Biopsy fluid and its use in laser cutting of porcine blastocysts

Abstract

The application provides a biopsy liquid and application thereof in laser cutting of pig blastocysts, and belongs to the field of animal biotechnology. The application provides a biopsy liquid and a preparation method thereof, and the biopsy liquid can be applied to biopsy of pig blastocysts by laser cutting, solves the problem that there is no suitable biopsy liquid for laser cutting of pig blastocysts at present, and further provides a method for laser cutting of pig blastocysts by using the biopsy liquid, and solves the problems of embryo cell damage and damage to embryo development potential caused by laser cutting of pig blastocysts in the prior art. The biopsy liquid and the using 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] This invention belongs to the field of animal biotechnology, specifically relating to a biopsy solution for laser cutting of pig blastocysts. Background Technology

[0002] In vitro production (IVP) technology, as a method for rapidly and massively producing high-quality embryos outside the body, has broad application value in the protection and utilization of high-quality genetic resources and in accelerating livestock breeding. Although significant progress has been made in livestock IVP production in recent decades, compared with in vivo embryos, pig IVP production faces problems such as polyspermia, early embryonic developmental arrest, and low-quality blastocysts. Therefore, efficiently assessing the quality of IVP embryos has become a key indicator for determining whether they are suitable for embryo transfer. Currently, livestock IVP embryo biopsy is the main means of pre-implantation quality assessment and control. IVP embryo biopsy can be used for sex determination in the early stages of embryonic development, allowing for the selection of embryos of a specific sex, thereby improving the breeding efficiency of livestock embryos. It can also be used for single-cell omics sequencing, which can more accurately evaluate the developmental status of embryos by detecting the expression of developmental genes, and can also be used for embryo genome selection. Therefore, livestock IVP embryo biopsy technology is an important animal biotechnology with broad application value in livestock breeding and basic research.

[0003] IVP embryo biopsy must ensure a sufficient number of cells for genetic testing while minimizing the impact on embryonic development and the retrieved cells. Therefore, developing efficient, stable, and minimally invasive micro-sample biopsy techniques is crucial for evaluating the quality of IVP embryos. Currently, there are three widely used methods for preimplantation genetic testing: retrieving one or two polar bodies (PB) from unfertilized oocytes or fertilized eggs; retrieving one or two blastomeres from cleavage-stage embryos; or retrieving a small number of trophoblast cells from blastocyst-stage embryos. Since the genetic material of polar bodies is entirely derived from oocytes, polar body biopsy can serve as an alternative to embryo biopsy. However, compared to embryonic cells, polar bodies lack a large amount of cytoplasmic RNA and sperm genetic material, thus having certain limitations. While the number of cells retrieved during the early cleavage stage of embryonic development is relatively small, embryos at this stage exhibit a high rate of chromosomal mosaicism and aneuploidy. Furthermore, embryos are more sensitive to manipulation during the early cleavage stage than later stages, and cleavage-stage embryo biopsy has a significant impact on in vitro blastocyst development and implantation rates. Trophoblastic cell biopsy is the most widely used embryo biopsy method today. On one hand, the trophoblastic cells of the blastocyst will eventually develop into the placenta or fetal membranes and do not participate in the formation of the fetus, thus avoiding damage to the fetus. On the other hand, obtaining trophoblastic cells for biopsy at the blastocyst stage allows for the collection of more cells, making the biopsy procedure easier compared to individual polar bodies and blastomeres. Furthermore, blastocyst biopsy has no impact on subsequent embryo implantation or full-term development rates.

