JP4899571B2 - A technique for securing high-quality eggs of Mahata parent fish considering the timing of artificial insemination after maturation and ovulation induction using LHRHa sustained release preparation - Google Patents

Description

  The present invention relates to hormonally-treated egg collection by artificial insemination from Mahata parent fish, after induction of maturation and ovulation using a LHRHa sustained-release preparation for the purpose of obtaining a high-quality fertilized egg with high fertilization rate stably and efficiently. It relates to a technique for securing high quality eggs in consideration of the timing of artificial insemination.

  In conventional hormonal treatment egg collection by artificial insemination for Mahata, regardless of the maturity of each parent fish, palpation of the abdomen was performed approximately 48 hours after hormonal treatment, and eggs were observed for parent fish that were confirmed to be ovulated. He was squeezing out and performing artificial insemination.

  However, if the timing of artificial insemination after induction of maturation and ovulation by hormonal treatment is set after 48 hours for all individuals, the presence or absence of ovulation of the parent fish and the egg quality of the obtained eggs are various, stable and efficient It was difficult to obtain high-quality fertilized eggs.

  Thus, one of the causes of unstable egg quality, such as the fertilization rate and hatching rate of eggs obtained by artificial insemination, may be due to the missed period of ovulation.

  In general, even in a parent fish group that carries out the same parent fish cultivation in an artificial environment, the maturity state among these individuals varies. Some parent fish have matured enough and the ovary egg has grown to a large diameter, while others are still immature. In yellowtail, ovarian eggs are collected with cannula tubes and the diameter and maturation status are investigated in order to select parent fish that can induce maturation and ovulation by hormone treatment. In yellowtail, the time from hormonal treatment to ovulation can be predicted for each individual based on the egg diameter of the parent fish. In Mahata, ovarian eggs are collected with cannula tubes and the diameter of the eggs is measured, so that there is a possibility of selecting a parent fish that can induce maturation and ovulation and predicting the ovulation time of the individual. Prediction of ovulation time for each individual is considered to play an important role in securing high quality eggs with high fertilization rate and hatching rate.

  In trough pufferfish, yellowtails, etc., the fertilization rate of eggs obtained by artificial insemination is closely related to the elapsed time after ovulation, and if the ovulated eggs stay in the ovarian cavity for a long time, the eggs are excessive. It has been confirmed that the egg quality such as fertilization rate and hatching rate is lowered due to ripening phenomenon. In Mahata, one of the reasons why it has been difficult to obtain high-quality fertilized eggs is that the timing of artificial insemination is inadequate, and there is a possibility that the eggs have already become overripe. is there.

  Therefore, the present invention, as a technique to ensure good quality eggs of Mahata parent fish, fertilization rate, hatching rate by performing the meditation at the optimal time in the artificial insemination timing after maturation and ovulation induction using LHRHa sustained release preparation It is an object to provide an egg collection method for stably and efficiently obtaining a high-quality fertilized egg.

  In order to achieve the above object, the inventors induced maturation and ovulation of Mahata parent fish using LHRHa sustained-release preparations, and variously investigated the ovulation situation and the relationship between post-ovulation elapsed time and egg quality in artificial insemination. As a result of clarification by the test, the present inventors have found a technique for securing high-quality eggs of Mahata parent fish in consideration of artificial insemination timing after induction of maturation and ovulation using the LHRHa sustained-release preparation of the present invention.

In the invention of claim 1, as a method for sustained release of synthetic luteinizing hormone-releasing hormone (LHRHa: des-Gly ¹⁰ , [D-Ala ⁶ ] -LHRH ethylamide), a cholesterol pellet solid preparation or a cacao butter oil preparation is used. The preparation method of the LHRHa cholesterol pellet solid preparation, which is administered by injection and implanted, is prepared by dissolving LHRHa in 50% ethyl alcohol, then adding the solution to cholesterol powder and mixing until it is pasty. The LHRHa cholesterol in paste form is stored in a vacuum desiccator, etc., dried thoroughly, and cocoa butter dissolved in the dried LHRHa cholesterol powder is added and mixed to homogenize the whole. Put LHRHa cholesterol powder with cocoa butter to serve in the hole of the pelletizer. The LHRHa cocoa butter oil preparation for injection administration is composed of a method obtained by compressing, cylindrical pellets, and storing the completed LHRHa cholesterol pellet solid preparation in a freezer at -20 ° C or lower. , LHRHa is dissolved in 50% ethyl alcohol, the mass ratio of cocoa butter to LHRHa solution is 95: 5 to 70:30, the dose of LHRHa is 20-100 μg / kg BW, Using cultivated parent fish cultivated in a natural environment, with an ovary egg diameter of 420-550 μm, egg collection after hormone treatment is performed by artificial insemination, and the timing of artificial insemination is determined immediately after ovulation. It is characterized by obtaining fertilized eggs of good quality stably and efficiently from Mahata parent fish by collecting and immediately fertilizing.

The invention of claim 2 relates to the method of inducing maturation and ovulation of a grouper parent fish according to claim 1 by injecting LHRHa using a cholesterol pellet solid preparation or injection of LHRHa using a cocoa butter oil preparation, and ovulation is administered by LHRHa. It occurs 36 to 54 hours later.

