Immune factor combination capable of improving development efficiency of bovine in-vitro embryos and application

Abstract

The invention relates to the technical field of livestock embryo engineering, in particular to an immune factor combination capable of improving bovine in-vitro embryo development efficiency and application, and the combination comprises IL-1alpha and IL-10. The culture medium is mainly composed of proinflammatory factor interleukin-1 alpha (IL-1 alpha) and anti-inflammatory factor interleukin-10 (IL-10) which are secreted by macrophages, and the deficiency of an immune regulation mechanism in a traditional in-vitro embryo culture system is effectively made up by simulating an immune microenvironment of in-vivo embryo development; the bovine in-vitro embryo culture process is obviously optimized, the excellent application potential is shown in the aspect of improving the embryo development efficiency and quality, and a brand new solution is provided for breaking through the bottleneck of the existing in-vitro embryo culture technology.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of livestock embryo engineering, in particular to an immune factor combination for improving the efficiency of bovine in vitro embryo development and application. BACKGROUND

[0002] The in vitro culture technology of livestock embryos has both important scientific research value and commercial development potential. The in vitro embryo culture and embryo transfer technology system based on superovulation technology has been widely used in the field of dairy cattle genetic improvement. Through this technology system, the number of offspring of excellent cows is significantly improved, and the reproductive efficiency is effectively optimized. However, compared with in vivo normally developed embryos, the quality and development efficiency of embryos obtained by in vitro culture still have significant differences. The in vitro culture process can cause changes in the DNA methylation patterns of follicular cells and embryos, and then cause adverse phenomena such as reduced embryo implantation rate and delayed development process. In addition, the newborn individuals developed from in vitro embryos are often accompanied by various congenital diseases, including glucose metabolism dysfunction, insulin resistance, macrosomia, and epigenetic disorders. The above problems seriously limit the application performance and promotion range of in vitro embryo production technology, and become the key bottleneck restricting the further expansion of embryo engineering technology.

[0003] Under physiological conditions, the ovulation process is regulated by the inflammatory response mediated by cytokines secreted by lymphocytes, macrophages and granulocytes; at the same time, the growth factors and chemotactic factors secreted by the female reproductive tract can promote the development process of pre-implantation embryos, and these growth factors are most likely to act as epigenetic determinants of post-implantation embryo development potential and pregnancy outcome. Compared with the microenvironment in which in vivo embryos grow, the in vitro embryo culture system lacks the synergistic participation of the immune system. Therefore, constructing and improving the immune system in the in vitro embryo culture system may be the key to solving the current problems of poor in vitro embryo culture efficiency and low quality. SUMMARY

[0004] The technical problem to be solved by the present application is to provide an immune factor combination for improving the efficiency of bovine in vitro embryo development and application, which is mainly composed of pro-inflammatory factor interleukin-1 alpha (IL-1 alpha) and anti-inflammatory factor interleukin-10 (IL-10) secreted by macrophages. By simulating the immune microenvironment of in vivo embryo development, the lack of immune regulation mechanism in the traditional in vitro embryo culture system is effectively made up; the bovine in vitro embryo culture process is significantly optimized, which shows excellent application potential in improving embryo development efficiency and quality, and provides a new solution to break through the bottleneck of existing in vitro embryo culture technology.

[0005] In order to solve the above technical problems, the technical scheme adopted by the present application is as follows:

[0006] The application discloses a combination of immune factors for improving the development efficiency of bovine embryos in vitro, and relates to the field of animal biotechnology.

[0007] Preferably, the concentration of IL-1α in the culture solution is 10-30 ng / mL, and the concentration of IL-10 in the culture solution is 10-30 ng / mL.

[0008] Preferably, the combination further comprises a basic culture medium, and the basic culture medium is an in-vitro embryo culture solution.

[0009] Preferably, the in-vitro embryo culture solution is IVC, which is from BO-IVF Company and has a product number of 71005.

[0010] The application discloses a combination of immune factors for improving the development efficiency of bovine embryos in vitro, and relates to the field of animal biotechnology.

[0011] Preferably, the preparation method of bovine in-vitro fertilized embryos comprises the following steps.

[0012] S1, mature culture of oocytes;

[0013] S2, thawing and purifying of sperm, and combining the mature oocytes in step S1 with the purified sperm to obtain fertilized embryos;

[0014] S3, culturing the fertilized embryos in step S2 in an embryo in-vitro culture solution containing IL-1α and IL-10;

[0015] S4, evaluating the quality of the embryos cultured in step S3: performing immunofluorescence positioning on the inner cell mass and the trophoblast cells by using the marker SOX2 of the early embryo inner cell mass and the marker CDX2 of the trophoblast cells, and evaluating the quality of the embryos by using the ratio of the number of the inner cell mass to the number of the trophoblast cells and the total number of all cells in the embryos.

