21 results about "Polar body" patented technology

The invention provides an oocyte in-vitro maturation culture solution additive and application thereof. The additive is tert-butylhydroquinone. By adding 100 [mu] g / mL of TBHQ into a basic culture solution, the first polar body discharge rate of oocytes is remarkably increased to 77.29%, the ROS level is reduced, and the blastocyst rate of in-vitro fertilized embryos is increased. The technology is suitable for breed conservation of livestock genetic resources such as sheep and can replace live livestock transportation to reduce the cost.

The invention belongs to the technical field of biology, and particularly relates to application of astragaloside in regulation of parthenogenetic activation embryo development. According to the method disclosed by the invention, the maturation rate of the oocytes can be obviously improved by adding substances such as astragaloside into the sheep oocyte in-vitro maturation liquid. The preparation method comprises the following steps: firstly, respectively adding lycopene, astragaloside, isoliquiritigenin, forsythin and baicalein into an oocyte maturation solution, and screening out the optimal dosage of each additive according to the first polar body discharge rate, the cleavage rate and the blastocyst rate; and finally, adding an additive combination into the oocyte maturation liquid according to the screened optimal dosage, detecting the first polar body discharge rate, the cleavage rate and the blastocyst rate to determine an optimal composition, and detecting the levels of ROS, GSH, mitochondrial membrane potential, oxidative stress and apoptosis related genes of the optimal composition to further determine the application effect of the composition.

The invention discloses a method for breeding land-sealed Malus salicina tetraploid. The method comprises the following steps: parent fish rearing; selecting parents; egg taking time; manually squeezing eggs; and inducing and activating, and hatching eggs. According to the method, by mastering the discharge time of the first polar bodies of the primary oocytes of the land-sealed Malus salicina and applying hydrostatic pressure induction to the first polar bodies, throwing of the first polar bodies is successfully inhibited, the first polar bodies are left in the oocytes to continue to be copied, the land-sealed Malus salicina tetraploid fry is successfully bred, and it is detected through a ploidy analyzer that 100% of the fry is tetraploid, so that the first polar bodies are completely separated from the oocytes. According to the method, the land-sealed type Malus salicina is bred to the adult fish stage at present, no malformation exists, the survival rate is high, and the method can provide theoretical and practical basis for land-sealed type Malus salicina fingerling resource protection, tetraploid population research and healthy triploid breeding, has important significance in promoting industrialization development of domestic land-sealed type Malus salicina and has wide market application prospects.

The invention discloses a method for inducing triploid of haliotis discus hannai by salinity, which comprises the following steps: (1) synchronously fertilizing sperms and ova of haliotis discus hannai to obtain fertilized ova; (2) when the appearance proportion of the first polar bodies of the fertilized eggs obtained in the step (1) is 60-70%, performing high-salt treatment or low-salt treatment on the fertilized eggs; (3) the fertilized eggs treated in the step (2) are subjected to salinity gradient sand filtration seawater re-infiltration treatment, the osmotic pressure is gradually adjusted through salinity gradient sand filtration seawater, damage to the fertilized eggs is relieved, then the fertilized eggs are resuspended in fresh sand filtration seawater with normal salinity, and conventional incubation and cultivation are conducted. According to the method, efficient triploid induction can be achieved through salinity optimization and re-infiltration steps, the problems of drug residues and equipment requirements of traditional chemical and physical methods are solved, and the method has the advantages of being safe, simple, convenient and efficient.

The invention provides application of endothelin in oocyte in-vitro maturation and embryo in-vitro culture. New application of endothelin is found, the endothelin can improve the polar body discharge rate of oocytes matured in vitro, promote cytoplasm maturing and improve the normal arrangement proportion of organelles after maturing, certain promotion is provided for improvement of oocyte fertilization, and meanwhile subsequent blastocyst development is promoted. The invention further provides an in-vitro maturation culture solution of the sheep oocytes and an in-vitro maturation method of the sheep oocytes, the in-vitro maturation culture solution contains endothelin, and after the sheep oocytes are treated with the in-vitro maturation culture solution containing the endothelin concentration, the mitochondrial membrane potential level of the oocytes can be remarkably improved, and the survival rate of the sheep oocytes is increased. ROS (reactive oxygen species) in the oocytes is remarkably reduced, and the ATP (adenosine triphosphate) and GSH (glutathione) levels of the oocytes and the normal proportion level of spindles are improved; the maturation rate, the fertilization efficiency, the blastocyst number and the blastocyst rate are remarkably increased, in-vitro maturation of sheep oocytes and in-vitro development of early embryos are promoted, and the production efficiency of the sheep in-vitro embryos is effectively improved.