The invention of claim 3 relates to the method of inducing maturation and ovulation of a grouper parental fish by the implantation of LHRHa using a cholesterol pellet solid preparation or the injection of LHRHa using a cocoa butter oil-based preparation in claim 1 , after administration of LHRHa. The optimal timing of artificial insemination is characterized by 0 to 6 hours after ovulation.

The invention of claim 4 is the invention according to claim 1, wherein the LHRHa cholesterol pellet solid preparation contains 50 to 400 μg of LHRHa per piece.

The invention of claim 5 is characterized in that, in claim 1 , the LHRHa cholesterol pellet solid preparation is embedded in the muscle of a Mahata parent fish .

The invention of claim 6 is characterized in that, in claim 1 , the LHRHa cholesterol pellet solid preparation is embedded in the abdominal cavity of a Mahata parent fish .

The invention of claim 7 is the oil-based preparation of LHRHa cocoa butter according to claim 1, wherein LHRHa is dissolved in 50% ethyl alcohol, and the mass ratio of the cocoa butter to the LHRHa solution is 90:10 .

The invention of claim 8 is the invention according to claim 1, wherein the LHRHa cocoa butter oily preparation contains 50 to 200 μg of LHRHa per 0.1 ml.

The invention of claim 9 is characterized in that, in claim 1 , the LHRHa cocoa butter oil-based preparation is injected and administered intramuscularly to a Mahata parent fish .

The invention of claim 10 is characterized in that, in claim 1 , the LHRHa cocoa butter oil-based preparation is injected into the abdominal cavity of a Mahata parent fish .

The invention according to claim 11 is the invention according to claim 1, wherein the LHRHa cacao butter oily preparation is injected into the muscle of the Mahata parent fish and then coagulated at 17 to 23 ° C. of the maturing water temperature of the Mahata parent fish. It is characterized by being a sustained-release preparation that is eluted for a period of time.

The invention according to claim 12 is the invention according to claim 1, wherein the LHRHa cocoa butter oily preparation is injected into the abdominal cavity of the Mahata parent fish and then coagulated at 17-23 ° C. of the maturing water temperature of the Mahata parent fish. It is characterized by being a sustained-release preparation that is eluted for a period of time.

The invention of claim 13 is the invention of claim 1 , wherein the needle used for injection administration of the LHRHa cocoa butter oil-based preparation is a needle having a relatively large needle hole of 18 to 21G in order to prevent clogging.

The invention of claim 14 is the invention according to claim 1, wherein the LHRHa cholesterol pellet solid preparation and the LHRHa cacao butter oil preparation are administered to the Mahata parent fish once.

According to a fifteenth aspect of the present invention, in the first aspect , the Mahata parent fish uses an individual that has not yet ovulated in the natural environment in the spawning season.

The invention according to claim 16 is the invention according to claim 1, wherein the mahata parent fish uses an individual that is 5 years old or older and capable of inducing ovulation and has a weight of 2 kg or more.

  According to the technique for securing high-quality eggs of Mahata parent fish considering the timing of artificial insemination after induction of maturation and ovulation using the LHRHa sustained-release preparation of the present invention, it has been difficult to secure fertilized eggs with high fertilization rate, hatching rate, etc. This has the advantageous effect of ensuring stable and efficient high-quality fertilized eggs for seedling production.

  In addition, according to the technique for securing high-quality eggs of mahata parent fish that takes into account the timing of artificial insemination after maturation and ovulation induction using the LHRHa sustained-release preparation of the present invention, it is possible to stably secure fertilized eggs for seedling production Therefore, it has an advantageous effect of enabling more planned and stable production in larval and juvenile breeding where production is carried out from hatching to fry.

  In addition, according to the technique for securing high-quality eggs of Mahata parent fish that takes into account the timing of artificial insemination after maturation and ovulation induction using the LHRHa sustained-release preparation of the present invention, it is possible to collect eggs with high reproducibility from Mahata parent fish Therefore, there is an advantageous effect that it is possible to efficiently use expensive mahata parent fish, to reduce the number of parent fish used and to reduce the costs associated therewith.

  In addition, according to the method for securing a good quality egg of a mahata parent fish that takes into account the timing of artificial insemination after induction of maturation and ovulation using the LHRHa sustained-release preparation of the present invention, it is possible to stably and efficiently obtain a high-quality fertilized egg. As well as the subsequent breeding of juvenile fish, it is possible to produce a stable and stable production of mahata artificial seedlings that accompany it, and the aquaculture industry that has longed for mahata seedlings for aquaculture production. There is an advantageous effect that development is expected.

  In the method for securing high-quality eggs of the mahata parent fish of the present invention, as a method for inducing maturation and ovulation, an embedded administration of a cholesterol pellet solid preparation or an injection administration of a cacao butter oil preparation that enables sustained release administration of LHRHa is performed.

  First, the maturation / ovulation induction method by implanting LHRHa cholesterol pellet solid preparation will be described.

  The LHRHa cholesterol pellet solid preparation is prepared by dissolving LHRHa in 50% ethyl alcohol, and then adding the solution to cholesterol powder and mixing it with a glass rod or the like until it is pasted to make the whole uniform.

  Place the paste-like LHRHa cholesterol in a vacuum desiccator, etc., and dry it thoroughly.

