A method for cultivating all-male hybrid procyprinus palatus by hybridization of double super-male population

Abstract

The application provides a method for cultivating all-male hybrid yellow-head catfish by using double super-male population hybridization, comprising the following steps: S1, constructing super-male maintenance lines of yellow-head catfish and Pelteobagrus vishnui by using sex reversal technology and gender molecular markers; S2, performing artificial propagation on the maintenance lines to expand the super-male fish in batches; S3, using androgen antagonists to partially replace estrogen for the expanded super-male fish to cooperatively induce super-female fish; S4, applying specific nutrient enrichment culture to the super-female fish and super-male fish parents; and S5, performing positive and negative cross artificial propagation between the super-female fish and super-male fish of the two super-male populations to obtain a heterogamy super-male population, i.e., commercial fry of all-male hybrid yellow-head catfish. The technical scheme of the application can efficiently and in batches induce super-female fish, realize high-density culture of parent fish, and significantly improve the effect and efficiency of artificial propagation; the hybrid all-male yellow-head catfish has high uniformity, a growth speed of 20% and a male rate of 100%.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of aquaculture, and particularly relates to a method for cultivating all-male hybrid Pelteobagrus fulvidraco by using double super-male population hybridization. BACKGROUND

[0002] Many fish species have the characteristic of gender dimorphism, such as Pelteobagrus fulvidraco, Pelteobagrus vachelli and Channa argus, the growth rate of males is significantly higher than that of females; while for Cyprinus carpio, Oncorhynchus mykiss and Cynoglossus semilaevis, the growth rate of females is faster. Therefore, in aquaculture practice, by means of gender control technology to cultivate all-male or all-female population, the growth advantage of gender can be fully utilized to improve the yield of aquaculture, shorten the breeding cycle or control overpopulation, etc. These gender control technologies involve hormone induction, interspecific hybridization, gynogenesis, androgenesis, gender molecular markers and gene editing, etc. Among them, the construction of super-male fish by gender molecular markers is the most efficient, safe way of all-male fish production, which has the advantages of accuracy, rapidness and no environmental interference. For the young and precocious Pelteobagrus fulvidraco and Pelteobagrus vachelli, another significant advantage of this technology is that it can greatly reduce the consumption of nutrients caused by the development of female ovaries during the breeding process, thereby reducing the feed coefficient, saving the cost of aquaculture and improving the efficiency of aquaculture.

[0003] Pelteobagrus fulvidraco and Pelteobagrus vachelli, both belonging to the Bagridae family, are important representative varieties in pond aquaculture in China, among which Pelteobagrus fulvidraco has the highest economic value and Pelteobagrus vachelli has the fastest growth rate. Chinese fish breeding experts ingeniously use the advantages of gender dimorphism and their respective traits, and use gender molecular markers and hybridization technology to successfully breed a series of new all-male, hybrid and all-male hybrid varieties with excellent traits. From ordinary varieties to all-male, hybrid, and then to all-male hybrid, the growth rate of each generation has been significantly improved, especially the all-male hybrid new varieties, which have shown the double advantages of all-male and hybrid. The upgrading of these varieties has brought revolutionary technological progress to the Pelteobagrus fulvidraco industry. However, research has shown that gender molecular marker technology cannot completely rule out the potential influence of environmental factors and other genetic factors on the male rate. Therefore, even a batch of super-male fish, the offspring of all-male or all-male hybrid, still shows a certain degree of instability in male rate, most of which can reach 100%, and a small part of which is only 70-80%. In addition, the offspring produced by the cross of Pelteobagrus vachelli females and Pelteobagrus fulvidraco super-males show phenotypic differentiation, about 10% of the individuals show short and thick type characteristics.

[0004] The production of all-male or all-male hybrid yellow catfish is mainly limited to the reproduction between the common female population and the single super-male population. However, it is of great research value and application potential to explore the positive and negative crosses between the super-female (genetically still YY genotype, but physiologically female) and super-male of two different super-male populations, whether it can achieve more stable male rate and individual phenotype, and whether there is room for growth potential. Unlike the single population, which can achieve large-scale production of all-male with only a few hundred super-male fish, the use of double super-male populations to breed all-male offspring needs to break through the problem of batch super-female induction. However, the dose effect of estrogen-induced sex reversal is negatively correlated with the survival rate, especially in the late induction period, the mortality rate will rise sharply, resulting in a low survival rate, which brings a major challenge to large-scale super-female induction. In addition, due to the heterogeneity of inter-specific mitochondria, if the cell autophagy cannot timely remove the paternal mitochondria, it will produce 10-30% of the abnormal fry. However, when the density of parent fish is reduced to 75-100 kg / acre, this condition is significantly improved, and the abnormal fry is reduced to less than 10%. However, this still seriously affects the production efficiency of artificial propagation. SUMMARY

[0005] To overcome the limitations and defects of the prior art, the present application provides a method for breeding all-male hybrid yellow catfish by using double super-male populations.

