30 results about "Bovine embryo" patented technology

The invention discloses a preparation method and application of a conditioned culture solution for improving the development rate of bovine in-vitro embryos. The preparation method comprises the following steps: culturing immortalized bovine endometrial epithelial cells by using a DMEM / F12 cell culture solution containing 10% FBS, collecting the cell culture solution in the culture process, and filtering to obtain an immortalized bovine endometrial epithelial cell conditional culture solution, namely the conditional culture solution capable of improving the development rate of the bovine in-vitro embryos. The prepared conditioned culture solution can simulate a liquid environment of an in-vivo environment, is used for culturing in-vitro embryos such as parthenogenetic activated embryos and in-vitro fertilized embryos, improves the cleavage rate and blastocyst rate of the in-vitro embryos and improves the development rate of the in-vitro embryos of cattle.

The invention relates to the field of computer vision, in particular to a bovine embryo quality evaluation method and system based on a time sequence microscopic image, and the method comprises the steps: collecting a microscopic image and a timestamp, carrying out the embryo segmentation and inter-frame registration of the image, and obtaining the frame quality weight, morphological characteristics, image block embedding and topological data; respectively calculating a topological distance and an optimal transmission distance, determining a change point generation event sequence in combination with a frame quality weight, and aligning and outputting a penalty term based on a time Petri network; inferring a stage sequence and a quality posteriori based on the event sequence and a penalty term, and constructing a multi-instance set based on an event time window to obtain a multi-instance learning score; and fusing quality posteriori and multi-instance learning score to obtain an evaluation result, calculating a conflict coefficient, and relocating an event time window when a threshold value is exceeded to update the evaluation result. According to the method, rating consistency, noise immunity and traceability are improved.

The present disclosure relates to methods, products, and compositions useful for deriving bovine induced blastoids. Methods are described that allow for juvenile stem cell aggregation and induced blastocyst formation. The invention further provides an aggregation culture medium and an induced blastocyst-like culture medium which are suitable for promoting aggregation of the young stem cells and preparing the induced blastocyst-like. The bovine induced blastoids can be used in selection and breeding procedures, including the replication of pre-implant embryos with desired characteristics and in vitro breeding including a cross-generation breeding cycle from parent germ cells and / or embryos to progeny embryos.

The present invention belongs to the field of molecular biology and genetics, and in particular relates to a method for editing a bovine gene based on Pro-iCHI. The present invention provides a method for editing a bovine gene based on Pro-iCHI. Protamine is transiently expressed in gene-edited b-haSCs, which are then injected to mature oocytes to obtain reconstructed embryos. Protamine can eliminate abnormal DNA methylation resulting from oocyte intracytoplasmic haSCs injection and enable the nucli to compress into sperm-like structures, and the obtained bovine Pro-iCHI embryos can successfully develop into blastocysts, with a blastocyst rate comparable to that of the embryos obtained by in vitro fertilization. Moreover, in the present invention, a protamine-encoding gene is inserted into a Saccharomyces cerevisiae protein expression vector for transient expression, which ensures that abnormal DNA methylation is erased, without integration into the genome resulting in the insertion of exogenous genes.

The present application relates to the field of animal embryo engineering technology, and provides a method for improving the development ability of bovine IVP embryos. In the production of bovine in vitro embryos, by adding PAPPA, ergothioneine and selenocysteine to the bovine oocyte in vitro maturation culture solution, early embryo culture solution, late embryo culture solution, freezing solution and thawing solution, the in vitro maturation performance of oocytes and the development performance and vitality of the subsequent obtained embryos can be effectively improved. Studies have shown that after adding the above additives, the maturation rate of oocytes, the cleavage rate, the blastocyst rate of embryos, and the freezing survival rate, the hatching rate and the ATP content of embryos after freezing preservation are significantly improved, which has important application value.

