JP2019147764A - Preservation liquid for semen and semen transport method using same - Google Patents

Abstract

To provide a preservation liquid in which the sperm motility is maintained at a high level in order to provide sperm with a high product acceptance rate to remote locations.SOLUTION: Mammalian semen is stored using a semen preservation liquid containing a sperm protein dephosphorylation inhibitor. The stored semen is transported in a contact state with mineral oil or a gas containing more inert gas than air.SELECTED DRAWING: None

Description

  The present invention relates to a semen preservation solution and a semen transport method using the same.

  Artificial insemination is popular not only from the treatment of infertility in humans but also from the viewpoints of producing excellent livestock, breeding, and prevention of infectious diseases. In particular, the penetration rate of artificial insemination in the field of livestock production is very high at approximately 100% for cattle and approximately 40% for pigs (Non-patent Document 1).

  In general, livestock artificial insemination laboratories that produce and supply semen for artificial insemination of livestock have artificial sperm motility or viability that can be examined relatively easily using a microscope, an automatic cell analyzer, etc. Established as an acceptable standard for product quality of semen for insemination. When producing frozen semen, sperm motility or survival after freezing and thawing after diluting and cooling semen with diluted solution when producing semen motility or survival beyond a certain level after freezing and thawing Supplies a certain number of products to the market as acceptable.

  In general, a facility for producing semen for artificial insemination of mammals such as livestock is adjacent to a barn for feeding seedstock. Semen is diluted and cooled with diluent in the production facility immediately after collection, and refrigeration or cryopreservation delays sperm metabolic reaction, maintaining motility and viability, and thus fertility by artificial insemination. It is believed that. In order to improve the quality of frozen semen, artificial insemination straws (Patent Document 1) and dilution liquids during freezing (Patent Document 2) have been improved.

Japanese Patent No. 5738314 International Publication No. 2017/159737

Livestock artificial insemination class text, March 2016 revised version

  However, semen storage technology and transport technology are not sufficient when semen is collected and not immediately cooled or frozen after handling semen from a large number of breeding animals at one time or having facilities with production facilities in a remote location from a barn. The situation has not been established.

  That is, when semen from mammals such as livestock is stored with a semen preservation solution containing a pH buffer or saccharide, the sperm motility is significantly lower than that immediately after semen collection. In addition, sperm motility and viability decrease due to stress due to transport. For this reason, the conventional semen preservation solution and the transport method using the same have not been able to provide a sufficient effect for preventing sperm motility and viability reduction.

  An object of the present invention is to provide a semen preservation solution and a semen transport method for transporting high-quality semen used for artificial insemination to a remote place.

  In order to stably supply high-quality semen, the present inventor has studied a semen preservation solution that prevents a decrease in sperm motility observed during semen preservation.

  We focused on sperm metabolism and oxidative stress associated with storage and transport of semen. By adding a protein dephosphorylation inhibitor to the semen preservation solution, sperm motility was maintained at a high level. And found that it can be stored and transported.

  In addition, by replacing the air layer of the storage container with substances such as hydrogen gas, nitrogen gas, argon gas, mineral oil, etc., not only the sperm motility after transport but also the quality of frozen semen prepared after transport should be improved. The present invention was found.