The invention discloses application of a compound shown in a formula I in promoting in-vitro cultured oocyte development or in-vitro embryo development, and in the formula I, R1 is selected from halogenated alkyl; r2 is selected from H, halogen or C1-6 alkyl; n is selected from 0, 1, 2 or 3. According to the application, the expansion area of cumulus cells in the cumulus-oocyte complex can be increased; the oxidative damage of the cumulus-oocyte complex is relieved; the first polar body discharge rate of the oocytes is improved; the abnormal distribution rate of mitochondria in oocytes is reduced; the membrane potential of mitochondria in the oocyte is improved; and the cleavage rate and blastocyst formation rate of fertilized eggs can be improved, and the apoptosis rate of oocytes can be reduced.

The invention belongs to the technical field of biology, and particularly relates to application of lycopene in sheep oocyte in-vitro culture. It is determined that the maturation rate of the oocytes can be remarkably increased by adding the lycopene and astragaloside composition into the sheep oocyte in-vitro maturation liquid. The preparation method comprises the following steps: firstly, respectively adding lycopene, astragaloside, isoliquiritigenin, forsythin and baicalein into an oocyte maturation solution, and screening out the optimal dosage of each additive according to the first polar body discharge rate, the cleavage rate and the blastocyst rate; and finally, adding an additive combination into the oocyte maturation liquid according to the screened optimal dosage, detecting the first polar body discharge rate, the cleavage rate and the blastocyst rate to determine an optimal composition, and detecting the levels of ROS, GSH, mitochondrial membrane potential, oxidative stress and apoptosis related genes of the optimal composition to further determine the application effect of the composition.

The invention discloses a novel application of myristic acid and a culture solution for promoting maturation of oocytes of pig small cavity follicles, and relates to the technical field of reproductive biology. The method can be used for preparing products for promoting maturation of the oocytes of the small cavity follicles of the pigs. The method comprises the following steps: culturing a cumulus-oocyte complex (COC) in a pig small cavity follicle under an in-vitro condition, and adding a proper concentration of MA (Maleic Anhydride) into a culture solution. By detecting functional indexes such as first polar body discharge of oocytes, mitochondrial distribution of the oocytes and apoptosis of the oocytes, it is found for the first time that in-vitro maturation of the oocytes of the small follicles is promoted and the quality of the oocytes is improved by adding MA in vitro.

The invention belongs to the field of cell culture, and particularly discloses application of dominant follicle exosomes in oocyte in-vitro maturation culture, after the dominant follicle exosomes with the concentration of 10 ng / mL-200 ng / mL are added into a porcine oocyte in-vitro maturation culture solution for porcine oocyte in-vitro maturation culture, the first polar body discharge rate is remarkably increased, and the first polar body discharge rate is remarkably increased. Therefore, the dominant follicle exosome can effectively improve the in-vitro maturity of the porcine oocytes; the dominant follicle exosomes with different concentrations are added, so that the diffusion area of cumulus cells is remarkably increased; after the dominant follicle exosome is added, the ROS fluorescence intensity is obviously reduced, and the GSH fluorescence intensity is obviously increased; by adding the dominant exosome, the remodeling of the mitochondrial structure and function in the oocyte is accelerated; adding of the dominant exosome promotes functional remodeling of the mitochondrial complex I to increase ATP generation and reduce oxidative stress so as to promote nucleoplasm co-maturation of oocytes. The subsequent embryonic development can be promoted.

This invention provides a precise method for extracting cell polar bodies, belonging to the field of subcellular micromanipulation technology. The method includes the following steps: S1: Subtracting positive and negative defocused images, binarizing them, and using polar coordinate information to locate the polar body; S2: Performing force analysis on the polar body, and determining the motion trajectory of the microneedle approaching the polar body through finite element simulation modeling; S3: Inserting the microneedle into the cell, using an active disturbance rejection controller motion control algorithm to compensate for disturbances and errors during the microneedle's movement, correcting the microneedle's motion trajectory, and ensuring the microneedle approaches the polar body along the trajectory determined in S2; the microneedle then extracts the polar body. This invention manipulates cell polar bodies under a microscope while the microneedle is fixed in a suction-holding state. It allows the microneedle to be moved to a suitable position for polar body removal without staining the polar body, avoiding the difficulties caused by photobleaching and fluorescence fields resulting from staining, which can affect microneedle movement and the calculation of cytoplasm removal. It also reduces the damage to oocyte viability caused by excessive cytoplasm removal.

