100 results about "Blastula" patented technology

Methods, compositions and kits for determining the likelihood of reaching the blastocyst stage for one or more embryos or pluripotent cells are provided. These methods, compositions and kits find use in identifying embryos and oocytes in vitro that are most useful in treating infertility in humans.

An automated system and method of cryopreservation and reanimation of oocytes, embryos, or blastocysts is disclosed. One or more oocytes or embryos are positioned in a processing container, the processing container being configured to allow fluid to flow into and out of the processing container, where two or more fluids flow into and out of the processing container with oocytes or embryos therein. The temperature of the fluid may be controlled in the processing container according to predetermined requirements. The flowing of the fluids may be controlled by a central controller adapted to control one or more valves.

Methods, compositions and kits for determining the likelihood of reaching the blastocyst stage for one or more embryos or pluripotent cells are provided. These methods, compositions and kits find use in identifying embryos and oocytes in vitro that are most useful in treating infertility in humans.

The invention relates to canine embryonic stem cells, methods of cultivation and propagation of the cells, and production of differentiated cells. The embryonic stem cells may be obtained by isolating a canine embryo, culturing the embryo in the presence of a feeder layer and one or more proliferation agent, removing a blastocyst outgrowth, and culturing the outgrowth in the presence of a fresh feeder layer.

The invention provides a method of performing scRRBS (single-cell reduced-representation bisulfite sequencing) on an embryo culture solution. According to the method provided by the invention, medicalwaste (blastula culture solution) generated by 'test-tube baby' operation is adopted as a raw material, double analysis can be performed on the chromosome aneuploidy status and DNA methylation statusof the embryo, development potential of the embryo is evaluated from a brand new angle of epigenetics and reaction between the embryo and the culture environment, new reference is provided for selecting a 'correct' embryo in assisted reproduction, and powerful support is provided for increasing the success rate of the period of the test-tube baby.

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 method for improving the blastocyst rate of in-vitro embryos of animals. According to the method, the developmental capacity of a parthenogenetic embryo and an in-vitro fertilization embryo can be remarkably improved by adding type I interferon into an embryonic development culture solution for the in-vitro culture of the embryos; compared with the blastocyst rate obtained under the culture of the conventional culture solution, the blastocyst rate is improved by 7 to 13 percent; and the blastocyst hatching rate is also remarkably higher than the blastocyst hatching rate under the culture of the conventional culture solution. By the method, the early development capacity of in-vitro embryos of cattle and sheep is improved, so that the in-vitro production efficiency of embryos of the cattle and sheep is improved, and high-quality embryos are provided for the scientific research and production field in which the embryos of the cattle and sheep are taken as materials.

The invention relates to a novel bovine oocyte in vitro maturation (IVM) culture solution, belonging to the field of embryo biotechnolory. In the invention, the routine IVM culture solution is added with amniotic epithelia or the supernatant of the culture solution thereof to establish a novel bovine immature oocyte IVM culture system, so as to improve the quality and maturation rate of the IVM bovine oocyte, thus solving the problem of low fertilization rate and low formation rate of blastula in the existing culture method.

The invention provides a serum-free separating and culturing method for a sheep embryo stem cell, comprising the following steps of: removing a zona pellucida of sheep blastula cultured in vitro with a Tyrode's Solution and then inoculating the sheep blastula to a serum-free culture solution of the sheep embryo stem cell and fixing by using a needle head; placing under the conditions that the temperature is 38.6DEG C and the saturated humidity is 5 percent CO2 for culturing; during the culturing, replacing the culture solution in a half quantity every 48 hours with the pH value of 6.8-7.2 and primarily culturing for 7-9 days; and carrying out the transfer culture once according to the ratio of 1:2-1:4 by adopting a conventional mechanical method, that is to say, transferring the sheep embryo stem cell cultured in vitro to the 16th generation. The serum-free culture solution of the sheep embryo stem cell is prepared from D-MEM / F-12+80ng / mL bFGF+3muMCHIR 99021+10mu L / mL N2+20mu L / mL B27+10mu L / mL NEAA+2mM L-Glutamine+0.2mM beta-Mercaptoethanol. Bared embryos are fixed at the bottom of a culture dish by using a No.32 needle head so as to avoid the damage to the cells in the blastula, and a baked embryo trophocyte is stripped off. The preferable inoculating amount of the baked embryos is 45-60 / groups. The method can ensure that the formation rate of primary colony of sheep embryo stem cells is improved to 33 percent (14 / 42).

