An induction method for efficient sex reversal of adult female Nibea albiflora

Abstract

The invention discloses an efficient inducing method for sex reversal of adult female yellow croaker, belonging to the technical field of female fish sex reversal. The invention specifically relates to a method for ablating oocytes of adult female yellow croaker using busulfan, and then feeding the treated adult female yellow croaker with a compound feed containing an aromatase inhibitor for 6-12 months to obtain adult yellow croaker with successful sex reversal. The aromatase inhibitor is letrozole, and busulfan can be used in combination with a pyrimidine compound or a pyrimidine-based chalcone. Thus, the method has the following beneficial effects: a high survival rate of the adult yellow croaker, which is above 90%, and a high sex reversal success rate, which is above 70%.

Description

Technical Field

[0001] The invention belongs to the technical field of female fish sex reversal, and particularly relates to an efficient induction method for sex reversal of adult female yellow croaker. Background Art

[0002] Yellow croaker, belonging to the order Perciformes, suborder Perciformes, family Sciaenidae, and genus Sciaenidae, is an important commercial marine fish species in my country and is highly sought after by consumers. In actual aquaculture, yellow croaker exhibits rapid growth, strong stress tolerance, and a short period of sexual maturity (1-2 years of age). With appropriate artificial control, year-round reproduction is possible. Yellow croaker also exhibits typical sexually dimorphic growth characteristics, with females growing significantly faster than males. Therefore, the preparation of pseudo-male yellow croaker not only has potential economic benefits in yellow croaker aquaculture but also has important applications in research such as germ cell transplantation and gene editing breeding of Sciaenidae.

[0003] Artificially inducing pseudo-males is generally believed to be possible only during the specific sexual differentiation period of juvenile fish. Once sex differentiation is complete, the fish's sex cannot be changed. Therefore, current pseudo-male induction in fish begins during the juvenile stage. However, pseudo-males created during the juvenile stage require prolonged cultivation until sexual maturity, which can be affected by disease and other factors, leading to mortality during the rearing stage. Therefore, sex control in adult fish to generate pseudo-males can reduce aquaculture costs and risks. Summary of the Invention

[0004] The object of the present invention is to provide an efficient inducing method for sex reversal of adult female yellow croaker, which has a high survival rate and a high sex reversal success rate.

[0005] The technical solutions adopted by the present invention to achieve the above-mentioned purpose are:

[0006] A highly effective method for inducing sex reversal in adult female yellow croaker comprises: first ablating oocytes of adult female yellow croaker using busulfan, then feeding the treated female yellow croaker with a compound feed containing an aromatase inhibitor for 6-12 months to obtain adult yellow croaker with successful sex reversal, wherein the aromatase inhibitor is letrozole. Busulfan is a bifunctional alkylating agent of the bismethylsulfonate class and a non-specific drug for the cell cycle. Upon entering the body, the cyclic structure of its sulfonate group opens, and by alkylating guanine in cellular DNA, it destroys the structure and function of DNA. Letrozole is a new generation of highly selective aromatase inhibitors that inhibit aromatase, thereby reducing estrogen levels. The present invention prevents oocyte viability recovery by adding it to the compound feed. Under the coordinated action of busulfan and the aromatase inhibitor at different stages, the oocyte is successfully ablated, and the testis structure is obtained, thereby obtaining adult male yellow croaker.

[0007] Preferably, the single dosage of busulfan is 20-50 mg / kg.

[0008] Preferably, the number of injections of busulfan is 2-4 times.

[0009] Preferably, the amount of aromatase inhibitor in the formula feed is 80-120 mg / kg.

[0010] The invention adopts busulfan reagent to be injected into the gonad of adult female yellow croaker through the genital pore to ablate oocytes.

[0011] Preferably, for oocyte ablation, normal adult female yellow croaker are selected and anesthetized with the fish anesthetic MS-222. A busulfan solution is injected into the female yellow croaker via genital pore injection at a dose of 20-50 mg / kg, two to four times, seven days apart. After culturing for an additional 14-28 days, oocyte ablation is completed.

[0012] Preferably, during the ablation of the oocyte, a pyrimidine compound is added to the busulfan solution, and the pyrimidine compound is 20-40 wt % of the busulfan.

[0013] Preferably, during oocyte ablation, L-carnitine is added to the busulfan solution, and the amount of L-carnitine used is 3-12 wt % of busulfan. In oocyte ablation, it was found that the use of L-carnitine in the presence of busulfan and pyrimidinyl chalcone can increase the success rate of sex reversal in female yellow croaker.

