JP4899569B2 - Maturation and ovulation induction method of Mahata parent fish by LHRHa implantation using solid cholesterol pellets - Google Patents

Description

  The present invention relates to a novel maturation / ovulation induction method aimed at obtaining a high-quality fertilized egg for seed and seedling production stably and efficiently in hormonally 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, and also uses a technique that increases the sustainability and enables sustained release, thereby providing a stable and efficient egg collection method. Is to provide.

  In order to achieve the above object, the inventors prepared a cholesterol pellet as a sustained release preparation of LHRHa, and clarified its maturation / ovulation-inducing effect on Mahata parent fish by various tests. We have come up with a method for inducing maturation and ovulation in Mahata parent fish by embedding LHRHa using a solid pellet.

The invention according to claim 1, synthetic luteinizing hormone releasing hormone ^{(LHRHa: des-Gly 10,} [D-Ala 6] -LHRH ethylamide) Using cholesterol pellet solid preparation to permit a sustained release administration of the LHRHa The method for preparing a cholesterol pellet solid preparation is to dissolve LHRHa in 50% ethyl alcohol, add the solution to cholesterol powder, mix until it becomes paste, and homogenize the whole, and paste LHRHa cholesterol Is stored in a vacuum desiccator, etc., dried thoroughly, added cocoa butter dissolved in dried LHRHa cholesterol powder, mixed, homogenized, and added with cocoa butter acting as a binder LHRHa cholesterol powder Into the hole of the pelletizer, and then compressed into a cylindrical pellet and finished LHRHa cholesterol Consists method obtained Rett solid formulation stored at -20 ° C. or less of a freezer, the dosage of LHRHa is a 20-100 / kg BW, Mahata parent fish using training broodstock were cultured under artificial environment, the An individual with an ovary egg diameter of 420 to 550 μm is used, and egg collection after hormonal treatment is performed by artificial insemination, whereby a high-quality fertilized egg can be stably and efficiently obtained from a Mahata parent fish.

The invention of claim 2 is that, in claim 1, the LHRHa cholesterol pellets contain 50 to 400 μg of LHRHa per piece .

The invention of claim 3 is characterized in that, in claim 1, the LHRHa cholesterol pellet is embedded in the muscle of a Mahata parent fish .

The invention of claim 4 is characterized in that, in claim 1, the LHRHa cholesterol pellet is embedded in the abdominal cavity of a Mahata parent fish .

  According to a fifth aspect of the present invention, in the first aspect, the number of times of embedding the LHRHa cholesterol pellet in the mahata parent fish is one.

  The invention according to claim 6 is the invention according to claim 1, wherein the Mahata parent fish uses an individual that has not yet ovulated in the natural environment in the spawning season.

  According to a seventh aspect of the present invention, in the first aspect, 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 method of inducing maturation and ovulation of Mahata parent fish by embedding LHRHa using the solid cholesterol pellet preparation of the present invention, the conventional method (HCG / SP mixing) Compared with the injection method of liquid etc.), there is an advantageous effect that fertilized eggs for seedling production can be secured more stably.

  In addition, according to the maturation and ovulation induction method of Mahata parent fish by LHRHa implantation administration using the cholesterol pellet solid preparation of the present invention, it becomes possible to secure a stable fertilized egg for seed production, from hatching to fry In the breeding of larvae and larvae that produce, there is an advantageous effect of enabling more systematic and stable production.

  Further, according to the maturation and ovulation induction method of Mahata parent fish by embedding LHRHa using the cholesterol pellet solid preparation of the present invention, it is possible to collect eggs with high reproducibility from the Mahata parent fish. It has the advantageous effect of being able to use efficiently, reduce the number of parent fish used, and reduce costs associated therewith.

  In addition, according to the maturation and ovulation induction method of Mahata parent fish by LHRHa implantation administration using the cholesterol pellet solid preparation of the present invention, it becomes possible to stably and efficiently obtain high-quality fertilized eggs. With this, 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 cholesterol pellet solid preparation that enables sustained release administration of LHRHa is prepared and used.

  The LHRHa cholesterol pellet solid preparation method (Figure 1) consists of dissolving LHRHa in 50% ethyl alcohol, then adding the solution to cholesterol powder and mixing with a glass rod or the like until it is pasted to homogenize the whole. Let

  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 preparation, a pellet administration device (Fig. 2) 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 may be used.

  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 (FIG. 3). ).

  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 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 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.

  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
In the maturation and ovulation induction method of Mahata parent fish, a specific method for preparing a cholesterol pellet solid preparation that enables sustained release of LHRHa is shown below.

