JP5390484B2 - Puffer sex change method, blowfish breeding method, and sex change blowfish obtained by the method - Google Patents

Description

  The present invention relates to a technique for artificially transducing a genetic male to a functional female by administering estrogen, a kind of steroid hormone, to pufferfish.

  Conventionally, pufferfish is eaten favorably, but it has a poison commonly called pufferfish poison in the gonad, viscera, skin and the like. For example, it is widely known that there is a tetrodotoxin in the body of the tiger puffer, and there is also a paftoxin possessed by the boxfish. The location of the pufferfish poison varies depending on the pufferfish species. The pufferfish poisons the skin and gonads and can eat only the flesh (meat), the skin cannot eat but the gonads (male testis) and meat can eat, the skin , Gonads, and meat are all edible. There are 22 types of puffer fish that are currently approved for food use, based on the “New Standards for Puffer Hygiene Measures” issued by the Director of the Ministry of Health and Welfare in 1983. For example, pufferfish, common pufferfish, common pufferfish, sunfish pufferfish are listed as pufferfish that can only eat meat. If the species has no puffer venom on the skin or testis, the testicles are eaten as larvae and the skin is eaten as fillet. The typical example of a puffer fish that can eat the male testis is a trough puffer fish as a high-class food.

  In general, white tiger pufferfish is considered a delicacy and is offered to consumers at an expensive price. On the other hand, the tiger ovary of tiger puffer is toxic and must be discarded. In terms of safety, the testis (white cat) of a non-toxic tiger puffer is more important than the ovary of a toxic female tiger puffer. Therefore, the demand for trough puffer males is higher than that for trough puffer females. A trough, when grown under normal environmental conditions, generally has a ratio of about 1: 1 female to male. This sex ratio is the same in aquaculture production. In other words, about 50% of the farmed and produced tiger puffer is female, and 50% of the cultivated and produced tiger puffer's gonad is discarded. Moreover, the gonads of tiger puffer fish have a large proportion of fish body weight, that is, GSI (gonad index), and develop to about 20-30% by weight of fish body weight during the breeding season. In the case of trough females, since all of this is discarded, the yield of the edible portion is extremely reduced.

  Since troughs are difficult to distinguish between males and females, they are currently sold at the same price regardless of sex. There are some reports that males and females can be discriminated by abdominal palpation mainly in the breeding season, but it is not 100% accurate discrimination. In addition, since a large amount of trough puffers of several hundred to several tens of thousands are handled at the time of shipment, discrimination by abdominal palpation requires a lot of labor. Although it is conceivable to discriminate between males and females by DNA determination at the seedling production site in the seedling production site, it is realistic to perform DNA determination for all larvae and larvae handled in units of hundreds of thousands of fish in terms of cost and labor. Impossible. For this reason, the supply of trout pufferfish, which is useful, is unstable, and systematic and stable production is required. In the first place, the sex ratio of trough puffer fish is about 1: 1. Therefore, simply distinguishing between males and females results in the existence of females, which does not lead to a fundamental solution. Therefore, it is ideal to produce and supply only the trough puffer male. In response, the aim is to establish a technology for making all the trough fish male. Such issues are not as important as trough puffer fish, but the same applies to other types of puffer fish as long as the ovary is not edible and the male testis is edible.

  Non-patent document 1, for example, discloses a basic technique related to alligating a trough. According to Non-Patent Document 1, first, estrogen, which is a type of female hormone, is administered to a genetic (essentially) male troughfish whose sex chromosome is XY, and the sex chromosome remains XY. Sham females that have been transsexualized to have ovaries are produced. The genetic (normal) female sex chromosome is XX. When the obtained pseudo female matures over 2 to 3 years, an unfertilized egg is taken out from the pseudo female and fertilized with a normal male sperm whose sex chromosome is XY. Then, a super male whose sex chromosome is YY, which cannot normally exist in nature, is obtained. Next, when the second generation super male and a normal female whose sex chromosome is XX are mated, all the third generation puffer fish become male. As a result, it is said that all trough fish can be made male. Therefore, in order to establish a method for maleizing this trough puffer, a functional female (pseudo-female) having sex chromosomes of XY, which has been sex-transformed by administering estrogen to a genetic male having sex chromosomes of XY, is used. Production is essential.

