Application of lycopene in sheep oocyte in-vitro culture
By adding lycopene and astragaloside IV combination to the in vitro culture medium of sheep oocytes, the problem of poor quality of in vitro culture of sheep oocytes is solved, the maturation rate and embryo development rate are increased, and the health status of cells is improved.
Patent Information
- Application Number
- CN202510886206.7
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-06-30
- Publication Date
- 2025-09-19
AI Technical Summary
The quality of sheep oocytes cultured in vitro is poor and the maturation rate is low, which affects the efficiency of in vitro embryo production.
Adding a combination of lycopene and astragaloside IV to the in vitro culture medium of sheep oocytes can regulate the first polar body extrusion rate, cleavage rate and blastocyst rate of oocytes, and reduce the damage of oxidative stress to oocytes by improving mitochondrial membrane potential and reducing the level of reactive oxygen.
It significantly increased the first polar body extrusion rate, cleavage rate and blastocyst rate of oocytes, reduced the level of reactive oxygen species, and enhanced the health of oocytes.
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Abstract
Description
Technical Field
[0001] The invention belongs to the field of biotechnology, and particularly relates to application of lycopene in in vitro culture of sheep oocytes. Background Art
[0002] Strengthening germplasm resources is an important action in today's society. We must lay a solid foundation for germplasm resources and protect and utilize them well. Livestock and poultry genetic resources are an important part of agricultural germplasm resources. They are the source of breeding excellent varieties and are related to the stable production and supply of livestock products. Livestock and poultry breeding is one of the important aspects of the revitalization of my country's seed industry, and it is also an important support for achieving a strong pastoral country. The most in-demand meat in Xinjiang is mutton, especially in southern Xinjiang, where mutton consumption is a rigid demand. In order to improve the efficiency of in vitro embryo production in sheep, break the bottleneck of low production efficiency in sheep breeding, and cultivate offspring with excellent production performance, in vitro embryo production technology (IVP) is an important means to solve this problem.
[0003] IVP technology involves in vitro oocyte maturation, in vitro fertilization, and in vitro embryo development. In vitro oocyte maturation is the key step affecting IVP results. However, the quality of oocytes cultured in vitro is extremely poor, with a maturation rate of only 45% to 59%. Therefore, improving the quality of oocyte culture in vitro is a critical issue that needs to be addressed by those skilled in the art. Summary of the Invention
[0004] In view of the problems existing in the prior art, the present invention provides the use of lycopene in the in vitro culture of sheep oocytes, aiming to solve some of the problems in the prior art or at least alleviate some of the problems in the prior art.
[0005] The present invention is achieved by using lycopene in preparing a reagent for in vitro culture of sheep oocytes.
[0006] Furthermore, lycopene regulates the first polar body extrusion rate, cleavage rate, and blastocyst rate of oocytes cultured in vitro.
[0007] The present invention also provides an oocyte maturation fluid, which contains lycopene.
[0008] Furthermore, it also contains at least one of astragaloside IV, isoliquiritigenin, forsythiaside and baicalein.
[0009] Furthermore, the concentration of the lycopene is 5 μM-20 μM.
[0010] Furthermore, the concentration of astragaloside IV is 10 μM-40 μM.
[0011] Furthermore, the concentration of the isoliquiritigenin is 20 μM-60 μM.
[0012] Furthermore, the concentration of the forsythin is 20 μM-60 μM.
