CN110463687B - Preservation solution and preservation method for sheep semen solution - Google Patents
Preservation solution and preservation method for sheep semen solution Download PDFInfo
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- CN110463687B CN110463687B CN201910620798.2A CN201910620798A CN110463687B CN 110463687 B CN110463687 B CN 110463687B CN 201910620798 A CN201910620798 A CN 201910620798A CN 110463687 B CN110463687 B CN 110463687B
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- 241001494479 Pecora Species 0.000 title claims abstract description 97
- 238000004321 preservation Methods 0.000 title claims abstract description 67
- 239000003761 preservation solution Substances 0.000 title claims abstract description 65
- 238000000034 method Methods 0.000 title claims abstract description 31
- 239000000243 solution Substances 0.000 title claims abstract description 22
- ABBQHOQBGMUPJH-UHFFFAOYSA-M Sodium salicylate Chemical compound [Na+].OC1=CC=CC=C1C([O-])=O ABBQHOQBGMUPJH-UHFFFAOYSA-M 0.000 claims abstract description 36
- 229960004025 sodium salicylate Drugs 0.000 claims abstract description 36
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 claims abstract description 29
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- GVJHHUAWPYXKBD-UHFFFAOYSA-N (±)-α-Tocopherol Chemical compound OC1=C(C)C(C)=C2OC(CCCC(C)CCCC(C)CCCC(C)C)(C)CCC2=C1C GVJHHUAWPYXKBD-UHFFFAOYSA-N 0.000 claims abstract description 22
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 claims abstract description 20
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- WEEMDRWIKYCTQM-UHFFFAOYSA-N 2,6-dimethoxybenzenecarbothioamide Chemical compound COC1=CC=CC(OC)=C1C(N)=S WEEMDRWIKYCTQM-UHFFFAOYSA-N 0.000 claims abstract description 16
- 108091003079 Bovine Serum Albumin Proteins 0.000 claims abstract description 16
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- 239000005715 Fructose Substances 0.000 claims abstract description 16
- FCPVYOBCFFNJFS-LQDWTQKMSA-M benzylpenicillin sodium Chemical compound [Na+].N([C@H]1[C@H]2SC([C@@H](N2C1=O)C([O-])=O)(C)C)C(=O)CC1=CC=CC=C1 FCPVYOBCFFNJFS-LQDWTQKMSA-M 0.000 claims abstract description 16
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- WIGCFUFOHFEKBI-UHFFFAOYSA-N gamma-tocopherol Natural products CC(C)CCCC(C)CCCC(C)CCCC1CCC2C(C)C(O)C(C)C(C)C2O1 WIGCFUFOHFEKBI-UHFFFAOYSA-N 0.000 claims abstract description 11
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- FYFFGSSZFBZTAH-UHFFFAOYSA-N methylaminomethanetriol Chemical compound CNC(O)(O)O FYFFGSSZFBZTAH-UHFFFAOYSA-N 0.000 claims abstract description 3
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- 239000007788 liquid Substances 0.000 claims description 18
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- WSFSSNUMVMOOMR-NJFSPNSNSA-N methanone Chemical compound O=[14CH2] WSFSSNUMVMOOMR-NJFSPNSNSA-N 0.000 description 1
- 235000013336 milk Nutrition 0.000 description 1
- 230000035764 nutrition Effects 0.000 description 1
- 230000004792 oxidative damage Effects 0.000 description 1
- 239000001301 oxygen Substances 0.000 description 1
- 229910052760 oxygen Inorganic materials 0.000 description 1
- 239000012466 permeate Substances 0.000 description 1
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- NLJMYIDDQXHKNR-UHFFFAOYSA-K sodium citrate Chemical compound O.O.[Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O NLJMYIDDQXHKNR-UHFFFAOYSA-K 0.000 description 1
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- HRXKRNGNAMMEHJ-UHFFFAOYSA-K trisodium citrate Chemical compound [Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O HRXKRNGNAMMEHJ-UHFFFAOYSA-K 0.000 description 1
- 229940038773 trisodium citrate Drugs 0.000 description 1
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Classifications
-
- 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
- A01N1/0215—Disinfecting agents, e.g. antimicrobials for preserving living parts
-
- 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
-
- 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
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Dentistry (AREA)
- General Health & Medical Sciences (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Environmental Sciences (AREA)
- Biophysics (AREA)
- Physiology (AREA)
- Agricultural Chemicals And Associated Chemicals (AREA)
Abstract
The invention relates to a preservation solution and a preservation method for sheep sperm solution. The preservation solution comprises the following components in each 100 mL: 0.16-1.6 mg of sodium salicylate or 0.18-1.8 mg of acetylsalicylic acid, 3.5-4.5 mL of vitamin E, 19-21 mL of fresh egg yolk, 1.5-2.5 mL of dimethyl sulfoxide or 3.5-4.5 mL of glycerol, 1.2-1.3 g of fructose, 1.7-1.8 g of citric acid monohydrate, 3.5-3.6 g of trihydroxymethyl aminomethane, 0.4-0.6 g of vitamin C, 0.4-0.6 g of bovine serum albumin, 10000-15000 IU of penicillin sodium, 10000-15000 IU of streptomycin sulfate, and the balance of double distilled water. The invention effectively prolongs the preservation time of the sheep semen under the conditions of normal temperature and low temperature and improves the quality of preserved sperms.
Description
Technical Field
The invention relates to a semen preservation solution, in particular to a preservation solution and a preservation method for sheep semen.