  Add and mix cacao butter dissolved in a hot water bath or the like into the dried LHRHa cholesterol powder and homogenize the whole.

  LHRHa cholesterol powder to which cacao butter serving as a binder is added is weighed 30 mg each, put into a hole of a pellet making machine, and then compressed with a stainless steel rod or the like to form a pellet.

  The completed LHRHa cholesterol pellet solid preparation can be used immediately or stored in a freezer below -20 ° C.

  Various sizes and shapes of LHRHa cholesterol pellet solid preparations are conceivable, but those molded into a cylindrical shape of about 2 × 6 mm are particularly preferable. LHRHa cholesterol pellets are embedded in the body of Mahata parent fish, but if the size of the pellet is too large, it will cause great stress on the parent fish. If the shape is cylindrical, it can be easily loaded into a pellet dispenser, can be easily handled, and workability can be improved, and the working time can be shortened.

  Further, the LHRHa content per LHRHa cholesterol pellet solid preparation may be 50 to 400 μg when used for Mahata parent fish, and among them, 100 to 200 μg is preferable. Assuming that the dose of LHRHa to Mahata parent fish is 50 μg / kg BW, if the weight of the parent fish is 6 kg, LHRHa needs 300 μg. If the LHRHa content per pellet is 100 μg, 3 will be embedded, and if it is 200 μg, 1.5 will be embedded. Also, if the LHRHa content per pellet is reduced to 10 μg, the number of embeds will be 30 and stress on the parent fish will increase. Conversely, when the LHRHa content per pellet is increased to 600 μg, the number of embeddings becomes 0.5, making it difficult to handle and embed solid preparations. Of course, the conditions under which LHRHa elutes from the pellet will change drastically, and the expected sustained release amount and duration may not be realized.

  For embedding administration of LHRHa cholesterol pellet solid preparations, it is recommended to use a pellet dispenser made of a PFA tube with an inner diameter of 2 mm and an outer diameter of 3 mm and a stainless steel round bar with an outer diameter of 2 mm.

  The pellet dispenser can be pre-prepared with one, 1.5, or two pellets, and it will be administered according to the required number of pellets as needed depending on the fish weight of the Mahata parent fish. It becomes possible. For example, if the dose of LHRHa to Mahata parent fish is 50 μg / kg BW, 300 μg of LHRHa is required when the weight of the parent fish is 6 kg. When the LHRHa content per pellet is 200 μg, a pellet dispenser loaded with 1.5 pellets may be used and embedded. If the weight of another parent fish is 4 kg, 200 μg of LHRHa is required, and it can be embedded using a pellet dispenser loaded with one pellet. By preparing various numbers of pellet dispensers in advance, LHRHa can be implanted quickly, and the stress on mahata parent fish can be minimized.

  The embedded portion of the LHRHa cholesterol pellet solid preparation is considered to be intramuscular and intraperitoneal of Mahata parent fish, but it is particularly preferable to carry out at a depth of about 1 to 2 cm subcutaneously in the thick portion of the back muscle.

  When embedding the LHRHa cholesterol pellet solid preparation, first, after anesthetizing the Mahata parent fish, make a 5 mm incision with a scalpel in the part to be implanted and insert the pellet dispenser. When the tip of the pellet dispenser reaches about 2 cm subcutaneously, push the stainless steel rod of the pellet dispenser into the muscle. Thereafter, the pellet dispenser is quickly pulled out.

  The LHRHa cholesterol pellet solid preparation can be implanted and administered into the abdominal cavity, but it requires skill in the work and may damage the internal organs if the worker is immature. Therefore, in order to further reduce the stress on the Mahata parent fish and to perform the implantation administration work quickly, it is considered that the thick portion of the fish back muscle part is suitable for the administration part.

  One embedding of LHRHa cholesterol pellets into Mahata parent fish is sufficient. The period of time during which LHRHa continues to elute from cholesterol pellets at a high level is approximately 5 days, but it is 42 to 48 hours until Mahata parent fish ovulate by implantation of LHRHa, so one implantation administration is sufficient It is thought that. If the ovulation of the parent fish is not performed by a single implantation, it is highly possible that the maturity of the parent fish was immature.

  In the induction of maturation and ovulation of Mahata parent fish by embedding LHRHa using a cholesterol pellet solid preparation, the LHRHa dose to Mahata parent fish is considered to be 20-100 μg / kg BW, but among them, 50 μg / kg BW is stable Maturation and ovulation can be induced efficiently and efficiently, and high-quality fertilized eggs can be obtained. During the egg-laying season of May-June 2001, 2002, matured ovulation was achieved in 44 individuals by implanting LHRHa cholesterol pellet solid preparations using a total of 59 Mahata parent fish and LHRHa dosage of 50 μg / kg BW A total of 35.73 million floating eggs were obtained. The amount of floating eggs obtained from one Mahata parent was 812,000 grains, and the average fertilization rate was 80% or more, and high-quality fertilized eggs could be obtained.

  The parent fish used for egg collection can be derived from natural sources or aquaculture. However, if you want to obtain fertilized eggs of high quality stably and efficiently, it is better to use cultured parent fish cultured in an artificial environment.

