JP3947265B2 - Sperm cold injury prevention liquid and cell cold injury prevention liquid - Google Patents

Description

[0001]
BACKGROUND OF THE INVENTION
The present invention relates to an injury prevention solution useful for sperm cryopreservation or a cell injury preservation solution useful for cryopreservation of cells. In particular, the present invention relates to a sperm cold injury prevention solution effective for low-temperature preservation of porcine sperm.
[0002]
[Prior art]
Conventionally, several alcohols that have an effect of preventing freezing injury of cells are known as an injury prevention solution for sperm cryopreservation, and a typical example is glycerin. Moreover, egg yolk and its components are also used as an injury prevention liquid for sperm cryopreservation, and these are also known to have an effect of preventing cold shock caused by rapid cooling. It is also known that aromatic compounds such as naphthalene and BHT (2,6-di-tert-butyl-p-cresol) have the effect of preventing cold shock of sperm and preventing the physiological function thereof. (Bamba, K. and Miyagawa, N., Cryobiology, 29, pp. 533-536, 1992).
[0003]
[Problems to be solved by the invention]
However, none of the above-mentioned injury prevention liquids have sufficient protection performance, and in particular, BHT has a high action of suppressing sperm motility, which may impair the physiological function of sperm.
Accordingly, an object of the present invention is to provide an injury prevention solution useful for cryopreservation while substantially maintaining the physiological function of sperm. Another object of the present invention is to provide an injury-preventing solution useful for cryopreservation while substantially maintaining the physiological functions of cells of animals, plants, and the like.
[0004]
[Means for Solving the Problems]
What achieves the above object is a sperm cold injury prevention liquid containing a long-chain alcohol having 10 to 14 carbon atoms and a lipid.
Moreover, what achieves the said objective is the low-temperature cell injury prevention liquid containing C10-14 long-chain alcohol, lipid, and serum albumin.
The long chain alcohol is preferably lauryl alcohol. The lipid is, for example, vegetable oil or animal oil. The lipid is, for example, any of olive oil, soybean oil, egg yolk, milk, and lecithin. The lipid is preferably α-tocopherol. And it is preferable that the said sperm low temperature injury prevention liquid is ultrasonically treated.
[0005]
DETAILED DESCRIPTION OF THE INVENTION
The sperm cold injury prevention liquid of the present invention contains a long-chain alcohol having 10 to 14 carbon atoms and a lipid. For this reason, the physiological function of a sperm can be substantially hold | maintained, even after applying the cold shock by rapidly cooling a sperm to 2-3 degreeC.
Examples of sperm that can be suitably cooled and stored with the injury-preventing solution of the present invention include spermatozoa derived from mammals such as humans, monkeys, pigs, cows, and horses. In particular, it is effective against pig sperm.
[0006]
Long chain alcohols having 10 to 14 carbon atoms are decyl alcohol (carbon number 10), undecyl alcohol (carbon number 11), lauryl alcohol (carbon number 12), dodecyl alcohol (carbon number 13), myristyl alcohol (carbon number 14). ). The long-chain alcohol may have a side chain, but a straight-chain alcohol is preferred. In addition, the effect of this invention was not acquired in C9 nonyl alcohol.
[0007]
As lipids, vegetable oils such as olive oil and soybean oil, animal oils such as egg yolk, milk, lecithin and squalane, mineral oils such as mineral oil, liquid paraffin, and α-tocopherol can be used. In particular, α-tocopherol is preferable.
[0008]
A sperm cold injury prevention liquid can be produced by adding a long-chain alcohol and a lipid to a diluent and sonicating.
As a diluent, BTS diluent (Pursel, VG and Johnson, LA, J. Anim. Sci., 40, pp. 99-102, 1975, component (mg / 100 ml): D-glucose: 3700; sodium citrate NaHCO ₃ , EDTA-2Na: 125; KCl: 75; antibiotics: 10 (for example, amino sugar antibiotics such as gentamicin and amikacin, dibekacin sulfate, etc. are used).
[0009]
The long chain alcohol concentration of 10 to 14 carbon atoms in the injury preventing liquid of the present invention is preferably about 0.01 to 0.1%, and the lipid is preferably about 0.1 to 1%. In particular, the concentration of the long-chain alcohol having 10 to 14 carbon atoms, which is an active ingredient in the injury preventing liquid, is preferably about 0.02%, and the lipid is preferably about 0.4%.
[0010]
The low-temperature disorder preventing solution of the present invention is adjusted so that, for example, the sperm concentration of the collected semen is about 4 × 10 ⁸ per ml, and the injury preventing solution of the present invention is adjusted to an appropriate concentration. It is particularly effective when added and stored at a low temperature of about 2 to 10 ° C., preferably about 5 ° C. For adjusting the sperm concentration of semen, seminal plasma, physiological saline, or the like can be used as a diluent. As a method for diluting the semen, for example, the concentrated part semen and the diluted part semen are fractioned and collected, and the concentrated part semen is diluted with the seminal plasma obtained by centrifuging the diluted part semen at about 1200 × G for 15 minutes. The method of doing can be mentioned. The concentration adjusting step may be performed at 20 to 30 ° C, preferably 25 ° C.
