Serum-free embryo cutting biopsy culture solution and application thereof

Abstract

The application provides a serum-free embryo cutting biopsy culture solution and application thereof, and belongs to the technical field of embryo cutting biopsy. The serum-free embryo cutting biopsy culture solution comprises a basic culture medium component, an antioxidant component such as a vitamin, a protein growth factor component, a small molecule inhibitor component and an other molecular compound component. The biopsy culture solution is carefully screened and optimized in combination, so that the components can efficiently synergize, provide key nutritional support and anti-apoptotic protection, effectively reduce the operation damage of embryo biopsy, and maintain the efficient development potential and implantation rate of embryos. The chemical components of the application are clear, and the instability caused by serum factors is excluded, so that the problem of long-term lack of efficient, stable and standardized serum-free cutting biopsy culture solution in the field of embryo cutting biopsy is solved. The application has a wide application prospect in the field of livestock embryo genomic selection breeding and embryo cutting biopsy.

Description

Technical Field

[0001] This invention relates to the field of embryo biopsy technology, and more particularly to a serum-free embryo biopsy culture medium and its application. Background Technology

[0002] Genomic selection technology for livestock embryos, a cutting-edge technique in modern animal breeding, focuses on the precise assessment of the genetic potential of fertilized eggs or early embryos through high-throughput genotyping and bioinformatics analysis. This technology integrates genome-wide association studies, quantitative trait locus mapping, and machine learning algorithms, overcoming the spatiotemporal limitations of traditional phenotypic selection and significantly shortening generation intervals. In dairy cattle, genomic selection has increased the rate of genetic progression in milk yield by 2.3 times compared to traditional methods, and achieved a prediction accuracy of 0.78 for backfat thickness. In pig breeding, by integrating transcriptome data, key SNP molecular markers for resistance to porcine reproductive and respiratory syndrome (PRRS) have been successfully screened, reducing the breeding cycle of pigs to 14 months.

[0003] Livestock embryo genomic selection technology mainly includes embryo production, embryo development, embryo biopsy, genome sequencing, and embryo transfer. Among these, the embryo biopsy step is a key step affecting the efficiency of the entire process. Embryo biopsy refers to the extraction and analysis of a small number of cells from an early embryo during in vitro fertilization. Performing embryo biopsy requires extremely high standards: ensuring sufficient cells are obtained for DNA sequencing while preserving the normal development of the embryo and its fertility after transfer.

[0004] The culture medium used for embryo dissection biopsy is crucial to the efficiency of embryo dissection. The embryo dissection process inevitably causes damage to the embryo, leading to decreased developmental capacity. A suitable embryo dissection biopsy culture medium can mitigate this adverse effect to some extent. Furthermore, currently used embryo culture media are almost all serum-added types. Serum, derived from fetal bovine blood, varies in composition and quality between different batches, thus having an unstable impact on embryo development. However, there is currently no specific, standardized serum-free culture medium for livestock embryo dissection biopsy, resulting in problems such as poor embryo survival rate after dissection, low re-cavitation rate, and low embryo transfer conception rate.

[0005] Therefore, establishing a serum-free embryo biopsy culture medium with clearly defined components, high efficiency, and standardization is an urgent problem to be solved in the fields of embryo genome selection technology and livestock breeding. Summary of the Invention

[0006] In view of this, the present invention provides a serum-free embryo biopsy culture medium and its application to solve the above-mentioned problems. The biopsy culture medium of the present invention, through careful screening and optimized combination, enables each component to work synergistically and efficiently, effectively reducing operative damage during embryo biopsy and maintaining the high developmental potential of the embryo and the implantation success rate. The chemical composition of the present invention is clearly defined, eliminating the instability of serum culture and solving the long-standing problem of the lack of efficient, stable, and standardized serum-free embryo biopsy culture medium in the field of embryo biopsy. The present invention has broad application prospects in the fields of genomic selection breeding and embryo biopsy in livestock embryos.

[0007] In a first aspect, this invention provides a serum-free embryo biopsy culture medium. According to embodiments of the invention, the biopsy culture medium comprises the following components: basal culture medium components, antioxidant components, protein growth factor components, small molecule inhibitor components, and other molecular compound components. The biopsy culture medium according to this invention, by adding antioxidant components such as vitamins, protein growth factor components, and small molecule inhibitor components as core components, allows these three components to synergistically act on the embryo biopsy microenvironment, effectively reducing operative damage during embryo biopsy, providing crucial nutritional support and anti-apoptotic protection, maintaining the high developmental potential of the embryo, and increasing the implantation success rate. The chemical composition of this invention is clearly defined, eliminating the instability of serum culture. Thus, it solves the long-standing problem of a lack of efficient, stable, and standardized serum-free embryo biopsy culture media in the field of embryo biopsy.

[0008] According to embodiments of the present invention, the above-mentioned biopsy culture medium may further have the following additional technical features:

[0009] According to an embodiment of the present invention, the basal culture medium components include Neurobasal and DMEM / F12, wherein the concentrations of Neurobasal and DMEM / F12 in the serum-free embryo dissection biopsy culture medium are independently 45.00–50.00 v / v.

[0010] According to embodiments of the present invention, the antioxidant components include vitamin C, vitamin B6, vitamin B8, dimethyl sulfoxide, and N-Acetyl-L-cysteine; the concentrations of each component in serum-free embryo biopsy culture medium are, respectively, 0.005–0.008 g / 100 mL, 0.0846–0.0946 μg / 100 mL, 0.2440–0.2540 μg / 100 mL, 0.100–0.150 v / v%, and 0.0816–0.0916 g / 100 mL.

