KR101413081B1 - Composition for preservation of avian sperm and preserving method using the same - Google Patents

Abstract

The present invention relates to a composition for preserving algae sperm and a method of preserving sperm using the same. According to the present invention, it is possible to enhance the efficiency of conserving algae genetic resources by promoting viability and survival of spermatozoa in the process of conserving (albeit chilled, cryopreserved and thawed) the algae genetic resources.

Description

The present invention relates to a composition for preserving avian sperm and a method for preserving avian sperm using the same.

The present invention relates to a composition for preserving algae sperm and a method for preserving sperm using the same.

Sperm freezing and cryopreservation are useful for the preservation and breeding of varieties such as livestock and are actively researched in biotechnology. In particular, during freezing, sperm are damaged by intracellular ice formation, osmotic pressure differences, and cold stress. Glycerol is a typical cryoprotectant. Since the concentration of the original semen used in the freezing process is diluted with a diluent, the cryopreservative is dispensed with the diluent during the dilution process. Glycerol is a toxic substance in cells, and the spermatozoa of birds can be easily damaged by glycerol because they are highly susceptible to external stimuli.

In order to preserve the genetic resources of birds and to increase the availability of sperm, spermatozoa preserve viable spermatozoa in a liquid form at low temperature or by cryopreservation. In the former case, it is usually kept at 4 degrees Celsius and preserved for a relatively short time. In the latter case, it is frozen and melted if necessary. As a genetic resource of algae, sperm are sensitive to cold and cryopreservation and are limitedly used for refrigeration and cryopreservation. The present invention aims to develop a basic culture solution used for refrigeration, cryopreservation and fusion of avian spermatozoa, and to develop a culture solution for enhancing survival and vitality.

Prior arts in this field include Korean Registered Patent No. 0666595 and Korean Published Patent Application No. 2007-0007836.

Under these technical backgrounds, the present inventors have made intensive efforts to complete the present invention.

It is an object of the present invention to provide a composition for preserving algae sperm and a method of preserving sperm using the same.

According to one aspect of the present invention, there can be provided a composition for preserving sperm count comprising alanylglutamine as an active ingredient.

According to one embodiment, the preservative composition is selected from the group consisting of alanyl glutamine, glucose, fructose, inositol, sodium dihydrogen phosphate, sodium hydrogen phosphate Sodium hydrogen phosphate, and potassium citrate monohydrate.

According to one embodiment, the preservation composition is prepared by mixing 96 mM alanyl glutamine, 39 mM glucose, 11.1 mM fructose, 39 mM Inositol, sodium dihydrogen phosphate ) 17.5 mM, sodium hydrogen phosphate 70 mM, and potassium citrate monohydrate 4.31 mM.

According to one embodiment, the composition for preserving sperm may further comprise 0.1% (w / v) PVP (polyvinylpyrrolidone, MW = 1,000).

According to one embodiment, the alanylglutamine may be replaced with a mixture of glutamine and alanylglutamine so that the concentration of alanylglutamine is 48 mM.

According to one embodiment, the sperm may be a sperm of a bird.

According to another aspect of the present invention,

(1) cooling the original semen and the sperm storage composition;

(2) mixing the cooled original sperm and the cooled sperm storage composition to dilute the sperm to a frozen concentration; And

(3) preserving the diluted solution in a cooled state;

A method of preserving the sperm containing the sperm may be provided.

According to the present invention, it is possible to enhance the efficiency of conserving algae genetic resources by promoting viability and survival of spermatozoa in the process of conserving (albeit chilled, cryopreserved and thawed) the algae genetic resources.

