A method for constructing a zebrafish reproductive toxicity model, a method for evaluating reproductive toxicity of a chemical substance, and application thereof

Abstract

The application discloses a method for constructing a zebrafish reproductive toxicity model, a method for evaluating reproductive toxicity of a chemical substance and application, and relates to the technical field of reproductive development toxicity of a chemical substance. The method for evaluating reproductive toxicity of a chemical substance provided by the application can focus on changes in courtship behavior by taking behavior pictures or videos, does not need to sacrifice test animals, and does not need professional fluorescence imaging instruments and equipment and tissue section equipment; the method is more in line with animal ethics, saves cost, and can realize continuous observation by using the same batch of organisms. Test courtship behavior can be quickly analyzed by using software to obtain changes in zebrafish behavior. The evaluation method provided by the application can detect ovarian malformation of females and male reproductive toxicity, and has the advantage of more evaluation dimensions. Based on the method provided by the application, a zebrafish reproductive toxicity model can be constructed, and the model can be used as a drug screening platform for damage of reproductive function of zebrafish.

Description

Technical Field

[0001] This invention relates to the field of reproductive and developmental toxicity technology of chemical substances, and more specifically, to a method for constructing a zebrafish reproductive toxicity model, a method for evaluating the reproductive toxicity of chemical substances, and their applications. Background Technology

[0002] Currently, the main methods for evaluating the reproductive and developmental toxicity of chemical substances include: real-time fluorescence dynamic tracking in transgenic animal models, assessment of spermatogenesis in male zebrafish and oviposition in female zebrafish after anesthesia, and evaluation of ovarian malformation in fruit flies. Among these, fluorescence imaging technology primarily focuses on tissue development and / or early embryo implantation and / or in vitro embryonic developmental toxicity. This requires the observation of biological tissue sections, necessitating not only appropriate technical equipment but also the sacrifice of experimental animals. However, zebrafish anesthesia presents operational challenges, requiring the extraction of sperm and eggs after anesthesia, followed by manual counting. The anesthesia and statistical processes are time-consuming and prone to introducing random errors.

[0003] In view of this, the present invention is proposed. Summary of the Invention

[0004] The purpose of this invention is to provide a method and application for evaluating the reproductive toxicity of chemical substances to solve the above-mentioned technical problems.

[0005] This invention is implemented as follows:

[0006] In a first aspect, the present invention provides a method for constructing a zebrafish reproductive toxicity model, which includes the following steps:

[0007] Expose zebrafish to chemicals with reproductive toxicity;

[0008] The reproductive toxic chemicals are selected from methylmercury, alkaloid toxins, bisphenols or pyrethroid insecticides; the concentration of the chemical in the exposure environment is at least 0.5 μg / L, and the exposure time to the reproductive toxic chemical is 14-42 days;

[0009] Preferably, the alkaloid toxin is cylindrospermopsin (CAS: 143545-90-8).

[0010] Secondly, the present invention provides a method for evaluating the reproductive toxicity of chemical substances, which includes the following steps: evaluating the courtship behavior and fertility of zebrafish.

[0011] The courtship behavior evaluation includes: first, exposing female and male zebrafish to an environment containing reproductively toxic chemicals; then, placing the exposed female or male target zebrafish in one area of ​​the courtship behavior evaluation, and correspondingly placing the exposed male or female test zebrafish in another area of ​​the courtship behavior evaluation; the activity areas of the target zebrafish and the test zebrafish do not overlap, and capturing behavioral videos and / or images of the zebrafish over a period of time; and evaluating the movement behavior of male and female zebrafish during courtship.

[0012] The concentration of the chemical substance in the exposure environment is at least 0.5 μg / L; the concentration of the chemical substance in the courtship behavior evaluation area is the same as that in the exposure environment.

[0013] The kinetic behaviors during courtship are selected from at least one of the following:

[0014] The test zebrafish's movement distance during courtship, the total duration of the test zebrafish's stay in a designated area, the frequency of entering the designated area, the courtship trajectory, and the courtship index; the designated area is the area close to the target fish;

[0015] Fertility assessment includes the following steps: female and male zebrafish exposed to a chemical substance with reproductive toxicity are co-cultured, and then the zebrafish are evaluated for at least one of the following: assessing the egg production and / or fertilization rate of female zebrafish, and detecting the levels of reproductive-related hormones in female and / or male zebrafish.

