CN105647853A - Method for improving development quality of in vitro fertilization female embryos after implantation - Google Patents
Method for improving development quality of in vitro fertilization female embryos after implantation Download PDFInfo
- Publication number
- CN105647853A CN105647853A CN201610115860.9A CN201610115860A CN105647853A CN 105647853 A CN105647853 A CN 105647853A CN 201610115860 A CN201610115860 A CN 201610115860A CN 105647853 A CN105647853 A CN 105647853A
- Authority
- CN
- China
- Prior art keywords
- tretinoin
- liquid
- ivf
- added
- incubator
- 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
- C12N5/0604—Whole embryos; Culture medium therefor
-
- 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
- C12N2500/00—Specific components of cell culture medium
- C12N2500/30—Organic components
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Biomedical Technology (AREA)
- Genetics & Genomics (AREA)
- Zoology (AREA)
- Gynecology & Obstetrics (AREA)
- Biotechnology (AREA)
- Organic Chemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Developmental Biology & Embryology (AREA)
- Chemical & Material Sciences (AREA)
- Wood Science & Technology (AREA)
- Reproductive Health (AREA)
- Microbiology (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Cell Biology (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
Abstract
The invention provides a method for improving development quality of in vitro fertilization (IVF) female embryos after implantation and correcting sex ratio of IFV embryos. The method comprises: transferring mouse fertilized embryos obtained by IVF technology into a culture solution added with low-dose retinoic acid (RA) for short-term culturing, a female specific apparent modification event (namely x-chromosomal inactivation) in early development of IFV embryos is established normally so that an abnormal development ratio of mouse IFV female embryos after implantation is reduced and the mouse IVF birth sex ratio imbalance problem is finally corrected. In addition, the method is successfully applied in a bovine IVF system, effectively increasing development rate of bovine IFV female blastulas and correcting the sex ratio of bovine IFV blastulas. The RA in the invention is used as a fallopian tube to widely express an endogenous active matter, this matter is suitably added in the IVF system with endogenous ingredients not blindly added, and application safety is greatly ensured.
Description
Technical field
The present invention relates to sexual reproduction technique field, specifically, relate to a kind of improving the attached method planting rear development quality of external fertilization female embryo.
Background technology
External fertilization (invitrofertilization, IVF; I.e. test-tube baby) be widely used in treatment the mankind sterile. At present, the whole world is existing is born more than the test-tube baby of 5,400,000 examples, and the speed in 350,000 examples/year is increased sharply. Additionally, IVF is also widely used in the breeding expanding propagation of domestic animal, for instance in cattle embryo transfer, IVF embryo proportion has reached 40%. Although IVF exists so wide application space, but IVF still suffers from many restraining factors, for instance: developmental rate is low, pregnancy loss, premature labor, birth defect, gender imbalance, suffers from diabetes, risk of hypertension increases. At present, for these IVF defects, mainly through changing the physical environment of embryo in vitro operation, by seminal fluid, culture fluid composition, adding small-molecule substance etc., this progress is slow.
In recent years, gender im-balance's problem that IVF causes obtains extensive concern. From 1991, there is gender imbalance problem (male many, female few) in the reported first cattle IVF blastaea such as AveryB, cattle, pig IVF research afterwards repeatedly confirms this problem. Recently, there is Birth sex ratio unbalance (male many female are few) too in hundreds thousand of example people's Epidemiological studys display people IVF that Oceania, Britain launch respectively. At present, for this problem, a kind of improvement mode is interpolation small-molecule substance in culture fluid. Such as, the culture fluid of pig IVF embryo is replaced and adds different materials, find hyaluronic acid (hyaluronicacid, HA) it is remarkably improved blastocyst rate, but the more serious (TornerE of gender imbalance problem can be made, BussalleuE, BrizMD, YesteM, BonetSEmbryodevelopmentandsexratioofinvitro-producedporc ineembryosareaffectedbytheenergysubstrateandhyaluronicac idaddedtotheculturemedium.ReprodFertilDev2014;26:570-577.). Cattle IVF embryo medium adds zwischen-ferment inhibitive factor DHEA and 6-AN, the sex ratio of cattle IVF blastaea can be significantly improved, but the growth efficiency (KimuraK reducing cattle IVF embryo is comprehended in this interpolation place, SpateLD, GreenMP, RobertsRMEffectsofD-glucoseconcentration, D-fructose, andinhibitorsofenzymesofthepentosephosphatepathwayonthed evelopmentandsexratioofbovineblastocysts.MolReprodDev200 5; 72:201-207.). At present, caused by IVF technology, all solutions of gender im-balance's problem all do not produce a desired effect.
