Zebrafish hypoxia exposure parameter control method based on three-gas incubator

Abstract

The present application relates to the technical field of exposure parameter control, and particularly relates to a zebrafish hypoxia exposure parameter control method based on a three-gas incubator, which comprises the following steps: S1: after loading the exposure container loaded with zebrafish into the three-gas incubator, a low-oxygen atmosphere is formed in the three-gas incubator, and water hypoxia reaches a mark; S2: whether a stratified reaching state of surface-layer hypoxia first and bottom-layer hypoxia later exists in the culture water body is identified, if the stratified reaching state exists, the formal exposure calculation is delayed, and the air circulation intensity and the nitrogen replacement rhythm of the three-gas incubator are adjusted, and homogeneous hypoxia calculation conditions are generated; S3: when the homogeneous hypoxia calculation conditions are established, the zebrafish hypoxia exposure timing is started, and the oxygen, carbon dioxide and nitrogen supply parameters of the three-gas incubator are locked as hypoxia maintenance parameters in the exposure process, and zebrafish hypoxia exposure parameter control results are generated. The present application improves the accuracy of hypoxia exposure time calculation and the repeatability of experimental results.

Description

Technical Field

[0001] This invention relates to the field of exposure parameter control technology, and in particular to a method for controlling hypoxic exposure parameters in zebrafish based on a three-gas incubator. Background Technology

[0002] Zebrafish, due to their clear genetic background, short developmental cycle, high tissue transparency, and high genetic homology with higher vertebrates, have been widely used in research on hypoxia injury mechanisms, cardiovascular and cerebrovascular diseases, drug screening, and environmental toxicology evaluation. In zebrafish hypoxia exposure experiments, a three-gas incubator is typically used to adjust the supply ratio of oxygen, carbon dioxide, and nitrogen to achieve a preset low-oxygen level, thereby inducing corresponding physiological and molecular biological responses in the zebrafish. In current techniques, researchers mostly use the moment when the oxygen concentration in the three-gas incubator reaches the target value as the starting point of hypoxia exposure and control subsequent experiments according to the preset exposure time. However, zebrafish are actually in culture water, and the direct target of hypoxia stimulation is the dissolved oxygen environment in the culture water, not the gaseous oxygen concentration in the incubator. Therefore, changes in gaseous oxygen concentration cannot fully represent the formation process of the actual hypoxic state in the culture water.

[0003] Because of the gas-liquid mass transfer process between the culture water and the low-oxygen atmosphere inside the three-gas incubator, the dissolved oxygen concentration in the culture water usually reaches the target hypoxia level after a lag compared to the time it takes for the oxygen concentration in the incubator to reach the target value. Furthermore, during the establishment of hypoxia, the rate of oxygen concentration decrease between the surface and bottom layers of the culture water is often inconsistent, resulting in a stratification phenomenon where the surface layer becomes hypoxic first, followed by the bottom layer. Current technologies typically lack mechanisms for identifying the actual arrival time of hypoxia in the culture water, as well as mechanisms for determining and correcting hypoxia stratification. This leads to differences in the actual time and intensity of hypoxia stimulation received by zebrafish in different locations, thus affecting the consistency of hypoxia exposure conditions and the reproducibility of experimental results. Summary of the Invention

[0004] This invention provides a method for controlling the hypoxia exposure parameters of zebrafish based on a three-gas incubator. It can accurately identify the actual time when the culture water enters a hypoxic state and eliminate the hypoxia stratification phenomenon inside the culture water, thereby achieving precise control of the start time of hypoxia exposure and the hypoxia maintenance conditions.

[0005] A method for controlling hypoxia exposure parameters in zebrafish based on a three-gas incubator includes the following steps:

[0006] S1: After placing the exposed container containing zebrafish into the three-gas incubator, first control the three-gas incubator to form a low-oxygen atmosphere, and make the culture water in the exposed container undergo a pre-hypoxia balance process to generate a water hypoxia threshold corresponding to the exposed container.

[0007] S2: Based on the water body hypoxia arrival marker, identify whether there is a stratified arrival state in the culture water body where the surface layer is hypoxic first and the bottom layer is hypoxic later. If there is a stratified arrival state, delay the start of formal exposure and adjust the air circulation intensity and nitrogen replacement rhythm of the three-gas incubator to generate homogeneous hypoxia start conditions.

[0008] S3: Once the homogeneous hypoxia start-up condition is met, start the zebrafish hypoxia exposure timer, and during the exposure process, lock the oxygen, carbon dioxide and nitrogen supply parameters of the three-gas incubator to the hypoxia maintenance parameters corresponding to the homogeneous hypoxia start-up condition, and generate the zebrafish hypoxia exposure parameter control results.

[0009] Optionally, S1 specifically includes:

[0010] S11: After placing the exposed container containing zebrafish and culture water into the three-gas incubator, close the oxygen supply valve of the three-gas incubator, open the nitrogen supply valve, and at the same time adjust the carbon dioxide volume fraction in the chamber to the preset balance value, so that the oxygen volume fraction in the chamber decreases from the atmospheric oxygen concentration to the oxygen concentration of the hypoxic exposure at the first decreasing rate, thus forming a low-oxygen atmosphere.

[0011] S12: Install a dissolved oxygen sensor at the bottom or lower middle part of the culture water in the exposed container to continuously collect the dissolved oxygen concentration value of the water during the formation of a low-oxygen atmosphere.

[0012] S13: When the dissolved oxygen sensor first detects that the dissolved oxygen concentration in the water body is lower than or equal to the preset hypoxia threshold, the corresponding time is recorded as the time when hypoxia in the water body arrives, and timestamp data is generated as a marker for the arrival of hypoxia in the water body.

[0013] S14: After generating the water body hypoxia reached mark, maintain the current gas supply parameters, temperature and pressure of the three-gas incubator unchanged, and continue to run for a preset stabilization period of time, so that the dissolved oxygen concentration of the water body further approaches and stabilizes at the level of dissolved oxygen corresponding to the hypoxia exposure target oxygen concentration, and complete the pre-hypoxia balancing process.

[0014] Optionally, the hypoxic environment is a controlled gas environment formed when the oxygen volume fraction in the three-gas incubator is lower than the normal atmospheric oxygen concentration and reaches the preset hypoxic exposure target oxygen concentration. The hypoxic environment is used to drive oxygen exchange in the culture water in the exposure container and induce the culture water to gradually enter a hypoxic state.

[0015] Optionally, the pre-hypoxia balancing process refers to the transition process in which, after the culture water body first reaches the preset hypoxia threshold, the low-oxygen environment is maintained and the gas supply parameters remain unchanged, so that the oxygen concentration gradient inside the culture water body gradually weakens, the dissolved oxygen concentration tends to stabilize, and the spatial distribution tends to be uniform.