This invention belongs to the field of organoid culture technology, specifically involving BCL2 Application of genes in 3D cluster culture of stem cell-derived organoids. Starting on day 4 of bovine embryonic stem cell cluster culture, the cell cluster edges became indistinct, cells began to die and darken, and the cell clusters began to diffuse. By day 7, the cell cluster survival rate was only 14.2%. This invention utilizes the overexpression of genes in bovine embryonic stem cells... BCL2 Following gene expression, the cells were cultured in clusters using the same method. The overexpressed cell clusters showed clear edges, and their volume increased continuously with the number of culture days. Even on day 7, clear cell cluster edges were still observable, and no diffuse cell death was observed; the survival rate remained high at 85.54%. BCL2 Genes can improve the survival rate of early cultured cell clusters of organoids, which is of great significance for the normal morphogenesis and functional maturation of organoids and can greatly improve the efficiency of organoid preparation.

This disclosure provides ungulate embryonic stem cells (ESCs) derived from the inner cell mass of pre-implantation blastocysts or pluripotent cells from embryos. From an agricultural and biomedical perspectives, the derivation of stable ESCs from domestic ungulates is important for genomic testing and selection, genetic engineering, and providing an experimental tool for studying human diseases. Cattle are one of the most important domestic ungulates that are commonly used for food and bioreactors.

The utility model provides a yak embryo sterile preservation room, which relates to the technical field of sterile preservation and comprises a sterile room body consisting of four groups of coamings, an upper sealing plate and a lower sealing plate. A tooth type conveying belt is rotationally arranged in the middle of each movable display stand, a control motor is fixedly arranged outside each movable display stand, and the control motors are in transmission connection with rotating shafts of the tooth type conveying belts; a pick-and-place channel is fixedly arranged in the middle of the front side of the sterile room body; a bottom guide row is fixedly arranged in the middle of the bottom of the sterile room body, and a vertical sliding frame matched with the guide rail is slidably arranged at the top; an arc-shaped clamping base is arranged, a three-axis moving storage function is provided, through three-axis moving control, storage vessels can be automatically grabbed and fixed and placed above a conveying belt, then a control motor is started to move the tooth type conveying belt by one station, the storage vessels can continue to be placed, and large-scale unmanned storage is achieved; and the storage vessel can be taken out through reverse operation, control is convenient, and the problem that an existing storage chamber is inconvenient to store automatically is solved.

The utility model belongs to the technical field of cattle embryo refrigeration preservation, and particularly relates to a cattle embryo refrigeration preservation device which comprises a liquid nitrogen tank, the upper end of the liquid nitrogen tank is in contact with a tank cover, the outer side of the end of the tank cover is fixedly sleeved with a sealing ring, and the sealing ring is connected with the liquid nitrogen tank in a sliding mode. Two symmetrically-distributed connecting rods are fixedly connected to the end of the tank cover, a fixing frame is fixedly connected to the lower ends of the two connecting rods together, an embryo tank is fixedly connected to the bottom of the inner side of the fixing frame, and a supporting frame is fixedly connected to the outer side of the liquid nitrogen tank. According to the scheme, by arranging a motor, a gear, a toothed plate and other structures, a tank cover can be driven to move so as to drive a fixing frame to move, then an embryo tank is driven to move, automatic taking and using are conducted on the embryo tank, and under the action of clamping holes, clamping rods, first springs and other structures, movement limiting treatment can be conducted on the tank cover; and the embryo tank can be accurately positioned and used.

The invention relates to the technical field of biological information processing, in particular to a cattle embryo pre-implantation genetic evaluation method and system based on whole genome sequencing, and the method comprises the following steps: obtaining whole genome sequencing data of a male parent and a female parent, embryo trophoblast live detection sequencing data and culture solution free desoxyribonucleic acid sequencing data; performing variation detection and haplotype phasing on male and female parent data to form haplotype data, and calculating a haplotype transmission posterior at an anchor point based on trophoblast data; performing genome segmentation based on reading depth observation and allele frequency observation of a trophoblast and a culture solution, and performing Bayesian inference by combining haplotype transfer posteriori as priori to obtain posteriori probabilities of aneuploid, copy number variation, chimera, pathogenic homozygosis and pollution events; and outputting a passing / rechecking / elimination conclusion, adding sequencing and updating the posteriori during rechecking until ending, and outputting a final conclusion and a genome breeding value, so that the evaluation reliability and the decision interpretability are improved.

