50 results about "Bovine oocyte" patented technology

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 relates to a method of producing a protein of interest, comprising making a non-human transgenic mammal that produces said protein in its milk, obtaining said milk from the non-human transgenic mammal and purifying said protein of interest from the milk. Transgenic bovine animals were generated, which are able to produce human growth hormone in mammary glands. The method involves cloning of a genetic construct encoding hGH gene and beta casein promoter conveniently in an expression vector. It also includes transfection procedures into fetal bovine somatic cells, generally fibroblasts, and the nuclear transfer into enucleated bovine oocytes, generating thus transgenic embryos. The method also includes other procedures to generate transgenic embryos for the further expansion of the transgenic herd, such as the subcloning of transgenic female bovines, the superovulation of transgenic cows and their insemination with semen from a non-transgenic or a transgenic male bovine, and the superovulation of non-transgenic cows and their insemination with semen from a transgenic male bovine. Afterwards, transgenic embryos give rise to transgenic cattle that produce human growth hormone in huge amounts in their milk, from which the hormone is completely purified and analysed to fulfill all the requirements for the manufacture of a pure biopharmaceutical product.

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 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 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 discloses a targeting vector and a reconstituted cell for the Cas9-mediated site-specific integration of FABP4 (adipose fatty acid-binding protein) gene and MSTN (myostatin) gene point mutation. The invention utilizes an electroporation method to co-transfect a Cas9 eukaryotic expression vector and a targeting vector targeting the MSTN gene to Luxi bovine fetal fibroblasts, consequently, gene targeting for the Luxi bovine fetal fibroblasts is realized, and bovine fetal fibroblasts with FABP4 gene and MSTN gene point mutation knocked in the MSTN site are obtained. By Junction of PCR (Polymerase Chain Reaction) screening and verification, targeted positive cloned cells are obtained. The positive cloned cells as donor cells are transplanted into denucleated bovine oocytes, so that a transgenic cloned embryo can be obtained, and thereby a solid foundation is laid for the rapid and efficient development of high-quality new genetically modified beef varieties.

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 relates to a method for in-vitro fertilization and embryo culture by collecting bovine oocytes in vivo. Specifically, on one hand, the bovine in-vitro fertilization embryo culture methodcomprises the following steps of: taking follicular fluid derived from ovum pick-up of bovine living body, picking out a cumulus-oocyte complex wrapped with at least containing three layers of cumulus cells under a stereoscopic microscope, putting the cumulus-oocyte complex into a transport culture solution, and transporting the cumulus-oocyte complex back to a laboratory within 24 hours; washingCOCs once in an oocyte maturation culture solution and then transferring into a new maturation culture solution for culturing for 22-24 h under the culture conditions that the temperature is 38.8 DEGC, the concentration of CO2 is 5.5-6.5% and the humidity is saturated; performing in vitro fertilization; and carrying out embryo in vitro culture and preservation. The invention also relates to a transport culture solution for oocytes. The method and the transportation culture solution disclosed by the invention show excellent technical effects as shown in the specification.

The present invention provides an in vitro mature culture fluid containing tea-polyphenol for bovine ovocyte and its culture method. The described in vitro mature culture flud uses conventional culture fluid as matrix, in the described matrix 20-150 microgram / mL of tea-polyphenol, 100 IU / mL of penicillin, 100 microgram / mL of streptomycin and serum whose volume percentape content is 8%-20% are also contained. Said invention can make tea-polyphenol be used in the field of bovine ovocyte in vitro mature culture so as to raise early embryonic in vitro development rate after the bovine ovocyte in vitro fertilization.

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 of producing a protein of interest, comprising making a non-human transgenic mammal that produces said protein in its milk, obtaining said milk from the non-human transgenic mammal and purifying said protein of interest from the milk. Transgenic bovine animals were generated, which are able to produce human growth hormone in mammary glands. The method involves cloning of a genetic construct encoding hGH gene and beta casein promoter conveniently in an expression vector. It also includes transfection procedures into fetal bovine somatic cells, generally fibroblasts, and the nuclear transfer into enucleated bovine oocytes, generating thus transgenic embryos. The method also includes other procedures to generate transgenic embryos for the further expansion of the transgenic herd, such as the subcloning of transgenic female bovines, the superovulation of transgenic cows and their insemination with semen from a non-transgenic or a transgenic male bovine, and the superovulation of non-transgenic cows and their insemination with semen from a transgenic male bovine. Afterwards, transgenic embryos give rise to transgenic cattle that produce human growth hormone in huge amounts in their milk, from which the hormone is completely purified and analysed to fulfill all the requirements for the manufacture of a pure biopharmaceutical product.