[0013] In summary, the advantages and positive effects of the present invention are: This study proposes an in vitro oocyte maturation culture medium containing a combination of lycopene and astragaloside IV. Extensive experiments have shown that this culture medium can increase the oocyte first polar body extrusion rate to 81.67±2.89%, the cleavage rate to 92.89±1.49%, and the blastocyst rate to 63.30±2.05%. It also significantly reduces reactive oxygen species (ROS) levels (P=0.0407) and increases glutathione content (P=0.0043), alleviating oxidative stress damage to oocytes. Furthermore, it effectively improves oocyte mitochondrial membrane potential (P<0.0001), maintains mitochondrial oxidative phosphorylation capacity, and improves oocyte health. BRIEF DESCRIPTION OF THE DRAWINGS
[0014] Figure 1 The effect of combined addition of lycopene and astragaloside IV on the oxidative stress level of sheep oocytes; Figure 2 The effect of combined addition of lycopene and astragaloside IV on the mitochondrial membrane potential of sheep oocytes. DETAILED DESCRIPTION
[0015] In order to make the purpose, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the examples. Unless otherwise specified, the equipment and reagents used in each example and test example can be obtained from commercial sources. The specific examples described herein are only used to illustrate the present invention and are not intended to limit the present invention.
[0016] Based on the information contained in this application, it will be readily apparent to those skilled in the art that various changes can be made to the precise description of the present invention without departing from the spirit and scope of the appended claims. It should be understood that the scope of the present invention is not limited to the defined processes, properties, or components, as these embodiments and other descriptions are provided merely to illustrate specific aspects of the present invention. In fact, various changes that a person skilled in the art or related fields would clearly be able to make to the embodiments of the present invention are encompassed within the scope of the appended claims.
[0017] In order to better understand the present invention and not to limit the scope of the present invention, all numbers used in this application to express amounts, percentages, and other numerical values should be understood to be modified by the word "approximately" in all cases. Therefore, unless otherwise specified, the numerical parameters listed in the specification and the appended claims are approximate values, which may be changed according to the different ideal properties attempted to be obtained. Each numerical parameter should at least be regarded as obtained based on the reported significant figures and by conventional rounding methods. In the present invention, "about" means within 10% of a given value or range, preferably within 5%.
[0018] Unless otherwise specified in the following embodiments of the present invention, room temperature conditions are assumed. Room temperature refers to natural room temperature conditions in all seasons without additional cooling or heating, and is generally controlled within a range of 10-30°C, preferably 15-25°C.
[0019] The present invention discloses the application of lycopene in the in vitro culture of sheep oocytes. The present invention determines that the addition of a combination of lycopene and astragaloside IV to the in vitro maturation fluid of sheep oocytes can significantly improve the oocyte maturation rate. First, lycopene, astragaloside IV, isoliquiritigenin, forsythiaside, and baicalein are added to the oocyte maturation fluid, and the optimal dose of each additive is screened based on the first polar body extrusion rate, cleavage rate, and blastocyst rate. Finally, the additive combination is added to the oocyte maturation fluid based on the screened optimal dose, and the first polar body extrusion rate, cleavage rate, and blastocyst rate are tested to determine the optimal combination. The levels of ROS, GSH, mitochondrial membrane potential, oxidative stress, and apoptosis-related genes in the optimal combination are also tested to further determine its application effect.
[0020] The egg collection solution, culture solution formula and operation method of the present invention are as follows: Egg collection medium: TCM199 basal medium (500 mL), sodium heparin (0.025 g), gentamicin (0.025 g), and fetal bovine serum (2%, added upon use). Filter and store at 4°C.
[0021] Table 1 Composition of basal medium for oocyte culture
[0022] Oocyte maturation fluid: When in use, add the processed drug to the oocyte basal fluid, and finally prepare oocyte maturation fluid of different concentrations.
