CN114304136A - Semen protection solution and preparation method and application thereof - Google Patents
Semen protection solution and preparation method and application thereof Download PDFInfo
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- CN114304136A CN114304136A CN202111496761.7A CN202111496761A CN114304136A CN 114304136 A CN114304136 A CN 114304136A CN 202111496761 A CN202111496761 A CN 202111496761A CN 114304136 A CN114304136 A CN 114304136A
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- Agricultural Chemicals And Associated Chemicals (AREA)
Abstract
The invention discloses a semen protection solution for improving the quality of frozen semen as well as a preparation method and application thereof. The semen protection solution contains active ingredients, wherein the active ingredients are prepared from rhodiola rosea polysaccharide and astragalus polysaccharide. The semen protection solution has the effects of resisting oxidation, improving the survival rate of frozen sperms and the like, can be effectively used for protecting semen freezing, can effectively improve the plasma membrane integrity rate, the sperm motility, the DNA integrity rate, the acrosome integrity rate and the like of thawed sperms, and improves the quality of frozen semen. Has the advantages of wide raw material source, safety, environmental protection, simple preparation process, low production cost and the like.
Description
Technical Field
The invention belongs to the field of animal genetic breeding, and particularly relates to a semen protection solution for improving the quality of frozen semen, and a preparation method and application thereof.
Background
With the development of artificial insemination technology, the semen freezing and storing technology is one of important biotechnological means for breeding mammals, can effectively reduce the feeding cost, ensures that the semen is not limited by time, place and objects, obviously increases the use time and the utilization rate of the semen of excellent breeding stock, and comprises a series of complex processes of sampling, diluting, cooling, freezing and the like. However, the semen freezing technology is still not completely mature due to the difficulties in producing frozen semen, the low survival rate and conception rate after freezing-thawing and the like. During the freezing-unfreezing process, semen can 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 cell membranes and cell internal structures caused by ice crystals generated by freezing. A series of damages caused by the solution effect due to ice crystallization are chemical damages: the redox balance in sperm cells is broken, Reactive Oxygen Species (ROS) generated by normal physiological metabolism are accumulated in a large quantity, cannot be neutralized by reaction, are easy to generate oxidation reaction with phospholipid on sperm cell membranes, and also can reduce the activity of intracellular enzyme substances, reduce the motility and even damage DNA in cell nuclei, thereby causing the fertilization capability of the sperm to be reduced.
In recent years, the prior art has studied in detail external conditions affecting the quality of frozen sperm, and can control the external conditions to be kept within an appropriate range during freezing, thereby minimizing the influence of the external conditions. The polysaccharide has antioxidant, antiaging and apoptosis preventing effects, and can inhibit the generation of active oxygen, improve peroxidase activity, prevent plasma membrane from lipid peroxidation damage, and prevent intracellular enzyme efflux. The traditional Chinese medicine composition can effectively improve the quality of frozen sperms, effectively ensure the safety of the sperms and improve the utilization rate of the sperms. Compared with animal-derived infiltrative protective agent, the traditional natural plant traditional Chinese medicine composition has the advantages of high safety, low toxicity, less residue and the like.
Disclosure of Invention
In order to solve the technical problems, the invention provides a semen protection solution for improving the quality of frozen semen, which has the following specific scheme:
the semen protection solution is prepared from a semen storage solution and a traditional Chinese medicine composition, wherein the semen storage solution is prepared from glucose, citric acid, tris (hydroxymethyl) aminomethane, penicillin and streptomycin, and the traditional Chinese medicine composition is prepared from rhodiola rosea polysaccharide and astragalus polysaccharide.