The invention relates to a method of producing a protein of interest, comprising making a non-human transgenic mammal that produces said protein in its milk, obtaining said milk from the non-human transgenic mammal and purifying said protein of interest from the milk. Transgenic bovine animals were generated, which are able to produce human growth hormone in mammary glands. The method involves cloning of a genetic construct encoding hGH gene and beta casein promoter conveniently in an expression vector. It also includes transfection procedures into fetal bovine somatic cells, generally fibroblasts, and the nuclear transfer into enucleated bovine oocytes, generating thus transgenic embryos. The method also includes other procedures to generate transgenic embryos for the further expansion of the transgenic herd, such as the subcloning of transgenic female bovines, the superovulation of transgenic cows and their insemination with semen from a non-transgenic or a transgenic male bovine, and the superovulation of non-transgenic cows and their insemination with semen from a transgenic male bovine. Afterwards, transgenic embryos give rise to transgenic cattle that produce human growth hormone in huge amounts in their milk, from which the hormone is completely purified and analysed to fulfill all the requirements for the manufacture of a pure biopharmaceutical product.

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 relates to a method of producing a protein of interest, comprising making a non-human transgenic mammal that produces said protein in its milk, obtaining said milk from the non-human transgenic mammal and purifying said protein of interest from the milk. Transgenic bovine animals were generated, which are able to produce human growth hormone in mammary glands. The method involves cloning of a genetic construct encoding hGH gene and beta casein promoter conveniently in an expression vector. It also includes transfection procedures into fetal bovine somatic cells, generally fibroblasts, and the nuclear transfer into enucleated bovine oocytes, generating thus transgenic embryos. The method also includes other procedures to generate transgenic embryos for the further expansion of the transgenic herd, such as the subcloning of transgenic female bovines, the superovulation of transgenic cows and their insemination with semen from a non-transgenic or a transgenic male bovine, and the superovulation of non-transgenic cows and their insemination with semen from a transgenic male bovine. Afterwards, transgenic embryos give rise to transgenic cattle that produce human growth hormone in huge amounts in their milk, from which the hormone is completely purified and analysed to fulfill all the requirements for the manufacture of a pure biopharmaceutical product.

The present invention provides an in vitro mature culture fluid containing tea-polyphenol for bovine ovocyte and its culture method. The described in vitro mature culture flud uses conventional culture fluid as matrix, in the described matrix 20-150 microgram / mL of tea-polyphenol, 100 IU / mL of penicillin, 100 microgram / mL of streptomycin and serum whose volume percentape content is 8%-20% are also contained. Said invention can make tea-polyphenol be used in the field of bovine ovocyte in vitro mature culture so as to raise early embryonic in vitro development rate after the bovine ovocyte in vitro fertilization.

The invention relates to a bovine oocyte in-vitro fertilization embryo culture method and a used culture medium. The basic culture medium is an aqueous solution comprising sodium chloride, sodium dihydrogen phosphate, sodium bicarbonate, sodium acetate and the like. The invention further relates to an ovum washing solution and a mature culture solution. The bovine oocyte in-vitro fertilization embryo culture method comprises the following steps: collection and in-vitro maturation of an oocyte; in-vitro fertilization; and in-vitro culture and storage of the embryo. The bovine oocyte in-vitro fertilization embryo culture method has the excellent technical effect as shown in specification.

The invention discloses a preparation method of a blastomere reconstructed embryo of a high productive dairy cow, and belongs to the field of embryology. The method specifically comprises the following steps: 1, preparing an in-vitro maturated dairy cow oocyte; 2, thawing frozen sperms of the high productive dairy cow; 3, preparing an in-vitro fertilized embryo, which is 32-cell stage or longer, of the high productive dairy cow; 4, constructing a blastomere reconstructed embryo of the high productive dairy cow embryo by a piezo operating system; 5, culturing the reconstructed embryo in vitro.The blastomere reconstructed embryo of the high productive dairy cow embryo is constructed by the piezo operating system, the step for preparing the reconstructed embryo by a laser system for assistedhatching or an inclined-port needle in the prior art is replaced, pulses produced by a voltage effect are directly applied to the surface of a cell for injection by the piezo operating system througha micro-injection needle, damage to the embryo is reduced greatly, and survival rate of the reconstructed embryo is increased.