[0011] According to an embodiment of the present invention, the protein growth factor components include RSPO1, WNT16, Relaxin, and Activin A; the concentrations of each component in serum-free embryo biopsy culture medium are 50.00–100.00 ng / mL, 50.00–100.00 ng / mL, 20.00–50.00 ng / mL, and 10.00–20.00 ng / mL, respectively.

[0012] According to embodiments of the present invention, the small molecule inhibitor components include Chroman, RKI-1447, Emricasan, Q-VD-OPh, AT-7867, Ruxolitinib, and CCT-241533; the concentrations of each component in serum-free embryo biopsy culture medium are, respectively, 0.00318–0.00418 μg / 100mL, 0.033843–0.043843 μg / 100mL, 0.0590–0.0690 μg / 100mL, 0.07289–0.08289 μg / 100mL, 0.03214–0.04214 μg / 100mL, 0.196735–0.206735 μg / 100mL, and 0.037242–0.047242 μg / 100mL.

[0013] According to embodiments of the present invention, the other molecular compound components include insulin, transferrin, sodium selenite, penicillin, and streptomycin; the concentrations of each component in serum-free embryo dissection biopsy culture medium are, respectively, 0.950–1.05 g / 100 mL, 0.5270–0.5670 g / 100 mL, 0.047–0.051 g / 100 mL, 0.009–0.011 g / 100 mL, and 0.011–0.013 g / 100 mL.

[0014] According to an embodiment of the present invention, the osmotic pressure of the serum-free embryo biopsy culture medium is 275–295 mOsm / L.

[0015] According to an embodiment of the present invention, the pH value of the serum-free embryo dissection biopsy culture medium is 7.2 to 7.4.

[0016] In a second aspect, the present invention provides an embryo biopsy method. According to an embodiment of the invention, the embryo biopsy method includes: placing a sample containing the embryo to be tested into the biopsy culture medium described in the first aspect, and performing detection or sampling detection; wherein the embryo biopsy method is for non-diagnostic and non-therapeutic purposes; the embryo to be tested is an early embryo in the mammalian fertilized egg to blastocyst stage. The embryo biopsy method according to the embodiments of the present invention, by placing the embryo to be tested into the aforementioned biopsy culture medium for detection or sampling detection, enables accurate detection or sampling analysis of the embryo without affecting its subsequent developmental potential and implantation success rate. Furthermore, if sampling detection is performed, the sample is highly suitable for genetic testing technology, and the trace amount of DNA is sufficient to meet the requirements of microarray detection and embryo genome selection; thus, it provides a safer and more efficient embryo biopsy method for fields such as in vitro embryo culture, embryo development research, and livestock breeding, and has extremely high application value.

[0017] In a third aspect of the invention, the invention proposes the application of the biopsy culture medium described in the first aspect and the embryo biopsy method described in the second aspect in embryonic genome selection, basic biology of embryonic development, and medical research; wherein the embryo biopsy is for non-diagnostic and non-therapeutic purposes; and the embryo is an early embryo in the blastocyst stage of a mammalian fertilized egg.

[0018] By adopting the above technical solution, the present invention has the following beneficial effects:

[0019] 1. When using this serum-free embryo biopsy culture medium for embryo biopsy, the embryo still maintains a high level of developmental potential, and the conception rate after embryo transfer is not affected; it effectively avoids DNA damage and cell apoptosis caused by embryo cutting; the serum-free embryo biopsy culture medium of this invention has the characteristics of being specific, efficient, and standardized.

[0020] 2. The embryonic cell cutting biopsy sampling method proposed in this invention is simple to operate, convenient and easy to learn, and easy for embryo operators to master and promote its use. Attached Figure Description

[0021] Figure 1 The embryo biopsy culture dish and cutting knife.

[0022] Figure 2 Images of embryos before, after, and 8 hours after cutting.

[0023] Figure 3 TUNEL staining and statistical results of apoptosis in cells after embryos were cut using different embryo biopsy culture media. Detailed Implementation

[0024] The embodiments of the present invention are described in detail below. The embodiments described below are exemplary and are only used to explain the present invention, and should not be construed as limiting the present invention.

[0025] It should be noted that the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of indicated technical features. Therefore, a feature defined as "first" or "second" may explicitly or implicitly include one or more of that feature. Furthermore, in the description of this invention, unless otherwise stated, "a plurality of" means two or more.

[0026] The endpoints and any values ​​of the ranges disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0027] In this document, the terms “comprising” or “including” are open-ended expressions, meaning that they include the contents specified in this invention, but do not exclude other aspects.

[0028] In this document, the terms “optionally,” “optionally,” or “optionally” generally refer to an event or condition that may, but may not, occur, and the description includes both cases in which the event or condition occurs and cases in which the event or condition does not occur.