Further, the semen storage solution contains 15-17 g of glucose, 19-21 g of citric acid, 34-36 g of Tris (hydroxymethyl) aminomethane (Tris) and 1 × 10 of penicillin and streptomycin respectively in every 1L of double distilled water6IU。
Further, the semen stock solution contains 16g of glucose, 20g of citric acid, 35g of Tris (hydroxymethyl) aminomethane (Tris) and 1 × 10 of penicillin and streptomycin respectively in each 1L of double distilled water6IU。
Furthermore, the traditional Chinese medicine composition contains 0.9-1.1g of rhodiola rosea polysaccharide and 0.5-0.7g of astragalus polysaccharide in each 1L of double distilled water.
Further, the traditional Chinese medicine composition contains 1.0g of rhodiola rosea polysaccharide and 0.6g of astragalus polysaccharide in each 1L of double distilled water.
The invention also provides a method for preserving animal semen by using the semen protection solution, which comprises the following steps:
(1) diluting the collected qualified sperms with a semen storage solution, carrying out equilibrium treatment at 25 ℃ for 1h, carrying out equilibrium treatment at 15 ℃ for 1h, and mixing uniformly at variable time;
(2) centrifuging the semen, removing the supernatant, adding the semen protection solution, and adjusting the final concentration of the semen to 3.2-4.8 × 108Performing equilibration treatment at 5 ℃ for 1h after resuspension;
(3) filling the mixed semen into a thin tube, sealing, gradient cooling to-6 deg.C, fumigating 3min at a position 3cm above liquid nitrogen, adding liquid nitrogen, and storing.
In the above method, the final concentration of the sperm to be adjusted in the step (2) is preferably 4X 108one/mL.
The invention also provides a preparation method of the semen protection solution, which comprises the steps of respectively weighing glucose, citric acid, tris (hydroxymethyl) aminomethane, penicillin and streptomycin, adding water to dissolve the glucose, the citric acid, the tris (hydroxymethyl) aminomethane, the penicillin and the streptomycin to prepare a semen storage solution, and adding the rhodiola rosea polysaccharide and the astragalus polysaccharide into the semen storage solution in proportion to prepare the semen protection solution.
The invention also provides application of the semen protection solution in animal semen storage and freezing protection, and particularly relates to application of the semen protection solution in animal semen freezing storage. For example, the semen is mixed with the semen protection solution, placed in a storage container, and then placed in a closed container for closed storage.
Furthermore, the animal semen is preferably semen of domestic animals such as pig, cattle, sheep, horse, etc.
As will be understood by those skilled in the art, the water in the semen protection solution of the present invention is preferably ultrapure water.
The person skilled in the art will understand that the preservation container used in the preservation method of the present invention may be any container that can be used for preserving semen, such as 0.2ml preservation tube, 0.5ml cryopreservation tube, etc.
Further, the semen protection solution can be stored in the form of: ampoules, pellets, aluminum foil (plastic) bags and fine (wheat) tubes.
The medicine of the invention has the advantages and beneficial effects that:
the medicine is healthy and nontoxic, and does not cause damage to sperms; the raw materials of the medicine are wide in source and low in cost, do not cause environmental pollution, and do not have medicine residues to harm the health of animal embryos; the semen protection solution provided by the invention reduces the damage of the centrifugal action on the activity of the semen, improves the centrifugal effect and avoids the loss of active ingredients in the semen.
The medicine expands the use of the raw materials of the semen protection solution, compared with the prior art, most of the raw materials of the traditional Chinese medicine used by the invention have lower price, and the effect of the technical scheme of the invention on the aspect of improving the quality of the frozen semen is also obviously better than that of the prior art while the cost is greatly reduced.
Detailed Description
Embodiments of the present invention will be described in detail below with reference to examples, but it will be understood by those skilled in the art that the following examples are only illustrative of the present invention and should not be construed as limiting the scope of the invention. The examples do not show the specific conditions, and the reagents or instruments used are not shown in the manufacturer, and all are conventional products commercially available.
