150 results about "In vitro maturation" patented technology

A method of in vitro maturation of adult human germ line cells in an artificial biological environment, which entails:a) isolating human spermatogonial stem cells (SSCs), and optionally purifying the same; andb) co-culturing the isolated and optionally purified SSCs with a suitably adjusted Sertoli cell environment to obtain haploid germ cells.

Described is the efficient and robust generation of oligodendrocyte progenitor cells (OPCs) and oligodendrocytes from pluripotent stem cells (PSCs). The protocols provided recapitulate the major steps of oligodendrocyte differentiation, from neural stem cells to OLIG2+ progenitors, and then to 04+ OPCs, in a significantly shorter time than the 120-150 days required by previous protocols. Furthermore, 04+ OPCs are able to differentiate into MBP+ mature oligodendrocytes in vitro, and to myelinate axons in vivo when injected into immuno-compromised Shiverer mice, providing proof of concept that transplantation of PSC-derived cells for remyelination is technically feasible.

The invention relates to a method for improving in-vitro production efficiency of buffalo embryos, comprising the following steps of: respectively adding 10 muL / mL of insulin-transferrin-selenium (ITS) and 20 muL / mL of insulin-transferrin-selenium (ITS) to an oocyte maturing solution and a culture solution, and then carrying out in-vitro maturation and in-vitro culture. The method indicates that the in-vitro maturation rate of buffalo oocyte is effectively improved by adding 10 muL / mL ITS to the maturing solution, and the exhaustion ratio of a first polar body is up to 65.61%; and adding 20 muL / mL of ITS to the embryo culture solution is beneficial to the growth of early embryos of a fertilized buffalo, and the growing rate of a blastaea is up to 29.93%. the method has the advantages of improving the in-vitro maturation rate of the buffalo oocyte and the growing rate of the blastaea, thereby improving the in-vitro production efficiency of the buffalo embryos and providing a large number of high-quality embryos for embryo transplantation.

The invention provides a method for in vitro culture of porcine oocytes, which comprises the steps of ovary acquisition, acquisition of oocytes from an ovary, screening of the oocytes, and maturation culture. The method is characterized in that the acquired ovary is stored in a container of physiological saline for preservation before the step of the acquisition of the oocytes; and the temperature in the container is maintained to between 32 and 34 DEG C; an egg washing liquid adopted in the step of the screening of the oocytes contains amphotericin B with a concentration of between 1 and 10mu g / mL, and serum with a volume percentage of between 2 and 20 percent v / v; and the maturation medium adopted in the step of the maturation culture of the oocytes contains a TCM-199[1X] basic medium with a volume percentage of between 50 and 90 percent v / v, hGG with an antibody titer of between 5 and 25IU / mL and PMSG with an antibody titer of between 5 and 20IU / mL. The method can solve the problems that the prior in vitro maturation technology of the porcine oocytes has long period, is not good for mass production, and has low maturation rate after the culture.

The invention relates to a method for culturing an in-vitro fertilization embryo of an oocyte collected from a living cow or in vitro. The method comprises the following steps: collection and in-vitromaturation of the oocyte, wherein the oocyte can be collected in vitro or from the living cow; in-vitro fertilization, wherein mature COCs is cultured in a fertilization culture solution and then themature COCs and sperm suspension are subjected to co-incubation culture; and in-vitro culture of the embryo and cryopreservation in liquid nitrogen. The method has the excellent technical effect as shown in specification.

The present invention relates to a method of producing an embryo from an oocyte by an assisted reproduction technology. The method includes (a) collecting an oocyte from an ovary of a subject in a collection medium comprising a first phosphodiesterase inhibitor and an agent that increases intracellular cAMP concentration in the oocyte, (b) culturing the oocyte in a maturation medium comprising a second phosphodiesterase inhibitor, and (c) producing an embryo from the oocyte by an assisted reproduction technology. The present invention also relates to methods of inducing oocyte maturation. For example a method of in vitro maturation of an oocyte is described which comprises steps (a) and (b) above. The present invention also relates to an oocyte maturation medium comprising a phosphodiesterase inhibitor and a ligand for inducing maturation of the oocyte. A combination product comprising an oocyte collection and maturation medium referred to above is also described.

