Method for efficiently inducing diploid rainbow trout pseudo-males and application thereof

Abstract

The present application belongs to the technical field of aquatic breeding, and relates to a method for efficiently inducing diploid rainbow trout pseudo-male fish and application thereof. The steps are as follows: from the first day when the young fish completely float to the opening after breaking the membrane, artificial feeding of inducing feed is carried out, and the accumulated temperature is 500 ± 20 ℃·d after fertilization; the inducing feed is feed added with methyltestosterone and letrozole, the mass ratio of methyltestosterone to the feed is 1-4 mg / kg, and the mass ratio of letrozole to the feed is 70-130 mg / kg; the feeding treatment time is 700 ± 30 ℃·d, and after the treatment is completed, the conventional feed is used for feeding, and the feeding is carried out 4 times a day. The present application relies on the physiological synergistic effect of the two agents, greatly reduces the induction failure individuals, intersex individuals and gonadal deformity individuals on the basis of controlling the dosage of methyltestosterone and reducing the toxic side effects of the drugs, and finally realizes that the effective induction rate of diploid rainbow trout pseudo-male fish tends to be close to 100%.

Description

Technical Field

[0001] This invention belongs to the field of aquatic breeding technology, specifically relating to a method for efficiently inducing diploid rainbow trout pseudo-males and its application. Background Technology

[0002] Sex-controlled breeding is a core technology in the field of aquaculture genetics and breeding. It utilizes the sex dimorphism of aquatic animals to obtain monosex aquaculture populations with better growth performance through sex regulation technology, thereby significantly improving aquaculture efficiency.

[0003] rainbow trout ( Oncorhynchus mykiss Rainbow trout are a major cold-water salmonid fish species in my country and an important cold-water aquaculture species worldwide. In the natural sex inheritance system of rainbow trout, sex is determined by sex chromosomes, with females having the genotype XX and males having the genotype XY. Under natural conditions, females have significant growth advantages: their growth rate is 15%-30% faster than males, and they reach sexual maturity later, avoiding problems such as growth stagnation and decreased meat quality that males experience due to sexual maturity. This results in shorter farming cycles and better-sized marketable fish. Therefore, all-female farming has become a key technological path for improving the efficiency of the rainbow trout industry.

[0004] In large-scale all-female rainbow trout farming, the most crucial and core technical aspect is obtaining pseudo-males with the genotype XX. These fish are genetically female with the genotype XX, but their physiological phenotype and reproductive function are completely transformed into male characteristics, enabling them to mate and release sperm with female fish. Artificial insemination is then performed between the successfully induced XX-type pseudo-males and naturally selected normal XX-type female rainbow trout. Because the pseudo-males have the genotype XX, they can only produce male gametes carrying the X chromosome. Similarly, the female parent fish only produce female gametes carrying the X chromosome. The offspring fry bred from these male and female gametes all have the same genotype XX, completely eliminating the birth of male individuals from the genetic source. This allows for the long-term, stable, and large-scale cultivation of a highly pure all-female rainbow trout farming population, a necessary prerequisite and key step for the commercial breeding of all-female rainbow trout fry and the widespread application of all-female farming models.

[0005] Currently, the mainstream methods for achieving sex reversal in bony fish include five main categories: interspecific hybridization, artificial induction of gynogenesis, environmental condition regulation, gene editing, and exogenous hormone induction. Among these, interspecific hybridization and artificial induction of gynogenesis not only have stringent requirements for aquaculture facilities, experimental conditions, and the professional skills of operators, but also involve complex implementation processes, resulting in low actual induction success rates and relatively high initial R&D and seedling cultivation costs. Furthermore, pseudo-male fish bred through these methods exhibit incomplete gonadal development, weak reproductive function, and poor stability of reproductive traits, making them unsuitable for the large-scale commercial production model of modern aquaculture seedlings. Since fish sex differentiation is the result of the interaction between endogenous genetic factors and the external environment, the internal differentiation regulation mechanism is highly complex and adaptable to changes in the external environment. Therefore, simply relying on altering external environmental conditions (such as aquaculture water temperature, pH, and photoperiod) to regulate sex is difficult to precisely guide the direction of sex development. At the same time, the effectiveness of these induction methods is highly susceptible to fluctuations in the aquaculture environment, seasonal changes, and water quality conditions, ultimately resulting in poor stability of the induction results, a narrow range of applicable fish species, and low universality. Gene editing technologies, such as CRISPR / Cas9, have not yet established a mature and stable application system in cold-water salmonids like rainbow trout. Research on the discovery and precise editing of related sex-determining genes remains in the laboratory exploration stage. Furthermore, gene-edited aquatic products are subject to strict national food safety laws and face significant ethical controversies. Limited by these multiple practical factors, technology transfer is unlikely in the short term, and commercial aquaculture promotion will require a considerable amount of time. Exogenous hormone induction, with its unique advantages of simple operation, low implementation threshold, and low aquaculture application cost, has become the most widely accepted sex regulation method in the industry.

[0006] Exogenous hormone induction methods primarily rely on the sex differentiation window periods of different fish species. By feeding them formulated diets supplemented with male hormones such as methyltestosterone, the gonadal differentiation process is artificially intervened, causing genetically female individuals to undergo sex reversal and develop into physiologically male individuals with normal male reproductive functions. This technique is relatively easy to implement and is currently the most commonly used induction technique for artificially inducing pseudo-males and conducting all-female breeding in salmonids.

