Bovine oocyte in-vitro maturation culture solution as well as preparation method and application thereof

Abstract

The invention discloses a bovine oocyte in-vitro maturation culture solution as well as a preparation method and application thereof, and belongs to the technical field of animal reproduction and embryo engineering. The preparation method of the bovine oocyte in-vitro maturation culture solution comprises the step of adding mitoquinine mesylate into a conventional IVM culture solution, wherein the adding concentration of the mitoquinine mesylate is preferably 50 nmol / L. The bovine oocyte in-vitro maturation culture solution provided by the invention can effectively reduce the oxidative stress reaction of the oocytes in the in-vitro culture process, enhance the mitochondrial function and remarkably improve the bovine oocyte in-vitro maturation quality and the embryonic development potential, so that the blastocyst rate is increased, and the problem that the development potential of the existing oocyte IVM culture solution is insufficient is solved.

Description

Technical Field

[0001] This invention relates to the field of animal reproduction and embryo engineering technology, and in particular to an in vitro maturation culture medium for bovine oocytes, its preparation method, and its application. Background Technology

[0002] Currently, in vitro embryo production (IVP) is a crucial method for the efficient propagation of superior breeding cattle populations. By utilizing the ovarian resources of culled or young cows, through ovarian extraction or live oocyte collection (OPU) at slaughterhouses, in vitro maturation culture (IVM), combined with in vitro fertilization (IVF), in vitro culture (IVC), and embryo transfer (IVF-ET) techniques, a large-scale population of superior breeding cattle can be obtained in a short period. Currently, 80% of the international bovine embryo trade uses this method to prepare embryos.

[0003] Although in vitro embryo production (IVP) technology has become the mainstream of the industry, compared with in vivo embryo production, in vitro embryos still have disadvantages such as low blastocyst rate and poor embryo quality. Currently, the most mature IVP blastocyst rate worldwide is around 40%. Optimizing the in vitro embryo production process and improving the blastocyst rate and quality will help promote the further development of the industry. In the entire in vitro embryo production technology process, in vitro maturation (IVM) is the most critical step in bovine IVP. Oocyte maturation in vitro involves nuclear maturation and cytoplasmic maturation. The physiological process of oocytes is extremely complex, and currently, no in vitro maturation culture system can completely simulate the in vivo maturation process. This is the fundamental reason for the continuous optimization of in vitro culture systems. During in vitro culture, oocytes often experience a decline in developmental potential due to mitochondrial dysfunction and oxidative stress (OS), which is one of the reasons for low oocyte maturation efficiency and low blastocyst rate. OS, as a recognized developmental limiting factor in in vitro embryo production, is essentially an imbalance in the generation and scavenging system of reactive oxygen species (ROS). Studies have found that cultured bovine oocytes exhibit reduced mitochondrial DNA (mtDNA) copy numbers, decreased ATP levels, and a significant increase in meiotic abnormalities, potentially leading to chromosomal errors and embryonic developmental failure. Furthermore, the hyperotropic environment in vitro causes excessive accumulation of reactive oxygen species, damaging DNA, proteins, and lipid membrane structures, and impairing mitochondrial function. This process is a key determinant of oocyte quality and subsequent embryonic development. Oocytes are susceptible to oxidative stress and mitochondrial dysfunction, resulting in decreased developmental potential.

[0004] The main strategy for preventing oxidative stress during in vitro maturation (IVM) is to add antioxidants to the maturation medium. Various antioxidants have shown neutralizing effects on reactive oxygen species (ROS), thereby improving the developmental potential of oocytes. While traditional antioxidants (such as resveratrol and melatonin) can alleviate some damage during in vitro maturation to a certain extent, their effects are limited due to their lack of mitochondrial targeting. Mitoquinone mesylate, as a mitochondrial-targeting derivative of coenzyme Q10, can effectively accumulate in the mitochondrial matrix and has functions such as ROS scavenging, maintaining membrane potential (MMP), and promoting ATP synthesis. Currently, the effects of mitochonone mesylate in bovine oocyte IVM have not been reported. Summary of the Invention

[0005] The purpose of this invention is to provide a bovine oocyte in vitro maturation culture medium supplemented with mitoxin mesylate, which can effectively reduce the oxidative stress response of oocytes during in vitro culture, enhance mitochondrial function, significantly improve the quality of bovine oocyte in vitro maturation and embryonic developmental potential, and thus increase the blastocyst rate, thereby solving the problem of insufficient developmental potential of existing oocyte IVM culture media.