[0016] Preferably, the specific operation of the mature culture of oocytes in step S1 comprises the following steps.

[0017] S11, collecting bovine ovaries, placing the bovine ovaries in a heat preservation container and sending the bovine ovaries to a laboratory within 1 h;

[0018] S12, removing connective tissue, adipose tissue and oviduct structures attached to the surface of the ovary and then rinsing;

[0019] S13, performing puncture operation on the follicles with a diameter of 2-10 mm on the surface of the ovary to obtain oocytes;

[0020] S14, screening and collecting cumulus-oocyte complexes COCs and cleaning;

[0021] S15, screening the morphologically normal COCs are rinsed twice in the in vitro maturation medium to remove residual impurities and potential inhibitors;

[0022] S16, transfer to the in vitro maturation medium system for 20-22h;

[0023] S17, after the completion of in vitro maturation culture, in the sperm in two washing operation to replace the culture medium composition;

[0024] S18, the mature oocytes are transferred to the pre-set in 38.5℃ incubator for more than 2 hours of sperm droplet system.

[0025] Preferably, the specific operation of the sperm thawing, purification and fertilization in step S2 includes the following steps:

[0026] S21, the frozen semen is taken out from the liquid nitrogen storage tank and immediately placed in a 37℃ constant temperature water bath environment for 45 seconds of thawing treatment;

[0027] S22, preparation of Percoll gradient centrifugation solution: in the centrifuge tube, first 1mL concentration of 80% Percoll solution is injected into the bottom of the tube, and then 1mL of 40% Percoll solution is gently added on the surface of the 80% Percoll solution by micro-volume sampling technology;

[0028] S23, the sperm sample after completing the thawing step in S21 is carefully spread on the surface of the gradient centrifugation Percoll solution to construct a complete gradient centrifugation system; the system is placed in a centrifuge with a centrifugal force of 500g for 10 minutes of centrifugation;

[0029] S24, after centrifugation, use a micro pipette to suck the semen sample at the bottom of the centrifuge tube and add it to the fertilization solution containing oocytes to obtain a fertilization system;

[0030] S25, the fertilization system is placed back in the incubator to promote the combination of sperm and egg, and the culture is continued for 22 hours.

[0031] Preferably, the specific operation of the culture of fertilized embryos in step S3 includes the following steps:

[0032] S31, the fertilized eggs obtained in step S2 are blown and sucked to remove the residual cumulus cells around the eggs and the sperm that do not participate in fertilization, to obtain the bovine in vitro fertilized embryos;

[0033] S32, the in vitro fertilized bovine embryos are transferred to a four-well culture plate, and an embryo in vitro culture solution containing IL-1α and IL-10 is added, and the culture plate is placed in an incubator for preliminary culture for 72 h;

[0034] S33, after the culture in S32 ends, the fertilized embryos that do not undergo cleavage are picked out and placed in the incubator again, and the culture is continued to the 8th day, and the subsequent development process of the embryos is observed.

[0035] Preferably, the culture environment in the incubator is set as follows: the temperature is 38.5℃, the gas composition is 5% CO2 and 7% O2, and the saturated humidity condition is maintained.

[0036] The present application has the following beneficial effects:

[0037] In the currently disclosed embryo culture solution system, the immune participation is lacking, and the immune factor combination of the present application is mainly composed of the pro-inflammatory factor interleukin-1α (IL-1α) and the anti-inflammatory factor interleukin-10 (IL-10) secreted by macrophages, which effectively makes up for the lack of immune regulation mechanism in the traditional in vitro embryo culture system by simulating the immune microenvironment of in vivo embryo development.

[0038] The immune factor combination of the present application has excellent application potential when applied to bovine in vitro embryo development, and experimental data show that the use of the immune factor combination of the present application for bovine in vitro embryo culture significantly optimizes the bovine in vitro embryo culture process, and significantly improves the embryo development efficiency and quality, thereby providing a new solution to break through the existing in vitro embryo culture technical bottleneck. BRIEF DESCRIPTION OF DRAWINGS

[0039] Figure 1 Statistical results of embryo development efficiency of Example 1 and Comparative Example 1 of the present application.

[0040] Figure 2 Statistical results of embryo development efficiency of Example 1 and Comparative Example 1 of the present application.

[0041] Figure 3 Statistical results of embryo development efficiency of Example 1 and Comparative Example 1 of the present application. DETAILED DESCRIPTION

[0042] In order to facilitate the understanding of those skilled in the art, the present application will be further described below in conjunction with the embodiments and the drawings, and the content mentioned in the embodiments is not a limitation of the present application.

[0043] Sources and preparation of reagents

[0044] IL-1α is a product of MedChemExpress Company (item number HY-P7027).