[0007] However, the traditional single-hormone induction method still suffers from numerous unavoidable technical drawbacks. For example, the dosage, initiation time, and duration of feeding for traditional single-methyltestosterone induction produce varying induction effects in different fish species. Single-methyltestosterone induction lacks an optimal dosage; low doses result in weak hormone effects, insufficient sex conversion induction rates, and gonadal malformations; high doses easily induce intersex individuals, significantly increase gonadal malformation rates, and excessive exogenous androgens can be converted into estrogens. It also leads to delayed sexual maturity in pseudo-males and low natural sperm production rates. Rainbow trout pseudo-males, and even normal males, obtained through traditional single-exogenous hormone induction typically require 2-3 years of rearing before reaching sexual maturity and releasing mature sperm, resulting in a prolonged breeding cycle for all-female fry and increased breeding costs. Furthermore, traditional high-concentration single-hormone induction disrupts the endocrine growth axis of rainbow trout juveniles, leading to generally inhibited body length and weight growth in induced pseudo-males. These problems severely restrict the standardization, efficiency, and industrialization of this technology. Currently, there is no suitable and standardized pseudo-male induction program for rainbow trout seedlings. Existing programs are complex to operate, costly, and carry a high risk of hormone residue.

[0008] While there are existing reports on sex control in yellow catfish (Pelteobagrus fulvidraco) using "androgens combined with aromatase inhibitors," such as Chinese patent CN113661948A which discloses a method for obtaining all-female homozygous yellow catfish clones through autofertilization, the method involves feeding XX-genotype yellow catfish juveniles with a high-dose diet containing a combination of 300-600 mg / kg letrozole and 5-10 mg / kg methyltestosterone for 60 consecutive days after they begin feeding, resulting in hermaphroditic XX individuals with simultaneously mature sperm and ovaries. However, since the purpose of this technology is to enable the fertilization of sperm and egg within a single fish, its core objective is the special intersex state of "sperm-egg coexistence," thus requiring the use of high-dose drug combinations. Yellow catfish, belonging to the order Siluriformes, are warm-water fish with a high tolerance threshold to exogenous steroid hormones. Therefore, high-dose combinations of drugs are required to disrupt their endocrine balance. Rainbow trout, on the other hand, belong to the order Salmoniformes and are typical cold-water fish with stringent aquaculture requirements. Their chromosome composition, evolutionary type, critical periods for sex differentiation, reproductive period, sexual maturity, and living habits and environments differ significantly from those of yellow catfish. Their endocrine system is extremely sensitive to external hormonal intervention. Based on these interspecies physiological differences, directly applying the induction start time, feeding duration, and high-dose combination of drugs used for yellow catfish would cause severe gonadal malformations and fatal toxic side effects in cold-water rainbow trout juveniles.

[0009] Unlike the existing techniques for inducing hermaphroditism in yellow catfish to achieve "sperm-egg coexistence," the goal of inducing pseudo-males in rainbow trout is to obtain completely sex-reversed pseudo-males with healthy reproductive function for subsequent all-female triploid breeding. Therefore, in the induction of pseudo-males in diploid rainbow trout, the presence of sperm-egg coexistence indicates induction failure, necessitating minimizing or even eliminating its occurrence. Based on this, this invention addresses the sex differentiation pathway, cold-water physiological characteristics, breeding season, sexual maturity time, and stringent aquaculture conditions of diploid rainbow trout, exploring a pseudo-male induction scheme with appropriate dosage, completely avoiding intersex individuals, and reducing exogenous hormone toxicity—a previously unsolved problem in the industry. Summary of the Invention

[0010] The purpose of this invention is to solve the aforementioned problems in the prior art and to propose a highly efficient method for inducing pseudo-males in diploid rainbow trout and its application. This invention addresses the dosage imbalance problem in hormone induction of rainbow trout using a single hormone. It employs a synergistic induction strategy using low-dose methyltestosterone combined with letrozole, effectively solving the technical drawbacks of single-dose hormones, such as insufficient effect of low doses, failure of some individuals to achieve sex reversal, low induction success rate, and high rate of gonadal malformation. Conversely, high doses can easily induce gonadal tissue development disorders, a high incidence of intersex individuals, and a significant increase in the rate of gonadal malformation. Relying on the physiological synergistic effect of the two agents, while controlling the dosage of methyltestosterone and reducing drug toxicity, this invention significantly reduces the number of individuals failing to induce sex, intersex individuals, and individuals with gonadal malformation, ultimately achieving an effective induction rate of nearly 100% for diploid rainbow trout pseudo-males.

[0011] The technical solution of this invention is: This invention provides a method for efficiently inducing pseudo-males in diploid rainbow trout, comprising the following steps: (1) Starting from the first day after the juvenile fish fully float to the surface and open their mouths after breaking through the membrane, artificial feeding of induction feed is carried out, with an accumulated temperature of 500 ± 20 ℃·d after fertilization; the feeding time is 8:00~8:20, 10:00~10:20, 12:00~12:20, 14:00~14:20, and 16:00~16:20, for a total of 5 times; The inducing feed is a feed supplemented with methyltestosterone and letrozole, wherein the mass ratio of methyltestosterone to feed is 1-4 mg / kg, and the mass ratio of letrozole to feed is 70-130 mg / kg. (2) The feeding treatment time is 700 ± 30 ℃·d. After the treatment is completed, the feed is switched to regular feed. The feeding time is 8:00~8:10, 11:00~11:10, 13:00~13:10, and 16:00~16:10, for a total of 4 times.

