A fish embryo screening method based on 1,6-hexanediol-induced intracellular phase change and application

Abstract

This application discloses a method and application for screening fish embryos based on 1,6-hexanediol-induced intracellular phase changes, belonging to the field of fish breeding technology. This method addresses the lack of non-destructive screening methods for early embryos in existing technologies. During the maternal zygote transition period, which includes fish embryonic development, 0.1% 1,6-hexanediol is used for stress treatment. Utilizing its ability to interfere with intracellular biomolecule condensation, it induces intracellular phase changes, thereby eliminating embryos with poor internal environmental stability and low robustness, while retaining high-quality embryos with high developmental potential. The screened embryos show a significantly reduced rate of deformities in the current generation, and superior traits are heritable to offspring. The fertilization rate and hatching rate of offspring are significantly improved, and the deformity rate is significantly reduced. This application is simple to operate, cost-effective, and suitable for large-scale seedling production, providing a new technical pathway for the breeding of superior fish breeds.

Description

Technical Field

[0001] This application relates to the field of fish breeding technology, specifically to a method and application for screening fish embryos based on intracellular phase changes induced by 1,6-hexanediol. Background Technology

[0002] Artificial breeding and seedling production of fish are fundamental aspects of the aquaculture industry. The quality of embryos directly determines hatching success rate, larval survival rate, and subsequent growth performance and environmental adaptability during rearing. Within embryo populations from the same parentage, significant differences exist in developmental potential and physiological state due to individual variations in maternal material reserves, intracellular homeostasis regulation, and early developmental regulatory mechanisms. These differences are largely determined early in embryonic development, but primarily occur at the cellular and molecular levels. Therefore, how to non-destructively and efficiently screen for high-quality embryos in the early stages of embryonic development is a critical technical challenge that urgently needs to be addressed in the field of aquaculture breeding.

[0003] Currently, the assessment of fish embryo quality mainly relies on two approaches: first, preliminary evaluation through microscopic observation of embryonic morphological characteristics, such as uniform cleavage and normal blastocyst development; second, post-hatching statistical methods, which analyze hatching rate, malformation rate, and early mortality rate after embryo hatching. However, both methods have significant technical limitations. While microscopic observation is intuitive, it is difficult to quantify and standardize, requires a high level of experience from operators, and cannot accurately assess the internal physiological state and developmental potential of the embryo. Post-hatching statistical methods, while providing objective data, require screening at the mid-to-late stages of embryonic development or even after hatching. This not only necessitates a long cultivation period to obtain evaluation criteria, leading to low screening efficiency, but also results in low-quality embryos not being promptly removed in the early stages, thus occupying cultivation space and resources that should belong to high-quality embryos. More importantly, even if some low-quality embryos manage to hatch, their offspring often exhibit unstable growth rates, stress resistance, and environmental adaptability, making it difficult to meet the needs of large-scale breeding and selective breeding. Long-term accumulation may even affect the genetic quality of the entire breeding population.

[0004] Therefore, developing a technical method that can effectively distinguish between high-quality and low-quality embryos in the early stages of fish embryonic development without damaging embryo survival, and that is applicable to large-scale breeding and production, is of great practical significance for improving the overall quality of seedlings, shortening the breeding cycle, reducing aquaculture risks, and promoting the high-quality development of the aquatic seed industry. Summary of the Invention

[0005] In view of this, the purpose of this application is to provide a method and application for screening fish embryos based on 1,6-hexanediol-induced intracellular phase changes. This screening method uses 1,6-hexanediol as the screening reagent and applies stress treatment during the maternal zygote transition window, which includes fish embryonic development. By inducing intracellular phase changes, it simulates environmental stress exceeding the embryonic homeostasis tolerance threshold, thereby eliminating embryos with poor internal environmental stability and low developmental potential, while retaining high-quality embryos with strong robustness and high developmental potential. The surviving embryos after screening exhibit advantages such as reduced malformation rate and increased hatching rate in contemporary development, and these superior traits can be inherited by offspring, significantly improving fertilization rate and hatching rate while reducing malformation rate, thus achieving early and rapid screening and breeding of high-quality fish embryos.

