KR102226182B1 - Composition for cryopreservating sperm comprising egg yolk extract and milk protein extract - Google Patents

Abstract

The present invention is a method for cryopreserving sperm containing egg yolk extract and milk protein extract, a method for cryopreserving sperm of cattle using a composition for cryopreservation of sperm containing the egg yolk extract and milk protein extract, and cryopreserved by the above method. Cattle breeding method comprising the step of thawing sperm and then injecting it into a cow during ovulation, and after thawing the frozen sperm by the above method, injecting a fertilized egg formed by in vitro fertilization into a cow during ovulation It's about the method. When using the cryopreservation composition provided in the present invention to cryopreserve sperm of cattle, the survival rate of sperm after thawing is improved, and thus, it will be widely used for effective breeding and breed management of cattle.

Description

Composition for cryopreservating sperm comprising egg yolk extract and milk protein extract

The present invention relates to a composition for cryopreservation of sperm comprising an egg yolk extract and a milk protein extract, and more specifically, the present invention relates to a composition for cryopreservation of sperm comprising an egg yolk extract and a milk protein extract, the yolk extract and a milk protein extract A method for cryopreserving bovine sperm using a composition for cryopreservation of sperm containing, a method for reproducing cattle comprising the step of thawing the sperm stored in the above method and then injecting it into a cow during an ovulation period, and cryopreservation by the method After thawing the sperm, it relates to a method of breeding cattle comprising the step of injecting a fertilized egg formed by in vitro fertilization into a cow during ovulation.

In the method of managing the variety of Korean cattle, it is practically difficult to collect sperm from seed hydrogen every time it is necessary to reproduce, so the technique of freezing and preserving sperm and defrosting it when necessary to use it for fertilization is a very important field. to be. In general, during the freezing process, sperm are damaged by the formation of ice crystals in cells, differences in osmotic pressure, and low temperature stress. A cryopreservative is a substance that reduces such damage, and glycerol is known as a representative substance. Since the raw semen used in the freezing process has a high concentration, it is diluted with a diluent. During the dilution process, a cryopreservative is administered together with the diluent. However, melted sperm after cryopreservation does not have the same fertilizing potential as fresh sperm, which is why the cooling process induces rearrangement of lipids and proteins in the cell membrane while cooling from 22°C to 1°C. Because it does. This transition is induced by a membrane that changes from liquid to gel at low temperatures. This is one of the main causes of reducing the viability and fertilization ability of sperm during cryopreservation. Among them, the cholesterol/phospholipid ratio of the sperm membrane is a major determinant in membrane fluidity and stability during cryopreservation. It has been reported that sperm obtained from species with very high cholesterol/phospholipid ratios, such as human and rabbit sperm, do not undergo this membrane damage when cooled. Typically, membrane damage is considered to be one of the major causes of cell death. Cholesterol, a major component of the membrane, plays an important role such as regulation of membrane function, and cholesterol in the cell membrane is known as a major determinant of membrane fluidity and permeability.

By minimizing such mutations, studies are actively being conducted to minimize damage to cells during cryopreservation. For example, WO 01/037655 discloses a diluent used in a method for cryopreserving mammalian sperm, and Korean Patent Publication No. 2009-0024029 discloses a cryopreservative for sperm containing amide. 2011-0020066 discloses a method of cryopreserving semen by using a semen specific cryopreservative, and Korean Patent Publication No. 2010-0117497 discloses an artificial suit for cryopreservation of sperm of japonica. However, when cryopreserved sperm are thawed by these various techniques, the survival rate of sperm is less than 50% in most cases, and in some cases, the survival rate is lower than the limiting survival rate (40%) at which pregnancy is possible. In addition, there is an urgent need to develop a method for cryopreservation of sperm that can further improve the survival rate after thawing.

Under this background, the present inventors have made intensive research efforts to develop a cryopreservative suitable for preserving bovine sperm, as a result of confirming that the cryopreservation solution including egg yolk extract and milk protein extract can improve the survival rate of sperm after thawing. The invention was completed.

