CN115918642A - Protective agent for preserving boar semen at 4-20 ℃, preparation and preservation method and application - Google Patents
Protective agent for preserving boar semen at 4-20 ℃, preparation and preservation method and application Download PDFInfo
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- 238000004321 preservation Methods 0.000 title claims abstract description 32
- 239000003223 protective agent Substances 0.000 title claims abstract description 19
- 238000000034 method Methods 0.000 title claims abstract description 18
- 238000002360 preparation method Methods 0.000 title claims abstract description 6
- XLOMVQKBTHCTTD-UHFFFAOYSA-N Zinc monoxide Chemical compound [Zn]=O XLOMVQKBTHCTTD-UHFFFAOYSA-N 0.000 claims abstract description 37
- 239000003085 diluting agent Substances 0.000 claims abstract description 26
- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 claims abstract description 24
- 239000011787 zinc oxide Substances 0.000 claims abstract description 19
- WCUXLLCKKVVCTQ-UHFFFAOYSA-M Potassium chloride Chemical compound [Cl-].[K+] WCUXLLCKKVVCTQ-UHFFFAOYSA-M 0.000 claims abstract description 16
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- NLJMYIDDQXHKNR-UHFFFAOYSA-K sodium citrate Chemical compound O.O.[Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O NLJMYIDDQXHKNR-UHFFFAOYSA-K 0.000 claims abstract description 8
- RDEIXVOBVLKYNT-VQBXQJRRSA-N (2r,3r,4r,5r)-2-[(1s,2s,3r,4s,6r)-4,6-diamino-3-[(2r,3r,6s)-3-amino-6-(1-aminoethyl)oxan-2-yl]oxy-2-hydroxycyclohexyl]oxy-5-methyl-4-(methylamino)oxane-3,5-diol;(2r,3r,4r,5r)-2-[(1s,2s,3r,4s,6r)-4,6-diamino-3-[(2r,3r,6s)-3-amino-6-(aminomethyl)oxan-2-yl]o Chemical compound OS(O)(=O)=O.O1C[C@@](O)(C)[C@H](NC)[C@@H](O)[C@H]1O[C@@H]1[C@@H](O)[C@H](O[C@@H]2[C@@H](CC[C@@H](CN)O2)N)[C@@H](N)C[C@H]1N.O1C[C@@](O)(C)[C@H](NC)[C@@H](O)[C@H]1O[C@@H]1[C@@H](O)[C@H](O[C@@H]2[C@@H](CC[C@H](O2)C(C)N)N)[C@@H](N)C[C@H]1N.O1[C@H](C(C)NC)CC[C@@H](N)[C@H]1O[C@H]1[C@H](O)[C@@H](O[C@@H]2[C@@H]([C@@H](NC)[C@@](C)(O)CO2)O)[C@H](N)C[C@@H]1N RDEIXVOBVLKYNT-VQBXQJRRSA-N 0.000 claims abstract description 7
- UIIMBOGNXHQVGW-UHFFFAOYSA-M Sodium bicarbonate Chemical compound [Na+].OC([O-])=O UIIMBOGNXHQVGW-UHFFFAOYSA-M 0.000 claims abstract description 6
- 235000017557 sodium bicarbonate Nutrition 0.000 claims abstract description 3
- 229910000030 sodium bicarbonate Inorganic materials 0.000 claims abstract description 3
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- UIIMBOGNXHQVGW-DEQYMQKBSA-M Sodium bicarbonate-14C Chemical compound [Na+].O[14C]([O-])=O UIIMBOGNXHQVGW-DEQYMQKBSA-M 0.000 claims description 5
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- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 1
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- Agricultural Chemicals And Associated Chemicals (AREA)
Abstract
The invention relates to the field of pig semen preservation, in particular to a protective agent for preserving pig semen at 4-20 ℃, a preparation and preservation method and application. The proportion of the nano zinc oxide in the basic diluent is 1-5 mg/L; wherein the proportion of the basic diluent is as follows: glucose 4.0g, sodium citrate 0.68g, EDTA0.15g, sodium bicarbonate 0.08g, citric acid 0.023g, potassium chloride 0.07g, gentamicin sulfate 0.03g are dissolved in 0.1L double distilled water. Has the advantages that: the invention uses the nano zinc oxide as the protective agent for preserving the boar semen at 4-20 ℃ for the first time, has reliable effect and easy popularization, the preserved boar semen is suitable for artificial insemination and artificial insemination after long-distance transportation, the survival rate, the acrosome integrity rate and the plasma membrane integrity rate of the boar semen preserved at 4-20 ℃ can be obviously improved, and the fertilization activity of the sperm is maintained.
