Method for producing genetically male salmonid fish with female gonads

Abstract

This invention provides a novel method for producing a high proportion of female salmonid fish, applicable to food resources. [Solution] The present invention provides a method for producing genetically male salmonid fish with female gonads, comprising rearing genetically male salmonid fish in the presence of equol. The present invention also provides a feminizing agent for genetically male salmonid fish in the presence of equol. According to the present invention, a high ratio of female salmonid fish can be produced in one generation without using hormones unsuitable for food resource production.

Description

Technical Field

[0001] The present invention relates to a method for producing a genetic male of salmonid fish having a female gonad.

Background Art

[0002] In the aquaculture industry of salmonid fish typified by masu salmon and cherry salmon, which are important fishery resources, eggs are traded at high prices, and females can have more uniform sizes and maturation ages compared to males. Therefore, so-called all-female production, in which only female individuals are raised, is desired.

[0003] As a method for all-female production of salmonid fish, a technique has been established in which a genetic female (XX female) is induced into a functional male (XX male, pseudo-male) with a male hormone, and all-female production (XX) is carried out by mating this pseudo-male (XX) with a normal female (XX) (Non-Patent Document 1). This method has the advantage that all fry are genetic females, but it takes 2 to 3 years of rearing in advance for the production and maturation of pseudo-males, and two generations are required to achieve all-female production. Therefore, there is a problem that it is laborious and costly to maintain. In addition, 17α-methyltestosterone used for hormone treatment for producing pseudo-males is a substance harmful to human health, so it cannot be used in aquaculture farms for food production.

[0004] As another method for all-female production of salmonid fish, there is also known a method in which genetically inactivated sperm and eggs are fertilized by irradiation with ultraviolet rays, and then female-generated diploids are produced by chromosomal manipulation by pressure treatment or high-temperature treatment to produce genetic females (Non-Patent Documents 2 and 3). While this method enables all-female production in one generation, it requires physical shocks such as high temperature and pressure treatment. In addition, the produced individuals may become homozygotes for recessive genes, and there is a problem that the survival rate is low. In addition, there is also known a method in which almost all genetic males are feminized by immersing fry continuously in estrone and estradiol 17β, which are female hormones, to produce functional females (XY females, pseudo-females) (Non-Patent Document 4). However, there is a problem that fish treated with such sex hormones cannot be used as food. [Prior art documents] [Non-patent literature]

[0005] [Non-Patent Document 1] Hokkaido Prefectural Fisheries Hatchery, "Application of Biotechnology in Salmonid Fishes," Hokkaido Prefectural Fisheries Hatchery Project Report, 1985, pp. 190-193. [Non-Patent Document 2] Yamamoto et al., "Studies on the production of all female Amago trout - V: Creation of diploid gynogenesis in Amago trout by inhibiting the first cleavage," Okayama Prefectural Fisheries Experiment Station Report, 1986, Vol. 4, pp. 93-96. [Non-Patent Document 3] Fujimoto et al., "Breeding through chromosome manipulation in aquatic organisms," Fisheries Breeding, 2012, Vol. 41, pp. 111-123. [Non-Patent Document 4] Nakamura, Feminization of masu salmon Oncorhynchus masou by administration of estradiol-17β. Bulletin of the Japanese Society of Scientific Fisheries, 1981, Vol. 47, pp. 1529. [Overview of the Initiative] [Problems that the invention aims to solve]

[0006] This invention provides a novel method for producing a high proportion of females in salmonid fish. [Means for solving the problem]

[0007] The inventors have discovered that by using equol, which can be safely ingested orally by humans, and rearing genetically male salmonid fish in the sexually undifferentiated stage in its presence, it is possible to produce genetically male fish with female gonads.