[0045] IL-10 is a product of MedChemExpress (Cat. No. HY-P70517).

[0046] IVC is a product of BO-IVF (Cat. No. 71005).

[0047] IVM is a product of BO-IVF (Cat. No. 71001).

[0048] IVF is a product of BO-IVF (Cat. No. 71004).

[0049] Example 1

[0050] Preparation of bovine in vitro fertilized embryos:

[0051] S1, maturation culture of oocytes:

[0052] The bovine ovarian samples used in this study were collected from the Laohexiang fixed slaughterhouse in Wuzhong, Ningxia Hui Autonomous Region from January 2025 to May 2025. After the ovary was removed, it was immediately placed in a pre-filled physiological saline incubator. The physiological saline system added 200 U / ml penicillin and 0.2 mg / ml streptomycin as antibacterial components, maintained the temperature at 20-25°C, and ensured that the sample was transported to the laboratory within 1 hour. After the sample arrived at the laboratory, sterile surgical instruments were used to accurately remove the connective tissue, adipose tissue and fallopian tube structure attached to the surface of the ovary, and then washed thoroughly in sterile physiological saline. With the help of a 10 mL syringe equipped with a 12G needle, follicles with a diameter of 2-10 mm on the surface of the ovary were punctured to obtain oocytes. Under the assistance of a microscope, cumulus-oocyte complexes (COCs) were selectively collected and washed three times in phosphate-buffered saline (PBS) containing 10% fetal bovine serum. Finally, according to the morphological standard of oocytes, normal COCs were selected as experimental samples for subsequent in vitro maturation culture, and the sample amount after screening was defined as the total number of oocytes.

[0053] The screened cumulus-oocyte complexes (COCs) were first rinsed twice in the in vitro maturation medium (IVM) to remove residual impurities and potential inhibitory factors. Then, they were transferred to the IVM system pre-equilibrated in a 38.5°C incubator for 2 hours. The culture process was strictly controlled in a 38.5°C constant temperature environment, and the gas component ratio of 5% CO2, 5% O2 was maintained, while the saturated humidity condition was maintained, and the culture was continued for 20-22 hours. After the COCs completed the in vitro maturation culture, they were washed twice in the fertilization medium (IVF medium) to replace the culture medium composition. Then, the mature oocytes were transferred to the IVF medium droplet system pre-equilibrated in a 38.5°C incubator for more than 2 hours to create a suitable environment for the subsequent fertilization process.

[0054] S2, thawing, purification, and fertilization of sperm:

[0055] In this study, the frozen semen was immediately placed in a 37°C constant temperature water bath environment after being taken out of the liquid nitrogen storage tank, and underwent thawing treatment for 45 seconds. At the same time, the preparation of Percoll gradient centrifugation solution was carried out: in the centrifuge tube, first 1 mL of 80% Percoll solution was accurately injected into the bottom of the tube, and then 1 mL of 40% Percoll solution was gently added on the surface of the 80% Percoll solution using micro-volume sampling technology. Then, the sperm sample after thawing step was carefully spread on the surface of 45% Percoll solution to construct a complete gradient centrifugation system. The system was placed in a centrifuge and centrifuged at 500g for 10 minutes. After centrifugation, a micro-volume pipette was used to carefully aspirate about 200 μL of semen sample from the bottom of the centrifuge tube and add it to the fertilization medium (IVF medium) containing oocytes. Finally, the fertilization system was re-placed in the incubator under suitable culture conditions to promote the combination of sperm and oocytes, and the culture was continued for 22 hours.

[0056] S3, culture of fertilized embryos

[0057] IL-1α and IL-10 were added to the IVC basal medium, respectively, so that the concentration of IL-1α and IL-10 in the culture medium was 20 ng / mL.

[0058] After the completion of the fertilization process, the oocyte is gently pipetted or aspirated using a pipette to remove the cumulus cells and sperm that did not participate in fertilization, thereby obtaining a presumptively fertilized bovine in vitro fertilized embryo. The obtained embryo is transferred to a four-well culture plate, about 40 embryos are placed in each well, and an in vitro culture (IVC) solution is added. Subsequently, the culture plate is placed in an incubator with precise environmental control, set to a temperature of 38.5°C, a gas composition of 5% CO2, 7% O2, and a saturated humidity condition, for a preliminary culture of 72 hours. After the end of the culture, the fertilized embryos that did not undergo cleavage are selected under a stereomicroscope and are re-placed in the above-mentioned incubator for a continuous culture until the 8th day (total culture time 192 hours) to observe the subsequent development of the embryo.

