A large-scale breeding method for all-female large yellow croaker

Abstract

The present invention relates to the technical field of fish breeding and discloses a large-scale breeding method for all-female large yellow croaker. The present invention provides a method for inducing sex reversal in genetically female large yellow croaker by feeding them a formula feed containing letrozole, producing pseudo-male fish. The pseudo-male fish are then identified through live marking and screening. The pseudo-male fish are then fertilized with genetically female large yellow croaker to produce all-female fish, thereby achieving large-scale breeding of all-female seedlings. The method has high induction efficiency for inducing female large yellow croaker to pseudo-male fish, does not use androgens, and is safe and simple.

Description

Technical Field

[0001] The present invention relates to the technical field of fish breeding, and in particular to a large-scale breeding method for all-female large yellow croaker. Background Art

[0002] In aquaculture, many fish exhibit significant sexual dimorphism, with females growing faster and larger than males. Therefore, cultivating all-female fish populations is crucial for increasing economic returns and shortening the aquaculture cycle. Both domestically and internationally, great importance is attached to the cultivation of monosexual populations, particularly all-female fish. Overseas, all-female seedlings have been successfully cultivated in fish species such as rainbow trout and salmon. my country has made significant progress in the large-scale cultivation of all-female seedlings in fish such as yellow catfish, yellow croaker, and tongue sole. However, limited progress has been made in the large-scale cultivation of all-female large yellow croaker, both domestically and internationally.

[0003] Large yellow croaker is China's most productive marine aquaculture fish, with an annual output of approximately 254,000 tons. Large yellow croaker, belonging to the Sciaenidae family and the genus Pseudocrocea, is commonly known as yellow croaker or yellow croaker. It is a nearshore warm-water migratory fish primarily found along the coast of East Asia. In my country, it is primarily found in the northern South China Sea, the East China Sea, and the southern Yellow Sea. It is one of my country's most important commercial marine fish. Large yellow croaker is highly valued by consumers for its golden color, delicate texture, and nutritious properties. However, overfishing and environmental degradation have severely depleted wild large yellow croaker stocks, making them difficult to capture. Large yellow croaker has an XY sex determination system, and studies have shown that female large yellow croaker grow 30% faster than males. Females can reach market size in 18 months, while males require at least two years.

[0004] Therefore, the cultivation of all-female large yellow croaker fry is of great significance for improving the aquaculture yield and economic benefits of large yellow croaker. However, there are no effective technologies and methods for large-scale cultivation of all-female large yellow croaker at home and abroad.

[0005] There are currently two approaches to cultivating all-female large yellow croaker fry: one involves using estrogen treatment to induce all-female fish for further cultivation; the other involves inducing gynogenesis to produce all-female fry for further cultivation. However, both methods have their limitations. The former, due to the use of hormones, is not feasible for widespread production; the latter, requiring physical or chemical methods during embryogenesis, results in very low fry survival rates and is not suitable for large-scale cultivation of all-female large yellow croaker fry.

[0006] Therefore, in this field, there has not yet been an effective and feasible method for large-scale cultivation of all-female large yellow croaker seedlings. Summary of the Invention

[0007] In order to solve the technical problem of large-scale cultivation of all-female large yellow croaker, the present invention provides a large-scale cultivation method of all-female large yellow croaker.

[0008] The specific technical solutions of the present invention are:

[0009] The present invention provides a large-scale breeding method for all-female large yellow croaker, comprising the following steps:

[0010] S1. Inducing pseudo-male fish: Feeding genetically female large yellow croaker with a formula feed containing letrozole to cause sex reversal in genetically female large yellow croaker;

[0011] S2. Screening pseudo-male fish: Screening the large yellow croaker obtained in step S1 for large yellow croakers identified as female by genotype, and selecting and screening the large yellow croakers identified as female by genotype to obtain pseudo-male fish;

[0012] S3, induced fertilization: taking the genetically female large yellow croaker and the pseudo-male fish obtained in step S2 as broodstock, using oxytocin to induce fertilization of the broodstock;

[0013] S4. Fry cultivation: collecting fertilized eggs discharged by parent fish, hatching them to obtain all-female large yellow croaker fry, and cultivating them.

