A method for producing snowflake beef live ovum in vitro fertilization embryo

Abstract

The present application relates to the field of animal reproductive biotechnology, and discloses a method for producing in vitro fertilized embryos by live ovum collection of snowflake beef cattle, aiming at solving the problems of uneven quality of oocytes, low efficiency of in vitro fertilization and loose connection of each technical link in the prior art. The method comprises the following steps: screening of donor cattle and superovulation treatment by decreasing injection of follicle stimulating hormone and luteinizing hormone; obtaining oocyte complex by transrectal ultrasonic guided puncture; in vitro maturation culture of the obtained oocyte; in vitro fertilization after capacitation treatment of semen by gradient centrifugation; sequential culture of the fertilized eggs to the stage of morula or blastocyst; morphological evaluation and vitrification preservation of the embryos cultured to the set stage. By the combined application of the standardized superovulation scheme, the precise ovum collection technology, the optimized maturation culture condition and the sequential culture strategy of embryos, the efficiency of embryo production can be significantly improved, and the quality of in vitro maturation of oocytes can be improved.

Description

Technical Field

[0001] This invention relates to the field of animal reproductive biotechnology, specifically to a method for producing in vitro fertilization embryos from live oocytes of Wagyu beef cattle. Background Technology

[0002] With the rapid development of modern biotechnology, embryo engineering has become a key technology in livestock and poultry breeding and genetic resource protection. In vitro fertilization (IVF), by simulating the in vivo fertilization environment, completes the fertilization of sperm and egg and early embryonic development under in vitro conditions, providing important technical support for the rapid propagation of superior breeds. In the beef cattle industry, high-grade Wagyu beef has extremely high economic value due to its unique intramuscular fat deposition characteristics. However, the propagation efficiency of high-quality Wagyu beef cattle has long been constrained by natural factors such as long reproductive cycles and limited calving numbers. How to fully utilize the genetic potential of high-quality breeding cattle has become a core issue that urgently needs to be addressed for the industry's development.

[0003] Among them, live oocyte retrieval technology, as an emerging assisted reproductive technology, involves collecting oocytes from visible follicles on the surface of the ovary via transrectal ultrasound-guided puncture. This allows for repeated oocyte retrieval without slaughtering the donor cattle. Compared to traditional slaughter-based oocyte retrieval, live oocyte retrieval significantly extends the donor lifespan of superior cows, making it particularly suitable for the protection and development of high-value germplasm resources. Based on live oocyte retrieval, combined with in vitro fertilization technology, embryos can be mass-produced under laboratory conditions, enabling the industrial-scale propagation of high-quality genetic material. This has significant application value in accelerating the beef cattle breeding process.

[0004] However, existing methods for live oocyte retrieval and in vitro fertilization (IVF) embryo production still have many shortcomings. In live oocyte retrieval, significant differences in follicle development between individuals lead to inconsistent oocyte quality. In IVF, the standardization of fertilization parameters and embryo culture conditions is low, making it difficult to achieve ideal oocyte maturation, fertilization, and blastocyst development rates. Furthermore, existing methods lack a systematic approach to optimizing embryo production efficiency, and the connections between different technical steps are not tight enough, making it difficult to meet the demands of large-scale high-quality embryo production. Therefore, developing an efficient, stable, and suitable live oocyte retrieval IVF embryo production method for Wagyu beef cattle is of significant practical importance for enhancing the core competitiveness of my country's beef cattle breeding industry. Summary of the Invention

[0005] The purpose of this invention is to provide a method for producing in vitro fertilization embryos from live oocytes retrieved from snowflake beef cattle, which can effectively solve the problems of inconsistent quality of oocytes retrieved from live oocytes, low efficiency of in vitro fertilization, and poor connection between various technical links in the existing technology.

[0006] To achieve the above objectives, the technical solution adopted by the present invention is as follows: A method for in vitro fertilization (IVF) embryo production using live oocytes from Wagyu beef cattle includes the following specific steps: Step 1: Donor cattle selection and superovulation treatment: Health checks and reproductive performance assessments are conducted on the donor Wagyu beef cattle to select individuals meeting the standards as donor cattle; superovulation treatment is performed on the donor cattle by continuously injecting follicle-stimulating hormone (FSH) and luteinizing hormone (LH) to induce the synchronous development of multiple follicles in the ovaries; Step 2: Preparation before oocyte retrieval: After superovulation treatment, the donor cattle to be retrieved are restrained within a preset time period. Ovarian response is checked via rectal examination, and after confirming the follicle development status, skin preparation and disinfection are performed; Step 3: Live oocyte retrieval: Transrectal ultrasound-guided puncture technique is used. An ultrasound probe with a puncture needle is inserted through the rectal wall into the ovarian follicles, and follicular fluid is aspirated under negative pressure to obtain the oocyte complex; Step 4: Oocyte body... In vitro maturation culture: The obtained oocyte complexes are screened under a stereomicroscope, granulosa cells are removed, and then transferred to maturation culture medium for culture under preset conditions; Step 5: Sperm processing and in vitro fertilization: Fresh semen from high-quality bulls is collected and subjected to gradient centrifugation for capacitation. The treated sperm is mixed with mature oocytes at a preset concentration and in vitro fertilization is performed in fertilization culture medium; Step 6: Embryo culture: Cumulus cells are removed at a preset time after fertilization, fertilization status is checked, and fertilized eggs are transferred to embryo culture medium for sequential culture. Cleavage embryo culture medium is used in the first preset period, and blastocyst culture medium is used in the subsequent period, for a total of preset number of days; Step 7: Embryo evaluation and preservation: Morula or blastocysts cultured to the preset stage are morphologically evaluated, and high-quality embryos are selected for vitrification and cryopreservation or direct transfer.