Description
Technical Field
The invention relates to the field of pig semen preservation, in particular to a protective agent for preserving pig semen at 4-20 ℃, a preparation and preservation method and application thereof.
Background
With the implementation of the strategy of sustainable development and the development of modern breeding industry, large-scale and intensive breeding becomes the whole trend of the pig industry. Under the trend, the artificial insemination technology of the pigs is particularly important. Semen preservation technology is an important component of artificial insemination technology. Semen preservation can prolong the survival time of the semen and maintain the fertilization capability of the semen so as to facilitate long-distance transportation or long-term preservation, thereby enlarging the application range of the semen, improving the utilization rate of the breeding male livestock, saving the cost and being not limited by regions and time. The preservation of the boar semen has important significance for researching boar reproductive medicine, promoting boar genetic breeding, preserving boar genetic resources and popularizing artificial insemination technology.
Methods for preserving semen can be classified into normal temperature preservation, low temperature preservation and freezing preservation. The temperature for normal temperature preservation is 15-25 ℃, the preservation by the method does not need special equipment, is simple and easy, is convenient for popularization, is suitable for short-term preservation of various livestock seminal fluids, and is particularly suitable for short-term preservation of the whole pig seminal fluid. The low-temperature preservation temperature is 0-5 ℃, the low temperature inhibits the metabolism of sperms, the sperm activity is weakened, and the sperms are in a dormant state, but in order to avoid the cold shock of the sperms, a certain amount of protective agent needs to be added into the diluent. The cryopreservation is a method for preserving semen in an ultralow temperature environment in a frozen manner for a long time by mainly using liquid nitrogen and the like as a cold source after the semen is specially treated, and can be used for preserving the semen of various livestock, but is commonly used for preserving the semen of cattle and sheep. Because the specificity of the cell structure of the pig sperms, particularly the cell membrane components, is sensitive to the temperature change, a large amount of pig sperms are damaged in the freezing process, the survival rate, the acrosome integrity rate and the plasma membrane integrity rate of the pig sperms are obviously lower than those of fresh sperms, the damage of sperm DNA, mitochondria and the like is also obviously higher than those of the fresh sperms, so that the pregnancy rate and the farrowing rate of sows are generally reduced in commercial pig raising production, and most pig farms are not provided with a freezing device, so that the artificial insemination by utilizing the frozen and stored pig sperms is difficult to popularize and apply in actual production. Therefore, the common preservation methods of the boar semen are low-temperature preservation and normal-temperature preservation.
Studies show that during the storage of pig semen at normal temperature and low temperature, pig sperm metabolism generates a large amount of Reactive Oxygen Species (ROS), and oxygen free radicals (O) carried by the ROS 2- 、H 2 O 2 OH) can cause the unsaturated fatty acid of the sperm cell membrane to generate lipid peroxidation, thereby causing the damage of the sperm cell membrane and finally leading the sperm fertilization capability of the pig to be reduced. The prior art has the following significant defects: the diluent formula in the market is independent at normal temperature and low temperature, but the constant temperature is difficult to ensure in application, particularly in the process of semen transportation.
Disclosure of Invention
The purpose of the invention is as follows: to solve the above technical problems. In the process of preserving the boar semen at normal temperature and low temperature, the antioxidant component is added into the diluent to play a role in protection. In the preservation process of the semen, the preservation temperature is not constant and generally changes at 4-20 ℃, so that the preservation effect is influenced. In order to improve the preservation efficiency of the boar semen and improve the success rate of artificial insemination of the preserved semen, it is necessary to find an antioxidant protective agent or semen diluent which is suitable for 4-20 ℃.