[0016] Optionally, S2 specifically includes:

[0017] S21: A first dissolved oxygen sensor and a second dissolved oxygen sensor are installed in the culture water in the exposed container. The first sensor is located at a first depth below the surface of the water, and the second sensor is located at a second depth above the bottom of the water. The dissolved oxygen concentration values ​​at the surface and the bottom are collected in real time.

[0018] S22: When the surface dissolved oxygen concentration value first falls below or equals the preset hypoxia threshold, record the time when surface hypoxia is reached; when the bottom dissolved oxygen concentration value first falls below or equals the preset hypoxia threshold, record the time when bottom hypoxia is reached.

[0019] S23: Calculate the time difference between the arrival time of bottom layer hypoxia and the arrival time of surface layer hypoxia. If the time difference is greater than the preset stratification tolerance time, it is determined that there is a stratification state in the culture water where the surface layer is hypoxic first and the bottom layer is hypoxic later.

[0020] S24: When it is determined that a stratification has been reached, delay the start of formal exposure, suspend the start of the hypoxia exposure timer, and perform adjustment operations.

[0021] Optionally, the adjustment operation specifically includes:

[0022] Adjustment Operation 1: Increase the air circulation intensity of the three-gas incubator from the current value to the first enhanced value and maintain it for the first duration to accelerate gas exchange between the upper and lower layers of the culture water.

[0023] Adjustment Operation 2: Switch the nitrogen supply rhythm of the three-gas incubator from continuous replacement mode to pulse replacement mode. The ratio of nitrogen on-time to off-time in each pulse cycle is the preset pulse ratio, which slows down the excessive stripping of surface oxygen and avoids aggravating stratification.

[0024] Adjustment Operation 3: After the first duration of action ends, restore the air circulation intensity to the second maintenance value and restore the nitrogen replacement rhythm to continuous replacement.

[0025] Optionally, S2 further includes continuously monitoring the dissolved oxygen concentration values ​​of the surface layer and the bottom layer after the adjustment operation is completed. When the absolute difference between the dissolved oxygen concentration values ​​of the surface layer and the bottom layer is less than the preset homogeneous allowable deviation and both are lower than or equal to the preset hypoxia threshold, it is determined that the homogeneous hypoxia start-up condition is met, a homogeneous hypoxia start-up flag is generated, and the zebrafish hypoxia exposure timer is allowed to start.

[0026] Optionally, S3 specifically includes:

[0027] S31: When the homogeneous hypoxia initiation condition is met, read the current time as the exposure start time, start the hypoxia exposure timer, and begin to accumulate the hypoxia exposure duration of the zebrafish;

[0028] S32: Read the actual values ​​of various environmental control parameters of the three-gas incubator at the moment when the homogeneous hypoxia start-up condition is established, and record them as hypoxia maintenance parameters. The hypoxia maintenance parameters include at least the oxygen volume fraction set value, the carbon dioxide volume fraction set value, and the nitrogen supply flow rate, as well as the incubator temperature set value and the incubator pressure set value.

[0029] S33: During the entire subsequent zebrafish hypoxia exposure process, the oxygen, carbon dioxide and nitrogen supply parameters of the three-gas incubator will be locked as hypoxia maintenance parameters. External interference or feedback adjustment will be prohibited from automatically correcting the oxygen, carbon dioxide and nitrogen supply parameters of the three-gas incubator. Only compensatory adjustment is allowed.

[0030] S34: Continuously run under the locked hypoxia maintenance parameters until the hypoxia exposure timer reaches the preset total exposure time. During the operation, the actual dissolved oxygen concentration value of the culture water is collected once every recording cycle as the exposure process record data.

[0031] Optionally, the compensatory adjustment includes real-time monitoring of the actual oxygen volume fraction, actual carbon dioxide volume fraction, and actual nitrogen supply status in the three-gas incubator. When the actual operating parameters are detected to deviate from the hypoxia maintenance parameters due to gas source pressure fluctuations, flow control errors, or equipment operation deviations, the corresponding gas supply device is adjusted to restore the actual operating parameters to the set range corresponding to the hypoxia maintenance parameters. The compensatory adjustment is only used to eliminate equipment operation deviations and does not change the target set value of the hypoxia maintenance parameters.

[0032] Optionally, S3 further includes, after the hypoxia exposure timer ends, packaging the exposure start time, the cumulative duration of the hypoxia exposure timer, the locked hypoxia maintenance parameters, and the exposure process recorded data together to generate zebrafish hypoxia exposure parameter control results.

[0033] The beneficial effects of this invention are:

[0034] This invention monitors the dissolved oxygen concentration in the culture water in real time, determines the arrival time of hypoxia using a preset hypoxia threshold, and generates a hypoxia arrival marker. This shifts the hypoxia exposure initiation point from the gaseous environment control level to the actual hypoxia state of the water, achieving a precise correspondence between hypoxia exposure time and the true hypoxia state of the culture water. Furthermore, the pre-hypoxia equilibration process further eliminates incomplete oxygen exchange processes within the culture water, ensuring that subsequent hypoxia exposure is based on stable hypoxia. This improves the authenticity, accuracy, and repeatability of the hypoxia exposure time calculation and experimental results.

[0035] This invention obtains the arrival times of surface and bottom hypoxia separately, calculates the time difference of hypoxia propagation, and determines whether stratification has occurred based on a preset stratification tolerance time. When stratification is detected, the oxygen concentration equilibrium process within the culture water is accelerated by increasing the air circulation intensity, switching to a pulsed nitrogen replacement mode, and implementing subsequent homogeneous correction control until the difference in dissolved oxygen concentration between the surface and bottom layers meets the preset homogeneous allowable deviation and reaches the hypoxia threshold. Only then is the hypoxia exposure timer allowed to start. Simultaneously, after the homogeneous hypoxia initiation conditions are met, the hypoxia maintenance parameters are locked to maintain a consistent hypoxic environment throughout the exposure phase. This not only improves the consistency of hypoxia stimulation experienced by zebrafish in different locations but also enhances the comparability, reliability, and accuracy of biological response analysis of experimental data. It provides a more standardized and reproducible experimental control method for zebrafish hypoxia mechanism research, drug screening, and hypoxia model construction. Attached Figure Description

[0036] To more clearly illustrate the technical solutions in this invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only for this invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0037] Figure 1 This is a schematic diagram of the method flow according to an embodiment of the present invention;

[0038] Figure 2 This is a schematic diagram of pre-hypoxia control according to an embodiment of the present invention. Detailed Implementation

[0039] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments. Those skilled in the art may employ other alternative methods to implement some well-known technologies; moreover, the accompanying drawings are only for more specific description of the embodiments and are not intended to specifically limit the present invention.

[0040] like Figures 1-2 As shown, a method for controlling hypoxia exposure parameters in zebrafish based on a three-gas incubator includes the following steps:

[0041] S1: After placing the zebrafish-containing exposure container into the three-gas incubator, instead of using the oxygen concentration in the incubator as the starting point for hypoxia exposure, the three-gas incubator is first controlled to form a low-oxygen atmosphere, and the culture water in the exposure container undergoes a pre-hypoxia balance process to generate a hypoxia threshold for the water in the exposure container.