[0012] Furthermore, the induction method is carried out in a flowing water system at 15~18℃, with dissolved oxygen content not less than 7.5 mg / L and ammonia nitrogen content <0.02 mg / L.

[0013] Furthermore, the method for preparing the inducing feed includes the following steps: (1) Weigh 20-25 mg of methyltestosterone and place it in a brown wide-mouth bottle. Add 1320 ml of 95% ethanol solution and stir until completely dissolved under the protection of light to obtain methyltestosterone hormone ethanol stock solution. Take 180 mL of methyltestosterone hormone ethanol stock solution and add 95% ethanol to make up to 680 mL to obtain methyltestosterone hormone ethanol solution. Weigh 70-130 mg of letrozole into a brown wide-mouth bottle, add 500 ml of 95% ethanol solution, and stir until completely dissolved under light-protected conditions to obtain letrozole ethanol mother liquor. (2) Weigh 1 kg of base feed and spray the prepared methyltestosterone hormone ethanol solution and letrozole ethanol mother liquor evenly onto the surface of the feed. (3) Spread the sprayed feed evenly, with a pile thickness not exceeding 1 cm, and dry it at 40℃ for 3-5 hours. Turn it over once every 1 hour of drying until the ethanol solvent is completely evaporated. Seal the dried feed and store it at 4℃.

[0014] Furthermore, the mass ratio of methyltestosterone to feed is 3 mg / kg, and the mass ratio of letrozole to feed is 100 mg / kg.

[0015] Furthermore, in step (1), the feeding of inducing feed should follow the principle of "small amounts and multiple times". Observe the feeding situation of the fry each time they are fed, and stop feeding when the fish's belly swells slightly.

[0016] Furthermore, the feeding principle is as follows: the amount of inducing feed for 1,000 fish on the first day is 8-10 g; the total accumulated temperature of 700±30℃·d is used as the complete drug administration and feeding cycle, and the average daily feeding amount is increased weekly to make a gradient feeding: 10-12 g of inducing feed on the first week, 15-18 g of inducing feed on the second week, 20-22 g of inducing feed on the third week, and 26-30 g of inducing feed on the fourth week, and so on.

[0017] Furthermore, the rainbow trout eggs used are eyed eggs of fully female diploid rainbow trout imported from Denmark.

[0018] The present invention also provides the application of the method for efficiently inducing diploid rainbow trout pseudo-males as described in any of the above-mentioned methods in the all-female breeding of rainbow trout.

[0019] The present invention also provides the application of the method for efficiently inducing diploid rainbow trout pseudo-males as described in any of the above-mentioned methods in promoting the growth of diploid rainbow trout juveniles.

[0020] Furthermore, statistical analysis of growth indicators of one-year-old fish revealed that the method can increase the weight and length of rainbow trout.

[0021] The present invention also provides the application of the method for efficiently inducing diploid rainbow trout pseudo-males as described above in improving the sperm motility of rainbow trout. The method can shorten the sexual maturity cycle of rainbow trout pseudo-males and make their sperm motility reach the level of natural three-year-old male parent fish.

[0022] The beneficial effects of this invention are: (1) Through the exploration of the ratio, this invention proposes a scheme of feeding low dose methyltestosterone and letrozole together. By using them together, the interference of methyltestosterone alone on the growth of diploid rainbow trout juveniles is reduced. Furthermore, by using the combined effect of methyltestosterone and letrozole to optimize the induction effect, the induction rate of diploid rainbow trout pseudo-males is finally close to 100%, the rate of gonadal malformation is significantly reduced, and the sperm activity is not significantly different from that of three-year-old natural females.

[0023] (2) The inducing feed provided by this invention overcomes the inherent technical shortcomings of existing methods for inducing diploid rainbow trout pseudomales with single methyltestosterone, optimizes the sex differentiation system, and significantly improves the stability and quality of diploid rainbow trout pseudomale breeding. This invention solves the problem of lacking an optimal dosage of single methyltestosterone, improves the problems of weak effects of low-dose hormones, insufficient sex induction conversion rate, and gonadal malformation; at the same time, it avoids the toxic side effects of high-dose hormones, effectively reduces the probability of intersex individuals, significantly reduces the proportion of gonadal malformation, and avoids the risk of high hormone residues.

[0024] (3) This invention provides a standardized pseudo-male induction and breeding process adapted to the large-scale treatment of diploid rainbow trout seedlings. Compared with gene editing sex modification technology represented by CRISPR / Cas9, the method of this invention is simple to operate, cost controllable, and has low risk of hormone residue, which can meet the needs of large-scale industrial application.

[0025] (4) The present invention also provides a specific scheme for evaluating the induction effect of diploid rainbow trout pseudo-male fish. Attached Figure Description

[0026] Figure 1 A schematic diagram illustrating the principle of the method for efficiently inducing diploid rainbow trout pseudo-males provided by this invention.

[0027] Figure 2 Statistical analysis of body length indicators at one year of age for all treatment groups and blank control group.

[0028] Figure 3The body weight indicators of all treatment groups and the blank control group at one year of age were statistically analyzed.

[0029] Figure 4 The images show the slice results of the five treatment groups and the blank control group.

[0030] Figure 5 The study compared the gonadal appearance of the blank control group, the LM3 group, and the MT3 group, and the proportion of males with gonadal malformation who were successfully induced in the five treatment groups.