[0023] Table 2 Composition of early embryonic development fluid
[0024] Oocyte collection and culture: Before egg collection, prepare the egg collection solution and oocyte maturation medium. Add the egg collection solution to a 90mm culture dish (two-thirds of the dish) for subsequent egg collection. Add 1mL of oocyte maturation medium to a 30mm culture dish to prepare two wash plates to wash away impurities around the oocytes. Take a four-well plate and add 600μL of oocyte maturation medium containing different concentrations of lycopene to each well. Move both the culture dish and the four-well plate to a 38.5°C, 5% CO2 incubator to equilibrate overnight. Remove the ovaries from the thermos and use scissors to trim away any excess material, including the uterus, leaving only the ovaries. Rinse the ovaries three to four times in sodium chloride solution. After rinsing, place the ovaries in a beaker containing sodium chloride solution to prevent them from drying out before egg retrieval. Remove the preheated egg retrieval solution from the incubator and place it on the workbench. Use forceps to remove four to five cleaned ovaries at a time, place them on gauze, wipe the sodium chloride solution off the surface of the ovaries, and place the ovaries in the egg retrieval solution. Use a scalpel to gently incise the surface of the ovaries where follicles are visible. Repeat this process to collect as many oocytes as possible. Remove the dish and place it on a hot plate. Once the oocytes are collected, move the dish containing the egg retrieval solution to a stereomicroscope and use a mouth pipette to select the oocytes. Select oocytes with uniform cytoplasm and surrounded by at least three layers of cumulus granulosa cells. Place the collected oocytes in the dish for washing. After selecting the oocytes, take out the four-well plate and place it on a heating plate. Move the washed oocytes to the four-well plate and repeat this step. After all the oocytes have been transferred, place the four-well plate back into a 38.5°C, 5% CO2 incubator and culture for 24 hours.
[0025] Mature oocyte processing: Prepare a four-well plate in advance. Add 500 μL of egg collection medium and 100 μL of 1% hyaluronidase (0.005 g of hyaluronidase, 5 mL of TCM199 medium, filtered, aliquoted into 100 μL, and stored at -20°C) to each well. Prewarm the plate on a thermostat. Remove the mature oocytes from the incubator and place them under a stereomicroscope. Use a mouth pipette to aspirate the mature oocytes from the four-well plate and transfer them to the prewarmed egg collection medium containing hyaluronidase (approximately 100-150 oocytes per well). Use a pipette to gently pipette the oocytes repeatedly (do not forcefully pipette the oocytes against the well wall) until the granulosa cells are completely detached.
[0026] Parthenogenetic activation: Prepare a four-well plate one day in advance and add 500 μL of SOFaa solution to well 1; add 500 μL of SOFaa solution containing 7% ethanol to well 2 (ethanol is added before activation); and add 500 μL of SOFaa solution containing 6-DMAP to wells 3 and 4. Preheat the plate to equilibrate overnight in a 38.5°C, 5% CO2 incubator. Transfer the oocytes, degranulosa cells removed, to well 1 and wash away the hyaluronidase with SOFaa solution. After washing, transfer them to well 2 and treat with 7% ethanol for 5 minutes. After the ethanol treatment, transfer the oocytes to well 3 and wash away the ethanol. After washing, transfer the oocytes to well 4 and activate with 6-DMAP. Activate the oocytes in the incubator for 4 hours.
[0027] In vitro development of early embryos: Prepare development medium in the afternoon of the day before oocyte maturation. Prepare a 60mm culture dish and use SOFaa solution to make 50μL maturation droplets in the culture dish. Use paraffin oil to completely cover each droplet. Add 2-3mL of SOFaa solution to a 30mm culture dish as a wash dish. After preparation, transfer both culture dishes to a 38.5℃, 5% CO2 incubator for overnight equilibrium.
[0028] After 6-DMAP activation for 4 hours, oocytes were transferred to a SOFaa wash dish using a mouth pipette to wash off the 6-DMAP. After washing, oocytes were transferred to SOFaa microdroplets in 60 mm culture dishes using a mouth pipette (20-25 oocytes per droplet) and cultured in a 38.5°C, 5% CO2 incubator. Blastocyst yield was calculated on the 7th day of culture.
[0029] Example 1 Effects of Different Concentrations of Lycopene Added to In Vitro Maturation Fluid on the Development of Parthenogenetically Activated Embryos (1) Experimental design Control group: Immature oocytes were cultured in in vitro maturation basal medium for 24 h.
[0030] H2O2 group: Immature oocytes were cultured in in vitro maturation basal medium containing 200 μM H2O2 for 24 h.
[0031] Treatment groups 1-4: Immature oocytes were cultured in in vitro maturation medium containing 5 μM (treatment group 1), 10 μM (treatment group 2), 15 μM (treatment group 3), and 20 μM (treatment group 4) lycopene for 24 h.