[0042] S1 specifically includes:

[0043] S11: The three-gas incubator controls the supply ratio of oxygen, nitrogen, and carbon dioxide separately through a gas flow control module. Before the exposure begins, the oxygen volume fraction in the three-gas incubator is adjusted to the atmospheric oxygen concentration level and kept stable. The incubator temperature and pressure are adjusted to preset constant values. Then, the oxygen supply valve is closed and the nitrogen supply valve is opened. By continuously introducing nitrogen to replace the air inside the chamber, while maintaining the carbon dioxide volume fraction at a preset balance value, a low-oxygen environment that meets the requirements of zebrafish culture is formed inside the chamber. The low-oxygen environment refers to the controlled gas environment formed by adjusting the supply ratio of oxygen, nitrogen, and carbon dioxide in the three-gas incubator to reduce the oxygen volume fraction inside the chamber from the normal atmospheric oxygen concentration to the preset hypoxic exposure target oxygen concentration. The low-oxygen environment is used to drive the gradual decrease of dissolved oxygen concentration in the culture water and provide gas-liquid mass transfer conditions for the subsequent formation of a hypoxic state in the water.

[0044] The preset balance value is the target value for the volume fraction of carbon dioxide in the three-gas incubator, which is 5%. Zebrafish are usually cultured using a culture medium containing a bicarbonate buffer system. The acid-base balance of the culture water is greatly affected by the carbon dioxide concentration. When the carbon dioxide concentration is too low, the pH of the culture water is prone to alkaline drift. When the carbon dioxide concentration is too high, it is easy to cause acidification of the culture water and cause additional stress to the zebrafish. Therefore, maintaining the volume fraction of carbon dioxide at about 5% ensures that the culture water maintains a relatively stable acid-base environment during the establishment of hypoxia, thereby avoiding interference from non-hypoxia factors caused by pH fluctuations and ensuring that the subsequent water hypoxia arrival markers mainly reflect the oxygen transfer process rather than the acid-base change process.

[0045] The three-gas incubator has temperature control and pressure adjustment functions. The temperature is adjustable from 25 to 28°C, and the pressure is adjustable from 0.95 to 1.05 atm. It is used to maintain the stability of the culture water and the physiological state of zebrafish during the establishment of a low-oxygen atmosphere and pre-hypoxia balance.

[0046] The change in oxygen volume fraction inside the chamber is represented as follows:

[0047] ;

[0048] when At the same time, oxygen concentration reduction control is continuously implemented;

[0049] when At that time, stop the descent process and maintain the target oxygen concentration;

[0050] in, For a moment The volume fraction of oxygen in the chamber. This represents the oxygen volume fraction corresponding to the initial atmospheric oxygen concentration. The duration of the descent process, This represents the oxygen volume fraction corresponding to the target oxygen concentration during hypoxic exposure. The first descent rate is the rate at which the oxygen volume fraction in the tank decreases from the atmospheric oxygen concentration to the target oxygen concentration for hypoxia exposure, and is set to 0.5% / min. If the oxygen concentration decreases too quickly, for example, above 1.0% / min, the atmosphere in the tank will rapidly reach the target oxygen concentration, while the culture water will remain in a state of high dissolved oxygen due to the lag in gas-liquid mass transfer, easily forming a significant oxygen concentration stratification phenomenon, thus prolonging the subsequent establishment time of homogeneous hypoxia. If the oxygen concentration decreases too slowly, for example, below 0.3% / min, the time required to establish a hypoxia environment will increase, which is not conducive to experimental efficiency. At the same time, it will cause zebrafish to be in a gradual oxygen decrease process for a long time, increasing the adaptation effect and affecting the hypoxia exposure results. Therefore, it is preferable to use a first descent rate of about 0.5% / min to keep the oxygen concentration decrease process in the tank relatively matched with the dissolved oxygen decrease process in the culture water. This ensures the efficiency of hypoxia establishment while reducing the degree of gas-liquid mass transfer mismatch, thereby improving the accuracy of the water hypoxia reaching the marker.

[0051] The three-gas incubator is equipped with a transparent viewing window, a sealed operating channel, and a light source module. The viewing window allows observation of the zebrafish's activity and survival status without opening the chamber. The sealed operating channel is used for medication, feeding, fluid replenishment, or sampling while maintaining stable oxygen, carbon dioxide, temperature, and pressure levels within the chamber. The light source module simulates the natural light conditions of zebrafish, including light intensity, light cycle, and spectral distribution, ensuring that the experiment meets both hypoxic exposure conditions and maintains a stable photophysiological environment. Through the above control process, the oxygen concentration within the chamber decreases steadily at a predetermined rate, avoiding drastic fluctuations in dissolved oxygen in the culture water due to sudden drops in oxygen concentration, thus creating a stable low-oxygen atmosphere.

[0052] S12: Due to the gas-liquid mass transfer process between the culture water and the atmosphere inside the chamber, when the oxygen concentration in the chamber reaches the target value, the dissolved oxygen concentration in the culture water usually has not yet reached the hypoxic state.

[0053] To accurately identify the actual time when the culture water enters a hypoxic state, a dissolved oxygen sensor is installed at the bottom or lower middle part of the exposed container. The dissolved oxygen concentration values ​​of the culture water are continuously acquired at a preset sampling period, forming a dissolved oxygen monitoring sequence, as shown below:

[0054] ;

[0055] in, This is a dissolved oxygen monitoring sequence. For the first Dissolved oxygen concentration value obtained from the second sampling This represents the total number of data collected during the monitoring period.

[0056] The preset sampling period is the time interval between continuous collection of dissolved oxygen concentration values ​​in the culture water by the dissolved oxygen sensor, ranging from 5s to 30s. During the hypoxia exposure of zebrafish, the change in dissolved oxygen concentration in the culture water is a slow process relative to electrical or pressure signals. If the sampling period is too short, such as less than 5s, it will increase data redundancy and the impact of sensor noise fluctuations on the judgment results. If the sampling period is too long, such as greater than 30s, it may cause the moment when the dissolved oxygen in the water first falls below the preset hypoxia threshold to be recorded late, affecting the accuracy of the hypoxia arrival marker. Therefore, setting the preset sampling period to 5s to 30s can balance the accuracy of identifying the arrival time of hypoxia in the water with the stability of data processing.

[0057] By continuously collecting dissolved oxygen monitoring sequences, the changes in dissolved oxygen within the culture water can be reflected in real time, providing a basis for determining the time when the water becomes hypoxic.

[0058] S13: The system reads the current dissolved oxygen concentration value from the dissolved oxygen monitoring sequence in real time and compares it with the preset hypoxia threshold, including:

[0059] S131: When satisfied When the above conditions are met for the first time during the monitoring process, the culture water is determined to be in a state of hypoxia.