  In Non-Patent Document 1, when estrogen is added to the feed at a rate of 100 μg / g (100 ppm) and orally administered, and estrogen is added to the feed at a rate of 0.1 μg / g (100 ppb) and orally administered. The test results for each case are also listed. When estrogen is added to a 100 ppm feed, a part of the testes of the tiger puffer male becomes ovary, most of which are neutral gonads in which sperm cells and oocytes are mixed. Moreover, it is unclear whether even partially ovarian fry will develop into normal ovaries. When estrogen is added to the 100 ppb diet, testicular ovarianization has not occurred.

“Is it possible to become masculine? Study on sex determination and sex differentiation of trough puffer fish” Akihiko Yamaguchi [General Information Paper for Making Fisheries] Aquaculture No567 Green Shobo 2008.7 P22-26

  Although the basic theory about trough masculinization is disclosed in Non-Patent Document 1, in fact, it has become possible to produce functional females whose sex chromosomes are the premise of this as described above. Not in. In the test of Non-Patent Document 1, even when 100 ppm of estrogen is added to the feed, only neutral gonads in which sperm cells and oocytes are mixed are obtained. This is presumed to be due to the fact that there is a variation in the feed intake rate, there is a limit in the amount of food intake, and in the case of oral administration, estrogen acts through the digestion / absorption system, so that the effect is attenuated. Moreover, it has not been confirmed that some spermatogonia develop into normal ovaries even if they become oocytes.

  Therefore, the present invention solves the above-mentioned problem, and a puffer's sex reversal method that can reliably transform a genetic male into a functional female by estrogen, a puffer's breeding method using this, and the like The purpose is to provide.

  First, a puffer sex change method can be proposed in which a pufferfish that belongs to the pufferfish and that can eat the testis is exposed to breeding water to which estrogen is added. As a result, the genetic male pufferfish whose sex chromosome is XY can be transsexualized into a functional female (pseudofemale) whose sex chromosome is XY but has an ovary. In addition, the ovary in this invention means the ovary in the strict meaning which has an ovum. In other words, a ovary having a structure having a ovarian cavity derived from a somatic cell or a thin plate but having no ovum derived from a germ cell may be a broad ovary, but in the present invention, an ovary having only the structure is excluded. Is done.

  The period during which the puffer is exposed to the breeding water to which estrogen has been added may continue until it matures after hatching, but is preferably a period having sexual plasticity. The estrogen concentration in the breeding water is preferably 0.1 to 100 weight ppb.

  The frequency of exposing puffer fish to breeding water to which estrogen is added is preferably 1 to 7 times / week. In this case, a constant effect can be expected even if exposed continuously or at irregular intervals, but it is preferable to expose periodically. For example, if you are exposed at a frequency of 3 times / week, you may be exposed 3 times a day, twice a day, once a different day, for 3 consecutive days, Although a certain effect can be expected even if it is irregular, such as exposure on Tuesdays and Saturdays, it is preferable to expose regularly, for example, on Mondays, Wednesdays, and Fridays.

  The time per exposure of the puffer to the breeding water to which estrogen is added is preferably 0.5 to 6 hours / time. As estrogen, there are estradiol, estrone, and estriol, but these may be used alone or in combination of two or more.