[0007] Preferably, in step 1, the superovulation treatment adopts a decreasing injection protocol. The total dose of follicle-stimulating hormone (FSH) is a preset dose, and it is injected in a preset number of times at preset intervals. The dose of each injection is a preset decreasing ratio. The total dose of luteinizing hormone (LH) is a preset dose, and it is injected once at a preset time after the last FSH injection.

[0008] Preferably, in step 2, before egg retrieval, the number and distribution of follicles of a predetermined size or larger on both ovaries are recorded by digital rectal examination and ultrasound exploration, and individuals with a predetermined number of follicles are selected for egg retrieval.

[0009] Preferably, in step 3, a single-lumen puncture needle of a preset specification is used for oocyte retrieval, the negative pressure is set to a preset negative pressure value, the number of follicles punctured on each ovary does not exceed a preset number, and the time for a single oocyte retrieval operation is controlled within a preset duration.

[0010] Preferably, in step 4, the oocyte selection criteria are: oocytes with a diameter that reaches a preset size, uniform cytoplasm, containing more than a preset number of complete granulosa cell layers, and with uniform light brown cytoplasm color are preferred.

[0011] Preferably, in step 4, the maturation culture medium is a TCM199 basal medium with a preset ratio of fetal bovine serum, a preset concentration of follicle-stimulating hormone, a preset concentration of luteinizing hormone, and a preset concentration of estradiol, and the pH and osmotic pressure of the culture medium are within a preset range.

[0012] Preferably, in step 5, the sperm capacitation treatment is performed by gradient centrifugation of collagen centrifuge fluid coated with silane at a preset density gradient, the centrifugation speed is a preset centrifugal force, the centrifugation time is a preset centrifugation duration, and the sperm motility reaches a preset motility threshold and the survival time reaches a preset survival duration after capacitation treatment.

[0013] Preferably, in step 6, the sequential culture process initially uses CR1aa medium with a preset proportion of serum, and the embryos are cultured to a preset time point where half of the medium is replaced. Subsequently, CR1aa medium with a preset proportion of serum and a preset concentration of glucose is used, and the embryos are cultured to a preset number of days to assess their grade.

[0014] Preferably, it also includes embryo quality assessment criteria: embryos are divided into three grades: A, B, and C. Grade A embryos have a cell count that meets the preset number, a fragmentation rate that is lower than the preset proportion, and uniform cell size. Grade B embryos have a cell count that is within the preset range, a fragmentation rate that is within the preset range, and relatively uniform cell size. Grade C embryos have a cell count that is lower than the preset number or a fragmentation rate that exceeds the preset proportion.

[0015] Preferably, it also includes embryo preservation record management: establishing a complete file for cryopreserved embryos, recording the donor's ear number, egg retrieval date, embryo development stage, embryo grade, cryopreservation medium number and storage location information, and ensuring that the survival rate of frozen embryos reaches a preset ratio.

[0016] Compared with the prior art, the beneficial effects achieved by the present invention are: 1. Significantly improves embryo production efficiency. Through the optimized combination of standardized superovulation treatment protocol and transrectal ultrasound-guided oocyte retrieval technology, a preset number of usable oocytes can be obtained from a single donor cow in a single oocyte retrieval. After in vitro fertilization, the blastocyst development rate reaches the preset ratio, which significantly increases embryo yield compared with traditional methods and effectively solves the problem of inconsistent oocyte quality caused by differences in follicle development among individuals.

[0017] 2. Comprehensively improve the quality of oocyte in vitro maturation. By strictly controlling oocyte selection criteria and optimizing maturation culture conditions, a compound hormone supplementation system containing follicle-stimulating hormone, luteinizing hormone, and estradiol is established. Combined with precise culture environment control parameters, the in vitro maturation rate and quality of oocytes are significantly improved, laying the foundation for subsequent in vitro fertilization and embryo development.

[0018] 3. Optimize in vitro fertilization and embryo culture effects. Ensure efficient fertilization through gradient centrifugation of semen for energy capture and precise control of fertilization parameters. Implement sequential embryo culture protocols to simulate the nutritional needs of each stage of in vivo embryo development, significantly improving blastocyst development rate and the proportion of high-quality embryos, and meeting the needs of large-scale production of high-quality embryos.

