JP7478406B2 - Sperm preservation solution and sperm preservation method - Google Patents

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 is a problem in that the fertilizing ability of frozen sperm is significantly reduced after freezing and thawing.

In response to these problems, cryopreservation agents have been improved to maintain the fertilizing capacity of sperm after freezing and thawing (see, for example, Patent Document 1).

JP 2009-022214 A

However, when transporting frozen sperm, the only option was to place the frozen sperm in liquid nitrogen using a dry zipper and transport it as luggage together with an expensive container, resulting in high transportation costs.

The present invention was made in consideration of the above circumstances, and provides a sperm preservation solution and a sperm preservation method that can preserve sperm for long periods at room temperature.

After examining the composition of sperm preservation solutions, the inventors identified a sperm preservation solution that can preserve fish sperm for more than one week and mammalian sperm for more than three days even at room temperature, thus completing the present invention.

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

According to the present invention, fish sperm can be stored for more than one week and mammalian sperm for more than four days at room temperature.

1 is a graph showing the fertilization rate of sperm stored using the media of Comparative Examples 1 and 2. 1 is a graph showing the fertilization rates of sperm stored using the media of Comparative Example 2 and Example 1. 1 is a graph showing the fertilization rate of sperm stored using the media of Examples 1 and 2. 1 is a graph showing the fertilization rate of sperm stored using the media of Examples 2 and 3. 1 is a graph showing the fertilization rate of sperm stored using the media of Examples 3 and 4. 1 is a graph showing the fertilization rates of sperm stored for 7 days and 28 days using the medium of Example 4. 1 is a graph showing the fertilization rates of sperm stored for 7 and 8 days using the media of Examples 4 to 6.

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

Examples of inorganic salts include 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, potassium dihydrogen phosphate, anhydrides thereof, and hydrates thereof.
Among these, it is preferable to contain at least one selected from the group consisting of 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, potassium dihydrogen phosphate, and their anhydrides and hydrates, and it is more preferable to contain at least one selected from the group consisting of calcium chloride, magnesium chloride, magnesium sulfate, potassium chloride, potassium dihydrogen phosphate, sodium chloride, and disodium hydrogen phosphate, and their anhydrides and hydrates, and it is even more preferable to contain at least one selected from the group consisting of calcium chloride dihydrate, magnesium chloride hexahydrate, anhydrous magnesium sulfate, potassium chloride, anhydrous potassium dihydrogen phosphate, sodium chloride, and anhydrous disodium hydrogen phosphate.

Sodium hydrogen sulfite, 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, their anhydrides, or their hydrates are preferably contained in an amount of 0.005 g to 2 g, more preferably 0.01 g to 1 g, and even more preferably 0.02 g to 0.5 g per liter of sperm preservation solution.
The amount of sodium chloride contained in 1 L of the sperm preservation solution is preferably 1 g to 40 g, more preferably 2 g to 20 g, and even more preferably 5 g to 10 g.

Inorganic salts in sperm preservation solutions for zebrafish or medaka include 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 anhydrous disodium hydrogen phosphate 0.15 g/L to 0.2 It is preferable that the solution contains 0.5 g/L of calcium chloride dihydrate, 0.1 g/L to 0.25 g/L of magnesium chloride hexahydrate, 0.15 g/L to 0.25 g/L of anhydrous magnesium sulfate, 0.06 g/L to 0.12 g/L of anhydrous magnesium sulfate, 0.3 g/L to 0.6 g/L of potassium chloride, 0.04 g/L to 0.08 g/L of anhydrous potassium dihydrogen phosphate, 6.0 g/L to 10.0 g/L of sodium chloride, and 0.15 g/L to 0.25 g/L of anhydrous disodium hydrogen phosphate.

Inorganic salts in mouse sperm preservation solution include calcium chloride dihydrate 0.04g/L to 0.075g/L, magnesium chloride hexahydrate 0.15g/L to 0.25g/L, anhydrous magnesium sulfate 0.06g/L to 0.12g/L, potassium chloride 0.09g/L to 0.18g/L, anhydrous potassium dihydrogen phosphate 0.04g/L to 0.08g/L, sodium chloride 6.0g/L to 10.0g/L, and anhydrous disodium hydrogen phosphate 0.15g/L to 0.25g/L. It is preferable that the solution contains 0.04 g/L to 0.075 g/L of calcium chloride dihydrate, 0.15 g/L to 0.25 g/L of magnesium chloride hexahydrate, 0.06 g/L to 0.12 g/L of anhydrous magnesium sulfate, 0.09 g/L to 0.18 g/L of potassium chloride, 0.04 g/L to 0.08 g/L of anhydrous potassium dihydrogen phosphate, 6.0 g/L to 10.0 g/L of sodium chloride, and 0.15 g/L to 0.25 g/L of anhydrous disodium hydrogen phosphate.