[0006] To achieve the above objectives, this application provides the following technical solution:

[0007] In a first aspect, this application provides a method for screening fish embryos based on 1,6-hexanediol-induced intracellular phase changes, comprising the following steps:

[0008] During maternal zygote conversion in fish embryonic development, fertilized embryos were subjected to stress treatment using a treatment solution containing 1,6-hexanediol.

[0009] After processing, the embryos were transferred to a clean culture medium free of 1,6-hexanediol for further cultivation.

[0010] Embryos that suffer irreversible damage or die during the processing are discarded, and surviving and normally developing embryos are retained, thus obtaining high-quality embryos after screening.

[0011] In some preferred embodiments, the maternal zygote conversion occurs 3.5-8 hours after fertilization.

[0012] In some embodiments, the concentration of 1,6-hexanediol in the treatment solution is from 0.01% to 1%.

[0013] In some preferred embodiments, the concentration of 1,6-hexanediol in the treatment solution is 0.1%.

[0014] In some preferred embodiments, the stress treatment involves immersing the fertilized embryo in a treatment solution containing 1,6-hexanediol, while maintaining a constant water temperature of 26-28 °C during the treatment.

[0015] In some preferred embodiments, the fish is the yellow catfish.

[0016] In some preferred embodiments, the fish embryo screening method further includes the steps of subsequently cultivating the surviving embryos after screening and evaluating the reproductive performance of their offspring; the reproductive performance of the offspring includes at least one of fertilization rate, hatching rate and malformation rate.

[0017] Secondly, this application provides an application of high-quality fish embryos obtained by the method described in the first aspect in the breeding of superior varieties.

[0018] Thirdly, this application provides the use of 1,6-hexanediol in the preparation of reagents for screening high-quality fish embryos, wherein the screening is achieved by stress treatment with 1,6-hexanediol during maternal zygote conversion in fish embryo development.

[0019] Fourthly, this application provides a kit for screening high-quality fish embryos, which contains a 1,6-hexanediol treatment solution at a concentration of 0.01% to 1%.

[0020] Compared with the prior art, this application has at least the following advantages and beneficial effects:

[0021] 1. This application achieves early non-destructive screening of fish embryos. By applying 1,6-hexanediol treatment within the time interval including the maternal zygote transition, high-quality and low-quality embryos can be rapidly distinguished in the early stages of embryonic development, significantly shortening the screening cycle and causing no subsequent developmental damage to surviving embryos. This overcomes the shortcomings of existing technologies, such as delayed screening nodes and low efficiency, and is suitable for large-scale seedling production scenarios.

[0022] 2. This application significantly improves the developmental quality of contemporary embryos. Experiments show that yellow catfish embryos treated with 0.1% 1,6-hexanediol exhibit a 4% lower rate of malformation compared to the control group, with a corresponding increase in hatchability. This stress treatment precisely eliminates embryos with poor intracellular environmental stability and low developmental potential, resulting in a more stable and higher-quality overall development of the surviving embryo population.

[0023] 3. The screening effect of this application is heritable across generations. When the surviving embryos after screening are cultured to sexual maturity, the reproductive performance of their offspring is significantly improved: the artificial insemination rate increases by 4%, the hatching rate increases by 4%, and the malformation rate decreases by 7%. This indicates that this application can retain individuals with excellent genetic endowments through screening, achieving continuous optimization of superior traits and overcoming the limitations of traditional screening methods that are confined to contemporary observation.

[0024] 4. This application is simple to operate, cost-effective, and has good prospects for industrial application. 1,6-Hexanediol is a conventional biochemical reagent, and the treatment method is only a simple soaking, requiring no complex equipment, making it easy to promote and apply in the production line. This method improves the overall quality of seedlings from the source, reduces aquaculture risks, shortens the breeding cycle, and provides a new technical path for the high-quality development of the aquatic seed industry. Attached Figure Description

[0025] Figure 1 A roadmap for yellow catfish embryo screening technology.

