A method and application of rapid and large-scale production of all-female Yellow River carp based on differentiated ovaries

Abstract

The present invention belongs to the technical field of genetic breeding of aquatic animals, and specifically relates to a method and application for rapid large-scale production of all-female Yellow River carp based on differentiated ovaries. The present invention feeds a feed containing letrozole to 120-day-old female carp whose gonads have differentiated into second-stage ovaries, and can obtain pseudo-male carp that can squeeze out semen within 3 months, without considering the difficulty of the key sensitive period of sex determination required by the traditional artificial induced sex reversal technology, and the survival rate is 100%, and the drug tolerance is strong; and the artificial induced sex reversal technology used is efficient, fast, simple, easy, and can continuously produce all-female carp on a large scale, and the obtained XX pseudo-male carp can squeeze out functional semen at the age of 7 months, and can be used for large-scale production of all-female carp in the second year breeding season. The method is used to treat 3-month-old XX female carp obtained by normal fertilization, and all female carp can be sex-reversed to XX pseudo-male carp, and all of them squeeze out functional semen in the second year breeding season.

Description

Technical Field

[0001] The invention belongs to the technical field of genetic breeding of aquatic animals, and specifically relates to a method and application for rapid and large-scale production of all-female Yellow River carp based on differentiated ovaries. Background Art

[0002] Common carp is a fish with XX / XY chromosomes. There is obvious sexual dimorphism in the growth traits of common carp. Females of the second year of life grow about 30% faster than males (Chen et al., Identification and comparison ofgonadal transcripts of testis and ovary of adult common carp Cyprinus carpiousing suppression subtractive hybridization. Theriogenology 2015, 83(9):1416-1427.), so the cultivation of monosexual all-female common carp has great economic value.

[0003] Currently, there are three ways to produce all-female carp: the first method is to use artificial gynogenesis technology to directly obtain all-female carp (Xiao et al., Microsatellite analysis of different ploidy offspring ofartificial gynogenesis in Cyprinus carpio. J Fish Biol 2011, 78(1):150-165.). However, there are reports showing that the gonads of gynogenetic fish show delayed development, and some fish even have intersexual gonads or infertility (Liu Wenhai, Research Progress on Artificial Gynogenesis in Fish [J]. Anhui Agricultural Sciences, 2008, 36 (35): 15519-15521.); Liu et al. also found that artificially gynogenetic carp would have delayed gonadal development in common carp (Liu et al., 2007). Moreover, experimental results in many fish have shown that the technology of artificially induced gynogenesis has the following problems: (1) The frequency of diploid seedlings is low; (2) The number of fish that can survive to sexual maturity is small due to problems such as chromosome deletion or breakage (Manan et al., A review of gynogenesis manipulation in aquatic animals. Aquaculture and Fisheries 2022, 7(1): 1-6.). Therefore, this method cannot meet the needs of large-scale production of all-female carp. The second method is to use CRISPR / Cas9 gene editing technology to knock out the cyp17a1a gene in carp. The homozygous knockout mutants of this gene all develop into males regardless of their chromosome type. Then, a pair of sex-specific molecular markers that can identify XX and XY male homozygous mutants are used to identify the XX male fish with homozygous knockout of the cyp17a1a gene. They are then artificially inseminated with normal XX female carp to obtain XX all-female carp (Zhai et al. Successful Production of an All-Female CommonCarp (Cyprinus carpio L.) Population Using cyp17a1-Deficient Neomale Carp. Engineering 2022, 8:181-189.), but gene-edited animals have not yet been approved by the country, and this method cannot be used in actual production for the time being.The third method is to treat normal XX female carp with exogenous hormones such as methyltestosterone or drugs such as aromatase inhibitors to reverse the sex of the normal XX female carp to XX physiological male carp, and then cross with normal XX female carp to obtain XX all-female carp (Wu et al., Production of all-female carp and its applications in fish cultivation. Aquaculture 1990, 85(1):327.). Compared with the first method of obtaining all-female carp through artificial gynogenesis, the third method avoids the above problems caused by the defects of artificial gynogenesis technology, and also avoids the impact of exogenous drugs or hormones on the safety of fish consumption (Mei & Gui, Genetic basis and biotechnological manipulation of sexual dimorphism and sex determination in fish. Science China Life Sciences 2015, 58(2):124-136; Liu Wenhai, 2008). However, such methods are all processed before the fish gonads are undifferentiated or during the sensitive period of sex determination. At this time, the animal individuals are small and the drug tolerance is poor. Hormone treatment easily causes the death of fry. Therefore, it is required that the time of starting treatment, the dosage of the hormone or drug used, and the duration of treatment are all appropriate to achieve the desired sex reversal effect, which limits the large-scale use of the method. Other studies have shown that the sex reversal adult fish obtained by using methyltestosterone treatment can produce semen in a very small number of individuals in the same year (Hulak et al., Water polluted by 17α-methyltestosterone provides successful male sex inversion of common carp (Cyprinus carpio L.) from gynogenetic offspring. Journal of Applied Ichthyology 2008, 24 (6): 707-710.), and can cause fish gonad atrophy. Therefore, it is urgent to establish a technical method for rapid and effective large-scale production of all-female carp. Summary of the invention