  When preparing a cholesterol pellet solid preparation containing 100 μg of LHRHa per grain, first dissolve 2.5 mg of LHRHa in 1 ml of 50% ethyl alcohol, add the solution to 625 mg of cholesterol powder, and paste into a paste using a glass rod or the like. Mix until until uniform. Place the paste-like LHRHa cholesterol in a vacuum desiccator, etc., and dry it thoroughly.

  Add 125 mg of cacao butter dissolved in hot water to dry LHRHa cholesterol powder and mix to homogenize the whole. LHRHa cholesterol powder to which cocoa butter has been added is weighed 30 mg each, put into a hole in a pelletizer, and then compressed with a stainless steel rod or the like to form a pellet. About 20 LHRHa cholesterol pellet solid preparations (100 μg / pellet) thus prepared are completed.

  Moreover, when preparing a cholesterol pellet solid preparation containing 200 μg of LHRHa per grain, the amount of LHRHa added may be 5 mg in the above preparation method. About 20 LHRHa cholesterol pellet solid preparations (100 μg / pellet) thus prepared are also completed.

Example 2
In the induction of maturation and ovulation of Mahata parent fish by LHRHa implantation using a cholesterol pellet solid preparation, the effect of the egg diameter at the time of LHRHa implantation on the parent fish's ovulation individual rate, egg volume and egg quality was investigated.

  For the test fish, 106 cultivated 7-year-old fish (weight: 4.1-5.8 kg) were used in an artificial environment, anesthetized, ovarian eggs were collected with cannula, and the diameter of 30 large eggs was reduced. It was measured. As a result of measuring the egg diameter, 8 individuals with an egg diameter of 520 μm or more, 14 individuals with 500-520 μm, 33 individuals with 480-500 μm, 17 individuals with 460-480 μm, 17 individuals with 440-460 μm There were 18 individuals, 7 individuals of 420-440 μm, and 9 individuals of 420 μm or less. LHRHa (50 μg / kg BW) cholesterol pellet solid preparation was implanted and administered to the back muscles of these 106 male grouper fish.

  Ovulation was confirmed by palpating the abdomen after anesthetizing the test fish at 42 hours after LHRHa implantation. After confirming ovulation, the eggs were squeezed out by pressing the abdomen. The obtained egg was immediately subjected to artificial insemination by the dry-conducting method using semen collected from two males for each individual egg.

  Eggs obtained by artificial insemination were washed, and floating eggs and sinking eggs were counted by the volumetric method (2,200 grains / ml) to calculate the floating egg rate. The fertilization rate was calculated as the percentage of individuals that had developed out of about 100 floating eggs.

  FIG. 4 shows the relationship between the egg diameter at the time of LHRHa implantation administration and the egg diameter after 42 hours. In individuals with an egg diameter of 420 μm or less, the egg diameter did not increase and ovulation was not induced even after 42 hours from the implantation of LHRHa. On the other hand, in individuals with an egg diameter of 420 μm or more, individuals with an egg diameter of 520 μm or more are 6 out of 8 animals, individuals with 500-520 μm are 13 out of 14 animals, individuals with 480-500 μm are 28 out of 33 animals, 460 ~ 480μm individuals are 13 out of 17; 440-460μm individuals are 13 out of 18; 420-440μm individuals are 3 out of 7; the egg diameter increases after 42 hours and induces ovulation We were able to. In particular, the ovulation individual rate of parent fish was high in individuals with an egg diameter of 440 μm or more, and the maturation / ovulation induction effect by LHRHa implantation administration was high.

  FIG. 5 shows the relationship between the egg diameter at the time of LHRHa implantation administration and the egg collection amount. The total amount of floating eggs and sinking eggs is 650,000 for individuals over 520 μm, 870,000 for individuals between 500 and 520 μm, 960,000 for individuals between 480 and 500 μm, and 110 for individuals between 460 and 480 μm. The number of grains was 530,000, 530,000 for 440-460 μm individuals, and 560,000 for 420-440 μm individuals. In particular, the amount of eggs collected was large in individuals with an egg diameter of 460 to 520 μm.

  FIG. 6 shows the relationship between the egg diameter at the time of LHRHa implantation administration, the ovulation individual rate, the floating egg rate, and the fertilization rate. Although no significant difference was observed in the floating egg rate and fertilization rate in individuals with an egg diameter of 420 to 520 μm, the ovulation individual rate was high in individuals with an egg diameter of 440 μm or more.

  Considering these results comprehensively, in the induction of maturation and ovulation of mahata parent fish by LHRHa implantation using a cholesterol pellet solid preparation, the egg size at the time of implantation of LHRHa was 460 to 520 μm, and the egg collection performance was It has become clear that good, fertilized eggs can be obtained stably and efficiently.

Example 3
In the induction of maturation and ovulation of Mahata parent fish by embedding LHRHa using a solid pellet of cholesterol pellets, the effect of inducing maturation and ovulation in Mahata parent fish was compared with the conventional HCG injection administration method and this method.