  Mahata's parent fish inhabiting the natural world are precious and few. In addition, even if the fish can be caught during the spawning season, the ovarian eggs vary in maturity, and it is very difficult to obtain high-quality fertilized eggs from these individuals due to adverse effects such as threading and stress from fishing. .

  On the other hand, cultured parent fish are accustomed to an artificial environment and are accustomed to human handling, so that the mature state of ovarian eggs is highly synchronized between individuals and is resistant to a certain amount of stress. From these things, the direction which uses the training parent fish can secure many individuals suitable for egg collection, and can obtain a high-quality fertilized egg.

  In the induction of ripening and ovulation of Mahata parent fish by LHRHa implantation using a cholesterol pellet solid preparation, parental fish at the time of LHRHa implantation administration can induce ovulation in individuals with an egg diameter of 420 to 550 μm, but among them It is better to use an individual whose ovary egg diameter is 460 to 520 μm.

  Individuals with an ovarian egg diameter of less than 420 μm are immature individuals in which maturation and ovulation are not induced by implantation of LHRHa, and individuals between 420 and 460 μm are not yet sensitive to hormones. It is an individual with a small number of mature and ovulated eggs.

  On the other hand, individuals with an ovary egg diameter of 550 μm or more are individuals who cannot expect to obtain a high-quality fertilized egg because there are many degenerated eggs and ovulation eggs in the ovary egg, and individuals with a diameter of 520 to 550 μm Is an individual whose egg quality is getting worse due to the appearance of some degenerate eggs and ovulatory eggs in the ovarian eggs. For these reasons, it is desirable that the parent fish used for egg collection is selected from individuals with an ovarian egg diameter of 460 to 520 μm.

  Mahata parent fish naturally matures in an artificial environment, and at the end of spawning in June, part of the ovarian egg is ovulated. In some ovulatory individuals seen in the late stage of egg laying, even if LHRHa is implanted and administered, the egg quality of the resulting egg is not stable. When it is desired to stably and efficiently obtain a high-quality fertilized egg, it is desirable to select and use an individual that has not yet ovulated in the natural environment of the early egg-laying stage.

  The age and weight of Mahata parent fish should be at least 5 years old, and the weight of fish should be at least 2 kg. Among them, it is desirable to select and use an individual of 7 to 10 years old and 4 to 6 kg that can synchronize the maturity between individuals and can secure a large amount of eggs.

  In addition, in the maturation and ovulation induction of Mahata parent fish by LHRHa implantation using a cholesterol pellet solid preparation, egg collection after LHRHa implantation must be performed by artificial insemination. Mahata parent fish do not lay eggs in an artificial environment, and eggs are not fertilized even if a female individual lays eggs. If the natural environment can be completely reproduced in the aquarium, it may not be impossible to induce natural spawning, but it is difficult with the current parent fish cultivation technology. For these reasons, when obtaining fertilized eggs from Mahata parent fish, it is necessary to collect eggs by artificial insemination using various hormone treatment techniques.

  Next, a method for inducing maturation and ovulation by injection administration of LHRHa cocoa butter oil preparation will be described.

  In the method for preparing an LHRHa cocoa butter oil-based preparation, first, LHRHa is dissolved in 50% ethyl alcohol.

  The cocoa butter is put in a container such as a sample bottle and dissolved by hot water (about 60 ° C.).

  The dissolved cocoa butter is cooled to about 40 ° C and then mixed with the LHRHa solution already prepared, stirred and homogenized.

  The finished LHRHa cocoa butter oil formulation can be used immediately or stored in a freezer below -20 ° C. The LHRHa cacao butter oil preparation that has been stored frozen can be used by re-dissolving in a hot water bath or the like, and exhibits the same effect of inducing ripening and ovulation as before freezing.

  The LHRHa cocoa butter oily formulation may have a mass ratio of cocoa butter to LHRHa ethyl alcohol solution of 95: 5 to 70:30, but considering the solidification after administration into the fish body, the mass ratio 90:10 is good. If the mass ratio of the LHRHa ethyl alcohol solution is too large, it will take time to solidify in the fish body, which may indicate an unexpected amount of LHRHa elution.

  In addition, the LHRHa content per 0.1 ml of the LHRHa cacao butter oil preparation may be 50 to 200 μg when used for Mahata parent fish, but 50 to 100 μg is preferable among them. Assuming that the dose of LHRHa to Mahata parent fish is 50 μg / kg BW, if the weight of the parent fish is 6 kg, LHRHa needs 300 μg. When the content of LHRHa per 0.1 ml of cocoa butter is 50 μg, 0.6 ml is injected, and when the content is 100 μg, 0.3 ml is injected. Also, if the LHRHa content per 0.1 ml of cocoa butter is reduced to 10 μg, the injection dose of LHRHa cocoa butter will be 3 ml, increasing the stress on the parent fish. On the contrary, when the LHRHa content per 0.1 ml of cocoa butter is increased to 600 μg, the injection dose becomes 0.05 ml, which makes it difficult to handle oily preparations and to perform accurate injection administration work. Of course, the conditions under which LHRHa elutes from cocoa butter oil-based preparations may change drastically, and the expected sustained release amount and duration may not be realized.