[0004] In view of the above problems, the object of the present invention is to provide a method and application for rapid and large-scale production of fully female Yellow River carp based on differentiated ovaries.

[0005] The technical contents of the present invention are as follows:

[0006] The present invention provides a method for rapid and large-scale production of all-female Yellow River carp based on differentiated ovaries, comprising the following steps:

[0007] 1) Parent breeding and screening: Female Yellow River carp with fast growth, beautiful body shape and good health are bred and screened as reserve parents;

[0008] 2) Artificial induction of gynogenesis in Yellow River carp;

[0009] ① Artificially squeeze the abdomen of the bighead bream to obtain its semen for inactivation. The specific operation is as follows: the semen is mixed and diluted with Hanks' solution, placed in a low-temperature environment, and then placed on a shaker and irradiated with an ultraviolet sterilization lamp until 20% of the sperm is inactivated;

[0010] ② Take the eggs of female Yellow River carp for artificial insemination, the specific operation is as follows: add the above-mentioned inactivated agglomerate bream sperm to the eggs of female Yellow River carp, gently stir and mix with dry and clean chicken feathers, add 23-25℃ aerated tap water and gently stir to stimulate the eggs, then take the eggs and place them in 4℃ aerated tap water for cold shock treatment, and place them in room temperature aerated water for incubation. Under normal circumstances, agglomerate bream sperm and Yellow River carp eggs cannot survive fertilization, and haploid Yellow River carp embryos cannot survive either, so the obtained seedlings are all XX fully female Yellow River carp;

[0011] 3) Artificially induce sex reversal in female carp whose gonads have differentiated into secondary ovaries;

[0012] After the XX all-female Yellow River carp seedlings start to hatch, they are normally cultivated until hatching for more than 100 days (can be 100-150 days). At this time, the gonads of the Yellow River carp have all differentiated to the second-stage ovarian stage, and compound feed mixed with letrozole is fed to them. Feeding is continued for more than 3 months (optionally 3-6 months). When sperm cells or sperm are detected in the gonads of the Yellow River carp, sex reversal occurs. Subsequently, commercial feed is used to continue feeding to obtain XX pseudo-male carp that are genetically female but physiologically male.

[0013] The dosage of letrozole is 100-300 mg / kg;

[0014] 4) Artificial breeding of all-female carp;

[0015] Select the female Yellow River carp that grows fast, has a beautiful body shape, and is healthy and is normally cultivated as the female parent, and the XX pseudo-male carp in step 3) as the male parent, and perform intensive cultivation on them. The specific operation is as follows:

[0016] When the water temperature reaches 18-20℃ (the water temperature in different provinces is different. If it is in Wuhan, breeding can generally start at the end of March and can last until the end of April. If it is in Henan, breeding can also be carried out in May. Breeding depends on the water temperature. If breeding is not done in time, the fish will miscarry naturally), inject the above-mentioned parents with oxytocin, carry out artificial breeding of Yellow River carp, put them in the oxytocin pool to wait for delivery, open the water inlet valve, and stimulate with a small water flow. When the water temperature is 20℃, the effect time is about 12 hours, and the effect time is negatively correlated with the water temperature; when chasing breeding behavior is observed in the oxytocin pool, check whether there are eggs in the breeding pool every 0.5 hours. If fish eggs are found in the breeding pool, separate the male parent fish from the female parent, collect semen and eggs respectively, and use artificial insemination to obtain fertilized eggs of Yellow River carp, and evenly spread them on 20 cm×30 cm 60-mesh plastic meshes, hang the meshes in the cement pool to naturally hatch the fry, and you can get all-female Yellow River carp fry;

[0017] The oxytocin is a mixed hormone of DOM+LHRH-A2+HCG, and the dosage is DOM 0.5~2 mg+LHRH-A2 2.5~10 μg+HCG 250~1000 IU / Kg body weight;

[0018] The method of using the oxytocin is to use back muscle injection to induce labor;

[0019] Female fish were injected twice, with an interval of 8-10 hours. The first injection was given at 11-12 noon, and the injection dose was 10% of the total dose; the second injection was given at 20-21 o'clock, and the injection dose was 90% of the total dose.

