Biopsy liquid and application thereof in pig blastocyst laser cutting

Abstract

The invention provides biopsy liquid and application thereof in pig blastocyst laser cutting, and belongs to the technical field of animal biology. The invention provides a biopsy solution and a preparation method thereof, the biopsy solution can be applied to pig blastocyst biopsy by laser cutting, the problem that at present, no adaptive biopsy solution exists for laser cutting of pig blastocyst is solved, and the invention further provides a method for laser cutting of pig blastocyst by using the biopsy solution. The problems of embryo cell injury, embryo development potential damage and the like possibly caused by laser cutting of the pig blastocyst in the prior art are solved. The biopsy liquid and the use method thereof can be efficiently and accurately applied to acquisition of pig blastocyst trophoblast cells, and are low in cost and good in repeatability.

Description

Technical Field

[0001] The invention belongs to the field of animal biotechnology, and in particular relates to a biopsy fluid used for laser cutting of pig blastocysts. Background Art

[0002] As a method for rapid and large-scale production of high-quality embryos in vitro, in vitro production (IVP) technology has a wide range of application values ​​in the protection and utilization of high-quality genetic resources and the acceleration of livestock breeding. Although livestock in vitro embryo production has made significant progress in recent decades, compared with in vivo embryos, pig in vitro embryo production faces the problems of polyspermy, early embryo development block and low-quality blastocysts. Therefore, efficient evaluation of the quality of IVP embryos has become a key indicator for judging whether embryo transfer is suitable. At present, livestock IVP embryo biopsy is the main means of quality evaluation and control before embryo transfer. On the one hand, IVP embryo biopsy can be used for sex identification. Sex identification in the early embryonic stage can screen embryos of specific sex, thereby improving the breeding efficiency of livestock embryos; on the other hand, it can also be used for single-cell omics sequencing, which can more accurately evaluate the developmental state of embryos by detecting the expression of development-related genes, and can also be used for embryo genome selection. Therefore, livestock IVP embryo biopsy technology is an important animal biotechnology, which has a wide range of application values ​​in livestock breeding and basic research.

[0003] IVP embryo biopsy should ensure that a sufficient number of cells for genetic testing are obtained, while minimizing the impact on embryo development and the cells obtained. Therefore, the development of efficient, stable, and less invasive micro-sample biopsy technology is crucial to evaluate the quality of IVP embryos. There are three widely used methods for preimplantation embryo biopsy, namely: removing one or two polar bodies (PB) from unfertilized oocytes or fertilized eggs, or removing 1-2 blastomeres from cleavage-stage embryos, or removing a small number of trophoblast cells from blastocyst-stage embryos. Since the genetic material of polar bodies is completely derived from oocytes, polar body biopsy can be used as an alternative to embryo biopsy, but compared with embryonic cells, polar bodies lack a large amount of cytoplasmic RNA and sperm genetic material, so they have certain limitations. Although the number of cells obtained in the early cleavage stage of embryonic development is small, embryos at this stage have a higher rate of chromosome mosaicism and aneuploidy. Moreover, embryos are more sensitive to manipulation in the early cleavage stage than in the later stages. Embryo biopsy in the cleavage stage has a significant effect on the in vitro blastocyst development rate and implantation rate. Blastocyst stage trophoblast cell biopsy is the most widely used embryo biopsy method today. On the one hand, the trophoblast cells of the blastocyst will develop into the placenta or fetal membranes in the future and will not participate in the formation of the fetus, thus avoiding damage to the fetus. On the other hand, obtaining trophoblast cells for biopsy at the blastocyst stage can obtain more cells, and the biopsy procedure is easier to perform than that of a single polar body and blastomere. Blastocyst stage biopsy has no effect on subsequent embryo implantation and subsequent full-term development rate.