Summary of the invention
It is an object of the invention to provide and a kind of improve the attached method planting rear development quality of external fertilization female embryo.
The present invention is based on the mechanism announcement of early-stage Study, in fetal development early stage, wide expression tretinoin synthesis related gene (Fig. 1) (MohanM in the embryo of the different plant species such as mice, cattle itself and residing fallopian tube, MalayerJR, GeisertRD, MorganGLExpressionpatternsofretinoidXreceptors, retinaldehydedehydrogenase, andperoxisomeproliferatoractivatedreceptorgammainbovinep reattachmentembryos.BiolReprod2002; 66:692-700.EvertsHB, SundbergJP, OngDEImmunolocalizationofretinoicacidbiosynthesissystems inselectedsitesinrat.ExpCellRes2005; 308:309-319.), it was shown that tretinoin (RA) is likely to play a significant role in fetal development in early days. According to the studies above result, the present invention optimizes IVF system targetedly, supplements the key molecule that IVF system lacks root, rather than selection external source active substance blindly is added processing.
In order to realize the object of the invention, present invention firstly provides tretinoin application in development quality after improving that external fertilization female embryo is attached and planting.
The present invention provides a kind of and improves the attached method planting rear development quality of external fertilization female embryo, the germ cell obtained by IVF technology is moved in the culture fluid being added with 1-50nM tretinoin and cultivates, grow to blastaea, then be transplanted in maternal uterine, grow and form fetus.
Described germ cell is to obtain mammal M II phase oocyte by hormone superovulation, then carries out the germ cell of external fertilization acquisition.
In one embodiment of the invention, the mouse fertilized egg obtained by IVF technology is moved in the culture fluid KSOM+AA being added with 1-20nM tretinoin, in 37 DEG C, 5%CO2, saturated humidity CO2In incubator, lucifuge cultivates 24-48h, then moves in the culture fluid KSOM+AA not containing tretinoin, in 37 DEG C, 5%CO2, saturated humidity CO2Incubator continues cultivate, grow to blastaea (mice embryonic amounts to 88-90h from by the precise and penetrating incubation time being developed to blastaea).
Preferably, the mouse fertilized egg obtained by IVF technology is moved in the culture fluid KSOM+AA being added with 5nM tretinoin, in 37 DEG C, 5%CO2, saturated humidity CO2In incubator, lucifuge cultivates 36h, then moves in the culture fluid KSOM+AA not containing tretinoin, in 37 DEG C, 5%CO2, saturated humidity CO2Incubator continues cultivate 52-54h, grow to blastaea.
Wherein, the preparation method of the culture fluid KSOM+AA being added with 5nM tretinoin described in is as follows: with DMSO, tretinoin is configured to the dense storage of 0.1mM ,-20 DEG C of preservations after 10 �� l/ pipe subpackages when lucifuge;Tretinoin adds culture fluid to be needed now with the current, before using, when lucifuge, draw 10 �� lKSOM+AA liquid and add in dense storage pipe, after mixing, obtain mixed liquor I, draw 10 �� l mixed liquors I and add in 990 �� lKSOM+AA liquid, continue mixing, obtain mixed liquor II, draw 20 �� l mixed liquors II again and add in 1980 �� lKSOM+AA liquid, after mixing, the culture fluid KSOM+AA of 5nM tretinoin must be added with. For making Medium drop. Above-mentioned repeatedly dilute operation, not only can ensure that tretinoin adds the accurate of concentration, and DMSO residual in culture fluid can be reduced.