[0014] The invention feeds letrozole to induce ovarian sex reversal.

[0015] Preferably, sex reversal is induced in oocyte-ablated female yellow croaker by feeding them a formula feed containing an aromatase inhibitor. Starting 14-28 days after the last busulfan injection, letrozole-containing feed is administered for 6-12 months. To prepare the formula feed, the aromatase inhibitor is weighed and dissolved in a small amount of ethanol. The aromatase inhibitor is added to the feed at a dose of 80-120 mg / kg, mixed, and then air-dried at room temperature. The aromatase inhibitor is letrozole.

[0016] Preferably, during sex reversal culture, a pyrimidine compound is added to the formula feed, and the amount of the pyrimidine compound used is 20-40 wt % of the aromatase inhibitor.

[0017] The present invention detects and verifies the sex-reversed yellow croaker.

[0018] Preferably, during the sexing of yellow croaker, fish are sampled monthly during the experiment, and their length and weight are measured. The gonads are then dissected and weighed, fixed with Bonn's solution, and observed by making tissue sections. During the experiment, the mortality rate of the fish is also measured and recorded.

[0019] Preferably, a pyrimidine compound or pyrimidinyl chalcone is used together with busulfan in oocyte ablation; the pyrimidine compound is prepared from thiourea, ethyl acetoacetate, and benzaldehyde; the pyrimidinyl chalcone is prepared by reacting a pyrimidine compound with a bromoalkane-substituted chalcone, which is prepared by reacting a 2'-hydroxychalcone with a dibromoalkane. In the present invention, busulfan is used to ablate oocytes of adult female yellow croaker. However, the amount of busulfan used affects the effect of oocyte ablation. If the amount of busulfan is too low, the effect is minimal, while if the amount of busulfan is too high, the effect on the organism is significant, and in severe cases, the subject organism may die. The present invention has found that using a pyrimidine compound or pyrimidinyl chalcone together with busulfan can better improve the ablation effect on oocytes and better obtain adult yellow croaker with successful sex reversal.

[0020] More preferably, the pyrimidine compound is 20-40 wt % of busulfan.

[0021] More preferably, the pyrimidinyl chalcone is 20-40 wt% of busulfan.

[0022] Preferably, in the preparation of the pyrimidine compound, thiourea, ethyl acetoacetate and benzaldehyde are added to anhydrous ethanol, and then potassium carbonate is added, and the mixture is stirred and mixed at 20-40°C for 15-60 minutes, and then heated under reflux for 2-6 hours. After the reaction is completed, ice water is added to the reaction system to dilute it, and then extracted with dichloromethane. The organic phase is separated and washed with hydrochloric acid solution, saturated sodium bicarbonate solution and saturated sodium chloride solution in sequence, dried over anhydrous magnesium sulfate, and dichloromethane is removed by distillation under reduced pressure to obtain the pyrimidine compound.

[0023] More preferably, in the preparation of pyrimidine compounds, the amount of thiourea used is 1-3 wt % of anhydrous ethanol.

[0024] More preferably, in the preparation of pyrimidine compounds, the molar amount of ethyl acetoacetate used is 100% of the molar amount of thiourea used.

[0025] More preferably, in the preparation of pyrimidine compounds, the molar amount of benzaldehyde used is 100% of the molar amount of thiourea used.

[0026] More preferably, in the preparation of pyrimidine compounds, the amount of potassium carbonate used is 200-400 wt % of thiourea.

[0027] More preferably, in the preparation of pyrimidine compounds, the amount of ice water used is at least 5 times the volume of the reaction system, and an appropriate amount is sufficient. The concentration of the hydrochloric acid solution used for washing is 0.5-2 M. The extractant and other washing liquids are used in appropriate amounts.

[0028] Preferably, in the preparation of the bromoalkane-substituted chalcone, 2'-hydroxychalcone is added to DMF, followed by potassium carbonate, and stirred in an ice-water bath for 1-3 hours. Then, dibromoalkane is added dropwise to react for 6-18 hours. After the reaction is completed, ice water is added, followed by extraction with ethyl acetate. The ethyl acetate layer is separated and then washed with hydrochloric acid solution, saturated sodium bicarbonate solution, and saturated sodium chloride solution in sequence, dried over anhydrous magnesium sulfate, and ethyl acetate is removed by distillation under reduced pressure to obtain the bromoalkane-substituted chalcone.

