CN114304139A - Semen preserving fluid for improving semen preserving quality and preparation method thereof - Google Patents
Semen preserving fluid for improving semen preserving quality and preparation method thereof Download PDFInfo
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- CN114304139A CN114304139A CN202210060757.4A CN202210060757A CN114304139A CN 114304139 A CN114304139 A CN 114304139A CN 202210060757 A CN202210060757 A CN 202210060757A CN 114304139 A CN114304139 A CN 114304139A
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- CJWQYWQDLBZGPD-UHFFFAOYSA-N isoflavone Natural products C1=C(OC)C(OC)=CC(OC)=C1C1=COC2=C(C=CC(C)(C)O3)C3=C(OC)C=C2C1=O CJWQYWQDLBZGPD-UHFFFAOYSA-N 0.000 claims abstract description 26
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- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 18
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- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 10
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- Meat, Egg Or Seafood Products (AREA)
Abstract
The invention discloses a semen preserving fluid for improving semen preserving quality and a preparation method thereof, wherein the semen preserving fluid comprises the following raw materials in parts by weight: 50-60 parts of glucose, 5-8 parts of glycine, 5-8 parts of citric acid, 10-15 parts of taurine, 3-5 parts of bacteriostatic agent, 5-10 parts of modified lecithin, 7-12 parts of glycerol, 5-10 parts of pentoxifylline, 5-10 parts of soybean isoflavone, 2-5 parts of homemade yolk and 15-20 parts of purified water. The invention can maintain the vitality of donkey sperms for a long time, can bring more sufficient artificial insemination operation time or operability of trans-regional artificial insemination, can be widely applied to donkey semen preservation and related embryo transplantation industries and embryo engineering, has wide market prospect, solves the problems of large-scale donkey breeding farms and popularization and application of artificial insemination to peripheral donkey breeding households in the concentrated estrus period of donkey, and can effectively improve the utilization efficiency of breeding male donkeys.
Description
Technical Field
The invention relates to the technical field of donkey breeding, in particular to a semen preserving fluid for improving semen preserving quality and a preparation method thereof.
Background
The cryopreservation liquid is a sperm protective agent and has a decisive influence on the semen freezing effect. Donkey is an equine animal and is a seasonal oestrus animal with long sunshine, the oestrus of female donkey usually appears intensively in large-scale donkey breeding farms, and if the male donkey is used for on-site semen collection, and then the collected semen is diluted on site by diluent and then is subjected to artificial insemination, the operation time is relatively short, and great inconvenience is brought to production. If the donkey semen collected is stored at low temperature in advance by using the semen low-temperature storage liquid in the long-day season, artificial insemination can be directly performed when the female donkey is in estrus, not only is the production convenient, but also the utilization efficiency of breeding male donkeys is effectively improved. However, due to poor breeding efficiency, the number of stockings is continuously reduced in recent years. The conception rate of donkey is obviously lower than that of fresh semen and liquid preserved semen, because the activity of frozen and unfrozen semen is reduced, and the number of effective sperms capable of keeping the fertilization capability is obviously reduced.
Disclosure of Invention
The invention aims to provide a semen preserving fluid for improving semen preserving quality and a preparation method thereof, which aim to solve the problems in the background technology.
In order to achieve the purpose, the invention provides the following technical scheme:
the semen preserving fluid for improving the semen preserving quality comprises the following raw materials in parts by weight: 50-60 parts of glucose, 5-8 parts of glycine, 5-8 parts of citric acid, 10-15 parts of taurine, 3-5 parts of bacteriostatic agent, 5-10 parts of modified lecithin, 7-12 parts of glycerol, 5-10 parts of pentoxifylline, 5-10 parts of soybean isoflavone, 2-5 parts of homemade yolk and 15-20 parts of purified water.
Preferably, the feed comprises the following raw materials in parts by weight: 55 parts of glucose, 6 parts of glycine, 7 parts of citric acid, 12 parts of taurine, 4 parts of bacteriostatic agent, 7 parts of modified lecithin, 10 parts of glycerol, 8 parts of pentoxifylline, 6 parts of soybean isoflavone, 3 parts of homemade yolk and 18 parts of purified water.
