A method for producing all-female triploid broodstock of large yellow croaker

Abstract

This invention provides a method for breeding large yellow croaker that is entirely female and triploid. First, pseudo-male large yellow croaker germplasm is obtained using sex reversal technology. Then, pseudo-males are identified using molecular markers for sex determination. These pseudo-males are mated with normal females to obtain wholly female fertilized eggs. Hydrostatic pressure treatment is then used to inhibit the expulsion of the second polar body from these fertilized eggs, inducing the formation of wholly female triploid large yellow croaker. Simultaneously, the concentration of the sex reversal reagent, the usage cycle, water temperature, accumulated temperature, pressurization time, pressure value, and hydrostatic pressure treatment duration were all optimized to achieve a 100% triploid rate and a 100% female rate, meeting the requirements for large-scale, industrialized breeding.

Description

Technical Field

[0001] This invention belongs to the field of aquatic genetic breeding technology, specifically the field of fish sex control and ploidy breeding technology, and specifically relates to a method for breeding large yellow croaker that is all female triploid. Background Technology

[0002] Large yellow croaker ( Larimichthys crocea Large yellow croaker is considered my country's "national fish" of marine life. In 2025, the national aquaculture production of large yellow croaker reached 292,615 tons, accounting for 13.53% of the national marine fish aquaculture production (2,162,149 tons, according to the "2025 China Fisheries Statistical Yearbook"), thus holding an important position in my country's marine fish aquaculture industry. The commercial traits of large yellow croaker, such as growth rate, meat quality, and stress resistance, are closely related to gonadal development and chromosome ploidy.

[0003] Large yellow croaker exhibits sexual dimorphism, with females showing significantly superior growth traits compared to males. Females are on average 30.77% heavier and 18.09% heavier in carcass than males, demonstrating a clear female growth advantage. Triploid large yellow croakers are sterile, with suppressed gonadal development, leading to a greater preference for nutrient allocation to body growth. This results in faster growth, firmer flesh, higher meat yield, and suitability for intensive aquaculture.

[0004] In existing technologies, there have been explorations into the breeding of all-female triploid fish. For example, Chinese invention patent CN111937783A provides a method for large-scale breeding of all-female triploid rainbow trout, and Chinese invention patent CN111937783A provides a method for efficiently inducing all-female triploid yellow croaker. Both methods first induce sex reversal in a gynogenetic population to obtain pseudo-males, and then collect sperm from the pseudo-males and eggs from the gynogenetic females for artificial insemination to screen the triploid fish. However, this method is not suitable for large yellow croaker because large yellow croaker have a strong gregarious nature and require a large population size to feed, grow, and develop normally. The population obtained through gynogenetic development is much smaller than the naturally bred population, which usually makes it difficult to guarantee normal feeding, growth, development, and survival rates. Therefore, it is difficult to obtain mature pseudo-males by sex reversing the gynogenetic population. At the same time, due to differences in fish morphology and performance, fertilized eggs also differ significantly, and induction conditions between different fish species cannot be mutually referenced. Summary of the Invention

[0005] This invention addresses the aforementioned problems by combining the effects of sex and ploidy to propose a targeted breeding technology for all-female triploid large yellow croaker. It organically integrates the growth advantages of females with the sterility and high meat yield characteristics of triploids. All-female fertilized eggs are obtained by mating pseudo-males with normal female large yellow croakers, and the technology is applied on a large scale using optimal hydrostatic pressure treatment. This achieves a synergistic improvement in growth rate and meat yield traits in the genetic improvement of large yellow croaker, providing a new technical pathway for the creation of high-quality large yellow croaker germplasm resources.

[0006] The technical solution of this invention is summarized as follows: First, pseudo-male large yellow croaker germplasm is obtained through sex reversal technology. Then, pseudo-males are identified using sex identification molecular markers. These pseudo-males are mated with normal females to obtain fully female fertilized eggs. Hydrostatic pressure treatment is then used to inhibit the expulsion of the second polar body from the fully female fertilized eggs, inducing the formation of fully female triploid large yellow croakers. The method has optimized the concentration of the sex reversal reagent, the usage cycle, water temperature, accumulated temperature, pressurization time, pressure value, and hydrostatic pressure treatment duration to achieve a triploid rate of 100% and a female rate of 100%, meeting the requirements for large-scale, industrialized seed production.

