KR100944145B1 - Cryopreservation compound of sperm having amide and freezing method of sperm - Google Patents

Abstract

The present invention relates to a cryopreservative and a freezing method of sperm containing an amide, more specifically, a freeze preservative and a freeze-preserving method using an amide (Mamideformamide, Dimethylformamide, Urea) It is about.

 In the present invention, instead of glycerol, which has a limit as a cryopreservative, it is toxic to living cells, and in particular, sperm having a lower molecular weight than glycerol, which has a negative effect on the quality of sperm in swine, which is more sensitive than the sperm of other mammalian animals. The application of amides (Methylformamide, Dimethylformamide, Urea), which are more effective than glycerol, while acting as a cryopreservative, can confirm the improved motility and survival rate of the crystallites. Therefore, the cryopreservative containing the amide substance of the present invention and the method of freezing sperm using the same have high value in the field of biotechnology, and in particular, may contribute to the development of the livestock industry through the preservation, breeding and maintenance of pig breeds. .

Sperm freezing, cryopreservatives, distillates for automatic freezing, amide

Description

Cryopreservation compound of sperm having amide and freezing method of sperm

The present invention The present invention relates to a cryopreservative comprising amide and a method for freezing sperm as a useful material for the use of frozen sperm.

Sperm freezing and low temperature storage are useful for the preservation and propagation of breeds such as livestock and are an active area of research in biotechnology. In particular, during the freezing process, sperm are damaged by intracellular ice crystal formation, difference in osmotic pressure and low temperature stress. The agent that reduces such damage is a cryopreservative and a representative material is glycerol. The raw semen used in the freezing process is dilute using a diluent because of its high concentration. The cryopreservative is administered together with the diluent during the dilution process. Glycerol is a toxic substance to cells and, in particular, sperm from pigs is more susceptible to external stimuli than sperm from other livestock animals and can be easily damaged by glycerol.

Sperm freezing is divided into different classes depending on the diluent used. The method used in this experiment was to use Beltsvile F5 (BF5; Pursel and Johnson, 1975) as the diluent. BF5 contains egg yolk-Tes-tris-glucose-OEP and is used for primary dilution of sperm. In the existing experiments by this freezing method, Glycerol was added to the BF5 diluent and used as a secondary diluent. Glycerol was found to be superior to other cryopreservatives after lysing of frozen sperm. Toxicity has a negative effect on the effects of high concentrations, but when used in high concentrations will result in deterioration of sperm quality. The other reagent used is Hulsen solution (Dtseh Tierarztl Wsher 82: 155-162, 1975), which contains glucose-lactose-citrate-EDTA-potassium, and is used for the segregation of raw semen.

Amide to demonstrate the effect in the present invention was applied to the sperm of the horse (Stallion) in the existing experiments, glycerol and five amide substances in skim milk-egg yolk (SEMY) diluent different from the diluent used in the present invention (Methylformamide, Dimethylformamide, Formamide, Acetamide, Methylacetamide) were added as cryopreservative. After applying the same concentration of six substances as cryopreservatives, we measured the motility after melting of the crystallites. The percent motility of the frozen sperm group using Methylformamide and Dimethylformamide was similar to that of the glycerol group. Indicated. This suggests that these two amide substances effectively reduce damage in horse sperm freezing (E.L. Squires, 2003).

In this regard, the present inventors applied the two amide substances and another amide substance, Urea, as a cryopreservative in the freezing process of swine sperm in consideration of the characteristics that the sperm of pigs are more susceptible to external stress than other livestock animals. As a result of comparing with the effect of the present invention has been achieved.

It is an object of the present invention to stably act on sperm with a lower molecular weight than glycerol, which is toxic to living cells and has a negative effect on the quality of sperm of swine, which is more sensitive than the sperm of other mammalian animals. It is to provide a cryopreservative of sperm containing a better amide material.

It is another object of the present invention to provide a distillation solution for automatic freezing, which is used for cryopreservation of sperm containing an amide substance.

Still another object of the present invention is to provide a method of freezing sperm by using the above-proven effect of amide.

This aims to contribute to the development of biotechnology using freezing technology of sperm and, in particular, the development of the livestock industry through the preservation, breeding and maintenance of pig breeds.

Three types of amides are used to replace glycerol, which is commonly used as a cryopreservative for sperm, but has a negative effect on the quality of sperm, and each amide instead of glycerol is used in the diluent used in the process of freezing sperm. I put (Amide). To demonstrate the cryopreservation effect of this substance, the motility, survival, and NAR after thawing of frozen sperm of pigs were measured and compared with the effects of conventional glycerol.