[0020] Male fish are injected once, the injection time is the same as the second injection time of female fish, and the dosage is half of that of female fish.

[0021] 5) Large-scale production of all-female carp

[0022] When the all-female Yellow River carp fry grow to be over 80 days old (can be 80-150 days old), the method of artificially induced sex reversal described in step 4) is used to breed more XX physiological male carp. In the breeding season of the third year, all the fry can squeeze out semen to varying degrees for large-scale production of all-female carp.

[0023] The present invention also provides a method for rapidly and massively producing all-female Yellow River carp based on differentiated ovaries and its application in large-scale and commercial breeding of Yellow River carp.

[0024] The beneficial effects of the present invention are as follows:

[0025] The method for rapid large-scale production of all-female Yellow River carp based on differentiated ovaries of the present invention feeds a feed containing letrozole to 120-day-old female carps whose gonads have differentiated into second-stage ovaries, and can obtain pseudo-male carps that can squeeze out semen within 3 months, without considering the difficulty of the key sensitive period of sex determination required by the traditional artificial induced sex reversal technology, and the survival rate is 100%, and the drug tolerance is strong; and the artificial induced sex reversal technology adopted by the present invention is efficient, rapid, simple, easy, and can continuously produce all-female carps on a large scale, and the obtained XX pseudo-male carps can squeeze out functional semen at the age of 7 months, and can be used for large-scale production of all-female carps in the second year of breeding season. The method is used to treat 3-month-old XX female carps obtained by normal insemination, so that all female carps can be sex-reversed into XX pseudo-male carps, and can squeeze out functional semen in the second year of breeding season, and there is no need to worry about the problems of insufficient pseudo-male carps and breeding maintenance costs, and the demand for continuous large-scale production of all-female carps can be well met. The obtained all-female carps are all from normal artificial insemination, avoiding the influence of exogenous drugs or hormones on the safety of fish consumption. In traditional cognition, fish with different sexes will not undergo sex reversal under natural conditions, and will only have one sex - male or female. It is generally believed that the gonads are no longer plastic after sex differentiation occurs. The method of the present invention can achieve sex reversal in carp that have undergone sex differentiation. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 The figure is a schematic diagram of the process of large-scale production of all-female Yellow River carp according to the present invention;

[0027] Figure 2 The gonadal anatomy of the Yellow River carp induced sex reversal by letrozole and methyltestosterone respectively;

[0028] Figure 3 These are H&E stained sections of the gonads of Yellow River carp induced by letrozole and methyltestosterone, respectively. DETAILED DESCRIPTION

[0029] The present invention is further described in detail below through specific implementation cases and accompanying drawings. It should be understood that these embodiments are only used to illustrate the present invention and are not used to limit the scope of protection of the present invention. After reading the present invention, various equivalent forms of modifications to the present invention by those skilled in the art are all within the scope of the claims attached to this application.

[0030] Unless otherwise specified, all raw materials and reagents of the present invention are raw materials and reagents from the conventional market.

[0031] Example 1

[0032] A method for rapidly and massively producing all-female Yellow River carp based on differentiated ovaries, the process is as follows Figure 1 Schematic diagram shown:

[0033] 1) Parent breeding and screening: Female Yellow River carp with fast growth, beautiful body shape and good health are bred and screened as reserve parents;

[0034] 2) Artificial induction of gynogenesis in Yellow River carp;

[0035] ① Artificially squeeze the abdomen of the bighead carp to obtain its semen for inactivation

[0036] Place the UV lamp in advance and turn it on to stabilize the wavelength;

[0037] 1 ml of semen was obtained by squeezing the abdomen of the bighead bream manually, and 2 ml of 1× Hanks' solution was added to mix and dilute, and then spread on a dry glass culture dish with a diameter of 6 cm. The thickness of the semen was 0.2 mm. The culture dish was placed on a dissecting tray containing crushed ice wrapped in tin foil to keep the semen in a low temperature environment; the dissecting tray was placed on a standard circular shaker and irradiated vertically with a 30W ultraviolet sterilization lamp with a wavelength of 254nm for 30 min, the irradiation distance was 17 cm, and the shaker speed was 60 rpm so that the sperm could be evenly irradiated; after 25 min of ultraviolet irradiation, the sperm inactivation was determined every 5 min under a microscope until about 20% of the sperm was inactivated, and the glass culture dish containing the inactivated semen was wrapped with tin foil and placed at 4℃ for use;