In another embodiment of the invention, the fertilized bovine oocytes obtained by IVF technology is moved into the early stage being added with 5-50nM tretinoin and grows in liquid, in 39 DEG C, 5%CO2, saturated humidity CO2In incubator, lucifuge cultivates 48h, then moves in the later stage growth liquid being added with 5-50nM tretinoin, in 39 DEG C, 5%CO2, saturated humidity CO2In incubator, lucifuge cultivates 24-48h, then moves in the later stage growth liquid not containing tretinoin, in 39 DEG C, 5%CO2, saturated humidity CO2Incubator continues cultivate, grow to blastaea (cattle embryo amounts to 7d from by the precise and penetrating incubation time being developed to blastaea).
It is CR1 liquid+6mg/mlBSA that described early stage grows liquid; It is CR1 liquid+10%FBS that the described later stage grows liquid.
Preferably, the fertilized bovine oocytes obtained by IVF technology is moved into the early stage being added with 20nM tretinoin and grows in liquid, in 39 DEG C, 5%CO2, saturated humidity CO2In incubator, lucifuge cultivates 48h, then moves in the later stage growth liquid being added with 20nM tretinoin, in 39 DEG C, 5%CO2, saturated humidity CO2In incubator, lucifuge cultivates 36h, then moves in the later stage growth liquid not containing tretinoin, in 39 DEG C, 5%CO2, saturated humidity CO2Incubator continues cultivate 3.5d, grow to blastaea.
The gender imbalance problem that the present invention causes around IVF technology, it is model animal first with mice, by disclosing its mechanism, find to have caused specific apparent modification mistake mainly due to IVF, namely x chromosome inactivation is not enough, cause the tendentious abnormal development of female embryo, ultimately result in Birth sex ratio unbalance. For this, utilize this endogenous material of tretinoin, by the optimization of the conditions such as a series of concentration for the treatment of, time, effectively reduce the abnormal development ratio of IVF female embryo, the correction of a final proof problem of IVF fetal birth gender imbalance. Further, it is added processing in cattle IVF system, successfully improves the developmental rate of the female blastaea of cattle IVF, and correct the sex ratio of cattle IVF blastaea.
The inventive method is applicable to optimize the IVF culture system in vitro of people and mammal (including mice, cattle etc.), improves the development quality of fetal female; Increase IVF and obtain the probability of female descendant, there is wide clinical practice and market prospect, produce considerable economic benefit and good social benefit. In culture fluid, add tretinoin be applied to human ancillary reproductive, it is possible to decrease the probability of abnormal development of fetus, make sterile family directly be benefited, effectively reduce the risk that Birth sex ratio is unbalance, be conducive to long-term social stability. The method is applied to domestic animal breeding expanding propagation, the sex ratio of active balance IVF offspring.
Accompanying drawing explanation
Fig. 1 is the PCR testing result of mice tretinoin synthesis related gene; Wherein, after bolt is shown in mice copulation, germ cell (copulation see bolt after 8-10h), 2-cell (copulation see bolt after 26-28h), 4-cell (copulation see bolt after 40-42h), 8-cell (copulation see bolt after 46-48h) is collected in different time points, and the fallopian tube of correspondence time point, carry out PCR detection.
Detailed description of the invention
Following example are used for illustrating the present invention, but are not limited to the scope of the present invention. If not specializing, the conventional means that technological means used in embodiment is well known to those skilled in the art, raw materials used it is commercial goods.
Culture fluid KSOM+AA purchased from American Millipore company, tretinoin available from Sigma, article No. R2500-100MG in following example.
Embodiment 1 mouse in vitro fertilization Embryo Production
1, external fertilization
Female Mus mouse peritoneal injection 5IUPMSG (Ningbo the second hormone factory) of ICR of 7��8 week old, injects 5IUhCG (Ningbo the second hormone factory) after 47��48h. 13h after injection hCG, cervical dislocation puts to death mice, dissect clip fallopian tube, the M2 being placed in preheating 37 DEG C operates in liquid (Sigma company), magnum tubae uterinae is scratched with syringe under stereomicroscope, collecting cumulus oocyte complex (ripe M II phase oocyte), the M2 moving to preheating 37 DEG C operates in liquid after abundant washing, stand-by. De-for the public Mus (10��12 week old) of adult healthy neck is put to death, cut off abdominal cavity with the shears of sterilizing, take tail of epididymis, with tweezers and shears, the sperm in tail of epididymis is extruded, and put it in the 100 �� lHTF liquid (SAGE company) that pre-equilibration is good, it is placed in 37 DEG C, 5%CO2Incubator is hatched 1h, completes capacitation, after sperm count, draw 1 �� 106The capacitated sperm of left and right, to the HTF liquid filling oocyte, carries out fertilization 4��6h.
