An antioxidant and antifreeze protein derived from Tibetan pigs and its application in cryopreservation semen diluent

Abstract

The present invention provides an antioxidant and antifreeze protein derived from Tibetan pigs. Adding the above protein to the cryopreservation solution can significantly increase the activities of peroxidase, catalase, superoxide dismutase, and glutathione peroxidase in Tibetan pig sperm after cryopreservation and thawing, significantly enhance the total antioxidant capacity of sperm, and significantly reduce the levels of intracellular reactive oxygen species and malondialdehyde content in sperm. The cryoprotectant provided by the present invention can greatly improve the survival rate and motility of thawed sperm and ensure the stability of the survival rate and motility of sperm after thawing; it improves the pregnancy rate of artificial insemination with cryopreserved semen of Tibetan pigs and greatly improves the quality of cryopreserved semen. Moreover, since no exogenous antifreeze protein needs to be added to the preservation solution, while ensuring the essential nutrients for sperm metabolism, it reduces the damage caused by exogenous substances to sperm and the risk of contamination.

Description

Technical Field

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

[0002] Tibetan pigs are primarily distributed in the middle reaches of the Yarlung Zangbo River valley and the high mountain valleys of the Three Rivers Basin in eastern Tibet. They are a uniquely Chinese breed that can adapt to high altitudes and are known as the "treasure of the plateau." Artificial insemination is essential for the rapid development of the Tibetan pig industry. Currently, the primary challenge facing core farms in the Tibetan pig farming industry is the inefficient utilization of boar semen. Most farms use semen collected on the same day or stored at 17°C for one to two days. Market share of medium-, medium-, and long-acting semen diluents is largely monopolized by developed pig-raising countries, resulting in high prices. This places significant financial pressure on the pig farming industry and increases production costs. Previous studies have shown that even in cases of same-day collection and insemination, storage of boar semen at 18°C ​​can induce lipid peroxidation, reduce mitochondrial membrane potential, and increase plasma membrane permeability. During the low-temperature storage of semen, pig sperm has a worse tolerance to low temperatures than other livestock and is more prone to cold shock. The sperm is in a state of oxidative stress, producing a large amount of reactive oxygen species (ROS), which damages the structure and function of the sperm and even induces its apoptosis.

[0003] Antifreeze proteins (AFPs) are a class of polypeptides or glycopeptides produced by organisms to adapt to extreme cold environments. They lower the freezing point of solutions while having minimal effect on their melting point. AFPs were first discovered in polar fish. To date, a variety of AFPs have been isolated from organisms including marine fish, terrestrial insects, plants, bacteria, and fungi. Although their structures vary, they all share the characteristics of preventing ice crystal formation, regulating the growth of extracellular ice crystals, inhibiting ice recrystallization, and maintaining a non-freezing state in liquids. While many researchers have studied AFPs in various animals in recent years, including the discovery that collagen from chicken, fish, and pig skin exhibits significant antifreeze activity, little research has been conducted on AFPs derived from Tibetan pigs. Furthermore, AFPs from these other animal sources can induce alloimmunization in insemination sows, leading to fertilization failure, making them unsuitable for direct application in Tibetan pig semen storage. Consequently, no truly applicable research results have been achieved, nor has their application in 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 defatting, hydrolysis, purification and other steps. 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 substituting, 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 antifreeze protein, which is used for preparing a diluent for frozen Tibetan pig semen.

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

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

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

[0015] The present invention is implemented by adopting the following technical solution: 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 motility, viability and density of sperm were assessed at 37°C. The volume of semen collected each time was calculated using a graduated semen collection cup. The apparent motility of fresh semen was observed under a microscope at 100x magnification and was graded from 0 to 5, with grade 0 indicating no fluctuation and grade 5 indicating rapid vortex-like fluctuation. The motility of fresh semen was assessed under a 400x magnification and the semen density was calculated on a hemocytometer after the semen was diluted 1000 times. Only semen with grade 5 motility, motility greater than 0.8 and density greater than 2.0×10 9 / mL can be used for frozen semen production;

[0017] (2) Add the basic diluent 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 100 ml / mL. Add half the required volume of Solution I to dilute the fresh semen. Mix thoroughly, transfer to a refrigerator or 4°C environment, and cool in a water bath to 0-4°C for 1.5-2 hours 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, and the cap was sealed with polyvinyl alcohol powder. The first step was to cool the semen and quickly remove the capillary tube. Tube placement Place the tube on a freezing rack 4-5 cm away from the surface of liquid nitrogen for 4-5 minutes. In the second step of cooling, place the tube into liquid nitrogen for long-term storage.

