Method for improving developmental capacity of frozen pig IVF embryos

Abstract

The invention provides a method for improving the developmental capacity of a frozen pig IVF (in vitro fertilization) embryo, which comprises the following steps: in the production of a pig in vitro embryo, before the pig embryo obtained by in vitro fertilization is cryopreserved, carrying out combined treatment by using fenofibric acid, sodium nitroprusside, FTY720-P and dopamine, then carrying out vitrification and unfreezing on the embryo, and during the freezing and unfreezing treatment, carrying out freeze-drying on the embryo to obtain the frozen pig IVF embryo. Z-VAD-FMK and TGF-beta are added into an embryo pretreatment solution, a vitrification freezing solution, an unfreezing solution and an embryo culture solution, so that the in-vitro developmental ability of the porcine embryo after vitrification freezing is improved. Experiments show that the embryo survival rate and the hatching rate of a group in which fenofibric acid, sodium nitroprusside, FTY720-P, dopamine, Z-VAD-FMK and TGF-beta are jointly added are obviously higher than those of a group in which fenofibric acid, sodium nitroprusside, FTY720-P, dopamine, Z-VAD-FMK and TGF-beta are added and a control group. The invention provides an effective, safe and feasible strategy for improving the pig in-vitro embryo production efficiency and embryo quality, and has great popularization value.

Description

Technical Field

[0001] This invention relates to the field of animal embryo engineering technology, and more specifically, to a method for improving the developmental capacity of frozen porcine IVF embryos. Background Technology

[0002] China is a major global producer and consumer of pork (Tong, B et al., 2022). Pig farming is the mainstay of the domestic animal husbandry industry, and pork consumption has long dominated my country's meat consumption market (Liang, Y et al., 2022). In the past, my country could only import pork to ensure the supply of pork to the market, which led to the near extinction of pig breeds in some areas (He, J. et al., 2020).

[0003] In the preservation of animal germplasm resources, the two most feasible methods are currently live preservation and cryopreservation. However, live preservation has many disadvantages, such as high cost and long cycle. Cryopreservation technology mainly establishes genomic libraries (CRBs) by freezing and storing various biological samples with genetic information (including gametes, embryos, etc.) to strengthen the protection of endangered species in a systematic and safe way (Biasetti et al., 2024). Among them, embryos contain the complete genetic information of both parents of the developing individual, and are one of the most direct and effective ways of germplasm conservation using livestock germplasm resource gene banks as a platform (Dobrinsky et al., 2000). Embryo cryopreservation plays an indispensable role in the construction of germplasm resource banks, the propagation of improved breeds, and international seed circulation (Cao Xuyang et al., 2023). Embryo cryopreservation not only breaks down the limitations of reproductive physiology, geography, and time on the reproductive potential of female animals, enabling long-distance embryo transfer and the preservation of genetic resources, but it is also crucial for animal husbandry and biomedical research (Xingzhu et al., 2021). Porcine embryo cryopreservation technology has multiple important implications for improving livestock production efficiency and protecting genetic resources (Xingzhu et al., 2021). Frozen embryos enable cross-regional and cross-temporal matching of high-quality genes, avoiding the high costs and disease risks associated with live animal introduction (Vining et al., 2021). This technology can significantly improve the reproductive efficiency of female animals, promote the cross-regional exchange and sharing of high-quality germplasm resources, and effectively reduce production losses caused by disease transmission. Simultaneously, it provides a key means for constructing large-scale germplasm banks and protecting the genetic diversity of endangered animals, and helps improve animal welfare and reduce the environmental impact and costs of live animal transportation (Lópezet et al., 2021). Compared to oocytes, embryos have greater advantages in cryopreservation. Embryos are more likely to be completely dehydrated during the cooling process, and their membrane lipid composition is rich in polyunsaturated fatty acids (PUFAs), which significantly improves their cryogenic tolerance (Amstislavsky et al., 2019).

[0004] Despite advancements in embryo cryopreservation technology, the survival rate and developmental potential of frozen embryos remain significantly lower than those of fresh embryos (Tajima et al., 2020). Understanding the cryopreservation and thawing process and how to improve post-thawing survival rates is crucial. The effectiveness of porcine embryo cryopreservation is influenced by multiple complex mechanisms, including apoptosis, mitochondrial damage, and abnormal gene expression (Tajima et al., 2020), all of which reduce the developmental capacity of frozen embryos after thawing. This reduced developmental capacity after freezing severely impacts subsequent embryo transfer pregnancy rates, representing the biggest challenge currently facing research. In practical applications, it is necessary to comprehensively consider these factors, optimize cryopreservation protocols, and improve the success rate of porcine embryo cryopreservation. Summary of the Invention

[0005] The purpose of this invention is to provide a method for improving the developmental ability of pig IVF embryos after freezing.