The molecular formula and molecular structure of the three amide materials used in the present invention are as follows.

Methylformamide (C ₂ H ₅ NO)

Dimethylformamide (C ₃ H ₇ NO)

3.Urea (CO (NH ₂ ) ₂₎

The present invention relates to a cryopreservative of sperm comprising an amide (Amide) as an active ingredient, wherein the amide is a cryopreservative, which is any one of methylformamide, dimethylformamide or urea. Include. Preferably the methyl formamide, dimethylformamide or urea is characterized in that it comprises a concentration of 2 to 4% (w / v), and also relates to a cryopreservative, characterized in that the sperm is a sperm of pig It includes more.

The present invention also relates to a diluent for freezing sperm added to the first diluent for freezing sperm, the first diluent is TES 12g / 1000mL, Tris 2g / 1000mL, glucose 32g / 1000mL, OEP 5g / 1000mL, Gentamycin It further comprises a diluent for freezing sperm, characterized in that consisting of 0.02g / 1000mL sulfate and 200g / 1000mL egg yolk (egg yolk) mixed in purified water. Preferably, the diluent for autoclaving includes the above cryopreservative including amide.

On the other hand, the present invention comprises the steps of: primary cooling the first diluent for sperm frozen semen and sperm; (C) mixing the cooled raw semen with a formal separation solution and centrifuging to remove the supernatant (suit) while preserving sperm; 희석 first diluting the semen from which the intestinal tract was removed with the first diluent for freezing sperm to a sperm freezing concentration; 2 second cooling the second diluent for freezing sperm containing the first diluted sperm and cryopreservative; ⑸ diluting the second cooled sperm in a secondary diluent at a ratio of volume by 1: 1 to 2 times, and leaving it on ice until frozen by mixing; and sperm freezing method comprising the above. Sperm is preferably swine sperm.

In the sperm freezing method, the first dilution solution is 12g / 1000mL, TES 2g / 1000mL, glucose 32g / 1000mL, OEP 5g / 1000mL, Gentamycin sulfate 0.02g / 1000mL and egg yolk (egg yolk) 200g / 1000mL in purified water It further comprises a method characterized by consisting of a mixture, the formal separation solution is glucose (glucose) 28.75 g / 500mL, α-lactose (α-lactose) 1.25 g / 500mL, C ₆ H ₅ Na ₃ O ₇ 2.25 g / 500 mL of 2H ₂ O (Sodium citrate, 2H ₂ O), 1.75 g / 500 mL of Na ₂ -EDTA (disodium ethylenediaminetetraacetic acid), 0.60 g / 500 mL of NaHCO ₃ , 0.20 g / 500 mL of KCl and 0.01 g / 500 mL of Gentamycin sulfate It further comprises a method characterized by consisting of mixed with purified water.

Preferably, in the automatic self-determination method, the primary cooling comprises cooling to 15 ℃, and the step 혼합 at 1: 17 ℃ by mixing the raw semen and formal separation solution in a ratio of 1: 2 by volume Further comprising centrifuging for 3 minutes, and the secondary cooling further comprises cooling to 5 ° C.

In addition, in the automatic crystallization method, the concentration of the sperm that can be frozen preferably comprises 10 ⁹ / mL, the second diluent further comprises that the cryopreservative is added to the first diluent, and The cryopreservative contained in the second diluent further includes the cryopreservative including the amide.

More preferably, the automatic crystallization method relates to a sperm of a pig.

In the present invention, instead of glycerol, which has a limitation as a cryopreservative, amide having a smaller molecular weight and a smaller molecular size than glycerol is easily penetrated through the sperm cell membrane and less toxic to living cells, is an excellent cryopreservative than glycerol in sperm freezing. This can be useful. In addition, it can be used to freeze sperm more efficiently. The improved motility and survival rate of frozen sperm by this material is very high in value, and it is expected that freezing of sperm and its application will be easier than before.

Hereinafter, embodiments of the present invention will be described in detail. The following examples are intended to illustrate the invention, but the invention is not limited thereto.

<Example 1>

1. Pig for sperm freezing Dilution  Produce

(1) primary Dilution  Produce

The diluent used in the present invention is Beltsvile F5 (BF5; Pursel and Johnson, 1975), where the raw semen was first diluted. The components and ratios of the BF5 dilutions are shown in a simple table. The preparation of the solution used in the experiment was made on the day before the preparation of the frozen semen to maintain the freshness of the solution.