The invention discloses a method for evaluating the sperm intracorporal conception rate of a breeding bull according to the extracorporal fertilization rate of the breeding bull, and belongs to the technical field of animal reproduction. The method includes the steps that COCs is washed by bull oocyte egg picking liquid and bull oocyte mature liquid respectively, placed in the bull oocyte mature liquid for culture, and placed in bull sperm drips capacitated by BO-AB liquid for fertilization; then granular cells are taken off by IVC SOF liquid, then eggs with the granular cells which are taken off are placed in SOF liquid for culture, and the extracorporal fertilization cleavage rate is measured; artificial insemination is conducted, the conception rate of the breeding bull is measured, and the breeding bull is evaluated according to the relevance of the cleavage rate and the conception rate. The method has the advantages that by the utilization of sperm extracorporal fertilization rate of the breeding bull, the practical conception capacity of the breeding bull can be rapidly estimated, the breeding capacity of the breeding bull can be known in time, the problem that a large amount of time, labor and money need to be wasted for detecting the breeding capacity of a breeding bull individual through manual insemination is solved, and resources are saved.

The invention relates to an in-vitro culture system for Tibetan yak oocyte. Researches show that in an in-vitro culture process of cumulus-oocyte complexes (COC) of yak, proliferation of granule cellsof the yak can be obviously improved by adding exogenous BMP15, and an effect of obviously improving the oocyte maturation rate is achieved. According to the culture system disclosed by the invention, the influence of the BMP15 on yak COC in-vitro culture and a molecular regulation mechanism thereof in an in-vitro maturation process can be explored, and a developmental mechanism of the yak oocytein the maturation process can be further known, so that the in-vitro maturation culture quality of the yak oocyte and the developmental capacity of early embryos can be improved.

The invention relates to a method for improving bovine oocyte maturation in vitro. Particularly, the method for improving bovine oocyte maturation in vitro comprises the following steps: (1) providing oocytes; and (2) maturation of oocytes in vitro: putting the occytes in mature culture solution to perform maturation cultivation in vitro for 22-24 h under culture conditions of 100% humidity, 38.5DEG C and 5% CO2. The mature culture solution comprises: ECS, NaHCO3, EGCG, Hepe, estradiol, BFF, luteinizing hormone (LH), cysteine, follicle stimulating hormone (FSH) and TCM-199. The method for improving bovine oocyte maturation in vitro can effectively promote performance of bovine occyte maturation in vitro.

The invention discloses a method for breeding cattle capable of producing hypoallergenic milk and application of the method. The method comprises the following steps: leading a zinc finger nuclease carrier pZFN into fibroblasts of the cattle, so as to obtain donor cells with a gene type which is a biallelic mutation type, wherein the biallelic mutation type is that nucleotide molecules of beta-LGgenes on two homologous chromosomes of the cattle are subjected to frame shift mutation; transferring cell nucleuses of the donor cells into cattle oocytes without the cell nucleuses and developing toform reconstructed embryos; then transferring the reconstructed embryos into uteri of cows and delivering to obtain the cattle capable of producing the hypoallergenic milk. According to the method disclosed by the invention, beta-LG biallelic gene knockout cattle which have no exogenous DNA (Deoxyribonucleic Acid) integration, can produce beta-lactoglobulin-free milk and deliveries mutation to the next generation through normal breeding are successfully bred. By adopting the method disclosed by the invention, an important foundation is laid for allergenic problems of the milk and also provides infinite possibility for preparing humanized milk. The method has important application value.

The invention provides a bovine oocyte in vitro pre-maturation solution. The pre-maturation solution uses a bovine oocyte in vitro culture solution as a ground substance, and octanoyl Ghrelin is contained in the ground substance. The recovery of bovine oocyte meiosis is restricted through octanoyl Ghrelin in vitro culture, so that oocyte nucleuses and cytoplasm are matured simultaneously, and then the quality and in vitro developmental capacity of bovine oocyte are improved. The bovine oocyte in vitro pre-maturation solution can become a novel meiosis preparation drug for popularization and application in livestock production, and has good market effect and social effect.

