CN114009427A - Rabbit semen cryopreservation diluent, preparation method, rabbit semen cryopreservation method and application - Google Patents
Rabbit semen cryopreservation diluent, preparation method, rabbit semen cryopreservation method and application Download PDFInfo
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- CN114009427A CN114009427A CN202111510937.XA CN202111510937A CN114009427A CN 114009427 A CN114009427 A CN 114009427A CN 202111510937 A CN202111510937 A CN 202111510937A CN 114009427 A CN114009427 A CN 114009427A
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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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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
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- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
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Abstract
The invention provides a diluent for preserving rabbit semen in a freezing way, a preparation method thereof, a rabbit semen preserving in a freezing way and application thereof, belonging to the technical field of artificial insemination of livestock. The invention provides a rabbit semen cryopreservation diluent, which takes water as a solvent, wherein every 100mL of water is added with: 3-4 g of trihydroxymethyl aminomethane, 1-2 g of citric acid, 0.5-1 g of glucose, 2-3 g of trehalose, 0.5-1 g of L-glutamic acid, 10-20 mL of dimethyl sulfoxide and 2-4 mL of glycerol; the pH value of the rabbit semen cryopreservation diluent is 6.5-7.5. The rabbit semen cryopreservation diluent provided by the invention obviously improves the survival rate, plasma membrane integrity rate and acrosome integrity rate of frozen sperms, and is beneficial to improving the breeding efficiency and breeding speed of rabbits.
Description
Technical Field
The invention belongs to the technical field of artificial insemination of livestock, and particularly relates to a rabbit semen cryopreservation diluent, a preparation method, a rabbit semen cryopreservation method and application.
Background
The rabbit belongs to a polyembryony animal, and the fresh essence can basically meet the daily artificial insemination requirement. But aiming at the long-distance transportation of breeding male rabbits, the frozen semen can avoid the problems of quarantine, isolation, breeding of breeding stock and the like when living animals are introduced. And sperm freezing aiming at endangered strains and transgenic strains is more convenient, simpler and easier than embryo seed preservation. Meanwhile, the semen freezing and storing technology can be used for storing the semen of various male rabbits with good quality for a long time, so that the problem caused by the neck clamping of germplasm resources is effectively solved. The phenomenon of male rabbit 'sterility in summer' takes place occasionally, and the cryopreservation of seminal fluid can solve and avoid causing the not good problem of the temporary seminal fluid quality of male rabbit because of other reasons for male and female rabbit raises the proportion and reduces male rabbit and raises the volume, reduces the plant and raises the cost simultaneously, improves economic benefits.
At present, the research on freezing, diluting and preserving fluid of the semen of pigs, horses, cattle, sheep and the like is more, products are completely commercialized, but the research on freezing and preserving the semen of rabbits is less. At present, the commercial products of the diluent for preserving rabbit semen in a freezing way are almost not available in the market, the quality is uneven, the vitality of the recovered sperms is not high, and the artificial insemination production and propagation work is difficult to be effectively carried out.
Disclosure of Invention
In order to solve the problems, the invention provides a rabbit semen cryopreservation diluent, a preparation method thereof, a rabbit semen cryopreservation method and application thereof. The rabbit semen cryopreservation diluent provided by the invention greatly improves the survival rate, plasma membrane integrity rate and acrosome integrity rate of frozen sperms, has stable quality, and is beneficial to improving the breeding efficiency and breeding speed of rabbits.
In order to achieve the above purpose, the invention provides the following technical scheme:
the rabbit semen cryopreservation diluent takes water as a solvent, and every 100mL of water is added with: 3-4 g of trihydroxymethyl aminomethane, 1-2 g of citric acid, 0.5-1 g of glucose, 2-3 g of trehalose, 0.5-1 g of L-glutamic acid, 10-20 mL of dimethyl sulfoxide and 2-4 mL of glycerol; the pH value of the rabbit semen cryopreservation diluent is 6.5-7.5.
Preferably, in the rabbit semen cryopreservation diluent, per 100mL of water are added: 3.03g of trihydroxymethyl aminomethane, 1.69g of citric acid, 0.85g of glucose, 2.05g of trehalose, 0.74g of L-glutamic acid, 16mL of dimethyl sulfoxide and 3mL of glycerol; the pH value of the rabbit semen cryopreservation diluent is 7.3.