[0006] The present application is implemented by the following technical solutions:

[0007] The first aspect of the present application provides a method for breeding all-male hybrid yellow catfish by using double super-male populations, comprising the following steps:

[0008] S1, using sex reversal technology and gender molecular markers to construct super-male maintenance lines of yellow catfish and Amur catfish, respectively, to obtain YY genotype super-male fish and super-female fish;

[0009] S2, crossbreeding YY super-male fish in the same species maintenance line;

[0010] S3, feeding the super-male fish with feed containing pukaluramine and estradiol valerate for synergistic induction to make them sex-reversed, to obtain YY genotype super-female fish with normal reproductive ability;

[0011] S4, through feeding specific nutritionally enhanced feed, the YY genotype super-female fish obtained in S3 and the YY genotype super-male fish obtained in S2 are subjected to parent fish nutritionally enhanced cultivation;

[0012] S5, the super-male population is artificially propagated by positive and negative hybridization between super-female and super-male fish, and the all-male hybrid yellow-head catfish commodity fry is obtained, wherein the positive hybridization is super-female yellow-head catfish and super-male Wuyang yellow-head catfish, and the negative hybridization is super-female Wuyang yellow-head catfish and super-male yellow-head catfish.

[0013] Further, in step S1, the process of constructing the super-male maintenance line comprises the following steps: 7-10-day-old yellow-head catfish and Wuyang yellow-head catfish newly hatched fry are fed with powder feed containing a specific estrogen concentration to induce female sex reversal. Then, individuals with typical female characteristics are selected from the sex-reversed population, and XY pseudo-female fish are identified using gender molecular markers; the pseudo-female fish are cultured to maturity, and are mated with XY male fish of the same species to produce a super-male candidate population. Two-thirds of the individuals in the candidate population continue to undergo female sex reversal, while the remaining one-third are normally cultured. Then, gender molecular markers are used again to identify and screen YY super-female fish from the sex-reversed super-male candidate population and YY super-male fish from the normally cultured super-male candidate population, and a super-male maintenance line is established by using super-female and super-male fish of the same species.

[0014] Further, the type of estrogen is estradiol valerate. The concentration of the yellow-head catfish powder feed is 30-50 mg / kg, and the feeding lasts for 30-45 days; and the concentration of the Wuyang yellow-head catfish powder feed is 10-40 mg / kg, and the feeding lasts for 55-75 days.

[0015] Further, in step S2, during propagation, super-female and super-male fish of the same species are mated to avoid mixing of the two.

[0016] Further, in step S3, the process of synergistically inducing female sex reversal by replacing estrogen with an androgen antagonist comprises the following steps: first, the androgen antagonist is dissolved in dimethyl sulfoxide to prepare a mother liquor with a concentration of 10-20 mg / L; then, the estrogen is dissolved in 95% ethanol solution or anhydrous ethanol to prepare a mother liquor with a concentration of 2.5-5.0 mg / L. Next, according to the required mass of the feed and the preparation concentration, the required two kinds of mother liquor are accurately measured, and the mother liquor is diluted into the required ethanol solution at a ratio of 500 mL of 95% ethanol per kilogram of feed, mixed, and then the diluted solution is mixed with the powder feed, uniformly dried in the shade, and stored in the dark at 4°C or below. Subsequently, the powder feed containing the specific concentrations of the two drugs is kneaded into a mass with water, and is fed to 7-10-day-old newly hatched fry twice a day. The feeding rate of the yellow-head catfish is 6-8% of the body weight, and the feeding lasts for 30-45 days; and the feeding rate of the Wuyang yellow-head catfish is 8-12%, and the feeding lasts for 55-75 days.

[0017] Further, the androgen antagonist is proxalutamide, and the estrogen is estradiol valerate, and the ratio of proxalutamide to estradiol valerate is 1 / 3 to 2 / 3.