  The above-mentioned means is a basic means for transsexualizing a genetic male puffer having a sex chromosome of XY into a functional female having a sex chromosome of XY, basically a tank isolated from the aquaculture production site, etc. Done in However, in reality, puffer fish are produced in aquaculture. Therefore, as a practical invention suitable for farming and producing puffer fish, a puffer breeding method in which estrogen is periodically added to the breeding water and administered to puffer fish can be proposed. Of course, puffer fish can be reared in an aquarium isolated from the aquaculture production site. In consideration of safety and the like, it is preferable to breed the pufferfish in an isolated environment such as a water tank.

  In this case as well, the exposure may continue until it matures after hatching, but it is preferable to expose only for a period having sexual plasticity. Moreover, the addition amount of estrogen is preferably 0.1 to 100 weight ppb, and the addition frequency is preferably 1 to 7 times / week. Depending on the amount of estrogen added, it is possible to use the estrogen-added breeding water as it is. However, estrogen is added to the estrogen-added breeding water every 0.5 to 6 hours after estrogen is added to the breeding water. It is preferable to replace it with breeding water that has not been added.

  One kind of estrogen may be used alone, or two or more kinds may be mixed and used, but at least estradiol is preferably used. The present invention can be applied to general pufferfish belonging to the order of pufferfish and edible testis, but it is preferably applied to breeding of typical pufferfish as trough pufferfish and edible pufferfish.

  Furthermore, a genetic male whose sex chromosome is XY obtained by the above-mentioned method for sex transformation and breeding of puffer fish is transformed into a functional female whose chromosomal chromosome remains XY but has an ovary. Is also proposed.

  In the present invention, an estrogen is added to the breeding water and administered to the pufferfish, that is, the pufferfish is exposed to the breeding water to which the estrogen is added. The pufferfish can be accurately transsexualized into a functional female (pseudofemale) with an ovary while the sex chromosome remains XY. Moreover, the sex-shifted puffer fake female can be grown in a functional female state for a long period of time. That is, it can be grown in the state of a pseudo female having the same ovary as a normal female puffer fish. Even if estrogen is administered to pufferfish after the loss of sexual plasticity, sex change does not occur, and costs are wasted. On the other hand, if the exposure (addition) period is limited only to the period having sexual plasticity, not only waste of cost does not occur, but a physiological load is not imposed on the puffer fish.

  When estrogen is added to breeding water and puffer is exposed to this, estrogen is absorbed not only by oral absorption but also by sputum and skin, so that estrogen is efficiently and reliably administered to pufferfish. Therefore, when the estrogen is added to the feed and orally administered, it is greatly affected by the feed intake rate and the like, so the amount of addition must be increased to the ppm order as in Non-Patent Document 1, but the present invention According to the above, even if the amount of estrogen added is small and the concentration of estrogen in the breeding water is 0.1 to 100 wt ppb, the puffer can be accurately transsexualized. In addition, oral administration with feed addition has a large individual difference in estrogen intake based on feed intake, but if estrogen is added to the breeding water, it can be uniformly administered to all puffers. In addition, as a method of administering estrogen to puffer fish, direct injection is also conceivable, but direct injection has a great stress on puffer fish and has a large adverse effect such as growth stagnation. On the other hand, according to the method of adding estrogen to the breeding water, there is almost no stress given to the pufferfish.

  If too much estrogen is taken, there is a concern about the physiological load (side effects) on the puffer fish. Therefore, if a puffer is reared in an environment where estrogen is always present, depending on the concentration, there is a high possibility that a problem of side effects will occur due to excessive intake of estrogen. Therefore, if the frequency of exposure (addition) is 1 to 7 times / week in the range of 0.1 to 100 wt ppb of estrogen addition to the breeding water, it is possible to accurately change sex while avoiding excessive intake of estrogen, Moreover, it can be grown while maintaining the state of a pseudo female. The effect of setting the time per exposure to 0.5 to 6 hours / time is the same. If exposed (added) regularly, it is easy to adjust the estrogen concentration in the breeding water.

