental condition.
Ii. 0 day and 18 days peripheral blood total serum IgEs are extracted, and reverse transcription is into cDNA.Fluorescence using milk cow early pregnancy is determined
Measure PCR kit and detection method (granted patent before inventor：ZL201410657206.1 after) detecting milk cow artificial insemination
Pregnant state at 18 days.
With reference to progesterone (P in serum4) whether gestation carries out comprehensive diagnos to milk cow for changes of contents and kit testing result.
Basis for estimation is：Progesterone content is less than 1ng/ml in 0 day serum after artificial insemination, and 15-18 days progesterone contents continue to increase and tieed up
Hold higher level；Meanwhile kit testing result also for it is positive when, then be detected individual be judged to pregnant ox, be otherwise nonpregnant ox.
3. the acquisition of embryo and body early embryo is butchered, rushed to pregnant ox
Pregnant ox was delivered into slaughterhouse in 18-19 days after artificial insemination to be butchered, completely won in 30 minutes after butchering
Cow uteri (including cervix, corpus uteri and cornua uteri) and its related institutional framework (genital tract), are placed on ice, 1 is small in time
When interior take back laboratory.In laboratory, the cornua uteri for the side that physically well developed to corpus luteum carries out rushing embryo, rushes proembryo, will be sterile
PBS solution is placed in 37 DEG C of water-baths and preheated in advance, and by manually shear by No. 18 2 tunnel formulas rush the borehole enlargement half of embryonic tube with
On.Appropriate dredging is carried out with cervical dilatation rod first when rushing embryo, is inserted into and rushes embryo conduit, pours into 50-70ml PBS every time
Rinse, continuous flushing 6-7 times, the embryo washing water of recovery stands 10-15 minutes in liquid collecting cup, sediment fraction is transferred to a diameter of
In 9cm sterile glass culture dish.
4. the morphologic observation of body early embryo tissue and the separation of Trophoblast tissue
Glass culture dish is placed in the institutional framework of stereomicroscopy Microscopic observation embryo, the complete conceptus tissue of form is used
After 4% paraformaldehyde is fixed, HE dyeing is carried out.Trophoblast tissue, PBS solution cleaning are peeled off using sterile suction pipette head
It is used for the original cuiture of milk cow trophocyte after 3 times.
For utilizing many defects present in more than 45 days cow collection Trophoblast tissues of pregnancy, this hair in prior art
The bright a variety of reproductive hormones of use in conjunction first implement estrus induction to milk cow and superfecundation is handled, by cyesiognosis to pregnant
Cow of being pregnent is butchered and carries out embryo's flushing after winning uterus, is identified with reference to cellular morphology and obtains ox embryonic feeder layer tissue, to grow
The efficient original cuiture for supporting confluent monolayer cells provides material.Therefore, the inventive method be intended to more precisely, effectively and economically come
From the Trophoblast tissue that development time after milk cow artificial insemination is 18-19 days embryos, around this basic goal：1. use in conjunction
Reproductive hormone FSH, PG and GnRH, optimal use dosage and optimum organization strategy are established, implement accurately induction to cow and send out
Feelings and superfecundation；Innovative integration molecular detection technology, it is supporting to establish accurately extreme early milk cow cyesiognosis method；Now
Embryonic itality is high, and trophocyte is in differentiation initial stage, and the later stage is more easy to cultivate.2. it is conceived to maternal gestational identification period (breeding
18-19 days afterwards) embryonic feeder layer tissue.Now embryo is not yet in close contact with parent, and the purity of Trophoblast tissue is high, uses
The contaminating cell being mixed with when trophocyte is cultivated is few；Regulation and tissue morphology identification with reference to the embryonic tube pore size that liquidates,
The experimental procedure of later stage cell culture can be greatly simplified, effectively obtains the Trophoblast tissue of high quality；3. improve ox trophoderm
Organize the economy of separation.Present invention hypervelocity is ovulated, and the embryo's quantity obtained is more, maternal gestational judgement is accurate, can improve experiment and imitate
Rate, save experimentation cost.
Specifically, advantage for present invention and good effect include：
1. accuracy is strong.
