An ICSI operation method for improving the efficiency of in vitro OPU sexed embryo production in dairy cows

Abstract

The present invention discloses an ICSI operation method for improving the efficiency of producing sexed OPU embryos in vitro in dairy cows: frozen sexed sperm is treated using the BO flotation method, and the floated sperm is centrifuged and diluted with IVF. The treated sexed sperm is injected into OPU donor oocytes, activated twice with 5nM ionomycin and once with 2nM 6-DMAP, and then embryo culture is performed. The present invention significantly improves the blastocyst rate of embryos produced after ICSI eggs are activated, and can be used to stably produce high-quality sexed OPU embryos in dairy cows.

Description

Technical Field

[0001] The present invention belongs to the field of livestock breeding, relates to a biotechnology for improving the reproductive efficiency of dairy cows, and specifically relates to an ICSI operation for stably producing high-quality OPU sexed embryos. Background Art

[0002] Intracytoplasmic sperm injection (ICSI) involves injecting a single sperm directly into the cytoplasm of an oocyte. ICSI has enabled births in a variety of species. In cattle, ICSI effectively utilizes sperm for livestock improvement and breeding of superior animals. For example, in dairy cows, the blastocyst rate of ICSI embryos currently reaches approximately 25%.

[0003] Although sexed sperm (e.g., X sperm) can be injected directly into the cytoplasm through intracytoplasmic sperm injection (ISI), thereby improving the efficiency of sexed embryo production, due to the scarcity of high-quality dairy cattle, live oocyte retrieval (OPU) is often performed on genetically valuable cows without regard for oocyte quality. As a result, poor-quality oocytes are often retrieved and used for in vitro fertilization (IVF), which typically reduces embryo yield. IVF results are also affected by the bull's fertility, semen processing, and storage conditions. In particular, the efficiency of obtaining sexed embryos through IVF with cryopreserved sexed semen is low. Possible reasons include poor sperm motility and the inability of sperm to successfully penetrate the zona pellucida, leading to inability to achieve fertilization.

[0004] Given the long-term reliance on imported high-quality dairy cattle and frozen semen, and the impact of factors such as unstable quality of OPU oocytes on in vitro fertilization, ICSI can be used to complete single sperm injection, activate ICSI eggs, and culture embryos, thereby improving the utilization rate of low-quality oocytes to a certain extent. However, previous studies have shown that using only one activator for single treatment in cattle is insufficient to activate ICSI eggs. While the use of multiple activators can improve activation efficiency to a certain extent, abnormal pronuclear formation after activation can affect embryo development and quality, and lead to a decrease in blastocyst rate.

[0005] In addition, Chinese patent CN101492669A uses ionomycin and 6-DMAP to treat enucleated oocytes after sperm injection, and in Chinese patent CN101302497A using the same activation treatment, the blastocyst rate (reference number of cleavage embryos) can reach more than 30%. However, due to the low cleavage rate, the actual blastocyst rate (reference number of mature oocytes) is relatively low. Summary of the Invention

[0006] The object of the present invention is to provide an ICSI operation method for improving the production efficiency of in vitro OPU sex-controlled embryos in dairy cows.

[0007] In order to achieve the above object, the present invention adopts the following technical solutions:

[0008] A method for producing OPU sexed embryos, comprising the following steps:

[0009] Sexed sperm and matured OPU oocytes used as donors were microinjected into reconstructed bodies. The reconstructed bodies were activated with ionomycin and 6-DMAP in sequence to promote sperm disaggregation in the reconstructed bodies, and then embryo culture was performed to obtain OPU sexed embryos.

[0010] Preferably, the maturation culture comprises the following steps: culturing the cumulus-oocyte complexes (COCs) obtained by the OPU technique with oocyte maturation culture medium at 38.4-38.6° C., saturated humidity and 4.85%-5.15% CO 2 for 20-24 hours.

[0011] Preferably, the cumulus-oocyte complex (COCs) is cultured and then treated with hyaluronidase, and then recovered in oocyte maturation culture medium (for example, incubated at 38.4-38.6°C, saturated humidity and 4.85%-5.15% CO2 for more than 30 minutes) to obtain OPU oocytes (referred to as OPU donor oocytes) as donors after mature culture.

[0012] Preferably, the cumulus-oocyte complexes (COCs) are collected by filtering the follicular fluid of a cow (eg, a dairy cow) using an egg collection cup, and then washing the follicles with HEPES solution or physiological saline for multiple times before picking up the eggs.

[0013] Preferably, the preparation of the sexed sperm comprises the following steps: thawing sexed frozen semen of cattle (e.g., dairy cows), separating the corresponding sexed sperm from the thawed sexed semen using the BO flotation method, and diluting the separated sexed sperm with fertilization medium to obtain the sexed sperm as a donor.

[0014] Preferably, the BO flotation method specifically includes the following steps: taking 200-400 μL of sexed semen and transferring it to the bottom of 3-4 mL of supernatant, centrifuging after the sperm floats under the conditions of 38.4-38.6°C, saturated humidity and 4.85%-5.15% CO2, and washing the sperm sediment obtained by centrifugation (for example, centrifugation at 300×g-500×g for 5-10 minutes) to obtain sexed sperm.

[0015] Preferably, the microinjection is to inject the sexed sperm as a donor into the cytoplasm of the OPU donor oocyte through ICSI to form an ICSI egg as a reconstructed object.

[0016] Preferably, the preparation of the reconstructed body specifically includes the following steps: first, an OPU donor oocyte is placed in an operating droplet, and a sexed sperm as a donor is placed in the sperm droplet; a single sexed sperm in the sperm droplet is tail-broken using a single sperm injection needle and then sucked into the single sperm injection needle, a single OPU donor oocyte in the operating droplet is adsorbed using a fixed needle, the single sperm injection needle is inserted in a direction orthogonal to the first polar body of the OPU donor oocyte, and after a certain amount of cytoplasm is sucked back, the cytoplasm sucked into the single sperm injection needle and the single sexed sperm are uniformly injected into the OPU donor oocyte, and after confirming that the single sexed sperm has been injected into the OPU donor oocyte, the single sperm injection needle is withdrawn (for example, extracted from the OPU donor oocyte).

[0017] Preferably, the activation specifically includes the following steps: after recovery, the reconstructed body is transferred to a SOF containing 4.9-5.1nM ionomycin for activation treatment for 3-5 minutes, then washed with SOF, and then cultured in fertilization culture medium for 25-30 minutes, and then transferred to a SOF containing 4.9-5.1nM ionomycin for activation treatment for 3-5 minutes, then washed with SOF, and then cultured in fertilization culture medium for 1.5-2.0 hours, and then transferred to a SOF containing 1.9-2.1nM 6-DAMP for activation treatment for 3-4 hours; wherein each activation treatment and culture are carried out at 38.4-38.6°C, saturated humidity and 4.85%-5.15% CO2.

