Heart rate-based marsupenaeus japonicus heat resistance evaluation method

Abstract

According to the marsupenaeus japonicus heat resistance evaluation method based on the heart rate, after to-be-tested marsupenaeus japonicus is domesticated for one week, the marsupenaeus japonicus is moved into a heartbeat video recording device to track and record the change of the heart rate of And after the heart rate of the prawns recovers to a resting state, testing the high-temperature resistance of the prawns, stopping heating and ending video recording when the heart rate of the prawns starts to be greatly reduced, and finally transferring the prawns into seawater with normal temperature to keep the prawns alive. The method comprises the following steps: analyzing a prawn heartbeat video by adopting a remote photoplethysmograph method, extracting heart rate change data, drawing an Arrhenius curve graph, and calculating an individual heart rate ABT value; and carrying out heart rate test on a plurality of healthy prawn individuals in the group, and calculating the average number of heart rate ABT values as an index of group heat resistance. Compared with the traditional method, the method has the advantages that the temperature rise is stopped and the temperature is returned to keep alive when the heart rate of the prawns is greatly reduced, so that the heat shock injury of the prawns can be reduced, the heat resistance of the prawns can be accurately evaluated, and an important evaluation basis is provided for high-temperature resistance breeding.

Description

Technical Field

[0001] The invention relates to a heat resistance trait evaluation technology in the field of aquatic biological technology, and in particular to a heat resistance evaluation method for Japanese marsupials based on heart rate. Background Art

[0002] Japanese mackerel is of high quality and expensive, and is one of the important economic aquaculture shrimps in my country. The aquaculture of Japanese mackerel in my country began in the 1950s. The aquaculture areas are mainly distributed in coastal waters from Hainan to Liaoning Province, among which Shandong, Fujian and Guangdong Province are the main aquaculture areas (Mai Xianjie et al., 2009). According to the statistics of the Food and Agriculture Organization (FAO) in 2021, the aquaculture output of Japanese mackerel in my country has accounted for 94.1% of the global output, making it the largest aquaculture country of Japanese mackerel. However, Japanese mackerel has poor high temperature resistance, and the high mortality rate in summer is still one of the main factors restricting the development of the aquaculture industry. Therefore, it is of vital importance to accurately evaluate the heat resistance differences between individual shrimps and cultivate high temperature resistant strains based on this to improve the overall heat resistance of the group, in order to achieve the sustainable development of the shrimp aquaculture industry. In this process, the development of an effective method for evaluating the heat resistance of shrimp plays a key role in the breeding of new varieties.

[0003] The traditional method for evaluating the heat tolerance of shrimp is mainly based on the semi-lethal temperature (LT 50 ), which is to evaluate heat resistance by raising the temperature at a constant rate until half of the shrimp die (Xie Shutao et al., 2005). Critical Thermal Maxima (CTM) is another indicator also used in shrimp heat resistance research (Li Lijun et al., 2015). However, these methods are not only unable to accurately measure individual heat resistance, which is not conducive to the screening of individuals with excellent heat resistance (Chen, 2016; Yu Feng, 2021), but also may cause a large number of individuals to be difficult to recover or even die after the evaluation, making it impossible to retain individuals with excellent heat resistance for subsequent genetic improvement. Therefore, how to accurately measure the temperature resistance of individual shrimp with less damage has become one of the difficulties that need to be overcome in the field of shrimp breeding. Summary of the invention

[0004] The purpose of the present invention is to solve the shortcomings of traditional heat resistance evaluation methods in the prior art, and to provide a heat resistance evaluation method for Japanese marsupials based on heart rate. The method is easy to operate, can accurately evaluate the heat resistance of individual Japanese marsupials, and can be applied to the cultivation of new high-temperature resistant varieties of Japanese marsupials.

[0005] In order to achieve the above object, the present invention adopts the following technical scheme:

[0006] A method for evaluating the heat tolerance of Japanese shrimp based on heart rate comprises the following steps:

[0007] 1) First, the Japanese shrimp samples to be tested were temperature acclimated according to standard operations, and lugworms were fed regularly at 18:00 every evening. The remaining bait was removed in time and two-thirds of the fresh seawater was replaced to ensure that the Japanese shrimp grew in the best health;

[0008] In step 1), the temperature acclimation experiment is carried out in a water tank, the acclimation time is 7 days, the acclimation temperatures are 26°C, 30°C and 34°C respectively, the salinity of the acclimation experiment is 30‰ to 32‰, and the saturation of dissolved oxygen is greater than 90%.

