CN117158352B - Desmodium styracifolium female induction feed, induction method and application thereof - Google Patents

Abstract

The invention provides a feminization induction feed, an induction method and an application thereof, and belongs to the technical field of breeding seedlings of goldfish. The invention takes goldfish 60 days after hatching as the research object, and for the first time performs an induction treatment on the goldfish for about 90 days by feeding the feed in combination with 300 μg/g estrogen E2 and 300 μg/g androgen inhibitor TR, and explores the influence of the induction treatment method on the sex differentiation, sex steroid hormone and related gene expression of goldfish, and finally successfully obtains feminized male fish with normal ovaries, further improves the research on inducing feminization of goldfish, and provides theoretical guidance for the production practice of unisexual goldfish.

Description

A kind of goldfish feminization induction feed, induction method and application thereof

Technical Field

The invention belongs to the technical field of goldfish seedling breeding, and in particular relates to a goldfish feminization induction feed, an induction method and an application thereof.

Background Art

Fish sex is plastic. Its sex differentiation and development is controlled by genetic factors, as well as temperature, exogenous hormones, etc. to varying degrees. It is generally believed that sex hormones can induce gonadal differentiation in fish. Estrogens such as 17-β-estradiol and estrone are female inducers, and androgens such as methyltestosterone are male inducers (Devlin et al., 2002; Ge Xianping et al., 2002). For example, estrogen can be used to induce XY individuals to reverse into pseudo-female fish. XY pseudo-female fish can be mated with XY normal male fish to obtain YY super male fish. YY super male fish can be mated with normal XX female fish to obtain all-male offspring (Mei et al., 2014). Existing studies have shown that the use of sex hormones to treat fry and juvenile fish during the critical period of gonadal differentiation can produce monosexual or primary monosexual populations, such as Nile tilapia (Tilapia nilotica), Mozambique tilapia (Oreochromis mossambicus), medaka (Oryzias latipessinensis), Chinook salmon (Oncorhynchus tshawytscha), common carp (Cyprinus carpio) and mullet (Odontesthes bonariensis) (Devlin et al., 2002; Strüssmann et al., 1996). In the field of aquaculture, many economically cultivated species show differences in body color between male and female individuals. Therefore, under the same conditions, selecting all-sex seedlings with bright body colors for cultivation can achieve the diversification of the breeding scale and sales form of the same species and ensure the sustainable development of the breeding industry. Selecting and breeding fish for body color and choosing sexes with ornamental and economic value for single-sex breeding can not only reveal the genetic mechanisms related to the regulation of fish body color, but also produce significant economic and social benefits.

Scatophagus argus, commonly known as golden drum, belongs to the order Perciformes, the suborder Acanthuroidei, the family Scatophagidae, and the genus Scatophagius. It is mainly distributed in the southeastern coast of my country. It is the only species of the family Scatophagius recorded in my country. It has both edible and ornamental value and is a precious aquaculture variety that can meet multiple sales models (Barry et al., 1992; Yang et al., 2018). In view of the economic value of Scatophagus argus, a large number of studies have been carried out on its reproductive biology at home and abroad, and its artificial breeding technology has been successful (Cai et al., 2010; Cui et al., 2013; Zhang et al., 2013). However, the genetic breeding of Scatophagus argus has been relatively slow and there are few reports. Under the same breeding conditions, the body color of male Scatophagus argus is brighter. It is of great significance to carry out the cultivation of ornamental all-male Scatophagus fry and the cultivation of fry body color enhancement.