Example 1
The semen storage solution is prepared from the following components in parts by weight: 16 portions of glucose, 20 portions of citric acid, 35 portions of Tris (hydroxymethyl) aminomethane (Tris), 1 multiplied by 10 of each penicillin and streptomycin6IU, 1000 parts of double distilled water, pH value of the prepared semen storage solution is 7.3, and osmotic pressure is 310 mOsm/L;
the semen protection solution is prepared from the following components in parts by weight: 16 portions of glucose, 20 portions of citric acid, 35 portions of Tris (hydroxymethyl) aminomethane (Tris), 1 multiplied by 10 of each penicillin and streptomycin6IU, 1.0 part of rhodiola polysaccharide, 0.6 part of astragalus polysaccharide and 1000 parts of double distilled water.
Example 2
The semen storage solution is prepared from the following components in parts by weight: 15 parts of glucose, 19 parts of citric acid, 34 parts of Tris (hydroxymethyl) aminomethane (Tris), and 1 × 10 parts of each of penicillin and streptomycin6IU, 1000 parts of double distilled water, pH value of the prepared semen storage solution is 7.4, and osmotic pressure is 300 mOsm/L;
the semen protection solution is prepared from the following components in parts by weight: 15 parts of glucose, 19 parts of citric acid, 34 parts of Tris (hydroxymethyl) aminomethane (Tris), and 1 × 10 parts of each of penicillin and streptomycin6IU, rhodiola polysaccharide 0.9 parts, astragalus polysaccharide 0.5 parts and double distilled water 1000 parts.
Example 3
The semen storage solution is prepared from the following components in parts by weight: 17 parts of glucose, 21 parts of citric acid, 36 parts of Tris (hydroxymethyl) aminomethane (Tris), and 1 × 10 parts of each of penicillin and streptomycin6IU, 1000 parts of double distilled water, pH value of the prepared semen storage solution is 7.2, and osmotic pressure is 320 mOsm/L;
the semen protection solution is prepared from the following components in parts by weight: 17 parts of glucose, 21 parts of citric acid, 36 parts of Tris (hydroxymethyl) aminomethane (Tris), and 1 × 10 parts of each of penicillin and streptomycin6IU, 1.1 parts of rhodiola polysaccharide, 0.7 parts of astragalus polysaccharide and 1000 parts of double distilled water.
Example 4
The semen storage solution is prepared from the following components in parts by weight: 16 parts of glucose, 21 parts of citric acid, 34 parts of Tris (hydroxymethyl) aminomethane (Tris), and cyan1X 10 of each of streptomycin and streptomycin6IU, 1000 parts of double distilled water, pH value of the prepared semen storage solution is 7.2, and osmotic pressure is 310 mOsm/L;
the semen protection solution is prepared from the following components in parts by weight: 16 portions of glucose, 21 portions of citric acid, 34 portions of Tris (hydroxymethyl) aminomethane (Tris), 1 multiplied by 10 of each penicillin and streptomycin6IU, rhodiola polysaccharide 0.9 parts, astragalus polysaccharide 0.6 parts and double distilled water 1000 parts.
Example 5
The semen storage solution is prepared from the following components in parts by weight: 15 portions of glucose, 20 portions of citric acid, 36 portions of Tris (hydroxymethyl) aminomethane (Tris), 1 multiplied by 10 of each penicillin and streptomycin6IU, 1000 parts of double distilled water, pH value of the prepared semen storage solution is 7.3, and osmotic pressure is 310 mOsm/L;
the semen protection solution is prepared from the following components in parts by weight: 15 portions of glucose, 20 portions of citric acid, 36 portions of Tris (hydroxymethyl) aminomethane (Tris), 1 multiplied by 10 of each penicillin and streptomycin6IU, 1.1 parts of rhodiola polysaccharide, 0.5 parts of astragalus polysaccharide and 1000 parts of double distilled water.