[0018] Further, the yellow catfish powder feed contains proxalutamide and estradiol valerate at concentrations of 40-80 mg / kg and 10-20 mg / kg, respectively, and the Pelteobagrus vishniakovi powder feed contains proxalutamide and estradiol valerate at concentrations of 30-60 mg / kg and 5-15 mg / kg, respectively.

[0019] Further, in step S4, the parent fish nutrition strengthening cultivation process includes the following steps: before the parent fish cultivation, limited feeding should be implemented. According to the examination of the fat deposition in the viscera of the parent fish, different measures are taken. If fat accumulation is found in the viscera, feeding should be stopped until the fat in the viscera is completely consumed; if there is no fat accumulation, feeding should be carried out once every 2-3 days, and the feeding rate should be controlled at 0.8-1.0% of the body weight. During the parent fish nutrition strengthening cultivation, ice-fresh sandworms, egg yolk powder, vitamin E and vitamin C should be added to the compound feed to enhance the nutrition of the parent fish, and the feeding amount should be adjusted flexibly according to the change of water temperature. In the early stage of cultivation, the ovary development is in phase II, and the parent fish should be fed once a day, with a feeding rate of 0.8-1.2% of the body weight; at the same time, the viscera should be checked once a week, and if fat accumulation is found in the viscera, the feeding amount should be immediately reduced. In the middle stage of cultivation, the ovary development is in the yolk large growth period of phase III, i.e. the granular eggs are visible to the naked eye, and the parent fish should be fed twice a day, with satiation feeding, to promote the synchronicity of ovary development. In the late stage of cultivation, the feeding is consistent with that in the middle stage of cultivation, but the ovary should be checked once a week to observe the rate of ovum nucleus deviation with egg transparent liquid, and if the deviation rate is more than 80%, it indicates that the ovary has developed to phase IV, i.e. the ovary has developed to physiological maturity.

[0020] Further, the purpose of limited feeding before parent fish cultivation is to consume the fat in the viscera of the parent fish, because fat accumulation in the viscera not only reduces the number of eggs, but also delays the development speed of the ovary.

[0021] Further, during the parent fish nutrition strengthening cultivation, 100-200% of ice-fresh sandworms (wet weight), 0.5-1.0% of egg yolk powder, 250-500 mg / kg of vitamin E and 500-1000 mg / kg of vitamin C are added to the compound feed, and the feed is mixed uniformly during feeding.

[0022] Further, in step S5, during the production of all-male hybrid Pelteobagrus fulvidraco, the orthogonal production is carried out by crossing the super female Pelteobagrus fulvidraco with the super male Pelteobagrus vishniakovi, and the reverse cross is carried out by crossing the super female Pelteobagrus vishniakovi with the super male Pelteobagrus fulvidraco.

[0023] Further, the spawning temperature is regulated at 28-30 DEG C during artificial breeding, and the gonadotropin is injected twice at the base of the dorsal fin. The first injection is a single use of luteinizing hormone releasing hormone analogue (LHRH-A2) with a dosage of 10-15 ug / kg, and the second injection is a mixed injection, in which the dosage of LHRH-A2 is 10-15 ug / kg, the dosage of diethylstilbestrol (DOM) is 5-6 mg / kg, and the dosage of chorionic gonadotropin (HCG) is 800-1000 IU / kg. The interval between the two injections is 10-12 hours.

[0024] Further, the postpartum parent fish is again cultured with nutrition reinforcement, and artificial breeding can be performed again after 30-45 days of culture. According to different climate conditions in different regions, artificial breeding can be performed 3-5 times in one year.

[0025] The second aspect of the present application provides application of the method for culturing all-male hybrid Pseudobrama sinensis by using double super-male populations for hybridization in the field of Pseudobrama sinensis breeding.

[0026] The present application has the following advantages and positive effects:

[0027] 1. The present application can successfully culture all-male hybrid Pseudobrama sinensis by positive and reverse hybridization between two double super-male populations of Pseudobrama sinensis and Pseudobrama vishnui, the male rate is stable and reaches 100%, the individual uniformity is high, and the growth rate is 20% higher than that of all-male hybrid Pseudobrama sinensis produced by using a single super-male fish, so the application prospect is wide.

[0028] 2. The present application produces all-male hybrid Pseudobrama sinensis by reverse hybridization, the individual phenotype is consistent, and the problem of short and thick differentiation of part of the individuals in the offspring of hybridization between Pseudobrama vishnui female fish and Pseudobrama sinensis super-male fish is effectively solved. In addition, the Pseudobrama vishnui super-female fish retains the same advantage of larger body size than ordinary female fish, thereby effectively improving the breeding efficiency.