[0006] To achieve the objective of this invention, in a first aspect, this invention provides a method for improving the developmental capacity of porcine IVF embryos after freezing. In porcine in vitro embryo production, porcine embryos obtained through in vitro fertilization are treated with a combination of fenofibrate acid, sodium nitroprusside, FTY720-P, and dopamine before cryopreservation. Then, the embryos are vitrified and thawed. During the freezing and thawing processes, Z-VAD-FMK and TGF-β are added to the embryo pretreatment solution, vitrification solution, thawing solution, and embryo culture medium, thereby improving the in vitro developmental capacity of porcine embryos after vitrification.

[0007] Furthermore, the embryos were first treated in embryo culture medium containing 1.0 μM fenofibrate, 10 mM sodium nitroprusside and 100 nM FTY720-P for 2 h, and then treated in embryo culture medium containing 1.0 μM fenofibrate, 10 mM sodium nitroprusside, 100 nM FTY720-P and 2 mg / mL dopamine for another 10 min. After washing with embryo culture medium, they were vitrified and frozen. The embryo culture medium is PZM-3, and its formula is as follows:

[0008] The embryo fluid was purchased from Sigma, product number W1503.

[0009] Further, the vitrification process includes: first, placing the embryos in an embryo pretreatment solution containing Z-VAD-FMK and TGF-β at room temperature for 3 minutes; then, transferring the embryos to a vitrification solution containing Z-VAD-FMK and TGF-β at room temperature for 30 seconds; and finally, aspirating the embryos into an OPS tube and immersing them in liquid nitrogen.

[0010] The embryo pretreatment solution is formulated as follows: a cryoequilibration solution containing 7.5% dimethyl sulfoxide (DMSO) and 7.5% ethylene glycol (EG); The vitrification fluid is formulated as follows: a refrigeration equilibrium fluid containing 16% DMSO and 16% EG; The cryoequilibration solution is TL-PVA (Tyrode's lactate (TL)-HEPES-polyvinyl alcohol), and its formula and preparation method are as follows: 1000ml of TL-PVA cryogenic equilibration buffer contains: 7.264g NaCl (Sigma), 0.237g mM KCl (Sigma, P4504), 0.168g NaHCO3 (Sigma, S8875), 0.041g KH2PO4 (Sigma, S5011), 1.868ml sodium lactate (Sigma, L7900), 0.102g MgCl2·6H2O (Sigma, MO250), 0.297g CaCl2·2H2O (Sigma, C7902), 2.383g HEPES (Sigma, H3784), 0.022g sodium pyruvate (P4562), 2.186g sorbitol (Sigma, S1876), 1g... PVA (polyvinyl alcohol, Sigma, P8136), 100 IU / ml penicillin-streptomycin bispecific antibody; First, dissolve PVA in 100ml of pure water by heating. After cooling to room temperature, add 700ml of pure water, then add the above-mentioned components. Adjust the pH to 7.2-7.4, and bring the volume to 1000ml. The osmotic pressure should be 295-310mOsm. Filter and package using a 0.22um filter, and store at 4℃. Use within one month.

[0011] Preferably, the concentrations of Z-VAD-FMK and TGF-β added to the embryo pretreatment solution and vitrification cryosol are 40.0 μM and 6 ng / ml, respectively.

[0012] Further, the thawing method includes: removing the OPS tube from liquid nitrogen, blowing the embryos in the tube into thawing solution I containing Z-VAD-FMK and TGF-β, incubating for 5 min at 39°C, and then placing it in thawing solution II containing Z-VAD-FMK and TGF-β, incubating for 5 min at 39°C; then washing the embryos sequentially with TL-PVA and PZM-3, and after washing, placing the embryos into embryo culture medium containing Z-VAD-FMK and TGF-β for in vitro culture.

[0013] The formula and preparation method of thawing solution I are as follows: each 10 mL of thawing solution I contains 1.026 g of sucrose and 2 mL of FBS. The volume is adjusted to 10 mL with TL-PVA and mixed well. The formula and preparation method of thawing solution II are as follows: each 10 mL of thawing solution II contains 0.513 g of sucrose and 2 mL of FBS. The volume is adjusted to 10 mL with TL-PVA and mixed well.

[0014] Preferably, the concentrations of Z-VAD-FMK and TGF-β added to the thawing solution and embryo culture medium are 40.0 μM and 6 ng / ml, respectively.

[0015] Preferably, the in vitro culture conditions are: 38.5℃, 5% CO2.

[0016] Secondly, the present invention provides the application of combined treatment with fenofibrate acid, sodium nitroprusside, FTY720-P, dopamine, Z-VAD-FMK and TGF-β in porcine in vitro embryo production; The application aims to improve the in vitro development capability of porcine embryos after vitrification.