[0020] Beneficial effects

[0021] The invention extracts an antioxidant and antifreeze protein derived from Tibetan pigs themselves through the steps of defatting, hydrolyzing and purifying the pigskin. Adding the protein to a freezing preservation solution can significantly improve the activities of peroxidase, catalase, superoxide dismutase and glutathione peroxidase in 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 level of active oxygen and malondialdehyde content in sperm cells (P<0.05). At the same time, the semen preservation solution described in the present invention effectively removes "aging" sperm, weak and dead sperm, and non-essential and harmful substances in semen, thereby obtaining highly motile sperm. The motility of semen before freezing is above 0.8, and the motility after thawing is 0.69-0.74, fully meeting the requirements for artificial insemination of frozen semen from Tibetan pigs. This significantly improves the motility and vitality of sperm after thawing and ensures the stability of these motility and vitality after thawing. It also increases the pregnancy rate in artificial insemination of frozen semen from Tibetan pigs and significantly enhances the quality of frozen semen. Furthermore, since the preservation solution does not require the addition of exogenous antifreeze proteins, it ensures essential nutrients for sperm metabolism while reducing the risk of damage and contamination caused by exogenous substances. The antifreeze proteins and freezing reagents of the present invention address the problems of low motility and vitality of existing Tibetan pig frozen semen after thawing and unstable freezing results, and are therefore widely applicable to mass production of Tibetan pigs. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0023] The technical solutions of the present invention are described clearly and completely below. It is obvious that the embodiments described are only some embodiments of the present invention, not all embodiments. All other embodiments obtained by persons of ordinary skill in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention. The present invention is further described below with reference to the embodiments.

[0024] Experimental Animals and Location: Six healthy boars aged 3 to 5 years were used for the experiment. The experiment took place from May to July 2024 at the College of Animal Science, Tibet Agricultural and Animal Husbandry University.

[0025] Semen collection: Tibetan pig semen used in this experiment was obtained from the Tibet Agricultural and Animal Husbandry College practice base. Healthy Tibetan pig semen was collected by hand-holding. The mid-section of the semen was filtered through three layers of gauze. Semen with a motility of over 80% was isothermally diluted in a 1:1 ratio with sperm washing solution (30 g Tris, 10 g citric acid, 12 g glucose, and 6 mM cysteine ​​per 1 L) and placed in a thermos bottle for immediate transport back to the laboratory. The collected semen was stored at -20°C until ready for use.

[0026] Example 1 Preparation of Tibetan pig antifreeze protein

[0027] The Tibetan pig skin raw material was cut into pieces (0.2 cm × 0.2 cm), degreased with 10 times the volume of 10% butanol solution for 24 h, and the butanol solution was changed every 12 h, and then washed with distilled water; the defatted pig skin was soaked in 0.5 mol / L acetic acid solution for 12 h, and homogenized with 10 times the volume of 0.5 mol / L acetic acid solution. 1 g / 100 mL pepsin was added to the homogenate and hydrolyzed for 48 h. The mixture was centrifuged (5000 × g, 40 min) and the supernatant was collected. ; 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 (pH 8.6), 0.1 mol / L acetic acid, and distilled water as the dialysis external fluid in sequence, changing 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 protective activity against 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 a pH 7.0, 0.05 mol / L KH2PO4-NaOH buffer to prepare a 0.5 mg / mL catalase solution. The catalase solution and 2 mg / mL crude antifreeze peptide extract were mixed in equal volumes, and the initial enzyme activity before freeze-thaw was measured. The mixture of catalase and crude antifreeze peptide extract was frozen at -33°C for 24 hours, then thawed at 25°C and frozen again at -33°C for 6 hours; this procedure was repeated four times. The catalase activity was then measured after repeated freeze-thaw cycles. In a control group, the crude antifreeze peptide extract was replaced with buffer, and the catalase activity after repeated freeze-thaw cycles was measured.

