31results about How to "Increase cleavage rate" patented technology

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.

A method is provided which allows high yields of acylated insulin. The method comprises expressing a singe-chain insulin precursor, preferably in yeast, cleaving the single-chain insulin precursor with a suitable protease which will open the peptide bond between the C-terminal amino group in the B-chain and a connecting peptide connecting the B chain with the A-chain, acylating the two-chain insulin intermediate in the ε-amino group in LysB29 and subjecting the acylated two-chain insulin intermediate to a proteolytic enzyme which will cleave of the N-terminal extension on the B- and A-chains on the precursor molecule.

The invention provides a method for improving the moveability of sperm in frozen bull semen and the in vitro fertilization rate. The method is characterized in that when SP-TALP washing liquid is used for washing at a prior disposal stage of the frozen bull semen, or when PHE (phenylalanine) capacitation liquid is used for capacitation disposal at a capacitation disposal stage of the semen before fertilization, or when fertilization drips are prepared at an in vitro fertilization stage, glutathione and caffeine of which the purities are both higher than 98% are added; in the fertilization process of each piece of frozen bull semen, in the SP-TALP washing liquid containing bull semen or in the mixed liquid of IVF-TALP seminal fluid containing the bull semen and the PHE capacitation liquid or in the mixed liquid of the fertilization drips containing oocytes and the bull semen, the final concentration of the glutathione and the caffeine is 5mmol / L. According to the method disclosed by the invention, two substances are jointed to be respectively applied to three stages in the fertilization process of the frozen bull semen, the moveability of the sperm is greatly improved, and the fertilization rate of the sperm is improved.

he invention discloses a method for producing somatic cell cloned bovine blastocysts. According to the method, to bovine ear limbal fibroblasts are used as nuclear transfer donorcells, and mature bovine oocytes cultivated in vitro are used as nuclear transfer receptor cells; the oocytes are denucleated by extrusion and removed with the zona pellucida; the oocytes and the donor cells are adhered in an arrangement way of oocytes-donor cells-oocytes and treated with electro-fusion to obtain reconstructed embryos; and the reconstructed embryos are activated and cultured in vitro to obtain the cloned blastocysts. The method can improve the production efficiency of the cloned bovine blastocysts.

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 relates to a method for improving the developmental capacity of sheep mature oocytes after vitrification refrigeration, belonging to the technical field of biology. The method comprises the steps of micro-injection fertilization of sheep mature oocytes and activation of fertilized eggs, wherein the activation of the fertilized eggs adopts a direct current pulse-chemical activation-direct current pulse mode, and experiments prove that the embryonic developmental capacity of the vitrified sheep mature oocytes subject to is greatly improved after the vitrified sheep mature oocytes is subject to intracytoplasmic sperm injection and is activated by the activation method of the invention. Compared with the conventional external fertilization method, the cleavage rate and the blastocyst rate are respectively increased to 13.7% and 6.8%. At the same time, by comparing the chromosome ploidy of embryos subject to different treatments, the result indicates that the proportion of normal-ploidy embryos (71.5%) obtained by the ECE method is apparently higher than that (45.4%) obtained by the common chemical activation method and no obvious difference exists compared with the external fertilization groups.

The invention relates to the field of animal husbandry and veterinary and particularly discloses a method for delaying the reproduction function decline of female animals, wherein the animals take in melatonin through oral administration. The delaying of the reproduction function decline of female animals is shown by increasing the litter size of older female animals, the SOD level of ovary, the SOD level of uterus, the number of useful eggs, total egg number, cleavage rate or blastocyst rate or reducing the MDA level of the ovary of older female animals, the MDA level of uterus or the number of useless eggs. In the invention, through a large quantity of objective experiments, different optimal intakes are provided aiming at reproduction function decline of specific different female animals so as to realize the best effect of delaying the reproduction function decline of female animals.

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.