[0029] More preferably, in the preparation of the bromoalkane-substituted chalcone, the amount of 2'-hydroxychalcone used is 2-6 wt % of DMF.

[0030] More preferably, in the preparation of bromoalkane-substituted chalcone, the amount of potassium carbonate used is 100-300 wt % of 2'-hydroxychalcone.

[0031] More preferably, in the preparation of the bromoalkane-substituted chalcone, the molar amount of the dibromoalkane used is 200-500 wt % of the molar amount of the 2'-hydroxychalcone used.

[0032] More preferably, in the preparation of the bromoalkane-substituted chalcone, the amount of ice water used is at least 5 times the volume of the reaction system, and the concentration of the hydrochloric acid solution used for washing is 0.5-2M. The extractant and other washing liquids are used in appropriate amounts.

[0033] Preferably, in the preparation of the pyrimidinyl chalcone, a bromoalkane-substituted chalcone is added to DMF, followed by the addition of potassium carbonate, and the mixture is stirred at 20-40° C. for 0.5-2 h. Then, a pyrimidine compound is added, and the mixture is reacted at 90-110° C. for 4-16 h. After the reaction is completed, the mixture is cooled, ice water is added to the reaction system, and then dichloromethane is added for extraction. The dichloromethane layer is separated and washed with hydrochloric acid solution, saturated sodium bicarbonate solution, and saturated sodium chloride solution, in that order, and dried over anhydrous magnesium sulfate. The dichloromethane is removed by distillation under reduced pressure to obtain the pyrimidinyl chalcone.

[0034] More preferably, in the preparation of pyrimidinyl chalcone, the amount of bromoalkane-substituted chalcone used is 1.5-4.5 wt % of DMF.

[0035] More preferably, in the preparation of pyrimidinyl chalcone, the amount of potassium carbonate used is 100-300 wt % of the bromoalkane-substituted chalcone.

[0036] More preferably, in the preparation of pyrimidinyl chalcone, the molar amount of the pyrimidine compound used is 100% of the molar amount of the bromoalkane-substituted chalcone used.

[0037] More preferably, in the preparation of pyrimidinyl chalcone, the amount of ice water used is at least 5 times the volume of the reaction system, and the concentration of the hydrochloric acid solution used for washing is 0.5-2 M. The extractant and other washing liquids are used in appropriate amounts.

[0038] Preferably, pyrimidine compounds or pyrimidinyl chalcone and aromatase inhibitor are added to the compound feed; the pyrimidine compounds are prepared from thiourea, ethyl acetoacetate and benzaldehyde; the pyrimidinyl chalcone is prepared by reacting the pyrimidine compounds with bromoalkane-substituted chalcone, and the bromoalkane-substituted chalcone is prepared by reacting 2'-hydroxychalcone with dibromoalkane.

[0039] More preferably, the amount of the pyrimidine compound used is 20-40 wt % of the aromatase inhibitor.

[0040] More preferably, the amount of pyrimidinyl chalcone used is 20-40 wt % of the aromatase inhibitor.

[0041] The invention discloses a sex-reversed yellow croaker adult fish prepared by the method.

[0042] The gonadal tissue sections of the adult female yellow croaker treated by the method of the present invention were stained and observed to analyze the gonadal structure and morphology. It was found that the gonadal oocytes of the experimental fish were completely apoptotic, and testis-like structures appeared, including the production of mature sperm, confirming that the experimental fish were sex-reversed into male yellow croaker capable of producing sperm.

[0043] The present invention uses busulfan to ablate oocytes of adult female yellow croaker, and then feeds the treated adult female yellow croaker with a compound feed containing an aromatase inhibitor for 6-12 months to obtain adult yellow croaker with successful sex reversal. The aromatase inhibitor is letrozole. Furthermore, busulfan can be used in combination with a pyrimidine compound or a pyrimidinyl chalcone. This method has the following beneficial effects: a high survival rate of adult yellow croaker, exceeding 90%, and a high sex reversal success rate, exceeding 70%. Therefore, the present invention provides an efficient method for inducing sex reversal in adult female yellow croaker, with a high survival rate and a high sex reversal success rate. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] Figure 1 This is the survival rate of adult yellow croaker;

[0045] Figure 2 This is a graph of the success rate of sex reversal;

[0046] Figure 3 2. It is a comparison of gonad tissue sections of adult yellow croaker fish in the embodiment group and the control group. DETAILED DESCRIPTION

[0047] The technical solution of the present invention is further described in detail below with reference to the specific embodiments and the accompanying drawings:

[0048] Example 1:

[0049] A highly efficient induction method for sex reversal of adult female yellow croaker.