2, In vitro culture
Matched group: fully wash germ cell with the mice embryonic culture fluid KSOM+AA balanced, moves into the good KSOM+AA of pre-balance and cultivates the continuous cultivation of drop relaying, and every 30 pieces of embryos put in 60 �� l Medium drop, are placed in 37 DEG C, 5%CO2, saturated humidity CO2Incubator is cultivated 88-90h, grows to blastaea.
Experimental group: fully wash germ cell with the mice embryonic culture fluid KSOM+AA balanced, moves into pre-balance good, is added with in the KSOM+AA culture fluid microdroplet of 5nM tretinoin 37 DEG C, 5%CO2, saturated humidity CO2In incubator, lucifuge cultivates 36h, then moves in the culture fluid KSOM+AA not containing tretinoin, in 37 DEG C, 5%CO2, saturated humidity CO2Incubator continues cultivate 52-54h, grow to blastaea.
Wherein, the preparation method of the culture fluid KSOM+AA being added with 5nM tretinoin is as follows: with DMSO, tretinoin is configured to the dense storage of 0.1mM ,-20 DEG C of preservations after 10 �� l/ pipe subpackages when lucifuge; Tretinoin adds culture fluid to be needed now with the current, before using, when lucifuge, draw 10 �� lKSOM+AA liquid and add in dense storage pipe, after mixing, obtain mixed liquor I, draw 10 �� l mixed liquors I and add in 990 �� lKSOM+AA liquid, continue mixing, obtain mixed liquor II, draw 20 �� l mixed liquors II again and add in 1980 �� lKSOM+AA liquid, after mixing, the culture fluid KSOM+AA of 5nM tretinoin must be added with. For making Medium drop. Above-mentioned repeatedly dilute operation, not only can ensure that tretinoin adds the accurate of concentration, and DMSO residual in culture fluid can be reduced.
3, blastaea uterine transplantation
The ICR female mice of 8 weeks of picking spontaneous estrus and ligation hero Mus mate, and morning next day examines bolt. Having the female Mus of bolt for transplanting, the same day of inspection bolt is the 1st day (D1) of gestation, is implanted in D4 8:00-11:00 in the morning and carries out. Pseudo-fetus Mus intraperitoneal injection of anesthesia agent (Sigma company during transplanting, tribromoethanol, T48402) 0.4ml, after mouse anesthesia, operative site local cropping, sterilization, makes an opening at mouse back spinal column near last 1 root bone trailing edge place with surgery shears, tears muscle layer with tweezers and peritoneum exposes abdominal viscera, ovarian fat pad is clamped by external for the pull-out of ovary, fallopian tube and part uterus with tweezers, clamping fixed with fat.Sting an aperture location with syringe needle at cornua uteri end, draw the blastaea in M2 liquid with transplanting pin, insert along aperture, move gently up and down and transplant pin, when determining that the needle point transplanting pin is in cornua uteri, slowly embryo is injected. Every side cornua uteri transplants 6 pieces of embryos.
4, experimental result
In the present embodiment, mice embryonic is after 5nM retinoic acid treatments 36h, and blastocyst rate does not have significant change (matched group: 67.3% �� 2.8%; Interpolation group: 64.5% �� 2.2%), and the ratio of the inner cell mass of blastaea/trophoblastic cell number is absent from significantly changing (matched group: 2.54 �� 0.61; Interpolation group: 2.50 �� 0.58), this shows that the potentiality of development of blastaea is not damaged. Additionally, compared with matched group, the sex ratio of Mouse Blastocysts is also without the significantly affected (hero of matched group/female: 1.01 (n=524); The hero of interpolation group/female: 1.03 (n=262)). Result above shows to adopt retinoic acid treatments that the growth of IVF embryo is free from side effects.
