Culture solution for bovine oocyte in-vitro culture and application thereof

Abstract

The invention discloses a culture solution for in-vitro culture of bovine oocytes and application of the culture solution, and belongs to the technical field of livestock embryo engineering. In order to solve the problem that in the prior art, a culture solution for in-vitro culture of bovine oocytes lacks an immune system, so that the in-vitro culture of the oocytes is greatly different from normal development in vivo, a proinflammatory factor IL-1alpha and an anti-inflammatory factor IL-10 secreted by macrophages are added into the culture solution for in-vitro culture of the bovine oocytes; further, the immune requirement in the bovine oocyte in-vitro culture process is perfected, the development quality of bovine oocyte in-vitro culture can be effectively improved, and the embryo development efficiency after oocyte fertilization is improved.

Description

Technical Field

[0001] The present invention relates to the technical field of livestock embryo engineering, in particular to a culture medium for in vitro culture of bovine oocytes and application thereof. Background Art

[0002] Assisted reproductive technology based on superovulation has been widely used for genetic improvement in dairy cattle, significantly increasing the number of calves and the reproductive efficiency of high-quality females. However, the quality of oocytes cultured in vitro and the efficiency of subsequent embryo development still lag significantly behind those of oocytes and embryos that develop normally in vivo. For example, in vitro culture can alter DNA methylation in follicular cells and embryos, impair oocyte maturation, deteriorate oocyte quality, increase oocyte chromosomal aneuploidy, disrupt genomic imprinting, affect endometrial receptivity, reduce implantation rates, and delay embryonic development. These issues severely impact the application and efficiency of in vitro embryo production, hindering the widespread application of embryo engineering techniques.

[0003] Under physiological conditions, ovulation is regulated by an inflammatory response mediated by cytokines secreted by lymphocytes, macrophages, and granulocytes. Growth factors secreted in the female reproductive tract promote preimplantation embryonic development and may serve as epigenetic determinants of postimplantation developmental competence and pregnancy outcomes. Therefore, compared to the in vivo environment in which oocytes and embryos grow, in vitro culture systems lack the involvement of the immune system. Improving the immune system in in vitro culture systems is key to addressing the low efficiency of in vitro culture. Summary of the Invention

[0004] In response to the above-mentioned problems, the present invention aims to provide a culture medium for in vitro culture of bovine oocytes and its application. By adding the pro-inflammatory factor IL-1α and the anti-inflammatory factor IL-10 secreted by macrophages to the culture medium for in vitro culture of bovine oocytes, the immune requirements of bovine oocytes can be improved during in vitro culture, thereby effectively improving the development quality of bovine oocytes in vitro culture and enhancing the efficiency of embryonic development after oocyte fertilization.

[0005] In order to achieve the above object, the technical solution adopted by the present invention is as follows: In one aspect, the present invention provides a culture medium for culturing bovine oocytes in vitro, wherein the culture medium contains a pro-inflammatory factor IL-1α and an anti-inflammatory factor IL-10.

[0006] Specifically, the pro-inflammatory factor IL-1α and the anti-inflammatory factor IL-10 are derived from macrophages.

[0007] In the present invention, IL-1α and IL-10 are added to the culture medium, which fills the gap in the immune system in the bovine oocyte in vitro culture system and can promote the in vitro culture quality of oocytes and the embryonic development ability after fertilization.

[0008] Furthermore, the culture medium also includes basal culture medium, E2, HMG, FBS, ITS-G, sodium pyruvate, EGF, bFGF, serine and glycine.

[0009] In the present invention, basal culture medium, E2, HMG, FBS, ITS, sodium pyruvate, EGF, bFGF, serine and glycine serve as the basis of bovine oocyte in vitro culture medium, providing the required nutrients for bovine oocyte in vitro culture.

[0010] Preferably, the added concentrations of different components in the culture medium are: based on the volume of the basal culture medium, the added concentration of E2 is 0.5-1.5 μg / mL, the added concentration of HMG is 0.05-0.1 IU / mL, the volume fraction of FBS is 5-15%, the volume fraction of ITS-G is 0.5-1.5%, the added concentration of sodium pyruvate is 0.02-0.025 mg / mL, the added concentration of EGF is 8-13 ng / mL, the added concentration of bFGF is 10-15 ng / mL, the added concentration of serine is 22-28 μM, the added concentration of glycine is 5-7 mM, the added concentration of IL-1α is 18-24 ng / mL, and the added concentration of IL-10 is 18-24 ng / mL.