[0017] In this invention, Z-Val-Ala-Asp(OMe)-FMK (Z-VAD-FMK), as a broad-spectrum, irreversible caspase inhibitor, has long been widely used as a key experimental tool for inhibiting apoptosis. Caspase family proteins are the core executors of the apoptosis cascade. Z-VAD-FMK mimics caspase substrates through its peptide sequence and utilizes the fluoromethyl ketone (FMK) group to form a covalent bond with the cysteine ​​residues at the caspase active site, thereby irreversibly inhibiting the activity of almost all caspase members (Wang and Yang et al., 2013; Hai and Li et al., 2024). Although Z-VAD-FMK effectively inhibits apoptosis, its role in embryonic development is not simply "protective." Studies have found that administering Z-VAD-FMK during the 1-2 cell stage of early mouse embryonic development not only failed to promote development but also hindered the transition of the embryo from the 8-cell stage to the blastocyst, leading to significant embryonic malformations and even death (Zakeri and Lockshin et al., 2005; Busso and Dominguez et al., 2010). This suggests that caspases may play a crucial non-apoptotic role in early embryonic development, and Z-VAD-FMK intervention disrupts these vital physiological processes (Nakajima and Kuranaga, 2017). In addition to inhibiting the caspase family, Z-VAD-FMK is also a potent inhibitor of N-glycanase 1 (NGLY1) (Misaghi and Korbel et al., 2006; Needs and Bootman et al., 2022). NGLY1 is a cytoplasmic enzyme responsible for cleaving N-linked glycans from glycoproteins and is a key component of the endoplasmic reticulum-associated protein degradation (ERAD) pathway. When Z-VAD-FMK inhibits NGLY1, misfolded glycoproteins accumulate in the cell, thereby triggering strong endoplasmic reticulum stress and autophagy (Needs and Bootman et al., 2022).

[0018] The transforming growth factor-β (TGF-β) superfamily includes a variety of secreted signaling molecules such as TGF-βs, bone morphogenetic proteins (BMPs), activins, and nodal (Xu and Zheng et al., 2018). These molecules initiate signaling cascades by binding to type II and type I serine / threonine kinase receptors on the cell surface (Zhang, 2009). TGF-β is one of the most potent known endogenous cell proliferation inhibitors, especially for epithelial cells, endothelial cells, and hematopoietic cells (Zhang and Alexander et al., 2017). Its classic anti-proliferative mechanism is mainly achieved by regulating cyclin-dependent kinase (CDK) inhibitors. TGF-β can induce the expression of p21 (Cip1) and p15 (Ink4b) and inhibit the degradation of p27 (Kip1). These inhibitors can effectively block the cell cycle transition from G1 phase to S phase, leading to cell cycle arrest (Polyak and Kato et al., 1994). TGF-β signaling exhibits a significant duality in stem cell biology. On the one hand, in human pluripotent stem cells (hPSCs), the TGF-β / Activin / Nodal signaling pathway is essential for maintaining their undifferentiated and pluripotent state (Du and Wu, 2024; Fan and Yuan et al., 2024). This pathway locks cells into a pluripotent state by activating the expression of core pluripotency transcription factors such as NANOG, thereby inhibiting cell differentiation into the ectoderm (Wang and Li et al., 2016; Zhao and Chen et al., 2024). On the other hand, TGF-β signaling is also a key inducing factor driving stem cell differentiation into specific lineages. This indicates that TGF-β signaling is a crucial upstream regulator for maintaining the epigenetic landscape and gene expression program of specific cell lineages, and its absence directly leads to misclassification of cell fate (Mallo, 2025).

[0019] Fenofibrate is a PPAR-α agonist and a commonly used lipid-lowering drug. Its mechanism of action primarily involves regulating lipid metabolism by activating peroxisome proliferator-activated receptor α (PPARα). Studies have shown that PPARα may be involved in regulating cell differentiation, proliferation, and apoptosis during embryonic development (Mahmoudi A, et al., 2022). Furthermore, fenofibrate can reduce obesity in pregnant and virgin rats through different mechanisms (Gonzalez, et al., 2009): a) in virgin rats, by promoting fatty acid oxidation; b) in pregnant rats, by increasing fatty acid output. Fenofibrate formulations have been shown to have greater ecotoxicity in zebrafish embryos (Hering I, et al., 2021). Low to medium doses of fenofibrate have no adverse effects on embryonic development, while some fetal toxicity was observed at the highest doses (Ujházy E, et al., 2021).