[0016] Preferably, the distance the zebrafish travels in a designated area during courtship is tested;

[0017] Preferably, the female and male zebrafish are kept together for 7-28 days after exposure.

[0018] In a preferred embodiment of the present invention, if zebrafish exhibit decreased courtship behavior and reduced fertility in an exposed environment, the reproductive toxicity of the chemical substance to the zebrafish is assessed.

[0019] In a preferred embodiment of the present invention, female and male zebrafish are exposed to a reproductively toxic chemical environment for 14-42 days.

[0020] In a preferred embodiment of the present invention, the time for filming zebrafish behavioral videos and / or pictures is 10-20 minutes;

[0021] In a preferred embodiment of the present invention, the zebrafish behavioral videos and / or images are taken under continuous illumination.

[0022] In a preferred embodiment of the present invention, software is used to analyze zebrafish behavioral videos and / or images to obtain their movement behavior during courtship.

[0023] In a preferred embodiment of the present invention, the courtship index refers to the time it takes for a male or female zebrafish to chase another female or male zebrafish.

[0024] In a preferred embodiment of the present invention, the reproductive-related hormone is selected from testosterone, estradiol, or lutein.

[0025] In a preferred embodiment of the present invention, the reproductive toxic chemical substance is selected from methylmercury, alkaloid toxins, bisphenols, or pyrethroid insecticides. The alkaloid toxin is cylindrospermopsin (CAS: 143545-90-8).

[0026] Thirdly, the present invention also provides the application of the zebrafish reproductive toxicity model constructed by the above-described model construction method or the zebrafish reproductive toxicity model constructed by the method for evaluating the reproductive toxicity of chemical substances in screening drugs for the prevention and / or treatment of reproductive dysfunction. The application includes: assessing the reproductive toxicity of chemical substances to zebrafish according to the above-described method for evaluating the reproductive toxicity of chemical substances, and obtaining a zebrafish reproductive toxicity model.

[0027] Fourthly, the present invention also provides the application of the zebrafish reproductive toxicity model constructed by the above-described model construction method or the zebrafish reproductive toxicity model constructed by the method for evaluating the reproductive toxicity of chemical substances in screening drugs that improve or inhibit abnormal courtship behavior in zebrafish. The application includes: assessing the reproductive toxicity of chemical substances to zebrafish according to the above-described method for evaluating the reproductive toxicity of chemical substances, and obtaining a zebrafish reproductive toxicity model.

[0028] The present invention has the following beneficial effects:

[0029] The method for evaluating the reproductive toxicity of chemical substances provided by this invention can monitor changes in courtship behavior by taking behavioral pictures or videos, without sacrificing test animals or requiring specialized fluorescence imaging equipment or tissue sectioning equipment; it is also more ethical, cost-effective, and can be used to achieve continuous observation with the same batch of organisms, making the experimental process more flexible.

[0030] Testing courtship behavior can quickly analyze changes in zebrafish behavior using software, which can save a lot of manpower and time compared with traditional spawning statistics methods, and does not require any anesthesia of the fish. Courtship behavior indicates reproductive effects, and can also be observed in vivo, supporting long-term tracking or repeated experiments of the same batch of zebrafish.

[0031] Compared to fruit flies, this invention uses zebrafish, which has 87% genetic similarity to humans, as a model organism. Its reproductive system is less different from that of mammals, and the results have higher reference value for clinical and environmental risk assessment. This invention detects ovarian malformations in females and also focuses on male reproductive toxicity, which has the advantage of more evaluation dimensions.

