Method for efficiently creating xy or yy pseudo-female cyprioid and application thereof

Abstract

The application belongs to the technical field of aquatic animal genetic breeding and invasive species control, and discloses a method for efficiently creating XY or YY pseudo-female carps and application. The application significantly improves the sex reversal success rate of XY pseudo-female carps, and YY pseudo-female carps are created for the first time. By using the method, XY and YY carps can be induced to achieve a sex reversal efficiency of up to 89.89% in a short time, and the growth, survival and body shape of the carps are least affected. The XY and YY carp sex reversal method provided by the application is simple, efficient and easy to operate, and the created XY pseudo-female carps and YY pseudo-female carps have important application prospects in YY carp mass production and carp biological invasion prevention and control.

Description

Technical Field

[0001] This invention belongs to the fields of aquatic animal genetic breeding technology and invasive species control technology, and more specifically relates to a method for efficiently creating XY or YY pseudo-female carp and its application. This method can efficiently induce sex reversal from male to female in XY and YY carp, while minimizing interference with carp growth and development. The XY and YY pseudo-female carp created by this method are suitable for the mass production of YY carp and the prevention and control of carp invasive species. Background Technology

[0002] The carp (Cyprinus carpio) belongs to the genus Cyprinus in the family Cyprinidae. It is an XY sex-determined fish (Bongers et al., 1994; Bongers et al., 1999) and is one of the representative species of Asian carp. Due to its strong adaptability (Koehn, 2004; Li et al., 2021), it has been listed as one of the 100 most threatening invasive alien species in the world (Bajer and Sorensen, 2009; Hopf et al., 2023; Jiang et al., 2018; Luque et al., 2013; McCormick et al., 2020; Singh et al., 2010). Based on Hamilton's principle of regulating population size through sex ratio (Hamilton, 1967), Gutierrez and Teem proposed a "TrojanY chromosome" (TYC) strategy (Gutierrez and Teem, 2006; Teem and Gutierrez, 2010) for controlling invasive fish species. This strategy involves periodically releasing YY (yellow-sex) fish into the invasive waters to gradually shift the sex ratio towards 100% male, thereby eliminating the invasive species by blocking reproduction. Due to its eco-friendly and precise control methods, the TYC strategy is considered one of the most promising strategies for controlling XY sex-determined fish invasions (Cotton and Wedekind, 2007; Parshad and Gutierrez, 2010; Wang et al., 2014; Wang et al., 2016). Establishing a YY maintenance line is a crucial step in implementing the TYC strategy. To stably release YY fish into invading waters, it is necessary to obtain stable, self-breeding M-yy (supermale fish with YY genotype) and F-yy (sex-reversed female fish with YY genotype). These two species can be paired to sustainably produce a pure YY population, thus forming a continuous release system (Chen et al., 2025; Li et al., 2022).

[0003] M-yy and F-yy fish can be obtained through a series of technical processes, mainly including the preparation of M-yy fish and their female-transfer treatment to obtain F-yy individuals. Currently, the most feasible method for preparing M-yy fish is to first induce sex reversal in M-xy (male fish with genotype XY) using hormones to obtain F-xy (sex-reversed female fish with genotype XY), then pair them with M-xy fish for breeding, and combine this with the XX / XY / YY carp genetic identification method (Chen et al., 2025) to select approximately one-quarter of the offspring as M-yy individuals. However, previous studies have indicated that inducing sex reversal in carp is quite difficult (Jiang et al., 2018; Piferrer, 2001; Teem and Gutierrez, 2010), becoming a major limiting factor for the efficient preparation of M-yy carp. In the preparation of F-yy carp, estrogen induction is usually used to convert M-yy carp to females. However, studies on various fish species have found that sex reversal is more difficult for M-yy carp due to their stronger tendency towards male development and lower sex plasticity (Li et al., 2022; Schill et al., 2017; Zhang et al., 2024). Current research on the sex plasticity of M-yy carp is still very limited. While the applicant's previous proposal in WO2025107125A1, which suggested that "estrogen induction of 1 / 4 of the offspring YY population can yield an F-yy pseudo-female carp population," is theoretically feasible, there are currently no publicly reported successful preparations of F-yy carp. Therefore, to achieve stable, large-scale production of M-yy and F-yy carp, it is urgent to establish an efficient and reproducible hormone induction system suitable for both M-xy and M-yy carp.