The main object of the present invention is to provide a composition for cryopreservation of sperm comprising an egg yolk extract and a milk protein extract.

Another object of the present invention is to provide a method for cryopreserving bovine sperm using a composition for cryopreservation of sperm comprising the egg yolk extract and milk protein extract.

Another object of the present invention is to provide a method for breeding cows comprising the step of melting the sperm of cows cryopreserved by the above method and then injecting them into a cow during ovulation.

Another object of the present invention is to provide a method for breeding cows comprising the step of injecting a fertilized egg formed by in vitro fertilization into a cow during ovulation after melting the sperm of a cow that has been cryopreserved by the above method.

One embodiment of the present invention for achieving the above object is a composition for cryopreservation of bovine sperm comprising 1 to 30% (v/v) of egg yolk extract and 1 to 10% (v/v) of milk protein extract. to provide.

The term "yolk extract" of the present invention refers to a supernatant obtained by adding an equal volume of physiological saline to the egg yolk separated from an egg, stirring at room temperature, and then centrifuging.

In the present invention, the egg yolk extract can be interpreted as an active ingredient of the composition for cryopreservation of sperm. The content of the egg yolk extract contained in the cryopreservation composition is not particularly limited thereto, but as an example, it may be 1 to 30% (v/v), and as another example, 5 to 25% (v/v) May be, as another example, may be 10 to 20% (v/v).

The term "milk protein extract" of the present invention refers to a suspension obtained by centrifuging milk to obtain a precipitate, and mixing the precipitate and sterile water, and the main protein contained therein may be casein or the like.

In the present invention, the milk protein extract can be interpreted as an active ingredient of a composition for cryopreservation of sperm. The content of the milk protein extract contained in the cryopreservation composition is not particularly limited thereto, but as an example, it may be 1 to 10% (v/v), and as another example, 1 to 5% (v/v) May be, and as another example, it may be 3% (v/v).

The term "sperm" of the present invention, also referred to as spermatozoa, refers to a sex cell produced in the testes of a male animal, the length of which is about 0.02 mm, and consists of three parts: a head, a neck and a long tail. The head contains the DNA of a male animal, and the acrosome at the front of the head contains an enzyme capable of dissolving the egg membrane of the egg; The cervix contains mitochondria containing sugar, which is an energy storage; The tail is made of a bundle of fine threads, and the bundle of fine threads skillfully expands and contracts to move the tail.

In the present invention, the sperm is not particularly limited thereto, but as an example, it may be a mature sperm of a mammal, and as another example, it may be a sperm of a cow.

The term "freeze preservation" of the present invention refers to an act of stably maintaining cells over a long period of time through freezing.

In the present invention, the cryopreservation can be interpreted to mean cryopreservation of sperm.

The cryopreservation is not particularly limited as long as it can cryopreserve sperm, and may be performed by any known means, as an example, cryopreservation may be performed at -196°C using liquid nitrogen.

The term "freeze preservation composition" of the present invention refers to a composition containing a component capable of reducing freezing damage when the cells of an organism are stored alive in a frozen state.

In the present invention, the composition for cryopreservation can be interpreted as being a composition containing an intestinal fluid capable of reducing the freezing of bovine sperm.

In addition, the cryopreservation composition may further include various cryopreservatives added to the medium liquid for the purpose of alleviating freezing when sperm are preserved alive in a frozen state in addition to the egg yolk extract and milk protein extract. .

The added cryopreservative is not particularly limited as long as it can prevent sperm damage due to freezing damage and reduce the concentration of semen, so as to prevent damage that may occur due to collisions between sperm during cryopreservation, but salts, antioxidants It may be a buffer solution containing an agent, a sugar, an organic acid, an egg yolk component, an antibiotic, and the like, preferably, salts such as citrate and bicarbonate salts; Antioxidants such as taurine, hypotaurine, and trehalose; Sugars such as glycerol, glucose, sucrose, ethylene glycol, propylene glycol, fructose, and dextran; Organic acids such as hyaluronic acid, citric acid, acetic acid, butyric acid, palmitic acid, oxalic acid, and tartaric acid; Antibiotics such as streptomycin, penicillin, ampicillin, and kanamycin; Egg yolk protein, egg yolk extract, egg yolk-derived amino acids and the like may be yolk components, and each of these components may be used in combination by a person skilled in the art as needed.

