Method for optimizing maturation effect of cattle and sheep oocytes through CNP pretreatment

Abstract

The invention discloses a method for optimizing the maturation effect of cattle and sheep oocytes through CNP pretreatment, and relates to the technical field of biological breeding, the method comprises the following steps: S1, selecting and treating donor cells; s2, collection of oocytes; s3, in-vitro maturation culture of the oocytes: after the pre-maturation treatment, transferring the oocytes into a maturation culture solution for continuous culture under the in-vitro culture condition of air containing 5% CO2, keeping the culture temperature of the oocytes within 1-20 hours at 38.5-39 DEG C, and keeping the culture temperature of the oocytes within 20-28 hours at 39-39.5 DEG C; s4, culturing and screening reconstructed embryos; s5, preparing and transplanting a receptor animal; and S6, performing subsequent monitoring and management. According to the method, the culture temperature is kept at 38.5-39 DEG C within 1-20 hours, the culture temperature is kept at 39-39.5 DEG C within 20-28 hours, a series of enzymes and signal channels in the oocytes can be effectively activated by accurately controlling the culture temperature, and the survival rate and the development quality of the oocytes are improved.

Description

Technical Field

[0001] The invention relates to the technical field of biological reproduction, in particular to a method for optimizing the maturation effect of cattle and sheep oocytes by CNP pretreatment. Background Art

[0002] In the field of cattle and sheep breeding, improving reproductive efficiency has always been a key issue that researchers have focused on. The level of reproductive efficiency mainly depends on multiple key links such as the maturation quality of oocytes, fertilization rate, and the potential for subsequent embryonic development. As one of the core elements of the reproductive process, the maturation quality of oocytes is crucial to the entire reproductive process. High-quality oocytes can provide a good foundation for fertilization and embryonic development; After searching, China Patent Authorization Announcement No. CN118956735A discloses a method for accurately optimizing the maturation cycle of cattle and sheep oocytes with actinomycin as the core, including: the control solution is composed of 1-50 micrograms / ml actinomycin, the maturation solution is composed of M199 culture medium and 10% volume of serum or bovine serum albumin, or other similar substitutes, and the maturation solution does not contain FSH and LH hormones. The present invention can effectively improve the maturation rate of cattle and sheep oocytes. By accurately using actinomycin as a protein synthesis inhibitor, the protein synthesis of oocytes is regulated within a specific concentration range without affecting the synthesis of RNA, which creates unique conditions for the maturation of oocytes. After pretreatment and maturation solution culture, the improvement of oocyte maturity can be clearly seen through microscopic observation and staining methods. Mature oocytes have obvious characteristics, such as the discharge of the first polar body, uniform cytoplasm and clear nucleus, so that the maturation rate is significantly improved.

[0003] CNP pretreatment requires precise temperature and time control. The existing CNP pretreatment temperature and time are vague, which may lead to insufficient or excessive treatment of some oocytes, thus affecting their maturation quality and subsequent fertilization ability. In addition, the existing culture medium may have an imbalance in energy supply, affecting the mitochondrial function and energy production of the oocytes, which will increase the difficulty and error rate of the experiment. Summary of the invention

[0004] The purpose of the present invention is to solve the problem that CNP pretreatment requires precise operation steps and time control, CNP pretreatment requires precise temperature and time control, the existing CNP pretreatment temperature and time are general, which may lead to insufficient or excessive treatment of some oocytes, thereby affecting their maturation quality and subsequent fertilization ability, and the existing culture medium may have an imbalance in energy supply, affecting the mitochondrial function and energy production of the oocyte, and increasing the difficulty and error rate of the experiment. A method for optimizing the maturation effect of cattle and sheep oocytes by CNP pretreatment is provided.

[0005] To achieve the above object, the present invention provides the following technical solutions: The present invention is a method for optimizing the maturation effect of cattle and sheep oocytes by CNP pretreatment, comprising the following steps: S1. Selection and processing of donor cells: Select healthy donor animals with good genetic background and extract their skin cells or fibroblasts as donor cells; S2. Oocyte collection: Choose an appropriate method to collect oocytes from the ovaries after follicle puncture and slaughter under ultrasound guidance; S3. In vitro maturation culture of oocytes: After pre-maturation treatment, the oocytes are transferred to the maturation culture medium for further culture. The in vitro culture conditions are air containing 5% CO2. The culture temperature of the oocytes is maintained at 38.5 to 39 degrees Celsius within 1-20 hours and at 39 to 39.5 degrees Celsius within 20-28 hours. S4. Cultivation and screening of reconstructed embryos: Using micromanipulation techniques, donor cells are fused with enucleated oocytes to form reconstructed embryos; S5. Recipient animal preparation and transplantation: Select appropriate recipient animals and transplant the reconstructed embryos into the recipient animals; S6. Subsequent monitoring and management: After transplantation, the recipient animals should be examined regularly to monitor their pregnancy status.