[0059] Example 2

[0060] Preparation of bovine in vitro fertilized embryos:

[0061] S1, maturation culture of oocytes:

[0062] The bovine ovarian samples used in this study were collected from the Liangheqiao fixed slaughterhouse in Wuzhong, Ningxia Hui Autonomous Region from January 2025 to May 2025. After the ovary was removed, it was immediately placed in a pre-loaded incubation container with physiological saline, which was supplemented with 200 U / ml penicillin and 0.2 mg / ml streptomycin as antibacterial components, and the temperature was maintained at 20-25°C. The sample was transported to the laboratory within 1 hour. After the sample arrived at the laboratory, sterile surgical instruments were used to accurately remove the connective tissue, adipose tissue, and fallopian tube structures attached to the surface of the ovary, and then the ovary was thoroughly washed three times in sterile physiological saline. A 10 mL syringe equipped with a 12G needle was used to puncture the ovarian follicles with a diameter of 2-10 mm on the surface of the ovary to obtain oocytes. Under the assistance of a microscope, cumulus-oocyte complexes (COCs) were selectively collected and washed three times in phosphate-buffered saline (PBS) containing 10% fetal bovine serum. Finally, according to the morphological criteria of oocytes, normal COCs were selected as experimental samples for subsequent in vitro maturation culture, and the sample size after selection was defined as the total number of oocytes.

[0063] The screened cumulus-oocyte complexes (COCs) were first rinsed twice in the in vitro maturation medium (IVM) to remove residual impurities and potential inhibitory factors. Then, they were transferred to the IVM system pre-equilibrated in a 38.5°C incubator for 2 hours. The culture process was strictly controlled in a 38.5°C constant temperature environment, and the gas component ratio of 5% CO2, 5% O2 was maintained, while the saturated humidity condition was maintained, and the culture was continued for 20-22 hours. After the COCs completed the in vitro maturation culture, they were washed twice in the fertilization medium (IVF medium) to replace the culture medium composition. Then, the mature oocytes were transferred to the IVF medium droplet system pre-equilibrated in a 38.5°C incubator for more than 2 hours to create a suitable environment for the subsequent fertilization process.

[0064] S2, thawing, purification, and fertilization of sperm:

[0065] In this study, the frozen semen was immediately placed in a 37°C constant temperature water bath environment after being taken out of the liquid nitrogen storage tank, and the thawing process lasted for 45 seconds. At the same time, the preparation of Percoll gradient centrifugation solution was carried out: in the centrifuge tube, first, 1 mL of 80% Percoll solution was accurately injected into the bottom of the tube, and then 1 mL of 40% Percoll solution was gently overlaid on the surface of the 80% Percoll solution using micro-volume sampling technology. Then, the sperm sample that had completed the thawing step was carefully spread on the surface of the 45% Percoll solution to construct a complete gradient centrifugation system. The system was placed in a centrifuge and centrifuged at 500g for 10 minutes. After centrifugation, a micro-volume pipette was used to carefully aspirate about 200 μL of semen sample from the bottom of the centrifuge tube and add it to the fertilization medium (IVF medium) containing oocytes. Finally, the fertilization system was re-placed in the incubator under suitable culture conditions to promote the combination of sperm and oocytes, and the culture was continued for 22 hours.

[0066] S3, culture of fertilized embryos

[0067] IL-1α and IL-10 were added to the IVC basal medium, respectively, so that the concentration of IL-1α in the culture medium was 10 ng / mL, and the concentration of IL-10 in the culture medium was 30 ng / mL.

[0068] After the completion of the fertilization process, the oocyte is gently pipetted or aspirated using a pipette to remove the cumulus cells and sperm that did not participate in fertilization, thereby obtaining a presumptively fertilized bovine in vitro fertilized embryo. The obtained embryo is transferred to a four-well culture plate, about 40 embryos are placed in each well, and an in vitro culture (IVC) solution is added. Subsequently, the culture plate is placed in an incubator with precise environmental control, set to a temperature of 38.5°C, a gas composition of 5% CO2, 7% O2, and a saturated humidity condition, for a preliminary culture of 72 hours. After the end of the culture, the fertilized embryos that did not undergo cleavage are selected under a stereomicroscope and are re-placed in the above-mentioned incubator for a continuous culture until the 8th day (total culture time 192 hours) to observe the subsequent development of the embryo.