  For injection administration of the LHRHa cocoa butter oil-based preparation, an injection administration device using an 18-21G needle having a relatively large needle hole and a 1-5 ml syringe may be used. LHRHa cocoa butter oil solidifies at 17-23 ° C, so in order to administer quickly, cocoa butter solidifies in the needle hole during administration unless an injection needle with a relatively large needle hole is used, Cannot administer.

  The injection administration part of the LHRHa cacao butter oil preparation is considered to be intramuscular and intraperitoneal of Mahata parent fish, but it is particularly preferable to carry out at a depth of about 1-2 cm subcutaneously in the thick part of the back muscle part.

  LHRHa cacao butter oil formulation can be injected by intraperitoneal injection, but it may damage internal organs. Therefore, in order to further reduce the stress on the Mahata parent fish and to perform the injection administration quickly, it is considered that a thick portion of the back muscle of the fish body is suitable for the administration portion.

  When administering LHRHa cocoa butter oil formulation, first administer the mahata parent fish after anesthesia, but the cocoa butter oil formulation in the syringe is highly viscous and difficult to penetrate into the muscle. Be careful not to be fooled. Slowly inject the cocoa butter oil-based preparation, and after injection, hold the planned amount with your fingers if necessary, and pull out the syringe quickly while preventing backflow of the cocoa butter oil-based preparation.

  The LHRHa cocoa butter oil-based preparation is a sustained release preparation that solidifies at 17-23 ° C. at the maturing water temperature of the Mahata parent fish after injection and causes LHRHa to elute in the fish body for a long time.

  A single injection of LHRHa cacao butter oil preparation into Mahata parent fish is sufficient. After cocoa butter solidifies, the period during which LHRHa continues to elute at a high level is approximately 5 days, but since the time until the Mahata parent fish ovulates by injection of LHRHa is 42 to 48 hours, Injection administration is considered sufficient. If the parent fish is not ovulated by a single injection, it is highly likely that the parent fish has not yet matured.

  In the induction of maturation and ovulation of Mahata parent fish by injection of LHRHa using cocoa butter oil-based formulation, the dose of LHRHa to Mahata parent fish is considered to be 20-100 μg / kg BW, among which 50 μg / kg BW is stable Maturation and ovulation can be induced efficiently and efficiently, and high-quality fertilized eggs can be obtained.

  The parent fish used for egg collection can be derived from natural sources or aquaculture. However, if you want to obtain fertilized eggs of high quality stably and efficiently, it is better to use cultured parent fish cultured in an artificial environment.

  Mahata's parent fish inhabiting the natural world are precious and few. In addition, even if the fish can be caught during the spawning season, the ovarian eggs vary in maturity, and it is very difficult to obtain high-quality fertilized eggs from these individuals due to adverse effects such as threading and stress from fishing. .

  On the other hand, cultured parent fish are accustomed to an artificial environment and are accustomed to human handling, so that the mature state of ovarian eggs is highly synchronized between individuals and is resistant to a certain amount of stress. From these things, the direction which uses the training parent fish can secure many individuals suitable for egg collection, and can obtain a high-quality fertilized egg.

  In the induction of ripening and ovulation of Mahata parent fish by LHRHa injection administration using cocoa butter oil formulation, parent fish at the time of LHRHa injection administration can induce ovulation in individuals with an ovary egg diameter of 420 to 550 μm. It is better to use an individual whose ovary egg diameter is 460 to 520 μm.

  Individuals with an ovarian egg diameter of less than 420 μm are immature individuals whose maturation and ovulation are not induced by injection of LHRHa, and those between 420 and 460 μm are not yet sensitive to hormones. It is an individual with a small number of mature and ovulated eggs.

  On the other hand, individuals with an ovary egg diameter of 550 μm or more are individuals who cannot expect to obtain a high-quality fertilized egg because there are many degenerated eggs and ovulation eggs in the ovary egg, and individuals with a diameter of 520 to 550 μm Is an individual whose egg quality is getting worse due to the appearance of some degenerate eggs and ovulatory eggs in the ovarian eggs. For these reasons, it is desirable that the parent fish used for egg collection is selected from individuals with an ovarian egg diameter of 460 to 520 μm.

  Mahata parent fish naturally matures in an artificial environment, and at the end of spawning in June, part of the ovarian egg is ovulated. In some ovulation individuals seen in the late stage of egg laying, even if LHRHa is administered by injection, the egg quality of the resulting egg is not stable. When it is desired to stably and efficiently obtain a high-quality fertilized egg, it is desirable to select and use an individual that has not yet ovulated in the natural environment of the early egg-laying stage.

  The age and weight of the Mahata parent fish should be at least 5 years old, and the weight of the fish should be at least 2 kg. Among them, it is desirable to select and use an individual of 7 to 10 years old and 4 to 6 kg that can synchronize the maturity between individuals and can secure a large amount of eggs.

  In addition, in the maturation and ovulation induction of Mahata parent fish by LHRHa injection administration using cocoa butter oil-based preparation, egg collection after LHRHa injection administration must be performed by artificial insemination. Mahata parent fish do not lay eggs in an artificial environment, and eggs are not fertilized even if a female individual lays eggs. If the natural environment can be completely reproduced in the aquarium, it may not be impossible to induce natural spawning, but it is difficult with the current parent fish cultivation technology. For these reasons, when obtaining fertilized eggs from Mahata parent fish, it is necessary to collect eggs by artificial insemination using various hormone treatment techniques.