[0018] Preferably, the fertilization medium is IVF.

[0019] Preferably, the recovery of the reconstructed body specifically includes the following steps: transferring the OPU donor oocyte injected with sexed sperm by ICSI into an oocyte maturation culture medium and incubating for 20 to 30 minutes; wherein the incubation is carried out at 38.4 to 38.6° C., saturated humidity and 4.85% to 5.15% CO2.

[0020] Preferably, the embryo culture conditions include: using embryo culture medium (such as IVC) and culturing at 38.4-38.6° C., 4.85%-5.15% CO 2 and 6.9%-7.0% O 2 for 6-7 days.

[0021] A livestock breeding method comprising the following steps:

[0022] The above method for producing OPU sexed embryos is used to obtain OPU sexed embryos from donor livestock (eg, dairy cows), which are then transplanted into recipient livestock (eg, dairy cows), and the conception (eg, pregnancy) status of the recipient livestock is then examined and determined.

[0023] The beneficial effects of the present invention are embodied in:

[0024] The present invention injects sexed sperm into mature and cultured OPU oocytes, and then activates the resulting reconstructed body (e.g., ICSI oocyte) with ionomycin and 6-DMAP. It is then possible to culture sexed OPU embryos with a high blastocyst rate and a high pregnancy rate, providing a scientific reference and practical basis for the rapid propagation of sexed high-yielding livestock.

[0025] Furthermore, the present invention uses low-concentration activators (ionomycin, 6-DMAP) and selects appropriate activation treatment time intervals (i.e., culture time between different activation treatments), thereby improving the blastocyst rate of embryos produced by ICSI, activation and embryo culture of sexed bovine sperm. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 Schematic diagram of the operation process of constructing ICSI eggs; wherein: A shows a single sperm being drawn into the injection needle from the tail after the needle has been mechanically cut (before injection, the needle is fixed to the OPU donor oocyte, and the first polar body is located at the "12 o'clock" position); B shows the injection needle waiting to inject a single sperm (the injection needle retracts the cytoplasm); C shows the injection needle being withdrawn after a single sperm is injected into the OPU donor oocyte; 1-first polar body, 2-sperm.

[0027] Figure 2 This is the status diagram of ICSI eggs 48 hours after activation (cleavage).

[0028] Figure 3 This is the status of ICSI eggs 168 hours after activation (blastocyst).

[0029] Figure 4 This figure shows the pronuclear formation 16 hours after sperm was injected into the OPU donor oocyte (normal fertilization; the oocyte boundary is roughly marked by a dotted line in the figure). DETAILED DESCRIPTION

[0030] The present invention will be further described in detail below with reference to the accompanying drawings and examples, which are only used to explain the present invention and are not intended to limit the scope of protection of the present invention.

[0031] (1) Sources and preparation of materials and reagents

[0032] 1.1 Materials

[0033] (1) OPU oocytes were collected from Wuzhong City, Ningxia Hui Autonomous Region from September 2023 to January 2024.

[0034] (2) X sexed frozen semen was purchased from the United States in November 2022.

[0035] (3) The above-mentioned OPU oocytes and X-sexed frozen semen were all from Holstein cows.

[0036] 1.2 Reagents

[0037] (1) BJ-40 capillary glass tubes were purchased from Beijing Zhengtian Yi Science and Technology Co., Ltd., and borosilicate glass was purchased from Sutter Instrument (Cat. No. B100-75-15).

[0038] (2) IVF fertilization medium, IVC embryo culture medium, and TRANSFER embryo transfer medium were purchased from IVFBIOSCIENCE, gamete buffer was purchased from COOK MEDICAL (catalog number: K-SIGB-20), and sperm inhibitor PVP was purchased from Vitrolife.

[0039] (3) PureSperm 40 was purchased from Nidacon (catalog number: PS40-100), and PureSperm 80 was purchased from Nidacon (catalog number: PS80-100);

[0040] (4) 6-DMAP (6-dimethylaminopurine) was purchased from SIGMA (catalog number: D2629-250MG), and ionomycin was purchased from SIGMA (catalog number: 10634-1MG);

[0041] (5) Fetal bovine serum (FBS) was a product of Gibco, essential amino acids were a product of Thermo Fisher (Cat. No. 1831512), non-essential amino acids were a product of Thermo Fisher (Cat. No. 1958909), and insulin-transferrin-selenium supplement ITS-G was a product of Thermo Fisher (Cat. No. 1865342).

[0042] (6) Fixative solution was purchased from Beyotime (Cat. No.: P0098-500 mL);

[0043] (7) Others not marked are products of SIGMA;

[0044] (8) Oocyte maturation medium OM (self-prepared): M199 without Hepes as the base medium, supplemented with 10% (v / v) fetal bovine serum, 1% (v / v) ITS-G, 0.075 IU / mL HMG, 1 μg / mL 17β-estradiol, 10 ng / mL EGF, and 10 ng / mL bFGF;

[0045] (9) HEPEs wash solution (self-prepared): 133.38 mg NaCl, 4.78 mg KCl, 3.36 mg NaH2PO4·H2O, 283.2 μL sodium lactate (liquid reagent), 2.3426 mL 25.1 mg / mL CaCl2·2H2O, 2.04 mL 10 mg / mL MgCl2·6H2O, 47.68 mg Hepes, 60.00 mg BSA, 2 mL Na-py stock solution (sodium pyruvate stock solution, 2.2 mg / mL) and 150 μL gentamicin stock solution (10 mg / mL), dilute to 200 mL with deionized water;

[0046] (10) Floatant BO (self-prepared): SOF solution was used as the basal culture medium, and 48 mg / mL fatty acid-free bovine serum albumin, 0.1942 mg / mL caffeine, and 0.05 mg / mL heparin sodium were added;

[0047] (11) SOF (i.e., the above-mentioned SOF solution, self-prepared): 26.168 mg CaCl2·2H2O, 9.999 mg C6H5Na3O7·2H2O, 2.923 mg glutamine, 37.218 mg MgSO4·7H2O, 49.904 mg inositol, 53.378 mg KCl, 16.195 mg KH2PO4, 4.402 mg sodium pyruvate, 200.025 mg NaHCO3, 629.399 mg NaCl, and 0.0757 mg sodium lactate (solid reagent), dilute to 100 mL with deionized water;

[0048] (12) TALP fertilization medium (self-prepared): 33.33 mg NaCl, 1.19 mg KCl, 12.33 mg calcium lactate pentahydrate, 0.51 mg MgCl2·6H2O, 0.24 mg NaH2PO4·H2O, 10.53 mg NaHCO3, 70.8 μL sodium lactate (liquid reagent), 0.60 mg Na-py, 4.50 mg glucose, 1.94 mg caffeine, 15.00 mg BSA, 5.00 mg PVA, and 37.5 μL gentamicin stock solution (10 mg / mL), and the volume was adjusted to 50 mL with deionized water.