This invention discloses a kit for amplifying the whole genome of bovine embryonic cells and its applications. The invention provides a primer set for amplifying the whole genome of bovine embryonic cells, consisting of 20 primers, each 6 nt in length. This invention can utilize 4-8 bovine embryonic cells or genomic DNA down to the pg level to amplify and generate whole-genome DNA amplification products with high coverage and low mismatch rate. These products can be directly adapted to downstream applications such as next-generation sequencing and targeted capture sequencing, while reducing detection costs. Furthermore, the three-step operation process of this invention (cell lysis-lysis termination-isothermal amplification) can complete efficient amplification within 3.5 hours.

This invention relates to the field of bovine embryonic stem cell culture technology, and particularly to a long-term culture system for morphological bovine embryonic stem cells completely without a feeder layer and its applications. After trying various culture methods, this invention establishes a feeder-free bovine morphological embryonic stem cell line (FF-bESCs). Bovine morphological embryonic stem cells cultured under the feeder-free conditions provided by this invention can maintain stable proliferation capacity and pluripotency over a long period. The bovine morphological embryonic stem cells provided by this invention can undergo gene editing and can be effectively induced to differentiate into primordial germ cell-like cells (PGCLCs). This invention lays an important foundation for the standardized and large-scale culture of bovine embryonic stem cells under feeder-free conditions, and for their application in stem cell breeding and other fields.

This disclosure relates to methods, products, and compositions useful for inducing bovine induced blastoids. Methods enabling naive stem cell aggregation and induced blastoid formation are described. Aggregation and induced blastoid media suitable for promoting naive stem cell aggregation and preparing induced blastoids are also provided. Bovine induced blastoids may be used in selection and breeding programs, including the replication of pre-transplant embryos with desirable characteristics, as well as in vitro breeding, including cross-generation breeding cycles from parental germ cells and / or embryos to offspring embryos.

The application belongs to the technical field of cell biology, and particularly relates to a feeder-free and serum-free bovine embryonic stem cell culture medium, a culture system and a culture method. The bovine embryonic stem cell culture medium comprises a conditioned medium and an additive. The additive is IWR-1 and activin A. The concentrations of IWR-1 and activin A in the conditioned medium are 2.5-5 muM and 20 ng / mL-25 ng / mL respectively. The culture system comprises the bovine embryonic stem cell culture medium, 0.1%-0.2% gelatin in mass concentration and a subculture medium. The 0.1%-0.2% gelatin in mass concentration is used to provide an attachment matrix for the bovine embryonic stem cell. The subculture medium is prepared by adding Y27632 to the bovine embryonic stem cell culture medium. The application reduces the cost of extracellular matrix, and the feeder-free bovine embryonic stem cell has high single cell subculture efficiency and is convenient for gene editing operation.

The application discloses a culture solution for improving the blastocyst rate and quality of bovine embryos, application and an in-vitro culture method, and relates to the technical field of animal embryo engineering. The application provides a culture solution for in-vitro culture of bovine embryos, which comprises a first culture solution and a second culture solution with different osmotic pressure concentrations. By controlling the osmotic pressure concentrations of the first culture solution and the second culture solution in a suitable range, and by using the first culture solution in the early stage of embryo development and the second culture solution in the late stage of embryo development, the in-vitro culture system can better simulate the environment in the oviduct in the early stage of embryo development and better simulate the environment in the uterus in the late stage of embryo development, so that the osmotic pressure stress of the embryo in the development process is reduced, and the blastocyst rate and the blastocyst quality of the bovine embryo are improved. Compared with a traditional culture system with constant osmotic pressure, the application significantly improves the blastocyst rate by sequential regulation of the osmotic pressure, the total cell number of the obtained blastocysts is more, and the apoptosis index is lower.