[0009] 2) Before the heart rate measurement experiment, the Japanese shrimp samples were placed in low-temperature seawater for a short period of low-temperature anesthesia;

[0010] In step 2), the water temperature for low-temperature anesthesia of the Japanese shrimp is 12-14° C., and the anesthesia time is controlled at 5-20 minutes;

[0011] 3) Move the Japanese marsupial into the heart beat video recording device, turn on the camera to record the changes in the heart rate of the Japanese marsupial in real time, and wait until the heart rate of the Japanese marsupial returns to a resting state before starting the temperature increase experiment;

[0012] In step 3), the Japanese shrimp was placed in a heart beat video recording device filled with 26 ℃ seawater for at least 2 hours to ensure that the heart rate of the Japanese shrimp returned to a resting state before starting the temperature increase experiment.

[0013] 4) Use an artificial intelligence temperature control system to ensure that the seawater in the heartbeat recording device is heated at the target heating rate. When the heart rate of the Japanese shrimp begins to drop, the temperature is stopped and the recording ends. The Japanese shrimp is quickly transferred to seawater at normal temperature. The temperature changes are recorded in real time during the heart rate measurement.

[0014] In step 4), the target heating rate is 1°C / h, and the seawater temperature ranges from 26°C to 42°C.

[0015] 5) Use remote photoplethysmography to analyze the recorded heart beat video of Japanese marsupials to accurately obtain the heart rate change data of Japanese marsupials during temperature changes;

[0016] In step 5), the remote photoplethysmography (rPPG) technology is used to obtain the heart rate change data of Japanese shrimp based on python.

[0017] 6) Data analysis: Establish a coordinate system, set the temperature data as the horizontal axis after standardization, transform the acquired heart rate data into natural numbers and set it as the vertical axis, and then draw an Arrhenius curve. Perform linear fitting based on the relationship between heart rate and temperature. The horizontal coordinate of the intersection of the two linear equations is the heart rate ABT value of Japanese shrimp. ABT is the Arrhenius break temperatures (ABT) based on heart performance.

[0018] The standardization formula is as follows:

[0019]

[0020] in: is the actual temperature of the seawater in the device (°C); The absolute temperature is normalized to the actual temperature;

[0021] In step 6), the drawing of the Arrhenius curve and the calculation of ABT are completed based on R software.

[0022] Compared with the prior art, the technical solution of the present invention has the following beneficial effects:

[0023] 1. The present invention monitors the change of the heart rate of Japanese marsupenaeus during the temperature increase process, draws an Arrhenius curve using linear regression, calculates the ABT value when the heart rate of Japanese marsupenaeus is significantly reduced, and evaluates the heat resistance of different Japanese marsupenaeus samples by comparing the ABT values. The method can be completed in the laboratory, and the evaluation operation is simple.

[0024] 2. The method of the present invention focuses on the changes in the heart rate of Japanese marsupae, which is different from the traditional heat resistance evaluation method that uses survival as an indicator. After the heat resistance evaluation is completed, most Japanese marsupae samples can still survive.

[0025] 3. The method of the present invention uses the individual Japanese marsupenaeus as a unit and can accurately evaluate the heat resistance of the individual, which makes it possible to screen Japanese marsupenaeus individuals with excellent heat resistance, and may provide benefits for Japanese marsupenaeus breeding. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 The present invention is a schematic diagram of the device structure used in the method.

[0027] Figure 2 This is an Arrhenius curve diagram of the shrimp heart rate according to an embodiment of the present invention.

[0028] Figure 3 The figure shows the influence of different heating rates on the heart rate ABT value of Japanese shrimp in the embodiments of the present invention.

[0029] Figure 4 The figure shows the influence of different acclimation temperatures on the heart rate ABT value of Japanese shrimp in the embodiments of the present invention.

[0030] Figure 5 The heart rate ABT values ​​of different heat-resistant groups of Japanese shrimp in the embodiment of the present invention. Figure 3 In the figure, the pink solid circle represents the average heart rate ABT value of the Japanese marsupial population with lower heat resistance, the green solid circle represents the average heart rate ABT value of the Japanese marsupial population with medium heat resistance, and the blue solid circle represents the average heart rate ABT value of the Japanese marsupial population with higher heat resistance.