  Considering the troubles such as the number of times of estrogen administration at the aquaculture site and the replacement of breeding water, it is desirable to maintain an environment where estrogen continues to act at a low concentration so that there is no problem of side effects. However, the rate of decrease in estrogen concentration varies depending on the intake of pufferfish, the environment such as weather and temperature, and is not always uniform, so it is extremely difficult to keep estrogen concentration in the breeding water constant. Yes, practically impossible. Therefore, if the estrogen-added breeding water is replaced with breeding water to which no estrogen is added each time exposure is performed, the concentration can be adjusted from the state where the estrogen concentration is reset, so that the estrogen concentration in the breeding water can be adjusted reliably. .

  If at least estradiol with high physiological activity is used as the estrogen, sex change can be caused efficiently. If the present invention is applied to the breeding of tiger pufferfish and pufferfish which are typical as edible pufferfish and are well-cultivated, the benefits are great. Further, according to the functional female pufferfish obtained by the present invention, the pufferfish all-maleization technique can be finally established.

It is a graph which shows the functional sex ratio after estrogen administration. It is a gonad tissue photograph of a 200-day-old male puffer fish in Control Group 1. It is a gonad tissue photograph of a 200-day-old female puffer fish in Control Group 1. It is a reproductive line tissue photograph of a 500-day-old puffer fish in Test Zone 3. It is a reproductive line tissue photograph of a 500-day-old puffer in Test Zone 4. It is a reproductive tissue photograph of a 500-day-old puffer fish in Test Zone 6. It is a graph which shows the functional sex ratio after estrogen administration.

  The present invention artificially transforms a genetic male into a functional female (pseudo female) by exposing puffers to breeding water containing estrogen which is a kind of female hormone (steroid hormone). It is possible to add estrogen directly to the breeding water at the production site (culture farm), but from the viewpoint of safety, etc., add estrogen to the breeding water in a tank isolated from the production site. It is preferable to do.

  The pufferfish that are subject to sex change are all pufferfish that belong to the pufferfish and can eat testes. This is because, based on the pseudo female obtained in the present invention, finally, only males are cultured and produced to improve the productivity of white scabs with high commercial value. Therefore, even if it is applied to the pufferfish that cannot eat the testis, the sex change can occur, but the benefit is small. Specifically, among the pufferfish listed in the “New Measures Standard for Puffer Hygiene” notified by the Director-General of the Ministry of Health and Welfare in 1983, the pufferfish, pufferfish, pufferfish, pufferfish, pufferfish, pufferfish, and pufferfish , Crow, puffer puffer, puffer pufferfish, puffer pufferfish, white-puffer pufferfish, black-puffer pufferfish, Yorito pufferfish, puffy-puffy spider pufferfish, pike-puffer pufferfish, puffer puffer pufferfish, puffer puffer-puffer pufferfish Among them, typical trough pufferfish and mahugu are preferred for food use, and high-grade foods are most preferred.

  Estrogens (Estrone: E1, IUPAC name: 3-hydroxyestra-1,3,5 (10) -trien-17-one), estradiol (E2, IUPAC name: estra-1,3,5) 10) -triene-3,17β-diol) and estriol (Estriol: E3, IUPAC name: estra-1,3,5 (10) -triene-3,16α, 17β-triol). Any of these may be used, and only 1 type may be used independently, and 2 or more types may be mixed and used for it. These physiological activities are higher in the order of estradiol, estrone, and estriol. For example, the biological activity of estradiol is about 5 times that of estrone and about 10 times that of estriol. Thus, when only one estrogen is used, estradiol is most preferred, followed by estrone. Moreover, when using 2 or more types of estrogen in mixture, it is preferable to use at least estradiol.

  As the estrogen, any of a synthetic product, an extract extracted from a natural raw material containing estrogen, or an extracted purified product may be used, or only one kind may be used alone, or two or more kinds may be used in combination. The form of estrogen can be used in a liquid state dissolved in an organic solvent. Examples of the organic solvent include ethanol, methanol, propanol, butanol, acetone, chloroform, ether, ethyl acetate, methylene chloride, benzene, carbon tetrachloride, and petroleum ether. Of these, ethanol is desirable because of its good solubility and low toxicity.