[0032] The method provided by this invention can construct a zebrafish reproductive toxicity model, which can serve as a drug screening platform for zebrafish reproductive dysfunction. Potentially effective drugs can be screened by observing changes in zebrafish courtship behavior. Furthermore, utilizing the genetic background of zebrafish facilitates the study of the molecular mechanisms of reproductive dysfunction, providing a theoretical basis for future treatments. This invention has promising applications in medicine, drug development, and the screening of natural active ingredients. Attached Figure Description

[0033] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0034] Figure 1 A schematic diagram of the cruciform chamber structure for testing the social behavior of adult zebrafish.

[0035] Figure 2 A technology roadmap for evaluating the reproductive toxicity of chemical substances;

[0036] Figure 3 The effects of Cyclopodiatoxin on courtship distance (A, B) and frequency of entering the Region of Approaching (ROA) (C, D) in male and female zebrafish;

[0037] Figure 4 A diagram showing the effect of Cyclospora toxin on the courtship trajectories of male (A) and female zebrafish (B) throughout the region;

[0038] Figure 5 The effect of Cyclopodiatoxin on the duration of entry into the ROA in male and female zebrafish (A: duration of entry into the ROA and heatmap of males; B: duration of entry into the ROA and heatmap of females (**p<0.01; ***p<0.001));

[0039] Figure 6The results of the effect of Cylindricalotoxin on the fertility of adult zebrafish are shown in the figure ((A) cumulative average number of eggs laid per female 14 days after pre-exposure; (B) cumulative average number of eggs laid per female 14 days after Cylindricalotoxin exposure; (C) fertilization rate 14 days after pre-exposure and 14 days after Cylindricalotoxin exposure).

[0040] Figure 7 The levels of testosterone (T), estradiol (E2), and lutein (VTG) in male (A, B, and C) and female (D, E, and F) zebrafish after exposure to cyclotoxin. An asterisk indicates a significant difference (*, p < 0.05) and a highly significant difference (**, p < 0.01) compared to the control group. Detailed Implementation

[0041] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0042] In a first aspect, the present invention provides a method for constructing a zebrafish reproductive toxicity model, which includes the following steps:

[0043] Expose zebrafish to chemicals with reproductive toxicity;

[0044] The reproductive toxic chemicals are selected from methylmercury, alkaloid toxins, bisphenols or pyrethroid insecticides; the concentration of the chemical in the exposure environment is at least 0.5 μg / L, and the exposure time to the reproductive toxic chemical is 14-42 days;

[0045] Preferably, the alkaloid toxin is cylindrospermopsin (CAS: 143545-90-8).

[0046] Secondly, the present invention provides a method for evaluating the reproductive toxicity of chemical substances, which includes the following steps: evaluating the courtship behavior and fertility of zebrafish.

[0047] The courtship behavior evaluation includes: first, exposing female and male zebrafish to an environment containing reproductively toxic chemicals; then, placing the exposed female or male target zebrafish in one area of ​​the courtship behavior evaluation, and correspondingly placing the exposed male or female test zebrafish in another area of ​​the courtship behavior evaluation; the activity areas of the target zebrafish and the test zebrafish do not overlap, and capturing behavioral videos and / or images of the zebrafish over a period of time; and evaluating the movement behavior of male and female zebrafish during courtship.

[0048] The concentration of the chemical substance in the exposure environment is at least 0.5 μg / L; the concentration of the chemical substance in the courtship behavior evaluation area is the same as that in the exposure environment.

[0049] The kinetic behaviors during courtship are selected from at least one of the following:

[0050] The test zebrafish's movement distance during courtship, the total duration of the test zebrafish's stay in a designated area, the frequency of entering the designated area, the courtship trajectory, and the courtship index; the designated area is the area close to the target fish;

[0051] Fertility assessment includes the following steps: female and male zebrafish exposed to a chemical substance with reproductive toxicity are co-cultured, and then the zebrafish are evaluated for at least one of the following: assessing the egg production and / or fertilization rate of female zebrafish, and detecting the levels of reproductive-related hormones in female and / or male zebrafish.

[0052] Preferably, the distance the zebrafish travels in a designated area during courtship is tested;

[0053] Preferably, the female and male zebrafish are kept together for 7-28 days after exposure.