The invention discloses a detection kit for chromosome abnormality before embryo implantation. The kit is characterized by comprising a single cell amplification reagent, a fragmentation reagent, a library construction reagent, a DNA purification reagent, and negative and positive quality control substances. The invention also discloses a detection method of the aforementioned kit. Compared with the prior art, the invention establishes a method using embryo blastula stage cells for preimplantation chromosome detection, adopts innovative single cell amplification technology, improves the uniformity (97% or above) and coverage (92% or above) of single cell amplification, and has the advantages of rapid detection, wide coverage, high resolution, high accuracy and moderate flux.

The invention provides Lateolabrax japonicus stem cells and the culturing method, which comprises the steps of collecting Lateolabrax japonicus blastula stage cells, embryo stem cells primary culture and subculture, influencing growth of Lateolabrax japonicus embryo stem cells by temperature, culturing embryo stem cells in stationary phase, and identifying the Lateolabrax japonicus embryo stem cell features. The embryo stem cells are cultured with complete medium.

The invention discloses a method for breeding a transgenic pig for the over expression of a pig PBD-2 gene. The method comprises the following steps: (1) building and straightening a pc DNA 3.1 (+) / pCA-PBD2 carrier in such a manner that a primer is designed, the liver tissue RNA of a pig is extracted, RT-PCR is enlarged to obtain a PBD-2 gene, alcohol is used for precipitation, and sterilization water is used for dissolution; (2) transfecting lipidosome and screening cells in such a manner that a pig fibroblast for over-expression of the PBD-2 is built; (3), obtaining a recombined blastula according to a manual clone method; (4), transplanting an embryo in such a manner that the bred blastula is transplanted into the fallopian tube of a surrogate sow; and (5), identifying the transgenic pig. The method has the advantage that the operation is feasible, simple and convenient. After being cloned, the pig PBD-2 gene is inserted into the expression vector, and a pig fibroblast over expressing the pig PBD-2 is screened out through cell transfection; the pig fibroblast is transplanted to the megagametocyte of the sow through manual cloning to obtain a reconstructed blastula; and a masculine blastula is transplanted to the fallopian tube of the recipient sow to obtain the transgenic pig for over-expression of the PBD-2 gene. The method has a wide anti-bacterial capability, and provides a favorable material for disease-resistant breeding research.

The invention discloses a method for analyzing embryo quality based on molecular immunoassay of mammalian blastulas. The expressions of key transcription factors OCT4 (octamer-binding transcription factor 4) and CDX2 (caudal-related homeodomain transcription 2) respectively expressing in inner cell masses and trophectoderm cells are used as the basis for analysis of the mammalian blastulas, and cellular level and molecular level analyses are carried out based on the immunostaining results of OCT4 and CDX2, so that the conventional blastula quality assessment method is covered, and the lineage differentiation with higher accuracy is proposed to conduct assessment; the blastulas with a proper ratio of the number of the inner cell masses to the number of the trophectoderm cells and complete first lineage differentiation are used as quality blastulas. Compared with the conventional blastula quality assessment method, the method disclosed by the invention not only can assess the blastula quality at a cellular level, but also can accurately assess the blastula quality and subsequent development potential at a molecular level, and the method is simple and high in accuracy and can provide technical support for embryonic implantation.

The invention discloses a method for testing toxicity of environmental estrogen on whitebait embryonic development. The method of the invention utilizes the sensitivity of embryo to the simulation of environmental pollutant or chemical matter to perform a test under semi-static state on whitebait embryo which is exposed in standard diluted aqueous solution of subject having a series of concentrations. The test lasts about 30 days, begins as exposing a live embryo at the blastula stage in the aqueous solution of subject, and ends with hatching all embryos in a control group and an exposed group. The toxicity endpoint contains death, monstrosity and delayed hatching. The method comprises the steps of confirming the toxicity of the subject on the whitebait embryonic development by observing the toxicity endpoint of the exposed group and comparing with the control group; researching on the influence of two natural estrogens E2 and EE2 on the whitebait embryo-yolk sac stage so as to confirm the toxic dose-effect relationship of the natural estrogens to the whitebait embryonic development, and evaluating the potential risk of this type of fish exposed under the estrogen with environmental concentration.

The separation and cultivation method for fish embryonic cell comprises: collecting cell; in CM, separating the cell of blastula-archenteron late time with laceration method, and culturing the initial and passage cells; identifying the cell on morphology, proliferation feature and chromosome karyotype. Wherein, using 70% alcohol to dip and sterilize for embryo; culturing cell in common incubator with 24Deg to passage 2-3 times every week. The objective cell is litle, circle or polygon, and growing stably, and has high rate of diploid cell. This invention is simple and convenient, and can be applied into separation and cultivation for almost all fish embryonic cell.

