Method for determining feeding behavior of adult zebra fish

Abstract

The invention discloses a method for measuring the feeding behavior of adult zebrafish, which comprises the following steps: S1, experimental fish preparation: selecting healthy female TU zebrafish at the age of six months, and dividing the female TU zebrafish into a control group and an exposure group after the female TU zebrafish adapts to a system water environment; s2, long-term drug exposure: using system water in a control group, using system water containing a target drug in an exposure group, continuously exposing for 28 days, replacing exposure liquid every day and maintaining a constant environmental condition; s3, food intake behavior determination: transferring the single zebra fish to an independent water tank, putting quantitative feed after adaptation, recording the food intake process through a behavior analysis system, and analyzing the first food intake time, the incomplete food intake proportion and the total food intake duration; and S4, performing data statistics: performing difference significance analysis by adopting SPSS software, and judging the influence of the medicine on the ingestion behavior. The invention aims to detect the potential influence of long-term exposure of environment-related concentration drugs on the ingestion function of fishes, and is suitable for evaluating the interference effect of long-term exposure of low-concentration drugs on the ingestion function of aquatic organisms.

Description

Technical Field

[0001] The present invention relates to the technical field of environmental toxicology and biological behavior analysis, and in particular to a method for measuring the feeding behavior of adult zebrafish. Background Art

[0002] In recent years, the problem of drug and personal care product (PPCP) residues in aquatic environments has become increasingly prominent, and their chronic toxic effects have received widespread attention. Zebrafish has become a model organism for environmental toxicology research because of its clear genome and rich behavioral phenotypes. In the prior art, behavioral analysis of zebrafish mostly focuses on locomotor activity or acute toxicity testing, while systematic determination methods for feeding behavior under long-term low-concentration exposure are still imperfect. In addition, traditional feeding assessment relies on a single indicator (such as food intake), which is difficult to fully reflect complex behavioral changes such as appetite suppression and decreased feeding efficiency. The present invention fills the technical gap in this field by optimizing exposure conditions, standardizing feeding test procedures and conducting multi-indicator combined analysis. Summary of the Invention

[0003] 1. Technical problem to be solved by the invention

[0004] The purpose of the present invention is to solve the technical problems existing in the prior art and provide a highly sensitive and standardized method for measuring the feeding behavior of adult zebrafish, which is used to detect the potential impact of long-term exposure to environmentally relevant concentrations of drugs on the feeding function of fish and is suitable for evaluating the interference effect of long-term exposure to low concentrations of drugs on the feeding function of aquatic organisms.

[0005] 2. Technical solution

[0006] In order to solve the above problems, the technical solution provided by the present invention is:

[0007] A method for measuring the feeding behavior of adult zebrafish comprises the following steps: S1. experimental fish preparation: selecting healthy six-month-old female TU zebrafish, acclimating to the system water environment, and then dividing them into a control group and an exposure group; S2. long-term drug exposure: the control group uses system water, and the exposure group uses system water containing a target drug, and the exposure is continued for 28 days, with the exposure solution replaced daily and the environmental conditions maintained constant; S3. feeding behavior measurement: transferring a single zebrafish to an independent water tank, feeding it a fixed amount of feed after acclimation, recording the feeding process using a behavior analysis system, and analyzing the first feeding time, the proportion of incomplete feeding, and the total feeding time; S4. data statistics: using SPSS software to perform a significance analysis to determine the effect of the drug on the feeding behavior.

[0008] Preferably, the system water is prepared by adding 120 mg / L salt and 160 mg / L baking soda to filtered tap water, with a pH value of 7.0-7.5.

[0009] Preferably, the target drug is fluoxetine hydrochloride, and the exposure concentration is 50 ng / L, simulating the concentration in a real water environment.

[0010] Preferably, the long-term drug exposure conditions are a temperature of 28±1° C., a photoperiod of 14h:10h, feeding twice a day and cleaning up the residue.

[0011] Preferably, when measuring the feeding behavior, a single fish is tested independently, fed with 20 pellets of feed, and the time of first opening within 8 minutes, the proportion of individuals who have not completely eaten, and the total feeding time are recorded.

[0012] Preferably, the feeding behavior is measured and recorded using a ViewPoint ZebraCube system.

[0013] Preferably, in the feeding behavior test, the camera frame rate is set to 25fps, the resolution is 1920×1080, and the analysis software identifies the fish movement trajectory through the background difference method, with the fish mouth contacting the feed and the gill cover opening as the criteria for determining effective feeding action.

[0014] Preferably, the exposure group is set up with at least 3 parallel experimental groups, each parallel group contains 10 zebrafish, and the water temperature, pH and dissolved oxygen are measured synchronously when the exposure solution is changed, and the fluctuation range is ≤±5%.

