Induction method and feed for XX type pseudo male salmon of Atlantic salmon and application of feed

Abstract

The invention relates to an induction method and feed for XX type pseudo male fishes of Atlantic salmon and application of the induction method and the feed, and belongs to the technical field of aquaculture breeding, the method comprises the following steps: when genetic female fish fries of Atlantic salmon open mouths for ingestion, 17 alpha-methyltestosterone or letrozole is fed every day, the feeding time duration is greater than or equal to 60 days, and the XX type pseudo male fishes are obtained. The invention also provides a feed containing 17 alpha-methyltestosterone or letrozole and application of the feed to culture of pseudo male Atlantic salmon. According to the invention, the effective window period is accurately shortened to the 60th day from the beginning of exogenous ingestion to ingestion, and the induction success rate of 1.5-4.5 mg / kg of 17 alpha-methyltestosterone can reach 100%; the induction success rate of 150 mg / kg letrozole is 80%, the induction success rate of 200 mg / kg letrozole is 83%, and the induction success rate of 250 mg / kg letrozole is 100%.

Description

Technical Field

[0001] This invention belongs to the field of aquaculture breeding technology, and specifically relates to a method for inducing pseudo-male Atlantic salmon of type XX, feed and its application. Background Technology

[0002] Atlantic salmon ( Salmo salar Atlantic salmon is a major farmed fish species globally. As a representative species of salmon, its flesh is delicious and suitable for raw consumption. It is rich in nutrients such as Omega-3 fatty acids, vitamin D, and antioxidants, making it highly sought after in the market. Male Atlantic salmon typically reach sexual maturity earlier than females (especially precocious two-year-olds). After sexual maturity, males experience stunted growth, soft flesh, and darkening of body color, severely impacting economic efficiency. In contrast, all-female Atlantic salmon populations grow rapidly, reach sexual maturity later, and are of uniform size. Therefore, producing all-female fry is a consensus in the international Atlantic salmon farming industry. The core technology for producing all-female populations is obtaining XX-type pseudo-males.

[0003] Methods for artificially controlling the sex of fish include interspecific hybridization, artificial induction of gynogenetic (androgen) development, environmental regulation, genetic engineering, and hormone induction. The advantages and disadvantages of each method are as follows: (1) Interspecific hybridization: This technique utilizes the hybridization between different species to produce offspring with a skewed sex ratio or all of the same sex. It utilizes natural biological processes and requires no external chemical or physical intervention; in principle, it only requires raising two species together. However, it is suitable for specific species combinations (such as hybridization between certain tilapia species to produce all-male offspring) with a low success rate. Hybrid vigor may not be obvious, and the growth performance of the offspring may be unsatisfactory.

[0004] (2) Artificial induction of gynogenetic (androgenous) development: Sperm or eggs are inactivated using physical or chemical methods, and then chromosomes are doubled through cold or heat shock or stress treatment, resulting in offspring containing only the maternal or paternal genome. This method can achieve the production of all-female or all-male populations with significant effects and can quickly establish pure lines. However, the process causes significant damage to the embryo, resulting in a high rate of seedling deformities. Precise control of the timing and intensity of inactivation and chromosome doubling is required. Moreover, the offspring have low genetic diversity and require strict management to prevent germplasm degradation.

[0005] (3) Environmental factor regulation: During the sensitive period of sex differentiation, gonadal differentiation can be affected by regulating environmental conditions such as water temperature, pH, light, and density. This method does not require the addition of exogenous substances and is an ideal green technology with low cost, requiring only the control of aquaculture environmental parameters. However, the mechanism of action is complex, difficult to control and predict precisely, and the sex ratio fluctuates greatly, making it difficult to achieve 100%. Optimization is required for specific fish species, resulting in poor universality.