[0008] The present invention provides the following: Item 1. A method for producing genetically male salmonid fish with female gonads, comprising rearing genetically male salmonid fish in the presence of equol. Item 2. The method according to Item 1, wherein the rearing environment contains equol at concentrations of 10 μg / L or more, 20 μg / L or more, 30 μg / L or more, 40 μg / L or more, 50 μg / L or more, 60 μg / L or more, 70 μg / L or more, 80 μg / L or more, 90 μg / L or more, 100 μg / L or more, 110 μg / L or more, 120 μg / L or more, 130 μg / L or more, 140 μg / L or more, or 150 μg / L or more. Item 3. The method according to item 1 or 2, wherein the rearing environment contains equol at concentrations of 20 mg / L or less, 15 mg / L or less, 12 mg / L or less, 10 mg / L or less, 8 mg / L or less, 6 mg / L or less, 5 mg / L or less, 4 mg / L or less, 3 mg / L or less, 2 mg / L or less, 1000 μg / L or less, 900 μg / L or less, 800 μg / L or less, 700 μg / L or less, 600 μg / L or less, 500 μg / L or less, 400 μg / L or less, or 300 μg / L or less. Item 4. The method according to any one of items 1 to 3, wherein the salmonid fish is a cherry salmon, and equol is present from the time of eye formation until just before the juvenile fish emerges. Item 5. The method described in any one of items 1 to 4, wherein genetically males are reared in a mixed environment with genetically females. Item 6. The method according to any one of items 1 to 5, wherein the genetic male is diploid or triploid. Item 7. A feminizing agent containing equol for genetically undifferentiated male salmonid fish. [Effects of the Invention]

[0009] According to the present invention, it is possible to produce a high proportion of female salmonid fish in one generation without using hormones that are unsuitable for the production of food resources. [Brief explanation of the drawing]

[0010] [Figure 1]This graph shows the ratio of males and females (top row) and the survival rate (bottom row) of cherry salmon reared in water supplemented with equol-containing supplements or kinako (roasted soybean flour), as determined by macroscopic observation. Fisher's exact test was used to compare the untreated group with each test group, and P<0.05 was considered statistically significant and is indicated by an asterisk (*) in the figure. [Figure 2] This is an image of an electrophoresis gel showing the results of an SdY gene test. [Figure 3] This is a hematoxylin-eosin stained image of the gonads. [Figure 4] This graph shows the ratio of males to females in cherry salmon individuals raised in water supplemented with equol, as determined by visual observation. [Figure 5] This graph shows the survival rate of cherry salmon individuals raised in water to which equol has been added. [Figure 6] This graph shows the male-to-female ratio of cherry salmon individuals raised in water supplemented with genistein. [Modes for carrying out the invention]

[0011] The following descriptions may be based on representative embodiments or specific examples, but the present invention is not limited to such embodiments or specific examples. In this specification, numerical ranges represented using "~" or "-" mean ranges that include the numbers at both ends as the upper and lower limits, respectively, unless otherwise specified. The upper and lower limits of each numerical range exemplified in this specification can be combined in any way. All numbers include numbers with one less digit than the number to which it is rounded. For example, "1.0" includes numbers that are rounded to which it is, i.e., numbers between 0.95 and less than 1.05.

[0012] The salmonid fish used in the present invention is not particularly limited as long as it belongs to the family Salmonidae of the order Salmoniformes. Examples thereof include masu salmon, iwana, Atlantic salmon, brown trout, amago salmon, common salmon (white salmon), silver salmon, cherry salmon (yamame), early summer salmon (amago), rainbow trout, pink salmon (hime salmon), Biwa trout, masunosuke, Karafuto salmon, kunimasu, Apache trout, golden trout, Mexican golden trout, nodokiremasu, Gila trout, and the like. Salmonid fish are those for which it is expected to secure their growth numbers and thus catch amounts by means of aquaculture, stock enhancement, or other artificial means. For example, cherry salmon or silver salmon are preferred.

