JP2020092703A - Sperm preservation solution and sperm preservation method - Google Patents

Abstract

To provide a sperm preservation solution and a sperm preservation method that can preserve sperm for a long time at room temperature.SOLUTION: A sperm preservation solution contains inorganic salt, amino acid, vitamin, and fetal bovine serum. A sperm preservation method uses the sperm preservation solution.SELECTED DRAWING: None

Description

The present invention relates to a sperm preservation solution and a sperm preservation method.

When transporting sperm for therapeutic or research purposes, it is common to freeze the sperm. However, there has been a problem that the fertilization ability of frozen sperm after freeze-thawing is significantly reduced.

With respect to such a problem, a cryopreservation agent for maintaining the fertilizing ability of sperm after freeze-thawing has been improved (see, for example, Patent Document 1).

JP, 2009-022214, A

However, when transporting cryopreserved spermatozoa, it was inevitable to put the cryopreserved spermatozoa in liquid nitrogen using a dry zipper and transport it as a package with an expensive container. Needed.

The present invention has been made in view of the above circumstances, and provides a sperm storage solution and a sperm storage method capable of storing sperm at room temperature for a long period of time.

As a result of investigating the composition of the sperm preservation solution, the present inventors have completed the present invention by identifying a sperm preservation solution capable of preserving fish sperms for 1 week or longer and mammalian sperms for 3 days or longer even at room temperature.

That is, the present invention includes the following aspects.
[1] A sperm preservation solution containing inorganic salts, amino acids, vitamins, and fetal bovine serum.
[2] The sperm preservation solution according to [1], which further contains bovine serum albumin.
[3] The sperm preservation solution according to [1] or [2], which further contains a buffer and has a pH of 6.5 to 10.0.
[4] The sperm preservation solution according to any one of [1] to [3], which further contains glucose.
[5] The sperm preservation solution according to any one of [1] to [4], which further contains lactic acid.
[6] The sperm preservation solution according to any one of [1] to [5], further containing an antioxidant.
[7] The sperm preservation solution according to any one of [1] to [6], further containing penicillin and/or streptomycin.
[8] A method for preserving sperm, which comprises using the sperm preservation solution according to any one of [1] to [7].

According to the present invention, fish sperm can be stored for 1 week or longer and mammalian sperm can be stored for 4 days or longer even at room temperature.

It is a graph which showed the fertilization rate of the sperm preserved using the culture medium of Comparative Examples 1-2. 5 is a graph showing the fertilization rate of sperm stored using the mediums of Comparative Example 2 and Example 1. It is a graph which showed the fertilization rate of the sperm preserve|saved using the culture medium of Examples 1-2. It is a graph which showed the fertilization rate of the sperm preserved using the culture medium of Examples 2-3. It is a graph which showed the fertilization rate of the sperm preserved using the culture medium of Examples 3-4. 5 is a graph showing the fertilization rate of sperm stored for 7 days and 28 days using the medium of Example 4. It is a graph which showed the fertilization rate of the sperm preserved for 7 days and 8 days using the culture medium of Examples 4-6.

<< sperm preservation solution >>
The sperm preservation solution of the present invention contains inorganic salts, amino acids, vitamins, and fetal bovine serum.

As the inorganic salts, sodium hydrogen sulfite, sodium sulfite, potassium chloride, calcium chloride, sodium chloride, magnesium chloride, sodium hydrogen carbonate, sodium carbonate, sodium thiosulfate, magnesium sulfate, disodium hydrogen phosphate, sodium dihydrogen phosphate, Examples thereof include potassium dihydrogen phosphate, their anhydrides, and their hydrates.
Among them, sodium hydrogen sulfite, sodium sulfite, potassium chloride, calcium chloride, sodium chloride, magnesium chloride, sodium hydrogen carbonate, sodium carbonate, sodium thiosulfate, magnesium sulfate, disodium hydrogen phosphate, sodium dihydrogen phosphate, and phosphoric acid. It is preferable to contain at least one selected from the group consisting of potassium dihydrogen, their anhydrides, and their hydrates, and calcium chloride, magnesium chloride, magnesium sulfate, potassium chloride, potassium dihydrogen phosphate. , Sodium chloride and disodium hydrogen phosphate, and more preferably at least one selected from the group consisting of their anhydrides and their hydrates, calcium chloride dihydrate, magnesium chloride It is more preferable to contain at least one selected from the group consisting of hexahydrate, anhydrous magnesium sulfate, potassium chloride, anhydrous potassium dihydrogen phosphate, sodium chloride, and anhydrous disodium hydrogen phosphate.