[0026] Figure 2 Morphological comparison of embryos treated with different concentrations of 1,6-hexanediol.

[0027] Figure 3 The results show the effects of different concentrations of 1,6-hexanediol on embryonic development.

[0028] Figure 4 The results show the effect of different concentrations of 1,6-hexanediol on embryo survival rate.

[0029] Figure 5 The results show the effect of different concentrations of 1,6-hexanediol on the embryo malformation rate.

[0030] Figure 6 The results show the effect of 1,6-hexanediol treatment on intracellular phase separation structure. A represents the control sample without 1,6-hexanediol treatment, and B represents the sample treated with 0.1% 1,6-hexanediol.

[0031] Figure 7 The purpose of this study is to compare the reproductive performance of selected embryos as they develop to sexual maturity. In this study, A represents the comparison of fertilization rate, B represents the comparison of hatching rate, and C represents the comparison of malformation rate. Detailed Implementation

[0032] To make the objectives, technical solutions, and advantages of this application clearer, the following detailed description is provided in conjunction with embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the scope of this application.

[0033] The materials used in the following embodiments are not limited to those listed below, and other similar materials may be used instead. Unless otherwise specified, the instruments shall be used under conventional conditions or as recommended by the manufacturer. Those skilled in the art should have relevant knowledge of the use of conventional materials and instruments.

[0034] To better understand this teaching and without limiting its scope, all figures and other numerical values ​​used in the specification and claims to express quantities, percentages, or proportions should, in all cases, be understood to be modified by the term "about." Therefore, unless otherwise stated, the numerical parameters set forth in the following specification and appended claims are approximate values ​​that may vary depending on the desired properties sought. At a minimum, each numerical parameter should be interpreted based at least on the reported significant figures and by applying common rounding techniques.

[0035] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the subject matter of this application pertains. Before providing a detailed description of this application, the following terms and definitions are provided to better understand it.

[0036] 1. Intracellular phase change: refers to the reversible process by which intracellular biomolecules (such as proteins and nucleic acids) separate into membrane-free organelles or biomolecular condensates through liquid-liquid phase separation. In this application, it specifically refers to the dynamic changes induced by 1,6-hexanediol through interference with weak interactions (such as hydrophobic interactions and aromatic amino acid interactions) between low-complexity domains of proteins, leading to depolymerization or recombination of biomolecular condensates. (Affecting the stability of biomolecular condensates)

[0037] 2. Maternal-zygotic transition period: This refers to the critical period in the early stages of fish embryonic development when the embryo transitions from dependence on maternal mRNA and protein regulation to dependence on transcriptional regulation by the zygotic genome itself. In this application, it specifically refers to the developmental window from fertilization to approximately 8 hours post-fertilization, during which the embryo is highly sensitive to disturbances in the intracellular environment.

[0038] 3. 1,6-Hexanediol (HDO): A straight-chain aliphatic diol with the molecular formula C6H2O. 14 O2, CAS No. 629-11-8. In this application, 1,6-hexanediol is used as a phase separation regulator to induce reversible depolymerization of intracellular biomolecule condensates by interfering with hydrophobic interactions and aromatic amino acid interactions between low-complexity domains of proteins, thereby achieving selective screening of embryos of different quality.

[0039] 4. Robustness: refers to the ability of a biological system to maintain normal physiological functions and developmental processes when subjected to internal or external disturbances. In this application, it specifically refers to the ability of an embryo to maintain intracellular homeostasis and the stability of its molecular interaction network under 1,6-hexanediol stress. Embryos with high robustness can tolerate the treatment and develop normally, while embryos with low robustness suffer irreversible damage.