[0049] (13) Electroactivation solution (self-prepared): 100 μL MgSO4 concentrate (the concentrate was prepared by mixing 0.12 mg MgSO4 with 1 mL ddH2O), 100 μL CaCl2·2H2O concentrate (the concentrate was prepared by mixing 0.147 mg CaCl2·2H2O with 1 mL ddH2O), 1.19 mg Hepes, 0.1 mg PVA, and 54.65 mg mannitol, and the volume was adjusted to 10 mL with deionized water;

[0050] (14) PBS-PVA (self-prepared): 1.6012 g NaCl, 0.0402 g KCl, 0.284 g Na2HPO4, 0.0544 g KH2PO4 and 5.00 mg PVA, dilute to 200 mL with deionized water.

[0051] (2) Establish a technology for the stable production of high-quality dairy cow OPU sexed embryos

[0052] 2.1 Collection of OPU oocytes

[0053] The follicular fluid obtained from live egg collection is transferred to an egg collection cup with a syringe for filtration. The side net is repeatedly rinsed with saline until the follicular fluid in the egg collection cup is clear and eggs can be collected. The cumulus-oocyte complexes (COCs) are picked out with a handheld egg collection needle under a stereo microscope.

[0054] 2.2 OPU oocyte maturation culture

[0055] The picked COCs were repeatedly washed with HEPEs solution until there were no impurities and granulosa cells around the COCs. After cleaning, they were transferred to OM and rinsed once. The COCs with dead oocytes (loose cytoplasm, overmature) were removed and all were transferred to OM for maturation culture in a 5% CO2 incubator at 38.5℃ and saturated humidity.

[0056] 2.3 ICSI Procedure for Producing OPU Sexed Embryos in Dairy Cows

[0057] S1 Processing of dairy cow oocytes and sperm

[0058] Preparation of OPU donor oocytes: After 22 h of maturation culture, the cultured COCs were repeatedly blown with 0.5% hyaluronidase to detach the cumulus cells and obtain naked oocytes (the zona pellucida was not removed after hyaluronidase treatment). The oocytes were transferred to OM and placed in a 38.5°C, saturated humidity, 5% CO2 incubator to recover for 30 min before preparing for intracytoplasmic sperm injection.

[0059] Treatment of X-sexed sperm by BO flotation: Prepare 4 mL of BO and 2 mL of HEPES wash solution and equilibrate in an incubator for at least 1 hour. Thaw X-sexed frozen semen in a 38°C waterbath. Transfer an appropriate amount of the resulting X-sexed semen to the bottom of a centrifuge tube containing 4 mL of BO. Tilt the tube at a 45°C angle and place it in a 38.5°C, 5% CO2 incubator to allow the sperm to float. After 30 minutes of sperm floatation, remove the supernatant and centrifuge for 10 minutes (at room temperature and 500 × g). After centrifugation, discard the supernatant, transfer the sperm pellet to 1 mL of HEPES wash solution, mix thoroughly by pipetting, and wash by centrifugation twice, each for 5 minutes (at room temperature and 300 × g).

[0060] S2 sperm dilution

[0061] After centrifugation in HEPEs washing solution, the supernatant was removed, and a small amount of liquid was retained to suspend the precipitate to obtain a sperm suspension; 10 μL of the sperm suspension was drawn into 60 μL IVF with a micropipette for dilution, which could then be used for intracytoplasmic sperm injection.

[0062] S3 intracytoplasmic sperm injection

[0063] Before sexed sperm injection in dairy cows, first prepare microdroplets such as working drops (using gamete buffer) and sperm drops (using PVP) on a 35mm plastic dish and cover with oil. Assemble the fixed needle on the left micromanipulator arm, and the injection needle on the right micromanipulator arm. The fixed needle has an inner diameter of less than 20μm and an outer diameter between 150 and 200μm, and the injection needle has a diameter between 7 and 9μm. The fixed needle is made of a BJ-40 capillary glass tube, and the injection needle is made of borosilicate glass. After assembly, adjust the angle and distance until the fixed needle and injection needle are visible in the center of the microscope field of view.

[0064] The prepared OPU donor oocyte is transferred from the OM to the operating drop, and a small amount of diluted sperm is added to the sperm drop. The injection needle is transferred to the sperm drop and rinsed for 1 minute. After rinsing, a sperm with a complete head and tail without deformity and impurities is selected, and the sperm tail is mechanically cut off with an injection needle. After the tail is cut off, the cut sperm 2 is sucked into the injection needle from the end of the tail, and the cut sperm 2 is kept at the front end of the injection needle; the cut sperm 2 is transferred to the operating drop with the injection needle, and the fixing needle is adjusted to adsorb and fix an OPU donor oocyte. The oocyte is rotated up and down and forward and backward by the injection needle so that the first polar body 1 is in the "6 o'clock" or "12 o'clock" direction (such as Figure 1 Then adjust the injection needle to the same level as the oocyte, insert the needle at the "3 o'clock" direction of the oocyte, and insert the injection needle into the 2 / 3 of the oocyte, so that the injection needle slowly and evenly sucks back the cytoplasm (as shown in Figure 1). Figure 1When the cytoplasm in the injection needle is observed to be aspirated back suddenly faster, the cytoplasm aspiration operation is immediately stopped, and the cytoplasm in the injection needle and the sperm 2 after tail cutting are uniformly injected into the oocyte. After the injection is completed, the injection needle is withdrawn from the oocyte (as shown in FIG. Figure 1 C); complete the intracytoplasmic sperm injection of other OPU donor oocytes with sexed cow sperm according to the above operation (usually controlled within 1 hour).