[0004] There are several methods for blastocyst biopsy, including aspirating trophoblast cells after Piezo punching; or using Tyrode solution or pronase to remove the zona pellucida, and then directly tearing or aspirating trophoblast cells through a micromanipulator; or using a special blade to directly cut a part of the embryonic trophoblast for biopsy; or treating the blastocyst with enzymes to reduce the tight junctions between cells, making it easier to obtain embryonic trophoblast cells, but it has a certain impact on embryo survival. In addition to the above methods, laser cutting is another commonly used method. This method obtains cell samples through the precise heating and cutting function of the laser. This method can accurately and quickly cut the zona pellucida or trophoblast cells, and there is no chemical toxicity, which does not significantly affect the subsequent development of the embryo. However, the laser method also has the following problems: the cutting process will produce local high temperature, which may cause thermal damage to embryonic cells, especially in the case of improper operation or poor laser parameter settings, which may affect the developmental potential of the embryo; at the same time, the laser will produce light radiation, especially under long-term exposure, which may cause damage to embryonic cells. At present, embryo biopsy performed by laser cutting is performed in biopsy fluid. Different animals have different characteristics and are suitable for different biopsy fluids. In addition, whether the biopsy fluid is compatible with the embryo also seriously affects the damage caused by laser cutting to embryonic cells and the potential for embryonic development. However, there is no specially adapted biopsy fluid for the method of laser cutting for pig blastocyst biopsy. Therefore, in view of the problems existing in the use of laser cutting of pig blastocysts, it is very necessary to establish a biopsy fluid suitable for laser cutting of pig blastocysts. Summary of the invention

[0005] The present invention solves the problem that there is no suitable biopsy fluid for cutting pig blastocysts by laser at present, and reduces the problems of embryonic cell damage and damage to embryonic development potential that may be caused by cutting pig blastocysts by laser in the prior art.

[0006] A biopsy fluid comprises TCM199, NaHCO3, Hepes, NaCl, Penicillin G, Streptomycin, BSA, Cysteine ​​and betaine.

[0007] A method for preparing a biopsy fluid comprises dissolving TCM199 dry powder with ultrapure water, then sequentially adding the following reagents: NaHCO3, Hepes, NaCl, Penicillin G, Streptomycin, BSA, Cysteine ​​and betaine, adjusting the osmotic pressure and pH value after sufficient dissolution, and filtering with a 0.22 μm sterile filter.

[0008] The concentration of the TCM199 is 0.80-1.10 g / 100 mL.

[0009] The concentrations of the reagents are respectively NaHCO3 0.003-0.006 g / 100 mL, Hepes 0.065-0.075 g / 100 mL, NaCl 0.170-0.195 g / 100 mL, Penicillin G 0.005 g / 100 mL, Streptomycin 0.006 g / 100 mL, BSA 0.2-0.5 g / 100 mL, Cysteine ​​0.06-0.12 g / 100 mL and betaine 0.04 g-0.06 g / 100 mL.

[0010] The osmotic pressure is 275-290.

[0011] The pH value is 7.2-7.4.

[0012] The application of the above biopsy fluid in pig blastocyst biopsy using laser cutting.

[0013] A method for cutting pig blastocysts using laser, the method comprising: placing the pig blastocysts in the above-mentioned biopsy fluid, fixing the embryos on a fixing needle, planning the laser cutting route using a laser cutting system, and obtaining trophoblast cells through 2-3 times of laser cutting.

[0014] Beneficial Effects

[0015] The present invention provides a biopsy fluid suitable for pig blastocyst laser cutting, and successfully applies it to pig blastocyst biopsy. The biopsy fluid can protect the activity of embryos and cut cells, especially effectively prevent DNA damage and cell apoptosis, and can also protect pig blastocysts from affecting their developmental potential after laser cutting. The cells obtained after laser cutting can be used for sex identification and single-cell transcriptome sequencing. Therefore, the biopsy fluid and its use method can be efficiently and accurately applied to the acquisition of pig blastocyst trophoblast cells, and have low cost and good repeatability. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 The results of laser microdissection to efficiently obtain embryo biopsy cells, where A is a flow chart of the verification and application detection of laser cutting pig blastocyst biopsy technology; B is a schematic diagram of laser cutting to obtain a small amount of trophoblast cell samples; C is a picture of the blastocyst morphology before and after laser cutting of pig blastocysts under different magnifications; D is the number of cells in the laser-cut trophoblast cell sample detected by nuclear staining.

