Method for in-vitro production of high-quality embryos by using frozen boar semen

Abstract

The invention belongs to the technical field of embryo in-vitro culture, and discloses a method for in-vitro production of high-quality embryos by using frozen boar semen. In the in-vitro production process of the pig embryo, the fertilization rate and the quality of the in-vitro cultured embryo are improved by using the frozen semen treated by the antifreeze protein NT5C1B and the mature oocyte through in-vitro fertilization. Wherein the selected protein can reduce the generation of ice crystallization in the freezing process of the semen, improve the quality and fertilization ability of the frozen semen, and provide a basis for high-quality development of embryos after in-vitro fertilization. The application provided by the invention is simple and convenient to operate and remarkable in effect, and does not generate any harmful influence on animals. The invention provides a powerful theoretical basis for in-vitro production of high-quality embryos.

Description

Technical Field

[0001] This invention relates to the field of in vitro embryo culture technology, and more specifically, to a method for producing high-quality embryos in vitro using frozen porcine semen. Background Technology

[0002] In vitro embryo production in pigs is an important biotechnology for studying embryo cloning, transgenic animals, and the mechanisms of early embryonic development. Gamete quality, in vitro fertilization, and in vitro embryo culture are all key steps that are crucial for embryonic development.

[0003] During embryonic development, the male pronucleus is crucial in initiating the embryonic development process. High-quality sperm has a vital and continuous impact on early embryonic development, affecting whether the embryo can form normally, divide successfully, and even its long-term health. High sperm DNA fragmentation or chromosomal abnormalities can directly lead to fertilization failure, early embryonic developmental arrest, and low blastocyst formation rate.

[0004] In in vitro embryonic development, the in vitro environment cannot completely mimic the in vivo environment, leading to a decline in the quality of in vitro embryos. Therefore, in vitro embryonic development systems are crucial for successful in vitro embryonic development. The success rate of in vitro embryonic development is influenced by multiple factors, which collectively constitute the embryo's "in vitro microenvironment." Cytokines, as important signaling molecules, participate in embryonic development regulation by constructing regulatory networks similar to those in vivo. Establishing cytokine-based embryonic microenvironment regulation strategies in in vitro culture systems is a key approach to improving the efficiency of livestock embryo biotechnology. Summary of the Invention

[0005] The purpose of this invention is to provide a method for producing high-quality embryos in vitro using frozen porcine semen.

[0006] Studies have found that NT5C1B is abundant in the sperm of high-producing piglets, but low in the sperm of low-producing piglets, and it is associated with male fertility. In order to improve the quality of frozen sperm, this invention screens out the protein NT5C1B, which is related to sperm antifreeze, as an antifreeze protein and adds it to the freezing diluent to protect sperm, improve the quality of frozen pig semen, and prepare it as gametes to obtain high-quality embryos.

[0007] Furthermore, the addition of appropriate concentrations of cytokines in in vitro culture plays a crucial role: insulin-like growth factor promotes embryonic cell proliferation and glucose metabolism; leukemia inhibitory factor maintains the pluripotency of embryonic stem cells and regulates the expression of genes related to embryo implantation; fibroblast growth factor improves blastocyst formation quality by promoting trophoblast cell expansion; and epidermal growth factor enhances the embryo's antioxidant capacity and reduces apoptosis rate. These cytokines synergistically activate key signaling pathways such as PI3K / Akt and STAT3, jointly promoting blastocyst cavity expansion and cell lineage differentiation, ultimately significantly improving blastocyst development rate and cell number, and enhancing embryonic developmental performance.

[0008] To achieve the objectives of this invention, a method for producing high-quality embryos in vitro using frozen porcine semen is provided. The method involves fertilizing mature oocytes with frozen porcine semen treated with the antifreeze protein NT5C1B, followed by in vitro embryo maturation culture to obtain high-quality embryos. The reference sequence number for the antifreeze protein NT5C1B in NCBI is XP_005655317.1.

[0009] Frozen boar semen is produced by adding antifreeze proteins to the semen freezing process to improve the low-temperature tolerance of boar sperm; high-quality embryos are early embryos cultured in an in vitro culture (IVM) system to the morula and blastocyst stages and their quality is evaluated.

[0010] Further, the collected bovine semen was mixed with a preheated basal diluent at a 1:1 volume ratio and allowed to stand at room temperature for 1-2 hours. Then, the semen and solution I were simultaneously placed in a 17°C incubator for equilibration for 0.5-1 hours. The equilibrated semen was then aliquoted into 50mL centrifuge tubes, and the semen volume and density were measured. The total sperm count was calculated, and the mixture was centrifuged at 800g for 15 minutes at 17°C. After centrifugation, the supernatant was discarded. The amount of Solution I added was determined based on the total sperm count and target semen density. The resuspended semen was cooled from 17 °C to 4 °C, and then equilibrated with Solution II at 4 °C for 0.5 h. The equilibrated semen was then aliquoted into 0.5 mL capillary tubes, sealed, and frozen using a semen cryostat (freezing program: ①: freezing from 4 °C to -5 °C at a rate of -3 °C to -5 °C / min; ②: freezing from -5 °C to -80 °C at a faster rate of -10 °C to -40 °C / min; ③: freezing from -80 °C to -150 °C at a rate of -50 °C / min or faster. The entire process lasted approximately 3 to 5 minutes). The capillary tubes containing the frozen semen were then placed in a liquid nitrogen tank for storage.