This invention belongs to the field of biotechnology, specifically relating to an epigenetic regulation inhibitor, a culture medium, and their applications. The epigenetic regulation inhibitor is composed of 1 nM-10 nM of 3-Deazaneplanocin A, 1 nM-10 nM of Trichostatin A, and 10 nM-100 nM of 5-Azacytidine. This invention alters the epigenetic modification state of bovine embryonic stem cells by synergistically regulating histone modification and DNA methylation levels, thereby promoting the activation of pluripotency gene networks and improving the pluripotency and stability of bovine embryonic stem cells. The epigenetic regulation inhibitor of this invention is suitable for long-term culture, maintenance of pluripotency, and preservation of differentiation potential of bovine embryonic stem cells, and can be used in fields such as research on the regulatory mechanisms of bovine embryonic development, screening of important functional genes, and preparation of gene-edited animals.

The utility model discloses a transfer cabinet for bovine embryo transplantation, which relates to the technical field of transfer equipment and comprises a transfer cabinet body, and a test tube limiting mechanism and a cooling mechanism are arranged in the transfer cabinet body. The test tube limiting mechanism comprises three test tube limiting seats which are distributed in the transfer cabinet body in parallel, and the three test tube limiting seats are rotationally arranged on the inner bottom wall of the transfer cabinet body. According to the transfer cabinet disclosed by the utility model, the test tube limiting mechanism is arranged in the transfer cabinet, so that embryo test tubes can be limited by utilizing the cylindrical slots in the surfaces of the test tube limiting seats, and the test tubes can be ventilated by utilizing the first strip-shaped vent holes and the second strip-shaped vent holes in the surfaces of the test tube limiting seats and the cylindrical slots; cold air generated by the refrigeration box is conveyed into a cylindrical slot by utilizing a flow guide cavity of a test tube limiting seat, the test tubes are cooled by utilizing the cold air, a low-temperature storage effect is achieved, a uniform low-temperature effect can be kept on the surfaces of the test tubes, and transfer and transportation of embryos are facilitated.

The application discloses application of sodium crotonate in improving bovine embryo development potential. In the process of culturing bovine SCNT embryos in vitro, adding sodium crotonate (Nacr) with a proper concentration can promote the development of bovine SCNT cells, significantly improve the development rate and blastocyst rate of the SCNT embryos, improve the embryo quality, and further improve the somatic cell nuclear transfer efficiency of pre-implantation embryos. On this basis, the inventors construct a bovine SCNT early embryo in-vitro culture system, optimize the formula of the in-vitro embryo culture solution and the concentration of Nacr, and further establish a method for improving the development potential of early bovine embryos in vitro. The application can improve the development quality of bovine SCNT embryos, provides a research basis for the rapid propagation of high-yield dairy cattle, high-quality beef cattle and even disease-resistant fine breeds, provides a reference for the batch production of high-quality bovine embryos in vitro, and has important practical significance in the field of SCNT embryo in-vitro culture.

The application relates to the technical field of embryo culture, and discloses a biological-sensing-based optimization method for in-vitro culture of bovine embryos. The method acquires basic culture parameters for in-vitro culture of bovine embryos, sets a monitoring period, and collects biological sensing data in each period; the sensing data are processed to obtain a metabolic fluctuation amplitude, and the basic culture parameters are combined to calculate a culture environment optimization coefficient of each period; the optimization coefficient is compared with a preset safety threshold value, when the optimization coefficient is higher than the threshold value, the culture environment parameters are dynamically adjusted according to the metabolic fluctuation amplitude; the adjustment process is recorded, and when the adjustment times reach a preset threshold value, a warning is triggered; after the culture period ends, the environmental changes are continuously monitored and maintenance operation is performed; key parameters are monitored in real time, and when the key parameters exceed a safety range, emergency intervention is performed; finally, the basic culture parameters are updated according to the adjustment process and the emergency intervention result. The method realizes accurate adaptation of the culture environment, and guarantees the stability of the in-vitro culture process of bovine embryos.