[0020] FTY720-P (Fingolimod-P) is the active phosphorylated metabolite of the immunomodulatory drug FTY720. Treatment with FTY720 in pregnant inbred LM / Bc mice resulted in neural tube defects (NTDs) in approximately 60% of the embryos. FTY720-induced NTDs may involve multiple mechanisms, including: (1) persistent and / or altered S1P receptor activation and signal transduction by cytoplasmic FTY720-P; and (2) inhibition of HDAC and histone hyperacetylation by nuclear FTY720-P, potentially leading to epigenetic changes in gene regulation (Gardner NM, et al., 2016). Furthermore, FTY720-P may reverse the Th1 / Th2 and Treg / Th17 imbalance by blocking the S1P signaling pathway, ultimately inducing pregnancy immune tolerance and thus reducing embryo loss rates in spontaneous abortion models of pregnant mice (Xiong M, et al., 2019).

[0021] Dopamine plays a crucial signaling role in the formation of the embryonic nervous system. A 2023 study (WANG H et al., 2024) successfully generated functional human dopaminergic neurons by performing embryonic chimerism in mouse embryos using human embryonic stem cells. Although the integration efficiency was low, these TH... +Neurons exhibited typical electrophysiological properties and responded to α-synuclein aggregates, validating the plasticity of dopaminergic cells during embryonic development (WANGH et al., 2024). Furthermore, experiments in 2025 showed that, during in vitro induction of mesenchymal stem cell differentiation into a neural phenotype, appropriate addition of dopamine (2.5–5 µM) significantly enhanced the expression of neural markers (such as Nestin, MAP2, and DAT) and tended to convert to cholinergic neurons (KHASWANEH RR et al., 2025), further demonstrating that dopamine itself can act as a developmental signal to regulate the neural lineage differentiation of stem cells. These latest pieces of evidence collectively reveal the multiple regulatory functions of dopamine at the cellular level and during embryonic development.

[0022] Sodium nitroprusside (SNP) breaks down and releases NO under physiological conditions. NO plays a central role by activating the soluble guanylate cyclase (sGC)-cyclic guanosine monophosphate (cGMP)-protein kinase G (PKG) signaling axis (Chakrabarty and Kanai et al., 2024). At the cellular level, the effects of SNP are cell type specific. SNP (50-400 μM) can inhibit the survival rate of neural stem cells (NSCs) by activating the p38 phosphorylation pathway and induce apoptosis by upregulating Caspase-3 expression, but has no significant effect on neuronal differentiation markers (such as β-tubulin III) (Jiao and Xu et al., 2023). In mesenchymal stem cells (MSCs), short-term exposure to low-dose SNPs is safe for bone marrow mesenchymal stem cells (MSCs) but may alter their anaerobic metabolic pathways; high doses lead to apoptosis (Pari and Abnosi et al., 2017). For osteocytes and myoblasts, SNPs induce osteocyte apoptosis through the miR-1-HSP70 axis and trigger myoblast apoptosis through the p53-Bax-Caspase pathway (Lee and Jang et al., 2005; Lee and Hong et al., 2015). Furthermore, for hematopoietic cells, low concentrations of NO promote the proliferation of human bone marrow hematopoietic stem cells / progenitor cells (HSPCs), while high concentrations inhibit proliferation and induce myeloid differentiation (Hummer and Kraus et al., 2020). At the embryonic level, in bovine in vitro embryo production, SNP exposure reduces blastocyst formation rate and alters gene expression. Regarding amphibian and fish embryonic development, studies have found that SNP exposure delays embryonic development in Asian black-rimmed toads, significantly reducing body length and weight, markedly delaying embryonic development, and causing various morphological abnormalities such as tumors, tissue hyperplasia, and ascites. It also increases in vivo NO levels and interferes with thyroid hormone and cytochrome P450 metabolic pathways (Chen and Pang et al., 2023).

[0023] By employing the above technical solution, the present invention has at least the following advantages and beneficial effects: This invention improves the in vitro development capacity of porcine embryos after vitrification by adding fenofibrate acid, sodium nitroprusside, FTY720-P, dopamine, Z-VAD-FMK, and TGF-β, either alone or in combination, during the freezing, thawing, and late-stage embryo culture processes of porcine IVF embryos. Experiments show that the embryo survival rate and hatching rate of the group receiving the combined addition of fenofibrate acid, sodium nitroprusside, FTY720-P, dopamine, Z-VAD-FMK, and TGF-β are significantly higher than those of the single-substance addition groups and the control group. This invention lays the foundation for future in vitro fertilization embryo production and related scientific research, providing an effective, safe, and feasible strategy for improving the efficiency and quality of porcine in vitro embryo production, and has significant potential for widespread application. Attached Figure Description

[0024] Figure 1 In a preferred embodiment of the present invention, the effects of adding fenofibrate acid, SNP, FTY720-P, dopamine, Z-VAD-FMK, and TGF-β alone or in combination on frozen ATP and gene expression in porcine IVF embryos are described. Detailed Implementation

[0025] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art, and the raw materials used are all commercially available products.

