Method and system for influencing in-vitro maturation of porcine oocytes by EPA (Eicosapentaenoic Acid)

Abstract

The invention belongs to the technical field of biology, and discloses a method for influencing in-vitro maturation of porcine oocytes by EPA (Eicosapentaenoic Acid), and the result of the invention shows that 37 genes related to signaling pathways such as apoptosis and programmed death are obviously reduced at the transcriptional level, and the genes related to mitotic pathways are obviously improved at the transcriptional level. 1500 oocytes of the EPA treatment group and 1500 oocytes of the control group in the MII stage are collected for proteome sequencing, 28 significantly up-regulated proteins exist in the EPA treatment group, and 95 significantly down-regulated proteins exist in the EPA treatment group. Results of the invention show that 10 [mu] MEPA treatment can significantly improve the first polar body discharge rate of in-vitro maturation of porcine oocytes. The 10 [mu] m MEPA treatment group significantly improves the expansion condition of cumulus cells in the in-vitro maturation process, and also reduces the proportion of apoptotic cells in cumulus cells. The 10mu MEPA treatment can promote the cytoplasm maturation of the porcine oocytes, thereby coordinating the synchronous maturation of the karyoplasm.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to a method and a system for influencing EPA on in vitro maturation of pig oocytes. Background Art

[0002] With the development of modern animal husbandry, efficient breeding technology has become an important means to improve livestock production efficiency. Embryo transplantation technology plays a key role in pig breeding, especially in accelerating the spread of high-quality genetic resources and improving population quality. However, the current porcine oocyte in vitro maturation (IVM) system has not yet fully met the needs of high-quality embryo production, and the quality of mature oocytes fluctuates greatly, which directly affects the embryo development potential and transplantation success rate. In addition, pigs and humans are highly similar at the physiological, anatomical and genomic levels and are important biomedical model animals. Optimizing the IVM system is of great significance to pig transgenic technology, disease model construction and organ transplantation research. However, the limitation of the efficiency of the IVM system has become a bottleneck in the development of related research.

[0003] The imperfections of the porcine oocyte IVM system are mainly due to the inability of in vitro conditions to fully simulate the in vivo environment, asynchronous nuclear and cytoplasmic maturation, single culture medium components, oxidative stress, insufficient DNA damage repair, and lack of a comprehensive understanding of the regulatory mechanism. In-depth research and solutions to these problems will help build a more efficient and stable IVM system, further improve the oocyte maturation rate and embryo development quality, and promote the common development of animal husbandry and biomedicine.

[0004] In vivo, oocyte maturation depends on a complex follicular microenvironment, including precise regulation of hormones, cytokines, nutrients, and redox states. However, it is difficult for existing IVM systems to fully simulate these conditions, resulting in the quality of mature oocytes being lower than that under in vivo conditions. In in vitro culture, high oxygen concentrations and reactive oxygen species (ROS) produced during culture can induce oxidative stress, damage the DNA, proteins, and membrane structure of oocytes, and reduce the quality of maturation. Nuclear maturation (such as completion of meiosis) usually occurs relatively smoothly under in vitro conditions, but cytoplasmic maturation (such as mitochondrial function improvement and protein synthesis) is often incomplete. This asynchrony of nuclear and cytoplasmic maturation directly affects the subsequent developmental potential of the embryo. Mitochondria in the oocyte cytoplasm are important organs for providing energy and regulating metabolism. In in vitro culture, the mitochondrial distribution pattern and function are easily damaged, resulting in insufficient intracellular ATP levels, affecting the quality of maturation.

[0005] Traditional culture media usually contain only basic glucose, amino acids and hormones, but lack the diverse factors required to regulate oocyte maturation, such as antioxidants and signaling molecules. Oocyte maturation requires the activation of multiple signaling pathways, such as cAMP-PKA and MAPK signals. The lack of proper regulation of these signals in the culture medium leads to an inefficient maturation process. In vivo, cumulus cells provide important nutrition and signal support for oocytes. In in vitro culture, the function of cumulus cells is often limited, resulting in a decrease in their ability to support oocytes. Cumulus cells are prone to apoptosis or degeneration in in vitro culture, which not only affects the maturation of oocytes, but also limits their subsequent embryonic development ability. During in vitro culture, oxidative stress is an important factor affecting oocyte maturation. However, the current culture system lacks sufficient antioxidants to maintain intracellular redox balance. Excessive ROS not only damages oocyte DNA, but also affects organelle function (such as mitochondria and endoplasmic reticulum), leading to cellular metabolic disorders. During in vitro culture, oocytes are easily affected by the external environment (such as culture conditions and ROS) and DNA damage occurs, and the repair ability is weak. Key proteins that regulate DNA repair (such as BRCA1 and RAD51) are underexpressed in in vitro culture, further limiting the quality of oocytes. Improper culture conditions (such as nutritional imbalance or oxidative stress) can activate the apoptotic signaling pathway of oocytes, leading to a decrease in maturation rate. The apoptosis of cumulus cells not only affects their interaction with oocytes, but also further weakens the stability of the IVM system.

[0006] In order to alleviate the adverse effects of oxidative stress on oocyte maturation in vitro and improve the quality of oocyte development in vitro, this study added polyunsaturated fatty acid eicosapentaenoic acid (EPA) to conduct experiments. Different doses of EPA were added to the porcine oocyte in vitro maturation system to screen the optimal dose for improving porcine oocyte maturation. The effects of EPA treatment on porcine oocyte in vitro maturation were studied with nuclear maturation, expansion of cumulus cells, cytoplasmic maturation and ROS levels, mitochondrial and cortical granule distribution patterns, and embryonic development as key indicators; and the potential mechanism of EPA improving porcine oocyte in vitro maturation was explored from the two levels of cumulus cells and oocytes with the help of transcriptomics and proteomics.

[0007] Through the above analysis, the problems and defects of the prior art are as follows:

[0008] The in vitro conditions of porcine oocytes cannot completely simulate the in vivo environment, nuclear and cytoplasmic maturation is not synchronized, the culture medium components are single, oxidative stress, and DNA damage repair is insufficient. During the in vitro maturation process, the lack of protection of antioxidant enzymes in the follicular fluid leads to the accumulation of ROS molecules, which can attack other organelles and cause cell dysfunction, such as endoplasmic reticulum stress, spindle abnormalities, cytoskeleton damage and abnormal mitochondrial distribution, which will eventually lead to cell cycle arrest and apoptosis, thereby affecting the quality of oocytes and the subsequent embryonic development ability. Summary of the invention

[0009] In view of the problems existing in the prior art, the present invention provides a method for influencing EPA on in vitro maturation of pig oocytes.

[0010] The present invention is achieved in that a method for influencing EPA on in vitro maturation of porcine oocytes comprises:

[0011] Step 1, selection of oocytes;

[0012] After the oocytes were extracted, they were washed three times with egg washing solution, and then oocytes with cumulus granulosa cells and uniform cytoplasm were selected for culture under a stereoscope;

[0013] Step 2, 10 μM EPA in the in vitro maturation system;

[0014] 10 μM EPA was added to the maturation medium, GV stage oocytes were cultured, and the effects of different doses of EPA on oocyte maturation were compared;

[0015] Step 3, 10 μM EPA treatment of oocytes for subsequent embryonic developmental capacity testing;

[0016] After parthenogenetic activation of selected mature oocytes, the cleavage rate, blastocyst rate and number of blastocyst cells in each group were counted.

[0017] Step 4, 10 μM EPA was used to treat oocytes to detect cytoplasmic maturation levels;

[0018] (1) Mature oocytes were selected, and the spindles were labeled with anti-microtubule antibodies (such as anti-α-tubulin), the cortical granules were labeled with FITC-labeled PNA cortical granule-specific dye, and the mitochondria were labeled with mitochondrial-specific fluorescent probes (MitoTrackerRed CMXRos).

[0019] (2) The proportions of spindle morphology (normal and abnormal), cortical granule distribution pattern (peripheral, cortical, uniform, and abnormal), and mitochondrial distribution pattern (uniform, peripheral, and abnormal) were calculated respectively.

[0020] Step 5: transcriptome sequencing of cumulus cells and oocytes;

[0021] The oocyte samples were divided into EPA group and control group, with 3 samples in each group and 500 oocytes in each sample; the cumulus cells of EPA group and control group were collected respectively, with 3 samples in each group and 10 7 cells.

[0022] Step 6, oocyte TMT quantitative proteome;

[0023] The samples were divided into EPA group and control group, with 3 samples in each group and 500 oocytes in each sample for transfection protein sequencing;

[0024] Step 7, statistical analysis;

[0025] All experiments were repeated at least three times independently, and all experimental data were expressed as mean ± standard error (mean ± SD). GraphPad Prism 9 software was used for difference significance analysis and correlation analysis; p < 0.05 was considered statistically significant.

[0026] Further, the selection of the oocytes:

[0027] Ovaries with follicles of 3-8mm in diameter were collected from slaughterhouses and placed in a constant temperature incubator. They were rinsed three times with warm saline before being placed in the incubator. The egg retrieval needle was adjusted to a suitable suction pressure for egg extraction. The tube wall was first rinsed with buffer before egg extraction, and the follicular fluid was then sucked into a centrifuge tube for temporary storage. The centrifuge tube was placed in an incubator to keep warm.

[0028] Further, the in vitro maturation:

[0029] (1) the follicular fluid in the centrifuge tube is precipitated in the egg washing solution at least three times, and the sedimentation process is carried out in a water bath;

[0030] (2) Select COCs wrapped by three layers of granulosa cells under stereomicroscope;

[0031] (3) Mix the mature solution containing different concentrations of EPA in advance and add it to a four-well plate in CO 2 The COCs were then transferred to a four-well culture plate for culturing.

[0032] (4) Oocytes were maintained at 38.5°C and 5% CO 2 Culture in an incubator for 42 h;

[0033] (5) After 42 h of culture, COCs were digested by blowing in hyaluronidase to remove cumulus cells, and then mature oocytes were selected.