[0060] in, This represents the dissolved oxygen concentration at the current sampling time. The preset hypoxia threshold was set at 2.0 mg / L. In the zebrafish hypoxia exposure experiment, zebrafish began to show hypoxia stress response after the dissolved oxygen concentration in the culture water fell below the normal saturation level. When the dissolved oxygen concentration dropped to about 2.0 mg / L, a stable hypoxia exposure condition could be formed, while the risk of severe asphyxiation or acute death would not be reached too quickly. Therefore, 2.0 mg / L was selected as the preferred preset hypoxia threshold, which can better distinguish between water that has entered a hypoxic state and water that is still in a transitional state of decreasing oxygen concentration.

[0061] S132: Record the sampling time when the conditions are first met as the time when water anoxic conditions are reached, denoted as:

[0062] ;

[0063] in, This refers to the moment when the water body becomes oxygen-deficient. For the corresponding sampling time;

[0064] Generate timestamp data, represented as:

[0065] ;

[0066] The timestamp data is then output as a marker indicating the arrival of oxygen depletion in the water.

[0067] in, This is a marker indicating the arrival of oxygen deficiency in the water, used to characterize the starting moment when the culture water actually enters a hypoxic state, rather than the moment when the oxygen concentration in the chamber reaches the target value.

[0068] S14: After the water body reaches the hypoxia threshold, the zebrafish hypoxia exposure timer is not immediately started. Instead, the current gas supply parameters, temperature, and pressure of the three-gas incubator are maintained constant, allowing the culture water to continue gas-liquid mass exchange with the low-oxygen atmosphere, including:

[0069] S141: The system continuously monitors changes in dissolved oxygen concentration in the culture water and calculates the concentration change between adjacent sampling times, expressed as:

[0070] ;

[0071] in, This represents the change in dissolved oxygen concentration between adjacent sampling times. This represents the current dissolved oxygen concentration. This represents the dissolved oxygen concentration value at the previous moment.

[0072] S142: When multiple consecutive sampling periods satisfy At that time, it was determined that the dissolved oxygen concentration in the culture water had stabilized.

[0073] in, The threshold for determining dissolved oxygen stability;

[0074] S143: Continue running until the preset steady-state duration. End, completion of the pre-hypoxia balance process;

[0075] in, The preset stability duration.

[0076] The pre-hypoxia equilibration process refers to the process of maintaining the current oxygen, nitrogen, and carbon dioxide supply parameters of the three-gas incubator unchanged after the generated water reaches the hypoxia threshold. This allows the culture water to continue gas-liquid mass exchange with the hypoxic environment, while continuously monitoring changes in dissolved oxygen concentration until the dissolved oxygen concentration in the culture water tends to stabilize and its spatial distribution gradually becomes uniform. Because the culture water has a certain depth, and the diffusion rate of oxygen in water is much lower than that in the gas phase, when the monitoring point at the bottom of the water body first reaches the preset hypoxia threshold, there are often still local differences in oxygen concentration within the culture water. For example, some areas may have reached the target hypoxia level, while other areas may still be in the continuous deoxygenation phase. If the formal hypoxia exposure timer is started immediately, the actual time that zebrafish in different locations are exposed to hypoxia may vary, thus affecting the consistency of the experimental results. Therefore, after the hypoxia level is reached, this invention continues to maintain the low-oxygen environment for a preset stabilization period, further eliminating the residual oxygen concentration gradient inside the culture water, and causing the overall dissolved oxygen concentration of the culture water to gradually approach and stabilize at the level of dissolved oxygen corresponding to the hypoxia exposure target oxygen concentration, thereby forming a more uniform and stable hypoxia state.

[0077] After the pre-hypoxia equilibration process is completed, the dissolved oxygen concentration inside the culture water is sufficiently close to and stabilized at the level of dissolved oxygen corresponding to the target oxygen concentration of hypoxia exposure. This ensures that the subsequent hypoxia threshold can accurately reflect the time reference of the culture water entering a stable hypoxic state, providing a basic input for the establishment of subsequent homogeneous hypoxia starting conditions.

[0078] S2: Based on the water body hypoxia arrival marker, identify whether there is a stratified arrival state in the culture water body where the surface layer is hypoxic first and the bottom layer is hypoxic later. If a stratified arrival state exists, delay the start of formal exposure and adjust the air circulation intensity and nitrogen replacement rhythm of the three-gas incubator to generate homogeneous hypoxia start conditions.

[0079] S2 specifically includes:

[0080] S21: To identify whether oxygen concentration stratification exists within the culture water, a first dissolved oxygen sensor and a second dissolved oxygen sensor are respectively deployed inside the exposed container; wherein, the first dissolved oxygen sensor is used to monitor the dissolved oxygen status in the surface area of ​​the culture water, and the second dissolved oxygen sensor is used to monitor the dissolved oxygen status in the bottom area of ​​the culture water; during the monitoring process, the following data are generated respectively:

[0081] ;

[0082] ;

[0083] in, This is a surface dissolved oxygen monitoring sequence. For the first The surface dissolved oxygen concentration value obtained from the second sampling This is a low-level dissolved oxygen monitoring sequence. For the first The dissolved oxygen concentration value of the bottom layer obtained from the second sampling. This represents the total number of samples taken.

[0084] By simultaneously collecting surface and bottom dissolved oxygen concentration values, a data foundation is provided for subsequent identification of the stratification status in the culture water.

[0085] S22: The system continuously monitors surface dissolved oxygen and bottom dissolved oxygen sequences, including:

[0086] S221: When the first satisfaction is met At that time, the corresponding moment is recorded as the moment when surface hypoxia arrives, represented as:

[0087] ;

[0088] in, To preset the hypoxia threshold, This represents the time when surface hypoxia is achieved. This refers to the sampling time when the condition is first met.

[0089] S222: When the first satisfaction is met At that time, the corresponding moment is recorded as the moment when hypoxia in the lower stratum is reached, represented as:

[0090] ;

[0091] in, This indicates the moment when oxygen deprivation occurs at the lower layers. This refers to the sampling time when the condition is met for the first time.

[0092] The system continuously receives real-time monitoring data from the first and second dissolved oxygen sensors, and establishes surface dissolved oxygen change records and bottom dissolved oxygen change records respectively. During the monitoring process, the dissolved oxygen concentration value collected each time is compared with the preset hypoxia threshold. When the surface dissolved oxygen concentration value first drops to or below the preset hypoxia threshold, this moment is recorded as the arrival time of surface hypoxia. Thereafter, the system continues to monitor the changes in bottom dissolved oxygen concentration. When the bottom dissolved oxygen concentration value first drops to or below the preset hypoxia threshold, the corresponding moment is recorded as the arrival time of bottom hypoxia. The arrival time of surface hypoxia represents the time node when the upper region of the culture water actually enters the hypoxic state, and the arrival time of bottom hypoxia represents the time node when the lower region of the culture water actually enters the hypoxic state. By recording the time of reaching the hypoxic state at two different depths, the temporal characteristics of the hypoxia propagation process inside the culture water can be obtained, providing a basis for subsequent calculation of the arrival time difference of hypoxia between the upper and lower layers, identification of the stratified arrival state of hypoxia at the surface first and then at the bottom, and judgment of the uniformity of oxygen distribution inside the culture water.

