A culture solution free DNA efficient preparation and SNP site detection method for non-invasive genetic detection of bovine embryos

Abstract

The present application relates to the technical field of bovine in vitro embryo production, and provides a culture solution free DNA efficient preparation and SNP site detection method for bovine embryo non-invasive genetic detection.The present application takes bovine in vitro embryo as a research object, explores the influence of single drop culture starting time and culture volume on embryo development efficiency, blastocyst cell number, apoptosis level and metabolic characteristics, and optimizes the whole genome amplification of embryo culture medium cf-DNA and SNP detection scheme.The results show that from the fifth day (D5) of embryo culture, the embryo blastocyst rate and total cell number of the embryo cultured in a 20 muL culture volume to the eighth day (D8) are significantly better than those of other single drop culture conditions.The D5 starting, 20 muL single drop culture to D8 can significantly improve the bovine embryo development quality, and improve the SNP detection efficiency of embryo culture solution cf-DNA, which is up to 71.62%, and the present application provides an experimental basis and technical support for the establishment of bovine non-invasive embryo genetic detection technology system.

Description

Technical Field

[0001] This invention relates to the field of bovine in vitro embryo production technology, and more specifically, to a method for efficient preparation of cell-free DNA from culture medium and detection of SNP sites in bovine embryos for non-invasive genetic testing. Background Technology

[0002] Preimplantation genetic testing (PGT) is not only an important direction for the innovative development of assisted reproductive technology (ART), but also one of the core technologies for in vitro embryo quality screening. Its core principle is to obtain materials that represent all the genetic information of an embryo without affecting its developmental potential. By testing, the genetic characteristics of the embryo are clarified, providing a basis for the transfer of high-quality embryos (Zhang et al., 2016; Ana del et al., 2024). PGT also has important applications in animal husbandry, where it can be used to introduce desirable traits, enhance the genetic diversity of livestock, and improve the overall quality of the herd. Especially for single-birth animals like cattle with long gestation periods, early screening for embryos with superior genetic performance can effectively shorten the generation interval, increase genetic gain, and improve reproductive efficiency (Janine et al., 2023). However, the commonly used PGT biopsy methods (polar body biopsy, blastomere biopsy, trophoblast cell biopsy) are all invasive procedures, which have problems such as mechanical damage affecting embryo development, high operational requirements, low detection efficiency, and easy interference of detection results by chimerism, which limit their widespread application in assisted reproduction and livestock embryo production (Aoyama et al., 2020; Palini et al., 2013).

[0003] In animal husbandry, ni-PGT can be used to screen embryos with superior genetic traits, shortening the breeding cycle and accelerating the breeding process (Alves et al., 2022). Non-invasive preimplantation genetic testing (ni-PGT) is an important development direction that avoids the defects of invasive PGT. Its core is to obtain the genetic information of the embryo by detecting cell-free DNA (cf-DNA) in the blastocyst fluid or embryo culture medium without damaging the embryo structure, and can effectively maintain embryo viability (Amin et al., 2022). cf-DNA is a short double-stranded DNA fragment released into the microenvironment by cells through biological processes such as necrosis and apoptosis. It can serve as a biomarker reflecting the characteristics of cellular nucleic acids and is also the core detection target of ni-PGT (Amin et al., 2022). Single-drop culture can meet the needs of individual embryo assessment (Yang et al., 2023) and can effectively ensure the clear source of subsequent test samples (cell-free DNA in embryo culture medium) and avoid cross-contamination, becoming a key culture mode adapted to non-invasive PGT (Viotti, 2022). However, single-drop culture also has significant drawbacks. It lacks the paracrine / autocrine interactions between embryos in population culture, i.e., the population effect, which can easily lead to a decline in embryo development efficiency (Travaglione et al., 2024). Optimizing single-drop culture conditions and improving embryo development quality have become the current research focus in this field (Layek et al., 2024).

[0004] Currently, various technologies are available for SNP detection, including single-strand conformation polymorphism (SSCP), restriction fragment length polymorphism, DNA sequencing, and gene chip methods (Kim et al., 2016; Mekonnen et al., 2024). These technologies, with their high resolution and comprehensive data output capabilities, play a crucial role in individual diagnosis and treatment, population genetics, and especially in animal husbandry for screening superior livestock breeds, providing technical support for breeding (Gkioka et al., 2023; Maceda et al., 2021). Whole-genome amplification (WGA) technology, as an important auxiliary method for SNP detection, can non-selectively and uniformly amplify the entire genome sequence of a sample, effectively improving the quantity and quality of genetic data required for subsequent detection. Commonly used WGA techniques include multiple substitution amplification (MDA) and multiple annealing loop-based amplification cycle (MALBAC) (Glotov et al., 2024; Huang et al., 2024).

[0005] In summary, optimizing single-drop culture conditions, improving cf-DNA detection efficiency, and perfecting the SNP detection technology system are key to promoting the application of ni-PGT in animal husbandry and achieving rapid propagation of superior breeds. Summary of the Invention

[0006] The purpose of this invention is to provide a method for efficient preparation of cell-free DNA from culture medium and detection of SNP sites in bovine embryos for non-invasive genetic testing.

[0007] To achieve the objective of this invention, in a first aspect, this invention provides a method for the efficient preparation of cell-free DNA from culture medium and the detection of SNP sites in bovine embryos for non-invasive genetic testing, comprising the following steps: (1) After obtaining bovine embryos through in vitro fertilization, on the 5th day after fertilization, a single embryo that has developed to the morula stage is transferred to a 20 μL volume of embryo culture medium for single-drop culture until the 8th day after fertilization; among them, from the 0th to the 4th day after fertilization, the embryos are cultured in a population, with about 20 embryos cultured in 100 μL of single-drop culture medium; the conditions for population culture are: constant temperature 38.0℃~38.5℃, 4.5%~5.0% CO2 (preferably constant temperature 38.5℃, 5% CO2); (2) Collect the culture medium containing a single embryo from step (1), wherein the culture medium contains cell-free DNA released by the embryo; (3) Perform whole-genome amplification on the free DNA collected in the culture medium in step (2); (4) Perform SNP site detection on the product amplified in step (3).

[0008] Further, in step (1), the mulberry embryo is cultured to the expanded blastocyst stage.

[0009] Further, in step (1), the culture conditions for the single drop culture are 38.0℃~38.5℃ and 4.5%~5.0% CO2 (preferably 38.5℃ and 5% CO2).

[0010] Furthermore, prior to step (2), i.e. before collecting the culture medium, there is also a step of assessing the quality of embryonic development; The evaluation indicators include blastocyst rate, total number of blastocyst cells, and apoptosis rate.

[0011] Furthermore, the whole genome amplification described in step (3) employs the Multiple Displacement Amplification (MDA) method.

[0012] Furthermore, amplification was performed using a whole genome amplification kit, specifically the Yikang ChromLong kit. TMSingle-cell whole genome amplification kit or Huazhi cell culture medium amplification kit; Huazhi cell culture medium amplification kit is preferred.

[0013] Furthermore, after whole-genome amplification in step (3), the step also includes quality testing of the amplification products; Preferably, the quality detection includes agarose gel electrophoresis detection; More preferably, the amplification products exhibit diffuse bands in the range of 100-1000 bp as detected by agarose gel electrophoresis.

[0014] Furthermore, the SNP site detection in step (4) is performed using SNP chip detection or sequencing methods.

[0015] Furthermore, the SNP site detection described in step (4) is used for screening for superior genetic traits or sex determination in bovine embryos.

[0016] Secondly, the present invention provides the application of the method in non-invasive genetic testing of bovine embryos; The application is for purposes other than disease diagnosis and treatment.

[0017] Furthermore, the non-invasive genetic testing is used to assess the developmental potential of bovine embryos, screen for desirable hereditary traits, or determine sex.

[0018] By employing the above technical solution, the present invention has at least the following advantages and beneficial effects: This invention uses bovine in vitro embryos as the research object to systematically investigate the effects of single-drop culture initiation time and culture volume on embryonic development efficiency, blastocyst cell number, apoptosis level, and metabolic characteristics, and optimizes the whole-genome amplification and SNP detection protocol of cf-DNA in embryonic culture medium. The results showed that the blastocyst rate in the single-drop culture group was significantly lower than that in the population culture group at the same time. However, starting from day 5 (D5) of embryonic culture, using a 20 μL culture volume to day 8 (D8), the blastocyst rate (40.06 ± 6.82) and total cell count (119.20 ± 21.86) were significantly better than other single-drop culture conditions. In summary, starting at day 5 and using a 20 μL single-drop culture to day 8 can significantly improve the quality of bovine embryonic development. The embryonic development stage and quality directly affect the SNP detection efficiency of cf-DNA in the culture medium, with a detection efficiency reaching up to 71.62%. This invention provides experimental evidence and technical support for the establishment of a bovine non-invasive embryo genetic testing technology system. Attached Figure Description

[0019] Figure 1This is a statistical chart showing the blastocyst rate of embryos in different culture volumes at the same culture time in a preferred embodiment of the present invention. (A) Statistical analysis of blastocyst rate in different culture volumes from D3 to D7; (B) Statistical analysis of blastocyst rate in different culture volumes from D3 to D8; (C) Statistical analysis of blastocyst rate in different culture volumes from D5 to D7; (D) Statistical analysis of blastocyst rate in different culture volumes from D5 to D8; different letters indicate significant differences. （P<0.05） .