[0031] Figure 6 The figure shows the comparison of sperm activity between the LM3 group at 16 months of age and three-year-old natural males; where A is the sperm curve velocity (VCL) index, B is the linear velocity (VSL) index, C is the average velocity (VAP) index, D is the linear index (Lin) index, and E is the average amplitude of head lateral movement (ALH) index. Detailed Implementation

[0032] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0033] To further understand the present invention, it will be further described in conjunction with the accompanying drawings and embodiments.

[0034] The experimental material used in this invention was a wholly female diploid rainbow trout purchased from Denmark. Oncorhynchus mykiss Eyed eggs were obtained from this population using known techniques (such as inducing gynogenesis or using artificially created pseudo-males for mating) to ensure that their initial genetic sex was XX, thus reducing interference from natural male individuals in the induction experiment.

[0035] The inducing drugs used in this invention are methyltestosterone MT (CAS: 58-18-4) and letrozole LZ (CAS: 112809-51-5) raw materials.

[0036] The principle of the method for efficiently inducing diploid rainbow trout pseudo-males provided by this invention is as follows: Figure 1 As shown: The differentiation of rainbow trout primordial germ cells involves two distinct regulatory pathways under normal physiological conditions and under conditions of exogenous hormone interference. Under normal physiological conditions, testosterone (T) in the body is converted into 11-KT under the influence of cyp11c1 and hsd11b2 expression products, differentiating towards testicular development. Simultaneously, it is converted into E2 under the influence of aromatase, a product of cyp19a1a expression, differentiating towards ovarian development. Under conditions of exogenous hormone interference, conventional single methyltestosterone induction causes compensatory upregulation of cyp19a1a expression in fish, resulting in the large-scale conversion of exogenous androgens into estradiol (E2), leading to induction failure and the occurrence of sperm-egg coexistence (intersex individuals) and gonadal malformations. This invention uses 1-4 mg / kg methyltestosterone and 70-130 mg / kg letrozole (an aromatase inhibitor) for synergistic induction, which can effectively inhibit aromatase activity, cut off the physiological pathway of methyltestosterone and T to E2 conversion, so as to efficiently accumulate androgens, continuously activate the testis differentiation pathway, and finally achieve highly efficient pseudo-male induction with an induction rate of >90%. At the same time, it significantly reduces the rate of gonadal malformation, effectively reduces the probability of intersex individuals, and the sperm activity is no different from that of normal males.

[0037] Example 1 This embodiment provides a method for preparing inducing feed for diploid rainbow trout pseudo-male fish.

[0038] The base feed is rainbow trout fry starter feed with diameter specifications of 0.2 mm and 0.5 mm, and the feed is produced by Aile Aquatic Products (Qingdao) Co., Ltd.

[0039] The preparation method of induction feed includes the following steps: (1) Weigh 22 mg of methyltestosterone and place it in a brown wide-mouth bottle. Add 1320 ml of 95% ethanol solution to the bottle and stir with a magnetic stirrer under light protection until the drug is completely dissolved to obtain a uniform and transparent methyltestosterone hormone ethanol mother liquor. Weigh 100 mg of letrozole into a brown wide-mouth bottle, add 500 ml of 95% ethanol solution, and stir with a magnetic stirrer under light-protected conditions until the drug is completely dissolved to obtain a uniform and transparent letrozole ethanol stock solution. (2) Take 180 mL of methyltestosterone hormone ethanol stock solution, add 95% ethanol to make up to 680 mL, and obtain methyltestosterone hormone ethanol solution; (3) The letrozole ethanol mother liquor prepared in step (1) and the methyltestosterone hormone ethanol solution prepared in step (2) are evenly sprayed onto the surface of a 1 kg base feed. Multiple even spraying and timely stirring are used to ensure that the drug components carried by each feed particle reach molecular-level uniformity. (4) Spread the sprayed feed evenly on a stainless steel tray, with the feed pile thickness not exceeding 1 cm. Place it in a forced-air drying oven and dry it continuously at 40°C for 4 hours. Turn the feed over once every 1 hour of drying, spread it out and continue drying to ensure that the ethanol solvent evaporates completely (eliminating solvent toxicity), avoid the degradation of feed nutrients caused by high temperature, and retain nutrients to the maximum extent. (5) After drying, the feed is sealed and placed in a refrigerator at 4°C to prevent fat oxidation and hormone degradation, and to ensure that the price of the feed remains stable throughout the feeding cycle with an accumulated temperature of 700 ± 30 °C·d.

[0040] Example 2 The method for preparing the diploid rainbow trout pseudo-male induction feed provided in this embodiment includes the following steps: (1) Weigh 20 mg of methyltestosterone and place it in a brown wide-mouth bottle. Add 1320 ml of 95% ethanol solution to it and stir with a magnetic stirrer under light protection until the drug is completely dissolved to obtain a uniform and transparent methyltestosterone hormone ethanol mother liquor. Weigh 70 mg of letrozole into a brown wide-mouth bottle, add 500 ml of 95% ethanol solution, and stir with a magnetic stirrer under light-protected conditions until the drug is completely dissolved to obtain a uniform and transparent letrozole ethanol stock solution. (2) Take 180 mL of methyltestosterone hormone ethanol stock solution, add 95% ethanol to make up to 680 mL, and obtain methyltestosterone hormone ethanol solution; The remaining steps and parameters are the same as in Example 1.