[0031] Figure 6 FIG. 1 is the correlation between the heart rate ABT and the heart rate flat line temperature (Flat line temperature, FLT) of the Japanese shrimp according to an embodiment of the present invention. DETAILED DESCRIPTION

[0032] In order to make the technical problems, technical solutions and beneficial effects to be solved by the present invention clearer and more understandable, the present invention is further described in detail below in conjunction with the accompanying drawings and embodiments.

[0033] like Figure 1 As shown, the device used in the method of the present invention includes the following: a camera, a computer, a studio, a light source, a water tank, an acrylic test tube, a Japanese shrimp, a bubble plate, and an artificial intelligence temperature control system;

[0034] Camera: Fixed above the black studio, connected to the computer interface via USB. Function: To record video images of the heart beating area of ​​Japanese shrimp.

[0035] Computer: Placed on the left side outside the studio, connected to the camera. Function: Use the built-in software to see the heartbeat of the shrimp on the computer screen, and save the recorded video of the heartbeat to the mobile hard disk.

[0036] Photo studio: Place the water tank. Block natural light or external light to provide a stable and uniform light environment for recording the heartbeat of shrimps; slow down the heat loss of seawater in the water tank and have a certain constant temperature effect.

[0037] Light source: LED tube, placed on a bracket inside the studio. Function: Provide a stable and controllable light source for the video recording of the shrimp heart beating.

[0038] Water tank: Contains a certain volume of fresh seawater, with a length, width and height of 55cm, 66cm and 70cm respectively. Function: Provides a natural water environment for experimental shrimps to ensure their normal physiological activities.

[0039] Acrylic test tube: fixed in the water tank directly below the camera and completely submerged in seawater. Function: to fix the experimental shrimp to ensure that it does not move during the experiment and to ensure that the shrimp heart beat area is within the camera's recording field of view.

[0040] Japanese shrimp: As an experimental organism, it is fixed in a transparent acrylic test tube under non-destructive conditions. Function: Used to observe the changes in heart beats under different temperature conditions.

[0041] Bubble tray: One end is placed on one side of the bottom of the water tank, and the other end is connected to the power supply. Function: It is used to oxygenate the seawater in the water tank and provide sufficient oxygen for the shrimp.

[0042] Artificial intelligence temperature control system: It consists of a heating rod, a sensor and a black dashboard, and is placed on the right side outside the studio. The heating rod and the sensor are placed at the bottom of the water tank. The water temperature in the water tank is controlled by inputting different program segments through the control of the black dashboard.

[0043] The present invention describes a method for evaluating the heat resistance of Japanese marsupial based on heart rate, which is achieved by monitoring the heart rate changes of Japanese marsupial. The Japanese marsupial is moved into a heart beat video recording device, a camera is used to record the heart rate changes of the Japanese marsupial in real time, an artificial intelligence temperature control system is used to ensure that the seawater in the device is heated at a target heating rate, temperature changes are recorded in real time during heart rate measurement, and then a remote photoplethysmography technique is used to analyze the recorded Japanese marsupial heart beat video to accurately obtain the heart rate change data of the Japanese marsupial during the temperature change process, a linear fit is performed on the relationship between the heart rate and temperature data of the Japanese marsupial, and the abscissa of the intersection of the two linear equations is the heart rate ABT value of the Japanese marsupial.

[0044] In this embodiment, a method for evaluating the heat tolerance of Japanese shrimp based on heart rate is provided, using the above device, and specifically includes the following steps:

[0045] 1) First, the Japanese shrimp samples to be tested were temperature acclimated according to standard operations, and sandworms were fed regularly at 18:00 every evening. The remaining bait was removed in time and replaced with two-thirds of fresh seawater to ensure that the Japanese shrimp grew in the best health. Specifically, the temperature acclimation experiment was carried out in a water tank for 7 days. The acclimation temperatures were 26℃, 30℃ and 34℃ respectively. The salinity of the acclimation experiment was 30‰~32‰, and the saturation of dissolved oxygen was greater than 90%.