[0032] (2) Oocyte in vitro maturation, parthenogenetic activation and embryo culture Immature sheep oocytes were collected and cultured using the in vitro maturation medium of the above groupings. The oocyte cleavage rate and blastocyst rate were calculated.
[0033] (3) Test results Statistical results of oocyte development rates are shown in Table 3. The data showed that the first polar body extrusion rate in treatment group 1 (5 μM treatment group) (68.34 ± 1.86%) was significantly higher than that in the control group (51.10 ± 0.23%). There were no statistical differences between the control group and treatment groups 2 (50.98 ± 1.08%), 3 (51.80 ± 1.28%), and 4 (50.71 ± 1.47%). Compared with the control group (61.29 ± 1.41%), the cleavage rate in treatment group 1 (82.01 ± 0.88%) was significantly higher, while the cleavage rates in treatment groups 2, 3, and 4 were not statistically different from those in the control group. Furthermore, the blastocyst rate in treatment group 1 (54.40 ± 0.41%) was significantly higher than that in the control group (43.55 ± 1.40%). H2O2 significantly reduced the development rate of parthenogenetically activated embryos, and the addition of 5μM lycopene to the basal culture medium for in vitro maturation of oocytes had the most significant effect on improving the development rate of parthenogenetically activated embryos.
[0034] Table 3 Effects of adding lycopene to in vitro maturation medium on the development of parthenogenetically activated embryos
[0035] Note: Different superscript letters (a, b, c) in the same column indicate significant differences among the groups (P<0.05).
[0036] Example 2 Effects of Adding Different Concentrations of Astragaloside IV to In Vitro Maturation Fluid on the Development of Parthenogenetically Activated Embryos (1) Experimental design Control group: Immature oocytes were cultured in in vitro maturation basal medium for 24 h.
[0037] H2O2 group: Immature oocytes were cultured in in vitro maturation basal medium containing 200 μM H2O2 for 24 h.
[0038] Treatment groups 1-3: Immature oocytes were cultured in in vitro maturation medium containing 10 μM (treatment group 1), 20 μM (treatment group 2), and 40 μM (treatment group 3) of astragaloside IV for 24 h.
[0039] (2) Oocyte in vitro maturation, parthenogenetic activation and embryo culture Immature sheep oocytes were collected and cultured using the in vitro maturation medium of the above groupings. The oocyte cleavage rate and blastocyst rate were calculated.
[0040] (3) Test results Statistical results of oocyte development rates are shown in Table 4. The data showed that the first polar body extrusion rate in treatment group 2 (20 μM) (66.92 ± 1.35%) was significantly higher than that in the control group (52.70 ± 3.64%). There was no significant difference between the control group and treatment group 1 (52.01 ± 2.09%), while treatment group 3 (38.32 ± 0.91%) was significantly lower than that in the control group. Compared with the cleavage rate in the control group (62.51 ± 0.86%), the cleavage rate in treatment group 2 (85.15 ± 1.58%) was significantly higher. There was no statistical difference between the cleavage rate in treatment group 1 (61.84 ± 0.99%) and the control group, while the cleavage rate in treatment group 3 (48.65 ± 5.57%) was significantly lower than that in the control group. Furthermore, the blastocyst rate in the 20 μM treatment group (53.07 ± 0.47%) was significantly higher than that in the control group (42.18 ± 0.82%). H2O2 significantly reduced the development rate of parthenogenetically activated embryos, and the addition of 20 μM astragaloside IV to the basal culture medium for in vitro maturation of oocytes had the most significant effect on improving the development rate of parthenogenetically activated embryos.
[0041] Table 4 Effects of adding astragaloside IV to in vitro maturation medium on the development of parthenogenetic activated embryos
[0042] Note: Different superscript letters (a, b, c) in the same column indicate significant differences among the groups (P<0.05).