[0014] Pseudo-male fish refers to fish individuals with a female genotype that appear male in external morphology. The gonads of pseudo-male fish exhibit the morphological characteristics of normal male testes. The present invention provides a method for large-scale cultivation of all-female large yellow croaker, which induces genetically female large yellow croaker to become pseudo-male fish by feeding them with a formula feed containing letrozole, and uses live marking to screen out pseudo-male fish, and then further uses pseudo-male fish and genetically female large yellow croaker for fertilization to produce all-female fish, thereby achieving large-scale cultivation of all-female seedlings. The principle of the present invention for achieving large-scale cultivation of all-female large yellow croaker is specifically as follows:

[0015] This invention demonstrates for the first time that letrozole can be used to induce sex reversal in large yellow croaker. Letrozole is an aromatase inhibitor, currently used as a tumor suppressant. It is minimally harmful to fish and, therefore, can serve as a substitute for androgens in inducing sex reversal in large yellow croaker. Furthermore, the present invention demonstrates through experiments that using letrozole to induce sex reversal in large yellow croaker can achieve a 99% sex reversal rate in female fish, demonstrating that letrozole is a superior alternative to androgens for inducing sex reversal in large yellow croaker. Therefore, in step S1 of the present invention, genetically female large yellow croaker are fed a formulated feed containing letrozole, causing them to undergo sex reversal, becoming pseudo-males.

[0016] Since normal male fish may still be present in the large yellow croaker obtained after step S1 is induced, in step S2 of the present invention, marker screening is performed on the large yellow croaker obtained in step S1 to screen out individuals identified as pseudo-male fish and avoid the presence of normal male fish. Large yellow croaker is a fish with an XY sex determination system. The method for identifying and confirming pseudo-male fish in this step is that the genotype is identified as female. In this step, further, selection and screening are also performed to further ensure that the male broodstock that performs the next step of induced fertilization is pseudo-male fish. Avoiding the presence of normal male fish for fertilization is the basis for obtaining all-female large yellow croaker.

[0017] In step S3 of the present invention, genetically female large yellow croaker and the pseudo-male fish obtained in step S2 are used as broodstock, and oxytocin is used to induce fertilization of the broodstock. Since the broodstock are all XX genotypes, all-female fish can be obtained. Steps S1 and S2 are the basis for ensuring that all-female fish are obtained through fertilization.

[0018] In step S4 of the present invention, the fertilized eggs discharged by the parent fish are collected and hatched to obtain all-female large yellow croaker fry, which are then cultivated to achieve large-scale cultivation of all-female large yellow croaker.

[0019] Based on the above, the method provided by the present invention can achieve large-scale cultivation of all-female large yellow croaker. The present invention has been experimentally proven that in step S1, the proportion of genetically female large yellow croaker undergoing sex reversal can reach 99%, and the accuracy rate of screening out large yellow croakers identified as female by genotype in step S2 is 100%, thereby ensuring that the male parent undergoing induced fertilization and insemination is a pseudo-male fish with an accuracy rate of more than 99%. Further combined with the selection and screening of step S2, it is ensured that the male parent is a high-quality pseudo-male fish. Through the interaction of steps S1 to S4, a large-scale cultivation method of all-female large yellow croaker that is safe, simple, efficient, and easy to scale up is formed.

[0020] As a preferred embodiment of the above technical solution of the present invention, the method of feeding genetically female large yellow croaker with a compound feed containing letrozole is: starting from the age of genetically female large yellow croaker 38 to 45 days, the compound feed containing letrozole is fed by the method of incremental feeding of letrozole for a total of 80 to 102 days.

[0021] Feeding genetically female large yellow croaker with a formula feed containing letrozole to induce sex reversal and produce pseudo-male fish is the basis of the present invention's large-scale breeding method for all-female large yellow croaker. A high reversal rate for induced sex reversal is the key to ensuring this foundation. Inducing sex reversal in female large yellow croaker involves increasing the amount of letrozole in the formula feed starting at 38 to 45 days of age for 80 to 102 days, ensuring a 99% reversal rate.