[0011] Preferably, the added concentration of E2 is 1 μg / mL, the added concentration of HMG is 0.075 IU / mL, the volume fraction of FBS is 10%, the volume fraction of ITS-G is 1%, the added concentration of sodium pyruvate is 0.022 mg / mL, the added concentration of EGF is 10 ng / mL, the added concentration of bFGF is 12 ng / mL, the added concentration of serine is 25 μM, the added concentration of glycine is 6 mM, the added concentration of IL-1α is 20 ng / mL, and the added concentration of IL-10 is 20 ng / mL.

[0012] On the other hand, the present invention also provides a method for culturing bovine oocytes in vitro, comprising the following steps: Obtaining bovine oocytes; The bovine oocytes were cultured in the culture medium as described above.

[0013] Preferably, the bovine oocyte culture conditions are 38.5° C., 5% CO 2 , 7% O 2 and 100% humidity, and the culture time is 20-22 h.

[0014] On the other hand, the present invention also provides a method for culturing bovine in vitro fertilized embryos, comprising the following steps: Obtaining bovine oocytes; The bovine oocytes are placed in the culture medium as described above for culture, and after being cultured and matured, the bovine oocytes are transplanted into fertilization droplets; After the frozen sperm is thawed and purified, it is fertilized with bovine oocytes; The fertilized bovine in vitro fertilized embryos were placed in IVC solution and cultured in an incubator.

[0015] The beneficial effects of the present invention are: 1. The present invention adds IL-1α and IL-10 to the culture medium for in vitro culture of bovine oocytes, filling the gap in the immune system in the in vitro culture system of bovine oocytes, and can promote the quality of in vitro culture of oocytes and the developmental ability of embryos after fertilization.

[0016] 2. IL-10 inhibits macrophage overactivation and reduces ROS (reactive oxygen species) accumulation in oocyte culture systems, thereby improving embryo survival. Furthermore, IL-10 promotes trophoblast differentiation and embryo implantation by activating the STAT3 pathway. Therefore, in bovine in vitro embryo culture, the addition of IL-10 significantly improves blastocyst formation and enhances the ratio of inner cell mass (ICM) to trophoblast (TE). IL-1α, when present in oocyte culture medium, stimulates the secretion of EGF-like factors, improving synchronized nuclear and cytoplasmic maturation. Therefore, the addition of IL-1α and IL-10 significantly increases the rate of meiotic completion. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 It is the development result of the oocyte and fertilized embryo in the present invention.

[0018] Figure 2 This is the result of the granulosa cell proliferation rate detection in the present invention. DETAILED DESCRIPTION

[0019] In order to enable those skilled in the art to better understand the technical solution of the present invention, the technical solution of the present invention is further described below in conjunction with the accompanying drawings and embodiments.

[0020] Example 1: 1. Reagent Sources and Culture Medium Preparation IL-1α was a product of MedChemExpress (catalog number HY-P7027); IL-10 is a product of MedChemExpress (catalog number HY-P70517); Serine was a product of MedChemExpress (catalog number HY-N0650); Glycine was a product of MedChemExpress (catalog number HY-Y0966); EGF is a product of Gibco (catalog number PHG0311); bFGF was a product of Sino Biological (Cat. No. 10014-HNAE); FBS was a product of Invigentech (Cat. No. A6907FBS-500); ITS is a product of Gibco (item number 41400-045); M199 is a product of Gibco (item number 11150067); Sodium pyruvate was a product of Sigma (catalog number P4562-25g); E2 is a product of Sigma (catalog number 23-022-M-10 μg); HMG is a product of Sigma (catalog number 567020); In this study, bovine oocytes were divided into a control group and an experimental group. The culture medium configurations of the control group and the experimental group were as follows: Control group: M199 basal culture medium without Hepes was supplemented with 10% (v / v) fetal bovine serum (FBS), 1% (v / v) ITS-G, 0.075 IU / mL HMG, 1 μg / mL E2, 10 ng / mL EGF, 12 ng / mL bFGF, 0.022 mg / mL sodium pyruvate, 25 μM serine, and 6 mM glycine.

[0021] Experimental group: M199 basal culture medium without Hepes was supplemented with 10% (v / v) fetal bovine serum (FBS), 1% (v / v) ITS-G, 0.075 IU / mL HMG, 1 μg / mL E2, 10 ng / mL EGF, 12 ng / mL bFGF, 0.022 mg / mL sodium pyruvate, 25 μM serine, 6 mM glycine, 20 ng / mL IL-1α, and 20 ng / mL IL-10.

