JP4899570B2 - Maturation and ovulation induction of Mahata parent fish by LHRHa injection administration using cocoa butter oil formulation - Google Patents

Description

  The present invention relates to a novel maturation / ovulation induction method for the purpose of obtaining high-quality fertilized eggs for seedling production in a stable, efficient and simple manner in hormone-treated egg collection from Mahata parent fish.

  Conventional maturation and ovulation induction by administration of hormones for Mahata has been mainly performed by injection of HCG (human placental gonadotropic hormone) solution, SP (chum salmon pituitary) solution and HCG / SP mixed solution. I had a fertilized egg for seed production.

  However, it is difficult to obtain a high-quality fertilized egg stably and efficiently even after injection of HCG solution, SP solution and HCG / SP mixed solution. It was low.

  In general, in the method of injecting an aqueous solution of a hormonal agent into fish, the concentration in the body rapidly increases after absorption, but thereafter it is rapidly decomposed, so that the hormonal sustained effect is short. In Mahata, ovulation does not occur until 42-48 hours have passed after hormone treatment, but egg collection results may be improved by using a technique that maintains the hormone treatment effect until then.

On the other hand, in recent years, synthetic luteinizing hormone-releasing hormone (LHRHa: des-Gly ¹⁰ , [D-Ala ⁶ ] -LHRH ethylamide) has begun to be used to promote fish maturation and ovulation, and its maturation and ovulation induction effect is high. Has become clear.

  Therefore, the present invention adopts LHRHa as a maturation / ovulation-inducing hormone agent for Mahata parent fish, increases its sustainability, enables sustained-release administration, and facilitates production of hormone administration methods and sustained-release preparations. An object is to provide a novel stable, efficient and simple egg collection method.

  In order to achieve the above-mentioned object, the inventors made a cocoa butter solution as a sustained release preparation of LHRHa, and as a result of clarifying its maturation / ovulation inducing effect on mahata parent fish by various tests, We have found a method for inducing maturation and ovulation of Mahata parent fish by injection of LHRHa with cocoa butter oil.

The invention of claim 1 uses a cocoa butter oil-based preparation as a sustained-release preparation that enables a sustained-release administration of a synthetic luteinizing hormone-releasing hormone (LHRHa: des-Gly ¹⁰ , [D-Ala ⁶ ] -LHRH ethylamide) The LHRHa cocoa butter oil preparation is prepared by dissolving LHRHa in 50% ethyl alcohol, the mass ratio of cocoa butter and LHRHa solution is 95: 5 to 70:30, and the oil preparation containing LHRHa is administered by injection. The dosage of LHRHa is 20-100 μg / kg BW, and the Mahata parent fish is a cultured parent fish cultured in an artificial environment, using an individual whose ovary egg diameter is 420-550 μm, and after hormone treatment Egg collection is performed by artificial insemination to obtain a high-quality fertilized egg stably, efficiently and simply from a Mahata parent fish.

The invention of claim 2 is the invention according to claim 1, wherein the LHRHa cocoa butter oil-based preparation is prepared by dissolving LHRHa in 50% ethyl alcohol and having a mass ratio of cocoa butter and LHRHa solution of 90:10 .

  The invention of claim 3 is that, in claim 1, the LHRHa cocoa butter oil-based preparation contains 50 to 200 μg of LHRHa per 0.1 ml.

  The invention of claim 4 is the invention according to claim 1, wherein the LHRHa cacao butter oil preparation can be stored at −20 ° C. or lower after being stored in a sample bottle, etc. It can be used.

  The invention of claim 5 is characterized in that, in claim 1, the LHRHa cacao butter oily preparation is injected and administered intramuscularly or intraperitoneally of a Mahata parent fish.

  The invention of claim 6 is the invention according to claim 1, wherein the LHRHa cocoa butter oily preparation is injected and injected intramuscularly or intraperitoneally of the Mahata parent fish, and then coagulates at 17-23 ° C. at the maturing water temperature of the Mahata parent fish. It is characterized by being a sustained-release preparation that elutes LHRHa for a long period of time.

  The invention of claim 7 is the invention of claim 1, wherein the needle used for injection administration of the LHRHa cocoa butter oil preparation is a needle having a relatively large needle hole of 18 to 21G in order to prevent clogging.

