CN110012901B - Diluting preparation for preserving boar semen at normal temperature - Google Patents
Diluting preparation for preserving boar semen at normal temperature Download PDFInfo
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- 210000000582 semen Anatomy 0.000 title claims abstract description 184
- 238000002360 preparation method Methods 0.000 title claims abstract description 99
- 238000007865 diluting Methods 0.000 title description 24
- 238000004321 preservation Methods 0.000 claims abstract description 36
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 claims abstract description 18
- WCUXLLCKKVVCTQ-UHFFFAOYSA-M Potassium chloride Chemical compound [Cl-].[K+] WCUXLLCKKVVCTQ-UHFFFAOYSA-M 0.000 claims abstract description 18
- UIIMBOGNXHQVGW-UHFFFAOYSA-M Sodium bicarbonate Chemical compound [Na+].OC([O-])=O UIIMBOGNXHQVGW-UHFFFAOYSA-M 0.000 claims abstract description 16
- PAFLSMZLRSPALU-MRVPVSSYSA-N (2R)-3-(3,4-dihydroxyphenyl)lactic acid Chemical compound OC(=O)[C@H](O)CC1=CC=C(O)C(O)=C1 PAFLSMZLRSPALU-MRVPVSSYSA-N 0.000 claims abstract description 14
- PAFLSMZLRSPALU-UHFFFAOYSA-N Salvianic acid A Natural products OC(=O)C(O)CC1=CC=C(O)C(O)=C1 PAFLSMZLRSPALU-UHFFFAOYSA-N 0.000 claims abstract description 14
- PAFLSMZLRSPALU-QMMMGPOBSA-N Danshensu Natural products OC(=O)[C@@H](O)CC1=CC=C(O)C(O)=C1 PAFLSMZLRSPALU-QMMMGPOBSA-N 0.000 claims abstract description 13
- CEAZRRDELHUEMR-URQXQFDESA-N Gentamicin Chemical compound 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 CEAZRRDELHUEMR-URQXQFDESA-N 0.000 claims abstract description 10
- 229930182566 Gentamicin Natural products 0.000 claims abstract description 10
- 229960002518 gentamicin Drugs 0.000 claims abstract description 10
- 239000001103 potassium chloride Substances 0.000 claims abstract description 9
- 235000011164 potassium chloride Nutrition 0.000 claims abstract description 9
- 235000017557 sodium bicarbonate Nutrition 0.000 claims abstract description 6
- 229910000030 sodium bicarbonate Inorganic materials 0.000 claims abstract description 6
- 239000000203 mixture Substances 0.000 claims description 14
- 238000002156 mixing Methods 0.000 claims description 13
- 239000000126 substance Substances 0.000 claims description 6
- 239000001509 sodium citrate Substances 0.000 claims description 5
- 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 description 5
- 238000003756 stirring Methods 0.000 claims description 5
- BJHIKXHVCXFQLS-UYFOZJQFSA-N keto-D-fructose Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)C(=O)CO BJHIKXHVCXFQLS-UYFOZJQFSA-N 0.000 claims description 4
- 238000009461 vacuum packaging Methods 0.000 claims description 4
- 238000005303 weighing Methods 0.000 claims description 4
- 239000003814 drug Substances 0.000 claims 1
- 238000010790 dilution Methods 0.000 abstract description 37
- 239000012895 dilution Substances 0.000 abstract description 37
- 230000000694 effects Effects 0.000 abstract description 36
- 239000003085 diluting agent Substances 0.000 abstract description 13
- 239000003963 antioxidant agent Substances 0.000 abstract description 9
- 230000003078 antioxidant effect Effects 0.000 abstract description 8
- 241000282887 Suidae Species 0.000 abstract description 7
- 239000012154 double-distilled water Substances 0.000 abstract description 7
- RFSUNEUAIZKAJO-VRPWFDPXSA-N D-Fructose Natural products OC[C@H]1OC(O)(CO)[C@@H](O)[C@@H]1O RFSUNEUAIZKAJO-VRPWFDPXSA-N 0.000 abstract description 6
- RFSUNEUAIZKAJO-ARQDHWQXSA-N Fructose Chemical compound OC[C@H]1O[C@](O)(CO)[C@@H](O)[C@@H]1O RFSUNEUAIZKAJO-ARQDHWQXSA-N 0.000 abstract description 6
- 229910021642 ultra pure water Inorganic materials 0.000 abstract description 3
- 239000012498 ultrapure water Substances 0.000 abstract description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 abstract description 3
- OQUFOZNPBIIJTN-UHFFFAOYSA-N 2-hydroxypropane-1,2,3-tricarboxylic acid;sodium Chemical compound [Na].OC(=O)CC(O)(C(O)=O)CC(O)=O OQUFOZNPBIIJTN-UHFFFAOYSA-N 0.000 abstract 1
- 238000000034 method Methods 0.000 description 16
- 229960001484 edetic acid Drugs 0.000 description 14
- 235000013350 formula milk Nutrition 0.000 description 14
- 238000003860 storage Methods 0.000 description 14
- 239000002994 raw material Substances 0.000 description 12
- 230000009027 insemination Effects 0.000 description 11
- 230000014759 maintenance of location Effects 0.000 description 8
- WSMYVTOQOOLQHP-UHFFFAOYSA-N Malondialdehyde Chemical compound O=CCC=O WSMYVTOQOOLQHP-UHFFFAOYSA-N 0.000 description 6
- 230000035935 pregnancy Effects 0.000 description 6
