Method for evaluating the tolerance of sinonovacula constricta to ammonia-nitrogen by using infrared heartbeat

Abstract

This invention provides a method for assessing the ammonia nitrogen tolerance of razor clams using infrared heart rate measurement, comprising the following steps: S1: Raising and acclimatizing the razor clams to be tested under established acclimatization conditions to adapt them to the experimental environment; S2: Preparing the connection of the heart rate detection device before the assessment; S3: Using the acclimatized razor clams from step S1 for heart rate measurement to obtain an electrocardiogram (ECG); S4: Placing the razor clams with the sensor attached in a temporary holding tank filled with seawater to acclimatize them to the testing environment; S5: Adding ammonia nitrogen stock solution multiple times to the temporary holding tank and measuring the ECG, plotting a scatter plot, performing regression and curve fitting analysis, obtaining the corresponding curve equations, and solving the equations simultaneously to calculate the x-coordinate value of the intersection point, which is the required inflection point ammonia nitrogen concentration (BPAN) value, thus completing the determination of the ammonia nitrogen tolerance of razor clams. This invention solves the problems of high cost, low accuracy, and low intuitiveness and sensitivity in existing technologies for assessing ammonia nitrogen tolerance in razor clams.

Description

Technical Field

[0001] This invention relates to the field of aquaculture technology, and more specifically, to a method for assessing the ammonia nitrogen tolerance of razor clams using infrared heartbeat testing. Background Technology

[0002] Razor clams (Sinonovacula constricta) are one of the four traditional farmed shellfish in my country. In Zhejiang and Fujian provinces, polyculture with shrimp and crabs is the primary farming method, forming a large-scale and unique pond aquaculture industry. However, in this intensive farming system, high-density farming and overfeeding inevitably lead to the accumulation of ammonia nitrogen. As a burrowing shellfish, razor clams typically live in the mud and sand at a depth of 30-40 cm, often facing a more severe high-ammonia nitrogen environment than other aquatic animals. Furthermore, mass mortality of razor clams in summer is often induced by excessive ammonia nitrogen levels. Therefore, cultivating new ammonia nitrogen-tolerant varieties of razor clams is particularly important, and establishing accurate and rapid methods for evaluating ammonia nitrogen tolerance is key to developing new varieties.

[0003] In previous breeding practices, most methods involved subjecting shellfish to acute or chronic exogenous stress, assessing their tolerance to pollutants by measuring their survival rate or 50% lethal concentration (LC50). This method can directly reflect the shellfish's tolerance to pollutants. However, in actual production, this method has significant drawbacks, such as harming a large number of experimental animals and failing to accurately obtain physiological data. Subsequently, some researchers attempted to use physiological, biochemical, and molecular methods (such as measuring blood biochemical parameters, immune and antioxidant enzyme activities, and gene mRNA expression) to determine the tolerance of marine shellfish to pollutants. However, these indirect methods are costly and cannot comprehensively reflect the shellfish's tolerance to pollutants, lacking intuitiveness and sensitivity. Therefore, there is an urgent need for a convenient, intuitive, reliable, minimally invasive, or non-destructive method to determine the tolerance of marine shellfish to pollutants.

[0004] Using heart rate to reflect physiological changes in shellfish is a simple, rapid, and highly accurate technique. Stillman and Somero (1996) and Dong and Williams (2011) developed and refined non-invasive heart rate monitoring technology, and based on this, developed an index for measuring the heat tolerance of poikilothermic animals: the Arrhenius break temperatures (ABT). This means that within a certain temperature range, an animal's heart rate increases with increasing temperature; however, above a certain temperature, the heart rate decreases with further increases in temperature. This break temperature (ABT) represents the extreme value of heat tolerance in invertebrates; above this temperature, the organism's physiological metabolic functions decline, manifested as a decrease in heart rate. Thanks to the successful development of infrared heart rate detection technology, the measurement of shellfish heart rate indicators has emerged and is gradually being applied in marine shellfish genetic breeding. Bao Zhenmin et al. (2015) and Xing Qiang et al. (2020) conducted rapid breeding selection based on scallop heartbeat indices, using in vitro, non-invasive, and non-destructive heartbeat detection of scallops and taking the obtained heart rate frequency and waveform as the basis for scallop resistance selection. Boamah et al. (2022) determined the critical salinity of the wrinkled abalone (Haliotis discus hannai) through non-invasive heart rate measurement, indicating that the abalone heartbeat has two salinity breakpoints (BOS1 and BOS2), providing technical support for the cultivation of low-salinity new varieties. In wrinkled abalone (H. discus hannai), western abalone (H. discus hannai♀×H. fulgens♂), and green abalone (Haliotis gigantea♀×H. discus hannai♂), researchers successfully developed a parameter that can measure the abalone's hypoxia tolerance performance—the breakpoint for dissolved oxygen concentration—by non-invasively measuring the changes in heart rate under different dissolved oxygen levels. (Shen et al., 2019); however, there are few reports on using heart rate as a breeding index for ammonia nitrogen tolerance in the breeding of new marine shellfish varieties.