One of key technology of the present invention is the estrus induction and superfecundation of milk cow, this step be obtain more embryo and
The key of Trophoblast tissue.The optimum organization that the present invention passes through use in conjunction FSH, PG and GnRH and combination optimal use dosage
Acquired estrus induction performance more conforms to the actual reproductive physiology state of milk cow follicular development itself, ensure that outside heat
Performance is accurate consistent with actual follicular development.
The two of key technology are the diagnosis of early gestations of milk cow after artificial insemination, and only extreme early cyesiognosis differentiates to obtain
Pregnant ox could obtain the embryo of mesoderm growing early stage.It is different using traditional ultrasonic diagnosis or artificial examination per rectum from other people,
On the one hand both approaches more rely on the clinical position experience of inspection personnel, misdiagnosis rate is high, on the other hand due to itself
Technical limitation, extreme early cyesiognosis is not accomplished at all.This method has accomplished further Optimal improvements：It is pregnant in comprehensive peripheral blood
The expression of interferon-stimulated gene can in peripheral blood after the horizontal detection of ketone (P4) and fluorescence quantitative PCR method detection cow artificial insemination
To ensure to make more accurately cyesiognosis to milk cow gestation in 18 days after breeding, artificial insemination the latter heat week is realized
In phase (after breeding in 21 days) 100% cyesiognosis positive rate, and other cyesiognosis technologies are difficult to accomplish this point.
It is 2. more efficient.
The present invention compares two kinds of estrus inductions and superfecundation scheme and best practice is determined：The first is that joint should
With PMSG, PG and GnRH, second is use in conjunction FSH, PG and GnRH.Although two schemes have obvious heat table
Existing, PMSG dosage is difficult to hold in scheme one, and when PMSG dosage is excessive, test ox may shift to an earlier date heat, though
So there is the typical heat performance such as mounting and stream mucus, but conception rate is poor.The method that the present invention finally establishes is in estrus induction with surpassing
Effect is more notable in terms of number ovulation.
The present invention now knows using the successful pregnancy cow of 18-19 days because being in parent to the gestation of fetus
In other period, therebetween and do not set up physical link closely, compared with the pregnant ox of 45 days and the above, this period embryo
Tire Trophoblast tissue is relatively independent, easily separated；Importantly, now Trophoblast tissue vigor is high, does not have its excessive hetero-organization
The interference of type dopant cell, mesh can be ensured in conjunction with the embryo section and Trophoblast tissue form microscopy of HE dyeing and body early embryo
Effective acquisition of tissue is marked, more can guarantee that living cells yield and purity during for original cuiture.
In addition, in terms of the three of the key technology of the present invention i.e. acquisition of embryo and Trophoblast tissue, present invention experiment is compared
Directly rush embryo and cut off uterine tissue and pick up two methods of embryo, establish the respective optimal strategy in the case of different experiments purpose.
Although picking up embryo after cutting off uterus, embryonic tissue will not cause to crush (to ensure that the complete of embryo's subject organization because embryo is rushed
Property), but due to outside embryo Trophoblast tissue be adhered in uterine wall because very thin, be visually difficult to differentiate between causing to obtain
Yield reduction.But shown by the technology contrast test of this method, when directly rushing embryo, can ensure to rush embryo by manually shearing
Pipe pore size is suitable, and ensures that the importing of embryo washing water and export process are softly slow during operation, thus can also subtract
Few damage to embryo, the integrality of embryo is ensured, while also ensure that the yield of Trophoblast tissue.
More economical 3. (cost-effective).
Because test ox cost is high, test period length, thus improve operating efficiency, save the operating time, reduce it is unnecessary
Animal losses are to ensure the fundamental way that experimental cost reduces.The present invention has accomplished that experiment process is compacter, time-consuming less, separately
The outer economic loss risk that can also substantially reduce experiment.Can be in artificial insemination the latter using this comprehensive cyesiognosis scheme
The empty cow (failed cow in calf) after breeding is investigated out in time (after breeding in 21 days) in oestrous cycle, so as to not only may be used
To be compounded in the very first time according to the method for the invention, the Tu of test ox only caused by diagnosing inaccuracy can also be avoided
Kill and waste, according to current dairy market, experiment financial cost increase at least 1.2 ten thousand can be made by mistakenly massacring an empty cow
Yuan, do not include manpower and test consumable cost also among these.In addition, with utilizing 45 days even 8- of gestation in prior art
10 week old cows in calf are compared, and the present invention 18-19 days can be to be better achieved test objective, therefore test period gestation
Shorten 27-52 days；Test ox is generally the normal low-yield cattle of fecundity, and the output of milk is pressed according to 5.5 kg/days or so, milk valency
According to 4.2 yuan/kilogram, then the income given milk daily is 23.1 yuan；And the actual feeding cost that a current Grown cow is daily is (total
43.3 member/day) calculate：Average 30 yuan/day of feed, yuan/day of labour cost about 8, yuan/day of equipment depreciation 5,0.3 yuan of veterinary drug expense/
My god.Therefore, can be to save yuan/day of feeding management cost 20.2 for an experiment cow present invention, the whole test period can
Save feeding management cost 545.4-1050.4 members/head.