Preferably, the feed comprises the following raw materials in parts by weight: 50 parts of glucose, 5 parts of glycine, 5 parts of citric acid, 10 parts of taurine, 33 parts of bacteriostatic agent, 5 parts of modified lecithin, 7 parts of glycerol, 5 parts of pentoxifylline, 5 parts of soybean isoflavone, 2 parts of homemade yolk and 15 parts of purified water.
Preferably, the feed comprises the following raw materials in parts by weight: 60 parts of glucose, 8 parts of glycine, 8 parts of citric acid, 15 parts of taurine, 5 parts of bacteriostatic agent, 10 parts of modified lecithin, 12 parts of glycerol, 10 parts of pentoxifylline, 10 parts of soybean isoflavone, 5 parts of homemade yolk and 20 parts of purified water.
Preferably, the bacteriostatic agent is penicillin 10-20 ten thousand IU and streptomycin 10-20 ten thousand IU.
Preferably, the self-made egg yolk is egg, duck egg, goose egg or quail egg.
Preferably, the eggs of the chicken, the ducks, the geese or the quail are all from farmhouse eggs without epidemic diseases, and the farmhouse eggs are fresh, complete and clean in surface, in particular to fresh farmhouse eggs for 3-7 days.
Preferably, the preparation method of the homemade yolk comprises the following steps: sterilizing the surface of an eggshell by using a 75% alcohol cotton ball, opening a hole on the surface of one end of the eggshell after alcohol is volatilized completely, sterilizing an injector, and then penetrating a yolk membrane to extract yolk to obtain the self-made yolk.
On the other hand, the invention also provides a preparation method of the semen preservation solution for improving the semen preservation quality, which comprises the following steps:
s11: weighing the raw materials in parts by weight;
s12: adding the weighed glucose, glycine, citric acid, taurine, modified lecithin, glycerol, pentoxifylline, soybean isoflavone and self-made yolk into a sterilized container, and adding purified water to fully dissolve at normal temperature to obtain a mixed solution;
s13: adding modified lecithin, soybean isoflavone, pentoxifylline and bacteriostatic agent into the mixed solution in sequence, controlling the temperature at 20-25 ℃, stirring uniformly, and filtering through a 0.22-0.3 mu m filter membrane to obtain filtrate, namely the semen preservation solution;
s14: and subpackaging the semen preservation solution into 50-80ml centrifuge tubes, sealing and refrigerating for later use.
Preferably, steps S11 through S14 are all operated in a sterile environment.
Compared with the prior art, the invention has the beneficial effects that:
1. the semen preservation solution for improving semen preservation quality and the preparation method thereof can maintain the vitality of donkey sperms for a long time, can bring more sufficient artificial insemination operation time or operability of trans-regional artificial insemination, can be widely applied to donkey semen preservation and related embryo transplantation industries and embryo engineering, have wide market prospect, solve the problems of large-scale donkey breeding fields and popularization and application of artificial insemination to peripheral donkey breeding households in the time period of concentrated estrus of female donkeys, and can effectively improve the utilization efficiency of male donkeys.
2. According to the semen preserving fluid for improving semen preserving quality and the preparation method thereof, donkey sperms are protected from microorganisms and impurities carried in the semen collection and dilution process by the added bacteriostatic agent, the metabolism of the microorganisms can influence the quality of the semen diluent, and the added modified lecithin can be better dispersed in the solution than common lecithin, has the effects of regulating osmotic pressure, reducing the concentration of electrolyte in the semen and protecting a sperm membrane, and is beneficial to the survival of the sperms; by adding pentoxifylline, the activity of phosphodiesterase can be inhibited, degradation of adenosine cyclophosphate in sperm cells is reduced, the cAMP concentration in the sperm cytoplasm is increased, cAMP dependent protease is activated, the tail of the sperm is phosphorylated, and the movement capacity of the sperm is enhanced; the soybean isoflavone is added, so that the free radicals can be removed, the activity of antioxidant enzyme is improved, the soybean isoflavone can be used as an antioxidant to remove excessive free oxygen in the in vitro environment of sperms, the sperms are prevented from being oxidized and damaged, the vitality of the sperms is maintained, and the success rate of artificial insemination is improved.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and 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 semen preserving fluid for improving the semen preserving quality comprises the following raw materials in parts by weight: 55 parts of glucose, 6 parts of glycine, 7 parts of citric acid, 12 parts of taurine, 4 parts of bacteriostatic agent, 7 parts of modified lecithin, 10 parts of glycerol, 8 parts of pentoxifylline, 6 parts of soybean isoflavone, 3 parts of homemade yolk and 18 parts of purified water.