[0093] S23: Calculate the time difference of hypoxia propagation based on the arrival times of surface hypoxia and bottom hypoxia, expressed as:

[0094] ;

[0095] in, Due to the time difference in transmission caused by hypoxia, This indicates the moment when oxygen deprivation occurs at the lower layers. This is the time when surface hypoxia is achieved;

[0096] Compare the hypoxia transmission time difference with the preset stratification tolerance time. If so, it is determined that the culture water has reached a state of stratification where the surface layer is hypoxic first and the bottom layer is hypoxic later.

[0097] Conversely, when At that time, it was determined that the spread of hypoxia in the culture water was basically synchronous, and there was no significant stratified arrival state;

[0098] in, The preset stratification tolerance time represents the maximum time difference allowed between the surface and bottom layers of the culture water to reach an anoxic state. When the actual time difference exceeds this time interval, a significant vertical stratification of oxygen concentration is considered to exist within the culture water, meaning the surface layer reaches anoxic status first, followed by the bottom layer. When the actual time difference does not exceed this time interval, the propagation of anoxic conditions within the culture water is considered to be basically synchronous, not constituting a significant stratification. A value of 60 seconds is used because the oxygen diffusion process within the culture water has a certain lag. Even under normal air circulation conditions, the surface area usually reaches an anoxic state before the bottom area; therefore, a certain time difference between the upper and lower layers is normal. If the preset stratification tolerance time is set too small, such as less than 30 seconds, the minute propagation time difference generated during normal gas-liquid mass transfer may be misjudged as stratification. The system frequently initiates homogeneous correction processes, increasing experimental time and reducing control efficiency. If the preset stratification tolerance time is set too large, for example, exceeding 120 seconds, a significant oxygen concentration stratification may have already formed in the culture water, but the system may still consider it a normal propagation process. This causes some zebrafish to enter a hypoxic state prematurely, while others have not yet reached a hypoxic state, affecting the consistency of the hypoxic exposure start time. Therefore, a preset stratification tolerance time of 60 seconds is preferred. When the time difference between the arrival of hypoxia at the bottom layer and the arrival of hypoxia at the surface layer exceeds 60 seconds, it is considered that a hypoxic stratification phenomenon has formed in the culture water that is sufficient to affect the consistency of the experiment, and it is necessary to perform operations to increase the air circulation intensity and adjust the nitrogen replacement rhythm. When the time difference does not exceed 60 seconds, it is considered that the hypoxia propagation in the culture water is basically synchronous, and it can directly enter the subsequent homogeneous hypoxia determination stage.

[0099] S24: When a stratified state is detected, the zebrafish hypoxia exposure timer is not immediately started. Instead, the system state is switched to homogeneous correction mode. At this time, the hypoxia exposure timer startup logic is paused, and the current low-oxygen environment is maintained. Before adjusting the air circulation intensity and nitrogen replacement rhythm, the system synchronously reads the current temperature and pressure values ​​inside the three-gas incubator and compares them with the preset incubation temperature and pressure, respectively. If a deviation from the preset range is detected, compensation adjustment is first performed through the temperature control module and pressure control module to restore the incubator environment to the preset incubation state. Then, the air circulation intensity and nitrogen replacement rhythm adjustment operations are performed to reduce the oxygen concentration gradient inside the culture water, so that the culture water gradually changes from a stratified hypoxia state to a homogeneous hypoxia state, including:

[0100] S241: Control the internal circulation fan of the three-gas incubator to increase its operating power, so that the air circulation intensity is adjusted from the current value to the first enhanced value, as shown below:

[0101] ;

[0102] in, The current wind circulation intensity, As the first enhancement value, The enhancement coefficient, used to characterize the increase in air circulation intensity relative to the current air circulation intensity after the stratification state is reached, is set to 1.5. When the culture water exhibits a phenomenon of initial oxygen deficiency at the surface followed by oxygen deficiency at the bottom, it is necessary to appropriately increase the airflow velocity inside the incubator to enhance the convective exchange capacity at the gas-liquid interface and promote a uniformity of oxygen concentration between the upper and lower layers of the culture water. If the enhancement coefficient is lower than 1.3, the air circulation enhancement effect is limited and its improvement effect on eliminating oxygen concentration stratification is not significant. If the enhancement coefficient is higher than 2.0, the airflow disturbance inside the incubator is too strong, which can easily cause surface oscillation of the culture water or even form local turbulence, which is not conducive to maintaining a stable exposure state for zebrafish. Therefore, a value of 1.5 is chosen, which is a 50% increase based on the original air circulation intensity, which can improve the uniformity of oxygen exchange without generating excessive mechanical disturbance.

[0103] The duration of operation after the enhanced wind circulation intensity is: ;

[0104] in, The duration of the first action;

[0105] By enhancing air circulation, the uniformity of oxygen exchange at the gas-liquid interface is improved, promoting a more consistent oxygen concentration within the culture water.

[0106] S242: The nitrogen supply method is changed from continuous supply to periodic supply, and the preset pulse ratio is expressed as:

[0107] ;

[0108] in, For the preset pulse ratio, The duration of nitrogen gas operation within a single pulse cycle. This refers to the duration of nitrogen shut-off within a single pulse cycle.

[0109] By periodically alternating nitrogen supply, the rate of oxygen stripping in the surface area can be reduced, thus slowing down the further widening of the oxygen concentration difference between the surface and the bottom layer.

[0110] S243: After the first duration of action ends, the wind circulation intensity is restored to the second maintenance value, expressed as:

[0111] ;

[0112] in, This is the second maintenance value. To maintain the proportionality coefficient, which characterizes the retention rate when the air circulation intensity recovers from the first enhanced value to the long-term stable operating state after homogeneous correction, a value of 0.7 is set. After the first duration of enhanced circulation, the oxygen concentration gradient in the culture water has been significantly weakened. At this point, it is not necessary to continue maintaining the highest circulation intensity. If the proportionality coefficient is maintained below 0.6, the air circulation will decrease too quickly, which may cause slight stratification of the water body again. If the proportionality coefficient is maintained above 0.8, maintaining a high air circulation intensity for a long time will increase equipment energy consumption and may cause continuous disturbance to the zebrafish. Therefore, a value of 0.7 is set to ensure that the system maintains good operational stability while ensuring the uniformity of hypoxia.