[0040] 5. Surviving embryos: refers to embryos that meet all of the following criteria after treatment with 1,6-hexanediol: early yellow catfish embryos divide at the expected time, the blastospheres are of uniform and symmetrical size, the blastodisc structure is intact, the boundary between the embryo and the yolk below is clear, the peri-ovipositor structure is intact, and there is no fragmentation or other issues.

[0041] 6. Dead embryos: refers to embryos that exhibit one of the following characteristics after treatment with 1,6-hexanediol: cessation of embryonic cleavage, fragmentation of yolk, loss of cell boundaries, and whitening or turbidity of the embryo.

[0042] 7. Deformed embryos: refers to individuals that survive treatment with 1,6-hexanediol but exhibit obvious morphological abnormalities, including one or more of the following characteristics: body axis curvature, pericardial or yolk sac edema, shortened body length, abnormal head or tail development, etc.

[0043] The technical solution of this application and the technical effects achieved will be described in detail below through more specific embodiments.

[0044] Source of materials:

[0045] The parent yellow catfish used in the experiment were purchased from Tianjiahu Aquatic Seed Industry Base, Yueyang City, Hunan Province. 1,6-hexanediol was purchased from Merck (Darmstadt, Germany), product code 240117-50G. All other routine reagents were of analytical grade unless otherwise specified. The embryo culture water was tap water that had been aerated for 24 hours (hereinafter referred to as "aerated water"). Aeration was continuously performed during the aeration process to remove residual chlorine and achieve dissolved oxygen saturation.

[0046] Example 1: High-quality embryo screening experiment of yellow catfish

[0047] The overall technical approach of this embodiment is as follows: Figure 1 As shown, fertilized embryos were subjected to gradient stress treatment and then transferred to a normal environment for continued rearing. Individuals with poor developmental stability were culled. Surviving embryos were cultured to sexual maturity and their reproductive performance was tested to determine the final breeding parameters. Figure 1 This provides a visual framework for the experimental design of Example 1 of this application, clarifying the complete technical chain from gradient treatment → screening of surviving individuals → routine feeding → offspring performance verification, ensuring the operability and repeatability of the technical solution.

[0048] 1. Experimental Materials and Grouping

[0049] In this embodiment, healthy, mature, one-year-old wild yellow catfish with swollen abdomens were selected as parent fish in May and June. Gametes were obtained after artificial spawning and artificial insemination was performed. The fertilized embryos obtained from the same batch were used for the following screening and treatment experiments.

[0050] Fertilized embryos were randomly assigned to groups of approximately 100 embryos each and cultured in 1,6-hexanediol solutions of different treatment concentrations. The treatment concentrations included a control group (0%, aerated water only) and seven concentration gradients: 0.01%, 0.1%, 0.25%, 0.5%, 0.75%, and 1%. Each concentration group had six biological replicates, resulting in approximately 600 embryos per concentration group.

[0051] 2.1 Preparation of 6-hexanediol treatment solution

[0052] Weigh out 1,6-hexanediol and dissolve it in aerated water to prepare a stock solution with a concentration of 1 g / L. When using, dilute the stock solution to the target concentration with aerated water: all concentrations are diluted by volume of the stock solution; 1 ml of stock solution + 99 ml of aerated water equals a 100-fold dilution, i.e., a 1% concentration. Other concentrations (0.01%, 0.1%, 0.25%, 0.5%, 0.75%) are prepared in the same proportions.

[0053] 3. Treatment and Culture Methods

[0054] After fertilization, gently agitate the culture dish twice with aerated water to remove excess sperm and impurities. Then add 40 mL of prepared 1,6-hexanediol treatment solution, ensuring the liquid completely covers all embryos. Cover the culture dish and place it in a 28 ℃ incubator for static immersion culture. The incubator photocycle is set to 12 hours light / 12 hours dark.