Sodium bisulfite, sodium sulfite, potassium chloride, calcium chloride, magnesium chloride, sodium hydrogen carbonate, sodium carbonate, sodium thiosulfate, magnesium sulfate, disodium hydrogen phosphate, sodium dihydrogen phosphate, potassium dihydrogen phosphate, these As an anhydride or a hydrate of these, 0.005 g to 2 g is preferably contained in 1 L of the sperm preservation solution, 0.01 g to 1 g is more preferably contained, and 0.02 g to 0.5 g is contained. More preferably.
The sodium chloride is preferably contained in 1 L of the sperm preservation solution in an amount of 1 g to 40 g, more preferably 2 g to 20 g, still more preferably 5 g to 10 g.

As the inorganic salts in the sperm preservation solution for zebrafish or medaka, calcium chloride dihydrate 0.1 g/L to 0.25 g/L, magnesium chloride hexahydrate 0.15 g/L to 0.25 g/ L, anhydrous magnesium sulfate 0.06 g/L to 0.12 g/L, potassium chloride 0.3 g/L to 0.6 g/L, anhydrous potassium dihydrogen phosphate 0.04 g/L to 0.08 g/L, chloride It is preferable to contain sodium 6.0 g/L to 10.0 g/L, and anhydrous disodium hydrogen phosphate 0.15 g/L to 0.25 g/L, and calcium chloride dihydrate 0.1 g/L to 0. 0.25 g/L, magnesium chloride hexahydrate 0.15 g/L to 0.25 g/L, anhydrous magnesium sulfate 0.06 g/L to 0.12 g/L, potassium chloride 0.3 g/L to 0.6 g/L L, anhydrous potassium dihydrogen phosphate 0.04 g/L to 0.08 g/L, sodium chloride 6.0 g/L to 10.0 g/L, and anhydrous disodium hydrogen phosphate 0.15 g/L to 0.25 g More preferably, it consists of /L.

The inorganic salts in the sperm preservation solution for mice include calcium chloride dihydrate 0.04 g/L to 0.075 g/L, magnesium chloride hexahydrate 0.15 g/L to 0.25 g/L, and anhydrous sulfuric acid. Magnesium 0.06 g/L to 0.12 g/L, potassium chloride 0.09 g/L to 0.18 g/L, anhydrous potassium dihydrogen phosphate 0.04 g/L to 0.08 g/L, sodium chloride 6.0 g /L to 10.0 g/L and anhydrous disodium hydrogen phosphate 0.15 g/L to 0.25 g/L are preferable, and calcium chloride dihydrate 0.04 g/L to 0.075 g/L , Magnesium chloride hexahydrate 0.15 g/L to 0.25 g/L, anhydrous magnesium sulfate 0.06 g/L to 0.12 g/L, potassium chloride 0.09 g/L to 0.18 g/L, anhydrous phosphorus Consists of 0.04 g/L to 0.08 g/L potassium dihydrogen acid salt, 6.0 g/L to 10.0 g/L sodium chloride, and 0.15 g/L to 0.25 g/L anhydrous sodium dihydrogen phosphate. Is more preferable.

The total amount of inorganic salts in the sperm storage solution is preferably 0.01 to 10%, more preferably 0.1 to 10%, and even more preferably 0.1 to 5%.

As the amino acid, alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, valine, these L-forms, Examples thereof include D-form, DL-form, base-free form, and their anhydrides.
Among them, alanine, arginine, asparagine, cysteine, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, serine, threonine, tryptophan, tyrosine, and valine, and their L-form, D-form, DL-form, It is preferable to contain at least one selected from the group consisting of a base-free form and an anhydride thereof, DL-alanine, L-arginine (base-free), anhydrous L-asparagine, L-cysteine (base-free), L-glutamine, glycine, L-histidine, DL-isoleucine, L-leucine, L-lysine, DL-methionine, DL-phenylalanine, L-serine, DL-threonine, L-tryptophan, L-tyrosine (base-free), And at least one selected from the group consisting of DL-valine.

Alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, valine, their L-form, D-form, DL As a body, a base-free body, or an anhydride thereof, it is preferable to contain 0.0005 g to 4 g, more preferably 0.001 g to 2 g, and 0.003 g to 1 g in 1 L of sperm preservation solution. More preferably.