[0006] The above steps can improve the quality of CNP pretreatment to optimize the maturation of bovine and sheep oocytes. In an air environment containing 5% CO2, the culture temperature is maintained between 38.5 and 39 degrees Celsius for the first 20 hours, and then the culture temperature is increased to 39 to 39.5 degrees Celsius to promote the maturation of oocytes.

[0007] As a further solution of the present invention: in S1, selection and processing of donor cells, the operator should perform health monitoring on the donors one month in advance, with the monitoring frequency being twice a week.

[0008] By monitoring the donor in advance, we can ensure that the quality of the oocytes is optimal. High-quality oocytes can increase the success rate of in vitro fertilization and reduce the risk of abnormal embryo development.

[0009] As a further solution of the present invention: in S2, the collection of oocytes includes ovarian collection and follicle collection. The ovarian collection is oocyte collection, and a 5 mL syringe and a No. 12 needle are used to extract oocytes from follicles with a diameter of 2 to 6 mm.

[0010] By precisely controlling the size of the needle and the force of suction, mechanical damage to the oocyte can be reduced, the integrity of the oocyte can be effectively protected, and the success rate of subsequent fertilization and embryo development can be improved.

[0011] As a further solution of the present invention: the ovary collection is to aseptically collect cattle and sheep ovaries from slaughterhouses, remove excess fat tissue, wipe with sterile absorbent paper, and store in physiological saline at 25°C to 35°C, and add antibiotics to prevent contamination.

[0012] By storing oocytes in an appropriate temperature range (25°C to 35°C), their metabolic rate can be slowed down and the survival time of oocytes can be extended.

[0013] As a further scheme of the present invention: in the in vitro maturation culture of S3, oocytes, the operator uses M199 culture medium and adds 10% newborn calf serum (NCS), 1-2 mg / L 17β-estradiol (E2), 10 mmol / L Hepes, 0.12 mg / mL sodium pyruvate and double antibodies (0.1 mg / mL streptomycin and 0.125 mg / mL penicillin) to prepare the maturation culture medium. Adding GnRH to the culture medium can simulate the in vivo environment and promote the meiotic recovery of oocytes.

[0014] The M199 culture medium has good buffering capacity and pH stability, which helps maintain the stability of the culture environment and reduce the impact of external factors on the oocyte maturation process.

[0015] As a further scheme of the present invention: in the culture and screening of reconstructed embryos in S4, oocytes are collected and cultured in vitro to the metaphase of the second meiotic division. The nucleus in the oocyte is removed by micromanipulation. Since the position of the nucleus in the metaphase of the second meiotic division is close to the first polar body, the nucleus and the first polar body can be sucked out together with a micropipette. The donor cell is injected into the enucleated oocyte and the two cells are fused by electrical stimulation. The donor cell enters the recipient oocyte to construct a reconstructed embryo. The morphology of the embryo, including the number of cells, cell size, and cell arrangement, is observed under a microscope to evaluate the quality of the embryo. The measurement and evaluation rate is maintained every five hours, and the developmental stage of the embryo at different time points, the number of blastomeres and the formation of blastocysts are recorded to judge the developmental potential of the embryo. The oxygen consumption rate of the embryo during the culture process is measured every two hours to evaluate its metabolic activity.

[0016] The process of fusing two cells through electrical stimulation simulates the natural fertilization process, which helps maintain the normal development trajectory of the embryo, thereby improving the overall quality of the embryo. Observing the embryo morphology every five hours, including indicators such as cell number, size and arrangement, can promptly detect abnormal embryos and avoid using low-quality embryos for subsequent experiments or transplantation.

[0017] As a further solution of the present invention: in said S5, during the preparation and transplantation of the recipient animal, the recipient animal is subjected to estrus synchronization treatment while culturing the oocytes, so that its endometrium is in a state suitable for transplantation.

[0018] By arranging the experimental time reasonably, we can ensure that embryo transplantation is performed when the recipient animal is in a suitable transplantation state, thereby improving the experimental efficiency and accuracy.

[0019] As a further solution of the present invention: in the said S6, follow-up monitoring and management, the monitoring data are regularly analyzed, the pregnancy progress is evaluated, and the management strategy is adjusted.

