Method for mass production of all-male salmon trout

Abstract

This invention discloses a method for large-scale breeding of all-male salmon and trout, belonging to the field of fish sex regulation breeding technology. The method includes: collecting fertilized eggs from different families of the same species of salmon and trout, artificially incubating them to obtain fry, and inducing sex reversal in the fry by using 17β-estradiol when they begin feeding; subsequently, raising the fry to sexual maturity, selecting individuals with a female phenotype, and then using sdY as a sex identification gene, selecting pseudo-females with genotype XY by quantitative real-time PCR; pairing the sexually mature pseudo-females with genotype XY with normal males with genotype XY, and obtaining super-males with genotype YY through artificial insemination; pairing the sexually mature super-males with females with genotype XX, and producing all-male salmon and trout with genotype XY on a large scale through artificial insemination. This invention overcomes the problem that existing technologies cannot achieve large-scale breeding of all-male salmon and trout.

Description

Technical Field

[0001] This invention relates to the field of fish sex regulation breeding technology, and in particular to a method for large-scale breeding of all-male salmon and trout. Background Technology

[0002] Parthenogenesis in fish, as an important genetic breeding technology, has shown great potential in aquaculture. By controlling the sex of fish, aquaculture efficiency can be significantly improved, driving industrial upgrading. For salmonid fish, which have significant economic value, males and females often exhibit different growth rates, such as rainbow trout and Atlantic salmon. Male salmonids typically reach sexual maturity earlier than females, while females are larger. Therefore, when the production target is fish meat, farming only females yields greater profits. In recent years, polydeoxyribonucleotides (PDRNs) extracted from the testes of male salmonids have become a hot topic in anti-aging research. Because they are very similar to human DNA, when injected into the skin, these bioactive substances act as cellular messengers, stimulating collagen formation. They are often used for facial rejuvenation, particularly targeting fine lines, wrinkles, and decreased skin elasticity, showing great application potential in beauty and health products. Large-scale breeding of all-male fish can provide sufficient raw materials for these industries, opening up new application markets.

[0003] Currently, there are two commonly used methods abroad to obtain all-male fish populations. One method involves inducing female fish (genotype XX) from mixed sexes to develop gonads and testes into pseudo-males. This technique has been widely used for the large-scale production of all-female salmon and trout, where sperm from pseudo-males (genotype XX) is fertilized with eggs from normal females to achieve mass production of all females with the genotype XX. However, the induction rate of pseudo-males is not stable, requiring continuous feeding of each batch of juveniles with androgen-containing feed, which is cumbersome, time-consuming, and costly. Furthermore, the testes of pseudo-males are of far inferior quality to those of normal males, making it unsuitable for large-scale production of all-male fish. The other method involves creating a supermale population (genotype YY) and fertilizing the eggs of normal females with the sperm of supermales to obtain all-normal male fish (genotype XY). Internationally, theoretical research on the creation of super-male tilapia has been largely conducted on tilapia. As one of the world's most produced aquaculture species, super-male tilapia (YY) are considered to have better growth performance than mixed-sex or normal single-sex populations, thus yielding higher profits from their cultivation. Conversely, because male salmonids traditionally have less advantage in aquaculture than females, there is relatively less theoretical research on super-male salmonids abroad. The creation of super-male salmonids is primarily aimed at eliminating invasive species, and the related procedures and creation parameters are unstable and remain at the experimental level. Standardized and large-scale production of super-male and all-male salmonids has not yet been achieved.

[0004] my country's salmon and trout industry started relatively late compared to other countries. Single-sex breeding technology mainly focuses on the large-scale production of all-female rainbow trout, with no reports of large-scale production of super-male (YY) or all-male (XY) salmon and trout. The existing pseudo-male breeding technology is not designed to create truly male fish, let alone to create a standardized, large-scale all-male population. With the booming demand for PDRN products from the medical aesthetics and health industry in recent years, the added value of male salmon and trout has increased. A breakthrough in the large-scale production technology of all-male salmon and trout could bring new economic growth to the salmon and trout industry. Summary of the Invention

[0005] The purpose of this invention is to provide a method for large-scale breeding of all-male salmon and trout to solve the problems existing in the prior art. By using estrogen to reverse the sex of normal male fish with genotype XY into pseudo-female fish, artificially inseminating them with normal male fish (XY), and then using molecular markers to identify sex genotypes, super-male fish with genotype YY are created and screened out. These super-male fish are then artificially inseminated with ordinary female fish with genotype XX to obtain normal male fish with all genotypes XY. This method solves the problem that the prior art cannot achieve large-scale breeding of all-male salmon and trout.