- 102000016938 Catalase Human genes 0.000 description 5
- 108010053835 Catalase Proteins 0.000 description 5
- 210000000170 cell membrane Anatomy 0.000 description 5
- 229940118019 malondialdehyde Drugs 0.000 description 5
- 230000008901 benefit Effects 0.000 description 4
- 239000003153 chemical reaction reagent Substances 0.000 description 4
- 238000009472 formulation Methods 0.000 description 4
- 230000019100 sperm motility Effects 0.000 description 4
- HRXKRNGNAMMEHJ-UHFFFAOYSA-K trisodium citrate Chemical compound [Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O HRXKRNGNAMMEHJ-UHFFFAOYSA-K 0.000 description 4
- 238000009395 breeding Methods 0.000 description 3
- 230000001488 breeding effect Effects 0.000 description 3
- 238000005516 engineering process Methods 0.000 description 3
- 239000007788 liquid Substances 0.000 description 3
- 230000003647 oxidation Effects 0.000 description 3
- 238000007254 oxidation reaction Methods 0.000 description 3
- 230000036542 oxidative stress Effects 0.000 description 3
- DOUMFZQKYFQNTF-WUTVXBCWSA-N (R)-rosmarinic acid Chemical compound C([C@H](C(=O)O)OC(=O)\C=C\C=1C=C(O)C(O)=CC=1)C1=CC=C(O)C(O)=C1 DOUMFZQKYFQNTF-WUTVXBCWSA-N 0.000 description 2
- 230000007547 defect Effects 0.000 description 2
- 238000007710 freezing Methods 0.000 description 2
- 230000008014 freezing Effects 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- 239000000843 powder Substances 0.000 description 2
- 238000003825 pressing Methods 0.000 description 2
- 238000007789 sealing Methods 0.000 description 2
- 239000000243 solution Substances 0.000 description 2
- SNKFFCBZYFGCQN-UHFFFAOYSA-N 2-[3-[3-[1-carboxy-2-(3,4-dihydroxyphenyl)ethoxy]carbonyl-2-(3,4-dihydroxyphenyl)-7-hydroxy-2,3-dihydro-1-benzofuran-4-yl]prop-2-enoyloxy]-3-(3,4-dihydroxyphenyl)propanoic acid Chemical compound C=1C=C(O)C=2OC(C=3C=C(O)C(O)=CC=3)C(C(=O)OC(CC=3C=C(O)C(O)=CC=3)C(O)=O)C=2C=1C=CC(=O)OC(C(=O)O)CC1=CC=C(O)C(O)=C1 SNKFFCBZYFGCQN-UHFFFAOYSA-N 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- 208000035473 Communicable disease Diseases 0.000 description 1
- SNKFFCBZYFGCQN-VWUOOIFGSA-N Lithospermic acid B Natural products C([C@H](C(=O)O)OC(=O)\C=C\C=1C=2[C@H](C(=O)O[C@H](CC=3C=C(O)C(O)=CC=3)C(O)=O)[C@H](OC=2C(O)=CC=1)C=1C=C(O)C(O)=CC=1)C1=CC=C(O)C(O)=C1 SNKFFCBZYFGCQN-VWUOOIFGSA-N 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- ZZAFFYPNLYCDEP-HNNXBMFYSA-N Rosmarinsaeure Natural products OC(=O)[C@H](Cc1cccc(O)c1O)OC(=O)C=Cc2ccc(O)c(O)c2 ZZAFFYPNLYCDEP-HNNXBMFYSA-N 0.000 description 1
- 239000000654 additive Substances 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000005540 biological transmission Effects 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 239000003792 electrolyte Substances 0.000 description 1
- 230000037149 energy metabolism Effects 0.000 description 1
- 238000011049 filling Methods 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 210000004392 genitalia Anatomy 0.000 description 1
- 239000011521 glass Substances 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 230000000670 limiting effect Effects 0.000 description 1
- 244000144972 livestock Species 0.000 description 1
- 230000004899 motility Effects 0.000 description 1
- 230000003204 osmotic effect Effects 0.000 description 1
- 239000006174 pH buffer Substances 0.000 description 1
- 238000004806 packaging method and process Methods 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- 235000020610 powder formula Nutrition 0.000 description 1
- 239000003223 protective agent Substances 0.000 description 1
- 230000002829 reductive effect Effects 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- DOUMFZQKYFQNTF-MRXNPFEDSA-N rosemarinic acid Natural products C([C@H](C(=O)O)OC(=O)C=CC=1C=C(O)C(O)=CC=1)C1=CC=C(O)C(O)=C1 DOUMFZQKYFQNTF-MRXNPFEDSA-N 0.000 description 1
- TVHVQJFBWRLYOD-UHFFFAOYSA-N rosmarinic acid Natural products OC(=O)C(Cc1ccc(O)c(O)c1)OC(=Cc2ccc(O)c(O)c2)C=O TVHVQJFBWRLYOD-UHFFFAOYSA-N 0.000 description 1
- STCJJTBMWHMRCD-UHFFFAOYSA-N salvianolic acid B Natural products OC(=O)C(Cc1ccc(O)c(O)c1)OC(=O)C=Cc2cc(O)c(O)c3OC(C(C(=O)OC(Cc4ccc(O)c(O)c4)C(=O)O)c23)c5ccc(O)c(O)c5 STCJJTBMWHMRCD-UHFFFAOYSA-N 0.000 description 1
- 230000001954 sterilising effect Effects 0.000 description 1
- YMBCJWGVCUEGHA-UHFFFAOYSA-M tetraethylammonium chloride Chemical compound [Cl-].CC[N+](CC)(CC)CC YMBCJWGVCUEGHA-UHFFFAOYSA-M 0.000 description 1
Classifications
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N1/00—Preservation of bodies of humans or animals, or parts thereof
- A01N1/10—Preservation of living parts