[0004] There are several methods for blastocyst biopsy, including aspirating trophoblast cells after Piezopuncture; removing the zona pellucida using Tyrode solution or streptolytic enzyme, then directly tearing or aspirating trophoblast cells using a micromanipulation needle; directly cutting a portion of the trophoblast for biopsy using a special blade; or treating the blastocyst with enzymes to reduce the tight junctions between cells, making it easier to obtain trophoblast cells, but this can affect embryo viability. Besides these methods, laser ablation is another commonly used method. This method obtains cell samples through the precise heating and cutting capabilities of a laser. It can accurately and quickly cut the zona pellucida or trophoblast cells without chemical toxicity, and does not significantly affect subsequent embryonic development. However, the laser method also has the following problems: the cutting process generates localized high temperatures, which may cause thermal damage to embryonic cells, especially under improper operation or incorrect laser parameter settings, potentially affecting the embryo's developmental potential; simultaneously, the laser generates light radiation, which, especially with prolonged exposure, may damage embryonic cells. Currently, embryo biopsies performed using laser ablation are all conducted in biopsy solutions, and different animal embryos require different biopsy solutions due to their varying characteristics. Furthermore, the compatibility between the biopsy solution and the embryo significantly impacts the damage to embryonic cells caused by laser ablation and the embryo's developmental potential. However, there is currently no specifically formulated biopsy solution for laser ablation of porcine blastocysts. Therefore, it is essential to develop a biopsy solution suitable for laser ablation of porcine blastocysts, addressing the existing problems associated with this method. Summary of the Invention

[0005] This invention solves the problem of the lack of a suitable biopsy solution for laser cutting of porcine blastocysts, and reduces the problems of embryonic cell damage and impaired embryonic development potential that may be caused by laser cutting of porcine blastocysts in the prior art.

[0006] A biopsy solution comprising TCM199, NaHCO3, Hepes, NaCl, Penicillin G, Streptomycin, BSA, Cysteine, and betaine.

[0007] A method for preparing a biopsy solution, wherein TCM199 dry powder is dissolved in ultrapure water, and then the following reagents are added sequentially: NaHCO3, Hepes, NaCl, Penicillin G, Streptomycin, BSA, Cysteine ​​and betaine. After complete dissolution, the osmotic pressure and pH value are adjusted, and the solution is filtered using a 0.22 μm sterile filter.

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

[0009] The concentrations of the reagents are as follows: 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 above-mentioned biopsy solution is used in the biopsy of porcine blastocysts using laser cutting.

[0013] A method for cutting porcine blastocysts using a laser, the method comprising: placing the porcine blastocyst in the aforementioned biopsy solution, fixing the embryo on a fixation needle, planning the laser cutting path using a laser cutting system, and obtaining trophoblast cells through 2-3 laser cuttings.

[0014] Beneficial effects

[0015] This invention provides a biopsy solution suitable for laser cutting of porcine blastocysts and has been successfully applied to porcine blastocyst biopsy. This biopsy solution can protect the viability of the embryo and the cut cells, especially effectively preventing DNA damage and apoptosis. It also protects the porcine blastocyst from affecting its developmental potential after laser cutting. Furthermore, the cells obtained after laser cutting can be used for sex determination and single-cell transcriptome sequencing. Therefore, this biopsy solution and its application method can be used efficiently and accurately for obtaining trophoblast cells from porcine blastocysts, and it is low-cost and has good reproducibility. Attached Figure Description

[0016] Figure 1 The diagram shows the results of obtaining embryonic biopsy cells efficiently through laser microdissection. A is a flowchart of the verification and application of laser-dissected porcine blastocyst biopsy technology; B is a schematic diagram of obtaining a small amount of trophoblast cell sample through laser dissection; C is a blastocyst morphology diagram of porcine blastocysts before and after laser dissection under different magnifications; D is a cell count of the laser-dissected trophoblast cell sample detected by cell nuclear staining.

[0017] Figure 2The images show the results of in vitro cultured trophoblastocysts and IVF blastocysts. A shows the morphology of porcine blastocysts at different time points between 0 and 12 hours after trophoblastocyst cutting; B shows the percentage of porcine blastocysts that re-expanded at different time points between 0 and 12 hours after trophoblastocyst cutting; C shows the morphology of porcine blastocysts in the experimental and control groups at different time points between 0 and 48 hours; D shows the diameter of porcine blastocysts in the experimental and control groups at different time points between 0 and 48 hours; and E shows the developmental statistics of porcine blastocysts in the experimental and control groups at 24 and 48 hours.