Figure 1 shows the sperm motility and activity of glutamine and alanylglutamine treated groups immediately after dilution.
FIG. 2 shows the sperm motility and activity of the glutamine and alanylglutamine-treated groups under the culture conditions of 3 hours at 4 ° C after dilution.
FIG. 3 shows the results of comparison of sperm motility and activity of glutamine and alanylglutamine-treated groups under 24 hour culture conditions at 4 degrees Celsius after dilution.
FIG. 4 is a graph showing sperm motility and activity of glutamine and alanylglutamine-treated groups at 72 ° C in a 4 ° C environment after dilution.
FIG. 5 is a graph showing sperm motility and activity of glutamine and alanylglutamine-treated groups at 96 hours in a 4 ° C environment after dilution.
FIG. 6 is a graph showing the sperm motility (Gln) activity of glutamine (Gln), mixture of glutamine and alanylglutamine (Gln + Ala-Gln) and alanylglutamine (Ala-Gln) And activity.

Hereinafter, the present invention will be described in more detail.

Justice

In the present invention, the motility of the spermatozoa is expressed as a percentage of the sperm tail's advancement and absence.

In the present invention, the sperm having excellent sperm activity is alive, and the sperm motility of the moving sperm is active to express the relative value of the mobility. In the present invention, when the sperm activity is stored and analyzed as a moving image, Means a sperm that is at least twice the mean per unit time and is expressed as a percentage of the observed sperm.

The present invention relates to a composition for preserving algae sperm and a method of preserving sperm using the same.

According to one aspect of the present invention, there can be provided a composition for preserving sperm count comprising alanylglutamine as an active ingredient.

According to one embodiment, the preservative composition is selected from the group consisting of alanyl glutamine, glucose, fructose, inositol, sodium dihydrogen phosphate, sodium hydrogen phosphate Sodium hydrogen phosphate, and potassium citrate monohydrate.

According to one embodiment, the preservation composition is prepared by mixing 96 mM alanyl glutamine, 39 mM glucose, 11.1 mM fructose, 39 mM Inositol, sodium dihydrogen phosphate ) 17.5 mM, sodium hydrogen phosphate 70 mM, and potassium citrate monohydrate 4.31 mM.

According to one embodiment, the composition for preserving sperm may further comprise 0.1% (w / v) of PVP (polyvinylpyrrolidone, MW = 1,000), and the composition may be used for measuring motility and activity of sperm in the present invention .

According to one embodiment, the alanylglutamine may be replaced with a mixture of glutamine and alanylglutamine so that the concentration of alanylglutamine is 48 mM. More preferably 48 mM of the alanylglutamine and 48 mM of the glutamine.

According to one embodiment, the sperm may be a sperm of a bird.

According to another aspect of the present invention,

(1) cooling the original semen and the sperm storage composition;

(2) mixing the cooled original sperm and the cooled sperm storage composition to dilute the sperm to a frozen concentration; And

(3) preserving the diluted solution in a cooled state;

A method of preserving the sperm containing the sperm may be provided.

In the method for preserving sperm, the primary cooling includes cooling to 5 degrees Celsius.

The step (2) may be performed by mixing the cooled raw semen with the formal separation solution, centrifuging the supernatant, and removing the supernatant while preserving the sperm. In this case, it may further include centrifugation at 5 ° C. for 3 minutes by mixing the original semen and the formal separation solution at a ratio of 1: 1 to 3, more preferably 1: 2.

In the method for preserving sperm, the concentration of the sperm capable of freezing includes from 10 ⁸ to 10 ¹⁰ cells / mL, preferably from 10 ⁹ cells / mL.

The composition for preserving sperm of the present invention enhances the function of a seminal diluent for preserving algae sperm by replacing glutamine with alanyl glutamine or enhancing function. Although alanyl glutamine is known to be superior in physiological stability to glutamine by changes in temperature and pH, there is no report on the superiority of semen freezing and cryopreservation with sperm diluent composition.

The present invention provides a composition for preserving sperm that contains alanylglutamine as an effective ingredient in a basic dilution of bird's sperm.

The main components of the semen-diluted solution mentioned in the present invention are phosphoric acid, saccharides and citric acid. The composition of the phosphate buffer used in the present invention is 39 mM glucose, 11.1 mM fructose, 39 mM inositol, 17.5 mM sodium dihydrogen phosphate, and sodium hydrogen phosphate hydrogen phosphate 70 mM and potassium citrate monohydrate 4.31 mM.

