A lipo-chitinous culture solution for improving the efficiency of in vitro maturation of porcine oocytes and application thereof

Abstract

The application discloses a culture solution containing lupeol for improving in-vitro maturation efficiency of pig oocytes and application thereof. The application adds lupeol, especially 4 muM lupeol, to an in-vitro maturation culture solution of oocytes, which can effectively improve the oxidation-reduction state of oocytes, significantly reduce the intracellular ROS level, and meanwhile, improve the GSH content, thereby maximally relieving oxidative stress damage in the in-vitro maturation process, significantly improving the maturation rate of pig oocytes in the meiotic metaphase II, and effectively reducing the DNA fragmentation ratio, which indicates that the lupeol has excellent effects of promoting nuclear maturation and protecting genetic material. The lupeol is added to the in-vitro maturation culture solution of oocytes, which is easy to operate and can be applied to the existing culture process, and can stably improve the efficiency and quality of pig in-vitro embryo production, and has important application value for pig germplasm resource preservation, rapid propagation of excellent breeds and development of animal husbandry.

Description

Technical Field

[0001] This invention belongs to the field of animal reproduction technology, specifically relating to a lupeol-containing culture medium for improving the in vitro maturation efficiency of porcine oocytes and its application. Background Technology

[0002] In vitro maturation (IVM) of porcine oocytes is a crucial step in animal biotechnology processes such as in vitro fertilization, somatic cell nuclear transfer, and transgenics, and its maturation quality directly affects the developmental potential of subsequent embryos. While the porcine oocyte IVM culture system is relatively mature, problems remain, including low oocyte maturation rates, asynchronous cytoplasmic and nuclear maturation, and suboptimal developmental capacity of matured oocytes, limiting the further development and application of related biotechnologies. Traditional culture media are complex in composition and still fall short in mimicking the in vivo physiological environment, making it difficult to fully meet the nutritional requirements and signal regulation during the in vitro maturation process. Embryo development is highly dependent on oocyte quality; to produce high-quality in vitro embryos, an optimized IVM system must be established. In recent years, researchers have focused on improving the IVM system, but compared to in vivo matured oocytes, the developmental capacity of in vitro matured oocytes remains weaker. Many defects in the in vitro culture system, compared to the in vivo environment, can interfere with embryo development, primarily due to a lack of antioxidants and excessively high concentrations of reactive oxygen species (ROS). Therefore, finding novel culture medium additives (antioxidants) that can effectively improve the efficiency and quality of porcine oocyte in vitro maturation is of great significance for optimizing in vitro maturation systems and promoting the advancement of animal reproductive technology. Lupinol, as a natural triterpenoid compound, possesses various biological activities such as anti-inflammatory, antioxidant, and regulation of cell proliferation and apoptosis. Existing studies have shown that it may play a positive role in cell culture by influencing cell signaling pathways and improving the cell microenvironment, but its application in the porcine oocyte IVM system has not yet been reported. Summary of the Invention

[0003] To address the problems of low in vitro maturation rate and insufficient embryo production efficiency of porcine oocytes in existing technologies, as well as the lack of efficient and safe antioxidant application solutions, this invention aims to provide a culture medium containing lupeol to improve the in vitro maturation efficiency of porcine oocytes and its application. By adding lupeol as an antioxidant to the culture medium, the damage of oxidative stress to oocytes can be alleviated, thereby improving in vitro maturation and embryo production efficiency, and providing technical support for the protection of porcine germplasm resources and the breeding of superior breeds.

[0004] The first objective of this invention is to provide the application of lupeol in improving the efficiency of oocyte maturation in vitro.

[0005] Preferably, the application is the use of lupeol in improving the GVBD rate of oocytes, improving the MII stage maturation rate of oocytes, or reducing the DNA damage rate of oocytes.

[0006] Preferably, the application includes the following steps: culturing oocytes in an in vitro maturation culture medium containing lupeol.