[0034] Further, the embryo develops:

[0035] The selected oocytes were transferred to the preheated fusion solution and washed three times, and then transferred to the fusion tank with an electrode width of 1.0 mm, and the parameters were adjusted to 135V 30μs DC pulse, and the oocytes were activated by electric shock; after electric activation, the oocytes were washed three times with the balanced embryo culture medium, and then transferred to the balanced embryo culture medium covered with paraffin oil in advance, and incubated at 38.5℃, 5% CO 2 , culture under saturated humidity; observe the cleavage of the embryo under a stereoscope for 26-28 hours, and record the blastocyst status and count the blastocyst cell number on the seventh day of embryo culture.

[0036] Further, the porcine oocyte cytoplasmic maturation level is detected:

[0037] The selected oocytes were stained for tubulin, cortical granules and mitochondria.

[0038] Further, the freezing of porcine oocytes and cumulus cells:

[0039] Under a stereoscope, MII stage oocytes in good condition were selected, and then washed several times in PBS to ensure that no cumulus cells were attached. The oocytes were then transferred to RNase-free centrifuge tubes and quickly frozen in liquid nitrogen for subsequent transcriptome and TMT proteome sequencing. The cumulus cells that were blown off were collected, added to PBS and centrifuged several times, and then quickly frozen in liquid nitrogen for subsequent transcriptome sequencing.

[0040] Another object of the present invention is to provide a system for influencing EPA on in vitro maturation of porcine oocytes, comprising:

[0041] The selection module is used for selecting oocytes. After the oocytes are extracted, they are washed three times with egg washing solution, and then oocytes with cumulus granulosa cells and uniform cytoplasm are selected under a stereoscope for culture.

[0042] The culture module is used for in vitro maturation of oocytes; a concentration gradient is set in the maturation solution to add 10 μM EPA, and the GV stage oocytes are cultured for 42 hours;

[0043] The evaluation module is used to evaluate the nuclear maturation and cytoplasmic maturation of oocytes; the first polar body exclusion test is performed on oocytes cultured in vitro to evaluate the effect of EPA on their nuclear maturation; the spindle morphology, cortical granule distribution pattern, mitochondrial distribution pattern and ROS level in mature oocytes are detected to evaluate the effect of EPA treatment on their cytoplasmic maturation;

[0044] Sequencing module, used for reference transcriptome sequencing; the samples were grouped into EPA group and control group, with 3 samples in each group and 500 oocytes in each sample, and reference transcriptome analysis was performed to detect the effect of EPA treatment on the transcriptome of mature oocytes;

[0045] Quantitative module, used for TMT quantitative proteome; samples were divided into EPA group and control group, 3 samples in each group, 500 oocytes in each sample, and proteome sequencing was performed to detect the effect of EPA treatment on protein levels in mature oocytes;

[0046] The statistical analysis module was used to express the experimental data in the form of mean ± standard error (mean ± SD); GraphPad Prism 9 software was used for difference significance analysis and correlation analysis; P < 0.05 was considered to be statistically significant.

[0047] In combination with the above technical solutions and the technical problems solved, the advantages and positive effects of the technical solutions to be protected by the present invention are as follows:

[0048] First,

[0049] 1μM, 10μM, and 100μM EPA were added to the in vitro maturation system of porcine oocytes, and 10μM EPA treatment could significantly increase the in vitro maturation rate of porcine oocytes (87.6±1.19VS 79.2±2.84, P<0.05). Further studies found that 10μM EPA improved the abnormal proportion of spindle assembly (5.88%VS15.15%, P<0.05), and also significantly increased the proportion of fully expanded cumulus-oocyte complexes (COCs) (72.66±0.577%VS 66.70±2.055%, P<0.05) and significantly reduced the apoptosis rate of cumulus cells (14.07±1.670%VS20.93±1.768%, P<0.05). The significantly increased RvE1 level in the oocyte in vitro culture system is the potential mechanism by which EPA treatment reduces cumulus cell apoptosis. The results of cumulus cell transcriptome showed that the transcription levels of bone morphogenetic protein 15 (BMP15) and cytochrome C (CYP1A1) genes were significantly increased in the EPA-treated group, which is the potential mechanism by which EPA promotes the expansion and apoptosis inhibition of cumulus cells and promotes oocyte maturation. The developmental capacity of mature oocytes obtained by 10 μM EPA treatment was significantly improved, including cleavage rate (74.8±1.72 VS 71.2±1.68, P<0.05), blastocyst rate (24.16±1.21 VS 20.3±1.38, P<0.05), and blastocyst cell number (62.17±1.12 VS 54.58±1.70, P<0.05). Further studies found that EPA treatment significantly improved cytoplasmic maturation: the proportion of peripheral distribution patterns of cortical granules (54.84% VS 33.33%, P < 0.05), the proportion of mature oocytes with uniform mitochondrial distribution patterns was also significantly increased (64.1% VS 50%, P < 0.05), and EPA treatment also significantly reduced the level of ROS in mature oocytes. In order to analyze the potential mechanism by which EPA treatment improves the cytoplasmic maturation and embryonic development ability of porcine oocytes, 1,500 oocytes from the EPA treatment group and the control group at the MII stage were collected for transcriptome sequencing. The results showed that 37 genes related to signaling pathways such as apoptosis and programmed death were significantly reduced at the transcriptional level, while genes related to the mitotic pathway were significantly increased at the transcriptional level. 1,500 oocytes from the MII stage EPA-treated and control groups were collected for proteomic sequencing. In the proteomic sequencing, 28 proteins were significantly upregulated and 95 proteins were significantly downregulated in the EPA-treated group. The expression of genes related to ATP metabolism and synthesis in the EPA-treated group was significantly increased, while the expression of genes related to gametogenesis was significantly decreased, suggesting that EPA treatment can provide effective mRNA and protein reserves for subsequent embryonic development.

[0050] The results of this study showed that:

[0051] (1) Treatment with 10 μM EPA can significantly increase the first polar body extrusion rate of pig oocytes matured in vitro.

[0052] (2) The 10 μM EPA treatment group significantly improved the expansion of the cumulus during in vitro maturation and reduced the proportion of apoptotic cells in cumulus cells.

[0053] (3) Treatment with 10 μM EPA can promote the cytoplasmic maturation of pig oocytes and coordinate the synchronous maturation of nucleus and cytoplasm.

[0054] Second, as auxiliary evidence of the inventiveness of the claims of the present invention, it is also reflected in the following important aspects:

[0055] (1) The expected benefits and commercial value of the technical solution of the present invention after transformation are:

[0056] Expected Returns

[0057] Improve the reproductive efficiency of breeding pigs: By optimizing the in vitro maturation technology of pig oocytes, the success rate of pig embryo culture can be significantly improved, thereby improving the efficiency of artificial reproduction.

[0058] Improved embryo engineering technology: As a key link in embryo engineering, this technology provides strong support for the in vitro embryo production (IVP) technology chain and can provide higher quality embryos for biological breeding and embryo transplantation.

[0059] Business Value

[0060] Improved efficiency of animal husbandry: By improving the reproductive efficiency and survival rate of high-quality pig breeds, it can help farms and enterprises reduce production costs and increase economic benefits.

[0061] Commercial promotion: This technology can be transformed into a complete in vitro maturation optimization system or kit and sold to scientific research institutions, breeding pig farms, artificial insemination and embryo transplantation companies, with clear market value.

[0062] Technology licensing and cooperation: Establish cooperative relationships with related companies through technology patent licensing or joint development to obtain continuous technology transformation benefits.

[0063] (2) The technical solution of the present invention fills the technical gap in the industry at home and abroad:

[0064] Promote scientific research progress: This technical solution provides new tools and methods for studying the effects of EPA (arachidonic acid eicosapentaenoic acid) on the in vitro maturation of porcine oocytes, helps to deeply understand the relationship between lipid metabolism and oocyte development, and provides basic data for the field of animal husbandry science.

[0065] Platform technology: This method can be further extended to study the effects of other nutritional factors or drugs on oocyte development, and has broad scientific research application value.

[0066] (3) The technical solution of the present invention solves the technical problems that people have been eager to solve but have never been able to solve successfully:

[0067] Establish an EPA regulation system: optimize the in vitro maturation conditions of pig oocytes by precise EPA concentration control and addition timing, thereby significantly improving the maturation rate and embryo development potential.

[0068] Reveal biological mechanisms: clarify the molecular mechanism of EPA action and provide a theoretical basis for optimizing the IVM system of other species in the future.

[0069] Repeatability and practicality: Design a set of standardized and stable technical solutions to meet actual production needs and significantly improve the economic benefits of embryo culture.

[0070] (4) The technical solution of the present invention overcomes technical prejudice:

[0071] Breaking through the traditional perspective, introducing EPA as a key regulatory factor: If the present invention clarifies the positive effect of EPA on the in vitro maturation of oocytes, it will break through the bias of traditional research that relies too much on regulatory factors such as hormones and proteins, and expand the research perspective.

[0072] Innovation: Fatty acids are non-traditional biological regulators, and their optimization methods have a disruptive impact on the entire IVM system.

[0073] Based on the systematic and multi-dimensional study of the impact of EPA, the present invention breaks away from the technical limitations of verification under a single condition.

[0074] This technical solution not only verifies the mechanism of action of EPA, but also provides specific operating specifications for optimizing production, thus overcoming the bias of disconnection between basic research and practice. If the present invention is based on a large number of scientific experiments, uses statistical methods for rigorous verification, and excludes interference from noise factors, it reduces the bias caused by insufficient data samples or a single method. BRIEF DESCRIPTION OF THE DRAWINGS

[0075] Figure 1 It is a flow chart of the method for influencing EPA on in vitro maturation of porcine oocytes provided by an embodiment of the present invention.

[0076] Figure 2 It is a system structure diagram of the effect of EPA on in vitro maturation of porcine oocytes provided by an embodiment of the present invention.

[0077] Figure 3 This is the in vivo synthesis route map of RvE1 provided in the embodiments of the present invention.

[0078] Figure 4 The 10 μM EPA treatment provided by the embodiment of the present invention can significantly improve the nuclear maturation of porcine oocytes; A: GV stage COCs picture, (scale = 100 μm), MII stage oocyte picture, arrow points to the extruded polar body (scale = 50 μm). B: Pb1 extrusion rate of the control group and the EPA treatment group at 42 hours. C: Spindle staining in the second meiotic metaphase, the box part is the magnified part of the spindle, scale = 20 μm. D: Spindle staining statistics (n = 67) Percentage of normal and abnormal distribution. Data are shown as mean ± standard error. *P < 0.05, P < 0.01.