[0041] Example 3 The method for preparing the diploid rainbow trout pseudo-male induction feed provided in this embodiment includes the following steps: (1) Weigh 25 mg of methyltestosterone and place it in a brown wide-mouth bottle. Add 1320 ml of 95% ethanol solution to the bottle and stir with a magnetic stirrer under light protection until the drug is completely dissolved to obtain a uniform and transparent methyltestosterone hormone ethanol mother liquor. Weigh 130 mg of letrozole into a brown wide-mouth bottle, add 500 ml of 95% ethanol solution, and stir with a magnetic stirrer under light-protected conditions until the drug is completely dissolved to obtain a uniform and transparent letrozole ethanol stock solution. (2) Take 180 mL of methyltestosterone hormone ethanol stock solution, add 95% ethanol to make up to 680 mL, and obtain methyltestosterone hormone ethanol solution; The remaining steps and parameters are the same as in Example 1.

[0042] Example 4 The preparation method of diploid rainbow trout pseudo-male induction feed provided in this embodiment uses 20 mg of methyltestosterone and 130 mg of letrozole, and the remaining steps and parameters are the same as in Example 1.

[0043] Example 5 The preparation method of diploid rainbow trout pseudo-male induction feed provided in this embodiment uses 25 mg of methyltestosterone and 70 mg of letrozole, and the remaining steps and parameters are the same as in Example 1.

[0044] Example 6 This embodiment provides a method for inducing pseudo-male diploid rainbow trout. Eyed eggs of fully female diploid rainbow trout imported from Denmark are used; induction begins after all eggs have ruptured and floated to the surface. The pseudo-male induction feed prepared in Example 1 is used for induction feeding.

[0045] Delineation of the induction feeding period for hormone-treated feed: In this embodiment, the starting point for drug administration (i.e., the starting point of the induction feeding period) is when the rainbow trout eggs have completely floated to the surface after hatching, with an accumulated temperature of 500 ± 20 ℃・d post-fertilization. At this time, the yolk sac of the juvenile fish has been completely absorbed, exogenous feeding behavior has been established, and the intestines have complete hormone absorption capacity. This time point is not only a critical period for sex differentiation in rainbow trout juveniles, precisely activating the male-related pathways in the juvenile fish; it also avoids endogenous nutritional interference caused by yolk sac residue, making the drug intake highly linearly correlated with the feed intake, thus improving the induction rate.

[0046] Aquaculture conditions control: The induction experiment was conducted in a flowing water system at 15-18℃, with dissolved oxygen maintained above 7.5 mg / L and ammonia nitrogen content <0.02 mg / L to ensure that the fish were in the optimal physiological and metabolic state.

[0047] The induction method provided in this embodiment includes the following steps:

[0048] (1) Feeding strategy: From the first day the juvenile fish fully surfaced and began feeding, artificial feeding with hormone-treated feed was initiated. Following the principle of "small amounts, multiple times," the feeding principle for 1000 fish was as follows: 8 g of inducing feed was given on the first day. The feeding behavior of the fry was observed each time they were fed; feeding was stopped when the fish's bellies slightly swelled and they stopped swarming to feed. A complete drug administration and feeding cycle was defined as 700±30℃·d total accumulated temperature. The daily average feeding amount was increased weekly, with an average of 10 g of inducing feed on the first week, 15 g on the second week, 20 g on the third week, and 26 g on the fourth week, increasing sequentially.

[0049] Feeding times are 8:00-8:20, 10:00-10:20, 12:00-12:20, 14:00-14:20, and 16:00-16:20, for a total of 5 times, to ensure that the drug maintains an effective physiological concentration in the blood circulation of the juvenile fish.

[0050] Use a siphon to clean up any uneaten food after each feeding to prevent water pollution.

[0051] (2) Control of continuous feeding time: The feeding treatment was strictly continuous at 700 ± 30 ℃·d. The calculation method was: number of treatment days × average daily water temperature.

[0052] After treatment, remove the medicated feed and switch to regular nutritional feed for normal feeding. The feeding times are changed to 8:00-8:10, 11:00-11:10, 13:00-13:10, and 16:00-16:10, for a total of 4 times.

[0053] Experimental Example 1

[0054] (a) Induction effect experiment

[0055] 1. Experimental group: Eyed eggs of all-female diploid rainbow trout imported from Denmark were induced after all eggs hatched and floated to the surface. A total of 6,000 eyed eggs were randomly divided into 6 groups of 1,000 eggs each after hatching. There were 4 groups with single hormone treatment, 1 group with combined treatment, and 1 group with blank control.

[0056] Four single hormone treatment groups: Methyltestosterone was added to the feed of four groups of feed at doses of 1 mg / kg, 3 mg / kg, 6 mg / kg, and 9 mg / kg, respectively, and labeled as MT1, MT3, MT6, and MT9.

[0057] Combined treatment group: 3 mg / kg methyltestosterone and 100 mg / kg letrozole were added to the feed (i.e., the induction feed prepared in Example 1 was used for treatment), denoted as LM3.

[0058] Blank control group: fed with conventional feed without any drugs, denoted as NC.

[0059] 2. Definition of the induction feeding period for hormone-treated feed: The starting point for drug administration was when rainbow trout eggs fully floated to the surface after hatching, and the accumulated temperature was 500 ± 20 ℃·d after fertilization.