[0020] Figure 2 The following is a statistical chart showing the blastocyst rate of embryos cultured for different times in the same culture volume according to a preferred embodiment of the present invention. (A) Statistical analysis of blastocyst rate at different times in a 10 μL culture volume; (B) Statistical analysis of blastocyst rate at different times in a 15 μL culture volume; (C) Statistical analysis of blastocyst rate at different times in a 20 μL culture volume; different letters indicate significant differences (P<0.05).

[0021] Figure 3 This is a statistical chart showing the number of embryonic cells in different culture volumes at the same culture time in a preferred embodiment of the present invention. (A) Statistical analysis of the number of embryonic cells in different culture volumes from D3 to D7; (B) Statistical analysis of the number of embryonic cells in different culture volumes from D3 to D8; (C) Statistical analysis of the number of embryonic cells in different culture volumes from D5 to D7; (D) Statistical analysis of the number of embryonic cells in different culture volumes from D5 to D8; Different letters indicate significant differences (…). P <0.05).

[0022] Figure 4 This is a statistical chart showing the number of embryonic cells at different culture times within the same culture volume, as per a preferred embodiment of the present invention. (A) Statistical analysis of the number of embryonic cells at different culture times within a 10 μL culture volume; (B) Statistical analysis of the number of embryonic cells at different culture times within a 15 μL culture volume; (C) Statistical analysis of the number of embryonic cells at different culture times within a 20 μL culture volume; different letters indicate significant differences (…). P <0.05).

[0023] Figure 5 This invention provides a preferred embodiment of embryonic cell apoptosis at different culture volumes (D3-D7). (A) Representative graph of blastocyst cell number and apoptosis number at different culture volumes (D3-D7), scale bar = 100 μm; (B) Statistical analysis of apoptosis rate of embryonic cells at different culture volumes (D3-D7); different letters indicate significant differences (…). P <0.05).

[0024] Figure 6This invention provides a preferred embodiment of embryonic cell apoptosis at different culture volumes (D5-D7). (A) Representative graph of blastocyst cell number and apoptosis number at different culture volumes (D5-D7), scale bar = 100 μm; (B) Statistical analysis of apoptosis rate of embryonic cells at different culture volumes (D5-D7); different letters indicate significant differences (…). P <0.05).

[0025] Figure 7 This invention provides a preferred embodiment of embryonic cell apoptosis at different culture volumes (D3-D8). (A) Representative graph of blastocyst cell number and apoptosis number at different culture volumes (D3-D8), scale bar = 100 μm; (B) Statistical analysis of apoptosis rate of embryonic cells at different culture volumes (D3-D8); different letters indicate significant differences (…). P <0.05).

[0026] Figure 8 This invention provides a preferred embodiment of embryonic cell apoptosis at different culture volumes (D5-D8). (A) Representative graph of blastocyst cell number and apoptosis number at different culture volumes (D5-D8), scale bar = 100 μm; (B) Statistical analysis of apoptosis rate of embryonic cells at different culture volumes (D5-D8); different letters indicate significant differences (…). P <0.05).

[0027] Figure 9 This is a statistical chart showing the apoptosis rate of embryonic cells at different culture times within the same culture volume, as per a preferred embodiment of the present invention. (A) Statistical analysis of apoptosis rate of embryonic cells at different culture times with a culture volume of 10 μL; (B) Statistical analysis of apoptosis rate of embryonic cells at different culture times with a culture volume of 15 μL; (C) Statistical analysis of apoptosis rate of embryonic cells at different culture times with a culture volume of 20 μL; different letters indicate significant differences (…). P <0.05).

[0028] Figure 10 This is a representative agarose gel electrophoresis image of the free DNA amplification products in bovine embryo culture medium in a preferred embodiment of the present invention.

[0029] Figure 11 This is a statistical chart showing the number of detection sites in free DNA and blank control in a preferred embodiment of the present invention.

[0030] Figure 12 This is a statistical chart showing the number of SNP detection sites in embryo culture medium at different culture times in a preferred embodiment of the present invention.

[0031] Figure 13 This is a statistical chart showing the number of SNP detection sites in embryo culture medium at different embryonic development stages in a preferred embodiment of the present invention.

[0032] Figure 14This is a statistical chart showing the number of SNP detection sites in embryo culture medium under different amplification methods in a preferred embodiment of the present invention. Detailed Implementation

[0033] Cell-free DNA (cf-DNA) in embryo culture medium provides important evidence for non-invasive preimplantation genetic testing (ni-PGT). However, population culture modes make it difficult to trace the individual origin of cf-DNA, while single-drop culture systems often lead to decreased embryo development quality due to the lack of population effects. Furthermore, the content of cf-DNA in embryo culture medium is extremely low, and its efficient amplification and accurate detection are key issues restricting the application of ni-PGT technology in livestock embryo production. Therefore, this invention provides a method for the efficient preparation of cell-free DNA from culture medium and the detection of SNP loci for non-invasive genetic testing of bovine embryos.

[0034] The present invention adopts the following technical solution: Bovine oocytes and embryos were obtained through in vitro maturation (IVM) and in vitro fertilization (IVF). Single-drop culture groups with different culture times and volumes were set up, with population culture as a control. Embryonic development indicators and culture medium metabolic characteristics were detected to screen for optimal culture conditions. At the same time, the culture medium of single-drop embryos was collected, and whole genome amplification and SNP microarray detection were carried out in combination with different sampling times, embryonic development stages and amplification kits. The aim is to provide theoretical and practical support for the improvement and application of ni-PGT technology in livestock embryo production.

[0035] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art, and the raw materials used are all commercially available products.

[0036] The reagents used in the following examples: Semen washing solution (i.e., Bo solution): 3 mg / mL BSA + 138 μg / mL C3H3NaO3 + 0.33 mg / mL CaCl2·2H2O + 2.5 mg / mL glucose + 0.3 mg / mL KCl + 106 μg / mL MgCl2·6H2O + 6.55 mg / mL NaCl + 99 μg / mL NaH2PO4 + 3.10 mg / mL NaHCO3.

[0037] Semen washing: Bo solution + 3.87 mg / mL caffeine.

[0038] Fertilization fluid: Bo solution + 20 mg / mL BSA + 20 μg / mL Heparin sodium + 100 μg / mL Streptomycin.

[0039] Early embryo culture medium (MCR solution): 44 μg / mL C3H3NaNO3+ 0.33 mg / mL CaCl2·2H2O + 0.23 mg / mL KCl+ 0.1619 mg / mL KH2PO4+ 0.1626 mg / mL MgCl2·6H2O + 6.55 mg / mL NaCl+ 2.2 mg / mL NaHCO3+ 0.55 mg / mL calcium galactonate + 2 μL / mL phenol red + 0.27 mg / mL glucose.

[0040] Early embryo culture medium: MCR medium + 6 mg / mL BSA + 20 μL / mL essential amino acids (50×) + 10 μL / mL non-essential amino acids (100×) + 0.15 mg / mL glutamine.

[0041] Late embryo culture medium (late embryo culture medium): early embryo culture medium + 10% FBS.

[0042] The culture medium used for single-drop culture is the same as that used for late-stage embryo culture.

[0043] PVA (polyvinyl alcohol) was purchased from Sigma, P8136.

[0044] DPBS (phosphate-buffered saline) was purchased from Gibco, 14190-144.

[0045] Unless otherwise specified, the percentage sign "%" used in this invention refers to mass percentage. However, the percentage of a solution, unless otherwise specified, refers to the number of grams of solute contained in 100 mL of solution.

[0046] Example 1. Test Methods 1.1 Bovine oocyte collection and in vitro maturation Oocyte Collection: 2-3 hours before the experiment, prepare IVM culture trays using four-well plates in a laminar flow hood. Add 500 μL of IVM culture medium to each well and cover with 250 μL of mineral oil, then place in an incubator for pre-equilibration. Bovine ovaries are obtained from healthy Holstein cows at a local slaughterhouse. After the ovaries arrive at the laboratory, wash 3-4 times with sterile saline at 37°C containing penicillin and streptomycin, and then place in a 33°C water bath. Using a vacuum pump, extract cumulus oocyte complexes (COCs) from follicles with a diameter of 2-8 mm. Under a stereomicroscope, select COCs with at least 3 layers of cumulus cells using a glass needle, wash twice in oocyte washing solution, and then wash 3 times in an IVM washing tray. After cleaning, COCs were transferred to IVM culture trays, 40-50 per well, and matured in an incubator (38.5℃, 5% CO2) for 22-24 hours.

[0047] 1.2 In vitro fertilization Frozen semen was thawed in a water bath at 37℃-39℃ for 30 seconds. After thawing, the semen was mixed with 7 mL of pre-equilibrated semen washing solution in a laminar flow hood and centrifuged at 1800 r / min for 5 min. The supernatant was removed, and the mixture was resuspended in 7 mL of semen washing solution. The mixture was then centrifuged again, and the supernatant was removed after centrifugation. The mixture was then resuspended in pre-equilibrated fertilization solution, and the sperm density was adjusted to 1 × 10⁻⁶. 6 Fertilization culture drops were prepared using medium-sized culture dishes at a concentration of 1 / mL and placed in a constant temperature incubator (38.5℃, 5% CO2). The cumulus cells of mature COCs were removed using a 1 mg / mL hyaluronidase solution, leaving 1-2 layers. After removal, 400 μL of reaction stop solution (TCM199 + 10% FBS) was added to terminate the reaction. The oocytes with removed cumulus cells were washed three times in a wash drop prepared with fertilization solution, transferred to a fertilization drop, and incubated in a constant temperature incubator for 16-18 h for in vitro fertilization.