[0007] The core innovations include:

[0008] (1) Construct a sex reversal induction program specific to large yellow croaker to obtain high-quality pseudo-male fish;

[0009] (2) Combine sex control technology with polyploid breeding technology to obtain all-female triploids;

[0010] (3) Form a robust breeding system with a standardized rate of 100% triploidy and 100% female.

[0011] The technical solution adopted in this invention is as follows:

[0012] 1. Induction and identification of pseudo-male large yellow croaker

[0013] (1) Selection of broodstock and fry breeding: During the breeding season of large yellow croaker, select two-year-old broodstock with well-developed gonads, with a female-to-male ratio of 2:1 (e.g., 200 females and 100 males). Induce spawning by injecting luteinizing hormone-releasing hormone (GnRH-A3) into the broodstock at a dose of approximately 5 μg / kg of fish body weight. About 30 hours after injection, the broodstock will begin to spawn and fertilize. Collect high-quality buoyant fertilized eggs and incubate them in seawater at 25–26 ℃ for about 26 hours to obtain newly hatched fry. About 3 days later, the fry will start eating. They will be fed rotifers as their first food and gradually transitioned to feeding them brine shrimp, copepods, and formulated feed. During the fry rearing process, the water temperature should be maintained at 24–26 ℃.

[0014] (2) Sex reversal: When the large yellow croaker fry reach 1 month of age, 100,000 high-quality fry with good vitality and fast growth (approximately 1.5 cm in total length) are selected and temporarily raised for 10 days to induce pseudo-males. Letrozole (LTZ) is used to induce sex reversal in females by feeding, with a concentration of 10 mg of letrozole per kilogram of feed. The large yellow croaker fry are fed a compound feed containing 10 mg / kg of letrozole from 40 days of age, and this feeding continues for 80 to 120 days. Then, they are fed a compound feed without letrozole, and the sex-reversed group is further cultured until sexual maturity.

[0015] (3) Histological sex determination: The sex of the 120-day-old sex-reversed group was determined by histological sectioning. The fact that 100% of the fish were male indicated that pseudo-males were successfully obtained.

[0016] 2. Preparation of fertilized eggs from all female large yellow croakers

[0017] (1) Selection of candidate parents: Select parents that grow fast, have well-developed gonads, and are easy to extract sperm from the sex-reversed mature population cultured above. Inject each fish with an electronic marker to identify the individual and collect fin rays to extract DNA for genetic sex identification.

[0018] (2) Identification and screening of pseudo-male fish: Using molecular markers for sex identification of large yellow croaker, individuals with a female genotype were identified, and pseudo-male fish with a male phenotype and a female genotype were obtained by screening based on electronic marker codes.

[0019] (3) In a two-year-old population of common large yellow croaker that had not undergone sex reversal, mature females with rapid growth and well-developed gonads were selected as female parents; the pseudo-males selected above were used as male parents; luteinizing hormone-releasing hormone (GnRH-A3) was injected into the parents to induce spawning, with an injection dose of approximately 5 μg / kg of fish body weight. After spawning induction, all parents were placed in the same parent fish pond, and the seawater temperature was controlled at around 24–26 ℃.

[0020] (4) About 30 hours after the injection of oxytocin, first take the female fish from the parent fish pond, squeeze the abdomen of the female fish to lay the eggs in a clean beaker; then take the pseudo-male fish, squeeze the abdomen of the pseudo-male fish to collect the pseudo-male fish sperm into the beaker containing the eggs; throughout the process, water must be strictly prohibited from entering the beaker.

[0021] (5) After mixing the sperm and eggs in the beaker evenly with a brush or feather, add clean seawater at a temperature of 24-26 °C for fertilization and obtain fertilized eggs. Start the fertilization timer immediately after adding seawater.

[0022] 3. Induction of all-female triploidy in large yellow croaker

[0023] (1) Pressure treatment of fertilized eggs: When the fertilization time reaches 2 minutes, the fertilized eggs are placed in a hydrostatic pressure device for pressure treatment to prevent the polar body from being discharged. The pressure is 7000-8000 PSI and the treatment time is 3 minutes. The seawater temperature in the hydrostatic pressure device is controlled at 24-26 ℃.

[0024] (2) Hatching of fertilized eggs: After the hydrostatic treatment is completed, the fertilized eggs are immediately placed in a hatching tank with a water temperature of 25-26 ℃ for hatching.