[0060] 3. Control of breeding conditions: The induction experiment was conducted in a flowing water system at 15-18℃, with dissolved oxygen maintained above 7.5 mg / L and ammonia nitrogen content <0.02 mg / L to ensure that the fish were in the optimal physiological and metabolic state.

[0061] 4. Preparation of hormone-induced feed: (1) Single hormone treatment group: Methyltestosterone hormone was weighed according to the proportion of 1 mg / kg, 3 mg / kg, 6 mg / kg and 9 mg / kg of feed. The weighed methyltestosterone was dissolved in 95% ethanol. The ethanol solution was sprayed evenly on the same mass (1 kg) of compound feed in the four groups and labeled as MT1, MT3, MT6 and MT9.

[0062] (2) Combined treatment group: The induction feed prepared in Example 1 was used and labeled as LM3.

[0063] 5. Precise timing and management of guidance: The induction method of Example 6 of the present invention was used to feed four groups of single hormone treatment groups, one group of combined treatment group and one group of blank control group.

[0064] (II) Detection of induction effect

[0065] 1. Weight and body length were measured in 6 groups at age one.

[0066] The measurement method includes the following steps: (1) 100 healthy experimental fish without any external injuries were randomly selected from each group. MS-222 (m-aminobenzoic acid ethyl ester methanesulfonate) anesthetic solution of appropriate concentration was prepared in advance. The experimental fish were immersed in the anesthetic solution in batches for mild sedation. After the fish lost balance, swimming behavior weakened and respiratory rate slowed down, they were quickly taken out. The duration of anesthesia was controlled throughout the process to avoid deep anesthesia leading to fish injury or death. (2) Place the experimental fish stably on a white acrylic plate. A transparent scale with an accuracy of 1 mm is pre-fixed on the surface of the operating plate. The scale is laid out horizontally along the long side of the plate as the size reference for subsequent image measurement to ensure that the measurement scale of each group of samples is uniform. (3) Fix the camera with a fixed-focus lens on a tripod, adjust the height of the tripod so that the camera is vertically facing the acrylic control panel, and keep the shooting distance and camera parameters unchanged; before shooting, use the ruler as a reference to complete the focus, and then shoot each experimental fish to ensure that the fish body is parallel to the ruler and without bending or obstruction, so that the image can be used for accurate size extraction. (4) Use an electronic scale with an accuracy of 0.01 g to measure the weight; before weighing, gently wipe the water off the surface of the fish with absorbent paper, place the experimental fish gently in the weighing dish, and read and record the weight data after the balance value stabilizes; weigh each fish twice and take the average value as the final weight value to reduce the weighing error caused by manual operation. (5) After the growth index is measured, the experimental fish are immediately transferred to continuously aerated clean water for recovery and their swimming status is observed. After the fish recovers to normal swimming, they are returned to the corresponding group of breeding ponds. At the same time, the number of samples is checked to avoid confusion between fish from different groups. (6) Calculate the body length data of each group based on the photos.

[0067] Measurement results: Statistical analysis of growth indicators in one-year-old fish revealed that the growth rate of the LM3 group was significantly higher than that of the blank control group and other single hormone treatment groups (MT1, MT3, MT6, MT9). Figure 2 and Figure 3 As shown, their body length and weight were significantly higher than those of other groups, indicating that the induction scheme of the present invention not only improves the induction effect but also helps rainbow trout growth.

[0068] 2. Physiological phenotype and induction rate were determined by histological sections and HE staining. (1) Processing of gonadal tissue samples: Fresh gonadal tissue was placed in 4% PFA general-purpose tissue fixative and fixed at 4°C for 24 h. The tissue samples were then slowly rinsed under running water for 5 minutes to remove the tissue fixative, transferred to 75% ethanol, and stored at 4°C until sectioning and observation.

[0069] (2) Gradient ethanol dehydration: 85% I ethanol for 30 min, 85% II ethanol for 30 min, 95% I ethanol for 20 min, 95% II ethanol for 20 min, 100% I ethanol for 20 min, 100% II ethanol for 20 min, I ethanol xylene mixed solution (volume ratio 1:1) for 10 min, II ethanol xylene mixed solution (volume ratio 1:1) for 10 min, I xylene for 10 min, II xylene for 10 min.

[0070] (3) Tissue impregnation and embedding: I. Immerse in paraffin for 50 min, II. Immerse in paraffin for 50 min, III. Immerse in paraffin for 50 min. After the impregnation is completed, transfer the tissue and embedding box to the embedding machine, use a plastic mold for embedding, and place on a cold table overnight.

[0071] (4) Tissue sectioning: Trim the paraffin block to the appropriate size with a knife. Fix the paraffin block on the microtome and use a special trimming knife to coarsely cut the paraffin block. The section thickness is 20 μm. After the tissue is exposed, use a microtome knife to section it with a thickness of 5 μm.

[0072] (5) Spreading: Place the cut wax slices on a glass slide, tilt the slide slightly, and use a dropper to draw 75% ethanol and slowly drip it onto the glass slide from top to bottom to spread the wax slices initially on the glass slide. Adjust the water temperature of the spreader to 40℃ and carefully place the initially spread wax slices into the water.

[0073] (6) Drying: After the wax sheet is fully unfolded, carefully lift it with a glass slide and quickly place it in an oven at 37°C to dry overnight.