[0006] (4) Genetic engineering technology: By editing genes (such as CRISPR / Cas9) to knock out or silence key sex-determining genes (such as dmrt1, amh, etc.), sterile or single-sex populations can be created directly. This addresses the problem at its root and can create traits that are impossible to achieve using traditional methods. Once a breeding line is successfully created, it can be used permanently without repeated processing. However, it requires a strong foundation in molecular biology and expensive equipment, and its application in food production is subject to ethical controversies and regulatory restrictions, resulting in low consumer acceptance of genetically engineered foods.

[0007] (5) Hormone-induced method: During the critical period of sex differentiation, exogenous hormones (androgens or estrogens) are administered to induce the genetic sex to develop in the opposite direction of the phenotype. This technique is mature and efficient, and is the most classic and widely used method, with a sex reversal success rate often exceeding 95%. It is relatively simple, suitable for large-scale operations, and the cost of hormones is relatively low, making it suitable for commercial applications in aquaculture. However, precise control of dosage and treatment window is required, otherwise it is prone to failure or infertility. Summary of the Invention

[0008] The purpose of this invention is to provide a method, feed, and application for inducing XX-type pseudomale Atlantic salmon. Both 17α-methyltestosterone and letrozole can induce sex reversal in XX-type genetically modified female Atlantic salmon into XX-type pseudomales, with a high success rate, high survival rate of XX-type pseudomales, and low deformity rate, thus achieving all-female breeding of Atlantic salmon.

[0009] The first aspect of the present invention is to provide a method for creating a pseudo-male Atlantic salmon.

[0010] A second aspect of the invention is to provide a feed for creating pseudo-male Atlantic salmon.

[0011] A third aspect of the present invention is to provide a method for preparing feed for creating pseudo-male Atlantic salmon.

[0012] A fourth aspect of the present invention is to provide a method for hatching eyed eggs and cultivating seedlings of Atlantic salmon.

[0013] A fifth aspect of the present invention is to provide the application of the feed of the second aspect of the present invention in improving the efficiency of Atlantic salmon farming.

[0014] To achieve the above-mentioned objectives of this invention, the technical solution adopted by this invention is as follows: In a first aspect, the present invention provides a method for creating pseudo-male Atlantic salmon, comprising the following steps: feeding 17α-methyltestosterone or letrozole daily for 60 days or more when genetically female Atlantic salmon fry begin to feed, thereby obtaining XX-type pseudo-males.

[0015] In this invention, 17α-methyltestosterone or letrozole is added to feed for feeding, wherein the content of 17α-methyltestosterone in the feed is 1.5~4.5 mg / kg.

[0016] In this invention, 17α-methyltestosterone or letrozole is mixed into the feed and fed to the animal, wherein the content of letrozole in the feed is 150~250 mg / kg.

[0017] In this invention, feeding begins when the female Atlantic salmon begin to eat (approximately 800 degree-days). In some embodiments of this invention, the feeding period is 60 days (approximately 600 degree-days). In some embodiments of this invention, the feeding method is satiating feeding; satiating feeding means feeding every 3 hours when the fish first starts eating, with a daily feed amount of approximately 5% of the fish's body weight, which can be adjusted appropriately according to feeding behavior and the amount of uneaten food, gradually reducing to 3-4 times per day.

[0018] In this invention, the specifications of the feed are adjusted according to the growth stages of Atlantic salmon; specifically: when the larvae change from gathering at the bottom to floating upwards, they are fed with artificial feed of 0.2mm size, and as the larvae grow, they are gradually fed with feed of 0.3-0.4mm and 0.5-0.8mm size.

[0019] A second aspect of the invention provides a feed for creating pseudo-male Atlantic salmon, comprising: 17α-methyltestosterone or letrozole and Atlantic salmon base feed. In some embodiments of the invention, the letrozole content in the feed is 150-250 mg / kg. In some embodiments of the invention, the 17α-methyltestosterone content in the feed is 1.5-4.5 mg / kg. In some embodiments of the invention, the nutrient content of the Atlantic salmon base feed is: crude protein 64.0%, crude fat 12.0%, nitrogen-free extract 4.9%, cellulose 1%, ash 12.1%, etc.

