A method for cryopreservation of porcine embryos

Abstract

The present application relates to the technical field of animal breeding, and particularly relates to a freezing preservation method of pig embryos. The freezing preservation method comprises the following steps: freezing and preserving the pig embryos by using a Cryotop method; before a freezing preservation process in the Cryotop method, treating the pig embryos by using berberine; and in the freezing preservation process in the Cryotop method, adding Fe3O4 nanoparticles and anti-freeze protein type I into a freezing preservation solution. The present application provides a freezing preservation method of pig embryos, and the survival rate and hatching rate of the pig embryos after the freezing preservation are significantly improved by the synergistic effect of the combined treatment methods, which has important significance in the field of pig breeding.

Description

Technical Field

[0001] This invention relates to the field of animal husbandry technology, and in particular to a method for cryopreservation of pig embryos. Background Technology

[0002] As a vital livestock species in agriculture, the continuous development of pig reproductive technology is crucial for increasing meat production and improving genetics. The advancement of in vitro embryo cryopreservation technology for pigs can facilitate embryo transport, replacing the introduction of live herds, establishing animal gene banks, and preserving superior livestock and endangered animal resources. Against this backdrop, in vitro embryo cryopreservation technology plays a key role in pig reproduction and germplasm resource protection. Therefore, research on pig embryo cryopreservation technology is extremely important for both agricultural production and biomedical research.

[0003] However, it is noteworthy that porcine embryos have significantly higher cytoplasmic lipid content compared to sheep, bovine, and mouse embryos. This unique physiological characteristic presents greater challenges for the cryopreservation and thawing of porcine embryos. During cryopreservation, in vitro embryos may experience various hypothermic injuries, such as osmotic freezing, ice crystal formation, the appearance of polycrystalline structures, and fracture damage. These hypothermic injuries can adversely affect important structures related to embryonic development, including the zona pellucida, cytoskeleton, and mitochondria. These structures are crucial for normal embryonic development and subsequent survival; therefore, damage to these structures can negatively impact embryonic health. Summary of the Invention

[0004] To address the problems existing in the prior art, this invention provides a method for cryopreservation of pig embryos. By combining multiple cryopreservation methods, the survival rate and hatching rate of frozen pig embryos are effectively improved.

[0005] In a first aspect, the present invention provides a method for cryopreservation of pig embryos, comprising:

[0006] The Cryotope method was used for cryopreservation of pig embryos;

[0007] Before the cryopreservation process in the Cryotop method, the pig embryos are treated with berberine;

[0008] In the cryopreservation process of the Cryotop method, Fe3O4 nanoparticles and antifreeze protein type I are added to the cryopreservation solution.

[0009] The antifreeze protein type I described in this invention is Anti Freeze Protein type 1, which is commercially available (e.g., Wuhan Gaiyuntian).

[0010] This invention employs berberine for defatting treatment, reducing lipid levels in porcine embryos, preventing lipid crystal formation during cryopreservation, and minimizing its damage to cell membranes. Simultaneously, this invention adds Fe3O4 nanoparticles and antifreeze protein type I to the cryopreservation solution. The structural and biological properties provided by the Fe3O4 nanoparticles further maintain the stability of structures such as cell membranes, while antifreeze protein type I inhibits ice crystal formation. The combination of these three factors effectively improves the cryopreservation efficiency of porcine embryos, reduces the impact of the cryopreservation process on the embryos, and increases the survival and hatchability of frozen porcine embryos.

[0011] Furthermore, the amount of berberine used is 2-3 μM, and the treatment method includes adding berberine to the culture medium during the in vitro culture stage of the pig embryo.

[0012] Furthermore, the amount of Fe3O4 nanoparticles in the cryopreservation solution is 0.5–1.5 wt%; the amount of antifreeze protein type I in the cryopreservation solution is 250–700 ng / mL.

[0013] Furthermore, the cryopreservation solution is based on TL-PVA and also includes one or more of DMSO, EG, or sucrose.

[0014] The TL-PVA described in this invention is Tyrode's lactate (TL)-HEPES-polyvinyl alcohol.

[0015] Furthermore, the pig embryo is a parthenogenetic embryo.

[0016] Furthermore, the parthenogenetic embryos are prepared by the following method:

[0017] mature porcine oocytes were placed in a fusion solution for electroactivation under the following conditions: DC 120–180V, 80–120μs, 2–4 pulses.

[0018] The electroactivated mature porcine oocytes were placed in an auxiliary activation solution and activated for more than 4 hours under conditions of 3-10% CO2, 3-8% O2 and 38-39℃.

[0019] Furthermore, the auxiliary activation solution was obtained by adding 10 μg / ml of actinomycete CHX to PZM-3 medium.

[0020] Furthermore, the Cryotop method includes:

[0021] The pig embryos were placed in a cryopreservation solution and then placed in an environment below -190°C for 1 minute, preferably in liquid nitrogen.

