CN110959607A - Formula and dilution method of diluent for storing Hu sheep semen at normal temperature - Google Patents
Formula and dilution method of diluent for storing Hu sheep semen at normal temperature Download PDFInfo
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- CN110959607A CN110959607A CN201911343515.0A CN201911343515A CN110959607A CN 110959607 A CN110959607 A CN 110959607A CN 201911343515 A CN201911343515 A CN 201911343515A CN 110959607 A CN110959607 A CN 110959607A
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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
- A01N1/0226—Physiologically active agents, i.e. substances affecting physiological processes of cells and tissue to be preserved, e.g. anti-oxidants or nutrients
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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
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Abstract
The invention provides a diluent formula for preserving Hu sheep semen at normal temperature and a diluting method thereof, wherein five diluents are used for preserving the Hu sheep semen at normal temperature, and the methods such as a computer sperm auxiliary analyzer (CASA), Coomassie brilliant blue dyeing, hypotonic test and the like are adopted to detect the survival rate, the vitality, the plasma membrane integrity rate, the acrosome integrity rate, the survival time and some movement parameters during the semen preservation period. The invention aims to objectively evaluate and screen out a diluent formula with the best Hu sheep semen normal-temperature storage effect, so as to provide reference for popularization and application of the Hu sheep semen storage technology in production and further improve the breeding scale of Hu sheep.
Description
Technical Field
The invention relates to a diluent formula for preserving the sperm of Hu sheep at normal temperature and a diluting method thereof, belonging to the technical field of Hu sheep breeding.
Background
The Hu sheep is a world famous multiparous sheep variety and has the advantages of four seasons estrus, two fetuses in one year, fast growth and development and the like, so the breeding scale of the Hu sheep is rapidly expanding. 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 is applied by matching with the estrus synchronization technology, so that the breeding cost can be reduced, the disease transmission can be prevented, and the large-scale breeding and management can be realized. 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. The membrane of sheep sperms contains a large amount of high-density unsaturated fatty acid and is lack of antioxidant, and due to the special structure of the membrane, the membrane is very sensitive to the influence of freezing stimulation, so that the fertilization rate is low, and the application of semen freezing preservation on sheep is limited. 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 total survival time of the existing semen normal temperature preservation diluent formula is only 45 hours, and the time of activity over 0.3 is only 25 hours.
Therefore, a diluent formula capable of improving the normal-temperature preservation quality of the Hu sheep semen is needed to be invented, 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, and provides a diluent formula for preserving the Hu sheep semen at normal temperature and a diluting method thereof, which are used for solving the problem that the Hu sheep semen in the prior art is too short in normal-temperature preservation time and can not meet the requirement of actual production.
The invention aims to realize the formula of the diluent for preserving the sperm of the Hu sheep at normal temperature, which is characterized in that the formula is formed by 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;
firstly, 30.2-31.2 g of Tris, 15.9-16.9 g of citric acid and 19.5-20.5 g of fructose are dissolved in 1000mL of sterilized ultrapure water to be fully dissolved, then a 0.22-micron filter membrane is used for filtration sterilization, the filtration sterilization is carried out, the filtrate sterilization is stored in a refrigerator at 4 ℃, and finally 0.3-0.4 g of sodium penicillin and 0.6-0.7 g of streptomycin sulfate are added to obtain the diluted solution for the normal-temperature storage of the sheep sperm of a Hu sheep.
The Tris, the citric acid, the fructose, the penicillin sodium and the streptomycin sulfate are respectively 30.7g, 16.4g, 20g, 0.3118g and 0.6944 g.
A method for diluting Hu sheep semen by using a diluent formula stored at normal temperature is characterized in that collected Hu sheep semen is poured into a centrifugal tube for mixing treatment to eliminate individual difference, and diluent preheated at 37 ℃ is slowly added along the wall of the centrifugal tube to dilute the Hu sheep semen by 10 times; and finally, wrapping 3 layers with cotton, placing at the temperature of 16 ℃ for normal-temperature storage, and slightly shaking the centrifuge tube for storing the semen every 24 hours to dissolve the precipitated substances into the diluent as much as possible, so as to prevent the aggregation of metabolic wastes from generating toxic action on the sperm and finish the dilution of the sperm of the Hu sheep.
When the artificial vagina is used for collecting the sperm of the Hu sheep, the water temperature of the artificial vagina is controlled to be 40-45 ℃, the collected sperm of the Hu sheep is sucked into a 2mL centrifuge tube, the centrifuge tube is placed in a thermos cup filled with water at the temperature of 37 ℃, and the collected sperm is processed within 1 hour.