Background
The sheep raising industry in China has a long history, the germplasm resources of sheep and goats are rich, and the raising quantity accounts for the first place in the world. The sheep raising industry has important significance for the modern development of the country, has huge development prospects in broad grassland and rural sheep raising industry, and the rapid development of the sheep raising industry can not only open the contribution to the national reform, but also improve the living standard of people. With the continuous progress of science and technology, the sheep raising industry in China has long-term progress, but has a plurality of problems, has a large gap compared with developed countries, and is particularly suitable for the aspects of large-scale cultivation, intensive feeding and the like. In recent years, in order to improve the quality of mutton sheep varieties in China, excellent mutton sheep varieties are continuously introduced from abroad, wherein the DuPo sheep is one of the mutton sheep, has delicious meat quality, high feed conversion rate and high lean meat ratio, is a diamond-grade meat sheep variety introduced in China, and has high economic value.
Yamane et al discovered that the survival time of equine semen diluted with a glucose-phosphate diluent was significantly longer than that of undiluted sperm, reflecting the necessity of the diluent for sperm survival as early as 1928. The diluent formulation is also being perfected and Lopez et al compare skimmed milk diluent, Test and Tris-trehalose and find that skimmed milk diluent is able to preserve sperm viability for a longer period of time. Paulenz et al selected milk diluent, sodium citrate diluent, and Tris base in 2002, and found that the Tris base protects sperm better than milk and citric acid diluent, and prolongs the storage time of sperm. At present, the preservation solution for sheep semen is few in variety, high in requirement on refrigeration preservation condition and high in cost, and the wide popularization of the preservation solution for sheep semen is limited. Under the condition of normal temperature or low temperature preservation, the metabolism of the sperms is fast, the preservation time is short, the survival rate of the sperms after preservation is low, and the success rate of artificial insemination is low.
Disclosure of Invention
In order to solve the technical problems, the invention provides a preservation solution for sheep semen. The preservation solution is suitable for preservation of goat and sheep semen, and can effectively prolong semen preservation time and preservation quality.
The preservation solution for sheep semen solution is characterized in that: every 100mL of the preservation solution comprises the following components: the feed comprises a component A, 3.5-4.5 mL of vitamin E, 19-21 mL of fresh egg yolk, a component B, 1.2-1.3 g of fructose, 1.7-1.8 g of citric acid monohydrate, 3.5-3.6 g of trihydroxymethyl aminomethane, 0.4-0.6 g of vitamin C, 0.4-0.6 g of bovine serum albumin, 10000-15000 IU of penicillin sodium, 10000-15000 IU of streptomycin sulfate and the balance of double distilled water; the component A is 0.16-1.6 g of sodium salicylate or 0.18-1.8 g of acetylsalicylic acid, and the component B is 1.5-2.5 mL of dimethyl sulfoxide or 3.5-4.5 mL of glycerol.
Preferred embodiment 1: the preservation solution comprises the following components in each 100 mL: 0.3202g of sodium salicylate, 4mL of vitamin E, 20mL of fresh egg yolk, 2mL of dimethyl sulfoxide, 1.26g of fructose, 1.72g of citric acid monohydrate, 3.53g of tris (hydroxymethyl) aminomethane, 0.5g of vitamin C, 0.5g of bovine serum albumin, 15000IU of penicillin sodium, 15000IU of streptomycin sulfate and the balance of double distilled water.
Preferred embodiment 2: the preservation solution comprises the following components in each 100 mL: 0.9008g of acetylsalicylic acid, 4mL of vitamin E, 20mL of fresh egg yolk, 4mL of glycerol, 1.26g of fructose, 1.72g of citric acid monohydrate, 3.53g of tris (hydroxymethyl) aminomethane, 0.5g of vitamin C, 0.5g of bovine serum albumin, 15000IU of penicillin sodium, 15000IU of streptomycin sulfate and the balance of double distilled water.
The second purpose of the invention is to provide a first preservation method for preserving sheep semen by using the preservation solution of the preferred embodiment 1, which comprises the following steps:
and diluting the collected qualified sheep semen by 8 times by using the preservation solution, subpackaging the diluted semen into centrifugal tubes, and preserving at a constant temperature of 20 ℃.
The third purpose of the invention is to provide a second preservation method for preserving sheep semen by using the preservation solution of the preferred scheme 2, which comprises the following steps:
diluting the collected qualified sheep semen by 8 times by using the preservation solution, subpackaging the diluted semen into thin tubes, sealing the thin tubes by using polyvinyl alcohol powder, wrapping the thin tubes by absorbent cotton, and storing the thin tubes in a refrigerator at 4 ℃.
The fourth purpose of the invention is to provide a third preservation method for preserving sheep semen by using the preservation solution of the preferred embodiment 2, which comprises the following steps:
diluting the collected qualified sheep semen by 8 times by using the preservation solution, respectively loading the diluted semen into a thin tube, sealing the thin tube by using polyvinyl alcohol powder, wrapping absorbent cotton outside the thin tube, slowly cooling and balancing the thin tube in a refrigerator at 4 ℃, then placing the thin tube in prepared liquid nitrogen for fumigation, and finally placing the thin tube in the liquid nitrogen for preservation.
The invention has the beneficial effects that:
(1) the preservation solution of the invention has the following functions of the components:
vitamin C is an antioxidant substance, can effectively prevent sperm phospholipid from undergoing peroxidation to improve sperm motility and prolong the survival time of sperms in vitro, and has an antioxidant effect embodied as an effective free radical scavenger and capable of protecting genetic gene DNA in sperm cells from oxidative damage.
Bovine serum albumin is a protein protective agent, can compensate the reduction of protein motion activators in the seminal plasma caused by dilution, maintain the motion of sperms, reduce the head agglutination caused by opposite charges on the heads of the sperms caused by dilution, can neutralize the metabolites of the sperms, and plays a certain role in buffering and antioxidation.