[0029] Biopsy culture medium

[0030] This invention provides a serum-free embryo dissection biopsy culture medium containing the following components at the following concentrations: 45.00–50.00 v / v% Neurobasal, 45.00–50.00 v / v% DMEM / F12, 0.005–0.008 g / 100 mL Vitamin C, 0.0846–0.0946 μg / 100 mL Vitamin B6, 0.2440–0.2540 μg / 100 mL Vitamin B8, 0.100– 0.150 v / v% dimethyl sulfoxide, 0.950–1.05 g / 100 mL insulin, 0.5270–0.5670 g / 100 mL transferrin, 0.047–0.051 g / 100 mL sodium selenite, 0.0816–0.0916 g / 100 mL N-Acetyl-L-cysteine, 50.00–100.00 ng / mL RSPO1, 50.00–100.00 ng / mL WNT16, 20.00~50.00ng / mL Relaxin, 10.00~20.00ng / mL Activin A, 0.00318~0.00418μg / 100mL Chroman, 0.033843~0.043843μg / 100mL RKI-1447, 0.0590~0.0690μg / 100mL Emricasan, 0.07289~0.08289μg / 100mL Q-VD-OPh, 0.03214~0.04214μg / 100mLAT-7867, 0.196735~0.206735μg / 100mLRuxolitinib, 0.037242~0.047242μg / 100mL CCT-241533, 0.009–0.011 g / 100 mL penicillin and 0.011–0.013 g / 100 mL streptomycin. The volume fraction of Neurobasal is preferably 46.00–49.00 v / v%, more preferably 48.00 v / v%.

[0031] The volume fraction of DMEM / F12 is preferably 46.00 to 49.00 v / v%, more preferably 48.00 v / v%.

[0032] The vitamin C concentration is preferably 0.006–0.007 g / 100 mL, more preferably 0.006 g / 100 mL;

[0033] The concentration of vitamin B6 is preferably 0.0880–0.0920 μg / 100 mL, more preferably 0.09 μg / 100 mL;

[0034] The concentration of vitamin B8 is preferably 0.248–0.252 μg / 100 mL, more preferably 0.25 μg / 100 mL;

[0035] The volume fraction of dimethyl sulfoxide is preferably 0.12 to 0.14 v / v%, more preferably 0.13 v / v%.

[0036] The preferred concentration of insulin is 0.97–1.00 g / 100 mL, more preferably 0.98 g / 100 mL;

[0037] The concentration of transferrin is preferably 0.537–0.557 g / 100 mL, more preferably 0.547 g / 100 mL;

[0038] The concentration of sodium selenite is preferably 0.048–0.050 g / 100 mL, more preferably 0.049 g / 100 mL;

[0039] The concentration of N-Acetyl-L-cysteine ​​is preferably 0.0836–0.0886 g / 100 mL, more preferably 0.0856 g / 100 mL;

[0040] The concentration of RSPO1 is preferably 70.00–90.00 ng / mL, more preferably 80.00 ng / mL;

[0041] The concentration of WNT16 is preferably 70.00–90.00 ng / mL, more preferably 80.00 ng / mL;

[0042] The preferred concentration of Relaxin is 30.00–40.00 ng / mL, more preferably 35.00 ng / mL;

[0043] The concentration of Activin A is preferably 13.00–18.00 ng / mL, more preferably 15.00 ng / mL;

[0044] The concentration of Chroman is preferably 0.00338 to 0.00388 μg / 100 mL, more preferably 0.00358 μg / 100 mL;

[0045] The concentration of RKI-1447 is preferably 0.036843 to 0.040843 μg / 100mL, more preferably 0.038843 μg / 100mL;

[0046] The concentration of Emricasan is preferably 0.0620–0.0670 μg / 100 mL, more preferably 0.0650 μg / 100 mL;

[0047] The concentration of Q-VD-OPh is preferably 0.07589 to 0.07989 μg / 100 mL, more preferably 0.07789 μg / 100 mL;

[0048] The concentration of AT-7867 is preferably 0.03514 to 0.03914 μg / 100 mL, more preferably 0.03714 μg / 100 mL;

[0049] The concentration of Ruxolitinib is preferably 0.198735 to 0.204735 μg / 100 mL, more preferably 0.201735 μg / 100 mL;

[0050] The concentration of CCT-241533 is preferably 0.040242 to 0.044242 μg / 100 mL, more preferably 0.042242 μg / 100 mL;

[0051] The concentration of penicillin is more preferably 0.010 g / 100 mL; the concentration of streptomycin is more preferably 0.012 g / 100 mL.

[0052] According to an embodiment of the present invention, the osmotic pressure of the serum-free embryo biopsy culture medium is 275-295 mOsm / L, preferably 282-288 mOsm / L, and more preferably 285 mOsm / L;

[0053] According to embodiments of the present invention, the pH value of the serum-free embryo dissection biopsy culture medium is 7.2 to 7.4, more preferably 7.3;

[0054] According to embodiments of the present invention, the biopsy culture medium is suitable for embryos from fertilized egg to blastocyst stage, wherein the embryo is a mammalian embryo. Thus, the biopsy culture medium of the present invention is suitable for biopsies of mammalian embryos from fertilized egg to blastocyst stage; exemplaryly, the embryo may be derived from one or more mammals including humans, non-human primates, cattle, sheep, pigs, horses, and mice.

[0055] It should be noted that the scope of application of the biopsy culture medium of the present invention is not limited to the above-mentioned mammalian embryos, but also applicable to all other mammals, as long as the embryos of these animals can be obtained through in vitro fertilization, cloning, parthenogenesis or other in vitro techniques, they should all fall within the protection scope of the present invention.

[0056] It should be noted that in this application, when the type of embryo is human embryo, the "Ethical Guidelines for Human Embryonic Stem Cell Research" will be strictly followed to ensure that all operations are carried out within a legal and ethical framework and do not compromise human dignity and moral standards. Specifically, this application only involves the isolation or acquisition of human embryos that have not undergone in vivo development and are within 14 days after fertilization. These embryos are blastocysts obtained through in vitro fertilization, and their in vitro culture period from fertilization does not exceed 14 days, which meets ethical and legal requirements.

