Animal embryo biopsy sampling method and application thereof

Abstract

The invention discloses an animal embryo biopsy sampling method and application thereof, and belongs to the technical field of animal breeding. The animal embryo biopsy sampling method provided by the invention is safer, more accurate and more efficient, and can effectively reduce the difficulty of an embryo biopsy sampling technology, reduce mechanical damage to embryos, effectively avoid development retardation of the embryos after sampling and remarkably improve the ratio and utilization efficiency of available embryos on the premise of ensuring the stability of the detection rate of a genetic evaluation chip; the survival rate, the developmental ability and the embryo pregnancy success rate of the sampled embryos are remarkably improved, and the transplantation conception rate of the frozen embryos after sampling reaches 40%; the method can be used for hereditary performance detection and evaluation and embryo screening before embryo implantation, and has important significance in shortening generation intervals, accelerating the breeding process and reducing the overall production cost of animal breeding and breeding.

Description

Technical Field

[0001] This invention relates to the field of animal reproduction and breeding technology, and in particular to a method for obtaining animal embryo biopsy samples and its application. Background Technology

[0002] The significance of animal breeding is mainly reflected in: 1) Species continuation and gene optimization: By selecting superior gene combinations, animal disease resistance, production performance and adaptability can be improved, ensuring the continuous reproduction of species in natural or artificial environments; 2) Enhancing industrial economic benefits: Breeding directly promotes the large-scale development of animal husbandry and helps upgrade local characteristic breeding industries. At the same time, the combination of local species protection and industrialization can maintain biodiversity and meet the market demand for high-quality livestock products; 3) Ecosystem stability and sustainable development: Breeding can reduce dependence on wild populations and reduce the damage to the ecology caused by overgrazing or hunting. In addition, efficient breeding technology can reduce resource consumption and lower breeding costs.

[0003] Embryo transfer is a crucial method in animal breeding. Preimplantation genetic performance testing and embryo selection refer to the use of technical means to detect the normality of the genetic material of embryos before transfer and to select qualified embryos for transfer, thereby improving reproductive success rates and offspring quality, and accelerating the genetic improvement process. Embryo biopsy sampling is the foundation of preimplantation genetic performance testing and evaluation. Sufficient embryonic genome data is obtained through single-cell / small-cell genomic DNA amplification technology obtained from embryo biopsy. Whole-genome breeding microarray analysis is then used to obtain the genomic breeding value and comprehensive performance index of each embryo, based on which superior embryos are selected for transfer. The goal pursued by those skilled in the art is to reduce the technical difficulty of embryo biopsy sampling while ensuring a stable detection rate of genetic evaluation microarrays, improving the safety of embryo biopsy, and ultimately increasing the survival rate, developmental capacity, and pregnancy success rate of the sampled embryos. Summary of the Invention

[0004] The purpose of this invention is to provide an animal embryo biopsy sampling method and its application. The sampling method provided can reduce the technical difficulty of embryo biopsy sampling while ensuring the stable detection rate of genetic evaluation chips, and improve the safety of embryo biopsy and the pregnancy success rate of the embryos after sampling.

[0005] To achieve the above objectives, the present invention provides a method for obtaining animal embryo biopsy samples, comprising the following steps: Blastocyst-stage embryos are collected, and 1-10 trophoblast cells are cut from the blastocyst-stage embryos using a micromanipulator and assisted laser.

[0006] Preferably, the laser width adjustment range during laser cutting is determined by the width of the trophoblast cell junction, and the laser width on both the left and right sides does not exceed the zona pellucida of the blastocyst and the injection needle opening.

[0007] Preferably, the microscopic operating system is the PIEZO microscopic operating system.

[0008] Preferably, the zona pellucida of the blastocyst embryo is first perforated at the three o'clock position using a micro-operating system PIEZO, but without perforating the trophoblast cells. 1-10 trophoblast cells are aspirated, and the junctions of the trophoblast cells are cut with laser assistance. After the junctions are cut with laser, the trophoblast cells are mechanically torn off with an injection needle.

[0009] Application of an animal embryo biopsy sampling method as described above in the screening of embryos with superior genotypes.

[0010] Preferably, the application method is as follows: after lysing the obtained trophoblast cells, a whole genome amplification reaction is performed, the amplification product is purified, and the embryo genome is detected using a whole genome chip.

[0011] Application of an animal embryo biopsy sampling method as described above in animal breeding.