[0043] Example 3 Effects of adding different concentrations of isoliquiritigenin to in vitro maturation fluid on the development of parthenogenetically activated embryos (1) Experimental design Control group: Immature oocytes were cultured in in vitro maturation basal medium for 24 h.
[0044] H2O2 group: Immature oocytes were cultured in in vitro maturation basal medium containing 200 μM H2O2 for 24 h.
[0045] Treatment groups 1-3: Immature oocytes were cultured in in vitro maturation medium containing 20 μM (treatment group 1), 40 μM (treatment group 2), and 60 μM (treatment group 3) of isoliquiritigenin for 24 h.
[0046] (2) Oocyte in vitro maturation, parthenogenetic activation and embryo culture Immature sheep oocytes were collected and cultured using the in vitro maturation medium of the above groupings. The oocyte cleavage rate and blastocyst rate were calculated.
[0047] (3) Test results The statistical results of oocyte development rates are shown in Table 5. The data show that the first polar body extrusion rate in treatment group 2 (40 μM) (66.72 ± 1.55%) was significantly higher than that in treatment group 1 (59.14 ± 0.71%) and treatment group 3 (61.77 ± 1.08%). The first polar body extrusion rates in treatment groups 1 and 3 were also higher than those in the control group (53.20 ± 1.12%). Compared with the cleavage rate in the control group (63.08 ± 1.55%), the cleavage rates in treatment group 2 (81.26 ± 1.81%) and treatment group 3 (75.13 ± 1.49%) were significantly higher. Furthermore, the blastocyst rate in treatment group 2 (50.59 ± 1.03%) was significantly higher than that in the control group (42.40 ± 1.14%). H2O2 significantly reduced the development rate of parthenogenetically activated embryos, and the addition of 40 μM isoliquiritigenin to the basal culture medium for in vitro maturation of oocytes had the most significant effect on improving the development rate of parthenogenetically activated embryos.
[0048] Table 5 Effects of adding isoliquiritigenin to in vitro maturation medium on the development of parthenogenetically activated embryos
[0049] Note: Different superscript letters (a, b, c) in the same column indicate significant differences among the groups (P<0.05).
[0050] Example 4 Effect of adding forsythiaside to in vitro maturation fluid on the development of parthenogenetically activated embryos (1) Experimental design Control group: Immature oocytes were cultured in in vitro maturation basal medium for 24 h.
[0051] H2O2 group: Immature oocytes were cultured in in vitro maturation basal medium containing 200 μM H2O2 for 24 h.
[0052] Treatment groups 1-3: Immature oocytes were cultured in in vitro maturation medium containing 20 μM (treatment group 1), 40 μM (treatment group 2), and 60 μM (treatment group 3) for 24 h.
[0053] (2) Oocyte in vitro maturation, parthenogenetic activation and embryo culture Immature sheep oocytes were collected according to the method described in Example 1, and oocyte culture was performed using the in vitro maturation culture medium of the above groupings. At the same time, the oocyte cleavage rate and blastocyst rate were calculated according to the method described in Example 1.
[0054] (3) Test results The statistical results of oocyte development rates are shown in Table 6. The data showed that the first polar body extrusion rate in treatment group 2 (40 μM) (61.39 ± 1.27%) was significantly higher than that in the control group (51.11 ± 1.73%). There was no statistical difference between treatment groups 1 (54.17 ± 1.44%) and 3 (57.22 ± 1.27%). Compared with the cleavage rate in the control group (62.55 ± 3.31%), the cleavage rates in treatment groups 1 (71.87 ± 4.15%), 2 (76.48 ± 1.86%), and 3 (69.45 ± 2.25%) were significantly higher, with no statistical difference. Furthermore, the blastocyst rate in treatment group 2 (55.70 ± 3.30%) was significantly higher than that in the control group (41.90 ± 3.31%). H2O2 significantly reduced the development rate of parthenogenetically activated embryos, and the addition of 40 μM forsythiaside to the basal culture medium for in vitro maturation of oocytes had the most significant effect on improving the development rate of parthenogenetically activated embryos.