After the blastaea obtained carries out uterine transplantation, interpolation group abnormal development of fetus ratio significantly reduces (matched group: 10.1%; Interpolation group: 4.2%); The sex ratio of perinatal stage viable fetus significantly improves, the hero of matched group/female: 1.34 (n=502); The hero of interpolation group/female: 1.08 (n=296). These results show that retinoic acid treatments can improve the attached development quality planting rear mice fetal female.
2 Ns of IVF Embryos of embodiment produce
1, external fertilization
By the ripe M II phase oocyte obtained by hormone superovulation by putting into the 50 �� l balanced after seminal fluid (BO liquid+10mM caffeine+3mg/mlBSA) cleans 3 times by (15 pieces/drip) in seminal fluid microdroplet; Floating method is adopted to carry out capacitation, sperm is washing seminal fluid (BO liquid+20 �� g/ml heparin sodium+6mg/mlBSA) floating 20-30min, takes supernatant 600-800 �� l afterwards and puts into 1.5ml centrifuge tube centrifugal (1500 turns, centrifugal 5min), abandon supernatant, repeated washing 1 time. Remove supernatant after centrifugal, add and wash seminal fluid, sperm count, then take the seminal fluid after 50 �� l process add put into oocyte by seminal fluid, sperm final concentration of 1 �� 106Sperm/ml, condition of culture is 39 DEG C, 5%CO2, saturated humidity, fertilization time 8h.
2, In vitro culture
Matched group: germ cell grows liquid (CR1 liquid+6mg/mlBSA in the early stage balanced, four orifice plate two stage culture are adopted: first grow in 500 �� l early stages and liquid (about 50 pieces) is cultivated 2d after washing 3 times in 2ml), moving into for 500 �� l later stages after counting spilting of an egg embryo grows cultivation 5d in liquid (CR1 liquid+10%FBS), interval 2d half amount changes liquid, the 7th day counting blastaea of fetal development. Condition of culture is 39 DEG C, 5%CO2, saturated humidity, incubation time 7d.
Experimental group: germ cell grows liquid (CR1 liquid+6mg/mlBSA in the early stage balanced, four orifice plate two stage culture are adopted: first grow lucifuge in liquid (about 50 pieces) in the 500 �� l early stages adding 20nM tretinoin and cultivate 48h after washing 3 times in 2ml), the 500 �� l later stages that then spilting of an egg embryo moves into interpolation 20nM tretinoin grow lucifuge cultivation 36h in liquid (CR1 liquid+10%FBS), moving into all embryos to grow without 500 �� l later stages of tretinoin again and continue to cultivate 3.5d in liquid (CR1 liquid+10%FBS), fetal development counts blastaea on the 7th day. Condition of culture is 5%CO2Air, temperature 39 DEG C, saturated humidity.
3, experimental result
After being identified by PCR, sex carrying out ASSOCIATE STATISTICS (table 1), result is compared with matched group, and the male blastaea of experimental group accounts for the ratio of total spilting of an egg embryo and do not have significant change (P > 0.05);The female blastaea of experimental group accounts for the ratio of total spilting of an egg embryo and significantly raises (P < 0.05). Additionally, the sex ratio of blastaea significantly improves.
Table 1 retinoic acid treatments statistics to cattle IVF blastaea developmental state
Note: a, b represent significant difference (P < 0.05);Sex ratio is the result compared with expected value (1.00), and method is X 2 test (* P < 0.05).
Although, above the present invention is described in detail with a general description of the specific embodiments, but on basis of the present invention, it is possible to it is made some modifications or improvements, and this will be apparent to those skilled in the art. Therefore, these modifications or improvements without departing from theon the basis of the spirit of the present invention, belong to the scope of protection of present invention.
Claims (8)
1. tretinoin application in development quality after improving that external fertilization female embryo is attached and planting.
2. one kind is improved the attached method planting rear development quality of external fertilization female embryo, it is characterized in that, the germ cell obtained by IVF technology is moved in the culture fluid being added with 1-50nM tretinoin and cultivates, grow to blastaea, it is transplanted to again in maternal uterine, grows and form fetus.
3. method according to claim 2, it is characterised in that it is to obtain mammal M II phase oocyte by hormone superovulation, then carries out external fertilization and obtains germ cell.
