CN112640886A - Cattle frozen semen diluent and preparation method thereof - Google Patents
Cattle frozen semen diluent and preparation method thereof Download PDFInfo
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- CN112640886A CN112640886A CN202011510954.9A CN202011510954A CN112640886A CN 112640886 A CN112640886 A CN 112640886A CN 202011510954 A CN202011510954 A CN 202011510954A CN 112640886 A CN112640886 A CN 112640886A
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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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- 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
Abstract
The invention provides a cattle frozen semen diluent which comprises the following components in percentage by mass: 7-13.5% of tris (hydroxymethyl) aminomethane, 5-8.3% of citric acid, 3-9.3% of fructose, 0.2-1.2% of glycerol, 23-35% of egg yolk and the balance of deionized water. The preparation method comprises four steps of primary mixing, secondary mixing, disinfection and purification, primary fine dilution and the like. The sheep semen diluent and the preparation method thereof have low production cost, can effectively solve the problems of diluent pollution and short preservation time and low conception success rate caused by the high colony count of the frozen cow semen on the premise of ensuring that the function of the diluent is not influenced, thereby greatly improving the conception rate of artificial insemination and the efficiency of in vitro insemination, reducing the cost of artificial insemination and the labor intensity and difficulty, and being convenient for large-scale popularization and application.
Description
Technical Field
The invention relates to a cattle frozen semen diluent and a preparation method thereof, belonging to the technical field of breeding and artificial propagation.
Background
In livestock breeding, because the balance of intestinal flora of animals is broken, and harmful bacteria are bred and propagated, digestive system diseases of animals are easily caused, the absorption, transportation and transformation capacity of nutrient components of feed are seriously influenced, so that the growth and development speed of the livestock is slow, even the livestock is subjected to digestive system diseases such as diarrhea and the like in serious cases, and the life safety of the livestock is seriously threatened, so that the breeding risk and the cost are greatly increased, and aiming at the problem, although various medicines for treating the digestive system diseases and stopping dysentery of the animals exist at present, the medicines generally adopt western medicine antibiotics or single enzyme preparations or traditional Chinese medicine powder, the effects of the products are single, only a small part of intestinal problems can be solved, the effects are not obvious, and in use, the antibiotic products can kill the intestinal harmful bacteria and beneficial bacteria and simultaneously cause the imbalance of the intestinal flora, and a large amount of drug residues are left, so that the method is not suitable for the requirements of green culture and food health development.
Therefore, in order to solve the problem, the development of a novel medicament for regulating the intestinal tract of animals is urgently needed so as to meet the requirement of actual breeding work.
Disclosure of Invention
Aiming at the defects in the prior art, the invention provides a cattle frozen semen diluent and a preparation method thereof, so as to meet the requirements of practical use.
In order to achieve the purpose, the invention provides the following technical scheme:
the cattle frozen semen diluent consists of the following components in percentage by mass: 7-13.5% of tris (hydroxymethyl) aminomethane, 5-8.3% of citric acid, 3-9.3% of fructose, 0.2-1.2% of glycerol, 23-35% of egg yolk and the balance of deionized water.
Furthermore, the deionized water is additionally added with compound antibiotics accounting for 0.5-1.5 percent of the total amount of the deionized water, sodium chloride accounting for 1.5-2.3 percent of the total amount of the deionized water and vitamins accounting for 0.1-0.3 percent of the total amount of the deionized water.
Further, the compound antibiotic is any one of penicillin and gentamicin and any one of the penicillin and the gentamicin which are mixed according to the proportion of 1: 1-3.5; the vitamin is the mixture of vitamin C and vitamin B in the ratio of 1: 1-1.8.