[0017] Figure 2These are the results of in vitro culture of cut trophoblast blastocysts and IVF blastocysts, wherein A is the morphological diagram of pig blastocysts at different time points during the period from 0 to 12 hours after cutting the trophoblast cells; B is a statistical diagram of the ratio of pig blastocysts that re-expanded at different time points during the period from 0 to 12 hours after cutting the trophoblast cells; C is the morphological diagram of pig blastocysts in the experimental group and the control group at different time points during the period from 0 to 48 hours; D is a statistical diagram of the diameter of pig blastocysts in the experimental group and the control group at different time points during the period from 0 to 48 hours; E is a statistical diagram of the development of pig blastocysts in the experimental group and the control group at 24 hours and 48 hours.

[0018] Figure 3 The results show that laser cutting of trophoblast cells does not affect the quality of blastocysts in vitro, where A is the E-cadherin expression graph of pig blastocysts in the experimental group and the control group detected by IF; B is the F-actin expression graph of pig blastocysts in the experimental group and the control group detected by IF; C is the CDX2 expression graph of pig blastocysts in the experimental group and the control group detected by IF; D is the SOX2 expression graph of pig blastocysts in the experimental group and the control group detected by IF; E is the apoptotic cell graph of pig blastocysts in the experimental group and the control group detected by TUNEL; F is the statistical graph of the proportion of pig blastocysts expressing E-cadherin in the experimental group and the control group; G is the statistical graph of the proportion of pig blastocysts expressing F-actin in the experimental group and the control group; H is the statistical graph of the total number of cells in the pig blastocysts in the experimental group and the control group; I is the statistical graph of the number of trophoblast cells expressing CDX2 in the pig blastocysts in the experimental group and the control group; J is the statistical graph of the number of ICM cells expressing SOX2 in the pig blastocysts in the experimental group and the control group; K is the statistical graph of the number of apoptotic cells in the pig blastocysts in the experimental group and the control group.

[0019] Figure 4 This is the result of identifying the sex of IVF embryos by nested PCR using laser-dissected trophoblast cells.

[0020] Figure 5 The results of single-cell transcriptome sequencing analysis of the quality of laser-cut trophoblast cells, where A is the result of PCA analysis of D7.5 TE, E5.5 TE and ICM; B is the result of unsupervised clustering analysis of D7.5 TE, E08 TE and E5.5 TE; C is the statistical graph of the expression levels of marker genes in trophoblast cells and inner cell mass cells of D7.5 TE and E5.5 TE. DETAILED DESCRIPTION

[0021] Example 1. Biopsy was performed using the biopsy fluid and sex identification was successfully performed.

[0022] 1. In vitro maturation of porcine oocytes (IVM).

[0023] After fresh pig ovaries were collected from the slaughterhouse, they were placed in 37°C physiological saline containing antibiotics and transported to the laboratory within 4 hours. The follicles were aspirated using a syringe with an inner diameter of 1.2 mm, and impurities were removed after sedimentation and resuspending three times using Hepes buffer. Then, a glass needle with an inner diameter of 200 um was used to select oocyte complexes (COCs) that were wrapped with more than three layers of cumulus cells and were evenly wrapped. They were cultured in maturation medium (Maturation medium, MAT) for 38-42 hours, and then 0.5% hyaluronidase was vortexed for 4 minutes to remove the cumulus cells, and then mature oocytes containing the first polar body, regular shape and uniform cytoplasm were selected for subsequent operations.

[0024] 2. In vitro fertilization (IVF) embryo acquisition.