[0029] 3. The present application replaces 2 / 3 of the estrogen with androgen antagonists, and the survival rate and the survival rate of the induced female rate are up to 100% and more than 78% respectively, the induction efficiency is high and stable, and the induced super-female fish has normal reproductive capacity.

[0030] 4. The present application uses ice-fresh sandworm, egg yolk powder and vitamin E and C as the nutrition reinforcement agent for culturing parent fish, the parent fish culture density can be increased to 400 kg / mu, the egg carrying capacity is increased by more than 30%, artificial breeding can be performed 3-5 times in one year, the fertilization rate is significantly improved, the malformation rate is reduced, the efficiency and effect of breeding production are effectively improved, and the cost advantage is obvious. BRIEF DESCRIPTION OF DRAWINGS

[0031] In order to more clearly illustrate the technical solutions in the embodiments of the present application, the drawings needed to be used in the embodiments will be briefly introduced as follows. Obviously, the drawings in the following description are only some embodiments of the present application, and other drawings can be obtained by those skilled in the art without any creative effort on the basis of these drawings.

[0032] Figure 1 The technical roadmap of the method for breeding all-male hybrid yellow catfish by using double super-male population hybridization according to the embodiments of the present application.

[0033] Figure 2 The ovary and its section of the super-female Wushih yellow catfish induced by replacing 1 / 3 and 2 / 3 of the estrogen with estrogen and androgen antagonists in the embodiment 2 of the present application.

[0034] Figure 3 The figure of the deformed fry produced by the high-density breeding conditions of the parent fish without the implementation of nutritional fortification in the embodiment 3 of the present application. DETAILED DESCRIPTION

[0035] Based on the information provided by the present application, various changes can be easily made to the precise description of the present application by those skilled in the art without departing from the spirit and scope of the appended claims. It should be understood that the scope of the present application is not limited to the defined processes, properties or components, as these embodiments and other descriptions are only illustrative of the specific aspects of the present application. In fact, various changes made by those skilled in the art or related fields to the embodiments of the present application are all covered within the scope of the appended claims.

[0036] For the purpose of clearly setting forth the present application, the technical solutions and advantages, the following will be further described in detail in combination with the embodiments. Please note that the embodiments described herein are only used to explain the present application, but not to limit the present application.

[0037] Embodiment 1: A method for breeding all-male hybrid yellow catfish by using double super-male population hybridization

[0038] A method for breeding all-male hybrid yellow catfish by using double super-male population hybridization, referring to the attached Figure 1 , comprising the following steps:

[0039] 1. Obtaining XY type pseudo-female fish by female sex reversal

[0040] A. Parent fish source: The original populations of yellow catfish and Wushih yellow catfish are both collected from the natural populations in the Yangtze River from Honghu to Dongting Lake in 2018. Among them, the yellow catfish parent fish for constructing super-male maintenance line is selected from the group with the largest body size, and the Wushih yellow catfish is selected from a family with the fastest growth rate among the 20 families constructed.

[0041] B. Inducing sex reversal: Artificially breed the Pelteobagrus fulvidraco and Pelteobagrus vishnui by conventional methods, and raise the newly hatched fry separately. When the fry reaches 3 days old, at which time the yolk sac has completely disappeared, feed them with artificially hatched Artemia, 6-8 times a day, and implement satiation feeding, with the whole pool being splashed each time. When they reach 8 days old, switch to feeding them with powdered feed containing a specific concentration of estradiol valerate. When feeding, knead the powdered feed into a ball with water, and feed it at a fixed point, twice a day. The feeding rate for the Pelteobagrus fulvidraco is 6-8% of the body weight, and the feeding is continuous for 35 days; the feeding rate for the Pelteobagrus vishnui is 8-12%, and the feeding is continuous for 60 days. After that, switch to feeding them with feed that does not contain estrogen.

[0042] Among them, the concentration of estradiol valerate in the powdered feed for the Pelteobagrus fulvidraco is 30 mg / kg, and that for the Pelteobagrus vishnui is 20 mg / kg.

[0043] C. Identification of genetic sex: Select individuals with typical female characteristics from the sex-reversed population, and mark them with PIT chips. The genetic sex of the Pelteobagrus fulvidraco is identified according to the sex molecular marker and identification method in the public literature (see the literature Wang D, Mao H L, Chen H X, et al. Isolation of Y-and X-linked SCAR markers in yellow catfish and application in the production of all-male populations [J], Animal Genetics, 2009, 40(6): 978-981.), and the genetic sex of the Pelteobagrus vishnui is identified by using the family sex molecular marker and identification method independently developed by the inventors (see the invention patent CN 116064759 A, molecular marker for identifying the sex of Pelteobagrus vishnui, primer set, kit, method and application), and XY-type pseudo females are screened from them, respectively.