[0013] The sexually undifferentiated stage refers to the stage in the growth process of fry in which the sex of the gonads is morphologically undifferentiated, and corresponds to the period from immediately after hatching until the gonads begin to differentiate. In the case of cherry salmon, fry immediately after hatching have a large yolk sac in the abdomen and are in an inactive state of swimming, sinking to the bottom. The fry then become relatively active in swimming at the bottom while still having the yolk sac, and eventually rise to the surface and begin feeding. In the gonads of fry at the start of feeding, the division and proliferation of germ cells become active, and cysts of germ cells can be observed. It is considered that this period until the start of feeding corresponds to the sexually undifferentiated stage of cherry salmon (Nakamura et al., Hokkaido Salmon and Masu Fry Research Report, 1974, No. 28, pp. 1-8). The sexually undifferentiated stage of silver salmon is equivalent to that of cherry salmon.

[0014] Equol ((3S)-3-(4-Hydroxyphenyl)-7-chromanol, also known as 4’,7-isoflavandiol) is a water-soluble non-steroidal compound represented by the following formula 1.

Chemical formula

[0015] Equol is a metabolite of soy isoflavones. The glycoside form of daidzin contained in soybeans is converted into the aglycone form of daidzein by β-glucosidase or the like, and further metabolized by intestinal bacteria to produce equol via dihydrodaidzein and tetrahydrodaidzein. Industrially, equol is produced using microorganisms having the ability to produce equol, such as lactic acid bacteria, and is used as a raw material for supplements and foods. A number of microorganisms having the ability to produce equol have been reported, and examples thereof include Lactococcus 20-92 (FERM BP-10036), Streptococcus E-23-17 (FERM BP-6436), Streptococcus A6G225 (FERM BP-6437), and Bacteroides E-23-15 (FERM BP-6435).

[0016] In the present invention, by allowing a microorganism having the ability to produce equol to act on a raw material containing a compound convertible to equol such as daidzin and daidzein, for example, equol produced by culturing a microorganism having the ability to produce equol in a medium containing the raw material can be used. Equol may be used in the form of a culture of a microorganism having the ability to produce equol as long as it does not interfere with the rearing of salmonid fish, or may be recovered or purified from the culture and used. Also, commercially available products such as supplements containing equol may be used in their original form, or recovered or purified from the commercially available products and used. For example, equol can be a fermented product of soybean germ obtained by fermenting soybean germ with a microorganism having the ability to produce equol.

[0017] The method of the present invention includes rearing genetically male salmonid fish in the sexually undifferentiated stage in the presence of equol. Rearing in the presence of equol can be carried out by rearing genetically male salmonid fish in rearing water to which equol has been added (hereinafter referred to as rearing water), or by rearing them using feed containing equol. The concentration of equol in the rearing water may be, for example, 10 μg / L or more, 20 μg / L or more, 30 μg / L or more, 40 μg / L or more, 50 μg / L or more, 60 μg / L or more, 70 μg / L or more, 80 μg / L or more, 90 μg / L or more, 100 μg / L or more, 110 μg / L or more, 120 μg / L or more, 130 μg / L or more, 140 μg / L or more, or 150 μg / L or more. Furthermore, the concentration of equol in the rearing water may be, for example, 20 mg / L or less, 15 mg / L or less, 12 mg / L or less, 10 mg / L or less, 8 mg / L or less, 6 mg / L or less, 5 mg / L or less, 4 mg / L or less, 3 mg / L or less, 2 mg / L or less, 1000 μg / L or less, 900 μg / L or less, 800 μg / L or less, 700 μg / L or less, 600 μg / L or less, 500 μg / L or less, 400 μg / L or less, or 300 μg / L or less.

[0018] When genetically male cherry salmon are reared in the presence of equol, the concentration of equol in the rearing water may be, for example, 10 μg / L to 2 mg / L, 30 μg / L to 2 mg / L, 10 μg / L to 1000 μg / L, 30 μg / L to 1000 μg / L, 30 μg / L to 800 μg / L, 30 μg / L to 400 μg / L, 30 μg / L to 300 μg / L, 30 μg / L to 200 μg / L, or 30 μg / L to 150 μg / L.

[0019] When genetically male coho salmon are reared in the presence of equol, the concentration of equol in the rearing water may be, for example, 100 μg / L to 20 mg / L, 300 μg / L to 20 mg / L, 100 μg / L to 10 mg / L, 300 μg / L to 10 mg / L, 300 μg / L to 8 mg / L, 300 μg / L to 4 mg / L, 300 μg / L to 3 mg / L, 300 μg / L to 2 mg / L, or 300 μg / L to 1.5 mg / L.