The sodium salicylate can remove active oxygen, inhibit peroxidase, reduce synthesis of prostaglandin by inhibiting cyclooxygenase, has effects of anti-inflammation, antirheumatic, antipyretic and analgesic, and is commonly used for treating rheumatic fever and rheumatoid arthritis in daily life.
Acetylsalicylic acid, also known as aspirin, is the earliest, most widely and most common antipyretic analgesic antirheumatic drug. Has various pharmacological effects of relieving fever, easing pain, resisting inflammation, resisting rheumatism, resisting platelet aggregation and the like, and has quick and stable drug effect.
The sodium salicylate and the acetylsalicylic acid both have the function of strongly inhibiting the release of lysosomal enzyme and play a good role in protecting cytoplasmic membranes.
Fructose: the saccharide is an important energy substance in the preservation diluent of the sheep semen, has the main functions of providing nutrition and supplementing energy for sheep semen, and can stabilize a sperm cell membrane protein-lipid complex to play a role in protecting the sperm cell membrane.
Citric acid monohydrate, tris: the buffer substance is an essential component in the sheep semen preservation diluent. The purpose of adding buffer substances to the dilution is to adjust and maintain the normal pH of the semen to facilitate sperm survival.
Fresh egg yolk: the product is also widely applied to the dilution of sheep semen as an energy substance. In addition, the low-density lipoprotein in the yolk directly permeates into the plasma membrane, and can stabilize the plasma membrane and protect the sperm cell membrane.
Penicillin sodium, streptomycin sulfate: the sheep semen is inevitably polluted by certain microorganisms in the collection and preservation processes, and the addition of an appropriate amount of antibacterial substances into the diluent can inhibit the propagation of the microorganisms of the bacteria and improve the killing capability of harmful bacteria, thereby prolonging the survival time of the sheep semen.
Dimethyl sulfoxide, glycerin: as a permeability protective agent, the composition can lower the freezing point of cells, reduce the formation of ice crystals, relieve the damage of free radicals to the cells and change the permeability of biological membranes to electrolytes, medicaments, poisons and metabolites.
The sodium salicylate and acetylsalicylic acid are used as anti-inflammatory and antirheumatic drugs, but are not applied in the semen preservation process, in the experiment, the sodium salicylate or acetylsalicylic acid and other auxiliary components are added into the diluent, so that the preservation time of the sheep semen under the conditions of normal temperature and low temperature is effectively prolonged, the quality of preserved sperms is improved, a good foundation and a good premise are provided for long-time preservation and long-distance transportation of the sheep semen, and theoretical support can be provided for solving the problems in production.
According to the invention, different components are combined into the preservation solution according to a certain proportion, so that the preservation effect of the sheep semen is obviously improved, especially sodium salicylate or acetylsalicylic acid is added into the semen preservation solution for the first time, and experiments prove that the preservation time of the sheep semen under the conditions of normal temperature, low temperature and freezing is obviously prolonged by adding 0.16-1.6 g of sodium salicylate or 0.18-1.8 g of acetylsalicylic acid into the semen diluent, and the key indexes of sperm motility, plasma membrane integrity, acrosome integrity and the like after the sheep semen is preserved are improved.
(2) The preservation solution of the optimal scheme formula of the invention is used for preserving the sheep semen solution, so that the preservation time of the sheep semen under the conditions of normal temperature and low temperature can be longest, and the quality of the semen can be best.
(3) The first preservation method has the advantages that: in the invention, by researching the composition of the preservation solution and the proportion of each component, the higher-quality maintenance of the sheep semen solution under the normal-temperature preservation condition can be realized only by using the constant-temperature water bath to maintain the constant preservation temperature of the semen. The normal temperature preservation technology has the advantages of good preservation effect, no need of special temperature control and refrigeration equipment, simple and convenient treatment process, convenient popularization and application and the like.
The purpose of 8-fold dilution with a preservation solution before preservation is to achieve a sperm density of about 2X 108～2×109The number per mL of the cells is increased to maintain the osmotic pressure inside and outside the spermatids, reduce the damage to the plasma membrane of the sperms, maintain the permeability of the plasma membrane and simultaneously contribute to improving the effective insemination number and the estrus conception rate in production.
(4) The second preservation method has the advantages that: according to the invention, the sheep semen solution is maintained for a long time and at high quality under the low-temperature condition by researching the composition of the preservation solution and the proportion of each component. The low-temperature preservation effectively prolongs the utilization time of the sheep semen, more fully exerts the utilization value of excellent stud rams, and improves the utilization rate and the improvement effect of the stud rams. In addition, the low-temperature storage has low requirements on conditions, is easy to obtain, has low application threshold in production practice, and has extremely high utilization value. The preservation principle is as follows:
the low temperature preservation can reduce the metabolic efficiency of sperm, and sperm preservationThe components in the liquid can protect the sperm plasma membrane and provide the nutritional requirements for low temperature metabolism of sperm. The sperm density of the sperm cells was about 2X 10 as the result of 8-fold dilution with a preservative solution before preservation8～2×109The number per mL of the sperm cells is increased, so that the osmotic pressure inside and outside the sperm cells is maintained, the damage to the plasma membrane of the sperm is reduced, the permeability of the plasma membrane is maintained, and the dilution multiple of the sperm is proper, thereby being beneficial to improving the effective insemination number and the estrus conception rate in production.
The contact area of the semen and the external environment can be obviously increased by the fine tube split charging, so that the semen is uniformly heated; in addition, the tubule is convenient to use and operate and convenient to mark and distinguish. The outer layer is wrapped with absorbent cotton to slow down the cooling speed and the cooling process and prevent sperm from being damaged by stress.