[0057] Embryo biopsy methods

[0058] This invention proposes an embryo biopsy method. According to an embodiment of the invention, the embryo biopsy method includes: placing a sample containing the embryo to be tested into the aforementioned biopsy culture medium, and performing detection or sampling detection; wherein, the embryo biopsy method is for non-diagnostic and non-therapeutic purposes; the embryo to be tested is a mammalian embryo. The embryo biopsy method according to this invention, by placing the embryo to be tested into the aforementioned biopsy culture medium for detection or sampling detection, enables accurate detection or sampling analysis of the embryo without affecting its subsequent developmental potential and implantation success rate. Furthermore, if sampling detection is performed, the sample is highly suitable for genetic testing technologies, and the trace amount of DNA is sufficient to meet the requirements of microarray detection and embryo genome selection. Therefore, it provides a safer and more efficient embryo biopsy method for fields such as in vitro embryo culture, embryonic development research, and livestock breeding, and has extremely high application value.

[0059] It should be noted that the genetic testing technology refers to a series of techniques used to analyze the genetic information of embryonic cells. These techniques can detect and analyze the DNA of embryonic cells to obtain information about the genetic characteristics of the embryo. Specifically, the genetic testing technology includes, but is not limited to, the following: DNA chip sequencing, single-cell sequencing, multiple substitution amplification, polymerase chain reaction, fluorescence in situ hybridization, whole genome sequencing, whole transcriptome sequencing, single nucleotide polymorphism (SNP) detection, etc. It should be noted that although the embryo biopsy solution of the present invention is only verified to meet the requirements of DNA chip sequencing, the formulation of the embryo biopsy solution of the present invention is not limited to this specific detection technology. As long as the cell sample obtained by embryo biopsy meets the genetic testing requirements of this technology, including but not limited to the above-listed techniques, they all fall within the protection scope of the present invention.

[0060] It should be noted that the embryo biopsy method of the present invention is applicable to humans, non-human primates, cattle, sheep, pigs, horses, and mice, but its scope of application is not limited to the embryos of the aforementioned mammals. It is also applicable to all other mammals, as long as the embryos of these animals can be obtained through in vitro fertilization, cloning, parthenogenesis, or other in vitro techniques, they should all fall within the protection scope of the present invention.

[0061] It is important to note that when the type of embryo to be tested is a human embryo, the "Ethical Guidelines for Human Embryonic Stem Cell Research" will be strictly followed to ensure that all operations are conducted within a legal and ethical framework and do not compromise human dignity and moral standards. Specifically, this application only involves the isolation or acquisition of human embryos that have not undergone in vivo development and are within 14 days after fertilization. These embryos are blastocysts obtained through in vitro fertilization, and their in vitro culture period from fertilization does not exceed 14 days, which meets ethical and legal requirements.

[0062] application

[0063] This invention proposes the application of the aforementioned biopsy culture medium and the aforementioned embryo biopsy method in embryonic genome selection, basic biology of embryonic development, and medical research; wherein, the embryo biopsy is for non-diagnostic and non-therapeutic purposes; and the embryo is a mammalian embryo.

[0064] Those skilled in the art will understand that the features and advantages described above for biopsy culture media or embryo biopsy methods also apply to this application, and will not be repeated here.

[0065] For example, the application scenarios of the aforementioned biopsy culture medium and the aforementioned embryo biopsy method mainly include: (1) after sampling embryonic cells using the biopsy culture medium and live embryo biopsy method of the present invention, it is used for livestock embryo genome selection to accelerate the breeding of new livestock breeds; (2) after sampling embryonic cells using the biopsy culture medium and live embryo biopsy method of the present invention, it is combined with omics sequencing technology for livestock embryo quality identification, screening high-quality embryos for embryo transfer, increasing the conception rate, and improving the economic benefits of livestock breeding; (3) as a basic technical means for studying the early embryonic development mechanism of animals, such as the biopsy culture medium of the present invention. (3) After sampling embryonic cells using the live embryo biopsy method of the present invention, the gene expression change pattern of embryos at different developmental stages is investigated; (4) Embryonic cells are sampled using the live embryo biopsy method of the present invention to isolate single stem cells or specific stem cell populations from the embryo, and cultured in vitro to produce specific types of stem cell lines, such as pluripotent stem cells and trophoblast stem cells; (5) After sampling embryonic cells using the live embryo biopsy method of the present invention, the embryos are subjected to microdissection and gene editing operations to construct various disease models for studying the pathogenesis of diseases, drug screening and exploration of treatment methods.

[0066] The present invention will be explained below with reference to embodiments. Those skilled in the art will understand that the following embodiments are for illustrative purposes only and should not be considered as limiting the scope of the invention. Where specific techniques or conditions are not specified in the embodiments, they are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be obtained commercially.

[0067] Example 1. In vitro production of early bovine and ovine embryos

[0068] (1) In vitro maturation of oocytes

[0069] Ovaries were collected at the slaughterhouse and placed in physiological saline at 35°C, then transported back to the laboratory within 2 hours. After washing three times with physiological saline, the follicles were punctured using a disposable syringe with a 20G needle containing 5 mL of oocyte aspiration fluid, releasing the cumulus-oocyte complex. Under a stereomicroscope, oocytes with homogeneous cytoplasm and at least three layers of cumulus cells were collected. The selected cumulus-oocyte complexes were washed three times with oocyte aspiration fluid, and then three times with basal oocyte maturation culture medium pre-equilibrated in an incubator for 3 hours. The complexes were then transferred to four-well plates containing 600 μL of different in vitro maturation culture media and 300 μL of mineral oil per well, with 30 cumulus-oocyte complexes per well. The four-well plates were then incubated in a 5% CO2, 38.5°C saturated humidity incubator for 24 hours.