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

Examples of amino acids include alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, valine, their L-, D-, and DL-forms, base-free forms, and anhydrides thereof.
Among these, it is preferable to contain at least one selected from the group consisting of alanine, arginine, asparagine, cysteine, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, serine, threonine, tryptophan, tyrosine, and valine, as well as their L-, D-, and DL-forms, base-free forms, and anhydrides thereof, and it is more preferable to contain at least one selected from the group consisting of 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 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-, D-, or DL-forms, base-free forms, or their anhydrides are preferably contained in 1 L of sperm preservation solution at 0.0005 g to 4 g, more preferably 0.001 g to 2 g, and even more preferably 0.003 g to 1 g.

The amino acids in sperm preservation solutions for zebrafish or mice are: DL-alanine 0.3g/L to 0.6g/L, L-arginine (base free) 0.35g/L to 0.65g/L, anhydrous L-asparagine 0.15g/L to 0.35g/L, L-cysteine (base free) 0.08g/L to 0.2g/L, L-glutamine 0.2g/L to 0.4g/L, glycine 0.15g/L to 0.35g/L, L-histidine 0.15g/L to 0.35g/L, DL-isoleucine 0.13g/L to 0.2g/L, L-0.35g/L, L-leucine 0.08g/L-0.2g/L, L-lysine 0.055g/L-0.095g/L, DL-methionine 0.08g/L-0.25g/L, DL-phenylalanine 0.15g/L-0.35g/L, L-serine 0.15g/L-0.3g/L, DL-threonine 0.4g/L-0.8g/L, L-tryptophan 0.015g/L-0.035g/L, L-tyrosine (base free) 0.2g/L-0.4g/L, and DL-valine 0.15g/L-0.3 It is preferable that the total amount of the glycerol is 0.5 g/L, and the total amount of the glycerol is 0.5 g/L to ... More preferably, it is composed of 0.8 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 0.35 g/L.

The amino acids in sperm preservation solution for medaka are DL-alanine 0.07g/L to 0.6g/L, L-arginine (base free) 0.15g/L to 0.65g/L, anhydrous L-asparagine 0.08g/L to 0.35g/L, L-cysteine (base free) 0.045g/L to 0.2g/L, L-glutamine 0.085g/L to 0.4g/L, glycine 0.08g/L to 0.35g/L, L-histidine 0.07g/L to 0.35g/L, DL-isoleucine 0.07g/L to 0.35g/L, L,L-leucine 0.04g/L to 0.2g/L, L-lysine 0.02g/L to 0.095g/L, DL-methionine 0.045g/L to 0.25g/L, DL-phenylalanine 0.085g/L to 0.35g/L, L-serine 0.08g/L to 0.3g/L, DL-threonine 0.2g/L to 0.8g/L, L-tryptophan 0.075g/L to 0.035g/L, L-tyrosine (base free) 0.085g/L to 0.4g/L, and DL-valine 0.085g/L to 0.35g/L. and preferably contains 0.07 g/L to 0.6 g/L DL-alanine, 0.15 g/L to 0.65 g/L L-arginine (base free), 0.08 g/L to 0.35 g/L anhydrous L-asparagine, 0.045 g/L to 0.2 g/L L-cysteine (base free), 0.085 g/L to 0.4 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 0.35 g/L. More preferably, it is composed of 0.02g/L to 0.2g/L, L-lysine 0.02g/L to 0.095g/L, DL-methionine 0.045g/L to 0.25g/L, DL-phenylalanine 0.085g/L to 0.35g/L, L-serine 0.08g/L to 0.3g/L, DL-threonine 0.2g/L to 0.8g/L, L-tryptophan 0.075g/L to 0.035g/L, L-tyrosine (base free) 0.085g/L to 0.4g/L, and DL-valine 0.085g/L to 0.35g/L.