The present invention includes the following inventions.
[1] A semen preservation solution comprising a protein dephosphorylation inhibitor.
[2] The semen preservation solution according to [1], wherein the semen is a semen derived from a mammal.
[3] The semen preservation solution according to [1] or [2], wherein the dephosphorylation inhibitor is an inhibitor that inhibits dephosphorylation of a sperm protein contained in the semen.
[4] The semen preservation solution according to [3], wherein the sperm protein is a protein having a mass of 50 to 100 kilodaltons (kDa) that is related to sperm motility and is determined by SDS-PAGE.
[5] The semen preservation solution according to any one of [1] to [4], wherein the dephosphorylation inhibitor is an acid phosphatase inhibitor, an alkaline phosphatase inhibitor, or a tyrosine phosphatase inhibitor.
[6] The semen preservation solution according to any one of [1] to [5], wherein the dephosphorylation inhibitor is imidazole, sodium fluoride, or sodium molybdate.
[7] The semen preservation solution according to any one of [1] to [6], wherein the content of the dephosphorylation inhibitor is 1 to 100 mM.
[8] The semen preservation solution according to any one of [1] to [7], further containing a sugar that is an energy source for sperm.
[9] The semen preservation solution according to [8], wherein the sugar is glucose, fructose, sucrose, lactose, raffinose, fructooligosaccharide, isomaltooligosaccharide, gentio-oligosaccharide, or galactooligosaccharide.
[10] The semen preservation solution according to any one of [1] to [9], wherein the osmotic pressure measured using an osmometer (Osmometer) is 230 to 400 mmol / kg.
[11] The semen preservation solution according to any one of [1] to [10], further comprising a buffer.
[12] The semen preservation solution according to any one of [1] to [11], wherein the buffer is tris (hydroxymethyl) aminomethane or citric acid.
[13] The semen preservation solution according to any one of [2] to [12], wherein the mammal is a cow, buffalo, pig, goat, horse, or sheep.
[14] A semen-containing storage solution comprising the semen storage solution according to any one of [1] to [13], which contains semen.
[15] The semen-containing storage solution according to [14], wherein the semen is a semen obtained by thawing cryopreserved semen.
[16] A method for transporting semen, characterized in that the semen-containing preservative solution according to [14] is transported in contact with mineral oil.
[17] A method for transporting semen, characterized in that the semen-containing preservative solution according to [14] is brought into contact with a gas containing an inert gas and transported in a state where the oxygen volume ratio is 20.1% or less. .
[18] The semen transport method according to [17], wherein the inert gas is hydrogen gas, nitrogen gas, or argon gas.

According to the present invention, sperm motility after storage can be maintained high by using a sperm protein dephosphorylation inhibitor. Moreover, not only sperm motility but also semen for artificial insemination that is excellent in survival and acrosome normal sperm rate can be supplied.
As a mechanism for maintaining sperm motility and viability in the present invention, it is possible to suppress metabolism of sperm during storage and transport by controlling protein phosphorylation such as dephosphorylation inhibition, and to relate to sperm motility and viability. It may be possible to maintain sperm motility after storage / transport and further freeze-thaw by controlling specific phosphorylation of proteins.

The graph which shows the motor sperm rate after preserve | saving with the preservation | save liquid for semen containing imidazole. (Significant difference between different signs (p <0.05, Tukey ’s test)) The graph which shows the motor sperm rate after preserve | saving with the preservation solution for semen containing sodium molybdate. (Significant difference between different signs (p <0.05, Tukey ’s test)) The graph which shows the motor sperm rate after preserve | saving with the preservation | save liquid for semen containing sodium fluoride. (Significant difference between different signs (p <0.05, Tukey ’s test)) The graph which shows the motile sperm rate immediately after thawing | decompression and the culture | cultivation of the frozen semen produced after the preservation | save with the stock solution for semen containing imidazole. (*: Significantly different between 0 mM added group and 10 mM added group (p <0.05, Dunnett's test)) The graph which shows the motor sperm rate immediately after thawing | decompression and the culture | cultivation of the frozen semen produced after the preservation | save with the preservation solution for semen containing sodium molybdate. The graph which shows the motor sperm rate immediately after thaw | melting and the culture | cultivation of the frozen semen produced after storage with the preservation solution for semen containing sodium fluoride. (*: Significantly different between 0 mM added group and 10 mM added group (p <0.05, Dunnett's test))

The fluorescence micrograph and graph which show the tyrosine phosphorylation state change of the bovine sperm protein before and behind preservation | save with TC liquid. (* Significant difference between before and after storage (p <0.05, Paired t-test)) The graph which shows the survival, the acrosome normal rate, and the motor sperm rate of the sperm preserve | saved with the TC liquid which added the dephosphorylation inhibitor cocktail. (* Indicates significant difference in test interval (p <0.05, Paired t-test)) The graph which shows the motile sperm rate of the frozen semen produced after the preservation | save with the TC liquid which added the dephosphorylation inhibitor cocktail immediately after thaw | melting and after culture | cultivation. (* Indicates significant difference in test interval (p <0.05, Paired t-test)) The graph which shows the motor sperm rate after preserve | saving with the preservation | save liquid for semen containing imidazole. (Significant difference between different signs (p <0.05, Tukey ’s test))