Example 6
The semen preservation method comprises the following steps:
(1) diluting the collected qualified sperms with a semen storage solution, carrying out equilibrium treatment at 25 ℃ for 1h, carrying out equilibrium treatment at 15 ℃ for 1h, and mixing uniformly at variable time;
(2) centrifuging the semen, removing the supernatant, adding a semen protection solution, and adjusting the final concentration of the semen to 3.2-4.8 × 108piece/mL, after resuspension 5 ℃ balance treatment for 1 h.
(3) Filling the mixed semen into a thin tube, sealing, gradient cooling to-6 deg.C, fumigating 3min at a position 3cm above liquid nitrogen, adding liquid nitrogen, and storing.
Comparative example 1
The rhodiola rosea polysaccharide and the astragalus polysaccharide in the semen protection solution are replaced by equal amount of distilled water, and the rest operation steps are completely the same as the embodiment 1.
Comparative example 2
The rhodiola rosea polysaccharide and the astragalus polysaccharide in the semen protection solution are replaced by the same amount of rhodiola rosea polysaccharide, and the rest operation steps are completely the same as the embodiment 1.
Comparative example 3
The rhodiola rosea polysaccharide and the astragalus polysaccharide in the semen protection solution are replaced by the same amount of astragalus polysaccharide, and the rest operation steps are completely the same as the embodiment 1.
Comparative example 4
The traditional Chinese medicine composition in the semen protection solution is replaced by the astragalus extract, the acerola cherry extract and the bamboo leaf extract, and the rest operation steps are completely the same as the embodiment 1.
Comparative example 5
The traditional Chinese medicine composition in the semen protection solution is replaced by astragalus polysaccharide and resveratrol, and the rest operation steps are completely the same as those in the embodiment 1.
Comparative example 6
The semen protective solution is replaced by the glycerol-yolk-citrate cryoprotectant, and the rest of the operation steps are completely the same as the example 1.
Test example 1
The storage solution and the protective solution of the examples 1 to 5 and the comparative example are used for carrying out freezing and thawing treatment on the semen of the same pig according to the following method:
(1) diluting the collected qualified sperm (with fishy smell, milky white, and activity over 85% as qualified semen) with semen storage solution, balancing at 25 deg.C for 1 hr, balancing at 15 deg.C for 1 hr, and mixing at variable time;
(2) centrifuging semen, removing supernatant, adding semen protecting solution, and adjusting semen concentration to 4 × 108piece/mL, after resuspension 5 ℃ balance treatment for 1 h.
(3) Filling the mixed semen into a thin tube, sealing, gradient cooling to-6 deg.C, fumigating 3min at a position 3cm above liquid nitrogen, adding liquid nitrogen, and storing.
(4) When thawing, taking out the thin tube from the liquid nitrogen, placing the thin tube in a water bath at 36.9 ℃, shaking for 14s, transferring the thin tube to a semen deposition bottle preheated at 37.1 ℃, and completing thawing.
And then testing the plasma membrane integrity, sperm motility, DNA integrity, glutathione reductase concentration, mitochondrial integrity, lipid oxidation degree and acrosome integrity after thawing, wherein:
the invention adopts a sperm hyposmosis swelling test (HOST) to test the integrity rate of a sperm plasma membrane: preheating hypotonic solution at 37 deg.C, adding thawed semen, and adjusting final concentration to 3 × 106And (4) incubating for 5min, observing a smear, observing the curling condition of the tail part, and calculating the integrity rate of the plasma membrane of the sperm.
Sperm motility was detected by a computer assisted sperm analysis system (CASA).
And detecting the sperm DNA integrity rate by using an AO fluorescent staining method. The semen was thawed by washing with PBS three times and the sperm density was adjusted to 3X 106Smear, fixing with absolute ethyl alcohol-glacial acetic acid (3:1), sealing after AO staining solution staining, observing, and calculating the sperm DNA integrity.