[0031] The method for determining catalase activity 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, place in the sample cell of the spectrophotometer, and record the initial absorbance value. Record the absorbance value every 1 minute, and time for 5 minutes. Calculate the experimental results: a decrease 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, where A0 refers to the absorbance of the control tube with inactivated enzyme; 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 measurement, mL; t refers to the time from the addition of hydrogen peroxide to the last recording, min; and W refers to the mass of the enzyme in the sample solution, g.

[0033] The ratio of enzyme activity after freeze-thaw to enzyme activity before freeze-thaw (residual catalase activity) was used to indicate antifreeze activity. The greater the residual enzyme activity, the greater the antifreeze activity of the sample being tested. The results showed that the ZZ peak had the best effect, with a residual catalase activity of 80.34% of the initial enzyme activity.

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

[0035] E. coli primary culture was cultured at 37°C to an OD600 of approximately 0.8. The culture was then diluted 105-fold with culture medium. 100 μL of the diluted culture was transferred to an Eppendorf centrifuge tube, and 900 μL of a 1 mg / mL crude antifreeze peptide extract was added. After vortexing, 100 μL of the mixture was plated onto LB medium and incubated upside down at 37°C for 20 hours before counting. The remaining culture was refrigerated at -33°C for 24 hours, then thawed. 100 μL of the thawed culture was plated onto LB medium and incubated upside down at 37°C for 20 hours before counting. The culture medium was used instead of the crude antifreeze peptide extract to determine the number of surviving E. coli in the control group. A blank control experiment was performed using sterile water.

[0036] The survival rate of E. coli is expressed as the ratio of the number of E. coli cells after freeze-thaw to the number of E. coli cells before freeze-thaw. A higher survival rate indicates a higher cryoprotective activity against E. coli. The results also showed that the ZZ peak was the most effective, 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 point is called the retention temperature (Th). Cool it to -25°C at 1°C / min, and record the temperature at which the sample begins to crystallize at the retention temperature (T0). 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 isolated from the screening was further separated using a Sulfopropyl-Sepadex C-25 cation exchange chromatography column (55 cm long, 2.0 cm diameter). The eluent consisted of a 0-0.5 M NaCl gradient in 0.01 mol / L pH 7.0 phosphate buffer at a flow rate of 0.5 mL / min. Peak samples were collected and re-assayed for antifreeze activity. The sample with the highest antifreeze activity was further analyzed and fractionated using UPLC-MS. The chromatographic experimental parameters used are as follows: the chromatographic column is a Waters Acquity UPLC BEH C18 column (50×2.1 mm, 1.7 μm); the mobile phase A is deionized water (containing 0.1% formic acid), and the mobile phase B is acetonitrile; the (B) phase elution program is: 0-40% from 0 to 40 min, and 40-0% from 40 to 42 min; the elution flow rate is 0.3 mL / min; and the sample load is 5 μL.

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

[0043] The resulting single-stage component, XZ, exhibited the best antifreeze activity. Its amino acid sequence: His-Gly-Glu-Glu-Gly-Ala-Ser-Gly-Val-Arg-Gly-Ala-Phe (SEQ ID NO. 1). The residual catalase activity was 85.12% of the initial activity. The E. coli survival rate was 75.30%, and the thermal hysteresis activity reached 4.7°C, demonstrating promising 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, including 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, and set up a concentration gradient of 1 ml, 2 ml, 5 ml, and 10 ml. At the same time, set up a positive control with 5 g of egg yolk added alone and a negative control without any antifreeze added.

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

[0051] (3) Add the basic diluent 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 100 ml / mL. Add half the required volume of Solution I to dilute the fresh semen. Mix thoroughly, transfer to a refrigerator or 4°C environment, and cool in a water bath to 0-4°C for 1.5-2 hours 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, and the cap was sealed with polyvinyl alcohol powder. The first step was to cool the semen and quickly remove the capillary tube. Tube placement Place the tube on a freezing rack 4-5 cm away from the surface of liquid nitrogen for 4-5 minutes. In the second step of cooling, place the tube into liquid nitrogen for long-term storage.

[0054] (6) Liquid nitrogen freezing effect test. After removing the frozen sperm tube from liquid nitrogen with tweezers, expose it to air for 1-2 seconds, place it in a constant temperature water bath at 38°C for 30 seconds, and incubate it at 37°C for 5 minutes. Then test the semen motility, plasma membrane integrity, acrosome integrity, and antioxidant properties.