Western blot photograph and graph which show the tyrosine phosphorylation state change of the sperm protein preserve | saved with the preservation | save liquid for semen which preserve | saved with TC liquid and which added imidazole before a preservation | save. (* Indicates significant difference in test interval (p <0.05, Paired t-test)) The graph which shows the motor sperm rate and the average path | route speed | rate immediately after thaw | melting and the culture | cultivation of the frozen semen produced after filling mineral oil and preserve | saved. (* Indicates significant difference in test interval (p <0.05, Paired t-test)) Hydrogen gas _(H 2), nitrogen gas _(N 2), a graph showing the argon gas (Ar) or oxygen _{(O 2)} gas motile sperm rate after storage was filled with. (Significant difference between different signs (p <0.05, Tukey's test)) The rate of motile sperm immediately after thawing and after culturing of frozen semen prepared after filling with hydrogen gas (H ₂ ), nitrogen gas (N ₂ ), argon gas (Ar) or oxygen gas (O ₂ ) The graph which shows a maintenance rate (the value at the 6th culture time / the value at the 0th culture time) and the average route speed. (*: Significantly different from Air (p <0.05, Dunnett's test), Significantly different between different signs (p <0.05, Tukey's test)) The graph which shows the motor sperm rate after preserve | saving for 24 hours combining the addition of imidazole and nitrogen gas filling, and the motor sperm rate immediately after the melt | dissolution of the frozen semen produced after the preservation | save. (There is a significant difference between different signs (p <0.05, Paired t-test). The graph which shows the motor sperm rate immediately after conveying by courier using addition of imidazole and nitrogen gas filling together, and the motor sperm rate immediately after thawing | frozen of the frozen semen produced after storage, and after culture | cultivation. (* Indicates significant difference in test interval (p <0.05, Paired t-test))

  The present invention relates to a semen preservation solution containing a protein dephosphorylation inhibitor and a semen transport method using the same.

  In the present invention, mammal-derived semen can be used as the semen to be stored or transported. Mammals include domestic animals such as cattle, buffalo, pigs, goats, horses and sheep, pets such as dogs, cats and rabbits, endangered animals such as pandas, and experimental animals such as mice, hamsters and rats. Can be mentioned. The semen preservation solution of the present invention can be used for sperm preservation and sperm transport in birds and fish, in addition to mammals.

  The dephosphorylation inhibitor of the present invention is a compound that inhibits dephosphorylation of proteins contained in semen, particularly sperm proteins.

  A sperm protein whose dephosphorylation is inhibited is a protein associated with sperm motility. Preferably, a protein whose mass calculated | required by SDS-PAGE (Sodium Dodecyl Sulfate-PolyAcrylamide Gel Electrophoresis) is 50-100 kilodalton (kDa) is mentioned. In particular, a protein having a mass of 55 kDa and a protein having a mass of 77 kDa can be mentioned.

  Examples of dephosphorylation inhibitors include acid phosphatase inhibitors, alkaline phosphatase inhibitors, tyrosine phosphatase inhibitors and the like. Specific compounds that are dephosphorylation inhibitors are preferably imidazole, sodium orthovanadate, sodium dihydrogen pyrophosphate, disodium β-glycerophosphate, sodium tartrate, sodium molybdate, and sodium fluoride. Sodium and sodium molybdate are more preferred.

The content of the dephosphorylation inhibitor is preferably 1 to 100 mM, more preferably 1 to 20 mM, and particularly preferably 4 to 12 mM.
The sperm preservation solution of the present invention may contain a sugar component as an energy source for sperm. Examples of the sugar include oligosaccharides such as raffinose, fructooligosaccharide, isomaltooligosaccharide, gentiooligosaccharide, and galactooligosaccharide, in addition to glucose, fructose, sucrose, lactose and the like.
The concentration of the sugar component is preferably 1 to 150 mM.

  When fructose or lactose is used as the sugar component, the concentration of fructose is preferably 1 to 50 mM, and more preferably 5 to 30 mM. The concentration of lactose is preferably 1 to 100 mM, and more preferably 20 to 60 mM. When sucrose or lactose is used as the sugar component, the sucrose concentration is preferably 10 to 150 mM, more preferably 30 to 70 mM, and the lactose concentration is preferably 10 to 150 mM, more preferably 30 to 70 mM.

  When oligosaccharide is used as the sugar component, the concentration of oligosaccharide is preferably 20 to 40 g / L (w / v), more preferably 25 to 35 g / L (w / v).

The osmotic pressure of the sperm preservation solution of the present invention may be any osmotic pressure as long as the sperm motility and fertilization activities can be maintained, but is usually 230 to 3414 mmol / kg (Guthrie et al., Biology of Reproduction 67, 1811-1816 (2002)). The osmotic pressure can be calculated theoretically from the concentration of solute, the degree of dissociation, etc., but is measured using an osmometer (osmometer) in consideration of the interaction of substances constituting the solution.
The osmotic pressure of the sperm preservation solution of the present invention is preferably 230 to 400 mmol / kg, more preferably 250 to 350 mmol / kg.

