Method for increasing normal fertilization rate of in-vitro ICSI embryo of dairy cow and reducing abortion rate

Abstract

The invention discloses a method for improving the normal fertilization rate of an in-vitro ICSI embryo of a dairy cow and reducing the abortion rate, which comprises the following steps: preparing an ICSI egg from X-sex controlled sperm and an oocyte which is cultured by a mature oocyte and is used as a donor by adopting a microinjection mode, the ICSI eggs are incubated in an oocyte maturation culture solution (OM) for 3-5 h and then transferred to ETH to be activated for 4-6 min so as to promote normal fertilization of the ICSI eggs, then embryo culture is conducted, and then sexual control embryos are obtained; the ETH refers to an oocyte maturation culture solution containing 6 to 8 percent of ethanol. The normal fertilization rate of the ICSI embryos produced by the method is remarkably improved, and the early abortion rate is remarkably reduced in inspection after transplantation.

Description

Technical Field

[0001] This invention belongs to the field of livestock breeding technology, specifically relating to a method for improving the normal fertilization rate of in vitro ICSI embryos in dairy cows and reducing the abortion rate. Background Technology

[0002] The activation stage of ICSI technology in dairy cows faces significant challenges. Studies have confirmed that a single activator is insufficient to fully activate ICSI oocytes, while the combined use of multiple activators, although improving activation efficiency, can lead to abnormal pronucleus formation, thereby affecting normal fertilization efficiency and embryo development quality, resulting in reduced blastocyst rates and post-implantation pregnancy rates. Overcoming these technical bottlenecks is crucial to improving the effectiveness of dairy cow ICSI technology.

[0003] The literature "Production of female bovine embryos with sex-sorted sperm using intracytoplasmic sperm injection: Efficiency and in vitro developmental competence" describes ICSI (without Piezo) using sex-sorted sperm. The activation method after ICSI was 2 mM ION activation for 5 min followed by 5 mM 6D incubation for 4 h. The resulting sex-sorted embryos showed a pronucleus formation rate of 54%, a cleavage rate of 79.1%, and a blastocyst rate of 24.7%, demonstrating that sex-sorted bovine sperm can successfully produce bovine embryos of the desired sex using ICSI technology. Furthermore, the developmental ability of pre-selected female embryos from sperm sex-sorted ICSI was significantly higher than that of IVF embryos from sperm sex-sorted IVF. However, the resulting embryos were not transferred. This patent aims to explore an optimal ICSI activation protocol that is independent of Piezo, has a high normal fertilization rate, and high transfer efficiency.

[0004] Chinese patent application CN118995579A describes a method for treating cryopreserved sex-controlled sperm using the Brackett and Oliphant (BO) flotation method. The flotated sperm are centrifuged and diluted with in vitro fertilization (IVF) fluid. The treated sex-controlled sperm are then injected into oocytes from live oocyte retrieval units (OPU) donors. The oocytes are first activated twice with 5 nM iomycin, then once with 2 nM 6-DMAP, followed by embryo culture. This invention significantly increases the blastocyst rate of embryos produced after ICSI oocyte activation, and can be used for the stable production of high-quality dairy cow OPU sex-controlled embryos.

[0005] Intracytoplasmic sperm injection (ICSI), a precision fertilization technique using micromanipulation devices, directly injects a single sperm into the cytoplasm of an oocyte via mechanical injection. In dairy cattle breeding, this technology has become a key means of improving breeds and cultivating high-quality dairy cows without relying on gamete quality. Currently, the success rate of dairy cow embryos developing to the blastocyst stage under Piezo-ICSI manipulation can reach approximately 30%, effectively improving the production efficiency of high-quality breeding stock. However, when reducing reliance on Piezo equipment to control costs, the oocyte microinjection damage rate increases from 8% to 12%, and the blastocyst rate also decreases accordingly, further exacerbating the complexity of embryo development after activation. How to maintain the advantages of low-cost operation while constructing an efficient and stable activation system has become the core challenge in advancing the application of ICSI technology. Therefore, developing methods to improve the normal fertilization rate of dairy cow in vitro ICSI embryos and reduce the abortion rate is of great significance. Summary of the Invention

[0006] For the reasons stated above, this invention proposes a method to improve the normal fertilization rate of in vitro ICSI embryos in dairy cows and reduce the abortion rate. Specifically, to achieve the objectives of this invention, the following technical solution is proposed:

[0007] This invention relates to a method for improving the normal fertilization rate of in vitro ICSI embryos in dairy cows and reducing the abortion rate, comprising the following steps:

[0008] Intracytoplasmic sperm injection (ICSI) oocytes were prepared by microinjection using X-sex-controlled sperm and mature oocytes as donor oocytes. The ICSI oocytes were incubated in oocyte maturation medium (OM) for 3-5 hours and then transferred to ETH for 4-6 minutes to promote normal fertilization. Embryo culture was then performed to obtain sex-controlled embryos. ETH refers to oocyte maturation medium containing 6-8 v / v% ethanol.

[0009] Preferably, the activation specifically includes the following steps: wherein each activation treatment and culture is carried out at 38.4–38.6°C, saturated humidity, and 4.85%–5.15% CO2.

[0010] Preferably, the oocyte maturation culture medium is based on Hepes-free M199 medium, with the addition of 8-12% (v / v) fetal bovine serum, 0.08-0.12% (v / v) insulin-transferrin-selenium supplement (ITS-G), 0.07-0.08 IU / mL human gonadotropin (HMG), 0.8-1.2 μg / mL 17β-estradiol, 8-12 ng / mL epidermal growth factor (EGF), and 8-12 ng / mL basic fibroblast growth factor (bFGF).

[0011] Preferably, the activation specifically includes the following steps: the ICSI oocytes are transferred to maturation culture medium (OM) and incubated for 3.5 to 4.5 hours, then transferred to OM containing 7% ethanol for activation treatment for 4 to 6 minutes, and then washed clean with SOF; wherein each activation treatment and culture is carried out at 38.4 to 38.6°C, saturated humidity and 4.85% to 5.15% CO2.

[0012] Preferably, the incubation of ICSI oocytes specifically includes the following steps: transferring donor oocytes injected with sex-controlled sperm via ICSI into oocyte maturation culture medium (OM) and incubating for 3.5 to 4.5 hours; wherein the incubation is carried out at 38.4 to 38.6°C, saturated humidity, and 4.85% to 5.15% CO2.

[0013] Preferably, the activation process takes 5 minutes.

[0014] Preferably, the activation process does not include other activation processes.

[0015] Preferably, the embryo culture conditions include: culturing in embryo culture medium (e.g., IVC) at 38.4–38.6°C, 4.85%–5.15% CO2, and 6.9%–7.0% O2 for 6–7 days.