A disinfection preparation method of cattle frozen semen diluent comprises the following steps:
s1, primarily mixing, namely adding deionized water into a mixing cavity, regulating the temperature to 20-50 ℃, preserving at constant temperature, adding tris (hydroxymethyl) aminomethane, citric acid and fructose into the deionized water, and uniformly stirring to obtain a primarily mixed solution;
s2, mixing for the second time, adding glycerol and yolk into the primary mixed solution, performing ultrasonic homogenization to obtain a finished product diluent, and then preserving the diluent at constant temperature for later use;
s3, sterilizing and purifying, namely, performing bus sterilization and ultraviolet irradiation sterilization on the sterilization cavity in sequence, heating the sterilization cavity to 20-30 ℃, adding the diluent prepared in the step S2 into the sterilization cavity, sealing the sterilization cavity, heating the sterilization cavity in a water bath until the temperature of the diluent in the sterilization cavity and the temperature of the diluent in the sterilization cavity are constant to 61-64 ℃, preserving the heat for 25-40 minutes, and then placing the sterilized cavity and the diluent in the sterilization cavity which are preserved in the water bath for cooling until the temperature of the diluent in the sterilization cavity reaches the normal temperature to obtain the finished product diluent;
s4, diluting the original essence, preserving and sampling the collected original essence at constant temperature of 38 ℃, and observing and analyzing the original essence under the microscope in an environment amplified by 400 times of 200 times, wherein the activity is more than or equal to 65 percent, and the density is more than or equal to 6 hundred million/ml, and the original essence is a qualified product; and then mixing the diluent prepared in the step S3 with the qualified product raw essence, homogenizing for 1-5 minutes at the temperature of 3-5 ℃, standing for 3-10 minutes, filling by using a 0.25ml plastic tube container, cooling the filled plastic tube container to-140 ℃ at the temperature of 3-5 ℃ for 7-9 minutes, packaging the cooled plastic tube container, and storing at the constant temperature of-196 ℃ to obtain a finished product.
Further, in the step S3, when the water bath heating and cooling is performed, the liquid level of the water bath water body is 0 to 10 mm higher than the liquid level of the diluent in the disinfection chamber.
Further, in the step S3, the prepared finished product diluent is stored at the constant temperature of 25-40 ℃ in a dark place, and the storage time is not more than 24 hours after the sealing state is broken.
Further, in the step S4, the activity of the prepared finished product is more than or equal to 35%; the number of linearly moving sperms is more than or equal to 800 ten thousand per dose; the colony number is less than 800/dose; the distortion rate is less than 18 per dose; the integrity rate of the top body is more than or equal to 40, and the plastic pipe container has complete sealing, no crack and clear mark and is a qualified product.
The sheep semen diluent and the preparation method thereof have low production cost, can effectively solve the problems of diluent pollution and short preservation time and low conception success rate caused by the high colony count of the frozen cow semen on the premise of ensuring that the function of the diluent is not influenced, thereby greatly improving the conception rate of artificial insemination and the efficiency of in vitro insemination, reducing the cost of artificial insemination and the labor intensity and difficulty, and being convenient for large-scale popularization and application.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the drawings without creative efforts.
FIG. 1 is a flow chart of the manufacturing process of the present invention.
Detailed Description
The technical solutions of the present invention will be described clearly and completely with reference to the accompanying drawings of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example 1
The cattle frozen semen diluent consists of the following components in percentage by mass: 7% of trihydroxymethyl aminomethane, 5% of citric acid, 3% of fructose, 0.2% of glycerol, 23% of yolk and the balance of deionized water.
Wherein, the deionized water is additionally added with compound antibiotics accounting for 0.5 percent of the total amount of the deionized water, sodium chloride accounting for 1.5 percent of the total amount of the deionized water and vitamins accounting for 0.1 percent of the total amount of the deionized water.
In addition, the compound antibiotic is penicillin; the vitamin is vitamin C and vitamin B mixed in a ratio of 1:1.
A disinfection preparation method of cattle frozen semen diluent comprises the following steps:
s1, primarily mixing, namely adding deionized water into a mixing cavity, regulating the temperature to 20 ℃, preserving at a constant temperature, adding tris (hydroxymethyl) aminomethane, citric acid and fructose into the deionized water, and uniformly stirring to obtain a primary mixed solution;
s2, mixing for the second time, adding glycerol and yolk into the primary mixed solution, performing ultrasonic homogenization to obtain a finished product diluent, and then preserving the diluent at constant temperature for later use;
and S3, sterilizing and purifying, namely, performing bus sterilization and ultraviolet irradiation sterilization on the sterilization cavity in sequence, heating the sterilization cavity to 20 ℃, adding the diluent prepared in the step S2 into the sterilization cavity, sealing the sterilization cavity, heating the sterilization cavity in water bath until the temperature of the diluent in the sterilization cavity and the temperature of the diluent in the sterilization cavity are constant to 61, preserving the heat for 25 minutes, placing the sterilized cavity and the diluent in the sterilization cavity which are preserved in the water bath at normal temperature, and cooling the sterilized cavity and the diluent in the sterilization cavity in the water bath until the temperature of the diluent in the sterilization cavity reaches the normal temperature to obtain the finished product diluent.