The present invention relates to a method for cultivating tetraploid Haliotis discus hannai, belonging to the technical field of genetic breeding of aquaculture varieties. The method includes the selection of broodstock, the ripening promotion of broodstock, the spawning induction of broodstock, artificial insemination, and the inhibition of polar body release of fertilized eggs; the selected male and female broodstock are separately ripened; the female broodstock is induced to spawn by means of air-drying, running water stimulation, and soaking in hydrogen peroxide seawater; the eggs are preferably selected and fertilized by adding high-concentration sperm; cytochalasin B is used to inhibit the release of the first polar body of the fertilized eggs. The unique three-stage gonad ripening promotion method and spawning induction method for triploid female abalones of the present invention can obtain more high-quality eggs; the present invention uses cytochalasin B to inhibit the release of the second polar body of fertilized eggs to obtain tetraploids, with a stable induction rate of more than 96%, and a large number of tetraploid Haliotis discus hannai can be obtained.

The present invention provides a method for preparing allodiploid scallops, which is to screen for all-female individuals among the hybrid offspring of Argopecten irradians (♀) and Argopecten purpuratus (♂), fertilize the eggs of the all-female individuals with the sperm of Argopecten irradians, and cultivate the fertilized eggs to obtain allodiploid scallops; the screening is to screen for all-female individuals with a ratio of the nuclear genome derived from Argopecten irradians to the nuclear genome derived from Argopecten purpuratus in the genome higher than 1.5. The method of the present invention is different from the currently mainstream methods of inhibiting the release of polar bodies of diploid fertilized eggs by chemical reagents, salinity adjustment, cold and heat shock, etc., and the method of obtaining triploid offspring by crossing tetraploid and diploid parent scallops, and has opened up a brand-new allodiploid scallop breeding technology.

The present invention discloses a chemical induction method for the triploid of Apostichopus japonicus Huachun No. 1, belonging to the technical field of genetic breeding of marine echinoderms. The chemical induction method comprises the following steps: promoting the maturation of broodstock, artificially inducing ovulation or sperm ejaculation, treating fertilized eggs with staurosporine, hatching the fertilized eggs to obtain juvenile sea cucumbers, and continuing to cultivate the juvenile sea cucumbers. The induction drug used in the present invention is staurosporine, which is a reversible inhibitor related to microfilament assembly and can inhibit the emission of the first polar body of fertilized eggs. Compared with traditional induction drugs such as cytochalasin B and 6-DMAP, staurosporine has the advantages of rapid inhibitory effect, low toxicity and easy cleaning. In addition, the staurosporine induction method provided by the present invention is completely different from the previous physical induction methods, not only reducing the operation difficulty, but also improving the survival rate of larvae and the triploid yield.

The invention discloses a novel application of a compound MLN4924 (Pevonedat) in preparation of a medicine for improving oocyte quality and treating related diseases, and belongs to the technical field of biological medicines. PCOS animal model experiments prove that the reproductive endocrine indexes of model animals can be remarkably improved by applying the MLN4924, and the reproductive endocrine indexes are specifically represented by restoring the regularity of an estrus cycle and reducing the levels of testosterone and luteinizing hormone in serum; meanwhile, the fertility is improved, and the birth number is increased. In the aspect of oocyte quality, the treatment can increase the number of normal oocytes in an ovary, improve the first polar body discharge rate of in-vitro maturation of the oocytes and improve the blastocyst formation rate after in-vitro fertilization. A further mechanism research shows that the MLN4924 can inhibit the plasmosis of the TRIM21 in the ovarian granular cells and down-regulate the expression level of the protein of the TRIM21. The results show that the MLN4924 provides a new drug choice for developing related diseases for treating polycystic ovarian syndrome (PCOS) and improving oocyte quality.

The application develops a quantitative induction method for tetraploid time point of Fujian oyster by taking the development stage of fertilized egg as a biological index. The application effectively eliminates the defects of poor gamete synchrony caused by traditional mixed gametes of multiple pairs of oysters by means of optimizing sperm and egg, obtaining sperm and egg by dry stripping method, and single pair fertilization. The application is different from the traditional method (after 8-10 min of fertilization, using reagent for continuous treatment for 15-20 min), and takes the development status of fertilized egg as a biological index. The time period from the beginning of fertilization of the control group to the appearance of the first first polar body (PB1) of the fertilized egg is defined as A min, and the time period from the appearance of the first PB1 of the fertilized egg of the control group to the development of PB1 of 50% of the fertilized eggs is defined as B min. When the fertilization time of the treatment group is (A-3) min, the inducer is added for continuous treatment for B min, which effectively avoids the problems of low and unstable induction rate caused by fixed induction time. The tetraploid rate of the Fujian oyster cultivated by the application is high and stable to the juvenile stage, which provides a new idea for polyploid breeding of Fujian oyster.