[0038] ② Take eggs from female Yellow River carp for artificial insemination

[0039] Add 2 mL of the above-mentioned inactivated sperm of Megalobrama amblycephala to 100 mL of female Yellow River carp eggs, stir gently with dry and clean chicken feathers to mix, add 80 mL of 25°C aerated tap water and stir gently to stimulate the eggs, and after 2 minutes, evenly spread the Yellow River carp eggs stimulated by the inactivated sperm on a 20 cm×30 cm 60-mesh plastic mesh, and then quickly put the mesh into the 4°C aerated tap water prepared in advance (prepared by a large 1.5-horsepower fish tank cold water machine) for cold shock treatment for 30 minutes, and then place it in room temperature aerated tap water or pond water for incubation;

[0040] The amount of the amblycephala bream sperm can be adjusted to 1-2 mL, the tap water added can be at a temperature of 23-25° C., and the amount used is 50-100 mL;

[0041] Under normal circumstances, the sperm of the bighead bream cannot survive fertilization with the eggs of the Yellow River carp, and the haploid Yellow River carp embryos cannot survive either, so the obtained seedlings are all XX all-female Yellow River carp;

[0042] 3) Artificially induce sex reversal in female carp whose gonads have differentiated into secondary ovaries;

[0043] After the XX all-female Yellow River carp seedlings start to hatch, they are cultivated normally (fed with Artemia for the first 15 days, and then switched to commercial feed) until 120 days after hatching. At this time, the gonads of the Yellow River carp have all differentiated to the second-stage ovarian stage, and compound feed mixed with letrozole is started to be fed for more than 3 months until sperm cells or sperm are detected in the gonads of the Yellow River carp, that is, sex reversal occurs. Subsequently, commercial feed is continued to be used to obtain XX pseudo-male carp that are genetically female but physiologically male;

[0044] The dosage of letrozole is 100-300 mg / kg;

[0045] The compound feed is prepared by dissolving 200 mg of letrozole in 500 mL of anhydrous ethanol, and then spraying the mixture evenly on the surface of the commercial feed, stirring the mixture while spraying to fully mix the letrozole and the feed, and then drying and storing the mixture for later use;

[0046] The commercial feed was purchased from Tongwei's extruded compound feed for fish, with a particle size of 2 mm and a floating feed;

[0047] The feeding is a fixed-point, fixed-time, and fixed-quantity feeding method using an automatic fish feeder (Iverace Koi automatic fish feeder), three times a day, at 7:00 a.m., 11:00 a.m., and 17:30 p.m.;

[0048] 4) Artificial breeding of all-female carp;

[0049] Select the female Yellow River carp with fast growth rate, beautiful body shape and health from normal breeding as the female parent, and the XX pseudo male carp from step 3) as the male parent, and carry out intensive breeding on them:

[0050] In early April, when the water temperature reached 18-20℃, the above parents were injected with oxytocin DOM 1mg+LHRH-A2 5 μg+HCG 500IU / Kg body weight, and the dorsal muscle injection was used to induce labor. The female fish was injected twice, with an interval of 8-10 hours. The first injection was performed at 11-12 noon, and the injection dose was 10% of the total dose; the second injection was performed at 20-21 pm, and the injection dose was 90% of the total dose. The male fish was injected once, and the injection time was the same as the second injection time of the female fish, and the dose was half of that of the female fish. The Yellow River carp was artificially bred and placed in the oxytocin pool to wait for delivery. The water inlet valve was opened and stimulated with a small water flow. When the water temperature was 20℃, the effect time was about 12 hours, and the effect time was negatively correlated with the water temperature.