The nano zinc oxide is used as an antioxidant substance, and can reduce the damage of free radicals to cell membranes and cell genetic materials.
So far, no relevant report is found at home and abroad about the research of using nano zinc oxide as a protective agent for preserving the boar semen at 4-20 ℃.
In order to achieve the purpose, the invention adopts the following technical scheme:
the protective agent for preserving the boar semen at 4-20 ℃ is characterized in that the proportion of the nano zinc oxide in the basic diluent is 1-5 mg/L; wherein the proportion of the basic diluent is as follows: glucose 4.0g, sodium citrate 0.68g, EDTA0.15g, sodium bicarbonate 0.08g, citric acid 0.023g, potassium chloride 0.07g, gentamicin sulfate 0.03g are dissolved in double distilled water 0.1L.
The further technical scheme of the invention is that the solution has pH =6.30 and osmotic pressure of 360mosM.
The further technical scheme of the invention is that the preservation temperature is 4-20 ℃.
The preparation method of the protective agent for preserving the boar semen at the temperature of 4-20 ℃ is characterized by comprising the following steps:
1) Dissolving 4.0g of glucose, 0.68g of sodium citrate, 0.15g of EDTA0, 0.08g of sodium bicarbonate, 0.023g of citric acid, 0.07g of potassium chloride and 0.03g of gentamicin sulfate in 100mL of double distilled water to prepare a basic diluent;
2) Adding 0.1-0.5 mg of nano zinc oxide into the basic solution, wherein the pH value of the solution is 6.00-6.20, the osmotic pressure is 0.340-0.360 Osmol/kg, the pH value of the basic diluent is =6.30, and the osmotic pressure is 360mosM; filtering, sterilizing, cooling to room temperature, and storing in 17 deg.C refrigerator.
5. The method for preserving the boar semen at the temperature of between 4 and 20 ℃ is characterized by comprising the following steps:
1) Centrifuging fresh pig semen, discarding the supernatant, and mixing the fresh pig semen with the supernatant according to the weight ratio of the pig semen: the volume ratio of the protective agent 1 of claim 1 to 2 is that the treated boar semen is added into a pre-warmed preservation diluent, and after being wrapped by a warp cloth, the mixture is balanced in a thermostat at 17 ℃ for 1 to 2 hours;
2) Storing the semen after the balance treatment in the step 1) in a refrigerator at 4 ℃;
3) And (3) storing the semen after the balance treatment in the step 1) at room temperature.
The further technical scheme of the invention is that the room temperature is 4-20 ℃.
The application of the nano zinc oxide as a protective agent for preserving the boar semen at 4-20 ℃.
Compared with the prior art, the invention adopting the technical scheme has the following beneficial effects: the invention uses the nano zinc oxide as the protective agent for preserving the boar semen at 4-20 ℃ for the first time, has reliable effect and easy popularization, the preserved boar semen is suitable for artificial insemination and insemination after long-distance transportation, the survival rate, the acrosome integrity rate and the plasma membrane integrity rate of the boar semen preserved at 4-20 ℃ can be obviously improved, and the fertilization activity of the sperm is maintained.
Detailed Description
The present invention is further described below with reference to specific examples so that those skilled in the art can better understand the present invention and can practice the present invention, but the examples are not intended to limit the present invention.
Example 1
Accurately measuring 4.0g of glucose, 0.68g of sodium citrate, 0.15g of EDTA0, 0.08g of sodium bicarbonate, 0.023g of citric acid, 0.07g of potassium chloride and 0.03g of gentamicin sulfate, dissolving in 100mL of double distilled water, and preparing into a basic diluent; accurately measuring 0.1mg of nano zinc oxide, and uniformly mixing with a basic diluent to prepare a diluent; filtering, sterilizing, cooling to room temperature, and placing into a 17 deg.C refrigerator.