[0019] 4. Achieve standardized management throughout the entire embryo production process. By establishing a complete embryo evaluation system and standardized record management, standardized control can be achieved from donor selection to embryo preservation. All technical links are closely connected, effectively improving the stability and repeatability of embryo production, and providing reliable technical support for the protection and industrial propagation of Wagyu beef cattle germplasm resources. Attached Figure Description

[0020] Figure 1 This is a schematic diagram of the overall process of the in vitro fertilization embryo production method for live oocytes retrieved from snowflake beef cattle according to an embodiment of this application; Figure 2 This is a schematic diagram of the in vitro fertilization and sequential embryo culture process in the method for producing in vitro fertilization embryos from live oocytes of Wagyu beef cattle according to an embodiment of this application. Figure 3 This is a schematic diagram of the process of superovulation treatment and live oocyte retrieval in the in vitro fertilization embryo production method of live oocyte retrieval from snowflake beef cattle according to an embodiment of this application. Figure 4 This is a schematic diagram of the standardized management process for embryo quality assessment and preservation records in the in vitro fertilization embryo production method using live oocytes retrieved from snowflake beef cattle according to an embodiment of this application. Detailed Implementation

[0021] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. 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 skilled in the art without creative effort are within the scope of protection of the present invention.

[0022] Example 1 This invention provides a method for producing in vitro fertilization embryos from live oocytes retrieved from Wagyu beef cattle. The method includes the following technical steps: donor cattle screening and superovulation treatment, pre-retrieval preparation, live oocyte retrieval, in vitro maturation culture of oocytes, semen processing and in vitro fertilization, embryo culture, and embryo evaluation and preservation. Each step is described in detail below.

[0023] Step 1: Donor cow selection and superovulation treatment The selection of donor Wagyu cattle is a fundamental step in embryo production, directly determining the quality of subsequent oocytes and the developmental potential of embryos. This invention implements a rigorous screening procedure for donor cattle, with screening criteria covering two dimensions: health status assessment and reproductive performance evaluation.

[0024] Health assessment includes: body temperature monitoring, requiring a temperature maintained within the normal physiological range of 37.5℃ to 39.0℃; respiratory rate monitoring, with a normal range of 12 to 30 breaths per minute; and heart rate monitoring, with a normal range of 50 to 80 beats per minute. A comprehensive clinical examination is conducted to rule out infectious diseases, reproductive system infections, endocrine disorders, and other conditions. A complete blood count is performed on the donor cattle, focusing on indicators such as white blood cell count, hematocrit, and hemoglobin content to ensure that hematological parameters are within the normal reference range.

[0025] Reproductive performance evaluation includes: recording the age information of donor cattle, selecting individuals aged 3 to 8 years with a good reproductive history; examining the integrity of the reproductive tract anatomical structure, confirming the symmetry of the uterine horns, the elasticity of the uterine body, and the position and shape of the cervix through rectal palpation; assessing the ovarian function status, detecting the volume, number and distribution of follicles in both ovaries through ultrasound, and selecting individuals with clear ovarian outlines and normal follicle development as candidate donor cattle.

[0026] Selected donor cows undergo superovulation treatment. The purpose of superovulation treatment is to induce the synchronous development of multiple follicles in the ovary, thereby obtaining a sufficient number of oocytes. This invention employs a tapering injection protocol to implement superovulation treatment, and the specific operation is as follows: The total dose of follicle-stimulating hormone (FSH) is set at 300 to 400 IU, administered in 6 to 8 progressively decreasing injections. Each injection is spaced 12 hours apart, and the dose is adjusted according to a pre-defined decreasing ratio: the initial injection dose is 20% to 25% of the total dose, with subsequent injections decreasing to 15%, 12%, 10%, 8%, 6%, and 4% of the total dose respectively. High-purity lyophilized FSH powder is used, prepared to the required concentration with physiological saline.

[0027] The total dose of luteinizing hormone (LH) is set at 150 to 200 IU, administered as a single intramuscular injection 18 to 20 hours after the last follicle-stimulating hormone (FSH) injection. The LH preparation used is human chorionic gonadotropin (hCG) or animal-derived LH, diluted to an appropriate concentration with physiological saline.

[0028] During the superovulation treatment, the donor cow's vulva and behavior were observed daily, and estrus signs were recorded. Within 48 to 72 hours after the superovulation treatment, the donor cow entered estrus, and multiple mature follicles could be seen on the ovaries.

[0029] Step 2: Preparation before egg retrieval After superovulation treatment, the donor cows to be retrieved are restrained within a predetermined time period. The restraint area is a dry, flat, and well-ventilated dedicated restraint pen. During restraint, the donor cows are kept in a natural standing position with their backs horizontal.

[0030] After the procedure is completed, a rectal examination is performed. The specific procedure includes: technicians wearing long gloves palpating both ovaries through the rectal wall, recording the size, texture, and follicle distribution of the ovaries. After superovulation treatment, the ovaries can reach 2 to 3 times their normal size, and multiple follicles with a diameter of 8 to 12 millimeters can be palpated on the surface. Care must be taken to be gentle during palpation to avoid mechanical damage to the ovaries.

[0031] After digital rectal examination, a portable ultrasound diagnostic instrument is used to confirm ovarian response. The ultrasound probe is inserted through the rectal wall to examine both ovaries, scanning and recording the number and distribution of follicles larger than 5 mm in diameter. Typically, after superovulation treatment, each ovary of the donor cow can produce 8 to 15 retrievable follicles. Based on ultrasound image analysis, individuals with a total of 12 or more follicles in both ovaries are selected for the oocyte retrieval procedure.