[0079] Figure 5 The 10μM EPA treatment provided in the embodiment of the present invention can reduce the proportion of apoptotic cells in cumulus cells; Figure 5 Map of 10μM EPA treatment reduces the proportion of cumulus cell; A: Cumulus cell expansion picture, scale 100=μm. B: Cumulus cell expansion statistics. C: COCs in the control group and EPA treatment group were cultured to maturity and then cumulus cells were removed to detect the apoptosis ratio by flow cytometry. D: Apoptosis ratio of cumulus granulosa cells. The content of sTNF-α in the culture medium of the control group and EPA treatment group at different time points. E: The content data of RvE1 in the culture medium of the control group and EPA treatment group at different time points are mean ± standard error. *P<0.05, P<0.01.

[0080] Figure 6 It is a 10μM EPA-treated granulocyte pattern of action plot provided in an embodiment of the present invention; Figure 6 Map of 10μM EPA-treated granulocyte pattern of action plot; A: principal component analysis. B: Volcano plot, 103 significantly upregulated genes and 63 significantly downregulated genes in the EPA-treated group. C: GO enrichment analysis of the 103 significantly upregulated genes and 63 significantly downregulated genes obtained by EPAVSCtrl. D: The genes of the obtained key pathways are presented in a heat map (*P<0.05, P<0.01).

[0081] Figure 7The 10 μM EPA treatment provided in the embodiment of the present invention can improve the subsequent blastocyst development; Figure 7 Map of 10 μM EPA treatment improves subsequent blastocyst development; A: 26-hour cleavage picture, scale bar = 50 μm. Blastocyst number picture, scale bar = 200 μm. Blastocyst cell number picture, scale bar = 50 μm. B: 26-hour cleavage rate of the control group and the EPA-treated group after parthenogenetic activation. C: Blastocyst rate on the sixth day after parthenogenetic activation D: Blastocyst cell number (n = 25) Data are shown as mean ± standard error. *P < 0.05, P < 0.01.

[0082] Figure 8 The 10 μM EPA treatment provided in the embodiment of the present invention can improve the cytoplasmic maturation of porcine oocytes by improving the distribution of cortical granules, mitochondrial distribution and ROS level; A: Cortical granule distribution pattern of porcine oocytes after in vitro maturation. (A1) Peripheral distribution. (A2) Cortical distribution. (A3) Uniform distribution. (A4) Abnormal distribution. (n=76). Scale bar=20 μm. B: Percentages of peripheral distribution, cortical distribution, uniform distribution and abnormal distribution of cortical granules in the control group and EPA-treated group. C: Distribution of mitochondria in porcine oocytes after in vitro maturation under a confocal microscope, including uniform distribution, perinuclear distribution and abnormal distribution. D: Percentages of uniform distribution, perinuclear distribution and atrophic distribution of mitochondria in the control group and EPA-treated group (n=77). E: Detection of reactive oxygen levels. Scale bar, 100 μm. F: The figure shows the relative fluorescence intensity of mature oocytes (n=94). The ROS level in the EPA-treated group was significantly lower than that in the control group. Data are mean ± standard error. *P<0.05, P<0.01.

[0083] Fig. 9 This is a diagram of the effect of 10 μM EPA treatment on the transcriptome pattern of mature oocytes provided by an embodiment of the present invention; A: principal component analysis. B: There are 142 significantly upregulated genes and 774 significantly downregulated genes in the EPA treatment group. C: KEGG enrichment of the 142 upregulated genes obtained by EPAVSCtrl. D: The KEGG enriched pathways and the key genes therein are specifically displayed. E: KEGG enrichment of the 774 downregulated genes obtained by EPAVSCtrl. F: The KEGG enriched pathways and the key genes therein are displayed.

[0084] Fig.10It is the effect of 10μM EPA treatment on the proteomic pattern of mature oocytes provided by an embodiment of the present invention; Figure 10 Map of 10μM EPA treatment on the proteomic pattern of mature oocytes; A: principal component analysis. B: Volcano plot, 26 significantly upregulated proteins and 95 significantly downregulated proteins in the EPA treatment group. C: The 26 significantly upregulated proteins and 95 significantly downregulated proteins obtained by EPAVSCTRL were subjected to differential heat map analysis. D: The 26 upregulated proteins obtained by EPAVSCTRL were enriched in GO. E: The GO pathways enriched by the upregulated proteins and the key proteins therein were specifically displayed. F: The 95 downregulated proteins obtained by EPAVSCTRL were enriched in GO. G: The GO pathways enriched by the downregulated proteins and the key proteins therein were specifically displayed. DETAILED DESCRIPTION

[0085] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0086] Example 1: Dose response experiment of EPA on in vitro maturation of porcine oocytes

[0087] Step 1: Oocyte selection and culture

[0088] Oocytes were extracted from healthy pig ovaries, washed three times with egg washing solution, and oocytes with intact cumulus granulosa cells and uniform cytoplasm were selected under a stereomicroscope. The selected cells were placed in basic maturation medium and prepared for subsequent processing.

[0089] Step 2: Add different concentrations of EPA

[0090] The selected oocytes were randomly divided into 4 groups (500 cells in each group), and EPA at concentrations of 0 μM (control group), 1 μM, 10 μM, and 100 μM was added to the basal culture medium. The GV stage oocytes were cultured to observe the effect of EPA on cell maturation.

[0091] Step 3: Measure maturity

[0092] After 48 hours of culture, the polar body emission of oocytes was observed by stereomicroscope, and the maturation rate of each group was recorded. The results showed that EPA at concentrations of 1 μM and 10 μM significantly increased the maturation rate of oocytes, while 100 μM concentration led to a decrease in the maturation rate, and the difference was statistically significant compared with the control group (P<0.05).

[0093] Example 2: Effects of EPA on Oocyte Gene Expression and Protein Regulation

[0094] Step 1: Experimental grouping and cultivation

[0095] High-quality oocytes were selected from the ovaries and randomly divided into an EPA-treated group and a control group, with 3 samples in each group and each sample containing 500 oocytes. In the EPA-treated group, 10 μM EPA was added to the culture medium, while EPA was not added to the control group. Cell samples were collected after 48 hours of culture.

[0096] Step 2: Transcriptome sequencing and proteome analysis

[0097] The cell samples of the treatment group and the control group were subjected to reference transcriptome sequencing and TMT quantitative proteome analysis. The results showed that the expression levels of key genes related to oocyte maturation (such as GDF9 and BMP15) in the EPA-treated group were significantly upregulated, while the expression of genes related to oxidative stress was downregulated. At the same time, proteome analysis found that the expression levels of proteins responsible for energy metabolism and cell cycle regulation (such as Cyclin B and CDK1) in the EPA group were significantly increased.

[0098] Step 3: Statistical analysis

[0099] The data were analyzed by GraphPad Prism 9 software, and the significance of differences in gene and protein expression levels was set at P < 0.05. The analysis results showed that EPA, when added in appropriate amounts, could significantly promote the expression of genes and proteins related to oocyte maturation, thereby improving cell maturation efficiency, supporting its use as a potential additive to promote oocyte maturation in vitro.

[0100] like Figure 1 As shown, a method for influencing EPA on in vitro maturation of porcine oocytes provided in an embodiment of the present invention comprises the following steps:

[0101] S101, oocyte selection;

[0102] After the oocytes were extracted, they were washed three times with egg washing solution, and then oocytes with cumulus granulosa cells and uniform cytoplasm were selected for culture under a stereoscope. Samples were collected at different time periods according to the in vitro development of porcine oocytes.

[0103] S102, different doses of EPA were added to the in vitro maturation fluid;

[0104] A concentration gradient was set in the maturation solution to add EPA at different doses of 0 μM, 1 μM, 10 μM, and 100 μM, and the GV stage oocytes were cultured;

[0105] S103, transcriptome sequencing with reference;

[0106] The samples were divided into EPA group and control group, with 3 samples in each group and 500 oocytes in each sample;

[0107] S104, TMT quantitative proteome;

[0108] The samples were divided into EPA group and control group, with 3 samples in each group and 500 oocytes in each sample for transfection protein sequencing;

[0109] S105, statistical analysis;

[0110] All experiments were repeated at least three times independently, and all experimental data were expressed as mean ± standard error (mean ± SD). GraphPad Prism 9 software was used for significance analysis and correlation analysis; P < 0.05 was considered statistically significant.

[0111] Pig oocytes were extracted from the experimental subjects and washed three times with egg washing solution to ensure the purity of the cell samples. Under the stereoscope, high-quality oocytes with uniform cumulus granulosa cells and cytoplasm were selected, and contaminated or abnormally developed cells were excluded. During the culture process, these high-quality oocytes were classified according to their developmental stages, and samples were collected in different time periods to provide accurate initial materials for subsequent experiments.

[0112] Different concentrations of EPA (0μM, 1μM, 10μM, 100μM) were set in the in vitro maturation medium to simulate the effects of different doses of EPA on the oocyte maturation process. The addition of EPA directly acts on oocytes in the GV stage (initial growth stage of oocytes), and by regulating their metabolism and signaling pathways, the effects of EPA at different doses on the promotion or inhibition of cell maturation in vitro were observed, providing data support for the optimization of the dosage.

[0113] The EPA group and the control group were subjected to reference transcriptome sequencing and TMT quantitative proteome analysis, with each group containing 3 samples, and each sample consisting of 500 oocytes. Transcriptome sequencing was used to analyze the changes in gene expression in oocytes after EPA treatment, revealing the role of EPA in signaling pathways and gene regulation during cell maturation. TMT quantitative proteome analysis explored the regulatory effect of EPA on key functional proteins by comparing protein expression levels, revealing its deep-seated impact on cell metabolism and maturation.

[0114] The experimental data were statistically analyzed by GraphPad Prism 9 software, and all data were expressed as mean ± standard error. The significance of the differences between the experimental groups was evaluated by correlation analysis and statistical tests, with P < 0.05 considered statistically significant. The stability and reliability of the results were verified by repeated experiments, and the specific effects and mechanism of EPA on in vitro maturation of porcine oocytes were finally obtained, providing a scientific basis for optimizing the in vitro maturation conditions of oocytes.