  In the method of securing good quality eggs of the mahata parent fish of the present invention, maturation and ovulation induction are performed by implanting LHRHa cholesterol pellet solid preparation or injection administration of LHRHa cocoa butter oil preparation, and the timing of artificial insemination is collected immediately after ovulation. Immediately fertilizing can yield high-quality fertilized eggs stably and efficiently.

  In Mahata, the egg quality of eggs obtained by hormonal treatment decreased with the passage of time after ovulation. It is necessary to check the ovulation status of the parent fish at an appropriate time and to perform artificial insemination immediately after ovulation.

  Furthermore, compared to other fish species, Mahata has a rapid decline in egg quality that progresses over time after ovulation, so in order to obtain high-quality fertilized eggs stably and efficiently, This technology is important.

  In the method of maturation and ovulation induction of Mahata parent fish by implantation of LHRHa using a solid pellet of cholesterol pellets or injection of LHRHa using a cacao butter oil preparation, parental fish with an ovarian egg size of 420-550 μm are treated with LHRHa. Ovulation occurs between 36 and 54 hours, but it is concentrated in 42 hours after LHRHa administration.

  In the method of inducing maturation and ovulation of mahata parent fish by implantation of LHRHa using a solid cholesterol pellet formulation or injection of LHRHa using a cocoa butter oil formulation, the optimal timing of artificial insemination after administration of LHRHa is 0 to 0 after ovulation. Although it is 6 hours, fertilized eggs with higher egg quality such as fertilization rate and hatching rate can be obtained by artificial insemination immediately after ovulation.

  Therefore, in order to stably and efficiently secure high-quality fertilized eggs from Mahata parent fish, an egg collection schedule for artificial insemination after 42 hours from administration of the LHRHa sustained-release preparation may be planned.

  In other words, the administration of LHRHa sustained-release preparation to Mahata parent fish was carried out at 16:00, and at 10:00 the next day, the abdomen of the parent fish was palpated and ovulation was confirmed. Artificial insemination may be performed.

  In this way, by taking into account the timing of artificial insemination after induction of ripening and ovulation using LHRHa sustained-release preparations, sophistication of hormonal processing egg collection technology has been achieved, and stable and efficient production of high-quality eggs from Mahata parent fish. It can be secured.

  EXAMPLES Hereinafter, the present invention will be described more specifically with reference to examples. However, the present invention is not intended to be interpreted as being limited to the following examples.

Example 1
For the purpose of improving fertilization rate in egg collection by artificial insemination using Mahata-trained parent fish, we investigated the relationship between the elapsed time after ovulation and the fertilization rate using parent fish that have induced maturation and ovulation by LHRHa administration. A study was made on the optimal time for the spermatozoon.

Implantation of a cholesterol pellet solid preparation of synthetic luteinizing hormone-releasing hormone (LHRHa: des-Gly ¹⁰ , [D-Ala ⁶ ] -LHRH ethylamide) was carried out on 11 cultured female parent fish, and induction of maturation and ovulation . The dose of LHRHa was 50 μg / kg BW. Ovarian eggs were collected with a cannula tube at the time of LHRHa administration and the egg diameter was measured. As a result, the egg diameter of the ovary egg of the test fish was 457 to 516 μm. The administration of the LHRHa sustained-release preparation was carried out by embedding a cholesterol pellet solid preparation in Example 1, but the LHRHa injection administration using a cocoa butter oil-based preparation was different from the contents of Example 1 instead. Does not occur. For this reason, the example by the injection administration of LHRHa using a cacao butter oil formulation was abbreviate | omitted.

  FIG. 1 shows an experimental schedule for examining the optimum timing of artificial insemination of Mahata parent fish in this test. Parents were anesthetized at 12, 18, 24, 30, 36, 42, 48 and 54 hours after LHRHa administration, and then examined for ovulation by palpation of the abdomen. The ovulation time was confirmed by pressing the abdomen of the test fish and checking whether or not the ovulated egg flows out from the genital mouth, and the time when the ovulated egg began to flow out was immediately after ovulation. For individuals confirmed to be ovulated, immediately after ovulation (set to 0 hour) from the same individual, about 2,000 eggs were squeezed every 6 hours, semen was added, and artificial insemination was performed by the dry-conduction method .

  Eggs (about 2,000 grains) obtained by artificial insemination were washed and then separated into floating eggs and sinking eggs, and the floating egg rate (number of floating eggs / total number of eggs) was calculated. The fertilization rate was calculated as the percentage of individuals with an advanced development out of about 100 floating eggs in the 16-32 cell stage (water temperature 20 ° C.) after 4 hours of artificial insemination. The hatching rate was calculated as the proportion of hatched eggs that were separately contained in a 1 L container in an incubator at 20 ° C. with about 200 floating eggs.

  FIG. 2 shows the ovulation situation after administration of LHRHa of the Mahata parent fish. The time when ovulation was confirmed earliest after LHRHa administration was 36 hours later, and ovulation was confirmed in 1 out of 11 individuals. Thereafter, ovulation was confirmed in 8 individuals after 42 hours, 1 individual after 48 hours, and 1 individual after 54 hours. Thus, it was confirmed that ovulation was concentrated 42 hours after administration of LHRHa in Mahata parent fish.