[0020] Through monitoring data, management strategies can be adjusted in a timely manner, and appropriate measures can be taken to solve problems and improve the success rate of pregnancy.

[0021] Compared with the prior art, the present invention has the following beneficial effects: During the in vitro maturation of oocytes, the culture temperature is maintained at 38.5 to 39 degrees Celsius within 1-20 hours and at 39 to 39.5 degrees Celsius within 20-28 hours. Accurately controlling the culture temperature can promote the normal development of oocytes, effectively activate a series of enzymes and signal pathways in oocytes, and promote their maturation and development. Accurately controlling the temperature can prevent oocytes from being damaged by excessively high or low temperatures, thereby improving the survival rate and development quality of oocytes. In addition, adding newborn calf serum (NCS) to the culture medium, the protein in the serum can help oocytes better adhere to the bottom of the culture dish, which is beneficial to the normal development of cells. Adding 1-2 mg / L 17β-estradiol (E2) to in vitro culture can simulate the hormone environment in the body and promote the meiotic recovery of oocytes. 10 mmol / L Hepes is an effective buffer that can maintain the pH value of the culture medium stable within a certain range and prevent changes in the pH of the culture medium caused by the accumulation of metabolites. Through precise culture temperature and time, combined with M199 culture medium, and the addition of 10% newborn calf serum (NCS), 1-2 mg / L 17β-estradiol (E2), 10 mmol / L Hepes, 0.12 mg / mL sodium pyruvate and double antibodies (0.1 mg / mL streptomycin and 0.125 mg / mL penicillin) to prepare mature culture medium, the quality of maturation of bovine and sheep oocytes is improved. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 The present invention is a flow chart of the method for optimizing the maturation effect of bovine and sheep oocytes by CNP pretreatment. DETAILED DESCRIPTION

[0023] In order to make the technical problems, technical solutions and advantages to be solved by the present invention more clear, a detailed description will be given below with reference to the accompanying drawings and specific embodiments.

[0024] See also Figure 1In an embodiment of the present invention, the method for optimizing the maturation effect of cattle and sheep oocytes by CNP pretreatment comprises the following steps: S1. Selection and processing of donor cells: Select healthy donor animals with good genetic background and extract their skin cells or fibroblasts as donor cells; S2. Oocyte collection: Choose an appropriate method to collect oocytes from the ovaries after follicle puncture and slaughter under ultrasound guidance; S3. In vitro maturation culture of oocytes: After pre-maturation treatment, the oocytes are transferred to the maturation culture medium for further culture. The in vitro culture conditions are air containing 5% CO2. The culture temperature of the oocytes is maintained at 38.5 to 39 degrees Celsius within 1-20 hours and at 39 to 39.5 degrees Celsius within 20-28 hours. S4. Cultivation and screening of reconstructed embryos: Using micromanipulation techniques, donor cells are fused with enucleated oocytes to form reconstructed embryos; S5. Recipient animal preparation and transplantation: Select appropriate recipient animals and transplant the reconstructed embryos into the recipient animals; S6. Subsequent monitoring and management: After transplantation, the recipient animals should be examined regularly to monitor their pregnancy status.

[0025] In S1, selection and processing of donor cells, operators should monitor the health of donors one month in advance and twice a week.

[0026] In S2, oocyte collection, including ovarian collection and follicle collection, ovulation collection is oocyte collection. Using a 5mL syringe and a No. 12 needle, oocytes are extracted from follicles with a diameter of 2 to 6mm. This can significantly improve the collection efficiency. The method is simple and fast, suitable for large-scale operations, and can meet the needs of scientific research and production. Through microscopic examination, cumulus-oocyte complexes (COCs) with normal morphology and good development can be screened out, thereby ensuring the reliability of the experimental results.

[0027] Ovarian collection is the process of aseptically collecting cattle and sheep ovaries from slaughterhouses, removing excess fat tissue, wiping them with sterile absorbent paper, and storing them in normal saline at 25°C to 35°C. Antibiotics are added to prevent contamination. Aseptically collecting cattle and sheep ovaries and removing excess fat tissue can reduce the impact of microbial contamination and impurities. After wiping them dry with sterile absorbent paper, they are stored in normal saline at 25°C to 35°C. Antibiotics are added to prevent contamination, which helps maintain the quality and activity of oocytes, slows down their metabolic rate, and prolongs the survival time of oocytes.