[0025] (3) Ploidy detection: After the fertilized eggs hatch into newly hatched larvae, the triploid rate was detected. Fifty newly hatched larvae were randomly selected and placed one by one in a petri dish. 800 μL of lysis-staining solution was added, and the fins were cut 20-30 times with a blade. The mixture was then filtered through a screen into a test tube. Another 800 μL of lysis-staining solution was added to the petri dish, and the mixture was again filtered through a screen into the same test tube. The DNA content of the samples was detected using a ploidy analyzer. The optimal gain value for the ploidy analyzer was set to 597.0 V, the threshold value to 61.89%, and the fluidics value to 0.5 μL / sec. The ploidy of the large yellow croaker diploid newly hatched larvae was used as a reference standard to calculate the ploidy of the samples.

[0026] (4) Sex determination: 50 newly hatched fry were randomly selected, and DNA was extracted from each fry for genetic sex determination. Molecular markers for sex determination of large yellow croaker were used to identify the genetic sex of each newly hatched fry.

[0027] Invention Function and Effect

[0028] This invention establishes a pseudo-male induction technology for large yellow croaker. Based on a natural breeding population of large yellow croaker, sex reversal is induced, and pseudo-males are screened using molecular markers for sex identification. These pseudo-males are then mated with normal female large yellow croaker to obtain fully female fertilized eggs. Optimal hydrostatic pressure treatment is then used to achieve large-scale production of fully female triploid large yellow croaker. This invention opens up a new technical solution for the organic combination of sex-controlled breeding and polyploid breeding of large yellow croaker, possessing strong innovation and practicality. The transformation and application of this technology is expected to enable large-scale production and commercial promotion of fully female triploid large yellow croaker, providing innovative impetus for the development of my country's large yellow croaker seed industry. Attached Figure Description

[0029] Figure 1 This shows a technical roadmap for the breeding of pseudo-male large yellow croaker;

[0030] Figure 2 This shows the technical roadmap for the all-female triploid breeding of large yellow croaker;

[0031] Figure 3 The diagram shows a triploid ploidy test result for large yellow croaker. Detailed Implementation

[0032] The implementation of the present invention will be described in detail below with reference to the accompanying drawings and embodiments. The following embodiments are implemented under the premise of the technical solution of the present invention, and detailed implementation methods and specific operation processes are given. However, the protection scope of the present invention is not limited to the following embodiments.

[0033] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Every smaller range between any stated value or intermediate value within a stated range, and any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0034] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0035] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be readily apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0036] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0037] Figures 1-3 The combined presentation illustrates the complete technical route for the all-female triploid breeding of large yellow croaker in this invention, with the specific steps as follows:

[0038] 1. Parent selection and seedling propagation

[0039] During the breeding season of large yellow croaker, two-year-old broodstock with well-developed gonads and a weight of 0.8–1.5 kg were selected (400 females and 200 males). The broodstock were injected with luteinizing hormone-releasing hormone (GnRH-A3) at a dose of approximately 5 μg / kg body weight to induce spawning. About 30 hours after injection, the broodstock began spawning and fertilization. High-quality, buoyant fertilized eggs were collected and incubated in seawater at 25–26℃ for approximately 26 hours to obtain newly hatched larvae. After about 3 days, the larvae began to eat. They were initially fed rotifers as their first food, gradually transitioning to a diet of brine shrimp, copepods, and formulated feed. Throughout the larval rearing process, the water temperature was maintained at 24–26℃.

[0040] 2. The process of inducing sex reversal

[0041] When the large yellow croaker fry reach one month of age, 100,000 high-quality fry with good vitality and rapid growth (approximately 1.5 cm in total length) are selected and temporarily raised for 10 days before being used to induce pseudo-males. Letrozole (LTZ), an androgen, is used to induce sex reversal in the females via feeding at a concentration of 10 mg of letrozole per kilogram of feed. From 40 days of age, the large yellow croaker fry are fed a formulated feed containing 10 mg / kg letrozole, continuously for 80 to 120 days. Then, they are fed a formulated feed without letrozole, and the sex-reversed group continues to be cultured until sexual maturity.

[0042] 3. Histological determination of sex

[0043] The sex of the 120-day-old sex-reversed group was identified by histological sectioning, and the results showed that males accounted for 100%, indicating that pseudo-males had been successfully induced.

[0044] 4. Selection of candidate pseudomale parents

[0045] From the sex-reversed mature population cultivated above, 500 parent fish with fast growth, well-developed gonads, and easy sperm extraction were selected. Each fish was injected with an electronic marker to identify the individual, and fin rays were collected to extract DNA for genetic sex determination.