[0069] Example 3

[0070] Preparation of bovine in vitro fertilized embryos:

[0071] S1, maturation culture of oocytes:

[0072] The bovine ovarian samples used in this study were collected from the Liangheqiao fixed slaughterhouse in Wuzhong, Ningxia Hui Autonomous Region from January 2025 to May 2025. After the ovary was removed, it was immediately placed in a pre-loaded incubation container with physiological saline, which was supplemented with 200 U / ml penicillin and 0.2 mg / ml streptomycin as antibacterial components, and the temperature was maintained at 20-25°C. The sample was transported to the laboratory within 1 hour. Upon arrival at the laboratory, sterile surgical instruments were used to precisely remove the connective tissue, adipose tissue, and fallopian tube structures attached to the surface of the ovary, followed by three thorough rinses in sterile physiological saline. A 10 mL syringe equipped with a 12G needle was used to puncture the ovarian follicles with diameters between 2-10 mm to obtain oocytes. Under the assistance of a microscope, cumulus-oocyte complexes (COCs) were selectively collected and washed three times in phosphate-buffered saline (PBS) containing 10% fetal bovine serum. Finally, based on the morphological criteria of oocytes, morphologically normal COCs were selected as experimental samples for subsequent in vitro maturation culture, and the sample size after this selection was defined as the total number of oocytes.

[0073] The screened cumulus-oocyte complexes (COCs) were first rinsed twice in the in vitro maturation medium (IVM) to remove residual impurities and potential inhibitory factors. Then, they were transferred to the IVM system pre-equilibrated in a 38.5°C incubator for 2 hours. The culture process was strictly controlled in a 38.5°C constant temperature environment, and the gas component ratio of 5% CO2, 5% O2 was maintained, while the saturated humidity condition was maintained, and the culture was continued for 20-22 hours. After the COCs completed the in vitro maturation culture, they were washed twice in the fertilization medium (IVF medium) to replace the culture medium composition. Then, the mature oocytes were transferred to the IVF medium droplet system pre-equilibrated in a 38.5°C incubator for more than 2 hours to create a suitable environment for subsequent fertilization.

[0074] S2, thawing, purification, and fertilization of sperm:

[0075] In this study, the frozen semen was immediately placed in a 37°C constant temperature water bath environment after being taken out of the liquid nitrogen storage tank, and the thawing process lasted for 45 seconds. At the same time, the preparation of Percoll gradient centrifugation solution was carried out: in the centrifuge tube, first, 1 mL of 80% Percoll solution was accurately injected into the bottom of the tube, and then 1 mL of 40% Percoll solution was gently overlaid on the surface of the 80% Percoll solution using micro-volume sampling technology. Then, the sperm sample that had completed the thawing step was carefully spread on the surface of the 45% Percoll solution to construct a complete gradient centrifugation system. The system was placed in a centrifuge and centrifuged at 500g for 10 minutes. After centrifugation, a micro-volume pipette was used to carefully aspirate about 200 μL of semen sample from the bottom of the centrifuge tube and add it to the fertilization medium (IVF medium) containing oocytes. Finally, the fertilization system was re-placed in the incubator under suitable culture conditions to promote the combination of sperm and oocytes, and the culture was continued for 22 hours.

[0076] S3, culture of fertilized embryos

[0077] IL-1α and IL-10 were added to the IVC basal medium, respectively, so that the concentration of IL-1α in the culture medium was 30 ng / mL, and the concentration of IL-10 in the culture medium was 10 ng / mL.

[0078] After the completion of the fertilization process, the oocyte is gently pipetted or aspirated using a pipette to remove the cumulus cells and sperm that did not participate in fertilization, thereby obtaining the presumptively fertilized bovine in vitro fertilized embryo. The obtained embryo is transferred to a four-well culture plate, about 40 embryos are placed in each well, and an in vitro culture (IVC) solution is added. Subsequently, the culture plate is placed in an incubator with precise environmental control, set to a temperature of 38.5°C, a gas composition of 5% CO2, 7% O2, and a saturated humidity condition, for a preliminary culture of 72 hours. After the end of the culture, the fertilized embryos that did not undergo cleavage are selected under a stereomicroscope and are re-placed in the above-mentioned incubator for a continuous culture until the 8th day (total culture time 192 hours) to observe the subsequent development of the embryo.

[0079] Example 4

[0080] S1, maturation culture of oocytes:

[0081] The bovine ovary samples used in this study were collected from the Laohexiang fixed slaughterhouse in Wuzhong, Ningxia Hui Autonomous Region from January 2025 to May 2025. After the ovary was removed, it was immediately placed in a pre-loaded incubation container with physiological saline, which was supplemented with 200 U / ml penicillin and 0.2 mg / ml streptomycin as antibacterial components, maintained at a temperature of 20-25°C, and ensured that the sample was transported to the laboratory within 1 hour. After the sample arrived at the laboratory, sterile surgical instruments were used to accurately remove the connective tissue, adipose tissue, and fallopian tube structures attached to the surface of the ovary, and then it was thoroughly rinsed in sterile physiological saline three times. With the aid of a 10 mL syringe equipped with a 12G needle, follicles with a surface diameter of 2-10 mm were punctured to obtain oocytes. Under the assistance of a microscope, cumulus-oocyte complexes (COCs) were selectively collected and washed three times in phosphate-buffered saline (PBS) containing 10% fetal bovine serum. Finally, according to the morphological criteria of oocytes, morphologically normal COCs were selected as experimental samples for subsequent in vitro maturation culture, and the sample size after this selection was defined as the total number of oocytes.

