A kind of semen protection liquid and its preparation method and application

Abstract

The present invention discloses a semen protection liquid for improving the quality of semen after freezing, and a preparation method and application thereof. The semen protection liquid contains active ingredients, and the active ingredients are made of rhodiola rosea polysaccharide and astragalus polysaccharide. The semen protection liquid of the present invention has the effects of anti-oxidation, improving the survival rate of frozen sperm, etc., can be effectively used for the protection of semen freezing, can effectively improve the plasma membrane integrity rate, sperm motility, DNA integrity rate, acrosome integrity rate, etc. of sperm after thawing, and improve the quality of semen after freezing. It has many advantages such as wide source of raw materials, safety, environmental protection, simple preparation process, low production cost, etc.

Description

Technical Field

[0001] The invention belongs to the field of animal genetic breeding, and particularly relates to a semen protection liquid for improving the quality of semen after freezing, and a preparation method and application thereof. Background Art

[0002] With the development of artificial insemination technology, semen cryopreservation technology is one of the important biotechnological means of mammalian breeding. It can effectively reduce the cost of breeding, make the use of semen not limited by time, place and user, and significantly increase the use time and utilization rate of semen of excellent breeding animals. It includes a series of complex processes such as sampling, dilution, cooling, and freezing. However, due to the difficulties in the production of frozen semen, the low survival rate and conception rate after freezing-thawing, etc., the semen freezing technology is still not fully mature. In the process of freezing-thawing, semen will be damaged to a certain extent, and the damage mechanism can be divided into physical damage and chemical damage. Physical damage is the mechanical damage of ice crystals produced by freezing to the cell membrane and internal structure of the cell. A series of damages caused by the solution effect caused by ice crystallization are chemical damage: the redox balance in sperm cells is broken, and the reactive oxygen species (ROS) produced by normal physiological metabolism accumulate in large quantities, which cannot be neutralized by reaction. It is easy to react with phospholipids on the sperm cell membrane, and it will also reduce the activity of enzymes in the cell, reduce motility, and even damage DNA in the cell nucleus, thereby reducing the fertilization ability of sperm.

[0003] The purpose of sperm protection fluid is to reduce ice crystals and anti-oxidation. In recent years, the existing technology has made detailed studies on the external conditions that affect the quality of frozen sperm. The external conditions can be controlled to be kept within an appropriate range during the freezing process to minimize the impact of external conditions. Regarding the study of internal damage, the products currently available on the market are mainly based on the addition of osmotic protective agents such as egg yolk, but there are concerns such as complex animal-derived ingredients and low safety. Polysaccharides have the characteristics of antioxidant, anti-aging and anti-apoptosis, which inhibit the production of reactive oxygen, increase the activity of peroxidase, protect the cytoplasmic membrane from lipid peroxidation damage, and prevent the outflow of intracellular enzymes. The use of Chinese medicine compositions can not only effectively improve the quality of sperm after freezing, but also effectively ensure the safety of sperm and improve sperm utilization. Compared with animal-derived osmotic protective agents, traditional natural plant Chinese medicine compositions have the advantages of high safety, relatively low toxicity and less residue. Summary of the invention

[0004] In order to solve the above technical problems, the present invention provides a semen protection solution for improving the quality of semen after freezing. The specific scheme is as follows:

[0005] The semen protection solution is prepared from semen storage solution and a traditional Chinese medicine composition. The semen storage solution is prepared from glucose, citric acid, tris(hydroxymethyl)aminomethane), penicillin and streptomycin. The traditional Chinese medicine composition is prepared from rhodiola rosea polysaccharide and astragalus polysaccharide.

[0006] Furthermore, the semen storage solution contains 15-17 g of glucose, 19-21 g of citric acid, 34-36 g of tris (hydroxymethyl)aminomethane (Tris), 1×10 6 IU.

[0007] Furthermore, the semen storage solution contains 16 g of glucose, 20 g of citric acid, 35 g of tris (hydroxymethyl)aminomethane (Tris), 1×10 6 IU.

[0008] Furthermore, the Chinese medicine composition contains 0.9-1.1 g of Rhodiola rosea polysaccharide and 0.5-0.7 g of Astragalus polysaccharide in every 1 L of double distilled water.