(5) The third preservation method has the advantages that: according to the invention, the long-time preservation of the sheep semen liquid under the freezing condition is realized by researching the composition of the preservation liquid and the proportion of the components. The preservation method can preserve sheep semen for a long time without time and region limitation, thereby enlarging the number of breeding female animals and improving the breeding efficiency of breeding male animals. The basic principle of operation is as follows:
before preservation, the preservation solution is diluted by 8 times, so that the osmotic pressure and the pH value of the environment where the sperms are located are optimized, energy is provided for the sperms, the loss of components of cell membranes of the sperms is prevented, and the effect of an antifreezing agent is achieved. The aim of being respectively arranged in the thin tubes is to increase the contact area of the semen and the external environment as much as possible, thus being beneficial to the balance of temperature change and enabling the semen to be heated uniformly; in addition, the tubule is convenient to use and operate and convenient to mark and distinguish. The purpose of wrapping the absorbent cotton is to slow down the temperature reduction process of the sperms and prevent the sperms from being damaged by stress. The purpose of fumigating before adding liquid nitrogen is to pre-cool semen in advance and prevent the stress injury of sperm caused by the ultralow temperature of liquid nitrogen.
After balancing, the tubule is put into liquid nitrogen prepared in advance for fumigation, and finally is put into the liquid nitrogen for preservation. The step adopts a rapid cooling method, and aims to prevent the formation of ice crystals in the sperms during the cooling process, wherein the formation of the ice crystals can damage the sperms to a great extent, so that the sperms die or do not have fertilization capability after being thawed.
Detailed Description
The technical scheme of the invention is more specifically explained by combining the following embodiments, wherein the raw materials involved in the following embodiments are all from common commercial products:
example 1
A preservation solution for sheep semen solution, wherein each 100mL of the preservation solution comprises the following components: sodium salicylate, vitamin E4 mL, fresh egg yolk 20mL, dimethyl sulfoxide 2mL, fructose 1.26g, citric acid monohydrate 1.72g, tris (hydroxymethyl) aminomethane 3.53g, vitamin C0.5 g, bovine serum albumin 0.5g, penicillin sodium 15000IU, streptomycin sulfate 15000IU, and the balance of double distilled water. Preparation of 5 dilutions, group 1: blank control, 0g of sodium salicylate; group 2: 0.1601g/100mL sodium salicylate (corresponding to 10mM in the table); group 3: 0.3202g/100mL sodium salicylate (20 mM in the corresponding table); group 4: 0.8005g/100mL sodium salicylate (corresponding to 50mM in the table); group 5: sodium salicylate 1.6010g/100mL (corresponding to 100mM in the table).
Example 2
A preservation solution for sheep semen solution, wherein each 100mL of the preservation solution comprises the following components: sodium salicylate, vitamin E4 mL, fresh egg yolk 20mL, glycerol 4mL, fructose 1.26g, citric acid monohydrate 1.72g, tris (hydroxymethyl) aminomethane 3.53g, vitamin C0.5 g, bovine serum albumin 0.5g, penicillin sodium 15000IU, streptomycin sulfate 15000IU, and the balance of double distilled water. Preparation of 5 dilutions, group 1: 0g of sodium salicylate; group 2: 0.1601g/100mL sodium salicylate (corresponding to 10mM in the table); group 3: 0.3202g/100mL sodium salicylate (20 mM in the corresponding table); group 4: 0.8005g/100mL sodium salicylate (corresponding to 50mM in the table); group 5: sodium salicylate 1.6010g/100mL (corresponding to 100mM in the table).
Example 3
A preservation solution for sheep semen solution, wherein each 100mL of the preservation solution comprises the following components: acetylsalicylic acid, vitamin E4 mL, fresh egg yolk 20mL, dimethyl sulfoxide 2mL, fructose 1.26g, citric acid monohydrate 1.72g, tris (hydroxymethyl) aminomethane 3.53g, vitamin C0.5 g, bovine serum albumin 0.5g, penicillin sodium 15000IU, streptomycin sulfate 15000IU, and the balance of double distilled water. Preparation of 5 groups of preservation solution group 1: 0g of acetylsalicylic acid; group 2: acetylsalicylic acid 0.1601g/100mL (corresponding to 10mM in the table); group 3: acetylsalicylic acid 0.3202g/100mL (corresponding to 20mM in the table); group 4: acetylsalicylic acid 0.8005g/100mL (corresponding to 50mM in the table); group 5: acetylsalicylic acid 1.6010g/100mL (corresponding to 100mM in the table).
Example 4
A preservation solution for sheep semen solution, wherein each 100mL of the preservation solution comprises the following components: acetylsalicylic acid, vitamin E4 mL, fresh egg yolk 20mL, glycerol 4mL, fructose 1.26g, citric acid monohydrate 1.72g, tris (hydroxymethyl) aminomethane 3.53g, vitamin C0.5 g, bovine serum albumin 0.5g, penicillin sodium 15000IU, streptomycin sulfate 15000IU, and the balance of double distilled water. Preparation of 5 groups of preservation solution group 1: 0g of acetylsalicylic acid; group 2: acetylsalicylic acid 0.1601g/100mL (corresponding to 10mM in the table); group 3: acetylsalicylic acid 0.3202g/100mL (corresponding to 20mM in the table); group 4: acetylsalicylic acid 0.8005g/100mL (corresponding to 50mM in the table); group 5: acetylsalicylic acid 1.6010g/100mL (corresponding to 100mM in the table).