[0082] The screened cumulus-oocyte complexes (COCs) were first rinsed twice in the in vitro maturation medium (IVM) to remove residual impurities and potential inhibitory factors. Then, they were transferred to the IVM system which had been equilibrated in a 38.5°C incubator for 2 hours. The culture process was strictly controlled in a 38.5°C constant temperature environment, and the gas component ratio of 5% CO2, 5% O2 was maintained, while the saturated humidity condition was maintained, and the culture was continued for 20-22 hours. After the COCs completed the in vitro maturation culture, they were washed twice in the fertilization medium (IVF medium) to replace the culture medium composition. Then, the mature oocytes were transferred to the IVF medium droplet system which had been equilibrated in a 38.5°C incubator for more than 2 hours to create a suitable environment for the subsequent fertilization process.

[0083] S2, thawing, purification and fertilization of sperm:

[0084] In this study, the frozen semen was immediately placed in a 37°C constant temperature water bath environment after being taken out from the liquid nitrogen storage tank, and the thawing process lasted for 45 seconds. At the same time, the preparation of Percoll gradient centrifugation solution was carried out: in the centrifuge tube, first 1 mL of 80% Percoll solution was accurately injected into the bottom of the tube, and then 1 mL of 40% Percoll solution was gently added on the surface of the 80% Percoll solution using micro-volume sampling technology. Then, the sperm sample after thawing step was carefully spread on the surface of 45% Percoll solution to construct a complete gradient centrifugation system. The system was placed in a centrifuge with a centrifugal force of 500g for 10 minutes. After centrifugation, a micro-volume pipette was used to carefully suck about 200μL of semen sample from the bottom of the centrifuge tube and add it to the fertilization medium (IVF medium) containing oocytes. Finally, the fertilization system was placed back in the incubator under suitable culture conditions to promote the combination of sperm and oocyte, and the culture was continued for 22 hours.

[0085] S3, culture of fertilized embryos

[0086] IL-1α and IL-10 were added to the IVC basal medium, respectively, so that the concentration of IL-1α in the culture medium was 10 ng / mL, and the concentration of IL-10 in the culture medium was 10 ng / mL.

[0087] Example 5

[0088] S1, maturation culture of oocytes:

[0089] The bovine ovarian samples used in this study were collected from the fixed slaughterhouse of Liangheqiao in Wuzhong City, Ningxia Hui Autonomous Region, from January 2025 to May 2025. After the ovaries were removed, they were immediately placed in a pre-filled physiological saline incubator. The physiological saline system added 200 U / ml penicillin and 0.2 mg / ml streptomycin as bacteriostatic components, maintained at 20-25°C, and ensured that the samples were transported to the laboratory within 1 hour. After the samples arrived at the laboratory, sterile surgical instruments were used to accurately remove the connective tissue, adipose tissue and fallopian tube structure attached to the surface of the ovaries, and then washed thoroughly in sterile physiological saline. With the help of a 10 mL syringe equipped with a 12G needle, follicles with a diameter of 2-10 mm on the surface of the ovary were punctured to obtain oocytes. Under the assistance of a microscope, cumulus-oocyte complexes (COCs) were selectively collected and washed three times in phosphate-buffered saline (PBS) containing 10% fetal bovine serum. Finally, according to the morphological criteria of oocytes, normal COCs were selected as experimental samples for subsequent in vitro maturation culture, and the sample size after screening was defined as the total number of oocytes.

[0090] After screening, the cumulus-oocyte complexes (COCs) were first washed twice in the oocyte in vitro maturation medium to remove residual impurities and potential inhibitors. Then, they were transferred to the oocyte in vitro maturation medium (IVM) system that had been equilibrated at 38.5°C for 2 hours in a constant temperature incubator. The culture process was strictly controlled at 38.5°C in a constant temperature environment, with a gas composition ratio of 5% CO2, 5% O2, and saturated humidity conditions, and cultured for 20-22 hours. After the COCs completed in vitro maturation culture, they were washed twice in the fertilization medium (IVF medium) to replace the culture medium components. Then, the mature oocytes were transferred to the fertilization medium droplet system that had been equilibrated at 38.5°C for more than 2 hours in a constant temperature incubator to create a suitable environment for subsequent fertilization.