[0055] Semen motility: Use the 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 welling method (HOST) is used to assess plasma membrane integrity. Sperm is mixed with a hypotonic fructose sodium citrate solution (13.5 g / L fructose, 7.35 g / L sodium citrate) and incubated at 37°C for 60 minutes. After incubation, the sperm is observed under a phase contrast microscope at 1000x 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: FITC-PNA (Peanut lectin labeled with isothiocyanate) was used to assess acrosome integrity. The FITC-PNA kit was purchased from Beyotime Biotechnology. 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 and dehydrated with 95% ethanol for 30 seconds. 15 μL of FITC-PNA solution was then added dropwise. The mixture was incubated at 4°C for 30 minutes, followed by rinsing with PBS. Observation was performed using a fluorescence microscope at 1000x magnification. The number of spermatozoa observed was no less than 200, and at least five fields of view were included.

[0058] Antioxidant properties: ROS kit (Beyotime Biotechnology Co., Ltd.), MDA kit, SOD kit, GSH-PX kit (Beyotime 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] The results indicate that the addition of Tibetan pig antifreeze protein significantly improves the preservation of frozen semen. Semen motility, vitality, VSL, VCL, VAP, plasma membrane integrity, and acrosome integrity were significantly higher than those in the negative control group (P>0.05). Motility, VCL, VAP, and plasma membrane integrity were comparable to those of traditional egg yolk diluent, with no significant differences. Furthermore, the protein exhibited a dose-dependent effect, with higher doses significantly more effective than lower doses. This may be due to the small molecular weight of the peptide, which makes it difficult to fully protect sperm at low concentrations. However, the specific mechanism requires further investigation. These results suggest that the addition of appropriate amounts of Tibetan pig antifreeze protein can improve sperm motility and vitality, protect the integrity of the sperm plasma membrane and acrosome, and reduce freezing damage. Because the sperm plasma membrane, which contains a high concentration of unsaturated fatty acids, and the sperm cytoplasm, contains only a small amount of inhibitory enzymes, sperm are particularly sensitive to damage caused by reactive oxygen species (ROS). Analysis showed that the above proteins may increase the activities of CAT and SOD enzymes to varying degrees, thereby reducing the oxidative damage caused by excessive ROS.

[0062] Sperm fertilization ability test: Using the method of culturing in vitro embryos, select mature oocytes and incubate them with thawed sperm. Use a stereomicroscope to observe the ratio of the sperm head 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 medium to a four-well plate, cover it with 300μl of embryo-grade mineral oil, and place it in an incubator for at least 2 hours in advance. Wash the oocytes 24 hours after in vitro fertilization with the equilibrated culture medium three times, then transfer them to a four-well plate, about 30 per well, put them in an air bag, culture them with a mixed gas (5% O2+5% CO2+90% N2), and place them in a saturated humidity incubator at 38.5°C and 5% CO2. The cleavage rate is calculated at 48 hours, and the blastocyst rate is calculated at 7 days.

[0063] Table 2 Effects 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] The above results demonstrate that the Tibetan pig antifreeze protein provided in this application can effectively enhance sperm motility after freezing, effectively prolonging sperm survival in vitro while ensuring sperm fertilization ability, and improving the breeding efficiency of high-quality boar Tibetan pigs. This experiment compared the effects of antifreeze protein added at 1ml, 2ml, 5ml, and 10ml doses. It can be seen that with increasing concentrations, the cleavage rate and blastocyst rate also increased, both significantly better than the egg yolk in the control group. This demonstrates that, in the absence of exogenous protein, it helps enhance sperm fertilization ability and can serve as a good preservation additive for Tibetan pig sperm cryopreservation.

[0066] In the description of this specification, reference to the terms "one embodiment," "some embodiments," "examples," "specific examples," 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 suitable manner in any one or more embodiments or examples. In addition, those skilled in the art may combine and integrate different embodiments or examples described in this specification, as well as features of different embodiments or examples, unless otherwise inconsistent.

[0067] The above are merely specific embodiments of the present invention, but the scope of protection of the present invention is not limited thereto. Any modifications or substitutions that can be easily conceived by a person skilled in the art within the technical scope disclosed in the present invention should be included within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be based on the scope of protection of the claims.