The invention relates to the technical field of assisted reproduction, and discloses a cryoprotectant and an application thereof, a sperm refrigerating fluid and a preparation method of the sperm refrigerating fluid. The sperm refrigerating fluid comprises a cryoprotectant, a composition and a basic culture solution, wherein the cryoprotectant comprises 0.3 to 1.1 mg / L of paclitaxel, 16 to 22 g / Lof trehalose, 7 to 13 g / L of cane sugar and 80 to 250 mL / L of glycerinum; the composition comprises 12 to 26 mg / L of resveratrol, 0.2 to 1.9 mg / L of reduced glutathione, 436 to 775 mg / L of lecithin, 0.5 to 1.6 mg / L of cholesterol, 9 to 17 mg / L of ATP sodium salt, 11 to 28 mg / L of heparin, 93 to 299 mg / L of phosphatidylserine and 5 to 25 mg / L of coenzyme Q10. The sperm refrigerating fluid can reduce damage of sperms in low-temperature freezing and freezing resuscitation processes, improve vitality of the sperms after freezing resuscitation, prolong survival time of the sperms after freezing resuscitation, promote sperm capacitation and enhance penetrating fertilization capacity of the sperms to egg cells. Thus, the fertilization success rate and blastocyst formation rate of the ova are improved, and finally the effect of improving the pregnancy success rate of the in-vitro fertilization assisted reproduction technology is achieved.

The invention belongs to the field of assisted reproduction and particularly relates to vitrification refrigerating liquid. According to the vitrification refrigerating liquid provided by the invention, taxol is added to serve as a cryoprotectant and the vitrification refrigerating liquid obtained with reference to the taxol adding mode is treated, so that the development rate of in vitro fertilized blastocyst after the oocyte is thawed can be increased. The taxol serving as an additive in the refrigerating liquid can achieve the effects of reducing freezing injury of the mature oocytes and improving the development capability of the embryo after fertilization. The optimal concentration of the adding mode is obtained by researching the adding concentration of the taxol. The vitrification refrigerating liquid provided by the invention can greatly increase recovery rate after the frozen mature oocytes are thawed and the development rate of the blastula and achieves the protective effecton the cell ultrastructure.

The invention provides a method for detecting chromosome abnormality of embryos by the aid of blastocyst cultivation solution without zona pellucida. The method which is particularly an in-vitro non-therapeutic method for detecting the chromosome abnormality of the embryos by the aid of the blastocyst cultivation solution includes steps of (a), providing the cultivation solution from blastocyst cultivation systems; (b), carrying out gene detection on the cultivation solution so as to authenticate whether chromosomes of the embryos are abnormal or not. The zona pellucida of the embryos in the blastocyst cultivation systems is removed before the embryos are cultivated, the embryos are cultivated in the blastocyst systems for 3-6 days, and preferably, the cultivation solution is separated from the cultivation systems after 4 days. The method has the advantages that contamination and interference risks due to redundant sperms and parent-source granular cells in IVF (in-vitro fertilization) and ICSI (intracytoplasmic sperm injection) technical procedures can be eliminated, and whether the chromosomes of the embryos are abnormal or not can be accurately authenticated.

The invention discloses an optimized mice somatic cell nuclear transplantation method. According to the mice somatic cell nuclear transplantation method, three kinds of cells, including cumulus cell, fetal fibroblasts, and embryonic stem cell, are adopted to construct reconstructed embryos; the activation efficiencies of three reconstructed embryos in different activation mediums are compared, development efficiencies in different solutions are compared, and pregnancy efficiencies of different embryo transplantation methods are compared; it is concluded that calcium-free KSOM activation medium is a universal high efficiency activation medium, and the activation efficiency is about 93.5%; KSOM-AA culture medium is suitable for in vitro culture of the reconstructed embryos of cumulus cells and embryonic stem cells, and the best culture medium for fetal fibroblasts is alpha MEM culture medium; when the reconstructed embryos are at 2-cell period, nuclear transplanted animals can be obtained via transplantation of embryos through bilateral fallopian tubes, wherein for each fallopian tube, transplantation of 15 embryos is carried out. The optimized mice somatic cell nuclear transplantation method is capable of increasing reconstructed embryo cleavage rate, blastula development rate, cloning animal birth rate, and survival rate, so that the success rate of obtaining embryonic stem cells from the reconstructed embryos is increased.