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

Examples of vitamins include biotin, choline, folic acid, inositol, nicotinic acid, pantothenic acid, pyridoxine, riboflavin, thiamine, vitamin B12, para-aminobenzoic acid (PABA), ascorbic acid, ergocalciferol (vitamin D2), vitamin A, α-tocopherol, derivatives thereof, salts thereof, and hydrates thereof.
Among these, it is more preferable that the composition contains at least one selected from the group consisting of choline chloride, flavin mononucleotide sodium salt, folic acid, myo-inositol, nicotinamide, DL-pantothenic acid hemicalcium salt, pyridoxine hydrochloride, and thiamine monophosphate hydrochloride, and it is even more preferable that the composition contains at least one selected from the group consisting of choline chloride, flavin mononucleotide sodium salt, folic acid, myo-inositol, nicotinamide, DL-pantothenic acid hemicalcium salt, pyridoxine hydrochloride, and thiamine monophosphate hydrochloride.

Biotin, choline, folic acid, inositol, nicotinic acid, pantothenic acid, pyridoxine, riboflavin, thiamine, vitamin B12, para-aminobenzoic acid (PABA), ascorbic acid, ergocalciferol (vitamin D2), vitamin A, α-tocopherol, their derivatives, their salts, or their hydrates are preferably contained in 1 L of sperm preservation solution at 0.01 mg to 5 mg, more preferably 0.02 mg to 2 mg, and even more preferably 0.03 mg to 1 mg.

Vitamins in sperm preservation solutions for zebrafish or mice include choline chloride 0.0008g/L to 0.0012g/L, flavin mononucleotide sodium salt 0.00008g/L to 0.00012g/L, folic acid 0.0008g/L to 0.0012g/L, myo-inositol 0.0015g/L to 0.0025g/L, nicotinamide 0.0008g/L to 0.0012g/L, DL-pantothenic acid hemicalcium salt 0.0008g/L to 0.0012g/L, pyridoxine hydrochloride 0.0008g/L to 0.0012g/L, and thiamine monophosphate hydrochloride 0.0008g/L to 0.00 It is preferable that the composition contains 12 g/L, and more preferably, the composition is made up of 0.0008 g/L to 0.0012 g/L of choline chloride, 0.00008 g/L to 0.00012 g/L of flavin mononucleotide sodium salt, 0.0008 g/L to 0.0012 g/L of folic acid, 0.0015 g/L to 0.0025 g/L of myo-inositol, 0.0008 g/L to 0.0012 g/L of nicotinamide, 0.0008 g/L to 0.0012 g/L of DL-pantothenic acid hemicalcium salt, 0.0008 g/L to 0.0012 g/L of pyridoxine hydrochloride, and 0.0008 g/L to 0.0012 g/L of thiamine monophosphate hydrochloride.

Vitamins in sperm preservation solution for medaka include choline chloride 0.0004g/L to 0.0012g/L, flavin mononucleotide sodium salt 0.00004g/L to 0.00012g/L, folic acid 0.0004g/L to 0.0012g/L, myo-inositol 0.0007g/L to 0.0025g/L, nicotinamide 0.0004g/L to 0.0012g/L, DL-pantothenic acid hemicalcium salt 0.0004g/L to 0.0012g/L, pyridoxine hydrochloride 0.0004g/L to 0.0012g/L, and thiamine monophosphate hydrochloride 0.0004g/L to 0.0012g/L. It is preferable that the composition contains 0.0004g/L to 0.0012g/L of choline chloride, 0.00004g/L to 0.00012g/L of flavin mononucleotide sodium salt, 0.0004g/L to 0.0012g/L of folic acid, 0.0007g/L to 0.0025g/L of myo-inositol, 0.0004g/L to 0.0012g/L of nicotinamide, 0.0004g/L to 0.0012g/L of DL-pantothenic acid hemicalcium salt, 0.0004g/L to 0.0012g/L of pyridoxine hydrochloride, and 0.0004g/L to 0.0012g/L of thiamine monophosphate hydrochloride.

The total amount in the sperm preservation solution is preferably 0.1 mg to 40 mg per liter of sperm preservation solution, more preferably 0.3 mg to 20 mg, and even more preferably 0.6 mg to 10 mg.

The sperm preservation solution preferably contains 0.2% (v/v) to 15% (v/v) of fetal bovine serum, more preferably 0.5% (v/v) to 10% (v/v), and even more preferably 1% (v/v) to 5% (v/v).
A 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 at a concentration of 0.05% to 10%, more preferably 0.1% to 5%, and even more preferably 0.2% to 2%.
A 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 preferably further contains a buffer such as Hepes (2-[4-(2-hydroxyethyl)-1-piperazinyl]ethanesulfonic acid) and has a pH of 6.5 to 10.0. The sperm preservation solution preferably contains 1 to 100 mM Hepes, more preferably 2 to 50 mM, and even 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.
A sperm preservation solution for zebrafish or medaka preferably contains 8 mM to 15 mM Hepes (pH 7 to 8).
The sperm preservation solution for mice preferably contains 15 mM to 25 mM Hepes (pH 8 to 9).