[0025] In vitro fertilization of mature porcine oocytes. After washing the mature oocytes 4 times in mTBM liquid at 39°C, every 30 oocytes were placed in 50 μL mTBM liquid for later use. Fresh semen was preheated in a 39°C incubator for 10 minutes, and then washed twice in DPBS containing 0.1% BSA at 39°C at a ratio of 1:99. Each wash was centrifuged at 2000 rpm for 4 minutes, and then the sperm was resuspended in the diluent mTBM to a final sperm concentration of 3×10 5 / mL to obtain sperm diluent. Then mix the sperm diluent with the oocyte to achieve the optimal fertilization density of 500:1 for the optimal sperm-egg ratio to obtain fertilized drops. Take out the fertilized drops, take 50μL of diluted semen and add it to 50μL of mTBM, and culture it in a 39℃ incubator containing 5% CO2 and saturated humidity for 4-6 hours for in vitro fertilization to obtain fertilized embryos. Then thoroughly wash the fertilized embryos with embryo culture medium PZM3, and then add the washed embryos to a four-well plate containing PZM3 for culture.

[0026] 3. Preparation of pig blastocyst biopsy fluid

[0027] Weigh 0.80-1.10 g of TCM199 dry powder, dissolve it in 90-95 mL of ultrapure water, and then add the following concentrations: NaHCO3 0.003-0.006 g / 100 mL, Hepes 0.065-0.075 g / 100 mL, NaCl 0.170-0.195 g / 100 mL, Penicillin G 0.005 g / 100 mL, Streptomycin 0.006g / 100mL, BSA0.2-0.5g / 100mL, Cysteine0.06-0.12g / 100mL, betaine 0.04g-0.06 / 100mL are added in sequence, and the volume is adjusted to 100mL after being fully dissolved and mixed, and the osmotic pressure is adjusted to 275-290, and the pH value is 7.2-7.4. The mixed solution is filtered with a 0.22μm sterile filter, packaged, labeled, and stored in a refrigerator at 4℃ to obtain porcine blastocyst biopsy fluid. The prepared porcine blastocyst biopsy fluid should not be used for more than two weeks. When using the porcine blastocyst biopsy fluid, the biopsy fluid must be heated at 37℃ 1 hour in advance, and then made into an operating drop, and covered with paraffin oil on the operating drop.

[0028] 4. Use pig blastocyst biopsy fluid to perform laser cutting of pig blastocyst.

[0029] Flow chart of the in vitro biopsy technique for laser cutting of porcine blastocysts using porcine blastocyst biopsy fluid ( Figure 1 A). After culturing the fertilized embryo in vitro for about 7 days, the porcine blastocyst is obtained, and the porcine blastocyst is placed in a porcine blastocyst biopsy fluid operation drop. The porcine blastocyst is fixed on the fixing needle with the help of a micromanipulator, and a small piece of trophoblast cells is sucked with an injection needle. The laser cutting system is used to plan the laser cutting route. When laser cutting is performed, the porcine blastocyst will shrink. At this time, the trophoblast cells must be sucked slowly to prevent them from escaping, and the trophoblast cells must be completely cut through 2-3 laser cuttings. The TE cells obtained by cutting are thoroughly washed with DPBS for subsequent operations. TE cells of each embryo are obtained through the micromanipulation system. The results showed that after sucking the trophoblast cells away from the ICM with a micromanipulator glass pipette, laser cutting can successfully cut a small amount of trophoblast cells ( Figure 1 B). At the same time, due to the damage of trophoblast cells caused by laser cutting, the blastocyst cavity shrinks rapidly within 3-5 seconds, forming a shrinkage and dense cell mass ( Figure 1 C). To further confirm the amount of trophoblast cells obtained, Hoechse was used to stain the trophoblast cell samples. The results showed that among the 28 trophoblast microsamples obtained, 5 trophoblast cell samples did not contain cell nuclei, accounting for 17.86% of the total sample volume; 3 trophoblast cell samples contained more than 2 cell nuclei, accounting for 10.72 of the sample volume; and 20 trophoblast cell samples contained only 1 cell nucleus, accounting for 71.43% of the total sample volume ( Figure 1 D) Therefore, embryo biopsy using micro-laser dissection can efficiently obtain trophoblast cells from porcine blastocysts.