Other features and advantages of the present invention will be described in detail in subsequent specific embodiment part.
Brief description of the drawings
Exemplary embodiment of the invention is described in more detail in conjunction with the accompanying drawings, it is of the invention above-mentioned and its
Its purpose, feature and advantage will be apparent.
Fig. 1 is a kind of method flow diagram of preferred embodiment of the present invention.
Fig. 2 shows the fluorogenic quantitative detection result of test ox early pregnancy；A:1208-1 amplification curve diagrams；B:1127 expand
Increase curve map；C:1208-2 amplification curve diagrams；D:1089 amplification curve diagrams.
Fig. 3 is the HE colored graphs of the gestation cow embryo tissue of 19 days.
Embodiment
The preferred embodiment of the present invention is described in more detail below.Although the following describe being preferable to carry out for the present invention
Mode, however, it is to be appreciated that may be realized in various forms the present invention without should be limited by embodiments set forth herein.
(1) experimental animal：
3 test ox are from Wuhan City's standardization milk cow demonstration cultivation base, and body condition health, Milk Production are low, reproduction
Ability is normal.
(2) main agents and instrument：
Follicle-stimulating hormone (FSH), cloprostenol (PG), injection gonadotropin-releasing hormone (GRH) (GnRH) and pregnant mare serum
Promoting sexual gland hormone (PMSG) is purchased from Ningbo Sansheng Pharmaceutical Co., Ltd..Rush embryo conduit and be purchased from the river scientific and technological development of Beijing pool
Center, RNA extracts kits are purchased from TIANGEN Biotech (Beijing) Co., Ltd., and reverse transcription reagent box is purchased from ThermoFisher
Scientific companies, the premix solution of real-time quantitative PCR amplification spin (Shanghai) bio tech ltd purchased from Japan, its
Its PCR related reagent is purchased from Takara companies.Key instrument platform is：The Chemiluminescence Apparatus of Maglumi 800 (Snibe),
BioRad CFX ConnectTM real-time fluorescence quantitative PCR instrument.
(3) test method：
1. heat induction is handled with superfecundation
Scheme 1：Intramuscular injection pregnant mare serum gonadotrop(h)in (PMSG) (PMSG) 2000IU one afternoon, the 3rd day P.M. injection
Artificial insemination one after cloprostenol (PG) 0.5mg, the 5th day P.M. injection gonadotropin-releasing hormone (GRH) (GnRH) 200 μ g, 2h
Secondary, morning next day carries out artificial insemination again.
Scheme 2：In the Superovluation that proceeds by for the 9th day in experiment cow oestrus cycle, continuous intramuscular injection in four days promotees ovum
Bubble is plain (FSH), once in the morning and once at night.Sooner or later the units of FSH 70, each injection sooner or later in second day and the 3rd day are respectively injected within first day
60 units, 50 units are respectively injected sooner or later within the 4th day.Wherein, the 3rd day evening other injection cloprostenol (PG) 0.5mg, the 5th day
Injection gonadotropin-releasing hormone (GRH) (GnRH) 200 μ g at night.5th day evening and the 6th day morning respectively carry out artificial insemination one
It is secondary.