Example 5
1. Collection of semen
The method comprises the following steps of fixing a semen collection place in an affiliated sheep farm of an experimental station, placing a ram in a house before semen collection, placing a fixing frame for fixing a ewe in the house, and cleaning sanitation in the house before semen collection. A healthy and disease-free oestrus ewe with strong physique and mild temperament is found in a ewe shed and is fixed on a retaining frame, so that female animals are ensured to be in the visual field of male animals, and the sexual desire of the male animals is caused. Cleaning the false vagina, the semen collection cup and the glass rod, installing the prepared semen collection cup at one end of the false vagina, then pouring warm water into the interlayer of the false vagina, and installing the air nozzle with the piston; a small amount of vaseline is taken by a glass rod, a layer of vaseline is uniformly coated on the inner tube, and after air blowing and pressurization, one end of the vaseline coated part is preferably triangular.
2. Normal-temperature preservation method of sheep semen
And diluting the collected qualified sheep semen by 8 times by using the preservation solutions prepared in the embodiments 1 and 3, subpackaging the diluted semen into centrifuge tubes, and preserving at a constant temperature of 20 ℃.
3. Refrigeration preservation method of sheep semen
Diluting the collected qualified sheep semen by 8 times by using the preservation solutions prepared in the embodiments 2 and 4, subpackaging the diluted semen into thin tubes, sealing the thin tubes by using polyvinyl alcohol powder, wrapping the thin tubes by using absorbent cotton, and storing the thin tubes in a refrigerator at 4 ℃.
4. Cryopreservation method of sheep semen
Diluting the collected qualified sheep semen by 8 times by using the preservation solutions prepared in the embodiments 2 and 4, respectively loading the diluted semen into a thin tube, sealing the thin tube with polyvinyl alcohol powder, wrapping absorbent cotton, slowly cooling and balancing the thin tube in a refrigerator at 4 ℃, then putting the thin tube in prepared liquid nitrogen for fumigation, and finally putting the thin tube in the liquid nitrogen for preservation.
5. Semen index detection
And detecting the sperm motility, the plasma membrane integrity and the acrosome integrity at intervals.
The method for measuring sperm motility: and (3) diluting the semen with a preserving solution, preserving the diluted semen, and performing sperm quality analysis through a CASA system at different times.
Method for determining integrity of plasma membrane: using the osmotic swelling test (HOST), 200. mu.L of hypotonic solution (formulation: 4.9g trisodium citrate, 9.0g distilled fructose water to 1000mL) was added to 20. mu.L of semen, mixed and incubated at 37 ℃ for 35min, then smeared and air-dried, fixed with 2% glutaraldehyde for 15min and then washed with water to dryness. The examination was performed under a microscope, and 200 sperm were counted, and the number of sperm that met the examination criteria was divided by the total number of sperm. The sperm plasma membrane integrity rate ═ (plasma membrane intact sperm count/total number of sperm) × 100%.
The method for measuring the acrosome integrity rate comprises the following steps: adopting a Jiemsa staining method, taking 20 mu L of diluted semen smeared sheets, air-drying, fixing for 15min by using 4% formaldehyde, washing, air-drying, sucking newly prepared Jiemsa staining solution by using a liquid transfer gun for staining, and ensuring that the staining solution is uniformly distributed on the smeared sheets for staining for 2 h. And finally, washing with water, air-drying, detecting under a microscope according to a detection standard, and counting 200 sperms. The sperm acrosome integrity rate is (number of normal acrosome sperm/total number of sperm) × 100%.
6. Analysis of results
(1) Effect of the preservation solution prepared in example 1 on sheep sperm preservation at Normal temperature
A. The data of sperm motility measured in example 1 are shown in Table 1.
TABLE 1 Effect of different preserving fluids on sperm motility (%) of sheep at a preserving temperature of 20 ℃ in example 1
Note: a, b, c, d represent differential significance between different groups at each time point, alphabetical differences represent differential significance (p <0.05), and alphabetical identity represents no differential significance (p > 0.05).
The results show that when the sheep sperms are preserved at the temperature of 20 ℃, the addition of the sodium salicylate in the diluent can effectively improve the sperm preservation survival rate. Meanwhile, the use of sodium salicylate with the concentration of 20mM can effectively prolong the preservation time of the semen at the temperature of 20 ℃. In addition, from the analysis of the results, the sperm motility rate does not increase along with the increase of the concentration of the sodium salicylate, and the sodium salicylate is a non-dosage-dependent semen preservation additive.
B. The data of the plasma membrane integrity of sperm measured in example 1 are shown in Table 2
TABLE 2 Effect of different preservation solutions on the plasma membrane integrity (%) of sheep sperm cells at 20 ℃ preservation temperature in example 1
Note: a, b, c represent the significance of difference between different groups at each time point, with different letters representing the significance of difference (p <0.05) and the same letters representing no significance of difference (p > 0.05).
As can be seen from Table 2, there was no significant difference in the plasma membrane integrity of sperm in each treatment group after the treatment of the sheep sperm solution in 5 preservation solutions for 0 h. After the sperm plasma membrane is stored for 28-76 h at 20 ℃ in a dark place, the integrity of the sperm plasma membrane of the 20mM treatment group and the integrity of the sperm plasma membrane of the 50mM treatment group are mostly obviously higher than that of the rest groups (P <0.05), wherein the integrity of the sperm plasma membrane of the 20mM treatment group is mostly obviously higher than that of the rest groups (P <0.05), and the integrity of the sperm plasma membrane of the 10mM test group, the 50mM test group and the 100mM test group is mostly not obviously different from that of the control group.