[0065] S4 Activation

[0066] The ICSI eggs obtained after the completion of the cow sexed sperm injection were transferred to OM and placed in a 38.5°C, 5% CO2 incubator for recovery for 30 minutes; then transferred to SOF containing 5nM ionomycin (abbreviated as ION) for activation for 5 minutes (specifically, the activation was carried out in a 38.5°C, 5% CO2 incubator for 5 minutes), cleaned with SOF, and cultured in IVF for 30 minutes (specifically, the culture was carried out in a 38.5°C, 5% CO2 incubator for 5 minutes), and then Transfer to SOF containing 5nM ionomycin (abbreviated as ION) and activate for 5 minutes (specifically, the activation treatment is carried out in a 5% CO2 incubator at 38.5°C and saturated humidity), wash with SOF and culture in IVF for 2 hours (specifically, culture is carried out in a 5% CO2 incubator at 38.5°C and saturated humidity), and then transfer to SOF containing 2nM 6-DAMP (abbreviated as 6D) and activate for 3 hours (specifically, the activation treatment is carried out in a 5% CO2 incubator at 38.5°C and saturated humidity).

[0067] S5 embryo culture

[0068] After activation, the non-viable ICSI eggs were rinsed with SOF and removed, and then transferred to the IVC and cultured in a three-gas incubator at 38.5°C, 5% CO2, and 7% O2. Cleavage occurred after 1 to 2 days of culture (e.g. Figure 2 ), blastocysts are formed on day 6 to 7 (as shown in Figure 3 shown).

[0069] 2.4 Testing of OPU sexed embryos (cleavage rate, blastocyst rate) and reproductive performance of dairy cows after embryo transfer (pregnancy rate)

[0070] Cleavage rate = number of cleavages / number of surviving eggs × 100%

[0071] Blastocyst rate = number of blastocysts / number of surviving eggs × 100%

[0072] Embryo transfer and pregnancy test: blastocysts formed after 7 days of culture are selected and placed in a TRANSFER, loaded into a straw, placed in a sterile centrifuge tube, and finally placed in a 38.5℃ constant temperature transport box and taken to the cattle farm; the recipient cow is given epidural anesthesia at 1 to 2 caudal intervertebral spaces, and the vulva is cleaned and disinfected; the straw containing the blastocyst is placed in the transplant gun, then covered with a transplant hard coat, and finally covered with a sterile isolation coat to keep the front end of the transplant gun sterile; the blastocyst is transplanted into the uterine horn on the corpus luteum side of the recipient cow, and a transplant record of the recipient cow is kept; within 14 days after embryo transfer, the pregnancy of the recipient cow is preliminarily judged based on the return of estrus. If the recipient cow returns to estrus after 14 days and has obvious vaginal bleeding, it is considered to have miscarried. The final determination of pregnancy is based on the B-ultrasound examination at 28 days, and a record of pregnant cows is kept; the pregnancy rate can be calculated based on the record.

[0073] In addition, the effects of activation and other operations on ICSI eggs were investigated through pronuclear detection and calculation of pronuclear formation rate, fertilization rate and other indicators.

[0074] Pronuclear formation rate = number of eggs with "1MPN+1FPN" and no agglutinated sperm heads / number of viable eggs × 100%

[0075] Fertilization rate (diode rate) = number of eggs that produce diodes after activation / number of surviving eggs × 100%

[0076] Pronuclear detection: 16 hours after ISI, the zona pellucida was digested with 0.2% pronase (purchased from SIGMA), fixed with fixative for 4 hours, and then washed with PBS-PVA. Fluorescent light-proof staining was performed with 10 μg / mL Hochest33342. After 10 minutes, the dye was washed with PBS-PVA, and the pronuclear formation was observed under a fluorescence microscope. The standard for normal fertilization was 1 male pronucleus and 1 female pronucleus (1MPN+1FPN) with no agglutinated sperm heads (e.g. Figure 4 shown).

[0077] In 2023, a total of 588 dairy bovine OPU donor oocytes were subjected to intracytoplasmic sperm injection, activation, embryo culture, and embryo transfer. The following results were obtained: the pronuclear formation rate after activation was 34.44%, the cleavage rate was 74.66%, and 254 blastocysts were obtained in vitro, with a blastocyst rate of 43.19%. A total of 90 blastocysts were transferred, resulting in 35 pregnancies with a pregnancy rate of 38.89%. These results demonstrate that the ICSI method for producing sexed OPU bovine embryos is feasible and can consistently produce high-quality sexed OPU bovine embryos.

[0078] (3) Effects of different sexed sperm processing methods on ICSI embryo development

[0079] 3.1 Comparative test of different sexed sperm treatment methods (Comparative Example 1)

[0080] Collection of OPU oocytes: The follicular fluid obtained from live egg collection is transferred to the egg collection cup with a syringe for filtration. The side net is repeatedly rinsed with saline until the follicular fluid in the egg collection cup is clear and the eggs can be collected. The COCs are picked out with a handheld egg collection needle under a stereo microscope.

[0081] OPU oocyte maturation culture: The picked COCs were repeatedly washed with HEPEs solution until there were no impurities and granulosa cells around the COCs. After cleaning, they were transferred to OM and rinsed once. The dead COCs (loose cytoplasm, overmature) were removed and all were transferred to OM for maturation culture in a 5% CO2 incubator at 38.5℃ and saturated humidity.

[0082] Preparation of OPU donor oocytes: After 22 h of maturation culture, the cultured COCs were repeatedly blown with 0.5% hyaluronidase to detach the cumulus cells and obtain naked oocytes, which were transferred to OM and placed in a 38.5°C, 5% CO2 incubator with saturated humidity to recover for 30 min before preparing for intracytoplasmic sperm injection.

[0083] Sperm was processed by BO flotation method and Porcoll density gradient centrifugation method respectively:

[0084] (1) Treatment of X-sexed sperm by BO flotation method

[0085] Prepare 4 mL of BO and 2 mL of HEPES wash solution and equilibrate in an incubator for at least 1 hour. Thaw X-sexed frozen semen in a 38°C waterbath. Transfer an appropriate amount of the resulting X-sexed semen to the bottom of a centrifuge tube containing 4 mL of BO. Tilt the tube at a 45°C angle and place it in a 38.5°C, 5% CO2 incubator to allow the sperm to float. After 30 minutes of sperm floating, remove the supernatant and centrifuge for 10 minutes (room temperature, 500 × g). After centrifugation, discard the supernatant and transfer the sperm pellet to 1 mL of HEPES wash solution, mix thoroughly by pipetting, and wash by centrifugation twice, each for 5 minutes (room temperature, 300 × g).