[0009] Furthermore, the Chinese medicine composition contains 1.0 g of Rhodiola rosea polysaccharide and 0.6 g of Astragalus polysaccharide in every 1 L of double distilled water.

[0010] The present invention also provides a method for preserving animal semen using the semen protection liquid, which comprises the following steps:

[0011] (1) Dilute the collected qualified sperm with semen storage solution, balance at 25°C for 1 hour, and balance at 15°C for 1 hour, mixing at irregular intervals;

[0012] (2) Centrifuge the semen, discard the supernatant, add the semen protection solution, and adjust the final sperm concentration to 3.2-4.8×10 8 / mL, and then resuspended and equilibrated at 5℃ for 1h;

[0013] (3) The mixed semen is filled into a straw tube, which is sealed. The straw tube is then cooled to -6°C and placed 3 cm above liquid nitrogen for fumigation for 3 min. Finally, it is placed in liquid nitrogen for storage.

[0014] In the above method, the final sperm concentration in step (2) is preferably adjusted to 4×10 8 Pieces / mL.

[0015] The present invention also provides a method for preparing the semen protection solution, which specifically comprises weighing glucose, citric acid, tris(hydroxymethylaminomethane), penicillin and streptomycin respectively, dissolving them in water to prepare semen storage solution, and adding rhodiola rosea polysaccharide and astragalus polysaccharide into the semen storage solution in proportion to prepare the semen protection solution.

[0016] The present invention also provides an application of the semen protection liquid in the cryopreservation of animal semen, specifically using the semen protection liquid for cryopreservation of animal semen. For example, after the semen is mixed with the semen protection liquid, it is placed in a storage container, and then placed in a sealed container for sealed storage.

[0017] Furthermore, the animal semen used in the above application is preferably semen from livestock such as pigs, cattle, sheep, and horses.

[0018] Those skilled in the art will appreciate that the water in the semen protection solution of the present invention is preferably ultrapure water.

[0019] Those skilled in the art will appreciate that the storage container used in the storage method described in the present invention may be any container that can be used to store semen, such as a 0.2 ml storage tube, a 0.5 ml cryopreservation tube, etc.

[0020] Furthermore, the semen preservation solution can be stored in the form of: ampoules, granules, aluminum foil (plastic) bags and thin (wheat) tubes.

[0021] The advantages and beneficial effects of the medicine of the present invention are:

[0022] The medicine of the present invention is healthy and non-toxic, and does not cause harm to sperm itself; the raw materials of the medicine of the present invention have a wide range of sources and are low in cost, do not cause environmental pollution, and have no drug residues that endanger the health of animal embryos; the semen protection liquid provided by the present invention reduces the damage of centrifugation to sperm activity, improves the centrifugation effect, and avoids the loss of active ingredients in semen.

[0023] The drug of the present invention expands the use of raw materials for semen preservation fluid. Compared with the prior art, most of the Chinese medicinal raw materials used in the present invention are relatively low in price. While greatly reducing the cost, the technical solution of the present invention is also significantly better than the prior art in improving the quality of semen after freezing. DETAILED DESCRIPTION

[0024] The embodiments of the present invention will be described in detail below in conjunction with the examples, but those skilled in the art will appreciate that the following examples are only used to illustrate the present invention and should not be considered to limit the scope of the invention. Where specific conditions are not specified in the examples, the experiments were carried out under conventional conditions or conditions recommended by the manufacturer, and where the manufacturers of the reagents or instruments used are not specified, they are all conventional products that can be purchased commercially.

[0025] Example 1

[0026] The semen storage solution is made of the following components by weight: 16 parts of glucose, 20 parts of citric acid, 35 parts of tris (hydroxymethyl)aminomethane (Tris), 1×10 each of penicillin and streptomycin. 6IU, 1000 parts of double distilled water, the pH value of the prepared semen storage solution is 7.3, and the osmotic pressure is 310mOsm / L;

[0027] The semen protection solution is made of the following components by weight: 16 parts of glucose, 20 parts of citric acid, 35 parts of tris (hydroxymethyl)aminomethane (Tris), 1×10 each of penicillin and streptomycin. 6 IU, 1.0 part of Rhodiola rosea polysaccharide, 0.6 part of Astragalus polysaccharide, and 1000 parts of double distilled water.