[0113] At the same time, depending on the current uniformity of oxygen concentration in the culture water, the nitrogen supply mode is restored to continuous replacement mode, or a low-frequency pulse replacement mode is maintained to keep the hypoxic environment stable.

[0114] The continuous replacement mode means that the nitrogen supply valve is continuously open, that is, the nitrogen supply system remains open during operation, and nitrogen is continuously input into the three-gas incubator at a constant flow rate. Through continuous gas replacement, the oxygen volume fraction in the chamber is maintained near the target oxygen concentration for hypoxia exposure. Its characteristics are a stable oxygen concentration decrease rate and simple control logic, which is suitable for the long-term maintenance stage after the hypoxia state has been uniformly established. The lower frequency pulse replacement mode means that the nitrogen supply valve is intermittently opened and closed according to a preset cycle, but the pulse frequency is lower than that in the homogeneous correction stage in S242. That is, after the stratification correction is completed, the nitrogen pulse supply frequency is reduced, so that there is a longer interval between two adjacent nitrogen supplies. This reduces the rapid oxygen stripping effect in the surface area while maintaining the stability of the hypoxia environment, and avoids the reformation of oxygen concentration stratification in the culture water.

[0115] After adjusting the air circulation intensity and nitrogen replacement rhythm, the system continuously monitors the temperature and pressure changes inside the three-gas incubator. When both temperature and pressure fluctuations are within the preset stable range, the current incubation environment is considered to meet the prerequisites for homogeneous hypoxia determination. If temperature or pressure fluctuations still exceed the limits, environmental stability control is maintained until the temperature and pressure return to the preset range. By using temperature and pressure stability as auxiliary constraints before determining homogeneous hypoxia, the redistribution of dissolved oxygen in the upper and lower layers of the culture water can be avoided due to fluctuations in environmental parameters, thus improving the reliability of establishing the starting conditions for homogeneous hypoxia.

[0116] S25: Continuously calculate the difference between the surface dissolved oxygen concentration and the bottom dissolved oxygen concentration, expressed as:

[0117] ;

[0118] in, This represents the difference in dissolved oxygen concentration between the upper and lower layers. This represents the current dissolved oxygen concentration at the surface. This represents the current dissolved oxygen concentration at the bottom layer.

[0119] When both conditions are met as well as and When the condition for initiating homogeneous hypoxia is met, a homogeneous hypoxia initiation flag is generated, which is represented as:

[0120] ;

[0121] in, To preset the hypoxia threshold, The preset homogeneous allowable deviation is the maximum allowable deviation in the dissolved oxygen concentration difference between the surface and bottom layers of the culture water when a uniform hypoxia state is determined. This value is set at 0.1-0.3 mg / L. When the difference between the surface and bottom dissolved oxygen concentrations is less than 0.1 mg / L, the judgment standard is too strict and easily affected by instantaneous fluctuations in the dissolved oxygen sensor, probe response delays, and minor disturbances in the water, resulting in the homogeneous hypoxia starting condition not being met for a long time. When the difference is greater than 0.3 mg / L, although the exposure timer can be started relatively quickly, there may still be significant differences in the degree of hypoxia between the upper and lower layers of the culture water, affecting the consistency of the actual hypoxia exposure intensity of the zebrafish. This serves as the starting point for homogeneous hypoxia.

[0122] Once the homogeneous hypoxia start marker is generated, the zebrafish hypoxia exposure timer can be started, and the subsequent hypoxia maintenance phase can begin.

[0123] S3: Once the homogeneous hypoxia starting condition is met, start the zebrafish hypoxia exposure timer and lock the oxygen, carbon dioxide and nitrogen supply parameters of the three-gas incubator to the hypoxia maintenance parameters corresponding to the homogeneous hypoxia starting condition during the exposure process, and generate the zebrafish hypoxia exposure parameter control results.

[0124] S3 specifically includes:

[0125] S31: After generating the homogeneous hypoxia start flag, the system determines that the culture water has reached a uniform and stable hypoxic state. At this time, the current system time is read as the start time of the formal hypoxia exposure, and the hypoxia exposure timer is started, including:

[0126] S311: The exposure start time is defined as:

[0127] ;

[0128] in, To expose the start time, This is the current system time corresponding to the condition for the start of homogeneous hypoxia.

[0129] S312: During hypoxia exposure, the hypoxia exposure timer continuously accumulates the duration of hypoxia exposure, as shown below:

[0130] ;

[0131] in, This represents the current cumulative duration of hypoxia exposure. For the current moment, The exposure start time;

[0132] By using the above method, the statistical starting point for hypoxia exposure time is established after the culture water body as a whole enters a uniform hypoxic state, thereby ensuring that the exposure time between different batches of experiments is consistent and comparable.

[0133] S32: When the homogeneous hypoxia initiation condition is met, the system synchronously reads the actual values ​​of various environmental control parameters under the current operating state of the three-gas incubator and saves them as hypoxia maintenance parameters for subsequent hypoxia exposure stages, expressed as:

[0134] ;

[0135] in, For hypoxia maintenance parameters, Set a value for the oxygen volume fraction. Set a value for the volume fraction of carbon dioxide. To supply nitrogen flow rate, The temperature setpoint is used to maintain the thermal balance of the culture water. The setpoint is 28℃. At this temperature, the metabolic activity, growth and development and behavior of zebrafish are relatively stable. Too low a temperature will reduce the metabolic level, and too high a temperature may cause additional heat stress. Therefore, it is preferable to maintain it at 28℃. The pressure setpoint is used to maintain a stable partial pressure environment of gases inside the incubator, ensuring that the dissolved oxygen state of the culture water during the hypoxic exposure process remains consistent with the initial conditions of homogeneous hypoxia. The value is set to 1.0 atm, maintaining a near-standard atmospheric pressure inside the incubator. This ensures a stable partial pressure relationship between oxygen, carbon dioxide, and nitrogen, preventing pressure fluctuations from affecting the dissolved oxygen balance in the culture water. At the same time, it can simulate the normal living environment of zebrafish to the greatest extent possible, reducing interference from non-hypoxic factors.

[0136] The hypoxia maintenance parameters reflect the gas environment control conditions corresponding to the homogeneous hypoxia state of the culture water, and serve as the control benchmark for subsequent exposure processes.

[0137] S33: After the hypoxia maintenance parameters are generated, the system enters a parameter-locked operating state, which is represented as:

[0138] ;

[0139] in, Provide a set of parameters for the actual gas at any given time. For parameters used to maintain hypoxia;

[0140] Throughout the hypoxic exposure period, the system maintains the oxygen volume fraction setpoint, carbon dioxide volume fraction setpoint, and nitrogen supply flow rate without any active changes, while locking the temperature setpoint and pressure setpoint.

[0141] When deviations occur due to equipment operating errors, flow fluctuations, or changes in air source pressure, only compensatory adjustments are performed, as indicated by:

[0142] ;

[0143] in, For parameter compensation amount, These are the actual operating parameters detected in real time.