[0004] In aquatic breeding and sex control practices, exogenous sex hormone induction is currently the most common method for sex reversal. Among these, 17β-estradiol (E2) is the "standard hormone" for inducing feminization in most bony fishes due to its high receptor affinity and biological activity, which promotes ovarian development and inhibits male gonad formation (Pandian and Sheela, 1995; Piferrer, 2001). In addition, some studies have used synthetic estrogens such as diethylstilbestrol (DES) and ethinylestradiol (EE2) to achieve good feminization effects in species such as tilapia (Oreochromis spp.) and rainbow trout (Oncorhynchus mykiss) (Guerrero, 1975; Johnstone et al., 1979; Piferrer and Donaldson, 1991). However, significant differences exist among different fish species in terms of sex differentiation time, hormone sensitivity, and endocrine feedback mechanisms, exhibiting marked "species specificity" in their hormonal responses (Yamamoto, 1969; Devlin and Nagahama, 2002; Zhang et al., 2023). In carp, relevant research has mainly focused on induction protocols primarily based on E2, with some studies attempting to combine it with the androgen receptor antagonist flutamide (Jiang et al., 2018), achieving a maximum feminization rate of 62.5%. This research provides an important technical foundation for establishing a carp sex reversal system. However, long-term E2 treatment is often accompanied by negative effects such as slowed growth, increased deformity rates, and decreased survival rates (Król et al., 2014; Hanson et al., 2012). Meanwhile, existing research on carp sex reversal is mostly limited to a few hormone types, and systematic comparisons of different hormone types, dosages, and combinations are still relatively lacking.

[0005] The hormone treatment window is another important factor affecting the efficiency of sex reversal. Previous studies have shown that the sensitive period for sex differentiation in carp and most cyprinid fishes typically occurs 45–120 days post-hatching (dpf) (Piferrer, 2001; Komen and Thorgaard, 2007; Jiang et al., 2018). During this stage, the reproductive primordia have formed but have not yet undergone directional differentiation, making it a critical window for exogenous hormone-induced sex reversal. However, the definition of this time window remains somewhat coarse, lacking more refined histological developmental evidence. The specific induction start time and treatment duration have not been standardized, leading to significant differences in results among different studies. In carp sex reversal treatment practices, most studies choose to begin treatment at 2 to 3 months of age and continue for 3 to 6 months (Jiang et al., 2020; Jiang et al., 2018). However, later start times and longer treatment cycles may lead to lower sex reversal efficiency and cause undesirable phenotypes such as growth retardation and developmental abnormalities. Therefore, optimizing hormone types and treatment methods, as well as accurately determining the start time and window period of hormone treatment, are of great significance for improving the sex reversal efficiency of carp and reducing individual physiological risks.

[0006] In summary, in the practical application of the TYC strategy to control invasive carp species, in order to develop F-xy, M-yy and F-yy carp in large quantities, it is urgent to establish a technical method that is minimally harmful to carp and can efficiently induce sex reversal in M-xy and M-yy carp through short-term treatment. Summary of the Invention

[0007] The purpose of this invention is to provide an efficient method for creating XY or YY pseudo-female carp. The method of this invention significantly improves the success rate of XY pseudo-female carp and, for the first time, successfully produces YY pseudo-female carp. The XY and YY carp conversion methods provided by this invention are simple and efficient, with minimal harm to the carp, and the resulting XY and YY pseudo-female carp grow well, have a high survival rate, and a low deformity rate.

[0008] Another objective of this invention is to provide an application of a method for efficiently creating XY or YY pseudo-female carp, including applications in carp breeding, aquaculture, or prevention and control of carp invasive species.

