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 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 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 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 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 novel methods for IVM of bovine oocytes. These methods can be used in efficient propagation of superior animals at low cost, which is very helpful in improving ecological and economic performance in the cattle industry.

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 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 an application of sulforaphane in preparation of a bovine oocyte in-vitro maturation solution. The invention provides the culture solution for promoting in-vitro maturation of the bovine oocytes, and the sulforaphane in the culture solution has an obvious promoting effect on in-vitro maturation and in-vitro developmental ability of the bovine oocytes.