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

The sperm preservation solution of the present invention preferably further contains lactic acid. The sperm preservation solution preferably contains 0.01 mM to 2 mM of lactic acid, more preferably 0.02 mM to 1 mM, and even more 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 lactic acid.
The sperm preservation solution for medaka preferably contains 0.06 mM to 0.35 mM of lactic acid.

The sperm preservation solution of the present invention preferably further contains an antioxidant, such as vitamin C, hydroquinone, arbutin, astaxanthin, dibutylhydroxytoluene, coenzyme Q10, butylhydroxytoluene, butylhydroxyanisole, citric acid, glutathione, selenium, lycopene, vitamin A, or vitamin E.
The antioxidant is preferably contained in the sperm preservation solution at 0.01 mM to 2 mM, more preferably 0.02 mM to 1 mM, and even more 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 contains penicillin and streptomycin. In the case of 5,000 units/ml penicillin and 5,000 μg/ml streptomycin, it is preferable that they are diluted 50 to 1000 times, more preferably 50 to 300 times, and even more preferably 50 to 150 times before use in the sperm preservation solution.

In addition, the sperm preservation solution of the present invention preferably contains D-galactose, sodium phenol red, sodium pyruvate, and the like.
A sperm preservation solution for zebrafish or mouse preferably contains 0.7 g/L to 1.2 g/L of D-galactose, 0.008 g/L to 0.013 g/L of phenol red sodium salt, and 0.45 g/L to 0.6 g/L of pyruvate sodium salt.
A sperm preservation solution for medaka preferably contains 0.3 g/L to 1.2 g/L of D-galactose, 0.004 g/L to 0.013 g/L of phenol red sodium salt, and 0.2 g/L to 0.6 g/L of pyruvate sodium salt.

＜＜How to store sperm＞＞
The sperm preservation method of the present invention is a method using the sperm preservation solution described above.
Examples of sperm to be preserved include sperm from mammals such as humans, monkeys, dogs, cats, rabbits, pigs, cows, mice, rats, and hamsters. In addition, sperm from fish, amphibians, birds, reptiles, etc. Examples of fish sperm include sperm from medaka and zebrafish.

The sperm preservation solution and sperm preservation method of the present invention allow sperm to be preserved for long periods at room temperature, so a small amount of sperm suspension can be placed in a tube and transported in an envelope. The sperm preservation solution itself can be prepared at low cost, so the present invention allows for a significant reduction in transportation costs.

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

[Antibiotics review]
Sperm 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 sperm was counted. Next, the sperm suspension was adjusted to 74,000 sperm/μL using the medium and stored in a refrigerator at 4° C. for 4 days. On the day of fertilization, eggs were squeezed out from female zebrafish into a glass dish, and 40 μL of the sperm suspension was poured onto about 100 eggs, followed by further pouring 800 μL of sterilized fish breeding water and leaving it for 1.5 minutes. Furthermore, an appropriate amount of sterilized fish breeding water was added to the tube, and the eggs were cultured until the sac-embryo stage, after which the fertilization rate was counted. The results are shown in FIG. 1.

In Figure 1, the vertical axis indicates the fertilization rate (%). As shown in Figure 1, it was confirmed that the inclusion of penicillin/streptomycin had no effect on the fertilization rate.

[Study of fetal bovine serum (FBS)]
The effect of adding FBS on the fertility rate was examined in the same manner as in [Study of antibiotics], except that ice-cooled media of Example 1 and Comparative Example 2 shown in Table 2 were used. The results are shown in FIG.

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

[Bovine serum albumin (BSA) and pH study]
The effects of adding BSA and adjusting the pH on the fertility rate were examined in the same manner as in [Study of antibiotics], except that ice-cooled media of Examples 1 and 2 shown in Table 3 were used. The results are shown in Figure 3.

In Figure 3, the vertical axis indicates the fertilization rate (%), and * indicates p<0.05. As shown in Figure 3, it was confirmed that the addition of BSA further increased the fertilization rate.

[Glucose study]
The effect of glucose addition on fertility was examined in the same manner as in [Study of antibiotics], except that ice-cooled media of Examples 2 and 3 shown in Table 4 were used. The results are shown in FIG.