[0006] To achieve the above objectives, the present invention provides a method for preparing bovine oocyte in vitro maturation culture medium, comprising the following steps: adding mitoxin mesylate to conventional IVM culture medium.

[0007] Preferably, the concentration of mitoxin mesylate added is 10–200 nmol / L.

[0008] Preferably, the concentration of mitoxin mesylate added is 50 nmol / L.

[0009] Preferably, the conventional IVM culture medium comprises: 86.925% TCM-199 culture medium, 10% fetal bovine serum, penicillin 100 IU / mL, streptomycin 100 μg / mL, sodium pyruvate 0.022 mg / mL, GlutaMAX 0.292 mg / mL, recombinant human EGF 50 ng / mL and follicle-stimulating hormone (FSH) 5 μg / mL.

[0010] In addition, the present invention also provides a bovine oocyte in vitro maturation culture medium prepared according to the above-described method for preparing bovine oocyte in vitro maturation culture medium.

[0011] This invention also provides the application of the above-mentioned bovine oocyte in vitro maturation culture medium for improving the blastocyst rate after in vitro fertilization.

[0012] This invention also provides the application of the above-mentioned bovine oocyte in vitro maturation culture medium for improving the cleavage rate after in vitro fertilization.

[0013] This invention also provides the application of the above-mentioned bovine oocyte in vitro maturation culture medium for increasing GSH levels in oocytes.

[0014] The present invention also provides the application of the above-mentioned bovine oocyte in vitro maturation culture medium for increasing the relative level of ATP in oocytes.

[0015] Beneficial effects of the present invention

[0016] This invention discloses a culture medium for improving the in vitro maturation and blastocyst rate of bovine oocytes, and clarifies the effect of adding mitoxin mesylate on the development of in vitro matured bovine oocytes: it can effectively reduce the oxidative stress response of oocytes during in vitro culture, enhance mitochondrial function, significantly improve the quality of in vitro maturation of bovine oocytes and embryonic developmental potential, thereby increasing the blastocyst rate. Specifically, after conventional IVM culture, the first polar body extrusion rate (PBE) of oocytes was 70.35%, the cleavage rate after in vitro fertilization was 67.79%, the 8-cell rate was 36.57%, and the blastocyst rate was 23.40%; after adding mitoxin mesylate (50 nmol / L) to the IVM culture medium, the PBE was 76.14%, the cleavage rate after in vitro fertilization was 75.94%, the 8-cell rate was 45.55%, and the blastocyst rate was 30.37%, all of which significantly improved the blastocyst rate compared to the control group. Attached Figure Description

[0017] Figure 1 The image shows the effect of mitoxin mesylate on the reactive oxygen species (ROS) level in bovine oocytes. In the image, A is a representative image of ROS staining in oocytes of the control group and the treatment group, with a scale bar of 200 μm. B is a fluorescence intensity analysis image of ROS in the control group and the treatment group, where n represents the number of oocytes.

[0018] Figure 2 The image shows the effect of mitoxin mesylate on GSH levels in bovine oocytes. In this image, A represents representative images of GSH staining in oocytes from the control and treatment groups (scale bar: 200 μm). B shows the fluorescence intensity analysis of GSH in the control and treatment groups, where n represents the number of oocytes.

[0019] Figure 3 Figure 1 shows the effect of mitoxin mesylate on MMP and ATP levels in bovine oocytes. In Figure 2, A represents representative images of JC-1 staining in oocytes from the control and treatment groups; red: JC-1 aggregates; green: JC-1 monomers; scale bar: 200 μm; B shows the relative fluorescence intensity of JC-1 (aggregates / monomers) between the control and treatment groups; C shows the quantitative analysis of ATP levels in the control and treatment groups. In Figures B and C, n represents the number of oocytes.