[0006] To achieve the above objectives, the present invention provides the following solution:

[0007] This invention provides a method for large-scale breeding of all-male salmon and trout, comprising the following steps:

[0008] (1) Collect fertilized eggs from different families of the same species of salmon and trout, artificially incubate them to obtain fry, and when the fry start feeding, use estrogen-containing feed to induce sex reversal in order to obtain pseudo-female fish.

[0009] (2) The induced juvenile fish were raised to sexual maturity, and individuals with female phenotypes were screened. Then, sdY was used as the sex identification gene, and pseudo-female fish with genotype XY were screened by real-time PCR.

[0010] (3) Pair sexually mature pseudo-female fish with genotype XY with normal male fish with genotype XY, and obtain super-male fish with genotype YY through artificial insemination.

[0011] (4) Pair sexually mature super-male fish with genotype YY with female fish with genotype XX, and produce all-male salmon and trout with genotype XY on a large scale through artificial insemination.

[0012] Preferably, the salmon and trout include rainbow trout, brown trout, mountain trout, Atlantic salmon, king salmon, American red-spotted salmon, and flower cake red-spotted salmon.

[0013] Preferably, the fertilized eggs are produced by artificial insemination of healthy, mature male and female salmonids from no fewer than 10 families, wherein the ratio of male to female fish is 1:1.

[0014] Preferably, the conditions for artificial incubation or artificial insemination are: water temperature maintained at 8-12℃, dissolved oxygen >8mg / L, and pH value of 7.0-7.5.

[0015] Preferably, the estrogen-containing feed is a feed containing 17β-estradiol, and the content of 17β-estradiol in the feed is 4-16 mg / kg.

[0016] Preferably, the primer pair for the sdY gene in the real-time PCR is the upstream primer shown in SEQ ID NO: 1 and the downstream primer shown in SEQ ID NO: 2.

[0017] Preferably, the method for determining the genotype of the target fish is as follows: using RPL8 as an internal reference gene, if only RPL8 signal is present, it is XX type; if the difference in CT value between sdY and RPL8 is in the range of 1 to 3, it is XY type; if the difference in CT value between sdY and RPL8 is in the range of -2 to 0, it is YY type.

[0018] Preferably, the ratio of the pseudo-female fish with genotype XY to the normal male fish with genotype XY is 1:1.

[0019] Preferably, the ratio of super-male fish with genotype YY to female fish with genotype XX is 1:5.

[0020] The present invention discloses the following technical effects:

[0021] This invention provides a method for large-scale breeding of all-male salmon and trout. Utilizing a sex-identifying molecular marker in this method, male fish with genotype XY are induced with estrogen to reverse gonadal development into pseudo-females (XY). These pseudo-females (XY) are then artificially inseminated with normal male fish (XY) to produce super-males containing genotype YY. Some of these super-males (YY) are further induced with estrogen to become super-male pseudo-females (YY) for super-male breeding. Normal super-males (YY) can then be artificially inseminated with females (XX) from any source to mass-produce all-male (XY) salmon and trout. (The entire breeding process is described in [link to process details]). Figure 1 This invention achieves efficient and stable large-scale breeding of all-male salmon and trout. The induction rate of super-male pseudo-female fish with genotype YY reaches 82%, and the all-male rate (XY) is as high as 100%. The survival rates of super-male (YY) and all-male (XY) fish are 86% and 95%, respectively. All of the above key technical indicators have reached the international leading level. The transformation and application of the technological achievements of this invention can provide a large number of PDRN product raw materials for the medical aesthetics and health care industry, greatly increasing the added value of male salmon and trout, and bringing new economic growth points for fishermen and the salmon and trout industry. Attached Figure Description

[0022] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0023] Figure 1 This invention provides a flow chart of a method for large-scale breeding of all-male salmon and trout. Detailed Implementation

[0024] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0025] 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. Any stated value or intermediate value within a stated range, as well as each smaller range between 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.

