KR20180082275A - Freeze conservative solution of the Epinephelus septemfasciatus sperm and freezing preservation method using the same - Google Patents

Abstract

The present invention relates to a cryopreservation liquid for Epinephelus septemfasciatus sperms for improving preservative effects of Epinephelus septemfasciatus sperms; and a cryopreservation method for Epinephelus septemfasciatus sperms using the same. Since the mobility and survival rate of Epinephelus septemfasciatus sperms even after cryopreservation are excellent, the cryopreservation liquid has excellent refrigeration preservation effects for the sperms, and the present invention may resolve the problem of fishery breeding that may occur due to a lack of Epinephelus septemfasciatus sperms, an inconsistent spawning time, or an imbalance in sex ratio in Epinephelus septemfasciatus adults. In addition, by using a cryopreservation liquid that is species-specific to Epinephelus septemfasciatus, the present invention can preserve Epinephelus septemfasciatus sperms for a long period of time, and also can simplify a species preservation method for Epinephelus septemfasciatus, and thus can be expected to contribute greatly to the growth of the fish farming industry.

Description

[0001] The present invention relates to a cryopreservation solution for spermatozoa, and a method for cryopreservation of a spermatozoa using the same,

The present invention relates to a frozen stock solution of spermatozoa and a method for cryopreserving spermatozoa using the same to improve the preservation effect of the spermatozoa.

Epinephelus septemfasciatus is a marine fish of barracuda and barrette. It is also known as a nine-ton evacuation, seven tonal bari, or a parasite in Korea, and it is known that there are about 159 species in 15 branches. The color of the body of the greens is purple-toned, light brownish brown, with seven lines of black-brown stripes on the sides.

The larvae generally live in sub-tropics and tropical reef development areas and reef areas, coastal seabed and do not move largely due to spawning, feeding or water temperature changes. Usually, there are 50 ~ 60m deep reefs Inhabited places. During the day, it is nocturnal to live mainly in rock groves. Feeds include horse mackerel, small mackerel fish, squid, shrimp, crab, and more.

In addition, because of its excellent taste and flavor, it has high preference of consumers, and therefore it is a high-grade fish with a very high price. It has strong resistance to the environment and grows fast as a large fish species. It is a highly profitable fish species in Jeonnam province and is aquaculture species that is envied by aquaculture farmers.

However, research and development on mass production technology for such a lentil seedlings has been limited. Therefore, it is difficult to obtain high quality embryos by artificial mother rearing. In addition, due to differences in sex ratio or maturity period, It is difficult to secure large quantities. Therefore, it is necessary to produce seedlings by using embryos collected from wild-born mothers in the case of lethal fishes. In addition, not only is the difficulty in securing embryos derived from wild-born moths due to the drastic reduction of natural resources, And it is difficult to obtain high quality eggs and sperm at the same time due to premature germination or overgrowth of the gonads. To solve this problem, researches have been conducted to collect seeds and spermatozoa to secure seeds at any time.

In general, animal sperm are not able to survive for a certain period of time when they are released to the outside of the body. Therefore, in order to preserve the spermatozoa of the animal for a certain period of time in vitro, the sperm does not exercise in vitro, It is known that the species of spermatozoa are different from each other depending on the species because the species is very diverse in that the preservation of the spermatozoa is very different due to the speciality of the species environment. have.

The need for conservation of fish sperm is known for the artificial insemination for the production of new artificial seeds which are difficult to cultivate. Especially, in the case of fish which can not be picked up and spermatized, It is also economically very useful to preserve and use spermatozoa for breeding or hybrid production in the same species of fish.

In order to preserve the sperm of such fish, there is a method of cryopreserving the spermatozoa of the fish. The sperm cryopreservation of these fishes is capable of selectively crossing, preserving the genes of the superior species, Can be used all year round, can create a gene pool of endangered species, is advantageous for interspecific hybridization and species conservation, can be used to collect large amounts of semen during the breeding season, It is easy to take semen.

However, the method of cryopreserving these spermatozoa can not be applied to the spermatozoa without consideration of the characteristics of the sperm according to the habitat environment, breeding habit, It is known that freezing and cryopreservation are maintained for a certain period of time while fertilization ability is maintained. It is necessary to research and develop semen characteristics and sperm activity of the target species to be preserved, or to maintain sperm viability after thawing of frozen-preserved sperm to be.