[0055] Treatment began immediately after fertilization and continued until 8 hours post-fertilization. During this period, observations were conducted according to the embryonic developmental stages: 1-cell stage (approximately 20 minutes post-fertilization), 4-cell stage (approximately 1 hour post-fertilization), 64-cell stage (approximately 2 hours post-fertilization), and early gastrulation stage (approximately 8 hours post-fertilization). The developmental morphology and survival status of each group of embryos were recorded.

[0056] 4. Post-treatment culture and observation

[0057] After treatment (8 hours post-fertilization), dead embryos were removed, rinsed three times with aerated water, and then replaced with clean aerated water at 28°C. The embryos were then placed in a 28°C incubator for further culture. The aerated water was changed every 8 hours thereafter until 72 hours post-fertilization, at which point the hatching rate and deformity rate were calculated.

[0058] 5. Judgment Criteria

[0059] The embryonic status is determined based on the following criteria:

[0060] Surviving embryos: The early embryos of yellow catfish divided at the expected time, the blastospheres were uniform in size and symmetrical, the embryonic disc structure was intact, the boundary between the embryo and the yolk below was clear, the structure around the ovipositor was intact, and there was no fragmentation or other issues.

[0061] Dead embryo: Embryo cleavage stops, yolk breaks apart, cell boundaries are lost, and the embryo turns white or cloudy.

[0062] Abnormal embryos: Individuals that survive but exhibit obvious morphological abnormalities, including body axis curvature, pericardial or yolk sac hydrops, shortened body length, abnormal head or tail development, etc.

[0063] 7. Microscopic verification

[0064] Embryos from the WT group and the 0.1% treatment group at 4 hpf were collected, fixed in tissue fixative at 4 ℃ for 24 hours, and then peeled off. ANP32E immunofluorescence staining was performed. Morphological differences between the two groups were observed using a Leica TCS SP8 DLS confocal microscope (20x magnification).

[0065] Example 2: Effects of different concentrations of 1,6-hexanediol on embryonic development

[0066] Embryos from different treatment groups were systematically observed and statistically analyzed according to the method in Example 1. All data are expressed as mean ± standard error (SEM). One-way ANOVA was used for comparisons among multiple groups, followed by Tukey's HSD multiple comparison test. The significance level was set at P < 0.05. Different letters (a, b, c, etc.) in the figure indicate significant differences between groups. The results are as follows:

[0067] 1. Morphological observation

[0068] The embryos of yellow catfish were observed at the 1-cell, 4-cell, 64-cell, and early gastrulation stages. The morphological characteristics of normal embryos at each developmental stage are as follows:

[0069] 1. Cell stage: The cytoplasm of the animal pole concentrates upward to form a round cell with a clear boundary. The cell surface is smooth, no cleavage groove is seen, and the boundary between the cell and the yolk below is obvious. The polarity is clear, the embryonic structure is complete, and no collapse or abnormal morphology is seen.

[0070] 4-cell stage: The cells at the animal pole undergo two consecutive cleavages, forming four blastomeres of roughly the same size. The cleavage grooves are clear and crisscrossed, dividing the blastodisc into four equal regions. The blastodisc boundaries are intact, clearly demarcated from the underlying yolk, and the perifollicular structures are intact, with no obvious morphological abnormalities observed.

[0071] 64-cell stage: The animal pole blastodisc has formed 64 relatively uniform blastomeres. The blastomeres are closely packed, with clear boundaries, and exhibit a regular polygonal structure. The blastodisc region is significantly thickened, but no significant enveloping movement has yet occurred. The yolk structure is homogeneous, and the perifollicular space is intact. The overall morphology is regular, and no obvious developmental abnormalities are observed.

[0072] Early gastrulation: The embryo is generally round and regular, with an intact and transparent egg membrane and a clearly defined yolk outline. The blastodisc extends significantly towards the yolk surface and gradually envelops it, exhibiting typical enveloping movement characteristics. The germ layer cells are relatively uniformly arranged, the embryo has high transparency, and no obvious granulation or cell fragmentation is observed. The blastodisc boundary is clear, with a distinct boundary between it and the yolk.

