CN110622958B - Animal sperm diluent - Google Patents
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- CN110622958B CN110622958B CN201910985506.5A CN201910985506A CN110622958B CN 110622958 B CN110622958 B CN 110622958B CN 201910985506 A CN201910985506 A CN 201910985506A CN 110622958 B CN110622958 B CN 110622958B
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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 an animal sperm diluent which is composed of 50mL-100mL of glycerol, 10g-20g of glucose, 10g-20g of fructose, 20g-30g of tris (hydroxymethyl) aminomethane, 10g-20g of citric acid, 0.1g-0.5g of defatted wheat protein powder, 0.1g-1g of defatted pea protein powder, 0.5g-1g of defatted peanut protein powder, 0.5g-1g of defatted soybean protein powder and 180mL-190mL of sterilized distilled water. The animal sperm diluent is used for preserving animal sperm. The invention solves the problems of unclear observation visual field, easy pollution, short survival time and nonstandard antibiotic use of the animal sperm diluent in the traditional method so as to cause the generation of super bacteria.
Description
The invention is a divisional application, and the original Chinese patent application number is: 201610494390.1, filing date: 2016, 6/29, original patent application with the name: an animal sperm diluent, a preparation method thereof and a method for freezing and refrigerating sperm by using the diluent.
Technical Field
The invention belongs to the technical field of animals, and particularly relates to an animal sperm diluent.
Background
In the prior art, animal sperm is generally stored by using animal source diluent, and other animal source pathogenic microorganisms can be introduced into the animal source diluent such as yolk diluent, so that diseases are brought to artificial insemination of female herds, particularly, a large amount of antibiotics is added during the preparation of the diluent, and the production of super bacteria is caused by the non-standard use of the antibiotics. The egg yolk diluent is prepared at present, the unused egg white or eggshell is not easy to be stored in a garbage can, the smell of smelly eggs is easy to be generated in a laboratory, and the environment is polluted. The yolk diluent is used for observing sperm motility under a microscope, and cannot be easily observed due to unclear visual field caused by the yolk particles.
Researchers also use substances without animal sources to prepare animal sperm diluent, but substances used by the diluent such as caffeine belong to forbidden drugs, are not suitable for large-scale popularization and application, and have the problems of non-lasting sperm motility and short survival time.
Disclosure of Invention
In view of the problems of the prior art, the present invention provides a diluent for animal sperm, a method for preparing the same, and a method for preserving animal sperm by refrigeration or freezing.
The invention aims to solve the problems that the observation visual field of the animal sperm diluent prepared by the traditional method is not clear, the animal sperm diluent is easy to pollute, the survival time is short, and the use of antibiotics is not regulated, so that super bacteria are generated, and provides the animal sperm diluent, the preparation method thereof and a method for freezing and refrigerating sperm by using the diluent.
The animal sperm diluent consists of glycerol, glucose, fructose, tris (hydroxymethyl) aminomethane, citric acid, defatted wheat protein powder (protein and wheat protein peptide), defatted pea protein powder (protein, K factor, carotene, vitamin Bt, vitamin E and vitamin C), defatted peanut protein powder (protein, calcium, phosphorus, iron, thiamine, palmitic acid and vitamin A, B, C, E, K), defatted soybean protein powder (protein, lecithin, plant immune factor and plant trace elements) and sterilized distilled water.
The invention provides an animal sperm diluent which comprises 50mL-100mL of glycerol, 10g-20g of glucose, 10g-20g of fructose, 20g-30g of tris (hydroxymethyl) aminomethane, 10g-20g of citric acid, 0.1g-0.5g of defatted wheat protein powder, 0.1g-1g of defatted pea protein powder, 0.5g-1g of defatted peanut protein powder, 0.5g-1g of defatted soybean protein powder and 180mL-190mL of sterilized distilled water.