[0022] Furthermore, the Cryotop method includes:

[0023] The porcine embryos were placed in vitrification cryopreservation solution I at an environment of 40–42°C. After 2–4 minutes, the porcine embryos were placed in vitrification cryopreservation solution II, and then placed in an environment below -190°C for 1 minute.

[0024] Furthermore, the vitrification cryopreservation solution I is based on TL-PVA with 5-10% DMSO and 5-10% EG.

[0025] Furthermore, the vitrification cryopreservation solution II is based on TL-PVA with 14-18% DMSO, 14-18% EG, and 0.3-0.5M sucrose.

[0026] All percentages in vitrification cryopreservation I / II solutions are by mass-volume percentages (wt%).

[0027] In this invention, EG refers to ethylene glycol, and sucrose refers to sucrose.

[0028] The present invention further provides the application of the method in improving the cryopreservation efficiency of porcine embryos.

[0029] Secondly, the present invention provides the application of the method described therein in improving the cryopreservation efficiency of porcine embryos.

[0030] The present invention further provides the application of the method in reducing the level of apoptosis in pig embryos after freezing.

[0031] The present invention further provides the application of the method in reducing ROS levels in frozen porcine embryos.

[0032] The present invention has the following beneficial effects:

[0033] This invention provides a method for cryopreserving porcine embryos by combining berberine treatment with the addition of Fe3O4 nanoparticles and antifreeze protein type I to the cryopreservation solution. This cryopreservation method can effectively improve the cryopreservation efficiency of porcine embryos, increase the survival rate of frozen porcine embryos, and reduce the apoptosis and ROS levels of porcine embryos, which has important application value in the field of pig farming. Detailed Implementation

[0034] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of this invention, not all embodiments. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention.

[0035] Unless otherwise specified, the experimental methods used in the following embodiments can be implemented using conventional methods in the art. Unless otherwise specified, the experimental materials used are commercially available.

[0036] Example 1

[0037] This embodiment provides a basic method for cryopreservation of porcine embryos in vitro, specifically including the following steps:

[0038] 1. Collection and maturation of oocytes

[0039] (1) Within 30 minutes of slaughtering a sow at the slaughterhouse, cut off both ovaries and place them in a sterile saline wide-mouth thermos bottle at 35°C (with penicillin and streptomycin added at the same time). Transport the ovaries back to the laboratory within 2 hours.

[0040] (2) Using an 18-gauge needle connected to a filtration pump, select follicles with a diameter of 3-6 mm to extract and collect follicular fluid, and let it stand for 15 minutes.

[0041] (3) Discard the supernatant, collect the precipitate, add the ovum flushing solution, invert and resuspend, let stand for 15 minutes, repeat twice, take an appropriate amount of suspension in a 10cm culture dish, and use a pipette under a stereomicroscope to pick up the intact COCs wrapped by the cumulus cells.

[0042] (4) After washing once with rinsing solution in a 35mm dish, transfer and wash twice in maturation solution (IVM), then transfer to a four-well plate that has been equilibrated in an embryo culture incubator for more than 4 hours, and place 50 COCs in each well.

[0043] (5) Place the petri dish in an incubator with 5% CO2 at 38.5℃ and incubate for about 40 hours.

[0044] (6) After maturing, COCs are placed in M199 solution of 1 mg / mL hyaluronidase preheated at 38.5℃. The cumulus cells are gently removed by pipetting with a 100 μL pipette. The removal is confirmed to be complete under a stereomicroscope.

[0045] (7) Select mature oocytes that have expelled the first polar body, have a clear periovarian space, and have uniform cytoplasm, and place them in a T2 culture drop preheated at 38.5℃ for later use.

[0046] 2. Parthenogenetic embryo preparation

[0047] (1) Add fusion liquid to the fusion tank connected to the BLS fusion instrument and set the electrical activation parameters: DC 150V, 100μs, 2 pulses.

[0048] (2) Place the mature oocytes selected in step 1 into the fusion droplet and equilibrate for about 1 minute.

[0049] (3) Then, transfer the oocytes into the fusion medium of the fusion tank, arrange them in a row parallel to the electrodes, and place about 50 oocytes at a time to avoid overlapping. After pressing the start button to activate the electrode, transfer them into the T2 culture medium.

[0050] (4) After electro-activation, the oocytes were transferred into droplets of PZM-3 medium containing 10 μg / ml actinomycete CHX in an auxiliary activation medium that had been balanced for more than 2 hours in advance, and then placed in a 5% CO2, 5% O2 hypoxic incubator at 38.5℃.

[0051] (5) After 4 hours of assisted activation, the oocytes were transferred to four-well plates of overnight balanced PZM-3 medium and cultured in an incubator with 5% CO2, 5% O2 and 38.5°C.

