CN110604129A - Hu sheep semen cryopreservation liquid and application thereof - Google Patents
Hu sheep semen cryopreservation liquid and application thereof Download PDFInfo
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- CN110604129A CN110604129A CN201911031372.XA CN201911031372A CN110604129A CN 110604129 A CN110604129 A CN 110604129A CN 201911031372 A CN201911031372 A CN 201911031372A CN 110604129 A CN110604129 A CN 110604129A
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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
- A01N1/0226—Physiologically active agents, i.e. substances affecting physiological processes of cells and tissue to be preserved, e.g. anti-oxidants or nutrients
Abstract
The invention discloses a Hu sheep semen cryopreservation solution, which comprises the following components: tris, glucose, raffinose, wheat germ oil, sodium citrate, glycerol, cryoprotectants, penicillin and streptomycin; the cryoprotectant comprises an astragalus extract, an acerola extract and a bamboo leaf extract. The invention also discloses a preparation method and application of the Hu sheep semen cryopreservation liquid. The cryopreservation solution provided by the invention can effectively improve the survival rate, activity and integrity of the Hu sheep semen in the preservation process, the preserved semen still has good insemination capability after being thawed, the conception effect is stable, the requirement of Hu sheep frozen semen artificial insemination can be met, and the application prospect is wide.
Description
Technical Field
The invention belongs to the technical field of Hu sheep breeding, and particularly relates to Hu sheep semen cryopreservation liquid and application thereof.
Background
The Hu sheep is one of important domestic animals in Taihu plain and is a first-level protective local livestock and poultry variety in China. In Hu sheep physique, there are no corners on male and female, long and narrow head, humped nose bridge, large and drooping ears, long and thin neck, long and narrow trunk, flat back and waist, slight drooping abdomen, flat and round tail, upwarping tail tip, thin and high limbs. White quilt hair, thick, thin and short abdomen hair, and strong physique. The Hu sheep is a rare white lamb skin sheep variety, has the excellent properties of precocity, four-season estrus, two fetuses in one year, more lambs in each fetus, good lactation performance, quick growth and development, ideal meat production performance after improvement, high temperature and high humidity resistance and the like, is distributed in Taihu areas in China, is fed with the Chinese lamb skin sheep variety in houses in the last year, and is attractive in pattern of the small lake lamb skin which is peeled 1-2 days after delivery and is famous for the life.
The semen freezing preservation and artificial insemination technology plays a very important role in the production of modern animal husbandry, the artificial insemination by using the frozen semen can be free from the limitation of time, regions, varieties and individuals, the feeding quantity of the male livestock is greatly reduced, the production cost is reduced, the utilization rate of the male livestock with excellent varieties is improved, and the economic benefit of the breeding industry is improved. Meanwhile, the technology has important significance in the aspects of variety introduction, hybridization improvement, reproductive performance improvement, disease control and protection of genetic diversity. Sheep sperms are weaker than chicken sperms and cattle sperms, and damage is easily caused in the freezing and unfreezing processes, so that the fertilization capability is lost. The existing frozen sheep semen has low survival rate after being thawed, has a plurality of aged sperms and weak dead sperms, has less effective sperms, low vitality and poor quality, and is difficult to meet the requirement of artificial insemination.
Disclosure of Invention
Based on the above, the invention aims to overcome the defects of the prior art and provide the Hu sheep semen cryopreservation liquid, which can effectively improve the survival rate, activity and integrity of the Hu sheep semen in the preservation process, the preserved semen still has good insemination capability after being thawed, the conception effect is stable, and the requirement of Hu sheep frozen semen artificial insemination can be met.
In order to achieve the purpose, the invention adopts the technical scheme that: a Hu sheep semen cryopreservation liquid comprises the following components: tris, glucose, raffinose, wheat germ oil, sodium citrate, glycerol, cryoprotectants, penicillin and streptomycin; the cryoprotectant comprises an astragalus extract, an acerola extract and a bamboo leaf extract.
Preferably, the mass ratio of the astragalus extract, the acerola extract and the bamboo leaf extract in the cryoprotectant is 2:1: 1.