  FIG. 3 shows the change in egg diameter after administration of LHRHa in Mahata parent fish. Ovulation was concentrated in 8 out of 11 individuals 42 hours after LHRHa administration, and the egg diameter in that case increased rapidly over 30 to 42 hours.

  FIG. 4 shows the relationship between the elapsed time after ovulation and the fertilization rate monitored for each individual. The fertilization rate of each individual immediately after ovulation was as high as 88.2% (74.3% to 100%) on average, and the average value was as high as 68.0% after 6 hours. However, after that, the fertilization rate decreased rapidly with the elapsed time after ovulation, reaching 28.9% at 12 hours after ovulation and 12.3% at 18 hours. It was confirmed that the fertilization rate decreased with the elapsed time after ovulation in all the test fish.

  FIG. 5 shows the relationship between the elapsed time after ovulation and the hatching rate monitored for each individual. The hatching rate examined from the eggs immediately after ovulation of each individual showed a very high value of 68.2% (29.5-98.2%) on average. However, after that, the hatching rate decreased rapidly with the elapsed time after ovulation and became 0.9% at 12 hours after ovulation. From these results, it was confirmed that a high hatching rate could not be expected unless the eggs were just after ovulation.

  FIG. 6 shows changes in the average floating egg rate, average fertilization rate, and average hatching rate of 11 individuals. The floating egg rate, fertilization rate, and hatching rate were high immediately after ovulation and then decreased with time. In particular, the hatching rate decreased rapidly at 12 hours and showed a value of 0.9%. From this, it became clear that high-quality fertilized eggs cannot be secured unless artificial insemination is performed within 6 hours of ovulation.

  From the above results, it has been clarified that the fertilization rate of Mahata artificially inseminated eggs depends on the time elapsed since the ovulation of the eggs, that is, the residence time in the ovarian cavity. In other words, when fertilization was performed within 6 hours after ovulation, the average fertilization rate was 68.0% or higher, but then the fertilization rate decreased rapidly and became 10% or less after 24 hours from ovulation. . The same applies to the floating egg rate and hatching rate, and these values were highest immediately after ovulation and then decreased with time. When artificial insemination is performed at the actual seed production site, it is desirable to perform artificial insemination immediately after ovulation, considering the floating egg rate, fertilization rate, and hatching rate of the obtained egg, and it is necessary to do it within 6 hours at the latest It has been found.

  Therefore, when performing artificial insemination of Mahata as a business, predicting the ovulation time and promptly fertilizing is the decisive factor for efficiently obtaining good quality eggs. Since ovulation was concentrated in most individuals at 42 hours after administration of LHRHa, Mahata performed ovulation confirmation work at that time, and artificial insemination should be carried out immediately in individuals confirmed ovulation Thus, it was revealed that high-quality fertilized eggs showing a high fertilization rate and hatching rate can be stably secured.

  The present invention enables the sophistication of egg collection technology from Mahata parent fish and the stable production of artificial seedlings of this species, and has an extremely high contribution in the aquaculture field (aquaculture and cultivated fisheries) in the fishery industry.

It is a figure which shows the experiment schedule for examining the optimal artificial insemination timing of a Mahata parent fish. It is a figure which shows the ovulation state after LHRHa administration of Mahata parent fish. It is a figure which shows the change of the egg diameter after LHRHa administration of Mahata parent fish. It is a figure which shows the relationship between the elapsed time after ovulation and fertilization rate of Mahata parent fish. It is a figure which shows the relationship between the elapsed time after ovulation and hatching rate of Mahata parent fish. It is a figure which shows the relationship between the elapsed time after ovulation and egg quality (floating egg rate, fertilization rate, hatching rate) in artificial insemination of Mahata parent fish.

Claims (16)