  The added amount of estrogen, that is, the estrogen concentration in the breeding water is preferably 0.1 to 100 weight ppb (0.1 to 100 ng / g). If the estrogen concentration is less than 0.1 wt ppb, it will not be possible to accurately transsexual. The estrogen concentration may exceed 100 weight ppb, but the possibility of side effects increases. More preferably, it is 0.5-40 weight ppb, More preferably, it is 1.0-20 weight ppb. If the estrogen concentration is 1.0 weight ppb or more, 100% of males can be transsexualized to pseudo females.

  The period during which the estrogen is added to the breeding water, that is, the period during which the pufferfish is exposed to the estrogen-containing breeding water is preferably a period during which the puffer's gonads have sexual plasticity. This is because the administration of estrogen beyond the period when the puffer's gonads have sexual plasticity has no effect. Even if the gonad begins to be administered after a period of sexual plasticity, it is difficult to perform sex change afterwards because the gonad has already undergone sex differentiation and sex has been determined. Specifically, about 7 to 100 days of age after hatching is preferable. Although it may be administered before the age of 7 days after hatching, the gonad is not yet developed and the action effect of estrogen is low. More preferably, it is about 10 to 100 days of age after hatching, and more preferably about 20 to 100 days of age.

  The frequency of adding estrogen to the breeding water, that is, the frequency of exposing puffer fish to the breeding water containing estrogen is preferably 1 to 7 times / week. The frequency of estrogen administration may exceed 7 times / week, but there are concerns about side effects caused by excessive intake of estrogen. More preferably, it is 3-7 times / week. Moreover, when administering 2-7 times per week, although depending on exposure time, once a day is preferable. It may be multiple times a day. Moreover, it is preferable to periodically perform the frequency of adding estrogen to the breeding water, that is, the frequency of exposing puffer fish to the breeding water containing estrogen.

  The time per exposure of the puffer fish to the estrogen-containing breeding water is preferably 0.5 to 6 hours / time. The estrogen exposure time may exceed 6 hours / dose, but there are concerns about side effects due to excessive intake of estrogen. More preferably, it is 1-4 hours / time, More preferably, it is 2-3 hours / time.

  The total exposure time for exposing puffer fish to the estrogen-containing breeding water is at least 7 hours, preferably 15 hours or more, and more preferably 30 hours or more. If the total exposure time for exposing pufferfish to estrogen-containing breeding water is less than 7 hours, accurate sex change is difficult. If the total exposure time is 30 hours or more, all individuals can be 100% transsexual. The upper limit of the total exposure time is not particularly limited, but about 100 hours is preferable in view of physiological load.

  In addition, after adding estrogen to breeding water, after the said exposure time progress, it is preferable to replace estrogen containing breeding water with breeding water which does not contain estrogen each time. Although it is not always necessary to replace the breeding water, in this case, it is assumed that estrogen is always present in the breeding environment although the concentration is high and low, which is not preferable in terms of side effects and germ cell cultivation. If the breeding water is changed every time after exposure for a certain period of time, the estrogen concentration adjustment can be stabilized. As for replacement of breeding water, total replacement is most preferable, but at least 50% or more is preferable. More preferably, it is 80% or more. The temperature of the breeding water is preferably 16 to 24 ° C. where feeding of puffer fish is active and shows good growth.