[0011]
The inventor took long-chain alcohol in a test tube, added various basic diluents, and sonicated to dissolve it. Porcine semen (400 million / ml) was diluted 1: 1, rapidly cooled to 2-3 ° C., allowed to stand for 10 minutes, treated with cold shock (CS), and examined for sperm vitality and cap morphology. Lauryl alcohol (LA) showed toxicity only when added to a BTS diluent that did not contain lipids, but the diluent that added lipids at the same time showed a very good CS prevention effect. Almost the same effect was observed in the combination of vegetable oils (olive oil, soybean oil), animal oils (egg yolk, milk, lecithin, etc.) or alpha (α-) tocopherol and LA that have no harmful effect on sperm (combination) Example: 0.4% olive oil + 0.02% LA). The same effect as LA was observed with alcohols having 10 to 14 carbon atoms, but 9 was ineffective.
From this, it was found that long-chain alcohols can be combined with a small amount of lipids to mask their toxicity and obtain a low-temperature injury prevention effect.
[0012]
Surfactant Orvus Es Paste (OEP) is known to improve the freezing resistance of sperm when added to a dilute solution containing egg yolk. For this reason, only a limited number of diluents can be used. In contrast, long-chain alcohols have the advantage that only a very small amount of lipid is required to mask toxicity. The combination of egg yolk, milk, and tocopherol, which can be expected to improve sperm viability, masks LA toxicity and lowers the temperature. Its application can be expected in that it can provide an injury prevention effect. The effectiveness of LA was confirmed even when the porcine sperm was stored at 5 ° C or frozen.
[0013]
Next, the cell cold injury preventing solution of the present invention will be described.
The cell cold injury prevention liquid of the present invention contains a long-chain alcohol having 10 to 14 carbon atoms, a lipid, and serum albumin. For this reason, the physiological function of the cell can be substantially retained even after a cold shock is applied by rapidly cooling the cell to 2 to 3 ° C.
[0014]
Examples of the cells that can be suitably cooled and stored with the injury-preventing solution of the present invention include sperm, eggs, erythrocytes, platelets, stem cells and the like derived from mammals such as humans, monkeys, pigs, cows, and horses. In particular, it is effective against sperm, particularly pig sperm.
Long chain alcohols having 10 to 14 carbon atoms are decyl alcohol (carbon number 10), undecyl alcohol (carbon number 11), lauryl alcohol (carbon number 12), dodecyl alcohol (carbon number 13), myristyl alcohol (carbon number 14). ). The long-chain alcohol may have a side chain, but a straight-chain alcohol is preferred.
[0015]
As lipids, vegetable oils such as olive oil and soybean oil, animal oils such as egg yolk, milk, lecithin and squalane, mineral oils such as mineral oil, liquid paraffin, and α-tocopherol can be used. In particular, α-tocopherol is preferable. Most preferred is dl-α-tocopherol.
[0016]
As serum albumin, bovine serum albumin is suitable. By adding this serum albumin, the motility of the cells can be improved and the adhesion of the cells to the test tube wall can be suppressed, and the workability is improved.
The cell cold injury prevention liquid can be produced by adding a long-chain alcohol and a lipid to a diluent and sonicating.
[0017]
As a diluent, BTS diluent (Pursel, VG and Johnson, LA, J. Anim. Sci., 40, pp. 99-102, 1975, component (mg / 100 ml): D-glucose: 3700; sodium citrate NaHCO ₃ , EDTA-2Na: 125; KCl: 75; antibiotics: 10 (for example, amino sugar antibiotics such as gentamicin and amikacin, dibekacin sulfate, etc. are used).
[0018]
The long chain alcohol concentration of 10 to 14 carbon atoms in the injury preventing liquid of the present invention is preferably about 0.01 to 0.1%, and the lipid is preferably about 0.1 to 1%. In particular, the concentration of the long-chain alcohol having 10 to 14 carbon atoms, which is an active ingredient in the injury preventing liquid, is preferably about 0.02%, and the lipid is preferably about 0.4%. The serum albumin concentration is preferably about 0.2 to 1%.
[0019]
【Example】
(1) Semen collection Rich fractional semen and dilute fractional semen were collected from large Yorkshire, Landrace, and Duroc species by palm compression. After collecting semen, it was kept at about 30 ° C. and subjected to the following operation within 2 hours.