[0144] Compensatory adjustments are only used to restore hypoxia maintenance parameters without changing the hypoxia maintenance parameters themselves, thereby ensuring that the entire hypoxia exposure process is in a stable and consistent hypoxic environment.

[0145] S34: The system continuously monitors the hypoxia exposure timer's operating status, and when the condition is met... During this period, maintain hypoxia maintenance parameters.

[0146] in, The preset total exposure time is the target time for zebrafish to continuously receive hypoxic stimulation after meeting the homogeneous hypoxia starting condition. If the exposure time is too short, it is difficult to form a stable physiological or behavioral response, while if the exposure time is too long, it may cause zebrafish to enter the risk range of irreversible damage or death. Therefore, it should be set according to the experimental purpose, the developmental stage of zebrafish, and the target hypoxia intensity, with a value of 30 min to 24 h. Acute hypoxia exposure can be set to 30 min to 2 h, and subacute hypoxia exposure can be set to 6 h to 24 h. As a preferred value for general hypoxia stress experiments, 2 h can be used.

[0147] Simultaneously, the actual dissolved oxygen concentration in the culture water is collected according to the preset recording cycle to form exposure process record data, represented as follows:

[0148] ;

[0149] in, To record data during the exposure process, For the first Dissolved oxygen concentration values ​​corresponding to each recording period, To record the total number of times; the changes in dissolved oxygen in the culture water during the hypoxic exposure phase are usually relatively slow. If the recording period is too short, it will generate a large amount of duplicate data and increase the impact of sensor noise. If the recording period is too long, it will not be conducive to detecting abnormal fluctuations during the hypoxic maintenance process. Therefore, a recording period of 1 min to 5 min can balance the integrity of the recording and the stability of the data. The data recorded during the exposure process is used to reflect the changes in the dissolved oxygen status of the culture water during the hypoxic exposure.

[0150] During the hypoxic exposure, the dissolved oxygen sensor installed inside the exposure container continues to be used, and the monitoring values ​​of the first and second dissolved oxygen sensors are read simultaneously. At each preset recording period, the current surface dissolved oxygen concentration value and the bottom dissolved oxygen concentration value are collected respectively, and the average of the two can be used as the actual dissolved oxygen concentration value corresponding to that recording period. , represented as:

[0151] ;

[0152] in, For the first The surface dissolved oxygen concentration values ​​collected in each recording period, For the first The dissolved oxygen concentration value at the bottom layer collected in each recording cycle.

[0153] While collecting the actual dissolved oxygen concentration in the culture water, the system simultaneously records the temperature and pressure values ​​inside the chamber at the corresponding time, and stores them as part of the exposure process data. By recording the correspondence between temperature, pressure and dissolved oxygen, the complete traceability and result verification of the subsequent hypoxia exposure experiment process can be realized.

[0154] During hypoxia exposure, researchers can observe the swimming behavior, aggregation state, respiratory rate changes, and survival status of zebrafish in the exposure container in real time through a visual observation window without opening the incubator door. When observing through the visual observation window, the hypoxic atmosphere, temperature field, pressure field, and gas supply status inside the incubator remain unchanged, thus avoiding interference from oxygen concentration rebound, temperature fluctuations, or pressure changes caused by opening the incubator, and improving the accuracy and repeatability of hypoxia exposure experimental results.

[0155] During hypoxia exposure, when drug intervention, nutritional supplementation, biological sample delivery, or culture medium treatment is required, the experimenters can complete the corresponding operations through the sealed operation channel without opening the incubator door. The sealed operation channel can carry out experimental intervention operations while maintaining the hypoxia maintenance parameters unchanged, so that the hypoxia exposure process can continue and avoid the impact on the hypoxia exposure results caused by oxygen concentration rebound, temperature changes, or pressure fluctuations due to opening the chamber.

[0156] S35: When the hypoxia exposure timer reaches the preset total exposure time, that is, when At that time, the system stops the hypoxia exposure timer and summarizes the key parameters generated throughout the exposure process. These key parameters include the exposure start time, the cumulative duration of the hypoxia exposure timer, the locked hypoxia maintenance parameters, and the data recorded during the exposure process. The zebrafish hypoxia exposure parameter control results are expressed as follows:

[0157] ;

[0158] in, Results of controlled parameters for hypoxia exposure in zebrafish. The exposure start time is used to characterize the time point at which hypoxia exposure officially begins. The cumulative hypoxia exposure duration is used to characterize the actual duration of hypoxia stimulation experienced by zebrafish. These are locked hypoxia maintenance parameters used to characterize the gas control conditions for maintaining a stable hypoxic environment during hypoxic exposure. Data was recorded during the exposure process to characterize changes in dissolved oxygen levels in the culture water during hypoxia exposure.

[0159] The results of zebrafish hypoxia exposure parameter control are output and stored for subsequent experimental analysis, exposure condition tracing, and consistency verification of hypoxia exposure experimental results of different batches of zebrafish.

[0160] This invention encompasses any substitutions, modifications, equivalent methods, and solutions made within the spirit and scope of this invention. To provide the public with a thorough understanding of this invention, specific details are described in detail in the following preferred embodiments; however, those skilled in the art will fully understand the invention even without these details. Furthermore, to avoid unnecessary misunderstanding of the essence of this invention, well-known methods, processes, procedures, components, and circuits are not described in detail.

[0161] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for controlling hypoxia exposure parameters in zebrafish based on a three-gas incubator, characterized in that, Includes the following steps: S1: After placing the exposed container containing zebrafish into the three-gas incubator, first control the three-gas incubator to form a low-oxygen atmosphere, and make the culture water in the exposed container undergo a pre-hypoxia balance process to generate a water hypoxia threshold corresponding to the exposed container. S2: Based on the water body hypoxia arrival marker, identify whether there is a stratified arrival state in the culture water body where the surface layer is hypoxic first and the bottom layer is hypoxic later. If there is a stratified arrival state, delay the start of formal exposure and adjust the air circulation intensity and nitrogen replacement rhythm of the three-gas incubator to generate homogeneous hypoxia start conditions. S3: Once the homogeneous hypoxia start-up condition is met, start the zebrafish hypoxia exposure timer, and during the exposure process, lock the oxygen, carbon dioxide and nitrogen supply parameters of the three-gas incubator to the hypoxia maintenance parameters corresponding to the homogeneous hypoxia start-up condition, and generate the zebrafish hypoxia exposure parameter control results.