After the Hu sheep semen is diluted, the survival rate, the vitality, the plasma membrane integrity rate, the acrosome integrity rate, the survival time and some set movement parameters of the Hu sheep semen during the preservation period are detected by adopting a Computer Assisted Sperm Analyzer (CASA) or Coomassie brilliant blue staining and hypotonic test method.
The method is scientific and reasonable, and the invention uses five kinds of diluents to store the Hu sheep semen at normal temperature, and adopts the methods of a computer sperm auxiliary analyzer (CASA), Coomassie brilliant blue dyeing, hypotonic test and the like to detect the survival rate, the vitality, the plasma membrane integrity rate, the acrosome integrity rate, the survival time and some movement parameters during the semen storage period. The invention aims to objectively evaluate and screen out a diluent formula with the best Hu sheep semen normal-temperature storage effect, so as to provide reference for popularization and application of the Hu sheep semen storage technology in production and further improve the breeding scale of Hu sheep.
The formula of the diluent in the invention adds a proper amount of nutrient substances: fructose and substances to maintain PH stability: the sperm viability preserved by Tris and citric acid solution I can be kept for more than 72 hours when the sperm viability is more than 0.6, and the total survival time can be kept for more than 17 days. The diluent obtained by the invention has low formula cost, simple preparation process, no need of expensive equipment for storage, reduction of breeding cost to a certain extent, good storage effect, profound influence on the development of Hu sheep breeding scale and 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.
Drawings
FIG. 1 is a graph showing the effect of different dilutions on sperm motility preservation (dilution No. I is the formula of the dilution provided by the present invention);
FIG. 2 is a graph showing the effect of different dilutions on sperm motility preservation (dilution No. I is the diluent formulation 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 hu sheep semen normal temperature preservation diluent: 30.7g Tris, 16.4g citric acid and 20g fructose were accurately weighed by a balance, dissolved in 1000mL sterilized ultrapure water, sufficiently dissolved, filtered through a 0.22 μm filter for sterilization, stored in a refrigerator at 4 ℃ and added with 0.3118g penicillin sodium and 0.6944g streptomycin when ready to use.
Semen collection and semen dilution preservation: when semen is collected, the temperature of the water in the artificial vagina is controlled to be between 40 ℃ and 45 ℃, the collected semen is sucked into a 2mL centrifuge tube, placed into a thermos cup filled with water at 37 ℃, and the collected semen is processed within 1 hour. The semen collected is first subjected to a mixing treatment to eliminate individual differences, and then the sperm of the Hu sheep is diluted 10 times, for example, 5mL of the semen is added to a 50mL centrifuge tube, and 45mL of the diluted solution which has been preheated at 37 ℃ is slowly added along the wall of the centrifuge tube. And finally, wrapping 3 layers with cotton, placing at the temperature of 16 ℃ for normal-temperature storage, and slightly shaking the centrifugal tube for storing the semen every 24 hours to dissolve the precipitated substances into the diluent seeds as much as possible so as to prevent the metabolic waste from gathering to generate toxic action on the sperms.
And (3) detecting the semen quality: the invention uses five kinds of diluent to store the Hu sheep semen at normal temperature, and adopts the methods of a computer sperm auxiliary analyzer (CASA), Coomassie brilliant blue dyeing, hypotonic test and the like to detect the survival rate, the vitality, the plasma membrane integrity rate, the acrosome integrity rate, the survival time and some movement parameters during the semen storage period.
TABLE 15 different Diluent formulations
TABLE 2 Effect of different dilutions on the viability of preserved sperm%
Note: the data from the same row, marked with lower case letters, are significantly different (P < 0.05), identically are not significant (P > 0.05), and-indicate a loss of statistical significance.
TABLE 3 Effect of different dilutions on preserving sperm motility%
Note: the data from the same row, marked with lower case letters, are significantly different (P < 0.05), identically are not significant (P > 0.05), and-indicate a loss of statistical significance.
TABLE 4 Effect of different dilutions on preserving sperm motility
Note: the data from the same row, marked with lower case letters, are significantly different (P < 0.05), identically are not significant (P > 0.05), and-indicate a loss of statistical significance.
TABLE 5 Effect of different dilutions on the survival time of preserved sperm h
Note: the data in the same row with the lower case letters in shoulder marks represent significant difference (P < 0.05), and the same data represent insignificant difference (P > 0.05).
TABLE 6 Effect of different dilutions on preservation of sperm plasma Membrane integrity%
Note: the data from the same row, marked with lower case letters, are significantly different (P < 0.05), identically are not significant (P > 0.05), and-indicate a loss of statistical significance.
TABLE 7 Effect of different dilutions on preservation of sperm acrosome integrity%
Note: the data from the same row, marked with lower case letters, are significantly different (P < 0.05), identically are not significant (P > 0.05), and-indicate a loss of statistical significance.
The invention relates to a diluent formula for normal-temperature preservation of Hu sheep semen, which is characterized in that firstly, 30.7g of Tris, 16.4g of citric acid and 20g of fructose are accurately weighed by a balance and dissolved in 1000mL of sterilized ultrapure water to be fully dissolved, then, a 0.22-micron filter membrane is used for filtration sterilization, the Hu sheep semen is preserved in a refrigerator at the temperature of 4 ℃, and finally, 0.3118g of penicillin sodium and 0.6944g of streptomycin sulfate are added when the Hu sheep semen is used.
After semen collection is finished, mixing the collected semen to eliminate individual difference, diluting the semen of the Hu sheep by 10 times, wrapping 3 layers of cotton, placing the mixture at the normal temperature of 16 ℃ for storage, and slightly shaking a centrifugal tube for storing the semen every 24 hours to dissolve the precipitated substances into the diluent as much as possible so as to prevent metabolic waste from gathering to generate toxic action on the semen.
Claims (5)
1. The formula of the diluent for normal-temperature preservation of the Hu sheep semen is characterized by being prepared by 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;
firstly, dissolving 30.2-31.2 g of Tris, 15.9-16.9 g of citric acid and 19.5-20.5 g of fructose in 1000mL of sterilized ultrapure water to fully dissolve the Tris, then performing filtration sterilization by using a 0.22 mu m filter membrane, storing the filtrate in a refrigerator at 4 ℃ after the filtration sterilization, and finally adding 0.3-0.4 g of sodium penicillin and 0.6-0.7 g of streptomycin sulfate to obtain the diluted solution for normal-temperature storage of the sperm of the Hu sheep.
2. The formulation of claim 1, wherein the amount of Tris, citric acid, fructose, penicillin sodium and streptomycin sulfate is 30.7g, 16.4g, 20g, 0.3118g and 0.6944g, respectively.
3. The method for diluting the hu-sheep semen according to the formula of the diluent for the normal-temperature preservation of the hu-sheep semen as claimed in any one of claims 1 to 2, wherein the collected hu-sheep semen is poured into a centrifuge tube for mixing treatment to eliminate individual differences, and the diluent preheated at 37 ℃ is slowly added along the wall of the centrifuge tube to dilute the hu-sheep semen by 10 times; and finally, wrapping 3 layers with cotton, placing at the temperature of 16 ℃ for normal-temperature storage, and slightly shaking the centrifuge tube for storing the semen every 24 hours to dissolve the precipitated substances into the diluent as much as possible, so as to prevent the aggregation of metabolic wastes from generating toxic action on the sperm and finish the dilution of the sperm of the Hu sheep.
4. The method as claimed in claim 3, wherein when the artificial vagina is used for the collection of the sperm of the Hu sheep, the water temperature of the artificial vagina is controlled to be 40-45 ℃, the collected sperm of the Hu sheep is sucked into a 2mL centrifuge tube and placed in a thermos cup containing water at 37 ℃, and the collected sperm is processed within 1 hour.
5. The method as claimed in claim 3, wherein after the dilution of the sperm of the Hu sheep, the sperm-assisted computer analyzer (CASA) or Coomassie brilliant blue staining and hypotonic test method is used to detect the survival rate, vitality, plasma membrane integrity, acrosome integrity, survival time and some set kinetic parameters during the preservation of the sperm of the Hu sheep.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111820209A (en) * | 2020-08-12 | 2020-10-27 | 扬州大学 | Screening method for Hu sheep semen at optimum temperature for normal-temperature storage |
| CN111919839A (en) * | 2020-09-11 | 2020-11-13 | 扬州大学 | Preparation method and use method of Hu sheep semen low-temperature preservation diluent formula |
| CN112021307A (en) * | 2020-10-15 | 2020-12-04 | 扬州大学 | Method for improving normal-temperature storage quality of sheep semen |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111820209A (en) * | 2020-08-12 | 2020-10-27 | 扬州大学 | Screening method for Hu sheep semen at optimum temperature for normal-temperature storage |
| CN111919839A (en) * | 2020-09-11 | 2020-11-13 | 扬州大学 | Preparation method and use method of Hu sheep semen low-temperature preservation diluent formula |
| CN112021307A (en) * | 2020-10-15 | 2020-12-04 | 扬州大学 | Method for improving normal-temperature storage quality of sheep semen |
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