Kit for improving cattle embryo freezing quality and application thereof

Abstract

The invention provides an anti-freezing polypeptide derived from Yanbian cattle collagen, the polypeptide has obvious protection and improvement effects on cattle embryo freezing quality, and the amino acid sequence of the polypeptide is shown as SEQ ID NO.1 or 2. On the basis, the invention provides a bovine embryo vitrification freezing kit using the polypeptide and a freezing method of the bovine embryo vitrification freezing kit. The kit can effectively protect the embryo, so that the embryo to be preserved is put into liquid nitrogen for cryopreservation, the operation is simple, and expensive instruments and equipment are not needed.

Description

Technical Field

[0001] The present invention belongs to the fields of biotechnology and medicine, and relates to the in-depth application of an antifreeze protein. Specifically, it relates to the application of an antifreeze polypeptide derived from bovine collagen as a biological cryoprotectant, and further, as an embryo diluent. Background Art

[0002] With the continuous improvement of the living standards of the people in our country, beef is becoming more and more popular. The yellow cattle industry is currently in a strategic transformation stage. Scattered households are gradually withdrawing from the market, and large-scale breeding will become the mainstream. However, the basic number of yellow cattle during the transformation period is the primary problem that needs to be faced at present. Yanbian yellow cattle, together with Qinchuan cattle, Nanyang cattle, Luxi yellow cattle, and Jinnan cattle, are known as the five excellent yellow cattle breeds in China. They are precious yellow cattle breeds in our country, with the characteristics of being cold-resistant, roughage-tolerant, tall in stature, deep and wide in chest, solid in bones, strong in disease resistance, delicate in meat quality, and delicious in taste. However, at present, the coverage rate of improved varieties in the livestock production in our country is only about 40%, and a large part depends on imports from abroad. Exploring and utilizing the germplasm resources of yellow cattle and realizing efficient and rapid propagation are the main countermeasures to solve the current problem of yellow cattle germplasm resources.

[0003] The technology of ovum pick-up (OPU) from live cattle has now become an important technology for many developed livestock husbandry countries to expand the herds of improved cattle. Live ovum pick-up is beneficial to the efficient propagation of excellent breeding livestock and plays a positive role in promoting the development of the livestock industry. The number of embryos obtained by the OPU technology and in vitro embryo production (IVP) has far exceeded the number of embryos obtained by the conventional superovulation technology. According to the annual statistical data of the International Embryo Technology Society (IETS), as of 2020, about 72% of the embryos produced in the world are IVP embryos. At present, about 33% of the IVP embryos need to be cryopreserved at low temperature, and more than 50% of the IVD embryos need to be cryopreserved. Although a good in vitro production system has been established, at present, embryos with the same quality and ability as those produced by natural pregnancy cannot be produced. In contrast, the quality of embryos produced in vitro is lower than that of in vivo embryos, and there are problems such as vacuoles in trophoblast cells, sparse microvilli, loose inner cell mass, gene expression differences, and lipid metabolism changes. Therefore, one of the challenges in assisted reproduction is to improve the embryo production system and optimize the cryopreservation technology in order to obtain relatively high survival rates and pregnancy rates after cryopreservation of embryos. Summary of the Invention

[0004] In previous research, the applicant extracted an antifreeze polypeptide from bovine collagen, and it has been confirmed that this antifreeze polypeptide can reduce the solute concentration difference inside and outside cells during the cooling process and plays an important protective role in the cryopreservation of semen (CN118530337A). Based on this, the applicant further studied and proposed the present invention.

[0005] The present invention provides an antifreeze polypeptide derived from Yanbian cattle collagen, which has an obvious protective and enhancing effect on the freezing quality of cattle embryos. The amino acid sequence of the polypeptide is: GPKGDTGPRGPRGPGPPG (SEQ ID NO.1) and / or GGKPGDQGVPGEGAPGLVGPR (SEQ ID NO.2).

[0006] Furthermore, the present application provides a nucleotide encoding the above-mentioned antifreeze polypeptide.

[0007] Furthermore, the present application provides an application of the above-mentioned antifreeze protein in the preparation of a cryoprotectant for cattle embryos. The cryoprotectant for cattle embryos includes:

[0008] Base solution: PBS solution added with antifreeze polypeptide;

[0009] FS solution: 300 g / L of polysucrose and 0.5 mol / L of sucrose in PBS solution, which is the FS solution.

[0010] Vitrification solution: Ethylene glycol and FS solution are mixed evenly according to the ratio of 35:65 (v / v) to prepare the EFS35 vitrification solution.