[0022] As a preferred embodiment of the above technical solution of the present invention, the method of incremental feeding of letrozole is as follows: when the genetic female large yellow croaker is 38 to 60 days old, the letrozole content in the compound feed containing letrozole is 10 to 20 mg / kg; when the genetic female large yellow croaker is 60 to 80 days old, the letrozole content in the compound feed containing letrozole is 40 to 50 mg / kg; when the genetic female large yellow croaker is 80 to 140 days old, the letrozole content in the compound feed containing letrozole is 80 to 100 mg / kg.

[0023] The present invention first demonstrates that letrozole can be used to induce sex reversal in large yellow croaker, and provides a method for achieving a high sex reversal rate in female large yellow croaker using letrozole. The fact that letrozole can be used to induce sex reversal in other fish cannot be used as a reference in large yellow croaker.

[0024] A high sex reversal rate is crucial for the large-scale breeding of all-female large yellow croaker. The use of incremental letrozole feeding can further increase the sex reversal rate in genetically female large yellow croaker and reduce the effects of letrozole feeding on their growth and survival.

[0025] As a preferred embodiment of the above technical solution of the present invention, the large yellow croaker identified as female by genotype is screened out:

[0026] Using PIT live electronic tagging technology, a radio frequency tag is injected into the body of the large yellow croaker obtained in step S1 to achieve single individual identification; at the same time, the fin ray of each large yellow croaker corresponding to the radio frequency tag is taken, and genomic DNA is extracted for PCR amplification and gel electrophoresis. The large yellow croaker with a genotype identified as female is screened out through the difference in the amplified DNA bands.

[0027] The above method uses PIT electronic markers and molecular markers for screening. By screening out large yellow croakers identified as female by the above method, live screening can be achieved. PCR amplification and gel electrophoresis are performed using existing large yellow croaker sex-specific primers, and the sex of males and females is identified by the difference in amplified DNA bands. Fin ray DNA amplification of genetically female large yellow croaker only produces one band at 500bp, while fin ray DNA amplification of genetically male large yellow croaker produces two bands at 500bp and 390bp, thereby screening out pseudo-male large yellow croakers. In the large-scale cultivation of different batches of all-female large yellow croaker, the identification of female genotypes with 100% accuracy can be achieved every time.

[0028] As a preferred embodiment of the above technical solution of the present invention, in step S2, the selection and screening conditions are: normal body color, lively and healthy, and body weight greater than average weight.

[0029] Further combined with the selection and screening in step S2, it is ensured that the male parents are high-quality pseudo-male fish. By screening out high-quality pseudo-male fish with normal body color, lively and healthy body, and weight greater than the average weight as parents, it can be ensured that the fry cultivated on a large scale have the advantages of fast growth and stable traits.

[0030] Among them, the normal body color in the present invention refers to the body color of a healthy large yellow croaker, and the measure of liveliness and health can be compared among fish schools. The relatively lively ones in the fish school are lively and healthy individuals.

[0031] As a preferred embodiment of the above technical solution of the present invention, step S3 is:

[0032] Sexually mature genetically female large yellow croaker and the pseudo-male fish obtained in step S2 are placed together as broodstock, and are fed twice a day with green croaker and sandworm as bait, wherein the bait contains 3-5wt% vitamin C and 1-2wt% vitamin E; at the same time, the lighting in the breeding environment is controlled by an incandescent lamp, the incandescent lamp intensity is 60-650 lux, the lighting time is 12-16 hours / day, and the water temperature is controlled at 18-22°C; then, oxytocin is injected to cause the broodstock to lay eggs and fertilize.

[0033] The sexually mature genetically female large yellow croaker is placed together with the pseudo male fish obtained in step S2 and raised together, and the parent fish are nutritionally regulated under the condition of "feeding with green fish and sandworms twice a day, and the bait containing 3-5wt% vitamin C and 1-2wt% vitamin E". At the same time, the light and temperature of the induced fertilization environment of the parent fish are regulated by "controlling the lighting of the breeding environment with an incandescent lamp, the incandescent lamp intensity is 60-650 lux, the lighting time is 12-16 hours / day, and the water temperature is controlled at 18-22°C", which can improve the quality of fertilized eggs and increase the hatching rate.

[0034] More preferably, the oxytocin is oxytocin LHRH-A3.