[0007] Preferably, the application includes the following steps: S1. Extract antral follicular fluid from healthy ovaries, centrifuge, select cumulus-oocyte complexes, and wash the cumulus-oocyte complexes three times with in vitro maturation culture medium containing human chorionic gonadotropin. S2. The cumulus-oocyte complexes treated in step S1 were sequentially transferred to in vitro maturation culture medium containing human chorionic gonadotropin (hCG) and in vitro maturation culture medium without hCG, covered with mineral oil, and cultured for 22 h respectively.

[0008] Preferably, the cumulus-oocyte complex is a morphologically intact cumulus-oocyte complex with at least three cumulus cell layers, uniform cytoplasm distribution, and no obvious damage.

[0009] Preferably, the in vitro maturation culture medium containing human chorionic gonadotropin (hCG) consists of: 10% PFF (volume fraction), 50 μM sodium pyruvate, 2 mg / mL D-sorbitol, 10 IU / mL equine hCG, 10 IU / mL human hCG, 50 μg / mL gentamicin, 2-8 μM lupeol, with the remainder being TCM199 medium containing 25 mM HEPES. The in vitro maturation culture medium that does not contain human chorionic gonadotropin consists of: 10% PFF (volume fraction), 50 μM sodium pyruvate, 2 mg / mL D-sorbitol, 50 μg / mL gentamicin, 2-8 μM lupeol, and the remainder is TCM199 medium containing 25 mM HEPES.

[0010] Preferably, the in vitro maturation culture medium containing human chorionic gonadotropin (hCG) consists of: 10% PFF (volume fraction), 50 μM sodium pyruvate, 2 mg / mL D-sorbitol, 10 IU / mL equine hCG, 10 IU / mL human hCG, 50 μg / mL gentamicin, 4 μM lupeol, with the remainder being TCM199 medium containing 25 mM HEPES. The in vitro maturation culture medium that does not contain human chorionic gonadotropin consists of: 10% PFF (volume fraction), 50 μM sodium pyruvate, 2 mg / mL D-sorbitol, 50 μg / mL gentamicin, 4 μM lupeol, and the remainder is TCM199 medium containing 25 mM HEPES.

[0011] Preferably, the culture is carried out at 39°C, 5% CO2, and saturated humidity.

[0012] Preferably, the oocyte is a porcine oocyte.

[0013] The beneficial effects of this invention are: This invention applies lupeol to the in vitro maturation culture medium of porcine oocytes. Lupeol, as a natural triterpenoid antioxidant, possesses stronger free radical scavenging and anti-apoptotic activities. By establishing CT and DMSO controls and using multiple concentration gradients (1, 2, 4, 8 μM), the study scientifically verified that its effects originate from lupeol itself, and determined that 4 μM is the optimal concentration. Culture medium at this concentration significantly improves the in vitro maturation rate (MII stage proportion), fertilization rate, and blastocyst formation rate of porcine oocytes, while significantly reducing intracellular reactive oxygen species levels and apoptosis rate, effectively solving the problem of low efficiency in existing in vitro culture systems. This invention is simple and easy to implement, directly optimizing existing in vitro porcine oocyte culture processes and improving in vitro embryo production efficiency. It provides a new technical pathway for the protection of porcine germplasm resources, the breeding of superior germplasm, and large-scale livestock farming, with broad application prospects. Attached Figure Description

[0014] Figure 1 Effects of lupeol on in vitro maturation of porcine oocytes; where A represents the effect of lupeol on the incidence of GVBD in porcine oocytes, B represents the effect of lupeol on the MII stage maturation rate of porcine oocytes, and C represents the effect of lupeol on the percentage of DNA fragmentation in the nuclei of porcine oocytes; different letters in the figure indicate significant differences between groups (P<0.05), and the same letter indicates no significant differences between groups (P>0.05).

