CN111226907A - Preparation method of diluent for normal-temperature preservation of sheep semen - Google Patents
Preparation method of diluent for normal-temperature preservation of sheep semen Download PDFInfo
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- CN111226907A CN111226907A CN202010068814.4A CN202010068814A CN111226907A CN 111226907 A CN111226907 A CN 111226907A CN 202010068814 A CN202010068814 A CN 202010068814A CN 111226907 A CN111226907 A CN 111226907A
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- 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
- A01N1/02—Preservation of living parts
- A01N1/0205—Chemical aspects
- A01N1/021—Preservation or perfusion media, liquids, solids or gases used in the preservation of cells, tissue, organs or bodily fluids
- A01N1/0221—Freeze-process protecting agents, i.e. substances protecting cells from effects of the physical process, e.g. cryoprotectants, osmolarity regulators like oncotic agents
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Abstract
The invention relates to a preparation method of a diluent for preserving sheep semen at normal temperature, which comprises the following steps: step 1), dissolving 30.2-31.2 g of Tris, 15.9-16.9 g of citric acid, 19.5-20.5 g of fructose, 0.3-0.4 g of penicillin sodium and 0.6-0.7 g of streptomycin sulfate in 1000mL of sterilized ultrapure water to obtain a basic diluent; step 2), when 95mL of basic diluent is taken, 5mL of yolk is added into 95mL of basic diluent to obtain first diluent; when 85mL of basic diluent is taken, 15mL of yolk is added into 85mL of basic diluent to obtain second diluent; when 80mL of basic diluent is taken, 20mL of yolk is added into the 80mL of basic diluent to obtain a third diluent; and (3) putting the first diluent, the second diluent and the third diluent into a refrigerator to be fully dissolved, then centrifuging for 20min at the temperature of 4 ℃ at 10000r/min, and taking out supernate after centrifugation to obtain the yolk diluents with the concentration of 5%, 15% and 20%. The diluent provided by the invention improves the semen preservation effect.
Description
Technical Field
The invention relates to a preparation method of a diluent for preserving sheep semen at normal temperature, belonging to the technical field of sheep breeding.
Background
The artificial fertilization technology is a basic technology in livestock breeding, can fully utilize semen of male livestock of superior variety, and is an important means for improving reproductive efficiency and productivity of livestock. The artificial insemination technology can reduce the breeding cost, prevent the spread of diseases and realize the large-scale breeding and management. Semen preservation is a key link in the artificial insemination technology. Semen preservation is generally classified into normal temperature preservation, low temperature preservation and freezing preservation. In the actual production, the semen is mostly stored at normal temperature, and the method has the advantages of simple processing operation process, suitability for popularization and application, no need of special temperature control and refrigeration equipment and the like, and has actual application value. The sperm motility of the conventional diluent formula for preserving sheep semen at normal temperature is rapidly reduced after being preserved for 96 hours, and the semen quality is seriously influenced.
Yolk is usually added in the semen cryopreservation and the ultra-low temperature freezing preservation, and the yolk can be added in the semen diluent of the pig, the dog, the rabbit and other species. Different egg yolks such as eggs, duck eggs, etc. are also used in the preservation of semen of different mammals. The yolk can play two roles of an anti-freezing protective agent and energy supply. Phihphs adds egg yolk as an antifreeze protective agent to the diluent at the earliest, effectively reduces the damage to sperm cells in the cooling process and obtains better effect. The low-density lipoprotein in the yolk is gathered on the surface of a sperm membrane, so that the cold shock of the sperm is reduced, and the yolk plays a key role in the semen cryopreservation. In addition, the yolk can regulate osmotic pressure and electrolyte concentration in the diluent, so that the environment is suitable for semen preservation, the influence on a sperm membrane is reduced, and the effective survival time and the total survival time are prolonged. Since the yolk can play a certain role in cryopreservation and freezing preservation of semen, the research on the cryopreservation of sheep semen is rarely reported.
Therefore, the diluent formula capable of improving the normal-temperature preservation quality of the sheep semen needs to be invented by researching the action of the yolk, so that the efficiency of the artificial fertilization technology is improved, the semen preservation cost is reduced, and the requirement of actual production is met.
Disclosure of Invention
The invention aims to solve the existing problems, provides a preparation method of a diluent for preserving sheep semen at normal temperature, provides an application effect of egg yolk in a diluent formula for preserving sheep semen at normal temperature, is used for solving the problem that the sperm motility is rapidly reduced in the later stage of preserving sheep semen in the prior art, prolongs the semen preservation time, and selects diluent formulas with different effects according to different time requirements.