[0020] A third aspect of the present invention provides a method for preparing the Atlantic salmon pseudo-male feed according to the second aspect of the present invention, comprising the following steps: dissolving 17α-methyltestosterone powder or letrozole in 95% ethanol (by volume), then uniformly spraying the solution onto the Atlantic salmon base feed, mixing it evenly so that each kg of Atlantic salmon base feed contains 1.5-4.5 mg of 17α-methyltestosterone or 150-250 mg of letrozole. In this invention, to avoid degradation of 17α-methyltestosterone and letrozole affecting the effect, the feed is placed in a cool, dark, and ventilated place to dry, and the dried feed is stored in a refrigerator at 4°C.

[0021] The fourth aspect of this invention provides a method for cultivating Atlantic salmon pseudo-male fry. Specifically, it includes the following steps: Atlantic salmon eyed eggs are hatched at a water temperature of 6.0±0.5℃, a pH of 7.0-8.0, and dissolved oxygen >6.0mg / L. After hatching, the water temperature is increased to 10.0±0.5℃ at a rate not exceeding 0.5℃ per day. When the fry change from gathering at the bottom to floating upwards, they are fed artificial feed containing 1.5-4.5mg of 17α-methyltestosterone or 150-250mg of letrozole per kilogram, with a particle size of 0.2mm. As the fry grow, the feed size is gradually changed to 0.3-0.4mm and 0.5-0.8mm. After the fry become more active and begin to swim, a circular water flow is provided in the rearing pond to promote their growth, and the water temperature is gradually adjusted to 12.0±0.5℃.

[0022] A fifth aspect of the invention provides the use of the feed described in the second aspect of the invention in the preparation of Atlantic salmon pseudo-males.

[0023] In this invention, the induction refers to adding 17α-methyltestosterone or letrozole to the feed, and then feeding it to all-female Atlantic salmon with XX chromosomes; the female Atlantic salmon undergoes a physiological sex reversal to male.

[0024] Here, "female Atlantic salmon" refers to Atlantic salmon that are genetically female; genetic sex refers to sex directly indicated by sex chromosomes; physiological sex refers to sex exhibiting specific sexual physiological structures under the combined influence of multiple conditions such as heredity, environment (e.g., temperature), behavior, and physiological factors. Specifically, male Atlantic salmon are Atlantic salmon that possess testes and are capable of producing mature sperm.

[0025] In this invention, the Atlantic salmon that is genetically female but undergoes induction to become physiologically male is called a pseudo-male.

[0026] The beneficial effects of this invention compared to the prior art are as follows: The feed described in this invention can induce genetically female Atlantic salmon to become physiologically male. By mating the induced XX pseudo-male Atlantic salmon with normal XX-type females, all-female offspring can be obtained, enabling autonomous breeding of Atlantic salmon. Furthermore, all-female Atlantic salmon reach sexual maturity later than males, allowing for a longer growth cycle, significantly reducing aquaculture management costs, and substantially improving production efficiency, thereby increasing the profitability of Atlantic salmon farming.

[0027] This invention provides a method for inducing sex reversal in Atlantic salmon, involving the induction of sex reversal in Atlantic salmon using either 17α-methyltestosterone or letrozole, respectively. This method has the following significant advantages: (1) More precise: Through systematic research on the explosive expression sequence of SdY, a key gene for gonadal histological differentiation and sex determination, we have precisely narrowed the effective window period to the period from the start of exogenous feeding to 60 days after feeding (approximately 600 degree-days), during which gonadal cells are most sensitive to hormone signals. Due to intervention during the most sensitive period, the success rate of sex reversal is high. The induction success rate of 17α-methyltestosterone at three concentrations (1.5 mg / kg, 3 mg / kg, and 4.5 mg / kg) can all reach 100%; the induction success rate of letrozole at 150 mg / kg is 80%, at 200 mg / kg it is 83%, and at 250 mg / kg it is 100%.