[0055] Table 6 Effects of forsythiaside supplemented with in vitro maturation solution on the development of parthenogenetically activated embryos
[0056] Note: Different superscript letters (a, b, c) in the same column indicate significant differences among the groups (P<0.05).
[0057] Example 5 Effects of adding different concentrations of baicalein to in vitro maturation fluid on the development of parthenogenetically activated embryos (1) Experimental design Control group: Immature oocytes were cultured in in vitro maturation basal medium for 24 h.
[0058] H2O2 group: Immature oocytes were cultured in in vitro maturation basal medium containing 200 μM H2O2 for 24 h.
[0059] Treatment groups 1-3: Immature oocytes were cultured in in vitro maturation medium containing 20 μM (treatment group 1), 30 μM (treatment group 2), and 40 μM (treatment group 3) baicalein for 24 h.
[0060] (2) Oocyte in vitro maturation, parthenogenetic activation and embryo culture Immature sheep oocytes were collected according to the method described in Example 1, and oocyte culture was performed using the in vitro maturation culture medium of the above groupings. At the same time, the oocyte cleavage rate and blastocyst rate were calculated according to the method described in Example 1.
[0061] (3) Test results Statistical results of oocyte development rates are shown in Table 7. The data showed that the first polar body extrusion rates in treatment groups 1 (20 μM treatment group) (55.56 ± 1.27%) and 2 (55.56 ± 0.48%) were significantly higher than those in the control group (51.94 ± 0.96%), while no statistical difference was observed between treatment group 3 (52.22 ± 1.27%) and the control group. The cleavage rates in treatment groups 1 (61.02 ± 2.52%) and 2 (61.01 ± 3.75%) were not significantly different from those in the control group (60.46 ± 3.40%), while those in treatment group 3 (57.97 ± 0.69%) were significantly lower than those in the control group. Furthermore, the blastocyst rates in treatment groups 1 (41.49 ± 1.17%) and 2 (41.01 ± 1.23%) were not significantly different from those in the control group (40.61 ± 2.64%). H2O2 significantly reduced the development rate of parthenogenetically activated embryos. Adding 30 μM baicalein to the basal culture medium for in vitro maturation of oocytes increased the rate of first polar body extrusion of oocytes, but there was no significant difference in the blastocyst rate.
[0062] Table 7 Effects of adding baicalein to in vitro maturation solution on the development of parthenogenetic activated embryos
[0063] Note: Different superscript letters (a, b, c) in the same column indicate significant differences among the groups (P<0.05).
[0064] Example 6 Effects of Adding Different Compositions to In Vitro Maturation Fluid on the Development of Parthenogenetically Activated Embryos (1) Experimental design Control group: Immature oocytes were cultured in in vitro maturation basal medium for 24 h.
[0065] Treatment groups 1-10: Immature oocytes were cultured in in vitro maturation medium containing the following compositions for 24 h.
[0066] Table 8 Compositions added to different treatment groups
[0067] (2) Oocyte in vitro maturation, parthenogenetic activation and embryo culture Immature sheep oocytes were collected and cultured using the in vitro maturation medium of the above groupings. The oocyte cleavage rate and blastocyst rate were calculated.
[0068] (3) Test results Table 9 shows the statistical results of oocyte development rates. The data indicate that among the treatments supplemented with both components, Treatment 1 (5μM lycopene + 20μM astragaloside IV) achieved the highest first polar body extrusion rate, cleavage rate, and blastocyst rate. These results demonstrate that the addition of 5μM lycopene and 20μM astragaloside IV to the basal medium for in vitro oocyte maturation significantly improves the development rate of parthenogenetically activated embryos.
[0069] Table 9 Effects of adding different compositions to in vitro maturation solution on the development of parthenogenetically activated embryos
[0070] Note: Different superscript letters (a, b, c) in the same column indicate significant differences among the groups (P<0.05).
[0071] Example 7 Effects of Lycopene and Astragaloside IV Added to In Vitro Maturation Fluid on Oocyte Maturation Quality (1) Experimental design Control group: Immature oocytes were cultured in vitro in the in vitro maturation basal culture medium for 24 h.