[0046] 2) Before the heart rate measurement experiment, the Japanese shrimp samples were placed in low-temperature seawater for a short period of low-temperature anesthesia; specifically, the water temperature for low-temperature anesthesia was 12-14°C, and the anesthesia time was controlled at 5-20 minutes;

[0047] 3) Move the Japanese marsupial into the heart beat video recording device, turn on the camera to record the changes in the heart rate of the Japanese marsupial in real time, and wait for the heart rate of the Japanese marsupial to return to a resting state before starting the temperature increase experiment; specifically, the Japanese marsupial is placed in the heart beat video recording device filled with 26°C seawater for at least 2 hours to ensure that the heart rate of the Japanese marsupial returns to a resting state before starting the temperature increase experiment;

[0048] 4) Using an artificial intelligence temperature control system to ensure that the seawater in the heartbeat video recording device is heated at a target heating rate, and when the heart rate of the Japanese marsupial begins to decrease, the heating is stopped and the video recording is ended, and the Japanese marsupial is quickly transferred to seawater at normal temperature, and the temperature changes are recorded in real time during the heart rate measurement process; specifically, the target heating rate is 1°C / h, and the seawater temperature range is 26-42°C;

[0049] 5) Using remote photoplethysmography technology to analyze the recorded heart beat video of Japanese marsupial, so as to accurately obtain the heart rate change data of Japanese marsupial during temperature changes; specifically, the remote photoplethysmography technology to obtain the heart rate change data of Japanese marsupial is completed based on Python;

[0050] 6) Data analysis: Establish a coordinate system, set the temperature data as the horizontal axis after standardization, transform the acquired heart rate data into natural numbers and set it as the vertical axis, and then draw an Arrhenius curve; perform linear fitting based on the relationship between heart rate and temperature, and the horizontal coordinate of the intersection of the two linear equations is the ABT value of the heart rate of Japanese shrimp;

[0051] The standardization formula is as follows:

[0052]

[0053] in: is the actual temperature of the seawater in the device (°C); The absolute temperature is normalized to the actual temperature;

[0054] In step 6), the drawing of the Arrhenius curve and the calculation of ABT are completed based on R software.

[0055] Arrhenius curve of the heart rate of Japanese shrimp ( Figure 2 ), as shown in Figure 2, the heart rate changes of Japanese marsupials can be divided into two stages as the temperature rises. In the first stage, the heart rate of Japanese marsupials increases with the increase of temperature; in the second stage, after the temperature exceeds ABT, the heart rate begins to decrease as the temperature continues to rise.

[0056] The stability of the heart rate ABT value was evaluated by measuring the heart rate ABT of Japanese shrimp under different heating rate conditions. Figure 3 The ABT of the 1℃ / h experimental group was lower than that of the other experimental groups, and the differences were extremely significant (p <0.01). The ABT differences between the 2-6℃ / h experimental groups were not significant (p>0.05). This evaluation result showed that the ABT value of the heart rate of Japanese shrimp had good stability.

[0057] The ABT of the heart rate of Japanese marsupials was measured under different acclimation temperatures to explore the effect of different acclimation temperatures on the ABT of the heart rate of Japanese marsupials. The seawater temperatures for acclimation of Japanese marsupials were 26°C, 30°C and 34°C, and the acclimation time was one week. During the acclimation period, the aquaculture water was seawater filtered by ultrafiltration membrane assembly equipment with a salinity of 30‰ to 32‰. Figure 4 There was no significant difference in the ABT of the heart rate of Japanese shrimp under different acclimation temperature conditions (p>0.05), which further verified that the ABT value of the heart rate of Japanese shrimp has good stability.

[0058] like Figure 5 As shown in the data, the ABT values ​​of different heat-resistant groups of Japanese marsupenaeus were significantly different (p<0.05), among which the ABT value of Japanese marsupenaeus with lower heat tolerance was 35.5±0.76℃, the ABT value of Japanese marsupenaeus with medium heat tolerance was 37.7±0.64℃, and the ABT value of Japanese marsupenaeus with higher heat tolerance was 39.7±1.19℃. This indicates that the determination method of heart rate ABT can be used to distinguish the differences in heat tolerance between different Japanese marsupenaeus groups.