[0012] Therefore, the present invention provides a method for animal embryo biopsy sampling and its application, the specific technical effects of which are as follows: (1) The animal embryo biopsy sampling method provided by the present invention is safer, more accurate and more efficient. Under the premise of ensuring the stable detection rate of genetic evaluation chip, it can effectively reduce the technical difficulty of embryo biopsy sampling, reduce mechanical damage to the embryo, effectively avoid developmental arrest of the embryo after sampling, and significantly improve the ratio and utilization efficiency of usable embryos. (2) The animal embryo biopsy sampling method provided by the present invention can significantly improve the survival rate, developmental capacity and embryo pregnancy success rate of the sampled embryos. The implantation pregnancy rate of the frozen embryos after sampling reaches 40%. It can be used for genetic performance testing and evaluation and embryo screening before embryo implantation. It is of great significance for shortening the generation interval, accelerating the breeding process and reducing the overall production cost of animal breeding.

[0013] The technical solution of the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. Attached Figure Description

[0014] To more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings used in the description of the embodiments of the present invention will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0015] Figure 1This is a schematic diagram of the embryo biopsy sampling process in Embodiment 1 of the present invention; wherein A is fixing the blastocyst for biopsy and TE cell collection; B is using a micro-operating system PIEZO to make a hole at the three o'clock position of the zona pellucida to pierce the zona pellucida, but without piercing the TE cells of the blastocyst; C is aspirating 1-10 TE cells and adjusting the microscope to make the TE cell junctions clearest; D is using laser-assisted cutting of the TE cell junctions; E is cutting the TE cell junctions with a laser and then mechanically tearing the TE cells with an injection needle; F is aspirating the torn TE cells. Figure 2 These are partial process photos of the embryo biopsy sampling process in Embodiment 1 of the present invention; wherein A is fixing the blastocyst for biopsy and TE cell collection; B is aspirating 1-10 TE cells, adjusting the microscope to make the TE cell junctions clearest, and using laser-assisted cutting of the TE cell junctions; C is after the TE cell junctions are cut open with a laser; D is mechanically tearing the TE cells with an injection needle and aspirating the torn TE cells for subsequent testing; Figure 3 These are partial photographs of the sampling process in Embodiment 2 of the present invention; wherein A is the preparation of embryonic cells for biopsy from morula before fixation and densification; B is the perforation of the zona pellucida at the three o'clock position using a micro-operating system PIEZO to puncture the zona pellucida; C is the aspiration of embryonic cells; and D is the removal of 1-10 embryonic cells for subsequent testing. Figure 4 This is the statistical result of the chip detection rate of each embryo in Examples 1 and 2 of Example 4 of the present invention. Detailed Implementation

[0016] The technical solution of the present invention will be further described below with reference to the accompanying drawings and embodiments.

[0017] To make the objectives, technical solutions, and advantages of this application clearer, more thorough, and more complete, the technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings and embodiments. The following detailed descriptions are all illustrations of embodiments, intended to provide further detailed explanation of the present invention. Unless otherwise specified, all technical terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains.

[0018] The instruments, equipment, and reagents used in the examples were all obtained through commercial means; the methods and steps not described in detail in the examples are all conventional techniques in the field; the donor cows used in the examples were all from Inner Mongolia Saikexing Ranch in Inner Mongolia Autonomous Region, and were over 12.5 months old, or Holstein cows that had calved for more than 80 days, with normal estrous cycles and normal uterine and ovarian reproductive organ structures.

[0019] Example 1 The specific steps for obtaining breeding embryos using the animal embryo biopsy sampling method provided by this invention are as follows: (1) Inducing donor cows to undergo superovulation (superovulation).

[0020] A progesterone implant (CIDR) was placed in the cow's vagina in the morning. The day of CIDR placement was designated as day 0 of estrus. Vitamin B1 was injected simultaneously. AD Administer 10 mL of vitamin AD injection solution per head, starting on day 5 of estrus. Administer follicle-stimulating hormone (FSH) for four consecutive days: 100 units per head on day 1, 75 units per head on day 2, 50 units per head on day 3, and 25 units per head on day 4, twice daily, 12 hours apart. On day 7 (day 3 of FSH injection), administer 0.4-0.6 mg of prostaglandin (PG) in the morning and 0.4-0.5 mg of PG in the afternoon to dissolve the functional corpus luteum (CL) on the donor cow's ovary, thereby synchronously inducing estrus and ovulation. On day 8 of estrus (day 4 of FSH injection), remove the thrombus in the morning, and administer FSH and 10 mL of VAD injection solution in the afternoon. On day 9, administer 100 µg of gonadotropin-releasing hormone (GnRh) per head in the morning.