The wheat germ oil contains nutrient substances such as vitamin E, choline, phytosterol and the like, and also contains various trace elements such as glutathione and the like. The dioctadecyl alcohol contained in the compound is an internationally recognized anti-fatigue substance, and has the functions of enhancing physical strength, improving muscle function, improving response sensitivity and improving exercise durability as a natural nutrient. The wheat germ oil has effects of resisting infertility, resisting aging, promoting metabolism, enhancing physical strength and memory, reducing cholesterol, preventing and treating arteriosclerosis, improving blood circulation and enhancing immunity.
Radix astragali extract, root extract of radix astragali of Leguminosae, contains saponin, flavone, polysaccharide and amino acid, and has effects of enhancing immunity, enhancing energy, relieving fatigue, resisting mutation, protecting liver and inhibiting osteoclast. The astragalus polysaccharides have the functions of reducing blood fat, namely reducing cholesterol and triglyceride and increasing high-density lipoprotein; can be used for preventing and treating cardiovascular and cerebrovascular diseases, such as atherosclerosis, coronary artery disease, peripheral angiopathy, and hyperlipidemia. Astragaloside IV has effects of remarkably reducing blood sugar, glycosylated hemoglobin and urine protein, and reducing AGEs in renal cortex and blood serum, and has effects of resisting oxidation, inhibiting aldose reductase, inhibiting mesangial cell proliferation, and relieving renal hypertrophy.
The acerola cherry extract is extract of acerola cherry fruit of acerola, contains various nutrients such as vitamins C, A, B1, B2, E, P, nicotinic acid, anti-aging factor (SOD), calcium, iron, zinc, potassium and protein, and has high nutritive value. Wherein vitamin P can prevent vitamin C from being damaged by oxidation, is a good antioxidant, has good antianemia, antifungal and anti-genetic toxicity effects, and also has strong scavenging ability of superoxide anion free radical, hydroxyl free radical and DPPH free radical.
A bamboo leaf extract is obtained by extracting leaf of Lophatherum gracile belonging to family Gramineae, and contains bamboo leaf flavone as main active ingredient, which has SOD and GSH-Px similar effects. The effective components of bamboo leaf flavonoid are mainly flavone glycoside and coumarin lactone. Has immunity regulating, fatigue relieving, peroxide eliminating, liver protecting, metabolism promoting, and life activity enhancing effects, and has antioxidant, antibacterial, and antiseptic effects.
The inventor of the application discovers that the cryoprotectant obtained by compounding the astragalus extract, the acerola extract and the bamboo leaf extract according to the mass ratio of 2:1:1 can effectively improve the cryopreservation effect of the cryoprotectant on the Hu sheep semen, and the cryoprotectant has the best cryopreservation effect when the concentration of the cryoprotectant in the cryopreservation liquid is 0.1 g/L.
Preferably, the Hu sheep semen cryopreservation solution comprises the following components in concentration: 30-35 g/L of Tris, 8-15 g/L of glucose, 30-50 g/L of raffinose, 2-5% (v/v) of wheat germ oil, 15-20 g/L of sodium citrate, 5-8% (v/v) of glycerol, 0.05-0.15 g/L of cryoprotectant, 80-100 ten thousand IU/L of penicillin and 0.8-1 g/L of streptomycin.
Preferably, the concentration of the cryoprotectant in the Hu sheep semen cryopreservation solution is 0.1 g/L.
The invention also provides a preparation method of the Hu sheep semen cryopreservation liquid, which comprises the following steps: (1) weighing Tris, glucose, raffinose, sodium citrate and a cryoprotectant according to a proportion, adding into double distilled water, fully dissolving and uniformly mixing, filtering with a 0.2um filter membrane, then adding penicillin and streptomycin, and uniformly mixing to obtain a solution A; (2) adding the wheat germ oil into the solution A according to a proportion, and uniformly mixing to obtain a solution B; (3) and (3) adding glycerol into the solution B according to a proportion, and uniformly mixing to obtain the Hu sheep semen cryopreservation solution.