[0020]
(2) Sperm concentration adjustment After measuring the sperm concentration of the concentrated semen with a toma hemocytometer, the sperm obtained by centrifuging the diluted semen for 15 minutes at 1200 × G was added, and the sperm concentration was 4 × 10 cells / Adjusted to ml.
[0021]
(3) BTS (Pursel, VG and Johnson, LA, J. Anim. Sci., 40, pp. 99-102, 1975, component (mg / 100 ml): D-glucose : 3700; sodium _{citrate: 600; NaHCO 3, EDTA-} 2Na: 125; KCl: 75; gentamicin: 10) was used.
The sperm of the present invention is added to BTS so that the long-chain alcohol concentration of 10 to 14 carbon atoms is 0.02% and the lipid concentration is 0.4%, sonicated and dissolved (dispersed). A low temperature failure prevention liquid was prepared.
And the sperm low temperature disorder | damage | failure prevention liquid of this invention was diluted so that it might become a ratio of 1: 1 with an original semen at 37 degreeC. While incubating at 37 ° C. for 5 minutes, it was shaken vigorously by hand for 1 minute.
[0022]
(4) Cold shock 1 ml of semen at 37 ° C. was taken into a small glass test tube (10 × 60 mm), transferred directly to a constant temperature water bath at 2-3 ° C., and incubated for 10 minutes to give a cold shock.
[0023]
(5) Evaluation of sperm Before and after the cold shock, the sperm motility rate and the normal cap rate were examined by the following methods. Sperm motility: 10 μl of semen was placed on a slide glass coated with anhydrous caffeine (about 20 μg / ml), covered with a cover glass (18 × 18 mm), and the sperm motility was controlled under a phase contrast microscope controlled at 37 ° C. The percentage was determined. Normal cap rate: Semen was diluted with a BTS solution containing 1% (v / v) formalin at 37 ° C. and observed under a phase contrast microscope (× 750). 200 spermatozoa were observed in each section, and classified into normal caps and abnormal caps, and the ratio of normal cap spermatozoa (%) was defined as the normal cap rate.
Several experiments were performed using the above method.
[0024]
(Experiment 1)
Lauryl alcohol was used as the long-chain alcohol, and egg yolk PC was used as the lipid. As a comparative example, comparison was made with a combination of naphthalene, decane, egg yolk PC and 0.2% OEP. Naphthalene was dissolved in dimethyl sulfoxide (DMSO) and added to BTS at 2% (v / v). The raw semen was incubated with an equal amount of naphthalene-added BTS solution at 37 ° C. for 5 minutes. The sperm treatment concentration of naphthalene was 0.5 mM. The result was as shown in FIG.
[0025]
(Experiment 2) Examination of utilization for storage at 5 ° C. Preservation at 5 ° C. was compared with the BHT method in a diluted solution combining egg yolk, milk, olive oil, tocopherol and lauryl alcohol. Egg yolk and milk were added so that their fat was 0.4%. The result was as shown in FIG.
The treatment method was the same as that for cold shock, diluted twice with BTS, cooled to 5 ° C. over about 2 hours, and stored for 10 days.
In any of the added groups, the motility rate and the normal cap rate were significantly higher than those in the non-added group, and the survival rate was significantly higher in the α-tocopherol group. Among the fats and oils, the combination with α-tocopherol was particularly excellent, and the storage stability comparable to that of the BHT treated area was shown. This is thought to be due to the involvement of both antioxidant effects.
[0026]
(Experiment 3) Effects of various long-chain alcohols Next, fats and oils were fixed to olive oil, and various alcohols were combined to compare the effects of cold shock prevention. The result was as shown in FIG.
As a result, the alcohol having 10 to 15 carbon atoms showed a significantly higher value in both the motility and cap indicators than the non-added group. Particularly, those having 11 to 14 carbon atoms were excellent, and the normality rate of the cap was significantly higher than that of naphthalene, which is the most effective protective compound among aromatic compounds.
[0027]
(Experiment 4) Use for Cryopreservation BF5 diluted solution is widely used for cryopreserving swine semen, and 0.5% of surfactant ORVUS ES PASTE (OEP) is added to this diluted solution. In this experiment, BF5 excluding OEP was used as a basic diluent, and a comparison was made between the 0.5% EP addition group and the non-addition group as in the original method with the lauryl alcohol addition group of various concentrations. Freezing was performed by the pellet method in the presence of 2.5% glycerin at the final concentration. The result was as shown in FIG. From this, the group to which 0.05% lauryl alcohol was added and the 0.5% OEP group showed values significantly higher than the non-added group in any index (survival rate was 5% risk).
[0028]
Based on the above results, lauryl alcohol has excellent cold shock when combined with various oils and fats (olive oil and other vegetable oils, various neutral fats, egg yolk, lecithin, milk, tocopherol, squalane, mineral oil, liquid paraffin, etc.) It was found to show a preventive effect.