In the laboratory and fishery production, estrogen or androgen can be used to produce all-male or all-female groups as needed, which shows that endogenous steroid hormones play an important role in the differentiation of fish gonads (Ankley et al., 2009). KitanoT (Kitano et al., 2000) and others have proved that sex steroid hormones can affect the development of organisms in many ways. It has been reported that _E2 can affect the direction of early differentiation of fish gonads and has been successfully used to induce feminization of aquatic animals (Piferrer et al., 2001). For example, after yellowfin mackerel (Torputitora) was induced with _E2 on the 30th day after hatching, the female fish ratio increased from 24.2% to 69.5% (Singh et al., 2013); Odontesthes bonariensis was induced with _E2 6 weeks after hatching, and the female fish ratio increased from 27% to 42%~46% (Perezm et al., 2012). At present, the sex reversal of XY fish from male to female is limited to XY fish with undifferentiated gonads induced by exogenous estrogen (Yamamoto et al., 1953; Chen et al., 2016). Once the gonads begin to differentiate morphologically, androgens gradually increase with the differentiation of the testes and the expression level of hormone-related genes, it becomes more difficult to induce the feminization sex reversal of XY fish. Therefore, androgens in the testes will antagonize the sex reversal of XY fish from male to female induced by estrogen. TR (Trilostane, an inhibitor of 3β-hydroxysteroid dehydrogenase) is an inhibitor of androgen synthesis enzymes. Trilostane inhibits androgen synthesis by reducing the expression level of androgen synthesis-related genes. Studies have shown that when XY tilapia were treated with TR alone for three months, the level of 11-KT (11-ketotestosterone) in serum and the expression of androgen synthesis enzyme gene cyp11b2 were significantly downregulated. However, the expression of estrogen-related genes foxl2 and cyp19a1a and the level of _E2 did not change. Moreover, immunohistochemistry did not detect the expression of cyp19a1a in the gonads of XY fish in these treatment groups, indicating that simply inhibiting androgens cannot induce sex reversal in XY fish (Shi Hongjuan et al., 2018). Although both _E2 and TR can inhibit the synthesis of 11-KT, _E2 can also promote ovarian differentiation (Yamamoto et al., 1953). Although tilapia treated with _E2 alone can successfully reverse sex, the sex reversal rate is low.

At present, in the study of artificial sex reversal in goldfish, Mustapha et al. (Mustapha et al., 2021) used 300 (μg/g) concentration of estrogen (E ₂ ) to induce sex reversal in goldfish, and obtained different degrees of reversal. Based on gonadal histology, its sex reversal can be divided into incomplete sex reversal and complete sex reversal: incomplete sex reversal, male and female germ cells coexist; complete sex reversal, only female germ cells are observed. However, the induction effect was not good, and the gonadal morphology of the obtained sex-reversed fish was observed to be deformed after dissection.

Summary of the invention

In order to solve the above technical problems, the present invention provides a feminization induction feed, induction method and application of goldfish. The present invention takes goldfish 60 days after hatching as the research object, and for the first time performs an induction treatment for about 90 days by feeding the feed with 300 μg/g estrogen E2 and 300 μg/g androgen inhibitor TR in combination, and explores the effects of the induction treatment on the sex differentiation, sex steroid hormone and related gene expression of goldfish, and finally successfully obtains feminized male fish with normal ovaries, further improves the research on inducing feminization of goldfish, and provides theoretical guidance for the production practice of unisexual goldfish.

In order to achieve the above object, the present invention adopts the following technical solutions:

The present invention provides a method for inducing feminization of golden croaker, comprising the following steps:

(1) Domestication before induction: Place the golden croaker fry in a container and domesticate them until 58 to 62 days after hatching;

(2) Preparation of induction feed: First, estradiol (E2) and trilostane (TR) are dissolved in anhydrous ethanol to prepare a 3-5 mg/mL mother solution, and then the mother solution is mixed with anhydrous ethanol solution again, and after mixing, it is added to the feed and fully stirred, and then dried in an oven at 58-65°C to make the content of estradiol (E2) and trilostane (TR) in the feed 280-320 μg/g, thereby preparing the induction feed;

(3) Induction breeding: feeding the goldfish tamed in step (1) with the induction feed until the goldfish is 5 months old, and the induction is completed;

(4) Cultivation after induction: The induced goldfish is transferred to the outdoors and fed with commercial feed for further cultivation.

Preferably, healthy golden croaker fry are selected in step (1).

Preferably, in the step (1), the goldfish fry are domesticated until 60 days after hatching.

Preferably, the domestication method of the goldfish fry in the step (1) is: first feed rotifers, then switch to Artemia after 10 days, until 20 days after hatching, then start feeding artificial compound feed, feed twice a day, then gradually reduce the amount of Artemia feeding every day, increase the amount of artificial compound feed, until all artificial compound feed is fed.

Preferably, in step (2), estradiol (E2) and trilostane (TR) are first dissolved in anhydrous ethanol to prepare a 4 mg/mL stock solution.