The invention aims to realize the purpose, and the preparation method of the diluent for preserving the sheep semen at normal temperature is characterized by comprising the following steps of:
step 1), dissolving 30.2-31.2 g of Tris, 15.9-16.9 g of citric acid, 19.5-20.5 g of fructose, 0.3-0.4 g of penicillin sodium and 0.6-0.7 g of streptomycin sulfate in 1000mL of sterilized ultrapure water to obtain a basic diluent;
step 2), when 95mL of the basic diluent prepared in the step 1) is taken, adding 5mL of egg yolk into 95mL of the basic diluent to obtain a first diluent, then putting the first diluent into a refrigerator to be fully dissolved, centrifuging for 20min at 10000r/min and 4 ℃, and taking out supernatant after centrifuging to obtain the egg yolk diluent with the egg yolk concentration of 5%;
taking 85mL of the basic diluent prepared in the step 1), adding 15mL of egg yolk into 85mL of the basic diluent to obtain a second diluent, then putting the second diluent into a refrigerator to be fully dissolved, centrifuging for 20min at 10000r/min and 4 ℃, and taking out a supernatant after centrifuging to obtain the egg yolk diluent with the egg yolk concentration of 15%;
taking 80mL of the basic diluent prepared in the step 1), adding 20mL of egg yolk into 80mL of the basic diluent to obtain a third diluent, putting the third diluent into a refrigerator to be fully dissolved, centrifuging for 20min at 10000r/min and 4 ℃, and taking out a supernatant after centrifuging to obtain the egg yolk diluent with the egg yolk concentration of 20%;
the yolk diluent with the yolk concentration of 5 percent, the yolk diluent with the yolk concentration of 15 percent and the yolk diluent with the yolk concentration of 20 percent are all diluents stored at the sheep semen at normal temperature.
In the step 1), firstly, 30.2-31.2 g of Tris, 15.9-16.9 g of citric acid, 19.5-20.5 g of fructose, 0.3-0.4 g of sodium penicillin and 0.6-0.7 g of streptomycin sulfate are fully dissolved in 1000mL of sterilized ultrapure water, then a 0.22-micron filter membrane is used for filtration sterilization, and 0.3-0.4 g of sodium penicillin and 0.6-0.7 g of streptomycin sulfate are added after filtration sterilization to be fully dissolved, so that a basic diluent is obtained.
The basic diluent in the step 1) is prepared by dissolving 30.7g of Tris, 16.4g of citric acid, 20g of fructose, 0.3118g of penicillin sodium and 0.6944g of streptomycin sulfate in 1000mL of sterilized ultrapure water.
The method is advanced and scientific, and the diluent for preserving the sheep semen at the normal temperature comprises three types of yolk diluent with the concentration of 5%, yolk diluent with the concentration of 15% and yolk diluent with the concentration of 20%, and if artificial insemination is carried out within 6 days before preservation, the effect of the diluent I, namely the addition amount of the yolk, is the best if 5%. If the artificial insemination is carried out within 6 to 9 days of storage, the diluent II, namely the egg yolk addition amount is 15 percent, and the best effect is achieved. If the artificial insemination is carried out within 9-12 days of storage, the diluent III, namely the yolk addition amount is 20%, and the best effect is achieved.
After semen collection is finished, diluting the semen of the sheep by 10 times, inserting a centrifugal tube into a floating plate, placing the centrifugal tube into a beaker filled with warm water at 25 ℃, placing the beaker into a beaker at 16 ℃ for slow cooling so as to carry out normal-temperature preservation, and slightly shaking the centrifugal tube for preserving the semen at intervals so as to prevent the sperm from precipitating. After the sheep semen is diluted, a Computer Assisted Sperm Analyzer (CASA) is adopted to detect the survival rate and the vitality of the sheep semen during the storage period and some set kinetic parameters.
According to the diluent formula for preserving the sheep semen at the normal temperature, five yolk diluents with different concentrations are used for preserving the sheep semen at the normal temperature, and a computer assisted analyzer (CASA) is adopted for detecting the survival rate and the vitality as well as some motion parameters during the semen preservation period. If artificial insemination is performed within 6 days before storage, the addition amount of the diluent, i.e., yolk, is 5% at the best. If the artificial insemination is carried out within 6 to 9 days of storage, the best effect is achieved when the addition amount of the second diluent, namely the yolk is 15 percent. If the artificial insemination is carried out within 9 to 12 days of storage, the effect is best when the addition amount of the diluent, namely the three yolk is 20 percent. The invention aims to objectively evaluate and screen out a diluent formula capable of improving the normal-temperature preservation effect of the sheep semen so as to provide reference for popularization and application of a sheep semen preservation technology in production and further improve the breeding scale of sheep.
According to the diluent formula, the yolk with different concentrations is added, so that the semen preservation effect is improved, the survival rate and the vitality of the yolk-added group are slowly and stably reduced during semen preservation, the effective survival time is prolonged, and the total survival time is obviously prolonged. The formula of the diluent obtained by the invention has low cost, the preparation process is simple and convenient, expensive equipment is not needed for storage, the breeding cost is reduced to a certain extent, and meanwhile, the diluent has a good storage effect, has profound influence on the development of sheep breeding scale and has profound influence on practical application.
After the diluent formula is used, the complex preparation work during semen cryopreservation and ultralow temperature preservation can be reduced, the unfreezing operation process can be removed, the operation time is greatly reduced, the purchase of expensive equipment such as a refrigerator, a liquid nitrogen tank and a program-controlled cooling instrument can be reduced, the operation of professional technicians is not needed, and the breeding cost is greatly reduced.
If the formula is used for preserving semen at normal temperature, a good preservation effect can be achieved, the damage of ultralow temperature cryopreservation to the sperm structure can be avoided, the efficiency of the artificial insemination technology can be improved, the conception rate is improved, certain economic benefits are generated, and the formula has a far-reaching application prospect in actual production and experiments.
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FIG. 1 is a graph showing the effect of different dilutions on sperm preservation survival rate (three dilutions, i.e., 5% egg yolk addition amount, 15% egg yolk addition amount, and 20% egg yolk addition amount, are the formulations of the dilutions provided by the present invention);
FIG. 2 is a graph showing the effect of different dilutions on sperm motility preservation (three dilutions, i.e., 5% egg yolk addition, 15% egg yolk addition, and 20% egg yolk addition, are the formulations of the dilutions provided by the present invention).
Detailed Description
The invention will be further illustrated with reference to specific embodiments.
Materials and methods;
1. test reagent and consumable
Tris (A100826-0500), fructose (A100226-0500), citric acid (A100529-0250) and 0.22 μm filter (F513142-0001) were purchased from Biotechnology engineering (Shanghai) GmbH; penicillin sodium (S17030-100g) and streptomycin sulfate (S17058-100g) were obtained from Phyllostachys (Shanghai) science and technology, Inc.; electronic analytical balances (AB204-N) were purchased from Mettlerltoni Multi-measurement devices (Shanghai) Inc.
2. A specific operation process;
preparing a sheep semen normal-temperature preservation diluent: firstly, fully dissolving 30.2-31.2 g of Tris, 15.9-16.9 g of citric acid, 19.5-20.5 g of fructose, 0.3-0.4 g of penicillin sodium and 0.6-0.7 g of streptomycin sulfate in 1000mL of sterilized ultrapure water, then carrying out filtration sterilization by using a 0.22 mu m filter membrane, and adding 0.3-0.4 g of penicillin sodium and 0.6-0.7 g of streptomycin sulfate after filtration sterilization to fully dissolve the substances. Diluting a first solution: 95mL of the diluted solution prepared above was taken, and 5mL of egg yolk was added thereto. And (2) diluting a second solution: 85mL of the diluted solution prepared above was taken, and 15mL of egg yolk was added thereto. Diluent III: 80mL of the diluted solution prepared above was taken, and 20mL of egg yolk was added thereto. Putting the three dilutions into a refrigerator overnight to be fully dissolved, then centrifuging for 20min at 10000r/min and 4 ℃, subpackaging supernate after centrifuging to obtain three yolk dilutions with yolk concentrations of 5%, 15% and 20%, adding the yolk, then paying attention to placing the dilutions at 4 ℃ for balancing to fully dissolve the yolk, then centrifuging completely without shaking a centrifuge tube, and reducing large yolk particles from entering the dilutions as much as possible to obtain three dilutions stored in sheep semen at normal temperature.
Semen collection and semen dilution preservation: semen collection was performed by the pseudo-vaginal method, the temperature of water in the pseudo-vagina was 42 ℃, the collected semen was added to a 5mL centrifuge tube, stored in a 37 ℃ beaker, and taken back to the laboratory. Diluting the semen of the sheep by 10 times, inserting a centrifugal tube into a floating plate, placing the centrifugal tube into a 500mL beaker filled with warm water at 25 ℃, placing the beaker at 16 ℃ for slow cooling to store at normal temperature, and slightly shaking the centrifugal tube for storing the semen at intervals to prevent the sperm from precipitating.
And (3) detecting the semen quality: the invention uses five kinds of diluent to store the sheep semen at normal temperature, and uses a computer sperm auxiliary analyzer (CASA) to detect the survival rate, the vitality and some movement parameters during the semen storage period.
TABLE 1 Effect of yolk at different concentrations on sheep sperm motility% at ambient temperature storage%
TABLE 2 Effect of different concentrations of egg yolk on sheep sperm motility at ambient temperature storage%
TABLE 3 Effect of different concentrations of yolk on sheep sperm motility at ambient temperature storage
Claims (3)
1. A preparation method of a diluent for preserving sheep semen at normal temperature is characterized by comprising the following steps:
step 1), dissolving 30.2-31.2 g of Tris, 15.9-16.9 g of citric acid, 19.5-20.5 g of fructose, 0.3-0.4 g of penicillin sodium and 0.6-0.7 g of streptomycin sulfate in 1000mL of sterilized ultrapure water to obtain a basic diluent;
step 2), when 95mL of the basic diluent prepared in the step 1) is taken, adding 5mL of egg yolk into 95mL of the basic diluent to obtain a first diluent, then putting the first diluent into a refrigerator to be fully dissolved, centrifuging for 20min at 10000r/min and 4 ℃, and taking out supernatant after centrifuging to obtain the egg yolk diluent with the egg yolk concentration of 5%;
taking 85mL of the basic diluent prepared in the step 1), adding 15mL of egg yolk into 85mL of the basic diluent to obtain a second diluent, then putting the second diluent into a refrigerator to be fully dissolved, centrifuging for 20min at 10000r/min and 4 ℃, and taking out a supernatant after centrifuging to obtain the egg yolk diluent with the egg yolk concentration of 15%;
taking 80mL of the basic diluent prepared in the step 1), adding 20mL of egg yolk into 80mL of the basic diluent to obtain a third diluent, putting the third diluent into a refrigerator to be fully dissolved, centrifuging for 20min at 10000r/min and 4 ℃, and taking out a supernatant after centrifuging to obtain the egg yolk diluent with the egg yolk concentration of 20%;
the yolk diluent with the yolk concentration of 5 percent, the yolk diluent with the yolk concentration of 15 percent and the yolk diluent with the yolk concentration of 20 percent are all diluents stored at the sheep semen at normal temperature.
2. The preparation method of the diluent for preserving sheep semen at normal temperature according to claim 1, wherein in the step 1), 30.2-31.2 g of Tris, 15.9-16.9 g of citric acid, 19.5-20.5 g of fructose, 0.3-0.4 g of penicillin sodium and 0.6-0.7 g of streptomycin sulfate are fully dissolved in 1000mL of sterilized ultrapure water, then 0.22μm filter membrane is used for carrying out filtration sterilization, 0.3-0.4 g of penicillin sodium and 0.6-0.7 g of streptomycin sulfate are added after filtration sterilization, and the base diluent is fully dissolved to obtain the base diluent.
3. The method as claimed in claim 1, wherein the basic diluent in step 1) is prepared by dissolving 30.7g of Tris, 16.4g of citric acid, 20g of fructose, 0.3118g of sodium penicillin and 0.6944g of streptomycin sulfate in 1000mL of sterilized ultrapure water.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN114788516A (en) * | 2022-04-08 | 2022-07-26 | 扬州大学 | Sheep semen diluent and preparation method thereof |
| CN115428786A (en) * | 2022-09-27 | 2022-12-06 | 湖北省农业科学院畜牧兽医研究所 | Normal-temperature storage diluent for improving semen storage quality and preparation and application thereof |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114788516A (en) * | 2022-04-08 | 2022-07-26 | 扬州大学 | Sheep semen diluent and preparation method thereof |
| CN115428786A (en) * | 2022-09-27 | 2022-12-06 | 湖北省农业科学院畜牧兽医研究所 | Normal-temperature storage diluent for improving semen storage quality and preparation and application thereof |
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