147 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.

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 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 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 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 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 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 provides a method for enhancing the ectogenesis of sheep oocyte. The method adopts an adenylate cyclase activator Forskolin (FSK), phosphodiesterase inhibitor cilostamide (CIL) and 3-isobutyl-1-methylxanthine (IBMX), treats the sheep oocyte in a combining manner and improves the capability of the ectogenesis of the sheep oocyte. The method comprises the following detailed steps of: picking the ovary of the killed sheep, putting the ovary of the killed sheep into normal saline for cleaning for 3-4 times; pumping the 2-8mm follicles on the surface of the ovary by using a 10ml syringe which absorbs 1ml ovum-pumping liquid containing 100mu mol / L FSK and 500mumol / L IBMX and is provided with a number-9 needle head, and injecting the follicular fluid recovered in the syringe into a35-60mm vessel; picking the oocyte under a microscope, putting the oocyte into the pumped follicular fluid, cleaning the oocyte for three times with in-vitro mature fluid containing 20mu mol / L CIL, and putting the oocyte in a CO2 incubator for culture; then cleaning the oocyte for three times with in-vitro mature fluid which does not contain CIL, putting the oocyte in the CO2 incubator for culture and adding into in-vitro fertilization fluid drop; unfreezing the seminal fluid with water bathing, and commonly incubating with the oocyte; and calculating the cleavage rate after 24 hours and calculating the blastocyst rate after 168 hours.