[0028] (2) Lower dosage: Based on the precise window period mentioned above, we found that only 1.5 mg / kg of 17α-methyltestosterone or 200 mg / kg of 17α-methyltestosterone was needed to effectively induce reversal of hormone levels, both of which are lower than the traditionally reported dosages. A short-term, concentrated treatment strategy was adopted, with the feeding cycle controlled within 60 days. This ensured that hormone treatment was timely and decisive, minimizing unnecessary drug exposure time for the fish.

[0029] (3) Safer: Too high or too low doses of hormones can affect the sex conversion effect and the health of the fish. This method uses different concentrations of letrozole and methyltestosterone in the feed, and no significant impact was found on the survival rate of Atlantic salmon from 110 to 240 days of age. The health of the Atlantic salmon was not suppressed. Moreover, only the first generation needs to be treated with hormones. Subsequent generations can be bred using this pseudo-male fish without further hormone treatment. The hormone levels of the offspring are normal, there is no safety risk, and there is less pollution to the water.

[0030] This invention establishes a systematic method for creating pseudo-male Atlantic salmon by selecting 17α-methyltestosterone or letrozole as an inducer, determining the optimal dosage, and precisely controlling the treatment time. The effectiveness is verified through observation of gonadal tissue cells. Compared to existing technologies, these key technical points improve the efficiency and stability of sex conversion, ensuring the growth and health of the fish. This lays the foundation for the subsequent localized independent breeding of Atlantic salmon and the creation of all-female Atlantic salmon varieties, possessing significant scientific importance and remarkable application value. Attached Figure Description

[0031] Figure 1The images show Atlantic salmon gonad sections and H&E staining in Example 3 of this invention, where A is the control group (genetically female - physiological female); B is the 17α-methyltestosterone 1.5 mg / kg group (genetically female - physiological male); C is the 17α-methyltestosterone 3 mg / kg group (genetically female - physiological male); D is the 17α-methyltestosterone 4.5 mg / kg group (genetically female - physiological male); E is the control group (genetically female - physiological female); F is the letrozole 150 mg / kg group (genetically female - physiological male); G is the letrozole 200 mg / kg group (genetically female - physiological male); and H is the letrozole 250 mg / kg group (genetically female - physiological male).

[0032] Figure 2 This is a graph showing the effect of 17α-methyltestosterone and letrozole treatment on the growth performance of Atlantic salmon at 225 days post-hatching, as described in Example 3 of this invention. Figure A shows a comparison of body weight, and figure B shows a comparison of total length. Detailed Implementation

[0033] The following will describe the concept and technical effects of the present invention clearly and completely with reference to embodiments, so as to fully understand the purpose, features and effects of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are all within the scope of protection of the present invention.

[0034] Example 1: Hatching and rearing of eyed eggs of Atlantic salmon An isolation breeding farm and a visible egg hatching system were constructed. The isolation breeding farm included an isolation breeding area, disinfection channel, tool storage area, sick animal isolation area, and dead animal disposal area. The Atlantic salmon visible egg hatching system adopted a flow-through culture mode, including a high-level water storage tank and two hatching tanks, and was equipped with disinfection, oxygenation, air source heat pumps, and online water quality monitoring instruments. In August 2024, a batch of visible eggs with a daily temperature of 380-400 degrees Celsius were imported. All were all female diploids. Under customs supervision, the visible eggs were transferred to the hatching system for a 14-day quarantine period. During the incubation period, the water temperature was controlled at 6.0±0.5℃, the pH at 7.0-8.0, and the dissolved oxygen at >6.0mg / L. The visible eggs were regularly disinfected by soaking in a 200mg / L iodine solution and then rinsed with clean water. Dead visible eggs were removed from the hatching tanks daily using a pipette to ensure that the visible eggs were evenly distributed in the hatching tanks, and handling time and light interference were minimized. After the large-scale hatching of Atlantic salmon eyed eggs, eggshells and dead larvae are promptly removed daily. No feeding is required after hatching; nutrients are provided by the yolk sac. The rearing ponds are regularly cleaned and disinfected, with the water temperature increased to 10.0±0.5℃ at a rate not exceeding 0.5℃ per day. When the larvae change from bottom-gathering to surface swimming, they are fed 0.2mm artificial feed, gradually switching to 0.3-0.4mm and 0.5-0.8mm feed as they grow. Once the larvae become more active and begin swimming, a circular water flow is provided in the rearing pond to promote growth, and the water temperature is gradually adjusted to 12.0±0.5℃. Through artificial feeding, large-scale rearing is achieved at a water temperature of 12.0±0.5℃, with a survival rate exceeding 98%, providing healthy fry for research on Atlantic salmon factory-scale recirculating aquaculture and pseudo-male induction technology.