As amino acids in the sperm storage solution for zebrafish or mouse, DL-alanine 0.3 g/L to 0.6 g/L, L-arginine (base-free) 0.35 g/L to 0.65 g/L, anhydrous L-asparagine 0.15 g/L to 0.35 g/L, L-cysteine (base free) 0.08 g/L to 0.2 g/L, L-glutamine 0.2 g/L to 0.4 g/L, glycine 0.15 g/L to 0.35 g/L, L-histidine 0.15 g/L to 0.35 g/L, DL-isoleucine 0.13 g/L to 0.35 g/L, L-leucine 0.08 g/L -0.2 g/L, L-lysine 0.055 g/L-0.095 g/L, DL-methionine 0.08 g/L-0.25 g/L, DL-phenylalanine 0.15 g/L-0.35 g/L L, L-serine 0.15 g/L to 0.3 g/L, DL-threonine 0.4 g/L to 0.8 g/L, L-tryptophan 0.015 g/L to 0.035 g/L, L-tyrosine (Base-free) 0.2 g/L to 0.4 g/L, and DL-valine 0.15 g/L to 0.35 g/L are preferably contained, and DL-alanine 0.3 g/L to 0.6 g/ L, L-arginine (base free) 0.35 g/L to 0.65 g/L, anhydrous L-asparagine 0.15 g/L to 0.35 g/L, L-cysteine (base free) 0.08 g/L to 0.2 g/L, L-glutamine 0.2 g/L to 0.4 g/L, glycine 0.15 g/L to 0.35 g/L, L-histidine 0.15 g/L to 0.35 g/L, DL -Isoleucine 0.13 g/L to 0.35 g/L, L-leucine 0.08 g/L to 0.2 g/L, L-lysine 0.055 g/L to 0.095 g/L, DL-methionine 0.08 g /L to 0.25 g/L, DL-phenylalanine 0.15 g/L to 0.35 g/L, L-serine 0.15 g/L to 0.3 g/L, DL-threonine 0.4 g/L to 0. 8 g/L, L-tryptophan 0.015 g/L to 0.035 g/L, L-tyrosine (base-free) 0.2 g/L to 0.4 g/L, and DL-valine 0.15 g/L to 0. More preferably, it consists of 35 g/L.

As amino acids in the sperm preservation solution for medaka, DL-alanine 0.07 g/L to 0.6 g/L, L-arginine (base-free) 0.15 g/L to 0.65 g/L, anhydrous L-asparagine 0 0.08 g/L to 0.35 g/L, L-cysteine (base free) 0.045 g/L to 0.2 g/L, L-glutamine 0.085 g/L to 0.4 g/L, glycine 0.08 g/L L-0.35 g/L, L-histidine 0.07 g/L-0.35 g/L, DL-isoleucine 0.07 g/L-0.35 g/L, L-leucine 0.04 g/L-0.2 g /L, L-lysine 0.02 g/L to 0.095 g/L, DL-methionine 0.045 g/L to 0.25 g/L, DL-phenylalanine 0.085 g/L to 0.35 g/L, L- Serine 0.08 g/L to 0.3 g/L, DL-threonine 0.2 g/L to 0.8 g/L, L-tryptophan 0.075 g/L to 0.035 g/L, L-tyrosine (base-free) It is preferable to contain 0.085 g/L to 0.4 g/L and DL-valine 0.085 g/L to 0.35 g/L, and DL-alanine 0.07 g/L to 0.6 g/L, L-. Arginine (base free) 0.15 g/L to 0.65 g/L, anhydrous L-asparagine 0.08 g/L to 0.35 g/L, L-cysteine (base free) 0.045 g/L to 0.2 g/ L, L-glutamine 0.085 g/L to 0.4 g/L, glycine 0.08 g/L to 0.35 g/L, L-histidine 0.07 g/L to 0.35 g/L, DL-isoleucine 0. 07 g/L to 0.35 g/L, L-leucine 0.04 g/L to 0.2 g/L, L-lysine 0.02 g/L to 0.095 g/L, DL-methionine 0.045 g/L to 0 0.25 g/L, DL-phenylalanine 0.085 g/L to 0.35 g/L, L-serine 0.08 g/L to 0.3 g/L, DL-threonine 0.2 g/L to 0.8 g/L, From L-tryptophan 0.075 g/L to 0.035 g/L, L-tyrosine (base free) 0.085 g/L to 0.4 g/L, and DL-valine 0.085 g/L to 0.35 g/L It is more preferable that

The total amount of amino acids in the sperm storage solution is preferably 0.3 to 200 mM, more preferably 1 to 100 mM, still more preferably 3 to 50 mM.

Examples of vitamins include biotin, choline, folic acid, inositol, nicotinic acid, pantothenic acid, pyridoxine, riboflavin, thiamine, vitamin B12, paraaminobenzoic acid (PABA), ascorbic acid, ergocalciferol (vitamin D2), vitamin A, α. -Tocopherols, their derivatives, their salts, their hydrates.
Among them, it contains at least one selected from the group consisting of choline chloride, flavin mononucleotide sodium salt, folic acid, myo-inositol, nicotinamide, DL-pantothenic acid hemi-calcium salt, pyridoxine hydrochloride, and thiamine monophosphate hydrochloride. More preferably, at least one selected from the group consisting of choline chloride, flavin mononucleotide sodium salt, folic acid, myo-inositol, nicotinamide, DL-pantothenic acid hemi-calcium salt, pyridoxine hydrochloride, and thiamine monophosphate. It is more preferable to contain

Biotin, choline, folic acid, inositol, nicotinic acid, pantothenic acid, pyridoxine, riboflavin, thiamine, vitamin B12, paraaminobenzoic acid (PABA), ascorbic acid, ergocalciferol (vitamin D2), vitamin A, α-tocopherol, these As the derivative, the salt thereof, or the hydrate thereof, 0.01 mg to 5 mg is preferably contained in 1 L of the sperm preservation solution, more preferably 0.02 mg to 2 mg is contained, and 0.03 mg to 1 mg is contained. More preferably.