[0018] Figure 3 To ensure that laser cutting of trophoblast cells does not affect the quality of in vitro blastocysts, the following graphs are presented: A) E-cadherin expression in porcine blastocysts of the experimental and control groups as detected by IF; B) F-actin expression in porcine blastocysts of the experimental and control groups as detected by IF; C) CDX2 expression in porcine blastocysts of the experimental and control groups as detected by IF; D) SOX2 expression in porcine blastocysts of the experimental and control groups as detected by IF; E) apoptotic cells in porcine blastocysts of the experimental and control groups as detected by TUNEL assay; F) the percentage of E-cadherin expression in porcine blastocysts of the experimental and control groups; G) the percentage of F-actin expression in porcine blastocysts of the experimental and control groups; H) the total number of cells in porcine blastocysts of the experimental and control groups; I) the number of trophoblast cells expressing CDX2 in porcine blastocysts of the experimental and control groups; J) the number of ICM cells expressing SOX2 in porcine blastocysts of the experimental and control groups; and K) the number of apoptotic cells in porcine blastocysts of the experimental and control groups.

[0019] Figure 4 This image shows the results of using nested PCR to identify the sex of IVF embryos by laser-cutting trophoblast cells.

[0020] Figure 5 The figures show the results of single-cell transcriptome sequencing analysis of laser-cut trophoblast cell quality, where A is the result of PCA analysis of D7.5 TE, E5.5 TE, and ICM; B is the result of unsupervised cluster analysis of D7.5 TE, E08 TE, and E5.5 TE; and C is a 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 Implementation

[0021] Example 1. The biopsy solution was used to perform a biopsy and successfully determine the sex of the animal.

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

[0023] After collecting fresh pig ovaries from the slaughterhouse, the ovaries were placed in physiological saline containing antibiotics at 37°C and transported to the laboratory within 4 hours. Follicles were aspirated using a syringe with an inner diameter of 1.2 mm. After sedimentation and resuspension three times with Hepes buffer, impurities were removed. Oocyte complexes (COCs) with more than three layers of uniformly encapsulated cumulus cells were selected using a glass needle with an inner diameter of 200 μm and cultured in maturation medium (MAT) for 38-42 h. Then, cumulus cells were removed by vortexing with 0.5% hyaluronidase for 4 min. Mature oocytes containing the first polar body, with regular morphology and uniform cytoplasm were selected for subsequent operations.

[0024] II. Embryo Acquisition for In Vitro Fertilization (IVF)

[0025] In vitro fertilization of mature porcine oocytes was performed. Mature oocytes were washed four times in mTBM solution at 39°C, and then 30 oocytes were placed in 50 μL of mTBM solution for later use. Fresh semen was preheated at 39°C for 10 minutes, then washed twice in DPBS containing 0.1% BSA at a ratio of 1:99 at 39°C. Each wash was performed by centrifugation at 2000 rpm for 4 minutes. The sperm were then resuspended in mTBM dilution buffer to a final sperm concentration of 3 × 10⁻⁶. 5 / mL, to obtain sperm diluent. Then, the sperm diluent is mixed with oocytes to achieve the optimal sperm-to-egg ratio of 500:1, obtaining fertilized droplets. The fertilized droplets are removed, and 50μL of the diluted sperm is added to 50μL of mTBM. The mixture is then incubated at 39℃ with 5% CO2 and saturated humidity for 4-6 hours for in vitro fertilization to obtain fertilized embryos. The fertilized embryos are then thoroughly washed with embryo culture medium PZM3, and then transferred to four-well plates containing PZM3 for further culture.

[0026] III. Preparation of porcine blastocyst biopsy solution

[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 Add 0.006 g / 100 mL of BSA, 0.2-0.5 g / 100 mL of Cysteine, and 0.04 g-0.06 g / 100 mL of Betaine sequentially. After thorough dissolution and mixing, bring the volume to 100 mL. Adjust the osmotic pressure to 275-290 and the pH to 7.2-7.4. Filter the mixture through a 0.22 μm sterile filter, dispense, label, and store at 4°C to obtain porcine blastocyst biopsy solution. The prepared porcine blastocyst biopsy solution should not be used for more than two weeks. Before use, heat the biopsy solution at 37°C for one hour, then prepare operating drops, covering the drops with paraffin oil.