[0059] The correlation analysis between the heart rate ABT and the heart rate flatline temperature (FLT) of Japanese shrimp was carried out to explore the correlation between the heart rate ABT and the heat tolerance of Japanese shrimp. Figure 6 , the correlation coefficient R between heart rate ABT and FLT 2 =0.99, which shows that ABT and FLT have a strong correlation. This evaluation result shows that the ABT value of the heart rate of Japanese shrimp has a high accuracy and can be used as a heat resistance index for the heat resistance evaluation of Japanese shrimp.

[0060] The present invention monitors the heart rate changes of Japanese marsupials based on computer vision technology, and has the characteristics of less damage and individual analysis. Compared with the traditional method, when the heart rate of the shrimp drops sharply, the temperature is stopped and the shrimp is returned to the temperature to keep alive, which can reduce the heat shock damage of the shrimp and accurately evaluate its heat resistance, providing an important evaluation basis for the breeding of high temperature resistance traits.

Claims

1. A method for evaluating the heat tolerance of Japanese shrimp based on heart rate, characterized in that The following steps are involved: 1) First, the shrimp samples to be tested are temperature acclimated. During the acclimation period, lugworms are fed every night, the remaining bait is cleaned in time, and some fresh seawater is replaced to ensure that the shrimp are in a healthy growth state; 2) Before the heart rate measurement experiment, the shrimp samples were placed in low-temperature seawater for a short period of anesthesia; 3) The shrimps need to be placed in the heart beat video device and left to rest for at least several hours, and the temperature rise experiment can be started after the heart rate returns to the resting state; 4) Use an artificial intelligence temperature control system to ensure that the seawater in the heartbeat recording device is heated at the target heating rate. When the shrimp's heart rate begins to drop, the temperature is stopped and the recording ends. The shrimp is quickly transferred to seawater at normal temperature. The temperature changes are recorded in real time during the heart rate measurement. 5) Use remote photoplethysmography technology to analyze the recorded shrimp heartbeat video, and then accurately obtain the shrimp heart rate change data during temperature changes; 6) Data analysis: Establish a coordinate system, set the temperature data as the horizontal axis after standardization, transform the acquired heart rate data into natural numbers and set it as the vertical axis, and then draw an Arrhenius curve; perform linear fitting based on the relationship between heart rate and temperature, and the horizontal coordinate of the intersection of the two linear equations is the ABT value of the shrimp's heart rate; 7) Randomly select multiple healthy shrimps to perform the above heart rate test, calculate the average ABT value of the shrimp heart rate, and thus evaluate the thermal tolerance of the entire group.

2. The method for evaluating the heat tolerance of Japanese shrimp based on heart rate according to claim 1, characterized in that: In step 1), the duration of the temperature acclimation experiment was one week, and the acclimation temperatures were 26°C, 30°C, and 34°C, respectively.

3. The method for evaluating the heat tolerance of Japanese shrimp based on heart rate according to claim 1, characterized in that: In step 1), the salinity of the acclimation experiment is 30‰ to 32‰, and the saturation of dissolved oxygen is greater than 90%.

4. The method for evaluating the heat tolerance of Japanese shrimp based on heart rate according to claim 1, characterized in that: In step 2), the temperature of the low-temperature seawater is 12 to 14°C, and the anesthesia time is controlled at 5 to 20 minutes.

5. The method for evaluating the heat tolerance of Japanese shrimp based on heart rate according to claim 1, characterized in that: In step 3), the shrimps must be placed in a heart beat recording device filled with 26°C seawater for at least 2 hours, and the temperature increase experiment begins when the heart rate returns to a resting state.

6. The method for evaluating the heat tolerance of Japanese shrimp based on heart rate according to claim 1, characterized in that: In step 4), the target heating rate is 1°C / h, and the seawater temperature ranges from 26 to 42°C.

7. The method for evaluating the heat tolerance of Japanese shrimp based on heart rate according to claim 1, characterized in that: In step 5), the remote photoplethysmography technology is used to obtain the heart rate change data of the shrimp based on Python.

8. The method for evaluating the heat tolerance of Japanese shrimp based on heart rate according to claim 1, characterized in that: In step 6), the drawing of the Arrhenius curve and the calculation of ABT are completed based on R software.

9. The method for evaluating the heat tolerance of Japanese shrimp based on heart rate according to claim 1, characterized in that: In step 6), the standardization is as follows: in: is the actual temperature of the seawater in the device (°C); It is the absolute temperature normalized to the actual temperature.

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