[0035] As a preferred embodiment of the above technical solution of the present invention, the method for preparing the compound feed containing letrozole comprises the following steps:

[0036] Letrozole is dissolved in ethanol to obtain a letrozole solution, the letrozole solution is mixed with a compound feed, and then dried to obtain a compound feed containing letrozole.

[0037] More preferably, the drying method is natural drying.

[0038] As a preferred embodiment of the above technical solution of the present invention, in step S4, the cultivation conditions include: water temperature of 22-26°C, salinity of 22-30%, and pH of 8.0-8.2.

[0039] Further preferably, in step S4, the cultivation conditions further include: the water change amount and the aeration amount are gradually increased as the fry grows.

[0040] In the present invention, the water temperature for the larval, juvenile, and young stages of large yellow croaker cultivation is 22-26°C, the salinity is 22-30%, and the pH is 8.0-8.2. The amount of water exchange and aeration is gradually increased as the fry grow. The bait used for fry cultivation is, in order, rotifers, Artemia nauplii, and compound feed.

[0041] The compound feed for large yellow croaker used in the present invention refers to the standard GB / T 36206-2018.

[0042] Compared with the prior art, the present invention has the following technical effects:

[0043] (1) The present invention provides a method for large-scale breeding of all-female large yellow croaker, wherein genetically female large yellow croaker are induced to become pseudo-male fish by feeding them with a formula feed containing letrozole, the pseudo-male fish are screened out by live marking, and then the pseudo-male fish are further fertilized with genetically female large yellow croaker to produce all-female fish, thereby achieving large-scale breeding of all-female large yellow croaker seedlings. This method does not use androgens and is safe and simple.

[0044] (2) The present invention uses letrozole to induce sex reversal in large yellow croaker fry, achieving a sex reversal rate of over 99% in female fish. After induction, sex-specific molecular markers are used to screen for pseudo-male fish, with an accuracy rate of 100% for identifying pseudo-male fish. The present invention has high efficiency in inducing female large yellow croaker to pseudo-male fish, and is simple and feasible to operate.

[0045] (3) Furthermore, the present invention uses a letrozole-containing formula feed method to feed genetically female large yellow croaker starting from 40 to 60 days of age, using a letrozole-increasing feeding method, for a total of 80 to 100 days, to ensure that the reversal rate of sex reversal in genetically female large yellow croaker reaches 99%. A specific method for using letrozole to induce sex reversal to pseudo-male in genetically female large yellow croaker is provided, and the reversal rate is high. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] Figure 1 This is an electrophoresis detection diagram of genomic DNA of different large yellow croakers in Example 6 of the present invention;

[0047] Figure 2 This is a section of gonadal tissue from an 8-month-old female large yellow croaker obtained through large-scale cultivation in Example 7 of the present invention. DETAILED DESCRIPTION

[0048] The present invention will be further described below with reference to the embodiments and accompanying drawings.

[0049] In the embodiment of the present invention, the normal body color refers to the body color of a healthy large yellow croaker, and the measure of liveliness and health can be compared among fish schools. The relatively lively fish in the school are lively and healthy individuals.

[0050] In the embodiments of the present invention, the compound feed used for large yellow croaker is based on the standard GB / T 36206-2018.

[0051] In an embodiment of the present invention, the preparation method of the compound feed containing letrozole is as follows: letrozole is dissolved in 95% ethanol to obtain a letrozole solution, the letrozole solution is mixed with the compound feed, and the mixture is naturally dried to obtain the compound feed containing letrozole.

[0052] Example 1 Optimization of pseudo-male fish induction conditions

[0053] 200 vigorous, well-grown, 35-day-old common female large yellow croaker seedlings were selected and transferred to a cement pond. When they were 40 days old, they were fed a feed containing letrozole. At 40 to 60 days of age, the letrozole dosage was 15 mg / kg; at 60 to 80 days of age, the letrozole dosage was 45 mg / kg; and at 80 to 140 days of age, the letrozole dosage was 90 mg / kg. The letrozole treatment lasted for 100 days, after which the fish were fed a normal feed. During feeding, the fish were fed as evenly as possible and were fed as much as possible. The water was changed on time every day, with each water change of 200%. The letrozole dosage refers to the amount of letrozole in the feed, which is the same in the following examples.