2. cyesiognosis
Milk cow peripheral blood is gathered through tail vein and is separated within 0 day, 7 days, 15 days, 16 days, 17 days and 18 days after breeding
Serum, the progesterone content in serum is detected with chemoluminescence method.0 day and 18 days peripheral blood total serum IgEs and reverse transcription are extracted simultaneously
Into cDNA, operating procedure in commercial reagents box specification is purchased in specific method reference.Utilize the milk cow early stage of independent research early stage
PCR kit for fluorescence quantitative and the detection method (granted patent before inventor of gestation：ZL201410657206.1 milk) is detected
The pregnant state of ox：The expression quantity of ISG15 and RSAD2 genes in 0 day and 18 days milk cow peripheral bloods after artificial insemination is detected, by milk
The relative expression quantity of 0 day each gene is corrected to 1 after ox artificial insemination, and gene relative expression quantity is：The table of 18 days after artificial insemination
Up to amount relative to expression quantity at 0 day, represented with multiple.If ISG15 expression quantity is in 18 days milk cow peripheral bloods after artificial insemination
X1, RSAD2 expression quantity are X2, bring X1 and X2 into formula P=1/ [1+e respectively-(-6.53+2.830X1+0.866X2)] in, P values are more than
Positive findings (i.e. pregnant) is judged to when 0.680.To milk cow, whether gestation is carried out for progesterone content change in compbined test cow's serum
Judge.
3. the collection and identification of Trophoblast tissue
Pregnant ox is transported into Wuhan City Wu Feng slaughterhouses for 19 days after artificial insemination to be butchered, after butchering in 30 minutes
Complete cow uteri (including cervix, corpus uteri and cornua uteri) and its related institutional framework (genital tract) uterus and timely of winning
It is placed on ice, institute's collecting sample is taken back into laboratory in 1 hour.In laboratory, in the well-developed cornua uteri one of corpus luteum
Embryo conduit is rushed in side insertion, every time with 70ml PBS solutions (PBS volumes specifically adjust according to the size in uterus, and 37 DEG C preheat in advance)
Rinse, continuous flushing 6-7 times, the conceptus of collection in stereomicroscopy Microscopic observation and separates Trophoblast tissue.Conceptus tissue is used
HE dyeing is carried out after being fixed with 4% paraformaldehyde, the identification for further tissue morphology.
(4) result of the test
1. test ox heat effect
Test ox 1208 (ox is only numbered, similarly hereinafter) and 1127 carried out according to aforementioned schemes 1 at estrus induction and super row
Reason.1208 occurred mounting symptom the 3rd day morning, were bred according to examination per rectum and follicular development situation the 4th day morning.Root
Further the hormone dosage of the estrus induction of test ox 1127 is adjusted according to the heat performance of test ox 1208, PMSG is used
Dosage halves, and after all heat drug injections are complete, that is, the 6th day No. 1127 milk cows tested start mounting symptom occur, evening
Upper breeding.
Feelings (Pregnancy failure) are returned after being bred due to test ox 1208 within the 19th day, treat that it enters (distance after next feelings phase
Last time breeding 19 days) carry out estrus induction according to aforementioned schemes 2 with other one first No. 1089 newly-increased experiment cows to 1208 and surpass
Row is handled.Two ox observes obvious heat symptom (mounting and stream mucus) in the 5th day morning of experiment, is injecting
Breed and bred again in next day within 2 hours after GnRH.
2. test progesterone content in cow's serum
Progesterone content (ng/ml) in serum after the breeding of the test ox of table 1
From table 1,17 days and 18 days progesterone content urgency after test ox 1208-1 first times estrus induction and artificial insemination
Play declines, and is down to below 1ng/ml (being the omen for returning feelings).Progesterone in 15 days serum after No. 1127 test ox artificial insemination
Content highest, 15-18 days progesterone content continuous decreases, but the 1208-1 from returning feelings after breeding is different, is still maintained at higher
Level.Using scheme 2, the progesterone after second of estrus induction of test ox 1208-2 and artificial insemination in 15-18 days serum contains
Measure in rising trend, and the progesterone level of significantly larger than No. 1127 milk cows.In the serum of test ox 1089 expression rule of progesterone with
1208-2 situation is similar.
3. the fluorogenic quantitative detection result of milk cow early pregnancy
As shown in Fig. 2 being shown using the fluorescence quantitative PCR detection result of milk cow early pregnancy, 1208-1 is feminine gender (P=
0.005), 1127 be positive (P=1.00), and 1208-2 (P=1.00) is the positive, and 1089 (P=0.99) are the positive.Due to this
Test as estrus induction ox, may influence to carry out by detecting interferon-stimulated gene expression using fluorescent quantitative PCR technique
The accuracy of cyesiognosis, therefore, scheme is further combined with progesterone testing result, synthetic determination 1208-2 and No. 1089 milk cows
Doubtful pregnant ox is judged to for pregnant ox, 1127.