[0011] The liquid I contains lactose, trehalose, and egg yolk (the egg yolk comes from eggs); The II solution contains glycerol, the I solution, and the antifreeze protein NT5C1B.

[0012] Preferably, the preparation method of solution I is as follows: weigh 4.25 g of lactose and 4.25 g of trehalose; add 50 mL of double-distilled water, dissolve and add to a volumetric flask, add 20 mL of egg yolk solution, and make up to 100 mL with double-distilled water.

[0013] Preferably, the preparation method of the II solution is as follows: add 6 mL of glycerol to a volumetric flask, then add the antifreeze protein NT5C1B, mix well, and then make up to 100 mL with the I solution; the concentration of the antifreeze protein NT5C1B in the II solution is 1 µg / mL.

[0014] The basic diluent contains the following components: 3.7 g glucose, 0.125 g EDTA, 0.6 g sodium citrate, 0.125 g sodium bicarbonate, 0.075 g potassium chloride, and 10 mL of a 100 IU penicillin-streptomycin mixture, diluted to 100 mL with double-distilled water.

[0015] Furthermore, the method for preparing mature oocytes includes: collecting pig ovaries from slaughterhouses, selecting follicles of 3-6 mm and oocytes from three layers of cumulus ova, culturing them in a maturation culture medium for 47 h, and selecting mature oocytes that have excluded the first polar body; The formula for the mature culture medium is as follows: Prepared with TCM-199 basal medium, 100 IU of follicle-stimulating hormone, 100 IU of luteinizing hormone, 0.1 µg of epidermal growth factor, 1 mg of sodium pyruvate, 10% porcine follicular fluid, and 1% penicillin-streptomycin bispecific antibiotics are dissolved in TCM-199 basal medium according to a total volume of 10 mL. After filtration through a 0.22 µm filter membrane, it is stored at 4°C for later use.

[0016] Furthermore, the method includes the following steps: (1) Semen capacitation treatment: After thawing, the frozen bovine semen was diluted, centrifuged, and the sperm precipitate was resuspended, and then diluted to a concentration of 1×10⁻⁶. 6 Approximately 1000 mg / mL, after dilution, the sperm is placed in the mTBM capacitation solution for capacitation treatment for 30 min; (2) In vitro fertilization: After sperm capacitation treatment, sperm and mature oocytes are co-incubated in an in vitro environment to complete fertilization; (3) Early embryo in vitro culture: After fertilization, the fertilized eggs are transferred to a modified IVC culture medium and cultured in vitro to obtain high-quality embryos; (3) The modified IVC culture medium is a PZM-3 culture medium containing EGF (epidermal growth factor), FGF (basic fibroblast growth factor), and IGF (insulin-like growth factor). The addition of growth factors helps in vitro embryo development.

[0017] Furthermore, (3) the concentrations of EGF, FGF and IGF in the modified IVC culture medium are 10 ng / mL, 20 ng / mL and 20 ng / mL, respectively; The PZM-3 culture medium was prepared as follows: 0.63116 g of sodium chloride, 0.07456 g of potassium chloride, 0.00476 g of potassium dihydrogen phosphate, 0.00986 g of magnesium sulfate, 0.21082 g of sodium bicarbonate, 0.0022 g of sodium pyruvate, 0.06166 g of calcium lactate, 0.02924 g of L-glutamine, 0.05456 g of taurine, 0.005 g of gentamicin, 2 mL of non-essential amino acids, 1 mL of essential amino acids, and BSA were weighed, dissolved in ddH2O, and then brought to a final volume of 100 mL. The concentration of BSA was 3 mg / mL. The non-essential amino acids were sourced from Sigma-Aldrich (product number M7145), and the essential amino acids were sourced from Sigma-Aldrich (product number B6766).

[0018] Further, (1) the formula of the mTBM energy-generating solution is as follows: based on a total volume of 100mL, it contains 0.6611g of NaCl, 0.0224g of KCl, 0.2423g of Tris, 0.1982g of glucose, 0.0550g of sodium pyruvate, 0.1102g of CaCl2·2H2O, 0.0667g of caffeine and 0.2g of BSA.

[0019] Furthermore, (2) the incubation conditions are: fertilization at 38.5℃ in a 5% CO2 incubator for 5 hours.

[0020] Furthermore, (3) the in vitro culture conditions are: cultured in a constant temperature CO2 incubator at 38.5℃ and 5% CO2 saturated humidity for 2-7 days.