The sperm preservation solution of the present invention may contain a buffer to achieve the desired pH. As the buffer, any buffer can be selected as long as it has a buffering action near neutrality. Buffers such as tris (hydroxymethyl) aminomethane, female, hepes, tes, and tricine, phosphate Examples include a buffer solution, a citrate buffer solution, an acetate buffer solution, and a carbonate buffer solution.
As the buffer, tris (hydroxymethyl) aminomethane is preferred.

  Moreover, in order to adjust to desired pH, an acid or an alkali can be used. Examples of the acid include hydrochloric acid, sulfuric acid, phosphoric acid, acetic acid, citric acid, formic acid, gluconic acid, lactic acid, oxalic acid, tartaric acid, ascorbic acid and the like. Examples of the alkali include alkali metal hydroxides such as sodium hydroxide, potassium hydroxide, calcium hydroxide, sodium carbonate, sodium hydrogen carbonate, potassium carbonate, and potassium hydrogen carbonate.

When tris (hydroxymethyl) aminomethane or citric acid is used as the buffer, the concentration of tris (hydroxymethyl) aminomethane is preferably 50 to 200 mM, more preferably 80 to 150 mM. The concentration of citric acid is preferably 20 to 80 mM, and more preferably 25 to 60 mM.
As a buffer for adjusting pH, citric acid is preferable.

  The semen preservation solution of the present invention may contain antibiotics to prevent bacterial growth. May be included. Antibiotics include penicillin, streptomycin, gentamicin, tylosin, lincomycin, spectinomycin, dibekacin and the like.

  The semen preservation solution of the present invention may contain soy lecithin or egg yolk in order to protect sperm from cooling stress. On the other hand, when the distribution of poultry eggs is restricted due to the occurrence of bird flu, etc., or when the production facility stops functioning in a situation where semen can be collected from breeding stock due to natural disasters, Since it cannot be stored, semen can be stored for a long time without cooling and transported to a remote location.

  The semen-containing storage solution of the present invention is obtained by diluting semen with the semen storage solution. The dilution factor of semen is not particularly limited as long as it does not affect the viability of sperm, but is preferably 1.5 times to 10 times, more preferably 2 to 4 times.

The semen transport method of the present invention is not particularly limited as long as it does not affect the viability of sperm, but semen or a semen-containing storage solution is brought into contact with mineral oil or a gas containing more inert gas than air. It is preferable to transport in a state.
The mineral oil is not particularly limited as long as it is a purified paraffinic one, but is preferably used as a medical ointment base.
Examples of the inert gas include hydrogen gas, nitrogen gas, and argon gas.

  In the semen transporting method of the present invention, semen is preferably transported in a two-layer structure comprising a semen-containing preservation solution and mineral oil or inert gas.

  Hereinafter, the present invention will be specifically described with reference to examples. The present invention is not construed as being limited to these.

<1. Preparation of stock solution for semen>
Each substance shown in the following Table 1 was weighed so as to have the composition shown in Table 1, and distilled water was added to 100 mL to prepare 100 mL of triskelic acid buffered sugar solution (TC solution).

  To the TC solution, 0.1 mM imidazole (manufactured by Wako Pure Chemical Industries), sodium molybdate (manufactured by Sigma Aldrich), or sodium fluoride (manufactured by Wako Pure Chemical Industries) as a dephosphorylation inhibitor, A stock solution for semen was prepared by adding 1 mM or 10 mM.

<2. Preparation of semen-containing stock solution>
1 mL of fresh semen collected from 5 Holstein bulls (5-9 years old, 1 ejaculation each) was placed in a polyethylene round bottom tube (Corning), and then the above semen preservation solution (25 ° C.) 1 mL was added and diluted at a ratio of 1: 1 (2-fold dilution).
The obtained semen-containing stock solution was stored for 24 hours while shaking at a shaking speed of 30 rpm using a desktop shaking thermostat (manufactured by Taitec Co., Ltd.) with a water temperature set at 18 ° C.

Example 1 (Measurement of sperm motility)
Assuming that all the semen cannot be cooled immediately or transported from a barn to a facility with advanced manufacturing technology or expensive manufacturing equipment, store it for 24 hours, and then store the sperm concentration in the TC solution. The semen-containing stock solution was diluted to about 20 million / mL. After mounting the diluted semen on a slide chamber (Leja) with a thickness of 20μm preheated to 38 ° C, the sperm motility is analyzed by a sperm motility analysis system (Ceros, Hamilton Thorne) Was measured.
The measurement results are shown in FIGS. A dephosphorylation inhibitor concentration-dependent improvement in the rate of motile sperm was observed.