[0030] In order to morphologically analyze the effect of laser cutting of trophoblast cells on embryonic development, the blastocysts with cut trophoblast cells were continuously photographed within 12 hours of in vitro culture. The analysis of blastocyst re-expansion at different time points showed that after trophoblast cell cutting, the shrunken blastocysts could quickly re-expand. At 1 hour after cutting, 42%-71% of the blastocysts had re-expanded to form the blastocyst cavity. At 5 hours after cutting the trophoblast cells, all the shrunken blastocysts had re-expanded to form the blastocyst cavity ( Figure 2 AB). Statistics of blastocyst diameters at each time point showed that after 24 hours of culture, the diameter of the re-expanded blastocysts was not significantly different from that of the IVF blastocysts cultured in vitro (control group) ( Figure 2 CD). Subsequently, analysis of the blastocyst development rate at 24h and 48h of in vitro culture showed that there was no significant difference between the blastocyst development rate of the trophoblast cells and the IVF blastocyst cultured in vitro (control group) ( Figure 2 E). Therefore, there is no significant difference in blastocyst morphology and blastocyst development rate between laser-cut trophoblast blastocysts and IVF blastocysts during in vitro culture, that is, the development rate of pig blastocysts after laser cutting using the above biopsy fluid is not affected.

[0031] Further evaluation was conducted to determine whether laser cutting of the trophoblast cells of porcine blastocysts in the above-mentioned biopsy fluid had a significant effect on the in vitro development quality of the blastocysts. The blastocysts that underwent laser cutting in the biopsy fluid were cultured in vitro for 48 hours and then subjected to immunofluorescence detection of E-cadherin and F-actin. The results showed that after 48 hours of culture, the blastocysts that had cut trophoblast cells expressed the tight junction protein E-cadherin and the cytoskeletal protein F-actin normally. Compared with the control group, there was no significant difference in the number of blastocysts expressing E-cadherin and F-actin ( Figure 3AB, 3F-G). Subsequently, in order to detect the effect of laser cutting on the quality of blastocysts under this system, the number of blastocyst cells, the number of cells in the inner cell mass of the blastocyst, and the number of trophoblast cells were detected by immunofluorescence. The results showed that there was no significant difference in the number of blastocyst cells, the number of cells in the blastocyst cell mass, and the number of trophoblast cells between the blastocysts with trophoblast cells cut and the blastocysts in the control group (3C-D, 3H-J). At the same time, since laser cutting can generate heat and light damage, it will cause genomic damage and apoptosis of embryonic cells. The apoptosis of blastocyst cells was detected by TUNEL, and the results showed that there was no significant difference in the number of apoptotic cells in the blastocysts between the experimental group and the control group (3E, 3K). Therefore, laser cutting of blastocyst trophoblast cell biopsy will not affect the quality of blastocyst development in vitro, which further proves that laser cutting of the trophoblast cells of pig blastocysts in the above-mentioned biopsy fluid has no significant effect on the quality of blastocyst development in vitro.

[0032] 5. Sex determination of blastocyst biopsy cells

[0033] In order to verify that laser-cut microtrophoblast cells can be used for IVP embryo sex identification, 12 microtrophoblast cells obtained from IVP embryos were subjected to nested PCR. The results of agarose gel electrophoresis showed that among the 12 trophoblast cell samples obtained, samples 3 and 8 had no bands, indicating that the sex of the IVP embryos was not successfully identified. The remaining 10 samples all had obvious bands, among which samples 1, 2, 4, 7, and 12 were female, and samples 5, 6, 9, 10, and 11 were male ( Figure 4 ). Therefore, the micro-trophoblast cells obtained by laser cutting using pig blastocyst biopsy fluid can be used for sex identification of IVP embryos.