S4, diluting the original essence, preserving and sampling the collected original essence at constant temperature of 38 ℃, and observing and analyzing the original essence under the microscope in an environment amplified by 400 times of 200 times, wherein the activity is more than or equal to 65 percent, and the density is more than or equal to 6 hundred million/ml, and the original essence is a qualified product; and then mixing the diluent prepared in the step S3 with the qualified product raw essence, homogenizing for 1 minute at the temperature of 3 ℃, standing for 3 minutes, filling by using a 0.25ml plastic tube container, reducing the temperature of the filled plastic tube container to-140 ℃ within 7 minutes, packaging the cooled plastic tube container, and storing at the constant temperature of-196 ℃ to obtain a finished product.
In the step S3, when the water bath heating and cooling are performed, the liquid level of the water bath water is 0 mm higher than the liquid level of the diluent in the disinfection chamber.
Meanwhile, in the step S3, the prepared finished product diluent is stored at a constant temperature of 25 ℃ in a dark place, and the storage time is not more than 24 hours after the sealing state is broken.
In addition, in the step S4, the activity of the prepared finished product is more than or equal to 35 percent; the number of linearly moving sperms is more than or equal to 800 ten thousand per dose; the colony number is less than 800/dose; the distortion rate is less than 18 per dose; the integrity rate of the top body is more than or equal to 40, and the plastic pipe container has complete sealing, no crack and clear mark and is a qualified product.
Example 2
The cattle frozen semen diluent consists of the following components in percentage by mass: 13.5% of trihydroxymethyl aminomethane, 8.3% of citric acid, 9.3% of fructose, 1.2% of glycerol, 35% of yolk and the balance of deionized water.
In this embodiment, the deionized water is further added with a compound antibiotic accounting for 1.5% of the total amount of the deionized water, sodium chloride accounting for 2.3% of the total amount of the deionized water, and vitamins accounting for 0.3% of the total amount of the deionized water.
Preferably, the compound antibiotic is gentamicin; the vitamin is the mixture of vitamin C and vitamin B in the ratio of 1: 1.8.
A disinfection preparation method of cattle frozen semen diluent comprises the following steps:
s1, primarily mixing, namely adding deionized water into a mixing cavity, regulating the temperature to 50 ℃, preserving at a constant temperature, adding tris (hydroxymethyl) aminomethane, citric acid and fructose into the deionized water, and uniformly stirring to obtain a primary mixed solution;
s2, mixing for the second time, adding glycerol and yolk into the primary mixed solution, performing ultrasonic homogenization to obtain a finished product diluent, and then preserving the diluent at constant temperature for later use;
s3, disinfection and purification, namely, performing bus sterilization and ultraviolet irradiation sterilization on the disinfection cavity in sequence, heating the disinfection cavity to 30 ℃, adding the diluent prepared in the step S2 into the disinfection cavity, sealing the disinfection cavity, performing water bath heating on the disinfection cavity until the temperature of the diluent in the disinfection cavity and the temperature of the diluent in the disinfection cavity are constant to 64 ℃, performing heat preservation for 40 minutes, placing the disinfection cavity and the diluent in the disinfection cavity after heat preservation into a normal-temperature water body for water bath cooling, and obtaining a finished product diluent when the temperature of the diluent in the disinfection cavity reaches the normal temperature;
s4, diluting the original essence, preserving and sampling the collected original essence at constant temperature of 38 ℃, and observing and analyzing the original essence under the microscope in an environment amplified by 400 times of 200 times, wherein the activity is more than or equal to 65 percent, and the density is more than or equal to 6 hundred million/ml, and the original essence is a qualified product; and then mixing the diluent prepared in the step S3 with the qualified product original essence, homogenizing for 5 minutes at the temperature of 3-5 ℃, standing for 10 minutes, filling by using a 0.25ml plastic tube container, cooling the filled plastic tube container to-140 ℃ at the temperature of 9 minutes and 5 ℃, packaging the cooled plastic tube container, and storing at the constant temperature of-196 ℃ to obtain the finished product.
In this embodiment, in the step S3, when the water bath heating and cooling is performed, the liquid level of the water bath water is 10 mm higher than the liquid level of the diluent in the disinfection chamber.
Meanwhile, in the step S3, the prepared finished product diluent is preserved at the constant temperature of 40 ℃ in a dark place, and the preservation time is not more than 24 hours after the sealing state is broken.