In Figure 4, the vertical axis indicates the fertilization rate (%). As shown in Figure 4, it was confirmed that the addition of glucose further increased the fertilization rate.

[Lactic acid study]
The effect of adding lactic acid on the fertilization rate was examined in the same manner as in [Study of antibiotics], except that the ice-cooled media of Example 3 and Example 4 shown in Table 5 were used and the sperm suspension prepared using the medium of Example 4 was stored at 23° C. for 4 days. The results are shown in FIG.

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

[Study of long-term storage effect]
The effect of adding lactic acid on the fertilization rate was examined in the same manner as in [Study of antibiotics], except that the medium of Example 4 was used on ice and the sperm suspension prepared using the medium of Example 4 was stored at 23° C. for 7 and 28 days. The results are shown in FIG.

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

[Preservation of medaka sperm]
As shown in Table 6, in addition to the medium of Example 4, a medium was prepared in which only lactic acid was increased to 0.2 mM (Example 5). In addition, the amounts of 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 those in the medium used for zebrafish in Example 4, and the amounts of amino acids, vitamins, D-galactose, sodium phenol red, and sodium pyruvate were half those in the medium used for zebrafish (Example 6). A testis (one) was removed, sterilized with 0.5% bleach for 30 seconds, washed twice with medaka Ringer's solution (BSS), and sperm were squeezed out using tweezers in each of the prepared media (66 μl). 10 μl of the sperm suspension was transferred to a 1.5 ml tube, and 90 μl of medium was added thereto, and the sperm concentration was maintained at 450,000/ml.
After storage at room temperature for 7 days or at 4°C for 8 days, fertilization experiments were performed using eggs prepared from female medaka, with 100 μl (45,000 sperm) per tube. The eggs were cultured until the sac-embryo stage, and the fertilization rate was counted. The results are shown in Figure 7. In Figure 7, the vertical axis indicates the fertilization rate (%). In some of the sperm stored at room temperature using the medium of Example 4, the color of the sperm storage solution became yellow to orange after about one week. This is presumed to be due to proliferation of somatic cells derived from the testes. It was confirmed that the fertilization ability of the sperm was otherwise 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, sodium phenol red, and sodium pyruvate were used in the same amounts as those used for the zebrafish in Example 4, and calcium chloride dihydrate and potassium chloride were used in 1/3 the amounts of those used for the zebrafish in Example 4. A 20 mM Hepes (pH 8.8) medium was prepared (Example 7). The mouse cauda epididymis was cut with scissors, and the sperm squeezed out were suspended in 0.5 ml of the above culture solution to prepare a sperm suspension.
After storing at room temperature (22-23°C) for 4 days, about 0.02 ml of the lower layer was added to a 0.2 ml drop of CARD FERTIUP mouse sperm preculture medium in a sperm preculture dish, precultured at 37°C for 60 minutes, and the sperm was added to an mHTF drop for fertilization containing unfertilized eggs for insemination. Three hours after insemination, only morphologically normal eggs were washed with the mHTF drop. The next day, embryos that had developed into 2 cells were transferred to embryo culture medium KSOM, and the eggs were cultured to blastocysts, after which 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 five repeated experiments using the medium of Example 4, and Examples 7-1 to 7-5 show the results of five repeated experiments using the medium of Example 7.
As shown in Table 8, five repeated experiments confirmed that the medium of Example 7 has excellent preservation ability.

Claims (7)