Further, it was found that the effect of preventing cold shock was recognized by the combination of a linear alcohol having 9 to 15 carbon atoms and olive oil, and the effect of alcohol having 11 to 14 carbon atoms was particularly high.
Furthermore, it was found that the storage stability at 5 ° C. was enhanced by a combination of lauryl alcohol and α-tocopherol.
[0029]
Next, an example of the cell cold injury prevention liquid will be described.
BTS (Pursel, VG and Johnson, LA, J. Anim. Sci., 40, pp. 99-102, 1975, component (mg / 100 ml): D-glucose: 3700; sodium citrate: 600 NaHCO ₃ , EDTA-2Na: 125; KCl: 75; gentamicin: 10) were used.
[0030]
Add to BTS so that the lauryl alcohol concentration is 0.02%, the α-tocopherol concentration is 0.4%, and the bovine serum albumin concentration is 0.3%, sonicated, and dissolved (dispersed). Then, the cell cryogenic injury prevention solution of the present invention was prepared.
Then, the cell cold injury prevention solution of the present invention was diluted to a raw semen prepared in the same manner as in the above-described example so that the ratio was 1: 1 with the raw semen at 37 ° C. While incubating at 37 ° C. for 5 minutes, it was shaken vigorously by hand for 1 minute.
A cold shock was applied by taking 1 ml of semen at 37 ° C. into a small glass test tube (10 × 60 mm), transferring directly to a constant temperature water bath at 2-3 ° C., and incubating for 10 minutes.
[0031]
Then, using the same method as the sperm evaluation described above, when compared with the one without the addition of bovine serum albumin, the one with the addition of bovine serum albumin had better cell motility. Further, the number of cells adhering to the test tube wall was small, and the workability was good.
[0032]
【The invention's effect】
The sperm cold injury prevention liquid of the present invention contains a long-chain alcohol having 10 to 14 carbon atoms and a lipid. For this reason, the physiological function of a sperm can be substantially hold | maintained, even after applying the cold shock by rapidly cooling a sperm to 2-3 degreeC.
The cell cold injury prevention liquid of the present invention contains a long-chain alcohol having 10 to 14 carbon atoms, a lipid, and serum albumin. For this reason, the physiological function of the cell can be substantially retained even after a cold shock is applied by rapidly cooling the cell to 2 to 3 ° C. Furthermore, since serum albumin is contained, it is possible to improve the motility of the cells and to suppress the adhesion of the cells to the instrument used for adjustment, for example, the test tube wall, and the workability is improved.
[Brief description of the drawings]
FIG. 1 is a graph showing the results of an experiment using a sperm low-temperature damage prevention liquid of an example of the present invention and a sperm low-temperature damage prevention liquid of a comparative example.
FIG. 2 is a graph showing the results of an experiment using the sperm low-temperature damage prevention liquid of an example of the present invention and the sperm low-temperature damage prevention liquid of a comparative example.
FIG. 3 is a graph showing the results of an experiment using the sperm low temperature damage prevention liquid of the example of the present invention and the sperm low temperature damage prevention liquid of the comparative example.
FIG. 4 is a graph showing the results of an experiment using the sperm low-temperature damage prevention liquid of an example of the present invention and the sperm low-temperature damage prevention liquid of a comparative example.

Claims (12)

A sperm low-temperature injury prevention liquid comprising a long-chain alcohol having 10 to 14 carbon atoms and a lipid. The sperm cold injury prevention liquid according to claim 1, wherein the long-chain alcohol is lauryl alcohol. The sperm cold injury prevention liquid according to claim 1 or 2, wherein the lipid is vegetable oil or animal fat. The sperm cold injury prevention liquid according to claim 1 or 2, wherein the lipid is any one of olive oil, soybean oil, egg yolk, milk, and lecithin. The sperm cold injury prevention solution according to claim 1 or 2, wherein the lipid is α-tocopherol. The sperm cold injury prevention liquid according to any one of claims 1 to 5, wherein the sperm cold injury prevention liquid is subjected to ultrasonic treatment. A low-temperature cell injury prevention liquid, comprising a long-chain alcohol having 10 to 14 carbon atoms, a lipid, and serum albumin. The cell cryoprotective solution according to claim 7, wherein the long-chain alcohol is lauryl alcohol. The cell cold injury prevention liquid according to claim 7 or 8, wherein the lipid is vegetable oil or animal oil. The cell cold injury prevention liquid according to claim 7 or 8, wherein the lipid is any one of olive oil, soybean oil egg yolk, milk, and lecithin. The cell cold injury prevention solution according to claim 7 or 8, wherein the lipid is α-tocopherol. The cell cold injury prevention liquid according to any one of claims 7 to 11, wherein the cell cold injury prevention liquid is subjected to ultrasonic treatment.

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