[0015] Figure 2 Effects of lupeol on reactive oxygen species (ROS) and glutathione (GSH) levels in porcine oocytes; where A represents the effect of lupeol on ROS levels in porcine oocytes, and B represents the effect of lupeol on GSH levels in porcine oocytes; different letters in the figure indicate significant differences between groups (P<0.05), and the same letter indicates no significant differences between groups (P>0.05).

[0016] Figure 3 Effects of lupeol on the fertilization capacity of porcine oocytes; where A represents the effect of lupeol on the fertilization rate of porcine oocytes, and B represents the effect of lupeol on the fertilization rate of porcine intracytoplasmic sperm; different letters in the figure indicate significant differences between groups (P<0.05), and the same letter indicates no significant differences between groups (P>0.05). Detailed Implementation

[0017] The following embodiments are further illustrations of the present invention, but not limitations thereof.

[0018] To address the issues of significant oxidative stress damage, poor maturation quality, and insufficient potential for subsequent embryonic development in porcine oocytes during in vitro maturation, this invention improves oocyte quality by adding lupeol to the maturation culture medium to regulate the cellular redox environment.

[0019] (a) Experimental Samples Pig ovarian samples were collected from a local slaughterhouse. Immediately after collection, the samples were placed in a 0.9% NaCl aqueous solution containing penicillin (100 U / mL penicillin + 100 μg / mL streptomycin sulfate) at 37°C and transported back to the laboratory within 2 hours. The temperature was kept constant throughout the process to avoid mechanical damage and temperature fluctuations that could affect the activity of the samples.

[0020] (II) Experimental Reagents The reagents used in the experiment included: TCM199 medium containing 25 mM HEPES, porcine follicular fluid (PFF, PFF was obtained from 3-6 mm antral follicles in the ovaries of healthy sows, and the supernatant was collected after centrifugation at 3000 r / min for 10 min, 0.22 Filtered through a μm filter and aliquoted, stored at -20℃ for later use; lupeol (purity ≥94%, purchased from Sigma-Aldrich, L5632); dimethyl sulfoxide (DMSO, analytical grade); sodium pyruvate; D-sorbitol; gentamicin; equine chorionic gonadotropin (eCG); human chorionic gonadotropin (hCG); porcine in vitro fertilization medium (PFM); cleavage stage medium (PZM-5); blastocyst formation medium (PBM); mineral oil; glutathione (GSH) assay kit (purchased from Elabscience, E-EL-0026); reactive oxygen species (ROS) assay kit (purchased from Elabscience, E-BC-K138-F); TUNEL apoptosis assay kit (purchased from Roche Diagnostics, 11767305001). (III) Experimental Instruments Stereo microscope, carbon dioxide incubator, tri-gas incubator, high-speed refrigerated centrifuge, ELISA reader, inverted fluorescence microscope, water bath, cell counting plate, sterile four-well cell culture plate, and sterile syringe with a No. 10 needle.

[0021] Example 1: Verification of the effect of different concentrations of lupeol on in vitro maturation of porcine oocytes This embodiment investigates the correlation between lupeol concentration and porcine oocyte maturation quality by setting different concentration gradients of lupeol, and screens out the most suitable lupeol concentration for in vitro oocyte maturation.

[0022] 1. Experimental Grouping Blank control group (CT): only the basic culture system, without lupeol and DMSO.

[0023] Solvent control group (DMSO): An equal volume of DMSO was added to the basic culture system to eliminate the interference of the solvent on the experimental results.

[0024] Lupeol experimental group: Lupeol was added to the basic culture system to concentrations of 1 μM, 2 μM, 4 μM and 8 μM respectively, and then used for in vitro culture of oocytes.

