134 results about "Embryo transfer" patented technology

An intravaginal fertilization and culture container includes a main chamber and a microchamber. A culture medium, one or more oocytes and sperm are introduced through an access opening having an associated resealable closure device. The container is enveloped in a capsule of soft elastic material prior to lodgment in the posterior fornix. A loop on one capsule part adapts the length to anatomic variations. The microchamber has a channel which receives the catheter tip to facilitate embryo retrieval while eliminating risk of injury thereto. The microchamber permits embryos to be microscopically inspected in situ before transfer to the uterine cavity.

A method and apparatus for safer and more effective deep trans-cervical intra-uterine artificial insemination (AI) is provided. Such a deep AI catheter causes minimal discomfort and risk of trauma, and does not require the services of a highly trained AI professional. First, a catheter is inserted into the cervical tract of the animal. A membrane, initially positioned inside a tube section of the catheter, is then extended from an opening in the tube and into the tract under pressure. The membrane extends into the tract without friction thereby reducing the discomfort and the risk of trauma or injury to the animal. When the membrane is fully extended into the tract, pressure causes the tip of the membrane to open thereby releasing the AI fluid and depositing the genetic material suspended in the fluid into the reproductive tract. Deployment of the membrane is facilitated by tapering its wall thickness towards its tip. In addition to AI and embryo transplant, other applications for the pathway include therapeutic, diagnostic, or other procedures such as introducing fluoroscopic cameras, instruments, and drug delivery.

The invention provides a targeted sgRNA for editing a pig APN gene and a modified carrier as well as a preparation method and application thereof, and relates to the technical field of gene engineering. The sgRNA and the gene modified carrier provided by the invention are strong in speciality, and effectively edit the pig APN gene on cell level by a CRISPR / Cas9n system, and positive cells are edited by the obtained APN gene into donor cells to carry out somatic cell cloning and embryo transferring, so that the obtained APN gene edited cloning pig has ability of resisting piglet diarrhea. According to the preparation method of the piglet diarrhea resistant pig provided by the invention, a receptor of a virus causing the piglet diarrhea is destroyed, any other exogenous genes cannot be introduced except edition of a targeted gene APN, non-speciality edition on a non-APN gene area on a gene group cannot be carried out, a genetic background is clean and clear, and a late safety assessment work of transgenesis is greatly reduced.

Disclosed is a method for using a first polar body and a second polar body as well as a single-cell embryo to carry out whole-genome non-exponential amplification and high-throughput genome sequencing, so as to perform preimplantation genetic diagnosis for genetic disease and testing for pathogenic genes causing repeated miscarriages. The method of the present invention comprises the following steps: (1) obtaining oocytes and embryos, and carrying out separation and genome amplification of first and second polar bodies and single-cell embryos; (2) establishing a genome sequencing library and sequencing, and carrying out bioinformatic analysis of the genome to obtain a gene spectrum and information concerning the number of copies of chromosomes and fragments thereof; (3) determining the chromosome ploidy of the polar bodies and embryos as well as information concerning defects, replication and point mutation in the chromosome fragments; (4) selecting normal or suitable embryos for implantation.

The invention discloses a method for pig in-vitro fertilization and embryo transplantation, which comprises the following steps: oocyte preparation; sperm capacitation; adjustment of sperm concentration through adding sperm receiving fluid and capacitation through placing the adjusted mixture into an incubator; in-vitro fertilization and in-vitro oosperm culture; and embryo transplantation which adopts an embryo transplantation tube for transplantation. The method for pig in-vitro fertilization and embryo transplantation provided by the invention can be operated simply, the in-vitro fertilization and embryo transplantation can be realized without surgery, no damage can be caused to the pig, the success rate is high, the cost is low, and the method has great significance for promotion of pig in-vitro fertilization, pig cloning and transgenic embryo research and production.