The invention also provides application of the Hu sheep semen cryopreservation liquid in Hu sheep semen cryopreservation.
The invention also provides a method for freezing and storing the Hu sheep semen, which comprises the following steps: (1) taking fresh Hu sheep semen, and evaluating the semen survival rate, wherein the fresh Hu sheep semen can be used for cryopreservation only if the survival rate is more than 0.8; (2) adding the Hu sheep semen cryopreservation liquid into the semen obtained in the step (1), uniformly mixing, adding into a cryopreservation tube, placing the cryopreservation tube at 4 ℃ for 1-2 h, then placing at a position 4-6 cm away from the liquid level of liquid nitrogen for 8-10 min, and finally putting into the liquid nitrogen for preservation.
Preferably, theThe density of the semen in the freezing and storing tube in the step (2) is 6 multiplied by 108one/mL.
Compared with the prior art, the invention has the beneficial effects that: (1) the cryopreservation solution provided by the invention can effectively improve the survival rate, activity and integrity of the Hu sheep semen in the preservation process, the preserved semen still has good insemination capability after being thawed, the conception effect is stable, the requirement of Hu sheep frozen semen artificial insemination can be met, and the application prospect is wide; (2) the inventor of the application discovers that the cryoprotectant obtained by compounding the astragalus extract, the acerola extract and the bamboo leaf extract according to the mass ratio of 2:1:1 can effectively improve the cryopreservation effect of the cryoprotectant on the Hu sheep semen, and the cryoprotectant has the best cryopreservation effect when the concentration of the cryoprotectant in the cryopreservation liquid is 0.1 g/L.
Detailed Description
In order that the invention may be more fully understood, reference will now be made to the following description. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.
Example 1
The invention discloses an embodiment of a Hu sheep semen cryopreservation solution, which comprises the following components in concentration: 30g/L Tris, 10g/L glucose, 40g/L raffinose, 3% (v/v) wheat germ oil, 18g/L sodium citrate, 5% (v/v) glycerol, 0.1g/L cryoprotectant, 100 ten thousand IU/L penicillin and 1g/L streptomycin; the cryoprotectant comprises an astragalus extract, an acerola extract and a bamboo leaf extract, and the mass ratio of the astragalus extract to the acerola extract to the bamboo leaf extract is 2:1: 1.
The preparation method of the Hu sheep semen cryopreservation liquid comprises the following steps: (1) weighing Tris, glucose, raffinose, sodium citrate and a cryoprotectant according to a proportion, adding into double distilled water, fully dissolving and uniformly mixing, filtering with a 0.2um filter membrane, then adding penicillin and streptomycin, and uniformly mixing to obtain a solution A; (2) adding the wheat germ oil into the solution A according to a proportion, and uniformly mixing to obtain a solution B; (3) and (3) adding glycerol into the solution B according to a proportion, and uniformly mixing to obtain the Hu sheep semen cryopreservation solution.
Example 2
The invention discloses an embodiment of a Hu sheep semen cryopreservation solution, which comprises the following components in concentration: tris 33g/L, glucose 15g/L, raffinose 30g/L, wheat germ oil 5% (v/v), sodium citrate 20g/L, glycerol 6% (v/v), cryoprotectant 0.06g/L, penicillin 100 ten thousand IU/L and streptomycin 1 g/L; the cryoprotectant comprises an astragalus extract, an acerola extract and a bamboo leaf extract, and the mass ratio of the astragalus extract to the acerola extract to the bamboo leaf extract is 2:1: 1.
The preparation method of the Hu sheep semen cryopreservation solution is the same as that in example 1.
Example 3
The invention discloses an embodiment of a Hu sheep semen cryopreservation solution, which comprises the following components in concentration: tris 35g/L, glucose 12g/L, raffinose 35g/L, wheat germ oil 2% (v/v), sodium citrate 16g/L, glycerol 8% (v/v), cryoprotectant 0.13g/L, penicillin 80 ten thousand IU/L and streptomycin 0.8 g/L; the cryoprotectant comprises an astragalus extract, an acerola extract and a bamboo leaf extract, and the mass ratio of the astragalus extract to the acerola extract to the bamboo leaf extract is 2:1: 1.