Japanese Patent Application Laid-Open No. 10-0604262 (Registered on Jul. 18, 2006) relates to artificial suits for preserving cold storage of flounder sperm, and more particularly, to an artificial suit capable of preserving flounder spermatozoa in vitro, The artificial suit for preserving the cold storage of the flounder sperm which can breed the spermatozoa of the flounder by allowing the spermatozoa of the flounder to be stored for more than 3 weeks in the artificial suit is obtained.

However, in the case of the above-mentioned conventional art, artificial suits for preserving flounder and sperm among fish are used, and the same artificial suits and preservation liquids are used depending on species of fish, such as reproductive phenomena, blood composition, semen composition, Therefore, in order to preserve spermatozoa and spermatozoa of the spermatozoa using the artificial suits of the prior art as described above, the survival rate and activity of the spermatozoa are significantly lowered.

Registration No. 10-0604262 (Registered on July 18, 2006)

The present invention relates to a cryopreservation solution of spermatozoa capable of cryopreserving spermatozoa or spermatozoa for a long period of time in accordance with the characteristics of the forefingers and to a method for cryopreservation of the spermatozoa using the same for improving the survival rate and activity of spermatozoa Which is capable of improving the efficiency of production of the seedlings of the lucerne sperm, and a method for cryopreserving the spermatozoa using the same.

In order to achieve the above object, the frozen storage solution of the spermatozoa of the present invention may include glucose and dimethyl sulfoxide (DMSO), preferably 0.2 to 0.4 M glucose For 100 parts skin, 10 to 30 parts of dimethyl sulfoxide can be included.

According to another aspect of the present invention, there is provided a method for cryopreserving a spermatozoon, and more particularly, A mixing step of injecting the collected frozen louse semen into a frozen storage liquid and mixing the same; A first freezing step of freezing the frozen storage liquid containing the live fish semen obtained by performing the mixing step; And a second freezing step of freezing the frozen storage liquid containing the frozen groundnut semen through the first freezing step at a temperature lower than the temperature of the first freezing step. At this time, the used frozen storage liquid may include glucose and dimethyl sulfoxide (DMSO).

Preferably, after the mixing step, the equilibrium inducing step may be performed by allowing the frozen storage liquid containing the spermatogenic sperm to remain at room temperature for 10 to 20 minutes.

Specifically, the collecting step may include pressing the abdomen of the tofu to remove the excrement; Urging the abdomen of the asperity with the excrement removed to excrete the semen; And storing the excreted lump semen at a temperature of 0 to 5 캜.

It is preferable that the frozen storage liquid contains 10 to 30 parts by mass of dimethyl sulfoxide per 100 parts by mass of 0.2 to 0.4 M glucose.

In the mixing step, the collected juvenile louse semen can be mixed with the frozen storage liquid at a volume ratio of 1: 2 to 4.

Wherein the first freezing step is carried out by freezing the frozen storage liquid containing the frozen lyse sperm into the container containing the liquid nitrogen and freezing the frozen storage liquid by 4 to 6 cm from the surface of the liquid nitrogen, It is preferable that the frozen storage liquid containing frozen groundnut semen is immersed in liquid nitrogen for cryopreservation.

The frozen stock solution of the spermatozoa of the present invention has excellent mobility and survival rate of the spermatozoa after freezing and then thawing and is excellent in the ability to freeze and preserve the spermatozoa, The problem of seedling production caused by the sex ratio imbalance of the seeds can be solved.

In addition, because of the species - specific frozen - preserved solution of the lentil, it is possible not only long - term preservation of the lentil spermatozoa but also the method of preserving the species of lentilfish, which is expected to contribute greatly to the development of the aquaculture industry.

1 is a graph showing a result of measuring the motility of a spermatozoon according to an embodiment of the present invention.
2 is a graph showing the results of measurement of the survival rate of the spermatozoa according to an embodiment of the present invention.
3 is a graph showing a result of measuring mobility of spermatogonium javanis according to an embodiment of the present invention.
4 is a graph showing the results of measurement of the survival rate of spermatogonium javanis according to an embodiment of the present invention.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS The preferred embodiments of the present invention will now be described in detail with reference to the accompanying drawings. Hereinafter, preferred embodiments of the present invention will be described in detail with reference to the accompanying drawings. As well as the fact that

Throughout this specification, when an element is referred to as "including" an element, it is understood that it may include other elements as well, without departing from the other elements unless specifically stated otherwise.