[0028] Example 2

[0029] The semen storage solution is made of the following components by weight: 15 parts of glucose, 19 parts of citric acid, 34 parts of tris (hydroxymethyl)aminomethane (Tris), 1×10 each of penicillin and streptomycin. 6 IU, 1000 parts of double distilled water, the pH value of the prepared semen storage solution is 7.4, and the osmotic pressure is 300mOsm / L;

[0030] The semen protection solution is made of the following components by weight: 15 parts of glucose, 19 parts of citric acid, 34 parts of tris (hydroxymethyl)aminomethane (Tris), 1×10 each of penicillin and streptomycin. 6 IU, 0.9 parts of Rhodiola rosea polysaccharide, 0.5 parts of Astragalus polysaccharide, and 1000 parts of double distilled water.

[0031] Example 3

[0032] The semen storage solution is made of the following components by weight: 17 parts of glucose, 21 parts of citric acid, 36 parts of tris (hydroxymethyl)aminomethane (Tris), 1×10 each of penicillin and streptomycin. 6 IU, 1000 parts of double distilled water, the pH value of the prepared semen storage solution is 7.2, and the osmotic pressure is 320mOsm / L;

[0033] The semen protection solution is made of the following components by weight: 17 parts of glucose, 21 parts of citric acid, 36 parts of tris (hydroxymethyl)aminomethane (Tris), 1×10 each of penicillin and streptomycin. 6 IU, 1.1 parts of Rhodiola rosea polysaccharide, 0.7 parts of Astragalus polysaccharide, and 1000 parts of double distilled water.

[0034] Example 4

[0035] The semen storage solution is made of the following components by weight: 16 parts of glucose, 21 parts of citric acid, 34 parts of tris (hydroxymethyl)aminomethane (Tris), 1×10 each of penicillin and streptomycin. 6 IU, 1000 parts of double distilled water, the pH value of the prepared semen storage solution is 7.2, and the osmotic pressure is 310mOsm / L;

[0036] The semen protection solution is made of the following components by weight: 16 parts of glucose, 21 parts of citric acid, 34 parts of tris (hydroxymethyl)aminomethane (Tris), 1×10 6 IU, 0.9 parts of Rhodiola rosea polysaccharide, 0.6 parts of Astragalus polysaccharide, and 1000 parts of double distilled water.

[0037] Example 5

[0038] The semen storage solution is made of the following components by weight: 15 parts of glucose, 20 parts of citric acid, 36 parts of tris (hydroxymethyl)aminomethane (Tris), 1×10 each of penicillin and streptomycin. 6 IU, 1000 parts of double distilled water, the pH value of the prepared semen storage solution is 7.3, and the osmotic pressure is 310mOsm / L;

[0039] The semen protection solution is made of the following components by weight: 15 parts of glucose, 20 parts of citric acid, 36 parts of tris (hydroxymethyl)aminomethane (Tris), 1×10 each of penicillin and streptomycin. 6 IU, 1.1 parts of Rhodiola rosea polysaccharide, 0.5 parts of Astragalus polysaccharide, and 1000 parts of double distilled water.

[0040] Example 6

[0041] Semen storage method:

[0042] (1) Dilute the collected qualified sperm with semen storage solution, balance at 25°C for 1 hour, and balance at 15°C for 1 hour, mixing at irregular intervals;

[0043] (2) Centrifuge the semen, discard the supernatant, add semen protection solution, and adjust the final sperm concentration to 3.2-4.8×10 8 / mL, and equilibrate at 5℃ for 1h after resuspending.

[0044] (3) The mixed semen is filled into a straw tube, which is sealed. The straw tube is then cooled to -6°C and placed 3 cm above liquid nitrogen for fumigation for 3 min. Finally, it is placed in liquid nitrogen for storage.

[0045] Comparative Example 1

[0046] The Rhodiola rosea polysaccharide and Astragalus polysaccharide in the semen protection solution were replaced with an equal amount of distilled water, and the remaining operating steps were exactly the same as in Example 1.