[0020] Figure 4This figure shows the effect of mitoxin mesylate on early apoptosis in bovine oocytes. In Figure A, representative images of early apoptosis staining in oocytes from the control and treatment groups are shown (scale bar: 200 μm; magnification scale bar: 20 μm). Figure B shows the proportion of early apoptosis in the control and treatment groups, where n represents the number of oocytes.

[0021] Figure 5 The figure shows the effect of mitoxin mesylate on the mRNA levels of antioxidant-related genes (SOD3, SIRT3, and SIRT2) in bovine oocytes. In the figure, A represents the relative mRNA levels of SOD3, B represents SIRT3, and C represents the relative mRNA levels of SIRT2.

[0022] Figure 6 The figure shows the effect of mitoxin mesylate on the mRNA levels of mitochondrial-related genes (MFN2, DNM1, and DNM2) in bovine oocytes. In the figure, A represents the relative mRNA levels of MFN2, B represents DNM1, and C represents DNM2.

[0023] Figure 7 The figure shows the effect of mitoxin mesylate on the mRNA levels of apoptosis-related genes (BCL-2 and BAX) in bovine oocytes. In the figure, A represents the relative mRNA levels of BCL-2 and B represents the relative mRNA levels of BAX. Detailed Implementation

[0024] This invention significantly reduces oxidative stress during in vitro maturation of bovine oocytes by adding mitoxin mesylate to the bovine oocyte in vitro maturation (IVM) culture medium, improves mitochondrial functional status, and enhances intracellular antioxidant capacity and energy metabolism levels.

[0025] First, this invention provides a method for preparing an in vitro maturation culture medium for bovine oocytes, comprising the following steps: adding mitoxin mesylate to a conventional IVM culture medium. Preferably, the concentration of mitoxin mesylate added is 10–200 nmol / L. More preferably, the concentration of mitoxin mesylate added is 50 nmol / L. The conventional IVM culture medium comprises: 86.925% TCM-199 culture medium, 10% fetal bovine serum, penicillin 100 IU / mL, streptomycin 100 μg / mL, sodium pyruvate 0.022 mg / mL, GlutaMAX 0.292 mg / mL, recombinant human EGF 50 ng / mL, and follicle-stimulating hormone (FSH) 5 μg / mL.

[0026] In addition, the present invention also provides a bovine oocyte in vitro maturation culture medium prepared according to the above-described method for preparing bovine oocyte in vitro maturation culture medium.

[0027] This invention also provides the application of the above-mentioned bovine oocyte in vitro maturation culture medium for improving the blastocyst rate after in vitro fertilization.

[0028] This invention also provides the application of the above-mentioned bovine oocyte in vitro maturation culture medium for improving the cleavage rate after in vitro fertilization.

[0029] This invention also provides the application of the above-mentioned bovine oocyte in vitro maturation culture medium for increasing GSH levels in oocytes.

[0030] The present invention also provides the application of the above-mentioned bovine oocyte in vitro maturation culture medium for increasing the relative level of ATP in oocytes.

[0031] Example 1

[0032] 1.1 In vitro maturation of bovine oocytes and embryonic development

[0033] (1) Oocyte collection

[0034] Bovine ovaries were obtained from a local slaughterhouse and transported to the laboratory within 3 hours after being placed in 37°C physiological saline. The ovaries were repeatedly rinsed with physiological saline until the saline solution became clear and no blood remained on the surface. Follicular fluid was extracted from follicles with a diameter of 2-8 mm using a 10mL syringe with an 18G needle. The follicular fluid was collected into a 50mL sterile centrifuge tube and placed in a 38.5°C water bath until collection was complete. Cumulooocyte-oophoroid complexes (COCs) were collected from the follicular fluid in the centrifuge tube and allowed to stand for 15 minutes. The remaining 4ml of supernatant was extracted and added to oocyte recovery medium (OCM), and allowed to stand for another 15 minutes. Subsequently, the mixture was transferred to a culture dish and observed under a stereomicroscope. COCs with at least three dense layers of cumulus cells were selected for in vitro endoscopic microscopy (IVM).

[0035] (2) In vitro maturation of oocytes

[0036] Selected COCs were washed four times in IVM medium. Subsequently, 30 COCs were randomly allocated into 500 μL droplets of IVM medium. They were then incubated in a saturated humidity incubator at 38.5 °C and 5% CO2 for 22–24 hours.