[0028] IV. Laser cutting of porcine blastocysts using porcine blastocyst biopsy fluid.

[0029] Flowchart of in vitro biopsy technique using porcine blastocyst biopsy fluid for laser cutting of porcine blastocysts ( Figure 1 A). After culturing fertilized embryos in vitro for approximately 7 days, porcine blastocysts were obtained. These blastocysts were placed in a porcine blastocyst biopsy solution droplet and fixed to a pin using a micromanipulator. A small piece of trophoblast cells was aspirated using an injection needle. A laser cutting system was used to plan the laser cutting path. During laser cutting, the porcine blastocyst would shrink; at this point, the trophoblast cells needed to be slowly aspirated to prevent escape. The trophoblast cells were completely removed through 2-3 laser cuts. The obtained TE cells were thoroughly washed with DPBS and used for subsequent operations. TE cells were obtained from each embryo using a micromanipulator. Results showed that after aspirating trophoblast cells far from the ICM using a glass pipette under micromanipulation, laser cutting could successfully remove a small amount of trophoblast cells. Figure 1 B). Simultaneously, due to the damage to the trophoblast cells caused by laser cutting, the blastocoel rapidly shrinks within 3-5 seconds, forming a shrunken, dense cluster of cells. Figure 1 C). To further confirm the obtained trophoblast cell quantity, Hoechse staining was used to detect the trophoblast cell samples. The results showed that among the 28 trophoblast cell samples obtained, 5 trophoblast cell samples contained no cell nucleus, accounting for 17.86% of the total sample; 3 trophoblast cell samples contained more than 2 cell nuclei, accounting for 10.72% of the sample; and 20 trophoblast cell samples contained only 1 cell nucleus, accounting for 71.43% of the total sample. Figure 1 D). Therefore, embryo biopsy using microlaser cutting can efficiently obtain trophoblast cells from porcine blastocysts.

[0030] To morphologically analyze the effects of laser-cut trophoblast cells on embryonic development, continuous imaging of trophoblastocysts cultured in vitro for 12 hours was performed. Analysis of blastocyst re-expansion at different time points showed that after trophoblast cell cutting, the shrunken blastocysts rapidly re-expanded. One hour after cutting, 42%-71% of the blastocysts had re-expanded to form a blastocoel. By five hours after trophoblast cell cutting, all the shrunken blastocysts had re-expanded to form a blastocoel. Figure 2 AB). Statistical analysis of blastocyst diameters at various time points showed that, after 24 hours of culture, the diameter of the re-expanded blastocysts was not significantly different from that of the in vitro cultured IVF blastocysts (control group). Figure 2 CD). Subsequently, blastocyst development rate analysis at 24h and 48h in vitro culture showed no significant difference in the development rate of blastocysts with cut trophectoderm cells compared to the development rate of IVF blastocysts cultured in vitro (control group). Figure 2 E). Therefore, there was no significant difference in blastocyst morphology and development rate between laser-cut trophoblastocysts and IVF blastocysts during in vitro culture, meaning that the development rate of porcine blastocysts after laser cutting using the aforementioned biopsy solution was not affected.

[0031] Further evaluation was conducted to determine whether laser-dissecting the trophoblast cells of porcine blastocysts in the aforementioned biopsy solution significantly affected the in vitro developmental quality of the blastocysts. Blastocysts laser-dissected in the biopsy solution were cultured in vitro for 48 hours, followed by immunofluorescence detection of E-cadherin and F-actin. The results showed that after 48 hours of culture, blastocysts with dissected trophoblast cells normally expressed the intercellular tight junction protein E-cadherin and the cytoskeletal protein F-actin. Compared with the control group, there was no significant difference in the number of blastocysts expressing E-cadherin and F-actin. Figure 3(AB, 3F-G). Subsequently, to investigate the effect of laser cutting on blastocyst quality under this system, the number of blastocyst cells, the number of cells in the inner cell mass, and the number of trophoblast cells were detected by immunofluorescence. The results showed that there were no significant differences in the number of blastocyst cells, the number of cells in the inner cell mass, and the number of trophoblast cells between the experimental and control groups after trophoblast cell cutting (3C-D, 3H-J). Meanwhile, because laser cutting generates heat and photodamage, it can lead to genomic damage and apoptosis in embryonic cells. TUNEL assays of blastocyst cell apoptosis showed no significant difference in the number of apoptotic cells between the experimental and control groups (3E, 3K). Therefore, laser cutting of trophoblast cells does not affect the in vitro development quality of blastocysts, further demonstrating that laser cutting of trophoblast cells in porcine blastocysts in the above biopsy solution has no significant impact on the in vitro development quality of blastocysts.