Hereinafter, the frozen stock solution of the spermatozoa of the present invention and the method of cryopreserving the spermatozoa using the same will be described in detail.

In general, the technique of cryopreserving fish sperm in a living state can solve the problem that occurs when the seedling breeding of the aquaculture fish is not coincident with the breeding period of the mother or the males, or when the sex ratio is uneven. In addition, it not only saves effort and expense required for the management of male breeding, but also enables selective breeding of superior breeds and facilitates preservation of domesticated species and native species.

Factors affecting the cryopreservation effect and survival of sperm after storage include antiseptic, diluent, equilibration time, freezing rate and thawing temperature.

First, the frozen stock solution of the spermatozoa of the present invention is to improve the preservability of spermatozoa by freezing and storing the spermatozoa in order to improve efficiency of seedling production in the fish among the fishes. The spermatozoa and the spermatozoa which dilute the semen are frozen, , And may contain a lot of damage during the defrosting process, that is, an antifungal agent to prevent the frost damage.

The diluent serves to dilute the semen, which not only controls the osmotic pressure, pH and ion concentration of the sperm but also increases the amount of semen during fertilization. The diluted solution contained in the frozen preserved solution of the spermatozoa of the present invention can be prepared by dissolving the diluted solution in water, preferably using glucose as a diluent. At this time, by controlling the concentration and content of glucose used as a diluent, In addition to creating an environment in which spermatozoa can survive, it is necessary to control the spermatozoa concentration by controlling the osmotic quality and to lower the sperm motility. The glucose may be a mixture of D-glucose or L-glucose, D-glucose and L-glucose, but glucose, preferably D-glucose, may be used.

Therefore, the frozen stock solution of the spermatozoa containing the diluent solution can control the concentration and amount of glucose to be similar to the osmotic concentration of the seminal fluid, thereby preventing activation of the spermatozoa, It is preferable to lower the motility of the sperm.

The antidisaster agent is used to prevent many damages, that is, frost damage during the freezing, freezing and thawing processes in preserving sperm. In the frozen stock solution of the spermatozoa of the present invention, dimethyl sulfoxide (DMSO) Can be used.

Preferably, the frozen stock solution of the spermatozoa of the present invention may contain glucose and dimethyl sulfoxide (DMSO), more preferably 0.2 to 0.4 M glucose, 10 to 30 parts of sulphoxydicarboxylate, and most preferably about 15 to 25 parts of dimethyl sulfoxide per 100 parts of 0.3 M glucose.

The ratio of glucose and dimethyl sulfoxide to be contained in the frozen stock solution of the spermatozoa spermatozoa is such that the spermatozoa of the spermatozoa in the fish and fish are effectively cryopreserved and frozen and thawed to obtain the survival rate of the spermatozoa , Mobility and activation of the spermatozoa of the spermatozoa of the spermatozoa of the present invention. If the ratio exceeds the above range, the survival rate and motility of the spermatozoan spermatozoa may be lowered, resulting in deterioration of storage stability. The sperm survival rate and motility of other fish may also be lowered.

According to another embodiment of the present invention, there is provided a method for cryopreserving a spermatozoon, comprising: a sampling step of collecting sperm from a fish; A mixing step of injecting the collected frozen louse semen into a frozen storage liquid and mixing the same; A first freezing step of freezing the frozen storage liquid containing the live fish semen obtained by performing the mixing step; And a second freezing step of freezing the frozen storage liquid containing the frozen groundnut semen through the first freezing step at a temperature lower than the temperature of the first freezing step.

First, the sampling step of collecting the spermatozoa semen can be used generally as long as it is a method of collecting semen in a fish. Preferably, the collecting step is performed by pressing the abdomen of the fish to remove feces. Urging the abdomen of the asperity with the excrement removed to excrete the semen; And storing the excreted lump semen at a temperature of 0 to 5 캜.

Specifically, after maturedly grounded fishes are cleaned with clean gauze, the abdomen of the larvae is lightly pressed to remove the excretion of the fishes such as urine, and then the excrement is removed. Then, , And then the accumulated luteal semen is stored at a temperature of 0 to 5 ° C using an ice box to collect the luteal semen. When the excreted juvenile premature ejaculate is stored, if the temperature is out of the temperature range, the survival rate of the spermatozoa sperm rapidly decreases or the spermatozoa may be damaged. Therefore, It is preferable to mix with a preserving solution and preserve in a frozen state.