Preferably, the content of estradiol (E2) and trilostane (TR) in the induction feed prepared in step (2) is both 300 μg/g.

Preferably, the feeding method of the induction feed in step (3) is: feeding twice a day, respectively at 8:30-9:30 in the morning and 15:30-16:30 in the afternoon; the feeding amount each time is about 0.5g-1.0g per fish, and the amount is adjusted appropriately in the later stage according to the increase of fish body.

Preferably, the commercial feed in step (4) is fed once in the morning and once in the afternoon every day, and the remaining bait and feces are cleaned up after the feed is completely eaten.

The present invention also provides an induction feed prepared in the method for inducing feminization of golden croaker.

The present invention also provides a method for inducing feminization of golden croaker or application of the inducing feed in inducing feminization of golden croaker.

Compared with the prior art, the present invention has the following technical effects:

The present invention uses trilostane (TR) and estradiol ( _E2 ) 300 μg/g each to jointly treat the golden croaker 60 days after hatching, treat for about 90 days, then feed normally, and take samples after raising to 1 year old. 28 XY individuals were identified by sex molecular markers, and sections found that 4 were completely reversed to ovaries and 3 were incompletely reversed sperm ovaries. The key hormone content in the serum of the treated fish was determined by ELISA, and the gonad gene expression characteristics were tested by qRT-PCR and immunohistochemistry. It was found that the _E2 content related to female hormones was significantly higher in female fish than in male fish, while the 11-KT content related to male hormones was significantly higher in male fish than in female fish. qRT-PCR detection of the expression of each feminization-related gene in the gonad of the golden croaker showed that the female-related genes 42sp50, foxl2, figla, zar1, zp2, etc. were significantly highly expressed in male fish and incomplete sex-reversed fish in female fish and complete sex-reversed fish, and the incomplete sex-reversed fish was higher than the male fish; the male-related genes dmrt1, gsdf, amh, cyp11b2, etc. were significantly highly expressed in male fish in incomplete sex-reversed fish, complete sex-reversed fish and female fish, and the expression level of female fish was the lowest. The results of immunohistochemistry (IHC) showed that the 42sp50 staining signal was highlighted in the egg cells in incomplete sex-reversed fish, complete sex-reversed fish and female fish, while the gsdf staining signal was only displayed in the sperm cells of incomplete sex-reversed and male fish. The present invention uses E ₂ and TR to jointly treat the successfully feminized golden croaker XY pseudo-female fish, which provides theoretical guidance for the production practice of unisexual golden croaker.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG1 is an electrophoretic diagram of molecular marker sex identification in Example 1 of the present invention, wherein the fish with two bands, 593 and 693, are male fish, and the fish with only one band, 593, are female fish;

Fig. 2 is a diagram of the morphology and tissue sections of the gonads of the fish treated in Example 1 of the present invention, wherein a, d, i are anatomical diagrams of the gonads of female fish, incomplete sex-reversed fish, and complete sex-reversed fish, respectively; b, f, k are appearance diagrams of the gonads of female fish, incomplete sex-reversed fish, and complete sex-reversed fish, respectively; c, g, l are gonad sections of female fish, incomplete sex-reversed fish, and complete sex-reversed fish, respectively; SC is spermatocyte, and OC is oocyte;

Figure 3 is a comparison of gonadal morphology of fish treated with different drugs in Example 1 of the present invention, wherein a and b are gonadal morphology of fish with complete and incomplete sex reversal after _E2 treatment alone at the same concentration and treatment time, respectively; c and d are gonadal morphology of fish with complete and incomplete sex reversal after _E2 /TR combined treatment at the same concentration and treatment time, respectively;

Figure 4 is a comparison of the hormone levels of estradiol, ketone testosterone and testosterone in the serum of individual goldfish of different genders in Example 1 of the present invention, wherein A is male, B is incomplete sex reversal, C is complete sex reversal, and D is female; statistical significance is p<0.05 (*), statistically extremely significant is P<0.01 (**) and P<0.001 (***), and no mark (p>0.05) indicates no significant difference;

Figure 5 shows the expression changes of various sex differentiation-related genes in the gonads of individual goldfish of different sexes in Example 1 of the present invention, wherein A is male, B is incomplete sex reversal, C is complete sex reversal, and D is female; statistical significance is p<0.05 (*), statistically extremely significant is P<0.01 (**) and P<0.001 (***), and no mark (p>0.05) indicates no significant difference;

Figure 6 is a key gene expression localization analysis diagram detected by immunohistochemistry (IHC) in Example 1 of the present invention, wherein a, d, g, j are blank control groups, b, e, h, k are gsdf groups, and c, f, i, l are 42sp50 groups.