[0009] To achieve the above objectives, the present invention employs the following technical measures:

[0010] A method for efficiently creating XY or YY pseudo-female carp includes the following steps:

[0011] (1) Formulation of hormone-fed feed:

[0012] After dissolving estrone in anhydrous ethanol, it was sprayed into carp feed to achieve a hormone concentration of 150-250 mg / kg in the feed.

[0013] (2) Hormone feeding and daily management: During the breeding season, F-xy and M-xy carp are used to breed to obtain a full-sib population composed of 1 / 4 XX, 2 / 4 XY, and 1 / 4 YY genotypes. After hatching at room temperature, the fish are fed with Artemia for 1-1.5 weeks indoors. Then, they are transferred to outdoor fish ponds for further cultivation. When they grow to 25 dpf, M-xy and M-yy carp are identified. They are transferred to outdoor cement ponds at a ratio of 1:1 to 1.3 per pond of M-xy:M-yy carp. After the juvenile fish are acclimatized to suitable feed for 3-10 days, hormone treatment is carried out. Hormone feed is given at 4-6% of body weight starting at 28-35 dpf, twice a day, at 7:00-8:00 am and 4:00-5:00 pm, with a 9-hour interval between the two times. After 2 to 4 months of feeding, XY or YY pseudo-female carp can be obtained.

[0014] The M-xy refers to male fish with genotype XY, and the F-xy carp refers to female fish with genotype XY that have undergone sex reversal; M-yy refers to supermale fish with genotype YY.

[0015] The method described above preferably involves preparing hormone-containing feed in step (1) by re-preparing the hormone solution or feed containing hormones every 7-15 days. The prepared feed should be stored in a dark, sealed, moisture-proof, and low-temperature environment throughout the storage and use process.

[0016] In the method described above, preferably, the hormone concentration in the hormone feed in step (1) is 200 mg / kg.

[0017] In the method described above, preferably, the acclimatization time for palatable feed in step (1) is 5 days.

[0018] In the method described above, preferably, in step (1), hormone feed is given at 4% of body weight starting at 30 dpf.

[0019] In the method described above, preferably, in step (2), the hormone feed is administered at 7:00 AM and 4:00 PM respectively.

[0020] In the method described above, preferably, the hormone feed is fed for 4 months in step (2).

[0021] Preferably, the outdoor cement pool described in step (2) of the method described above has a built-in circulating water system.

[0022] An application of a method for efficiently creating XY or YY pseudo-female carp, which is any of the following applications:

[0023] 1) Carp breeding;

[0024] 2) Carp farming;

[0025] 3) Prevention and control of carp invasion.

[0026] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0027] Current methods for converting carp to females suffer from low sex reversal efficiency, significant harm to carp, and high mortality and deformity rates. Furthermore, limitations in F-xy carp development technology hinder the mass production of M-yy carp, resulting in no reports of successful F-yy carp development to date. This invention significantly improves the success rate of XY pseudo-female carp and, for the first time, successfully develops YY pseudo-female carp. It can induce sex reversal in both XY and YY carp with an efficiency of 89.89% under short-term treatment, while minimizing impact on carp growth, survival rate, and body shape. The XY and YY carp conversion methods provided by this invention are simple, efficient, and highly operable. The developed XY and YY pseudo-female carp show significant application potential in the mass production of YY carp and the control of invasive carp species. Attached Figure Description

[0028] Figure 1 M-yy Carp Batch Creation Flowchart.

[0029] Figure 2 This is a schematic diagram of the overall experimental design scheme for this invention;

[0030] Wherein: A represents the gonadal development process of carp, marking the time nodes of each key event in the gonadal development of carp; B represents the time window for the female conversion treatment of the present invention, starting from 1 month of age and continuing for 2-4 months; C represents the hormone treatment group setup and phenotypic analysis process of the present invention.

[0031] Figure 3 The effects of different types of hormone treatments on body length, weight, and survival rate of M-xy and M-yy in Example 2;

[0032] Where: A and B are the body lengths of M-xy and M-yy in each hormone-treated group at 2 months and 4 months of age, respectively; C and D are the body weights of M-xy and M-yy in each hormone-treated group at 2 months and 4 months of age, respectively; E to L are the survival rates of M-xy and M-yy in each hormone-treated group at 4 months of age.