The invention provides a method for improving in vitro fertilizable competence of vitrification freezing oocyte. The method disclosed by the invention comprises the following steps: performing combined treatment on bovine oocyte with a solution containing gadolinium and CLC before vitrification freezing treatment, and performing vitrification freezing; treating with methyl-beta-cyclodextrin afterunfreezing, and performing in vitro fertilization. Results prove that the method is capable of obviously improving the cleavage rate and blastocyst rate of vitrification freezing bovine oocyte and achieving in vitro fertilization efficiency higher than that of fresh bovine oocyte, has a wide application prospect and is worthy of popularization.

The invention discloses a making method of an operation vessel for preparation of pig ICSI zygotes. The making method comprises the steps that an operation liquid microdroplet of a set size, a PVP microdroplet for braking, an injection needle treatment microdroplet and a semen culture microdroplet are sequentially made on a phi 60-mm disposable cell culture dish; the semen culture microdroplet comprises a first semen culture microdroplet of an H-shaped structure, a semen screening bridge and a second semen culture microdroplet, and a large quantity of high-quality sperms are selected under the action of the semen screening bridge. The purpose of realizing a large amount of pig ICSI operation in a short time is achieved, time for selecting the sperms in the pig ICSI operation process is shortened, air exposure time of oocytes is shortened, and the survival rate of pig ICSI embryos is increased.

The invention provides an extraction method of a mouse ovarian tissue fluid. The method comprises the following steps: placing mouse ovaries in a basal medium; mashing and dissloving the ovarian tissue fluid in the basal medium to form a pre-culture solution. The invention also provides an in-vitro maturation culture method of mouse oocyte, and the method comprises: transferring an immature mouse oocyte into a mature culture solution for in-vitro culture, wherein the mature culture solution is formed by adding the pre-culture solution in the basal medium of oocytes. In addition, the invention also provides a method for obtaining a test-tube mouse through embryo transplantation, and the method comprises: carrying out in-vitro fertilization and embryo transplantation on the oocyte matured by the in-vitro maturation culture method. By using the ovarian tissue fluid provided by the invention in the in-vitro maturation of the mouse oocyte, the cleavage rate of the in-vitro fertilized mature egg can be effectively increased, and is nearly the same as the that of the in-vivo mature egg; and finally, the test-tube mouse can be obtained through embryo transplantation.

The invention relates to an intracytoplasmic sperm injection activation method, which belongs to the embryo engineering field. The invention uses ionomycin and DMAP to activate a fertilized ovum together, thereby the activation rate of oocytes and the cleavage rate are improved; after the ionomycin with a final concentration of 5 mu m and the DMAP with a final concentration of 2 mu m are used for activating the fertilized ovum, the cleavage rate is 60 to 65 percent.

The invention discloses a human oocyte in-vitro mature culture solution and a preparing method and culture method thereof, and relates to the technical field of medicines. The formula of the culture solution includes, by mass, 70-95 parts of 75IU / L rFSH, 60-70 parts of 150IU / L rHCG, 50-55 parts of 20% SPS, 30-40 parts of EGF with the concentration of 2 mg / L, 20-25 parts of sodium pyruvate with theconcentration of 25 mmol / L and 150-200 parts of a basic cell culture solution Qiunn's 1029. According to the human oocyte in-vitro mature culture solution and the preparing method and culture methodthereof, the cost for purchasing a commercial in-vitro mature culture medium can be effectively reduced; in the culture medium, the human blastocyst culture solution Qiunn's 1029 serves as the basic culture medium, the culture medium is safe and free of toxic and side effects, the meiosis of immature oocytes, especially oocytes in a third-stage cumulus oophorus and oocyte complex, can be promoted,the in-vitro development ability of this type of cells is improved, the maturity of cytoplasm is promoted, the after-cell-maturity cleavage rate and blastocyst development rate are increased, and theembryo quality is further improved.

The invention provides a method for improving the quality and efficiency of in vitro maturation of an oocyte, which includes the step of changing an in vitro maturation fluid at least once during in vitro maturation, and further comprising adding an amount of FGF to the in vitro maturation fluid. The oocyte prepared by the method of the invention has remarkably improved nuclear maturation efficiency and cytoplasmic maturation efficiency, the MII oocyte is up to 95%, the proportion of oocytes with normal distribution of cortical granules and mitochondria, the levels of glutathione and ATP are significantly higher than those of the normal maturation group; Adding a certain amount of FGF to IVM can further improve the maturation quality and efficiency of oocytes. After in vitro fertilization,the in vitro fertilization efficiency of the oocytes prepared by the method of the invention is significantly higher than that of the normal maturation group, which is embodied in higher cleavage rate, blastocyst rate and more blastocyst cells, and has great application value.