[0021] Furthermore, after fertilization with frozen semen, indicators of its fertilization capacity (such as sperm penetration rate) are detected; embryo quality is evaluated by detecting indicators such as cleavage rate, blastocyst rate, blastocyst diameter, and number of cells within the blastocyst.

[0022] By employing the above technical solution, the present invention has at least the following advantages and beneficial effects: (i) This invention provides a method for producing high-quality embryos in vitro using frozen porcine semen, namely, obtaining fertilized eggs by in vitro fertilization of frozen porcine semen treated with antifreeze protein NT5C1B, and culturing the fertilized eggs into high-quality embryos in an IVC optimized system.

[0023] (II) The method for producing high-quality embryos in vitro using frozen semen provided by this invention can significantly improve sperm penetration rate and early embryo quality during fertilization. This invention is simple to operate, has significant effects, and does not produce any harmful effects on pigs.

[0024] (III) The high-quality embryos obtained by this invention can be widely used in the embryo transfer industry and embryo engineering, improve the survival rate of in vitro embryo transfer, and have broad market prospects. Attached Figure Description

[0025] Figure 1 The effects of NT5C1B on sperm fertilization capacity and embryonic development in a preferred embodiment of the present invention include: the penetration rate of sperm in vitro fertilization through oocytes, the cleavage rate of early embryos, the morula rate of early embryos, and the blastocyst rate of early embryos.

[0026] Figure 2 These are quality indicators of blastocysts during early embryonic development in a preferred embodiment of the present invention. A: DAPI staining image of blastocyst cells; B: Number of cells within the blastocyst; C: Image of blastocyst cells; D: Blastocyst diameter ratio. express P <0.01. Detailed Implementation

[0027] To address the problems of early embryo development after in vitro fertilization with boar semen in existing technologies, this invention aims to verify the fertilization capacity of frozen boar semen and improve the impact of frozen boar sperm after in vitro fertilization on the developmental potential of early embryos in vitro, so as to facilitate embryo transfer for practical application.

[0028] The present invention adopts the following technical solution: In a first aspect, the present invention provides a method for in vitro production of high-quality embryos using frozen porcine semen, as detailed below: In the sperm freezing process, the protein NT5C1B was selected for addition. This protein was a differentially expressed protein related to cryotolerance, which was identified in the previous sperm proteomics screening and was ultimately determined based on its function.

[0029] The cryopreservation solution for porcine semen is prepared from a base diluent, solution I, solution II, protein NT5C1B, etc.; among which, protein NT5C1B is added to solution II to play a role, and the concentration of protein NT5C1B is 1µg / ml.

[0030] The cryopreservation process is as follows: After semen collection, the semen viability, volume, and density are measured. The qualified semen is diluted with preheated basal diluent at a 1:1 volume ratio in a 500mL reagent bottle. The solution is allowed to stand at room temperature for 1-2 hours. Then, the semen and solution I are simultaneously placed in a 17℃ incubator for equilibration for 0.5-1 hours. The equilibrated semen is aliquoted into 50mL centrifuge tubes and centrifuged at 17℃ and 800g for 12-20 minutes. The volume and density of the supernatant are measured, and the total sperm count is calculated. After centrifugation, the supernatant is discarded. The sperm is resuspended in preheated solution I. Solution I is slowly added to the bottom of the centrifuged sperm precipitate and mixed using aspiration. The resuspended semen is slowly cooled from 17℃ to 4℃ (1.5-2 hours), and the semen and solution II are equilibrated together at 4℃ for 0.5-1 hours. After semen equilibration, an equal volume of solution II at 4°C was added for isothermal dilution. The solution was then filled using an automatic filling machine, sealed, and the sealed caps were placed on a tray. The tray was then placed in a programmed freezing apparatus to freeze the semen (freezing program: ①: freezing from 4°C to -5°C at a rate of -3°C to -5°C / minute; ②: freezing from -5°C to -80°C at a faster rate of -10°C to -40°C / minute; ③: freezing from -80°C to -150°C at a rate of -50°C / minute or faster. The entire process lasted approximately 3 to 5 minutes). After freezing, the caps containing the frozen semen were placed in a cloth bag, labeled, and stored in a liquid nitrogen tank.

[0031] Secondly, the present invention provides a method for in vitro embryo culture, the method comprising: adding cell growth factors to embryo culture medium PZM-3.

[0032] The embryo culture medium PZM-3 comprises: PZM-3 embryo culture medium stock solution, EGF (10 ng / mL), FGF (20 ng / mL), IGF (20 ng / mL), and bovine serum albumin (BSA) (3 mg / mL).

[0033] The specific methods for in vitro embryo culture are as follows: Sperm treatment: Thaw the frozen semen prepared in step one in basal solution (basal dilution buffer). Add the thawed semen to PBS containing 0.1% BSA and centrifuge to precipitate. Wash three times by centrifugation at 1900g for 3 min. At the end of the washing cycle, resuspend the sperm pellet in 1 mL of sperm capacitation solution. Dilute the semen again in the capacitation solution to a concentration of approximately 1 × 10⁻⁶. 6 / mL, place the diluted semen into a cell culture incubator for capacitation for 30 minutes.