And (3) measuring the content of glutathione reductase: 1mL of the solution was taken at a concentration of 3.0X 108Adding 1mL of 50% trichloroacetic acid solution into semen per mL, centrifuging to obtain supernatant, adding 2mL of 0.2mol/L tris-buffer solution (pH 8.9) and 0.5mL of 0.01 mol/L55-dithio-bis-nitro-p-benzoic acid, mixing, standing for 5min, measuring absorbance at 412nm, and calculating the concentration (nmol/mg) of glutathione reductase.
The method for measuring the integrity of the sperm mitochondria by using the combined dyeing method of rhodamine 123 and propidium iodide is adopted, 3 mu L of 1mg/mL rhodamine-123 solution is added into 1mL of the 2 multiplied by 10 concentration sperm7And (3) incubating the semen in each mL of semen for 20min at 37 ℃ in the dark, adding 10 mu L of 0.5mg/mL propidium iodide, incubating the semen for 10min at 37 ℃, centrifuging the semen for 6min at the rotating speed of 2500r/min, removing the supernatant, and adding 1mL of PBS for resuspension. And (3) preheating a glass slide, adding 10 mu L of resuspension, mounting, observing at 490/515nm (rhodamine-123) and 545/590nm (propidium iodide) by using a fluorescence microscope respectively, and calculating the integrity of the sperm mitochondria.
And (4) performing oxidation determination of the plasma membrane of the sperm by using an ultraviolet color development method. Take 3.0X 108Adding precooled 20% (W: V) trichloroacetic acid into 1mL of semen per mL, centrifuging to obtain a supernatant, adding 0.67% (W: V) thiobarbituric acid (TBA) into 1mL of the semen, heating in a water bath for 10min, cooling, measuring the absorbance at 534nm, and calculating the concentration (nmol/mL) of Malondialdehyde (MDA).
Peanut agglutinin (FITC-PNA) marked by fluorescein isothiocyanate is selected for detection by a fluorescent staining method, and a semen processing method is the same as a processing method for measuring the integrity of DNA. Sampling smear, air-drying the smear, fixing for 10min, using FITC-PNA dye solution smear, incubating for 30min at 37 ℃ in the dark, washing with PBS, air-drying in the dark, exciting light at 480nm, emitting light at 530nm, and calculating the complete rate of sperm displacement. The test results are shown in Table 1.
TABLE 1 results of the experiment
As can be seen from table 1, the preparation method of the semen protection solution for improving the frozen sperm quality provided by the invention can effectively improve the frozen sperm quality, the protection effect of the frozen-thawed sperm by using polysaccharide as a protection additive is stronger than that of additives such as egg yolk and glycerol, and the rhodiola rosea polysaccharide and the astragalus polysaccharide have a synergistic effect and the effect is better than that of using single polysaccharide.
Test example 2
In order to further verify the effect of the medicine, 100 sows which are mature, normal in sexual cycle rhythm, good in health condition, abnormal in reproductive organs and complete in immune program record are selected in a certain pig farm of Dabei agricultural group in 3 months in 2021, and are averagely divided into 5 test groups, wherein each group comprises 20 sows, and the weight of each sow is as follows:
experiment 1 group used the semen protective solution prepared in example 1, added to semen qualified for collection and determination, and bred sows according to the freezing-thawing-artificial insemination method;
experiment 2, the semen protection solution prepared in the comparative example 1 is added into semen qualified in collection and determination, and the sow is bred according to the freezing-unfreezing-artificial insemination method;
experiment 3 groups used the semen protection solution prepared in comparative example 2, added into semen qualified in collection and determination, and bred sows according to the method of freezing-unfreezing-artificial insemination;
in the experiment 4, the semen protection solution prepared in the comparative example 3 is added into semen qualified in collection and determination, and the sow is bred according to the freezing-unfreezing-artificial insemination method;
experiment 5 groups used the semen protective solution prepared in comparative example 4, added into semen qualified in collection and determination, and bred sows according to the method of freezing-unfreezing-artificial insemination;
the semen protection solution prepared in the comparative example 5 is added into semen qualified in collection and determination in the test 6 groups, and the sow is bred according to the freezing-unfreezing-artificial insemination method;
in test 7, a commercially available control sperm cryoprotectant (also known as a glycerol-yolk-citrate cryoprotectant, which mainly contains glucose, sodium citrate, glycerol, glycine, yolk, antibiotics and the like) was added to the semen qualified for collection and determination, and the sows were bred according to the freezing-thawing-artificial insemination method under substantially the same other conditions. The test results are shown in Table 2.