[0070] Oocyte aspiration fluid composition: 49mL TCM199 culture medium + 1mL FBS (fetal bovine serum).

[0071] Composition of the basal culture medium for in vitro maturation of oocytes: 90% TCM199 medium, 10 IU / mL pregnant mare serum gonadotropin, 10 IU / mL luteinizing hormone, 10 ng / mL estrogen, 100 IU / mL penicillin, 100 μg / mL streptomycin, 10% follicular fluid, 0.50 mg / mL glucose, 2.10 mg / mL sodium bicarbonate, 0.06 mg / mL sodium pyruvate, and 0.57 mg / mL cysteine.

[0072] (2) In vitro fertilization

[0073] The cumulus-oocyte complex, which matured in vitro for 24 hours, was placed in 0.5% hyaluronidase and gently and repeatedly pipetted to remove most of the cumulus cells. The cells were then washed three times with fertilization solution to obtain mature oocytes. These oocytes were then transferred into fertilization solution that had been equilibrated in an incubator for more than 2 hours and cultured in four-well plates. Each well contained 400 μL of fertilization solution, 300 μL of mineral oil for covering, and 30 mature oocytes for in vitro fertilization.

[0074] Frozen sperm was removed from liquid nitrogen and rapidly thawed in a 38°C water bath. The thawed sperm was then transferred to 600 μL of sperm suspension medium (purchased from IVF Bioscience) and incubated for 30 minutes to allow the sperm to float. Subsequently, 100 μL of the semen supernatant was added to a four-well plate containing mature oocytes, and the sperm and eggs were incubated for 21 hours. Fertilization conditions were saturated humidity, 38.5°C, 5% CO2, and 95% air.

[0075] The fertilization solution was prepared as follows: mSOFaa culture medium + 2% bovine serum (by volume) + 100 μg / mL streptomycin + 100 IU / mL penicillin.

[0076] The mSOFaa culture medium was prepared as follows: each 1L contained 6.34g NaCl, 0.54g KCl, 0.23g CaCl2·2H2O, 0.01g MgSO4·7H2O, 0.17g KH2PO4, 0.24g NaHCO3, 0.30g glucose, 0.04g sodium pyruvate, 5.8mL sodium lactate, 2% BME amino acid solution (v / v), and 1% MEM non-essential amino acid solution (v / v). All reagents were purchased from the Sigma-Aldrich website.

[0077] (3) Embryo culture

[0078] After co-incubation of sperm and eggs for 21 hours, oocytes were aspirated, and residual cumulus cells and sperm were gently removed by pipetting. The oocytes were washed three times with mSOFaa culture medium, then transferred to mSOFaa culture medium pre-equilibrated in an incubator for at least 2 hours, and placed in separate wells of a four-well plate. The plates were then cultured in a saturated humidity incubator at 38.5℃, 5% O2, 5% CO2, and 90% N2. Blastocysts were obtained after 7 days of culture and used for embryo biopsy.

[0079] Example 2: Cut biopsy of early bovine and ovine embryos

[0080] This embodiment demonstrates the effect of the serum-free embryo biopsy culture medium of the present invention on the survival, re-entrapment, and implantation of bovine and ovine embryos.

[0081] (1) In vitro embryo production in cattle and sheep

[0082] Collect cumulus-oocyte complexes according to the method described in Example 1, and perform in vitro maturation, in vitro fertilization, and embryo culture to obtain blastocysts.

[0083] (2) Preparation of serum-free embryo dissection biopsy culture medium

[0084] Accurately measure 48 mL of Neurobasal and 48 mL of DMEM / F12 culture medium, mix well, and then add the following components in sequence according to the following volumes or mass concentrations to prepare embryo biopsy culture solutions I–V:

[0085] Embryo biopsy culture medium I (lacking antioxidant components such as vitamins, protein growth factors, and small molecule inhibitors): 0.98g / 100mL insulin, 0.547g / 100mL transferrin, 0.049g / 100mL sodium selenite, 0.010g / 100mL penicillin, 0.012g / 100mL streptomycin.

[0086] Embryo biopsy culture medium II (lacking vitamins and other antioxidant components): 0.98 g / 100 mL insulin, 0.547 g / 100 mL transferrin, 0.049 g / 100 mL sodium selenite, 0.010 g / 100 mL penicillin, 0.012 g / 100 mL streptomycin, 80.00 ng / mL RSPO1, 80.00 ng / mL WNT16, 35.00 ng / mL Relaxin, 15.00 ng / mL Activin A, 0.00358 μg / 100 mL Chroman, 0.038843 μg / 100 mL RKI-1447, 0.0650 μg / 100 mL Emricasan, 0.07789 μg / 100 mL Q-VD-OPh, 0.03714 μg / 100 mL AT-7867, 0.201735μg / 100mL Ruxolitinib, 0.042242μg / 100mL CCT-241533.

[0087] Embryo biopsy culture medium III (lacking protein growth factor components): 0.98 g / 100 mL insulin, 0.547 g / 100 mL transferrin, 0.049 g / 100 mL sodium selenite, 0.010 g / 100 mL penicillin, 0.012 g / 100 mL streptomycin, 0.006 g / 100 mL vitamin C, 0.09 μg / 100 mL vitamin B6, 0.25 μg / 100 mL vitamin B8, 0.13% dimethyl sulfoxide, 0.0856 g / 100 mL N-Acetyl-L-cysteine, 0.00358 μg / 100 mL Chroman, 0.038843 μg / 100 mL LRKI-1447, 0.0650 μg / 100 mL Emricasan, 0.07789 μg / 100 mL Q-VD-OPh, 0.03714μg / 100mLAT-7867, 0.201735μg / 100mL Ruxolitinib, 0.042242μg / 100mL CCT-241533.