[0091] S2, sperm thawing, purification, and fertilization:

[0092] In this study, the frozen semen was taken out from the liquid nitrogen storage tank and immediately placed in a constant temperature water bath environment at 37°C for 45 seconds of thawing treatment. At the same time, the preparation of Percoll gradient centrifugation solution was carried out: in the centrifuge tube, first 1 mL of 80% concentration Percoll solution was accurately injected into the bottom of the tube, and then 1 mL of 40% concentration Percoll solution was gently superimposed on the surface of the 80% Percoll solution using micro-volume sampling technology. Then, the sperm sample after the thawing step was carefully spread on the surface of the 45% Percoll solution to construct a complete gradient centrifugation system. The system was placed in a centrifuge for 10 minutes of centrifugation at a centrifugal force of 500g. After centrifugation, a micro-volume pipette was used to carefully aspirate about 200 μL of semen sample from the bottom of the centrifuge tube and add it to the fertilization liquid (IVF liquid) containing the oocyte. Finally, the fertilization system was placed back in the incubator under suitable culture conditions to promote the combination of sperm and egg, and the culture was continued for 22 hours.

[0093] S3, culture of fertilized embryos

[0094] IL-1α and IL-10 were added to the IVC basic culture medium, so that the concentration of IL-1α in the culture medium was 30 ng / mL, and the concentration of IL-10 in the culture medium was 30 ng / mL.

[0095] Comparative Example 1

[0096] The same as Example 1, except that no IL-1α and IL-10 were added to the IVC basic culture medium.

[0097] Test Example 1

[0098] The development of embryos in Example 1 to Example 5 (experimental group) and Comparative Example 1 (control group) was observed and recorded. The observation results are shown in Table 1 as follows. Figure 1

[0099] Table 1 Statistics of embryo development efficiency

[0100] Group Number of oocytes / group % of cleavage rate % of blastocyst rate Example 1 (experimental group) 317 77.52±5.40 37.37 ± 3.33 b ]] Example 2 (experimental group) 265 75.85±7.21 34.82 ± 7.14 ab ]] Example 3 (experimental group) 217 77.42±6.33 36.41 ± 2.85 b ]] Example 4 (experimental group) 284 78.57±9.14 32.72 ± 4.77 a ]] Example 5 (experimental group) 258 75.18±6.82 36.46 ± 3.59 b ]] Comparative Example 1 (control group) 242 76.91±5.44 28.34 ± 1.67 a ]]

[0101] Wherein, a, b different letters represent significant, and the significance is detected by chi-square test.

[0102] Test Example 2

[0103] Evaluation of embryo quality of Example 1 (experimental group) and Comparative Example 1 (control group):

[0104] ​The quality of the embryo was evaluated by the ratio of the number of the inner cell mass to the number of the trophoblast and the total number of all cells in the embryo, and the test results are shown in Table 1. Figure 2

[0105] Specific method of immunofluorescence: the blastocyst was fixed in a culture dish with 4% paraformaldehyde. After washing with PBS / PVA, it was transferred into PBS / PVA containing 0.1% Triton X-100 for permeabilization for 30 minutes. After washing, the embryo was incubated in immunostaining blocking buffer (P0102, Beyotime) at room temperature for 3 hours, and the embryo was incubated with anti-SOX2 (#3579, Cell Signaling Technology, USA) and anti-CDX2 (AM392-5M, BioGenex, USA) at 4°C overnight. After washing, the embryo was incubated with secondary antibody (Alexa Fluor 488 labeled goat anti-rabbit IgG [1:500; A0423, Biyuntian] and Alexa Fluor 555 labeled donkey anti-mouse IgG [1:500, A0460, Biyuntian]) at 25°C in the dark for 2 hours. Subsequently, DNA was stained with 4', 6-diamidino-2-phenylindole (DAPI, C1006, Biyuntian) for 8 minutes. Finally, images were collected using a confocal microscope (Zeiss LSM 980 equipped with Airyscan2).

[0106] The quality of the embryo development of Examples 1-5 and Comparative Example 1 was analyzed and statistically processed, and the data processing results are shown in Table 1. Figure 3

[0107] All the technical features in the embodiments can be modified in appearance according to actual needs.

[0108] The above embodiments are the preferred implementation of the present application, in addition to this, the present application can be implemented in other ways, without departing from the technical solution concept of the present application, any obvious replacement within the protection scope of the present application.​​

Claims

1. An immune factor combination capable of improving the efficiency of bovine embryo development in vitro, characterized in that: The combination comprises IL-1α and IL-10. ​ 2. The immune factor combination of claim 1, which can improve the efficiency of bovine embryo development in vitro, characterized in that: The concentration of the IL-1α in the culture solution is 10-30 ng / mL, and the concentration of the IL-10 in the culture solution is 10-30 ng / mL.

3. The immune factor combination of claim 1, which can improve the efficiency of bovine embryo development in vitro, characterized in that: The combination further comprises a basic culture medium, which is an in vitro embryo culture solution.