[0021] The estrus status of the donor cow was then determined by on-site observation. The cows were observed every 3 to 4 hours, with each observation lasting more than 0.5 hours. The estrus time of the donor cow was determined by whether it could stably accept mounting.

[0022] (2) Artificial insemination (AI).

[0023] Quickly remove a vial of frozen semen from the liquid nitrogen tank, expose it to air for 5 seconds, then rapidly immerse it in 37°C warm water for 45 seconds. Remove it, wipe the surface of the vial clean with sterile paper, and insert it into the insemination gun. Cut off the sealed end of the vial with semen shears, then attach the insemination gun cover. Perform the first insemination approximately 12 hours after the donor cow comes into estrus, infusing one vial of frozen semen into each uterine horn. Perform a second insemination 10-12 hours later, infusing half a vial of frozen semen into each uterine horn.

[0024] (3) Non-surgical methods were used to collect embryos for seed.

[0025] Embryo collection at the blastocyst stage was performed on the 7th day after the donor cow came into heat (the day of estrus was recorded as day 0). A specially designed three-channel catheter was inserted through the cervix into the uterine horn using a rectal manipulation method. The balloon of the catheter was inflated and fixed inside the uterine horn to seal the uterine cavity. Subsequently, preheated flushing fluid (Dubor's phosphate-buffered saline containing serum, DPBS) was injected into the uterine horn through the catheter, and rectal massage was used to thoroughly flush the uterus, causing the embryos to detach from the uterine wall. Finally, using the return flow path within the catheter, the flushing fluid was collected into an embryo collection cup by the hydrostatic pressure difference of the fluid. Oocytes were retrieved under a stereomicroscope, and the retrieved embryos were washed three times with preheated and equilibrated IVC solution before being placed in embryo transfer tubes and brought back to the laboratory.

[0026] (4) Preparations for embryo biopsy.

[0027] 1) Place the necessary experimental equipment, such as pipettes, four-well plates, 60mm dishes, and pipette tips, in a clean bench and sterilize them with ultraviolet light for half an hour. Remove the required IVC solution from the 4℃ freezer and allow it to equilibrate to room temperature. Place the pre-prepared egg washing solution (HM) and sperm micromanipulation solution on a 37℃ dry bath for preheating.

[0028] HM components: 9mL HM199 (Gibco, 12340030) + 1mL FBS + 10µL Penicillin-Streptomycin, Liquid (Gibco, 15140122).

[0029] 2) Place the IVC solution in a clean bench. Use a 1mL syringe to draw 2mL of IVC solution and add 500μL of IVC to each well of a four-well plate. Then, use a pipette to add 300μL of mineral oil to each well, ensuring the mineral oil completely covers the IVC solution. Label the solution with the name and date, and incubate at 38.5℃ and 5% CO2 saturated humidity for at least 2 hours to equilibrate. Continue by drawing 0.6mL of IVC solution with a 1mL syringe. Label the bottom of each 60mm dish with numbers to clearly distinguish subsequent embryos. Use a pipette to make 6 drops, each containing 100mL of IVC. Use a pipette to add 6mL of mineral oil to cover each drop, label the solution with the name and date, and incubate at 38.5℃ and 5% CO2 saturated humidity for at least 2 hours to equilibrate.

[0030] 3) Place the egg washing solution and sperm micromanipulation solution in a clean bench to begin the microdissection operation. Take a 60mm dropper and place it in two rows. Place three drops in the first row using a pipette, each drop containing 50μL of sperm micromanipulation solution. Place three drops in the second row using a pipette, each drop containing 100μL of HM solution. Add 6mL of mineral oil using a pipette until the droplets are completely covered. Place the solution in a 38.5℃, 5% CO2 saturated humidity incubator for at least 2 hours to equilibrate.

[0031] (5) Embryo biopsy sampling procedure as follows: Figure 1 As shown.

[0032] Cut off the back end of the embryo transfer tube from the blastocysts collected in step (3) with scissors, observe under a stereomicroscope, slowly transfer the embryos into a pre-prepared 60mm dish, wash them three times with pre-heated IVC solution, and place them into a pre-balanced four-well plate containing IVC. Mark the embryo codes to be sampled with a marker pen.