C. The data of the sperm acrosome integrity measured in example 1 are shown in Table 3
TABLE 3 Effect of different preserving fluids on the percentage (%) of acrosome integrity of sheep sperm at a preserving temperature of 20 ℃ in example 1
Note: a, b, c represent the significance of difference between different groups at each time point, with different letters representing the significance of difference (p <0.05) and the same letters representing no significance of difference (p > 0.05).
As can be seen from table 3, the acrosome integrity was significantly higher in the 20mM treated group than in the remaining groups (P < 0.05). The results show that when the sheep sperms are stored at 20 ℃, the addition of 20mM sodium salicylate in the diluent can improve the integrity rate of the sperm acrosome and delay the decline trend of the integrity rate of the sperm acrosome, but the time for the integrity of the sperm acrosome cannot be prolonged when the sheep sperms are stored at 20 ℃.
(2) EXAMPLE 2 Effect of the preservative solution prepared in example 2 on sheep sperm preservation under refrigeration
A. The data of the sperm motility rate measured in example 2 are shown in Table 4
TABLE 4 Effect of different preserving fluids on sperm motility (%) of sheep at a preserving temperature of 4 ℃ in example 2
Note: a, b, c represent the significance of difference between different groups at each time point, with different letters representing the significance of difference (p <0.05) and the same letters representing no significance of difference (p > 0.05).
As can be seen from table 4, the sperm motility was consistently higher in the 10 and 20mM sodium salicylate treated groups than in the other treated groups throughout the storage period and was significantly higher than in the control group (p <0.05), with the 20mM sodium salicylate treated group having the highest sperm motility.
B. The data of the plasma membrane integrity of sperm as determined in example 2 are shown in Table 5
TABLE 5 Effect of different preservation solutions on the plasma membrane integrity (%) of sheep sperm cells at a preservation temperature of 4 ℃ in example 2
Note: a, b, c represent the significance of difference between different groups at each time point, with different letters representing the significance of difference (p <0.05) and the same letters representing no significance of difference (p > 0.05).
As can be seen from Table 5, plasma membrane integrity decreased with increasing storage time, but the addition of sodium salicylate helped to increase the integrity of the plasma membrane during storage of sheep sperm. Wherein, when the sperm plasma membrane integrity of the sodium salicylate treatment group is already obviously higher than that of the control group (p <0.05) when the sperm plasma membrane is preserved to 1 d; when the cells are preserved for 3d, the integrity rate of the plasma membrane of 10 and 20mM treatment groups is obviously higher than that of a control group; the 20mM treatment group has significantly higher plasma membrane integrity than the control group (p <0.05) when stored to 7 d; when the storage time was extended to 13d, plasma membrane integrity was significantly higher in the 10, 20 and 100mM treated groups than in the control group (p < 0.05). The results confirmed that the plasma membrane integrity of sperm in the 20mM treated group remained highest throughout the preservation process and was significantly higher than that in the control group (p < 0.05).
C. The data of the plasma membrane integrity of sperm determined in example 2 are shown in Table 6
TABLE 6 Effect of different preserving fluids on the percentage (%) of acrosome integrity of sheep sperm at a preserving temperature of 4 ℃ in example 2
Note: a, b, c represent the significance of difference between different groups at each time point, with different letters representing the significance of difference (p <0.05) and the same letters representing no significance of difference (p > 0.05).
As can be seen from Table 6, the integrity of the sperm acrosomes was gradually reduced in all treatment groups with the increase in storage time. The effect of the sodium salicylate treatment groups with different concentrations on the sperm acrosome integrity at different storage times is not obvious, and the effect of the 50mM and 100mM treatment groups is relatively good.
(3) EXAMPLE 3 Effect of the preservation solution prepared in example 3 on sheep sperm preservation at Normal temperature
A. The data of the sperm motility rates measured in example 3 are shown in Table 7
TABLE 7 Effect of different preserving fluids on sperm motility (%) of sheep at a preserving temperature of 20 ℃ in example 3
Note: a, b, c, d represent differential significance between different groups at each time point, alphabetical differences represent differential significance (p <0.05), and alphabetical identity represents no differential significance (p > 0.05).
As can be seen from the sperm motility index of Table 7, the addition of acetylsalicylic acid can improve the sperm motility of sheep when stored at 20 deg.C. Wherein, when the composition is stored for 4 hours, the storage effect of 10, 20 and 50mM acetylsalicylic acid treatment groups is good and is obviously higher than that of a control group (p < 0.05); the sperm motility of the 50mM acetylsalicylic acid treatment group is the highest and is obviously higher than that of other groups (p <0.05) when the storage time is prolonged to 52 hours; at 76h of storage, the sperm motility of the control group was less than 10%, no subsequent detection was possible, and the sperm motility of the 20 and 50mM acetylsalicylic acid-treated groups remained better and significantly higher than that of the other groups (p < 0.05). In summary, sperm cells from the 50mM acetylsalicylic acid treated group were most viable during storage and were mostly significantly higher than the other groups (p < 0.05).
B. The data of the plasma membrane integrity of the sperm cells measured in example 3 are shown in Table 8
TABLE 8 Effect of different preservative solutions in example 3 on the plasma membrane integrity (%) of sheep sperm cells at a preservation temperature of 20 deg.C
Note: a, b, c, d represent differential significance between different groups at each time point, alphabetical differences represent differential significance (p <0.05), and alphabetical identity represents no differential significance (p > 0.05).
As can be seen from Table 8, plasma membrane integrity decreased with increasing storage time, but the addition of acetylsalicylic acid helped to increase the integrity of the plasma membrane during storage of sheep sperm. Plasma membrane integrity of acetylsalicylic acid treated sperm was already significantly higher upon dilution than control (p <0.05), with 50mM treatment highest; the 50mM treated group was significantly higher than each of the other groups by 28h of storage (p < 0.05); the sperm plasma membrane integrity remained high in the 50mM treated groups when stored for 52h and 76h, and was mostly significantly higher than in each of the other groups (p < 0.05).