The invention provides a cultivation method of cross-bred wagy. The method comprises the steps of culturing cross-bred wagy embryo in vitro and transplanting the cultured embryo into uterus of receptor cow to breed, wherein the embryo in-vitro culture method comprises the following steps: (1) taking an ox with 99.9% of Japanese wagy hereditary characters as a paternal line, and Holstein cow as a maternal line, respectively extracting semen of the paternal line and ovocyte of the maternal line; (2) culturing the ovocyte in ovocyte follicle fluid; (3) performing in-vitro fertilization on mature ovocyte in an in-vitro fertilization culture solution; (4) culturing the fertilized ovum in the embryo culture solution; and (5) vitrifying the in-vitro embryo. The in-vitro cultured embryo is transplanted into the uterus of receptor cow by a bilateral uterine lining embryo transplantation method. At last, the cross-bred first filial generation wagy new strain with characters of Japanese wagy and Holstein cow is cultivated, and has high table purpose value and good production performance.

The present invention discloses a vector, a cell and a method for improving bovine cloning efficiency on the basis of histone methylation modifying level. The vector includes a repressor expression vector and a repressor-operon-containing expression vector, and in the transfection, the repressor expression vector and the repressor-operon-containing expression vector are cotransfected into donor cells; and bovine KDM4B or bovine KDM4C gene is inserted into the repressor-operon-containing expression vector. Positive transfected cells as donor cells are injected into enucleated oocytes for electro-fusion, and successfully-fused bovine somatic cell cloned embryos are selected for culturing for 36h for induced expression of the bovine KDM4B and bovine KDM4C. Histone H3K9me3 epigenetic modifying level of the somatic cell cloned embryos in zygotic activation period can be effectively reduced, the development and quality of post-bovine somatic cell cloned blastocysts can be significantly improved, efficient and in-vitro production of the bovine somatic cell cloned embryos can be achieved, and birth rate of cloned cattle after embryo transplant can be significantly increased.

The invention relates to a coronavirus resistant clone pig based on CRISPR / Cas9 gene editing technology and a preparation method thereof. The preparation method of the clone pig is characterized by comprising the following steps of: (1) obtaining gRNA sequences of a second exon and third exon of a specific targeting pAPN gene, wherein the nucleotide sequencesare shown as SEQ ID No. 1-2 in sequence; preparing CRISPR-Cas9 targeting vectors PX330-pAPN2 and PX330-pAPN3 corresponding to the gRNA sequences; (2) transferring the obtained targeting vector PX330-pAPN2 or targeting vector PX330-pAPN3 into porcine fetal fibroblasts to obtain a pAPN gene editing cell line; (3) carrying out somatic cell nuclear transfer operation on the pAPN gene editing cell line, transferring the reconstructed embryointo a substitute pregnancy receptor, and bearing the clone pig through natural pregnancy. The method can obtain the clone pig with complete deletion expression of porcine coronavirus receptor pAPN,and has the capability of resisting porcine coronavirus infection.

The invention discloses a new method for gene injection for a somatic cell nuclear transfer reconstructed embryo. The method comprises the following steps: target gene preparation, somatic cell nuclear transfer and a microinjection technology. An expression vector is constructed after a target gene is obtained, the cell nucleus of an oocyte is removed by virtue of micromanipulation, then a somatic cell is injected in the space of the denucleated oocyte, then an exogenous gene is directly injected in the somatic cell, the reconstructed embryo is constructed through one-step electric fusion and activation, and the embryo is transferred to obtain a transgenic progeny. According to the method disclosed by the invention, large-fragment cell gene transfection and screening are avoided, the transgenosis efficiency is increased, the time is saved, and the transgenosis application range is expanded. Importantly, the somatic cell is injected in a transpanent zone during a nuclear transfer operation process, thus microinjection for the exogenous target gene is facilitated.

The invention relates to sheep X / Y sperm-separated frozen semen, the production method and application thereof. The sheep X / Y sperm-separated frozen semen provided by the invention contains a frozen balance solution A, a frozen balance solution B including 12 percent glycerin cryoprotectant and a sheep X or Y sperm-separated frozen semen. The invention applies the difference of DNA content of sheep X and Y sperms and separates X and Y sperms through flow cytometry so as to manufacture the sex-control frozen semen and the frozen storage condition is 20 to 30min treatment under -90 to 120 DEG C. The frozen semen provided by the invention controls the lamb sex through artificial insemination or embryo transplantation. With high separation efficiency and sex control accuracy up to 90 percent, the frozen semen is suitable for industrialization production and culture, thus greatly increasing the culturing effect.