  The invention according to claim 8 is the invention according to claim 1, wherein the number of injections of the LHRHa cacao butter oil preparation to the mahata parent fish is one.

  According to a ninth 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.

  According to a tenth aspect of the present invention, in the first aspect, the mahata parent fish is 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 maturation and ovulation induction method of mahata parent fish by LHRHa injection administration using the cocoa butter oil-based preparation of the present invention, the conventional method (HCG / SP) Compared with injection methods such as a mixed solution, it has an advantageous effect that fertilized eggs for seedling production can be secured more stably.

  According to the method of inducing maturation and ovulation of Mahata parent fish by LHRHa injection administration using the cocoa butter oil-based preparation of the present invention, the hormonal administration work is easier compared to the LHRHa pellet implantation administration method, and the Since the preparation of the releasable preparation is simple and the cost burden is small, it has an advantageous effect that the technology can be widely spread to seedling production organizations currently producing on a business basis.

  Further, according to the maturation / ovulation induction method of Mahata parent fish by LHRHa injection administration using the cocoa butter oil-based preparation of the present invention, it becomes possible to obtain high-quality fertilized eggs stably, efficiently and simply. As a result, mass production of Mahata artificial seedlings is realized, and there is an advantageous effect that development of a fish farming industry that expects Mahata seedlings for aquaculture production is expected.

  In the method for inducing maturation and ovulation of Mahata parent fish of the present invention, a cocoa butter oil-based preparation that enables sustained release administration of LHRHa is prepared and used.

  In the method for producing an LHRHa cocoa butter oil-based preparation (FIG. 1), 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 (FIG. 2) using an 18 to 21G injection needle having a relatively large needle hole and a 1 to 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 (FIG. 3). ).

  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 coagulates at 17-23 ° C. at the maturing water temperature of the Mahata parent fish after injection and becomes a sustained-release preparation that elutes LHRHa within the fish body for a long time (FIG. 4).

  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 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 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.

  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
A specific method for preparing a cocoa butter oil-based preparation that enables the sustained release administration of LHRHa in the maturation / ovulation induction method of Mahata parent fish is shown below.

  When preparing a cocoa butter oil-based preparation containing 100 μg of LHRHa per 0.1 ml, first, 10 mg of LHRHa is dissolved in 1 ml of 50% ethyl alcohol.

  Cocoa butter is dissolved in a hot water bath (about 60 ° C.), and 9 ml is stored in a 20-30 ml sample bottle or the like. After the dissolved cocoa butter is cooled to about 40 ° C., the LHRHa solution prepared previously is added to the container, the cap is capped, and shaken up and down to stir and homogenize. In this case, the mass ratio of cocoa butter to LHRHa solution is 90:10.

  The LHRHa cocoa butter oil preparation (LHRHa: 100 μg / 0.1 ml) prepared in this way is completed for a 10 ml dose. In the case of processing LHRHa at a dose of 50 μg / kg BW for Mahata parent fish, this corresponds to a treatment amount of 200 kg body weight of the parent fish.

  In addition, when preparing a cacao butter oil-based preparation containing 50 μg of LHRHa per 0.1 ml, the amount of LHRHa added may be 5 mg in the above preparation method. The LHRHa cacao butter oil preparation (LHRHa: 50 μg / 0.1 ml) prepared in this way has a processing amount of 100 kg of the parent fish weight when processing LHRHa at a dose of 50 μg / kg BW against the Mahata parent fish. It will be completed.

Example 2
The purpose of this study group was to understand the change in the amount of LHRHa elution in the blood of fish body by both methods in the induction of maturation and ovulation of parental fish by LHRHa administration using cholesterol pellet solid preparation and cocoa butter oil preparation. Using testosterone, which has established a measurement system at the present time, administration experiments were conducted using both methods, and changes in the amount of elution in the blood were examined.

  In this experiment, Mahata should be used. However, it was difficult to secure the necessary experimental fish, and Magoi, which is easily available, was used. In addition, since testosterone is produced in a large amount in mature fish, immature fish having a low testosterone-producing ability and a body weight of about 100 g was used for the experiment.