[0033] Figure 4 The effects of different types of hormone treatments on the body shape and physique of M-xy and M-yy in Example 2;

[0034] Where: A represents the body shape of the control group at 4 months of age, B to F represent the deformed carp at 4 months of age in the other hormone-treated groups, and G to N represent the deformity rate statistics of each experimental group at 4 months of treatment.

[0035] Figure 5The sex reversal efficiency of M-xy and M-yy in different hormone treatment groups in Example 2;

[0036] Where: A to H represent the statistics of M-xy and M-yy carp sex reversal efficiency in each experimental group during February, and A' to H' represent the statistics of M-xy and M-yy carp sex reversal efficiency in each experimental group during April. Detailed Implementation

[0037] To better understand the technical solution of this invention, the following specific embodiments further describe the invention. Unless otherwise specified, the technical solutions described in this invention are conventional solutions in the art; unless otherwise specified, the reagents or materials are all from commercial sources. Conditions not specifically described in this invention, such as dissolved oxygen or water temperature, are conventional conditions for carp farming and need not be elaborated upon.

[0038] Example 1:

[0039] Application of a highly efficient method for creating XY or YY pseudo-female carp:

[0040] (1) Dissolve 0.4 g of E1 (Estrone, Macklin, E808824-5g) in 500 mL of anhydrous ethanol, place it in a constant temperature shaker at 37℃, and shake at 220 rpm / min for 2 hours to fully dissolve the hormone.

[0041] (2) The above hormone solution was sprayed and evenly mixed with 2 kg of 1 mm diameter commercial extruded feed (Tongwei Co., LTD, China), placed in a cool and ventilated place to dry thoroughly, sealed in bags and temporarily stored at 4℃. The hormone concentration in the obtained feed was 200 mg / kg. To prevent the active ingredients of the hormone from degrading or becoming ineffective during storage or feeding, and to ensure the stability of the sex reversal induction effect, the preparation of feed and hormone solution followed the principle of "small amount, multiple times, and freshly prepared and used". In actual operation, feed and hormone stock solution were prepared again every half month (about 15 days) to ensure the stability of hormone content and the consistency of biological effects. During storage and use, the feed was kept away from light, sealed, moisture-proof and low temperature, and exposed to high temperature, strong light or air environment to prevent hormone oxidation, photodegradation and thermal inactivation.

[0042] 3. Experimental group setup and feeding management

[0043] M-xy (male fish with genotype XY) and F-xy carp (sex-reversed female fish with genotype XY) were bred (Chen et al., 2025) to obtain a full-sib experimental population consisting of 1 / 4 XX, 2 / 4 XY, and 1 / 4 YY genotypes. After hatching at room temperature, the fish were fed with Artemia spp. indoors for one week, and then transferred to a half-acre outdoor fishpond for further cultivation. Once the fish reached 25 days past fertilization (dpf), M-xy and M-yy carp were identified using the method described in the aforementioned literature (Chen et al., 2025). Figure 1 The carp were transferred to well-equipped outdoor cement ponds (2.5 m * 2.5 m * 1.4 m) with a built-in circulation system, according to a ratio of 1:1 for the number of M-xy and M-yy carp per pond. In this embodiment, the total number of M-xy and M-yy carp per pond is 160.

[0044] The gonadal development process in carp exhibits a clear temporal pattern. In the preliminary research of this invention, through serial histological section observation (Figure 2A), it was found that in carp, bipotent gonads form at approximately 12 days postpartum (dpf), primordial germ cells colonize the gonadal crest at 15 dpf, and male and female gonadal morphological differentiation begins at 30-35 dpf. This study clearly established for the first time that gonadal differentiation in carp begins around 30 dpf. Based on this, this invention precisely determines the starting time of hormone treatment to 1 month of age, that is, the point at which the gonads have just entered the differentiation stage but have not yet fully oriented. Compared with the previously commonly used starting time of 2 months of age or later (Jiang et al., 2018; Teem and Gutierrez, 2010), the treatment time of this invention is significantly earlier and precisely aligned with the sex differentiation window, thereby achieving higher sex reversal efficiency within a shorter hormone exposure cycle. This time-series optimization not only reduces the hormone treatment cycle and potential toxic side effects, but also improves the stability of sex reversal results and the consistency of individual development, providing a scientific basis and technological breakthrough for the mass production of F-xy and F-yy carp.