The preparation method of the Hu sheep semen cryopreservation solution is the same as that in example 1.
Example 4
The invention discloses an embodiment of a Hu sheep semen cryopreservation solution, which comprises the following components in concentration: 30g/L of Tris, 8g/L of glucose, 50g/L of raffinose, 4% (v/v) of wheat germ oil, 20g/L of sodium citrate, 7% (v/v) of glycerol, 0.15g/L of cryoprotectant, 80 ten thousand IU/L of penicillin and 0.8g/L of streptomycin; the cryoprotectant comprises an astragalus extract, an acerola extract and a bamboo leaf extract, and the mass ratio of the astragalus extract to the acerola extract to the bamboo leaf extract is 2:1: 1.
The preparation method of the Hu sheep semen cryopreservation solution is the same as that in example 1.
Example 5
The invention discloses an embodiment of a Hu sheep semen cryopreservation solution, which comprises the following components in concentration: tris 35g/L, glucose 10g/L, raffinose 45g/L, wheat germ oil 2% (v/v), sodium citrate 20g/L, glycerin 5% (v/v), cryoprotectant 0.05g/L, penicillin 100 ten thousand IU/L and streptomycin 0.9 g/L; the cryoprotectant comprises an astragalus extract, an acerola extract and a bamboo leaf extract, and the mass ratio of the astragalus extract to the acerola extract to the bamboo leaf extract is 2:1: 1.
The preparation method of the Hu sheep semen cryopreservation solution is the same as that in example 1.
Example 6
The invention discloses an embodiment of a Hu sheep semen cryopreservation solution, which comprises the following components in concentration: tris 32g/L, glucose 15g/L, raffinose 40g/L, wheat germ oil 3.5% (v/v), sodium citrate 18g/L, glycerol 8% (v/v), cryoprotectant 0.08g/L, penicillin 100 ten thousand IU/L and streptomycin 1 g/L; the cryoprotectant comprises an astragalus extract, an acerola extract and a bamboo leaf extract, and the mass ratio of the astragalus extract to the acerola extract to the bamboo leaf extract is 2:1: 1.
The preparation method of the Hu sheep semen cryopreservation solution is the same as that in example 1.
EXAMPLE 7 Effect of preserving cryopreservation liquid
Collecting semen of male sheep of adult Hu sheep species by a pseudo-vaginal method, measuring the semen survival rate, and performing cryopreservation when the semen survival rate meets the requirement, wherein the preservation method comprises the following steps: adding the Hu sheep semen cryopreservation solution of example 1-6 into the collected semen to make the density of the semen 6 x 108And mixing the components in the solution per mL uniformly, adding the mixture into a freezing tube, placing the freezing tube at 4 ℃ for 2 hours, placing the tube at a position 4-6 cm away from the liquid level of liquid nitrogen for 10min, and finally putting the tube into the liquid nitrogen for preservation.
Unfreezing and detecting: taking out the frozen tube from liquid nitrogen, quickly putting the tube into a water bath kettle at 37 ℃, shaking until semen is dissolved into a transparent state, and taking out the semen for quality index detection.
The results are shown in table 1:
TABLE 1 quality index test results
| Group of | Rate of activity | Vitality of the body | Percentage of acrosomal integrity |
| Example 1 | 72.3％ | 58.1％ | 75.8％ |
| Example 2 | 65.5％ | 55.2％ | 72.3％ |
| Example 3 | 66.1％ | 54.3％ | 73.1％ |
| Example 4 | 64.8％ | 56.1％ | 72.6％ |
| Example 5 | 67.9％ | 55.8％ | 71.4％ |
| Example 6 | 66.2％ | 56.9％ | 71.9％ |
The results show that the cryopreservation liquid disclosed by the embodiments 1-6 of the invention has a good preservation effect on the Hu sheep semen, the survival rate, the vitality and the acrosome integrity rate of the thawed sperm are kept at high levels, and the frozen sperm quality is improved.