  First, a solid preparation of cholesterol pellets and a cocoa butter oil preparation containing testosterone were prepared, and testosterone was implanted and injected. The testosterone dose was 100 μg for each testosterone. The test fish were reared and managed at a water temperature of 20 ° C. during the experiment.

  Blood was collected 12 hours, 1 day, 2 days, 4 days and 7 days after testosterone administration. The collected blood was separated from serum by centrifugation, and the amount of testosterone elution was measured by ELISA.

  The change in the amount of testosterone eluted in the blood is shown in FIG. The amount of testosterone in the blood 12 hours after the testosterone implantation and injection was 3.4 ng / ml by the cholesterol pellet method and 12.3 ng / ml by the cocoa butter method. Thereafter, it decreased with the passage of time, and the blood elution amount after one week was 0.4 ng / ml by the cholesterol pellet method and 1.8 ng / ml by the cocoa butter method. The cocoa butter method always showed a higher elution amount (about 3 times) than the cholesterol pellet method.

  As a result of administering testosterone by the embedding administration method using the cholesterol pellet solid preparation and the injection administration method using the cacao butter oil preparation, there was a difference in the amount of testosterone dissolved in blood between the two methods. The elution amount of testosterone was higher in the cocoa butter method than in the cholesterol pellet method until one week later. It takes 42 to 48 hours for Mahata to ovulate after hormone treatment. During this period, it is sufficient if the hormone required for ovulation is eluted from the sustained release formulation. Therefore, both methods were considered to be effective sustained-release preparations with sufficient hormone elution period. In addition, the cacao butter oil-based formulation was about 3 times the amount eluted from the cholesterol pellet solid formulation, so even if the hormone dose was reduced to about 1/3, induction of ripening and ovulation equivalent to the cholesterol pellet method was achieved. There may be an effect.

Example 3
In the induction of maturation and ovulation of Mahata parent fish by LHRHa injection administration using cocoa butter oil formulation, LHRHa implantation method using cholesterol pellet solid formulation developed at the same time as the present invention and this method in Mahata parent fish The effect of inducing maturation and ovulation was compared by an egg collection test.

  MHR / ovulation induction methods by LHRHa administration that have been compared are the cholesterol pellet solid preparation implantation method and the cocoa butter oil preparation injection method.

  In the method of implanting a cholesterol pellet solid preparation, a cylindrical cholesterol pellet solid preparation having a length of 6 mm and a diameter of 2 mm containing 100 μg / kg of LHRHa per pellet was prepared. The LHRHa cholesterol pellet solid preparation was anesthetized with a parent fish, cut into the back muscle epidermis with a scalpel, and implanted into the back muscle using a pellet implanter.

  In the injection administration method using the cocoa butter oily preparation, a cocoa butter oily preparation containing 50 μg of LHRHa per 0.1 ml was prepared. The LHRHa cocoa butter oil-based preparation was injected into the back muscles using a syringe after anesthetizing the parent fish.

  The dose of LHRHa was 50 μg / kg BW in any administration method.

  For the test fish, 40 cultivated 7-year-old fish cultured in an artificial environment were used. The test fish was anesthetized, ovarian eggs were collected in advance with a cannula, and the diameter of 30 large eggs was measured. As a result, the egg diameter was 415 to 544 μm.

  For confirmation of ovulation, the abdomen was palpated 42 hours after administration of HCG and LHRHa, and for the individual confirmed to be ovulated, the egg was squeezed out by pressing the abdomen, and artificial insemination was immediately performed by the dry induction method.

Eggs obtained by artificial insemination were washed, separated into floating eggs and sinking eggs, and the amount of floating eggs was calculated by the volumetric method (2,200 grains / ml). In addition, the fertilization rate was calculated as the proportion of individuals that were developing out of about 100 floating eggs. The hatching rate was calculated by separately storing 200 grains in a 1 L container in an incubator at 20 ° C., and the ratio of hatched individuals among the 200 grains.