[0034] Mature oocyte acquisition: Take out mature oocytes cultured for 47 h from the maturation culture medium, gently blow away the cumulus granules, select mature oocytes that have excluded the first polar body, and place 30-40 oocytes in a pre-balanced 100 μl drop of fertilization solution until sperm is added for fertilization.

[0035] In vitro fertilization and early embryo culture: 40 μl of capacitated semen was added to a 100 μl droplet containing oocytes. The oocytes and sperm were co-incubated at 39 ℃ in a cell culture incubator containing 5% CO2 for 5 h. After co-incubation, sperm adhering to the oocytes were washed away in PZM-3 droplets, and then cultured in four-well culture dishes containing 500 μl of PZM-3 droplets prepared and equilibrated 2 h ago (50 fertilized oocytes per droplet). 350 μl of mineral oil was added to the surface of the liquid. The four-well culture dishes were placed in a constant temperature CO2 incubator at 38.5 ℃ and 5% CO2 saturated humidity for 2-7 days, after which the embryos were observed.

[0036] Thirdly, the present invention provides the application of improving the quality of frozen semen in enhancing the quality of early embryonic in vitro development.

[0037] Preferably, the frozen semen provided by the above method can be used as a high-quality gamete for in vitro fertilization, and the fertilized eggs can be cultured in the optimized early embryo culture medium to finally obtain high-quality early embryos.

[0038] 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.

[0039] The reagents used in the following examples are as follows: The basic diluent contains the following components: 3.7 g glucose, 0.125 g EDTA, 0.6 g sodium citrate, 0.125 g sodium bicarbonate, 0.075 g potassium chloride, and 10 mL of a 100 IU penicillin-streptomycin mixture, diluted to 100 mL with double-distilled water.

[0040] The preparation method of solution I is as follows: Weigh 4.25 g of lactose and 4.25 g of trehalose; add 50 mL of double-distilled water, dissolve and add to a volumetric flask, add 20 mL of egg yolk solution, and make up to 100 mL with double-distilled water.

[0041] The preparation method for solution II is as follows: add 6 mL of glycerol to a volumetric flask, then add the antifreeze protein NT5C1B, mix well, and then bring the volume to 100 mL with solution I; the concentration of antifreeze protein NT5C1B in solution II is 1 µg / mL.

[0042] The sperm washing solution is formulated as follows: PBS containing 0.1% BSA.

[0043] The formula for the energy-harvesting solution mTBM is as follows: based on a total volume of 100 mL, it contains 0.6611 g of NaCl, 0.0224 g of KCl, 0.2423 g of Tris, 0.1982 g of glucose, 0.0550 g of sodium pyruvate, 0.1102 g of CaCl2·2H2O, 0.0667 g of caffeine, and 0.2 g of BSA.

[0044] The modified IVC culture medium is PZM-3 culture medium containing 10 ng / mL EGF, 20 ng / mL FGF and 20 ng / mL IGF. The PZM-3 culture medium was prepared as follows: 0.63116 g of sodium chloride, 0.07456 g of potassium chloride, 0.00476 g of potassium dihydrogen phosphate, 0.00986 g of magnesium sulfate, 0.21082 g of sodium bicarbonate, 0.0022 g of sodium pyruvate, 0.06166 g of calcium lactate, 0.02924 g of L-glutamine, 0.05456 g of taurine, 0.005 g of gentamicin, 2 mL of non-essential amino acids, 1 mL of essential amino acids, and BSA were weighed, dissolved in ddH2O, and then brought to a final volume of 100 mL. The concentration of BSA was 3 mg / mL. The non-essential amino acids were sourced from Sigma-Aldrich (product number M7145), and the essential amino acids were sourced from Sigma-Aldrich (product number B6766).

[0045] 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.

[0046] Example 1: Preparation of frozen boar semen This embodiment provides a method for improving the quality of semen cryopreservation.

[0047] 1. Use products with a vitality greater than 80% and a density of 2×10⁻⁶. 8For qualified semen with a concentration of NT5C1B or higher, dilute the qualified semen with preheated basal diluent at a 1:1 volume ratio. Then, place the semen and solution I in a 17°C incubator for equilibration for 0.5-1 h. Aliquot the equilibrated semen into 50 mL centrifuge tubes, measure the semen volume and density, calculate the total sperm count, and centrifuge at 17°C, 800 g for 15 min. After centrifugation, discard the supernatant. Determine the amount of solution I to add based on the total sperm count and target semen density. Resuspend the sperm in solution I equilibrated to 17°C, and slowly add solution I to the bottom of the centrifuged sperm precipitate, mixing thoroughly by aspiration and exhalation. Slowly cool the resuspended semen from 17°C to 4°C (1.5-2 h), and equilibrate the semen with solution II containing NT5C1B at 4°C for 0.5-1 h.