[0033] The blastocyst is placed in HM micromanipulation solution. The inner cell mass (ICM) is fixed at the nine o'clock position using a fixation needle. A PIEZO micromanipulation system is used to puncture the zona pellucida at the three o'clock position, taking care not to puncture the TE cells (transferocytes) of the blastocyst. 1-10 trophoblast cells (TEs) are aspirated. The microscope is adjusted to ensure the TE cell junctions are at their clearest point. Laser-assisted cutting of the TE cell junctions is performed. After the junction is cut with the laser, the TE cells are mechanically severed using an injection needle. The severed TE cells are placed in a micro-genomic DNA extraction lysis buffer using an oocyte retrieval needle and clearly labeled for further experimental analysis. The laser width adjustment range is determined by the width of the TE cell junctions, ensuring that the laser does not extend beyond the zona pellucida and the injection needle on either side. Some images during the sampling process are shown below. Figure 2 As shown.

[0034] Example 2 The commonly used method was used to perform embryo biopsy sampling for breeding purposes. The specific steps were the same as in Example 1, except that in step (3), the pre-densified morula was collected on the 5th day after the donor cow came into estrus. The sampling method in step (5) is as follows: cut off the end of the embryo transfer tube of the pre-densified morula collected in step (3) with scissors, observe it under a stereomicroscope, slowly flow the embryo into a 60mm dish prepared in advance, wash it three times with the preheated IVC solution, put it into a four-well plate containing IVC that has been balanced in advance, and mark the embryo codes to be sampled with a marker pen.

[0035] Before densification, the morula was placed in the HM micromanipulation solution. One side of the embryo was fixed at the nine o'clock position with a fixation needle. The zona pellucida was then perforated at the three o'clock position using the PIEZO micromanipulation system to extract the morula cells for subsequent testing.

[0036] Some photos from the sampling process, such as Figure 3 As shown.

[0037] Example 3 The embryos sampled in Examples 1 and 2 were cryopreserved and transferred, and the specific steps are as follows: (1) After cutting, the embryos were transferred sequentially to the IVC solution in a 60 mm dish that had been balanced beforehand and allowed to recover for 4 hours. A 4-well plate was prepared with gradient freezing solutions. 1 mL of embryo buffer (Holding solution ABT) was added to well 1 with a pipette. 600 µL of embryo buffer and 300 µL of embryo freezing solution (Ethylene glycol freeze solution ABT) were added to well 2 with a pipette. 300 µL of embryo buffer and 600 µL of embryo freezing solution were added to well 3 with a pipette. 1 mL of embryo freezing solution was added to well 4 with a pipette. The plate was kept at room temperature. The donor cow number, frozen semen number, embryo development stage, embryo grade, and date were clearly marked on the plastic stopper with a marker.

[0038] (2) The embryos were restored in sequence in wells 1, 2, 3 and 4, with each well restored for 5 min 30 s. Then the embryos were loaded into cryovials in three stages. After the timing was completed, they were placed in a cryostat for programmed freezing and stored in liquid nitrogen. The programmed freezing settings were: initial cooling from 0℃ to -6 to -7℃, holding for 5-10 minutes, linear cooling at about 0.3℃ / min to -35℃, and then quickly immersed in liquid nitrogen (-196℃) for long-term storage.

[0039] (3) After thawing the biopsied embryos, the embryos were transferred to recipient cows that were in estrus at the same time. The embryos and recipient cows were recorded in detail before the transfer. The pregnancy status of the recipient cows was tracked and observed, and the pregnancy rate was calculated.

[0040] Example 1: After cutting and freezing, 20 embryos were transferred, one embryo per recipient cow. Example 2: After cutting and freezing, 17 embryos were transferred, one embryo per recipient cow. The conception rate of frozen embryo transfer was calculated using the following formula. The results showed that the conception rate of frozen embryo transfer using the method in Example 1 was 40.00%, and the conception rate of frozen embryo transfer using the method in Example 2 was 35.29%.

[0041] Frozen embryo transfer pregnancy rate = number of pregnant recipients / total number of transfer recipients.

[0042] Example 4 The embryo biopsy samples (TE cells) obtained in Examples 1 and 2 were subjected to whole-genome sequencing of embryos for seed production. The specific steps are as follows: (1) TE cell isolation and lysis. TE cells obtained from embryo biopsy were added to 3 μL of Buffer D2 (containing proteinase K) from the lysis kit, gently mixed, and incubated at 65°C for 10 minutes, followed by heat treatment at 95°C for 10 minutes to completely lyse the cells and inactivate the proteinase. After a brief centrifugation, the lysate was stored at 4°C for later use.

[0043] (2) Whole genome amplification. Add 9 μL of REPLI-g scReaction Buffer and 3 μL of REPLI-g sc DNA Polymerase to the lysis product obtained in step (1), mix gently and centrifuge briefly. Incubate the reaction system at 30°C for 4 hours to perform multiple displacement amplification (MDA) reaction.

[0044] (3) Reaction termination and product purification.