  Even during the addition of estrogen to the breeding water, the feed itself may be given a general known feed for fish farming or live feed such as moist pellets based on the usual breeding method. Animal feed such as fish meal, casein, squid meal, krill meal, vegetable materials such as soybean meal, corn gluten meal, wheat flour, defatted rice bran, starch, animals such as cod liver oil, squid liver oil Oils, soybean oil, rapeseed oil, palm oil, corn oil and other vegetable oils, pregelatinized starch, CMC (carboxymethylcellulose), caking agents such as sodium alginate, guar gum, vitamins, minerals, amino acids, antioxidants, etc. it can. Examples of raw food materials include sardines, anchovy, mackerel, saury, herring, hockey, cod, squid, krill, isazaami and the like. What is necessary is just to adjust a mixing | blending suitably according to a blowfish kind, a growth stage, etc.

  When estrogen is added to the breeding water and the pufferfish is exposed to the breeding water containing the estrogen, the estrogen is not only taken orally when the seawater is ingested for feeding and osmotic pressure control, but also from the puffer and the skin. It is also absorbed into the body. In the present invention, absorption from wrinkles and skin is preferential. Therefore, the estrogen can be caused to act accurately despite the addition amount with a low weight ppb (ng / g) level. Moreover, since it is added to the breeding water, even if a large number of puffer fish are cultured at once, estrogen can be uniformly administered to all puffers.

  The gonad consists of somatic cells and germ cells. Usually, a genetic male pufferfish having a sex chromosome of XY forms a structure called cyst from somatic cells, and sperm is formed from germ cells. On the other hand, a normal female female puffer having the sex chromosome XX has an ovary cavity and a thin plate formed from somatic cells, and an egg formed from germ cells. Germ cells at the time when sex change can occur, that is, when there is sexual plasticity, are oocytes in females and spermatogonia in males. However, when estrogen is administered to the genetic male pufferfish according to the above-mentioned conditions of the present invention, the sex chromosome remains XY, but the ovarian cavity and thin plate are formed from somatic cells by the action of estrogen, and the spermatogonia are oocyte cells. Convert to In this way, the gonad that should originally be the testis becomes a functional female (pseudofemale) that has changed into an ovary, and grows with the ovary intact.

  When the obtained pseudo female pufferfish has matured over 2 to 3 years, unfertilized eggs are taken out of the pseudo female pufferfish and fertilized with normal male puffer fish sperm whose sex chromosome is XY. Then, a super male pufferfish whose sex chromosome is YY, which cannot normally exist in nature, is obtained. Subsequently, when the second generation super male puffer and a normal female puffer having a sex chromosome consisting of XX are crossed, male puffer is obtained in the third generation. This establishes the all-muffling of the pufferfish. Thus, the gonads of pufferfish obtained by aquaculture are all testis, and the production volume of white babies sold and sold at a high price is doubled.

<Estrogen administration test 1 for juvenile puffer fish>
To confirm the effects on the gonads when estrogen is administered to puffer fish during the period when the gonads have sexual plasticity. Synthetic estradiol was used as estrogen, and pufferfish was used as trough puffer. Estradiol was periodically added to the breeding water in the form of an ethanol solution dissolved in ethanol. As test sections, a plurality of test sections with different amounts of added estrogen were set. Specifically, the estrogen concentration in the breeding water is 1 wt ppb (1 ng / g) test plot 1, 5 wt ppb (5 ng / g) test plot 2, 10 wt ppb (10 ng / g) test plot, respectively. 3. Set test group 4 of 25 weight ppb (25 ng / g), test group 5 of 50 weight ppb (50 ng / g), test group 6 of 100 weight ppb (100 ng / g), and nothing is added. A total of 8 sections were set, including the control section 1 of the treatment and the control section 2 to which only ethanol was added (estradiol concentration 0 wt ppb). The amount of ethanol in each compartment was the same.

  Troughfish female parent fish and male parent fish cultivated at Chubu Feed Co., Ltd., Oigawa Experiment Station were randomly selected, and eggs were collected and fertilized for artificial insemination. The hatched larvae were fed with biological feeds such as rotifer, artemia, and mixed feed, and were cultivated to 20-30 days of age, and then housed 500 each in a 500 L circular aquarium. Table 1 shows the test conditions in Test Groups 1 to 6 and Control Groups 1 and 2.