  FIG. 6 shows the results of maturation / ovulation induction by implantation using LHRHa cholesterol pellet solid preparation and injection administration using cocoa butter oil preparation. In the cholesterol pellet implantation method, 20 out of 26 parent fish ovulated, the ovulation individual rate was 76.9%, the floating egg amount was 636,000, the fertilization rate was 77.4%, and the hatching rate was 54.8%. On the other hand, in the cocoa butter injection method, 10 out of 14 parent fish ovulated, the ovulation individual rate was 71.4%, the floating egg amount was 605,000, the fertilization rate was 72.9%, and the hatching rate was 50.4%.

  From these results, it was clarified that in both maturation and ovulation induction of Mahata parent fish, good egg collection results were obtained by both methods, and high-quality fertilized eggs were obtained stably and efficiently from Mahata parent fish.

  In the injection administration method using a cocoa butter oil-based preparation, the LHRHa solvent can be easily prepared tens of minutes before administration, and no tools or equipment are required. Moreover, the administration method to Mahata parent fish is also simple. Furthermore, since a special embedding operation is not required, the load of the parent fish is small, and the work is easy in the field of seedling production, so that it is a widespread and practical method.

  Moreover, in the injection administration method using the cocoa butter oil-based preparation, a high maturation / ovulation induction effect was confirmed as in the case of the implantation administration method using the cholesterol pellet solid preparation. Furthermore, the fertilization rate and hatching rate of the obtained eggs were also good, and it was judged that they could be used sufficiently for seedling production.

  As described above, the injection administration method using the cocoa butter oil-based preparation is an epoch-making method that solves the problems while maintaining the excellent point of the cholesterol pellet embedding method developed at the same time as the present invention. It was evaluated.

Example 4
In the induction of maturation and ovulation of Mahata parent fish by LHRHa injection administration using cocoa butter oil-based preparations, we investigated the effect of induction of maturation and ovulation on the difference in LHRHa dose to the parent fish, and examined the appropriate LHRHa dose.

  The LHRHa doses studied for comparison are 50 and 10 μg / kg BW.

  In the injection administration method using the cocoa butter oily preparation, a cocoa butter oily preparation containing 50 μg of LHRHa per 0.1 ml was prepared. The LHRHa cocoa butter oil-based preparation was injected into the back muscles using a syringe after anesthetizing the parent fish.

  Thirteen cultivated 7-year-old fish cultured in an artificial environment were used as test fish. The test fish was anesthetized, ovarian eggs were collected in advance with a cannula, and the diameter of 30 large eggs was measured. As a result, the egg diameter was 422 to 507 μm.

  For confirmation of ovulation, the abdomen was palpated 42 hours after administration of HCG and LHRHa, and for the individual confirmed to be ovulated, the egg was squeezed out by pressing the abdomen, and artificial insemination was immediately performed by the dry induction method.

  Eggs obtained by artificial insemination were washed, separated into floating eggs and sinking eggs, and the floating egg rate and floating egg amount were calculated by the volumetric method (2,200 grains / ml). In addition, the fertilization rate was calculated as the proportion of individuals that were developing out of about 100 floating eggs.

  FIG. 7 shows the maturation and ovulation induction results of Mahata parent fish in the case of LHRHa injection administration using cocoa butter oil-based preparations when the dosage is 50 and 10 μg / kg BW. When the dose is 50μg / kg BW, 5 of the 6 parent fish ovulate, the ovulation rate is 83.3%, the floating egg rate is 71.2%, the floating egg amount is 1328,000, and the fertilization rate is 65.5% Met. On the other hand, when the dose is 10μg / kg BW, 4 out of 7 parent fish ovulate, the ovulation rate is 57.1%, the floating egg rate is 58.2%, the floating egg amount is 423,000, and the fertilization rate is It was 60.1%.

  From these results, the dose of LHRHa in the injection administration method of LHRHa using a cacao butter oil-based preparation is 50 μg / kg BW higher than that of 10 μg / kg BW, and the maturation / ovulation induction effect of Mahata parent fish is higher. It was revealed that fertilized eggs of good quality can be obtained stably, efficiently and simply.