[0045] After acclimatization to palatable feed (Tongwei Co., Ltd., China) for 5 days, juvenile fish are then treated with hormones, starting at 1 month of age. Figure 2(B) During the hormone treatment period, fish were fed equal amounts of untreated feed (WT) and hormone-treated feed (treatment group) daily, at a ratio of 4% of their body weight. Feeding was twice daily, at 7:00 AM and 4:00 PM, to maintain stable hormone intake and feed utilization. This setting was based on the diurnal rhythm and hormone metabolism characteristics of fish: hormone-treated feed is easily absorbed and degraded in water; feeding only once daily can easily lead to insufficient intake and fluctuations in plasma hormone concentrations (Yamaguchi et al., 2015; Martyniuk et al., 2020), thus affecting the induction effect. Feeding twice daily, morning and evening, matches the fish's natural feeding peaks, maintaining stable levels of exogenous and in vivo hormones, significantly improving the success rate of sex reversal induction and individual developmental consistency. Hormone treatment lasted for 4 months, during which constant water temperature and sufficient dissolved oxygen were maintained, and morphological and histological examinations were conducted regularly.

[0046] Example 2:

[0047] Analysis of the effects of different types of hormones on the growth and survival of M-xy and M-yy carp:

[0048] Following the method described in Example 1, the hormone in step (1) was replaced with other hormones or combinations of hormones, while the remaining steps remained unchanged, to examine the effects of different types of hormones on M-xy and M-yy sex conversion. Figure 2 (C)

[0049] The different hormone groups are set as follows:

[0050] E2 group: 0.4 g of E2 (17β-Estradiol, Macklin, MK-E808987-25g) was added to 500 mL of anhydrous ethanol;

[0051] E2+F group: 0.2 g E2 and 0.2 g F (Flutamide, Macklin, MK-F835355-25g) were added to 500 mL of anhydrous ethanol;

[0052] E2+Tri group: 0.2 g E2 and 0.2 g Tri (Trilostane, Macklin, T829829-1g) were added to 500 mL of anhydrous ethanol;

[0053] EE2 group: 0.4 g EE2 (17α-ethynylestradiol, Macklin, E829825-5g) was added to 500 mL of anhydrous ethanol;

[0054] DES group: Add 0.4 g DES (Diethylstilbestrol, Macklin, D807246-5g) to 500 mL of anhydrous ethanol;

[0055] Group H: Add 0.4 g H (Hexestrol, Macklin, H109882-1g) to 500 mL of anhydrous ethanol;

[0056] WT group: This is the control group, which received 500 mL of anhydrous ethanol.

[0057] The results are as follows:

[0058] (1) Statistics on growth and development of M-xy and M-yy carp in each group

[0059] To analyze the growth and development of XY and YY carp in different hormone treatment groups, we collected samples at 2 and 4 months after treatment to measure the body length and weight of each treatment group. Regarding body length, there were no significant differences in body length between XY and YY carp in each treatment group at 2 and 4 months after treatment, and the body length parameters were relatively uniform among the treatment groups. Figure 3 (A to B). Regarding body weight, although there were no significant differences in the weight indicators of XY and YY carp within different experimental groups at 2 and 4 months after treatment, there were fluctuations in body weight distribution among different treatment groups. At 4 months of age, the overall body weight ranking of each hormone treatment group was as follows: WT (43.07g) > E2+F (40.16g) > E1 (36.82g) > E2 (33.00g) > E2+Tri (31.22g) > H (26.92g) > DES (25.81g) > EE2 (20.27g) Figure 3 (C to D) Although all hormone treatments showed an inhibitory effect on carp growth compared to the WT group, E2+F and E1 performed the best, with the smallest decrease in average weight compared to the control group, while H, DES, and EE2 treatments showed the most significant inhibitory effect on carp weight gain.