[0086] (2) Treatment of X-sexed sperm by porcoll density gradient centrifugation

[0087] 1 mL each of PureSperm 40 and PureSperm 80 was added to a 15 mL centrifuge tube, with PureSperm 80 placed at the bottom. PureSperm 40 was slowly pipetted along the tube wall onto the PureSperm 80 solution to form porcoll. Once completed, the centrifuge tube was transferred to an incubator for equilibration. Simultaneously, a 4 mL tube of HEPES wash solution was prepared for equilibration. All liquids were equilibrated in a 38.5°C, 5% CO2 incubator at saturated humidity for at least 1 h. After thawing the X-sexed frozen semen in a 38°C water bath, an appropriate amount of the resulting X-sexed semen was slowly pipetted along the tube wall onto the pre-equilibrated porcoll. The tube was then centrifuged for 10 min (room temperature, 500 × g). The supernatant was discarded, and the sperm pellet was transferred to 4 mL of HEPES wash solution, mixed thoroughly by pipetting, and washed once by centrifugation for 5 min (room temperature, 300 × g).

[0088] Sperm dilution: After centrifugation in HEPEs wash solution, remove the supernatant and keep a small amount of liquid to suspend the precipitate to obtain a sperm suspension; use a micropipette to draw 10μL of sperm suspension into 60μL IVF for dilution, which can then be used for intracytoplasmic sperm injection.

[0089] Intracytoplasmic sperm injection: Before performing sexed sperm injection on dairy cows, first prepare microdrops such as operating drops (using gamete buffer) and sperm drops (using PVP) on a 35mm plastic dish and cover with oil; assemble the fixing needle on the left micromanipulator arm and the injection needle on the right micromanipulator arm. The fixing needle is made of a BJ-40 fine glass tube with an inner diameter of less than 20μm and an outer diameter of 150-200μm. The injection needle is made of borosilicate glass with a diameter of 7-9μm. After assembly, adjust the angle and distance until the fixing needle and injection needle are visible in the center of the microscope field of view; transfer the prepared OPU donor oocyte from the OM to the operating drop, and add a small amount of diluted sperm (X-sexed sperm processed by BO flotation method or porcoll density gradient centrifugation method) to the sperm drop. Transfer the injection needle to the sperm drop and rinse for 1 minute. After rinsing, select an oocyte with a complete head and tail without deformity. For sperm without impurities adhering to them, use an injection needle to mechanically cut off the tail of the sperm. After ensuring that the tail cutting is completed, the cut sperm is sucked into the injection needle from the end of the tail and the cut sperm is kept at the front end of the injection needle. The cut sperm is transferred to the operating drop with the injection needle. The fixing needle is adjusted to adsorb and fix an OPU donor oocyte. The oocyte is rotated up and down and back and forth with the injection needle so that the first polar body is at the "6 o'clock" or "12 o'clock" direction. The injection needle is then adjusted to the same level as the oocyte and inserted at the "3 o'clock" direction of the oocyte. The injection needle is inserted into the oocyte 2 / 3 of the way and the injection needle is slowly and evenly aspirated back to the cytoplasm. When the cytoplasm in the injection needle is observed to suddenly increase in the aspiration speed, the cytoplasm aspiration operation is immediately stopped and the cytoplasm and the cut sperm in the injection needle are uniformly injected into the oocyte. After confirming that the injection is complete, the injection needle is withdrawn from the oocyte.

[0090] Activation: The ICSI eggs obtained after the completion of the cow sexed sperm injection were transferred to OM and placed in a 5% CO2 incubator at 38.5°C and saturated humidity to recover for 30 minutes; then transferred to SOF containing 5nM ionomycin for activation treatment for 5 minutes (specifically, the activation treatment was carried out in a 5% CO2 incubator at 38.5°C and saturated humidity), cleaned with SOF, and cultured in IVF for 5 hours (specifically, cultured in a 5% CO2 incubator at 38.5°C and saturated humidity), then transferred to SOF containing 5nM ionomycin for activation treatment for 5 minutes (specifically, the activation treatment was carried out in a 5% CO2 incubator at 38.5°C and saturated humidity), cleaned with SOF, and cultured in IVF for 5 hours (specifically, cultured in a 5% CO2 incubator at 38.5°C and saturated humidity).

[0091] Embryo culture: After activation, rinse with SOF and remove non-viable ICSI eggs. Then transfer them to the IVC and culture them in a three-gas incubator at 38.5°C, 5% CO2, and 7% O2. The dipole rate is the fertilization rate. After 2 days of culture, the cleavage status is observed and recorded. After 6-7 days of culture, the blastocyst formation status is observed and recorded.

[0092] 3.2 Analysis of test results

[0093] All experimental data were statistically analyzed using SPSS, and the significance of the differences was determined. As shown in Table 1, the BO flotation method significantly increased the blastocyst rate of embryos produced after sexed sperm treatment in ICSI procedures compared to the porcoll density gradient centrifugation method.

[0094] Table 1. Effects of different sexed sperm processing methods on ICSI embryo development

[0095] Total number of injections Number of viable eggs Fertilization rate (%) Cleavage rate (%) Blastocyst rate (%) BO floatation method 80 63 <![CDATA[87.04±17.18 a ]]> <![CDATA[57.43±5.14 b ]]> <![CDATA[20.88±3.69 a ]]> Porcoll density gradient centrifugation 115 86 <![CDATA[85.35±12.37 a ]]> <![CDATA[73.96±22.45 a ]]> <![CDATA[14.10±4.32 b ]]>

[0096] Note: This experiment was repeated 4 times. In the same column, the same letters indicate no significant difference (P>0.05), and different letters indicate significant difference (P<0.05).

[0097] (IV) Effects of different sperm diluents on ICSI embryo development

[0098] 4.1 Comparative test of different sperm diluents (Comparative Example 2)

[0099] Collection of OPU oocytes: The follicular fluid obtained from live egg collection is transferred to the egg collection cup with a syringe for filtration. The side net is repeatedly rinsed with saline until the follicular fluid in the egg collection cup is clear and the eggs can be collected. The COCs are picked out with a handheld egg collection needle under a stereo microscope.

[0100] OPU oocyte maturation culture: The picked COCs were repeatedly washed with HEPEs solution until there were no impurities and granulosa cells around the COCs. After cleaning, they were transferred to OM and rinsed once. The dead COCs (loose cytoplasm, overmature) were removed and all were transferred to OM for maturation culture in a 5% CO2 incubator at 38.5℃ and saturated humidity.

[0101] Preparation of OPU donor oocytes: After 22 h of maturation culture, the cultured COCs were repeatedly blown with 0.5% hyaluronidase to detach the cumulus cells and obtain naked oocytes, which were transferred to OM and placed in a 38.5°C, 5% CO2 incubator with saturated humidity to recover for 30 min before preparing for intracytoplasmic sperm injection.