[0032] The skin preparation area includes the base of the donor cow's tail and the area around the anus. Hair is removed using an electric razor to fully expose the skin. After preparation, the skin is disinfected with povidone-iodine solution, wiping in a spiral motion from the center outwards, covering the prepared area and a 3-5 cm perimeter. After disinfection, the area is rinsed with sterile saline to remove residual povidone-iodine, awaiting the surgical procedure.

[0033] Step 3: Live egg retrieval This invention employs transrectal ultrasound-guided puncture technology for live oocyte retrieval. This technique combines ultrasound imaging positioning and negative pressure aspiration with a puncture needle to achieve safe and efficient recovery of oocytes from follicles.

[0034] The oocyte retrieval equipment includes: an ultrasound diagnostic instrument with a puncture adapter, the probe frequency being set to 5 to 7.5 MHz; a single-lumen puncture needle, 17 to 18 Gauge, with a length selected according to the depth of the donor bovine rectum, typically 50 to 60 cm; a negative pressure suction device with an adjustable vacuum range of 50 to 120 mmHg; and an oocyte collection cup equipped with a collection solution preheated to 37°C.

[0035] Before oocyte retrieval, the puncture needle is inserted into the puncture guide groove of the ultrasound probe, and the negative pressure suction device is adjusted to the preset negative pressure value of 75 mmHg. Preheated oocyte collection solution at 37°C is added to the oocyte collection cup; the solution is a phosphate buffer solution supplemented with 10% fetal bovine serum.

[0036] During egg retrieval, the technician uses one hand to hold the ultrasound probe close to the ovary through the rectal wall, while the other hand manipulates the puncture needle to penetrate along the guide groove. The needle passes through the rectal wall and pelvic cavity into the ovarian parenchyma. Once the needle tip reaches the inside of the follicle, the negative pressure suction device is activated to aspirate the follicular fluid and oocyte complex. After each follicle is aspirated, the needle is withdrawn to the ovarian surface, the angle is adjusted, and the needle is inserted into the next follicle.

[0037] During oocyte retrieval, the number of follicles retrieved from each ovary should be controlled to no more than 8 to 10, and the total time for a single oocyte retrieval operation should be controlled to no more than 45 to 60 minutes. The donor cow's condition should be closely monitored during oocyte retrieval; if a significant stress response occurs, the operation should be paused appropriately and the animal should be calmed.

[0038] Follicular fluid obtained by negative pressure aspiration was collected in an oocyte collection cup, and oocyte complexes were immediately searched under a stereomicroscope. The searched oocyte complexes were transferred to a pre-equilibrated washing solution and washed in a 37°C, 5% CO2 incubator to remove blood cells and debris.

[0039] Step 4: In vitro maturation culture of oocytes The obtained oocyte complexes were rigorously screened under a stereomicroscope using the following criteria: The oocyte diameter should be between 110 and 130 micrometers to ensure appropriate maturity. The cytoplasm should be uniform and dense, without vacuolization or granulation. The granulosa cell layer should be intact, with at least three layers arranged closely. Oocytes with uniformly light brown cytoplasm are preferred. The zona pellucida should be intact and of uniform thickness.

[0040] Oocytes that do not meet the above standards are discarded, and qualified oocytes proceed to the maturation culture stage. The maturation culture medium is formulated based on TCM199, with the following components added: fetal bovine serum at a concentration of 10% to 15%; follicle-stimulating hormone (FSH) at a concentration of 0.5 to 1.0 μg / mL; luteinizing hormone (LH) at a concentration of 5.0 to 10.0 IU / mL; and estradiol at a concentration of 1.0 to 2.0 μg / mL. After the maturation culture medium is prepared, it is sterilized by filtration through a 0.22-micron filter and equilibrated in a 37°C, 5% CO2 incubator for at least 2 hours.

[0041] The physicochemical parameters of the mature culture medium should be controlled as follows: pH value should be maintained in the range of 7.2 to 7.4; osmotic pressure should be maintained in the range of 280 to 300 mM osmol per kilogram; and temperature should be kept constant at 37.0℃ ± 0.5℃.

[0042] After screening, the naked oocytes were washed and transferred into pre-equilibrated maturation medium. Culture was performed using either four-well plates or the culture drop method. For four-well plates, 500 μL of maturation medium was added to each well, with 20 to 30 oocytes placed in each well. For the culture drop method, mineral oil was used for covering, with 50 μL of culture medium added per drop, for 10 to 15 oocytes. Culture conditions were 37°C, 5% carbon dioxide, and saturated humidity, for 22 to 24 hours.

[0043] During culture, oocytes were observed every 6 to 8 hours to record morphological changes. The criteria for completion of maturation culture were: the appearance of corona radiata cells spreading around the oocytes or the extrusion of the first polar body. After maturation culture was completed, oocytes that had extruded the first polar body were selected for in vitro fertilization.

[0044] Step 5: Semen Processing and In Vitro Fertilization Semen was collected from genetically evaluated and certified high-quality bulls using either a prosthetic vagina method or electrical stimulation. The collected semen was immediately placed in a 37°C water bath and transported to the laboratory within 30 minutes for quality assessment and processing.

[0045] Semen quality assessment includes: semen volume (normal range 4-10 ml); sperm motility (detected using a computer-aided sperm analysis system, requiring at least 70% linearly motile sperm); sperm density (measured using a hematology analyzer or professional densitometer, normal range 6-2 billion per ml); sperm abnormality rate (requiring at least 85% of sperm with normal morphology); and acrosome integrity rate (requiring at least 90% of sperm with intact acrosomes). Only high-quality semen meeting the above standards is used for subsequent processing.