[0115] Selection of oocytes provided by the embodiments of the present invention:

[0116] Ovaries with follicles of 3-8mm in diameter were collected from slaughterhouses and placed in a constant temperature incubator. They were rinsed three times with warm saline before being placed in the incubator. The egg retrieval needle was adjusted to a suitable suction pressure for egg extraction. The tube wall was first rinsed with buffer before egg extraction, and the follicular fluid was then sucked into a centrifuge tube for temporary storage. The centrifuge tube was placed in an incubator to keep warm.

[0117] In vitro culture of porcine oocytes provided by the embodiment of the present invention:

[0118] (1) In vitro maturation;

[0119] (2) Embryonic development.

[0120] In vitro maturation provided by the embodiments of the present invention:

[0121] (1) the follicular fluid in the centrifuge tube is precipitated in the egg washing solution at least three times, and the sedimentation process is carried out in a water bath;

[0122] (2) After washing three times in the egg washing solution, draw about 1 mL of the mixed solution into a large dish and then add 4 mL of the egg washing solution to mix and shake well, and then select the COCs wrapped by three layers of granulosa cells under a stereoscope;

[0123] (3) Mix the EPA containing different concentrations with the maturation solution in advance, then add it to a four-well plate and equilibrate it in a CO2 incubator for at least two hours; then wash the COCs with the maturation solution for at least three times for five minutes each time and then transfer them to the four-well culture plate for culture;

[0124] (4) Oocytes were maintained at 38.5°C and 5% CO 2 Culture in an incubator for 42 h;

[0125] (5) After culturing in the incubator for 42 h, COCs were extracted with a medium-sized pipette tip and digested by blowing in hyaluronidase. After digestion was complete, the mixture was extracted into a small dish and mature oocytes were selected in the operating solution.

[0126] Embryonic development provided by the embodiments of the present invention:

[0127] The oocytes matured in vitro were taken out from the carbon dioxide incubator, collected from the four-well plate with a pipette and transferred to the hyaluronidase preheated in a 38.5°C oven, and then blown slowly and evenly to degranulate. When the granulosa cells around the oocytes were blown clean, the blowing was stopped, and then the degranulated oocytes were transferred to the preheated operating solution for washing three times, and the oocytes with intact cell membrane, clear perivitelline space, uniform cytoplasm, and clear PB1 were selected under a stereoscope, and the mature oocytes that developed to the MII stage were selected; the selected oocytes were transferred to the preheated fusion solution for washing three times, and then the oocytes were transferred to the fusion tank with an electrode width of 1.0mm, and the parameters were adjusted to 135V30μs DC pulse, and parthenogenetic activation was performed by electric shock; after electric activation, the oocytes were washed three times with the balanced embryo culture solution, and then transferred to the embryo culture solution covered with paraffin oil in advance and balanced, and 38.5℃, 5% CO 2 , culture under saturated humidity; observe the cleavage of the embryo under a stereoscope for 26-28 hours, and record the blastocyst status and count the blastocyst cell number on the seventh day of embryo culture.

[0128] The freezing of porcine oocytes provided by the embodiment of the present invention:

[0129] Under a stereoscope, select the MII stage oocytes in good condition, add preheated permeabilization enzyme to a 1.5mL centrifuge tube and blow repeatedly, then wash several times in 0.01% PVA PBS to ensure that no cumulus cells are attached, then transfer the oocytes to an RNase-free centrifuge tube and pull one with a thin glass tube.

[0130] like Figure 2 As shown, a system for influencing EPA on in vitro maturation of porcine oocytes provided by an embodiment of the present invention comprises:

[0131] The selection module is used for selecting oocytes. After the oocytes are extracted, they are washed three times with egg washing solution, and then oocytes with cumulus granulosa cells and uniform cytoplasm are selected for culture under a stereoscope. Samples are collected at different time periods according to the in vitro development of pig oocytes.

[0132] A culture module is used for adding different doses of EPA to the in vitro maturation solution; a concentration gradient is set in the maturation solution to add different doses of EPA, such as 0 μM, 1 μM, 10 μM, and 100 μM, to culture the GV stage oocytes;

[0133] Sequencing module, used for transcriptome sequencing with references; the samples were grouped into EPA group and control group, with 3 samples in each group and 500 oocytes in each sample;

[0134] Quantitative module, used for TMT quantitative proteome; samples were divided into EPA group and control group, 3 samples in each group, 500 oocytes in each sample, and proteome sequencing was performed;

[0135] The statistical analysis module was used to express the experimental data in the form of mean ± standard error (mean ± SD); GraphPad Prism 9 software was used for difference significance analysis and correlation analysis; P < 0.05 was considered to be statistically significant.

[0136] Another object of the present invention is to provide a computer device, which includes a memory and a processor, wherein the memory stores a computer program, and when the computer program is executed by the processor, the processor executes the steps of the method for influencing EPA on in vitro maturation of pig oocytes.

[0137] Another object of the present invention is to provide a computer-readable storage medium storing a computer program, which, when executed by a processor, enables the processor to execute the steps of the method for influencing EPA on in vitro maturation of pig oocytes.

[0138] Another object of the present invention is to provide an information data processing terminal, which is used to implement the system for the effect of EPA on in vitro maturation of pig oocytes.

[0139] The present invention is specifically implemented:

[0140] EPA is converted into 18R-HpEPE (18R-hydroperoxy-eicosapentaenoic acid, 18R-HpEPE) by CYP450, and then 18R-HpEPE is converted into 18R-HEPE for subsequent reactions to generate RvE1 ( Figure 3 )

[0141] 1. Experimental research

[0142] 1 Test materials

[0143] 1.1 Experimental animal samples

[0144] Sow ovaries were collected from Qingdao Wanfu Slaughterhouse in Laixi City, Shandong Province.

[0145] 1.2 Test reagents

[0146] The reagents used in this experiment were purchased from Sigma, Gibco BRL, Aladdin, Beyotime, Merck, Jingmei, Dalian Meilun, and Wuhan Eliorite Biological Company.

[0147] The specific reagent information is as follows (Table 1):

[0148] Table 1 Reagent names and manufacturers

[0149]

[0150]

[0151] 1.3 Antibody information

[0152] Table 2 Antibody name, product number and manufacturer

[0153]

[0154] 1.4 Main test instruments

[0155] Table 3 Instrument name and manufacturer

[0156]

[0157]

[0158] 1.5 Reagent preparation

[0159] TL-Hepes egg washing solution: NaH 2 PO 4 0.041g, KCl 0.237g, NaHCO 3 0.168g, NaLactatc1.868mL, Hepes2.383g, Phenolred0.010g, CaCl 2 ·2H 2 O0.294g, MgCl 2 6H 2 O102mg, Polyvinylalcohol0.10g, PenicillinG0.06g, D-Sorbitol2.1856g, Gentamicin0.025g, NaCl6.66g, Sodiumpyruvate0.022g, MilliQH 2 O1000mL.

[0160] Basic IVM maturation culture medium: 2 mL of polyvinyl alcohol (PVA) (5%), NaHCO 3 0.25g, Streptomycin0.005g, D-glucose0.0548g, Sodiumpyruvate0.01g, TCM-1990.95g, PenicillinG0.0075g, MilliQH 2 O98mL.

[0161] MM operating solution: Streptomycin 0.060g, Hepes 0.750g, Penicillin G 0.050g, TCM-1999.500g, NaCl 1.755g, BSA 3.00g, NaHCO 3 0.050g, MilliQH 2 O1000mL.

[0162] Fusion solution: Hepes 0.065g, mannitol 27.29g, CaCl 2 ·2H 2 O0.07g, MgCl 2 6H 2 O0.01g.

[0163] PZM-3 culture medium: KCl 0.060g, KH 2 PO 4 0.005 g, NaHCO 3 0.20g, NaCl0.5831g, Napyruvate0.0022g, glutamax0.0146g, Hypotaurine0.05g, MgSO 4 7H 2 O0.01g, BME aminoacids solution 2mL, calcium lactate 0.062g, MEM non-essential amino acids gentamicin 1mL, BSA 0.3g, H 2 O 100mL, filter and store at 4℃.

[0164] PBS Phosphate Buffered Saline: KH 2 PO 4 0.24g, KCl 0.2g, Na 2 HPO 4 1.44 g, 0.01% polyvinyl alcohol, 8.00 g NaCl, MilliQH 2 O1000mL.

[0165] Hyaluronidase digestion solution: TL-Hepes egg washing solution 5mL, hyaluronidase 100mg, Mannitol 6g, BSA 0.001g, MilliQ H 2 O95mL. (Stored in -20℃ refrigerator)

[0166] EPA solution: Dissolve 10 mg EPA in 661.2 μL IVM. The concentration of the stock EPA solution is 50 mM.

[0167] The above solutions all need to be adjusted to a pH between 7.2 and 7.4 and need to be sterilized by filtration through a 0.22 μm filter membrane.

[0168] 2 Test methods

[0169] 2.1 Experimental design

[0170] 2.1.1 Selection of oocytes

[0171] After the oocytes were extracted, they were washed three times with egg washing solution, and then the oocytes with cumulus granulosa cells (COCs with more than three layers of granulosa cells) and uniform cytoplasm were selected for culture under a stereoscope. Samples were collected at different time periods according to the in vitro development of pig oocytes.

[0172] 2.1.2 Addition of different doses of EPA to in vitro maturation fluid

[0173] A concentration gradient was set up in the maturation medium to add different doses of EPA, including 0 μM, 1 μM, 10 μM, and 100 μM, and the GV stage oocytes were cultured.

[0174] 2.1.3 Transcriptome sequencing with references

[0175] The samples were divided into EPA group and control group, with 3 samples in each group and 500 oocytes in each sample.

[0176] 2.1.4 TMT quantitative proteomics

[0177] The samples were divided into EPA group and control group, with 3 samples in each group and 500 oocytes in each sample for proteome sequencing.

[0178] 2.1.5 Statistical analysis

[0179] All experiments in this paper were repeated at least three times, and all experimental data were expressed as mean ± standard error (mean ± SEM). GraphPad Prism 9 software was used for difference significance analysis and correlation analysis. P < 0.05 was considered statistically significant.