[0050] Oocyte ablation: Normal adult female yellow croakers were selected and anesthetized with the fish anesthetic MS-222. Two injections of busulfan solution at a dose of 20 mg / kg were injected into the genital pore, 7 days apart. After 14 days of incubation, oocyte ablation was completed.

[0051] Sex Reversal Culture: Sex reversal was induced in oocyte-ablated female yellow croaker by feeding them a formula feed containing an aromatase inhibitor. Starting 14 days after the last busulfan injection, letrozole was fed for four months. To prepare the formula feed, the aromatase inhibitor was weighed and dissolved in a small amount of ethanol. The aromatase inhibitor was added to the feed at a dose of 100 mg / kg, mixed thoroughly, and then air-dried at room temperature. The aromatase inhibitor was letrozole.

[0052] Sexing of yellow croaker: During the experiment, fish were sampled monthly, and their length and weight were measured. The gonads were removed and weighed, and the fish were fixed with Bonn's solution and observed by making tissue sections. Mortality rates were also measured and recorded.

[0053] Example 2:

[0054] A highly efficient induction method for sex reversal of adult female yellow croaker.

[0055] Oocyte ablation: Normal adult female yellow croakers were selected and anesthetized with the fish anesthetic MS-222. Two injections of busulfan solution at a dose of 40 mg / kg were injected into the genital pore, 7 days apart. After 14 days of incubation, oocyte ablation was completed.

[0056] Sex Reversal Culture: Sex reversal was induced in oocyte-ablated female yellow croaker by feeding them a formula feed containing an aromatase inhibitor. Starting 14 days after the last busulfan injection, letrozole was fed for four months. To prepare the formula feed, the aromatase inhibitor was weighed and dissolved in a small amount of ethanol. The aromatase inhibitor was added to the feed at a dose of 100 mg / kg, mixed thoroughly, and then air-dried at room temperature. The aromatase inhibitor was letrozole.

[0057] Sexing of yellow croaker: During the experiment, fish were sampled monthly, and their length and weight were measured. The gonads were removed and weighed, and the fish were fixed with Bonn's solution and observed by making tissue sections. Mortality rates were also measured and recorded.

[0058] Example 3:

[0059] A highly efficient method for inducing sex reversal in adult female yellow croaker. Compared with Example 1, this embodiment differs in that a pyrimidine compound is added to the busulfan solution during the ablation of oocytes.

[0060] Preparation of pyrimidine compounds: Thiourea, ethyl acetoacetate, and benzaldehyde were added to anhydrous ethanol, followed by the addition of potassium carbonate. The mixture was stirred at 30°C for 30 minutes, then heated under reflux for 4 hours. After completion of the reaction, the reaction system was diluted with ice water, then extracted with dichloromethane. The organic phase was separated and washed sequentially with hydrochloric acid solution, saturated sodium bicarbonate solution, and saturated sodium chloride solution, dried over anhydrous magnesium sulfate, and dichloromethane was removed by distillation under reduced pressure to obtain pyrimidine compounds. The amount of thiourea used was 2 wt % of the anhydrous ethanol, the molar amount of ethyl acetoacetate used was 100% of the molar amount of thiourea used, the molar amount of benzaldehyde used was 100% of the molar amount of thiourea used, and the amount of potassium carbonate used was 300 wt % of the molar amount of thiourea used. The amount of ice water used was 10 times the volume of the reaction system. The concentration of the hydrochloric acid solution used for washing was 1 M. The extractant and other washing solutions were used in appropriate amounts.

[0061] Oocyte ablation: Normal adult female yellow croaker were selected and anesthetized with the fish anesthetic MS-222. Two injections of busulfan solution (20 mg / kg) were administered via genital pore injection, 7 days apart. After 14 days of incubation, oocyte ablation was completed. A pyrimidine compound was added to the busulfan solution at a concentration of 30% by weight of the busulfan.

[0062] Example 4:

[0063] A highly efficient method for inducing sex reversal in adult female yellow croaker. Compared with Example 1, this embodiment differs in that pyrimidine-based chalcone is added to the busulfan solution during oocyte ablation.