The invention provides a preparation method of a rabbit semen cryopreservation diluent in the technical scheme, which comprises the following steps: mixing trihydroxymethyl aminomethane, citric acid, glucose, trehalose, L-glutamic acid, dimethyl sulfoxide, glycerol and water.
The invention provides a rabbit semen cryopreservation method of the rabbit semen cryopreservation diluent in the technical scheme or the rabbit semen cryopreservation diluent prepared by the preparation method in the technical scheme, which comprises the following steps: refrigerating the rabbit semen to obtain the refrigerated rabbit semen; carrying out isothermal mixing on the refrigerated rabbit semen and the rabbit semen cryopreservation diluent to obtain a mixed solution; and (4) balancing the mixed solution, fumigating the mixed solution by liquid nitrogen, and freezing and storing the mixed solution.
Preferably, before refrigeration, the rabbit semen is rabbit semen from which gel components are removed; the survival rate of the rabbit semen is more than 0.8.
Preferably, the cold storage temperature of the rabbit semen is 2-8 ℃, and the cold storage time is 60-120 min.
Preferably, the volume ratio of the rabbit semen cryopreservation diluent to the refrigerated rabbit semen is less than or equal to 5: 1.
Preferably, the temperature of the equilibrium is 2-8 ℃, and the time of the equilibrium is 30-60 min.
Preferably, the fumigating is carried out by fumigating the balanced mixed liquid 3-10 cm above the liquid level of the liquid nitrogen; the fumigating time is 5-20 min.
The invention provides the rabbit semen cryopreservation diluent in the technical scheme or the application of the rabbit semen cryopreservation method in the technical scheme in rabbit semen cryopreservation.
Has the advantages that:
the invention provides a rabbit semen cryopreservation diluent. In the diluent, the trihydroxymethyl aminomethane and the citric acid can prevent the sperm cell structure from being damaged, and have good buffering effect on sperm enzyme reaction and neutralization of metabolic acidic substances thereof; the glucose can provide nutrients required by the sperm for survival in vitro and supplement energy for the sperm; glycerol has the ability to replace water under dehydration conditions, and can increase the stability of the membrane; in addition, glycerol has the effect of increasing viscosity, can prevent solute symbiotic crystallization, promote cell vitrification formation, change the shape of unfrozen solution channel, increase the plasticity of unfrozen solution, reduce mechanical damage to sperm cells, and improve the survival rate of frozen sperm. DMSO can enter cells to replace water in the cells, the replacement enables the cells to be dehydrated or combined with cell water to prevent ice crystals from forming in the cells, the water is in a supercooled state, the upper limit of a dangerous area is reduced, the range of a dangerous temperature area is narrowed, and the glycerol and the DMSO are added in a compounding manner, so that the DMSO not only has a protection effect on the interior of spermatids, but also has a protection effect on plasma membranes, and the survival rate, the plasma membrane integrity rate and the acrosome integrity rate of frozen sperms are obviously improved. L-glutamic acid can remove ROS generated by the respiratory metabolism of sperms, so that peroxide and antioxidant in cells are kept in balance, the aim of protecting spermatids is fulfilled, and the survival rate of the frozen sperms is improved. Trehalose molecules can produce a glassy protective layer outside the cell membrane, thereby improving the tolerance of the cells to high osmotic pressure, and keeping the cells resuscitative and continuing to maintain the activity after thawing. The rabbit semen cryopreservation diluent provided by the invention obviously improves the survival rate, plasma membrane integrity rate and acrosome integrity rate of frozen sperms, and is beneficial to improving the breeding efficiency and breeding speed of rabbits.
Detailed Description
The invention provides a rabbit semen cryopreservation diluent, which takes water as a solvent, wherein every 100mL of water is added with: 3-4 g of trihydroxymethyl aminomethane, 1-2 g of citric acid, 0.5-1 g of glucose, 2-3 g of trehalose, 0.5-1 g of L-glutamic acid, 10-20 mL of dimethyl sulfoxide and 2-4 mL of glycerol; the pH value of the rabbit semen cryopreservation diluent is 6.5-7.5.