BF5 solution (first diluent, J Anim Sci 40: 99-102, 1975) Component g / 1,000 mL Manufacture TES Trizma Base (Tris) D (+)-Glucose OEP Gentamycin sulfate Egg yolk 12.00 2.00 32.00 5.00 (5 mL) 0.02 200 mL Sigma T-1375 Sigma T-1503 Ishizu 012-3333 Sigma G 1264

#One. Dissolve TES, Tris, glucose, and Gentamycin sulfate in 800 mL of purified water, add OEP using a plastic syringe, and mix them together.

#2. If the OEP is as viscous as grease, add it to the warm bath.

# 3. Break the egg and roll only egg yolk on the filter paper to remove excess egg whites.

(2) secondary Dilution  Produce

Diluted solution about 1L was prepared in consideration of the difference in the concentration of raw semen used in each experiment. Three Amide materials and a control group (no addition / glycerol) were added to the prepared BF5 to prepare a solution for secondary dilution. At this time, the concentration of Amide substances added to BF5 was equal to 2% (v / v), the optimal concentration of glycerol as a cryopreservative.

The components and ratios of the secondary dilutions are shown in a simple table.

Second Diluent (J Anim Sci 40: 99-102, 1975) Component Volume group 1.BF5 500 mL group 2.BF5 + Glycerol (2%) 490mL + 10mL group 3.BF5 + Methylformamide (2%) 490mL + 10mL group 4.BF5 + Dimethylformamide (2%) 490mL + 10mL group 5.BF5 + Urea (2%) 490mL + 10mL

2. Preparation of Formal Separation Solution

The seminal fluid of semen decreases the freezing ability of sperm and should be removed. For this purpose, a Hulsen solution was prepared. The components and ratios of the Hulsen solution are shown in [Table 3].

Hulsen solution (Dtseh Tierarztl Wsher 82: 155-162, 1975) Component g / 500 mL Manufacturer D (+)-Glucose 28.75 Ishizu 012-3333 α-Lactose 1.25 Sigma L2643 Sodium citrate2H₂O (C ₆ H ₅ Na₃O ₇ · 2H₂O) 2.25 Ishizu 014-4123 Na₂-EDTA 1.75 Sigma ED₂SS NaHCO₃ 0.60 Ishizu KCl 0.20 Ishizu Gentamycin sulfate 0.01 Sigma G1264

3. Freezing method of pig sperm

(1) Conditions of semen used

Sperm concentration and sperm activity of raw semen from boar were examined prior to freezing of swine sperm. Sperm concentration was diluted 400 times (volume to volume) with purified water, counted three times with hematocytometer, and then calculated as the average value. Sperm vitality was determined based on 80% or more. Inferior raw semen was not used in the experiment.

(2) freezing method of pig sperm

The water temperature of the water-bath was adjusted to the temperature of the collected semen, and the mixture was first cooled to 15 ° C. over 1 hour. This BF5 diluent was also placed in a bath with semen and cooled to the same temperature as semen.

In order to separate the suit, 10 mL of primary-cooled raw semen was divided into 5 tubes (None, Glycerol, Methylformamide, Dimethylformamide, and Urea), and 20 mL of the prepared Hulsen solution was added to 30mL in total. The semen mixed with Hulsen solution was centrifuged at 2400C for 3 minutes at 2400g.

After centrifugation, the supernatant (suit) was removed while preserving sperm pellets using a Pasteur pipette.

Each of the five tubes from which the suit was removed was first diluted with a BF5 dilution. The first dilution step is to achieve a suitable sperm concentration (10 ⁹ / mL) for freezing. For example, if the concentration of the counted raw semen is 2 × 10 ⁹ / mL, if the amount of semen in each tube was 10mL during the formal separation process, the sperm count of one tube would be 2 × 10 ^ 10 / centrifuge. If 20 mL of BF5 diluent is added to the centrifuge tube, the sperm concentration will be 10 ⁹ / mL, but the second dilution will be diluted 1: 1 so that only 10 mL of BF5 is added to make the sperm concentration 2 × 10 ⁹ / mL. Dilute the BF5 solution into each tube and mix well with sperm with a Pasteur pipette.