[0032] V. Blastocyst biopsy cell sex determination

[0033] To verify that laser-cut microtrophoblast cells could be used for sex determination in IVP embryos, nested PCR was performed on 12 microtrophoblast cells obtained from IVP embryos. Agarose gel electrophoresis results showed that samples 3 and 8 showed no bands, indicating unsuccessful sex determination of the IVP embryos. The remaining 10 samples all showed clear bands, with samples 1, 2, 4, 7, and 12 being female and samples 5, 6, 9, 10, and 11 being male. Figure 4 Therefore, the trace amounts of trophoblast cells obtained by laser cutting using porcine blastocyst biopsy fluid can be used for sex determination of IVP embryos.

[0034] The specific sex determination procedure is as follows: After washing the cut feeder cells three times in RNase-free dPBS using a glass pipette, add lysis buffer. Note that the volume of dPBS containing feeder 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 RNase A (RNase A, TaKaRa). After adding the feeder cells, mix thoroughly, then incubate at 58°C for 60 min, then at 95°C for 15 min, and finally store at 4°C. Nested PCR is then performed for amplification using 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, and then add distilled water to a final volume of 20 μl. The PCR reaction parameters were set as follows: 98℃ pre-denaturation for 3 min, 98℃ denaturation for 10 s, 58℃ annealing for 5 s, 68℃ extension for 10 s, and 68℃ final extension for 7 min, with a cycle count of 35. The annealing temperature was determined by the primers, which were synthesized by Jilin Kemei Biotechnology Co., Ltd. DNA from trophoblast cell samples was obtained after three rounds of nested PCR. Positive control DNA from female and male cells was extracted from porcine ear-derived fibroblasts using a commercial DNA extraction kit (TaKaRa, 9765). The sample DNA obtained from nested PCR was added to a pre-prepared 1% agarose gel, electrophoresed at 130V for 30 min, and then observed using a gel imaging system.