[0052] 3. Freeze for preservation

[0053] (1) Freezing: The temperature of the heating table is set to 41 degrees. About 5 embryos are frozen at a time. They are then transferred into vitrification freezing solution I (7.5wt% DMSO + 7.5wt% EG on the basis of TL-PVA) for 3 minutes.

[0054] (2) The pig embryos were transferred into vitrification II solution (TL-PVA + 16wt% DMSO + 16wt% EG + 0.4M sucrose) and loaded into the cryogenic carrier after 30 seconds. Liquid nitrogen was rapidly added within 1 minute.

[0055] (3) After putting on the cannula, move it to the wheat straw thumb tube (mark according to the individual pig).

[0056] (4) Thawing: Preheat the thawing solution to 39 degrees Celsius. 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 the thawing solution (ensuring the entire flexible part of the CRYOTOP is submerged). Gently agitate the tube. Once the embryo has detached, remove the tube (approximately 10 seconds). After 5 minutes, remove the thawing solution. Clean the tube with TL-PVA and NCSU23 respectively before use for subsequent culture. Observe the hatching rate after 24 hours of NCSU23 culture.

[0057] The aforementioned TL-PVA is Tyrode's lactate (TL)-HEPES-polyvinyl alcohol, comprising the following (1000ml): 7.264g NaCl (Sigma), 0.237g mM KCl (Sigma, P4504), 0.168g NaHC O3 (Sigma, S8875), 0.041g KH2PO4 (Sigma, S5011), 1.868ml Na-lactate (sodium lactate Sigma, L7900), 0.102g MgCl2·6H2O (Sigma, Mo250), 0.297g CaCl2·2H2O (Sigma, C7902), 2.383g HEPES (Sigma, H3784), 0.022g Na-pyruvate (P4562), 2.186gs orbitol (sigma, S1876), 1g PVA (sigma, P8136), 100IU / ml bispecific antibody.

[0058] 4. ROS staining steps

[0059] According to the manufacturer's instructions, the ROS levels in cryopreserved porcine parthenogenetic embryos were detected using a reactive oxygen species (ROS) detection kit (S0033S, Beyotime). The embryos were washed three times with 0.1% PVA-PBS and then placed in a 10 mmol / L solution. -1 The embryos were stained in DCFH-DA staining solution at 37°C in the dark for 20 min. After staining, the embryos were washed three times with 0.1% PVA-PBS solution and photographed under an inverted fluorescence microscope (Nikon, Japan). The fluorescence values ​​of the acquired fluorescence images were statistically analyzed using EZ-C1 Free Viewer software (Nikon, Japan).

[0060] 5. TUNEL staining:

[0061] According to the manufacturer's instructions, the apoptosis level of cryopreserved porcine parthenogenetic embryos was detected using a TUNEL assay kit (T2130, Solarbio). Embryos were fixed in 4% paraformaldehyde for 30 min, followed by permeabilization with 0.2% Triton X-100 at room temperature for 20 min. TdT enzyme was thoroughly mixed with TUNEL Reaction Buffer to prepare the TUNEL reaction solution. Embryos were incubated in the TUNEL reaction solution at 37°C in the dark for 1 h. After washing with 0.1% Triton X-100, the embryos were stained with 5 μg / mL DAPI and incubated at room temperature in the dark for 5 min. Images were then taken under an inverted fluorescence microscope (Nikon, Japan). The fluorescence intensity of the TUNEL staining was analyzed using ImageJ software.

[0062] Example 2

[0063] To obtain a better method for cryopreservation of porcine embryos in vitro, this invention compared the effects of different cryopreservation methods on the survival rate of porcine parthenogenetic activated embryos, as detailed below:

[0064] In addition to the Cryotop method in Example 1, this invention compared the effects of the thin tube method (IMV, France) and the OPS method (manual pulling) on ​​the survival rate of porcine parthenogenetic activated embryos, and obtained the results shown in the table below:

[0065] Table 1. Effects of different freezing methods on the survival rate of porcine parthenogenetic activated embryos.

[0066] capillary method <![CDATA[52.94±6.35%(18 / 34) c ]]> OPS method <![CDATA[65.85±7.52%(27 / 41) b ]]> Cryotop method <![CDATA[70.27±7.16%(26 / 37) a ]]>

[0067] Note: Different letters indicate significant differences.

[0068] The results show that the survival rate of parthenogenetic activated porcine embryos obtained by the Cryotop method is higher than that obtained by the thin tube method and the OPS method.

[0069] Example 3

[0070] This invention further verifies the effects of defatting, protecting the cytoskeleton, increasing the viscosity of the cryopreservation solution, nanoparticles, and antifreeze protein type I on the cryopreservation survival rate of porcine parthenogenetic activated embryos, with specific comparisons as follows:

[0071] 1. Based on the method in Example 1, a defatting treatment was adopted in the in vitro culture process of step 2, stage (5) (2.5 μM berberine was added to the culture medium).