[0046] 5. Identification and screening of pseudo-male fish

[0047] By applying molecular markers for sex identification of large yellow croaker, individuals with a genetically female genotype were identified, and pseudo-males with a male phenotype and a female genotype were obtained by screening based on electronic marker codes.

[0048] 6. Phenotype and genotype are both used for parental selection and induced labor in females.

[0049] In a two-year-old population of common large yellow croaker that had not undergone sex reversal, mature females with rapid growth and well-developed gonads were selected as female broodstock; the pseudo-males selected above were used as male broodstock; luteinizing hormone-releasing hormone (GnRH-A3) was injected into the broodstock to induce spawning, with an injection dose of approximately 5 μg / kg of fish body weight. After spawning induction, all broodstock were placed in the same broodstock tank, and the seawater temperature was controlled at approximately 24–26 ℃.

[0050] 7. Artificial insemination

[0051] Approximately 30 hours after induced spawning, female fish are first removed from the broodstock tank, and their abdomens are squeezed to extract eggs into a clean beaker. Then, pseudo-male fish are removed, their abdomens are squeezed, and their sperm is collected into the beaker containing the eggs. Water must be strictly prohibited from entering the beaker throughout the entire process. The sperm and eggs in the beaker are mixed thoroughly with a brush or feather, and then clean seawater at 24–26 °C is added for fertilization to obtain fertilized eggs. Fertilization timing begins immediately after the seawater is added.

[0052] 8. Stress treatment of the fertilized egg to prevent expulsion of polar bodies.

[0053] At 2 minutes into the fertilization time, the fertilized eggs are placed in a hydrostatic pressure device for pressure treatment to prevent the polar body from being expelled. The pressure is 7000–8000 PSI, and the treatment time is 3 minutes. The seawater temperature in the hydrostatic pressure device is controlled at 24–26 °C.

[0054] 9. Hatching of fertilized eggs

[0055] After the hydrostatic treatment is completed, the fertilized eggs are immediately placed in an incubation tank with a water temperature of 25-26 ℃ for incubation and further cultivation.

[0056] 10. Plumpness detection

[0057] After the fertilized eggs hatched into newly hatched larvae, the triploidity rate was determined. Fifty newly hatched larvae were randomly selected and placed one by one in a petri dish. 800 μL of lysis-staining solution was added first, and the fins were cut 20-30 times with a blade. The mixture was then filtered through a screen into a test tube. Another 800 μL of lysis-staining solution was added to the petri dish, and the mixture was again filtered through a screen into the same test tube. The DNA content of the samples was measured using a ploidy analyzer. The optimal gain value for the ploidy analyzer was set to 597.0 V, the threshold value to 61.89%, and the fluidics value to 0.5 μL / sec. Using diploid newly hatched larvae of large yellow croaker as a reference standard, the ploidy of the measured samples was calculated. The results showed a triploidity rate of 100%.

[0058] 11. Gender identification

[0059] Fifty newly hatched fry were randomly selected, and their DNA was extracted for genetic sex determination. Molecular markers for sex determination of large yellow croaker were applied to identify the genetic sex of each newly hatched fry, and the results showed that 100% of the fry were female.

[0060] The undescribed parts of this invention are the same as or implemented using existing technology. The applicant declares that this invention is illustrated through the above embodiments, but the invention is not limited to the above detailed methods, i.e., it does not mean that the invention must rely on the above detailed methods to be implemented. Those skilled in the art should understand that any improvements to this invention, equivalent substitutions of raw materials for the product of this invention, additions of auxiliary components, and selection of specific methods all fall within the protection and disclosure scope of this invention.