[0180] 2.2 Processing of pig ovaries and collection of oocytes

[0181] Ovaries with follicle diameters of 3-8mm are collected from slaughterhouses and placed in a constant temperature box. Rinse three times with warm saline before placing in the incubator. Use an egg retrieval needle to adjust the appropriate suction pressure to extract eggs. Before extracting eggs, rinse the tube wall with buffer, then draw the follicular fluid into a centrifuge tube for temporary storage, and place the centrifuge tube in an incubator to keep warm.

[0182] 2.3 In vitro culture of porcine oocytes

[0183] 2.3.1 In vitro maturation

[0184] (1) The follicular fluid in the centrifuge tube is precipitated in the egg washing solution at least three times in a water bath.

[0185] (2) After washing three times in the egg washing solution, about 1 mL of the mixed solution was drawn into a large dish and then 4 mL of the egg washing solution was added to mix and shake well. COCs wrapped in three layers of granulosa cells were selected under a stereomicroscope.

[0186] (3) Mix the mature solution with different concentrations of EPA in advance and then add it to the four-well plate in CO 2 The COCs were then washed at least three times with the maturation solution for five minutes each time and then transferred to a four-well culture plate for culture.

[0187] (4) Oocytes were maintained at 38.5°C and 5% CO 2 Culture in an incubator for 42 h.

[0188] (5) After 42 h of incubation in the incubator, extract the COCs with a medium-sized pipette tip and digest them by blowing in hyaluronidase (the hyaluronidase must be thawed in the incubator in advance). After digestion is complete, extract the mixture into a small dish and select mature oocytes in the operating solution.

[0189] 2.3.2 Embryonic development

[0190] Take out the mature oocytes cultured in vitro from the carbon dioxide incubator, collect them from the four-well plate with a pipette and transfer them to the hyaluronidase preheated in a 38.5°C oven, blow and degranulate them at a uniform speed and slowly, and stop blowing until the granulosa cells around the oocytes are blown clean. Then transfer the degranulated oocytes to the preheated operating solution and wash them three times. Select the oocytes with intact cell membrane, clear perivitelline space, uniform cytoplasm, and clear PB1 discharge under the stereoscope, and the mature oocytes that have developed to the MII stage. Transfer the selected oocytes to the preheated fusion solution and wash them three times, and then transfer the oocytes to the fusion tank with an electrode width of 1.0mm, adjust the parameters to 135V30μs DC pulse, and perform electric shock for parthenogenetic activation. After electrical activation, wash the oocytes three times with the balanced embryo culture solution, and then transfer them to the embryo culture solution covered with paraffin oil and balanced in advance, and mix with 38.5℃, 5% CO 2 , saturated humidity. Observe the cleavage of the embryo under a stereoscope for 26-28 hours, and record the blastocyst status and count the blastocyst cell number on the seventh day of embryo culture.

[0191] 2.4 Porcine oocyte cryopreservation

[0192] Under a stereoscope, select the MII stage oocytes in good condition, add preheated permeabilization enzyme to a 1.5mL centrifuge tube, blow repeatedly, and then wash several times in 0.01% PVA PBS to ensure that there are no cumulus cells attached. Then transfer the oocytes to an RNase-free centrifuge tube, use a thin glass tube to pull a very thin needle to suck out the excess PBS, and then cool the centrifuge tube to prevent the tube from exploding and then put it into liquid nitrogen for quick freezing and store it in a -80℃ refrigerator.

[0193] 2.5 Immunofluorescence staining

[0194] (1) Fixation: Transfer the eggs to 4% paraformaldehyde (PFA) and fix them at room temperature for 30 min or in a refrigerator at 4°C overnight.

[0195] (2) Permeabilization: Transfer the oocytes in PFA into the permeabilization solution for three times to clean the residual PFA, and then transfer the oocytes into the permeabilization solution and treat at room temperature for 20 minutes.

[0196] (3) Blocking: Wash the oocytes three times with elution buffer and transfer to blocking buffer for 2 h at room temperature. (4) Primary antibody: Dilute the primary antibody with blocking buffer at a ratio of 1:500-1:1000 and transfer the oocytes to the primary antibody for 2 h at room temperature or overnight at 4°C.

[0197] (5) Elution: Wash three times with elution buffer, 5 min each time.

[0198] (6) Secondary antibody: Dilute the secondary antibody in blocking solution at a ratio of 1:500-1:1000 and incubate at room temperature in a dark box for 1 h.

[0199] (7) Elution: Elute with elution buffer 3-5 times in a light-proof condition, each time for 5 min.

[0200] (8) Nuclear staining and sealing: Hoechst staining for 15 minutes or PI staining for 10 minutes, drop anti-fluorescence quenching agent on the slide, transfer the eggs into it, and then seal the slide with a coverslip coated with lanolin on the four corners.

[0201] 2.6 Enzyme-linked immunosorbent assay

[0202] (1) Dilution of standard: refer to the original standard and dilute it according to the requirements.

[0203] (2) Sample addition: Set up blank wells, standard wells, and test sample wells. Accurately add 50uL of the standard to the ELISA plate, and first add 40uL of the diluent to the test sample well and then add 10uL of the test sample.

[0204] (3) Incubation: After sealing the plate, incubate at 37°C for 30 min.

[0205] (4) Liquid preparation: dilute the washing solution for later use

[0206] (5) Washing: Wash each well for 30 seconds, pat dry, and repeat five times

[0207] (6) Add enzyme: Add 50uL of enzyme-labeled reagent to each well.

[0208] (7) Incubation: Same as above

[0209] (8) Washing: Same as above

[0210] (9) Color development: Add color development solution and incubate for 10 minutes

[0211] (10) Termination: Add stop solution to terminate the reaction

[0212] (11) Determination: Determination should be carried out 15 minutes after adding the stop solution.

[0213] 2.7 Transcriptome analysis with references

[0214] 2.7.1 RNA extraction from animal cells

[0215] 4℃, 12000rpm, centrifuge for 15min; then transfer the aqueous phase to a new 2.0mL centrifuge tube, take about 600uL of supernatant (if the supernatant is less than 600uL, add NF water to 600uL), add 300uLRL and 450uL isopropanol (or 450uL anhydrous ethanol) to the supernatant, take 700uL of aqueous phase onto the CR3 column (provided by the kit), centrifuge at room temperature 12000rpm for 30s, discard the downstream liquid, and reassemble the column. Subsequent experiments were performed according to the different requirements of the kit.

[0216] 2.7.2 Separation and purification

[0217] RNA of the total sample was isolated and purified using TRIzol (thermofisher, 15596018) according to the protocol provided by the manufacturer.

[0218] 2.7.3 Quality Control

[0219] The amount and purity of total RNA were controlled by NanoDrop ND-1000 (NanoDrop, Wilmington, DE, USA) and the integrity of RNA was tested by Bioanalyzer 2100 (Agilent, CA, USA); the concentration was >50 ng / μL, RIN value was >7.0, and total RNA >1 μg, which was sufficient for downstream experiments.

[0220] The quality control samples were sequenced

[0221] 2.7.5 Analysis of test results

[0222] After using StringTie to quantify the genes or transcripts of each sample, the quantitative status of genes and transcripts was statistically analyzed.

[0223] Table 4 Database

[0224]

[0225] Table 5 Data analysis procedures

[0226]

[0227] 2.8 TMT proteome sequencing

[0228] 2.8.1 Protein extraction from animal cells

[0229] After taking out the sample from the ultra-low temperature refrigerator, transfer it to a centrifuge tube, add protein lysis buffer and shake to mix, centrifuge it in a low-temperature high-speed centrifuge, take the supernatant, add appropriate amount of 1M DTT and react in a heating tube for 2 hours and then water bath, add IAM after water bath to react in the dark.

[0230] 2.8.2 Protein detection

[0231] The obtained protein is subjected to quality inspection and then sequenced

[0232] 2.8.3 On-the-machine

[0233] The samples that passed the quality inspection were subjected to protease desalting, followed by peptide segment labeling, fraction separation, and finally liquid chromatography and mass spectrometry analysis.

[0234] 2.8.4 Data Analysis

[0235] The search software ProteomeDiscoverer (PD, Thermo, HFX and 480) was used to search the result spectrum of each run. The protein quantification results were statistically analyzed using the T-test, and the proteins with significant quantitative differences between the experimental group and the control group (P<0.05, FC>1.5 or FC<0.67) were defined as differentially expressed proteins (DEP).

[0236] 3 Results and analysis

[0237] 3.1. 10 μM EPA treatment can significantly improve the in vitro maturation of porcine oocytes

[0238] Table 6 The first polar body of porcine oocytes

[0239]

[0240] Note: The values ​​in brackets are the Mean±SEM of two groups of experimental repetitions, expressed as percentages. a, b Values ​​with different superscripts in the same column indicate significant differences when P<0.05. In order to study the effect of adding EPA to the in vitro maturation system on the maturation of porcine oocytes, a control group, 1μM, 10μM and 100μM EPA treatment groups were set up (Table 6). COCs tightly wrapped with more than three layers of cumulus cells were selected and randomly divided into four groups. After culturing for 42 hours, the extrusion rate of the first polar body of the oocytes was counted. The results showed that 10μM EPA treatment can significantly increase the extrusion rate of the first polar body during the in vitro maturation of oocytes (87.6±1.19% VS79.2±2.84%, P<0.05) ( Figure 4 A and B). The first polar body extrusion rate of oocytes treated with 1 μM EPA was measured after seven repeated tests (79.5±2.94%), and then the maturation rate of oocytes was reduced after treatment with 100 μM EPA (74.5±2.97%). The maturation rate of the control group after multiple repeated tests (79.2±2.84%) was significantly lower than that of the 10 μM EPA treatment group.

[0241] In vitro maturation of mammalian oocytes often causes abnormal spindle assembly. This study found that treatment with 10 μM EPA can significantly improve spindle assembly during in vitro maturation of porcine oocytes ( Figure 4 C), the proportion of abnormal assembly decreased from 15.15% to 5.88% (P<0.05) ( Figure 4 D). These results indicate that 10 μM EPA treatment can significantly improve the nuclear maturation of porcine oocytes during in vitro maturation.