4. The immune factor combination of claim 3, which can improve the efficiency of bovine embryo development in vitro, characterized in that: The in vitro embryo culture solution is specifically IVC, which is sourced from BO-IVF Company and has a product number of 71005.

5. Use of the immune factor combination capable of improving the development efficiency of bovine in vitro embryos according to any one of claims 1-4 in the culture process of bovine in vitro embryos.

6. The application of the immune factor combination for improving the efficiency of bovine embryo development in vitro according to claim 5, characterized in that: The preparation method of bovine in vitro fertilized embryos comprises the following steps: S1, maturation culture of oocytes; S2, thawing and purification of sperm, and combining the mature oocytes in step S1 with the purified sperm to obtain fertilized embryos; S3, culturing the fertilized embryos in step S2 in an embryo in vitro culture solution containing IL-1α and IL-10; S4, evaluating the quality of the embryos cultured in step S3: performing immunofluorescence localization of the inner cell mass and trophoblast cells by using the marker SOX2 of the early embryo inner cell mass and the marker CDX2 of the trophoblast cells, and evaluating the quality of the embryos by using the ratio of the number of the inner cell mass to the number of the trophoblast cells and the total number of all cells in the embryos.

7. The application of the immune factor combination for improving the efficiency of bovine embryo development in vitro according to claim 5, characterized in that: The specific operation of the maturation culture of oocytes in step S1 comprises the following steps: S11, collecting bovine ovaries, placing them in a heat preservation container, and delivering them to the laboratory within 1 h; S12, removing the connective tissue, adipose tissue and oviduct structure attached to the surface of the ovary, and then rinsing; S13, performing puncture operation on the follicles with a diameter of 2-10 mm on the surface of the ovary to obtain oocytes; S14, screening and collecting cumulus-oocyte complexes COCs, and washing; S15, screening the COCs with normal morphology, and performing twice rinsing treatment in the oocyte in vitro maturation culture solution to remove residual impurities and potential inhibitory factors; S16, transferring to the oocyte in vitro maturation culture solution system for culture for 20-22 h; S17, after the in vitro maturation culture of the COCs is completed, performing twice washing operation in the fertilization solution to replace the culture solution components; S18, transferring the mature oocytes to the fertilization solution droplet system pre-equilibrated in a 38.5℃ incubator for more than 2 h.

8. The application of the immune factor combination for improving the efficiency of bovine embryo development in vitro according to claim 5 in the process of bovine embryo culture in vitro, characterized in that: The specific operation of the thawing, purification and fertilization of sperm in step S2 comprises the following steps: S21, immediately placing the frozen semen taken out from the liquid nitrogen storage tank in a 37℃ constant temperature water bath environment for thawing treatment for 45 s; S22, preparing Percoll gradient centrifugation solution: first accurately injecting 1 mL of Percoll solution with a concentration of 80% into the bottom of the centrifuge tube, and then gently stacking 1 mL of Percoll solution with a concentration of 40% on the surface of the 80% Percoll solution by using micro-volume sample addition technology; S23, the sperm sample which has finished the thawing step in S21 is carefully spread on the surface of the gradient centrifugation Percoll solution to construct a complete gradient centrifugation system; the system is placed in a centrifuge and centrifuged at 500g for 10 minutes; S24, after the centrifugation, the semen sample at the bottom of the centrifuge tube is taken by a micropipette and added to the fertilization solution containing the oocyte to obtain a fertilization system; S25, the fertilization system is placed in the incubator again to promote the combination of the sperm and the oocyte, and the culture is continued for 22 hours.

9. The application of the immune factor combination for improving the efficiency of bovine embryo development in vitro according to claim 5 in the process of bovine embryo culture in vitro, characterized in that: The specific operation of the culture of the fertilized embryo in step S3 includes the following steps: S31, the fertilized oocyte obtained in step S2 is blown and sucked to remove the cumulus cells around the oocyte and the sperm that do not participate in fertilization, and a presumptive fertilized bovine in vitro fertilized embryo is obtained; S32, the presumptive fertilized bovine in vitro fertilized embryo is transferred to a four-well culture plate, and an embryo in vitro culture solution containing IL-1α and IL-10 is added, and the culture plate is placed in an incubator for preliminary culture for 72h; S33, after the culture in S32 is finished, the fertilized embryo that does not occur cleavage is picked out and placed in the incubator again, and the culture is continued to the 8th day, and the subsequent development process of the embryo is observed.

10. The application of the immune factor combination for improving the efficiency of bovine embryo development in vitro according to claim 9, characterized in that: The culture environment in the incubator is set as follows: the temperature is 38.5℃, the gas composition is 5% CO2 and 7% O2, and the saturated humidity condition is maintained.

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