[0102] Sperm processing (X-sexed sperm by BO flotation): Prepare 4 mL of BO and 2 mL of HEPES wash solution and equilibrate in an incubator for at least 1 hour. Thaw X-sexed frozen semen in a 38°C water bath. Transfer an appropriate amount of the resulting X-sexed semen to the bottom of a centrifuge tube containing 4 mL of BO. Tilt the tube at a 45° angle and place it in a 38.5°C, 5% CO2 incubator to allow the sperm to float. After the sperm has floated for 30 minutes, remove the supernatant and centrifuge for 10 minutes (room temperature, 500 × g). After centrifugation, discard the supernatant, transfer the sperm pellet to 1 mL of HEPES wash solution, mix thoroughly by pipetting, and wash by centrifugation twice, each for 5 minutes (room temperature, 300 × g).

[0103] Sperm dilution: After centrifugation in HEPEs wash solution, remove the supernatant and retain a small amount of liquid to suspend the precipitate to obtain a sperm suspension; use a micropipette to draw 10μL of sperm suspension into 60μL IVF or TALP for dilution, which can then be used for intracytoplasmic sperm injection.

[0104] Intracytoplasmic sperm injection: Before performing sexed sperm injection on dairy cows, first prepare microdrops such as operating drops (using gamete buffer) and sperm drops (using PVP) on a 35mm plastic dish and cover with oil; assemble the fixing needle on the left micromanipulator arm, and assemble the injection needle on the right micromanipulator arm. The fixing needle is made of a BJ-40 fine glass tube with an inner diameter of less than 20μm and an outer diameter of 150-200μm. The injection needle is made of borosilicate glass with a diameter of 7-9μm. After assembly, adjust the angle and distance until the fixing needle and injection needle are visible in the center of the microscope field of view; transfer the prepared OPU donor oocyte from OM to the operating drop, and add a small amount of sperm diluted with different diluents (IVF or TALP) to the sperm drop. Transfer the injection needle to the sperm drop and rinse for 1 minute. After rinsing, select a sperm with a complete head and tail without deformities and no impurities attached. Use an injection needle to mechanically cut the sperm tail. After the tail is cut, draw the cut sperm into the injection needle from the end of the tail and keep the cut sperm at the front end of the injection needle. Transfer the cut sperm to the operating drop with the injection needle. Adjust the fixing needle to adsorb and fix an OPU donor oocyte. Use the injection needle to rotate the oocyte up and down and back and forth so that the first polar body is at the "6 o'clock" or "12 o'clock" direction. Then adjust the injection needle to the same level as the oocyte, insert the needle at the "3 o'clock" direction of the oocyte, and insert the injection needle into the oocyte 2 / 3 of the way. Slowly and evenly aspirate the cytoplasm with the injection needle. When the cytoplasm in the injection needle suddenly accelerates, stop aspirating the cytoplasm immediately and inject the cytoplasm and the cut sperm in the injection needle into the oocyte at a uniform speed. After confirming that the injection is complete, withdraw the injection needle from the oocyte.

[0105] Activation: The ICSI eggs obtained after the completion of the cow sexed sperm injection were transferred to OM and placed in a 5% CO2 incubator at 38.5°C and saturated humidity to recover for 30 minutes; then transferred to SOF containing 5nM ionomycin for activation treatment for 5 minutes (specifically, the activation treatment was carried out in a 5% CO2 incubator at 38.5°C and saturated humidity), cleaned with SOF, and cultured in IVF for 5 hours (specifically, cultured in a 5% CO2 incubator at 38.5°C and saturated humidity), then transferred to SOF containing 5nM ionomycin for activation treatment for 5 minutes (specifically, the activation treatment was carried out in a 5% CO2 incubator at 38.5°C and saturated humidity), cleaned with SOF, and cultured in IVF for 5 hours (specifically, cultured in a 5% CO2 incubator at 38.5°C and saturated humidity).

[0106] Embryo culture: After activation, rinse with SOF and remove non-viable ICSI eggs. Then transfer them to the IVC and culture them in a three-gas incubator at 38.5°C, 5% CO2, and 7% O2. The dipole rate is the fertilization rate. After 2 days of culture, the cleavage status is observed and recorded. After 6-7 days of culture, the blastocyst formation status is observed and recorded.

[0107] 4.2 Analysis of test results

[0108] All experimental data were statistically analyzed using SPSS, and the significance of the differences was determined. As shown in Table 2, using IVF as a sperm dilution fluid significantly increased the blastocyst rate of embryos produced after sexed sperm dilution in ICSI procedures compared to TALP.

[0109] Table 2. Effects of different sperm diluents on the development of OPU sexed embryos in dairy cows

[0110] Total number of injections Number of viable eggs Fertilization rate (%) Cleavage rate (%) Blastocyst rate (%) IVF 79 74 <![CDATA[89.47±9.08 a ]]> <![CDATA[71.80±18.41 a ]]> <![CDATA[21.53±6.57 a ]]> TALP 105 98 <![CDATA[92.52±5.99 a ]]> <![CDATA[88.35±8.51 a ]]> <![CDATA[6.44±3.71 b ]]>

[0111] Note: This experiment was repeated 4 times. In the same column, the same letters indicate no significant difference (P>0.05), and different letters indicate significant difference (P<0.05).

[0112] (V) Effects of different activation schemes on ICSI embryo development

[0113] 5.1 Comparative test of different activation schemes (Comparative Example 3)

[0114] Collection of OPU oocytes: The follicular fluid obtained from live egg collection is transferred to the egg collection cup with a syringe for filtration. The side net is repeatedly rinsed with saline until the follicular fluid in the egg collection cup is clear and the eggs can be collected. The COCs are picked out with a handheld egg collection needle under a stereo microscope.

[0115] OPU oocyte maturation culture: The picked COCs were repeatedly washed with HEPEs solution until there were no impurities and granulosa cells around the COCs. After cleaning, they were transferred to OM and rinsed once. The dead COCs (loose cytoplasm, overmature) were removed and all were transferred to OM for maturation culture in a 5% CO2 incubator at 38.5℃ and saturated humidity.

[0116] Preparation of OPU donor oocytes: After 22 h of maturation culture, the cultured COCs were repeatedly blown with 0.5% hyaluronidase to detach the cumulus cells and obtain naked oocytes, which were transferred to OM and placed in a 38.5°C, 5% CO2 incubator with saturated humidity to recover for 30 min before preparing for intracytoplasmic sperm injection.

[0117] Sperm processing (X-sexed sperm by BO flotation): Prepare 4 mL of BO and 2 mL of HEPES wash solution and equilibrate in an incubator for at least 1 hour. Thaw X-sexed frozen semen in a 38°C water bath. Transfer an appropriate amount of the resulting X-sexed semen to the bottom of a centrifuge tube containing 4 mL of BO. Tilt the tube at a 45° angle and place it in a 38.5°C, 5% CO2 incubator to allow the sperm to float. After the sperm floats for 30 minutes, remove the supernatant and centrifuge for 10 minutes (room temperature, 500 × g). After centrifugation, discard the supernatant, transfer the sperm pellet to 1 mL of HEPES wash solution, mix thoroughly by pipetting, and wash by centrifugation twice, each for 5 minutes (room temperature, 300 × g).