[0035] Example 2 Feed Preparation The base feed consisted of Aller Futura fry feed with a particle size of 0.2mm, 0.3-0.4mm, and 0.5-0.8mm, produced by Aller Aquatic Products (Qingdao) Co., Ltd., model number ALLER FUTURA EX GR. 0.50g of 17α-methyltestosterone was accurately weighed and diluted to 500ml with 95% ethanol, mixed thoroughly to completely dissolve the 17α-methyltestosterone, yielding a stock solution of 1mg / ml, which was stored at a low temperature and protected from light. 0.50g of letrozole was accurately weighed and diluted to 500ml with 95% ethanol, mixed thoroughly to completely dissolve the letrozole, yielding a stock solution of 1mg / ml, which was stored at a low temperature and protected from light. The feed was thoroughly mixed with the designed feeding concentration and air-dried overnight in a ventilated system to ensure complete ethanol evaporation. The prepared feed was stored at 4°C under light-protected conditions.

[0036] Example 3 Atlantic salmon induction In this embodiment, eyed eggs (chromosome type XX) of all-female diploid Atlantic salmon imported from Iceland were selected as the experimental subjects. The eyed eggs were incubated in a constructed eyed egg incubation system under the same conditions as in Example 1. After the yolk sac was consumed, 7000 healthy, vigorous Atlantic salmon fry of similar size were selected and divided into 7 groups for rearing in rectangular tanks with internal dimensions of 55×35×30 mm. During the feeding period, the fry were reared in a constant flow of slightly flowing water, with a daily water change volume of 1-2 times the total volume. The water temperature was maintained at 12℃-15℃, the pH at 6.5-8.0, and the dissolved oxygen at no less than 7.0 mg / L. Fluorescent lamps were used as the light source, with a surface light intensity of 300-500 lx, under dim lighting conditions (24:0 LD).

[0037] Seven groups of Atlantic salmon were fed different diets. The first group was fed a normal diet, the second to fourth groups were fed diets containing different concentrations (1.5 mg / kg, 3 mg / kg, 4.5 mg / kg) of methyltestosterone, and the fifth to seventh groups were fed diets containing different concentrations (150 mg / kg, 200 mg / kg, 250 mg / kg) of letrozole. The fish were fed to satiety and evenly throughout the 24 hours. Initially, they were fed every four hours, gradually increasing to three times a day. The daily feed amount was about 5% of the fish's body weight. The feeding period was 60 days (about 600 degree-days), after which they were fed a normal diet.

[0038] When the fry first start feeding, they are fed with artificial feed of 0.2mm size. As the fry grow, they are gradually fed with feed of 0.3-0.4mm and 0.5-0.8mm size.

[0039] When the experimental Atlantic salmon were raised to 240 days after hatching, 10 Atlantic salmon weighing 10-15 grams were randomly selected from each of the seven groups. The tail fin rays were cut off and placed in anhydrous alcohol, and the gonadal tissue was taken and fixed in paraformaldehyde.

[0040] 1. Genetic sex determination: Since this batch of eggs were all female diploid eggs imported from Iceland, their genetic sex is XX. After extracting the genome from the tail fin of Atlantic salmon, sex-related DNA markers were used to determine the genetic sex of each fish for verification. The specific method is as follows: Genomic DNA was extracted from the tail fin of Atlantic salmon using the TIANGEN Marine Animal Tissue Genomic DNA Extraction Kit.