The invention discloses a pig oocyte in-vitro maturation culture solution and a preparation method and application thereof, and belongs to the technical field of oocyte in-vitro maturation culture. The problems of low in-vitro maturation rate and development rate of porcine oocytes currently are solved. The pig oocyte in-vitro maturation culture solution is prepared from a basal culture solution TCM-199, penicillin, streptomycin, NaHCO3, 4-hydroxyethylpiperazine ethane sulfonic acid, polyvinyl alcohol, sodium pyruvate, insulin, cysteine, epidermal growth factor, porcine follicular fluid, pregnant mare serum gonadotropin, human chorionic gonadotropin and tannic acid. According to the pig oocyte in vitro maturation culture solution, the oocyte in-vitro maturation quality, the cumulus cell diffusion index, the parthenogenetic embryo blastocyst rate and blastocyst total cell number, and the in-vitro fertilization embryo cleavage rate, blastocyst rate and blastocyst total cell number can beimproved.

A method of enhancing in vitro development of a mammalian embryo is disclosed which comprises supplementing the culture medium with a prostaglandin, or a prostaglandin analog, in an amount effective to promote complete hatching of the embryo (i.e., freeing of the embryo from the zona pellucida). The quality of human blastocysts is enhanced in vitro by culturing with a prostacyclin agonist, Iloprost. The in vivo implantation potential and live birth potential of an in vitro fertilization embryo is thereby enhanced and establishment of a viable pregnancy is facilitated.

A method for isolating an inner cell mass comprising the steps of immobilizing a blastocyst stage embryo having a zona pellucida, trophectoderm, and inner cell mass, creating an aperture in the blastocyst stage embryo by laser ablation, and removing the inner cell mass from the blastocyst stage embryo through the aperture. The aperture is through the zona pellucida and the trophectoderm. The laser ablation is acheived using a non-contact diode laser. The inner cell mass removed from the blastocyst stage embryo is used to establish human Embryonic Stem Cell lines.

The invention discloses a special culture medium and method for culturing porcine trophoderm stem cells. The special culture medium is composed of bFGF, activin A, Y27632, Knockout SR and beta-mercaptoethanol. The culture method comprises the following steps: previously coating a stem cell culture dish with an extracellular matrix; digesting porcine blastula with proteinase to remove the zona pellucida, transferring into the stem cell culture dish, and adding the special culture medium; and culturing until a trophoderm stem cell cloning group with the total cell count of 1000-2000 is formed, digesting into unicells, transferring into a new extracellular-matrix-coated culture dish, and carrying out cell subculture. The special culture medium has the advantages of definite components and high use safety, and avoids the pollution of heterogenous cells. The culture method is simple and efficient, and has the advantages of high cloning formation rate, low apoptosis rate, high proliferation and subculture capacity, high safety and high stability when being used for porcine trophoderm stem cell culture.

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.

The invention discloses a culture solution for promoting in-vitro maturation of porcine oocyte, the culture solution takes an M199 culture solution without HEPES as a basic culture solution, and porcine follicular fluid, L-cysteine, sodium pyruvate, epidermal growth factor, insulin, gonadotropin, chorionic gonadotropin and WNT5A cytokine are also added. When the culture solution is used for carrying out in-vitro culture on the porcine oocytes, the in-vitro maturation efficiency and quality of the oocytes can be effectively improved, the obtained mature oocytes are used for carrying out porcinein-vitro fertilization and somatic cell nuclear transfer embryo production, and the blastocyst development rate and quality of the mature oocytes are also remarkably improved.

The invention belongs to the technical field of bioengineering, and relates to a method for promoting sheep in-vitro embryo development by using follistatin, which comprises the following steps: (1) acquiring a slaughtered sheep ovary; (2) extracting follicles for later use; (3) culturing selected oocytes in a CO2 incubator, and adding into in-vitro fertilization solution drops; (4) adding the in-vitro fertilization drops, and carrying out co-incubation with the oocytes; (5) taking out the early embryo, transferring into a 10 mu g / L follistatin in-vitro culture solution, washing 3-4 times, and culturing for 24-26 hours; and (6) taking out the embryo, washing with the in-vitro culture solution, calculating the merogenesis rate, and calculating the blastula rate after culturing for 144-168 hours. By using the follistatin to treat the early embryo subjected to sheep oocyte in-vitro fertilization, the method can obviously enhance the in-vitro embryo development blastula rate.