[0088] Embryo biopsy culture medium IV (lacking small molecule inhibitor components): 0.98g / 100mL insulin, 0.547g / 100mL transferrin, 0.049g / 100mL sodium selenite, 0.010g / 100mL penicillin, 0.012g / 100mL streptomycin, 0.006g / 100mL vitamin C, 0.09μg / 100mL vitamin B6, 0.25μg / 100mL vitamin B8, 0.13% dimethyl sulfoxide, 0.0856g / 100mL N-Acetyl-L-cysteine, 80.00ng / mL RSPO1, 80.00ng / mL WNT16, 35.00ng / mL Relaxin, 15.00ng / mL Activin A.

[0089] Embryo biopsy culture medium V (containing all components): 0.98g / 100mL insulin, 0.547g / 100mL transferrin, 0.049g / 100mL sodium selenite, 0.010g / 100mL penicillin, 0.012g / 100mL streptomycin, 0.006g / 100mL vitamin C, 0.09μg / 100mL vitamin B6, 0.25μg / 100mL vitamin B8, 0.13% dimethyl sulfoxide, 0.0856g / 100mL N-Acetyl-L-cysteine, 80.00ng / mL RSPO1, 80.00ng / mL WNT16, 35.00ng / mL L-elaxin, 15.00ng / mL Activin A, 0.00358μg / 100mL Chroman, 0.038843μg / 100mL RKI-1447, 0.0650μg / 100mL Emricasan, 0.07789μg / 100mL Q-VD-OPh, 0.03714μg / 100mL AT-7867, 0.201735μg / 100mL Ruxolitinib, 0.042242μg / 100mL CCT-241533.

[0090] After fully dissolving and mixing, adjust the osmotic pressure to 285 mOsm / L and the pH value to 7.3. Filter using a 0.22 μm sterile filter and store at 4°C. For long-term storage, store at -80°C.

[0091] (3) Cut biopsy of bovine and ovine blastocysts

[0092] Blastocysts on day 7 of development were selected for cutting. The embryos were washed three times with embryo biopsy culture medium and then placed in the medium for later use. During cutting and sampling, individual embryos were first transferred into a cutting droplet (i.e., drops of embryo biopsy culture medium I-V and commercially available cutting biopsy culture medium) in a cutting dish with a slit at the bottom. Under a stereomicroscope, a self-made cutting scalpel was used for freehand cutting and sampling. Figure 1 After cutting, the embryos were transferred to embryo cutting biopsy culture medium and placed for 8 hours. The embryo re-entrapment rate (survival rate) was then calculated. Figure 2 ).

[0093] Control group: Bovine and ovine blastocysts were biopsied using the same procedure with commercially available embryo biopsy culture medium, Biopsy Medium (purchased from IVF Bioscience).

[0094] Blastocoel recovery rate (survival rate) = (number of embryos with restored blastocoel after cutting / number of embryos cut) × 100%.

[0095] (4) Embryo transfer and pregnancy detection

[0096] Embryos that had recovered for 8 hours after cutting were washed three times and transferred to fresh embryo cutting biopsy culture medium for later use. The recipient animal was strapped to a surgical frame, and the tip of the uterine horn on the side of the ovary with the corpus luteum or the side with a good corpus luteum was fixed. The uterine horn wall was punctured in the upper 1 / 3 of the uterine horn wall at the avascular point using a paperclip. Then, the transfer tube tip was inserted into the uterine cavity through the puncture hole. The tip was moved to confirm that it was in the uterine cavity, and the plunger of the syringe connected to the transfer tube was pushed to inject the embryo. The transfer tube was then withdrawn, and finally the uterine horn was returned to the abdominal cavity and disinfected.

[0097] At the same time, the same method was used to transfer uncut embryos.

[0098] Pregnancy was detected by ultrasound on day 45 after sheep embryo transfer and on day 35 after bovine embryo transfer. The pregnancy rate after embryo transfer was then calculated.

[0099] The pregnancy rate is calculated using the following formula: Pregnancy rate = (Number of pregnant recipients / Total number of transplant recipients) × 100%.

[0100] (5) Detection of embryonic cell apoptosis level

[0101] Embryos were collected and washed three times with 0.1% PVA-PBS. The number of apoptotic cells was detected using a one-step TUNEL apoptosis detection kit, and the total number of embryonic cells was determined by DAPI staining. The specific steps were as follows: the zona pellucida was removed with tamperontine, and the embryos were fixed in 4% PFA solution for 30 min. They were then permeabilized for 20 min at room temperature using 0.1% PVA-PBS solution containing 0.5% Triton X100. The embryos were then incubated in TUNEL reaction solution in the dark for 1 h, and washed three times with 0.1% PVA-PBS. Finally, the embryonic nuclei were stained with 1 μg / mLDAPI, and the embryos were incubated in the dark for 5 min before mounting. The number of DAPI- and TUNEL-labeled positive cells was observed and counted under a fluorescence microscope, and photographs were taken. Cells were counted using blue (DAPI) and green (TUNEL) signals, and the total number of cells, the number of apoptotic cells, and the apoptosis rate for each embryo were recorded.

[0102] The formula for calculating the apoptosis rate is: apoptosis rate = (number of apoptotic cells / total number of cells) × 100%. (6) Statistics on the re-entrapment, apoptosis, and embryo transfer of bovine and ovine embryos after biopsy.