[0005] Therefore, without harming the parent shellfish, we explored a method for rapid selection using heart rate and waveform as indicators of ammonia nitrogen tolerance. This method can achieve non-destructive infrared sensing detection and provide technical support for the breeding of new stress-resistant marine shellfish varieties. Summary of the Invention

[0006] The technical problem to be solved by the present invention is to provide a method for evaluating the ammonia nitrogen tolerance of razor clams using infrared heartbeat measurement, so as to solve the problems of high cost, inability to fully reflect the tolerance of shellfish to pollutants, and low intuitiveness and sensitivity of the existing technology for ammonia nitrogen tolerance of razor clams.

[0007] To address the above problems, this invention provides a method for assessing the ammonia nitrogen tolerance of razor clams using infrared heartbeat testing, comprising the following steps:

[0008] S1: The razor clams to be tested were raised and domesticated under the established domestication conditions to adapt them to the experimental environment;

[0009] S2: Prepare the connection of the heart rate detection device before the assessment;

[0010] S3: Take the razor clam that has been domesticated in step S1 for heart rate assessment. After cleaning the razor clam, turn on the power and the Power Lab data acquisition host of the heartbeat detection device, set the relevant parameters of the Lab Chart software, and then attach an infrared probe to the shell surface above the heart of the razor clam. Adjust the position of the infrared probe to obtain a clear and stable electrocardiogram.

[0011] S4: Place the razor clams with the attached sensors into a temporary holding tank filled with seawater to allow them to adapt to the detection environment;

[0012] S5: Control the seawater temperature, add ammonia nitrogen stock solution with a concentration of 1000 mg / L (national standard) to the temporary holding tank for the first time, stir the seawater, and measure the ammonia nitrogen concentration of the seawater to make the ammonia nitrogen concentration in the seawater reach 90-110 mg / L;

[0013] After the heartbeat waveform of the razor clam stabilizes for 30 minutes, ammonia nitrogen stock solution is added to the temporary holding tank for the second time to make the ammonia nitrogen concentration of the seawater reach 130-160 mg / L;

[0014] Similarly, with a 30-minute interval after the heartbeat of the razor clam stabilizes following the addition of ammonia nitrogen stock solution, ammonia nitrogen stock solution was continuously added to the temporary holding tank, with each addition being 40 mg / L more than the previous addition, until the heartbeat waveform of the razor clam became disordered.

[0015] During the measurement process, the heart rate changes of the razor clams were recorded in real time using Lab Chart software. A scatter plot was drawn in Excel with ammonia nitrogen concentration as the X-axis and the heart rate of the razor clams as the Y-axis. Regression and curve fitting analysis were performed to obtain the corresponding curve equations. The x-coordinate value of the intersection point was calculated, which is the required inflection point ammonia nitrogen concentration BPAN value, thus completing the measurement of ammonia nitrogen tolerance of the razor clams.

[0016] Compared to existing technologies, the method for assessing the ammonia nitrogen tolerance of razor clams using infrared heartbeat measurement, as described in this invention, offers the following technological advancements: The entire detection process can be completed in a laboratory under controlled conditions, avoiding the influence of factors such as seawater temperature, salinity, dissolved oxygen, and feed in natural waters. Furthermore, this method combines infrared sensing, filtering, and waveform amplification technologies, enabling rapid and sensitive capture of the razor clam's heartbeat waveform. It offers advantages such as accuracy, speed, minimal sample damage, and individual measurement capability, saving significant manpower and resources and filling a gap in existing technologies. Compared to traditional methods, this method is non-invasive and non-destructive, and it is the first time that the razor clam's heartbeat has been used as an indicator for tolerance assessment. The method of this invention can provide a research foundation for the breeding of ammonia nitrogen-tolerant razor clam varieties and the establishment of a resistance index system.

[0017] In one possible implementation, in step S1, the acclimatization conditions include temperature, dissolved oxygen, and salinity levels, wherein the temperature is 20-25°C, the dissolved oxygen saturation is above 90%, the salinity is 20-22, the pH is 8, and the acclimatization time is 7 days.