  After 2 hours from the addition of estrogen to the breeding water, the entire amount of breeding water was changed each time until the estrogen concentration in the breeding water reached 0 wt ppb. Such an exposure treatment was performed in a period of 20 to 100 days. During the breeding period, the water temperature was set to 16 to 24 ° C., and the breeding conditions such as the feed, feed, number of feeds, feed amount, breeding water temperature, breeding density were the same in all sections. After the test was completed, the animals were reared until the age of 200 days so that the sexes could be easily discriminated, 50 juveniles were randomly sampled from each section, and the gonads were removed after laparotomy and fixed with Buan's solution. Then, paraffin sections were prepared and sexes were discriminated by observing gonad tissues. The criterion for discrimination was that an individual having an oocyte, which is a characteristic of a female, was a female, and the other was a male. The result is shown in FIG. Moreover, a normal male gonad tissue photograph of the control group 1 is shown in FIG. 2, and a normal female gonad tissue photograph of the control group 1 is shown in FIG.

  As is apparent from the results of FIG. 1, the sex ratio in the untreated control group 1 was about 1: 1. Further, in the control group 2 to which only ethanol was added (estrogens concentration 0 wt ppb), the male / female ratio was about 1: 1 as in the control group 1, and it was confirmed that ethanol did not affect the sex ratio of trough puffer fish. . In addition, in all test groups 1 to 6 to which estrogen was added, ovaries having oocytes as shown in FIG. 3 were confirmed in all individuals. Regarding survival rate, there was no big difference in each test section. That is, we succeeded in transsexualizing all genetic males to functional females (pseudo females). From the above results, if estrogen is added to the breeding water at a concentration of 1.0 wt ppb or more and the pufferfish is exposed, all individuals have ovaries that are characteristic of females, and pufferfish can be completely feminized I understood it.

<Trend test of gonads by long-term rearing of fake females>
According to the above test, it was confirmed that if estrogen was added to the breeding water, the genetic male pufferfish was transsexualized to a functional female. Become. Therefore, long-term breeding of the fry obtained from the above test was conducted, the trends of the gonads (ovaries) of pseudo females and genetic females were investigated, and the pseudo females again became males with testis due to the genetic influence from sex chromosomes. It was confirmed whether or not sex changes.

  In this test, pseudo females produced in test group 3, test group 4 and test group 6 in the above test and normal male puffer fish produced in control group 1 and control group 2 were used. 300 troughs produced in these compartments were housed in a 5-ton circular aquarium, and were reared at a water temperature of 16-24 ° C. until 500 days of age. During the breeding period, breeding conditions such as feeding feed, number of feedings, feeding amount, breeding water temperature, breeding density, and breeding method were the same in all sections. Thirty fish were randomly sampled every 300 days, 400 days, and 500 days, and after laparotomy, the gonads were removed and fixed with Buan's solution, then paraffin sections were prepared, and functional by observation of gonad tissues Sexual discrimination was performed. The criterion for discrimination was that an individual having an ovary characteristic of a female was a female and the other was a male. The results are shown in Table 2. In addition, photographs of gonad tissues of 500-day-old puffers in test sections 3, 4, and 6 are shown in FIGS. The numerical values in Table 2 are%.

  From the results of Table 2, in the control groups 1 and 2, the sex ratio is about 1: 1 for all of 300-day-old, 400-day-old, and 500-day-old, and the breeding is carried out by a general breeding method for a long time. However, it was confirmed that the functional sex ratio did not change. Further, it was confirmed that the puffers in the test groups 3, 4 and 6 to which estrogen was added all possessed ovaries at 300 days, 400 days and 500 days. That is, the sex of the sex-changed individual was maintained even after 500 days. In particular, as is clear from FIGS. 4, 5, and 6, the gonads of the 500-day-old puffer fish in Test Zones 3, 4, and 6 all maintain the same gonad tissue as the normal female gonads shown in FIG. 3. It was. Based on the above results, if estrogen is added to the breeding water, the genetic male puffer fish changes into a functional female, and even if the breeding is carried out for a long time using the usual breeding method, It was confirmed that the ovary grew without being reverted to male due to the influence.