Example 2 (Moving sperm rate immediately after thawing and after culturing of frozen semen)
[Preparation of frozen semen]
The semen stored for 24 hours was washed twice with TC solution. The washed semen was frozen by the method described on pages 310 to 311 of Non-Patent Document 1, and stored in liquid nitrogen until the culture test was performed.
[Semen thawing and sperm culture]
The frozen semen was thawed with hot water at 38 ° C. over 15 seconds. Sperm contained in the melted semen was washed using the sperm washing solution described in Example 1 of Patent Document 1. While culturing at 38 ° C. after washing, sperm motility was analyzed with a sperm motility analyzer (CASA) immediately after thawing, 0 hours, 3 hours, and 6 hours, and the rate of motility was measured.
The measurement results are shown in FIGS. It was observed that the addition of dephosphorylation inhibitors improved the rate of motile sperm.

Example 3 (Confirmation of sperm tyrosine phosphorylation state before and after storage using immunostaining)
Semen was collected from 5 Holstein bulls (5-7 years old, 1 ejaculation each), and sperm tyrosine phosphorylation status was confirmed using semen stored for 24 hours in TC solution and semen before storage did.
The sperm in the semen was washed twice with phosphate buffered saline (PBS). The washed sperm was fixed with 1% formaldehyde (Wako Pure Chemical Industries, Ltd.)-PBS for 15 minutes at room temperature, and 1 mL of 200 mM glycine (Wako Pure Chemical Industries, Ltd.) was added to stop the reaction. The supernatant was removed by centrifugation and washed with PBS twice to recover the fixed sperm. The washed sperm was smeared on a slide glass (manufactured by Matsunami Glass Kogyo Co., Ltd.), air-dried, and used for immunostaining.

The smeared sperm was treated with 0.1% Triton X-100 (SERVA Electrophoresis) -PBS for 15 minutes at room temperature, and blocked with 3% BSA (Wako Pure Chemical Industries) -PBS for 30 minutes. It was treated with an anti-tyrosine phosphorylated antibody (Clone 4G10; manufactured by Merck) diluted 1000 times with PBS for 1 hour. After washing 3 times with PBS, it was treated with Alexa488-labeled anti-mouse IgG antibody (manufactured by Thermo Fisher Scientific) diluted 300 times for 1 hour. After washing with PBS three times, a cover glass (manufactured by Matsunami Glass Kogyo Co., Ltd.) was applied and observed with an upright fluorescent microscope system (DM5500 B; manufactured by Leica Microsystems).
100 sperm of each individual was counted, and the number of sperm having high fluorescence intensity in the middle part of the sperm among phosphorylated tyrosine (pY) positive sperm was compared.
The observation results are shown in FIG.

Sperm after storage for 24 hours confirmed a tendency for the intensity of the phosphorylated tyrosine (pY) positive signal to decrease.
During storage, the number of sperm from which pY was dephosphorylated increased, suggesting that inhibition of dephosphorylation contributes to maintenance of sperm motility.

Example 4 (Effect of Dephosphorylation Inhibitor Cocktail on Sperm Viability, Acrosome Normality, and Sperm Motility after Storage)
Semen was collected from 6 Holstein bulls (7-9 years old, 1 ejaculation each), and Phosphatase Inhibitor Cocktail Solution II (hereinafter dephosphorylation inhibitor cocktail) manufactured by Wako Pure Chemical Industries, Ltd. was inhibited from dephosphorylation. Except for the point used as an agent, a semen preservation solution was prepared in the same manner as in 1 and 2 above to obtain a semen-containing preservation solution. Using the obtained semen-containing storage solution, sperm survival, acrosome normality, and sperm motility after 24 hours storage were confirmed.