  There are two types of hormonal agents used in the comparative egg collection test: HCG (human placental gonad-stimulating hormone) and LHRHa (synthetic luteinizing hormone-releasing hormone).

  Regarding the administration method of the hormonal agent, HCG was dissolved in a 0.6% NaCl solution and adjusted so that the amount injected into the test fish was 0.2 ml / kg. HCG was administered by an injection method in which the parent fish was anesthetized and then administered to the back muscle using a syringe. On the other hand, LHRHa prepared a columnar cholesterol pellet solid preparation having a length of 6 mm and a diameter of 2 mm containing 100 μg / kg of LHRHa per pellet. After anesthetizing the parent fish, the LHRHa cholesterol pellet was cut into the back muscle epidermis with a scalpel and implanted into the back muscle using a pellet implanter.

  The dose of the hormonal agent was HCG 500 IU / kg for the HCG injection method, and LHRHa 50 μg / kg for the LHRHa implantation method.

  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 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 results of maturation / ovulation induction by implantation of LHRHa cholesterol pellet solid preparation and administration of HCG injection. In LHRHa implantation, 10 of 14 parent fish ovulated, the ovulation rate was 71.4%, the floating egg rate was 72.6%, the floating egg amount was 972,000 and the fertilization rate was 83.5%. On the other hand, in the HCG injection administration, 8 out of 13 parent fish ovulated, the ovulation individual rate was 61.5%, the floating egg rate was 72.5%, the floating egg amount was 464,000 grains, and the fertilization rate was 61.7%.

  Considering these results comprehensively, in terms of maturation and ovulation induction of Mahata parent fish, the LHRHa cholesterol pellet implantation method has better egg collection results than the HCG injection method, and stable and efficient high-quality fertilized eggs It became clear that

Example 4
Use cholesterol pellet solid preparations using Mahata parent fish that have not yet ovulated in the natural environment of the early stage of laying (hereinafter referred to as ovulation individual) and Mahata parent fish that has already partially ovulated in the late stage of laying (partially ovulated solid) LHRHa was administered by implantation, and the effects of inducing maturation and ovulation were compared and investigated.

  In the case of LHRHa implantation, a solid cholesterol pellet of 6 mm in length and 2 mm in diameter containing 100 μg / kg of LHRHa per pellet was prepared, and LHRHa was administered at a dose of 50 μg / kg into the back muscles. Embedded.

  For confirmation of ovulation, palpation of the abdomen was performed 42 hours after the LHRHa cholesterol pellet solid preparation was implanted, and for those confirmed ovulation, the egg was squeezed out by pressing the abdomen, and immediately after artificial insemination by dry-conveyance method Went.

Eggs obtained by artificial insemination were 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. 8 shows the results of maturation and ovulation induction by embedding LHRHa cholesterol pellet solid preparations using non-ovulatory and partially ovulated individuals of Mahata parent fish. In maturation and ovulation induction using unovulated solids, all 9 of the 9 parent fish ovulate, the ovulation rate is 100%, the floating egg rate is 72.6%, the floating egg amount is 365,000 grains and the fertilization rate Was 68.8%. On the other hand, in maturation and ovulation induction using some ovulation individuals, 6 out of 8 parent fish ovulate, ovulation rate is 75%, floating egg rate is 70.0%, floating egg amount is 30 million grains and fertilization rate Was 45.9%.

  Considering these results comprehensively, in the maturation and ovulation induction of the Mahata parent fish, the parent fish that has not yet ovulated in the early spawning period is better than the parent fish that has already partially ovulated in the natural environment in the late spawning period. It was clarified that the selection and the LHRHa cholesterol pellet implantation administration gave better egg collection results and stable and efficient high-quality fertilized eggs were obtained.

Example 5
We investigated the relationship between the body weight and the amount of eggs collected in the induction of maturation and ovulation by embedding LHRHa cholesterol pellets.

  Mahata parent fish was a cultured parent fish cultured in an artificial environment, and a total of 61 individuals weighing 2-7 kg were used.

  In the case of LHRHa implantation, a solid cholesterol pellet of 6 mm in length and 2 mm in diameter containing 100 μg / kg of LHRHa per pellet was prepared, and LHRHa was administered at a dose of 50 μg / kg into the back muscles. Embedded.

  For confirmation of ovulation, palpation of the abdomen was performed 42 hours after the LHRHa cholesterol pellet solid preparation was implanted, and for those confirmed ovulation, the egg was squeezed out by pressing the abdomen, and immediately after artificial insemination by dry-conveyance method Went. The egg yield of the eggs obtained by artificial insemination was calculated by the volume method (2,200 grains / ml).