[0028] S3. In the in vitro maturation culture of oocytes, the operator used M199 medium and added 10% newborn calf serum (NCS), 1-2 mg / L 17β-estradiol (E2), 10 mmol / L Hepes, 0.12 mg / mL sodium pyruvate and double antibodies (0.1 mg / mL streptomycin and 0.125 mg / mL penicillin) to prepare the maturation culture medium. Adding GnRH to the culture medium can simulate the in vivo environment and promote the meiotic recovery of oocytes. Among them, M199 is a commonly used cell culture medium, which provides the basic nutrients and trace elements required for cell growth. For the in vitro maturation culture of oocytes, M199 can provide the necessary environmental conditions to support the development of oocytes. NCS is rich in a variety of growth factors, hormones and other bioactive substances, which can promote the growth and development of oocytes and improve their maturation rate. E2 is one of the important sex hormones in the female reproductive system, which regulates the maturation process of oocytes in vivo. Adding an appropriate amount of E2 in in vitro culture can simulate the hormone environment in the body and promote the meiotic recovery of oocytes. Hepes is an effective buffer that can maintain the pH value of the culture medium stable within a certain range and prevent the pH change of the culture medium caused by the accumulation of metabolites. Sodium pyruvate is an important intermediate product in the cell metabolism process. It can provide energy for oocytes and support their normal life activities. GnRH is a hormone secreted by the hypothalamus, which regulates the release of gonadotropin in the body. Adding GnRH in in vitro culture can simulate the endocrine environment in the body and promote the meiotic recovery of oocytes.

[0029] S4. In the culture and screening of reconstructed embryos, oocytes are collected and cultured in vitro to the metaphase of the second meiotic division. The nucleus in the oocyte is removed by micromanipulation. Since the nucleus of the metaphase of the second meiotic division is close to the first polar body, the nucleus and the first polar body can be sucked out together with a micropipette. The donor cell is injected into the enucleated oocyte and the two cells are fused by electrical stimulation. The donor cell enters the recipient oocyte to construct a reconstructed embryo. The morphology of the embryo, including the number of cells, cell size, cell arrangement, etc., is observed under a microscope to evaluate the quality of the embryo. The measurement and evaluation rate is maintained every five hours, and the developmental stage of the embryo at different time points, the number of blastomeres and the formation of blastocysts are recorded to judge the developmental potential of the embryo. The oxygen consumption rate of the embryo during the culture process is measured every two hours to evaluate its metabolic activity. Among them, observing the embryo morphology every five hours, including indicators such as cell number, size and arrangement, can timely detect abnormal embryos and avoid using low-quality embryos for subsequent experiments or transplantation. Recording the number of blastomeres and blastocyst formation at different time points will help evaluate the development speed and potential of the embryo and provide a basis for choosing the best time for transplantation.

[0030] S5. During the preparation and transplantation of recipient animals, the recipient animals are subjected to estrus synchronization while culturing oocytes so that their endometrium is in a state suitable for transplantation. Synchronous estrus treatment can make the culture process of recipient animals and oocytes more coordinated.

[0031] S6. During follow-up monitoring and management, regularly analyze monitoring data, evaluate pregnancy progress, and adjust management strategies.

[0032] During the in vitro maturation of oocytes, the culture temperature is maintained at 38.5 to 39 degrees Celsius within 1-20 hours and at 39 to 39.5 degrees Celsius within 20-28 hours. Accurately controlling the culture temperature can promote the normal development of oocytes, effectively activate a series of enzymes and signal pathways in oocytes, and promote their maturation and development. Accurately controlling the temperature can prevent oocytes from being damaged by excessively high or low temperatures, thereby improving the survival rate and development quality of oocytes. In addition, adding newborn calf serum (NCS) to the culture medium, the protein in the serum can help oocytes better adhere to the bottom of the culture dish, which is beneficial to the normal development of cells. Adding 1-2 mg / L 17β-estradiol (E2) to in vitro culture can simulate the hormone environment in the body and promote the meiotic recovery of oocytes. 10 mmol / L Hepes is an effective buffer that can maintain the pH value of the culture medium stable within a certain range and prevent changes in the pH of the culture medium caused by the accumulation of metabolites. Through precise culture temperature and time, combined with M199 culture medium, and the addition of 10% newborn calf serum (NCS), 1-2 mg / L 17β-estradiol (E2), 10 mmol / L Hepes, 0.12 mg / mL sodium pyruvate and double antibodies (0.1 mg / mL streptomycin and 0.125 mg / mL penicillin) to prepare mature culture medium, the quality of maturation of bovine and sheep oocytes is improved.