The invention relates to a frozen semen deer X / Y sperm separation and a production method and application thereof. The invention provides a frozen semen deer X / Y sperm separation, comprising frozen equilibrium liquid A, frozen equilibrium liquid B containing 12% of glycerin cryoprotectant and frozen semen of deer X or Y sperm separation. The invention uses TALP liquid for protecting the fresh sperms of deer, and applies the difference of DNA contents with deer X and Y sperms to separate X and Y sperms by a flow cytometry mode, so that frozen semen of sexual control is produced in the frozen conserving conditions of -90 DEG C and 60 minutes of processing. The frozen semen obtained by the invention is used for controlling the sex of calved deer in the way of artificial insemination or embryo transplantation with high separation efficiency, and the accuracy of sexual control can be more than 88%. The invention can be applied to industrialized production and cultivation, thereby greatly improving the cultivation effects of deer.

The method of producing extracorporeal buffalo embryo in controlled sex includes recovering buffalo egg in butchery or from living buffalo body, extracorporeal culturing to mature, external fertilization with fresh or frozen X or Y sperm of fine breed bull in a flow cytometer; and recovering embryo of the fertilized egg via external culture to blastula stage for transplanting or further freeze maintaining. The embryo is used in generating posterity of the expected sex. The present invention has the advantages of industrial embryo production in controlled sex and raised propagation speed of high yield milk buffalo.

The invention discloses an equus oocyte vitrification cryopreservation fluid, a cryopreservation method and an application. The equus oocyte vitrification cryopreservation fluid comprises a base solution (BS), an equilibrium solution (ES) and a vitrification solution (VS), wherein the VS is composed of the following components by volume percent: 10-20% of glycol (EG), 6-10% of dimethyl sulfoxide (DMSO), 6-10% of propylene glycol (PROH), 15-25% of inactivated fetal calf serum (NCS), 8-15% of carboxylate E-polylysine (COOL-PLL) and the balance of an HEPES solution. Each liter of the VS includes 0.3-0.8mol of saccharose. The invention has the beneficial effects that the vitrification cryopreservation fluid has low cytotoxicity, high permeability and obvious vitrification effect, meanwhile can obviously reduce the generation of ice crystals in freezing and re-warming processes, can protect cells from physical injury, can increase the survival rate of oocyte, is suitable for the vitrification cryopreservation of equus oocytes or embryos of horses, donkeys and the like, and is beneficial to the popularization and application of artificial insemination and embryo transplantation technology.

The invention belongs to the technical field of pig transgenosis and particularly relates to a method for producing transgenic pigs through overexpression HOXA10 genes. The method is characterized in that constructed HOXA10 gene expression carriers are subjected to lipofection and sulfate (G418) screening to obtain HOXA10 transgenic cell lines with stable expression; the HOXA10 transgenic cell lines are transplanted into denucleated pig oocytes with HOXA10 transgenic pig fibroblasts as nuclear donors through an artificial nuclear transplantation method to form reconstructed embryos; the reconstructed embryos are transplanted into a receptor sow oviduct, and after a sow gives birth to the HOXA10 transgenic pigs and molecular identification is conducted, masculine HOXA10 transgenic pigs are screened out. The transgenic pigs obtained through the method provide materials for research about the mechanism of improving the reproductive capacity through the HOXA10 genes and provide an animal model for revealing other functions of the HOXA10 genes.

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 present invention provides a preparation method for an anti-porcine reproductive and respiratory syndrome cloned pig, comprising: transferring CRISPR / Cas9 targeting vectors and CD163 gene homologous recombination modification vectors into fibroblasts of a pig to obtain positive clone cells, a seventh exon of porcine endogenous CD163 gene being replaced with a eleventh exon of human CD163-L1 gene, so that the positive clone cells are incapable of mediating invasions of PRRSV; taking the positive cell as nuclear transfer donor cells and oocytes as nuclear transfer recipient cells to obtain a cloned embryo by adopting a somatic cell nuclear transfer technology; and impregenating a pig by transferring the cloned embryo into the uterus of the pig, to obtain a cloned pig.