[0041] 1) Place 30 mg of fins into a centrifuge tube containing 200 µL of GA buffer and vortex for 15 seconds; 2) Add 20µL Proteinase K solution, vortex to mix, place at 56℃, shake to mix once every 20 min, and let the tissue completely dissolve in 1 h. Briefly centrifuge to remove water droplets from the inner wall of the tube cap. 3) Add 200µL buffer GB, mix thoroughly by inverting, place at 70℃ for 10 min. After the solution becomes clear, add 200µL anhydrous ethanol, mix thoroughly by inverting, add the resulting solution and flocculent precipitate to an adsorption column CB3, centrifuge at 12,000 rpm for 30 sec, discard the waste liquid, and put the adsorption column CB3 back into the collection tube. 4) Add 500 µL of buffer GD to the adsorption column CB3, centrifuge at 12,000 rpm for 30 sec, discard the waste liquid, and put the adsorption column CB3 into the collection tube; 5) Add 600 µL of washing buffer PW to the adsorption column CB3, centrifuge at 12,000 rpm for 30 sec, discard the waste liquid, put the adsorption column CB3 into the collection tube, and repeat the operation once. 6) Place the adsorption column CB3 back into the collection tube, centrifuge at 12,000 rpm for 2 min, and discard the waste liquid. Place the adsorption column CB3 at room temperature for several minutes to thoroughly dry any residual washing liquid in the adsorption material.

[0042] 7) After the rinsing buffer has dried, transfer the adsorption column CB3 into a clean centrifuge tube, add 50 µL of elution buffer TE to the middle of the adsorption membrane, incubate at room temperature for 5 min, centrifuge at 12,000 rpm for 2 min, collect the solution into the centrifuge tube, detect the DNA concentration, and store in a 4℃ refrigerator for later use.

[0043] Sex determination method: Identification is performed using polymerase chain reaction (PCR) experiment. The PCR system is shown in Table 1; Table 1 PCR system ; Primer sequences for PCR amplification of the sdY gene: Primer 1F: CTCTCCTGGAGTCTGAAA (SEQ ID NO. 1); Primer 2R: GGAGGAGAGGTGATTAGG (SEQ ID NO. 2).

[0044] Genetic sex determination results showed that all experimental samples only amplified the internal reference gene band, and did not show the Y chromosome-specific sdY gene band. Therefore, it was confirmed that the genetic sex of this batch of samples was entirely XX (female).

[0045] 2. Physiological sex identification: In this embodiment, the physiological sex of fish gonadal tissue is accurately identified by microscopic morphological observation of tissue paraffin sections and hematoxylin-eosin (H&E) staining techniques.

[0046] 2.1 Preparation of tissue paraffin blocks 1) Tissue fixation: The obtained gonadal tissue samples were immediately and completely immersed in a sufficient amount of 4% (w / v) paraformaldehyde phosphate buffer (pH 7.4) and fixed at room temperature of 20-25°C for 24 to 48 hours to ensure that the tissue cell structure was preserved intact.

[0047] 2) Tissue rinsing: After fixation, place the tissue in the tissue sample box and rinse continuously for 5 minutes with running tap water or phosphate buffered saline (PBS) at a low flow rate. This is to thoroughly remove any residual fixative and avoid interference with subsequent staining steps.

[0048] 3) Gradient Dehydration of Tissue: The rinsed tissue is sequentially immersed in ethanol solutions of progressively increasing concentrations for dehydration. The specific process is as follows: 60% ethanol for 90 minutes → 75% ethanol for 90 minutes → 90% ethanol for 90 minutes → 95% ethanol for 60 minutes → 100% anhydrous ethanol (I) for 60 minutes → 100% anhydrous ethanol (II) for 60 minutes. Subsequently, the tissue is transferred to xylene or its environmentally friendly alternative (such as limonene) for clearing treatment to displace the ethanol in the tissue and facilitate paraffin impregnation. The process is as follows: Xylene (I) for 10 minutes → Xylene (II) for 20 minutes → Xylene (III) for 10 minutes, until the tissue becomes transparent.