According to an embodiment of the present invention, when using a cryopreservation solution containing 10 to 15% (v/v) egg yolk extract provided by the present invention, it is more than the limiting survival rate (40%) of sperm of cattle capable of artificial insemination. In addition to showing a high level of survival after thawing, it can also show a higher level of survival after thawing than that of the basic semen dilution (Table 1), and a cryopreservation solution containing 1 to 5% (v/v) milk protein extract is used. In this case, the survival rate after thawing is higher than the limiting survival rate (40%) of bovine sperm capable of artificial insemination, and the survival rate after thawing is higher than that of using a cryopreservation solution containing only egg yolk extract as well as basic semen dilution. Can be represented (Table 2).

Another embodiment of the present invention for achieving the above object is the steps of (a) mixing the composition for cryopreservation of cattle and sperm of cattle to obtain an automatic condensation solution; And (b) freezing and preserving the sperm of the sperm.

At this time, the cryopreservation composition and sperm are the same as described above.

The concentration of sperm contained in the cryopreservation composition is not particularly limited thereto, but as an example, 1 X 10 ⁶ to 7 X 10 ⁶ per unit volume (ml) of cryopreservation solution, and as another example, unit volume (ml) It is 2 X 10 ⁶ to 4 X 10 ⁶ per unit, and as another example, it may be ^{3 X 10 6 per unit volume (ml).}

In addition, the method of freezing bovine sperm may further include the step of thawing the frozen bovine sperm.

At this time, the defrosting method is not particularly limited as long as it can restore the frozen bovine sperm to a normal state. Preferably, the frozen bovine sperm contained in the bovine sperm is allowed to stand at room temperature for a certain period of time. The refrigerant is removed by volatilization, and the sperm from which the refrigerant has been removed are immersed in water at 1 to 10° C. for 2 to 10 minutes, or immersed in water at 30 to 40° C. for 10 to 30 seconds, or water at 60 to 80° C. It may be performed by immersing in 1 to 5 seconds, and more preferably, freezing-preserved semen is allowed to stand at room temperature for 10 to 20 seconds to remove the refrigerant, and the sperm from which the refrigerant has been removed is placed in water at 4° C. for 5 minutes It can be immersed for 20 seconds, immersed in water at 37°C for 20 seconds, or immersed in water at 70°C for 3 seconds, and most preferably, freeze-preserved sperm are allowed to stand at room temperature for 20 seconds to remove the refrigerant. And, it may be performed by immersing the sperm from which the refrigerant has been removed in hot water at 37° C. for 20 seconds.

Another embodiment of the present invention for achieving the above object is the steps of (a) thawing mature sperm of cattle cryopreserved by the above method; And (b) injecting the thawed mature sperm into the cow during ovulation.

At this time, the cryopreservation and sperm are the same as described above.

The injection may be performed by injecting the mature sperm into the deep of the uterus for a period from 1 day before ovulation to 2 days after ovulation of a cow, which is generally subjected to artificial insemination, in consideration of the survival period of sperm and egg.

Another embodiment of the present invention for achieving the above object is the steps of (a) thawing mature sperm of cattle cryopreserved by the above method; (b) obtaining a bovine fertilized egg by in vitro fertilization of the thawed bovine mature sperm; And (c) injecting the fertilized egg into a cow during ovulation.

At this time, the cryopreservation, sperm and injection are the same as described above.

When using the cryopreservation composition provided in the present invention to cryopreserve sperm of cattle, the survival rate of sperm after thawing is improved, and thus, it will be widely used for effective breeding and breed management of cattle.

Hereinafter, the present invention will be described in more detail through examples. However, these examples are for illustrative purposes only, and the scope of the present invention is not limited to these examples.