As vitamins in the sperm preservation solution for zebrafish or mouse, choline chloride 0.0008 g/L to 0.0012 g/L, flavin mononucleotide sodium salt 0.00008 g/L to 0.00012 g/L, folic acid 0 0.0008 g/L to 0.0012 g/L, myo-inositol 0.0015 g/L to 0.0025 g/L, nicotinamide 0.0008 g/L to 0.0012 g/L, DL-pantothenic acid hemi-calcium salt 0.0008 g/ L-0.0012 g/L, pyridoxine hydrochloride 0.0008 g/L-0.0012 g/L, and thiamine monophosphate hydrochloride 0.0008 g/L-0.0012 g/L are preferable, and choline chloride 0 0.0008 g/L to 0.0012 g/L, flavin mononucleotide sodium salt 0.00008 g/L to 0.00012 g/L, folic acid 0.0008 g/L to 0.0012 g/L, myoinositol 0.0015 g/L to 0 .0025 g/L, nicotinamide 0.0008 g/L to 0.0012 g/L, DL-pantothenic acid hemi-calcium salt 0.0008 g/L to 0.0012 g/L, pyridoxine hydrochloride 0.0008 g/L to 0.0012 g /L and thiamine monophosphate hydrochloride 0.0008 g/L to 0.0012 g/L are more preferable.

As vitamins in the sperm preservation solution for medaka, choline chloride 0.0004 g/L to 0.0012 g/L, flavin mononucleotide sodium salt 0.00004 g/L to 0.00012 g/L, folic acid 0.0004 g/L to 0.0012 g/L, myo-inositol 0.0007 g/L to 0.0025 g/L, nicotinamide 0.0004 g/L to 0.0012 g/L, DL-pantothenic acid hemi-calcium salt 0.0004 g/L to 0.0012 g /L, pyridoxine hydrochloride 0.0004 g/L to 0.0012 g/L, and thiamine monophosphate hydrochloride 0.0004 g/L to 0.0012 g/L are preferable, and choline chloride 0.0004 g/L to 0.0012 g/L, flavin mononucleotide sodium salt 0.00004 g/L to 0.00012 g/L, folic acid 0.0004 g/L to 0.0012 g/L, myoinositol 0.0007 g/L to 0.0025 g/L, Nicotinamide 0.0004 g/L to 0.0012 g/L, DL-pantothenic acid hemi-calcium salt 0.0004 g/L to 0.0012 g/L, pyridoxine hydrochloride 0.0004 g/L to 0.0012 g/L, and thiamine More preferably, it is comprised of 0.0004 g/L to 0.0012 g/L of monophosphate.

As the total amount in the sperm storage solution, 1 mg of sperm storage solution is preferably contained in an amount of 0.1 mg to 40 mg, more preferably 0.3 mg to 20 mg, and further preferably 0.6 mg to 10 mg. ..

The fetal bovine serum is preferably contained in the sperm preservation solution at 0.2% (v/v) to 15% (v/v), and 0.5% (v/v) to 10% (v/v). ) Is more preferable, and 1% (v/v) to 5% (v/v) is further preferable.
The sperm preservation solution for zebrafish, medaka or mouse preferably contains 2% (v/v) to 4% (v/v) of fetal bovine serum.

The sperm preservation solution of the present invention preferably further contains bovine serum albumin. The bovine serum albumin is preferably contained in the sperm preservation solution in an amount of 0.05% to 10%, more preferably 0.1% to 5%, further preferably 0.2% to 2%. preferable.
The sperm preservation solution for zebrafish, medaka or mouse preferably contains 0.3% (w/v) to 0.6% (w/v) of bovine serum albumin.

The sperm preservation solution of the present invention further contains a buffering agent such as Hepes (2-[4-(2-Hydroxyethyl)-1-piperazinyl]ethane sulfonic acid) and has a pH of 6.5 to 10.0. Is preferred. Hepes are preferably contained in the sperm storage solution in an amount of 1 to 100 mM, more preferably 2 to 50 mM, still more preferably 5 to 20 mM. The pH is 6.5 to 10.0, preferably 7.0 to 9.5, and more preferably 7.5 to 9.0.
The sperm storage solution for zebrafish or medaka preferably contains Hepes (pH 7 to 8) at 8 mM to 15 mM.
The sperm preservation solution for mice preferably contains Hepes (pH 8-9) in an amount of 15 mM to 25 mM.