TABLE 2 test results
The test result shows that the semen protection solution has obvious effect on improving the quality of frozen semen, wherein the effect of the combination of rhodiola rosea polysaccharide and astragalus polysaccharide is better than that of a single component, and the effect is also obvious in the current similar products.
Test example 3
In a certain pig farm paid in Hebei province Heshui city, the frozen sperms treated by the semen protection solution in the embodiment 1 are unfrozen, transferred to a centrifuge tube, placed in a centrifuge for centrifugation for 2min at the rotating speed of 3000r/min, the supernatant is discarded, the semen storage solution is added and mixed uniformly, then the centrifugation is continued for 2min, the supernatant is discarded, the semen storage solution is added and mixed uniformly for artificial insemination of pigs, and compared with the frozen sperms only using the semen storage solution, the conception rate and the survival rate of piglets are observed and recorded.
The results of the return visit after four months show that the total conception rate reaches 58% and the survival rate of piglets also reaches 86% after the sperm treated by the semen protection solution is matched. Compared with sperm treated only by the semen storage solution, the total conception rate and the survival rate of piglets are both obviously improved.
Test example 4
When frozen semen is adopted for artificial breeding, 500 basic sows bred in Liu of Yikou city in Liaoning province use semen refrigerating fluid purchased from the market, and the final conception effect and the survival rate of piglets are found to be not ideal. After a technician is found in Liu, a new protective solution is expected to be found to improve the activity of frozen semen and improve the farrowing rate. To this end, the inventors used the sperm cell preservation solution prepared in accordance with the method of example 2 to replace the previously used sperm cell preservation solution in a conventional freeze-thaw procedure. Then artificial insemination is carried out on the sow. The semen protection solution is used for the piglets, the conception rate of the sows is improved to 59 percent from the original 35 percent, the survival rate of the piglets is also improved to 87 percent from 62 percent, the breeding cost is greatly reduced, and the efficiency is improved. The medicine feedback of Liu' e shows that the invention has good improvement effect on the quality of frozen sperms.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the technical principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Claims (10)
1. The semen protection solution is characterized by being prepared from a semen storage solution and a traditional Chinese medicine composition, wherein the semen storage solution is prepared from glucose, citric acid, tris (hydroxymethyl) aminomethane, penicillin and streptomycin, and the traditional Chinese medicine composition is prepared from rhodiola rosea polysaccharide and astragalus polysaccharide.
2. The semen protection solution as claimed in claim 1, wherein the active ingredient of the storage solution is 15-17% glucose in 1L double distilled waterg, 19-21 g of citric acid, 34-36 g of trihydroxymethyl aminomethane, 1 × 10 of each of penicillin and streptomycin6IU。
3. The semen protection solution as claimed in claim 2, wherein the active ingredients of the stock solution comprise 16g of glucose, 20g of citric acid, 35g of tris (hydroxymethyl) aminomethane, and 1 x 10 of each of penicillin and streptomycin per 1L of double distilled water6IU。
4. The semen protection solution according to any one of claims 1 to 3, wherein the pH value of the storage solution is 7.2 to 7.4, and the osmotic pressure is 300 to 320 mOsm/L.
5. The semen protection solution according to claim 1, wherein the active ingredients of the Chinese medicinal composition comprise rhodiola rosea polysaccharide 0.9-1.1g and astragalus polysaccharide 0.5-0.7g in per 1L of double distilled water.