DETAILED DESCRIPTION

The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention. Without departing from the spirit and essence of the present invention, modifications or substitutions made to the methods, steps or conditions of the present invention are within the scope of the present invention. The reagents, kits and instruments used in the following examples can all be obtained commercially, and the methods used in the examples are consistent with the conventional methods unless otherwise specified.

The technical solution of the present invention is further described in detail below in conjunction with embodiments.

Example 1

1. Materials and Methods

1.1 Experimental fish selection and domestication

The experimental goldfish were obtained from the Marine Biology Research Base of Guangdong Ocean University in Zhanjiang, Guangdong Province, and all experiments were conducted in accordance with the Regulations on the Administration of Experimental Animals of the Science and Technology Bureau ((Ministry of Science and Technology (MST)), 1988). Healthy goldfish fry from the same batch were selected and placed in a 2000L drum for domestication. Feed rotifers first, then switch to Artemia after 10 days, until 20 days after hatching, start feeding Haitong brand prosperous food series juvenile fish seedling compound feed 3# (0.36mm-0.58mm), feed twice a day, then gradually reduce the amount of Artemia feeding every day, and increase the amount of artificial compound feed feeding, until all artificial compound feed is fed until 30 days after hatching, feed Haitong brand prosperous food series juvenile fish seedling compound feed 4# (0.58mm-0.84mm) until 40 days after hatching, feed Haitong brand prosperous food series juvenile fish seedling compound feed 5# (0.84mm-1.1mm) until 50 days after hatching, feed Haitong brand prosperous food series juvenile fish seedling compound feed 5# (1.1mm-1.4mm) until 60 days after hatching, and the domestication is completed.

1.2 Induction feed preparation and induction process

17b-E ₂ (17b-Estrodiol, E ₂ ) and TR were purchased from MCE (USA). E ₂ and TR were first dissolved in anhydrous ethanol to prepare a 4 mg/mL mother solution, and then the E ₂ /TR mother solution was mixed with the anhydrous ethanol solution again and added to the Haitong brand Wangshi series of juvenile fish breeding compound feed EP material 1.2# (1.2mm), and after being fully stirred, it was dried in an oven at 60°C to make the content of estradiol (E2) and trilostane (TR) in the feed 300μg/g, and the induction feed was prepared and sealed in a bag for storage.

The experimental group was set up with 2 parallels, and 150 fish were stocked in each parallel. The fry in each group were fed with the above-mentioned induction feed twice a day, at 9:00 am and 16:00 pm, respectively. The amount of feed per fish was about 0.5g to 1.0g, and the amount was adjusted appropriately according to the increase of fish body in the later stage. The induction feed was made into micro-particles, and the particle size of micro-particles was adjusted according to the body length of fry. After the experimental fish were cultured to 5 months old, they were transferred to an outdoor cement pool (5m×5m×1m) for further culture and fed with commercial feed (Guangdong Yuequn Biotechnology Co., Ltd.). Feed once a day at 9:00 am and 16:00 pm, and each feeding lasted about 30 minutes to ensure that the feed was completely ingested, and then clean up the remaining bait and feces.

1.3 Sampling

The experimental fish were collected when they were 1 year old and fasted for 24 hours before sampling. The experimental fish were anesthetized with 1% eugenol (E809010, Macklin Biochemical, Shanghai, China). A small amount of tail fin was cut and stored in anhydrous ethanol, and stored at -20°C for use in DNA extraction. Genomic DNA was extracted using a DNA extraction kit (AD1173, Guangzhou Aitel Biotechnology Co., Ltd., China), and the sex identification markers of the money fish obtained by this research group (a method for rapid identification of the genetic sex of money fish, Huang Yang et al., 2022, Chinese Fisheries Science) were used to accurately identify the sex of the collected money fish. Blood was collected through the tail vein, left to stand for 4 hours, centrifuged at 3000g for 20 minutes, and the supernatant was collected and stored at -80°C for use in serum hormone determination. The gonads were then dissected out and photographed for morphology and appearance. The gonads were then washed with physiological saline. Part of the gonads were quickly frozen in liquid nitrogen and then stored at -80°C for use in extracting total RNA. Part of the gonads were fixed in Bouin's fixative for 24 hours and then stored in 70% ethanol for use in making tissue sections.