The mixing step of mixing the frozen-thawed semen to the frozen-thawed semen collected through the sampling step prevents the frozen-thawed semen contained in the semen when the frozen-thawed frozen semen is frozen, In order to improve the survival rate of the spermatozoa and maximize the mobility before freezing, it is preferable to mix the sperm in the frozen storage solution of the spermatozoa containing glucose and dimethyl sulfoxide (DMSO). Since the specific components and mixing ratios of the frozen storage liquid used in this case are mentioned above, they will not be described here.

In the mixing step, it is preferable to mix the lion fish semen collected preferably in a volume ratio of 1: 2 to 4 volume of the frozen storage liquid. When the mixing ratio is exceeded, the osmotic pressure and pH of the lion sperm are not balanced, Can be remarkably deteriorated.

The frozen storage solution containing the semen-containing semen is left to stand for 10 to 20 minutes at room temperature to completely dissolve the frozen storage liquid into the sperm cells contained in the lupus semen or lupus semen before freezing the frozen storage solution containing the lupus semen through the mixing step And an equilibrium inducing step to be performed. It is preferable to keep the frozen stock solution in the frozen sperm to be sufficiently dispersed in order to prevent the frozen sperm from being damaged during freezing or freezing and to improve the survival rate of the frozen spermatozoa.

After the frozen storage liquid is sufficiently diffused into the spermatozoa through the equilibrium induction step, the frozen storage liquid containing the spermatic sperm can be frozen through the first freezing step. In the first freezing step, it is preferable that the freezing rate is frozen at a rate that can prevent ice crystals from being formed between the sperm contained in the lupus semen and the oocyte cell of the intramuscular lupus. Specifically, The included frozen storage liquid is placed in a container containing liquid nitrogen and frozen. It can be frozen first by floating 4 to 6 cm from the surface of liquid nitrogen, and the first freezing time is preferably 15 to 25 minutes.

In this case, when the frozen storage liquid containing the spermatozoa in the first freezing step is rapidly frozen, ice crystals are formed between the spermatozoa of the spermatids and the inner cells of the suits and the cell-cells, The sperm survival rate and motility of the spermatozoa may be markedly lowered. If the freezing rate is slower than the freezing rate, the osmotic concentration of the frozen storage liquid containing the luteal semen may be too high or the pH value may be changed. .

Wherein the frozen storage liquid containing the frozen liquor semen is frozen at a temperature lower than the temperature of the first freezing step, and the frozen storage liquid containing frozen lunase semen is stored in the liquid nitrogen It is desirable to immerse and freeze.

It is preferable that the frozen storage liquid containing frozen-stored frozen-frozen semen in the vapor of liquid nitrogen or liquid nitrogen through the two-person freezing step is immersed in fresh water at 25 to 40 ° C for thawing using a constant temperature water tank, In order to minimize the regeneration of the intracellular ice crystals of the spermatozoa in the luteal spermatozoa, it is preferable to immerse the spermatozoa in a constant temperature water bath and thaw them in a short time.

Hereinafter, an embodiment of the present invention will be described. However, the scope of the present invention is not limited to the following preferred embodiments, and a person skilled in the art can carry out various modifications of the contents described in the present invention within the scope of the present invention.

[Example]

Changes in contents and contents of frozen storage liquid A fagot  Measurement of survival rate and mobility of spermatozoa

Using the gauze to remove the foreign material on the surface of the fish body, the abdomen was pressed to remove urine and other excreta. The larvae removed from the slurry were discharged to the outside of the living body through the abdominal compression method, and then put into the ice box maintained at a temperature of 4 ° C or lower to collect the louse semen.

Spermatic sperm were collected from frozen stock solution of dimethyl sulfoxide (DMSO) in 0.3 M glucose and mixed with 3: 1 volume ratio.

The frozen storage solution was prepared by mixing dimethyl sulphoxide (DMSO) with 15 v / v% of the whole frozen storage solution containing semen.

The frozen semen was mixed with the frozen storage solution for 15 minutes at room temperature and equilibrated for 15 minutes. The frozen semen was first frozen for 20 minutes at the surface of liquid nitrogen and 5 cm from the surface of liquid nitrogen, and then immersed in liquid nitrogen. Respectively.