[0048] 2. Semen Freezing: After semen equilibration, add an equal volume of 4°C solution II for isothermal dilution in a cryogenic operating cabinet. Gently mix and fill using an automatic filling machine. Seal the tubes and place them on a tray. Then, place the tray in a programmable freezing instrument to freeze the semen. After freezing, place the tubes in a cloth bag, label it, and store it in a liquid nitrogen tank.

[0049] The advantages of this embodiment are: I. In this embodiment, protein NT5C1B was selected as the protein screened from cryogenically frozen semen. It is an endogenous protein and has no toxicological effect on porcine sperm.

[0050] II. The semen cryopreservation method in this embodiment can preserve the fertilization capacity of frozen semen and improve the quality of boar semen after freeze-thaw.

[0051] Third, the embryos obtained after in vitro fertilization with frozen semen in this embodiment can develop into high-quality embryos after culture.

[0052] Example 2: Oocyte Maturation Culture This embodiment provides a method for oocyte maturation culture, the specific steps of which are as follows: 1. Obtaining COCs Swine ovaries collected from the slaughterhouse were washed 2-3 times with preheated PBS (38.5°C) to remove impurities and blood from the ovarian surface. A penicillin-streptomycin mixture could be added to the PBS for antibacterial effect. 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 placed in a 60 mm culture dish preheated to 38.5°C with Hepes buffer for selection.

[0053] 2. In vitro maturation of oocytes COCs with homogeneous cytoplasm, surrounded by three layers of cumulus cells, were selected from the culture dish under a stereomicroscope and collected. The collected COCs were then cultured in four-well culture dishes that had been pre-equilibrated in a constant temperature incubator for 2-3 hours. Finally, the four-well culture dishes were placed in a constant temperature carbon dioxide incubator at 38.5℃, 5% CO2, and saturated humidity for 46-48 hours.

[0054] The advantages of this embodiment are: I. The ovaries selected in this embodiment are all from young, healthy sows, whose oocytes are of high quality and have better developmental potential.

[0055] Second, in this embodiment, follicles of 3-6 mm in size and oocytes with three layers of cumulus ova were selected as experimental samples. These oocytes are more likely to mature and have excellent quality after culture.

[0056] Third, in this embodiment, oocytes were cultured for about 47 hours. At this time, the nucleus and cytoplasm of the oocytes have matured and have greater developmental potential after in vitro fertilization.

[0057] Example 3: In vitro culture of early embryos This embodiment provides a method for producing high-quality embryos in vitro using frozen porcine semen.

[0058] 1. In vitro fertilization Thaw the frozen semen in a 50°C water bath for 16 seconds. Add it to 4 ml of preheated (37°C) frozen semen basal diluent and mix gently. Take 2 ml of this solution. Add 8 ml of preheated (38°C) sperm washing buffer and centrifuge at 1900 rpm for 4 minutes. Discard the supernatant, add 10 ml of sperm washing buffer, and centrifuge at 1900 rpm for 4 minutes. Discard the supernatant, resuspend the semen in 1 ml of mTBM, and count the sperm. Based on the count, take an appropriate amount of the diluted semen and add it to 1 ml of mTBM to a final concentration of 1×10⁻⁶. 6 / ml, then place the semen in a cell culture incubator for capacitation for 30 min. Wash mature oocytes once in mTBM, and add 50 mature oocytes to a 100µL mTBM fertilization droplet. Aspirate 50µL of diluted semen (1×10⁻⁶). 6 Add ( / ml) to the fertilization droplet and incubate at 38.5℃ in a 5% CO2 incubator for 5 hours.

[0059] 2. In vitro early embryo culture After fertilization, the fertilized eggs were rinsed three times with a modified IVC culture medium and then transferred to four-well culture dishes. Each well of the four-well dish contained 500 µL of IVC culture medium and was covered with 350 µL of paraffin oil. Approximately 40 embryos were cultured in each well. Finally, the four-well culture dishes were placed in a constant temperature carbon dioxide incubator at 38.5°C, 5% CO2, and saturated humidity.

[0060] The advantages of this embodiment are: In this embodiment, during sperm capacitation treatment, all instruments and test tubes were preheated to 38°C to ensure that sperm were not subjected to cold shock, which could lead to a decline in semen quality.

[0061] II. In this embodiment, the sperm density is adjusted to a suitable level of 1×10 during fertilization. 6 / ml, to reduce the occurrence of polysperm entry into the egg, thereby reducing the occurrence of embryonic developmental arrest during embryo culture.

[0062] Third, this embodiment uses an optimized IVC culture medium. The cytokines in this culture medium are all key factors in the embryonic development process. The combined addition can increase the probability of the blastocyst developing into the embryonic stage. The blastocyst rate of this method for in vitro fertilization with frozen sperm can reach up to about 30%, and the average blastocyst rate is about 24%.