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

[0036] First, porcine blastocysts at approximately 7 days of age were obtained for IVF. Then, laser cutting was performed in the biopsy fluid, following the same steps as in Example 1. In this case, single-cell transcriptome sequencing was performed on the four trophoblast cells after cutting, and three high-quality sequencing data were obtained after quality control. Transcriptome data analysis of the obtained E5.5 trophoblast cells, trophoblast cells from in vivo embryos, and ICM cells showed that the trophoblast cells cultured in vitro to D7.5 blastocysts are similar to the trophoblast cells of in vivo at 5-6 days of age. Figure 5 AB). Subsequently, a comparative analysis of marker gene expression levels in trophoblast cells was performed. The results showed that the marker gene expression level in trophoblast cells obtained by laser dissection after 7-8 days in vitro was higher than that in ICM cells, while the marker gene expression level in ICM cells was lower than that in E5.5 ICM cells (AB). Figure 5C). Therefore, the transcriptome data of 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 for transcriptome sequencing and analysis are as follows: First, amplification was performed using the Single Cell Full Length mRNA-Amplification Kit (N712). The isolated single feeder cells were transferred into Sample Buffer for lysis, with a reaction volume of 3.5 μl. An annealing reaction mixture was prepared, consisting of 3.5 μl of lysis reaction mixture, 1 μl of Oligo(dT)VN Primer, and 1 μl of dNTP Mix. Annealing was performed at 72℃ for 3 min, followed by incubation on ice for 2 min to complete the synthesis of the first strand, yielding the annealed first strand product. Immediately afterwards, reverse transcription was performed, with the following reaction mixture: 5.5 μl of the annealed first strand product, 2 μl of 1st Strand Buffer, 0.5 μl of DTT, 0.5 μl of RNase Inhibitor, 0.5 μl of 5'TS Oligo Primer, and 1 μl of Sc Reverse Transcriptase. Reaction conditions: 42℃ for 90 min; 70℃ for 15 min, to obtain the first-strand cDNA synthesis product. Subsequently, full-length cDNA amplification was performed. The reaction system consisted of 10 μl of the first-strand cDNA synthesis product, 2 μl of nuclease-free H2O, 0.5 μl of PCR Primer, and 12.5 μl of 2×Amplification Mix. The reaction conditions were: 98℃ for 1 min; 98℃ for 10 s, 65℃ for 15 s, 72℃ for 6 min, 15 cycles, 72℃ for 5 min, to obtain the cDNA product. After the reaction, the cDNA product was purified to obtain 15 μl of solution. Then, libraries were constructed using the TruePrep DNA Library Prep Kit V2 for Illumina (TD503) and TruePrep Index Kit V2 for Illumina (TD202). The DNA was then fragmented to obtain the product. PCR enrichment was then performed using a mixture of 4 μl ddH₂O, 25 μl product, 10 μl 5×TAB, 5 μl N₅XX, 5 μl N₇XX, and 1 μl TAE. The reaction conditions were: 72℃ for 3 min; 98℃ for 30 s; 98℃ for 15 s; 60℃ for 30 s; 72℃ for 3 min; 72℃ for 5 min; and held at 4℃ to obtain the enriched product. After the reaction, the enriched product was length-sorted using VAHTSDNA CleanBeads, yielding 20 μl of the reaction product. Finally, fragment length verification was performed using an Agilent 2100 bioanalyzer, and library concentration was measured using an Invitrogen Qubit 4 fluorescence analyzer. After passing the verification, the sample was sent for Illumina sequencing.Data analysis was based on Cleandata (Fastq files). After obtaining the raw data, FsatQC software was used for quality control of the sequencing data. After the data passed quality control, Hisat2 or Bowtie2 software was used for data alignment. Then, samtools was used for data transformation. Finally, Stringtie software was used to quantify gene expression levels to obtain TPM values, or Subread software was used to count the number of expressed RNAs to obtain ReadsCount values. Throughout the analysis workflow, Ensembl Sus scrofa version 11.1 (version 105) was used as the reference genome, and a custom annotation file based on Ensembl Sus scrofa version 11.1 was used.

Claims

1. A porcine blastocyst biopsy solution, characterized in that, The biopsy solution comprises the following components: TCM199, NaHCO3, Hepes, NaCl, Penicillin G, Streptomycin, BSA, Cysteine, and betaine; the concentration of TCM199 is 0.80-1.10 g / 100 mL, the concentration of NaHCO3 is 0.003-0.006 g / 100 mL, the concentration of Hepes is 0.065-0.075 g / 100 mL, the concentration of NaCl is 0.170-0.195 g / 100 mL, the concentration of Penicillin G is 0.005 g / 100 mL, the concentration of Streptomycin is 0.006 g / 100 mL, and the concentration of BSA is 0.2-0.5 g / 100 mL. The concentrations of Cysteine ​​and betaine are 0.06-0.12 g / 100 mL, respectively.

2. A method for preparing the porcine blastocyst biopsy solution according to claim 1, characterized in that, The method involves dissolving TCM199 dry powder in ultrapure water, then sequentially adding the following reagents: NaHCO3, Hepes, NaCl, Penicillin G, Streptomycin, BSA, Cysteine, and betaine. After complete dissolution, the osmotic pressure and pH value are adjusted, and the mixture is filtered using a 0.22μm sterile filter.

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

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

4.

5. The use of the biopsy solution of claim 1 in the biopsy of porcine blastocysts using laser cutting for non-diagnostic and non-therapeutic purposes.

6. A method for laser-cutting porcine blastocysts for non-diagnostic and non-therapeutic purposes, characterized in that, The method is as follows: the porcine blastocyst is placed in the biopsy solution described in claim 1, the porcine blastocyst is fixed on a fixation needle, a laser cutting path is planned using a laser cutting system, and trophoblast cells are obtained through 2-3 laser cuttings.

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