To analyze the motility and activity of sperm, 0.1% of PVP (polyvinylpyrrolidone, MW = 1,000) was further added to the basic diluent.

For selection of avian sperm, chick sperm were used which are known to be most sensitive to cold storage and cryopreservation.

Glutamine is a major component of algal fluid and is known to be important for the survival and motility of sperm, unlike mammals, and is widely used for algal dilution. The alanyl glutamine used in the present invention is a two peptide complex in which alanine and glutamine are combined and is known as a substance participating in glutamine metabolism by decomposition in a cell and has excellent heat stability.

Hereinafter, the present invention will be described in more detail with reference to Examples. It should be understood, however, that these examples are for illustrative purposes only and are not to be construed as limiting the scope of the present invention.

Example

In the present invention, sperm were preserved through the following steps. First, the original semen and the composition for preserving the sperm were first cooled to 5 degrees Celsius respectively, and then the cooled original sperm was mixed with the formal separation solution and stored at the refrigeration temperature.

Then, semen containing formalin was diluted with 10 ⁹ cells / mL of the cooled spermatogonium composition containing alanylglutamine as an active ingredient, and the diluted solution was cooled to 4 degrees Celsius and stored.

The following Examples 1 to 6 show different examples of sperm motility and activity difference according to the time and the amount of alanylglutamine stored after cooling at 4 degrees Celsius, and the results are shown graphically in the figure.

Example  One

To compare the effects of glutamine and alanylglutamine, 96 mM of each of these substances was added to the basic diluent, and fresh chicken semen collected from the same individual was diluted 1: 8, and the activity of the sperm was measured after 1 hour .

As shown in FIG. 1, the proportion of motile spermatozoa in the glutamine-treated group was 84.46 ± 3.26%, and the ratio of spermatozoa with activity was 31.22 ± 6.76%. The percentage of motile sperm in the alanylglutamine - treated group was 82.86 ± 5.11% and the percentage of spermatozoa with activity was 24.4 ± 4.83%.

That is, there was no significant difference between the two treatment groups immediately after dilution. (p = 0.05)

Example  2

Sperm was diluted in the same ratio as in Example 1, and then the results of the motility of the spermatozoa stored for 3 hours at a temperature of 4 degrees Celsius were observed.

Referring to FIG. 2, the percentage of motile spermatozoa in the glutamine-treated group was 90.24 ± 2.94%, and the ratio of spermatozoa with activity was 59.58 ± 7.33%. The ratio of motile spermatozoa in the alanylglutamine treated group was 96.54 ± 0.36%, and the ratio of spermatozoa with activity was 72.82 ± 1.77%.

In other words, it can be seen that there is a significant difference in the proportion of motile sperm and the activity and the ratio of excellent sperm between the two treatment groups during low temperature preservation, and the sperm motility and activity of the sperm in the treatment with alanylglutamine are higher appear. (p = 0.05)

Example  3

Sperm was diluted in the same ratio as in Example 1, and the results of the motility of the spermatozoa stored for 24 hours at a temperature of 4 degrees Celsius were observed.

Referring to FIG. 3, the proportion of motile spermatozoa in the glutamine-treated group was 62.04 ± 7.2%, and the ratio of spermatozoa with activity was 40.58 ± 2.29%. The percentage of motile sperm in the alanylglutamine - treated group was 75.44 ± 2.59% and the percentage of spermatozoa with activity was 46.3 ± 2.42%.

In other words, it can be seen that there is a significant difference in the proportion of motile sperm and the activity and the ratio of excellent sperm between the two treatment groups during low temperature preservation, and the sperm motility and activity of the sperm in the treatment with alanylglutamine are higher appear. (p = 0.05)

Example  4

Sperm was diluted in the same ratio as in Example 1, and the results of the motility of the spermatozoa stored for 72 hours in a 4 ° C environment were observed.

Referring to FIG. 4, the percentage of motile spermatozoa in the glutamine-treated group was 55.06 ± 5.06%, and the ratio of spermatozoa with activity was 24.64 ± 8.6%. The ratio of motile spermatozoa in the alanylglutamine treated group was 75.66 ± 3.92%, and the ratio of spermatozoa with activity was 39.44 ± 7.19%.