Claims

1. A method for breeding large yellow croaker that is entirely female and triploid, characterized in that, Includes the following steps: A. Induction and identification of pseudo-male fish in large yellow croaker (1) Selection of parent fish and breeding of seedlings: During the breeding season of large yellow croaker, two-year-old male and female parent fish with well-developed gonads are selected. The parent fish are injected with luteinizing hormone-releasing hormone to induce spawning. After fertilization, high-quality floating fertilized eggs are collected and placed in seawater at 25-26℃ to hatch and obtain newly hatched fry for seedling cultivation. (2) Sex reversal: When the large yellow croaker fry are raised to 1 month old, select high-quality fry with good vitality and fast growth, temporarily raise them for 10 days, and use them to induce pseudo-male fish; use the androgen letrozole to induce sex reversal in female fish by feeding. The large yellow croaker fry are fed with a compound feed containing 10 mg / kg letrozole from 40 days old, and continue to be fed for 80 to 120 days; then feed them with a compound feed without letrozole, and continue to raise the sex reversal group to sexual maturity. (3) Histological sex determination: The sex of the 120-day-old sex-reversed group was determined by histological sectioning. A 100% male ratio indicated that pseudo-males were successfully obtained. B. Preparation of fertilized eggs from all female large yellow croakers (1) Selection of candidate parents: Select parents that grow fast, have well-developed gonads, and are easy to extract sperm from the sex-reversed mature population cultured above. Inject each fish with an electronic marker to identify the individual and collect fin rays to extract DNA for genetic sex identification. (2) Identification and screening of pseudo-male fish: Using molecular markers for sex identification of large yellow croaker, individuals with a female genotype were identified, and pseudo-male fish with a male phenotype and a female genotype were obtained by screening based on electronic marker codes. (3) In the two-year-old population of common large yellow croaker without sex reversal operation, mature female fish with fast growth and good gonad development were selected as female parents; the pseudo-male fish selected above were used as male parents; luteinizing hormone-releasing hormone was used, and then all parents were placed in the same parent fish pond, and the seawater temperature was controlled at 24-26℃. (4) 30 hours after the injection of oxytocin, first take the female fish from the parent fish pond, squeeze the abdomen of the female fish to lay the eggs into a clean container; then take the pseudo-male fish, squeeze the abdomen of the pseudo-male fish to collect the pseudo-male fish sperm into the container containing the eggs; throughout the process, water must be strictly prohibited from entering the beaker. (5) After gently mixing the sperm and eggs evenly, add clean seawater at a temperature of 24-26℃ for fertilization, obtain fertilized eggs, and start the fertilization timer immediately after adding seawater. C. The large yellow croaker all-female triploid induction protocol includes the following steps: (1) Pressure treatment of fertilized eggs: When the fertilization time reaches 2 minutes, the fertilized eggs are placed in a hydrostatic pressure device for pressure treatment to prevent the polar body from being discharged. The pressure is 7000-8000 PSI and the treatment time is 3 minutes. The seawater temperature in the hydrostatic pressure device is controlled at 24-26 ℃. (2) Hatching of fertilized eggs: After the hydrostatic treatment is completed, the fertilized eggs are immediately placed in a hatching tank with a water temperature of 25-26 ℃ for hatching; (3) Ploidy detection and sex identification: After the fertilized eggs hatch into newly hatched fry, the triploid rate is detected and the sex is identified.

2. The method for breeding large yellow croaker that is entirely female and triploid according to claim 1, characterized in that: in, In step A, the ratio of female to male fish in the parent selection and seedling breeding steps is 2:1; The luteinizing hormone-releasing hormone was selected from GnRH-A3, and the injection dose was 5 μg / kg fish body weight; The fry rearing method is as follows: Three days after hatching, the fry are fed with rotifers as their first food, gradually transitioning to feeding them brine shrimp, copepods, and formulated feed. Throughout the rearing process, the water temperature should be maintained at 24–26℃. In the sex reversal process, fish fry with a total length of 1.5cm are selected.

3. The method for breeding large yellow croaker that is entirely female and triploid according to claim 1, characterized in that: in, In step B, the sperm and egg are gently mixed evenly using a brush or feather.

4. The method for breeding large yellow croaker that is entirely female and triploid according to claim 1, characterized in that: in, In step C, the method for detecting triploidy rate is as follows: Randomly select a certain number of newly hatched larvae, place them one by one in a petri dish, add lysis-staining mixture first, cut the fins multiple times with a blade, filter through a screen into a test tube, add the same volume of lysis-staining mixture to the petri dish, filter through a screen into the same test tube, and use a ploidy analyzer to detect the DNA content of the sample: the optimal gain value of the ploidy analyzer is set to 597.0V, the threshold value to 61.89%, and the fluidics value to 0.5ul / sec. Using large yellow croaker diploid newly hatched larvae as a reference standard, calculate and determine the ploidy of the sample.

5. The method for breeding large yellow croaker that is entirely female and triploid according to claim 4, characterized in that: in, Cut the fins 20-30 times with a blade.

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