The invention relates to a bovine oocyte in-vitro fertilization and embryo culture method, and a transport culture solution. On one hand, the transport culture solution comprises glycine, alanine, arginine hydrochloride, aspartic acid, cystine dihydrochloride, glutamic acid, L-glutamine, histidine, threonine, tryptophan, tyrosine, valine, ascorbic acid, biotin, choline chloride, calcium pantothenate, folic acid, menadione and the like. The bovine oocyte in-vitro fertilization and embryo culture method comprises the following steps: oocyte collection and in-vitro maturation, in-vitro fertilization, and embryo in-vitro culture and preservation. The method and the related transport culture solution have excellent technical effects as described in the specification.

The invention discloses a porcine oocyte in vitro maturation medium and methods of preparation and culture. The medium comprises a TCM-199 base medium and the added porcine follicular fluid, fetal bovine serum, eCG, hCG, EGF and IGF-1. The porcine oocyte in vitro maturation medium of the invention can significantly increase the rate of maturation of porcine oocytes in vitro. Experiments confirm that after the oocyte collection and 42-44h maturation, the medium of the invention can have a polar body rate stabilized at 80% and a parthenogenetic embryo development rate up to 85%, and the entire maturation process does not require replacement of the medium and is easy to operate.

The invention discloses an in-vitro maturating method for sheep oocyte, a pretreatment solution and a kit. The related pretreatment solution applied to in-vitro maturating of sheep oocyte is characterized by comprising sheep small follicle fluid and routine culture-solution compositions for in-vitro maturating of sheep oocyte. The disclosed in-vitro maturating method for sheep oocyte is characterized in that a pretreatment step is increased on the basis of a conventional in-vitro maturating method. Also, experiments in the invention verify that oosperm obtained after an oocyte which is cultured by employing the maturating method is subjected to in-vitro fertilization has the cleavage rate and the blastocyst rate both substantially higher than those of an oocyte cultured by employing a conventional maturating method. The provided pretreatment solution and the in-vitro maturating culturing method are capable of effectively promoting in-vitro maturation of sheep oocyte, and have the characteristics of no hazard to occyte, few limits, good effect and the like.

The invention relates to a serum free culture medium for in vitro maturation of bovine oocyte, belonging to the field of cytobiology, in particular to a serum free medium IVMBM-I for in vitro maturation of bovine oocyte, comprising a TCM199 medium, hormone for oocyte maturation and antibacterial agent, which is characterized in that: Sodium pyruvate, Taurine, Insulin, Selenium, BSA, HEPES, TGF-a, L-Glutamine and EGF are added, and the serum is not contained. The serum free culture medium for in vitro maturation of bovine oocyte has the advantages of avoiding the adverse effect of some components in the serum on the oocyte, exact quantification and good repeatability because the conventional serum is substituted by the added components, and that the function of the serum in the maturation process of the oocyte can be completely substituted by the added components is proved by the experiments of in vitro maturation, in vitro fertilization and in vitro development.

The present invention provides a composition and method for making a modified germ cell (MGC) comprising a primordial sex cell, (PSC) or nucleus thereof, combined with an enucleated ovum. The PSC is a spermatogonia or an oogonia from a donor animal or mammal. Similarly, the enucleated ovum is from the same species donor animal or mammal as the donor of the PSC. The present invention also provides a method of maturing the MGC in vitro using a specialized cell culture apparatus or bioreactor chamber. The present invention also provides for a method of screening the MGCs for receptor sites, to determine if they are sufficiently matured to be translocated into a host animal or mammal in vivo and / or tissue in vitro. The present invention also provides for a specialized cell culture chamber or bioreactor chamber to grow, expand, maintain, sustain and mature the MGCs, or other types of cells.