[0242] Figure 4 The 10 μM EPA treatment provided by the embodiment of the present invention can significantly improve the nuclear maturation of porcine oocytes; A: GV stage COCs picture, (scale = 100 μm), MII stage oocyte picture, arrow points to the extruded polar body (scale = 50 μm). B: Pb1 extrusion rate of the control group and the EPA treatment group at 42 hours. C: Spindle staining in the second meiotic metaphase, the box part is the magnified part of the spindle, scale = 20 μm. D: Spindle staining statistics (n = 67) Percentage of normal and abnormal distribution. Data are shown as mean ± standard error. *P < 0.05, P < 0.01.

[0243] 3.2. Treatment with 10μM EPA can reduce the proportion of apoptotic cells in cumulus cells.

[0244] In order to study the potential mechanism of 10 μM EPA in improving the in vitro maturation of porcine oocytes, the effect of adding 10 μM EPA to the culture system on the expansion of porcine cumulus cells was first studied. The results showed that 10 EPA treatment could significantly increase the proportion of fully expanded COCs (72.66±0.577% VS 66.70±2.055% P<0.05) ( Figure 5 A, 5B), reduced the proportion of unexpanded and incompletely expanded cumulus cells. In addition, flow cytometry results showed that 10 μM EPA treatment could significantly reduce the apoptosis rate of cumulus granulosa cells cultured in vitro after 42 hours (14.07 ± 1.670% VS 20.93 ± 1.768%, P < 0.05) ( Figure 5 C, 5D). Further studies found that the addition of EPA to the culture system can increase the level of its metabolite RvE1. As shown in Figure E, the secretion level of RvE1 was detected at 0h, 24h, 30h, 36h, and 42h according to the time course of oocyte maturation. The results showed that the increased RvE1 helped to improve the apoptosis of cumulus cells. Therefore, the treatment of 10μM EPA significantly increased the proportion of fully expanded COCs and the secretion level of RvE1, while the increase in RvE1 levels reduced the apoptosis level of cumulus cells.

[0245] Figure 5 The 10 μM EPA treatment provided in the embodiment of the present invention can reduce the proportion of apoptotic cells in cumulus cells; Figure 5 Map of 10 μM EPA treatment reduces the proportion of cumulus cell; A: Cumulus cell expansion picture, scale 100 = μm. B: Cumulus cell expansion statistics. C: COCs in the control group and EPA treatment group were cultured to maturity and then cumulus cells were removed to detect the apoptosis ratio by flow cytometry. D: Apoptosis ratio of cumulus granulosa cells. The content of sTNF-α in the culture medium of the control group and EPA treatment group at different time points. E: The content data of RvE1 in the culture medium of the control group and EPA treatment group at different time points are mean ± standard error. *P < 0.05, P < 0.01.

[0246] 3.3. Effect of 10μM EPA treatment on the transcriptome pattern of cumulus cells.

[0247] In order to study the effect of EPA treatment on the transcriptional pattern of cumulus cells, the mature COCs were degranulated and then the degranulated cumulus cells were sequenced. 3×10^6 cumulus cells were collected from the 10μM EPA treatment group and the control group for subsequent sequencing experiments. The PCA results showed that there was a large difference between the 10μM EPA treatment group and the control group ( Figure 6A). Among them, there were 103 significantly up-regulated genes and 63 significantly down-regulated genes in the 10μM EPA treatment group. BMP15, which has a significant effect on the construction of the extracellular matrix, was significantly up-regulated, and CYP1A1, which is directly related to the metabolism of EPA into RvE1, was also directly up-regulated ( Figure 6 B) GO enrichment of significantly up- and down-regulated genes ( Figure 6 C) It was found that seven genes related to the interaction between cumulus cells and zona pellucida and female gametogenesis were significantly upregulated, four genes related to the positive regulation of growth and development were significantly upregulated, and two significantly downregulated genes were also enriched here ( Figure 6 D).

[0248] The significant increase in BMP15 at the transcriptome level is directly related to the expansion phenotype of cumulus cells, and the key enzyme cytochrome C (CYP1A1) produced by RvE1 also increased at the transcriptome level ( Figure 6 B), which is mutually verified with the phenotypic study in the previous part. This suggests that EPA treatment will help reduce the apoptosis rate of cumulus cells and promote the expansion of oocytes and the establishment of extracellular matrix. At the same time, the enhanced interaction between cumulus cells and zona pellucida is conducive to the maturation of cytoplasm.

[0249] Figure 6 It is a 10μM EPA-treated granulocyte pattern of action plot provided in an embodiment of the present invention; Figure 6 Map of 10μM EPA-treated granulocyte pattern of action plot; A: principal component analysis. B: Volcano plot, 103 significantly upregulated genes and 63 significantly downregulated genes in the EPA-treated group. C: GO enrichment analysis of the 103 significantly upregulated genes and 63 significantly downregulated genes obtained by EPAVSCtrl. D: The genes of the obtained key pathways are presented in a heat map (*P<0.05, P<0.01).

[0250] 3.4. Treatment with 10μM EPA can improve subsequent blastocyst development.

[0251] In order to further explore the effect of 10μM EPA treatment on oocyte development, EPA-treated oocytes and control oocytes were parthenogenetically activated and then the cleavage rate, blastocyst rate and blastocyst cell number were statistically analyzed (Table 7). The results showed that 10μM EPA treatment significantly increased the 26h cleavage rate (74.8±1.72VS71.2±1.68, P<0.05), the blastocyst rate on the sixth day (24.16±1.21VS20.3±1.38, P<0.05) and the number of blastocyst cells on the sixth day (62.17±1.12VS54.58±1.70, P<0.05) ( Figure 7 7A, 7B, 7C and 7D).

[0252] Table 7 In vitro maturation and developmental capacity of porcine oocytes

[0253]

[0254] Note: The values ​​in brackets are Mean ± SEM of two experimental replicates, expressed as percentages. a, b Values ​​with different superscripts in the same column indicate significant differences at P < 0.05.

[0255] Figure 7 The 10 μM EPA treatment provided in the embodiment of the present invention can improve the subsequent blastocyst development; Figure 7 Map of 10 μM EPA treatment improves subsequent blastocyst development; A: 26-hour cleavage picture, scale bar = 50 μm. Blastocyst number picture, scale bar = 200 μm. Blastocyst cell number picture, scale bar = 50 μm. B: 26-hour cleavage rate of the control group and the EPA-treated group after parthenogenetic activation. C: Blastocyst rate on the sixth day after parthenogenetic activation D: Blastocyst cell number (n = 25) Data are shown as mean ± standard error. *P < 0.05, P < 0.01.

[0256] 3.5. 10 μM EPA treatment can improve porcine oocyte cytoplasmic maturation by improving cortical granule distribution, mitochondrial distribution and ROS levels

[0257] The cytoplasmic maturation of oocytes is crucial for the subsequent development of embryos. In order to explore whether 10μM EPA treatment can improve the distribution of cortical granules, porcine oocytes were cultured in vitro for 42 hours and then the cortical granules were stained and divided into four distribution patterns; peripheral distribution of cortical granules distributed near the plasma membrane is a manifestation of complete cytoplasm maturity. Cortical distribution of cortical granules distributed near the cortical area is a manifestation of partial cytoplasm maturity. Uniform distribution of cortical granules evenly distributed in the cytoplasm of oocytes is a manifestation of immature cytoplasm. Abnormal distribution of cortical granules abnormal distribution in the cytoplasm usually represents poor or degenerated oocyte cytoplasm. This study found that 10μM EPA treatment can significantly improve the distribution pattern of cortical granules in mature oocytes, especially significantly increase the proportion of peripheral distribution pattern of cortical granules (54.84% VS33.33%, P < 0.05) ( Figure 8 A, 8B).

[0258] Secondly, the distribution of mitochondria is also an important criterion for measuring whether the cytoplasm is mature. The pig oocytes were cultured in vitro and then stained for mitochondria. The uniform distribution of mitochondria in the cytoplasm is a manifestation of cytoplasmic maturity, the perinuclear distribution of mitochondria is a manifestation of partial cytoplasmic maturity, and the chaotic distribution of mitochondria is a manifestation of poor cytoplasmic quality or degeneration. Figure 8C). The experimental results showed that the proportion of uniformly distributed mitochondria in oocytes was significantly increased (64.1% VS 50%, P < 0.05 Figure 8 In addition, various factors may cause oxidative stress in cells during in vitro culture. Therefore, the level of ROS in mature oocytes was examined. The treatment of 10 μM EPA also significantly reduced the level of ROS in mature oocytes (P < 0.05, Figure 8 E, 8F). In summary, 10 μM EPA treatment enhanced cytoplasmic maturation during in vitro maturation of porcine oocytes by improving cortical granule distribution, mitochondrial distribution and ROS levels.

[0259] Figure 8 The 10 μM EPA treatment provided in the embodiment of the present invention can improve the cytoplasmic maturation of porcine oocytes by improving the distribution of cortical granules, mitochondrial distribution and ROS level; A: Cortical granule distribution pattern of porcine oocytes after in vitro maturation. (A1) Peripheral distribution. (A2) Cortical distribution. (A3) Uniform distribution. (A4) Abnormal distribution. (n=76). Scale bar=20 μm. B: Percentages of peripheral distribution, cortical distribution, uniform distribution and abnormal distribution of cortical granules in the control group and EPA-treated group. C: Distribution of mitochondria in porcine oocytes after in vitro maturation under a confocal microscope, including uniform distribution, perinuclear distribution and abnormal distribution. D: Percentages of uniform distribution, perinuclear distribution and atrophic distribution of mitochondria in the control group and EPA-treated group (n=77). E: Detection of reactive oxygen levels. Scale bar, 100 μm. F: The figure shows the relative fluorescence intensity of mature oocytes (n=94). The ROS level in the EPA-treated group was significantly lower than that in the control group. Data are mean ± standard error. *P<0.05, P<0.01.

[0260] 3.6. Effect of 10 μM EPA treatment on the transcriptome of mature oocytes.

[0261] In order to study the potential mechanism of 10μM EPA treatment on the improvement of subsequent embryonic development of pig oocytes, the project team collected 1500 oocytes from the EPA treatment group and the control group for transcriptome sequencing. Since the resumption and progress of meiosis requires the chromatin in the oocyte to condense into chromosomes, the transcription events in the oocyte almost stagnate with the formation of chromosomes, and the mRNA stored in the oocyte will be continuously consumed and decomposed as the oocyte develops. Therefore, it is very necessary to discuss the mechanism of the effect of EPA treatment on the subsequent embryo of pig oocytes at the mRNA level in the MII stage.