1.3.1 Tissue sections

The gonadal tissue samples were dehydrated with gradient alcohol, transparentized with xylene, and embedded in paraffin before continuous sectioning (5-8 μm in thickness). The gonads of each fish were cross-sectioned at least three different sections, stained with hematoxylin-eosin (HE), and sealed with neutral gum. The sections were observed and photographed using a Nikon Eclipse Ti-E microscope (Nikon Eclipse Ti-E, Tokyo, Japan). The phenotypic sex was determined by histological characteristics.

1.3.2 Determination of serum estradiol, ketone testosterone and testosterone levels

Serum estradiol (E ₂ ), 11-ketotestosterone (11-KT) and testosterone (T) levels were determined by enzyme-linked immunosorbent assay. The kits were purchased from Shanghai Jianglai Biotechnology Co., Ltd. and the determination was performed according to the instructions.

1.3.3 RNA sample extraction and gene expression analysis

The total RNA of gonads was extracted by Trizol (purchased from Guangzhou Weijia Technology Co., Ltd.), and the quality of the extracted RNA samples was tested by 1% agarose gel electrophoresis, and then the concentration was detected by a microplate reader (BiotekCytation 5).

According to the kit instructions, 1 μg of total RNA was reversed into cDNA using the PrimeScript RT reagent Kit with gDNA Eraser commercial kit (TaKaRa). The reverse transcription system was 20 μL, and the reaction conditions were 42°C for 2 min, 37°C for 15 min, and 85°C for 5 s. After the cDNA was diluted 10 times, it was used for Real-time PCR to detect gene expression. Green real-time PCR Master Mix plus kit, Real-time PCR reaction system (20 μL) includes: 2 μL cDNA, 10 μL SYBR, 7.2 μL ddH ₂ O, 0.4 μL forward and reverse primers. Real-time PCR detection was performed using a real-time fluorescence quantitative PCR analyzer (Roche, Switzerland), and the reaction conditions were: 94°C pre-denaturation for 300 s; then 94°C denaturation for 30 s, 60°C low-temperature renaturation for 20 s and 72°C suitable temperature extension for 20 s, 40 cycles; finally 72°C extension for 10 min and then stored at 4°C. β-actin was used as the internal reference gene, and R=2- ^△△Ct was used to calculate the relative expression abundance of the gene. All primers were commissioned to Guangzhou Aiji Biotechnology Co., Ltd. for synthesis, and the specific sequences are shown in Table 1.

Table 1 Primer sequences used in the present invention

1.3.4 Immunohistochemistry analysis (IHC)

In order to locate Gsdf and 42sp50, IHC detection was performed on the ovaries of the treated goldfish. The specific steps refer to the paper of Zhi Fei et al. (2022).

1.4 Statistics

All data were expressed as mean ± standard deviation (X ± SD). The results were statistically analyzed using SPSS19.0 (USA) software. Two independent sample t-tests were used to test the differences between the groups. P < 0.05 indicated a significant difference.

2. Results

2.1 Genetic sex identification of golden croaker

In the present invention, 28 XY fish collected from the treatment group were subjected to genetic sex identification, and the electrophoresis detection results are shown in FIG1 .

The results showed that both genetic females and males had a 593 bp band, while genetic males also had a 693 bp amplified band.

2.2 Gonadal histology

The gonadal morphology and tissue sections are shown in Figure 2 .

The results showed that the gonads of male fish with complete sex reversal have fully developed into ovaries and are highly similar in appearance to the ovaries of normal female fish, and the ovarian structure can be observed in the gonad sections. The gonads of male fish with incomplete sex reversal have both testicular and ovarian characteristics in appearance, and the structure of spermatozoa and ovaries can be observed in the gonad sections.

The differences in gonadal morphology between experimental fish treated with E2 alone and E2/TR combined treatment are shown in Figure 3 .