[0063] Experimental Example 1 This experimental example provides a method for producing high-quality embryos in vitro using frozen porcine semen. The method involves using antifreeze proteins to enhance the quality of cryopreserved semen and combining it with an optimized early embryo culture medium to produce high-quality embryos in vitro.

[0064] The specific steps are as follows: 1. Semen collection Semen was collected using an automated boar semen collection system. The collector, wearing gloves, held and secured the artificial vagina, stimulating the boar until its penis became erect and ejaculated. The first portion of semen was discarded, and the semen collection cup was then fixed and connected to the artificial vagina. The artificial vagina maintained a suitable temperature and provided stimulation pressure to ensure continuous ejaculation. After ejaculation, the collector removed and discarded the filter bag from the semen collection bag, and then insulated the remaining semen. The collected semen was examined under a microscope; motility was greater than 80%, and the density reached 2 × 10⁻⁶. 8 Semen with a concentration of 100 ml or higher is considered qualified and will be used for subsequent testing.

[0065] 2. Semen dilution and balancing Before semen collection, preheat the basal diluent in a 37°C water bath. Dilute the qualified semen with the preheated 37°C basal diluent at a 1:1 volume ratio in a 500mL reagent bottle. Let it stand at room temperature for 1-2 hours, then simultaneously place the semen and solution I in a 17°C incubator for 0.5-1 hours to equilibrate. Aliquot the equilibrated semen into 50mL centrifuge tubes (weigh the empty centrifuge tubes with an electronic balance and label them before centrifugation), and centrifuge at 17°C, 800g for 12-20 minutes. If there are many sperm in the supernatant, the centrifugation time can be extended appropriately. Measure the volume and density of the supernatant and calculate the total sperm count. If it exceeds 1 billion, the supernatant needs to be centrifuged again. After centrifugation, discard the supernatant. Slowly add solution I to the bottom of the centrifuged sperm precipitate and mix by aspiration. Slowly cool the resuspended semen from 17°C to 4°C (1.5-2 hours), and equilibrate the semen with solutions II of different concentrations at 4°C for 0.5-1 hours.

[0066] 3. Semen cryopreservation After semen equilibration, equal volumes of different concentrations of solution II at 4°C were added isothermally to the cryogenic operating cabinet for dilution. The mixture was gently mixed and then filled using an automatic filling machine. The tubes were sealed, and the sealed caps were placed on a rack. The rack was then placed in a programmable cryogenic apparatus to freeze the semen. After freezing, the caps were placed in cloth bags, labeled, and stored in a liquid nitrogen tank.

[0067] 4. Semen thawing Remove the cloth bag from the liquid nitrogen container and place it in a foam box containing liquid nitrogen. Use long-handled tweezers to pick up the thin tube and quickly place it in a 37°C water bath, gently shaking for 30 seconds. Remove it and wipe the water off the thin tube with sterile gauze. Then, use special scissors to cut off the sealed end and quickly place the cut end into a preheated centrifuge tube at 37°C, allowing it to slowly flow into the centrifuge tube. Add the preheated basal diluent to the centrifuge tube and gently shake to mix. The diluted semen can be used for subsequent testing.

[0068] 5. Obtaining COCs Swine ovaries collected from the slaughterhouse were washed 2-3 times with preheated PBS (38.5°C) 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 Hepes buffer.

[0069] 6. In vitro maturation and collection of oocytes The collected cumulus cells (COCs) were washed 2-3 times with IVM solution pre-equilibrated in a constant-temperature incubator for 2-3 hours, and then transferred to four-well culture dishes. The four-well dishes were then incubated in a constant-temperature CO2 incubator at 38.5℃, 5% CO2, and saturated humidity for 46-48 hours. Mature COCs were then transferred to pre-warmed 0.1% hyaluronidase solution using a pipette, gently aspirating and retracting 20-30 times. When almost all cumulus cells were observed to have detached under a microscope, the oocytes were transferred to pre-warmed PBS and washed 2-3 times until the oocytes were completely separated from the cumulus cells. The resulting mature oocytes were then transferred to fertilization medium for subsequent experiments.

[0070] 7. In vitro fertilization Thaw the frozen semen in a 50°C water bath for 16 seconds. Add it to 4 ml of preheated (37°C) frozen semen basal diluent, mix gently, and take 2 ml. Add 8 ml of preheated (37°C) sperm washing buffer; centrifuge at 1900 rpm for 4 min. Discard the supernatant, add 10 ml of sperm washing buffer; centrifuge at 1900 rpm for 4 min. Discard the supernatant, resuspend in 1 ml of mTBM, and count the sperm. Based on the count, take an appropriate amount of the diluted semen and add 1 ml of mTBM to a final concentration of 1×10⁻⁶. 6 / ml, then place the semen in a cell culture incubator for capacitation for 30 min. Wash mature oocytes once in mTBM, and add 50 mature oocytes to a 100µL mTBM fertilization droplet. Add 50µL of diluted semen to the fertilization droplet to achieve a final sperm concentration of 5×10⁻⁶. 5 Fertilization was performed in a 38.5℃, 5% CO2 incubator for 5 hours.