[0048] 1.3 In vitro embryo culture After fertilization, the fertilized eggs were transferred to wash drops prepared with pre-embryo culture medium and washed three times. The eggs were gently agitated with a pipette until no sperm or granulosa cells remained attached. They were then cultured in pre-embryo culture medium, and the cleavage rate was calculated after 48 hours. In the population culture group (conditions: 20 embryos per 100 μL culture drop; 38.5℃, 5% CO2), well-formed 8-cell embryos were transferred to post-embryo culture medium, with half the medium changed every 48 hours. The blastocyst rate was calculated on days 7 and 8 post-fertilization. In the single-drop culture group, a sufficient number of well-formed 8-cell / morula embryos were selected on days 3 and 5 post-fertilization and cultured in single drops of 10 μL, 15 μL, and 20 μL. The blastocyst rate was calculated on days 7 and 8 post-fertilization.

[0049] 1.4 Embryonic cell count detection Blastocysts collected on days 7 and 8 post-fertilization from single-drop and population cultures were washed three times with DPBS / PVA (0.1% DPBS / PVA, i.e., 0.01g PVA + 10 mL DPBS), fixed in 4% paraformaldehyde for 1 h, washed three times with DPBS / PVA after fixation, permeabilized in 0.5% Triton X-100 solution at room temperature for 25 min, washed three times with DPBS / PVA containing 0.1% Triton X-100 after permeabilization, incubated in Hoechst 33342 staining solution for 30 min, washed three times with DPBS / PVA, transferred to slides containing 3 μL of fluorescence quencher, covered with coverslips for mounting, photographed under an inverted fluorescence microscope, and statistically analyzed using ImageJ software.

[0050] 1.5 Detection of blastocyst cell apoptosis Blastocysts collected on days 7 and 8 post-fertilization from single-drop and population cultures were washed three times with DPBS containing 1% BSA, fixed in 4% paraformaldehyde for 1 h, washed three times with DPBS / BSA (0.1% DPBS / PVA, i.e., 0.01 g PVA + 10 mL DPBS), permeabilized at room temperature for 25 min in 0.5% Triton X-100 solution, washed three times with DPBS / BSA containing 0.1% Triton X-100, and incubated in TUNEL working solution at 38.5℃ for 1 h. After incubation, washed three times with DPBS / BSA containing 0.1% Triton X-100, incubated in Hoechst 33342 staining solution for 30 min, washed three times with DPBS / PVA, and then transferred to a solution containing 3% paraformaldehyde. The sample was placed on a glass slide containing μL of fluorescent antiquenching agent, covered with a coverslip to complete the mounting, and then observed and images were acquired under a laser confocal microscope. The results were then statistically analyzed using ImageJ software.

[0051] 1.6 Collection of Embryo Culture Media In the experiment, morphologically sound single-drop cultured morulae (D5-D7, M1-M10), early blastocysts (D5-D7, M11-M22), unexpanded blastocysts (D5-D8, M23-M36), and expanded blastocysts (D5-D8, M37-M47) were selected on days 5-D7 and 5-D8. Using a 10 μL pipette under a microscope, the pipette tip was placed at the edge of the droplet (avoiding the location of the embryo) and the culture medium was slowly aspirated and transferred to 200 μL nuclease-free centrifuge tubes, which were then stored on ice for later use.

[0052] 1.7 Yikang ChromLong TM Single-cell whole genome amplification kit amplification method Remove all kit components. Gently tap the tube wall to mix the DNA polymerase and place it in an ice box for later use. Thaw the remaining components at room temperature, briefly vortex to mix, and then centrifuge for 3-5 seconds before transferring them to the ice box as well. Next, perform sample lysis. Prepare a lysis mixture by mixing 2.7 μl of lysis buffer and 0.3 μl of DTT per reaction system. This mixture should be prepared fresh and sufficient for at least 4 reactions. Then, add 3 μl of the lysis mixture to each PCR tube (each containing 15 μl of culture medium), gently tap the tube wall to mix, and then centrifuge for 3-5 seconds. Avoid touching the liquid surface and vigorous shaking during sample addition to prevent cell sample loss. Place the PCR tubes in the PCR instrument, set the "heat cap" to "On, 70℃", and lyse at 70℃ and 65℃ for 10 minutes. After lysis, perform a neutralization reaction by adding 3 μl of the mixture to each tube. Neutralize the sample tube, gently tap the tube wall to mix, and then centrifuge briefly for 3-5 seconds. Note that the sample tube must be kept on an ice box until the amplification mixture is added. Finally, perform the amplification reaction. Prepare the amplification mixture by taking 37.5 μl of amplification buffer and 2.5 μl of DNA polymerase per reaction system. Immediately add 40 μl of the amplification mixture to the neutralized mixture, vortex to mix, and then centrifuge briefly for 3-5 seconds. Place the PCR tube in the PCR instrument, set the "heat cap" to "On, 70℃", and run the program at 30℃ for 8 hours, 65℃ for 10 minutes, and 4℃ in a cycle until the tube is removed.

[0053] 1.8 Amplification Methods Using Huazhi Biotechnology Reagent Kit When using the Huazhi Cell Culture Amplification Kit, first centrifuge the culture medium sample and collect 15 μL of the supernatant for subsequent reactions. Prepare a cell lysis buffer mixture (19.2 μL lysis buffer, 0.8 μL lysis enzyme) according to the specified ratio. Add 20 μL of this mixture to the sample, mix with a pipette, centrifuge, and then perform a temperature control program of incubation at 75℃ for 10 min, 95℃ for 4 min, and maintain a constant temperature of 22℃. Subsequently, prepare a pre-amplification reaction mixture (19.2 μL pre-amplification buffer, 0.8 μL pre-amplification enzyme) according to the specified ratio. Add 20 μL of this mixture to the lysis product, mix, centrifuge, and then perform pre-denaturation at 95℃ for 2 min (1 cycle). Then perform 12 cycles of gradient amplification (95℃ for 15 sec, 15℃ for 50 sec, 25℃ for 40 sec, 35℃ for 30 sec, 65℃ for 40 sec, 75℃ for 40 sec). After 15 sec, the amplification reaction mixture (35 μL amplification buffer, 1.12 μL amplification enzyme, and 2.88 μL nuclease-free water) was prepared according to the specified ratio. 39 μL of this mixture was added to the pre-amplification product, mixed, centrifuged, and then pre-denatured at 95℃ for 2 min (1 cycle). Then, 10 cycles of amplification were performed (95℃ for 15 sec, 65℃ for 1 min, and 75℃ for 1 min). Finally, the mixture was kept at 4℃. After the reaction, the double-stranded DNA concentration and integrity of the amplification product were tested. Qualified amplification products were stored at -20℃.

[0054] 1.9 Agarose gel electrophoresis Clean the gel casting mold and comb with distilled water, place them on the gel casting plate, seal the edges of the mold, and insert the comb. Prepare a 1% agarose gel using gel buffer: accurately weigh 0.3 g of agarose powder into an Erlenmeyer flask, add 60 mL of electrophoresis buffer, and then heat in a microwave oven for 1 min; add 3 μL of fluorescent dye, gently shake the flask to mix the gel solution thoroughly, cool slightly, pour into the electrophoresis tank, and let stand until completely solidified. After the gel solidifies, carefully remove the comb, place the gel in the electrophoresis tank, pour electrophoresis buffer into the tank until the liquid level is about 1 mm above the gel surface, and remove any air bubbles from the sample wells. Take 2 μL of amplified product sample, mix it with 10 μL of loading buffer, gently tap the tube wall to mix, and centrifuge; use a pipette to draw 10 μL of the above mixture and slowly inject it into the sample wells of the gel immersed in the buffer. Connect the power supply (red wire is positive, black wire is negative), set the voltage to 120 V, and determine whether to terminate electrophoresis based on the migration position of the indicator. Control the electrophoresis time to 30–40 min. Finally, place the completed agarose gel in a gel imaging system for imaging.

[0055] 1.10 SNP Chip Testing DNA was obtained from samples using a high-throughput DNA extraction kit. An appropriate amount of DNA was fragmented using restriction endonucleases, and the fragmented products were end-repaired and A-tailed. The A-tailed fragments were then ligated to sequencing adapters using ligase, followed by library purification using carboxyl-modified magnetic beads. The ligation product was mixed with barcode-labeled sequencing primers and a high-fidelity PCR reaction system for PCR amplification; different barcodes were used to distinguish different samples. After purification with carboxyl-labeled magnetic beads, the amplified product was ready for probe hybridization experiments. 500 ng of the constructed sequencing library was lyophilized, and probes and hybridization reagents were added. The mixture was denatured and incubated at 65°C for 2 hours to complete hybridization. After washing, the hybridization product was subjected to another round of PCR to construct a hybridization capture library. Multiplex PCR panel mix and multiplex PCR amplification enzyme were added to quantitative DNA, and amplification was performed in a PCR instrument. After purification with carboxyl-labeled magnetic beads, the amplified product was again amplified with barcode-labeled sequencing primers and a high-fidelity PCR reaction system; the barcodes were used to distinguish different samples. The purified amplified product constituted the multiplex PCR capture and library construction. Using the aforementioned DNA samples and appropriate products, targeted sequencing libraries were constructed, completing the library preparation for all samples. After library construction, preliminary quantification was performed using Qubit 2.0, followed by accurate determination of the effective library concentration using qPCR to ensure library quality. Once the libraries passed inspection, they proceeded to the sequencing stage. Information analysis included data quality control (removal of adapters and low-quality sequences) and alignment with a reference genome.