- A01N1/12—Chemical aspects of preservation
- A01N1/122—Preservation or perfusion media
- A01N1/126—Physiologically active agents, e.g. antioxidants or nutrients
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Dentistry (AREA)
- General Health & Medical Sciences (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Environmental Sciences (AREA)
- Agricultural Chemicals And Associated Chemicals (AREA)
Abstract
本发明公开了一种用于猪精液常温保存的稀释制剂，制得的该制剂由以下组分按重量份组成：D‑果糖：35.0～39.0份，碳酸氢钠：5.5～6.5份，柠檬酸钠：1.2～1.3份，乙二胺四乙酸(EDTA)：1.2～1.3份，氯化钾：0.7～0.8份，庆大霉素：0.18～0.22份，丹参素：5.5～6.5份。使用时，先将猪精液常温保存的稀释制剂溶解于1000mL双蒸水或超纯水中，在35℃～37℃的恒温水浴锅中平衡30‑45分钟，然后调节pH至6.8～7.0，制得稀释液。然后再对猪精液用于常温保存的常规或者高倍稀释，分装入瓶，保存。其配方简单，成本低，可以提高精子的抗氧化能力，保存效果好，可以提高优秀种公猪的利用效率。The invention discloses a dilution preparation for preservation of pig semen at room temperature. The prepared preparation consists of the following components by weight: D-fructose: 35.0-39.0 parts, sodium bicarbonate: 5.5-6.5 parts, citric acid Sodium: 1.2-1.3 parts, ethylenediaminetetraacetic acid (EDTA): 1.2-1.3 parts, potassium chloride: 0.7-0.8 parts, gentamicin: 0.18-0.22 parts, Danshensu: 5.5-6.5 parts. When using, first dissolve the diluted preparation of pig semen stored at room temperature in 1000mL of double distilled water or ultrapure water, equilibrate in a constant temperature water bath at 35°C to 37°C for 30-45 minutes, and then adjust the pH to 6.8 to 7.0 to prepare. Diluent is obtained. Then, the pig semen is used for routine or high-fold dilution for normal temperature preservation, and is divided into bottles and stored. The formula is simple, the cost is low, the antioxidant capacity of sperm can be improved, the preservation effect is good, and the utilization efficiency of excellent boars can be improved.
Description
Technical Field
The invention belongs to the technical field of artificial insemination of livestock, and particularly relates to a diluting preparation for preserving boar semen at normal temperature.
Background
The pig demand in China is very large, and the pig industry is an important component of the animal husbandry in China. With the development of the pig raising industry in recent years, the artificial insemination technology of pigs is widely applied in production practice. The artificial insemination technology can fully exert the advantages of excellent boars; the cost for raising the breeding boars is saved; is beneficial to accelerating the variety popularization; effectively preventing the transmission of the genital infectious diseases. However, the quality of the pig semen preservation effect is always an important factor for limiting the superiority of excellent breeding boars.
The known methods for preserving the boar semen mainly comprise liquid preservation and freezing preservation, wherein the freezing preservation can preserve the semen for a long time, but the semen is sensitive to temperature, so that the semen is iced and crystallized, and the semen quality is rapidly reduced. The frozen storage is adopted, and the technical and equipment requirements are higher. The normal temperature storage has the advantages of simplicity, convenience and the like. The existing common normal-temperature preservation diluent mainly comprises D-fructose (meeting the energy requirement of sperms), sodium citrate (a protective agent for reducing electrolyte in semen), gentamicin and the like (inhibiting bacteria and sterilizing), and sodium bicarbonate (regulating osmotic pressure balance). There are studies that show that the influence of oxidative stress on semen quality is larger, but the current semen diluent formula has little protection on the sperm oxidative stress, and there are studies that show that the addition of antioxidant can compensate the defect. Therefore, the development of a novel diluted preparation for preserving the boar semen at normal temperature based on the addition of the antioxidant becomes the key for popularizing the application of the artificial insemination technology.
Disclosure of Invention
Aiming at the defects or shortcomings of the existing pig semen preservation preparation, the invention aims to provide a novel pig semen normal-temperature preservation dilution preparation based on addition of an antioxidant, so as to make up the shortcomings of the existing pig semen dilution powder formula, improve the quality of a pig semen preservation sample, prolong the semen preservation time, improve the utilization rate of excellent boars, reduce the raising cost and improve the economic benefit.