  FIG. 9 shows the relationship between the body weight and the amount of eggs collected in the maturation of ovulation in ripening and ovulation induction by embedding LHRHa cholesterol pellet solid preparation. By embedding the LHRHa cholesterol pellet solid preparation, ovulation was induced in parent fish weighing 2 kg or more, and eggs could be collected. The amount of eggs collected increased as the weight of the parent fish increased. However, Mahata was female and matured and hermaphroditic. When the body weight exceeded 6 kg, the ratio of males increased, making it difficult to secure female parent fish.

  Considering these results comprehensively, in the induction of maturation and ovulation by embedding administration of LHRHa cholesterol pellet solid formulation of Mahata parent fish, ovulation induction is possible in parent fish whose body weight is 2 kg or more. From the aspect of selection, it has been clarified that high-quality fertilized eggs can be obtained stably and efficiently when individuals with a body weight of 4 to 6 kg are used.

  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 cholesterol pellet solid formulation. It is a figure which shows the cholesterol pellet solid formulation and implantation administration device which enable the sustained release administration of LHRHa. It is a figure which shows the embedding condition of the parent fish for egg collection of Mahata and the solid preparation of a LHRHa cholesterol pellet. It is a figure which shows the relationship between the egg diameter at the time of implantation administration of the LHRHa cholesterol pellet solid formulation, and the egg diameter after 42 hours. It is a figure which shows the relationship between the egg diameter at the time of implantation administration of the LHRHa cholesterol pellet solid formulation, and the amount of eggs collected. It is a figure which shows the relationship between the egg diameter at the time of implantation administration of the LHRHa cholesterol pellet solid formulation, an ovulation individual rate, a floating egg rate, and a fertilization rate. It is a figure which shows the maturation and ovulation induction effect by the implantation administration of a LHRHa cholesterol pellet solid formulation, and HCG injection administration. It is a figure which shows the maturation and ovulation induction effect by the implantation administration of the LHRHa cholesterol pellet solid preparation using the unovulated solid and partial ovulated solid of Mahata parent fish. It is a figure which shows the relationship between the body weight and the egg-collecting amount of a Mahata parent fish in the induction | guidance | derivation of maturation and ovulation by implantation administration of the LHRHa cholesterol pellet solid preparation.

Claims (7)

Method for producing LHRHa cholesterol pellet solid preparation using cholesterol pellet as a solid preparation that enables sustained release of synthetic luteinizing hormone-releasing hormone (LHRHa: des-Gly ¹⁰ , [D-Ala ⁶ ] -LHRH ethylamide) After dissolving LHRHa in 50% ethyl alcohol, the solution is added to cholesterol powder and mixed until it becomes a paste to homogenize the whole, and the paste LHRHa cholesterol is stored in a vacuum desiccator or the like. Add the cocoa butter dissolved in the fully dried and dried LHRHa cholesterol powder, mix and homogenize the whole, and put the LHRHa cholesterol powder with cocoa butter acting as a binder into the hole of the pelletizer Put, then compressed into cylindrical pellets, and finished LHRHa cholesterol pellets solid preparation- It consists of a method obtained by storage in a freezer at 20 ° C or lower. The dose of LHRHa is 20-100 μg / kg BW, and the Mahata parent fish is a cultured parent fish cultured in an artificial environment. Using a solid pellet of cholesterol pellets, which is characterized by obtaining high-quality fertilized eggs stably and efficiently from mahata parent fish by using an individual of 420-550 μm and collecting eggs after hormone treatment by artificial insemination A method for inducing maturation and ovulation of Mahata parent fish by LHRHa implantation. 2. The method for inducing maturation and ovulation induction of Mahata parent fish by embedding LHRHa using the cholesterol pellet solid preparation according to claim 1, wherein each LHRHa cholesterol pellet contains 50 to 400 μg of LHRHa . The method for inducing maturation and ovulation of mahata parent fish by embedding LHRHa using the cholesterol pellet solid preparation according to claim 1, wherein the LHRHa cholesterol pellet is embedded in muscle of mahata parent fish. The method for inducing maturation and ovulation of mahata parent fish by embedding LHRHa using the cholesterol pellet solid preparation according to claim 1, wherein the LHRHa cholesterol pellet is implanted into the abdominal cavity of mahata parent fish.   2. The method of inducing maturation and ovulation of mahata parent fish by embedding LHRHa using the cholesterol pellet solid preparation according to claim 1, wherein the number of embeddings of LHRHa cholesterol pellets into mahata parent fish is one.   2. The method for inducing maturation and ovulation of Mahata parent fish by embedding LHRHa using the cholesterol pellet solid 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.   2. The method of inducing maturation / ovulation of mahata parent fish by embedding LHRHa using a cholesterol pellet solid preparation according to claim 1, wherein the mahata parent fish is an individual over 5 years old and capable of inducing maturation / ovulation and weighing 2 kg or more.