[0026] The fenofibrate acid, sodium nitroprusside, FTY720-P, dopamine, Z-VAD-FMK, and TGF-β used in the following examples were all purchased from Sigma-Aldrich.

[0027] Hyaluronidase, TL-PVA, and PZM-3 were all purchased from Sigma.

[0028] The formula for IVM fluid (oocyte maturation fluid) is as follows:

[0029] Note: The above dosage refers to the amount of each ingredient added to 100 ml of TCM-199.

[0030] The embryo culture medium was PZM-3.

[0031] The embryo pretreatment solution was a cryoequilibration solution (TL-PVA) containing 7.5% DMSO and 7.5% EG.

[0032] The vitrification fluid is a thaw balance fluid (TL-PVA) containing 16% DMSO and 16% EG.

[0033] The thawing fluid includes thawing fluid I and thawing fluid II; their formulations and preparation methods are as described above.

[0034] Unless otherwise specified, the percentage sign "%" used in this invention refers to mass percentage. However, the percentage of a solution, unless otherwise specified, refers to the number of grams of solute contained in 100 mL of solution.

[0035] Example 1. Obtaining COCs Swine ovaries were washed 2-3 times with preheated 0.9% physiological saline to remove impurities and blood from the ovarian surface. Follicular fluid was extracted from follicles measuring 3-6 mm using a 10 mL syringe and collected in 15 mL centrifuge tubes for settling. The precipitate was washed three times with HEPES buffer and then, under a stereomicroscope, COCs (coccus cells) surrounded by three or more layers of cumulus cells with homogeneous cytoplasm were selected and collected into fresh IVM solution.

[0036] 2. In vitro maturation of oocytes The collected COCs were washed 2-3 times with IVM solution pre-equilibrated in a constant temperature incubator for 2-3 h, and then transferred to four-well culture dishes. Finally, the four-well culture dishes were placed in a constant temperature carbon dioxide incubator at 38.5℃, 5% CO2 and saturated humidity for 42-44 h.

[0037] 3. Acquisition and collection of cumulus cells Mature cumulus cells (COCs) were transferred to preheated 0.1% hyaluronidase solution using a pipette, and gently aspirated and pipetted 20-30 times. When almost all cumulus cells were observed to have detached under a microscope, the oocytes were transferred to preheated IVM solution and washed 2-3 times until the oocytes were completely separated from the cumulus cells. The oocyte maturation rate was then calculated.

[0038] 4. Preparation of embryos for in vitro fertilization (1) Prepare mTBM fertilization solution two days in advance: Take 15 mL of mTBM stock solution and add it to a 15 mL tube. Add 0.01 g of caffeine and 0.03 g of BSA (bovine serum albumin). After it is completely dissolved, filter it through a 0.22 μm filter membrane, loosen the centrifuge tube cap, and place it in an incubator to equilibrate overnight.

[0039] The formula for porcine in vitro fertilization fluid (mTBM) is as follows:

[0040] Note: No pH adjustment is required. Filter, dispense, and store at 4°C for later use.

[0041] (2) Prepare the semen washing solution one day in advance; filter 50 mL of DPBS solution containing 1% BSA through a 0.22 μm filter membrane and preheat at 38.5 °C.

[0042] (3) On the day of fertilization, mature COCs are desquamated and mature oocytes are selected and placed in the washing drop. Then, 30 oocytes / drop are transferred into the fertilization drop for equilibration.

[0043] (4) Sperm thawing: Add 10 mL of DPBS to a 15 mL centrifuge tube, remove the frozen sperm particles from the solution, quickly add one, gently shake to mix, centrifuge at 1800 rpm for 5 min, and discard the supernatant; repeat twice. Aspirate the supernatant as thoroughly as possible, resuspend the precipitate in 100 mL of mTBM fertilization solution, and adjust the sperm concentration to 20-50 sperm / mL with mTBM fertilization solution.

[0044] (5) Add 50 mL of sperm to the fertilization drop containing oocytes in step (4).

[0045] (6) After culturing the fertilized droplet in an incubator with 5% CO2, 5% O2 and 38.5℃ for 5-6 hours, wash it 3 times with PZM-3 medium and then transfer it to PZM-3 medium (i.e. embryo culture medium PZM-3) for further culture.

[0046] (7) Observe the cleavage rate two days later and the blastocyst rate five days later and record the results.