The application relates to an image recognition-based method for positioning an oocyte polar body in an intracytoplasmic sperm injection, obtaining a microscopic digital image of an oocyte; determining an inner edge of a zona pellucida, an outer edge of a cytoplasm and a peripheral gap region located between the two according to the microscopic digital image; extracting a candidate region in the peripheral gap region; respectively judging whether each candidate region is located in the peripheral gap region, is not connected with the cytoplasm region and is not connected with the zona pellucida outer side region; determining a valid candidate region which is located in the peripheral gap region, is not connected with the cytoplasm region and is not connected with the zona pellucida outer side region; determining a target polar body region according to the valid candidate region; determining a polar body center position and polar body orientation information according to the target polar body region, and outputting a polar body positioning result. The method can be implemented based on a microscopic digital image, and is favorable for improving the stability, consistency and automation application value of the polar body positioning result.

The invention discloses a method for constructing a linkage analysis library based on single sperms or polar bodies. The method comprises the following steps: splitting a single sperm or a polar body to prepare a split single sperm or polar body; and carrying out multiple PCR amplification, terminal repair, linker connection and PCR enrichment and purification to prepare the target library. The method at least has the advantages that single cell whole genome amplification does not need to be carried out, library construction can be directly carried out on a polar body or a single sperm, the detection period is shortened, and the detection cost is reduced.

The application discloses a method for inducing triploid of Haliotis discus hannai Ino by salinity, comprising the following steps: (1) synchronously fertilizing sperm and ovum of Haliotis discus hannai Ino to obtain fertilized eggs; (2) when the proportion of the first polar body appearing in the fertilized eggs obtained in step (1) is 60-70%, the fertilized eggs are subjected to high-salt treatment or low-salt treatment; and (3) the fertilized eggs treated in step (2) are subjected to reperfusion treatment of salinity gradient sand-filtered seawater, so as to gradually adjust the osmotic pressure by the salinity gradient sand-filtered seawater, slow down the damage to the fertilized eggs, and then resuspend in fresh sand-filtered seawater with normal salinity, and perform conventional incubation and cultivation. The application can realize efficient induction of triploid by optimizing the salinity and reperfusion steps, avoids the problems of drug residues and equipment requirements in traditional chemical and physical methods, and has the advantages of safety, simplicity and high efficiency.

The invention discloses a novel application of palmitoleic acid and a culture solution for promoting in-vitro development and maturation of porcine oocytes, and belongs to the field of reproductive biology in developmental biology. The method can be used for preparing products for promoting in-vitro development and maturation of oocytes. According to the present invention, pig cumulus-oocyte complexes (COCs) are cultured under the in vitro condition, and the palmitoleic acid is added to the culture solution to explore the influence of the palmitoleic acid on the first polar body discharge rate, the cortical particle distribution, the mitochondrial distribution and the oocyte reactive oxygen species (ROS) level of the oocytes, such that the results show that the in vitro addition of the palmitoleic acid can improve the oocyte quality, and the in vitro addition of the palmitoleic acid can provide the research on the oocyte first polar body discharge rate, the cortical particle distribution, the mitochondrial distribution and the oocyte reactive oxygen species (ROS) level; and a good effect is achieved.

The present invention relates to preimplantation genetic testing for aneuploidy (PGT-A). Specifically, the present invention identifies granulosa cell-specific differentially methylated regions (C-DMRs) and oocyte / polar body cell-specific differentially methylated regions (O-DMRs) for assessing maternal DNA contamination in blastocyst culture fluid, and provides a method for assessing maternal DNA contamination in blastocyst culture fluid based on the above-mentioned differentially methylated regions. Compared with the traditional SNP sequencing method, the method for determining maternal DNA contamination of the present invention is simpler, more economical, and time-saving, and is suitable for large-scale clinical applications. The present invention also provides a method for non-invasive preimplantation aneuploidy genetic testing based on blastocyst culture fluid, which simultaneously detects aneuploidy and maternal contamination rates, thereby achieving improved detection accuracy.