6. The semen protection solution according to claim 5, wherein the active ingredients of the Chinese medicinal composition comprise 1.0g of rhodiola rosea polysaccharide and 0.6g of astragalus polysaccharide in each 1L of double distilled water.
7. The method for preserving animal semen by using the semen protection solution as claimed in any one of claims 1 to 6, characterized by comprising the following steps:
(1) diluting the collected qualified sperms with a semen storage solution, carrying out equilibrium treatment at 25 ℃ for 1h, carrying out equilibrium treatment at 15 ℃ for 1h, and mixing uniformly at variable time;
(2) centrifuging the semen, removing the supernatant, adding the semen protection solution, and adjusting the final concentration of the semen to 3.2-4.8 × 108Performing equilibration treatment at 5 ℃ for 1h after resuspension;
(3) filling the mixed semen into a thin tube, sealing, gradient cooling to-6 deg.C, fumigating 3min at a position 3cm above liquid nitrogen, adding liquid nitrogen, and storing.
8. The method of claim 7, whichCharacterized in that the final concentration of the sperms is adjusted to be 4 multiplied by 10 in the step (2)8one/mL.
9. Use of a semen protection solution according to any one of claims 1 to 6 for the storage and cryopreservation of animal semen.
10. Use according to claim 9, wherein the animal semen is semen of a domestic animal, including but not limited to pigs, cattle, sheep, horses.
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|---|---|---|---|---|
| CN115624024A (en) * | 2022-08-22 | 2023-01-20 | 甘肃润牧生物工程有限责任公司 | Application of codonopsis pilosula polysaccharide as additive in sheep semen low-temperature diluent |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP6728463B1 (en) * | 2019-10-28 | 2020-07-22 | 浙江大学Ｚｈｅｊｉａｎｇ Ｕｎｉｖｅｒｓｉｔｙ | Goat semen cryopreservation liquid and its use |
| CN113396895A (en) * | 2021-07-14 | 2021-09-17 | 西北农林科技大学 | Application of aloe polysaccharide in preparation of livestock cryopreservation agent |
-
2021
- 2021-12-09 CN CN202111496761.7A patent/CN114304136A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP6728463B1 (en) * | 2019-10-28 | 2020-07-22 | 浙江大学Ｚｈｅｊｉａｎｇ Ｕｎｉｖｅｒｓｉｔｙ | Goat semen cryopreservation liquid and its use |
| CN113396895A (en) * | 2021-07-14 | 2021-09-17 | 西北农林科技大学 | Application of aloe polysaccharide in preparation of livestock cryopreservation agent |
Non-Patent Citations (3)
| Title |
|---|
| 柳立军;马鹏程;张爱萍;王燕超;周兰霞;王晶;: "黄芪多糖对人精子冻存质量保护作用的研究", 时珍国医国药, no. 10, 20 October 2015 (2015-10-20), pages 2386 - 2387 * |
| 胡传活;许春荣;陆媚;韦干显;刘德玉;王英群;彭夏云;韦永强;: "黄芪多糖对猪精液低温保存的影响", 中国兽医学报, no. 01, 15 January 2015 (2015-01-15), pages 150 - 154 * |
| 陈晓英;马金英;宋天增;唐建华;赵彦玲;任子利;朱彦宾;王良润;: "红景天多糖对藏猪精液4℃保存效果的研究", 养猪, no. 06, 10 December 2016 (2016-12-10), pages 33 - 36 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115624024A (en) * | 2022-08-22 | 2023-01-20 | 甘肃润牧生物工程有限责任公司 | Application of codonopsis pilosula polysaccharide as additive in sheep semen low-temperature diluent |
| CN115624024B (en) * | 2022-08-22 | 2023-08-22 | 甘肃润牧生物工程有限责任公司 | Application of codonopsis pilosula polysaccharide as additive in sheep semen low-temperature diluent |
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