[0047] 5. Frozen and thawed porcine embryos 1) Freezing: Set the heating plate temperature to 41℃, freeze about 5 embryos at a time, transfer them into the embryo pretreatment solution, and treat at room temperature for 3 minutes. 2) Then transfer them into the vitrification solution (freezing, VS), treat at room temperature for 30 seconds, and then start loading them into the freezing carrier. Control the rapid addition of liquid nitrogen within 1 minute. 3) After covering with a cannula, transfer them to a straw thumb tube (mark according to the individual pig).

[0048] Thawing: Preheat the thawing solution to 39°C. Place 2 ml of thawing solution (TS) in a 3 mm culture dish (multiple pig embryos can be thawed simultaneously in one dish). First, remove the thumb tube and the CRYOTOP sleeve in liquid nitrogen. Quickly immerse the CRYOTOP tube in thawing solution I (ensuring the entire soft part of the CRYOTOP tube is submerged). Gently agitate the tube. Once the embryo has detached, remove the tube (approximately 10 seconds). After treating the embryo in thawing solution I for 5 minutes, remove it and then incubate it in thawing solution II for 5 minutes. Then, wash the embryos with TL-PVA and PZM-3 respectively before placing them in embryo culture medium for further culture. Observe the hatching rate after 24 hours of PZM-3 culture.

[0049] 6. Quantitative Real-Time PCR Blastocysts were placed in streptomycin solution (5 mg / mL), pipetted to remove the zona pellucida, and then washed three times with DPBS. The blastocysts were then placed in 1.5 mL nuclease-free centrifuge tubes and stored at -80°C for later use. cDNA was synthesized using the Cells-to-cDNA II Kit (Invitrogen, USA), and gene expression levels were detected using the PowerUp™ SYBR™ Green Master Mix Kit (Applied Biosystems, USA). The specific steps are as follows: (1) Sample processing Add 100 μL of pre-chilled Cell Lysis II Buffer to the collected sample, mix well, incubate at 75°C for 10 min, and then place on ice for later use. Then, add 2 μL of DNase I, mix gently, centrifuge briefly, incubate at 37°C for 15 min, and then incubate at 75°C for 5 min.

[0050] (2) Reverse transcription PCR reaction Add 5 μL of cell lysate, 4 μL of dNTP Mix, 2 μL of Random Decamers (random decameric primers, Thermo Fisher, catalog number AM5722G), and 5 μL of Nuclease-free Water to the tube sequentially. After a brief centrifugation, incubate on ice for 1 min. Then, add 2 μL of 10 × RT Buffer, 1 μL of M-MLV Reverse Transcriptase, and 1 μL of RNase Inhibitor. Mix gently and centrifuge briefly. Incubate at 42 °C for 1 h, followed by incubation at 95 °C for 10 min. Store the reaction product at -20 °C for later use.

[0051] (3) Real-time quantitative PCR reaction This experiment used bovine gene sequences from NCBI as a reference. Primers were designed using primer design software (https: / / bioinfo.ut.ee / primer3-0.4.0 / ) and synthesized by BGI Genomics. Reaction conditions: 95℃ pre-denaturation for 2 min; 95℃ for 10 s, 60℃ for 30 s, 40 cycles. β-actin was used as an internal reference gene, and the relative gene expression level was calculated using 2−ΔΔCt.

[0052] Experimental Design: Pre-freezing treatment: 1. Fenofibrate acid treatment group: Embryos were treated in embryo culture medium containing 1.0 μM fenofibrate acid for 2 hours, then washed with embryo culture medium and frozen.

[0053] 2. SNP treatment group: Embryos were treated in embryo culture medium containing 10 mM sodium nitroprusside for 2 hours, then washed with embryo culture medium and frozen.

[0054] 3. FTY720-P treatment group: Embryos were cultured in embryo culture medium containing 100 nM FTY720-P at 38.5℃ for 2 hours, then washed with embryo culture medium and frozen.

[0055] 4. Dopamine treatment group: Embryos were incubated in embryo culture medium containing 2 mg / mL dopamine monomer for 10 minutes and then frozen.

[0056] Freezing, thawing, and subsequent culture treatment: 5. Z-VAD-FMK treatment group: Z-VAD-FMK is added to embryo pretreatment solution, freezing solution, thawing solution and embryo culture medium.

[0057] 6. TGF-β treatment group: TGF-β was added to the embryo pretreatment solution, freezing solution, thawing solution and embryo culture medium.

[0058] Experimental results: 1. Effects of fenofibrate acid, sodium nitroprusside, FTY720-P, and dopamine, alone or in combination, on the development of frozen porcine IVF embryos. As shown in Table 1, the embryo survival rate and hatching rate of the combined treatment group (1.0 μM fenofibrate acid for 2 h + 10 mM sodium nitroprusside for 2 h + 100 nMFTY720-P for 2 h + 2 mg / mL dopamine monomer for 10 min) were significantly higher than those of the single treatment group (74.42±3.85%-76.60±4.24%, 75.00±3.47%-76.67±2.08%) and the frozen control group (71.11±7.58%; 71.88±6.48%) (P <0.05).