[0064] Preparation of bromoalkane-substituted chalcone: 2'-hydroxychalcone is added to DMF, followed by potassium carbonate. The mixture is stirred in an ice-water bath for 2 hours, followed by the dropwise addition of dibromoalkane and the reaction is continued for 12 hours. After the reaction is complete, ice water is added, followed by extraction with ethyl acetate. The ethyl acetate layer is separated and washed sequentially with hydrochloric acid solution, saturated sodium bicarbonate solution, and saturated sodium chloride solution, dried over anhydrous magnesium sulfate, and the ethyl acetate is removed by vacuum distillation to obtain the bromoalkane-substituted chalcone. The amount of 2'-hydroxychalcone used is 4wt% of the DMF, the amount of potassium carbonate used is 200wt% of the 2'-hydroxychalcone, and the molar amount of dibromoalkane used is 300wt% of the molar amount of 2'-hydroxychalcone used. The amount of ice water used is 10 times the volume of the reaction system. The concentration of the hydrochloric acid solution used for washing is 1M. The extractant and other washing liquids are used in appropriate amounts.

[0065] Preparation of pyrimidine compounds: Thiourea, ethyl acetoacetate, and benzaldehyde were added to anhydrous ethanol, followed by the addition of potassium carbonate. The mixture was stirred at 30°C for 30 minutes, then heated under reflux for 4 hours. After completion of the reaction, the reaction system was diluted with ice water, then extracted with dichloromethane. The organic phase was separated and washed sequentially with hydrochloric acid solution, saturated sodium bicarbonate solution, and saturated sodium chloride solution, dried over anhydrous magnesium sulfate, and dichloromethane was removed by distillation under reduced pressure to obtain pyrimidine compounds. The amount of thiourea used was 2 wt % of the anhydrous ethanol, the molar amount of ethyl acetoacetate used was 100% of the molar amount of thiourea used, the molar amount of benzaldehyde used was 100% of the molar amount of thiourea used, and the amount of potassium carbonate used was 300 wt % of the molar amount of thiourea used. The amount of ice water used was 10 times the volume of the reaction system. The concentration of the hydrochloric acid solution used for washing was 1 M. The extractant and other washing solutions were used in appropriate amounts.

[0066] Preparation of pyrimidinyl chalcone: Add a bromoalkyl chalcone to DMF, then add potassium carbonate. Stir and mix at 30°C for 1 hour. Then add a pyrimidine compound and react at 100°C for 8 hours. After completion of the reaction, cool the reaction system, add ice water, and extract with dichloromethane. Separate the dichloromethane layer, wash it with hydrochloric acid, saturated sodium bicarbonate, and saturated sodium chloride, and dry it over anhydrous magnesium sulfate. Remove the dichloromethane by vacuum distillation to obtain the pyrimidinyl chalcone. The amount of bromoalkyl chalcone used is 3wt% of the DMF, the amount of potassium carbonate used is 200wt% of the bromoalkyl chalcone, and the molar amount of the pyrimidine compound is 100% of the molar amount of the bromoalkyl chalcone used. The amount of ice water used is 10 times the volume of the reaction system, and any amount is sufficient. The concentration of the hydrochloric acid solution used for washing is 1M. The extractant and other washing solutions are used in appropriate amounts.

[0067] Oocyte ablation: Normal adult female yellow croaker were selected and anesthetized with the fish anesthetic MS-222. Two injections of busulfan solution (20 mg / kg) were administered via genital pore injection, 7 days apart. After 14 days of incubation, oocyte ablation was completed. Pyrimidinyl chalcone was added to the busulfan solution at a concentration of 30% by weight of the busulfan.

[0068] Example 5:

[0069] A highly efficient method for inducing sex reversal in adult female yellow croaker. Compared with Example 1, this embodiment differs in that pyrimidine compounds are added to the formula feed during sex reversal culture.

[0070] Preparation of pyrimidine compounds: Thiourea, ethyl acetoacetate, and benzaldehyde were added to anhydrous ethanol, followed by the addition of potassium carbonate. The mixture was stirred at 30°C for 30 minutes, then heated under reflux for 4 hours. After completion of the reaction, the reaction system was diluted with ice water, then extracted with dichloromethane. The organic phase was separated and washed sequentially with hydrochloric acid solution, saturated sodium bicarbonate solution, and saturated sodium chloride solution, dried over anhydrous magnesium sulfate, and dichloromethane was removed by distillation under reduced pressure to obtain pyrimidine compounds. The amount of thiourea used was 2 wt % of the anhydrous ethanol, the molar amount of ethyl acetoacetate used was 100% of the molar amount of thiourea used, the molar amount of benzaldehyde used was 100% of the molar amount of thiourea used, and the amount of potassium carbonate used was 300 wt % of the molar amount of thiourea used. The amount of ice water used was 10 times the volume of the reaction system. The concentration of the hydrochloric acid solution used for washing was 1 M. The extractant and other washing solutions were used in appropriate amounts.