[0020] When rearing genetically male cherry salmon or coho salmon in the presence of equol, they should be reared in rearing water supplemented with equol from the time of eye development before hatching until just before they emerge as juveniles after hatching. Expressed in terms of cumulative water temperature (cumulative value of rearing water temperature after fertilization), the eye development period corresponds to approximately 300-400°C days, and the period just before they emerge corresponds to approximately 750-850°C days.

[0021] Genetically male salmonid fish may be derived from naturally occurring fertilized eggs or from artificially inseminated individuals. Furthermore, genetically male salmonid fish may be diploid or triploid. Triploid individuals can be produced by methods known to those skilled in the art, for example, by applying physical stimuli such as water temperature and water pressure to fertilized eggs of genetically male and genetically female fish.

[0022] As shown in the examples described later, equol does not adversely affect the growth and sexual differentiation of genetically female fish. Genetically female fish remain as they are, while genetically male fish can be feminized and differentiated into functionally female fish for growth. Therefore, salmonid fish can be reared in the presence of equol without separating the genetic sex of each individual, and genetically male and genetically female fish can be mixed together. Even eggs and juvenile fish whose sex is difficult to determine can be used directly in the method of the present invention.

[0023] The rearing conditions for juvenile fish, including egg hatching, such as the composition of the rearing water, temperature, and rearing container, can be those commonly used in the rearing of salmonid fish, except that they contain equol. For example, river water, lake water, spring water, groundwater, or dechlorinated tap water can be used, and the water temperature should be maintained at 5-15°C, preferably around 8-10°C. Rearing should preferably be carried out in a dark place, in flowing water, or with fresh rearing water changed every few days. Egg hatching can be done using an incubator, and juvenile fish can be reared using a suitable aquarium.

[0024] The fact that salmonid fish individuals produced by the method of the present invention are genetically male with female gonads can be confirmed by the presence of XY sex chromosomes and ovaries containing oocytes.

[0025] XY sex chromosomes can be identified by detecting the Y chromosome using a suitable gene located on the Y chromosome, such as the sdY gene (sexually dimorphic on the Y-chromosome gene, a testicular formation-related gene located on the Y chromosome), which is known as a sex determination marker.

[0026] The presence of ovaries containing oocytes can be confirmed by observing the external appearance of the gonads of salmonid fish individuals after rearing them until the gonads differentiate, for example, until approximately 20 weeks after hatching in cherry salmon and coho salmon. It can also be confirmed earlier by tissue observation.

[0027] Furthermore, the present invention provides a feminizing agent for genetically male salmonid fish in the sexually undifferentiated stage, which contains equol. Here, "feminization" refers to the transformation of genetically male salmonid fish in the sexually undifferentiated stage into genetically male fish having female gonads. Therefore, the "feminizing agent" can be described as an agent containing equol as an active ingredient for transforming genetically male salmonid fish in the sexually undifferentiated stage into genetically male fish having female gonads. The feminizing agent can be used in the method for producing genetically male fish having female gonads as described above.

[0028] The feminizing agent may be a composition containing components other than equol. Examples of such components include additives that may be found in commercially available supplements containing equol, such as excipients, binders, lubricants, solvents, disintegrants, solubilizers, suspending agents, emulsifiers, isotonic agents, stabilizers, preservatives, antioxidants, flavoring agents, colorants, buffers, and other components; components found in fermented soybean germ products obtained by fermenting soybean germ with microorganisms capable of producing equol; and components found in feeds commonly used for raising salmonid fish. It is preferable that components other than equol do not hinder the growth or feminization of salmonid fish. The feminizing agent may be in any form that can be added to rearing water and used, for example, in the form of a powder, granules, or liquid.