[0059] Table 1. Effects of fenofibrate acid, sodium nitroprusside, FTY720-P, and dopamine, alone or in combination, on the development of frozen porcine IVF embryos.

[0060] Note: Different superscripts indicate significant differences between groups. p < 0.05). The same applies below.

[0061] Tables 2-5 show the effects of different concentrations of fenofibrate acid, sodium nitroprusside, FTY720-P, and dopamine monomer on the development of frozen porcine IVF embryos.

[0062] Table 2. Effects of different concentrations of fenofibrate acid on the development of frozen porcine IVF embryos.

[0063] Table 3. Effects of different concentrations of sodium nitroprusside on the development of frozen porcine IVF embryos.

[0064] Table 4. Effects of different concentrations of FTY720-P on the development of frozen porcine IVF embryos.

[0065] Table 5. Effects of different concentrations of dopamine monomer addition on the development of frozen porcine IVF embryos.

[0066] 2. Effects of Z-VAD-FMK and TGF-β, alone or in combination, on the development of frozen porcine IVF embryos. As shown in Table 6, the embryo survival rate and hatching rate of the combined Z-VAD-FMK + TGF-β group (81.25±2.82%; 84.62±3.15%) were significantly higher than those of the Z-VAD-FMK group (78.38±3.65%; 75.86±3.48%), the TGF-β group (75.00±3.74%; 77.78±4.71%), and the frozen control group (71.11±4.48%; 71.43±3.35%) (P < 0.05).

[0067] Table 6. Effects of Z-VAD-FMK and TGF-β, alone or in combination, on the development of frozen porcine IVF embryos.

[0068] Tables 7 and 8 show the effects of different concentrations of Z-VAD-FMK and TGF-β addition on the development of frozen porcine IVF embryos.

[0069] Table 7 Effects of Z-VAD-FMK addition on the development of frozen porcine IVF embryos

[0070] Table 8 Effects of TGF-β addition on the development of frozen porcine IVF embryos

[0071] 3. Effects of fenofibrate acid, sodium nitroprusside, FTY720-P, dopamine, Z-VAD-FMK, and TGF-β, alone or in combination, on the development of frozen porcine IVF embryos. As shown in Table 9, the embryo survival rate and hatching rate of the group with combined addition of 1.0 μM fenofibrate acid for 2h + 10 mM sodium nitroprusside for 2h + 100 nMFTY720-P for 2h + 2 mg / mL dopamine monomer for 10 min + Z-VAD-FMK + TGF-β (96.23±4.34%; 96.08±3.52%) were significantly higher than those of the group with combined addition of 1.0 μM fenofibrate acid for 2h + sodium nitroprusside for 2h + FTY720-P for 2h + 2 mg / mL dopamine monomer for 10 min (82.35±4.06%; 83.33±4.75%) and Z-VAD-FMK + TGF-β (82.35±4.06%; 83.33±4.75%). The hatching rates of the control group (83.33±3.68%; 82.50±5.48%) and the frozen control group (73.08±3.72%; 71.05±4.46%), as well as the hatching rate of the fresh control group (96.55±3.58%) (P < 0.05).

[0072] Table 9. Effects of fenofibrate acid, sodium nitroprusside, FTY720-P, dopamine, Z-VAD-FMK, and TGF-β, alone or in combination, on the development of frozen porcine IVF embryos.

[0073] 4. Effects of fenofibrate acid, sodium nitroprusside, FTY720-P, dopamine, Z-VAD-FMK, and TGF-β, alone or in combination, on frozen ATP and gene expression in porcine IVF embryos. As shown in Table 10 and Figure 1 As shown, the expression levels of ITG, Bcl2, and OCT4 genes in embryos of the group treated with 1.0 μM fenofibrate acid for 2 h + 10 mM sodium nitroprusside for 2 h + 100 nM FTY720-P for 2 h + 2 mg / mL dopamine monomer for 10 min + Z-VAD-FMK + TGF-β were significantly higher than those in the group treated with 1.0 μM fenofibrate acid for 2 h + 10 mM sodium nitroprusside for 2 h + 100 nM FTY720-P for 2 h + 2 mg / mL dopamine monomer for 10 min, the group treated with Z-VAD-FMK + TGF-β, the frozen control group, and the fresh control group; the expression level of the Bax gene was significantly lower than that in all other groups (P<0.05). Primers used to detect the expression levels of each gene are shown in Table 11.