Example 1: Cryopreservation of bovine semen using egg yolk extract

First, as a cryopreservation solution for bovine semen, a basic semen dilution solution (Tris 30.25g/ℓ, Citric acid 17g/ℓ, Fructose 12.5g/ℓ, and glycerol 64mL/ℓ) was prepared, and then various concentrations (5, 10 or 20) were prepared. % (v/v)) of egg yolk extract (egg yolk plasma) and 3% (v/v) milk protein extract (casein) were prepared by adjusting the volume of sterilized water to prepare each cryopreservation solution. At this time, as the egg yolk extract, the same volume of physiological saline (0.9%, w/v) was added to the egg yolk separated from the egg, stirred at room temperature for 40 minutes, and then centrifuged (5° C., 10,000 xg, 45 minutes). The obtained supernatant was used. In addition, as the milk protein extract, a precipitate was obtained by centrifuging milk (5° C., 30,000 x g, 60 minutes), and a suspension obtained by mixing the precipitate and 10% volume of sterilized water thereof was used.

Next, by diluting the sperm of Korean beef in each of the prepared cryopreservation solutions, each self-contained solution was obtained, and then gradually cooled over 3 hours from room temperature to 5 degrees Celsius, and the self-contained solution was placed over liquid nitrogen. It was frozen by exposure for 10 minutes on 5 cm. Prior to the cryopreservation, the number of sperm contained in the automatic sperm solution was measured using a Markler chamber.

Finally, after thawing each of the frozen automatic condensation solutions at 37°C for 1 minute, the number of sperm contained in the thawed automatic condensation solution is measured again, and the number of sperm contained in the thawed automatic condensation solution The survival rate of sperm was calculated (Table 1). At this time, the survival rate of sperm before freezing was calculated as a control group, which was calculated by cooling each of the prepared sperm condensation solutions, and then observing it on the Markler chamber again, and measuring the ratio of the number of sperm showing motility to the total number of sperm. I did.

Survival rate of sperm contained in thawed sperm solution according to the concentration of egg yolk extract Sample additive Survival rate before freezing (%) Survival rate after melting (%) 20% yolk
5% egg yolk extract
10% egg yolk extract + 3% milk protein extract
20% egg yolk extract + 3% milk protein extract 88.3±4.1
92.3±2.9
92.5±2.7
94.1±2.8 65.0±8.2
38.4±5.8
81.3±6.8
70.4±8.7

As shown in Table 1 above, rather than the basic semen dilution that does not contain egg yolk extract and milk protein extract. When using a cryopreservation solution containing egg yolk extract and milk protein extract, it was confirmed that the survival rate of bovine sperm was increased during cryopreservation.

In particular, when a cryopreservation solution containing 10 to 15% (v/v) of egg yolk extract is used, the survival rate after thawing is higher than the limiting survival rate (40%) of sperm of cattle capable of artificial insemination. It was confirmed that the survival rate after thawing was higher than that of the semen dilution.

Example 2: Cryopreservation of bovine semen using milk protein extract

Instead of a cryopreservation solution containing various concentrations (5, 10 or 20% (v/v)) of egg yolk extract and 3% (v/v) milk protein extract (casein), 10% (v/v) of egg yolk extract and The same experiment as in Example 1 was performed, except that a cryopreservation solution containing a milk protein extract (casein) of various concentrations (1, 3 or 5% (v/v)) was used, and thawed automatic crystallizing solution The survival rate of sperm contained in was calculated (Table 2).

Survival rate of sperm contained in thawed sperm solution according to the concentration of milk protein extract Sample additive Survival rate before freezing (%) Survival rate after melting (%) 10% egg yolk extract
10% egg yolk extract + 1% milk protein extract
10% egg yolk extract + 3% milk protein extract
10% egg yolk extract + 5% milk protein extract 91.1±5.5
93.3±3.6
90.4±3.3
89.4±1.5 58.2±4.2
70.9±5.3
78.0±3.5
75.4±5.5

As shown in Table 2, it was confirmed that even if the egg yolk extract was included at 10% (v/v), in the case of the cryopreservation solution without the milk protein extract, the survival rate of bovine sperm was low during cryopreservation. .