[0051] The dosage of the catalyst can be adjusted according to the actual situation, DOM 0.5~2 mg+LHRH-A2 2.5~10 μg+HCG250~1000 IU / Kg body weight;

[0052] When chasing breeding behavior is observed in the induced spawning pond, check whether there are eggs in the breeding pond every 0.5 hours. If eggs are found in the breeding pond, separate the male and female parents, collect semen and eggs respectively, use artificial insemination to obtain fertilized eggs of Yellow River carp, and evenly spread them on 20 cm × 30 cm 60-mesh plastic meshes, hang the meshes in the cement pool to naturally hatch the fry, and you can get all-female Yellow River carp fry;

[0053] 5) Large-scale production of all-female carp

[0054] When the all-female Yellow River carp fry grow to 120 days old, the artificial induction sex reversal method described in step 4) is used to breed more XX physiological male carp. In the breeding season of the third year, all the fry can squeeze out semen to varying degrees for large-scale production of all-female carp.

[0055] As can be seen from the above, by feeding the 120-day-old female carp whose gonads have differentiated into second-stage ovaries with feed mixed with the aromatase inhibitor letrozole, the present invention can obtain pseudo-male carp that can squeeze out semen within 3 months, without considering the difficulty of the key sensitive period of sex determination required by the traditional artificial induced sex reversal technology. In addition, there is no death in the 120-day-old female carp, and the survival rate is 100%, which shows that the drug tolerance is strong.

[0056] Comparative Example 1

[0057] The steps are the same as step 1) to step 3) of Example 1, except that methyltestosterone is used to replace letrozole, the other steps and conditions remain unchanged, and the feed prepared using methyltestosterone is the same as that of letrozole.

[0058] After feeding with letrozole or methyltestosterone for 90 days, 12 Yellow River carp were sampled from the control group, the letrozole treatment group (Example 1) and the methyltestosterone treatment group (Comparative Example 1). Figure 2 Anatomy diagram, Figure 3 The HE staining results and gonadal index in Table 1 show:

[0059] like Figure 2As shown in the figure, after 90 days of letrozole treatment, all Yellow River carp in the letrozole treatment group underwent varying degrees of sex reversal, among which 33.33% of the Yellow River carp in the letrozole treatment group had a large amount of white testis-like tissue and a small amount of eggs in the gonads, and 25% of the Yellow River carp in the letrozole treatment group had complete sex reversal to white testis, with a large amount of semen flowing out; and after dissection of the Yellow River carp in the methyltestosterone treatment group, it was found that after 90 days of methyltestosterone treatment, a small amount of white testis-like tissue surrounding the ovaries was found in the gonads of 16.7% of the Yellow River carp in the methyltestosterone treatment group, and a large amount of white testis-like tissue and a small amount of eggs were found in the gonads of 16.7% of the Yellow River carp in the methyltestosterone treatment group. It can be seen that letrozole has a promoting effect on gonadal development, and the gonads of the Yellow River carp in the letrozole treatment group are significantly larger than those in the control group during the same period, and methyltestosterone has an inhibitory effect on gonadal development, and the gonads of the Yellow River carp in the methyltestosterone treatment group are significantly smaller than those in the control group during the same period.

[0060] like Figure 3 As shown, the results of gonad HE staining showed that 41.67% of the Yellow River carp in the letrozole treatment group had a moderate degree of sex reversal in the gonads, and a large number of spermatocytes could be seen in the gonads; 33.3% of the Yellow River carp in the letrozole treatment group had a high degree of sex reversal in the gonads; 25% of the Yellow River carp in the treatment group had complete sex reversal in the gonads to testis, and the gonads mainly contained sperm, spermatids and a small number of spermatocytes; while 66.6% of the Yellow River carp in the methyltestosterone treatment group had a low degree of sex reversal in the gonads, and a small number of spermatocytes could be seen in the gonads; 16.7% of the Yellow River carp in the treatment group had a moderate degree of sex reversal in the gonads, and a large number of spermatocytes could be seen in the gonads; 16.7% of the Yellow River carp in the methyltestosterone treatment group had a high degree of sex reversal in the gonads, and a small number of oocytes and a large number of spermatocytes, spermatids and sperm could be seen in the gonads.

[0061] Table 1 Comparison of the effects of letrozole and methyltestosterone on inducing sex reversal in carp

[0062]

[0063] As shown in Table 1, the gonad index results showed that letrozole promoted gonad development, and the gonad index of the Yellow River carp in the treatment group after 90 days of letrozole treatment was significantly higher than that in the control group; while methyltestosterone inhibited gonad development, and the gonad index of the Yellow River carp in the treatment group after 90 days of methyltestosterone treatment was significantly lower than that in the control group; it can be seen from the above that for the Yellow River carp, the proportion and degree of sex reversal of female fish treated with letrozole were higher than those treated with methyltestosterone.