[0071] Sex Reversal Culture: Sex reversal was induced in oocyte-ablated female yellow croaker by feeding them a formula feed containing an aromatase inhibitor. Starting 14 days after the last busulfan injection, letrozole-containing feed was fed for four months. To prepare the formula feed, the aromatase inhibitor was weighed and dissolved in a small amount of ethanol. The aromatase inhibitor was added to the feed at a dose of 100 mg / kg, mixed, and then allowed to dry at room temperature. The aromatase inhibitor was letrozole. A pyrimidine compound was added to the formula feed at a level of 30 wt% of the aromatase inhibitor.

[0072] Example 6:

[0073] A highly efficient method for inducing sex reversal in adult female yellow croaker. Compared with Example 1, this embodiment differs in that pyrimidine-based chalcone is added to the formula feed during sex reversal culture.

[0074] Preparation of bromoalkane-substituted chalcone: 2'-hydroxychalcone is added to DMF, followed by potassium carbonate. The mixture is stirred in an ice-water bath for 2 hours, followed by the dropwise addition of dibromoalkane and the reaction is continued for 12 hours. After the reaction is complete, ice water is added, followed by extraction with ethyl acetate. The ethyl acetate layer is separated and washed sequentially with hydrochloric acid solution, saturated sodium bicarbonate solution, and saturated sodium chloride solution, dried over anhydrous magnesium sulfate, and the ethyl acetate is removed by vacuum distillation to obtain the bromoalkane-substituted chalcone. The amount of 2'-hydroxychalcone used is 4wt% of the DMF, the amount of potassium carbonate used is 200wt% of the 2'-hydroxychalcone, and the molar amount of dibromoalkane used is 300wt% of the molar amount of 2'-hydroxychalcone used. The amount of ice water used is 10 times the volume of the reaction system. The concentration of the hydrochloric acid solution used for washing is 1M. The extractant and other washing liquids are used in appropriate amounts.

[0075] Preparation of pyrimidine compounds: Thiourea, ethyl acetoacetate, and benzaldehyde were added to anhydrous ethanol, followed by the addition of potassium carbonate. The mixture was stirred at 30°C for 30 minutes, then heated under reflux for 4 hours. After completion of the reaction, the reaction system was diluted with ice water, then extracted with dichloromethane. The organic phase was separated and washed sequentially with hydrochloric acid solution, saturated sodium bicarbonate solution, and saturated sodium chloride solution, dried over anhydrous magnesium sulfate, and dichloromethane was removed by distillation under reduced pressure to obtain pyrimidine compounds. The amount of thiourea used was 2 wt % of the anhydrous ethanol, the molar amount of ethyl acetoacetate used was 100% of the molar amount of thiourea used, the molar amount of benzaldehyde used was 100% of the molar amount of thiourea used, and the amount of potassium carbonate used was 300 wt % of the molar amount of thiourea used. The amount of ice water used was 10 times the volume of the reaction system. The concentration of the hydrochloric acid solution used for washing was 1 M. The extractant and other washing solutions were used in appropriate amounts.

[0076] Preparation of pyrimidinyl chalcone: A bromoalkyl-substituted chalcone is added to DMF, followed by potassium carbonate. The mixture is stirred at 30°C for 1 hour, followed by the addition of a pyrimidine compound. The reaction is allowed to react at 100°C for 8 hours. After completion of the reaction, the mixture is cooled, ice water is added to the reaction system, and then extracted with dichloromethane. The dichloromethane layer is separated and washed sequentially with hydrochloric acid solution, saturated sodium bicarbonate solution, and saturated sodium chloride solution, dried over anhydrous magnesium sulfate, and the dichloromethane is removed by distillation under reduced pressure to yield the pyrimidinyl chalcone. The amount of bromoalkyl-substituted chalcone is 3 wt% of the DMF, the amount of potassium carbonate is 200 wt% of the bromoalkyl-substituted chalcone, and the molar amount of the pyrimidine compound is 100% of the molar amount of the bromoalkyl-substituted chalcone. The amount of ice water used is 10 times the volume of the reaction system. The concentration of the hydrochloric acid solution used for washing is 1 M. The extractant and other washing solutions are used in appropriate amounts.