[0026] 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.

[0027] 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.

[0028] 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.

[0029] The breeding method provided by this invention is widely applicable to salmonids and trout belonging to the genera *Ocorhynchus*, *Salmo*, and *Salvelinus*, such as rainbow trout, brown trout, mountain trout, Atlantic salmon, king salmon, American red-spotted salmon, and spotted red-spotted salmon. Rainbow trout will be used as an example for specific explanation below.

[0030] Example 1: A method for large-scale breeding of all-male salmon and trout

[0031] This embodiment uses rainbow trout as an example, and the specific operation steps are as follows:

[0032] (1) Normal broodstock pairing: Five healthy male rainbow trout (2 years old) and five female rainbow trout (3 years old) from 10 different families were selected from the breeding pond and transferred to the flow-through holding tank in the hatchery production workshop for one week. During this period, feeding was stopped. The water temperature in the holding tank was 10.5℃, dissolved oxygen was 10.8mg / L, and pH was 7.3.

[0033] (2) Artificial insemination and hatching of fertilized eggs: After temporarily holding the parent fish for one week, anesthetize 10 parent fish in a tank containing 300 ppm ethylene glycol phenyl ether for 2-3 minutes until the parent fish stop swimming (to prevent the parent fish from struggling violently during artificial collection of sperm and eggs). After anesthesia, place the female fish to be spawning on the egg collection platform, gently dry the area around the genital opening with a dry towel, then lift the fish by the tail with the left hand and gently squeeze the abdomen with the right hand to squeeze the eggs of 5 female parent fish into 5 dry basins (each basin contains approximately 4000 eggs). Collect the sperm of mature male fish in the same way. Squeeze the sperm into the basins containing the eggs, and gently stir the sperm-egg mixture with a clean feather for 2-3 minutes to ensure thorough mixing. After thorough mixing, pour 1 / 2 basin of clean water into the basins and let it stand for 3 minutes for full fertilization. After fertilization, rinse the fertilized eggs with clean water 2-3 times to remove excess sperm and egg fluid. The fertilized eggs were transferred to five horizontal hatching tanks at a water temperature of 10.5°C, a flow rate of 0.5 L / min, dissolved oxygen of 10.8 mg / L, and pH 7.3. Dead eggs were checked and removed daily.

[0034] (3) Induction of pseudo-female (XY) fish: After the fertilized eggs in the 5 hatching tanks hatch and the fry rise to the surface to open their mouths, 50% of the fry are randomly selected from each hatching tank and transferred to 5 independent hatching tanks. They are then fed an initial feed containing 6 mg / kg of 17β-estradiol three times a day (morning, noon, and evening) for 7 weeks. The rearing water temperature is 10.5℃, dissolved oxygen is 10.8 mg / L, and pH is 7.3. The other 50% of the fry are fed normal feed separately. After induction, all fry, including the remaining 50% that were not fed 17β-estradiol feed, are divided into 10 independent strip-shaped flow-through culture ponds and raised individually using conventional methods until sexual maturity. During the rearing period, unhealthy and smaller individuals are gradually culled, ultimately retaining approximately 100 healthy individuals in each strip-shaped pond, all of whom are marked with PIT. During the rearing period, it is essential to monitor the water environment, including water temperature, dissolved oxygen, and pH, to ensure the water is clean and unpolluted.

[0035] (4) Development of molecular markers for sex identification: Rainbow trout sdY (Y chromosome-specific gene, gene number: NC_048593.1) and RPL8 (internal reference gene, gene number: NC_048586.1) sequences were downloaded from the NCBI GenBank database as sex identification genes to distinguish rainbow trout with genotypes of XX / XY / YY. Primers were designed using the Primer-BLAST tool on the NCBI website. The primer design principles were: primer length 18-25 bp, amplicon length 50-200 bp, GC content 40%-60%, annealing temperature 58-66℃, avoiding the appearance of four consecutive identical bases, and avoiding primer dimers and hairpin structures. The final selected fluorescent quantitative PCR primer sequences are shown in Table 1 below:

[0036] Table 1 Primer sequences

[0037]

[0038] When sex genotyping is required, a small amount of fin tissue is collected from the target fish, and DNA is extracted. Using two pairs of pre-designed primers, quantitative real-time PCR is performed to amplify the DNA and collect the fluorescence signal.