The bacteriostatic agent in the embodiment is penicillin 15 ten thousand IU and streptomycin 15 ten thousand IU.
In the embodiment, the self-made egg yolk is goose eggs which are from farmhouse eggs without epidemic diseases, and the farmhouse eggs are fresh, complete and clean in surface, and particularly are fresh farmhouse eggs for 3 days.
Further, the preparation method of the self-made yolk in this embodiment includes the following steps: sterilizing the surface of an eggshell by using a 75% alcohol cotton ball, opening a hole on the surface of one end of the eggshell after alcohol is volatilized completely, sterilizing an injector, and then penetrating a yolk membrane to extract yolk to obtain the self-made yolk.
The preparation method of the semen preservation solution for improving the semen preservation quality in the embodiment specifically comprises the following steps:
s11: weighing the raw materials in parts by weight;
s12: adding the weighed glucose, glycine, citric acid, taurine, modified lecithin, glycerol, pentoxifylline, soybean isoflavone and self-made yolk into a sterilized container, and adding purified water to fully dissolve at normal temperature to obtain a mixed solution;
s13: adding modified lecithin, soybean isoflavone, pentoxifylline and bacteriostatic agent into the mixed solution in sequence, controlling the temperature at 22 ℃, stirring uniformly, and filtering through a 0.25-micrometer filter membrane to obtain filtrate, namely the semen preservation solution;
s14: and subpackaging the semen preservation solution into 60ml centrifuge tubes, sealing and refrigerating for later use.
It should be noted that steps S11 to S14 are all performed in a sterile environment.
Example 2
The semen preserving fluid for improving the semen preserving quality comprises the following raw materials in parts by weight: 50 parts of glucose, 5 parts of glycine, 5 parts of citric acid, 10 parts of taurine, 33 parts of bacteriostatic agent, 5 parts of modified lecithin, 7 parts of glycerol, 5 parts of pentoxifylline, 5 parts of soybean isoflavone, 2 parts of homemade yolk and 15 parts of purified water.
The bacteriostatic agent in the embodiment is 20 ten thousand IU of penicillin and 20 ten thousand IU of streptomycin.
In the embodiment, the self-made egg yolk adopts quail eggs, the quail eggs are from farmhouse eggs without epidemic diseases, and the farmhouse eggs are fresh, complete and clean in surface, in particular to fresh farmhouse eggs for 5 days.
Further, the preparation method of the self-made yolk in this embodiment includes the following steps: sterilizing the surface of an eggshell by using a 75% alcohol cotton ball, opening a hole on the surface of one end of the eggshell after alcohol is volatilized completely, sterilizing an injector, and then penetrating a yolk membrane to extract yolk to obtain the self-made yolk.
The preparation method of the semen preservation solution for improving the semen preservation quality in the embodiment specifically comprises the following steps:
s11: weighing the raw materials in parts by weight;
s12: adding the weighed glucose, glycine, citric acid, taurine, modified lecithin, glycerol, pentoxifylline, soybean isoflavone and self-made yolk into a sterilized container, and adding purified water to fully dissolve at normal temperature to obtain a mixed solution;
s13: adding modified lecithin, soybean isoflavone, pentoxifylline and bacteriostatic agent into the mixed solution in sequence, controlling the temperature at 25 ℃, stirring uniformly, and filtering through a 0.22-micron filter membrane to obtain filtrate, namely the semen preservation solution;
s14: and subpackaging the semen preservation solution into 50ml centrifuge tubes, sealing and refrigerating for later use.
It should be noted that steps S11 to S14 are all performed in a sterile environment.
Example 3
The semen preserving fluid for improving the semen preserving quality comprises the following raw materials in parts by weight: 60 parts of glucose, 8 parts of glycine, 8 parts of citric acid, 15 parts of taurine, 5 parts of bacteriostatic agent, 10 parts of modified lecithin, 12 parts of glycerol, 10 parts of pentoxifylline, 10 parts of soybean isoflavone, 5 parts of homemade yolk and 20 parts of purified water.
The bacteriostatic agent in the embodiment is 20 ten thousand IU of penicillin and 20 ten thousand IU of streptomycin.