[0262] The control group and the EPA-treated group were subjected to principal component analysis (PCA). The PCA results showed that the transcriptome patterns of the two groups were quite different ( Fig. 9A); This indicates that the treatment with 10 μM EPA had a significant effect on the mRNA level of oocytes, among which 142 genes were significantly upregulated and 774 genes were significantly downregulated in the EPA treatment group ( Fig. 9 B); The results showed that the transcription levels of 37 genes related to apoptosis and programmed cell death signaling pathways were significantly reduced ( Fig. 9 E, 9F). EIF3, which is associated with transcription initiation, was significantly upregulated.

[0263] Fig. 9 This is a diagram of the effect of 10 μM EPA treatment on the transcriptome pattern of mature oocytes provided by an embodiment of the present invention; A: principal component analysis. B: There are 142 significantly upregulated genes and 774 significantly downregulated genes in the EPA treatment group. C: KEGG enrichment of the 142 upregulated genes obtained by EPAVSCtrl. D: The KEGG enriched pathways and the key genes therein are specifically displayed. E: KEGG enrichment of the 774 downregulated genes obtained by EPAVSCtrl. F: The KEGG enriched pathways and the key genes therein are displayed.

[0264] 3.7. Effect of 10 μM EPA treatment on the proteome of mature oocytes.

[0265] In the above experiment, it was found that EPA treatment can improve the cytoplasmic maturation of pig oocytes by improving the distribution of cortical granules, the distribution of mitochondria and reducing the level of ROS in the cytoplasm. In order to further explore the cause of the above phenotype, the protein level exploration was carried out. The project team collected 1500 oocytes from the EPA treatment group and the control group for proteome sequencing. The PCA results showed that there was a large difference in the protein expression patterns of the two groups ( Fig.10 A); 28 proteins were significantly up-regulated and 95 proteins were significantly down-regulated in the EPA treatment group ( Fig.10 B); In order to further explore the overall expression pattern of differentially expressed proteins, a heat map was drawn ( Fig.10 C) and GO enrichment analysis was performed; the expression of genes related to ATP metabolism and synthesis was significantly increased in the EPA-treated group ( Fig.10 D, 10E), while the expression of genes related to gametogenesis was significantly decreased ( Fig.10 F, 10G), suggesting that EPA treatment improved intracellular energy production and thus had a beneficial effect on oocyte development.

[0266] Fig.10It is the effect of 10 μM EPA treatment on the proteomic pattern of mature oocytes provided by an embodiment of the present invention; Figure 10 Map of 10 μM EPA treatment on the proteomic pattern of mature oocytes; A: Principal component analysis. B: Volcano plot, 26 significantly upregulated proteins and 95 significantly downregulated proteins in the EPA treatment group. C: The 26 significantly upregulated proteins and 95 significantly downregulated proteins obtained by EPAVSCTRL were subjected to differential heat map analysis. D: The 26 upregulated proteins obtained by EPAVSCTRL were enriched in GO. E: The GO pathways enriched by the upregulated proteins and the key proteins therein were specifically displayed. F: The 95 downregulated proteins obtained by EPAVSCTRL were enriched in GO. G: The GO pathways enriched by the downregulated proteins and the key proteins therein were specifically displayed.

[0267] The results of the study showed that compared with the control group, 10μM EPA treatment significantly increased the nuclear maturation rate of oocytes; after 10μM EPA treatment, the Pb1 extrusion rate of oocytes was increased, and the abnormal distribution of spindles was significantly reduced. This is because the addition of EPA improved the energy metabolism in the oocyte and thus improved the cytoplasmic maturation, which is reflected in the proteomic data, but the specific reasons still need to be further explored.

[0268] At the same time, 10μM EPA treatment can affect the distribution of cortical granules and mitochondria, helping them to locate correctly, which is also closely related to energy supply (Bahety et al., 2024). At the same time, EPA treatment reduces the level of reactive oxygen in in vitro matured oocytes, thereby improving the cytoplasmic maturation of oocytes during in vitro maturation, which is also a direction that should be considered. After all, oxidative stress is inevitable during in vitro culture (Jiang et al., 2023). 10μM EPA treatment also reduces the proportion of apoptotic cells in cumulus cells, and high expression of RvE1 was detected in the in vitro maturation culture medium of oocytes treated with 10μM EPA. Then, in order to explore the effects of 10μM EPA treatment on oocyte development from multiple angles, cumulus cells treated with 10μM EPA were collected for transcriptome sequencing. The results showed that cumulus cells interact with the zona pellucida, and genes related to female gametogenesis are significantly upregulated. This result is very important for the material and energy supply during in vitro maturation of oocytes, but it is still necessary to clarify which of EPA and its metabolite RvE1 plays a major role in this process. Four genes related to the positive regulation of growth and development were significantly upregulated, and two other significantly downregulated genes were also enriched here. The transcription levels of the BMP15 gene related to the expansion of cumulus cells and the CYP1A1 gene related to the production of RvE1 were significantly increased. Further exploration of the transcriptome and proteome patterns of mature oocytes treated with 10μM EPA revealed that there were significant differences between the 10μM EPA-treated group and the control group both at the transcriptome and proteome levels. At the same time, the transcriptome results showed that the transcription levels of the signal pathway genes related to mitosis in the oocytes treated with 10μM EPA were significantly increased, while the transcription levels of the genes related to the signal pathways such as apoptosis and programmed cell death were significantly reduced. Proteomic data showed that the levels of proteins related to ATP metabolism and synthesis were significantly increased in oocytes treated with 10 μM EPA, while the levels of proteins related to gametogenesis were significantly decreased. This was due to the combined effects of EPA and RvE1 on oocytes. Although experiments have shown that there is no RvE1 receptor chemR23 in oocytes (Zhang et al., 2020), it is still unknown whether RvE1 will affect oocytes through other channels.

[0269] By correlating the transcriptome data with the degradation of maternal mRNA, it was found that the consumption of maternal mRNA should be the mainstream event due to the stagnation of transcription events. The consumption of maternal mRNA represents the synthesis of functional proteins, but a considerable portion of maternal mRNA is degraded during the maturation of oocytes. Therefore, in subsequent experiments, the down-regulation events of the transcriptome were jointly analyzed and summarized with the proteome. The up-regulated genes in the transcriptome tend to be reserves for subsequent embryonic development, so the up-regulated part of the transcriptome should be discussed in the blastocyst stage or the earlier two-cell stage or four-cell stage. Based on the above results, the mechanism of action of EPA on oocytes is that EPA enters into cumulus cells and produces a large amount of RvE1 through the CYP450 pathway (Arita et al., 2005) and is secreted into ECM to improve the development quality of oocytes. Due to the anti-apoptotic effect of RvE1 itself, the apoptosis of cumulus cells is improved. The reduction of ROS levels in oocytes in the EPA-treated group compared with the control group should be the direct effect of EPA on oocytes. The process involves the metabolism of polyunsaturated fatty acids in oocytes (Kim et al., 2010), and the uniform distribution of mitochondria has also been significantly improved (Hecker et al., 2018). At the same time, spindle abnormalities have been improved, which is due to the combined action of EPA and cumulus cells. At the oocyte level, it was found that EPA treatment improved ATP metabolism and the level of synthesis-related proteins, which is also consistent with the mitochondrial distribution phenotype.

[0270] in conclusion

[0271] The results of this study showed that:

[0272] (1) Treatment with 10 μM EPA can significantly increase the first polar body extrusion rate of pig oocytes matured in vitro.

[0273] (2) The 10 μM EPA treatment group significantly improved the expansion of the cumulus during in vitro maturation and reduced the proportion of apoptotic cells in cumulus cells.

[0274] (3) Treatment with 10 μM EPA can promote the cytoplasmic maturation of pig oocytes and coordinate the synchronous maturation of nucleus and cytoplasm.

[0275] The specific application fields or related products of the present invention.

[0276] (1) Animal Husbandry and Pig Breeding

[0277] Breeding of high-quality breeding pigs: By optimizing the in vitro maturation process of pig oocytes, the efficiency of artificial breeding of breeding pigs can be improved, which helps to quickly expand the breeding of high-quality breeding pigs.

[0278] Embryo transfer: In embryo engineering, high-quality in vitro matured oocytes are provided as the basic material for embryo development.

[0279] (2) Embryo engineering and biological breeding

[0280] In vitro embryo production (IVP): supports the full chain optimization of embryo engineering technology, including in vitro maturation, fertilization, embryo culture and transplantation.

[0281] Gene editing and cloning: Provide high-quality oocytes for gene editing and cloning technology to improve the success rate.

[0282] (3) Life science research

[0283] Basic research: used to study the molecular mechanisms of EPA and other fatty acids on mammalian germ cell development and explore the potential application of fatty acids in reproductive health.

[0284] Drug screening: Construct EPA-related biological models for screening nutritional supplements or drugs related to reproduction.

[0285] (4) Food and nutrition industry

[0286] Functional feed development: Develop functional feed additives with EPA as the core to improve reproductive performance.

[0287] The effects of adding 10 μM EPA to the in vitro culture system of porcine oocytes and its mechanism of action. The main research results are as follows:

[0288] 1.10 μM EPA increases the nuclear maturation rate of oocytes

[0289] By detecting the extrusion rate of the first polar body (Pb1) of oocytes, it was found that the maturation rate of the 10μM EPA treatment group was significantly higher than that of the control group (87.6±1.19% vs. 79.2±2.84%, P<0.05). At the same time, 10μM EPA significantly reduced the abnormal assembly rate of spindles (from 15.15% to 5.88%, P<0.05), indicating that EPA can significantly improve the nuclear maturation of oocytes.

[0290]

[0291] 2.10μM EPA reduces the apoptotic rate of cumulus cells

[0292] 10μM EPA treatment increased the proportion of fully expanded cumulus-oocyte complexes (COCs) (72.66±0.577% vs. 66.70±2.055%, P<0.05), and significantly reduced the apoptosis of cumulus granulosa cells (14.07±1.670% vs. 20.93±1.768%, P<0.05). At the same time, EPA increased the secretion level of its metabolite RvE1, and the increase of RvE1 was closely related to the reduction of cumulus cell apoptosis.