[0016] Another aspect of the present invention relates to a method for livestock breeding, comprising the following steps:

[0017] Using the above-described method for producing sex-controlled embryos, sex-controlled embryos are obtained from donor livestock (e.g., dairy cows), and the sex-controlled embryos are transferred into recipient livestock (e.g., dairy cows). Afterward, the recipient livestock are examined and assessed for pregnancy (e.g., gestation) and early abortion.

[0018] The beneficial effects of this invention are as follows: This invention optimizes an activation protocol in which treated sex-controlled sperm are injected into oocytes, incubated in an OM (Oocyte Oocyte), and activated once with ETH (Electro-Effective Toxic Agent) for 5 minutes. This activation protocol significantly improves the normal fertilization rate of ICSI embryos and significantly reduces the early miscarriage rate in post-implantation examinations. Attached Figure Description

[0019] Figure 1 A schematic diagram of the ICSI oocyte construction process; where: A is the injection needle mechanically cutting off the tail of the sperm, and a single sperm is drawn into the injection needle from the tail end and held at the tip of the injection needle; B is the fixation needle fixing the donor oocyte before injection, and positioning the first polar body at the "6 o'clock" position; C is the injection needle aspirating cytoplasm; D is the injection needle withdrawing after the single sperm and the aspirated cytoplasm are injected into the donor oocyte; 1-first polar body, 2-sperm.

[0020] Figure 2 Cleavage diagram of eggs 48 hours after activation with the ICSI egg-using protocol OM+ETH (5 min).

[0021] Figure 3 Image of a blastocyst 168 hours after activation with the ICSI egg using the OM+ETH (5 min) protocol.

[0022] Figure 4 The pronucleus formation was observed 18 hours after sperm injection into donor oocytes for activation using the OM+ETH (5 min) protocol (for normal fertilization, 1 FPN+1 MPN; the dashed line in the figure represents the approximate oocyte boundary). Detailed Implementation

[0023] To further understand the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0024] Unless otherwise specified, all reagents involved in the embodiments of this invention are commercially available products and can be purchased through commercial channels.

[0025] Example 1:

[0026] (I) Sources and preparation of materials and reagents

[0027] 1.1 Materials

[0028] (1) Oocytes were collected from ovaries at a slaughterhouse in Wuzhong City, Ningxia Hui Autonomous Region, between September 2024 and May 2025.

[0029] (2) X-sex-controlled frozen sperm was purchased from the United States in November 2022.

[0030] (3) The oocytes and X-sex controlled frozen sperm mentioned above were all from Holstein cows.

[0031] 1.2 Reagents

[0032] (1) BJ-40 thin glass tubes were purchased from Beijing Zhengtian Yike Trade Co., Ltd., and borosilicate glass was purchased from Sutter Instrument Co., Ltd. (item number: B100-75-15);

[0033] (2) The fertilization culture medium IVF, embryo culture medium IVC, and embryo transfer medium TRANSFER were purchased from "IVFBIOSCIENCE", the gamete buffer was purchased from COOK MEDICAL (catalog number: K-SIGB-20), and the sperm immobilizer PVP was purchased from Vitrolife.

[0034] (3) 6-DMAP (6-dimethylaminopurine) was purchased from SIGMA (catalog number: D2629-250MG), Ionomycin was purchased from SIGMA (catalog number: 10634-1MG), and anhydrous ethanol (ETH) was purchased from Sangon Biotech (catalog number: A500737-0500).

[0035] (4) Fetal bovine serum (FBS) is a product of Gibco, essential amino acids are products of Thermo Fisher (product number: 1831512), non-essential amino acids are products of Thermo Fisher (product number: 1958909), and insulin-transferrin-selenium additive ITS-G is a product of Thermo Fisher (product number: 1865342).

[0036] (5) The fixative was purchased from Beyotime (item number: P0098-500mL);

[0037] (6) All other products not specified are SIGMA products;

[0038] (7) Oocyte maturation culture medium OM (self-prepared): Based on Hepes-free M199 as the basic culture medium, and additionally 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;

[0039] (8) HEPEs washing 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), and bring the volume to 200 mL with deionized water;

[0040] (9) BO (self-prepared): SOF solution was used as the basic 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.

[0041] (10) SOF (i.e., the above SOF solution, self-prepared): 26.168mg CaCl2·2H2O, 9.999mg C6H5Na3O7·2H2O, 2.923mg glutamine, 37.218mg MgSO4·7H2O, 49.904mg inositol, 53.378mg KCl, 16.195mg KH2PO4, 4.402mg sodium pyruvate, 200.025mg NaHCO3, 629.399mg NaCl and 0.0757mg sodium lactate (solid reagent), diluted to 100mL with deionized water;

[0042] (11) ETH (self-prepared): 7% anhydrous ethanol + oocyte maturation culture medium OM;

[0043] (12) PBS-PVA (self-prepared): 1.6012g NaCl, 0.0402g KCl, 0.284g Na2HPO4, 0.0544g KH2PO4 and 5.00mg PVA, diluted to 200mL with deionized water.

[0044] (II) Establish a technology for the stable production of high-quality sex-controlled dairy cow embryos.

[0045] 2.1 Collection of oocytes

[0046] Ovaries collected from the slaughterhouse were transported to the laboratory at a constant temperature. Excess connective tissue around the ovaries was removed, and the ovaries were cleaned with 75% alcohol and then repeatedly rinsed with physiological saline. Follicular fluid was extracted using a syringe, and cumulus-oocyte complexes (COCs) were picked out from the follicular fluid under a stereomicroscope using a hand-held ovum-collecting needle.

[0047] 2.2 Oocyte maturation culture

[0048] The collected COCs were repeatedly washed with HEPEs washing solution until there were no impurities or granular cells around them. After cleaning, they were transferred to OM for rinsing once. COCs that were dead (loose cytoplasm, over-mature) were removed and all were transferred to OM for maturation culture in a 38.5℃, 5% CO2 incubator with saturated humidity.

[0049] 2.3 ICSI activation method for producing sex-controlled embryos in dairy cows

[0050] Processing of S1 dairy cow oocytes and sperm

[0051] Preparation of donor oocytes: After 22 hours of maturation culture, the cultured COCs were repeatedly pipetted with 0.5% hyaluronidase to detach the cumulus cells and obtain naked oocytes (the zona pellucida was not removed after hyaluronidase treatment). The naked oocytes were transferred to OM and placed in a 38.5℃, 5% CO2 incubator with saturated humidity for 30 minutes to recover, and then prepared for intracytoplasmic sperm injection.

[0052] X-sex controlled sperm were treated by BO flotation: 4 mL of BO and 2 mL of HEPEs washing solution were prepared and equilibrated in an incubator for at least 1 hour; after thawing the X-sex controlled frozen sperm in a 38°C water bath, an appropriate amount of the obtained X-sex controlled sperm solution was transferred to the bottom of a centrifuge tube containing 4 mL of BO. The centrifuge tube was tilted at 45° and placed in a 5% CO2 incubator at 38.5°C with saturated humidity for sperm flotation. After the sperm had floated for 30 minutes, the supernatant was aspirated and centrifuged for 10 minutes (room temperature, centrifugation speed of 500×g); after centrifugation, the supernatant was discarded, and the sperm precipitate was transferred to 1 mL of HEPEs washing solution, mixed by pipetting, and centrifuged twice, each time for 5 minutes (room temperature, centrifugation speed of 300×g).