[0054] Samples of 50 large yellow croaker were collected at 140 and 180 days of age, and the gonads were fixed and histological sections were taken. The results of the tissue sections showed that 49 of the 50 large yellow croaker sampled at 140 days of age were male, and the remaining one could not be identified. The results of the 180-day-old sample showed that all 50 large yellow croaker were male.

[0055] This shows that, in this embodiment, the induction rate of sex reversal in genetically female large yellow croaker was 99% when the formula feed containing letrozole was fed to the genetically female large yellow croaker.

[0056] Example 2 Optimization of pseudo-male fish induction conditions

[0057] Two hundred vigorous, well-grown, 35-day-old common yellow croaker fry were selected and transferred to a cement pond. From 50 days of age, they were fed a diet containing letrozole. The letrozole dosage was 15 mg / kg between 50 and 60 days of age; 45 mg / kg between 60 and 80 days of age; and 90 mg / kg between 80 and 140 days of age. The letrozole treatment lasted for 90 days, after which the fish were fed a normal diet. Feeding was performed as evenly as possible and to the point of satiation. Water was changed daily, with each change exceeding 200%.

[0058] Samples of 140-day-old and 180-day-old large yellow croaker were collected, with 50 fish sampled each time. After fixing the gonads, tissue sections were taken. The tissue section results showed that of the 50 large yellow croaker sampled at 140 days of age, 33 were male, 10 could not be identified, and 7 were female. The results of the 180-day-old sample showed that 28 were male, 12 could not be identified, and 10 were female.

[0059] This shows that, in this embodiment, the induction rate of sex reversal in genetically female large yellow croaker was 61% when the formula feed containing letrozole was fed to the genetically female large yellow croaker.

[0060] Further analysis showed that the induction start time of this embodiment was different from that of Example 1. Induction was started at 50 days of age in this embodiment, which resulted in a significant decrease in the induction rate of this embodiment. This indicates that when using a formula feed containing letrozole to induce sex reversal in genetically female large yellow croaker, if the induction is started too late, the induction rate will be greatly reduced.

[0061] Example 3 Optimization of pseudo-male fish induction conditions

[0062] Two hundred vigorous, well-grown, 35-day-old common yellow croaker fry were selected and transferred to a cement pond. At 30 days of age, they were fed a diet containing letrozole. From 30 to 60 days of age, the letrozole dosage was 15 mg / kg; from 60 to 80 days of age, the letrozole dosage was 45 mg / kg; and from 80 to 140 days of age, the dosage was 90 mg / kg. The letrozole treatment lasted for 80 days, after which the fish were fed a normal diet. Feeding was performed as evenly as possible and to the point of satiation. Water was changed daily, with each water change exceeding 200%.

[0063] During the induction process, abnormal mortality was found in the fry. The survival rate of large yellow croaker at 140 days of age was 60%.

[0064] Samples of 50 large yellow croaker were collected at 140 and 180 days of age, and the gonads were fixed and then histologically sectioned. The results of the histological sections showed that 47 of the 50 large yellow croaker sampled at 140 days of age were male, with the remaining three unable to be identified. The results of the 180-day-old sampled fish showed that 48 of the 50 were male, with the remaining two unable to be identified.

[0065] This shows that, in this embodiment, the induction rate of sex reversal in genetically female large yellow croaker was 95% when the formula feed containing letrozole was fed to the genetically female large yellow croaker.

[0066] Further analysis showed that compared with the start time of induction in Example 1, the start time of induction in this example was 30 days of age. The start time of induction was too early, resulting in abnormal death of the fry in this example. At the same time, the induction rate of the surviving fry decreased, indicating that in the process of using compound feed containing letrozole to induce sex reversal in genetically female large yellow croaker, the start time of induction cannot be too early. Starting the induction too early will not only lead to a decrease in the induction rate, but also cause the death of the fry in the process.

[0067] Example 4 Optimization of pseudo-male fish induction conditions

[0068] Two hundred vigorous, well-grown, 35-day-old common large yellow croaker fry were selected and transferred to a 3-ton cement pond. At 40 days of age, they were fed a diet containing letrozole. The letrozole dosage was 5 mg / kg from 40 to 60 days of age; 35 mg / kg from 60 to 80 days of age; and 70 mg / kg from 80 to 140 days of age. The letrozole treatment lasted for 100 days, after which the fish were fed a normal feed. Feeding was performed as evenly as possible and to the point of satiation. Water was changed daily, with each change exceeding 200%.