2. The method for controlling hypoxia exposure parameters in zebrafish based on a three-gas incubator according to claim 1, characterized in that, S1 specifically includes: S11: After placing the exposed container containing zebrafish and culture water into the three-gas incubator, close the oxygen supply valve of the three-gas incubator, open the nitrogen supply valve, and at the same time adjust the carbon dioxide volume fraction in the chamber to the preset balance value, so that the oxygen volume fraction in the chamber decreases from the atmospheric oxygen concentration to the oxygen concentration of the hypoxic exposure at the first decreasing rate, thus forming a low-oxygen atmosphere. S12: Install a dissolved oxygen sensor at the bottom or lower middle part of the culture water in the exposed container to continuously collect the dissolved oxygen concentration value of the water during the formation of a low-oxygen atmosphere. S13: When the dissolved oxygen sensor first detects that the dissolved oxygen concentration in the water body is lower than or equal to the preset hypoxia threshold, the corresponding time is recorded as the time when hypoxia in the water body arrives, and timestamp data is generated as a marker for the arrival of hypoxia in the water body. S14: After generating the water body hypoxia reached mark, maintain the current gas supply parameters, temperature and pressure of the three-gas incubator unchanged, and continue to run for a preset stabilization period of time, so that the dissolved oxygen concentration of the water body further approaches and stabilizes at the level of dissolved oxygen corresponding to the hypoxia exposure target oxygen concentration, and complete the pre-hypoxia balancing process.

3. The method for controlling hypoxia exposure parameters in zebrafish based on a three-gas incubator according to claim 2, characterized in that, The hypoxic environment is a controlled gas environment formed when the oxygen volume fraction in the three-gas incubator is lower than the normal atmospheric oxygen concentration and reaches the preset hypoxic exposure target oxygen concentration. The hypoxic environment is used to drive oxygen exchange in the culture water in the exposure container and induce the culture water to gradually enter a hypoxic state.

4. The method for controlling hypoxia exposure parameters in zebrafish based on a three-gas incubator according to claim 2, characterized in that, The pre-hypoxia balance process refers to the transition process in which, after the culture water body first reaches the preset hypoxia threshold, the low-oxygen environment is maintained and the gas supply parameters remain unchanged, so that the oxygen concentration gradient inside the culture water body gradually weakens, the dissolved oxygen concentration tends to stabilize, and the spatial distribution tends to be uniform.

5. The method for controlling hypoxia exposure parameters in zebrafish based on a three-gas incubator according to claim 1, characterized in that, S2 specifically includes: S21: A first dissolved oxygen sensor and a second dissolved oxygen sensor are installed in the culture water in the exposed container. The first sensor is located at a first depth below the surface of the water, and the second sensor is located at a second depth above the bottom of the water. The dissolved oxygen concentration values ​​at the surface and the bottom are collected in real time. S22: When the surface dissolved oxygen concentration value first falls below or equals the preset hypoxia threshold, record the time when surface hypoxia is reached; when the bottom dissolved oxygen concentration value first falls below or equals the preset hypoxia threshold, record the time when bottom hypoxia is reached. S23: Calculate the time difference between the arrival time of bottom layer hypoxia and the arrival time of surface layer hypoxia. If the time difference is greater than the preset stratification tolerance time, it is determined that there is a stratification state in the culture water where the surface layer is hypoxic first and the bottom layer is hypoxic later. S24: When it is determined that a stratification has been reached, delay the start of formal exposure, suspend the start of the hypoxia exposure timer, and perform adjustment operations.

6. The method for controlling hypoxia exposure parameters in zebrafish based on a three-gas incubator according to claim 5, characterized in that, The adjustment operation specifically includes: Adjustment Operation 1: Increase the air circulation intensity of the three-gas incubator from the current value to the first enhanced value and maintain it for the first duration to accelerate gas exchange between the upper and lower layers of the culture water. Adjustment Operation 2: Switch the nitrogen supply rhythm of the three-gas incubator from continuous replacement mode to pulse replacement mode. The ratio of nitrogen on-time to off-time in each pulse cycle is the preset pulse ratio, which slows down the excessive stripping of surface oxygen and avoids aggravating stratification. Adjustment Operation 3: After the first duration of action ends, restore the air circulation intensity to the second maintenance value and restore the nitrogen replacement rhythm to continuous replacement.

7. The method for controlling hypoxia exposure parameters in zebrafish based on a three-gas incubator according to claim 5, characterized in that, S2 further includes continuously monitoring the dissolved oxygen concentration values ​​of the surface layer and the bottom layer after the adjustment operation is completed. When the absolute difference between the dissolved oxygen concentration values ​​of the surface layer and the bottom layer is less than the preset homogeneous allowable deviation and both are lower than or equal to the preset hypoxia threshold, it is determined that the homogeneous hypoxia start-up condition is met, a homogeneous hypoxia start-up flag is generated, and the zebrafish hypoxia exposure timer is allowed to start.

8. The method for controlling hypoxia exposure parameters in zebrafish based on a three-gas incubator according to claim 1, characterized in that, S3 specifically includes: S31: When the homogeneous hypoxia initiation condition is met, read the current time as the exposure start time, start the hypoxia exposure timer, and begin to accumulate the hypoxia exposure duration of the zebrafish; S32: Read the actual values ​​of various environmental control parameters of the three-gas incubator at the moment when the homogeneous hypoxia start-up condition is established, and record them as hypoxia maintenance parameters. The hypoxia maintenance parameters include at least the oxygen volume fraction set value, the carbon dioxide volume fraction set value, and the nitrogen supply flow rate. S33: During the entire subsequent zebrafish hypoxia exposure process, the oxygen, carbon dioxide and nitrogen supply parameters of the three-gas incubator will be locked as hypoxia maintenance parameters. External interference or feedback adjustment will be prohibited from automatically correcting the oxygen, carbon dioxide and nitrogen supply parameters of the three-gas incubator. Only compensatory adjustment is allowed. S34: Continuously run under the locked hypoxia maintenance parameters until the hypoxia exposure timer reaches the preset total exposure time. During the operation, the actual dissolved oxygen concentration value of the culture water is collected once every recording cycle as the exposure process record data.

9. A method for controlling hypoxia exposure parameters in zebrafish based on a three-gas incubator according to claim 8, characterized in that, The compensatory adjustment includes real-time monitoring of the actual oxygen volume fraction, actual carbon dioxide volume fraction, and actual nitrogen supply status in the three-gas incubator. When the actual operating parameters are detected to deviate from the hypoxia maintenance parameters due to gas source pressure fluctuations, flow control errors, or equipment operation deviations, the corresponding gas supply device is adjusted to restore the actual operating parameters to the set range corresponding to the hypoxia maintenance parameters. The compensatory adjustment is only used to eliminate equipment operation deviations and does not change the target set value of the hypoxia maintenance parameters.

10. A method for controlling hypoxia exposure parameters in zebrafish based on a three-gas incubator according to claim 8, characterized in that, S3 further includes, after the hypoxia exposure timer ends, packaging the exposure start time, the cumulative duration of the hypoxia exposure timer, the locked hypoxia maintenance parameters, and the exposure process recorded data together to generate the zebrafish hypoxia exposure parameter control results.

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