In order to realize the task, the invention is realized by the following technical scheme:
a diluting preparation for preserving boar semen at normal temperature is characterized in that the prepared preparation consists of the following components in parts by weight: d-fructose: 35.0-39.0 parts of sodium bicarbonate: 5.5-6.5 parts of sodium citrate: 1.2-1.3 parts of Ethylene Diamine Tetraacetic Acid (EDTA): 1.2-1.3 parts, potassium chloride: 0.7-0.8 parts of gentamicin: 0.18-0.22 parts, danshensu: 5.5 to 6.5 portions.
Further, the prepared formula preparation comprises the following components in parts by weight: d-fructose: 37.0 parts, sodium bicarbonate: 6.0 parts, sodium citrate: 1.25 parts, ethylenediaminetetraacetic acid (EDTA): 1.25 parts, potassium chloride: 0.75 part, gentamicin: 0.20 part, danshensu: 6.0 parts.
The components are all analytical pure chemicals which are sieved by a sieve of 80 meshes.
The preparation method of the diluted preparation for preserving the boar semen at normal temperature accurately weighs the components under the conditions that the temperature is 18-26 ℃ and the relative humidity is 45-65%, and the diluted preparation is obtained by mixing, stirring and vacuum packaging according to the proportion.
When the diluted preparation for preserving the boar semen at the normal temperature is applied, the diluted preparation for preserving the boar semen at the normal temperature is dissolved in 1000mL of double distilled water or ultrapure water, is balanced for 30-45 minutes in a constant-temperature water bath kettle at 35-37 ℃, and then the pH is adjusted to 6.8-7.0 to prepare the diluted solution. Then the boar semen is used for routine or high-power dilution for normal temperature preservation, and is subpackaged into bottles for preservation.
The diluting preparation for preserving the boar semen at normal temperature can effectively reduce the damage of oxidative stress to the semen in the preservation process, improve the preservation quality of the semen and prolong the preservation time of the semen. The boar semen adopts the diluted preparation stored at normal temperature, is stored at 17 ℃ after being subpackaged, has the sperm activity of more than 70 percent after 5 days, can meet the production requirement, and can improve the utilization efficiency of excellent boars. The composition is simple in components and low in cost, can improve the oxidation resistance of sperms, is long in preservation time and good in preservation effect, is expected to improve the benefits of pig farms, and promotes the development of the pig industry and breeding science in China.
Detailed Description
The present invention will be described in further detail with reference to specific examples given by the inventors.
The embodiment provides a diluted preparation for preserving boar semen at normal temperature, and the prepared preparation consists of the following components in parts by weight: d-fructose: 35.0-39.0 parts of sodium bicarbonate: 5.5-6.5 parts of sodium citrate: 1.2-1.3 parts of Ethylene Diamine Tetraacetic Acid (EDTA): 1.2-1.3 parts, potassium chloride: 0.7-0.8 parts of gentamicin: 0.18-0.22 parts, danshensu: 5.5 to 6.5 portions.
The components are all analytical pure chemicals which are sieved by a sieve of 80 meshes;
the preparation method of the diluted preparation preserved at normal temperature by the pig semen comprises the steps of accurately weighing the components, mixing the components according to the proportion, uniformly stirring the components and carrying out vacuum packaging to obtain the diluted preparation; the operations of weighing, mixing, stirring and vacuum packaging are carried out at the temperature of 18-26 ℃ and the relative humidity of 45-65%.
When the diluent is used, the diluent stored at normal temperature of the boar semen is dissolved in 1000mL of double distilled water or ultrapure water, the solution is balanced in a constant-temperature water bath kettle at the temperature of 35-37 ℃ for 30-45 minutes, and then the pH value is adjusted to 6.8-7.0 to prepare the diluent. Then the boar semen is used for routine or high-power dilution for normal temperature preservation, and is subpackaged into bottles for preservation.
When the diluted preparation for preserving the boar semen at the normal temperature is applied to low-power dilution of the boar semen (such as 1: 1-1: 4), the diluted preparation can be prepared by the following steps: the dilution ratio of the diluted preparation stored at the normal temperature of the boar semen is directly added into the semen; when the method is applied to high-power dilution of the pig semen (for example, the pig semen: a diluted preparation stored at the normal temperature of the pig semen is 1: 5 or more), the dilution is carried out according to the following steps:
(1) according to the pig semen: 1, diluting preparation for preserving boar semen at normal temperature: 1, slowly adding the diluent into the semen;
(2) diluting the liquid diluted in the step (1) step by step according to a high-fold dilution ratio to obtain the product. For example, when semen: 1, diluting preparation for preserving boar semen at normal temperature: a high dilution of 5 requires two dilutions, namely: firstly, semen is carried out: 1, diluting preparation for preserving boar semen at normal temperature: 1 dilution, then diluting again to 1: 5. the formula of the diluting preparation for preserving the boar semen at normal temperature mainly provides a better energy metabolism and pH buffer system for the semen, and is used for producing the semen at normal temperature together with other additives.