  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 preparation methods of a LHRHa cocoa butter oil-based formulation. It is a figure which shows the cocoa butter oil-based formulation and injection administration device which enable the sustained release administration of LHRHa. It is a figure which shows the injection administration situation of the parent fish for egg-collecting of a mahata, and a LHRHa cacao butter oil preparation. It is a figure which shows the solidification condition in the fish body after injection administration of a LHRHa cocoa butter oil-based formulation. It is a figure which shows the change of the amount of blood elution in the Magoi body of the testosterone administered by the injection administration method of a cacao butter oil-based preparation, and the implantation administration method of a cholesterol pellet solid preparation. It is a figure which shows the maturation and ovulation induction effect of the mahata parent fish by the LHRHa cholesterol pellet embedding administration method and the LHRHa cacao butter injection administration method. It is a figure which shows the difference in LHRHa dosage in the LHRHa cacao butter injection administration method, and its maturation and ovulation induction effect.

Claims (10)

Synthetic luteinizing hormone releasing hormone ^{(LHRHa: des-Gly 10,} [D-Ala 6] -LHRH ethylamide) Using the cocoa butter oil formulation into sustained-release preparations to permit a sustained release administration of the LHRHa cocoa butter oil The formulation is LHRHa dissolved in 50% ethyl alcohol, and the mass ratio of cocoa butter to LHRHa solution is 95: 5 to 70:30. The oily formulation containing LHRHa is administered by injection, and the dosage of LHRHa Is 20 ~ 100μg / kg BW, Mahata parent fish is a cultured parent fish cultured in an artificial environment, its ovary egg diameter is 420 ~ 550μm, and egg collection after hormone treatment is performed by artificial insemination A method for inducing maturation and ovulation of mahata parent fish by injection of LHRHa using a cacao butter oil-based preparation, which is characterized in that it provides stable, efficient and simple fertilized eggs in a stable, efficient and simple manner. The LHRHa cocoa butter oil preparation is prepared by dissolving LHRHa in 50% ethyl alcohol, and the mass ratio of the cocoa butter and the LHRHa solution is 90:10. The injection of LHRHa using the cocoa butter oil preparation according to claim 1 Maturation and ovulation induction method of Mahata parent fish by   2. The method for inducing maturation and ovulation of mahata parent fish by injection administration of LHRHa using the cocoa butter oil-based preparation according to claim 1, wherein the LHRHa cocoa butter oil-containing preparation contains 50 to 200 μg of LHRHa per 0.1 ml.   The cocoa butter oil-based preparation according to claim 1, wherein the LHRHa cocoa butter oil-based preparation can be stored at -20 ° C or lower after being stored in a sample bottle or the like, and can be used by dissolving in a hot water bath or the like when used again. A method for inducing maturation and ovulation of Mahata parent fish by injection of LHRHa using a pharmaceutical preparation.   2. The method for inducing maturation and ovulation of mahata parent fish by injection administration of LHRHa using the cocoa butter oily preparation according to claim 1, wherein the LHRHa cocoa butter oil preparation is administered intramuscularly or intraperitoneally of the mahata parent fish.   LHRHa cacao butter oil formulation is injected into intramuscularly or intraperitoneally of Mahata parent fish, and then coagulates at 17-23 ° C at the maturing water temperature of Mahata parent fish to release LHRHa for a long period of time. 2. A method for inducing maturation and ovulation of a mahata parent fish by LHRHa injection administration using the cocoa butter oil-based preparation according to claim 1, wherein the preparation is a preparation.   The injection of LHRHa using the cocoa butter oil preparation according to claim 1, wherein the needle used for injection administration of the LHRHa cocoa butter oil preparation is a needle having a relatively large needle hole of 18 to 21G in order to prevent clogging. Maturation and ovulation induction method of mahata parent fish by administration.   2. The method for inducing maturation and ovulation of mahata parent fish by LHRHa injection administration using the cocoa butter oil-based preparation according to claim 1, wherein the LHRHa cocoa butter oil-form preparation is injected once.   2. The method for inducing maturation and ovulation of mahata parent fish by LHRHa injection administration using the cacao butter oil-based preparation according to claim 1, wherein the mahata parent fish is an individual that has not yet ovulated in the natural environment in the spawning season.   The method for inducing maturation and ovulation of mahata parent fish by injection administration of LHRHa using the cacao butter oil preparation according to claim 1, wherein the mahata parent fish is an individual of 5 years old or older who can induce maturation and ovulation and the fish weight is 2 kg or more. .