The rabbit semen cryopreservation diluent takes water as a solvent. In the present invention, the water is preferably purified water. The invention has no special requirement on the source of the purified water, and the purified water can be obtained by common commercial products or conventional preparation methods.
In the rabbit semen cryopreservation diluent, 3-4 g of tris (hydroxymethyl) aminomethane is added to every 100mL of water, more preferably 3-3.4 g, and still more preferably 3.03 g. In the present invention, 1 to 2g, more preferably 1.69g, of citric acid is added to 100mL of water. The invention has no special requirements on the sources of the trihydroxymethyl aminomethane and the citric acid, and can be prepared by adopting common commercial products. The Tris and citric acid can prevent the sperm cell structure from being damaged, and have good buffering effect on sperm enzyme reaction and neutralization of metabolic acidic substances thereof.
In the rabbit semen cryopreservation diluent, 0.5-1 g of glucose is added to every 100mL of water, more preferably 0.7-0.9 g, and still more preferably 0.85 g. The invention has no special requirement on the source of the glucose and can be prepared by adopting common commercial products. The glucose can provide nutrients required by the sperm for survival in vitro and supplement energy for the sperm.
In the rabbit semen cryopreservation diluent, 2-3 g of trehalose is added to every 100mL of water, preferably 2-2.5, and more preferably 2.05 g. The invention has no special requirement on the source of the trehalose and can be prepared by adopting common commercial products. The trehalose is a non-permeable anti-freezing protective agent and can improve the motility, acrosome integrity, plasma membrane integrity and mitochondrial activity of rabbit sperms.
In the rabbit semen cryopreservation diluent, 0.5-1 g, more preferably 0.7-0.8 g, and even more preferably 0.74g of L-glutamic acid is added to every 100mL of water. The invention has no special requirements on the source of the L-glutamic acid, and can be prepared by adopting common commercial products. The L-glutamic acid can remove oxygen free radicals, inhibit lipid peroxidation, prevent the level of the L-glutamic acid in sperm cells from being reduced after freezing and thawing, and improve the survival rate of the sperm after freezing and thawing.
In the rabbit semen cryopreservation diluent, 10-20 mL of dimethyl sulfoxide (DMSO) is added to every 100mL of water, more preferably 14-18 mL, and still more preferably 16 mL. The invention has no special requirement on the source of the dimethyl sulfoxide, and can be prepared by adopting common commercial products. The dimethyl sulfoxide is a permeable antifreeze protective agent, and can be used for replacing water in cells by entering the cells, dehydrating the cells or combining the cells with the water to prevent ice crystals from forming in the cells, so that the water is in a supercooled state, the upper limit of a dangerous area is reduced, the range of the dangerous area is narrowed, and the damage of ice crystallization to sperms in the freezing process can be reduced.
In the rabbit semen cryopreservation diluent, 2-4 mL of glycerol is added to every 100mL of water, preferably 2.5-3.5, and more preferably 3 mL. The invention has no special requirement on the source of the glycerol and can be prepared by adopting common commercial products. The glycerol is a permeable anti-freezing protective agent, has the capability of replacing water under the dehydration condition, and increases the stability of the membrane; in addition, the viscosity of the glycerol is increased, so that the solute symbiotic crystallization can be prevented, the cell vitrification formation can be promoted, the shape of a channel of the unfrozen solution can be changed, the plasticity of the unfrozen solution can be increased, the glycerol is compounded with dimethyl sulfoxide, the glycerol not only has a protection effect on the interior of sperm cells, but also has a protection effect on a plasma membrane, and the survival rate, the plasma membrane integrity rate and the acrosome integrity rate of frozen sperms are obviously improved.
The pH value of the rabbit semen cryopreservation diluent is 6.5-7.5, more preferably 7.1-7.3, and still more preferably 7.3. The proper pH value of the invention ensures that the sperm cells maintain normal shape and function and prevents the sperm cell structure from being damaged.