Primary diluted sperm (15 ° C.) was secondary cooled to 5 ° C. in the refrigerator for 1 hour. At this time, the secondary diluent is also cooled to the same temperature. Sperm cooled to 5 ° C. were diluted 1: 1 to 2 times with a secondary diluent to which a cryopreservative was added, and left to stand on ice until frozen by mixing.

4. Secondary In diluent  Included in the added cryopreservative Amide  effect

(1) Samples were prepared using a dilution of five groups of sperm and 0.5 mL straw. (15 for each tube). Put the straw in the syringe, soak the final diluent, leaving only about 1cm, seal it carefully to prevent air from entering the straw, and then leave it in ice water.

Before entering the cryogenic freezing process with liquid nitrogen, the liquid nitrogen vapor was preliminarily frozen and the colored straws were immersed in liquid nitrogen.

When the work is done, place the straw in the canister and store it in a liquid nitrogen tank.

(2) measurement of sperm motility

After the addition to the liquid nitrogen tank and time passed, the straw sample was taken out and melted at 50 ° C. for 15 seconds. After thawing each group of five straws, motility was observed using a phase contrast microscope, and% values were calculated from the number of moving sperm per 100 frozen sperm.

Cryopreservative added to the secondary diluent Motility (%) None 3% Glycerol 35% Methylformamide 32% Dimethylformamide 45% Urea 40%

Dimethylformamide (2% (w / v)) was the best after fusion and showed better cryopreservation effect than glycerol. In addition, the group using Urea (2% (v / v)) showed higher motility than glycerol, and Methylformamide (2% (w / v)) was lower than glycerol but showed similar motility. Meanwhile, the group without any cryopreservatives showed low motility of less than 3%.

(3) sperm Survival rate  Measure

Flow cytometry was used to measure the survival after freezing of frozen sperm stained with SYBR-14 and PI (control staining), a membrane-permeable nuclear dye proven by Molecular Probes in 1993. As shown in Figure 1, SYBR-14 stains the DNA of surviving sperm green after fusion, and PI stains the dyed sperm DNA as a control stain of SYBR-14. .

Figure 1. Survival measurement after freezing of frozen sperm>

Through flow cytometry, the number of viable crystallites (stained in green) after fusion was measured and calculated as% values. It is expressed as the percentage of viable sperm per 100 frozen frozen sperm.

Cryopreservative added to the secondary diluent Viability (%) None 6% Glycerol 45% Methylformamide 43% Dimethylformamide 47% Urea 45%

Among the three amides, the survival rate of the dimethylformamide group was highest (47%) compared to the group using glycerol as a cryopreservative. %). The group without any cryopreservatives (6%) had extremely low survival rates.

These results confirm that cryopreservatives reduce damage to sperm during the freezing process.

(4) normality of sperm Acrosome  Rate measurement

NAR (Normal Acrosome Ridge) is the ratio of normal acrosome after thawing of frozen sperm and can be measured to determine the degree of sperm damage. Spermac stain was used to measure NAR. Staining was performed according to Spermac kit manufaturer's guidelines (Stain Enterprises, Onderstepoort, South Africa). The frozen frozen sperm was smeared on a slide, dried in air, fixed with Fixin containing formalin, stained for 5 minutes in stain solution A, 1 minute in stain solution B, and 1 minute 3 steps in stain solution C. After staining, a total of 100 sperm were observed under an optical microscope.

Normal acrosome of sperm head is dyed green, thin dark green band at the tip of head and equatorial band is dyed red. However, if the acrosome is damaged, the green strip at the tip of the head is broken.

Figure 2. Results of Spermac stain for NAR measurement>

The sperm with normal acrosome and damaged acrosome were distinguished from each other, and the percentage of sperm with normal acrosome out of 100 sperm was calculated as% value.

Cryopreservative added to the secondary diluent NAR (%) None 17% Glycerol 30% Methylformamide 37% Dimethylformamide 38% Urea 33%

Higher NAR levels indicate less damage to the acrosome from freezing. In the present invention, the results of the group using the three amides (Amide) is slightly higher than the NAR value of the group using glycerol as a cryopreservative.

The group without any cryopreservatives had low NAR levels, indicating that cryopreservatives reduce sperm damage.

<Example 2>

This example relates to the determination of the appropriate concentration of Urea in the three amide (amide) materials used as cryopreservatives.

Different secondary dilutions were prepared at concentrations of 2%, 4%, 6%, and 8% of urea, and the control group used secondary dilutions without the addition of cryopreservatives. It was divided into five groups (0%, 2%, 4%, 6%, 8% (w / v)).