[0053] S2 sperm dilution

[0054] After centrifugation in HEPEs washing solution, the supernatant was removed, and a small amount of liquid was left to suspend the precipitate, resulting in a sperm suspension. 10 μL of the sperm suspension was then diluted in 60 μL IVF solution using a micropipette and subsequently used for intracytoplasmic sperm injection (ICSI).

[0055] S3 intracytoplasmic sperm injection

[0056] Before performing sex-controlled sperm injection in dairy cows, microdroplets such as operation droplets (using gamete buffer) and sperm droplets (using PVP) are first prepared on a 35mm plastic petri dish and covered with oil. The fixation needle is assembled on the left micromanipulation arm, and the injection needle is assembled on the right micromanipulation arm. The inner diameter of the fixation needle is within 20μm and the outer diameter is between 150 and 200μm. The diameter of the injection needle is between 7 and 9μm. The fixation needle is made of BJ-40 fine glass tube, and the injection needle is made of borosilicate glass. After assembly, the angle and distance are adjusted so that the fixation needle and injection needle are visible in the center of the microscope field of view.

[0057] The prepared donor oocytes were transferred from the OM to the operating drop. Simultaneously, a small amount of diluted sperm was added to the sperm drop. The injection needle was then transferred to the sperm drop for 1 minute of rinsing. After rinsing, a sperm with a complete head and tail, free from abnormalities and adhering impurities was selected. The sperm tail was mechanically severed using the injection needle. After successful tail severance, the severed sperm (sperm 2) was aspirated into the injection needle from the tail tip, and held at the tip of the needle. The severed sperm (sperm 2) was then transferred to the operating drop along with the injection needle. The fixation needle was adjusted to hold one donor oocyte in place. The oocyte was rotated up and down and back and forth using the injection needle until the first polar body 1 was positioned at the "6 o'clock" position (e.g., ...). Figure 1 (As shown in A); then adjust the injection needle to the same level as the oocyte, insert the needle at the "3 o'clock" position of the oocyte, inserting the needle into the oocyte to 2 / 3 of its depth, and slowly and evenly aspirate the cytoplasm (as shown in A). Figure 1 (As shown in B); once the rate of cytoplasmic reabsorption from the injection needle suddenly increases, immediately stop the cytoplasmic reabsorption operation, and inject the cytoplasm and the severed sperm 2 from the injection needle into the oocyte at a uniform speed. After confirming the completion of the injection, withdraw the injection needle from the oocyte (as shown in B). Figure 1 (As shown in C); Perform the above procedure to complete the intracytoplasmic sperm injection (ICSI) of other donor oocytes (usually 50 oocytes are controlled within 1 hour).

[0058] S4 Activation

[0059] ICSI oocytes obtained after sex-controlled sperm injection in dairy cows were transferred to OM and incubated in a 38.5℃, 5% CO2 incubator with saturated humidity for 4 hours; then activated in 7% ETH for 5 minutes (specifically, the activation treatment was carried out in a 38.5℃, 5% CO2 incubator with saturated humidity).

[0060] S5 embryo culture

[0061] After activation, the eggs were rinsed with SOF to remove any non-viable ICSI oocytes, then transferred to an IVC and cultured in a three-gas incubator at 38.5℃, 5% CO2, and 7% O2. Cleavage occurred after 1–2 days of culture (e.g., cleavage occurred afterward). Figure 2 As shown), the blastocyst forms in 6-7 days (as shown). Figure 3 (As shown).

[0062] 2.4 Detection of sex-controlled embryos in dairy cows (cleavage rate, blastocyst rate) and detection of reproductive performance in dairy cows after embryo transfer (pregnancy rate, early abortion rate)

[0063] Fertilization rate (dipolar body rate) = Number of eggs that produced dipolar bodies after activation / Number of viable eggs × 100%

[0064] Cleavage rate = (Number of cleaved eggs / Number of surviving eggs) × 100%

[0065] Blastocyst rate = (Number of blastocysts / Number of surviving eggs) × 100%

[0066] Embryo transfer and pregnancy monitoring: Blastocysts cultured to 7 days are selected and placed in a transferry tube, then into a thin tube, which is then placed in a sterile centrifuge tube and finally transported to the cattle farm in a 38.5℃ constant temperature transport box. Recipient cows undergo epidural anesthesia at the 1st to 2nd caudal vertebral intervertebral space, and their vulva is cleaned and disinfected. The thin tube containing the blastocyst is placed into the transfer gun, then covered with a transfer hard cover, and finally with a sterile isolation cover to keep the tip of the transfer gun sterile. The blastocyst is transferred into the uterine horn on the side with the corpus luteum of the recipient cow, and a transfer record is made. Within 14 days after embryo transfer, the return to estrus is used as a preliminary indicator of pregnancy in recipient cows. Return to estrus after 14 days with obvious vaginal bleeding is considered an abortion. Final confirmation of pregnancy or abortion is based on ultrasound examinations at 35 days, 70 days, and 150 days, and a record of the pregnant cow is made. The pregnancy rate and early abortion rate can be calculated based on these records.

[0067] Pregnancy rate = (Number of pregnancies / Number of embryo transfers) × 100%

[0068] Early miscarriage rate = (Number of early miscarriages / Number of pregnancies) × 100%

[0069] In addition, the effects of activation and other procedures on ICSI oocytes were examined by pronuclear detection and calculation of indicators such as normal fertilization rate and sperm disaggregation rate.

[0070] "1MPN+1FPN" rate = (Number of eggs with only "1MPN+1FPN" / Number of viable eggs) × 100%

[0071] "1PN+PDSH" rate = (Number of eggs with only "1PN+PDSH" / Number of surviving eggs) × 100%

[0072] "1PN+CSH" rate = (Number of eggs with only "1PN+CSH" present) / (Number of surviving eggs) × 100%

[0073] "1PN" rate = (Number of eggs with only "1PN" fertilization) / (Number of viable eggs) × 100%

[0074] "3PN" rate = (Number of eggs with only "3PN" phenotype) / (Number of surviving eggs) × 100%

[0075] "2PN+CSH" rate = (Number of eggs with only "2PN+CSH" present) / (Number of surviving eggs) × 100%

[0076] Clotted nucleus rate = (Number of oocytes with lectin nuclei / Number of viable oocytes) × 100%

[0077] Normal fertilization rate (pronucleus rate) = "1MPN + 1FPN" / total number of eggs × 100%

[0078] Sperm depolymerization rate = ("1MPN + 1FPN" + "1PN + PDSH" + "3PN") / total number of eggs × 100%

[0079] Inactivation rate = (condensed nuclei / total number of eggs) × 100%

[0080] Parthenogenesis activation rate = "2PN + CSH" / total number of eggs × 100%

[0081] Pronucleus detection: 16 hours after intracytoplasmic sperm injection (ICSI), the zona pellucida was digested with 0.2% streptoprotein (SIGMA). The zona pellucida was fixed with fixative for 4 hours, then washed with PBS-PVA. Pronucleus formation was observed under a fluorescence microscope after staining with 10 μg / mL Hochest 33342 in the dark. After 10 minutes, the stain was washed off with PBS-PVA. Figure 4 (As shown).