[Measurement of survival and acrosome normality]
One million spermatozoa were collected from the semen stored for 24 hours, and FITC-labeled peanut lectin (Sigma, PNA) and propidium iodide (Sigma, PI) were each 2 μg / ml in the diluent described in Patent Document 1. Double staining was carried out at 25 ° C. for 10 minutes using a solution dissolved in mL. After staining, non-stained spermatozoa were counted as viable and acrosome normal spermatozoa with a cell analyzer (Quanta SC; manufactured by Beckman Coulter), the percentage was calculated, and the measurement of the viable and acrosome normal rate was measured.
The measurement results are shown in FIG.
Sperm survival and acrosome prevalence were improved when stored in a semen stock containing a dephosphorylation inhibitor cocktail compared to a control stored in a stock containing no dephosphorylation inhibitor cocktail.
[Measurement of motor sperm rate]
The semen after storage for 24 hours was diluted with a TC solution so that the sperm concentration was about 20 million / mL. Mount the diluted semen on a slide chamber (Leja) with a thickness of 20μm preheated to 38 ° C, then analyze the sperm motility with a sperm motility analysis system (Ceros, Hamilton Thorne) Was measured.
The measurement results are shown in FIG.

  Sperm motility was improved when stored in a semen stock containing a dephosphorylation inhibitor cocktail as compared to a control stored in a stock containing no dephosphorylation inhibitor cocktail.

[Moving sperm rate and average sperm speed after freezing and thawing semen after storage]
After freezing and thawing the semen stored for 24 hours by the method described in Example 1, it was cultured for 0 to 6 hours, and the rate of motile spermatozoa and the average pathway speed were measured.
Motility sperm rate and average path velocity are indicators of livestock semen quality, and high values indicate that the product is likely to pass.
The measurement results are shown in FIG.
Sperm motility after freezing and thawing improved when stored in a semen storage solution containing a dephosphorylation inhibitor cocktail, compared to a control group stored in a storage solution without a dephosphorylation inhibitor cocktail.

Example 5 (Influence of imidazole on sperm motility and sperm viability after storage)
A semen-containing stock solution was prepared in the same manner as in 1 and 2 above except that imidazole was added to 0, 2, 4, 6, 8, or 10 mM to obtain a semen-containing stock solution. Using the obtained semen-containing storage solution, sperm motility after storage for 24 hours was measured.
The measurement results are shown in FIG. 10 (n = 14).
Motile sperm rate increased in an imidazole concentration-dependent manner and was highest at an imidazole concentration of 10 mM.
Moreover, compared with the control group (0 mM) preserve | saved using the semen preservation solution which does not contain imidazole, the test group preserve | saved using the semen preservation solution containing 10 mM imidazole had a high motor sperm rate ( FIG. 1).

Furthermore, in order to confirm the influence of sperm on the tyrosine phosphorylation state, sperm preserved using a semen preservation solution containing 10 mM imidazole was compared in phosphorylated tyrosine oxidation state by Western blotting. As a result, the emission intensity of bands near 77 kilodalton (kDa) and 55 kDa was high in the test section (FIG. 11, n = 8). In FIG. 11, “on the day” indicates the result when the semen before storage is used.
This result suggests that imidazole inhibits tyrosine dephosphorylation of proteins around 77 kDa and 55 kDa.

The Western blot was performed according to the following procedure.
1. Semen before and after storage was washed twice with PBS, diluted with 4x Laemmli Sample Buffer (200 mM Tris, 8% SDS, 24% β-mercaptoethanol, 40% glycerol, 1% bromophenol blue) at 95 ° C For 5 minutes.
2. For SDS-PAGE, electrophoresis was performed for 1 hour and a half at 100 mm using a precast gel for electrophoresis (Miniprotian TGX gel 4-20%, manufactured by Biorad).
3. Thereafter, transcription onto a PVDF membrane (minitrans blot cell, manufactured by Biorad) was performed at 100 mm for 1 hour. The transferred PVDF membrane was treated with 3% BSA-added TBS-T (25 mM Tris, 150 mM sodium chloride, 0.1% Tween 20, pH 7.6) for 1 hour at room temperature to perform a blocking reaction.
4). Subsequently, the primary antibody reaction was performed at 4 ° C. overnight. As the primary antibody, an anti-phosphotyrosine antibody (Clone 4G10; manufactured by Merck) was used.
5. After washing with TBS-T, the secondary antibody reaction was carried out for 1 hour at room temperature. As the secondary antibody, an HRP-labeled antibody (Jackson ImmunoResearch) of an animal species of the primary antibody was diluted with TBS-T. The treated PVDF membrane was washed with TBS-T, then subjected to a luminescence reaction using ECL prime (manufactured by GE lifescience), and detected using a chemirmi imaging apparatus (MultiImager II; manufactured by Bio tools Inc.).
6). Luminescence intensity of the obtained bands around 77 kDa and 55 kDa was measured using image processing software (ImageJ, National Institutes of Health, USA), and the relative values of the control group and the test group with respect to “the same day” were compared.