[0039] The reaction system for two pairs of primers in real-time PCR amplification is as follows:

[0040] Table 2 Reaction System

[0041] reagents Dosage 2×S6 Universal SYBR qPCR mix 5μL Forward primer (10 μM) 0.2μL Reverse primer (10 μM) 0.2μL Template DNA 0.01ng ddH2O Make up to 10 μL

[0042] The procedure for quantitative real-time PCR is as follows:

[0043] Table 3 Reaction Procedure

[0044]

[0045] The results are interpreted based on the CT value pattern: when only RPL8 signal is present, it is type XX; when both genes have signals, if the difference in CT values ​​between sdY and RPL8 is in the range of 1 to 3, it is type XY; if the difference in CT values ​​between sdY and RPL8 is in the range of -2 to 0, it is type YY.

[0046] (5) Identification and screening of pseudo-females (XY): The pseudo-females induced in step (3) and normally fed individuals reached sexual maturity in the third year. External sexual characteristics of the induced fish were observed. 20 healthy females with protruding genital openings and soft abdomens, ready to give birth, were selected from each of the five induction ponds (100 in total). A small amount of fin tissue was harvested from each of the five induction ponds, and these females were temporarily housed in the five temporary holding ponds. 20 healthy males, ready to give birth, were selected from each of the other five non-induction ponds (100 in total). A small amount of fin tissue was harvested from each of the five males, and these males were temporarily housed in the five temporary holding ponds. DNA was extracted from all fin tissues, and quantitative real-time PCR amplification was performed using the primers from step 4. The genotype results were then interpreted. Statistics showed that 6, 5, 9, 6, and 7 pseudo-female fish with genotype XY were detected in the five pseudo-female fish holding ponds, respectively. In the end, only one healthy pseudo-female fish with the largest size and genotype XY was kept in each holding pond. In the five non-induced holding ponds, the male fish all had the genotype XY, and in the end, only one healthy male fish with the largest size was kept in each holding pond.

[0047] (6) Pairing of pseudo-females (XY): Referring to step (1), pair 5 pseudo-females and 5 males that have reached sexual maturity and are ready to spawn in step (5) at a 1:1 ratio. Repeat step (2) for artificial insemination. Theoretically, the genotype ratio of the fertilized eggs obtained should be 25% XX, 50% XY, and 25% YY. Repeat step (3) for estrogen induction. Repeat step (5) for genotype identification of super-male pseudo-females (YY) and super-males (YY). The induced super-male pseudo-females (YY) and non-induced super-males (YY) are raised in separate breeding ponds and cultivated to sexual maturity using conventional methods. The remaining individuals with non-YY genotypes are culled.

[0048] Statistics showed that 3, 5, 2, 4, and 2 super-male pseudo-females with the genotype YY were detected in the five super-male pseudo-female holding ponds, respectively. Ultimately, only one healthy, maximum-sized super-male pseudo-female with the genotype YY was retained in each holding pond. In addition, 5, 4, 5, 6, and 3 super-males with the genotype YY were detected in the five non-induced holding ponds, respectively. Ultimately, only one healthy, maximum-sized super-male with the genotype YY was retained in each holding pond.

[0049] (7) Production of all-male (XY) fish: Ten female common rainbow trout that are about to reach sexual maturity and are ready to spawn (approximately 2-3 years old) were selected from the breeding pond and paired with the two super-male (YY) fish from step (6) at a ratio of 1 male to 5 females. Artificial insemination and hatching were performed according to steps (1) and (2). After the fertilized eggs hatched to the eye-developing stage, 100 eye-developing eggs were randomly collected for DNA extraction and sex genotype identification. It was found that the sex genotype of all 100 eye-developing eggs was XY.