[0082] In 2024, a total of 727 bovine oocytes underwent intracytoplasmic sperm injection (ICSI), activation, embryo culture, and embryo transfer. The following results were obtained: the pronucleus rate after activation was 60.71%, the cleavage rate was 55.65%, and the blastocyst rate was 11.14%. A total of 8 blastocysts produced using this activation method were transferred, resulting in 4 pregnancies and 0 abortions, with a pregnancy rate of 50.00% and a miscarriage rate of 0.00%. These results demonstrate that the above ICSI procedure for producing sex-controlled embryos in dairy cows is feasible and can achieve a high pregnancy rate and a low miscarriage rate.

[0083] (III) Effects of different activation protocols on ICSI embryo development

[0084] 3.1 Comparative Experiment of Different Activation Schemes (Comparative Example 1)

[0085] Oocyte collection: Ovaries collected from the slaughterhouse are transported to the laboratory at a constant temperature. Excess connective tissue around the ovary is removed, the ovary is cleaned with 75% alcohol, and then repeatedly rinsed with physiological saline. Follicular fluid is extracted using a syringe, and cumulus-oocyte complexes (COCs) are picked out from the follicular fluid under a stereomicroscope using an oocyte retrieval needle.

[0086] Oocyte maturation culture: The collected COCs were repeatedly washed with HEPEs washing solution until there were no impurities and granular cells around the COCs. After cleaning, they were transferred to OM for rinsing once. The COCs that were dead (loose cytoplasm, over-mature) were removed and all were transferred to OM for maturation culture in a 5% CO2 incubator with saturated humidity at 38.5℃.

[0087] Preparation of donor oocytes: After 22 hours of maturation culture, the cultured COCs were repeatedly pipetted with 0.5% hyaluronidase to detach the cumulus cells and obtain naked oocytes. These were then transferred to OM and placed in a 38.5℃, 5% CO2 incubator with saturated humidity for 30 minutes to recover before being prepared for intracytoplasmic sperm injection (ICSI).

[0088] Processing of sex-controlled sperm: X-sex-controlled sperm were processed using the BO flotation method. 4 mL of BO and 2 mL of HEPEs washing buffer were prepared and equilibrated in an incubator for at least 1 hour. After thawing the X-sex-controlled frozen sperm in a 38°C water bath, an appropriate amount of the resulting X-sex-controlled sperm solution was transferred to the bottom of a centrifuge tube containing 4 mL of BO. The centrifuge tube was tilted at 45° and placed in a 5% CO2 incubator at 38.5°C with saturated humidity for sperm flotation. After the sperm had floated for 30 minutes, the supernatant was collected and centrifuged for 10 minutes (room temperature, centrifugation speed 500×g). After centrifugation, the supernatant was discarded, and the sperm precipitate was transferred to 1 mL of HEPEs washing buffer, mixed thoroughly by pipetting, and centrifuged twice, each time for 5 minutes (room temperature, centrifugation speed 300×g).

[0089] Sperm dilution: After centrifugation in HEPEs washing solution, the supernatant is removed, and a small amount of liquid is left to suspend the precipitate, resulting in a sperm suspension. 10 μL of the sperm suspension is then diluted in 60 μL IVF solution using a micropipette and can subsequently be used for intracytoplasmic sperm injection.

[0090] Intracytoplasmic sperm injection (ICSI): Before performing sex-controlled sperm injection in dairy cows, prepare an operating drop (gamete buffer) and a sperm drop containing PVP in a 35mm plastic petri dish, covering them with mineral oil. A fixation needle with an inner diameter of 20μm and an outer diameter of 150–200μm, prepared from a BJ-40 fine glass tube, is mounted on the left micromanipulation arm, and an injection needle with a diameter of 7–9μm, prepared from borosilicate glass, is mounted on the right. The angle and distance are adjusted so that both are visible in the center of the microscope's field of view. Next, the donor oocyte is transferred from the OM to the operating drop. X-sex-controlled sperm, treated with the BO flotation method, is diluted and added to the sperm drop. The sperm drop is rinsed with the injection needle for 1 minute. Sperm with intact heads and tails, free from deformities and impurities, are selected. The tails are mechanically decapitated, and the decapitated sperm is drawn into the injection needle from the tail end and held at the tip. The severed sperm was then transferred to the operating droplet via the injection needle. An oocyte was fixed in place with a fixation needle. The oocyte was rotated with the injection needle until the first polar body was in the "6 o'clock" position. The injection needle was adjusted to the same horizontal plane and inserted into the oocyte at the "3 o'clock" position, penetrating 2 / 3 of the way in. The cytoplasm was slowly and evenly aspirated back. When the aspiration speed suddenly increased, the aspiration was stopped. The cytoplasm and sperm were injected into the oocyte at a uniform speed. After confirming that the injection was complete, the injection needle was withdrawn.

[0091] Activation: ICSI oocytes obtained after sex-controlled sperm injection from dairy cows are transferred to an OM (Ovarian Oocyte) incubator and placed in a 38.5℃, 5% CO2 incubator with saturated humidity for recovery, followed by activation. The specific operational steps for different activation protocols are as follows:

[0092] (1) ETH solution

[0093] ICSI oocytes obtained after sex-controlled sperm injection in dairy cows were transferred to OM and incubated in a 38.5℃, 5% CO2 incubator with saturated humidity for 4 hours; they were then activated in OM containing 7% ETH for 5 minutes (specifically, the activation was carried out in a 38.5℃, 5% CO2 incubator with saturated humidity).

[0094] (2) ION+ETH solution

[0095] After ICSI oocytes were restored for 30 minutes, they were transferred to SOF containing 5 μM ionomycin for 5 minutes of activation treatment (specifically, activation treatment was carried out in a 5% CO2 incubator at 38.5℃ and saturated humidity). After being cleaned with SOF, they were incubated in OM for 4 hours (specifically, cultured in a 5% CO2 incubator at 38.5℃ and saturated humidity). Then, they were transferred to OM containing 7% ETH for 5 minutes of activation treatment (specifically, activation treatment was carried out in a 5% CO2 incubator at 38.5℃ and saturated humidity).