[0025] 2. Experimental Methods and Procedures (1) Preparation of culture medium In vitro maturation culture medium should be prepared and used immediately. Preparation of the basic culture system: Add 10% PFF, 50 μM sodium pyruvate, 2 mg / mL D-sorbitol, 10 IU / mL eCG, 10 IU / mL hCG, and 50 μg / mL gentamicin sequentially to TCM199 medium containing 25 mM HEPES. Mix thoroughly, filter through a 0.22 μm filter for sterilization, and store at 4°C. Preheat in a preheated water bath at 39°C for 1 h. Lupinol stock solution is 10 mM, prepared by dissolving in DMSO, and frozen at -20°C. Limit freeze-thaw cycles to no more than 3 times to prevent loss of activity. Lupinol experimental groups should be serially diluted according to their corresponding concentrations to ensure consistent solvent concentrations across groups.

[0026] (2) Oocyte collection and pretreatment The surface of the pig ovary was rinsed three times with sterile saline at 37°C to remove fat and connective tissue and wash away residual blood. Antral follicular fluid (3-6 mm) was extracted from the ovarian surface using a sterile syringe with a 10-gauge needle and injected into a sterile centrifuge tube. After standing for 15 minutes, the follicular fluid containing more sediment was aspirated and screened for cumulus-oocyte complexes (COCs) under a stereomicroscope at 37°C. The selection criteria were: intact morphology, at least three cumulus cell layers, uniform cytoplasmic distribution, and no obvious damage. The screened COCs were washed three times with different experimental groups of in vitro maturation culture medium, with gentle agitation each time to avoid mechanical damage, and then set aside for use.

[0027] (3) In vitro maturation culture Add 500 μL of the in vitro maturation culture medium for each experimental group to each well of a sterile four-well plate, cover with 250 μL of sterile mineral oil (autoclaved at 121℃ for 30 min, and used after cooling), and place in a carbon dioxide incubator at 39℃, 5% CO2, and saturated humidity for 1 h to equilibrate the culture medium to a stable culture environment. Inoculate 45-50 COCs per well, incubate for 22 h, and then transfer to a basal culture system without eCG and hCG but with the same other components. Continue to incubate under the same conditions for another 22 h, for a total culture time of 44 h.

[0028] (4) Detection of maturation indicators and statistics of the proportion of cell nuclei with DNA fragmentation After in vitro maturation culture of oocytes, equal amounts of cumulus cells (COCs) were selected from each experimental group and treated with 0.1% hyaluronidase at 37°C for 5 minutes. Cumulus cells were then gently removed by pipetting. The naked oocytes were washed three times with PBS-PVA (PBS solution containing 0.1% PVA, filtered and sterilized, then stored at 4°C). The maturation status and DNA integrity were then assessed using nuclear staining combined with terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling. The specific procedure was as follows: After cumulus cell removal, the oocytes were first fixed with 4% paraformaldehyde at room temperature for 15 minutes. After fixation, they were washed three times with PBS-PVA and then permeabilized in DPBS containing 0.1% (v / v) Triton-X100 for 15 minutes. The permeabilized oocytes were washed with PBS-PVA and then transferred to DPBS containing 10 mg / mL bovine serum albumin, incubated overnight at 4°C. The following day, oocytes were retrieved, washed with PBS-PVA, and then processed according to the TUNEL apoptosis detection kit instructions: fluorescein-labeled dUTP and TdT enzyme were added, and the cells were incubated at 38.5°C in the dark for 1 h; followed by washing with PBS-PVA and then counterstaining with DAPI for 10 minutes. After counterstaining, the cells were washed again with PBS-PVA, mounted, and observed under a fluorescence microscope. The incidence of GVBD, MII stage maturation rate, and DNA fragmentation were observed under the fluorescence microscope; TUNEL-positive cells were identified as DNA-fragmented cells. The percentages were counted and calculated using ImageJ software.