[0037] In vitro maturation culture media with final concentrations of mitoxin mesylate of 50, 100 and 150 nmol / L were used as experimental groups, while in vitro maturation culture media without mitoxin mesylate were used as control groups.

[0038] (3) In vitro fertilization

[0039] After culturing in IVM for 22 to 24 hours, the sperm was washed four times with IVF solution and placed in 50 μL droplets of pre-equilibrated IVF culture medium coated with mineral oil. Frozen semen (0.25 mL per pipette) was removed from the liquid nitrogen tank, allowed to stand in air for approximately 5 seconds, and then thawed in a 37°C water bath. Sperm were centrifuged at 1200 rpm for 5 min at 25°C with IVF solution, the supernatant was discarded, and IVF solution was added again for centrifugation for another 5 min. Finally, oocytes and sperm were homogenized in a saturated humidity environment of 38.5°C and 5% CO2 for 6–8 hours. The sperm density was approximately 1 × 10⁶ cells / mL. Frozen semen used in each experiment was from the same bull.

[0040] (4) Embryo culture

[0041] The day of fertilization was recorded as day 0. Six to eight hours later, cumulus cells were removed with 0.01% hyaluronidase, and the embryos were washed four times with culture medium equilibrated for at least 2 hours before being placed in culture drops covered with mineral oil. Embryo cleavage rate was observed after 48 hours (day 2) incubation at 38.5℃ and 5% CO2, 8-cell embryo rate was observed after 72 hours (day 3), and blastocyst rate was observed after 7-8 days.

[0042] Table 1 shows the data of cumulus-oocyte complexes (COCs) 22-24 hours after in vitro maturation. In this study, oocyte first polar body extrusion (PBE) was used as the maturation standard. The PBE rate in the experimental group treated with 50 nmol / L mitoxin mesylate was significantly higher than that in the other groups (p<0.05). Furthermore, the PBE rates in the 100 nmol / L and 150 nmol / L treatment groups were significantly higher than those in the 0 nmol / L control group (p<0.05). There was no statistically significant difference between the 100 nmol / L and 150 nmol / L groups (p>0.05).

[0043] To further investigate the effect of mitoxin mesylate on bovine oocyte development, statistical analysis was performed on cleavage rate, 8-cell embryo ratio, and blastocyst formation rate. The results are shown in Table 2. Cleavage rate analysis showed that the 50 nmol / L and 100 nmol / L mitoxin mesylate treatment groups were significantly higher than the 150 nmol / L group and the 0 nmol / L control group (p<0.05). However, there was no significant difference in cleavage rate between the 150 nmol / L group and the control group (p>0.05). Regarding the 8-cell embryo ratio, the 50 nmol / L mitoxin mesylate treatment group was significantly higher than the other groups (p<0.05). The 100 nmol / L mitoxin mesylate treatment group was significantly higher than the 150 nmol / L group and the control group (p<0.05). Notably, the 8-cell embryo ratio in the 150 nmol / L treatment group was also significantly higher than that in the control group (p<0.05). The blastocyst formation rate was significantly higher in the 50 nmol / L mitoxin mesylate treatment group than in the 150 nmol / L group and the control group (p<0.05). The blastocyst formation rate in the 100 nmol / L treatment group was also significantly higher than that in the control group (p<0.05). However, there was no significant difference in blastocyst formation rate between the 150 nmol / L group and the control group (p>0.05). In summary, these results indicate that the addition of mitoxin mesylate during in vitro maturation (IVM) can improve the subsequent embryonic developmental potential of bovine oocytes. The optimal concentration of mitoxin mesylate was determined to be 50 nmol / L; therefore, this concentration will be used in subsequent experiments.

[0044] Table 1. Effects of different concentrations of mitoxin mesylate on in vitro maturation of bovine oocytes.

[0045]

[0046]

[0047] Note: Data in the table are mean ± standard error. Different superscripts indicate significant differences (p<0.05), while the same superscript indicates no significant differences (p>0.05).

[0048] Table 2. Effects of different concentrations of mitoxin mesylate on early bovine embryonic development.