The diluent for preserving and freezing rabbit semen obviously improves the survival rate, the plasma membrane integrity rate and the acrosome integrity rate of the frozen semen, has high freezing speed and stable quality, is beneficial to the popularization of a semen preserving and freezing technology in the rabbit breeding industry and is beneficial to the improvement of the breeding efficiency and the breeding speed of rabbits.
The invention provides a preparation method of a rabbit semen cryopreservation diluent in the technical scheme, which comprises the following steps: mixing trihydroxymethyl aminomethane, citric acid, glucose, trehalose, L-glutamic acid, dimethyl sulfoxide, glycerol and water. The invention has no special requirements on the mixing method, and the mixing method is conventional in the field. After the rabbit semen freezing and preserving diluent is mixed, the rabbit semen freezing and preserving diluent is obtained. The rabbit semen cryopreservation diluent has no special storage condition, and can be stored at low temperature. In the present invention, the temperature for the low-temperature storage is preferably 2 to 8 ℃, and more preferably 4 ℃. The preparation method of the rabbit semen cryopreservation diluent provided by the invention is simple, the quality is easy to control, and the rabbit semen cryopreservation diluent is suitable for large-scale production.
The invention provides a rabbit semen cryopreservation method of the rabbit semen cryopreservation diluent in the technical scheme or the rabbit semen cryopreservation diluent prepared by the preparation method in the technical scheme, which comprises the following steps: refrigerating the rabbit semen to obtain the refrigerated rabbit semen; carrying out isothermal mixing on the refrigerated rabbit semen and the rabbit semen cryopreservation diluent to obtain a mixed solution; and (4) balancing the mixed solution, fumigating the mixed solution by liquid nitrogen, and freezing and storing the mixed solution.
The invention refrigerates rabbit semen to obtain the refrigerated rabbit semen. In the present invention, the rate of viability of the rabbit semen is preferably above 0.8. The rabbit semen survival rate meets the requirements, and the resuscitation effect is better after the freezing operation. In the present invention, the rabbit semen is preferably rabbit semen from which gel components are removed. The method for removing the gel component has no special requirements, and the gel component can be removed under the condition of ensuring the good activity of the rabbit semen. The invention removes gel components, which is beneficial to freezing and diluting rabbit semen and increasing the activity of the recovered rabbit sperm. After the gel components are removed, the rabbit semen after the gel components are removed is refrigerated to obtain the refrigerated rabbit semen. In the invention, the cold storage temperature of the rabbit semen is preferably 2-8 ℃, and further preferably 4 ℃. In the present invention, the time for cold storage is preferably 60 to 120min, and more preferably 90 min.
The method comprises the step of carrying out isothermal mixing on the refrigerated rabbit semen and the rabbit semen cryopreservation diluent to obtain a mixed solution. The rabbit semen cryopreservation diluent is preferably refrigerated, so that the refrigerated rabbit semen cryopreservation diluent and the refrigerated rabbit semen are consistent in temperature and are subjected to isothermal mixing, and the influence of cold shock caused by temperature difference on sperm motility is reduced. In the invention, the volume ratio of the rabbit semen freezing and storing diluent to the refrigerated rabbit semen is preferably less than or equal to 5: 1; more preferably ≦ 2: 1; more preferably (1-2): 1; still more preferably 1:1, the specific proportion of the invention has good protection effect on rabbit spermatids, and improves the survival rate of the rabbit sperms after freezing recovery.
After the mixed solution is obtained, the mixed solution is balanced, fumigated by liquid nitrogen and then frozen for storage. In the invention, the temperature of the balance is preferably 2-8 ℃; further preferably 4 ℃. In the invention, the balancing time is preferably 30-60 min; further preferably 45 min. The balance of the invention can ensure that the rabbit semen cryopreservation diluent fully permeates into sperms to realize balance so as to achieve the anti-freezing protection effect and cold shock resistance; and the sperm can enhance the freezing resistance after being balanced at proper low temperature, and the damage of the ice crystal to the sperm in the freezing process can be reduced.