4. method according to claim 3, it is characterised in that the mouse fertilized egg obtained by IVF technology is moved in the culture fluid KSOM+AA being added with 1-20nM tretinoin, in 37 DEG C, 5%CO2, saturated humidity CO2In incubator, lucifuge cultivates 24-48h, then moves in the culture fluid KSOM+AA not containing tretinoin, in 37 DEG C, 5%CO2, saturated humidity CO2Incubator continues cultivate, grow to blastaea.
5. method according to claim 4, it is characterised in that the mouse fertilized egg obtained by IVF technology is moved in the culture fluid KSOM+AA being added with 5nM tretinoin, in 37 DEG C, 5%CO2, saturated humidity CO2In incubator, lucifuge cultivates 36h, then moves into the culture fluid KSOM+AA not containing tretinoin, in 37 DEG C, 5%CO2, saturated humidity CO2Incubator continues cultivate 52-54h, grow to blastaea.
6. method according to claim 5, it is characterised in that described in be added with 5nM tretinoin the preparation method of culture fluid KSOM+AA as follows:
With DMSO, tretinoin is configured to the dense storage of 0.1mM ,-20 DEG C of preservations after 10 �� l/ pipe subpackages when lucifuge; Tretinoin adds culture fluid to be needed now with the current, before using, when lucifuge, draw 10 �� lKSOM+AA liquid and add in dense storage pipe, after mixing, obtain mixed liquor I, draw 10 �� l mixed liquors I and add in 990 �� lKSOM+AA liquid, continue mixing, obtain mixed liquor II, draw 20 �� l mixed liquors II again and add in 1980 �� lKSOM+AA liquid, after mixing, the culture fluid KSOM+AA of 5nM tretinoin must be added with.
7. method according to claim 3, it is characterised in that the fertilized bovine oocytes obtained by IVF technology is moved into the early stage being added with 5-50nM tretinoin and grows in liquid, in 39 DEG C, 5%CO2, saturated humidity CO2In incubator, lucifuge cultivates 48h, then moves in the later stage growth liquid being added with 5-50nM tretinoin, in 39 DEG C, 5%CO2, saturated humidity CO2In incubator, lucifuge cultivates 24-48h, then moves in the later stage growth liquid not containing tretinoin, in 39 DEG C, 5%CO2, saturated humidity CO2Incubator continues cultivate, grow to blastaea;
It is CR1 liquid+6mg/mlBSA that described early stage grows liquid; It is CR1 liquid+10%FBS that the described later stage grows liquid.
8. method according to claim 7, it is characterised in that the fertilized bovine oocytes obtained by IVF technology is moved into the early stage being added with 20nM tretinoin and grows in liquid, in 39 DEG C, 5%CO2, saturated humidity CO2In incubator, lucifuge cultivates 48h, then moves in the later stage growth liquid being added with 20nM tretinoin, in 39 DEG C, 5%CO2, saturated humidity CO2In incubator, lucifuge cultivates 36h, then moves in the later stage growth liquid not containing tretinoin, in 39 DEG C, 5%CO2, saturated humidity CO2Incubator continues cultivate 3.5d, grow to blastaea.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610115860.9A CN105647853B (en) | 2016-03-01 | 2016-03-01 | A method of improving development quality after the attached plant of female embryo in vitro fertilization |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610115860.9A CN105647853B (en) | 2016-03-01 | 2016-03-01 | A method of improving development quality after the attached plant of female embryo in vitro fertilization |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN105647853A true CN105647853A (en) | 2016-06-08 |
| CN105647853B CN105647853B (en) | 2019-07-05 |
Family
ID=56492057
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201610115860.9A Active CN105647853B (en) | 2016-03-01 | 2016-03-01 | A method of improving development quality after the attached plant of female embryo in vitro fertilization |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN105647853B (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107523538A (en) * | 2017-09-30 | 2017-12-29 | 江苏农林职业技术学院 | A kind of placenta in vitro culture method |
| CN113801840A (en) * | 2021-08-26 | 2021-12-17 | 武汉纤然生物科技有限公司 | Operating fluid for improving mouse in-vitro fertilization efficiency and using method thereof |