[0096] (3)ION*2+ETH

[0097] After ICSI oocytes were restored, they were transferred to SOF containing 5 μM iomycin for 5 min of activation treatment (specifically, activation treatment was carried out in a 5% CO2 incubator at 38.5℃ with saturated humidity). After being cleaned with SOF, they were cultured in OM for 30 min (specifically, cultured in a 5% CO2 incubator at 38.5℃ with saturated humidity). They were then transferred to SOF containing 5 μM iomycin for 5 min of activation treatment (specifically, activation treatment was carried out in a 5% CO2 incubator at 38.5℃ with saturated humidity). After being cleaned with SOF, they were transferred to OM for 4 h of culture (specifically, cultured in a 5% CO2 incubator at 38.5℃ with saturated humidity). Finally, they were transferred to OM containing 7% ETH for 5 min of activation treatment (specifically, activation treatment was carried out in a 5% CO2 incubator at 38.5℃ with saturated humidity).

[0098] (4) ION*2+6D scheme

[0099] After ICSI oocytes were restored, they were transferred to SOF containing 5 μM iomycin for 5 min of activation treatment (specifically, activation treatment was carried out in a 5% CO2 incubator at 38.5℃ with saturated humidity). After being cleaned with SOF, they were cultured in IVF for 30 min (specifically, cultured in a 5% CO2 incubator at 38.5℃ with saturated humidity). They were then transferred to SOF containing 5 μM iomycin for 5 min of activation treatment (specifically, activation treatment was carried out in a 5% CO2 incubator at 38.5℃ with saturated humidity). After being cleaned with SOF, they were transferred to IVF for 2 h of culture (specifically, cultured in a 5% CO2 incubator at 38.5℃ with saturated humidity). Finally, they were transferred to SOF containing 2 μM 6-DAMP for 3 h of activation treatment (specifically, activation treatment was carried out in a 5% CO2 incubator at 38.5℃ with saturated humidity).

[0100] Embryo culture: After activation, the embryos were rinsed with SOF and the non-viable ICSI oocytes were removed. They were then transferred to an IVC and cultured in a three-gas incubator at 38.5℃, 5% CO2, and 7% O2. The bipolar body rate was recorded as the fertilization rate. After 2 days of culture, the cleavage was observed and recorded. After 6-7 days, the blastocyst formation was observed and recorded.

[0101] 3.2 Analysis of Experimental Results

[0102] All experimental data were statistically analyzed using SPSS, and the significance of differences was determined. See Table 1. Compared to the “ETH,” “ION+ETH,” and “ION*2+ETH” protocols, the “ION*2+6D” protocol significantly improved the fertilization rate, cleavage rate, and blastocyst rate after activation during ICSI. Among the ethanol-activated protocols, the “ION+ETH” protocol significantly improved the blastocyst rate compared to the “ETH” and “ION*2+ETH” protocols; while the “ETH” protocol significantly improved the fertilization rate compared to the “ION+ETH” and “ION*2+ETH” protocols.

[0103] Table 1. Effects of different activation protocols on ICSI embryonic development

[0104]

[0105] Note: This experiment was repeated n>4 times. The same letter superscript in the same column was considered to have no significant difference (P>0.05), different lowercase letter superscripts were considered to have significant differences (P<0.05), and different uppercase letter superscripts were considered to have extremely significant differences (P<0.01).

[0106] (iv) The impact of different activation protocols on embryo transfer efficiency

[0107] In the comparative experiment of different activation protocols, the protocols "ETH", "ION+ETH", and "ION*2+6D" with higher blastocyst production efficiency after activation were used for embryo transfer and pregnancy checks: blastocysts formed after 7 days of culture were selected, placed in a transfer, loaded into a thin tube, placed in a sterile centrifuge tube, and finally transported to the cattle farm in a 38.5℃ constant temperature transport box; recipient cows underwent epidural anesthesia at the 1st to 2nd caudal vertebral intervertebral space, and the vulva was cleaned and disinfected; the thin tube containing the blastocyst was loaded into the transfer gun, then covered with a transfer hard cover, and finally covered with a sterile isolation cover to keep the tip of the transfer gun sterile; the blastocyst was transferred into the uterine horn on the side with the corpus luteum of the recipient cow, and the transfer record of the recipient cow was made; within 14 days after embryo transfer, the return of estrus was used as the initial basis for judging the recipient cow's pregnancy; after 14 days, the return of estrus and the observation of obvious vaginal bleeding were considered as abortion; the final determination of pregnancy and abortion was based on ultrasound examinations at 35 days, 70 days, and 150 days, and the pregnancy status and early abortion status were statistically analyzed.

[0108] Referring to Table 2, compared with the protocols "ION+ETH" and "ION*2+6D", the early miscarriage rate after embryo transfer of the activation protocol "ETH" was significantly lower. The pregnancy rate after embryo transfer of protocol "ETH" was somewhat higher than that of protocols "ION+ETH" and "ION*2+6D", but there was no significant difference.

[0109] Table 2. Effects of different activation protocols after ICSI on embryo transfer efficiency

[0110] transplant Pregnant Early miscarriage Pregnancy rate (%) Early miscarriage rate (%) ETH 8 4 0 <![CDATA[50.00 a ]]> <![CDATA[0.00 b ]]> ION+ETH 12 3 1 <![CDATA[25.00 a ]]> <![CDATA[33.33 ab ]]> ION*2+6D 14 5 4 <![CDATA[35.71 a ]]> <![CDATA[80.00 a ]]>

[0111] Note: Pregnancy rate = number of pregnancies / number of embryo transfers × 100%; Early miscarriage rate = number of early miscarriages / number of pregnancies × 100%;

[0112] Different lowercase superscripts in the same column were considered statistically significant (P<0.05), while different uppercase superscripts were considered extremely significant (P<0.01).

[0113] (V) The impact of different activation schemes on ICSI pronucleus formation

[0114] In the comparative experiments of different activation protocols, the protocols "ETH", "ION+ETH", and "ION*2+6D" with higher blastocyst production efficiency after activation were selected. Samples were taken 16 hours after intracytoplasmic sperm injection (ICSI) for pronuclear staining: the zona pellucida was digested with 0.2% streptoprotein, fixed with fixative for 4 hours, then washed clean with PBS-PVA, and stained with 10 μg / mL H33342 in the dark. After 10 minutes, the stain was washed clean with PBS-PVA, and pronuclear formation was observed under a fluorescence microscope. Data were recorded and grouped according to pronuclear formation type. All data were statistically analyzed using SPSS to determine the significance of differences.

[0115] Referring to Table 3, after activation using the "ETH" protocol, more male and female pronuclei (referring to "1MPN+1FPN") can be formed compared to the "ION*2+6D" protocol, which is beneficial for obtaining a higher pregnancy rate after embryo transfer.