[0056] The samples were divided according to culture time (D7, D8), embryonic development stage (morula, early blastocyst, expanded blastocyst), and different amplification methods (ChromLong). TM The samples were grouped into two groups (e.g., Huazhi) and the number and detection rate of SNP sites were compared.

[0057] 1.11 Data Statistics and Analysis All experiments in this embodiment were performed at least three times independently. Data from embryonic development-related experiments were analyzed using SPSS 20.0 software for one-way ANOVA, while data from metabolomics experiments were analyzed using SAS 20.0 software for multivariate ANOVA. If differences between groups reached a significant level, Duncan's method was used for multiple comparisons. All results are expressed as mean ± standard deviation, and the significance level was set at [value missing]. P<0.05. The raw metabolomics data were first converted to .mzXML format using ProteoWizard software, and then peak alignment, retention time correction, and peak area extraction were performed using XCMS software. After extraction, the data underwent metabolite structure identification, preprocessing (removal of ion peaks with a missing rate >50%, KNN method to fill missing values, and filtering of characteristic peaks with RSD >50%), and quality assessment before subsequent data analysis was carried out.

[0058] 2. Results and Analysis 2.1 Effects of single-drop culture time and single-drop culture volume on blastocyst development efficiency To investigate the effects of single-drop culture time and volume on bovine embryo development efficiency, this study statistically analyzed the blastocyst rate under different culture conditions. As shown in Table 1, the blastocyst rates of each culture group (D7: 36.45%; D8: 55.61%) were significantly higher than those of the single-drop culture groups at the corresponding culture times. Under the same culture time, there were no significant differences in blastocyst rates among groups with different single-drop culture volumes. Figure 1 Under the same culture volume, delaying the start of single-drop culture to D5 and extending the culture time to D8 both significantly improved the blastocyst rate, especially the blastocyst rate in the D5-D8 group, which was significantly higher than that in other single-drop culture groups. Figure 2 ).

[0059] Table 1. Statistical analysis of blastocyst rate at different single-drop culture times and culture volumes.

[0060] Note: Different lowercase letters in the same column indicate significant differences. P <0.05), the same below. D5-D7: Day 5-7 after fertilization; D5-D8: Day 5-8 after fertilization.

[0061] 2.2 Effects of single-drop culture time and culture volume on embryo quality To investigate the effects of single-drop culture time and volume on bovine embryo quality, embryos with good morphology from each group were selected for TUNEL and Hoechst 33342 staining and photographed using a laser confocal scanning microscope. As shown in Table 2, the cell count of blastocysts in the population culture group (D7: 111.75; D8: 143.60) was significantly higher than that in the single-drop culture group at the corresponding culture time. Figure 3 As shown, under the same culture time, there was no significant difference in the number of embryonic cells among the single-drop culture groups with different culture volumes. When the culture time was D5-D7, there was no significant difference between the 20 μL single-drop culture group (102.00) and the population culture group (111.75); at culture volumes of 10 μL and 15 μL ( Figure 4(A and B), the number of blastocyst cells cultured for D5-D8 was significantly higher than in other groups; at a culture volume of 20 μL ( Figure 4 The number of blastocyst cells was highest in the D5-D8 group (119.20±21.86), and there was no significant difference from the D5-D7 group (102.00), but both were significantly higher than the D3-D7 group (59.00) and the D3-D8 group (88.67).

[0062] During the culture period of D3-D7, the apoptosis rate of single-drop culture groups with different culture volumes was significantly higher than that of the population culture group, and there was no significant difference between the groups. Figure 5 When the culture time was D5-D7, there were no significant differences between the single-drop culture groups with different culture volumes. There was no significant difference between the 20 μL single-drop culture group and the population culture group, while there were significant differences between the 10 μL and 15 μL single-drop culture groups and the population culture group. Figure 6 When the culture time was D3-D8, there were no significant differences between the single-drop culture groups with different culture volumes. There was no significant difference between the 20 μL single-drop culture group and the population culture group, while there were significant differences between the 10 μL and 15 μL single-drop culture groups and the population culture group. Figure 7 When the culture time was D5-D8, there were no significant differences between the single-drop culture groups with different culture volumes. The 15 μL and 20 μL single-drop culture groups were not significantly different from the population culture group, while the 10 μL single-drop culture group was significantly different from the population culture group. Figure 8 Under the same culture volume, there were no significant differences between groups with different culture times. Figure 9 ).

[0063] Table 2. Effects of single-drop culture time and culture volume on embryonic cell number.

[0064] 2.3 Results of cell-free DNA whole-genome amplification Considering the optimization conditions and limitations of the single-drop culture system, this study selected embryo culture media from D5-D7 morula, early blastocyst, and D5-D8 blastocyst stages under a 20 μL single-drop culture system as the sample source for cf-DNA amplification. The amplified embryo culture media samples (single-drop cultured morula (D5-D7, M1-M9)) were subjected to agarose gel electrophoresis. Diffuse bands of DNA were observed in the 100-1000 bp range. SNP microarray detection was performed on the amplified culture media samples and the blank control samples. The detection rate of loci in the embryo culture media samples was significantly higher than that in the blank control samples (culture media without embryos), indicating that cf-DNA is released into the culture medium during embryo culture and can be amplified and detected. Figures 10-11 ).

[0065] 2.4 Sequencing Data Quality Assessment To ensure the accuracy and reliability of subsequent SNP detection and analysis, this study performed quality assessment on the sequencing data of cell-free DNA samples from 47 embryos (single-drop cultured morula (D5-D7, M1-M10), early blastocysts (D5-D7, M11-M22), unexpanded blastocysts (D5-D8, M23-M36), and expanded blastocysts (D5-D8, M37-M47) culture media. The results are shown in Table 3. The raw read lengths of the 47 samples ranged from 12,800,650 to 74,953,270, and the raw bases ranged from 1.92 × 10⁻⁶. 9 - 1.12×10 10 After filtering, the effective read length (Clean Reads) is basically consistent with the original read length, and the effective base number (Clean Bases) ranges from 1.79 × 10⁻⁶. 9 - 1.07×10 10 The effective rate of the samples ranged from 85.96% to 97.60%, with an average effective rate of 93.20%. The quality control process removed a small number of low-quality or adapter sequences, retaining the vast majority of effective data. The base quality values ​​Q20 (base identification accuracy ≥99%) for all samples were above 98.65%, with an average Q20 of 99.21%; the Q30 (base identification accuracy ≥99.9%) were all above 95.27%, with an average Q30 of 97.18%. The sequencing data quality was high and met the requirements for subsequent bioinformatics analysis. The effective data rates of samples M37, M39, and M40 were relatively low (86.12%, 86.54%, and 85.96%, respectively), but their Q20 and Q30 values ​​were still very high (Q20 > 99.5% and Q30 > 98.4%), indicating that the original data of the above samples had a high proportion of impurities, and the data quality after quality control was excellent and would not affect subsequent analysis.

[0066] Table 3. Statistical table of sequencing data quality assessment results

[0067] Note: Raw Reads Number: Number of reads in the sample before quality control; Clean Reads Number: Number of reads in the sample after quality control; Raw Bases (bp): Number of bases in the sample before quality control, calculated by multiplying the number of reads in the sequencing sequence by the length of the sequencing sequence, in bp; Clean Bases (bp): Number of bases in the sample after quality control, calculated by summing the lengths of clean reads, in bp; Effective Rate (%): Ratio of the number of bases in the sample after quality control to the number of bases in the sample before quality control; Q20, Q30: Ratio of the number of bases in samples with a quality score greater than 20 and 30 after quality control to the total number of bases in the sample after quality control.

[0068] 2.5 Comparison and analysis of sample and reference sequences The clean reads after quality control were compared with the reference genome to assess data usability, and the results are shown in Table 4. The aligned reads of the 47 samples ranged from 12,353,846 to 72,352,550, with alignment rates ranging from 90.50% to 99.36%, and an average alignment rate of 96.61%. The highest alignment rate was for sample M38 (99.36%), while the lowest were for samples M4 (90.50%) and M1 (90.67%). The vast majority of samples (43 / 47) had alignment rates above 95%, indicating high homology between the sequencing data and the reference genome, reliable sample sources, and no significant contamination during the sequencing process. This high alignment rate provides a solid data foundation for subsequent SNP locus identification and genotyping. In summary, the sequencing data obtained in this study are of excellent quality (average Q30 > 97%, average effective data rate > 93%), and the alignment rate with the reference genome is high (average > 96%), proving that the obtained data are accurate and reliable and can be used for subsequent SNP analysis.

[0069] Table 4. Statistical table of sample and reference sequence alignment analysis

[0070] 2.6 SNP sample detection results This study performed SNP locus detection on 47 samples (single-drop cultured mulberry embryos (D5–D7, M1–M10), early blastocysts (D5–D7, M11–M22), unexpanded blastocysts (D5–D8, M23–M36), and expanded blastocysts (D5–D8, M37–M47)). The number of detected sites (Called_site_number), detection rate (Call_rate), number of heterozygous mutation sites (Het_alt_number) and ratio (Het_alt_rate), number of homozygous mutation sites (Hom_alt_number) and ratio (Hom_alt_rate), and number of reference genotype sites (Ref_number) and ratio (Ref_rate) for each sample were statistically analyzed. The results are shown in Table 5.