The invention relates to a simple, economic and efficient method for the in-vitro maturity of porcine oocytes, which comprises the steps of collecting an ovary, collecting oocytes from the ovary, sieving the oocytes and maturing and culturing in vitro. The method is characterized in that the entire process of maturing and culturing the oocytes in vitro is carried out in a manual lung gas expiration environment, 40 of the sieved ovarian cumulus-oocyte complexes are in one hole on average and are transplanted into a four-hole plate which is transplanted into an inflation bag, a bag opening is sealed by a sealing machine, respiratory gas exchange is carried out by testing personnel, the gas of the lung is blown into the inflation bag by an air blowing needle, air in the inflation bag is exhausted completely, the inflation bag is filled with the gas of the lung, sealed, put into a constant temperature tank with the temperature of 38 DEG C and cultured, and maturing and culturing are carried out for 42-44h.

The invention belongs to the technical field of pig genetic breeding, and specifically relates to a culture solution capable of improving in vitro maturation rate of pig oocytes and application thereof. The culture solution capable of improving the in vitro maturation rate of pig oocytes is prepared from the following raw materials with the following concentrations: 9.5 g / L of TCM-199, 2.2 mg / mL of NaHCO3, 3.05 mmol / L of D-glucose, 0.91 mmol / L of Sodium Pyruvate, 0.5 g / L of Streptomycin sulfate, 0.05% of PVA, 0.57 mmol / L of Cysteine and 0.075 g / L of Penicillin G. The 0.05% of PVA and 5% of a follicular fluid are added on the basis of pig oocyte in vitro maturation culture solution widely used in our country, so that the in vitro maturation rate of the pig oocytes can be improved to more than 90%, and matured oocytes with higher quality and more quantity can be provided for porcine somatic cell nuclear transfer technology.

The invention provides a bovine oocyte in vitro maturation culture solution and a culture method. The maturation culture solution consists of 10% of fetal calf serum and 1% of ITS-G, as well as 0.075IU / mL of HMG, 1[mu]g / mL of 17[beta]-estradiol, 10ng / mL of EGF, 2.2mg / mL of bFGF and 50ng / mL of CXCL12. The culture solution, when applied to bovine oocyte in vitro maturation culture, can improve maturation quality of bovine oocyte in vitro culture, a development rate of in vitro fertilized embryos as well as a development rate of somatic cell nuclear transplantation embryos and blastocyst quality.

The present invention is to provide a method of constructing a nucleus-implanted egg, a parthenogenetic embryo and a parthenogenetic mammal each having 2 haploid genome sets originating in mammarian ova, and provides methods of constructing a nucleus-implanted egg having a haploid genome set derived from ng ovum and a haploid genome set from fg ovum, a parthenogenetic embryo and a parthenogenetic mammal, which includes the steps of (1) introducing a primitive ovarian follicle egg (ng ovum) into a nucleus-deleted egg in a germinal vesicle stage (GV stage egg) and then developing them to MII phase (second meiosis metaphase) by in vitro maturing and culturing to prepare a first nucleus-implanted egg, and (2) extracting MII phase chromosome from said first nucleus-implanted egg and introducing it into other MII phase egg (fg ovum) to prepare a second nucleus-implanted egg, wherein ovum from which an imprinted gene that undergoes gene modification posteriori during the generation of sperm is deleted is used as the ng ovum or fg ovum.

The invention discloses an in vitro maturation culture method of denuded oocytes of mice. The method comprises the following steps: (1) performing in vitro maturation culture on the denuded oocytes; (2) performing parthenogenetic activation on matured denuded oocytes; (3) performing in vitro fertilization on the matured denuded oocytes. According to the method, an environment for artificially simulating natural maturation of oocytes can be created, so that the denuded oocytes can efficiently maturate and grow in vitro and can be subjected to in vitro fertilization and embryonic development as same as normally growing oocytes after in vitro maturation culture. The practice proves that the in vitro maturation culture efficiency of the denuded oocytes of the system is high, the system is mature and stable, and the embryonic development quality of the denuded oocytes is high after in vitro natural fertilization, so that the application and popularization of the denuded oocytes in reproductive health of human beings and genetic breeding of modern livestock can be greatly facilitated. In addition, the operation of obtaining blastulae from the denuded oocytes is simplified and the denuded oocytes serving as ovum sources can be conveniently put into actual production.