[0103] Table 1 shows the embryo re-entrapment rate after amniotic blastocyst biopsy using different embryo culture media; Table 2 shows the embryo transfer conception rate; and Table 3 shows the embryo cell apoptosis level. Figure 3 As shown.

[0104] Table 1. Embryo re-cavity status after sheep blastocyst resection and biopsy.

[0105] Grouping Number of embryos cut / piece Number of double-cavity embryos / piece Re-cavity rate / % Control group (commercial liquid) 192 135 <![CDATA[70.31 c <!-- 8 -->]]> Embryo biopsy culture medium I of the present invention 184 82 <![CDATA[44.57 d ]]> Embryo biopsy culture medium II of this invention 186 143 <![CDATA[76.88 c ]]> Embryo biopsy culture medium III of this invention 190 153 <![CDATA[80.53 b ]]> Embryo biopsy culture medium IV of this invention 188 135 <![CDATA[71.81 c ]]> Embryo biopsy culture medium V of this invention 189 184 <![CDATA[97.35 a ]]>

[0106] In Table 1, different superscript letters (a, b, c, d) in the same column indicate significant differences between groups (P < 0.05).

[0107] Table 2. Embryo Transfer Recognition After Sheep Blastocyst Resection and Biopsy

[0108] Grouping Number of transplant recipients / animal Conception rate / % Control group (commercial liquid) 173 <![CDATA[31.21 c ]]> Embryo biopsy culture medium I of this invention 157 <![CDATA[19.11 d ]]> Embryo biopsy culture medium II of this invention 166 <![CDATA[36.75 c ]]> Embryo biopsy culture medium III of this invention 182 <![CDATA[54.95 b ]]> Embryo biopsy culture medium IV of this invention 178 <![CDATA[34.83 c ]]> Embryo biopsy culture medium V of this invention 168 <![CDATA[64.29 a ]]> Uncut embryos 162 <![CDATA[62.35 a ]]>

[0109] In Table 2, different superscript letters (a, b, c, d) in the same column indicate significant differences between groups (P < 0.05).

[0110] The results in Table 1 show that the re-entrapment rate (survival rate) of sheep blastocysts after resection biopsy using the serum-free embryo resection biopsy culture medium I-V of the present invention were 44.57%, 76.88%, 80.53%, 71.81%, and 97.35%, respectively. Among them, almost all embryos survived after resection using embryo resection biopsy culture medium V, while the re-entrapment rate (survival rate) of the control group (commercial medium) was 70.31%, and the difference was significant (P<0.05).

[0111] Table 2 shows that the embryo transfer conception rates of sheep blastocysts after cutting biopsy using the serum-free embryo cutting biopsy culture medium I-V of this invention were 19.11%, 36.75%, 54.95%, 34.83%, and 64.29%, respectively, while the embryo transfer conception rate of the control group (commercial medium) was 31.21%. The embryo transfer conception rate of embryos after cutting with embryo cutting biopsy culture medium V was similar to that of uncut embryos (62.35%), indicating that embryo cutting with embryo cutting biopsy culture medium V caused the least damage to the embryos.

[0112] Figure 3 The results showed that the apoptosis rate of sheep blastocysts after cutting and biopsy using the serum-free embryo cutting biopsy culture medium V of the present invention was 5.32%, and the apoptosis rate of uncut embryos was 7.24%, both of which were significantly lower than the apoptosis rate of embryos in the control group (commercial medium) (32.18%) (P<0.01).

[0113] In summary, the serum-free embryo cutting culture medium provided by this invention is significantly superior to commercially available cultures for cutting sheep blastocysts. The survival and development of sheep blastocysts are not affected after cutting with the serum-free embryo cutting culture medium V provided by this invention.

[0114] The embryo re-entrapment rate after bovine blastocyst biopsy using different embryo culture media is shown in Table 3, and the embryo transfer conception rate is shown in Table 4.

[0115] Table 3 Embryo re-cavity status after bovine blastocyst resection biopsy

[0116] Grouping Number of embryos cut / piece Number of double-column embryos / piece Re-cavity rate / % Control group (commercial liquid) 117 72 <![CDATA[61.54 b ]]> Embryo biopsy culture medium V of this invention 121 118 <![CDATA[97.52 a ]]>

[0117] In Table 3, different superscript letters (a, b, c, d) in the same column indicate significant differences between groups (P < 0.05).

[0118] The results in Table 3 show that the re-entrapment rate (survival rate) of bovine blastocysts after re-entrapment biopsy using the serum-free embryo culture medium V of the present invention was 97.52%, while the re-entrapment rate (survival rate) of the control group (commercial medium) was 61.54%, with a significant difference (P < 0.05).

[0119] Table 4. Embryo Transfer Success Rates After Bovine Blastocyst Resection Biopsy

[0120] Grouping Number of transplant recipients / animal Conception rate / % Control group (commercial liquid) 73 <![CDATA[36.99 b ]]> Embryo biopsy culture medium V of this invention 78 <![CDATA[56.41 a ]]> Uncut embryos 73 <![CDATA[54.79 a ]]>

[0121] In Table 4, different superscript letters (a, b, c, d) in the same column indicate significant differences between groups (P < 0.05).

[0122] The results in Table 4 show that the embryo transfer conception rate of bovine blastocysts after cutting biopsy using the serum-free embryo cutting biopsy culture medium V of the present invention was 56.41%, while the conception rate of uncut embryos was 54.79%, which was significantly higher than the embryo transfer conception rate of the control group (commercial medium) (36.99%) (P<0.05).