Example 8
The cryopreservation liquid comprises a cryoprotectant which is obtained by compounding an astragalus extract, an acerola extract and a bamboo leaf extract according to the mass ratio of 2:1:1, and the cryoprotectant comprises multiple antioxidant components, so that the cryopreservation effect of the Hu sheep semen can be effectively improved.
Experimental groups 1 and 2 were designed, and experimental group 1 was used to preserve sperm of a hu sheep using the cryopreservation solution described in example 1, and the cryopreservation solution used in experimental group 2 was identical to example 1 except that it did not contain a cryoprotectant.
Collecting semen of male sheep of adult Hu sheep species by a pseudo-vaginal method, measuring the semen survival rate, and performing cryopreservation when the semen survival rate meets the requirement, wherein the preservation method comprises the following steps: adding the Hu sheep semen cryopreservation liquid of the experimental groups 1 and 2 into the collected semen to ensure that the density of the semen is 6 multiplied by 108And mixing the components per mL uniformly, adding the mixture into a freezing tube, placing the freezing tube at 4 ℃ for 1.5h, placing the tube at a position 4-6 cm away from the liquid level of liquid nitrogen for 8min, and finally putting the tube into the liquid nitrogen for preservation.
Unfreezing and detecting: taking out the frozen tube from liquid nitrogen, quickly putting the tube into a water bath kettle at 37 ℃, shaking until semen is dissolved into a transparent state, and taking out the semen for quality index detection.
The results are shown in table 2:
TABLE 2 quality index test results
| Group of | Rate of activity | Vitality of the body | Percentage of acrosomal integrity |
| Experimental group 1 | 71.8％ | 58.6％ | 74.9％ |
| Experimental group 2 | 50.2％ | 35.1％ | 48.7％ |
The results show that the preservation effect of the cryopreservation solution of the experimental group 1 containing the cryoprotectant on the sperm of the Hu sheep is obviously superior to that of the experimental group 2 containing no cryoprotectant, the sperm motility rate, the vitality and the acrosome integrity rate of the Hu sheep sperm preserved by the cryopreservation solution of the experimental group 1 after thawing are obviously higher than those of the experimental group 2, and the cryoprotectant can effectively improve the cryopreservation effect of the cryopreservation solution on the sperm of the Hu sheep.
Example 9
The concentration of the cryoprotectant in the cryopreservation liquid is 0.05 to 0.15g/L, and the influence of the concentration of the cryoprotectant on the preservation effect of the cryopreservation liquid is studied in the example.
Experiment groups 3-9 are designed, and the components of the cryopreservation solution in each experiment group except for different concentrations of the cryoprotectant are the same, and are specifically shown in table 3:
TABLE 3 design of cryoprotectant concentration in cryopreservation fluids
Collecting semen of male sheep of adult Hu sheep species by a pseudo-vaginal method, measuring the semen survival rate, and performing cryopreservation when the semen survival rate meets the requirement, wherein the preservation method comprises the following steps: adding the Hu sheep semen cryopreservation liquid of the experimental group 3-9 into the collected semen to ensure that the density of the semen is 6 multiplied by 108And mixing the components in the solution per mL uniformly, adding the mixture into a freezing tube, placing the freezing tube at 4 ℃ for 1h, placing the tube at a position 4-6 cm away from the liquid level of liquid nitrogen for 10min, and finally putting the tube into the liquid nitrogen for preservation.
Unfreezing and detecting: taking out the frozen tube from liquid nitrogen, quickly putting the tube into a water bath kettle at 37 ℃, shaking until semen is dissolved into a transparent state, and taking out the semen for quality index detection.