The invention provides a purpose for preparing supplementary reproduction medicine from the following raw material medicine in percentage by weight: 18 to 30 parts of radix rehmanniae preparata, 9 to 12 parts of dogwood, 8 to 16 parts of Chinese yam, 9 to 12 parts of fructus lycii, 8 to 16 parts of semen cuscutae and 8 to 16 parts of antler gum. The invention also provides a method for promoting ovarian ovum in vitro maturation. The method comprises the following steps that a, an immature ovarian ovum is taken; and b, the immature ovarian ovum is placed in M199 culture liquid, a microdrop method is adopted for culture for 24 hours, and a mature ovarian ovum is obtained, wherein 2 percent of medicine is contained in the culture liquid. The medicine can promote the growth and development of the immature ovarian ovum in vitro and mainly promotes the ovarian ovum core maturation, and in addition, the cytoskeleton abnormality probability of the mature ovarian ovum is not increased. The medicine can also generate the influence on the ovum passing capability of sperms, and the reliable basis is provided for clinically preventing and treating the male sperm dysfunction, improving the IVF (in vitro fertilization) success ratio and reducing the use rate of ICSI (intracytoplasmic sperm injection).

The invention discloses a rotary-type micro-dynamic incubator which comprises an incubator box, a motor, a transmission mechanism and a motor speed-adjusting device. A tray is arranged in the incubator box and provided with a central shaft and a retaining groove for placement of an incubation dish, the central shaft is fixedly connected with the center of the tray, the retaining groove is arranged on a face of the tray, the central shaft of the tray is connected with the motor through the transmission mechanism, and the tray is driven by the motor which is connected with the motor speed-adjusting device. State of motion of oocytes or embryos in parents is simulated by slowly rotating the tray to enable the oocytes or the embryos to be in a dynamic incubation environment in in-vitro culture, so that in-vitro maturation of the oocytes or development quality of the embryos can be remarkably improved, later implantation of the embryos is promoted, and rate of pregnancy is increased.

The invention discloses a simple in-vitro maturation culture method for oocytes, which comprises the following steps: A1, collecting oocytes; A2, preparing a closed air bag and performing in-vitro maturation culture of oocytes; and A3, performing parthenogenetic activation and in-vivo culture of oocytes, namely after 22 to 24 hours of in-vitro maturation culture, selecting mature oocytes discharging first polar bodies, activating by a chemical process in the 28th hour, culturing for 3 days in merogenesis culture solution, culturing for 4 days in blastaea culture solution, and observing and counting a blastaea rate after 7 to 9 days. When a self-prepared closed air bag system is used, the in-vitro culture of the oocytes and embryos can be accomplished by using a common biochemical culture tank or water bath pot, so the dependency on expensive instruments is reduced and a research doorsill is reduced. When the simple closed air bag is used for in-vitro culture of the oocytes, blastaea can be grown, and the in-vitro maturation rate, merogenesis rate and blastaea rate are higher than those of oocytes cultured in a normal CO2 culture tank.

The invention discloses a method for improving in vitro maturation of oocytes in a foaming stage after vitrification freezing. The method includes freezing the oocytes in a freezing solution containing melatonin, thawing the oocytes in a sucrose solution containing melatonin, and finally performing in vitro maturation culture on the oocytes in a culture solution containing melatonin. The method has simple process and short time consumption, and can effectively improve the in vitro maturation of oocytes in the foaming stage after vitrification freezing.

The invention discloses a nutrient solution for maturation of oocyte in vitro and a method for culturing oocyte using the nutrient solution, and relates to a nutrient solution for nutrient solution for oocyte and a method for culturing the oocyte with the nutrient solution. The nutrient solution and the culturing method using the nutrient solution solve the problem that the viability of oocyte culturing in vitro is low. The nutrient solution for maturation of oocyte in vitro is composed of normal nutrient solution of oocyte, vitamin B12, fetal calf serum, FSH, LH, 17 betas estradiol, sodium pyruvate, EGF, penicillin and streptomycin sulphate. The oocyte is placed into the utrient solution for maturation of oocyte in vitro, the maturation culture on the oocyte lasts for 20-24 hours in the environment in which the volume concentration of CO2 is 5%, and a mature oocyte is acquired. The nutrient solution for maturation of oocyte in vitro can decrease stress injury caused by the external environment, the cytoplasm maturing of the oocyte is promoted, and the parthenogenetic activation of the matured oocyte matured in vitro and the embryos developmental potential after being fertilized are improved.