[0077] Sex Reversal Culture: Sex reversal was induced in oocyte-ablated female yellow croaker by feeding them a formula feed containing an aromatase inhibitor. Starting 14 days after the last busulfan injection, letrozole-containing feed was fed for four months. In the formulation of the formula feed, the aromatase inhibitor was weighed and dissolved in a small amount of ethanol. The aromatase inhibitor was added to the feed at a dose of 100 mg / kg and mixed thoroughly, and then allowed to dry at room temperature. The aromatase inhibitor was letrozole. Pyrimidinyl chalcone was added to the formula feed at a level of 30 wt% of the aromatase inhibitor.

[0078] Example 7:

[0079] A highly efficient method for inducing sex reversal in adult female yellow croaker. Compared with Example 4, this embodiment differs from Example 1 in that L-carnitine is added to the busulfan solution during oocyte ablation.

[0080] Oocyte ablation: Normal adult female yellow croaker were selected and anesthetized with the fish anesthetic MS-222. Two genital injections of busulfan solution (20 mg / kg) were administered, separated by 7 days. After 14 days of incubation, oocyte ablation was completed. Pyrimidinyl chalcone and L-carnitine were added to the busulfan solution; the amount of pyrimidinyl chalcone was 30% by weight of the busulfan, and the amount of L-carnitine was 5% by weight of the busulfan.

[0081] Example 8:

[0082] A highly efficient method for inducing sex reversal in adult female yellow croaker. Compared with Example 4, this embodiment differs from Example 1 in that L-carnitine is added to the busulfan solution during oocyte ablation.

[0083] Oocyte ablation: Normal adult female yellow croaker were selected and anesthetized with the fish anesthetic MS-222. Two genital injections of busulfan solution (20 mg / kg) were administered, separated by 7 days. After 14 days of incubation, oocyte ablation was completed. Pyrimidinyl chalcone and L-carnitine were added to the busulfan solution; the amount of pyrimidinyl chalcone was 30% by weight of the busulfan, and the amount of L-carnitine was 9% by weight of the busulfan.

[0084] Test example:

[0085] 1. Survival rate of adult yellow croaker

[0086] The survival rate of adult female yellow croakers after the induction method of each embodiment was calculated by statistics.

[0087] The survival rate of female yellow croaker after treatment by various embodiments of the present invention is as follows: Figure 1 As shown, S1 is Example 1, S2 is Example 2, S3 is Example 3, S4 is Example 4, S5 is Example 5, S6 is Example 6, S7 is Example 7, and S8 is Example 8. The various embodiments of the present invention cause basically no harm to adult female yellow croaker after treatment, and the survival rate of adult yellow croaker in each method is higher than 90%, and more preferably, the survival rate of adult yellow croaker is higher than 95%.

[0088] 2. Success rate of testicular structure transformation

[0089] After the induction method of each embodiment is completed, the gonads of the female yellow croaker are removed, tissue sections are made, and the presence of testicular structure and spermatogenesis is observed. The presence of spermatogenesis indicates successful transformation. Untreated female yellow croaker that is normally cultured is used as a control.

[0090] The results of sex reversal of female yellow croaker after treatment by various embodiments of the present invention are as follows: Figure 2 As shown, wherein S1 is Example 1, S2 is Example 2, S3 is Example 3, S4 is Example 4, S5 is Example 5, S6 is Example 6, S7 is Example 7, and S8 is Example 8. The sex conversion success rate of female yellow croaker after treatment by each embodiment of the present invention is higher than 70%, which is the sex conversion success rate obtained under the specific method of each embodiment. It can be found that adding busulfan to oocyte ablation and adding aromatase inhibitor letrozole to the formula feed in sex reversal culture, and their combined use at different stages can successfully reverse the sex of female yellow croaker; It is worth noting Interestingly, busulfan and the aromatase inhibitor letrozole, at varying dosages, successfully reversed the sex of adult female yellow croaker. Increasing the busulfan dosage increased the success rate of sex reversal in female yellow croaker. During oocyte ablation, the addition of pyrimidine compounds or pyrimidinyl chalcone at low busulfan dosages also improved the sex reversal rate, with pyrimidinyl chalcone being more effective than pyrimidine compounds. Furthermore, the addition of L-carnitine to the pyrimidinyl chalcone further enhanced the sex reversal rate. While the addition of pyrimidine compounds or pyrimidinyl chalcone to sex reversal culture has some success, it is not as effective as their use during oocyte ablation.