It should be noted that in the step S4, the activity of the prepared finished product is more than or equal to 35%; the number of linearly moving sperms is more than or equal to 800 ten thousand per dose; the colony number is less than 800/dose; the distortion rate is less than 18 per dose; the integrity rate of the top body is more than or equal to 40, and the plastic pipe container has complete sealing, no crack and clear mark and is a qualified product.
Example 3
The cattle frozen semen diluent consists of the following components in percentage by mass: 11.5% of trihydroxymethyl aminomethane, 7.3% of citric acid, 6.3% of fructose, 0.8% of glycerol, 31% of yolk and the balance of deionized water.
It is important to point out that the deionized water is additionally added with compound antibiotics accounting for 0.8 percent of the total amount of the deionized water, sodium chloride accounting for 2.0 percent of the total amount of the deionized water and vitamins accounting for 0.2 percent of the total amount of the deionized water.
Further, the composite antibiotic is penicillin and gentamicin which are mixed according to the proportion of 1: 1-3.5; the vitamin is the mixture of vitamin C and vitamin B in the ratio of 1: 1.5.
A disinfection preparation method of cattle frozen semen diluent comprises the following steps:
s1, primarily mixing, namely adding deionized water into a mixing cavity, regulating the temperature to 37 ℃, preserving at a constant temperature, adding trihydroxymethyl aminomethane, citric acid and fructose into the deionized water, and uniformly stirring to obtain a primary mixed solution;
s2, mixing for the second time, adding glycerol and yolk into the primary mixed solution, performing ultrasonic homogenization to obtain a finished product diluent, and then preserving the diluent at constant temperature for later use;
s3, disinfection and purification, namely, performing bus sterilization and ultraviolet irradiation sterilization on the disinfection cavity in sequence, heating the disinfection cavity to 25 ℃, adding the diluent prepared in the step S2 into the disinfection cavity, sealing the disinfection cavity, performing water bath heating on the disinfection cavity until the temperature of the diluent in the disinfection cavity and the temperature of the diluent in the disinfection cavity are constant to 63 ℃, performing heat preservation for 30 minutes, placing the disinfection cavity and the diluent in the disinfection cavity after heat preservation into a normal-temperature water body for water bath cooling, and obtaining a finished product diluent when the temperature of the diluent in the disinfection cavity reaches the normal temperature;
s4, diluting the original essence, preserving and sampling the collected original essence at constant temperature of 38 ℃, and observing and analyzing the original essence under the microscope in an environment amplified by 400 times of 200 times, wherein the activity is more than or equal to 65 percent, and the density is more than or equal to 6 hundred million/ml, and the original essence is a qualified product; and then mixing the diluent prepared in the step S3 with the qualified product original essence, homogenizing for 3 minutes at the temperature of 3-5 ℃, standing for 5 minutes, filling by using a 0.25ml plastic tube container, cooling the filled plastic tube container to-140 ℃ at the temperature of 8 minutes and 4 ℃, packaging the cooled plastic tube container, and storing at the constant temperature of-196 ℃ to obtain a finished product.
In this embodiment, in the step S3, when the water bath heating and cooling is performed, the liquid level of the water bath water body is 5 mm higher than the liquid level of the diluent in the disinfection chamber.
Meanwhile, in the step S3, the prepared finished product diluent is stored at a constant temperature of 30 ℃ in a dark place, and the storage time is not more than 24 hours after the sealing state is broken.
In addition, in the step S4, the activity of the prepared finished product is more than or equal to 35 percent; the number of linearly moving sperms is more than or equal to 800 ten thousand per dose; the colony number is less than 800/dose; the distortion rate is less than 18 per dose; the integrity rate of the top body is more than or equal to 40, and the plastic pipe container has complete sealing, no crack and clear mark and is a qualified product.
The sheep semen diluent and the preparation method thereof have low production cost, can effectively solve the problems of diluent pollution and short preservation time and low conception success rate caused by the high colony count of the frozen cow semen on the premise of ensuring that the function of the diluent is not influenced, thereby greatly improving the conception rate of artificial insemination and the efficiency of in vitro insemination, reducing the cost of artificial insemination and the labor intensity and difficulty, and being convenient for large-scale popularization and application.
The above description is only for the specific embodiments of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art can easily conceive of the changes or substitutions within the technical scope of the present invention, and all the changes or substitutions should be covered within the scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the protection scope of the claims.