[0034] The specific sex identification process is as follows: the cut trophoblast cells are rinsed three times in RNase-free dPBS with a glass pipette and then the lysis buffer is added. Note that the volume of dPBS containing trophoblast cells must be less than 0.5 μl. The lysis buffer contains 10 μl RIPA tissue lysis buffer (Beyotime), 1 μl proteinase K (TaKaRa), and 0.5 μl RNaseA (RNaseA, TaKaRa). After adding the trophoblast cells, mix thoroughly, then incubate at 58°C for 60 minutes, incubate at 95°C for 15 minutes, and finally store at 4°C. Then nested PCR is used for amplification, and the nested PCR amplification uses a high-efficiency and high-fidelity PCR enzyme (TOYOBO, KMM-201). The nested PCR reaction system is 20 μl, including 10 μl PCR amplification enzyme, 1 μl forward primer, 1 μl reverse primer and 1 μl purified DNA or 5 μl single cell lysis buffer, supplemented with distilled water to 20 μl. The PCR reaction parameters were set as follows: 98°C pre-denaturation for 3 min, 98°C denaturation for 10 s, 58°C annealing for 5 s, 68°C extension for 10 s, 68°C final extension for 7 min, and the number of cycles was set to 35 times. The annealing temperature was determined by the primers, which were synthesized by Jilin Kemei Biological Company. After 3 rounds of nested PCR, trophoblast cell sample DNA was obtained. The positive control female and male DNA were extracted from pig ear fibroblasts using a commercial DNA extraction kit (TaKaRa, 9765). The sample DNA obtained by nested PCR was added to a pre-prepared 1% agarose gel, electrophoresed at 130 V, and observed with a gel imager after 30 min.

[0035] Example 2: Transcriptome sequencing of blastocyst biopsy cells.

[0036] First, a pig blastocyst of about 7 days of IVF was obtained, and then laser cutting was performed in the biopsy fluid. The specific steps were the same as in Example 1. In this case, single-cell transcriptome sequencing was performed on the 4 trophoblast cells after cutting, and 3 high-quality sequencing data were obtained after quality control. First, the transcriptome data of the obtained E5.5 trophoblast cells and the trophoblast cells and ICM cells of the in vivo embryo were analyzed. The results showed that the trophoblast cells of the blastocyst cultured in vitro to D7.5 were similar to the trophoblast cells of the blastocysts of 5-6 days in vivo ( Figure 5 AB). Subsequently, the expression levels of marker genes in trophoblast cells were compared and analyzed. The results showed that the marker gene expression levels of 7-8d trophoblast cells obtained by laser cutting in vitro were higher than those of ICM cells, while the marker gene expression levels of ICM cells were lower than those of E5.5 ICM cells ( Figure 5C). Therefore, the transcriptome data quality of the trophoblast cells obtained by laser cutting can be used for subsequent analysis, and the D7.5 trophoblast cells obtained by laser cutting highly express trophoblast cell marker genes, similar to the E5.5 blastocyst trophoblast cells in vivo.