After balancing, the invention carries out liquid nitrogen fumigation on the balanced mixed solution. In the present invention, the fumigation is preferably carried out by fumigating the mixture obtained after the equilibration above the liquid nitrogen level. In the present invention, the distance above the liquid surface is preferably 3 to 10cm, and more preferably 5 cm. In the invention, the fumigating time is preferably 5-20 min, and more preferably 10 min. Fumigation can allow the semen to be frozen in a better glassy state, reducing the death of sperm due to crystallization. In the present invention, the fumigation is preferably performed after the mixed solution is separately packed. In the present invention, the size of the portion is preferably 0.25 mL. The split charging is convenient for taking in the future, and the freezing speed can be accelerated. After fumigation, the fumigated mixed solution is frozen and stored in liquid nitrogen.
After freezing and preservation, the frozen semen is thawed by adopting water bath high-temperature thawing preferably when in use. In the invention, the water bath high-temperature thawing temperature is preferably 50-60 ℃, more preferably 50-55 ℃, and even more preferably 50 ℃. In the invention, the time for thawing in the water bath at high temperature is preferably 10-20 s, and more preferably 10 s. During the high-temperature thawing in the water bath, the container with frozen semen is preferably pressed below the water surface and shaken continuously to ensure that the container is thawed evenly under heat, and ice crystals are prevented from forming again in the thawing process to cause irreversible damage to the semen.
According to the invention, by optimizing the formula of the freezing preservation diluent and optimizing the freezing and thawing conditions, the damage of the freezing and thawing operation to sperms is minimized, the sperm motility rate, the plasma membrane integrity rate and the acrosome integrity rate after freezing and thawing are obviously improved, the sperm motility rate and the sperm motility after freezing and thawing are strong, the conception rate, the parturition rate and the number of born rabbits are high when the female rabbits are bred, and compared with fresh sperms, the invention has no obvious difference, is convenient for the effective popularization of artificial insemination, and is beneficial to improving the breeding efficiency and the breeding speed of the rabbits. In addition, the formula and the operation flow of the rabbit semen cryopreservation diluent are optimized, so that the total time consumption of the freezing process is shortened, the freezing process can be completed only within 2-3 hours, the effect is kept to be optimal after the semen is frozen and recovered, and the work efficiency of semen freezing is obviously improved. Furthermore, the rabbit semen cryopreservation method provided by the invention has the advantages of long storage period, high safety and the like.
The invention provides the rabbit semen cryopreservation diluent in the technical scheme or the application of the rabbit semen cryopreservation method in the technical scheme in rabbit semen cryopreservation.
In order to further illustrate the present invention, the rabbit semen cryopreservation diluent and preparation method, the rabbit semen cryopreservation method and application provided by the present invention are described in detail below with reference to examples, but they should not be construed as limiting the scope of the present invention.
Example 1
A rabbit semen cryopreservation diluent is prepared by adding the following components in each 100mL of purified water: tris (hydroxymethyl) aminomethane 3.03g, citric acid 1.69g, glucose 0.85g, dimethyl sulfoxide 16mL, trehalose 2.05g, L-glutamic acid 0.74g, and glycerol 3mL, and pH 7.3.
The preparation method comprises the following steps: adding purified water into the above materials, stirring, mixing, and storing at 4 deg.C.
Examples 1 to 1
A rabbit semen cryopreservation diluent is prepared by adding the following components in each 100mL of purified water: tris (hydroxymethyl) aminomethane 3.4g, citric acid 1.5g, glucose 0.7g, dimethyl sulfoxide 14mL, trehalose 2.2g, L-glutamic acid 0.7g, and glycerol 2.5mL, pH 7.2.
The preparation method is the same as that of example 1.
Examples 1 to 2
A rabbit semen cryopreservation diluent is prepared by adding the following components in each 100mL of purified water: tris (hydroxymethyl) aminomethane 3.2g, citric acid 1.7g, glucose 0.9g, dimethyl sulfoxide 18mL, trehalose 2.3g, L-glutamic acid 0.8g, and glycerol 3.5mL, pH 7.1.
The preparation method is the same as that of example 1.
Comparative example 1
A semen cryopreservation diluent is prepared by the following method: 1.59g of glucose, 1.96g of citric acid, 8 ten thousand IU of penicillin sodium, 0.1g of streptomycin sulfate, 20mL of fresh egg yolk and 5% (v/v) of glycerol are dissolved in 100mL of double distilled water and stirred uniformly.