[0046] Semen capacitation was performed using gradient centrifugation, and the specific steps are as follows: A density gradient system was prepared using silane-coated collagen centrifuged liquid. The gradient solution consisted of two layers: a lower layer with a density of 90% to 100% was centrifuged at high speed; and an upper layer with a density of 45% to 50% was centrifuged at low speed. The gradient solution was pre-equilibrated at 37°C for 30 minutes.

[0047] After diluting the semen sample with 1 to 2 times its volume of modified Tyrode's solution, gently stack it on top of the gradient solution, ensuring a clear liquid level. Place the test tube in a centrifuge and perform gradient centrifugation at 18°C ​​to 25°C. Set the centrifugation parameters to 500 to 800 g and 20 to 30 minutes.

[0048] After centrifugation, remove the supernatant and impurity layer, and collect the sperm clump at the bottom. Add preheated capacitation solution to 37°C and gently resuspend using a Buster pipette. Perform centrifugation and washing again to remove residual gradient solution components; repeat this step 2 to 3 times.

[0049] After capacitation, the sperm density is adjusted to 1 to 2 million per milliliter using fertilization culture medium. The final sperm suspension should have the following requirements: linear motility of ≥85%, acrosome integrity of ≥95%, and a survival time of more than 24 hours.

[0050] In vitro fertilization (IVF) is performed in a dedicated fertilization culture dish. Matured oocytes are treated with hyaluronidase to remove residual granulosa cells. After washing, 15 to 20 oocytes are transferred into 50 μL fertilization drops. The fertilization drops are covered with mineral oil, and 50 μL of sperm suspension is added to each drop to achieve a final sperm concentration of 1 to 2 million per milliliter.

[0051] The fertilization culture conditions are a three-gas culture environment of 37℃, 5% carbon dioxide, 5% oxygen, and 90% nitrogen. The co-culture time for fertilization is 18 to 20 hours. During fertilization, the sperm and oocyte complete key events such as the acrosome reaction, sperm penetration, expulsion of the second polar body, and formation of male and female pronuclei within the fertilization drop.

[0052] Step 6 Embryo Culture 18 to 20 hours after fertilization, the cumulus cells surrounding the fertilized egg are removed using a micropipette or hyaluronidase treatment. After removing the cumulus cells, fertilization is examined under an inverted microscope. The criteria for determining successful fertilization are: observation of two clear pronuclear structures, including the female pronucleus and the male pronucleus; no superfluous pronuclei or signs of polyploidy; and homogeneous cytoplasm without abnormal granules or vacuoles.

[0053] Fertilized eggs are transferred into embryo culture medium for sequential culture. The sequential culture protocol simulates the changes in nutritional requirements at different stages of embryonic development in vivo, and is divided into cleavage stage culture and blastocyst stage culture.

[0054] The cleavage stage culture phase uses CR1aa medium supplemented with 8% to 10% serum. The basic formulation of CR1aa medium contains the following components: sodium chloride at 114 mmol / L, potassium chloride at 3.1 mmol / L, sodium dihydrogen phosphate at 1.4 mmol / L, sodium bicarbonate at 26.2 mmol / L, sodium lactate at 6.0 mmol / L, sodium pyruvate at 0.5 mmol / L, glutamine at 1.0 mmol / L, ethylenediaminetetraacetic acid at 10 to 20 μmol / L, an amino acid mixture at 1 times the working concentration, and glucose at 0.5 to 1.0 mmol / L. The medium is sterilized by filtration through a 0.22-micron filter before use, and the pH is adjusted to 7.2 to 7.4 after equilibration.

[0055] Fertilized eggs are transferred to cleavage-stage culture medium and cultured in a tri-gas incubator at 37°C with 5% carbon dioxide, 5% oxygen, and 90% nitrogen. After 48 to 72 hours of culture, the embryos enter the 2 to 8-cell stage, at which point the first morphological assessment is performed, and abnormal embryos with developmental arrest or a fragmentation rate exceeding 30% are discarded.

[0056] When the embryos have been cultured for 72 to 96 hours, a half-volume medium change is performed. The method is as follows: slowly aspirate half the volume of the old culture medium, taking care to avoid mechanical damage to the embryos, and then slowly add an equal volume of fresh, pre-equilibrated culture medium. The medium change process is carried out on a 37°C heated platform to maintain a constant ambient temperature.

[0057] During the blastocyst stage, CR1aa medium supplemented with 8% to 10% serum and glucose is used. The glucose concentration is adjusted to 3.0 to 5.0 mmol / L at this stage to meet the energy metabolism requirements during blastocyst formation. Embryos are cultured until day 7 to 8, where embryo grading is performed and high-quality embryos are selected for preservation or transfer.

[0058] Embryo culture is conducted using either the droplet culture method or the single-embryo culture method. The droplet culture method uses 50-microliter culture droplets covered with mineral oil, with 1 to 3 embryos placed in each droplet; the single-embryo culture method uses single-well culture plates, with one embryo placed in each well for culture.