[0044] 2. Constructing supermale maintenance lines from supermale candidate populations

[0045] The identified XY-type pseudo females are respectively bred to maturity, and are mated with individuals of the same species, i.e., the XY-type pseudo females of the Pelteobagrus fulvidraco are mated with XY-type males, and the same applies to the Pelteobagrus vishnui. Artificially breed them according to conventional methods, and obtain supermale candidate populations, respectively. Half of the individuals in the candidate populations continue to be induced for sex reversal according to the previous method, and the other half are normally bred. Mark them with PIT chips when they reach maturity, and identify their genetic sex again by using the previous method. Screen YY-type super females from the supermale candidate populations that are induced for sex reversal, and screen YY-type super males from the supermale candidate populations that are normally bred, and then group the super females and super males of the same species to construct supermale maintenance lines of the Pelteobagrus fulvidraco and the Pelteobagrus vishnui, respectively.

[0046] 3. Propagation of super-male fish among the same species and inducing feminization

[0047] A. Propagation of super-male fish: The parent fish of super-male maintenance line is bred to maturity, and artificial propagation is carried out according to the principle of cross between the same species, i.e. super-female and super-male of P. londinensis and P. walsingsi, to propagate the required number of super-male fish of P. londinensis and P. walsingsi for production.

[0048] B. Inducing feminization sex reversal: The super-male fish of P. londinensis and P. walsingsi is induced to reverse sex to super-female fish according to the method optimized in Example 2, which is used as the female fish required for the large-scale production of P. londinensis.

[0049] 4. Large-scale production of P. londinensis by all-male cross

[0050] A. Cross method: The cross between super-female of P. londinensis and super-male of P. walsingsi is called positive cross, and the cross between super-female of P. walsingsi and super-male of P. londinensis is called reverse cross.

[0051] B. Large-scale production: The parent fish breeding and artificial propagation are carried out according to the method optimized in Example 3, except that the artificial insemination of positive cross uses 0.3% physiological saline, and the reverse cross needs to increase the concentration of physiological saline to 0.45%.

[0052] Example 2: Method for high-efficiency synergistic induction of feminization sex reversal by partial replacement of estrogen with androgen antagonists

[0053] The method for high-efficiency synergistic induction of feminization sex reversal by partial replacement of estrogen with androgen antagonists comprises the following steps:

[0054] A. The androgen antagonist is prasterone, and the estrogen is estradiol valerate, wherein the proportion of prasterone replacing estradiol valerate is 1 / 3 to 2 / 3.

[0055] B. Feed preparation: First, prasterone is dissolved with dimethyl sulfoxide to prepare a mother liquor with a concentration of 10-20 mg / L, and estradiol valerate is also prepared into a mother liquor with a concentration of 2.5-5.0 mg / L using 95% ethanol solution or anhydrous ethanol. Then, according to the mass of the feed and the preparation concentration, the required two kinds of mother liquor are accurately measured, and the mother liquor is diluted and mixed in 95% ethanol solution according to the proportion of 500 mL of 95% ethanol per kilogram of feed. Subsequently, the diluted solution is uniformly sprayed onto the surface of the powdered feed, and mixed thoroughly while spraying. The dried feed is stored in a refrigerator at 4°C for use.

[0056] C. Synergistic induction: In order to reflect the actual application effect, the present application designs 6 test groups, respectively feeding androgen antagonists with different replacement ratios of estrogen for comparison and evaluation, as shown in Table 1. Among them, the first, second and third groups are super male fish of yellow catfish and the fourth, fifth and sixth groups are super male fish of Pseudobagrus vachelli.

[0057] Table 1 Effect of partial replacement of progulam with estradiol valerate on sex reversal of feminization

[0058]

[0059] After stocking, live enriched rotifers are fed, 6-8 times a day, and the rotifers are evenly distributed in the pool to ensure that the larvae can eat evenly. When reaching 8 days old, the larvae are fed with powdered feed containing specific drug concentrations according to the grouping set. The powdered feed is kneaded into a ball with water, and the larvae are fed freely. The feeding rate of yellow catfish is 6-8% of body weight, and the feeding rate of Pseudobagrus vachelli is 8-12%, and the feeding lasts for 35 days and 60 days respectively. Then, the larvae are transferred to the pond and fed with feed without drugs.