Comparative examples 1 to 1
A semen cryopreservation diluent is prepared by the following method: 8% of soybean lecithin, 1.1g of glucose, 1.48g of citric acid, 2.42g of tris (hydroxymethyl) aminomethane and 6% of glycerol (v/v is dissolved in 100mL of double distilled water and stirred uniformly.
Example 2
Freezing preservation method for rabbit semen
1) Semen collection and viability assessment
The male rabbit semen was collected by the pseudo-vaginal method. Under the microscope, the survival rate of the sperms detected by the microscopic examination method reaches more than 0.8, and the sperms are used for the cryopreservation test. 0.2-2 mL of collected semen containing gel components is removed before a semen freezing test, so that the semen freezing effect is improved.
2) Semen freezing procedure
Collecting rabbit semen, and cold preserving in refrigerator at 4 deg.C for 90 min; after refrigeration, slowly pouring the isothermal semen cryopreservation diluent of the example 1 into rabbit semen, and slightly shaking while adding the diluent to fully mix the cryopreservation diluent and the rabbit semen in a volume ratio of 1: 1; subpackaging the mixed rabbit semen by a thin tube, and balancing at 4 ℃ for 45 min; wrapping the obtained tubule with gauze, and fumigating at the position 5cm above liquid nitrogen liquid level for 10 min; and finally placing the mixture in a liquid nitrogen tank for freezing and storing.
Example 2-1
The rabbit semen cryopreservation method is the same as the rabbit semen cryopreservation method in example 2, except that: the semen of example 1-1 was used to cryopreserve the diluent.
Examples 2 to 2
The rabbit semen cryopreservation method is the same as the rabbit semen cryopreservation method in example 2, except that: the semen of example 1-2 was used to cryopreserve dilutions.
Examples 2 to 3
The rabbit semen cryopreservation method is the same as the rabbit semen cryopreservation method in example 2, except that: the volume ratio of the frozen and stored diluent to the rabbit semen is 3: 1; equilibrating at 2 deg.C for 30min, and fumigating at 3cm above liquid nitrogen for 5 min.
Examples 2 to 4
The rabbit semen cryopreservation method is the same as the rabbit semen cryopreservation method in example 2, except that: the volume ratio of the frozen and stored diluent to the rabbit semen is 5: 1; equilibrating at 8 deg.C for 60min, and fumigating at 10cm above liquid nitrogen for 20 min.
Comparative example 2
A method for preserving frozen rabbit semen, which is the same as the method in example 2, except that: the volume ratio of the frozen and stored diluent to the rabbit semen is 6: 1; equilibrating at 1 deg.C for 1.5h, and fumigating 2cm above liquid nitrogen for 2 min.
Comparative example 2-1
A method for preserving frozen rabbit semen, which is the same as the method in example 2, except that: the volume ratio of the frozen and stored diluent to the rabbit semen is 10: 1; equilibrating at 0 deg.C for 2h, and fumigating for 30min 15cm above liquid nitrogen.
Comparative examples 2 to 2
A method for preserving frozen rabbit semen, which is the same as the method in example 2, except that: the semen cryopreservation dilutions of comparative example 1 were used.
Comparative examples 2 to 3
A method for preserving frozen rabbit semen, which is the same as the method in example 2, except that: the semen cryopreservation diluent of comparative example 1-1 was used.
Example 3
The rabbit semen is subjected to cryopreservation by using the rabbit semen cryopreservation methods of example 2, example 2-1, example 2-2, example 2-3, example 2-4, comparative example 2-1, comparative example 2-2 and comparative example 2-3. Investigating the influence of different semen cryopreservation diluents and different rabbit semen cryopreservation conditions on the sperm motility rate and the plasma membrane integrity rate of the frozen and recovered rabbit semen; and breeding the mother rabbit with the thawed semen, and inspecting the influence of the thawed semen on the production efficiency of breeding the mother rabbit by taking the fresh rabbit semen as a control.