The invention has the beneficial effects that: the invention has the advantages of lasting sperm motility, long survival time and the like by reasonably setting the components and the proportion of the components. The invention solves the problems of unclear observation visual field, easy pollution, short survival time and nonstandard antibiotic use of the animal sperm diluent in the traditional method so as to cause the generation of super bacteria.
The animal sperm diluent can be prepared by the following steps:
(1) weighing 20g-30g of tris (hydroxymethyl) aminomethane by using an analytical balance, and then mixing and dissolving 180mL-190mL of sterilized distilled water in a volumetric flask to obtain a mixture;
(2) weighing respectively: respectively weighing 0.5g-1g of defatted soybean protein powder, 0.1g-1g of defatted pea protein powder, 0.5g-1g of defatted peanut protein powder and 0.1g-0.5g of defatted wheat protein powder, dissolving in the mixture obtained in the step (1), and performing ultrasonic cracking on macromolecular protein, bacteria and viruses to obtain a sterile solution mixed with low-molecular protein or polypeptide; refrigerating at 4 deg.C for 24-48 h;
(3) discarding the precipitate in the step (2) to obtain a supernatant; respectively weighing 10g-20g of citric acid, 10g-20g of glucose, 10g-20g of fructose and 50mL-100mL of glycerol on an analytical balance, adding into the supernatant, ultrasonically mixing and cracking bacteria and viruses, and refrigerating at 4 ℃ to obtain the animal sperm diluent.
Adopt the beneficial effect of above-mentioned scheme: the animal sperm diluent prepared by the method has the advantages of lasting sperm motility, long survival time and the like. The invention solves the problems of unclear observation visual field, easy pollution, short survival time and nonstandard antibiotic use of the animal sperm diluent in the traditional method so as to cause the generation of super bacteria.
Further, carrying out ultrasonic lysis on macromolecular proteins, bacteria and viruses in the step (2) to obtain a sterile solution mixed with low-molecular proteins or polypeptides; refrigerating at 4 deg.C and settling for 24 hr.
Adopt the beneficial effect of above-mentioned scheme: the quality of the animal sperm diluent can be further improved by adopting the parameters.
Furthermore, the mass of the defatted pea protein powder is 0.5g-1g or 0.1g-0.5 g.
The preparation method of the animal sperm diluent comprises the following steps:
(1) weighing 20g-30g of tris (hydroxymethyl) aminomethane by using an analytical balance, and then mixing and dissolving 180mL-190mL of sterilized distilled water in a volumetric flask to obtain a mixture;
(2) weighing respectively: respectively weighing 0.5g-1g of defatted soybean protein powder, defatted pea protein powder (0.5 g-1g or 0.1g-0.5 g), 0.5g-1g of defatted peanut protein powder and 0.1g-0.5g of defatted wheat protein powder, dissolving in the mixture obtained in the step (1), and ultrasonically cracking macromolecular protein, bacteria and viruses to obtain a sterile solution mixed with low-molecular protein or polypeptide; refrigerating at 0-8 deg.C for 24-48 h;
(3) discarding the precipitate in the step (2) to obtain a supernatant; respectively weighing 10g-20g of citric acid, 10g-20g of glucose, 10g-20g of fructose and 50mL-100mL of glycerol on an analytical balance, adding into the supernatant, ultrasonically mixing and cracking bacteria and viruses, and refrigerating at 4 ℃ to obtain the animal sperm diluent.
Adopt the beneficial effect of above-mentioned scheme: can further improve the quality of the animal sperm diluent.
Preferably, the mass of the defatted pea protein powder is 0.1 g.
Preferably, the tris (hydroxymethyl) aminomethane has a mass of 20g and the sterilized distilled water has a volume of 180 mL.
Preferably, the citric acid has a mass of 10g or 12 g.
Preferably, the glucose mass is 10g or 15 g.
Preferably, the mass of the defatted soybean protein powder is 0.5g, the mass of the defatted peanut protein powder is 0.5g, and the mass of the defatted wheat protein powder is 0.2 g.