[0074] (7) Hematoxylin-eosin staining (H&E staining): Before H&E staining, the sections were dried in a 60℃ oven for 30 min. After removing the sections from the oven, they were quickly immersed in xylene I for 10 min and xylene II for 10 min to dewax the tissue. To remove residual xylene, the sections were immersed in 100% anhydrous ethanol I for 3 min, 95% ethanol I for 2 min, 80% ethanol I for 2 min, 70% ethanol I for 2 min, and 50% ethanol for 1 min in sequence. The sections were then soaked in tap water for 2 min to remove residual ethanol. After soaking the sections in hematoxylin staining solution for 4 min, rinse them twice quickly with tap water to remove excess hematoxylin staining solution, each rinse lasting 3-5 s. Immerse the sections in 1% hydrochloric acid-alcohol separation solution for 3-5 s, then rinse quickly with tap water to remove the separation solution. Next, soak them in 1% ammonia-inverted blue solution for 4 min, then rinse quickly with tap water to remove the inverted blue solution. Then, sequentially immerse them in 70% ethanol (II) for 1 min, 80% ethanol (II) for 1 min, 95% ethanol (II) for 1 min, eosin staining solution for 1 min, 95% ethanol (III) for 1 min, and 100% ethanol (II) for 3 min. Finally, immerse them in xylene (III) for 10 min and xylene (IV) for 10 min to make the tissue clear. Mount the sections with neutral resin and coverslips.

[0075] (8) Observe and photograph under a microscope.

[0076] Experimental results: The gonadal sections of the blank control group (NC) were compared with those of the single hormone treatment group and the combined treatment group. The results are as follows: (1) Fifteen individuals from the NC group and 15 individuals from the other treatment groups were tested; all 15 individuals in the NC group had gonads with typical female structures (see Figure 4 In the gonadal section (A), only oogonia and primary oocytes were arranged in an orderly manner, with no testicular tissue or male germ cells present, and females accounted for 100%.

[0077] (2) Among the four groups MT1, MT3, MT6, and MT9, both groups MT6 and MT9 showed varying degrees of incomplete sex reversal, termed intersex individuals. This meant that the gonadal tissue simultaneously contained both testicular and ovarian structures, with one half exhibiting testicular morphology and the other half ovarian morphology. Four individuals in group MT6 showed intersex gonadal characteristics (see [link to article]). Figure 4 In the middle D group, 3 tails in the MT9 group showed interstitial gonads (see [reference]). Figure 4 (E).

[0078] Individuals in the MT1, MT3, and LM3 groups experienced unsuccessful induction, but no intersex individuals with sperm-egg coexistence were observed. The induction rate was consistent between the MT3 and LM3 groups (see [link to MT1 group]). Figure 4 China B and Figure 4 In the middle (C), the percentage can reach over 90%.

[0079] The induction rate in the MT1 group was lower than that in the MT3 and LM3 groups (see [link]). Figure 4 (Middle F).

[0080] 3. Determining the rate of gonadal malformation by comparing the external appearance of the gonads. (1) 15 experimental fish were randomly selected from each experimental group; an excess concentration of MS-222 solution was prepared, and the experimental fish were immersed in the solution for deep anesthesia and death. The fish were observed until the gill covers of the fish completely stopped moving and there was no stress response when touched. After confirming that the fish were dead, the dissection was carried out. (2) Place the experimental fish on a dissecting tray and use surgical scissors to cut open the abdominal cavity from the vent along the midline of the abdomen, extending the cut to below the base of the pectoral fin; gently peel off the abdominal wall muscles, visceral mesentery and adipose tissue with dissecting forceps to fully expose the bilateral gonadal tissue in the abdominal cavity. Avoid pulling the gonads during the operation to ensure the integrity of the overall gonadal structure. (3) Use surgical forceps to remove both gonads from the head to the tail end, preserving the natural developmental morphology of the gonads to the greatest extent possible; (4) Place the freshly picked gonadal tissue immediately on a white acrylic plate with a transparent ruler; gently absorb the tissue fluid, blood and impurities on the surface of the gonads with absorbent paper, and adjust the position of both gonads so that they are parallel to the ruler, without overlapping or curling. (5) Use a fixed-focus camera with a tripod to photograph the gonadal samples at a fixed height; complete the scale focusing before shooting to ensure that the shooting parameters, lighting conditions and shooting distance of all groups of samples are completely consistent, and eliminate systematic errors in the shooting process; (6) Calculate the gonadal malformation rate of each group based on the photos.

[0081] Experimental results: Comparison of gonadal appearance between the NC group and the LM3 and MT3 groups: Through observation, it was found that, for example Figure 5 As shown in Figure A, the gonads of the 15 individuals in the control group all exhibited typical female characteristics, with an orange or pale yellow appearance. like Figure 5 As shown in Figure B, among the 14 males successfully induced in the LM3 group, 4 had gonadal malformations, while the rest were morphologically normal. like Figure 5 As shown in Figure C, among the 14 males successfully induced in the MT3 group, 10 had gonadal malformations, while the rest were morphologically normal.

[0082] from Figure 5 The bar chart shows that, with the same induction rate, the gonadal malformation rate in the LM3 group was significantly lower than that in the MT3 group.

[0083] 4. Sperm production screening was conducted in each experimental group at 16 months of age. (1) Anesthesia and sedation were performed on 5 experimental groups using appropriate doses of MS222.

[0084] (2) Based on the body color, lateral line color and thickness, and relative length of the upper and lower jaws, the experimental fish were initially judged to be sexually mature. The experimental fish that were initially selected were temporarily placed in a new breeding pond.