[0073] Figure 2 Microscopic morphological photographs of the control group and each treatment group at the 1-cell stage, 4-cell stage, 64-cell stage, and early gastrulation stage are shown. Red arrows indicate areas of developmental or structural abnormalities. Observational results indicate that:

[0074] Control group (aerated water): Normal morphology at each developmental stage, used as a baseline reference.

[0075] 0.01% treatment group: Most embryos showed premature development.

[0076] 0.1% treatment group: The developmental process was basically the same as that of the control group, and the morphology was normal.

[0077] 0.25% treatment group: Some embryos showed developmental delay.

[0078] In the 0.5% and 0.75% treatment groups, most embryos showed developmental delay.

[0079] 0.75% and 1% treatment groups: widespread embryonic malformation and death occurred.

[0080] Morphological observations showed that treatment at a concentration of 0.1% had the least impact on embryonic morphological development, while high concentrations (≥0.5%) led to significant developmental abnormalities and death.

[0081] 2. Developmental Process Analysis

[0082] Figure 3 The proportions of embryos with early development (red bars) and delayed development (blue bars) are shown in each treatment group. Data are expressed as mean ± SEM, with different letters representing significant differences (P < 0.05). The results indicate that:

[0083] The 0.01% treatment group had a significantly higher rate of precocious puberty than other groups.

[0084] 0.1% treatment group: The proportion of early and delayed development was not significantly different from that of the control group.

[0085] Treatment groups with concentrations of 0.25% and above: The proportion of developmental delays increased significantly with increasing concentration.

[0086] Developmental process analysis further confirmed that the 0.1% concentration treatment group was able to maintain a normal embryonic development rhythm and was the preferred condition for maintaining developmental stability.

[0087] 3. Survival rate statistics

[0088] Embryo viability was assessed in each treatment group 3 days post-fertilization (3 dpf), and the results are as follows: Figure 4 As shown in the figure. Each point represents a biological replicate (n=6), and different letters (af) indicate significant differences (P<0.05). The results indicate that:

[0089] The control group, the 0.01% treatment group, and the 0.1% treatment group all had high survival rates, with no significant difference among the three groups (labeled as a / b).

[0090] 0.25% treatment group: survival rate was significantly reduced, approximately half that of the control group (labeled c / d).

[0091] 0.5%, 0.75%, and 1% treatment groups: survival rate decreased significantly and continuously with increasing concentration (labeled as e / f).

[0092] Survival data indicate that a 0.1% concentration can be used for screening while maintaining a high survival rate, while a concentration of ≥0.25% leads to significant embryo death.

[0093] 4. Deformity rate statistics

[0094] Analysis of the ratio of normal to abnormal embryos in each treatment group is as follows: Figure 5 As shown, blue bars represent normal embryos, and red bars represent malformed embryos. Different letters indicate significant differences between groups (P<0.05). The results indicate that:

[0095] 0.1% treatment group: The malformation rate was the lowest among all treatment groups, significantly lower than that of the control group.

[0096] The 0.01% and 0.25% treatment groups had relatively high rates of deformity.

[0097] Treatment group with a mortality rate of 0.5% or higher: Due to the excessively high mortality rate, the sample size for the malformation rate statistics was insufficient.

[0098] Analysis of the malformation rate showed that treatment with a concentration of 0.1% could significantly reduce the malformation rate of embryos, which is the optimal condition for screening high-quality embryos.

[0099] 5. Screening Mechanism Analysis

[0100] The screening method described in this application is based on the following scientific principles to differentiate embryo quality:

[0101] Cells contain a large number of proteins with low-complexity domains and hydrophobic amino acids. Many liquid condensates (i.e., biomolecular condensates) are formed by weak interactions between these low-complexity protein domains. 1,6-Hexanediol can interfere with hydrophobic interactions and interactions associated with aromatic amino acids, and these liquid condensates usually undergo rapid depolymerization after treatment.