The invention provides a method for improving the human in-vitro fertilization embryo development rate. The method is characterized in that an in-vitro fertilization culture solution is prepared by adding a traditional Chinese medicine monomer in a human oocyte conventional culture solution to improve the human in-vitro fertilization embryo development rate, wherein the traditional Chinese medicine monomer is any of baicalin, icariin and ligustrazine, and the mass ratio (or volume ratio) range of the traditional Chinese medicine monomer and the human oocyte conventional culture solution is 6.0ug / mL, 2.0 ug / mL and 3.0 ug / mL. The method provided by the invention has the benefits that studies show that the fertilization culture solution prepared by adding the baicalin as the traditional Chinese medicine monomer in the conventional culture solution has a good promoting effect on human in-vitro fertilization embryo development and is better than a control group without a traditional Chinese medicine ingredient in aspects of cleavage rate, blastocyst development rate and the like.

The invention discloses a culture solution for improving maturity quality and development potential of in-vitro cultured porcine oocytes and its cultivating method. The culture solution is prepared from, by weight, 3-7 mu mol / L of forskolin, 3-7 mu mol / L of Roscovitine, 8-12g / L of TCM-199, 1-4g / L of NaHCO3, 0.4-0.7g / L of D-glucose, 0.05-0.15g / L of sodium pyruvate, 0.05-0.1g / L of penicillin sodium, 0.03-0.07g / L of streptomycin sulphate, 1-4g / L of polyving akohol, 0.3-0.7mu g / mL of luteotropin, 0.3-0.7mu g / mL of follicle-stimulating hormone, 8-12ng / mL of epidermal growth factor, 0.3-0.7mmol / L of L- cysteine, and 8-12% of follicular fluid. The culture solution has the advantages of improving cleavage rate, blastaea number and blastaea cell population of porcine oocytes, and effectively improving the development potential of the in-vitro cultured porcine oocytes; so the culture solution has great application prospect.

The invention relates to the technical field of pharaoh cuttlefish breeding, in particular to a breeding method beneficial to improvement of the spawning quantity and quality of pharaoh cuttlefish. The breeding method comprises the steps: (1) parent domestication, wherein adult pharaoh cuttlefish parents are put into a pond for domestication and fed with live shrimps and / or live crabs and / or livefish mixed with marine algae; (2) reproduction regulation, wherein periodic starvation and refeeding are sequentially carried out, and temperature change culture is carried out in a gradient temperature change mode; (3) spawn collection; (4) keeping of fresh and alive pharaoh cuttlefish, wherein hatching of fertilized spawns cultured on a large scale is inhibited, alive and fresh keeping is carried out by combining a C-type natriuretic peptide with cysteamine, and finally hatching and larva breeding are carried out. The method is beneficial to improvement of the physique of the pharaoh cuttlefish parents, promotes earlier and faster sexual maturation of the parents, enhances the mating probability of the pharaoh cuttlefish parents, realizes synchronous sexual maturation of the parents andimprovement of the quality of reproductive gametes, remarkably reduces the conversion ratio of normal fertilized spawns to fertilized inkjet spawns, accelerates the hatching of cuttlefish larvae through alive and fresh keeping, promotes the larval development and further improves the survival rate of larvae.

The invention provides a living cell vitrification freezing fluid and a freezing thawing method. The freezing fluid is prepared by that on the basis of an MEM culture medium and an F12 culture mediumin a volume ratio of 1:1, 50-60nmol / L of isofraxidin, 20-30nmol / L of chlorogenic acid and 40-48Mumol / L of a protective agent are added, wherein the protective agent comprises one or more of hydroxyethyl starch, tea polyphenol, 4-hydroxyethyl piperazine ethanesulfonic acid, lentinan and adenosine triphosphate. The living cell vitrification freezing fluid has the advantage that the living cell survival rate, the in-vitro fertilization and development rate, the cleavage rate and the blastocyst rate can be increased.