The invention relates to a method for breeding a new Yangtze River Delta meat goat strain. According to the method, Yangtze River Delta white goats (also referred to as Haimen goats) are used as a female parent breeding material, Saanen goats are introduced as first male parents, and Boer goats are used as terminal male parents; and a new meat goat strain suitable for being raised in Yangtze River Delta districts is finally bred by means of cross breeding, embryo transplantation and propagation and the like in combination with a BLUP (Best Linear Unbiased Prediction) method, a marker-assisted selection (MAS) technology and a conventional breeding method. The new Yangtze River Delta meat goat strain bred by the method has high propagation rate and good meat performance, has the advantages of the Yangtze River Delta white goats, the Saanen goats and the Boer goats, and is maximum in heterosis.

The invention discloses a Bama miniature pig somatic cell cloning method, which comprises the following steps: 1) preparing related agents; 2) cultivating Bama miniature pig somatic cells; 3) freezing the Bama miniature pig somatic cells; 4) unfreezing the frozen somatic cells; 5) acquiring and externally cultivating pig oocytes; 6) transplanting nucleus; and 7) transplanting embryos. The invention has the advantages of speeding up the research on domestic transgenosis pig technology and human heterogeneous organ transplantation, accelerating the development of animal husbandry in Guangxi even the whole country, and academically filling the gap in China.

The present invention belongs to the field of auxiliary reproduction technology. Said invention described culture solution containing human mature follicular fluid is used for external culture technique of human immature ovum, and its composition is formed from Ham'e F-10 or TCM199, human serum substitute SSS, rFSH, HCG, 17 beta-estradiol and human mature follicular fluid. The in-vitro mature (IVM) technique of immature ovum can be used to take out the immature ovum of sterility woman with ovum maturation disturbance, and the culture solution prepared by said invention is used to culture it, and make it mature and make in-vitro fertilization and embryo transplantation to help gestation. It can obtain 65% of ovum mature rate and 33.3% of gestatino rate.

The invention belongs to the technical field of assisted reproduction, in particular to a one-step embryo culture medium. A composition for preparing the one-step embryo culture medium is prepared from(+ / -)-alphal-lipoic acid, N-acetyl-L-cysteine according to a molar ratio of (0.055-0.098) : (0.702 to 0.858). The one-step embryo culture medium prepared by using the composition keeps effective components secreted into the culture medium by an embryo, plays a role in protecting cells and restraining, decomposing and utilizing harmful products, and the problem of low blastocyst forming rate caused by mechanical injury in the embryo transplanting process and stress reaction due to different culture environments is solved.

A preventive agent of premature ovulation which may occur during in vitro fertilization or embryo transplantation, the agent comprising a non-peptide compound having an antagonistic effect on a gonadotropin-releasing hormone. The preventive agent shows a low toxicity, can be administered through an oral route, and has an excellent preventive effect on the premature ovulation during in vitro fertilization or embryo transplantation.

The present invention relates to a method for preparing a gene knock-out canine with use of somatic cell cloning technology, in particular relates to a method for preparing a gene knock-out canine with use of somatic cell cloning technology using a fusion liquid of low osmotic pressure together with autologous embryo transplanting.

The invention discloses an equine embryo-flushing solution as well as a preparation method and applications of the solution. The embryo-flushing solution contains sodium lactate mixed liquor, penicillin, streptomycin and one, two or three of bovine serum albumin, neonatal bovine serum and inactivated horse serum. The preparation method comprises the following steps: placing the sodium lactate mixed liquor in thermostat with the temperature of 37 DEG C for 0.5-6 hours for standby; and adding the penicillin, streptomycin and one, two or three of bovine serum albumin, neonatal bovine serum and inactivated horse serum in the sodium lactate mixed liquor according to the component contents to obtain the embryo-flushing solution. The preparation method is suitable for the non-surgical embryo transfer technology of horses, cattle and donkeys. The equine embryo-flushing solution has the following beneficial effects that the solution has the advantages that raw materials are available, cost is low, preparation method is simple and use is convenient, is very suitable for the equine large-scale non-surgical embryo transfer and is beneficial to the popularization and application of the embryo transfer technology.