Example 2
Accurately measuring 4.0g of glucose, 0.68g of sodium citrate, 0.15g of EDTA0, 0.08g of sodium bicarbonate, 0.023g of citric acid and 0.07g of potassium chloride, and dissolving 0.03g of gentamicin sulfate in 100mL of double distilled water to prepare a basic diluent; accurately measuring 0.5mg of nano zinc oxide, and uniformly mixing with the basic diluent to prepare a diluent; filtering, sterilizing, cooling to room temperature, and placing in a 17 deg.C refrigerator for use.
In the comparative example, 4.0g of glucose, 0.68g of sodium citrate, 0.15g of EDTA0, 0.08g of sodium bicarbonate, 0.023g of citric acid and 0.07g of potassium chloride are accurately measured and dissolved in 100mL of double distilled water to prepare a basic diluent, the pH value of the solution is adjusted to be 6.00-6.20, the solution is filtered, sterilized, cooled to room temperature and placed into a 17 ℃ refrigerator for later use.
The invention is described below with respect to the steps of cryopreservation of porcine semen and the method of sperm quality assessment.
Semen collection
Semen is collected by hand method, sperm-rich segment semen is selected, jelly is removed by filtering through 4 layers of sterile gauze, and after dilution by the basic diluent 1, routine quality inspection is carried out by a microscope at 37 ℃. Semen with no foreign odor, milky white color, normal sperm morphology, a survival rate of above 0.8, and a dense density is selected for the test.
(II) semen treatment and preservation
Centrifuging fresh semen (800 Xg, 10 min), removing supernatant, adding pre-warmed preservation diluent according to the proportion of the semen to the diluent of 1. And preserving the semen subjected to the balanced treatment at 4-20 ℃.
(III) evaluation of sperm quality after preservation
1. Motility rate of sperm
Incubating 10mL semen sample at 37 deg.C for 30min, mixing, dripping 10 μ L semen sample on glass slide for microscopic examination, and detecting sperm motility rate by computer-assisted semen analysis system (CASA). At each time, 3 fields were randomly selected for measurement, each field required a sperm count of greater than 200, and the results were averaged.
2. Sperm acrosome integrity
And (3) uniformly mixing semen samples, putting 50 mu L into a centrifuge tube, adding 1mL of 4% paraformaldehyde, uniformly mixing, fixing for 10min, centrifuging for 5min by a centrifuge (1500 r/min), discarding the supernatant, preparing 10 mu L of lower-layer semen into smears, naturally air-drying for 5min, dyeing for 30min by using Coomassie brilliant blue dye solution, washing off the redundant dye solution by water, performing microscopic examination after air drying, randomly recording at least 200 sperms in different visual fields, counting the number of the contained sperms with complete acrosomes, and calculating the completeness of the acrosomes.
3. Integrity rate of sperm plasma membrane
Sperm plasma membrane integrity was tested using the hypotonic tolerance test (HOST). If the plasma membrane of the sperm is structurally intact, the plasma membrane may swell in a hypotonic environment to allow the sperm tails to bend and form a ring, which is not the case with sperm with an incomplete plasma membrane. Taking 10mL semen samples to incubate for 30min at 37 ℃, uniformly mixing, taking 10 mu L, adding 100 mu L hypotonic buffer solution, and balancing for 30min in 37 ℃ constant temperature water bath. After mixing, 10 mu L of the mixture is dripped on a glass slide for microscopic examination, at least 200 sperms with different visual fields are randomly recorded, the number of sperms with bent tails is counted, and the plasma membrane integrity rate is calculated.
4. Rate of sperm teratogenesis
Incubating 10mL semen sample at 37 deg.C for 30min, mixing, dripping 10 μ L semen onto glass slide, making smear by pulling the sample droplet, naturally air drying for 5min, dyeing with 0.5% gentian violet alcohol solution or blue ink for 5min, naturally air drying for 10min, washing with water to remove excess dye solution, air drying, and performing microscopic examination. Randomly recording at least 200 sperms in different visual fields, counting the number of teratospermia contained in the sperms, and calculating the teratogenesis rate.