[0045] After amplification, the reaction system was incubated at 65°C for 3 minutes to terminate the reaction. The concentration and purity of the obtained genomic DNA samples were determined using a nucleic acid analyzer. The A260 / A280 ratio was between 1.8 and 2.0, and the A260 / A230 ratio was above 2.0. The yield and concentration reached 1 µg and 100 ng / µL or higher, respectively, meeting the requirements for microarray detection. The DNA concentration information of the embryos is shown in Table 1.

[0046] Table 1. Statistics on Genomic DNA Extraction and Microarray Detection Rate of Embryos

[0047] (4) Whole-genome analysis of embryos for breeding. Genomic DNA from 20 embryos was analyzed using the Boredi 140K whole-genome chip (catalog number PHR0105_Bt140K) to obtain genomic chip genotype data for each embryo. The chip detection rate for each embryo is shown in Table 1. The statistical results of the chip detection rates for each embryo in Examples 1 and 2 are as follows: Figure 4 As shown.

[0048] (5) Genomic breeding value of embryos used for breeding.

[0049] Based on genomic microarray data and inverse regression breeding values ​​from the Chinese bovine genome selection reference population (Zhang Qi, Research on Genomic Genetic Assessment of Dairy Bovines Integrating GWAS Prior Information, Master's Thesis, China Agricultural University, 2022), and using quality-controlled genomic microarray genotype data, the GBLUP method was used to calculate the GCPI and genomic breeding values ​​of traits such as milk yield, milk protein content, milk fat content, milk protein percentage, milk fat percentage, total body size score, lactation system, limb and hoof score, and somatic cell score of the aforementioned 20 embryos. The accuracy of the genomic breeding values ​​was then evaluated. The statistical results of the genomic breeding values ​​are shown in Table 2. The statistical results of the accuracy of the estimated genomic breeding values ​​are shown in Table 3.

[0050] Table 2 Statistical results of genomic breeding values

[0051] Table 3. Results of accuracy estimation for genomic breeding values

[0052] Therefore, the animal embryo biopsy sampling method provided by this invention is safer, more accurate, and more efficient. While ensuring a stable detection rate of the genetic evaluation chip, it can effectively reduce the technical difficulty of embryo biopsy sampling, reduce mechanical damage to the embryo, effectively avoid developmental arrest of the embryo after sampling, and significantly improve the ratio and utilization efficiency of usable embryos. It can also significantly improve the survival rate, developmental capacity, and embryo pregnancy success rate of the sampled embryos, with a 40% conception rate for frozen embryo transfer after sampling. It can be used for preimplantation genetic performance testing and embryo screening, which is of great significance for shortening the generation interval, accelerating the breeding process, and reducing the overall production cost of animal breeding.

[0053] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can still be made to the technical solutions of the present invention, and these modifications or equivalent substitutions cannot cause the modified technical solutions to deviate from the spirit and scope of the technical solutions of the present invention.

Claims

1. A method for obtaining animal embryo biopsy samples, characterized in that, The steps are as follows: Blastocyst-stage embryos are collected, and 1-10 trophoblast cells are cut from the blastocyst-stage embryos using a micromanipulator and assisted laser.

2. The method for animal embryo biopsy sampling according to claim 1, characterized in that: The laser width adjustment range during laser cutting is determined by the width of the trophoblast cell junction, and the laser width on both sides does not exceed the zona pellucida of the blastocyst and the injection needle opening.

3. The method for animal embryo biopsy sampling according to claim 1, characterized in that: The microscopic operating system is the PIEZO microscopic operating system.

4. The method for animal embryo biopsy sampling according to claim 3, characterized in that: First, use the PIEZO micromanipulation system to make a hole at the three o'clock position in the zona pellucida of the blastocyst embryo to puncture the zona pellucida, but do not puncture the trophoblast cells of the blastocyst. Aspirate 1-10 trophoblast cells, use laser-assisted cutting at the junction of the trophoblast cells, and after the junction is cut open with laser, use an injection needle to mechanically tear off the trophoblast cells.

5. The application of an animal embryo biopsy sampling method as described in any one of claims 1-4 in the screening of embryos with superior genotypes.

6. The application of the animal embryo biopsy sampling method according to claim 5 in the screening of superior genotype embryos, characterized in that, The application method is as follows: after lysing the obtained trophoblast cells, a whole genome amplification reaction is performed. After purifying the amplification products, the whole genome is used for embryo genome detection.

7. The application of an animal embryo biopsy sampling method as described in any one of claims 1-4 in the breeding of superior animal breeds.

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