Synthetic luteinizing hormone releasing hormone ^{(LHRHa: des-Gly 10,} [D-Ala 6] -LHRH ethylamide) as a sustained release dosage regimen performs injection administration of the embedded administration or cocoa butter oil preparation cholesterol pellet solid preparation, embedding The LHRHa cholesterol pellet solid preparation to be administered is prepared by dissolving LHRHa in 50% ethyl alcohol, then adding the solution to cholesterol powder, mixing until a paste is formed, and homogenizing the whole to obtain a paste The LHRHa cholesterol is stored in a vacuum desiccator, etc., dried thoroughly, and the cocoa butter dissolved in the dried LHRHa cholesterol powder is added and mixed, and the whole is homogenized, and the cocoa butter that acts as a binder is added. Place the LHRHa cholesterol powder into the hole of the pelletizer, and then compress it into a cylindrical shape And the finished LHRHa cholesterol pellet solid preparation is stored in a freezer at -20 ° C or lower, and the LHRHa cocoa butter oil preparation for injection administration contains 50% ethyl alcohol in LHRHa. The mass ratio of cacao butter and LHRHa solution is 95: 5 to 70:30, the dose of LHRHa is 20-100 μg / kg BW, and the mahata parent fish is cultured under artificial conditions Use a parent fish and use an individual whose ovary egg diameter is 420 to 550 μm. Egg collection after hormonal treatment is performed by artificial insemination. The timing of artificial insemination is immediately after ovulation, collecting the egg and immediately fertilizing it. To secure good quality eggs of Mahata parent fish in consideration of the timing of artificial insemination after induction of maturation and ovulation using LHRHa sustained-release preparation, which is characterized by obtaining stable fertilized eggs stably and efficiently . In mature ovulation induction methods Mahata broodstock by injection administration of LHRHa with embedded administration or cocoa butter oil formulation LHRHa with cholesterol pellet solid preparation, ovulation of claim 1, wherein the occurring 36-54 hours after LHRHa administration A technique for securing high-quality eggs in Mahata parent fish in consideration of the timing of artificial insemination after maturation and ovulation induction using LHRHa sustained-release preparations. In the method of inducing maturation and ovulation of mahata parent fish by implantation of LHRHa using a solid cholesterol pellet formulation or injection of LHRHa using a cocoa butter oil formulation, the optimal timing of artificial insemination after administration of LHRHa is 0 to 0 after ovulation. A method for securing a high-quality egg of a Mahata parent fish in consideration of the timing of artificial insemination after maturation and ovulation induction using the LHRHa sustained-release preparation according to claim 1, which is 6 hours. The method for securing a good quality egg of a Mahata parent fish in consideration of the timing of artificial insemination after induction of maturation and ovulation using the LHRHa sustained release preparation according to claim 1, wherein each LHRHa cholesterol pellet solid preparation contains 50 to 400 µg of LHRHa. A solid preparation of LHRHa cholesterol pellets is embedded in the muscles of Mahata parent fish, ensuring high quality eggs of Mahata parent fish in consideration of the timing of artificial insemination after induction of maturation and ovulation using the LHRHa sustained release preparation according to claim 1 Technique. The solid preparation of LHRHa cholesterol pellets is embedded in the abdominal cavity of Mahata parent fish, ensuring high quality eggs of Mahata parent fish in consideration of the timing of artificial insemination after induction of maturation and ovulation using the LHRHa sustained release preparation according to claim 1 Technique. The LHRHa cocoa butter oil preparation is prepared by dissolving LHRHa in 50% ethyl alcohol, and the mass ratio of cocoa butter to LHRHa solution is 90:10 . Maturation / ovulation using the LHRHa sustained release preparation according to claim 1 A technique for securing high-quality eggs of Mahata parent fish in consideration of the timing of artificial insemination after induction. The method for securing a good quality egg of a mahata parent fish in consideration of the timing of artificial insemination after induction of maturation and ovulation using the LHRHa sustained-release preparation according to claim 1 , wherein the LHRHa cacao butter oil preparation contains 50 to 200 µg of LHRHa per 0.1 ml. The LHRHa cacao butter oil-based preparation is injected intramuscularly into Mahata parent fish, and the quality of the Mahata parent fish in consideration of the timing of artificial insemination after induction of ripening and ovulation using the LHRHa sustained release preparation according to claim 1 Egg securing technique. The LHRHa cacao butter oil-based preparation is injected intraperitoneally into the Mahata parent fish, and the quality of the Mahata parent fish in consideration of the timing of artificial insemination after induction of maturation and ovulation using the LHRHa sustained release preparation according to claim 1 Egg securing technique. The LHRHa cacao butter oil preparation is injected into the muscles of Mahata parent fish and then solidifies at 17-23 ° C at the maturing water temperature of the Mahata parent fish, resulting in a sustained release formulation that elutes LHRHa continuously over a long period of time. A method for securing high-quality eggs of Mahata parent fish in consideration of the timing of artificial insemination after induction of ripening and ovulation using the LHRHa sustained release preparation according to claim 1 . LHRHa cacao butter oil formulation is injected into the abdominal cavity of Mahata parent fish and then coagulates at 17-23 ° C at the maturing water temperature of Mahata parent fish, resulting in a sustained release formulation that elutes LHRHa continuously over a long period of time. A method for securing high-quality eggs of Mahata parent fish in consideration of the timing of artificial insemination after induction of ripening and ovulation using the LHRHa sustained release preparation according to claim 1 . The needle used for injection administration of the LHRHa cocoa butter oil-based preparation is a mature one using the LHRHa sustained-release preparation according to claim 1 , wherein a needle having a relatively large needle hole of 18 to 21G is used to prevent clogging. A technique for securing high-quality eggs in Mahata parent fish, taking into account the timing of artificial insemination after ovulation induction. 2. The Mahtha parent fish in consideration of the timing of artificial insemination after induction of maturation and ovulation using the LHRHa sustained release preparation according to claim 1 , wherein the LHRHa cholesterol pellet solid preparation and the LHRHa cacao butter oil preparation are administered once. To secure good quality eggs. The high-quality Mahata parent fish in consideration of the timing of artificial insemination after induction of maturation and ovulation using the LHRHa sustained release preparation according to claim 1 , wherein the Mahata parent fish is an individual that has not yet ovulated in the natural environment during the spawning season. Egg securing technique. The Mahata parent fish is a mahata that takes into account the timing of artificial insemination after induction of maturation and ovulation using the LHRHa sustained-release preparation according to claim 1, wherein an individual with a weight of 2 kg or more is used. Securing high quality eggs for parent fish.