In cryopreservatives Urea Check the effect according to the concentration

(1) measurement of sperm motility

The motility of the frozen frozen sperm in each group was observed by phase contrast microscope, and the motility was measured by the ratio of sperm having motility per 100 sperm. The motility after thawing of frozen sperm for each of the five groups is shown in Table 7.

% Concentration of urea (w / v) Motility (%) None 2% 2% 40% 4% 25% 6% 2% 8% 2%

The table shows that the group using 2% (w / v) Urea as cryopreservative showed the best freeze sperm motility. The degree of motility does not differ significantly from sperm before freezing. The group without urea showed the lowest motility, and the sperm motility decreased as the concentration of urea increased.

(2) sperm Survival rate  Measure

Flow cytometry using SYBR-14 and PI staining is performed to measure the survival rate after freezing of frozen sperm. The method is the same as in Example 1, and the survival rate of each group is shown in [Table 8].

% Concentration of urea (w / v) Viability (%) None 6% 2% 40% 4% 28% 6% 10% 8% 9%

As a result of motility after thawing frozen sperm according to the difference in concentration of urea, the survival rate was the highest in the group using 2% (w / v) urea. The group without any cryopreservatives showed extremely low survival rates.

(3) normality of sperm Acrosome  Rate measurement

Next, the results of NAR measurement after thawing of five groups of frozen sperm are shown in [Table 9]. The method is the same as in Example 1.

% Concentration of urea (w / v) NAR (%) None 13% 2% 28% 4% 14% 6% 10% 8% 10%

As described in Example 1, a high NAR value means that sperm suffered less damage during freezing. Just as Urea of 2% (w / v) showed the highest motility among the five groups, NAR was also the highest. The results of the rest of the groups showed no significant difference, but NAR levels tended to decrease with increasing Urea concentration.

Claims (17)

Cryopreservative of pig sperm containing urea (Urea) as an active ingredient. delete The method of claim 1, The urea is a cryopreservative, characterized in that contained at a concentration of 2 ~ 4% (w / v). delete A diluent for freezing swine sperm to which a cryopreservative according to claim 1 is added to the first diluent for freezing sperm. The method of claim 5, The first diluent is composed of a mixture of TES 12g / 1000mL, Tris 2g / 1000mL, glucose 32g / 1000mL, OEP 5g / 1000mL, Gentamycin sulfate 0.02g / 1000mL and egg yolk (egg yolk) 200g / 1000mL in purified water Diluent for freezing sperm. delete (B) first cooling each of the primary diluent for sperm and sperm freezing; (C) mixing the cooled raw semen with a formal separation solution and centrifuging to remove the supernatant (suit) while preserving sperm; 희석 first diluting the semen from which the intestinal tract was removed with the first diluent for freezing sperm to a sperm freezing concentration; Secondly cooling the first diluted sperm and the second diluent for freezing sperm containing the cryopreservative according to claim 1 or 3; 희석 distilling the second cooled sperm in a second diluent in a ratio of volume 1-2: 1 and mixing and leaving it on ice until freezing; swine sperm freezing method comprising a. The method of claim 8, The first diluent is composed of a mixture of TES 12g / 1000mL, Tris 2g / 1000mL, glucose 32g / 1000mL, OEP 5g / 1000mL, Gentamycin sulfate 0.02g / 1000mL and egg yolk (egg yolk) 200g / 1000mL in purified water How to. The method of claim 8, The formal separation solution was glucose 28.75 g / 500 mL, α-lactose 1.25 g / 500 mL, C ₆ H ₅ Na ₃ O ₇ 2H ₂ O (Sodium citrate, 2H ₂ O) 2.25 g / 500 mL, Na ₂ -EDTA (disodium ethylenediaminetetraacetic acid) 1.75 g / 500 mL, NaHCO ₃ 0.60 g / 500 mL, KCl 0.20 g / 500 mL, and Gentamycin sulfate 0.01 g / 500 mL are mixed with purified water. The method of claim 8, The primary cooling is characterized in that the cooling to 15 ℃. The method of claim 11, The step 는 is characterized in that the mixture of raw semen and intestinal separation solution 1: 2 in a ratio to the volume and centrifuged for 3 minutes at 17 ℃. The method of claim 12, Secondary cooling is characterized by cooling to 5 ° C. The method of claim 8, The freezing sperm concentration is 10 ⁹ / mL method. delete delete delete