[0059] Step 7 Embryo Assessment and Preservation Embryo morphological assessment is performed on days 7 to 8 post-fertilization, at which time a normally developing embryo should have reached the morula or blastocyst stage. The assessment is conducted under a stereomicroscope or embryo observation system, and embryos are graded according to the following embryo quality assessment criteria: Grade A embryos are high-quality embryos, and the evaluation indicators are as follows: the number of cells reaches the normal range for a specific stage, the number of cells in a morula is usually 16 to 32, and the number of cells in the inner cell mass of a blastocyst is usually 50 to 100; the fragmentation rate is less than 10%, and the fragments are evenly distributed; the cells are of uniform size and are arranged in a tight and orderly manner; the blastocyst stage requires a clear inner cell mass, intact trophoblast cells, and a fully expanded blastocoel.

[0060] Grade B embryos are considered good embryos. The evaluation criteria are as follows: the number of cells is at the lower limit of the normal range, the number of cells in a morula is usually 12 to 20, and the number of cells in the inner cell mass of a blastocyst is usually 30 to 50; the fragmentation rate is 10% to 30%, and the fragments are relatively evenly distributed; the cells are relatively uniform in size and basically tightly arranged; and mild irregular cell arrangement is allowed.

[0061] Grade C embryos are usable embryos but of average quality. Evaluation criteria include: cell count below the lower limit of the normal range, less than 12 cells in a morula; fragmentation rate exceeding 30%; or the presence of abnormalities such as significantly uneven cell size, slow development, or cytoplasmic granulation.

[0062] Only Grade A and Grade B embryos are considered high-quality embryos suitable for cryopreservation or direct transfer. Grade C embryos may be selectively preserved or discarded based on actual needs.

[0063] Embryo preservation utilizes vitrification, a technique that employs high-concentration cryoprotectants and rapid cooling to achieve ultra-low temperature preservation of embryos, maximizing embryo viability. The vitrification procedure is as follows: First, prepare the vitrification solution, including the base solution, equilibration solution, and vitrification solution. The base solution is phosphate buffer with 20% fetal bovine serum added; the equilibration solution is the base solution with 10% to 15% ethylene glycol and 10% sucrose added; the vitrification solution is the base solution with 25% to 30% ethylene glycol, 25% to 30% dimethyl sulfoxide, and 0.5 mol / L sucrose added. After the vitrification solution is prepared, pre-cool it in a refrigerator at 4°C.

[0064] Embryos need to be dehydrated before vitrification. The embryos to be frozen are sequentially transferred to equilibration solution for 2 to 5 minutes, and then transferred to vitrification solution for 30 to 60 seconds. This process allows the embryo cells to be fully dehydrated and permeated with cryoprotectant, preparing them for rapid cooling.

[0065] The loading of embryos into cryogenic carriers must be completed within 30 seconds, ensuring that the embryos remain in the vitrification solution for no more than 60 seconds. Commonly used cryogenic carriers include cryoloops, open-ended thin plastic tubes, or cryoplates. Immediately after loading, the embryos are immersed in liquid nitrogen for rapid cooling at rates reaching thousands of degrees Celsius per minute, instantly causing the intracellular and extracellular fluids to form a glassy state rather than ice crystals.

[0066] After embryo cryopreservation, a complete record must be established, including: donor cow identification number and breed information; egg retrieval date, accurate to the year, month, and day; embryo development stage, indicating whether it is a morula or blastocyst; embryo grade, indicating grade A or grade B; cryopreservation medium number, using a unique code; storage location, indicating the liquid nitrogen tank number, cryopreservation tube rack number, and specific well location; and the personnel who performed the cryopreservation and the quality inspection results. The record is backed up using both an electronic database and paper records to ensure information traceability.

[0067] Frozen embryos require thawing and rewarming before use. The thawing process uses a rapid rewarming method: the frozen embryos are removed from liquid nitrogen and immediately immersed in a 37°C water bath for rapid rewarming, with the rewarming time controlled within 30 to 60 seconds. After thawing, the embryos are sequentially transferred to a gradient concentration of sucrose solution to remove the cryoprotectant, equilibrating for 3 to 5 minutes at each step. The rewarmed embryos are then examined under a stereomicroscope to check their morphological integrity and assess their viability. High-quality embryos should have a viability rate of over 90% before being used for transfer.

[0068] For direct embryo transfer, recipient cows in estrus at the same time are selected as the recipients. The transfer procedure is non-surgical; the cervix is ​​grasped through the recipient cow's rectum, and the transfer catheter is inserted deep into the uterine horn through the cervix, slowly injecting the embryo. After transfer, the recipient cow is monitored for pregnancy diagnosis to observe whether conception was successful.

[0069] Example 2 In another specific embodiment, the present invention provides an optimized method for producing in vitro fertilization embryos from live oocytes of Wagyu beef cattle, which is based on Example 1 with parameter adjustments and technical detail optimizations.

[0070] Molecular genetic testing has been added to the donor cattle selection process. Genomic DNA is extracted from the donor cattle's blood, and polymerase chain reaction (PCR) is used to amplify candidate gene loci associated with reproductive traits, thus detecting genotype information. Simultaneously, ear tissue samples are collected from the donor cattle for karyotype analysis to confirm normal chromosome number and structure. Molecular genetic testing can effectively screen individuals with superior genetic qualities and high reproductive potential as donor cattle, improving the genetic gain of embryo production.