[0069] Samples of 50 large yellow croaker were collected at 140 and 180 days of age, and the gonads were fixed and then histologically sectioned. The results of the histological sections showed that of the 50 large yellow croaker sampled at 140 days of age, 46 were male, 3 of which could not be identified, and 1 was female. The results of the sampled fish at 180 days of age showed that 47 were male, 3 of which could not be identified.

[0070] This shows that, in this embodiment, the induction rate of sex reversal in genetically female large yellow croaker was 93% when the formula feed containing letrozole was fed to the genetically female large yellow croaker.

[0071] Further analysis showed that the amount of letrozole fed for induction in this embodiment was different from that in Example 1. Compared with Example 1, the feeding amount in this embodiment was less and the induction rate was significantly decreased, indicating that in the process of using a formula feed containing letrozole to induce sex reversal in genetically female large yellow croaker, the amount of letrozole fed for induction has an impact on the induction results.

[0072] Example 5 Optimization of pseudo-male fish induction conditions

[0073] Two hundred vigorous, well-grown, 35-day-old common large yellow croaker fry were selected and transferred to a 3-ton cement pond. At 40 days of age, they were fed a diet containing letrozole. The letrozole dosage was 25 mg / kg from 40 to 60 days of age; 55 mg / kg from 60 to 80 days of age; and 110 mg / kg from 80 to 140 days of age. The letrozole treatment lasted for 100 days, after which the fish were fed a normal diet. Feeding was performed as evenly as possible and to the point of satiation. Water was changed daily, with each change exceeding 200%.

[0074] Samples of 50 large yellow croaker were collected at 140 and 180 days of age, and the gonads were fixed and histological sections were taken. The results of the histological sections showed that 48 of the 50 large yellow croaker sampled at 140 days of age were male, of which 2 could not be identified; and 48 of the 180-day-old sampled were male, of which 2 could not be identified.

[0075] This shows that, in this embodiment, the induction rate of sex reversal in genetically female large yellow croaker was 96% when the formula feed containing letrozole was fed to the genetically female large yellow croaker.

[0076] Further analysis showed that the amount of letrozole used for induction feeding in this embodiment was different from that in Example 1. Compared with Example 1, the feeding amount in this embodiment was larger and the induction rate was slightly decreased, indicating that in the process of using a formula feed containing letrozole to induce sex reversal in genetically female large yellow croaker, the amount of letrozole used for induction feeding had a certain effect on the induction result.

[0077] Based on Examples 4 and 5, it is reasonable to infer that the letrozole content in the formula feed containing letrozole needs to be controlled, as both excessive and insufficient letrozole content will affect the induction rate. Therefore, our team further verified through experiments that controlling the letrozole content was the only variable and found that "when genetically female large yellow croaker is 38 to 60 days old, the letrozole content in the formula feed containing letrozole is 10 to 20 mg / kg; when genetically female large yellow croaker is 60 to 80 days old, the letrozole content in the formula feed containing letrozole is 40 to 50 mg / kg; when genetically female large yellow croaker is 80 to 140 days old, the letrozole content in the formula feed containing letrozole is 80 to 100 mg / kg." This has the best effect in inducing female fish to reverse to pseudo-male fish, with an induction rate of over 99%.

[0078] Example 6 Screening of high-quality pseudo-male fish

[0079] S1. 50,000 vigorous, well-grown, 35-day-old common yellow croaker seedlings were selected and transferred to a 15-ton cement pond. Sex reversal of the genetically female yellow croaker was induced using a formula feed containing letrozole, using the same method as in Example 1. Gonadal analysis of 140- and 180-day-old yellow croaker revealed a 99% male sex ratio.

[0080] In practice, it's impossible to directly obtain a large number of female fry. The 50,000 fry in this particular case were a mixed group of males and females, so the induction results here can only be expressed as a "maleness ratio." A 99% maleness ratio among the fry obtained after step S1 means that 99% of the fry are externally male, but have a mixed genotype. Therefore, subsequent live individuals must be identified to identify genotypically female individuals.