[0060] (2) Survival rate statistics of M-xy and M-yy carp in each group

[0061] To analyze the survival rates of XY and YY carp treated with different hormones, we performed genetic identification and statistical analysis on the number of surviving individuals in each experimental group at 4 months of age after treatment. The results showed that, compared to the control group's near 100% survival rate, all hormone-treated groups exhibited lethality. The overall survival rate ranking was as follows: WT (98.89%) > DES (93.33%) > E1 (86.11%) > E2 (79.44%) > E2+Tri (50%) > E2+F (47.78%) > EE2 (16.11%) > H (11.11%). Figure 3 Among the treatments (E to L), the DES and E1 treatments had the highest survival rates, both exceeding 80%. The survival rates of E2+Tri and E2+F were relatively low, around 50%, while the EE2 and H treatments had the highest mortality rates, both below 20%. Although there were significant differences in survival rates among the experimental groups, the survival rates of XY and YY carp within each treatment group were similar, with no significant differences.

[0062] The survival rate was calculated as (number of surviving individuals / number of individuals initially fed) * 100%. Here, "number of surviving individuals" refers to the number of individuals confirmed to be alive by statistics and genetic identification at the end of the treatment, and "number of individuals initially fed" refers to the total number of individuals released into the wild when hormone feeding began in each treatment group.

[0063] (3) Analysis of the effects of different types of hormone treatment on the body shape and morphology of M-xy and M-yy carp:

[0064] At 4 months of age after treatment, the appearance of XY and YY carp in each experimental group was statistically analyzed, and it was found that compared with the control group carp, the spindle-shaped feature ( Figure 4 In group A), the remaining hormone-treated groups all exhibited varying degrees of physical deformities, mainly manifested as irregular shapes such as protruding abdomen and buttocks, and upturned tail. Figure 4 (From B to F), the overall malformation rates of each experimental group were ranked as follows: EE2 (83.13%) > E2+F (72.33%) > E2+Tri (69.58%) > E2 (49.65%) > H (24.12%) > DES (17.92%) > E1 (11.51%) > WT (0.00%). Figure 4(From G to N), the deformity rates in the E1 and DES treatment groups were both below 20%, showing the least impact on carp body shape. The deformity rate in the H group was 24.12%, also exhibiting a relatively small body shape interference effect. The deformity rate in the E2 group increased sharply compared to the H group, reaching 49.65%, with the interference from the three hormones EE2, E2+F, and E2+Tri being the most significant, causing deformity rates of 83.13%, 72.33%, and 69.58%, respectively. Further statistical comparisons showed that although the overall deformity rate fluctuated significantly among the experimental groups, the deformity rates of XY and YY carp within each treatment group were similar, with no significant differences.

[0065] The deformity rate is calculated as (number of deformed individuals / total number of individuals) * 100%.

[0066] Among them, "number of deformed individuals" refers to the number of individuals whose appearance was confirmed to be abnormal after morphological observation after treatment, and "total number of individuals" refers to the total number of surviving individuals in the experimental group who actually participated in the morphological assessment.

[0067] (4) Analysis of the effects of different types of hormone treatment on gonadal development in M-xy and M-yy carp:

[0068] To investigate the effects of different hormone treatments on gonadal development in carp, we dissected and analyzed the gonads of carp in each experimental group at 2 and 4 months after treatment. The results showed that all hormone treatments effectively induced sex reversal in M-xy and M-yy carp, causing them to transition from testes to ovaries. At 2 months of age, the overall sex reversal efficiency of each experimental group was ranked as follows: E2+F (91.67%) > E1 (89.89%) > E2 (69.69%) > EE2 (68.63%) > E2+Tri (63.75%) > DES (48.21%) > H (35.42%) > WT (0.00%). Figure 5 (From A to H), the E2+F, E1, and E2 groups showed the highest sex reversal efficiency, while the DES and H treatments had lower conversion efficiency, both below 50%. The statistical results at 4 months of age were similar to those at 2 months of age. The overall sex reversal efficiency of each treatment group was ranked as follows: E2+F (97.37%) > E1 (90.21%) > E2 (87.29%) > E2+Tri (65.69%) > EE2 (57.44%) > DES (47.72%) > H (28.59%) > WT (0.00%). Figure 5(From A' to H'), statistical results at 4 months of age showed that the E2+F, E1, and E2 groups had the best overall sex reversal efficiency, all achieving at least 87.29% female reversal efficiency. The E2+Tri and EE2 groups had intermediate female reversal efficiency, ranging from 65.69% to 57.44%, while the DES and H groups showed poor female reversal efficiency, both below 50%. Although there were significant differences in sex reversal efficiency among the various hormone treatment groups, our statistical analysis showed that under different hormone treatment methods and treatment durations, the sex reversal efficiency of M-xy and M-yy carp within each treatment group was relatively similar, with no significant differences. The sex reversal efficiency of M-yy carp was also relatively similar.