The invention provides a purpose for preparing supplementary reproduction medicine from the following raw material medicine in percentage by weight: 18 to 30 parts of radix rehmanniae preparata, 9 to 12 parts of dogwood, 8 to 16 parts of Chinese yam, 9 to 12 parts of fructus lycii, 8 to 16 parts of semen cuscutae and 8 to 16 parts of antler gum. The invention also provides a method for promoting ovarian ovum in vitro maturation. The method comprises the following steps that a, an immature ovarian ovum is taken; and b, the immature ovarian ovum is placed in M199 culture liquid, a microdrop method is adopted for culture for 24 hours, and a mature ovarian ovum is obtained, wherein 2 percent of medicine is contained in the culture liquid. The medicine can promote the growth and development of the immature ovarian ovum in vitro and mainly promotes the ovarian ovum core maturation, and in addition, the cytoskeleton abnormality probability of the mature ovarian ovum is not increased. The medicine can also generate the influence on the ovum passing capability of sperms, and the reliable basis is provided for clinically preventing and treating the male sperm dysfunction, improving the IVF (in vitro fertilization) success ratio and reducing the use rate of ICSI (intracytoplasmic sperm injection).

The invention relates to a method for promoting cow in-vitro embryo culture germ cell development. On one hand, the invention provides an embryo culture solution which contains sodium chloride, potassium chloride and other substances. On the other hand, the invention provides a method for cow in-vitro fertilization embryo culture. The method comprises the steps of 1 collecting oocytes and performing in-vitro maturation, 2 performing in-vitro fertilization and 3, performing embryo in-vitro culture and preservation. Germ cells are frozen and stored after being cultured in the embryo culture solution. The method has the excellent technological effect, for example, the germ cell development degree can be greatly increased.

The invention provides a screening culture method for sheep oocytes in vitro, which comprises the following steps: step 1: ovaries which were killed less than half an hour ago are put into saline water at the temperature of 30DEG C to 40DEG C and containing penicillin and streptomycin, washed for 3 to 4 times within 3 hours, and oocytes are picked out; 25 to 30mu mol / L brilliant cresyl blue is put into 35DEG C to 39DEG C water bath to be dyed for 85 to 92min, the cytoplasm is blue and is washed for 3 to 4 times by in vitro maturation fluid, is put into 55 to 78mul / drip in vitro maturation culture fluid by 25 to 30m / drip, and is cultured in 5 percent CO2 by 95 percent; step 2: cumulus cells are removed from oocytes which are maturated in vitro for 25 to 28 hours; IVF washing liquid is used to wash for 2 to 4 times, and is dripped into 50 to 70mu l fertilization fluid by 25 to 30m / drop; semen is unfrozen, the fertilization fluid is moved in, supernatant fluid is centrifugurated for 4 to 5min and removed, the precipitated sperms are added into fertilization fluid drips by the density of 2 to 4*106 / ml and incubated; eggs which are fertilized for 12 to 18 hours are treated in the above step and then moved into a four-hole culture plate to be cultured; and step 3: reagent egg absorption liquid, brilliant cresyl blue maturation liquid, dyeing liquor SOF, the fertilization fluid, the culture fluid and sheep serum are prepared.