[0047] Comparative Example 2

[0048] The Rhodiola rosea polysaccharide and Astragalus polysaccharide in the semen protection solution were replaced with an equal amount of Rhodiola rosea polysaccharide, and the remaining operating steps were exactly the same as in Example 1.

[0049] Comparative Example 3

[0050] The Rhodiola rosea polysaccharide and Astragalus polysaccharide in the semen protection solution were replaced with an equal amount of Astragalus polysaccharide, and the remaining operating steps were exactly the same as in Example 1.

[0051] Comparative Example 4

[0052] The Chinese medicine composition in the semen protection solution was replaced with astragalus extract, acerola cherry extract and bamboo leaf extract, and the remaining operating steps were exactly the same as those in Example 1.

[0053] Comparative Example 5

[0054] The Chinese medicine composition in the semen protection solution was replaced with astragalus polysaccharide and resveratrol, and the remaining operation steps were exactly the same as those in Example 1.

[0055] Comparative Example 6

[0056] The sperm protection solution was replaced with glycerol-egg yolk-citrate cryoprotectant, and the remaining operation steps were exactly the same as those in Example 1.

[0057] Test Example 1

[0058] The storage solution and protective solution of Examples 1 to 5 and the comparative example were used to freeze and thaw the semen of the same pig according to the following method:

[0059] (1) Dilute the collected qualified sperm (slightly fishy, ​​milky white, and with a motility of more than 85%) with semen storage solution, balance at 25°C for 1 hour, and balance at 15°C for 1 hour, mixing at irregular intervals;

[0060] (2) Centrifuge the semen, discard the supernatant, add semen protection solution, and adjust the final sperm concentration to 4×10 8 / mL, and equilibrate at 5℃ for 1h after resuspending.

[0061] (3) The mixed semen is filled into a straw tube, which is sealed. The straw tube is then cooled to -6°C and placed 3 cm above liquid nitrogen for fumigation for 3 min. Finally, it is placed in liquid nitrogen for storage.

[0062] (4) When thawing, remove the straw from the liquid nitrogen, place it in a 36.9℃ water bath and shake it for 14 seconds, then transfer it to a preheated insemination bottle at 37.1℃. Thawing is complete.

[0063] Then the plasma membrane integrity rate, sperm motility, DNA integrity rate, glutathione reductase concentration, mitochondrial integrity, lipid oxidation degree, and acrosome integrity rate after thawing were tested, among which:

[0064] The present invention uses the sperm hypoosmotic swelling test (HOST) to test the integrity of sperm plasma membrane: the hypotonic solution is preheated at 37°C, and the thawed semen is added to adjust the final concentration to 3×10 6 / mL, incubate for 5 minutes, smear and observe the curling of the tail, and calculate the sperm plasma membrane integrity rate.

[0065] Sperm motility was detected by computer-assisted sperm analysis (CASA).

[0066] AO fluorescence staining was used to detect sperm DNA integrity. The thawed semen was rinsed with PBS and repeated three times to adjust the sperm density to 3×10 6 / mL, smear, fix with anhydrous ethanol-glacial acetic acid (3:1), stain with AO dye and seal the slide, observe, and calculate the sperm DNA integrity rate.

[0067] Determination of glutathione reductase content: Take 1mL of 3.0×10 8 To the semen of 100 mg / mL, add 1 mL of 50% trichloroacetic acid solution, centrifuge and take the supernatant, add 2 mL of 0.2 mol / L tris-buffer (PH 8.9) and 0.5 mL of 0.01 mol / L 5,5-dithiobisnitrobenzoic acid, mix well, let stand for 5 min, measure the absorbance at 412 nm, and calculate the glutathione reductase concentration (nmol / mg).