The sperm preservation solution of the present invention preferably further contains glucose. As glucose, 0.5 g to 40 g is preferably contained in 1 L of the sperm preservation solution, more preferably 1 g to 20 g, and further preferably 2 g to 10 g.
The sperm preservation solution for zebrafish, medaka or mouse preferably contains glucose in an amount of 3 g/L to 5.5 g/L.

The sperm preservation solution of the present invention preferably further contains lactic acid. Lactic acid is preferably contained in the sperm storage solution in an amount of 0.01 mM to 2 mM, more preferably 0.02 mM to 1 mM, and further preferably 0.05 mM to 0.5 mM.
The sperm preservation solution for zebrafish or mouse preferably contains 0.06 mM to 0.15 mM of lactic acid.
It is preferable that the sperm storage solution for medaka contains 0.06 mM to 0.35 mM of lactic acid.

The sperm preservation solution of the present invention preferably further contains an antioxidant. Examples of the antioxidant include vitamin C, hydroquinone, arbutin, astaxanthin, dibutylhydroxytoluene, coenzyme Q10, butylhydroxytoluene, butylhydroxyanisole, citric acid, glutathione, selenium, lycopene, vitamin A and vitamin E.
The antioxidant is preferably contained in the sperm storage solution in an amount of 0.01 mM to 2 mM, more preferably 0.02 mM to 1 mM, and further preferably 0.05 mM to 0.5 mM.

The sperm preservation solution of the present invention preferably further contains penicillin and/or streptomycin, and more preferably penicillin and streptomycin. With 5,000 units/ml penicillin and 5,000 μg/ml streptomycin, it is preferable to use it in a sperm storage solution after diluting it 50 to 1000 times, more preferably 50 to 300 times. It is preferable to use it after diluting 50 to 150 times.

In addition, the sperm preservation solution of the present invention preferably contains D-galactose, phenol red sodium salt, pyruvate sodium salt and the like.
The sperm preservation solution for zebrafish or mouse preferably contains 0.7 g/L to 1.2 g/L of D-galactose, and 0.008 g/L to 0.013 g/L of phenol red sodium salt. It is preferable that the pyruvic acid sodium salt is contained in an amount of 0.45 g/L to 0.6 g/L.
The sperm preservation solution for medaka preferably contains D-galactose in an amount of 0.3 g/L to 1.2 g/L, preferably contains phenol red sodium salt in an amount of 0.004 g/L to 0.013 g/L, and pyruvin. The acid sodium salt is preferably contained in an amount of 0.2 g/L to 0.6 g/L.

<< sperm storage method >>
The sperm storage method of the present invention is a method using the sperm storage solution described above.
Examples of sperm to be stored include sperm of mammals such as human, monkey, dog, cat, rabbit, pig, cow, mouse, rat and hamster. Furthermore, sperm such as fish, amphibians, birds, and reptiles can also be mentioned. Examples of fish sperm include medaka and zebrafish sperm.

According to the sperm storage solution and sperm storage method of the present invention, sperm can be stored at room temperature for a long period of time, and thus a small amount of sperm suspension can be put in a tube and transported by envelope. Since the sperm storage solution itself can be prepared at a low price, according to the present invention, it is possible to significantly reduce the transportation cost.

Hereinafter, the present invention will be described with reference to examples, but the present invention is not limited to the following examples.

[Examination of antibiotics]
Sperms collected from 2-3 zebrafish were suspended in ice-cooled medium of Comparative Example 1 and Comparative Example 2 shown in Table 1 to prepare a sperm suspension. 10 μL of this sperm suspension was sampled and the number of sperms was counted. Then, the sperm suspension adjusted to 74000/μL using the medium was stored in a refrigerator at 4°C for 4 days. On the day of fertilization, eggs were squeezed from a zebrafish female into a glass dish, 40 μL of sperm suspension was applied to about 100 of these eggs, and 800 μL of sterilized fish breeding water was further applied and left for 1 and a half minutes. Further, an appropriate amount of sterilized fish breeding water was added to this tube, this egg was cultured to the cyst embryo stage, and then the fertilization rate was counted. The results are shown in Figure 1.

In FIG. 1, the vertical axis represents the fertilization rate (%). As shown in FIG. 1, it was confirmed that the fertilization rate was not affected even if penicillin/streptomycin was contained.

[Study of fetal bovine serum (FBS)]
The effect of FBS addition on the fertilization rate was examined by the same method as in [Investigation of Antibiotics] except that the ice-cooled mediums of Example 1 and Comparative Example 2 shown in Table 2 were used. The results are shown in Figure 2.

In FIG. 2, the vertical axis represents the fertilization rate (%), and *: p<0.01. As shown in FIG. 2, it was confirmed that the addition of FBS dramatically increased the fertilization rate.

[Study of bovine serum albumin (BSA) and pH]
The effect of addition of BSA and pH adjustment on the fertilization rate was examined in the same manner as in [Investigation of Antibiotics] except that the ice-cooled mediums of Example 1 and Example 2 shown in Table 3 were used. Results are shown in FIG.