[0029] The present invention will be described in more detail by the following examples, but the present invention is not limited thereto. [Examples]

[0030] [Example 1. Test using cherry salmon 1] A rearing experiment of cherry salmon was conducted twice using commercially available equol and kinako (roasted soybean flour, Sakaguchi Flour Mill, using soybeans from Hokkaido) according to the method described below. The equol used was a commercially available equol-containing supplement (soybean germ ferment product obtained by fermenting soybean germ with Lactococcus 20-92, a lactic acid bacterium with equol-producing ability, trade name Equelle®, Otsuka Pharmaceutical Co., Ltd.), which contained 246 mg of soy isoflavones and 385 mg of S-equol per 100 g of dry weight. As an example of analysis of kinako made from Hokkaido soybeans, the average isoflavone composition (after hydrolysis) was reported to be daidzein 604 μg / g, glycitein 155 μg / g, genistein 522 μg / g, and total isoflavones 1280 μg / g (Takahashi et al., Hokkaido Institute of Public Health Report, No. 52, 2002, Table 1 on pp. 29-36).

[0031] Fertilized eggs obtained by mating a normal male (XY type) and female (XX type) cherry salmon were placed in an incubator filled with spring water at 8-10°C and left undisturbed until eye development was visible through the egg membrane. After eye development was observed, 100-200 eggs were transferred to a tank containing 10L of rearing water supplemented with either equol or kinako (roasted soybean flour) as shown in Table 1, and hatched. The fry were then reared until just before they floated to the surface. Rearing in equol-supplemented water (also called equol treatment) was carried out for 38-52 days, during which the water temperature was 8-10°C, and the water was changed every three days. [Table 1]

[0032] Subsequently, the juvenile fish were reared in 7-9°C flowing water without equol or kinako (roasted soybean flour). When they reached a size of 5-6 cm, where the gonads were visible, their abdomens were opened, and the gonads were observed visually to determine their sex. The number of male and female fish in each test group was compared to the sex ratio of the untreated group (control) using Fisher's exact test (5% significance level). Fish whose sex could not be determined due to gonad development were classified as unknown and excluded from the test.

[0033] Furthermore, DNA was extracted from 20 individuals identified as female by the naked eye in Lot No. 2 of Experiment 1, and from 1 individual identified as female (Individual No. 25) and 1 individual identified as male (Individual No. 34) in the untreated group. The sex determination marker sdY was amplified by PCR, and genetic testing was performed. Genetic testing was carried out by PCR according to the method described in Yano et al., Evolutionary Applications, 2013, Vol. 6, pp. 486-496. As primers, sdY E1S1, sdY E2AS5, 18S S, and 18S AS listed in Table 2 of the same literature were used, and the following temperature cycle was applied: denaturation at 95°C for 3 minutes, followed by 35 repetitions of amplification cycles of 95°C for 30 seconds, 60°C for 30 seconds, and 72°C for 30 seconds, and finally extension at 72°C for 3 minutes. The PCR products were subjected to electrophoresis on a 2% agarose gel.

[0034] Furthermore, for pseudo-female individuals that were determined to be genetically male but appear female to the naked eye through genetic testing, tissue sections of the gonads were prepared, stained with hematoxylin and eosin, and the differentiation status of the gonadal cells was observed under a microscope.

[0035] Figure 1 shows the ratio of males and females and the survival rate of cherry salmon, determined by visual observation, combining the results of two tests. The male-to-female ratio was approximately 1:1 in the untreated group, while in the equol-treated group, 93-100% of the individuals whose sex was determined were female. On the other hand, the kinako (roasted soybean flour)-treated group had a male-to-female ratio of around 1:1, with no significant change observed. In the equol-treated group, the survival rate tended to decrease with increasing equol concentration, and in the group treated with 0.3 g / L of Equelle (equol equivalent concentration 1154 μg / L), all individuals died during rearing after the end of the equol treatment period. The survival rate was also low in the untreated group, but this is presumed to be due to experimental procedure errors. Normally, the survival rate of the untreated group is around 80%.