[0102] Embryos of different qualities exhibit differences in intracellular environmental stability and molecular interaction network structure. Under the same treatment conditions, embryos with relatively poor intracellular environments have looser, smaller, or less stable biomolecular condensate structures, making them more prone to depolymerization under the influence of 1,6-hexanediol. This leads to impaired biological functions, resulting in developmental restriction, malformation, or death. In contrast, embryos with stronger intracellular homeostasis have more stable molecular condensate network structures. Within appropriate concentrations and treatment durations, they can maintain some of their condensed structures and functions, thus tolerating the disturbance and maintaining normal development.

[0103] Therefore, under preset concentration (0.1%) and treatment time (from fertilization to 8 hours), the selective perturbation effect of 1,6-hexanediol can amplify the differences in developmental outcomes between embryos of different qualities, achieving functional screening of low-quality embryos. The phenomenon observed in this embodiment, with a higher survival rate and the lowest malformation rate in the 0.1% treatment group, is a concrete manifestation of this screening mechanism: embryos with strong intracellular environmental stability and high robustness can tolerate the treatment and develop normally, while embryos with poor intracellular environmental stability are selectively eliminated.

[0104] 6. Microscopic verification of the screening mechanism

[0105] To further verify the above screening mechanism, the direct effect of 1,6-hexanediol treatment on intracellular phase separation structure was observed using a Leica TCS SP8 DLS confocal microscope (20x magnification). The results are as follows: Figure 6 As shown.

[0106] Figure 6 Sample A is the control group (without 1,6-hexanediol treatment). Numerous small and densely distributed phase separation points can be seen within the cells. These phase separation points correspond to biomolecular condensates formed by weak interactions between low-complexity protein domains. Figure 6 Sample B is the sample treated with 0.1% 1,6-hexanediol. It can be seen that the small phase separation points are selectively dissolved, leaving only a small number of larger phase separation structures.

[0107] This phenomenon directly confirms the mechanism of action of 1,6-hexanediol: it can selectively interfere with weak interactions, causing the depolymerization of small, loosely structured aggregates with lower stability, while larger, more stable aggregates with a compact structure are retained. This is completely consistent with the principle of the screening method in this application—embryos with poor intracellular environment stability and loose biomolecular aggregate structure suffer irreversible damage under the action of 1,6-hexanediol, while embryos with strong intracellular environment stability and compact aggregate structure can tolerate the treatment and maintain normal development.

[0108] Conclusion: Comprehensive Figure 2-6 Based on the results and the above mechanistic analysis, treatment with 0.1% 1,6-hexanediol can maintain normal development while ensuring embryo survival rate, resulting in embryos with normal morphology and a significant reduction in malformation rate. The scientific essence of this screening effect lies in the selective perturbation effect of 1,6-hexanediol on intracellular phase, making a 0.1% concentration the optimal treatment condition for yellow catfish embryo screening.

[0109] Example 3: Evaluation of transgenerational genetic effects of embryos after screening

[0110] 1. Experimental Design

[0111] Embryos from surviving offspring of the control group (wt) and the 0.1% treatment group were transferred to the same environment and reared until sexual maturity. For the first 60 days after hatching, 150 juvenile fish from each treatment group were reared in a 61×48×36 cm rearing tank, placed in the same recirculating aquaculture system. After 60 days, they were transferred to cylindrical rearing tanks (1 meter in diameter, 1.5 meters deep) for flow-through rearing, and fed using the same brand of automatic feeder with identical feeding parameters.

[0112] 2. Reproductive performance test

[0113] After the yellow catfish in each group reached sexual maturity, females were randomly selected for induced spawning. For each breeding cycle, three females and one male from each group were paired. The male was anesthetized, dissected, and its sperm was ground and preserved in a sperm preservation solution. To amplify phenotypic differences between treatment groups, fertilization was performed using the same male-female pairing method, with a total of six independent replicates. The control group consisted of embryos raised in aerated water during the treatment process.