[0049] 4) Tissue impregnation and embedding: The transparentized tissue is transferred into molten paraffin (melting point 56-58℃) and impregnated three times, each time for no less than 60 minutes, to ensure that the paraffin fully penetrates into the tissue. Subsequently, the tissue is embedded in a pre-made paraffin block using a paraffin embedding machine, left to stand at room temperature until the paraffin has completely solidified, and then removed for use.

[0050] 2.2 Tissue Sections 1) Set the water temperature of the sheet spreader to 42ºC, carefully trim the wax block with a scalpel, and set aside.

[0051] 2) Fix the wax block onto the paraffin microtome and slowly trim it (20μm) with the microtome. After cutting into the tissue, proceed with the formal sectioning with a thickness of 3-4μm.

[0052] 3) Place the tissue sections in the water tank of the slide spreader to flatten them, retrieve them with a glass slide, observe the tissue morphology under a microscope, and select tissue samples with intact morphology.

[0053] 4) Place the slides containing tissue sections in a 65ºC oven for 2 hours and store at room temperature for later use.

[0054] 2.3 Dewaxing and hematoxylin-eosin staining 1) Place the gonadal tissue sections in a 65ºC oven for 30 minutes, and then perform tissue dewaxing by placing the sections in xylene (Ⅰ) for 10 minutes and xylene (Ⅱ) for 10 minutes.

[0055] 2) To remove the dewaxing agent, the slide was placed in anhydrous ethanol (Ⅰ) for 5 min, anhydrous ethanol (Ⅱ) for 5 min, and 75% ethanol for 5 min in sequence. Then, it was slowly rinsed with distilled water for 3 min to remove the residual ethanol from the slide.

[0056] 3) Immerse the cell nuclei in a dye bath containing hematoxylin for 3 minutes to stain them, then rinse slowly with distilled water for 3 minutes to remove any remaining hematoxylin from the slide.

[0057] 4) Place in the differentiation solution (0.5% hydrochloric acid, tap water and concentrated ammonia water volume ratio 1:99) and differentiate for 6 seconds.

[0058] 5) After rinsing with tap water for 3 minutes, immerse it in the blueing solution (0.5% ammonia water, tap water to concentrated ammonia water volume ratio 1:99) for 2 seconds.

[0059] 6) Rinse slowly with tap water for 2 minutes, then place in 95% anhydrous ethanol for 5 minutes to dehydrate, followed by eosin staining for 1 minute.

[0060] 7) Dehydrate with 95% ethanol for 4 min, then dehydrate with anhydrous ethanol for 4 min.

[0061] 8) Clear the paraffin sections with xylene (Ⅰ) for 5 minutes and xylene (Ⅱ) for 5 minutes. Place a drop of mounting adhesive in the center of the paraffin section and seal it. Place it in a fume hood for 24 hours, then observe and photograph it under a microscope.

[0062] 3. Experimental Results The results of Atlantic salmon gonad sections and H&E staining in the control and experimental groups are as follows: Figure 1 As shown.

[0063] In the control group, 10 animals were examined, and their physiological sex was determined by paraffin sectioning and HE staining. The results showed that all of them were female (e.g., ...). Figure 1 (As shown in A and E).

[0064] Ten fish from each of the 17α-methyltestosterone-fed groups were examined. Their physiological sex was determined by paraffin sectioning and HE staining. The results showed that all groups were male (e.g., ...). Figure 1 (B, C, and D) Ten fish from each letrozole feeding group were tested, and their physiological sex was determined by paraffin sectioning and HE staining. In the letrozole 150 mg / kg group, 8 fish were male (e.g., ...). Figure 1 In the F1 group, 2 genetic lines indicated female sex. In the letrozole 200mg / kg group, 9 physiological sexes were male (e.g., F1). Figure 1 In the G group, one genetic line indicates female sex. In the letrozole 250mg / kg group, all 10 physiological sexes were male (e.g., G). Figure 1 (H in the text).