[0060] During the induction period, the pool is pumped once a day, and micro-flow water and micro-porous oxygenation are used to ensure fresh water quality and sufficient dissolved oxygen.

[0061] D. Implementation effect: The survival rate is counted at the end of the induction, and 100 tails are randomly selected from each group for dissection after 90 days of rearing, and observed with Bohn's liquid. The female rate is calculated, and the results are shown in Table 1.

[0062] Table 1 shows that the survival rate increases with the increase of the replacement ratio of progulam to estradiol valerate, and the survival rate of the replacement group is significantly higher than that of the estrogen group. Especially the 2 / 3 replacement group of yellow catfish and Pseudobagrus vachelli, the survival rate is the highest, reaching 78.6% and 81.2% respectively.

[0063] In addition, Table 1 also shows that the female rate of each group is high, except for the first group, the female rate reaches 100%, that is, all YY type super male fish are successfully induced into super female fish.

[0064] As shown in the accompanying Figure 2 Figures, the ovaries of the super female fish of Pseudobagrus vachelli induced in the fourth, fifth and sixth groups are clearly visible, and the developing oocytes can be observed by sectioning.

[0065] In summary, the androgen antagonist partially replaces estrogen to synergistically induce sex reversal, and the survival rate is higher than that induced by estrogen alone, even exceeding the general level of traditional pond breeding of yellow catfish fry, and the induced female rate is more stable, reaching 100%. Therefore, the demand for large-scale production can be fully met.

[0066] Example 3: Method for improving the breeding performance of parent fish by specific nutritional enhancement technology

[0067] The method for improving the breeding performance of parent fish by specific nutritional enhancement technology comprises the following steps:

[0068] A. Limited feeding: Before the cultivation of parent fish, limited feeding should be performed. The purpose of limited feeding is to consume the fat in the viscera of the parent fish or prevent the accumulation of fat in the viscera. Because the accumulation of fat in the viscera not only reduces the number of eggs, but also delays the development speed of the ovary. Therefore, 2-3 months before the cultivation of parent fish, the accumulation of fat in the viscera of parent fish should be checked.

[0069] If there is no fat accumulation, feeding should be performed once every 2-3 days, and the feeding rate should be controlled at 0.8-1.0% of the body weight, and the accumulation of fat in the viscera should be checked regularly.

[0070] If fat accumulation in the viscera is found, feeding should be stopped immediately until the fat in the viscera is consumed.

[0071] B. Parent fish are divided into pools: Parent fish should be re-divided into pools during cultivation. In order to reflect the actual application effect, two kinds of stocking densities are designed for comparison. One is low density, with a stocking density of 100 kg / acre; the other is high density, with a stocking density of 400 kg / acre, as shown in Table 2.

[0072] C. Build a warm shed: After the parent fish are released, a warm shed is built. When building, steel cables with a diameter of 3-5 mm are arranged horizontally and vertically above the parent fish pool to fix the covering plastic film. Building a warm shed can raise the water temperature and promote the development of sex glands, which can advance artificial breeding by 30-60 days.

[0073] This step is determined according to the actual needs of production. If artificial breeding is needed in advance, a warm shed is built; if there is no need to advance, no warm shed is built.

[0074] D. Intensive cultivation: In order to reflect the actual application effect, the present application designs two cultivation modes of nutritional enhancement and non-nutritional enhancement to cultivate super female parent fish of yellow catfish. In the nutritional enhancement mode, two different feeding schemes are combined with different stocking densities to form four test groups:

[0075] The first group is the non-enhanced group, which only feeds yellow catfish feed, and the parent fish are released at a low density of 100 kg / acre;

[0076] Group 2 is the non-strengthened group, only feeding yellow catfish feed, high-density stocking of 400 kg / acre of parent fish;

[0077] Group 3 is the strengthened group, using yellow catfish feed added with 30% of ice fresh fish (wet weight), 500 mg / kg of vitamin E and 1000 mg / kg of vitamin C combined feeding, high-density stocking of 400 kg / acre of parent fish;

[0078] Group 4 is the strengthened group, using yellow catfish feed added with 100-200% of ice fresh sandworm (wet weight), 500 mg / kg of vitamin E and 1000 mg / kg of vitamin C combined feeding, high-density stocking of 400 kg / acre of parent fish.