[0114] 3. Performance statistics of offspring

[0115] The fertilization rate, hatching rate, and malformation rate of the obtained embryos were statistically analyzed, and the results are as follows: Figure 7 As shown:

[0116] Fertilization rate ( Figure 7 A): Compared with the control group, the fertilization rate of offspring produced by fish treated with 0.1% 1,6-hexanediol was significantly improved (*P<0.05). This indicates that the surviving embryos after selection with 0.1% 1,6-hexanediol can pass on their superior traits to their offspring, exhibiting a higher success rate in the fertilization stage of the offspring.

[0117] Hatching rate ( Figure 7 B): The hatching rate of offspring in the 0.1% treatment group was significantly higher than that in the control group (*P<0.05). This indicates that the screening treatment can increase the proportion of offspring embryos that complete the normal hatching process, demonstrating the intergenerational continuation of the screening effect.

[0118] Deformity rate ( Figure 7C): The malformation rate of offspring in the 0.1% treatment group was significantly lower than that in the control group (*P<0.05). This indicates that screening treatment can reduce the risk of morphological abnormalities and developmental defects in offspring embryos and improve the overall developmental stability of the population.

[0119] The above results demonstrate that early embryonic screening with 0.1% 1,6-hexanediol not only optimizes the quality of current-generation embryos but also transmits superior traits to offspring, significantly improving fertilization and hatching rates while reducing malformation rates. This achieves a complete technical loop from current-generation screening to intergenerational inheritance. The screening method described in this application has significant application value in fish embryo quality control and breeding.

[0120] The present application has been described in detail above. Specific examples have been used to illustrate the principles and implementation methods of the present application. The descriptions of the embodiments above are only for the purpose of helping to understand the present application and its core ideas. It should be noted that those skilled in the art can make several improvements and modifications to the present application without departing from the principles of the present application, and these improvements and modifications also fall within the protection scope of the claims of the present application.

Claims

1. A method for screening fish embryos based on 1,6-hexanediol-induced intracellular phase changes, characterized in that, Includes the following steps: During maternal zygote conversion in fish embryonic development, fertilized embryos were subjected to stress treatment using a treatment solution containing 1,6-hexanediol. After processing, the embryos were transferred to a clean culture medium free of 1,6-hexanediol for further cultivation. Embryos that suffer irreversible damage or die during the processing are discarded, and surviving and normally developing embryos are retained, thus obtaining high-quality embryos after screening.

2. The fish embryo screening method according to claim 1, characterized in that, The maternal zygote conversion period is 3.5 to 8 hours after fertilization.

3. The fish embryo screening method according to claim 1, characterized in that, The concentration of 1,6-hexanediol in the treatment solution is from 0.01% to 1%.

4. The fish embryo screening method according to claim 3, characterized in that, The concentration of 1,6-hexanediol in the treatment solution is 0.1%.

5. The fish embryo screening method according to claim 1, characterized in that, The stress treatment involves immersing the fertilized embryos in a treatment solution containing 1,6-hexanediol, while maintaining a constant water temperature of 26-28 °C during the treatment process.

6. The fish embryo screening method according to claim 1, characterized in that, The fish in question is the yellow catfish.

7. The fish embryo screening method according to claim 1, characterized in that, The fish embryo screening method further includes the steps of further cultivating the surviving embryos after screening and evaluating the reproductive performance of their offspring; the reproductive performance of the offspring includes at least one of fertilization rate, hatching rate and malformation rate.

8. The application of high-quality fish embryos obtained by any of the fish embryo screening methods described in claims 1-7 in the breeding of superior varieties. 9.1,6-Hexanediol's application in the preparation of reagents for screening high-quality fish embryos, characterized in that, The screening was achieved by stress treatment with 1,6-hexanediol during maternal zygote conversion in fish embryonic development.

10. A kit for screening high-quality fish embryos, characterized in that, It contains a treatment solution of 1,6-hexanediol at a concentration of 0.01% to 1%.

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