[0029] Germinal vesicle rupture (GVBD) is a key marker of oocyte maturation. A higher GVBD rate indicates a larger number of oocytes initiating meiosis and entering the maturation process, resulting in better overall oocyte maturation efficiency. Metaphase II (MII) marks complete oocyte maturation and fertilization capacity. A higher MII maturation rate indicates a higher proportion of oocytes completing the entire maturation process and acquiring normal fertilization capacity, resulting in better oocyte maturation quality. The percentage of DNA-fragmented nuclei reflects the degree of genetic material damage and apoptosis in oocytes. A lower percentage indicates higher DNA integrity, less nuclear damage and apoptosis, and better oocyte developmental potential.

[0030] 3. Experimental Results and Analysis This experiment investigated the effects of different concentrations of lupeol (0 μM (DMSO), 1 μM, 2 μM, 4 μM, and 8 μM) on the incidence of GVBD, MII maturation rate, and proportion of DNA-fragmented nuclei in porcine oocytes.

[0031] The results are as follows Figure 1 As shown: Figure 1 In the figure, A represents the GVBD rate of porcine oocytes. The GVBD rate of the low concentration (1 μM, 2 μM) experimental group was not significantly different from that of the control group (CT, DMSO), remaining at about 95%. However, the GVBD rate of the high concentration (4 μM, 8 μM) experimental group increased significantly to about 98%, which was significantly higher than that of the control group (p<0.05), indicating that high concentration of lupeol significantly promoted the recovery of oocyte meiosis.

[0032] Figure 1 B in the figure represents the MII stage maturation rate of porcine oocytes: the MII stage maturation rate of porcine oocytes treated with lupeol was significantly higher than that of the CT and DMSO control groups. As the lupeol concentration increased, the maturation rate initially increased and then decreased. The 4 µM treatment group achieved the highest MII stage maturation rate. This indicates that the optimal concentration (4 µM) of lupeol can effectively promote the final maturation of oocytes.

[0033] Figure 1 In the figure, C represents the proportion of DNA-fragmented nuclei. The addition of lupeol significantly reduced the proportion of DNA-fragmented nuclei (i.e., reduced DNA damage rate), and the DNA damage rate decreased significantly with increasing lupeol concentration. The 4 µM and 8 µM treatment groups showed the lowest DNA damage rates, significantly lower than the control group and the 1 µM and 2 µM low-concentration treatment groups. This indicates that appropriate concentrations of lupeol can effectively protect oocytes and reduce damage to their genetic material during maturation.

[0034] Example 2: Verification of the antioxidant properties of lupeol on porcine oocytes This embodiment focuses on investigating the antioxidant effect of lupeol on porcine oocytes, clarifying its effect on regulating the cellular redox environment and improving cell quality.

[0035] 1. Experimental Grouping Blank control group (CT): only the basic culture system, without lupeol and DMSO.

[0036] Solvent control group (DMSO): An equal volume of DMSO was added to the basic culture system to eliminate the interference of the solvent on the experimental results.

[0037] Lupeol experimental group: Lupeol was added to the basic culture system to concentrations of 1 μM, 2 μM, 4 μM and 8 μM.

[0038] Three independent replicate experiments were set up to ensure data reliability.

[0039] 2. Experimental Methods and Procedures The methods for oocyte collection, pretreatment, and in vitro maturation culture were the same as in Example 1. After 44 h of culture, two antioxidant-related indicators, reactive oxygen species and glutathione activity, were detected to evaluate the antioxidant effect of lupeol. After in vitro maturation culture of oocytes, equal amounts of cumulus cells (COCs) were selected from each experimental group and treated with 0.1% hyaluronidase at 37 °C for 5 minutes. Cumulus cells were then gently removed by pipetting. The naked oocytes were washed three times in PBS-PVA. Following the instructions of the reactive oxygen species (ROS) and glutathione (GSH) detection kits, the oocytes were carefully transferred to 10 μM fluorescent dye H2DCFDA (Thermo Fisher Scientific, D399) or 10 µM CMF2HC (Thermo Fisher Scientific, C12881) and incubated for 15-30 minutes. After incubation, the oocytes were rinsed with PBS-PVA and placed in 10 μL of PBS-PVA droplets. Fluorescence intensity was detected under a fluorescence microscope equipped with an ultraviolet filter. Green and blue represent ROS and GSH, respectively.