[0011] Thawing solution: PBS solution containing 10% (V / V) fetal bovine serum (FBS)

[0012] Among them, the antifreeze polypeptide is selected from one or more of SEQ ID NO.1-2, or a fusion protein containing one or more of the polypeptides shown in SEQ ID NO.1-2.

[0013] Furthermore, the present application provides an application of the above-mentioned cryoprotectant for cattle embryos in the cryopreservation of cattle embryos.

[0014] Furthermore, the present application provides a method for cryopreserving cattle embryos using the above-mentioned cryoprotectant for cattle embryos. The method includes the following steps:

[0015] Sequentially suck thawing solution (about 5-10 cm), air (about 1 cm), and cryoprotectant (about 0.5-1 cm) into the bovine embryo freezing straw, then place it parallel on the operating table. Transfer the bovine embryo into the vitrification solution at 25°C and equilibrate for 3 minutes. After the embryo is transferred, suck air (about 1 cm) and thawing solution (about 5-10 cm) and then seal it. Then directly put the straw into liquid nitrogen for cryopreservation. The order from left to right is: stopper - thawing solution - air - vitrification solution containing embryo - air - vitrification solution - air - thawing solution - stopper.

[0016] Further, the method further includes a step of thawing the embryo, and the steps are as follows: Take out the straw from liquid nitrogen, stay in an air bath for 5 - 10 seconds, then immerse it in a water bath at 20 - 25°C and shake it parallelly. After the solution in the straw melts, take out the straw, dry the surface moisture, and then invert and shake the straw back and forth 3 - 4 times. After the cryoprotectant in the tube is evenly mixed with the thawing solution, embryo examination and embryo transfer can be performed. It is advisable to complete the process within 5 - 10 minutes from embryo thawing to transplantation.

[0017] Beneficial effects

[0018] The present invention provides an antifreeze polypeptide derived from Yanbian yellow cattle collagen, which has an obvious protective and enhancing effect on the freezing quality of yellow cattle embryos. The amino acid sequence of the polypeptide is shown in SEQ ID NO.1 or 2. On this basis, the present application provides a vitrification freezing kit for bovine embryos using the above polypeptide and its freezing method. The kit can effectively protect embryos, enabling the embryos to be cryopreserved by being put into liquid nitrogen for storage. The operation is simple and does not require expensive instruments and equipment. Description of the drawings

[0019] Figure 1 HAT schematic diagram of the crude extract of Yanbian yellow cattle collagen polypeptide;

[0020] Figure 2 HAT schematic diagram of the refined component of Yanbian yellow cattle collagen polypeptide;

[0021] Figure 3 Schematic diagram of bovine frozen embryo loading into a tube, where 1 is a stopper, 2 is a thawing solution, 3 is air, 4 is a vitrification solution containing embryos, and 5 is a vitrification solution. Detailed implementation manners

[0022] Next, in combination with the embodiments of the present invention, the technical solutions of the present invention will be clearly and completely described. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts fall within the scope of protection of the present invention.

[0023] Example 1 Screening of antifreeze polypeptide from Yanbian yellow cattle collagen and analysis of its antifreeze activity

[0024] Referring to the method in CN118530337A, the antifreeze polypeptide of Yanbian yellow cattle collagen was screened, and some of the parameters were adjusted, which are summarized as follows: Select raw materials of Yanbian yellow cattle hides frozen at -18°C. First, rinse the impurities with clean water, then crush them through a crusher. After crushing, the particle size is controlled at 10 mm. After rinsing with clean water and removing impurities, carry out low-temperature reflux degreasing with ether. Carry out decalcification treatment with 0.25 mol / L EDTA. After the treatment, add ficin and alkaline protease (the enzyme activity ratio is 2:1) according to the proportion of 3% of the dry weight, stir evenly, and enzymolyze at 50°C for 2 h. After the enzymolysis is completed, centrifuge at 12,000 rpm, collect the supernatant, filter the supernatant through a 10 kDa membrane, and filter the filtrate through a 1000 Da membrane, and collect the retentate; the polypeptide solution is spray-dried to obtain the crude extract of Yanbian yellow cattle collagen polypeptide. Use macroporous adsorption resin (LSP-21:LSC200 = 1:1) to separate the polypeptide, and elute with methanol respectively. Collect the 20% methanol elution fraction F1, 40% methanol elution fraction F2, and 80% methanol elution fraction F3 respectively. After drying the collected different parts, carry out component analysis and activity detection. Use differential scanning calorimetry (DSC) method to measure the thermal hysteresis activity (THA) of the enzymolysis product. The results are as Figure 1 shown. The curves of BSA before and after temperature change are smoothly connected, and there is no phenomenon of freezing hysteresis, indicating that the BSA solution does not have a thermal hysteresis effect. The heat flow-temperature curves of F1 and F3 are similar to that of BSA, and there is no obvious freezing hysteresis phenomenon, so it also does not have an obvious thermal hysteresis effect. However, the crystallization peak of F2 appears significantly delayed, and the thermal hysteresis activity is 2.37°C.