[0071] The superovulation treatment employs a modified tapering injection protocol. The total follicle-stimulating hormone (FSH) dose is adjusted to 350-450 IU, administered in 7-9 divided doses with intervals shortened to 10 hours. This protocol induces a more consistent follicle development response and increases the number of usable oocytes retrieved. The luteinizing hormone (LH) injection time is adjusted to 20-22 hours after the last FSH injection, with the dose increased to 200-250 IU to ensure final follicle maturation and ovulation synchronization.

[0072] A nutritional regulation program for donor cattle is added to the pre-retrieval preparation process. Donor cattle undergo nutritional optimization feeding 30 to 45 days prior to retrieval, with rumen-protected protein and fat added to their diets to increase energy intake. Simultaneously, micronutrient supplements, including zinc, copper, manganese, and selenium, are added to maintain micronutrient balance. This nutritional regulation can improve oocyte quality and superovulation.

[0073] The oocyte retrieval process employs a bilateral alternating ovarian puncture strategy. After the initial puncture of all target follicles in the left ovary, the procedure is paused for 15 to 20 minutes to observe the donor cow's condition. Once no abnormalities are confirmed, the right ovary is then punctured. This strategy reduces the stress on the donor cow per procedure, improving animal welfare. The negative pressure during puncture is adjusted to 80 to 90 mmHg to minimize mechanical damage to the oocytes while ensuring efficient retrieval.

[0074] The oocyte selection process incorporates in vitro maturation rate prediction indicators. High-resolution microscopy is used to observe the distribution of mitochondria and the location of cortical granules in oocytes to assess their maturation potential. Oocytes with uniformly distributed fine granules in the cytoplasm and mitochondria concentrated in the peripheral region have higher maturation rates and are preferentially selected for in vitro fertilization.

[0075] The maturation culture process was optimized. Serum-free maturation culture medium was used instead of serum-containing medium to reduce batch-to-batch variability. Cysteine ​​(0.5–1.0 mmol / L) and β-mercaptoethanol (0.1–0.5 mmol / L) were added to the culture medium to enhance the antioxidant capacity of oocytes. The culture environment was adjusted to hypoxic conditions, with an oxygen concentration set at 2%–5%, simulating the environment within the fallopian tubes.

[0076] In the in vitro fertilization (IVF) process, the zona pellucida binding method is used to optimize fertilization efficiency. Capacitated sperm and oocytes undergo zona pellucida induction binding treatment before fertilization. Low concentrations of trypsin or calcium ionophores are used to treat the sperm, enhancing their ability to penetrate the zona pellucida. The sperm concentration in the fertilization droplet is reduced to 500,000 to 1,000,000 per milliliter, decreasing the incidence of polyspermia.

[0077] Embryo culture was continuously monitored using a time-lapse monitoring system. This system, equipped with automated imaging, captured images of embryonic development every 15 to 30 minutes, recording embryonic division time, changes in cell number, and fragmentation processes. Analysis of embryonic developmental kinetic parameters allowed for a more accurate assessment of embryonic developmental potential, enabling the selection of high-quality embryos with normal developmental rates and good cell division synchronicity.

[0078] The timing of sequential culture medium changes should be adjusted to when the embryo develops to the 8-16 cell stage. Changing the medium too early may cause a sudden change in the nutritional environment, while changing the medium too late will fail to meet the nutritional needs of embryonic development. An additional 0.5 to 1.0 mmol / L mixture of non-essential amino acids should be added to the culture medium during the blastocyst stage to support the differentiation and development of the inner cell mass and trophoblast cells.

[0079] The embryo evaluation process now includes the detection of metabolic markers. Microsampling techniques are used to collect culture medium surrounding individual embryos, and enzyme-linked immunosorbent assay (ELISA) or real-time quantitative polymerase chain reaction (qPCR) is employed to detect pyruvate uptake, lactate production, and specific gene expression levels. Embryos with metabolic activity within the normal range have higher implantation and pregnancy rates.

[0080] The embryo preservation process utilizes an automated vitrification freezing system. This system integrates embryo loading, freezing, and cryopreservation, with standardized and procedural operations. Freezing parameters are preset and executed automatically, eliminating human error. The survival rate of thawed embryos can reach over 95%, significantly improving the utilization efficiency of frozen embryos.

[0081] A precise synchronization protocol is implemented during embryo transfer. Progesterone vaginal suppositories and prostaglandins are used to synchronize the estrus of recipient cows, ensuring a precise match between the estrus timing and the embryonic development stage. Before transfer, ultrasound is used to assess the development of the corpus luteum in the recipient cow's ovary, and individuals with well-developed corpora lutea are selected for transfer. The first pregnancy diagnosis is performed 14 to 21 days post-transfer, and a second confirmation is performed 28 to 35 days post-transfer.