[0049]

[0050] Note: Data in the table are mean ± standard error. Different superscripts indicate significant differences (p<0.05), while the same superscript indicates no significant differences (p>0.05).

[0051] 1.2 Functional verification of the effects of mitoxin mesylate on bovine oocytes

[0052] (1) ROS level assessment

[0053] ROS levels were measured using a ROS detection kit from Beyotime (Shanghai, China). Cumulus cells (COCs) were removed with 0.01% hyaluronidase (Hy) 22-24 hours after COC maturation. Oocytes from both the control and mitoxin mesylate treatment groups were washed three times with 1% BSA-PBS. Subsequently, the oocytes were incubated for 20 minutes in a solution containing 10 μmol / L DCFH-DA dye at 38.5°C in the dark. After incubation, the oocytes were washed four times with 1% BSA-PBS. Images were captured under a fluorescence microscope (Olympus Corporation, Tokyo, Japan). Images were collected, and the green fluorescence intensity of ROS levels was analyzed using ImageJ software. Results are as follows: Figure 1 As shown, mitoxin mesylate significantly reduced the reactive oxygen species (ROS) level in oocytes (P < 0.01), indicating that mitoxin mesylate, as an effective mitochondrial-targeted antioxidant, can reduce intracellular ROS levels.

[0054] (2) GSH Level Assessment

[0055] Glutathione (GSH), an important intracellular antioxidant, is a key indicator for assessing the redox status and cytoplasmic maturity of oocytes. To systematically evaluate the effect of mitoxin mesylate on the antioxidant reserve in bovine oocytes, this study used a GSH-specific fluorescent probe (CellTracker). TM The detection was performed using Blue CMF2HC. This probe can penetrate the cell membrane and specifically binds to intracellular GSH, producing blue fluorescence. The fluorescence intensity is directly proportional to the GSH content. Specifically, after COCs matured for 22-24 hours, cumulus cells were removed with 0.01% hyaluronidase (Hy). Oocytes from the control group and the mitoxin mesylate treatment group were washed three times with 1% BSA-PBS. Subsequently, the oocytes were incubated in a solution containing 10 μmol / L CMF2HC dye in the dark at 38.5°C for 20 minutes. After incubation, the oocytes were washed four times with 1% BSA-PBS. Images were captured under a fluorescence microscope (Olympus Corporation, Tokyo, Japan). Images were collected, and the blue fluorescence intensity of GSH levels was analyzed using ImageJ software. The results are as follows: Figure 2 As shown, compared with the control group, oocytes supplemented with 50 nmol / L mitoxin mesylate exhibited significantly enhanced blue fluorescence intensity (P<0.05), indicating that mitoxin mesylate treatment effectively increased GSH levels in oocytes. This result suggests that mitoxin mesylate may improve intracellular redox homeostasis by enhancing the antioxidant defense capacity of oocytes.

[0056] (3) Assessment of MMP and ATP levels

[0057] Mitochondria, as the energy factories of cells, are crucial for the maturation quality and developmental potential of oocytes due to their functional integrity. Mitochondrial membrane potential (MMP) is a core indicator for assessing mitochondrial functional status, and its stable maintenance is a prerequisite for efficient ATP synthesis. To comprehensively evaluate the ameliorative effect of mitochondrial function in bovine oocytes, this study used the JC-1 fluorescent probe and an enhanced ATP assay kit to quantitatively analyze MMP and ATP levels. The JC-1 probe exists in mitochondria in two forms: polymer (red fluorescence) and monomer (green fluorescence), and the ratio of its red / green fluorescence intensity can sensitively reflect the level of MMP. First, MMP levels were measured using the enhanced mitochondrial membrane potential assay kit (JC-1) from Beyotime (Shanghai, China). Specifically, cumulus cells were removed with 0.01% hyaluronidase (Hy) 22-24 hours after COC maturation. Oocytes in both the control group and the mitochondrial mesylate treatment group were washed three times with 1% BSA-PBS. Subsequently, oocytes were placed in a solution containing 10 μmol / L JC-1 dye and incubated for 20 minutes in the dark at 38.5°C. After incubation, the oocytes were washed four times with 1% BSA-PBS. Images were then captured under a fluorescence microscope (Olympus Corporation, Tokyo, Japan). Images were collected, and the ratio of red fluorescence intensity to green fluorescence intensity at MMP levels was analyzed using ImageJ software, where red fluorescence represents high mitochondrial potential and green fluorescence represents low mitochondrial potential. Results are as follows: Figure 3 As shown in A and 3B, oocytes treated with 50 nmol / L mitoxin mesylate showed significantly enhanced fluorescence signals in the JC-1 polymer (red) and relatively weakened signals in the monomer (green). The red-green fluorescence ratio was significantly higher than that of the control group (P<0.05), indicating that mitoxin mesylate effectively maintained the stability of mitochondrial membrane potential.