In the embodiment, the self-made yolk adopts duck eggs, the duck eggs are derived from farmhouse eggs without epidemic diseases, and the farmhouse eggs are fresh, complete and clean in surface, and particularly are fresh farmhouse eggs for 3 days.
Further, the preparation method of the self-made yolk in this embodiment includes the following steps: sterilizing the surface of an eggshell by using a 75% alcohol cotton ball, opening a hole on the surface of one end of the eggshell after alcohol is volatilized completely, sterilizing an injector, and then penetrating a yolk membrane to extract yolk to obtain the self-made yolk.
The preparation method of the semen preservation solution for improving the semen preservation quality in the embodiment specifically comprises the following steps:
s11: weighing the raw materials in parts by weight;
s12: adding the weighed glucose, glycine, citric acid, taurine, modified lecithin, glycerol, pentoxifylline, soybean isoflavone and self-made yolk into a sterilized container, and adding purified water to fully dissolve at normal temperature to obtain a mixed solution;
s13: adding modified lecithin, soybean isoflavone, pentoxifylline and bacteriostatic agent into the mixed solution in sequence, controlling the temperature at 20 ℃, stirring uniformly, and filtering through a 0.25-micrometer filter membrane to obtain filtrate, namely the semen preservation solution;
s14: and subpackaging the semen preservation solution into 80ml centrifuge tubes, sealing and refrigerating for later use.
It should be noted that steps S11 to S14 are all performed in a sterile environment.
Experimental testing
1. Selecting healthy male donkey seeds, putting the semen diluent into a 38 ℃ water bath kettle in advance before semen collection, and requiring the survival rate of the semen in the semen to be more than 95% according to the observed semen quality after the semen is collected:
2. semen collection is carried out on the same male donkey, semen preservation solutions of the three embodiments of the invention and INRA9 on the market are used for diluting semen under the condition of similar semen quality analysis, and the proportion of the semen to the diluent is 1: 3, artificial insemination is respectively carried out on 30 estrus female donkeys by semen diluted by each diluent, the survival rate (the heat preservation temperature is 22 ℃) and the initial preparation power (within 20 min) of 5h, 10h and 20h are counted and stored, and the following table shows that:
| 5h survival/%) | 10h survival/%) | 20h survival/%) | Initial formulation power/%) | |
| Example 1 | 86.20 | 79.83 | 73.85 | 90 |
| Example 2 | 85.78 | 80.12 | 74.25 | 88 |
| Example 3 | 86.08 | 80.46 | 74.15 | 90 |
| INRA9 | 53.3 | 40.36 | 30.85 | 70 |
The semen preservation solution for improving the semen preservation quality and the preparation method thereof can maintain the vitality of donkey sperms for a long time, can bring more sufficient artificial insemination operation time or operability of trans-regional artificial insemination, can be widely applied to donkey semen preservation and related embryo transplantation industries and embryo engineering, have wide market prospects, solve the problems of large-scale donkey breeding farms and popularization and application of artificial insemination to peripheral donkey breeding households in the concentrated estrus time period of a donkey, and can effectively improve the utilization efficiency of breeding male donkeys.
According to the semen preserving fluid for improving semen preserving quality and the preparation method thereof, donkey sperms are protected from microorganisms and impurities carried in the semen collection and dilution process by the added bacteriostatic agent, the metabolism of the microorganisms can influence the quality of the semen diluent, and the added modified lecithin can be better dispersed in the solution than common lecithin, has the effects of regulating osmotic pressure, reducing the concentration of electrolyte in the semen and protecting a sperm membrane, and is beneficial to the survival of the sperms; by adding pentoxifylline, the activity of phosphodiesterase can be inhibited, degradation of adenosine cyclophosphate in sperm cells is reduced, the cAMP concentration in the sperm cytoplasm is increased, cAMP dependent protease is activated, the tail of the sperm is phosphorylated, and the movement capacity of the sperm is enhanced; the soybean isoflavone is added, so that the free radicals can be removed, the activity of antioxidant enzyme is improved, the soybean isoflavone can be used as an antioxidant to remove excessive free oxygen in the in vitro environment of sperms, the sperms are prevented from being oxidized and damaged, the vitality of the sperms is maintained, and the success rate of artificial insemination is improved.