Example 6 (Effect of mineral oil on sperm motility after semen storage)
2 mL of semen collected from 6 Holstein bulls (5-9 years old, 1 ejaculation each) was dispensed into a round bottom tube made of polystyrene (manufactured by Corning), and 2 mL of TC solution was added to make 1: 1 Diluted in proportion (2-fold dilution).
Thereafter, the space (air layer) other than the semen-containing storage solution in the round bottom tube was filled with mineral oil (D05042; manufactured by Yoshida Pharmaceutical) and sealed. Place the sealed tube on its long side on a shaker (NR-30; manufactured by Taitec) installed in a chromatography refrigerator (REC-5004; manufactured by Revco) set at a temperature of 18 ° C. It was installed so that it could be shaken in parallel, and stored for 24 hours while shaking at a shaking speed of 120 rpm.

The semen after storage for 24 hours was frozen and thawed by the method described in Example 1. Immediately after thawing, sperm motility was analyzed with a sperm motility analyzer (CASA) at 0 hours, 3 hours, and 6 hours of culture, and the motility sperm rate and average path velocity were measured.
The measurement results are shown in FIG.
When stored in a sealed state filled with mineral oil in the air layer, an improvement in sperm motility after thawing of frozen semen prepared after storage was observed.

Example 7 (Effect of gas filling on sperm motility after semen storage)
1 mL of semen collected from 5 Holstein bulls (5-9 years old, 1 ejaculation each) was dispensed into a round bottom tube made of polystyrene (manufactured by Corning), and 1 mL of TC solution was added to make 1: 1 Diluted in proportion (2-fold dilution).
After that, the space (air layer) other than the semen-containing preservative in the round bottom tube is filled with purified air, hydrogen gas, nitrogen gas, argon gas or oxygen gas using a gas spray can made by Taiyo Nippon Sanso And immediately sealed. Place the sealed tube on its long side on a shaker (NR-30; manufactured by Taitec) installed in a chromatography refrigerator (REC-5004; manufactured by Revco) set at 18 ° C. It was installed so that it could be shaken in parallel, and stored for 24 hours while shaking at a shaking speed of 120 rpm.

The results of measuring sperm motility after storage for 24 hours by the method described in Example 1 are shown in FIG. 13 (n = 5).
It was observed that when the air layer was filled with oxygen gas and stored for 24 hours, the sperm motility after storage decreased.
When the air layer was filled with argon gas, hydrogen gas, nitrogen gas, or argon gas and stored for 24 hours, it was observed that the sperm motility after storage was improved as compared with the case of filling with purified air.

The semen after storage for 24 hours was frozen and thawed by the method described in Example 1. Immediately after thawing, sperm motility was analyzed with a sperm motility analyzer (CASA) at 0 hours, 3 hours, and 6 hours of culture, and the motility sperm rate and average path velocity were measured.
The measurement results are shown in FIG. 14 (n = 5).
It was observed that when the air layer was filled with oxygen gas and stored for 24 hours, both the sperm motility and the motility maintenance rate after thawing the frozen sperm produced after storage were reduced.
When the air layer was filled with argon gas, hydrogen gas, nitrogen gas, or argon gas and stored for 24 hours, the sperm motility and motility maintenance rate after thawing the frozen sperm produced after storage was filled with purified air. An improvement was observed compared to the case.

Example 8 (Influence of combined use of imidazole and nitrogen gas filling on sperm motility after storage)
1 mL of semen collected from 8 Holstein bulls (5-9 years old, 1 ejaculation each) is dispensed to a round bottom tube made of polystyrene (manufactured by Corning) for semen containing TC solution or 10 mM imidazole 1 mL of the stock solution was added and diluted at a ratio of 1: 1 (2-fold dilution).
Thereafter, the space (air layer) other than the semen-containing preservative solution in the round bottom tube was filled with purified air or nitrogen gas using a gas spray can made by Taiyo Nippon Sanso and immediately sealed. Place the sealed tube on its long side on a shaker (NR-30; manufactured by Taitec) installed in a chromatography refrigerator (REC-5004; manufactured by Revco) set at 18 ° C. It was installed so that it could be shaken in parallel, and stored for 24 hours while shaking at a shaking speed of 120 rpm.