[0054] The inventors discovered that by evaluating courtship behavior and fertility in zebrafish as described above, the reproductive toxicity of chemical substances can be evaluated rapidly, at low cost, and continuously on the same batch of zebrafish. By capturing behavioral images or videos to monitor changes in courtship behavior, no experimental animals need to be sacrificed, nor are specialized fluorescence imaging equipment or tissue sectioning equipment required.

[0055] Testing courtship behavior allows for rapid analysis of zebrafish behavioral changes using software, significantly saving manpower and time compared to traditional spawning statistics methods, and eliminating the need for any anesthesia of the fish. Courtship behavior also indicates reproductive impact through live observation, supporting long-term tracking or repeated experiments with the same batch of zebrafish. This invention detects both ovarian malformations in females and male reproductive toxicity, offering the advantage of more evaluation dimensions.

[0056] When testing the courtship behavior of zebrafish, the courtship behavior towards the opposite sex is determined by observing the distance the test fish gets close to the target fish, the frequency of its appearance in a designated area, and the time of its appearance.

[0057] Within the same courtship behavior evaluation experimental apparatus or container, the target fish and the test zebrafish are of different sexes. For example, when testing the courtship behavior of male zebrafish, the target fish is female, and the male and female are separated by a physical barrier, such as a partition.

[0058] The method provided by this invention can construct a zebrafish reproductive toxicity model, which can serve as a drug screening platform for zebrafish reproductive dysfunction. Potentially effective drugs can be screened by observing changes in zebrafish courtship behavior. Furthermore, utilizing the genetic background of zebrafish facilitates the study of the molecular mechanisms of reproductive dysfunction, providing a theoretical basis for future treatments. This invention has promising applications in medicine, drug development, and the screening of natural active ingredients.

[0059] When the concentration of chemicals in the exposure environment is at least 0.5 μg / L, it can affect the reproductive function of zebrafish and has reproductive toxicity. This is manifested by: a significant reduction in the total duration of male or female test zebrafish entering the designated area, a significant reduction in the frequency of test zebrafish entering the designated area, a shortened courtship distance for male and female zebrafish, a more dispersed courtship trajectory for test zebrafish entering the designated area, and a lower courtship index.

[0060] The concentration of the chemical substance in the exposure environment is, for example, 0.5 μg / L, 1 μg / L, 1.5 μg / L, 2.5 μg / L, 3 μg / L, 3.5 μg / L, 4 μg / L, 4.5 μg / L, 5 μg / L, 5.5 μg / L, or 6 μg / L. Experiments of this invention have demonstrated that exposure concentrations of 0.5 μg / L and 5 μg / L can cause zebrafish reproductive toxicity, and higher exposure concentrations should also have zebrafish reproductive toxicity. Therefore, the concentration of the chemical substance in the exposure environment should be at least 0.5 μg / L.

[0061] The frequency of entering a designated area refers to the number of times a male or female zebrafish enters a designated area.

[0062] The "designated area" mentioned in this invention refers to an area designated for the reproductive toxicity assessment of female and / or male zebrafish, allowing the tested zebrafish (male or female) to move and remain, adjacent to the release area of ​​the target fish. The designated area can be a single area, a combination of several sub-areas, or multiple areas. For example, the area for evaluating courtship behavior can be divided into area 1, area 2, area 3, area 4, and area 5, where area 1 is used for releasing female zebrafish, area 2 for releasing male zebrafish, and area 3 is the "designated area." Courtship behavior is evaluated by assessing the total duration and frequency of female and male zebrafish entering area 3.

[0063] In this application, “ROA region” refers to “a specific region”.

[0064] In a preferred embodiment of the present invention, if zebrafish courtship behavior and fertility decrease in an exposed environment, the reproductive toxicity of the alkaloid toxins to zebrafish is assessed.

[0065] In a preferred embodiment of the present invention, female and male zebrafish are exposed to a reproductively toxic chemical environment for 14-42 days.

[0066] In a preferred embodiment of the present invention, the time for filming zebrafish behavioral videos and / or pictures is 10-20 minutes;

[0067] In a preferred embodiment of the present invention, the zebrafish behavioral videos and / or images are taken under continuous illumination.