[0069] The conversion efficiency is calculated as (number of converted individuals / total number of individuals) * 100%.

[0070] Among them, "number of individuals converted to female" refers to the number of individuals whose male gonads (testes) have been successfully reversed to female gonads (ovaries) through histological observation or gonadal morphology identification; "total number of individuals" refers to the total number of individuals in the experimental group whose tissue sections were completed and identified.

[0071] Based on the statistical results of growth, survival rate, deformity rate, and sex reversal efficiency of different treatment groups, it was found that the method described in Example 1 had the best overall effect. It can induce XY and YY carp to achieve a sex reversal efficiency of up to 89.89% under short-term treatment, while having the least impact on carp growth, survival rate, and body shape.

Claims

1. A method for efficiently creating XY or YY pseudo-female carp, comprising the following steps: (1) Formulation of hormone-fed feed: After dissolving estrone in anhydrous ethanol, it was sprayed into carp feed to achieve a hormone concentration of 150-250 mg / kg in the feed. (2) Hormone feeding and daily management: During the breeding season, F-xy and M-xy carp are used to breed and obtain a full-sib population composed of 1 / 4 XX, 2 / 4 XY, and 1 / 4 YY genotypes. After hatching at room temperature, they are fed with Artemia for 1-1.5 weeks indoors. Then they are transferred to outdoor fish ponds for further cultivation. When they grow to 25 dpf, M-xy and M-yy carp are identified. They are transferred to outdoor cement ponds at a ratio of 1:1 to 1.3 per pond of M-xy:M-yy carp. After the juvenile fish are acclimatized to suitable feed for 3-10 days, hormone treatment is carried out. Hormone feed is given at 4-6% of body weight starting at 28-35 dpf, twice a day, at 7:00-8:00 am and 16:00-17:00 pm, with a 9-hour interval between the two times. After 2 to 4 months of feeding, XY or YY pseudo-female carp can be obtained. The M-xy refers to male fish with genotype XY, and the F-xy carp refers to female fish with genotype XY that have undergone sex reversal; M-yy refers to supermale fish with genotype YY.

2. The method according to claim 1, characterized in that, In step (1), when preparing hormone feed, the hormone solution or feed containing hormones should be prepared again every 7-15 days. The prepared feed should be kept away from light, sealed, protected from moisture, and kept at low temperature throughout the storage and use process.

3. The method according to claim 1, characterized in that, In step (1), the hormone concentration in the hormone feed is 200 mg / kg.

4. The method according to claim 1, characterized in that, In step (1), the acclimatization time for palatable feed is 5 days.

5. The method according to claim 1, characterized in that, In step (1), hormone feed is given at 4% of body weight starting at 30 dpf.

6. The method according to claim 1, characterized in that, In step (2), hormone feed was administered at 7:00 AM and 4:00 PM.

7. The method according to claim 1, characterized in that, In step (2), the hormone feed is fed for 4 months.

8. The method according to claim 1, characterized in that, The outdoor cement pool described in step (2) has a built-in circulating water system.

9. An application of the method for efficiently creating XY or YY pseudo-female carp as described in any one of claims 1-8, wherein the application is any one of the following: 1) Carp breeding; 2) Carp farming; 3) Prevention and control of carp invasion.

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