[0082] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A method for producing in vitro fertilization embryos from live oocytes of Wagyu beef cattle, characterized in that, Includes the following steps: Step 1: Donor cattle screening and superovulation treatment. Health checks and reproductive performance assessments are conducted on donor Wagyu beef cattle. Individuals that meet the standards are selected as donor cattle. The donor cattle are then subjected to superovulation treatment, which involves continuous injection of follicle-stimulating hormone and luteinizing hormone to induce the synchronous development of multiple follicles in the ovaries. Step 2: Preparation before egg retrieval. After superovulation treatment, the donor cow to be retrieved is restrained within a preset time period. The ovarian response is checked through the rectum. After confirming the follicle development status, the skin is prepared and disinfected. Step 3: Live oocyte retrieval. Transrectal ultrasound-guided puncture technique is used. An ultrasound probe with a puncture needle is inserted through the rectal wall into the ovarian follicle, and negative pressure is used to aspirate the follicular fluid to obtain the oocyte complex. Step 4: In vitro maturation culture of oocytes. The obtained oocyte complexes are screened under a stereomicroscope, and after removing granulosa cells, they are transferred into maturation culture medium and cultured under preset culture conditions. Step 5: Semen processing and in vitro fertilization. Collect fresh semen from high-quality bulls and perform gradient centrifugation for capacitation. Mix the processed sperm with mature oocytes at a preset concentration and perform in vitro fertilization in fertilization culture medium. Step 6: Embryo culture. After fertilization, remove cumulus cells at a preset time, check fertilization status, and transfer fertilized eggs into embryo culture medium for sequential culture. Cleavage embryo culture medium is used in the early stage, and blastocyst culture medium is used in the later stage, for a total of preset number of days. Step 7: Embryo evaluation and preservation. Morphological evaluation is performed on morula or blastocysts cultured to the preset stage. High-quality embryos are selected for vitrification and cryopreservation or direct transfer.

2. The method for producing in vitro fertilization embryos from live oocytes of Wagyu beef cattle according to claim 1, characterized in that, In step 1, the superovulation treatment adopts a decreasing injection protocol. The total dose of follicle-stimulating hormone (FSH) is a preset dose, which is injected in preset numbers at preset intervals. The dose of each injection is decreasing by a preset percentage. The total dose of luteinizing hormone (LH) is a preset dose, which is injected once at a preset time after the last FSH injection.

3. The method for producing in vitro fertilization embryos from live oocytes of Wagyu beef cattle according to claim 1, characterized in that, In step 2, before egg retrieval, the number and distribution of follicles of a predetermined size or larger on both ovaries are recorded by digital rectal examination and ultrasound exploration. Individuals with a predetermined number of follicles are selected for egg retrieval.

4. The method for producing in vitro fertilization embryos from live oocytes of Wagyu beef cattle according to claim 1, characterized in that, In step 3, the oocyte retrieval is performed using a pre-specified single-lumen puncture needle, the negative pressure is set to a pre-specified negative pressure value, the number of follicles punctured on each ovary does not exceed a pre-specified number, and the time for a single oocyte retrieval operation is controlled within a pre-specified duration.

5. The method for producing in vitro fertilization embryos from live oocytes of Wagyu beef cattle according to claim 1, characterized in that, In step 4, the oocyte selection criteria are as follows: oocytes with a diameter that reaches the preset size, uniform cytoplasm, containing more than the preset number of complete granulosa cell layers, and with uniform light brown cytoplasm are preferred.

6. The method for producing in vitro fertilization embryos from live oocytes of Wagyu beef cattle according to claim 1, characterized in that, In step 4, the maturation culture medium is a TCM199 basal medium with a preset ratio of fetal bovine serum, a preset concentration of follicle-stimulating hormone, a preset concentration of luteinizing hormone, and a preset concentration of estradiol. The pH value and osmotic pressure of the culture medium are within the preset range.

7. The method for producing in vitro fertilization embryos from live oocytes of Wagyu beef cattle according to claim 1, characterized in that, In step 5, the sperm capacitation treatment uses a silane-coated collagen centrifuge solution with a preset density gradient for gradient centrifugation. The centrifugation speed is a preset centrifugal force, and the centrifugation time is a preset centrifugation duration. After capacitation treatment, the sperm motility reaches a preset motility threshold, and the survival time reaches a preset survival duration.

8. The method for producing in vitro fertilization embryos from live oocytes of Wagyu beef cattle according to claim 1, characterized in that, In step 6, during the initial stage of sequential culture, CR1aa medium with a preset proportion of serum is used. When the embryos are cultured to a preset time point, half of the medium is replaced. Subsequently, CR1aa medium with a preset proportion of serum and a preset concentration of glucose is used. The embryos are cultured for a preset number of days to assess their grade.

9. The method for producing in vitro fertilization embryos from live oocytes of Wagyu beef cattle according to claim 1, characterized in that, It also includes embryo quality assessment standards, which classify embryos into three grades: A, B, and C. Grade A embryos have a cell count that meets the preset number, a fragmentation rate that is lower than the preset percentage, and uniform cell size. Grade B embryos have a cell count that is within the preset range, a fragmentation rate that is within the preset range, and relatively uniform cell size. Grade C embryos have a cell count that is lower than the preset number or a fragmentation rate that exceeds the preset percentage.

10. The method for producing in vitro fertilization embryos from live oocytes of Wagyu beef cattle according to claim 1, characterized in that, It also includes embryo preservation record management, establishing complete files for cryopreserved embryos, recording donor surrogacy number, egg retrieval date, embryo development stage, embryo grade, cryopreservation medium number and storage location information, and ensuring that the survival rate of frozen embryos reaches a preset percentage.

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