[0068] In a preferred embodiment of the present invention, software is used to analyze zebrafish behavioral videos and / or images to obtain their movement behavior during courtship.

[0069] In a preferred embodiment of the present invention, the courtship index refers to the time it takes for a male or female zebrafish to chase another female or male zebrafish.

[0070] In a preferred embodiment of the present invention, the reproductive-related hormones are selected from testosterone (T), estradiol (E2), or lutein (VTG). If the levels of reproductive-related hormones in zebrafish are significantly lower than those in the control group (i.e., the group not exposed to alkaloid toxins), it indicates that alkaloid toxins affect the fertility of zebrafish.

[0071] The reproductive toxic chemical is selected from methylmercury, alkaloid toxins, bisphenols, or pyrethroid insecticides. The alkaloid toxin is cylindrospermopsin (CAS: 143545-90-8). In other embodiments, other compounds with similar effects may also be used.

[0072] Thirdly, the present invention also provides the application of a zebrafish reproductive toxicity model constructed by a method for constructing a zebrafish reproductive toxicity model or a method for evaluating the reproductive toxicity of chemical substances in screening drugs for the prevention and / or treatment of reproductive dysfunction. The application includes: assessing the reproductive toxicity of chemical substances to zebrafish according to the above-described method for evaluating the reproductive toxicity of chemical substances, and obtaining a zebrafish reproductive toxicity model.

[0073] Fourthly, the present invention also provides the application of a zebrafish reproductive toxicity model constructed by a method for constructing a zebrafish reproductive toxicity model or a method for evaluating the reproductive toxicity of chemical substances in screening drugs that improve or inhibit abnormal courtship behavior in zebrafish. The application includes: assessing the reproductive toxicity of chemical substances to zebrafish according to the above-described method for evaluating the reproductive toxicity of chemical substances, and obtaining a zebrafish reproductive toxicity model.

[0074] The features and performance of the present invention will be further described in detail below with reference to embodiments.

[0075] Example 1

[0076] This embodiment provides a method for evaluating the reproductive toxicity of chemical substances, and the technical route is as follows: Figure 2 As shown, zebrafish, which has 87% genetic similarity to humans, is used as a model organism to establish an animal model of zebrafish courtship behavior that indicates reproductive toxicity, which is quick, has a high success rate, and is simple and convenient to establish.

[0077] 1. Conduct the exposure experiment according to the following method.

[0078] The stock solution of *Strombyx mori* toxin was prepared using acetone as a solvent, and then further diluted to a final concentration of 0.5 μg / L. A blank control group and a treatment group (0.5 μg / L *Strombyx mori* toxin) were included in this experiment. Each 30L glass jar contained 18 adult zebrafish (male and female zebrafish were kept separate) and 20L of the test solution. Each treatment was exposed three times, repeated for 42 days. During the exposure period, the exposure solution was changed every 24 hours, and the fish were fed normal live brine shrimp twice daily and molted brine shrimp feed once daily.

[0079] 2. Social Behavior Test of Adult Zebrafish

[0080] Five minutes after the adult zebrafish adapted to the experimental environment, the following social behavior tests were conducted on the adult zebrafish:

[0081] like Figure 1 As shown, in Figure 1 The social behavior experiment of adult zebrafish was conducted in the chamber shown in the diagram. Figure 1 The chamber is cruciform in shape, with four areas designated as Arena 1, Arena 2, Arena 3, and Arena 4, and Arena 5 in the center. Arena 1 has an extension end, at which Arena A is located. In this embodiment, Arena A (i.e., area A) is the area for female fish (target fish), Arena 1 is the designated area, i.e., the ROA area, and Arena 5 is the area for male fish (test fish). Each chamber contains a culture medium for zebrafish, which contains 0.5 μg / L of *Strombophytoxin*.