The invention discloses miRNA-378 and application thereof. The miRNA-378 is differentially expressed in the bovine oocytes of different sizes. Proofed by verification, a miRNA-378 inhibitor can be used for promoting the maturing of the bovine oocytes. Based on this, the invention also discloses a culture medium containing the miRNA-378 inhibitor for culturing the bovine oocytes and a method for correspondingly promoting the maturing of the bovine oocytes. The miRNA-378 and the application thereof disclosed by the invention have important meaning on promoting the growth and development of embryos outside the bovine body.

The invention discloses a serum-free bovine oocyte in-vitro maturation culture solution and an oocyte culture method, and belongs to the technical field of cell culture. The serum-free bovine oocyte in-vitro maturation culture solution disclosed by the invention is prepared from Hepes-free M199 basic culture solution, fatty acid-free BSA (bovine serum Albumin), HMG (high mobility group box), 17 beta-estradiol, EGF (epidermal growth factor), L-cysteine, bFGF (basic fibroblast growth factor), 100xGlutamina, folic acid, cholic acid and CXCL12. The bovine oocyte in-vitro maturation culture solution disclosed by the invention is used for culturing bovine oocytes under the condition of not using serum; the maturation rate of the bovine oocytes is improved; and the early embryo development rate of in-vitro fertilization embryos and somatic cell nuclear transplantation embryos is effectively improved.

The invention provides an efficient vitrification freezing method for bovine in-vitro embryo production. According to the method, beta-nicotinamide mononucleotide is applied to a bovine IVF embryo vitrification technology in bovine in-vitro embryo production for the first time, the beta-nicotinamide mononucleotide with a certain concentration is added into a bovine oocyte in-vitro maturation solution and a bovine in-vitro fertilization embryo culture solution, and then the bovine in-vitro embryo is subjected to vitrification. The vitrification efficiency of the bovine IVF embryo can be greatly improved. The vitrification efficiency can be further improved by further adding growth factors and gap-linked proteins into a bovine oocyte in-vitro maturation solution and a bovine in-vitro fertilized embryo culture solution according to a proportion. Furthermore, by adding the nanoparticles with a certain concentration into the vitrification freezing liquid, the vitrification freezing efficiency can be further improved.

The invention provides bovine oocyte in-vitro mature culture solution containing mycose and a cultural method. The bovine oocyte in-vitro mature culture solution comprises 0.1-1.0mg / mL of mycose, 0.1-10mu g / mL of daidzein, 100IU / mL of penicillin, 100mu g / mL of streptomycin and 8% to 20% serum, wherein the solvent is regular nutrient solution which is one or more of M199, DMEM, F12 and MEM. The combination of mycose and daidzein is applied to bovine oocyte in-vitro mature culture, the cytoplasm maturing of oocyte can be remarkably promoted in comparison with solely using mycose or daidzein, and the problem of low embryo in-vitro developmental rate after the in-vitro mature oocyte is fertilized. The bovine oocyte in-vitro mature culture solution is low in economical cost and convenient to popularize and applied to production.

The invention discloses a method for evaluating the sperm intracorporal conception rate of a breeding bull according to the extracorporal fertilization rate of the breeding bull, and belongs to the technical field of animal reproduction. The method includes the steps that COCs is washed by bull oocyte egg picking liquid and bull oocyte mature liquid respectively, placed in the bull oocyte mature liquid for culture, and placed in bull sperm drips capacitated by BO-AB liquid for fertilization; then granular cells are taken off by IVC SOF liquid, then eggs with the granular cells which are taken off are placed in SOF liquid for culture, and the extracorporal fertilization cleavage rate is measured; artificial insemination is conducted, the conception rate of the breeding bull is measured, and the breeding bull is evaluated according to the relevance of the cleavage rate and the conception rate. The method has the advantages that by the utilization of sperm extracorporal fertilization rate of the breeding bull, the practical conception capacity of the breeding bull can be rapidly estimated, the breeding capacity of the breeding bull can be known in time, the problem that a large amount of time, labor and money need to be wasted for detecting the breeding capacity of a breeding bull individual through manual insemination is solved, and resources are saved.

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.