Claims (7)
1. The utility model provides a frozen bovine semen diluent which characterized in that: the cattle frozen semen diluent consists of the following components in percentage by mass: 7-13.5% of tris (hydroxymethyl) aminomethane, 5-8.3% of citric acid, 3-9.3% of fructose, 0.2-1.2% of glycerol, 23-35% of egg yolk and the balance of deionized water.
2. The preparation method of the sheep fresh essence normal-temperature short-term preservation diluent according to claim 1 is characterized by comprising the following steps of: the deionized water is additionally added with compound antibiotics accounting for 0.5-1.5% of the total amount of the deionized water, sodium chloride accounting for 1.5-2.3% of the total amount of the deionized water and vitamins accounting for 0.1-0.3% of the total amount of the deionized water.
3. The preparation method of the sheep fresh essence normal-temperature short-term preservation diluent according to claim 2 is characterized by comprising the following steps: the compound antibiotic is any one of penicillin and gentamicin and any one of the penicillin and gentamicin which are mixed according to the proportion of 1: 1-3.5; the vitamin is the mixture of vitamin C and vitamin B in the ratio of 1: 1-1.8.
4. A disinfection preparation method of cattle frozen semen diluent is characterized in that: the disinfection preparation method of the cattle frozen semen diluent comprises the following steps:
s1, primarily mixing, namely adding deionized water into a mixing cavity, regulating the temperature to 20-50 ℃, preserving at constant temperature, adding tris (hydroxymethyl) aminomethane, citric acid and fructose into the deionized water, and uniformly stirring to obtain a primarily mixed solution;
s2, mixing for the second time, adding glycerol and yolk into the primary mixed solution, performing ultrasonic homogenization to obtain a finished product diluent, and then preserving the diluent at constant temperature for later use;
s3, sterilizing and purifying, namely, performing bus sterilization and ultraviolet irradiation sterilization on the sterilization cavity in sequence, heating the sterilization cavity to 20-30 ℃, adding the diluent prepared in the step S2 into the sterilization cavity, sealing the sterilization cavity, heating the sterilization cavity in a water bath until the temperature of the diluent in the sterilization cavity and the temperature of the diluent in the sterilization cavity are constant to 61-64 ℃, preserving the heat for 25-40 minutes, and then placing the sterilized cavity and the diluent in the sterilization cavity which are preserved in the water bath for cooling until the temperature of the diluent in the sterilization cavity reaches the normal temperature to obtain the finished product diluent;
s4, diluting the original essence, preserving and sampling the collected original essence at constant temperature of 38 ℃, and observing and analyzing the original essence under the microscope in an environment amplified by 400 times of 200 times, wherein the activity is more than or equal to 65 percent, and the density is more than or equal to 6 hundred million/ml, and the original essence is a qualified product; and then mixing the diluent prepared in the step S3 with the qualified product raw essence, homogenizing for 1-5 minutes at the temperature of 3-5 ℃, standing for 3-10 minutes, filling by using a 0.25ml plastic tube container, cooling the filled plastic tube container to-140 ℃ at the temperature of 3-5 ℃ for 7-9 minutes, packaging the cooled plastic tube container, and storing at the constant temperature of-196 ℃ to obtain a finished product.
5. The preparation method of the sheep fresh essence normal-temperature short-term preservation diluent according to claim 4 is characterized by comprising the following steps: in the step S3, when the water bath heating and cooling is performed, the liquid level of the water bath water is 0 to 10 mm higher than the liquid level of the diluent in the disinfection chamber.
6. The preparation method of the sheep fresh essence normal-temperature short-term preservation diluent according to claim 4 is characterized by comprising the following steps: and in the step S3, the prepared finished product diluent is stored at the constant temperature of 25-40 ℃ in a dark place, and the storage time is not more than 24 hours after the sealing state is broken.
7. The preparation method of the sheep fresh essence normal-temperature short-term preservation diluent according to claim 4 is characterized by comprising the following steps: in the step S4, the activity of the prepared finished product is more than or equal to 35 percent; the number of linearly moving sperms is more than or equal to 800 ten thousand per dose; the colony number is less than 800/dose; the distortion rate is less than 18 per dose; the integrity rate of the top body is more than or equal to 40, and the plastic pipe container has complete sealing, no crack and clear mark and is a qualified product.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114208813A (en) * | 2021-12-23 | 2022-03-22 | 北京奶牛中心 | Cow frozen semen diluent, preparation method thereof and semen freezing method |
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| CN114208813A (en) * | 2021-12-23 | 2022-03-22 | 北京奶牛中心 | Cow frozen semen diluent, preparation method thereof and semen freezing method |
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