[0082] Randomly insert the female (or male) fish from the blank group into the blank group. Figure 1The test area is shown in the terminal chamber (Arena A) with a cross-shaped groove. Arena A (i.e., region A) is separated from region 1 (Arena 1) by a transparent Plexiglas plate (acrylic plate). Regions 1, 2, 3, 4, and 5 constitute the total test area for the fish. Male fish were placed in the central chamber (Arena 5) with a water depth of 8 cm, similar to the control group, to measure courtship behavior.

[0083] When evaluating the social behavior of female fish, male fish were placed in Arena A as the target fish, and female fish were placed in Arena 5 as the test fish. The behavior of female fish in Arena 1 (i.e., the designated area—ROA area) near male fish was observed.

[0084] The measurement involved recording zebrafish behavior videos under continuous light for 10 minutes using a LogiHD1080p camera.

[0085] The following kinetic behaviors of zebrafish during courtship were recorded:

[0086] The distance traveled into the ROA area during courtship, the total duration of female and male zebrafish entering the ROA area, the frequency of entering the ROA area, courtship trajectories, and courtship index.

[0087] The time spent entering the ROA area refers to the total time (moving and stationary time) spent by the zebrafish in Arena 1. Area 1 is the area closest to the opposite sex fish. The time of entry into the ROA area by the zebrafish in the behavioral video was extracted using EthoVision XT 15 software.

[0088] The movement distance during courtship, in this embodiment specifically refers to the total distance traveled by zebrafish within the Arena 1 region. This parameter is related to the degree to which an individual zebrafish participates in courtship behavior. The total distance traveled by zebrafish entering the Arena 1 region from behavioral videos was extracted using X software.

[0089] Frequency in ROA: Total frequency of zebrafish entering Arena 1. The total frequency of zebrafish entering the Arena 1 region from behavioral videos was extracted using X software.

[0090] Courtship index: The time (movement time) it takes for a male zebrafish (or female zebrafish) to chase another female zebrafish (or another male zebrafish).

[0091] 3. Assessment of Fertility in the F0 Generation

[0092] Female and male zebrafish were exposed to the reproductive toxin *Cyclopodium tumefaciens* for 14 days, with three parallel groups for each exposure concentration. Eight zebrafish were released into each parallel group. The zebrafish were then evaluated as follows: the egg production and fertilization rate of female zebrafish were assessed, and the levels of reproductive hormones in female and male zebrafish were measured.

[0093] (1) Artificial statistics on the number of eggs laid and fertilization rate of female zebrafish.

[0094] (2) Measure the levels of relevant reproductive hormones to verify the effects of chemical substances on the reproduction of zebrafish.

[0095] Example 2

[0096] Compared to Example 1, the only difference is that the sample was exposed to a 5 μg / L *Cyclopyralid* toxin solution in the exposure environment, and the stock solution was diluted with acetone to a final concentration of 5 μg / L. All other steps were the same as in Example 1.

[0097] Experimental Example 1

[0098] An evaluation experiment was conducted on the courtship behavior of zebrafish using the methods for evaluating the reproductive toxicity of chemical substances provided in Examples 1 and 2.

[0099] The results showed that, compared with the control group, the 5 μg / L columnar alginate treatment in Example 2 reduced the migration distance of male and female zebrafish in the ROA (Responsive Occurrence Area). Figure 3 (A and B) and the frequency of entering the ROA region ( Figure 3 (C and D in the middle).

[0100] Treatment with 0.5 μg / L Cyclospora toxin in Example 1 reduced the number of males entering the ROA and the distance female zebrafish traveled within the ROA area. Figure 3 (C and B).

[0101] Similarly, trajectory diagram ( Figure 4 This further demonstrates the effect of exposure to columnospirotoxin on the locomotor ability of males and females. Compared with the control group, males exposed to columnospirotoxin showed significantly improved locomotor ability. Figure 4 (A) and female ( Figure 4 (B) Zebrafish no longer concentrate their movements in the ROA region, but instead disperse more in other regions besides the ROA region. This phenomenon shows a clear dose-response relationship.