[0071] The detection rates varied considerably, ranging from 0.90% to 98.46%. Samples M25, M26, and M27 had the highest detection rates, at 98.46%, 98.25%, and 98.46% respectively, with over 110,000 detected loci, indicating high SNP detection quality and good coverage in these samples. Sample M23 also had a high detection rate of 95.69%, with 107,350 detected loci. In contrast, samples M3, M4, M5, M6, and M10 had lower detection rates, all below 1.5%, with fewer than 1,500 detected loci, suggesting potential issues such as poor DNA quality, amplification failure, or insufficient sequencing depth in these samples. Overall, there is significant heterogeneity in the detection rate and mutation type distribution among samples. Some samples (such as M23, M25, M26, and M27) ​​have high detection quality and rich mutation information, making them suitable for subsequent genetic analysis. However, the results of samples with low detection rates (such as M3, M4, M5, and M10) should be interpreted with caution.

[0072] Table 5 Statistical Table of Sample Detection Results

[0073] Note: Called_site_number: Number of sites detected in the sample; Call_rate: Proportion of sites detected in the sample; NA_number: Number of missing sites in the sample; NA_rate: Proportion of missing sites in the sample; Het_alt_number: Number of heterozygous sites in the sample; Het_alt_rate: Proportion of heterozygous sites in the sample; Hom_alt_number: Number of homozygous mutation sites in the sample; Hom_alt_rate: Proportion of homozygous mutation sites in the sample; Ref_number: Number of non-mutated sites in the sample; Ref_rate: Proportion of non-mutated sites in the sample.

[0074] 2.7 Effect of different culture times on SNP detection rate To compare the effects of different culture times on the amplification of cell-free DNA, SNP detection was performed on culture medium samples collected on days 7 and 8. The results are shown in Table 6 and 7. Figure 12 As shown. Statistical results indicate that extending the culture time can effectively improve the detection rate of cell-free DNA amplification in the culture medium. The number of detection sites (78885) and the site detection rate (70.32%) in group D8 were significantly higher than those in group D7 (3465; 3.09). P <0.05). The large standard deviation of the data indicates significant variability in test results among different samples, which may be related to differences in embryo quality.

[0075] Table 6. Statistical table of SNP detection sites and detection rates in embryo culture medium at different culture times.

[0076] 2.8 Effects of different developmental stages on SNP detection rate To further investigate the relationship between embryonic developmental stage and the detection rate of cell-free DNA SNPs in the culture medium, this study divided the samples into morula, early blastocyst, and expanded blastocyst groups based on embryonic morphology, and compared the differences in SNP detection rates among them. The results are shown in Table 7 and... Figure 13 As shown, the expanded blastocyst group exhibited the best SNP detection performance, with 80,340 detected loci and a detection rate as high as 71.62%, both significantly higher than the morula group (2,225; 1.98%) and the early blastocyst group (13,103; 11.68%). P <0.05. There was no significant difference in the number of detection sites and detection rate between the morula group and the early blastocyst group. P >0.05), but both were significantly lower than the expanded blastocyst group. This result indicates that embryonic developmental potential is positively correlated with the amount of free DNA released into the culture medium. High-quality embryos that develop to the expanded blastocyst stage have more abundant SNP site information in their culture medium.

[0077] Table 7. Statistical table of SNP detection sites and detection rates in embryo culture medium at different embryonic development stages.

[0078] 2.9 Effect of different amplification methods on SNP detection rate To compare the detection performance of different amplification kits, this study selected culture medium samples of D8 expanded blastocysts and used Econ ChromLong amplification kits. TMSNP detection rates were compared between a single-cell whole-genome amplification kit and a Huazhi self-developed single-cell whole-genome amplification kit. Results are shown in Table 8. Figure 14 As shown, the number of SNPs detected in samples amplified using the Yikang reagent kit was 80,340, with a detection rate of 71.62%; the number of SNPs detected in samples amplified using the Huazhi reagent kit was 71,362, with a detection rate of 63.61%. There was no significant difference in SNP detection rate between the two amplification methods. P >0.05). The two amplification methods showed comparable SNP detection performance for cell-free DNA in D8 expanded blastocyst culture medium, but the standard deviation of the Huazhi kit was lower than that of the Yikang kit, indicating that the amplification stability of the Huazhi kit was better than that of the Yikang kit.

[0079] Table 8. Statistical table of SNP detection sites and detection rates in embryo culture media of different amplification methods.

[0080] Most studies have shown that population culture is superior to single-drop culture in blastocyst development (Yuka et al., 2014). The core reason is that embryos cultured in vitro secrete paracrine factors such as growth factors and cytokines, which are beneficial to embryonic development (Nadia et al., 2006). In single-drop culture, the concentration of growth factors secreted by a single embryo is insufficient, leading to slow or arrested embryonic development (Dai et al., 2012). This study shows that when using 10 μL, 15 μL, and 20 μL volumes for single-drop culture, there were no significant differences in blastocyst rates among the groups, and all were significantly lower than those in the population culture group. This is consistent with the findings of Wydooghe et al. in bovine embryos (Wydooghe et al., 2014) and Ebner et al. in human embryos (Ebner et al., 2010). Herreros et al.'s study showed no difference in the rate of high-quality blastocysts and pregnancy rates between population culture and single-drop culture (Herreros et al., 2024). Population culture in microplates significantly improves the rate of high-quality blastocysts, but pregnancy and live birth rates are not significantly different from those of single-drop culture (Ergin et al., 2025). Layek et al. used 30 μL microdrops for single-drop culture and compared it with population culture, finding no significant difference in blastocyst rate between single-drop culture (40.6%) and population culture (27.5%) (Layek et al., 2024). Travaglione et al. reported a method for single-drop culture in a very small, closed environment, culturing single embryos in approximately 70 nL microwells for 7 days. The results showed that this culture method had no effect on blastocyst rate compared with conventional population culture and semi-restricted population culture, suggesting that small-volume single-drop culture systems may improve embryo quality by influencing embryo metabolic characteristics (Travaglione et al., 2024).

[0081] Culture time and culture volume are two important factors affecting embryonic development in single-drop culture. Bovine embryonic zygotic genome activation (ZGA) occurs between the 8-16 cell stage, during which in vitro embryos are prone to developmental arrest (Montjean et al., 2021; Raffaella et al., 2023). Our results indicate that the D5-D8 group significantly increased the blastocyst rate compared to the D3-D8 group, which may be related to embryonic arrest. D5 embryos have already passed the ZGA stage and reached the morula stage, thus exhibiting better developmental capabilities. Population culture helps embryos undergo zygotic genome activation to overcome embryonic arrest, thereby achieving a higher blastocyst rate. This is consistent with the findings of Tan et al., who demonstrated that population culture can alleviate in vitro embryonic developmental barriers, improve embryo quality, and increase pregnancy rates (Tanet et al., 2003).

[0082] Total cell count and apoptosis rate in blastocysts are important indicators for assessing embryo quality (Imran et al., 2016; Tao et al., 2019). This study showed that the number of blastocyst cells in the single-drop culture group was generally lower than that in the population culture group. The apoptosis rate was significantly higher in the single-drop culture groups during days 3-7 and 3-8. This suggests that initiating single-drop culture too early may exacerbate oxidative stress and metabolic disorders, leading to apoptosis, or that single-drop culture may hinder genome activation in embryos, resulting in apoptosis (Miguel et al., 2022). Insufficiently small culture drop size may also be a significant factor affecting embryo quality (Zhu et al., 2023). In vitro embryos should be covered with mineral oil in the culture drop to minimize fluctuations in culture medium temperature, osmotic pressure, and pH (Ebrahimi et al., 2022). Studies have shown that mineral oil may have a negative impact on embryo quality, especially when embryos are cultured in microdroplets, where the increased surface area in contact with oil exacerbates the adverse effects of mineral oil (Roberto et al., 2024). This is consistent with the results of this study. When the culture medium volume was increased to 20 μL, the cell number (119.20) during D5-D8 culture was not significantly different from that of the population culture group (143.60), and the cell apoptosis rate was also close to that of the population culture level.

[0083] This study compared and investigated the effects of different culture times, embryonic development stages, and amplification methods on the detection sites of SNPs in cell-free DNA amplification products in bovine embryo culture medium. Agarose gel electrophoresis and SNP microarray detection confirmed that bovine embryos release cf-DNA into the culture medium during culture, and this cf-DNA can be successfully amplified. The amplification products are diffuse bands of 100-1000 bp, which is largely consistent with the results of studies on human embryo cf-DNA (Leiet al., 2022). In addition, several studies have yielded similar results. For example, Stigliani et al.'s analysis of over 800 embryo culture medium samples found that cf-DNA fragment sizes ranged from 100 to 1000 bp, with an average of about 400 bp, and for the first time confirmed the simultaneous presence of genomic DNA and mitochondrial DNA in embryo culture medium (Stiglianie et al., 2013). Galluzzi et al.'s quantitative analysis of Day 3 and Day 5 / 6 culture medium samples showed that cf-DNA content was 80±70 pg and 99±113 pg, respectively, which was sufficient to meet the needs of subsequent genetic testing (Luca et al., 2015). Pan et al.'s latest study further used the TDual-Seq strategy to reveal the fragment omics characteristics of cf-DNA during early embryonic development, finding that long cf-DNA fragments (>500 bp) mainly originated from intracellular fragmentation in apoptotic cells, while short cf-DNA fragments underwent fragmentation both intracellularly and extracellularly (Panet et al., 2024). These studies provide an important foundation for understanding the generation mechanism and biological characteristics of cf-DNA.