[0123] In summary, using the serum-free embryo cutting biopsy culture medium V provided by this invention to cut bovine blastocysts yields significantly better results than commercial solutions, and the cut embryos still maintain a high level of viability and developmental capacity.

[0124] As can be seen from the above embodiments, the present invention provides a serum-free embryo cutting biopsy culture medium and its application. Placing early embryos in the serum-free embryo cutting biopsy culture medium of the present invention for cutting can improve the survival rate, re-cavity rate and embryo transfer conception rate after cutting.

[0125] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A serum-free embryo biopsy culture medium, comprising basal culture medium components, antioxidant components, protein growth factor components, small molecule inhibitor components, and molecular compound components, characterized in that, The basal culture medium components include Neurobasal and DMEM / F12, with the concentrations of Neurobasal and DMEM / F12 in the serum-free embryo dissection biopsy culture medium independently ranging from 45.00 to 50.00 v / v%. The antioxidant components include vitamin C, vitamin B6, vitamin B8, dimethyl sulfoxide, and N-Acetyl-L-cysteine; the concentrations of each component in serum-free embryo biopsy culture medium are 0.005~0.008 g / 100mL, 0.0846~0.0946 μg / 100mL, 0.2440~0.2540 μg / 100mL, 0.100~0.150 v / v%, and 0.0816~0.0916 g / 100mL, respectively. The protein growth factor components include RSPO1, WNT16, Relaxin, and Activin A; the concentrations of each component in serum-free embryo biopsy culture medium are 50.00~100.00 ng / mL, 50.00~100.00 ng / mL, 20.00~50.00 ng / mL, and 10.00~20.00 ng / mL, respectively. The small molecule inhibitor components include Chroman, RKI-1447, Emricasan, Q-VD-OPh, AT-7867, Ruxolitinib, and CCT-241533; the concentrations of each component in serum-free embryo biopsy culture medium are 0.00318~0.00418 μg / 100mL, 0.033843~0.043843 μg / 100mL, 0.0590~0.0690 μg / 100mL, 0.07289~0.08289 μg / 100mL, 0.03214~0.04214 μg / 100mL, 0.196735~0.206735 μg / 100mL, and 0.037242~0.047242 μg / 100mL, respectively. The molecular compound components include insulin, transferrin, sodium selenite, penicillin, and streptomycin; the concentrations of each component in serum-free embryo biopsy culture medium are 0.950~1.05 g / 100mL, 0.5270~0.5670 g / 100mL, 0.047~0.051 g / 100mL, 0.009~0.011 g / 100mL, and 0.011~0.013 g / 100mL, respectively.

2. A serum-free embryo biopsy culture medium, comprising basal culture medium components, antioxidant components, protein growth factor components, small molecule inhibitor components, and molecular compound components, characterized in that, The basal culture medium components include Neurobasal and DMEM / F12, with the concentrations of Neurobasal and DMEM / F12 in the serum-free embryo dissection biopsy culture medium being 48.00 v / v% and 48.00 v / v%, respectively. The antioxidant component comprises: 0.006 g / 100mL Vitamin C, 0.09 μg / 100mL Vitamin B6, 0.25 μg / 100mL Vitamin B8, 0.13 v / v% Dimethyl sulfoxide, and 0.0856 g / 100mL N-Acetyl-L-cysteine; The protein growth factor components include RSPO1, WNT16, Relaxin, and Activin A, with concentrations of 80.00 ng / mL, 80.00 ng / mL, 35.00 ng / mL, and 15.00 ng / mL, respectively, in serum-free embryo biopsy culture medium. The small molecule inhibitor components include Chroman, RKI-1447, Emricasan, Q-VD-OPh, AT-7867, Ruxolitinib, and CCT-241533; the concentrations of each component in serum-free embryo biopsy culture medium are 0.00358 μg / 100mL, 0.038843 μg / 100mL, 0.0650 μg / 100mL, 0.07789 μg / 100mL, 0.03714 μg / 100mL, 0.201735 μg / 100mL, and 0.042242 μg / 100mL, respectively. The molecular compound components include insulin, transferrin, sodium selenite, penicillin, and streptomycin; the concentrations of each component in serum-free embryo biopsy culture medium are 0.980 g / 100mL, 0.547 g / 100mL, 0.049 g / 100mL, 0.010 g / 100mL, and 0.012 g / 100mL, respectively.

3. The serum-free embryo dissection biopsy culture medium according to claim 1 or 2, characterized in that, The osmotic pressure of the serum-free embryo biopsy culture medium is 275~295 mOsm / L.

4. The serum-free embryo dissection biopsy culture medium according to claim 1 or 2, characterized in that, The pH value of the serum-free embryo dissection biopsy culture medium is 7.2~7.

4.

5. A method for embryo biopsy, characterized in that, The embryo biopsy method includes: placing a sample containing the embryo to be tested into the biopsy culture medium according to any one of claims 1 to 4, and performing testing or sampling on it; The embryo biopsy method described herein is for non-diagnostic and non-therapeutic purposes. The embryos to be tested are early embryos in the blastocyst stage of mammalian fertilized eggs.

6. The application of the biopsy culture medium according to any one of claims 1 to 4 and the embryo biopsy method according to claim 5 in embryonic genome selection, basic biology of embryonic development and medical research; in, The embryo biopsy is used for non-diagnostic and non-therapeutic purposes. The embryo is an early embryo in the blastocyst stage of a mammalian fertilized egg.

Images