The invention discloses application of tetracycline drugs in synchronous separation and maintenance of bovine embryonic stem cells and bovine trophoblast stem cells. Belongs to the technical field of cytobiology and animal embryo engineering. According to the method, cattle blastocysts or cell clusters in the cattle blastocysts are cultured in a culture medium containing tetracycline drugs, so that the dryness of bESCs and bTSCs is synchronously separated and maintained in the same culture system. The technical problem of synchronous separation of bESCs and bTSCs is solved by utilizing the bidirectional regulation and control capability of tetracycline drugs for the first time, and an efficient and low-cost standardized platform is provided for artificial embryos, regenerative medicine and agricultural bioengineering.

The invention provides an application of a metabolic small molecule additive in promoting the development process of a bovine in-vitro embryo, and belongs to the technical field of embryo in-vitro production. In particular to application of a polypeptide substance N-acetylaspartic acid-glutamine (NAAG) in promoting blastocyst formation in a bovine in-vitro embryo culture process. The invention further relates to a preparation method of the polypeptide substance NAAG, and particularly relates to application of the polypeptide substance NAAG in promoting blastocyst formation in a bovine in-vitro embryo culture process. Metabonomics analysis and experimental results prove that by adding the NAAG into an in-vitro embryo culture system, the blastocyst formation rate of the bovine in-vitro fertilized embryo can be remarkably increased, and the development efficiency and development quality of the in-vitro embryo are improved. The invention provides a new technical means for optimizing a cattle in-vitro embryo culture system, improving the embryo breeding efficiency and promoting the rapid propagation of improved cattle breeds, and has important application value and popularization significance in the fields of animal husbandry genetic breeding and animal embryo engineering.

The invention provides a proliferation culture medium for long-term stable passage of bovine-derived cells, a method for promoting long-term stable passage of bovine-derived cells and application of the proliferation culture medium. The proliferation culture medium comprises the following components: a complete culture medium, cycloastragenol and metformin hydrochloride. Researches show that the proliferation culture medium can make bovine embryo fibroblasts (BFFs) break through the proliferation limit in the in-vitro subculture process, a continuous cell line is formed, and production of cell culture meat products with excellent quality is facilitated. The culture medium provided by the invention can support continuous passage of BFFs to 145 generations, and the cell type of the bovine embryo fibroblast cell line subjected to passage to 145 generations is unchanged, so that the culture medium has the advantage of long-term stable passage. Through induction of the proliferation culture medium, the BFFs can break through aging limitation, long-term stable passage (more than or equal to 100 generations) is realized, the waveform protein positive rate of more than 90% is maintained, the requirement of mass production of cell culture meat on the cell number is met, and the proliferation culture medium has a very wide application prospect.

The present application relates to the technical field of exosome, and particularly discloses a stable production method of exosome for promoting bovine embryo development and application thereof. The exosome is prepared by a bovine endometrial epithelial cell line or a bovine oviduct epithelial cell, and stable, large and pure exosome can be obtained, which can be used for large-scale production. Meanwhile, the addition of 100 ug / ml of exosome into the culture medium at the 16-cell stage of bovine embryo development can significantly improve the blastocyst rate and blastocyst quality, and provide support for in vitro culture of embryos.

The present application belongs to the field of embryo transplantation technology, and particularly relates to a cervix dilating device for bovine embryo transplantation, which comprises a hand-held part, a dilating part, a wiping mechanism, a liquid supply mechanism and a storage mechanism. The hand-held part comprises a mounting shell and a storage box fixedly installed at the bottom of the mounting shell. The dilating mechanism comprises a supporting tube, a control wheel and a squeezing wheel. A through hole for the sliding of the supporting tube is formed in the outer wall of the mounting shell. The squeezing wheel is used for squeezing the supporting tube and is elastically arranged in the interior of the mounting shell. The control wheel is rotationally installed in the interior of the mounting shell, and the radial outer wall thereof is attached to the outer wall of the supporting tube. The wiping mechanism comprises a mounting ring and a wiping layer. The mounting ring is a hollow structure and is rotationally installed on the inner wall of the mounting shell. The wiping layer is fixedly installed on the inner wall of the mounting ring. The liquid supply mechanism is used for providing the inner cavity of the mounting ring with disinfectant. The storage mechanism comprises a storage box and an elastic strip. The above-mentioned structure cooperation effectively reduces the stress reaction of the mother body, improves the success rate of embryo transplantation and the operation safety.