[0118] Sperm dilution: After centrifugation in HEPEs wash solution, remove the supernatant and retain a small amount of liquid to suspend the precipitate to obtain a sperm suspension; use a micropipette to draw 10μL of sperm suspension into 60μL IVF for dilution, which can then be used for intracytoplasmic sperm injection.

[0119] Intracytoplasmic sperm injection: Before performing sexed sperm injection on dairy cows, first prepare microdrops such as operating drops (using gamete buffer) and sperm drops (using PVP) on a 35mm plastic dish and cover them with oil; assemble the fixing needle on the left micromanipulator arm, and assemble the injection needle on the right micromanipulator arm. The fixing needle is made of a BJ-40 fine glass tube with an inner diameter of less than 20μm and an outer diameter of 150-200μm. The injection needle is made of borosilicate glass with a diameter of 7-9μm. After assembly, adjust the angle and distance until the fixing needle and injection needle are visible in the center of the microscope field of view; transfer the prepared OPU donor oocyte from the OM to the operating drop, and add a small amount of diluted sperm to the sperm drop. Transfer the injection needle to the sperm drop and rinse for 1 minute. After rinsing, select a sperm with a complete head and tail without deformities and no impurities attached, and use the injection needle to inject the sperm. The tail is mechanically cut off. After the tail is cut off, the sperm with the cut tail is sucked into the injection needle from the end of the tail, and the sperm with the cut tail is kept at the front end of the injection needle; the sperm with the cut tail is transferred to the operating drop with the injection needle, and the fixing needle is adjusted to adsorb and fix an OPU donor oocyte. The oocyte is rotated up and down and back and forth with the injection needle so that the first polar body is at the "6 o'clock" or "12 o'clock" direction; then the injection needle is adjusted to the same horizontal plane as the oocyte, and the needle is inserted at the "3 o'clock" direction of the oocyte. The injection needle is inserted into the 2 / 3 of the oocyte, and the injection needle is slowly and evenly aspirated back the cytoplasm; when the cytoplasm in the injection needle is observed to suddenly accelerate, the cytoplasm aspiration operation is immediately stopped, and the cytoplasm in the injection needle and the sperm with the cut tail are injected into the oocyte at a uniform speed. After observation and confirmation that the injection is complete, the injection needle is withdrawn from the oocyte.

[0120] After completing the ICSI procedure, the eggs were transferred to OM and placed in a 38.5°C, 5% CO2 incubator with saturated humidity for 30 minutes before activation. The specific steps for the different activation protocols are as follows:

[0121] (1) Electrical activation scheme

[0122] 500 μL of electroactivation solution and several 100 μL SOF droplets prepared in 60 mm plastic dishes were pre-equilibrated in a 38.5°C, 5% CO2 incubator at saturated humidity for at least 20 minutes. After ICSI eggs recovered, they were placed in a dish containing electroactivation solution and washed for 1 minute. Then, the ICSI eggs were transferred to the groove of the electroactivation plate, which had been rinsed with electroactivation solution in advance, and filled with electroactivation solution. After one electroactivation (1.6 kV / cm DC pulse, activation for 20 μs), the ICSI eggs were transferred to the SOF droplets and washed twice. After washing, they were cultured in an IVF incubator for 5 hours (specifically, cultured in a 38.5°C, 5% CO2 incubator at saturated humidity).

[0123] (2) ION*2 solution

[0124] After recovery, the ICSI eggs were transferred to SOF containing 5nM ionomycin for activation treatment for 5 minutes (specifically, the activation treatment was carried out in a 5% CO2 incubator at 38.5°C and saturated humidity). After being cleaned with SOF, they were cultured in IVF for 5 hours (specifically, they were cultured in a 5% CO2 incubator at 38.5°C and saturated humidity). Then, they were transferred to SOF containing 5nM ionomycin for activation treatment for 5 minutes (specifically, the activation treatment was carried out in a 5% CO2 incubator at 38.5°C and saturated humidity). After being cleaned with SOF, they were cultured in IVF for 5 hours (specifically, they were cultured in a 5% CO2 incubator at 38.5°C and saturated humidity).

[0125] (3)ION*2+6D regimen

[0126] After recovery, the ICSI eggs were transferred to SOF containing 5nM ionomycin for activation treatment for 5 minutes (specifically, the activation treatment was carried out in a 5% CO2 incubator at 38.5°C and saturated humidity). After being cleaned with SOF, they were cultured in IVF for 30 minutes (specifically, they were cultured in a 5% CO2 incubator at 38.5°C and saturated humidity). Then, they were transferred to SOF containing 5nM ionomycin for activation treatment for 5 minutes (specifically, they were cultured in a 5% CO2 incubator at 38.5°C and saturated humidity). After being cleaned with SOF, they were transferred to IVF for culture for 2 hours (specifically, they were cultured in a 5% CO2 incubator at 38.5°C and saturated humidity). Then, they were transferred to SOF containing 2nM 6-DAMP for activation treatment for 3 hours (specifically, they were activated in a 5% CO2 incubator at 38.5°C and saturated humidity).

[0127] Embryo culture: After activation, rinse with SOF and remove non-viable ICSI eggs. Then transfer them to the IVC and culture them in a three-gas incubator at 38.5°C, 5% CO2, and 7% O2. The dipole rate is the fertilization rate. After 2 days of culture, the cleavage status is observed and recorded. After 6-7 days of culture, the blastocyst formation status is observed and recorded.

[0128] 5.2 Analysis of test results

[0129] All experimental data were statistically analyzed using SPSS, and the significance of differences was determined. As shown in Table 3, compared with the electrical activation protocol, the fertilization rate, cleavage rate, and blastocyst rate after activation in ICSI procedures using the "ION*2" or "ION*2+6D" protocols were significantly (or extremely significantly) higher. The "ION*2+6D" protocol was more efficient in producing blastocysts than the "ION*2" protocol, and the difference was extremely significant.