The foregoing shows and describes the general principles, essential features, and advantages of the invention. It will be understood by those skilled in the art that the present invention is not limited to the embodiments described above, and the preferred embodiments of the present invention are described in the above embodiments and the description, and are not intended to limit the present invention. The scope of the invention is defined by the appended claims and equivalents thereof.
Claims (10)
1. The utility model provides an improve semen preservation solution of semen preservation quality which characterized in that: the composite material comprises the following raw materials in parts by weight: 50-60 parts of glucose, 5-8 parts of glycine, 5-8 parts of citric acid, 10-15 parts of taurine, 3-5 parts of bacteriostatic agent, 5-10 parts of modified lecithin, 7-12 parts of glycerol, 5-10 parts of pentoxifylline, 5-10 parts of soybean isoflavone, 2-5 parts of homemade yolk and 15-20 parts of purified water.
2. The semen preserving fluid for improving semen preserving quality as claimed in claim 1, wherein: the composite material comprises the following raw materials in parts by weight: 55 parts of glucose, 6 parts of glycine, 7 parts of citric acid, 12 parts of taurine, 4 parts of bacteriostatic agent, 7 parts of modified lecithin, 10 parts of glycerol, 8 parts of pentoxifylline, 6 parts of soybean isoflavone, 3 parts of homemade yolk and 18 parts of purified water.
3. The semen preserving fluid for improving semen preserving quality as claimed in claim 1, wherein: the composite material comprises the following raw materials in parts by weight: 50 parts of glucose, 5 parts of glycine, 5 parts of citric acid, 10 parts of taurine, 33 parts of bacteriostatic agent, 5 parts of modified lecithin, 7 parts of glycerol, 5 parts of pentoxifylline, 5 parts of soybean isoflavone, 2 parts of homemade yolk and 15 parts of purified water.
4. The semen preserving fluid for improving semen preserving quality as claimed in claim 1, wherein: the composite material comprises the following raw materials in parts by weight: 60 parts of glucose, 8 parts of glycine, 8 parts of citric acid, 15 parts of taurine, 5 parts of bacteriostatic agent, 10 parts of modified lecithin, 12 parts of glycerol, 10 parts of pentoxifylline, 10 parts of soybean isoflavone, 5 parts of homemade yolk and 20 parts of purified water.
5. The semen preserving fluid for improving semen preserving quality as claimed in claim 1, wherein: the bacteriostatic agent is 10-20 ten thousand IU of penicillin and 10-20 ten thousand IU of streptomycin.
6. The semen preserving fluid for improving semen preserving quality as claimed in claim 1, wherein: the self-made yolk is egg, duck egg, goose egg or quail egg.
7. The semen preserving fluid for improving semen preserving quality as claimed in claim 1, wherein: the eggs, the duck eggs, the goose eggs or the quail eggs are all from farmhouse eggs without epidemic diseases, and the farmhouse eggs are fresh, complete and clean in surface, in particular to fresh farmhouse eggs for 3-7 days.
8. The semen preserving fluid for improving semen preserving quality as claimed in claim 1, wherein: the preparation method of the self-made yolk comprises the following steps: sterilizing the surface of an eggshell by using a 75% alcohol cotton ball, opening a hole on the surface of one end of the eggshell after alcohol is volatilized completely, sterilizing an injector, and then penetrating a yolk membrane to extract yolk to obtain the self-made yolk.
9. A method for preparing a semen preservation solution for improving semen preservation quality, comprising the semen preservation solution for improving semen preservation quality according to any one of claims 1 to 8, characterized in that: the method specifically comprises the following steps:
s11: weighing the raw materials in parts by weight;
s12: adding the weighed glucose, glycine, citric acid, taurine, modified lecithin, glycerol, pentoxifylline, soybean isoflavone and self-made yolk into a sterilized container, and adding purified water to fully dissolve at normal temperature to obtain a mixed solution;
s13: adding modified lecithin, soybean isoflavone, pentoxifylline and bacteriostatic agent into the mixed solution in sequence, controlling the temperature at 20-25 ℃, stirring uniformly, and filtering through a 0.22-0.3 mu m filter membrane to obtain filtrate, namely the semen preservation solution;
s14: and subpackaging the semen preservation solution into 50-80ml centrifuge tubes, sealing and refrigerating for later use.
10. The method for preparing semen preserving fluid for improving semen preserving quality as claimed in claim 9, wherein: steps S11 through S14 are all operated in a sterile environment.
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