The application method of the diluted preparation for preserving the boar semen at the normal temperature in the embodiment comprises the following steps:
1. filling the diluted semen into a bottle, sealing the bottle cap, keeping flat in the dark, and storing in a refrigerator at the normal temperature of 17 ℃;
2. the sperm is preferably turned over once every 12 hours during the preservation period so as to prevent the damage caused by the mutual stacking and pressing of the standing sperm for a long time;
3. the diluted preparation stored at normal temperature of the boar semen can be prepared half an hour before the semen is diluted.
The following are specific examples given by the inventors.
Example 1:
the raw materials were prepared according to the diluted preparation for normal-temperature preservation of porcine semen given in table 1:
table 1: diluent formula for preserving boar semen at normal temperature
| Reagent | Dosage of |
| D-fructose | 37.00g |
| Citric acid sodium salt | 6.00g |
| Carbonic acid sodium salt | 1.25g |
| Potassium chloride | 0.75g |
| Gentamicin | 0.20g |
| Salvianic acid A | 6.00g |
| Ethylenediaminetetraacetic acid (EDTA) | 1.25g |
Various chemicals (analytically pure), large particles or already agglomerated chemicals in the formulation of the commercial formulation need to be crushed and sieved through an 80 mesh sieve to form a powder.
The preparation operation process comprises the steps of accurately weighing various components, mixing according to the proportion in the formula, fully stirring, packaging into a bag, vacuumizing and sealing to obtain a bag of the diluted boar semen powder, wherein the temperature is generally controlled to be 18-26 ℃, and the relative humidity is controlled to be 45-65%.
Under normal conditions, a diluted preparation stored at the normal temperature of the boar semen is prepared half an hour before the dilution of the boar semen, a pack of the diluted preparation stored at the normal temperature of the boar semen is put into 1000ml of double distilled water, the mixture is continuously stirred by a magnetic stirrer until the diluted preparation is completely dissolved, the pH value is adjusted to be 6.8, and the mixture is heated to 37 ℃.
According to the sperm motility and density, the dilution ratio is adjusted, the diluted preparation stored at the normal temperature of the boar semen is slowly added into the semen, and if high-power dilution is carried out (for example, the diluted preparation stored at the normal temperature of the boar semen: the semen is 1: 5), the dilution is carried out in two steps:
(1) according to the following steps of 1: 1, slowly adding a diluted preparation preserved at normal temperature into the semen;
(2) gradually diluting the liquid diluted in the step (1) according to a high-fold dilution ratio.
The diluted preparation and the semen which are stored at normal temperature are respectively bottled, screwed, protected from light and laid flat, and then are stored at normal temperature of 17 ℃ in a refrigerator. Turning over once every 12 hours to prevent the sperms from being placed still for a long time and being damaged by mutual stacking and pressing.
Example 2:
the raw materials were prepared according to the diluted preparation for normal-temperature preservation of porcine semen given in table 2 below:
table 2: diluent formula for preserving boar semen at normal temperature
| Reagent | Dosage of |
| D-fructose | 37.00g |
| Citric acid sodium salt | 6.00g |
| Carbonic acid sodium salt | 1.25g |
| Potassium chloride | 0.75g |
| Gentamicin | 0.20g |
| Ethylenediaminetetraacetic acid (EDTA) | 1.25g |
The preparation operation is the same as that in example 1, diluted preparation stored at normal temperature of the boar semen is prepared half an hour before the dilution of the semen, a packet of diluted preparation stored at normal temperature of the boar semen is put into 1000ml of double distilled water, the mixture is continuously stirred by a magnetic stirrer until the diluted preparation is completely dissolved, the pH value is adjusted to 7.0, and the mixture is heated to 37 ℃.
According to the sperm activity and density, the dilution ratio of the semen and the diluted preparation stored at the normal temperature of the boar semen is adjusted, and the diluted preparation stored at the normal temperature of the boar semen is slowly added into the semen.
Example 3:
the diluted semen and the diluted preparation stored at the normal temperature of the boar semen in the embodiment 1-2 are adopted to test the activity change condition of the diluted preparation stored at the temperature of 17 ℃.
The specific implementation process is as follows:
taking the pig semen and a diluted preparation stored at the normal temperature of the pig semen, and firstly, mixing the raw materials according to the weight ratio of 1: 1, diluting according to the proportion of 1: 5 and stored at 17 ℃, sperm motility was observed every 24 hours and motility changes were recorded (10 replicates). The results are shown in Table 4:
table 3: change in Normal temperature (17 ℃ C.) storage Activity
| Retention time | Example 1 | Example 2 |
| 2h | 90.56±0.63 | 87.62±1.07 |
| 24h | 86.15±0.68 | 77.46±1.34 |
| 48h | 80.35±0.58 | 77.43±0.81 |
| 72h | 79.13±0.71 | 73.18±0.18 |
| 96h | 76.59±0.67 | 71.83±0.78 |
| 120h | 72.21±1.63 | 63.31±1.98 |
As can be seen from Table 3, the sperm motility of example 1 is still above 70% after the storage for 120 hours (5 days), which indicates that the formula has good effect on diluting and storing the boar semen due to the addition of danshensu.