[0072] 2. Based on the method in Example 1, in step 3, in stages (1) and (2), cytoskeleton protection treatment is adopted (7.5 mg / mL of cytochalasin B is added to vitrification solution I and vitrification solution II).

[0073] 3. Based on the method in Example 1, in step 3, in stages (1) and (2), the viscosity of the cryosol is increased (50 μg / mL HPC is added to the vitrification cryosol I and vitrification cryosol II).

[0074] 4. Based on the method in Example 1, 1% by mass / volume Fe3O4 nanoparticles were added to the cryopreservation solution I / II in step 3.

[0075] 5. Based on the method in Example 1, add 500 ng / ml of antifreeze protein type I to the cryopreservation solution I / II in step 3.

[0076] The results are shown in the table below:

[0077] Table 2. Effects of different pre-freezing treatments on the survival rate of porcine parthenogenetic activated embryos.

[0078] Cryotop method 71.11±4.44％(32 / 45)b 2.5 μM berberine + Cryotop method 81.57±8.46％(31 / 38)a 7.5 mg / mL cytochalasin B + Cryotop method 80.48±6.77％(33 / 41)a 50 μg / mL hydroxypropyl cellulose (HPC) + Cryotop method 81.08±7.74％(30 / 37)a <![CDATA[1.0% Nanoparticle Fe3O4 + Cryotop Method]]> 80.48±6.48％(33 / 41)a 500 ng / ml antifreeze protein type I + Cryotop method 83.33±8.42％(35 / 42)a

[0079] Note: Different letters indicate significant differences.

[0080] The above treatment methods were all optimized and tested at the optimal concentration. The results show that degreasing, protecting the cytoskeleton, increasing the viscosity of the cryopreservation solution, and adding nanoparticles and antifreeze protein type I to the cryopreservation solution can all improve the cryopreservation survival rate of porcine parthenogenetic activated embryos.

[0081] Example 4

[0082] This invention provides a combined treatment method that can significantly improve the cryopreservation survival rate of porcine embryos, and this method can also effectively reduce the apoptosis level and ROS level of porcine embryos. Specifically, by combining methods 1, 4, and 5 in Example 3, and based on the experimental method in Example 1, the results are shown in the table below:

[0083] Table 3. Effects of different combined treatments on the cryopreservation survival rate of porcine parthenogenetic activated embryos.

[0084]

[0085]

[0086] The results above show that the combined treatment with berberine, nanoparticles, and antifreeze protein type I significantly improved the cryopreservation survival rate of porcine embryos. However, when other combined treatments were used, the results were similar to those achieved by using a single treatment method.

[0087] Table 4. Effects of different combined treatments on apoptosis and ROS levels in frozen porcine parthenogenetic activated embryos.

[0088]

[0089] The results above show that the combined treatment with berberine, nanoparticles, and antifreeze protein type I can significantly reduce the apoptosis and ROS levels of porcine embryos after freezing.

[0090] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for cryopreservation of pig embryos, characterized in that, include: The Cryotope method was used for cryopreservation of pig embryos; Before the cryopreservation process in the Cryotop method, the porcine embryos were treated with 2-3 μM berberine. In the cryopreservation process of the Cryotop method, Fe3O4 nanoparticles and antifreeze protein type I are added to the cryopreservation solution. The amount of Fe3O4 nanoparticles in the cryopreservation solution is 0.5~1.5wt%, and the amount of antifreeze protein type I in the cryopreservation solution is 250~700ng / mL. The pig embryos were parthenogenetic embryos.

2. The cryopreservation method according to claim 1, characterized in that, The berberine treatment method includes adding berberine to the culture medium during the in vitro culture stage of the pig embryo.

3. The cryopreservation method according to claim 1, characterized in that, The cryopreservation solution is based on TL-PVA and also includes one or more of DMSO, EG, or sucrose.

4. The method according to any one of claims 1-3, characterized in that, The Cryotop method includes: The pig embryos were placed in a cryopreservation solution and then placed in an environment below -190°C for 1 minute.

5. The method according to claim 4, characterized in that, The environment below -190°C is liquid nitrogen.

6. The method according to claim 4, characterized in that, The Cryotop method includes: The porcine embryos were placed in cryopreservation solution I at an environment of 40-42°C. After 2-4 minutes, the porcine embryos were placed in cryopreservation solution II, and then placed in an environment below -190°C for 1 minute.

7. The application of the method according to any one of claims 1-6 in improving the cryopreservation efficiency of porcine embryos.

8. The application of the method according to any one of claims 1-6 in reducing the level of apoptosis in frozen porcine embryos.

9. The application of the method according to any one of claims 1-6 in reducing ROS levels in frozen porcine embryos.

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