The results showed that the gonad morphology of the experimental fish treated with E2 alone was deformed, while the gonad morphology of the experimental fish treated with E2/TR combined had no significant difference from that of normal female fish.

2.3 Changes in serum estradiol, ketone testosterone, and testosterone levels

The changes in the levels of estradiol, ketone testosterone and testosterone in serum are shown in FIG4 .

The results showed that the content of _E2 related to female hormones was significantly higher in female fish than in male fish, while the content of 11-KT related to male hormones was significantly higher in male fish than in female fish.

2.4 Expression changes of genes related to sex differentiation

The expression levels of various feminization-related genes in the gonads of golden croaker detected by qRT-PCR are shown in Figure 5.

The results showed that the female-related genes 42sp50, foxl2, figla, zar1, zp2, etc. were significantly highly expressed in female fish and completely sex-reversed fish than in male fish and incomplete sex-reversed fish, and the expression level in incomplete sex-reversed fish was higher than that in male fish; the male-related genes dmrt1, gsdf, amh, cyp11b2, etc. were significantly highly expressed in male fish than incomplete sex-reversed, completely sex-reversed and female fish, and the expression level in female fish was the lowest.

2.5 Analysis of key gene expression by immunohistochemistry (IHC)

The immunohistochemistry (IHC) results are shown in FIG6 .

The results showed that the 42sp50 staining signal was highlighted in the oocytes of incomplete sex reversal, complete sex reversal and female fish, while the gsdf staining signal was only shown in the sperm cells of incomplete sex reversal and male fish.

The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims (10)

1. A method for inducing feminization of golden croaker, characterized in that it comprises the following steps: (1) Domestication before induction: Place the golden croaker fry in a container and domesticate them until 58-62 days after hatching; (2) Preparation of induction feed: First, dissolve estradiol (E2) and trilostane (TR) in anhydrous ethanol to prepare a 3-5 mg/mL stock solution, then mix the stock solution with anhydrous ethanol solution again, add it to the feed and stir it thoroughly, then dry it in an oven at 58-65°C to make the content of estradiol (E2) and trilostane (TR) in the feed 280-320 μg/g, and prepare the induction feed; (3) Induction breeding: feeding the goldfish tamed in step (1) with the induction feed until the goldfish are 5 months old, and the induction is completed; (4) Post-induction culture: The induced goldfish are transferred to outdoor areas and fed with commercial feed for continued culture. 2. The method for inducing feminization of golden croaker according to claim 1, characterized in that healthy golden croaker fry are selected in step (1). 3. The method for inducing feminization of golden croaker according to claim 2, characterized in that in the step (1), the golden croaker fry are domesticated until 60 days after hatching. 4. The method for inducing feminization of gold croaker according to claim 3, characterized in that the domestication method of gold croaker fry in the step (1) is: first feed rotifers, then switch to Artemia spp. after 10 days, until 20 days after hatching, then start feeding artificial compound feed, feed twice a day, then gradually reduce the amount of Artemia spp. fed every day, increase the amount of artificial compound feed, until all artificial compound feed is fed. 5. The method for inducing feminization of golden croaker according to claim 4, characterized in that in the step (2), estradiol (E2) and trilostane (TR) are first dissolved in anhydrous ethanol to prepare a 4 mg/mL mother solution. 6. The method for inducing feminization of goldfish according to claim 5, characterized in that the content of estradiol (E2) and trilostane (TR) in the induction feed prepared in step (2) is both 300 μg/g. 7. The method for inducing feminization of goldfish according to claim 6, characterized in that the induction feed in step (3) is fed twice a day, at 8:30-9:30 in the morning and 15:30-16:30 in the afternoon; the feeding amount each time is 0.5g-1.0g per fish, and the amount is adjusted appropriately according to the increase in fish body size in the later stage. 8. The method for inducing feminization of gold coin fish according to claim 7, characterized in that the commercial feed in step (4) is fed once in the morning and once in the afternoon every day, and the remaining bait and feces are cleaned up after the feed is completely eaten. 9. The induction feed prepared by the method for inducing feminization of golden croaker according to claim 1. 10. Use of the method for inducing feminization of golden croaker according to any one of claims 1 to 8 or the inducing feed according to claim 9 in inducing feminization of golden croaker.