  The result of measuring the sperm motility after storage for 24 hours by the method described in Example 1 is shown in FIG. “Control” is diluted with TC solution and filled with purified air and sealed and stored. “Imidazole” is diluted with stock solution for semen containing 10 mM imidazole and then filled with purified air and sealed. -Stored, "Nitrogen gas" is diluted with TC solution, filled with nitrogen gas, sealed and stored, "Combination" is diluted with semen storage solution containing 10 mM imidazole And then filled and sealed with nitrogen gas.

The percentage of motile spermatozoa after diluting semen with a semen preservation solution containing 10 mM imidazole, filling the air layer with nitrogen gas and storing for 24 hours was higher than when each was treated alone.
The semen after storage for 24 hours was frozen and thawed by the method described in Example 1. Immediately after thawing, sperm motility was analyzed with a sperm motility analyzer (CASA) at 0 hours, 3 hours, and 6 hours of culture, and the motility sperm rate and average path velocity were measured.
The measurement results are shown in FIG.
When the sperm was washed and stored frozen after 24 hours of storage, the motile sperm rate during culturing after thawing was highest in the combination group.

Example 9 (Influence of combined use of imidazole and nitrogen gas filling on sperm motility after delivery by courier)
Divide 2 mL of semen collected from 4 Holstein bulls (5-9 years old, 2 ejaculations each) connected to the Morioka Bull Center (Morioka City) into polystyrene round-bottom tubes (Corning) Then, 2 mL of a semen stock solution containing TC solution or 10 mM imidazole was added and diluted at a ratio of 1: 1 (2-fold dilution).
The round bottom tube was further sealed in a 50 mL conical tube (Corning) and placed in a thermos (MWO-K150; Tiger Thermos) adjusted to a water temperature of 18 ° C. The thermos was packed in polystyrene foam and transported to the Livestock Improvement Technology Laboratory (Maebashi City) by courier (transport distance was about 600 km).
The result of having measured the sperm motility after conveyance by the method of Example 1 is shown to A of FIG.

The test group had a higher rate of motor sperm compared to the control group.
The semen after transportation was frozen and thawed by the method described in Example 1. Immediately after thawing, sperm motility was analyzed with a sperm motility analyzer (CASA) at 0 hours, 3 hours, and 6 hours of culture, and the rate of motility was measured.
The measurement results are shown in FIG.
The test group showed a higher rate of motile spermatozoa during culture than the control group.

  When semen is stored using the semen preservation solution of the present invention, motility of sperm can be maintained at a high level, so that high-quality semen can be transported to facilities located in remote locations.

Claims (15)

  A preservation solution for semen characterized by containing a protein dephosphorylation inhibitor.   The semen preservation solution according to claim 1, wherein the semen is a semen derived from a mammal.   The semen preservation solution according to claim 1 or 2, wherein the dephosphorylation inhibitor is an inhibitor that inhibits dephosphorylation of a sperm protein contained in the semen.   The sperm preservation solution according to claim 3, wherein the sperm protein is a protein associated with sperm motility and having a mass of 50 to 100 kilodaltons (kDa) as determined by SDS-PAGE.   The semen preservation solution according to any one of claims 1 to 4, wherein the dephosphorylation inhibitor is an acid phosphatase inhibitor, an alkaline phosphatase inhibitor, or a tyrosine phosphatase inhibitor.   The semen preservation solution according to any one of claims 1 to 5, wherein the dephosphorylation inhibitor is imidazole, sodium fluoride, or sodium molybdate.   The semen preservation solution according to any one of claims 1 to 6, wherein the content of the dephosphorylation inhibitor is 1 to 100 mM.   The semen preservation solution according to any one of claims 1 to 7, further comprising a sugar that serves as an energy source for sperm.   The preservation | save liquid for semen as described in any one of Claims 1-8 whose osmotic pressure measured using an osmometer (Osmometer) is 230-400 mmol / kg.   The semen preservation solution according to any one of claims 1 to 9, comprising a buffer.   The semen preservation solution according to any one of claims 2 to 10, wherein the mammal is a cow, buffalo, pig, goat, horse or sheep.   A semen-containing storage solution comprising the semen storage solution according to any one of claims 1 to 11, which contains semen.   A semen transporting method comprising transporting the semen-containing preservation solution according to claim 12 in contact with mineral oil.   A method for transporting semen, wherein the semen-containing preservative solution according to claim 12 is brought into contact with a gas containing an inert gas and transported in a state where the oxygen volume ratio is 20.1% or less.   The semen transport method according to claim 14, wherein the inert gas is hydrogen gas, nitrogen gas or argon gas.

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