In order to confirm the survival rate and motility changes of the spermatozoa before and after the cryopreservation, the cryopreserved spermatozoa before freezing (before freezing), after freezing 2 hours after cryopreservation (after 1 hour of freezing) and 2 Five months after cryopreservation (after 5 months of freezing), the survival rate and motility of the spermatozoa spermatozoa were measured after rapidly melting them in distilled water at 20 ° C.

The motility of the sperm was diluted 200 times and the motility of the sperm was observed on a slide glass without a cover slide. Sperm motility was measured using a video camera attached to a microscope (Nikon, ECLIPSE E600, JAPAN). The results are shown in Fig.

The survival rate of sperm was measured by sperm staining with 5% eosin and 10% nigrosin. The results are shown in Fig.

In addition, mabalii semen, which is similar in appearance to that of the wild-type fish, is visually difficult to identify. Using the prepared frozen stock solution of the present invention, the mobility and survival rate of the larval spermatozoa were evaluated through the same experimental method. The results are shown in FIG. 3 and FIG.

As shown in FIG. 1, the mobility of the spermatozoa of the spermatozoa showed an average of 80% mobility before freezing using 0.3 M glucose and 15 v / v% DMSO. After one hour of freezing, the sperm motility was 70% After a mean of 60% of mobility showed.

2, the survival rate of the spermatozoan spermatozoon was 94% before freezing, and 92% after one hour of freezing and 90% after 5 months of freezing.

In addition, the frozen storage solution of the present invention was used to cryopreserve spermatozoa which is similar in appearance to that of visceral and not easily distinguishable from the naked eye, and the test results are shown in FIGS. 3 and 4. In detail, The survival rate was 99% before freezing, 99% after 1-hour freezing, 98% after 5-month freezing, and 96% after 1-month freezing. And the survival rate was maintained constantly.

Therefore, the frozen stock solution of the spermatozoa of the present invention is prepared by using 0.2 to 0.5 M glucose as a diluent, and using a frozen storage liquid containing 10 to 30 parts of dimethyl sulfoxide as a diluent to 100 parts of 0.2 to 0.4 M glucose, Not only semen but also cryopreserved spermatozoa are expected to be useful for long-term preservation of spermatozoa and albatrosses, as well as enhancing survival rate and mobility after cryopreservation.

Claims (9)

Frozen storage solution of spermatozoa containing glucose and dimethyl sulfoxide (DMSO). The method according to claim 1,
The frozen stock solution of the spermatozoa of the above-
0.2 to 0.4 M Glucose 100 parts The frozen preserved solution of the spermatozoa of the present invention is characterized by containing 10 to 30 parts by mass of dimethyl sulfoxide on the skin. A sampling step of collecting the semen of the fish;
A mixing step of injecting the collected frozen louse semen into a frozen storage liquid and mixing the same;
A first freezing step of freezing the frozen storage liquid containing the live fish semen obtained by performing the mixing step; And
And a second freezing step of freezing the frozen preserved liquid containing the frozen groundnut semen through the first freezing step at a temperature lower than the temperature of the first freezing step,
The above-
A method for cryopreserving spermatozoa according to claim 1, characterized by comprising glucose and dimethyl sulfoxide (DMSO). 5. The method of claim 4,
After the mixing step,
The method according to claim 1, further comprising a step of allowing the frozen storage liquid containing the spermatogenic sperm to remain at room temperature for 10 to 20 minutes. 5. The method of claim 4,
The sampling step includes:
Pressing the abdomen of the tofu to remove feces;
Urging the abdomen of the asperity with the excrement removed to excrete the semen; And
And storing the excreted spermatozoa semen at a temperature of 0 to 5 ° C. 5. The method of claim 4,
The above-
A method for cryopreserving spermatozoa according to claim 1, wherein the skin contains 10 to 30 parts by weight of dimethyl sulfoxide per 100 parts by weight of 0.2 to 0.4 M glucose. 5. The method of claim 4,
Wherein the mixing step comprises:
Wherein the spermatozoa are collected in a 1: 2 to 4 volume ratio to a frozen storage solution. 5. The method of claim 4,
In the primary freezing step,
Characterized in that the frozen storage solution containing the above-mentioned frozen-thawed semen is placed in a container containing liquid nitrogen, frozen by 4 to 6 cm from the surface of liquid nitrogen and frozen. 5. The method of claim 4,
Wherein the second freezing step comprises:
Wherein the frozen storage liquid containing the frozen groundnut semen is immersed in liquid nitrogen to be frozen and preserved.

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