[0040] ROS refers to reactive oxygen species, a key indicator reflecting the degree of oxidative stress damage in cells. Higher ROS levels indicate more severe oxidative stress and greater cellular oxidative damage in oocytes. GSH refers to glutathione, an important intracellular antioxidant representing the cell's own antioxidant capacity. Higher GSH levels indicate stronger antioxidant capacity in oocytes and better effectiveness in scavenging free radicals and resisting oxidative damage. 3. Experimental Results and Analysis Compared with the CT and DMSO control groups, the reactive oxygen species levels in all lupeol treatment groups were significantly reduced, with the 4-8 µM lupeol treatment group showing the most significant reduction. Figure 2 (A) Meanwhile, glutathione activity was increased to varying degrees in all lupeol treatment groups, with the 4 µM treatment group reaching the highest level (A). Figure 2 (B in the text) Treatment with 4 µM lupeol simultaneously achieved lower reactive oxygen species (ROS) levels and the highest glutathione activity. This optimized redox state produced multiple synergistic effects: the reduced ROS directly decreased the risk of DNA oxidative damage, corresponding to... Figure 1The group exhibited good DNA integrity; the elevated glutathione levels not only enhanced antioxidant defense but also provided essential biochemical support for oocyte maturation and embryonic development. While the 8 µM lupeol treatment showed similar DNA protection to the 4 µM treatment, its glutathione activity decreased, indicating that lupeol concentrations exceeding 4 µM may interfere with normal antioxidant regulation, resulting in weaker support for subsequent developmental functions compared to the 4 µM group. Therefore, a lupeol concentration of 4 µM establishes an optimal balance between oxidative damage control and antioxidant system activation, thereby optimizing oocyte quality and embryonic developmental potential.

[0041] Example 3: Verification of the effect of 4 μM lupeol on early embryonic development after in vitro fertilization of porcine oocytes This embodiment verifies the long-term protective effect of lupeol on the early embryonic development potential after in vitro fertilization of porcine oocytes.

[0042] 1. Experimental Grouping Blank control group (CT): only the basic culture system, without lupeol and DMSO.

[0043] Solvent control group (DMSO): An equal volume of DMSO was added to the basic culture system to eliminate the interference of the solvent on the experimental results.

[0044] Lupeol experimental group: Lupeol was added to the basic culture system to concentrations of 1 μM, 2 μM, 4 μM and 8 μM.

[0045] Three independent replicate experiments were set up to ensure data reliability.

[0046] 2. Experimental Methods and Procedures The methods for oocyte collection, pretreatment, and in vitro maturation culture were the same as in Example 1. After oocyte maturation, in vitro fertilization and early embryo culture were performed, as follows: (1) In vitro fertilization Sperm processing and fertilization: Remove the frozen bovine semen tubes from liquid nitrogen and thaw them in a 37°C water bath for 8-10 seconds. Transfer the semen to 5 mL centrifuge tubes and centrifuge at 550×g for 10 min. After centrifugation, discard the supernatant, add fertilization medium (PFM), and count sperm using a cell counting chamber to adjust the sperm concentration to 4×10⁻⁶. 6 Sperm suspension was obtained by measuring sperm count / mL. Mature cocci were transferred to 250 μL LPFM medium, with 40-50 cocci placed in each well. 250 μL of sperm suspension was added, and the mixture was incubated in a 39℃, 5% CO2, 5% O2, 90% N2 triple gas incubator for 5-6 h to complete fertilization.

[0047] (2) Early embryo culture After fertilization, fertilized eggs were washed three times with PZM-5 cleavage-stage medium, and residual cumulus cells and excess sperm were gently removed by pipetting to avoid damage to the fertilized eggs. The washed fertilized eggs were then transferred to PZM-5 medium, 30 eggs per well, covered with sterile mineral oil, and cultured in a tri-gas incubator for 72 h. The cleavage rate was then calculated. Subsequently, the blastomeres were transferred to PBM medium and cultured under the same conditions for another 4 days, and the blastocyst formation rate was calculated.