[0084] Sequencing data quality assessment showed that the average effective data rate of 47 embryo culture medium samples was 93.20%, the average Q30 reached 97.18%, and the average alignment rate with the reference genome reached 96.61%, proving that the obtained data was of good quality and accurate and reliable. Regarding culture time, the number of detected loci (48623±49187) and detection rate (43.34±43.84%) in group D8 were significantly higher than those in group D7 (12131±20641, 10.81±18.40%), indicating that extending the culture time to day 8 helps accumulate sufficient cf-DNA for detection and obtains higher coverage of detection results. This result is consistent with the trend reported in several studies. For example, Shamonki et al. detected a DNA concentration of 2-642 ng / μL after amplification of Day 5 culture medium samples, confirming that extending the culture time can obtain sufficient cf-DNA for various genetic tests (Shamonki et al., 2016); Lei et al. pointed out that culture time may affect the amplification success rate of cf-DNA in embryo culture medium. The longer the culture time, the more cf-DNA can be obtained in the culture medium, and the higher the amplification success rate (et al., (2022); Lane et al.'s research also pointed out that cf-DNA in most culture medium samples could be amplified, and the accuracy of sample examination was significantly improved after extending the culture time, indicating that extending the culture time can increase the concentration of cf-DNA in the culture medium and can effectively reduce maternal granulocyte contamination and reduce the possibility of polar body DNA detection (Lane et al., 2017). In this study, the standard deviation of the D7 / D8 group data was relatively large, indicating that there were large differences between different samples. This is consistent with the phenomenon observed by Hammond et al. - they found that there were significant differences in the ability of different embryos to release cf-DNA, which may be partly related to the quality of the embryo itself, with better quality embryos releasing more cf-DNA (Hammond et al., 2017).

[0085] Regarding embryonic development stages, the expanded blastocyst group showed the best detection results, with significantly higher detection rates (80340±25941) and (71.62±23.12%) than the morula group (2225±1238, 1.98±1.10%) and the early blastocyst group (13103±10803, 11.68±9.63%). This indicates that embryonic developmental potential is closely related to the amount of cf-DNA released, and high-quality embryos that develop to the expanded blastocyst stage can release more cf-DNA. As the embryo develops to the blastocyst stage, cells begin to differentiate into the inner cell mass and trophectoderm (Paulina et al., 2023), a process accompanied by a certain degree of apoptosis (Alina et al., 2022). Studies on embryonic self-correction capabilities have shown that embryos can eliminate abnormal cells through apoptosis. These eliminated cells are expelled as cellular debris, becoming an important source of cf-DNA in the culture medium (Shrutiet et al., 2020). Giovanni et al.'s research confirmed that the proliferation and apoptosis of trophoblast cells are in dynamic equilibrium during blastocyst formation, with high-quality embryos exhibiting a higher total cell count and a lower apoptosis rate (Giovanniet et al., 2024). Hammond et al., through quantitative analysis of mitochondrial DNA, found that cf-DNA in the culture medium contains not only embryonic genomic DNA but also a certain proportion of mitochondrial DNA, and that the embryonic developmental stage is positively correlated with the amount of cf-DNA released (Hammond et al., 2017). Lane et al.'s research indicated that the ability to collect cf-DNA from the culture medium to detect aneuploidy depends on the embryonic developmental stage, with cf-DNA detection results superior to those from blastocyst-stage embryos (Lane et al., 2017).

[0086] In comparing amplification methods, there was no significant difference in SNP detection rates between the Yikang kit (detection rate 71.62±23.12%) and the Huazhi kit (detection rate 63.61±9.78%). However, the standard deviation of the Yikang kit was higher, indicating that its detection stability was worse than that of the Huazhi kit. Leaver's study compared the amplification effects of various whole-genome amplification methods on culture medium cf-DNA and found that the SurePlex kit achieved the highest amplification level and the best diagnostic concordance with trophectoderm biopsy samples (Leaver et al., 2020). A prospective study by Kuznyetsov et al. also confirmed that regardless of whether a cell lysis step was used before amplification, cf-DNA amplification products achieved high concordance (over 97%) with corresponding trophectoderm biopsy samples for aneuploidy detection. This study also pointed out that the stability of the amplification method is crucial for reducing the risk of false negative results (Kuznyetsov et al., 2020). A study by Vera-Rodriguez et al., using SNP sequencing to identify maternal contamination, found that the proportion of embryonic DNA in culture medium cf-DNA ranged from a minimum of 0% to a maximum of 100%, with a median of only 8%. This significant variation places higher demands on the stability and sensitivity of amplification methods. (Vera-Rodriguez) (et al., 2018). The results of this study indicate that when selecting a cf-DNA amplification method, in addition to considering the average detection rate, the stability and reproducibility of the method should also be given priority.

[0087] This study found that the SNP detection rate in the expanded blastocyst group (71.62%) was significantly higher than that in the early blastocyst group (11.68%) and the morula group (1.98%), a difference exceeding initial expectations. Although previous studies have suggested that embryonic developmental stages may affect cf-DNA release, this study is the first to quantitatively reveal the specific magnitude of this stage-specific difference in bovine embryos. Further analysis of its biological mechanisms suggests it may be closely related to cell proliferation and differentiation following embryonic genome activation. Fu et al.'s research indicates that major embryonic genome activation in buffalo embryos occurs at the 16-cell stage, followed by dramatic remodeling of gene expression and DNA methylation (Fu et al., 2023). As development progresses to the blastocyst stage, trophoblast cells begin to form and undergo programmed apoptosis, a process that may be the primary cause of the massive release of cf-DNA (Kemalet et al., 2024). Research on embryonic self-correction provides more direct evidence—they found that 64% of blastocysts expel cell debris during culture, which contains additional chromosomal abnormalities, indicating that embryos achieve self-correction by actively expelling abnormal cells (Brezina et al., 2011; Orvieto et al., 2018; Wang et al., 2023). This mechanism well explains why high-quality embryos that develop to the blastocyst stage release more cf-DNA.

[0088] In this study, some samples had low sequencing efficiency (e.g., 86.12%, 86.54%, and 85.96% for M37, M39, and M40, respectively), but their Q20 and Q30 values ​​remained high, suggesting that these samples may have some degree of contamination. Vera-Rodriguez et al., through haplotype analysis, found that the median proportion of embryonic DNA in culture medium cf-DNA was only 8%, with maternal cumulus cell contamination being the main source (Vera-Rodriguez et al., 2018). Chen et al. traced the origin of SCM cf-DNA by detecting DNA methylation, indicating that cf-DNA originated not only from blastocyst cells but also from cumulus cells, and most likely from polar bodies (Chen et al., 2021). Kuznyetsov et al. found that avoiding cell lysis before amplification helped reduce the risk of maternal contamination from residual cumulus cells (Kuznyetsov et al., 2020). These studies highlight the importance of identifying and controlling maternal contamination in preimplantation genetic testing.

[0089] Although the detection rate of loci in the blank control samples was significantly lower than that in the embryo culture medium samples in this study, a certain level of amplification signal was still present. The results of Hammond et al. showed that extremely low levels of DNA contamination were also detected in the culture medium controls that had not been exposed to embryos (Hammond et al., 2017), which is speculated to originate from human serum albumin added to the culture medium, as albumin has a high DNA-binding affinity (Malonga et al., 2006). A review by Yang et al. also pointed out that the complex origins of cf-DNA are one of the major challenges limiting the clinical application of non-implantable genetic testing (Yang et al., 2024). This study used bovine embryos as experimental material, and the culture medium used contained fetal bovine serum and bovine serum albumin; therefore, a blank control was necessary during the testing process to eliminate the influence of residual DNA in the culture medium.

[0090] In addition, the detection rate of the D8 group samples had a very large standard deviation (±43.84%), and the expanded blastocyst group also showed large individual differences (±23.12%), which may be related to embryo quality. Studies by Stigliani et al. showed a significant positive correlation between embryo fragmentation rate and dsDNA content in the culture medium, meaning that embryos with high fragmentation rates may release more cf-DNA (Stiglianie et al., 2013). Studies by Lei et al. and Jiao et al. showed that SECM cf-DNA is highly consistent with the embryonic genome, but whether SECM cf-DNA is more representative of the embryo than trophoblast cell biopsy remains an open question (Lei et al., 2019; Jiao et al., 2019). A review by Mrugacz et al. also pointed out that the main challenges of non-implantation genetic testing include amplification failure (10-50%) due to low cf-DNA production and result instability (Mrugacze et al., 2025). Therefore, the high detection rate of some samples in this study may be due to abnormal apoptosis or fragmentation during embryonic development, rather than a true reflection of embryonic quality. The study by Nguyen et al. also pointed out that cf-DNA in SCM has the potential of NiPGT-A, but the reliability of NiPGT-A is insufficient to replace traditional PGT-A through trophoblast cell biopsy. NiPGT-A results still partially reflect embryonic chromosomes. Its advantage is that it is completely non-invasive and does not adversely affect the embryo. This method can be used to screen embryos whose morphological characteristics are unsuitable for biopsy. Therefore, NiPGT-A still has high reference value in embryo classification (Nguyen et al., 2025). These results indicate that using cf-DNA detection rate alone as an embryo screening indicator has significant limitations and still needs to be combined with other assessment methods such as embryo morphology scoring for comprehensive judgment.