C. The data of the plasma membrane integrity of the sperm cells measured in example 3 are shown in Table 9
TABLE 9 Effect of different preservative solutions in example 3 on the percentage (%) of acrosome integrity of sheep sperm at a preservation temperature of 20 deg.C
Note: a, b, c, d represent differential significance between different groups at each time point, alphabetical differences represent differential significance (p <0.05), and alphabetical identity represents no differential significance (p > 0.05).
As can be seen from Table 9, after the semen is diluted by the diluent added with acetylsalicylic acid with different concentrations, the integrity rate of the sperm acrosome is gradually reduced along with the prolonging of the preservation time, and when the semen is preserved for 28 hours, the integrity rate of the sperm acrosome of the 50mM acetylsalicylic acid treatment group is best and is obviously higher than that of the control group (p <0.05), but the difference with the other three treatment groups is not obvious; the 10mM acetylsalicylic acid treatment group has the highest sperm acrosome integrity rate when being stored for 52 h; when the sperm were stored for 76h, the 50mM acetylsalicylic acid-treated group showed the highest acrosome integrity, but the difference from the other groups was not significant.
(4) EXAMPLE 4 Effect of the preservative solution prepared in example 4 on sheep sperm preservation under refrigeration
A. The data of the sperm motility rate measured in example 4 are shown in Table 10
TABLE 10 Effect of different preservative solutions in example 4 on sperm motility (%) of sheep at a preservation temperature of 4 ℃%
Note: a, b, c, d represent differential significance between different groups at each time point, alphabetical differences represent differential significance (p <0.05), and alphabetical identity represents no differential significance (p > 0.05).
As can be seen from Table 10, the addition of acetylsalicylic acid improved the viability of the sheep sperm at 4 ℃ storage conditions. Sperm motility was significantly higher in the 20 and 50mM acetylsalicylic acid treated groups than in the control group (P <0.05) immediately after semen dilution; sperm viability was significantly higher in the 20, 50 and 100mM treated groups than in the control group (P <0.05) during storage. Wherein, when the sperm cells are preserved to 5, 9 and 11 days, the sperm cell activity of 50 and 100mM processing groups is obviously higher than that of other groups; sperm viability was highest in the 50mM treated group and significantly higher than in the other groups by 13d (P < 0.05). The results demonstrate that 50mM acetylsalicylic acid maintains high sperm motility throughout the preservation process.
B. The data of the plasma membrane integrity of sperm measured in example 4 are shown in Table 11
TABLE 11 Effect of different preservative solutions in example 4 on the plasma membrane integrity (%) of sheep sperm cells at a preservation temperature of 4 deg.C
Note: a, b, c, d represent differential significance between different groups at each time point, alphabetical differences represent differential significance (p <0.05), and alphabetical identity represents no differential significance (p > 0.05).
As can be seen from Table 11, plasma membrane integrity decreased with increasing storage time, but the addition of acetylsalicylic acid helped to increase the integrity of the plasma membrane during storage of sheep sperm. The plasma membrane integrity of sperms in the 50mM acetylsalicylic acid treatment group is obviously higher than that in other groups (p <0.05) when diluted and stored to 1 d; when the composition is stored for 3 and 5 days, the acetylsalicylic acid treatment group is obviously higher than the control group (P < 0.05); the 50mM treated group was significantly higher than the control group (P <0.05) when stored to 9 and 11 d; at 13d, 10, 20 and 50mM treated groups were significantly higher than the control group (P < 0.05). The results show that the plasma membrane integrity of sperm in the 50mM treated group remained highest throughout the preservation process and was significantly higher than that in the control group (P < 0.05).
C. The data of the plasma membrane integrity of sperm measured in example 4 are shown in Table 12
TABLE 12 Effect of different preserving fluids on the percentage (%) of acrosome integrity of sheep sperm at a preserving temperature of 4 ℃ in example 4
Note: a, b, c, d represent differential significance between different groups at each time point, alphabetical differences represent differential significance (p <0.05), and alphabetical identity represents no differential significance (p > 0.05).
As can be seen from Table 12, the sperm acrosome integrity decreased with the duration of storage after the semen was diluted with the diluent containing acetylsalicylic acid at various concentrations. Wherein the sperm acrosome integrity rate does not change much when diluted; the acrosome integrity of all the acetylsalicylic acid-treated sperm was significantly higher than that of the control group (p <0.05) when stored for 1, 3 and 5 d; when the sperm cells are preserved for 7 days, the acrosome integrity rates of 10mM, 20mM and 50mM acetylsalicylic acid treatment groups are higher than that of a control group; the 50mM acetylsalicylic acid treated group had the highest rate of sperm acrosome integrity when stored at 11 and 13d, and was significantly higher at 13d than the other groups. Experiments confirm that the acrosome integrity of the sperm in the 50mM acetylsalicylic acid treated group is kept highest all the time during the preservation process and gradually shows the difference significance with other treated groups in the later period.
Through the examples 1 to 5, it was confirmed that a proper amount of sodium salicylate or acetylsalicylic acid not only can increase the survival rate of the sheep semen when the sheep semen is stored at normal temperature or low temperature, but also can greatly prolong the storage time and the storage quality of the sheep semen.