4. the collection of Trophoblast tissue
I. the cyesiognosis of test ox is with butchering
Carry out integrating cyesiognosis using genetic test and progesterone content.It is determined that after gestation, 220V is utilized in slaughterhouse
The of short duration electric shock of voltage 5 seconds, bloodletting after stunning falls down to the ground, splits abdominal cavity collection target sample i.e. intact uteri and its affiliated group.
Ii. the collection and observation of milk cattle early embryo tissue
Iii. the acquisition of the body early embryo tissue of test ox 1208
No. 1208 milk cows take importing to rush embryonic tube, and the taste of milk cattle early embryo is obtained with the mode in PBS solution flushing uterus
Layer tissue is supported, this method acquisition embryonic tissue is more, and Trophoblast tissue and embryo section part are easily separated, further, it is possible to ensure to nourish
The quality of layer tissue.And what No. 1089 milk cows were then taken is the uterus for directly splitting side, observe by the naked eye, chosen with ophthalmic tweezers
Go out embryonic tissue, the embryonic tissue morphosis that this method obtains is more complete, but Trophoblast tissue is relatively fewer.
5. cow embryo histology and morphology structure
Fig. 3 is the HE colored graphs of the gestation cow embryo tissue of 19 days.
It is described above various embodiments of the present invention, described above is exemplary, and non-exclusive, and
It is not limited to disclosed each embodiment.In the case of without departing from the scope and spirit of illustrated each embodiment, for this skill
Many modifications and changes will be apparent from for the those of ordinary skill in art field.
Sequence table
<110>Wuhan Polytechnic University
<120>A kind of preparation method of cattle early embryo Trophoblast tissue
<130> 1700581
<160> 6
<170> SIPOSequenceListing 1.0
<210> 1
<211> 21
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 1
ggtatccgag ctgaagcagt t 21
<210> 2
<211> 19
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 2
acctccctgc tgtcaaggt 19
<210> 3
<211> 20
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 3
ctggacttcg agcaggagat 20
<210> 4
<211> 20
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 4
ggatgtcgac gtcacacttc 20
<210> 5
<211> 21
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 5
gctgaaagaa gcaggtatgg a 21
<210> 6
<211> 21
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 6
cgtacttctg gaaccacctc t 21
Claims (8)
1. a kind of preparation method of cattle early embryo Trophoblast tissue, it is characterised in that this method comprises the following steps：
(1) acquisition of embryo and body early embryo is won, rushed to pregnant cattle uterus
The uterus of the pregnant ox of pregnant 18-44 days and its related institutional framework are won, the son for the side that physically well developed to corpus luteum
The cornua uteri in palace carries out rushing embryo, obtains body early embryo；
(2) morphologic observation of body early embryo tissue and the separation of Trophoblast tissue
The institutional framework of embryo is observed under the microscope, after the complete conceptus tissue of form is fixed with 4% paraformaldehyde, is carried out
HE is dyed, and is determined Trophoblast tissue and is peeled off Trophoblast tissue.
2. the preparation method of cattle early embryo Trophoblast tissue according to claim 1, wherein, this method also includes obtaining
The step of pregnant ox, the step include：
(1) the heat induction of ox is handled with superfecundation
The superfecundation processing is proceeded by for 8-12 days after heat to oestrous cycle normal ox, then advance to it is few once
Artificial insemination；
(2) cyesiognosis of ox
After artificial insemination, judge whether ox is pregnant ox.
3. the preparation method of cattle early embryo Trophoblast tissue according to claim 2, wherein, the heat induction of ox is with surpassing
Number ovulation processing includes：Continuous 4 days intramuscular injection follicle-stimulating hormone, once in the morning and once at night, it is specially：Sooner or later each injection in first day
Follicle-stimulating hormone 68-72 units, 58-62 units are respectively injected within second day and the 3rd day sooner or later, respectively injection 48-52 is mono- sooner or later within the 4th day
Position；Wherein, cloprostenol 0.4-0.6mg is injected while the 3rd day injects FSH at night, injects promoting sexual gland hormone at night within the 5th day
Artificial insemination is once after releasing hormone 180-220 μ g, 2-3 hours；6th day morning carried out artificial insemination once again.