[0065] It is evident that the sex reversal efficiency was 100% in the 17α-methyltestosterone-fed groups and 80%–100% in the letrozole-fed groups. Therefore, the optimal treatment groups are the methyltestosterone 1.5 mg / kg-fed group and the letrozole 250 mg / kg-fed group.

[0066] Furthermore, there was no significant difference in survival rate between the 17α-methyltestosterone and letrozole treatment groups and the normal control group. Growth experiments on Atlantic salmon showed that after treatment with different concentrations of methyltestosterone and letrozole, there were no significant differences in average body weight and average total length at 225 days post-hatching between the experimental groups and the control group. P >0.05, see details Figure 2 This indicates that, within the concentration and treatment period designed in this study, neither hormone showed a significant promoting or inhibiting effect on Atlantic salmon growth, and their safety profile was high.

[0067] As further verification, when Atlantic salmon were fed diets containing different concentrations of methyltestosterone (1.5 mg / kg, 3 mg / kg, 4.5 mg / kg) or different concentrations of letrozole (150 mg / kg, 200 mg / kg, 250 mg / kg) for more than 60 days (approximately 600 degree-days) after they were first fed, they could be induced to transform into pseudo-males. Therefore, the 60 days (approximately 600 degree-days) specified in this invention is the minimum lower limit and also the most economical technical solution.

Claims

1. A method for creating pseudo-male Atlantic salmon, characterized in that, The method includes the following steps: feeding 17α-methyltestosterone or letrozole daily for 60 days or longer when the genetically female Atlantic salmon fry begin to feed, thereby obtaining XX-type pseudo-male fish.

2. The method according to claim 1, characterized in that, 17α-methyltestosterone or letrozole is mixed into the feed and fed to the animal. The content of 17α-methyltestosterone in the feed is 1.5~4.5 mg / kg.

3. The method according to claim 1, characterized in that, 17α-methyltestosterone or letrozole is mixed into the feed and fed to the animal, wherein the letrozole content in the feed is 150~250 mg / kg.

4. The method according to claim 1, characterized in that, The female Atlantic salmon were fed 17α-methyltestosterone or letrozole starting at 800 degrees day when they were first introduced to food.

5. The method according to claim 4, characterized in that, The feeding period is 60 days.

6. The method according to claim 4, characterized in that, The feeding method is full feeding.

7. A feed for creating pseudo-male Atlantic salmon, characterized in that, The feed consists of 17α-methyltestosterone or letrozole and Atlantic salmon base feed.

8. The method for preparing the feed according to claim 7, characterized in that, The method includes the following steps: dissolving 17α-methyltestosterone powder or letrozole in 95% ethanol by volume, then spraying it evenly onto the Atlantic salmon base feed, mixing it evenly, so that each kg of Atlantic salmon base feed is coated with 1.5~4.5 mg of 17α-methyltestosterone or 150~250 mg of letrozole.

9. The use of the feed according to claim 7 in the preparation of Atlantic salmon pseudo-males.

10. The application according to claim 9, characterized in that, The method of application includes the following steps: Atlantic salmon eyed eggs are hatched at a water temperature of 6.0±0.5℃, a pH of 7.0-8.0, and dissolved oxygen >6.0mg / L. The water temperature is increased to 10.0±0.5℃ at a rate not exceeding 0.5℃ per day. When the larvae change from gathering at the bottom to floating and swimming, they are fed with artificial feed containing 1.5~4.5mg of 17α-methyltestosterone or 150~250mg of letrozole per kilogram and a particle size of 0.2mm. As the larvae grow, the feed is gradually changed to 0.3-0.4mm and 0.5-0.8mm size feed. After the larvae become more active and start swimming, a circular water flow is provided in the rearing pond to promote their growth, and the water temperature is gradually adjusted to 13.0±0.5℃.

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