[0091] The tissue sections of the gonads of the female yellow croaker adult fish of Example 1 and the control group are as follows: Figure 3As shown, A represents the tissue section of the gonad of the female yellow croaker of Example 1, B represents the tissue section of the gonad of the female yellow croaker of the control group, the female yellow croaker in group B were cultured normally and were not treated. The actual difference lies in the different treatments of group A, A1 represents the tissue section of the gonad of the female yellow croaker before the start of the experiment, so A1 and B1 are tissue sections of the gonad of the same female yellow croaker, A2 is the tissue section of the gonad of the yellow croaker 14 days after the injection of busulfan, and B2 is the tissue section of the gonad of the female yellow croaker after 14 days of normal culture. It can be found that the oocytes in A2 are ablated. But it was not completely ablated; A3 is a tissue section of the gonad of an adult yellow croaker after being fed the aromatase inhibitor letrozole for 9 months, and B3 is a tissue section of the gonad of an adult female yellow croaker after 9 months of normal culture. It can be found that the oocytes in A2 are completely ablated; A4 is a tissue section of the gonad of an adult yellow croaker after being fed the aromatase inhibitor letrozole for 10 months, and A5 is a magnified view of a part in A4. B4 is a tissue section of the gonad of an adult female yellow croaker after 10 months of normal culture. B5 is a magnified view of a part in B4. It can be found that sperm are present in the gonad in group A, i.e. A5, indicating that the sex of the adult yellow croaker has been successfully reversed.

[0092] The above embodiments are intended to illustrate the present invention only and are not intended to limit the present invention. Persons skilled in the art may make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, all equivalent technical solutions also fall within the scope of the present invention, and the scope of patent protection of the present invention shall be defined by the claims.

Claims

1. A highly effective method for inducing sex reversal in adult female yellow croaker, comprising: First, busulfan, pyrimidinyl chalcone, and L-carnitine are used to ablate oocytes of adult female yellow croakers. Then, the treated female yellow croakers are fed with a compound feed containing an aromatase inhibitor for 6-12 months to obtain adult yellow croakers with successful sex reversal. The aromatase inhibitor is letrozole. In the preparation of the pyrimidinyl chalcone, a bromoalkane-substituted chalcone is added to DMF, followed by potassium carbonate, and the mixture is stirred at 20-40°C for 0.5-2 hours. A pyrimidine compound is then added, and the mixture is reacted at 90-110°C for 4-16 hours. After the reaction is completed, the mixture is cooled, ice water is added to the reaction system, and then dichloromethane is added for extraction. The dichloromethane layer is separated and washed with a hydrochloric acid solution, a saturated sodium bicarbonate solution, and a saturated sodium chloride solution in sequence, dried over anhydrous magnesium sulfate, and the dichloromethane is removed by distillation under reduced pressure to obtain the pyrimidinyl chalcone. In the preparation of the bromoalkane-substituted chalcone, 2'-hydroxychalcone is added to DMF, followed by potassium carbonate, and stirred in an ice-water bath for 1-3 hours. Then, dibromoalkane is added dropwise to react for 6-18 hours. After the reaction is complete, ice water is added, followed by extraction with ethyl acetate. The ethyl acetate layer is separated and washed sequentially with hydrochloric acid solution, saturated sodium bicarbonate solution, and saturated sodium chloride solution, dried over anhydrous magnesium sulfate, and the ethyl acetate is removed by distillation under reduced pressure to obtain the bromoalkane-substituted chalcone. In the preparation of the pyrimidine compound, thiourea, ethyl acetoacetate and benzaldehyde are added to anhydrous ethanol, and potassium carbonate is added, and the mixture is stirred and mixed at 20-40° C. for 15-60 minutes, and then heated under reflux for reaction for 2-6 hours. After the reaction is completed, ice water is added to dilute the reaction system, and then extracted with dichloromethane. The organic phase is separated and washed with hydrochloric acid solution, saturated sodium bicarbonate solution and saturated sodium chloride solution in sequence, dried over anhydrous magnesium sulfate, and dichloromethane is removed by distillation under reduced pressure to obtain the pyrimidine compound. The single dosage of busulfan is 20 mg / kg, and the number of injections of busulfan is 2-4 times, with an interval of 7 days between each injection; the pyrimidine compound is 20-40 wt % of busulfan, and the pyrimidine-based chalcone is 20-40 wt % of busulfan; and the amount of L-carnitine used is 3-12 wt % of busulfan.

2. The method for inducing sex reversal of an efficient female adult yellow croaker according to claim 1, wherein: The amount of the aromatase inhibitor in the compound feed is 80-120 mg / kg.

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