[0050] (8) Preservation of Super Male Fish (YY): The 5 super male pseudo-female fish (YY) from step (6) were mixed with the remaining 3 super male fish. The eggs of the 5 super male pseudo-female fish (YY) were mixed together and artificially fertilized with the sperm of the 3 super male fish (YY) using the method in step (2). Finally, 80% of the fertilized eggs were randomly selected and induced into super male pseudo-female fish using the method in step (3), and then independently cultured to sexual maturity using conventional methods. The remaining 20% ​​of the fertilized eggs were also cultured to sexual maturity using conventional methods. After the super male pseudo-female fish (YY) reached sexual maturity, 100 fish were randomly selected for dissection and observation. The induction success rate was confirmed to be 82%. These super male pseudo-female fish (YY) capable of producing mature eggs and the mature super male fish (YY) can be used as breeding stock for the next generation of super male fish (YY).

[0051] (9) Collect sperm from mature super-male (YY) fish from step (7) or (8), and artificially inseminate and hatch normal female (XX) mature broodstock according to the methods in steps (1) and (2). Theoretically, all offspring should be XY males. Note: Since sex gene mutations exist in natural salmonid populations, such as mutant individuals with the genotype XY, their natural phenotype may be female. Therefore, in actual operation, it is essential to combine molecular identification results and phenotypic information to accurately select broodstock for mating.

[0052] According to statistics, the induction rate of pseudo-female fish with genotype YY in this embodiment reached 82%, the all-male rate (XY) was as high as 100%, and the survival rates of super-male (YY) and all-male (XY) fish were 86% and 95%, respectively.

[0053] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A method for large-scale breeding of all-male salmon and trout, characterized in that, Includes the following steps: (1) Collect fertilized eggs from different families of the same species of salmon and trout, artificially incubate them to obtain fry, and when the fry start feeding, use estrogen-containing feed to induce sex reversal in order to obtain pseudo-female fish; (2) The induced juvenile fish were raised to sexual maturity, and individuals with female phenotypes were screened. Then, sdY was used as the sex identification gene, and pseudo-female fish with genotype XY were screened by real-time PCR. (3) Pair sexually mature pseudo-female fish with genotype XY with normal male fish with genotype XY, and obtain super-male fish with genotype YY through artificial insemination; (4) Pair sexually mature super-male fish with genotype YY with female fish with genotype XX, and produce all-male salmon and trout with genotype XY on a large scale through artificial insemination. The fertilized eggs are produced by artificial insemination of healthy, mature male and female salmonids from no fewer than 10 families. The conditions for artificial incubation or artificial insemination are: water temperature maintained at 8-12℃, dissolved oxygen >8mg / L, and pH value of 7.0-7.5; The primer pair for the sdY gene in the quantitative real-time PCR is the upstream primer shown in SEQ ID NO: 1 and the downstream primer shown in SEQ ID NO: 2; The method for determining the genotype of the target fish is as follows: using RPL8 as an internal reference gene, if only RPL8 signal is present, it is XX type; if the difference in CT value between sdY and RPL8 is in the range of 1 to 3, it is XY type; if the difference in CT value between sdY and RPL8 is in the range of -2 to 0, it is YY type.

2. The method for large-scale breeding of all-male salmon and trout as described in claim 1, characterized in that, The salmon and trout include rainbow trout, brown trout, mountain trout, Atlantic salmon, king salmon, American red-spotted salmon, and flower cake red-spotted salmon.

3. The method for large-scale breeding of all-male salmon and trout as described in claim 1, characterized in that, The ratio of male to female fish is 1:

1.

4. The method for large-scale breeding of all-male salmon and trout as described in claim 1, characterized in that, The estrogen-containing feed is a feed containing 17β-estradiol, and the content of 17β-estradiol in the feed is 4-16 mg / kg.

5. The method for large-scale breeding of all-male salmon and trout as described in claim 1, characterized in that, The ratio of pseudo-female fish with genotype XY to normal male fish with genotype XY is 1:

1.

6. The method for large-scale breeding of all-male salmon and trout as described in claim 1, characterized in that, The ratio of super-male fish with genotype YY to female fish with genotype XX is 1:5.

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