[0015] 3. Beneficial effects

[0016] Compared with the prior art, the technical solution provided by the present invention has the following beneficial effects:

[0017] 1. Simulate real-world chronic toxicity through 28-day long-term exposure to improve the ecological relevance of the test.

[0018] 2. Combined analysis of the first feeding time, incomplete feeding ratio and total feeding duration was used to comprehensively evaluate the feeding inhibition effect.

[0019] 3. Standardized system water preparation and independent testing environment reduce experimental errors and ensure the reliability of results. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 This is a graph showing the effect of fluoxetine on the feeding behavior of zebrafish in the present invention. DETAILED DESCRIPTION

[0021] In order to make the purpose, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the accompanying drawings and specific embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and do not limit the scope of protection of the present invention.

[0022] It should be noted that when an element is referred to as being "fixed on", "set on", "fixed on" or "installed on" another element, it may be directly on the other element or there may be a central element. When an element is considered to be "connected" to another element, it may be directly connected to the other element or there may be a central element at the same time. Furthermore, when an element is considered to be "fixedly connected" to another element, the two may be fixed in a detachable manner or in a non-detachable manner, such as socketing, snap-fitting, integrally molded fixing, welding, etc., which can be achieved in the prior art and will not be described in detail here. When an element is perpendicular or approximately perpendicular to another element, it means that the ideal state of the two is vertical, but due to the influence of manufacturing and assembly, there may be a certain vertical error. The terms "vertical", "horizontal", "left", "right" and similar expressions used herein are for illustrative purposes only and do not represent the only implementation method.

[0023] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention pertains. The terms used herein in the specification of the present invention are for the purpose of describing specific embodiments only and are not intended to limit the present invention. The term "and / or" as used herein includes any and all combinations of one or more of the associated listed items.

[0024] The “first” and “second” involved in the present invention do not represent specific quantities and orders, but are only used to distinguish names.

[0025] Combined with attachment Figure 1 The present invention provides a method for measuring feeding behavior of adult zebrafish, comprising:

[0026] Experimental system construction

[0027] System water preparation: Tap water is filtered through sand filtration (particle size 0.5-1 mm), activated carbon adsorption (iodine value ≥ 900 mg / g) and reverse osmosis device (salt rejection rate ≥ 98%) for three stages, and then 120 mg / L sodium chloride (NaCl) and 160 mg / L sodium bicarbonate (NaHCO3) are added to adjust the pH to 7.0-7.5 to prepare standardized system water to minimize the impact of water quality fluctuations on behavior.

[0028] Exposure solution preparation: Dissolve fluoxetine hydrochloride (CAS 56296-78-7) with a purity >98.0% in system water to prepare a 50 ng / L exposure solution (concentration based on actual water environment testing values). Prepare and use immediately, referencing actual water environment testing values to enhance the practical significance of ecotoxicology studies.

[0029] Long-term exposure experiment

[0030] Screening of experimental fish: Six-month-old female TU strain zebrafish were selected, and individuals with body length deviation >5%, slow swimming, or body surface damage were eliminated to ensure the homogeneity of the experimental group and control individual differences.

[0031] Grouping and exposure:

[0032] Control group: 3 biological replicates, 10 fish per replicate, housed in system water, 3 biological replicates (n=30) were set to ensure statistical power;

[0033] Exposure group: 3 biological replicates, 10 fish per replicate, reared in 50 ng / L fluoxetine exposure solution;

[0034] Exposure conditions: constant temperature of 28±1℃, photoperiod of 14h light:10h dark, feeding brine shrimp at 09:00 and 17:00 daily, and removal of leftover bait and feces within 1 hour after feeding;

[0035] Exposure management: Fresh exposure solution was replaced every 24 hours for 28 consecutive days to simulate chronic environmental exposure scenarios.

[0036] Feeding behavior assay

[0037] Test environment: A single zebrafish was transferred to an independent test tank (21 cm × 10 cm × 10 cm), filled with 8 cm deep system water, and allowed to acclimate for 5 min. The independent test tank avoided group interference, and the 8 cm water depth provided a standardized behavioral observation space.

[0038] Feeding and recording: 20 HIKARI pellets (made in Japan, diameter 0.8-1.2 mm) were added, and the ViewPoint ZebraCube ZEB6118-1 behavioral analysis system (France) was used to record feeding behavior under continuous visible light for 8 min. A total of 30 fish were tested in each group (10 / replicate × 3 replicates).