Claims

1. An antioxidant and cryoprotective polypeptide derived from Tibetan pigs themselves, which is extracted from Tibetan pig skin through degreasing, hydrolysis, and purification steps, and the amino acid sequence of the antioxidant and cryoprotective polypeptide is shown in SEQ ID No.

1.

2. Use of the antioxidant and cryoprotective polypeptide according to claim 1, wherein the use is to prepare a cryopreservation diluent for Tibetan pig semen.

3. A cryopreservation semen diluent for Tibetan pigs, characterized in that The cryopreservation diluent for Tibetan pig semen comprises the following components: basic cryopreservation solution, calculated on a basis of 100 ml: 3 - 5 g of trehalose, 3 - 5 g of glucose, 1 - 3 g of lactose, 0.2 - 0.5 g of vitamin C, 1.6 g of sodium citrate, an antibacterial agent with a final concentration of 100 IU / L in the basic cryopreservation solution, 0.05 - 0.1 g of adenosine triphosphate, 0.02 - 0.04 g of glutathione; Solution I: Add 1 - 10 ml of the antioxidant and cryoprotective polypeptide solution according to claim 1 to the basic cryopreservation solution. The antioxidant and cryoprotective polypeptide solution is the antioxidant and cryoprotective polypeptide dissolved in 0.01 M PBS solution at a concentration of 1 mg / ml. Solution II: Add glycerol accounting for 6% of the total volume of Solution I to Solution I.

4. The cryopreservation diluent for Tibetan pig semen according to claim 3, wherein the antibacterial agent is selected from one or more of penicillin, gentamicin, amphotericin B, and penicillin - streptomycin.

5. Use of a cryopreservation diluent for Tibetan pig semen according to claim 3 or 4 in the preservation of Tibetan pig semen, wherein the use is: performing cryopreservation of semen using the cryopreservation diluent for Tibetan pig semen.

6. A method for cryopreserving semen of Tibetan pigs, characterized in that The method comprises the following steps: (1) Collect fresh semen from Tibetan pigs, and conduct quality assessment on the apparent motility, motility and density of sperm at 37 °C: Use a graduated semen collection cup to calculate the volume of each semen collection; Observe the apparent motility of fresh semen under a 100-fold field of view of a microscope, which is divided into levels 0-5, level 0 means no fluctuation, and level 5 means rapid vortex-like fluctuation; Assess the motility of fresh semen under a 400-fold field of view, dilute the fresh semen 1000 times and calculate the semen density on a hemocytometer; Only when the apparent motility of the semen is level 5, the motility is greater than 0.8 and the density is greater than 2.0×10 9 cells / mL can it be used for the production of frozen semen; (2) Add the basic cryoprotectant isothermally. Calculate the total amount of cryoprotectant required according to the dilution concentration of sperm density of 8×10 8 -10×10 8 per mL; Add half of the required total volume of Solution I to dilute the fresh semen, mix well and transfer it to the refrigerator freezer or a low-temperature environment of 4°C, and cool it to 0-4°C in a water bath for 1.5-2 hours to complete the first dilution; (3) Add Solution II with the same volume as Solution I at the same temperature to complete the second - step dilution, and equilibrate at 4°C for 2 - 6 h; (4) Aspirate the diluted and equilibrated semen into a 0.25 mL straw at a temperature of 0 - 4°C, seal it with polyvinyl alcohol powder, and for the first - step cooling, quickly place the straw on a freezing rack 4 - 5 cm above the liquid nitrogen surface and fumigate for 4 - 5 min, and for the second - step cooling, put the straw into liquid nitrogen for long - term preservation; Among them, the basic cryopreservation solution, calculated on a basis of 100 ml: 3 - 5 g of trehalose, 3 - 5 g of glucose, 1 - 3 g of lactose, 0.2 - 0.5 g of vitamin C, 1.6 g of sodium citrate, an antibacterial agent with a final concentration of 100 IU / L in the basic cryopreservation solution, 0.05 - 0.1 g of adenosine triphosphate, 0.02 - 0.04 g of glutathione; Solution I: Add 1 - 10 ml of the antioxidant and cryoprotective polypeptide solution according to claim 1 to the basic cryopreservation solution. The antioxidant and cryoprotective polypeptide solution is the antioxidant and cryoprotective polypeptide dissolved in 0.01 M PBS solution at a concentration of 1 mg / ml. Solution II: Add glycerol accounting for 6% of the total volume of Solution I to Solution I.

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