[0085] (3) Further examination of individuals after initial screening: after anesthesia, squeeze the abdominal gonads of the fish to see if semen is produced.

[0086] (4) Determine the experimental group to which each fish belongs based on the biotags in its body and record them.

[0087] (5) Preserve semen samples, use the computer-aided semen analysis (CASA) system to detect sperm motility, and compare them with three-year-old natural male sperm.

[0088] The results of sperm motility testing in 16-month-old sperm-producing individuals are as follows: Biomarker identification of sperm-producing individuals revealed that all sperm-producing individuals belonged to the LM3 group.

[0089] CASA results are as follows Figure 6 As shown, the sperm velocity curve VCL of the LM3 group experimental fish ( Figure 6 (A) Linear velocity VSL ( Figure 6 (B) Average speed VAP ( Figure 6 (C) Linear exponential Lin ( Figure 6 (middle D) and the mean amplitude of head lateral movement ALH ( Figure 6 The core indicators, such as E, showed no significant difference compared with natural three-year-old male parent fish (NC).

[0090] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Any modifications, equivalent substitutions, alterations, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for efficiently inducing pseudo-male diploid rainbow trout, characterized in that, Includes the following steps: (1) Starting from the first day after the juvenile fish fully float to the surface and open their mouths after breaking through the membrane, artificial feeding of induction feed is carried out, with an accumulated temperature of 500 ± 20 ℃·d after fertilization; the feeding time is 8:00~8:20, 10:00~10:20, 12:00~12:20, 14:00~14:20, and 16:00~16:20, for a total of 5 times; The inducing feed is a feed supplemented with methyltestosterone and letrozole, wherein the mass ratio of methyltestosterone to feed is 1-4 mg / kg, and the mass ratio of letrozole to feed is 70-130 mg / kg. (2) The feeding treatment time is 700 ± 30 ℃·d. After the treatment is completed, the feed is switched to regular feed. The feeding time is 8:00~8:10, 11:00~11:10, 13:00~13:10, and 16:00~16:10, for a total of 4 times.

2. The method for efficiently inducing diploid rainbow trout pseudo-males according to claim 1, characterized in that, The induction method is carried out in a flowing water system at 15~18℃, with dissolved oxygen content not less than 7.5 mg / L and ammonia nitrogen content <0.02 mg / L.

3. The method for efficiently inducing diploid rainbow trout pseudo-males according to claim 1, characterized in that, The method for preparing the inducing feed includes the following steps: (1) Weigh 20-25 mg of methyltestosterone and place it in a brown wide-mouth bottle. Add 1320 ml of 95% ethanol solution and stir until completely dissolved under the protection of light to obtain methyltestosterone hormone ethanol stock solution. Take 180 mL of methyltestosterone hormone ethanol stock solution and add 95% ethanol to make up to 680 mL to obtain methyltestosterone hormone ethanol solution. Weigh 70-130 mg of letrozole into a brown wide-mouth bottle, add 500 ml of 95% ethanol solution, and stir until completely dissolved under light-protected conditions to obtain letrozole ethanol mother liquor. (2) Weigh 1 kg of base feed and spray the prepared methyltestosterone hormone ethanol solution and letrozole ethanol mother liquor evenly onto the surface of the feed. (3) Spread the sprayed feed evenly, with a pile thickness not exceeding 1 cm, and dry it at 40℃ for 3-5 hours. Turn it over once every 1 hour of drying until the ethanol solvent is completely evaporated. Seal the dried feed and store it at 4℃.

4. The method for efficiently inducing diploid rainbow trout pseudo-males according to claim 1, characterized in that, The mass ratio of methyltestosterone to feed is 3 mg / kg, and the mass ratio of letrozole to feed is 100 mg / kg.

5. The method for efficiently inducing diploid rainbow trout pseudo-males according to claim 1, characterized in that, In step (1), the feeding of inducing feed should follow the principle of "small amounts and multiple times". Observe the feeding situation of the fry each time they are fed. Stop feeding when the fish's abdomen swells slightly and they no longer gather to compete for food.

6. The method for efficiently inducing diploid rainbow trout pseudo-males according to claim 5, characterized in that, The feeding principle is as follows: the amount of inducing feed for 1,000 fish on the first day is 8-10 g; the total accumulated temperature of 700±30℃·d is used as the complete drug administration and feeding cycle, and the average daily feeding amount is increased weekly. The average daily feeding amount is 10-12 g inducing feed on the first week, 15-18 g inducing feed on the second week, 20-22 g inducing feed on the third week, and 26-30 g inducing feed on the fourth week, and so on.

7. The method for efficiently inducing diploid rainbow trout pseudo-males according to claim 1, characterized in that, The rainbow trout eggs used are eyed eggs of fully female diploid rainbow trout imported from Denmark.

8. The application of the method for efficiently inducing diploid rainbow trout pseudo-males as described in any one of claims 1 to 7 in the all-female rainbow trout farming.

9. The application of the method for efficiently inducing pseudo-male diploid rainbow trout according to any one of claims 1 to 7 in promoting the growth of diploid rainbow trout juveniles, characterized in that, The method described can increase the weight and length of rainbow trout.

10. The application of the method for efficiently inducing diploid rainbow trout pseudo-males according to any one of claims 1 to 7 in improving rainbow trout sperm motility, characterized in that, Shorten the sexual maturity cycle of rainbow trout pseudo-males and bring their sperm motility up to the level of natural three-year-old male parent fish.

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