[0081] S2. The large yellow croaker obtained in step S1 is cultivated and the first selection is carried out at the age of 6 months. The average body weight of the 6-month-old fish is 19.9±6.92 and the average body length is 10.5±1.26. The selection criteria are normal body color, lively and healthy, and a body weight greater than 20g, with a retention rate of 20%. At the age of 8 months, 3,000 large yellow croakers with an average body weight above the average are selected. Using PIT live electronic marking technology, a radio frequency tag is injected into the body of the marked fish to realize a single individual identification method; and the fin rays are preserved in anhydrous ethanol and stored at -20°C, and correspond one to one with the radio frequency tag serial number. Since the large yellow croaker population has individuals with genotypes XX and XY, molecular specific markers are used to distinguish XX and XY individuals. The genomic DNA of the test samples was extracted using the phenol-chloroform extraction method; PCR amplification was performed using sex-specific primers for large yellow croaker, and the PCR amplification products were subjected to 2% agarose gel electrophoresis for sex identification. Females showed only one band at 500 bp, while males showed two bands at 500 bp and 390 bp, which were used to screen and confirm pseudo-male fish. Figure 1 The electrophoresis detection diagram is shown.

[0082] Primer sequences are: LC-SEQ-F: 5'-TATTTCTACAGAAGCCCAGCACAG-3'; LC-SEQ-R: 5'-GTCCTCCTTGTACTCATCAAGG-3'. The PCR reaction volume was 20 μL and consisted of: 10 μL 2× PCR Taq mix, 0.5 μL each of primers F and R, 1 μL 50 ng / μL DNA, and 8 μL double-distilled water. The PCR protocol was as follows: 94°C denaturation for 5 minutes, followed by 35 cycles of denaturation at 94°C for 30 seconds, annealing at 61°C for 30 seconds, and extension at 72°C for 30 seconds, followed by extension at 72°C for 10 minutes. Electrophoresis was performed on a 2% agarose gel at 180 V for 20 minutes.

[0083] Example 7 Oxygenation and Fertilization and All-Female Fish Cultivation

[0084] At the age of 12 months, the average weight of the pseudo-male fish obtained in Example 6 was 84.5±4.52g. 300 pseudo-male fish weighing more than 85g were selected and continued to be cultivated until sexual maturity. 30 2-year-old pseudo-male fish and 30 normal female fish were selected and placed in a 20-ton cement pool. Nutritional regulation and light and temperature regulation were performed on the pseudo-male fish and normal female fish. Nutritional regulation refers to using ice-fresh green fish and sandworms as bait, adding 4% vitamin C and 1.5% vitamin E of the bait weight, and feeding twice a day; light and temperature regulation is to use a 65W incandescent lamp to control the surface illumination of the broodstock pond, the illumination intensity is 500lux, the illumination time is transitioned from 12 hours / day to 16 hours / day, and the water temperature is controlled within the range of 18-22°C. Then, the oxytocin LHRH-A3 was injected to make them spawn and fertilize, and a total of 2kg of fertilized eggs were collected. The fertilized eggs were cultivated, and a total of 156,000 seedlings with a total length of 3cm were cultivated. During the larval, juvenile, and fingerling stages, the water temperature is controlled between 22 and 30°C, the salinity is between 22 and 30%, and the pH is between 8.0 and 8.2. Water changes and aeration levels are gradually increased as the fry grow. Rotifers, Artemia nauplii, and compound feed are used, in that order.

[0085] When the fry were cultured to 140 days of age, 50 fish were sampled and dissected to remove the gonads. All were females as observed by tissue section. At the same time, fin rays were taken to extract DNA to verify genetic sex using the same method as in Example 6. The results showed that all were females.

[0086] At 8 months old, 50 fish were sampled and dissected to remove the gonads. All of them were female fish. Figure 2 shown.

[0087] In the present invention, the larval stage of large yellow croaker refers to the stage of large yellow croaker after hatching, which is the larval stage, and this stage usually lasts from several weeks to several months. The juvenile stage of large yellow croaker refers to the stage of large yellow croaker gradually growing and developing, entering the juvenile stage, generally, the juvenile stage of large yellow croaker lasts from several months to one year. Juvenile stage: Large yellow croaker gradually enters the juvenile stage over time, and this stage usually lasts from one to two years.