[0022] 2. Oocyte Culture Method In this study, bovine ovaries were collected from the Laoheqiao designated slaughterhouse in Wuzhong City, Ningxia Hui Autonomous Region (collection period: October 2024 to April 2025). Ovaries were placed in a thermos containing 200 U / ml penicillin and 0.2 mg / ml streptomycin in normal saline at 20-25°C and transported to the laboratory within 5 hours. After transport, the ovaries were removed from the surface connective tissue, fat, and attached fallopian tubes using sterile scissors. The ovaries were rinsed three times in sterile saline. Oocytes from 2-8 mm follicles on the ovarian surface were extracted using a 10 mL syringe fitted with a 12G needle and placed into a 6 cm glass dish. Cumulus-oocyte complexes (COCs) were collected under a stereomicroscope. Following collection, the ovaries were rinsed three times in PBS supplemented with 10% fetal bovine serum. COCs with normal morphology were selected and divided into control and experimental groups for in vitro maturation (total oocyte counts were calculated in each group).

[0023] COCs from the control and experimental groups were washed twice in the corresponding culture medium and then transferred to 3 cm dishes containing 3 mL of culture medium (previously equilibrated in a 38.5°C incubator for 2 hours). The oocytes were incubated at 38.5°C, 5% CO₂, 5% O₂, and saturated humidity for 20–22 hours. Mature COCs were repeatedly pipetted with a 1000 mL pipette to remove any cumulus cells that had diffused outside the oocyte. Once only four to five tightly packed layers of cumulus cells remained around the COCs, the oocytes were washed twice in fertilization solution (IVF solution) to remove any free cumulus cells. The oocytes, which were then tightly packed with four to five layers of cumulus cells, were then transferred to a drop of fertilization solution (previously equilibrated in a 38.5°C incubator for at least 2 hours).

[0024] 3. Culture Methods of Fertilized Embryos 3.1 Oocyte culture: The method is exactly the same as in 2 above.

[0025] 3.2 Sperm Thawing, Purification, and Fertilization After removing the frozen semen from the liquid nitrogen tank, thaw it in a 37°C water bath and layer it with Percoll in a centrifuge tube. Place 1 mL of 80% Percoll at the bottom of the centrifuge tube. Carefully add 1 mL of 40% Percoll to the 80% Percoll level. Place the thawed sperm on the 45% Percoll level and centrifuge at 1500 rpm for 10 minutes. Carefully pipette approximately 200 μL of semen from the bottom and add it to the fertilization solution (IVF solution) containing the oocytes. Return the tube to the incubator to allow the sperm and egg to combine for 22 hours.

[0026] 3.3 Culture of fertilized embryos After fertilization, the remaining cumulus cells and sperm around the eggs were blown away with a mouth pipette or a gun tip to obtain presumed fertilized bovine in vitro fertilized embryos. The embryos were placed in IVC solution in a four-well plate, with about 40 embryos per well, and cultured in an incubator at 38.5°C, 5% CO2, 7% O2, and saturated humidity for 72 hours. The fertilized embryos that did not cleave were picked out and returned to the incubator for continued culture until the eighth day (192 hours).

[0027] Observe and record the development of oocytes and fertilized embryos in the control group and experimental group. The results are shown in the attached figure. Figure 1 shown.

[0028] A systematic analysis of the experimental data presented in Figure 1 shows that, compared to the control group, the oocytes in the experimental group that underwent in vitro maturation and fertilization showed statistically significant increases in both cleavage and blastocyst formation rates. This result strongly demonstrates that the combination of interleukin-1α (IL-1α) and interleukin-10 (IL-10) introduced into the in vitro culture system can effectively optimize the oocyte developmental microenvironment, significantly promote oocyte development, and positively impact oocyte quality.

[0029] Example 2: To further investigate the different effects of adding different concentrations of IL-1α and IL-10 to the culture medium of bovine oocytes in vitro, this study co-cultured bovine oocytes with cumulus cells during maturation, using granulosa cells as the research subjects. Different concentrations of IL-1α and IL-10 were added to the culture medium of granulosa cells, and the concentrations of IL-1α and IL-10 were optimized based on the proliferation capacity of granulosa cells. Specific research contents include: Follicular fluid from ovarian follicles was extracted with a syringe and collected in a centrifuge tube. The tube was then filtered through a 40 μm cell sieve and centrifuged. Granulosa cells were washed with PBS and resuspended in DMEM containing 10% FBS and 5% follicular fluid. Granulosa cells were seeded in a cell dish and cultured in a saturated humidity incubator at 37°C and 5% CO2. Granulosa cells were seeded in a 96-well plate and treated with 0 ng / mL, 10 ng / mL, 20 ng / mL, 40 ng / mL, and 80 ng / mL of IL-1α and IL-10, respectively. CCK8 (CA1210, Solarbio) was used to detect the proliferation rate of granulosa cells according to the manufacturer's instructions. The OD value represented the number of cells. Each concentration was tested for 7 days, with 3 replicates per day. The experimental results are shown in the attached figure. Figure 2 shown.