4. the preparation method of cattle early embryo Trophoblast tissue according to claim 2, wherein, it is described judge ox whether be
The method of pregnant ox includes：
I. gather ox peripheral blood through tail vein within 0 day, 15 days, 16 days, 17 days and 18 days after artificial insemination, separate serum, detection
Progesterone content in serum；
Ii. 0 day and 18 longicorn peripheral blood total serum IgEs are extracted, and reverse transcription detects ox into cDNA using PCR kit for fluorescence quantitative
Pregnant state after artificial insemination at 18 days；The PCR kit for fluorescence quantitative include ox ISG15 genes, reference gene β-
Actin specific primers pair and ox RSAD2 gene-specific primers pair, wherein, the primer pair of the ox ISG15 genes is：SEQ
ID NO：1 and SEQ ID NO：2, β-Actin primer pair is：SEQ ID NO：3 and SEQ ID NO：4, ox RSAD2 gene are special
Specific primer is to being：SEQ ID NO：5 and SEQ ID NO：6；
With reference to progesterone content change in serum, to ox, whether gestation integrate sentencing with PCR kit for fluorescence quantitative testing result
It is disconnected.
5. the preparation method of cattle early embryo Trophoblast tissue according to claim 4, wherein, the foundation of comprehensive descision
For：Progesterone content is less than 1ng/ml in 0 day serum after artificial insemination, and 15-18 days progesterone contents continue to increase and maintained compared with Gao Shui
It is flat；Meanwhile kit testing result is the positive.
6. the preparation method of cattle early embryo Trophoblast tissue according to claim 1, wherein, the uterus of the pregnant ox
Including cervix, corpus uteri and cornua uteri, the related institutional framework includes genital tract.
7. the preparation method of cattle early embryo Trophoblast tissue according to claim 1, wherein, described the step of rushing embryo, wraps
Include：Proembryo is rushed, sterile PBS solution is placed in into 37 DEG C of water-baths is preheated in advance, and No. 18 2 tunnel formulas are rushed into embryonic tube by manually shearing
Borehole enlargement more than half；When rushing embryo, appropriate dredging is carried out with cervical dilatation rod first, is inserted into and rushes embryo conduit, is filled
Enter the more flushings of PBS of preheating, the embryo washing water of recovery is stood in liquid collecting cup, and sediment fraction is transferred to sterile glass culture dish
It is interior.
8. the preparation method of cattle early embryo Trophoblast tissue according to claim 1, the ox is milk cow, beef cattle or water
Ox.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710846496.8A CN107723272B (en) | 2017-09-19 | 2017-09-19 | Method for obtaining cattle early embryo trophoblast tissue |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710846496.8A CN107723272B (en) | 2017-09-19 | 2017-09-19 | Method for obtaining cattle early embryo trophoblast tissue |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN107723272A true CN107723272A (en) | 2018-02-23 |
| CN107723272B CN107723272B (en) | 2021-05-18 |
Family
ID=61206635
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201710846496.8A Active CN107723272B (en) | 2017-09-19 | 2017-09-19 | Method for obtaining cattle early embryo trophoblast tissue |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN107723272B (en) |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2000017325A1 (en) * | 1998-09-23 | 2000-03-30 | Mount Sinai Hospital | Trophoblast cell preparations |
| CN104328202A (en) * | 2014-11-18 | 2015-02-04 | 武汉市畜牧兽医科学研究所 | Fluorescent quantitative PCR detection kit and detection method for early pregnancy of dairy cow |
| CN106137448A (en) * | 2015-03-19 | 2016-11-23 | 河北工程大学 | A kind of method inducing high yield cow timing to ovulate and improve conception rate |
| CN106399469A (en) * | 2016-06-15 | 2017-02-15 | 武汉市农业科学技术研究院畜牧兽医科学研究所 | Dairy cow early-gestation diagnosis primer pairs, probes and method based on duplex quantitative PCR |
-
2017
- 2017-09-19 CN CN201710846496.8A patent/CN107723272B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2000017325A1 (en) * | 1998-09-23 | 2000-03-30 | Mount Sinai Hospital | Trophoblast cell preparations |