Claims

1. A method for rapid and large-scale production of all-female Yellow River carp based on differentiated ovaries, characterized in that: The steps include: 1) Parent breeding and screening: Female Yellow River carp with fast growth, beautiful body shape and good health are bred and screened as reserve parents; 2) Artificial induction of gynogenesis in Yellow River carp; ① Artificially squeeze the abdomen of the bighead bream to obtain its semen for inactivation. The specific operation is as follows: the semen is mixed and diluted with Hanks' solution, placed in a low-temperature environment, and then placed on a shaker and irradiated with an ultraviolet sterilization lamp until 20% of the sperm is inactivated; ② Take eggs from female Yellow River carp for artificial insemination; 3) Artificially induce sex reversal in female carp whose gonads have differentiated into secondary ovaries; After the XX all-female Yellow River carp seedlings have opened, they are normally cultivated for more than 100 days, and the gonads of the Yellow River carp have all differentiated to the second-stage ovarian stage. The compound feed mixed with letrozole is fed to the seedlings, and the feeding is continued until sperm cells or sperm are detected in the gonads of the Yellow River carp, that is, sex reversal occurs. Subsequently, the seedlings are continued to be raised with expanded compound feed for fish, and XX pseudo-male carp that are genetically female but physiologically male are obtained; The dosage of letrozole is 100-300 mg / kg; 4) Artificial breeding of all-female carp; Selecting normally bred female Yellow River carp with fast growth rate, beautiful body shape and health as female parents, and the XX pseudo male carp in step 3) as male parents, and conducting intensive breeding on them; 5) Large-scale production of all-female carp When the all-female Yellow River carp fry grow to over 80 days old, the artificial induction sex reversal method described in step 4) is used to breed more XX physiological male carp. In the breeding season of the third year, all fry can squeeze out semen to varying degrees for large-scale production of all-female carp.

2. The method for rapid large-scale production of all-female Yellow River carp based on differentiated ovaries according to claim 1, characterized in that: The specific operation of artificial insemination described in step 2) is as follows: add the above-mentioned inactivated sperm of Megalobrama amblycephala to the eggs of female Yellow River carp, gently stir and mix with dry and clean chicken feathers, add 23-25°C aerated tap water and gently stir to stimulate the eggs, then take the eggs and place them in 4°C aerated tap water for cold shock treatment, and place them in aerated water at room temperature for incubation. The resulting seedlings are XX all-female Yellow River carp.

3. The method for rapid large-scale production of all-female Yellow River carp based on differentiated ovaries according to claim 1, characterized in that: The specific operation of step 4) is as follows: when the water temperature is 18-20°C, the above-mentioned parents are injected with an oxytocin agent for artificial breeding of Yellow River carp, and the parents are placed in an oxytocin pool to wait for delivery. The water inlet valve is opened and a small water flow is used for stimulation. When the water temperature is 20°C, the effect time is about 12 hours, and the effect time is negatively correlated with the water temperature. When chasing breeding behavior is observed in the oxytocin pool, the breeding pool is checked every 0.5 hours for the presence of eggs. If eggs are found in the breeding pool, the male parent fish is separated from the female parent, and semen and eggs are collected respectively. Fertilized eggs of Yellow River carp are obtained by artificial insemination, and the eggs are evenly spread on 20 cm×30 cm 60-mesh plastic meshes, and the meshes are hung in a cement pool to naturally hatch the fry, so that all-female Yellow River carp fry can be obtained.

4. The method for rapid large-scale production of all-female Yellow River carp based on differentiated ovaries according to claim 3, characterized in that: The oxytocin is a mixed hormone of DOM+LHRH-A2+HCG, and the dosage is DOM 0.5~2 mg+LHRH-A2 2.5~10 μg+HCG 250~1000 IU / Kg body weight.

5. The method for rapid large-scale production of all-female Yellow River carp based on differentiated ovaries according to claim 3, characterized in that: The method of using the oxytocin is to use back muscle injection to induce labor; Female fish were injected twice, with an interval of 8-10 hours. The first injection was given at 11-12 noon, with an injection dose of 10% of the total dose; the second injection was given at 20-21 o'clock, with an injection dose of 90% of the total dose. Male fish are injected once, the injection time is the same as the second injection time of female fish, and the dosage is half of that of female fish.

6. Use of the method for rapid large-scale production of all-female Yellow River carp based on differentiated ovaries as described in any one of claims 1 to 5 in large-scale and commercial breeding of Yellow River carp.

Images