The results are shown in Table 4:
TABLE 4 quality index test results
| Group of | Rate of activity | Vitality of the body | Percentage of acrosomal integrity |
| Experimental group 3 | 61.7％ | 49.1％ | 65.9％ |
| Experimental group 4 | 66.7％ | 54.4％ | 71.5％ |
| Experimental group 5 | 65.2％ | 55.3％ | 72.0％ |
| Experimental group 6 | 71.9％ | 58.3％ | 74.3％ |
| Experimental group 7 | 67.4％ | 53.8％ | 71.7％ |
| Experimental group 8 | 66.1％ | 54.7％ | 71.2％ |
| Experimental group 9 | 60.3％ | 50.3％ | 66.5％ |
The results show that when the concentration of the cryoprotectant in the cryopreservation liquid is 0.05-0.15 g/L (experiment group 4-8), the cryopreservation effect of the cryopreservation liquid on the Hu sheep semen is better, and especially when the concentration of the cryoprotectant is 0.1g/L (experiment group 6), the semen quality index effect after thawing is optimal; when the concentration of the cryoprotectant is less than 0.05g/L (experiment group 3) or more than 0.15g/L (experiment group 9), the preservation effect of the cryopreservation liquid is affected, and the preservation quality is reduced.
Example 10
In this example, the sperm of a hu sheep preserved by the cryopreservation solution described in example 1 was taken as an example, and the insemination effect of the thawed sperm of the hu sheep preserved by the cryopreservation solution of the present invention was studied.
Collecting semen of male sheep of adult Hu sheep species by a pseudo-vaginal method, measuring the semen survival rate, and performing cryopreservation when the semen survival rate meets the requirement, wherein the preservation method comprises the following steps: the Hu sheep semen cryopreservation solution described in example 1 was added to the collected semen so that the semen density was 6X 108And mixing the components in the solution per mL uniformly, adding the mixture into a freezing tube, placing the freezing tube at 4 ℃ for 2 hours, placing the tube at a position 4-6 cm away from the liquid level of liquid nitrogen for 10min, and finally putting the tube into the liquid nitrogen for preservation.
Unfreezing: taking out the frozen tube from liquid nitrogen, quickly putting the tube into a water bath kettle at 37 ℃, shaking until semen is dissolved into a transparent state, and taking out the semen for artificial insemination.
Through detection, the time of the insemination capability of the unfrozen Hu sheep semen is up to 26h, so that the conception rate of the Hu sheep ewe in estrus is up to 85%.
Finally, it should be noted that the above embodiments are only used for illustrating the technical solutions of the present invention and not for limiting the protection scope of the present invention, and although the present invention is described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions can be made on the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention.
Claims (8)
1. The Hu sheep semen cryopreservation liquid is characterized by comprising the following components: tris, glucose, raffinose, wheat germ oil, sodium citrate, glycerol, cryoprotectants, penicillin and streptomycin; the cryoprotectant comprises an astragalus extract, an acerola extract and a bamboo leaf extract.
2. The Hu sheep semen cryopreservation liquid as claimed in claim 1, wherein the mass ratio of the Astragalus membranaceus extract, the acerola cherry extract and the bamboo leaf extract in the cryoprotectant is 2:1: 1.
3. The Hu sheep semen cryopreservation liquid as claimed in claim 1 or 2, which comprises the following components in concentration: 30-35 g/L of Tris, 8-15 g/L of glucose, 30-50 g/L of raffinose, 2-5% (v/v) of wheat germ oil, 15-20 g/L of sodium citrate, 5-8% (v/v) of glycerol, 0.05-0.15 g/L of cryoprotectant, 80-100 ten thousand IU/L of penicillin and 0.8-1 g/L of streptomycin.
4. The Hu sheep semen cryopreservation liquid as claimed in claim 3, wherein the concentration of the cryoprotectant is 0.1 g/L.
5. The preparation method of the Hu sheep semen cryopreservation solution as claimed in any one of claims 1 to 4, characterized by comprising the following steps: (1) weighing Tris, glucose, raffinose, sodium citrate and a cryoprotectant according to a proportion, adding into double distilled water, fully dissolving and uniformly mixing, filtering with a 0.2um filter membrane, then adding penicillin and streptomycin, and uniformly mixing to obtain a solution A; (2) adding the wheat germ oil into the solution A according to a proportion, and uniformly mixing to obtain a solution B; (3) and (3) adding glycerol into the solution B according to a proportion, and uniformly mixing to obtain the Hu sheep semen cryopreservation solution.
6. The use of the Hu sheep semen cryopreservation solution as defined in any one of claims 1 to 4 in Hu sheep semen cryopreservation.