[0088] The compound feed for large yellow croaker used in the present invention refers to the standard GB / T 36206-2018.

[0089] Unless otherwise specified, the raw materials and equipment used in the present invention are commonly used in the art; the methods used in the present invention are conventional methods in the art unless otherwise specified.

[0090] The above description is only a preferred embodiment of the present invention and does not limit the present invention in any way. Any simple modification, change and equivalent transformation made to the above embodiment based on the technical essence of the present invention still fall within the scope of protection of the technical solution of the present invention.

Claims

1. A large-scale breeding method for all-female large yellow croaker, characterized by: The following steps are involved: S1. Inducing pseudo-male fish: Starting from 38 to 45 days of age, genetically female large yellow croaker are fed a formula feed containing letrozole by increasing the amount of letrozole for a total of 80 to 102 days to induce sex reversal. The method of incremental feeding of letrozole is as follows: when the genetic female large yellow croaker is 38 to 60 days old, the letrozole content in the compound feed fed is 10 to 20 mg / kg; when the genetic female large yellow croaker is 60 to 80 days old, the letrozole content in the compound feed fed is 40 to 50 mg / kg; when the genetic female large yellow croaker is 80 to 140 days old, the letrozole content in the compound feed fed is 80 to 100 mg / kg; S2. Screening pseudo-male fish: Screening the large yellow croaker obtained in step S1 for large yellow croakers identified as female by genotype, and selecting and screening the large yellow croakers identified as female by genotype to obtain pseudo-male fish; S3, induced fertilization: taking the genetically female large yellow croaker and the pseudo-male fish obtained in step S2 as broodstock, using oxytocin to induce fertilization of the broodstock; S4. Fry cultivation: collecting fertilized eggs, hatching to obtain all-female large yellow croaker fry, and cultivating them.

2. The large-scale breeding method for all-female large yellow croaker as claimed in claim 1, wherein: The method for screening out large yellow croakers identified as female by genotype is: Using PIT live electronic tagging technology, a radio frequency tag is injected into the body of the large yellow croaker obtained in step S1 to achieve single individual identification; at the same time, the fin ray of each large yellow croaker corresponding to the radio frequency tag is taken, and genomic DNA is extracted for PCR amplification and gel electrophoresis. The large yellow croaker with a genotype identified as female is screened out through the difference in the amplified DNA bands.

3. The large-scale breeding method of a fully female large yellow croaker as claimed in claim 1, wherein: In step S2, the selection and screening conditions are: normal body color, active and healthy, and body weight greater than average.

4. The large-scale breeding method of a fully female large yellow croaker as claimed in claim 1, wherein: The step S3 is: A sexually mature genetically female large yellow croaker and the pseudo-male fish obtained in step S2 are placed together as broodstock, and fed twice a day with green croaker and sandworm as bait, wherein the bait contains 3-5wt% vitamin C and 1-2wt% vitamin E; at the same time, the lighting in the breeding environment is controlled by an incandescent lamp, the incandescent lamp illumination intensity is 60-650 lux, the lighting time is 12-16 hours / day, and the water temperature is controlled at 18-22°C; then, oxytocin is injected to cause the broodstock to lay eggs and fertilize.

5. The large-scale breeding method of an all-female large yellow croaker as described in claim 1 or 4, characterized in that: The oxytocin is oxytocin LHRH-A3.

6. The large-scale breeding method of an all-female large yellow croaker as claimed in claim 1, wherein: The preparation method of the compound feed containing letrozole comprises the following steps: Letrozole is dissolved in ethanol to obtain a letrozole solution, and the letrozole solution is mixed with a compound feed, followed by drying to obtain a compound feed containing letrozole.

7. The large-scale breeding method of an all-female large yellow croaker as claimed in claim 1, wherein: In step S4, the cultivation conditions include: water temperature of 22-26° C., salinity of 22-30%, and pH of 8.0-8.

2.

8. The large-scale breeding method for all-female large yellow croaker as claimed in claim 7, wherein: In step S4, the cultivation conditions also include: the water change volume and the aeration volume gradually increase as the fry grows.

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