[0025] Take the F2 polypeptide and prepare a solution of 100 mg / mL, and refine it using preparative liquid chromatography (Dubhe C18 preparative column (250×20 mm, 10 μm), flow rate 6 mL / min, injection volume 1 mL, 100 mg / mL, column temperature is room temperature, detection wavelength 220 nm). The mobile phase is a mixed solution of water and methanol containing 0.1% trifluoroacetic acid, and the elution conditions are: 0 min, 5% methanol; 5 min, 5% methanol; 180 min, 80% methanol. Collect each component Fn-1 / 2 / 3 / 4 respectively, and carry out activity detection and component analysis after drying. The F2-1 component is eluted with 5% methanol and 0.1% trifluoroacetic acid aqueous solution, the F2-2 component is eluted with 5%-15% methanol and 0.1% trifluoroacetic acid aqueous solution, F2-3 is eluted with 15%-30% methanol and 0.1% trifluoroacetic acid aqueous solution, and F2-4 is eluted with 30%-45% methanol and 0.1% trifluoroacetic acid aqueous solution.

[0026] As Figure 2As shown, the heat flow-temperature curves of the refined components F2-1 and F2-4 of Yanbian yellow cattle collagen polypeptide are smoothly connected without obvious freezing hysteresis phenomenon, so there is no thermal hysteresis activity. The crystallization peaks of the refined components F2-2 and F2-3 appear significantly delayed, and the thermal hysteresis activities are 3.31 °C and 2.54 °C respectively; the above two polypeptide components F2-2 and F2-3 were refined and then sent to BGI for sequencing, and their amino acid sequences were obtained as: GPKGDTGPRGPRGPGPPG (SEQ ID NO.1) and / or GGKPGDQGVPGEGAPGLVGPR (SEQ ID NO.2).

[0027] Example 2 Preparation and Optimization of Cryoprotectant for Yellow Cattle Embryos

[0028] Base solution: PBS solution;

[0029] FS solution: 300 g / L of Ficoll and 0.5 mol / L of sucrose in PBS solution, which is the FS solution.

[0030] Vitrification solution: Mix ethylene glycol and FS solution in a ratio of 35:65 (v / v) to prepare the EFS35 vitrification solution.

[0031] Thawing solution: PBS solution containing 10% (V / V) fetal bovine serum (FBS).

[0032] In order to verify the screened antifreeze polypeptides, 3 mg / mL of F3-2, F3-3 and the bovine collagen antifreeze polypeptide (PYPGAANVPGPAATAGAGG) screened in previous studies were added to the base solution as positive controls for parallel tests.

[0033] The open pulled straw (OPS) method was used for vitrification freezing to reduce the cold sensitivity of bovine embryos.

[0034] The specific operation is as follows:

[0035] (1) Preparation and Selection of Yellow Cattle Embryos

[0036] Referring to "Animal Reproduction Technology", conventional in vivo oocyte retrieval was used. The oocyte retrieval process was carried out at the applicant's cooperative yellow cattle breeding base. The donor cows were 15 non-pregnant female Yanbian yellow cattle. The oocytes obtained by in vivo oocyte retrieval were in vitro fertilized with frozen semen of Yanbian yellow cattle breeding bulls, and a total of 368 qualified blastocysts were obtained for freezing experiments.

[0037] (2) Embryo Loading, Freezing and Thawing

[0038] As Figure 3As shown, suck thawing solution (about 5 - 10 cm), air (about 1 cm), and freezing solution (about 0.5 - 1 cm) into the fine tube for bovine embryo freezing in the order shown in the figure, then place it parallel on the operating table. Transfer the bovine embryo into the vitrification solution at 25°C and equilibrate for 3 minutes. After the embryo is transferred, suck in air (about 1 cm) and thawing solution (about 5 - 10 cm) and then seal the tube. Then directly put the fine tube into liquid nitrogen for cryopreservation.

[0039] Take out the fine tube from liquid nitrogen, stay in the air bath for 5 - 10 seconds, then immerse it in a 20 - 25°C water bath and shake it parallelly. After the solution in the fine tube melts, take out the fine tube, dry the surface moisture and then shake the fine tube back and forth 3 - 4 times to mix the freezing solution and thawing solution in the tube evenly. Then embryo examination and embryo transfer can be carried out. It is advisable to complete the process from embryo thawing to transplantation within 5 - 10 minutes.

[0040] The results are shown in Table 1. F3 - 2 at 3 mg / mL as an embryo cryoprotectant can achieve better protection effects, with the highest survival rate and hatching rate of cryopreserved embryos. While F3 - 3 at 3 mg / mL and the previous antifreeze protein also have certain protection effects on cryopreserved embryos, but the effects are significantly lower than that of F3 - 2, yet still significantly better than the blank control. It is speculated that the antifreeze protein can act as an antifreeze protectant, fully penetrate into the interior of bovine embryo cells, prevent the water inside from forming ice crystals at low temperatures, and the ice crystals will cause physical damage to the embryo, thus achieving the cryopreservation of the embryo. And it also shows from one aspect that the antifreeze protein which plays a good role in the cryopreservation of semen has inconsistent effects in the cryopreservation of embryos, and its specific mechanism and reasons need further study.

[0041] Table 1 Protection effects of different antifreeze proteins

[0042] Group Number of frozen embryos (pcs) Number of survivors (pcs) Survival rate (%) Number of hatched embryos (pcs) Hatching rate (%) PBS 30 12 40 3 10 PBS + F3-2 30 29 96.7 28 93.3 PBS + F3-3 30 18 60 15 50 Positive control 30 17 56.7 9 30

[0043] Thus, it can be seen that the screened antifreeze protein F3 - 2 has good protection effects on the embryo freezing of cattle, especially yellow cattle. The next step of research can focus on the specific addition concentration and its reasonable combination with other several cryoprotectants for the germplasm protection of yellow cattle resources.

[0044] The above description of the embodiments is for the ordinary technical personnel in the technical field to understand and use the present invention. It is obvious that those skilled in the art can easily make various modifications to these embodiments and apply the general principles described herein to other embodiments without creative labor. Therefore, the present invention is not limited to the above embodiments. The improvements and modifications made by those skilled in the art according to the principles of the present invention without departing from the scope of the present invention should be within the protection scope of the present invention.

Claims

1. An antifreeze polypeptide derived from Yanbian cattle collagen, characterized in that, The polypeptide has an obvious protective and enhancing effect on the freezing quality of yellow cattle embryos. The amino acid sequence of the polypeptide is: GPKGDTGPRGPRGPGPPG (SEQ ID NO.1) and / or GGKPGDQGVPGEGAPGLVGPR (SEQ ID NO.2).

2. A nucleotide encoding the antifreeze polypeptide according to claim 1.

3. Use of the antifreeze protein according to claim 1 in the preparation of a cryoprotectant for yellow cattle embryos.

4. A cryoprotectant for yellow cattle embryos containing the antifreeze protein described in claim 1, wherein, The cryoprotectant for yellow cattle embryos comprises: Base solution: PBS solution added with antifreeze polypeptide; FS solution: 300 g / L of Ficoll and 0.5 mol / L of sucrose in PBS solution, which is the FS solution. Vitrification solution: Prepared by mixing ethylene glycol and FS solution in a ratio of 35:65 (v / v) to form EFS35 vitrification solution. Thawing solution: PBS solution containing 10% (V / V) fetal bovine serum (FBS).

5. Use of the cryoprotectant for yellow cattle embryos according to claim 4 in the cryopreservation of bovine embryos.

6. A method for cryopreserving yellow cattle embryos using the cryoprotectant for yellow cattle embryos according to claim 4, the method comprising the following steps: Sequentially suck thawing solution (about 5 - 10 cm), air (about 1 cm), and cryoprotectant (about 0.5 - 1 cm) into a fine tube for freezing bovine embryos, then place it horizontally on the operating table. Transfer the bovine embryo into the vitrification solution at 25°C and equilibrate for 3 min. After the embryo is transferred, suck air (about 1 cm) and thawing solution (about 5 - 10 cm) again and then seal the tube. Then directly put the fine tube into liquid nitrogen for cryopreservation. The order from left to right is: stopper - thawing solution - air - vitrification solution containing embryo - air - vitrification solution - air - thawing solution - stopper.

7. As the method according to claim 6, the method further comprises an embryo thawing step, and the step is: Take out the fine tube from liquid nitrogen, stay in the air bath for 5 - 10 seconds, then immerse it in a 20 - 25°C water bath and shake it horizontally. After the solution in the fine tube melts, take out the fine tube, dry the surface moisture and then invert and shake the fine tube back and forth 3 - 4 times to mix the cryoprotectant and thawing solution in the tube evenly, and then embryo inspection and embryo transfer can be performed. It is advisable to complete the process from embryo thawing to transplantation within 5 - 10 minutes.

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