[0058] In addition, ATP levels were measured using the enhanced ATP assay kit from Beyotime (Shanghai, China). Specifically, after COCs matured for 22-24 hours, cumulus cells were removed with 0.01% hyaluronidase (Hy). Oocytes from the control group and the mitoxin mesylate treatment group were washed three times with 1% BSA-PBS. Subsequently, 10 mature oocytes were placed in each group, and 25 μL of ATP lysis buffer was added to fully lyse the oocytes. The ATP standard solution (0.5 mM) was diluted with ATP assay reagent diluent to concentrations of 0, 0.01, 0.03, 0.1, 0.3, 1, 3, and 10 μM. Then, 14 wells were selected in a 96-well plate, and 100 μL of ATP assay working solution was added to each well, and the plate was incubated at room temperature for 3-5 min. 20 μL of sample and standard solution diluent were added to the 96-well plate and thoroughly mixed with the ATP assay working solution. The luminescence was detected using a microplate reader (MOLECULAR), and ATP levels were analyzed according to the standard curve. The results are as follows Figure 3 As shown in Figure C, the relative ATP level in oocytes treated with mitoxin mesylate was significantly higher than that in the control group (P<0.05). As ATP is the direct energy source for cellular life activities, the increase in its level directly confirms the enhanced mitochondrial energy metabolism function.

[0059] (4) Apoptosis level assessment

[0060] Apoptosis is an important form of programmed cell death. During the in vitro maturation of oocytes, factors such as oxidative stress can trigger apoptosis signaling pathways, leading to a decrease in developmental potential. Phosphatidylserine (PS) is normally distributed on the inner side of the cell membrane lipid bilayer, but in the early stages of apoptosis, PS everts to the outer side of the cell membrane. This phenomenon can serve as a specific indicator for detecting early apoptosis.

[0061] Annexin V is a calcium-dependent phospholipid-binding protein with a high affinity for PS. FITC-labeled Annexin V can specifically bind to everted PS, thereby forming a green fluorescent ring signal on the outer side of the oocyte membrane. The fluorescence intensity can be quantitatively analyzed by fluorescence microscopy, thus accurately reflecting the incidence of early apoptosis.

[0062] Early apoptosis levels were measured using the Annexin V-FITC apoptosis detection kit from Betway Biotechnology (Shanghai, China). Specifically, cumulus cells were removed with 0.01% hyaluronidase (Hy) 22-24 hours after COC maturation. Oocytes from both the control and mitoxin mesylate-treated groups were washed three times with 1% BSA-PBS. Subsequently, the oocytes were incubated in a solution containing 10 μmol / L Annexin V-FITC dye at 38.5°C in the dark for 20 minutes. After incubation, the oocytes were washed four times with 1% BSA-PBS. Images were captured under a fluorescence microscope (Olympus Corporation, Tokyo, Japan). The green fluorescence intensity of the images was analyzed using ImageJ software to determine the early apoptosis level. Results are as follows: Figure 4 As shown, compared with the control group, oocytes treated with 50 nmol / L mitoxin mesylate exhibited significantly reduced green fluorescence intensity around the cell membrane and a significantly lower proportion of early apoptotic cells (P<0.01). These results indicate that adding mitoxin mesylate to the in vitro maturation culture system can effectively inhibit the early apoptosis process induced by in vitro environmental stress.