In FIG. 3, the vertical axis represents the fertilization rate (%), and *: p<0.05. As shown in FIG. 3, it was confirmed that the fertilization rate was further increased by adding BSA.

[Investigation of glucose]
The effect of glucose addition on the fertilization rate was examined by the same method as in [Investigation of Antibiotics] except that the ice-cooled mediums of Examples 2 and 3 shown in Table 4 were used. The results are shown in Fig. 4.

In FIG. 4, the vertical axis represents the fertilization rate (%). As shown in FIG. 4, it was confirmed that the fertilization rate was further increased by adding glucose.

[Study on lactic acid]
Using the ice-cooled mediums of Example 3 and Example 4 shown in Table 5, the sperm suspension prepared using the medium of Example 4 was stored at 23° C. for 4 days. ], the effect on the fertilization rate by the addition of lactic acid was examined. Results are shown in FIG.

In FIG. 5, the vertical axis represents the fertilization rate (%). As shown in FIG. 5, it was confirmed that the fertilization rate was further increased by using a medium supplemented with lactic acid and storing it at 23° C.

[Study of long-term preservation effect]
Using the ice-cooled medium of Example 4 and using the same method as in [Investigation of Antibiotics], except that the sperm suspension prepared using the medium of Example 4 was stored at 23° C. for 7 days and 28 days. The effect of lactic acid addition on the fertilization rate was investigated. Results are shown in FIG.

In FIG. 6, the vertical axis represents the fertilization rate (%). As shown in FIG. 6, by using the medium of Example 4 and storing at 23° C., a fertilization rate of about 80% was confirmed for 7 days storage, and about 30% for 28 days storage. It was

[Preservation of medaka sperm]
As shown in Table 6, in addition to the medium of Example 4, a medium in which lactic acid alone was increased to 0.2 mM was prepared (Example 5). In addition, inorganic salts (calcium chloride dihydrate, magnesium chloride hexahydrate, anhydrous magnesium sulfate, potassium chloride, anhydrous potassium dihydrogen phosphate, sodium chloride, anhydrous disodium hydrogen phosphate), penicillin/streptomycin, fetal bovine Serum (FBS), bovine serum albumin (BSA), 10 mM Hepes (pH 8.8), glucose, and lactic acid were the same as the amount of the medium used for the zebrafish of Example 4, and amino acids, vitamins, D -For galactose, phenol red sodium salt, and pyruvic acid sodium salt, a medium was prepared in an amount half that of the medium used for zebrafish (Example 6). Take out the testicle (1), sterilize it with 0.5% bleach for 30 seconds, wash it twice with Medaka's Ringer's solution (BSS), and use tweezers in each prepared medium (66 μl). Squeezed out sperm. 10 μl of the sperm suspension was transferred to a 1.5 ml tube, 90 μl of medium was added thereto, and the sperm concentration was stored at 450,000/ml.
After storing at room temperature for 7 days or at 4° C. for 8 days, the fertilization experiment was carried out using eggs prepared from medaka females, using 100 μl (45,000 sperms) per tube. After culturing the eggs up to the cyst embryo stage, the fertilization rate was counted. The results are shown in Fig. 7. In FIG. 7, the vertical axis represents the fertilization rate (%). A part of the culture medium stored at room temperature using the medium of Example 4 had a part of the sperm storage solution turned yellow to orange from about the first week. It is presumed that somatic cells derived from the testis proliferated. Other than that, it was confirmed that the fertility of sperm was maintained.

[Preservation of mouse sperm]
As shown in Table 7, magnesium chloride hexahydrate, anhydrous magnesium sulfate, anhydrous potassium dihydrogen phosphate, sodium chloride, anhydrous disodium hydrogen phosphate, penicillin/streptomycin, fetal bovine serum (FBS), bovine serum albumin ( BSA), glucose, lactic acid, amino acids, vitamins, D-galactose, phenol red sodium salt, and pyruvate sodium salt are the same as the amount of the medium used for the zebrafish of Example 4, and calcium chloride dihydrate is used. For the substances and potassium chloride, a medium of 20 mM Hepes (pH 8.8) was prepared in an amount 1/3 of the volume of the medium used for the zebrafish of Example 4 (Example 7). The cauda epididymis of the mouse was cut with scissors, and the squeezed sperm was suspended in 0.5 ml of the above culture solution to prepare a sperm suspension.
After storing at room temperature (22 to 23° C.) for 4 days, about 0.02 ml of the lower layer was added to CARD FERTIUP mouse sperm preculture medium in a sperm preculture dish. In addition to the 2 ml drop, preculture was carried out at 37° C. for 60 minutes, and the sperm was inseminated by adding to the fertilization mHTF drop containing unfertilized eggs. Three hours after the insemination, only morphologically normal eggs were washed with mHTF drops. The next day, the embryos developed in 2 cells were transferred to the embryo culture medium KSOM, and the eggs were cultured up to the blastocyst, and the fertilization rate was counted. The results are shown in Table 8.