[0071] 8. In vitro early embryo culture Embryos were rinsed three times with IVC culture medium and then transferred to four-well culture dishes. Each well contained 500 µL of IVC culture medium and was covered with 350 µL of paraffin oil. Approximately 50 embryos were cultured in each well. The four-well dishes were then placed in a constant-temperature carbon dioxide incubator at 38.5°C, 5% CO2, and saturated humidity. Timing was started after placing the four-well plates in the incubator, and embryo quality was assessed after 2-7 days of culture.

[0072] As shown in Table 1, when all growth factors were added together to the early embryo culture medium, the blastocyst rate of the group with the three growth factors added together was significantly higher than that of the other groups. This indicates that the combined action of EGF, FGF and IGF can provide a better in vitro growth environment for the early embryos, thus providing high-quality transferable embryos for production practice.

[0073] Table 1. Effects of various growth factors on the blastocyst rate of early embryos. Note: Different lowercase letters indicate significant differences.

[0074] Depend on Figure 1 It can be seen that the penetration rate of sperm in the NT5C1B group was significantly higher than that in the frozen sperm group during in vitro fertilization. P <0.05), which was significantly lower than that of the fresh sebum group ( Figure 1 China A, P <0.05); the early embryo cleavage rate, morula rate, and blastocyst rate of the NT5C1B group were significantly higher than those of the frozen sperm group ( P <0.05), significantly lower than the fresh sebum group ( Figure 1 B, C, and D P <0.05). This indicates that NT5C1B frozen semen possesses fertilization capacity, and its fertilization capacity is superior to that of the ordinary frozen semen group, demonstrating that NT5C1B can alleviate the problem of decreased fertilization capacity of sperm during cryopreservation, but there is still a certain gap compared with the fresh semen group. The cleavage of fertilized eggs demonstrates that the frozen sperm possesses reproductive and developmental potential. Using the method of this invention, the blastocyst rate of early embryos cultured in vitro in the NT5C1B frozen semen group reached approximately 24.58±0.55%, while the average blastocyst rate of fresh semen reached 26.88±0.68%.

[0075] Depend on Figure 2 It can be seen that the number of blastocyst cells and the diameter of blastocysts in the NT5C1B semen group were significantly higher than those in the control group. According to the current blastocyst grading standards: blastocyst expansion and hatching degree (grades 1-6), inner cell mass quality (grade AC), and trophoblast cell quality (grade AC), the three indicators were comprehensively evaluated and it was determined that about 90% of the blastocysts in the frozen semen NT5C1B group were high-quality embryos with grades of 3AA, 4AA, and 3AB.

[0076] In this invention, the combination of growth factors EGF, FGF, and IGF achieves a synergistic effect of "1+1+1>3". They cross-activate key pathways such as PI3K / Akt and MAPK / ERK, promoting cell proliferation, survival, and metabolism during embryonic development. Furthermore, the three factors are complementary: EGF primarily drives cell proliferation during the blastocyst stage, FGF maintains inner cell mass pluripotency to ensure quality, and IGF-1 provides core energy metabolism support and anti-apoptosis protection. This design aims to highly simulate the complex physiological environment in vivo, forming a cell growth factor regulatory network to obtain high-quality blastocysts with intact morphology and stronger developmental potential, thereby effectively improving blastocyst formation and implantation rates. In the experiment, embryonic cells were cultured after different concentrations of the three growth factors were combined, and the final concentrations of the three factors were determined based on the embryo division rate and blastocyst rate.

[0077] In summary, the method for producing high-quality embryos in vitro using frozen boar semen provided by this invention is feasible. Using frozen boar semen to produce high-quality embryos based on this method can reduce industry production costs, provide raw materials for frozen embryos, and also provide a new method for germplasm resource protection.

[0078] 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 producing high-quality embryos in vitro using frozen porcine semen, characterized in that, High-quality embryos were obtained by in vitro fertilization of mature oocytes with frozen porcine semen treated with the antifreeze protein NT5C1B, followed by in vitro embryo maturation culture.

2. The method according to claim 1, characterized in that, The method for preparing frozen boar semen includes: mixing the collected boar semen with a preheated basal diluent, then mixing the diluted semen with solution I and incubating, then centrifuging and discarding the supernatant, resuspending the semen with the preheated solution I, cooling the resuspended semen to 4 ℃, and mixing the semen with solution II at 4 ℃ for equilibration for 0.5-1 h before freezing. After freezing, the capillary tube containing the frozen semen is placed into a liquid nitrogen tank for storage. The I solution contains lactose, trehalose, and egg yolk. The II solution contains glycerol, an antifreeze agent, and the antifreeze protein NT5C1B.