[0030] From the attached Figure 2 It can be seen that 20 ng / mL of IL-1α and 20 ng / mL of IL-10 can be used as the optimal added concentration to promote the proliferation of granulosa cells.

[0031] In addition, it should be stated that the present invention is derived from the following scientific research projects: 1. National Key Scientific and Technological Project of Agricultural Biological Breeding, Project No. 2023ZD04075 2. Ningxia Hui Autonomous Region Key R&D Program, Project No. 2024BBF01005 3. Analysis of key regulatory signals for livestock embryonic development, project number 2023YFD1300503 4. Central Government Guidance for Local Science and Technology Development Fund, Project No. ZY-JCYJ-02-19 5. National Natural Science Foundation of China, Project No. 32472912.

[0032] The basic principles, main features, and advantages of the present invention are shown and described above. Those skilled in the art should understand that the present invention is not limited to the foregoing embodiments. The foregoing embodiments and descriptions are merely illustrative of the principles of the present invention. Various changes and modifications may be made to the present invention without departing from the spirit and scope of the present invention. Such changes and modifications are intended to fall within the scope of the present invention. The scope of protection claimed in the present invention is defined by the appended claims and their equivalents.

Claims

1. A culture medium for in vitro culture of bovine oocytes, characterized in that: The culture medium contains the pro-inflammatory factor IL-1α and the anti-inflammatory factor IL-10.

2. The culture medium for in vitro culture of bovine oocytes according to claim 1, wherein: The pro-inflammatory factor IL-1α and the anti-inflammatory factor IL-10 are derived from macrophages.

3. The culture medium for in vitro culture of bovine oocytes according to claim 1, wherein: The culture solution further comprises basal medium, E2, HMG, FBS, ITS-G, sodium pyruvate, EGF, bFGF, serine and glycine.

4. The culture medium for in vitro culture of bovine oocytes according to claim 3, wherein: The added concentrations of different components in the culture medium are: based on the volume of the basal culture medium, the added concentration of E2 is 0.5-1.5 μg / mL, the added concentration of HMG is 0.05-0.1 IU / mL, the volume fraction of FBS is 5-15%, the volume fraction of ITS-G is 0.5-1.5%, the added concentration of sodium pyruvate is 0.02-0.025 mg / mL, the added concentration of EGF is 8-13 ng / mL, the added concentration of bFGF is 10-15 ng / mL, the added concentration of serine is 22-28 μM, the added concentration of glycine is 5-7 mM, the added concentration of IL-1α is 18-24 ng / mL, and the added concentration of IL-10 is 18-24 ng / mL.

5. The culture medium for in vitro culture of bovine oocytes according to claim 4, wherein: The added concentrations of different components in the culture medium are: based on the volume of the basal medium, the added concentration of E2 is 1 μg / mL, the added concentration of HMG is 0.075 IU / mL, the volume fraction of FBS is 10%, the volume fraction of ITS-G is 1%, the added concentration of sodium pyruvate is 0.022 mg / mL, the added concentration of EGF is 10 ng / mL, the added concentration of bFGF is 12 ng / mL, the added concentration of serine is 25 μM, the added concentration of glycine is 6 mM, the added concentration of IL-1α is 20 ng / mL, and the added concentration of IL-10 is 20 ng / mL.

6. A method for culturing bovine oocytes in vitro, characterized in that: The following steps are included: Obtaining bovine oocytes; The bovine oocytes are placed in the culture medium according to any one of claims 1 to 5 for culture.

7. The method for culturing bovine oocytes in vitro according to claim 6, wherein: The bovine oocyte culture conditions were 38.5°C, 5% CO2, 7% O2, and 100% humidity, and the culture time was 20-22 h.

8. A method for culturing bovine in vitro fertilized embryos, characterized in that: The following steps are included: Obtaining bovine oocytes; placing bovine oocytes in the culture medium according to any one of claims 1 to 5 for culturing, and transplanting the bovine oocytes into fertilization droplets after maturation; After the frozen sperm is thawed and purified, it is fertilized with bovine oocytes; The fertilized bovine in vitro fertilized embryos are placed in IVC solution and cultured in an incubator.

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