The frozen semen thawing procedure comprises:
and thawing the frozen semen in a water bath at a high temperature of 50-60 ℃. Clamping required number of tubule frozen sperms from a liquid nitrogen tank, quickly putting into a constant temperature water bath kettle at 50-60 ℃ for 10-20 s, and then quickly transferring into a constant temperature water bath kettle at 37 ℃. And pressing the frozen semen tubules below the water surface during the thawing of the frozen semen in a constant-temperature water bath kettle at 50-60 ℃, and continuously shaking to ensure that the frozen semen is uniformly heated and thawed.
Sperm motility: after thawing in a frozen tubule water bath of rabbit semen, 10 μ L of semen is put on a glass slide, covered with a glass slide, and the percentage of the linearly forward-moving sperm in all the sperm is evaluated under a 400 Xinverted microscope.
Sperm plasma membrane integrity rate: the SYBR14/PI method is mainly adopted, the SYBR14/PI method is that sperms are added into a kit, the fluorescent reaction that live sperms emit green light and dead sperms emit red light is utilized to detect whether plasma membranes are complete through microscopic examination, and the plasma membrane complete rate of the sperms is calculated.
Sperm acrosome integrity rate: the FITC-PNA staining method is a sperm acrosome staining kit, can be used for observing the staining state of a sperm acrosome under a fluorescence microscope for judgment, and is mainly divided into grades I, II, III and IV which respectively represent acrosome integrity, acrosome slight injury, acrosome severe injury and acrosome complete injury. Wherein, the grade I is that the whole acrosome of the sperm shows uniform and bright fluorescence, the grade II is that part of the acrosome of the sperm shows fluorescence, the grade III is that the equator of the acrosome part of the sperm shows fluorescence, and the grade IV is that the acrosome of the sperm does not emit any fluorescence.
The results are shown in Table 1, tables 1-2 and Table 2.
TABLE 1 comparison of sperm motility after thawing of groups
Note: the difference of the same column in the lower case of the numerical subscript indicates that the difference is significant (P < 0.05).
As is clear from the results in Table 1, the survival rates of the frozen sperm cells of examples 2, 2-1, 2-2, 2-3 and 2-4 were significantly different from those of comparative examples 2, 2-1, 2-3 and 2-3. All examples of post-freezing sperm motility were superior to the comparative examples.
TABLE 1-1 comparison of plasma Membrane integrity after thawing for each group
Note: the difference of the same column in the lower case of the numerical subscript indicates that the difference is significant (P < 0.05).
The results in the table 1-1 show that the plasma membrane integrity of the rabbit semen cryopreservation diluent is significantly better than that of the rabbit semen cryopreservation diluent in the examples 2, 2-1 and 2-2 and 2-3, so that the rabbit semen cryopreservation diluent provided by the invention has a better formula, and the plasma membrane integrity of the frozen sperm is significantly improved. Meanwhile, the plasma membrane integrity rate of the sperms in the examples 2-3 and 2-4 is obviously superior to that in the comparative examples 2 and 2-1, which shows that the balance and fumigation conditions have important influence on sperm freezing, and the specific balance and fumigation conditions of the invention can obviously improve the plasma membrane integrity rate of the frozen rabbit sperms.
TABLE 1-2 comparison of percentage of acrosome integrity after thawing for each group
Note: the difference of the same column in the lower case of the numerical subscript indicates that the difference is significant (P < 0.05).
The results in the table 1-2 show that the acrosome integrity of the rabbit semen cryopreservation diluent in the examples 2, 2-1 and 2-2 is obviously better than that of the rabbit semen cryopreservation diluent in the comparative examples 2-2 and 2-3, so that the rabbit semen cryopreservation diluent provided by the invention has a better formula, and the integrity of the frozen sperm plasma membrane is obviously improved. Meanwhile, the acrosome integrity of the sperms of the examples 2-3 and 2-4 is obviously superior to that of the comparative examples 2 and 2-1, which shows that the balance and fumigation conditions have important influence on sperm freezing, and the specific balance and fumigation conditions of the invention can obviously improve the plasma membrane integrity of the frozen rabbit sperms.
Table 2: influence of mother Rabbit hybridization after thawing frozen semen
Note: the difference of the same column in the lower case of the numerical subscript indicates that the difference is significant (P < 0.05).