[0033] What is described above is only a preferred specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Any technician familiar with the technical field can make equivalent replacements or changes according to the technical scheme and inventive concept of the present invention within the technical scope disclosed by the present invention, which should be covered by the protection scope of the present invention.

Claims

1. A method for optimizing the maturation effect of cattle and sheep oocytes by CNP pretreatment, characterized in that: The steps include: S1. Selection and processing of donor cells: Select healthy donor animals with good genetic background and extract their skin cells or fibroblasts as donor cells; S2. Oocyte collection: Choose an appropriate method to collect oocytes from the ovaries after follicle puncture and slaughter under ultrasound guidance; S3. In vitro maturation culture of oocytes: After pre-maturation treatment, the oocytes are transferred to the maturation culture medium for further culture. The in vitro culture conditions are air containing 5% CO2. The culture temperature of the oocytes is maintained at 38.5 to 39 degrees Celsius within 1-20 hours and at 39 to 39.5 degrees Celsius within 20-28 hours. S4. Cultivation and screening of reconstructed embryos: Using micromanipulation techniques, donor cells are fused with enucleated oocytes to form reconstructed embryos; S5. Recipient animal preparation and transplantation: Select appropriate recipient animals and transplant the reconstructed embryos into the recipient animals; S6. Subsequent monitoring and management: After transplantation, the recipient animals should be examined regularly to monitor their pregnancy status.

2. The method for optimizing the maturation effect of cattle and sheep oocytes by CNP pretreatment according to claim 1, characterized in that: In S1, selection and processing of donor cells, operators should monitor the health of donors one month in advance and twice a week.

3. The method for optimizing the maturation effect of cattle and sheep oocytes by CNP pretreatment according to claim 1, characterized in that: In S2, oocyte collection includes ovarian collection and follicle collection. The follicle collection is oocyte collection. Oocytes are extracted from follicles with a diameter of 2 to 6 mm using a 5 mL syringe and a 12-gauge needle.

4. The method for optimizing the maturation effect of cattle and sheep oocytes by CNP pretreatment according to claim 3, characterized in that: The ovary collection is to collect cattle and sheep ovaries from slaughterhouses in a sterile manner, remove excess fat tissue, wipe with sterile absorbent paper, and store in physiological saline at 25°C to 35°C, and add antibiotics to prevent contamination.

5. The method for optimizing the maturation effect of cattle and sheep oocytes by CNP pretreatment according to claim 1, characterized in that: In the S3, in vitro maturation culture of oocytes, the operator uses M199 culture medium and adds 10% newborn calf serum (NCS), 1-2 mg / L 17β-estradiol (E2), 10 mmol / L Hepes, 0.12 mg / mL sodium pyruvate and double antibodies (0.1 mg / mL streptomycin and 0.125 mg / mL penicillin) to prepare the maturation culture medium. Adding GnRH to the culture medium can simulate the in vivo environment and promote the meiotic recovery of oocytes.

6. The method for optimizing the maturation effect of cattle and sheep oocytes by CNP pretreatment according to claim 1, characterized in that: In the culture and screening of reconstructed embryos in S4, oocytes are collected and cultured in vitro to the metaphase of the second meiotic division. The nucleus in the oocyte is removed by micromanipulation. Since the position of the nucleus in the metaphase of the second meiotic division is close to the first polar body, the nucleus and the first polar body can be sucked out together with a micropipette. The donor cell is injected into the enucleated oocyte and the two cells are fused by electrical stimulation. The donor cell enters the recipient oocyte to construct a reconstructed embryo. The morphology of the embryo, including the number of cells, cell size, and cell arrangement, is observed under a microscope to evaluate the quality of the embryo. The measurement and evaluation rate is maintained every five hours, and the developmental stage of the embryo at different time points, the number of blastomeres, and the formation of blastocysts are recorded to judge the developmental potential of the embryo. The oxygen consumption rate of the embryo during the culture process is measured every two hours to evaluate its metabolic activity.

7. The method for optimizing the maturation effect of cattle and sheep oocytes by CNP pretreatment according to claim 1, characterized in that: In the above-mentioned S5, during the preparation and transplantation of the recipient animal, the recipient animal is subjected to estrus synchronization while the oocytes are cultured so that its endometrium is in a state suitable for transplantation.

8. The method for optimizing the maturation effect of cattle and sheep oocytes by CNP pretreatment according to claim 1, characterized in that: In the follow-up monitoring and management of S6, the monitoring data are regularly analyzed, the pregnancy progress is evaluated, and the management strategy is adjusted.

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