[0102] In addition, compared with the control group, both 5 μg / L in Example 2 and 0.5 μg / L in Example 1 significantly reduced the levels of male ( Figure 5 (A) and female zebrafish ( Figure 5Total time spent in the ROA (Rich Angle Area) after exposure to columnar alginate. Compared with the control group, males (B) Figure 5 (A) and female ( Figure 5 The activity of B) in areas outside the ROA gradually increased, a phenomenon most pronounced in the 5 μg / L columnar alginate treatment group.

[0103] Experimental Example 2

[0104] The methods for evaluating the reproductive toxicity of chemical substances provided in Examples 1 and 2 were used to test the reproductive effects on zebrafish.

[0105] The study aimed to statistically analyze the egg production and fertilization rate of female zebrafish, and to measure the levels of related reproductive hormones to verify the effects of chemical substances on zebrafish reproduction. The zebrafish testosterone (T) ELISA kit was purchased from Jiangsu Jingmei (catalog number JM-08003F2); the zebrafish estradiol (E2) ELISA kit was purchased from Jiangsu Jingmei (catalog number JM-07997F2 48T); and the zebrafish vitellogenin (VTG) ELISA kit was purchased from Jiangsu Jingmei (catalog number JM-07790F2 48T).

[0106] Figure 6 The results showed no difference in oviposition and fertilization observed in the 14 days prior to exposure (i.e., under non-exposure conditions). Figure 6 (A and C in the text). After 14 days of exposure to *Cyclophorus spp.*, the cumulative average number of eggs laid by female fish in the 0.5 and 5 μg / L *Cyclophorus spp.* treatment groups was significantly lower than that in the control group. Figure 6 (B and C in the text). The oviposition and fertilization rates in the groups 14 days before exposure were 97.59%-98.42%, with no significant difference among the groups. However, after 14 days of exposure, the oviposition and fertilization rates in the 0.5 and 5 μg / L Cyclophyta toxin treatment groups were significantly reduced.

[0107] In male zebrafish exposed to *Cyclopodium clavatum* toxin, both testosterone and estrogen levels significantly increased. In female zebrafish, testosterone and estrogen levels did not change significantly, but VTG levels significantly decreased. These results indicate that *Cyclopodium clavatum* toxin acts as an estrogenic agent in zebrafish. Decreased VTG concentration affects mature oocytes and egg production. Therefore, it is speculated that long-term exposure to *Cyclopodium clavatum* toxin may affect reproduction by interfering with sex hormone levels in fish, thereby reducing fertilization and hatching success rates. Thus, the reproductive capacity of zebrafish exposed to *Cyclopodium clavatum* toxin is significantly affected.

[0108] In summary, zebrafish courtship behavior was significantly affected under exposure conditions of 0.5–5 μg / L cylindricalotoxin. To validate a model that uses courtship behavior to indicate the reproductive effects of zebrafish, the effects of the aforementioned chemical substance on zebrafish reproductive function were verified using traditional methods, namely, the detection of zebrafish egg production, fertilization rate, and reproductive hormones, under the same concentration conditions. The results showed that zebrafish reproductive function was significantly affected under exposure conditions of 0.5–5 μg / L, thus allowing the establishment of a model for assessing zebrafish courtship behavior as an indicator of chemical reproductive toxicity.

[0109] The experimental results of this invention show that after adult zebrafish were exposed to cylindrical toxins, the total duration of the reproductive activity area (ROA) and the frequency of entering the ROA in both male and female zebrafish were significantly reduced, resulting in a significant decrease in courtship behavior. The frequency and timing of entering the spawning area are key parameters for assessing fish reproductive behavior. After exposure to environmental pollutants, the spawning volume and fertilization rate of zebrafish decreased significantly, indicating that cylindrical toxins affect zebrafish reproductive function. Therefore, by influencing zebrafish courtship behavior, reproductive function damage can be caused. Thus, this reproductive toxicity model can be used to screen for drugs to prevent and treat reproductive dysfunction or abnormal courtship behavior. The method for evaluating the reproductive toxicity of chemical substances provided by this invention can be used in fields such as medicine, drug development, and screening of natural active ingredients.