3. The method according to claim 2, characterized in that, The preparation method of the solution I is as follows: Weigh 4.25 g of lactose and 4.25 g of trehalose; add 50 mL of double-distilled water, dissolve and add to a volumetric flask, add 20 mL of egg yolk solution, and make up to 100 mL with double-distilled water. The preparation method of the II solution is as follows: add 6 mL of glycerol to a volumetric flask, then add the antifreeze protein NT5C1B, mix well, and then make up to 100 mL with the I solution; the concentration of the antifreeze protein NT5C1B in the II solution is 1 µg / mL.

4. The method according to claim 2, characterized in that, The collected boar semen was mixed with a preheated basal diluent at a volume ratio of 1:1 and allowed to stand at room temperature for 1-2 hours. Then, the semen and solution I were placed in a 17°C incubator for equilibration for 0.5-1 hours. The equilibrated semen was aliquoted into 50mL centrifuge tubes, and the semen volume and density were measured. The total sperm count was calculated, and the semen was centrifuged at 800g for 15 minutes at 17°C. After centrifugation, the supernatant was discarded. The amount of solution I to add was determined based on the total sperm count and the target semen density. The resuspended semen was cooled from 17°C to 4°C, and the semen and solution II were mixed and equilibrated at 4°C for 0.5 hours. The equilibrated semen was then aliquoted into 0.5mL capillary tubes, sealed, and frozen using a semen cryostat. The capillary tubes containing the frozen semen were then placed in a liquid nitrogen tank for storage. The basic diluent contains the following components: 3.7 g glucose, 0.125 g EDTA, 0.6 g sodium citrate, 0.125 g sodium bicarbonate, 0.075 g potassium chloride, and 10 mL of a 100 IU penicillin-streptomycin mixture, diluted to 100 mL with double-distilled water.

5. The method according to claim 1, characterized in that, The preparation method of mature oocytes includes: collecting pig ovaries from slaughterhouse, selecting follicles of 3-6 mm and oocytes with three layers of cumulus ova, culturing them in a maturation culture medium for 47 h, and selecting mature oocytes that have excluded the first polar body; The preparation method of the mature culture medium is as follows: Using TCM-199 basal culture medium, based on a total volume of 10 mL, dissolve 100 IU of follicle-stimulating hormone, 100 IU of luteinizing hormone, 0.1 µg of epidermal growth factor, 1 mg of sodium pyruvate, 10% porcine follicular fluid, and 1% penicillin-streptomycin in the TCM-199 basal culture medium. After filtration through a 0.22 µm filter membrane, store at 4°C for later use.

6. The method according to claim 1, characterized in that, Includes the following steps: (1) Semen capacitation treatment: After thawing, the frozen bovine semen was diluted, centrifuged, and the sperm precipitate was resuspended, and then diluted to a concentration of 1×10⁻⁶. 6 / mL, after dilution, the sperm was placed in the mTBM capacitation solution for capacitation treatment for 30 min; (2) In vitro fertilization: After sperm capacitation treatment, sperm and mature oocytes are co-incubated in an in vitro environment to complete fertilization; (3) Early embryo in vitro culture: After fertilization, the fertilized eggs are transferred to a modified IVC culture medium and cultured in vitro to obtain high-quality embryos; (3) The improved IVC culture medium is PZM-3 culture medium containing EGF, FGF and IGF.

7. The method according to claim 6, characterized in that, (3) The concentrations of EGF, FGF and IGF in the modified IVC culture medium are 10 ng / mL, 20 ng / mL and 20 ng / mL, respectively; The PZM-3 culture medium was prepared as follows: 0.63116 g of sodium chloride, 0.07456 g of potassium chloride, 0.00476 g of potassium dihydrogen phosphate, 0.00986 g of magnesium sulfate, 0.21082 g of sodium bicarbonate, 0.0022 g of sodium pyruvate, 0.06166 g of calcium lactate, 0.02924 g of L-glutamine, 0.05456 g of taurine, 0.005 g of gentamicin, 2 mL of non-essential amino acids, 1 mL of essential amino acids, and BSA were weighed, dissolved in ddH2O, and then brought to a final volume of 100 mL; wherein the concentration of BSA was 3 mg / mL.

8. The method according to claim 6, characterized in that, (1) The formula of the mTBM energy-generating solution is as follows: based on a total volume of 100mL, it contains 0.6611 g of NaCl, 0.0224 g of KCl, 0.2423 g of Tris, 0.1982 g of glucose, 0.0550 g of sodium pyruvate, 0.1102 g of CaCl2·2H2O, 0.0667 g of caffeine and 0.2 g of BSA.

9. The method according to claim 6, characterized in that, (2) The incubation conditions are: fertilization at 38.5℃ and 5% CO2 for 5 hours.

10. The method according to claim 6, characterized in that, (3) The in vitro culture conditions are: 38.5℃, 5% CO2 saturated humidity constant temperature CO2 incubator for 2d-7d.

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