The invention discloses application of IMR-1A in isolated culture of bovine embryonic stem cells, and belongs to the technical field of embryo engineering. When IMR-1A with the final concentration of 0.5-2 [mu] M is added into a conventional bovine embryonic stem cell culture solution, the establishment efficiency of the bovine embryonic stem cells can be effectively improved to 80%, and the pluripotency of the separated stem cells can be effectively maintained. Compared with a conventional bovine embryonic stem cell culture solution, after the IMR-1A is added, the expression levels of pluripotent related genes and proteins of the stem cells are remarkably improved. The research result is of great significance in improving a breeding system, improving genetic screening efficiency and breaking through germplasm breeding bottlenecks of cattle.

The invention provides a method for improving the production efficiency of bovine in-vitro embryos, and belongs to the technical field of livestock breeding biology. According to the method, carnosine is added in the bovine oocyte in-vitro maturation stage, melatonin is added in the embryo culture stage after in-vitro fertilization, and the embryonic development effect is remarkably improved through the staged synergistic effect. According to the method, the characteristics of energy metabolism regulation and control of carnosine and oxidation resistance of melatonin are fully utilized, the defects of oxidative stress and insufficient energy supply in in-vitro culture are effectively overcome, the blastocyst development rate and the embryo quality are improved, and the method is high in safety, easy and convenient to operate and good in application prospect.

The invention relates to the technical field of biotechnology, and discloses a construction method of a bovine originating embryonic stem cell line, which comprises the following steps: (1) obtaining in vitro fertilized bovine early blastocyst; (2) trophoblast cells of the blastocyst are removed, an inner cell cluster is inoculated into a culture system containing a feeder layer, and the culture system is a basic culture medium added with FGF2 and WNT pathway inhibitors; and (3) culturing until an inner cell cluster forms a cell set drop, and carrying out digestion passage to obtain the stably proliferated bovine originated embryonic stem cell line. According to the construction method and the application of the bovine embryonic stem cell line, a specific initial induction combination of 'FGF2 + WNT inhibitor IWR1' is adopted, and a cell mass in an early blastocyst is used for establishing the line, so that the bovine embryonic stem cell line which is uniform in form, can be stably passaged for more than 26 generations and still keeps typical embryonic characteristics is successfully obtained; the problems of unstable state and easy differentiation of the bovine stem cell line in the prior art are solved.

The invention provides a method for improving the transfer developmental capacity of a frozen bovine IVF (in-vitro fertilization) embryo, which is characterized in that in the processes of freezing and unfreezing the bovine IVF embryo, culturing the embryo in a later period and the like, high-concentration sodium pyruvate, insulin, TLR2 (toll-like receptor 2) and a Cytosporone B are independently or jointly added, so that the survival rate and developmental capacity of the frozen embryo, the pregnancy rate after transplantation and the like are improved. Experiments show that the embryo survival rate, the hatching rate and the embryo transfer pregnancy rate of a group which is jointly added with sodium pyruvate, insulin, TLR2 and Nur77 agonist B are obviously higher than those of a double-substance adding group, a single-substance adding group and a control group. The method lays a foundation for production and related scientific research of in-vitro fertilized embryos in the future, provides an effective, safe and feasible strategy for improving in-vitro embryo production efficiency and embryo quality, and has great popularization value.

The application discloses a formula for promoting the development of bovine embryos by using KRY-21i and an application method thereof. By adding 10 muM of KRY-21i in the process of culturing bovine embryos after in vitro fertilization and combining the synergistic effect of LIF, FGF2 and IGF1, the formation rate of bovine blastocysts is significantly improved. The experimental results show that, compared with the control group, the blastocyst rate of the KRY-21i treatment group is significantly improved, proving that the addition of KRY-21i in the culture formula can effectively promote the normal development of bovine embryos. The formula and method provide a new technical approach for embryo engineering in the livestock industry, and can significantly improve the breeding efficiency and economic benefits.