[0039] Behavioral indicator analysis:

[0040] First feeding time: The time of feed introduction (t0) and the time of first effective feeding action (t1, defined as the time when the gill cover opens and contacts the feed) were manually marked, and Δt = t1-t0 was calculated. Simultaneously, the data were recorded using the ViewPoint ZebraCube system. Combining manual marking (t0 / t1) with the French ViewPoint automatic recording system, high-precision time series analysis (Δt down to the second level) was achieved.

[0041] Incomplete feeding ratio: Count the number of individuals that did not consume all 20 pellets within 8 minutes and calculate the ratio (number of incomplete feeding / 30 × 100%). The time of first feeding reflects the onset of appetite, the incomplete feeding ratio indicates the motivation to continue feeding, and the total duration evaluates feeding efficiency.

[0042] Total feeding time: record the time it takes for an individual to complete the feeding of 20 pellets (excluding those who did not complete the feeding).

[0043] Data Analysis

[0044] Data statistical analysis was performed using SPSS 27.0 statistical analysis software (SPSS, Chicago, IL, USA). Statistical significance was indicated by * (p < 0.05), ** (p < 0.01), and *** (p < 0.001). Figure 1 ).

[0045] The present study found that fluoxetine significantly increased the time from food input to the time zebrafish opened their mouths to eat ( Figure 1 A), while the proportion of fish that did not eat all their feed increased by 36.7% ( Figure 1 B), indicating that fluoxetine exposure caused zebrafish to lose their appetite (**(P<0.01) indicates a significant difference compared to the control group). Although the total time taken by fluoxetine-exposed fish to finish the fixed amount of feed was not much different from that of the control group ( Figure 1 C).

[0046] This method integrates environmental simulation, chronic exposure, and multidimensional behavioral analysis, providing a highly standardized and reproducible technical path for studying the mechanisms of the effects of environmental pollutants on the behavior of aquatic organisms. It has important application value, especially in the ecological risk assessment of neuroactive substances (such as antidepressants).

[0047] The above is a schematic description of the present invention and its embodiments, which is not restrictive. The drawings show only one embodiment of the present invention, and the actual structure is not limited thereto. Therefore, if a person skilled in the art is inspired by this and, without departing from the purpose of the present invention, designs a structure and embodiment similar to this technical solution without inventiveness, they shall fall within the scope of protection of the present invention.

Claims

1. A method for measuring feeding behavior of adult zebrafish, characterized by: The following steps are included: S1. Experimental fish preparation: Healthy six-month-old female TU zebrafish were selected and, after acclimatization to the system water environment, divided into a control group and an exposure group; S2. Long-term drug exposure: The control group used system water, and the exposure group used system water containing the target drug for 28 consecutive days. The exposure solution was changed daily and the environmental conditions were maintained constant. S3. Feeding behavior measurement: Individual zebrafish were transferred to independent tanks and fed a fixed amount of feed after adaptation. The feeding process was recorded using a behavioral analysis system, and the time of first feeding, proportion of incomplete feeding, and total feeding time were analyzed. S4. Data statistics: SPSS software was used to perform significance analysis to determine the effects of drugs on feeding behavior.

2. The method for measuring feeding behavior of adult zebrafish according to claim 1, wherein: The system water is prepared by adding 120 mg / L salt and 160 mg / L baking soda to filtered tap water, with a pH value of 7.0-7.

5.

3. The method for measuring feeding behavior of adult zebrafish according to claim 1, wherein: The target drug is fluoxetine hydrochloride, and the exposure concentration is 50 ng / L, simulating the real water environment concentration.

4. The method for measuring feeding behavior of adult zebrafish according to claim 1, wherein: The long-term drug exposure conditions were a temperature of 28±1°C, a photoperiod of 14h:10h, feeding twice a day, and cleaning of residues.

5. The method for measuring feeding behavior of adult zebrafish according to claim 1, wherein: When measuring the feeding behavior, each fish was tested independently and fed with 20 pellets of feed. The time of first opening of the mouth within 8 minutes, the proportion of individuals who did not fully eat, and the total feeding time were recorded.

6. The method for measuring feeding behavior of adult zebrafish according to claim 5, wherein: The feeding behavior was measured and recorded using the ViewPoint ZebraCube system.

7. The method for measuring feeding behavior of adult zebrafish according to claim 1, wherein: In the feeding behavior test, the camera frame rate was set to 25 fps and the resolution was 1920×1080. The analysis software identified the fish movement trajectory by background difference method, and the effective feeding action was determined by the fish mouth contacting the feed and the gill cover opening.

8. The method for measuring feeding behavior of adult zebrafish according to claim 1, wherein: The exposure group was set up with at least 3 parallel experimental groups, each parallel group contained 10 zebrafish, and the water temperature, pH and dissolved oxygen were measured synchronously when the exposure solution was changed, and the fluctuation range was ≤±5%.

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