Example 6
A preservation solution for sheep semen liquid comprises the following components in each 100 mL: 0.3202g of sodium salicylate, 3.5mL of vitamin E, 19mL of fresh egg yolk, 1.5mL of dimethyl sulfoxide, 1.2g of fructose, 1.7g of citric acid monohydrate, 3.5g of tris (hydroxymethyl) aminomethane, 0.4g of vitamin C, 0.4g of bovine serum albumin, 10000IU of penicillin sodium, 10000IU of streptomycin sulfate and the balance of double distilled water.
Example 7
A preservation solution for sheep semen liquid comprises the following components in each 100 mL: 0.9008g of acetylsalicylic acid, 4.5mL of vitamin E, 21mL of fresh egg yolk, 2.5mL of dimethyl sulfoxide, 1.3g of fructose, 1.8g of citric acid monohydrate, 3.6g of tris (hydroxymethyl) aminomethane, 0.6g of vitamin C, 0.6g of bovine serum albumin, 15000IU of penicillin sodium, 15000IU of streptomycin sulfate and the balance of double distilled water.
Example 8
A preservation solution for sheep semen liquid comprises the following components in each 100 mL: 0.3202g of sodium salicylate, 4mL of vitamin E, 20mL of fresh egg yolk, 3.5mL of glycerol, 1.25g of fructose, 1.75g of citric acid monohydrate, 3.55g of tris (hydroxymethyl) aminomethane, 0.5g of vitamin C, 0.5g of bovine serum albumin, 15000IU of penicillin sodium, 15000IU of streptomycin sulfate and the balance of double distilled water.
Example 9
A preservation solution for sheep semen liquid comprises the following components in each 100 mL: 0.9008g of acetylsalicylic acid, 4mL of vitamin E, 20mL of fresh egg yolk, 4.5mL of glycerol, 1.25g of fructose, 1.75g of citric acid monohydrate, 3.55g of tris (hydroxymethyl) aminomethane, 0.5g of vitamin C, 0.5g of bovine serum albumin, 15000IU of penicillin sodium, 15000IU of streptomycin sulfate and the balance of double distilled water.
Similar preservation effects to those of the preservation solutions in examples 1 to 4, namely, the preservation time and the preservation quality of the sheep semen are greatly prolonged, are obtained by respectively using the preservation solutions in examples 6 to 9 and preserving the sheep semen solution by the preservation method in example 5. The range of the components in the preservation solution is out of the range of the invention, and the preservation effect is lower than that of the invention.
Claims (6)
1. The preservation solution for sheep semen solution is characterized in that: every 100mL of the preservation solution comprises the following components: the feed comprises a component A, 3.5-4.5 mL of vitamin E, 19-21 mL of fresh egg yolk, a component B, 1.2-1.3 g of fructose, 1.7-1.8 g of citric acid monohydrate, 3.5-3.6 g of trihydroxymethyl aminomethane, 0.4-0.6 g of vitamin C, 0.4-0.6 g of bovine serum albumin, 10000-15000 IU of penicillin sodium, 10000-15000 IU of streptomycin sulfate and the balance of double distilled water; the component A is 0.16-1.6 g of sodium salicylate or 0.18-1.8 g of acetylsalicylic acid, and the component B is 1.5-2.5 ml of dimethyl sulfoxide or 3.5-4.5 ml of glycerol.
2. The preservation solution according to claim 1, characterized in that: the preservation solution comprises the following components in each 100 mL: 0.3202g of sodium salicylate, 4mL of vitamin E, 20mL of fresh egg yolk, 2mL of dimethyl sulfoxide, 1.26g of fructose, 1.72g of citric acid monohydrate, 3.53g of tris (hydroxymethyl) aminomethane, 0.5g of vitamin C, 0.5g of bovine serum albumin, 15000IU of penicillin sodium, 15000IU of streptomycin sulfate and the balance of double distilled water.
3. The preservation solution according to claim 1, characterized in that: the preservation solution comprises the following components in each 100 mL: 0.9008g of acetylsalicylic acid, 4mL of vitamin E, 20mL of fresh egg yolk, 4mL of glycerol, 1.26g of fructose, 1.72g of citric acid monohydrate, 3.53g of tris (hydroxymethyl) aminomethane, 0.5g of vitamin C, 0.5g of bovine serum albumin, 15000IU of penicillin sodium, 15000IU of streptomycin sulfate and the balance of double distilled water.
4. A preservation method for preserving sheep semen by using the preservation solution of claim 1 or 2, which is characterized by comprising the following steps:
and diluting the collected qualified sheep semen by 8 times by using the preservation solution, subpackaging the diluted semen into centrifugal tubes, and preserving at a constant temperature of 20 ℃.
5. A preservation method for preserving sheep semen by using the preservation solution of claim 1 or 3, which is characterized by comprising the following steps:
diluting the collected qualified sheep semen by 8 times by using the preservation solution, subpackaging the diluted semen into thin tubes, sealing the thin tubes by using polyvinyl alcohol powder, wrapping the thin tubes by absorbent cotton, and storing the thin tubes in a refrigerator at 4 ℃.
6. A preservation method for preserving sheep semen by using the preservation solution of claim 1 or 3, which is characterized by comprising the following steps:
diluting the collected qualified sheep semen by 8 times by using the preservation solution, respectively loading the diluted semen into a thin tube, sealing the thin tube by using polyvinyl alcohol powder, wrapping absorbent cotton outside the thin tube, slowly cooling and balancing the thin tube in a refrigerator at 4 ℃, then placing the thin tube in prepared liquid nitrogen for fumigation, and finally placing the thin tube in the liquid nitrogen for preservation.
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| CN111820209A (en) * | 2020-08-12 | 2020-10-27 | 扬州大学 | Screening method for Hu sheep semen at optimum temperature for normal-temperature storage |
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