[0036] Figure 2 shows the sdY gene test results for 13 of the 20 individuals identified as female with the naked eye (individuals No. 1-13) and 2 individuals from the untreated group (individuals No. 25 and 34). In this gene test, two bands are observed on the electrophoresis gel for genetically male individuals and one band for genetically female individuals. Two bands were observed in 5 of individuals No. 1-13, confirming that these individuals were pseudofemales, genetically male despite being identified as female with the naked eye.

[0037] Figure 3 shows gonadal tissue images of some of these individuals. Individuals No. 3, 5, 8, 11, and 13 in the equol-treated group, although genetically male, had oocytes in their gonads, and their condition and size showed good development comparable to that of genetically female individuals in the untreated group. From these results, it was confirmed that pseudofemales (genetically male, phenotypic female) can be produced by equol treatment. Furthermore, genetically female individuals treated with equol (corresponding to normal female 1 and normal female 2 in Figure 3, and No. 1 and 2 in Figure 2, respectively) also showed good development comparable to that of genetically female individuals in the untreated group, and no effect of equol treatment on genetically female individuals was observed.

[0038] [Example 2: Test using cherry salmon 2] Fertilized eggs obtained by mating a normal male (XY type) and female (XX type) cherry salmon were placed in an incubator filled with spring water at 8-10°C and left undisturbed until eye development was visible through the egg membrane. After eye development was observed (cumulative water temperature 333°C-days), 200 eggs were transferred to tanks containing 4.5-10 L of rearing water to which equol ((S)-equol, Daicel Corporation, manufacturer's product code 100152-001G, purity ≥98%) as shown in Table 2 had been added, and the eggs were hatched and reared until just before the juveniles surfaced. The equol treatment was carried out for 52 days (cumulative water temperature 805°C-days), during which the water temperature was 8.5-10°C. Water changes were performed every 3 days during the egg stage and every 1-2 days during the larval stage. [Table 2]

[0039] Subsequently, the juvenile fish were reared in flowing water at 7-9°C that did not contain equol. When they reached a size of 5-6 cm in which the gonads could be observed, their abdomens were opened, and the gonads were observed visually to determine their sex. The number of male and female fish in each test group was then compared to the sex ratio of the untreated group (control) using Fisher's exact test (5% significance level).

[0040] Figure 4 shows the male-to-female ratio of cherry salmon determined by visual observation. The male-to-female ratio was approximately 1:1 in the untreated group, whereas in the equol-treated groups of 1925 μg / L, 385 μg / L, 192.5 μg / L, and 38.5 μg / L, the proportion of females was higher than that of males.

[0041] Figure 5 shows the survival rates of cherry salmon. The group treated with 3850 μg / L of equol was completely wiped out, and the survival rate of the group treated with 1925 μg / L of equol was lower than that of the untreated group, but the survival rates of the other groups were not significantly lower than those of the untreated group.

[0042] [Example of test: Genistein treatment of cherry salmon] A rearing experiment of cherry salmon was conducted in the same manner as in Example 1, with equol-containing supplements replaced by genistein. Each test group received 100 treated eggs, and the genistein concentrations were 60 μg / L, 80 μg / L, 100 μg / L, or 120 μg / L. At the end of rearing, the number of surviving fish was 42 in the 60 μg / L group, 34 in the 80 μg / L group, 15 in the 100 μg / L group, and all juvenile fish died in the 120 μg / L group. The proportion of males was 53-55%, and the proportion of females was 45-47%, neither of which differed from the control groups of 54% and 46% (Figure 6).

Claims

1. A method for producing genetically male salmonid fish with female gonads, comprising rearing genetically male salmonid fish in the presence of equol.

2. The method according to claim 1, wherein equol is present in the rearing environment at a concentration of 10 μg / L to 20 mg / L.

3. The method according to claim 1, wherein the salmonid fish is a cherry salmon, and equol is present from the time of eye formation until just before the juvenile fish emerges.

4. The method according to claim 1, wherein genetically males are reared in a mixed environment with genetically females.

5. The method according to claim 1, wherein the genetically male is diploid or triploid.

6. A feminizing agent containing equol for genetically undifferentiated male salmonid fish.

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