Example 4:
the diluted preparation stored at normal temperature of the semen and the boar semen in the embodiment 1-2 is adopted to store the change condition of the plasma membrane integrity at 17 ℃.
The specific implementation process is as follows:
taking the pig semen and a diluted preparation stored at the normal temperature of the pig semen, and firstly, mixing the raw materials according to the weight ratio of 1: 1, diluting according to the proportion of 1: 5, storing at 17 ℃, observing and measuring the integrity rate of the plasma membrane of the semen at 2h, 24h, 72h and 120h, and recording the change condition of the integrity rate of the plasma membrane. The results are shown in Table 4:
table 4: change in plasma Membrane integrity Rate at Normal temperature (17 ℃ C.) storage
| Retention time | Example 1 | Example 2 |
| 2h | 79.64±0.69 | 74.34±0.99 |
| 24h | 71.91±0.85 | 65.79±1.54 |
| 72h | 68.59±0.84 | 59.40±1.09 |
| 120h | 58.32±1.01 | 52.20±3.19 |
As can be seen from Table 4, the integrity of the plasma membrane of the sperm in example 1 is still above 58% after the sperm is preserved for 120 hours (5 days), which indicates that the formulation has good effect on dilution and preservation of the boar semen due to the addition of danshensu.
Example 5:
the diluted preparation for storing the semen and the boar semen at normal temperature in the embodiment 1-2 is adopted to store the change condition of the top and bottom integrity rate at 17 ℃.
The specific implementation process is as follows:
taking the pig semen and a diluted preparation stored at the normal temperature of the pig semen, and firstly, mixing the raw materials according to the weight ratio of 1: 1, diluting according to the proportion of 1: 5, storing at 17 ℃, observing and measuring the integrity rate of the sperm acrosome at 2h, 24h, 72h and 120h, and recording the change condition of the integrity rate of the sperm acrosome. The results are shown in Table 5:
table 5: preservation of Change in roof integrity at Normal temperature (17 ℃ C.)
| Retention time | Example 1 | Example 2 |
| 2h | 88.57±1.37 | 89.77±0.73 |
| 24h | 85.26±0.39 | 84.09±1.09 |
| 72h | 79.68±0.33 | 73.76±1.14 |
| 120h | 67.83±0.92 | 62.61±0.86 |
As can be seen from Table 5, the sperm acrosome integrity of example 1 is still over 67% after being preserved for 120 hours (5 days), which indicates that the formula has good effect on dilution and preservation of the boar semen due to the addition of danshensu.
Example 6:
the diluted preparation of the semen and the boar semen stored at normal temperature in the embodiment 1-2 is adopted to test the change condition of the total oxidation resistance (T-AOC) stored at 17 ℃.
The specific implementation process is as follows:
taking the pig semen and a diluted preparation stored at the normal temperature of the pig semen, and firstly, mixing the raw materials according to the weight ratio of 1: 1, diluting according to the proportion of 1: 5, diluting, storing at 17 deg.C, observing semen total antioxidant capacity for 2h, 24h, 72h, and 120h, and recording total antioxidant capacity change. The total antioxidant capacity measured corresponds to the standard substance TEAC in mM. The results are shown in Table 6.
Table 6: changes in Total Oxidation resistance at Normal temperature (17 deg.C)
| Retention time | Example 1 | Example 2 |
| 0h | 1.76±0.0014 | 1.76±0.0022 |
| 24h | 1.76±0.0025 | 1.75±0.0040 |
| 72h | 1.75±0.0018 | 1.74±3.503e |
| 120h | 1.66±0.0134 | 1.58±0.0115 |
As can be seen from Table 6, after 120 hours (5 days) of storage, the semen of example 1 has higher total antioxidant capacity than the semen of example 2, indicating that the effect of adding danshensu in the formula to dilute and store the pig semen is very good.
Example 7:
the diluted preparations of the semen and the boar semen stored at normal temperature in the embodiments 1-2 are adopted to test the change condition of malondialdehyde content (MDA) stored at 17 ℃. The specific implementation process is as follows:
taking the pig semen and a diluted preparation stored at the normal temperature of the pig semen, and firstly, mixing the raw materials according to the weight ratio of 1: 1, diluting according to the proportion of 1: 5, storing at 17 ℃, observing the content of the malondialdehyde in the semen at 2h, 24h, 72h and 120h, and recording the change of the content of the malondialdehyde. The unit is μ M. The results are shown in Table 7.
Table 7: preservation of the Change in the malondialdehyde content at ambient temperature (17 ℃ C.)
| Retention time | Example 1 | Example 1 |
| 0h | 1.01±0.0912 | 1.14±0.1040 |
| 24h | 1.81±0.1649 | 1.68±0.2077 |
| 72h | 4.34±0.4849 | 4.71±0.7331 |
| 120h | 8.16±1.1395 | 12.50±0.6186 |
As can be seen from Table 7, the malondialdehyde content in the semen of example 1 is lower than that in the semen of example 2 after 120 hours (5 days) of storage, which indicates that the effect of adding danshensu in the formula on the dilution and storage of the boar semen is very good.
Example 8:
the diluted preparations of the semen and the boar semen stored at normal temperature in the embodiments 1-2 are adopted to test the change condition of Catalase Activity (CAT) stored at 17 ℃. The specific implementation process is as follows:
taking the pig semen and a diluted preparation stored at the normal temperature of the pig semen, and firstly, mixing the raw materials according to the weight ratio of 1: 1, diluting according to the proportion of 1: 5, storing at 17 ℃, observing the activity of the catalase in the semen at 2h, 24h, 72h and 120h, and recording the change of the catalase activity. The unit is μ M. The results are shown in Table 8.
Table 8: preservation of Catalase Activity changes at ambient temperature (17 ℃ C.)
| Retention time | Example 1 | Example 2 |
| 0h | 8.94±0.2347 | 12.96±0.7463 |
| 24h | 8.13±0.1565 | 11.20±0.4140 |
| 72h | 7.00±0.0808 | 5.28±0.1355 |
| 120h | 3.22±0.0694 | 2.64±0.1162 |
As can be seen from Table 8, the activity of catalase in the semen of example 1 after 120 hours (5 days) of storage is higher than that of the semen of example 2, indicating that the addition of danshensu in the formula has a good effect on the dilution storage of the pig semen.
Example 9:
the diluted preparation stored at normal temperature of the semen and the boar semen as described in the embodiment 1-2 is adopted, and the artificial insemination is carried out after the diluted preparation is stored at 17 ℃ for 5 days. The specific implementation process is as follows:
taking the pig semen and a diluted preparation stored at the normal temperature of the pig semen, and firstly, mixing the raw materials according to the weight ratio of 1: 1, diluting according to the proportion of 1: 5, storing at 17 ℃ until 5 days for artificial insemination. The 32 healthy primiparous sows are randomly divided into two groups, one group is subjected to artificial insemination by using the diluent preserved at the normal temperature of the boar semen of the embodiment 1 and the semen, the other group is subjected to artificial insemination by using the diluent preserved at the normal temperature of the boar semen of the embodiment 2 and the semen, and the pregnancy rate of the sows is checked after 28 days of insemination. The results are shown in Table 9.
Table 9: effect of Normal temperature (17 ℃) preservation of pregnancy Rate
As can be seen from table 9, when artificial insemination was performed after 120 hours (5 days) of storage, the pregnancy rate in example 1 was 87.5, the pregnancy rate in example 2 was 81.25, and the pregnancy rate in example 1 was 6.25 higher than that in example 2. The result shows that the effect of diluting and storing the boar semen by adding the danshensu in the formula is good, and the sow pregnancy rate is improved.
Example 10:
this example is a test of the effect of a diluted preparation stored at normal temperature on the normal temperature (17 ℃) storage activity of different breeds of porcine semen. The specific implementation process is as follows:
taking different pure boar semen (5 each), respectively using the boar semen of example 1 and the diluted preparation stored at normal temperature of the boar semen in the following proportion of 1: 5 dilution and storage at 17 ℃ and observation of sperm motility at 24-hour intervals, the results of which are shown in Table 10.
Table 10: influence of preparation on normal temperature (17 ℃) preservation activity of different breeds of boar semen
| Retention time | White spirit | Changbai (white and long) | Duroc |
| 2h | 90.56±0.63 | 93.58±1.22 | 94.61±0.95 |
| 24h | 86.15±0.68 | 89.80±0.94 | 93.06±1.33 |
| 48h | 80.35±0.58 | 85.90±0.38 | 88.27±1.70 |
| 72h | 79.13±0.71 | 83.81±1.33 | 83.40±1.64 |
| 96h | 76.59±0.67 | 75.52±0.83 | 77.27±2.21 |
| 120h | 72.21±1.63 | 65.20±1.68 | 70.85±2.58 |
As can be seen from Table 10, the diluted preparation preserved at normal temperature by using the boar semen of the present invention has a good effect on the preservation of the existing commercial boar semen after the boar semen is preserved for 120 hours (5 days).
Example 11:
the raw materials were prepared according to the diluted preparation for normal-temperature preservation of boar semen given in table 11 below:
table 11: diluent formula for preserving boar semen at normal temperature (17 ℃), and preparation method thereof
| Reagent | Dosage of |
| D-fructose | 37.00g |
| Citric acid sodium salt | 6.00g |
| Carbonic acid sodium salt | 1.25g |
| Potassium chloride | 0.75g |
| Gentamicin | 0.20g |
| Salvianolic acid b | 43.00g |
| Ethylenediaminetetraacetic acid (EDTA) | 1.25g |
The preparation operation is the same as that in example 1, diluted preparation stored at normal temperature of the boar semen is prepared half an hour before the dilution of the semen, a packet of diluted preparation stored at normal temperature of the boar semen is put into 1000ml of double distilled water, the mixture is continuously stirred by a magnetic stirrer until the diluted preparation is completely dissolved, the pH value is adjusted to 7.0, and the mixture is heated to 37 ℃.
According to the sperm activity and density, the dilution ratio of the semen and the diluted preparation stored at the normal temperature of the boar semen is adjusted, and the diluted preparation stored at the normal temperature of the boar semen is slowly added into the semen.
Example 12:
the raw materials were prepared according to the diluted preparation for normal-temperature preservation of boar semen given in table 12 below:
table 12: diluent formula for preserving boar semen at normal temperature (17 ℃), and preparation method thereof
| Reagent | Dosage of |
| D-fructose | 37.00g |
| Citric acid sodium salt | 6.00g |
| Carbonic acid sodium salt | 1.25g |
| Potassium chloride | 0.75g |
| Gentamicin | 0.20g |
| Rosmarinic acid | 27.00g |
| Ethylenediaminetetraacetic acid (EDTA) | 1.25g |
The preparation operation is the same as that in example 1, diluted preparation stored at normal temperature of the boar semen is prepared half an hour before the dilution of the semen, a packet of diluted preparation stored at normal temperature of the boar semen is put into 1000ml of double distilled water, the mixture is continuously stirred by a magnetic stirrer until the diluted preparation is completely dissolved, the pH value is adjusted to 7.0, and the mixture is heated to 37 ℃.
According to the sperm activity and density, the dilution ratio of the semen and the diluted preparation stored at the normal temperature of the boar semen is adjusted, and the diluted preparation stored at the normal temperature of the boar semen is slowly added into the semen.
Example 13:
this example is a test of the effect of different diluted preparations stored at room temperature on the normal temperature (17 ℃) storage activity of porcine semen.
The semen activity change condition test at 17 ℃ is carried out by using the diluted preparations stored at the normal temperature of the semen and the boar semen in the examples 1, 11 and 12.
The specific implementation process is as follows:
taking the pig semen and a diluted preparation stored at the normal temperature of the pig semen, and firstly, mixing the raw materials according to the weight ratio of 1: 1, diluting according to the proportion of 1: 5, storing at 17 ℃, detecting the activity of the semen at 2h, 24h, 48h, 72h, 96h and 120h, and recording the change of the activity of the semen, wherein the results are shown in Table 13.
Table 13: effect of different antioxidants on Activity of Normal temperature (17 ℃) preserved boar semen
| Retention time | Example 1 | Example 11 | Example 12 |
| 2h | 90.56±0.63 | 93.39±0.48 | 91.36±1.00 |
| 24h | 86.15±0.68 | 83.31±0.34 | 80.46±1.45 |
| 48h | 80.35±0.58 | 79.56±1.60 | 80.69±2.26 |
| 72h | 79.13±0.71 | 78.98±1.67 | 81.54±1.85 |
| 96h | 76.59±0.67 | 75.88±0.44 | 73.91±2.20 |
| 120h | 72.21±1.63 | 70.62±1.08 | 66.59±1.38 |
As can be seen from Table 13, the diluted preparation of the present invention, which is stored at normal temperature after 120 hours (5 days), has the best effect of improving semen quality due to the addition of danshensu.
The application method of the diluted preparation stored at the normal temperature by the boar semen in the embodiment 1 comprises the following steps:
1. semen collection and detection:
collecting semen from the middle concentrated part of semen by hand-held method, timely delivering the collected semen to laboratory for detecting activity, color, smell and density, and filtering qualified semen with sterile gauze.
2. And (3) semen dilution:
the filtered qualified semen is immediately diluted by the diluted preparation stored at normal temperature in the boar semen of the embodiment 1. Diluting by 1-10 times according to the density and activity of the semen, and ensuring that the density of the diluted semen is 106And/m L. The boar semen of example 1 is preserved at normal temperatureThe diluted formulation of (a) was slowly poured along a glass rod into the semen in several portions. The first time of the dilution is carried out in equal quantity, the second time of the dilution is carried out after one minute of the interval, the second time of the dilution is added with a dilution preparation which is twice of the boar semen and is preserved at normal temperature, and the dilution preparation which is preserved at normal temperature is completely added after one minute of the interval. The temperature of the semen is kept consistent with that of a diluted preparation stored at normal temperature of the boar semen as far as possible during dilution.
3. And (3) semen preservation:
wrapping the semen diluted by the diluted preparation stored at normal temperature with a towel, storing in a refrigerator at 17 deg.C, shaking gently every 12 hr, and slightly tilting the bottle when storing the semen.
When artificial insemination is performed, the bottle body is slightly shaken to enable semen to float upwards.
Claims (2)
1. A diluted preparation for preserving boar semen at normal temperature is characterized in that the prepared preparation comprises the following components in percentage by weight: d-fructose: 37.0 parts, sodium bicarbonate: 6.0 parts, sodium citrate: 1.25 parts, ethylenediaminetetraacetic acid (EDTA): 1.25 parts, potassium chloride: 0.75 part, gentamicin: 0.20 part, danshensu: 6.0 parts of (B);
accurately weighing the components at the temperature of 18-26 ℃ and the relative humidity of 45-65%, mixing the components according to the proportion, stirring the components uniformly, and carrying out vacuum packaging to obtain the traditional Chinese medicine composition.
2. The diluted preparation for ordinary-temperature preservation of boar semen according to claim 1, wherein the components are analytical pure chemicals which are sieved by a sieve of 80 meshes.
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| CN110574760A (en) * | 2019-08-22 | 2019-12-17 | 西北农林科技大学 | Application of rosmarinic acid as the active ingredient of boar semen antibacterial preservation solution at room temperature |
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