[0068] The mitochondrial integrity rate of sperm was determined by the combined staining method of rhodamine 123 and propidium iodide. 3 μL of 1 mg / mL rhodamine-123 solution was added to 1 mL of 2×10 7 / mL of semen, incubate at 37℃ in the dark for 20min, then add 10μL 0.5mg / mL propidium iodide, incubate at 37℃ for 10min, centrifuge at 2500r / min for 6min, discard the supernatant, and resuspend in 1mL PBS. Preheat the slide, add 10μL of the resuspension solution, seal the slide, and observe with a fluorescence microscope at 490 / 515nm (rhodamine-123) and 545 / 590nm (propidium iodide) to calculate the sperm mitochondrial integrity rate.

[0069] The ultraviolet colorimetric method was used to determine the sperm plasma membrane oxidation. 8 To 1 mL of semen, add pre-cooled 20% (W:V) trichloroacetic acid, centrifuge and take the supernatant, add 1 mL of 0.67% (W:V) thiobarbituric acid (TBA), heat in a water bath for 10 min, and after cooling, measure the absorbance at 534 nm and calculate the malondialdehyde (MDA) concentration (nmol / mL).

[0070] The detection was performed by the fluorescein isothiocyanate labeled peanut agglutinin (FITC-PNA) fluorescence staining method. The semen processing method was the same as the DNA integrity rate determination method. The sample was smeared, dried and then smeared with methanol, fixed for 10 minutes, smeared with FITC-PNA stain, incubated at 37°C in the dark for 30 minutes, rinsed with PBS, dried in the dark, the excitation light was 480nm, the emission light was 530nm, and the sperm replacement integrity rate was calculated. The test results are shown in Table 1.

[0071] Table 1 Experimental results

[0072]

[0073] As can be seen from Table 1, the method for preparing a semen protective solution for improving the quality of sperm after freezing provided by the present invention can effectively improve the quality of sperm after freezing. The protective effect of using polysaccharides as protective additives on sperm after freezing and thawing is stronger than the protective effect of additives such as egg yolk and glycerol. In addition, the combination of Rhodiola rosea polysaccharide and Astragalus polysaccharide has a synergistic effect, and the effect is better than using a single polysaccharide.

[0074] Test Example 2

[0075] In order to further verify the efficacy of the drug of the present invention, in March 2021, 100 sows with physical maturity, normal sexual cycle rhythm, good health, no abnormalities in reproductive organs, and complete immunization program records were selected from a pig farm of Da Bei Nong Group. They were evenly divided into 5 test groups, with 20 sows in each group, including:

[0076] In the first test group, the semen protection solution prepared in Example 1 was added to the semen collected and tested to be qualified, and the sows were bred according to the freeze-thaw-artificial insemination method;

[0077] In the second test group, the semen protection solution prepared in the comparative example 1 was added to the semen collected and tested to be qualified, and the sows were bred according to the method of freezing-thawing-artificial insemination;

[0078] In the third test group, the semen protection solution prepared in comparative example 2 was added to the semen collected and tested to be qualified, and the sows were bred according to the freeze-thaw-artificial insemination method;

[0079] In the experiment group 4, the semen protection solution prepared in comparative example 3 was added to the semen collected and tested to be qualified, and the sows were bred according to the freeze-thaw-artificial insemination method;

[0080] In the experiment group 5, the semen protection solution prepared in the comparative example 4 was added to the semen collected and tested to be qualified, and the sows were bred according to the method of freezing-thawing-artificial insemination;

[0081] In the experiment group 6, the semen protection solution prepared in comparative example 5 was added to the semen collected and tested to be qualified, and the sows were bred according to the freeze-thaw-artificial insemination method;

[0082] In the experimental group 7, a commercially available control sperm cryoprotectant (also known as glycerol-yolk-citrate cryoprotectant, mainly containing glucose, sodium citrate, glycerol, glycine, egg yolk, antibiotics, etc.) was added to the semen collected and tested to be qualified, and the sows were mated according to the method of freezing-thawing-artificial insemination, and other conditions were basically the same. The experimental results are shown in Table 2.

[0083] Table 2 Test results

[0084]

[0085]

[0086] The test results show that the semen protection solution of the present invention has a significant effect on improving the quality of semen after freezing, among which the combined effect of Rhodiola rosea polysaccharide and Astragalus polysaccharide is better than that of a single component, and the effect is also more significant among similar products currently available.