In Table 8, Examples 4-1 to 4-5 show the results of repeating the experiment 5 times using the medium of Example 4, and Examples 7-1 to 7-5 show Example 7 The results of 5 repeated experiments using the medium of
As shown in Table 8, it was confirmed that the culture medium of Example 7 was excellent in storage capacity in five repeated experiments.

Claims (11)

A sperm preservation solution containing inorganic salts, amino acids, vitamins, and fetal bovine serum. The sperm preservation solution according to claim 1, further comprising bovine serum albumin. The sperm preservation solution according to claim 1 or 2, further comprising a buffer and having a pH of 6.5 to 10.0. Furthermore, the sperm preservation solution according to any one of claims 1 to 3, further comprising glucose. The sperm preservation solution according to any one of claims 1 to 4, which further contains lactic acid. Furthermore, the sperm preservation solution according to any one of claims 1 to 5, further comprising an antioxidant. The sperm preservation solution according to any one of claims 1 to 6, further containing penicillin and/or streptomycin. For zebrafish,
As the final concentration, calcium chloride dihydrate 0.1 g/L to 0.25 g/L, magnesium chloride hexahydrate 0.15 g/L to 0.25 g/L, anhydrous magnesium sulfate 0.06 g/L to 0 .12 g/L, potassium chloride 0.3 g/L to 0.6 g/L, anhydrous potassium dihydrogen phosphate 0.04 g/L to 0.08 g/L, sodium chloride 6.0 g/L to 10.0 g/ L, and inorganic salts containing anhydrous disodium hydrogen phosphate 0.15 g/L to 0.25 g/L,
DL-alanine 0.3 g/L to 0.6 g/L, L-arginine (base free) 0.35 g/L to 0.65 g/L, anhydrous L-asparagine 0.15 g/L to 0.35 g/L, L-cysteine (base-free) 0.08 g/L to 0.2 g/L, L-glutamine 0.2 g/L to 0.4 g/L, glycine 0.15 g/L to 0.35 g/L, L -Histidine 0.15 g/L to 0.35 g/L, DL-isoleucine 0.13 g/L to 0.35 g/L, L-leucine 0.08 g/L to 0.2 g/L, L-lysine 0.055 g /L to 0.095 g/L, DL-methionine 0.08 g/L to 0.25 g/L, DL-phenylalanine 0.15 g/L to 0.35 g/L, L-serine 0.15 g/L to 0 .3 g/L, DL-threonine 0.4 g/L to 0.8 g/L, L-tryptophan 0.015 g/L to 0.035 g/L, L-tyrosine (base-free) 0.2 g/L to 0. 4 g/L, and an amino acid containing DL-valine 0.15 g/L to 0.35 g/L,
Choline chloride 0.0008 g/L to 0.0012 g/L, flavin mononucleotide sodium salt 0.00008 g/L to 0.00012 g/L, folic acid 0.0008 g/L to 0.0012 g/L, myoinositol 0.0015 g/L L-0.0025 g/L, nicotinamide 0.0008 g/L-0.0012 g/L, DL-pantothenic acid hemi-calcium salt 0.0008 g/L-0.0012 g/L, pyridoxine hydrochloride 0.0008-0. Vitamins containing 0012 g/L and thiamin monophosphate hydrochloride 0.0008 g/L to 0.0012 g/L;
D-galactose 0.7 g/L to 1.2 g/L,
Phenol red sodium salt 0.008 g/L to 0.013 g/L,
Pyruvate sodium salt 0.45 g/L to 0.6 g/L,
Fetal bovine serum (FBS) 2% (v/v) to 4% (v/v),
Bovine serum albumin (BSA) 0.3% (w/v) to 0.6% (w/v),
Hepes 8 mM-15 mM (pH 7-8),
Glucose 3 g/L to 5.5 g/L,
Lactic acid 0.06 mM to 0.15 mM,
Sperm preservation solution containing. For medaka,
As the final concentration, calcium chloride dihydrate 0.1 g/L to 0.25 g/L, magnesium chloride hexahydrate 0.15 g/L to 0.25 g/L, anhydrous magnesium sulfate 0.06 g/L to 0 .12 g/L, potassium chloride 0.3 g/L to 0.6 g/L, anhydrous potassium dihydrogen phosphate 0.04 g/L to 0.08 g/L, sodium chloride 6.0 g/L to 10.0 g/L An inorganic salt containing 0.15 g/L to 0.25 g/L of anhydrous disodium hydrogen phosphate,