[0116] Table 3. Prokaryotic formation under different activation protocols 16 hours after ICSI (distribution of different prokaryotic types)

[0117]

[0118] Note: MPN represents male pronucleus, FPN represents female pronucleus, PDSH represents depolymerized sperm head (indicating sperm depolymerization but not yet developed into male pronucleus), CSH represents undepolymerized sperm head, 1PN represents one pronucleus (e.g., female pronucleus), 3PN represents three pronuclei (e.g., parthenogenetic activation + male pronucleus); the number of replicates in this experiment was n=3. The same letter superscript in the same column indicates no significant difference (P>0.05), different lowercase letters superscript indicate significant difference (P<0.05), and different uppercase letters superscript indicate extremely significant difference (P<0.01).

[0119] Table 4. Pronucleus formation (sperm depolymerization) under different activation protocols 16 hours after ICSI

[0120] Total number of stains Normal fertilization (%) Sperm deaggregation (%) Not activated (%) ETH 33 <![CDATA[60.71±12.88 a ]]> <![CDATA[73.41±17.35 ab ]]> <![CDATA[14.29±14.29 a ]]> ION+ETH 33 <![CDATA[54.44±5.09a b ]]> <![CDATA[83.89±7.88 a ]]> <![CDATA[8.61±7.47 a ]]> ION*2+6D 41 <![CDATA[38.61±10.20 b ]]> <![CDATA[60.84±13.98 b ]]> <![CDATA[0.00±0.00 a ]]>

[0121] Note: Normal fertilization is 1MPN+1FPN, sperm depolymerization is 1MPN+1FPN, 1FPN+PDSH and 3PN, and unactivated sperm are condensed nuclei;

[0122] Different lowercase superscripts in the same column were considered statistically significant (P<0.05), while different uppercase superscripts were considered extremely significant (P<0.01).

[0123] Further analysis showed that activation using the "ETH" protocol can promote the formation of male and female pronuclei, improve the activation efficiency of ICSI oocytes (as shown in Tables 3 and 4), thereby generating more normally fertilized ICSI oocytes and promoting embryonic development and improving developmental quality.

[0124] (VI) Effects of different incubation solutions on embryonic development during ICSI egg activation

[0125] 4.1 Comparative experiment of different incubation liquids (Comparative Example 2)

[0126] Oocyte collection: Ovaries collected from the slaughterhouse are transported to the laboratory at a constant temperature. Excess connective tissue around the ovary is removed, the ovary is cleaned with 75% alcohol, and then repeatedly rinsed with physiological saline. Follicular fluid is extracted using a syringe, and cumulus-oocyte complexes (COCs) are picked out from the follicular fluid under a stereomicroscope using an oocyte retrieval needle.

[0127] Oocyte maturation culture: The collected COCs were repeatedly washed with HEPEs washing solution until there were no impurities and granular cells around the COCs. After cleaning, they were transferred to OM for rinsing once. The COCs that were dead (loose cytoplasm, over-mature) were removed and all were transferred to OM for maturation culture in a 5% CO2 incubator with saturated humidity at 38.5℃.

[0128] Preparation of donor oocytes: After 22 hours of maturation culture, the cultured COCs were repeatedly pipetted with 0.5% hyaluronidase to detach the cumulus cells and obtain naked oocytes. These were then transferred to OM and placed in a 38.5℃, 5% CO2 incubator with saturated humidity for 30 minutes to recover before being prepared for intracytoplasmic sperm injection (ICSI).

[0129] Processing of sex-controlled sperm: X-sex-controlled sperm were processed using the BO flotation method. 4 mL of BO and 2 mL of HEPEs washing buffer were prepared and equilibrated in an incubator for at least 1 hour. After thawing the X-sex-controlled frozen sperm in a 38°C water bath, an appropriate amount of the resulting X-sex-controlled sperm solution was transferred to the bottom of a centrifuge tube containing 4 mL of BO. The centrifuge tube was tilted at 45° and placed in a 5% CO2 incubator at 38.5°C with saturated humidity for sperm flotation. After the sperm had floated for 30 minutes, the supernatant was collected and centrifuged for 10 minutes (room temperature, centrifugation speed 500×g). After centrifugation, the supernatant was discarded, and the sperm precipitate was transferred to 1 mL of HEPEs washing buffer, mixed thoroughly by pipetting, and centrifuged twice, each time for 5 minutes (room temperature, centrifugation speed 300×g).

[0130] Sperm dilution: After centrifugation in HEPEs washing solution, the supernatant is removed, and a small amount of liquid is left to suspend the precipitate, resulting in a sperm suspension. 10 μL of the sperm suspension is then diluted in 60 μL IVF solution using a micropipette and can subsequently be used for intracytoplasmic sperm injection.

[0131] Intracytoplasmic sperm injection (ICSI): Before performing sex-controlled sperm injection in dairy cows, prepare an operating drop (gamete buffer) and a sperm drop containing PVP in a 35mm plastic petri dish, covering them with mineral oil. A fixation needle with an inner diameter of 20μm and an outer diameter of 150–200μm, prepared from a BJ-40 fine glass tube, is mounted on the left micromanipulation arm, and an injection needle with a diameter of 7–9μm, prepared from borosilicate glass, is mounted on the right. The angle and distance are adjusted so that both are visible in the center of the microscope's field of view. Next, the donor oocyte is transferred from the OM to the operating drop. X-sex-controlled sperm, treated with the BO flotation method, is diluted and added to the sperm drop. The sperm drop is rinsed with the injection needle for 1 minute. Sperm with intact heads and tails, free from deformities and impurities, are selected. The tails are mechanically decapitated, and the decapitated sperm is drawn into the injection needle from the tail end and held at the tip. The severed sperm was then transferred to the operating droplet via the injection needle. An oocyte was fixed in place with a fixation needle. The oocyte was rotated with the injection needle until the first polar body was in the "6 o'clock" position. The injection needle was adjusted to the same horizontal plane and inserted into the oocyte at the "3 o'clock" position, penetrating 2 / 3 of the way in. The cytoplasm was slowly and evenly aspirated back. When the aspiration speed suddenly increased, the aspiration was stopped. The cytoplasm and sperm were injected into the oocyte at a uniform speed. After confirming that the injection was complete, the injection needle was withdrawn.

[0132] Activation: ICSI oocytes obtained after sex-controlled sperm injection in dairy cows were transferred to OM or IVF and incubated in a 5% CO2 incubator at 38.5℃ and saturated humidity for 4 hours; then activated in 7% ETH for 5 minutes (specifically, the activation treatment was carried out in a 5% CO2 incubator at 38.5℃ and saturated humidity).

[0133] Embryo culture: After activation, the embryos were rinsed with SOF and the non-viable ICSI oocytes were removed. They were then transferred to an IVC and cultured in a three-gas incubator at 38.5℃, 5% CO2, and 7% O2. The bipolar body rate was recorded as the fertilization rate. After 2 days of culture, the cleavage was observed and recorded. After 6-7 days, the blastocyst formation was observed and recorded.

[0134] 4.2 Analysis of Experimental Results

[0135] All experimental data were statistically analyzed using SPSS, and the significance of differences was determined. See Table 5. Compared to IVF, using OM as the incubation medium during activation significantly improved the cleavage rate and blastocyst rate of embryos produced after ICSI.