In addition, when a cryopreservation solution containing 1 to 5% (v/v) of milk protein extract is used, the survival rate after thawing is higher than the limiting survival rate (40%) of sperm of cattle capable of artificial insemination. It was confirmed that the survival rate after thawing was higher than that of the case of using the semen diluted solution as well as the cryopreservation solution containing only egg yolk extract.

From the results of Tables 1 and 2 described above, for cryopreservation of bovine sperm, it is preferable to use a cryopreservation solution containing both egg yolk extract and milk protein extract, and the content of egg yolk extract contained in the cryopreservation solution is 10 to 15 It was found that% (v/v) is preferable, and the content of the milk protein extract contained in the cryopreservation solution is preferably 1 to 5% (v/v).

From the above description, those skilled in the art to which the present invention pertains will understand that the present invention can be implemented in other specific forms without changing the technical spirit or essential features thereof. In this regard, it should be understood that the embodiments described above are illustrative in all respects and not limiting. The scope of the present invention should be construed that all changes or modifications derived from the meaning and scope of the claims to be described later rather than the above detailed description and equivalent concepts are included in the scope of the present invention.

Claims (11)

A composition for cryopreservation of bovine sperm containing 10% (v/v) egg yolk extract, 3 to 5% (v/v) milk protein extract, and a cryopreservative,
The milk protein extract is obtained by centrifuging milk to obtain a precipitate and then mixing sterile water to obtain a composition.
delete The method of claim 1,
The cryopreservatives include citrate, bicarbonate salt, taurine, hypotaurine, trehalose, glycerol, glucose, sucrose, ethylene glycol, propylene glycol, fructose, dextran, hyaluronic acid, citric acid, acetic acid, butyric acid, palmitic acid, The composition for cryopreservation of bovine sperm is selected from the group consisting of oxalic acid, tartaric acid, streptomycin, penicillin, ampicillin, kanamycin, egg yolk protein, amino acids, and combinations thereof.
(a) Milk was centrifuged at 5°C for 60 minutes at 30,000×g to obtain a sediment, and a milk protein extract obtained by mixing the sediment and 10% volume of sterilized water thereof, an egg yolk extract, and a cryopreservative were mixed to obtain a cow Obtaining a composition for cryopreservation of the;
(b) mixing the composition for cryopreservation of bovine sperm and bovine sperm to obtain an automatic sperm solution; And
(c) freezing and preserving the sperm of bovine, comprising the step of freezing and preserving the automatic condensation solution.
The method of claim 4,
The content of bovine sperm in step (b) is 1 X 10 ⁶ to 7 X 10 ⁶ per unit volume (ml) of the cryopreservation composition, the method of cryopreservation of bovine sperm.
The method of claim 4,
The freezing and preservation of the step (c) is performed at -196°C using liquid nitrogen.
(a) defrosting the mature sperm of the frozen cow preserved by the method of claim 4; And
(b) Injecting the defrosted mature sperm into the cow during the ovulation phase, cattle breeding method.
The method of claim 7,
The thawing of step (a) is performed by immersing in water after allowing the frozen sperm of the cow to stand at room temperature.
The method of claim 8,
The immersion is performed for 2 to 10 minutes in water at 1 to 10°C, 10 to 30 seconds in water at 30 to 40°C, or 1 to 5 seconds in water at 60 to 80°C.
The method of claim 7,
The injection of step (b) is performed by injecting the mature sperm of the defrosted cow into the deep of the uterus for a period from 1 day before ovulation of the cow to 2 days after ovulation of the cow.
(a) defrosting the mature sperm of the frozen cow preserved by the method of claim 4;
(b) obtaining a bovine fertilized egg by in vitro fertilization of the thawed bovine mature sperm; And
(c) Injecting the fertilized egg into a cow during ovulation.