Preferably, the fructose mass is 10g, and the glycerol volume is 50 mL.
Adopt the beneficial effect of above-mentioned scheme: the quality of the animal sperm diluent can be further improved by adopting the parameters, and the using effect of the animal sperm diluent is improved.
The method for preserving sperm by freezing by using the animal sperm diluent comprises the following steps:
(1) putting the collected animal semen into a vacuum flask, and keeping the temperature at 20-35 ℃;
(2) measuring the density of the animal semen in the step (1) by using a spectrophotometer, diluting the animal semen by using animal semen diluent according to the sperm number of 300-1000 ten thousand/mL, filling the diluted semen into a thin tube by using a filling and sealing machine by using a frozen semen thin tube of 0.25 mL;
(3) putting the thin-tube semen in the step (2) into a cooling cabinet, cooling at the speed of 0.1-0.5 ℃/min to 0-4 ℃;
(4) balancing the thin tube semen in the step (3) for 1-2 h;
(5) putting the tubule semen of the step (4) above liquid nitrogen, fumigating at 100-120 deg.C for 5-15 min at a distance of 1.0-2.0 cm from the liquid nitrogen surface, directly throwing into liquid nitrogen, and freeze-drying in liquid nitrogen to obtain gauze bag.
The method for preserving sperms by cold storage of animal sperm diluent is carried out according to the following steps:
(1) putting the collected animal semen into a vacuum flask, keeping the temperature at 20-35 ℃, and bringing the animal semen to a laboratory;
(2) measuring the density of the semen in the step (1) by using a spectrophotometer, diluting the semen by using diluent according to the semen number of 300-1000 ten thousand/mL, filling the diluted semen into a frozen semen thin tube by using a 0.25mL frozen semen thin tube by using a filling and sealing machine, and sealing the opening of the frozen semen thin tube;
(3) putting the fine tube liquid in the step (2) into a cooling cabinet, cooling at the speed of 0.1-0.5 ℃/min to 0-4 ℃ for later use.
Compared with the prior art, the invention has the advantages that:
1. compared with the prior art, the animal sperm diluent is added with plant extracts (plant protein, plant polypeptide, plant trace elements, plant factors, lecithin, immune factors, vitamins, palmitic acid, sodium, potassium and other inorganic ions), plays an important role in improving the sperm motility and prolonging the preservation time of freezing or cold preservation, and avoids adding caffeine, which is an excitant, can improve the sperm motility but shorten the survival time, and is a forbidden medicine, thus preventing mass preparation and application and popularization; avoid the addition and abuse of antibiotics so that the superbacteria produce. The animal source diluent such as yolk diluent is prevented from introducing other animal source pathogenic microorganisms, and diseases are brought to artificial insemination female herds.
2. On the basis of adding soybean protein powder in the prior art, defatted soybean protein powder, defatted peanut protein powder, defatted pea protein powder and defatted wheat protein powder are added, compared with the prior art, the animal sperm diluent prepared by the method improves the activity of frozen sperm by 1-3 percent, and the survival time of the sperm stored by refrigeration is prolonged by 1-2 days.
3. By improving the existing preparation method of the animal sperm diluent, the sperm diluent with 5 times concentration and 1 year shelf life is obtained.
4. Can be used for producing animal sperm diluent in an industrialized way to freeze and refrigerate and preserve sperms, saves time and labor and has wide popularization and application prospect.
Detailed Description
The technical solution of the present invention is not limited to the specific embodiments listed below, and includes any combination of the specific embodiments.
The first embodiment is as follows: the animal sperm diluent of the embodiment is composed of 50mL-100mL of glycerol, 10g-20g of glucose, 10g-20g of fructose, 20g-30g of tris (hydroxymethyl) aminomethane, 10g-20g of citric acid, 0.1g-0.5g of defatted wheat protein powder, 0.5g-1g of defatted pea protein powder, 0.5g-1g of defatted peanut protein powder, 0.5g-1g of defatted soybean protein powder and 180mL-190mL of sterilized distilled water.
The second embodiment is as follows: the preparation method of the animal sperm diluent according to the first embodiment comprises the following steps:
(1) weighing 20g-30g of tris (hydroxymethyl) aminomethane by using an analytical balance, and then mixing and dissolving 180mL-190mL of sterilized distilled water in a volumetric flask to obtain a mixture;
(2) weighing respectively: respectively weighing 0.5g-1g of defatted soybean protein powder, 0.5g-1g of defatted pea protein powder, 0.5g-1g of defatted peanut protein powder and 0.1g-0.5g of defatted wheat protein powder, dissolving in the mixture obtained in the step (1), and performing ultrasonic cracking on macromolecular protein, bacteria and viruses to obtain a sterile solution mixed with low-molecular protein or polypeptide; refrigerating at 0-8 deg.C for 24-48 h;
(3) discarding the precipitate in the step (2) to obtain a supernatant; respectively weighing 10g-20g of citric acid, 10g-20g of glucose, 10g-20g of fructose and 50mL-100mL of glycerol on an analytical balance, adding into the supernatant, ultrasonically mixing and cracking bacteria and viruses, and refrigerating at 4 ℃ to obtain the animal sperm diluent.
The third concrete implementation mode: this embodiment is different from the second embodiment in that the tris (hydroxymethyl) aminomethane has a mass of 20 g. Other steps and parameters are the same as those in the second embodiment.
The fourth concrete implementation mode: the present embodiment is different from the second embodiment in that the volume of the sterilized distilled water is 180 mL. Other steps and parameters are the same as those in the second embodiment.
The fifth concrete implementation mode: the second difference between the present embodiment and the second embodiment is that, in the step (2), the ultrasonic wave cracks the macromolecular protein, bacteria and virus to obtain a sterile solution mixed with the low molecular protein or polypeptide; refrigerating at 4 deg.C and settling for 24 hr. Other steps and parameters are the same as those in the second embodiment.
The sixth specific implementation mode: the second difference between this embodiment and the second embodiment is that 10g of citric acid, 10g of glucose, 10g of fructose, and 50mL of glycerol were weighed in step (3) on an analytical balance. Other steps and parameters are the same as those in the second embodiment.
The seventh embodiment: the method for preserving sperm by freezing the animal sperm diluent according to the first embodiment comprises the following steps:
(1) putting the collected animal semen into a vacuum flask, and keeping the temperature at 20-35 ℃;
(2) measuring the density of the animal semen in the step (1) by using a spectrophotometer, diluting the animal semen by using animal semen diluent according to the sperm number of 300-1000 ten thousand/mL, filling the diluted semen into a thin tube by using a filling and sealing machine by using a frozen semen thin tube of 0.25 mL;
(3) putting the thin-tube semen in the step (2) into a cooling cabinet, cooling at the speed of 0.1-0.5 ℃/min to 0-4 ℃;
(4) standing the thin tube semen in the step (3) for 1-2 h;
(5) putting the tubule semen of the step (4) above liquid nitrogen, fumigating at 100-120 deg.C below zero for 5-15 min at a height of 1.0-2.0 cm above the liquid nitrogen surface, directly throwing into liquid nitrogen, and placing the tubule frozen semen into gauze bag in liquid nitrogen for use.
The specific implementation mode is eight: the method for preserving sperm by refrigeration by using the animal sperm diluent of the first embodiment comprises the following steps:
(1) putting the collected animal semen into a vacuum flask, keeping the temperature at 20-35 ℃, and bringing the animal semen to a laboratory;
(2) measuring the density of the semen in the step (1) by using a spectrophotometer, diluting the semen by using diluent according to the semen number of 300-1000 ten thousand/mL, filling the diluted semen into a frozen semen thin tube by using a 0.25mL frozen semen thin tube by using a filling and sealing machine, and sealing the opening of the frozen semen thin tube;
(3) putting the fine tube liquid in the step (2) into a cooling cabinet, cooling at the speed of 0.1-0.5 ℃/min to 0-4 ℃ for later use.
Example 1
The animal sperm diluent consists of 50mL of glycerol, 15g of glucose, 10g of fructose, 20g of tris (hydroxymethyl) aminomethane, 12g of citric acid, 0.2g of defatted wheat protein powder, 0.1g of defatted pea protein powder, 0.5g of defatted peanut protein powder, 0.5g of defatted soybean protein powder and 180mL of sterilized distilled water.
The preparation method of the animal sperm diluent of the embodiment is carried out according to the following steps:
(1) weighing tris (hydroxymethyl) aminomethane and sterilized distilled water by using an analytical balance, and mixing and dissolving in a volumetric flask to obtain a mixture; the volume ratio of the mass of the trihydroxymethyl aminomethane to the sterilized distilled water is 20.00 g: 180 mL;
(2) weighing respectively: 0.5g of defatted soybean protein powder, 0.1g of defatted pea protein powder, 0.5g of defatted peanut protein powder and 0.2g of defatted wheat protein powder are dissolved in the mixture obtained in the step (1), and microorganisms such as macromolecular protein, bacteria, viruses and the like are cracked by ultrasonic waves to obtain sterile solution mixed by low-molecular protein or polypeptide; refrigerating at 4 deg.C and settling for 24 hr;
(3) discarding the precipitate in the step (2) to obtain a supernatant; weighing 12g of citric acid, 15g of glucose, 10g of fructose and 50mL of glycerol on an analytical balance, adding into the supernatant, ultrasonically mixing with lysis proteins, bacteria, viruses and other microorganisms, and refrigerating at 4 ℃ to obtain the animal sperm diluent.
The method for preserving sperm by freezing by using the animal sperm diluent comprises the following steps:
(1) putting the collected semen of the dog into a thermos flask, and keeping the temperature at 28 ℃;
(2) measuring the density of the animal semen in the step (1) by using a spectrophotometer, diluting the animal sperm diluent provided by the invention according to the sperm number of 500 ten thousand/mL, using a frozen sperm tubule of 0.25mL, filling the diluted semen into the tubule by using a filling and sealing machine, and sealing;
(3) putting the thin-tube semen in the step (2) into a cooling cabinet, cooling at the speed of 0.2 ℃/min to 4 ℃;
(4) balancing the thin tube semen in the step (3) for 1 h;
(5) putting the fine tube liquid obtained in the step (4) above liquid nitrogen, fumigating for 10min at the temperature of about minus 110 ℃ and the height of 2.5cm away from the liquid nitrogen surface, directly throwing the fine tube liquid into the liquid nitrogen, and filling the fine tube liquid into a gauze bag for later use.
The method for preserving sperms by cold storage of animal sperm diluent is carried out according to the following steps:
(1) putting the collected semen of the dog into a thermos flask, and keeping the temperature at 28 ℃;
(2) measuring the density of the semen in the step (1) by using a spectrophotometer, diluting the semen by using diluent according to the number of the semen of 600 ten thousand/mL, filling the diluted semen into a frozen semen thin tube by using a 0.25mL frozen semen thin tube by using a filling and sealing machine, and sealing the opening of the frozen semen thin tube;
(3) putting the fine tube liquid in the step (2) into a cooling cabinet, cooling at the speed of 0.4 ℃/min, and cooling to 4 ℃ for later use.
The sperm viability of the animal sperm diluent obtained in the embodiment after freezing storage is 0.48, the survival time is 7-12 days, the sperm viability of the sperm refrigerated storage is 0.6, and the survival time is 10-15 days.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.
Claims (1)
1. An animal sperm diluent, which is characterized by being prepared from 50mL-100mL of glycerol, 10g-20g of glucose, 10g-20g of fructose, 20g-30g of tris (hydroxymethyl) aminomethane, 10g-20g of citric acid, 0.1g-0.5g of defatted wheat protein powder, 0.1g-1g of defatted pea protein powder, 0.5g-1g of defatted peanut protein powder, 0.5g-1g of defatted soybean protein powder and 180mL-190mL of sterilized distilled water;
the animal sperm diluent is prepared by the following steps:
(1) weighing 20g-30g of tris (hydroxymethyl) aminomethane by using an analytical balance, and then mixing and dissolving 180mL-190mL of sterilized distilled water in a volumetric flask to obtain a mixture;
(2) weighing respectively: respectively weighing 0.5g-1g of defatted soybean protein powder, 0.1g-1g of defatted pea protein powder, 0.5g-1g of defatted peanut protein powder and 0.1g-0.5g of defatted wheat protein powder, dissolving in the mixture obtained in the step (1), and performing ultrasonic cracking on macromolecular protein, bacteria and viruses to obtain a sterile solution mixed with low-molecular protein or polypeptide; refrigerating at 4 deg.C for 24-48 h;
(3) discarding the precipitate in the step (2) to obtain a supernatant; respectively weighing 10g-20g of citric acid, 10g-20g of glucose, 10g-20g of fructose and 50mL-100mL of glycerol on an analytical balance, adding into the supernatant, ultrasonically mixing and cracking bacteria and viruses, and refrigerating at 4 ℃ to obtain an animal sperm diluent;
step (2) ultrasonic cracking macromolecular protein, bacteria and virus to obtain sterile solution mixed with low molecular protein or polypeptide; refrigerating at 4 deg.C and settling for 24 hr.
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| CN201910985506.5A CN110622958B (en) | 2016-06-29 | 2016-06-29 | Animal sperm diluent |
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| CN201610494390.1A CN106135196B (en) | 2016-06-29 | 2016-06-29 | Animal sperm dilution and its preparation method and the method for utilizing dilution freezing, stored refrigerated sperm |
| CN201910985506.5A CN110622958B (en) | 2016-06-29 | 2016-06-29 | Animal sperm diluent |
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| CN201610494390.1A Active CN106135196B (en) | 2016-06-29 | 2016-06-29 | Animal sperm dilution and its preparation method and the method for utilizing dilution freezing, stored refrigerated sperm |
| CN201910990863.0A Active CN111066775B (en) | 2016-06-29 | 2016-06-29 | Method for cold storage of animal sperm |
| CN201910981400.8A Active CN111053079B (en) | 2016-06-29 | 2016-06-29 | Preparation method of animal sperm diluent |
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| CN201910990863.0A Active CN111066775B (en) | 2016-06-29 | 2016-06-29 | Method for cold storage of animal sperm |
| CN201910981400.8A Active CN111053079B (en) | 2016-06-29 | 2016-06-29 | Preparation method of animal sperm diluent |
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| CN108103012B (en) * | 2017-12-14 | 2021-04-02 | 内蒙古赛科星家畜种业与繁育生物技术研究院有限公司 | Production method and application of mouse X/Y sperm separation semen |
| CN115039766B (en) * | 2022-08-13 | 2022-11-18 | 中国农业科学院北京畜牧兽医研究所 | Normal-temperature boar semen diluent |
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Also Published As
| Publication number | Publication date |
|---|---|
| CN111066775B (en) | 2022-02-01 |
| CN110622958A (en) | 2019-12-31 |
| CN110742058B (en) | 2022-02-01 |
| CN111053079B (en) | 2022-02-01 |
| CN111066775A (en) | 2020-04-28 |
| CN106135196B (en) | 2019-11-19 |
| CN111053079A (en) | 2020-04-24 |
| CN106135196A (en) | 2016-11-23 |
| CN110742058A (en) | 2020-02-04 |
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