[0130] Table 3. Effects of different activation protocols on ICSI embryo development

[0131] Total number of injections Number of viable eggs Fertilization rate (%) Cleavage rate (%) Blastocyst rate (%) Electrical activation 67 67 <![CDATA[63.88±9.07 b ]]> <![CDATA[47.41±16.59 c ]]> <![CDATA[4.42±6.28 C ]]> ION*2 69 67 <![CDATA[90.62±11.97 a ]]> <![CDATA[77.43±18.44 a ]]> <![CDATA[18.588±2.62 B ]]> ION*2+6D 68 48 <![CDATA[85.89±9.87 a ]]> <![CDATA[73.47±8.52 b ]]> <![CDATA[47.27±13.87 A ]]>

[0132] Note: This experiment was repeated 4 times. In the same column, the same superscript letters indicate no significant difference (P>0.05), different lowercase superscript letters indicate significant difference (P<0.05), and different uppercase superscript letters indicate extremely significant difference (P<0.01).

[0133] (VI) Effects of different activation schemes on ICSI pronucleus formation

[0134] In the comparative study of different activation protocols, the protocols "ION*2" and "ION*2+6D," which showed the highest efficiency in producing blastocysts after activation, were sampled 16 hours after intracytoplasmic sperm injection for pronuclear staining. The zona pellucida was digested with 0.2% pronase, fixed with fixative for 4 hours, and then rinsed with PBS-PVA. The cells were then fluorescently stained with 10 μg / mL H33342 in the dark. After 10 minutes, the stain was rinsed with PBS-PVA. Pronuclear formation was observed under a fluorescence microscope and grouped by pronuclear type. All data were statistically analyzed using SPSS, and the significance of differences was determined.

[0135] As shown in Table 4, after activation using the "ION*2+6D" scheme, more male and female pronuclei (referring to "1MPN+1FPN") can be formed compared to the "ION*2" scheme, thereby significantly improving the blastocyst rate of embryos obtained under the "ION*2+6D" scheme and facilitating a higher pregnancy rate after embryo transplantation.

[0136] Table 4. Prokaryotic formation (distribution of different prokaryotic types) in different activation schemes 16 hours after ICSI

[0137]

[0138] Note: MPN stands for male pronucleus, FPN stands for female pronucleus, PDSH stands for disaggregated sperm head (indicating that the sperm has disaggregated but has not yet developed into the male pronucleus), CSH stands for non-disaggregated sperm head, 1PN stands for one pronucleus (e.g., female pronucleus), and 3PN stands for three pronuclei (e.g., parthenogenetic activation + male pronucleus). In the same column, the same superscript letters indicate no significant difference (P>0.05), different lowercase superscript letters indicate significant difference (P<0.05), and different uppercase superscript letters indicate extremely significant difference (P<0.01).

[0139] Table 5. Pronuclear formation (sperm disaggregation) of different activation schemes 16 hours after ICSI

[0140] Total number of staining Normal fertilization (%) Sperm disaggregation (%) Inactive (%) ION*2 47 <![CDATA[23.44±4.20 B ]]> <![CDATA[35.89±10.73 B ]]> <![CDATA[28.50±27.72 a ]]> ION*2+6D 49 <![CDATA[40.89±10.20 A ]]> <![CDATA[61.17±12.13 A ]]> <![CDATA[0.00±0.00 b ]]>

[0141] Note: Normal fertilization is 1MPN+1FPN, sperm depolymerization is 1MPN+1FPN, 1FPN+PDSH and 3PN, and unactivated is condensed nucleus;

[0142] In the same column, different lowercase letters in superscripts indicate significant differences (P<0.05), and different uppercase letters in superscripts indicate extremely significant differences (P<0.01).

[0143] Table 6. Pronuclear formation in different activation schemes 16 hours after ICSI (abnormal cases such as no activation)

[0144] ION*2 ION*2+6D 1MPN+1FPN(%) <![CDATA[23.44±4.20 a ]]> <![CDATA[40.89±10.20 A ]]> 1PN(%) <![CDATA[10.50±7.16 b ]]> <![CDATA[11.78±11.48 B ]]> 3PN (%) <![CDATA[0.00±0.00 c ]]> <![CDATA[4.00±8.94 C ]]> Condensation nucleus (%) <![CDATA[28.50±27.72 a ]]> <![CDATA[0.00±0.00 C ]]>

[0145] Note: In the same column, the same superscript letters indicate no significant difference (P>0.05), and different lowercase superscript letters indicate significant difference (P<0.05).

[0146] The data with different capital letters indicate extremely significant differences (P<0.01).

[0147] Further analysis showed that activation using the "ION*2+6D" protocol can promote sperm disaggregation and improve the activation efficiency of ICSI eggs (as shown in Tables 5 and 6), thereby forming more normal fertilized male and female pronuclei, promoting embryonic development, and improving developmental quality.

Claims

1. A method for producing OPU sexed embryos, characterized in that: The following steps are involved: Sexed sperm and matured OPU oocytes were microinjected into reconstructed bodies, which were activated with ionomycin and 6-DMAP and then cultured for embryos to obtain sexed bovine OPU embryos. The activation specifically includes the following steps: after the reconstructed body is recovered, it is activated with 4.9~5.1 nM ionomycin for 3~5 minutes, washed clean, and cultured in fertilization culture medium for 25~30 minutes; then it is activated with 4.9~5.1 nM ionomycin for 3~5 minutes, washed clean, and cultured in fertilization culture medium for 1.5~2.0 hours; then it is activated with 1.9~2.1 nM 6-DAMP for 3~4 hours.

2. The method for producing OPU sexed embryos according to claim 1, wherein: The preparation of sex-controlled sperm comprises the following steps: thawing sex-controlled frozen sperm, separating sex-controlled sperm from the obtained sex-controlled semen by using a flotation method, and diluting the separated sex-controlled sperm with a fertilization culture medium.

3. The method for producing OPU sexed embryos according to claim 2, wherein: The fertilization medium is IVF.

4. The method for producing OPU sexed embryos according to claim 2, wherein: The flotation method specifically includes the following steps: taking 200-400 μL of sexed semen and transferring it to the bottom of 3-4 mL of flotation solution, centrifuging after the sperm floats, and washing the sperm sediment obtained by centrifugation.

5. The method for producing OPU sexed embryos according to claim 1, wherein: The microinjection specifically adopts ICSI.

6. The method for producing OPU sexed embryos according to claim 1, wherein: The recovery specifically includes the following steps: transferring the OPU oocyte injected with sexed sperm into an oocyte maturation culture medium and incubating for 20 to 30 minutes.

7. The method for producing OPU sexed embryos according to claim 1, wherein: The embryo culture conditions include: culturing with embryo culture medium for 6 to 7 days.

8. A livestock breeding method, characterized in that: The following steps are involved: The method for producing OPU sexed embryos according to any one of claims 1 to 7 is used to obtain bovine OPU sexed embryos, and the OPU sexed embryos are transplanted into recipient bovines.

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