[0072] Treatment group: cultured in in vitro maturation medium containing 5 μM lycopene + 20 μM astragaloside IV for 24 h.
[0073] (2) Oocyte in vitro maturation, parthenogenetic activation and embryo culture Immature sheep oocytes were collected and cultured using the in vitro maturation culture medium of the above groupings, followed by in vitro fertilization and embryo culture.
[0074] (3) Detection of GSH and ROS DCFH-DA was used to detect ROS levels: DCFH-DA was diluted 1000-fold in TCM199 basal medium to a final concentration of 10 μmol / L. 80 μL of dye droplets were prepared, and cells were randomly selected and transferred into the droplets. The cells were then incubated in the dark at 38.5°C in an incubator. After 25 minutes, the cells were removed, washed twice with TCM199 medium, and observed under an inverted fluorescence microscope. Images were acquired using ZEN software.
[0075] CMF2HC was used to measure GSH levels: CMF2HC was diluted in TCM199 medium. 10 μL of CMF2HC was added to 990 μL of TCM199 medium. An 80 μL droplet of dye was prepared, and cells were randomly selected and transferred into the droplet. The cells were incubated in a dark incubator at 38.5°C for 25 minutes and washed twice with TCM199 medium. Images were acquired using an inverted fluorescence microscope using ZEN software.
[0076] The acquired fluorescence images were analyzed using Image J software.
[0077] (4) Detection of mitochondrial membrane potential Mitochondrial membrane potential was measured using JC-1: JC-1 was diluted at a ratio of 5 μL of JC-1 (200×) to 1 mL of JC-1 staining buffer. An 80 μL droplet of dye was prepared and randomly selected cells were placed in the droplet. Cells were incubated in the dark at 38.5°C, removed after 25 minutes, and washed twice with TCM199 medium. Images were acquired using an inverted fluorescence microscope using ZEN software and analyzed using Image J software for red and green fluorescence.
[0078] (5) Test results ROS and GSH levels: To evaluate the antioxidant effect of combined lycopene and astragaloside IV on in vitro matured oocytes, the ROS and GSH levels in the mature oocytes of the control and treatment groups were evaluated. Figure 1 As shown in the figure, according to the fluorescence intensity analysis, the ROS level in the oocytes of the treatment group was significantly decreased (P < 0.001) and the GSH level was significantly decreased (P = 0.0043) compared with the control group.
[0079] Mitochondrial membrane potential: Mitochondrial dysfunction and decreased mitochondrial membrane potential are important factors that lead to decreased oocyte maturation rate. Therefore, this experiment used JC-1 to detect changes in mitochondrial membrane potential. Figure 2 As shown in the figure, representative images collected after JC-1 staining showed that the mitochondrial membrane potential of the treatment group was significantly increased compared with the control group (P < 0.0001), indicating that the combined addition of lycopene and astragaloside IV can enhance mitochondrial function.
[0080] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.
Claims
1. Application of lycopene in the preparation of sheep oocyte in vitro culture reagents.
2. The use according to claim 1, characterized in that: Lycopene regulates the first polar body extrusion rate, cleavage rate, and blastocyst rate of oocytes cultured in vitro.
3. An oocyte maturation fluid, characterized in that: The oocyte maturation fluid contains lycopene.
4. The oocyte maturation solution according to claim 3, wherein: The invention also contains at least one of astragaloside IV, isoliquiritigenin, forsythiaside and baicalein.
5. The oocyte maturation solution according to claim 3, wherein: The concentration of lycopene is 5 μM-20 μM.
6. The oocyte maturation solution according to claim 4, characterized in that: The concentration of astragaloside IV is 10 μM-40 μM.
7. The oocyte maturation solution according to claim 4, characterized in that: The concentration of the isoliquiritigenin is 20 μM-60 μM.
8. The oocyte maturation solution according to claim 4, characterized in that: The concentration of the forsythin is 20 μM-60 μM.