[0063] (5) RNA extraction and quantitative reverse transcription PCR (RT-qPCR)

[0064] After culturing cocci (COCs) in vitro for 24 hours, 0.01% hyaluronidase was added to remove cumulus cells. Thirty oocytes were collected from each group, and total RNA was extracted from the oocytes using the TRIzol method. cDNA synthesis was performed using the DynaBeads mRNA Direct Kit (Life Technologies, Carlsbad, CA, USA). The assays were used to detect superoxide dismutase (SOD), deacetylase 2 (SIRT2), deacetylase 3 (SIRT3), dynein 1 (DNM1), dynein 2 (DNM2), mitochondrial fusion protein 2 (MFN2), Bcl-2-associated X protein (BAX), and B-cell lymphoma-2 (BCL-2). Results are as follows: Figures 5-7 As shown:

[0065] from Figure 5 The results showed that, compared with the control group, the expression of multiple antioxidant genes in bovine oocytes treated with mitoxin mesylate was significantly upregulated. Specifically, the mRNA expression levels of SOD3 (responsible for scavenging extracellular superoxide anions) and SIRT3 (mitochondrial deacetylase) were significantly increased (P<0.05); while the expression level of SIRT2, an important antioxidant regulatory node, was extremely significantly increased (P<0.01). These results indicate that mitoxin mesylate can synergistically enhance the antioxidant defense capacity of oocytes by upregulating the transcriptional expression of endogenous antioxidant genes at multiple levels.

[0066] from Figure 6 The results showed that, compared with the control group, the expression of the mitochondrial fusion gene MFN2 was significantly upregulated in the mitochondrial mesylate treatment group (P<0.01), while the expression of the mitochondrial division genes DNM1 and DNM2 was also significantly increased (P<0.001). These results indicate that mitochondrial mesylate can synergistically regulate mitochondrial fusion and division processes, enhancing mitochondrial function by optimizing mitochondrial dynamics. This provides a molecular-level explanation for the observations of mitochondrial membrane potential and ATP levels enhanced by mitochondrial mesylate.

[0067] from Figure 7 The results showed that, compared with the control group, the expression of the anti-apoptotic gene BCL-2 was significantly upregulated in the mitoxin mesylate treatment group (P<0.05), while the expression of the pro-apoptotic gene BAX was significantly downregulated (P<0.01). This indicates that mitoxin mesylate positively regulates the apoptosis pathway by controlling the BCL-2 / BAX ratio, confirming its mechanism of action in inhibiting oocyte apoptosis at the molecular level.

[0068] In summary, mitoxin mesylate reduces oocyte apoptosis and enhances antioxidant and mitochondrial function through gene regulation, thereby improving oocyte quality and developmental potential.

Claims

1. A method for preparing an in vitro maturation culture medium for bovine oocytes, characterized in that, Includes the following steps: Add mitoxin mesylate to the standard IVM culture medium.

2. The method for preparing an in vitro maturation culture medium for bovine oocytes according to claim 1, characterized in that, The concentration of mitoxin mesylate added is 10–200 nmol / L.

3. The method for preparing an in vitro maturation culture medium for bovine oocytes according to claim 2, characterized in that, The concentration of mitoxin mesylate added was 50 nmol / L.

4. The method for preparing an in vitro maturation culture medium for bovine oocytes according to claim 1, characterized in that, The conventional IVM culture medium consists of: 86.925% TCM-199 culture medium, 10% fetal bovine serum, penicillin 100 IU / mL, streptomycin 100 μg / mL, sodium pyruvate 0.022 mg / mL, GlutaMAX 0.292 mg / mL, recombinant human EGF 50 ng / mL, and follicle-stimulating hormone (FSH) 5 μg / mL.

5. The bovine oocyte in vitro maturation culture medium prepared by the preparation method according to any one of claims 1-4.

6. The application of the bovine oocyte in vitro maturation culture medium according to claim 5 for improving the blastocyst rate after in vitro fertilization.

7. The application of the bovine oocyte in vitro maturation culture medium according to claim 5 for improving the cleavage rate after in vitro fertilization.

8. The application of the bovine oocyte in vitro maturation culture medium according to claim 5 for increasing the GSH level in oocytes.

9. The application of the bovine oocyte in vitro maturation culture medium according to claim 5 for increasing the relative level of ATP in oocytes.

Images