As is clear from the results in Table 2, the production efficiency of the breeding mother rabbits after thawing of the frozen semen according to the present invention is weaker than that of the fresh semen, but examples 2, 2-3, and 2-4 are superior to comparative examples 2, 2-1, 2-2, and 2-3 in terms of number of live litter, and have significant differences. Meanwhile, in production, 1000 female rabbits are taken as an example, one production cycle is averaged, 230 healthy young rabbits are produced in the example 2-2 compared with the comparative example 2, and after fattening, at least 5.2 yuan of economic benefit can be produced in each nest of each female rabbit.
The results of the above examples show that the diluent for preserving frozen rabbit semen greatly improves the survival rate, plasma membrane integrity and acrosome integrity of the frozen semen, has stable quality, and is beneficial to improving the breeding efficiency and breeding speed of rabbits and preserving excellent germplasm resources. The male rabbit breeding quantity is saved, the use efficiency of the male rabbits is improved, and the male rabbit breeding method has the advantages of long storage period, high safety and the like.
Although the present invention has been described in detail with reference to the above embodiments, it is only a part of the embodiments of the present invention, not all of the embodiments, and other embodiments can be obtained without inventive step according to the embodiments, and the embodiments are within the scope of the present invention.
Claims (10)
1. A rabbit semen cryopreservation diluent is characterized in that water is used as a solvent; in the rabbit semen cryopreservation diluent, every 100mL of water is added: 3-4 g of trihydroxymethyl aminomethane, 1-2 g of citric acid, 0.5-1 g of glucose, 2-3 g of trehalose, 0.5-1 g of L-glutamic acid, 10-20 mL of dimethyl sulfoxide and 2-4 mL of glycerol; the pH value of the rabbit semen cryopreservation diluent is 6.5-7.5.
2. The rabbit semen cryopreservation diluent as claimed in claim 1, wherein per 100mL of water are added: 3.03g of trihydroxymethyl aminomethane, 1.69g of citric acid, 0.85g of glucose, 2.05g of trehalose, 0.74g of L-glutamic acid, 16mL of dimethyl sulfoxide and 3mL of glycerol; the pH value of the rabbit semen cryopreservation diluent is 7.3.
3. A method of preparing a rabbit semen cryopreservation diluent as claimed in claim 1 or 2, comprising the steps of: mixing trihydroxymethyl aminomethane, citric acid, glucose, trehalose, L-glutamic acid, dimethyl sulfoxide, glycerol and water.
4. A rabbit semen cryopreservation method based on the rabbit semen cryopreservation diluent of claim 1 or 2 or the rabbit semen cryopreservation diluent prepared by the preparation method of claim 3, comprising the following steps: refrigerating the rabbit semen to obtain the refrigerated rabbit semen; carrying out isothermal mixing on the refrigerated rabbit semen and the rabbit semen cryopreservation diluent to obtain a mixed solution; and (4) balancing the mixed solution, fumigating the mixed solution by liquid nitrogen, and freezing and storing the mixed solution.
5. The method for cryopreservation of rabbit semen according to claim 4, wherein the rabbit semen is rabbit semen from which gel components are removed before refrigeration; the survival rate of the rabbit semen is more than 0.8.
6. The rabbit semen cryopreservation method according to claim 4, wherein the temperature of the rabbit semen is 2-8 ℃, and the time of the refrigeration is 60-120 min.
7. The method for preserving rabbit semen by freezing as claimed in claim 4, wherein the volume ratio of the rabbit semen preserving diluent to the refrigerated rabbit semen is less than or equal to 5: 1.
8. The cryopreservation method of rabbit semen as claimed in claim 4, wherein the temperature of the equilibrium is 2-8 ℃ and the time of the equilibrium is 30-60 min.
9. The method for preserving frozen rabbit semen according to claim 4, wherein the fumigating is carried out by fumigating the balanced mixed solution 3-10 cm above the liquid level of liquid nitrogen; the fumigating time is 5-20 min.
10. The rabbit semen cryopreservation diluent of claim 1 or 2 or the rabbit semen cryopreservation method of any one of claims 4-9 for use in rabbit semen cryopreservation.
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