[0087] Test Example 3

[0088] At Fu's pig farm in Hengshui City, Hebei Province, the frozen sperm treated with the semen protection solution in Example 1 was thawed, transferred to a centrifuge tube, placed in a centrifuge and centrifuged at 3000r / min for 2 min, the supernatant was discarded, semen storage solution was added and mixed, and the mixture was centrifuged for another 2 min. The supernatant was discarded, and semen storage solution was added and mixed. The mixture was used for artificial insemination of pigs and compared with frozen semen treated with only semen storage solution. The conception rate and piglet survival rate were observed and recorded.

[0089] A follow-up visit four months later found that the total conception rate reached 58% and the piglet survival rate reached 86% after mating with sperm treated with semen preservation solution. Compared with sperm treated with semen storage solution alone, the total conception rate and piglet survival rate were significantly higher.

[0090] Test Example 4

[0091] Liu from Yingkou City, Liaoning Province, raised 500 basic sows. When using frozen sperm for artificial breeding, he used sperm freezing liquid purchased from the market, and found that the final conception effect and piglet survival rate were not ideal. After Liu found our technicians, he hoped to find a new protective liquid to improve the vitality of frozen sperm and increase the farrowing rate. In response to this, our technicians gave Liu the sperm protection liquid prepared by the method in Example 2, replacing the sperm protection liquid used before in the conventional freezing-thawing process. Afterwards, the test sows were artificially inseminated. After a return visit six months later, Liu said that after using the semen protection liquid, the sow conception rate increased from the original 35% to 59%, and the piglet survival rate also increased from 62% to 87%, greatly reducing the cost of breeding and improving efficiency. Liu's medication feedback shows that the present invention has a good improvement effect on the quality of sperm after freezing.

[0092] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the technical principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A semen protection solution, characterized in that: The semen protection solution is prepared from semen storage solution and a traditional Chinese medicine composition; The semen storage solution contains 15-17 g glucose, 19-21 g citric acid, 34-36 g tris(hydroxymethyl)aminomethane) and 1×10 each of penicillin and streptomycin in 1 L of double distilled water. 6 IU; The Chinese medicine composition contains 0.9-1.1g of Rhodiola rosea polysaccharide and 0.5-0.7g of Astragalus polysaccharide per 1L of double distilled water; The pH value of the semen storage solution is 7.2-7.4, and the osmotic pressure is 300-320 mOsm / L; The preparation method of the semen protection solution is as follows: glucose, citric acid, tris(hydroxymethylaminomethane), penicillin and streptomycin are weighed respectively and dissolved in double distilled water to prepare semen storage solution, and rhodiola rosea polysaccharide and astragalus polysaccharide are added to the semen storage solution in proportion to prepare semen protection solution.

2. The semen protection solution according to claim 1, characterized in that: The semen storage solution contains 16 g glucose, 20 g citric acid, 35 g tris(hydroxymethyl)aminomethane, 1×10 each of penicillin and streptomycin in 1 L of double distilled water. 6 IU.

3. The semen protection solution according to claim 1, characterized in that: The Chinese medicine composition contains 1.0 g of Rhodiola rosea polysaccharide and 0.6 g of Astragalus polysaccharide in every 1 L of double distilled water.

4. A method for preserving pig semen using the semen protection solution according to any one of claims 1 to 3, characterized in that: The steps include: (1) Diluting the collected qualified semen with the semen storage solution, equilibrate at 25° C. for 1 hour, and equilibrate at 15° C. for 1 hour, and mix at irregular intervals; (2) Centrifuge the semen, discard the supernatant, add the semen protection solution, and adjust the final sperm concentration to (3.2~4.8)×10 8 / mL, and then resuspended and equilibrated at 5℃ for 1h; (3) The mixed semen is filled into a straw tube, which is sealed. The straw tube is then cooled to -6°C and placed 3 cm above liquid nitrogen for fumigation for 3 minutes. Finally, it is placed in liquid nitrogen for storage.

5. The method according to claim 4, characterized in that The final sperm concentration in step (2) was adjusted to 4×10 8 Pieces / mL.

6. Use of the semen protection solution according to any one of claims 1 to 3 in the storage and cryopreservation of pig semen.

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