The invention provides a method for improving the fertilization ability of vitrified bovine oocytes. Before the vitrification treatment, the bovine oocytes are jointly treated with a solution containing gadolinium and CLC, and then vitrified; after thawing, they are treated with methylated-β-cyclodextrin, and then fertilized in vitro. The results show that this method can significantly increase the cleavage rate and blastocyst rate of vitrified bovine oocytes, and is higher than the in vitro fertilization efficiency of fresh bovine oocytes. It has broad application prospects and is worth promoting.

The invention provides a culture method of mammal embryonic cells. During culture, uterine fetal fluid of mammals during pregnancy is not added; the culture is divided into two stages, and during the two stages, culture times and medium formulas are different. The culture method provided by the invention is suitable for culture of the embryonic cells of the multiple mammals and has higher cleavage rate, fusion rate and blastocyst rate. After an embryo obtained by the culture method provided by the invention is directly transplanted, the birth rate is high.

The invention relates to a cell nucleus operation method based on the dynamic drift modeling of the cell nucleus position, comprising the following steps: off-line calibration of the three-dimensional distribution of the cell nucleus relative to a polar body; obtaining the dynamic drift track of the cell nucleus relative to the position of a microneedle through three-dimensional finite element modeling; The factorial design method is used to determine the dominant influencing factors of the dynamic drift of the nucleus position; the relationship between the intracellular movement parameters of the microneedle and the dynamic drift trajectory parameters of the nucleus position is fitted to determine the trajectory of the microneedle required to approach the nucleus, and the microneedle is controlled by the controller. The needle moves in the cell along the determined trajectory and approaches the nucleus and completes the corresponding operations. Using the method for operating the nucleus of the present invention, the enucleation operation can automatically control the trajectory of intracellular movement and the removal amount of cytoplasm. Compared with manual enucleation, the amount of cytoplasm removed is reduced by 60% under the same enucleation success rate, and the cleavage rate of pig cloned embryos is nearly doubled.

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.

The invention provides a method for improving the moveability of sperm in frozen bull semen and the in vitro fertilization rate. The method is characterized in that when SP-TALP washing liquid is used for washing at a prior disposal stage of the frozen bull semen, or when PHE (phenylalanine) capacitation liquid is used for capacitation disposal at a capacitation disposal stage of the semen before fertilization, or when fertilization drips are prepared at an in vitro fertilization stage, glutathione and caffeine of which the purities are both higher than 98% are added; in the fertilization process of each piece of frozen bull semen, in the SP-TALP washing liquid containing bull semen or in the mixed liquid of IVF-TALP seminal fluid containing the bull semen and the PHE capacitation liquid or in the mixed liquid of the fertilization drips containing oocytes and the bull semen, the final concentration of the glutathione and the caffeine is 5mmol / L. According to the method disclosed by the invention, two substances are jointed to be respectively applied to three stages in the fertilization process of the frozen bull semen, the moveability of the sperm is greatly improved, and the fertilization rate of the sperm is improved.

The invention discloses a porcine oocyte in-vitro maturation culture solution. The culture solution comprises an insulin transferrin selenium solution ITS, a fibroblast growth factor FGF, a leukemia inhibition factor LIF and inositol. When the culture solution is applied to oocyte in-vitro maturation culture, serum and follicular fluid do not need to be added, and the culture solution is safer, more stable and more efficient.

The invention relates to the technical field of reproductive medicine, in particular to a human oocyte in-vitro maturation culture solution containing a mesenchymal stem cell cytoplasm extract, and the culture solution is prepared by adding sex hormone, antibiotics and the mesenchymal stem cell cytoplasm extract on the basis of an oocyte basic culture solution. The invention also provides a human oocyte in-vitro maturation culture method. The invention provides a more practical and effective measure for improving an immature oocyte in-vitro maturation culture system. The culture solution disclosed by the invention can be used for remarkably improving the maturation rate, the fertilization rate, the cleavage rate and the blastocyst formation rate of immature oocytes in a foaming period.