7. A method for freezing and storing the sperm of Hu sheep is characterized by comprising the following steps: (1) taking fresh Hu sheep semen, and evaluating the semen survival rate, wherein the fresh Hu sheep semen can be used for cryopreservation only if the survival rate is more than 0.8; (2) adding the Hu sheep semen cryopreservation liquid as defined in any one of claims 1 to 4 into the semen obtained in the step (1), uniformly mixing, adding into a cryopreservation tube, standing the cryopreservation tube at 4 ℃ for 1-2 h, standing at a position 4-6 cm away from the liquid level of liquid nitrogen for 8-10 min, and finally putting into liquid nitrogen for storage.
8. The method for cryopreservation of Hu sheep semen as claimed in claim 7, wherein the density of semen in the cryopreservation tube of step (2) is 6 x 108one/mL.
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| JP2019223802A JP6728463B1 (en) | 2019-10-28 | 2019-12-11 | Goat semen cryopreservation liquid and its use |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111374124A (en) * | 2020-05-15 | 2020-07-07 | 辽宁省现代农业生产基地建设工程中心 | Formula and preparation method of donkey frozen semen diluent |
| CN111436421A (en) * | 2020-05-21 | 2020-07-24 | 新乡医学院三全学院 | Human sperm cryopreservation method |
| CN117256604A (en) * | 2023-11-23 | 2023-12-22 | 中国海洋大学三亚海洋研究院 | Dolphin semen cryopreservation agent and semen cryopreservation method |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102960336A (en) * | 2012-11-09 | 2013-03-13 | 镇江万山红遍农业园 | Frozen sperm diluent of Boer crossbred goats |
| CN103392692A (en) * | 2013-08-14 | 2013-11-20 | 镇江万山红遍农业园 | Cryopreservation solution for goat semen |
| WO2016009363A1 (en) * | 2014-07-14 | 2016-01-21 | Politécnico Colombiano Jaime Isaza Cadavid | Diluent for semen preservation |
-
2019
- 2019-10-28 CN CN201911031372.XA patent/CN110604129B/en active Active
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102960336A (en) * | 2012-11-09 | 2013-03-13 | 镇江万山红遍农业园 | Frozen sperm diluent of Boer crossbred goats |
| CN103392692A (en) * | 2013-08-14 | 2013-11-20 | 镇江万山红遍农业园 | Cryopreservation solution for goat semen |
| WO2016009363A1 (en) * | 2014-07-14 | 2016-01-21 | Politécnico Colombiano Jaime Isaza Cadavid | Diluent for semen preservation |
Non-Patent Citations (4)
| Title |
|---|
| 杨威: "三种抗氧化剂对猪精子冷冻保存效果的影响", 《中国优秀硕士学位论文全文数据库 农业科技辑》 * |
| 毛凤县: "波尔山羊精液冷冻稀释液及冷冻解冻方法的研究", 《中国优秀硕士学位论文全文数据库 农业科技辑》 * |
| 胡传活等: "黄芪多糖对猪精液低温保存的影响", 《中国兽医学报》 * |
| 陈世伟等: "绵羊冷冻精液研究进展", 《中国草食动物科学》 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111374124A (en) * | 2020-05-15 | 2020-07-07 | 辽宁省现代农业生产基地建设工程中心 | Formula and preparation method of donkey frozen semen diluent |
| CN111436421A (en) * | 2020-05-21 | 2020-07-24 | 新乡医学院三全学院 | Human sperm cryopreservation method |
| CN117256604A (en) * | 2023-11-23 | 2023-12-22 | 中国海洋大学三亚海洋研究院 | Dolphin semen cryopreservation agent and semen cryopreservation method |
| CN117256604B (en) * | 2023-11-23 | 2024-03-01 | 中国海洋大学三亚海洋研究院 | Dolphin semen cryopreservation agent and semen cryopreservation method |
Also Published As
| Publication number | Publication date |
|---|---|
| JP2021066719A (en) | 2021-04-30 |
| CN110604129B (en) | 2020-12-01 |
| JP6728463B1 (en) | 2020-07-22 |
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