[0074] Table 10 Effects of fenofibrate acid, sodium nitroprusside, FTY720-P, dopamine, Z-VAD-FMK, and TGF-β, alone or in combination, on ATP content and gene expression in frozen porcine IVF embryos.

[0075] Table 11 Primers used to detect gene expression levels

[0076] Although the present invention has been described in detail above with general descriptions and specific embodiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention.

Claims

1. A method for improving the developmental ability of frozen porcine IVF embryos, characterized in that, In porcine in vitro embryo production, porcine embryos obtained through in vitro fertilization are treated with a combination of fenofibrate acid, sodium nitroprusside, FTY720-P, and dopamine before cryopreservation. The embryos are then vitrified and thawed. During the freezing and thawing processes, Z-VAD-FMK and TGF-β are added to the embryo pretreatment solution, vitrification solution, thawing solution, and embryo culture medium to improve the in vitro development capacity of porcine embryos after vitrification.

2. The method according to claim 1, characterized in that, Embryos were first treated in embryo culture medium containing 1.0 μM fenofibrate, 10 mM sodium nitroprusside and 100 nM FTY720-P for 2 h, and then treated in embryo culture medium containing 1.0 μM fenofibrate, 10 mM sodium nitroprusside, 100 nM FTY720-P and 2 mg / mL dopamine for 10 min. After washing with embryo culture medium, they were vitrified and frozen. The embryo culture medium is PZM-3, and its formula is as follows: The embryo fluid was purchased from Sigma, product number W1503.

3. The method according to claim 2, characterized in that, The vitrification process includes: first, placing the embryos in an embryo pretreatment solution containing Z-VAD-FMK and TGF-β at room temperature for 3 minutes; then, transferring the embryos to a vitrification solution containing Z-VAD-FMK and TGF-β at room temperature for 30 seconds; finally, aspirating the embryos into an OPS tube and immersing them in liquid nitrogen. The embryo pretreatment solution is formulated as follows: a cryoequilibration solution containing 7.5% dimethyl sulfoxide and 7.5% ethylene glycol; The formulation of the vitrification fluid is: a freezing equilibrium fluid containing 16% dimethyl sulfoxide and 16% ethylene glycol; The equilibration solution is TL-PVA, and its formula and preparation method are as follows: 1000ml of TL-PVA cryoequilibration buffer contains: 7.264g NaCl, 0.237g mM KCl, 0.168g NaHCO3, 0.041g KH2PO4, 1.868ml sodium lactate, 0.102g MgCl2·6H2O, 0.297g CaCl2·2H2O, 2.383g HEPES, 0.022g sodium pyruvate (P4562), 2.186g sorbitol, 1g PVA, and 100IU / ml penicillin-streptomycin bispecific antibody; First, dissolve PVA in 100ml of pure water by heating. After cooling to room temperature, add 700ml of pure water, then add the above-mentioned components. Adjust the pH to 7.2-7.4, and bring the volume to 1000ml. The osmotic pressure should be 295-310mOsm. Filter and package using a 0.22um filter, and store at 4℃. Use within one month.

4. The method according to claim 3, characterized in that, The concentrations of Z-VAD-FMK and TGF-β added to the embryo pretreatment solution and vitrification solution were 40.0 μM and 6 ng / ml, respectively.

5. The method according to claim 3 or 4, characterized in that, The thawing method includes: removing the OPS tube from liquid nitrogen, blowing the embryos in the tube into thawing solution I containing Z-VAD-FMK and TGF-β, incubating for 5 min, then placing it in thawing solution II containing Z-VAD-FMK and TGF-β, incubating for 5 min; then washing the embryos sequentially with TL-PVA and PZM-3, and after washing, placing the embryos into embryo culture medium containing Z-VAD-FMK and TGF-β for in vitro culture; The formula and preparation method of thawing solution I are as follows: each 10 mL of thawing solution I contains 1.026 g of sucrose and 2 mL of FBS. The volume is adjusted to 10 mL with TL-PVA and mixed well. The formula and preparation method of thawing solution II are as follows: each 10 mL of thawing solution II contains 0.513 g of sucrose and 2 mL of FBS. The volume is adjusted to 10 mL with TL-PVA and mixed well.

6. The method according to claim 5, characterized in that, The concentrations of Z-VAD-FMK and TGF-β added to the thawing solution and embryo culture medium were 40.0 μM and 6 ng / ml, respectively.

7. The method according to claim 6, characterized in that, The in vitro culture conditions were: 38.5℃, 5% CO2.

8. Application of combined treatment with fenofibrate acid, sodium nitroprusside, FTY720-P, dopamine, Z-VAD-FMK and TGF-β in porcine in vitro embryo production; The application aims to improve the in vitro development capability of porcine embryos after vitrification.

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