A method for preserving sperm, comprising suspending sperm collected from fish in a sperm preservation solution and preserving the resulting sperm suspension at room temperature, comprising:
The sperm preservation solution contains inorganic salts, amino acids, vitamins, fetal bovine serum, bovine serum albumin, and 0.06 mM to 2 mM lactic acid. The sperm preservation method according to claim 1, wherein the sperm preservation solution further contains a buffer and has a pH of 6.5 to 10.0. The sperm preservation method according to claim 1 or 2, wherein the sperm preservation solution further contains glucose. The sperm preservation method according to any one of claims 1 to 3, wherein the sperm preservation solution further contains an antioxidant. The sperm preservation method according to any one of claims 1 to 4, wherein the sperm preservation solution further contains penicillin and/or streptomycin. The fish is a zebrafish,
The sperm preservation solution contains inorganic salts including, as final concentrations, 0.1 g/L to 0.25 g/L of calcium chloride dihydrate, 0.15 g/L to 0.25 g/L of magnesium chloride hexahydrate, 0.06 g/L to 0.12 g/L of anhydrous magnesium sulfate, 0.3 g/L to 0.6 g/L of potassium chloride, 0.04 g/L to 0.08 g/L of anhydrous potassium dihydrogen phosphate, 6.0 g/L to 10.0 g/L of sodium chloride, and 0.15 g/L to 0.25 g/L of anhydrous disodium hydrogen phosphate;
DL-alanine 0.3g/L to 0.6g/L, L-arginine (base free) 0.35g/L to 0.65g/L, anhydrous L-asparagine 0.15g/L to 0.35g/L, L-cysteine (base free) 0.08g/L to 0.2g/L, L-glutamine 0.2g/L to 0.4 g/L, glycine 0.15g/L to 0.35g/L, L-histidine 0.15g/L to 0.35g/L, DL-isoleucine 0.13g/L to 0.35g/L, L-leucine 0.08g/L to 0.2g/L, L-lysine 0.055g/L to 0.095g/L, DL-methionine 0.08g/L to 0.25g/L, DL-phenylalanine 0.15g/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 0.35 g/L;
vitamins including 0.0008g/L to 0.0012g/L choline chloride, 0.00008g/L to 0.00012g/L flavin mononucleotide sodium salt, 0.0008g/L to 0.0012g/L folic acid, 0.0015g/L to 0.0025g/L myo-inositol, 0.0008g/L to 0.0012g/L nicotinamide, 0.0008g/L to 0.0012g/L DL-pantothenic acid hemicalcium salt, 0.0008g/L to 0.0012g/L pyridoxine hydrochloride, and 0.0008g/L to 0.0012g/L thiamine monophosphate hydrochloride;
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;
Sodium pyruvate 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 8mM-15mM (pH 7-8);
Glucose 3 g/L to 5.5 g/L;
Lactic acid 0.06 mM to 0.15 mM,
The method for preserving sperm according to any one of claims 1 to 5, comprising: The fish is a medaka,
The sperm preservation solution contains inorganic salts including, as final concentrations, 0.1 g/L to 0.25 g/L of calcium chloride dihydrate, 0.15 g/L to 0.25 g/L of magnesium chloride hexahydrate, 0.06 g/L to 0.12 g/L of anhydrous magnesium sulfate, 0.3 g/L to 0.6 g/L of potassium chloride, 0.04 g/L to 0.08 g/L of anhydrous potassium dihydrogen phosphate, 6.0 g/L to 10.0 g/L of sodium chloride, and 0.15 g/L to 0.25 g/L of anhydrous disodium hydrogen phosphate;
DL-alanine 0.07g/L to 0.6g/L, L-arginine (base free) 0.15g/L to 0.65g/L, anhydrous L-asparagine 0.08g/L to 0.35g/L, L-cysteine (base free) 0.045g/L to 0.2g/L, L-glutamine 0.085g/L to 0.4 g/L, glycine 0.08g/L to 0.35g/L, L-histidine 0.07g/L to 0.35g/L, DL-isoleucine 0.07g/L to 0.35g/L, L-leucine 0.04g/L to 0.2g/L, L-lysine 0.02g/L to 0.095g/L, DL-methionine 0.045g/L to 0.25g/L, DL-phenylalanine 0.085g/L to 0.35g/L, L-serine 0.08g/L to 0.3g/L, DL-threonine 0.2 to 0.8g/L, L-tryptophan 0.01 to 0.02g/L, L-tyrosine (base free) 0.085 to 0.4g/L, and DL-valine 0.085 to 0.35g/L;
vitamins including choline chloride 0.0004g/L to 0.0012g/L, flavin mononucleotide sodium salt 0.00004g/L to 0.00012g/L, folic acid 0.0004g/L to 0.0012g/L, myo-inositol 0.0007g/L to 0.0025g/L, nicotinamide 0.0004g/L to 0.0012g/L, DL-pantothenic acid hemicalcium salt 0.0004g/L to 0.0012g/L, pyridoxine hydrochloride 0.0004g/L to 0.0012g/L, and thiamine monophosphate hydrochloride 0.0004g/L to 0.0012g/L;
D-galactose 0.3 g/L to 1.2 g/L,
Phenol red sodium salt 0.004 g/L to 0.013 g/L;
Sodium pyruvate 0.2 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 8mM-15mM (pH 7-8);
Glucose 3 g/L to 5.5 g/L;
Lactic acid 0.06 mM to 0.35 mM,
The method for preserving sperm according to any one of claims 1 to 5, comprising:

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