<Estrogen administration test 2 for juvenile puffer fish>
In the above estrogen administration test 1, if puffer is exposed to estrogen at a concentration of at least 1.0 wt ppb for a total exposure time of 30 hours or longer, all the puffers can be feminized in all individuals. It has been confirmed that increasing the time will reach a peak, and it is clear that these upper limits are not very important. On the other hand, lower limits on estrogen concentration and total exposure time are important. Therefore, in order to identify these lower limits, the estrogen administration test 2 was conducted under conditions that were less dilute than the estrogen administration test 1 described above.

Specifically, the test was performed under exactly the same conditions as in the estrogen administration test 1 except for the conditions shown in Table 3.

  After the test was completed, the sexes were discriminated by observing the gonad tissues in the same manner as in the estrogen administration test 1. The result is shown in FIG. As is clear from the results of FIG. 7, in the test group 7 having an estrogen concentration of 0.05 ppb, the male / female ratio was approximately 1: 1 even if the total exposure time was long. On the other hand, in the test group 8 having an estrogen concentration of 0.5 ppb, the female ratio was 20% or more higher than in the control groups 1 and 2 without addition of estrogen. As a result, it was found that the estrogen concentration needs to be at least 0.1 ppb or more.

On the other hand, even in the test groups 9 to 11 having an estrogen concentration of 10 ppb, oocytes were not confirmed in some individuals because the total exposure time was relatively short. However, the ratio of females was higher in the test group 9 than 30%, the test group 10 was about 40%, and the female ratio was higher than in the control groups 1 and 2 without estrogen. On the other hand, in Test Zone 11, it remained at just over 10%. Accordingly, it has been found that the total estrogen exposure time is preferably at least 7 hours or more, more preferably 15 hours or more. In addition, about survival rate, there was no big difference in each test section.

Claims (9)

  In the breeding water to which 0.1 to 100 ppb of estrogen is added, the pufferfish belonging to the pufferfish, which can eat the testis, is 1-7 times / week, 0.5-6 hours / time. A method of sex reversal of a pufferfish, wherein the male male puffer having a sex chromosome composed of XY is transsexualized to a functional female having a sex chromosome composed of XY but having an ovary by exposure at a frequency.   2. The method for sex reversal of puffer fish according to claim 1, wherein the pufferfish is exposed to breeding water to which the estrogen is added during a period when the gonad has sexual plasticity.   The method for sex-changing a pufferfish according to claim 1 or 2, wherein the pufferfish is periodically exposed to breeding water to which the estrogen is added.   4. The method for sex conversion of puffer fish according to claim 1, wherein the estrogen is one or more selected from estradiol, estrone, and estriol. 5. Genetic male whose sex chromosome consists of XY by adding estrogen to breeding water at a frequency of 0.1 to 100 weight ppb, 1 to 7 times / week and exposing at a frequency of 0.5 to 6 hours / time How to raise a pufferfish to be administered to a pufferfish.   The puffer breeding method according to claim 5, wherein the estrogen is added to the breeding water during a period when the gonad of the pufferfish has sexual plasticity.   The puffer breeding method according to claim 5 or 6, wherein the estrogen is added to breeding water, and the estrogen-added breeding water is replaced with breeding water to which estrogen is not added each time after the exposure time has elapsed.   The puffer breeding method according to any one of claims 5 to 7, wherein at least estradiol is used as the estrogen. The method for raising puffer fish according to any one of claims 5 to 8, wherein the pufferfish is a tiger puffer or a puffer puffer.