[0091] In livestock production, early embryo genetic assessment is crucial for accelerating breeding processes and improving the utilization rate of superior breeding stock (Janine et al., 2023). Currently, embryo genetic material detection mainly relies on trophoblast cell biopsy. While this method has high accuracy, it suffers from problems such as invasiveness, high technical requirements, expensive equipment, and potential impact on embryonic developmental potential (Mrugacz et al., 2025; Amin et al., 2025). Studies by Rubio et al. (Rubio et al., 2020) and Belen et al. (Belenet et al., 2023) have shown that non-implantation genetic testing based on cf-DNA can provide a new approach for embryo genetic assessment. This study demonstrates that extending the collection time of bovine embryo culture medium to day 8 can significantly improve the SNP detection rate, potentially further enhancing the clinical application value of non-implantation genetic testing in bovine embryo screening. This has significant practical implications for production scenarios with limited embryo numbers or where invasive testing cannot be performed due to technical limitations. This study found a close correlation between embryonic developmental stage and detection rate, providing a scientific reference for establishing a bovine embryo quality assessment system based on cf-DNA. The high SNP detection rate of 71.62% in the expanded blastocyst group suggests that for bovine blastocysts with excellent morphological scores, their SCMcf-DNA is sufficient to support relatively comprehensive genetic analysis, including sex determination, screening for genetic defects, and molecular breeding value assessment (Fu et al., 2023). Studies by Sheaba et al. have shown that mitochondrial DNA copy number in the culture medium is related to embryonic developmental potential (Sheaba et al., 2022). Based on the results of this study, it is hoped that a multi-dimensional bovine embryo screening system integrating embryonic morphological scoring and cf-DNA genetic analysis can be established in the future, which can improve the success rate of embryo transfer and thus improve overall breeding efficiency. In addition, this study provides a feasible solution for simplifying the bovine embryo genetic testing process and reducing reliance on micromanipulation equipment and professional personnel. Traditional embryo biopsy requires expensive micromanipulation equipment and highly trained technicians, limiting its widespread application in grassroots breeding units. The detection method based on SCM cf-DNA only requires the collection of culture medium samples, is simple to operate, and is easier to standardize and scale up. This is of great significance for promoting the modernization and intelligentization of my country's animal husbandry, especially cattle breeding technology.

[0092] This invention optimizes the in vitro culture mode of bovine embryos, establishing a culture system starting at D5 and progressing to D8 using a 20 μL single-drop culture. This effectively improves the problem of low developmental quality of bovine embryos under single-drop culture conditions, while simultaneously achieving efficient enrichment and whole-genome amplification of cell-free DNA (cf-DNA) in the embryo culture medium. Experimental results confirm that embryonic development stage and quality significantly affect the detection efficiency of SNP sites in cf-DNA. Under optimized conditions, the highest SNP detection rate can reach 71.62%, providing reliable data support for accurate genetic detection of cf-DNA. The optimized bovine embryo in vitro culture scheme and the methods for cell-free DNA extraction, amplification, and SNP detection established in this invention can acquire genetic information without damaging the embryo. This provides a stable, efficient, and scalable technical path for bovine non-invasive preimplantation genetic testing (ni-PGT), and has significant practical value and promising prospects for promoting the industrial application of in vitro production and non-invasive genetic screening technologies for improved livestock breeds.

[0093] Although the present invention has been described in detail above with general descriptions and specific embodiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention.