[0136] Table 5. Effects of different incubation solutions on the development of sex-controlled embryos in dairy cows

[0137] Total number of injections Number of surviving eggs Fertilization rate (%) Cleavage rate (%) Blastocyst rate (%) OM 688 525 <![CDATA[67.96±1.85 b ]]> <![CDATA[55.65±1.86 a ]]> <![CDATA[11.14±0.79 a ]]> IVF 731 554 <![CDATA[73.88±1.80 a ]]> <![CDATA[44.24±1.81 b ]]> <![CDATA[4.90±0.77 b ]]>

[0138] Note: This experiment was repeated n>4 times. The same letter superscript in the same column indicates no significant difference (P>0.05), and different letter superscripts indicate significant difference (P<0.05).

[0139] (VII) Effects of different incubation solutions on ICSI pronucleus formation during ICSI egg activation

[0140] In the comparative experiments above, ICSI embryos activated after incubation in different liquids following ICSI were sampled 16 hours later for pronuclear staining: the zona pellucida was digested with 0.2% streptoprotein, fixed with fixative for 4 hours, then the fixative was washed off with PBS-PVA, and fluorescently stained with 10 μg / mL H33342 in the dark. After 10 minutes, the dye was washed off with PBS-PVA, and pronuclear formation was observed under a fluorescence microscope. Data were grouped and recorded according to the type of pronuclear formation. All data were statistically analyzed using SPSS to determine the significance of differences.

[0141] Referring to Tables 6 and 7, there were no significant differences between using OM and IVF as incubation media in terms of male and female pronucleus formation (referring to "1MPN+1FPN") and sperm non-depolymerization (1FPN+CSH). However, in terms of depolymerized sperm (1FPN+PDSH), using IVF as the incubation media for activation significantly improved compared to OM, thus significantly affecting the fertilization rate of IVF. However, since "1FPN+PDSH" is not considered normal fertilization, the cleavage rate and blastocyst rate of ICSI sex-controlled embryos produced by IVF as the incubation media were significantly lower than those produced by OM.

[0142] Table 6. Prokaryotic formation in different incubation solutions (distribution of different prokaryotic types)

[0143]

[0144] Note: MPN represents male pronucleus, FPN represents female pronucleus, PDSH represents depolymerized sperm head (indicating sperm depolymerization but not yet developed into male pronucleus), CSH represents undepolymerized sperm head, 1PN represents one pronucleus (e.g., female pronucleus), 2PN+CSH represents two pronuclei and undepolymerized sperm head (e.g., parthenogenetic activation + undepolymerized sperm head), 3PN represents three pronuclei (e.g., parthenogenetic activation + male pronucleus); the number of replicates in this experiment was n=3. The same letter superscript in the same column indicates no significant difference (P>0.05), different lowercase letters superscript indicate significant difference (P<0.05), and different uppercase letters superscript indicate extremely significant difference (P<0.01).

[0145] Table 7. Pronucleus formation (sperm depolymerization) in different incubation solutions

[0146]

[0147] Note: Normal fertilization is 1MPN+1FPN, sperm depolymerization is 1MPN+1FPN, 1FPN+PDSH and 3PN, inactive sperm is condensed nucleus, parthenogenetic activation is 2PN+CSH; different lowercase superscripts in the same column indicate significant differences (P<0.05), and different uppercase superscripts indicate extremely significant differences (P<0.01).

[0148] (VIII) The effects of different ETH activation times on ICSI embryonic development

[0149] 8.1 Comparative Experiment of Different ETH Activation Times (Comparative Example 3)

[0150] Oocyte collection: Ovaries collected from the slaughterhouse are transported to the laboratory at a constant temperature. Excess connective tissue around the ovary is removed, the ovary is cleaned with 75% alcohol, and then repeatedly rinsed with physiological saline. Follicular fluid is extracted using a syringe, and cumulus-oocyte complexes (COCs) are picked out from the follicular fluid under a stereomicroscope using an oocyte retrieval needle.

[0151] Oocyte maturation culture: The collected COCs were repeatedly washed with HEPEs washing solution until there were no impurities and granular cells around the COCs. After cleaning, they were transferred to OM for rinsing once. The COCs that were dead (loose cytoplasm, over-mature) were removed and all were transferred to OM for maturation culture in a 5% CO2 incubator with saturated humidity at 38.5℃.

[0152] Preparation of donor oocytes: After 22 hours of maturation culture, the cultured COCs were repeatedly pipetted with 0.5% hyaluronidase to detach the cumulus cells and obtain naked oocytes. These were then transferred to OM and placed in a 38.5℃, 5% CO2 incubator with saturated humidity for 30 minutes to recover before being prepared for intracytoplasmic sperm injection (ICSI).

[0153] Processing of sex-controlled sperm: X-sex-controlled sperm were processed using the BO flotation method. 4 mL of BO and 2 mL of HEPEs washing buffer were prepared and equilibrated in an incubator for at least 1 hour. After thawing the X-sex-controlled frozen sperm in a 38°C water bath, an appropriate amount of the resulting X-sex-controlled sperm solution was transferred to the bottom of a centrifuge tube containing 4 mL of BO. The centrifuge tube was tilted at 45° and placed in a 5% CO2 incubator at 38.5°C with saturated humidity for sperm flotation. After the sperm had floated for 30 minutes, the supernatant was collected and centrifuged for 10 minutes (room temperature, centrifugation speed 500×g). After centrifugation, the supernatant was discarded, and the sperm precipitate was transferred to 1 mL of HEPEs washing buffer, mixed thoroughly by pipetting, and centrifuged twice, each time for 5 minutes (room temperature, centrifugation speed 300×g).

[0154] Sperm dilution: After centrifugation in HEPEs washing solution, the supernatant is removed, and a small amount of liquid is left to suspend the precipitate, resulting in a sperm suspension. 10 μL of the sperm suspension is then diluted in 60 μL IVF solution using a micropipette and can subsequently be used for intracytoplasmic sperm injection.

[0155] Intracytoplasmic sperm injection (ICSI): Before performing sex-controlled sperm injection in dairy cows, prepare an operating drop (gamete buffer) and a sperm drop containing PVP in a 35mm plastic petri dish, covering them with mineral oil. A fixation needle with an inner diameter of 20μm and an outer diameter of 150–200μm, prepared from a BJ-40 fine glass tube, is mounted on the left micromanipulation arm, and an injection needle with a diameter of 7–9μm, prepared from borosilicate glass, is mounted on the right. The angle and distance are adjusted so that both are visible in the center of the microscope's field of view. Next, the donor oocyte is transferred from the OM to the operating drop. X-sex-controlled sperm, treated with the BO flotation method, is diluted and added to the sperm drop. The sperm drop is rinsed with the injection needle for 1 minute. Sperm with intact heads and tails, free from deformities and impurities, are selected. The tails are mechanically decapitated, and the decapitated sperm is drawn into the injection needle from the tail end and held at the tip. The severed sperm was then transferred to the operating droplet via the injection needle. An oocyte was fixed in place with a fixation needle. The oocyte was rotated with the injection needle until the first polar body was in the "6 o'clock" position. The injection needle was adjusted to the same horizontal plane and inserted into the oocyte at the "3 o'clock" position, penetrating 2 / 3 of the way in. The cytoplasm was slowly and evenly aspirated back. When the aspiration speed suddenly increased, the aspiration was stopped. The cytoplasm and sperm were injected into the oocyte at a uniform speed. After confirming that the injection was complete, the injection needle was withdrawn.