[0293] 3.10 μM EPA improves oocyte cytoplasmic maturation

[0294] Cortical granule distribution: 10 μM EPA significantly increased the peripheral distribution ratio of cortical granules (54.84% vs. 33.33%, P < 0.05).

[0295] Mitochondrial distribution: The proportion of uniformly distributed mitochondria increased significantly (64.1% vs. 50%, P < 0.05).

[0296] ROS level: EPA significantly reduced the level of reactive oxygen species (ROS) in mature oocytes (P < 0.05).

[0297] 4.10μM EPA promotes subsequent blastocyst development

[0298] The cleavage rate, blastocyst rate and blastocyst cell number of the EPA-treated group after parthenogenetic activation were significantly higher than those of the control group (cleavage rate: 74.8±1.72% vs. 71.2±1.68%; blastocyst rate: 24.16±1.21% vs. 20.3±1.38%; blastocyst cell number: 62.17±1.12 vs. 54.58±1.70; P<0.05).

[0299]

[0300] 5. Transcriptome and proteome studies reveal the mechanism of action of EPA

[0301] Cumulus cell transcriptome: 10 μM EPA treatment significantly upregulated 103 genes (such as BMP15 and CYP1A1), downregulated 63 genes, and genes related to extracellular matrix construction and cumulus cell expansion were significantly upregulated.

[0302] Mature oocyte transcriptome: The EPA-treated group significantly upregulated 142 genes and downregulated 774 genes, involving key pathways such as energy metabolism, apoptosis inhibition and cytoplasmic maturation.

[0303] Proteomic analysis: EPA treatment significantly upregulated 28 ATP metabolism-related proteins and downregulated 95 gametogenesis-related proteins, suggesting that EPA improves cytoplasmic maturation by improving energy metabolism.

[0304] The study found that 10μM EPA treatment significantly increased the nuclear maturation rate and cytoplasmic maturation of porcine oocytes, while reducing the apoptosis rate of cumulus cells. This treatment also enhanced the subsequent blastocyst development potential of oocytes. Its mechanism of action includes improving energy metabolism efficiency, reducing oxidative stress levels, and regulating the expression of key genes and proteins, providing a theoretical basis for optimizing the in vitro maturation system of mammalian oocytes. This study lays the foundation for further exploring the potential application of EPA in in vitro maturation systems.

[0305] Through specific algorithms and models, software simulation effectively simulates the behavior of biological systems. In terms of parameter optimization, the simulation predicts the optimal concentration conditions of 10μM EPA for oocyte maturation by adjusting model parameters. In mechanism prediction, the simulation helps simulate the cellular metabolic pathways and signaling pathways of EPA, revealing the mechanism of action of its metabolites (such as RvE1). In addition, the visualization function generates images of molecular dynamics, energy distribution, and reaction processes, such as spindle assembly and mitochondrial distribution, providing researchers with intuitive analysis tools. These simulation results provide reliable guidance for experimental design.

[0306] Experimental verification is a real-life verification of simulation predictions to ensure the accuracy of the results. Based on the simulation results, the research team designed an experiment including a control group and a treatment group, and measured key indicators such as the polar body extrusion rate, spindle assembly ratio, and mitochondrial distribution of oocytes. The data were statistically analyzed by combining flow cytometry and fluorescent staining technology. The results showed that the simulation-predicted 10μM EPA treatment effect was highly consistent with the experimental data, confirming that EPA can improve oocyte maturity and subsequent embryonic development ability by improving intracellular energy metabolism, reducing oxidative stress levels, and reducing cell apoptosis.

[0307] The method combining software simulation and experimental verification significantly improves the research efficiency. This method not only confirms the specific mechanism of action of EPA on oocyte maturation, but also demonstrates great potential in optimizing in vitro maturation systems. In the future, this method can be extended to predict the effects of other additives or treatment methods on oocyte maturation. In addition, this method has broad application prospects in the fields of drug development, agricultural science, etc., and provides strong support for exploring new functional factors and their mechanisms of action. The above is only a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any technician familiar with the technical field shall be within the technical scope disclosed by the present invention, and any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention shall be covered within the scope of protection of the present invention.

Claims

1. A method for influencing EPA on in vitro maturation of porcine oocytes, characterized in that: The following steps are involved: Step 1, selection of oocytes; After the oocytes were extracted, they were washed three times with egg washing solution, and then oocytes with cumulus granulosa cells and uniform cytoplasm were selected for culture under a stereoscope. Samples were collected at different time periods according to the in vitro development of porcine oocytes. Step 2, adding different doses of EPA to the in vitro maturation fluid; A concentration gradient was set in the maturation solution to add EPA at different doses of 0 μM, 1 μM, 10 μM, and 100 μM, and the GV stage oocytes were cultured; Step 3, reference transcriptome sequencing; The samples were divided into EPA group and control group, with 3 samples in each group and 500 oocytes in each sample; Step 4, TMT quantification of proteome; The samples were divided into EPA group and control group, with 3 samples in each group and 500 oocytes in each sample for transfection protein sequencing; Step 5, statistical analysis; All experiments were repeated at least three times independently, and all experimental data were expressed as mean ± standard error (mean ± SEM). GraphPad Prism 9 software was used for significance analysis and correlation analysis; P < 0.05 was considered statistically significant.

2. The method for influencing EPA on in vitro maturation of porcine oocytes according to claim 1, characterized in that: Selection of the oocytes: Ovaries with follicles of 3-8mm in diameter were collected from slaughterhouses and placed in a constant temperature incubator. They were rinsed three times with warm saline before being placed in the incubator. The egg retrieval needle was adjusted to a suitable suction pressure for egg extraction. The tube wall was first rinsed with buffer before egg extraction, and the follicular fluid was then sucked into a centrifuge tube for temporary storage. The centrifuge tube was placed in an incubator to keep warm.

3. The method for influencing EPA on in vitro maturation of porcine oocytes according to claim 1, characterized in that: In vitro culture of the porcine oocytes: (1) In vitro maturation; (2) Embryonic development.

4. The method for influencing EPA on in vitro maturation of porcine oocytes as claimed in claim 3, characterized in that: The in vitro maturation: (1) the follicular fluid in the centrifuge tube is precipitated in the egg washing solution at least three times, and the sedimentation process is carried out in a water bath; (2) After washing three times in the egg washing solution, draw about 1 mL of the mixed solution into a large dish and then add 4 mL of the egg washing solution to mix and shake well, and then select the COCs wrapped by three layers of granulosa cells under a stereoscope; (3) Mix the EPA containing different concentrations with the maturation solution in advance, then add it to a four-well plate and equilibrate it in a CO2 incubator for at least two hours; then wash the COCs with the maturation solution for at least three times for five minutes each time and then transfer them to the four-well culture plate for culture; (4) Oocytes were cultured in a 38.5°C, 5% CO2 incubator for 42 h; (5) After culturing in the incubator for 42 h, COCs were extracted with a medium-sized pipette tip and digested by blowing in hyaluronidase. After digestion was complete, the mixture was extracted into a small dish and mature oocytes were selected in the operating solution.

5. The method for influencing EPA on in vitro maturation of porcine oocytes as claimed in claim 3, characterized in that: The embryonic development: The oocytes were taken out from the CO2 incubator and collected from the four-well plate with a pipette and transferred to the hyaluronidase preheated in a 38.5°C oven. The oocytes were degranulated by blowing at a uniform speed until the granulosa cells around the oocytes were blown clean. The blowing was stopped and the degranulated oocytes were transferred to the preheated operating solution and washed three times. The oocytes with intact cell membrane, clear perivitelline space, uniform cytoplasm and clear PB1 were selected under a stereoscope. These were mature oocytes that had developed to the MII stage. The selected oocytes were transferred to the culture medium. The oocytes were washed three times in the preheated fusion solution, and then transferred to a fusion tank with an electrode width of 1.0 mm, and the parameters were adjusted to a 135V 30μs DC pulse, and parthenogenetic activation was performed by electric shock; after electric activation, the oocytes were washed three times with the balanced embryo culture solution, and then transferred to the embryo culture solution covered with paraffin oil in advance and balanced, and cultured at 38.5°C, 5% CO2, and saturated humidity; the embryos were cultured for 26-28 hours, and the cleavage was observed under a stereoscope, and the blastocyst situation was recorded and the number of blastocyst cells was counted on the seventh day of embryo culture.

6. The method for influencing EPA on in vitro maturation of porcine oocytes according to claim 1, characterized in that: Cryopreservation of the porcine oocytes: Under a stereoscope, select the MII stage oocytes in good condition, add preheated permeabilization enzyme to a 1.5mL centrifuge tube and blow repeatedly, then wash several times in 0.01% PVA PBS to ensure that no cumulus cells are attached, then transfer the oocytes to an RNase-free centrifuge tube and pull one with a thin glass tube.

7. A system for influencing porcine oocyte in vitro maturation with EPA for implementing the method for influencing porcine oocyte in vitro maturation with EPA as claimed in any one of claims 1 to 6, characterized in that: The EPA influence system on porcine oocyte in vitro maturation includes: The selection module is used for selecting oocytes. After the oocytes are extracted, they are washed three times with egg washing solution, and then oocytes with cumulus granulosa cells and uniform cytoplasm are selected for culture under a stereoscope. Samples are collected at different time periods according to the in vitro development of pig oocytes. A culture module is used for adding different doses of EPA to the in vitro maturation solution; a concentration gradient is set in the maturation solution to add different doses of EPA, such as 0 μM, 1 μM, 10 μM, and 100 μM, to culture the GV stage oocytes; Sequencing module, used for transcriptome sequencing with references; the samples were grouped into EPA group and control group, with 3 samples in each group and 500 oocytes in each sample; Quantitative module, used for TMT quantitative proteome; samples were divided into EPA group and control group, 3 samples in each group, 500 oocytes in each sample, and proteome sequencing was performed; The statistical analysis module was used to express the experimental data in the form of mean ± standard error (mean ± SEM); GraphPad Prism 9 software was used for difference significance analysis and correlation analysis; P < 0.05 was considered to be statistically significant.

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