[0079] Ice fresh fish and ice fresh sandworm need to be first minced into pulp with a meat grinder, mixed evenly with feed and placed for 1 hour, so that the fish pulp or sandworm pulp is fully adsorbed on the surface of the feed, and then fed.

[0080] In the early stage of cultivation, the ovary development is in phase II, and the feeding rate is 0.8-1.2% of body weight per day; at the same time, the internal fat is checked once a week, and if fat accumulation is found, the feeding amount should be reduced.

[0081] In the middle stage of cultivation, the ovary development is in the yolk large growth period of phase III, i.e. the granular eggs are visible to the naked eye, and the feeding should be twice a day, with satiation feeding, to promote the synchronicity of ovary development.

[0082] In the late stage of cultivation, the feeding is the same as in the middle stage of cultivation, but the ovary is checked once a week, and the ovum transparent liquid is used to observe the ovum nucleus deviation, if the deviation rate is more than 80%, it indicates that the ovary has developed to phase IV, i.e. the ovary is mature.

[0083] During the cultivation period, the feeding amount of feed should be adjusted flexibly according to the change of water temperature.

[0084] E. Add new water: add new water every 15 days in the early and middle stages of cultivation, 5-10 cm each time; add new water once a week in the late stage of cultivation, 5-10 cm each time, to stimulate the development of gonads.

[0085] F. Artificial propagation: artificial propagation includes the following steps:

[0086] Artificial induction of labor: when inducing labor, the temperature is adjusted to 28-30℃, and the sex hormone is injected twice at the base of the dorsal fin. The first injection is the use of luteinizing hormone releasing hormone analogue (LHRH-A2) alone, the dose is 10-15 ug / kg; the second injection is mixed injection, in which the dose of LHRH-A2 is 10-1,5 ug / kg, the dose of diethylstilbestrol (DOM) is 5-6 mg / kg, and the dose of chorionic gonadotropin (HCG) is 800-1000 IU / kg. The interval between the two injections is 10-12 hours.

[0087] Artificial insemination: When the parent fish is in estrus, take the super-male fish of Pelteobagrus vishnui, kill the male and take the sperm, put the sperm into the sperm preservation solution and incubate for 20 minutes. Dilute and mix the sperm and the preservation solution at a ratio of 1:10. The preparation method of the sperm dilution solution is as follows: dissolve 10 g of sodium citrate, 0.3 g of potassium chloride, 2.0 g of sodium bicarbonate and 29.0 g of glucose into 1000 mL of distilled water, and then add L-arginine and L-leucine to make their concentrations reach 0.449 mmol / L and 0.968 mmol / L respectively. The purpose of adding L-arginine is to improve the capacitation ability of sperm, thereby enhancing the sperm motility, and L-leucine can reduce the malformation rate.

[0088] Extrude the matured eggs, take 10 mL of the diluted sperm for each batch of 2500 g of eggs, put the sperm into 1000 mL of 0.3% physiological saline and stir quickly to make it uniform, then pour it into the eggs and stir for 1 minute, and then pour away the supernatant and remove the adhesion in the yellow mud.

[0089] Artificial incubation: wash the de-adhesion eggs, put them into a conical incubation barrel with a diameter of 70 cm for incubation, and then transfer the larvae to a cement pool for temporary cultivation after 24 hours of membrane removal, until the yolk sac disappears.

[0090] Secondary breeding: put the postpartum parent fish back into the pond for cultivation according to the previous method. According to the water temperature, generally cultivate for 30-45 days for the second artificial breeding.

[0091] Implementation effect: the statistical results of the mature coefficient, spawning rate, fertilization rate, malformation rate and fry yield of the breeding performance evaluation are shown in Table 2.

[0092] Table 2 Effect of improving the breeding performance of parent fish by specific nutritional strengthening technology

[0093]

[0094]

[0095] Note: The spawning rate refers to the weight of the artificial extruded eggs at the time of induced spawning compared with the weight of the parent fish at the time of induced spawning.

[0096] Table 2 shows that in the mature coefficient and spawning rate indicators, the parent fish of the third and fourth groups of nutritional strengthening in high-density stocking is significantly higher than that of the second group without strengthening, and slightly higher than that of the first group of low-density and non-strengthening. Among the strengthening groups, the fourth group is slightly higher than the third group; while among the non-strengthening groups, the first group of low-density stocking is significantly higher than the second group of high-density stocking. These results show that through nutritional strengthening cultivation, the egg-carrying capacity of the parent fish can be significantly improved and the stocking density of the parent fish can be greatly increased.