DL-alanine 0.07 g/L to 0.6 g/L, L-arginine (base free) 0.15 g/L to 0.65 g/L, anhydrous L-asparagine 0.08 g/L to 0.35 g/L, L-cysteine (base-free) 0.045 g/L to 0.2 g/L, L-glutamine 0.085 g/L to 0.4 g/L, glycine 0.08 g/L to 0.35 g/L, L- Histidine 0.07 g/L to 0.35 g/L, DL-isoleucine 0.07 g/L to 0.35 g/L, L-leucine 0.04 g/L to 0.2 g/L, L-lysine 0.02 g/L L-0.095 g/L, DL-methionine 0.045 g/L-0.25 g/L, DL-phenylalanine 0.085 g/L-0.35 g/L, L-serine 0.08 g/L-0.3 g /L, DL-threonine 0.2 to 0.8 g/L, L-tryptophan 0.075 to 0.035 g/L, L-tyrosine (base free) 0.085 to 0.4 g/L, DL-valine 0 Amino acids containing 0.085 to 0.35 g/L,
Choline chloride 0.0004 to 0.0012 g/L, flavin mononucleotide sodium salt 0.00004 to 0.00012 g/L, folic acid 0.0004 to 0.0012 g/L, myoinositol 0.0007 to 0.0025 g/L, Nicotinamide 0.0004 to 0.0012 g/L, DL-pantothenic acid hemi-calcium salt 0.0004 to 0.0012 g/L, pyridoxine hydrochloride 0.0004 to 0.0012 g/L, thiamine monophosphate hydrochloride 0. Vitamins containing 0004 to 0.0012 g/L,
D-galactose 0.3 to 1.2 g/L,
Phenol red sodium salt 0.004 to 0.013 g/L,
Pyruvic acid sodium salt 0.2-0.6 g/L,
Fetal bovine serum (FBS) 2-4% (v/v),
Bovine serum albumin (BSA) 0.3-0.6% (w/v),
Hepes 8-15 mM (pH 7-8),
Glucose 3 to 5.5 g/L,
Lactic acid 0.06-0.35 mM,
Sperm preservation solution containing. For mice,
As the final concentration, calcium chloride dihydrate 0.04 g/L to 0.075 g/L, magnesium chloride hexahydrate 0.15 g/L to 0.25 g/L, anhydrous magnesium sulfate 0.06 g/L to 0 .12 g/L, potassium chloride 0.09 g/L to 0.18 g/L, anhydrous potassium dihydrogen phosphate 0.04 g/L to 0.08 g/L, sodium chloride 6.0 g/L to 10.0 g/L An inorganic salt containing 0.15 g/L to 0.25 g/L of anhydrous disodium hydrogen phosphate,
DL-alanine 0.3 g/L to 0.6 g/L, L-arginine (base free) 0.35 g/L to 0.65 g/L, anhydrous L-asparagine 0.15 g/L to 0.35 g/L, L-cysteine (base-free) 0.08 g/L to 0.2 g/L, L-glutamine 0.2 g/L to 0.4 g/L, glycine 0.15 g/L to 0.35 g/L, L-histidine 0.15 g/L to 0.35 g/L, DL-isoleucine 0.13 g/L to 0.35 g/L, L-leucine 0.08 g/L to 0.2 g/L, L-lysine 0.055 g/L -0.095 g/L, DL-methionine 0.08 g/L-0.25 g/L, DL-phenylalanine 0.15 g/L-0.35 g/L, L-serine 0.15 g/L-0.3 g/L L, DL-threonine 0.4 g/L to 0.8 g/L, L-tryptophan 0.015 g/L to 0.035 g/L, L-tyrosine (base-free) 0.2 g/L to 0.4 g/L , An amino acid containing DL-valine 0.15 g/L to 0.35 g/L,
Choline chloride 0.0008 g/L to 0.0012 g/L, flavin mononucleotide sodium salt 0.00008 g/L to 0.00012 g/L, folic acid 0.0008 g/L to 0.0012 g/L, myoinositol 0.0015 g/L L-0.0025 g/L, nicotinamide 0.0008 g/L-0.0012 g/L, DL-pantothenic acid hemi-calcium salt 0.0008 g/L-0.0012 g/L, pyridoxine hydrochloride 0.0008 g/L- Vitamins containing 0.0012 g/L, thiamine monophosphate hydrochloride 0.0008 g/L to 0.0012 g/L,
D-galactose 0.7 g/L to 1.2 g/L,
0.008 g/L to 0.013 g/L of phenol red sodium salt,
Pyruvate sodium salt 0.45 g/L to 0.6 g/L,
Fetal bovine serum (FBS) 2% (v/v) to 4% (v/v),
Bovine serum albumin (BSA) 0.3% (w/v) to 0.6% (w/v),
Hepes 15-25 mM (pH 8-9)
Glucose 3 to 5.5 g/L,
Lactic acid 0.06-0.15 mM,
A sperm preservation solution containing. A sperm storage method using the sperm storage solution according to any one of claims 1 to 10.

Images