| CN104328202A (en) * | 2014-11-18 | 2015-02-04 | 武汉市畜牧兽医科学研究所 | Fluorescent quantitative PCR detection kit and detection method for early pregnancy of dairy cow |
| CN106137448A (en) * | 2015-03-19 | 2016-11-23 | 河北工程大学 | A kind of method inducing high yield cow timing to ovulate and improve conception rate |
| CN106399469A (en) * | 2016-06-15 | 2017-02-15 | 武汉市农业科学技术研究院畜牧兽医科学研究所 | Dairy cow early-gestation diagnosis primer pairs, probes and method based on duplex quantitative PCR |
Non-Patent Citations (5)
| Title |
|---|
| HIROKI HIRAYAMA ET AL.: ""Enhanceme nt of maternal recognition of pregnancy with parthenogenetic embryos in bovine embryo transfer"", 《THERIOGENOLOGY》 * |
| YUTAKA HASHIYADA ET AL.: ""Transition of the Pregnancy Rate of Bisected Bovine Embryos after Co-transfer with Trophoblastic Vesicles Prepared from In Vivo-cultured In Vitro-fertilized Embryos"", 《JOURNAL OF REPRODUCTION AND DEVELOPMENT》 * |
| 任艳 等: ""牛胚胎滋养层巨细胞的体外分离培养"", 《细胞生物学杂志》 * |
| 左彦珍 等: ""不同分离纯化法建立人早孕滋养细胞模型的效果比较"", 《广东医学》 * |
| 王福兆: "《乳牛学》", 31 May 2004, 科学技术文献出版社 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN107723272B (en) | 2021-05-18 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Atkins et al. | Pregnancy establishment and maintenance in cattle | |
| Anouassi et al. | Development of a large commercial camel embryo transfer program: 20 years of scientific research | |
| Edwards | IVF, IVM, natural cycle IVF, minimal stimulation IVF–time for a rethink | |
| CN100998524A (en) | Method for generating sex controlled ox-embryo | |
| Soares et al. | Timing of insemination using sex-sorted sperm in embryo production with Bos indicus and Bos taurus superovulated donors | |
| CN103479746A (en) | New application of traditional Chinese medicine compound medicinal composition | |
| CN103184189A (en) | Cultivation method of cross-bred wagy | |
| CN110808099A (en) | System and method for detecting ectopic pregnancy | |
| Saad et al. | Effect of plasma progesterone on oocyte recovery, oocyte quality, and early in-vitro developmental competence of embryos in Bos indicus dairy cows | |
| Lee et al. | Expression of DAZ (deleted in azoospermia), DAZL1 (DAZ-like) and protamine-2 in testis and its application for diagnosis of spermatogenesis in non-obstructive azoospermia. | |
| CN103598146B (en) | A kind of method utilizing full-length genome to select to cultivate good species bull with sex control embryo technology | |
| CN110042078B (en) | Oocyte in-vitro maturation culture solution and preparation method and application thereof | |
| WO2012113124A1 (en) | New method for improving transgenic efficiency in animals | |
| Blawut et al. | Testicular collections as a technique to increase milt availability in sauger (Sander canadensis) | |
| CN103648435A (en) | Method for identifying a mammal receptive to embryo transfer | |
| Kawarasaki et al. | Sexing of porcine embryo by in situ hybridization using chromosome Y-and 1-specific DNA probes | |
| CN107723272A (en) | A kind of preparation method of cattle early embryo Trophoblast tissue | |
| Simões et al. | Nuclear maturation kinetics and in vitro fertilization of immature bovine oocytes injected into pre-ovulatory follicles | |
| Crowe et al. | A retrospective study of artificial insemination of 251 mares using chilled and fixed time frozen‐thawed semen | |
| CN109680074B (en) | Method for judging success or failure of cow artificial fertilization based on integrin expression | |
| CN109055574B (en) | Detection method for determining pregnancy of sow by using urine of sow | |
| CN101259043A (en) | Embryo transplantation acceptor cattle estrus synchronization method | |
| Afriani et al. | Response of Superovulation by Using FSH (Follicle Stimulating Hormone) and Sex Determination of Embryos Using PCR in Pesisir Cows of West Sumatra | |
| Mogase et al. | Applicability of Day 0 superovulation protocol in Boer goats | |
| Huang et al. | Generation of cynomolgus monkey fetuses with intracytoplasmic sperm injection based on the MII-stage oocytes acquired by personalized superovulation protocol |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |