阅读说明：本技术 一种冷冻保存动物***的方法 (Method for cryopreservation of animal sperm ) 是由 许红喜 卫喜明 宋伟红 甘文平 孙晓玉 张洪涛 韩志强 于 2016-06-29 设计创作，主要内容包括：本发明涉及一种冷冻保存动物精子的方法,包括以下步骤：(1)将采集到的动物精液放入保温瓶内,温度保持在20-35℃；(2)用动物精子稀释液按精子数300-1000万/mL稀释,灌装并封口,得到细管精液；(3)以0.1-0.5℃/min的速度降温至0-4℃；(4)静置1h-2h；(5)将细管精液放在液氮上方,距离液氮面高度1.0cm-2.0cm,温度为零下100℃-120℃,熏蒸5min-15min,直接投到液氮里,得到细管冻精,在液氮里把细管冻精装到纱布袋里备用。本发明解决了传统方法在保存动物精子时,观察视野不清晰、易污染、存活时间短和不规范使用抗生素以致超级细菌产生的问题。(The invention relates to a method for cryopreservation of animal sperm, comprising the following steps: (1) putting the collected animal semen into a vacuum flask, and keeping the temperature at 20-35 ℃; (2) diluting with animal sperm diluent according to sperm number of 300-; (3) cooling to 0-4 deg.C at a speed of 0.1-0.5 deg.C/min; (4) standing for 1-2 h; (5) putting the tubule semen above liquid nitrogen, with a height of 1.0-2.0 cm from the liquid nitrogen surface, fumigating at-100 deg.C to 120 deg.C for 5-15 min, directly adding into liquid nitrogen to obtain tubule frozen semen, and packing the tubule frozen semen into gauze bag in liquid nitrogen for use. The invention solves the problems that when animal sperms are preserved by the traditional method, the observation visual field is not clear, the pollution is easy, the survival time is short, and the antibiotic is not used in a standard way, so that the production of super bacteria is caused.)

1. A method of cryopreservation of animal sperm comprising the steps of:

(1) storing the collected animal semen at 20-35 deg.C;

(2) measuring the density of the animal semen in the step (1), diluting the animal semen with animal semen diluent according to the sperm number of 300-;

(3) cooling the thin tube semen in the step (2) to 0-4 ℃ at the speed of 0.1-0.5 ℃/min;

(4) standing the thin tube semen in the step (3) for 1-2 h;

(5) putting the tubule semen obtained in the step (4) above liquid nitrogen, fumigating at 100-120 ℃ below zero for 5-15 min at a distance of 1.0-2.0 cm from the liquid nitrogen surface, directly throwing into the liquid nitrogen to obtain tubule frozen semen, and filling the tubule frozen semen into a gauze bag in the liquid nitrogen for later use.

2. The method of claim 1, wherein the animal sperm diluent comprises glycerol 50mL to 100mL, glucose 10g to 20g, fructose 10g to 20g, tris (hydroxymethyl) aminomethane 20g to 30g, citric acid 10g to 20g, defatted wheat protein powder 0.1g to 0.5g, defatted pea protein powder 0.1g to 1g, defatted peanut protein powder 0.5g to 1g, defatted soybean protein powder 0.5g to 1g, and sterilized distilled water 180mL to 190 mL.

3. A method of cryopreservation of animal sperm cells as claimed in claim 1, wherein the animal sperm cell dilution is prepared by:

(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.

4. A method of cryopreservation of animal sperm as claimed in claim 2 or claim 3 characterised in that the defatted pea protein powder has a mass of 0.1g to 0.5 g.

5. A method of cryopreservation of animal sperm as claimed in claim 2 or claim 3 characterised in that the mass of defatted pea protein powder is 0.1 g.

6. The method of claim 2 or 3, wherein the amount of tris is 20g, the volume of sterilized distilled water is 180mL, the amount of citric acid is 10g or 12g, the amount of glucose is 10g or 15g, the amount of defatted soy protein powder is 0.5g, the amount of defatted peanut protein powder is 0.5g, the amount of defatted wheat protein powder is 0.2g, the amount of fructose is 10g, and the volume of glycerol is 50 mL.

7. A method of cryopreservation of animal sperm cells as claimed in any of claims 1 to 3, wherein in step (1) the temperature of storage is 28 ℃; in the step (2), animal sperm diluent is used for diluting according to the sperm number of 500 ten thousand/mL.

8. A method of cryopreservation of animal sperm according to any of claims 1 to 3 characterised in that in step (3) the temperature is reduced to 4 ℃ at a rate of 0.2 ℃/min.

9. A method of cryopreservation of animal sperm according to claims 1 to 3 characterised in that in step (4) the straw is equilibrated for 1 h.

10. A method for cryopreservation of animal sperm as claimed in any of claims 1 to 3, wherein in step (5) the animal sperm is fumigated for 10min at a temperature of-110 ℃ and a height of 2.5cm from the liquid nitrogen level.

Technical Field

The invention belongs to the technical field of animals, and particularly relates to a method for preserving animal sperms in a freezing mode.

Background

In the preservation of animal sperm, the preservation is generally performed by a freezing or refrigeration method. At the time of storage, an animal-derived diluent is generally added. In the prior art, the activity and the preservation time of the animal sperms are influenced because the operation is unreasonable or the formula of the animal source diluent is not proper in the preservation process. For example, the addition of egg yolk dilutions introduces pathogenic microorganisms of other animal origin, causing disease to artificial insemination herds, especially by adding large amounts of antibiotics when preparing the dilutions, which are not regulated so that superbacteria are produced. 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 method for cryopreservation of animal sperm.

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 invention provides a method for preserving sperm by freezing by using animal sperm diluent, which 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 0.25mL frozen semen thin tube by using a filling and sealing machine, and sealing to obtain the thin tube semen;

(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 invention has the beneficial effects that: the preservation method adopted by the invention has the advantages of lasting sperm motility, long survival time and the like.

Further, the animal sperm diluent consists 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 invention solves the problems that when animal sperms are preserved by the traditional method, the observation visual field is not clear, the pollution is easy, the survival time is short, and the use of antibiotics is not standardized, so that the production of super bacteria is caused.

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 the animal sperm diluent.

Furthermore, the mass of the defatted pea protein powder can be 0.1g-0.5 g. For example, the mass of defatted pea protein powder is 0.1 g. The weight of the trihydroxymethyl aminomethane is 20g, the volume of the sterilized distilled water is 180mL, the weight of the citric acid is 10g or 12g, the weight of the glucose is 10g or 15g, the weight of the defatted soybean protein powder is 0.5g, the weight of the defatted peanut protein powder is 0.5g, the weight of the defatted wheat protein powder is 0.2g, the weight of the fructose is 10g, and the volume of the glycerol is 50 mL.

The invention has the beneficial effects that: the animal sperm diluent can further improve the durability of the vitality of the preserved animal sperm and delay the survival time of the animal sperm.

Further, in the step (1), the temperature is kept at 28 ℃; in the step (2), animal sperm diluent is used for diluting according to the sperm number of 500 ten thousand/mL. In the step (3), the temperature is reduced to 4 ℃ at the speed of 0.2 ℃/min. In the step (4), the semen in the thin tube is balanced for 1 h. In the step (5), the height is 2.5cm away from the liquid nitrogen surface, the temperature is 110 ℃ below zero, and the fumigation lasts 10 min.

Adopt the beneficial effect of above-mentioned scheme: by adopting the parameters, the sperm motility and the storage time can be further improved.

The invention provides a method for preserving sperms by cold storage of animal sperm diluent, which 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 semen in the step (1) by using a spectrophotometer, diluting the semen by using animal sperm diluent according to the sperm number of 300-1000 ten thousand/mL (namely the ratio of the sperm number to the volume of the animal sperm diluent is 300-1000 ten thousand: 1 mL), filling the diluted semen into the frozen semen tubule by using a filling and sealing machine by using a frozen semen tubule of 0.25 mL;

(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.

The invention has the beneficial effects that: the preservation method adopted by the invention has the advantages of lasting sperm motility, long survival time and the like. The invention solves the problems that when animal sperms are preserved by the traditional method, the observation visual field is not clear, the pollution is easy, the survival time is short, and the use of antibiotics is not standardized, so that the production of super bacteria is caused.

Further, the animal sperm diluent consists 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 invention has the beneficial effects that: the animal sperm diluent can further improve the durability of the vitality of the preserved animal sperm and delay the survival time of the animal sperm.

Further, 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 the animal sperm diluent.

Furthermore, the mass of the defatted pea protein powder is 0.1g-0.5 g. Preferably, the mass of the defatted pea protein powder is 0.1 g. The weight of the trihydroxymethyl aminomethane is 20g, the volume of the sterilized distilled water is 180mL, the weight of the citric acid is 10g or 12g, the weight of the glucose is 10g or 15g, the weight of the defatted soybean protein powder is 0.5g, the weight of the defatted peanut protein powder is 0.5g, the weight of the defatted wheat protein powder is 0.2g, the weight of the fructose is 10g, and the volume of the glycerol is 50 mL.

Adopt the beneficial effect of above-mentioned scheme: further improve the quality of the animal sperm diluent.

Further, in step (1), the temperature was maintained at 28 ℃.

Further, in the step (2), the sperm is diluted with an animal sperm diluent in an amount of 600 ten thousand/mL.

Further, in the step (3), the temperature is reduced at a rate of 0.4 ℃/min.

Further, in the step (3), the temperature is reduced to 4 ℃ for standby.

Adopt the beneficial effect of above-mentioned scheme: by adopting the parameters, the sperm motility and the storage time can be further improved.

In summary, the advantages of the present invention over the prior art are:

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.