[0110] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for constructing a zebrafish model of reproductive toxicity, characterized by, It comprises the following steps: exposing zebrafish to a chemical substance with reproductive toxicity; the chemical substance with reproductive toxicity is cylindrospermopsin; the concentration of the chemical substance in the exposure environment is 0.5-5 μg / L; the time of exposure to the chemical substance with reproductive toxicity is 14-42 days.

2. A method for evaluating the reproductive toxicity of a chemical substance, characterized by, It comprises the following steps: evaluating the courtship behavior and fertility of zebrafish; the courtship behavior evaluation comprises the following steps: first, exposing female and male zebrafish to a chemical substance with reproductive toxicity; then, placing the exposed female or male target zebrafish in one of the courtship behavior evaluation areas, and placing the exposed male or female courtship behavior test zebrafish in the other courtship behavior evaluation area accordingly; the activity areas of the target zebrafish and the test zebrafish do not overlap; taking videos and / or pictures of the zebrafish behavior within a time period; and evaluating the movement behavior of male and female zebrafish during courtship; the concentration of the chemical substance in the exposure environment is 0.5-5 μg / L; the concentration of the chemical substance in the courtship behavior evaluation area is the same as that in the exposure environment; the movement behavior during courtship is selected from at least one of the following: the movement distance of the test zebrafish during courtship, the total duration of the test zebrafish entering a specified area, the frequency of entering the specified area, the courtship trajectory, and the courtship index; the specified area is an area close to the target zebrafish; the fertility evaluation comprises the following steps: mixing the exposed female and male zebrafish in a chemical substance with reproductive toxicity; and then evaluating at least one of the following: the spawning quantity and / or fertilization rate of female zebrafish, and detecting the reproductive hormone level of female and / or male zebrafish; the chemical substance with reproductive toxicity is cylindrospermopsin.

3. The method for evaluating the reproductive toxicity of a chemical substance according to claim 2, characterized by, the movement distance of the test zebrafish in the specified area during courtship; the time of mixing the exposed female and male zebrafish is 7-28 days.

4. The method for evaluating the reproductive toxicity of a chemical substance according to claim 2, characterized by, If the courtship behavior of zebrafish decreases and the fertility of zebrafish decreases in the exposure environment, it is evaluated that the chemical substance has reproductive toxicity to zebrafish.

5. The method for evaluating the reproductive toxicity of a chemical substance according to claim 2, characterized by, the time of exposing female and male zebrafish to a chemical substance with reproductive toxicity is 14-42 days.

6. The method for evaluating the reproductive toxicity of a chemical substance according to claim 5, characterized by, the time of taking videos and / or pictures of zebrafish behavior is 10-20 min; the taking of videos and / or pictures of zebrafish behavior is performed under continuous light conditions; the zebrafish behavior videos and / or pictures are analyzed by software to obtain the movement behavior during courtship.

7. The method for evaluating the reproductive toxicity of a chemical substance according to claim 2, characterized by, the courtship index refers to the time of a male zebrafish or a female zebrafish chasing another female zebrafish or another male zebrafish.

8. The method for evaluating the reproductive toxicity of a chemical substance according to claim 2, characterized by, the reproductive hormone is selected from testosterone, estradiol, or vitellogenin.

9. Use of the zebrafish model of reproductive toxicity constructed according to claim 1 or the zebrafish model of reproductive toxicity constructed according to the method of any one of claims 2 to 8 for the screening of drugs for the prevention and / or treatment of impairment of reproductive function, characterized in that, the application comprises: evaluating the reproductive toxicity of a chemical substance to zebrafish by the method for evaluating the reproductive toxicity of a chemical substance according to any one of claims 2-8, and obtaining a zebrafish reproductive toxicity model.

10. The use of the zebrafish model of reproductive toxicity constructed according to claim 1 or the zebrafish model of reproductive toxicity constructed according to the method of any one of claims 2 to 8 in the screening of drugs for ameliorating or inhibiting abnormal courtship behavior in zebrafish, characterized in that, The application comprises: evaluating the reproduction toxicity of the chemical substance to the zebrafish by the method for evaluating the reproduction toxicity of the chemical substance according to any one of claims 2-8, and obtaining a zebrafish reproduction toxicity model.

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