[0037] The specific steps of transcriptome sequencing and analysis are as follows: First, amplify using the Single Cell Full Length mRNA-Amplification Kit (N712), transfer the isolated single trophoblast cells into the Sample Buffer for lysis, and the reaction system is 3.5μl. Prepare the annealing reaction system, the lysis reaction system is 3.5ul, Oligo (dT) VN Primer 1ul, dNTP Mix 1μl. The annealing conditions are 72℃ 3min, and after annealing, stand on ice for 2min to complete the first chain synthesis and obtain the first chain annealing product. Then immediately perform the reverse transcription reaction, the reaction system: the first chain annealing product 5.5μl, 1st Strand Buffer 2μl, DTT 0.5μl, RNase Inhibitor 0.5μl, 5'TS Oligo Primer 0.5μl and Sc Reverse Transcriptase 1μl. Reaction conditions: 42℃90min; 70℃15min, to obtain the first-chain cDNA synthesis product. Then, full-length cDNA amplification was performed, and the reaction system was 10μl of the first-chain cDNA synthesis product, 2μl of Nuclease-free H2O, 0.5μl of PCR Primer and 12.5μl of 2×Amplification Mix. The reaction conditions were 98℃1min; 98℃10s, 65℃15s, 72℃6min, 15 cycles, and 72℃5min to obtain the cDNA product. After the reaction, the cDNA product was purified to obtain a 15μl solution. Then, the TruePrepDNALibrary Prep KitV2 forIllumina(TD503) and TruePrep Index Kit V2 for Illumina(TD202) were used to build the library. Then, the DNA was fragmented to obtain the product. Subsequently, a PCR enrichment reaction was performed, and the reaction system was ddH2O 4μl, product 25μl, 5×TAB 10μl, N5XX 5μl, N7XX 5μl and TAE 1μl. The reaction conditions were: 72℃3min; 98℃30s; 98℃15s, 60℃30s, 72℃3min; 72℃5min; 4℃ to obtain the enriched product. After the reaction, the enriched product was length-sorted using VAHTSDNA CleanBeads to obtain 20μl of reaction product. Finally, the fragment length was verified using an Agilent2100 bioanalyzer, and the library concentration was measured using an Invitrogen Qubit 4 fluorometer. After the test was qualified, the sample was sent for Illumina sequencing.Data analysis is based on Cleandata (Fastq files). After obtaining the raw data, the sequencing data is quality controlled using FsatQC software. After the data passes the quality control, the data is aligned using Hisat2 or Bowtie2 software, followed by data conversion using samtools. Finally, the gene expression level is quantitatively processed using Stringtie software to obtain the TPM value, or the number of gene expression RNAs is counted using Subread software to obtain the ReadsCount value. In the entire analysis process, the reference genome used Ensembl Sus scrofa 11.1 version 105, and the annotation file used a custom annotation file based on Ensembl Sus scrofa version 11.1.

Claims

1. A biopsy fluid, characterized in that: The biopsy fluid includes TCM199, NaHCO3, Hepes, NaCl, Penicillin G, Streptomycin, BSA, Cysteine ​​and betaine.

2. A method for preparing a biopsy fluid, characterized in that: The method comprises dissolving TCM199 dry powder with ultrapure water, and then sequentially adding the following reagents: NaHCO3, Hepes, NaCl, Penicillin G, Streptomycin, BSA, Cysteine ​​and betaine, adjusting the osmotic pressure and pH value after being fully dissolved, and filtering with a 0.22 μm sterile filter.

3. The method according to claim 2, characterized in that: The concentration of the TCM199 is 0.80-1.10 g / 100 mL.

4. The method according to claim 2, characterized in that: The concentrations of the reagents are respectively NaHCO3 0.003-0.006 g / 100 mL, Hepes 0.065-0.075 g / 100 mL, NaCl 0.170-0.195 g / 100 mL, Penicillin G 0.005 g / 100 mL, Streptomycin 0.006 g / 100 mL, BSA 0.2-0.5 g / 100 mL, Cysteine ​​0.06-0.12 g / 100 mL and betaine 0.04 g-0.06 g / 100 mL.

5. The method according to claim 2, characterized in that: The osmotic pressure is 275-290.

6. The method according to claim 2, characterized in that: The pH value is 7.2-7.

4.

7. Use of the biopsy fluid according to claim 1 in pig blastocyst biopsy using laser cutting.

8. A method for cutting pig blastocysts using laser, characterized in that: The method comprises: placing the pig blastocyst in the biopsy fluid of claim 1, fixing the pig blastocyst on a fixing needle, planning a laser cutting route using a laser cutting system, and obtaining trophoblast cells through 2-3 times of laser cutting.

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