| WO2024087350A1 (en) * | 2022-10-27 | 2024-05-02 | 南京医科大学 | Method for constructing short-telomere mouse model |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20020028849A1 (en) * | 2000-04-18 | 2002-03-07 | Godkin James D. | Use of retinol in assisted-reproduction protocols |
-
2016
- 2016-03-01 CN CN201610115860.9A patent/CN105647853B/en active Active
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20020028849A1 (en) * | 2000-04-18 | 2002-03-07 | Godkin James D. | Use of retinol in assisted-reproduction protocols |
Non-Patent Citations (3)
| Title |
|---|
| AHN JY等: "Retinoic acid accelerates downregulation of the Xist repressor, Oct4, and increases the likelihood of Xist activation when Tsix is deficient", 《BMC DEVELOPMENTAL BIOLOGY》 * |
| K TAN等: "IVF affects embryonic development in a sex-biased manner in mice", 《REPRODUCTION》 * |
| LS TAHAEI等: "Effects of retinoic acid on maturation of immature mouse oocytes in the presence and absence of a granulosa cell co-culture system", 《JOURNAL OF ASSISTED REPRODUCTION & GENETICS》 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107523538A (en) * | 2017-09-30 | 2017-12-29 | 江苏农林职业技术学院 | A kind of placenta in vitro culture method |
| CN107523538B (en) * | 2017-09-30 | 2020-08-25 | 江苏农林职业技术学院 | In-vitro culture method for embryo body |
| CN113801840A (en) * | 2021-08-26 | 2021-12-17 | 武汉纤然生物科技有限公司 | Operating fluid for improving mouse in-vitro fertilization efficiency and using method thereof |
| WO2024087350A1 (en) * | 2022-10-27 | 2024-05-02 | 南京医科大学 | Method for constructing short-telomere mouse model |
Also Published As
| Publication number | Publication date |
|---|---|
| CN105647853B (en) | 2019-07-05 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN109628380B (en) | Human body external receptor semen and preparation method thereof | |
| CN105052894B (en) | A kind of GV phases egg mother cell freezen protective liquid and freezing and storing method | |
| CN104688380B (en) | Method for improving superovulating quantity and quality of sheep | |
| CN103184189A (en) | Cultivation method of cross-bred wagy | |
| Brock et al. | The production of viable bovine ova | |
| CN102899286A (en) | Application of C-type natriuretic peptide to promotion on in vitro maturation of bovine oocyte | |
| CN105647853A (en) | Method for improving development quality of in vitro fertilization female embryos after implantation | |
| CN103898048A (en) | In vitro maturation culture method of denuded oocytes of mice | |
| CN107142239A (en) | The method for improving ox IVF Embryos culture efficiency | |
| CN110066763A (en) | Promote the method for ox embryo in vitro culture development of fertilized ova | |
| CN101962626B (en) | Calf in vitro embryo culture solution | |
| CN107460162A (en) | A kind of method for improving lamb extracorporeal embryo development ability | |
| CN113767896B (en) | Bovine in vitro fertilization embryo vitrification freezing method | |
| CN101555466B (en) | Sheep embryo in-vitro culture solution containing astragalus polysaccharide and culture method thereof | |
| CN104152404B (en) | Embryo heat resistance improving culture solution and using method thereof | |
| CN107043743A (en) | The method for in-vitro maturity of dog egg mother cell | |
| CN106520675A (en) | In-vitro embryo culture solution containing Clusterin protein and application of in-vitro embryo culture solution in embryo cryopreservation | |
| CN1226378A (en) | Technology for crossbreeding sheep in 'tubes' | |
| CN111919836A (en) | Serum-free cryopreservation protective agent for bovine testicular tissues, cryopreservation method and application | |
| CN103461323B (en) | One can be used for external time delay and preserves the bioactive method of egg mother cell | |
| CN101962627A (en) | Method for producing in-vitro calf embryo | |
| CN105316282A (en) | Acipenser dabryanus spermatogonium culture solution and application thereof | |
| Garcia-Garcia et al. | Culture of early stage ovine embryos to blastocyst enhances survival rate after cryopreservation | |
| CN102344907A (en) | Method for activating reconstructed embryo | |
| CN1226377A (en) | Process for industrializing technology of 'tube cattle' |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |