Antioxidant antifreeze protein derived from Tibetan pig and application of antioxidant antifreeze protein in frozen semen diluent

Abstract

According to the antioxidant antifreeze protein from Tibetan pigs, the protein is added into a cryopreservation solution, so that the activity of peroxidase, catalase, superoxide dismutase and glutathione peroxidase after the sperms of the Tibetan pigs are frozen and unfrozen can be remarkably improved, the total antioxidant capacity of the sperms is remarkably enhanced, and the antifreeze effect of the sperms is improved. The active oxygen level and malondialdehyde content in sperm cells are obviously reduced. The cryoprotectant provided by the invention can greatly improve the motility rate and activity of unfrozen sperms and ensure the stability of the motility rate and activity of the unfrozen sperms; the pregnancy rate of artificial insemination of the frozen semen of the Tibetan pigs is increased, and the quality of the frozen semen is greatly improved. In addition, exogenous antifreeze protein does not need to be added into the preserving fluid, so that essential nutrient substances for sperm metabolism are guaranteed, and meanwhile, damage and pollution risks caused by exogenous substances to sperms are reduced.

Description

Technical Field

[0001] The invention belongs to the technical field of genetic engineering, and particularly relates to an antioxidant antifreeze protein derived from Tibetan pigs and application of the protein in a frozen semen diluent. Background Art

[0002] Tibetan pigs are mainly distributed in the middle reaches of the Yarlung Zangbo River valley area and the middle reaches of the three rivers in eastern Tibet. They are an important breed unique to China that can adapt to high altitude areas and are known as the "treasure of the plateau". In order to promote the great development of the Tibetan pig industry, artificial insemination is an indispensable link. At present, the primary problem of the core farms in the Tibetan pig breeding industry is the insufficient utilization of boar semen. Most of the semen used for artificial insemination in most pig farms comes from semen collected on the same day or stored at 17℃ for 1 to 2 days; most of the medium-acting, medium-long-acting and long-acting pig semen diluents on the market are monopolized by developed pig-raising countries, and the prices are high, which brings great economic pressure to the pig breeding industry and increases the production costs of enterprises. In previous studies, it was found that even if the boar semen was collected and fertilized on the same day, the storage of boar semen at 18℃ would induce lipid peroxidation, reduce mitochondrial membrane potential, and increase plasma membrane permeability. During the cryopreservation of semen, pig sperm has a lower tolerance to low temperatures than other livestock and is more susceptible to cold shock. Sperm is in a state of oxidative stress, producing a large amount of reactive oxygen species (ROS), which damages the structure and function of sperm and even induces apoptosis.

[0003] Antifreeze proteins are a class of polypeptides or glycopeptides produced by organisms to adapt to extreme cold environments. They can lower the freezing point of solutions, but have little effect on melting points. Antifreeze proteins were first discovered in polar fish. So far, humans have isolated a variety of antifreeze proteins from organisms such as marine fish, terrestrial insects, plants, bacteria and fungi. Although the structures of different types of antifreeze proteins are different, they all have the characteristics of preventing ice crystal formation, regulating the growth of extracellular ice crystals, inhibiting ice recrystallization, and maintaining the non-freezing state of liquids. Although in recent years, many scholars have conducted research on antifreeze marker proteins in different animals, and found that collagen from chicken skin, fish skin, pig skin, etc. has good antifreeze activity, there is a lack of research on antifreeze proteins from Tibetan pigs. Moreover, the antifreeze proteins from other animal sources mentioned above can also induce allogeneic immunity in fertilized sows due to species differences, resulting in fertilization failure, and cannot be directly applied to the semen storage process of Tibetan pigs. Therefore, there are no real applied research results, nor have they been truly used in the practice of Tibetan pig semen freezing. Summary of the invention

[0004] In order to solve the problems of low viability and vitality of existing Tibetan pig frozen semen and unstable freezing effect, the present invention provides an antioxidant antifreeze protein derived from Tibetan pigs themselves and the application of the protein in Tibetan pig frozen semen diluent.

[0005] Specifically, the present invention first provides an antioxidant and antifreeze protein derived from Tibetan pigs themselves. The protein is extracted from Tibetan pig skin through the steps of defatting, hydrolysis, purification, etc. The amino acid sequence of the protein is shown in SEQ ID No.1.

[0006] Furthermore, the present invention provides an antifreeze protein, which is a protein with equivalent activity obtained by replacing, deleting or inserting one or more amino acids in the amino acid sequence shown in SEQ ID No.1.

[0007] Furthermore, the present invention provides an application of an antifreeze protein, which is used for preparing a diluent for Tibetan pig frozen semen.

[0008] Preferably, the Tibetan pig frozen semen diluent comprises the following components:

[0009] Basic freezing solution (100ml): 3-5g trehalose, 3-5g glucose, 1-3g lactose, 0.2-0.5g vitamin C, 1.6g sodium citrate, 100IU / L (final concentration) antibacterial agent, 0.05-0.1g adenosine triphosphate, 0.02-0.04g glutathione.

[0010] Solution I: Add 1-10 ml of the above-mentioned Tibetan pig antifreeze protein (1 mg / ml, dissolved in 0.01 M PBS) to the basic freezing solution.

[0011] Solution II: Add 6% glycerol to solution I.

[0012] Wherein, the antibacterial agent is selected from one or more of penicillin, gentamicin, amphotericin B, and penicillin-streptomycin.

[0013] Furthermore, the present application provides an application of a Tibetan pig frozen semen diluent in the preservation of Tibetan pig semen, and the application is: using the Tibetan pig frozen semen diluent to perform cryopreservation of semen.

[0014] Those skilled in the art can understand that the use of the above reagents to store Tibetan pig semen at room temperature or low temperature; and the storage of semen of other animals at room temperature, low temperature, or frozen storage all fall within the scope of protection of the present invention.

[0015] The present invention is implemented by adopting the following technical scheme: A method for freezing and preserving ultra-high-vitality Tibetan pig semen comprises the following steps:

[0016] (1) Fresh Tibetan pig semen was collected and the apparent viability, motility and density of sperm were evaluated at 37°C. The volume of each semen collection was calculated using a graduated semen collection cup. The apparent viability of fresh semen was observed under a microscope at a magnification of 100 times and was graded from 0 to 5, with grade 0 indicating no fluctuation and grade 5 indicating rapid vortex-like fluctuation. The fresh semen was evaluated at a magnification of 400 times and the semen density was calculated on a hemocytometer after the fresh semen was diluted 1000 times. Only semen with an apparent viability of grade 5, a motility greater than 0.8 and a density greater than 2.0×10 9 / mL can be used for frozen semen production;

[0017] (2) Add the basic diluent isothermally, and adjust the sperm density after dilution to 8×108-10×10 8 Calculate the total amount of freezing solution required based on the dilution concentration of sperm / mL. Add half the required total volume of I solution to dilute the fresh semen, mix well and transfer to a refrigerator or 4°C low temperature environment, and cool to 0-4°C in a water bath for 1.5-2h to complete the first dilution;

[0018] (3) At the same temperature, add an equal volume of Solution II to Solution I to complete the second dilution step and equilibrate at 4°C for 2-6 hours.

[0019] (4) The diluted and balanced semen was aspirated into a 0.25 mL capillary tube at 0-4°C, sealed with polyvinyl alcohol powder, and the first step was to cool the semen and quickly remove the capillary tube. Tube Placement Place the tubes on a freezing rack 4-5 cm away from the surface of liquid nitrogen for 4-5 minutes. The second step is to cool the tubes down and place them in liquid nitrogen for long-term storage.

[0020] Beneficial Effects

[0021] The invention obtains an antioxidant antifreeze protein derived from Tibetan pigs themselves by extracting the protein from Tibetan pig skin through the steps of defatting, hydrolyzing, purifying and the like. The protein is added into a freezing preservation solution, which can significantly improve the activities of peroxidase, catalase, superoxide dismutase and glutathione peroxidase of Tibetan pig sperm after freezing and thawing (P<0.05), significantly enhance the total antioxidant capacity of sperm (P<0.05), and significantly reduce the active oxygen level and malondialdehyde content in sperm cells (P<0.05). At the same time, the semen preservation solution of the present invention can effectively remove "aging" sperm, weak dead sperm and non-essential substances and harmful substances in semen to obtain high-activity sperm. The activity rate of semen before freezing is above 0.8, and the activity rate after thawing is 0.69-0.74, which fully meets the requirements of artificial insemination of frozen semen of Tibetan pigs, greatly improves the activity rate and vitality of sperm after thawing, and ensures the stability of the activity rate and vitality of sperm after thawing; improves the pregnancy rate of artificial insemination of frozen semen of Tibetan pigs, and greatly improves the quality of frozen semen. Moreover, since no exogenous antifreeze protein needs to be added to the preservation solution, while ensuring the necessary nutrients for sperm metabolism, the damage caused by exogenous substances to sperm and the pollution risk brought are reduced. The antifreeze protein and freezing reagent of the present invention solve the problems of low activity rate and vitality of existing Tibetan pig frozen semen after thawing, and unstable freezing effect, and can be widely used in mass production of Tibetan pigs. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 It is the elution curve of Sephadex G-50 gel chromatography column. DETAILED DESCRIPTION

[0023] The technical solution of the present invention is described clearly and completely below. Obviously, the described embodiments are only some embodiments of the present invention, rather than all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in the field without creative work are within the scope of protection of the present invention. The present invention is further described below in conjunction with the embodiments.

[0024] Experimental animals and experimental location: The experimental Tibetan pigs are 6 healthy boars aged 3 to 5 years old. The experimental time is May to July 2024, and the location is the College of Animal Science of Tibet Agricultural and Animal Husbandry University.

[0025] Semen collection The Tibetan pig semen used in this experiment came from the internship base of Tibet Agricultural and Animal Husbandry College. The semen of healthy Tibetan pigs was collected by hand-holding method, and the middle semen was taken and filtered through 3 layers of gauze. The semen with sperm motility above 80% was diluted isothermally at 1:1 with sperm washing solution (based on 1L system, Tris 30g, citric acid 10g, glucose 12g, cysteine ​​6mM) and then put into a thermos bottle and immediately transported back to the laboratory. The collected boar semen was stored at -20℃ for future use.

[0026] Example 1 Preparation of Tibetan pig antifreeze protein

[0027] Cut the Tibetan pig skin into pieces (0.2 cm × 0.2 cm), degrease with 10 times the volume of 10% butanol solution for 24 h, change the butanol solution every 12 h, and then wash with distilled water; soak the defatted pig skin in 0.5 mol / L acetic acid solution for 12 h, and homogenize with 10 times the volume of 0.5 mol / L acetic acid solution, add 1 g / 100 mL pepsin to the homogenate for hydrolysis for 48 h, centrifuge (5000 × g, 40 min), and collect the supernatant ; Add NaCl powder to the supernatant for salting out (so that the final concentration of NaCl is 0.9 mol / L), let it stand for 24 hours and then centrifuge (5000×g, 60 min); collect the precipitate and dissolve it in 0.5 mol / L acetic acid, and dialyze it with 0.02 mol / L NaH2PO4 (pH8.6), 0.1 mol / L acetic acid and distilled water as the dialysis external fluid in sequence, and change the dialysis external fluid every 2 hours; collect the sample solution in the dialysis bag for later use.

[0028] The supernatant was separated by Sephadex G-50 gel chromatography (16×600 mm), the eluent was deionized water, the flow rate was 2 mL / min, the elution peak was measured at 225 nm, and the detection was performed according to the following method, and the elution peak with the best antifreeze activity was collected ( Figure 1 ).

[0029] (1) Low-temperature protection activity of catalase

[0030] Referring to the prior art (Cao H, Zhao Y, Zhu YB, et al. Antifreeze and cryoprotective activities of ice-binding collagen peptides from pig skin [J]. Food Chemistry, 2016, 194 (MAR.1): 1245-1253), a certain amount of catalase was weighed and dissolved in pH 7.0, 0.05 mol / L KH2PO4-NaOH buffer to prepare a catalase solution with a concentration of 0.5 mg / mL. The catalase solution and 2 mg / mL of crude antifreeze peptide extract were mixed in equal volumes to determine the initial enzyme activity before freezing and thawing. The mixture of catalase and crude antifreeze peptide extract was frozen at -33°C for 24 hours, then thawed at 25°C, and then frozen at -33°C for 6 hours; the above operation was repeated 4 times. The catalase activity after repeated freezing and thawing was then determined. The crude antifreeze peptide extract was replaced with buffer to determine the catalase activity of the control group after repeated freezing and thawing.

[0031] The method for determining the activity of catalase is as follows: add 1.9 mL of distilled water to a quartz cuvette, then add 0.1 mL of a mixture of catalase and antifreeze peptide crude extract and 1 mL of 0.1 mol / mL hydrogen peroxide solution, shake well immediately, put into the sample cell of the spectrophotometer, and record the initial absorbance value. Record the absorbance value every 1 minute, time for 5 minutes, and calculate the experimental results: a reduction of 0.1 in A240nm within 1 minute is one enzyme activity unit (U).

[0032] Catalase activity (u / (g.min)) = (A0-A1) × Vt / 0.1 × Vs × t × W, wherein A0 refers to the absorbance of the control tube with inactivated enzyme added; A1 refers to the absorbance of the sample tube; Vt refers to the total volume of the sample solution, mL; Vs refers to the volume of the sample solution used for determination, mL; t refers to the time from the addition of hydrogen peroxide to the last recording, min; W refers to the mass of the enzyme in the sample solution, g.

[0033] The ratio of enzyme activity after freezing and thawing to enzyme activity before freezing and thawing (residual activity of catalase) is used to indicate antifreeze activity. The greater the residual activity of the enzyme, the greater the antifreeze activity of the sample to be tested. The results showed that the ZZ peak had the best effect, and the residual activity of catalase was 80.34% of the initial enzyme activity.

[0034] (2) Low-temperature protection activity against Escherichia coli

[0035] The primary bacterial solution of Escherichia coli was cultured at 37°C until OD600 was about 0.8, and the cultured bacterial solution was diluted 105 times with culture medium; 100 μL of the diluted bacterial solution was placed in an Eppendorf centrifuge tube, and then 900 μL of 1 mg / mL antifreeze peptide crude extract was added. After oscillation and mixing, 100 μL of the mixed solution was spread on LB culture medium, and inverted cultured at 37°C for 20 hours before counting. The remaining bacterial solution was placed in a -33°C refrigerator for 24 hours, then thawed, and 100 μL of the thawed bacterial solution was spread on LB culture medium, and inverted cultured at 37°C for 20 hours before counting. The culture medium was used instead of the antifreeze peptide crude extract to determine the number of Escherichia coli surviving in the control group. A blank control experiment was performed with sterile water.

[0036] The survival rate of E. coli is expressed by the ratio of the number of E. coli after freezing and thawing to the number of E. coli before freezing and thawing. The higher the survival rate of E. coli, the higher the low-temperature protection activity of the sample against E. coli. The results also show that the ZZ peak has the best effect, with an E. coli survival rate of 73.14%.

[0037] (3) Determination of thermal hysteresis activity (THA)

[0038] Weigh 5 mg of freeze-dried antifreeze peptide crude extract and seal it in an aluminum crucible. Using an empty crucible as a reference, cool the temperature from room temperature to -40°C at a rate of 5°C / min, keep it warm for 1 minute, then increase the temperature to 10°C at 1°C / min, then cool it to -40°C at 5°C / min, keep it warm for 1 minute, and then increase the temperature to a semi-molten state at 1°C / min. The temperature at this time is called the retention temperature (Th). Cool it to -25°C at 1°C / min, and record the temperature (T0) at which the sample begins to crystallize at the retention temperature. Calculate the thermal hysteresis activity of the sample using the following formula:

[0039] THA=Th-T0

[0040] The results confirmed that the thermal hysteresis activity of the ZZ peak could reach 4.1℃.

[0041] The crude ZZ peptide extract separated by the above screening was further separated by Sulfopropyl-Sepadex C-25 cation exchange chromatography (length 55 cm, diameter 2.0 cm), and the eluent was 0.01 mol / L pH 7.0 phosphate buffer with a NaCl concentration gradient of 0-0.5 M, and the flow rate was 0.5 mL / min. Each peak sample was collected and the antifreeze activity was measured again. The sample with the best antifreeze activity was further analyzed and fractionated by UPLC-MS. The chromatographic experimental parameters used are as follows: the chromatographic column is Waters Acquity UPLC BEH C18colum (50×2.1mm, 1.7μm); the mobile phase A is deionized water (containing 0.1% formic acid), and the mobile phase B is acetonitrile; (B) phase elution program: 0-40min is 0-40%, 40-42min is 40-0%; the elution flow rate is 0.3mL / min; the sample loading amount is 5μL.

[0042] The mass spectrometry experimental parameters used are as follows: electrospray ionization source, positive ion mode, scanning molecular weight range of 100-2000Da; capillary voltage of 3000V, cone voltage of 40V, spray 35psi, degassing temperature of 350℃, ion source temperature of 120℃, N2 flow rate of 900L / h.

[0043] Finally, the single-stage component XZ with the best antifreeze activity was obtained, and its amino acid sequence was: His-Gly-Glu-Glu-Gly-Ala-Ser-Gly-Val-Arg-Gly-Ala-Phe (SEQ ID NO.1), the residual activity of catalase was 85.12% of the initial enzyme activity, the survival rate of Escherichia coli was 75.30%, and the thermal hysteresis activity could reach 4.7°C, which has good application prospects.

[0044] Example 2 Preparation of Tibetan pig frozen semen diluent and verification of its effect

[0045] (1) Based on the applicant's previous research results, a Tibetan pig frozen semen diluent was prepared according to the following formula, which includes the following components:

[0046] Basic freezing solution (100 ml): 5 g trehalose, 5 g glucose, 1 g lactose, 0.2 g vitamin C, 1.6 g sodium citrate, 100 IU / L (final concentration) antibacterial agent, 0.05 g adenosine triphosphate, 0.02 g glutathione.

[0047] Solution I: Add the above-mentioned Tibetan pig antifreeze polypeptide XZ (1 mg / ml, dissolved in 0.01 M PBS) to the basic freezing solution, set up a concentration gradient of 1 ml, 2 ml, 5 ml, and 10 ml, and set up a positive control of adding 5 g of egg yolk alone and a negative control without adding any antifreeze agent.

[0048] Solution II: Add 6% glycerol to solution I.

[0049] Wherein, the antibacterial agent is selected from one or more of penicillin, gentamicin, amphotericin B, and penicillin-streptomycin.

[0050] (2) Fresh Tibetan pig semen was collected and the apparent viability, motility and density of sperm were evaluated at 37°C. The volume of each semen collection was calculated using a graduated semen collection cup. The apparent viability of fresh semen was observed under a microscope at a magnification of 100 times and was graded from 0 to 5, with grade 0 indicating no fluctuation and grade 5 indicating rapid vortex-like fluctuation. The fresh semen was evaluated at a magnification of 400 times and the semen density was calculated on a hemacytometer after the fresh semen was diluted 1000 times. Only semen with an apparent viability of grade 5, a motility greater than 0.8 and a density greater than 2.0×10 9 / mL can be used for frozen semen production;

[0051] (3) Add the basic diluent isothermally, and adjust the sperm density after dilution to 8×108-10×10 8 Calculate the total amount of freezing solution required based on the dilution concentration of sperm / mL. Add half the required total volume of I solution to dilute the fresh semen, mix well and transfer to a refrigerator or 4°C low temperature environment, and cool to 0-4°C in a water bath for 1.5-2h to complete the first dilution;

[0052] (4) At the same temperature, add an equal volume of Solution II to Solution I to complete the second dilution step and equilibrate at 4°C for 2-6 hours.

[0053] (5) The diluted and balanced semen was aspirated into a 0.25 mL capillary tube at 0-4°C, sealed with polyvinyl alcohol powder, and the first step was to cool the semen and quickly remove the capillary tube. Tube Placement Place the tubes on a freezing rack 4-5 cm away from the surface of liquid nitrogen for 4-5 minutes. The second step is to cool the tubes down and place them in liquid nitrogen for long-term storage.

[0054] (6) Liquid nitrogen freezing effect test. Use tweezers to take the frozen sperm tube out of liquid nitrogen and expose it to air for 1-2 seconds. Then put it into a constant temperature water bath at 38℃ for 30 seconds and incubate it at 37℃ for 5 minutes. Then test the semen motility, plasma membrane integrity, acrosome integrity, and antioxidant properties.

[0055] Semen motility: Use sperm analysis system (CASA) to analyze various sperm motility indicators, including TM, PM, LIN, VAP, VSL, VCL, and BCF.

[0056] Plasma membrane integrity: The hypotonic sperm swelling method (HOST) is used to detect the plasma membrane integrity. Sperm is mixed with a hypotonic fructose sodium citrate solution (13.5 g / L fructose, 7.35 g / L sodium citrate), incubated at 37°C for 60 minutes, and observed under a phase contrast microscope at 1000 times magnification. If the sperm plasma membrane is intact, the sperm head will absorb water and swell, and the sperm tail will swell and curl.

[0057] Acrosome integrity: isothiocyanate labeled peanut agglutinin (FITC-PNA) was used to detect acrosome integrity. The FITC-PNA kit was purchased from Bio-Technology. Semen was mixed with ethD-1 and incubated at 37°C for 15 minutes. 5 μl of the mixture was pipetted onto a glass slide, dehydrated with 95% ethanol for 30 seconds, and 15 μl of FITC-PNA solution was added. The reaction was carried out at 4°C for 30 minutes, and then rinsed with PBS. Observed with a fluorescence microscope at 1000 times. The number of sperm observed was not less than 200, and at least 5 fields of view.

[0058] Antioxidant properties: ROS kit (Biyuntian Biotechnology Co., Ltd.), MDA kit, SOD kit, GSH-PX kit (Biyuntian Biotechnology Co., Ltd.)

[0059] Table 1 Effect of Tibetan pig antifreeze protein on the quality of semen cryopreservation

[0060] index 1ml 2ml 5ml 10ml Yolk Negative Survival rate (%) 60.12±0.23 68.13±0.31 72.11±0.23 77.01±0.12 73.24±0.61 33.05±0.71 vitality(%) 44.31±0.38 44.98±0.15 52.39±0.51 58.84±0.23 53.08±0.31 30.91±1.31 VSL(μm / s) 31.22±0.61 35.11±0.21 37.25±0.21 42.12±0.22 39.11±0.54 22.04±0.90 VCL(μm / s) 52.44±0.20 60.72±0.43 67.30±0.23 68.14±0.41 65.46±0.81 24.31±093 VAP(μm / s) 33.18±0.59 36.24±0.73 45.01±0.42 46.11±0.72 45.81±1.02 20.02±1.48 Plasma membrane integrity rate (%) 62.12±0.17 64.12±0.10 72.18±0.23 75.51±0.26 73.31±0.61 25.80±0.93 Acrosome integrity rate (%) 62.03±0.80 67.35±0.47 72.23±0.62 77.11±0.39 76.57±0.28 29.84±0.52

[0061] From the results, it can be seen that the effect of adding Tibetan pig antifreeze protein can significantly improve the preservation effect of frozen semen. Its semen motility, vitality, VSL, VCL, VAP, plasma membrane integrity, and acrosome integrity are significantly higher than those of the negative control group (P>0.05). In terms of semen motility, VCL, VAP, and plasma membrane integrity, it is comparable to the traditional egg yolk diluent, with no significant difference. In terms of dosage, the above-mentioned protein is dose-dependent, and the effect of high dose is significantly better than that of low dose. This may be related to the small molecular weight of the obtained polypeptide, which is difficult to achieve comprehensive protection of sperm at low concentrations, but the specific mechanism needs further study. The above results show that the addition of an appropriate amount of Tibetan pig antifreeze protein can improve the motility and vitality of Tibetan pig sperm to a certain extent, protect the integrity of sperm plasma membrane and acrosome, and reduce freezing damage. Since the sperm plasma membrane containing a large amount of unsaturated fatty acids and the sperm cytoplasm contain only a small amount of inhibitory enzymes, sperm is particularly sensitive to damage caused by ROS. Analysis showed that the above proteins increased the activities of CAT and SOD enzymes to varying degrees, reducing the oxidative damage caused by excessive ROS.

[0062] Sperm fertilization ability test: Use the method of culturing in vitro embryos to select mature oocytes and incubate them with thawed sperm. Use a stereomicroscope to observe the ratio of sperm heads entering the oocyte cytoplasm, or observe the cleavage rate after 48 hours of embryo culture. The specific operation is: before in vitro culture, add 600μl of culture solution to the four-well plate, cover it with 300μl of embryo-grade mineral oil, and put it in the incubator for at least 2 hours in advance. Wash the oocytes that have been fertilized in vitro for 24 hours with the balanced culture solution three times, then move them into the four-well plate, about 30 per well, put them into the air bag, culture them with mixed gas (5% O2+5% CO2+90% N2), and place them in a saturated humidity incubator at 38.5℃ and 5% CO2. The cleavage rate is counted at 48 hours, and the blastocyst rate is counted at 7 days.

[0063] Table 2 Effect of Tibetan pig antifreeze protein on sperm fertilization ability

[0064] index 1ml 2ml 5ml 10ml Yolk Negative Cleavage rate (%) 60.31±0.64 63.14±0.49 74.39±0.94 88.31±0.71 72.46±+0.49 39.49±1.82 Blastocyst rate (%) 39.28±0.38 47.22±0.50 52.33±0.50 56.03±0.30 48.22±0.81 12.61±1.57

[0065] It can be seen from the above results that the Tibetan pig antifreeze protein provided by the present application can effectively improve the sperm motility after freezing, effectively prolong the sperm survival time in vitro while ensuring the sperm fertilization ability, and improve the breeding efficiency of excellent boar Tibetan pigs. This experiment compared the effects of antifreeze protein added in amounts of 1ml, 2ml, 5ml, and 10ml. It can be seen that with the increase in the added concentration, the cleavage rate and blastocyst rate also increase, which are significantly better than the yolk of the control group, indicating that in the absence of exogenous protein, it helps to improve the sperm fertilization ability and can be used as a good preservation additive in the cryopreservation of Tibetan pig sperm.

[0066] In the description of this specification, the description with reference to the terms "one embodiment", "some embodiments", "example", "specific example", or "some examples" means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. Moreover, the specific features, structures, materials or characteristics described may be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art may combine and combine different embodiments or examples described in this specification and the features of different embodiments or examples, unless they are contradictory.

[0067] The above are only specific embodiments of the present invention, but the protection scope of the present invention is not limited thereto. Any person skilled in the art who is familiar with the technical field can easily think of changes or substitutions within the technical scope disclosed by the present invention, which should be included in the protection scope of the present invention. Therefore, the protection scope of the present invention should be based on the protection scope of the claims.

Claims

1. An antioxidant and antifreeze polypeptide derived from Tibetan pigs themselves, wherein the antioxidant and antifreeze polypeptide is extracted from Tibetan pig skin through the steps of defatting, hydrolysis, purification, etc., and the amino acid sequence of the protein is shown in SEQ ID No.

1.

2. The use of the antioxidant and antifreeze polypeptide according to claim 1, wherein the use is for preparing a Tibetan pig frozen semen diluent.

3. A Tibetan pig frozen semen diluent, characterized in that The Tibetan pig frozen semen diluent comprises the following components: Basic freezing solution (100ml): 3-5g trehalose, 3-5g glucose, 1-3g lactose, 0.2-0.5g vitamin C, 1.6g sodium citrate, 100IU / L (final concentration) antibacterial agent, 0.05-0.1g adenosine triphosphate, 0.02-0.04g glutathione. Solution I: Add 1-10 ml of the antioxidant antifreeze polypeptide of claim 1 (1 mg / ml, dissolved in 0.01 M PBS) to the basic freezing solution. Solution II: Add 6% glycerol to solution I.

4. The Tibetan pig frozen semen diluent as claimed in claim 3, wherein the antibacterial agent is selected from one or more of penicillin, gentamicin, amphotericin B, and penicillin-streptomycin.

5. The use of a Tibetan pig frozen semen diluent as described in claim 3 or 4 in the preservation of Tibetan pig semen, wherein the use is: using the Tibetan pig frozen semen diluent to cryopreserve semen.

6. A method for freezing and preserving ultra-high-vitality Tibetan pig semen, characterized in that The method comprises the following steps: (1) Fresh Tibetan pig semen was collected and the apparent viability, motility and density of sperm were evaluated at 37°C. The volume of each semen collection was calculated using a graduated semen collection cup. The apparent viability of fresh semen was observed under a microscope at a magnification of 100 times and was graded from 0 to 5, with grade 0 indicating no fluctuation and grade 5 indicating rapid vortex-like fluctuation. The fresh semen was evaluated at a magnification of 400 times and the semen density was calculated on a hemocytometer after the fresh semen was diluted 1000 times. Only semen with an apparent viability of grade 5, a motility greater than 0.8 and a density greater than 2.0×10 9 / mL can be used for frozen semen production; (2) Add the basic freezing solution isothermally, and the sperm density after dilution is 8×108-10×10 8 Calculate the total amount of freezing solution required based on the dilution concentration of sperm / mL; add half the required total volume of I solution to dilute the fresh semen, mix well and transfer to a refrigerator or 4°C low temperature environment, and cool to 0-4°C in a water bath for 1.5-2h to complete the first dilution; (3) Add an equal volume of Solution II to Solution I at the same temperature to complete the second dilution step and equilibrate at 4°C for 2-6 hours; (4) The diluted and balanced semen is sucked into a 0.25 mL straw at a temperature of 0-4°C, sealed with polyvinyl alcohol powder, and the first step of cooling is to quickly place the straw on a freezing rack 4-5 cm away from the surface of liquid nitrogen and fumigate for 4-5 minutes. The second step of cooling is to put the straw into liquid nitrogen for long-term storage; Among them, the basic freezing solution (100 ml): 3-5g trehalose, 3-5g glucose, 1-3g lactose, 0.2-0.5g vitamin C, 1.6g sodium citrate, 100IU / L (final concentration) antibacterial agent, 0.05-0.1g adenosine triphosphate, 0.02-0.04g glutathione. Solution I: Add 1-10 ml of the antioxidant antifreeze polypeptide of claim 1 (1 mg / ml, dissolved in 0.01 M PBS) to the basic freezing solution. Solution II: Add 6% glycerol to solution I.

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