In other words, it can be seen that there is a significant difference in the proportion of motile sperm and the activity and the ratio of excellent sperm between the two treatment groups during low temperature preservation, and the sperm motility and activity of the sperm in the treatment with alanylglutamine are higher appear. (p = 0.05)

Example  5

Sperm was diluted in the same ratio as in Example 1, and the results of the motility of the spermatozoa stored for 96 hours at 4 ° C were observed.

Referring to FIG. 5, the proportion of motile spermatozoa in the glutamine-treated group was 52.36 ± 1.82%, and the ratio of spermatozoa with activity was 43.94 ± 14.45%. The ratio of motile spermatozoa in the alanylglutamine treated group was 81.02 ± 2.46%, and the percentage of spermatozoa with activity was 53.02 ± 10.09%.

In other words, it can be seen that there is a significant difference in the proportion of motile sperm and the activity and the ratio of excellent sperm between the two treatment groups during low temperature preservation, and the sperm motility and activity of the sperm in the treatment with alanylglutamine are higher appear. (p = 0.05)

Example  6

1) Glutamine 96mM,

2) a mixture of 48 mM glutamine and 48 mM alanyl,

3) alanylglutamine 96mM

The results of the mobility and activity of the spermatozoa stored for 168 hours in a 4 ° C. environment were observed by mixing the three above in the same manner as in Example 1.

6, 1) the proportion of motile spermatozoa in the glutamine-treated group was 21.82 ± 4.25%, and the ratio of spermatozoa with activity was 7.05 ± 1.98%.

2) The ratio of motile spermatozoa in the group treated with the same mixture of alanylglutamine and glutamine was 65.57 ± 4.08%, and the ratio of spermatozoa with activity was 34.33 ± 5.5%.

3) The ratio of motile spermatozoa in the alanylglutamine - treated group was 81.3 ± 6.4% and the percentage of spermatozoa with activity was 63.61 ± 4.62%.

In other words, it can be seen that there is a significant difference in the ratio of the motile sperm and the activity and the ratio of the excellent sperm between the two treatment groups during the low temperature preservation process, and the sperm motility and activity by the alanylglutamine increase remarkably It can be seen that it is high. (p = 0.05)

These results suggest that the addition of alanyl glutamine to the dilution of bird semen resulted in better efficacy in long - term storage of spermatozoa. The sperm motility was maintained at 80% or more even on the 7th day of culturing. It is likely to be used for long-term low-temperature storage for 7 days or longer. In preserving sperm as a bird genetic resource, it can be used for cold storage, .

As a result, according to the present invention, it is possible to enhance the efficiency of conserving algae genetic resources by promoting viability and survival of spermatozoa in the process of preserving (genetic) source of algae (refrigeration and cryopreservation and fusion).

While the present invention has been particularly shown and described with reference to specific embodiments thereof, those skilled in the art will appreciate that such specific embodiments are merely preferred embodiments and that the scope of the present invention is not limited thereby. something to do. It is therefore intended that the scope of the invention be defined by the claims appended hereto and their equivalents.

Claims (7)

Alanyl glutamine, glucose, fructose, inositol, sodium dihydrogen phosphate, sodium hydrogen phosphate, and potassium citrate monohydrate Potassium citrate monohydrate). ≪ / RTI >
The composition of claim 1, wherein the composition comprises 96 mM alanyl glutamine, 39 mM glucose, 11.1 mM fructose, 39 mM Inositol, 17.0 sodium dihydrogen phosphate mM, sodium hydrogen phosphate (70 mM) and potassium citrate monohydrate (4.31 mM).
The composition according to claim 2, further comprising 0.1% (w / v) of PVP (polyvinylpyrrolidone, MW = 1,000).
The composition according to claim 2, wherein the alanyl glutamine is replaced with a mixture of alanyl glutamine at a concentration of 48 mM and glutamine at 48 mM.
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