[0048] (3) Blastocyst quality testing Blastocyst quality is comprehensively assessed using multiple indicators, including cleavage rate, fertilization status, blastocyst formation rate, total number of blastocyst cells, and the proportion of DNA-fragmented cells in the blastocyst. The specific methods are as follows: ① Fertilization rate detection: The acetic acid staining method was used. Fertilized eggs 10 hours after in vitro fertilization were placed on a glass slide and fixed with acetic acid:ethanol (1:3 v / v) fixative for 48 hours. Then, they were stained with acetic lichen red (1% lichen red dissolved in 45% acetic acid) and examined under a phase contrast microscope. Oocytes with both female and male pronuclei were considered to have normal fertilization, and the fertilization rate and intracytoplasmic sperm fertilization rate were calculated accordingly.

[0049] ② Developmental progress monitoring: The cleavage rate was counted on day 3 after fertilization and the blastocyst formation rate was counted on day 7, which systematically reflects the developmental efficiency of the embryo from fertilization to the blastocyst stage.

[0050] ③ The analysis of blastocyst structure and genetic quality was carried out by the following methods: After the blastocysts were fixed with 4% paraformaldehyde, they were stained with DAPI and the cell nuclei were counted under a fluorescence microscope to obtain the total number of cells in the blastocysts; the proportion of DNA fragmented cells in the blastocysts was further detected by the TUNEL method to quantitatively assess the degree of apoptosis of blastocyst cells, thereby systematically judging the stability of the cellular composition and genetic integrity of the blastocysts.

[0051] Table 1. Effects of lupeol on blastocyst development and quality in early porcine embryos. The data in this table were obtained after six replicate experiments and are expressed as a percentage of mean ± SEM. CT represents the blank control group: only the basal culture system, without lupeol and DMSO; 0 µM represents the solvent control group (DMSO): an equal volume of DMSO was added to the basal culture system to eliminate the interference of the solvent on the experimental results. Different superscript letters in the same column indicate significant differences in data (P<0.05).

[0052] 3. Experimental Results and Analysis Fertilization rate refers to the proportion of oocytes that have successfully undergone fertilization out of the total number of oocytes. A higher fertilization rate indicates a stronger ability of the oocytes to combine with fertilization and a higher success rate of fertilization. Cleavage rate refers to the proportion of embryos that have undergone cleavage after fertilization. It is an important indicator of the early development of the embryo. A higher cleavage rate indicates smoother early division of the fertilized egg and a higher efficiency and proportion of embryonic development. Blastocyst formation rate refers to the proportion of fertilized eggs that have developed to the blastocyst stage. It is a core indicator for evaluating the overall developmental potential of the embryo. A higher blastocyst formation rate indicates a stronger embryonic developmental capacity and a higher proportion of embryos that can develop to the blastocyst stage. The total number of cells in the blastocyst is used to characterize the degree of blastocyst development and cell proliferation. A higher total number of cells indicates a more fully developed blastocyst with better overall structure and viability. The percentage of cell nuclei with fragmented DNA in the blastocyst reflects the degree of damage to genetic material and apoptosis in the blastocyst. A lower percentage indicates higher DNA integrity in the blastocyst, fewer apoptotic cells, and better blastocyst quality and subsequent implantation and developmental potential.

[0053] Experimental data show that lupeol exhibits a clear concentration-dependent effect in promoting the fertilization capacity of porcine oocytes and subsequent embryonic development. Figure 3 A in the study showed that lupeol significantly improved the fertilization rate of mature porcine oocytes, with the best effect observed at a concentration of 4 μM, which was significantly higher than that of the control group and the lupeol treatment groups at concentrations of 1-2 μM; however, lupeol treatment had no significant effect on the fertilization rate of single sperm. Figure 3 The B in the figure indicates that lupeol mainly works by improving overall fertilization efficiency, without changing the proportion of single sperm fertilization.