[0156] Activation: ICSI oocytes obtained after sex-controlled sperm injection in dairy cows were transferred to OM and incubated in a 5% CO2 incubator at 38.5℃ and saturated humidity for 4 hours; then activated in 7% ETH for 5 or 10 minutes (specifically, activation was performed in a 5% CO2 incubator at 38.5℃ and saturated humidity).

[0157] Embryo culture: After activation, the embryos were rinsed with SOF and the non-viable ICSI oocytes were removed. They were then transferred to an IVC and cultured in a three-gas incubator at 38.5℃, 5% CO2, and 7% O2. The bipolar body rate was recorded as the fertilization rate. After 2 days of culture, the cleavage was observed and recorded. After 6-7 days, the blastocyst formation was observed and recorded.

[0158] 8.2 Analysis of Experimental Results

[0159] All experimental data were statistically analyzed using SPSS to determine the significance of differences. See Table 8. Compared to activation with 7% ETH for 10 minutes, activation with 7% ETH for 5 minutes significantly improved the cleavage rate and blastocyst rate of ICSI embryos.

[0160] Table 8. Effects of different ETH activation times on bovine sex-controlled embryonic development

[0161]

[0162] Note: This experiment was repeated n>4 times. The same letter superscript in the same column was considered to have no significant difference (P>0.05), different lowercase letter superscripts were considered to have significant differences (P<0.05), and different uppercase letter superscripts were considered to have extremely significant differences (P<0.01).

[0163] (ix) The impact of different ETH activation times on ICSI pronucleus formation

[0164] In the above comparative experiments, ICSI embryos generated at different activation times after intracytoplasmic sperm injection (ICSI) were sampled 16 hours later for pronuclear staining: the zona pellucida was digested with 0.2% streptoprotein, fixed with fixative for 4 hours, then the fixative was washed off with PBS-PVA, and fluorescently stained with 10 μg / mL H33342 in the dark. After 10 minutes, the dye was washed off with PBS-PVA, and pronuclear formation was observed under a fluorescence microscope. Data were grouped and recorded according to the type of pronuclear formation. All data were statistically analyzed using SPSS to determine the significance of differences.

[0165] Referring to Tables 9 and 10, activation with 7% ETH for 5 minutes results in the formation of more male and female pronuclei (referring to "1MPN+1FPN") compared to activation for 10 minutes, which significantly increases the normal fertilization rate. This, in turn, significantly improves the cleavage rate and blastocyst rate of embryos obtained under the 7% ETH activation for 5 minutes protocol.

[0166] Table 9. Pronuclear formation at different activation times in ETH (distribution of different pronuclear types)

[0167]

[0168] Note: MPN represents male pronucleus, FPN represents female pronucleus, PDSH represents depolymerized sperm head (indicating sperm depolymerization but not yet developed into male pronucleus), CSH represents undepolymerized sperm head, 1PN represents one pronucleus (e.g., female pronucleus), 2PN+CSH represents two pronuclei and undepolymerized sperm head (e.g., parthenogenetic activation + undepolymerized sperm head), 3PN represents three pronuclei (e.g., parthenogenetic activation + male pronucleus); the number of replicates in this experiment was n=3. The same letter superscript in the same column indicates no significant difference (P>0.05), different lowercase letters superscript indicate significant difference (P<0.05), and different uppercase letters superscript indicate extremely significant difference (P<0.01).

[0169] Table 10. Pronucleus formation (sperm depolymerization) under different activation protocols 16 hours after ICSI

[0170]

[0171] Note: Normal fertilization is 1MPN+1FPN, sperm depolymerization is 1MPN+1FPN, 1FPN+PDSH and 3PN, inactive sperm is condensed nucleus, parthenogenetic activation is 2PN+CSH; different lowercase superscripts in the same column indicate significant differences (P<0.05), and different uppercase superscripts indicate extremely significant differences (P<0.01).

[0172] The preferred embodiments of the present invention have been described above, but are not intended to limit the invention. Those skilled in the art can make modifications and variations to the embodiments disclosed herein without departing from the scope and spirit of the invention.

Claims

1. A method to improve the normal fertilization rate of in vitro ICSI embryos in dairy cows and reduce the abortion rate, including the following steps: X-sex-controlled sperm and mature oocytes were cultured and used as donor oocytes to prepare intracytoplasmic sperm injection (ICSI) oocytes via microinjection. The ICSI oocytes were incubated in oocyte maturation culture medium (OM) for 3-5 hours and then transferred to ETH for 4-6 minutes to promote normal fertilization. Embryo culture was then performed to obtain sex-controlled embryos. ETH refers to oocyte maturation culture medium containing 6-8 v / v% ethanol.

2. The method according to claim 1, wherein the activation specifically includes the following steps: Among them each Activation and incubation were carried out at 38.4–38.6℃, saturated humidity, and 4.85%–5.15% CO2.

3. According to the method of claim 1, the oocyte maturation culture medium is based on Hepes-free M199 as the base culture medium, and additionally supplemented with 8-12% (v / v) fetal bovine serum, 0.08-0.12% (v / v) ITS-G, 0.07-0.08 IU / mL HMG, 0.8-1.2 μg / mL 17β-estradiol, 8-12 ng / mL EGF and 8-12 ng / mL bFGF.

4. The method according to claim 1, wherein the incubation of the ICSI oocyte specifically includes the following steps: Donor oocytes injected with sex-controlled sperm via ICSI were transferred to oocyte maturation culture medium (OM) and incubated for 3.5–4.5 h; the incubation was carried out at 38.4–38.6 °C, saturated humidity and 4.85%–5.15% CO2.

5. The method according to claim 1, wherein the activation processing time is 5 minutes.

6. The method according to any one of claims 1-5, wherein the activation process does not include other activation processes.

7. The method according to claim 1, wherein the conditions for embryo culture include: Embryo culture medium was used to culture the embryos at 38.4–38.6°C, 4.85%–5.15% CO2, and 6.9%–7.0% O2 for 6–7 days.

8. A method for breeding livestock, comprising the following steps: Using the method described in claims 1-7, sex-controlled embryos are obtained, which are then transplanted into recipient livestock. The pregnancy and early abortion status of the recipient livestock are then examined and assessed.

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