(IV) sperm quality assessment results
By applying the protective agent for preserving the boar semen at the temperature of 4-20 ℃ and the method for preserving the boar semen at the temperature of 4-20 ℃, the evaluation result is as follows:
when the addition of the nano zinc oxide is 0.1-0.3mg, the sperm motility rate reaches 75%, the acrosome integrity rate reaches 78%, and the plasma membrane integrity rate reaches 69% after the nano zinc oxide is stored for 6 days at 4-20 ℃; when the adding amount of the nano zinc oxide is 0.3-0.5mg, the sperm motility rate reaches 75%, the acrosome integrity rate reaches 79%, and the plasma membrane integrity rate reaches 68% after the nano zinc oxide is stored at 4-20 ℃.
Compared with common zinc oxide, the nano material has more advantages.
The results of the test examples are shown in the following table:
TABLE 1 influence of different concentrations of nano-zinc oxide on the preservation effect of pig sperm at 4-20 deg.C
| Treatment of | Motility rate of sperm | Percentage of acrosomal integrity | Plasma membrane integrity rate | Rate of deformity |
| Control group | 64％ | 65％ | 64％ | 11％ |
| Example 1 | 75％ | 78％ | 69％ | 7％ |
| Example 2 | 75％ | 79％ | 68％ | 6％ |
The foregoing shows and describes the general principles, principal features, and advantages of the invention. It will be understood by those skilled in the art that the present invention is not limited to the embodiments described above, which are intended to illustrate the principles of the invention, but that various changes and modifications may be made without departing from the spirit and scope of the invention, and the invention is to be limited to the embodiments described above.
Claims (6)
1. The protective agent for preserving the boar semen at 4-20 ℃ is characterized in that the proportion of the nano zinc oxide in the basic diluent is 1-5 mg/L; wherein the basic diluent comprises the following components in percentage by weight: glucose 4.0g, sodium citrate 0.68g, EDTA0.15g, sodium bicarbonate 0.08g, citric acid 0.023g, potassium chloride 0.07g, gentamicin sulfate 0.03g are dissolved in double distilled water 0.1L.
2. The protective agent for preserving porcine semen at 4-20 ℃ according to claim 1, wherein the basic diluent has a pH =6.30 and an osmotic pressure of 360mosM.
3. The preparation method of the protective agent for preserving the boar semen at 4-20 ℃ as claimed in claim 1 is characterized by comprising the following steps:
1) Dissolving 4.0g of glucose, 0.68g of sodium citrate, 0.15g of EDTA0, 0.08g of sodium bicarbonate, 0.023g of citric acid, 0.07g of potassium chloride and 0.03g of gentamicin sulfate in 100mL of double distilled water to prepare a basic diluent;
2) Adding 0.1-0.5 mg of nano zinc oxide into the basic liquid, wherein the pH of the solution is =6.30, the osmotic pressure is 360mosM, filtering, sterilizing, cooling to room temperature, and storing in a refrigerator at 17 ℃.
4. The method for preserving the boar semen at the temperature of 4-20 ℃ is characterized by comprising the following steps:
1) Centrifuging fresh pig semen, discarding the supernatant, and mixing the fresh pig semen with the supernatant according to the weight ratio of the pig semen: the volume ratio of the protective agent 1 of claim 1 to 2 is that the treated boar semen is added into a pre-warmed preservation diluent, and after being wrapped by a warp cloth, the mixture is balanced in a thermostat at 17 ℃ for 1 to 2 hours;
2) Storing the semen after the balance treatment in the step 1) in a refrigerator at 4 ℃;
3) And (2) storing the semen after the balance treatment in the step 1) at room temperature.
5. The method for preserving porcine semen at 4-20 ℃ as claimed in claim 4, wherein the room temperature is 4-20 ℃.
6. The application of the nano zinc oxide as a protective agent for preserving the boar semen at 4-20 ℃.
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| CN202211549735.0A CN115918642B (en) | 2022-12-05 | 2022-12-05 | Protective agent for preserving pig semen at 4-20 ℃, and preparation and preservation methods and applications thereof |
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| CN101301402A (en) * | 2008-07-03 | 2008-11-12 | 刘庆华 | Nutrition health care replenisher for reinforcing reproduction performance of boar |
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