The invention discloses a reconstructed oocyte of a deaf model pig and a construction method of the reconstructed oocyte, and also discloses the deaf model pig and a construction method and application of the deaf model pig. According to the reconstructed oocyte of the deaf model pig, the construction method of the reconstructed oocyte and the construction method of the deaf model pig, by knockingout a pig OSBPL2 gene, the reconstructed oocyte of a deaf mini-pig is obtained, and in combination with a CRISPR / Cas9 gene editing technology, a somatic cell nuclear transfer technology and an embryotransfer technology, high efficiency and feasibility of the construction method for the genetically engineered deaf model pig are proven. Through the construction method of the deaf model pig, genotypes and deafness phenotypes of human OSBPL2 gene mutations can be precisely duplicated, the pig of which an OSBPL2 gene is knocked out has typical characteristics of human autosomal dominant hereditary hearing loss (DFNA67), therefore, a reliable big animal model can be provided for disease research of the human hereditary hearing loss, and the deaf model pig can be applied to inner-ear gene repairing, inner-ear hair cell regeneration, hearing reconstruction and the like.

The invention discloses a method for efficiently preparing high-quality cloned embryos, belonging to the field of embryo transplanting methods. The method provided by the invention comprises the following steps of: agitating and screening to obtain sheep oocytes in the same cell period and transplanting a supplier cell into the sheep oocytes through utilizing microscopy injection; then treating by utilizing a fusion solution and putting the treated embryo into a mature solution to be cultured to form a fused embryo; and chemically activating and then starting and reconstructing embryo cleavage. According to the method provided by the invention, the sheep oocytes which are in the same cell period and have the uniform mature time through an agitating method are acquired, a new method for picking the good sheep oocytes is provided and the mature rate of the sheep oocytes is improved. According to the method provided by the invention, the operation efficiency of nuclear transfer is improved by utilizing a method of simultaneously denucleating through extrusion and carrying out nucleus injection by utilizing the same injection needle. The fusion efficiency is improved to more than 95% by using the fusion solution which is developed by the method provided by the invention, the successful rate of the cloned embryo is greatly improved and the subsequent embryo development is not influenced. The method provided by the invention has the advantages of reducing the loss of the embryos in the process of preparing the cloned embryo and improving the efficiency of preparing the sheep cloned embryos; and the method provided by the invention is a method for efficiently preparing the sheep cloned embryos with good quality and has a wide application prospect.

The invention discloses a method for increasing the cloning efficiency by increasing the embryo transplantation lambing percentage and survival rate, belonging to the field of embryo transplantation methods. The method comprises the following steps of: transplanting embryos with the embryonic ages 24-72 hours and uniform cleavage into synchronous estrus female sheep after transplantation activation, wherein 10-15 embryos are transplanted into every sheep; performing pregnancy management on female sheep which do not return to estrus within four continuous sexual periods after surgery; performing caesarean operation on surrogacy female sheep which surpass an expected date of childbirth by 5 days and of which the fetus body forms are too large; strictly controlling the nursing ambient temperature of borne lambs at 21-23 DEG C; and feeding the lambs with colostrum of healthy female sheep. Due to the adoption of the method provided by the invention, the pregnancy rate, lambing percentage and lamb survival rate of cloned sheep can be increased remarkably. After operation is performed in strict accordance with the invention, the pregnancy rate of cloned sheep is up to 30 percent, the lambing percentage is up to 31.67 percent, and the survival rate is up to 63 percent. The method for increasing the sheep cloning efficiency disclosed by the invention has wide application prospect.

A dual-hybridizing method for increasing the quality and output of mutton includes such steps as hybridization between at least two excellent varieties of foreign meat sheep to generate the first generation hybrid meat sheep, hybridizing with native meat sheep to obtain a generation of mictic hybrid meat sheep, and secondary hybridizing combination.