The invention discloses an in vitro maturation method and an in vitro maturation culture solution for a mouse naked ovum. The in vitro maturation method comprises the following steps: firstly, carrying out isolated culture on the mouse naked ovum; secondly, detecting the maturation of the mouse naked ovum; and thirdly, detecting the development capability of the matured mouse naked ovum. The in vitro maturation culture solution of the mouse naked ovum consists of alpha-MEM, 10 percent FBS (Fibroblast Serum), 10 ng / mlEGF (Epidermal Growth Factor), 1.5 U / mlhCG, 1 mM / mlGlu, 0.23 mM / ml sodium pyruvate and 10 muM Forskplin. According to the in vitro maturation method and the in vitro maturation culture solution for the mouse naked ovum disclosed by the invention, an in vitro high-efficiency mature development system of the mouse naked ovum is established by adding a chemical reagent correlated with oocyte in vitro maturation in a mouse oocyte culture system; according to the system, in vitro maturation of the mouse naked ovum can be effectively and manually regulated and controlled; in addition, the matured naked ovum can have the development capability of normal oocyte for forming embryon, thereby providing important data for the mouse to be used as an experimental animal model in the aspects of new variety culture, transgenic breeding, variety improvement and the like and providing the foundation for development of a human assisted reproductive technique in depth and breadth.

The invention relates to methods to be used in the maturation of ovarian follicles and oocytes. More specifically, the invention concerns the use of inhibitors of the phosphatase PTEN, such as oxovanadate and peroxovanadate complexes, in methods for in vitro and in vivo maturation of follicles and oocytes.

The present invention belongs to the field of auxiliary reproduction technology. Said invention described culture solution containing human mature follicular fluid is used for external culture technique of human immature ovum, and its composition is formed from Ham'e F-10 or TCM199, human serum substitute SSS, rFSH, HCG, 17 beta-estradiol and human mature follicular fluid. The in-vitro mature (IVM) technique of immature ovum can be used to take out the immature ovum of sterility woman with ovum maturation disturbance, and the culture solution prepared by said invention is used to culture it, and make it mature and make in-vitro fertilization and embryo transplantation to help gestation. It can obtain 65% of ovum mature rate and 33.3% of gestatino rate.

The invention discloses a preparation method of heterogeneous in-vitro fertilization embryos of cows and cattle. The preparation method includes that cow ovaries abandoned in a slaughter house are adopted and subjected to gradient washing through normal saline in a temperature increasing type, oocytes are extracted for in-vitro maturation, and high-quality in-vitro matured oocytes are obtained; high-yield cow sperms are unfreezed and placed into a fertilization medium to be gently patted through a pipette, the cow sperms float in an incubator after overturning up and down for several times, in-vitro fertilization is performed, and the in-vitro fertilization embryos of the cows and the cattle are prepared. The oocytes are obtained through the ovaries abandoned in the slaughter house, so that utilization rate of cow resources is increased; through improvement of technical details, good effect is achieved; utilization of good male herbs can be greatly increased, the good male herbs can obtain a great deal of offspring, and function of the preparation method in genetic modification of herbs is expanded.

The invention discloses a method for efficiently protecting the mitochondrial function of vitrified frozen bovine oocytes. According to the method, 10-9M melatonin (MT) is added into a bovine oocyte in vitro maturation medium and a vitrification solution. There is no significant difference between bovine oocytes frozen by using this method and fresh oocytes with respect to ATP content and in vitro development. Application of the method of the invention can improve the developmental capacity and application scope of frozen oocytes. The method of the invention is simple in execution and low in cost, and will play a great role in cryopreservation of bovine oocyte.

The invention relates to a method for improving the bovine in-vitro fertilization efficiency. On one hand, the invention relates to a bovine in-vitro fertilization embryo culture method. The method comprises the following steps of performing collection and in-vitro maturation of oocytes, performing in-vitro fertilization by using a fertilization culture solution and a seminal fluid preparation culture solution, and performing in-vitro embryo culture and preservation. A maturation culture solution used in the method is a BY basic culture solution added with 100 mL / L of FBS, 10 [mu]g / mL of FSH,10 [mu]g / mL of LH, 1 [mu]g / mL of E2 and 20 ng / mL of EGF, and the used fertilization culture solution and seminal fluid preparation culture solution contain sodium pyruvate, heparin and bovine serum albumin. The invention also relates to a bovine in-vitro fertilization method, and also relates to the fertilization culture solution and the seminal fluid preparation culture solution used in the methods. The method using a reagent shows the excellent technical effect of significantly improving the in-vitro fertilization efficiency.