[0097] Table 2 also shows that, in terms of fertilization rate, deformity rate and the number of seedlings, the parent fish in high-density culture, the third and fourth groups of which are nutritionally enhanced, are significantly better than the first and second groups which are not enhanced. Among the enhanced groups, the fourth group performs better than the third group; and among the non-enhanced groups, the first group of low-density culture is significantly better than the second group of high-density culture. Among them, the second group of high-density culture without enhancement has the worst effect, and the deformity rate rises sharply, as shown in the attached Figure 3 These results show that, under high-density conditions, the effect and efficiency of artificial propagation can still be significantly improved through nutritional enhancement.

[0098] In addition, Table 2 also shows that post-natal parent fish that have been nutritionally enhanced again can still maintain high reproductive performance in the second artificial propagation.

[0099] In summary, the implementation effect is that even under high-density culture conditions, parent fish can still maintain high reproductive performance in multiple artificial propagations through nutritional enhancement, and the production effect and efficiency are significantly improved, providing solid technical support for large-scale production.

[0100] Finally, it should be noted that the above examples are only the preferred embodiments of the present application and are not intended to limit the present application. Any modifications, equivalent replacements and improvements made within the spirit and principles of the present application shall be included in the protection scope of the present application.

Claims

1. A method for breeding all-male hybrid yellow catfish by using double super-male population hybridization, characterized in that, Comprise the following steps: S1, using sex reversal technology and gender molecular markers, respectively, to construct the super male maintenance line of yellow catfish and Wasi yellow catfish, and obtain YY genotype super male fish and super female fish; S2, crossbreed YY super male fish in the same maintenance line to expand; S3, feed the super male fish with feed containing pukruamine and estradiol valerate to induce sex reversal and obtain YY genotype super female fish with normal reproductive capacity; S4, feed the YY genotype super female fish obtained in S3 and the YY genotype super male fish obtained in S2 with specific nutrition-enhanced feed for nutrition-enhanced cultivation; S5, crossbreed super female fish and super male fish between two super male populations through orthogonal backcross to obtain commodity fry of all-male hybrid yellow catfish, wherein the orthogonal is yellow catfish super female fish x Wasi yellow catfish super male fish, and the backcross is Wasi yellow catfish super female fish x yellow catfish super male fish; In step S3, the feed containing pukruamine and estradiol valerate has a concentration of 40-80 mg / kg and 10-20 mg / kg, respectively, for yellow catfish; For Wasi yellow catfish, the feed contains pukruamine and estradiol valerate at a concentration of 30-60 mg / kg and 5-15 mg / kg, respectively; In step S3, the synergistic induction for sex reversal and feminization includes: Feed the initial hatched fry with feed containing pukruamine and estradiol valerate when they reach 7-10 days old, knead the powdered feed into lumps with water, and let the fry freely nibble, twice a day; The feeding rate for yellow catfish is 6-8% of body weight, and the feeding duration is 30-45 days; The feeding rate for Wasi yellow catfish is 8-12%, and the feeding duration is 55-75 days; In step S4, the specific nutrition-enhanced feed includes adding 100-200% of fresh frozen sandworms, 0.5-1.0% of egg yolk powder, 250-500 mg / kg of vitamin E, and 500-1000 mg / kg of vitamin C to the basic feed. Step S1 specifically includes:

2. The method for breeding all-male hybrid Pseudobrama sinensis by using double super-male population hybridization according to claim 1, characterized in that, Feed yellow catfish fry with feed containing 30 mg / kg of estradiol valerate for 35 days when they are 7-10 days old to screen XY pseudo female fish; Feed Wasi yellow catfish fry with feed containing 20 mg / kg of estradiol valerate for 60 days to screen XY pseudo female fish; Screen after same cross to obtain YY super male fish and super female fish. Step S2 specifically includes:

3. The method for breeding all-male hybrid Pseudobrama sinensis by using double super-male population hybridization according to claim 1, characterized in that, Artificially breed yellow catfish YY super female fish x YY super male fish and Wasi yellow catfish YY super female fish x YY super male fish to obtain batches of YY super male fish. In step S4, the cultivation has a cultivation density of 400 kg / acre, and the ovary III stage is fed twice a day.

4. The method for breeding all-male hybrid Pseudobrama sinensis by using double super-male population hybridization according to claim 1, characterized in that, 5. The use of the method for cultivating all-male hybrid yellow catfish by utilizing double super male populations in the field of yellow catfish breeding according to any one of claims 1-4. ​

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