[0094] References: 1. Zhang YX, Li N, Wang L, et al. Molecular analysis of DNA inblastocoele fluid using next-generation sequencing[J]. J Assist Reprod Genet, 2016, 33(5): 637-645. 2. Ana DADLP, Carla GR, Aikaterini S, et al. Advancements andChallenges in Preimplantation Genetic Testing for Aneuploidies: In thePathway to Non-Invasive Techniques[J]. Genes, 2024, 15(12): 1613. 3. Janine A, Henriette Z D V, Zarco G, et al. Diagnostic applicationsand limitations for the use of cell-free fetal DNA (cffDNA) in animalhusbandry and wildlife management[J]. Res Vet Sci, 2023, 158: 106-116. 4. Aoyama N, Kato K. Trophectoderm biopsy for preimplantation genetictest and technical tips: A review[J]. Reprod Med Biol, 2020, 19(3): 222-231. 5. Palini S, Galluzzi L, Stefani D S, et al. Genomic DNA in humanblastocoele fluid[J]. Reprod Biomed Online, 2013, 26(6): 603-610. 6. Alves G J F, Elisa P, Iana P S, et al. Current applications andperspectives of genomic selection in Bos indicus (Nellore) cattle[J]. LIVEST,2022, 263. 7. Amin A, Hassan M D, Nasrin S, et al. Application of cell free DNAin ART[J]. Placenta, 2022, 120: 18-24. 8. Yang L Y, Shi W H, Li Y Y, et al. SCM is potential resource fornon-invasive preimplantation genetic testing based on human embryos single-cell sequencing[J]. Gene, 2023, 882: 147647. 9. Viotti M. Preimplantation Genetic Testing for ChromosomalAbnormalities: Aneuploidy, Mosaicism, and Structural Rearrangements[J].Genes, 2020, 11(6): 602. 10. Travaglione A, Candela A, Gregorio D V, et al. IndividuallyCultured Bovine Zygotes Successfully Develop to the Blastocyst Stage in anExtremely Confined Environment[J]. Cells, 2024, 13(10): 868. 11. Layek S S, Kanani S, Doultani S, et al. Analyzing Cell-freeGenomic DNA in Spent Culture Media: Noninvasive Insight into the Blastocysts[J]. Glob Med Genet, 2024, 11(3). 12. Kim S Y, Chung B, Chang J H, et al. Simultaneous Identificationof 13 Foodborne Pathogens by Using Capillary Electrophoresis-Single StrandConformation Polymorphism Coupled with Multiplex Ligation-Dependent ProbeAmplification and Its Application in Foods[J]. Foodborne Pathog Dis, 2016, 13(10): 566-574. 13. Mekonnen T K, Lee H D, Cho G Y, et al. Genomic and ConventionalInbreeding Coefficient Estimation Using Different Estimator Models in KoreanDuroc, Landrace, and Yorkshire Breeds Using 70K Porcine SNP BeadChip[J].Animals (Basel), 2024, 14(17). 14. Gkioka V, Balaoura O, Goulielmaki M, et al. The Organization ofContemporary Biobanks for Translational Cancer Research[J]. Onco, 2023, 3(4):205-216. 15. Maceda I, Lao O. Analysis of the Batch Effect Due to SequencingCenter in Population Statistics Quantifying Rare Events in the 1000 GenomesProject[J]. Genes, 2021, 13(1). 16. Glotov O S, Saifitdinova A F, Pavlova O A, et al. Comparativeanalysis of the reliability of data on the molecular karyotype of the embryoobtained by whole-genome amplification methods based on NGS technology oftrophectoderm of individual cells[J]. Am J Obstet Gynecol, 2024(6): 66-74. 17. Huang P, Lan Y Y, Zhou H, et al. Comprehensive application ofmultiple molecular diagnostic techniques in pre-implantation genetic testingfor monogenic[J]. Mol Genet Genomic Med, 2024, 12(1): e2293. 18. Yuka A, Koji M. Mouse embryo motion and embryonic developmentfrom the 2-cell to blastocyst stage using mechanical vibration systems[J].Reprod Fertil Dev, 2014, 26(5): 733-741. 19. Nadia G, Henry J L. The effect of paracrine / autocrineinteractions on the in vitro culture of bovine preimplantation embryos[J].Reproduction, 2006, 131(2): 269-277. 20. Dai S J, Xu C L, Wang J, et al. Effect of culture medium volumeand embryo density on early mouse embryonic development: tracking thedevelopment of the individual embryo[J]. J Assist Reprod Genet, 2012, 29(7):617-623. 21. Wydooghe E, Vandaele L, Piepers S, et al. Individual commitmentto a group effect: strengths and weaknesses of bovine embryo group culture[J]. Reproduction, 2014, 148(5): 519-529. 22. Ebner T, Shebl O, Moser M, et al. Group culture of human zygotesis superior to individual culture in terms of blastulation, implantation andlife birth[J]. Reprod Biomed Online, 2010, 21(6): 762-768. 23. Herreros M, Martí L, Díaz N, et al. Impact of Group vs IndividualEmbryo Culture Strategies on Blastocyst and Clinical Outcomes[J]. Reprod Sci,2024, 31(6): 1705-1711. 24. Ergin E, Tavuz A I, Öztel Z, et al. Randomized Controlled TrialComparing Single Embryo Culture with Group Culture in a Micro-Well Dish:Impact on Embryo Development and Clinical Outcomes[J]. Anatolian Journal ofObstetrics and Gynecology Research, 2025, 2(1): 16-19. 25. Montjean D, Geoffroy-Siraudin C, Gervoise-Boyer M J, et al.Competence of embryos showing transient developmental arrest during in vitroculture[J]. J Assist Reprod Genet, 2021, 38(4): 857-863. 26. Raffaella S, Daniel Č, Susanne H, et al. In vitro production ofnaked mole-rats’ blastocysts from non-breeding females using in vitromaturation and intracytoplasmic sperm injection[J]. Sci Rep, 2023, 13(1):22355. 27. Tan X W, Tan J H. Co-culture of embryos: influencing factors andmechanisms of action[J]. Sheng Wu Gong Cheng Xue Bao, 2003, 19(4): 502-505. 28. Imran K, Kyeong-Lim L, Md F, et al. Coagulansin-A has beneficialeffects on the development of bovine embryos in vitro via HSP70 induction[J].Biosci Rep, 2016, 36(2): e00310. 29. Tao L, Eun J L, Yeong H S, et al. Production and development ofporcine tetraploid parthenogenetic embryos[J]. J Anim Sci Technol, 2019, 61(4): 225-233. 30. Miguel A G R, Constanza J A, Felipe N, et al. Effects of Extra-Long-Acting Recombinant Bovine FSH (bscrFSH) on Cattle Superovulation[J].Animals, 2022, 12(2): 153. 31. Zhu Y, Feng H L, Jiang M X. Evaluation of an automated dishpreparation system for IVF and embryo culture using a mouse mode[J]. Sci Rep,2023, 13(1): 16490. 32. Ebrahimi M, Mara L, Parham A, et al. Mineral Oil for in vitroEmbryo Production: What We Should Know?[J]. Arch Razi Inst, 2022, 77(4):1325-1330. 33. Roberto G, Vincenza D G, Andrea C, et al. In Vitro Culture ofMammalian Embryos: Is There Room for Improvement?[J]. Cells, 2024, 13(12):996. 34. Lei C X, Fu J, Li X, et al. Re-denudation of residual cumuluscells on day 3 increases the accuracy of cell-free DNA detection in spentembryo culture medium[J]. J Assist Reprod Genet, 2022, 39(7): 1653-1660. 35. Stigliani S, Anserini P, Venturini P L, et al. Mitochondrial DNAcontent in embryo culture medium is significantly associated with humanembryo fragmentation[J]. Hum Reprod, 2013, 28(10): 2652-2660. 36. Luca G, Simone P, De S S, et al. Extracellular embryo genomic DNAand its potential for genotyping applications[J]. Future science OA, 2015, 1(4): 62. 37. Pan M, Shi H, Qi T, et al. The biological characteristics of longcell-free DNA in spent embryos culture medium as noninvasive biomarker in in-vitro embryo selection[J]. Gene, 2024, 927: 148667. 38. Shamonki I M, Jin H, Haimowitz Z, et al. Proof of concept:preimplantation genetic screening without embryo biopsy through analysis ofcell-free DNA in spent embryo culture media[J]. Fertil Steril, 2016, 106(6):1312-1318. 39. Lane M, Zander-Fox D, Hamilton H, et al. Ability to detectaneuploidy from cell free DNA collected from media is dependent on the stageof development of the embryo[J]. Fertil Steril, 2017, 108(3S): e61. 40. Hammond R E, McGillivray C B, Wicker M S, et al. Characterizingnuclear and mitochondrial DNA in spent embryo culture media: geneticcontamination identified[J]. Fertil Steril, 2017, 107(1): 220-228. 41. Paulina L, Piotr P, Ewelina W. Species and embryo genome originaffect lipid droplets in preimplantation embryos[J]. Front Cell Dev Biol,2023, 11: 1187832. 42. Alina E O, Iris M, Tania G, et al. Exopolysaccharide ID1 ImprovesPost-Warming Outcomes after Vitrification of In Vitro-Produced Bovine Embryos[J]. Int J Mol Sci, 2022, 23(13): 7069. 43. Shruti S, K L I, Meng Z, et al. Autophagy-mediated apoptosiseliminates aneuploid cells in a mouse model of chromosome mosaicism[J]. NatCommun, 2020, 11(1): 2958. 44. Giovanni C, Cristina L, Roger S, et al. The enigmatic morula:mechanisms of development, cell fate determination, self-correction andimplications for ART[J]. Hum Reprod Update, 2019, 25(4): 422-438. 45. Leaver M, Wells D. Non-invasive preimplantation genetic testing(niPGT): the next revolution in reproductive genetics?[J]. Hum Reprod Update,2020, 26(1): 16-42. 46. Kuznyetsov V, Madjunkova S, Abramov R, et al. Minimally InvasiveCell-Free Human Embryo Aneuploidy Testing (miPGT-A) Utilizing Combined SpentEmbryo Culture Medium and Blastocoel Fluid -Towards Development of a ClinicalAssay[J]. Sci Rep, 2020, 10(1): 7244. 47. Vera-Rodriguez M, Diez-Juan A, Jimenez-Almazan J, et al. Originand Composition of Cell-Free DNA in Spent Medium From Human Embryo CultureDuring Preimplantation Development[J]. Obstet Gynecol Surv, 2018, 73(6): 355-356. 48. Fu P H, Zhang D, Yang C Y, et al. Whole-genome transcriptome andDNA methylation dynamics of pre-implantation embryos reveal progression ofembryonic genome activation in buffaloes[J]. J Anim Sci Biotechnol, 2023, 14(1): 94. 49. Kemal A H, Fitriayu A A, Atikah S, et al. Embryo response toaneuploidy through self-correction mechanism: a literature review[J]. MiddleEast Fertil Soc J, 2024, 29(1): 1-6. 50. Brezina P, Barker A, Benner A, et al. Genetic Normalization ofDifferentiating Aneuploid Human Embryos[J]. Nature Precedings, 2011: 1. 51. Orvieto R, Shimon C, Rienstein S, et al. Do human embryos havethe ability of self-correction?[J]. Reprod Biol Endocrinol, 2020, 18(1): 98. 52. Wang X X, Zhao J, Yao Z Y, et al. Arrested Cells / Cellular DebrisExpelled from Blastocysts Is Self-Correction Phenomenon During EarlyEmbryonic Development[J]. Reprod Sci, 2023, 30(7): 2177-2187. 53. Chen Y D, Gao Y, Jia J L, et al. DNA methylome reveals cellularorigin of cell-free DNA in spent medium of human preimplantation embryos[J].J Clin Invest, 2021, 131(12): 1-10. 54. Jiao J, Bei S, Matthew S, et al. Minimally invasivepreimplantation genetic testing using blastocyst culture medium[J]. HumReprod, 2019, 34(7): 1369-1379. 55. Lei H, Berhan B, Yaqiong T, et al. Noninvasive preimplantationgenetic testing for aneuploidy in spent medium may be more reliable thantrophectoderm biopsy[J]. Proc Natl Acad Sci U S A, 2019, 116(28): 14105-14112. 56. Mrugacz G, Mospinek A, Głowacka J, et al. NoninvasivePreimplantation Genetic Testing in Recurrent Pregnancy Loss and ImplantationFailure: Breakthrough or Overpromise?[J]. Cells, 2025, 14(20): 1591. 57. Nguyen T T H, Luu M T T, Đo T L, et al. Non-invasivepreimplantation genetic testing for aneuploidy using cell-free DNA inblastocyst culture medium[J]. J Assist Reprod Genet, 2025, 42(8): 1-9. 58. Rubio C, Navarro-Sánchez L, García-Pascual C M, et al.Multicenter prospective study of concordance between embryonic cell-free DNAand trophectoderm biopsies from 1301 human blastocysts[J]. Am J ObstetGynecol, 2020, 223(5): 751.e1-e13. 59. Belen L, Ruth M, Jose O A, et al. Noninvasive preimplantationgenetic testing using the embryo spent culture medium: an update[J]. CurrOpin Obstet Gynecol, 2023, 35(4): 294-299. 60. Sheaba M H, Gadel-Rab A G, EL-Bitar A M H, et al. Total AndMitochondrial Cell Free DNA Quantification in Day 5 Embryos Culture MediaReflect Embryos Quality[J]. Egypt J Hosp Med, 2022, 89: 4346-4352. 61. Malonga H, Neault J F, Arakawa H, et al. DNA interaction withhuman serum albumin studied by affinity capillary electrophoresis and FTIRspectroscopy[J]. DNA Cell Biol, 2006, 25(1): 63-68. 62. Yang S, Xu B, Zhuang Y, et al. Current research status andclinical applications of noninvasive preimplantation genetic testing: Areview[J]. Medicine (Baltimore), 2024, 103(40): e39964.

Claims

1. A method for efficient preparation of cell-free DNA from culture medium and detection of SNP sites in bovine embryos for non-invasive genetic testing, characterized in that, Includes the following steps: (1) After obtaining bovine embryos through in vitro fertilization, on the 5th day after fertilization, a single embryo that has developed to the morula stage is transferred to a 20 μL volume of embryo culture medium for single-drop culture until the 8th day after fertilization; (2) Collect the culture medium containing a single embryo from step (1), wherein the culture medium contains cell-free DNA released by the embryo; (3) Perform whole-genome amplification on the free DNA collected in the culture medium in step (2); (4) Perform SNP site detection on the product amplified in step (3).

2. The method according to claim 1, characterized in that, In step (1), the mulberry embryo is cultured to the expanded blastocyst stage.

3. The method according to claim 2, characterized in that, In step (1), the culture conditions for the single drop culture are 38.0℃~38.5℃ and 4.5%~5.0% CO2.

4. The method according to claim 1, characterized in that, Before step (2), that is, before collecting the culture medium, there is also a step of assessing the quality of embryonic development; The evaluation indicators include blastocyst rate, total number of blastocyst cells, and apoptosis rate.

5. The method according to claim 1, characterized in that, The whole genome amplification described in step (3) employs a multiple substitution amplification method; Amplification was performed using a whole-genome amplification kit, specifically the Yikang ChromLong single-cell whole-genome amplification kit or the Huazhi cell culture medium amplification kit; the Huazhi cell culture medium amplification kit was preferred.

6. The method according to claim 1, characterized in that, After whole-genome amplification in step (3), the process also includes quality testing of the amplification products. Preferably, the quality detection includes agarose gel electrophoresis detection; More preferably, the amplification products exhibit diffuse bands in the range of 100-1000 bp as detected by agarose gel electrophoresis.

7. The method according to claim 1, characterized in that, The SNP site detection in step (4) is performed using SNP chip detection or sequencing methods.

8. The method according to claim 1, characterized in that, The SNP site detection described in step (4) is used for screening for superior genetic traits or sex determination in bovine embryos.

9. The application of the method according to any one of claims 1-8 in non-invasive genetic testing of bovine embryos; The application is for purposes other than disease diagnosis and treatment.

10. The application according to claim 9, characterized in that, The non-invasive genetic testing is used to assess the developmental potential of bovine embryos, screen for desirable hereditary traits, or determine sex.

Images