[0054] As shown in Table 1, in terms of embryonic development, the blastocyst formation rate of the 4 µM treatment group was 14.8%, significantly higher than that of the other groups, and the proportion of DNA fragmented cells in the blastocysts was the lowest (2.0%), indicating that its embryonic development efficiency and genetic quality were the best. In contrast, although the 8 µM treatment group was still better than the control group, all indicators were lower than those of the 4 µM group, indicating that its development-promoting effect had entered the decay range.

[0055] Correlation analysis of the two sets of data showed that 4 μM lupeol treatment not only optimized the fertilization function of oocytes but also translated it into excellent embryonic development outcomes, achieving an optimal balance in both blastocyst production rate and blastocyst quality. This finding provides an important concentration reference for establishing an efficient in vitro maturation system for porcine oocytes.

Claims

1. Application of lupeol in improving the efficiency of oocyte maturation in vitro.

2. The application according to claim 1, characterized in that, This study investigates the application of lupeol in improving oocyte GVBD rate, increasing oocyte MII maturation rate, or reducing oocyte DNA damage rate.

3. The application according to claim 1, characterized in that, Includes the following steps: Oocytes were cultured in an in vitro maturation medium containing lupeol.

4. The application according to claim 3, characterized in that, Includes the following steps: S1. Extract antral follicular fluid from healthy ovaries, centrifuge, select cumulus-oocyte complexes, and wash the cumulus-oocyte complexes three times with in vitro maturation culture medium containing human chorionic gonadotropin. S2. The cumulus-oocyte complexes treated in step S1 were sequentially transferred to in vitro maturation culture medium containing human chorionic gonadotropin (hCG) and in vitro maturation culture medium without hCG, covered with mineral oil, and cultured for 22 h respectively.

5. The application according to claim 4, characterized in that, The cumulus-oocyte complex is a morphologically intact cumulus-oocyte complex with at least three cumulus cell layers, uniform cytoplasm distribution, and no obvious damage.

6. The application according to claim 4, characterized in that, The in vitro maturation culture medium containing human chorionic gonadotropin (hCG) consists of: 10% PFF (volume fraction), 50 μM sodium pyruvate, 2 mg / mL D-sorbitol, 10 IU / mL equine hCG, 10 IU / mL human hCG, 50 μg / mL gentamicin, 2-8 μM lupeol, with the remainder being TCM199 medium containing 25 mM HEPES. The in vitro maturation culture medium that does not contain human chorionic gonadotropin consists of: 10% PFF (volume fraction), 50 μM sodium pyruvate, 2 mg / mL D-sorbitol, 50 μg / mL gentamicin, 2-8 μM lupeol, and the remainder is TCM199 medium containing 25 mM HEPES.

7. The application according to claim 6, characterized in that, The in vitro maturation culture medium containing human chorionic gonadotropin (hCG) consists of: 10% PFF (volume fraction), 50 μM sodium pyruvate, 2 mg / mL D-sorbitol, 10 IU / mL equine hCG, 10 IU / mL human hCG, 50 μg / mL gentamicin, 4 μM lupeol, with the remainder being TCM199 medium containing 25 mM HEPES. The in vitro maturation culture medium that does not contain human chorionic gonadotropin consists of: 10% PFF (volume fraction), 50 μM sodium pyruvate, 2 mg / mL D-sorbitol, 50 μg / mL gentamicin, 4 μM lupeol, and the remainder is TCM199 medium containing 25 mM HEPES.

8. The application according to claim 4, characterized in that, The culture was carried out at 39°C, 5% CO2, and saturated humidity.

9. The application according to claim 1, characterized in that, The oocytes mentioned are porcine oocytes.

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