[0018] Ammonia nitrogen in water exists in a dissociation equilibrium under certain conditions, namely NH4+. + NH3 and H3O can interconvert in water, and the conversion equation is NH3 + H3O - =NH4 + +H₂O, the dissociation constant (pKa) is around 9.0–9.5 (Thomas et al., 2008). The toxicity of ammonia nitrogen is closely related to its form; NH₃, due to its strong lipid solubility, easily permeates cell membranes and exhibits strong toxicity (Mayes et al., 2010). Temperature and pH have the greatest impact on the dissociation of ammonia nitrogen. In our previous study, at temperatures below 25°C, *Razor clams* showed strong tolerance to ammonia nitrogen, with a 96 hLC... 50 The ammonia nitrogen concentration can reach 244.55 mg / L (Lv et al., 2022), which is far higher than that of other shellfish such as the hard clam (Meretrix meretrix) at 92.37 mg / L (Chen et al., 2010) and the green clam (Cyclinasinensis) at 65.79 mg / L (Ge et al., 2021). When the temperature is above 25℃, the proportion of ammonia nitrogen in the water that is converted into the more toxic NH3 increases, leading to increased tolerance of razor clams to ammonia nitrogen. This is also the main reason for the large-scale mortality of razor clams in intensive aquaculture waters during the summer.

[0019] In one possible implementation, in step S1, the cultivation conditions are: feeding Chaetoceros muelleri once every 12 hours, and promptly cleaning up food residue and changing the water.

[0020] Studies have shown that ammonia nitrogen in aquaculture water mainly comes from two sources: first, it is produced by the ammonification of nitrogenous organic matter such as food residue, aquatic animal feces and remains by microorganisms; second, it is produced by the ammonia production of proteins or other compounds in aquatic animals through a series of transammoniation and deammoniation processes (Wang Guohui, 2014). Therefore, during the temporary holding period before the start of the experiment, it is necessary to clean up food residue and change the water in a timely manner.

[0021] In one possible implementation, step S2 includes: connecting the other end of the infrared sensor to a signal amplifier via an RJ11 connection; connecting the signal amplifier to a data acquisition host via a BNC interface; and connecting the acquired data to a computer via a USB connection. The photocurrent generated by the heartbeat is filtered by the amplifier, and the voltage value is converted into a digital signal by the data acquisition and analysis system and input into the computer. The heart rate-related parameters are obtained using the Lab Chart Version 8.0 software.

[0022] In one possible implementation, step S3 includes cleaning the surface of the razor clam shell and wiping it dry.

[0023] In one possible implementation, in step S3, the relevant parameters of the Lab Chart software are set as follows: range 2-5V, low pass 1-10Hz, AC coupling, and acquisition rate of 1k / s.

[0024] In one possible implementation, the temporary incubation time in step S4 is 1-2 hours.

[0025] In one possible implementation, in step S5, the ammonia nitrogen concentration of the seawater after the first addition is 100±5.82 mg / L, and the ammonia nitrogen concentration of the seawater after the second addition is 140±6.13 mg / L.

[0026] In one possible implementation, in step S5, the seawater temperature is 20-25°C; the changes in the razor clam's heart rate include a normal heartbeat phase and an arrhythmic heartbeat phase.

[0027] During the normal heartbeat phase, the heart rate of the razor clam showed an upward trend. The linear fitting equation between the heart rate and ammonia nitrogen concentration during this phase was y = (0.05245 ± 0.01475)x + (18.8945 ± 0.6385).

[0028] During the arrhythmia phase, the heart rate of the razor clam decreased linearly with increasing exposure time. The linear regression equation between the heart rate and ammonia nitrogen concentration during this phase was y = -(0.05895 ± 0.00585)x + (42.399 ± 2.474).

[0029] In one possible implementation, in step S5, the ammonia nitrogen concentration BPAN value is 201.58–224.58 mg / L.

[0030] This invention, through the above-mentioned linear fitting equations of razor clam heart rate and ammonia nitrogen concentration at conventional temperatures (20-25℃), yields the ammonia nitrogen concentration BPAN value, which has universality within a specific temperature range. This facilitates selection during razor clam breeding and lays the foundation for the establishment of subsequent systems. Attached Figure Description

[0031] Figure 1 This is a schematic diagram of the process and structure of the method for evaluating the ammonia nitrogen tolerance of razor clams using infrared heartbeat measurement according to the present invention.

[0032] Figure 2 The waveforms of normal and abnormal heartbeats of the razor clam of the present invention are shown; where a represents a normal heart rate, the waveform is "mountain" shaped, showing 3 peaks in one cycle; b, c, and d represent abnormal heartbeats, the waveforms are "V" shaped and "wave" shaped, different from the normal waveforms, showing 2 or 4 peaks in one cycle.

[0033] Figure 3 This is a schematic diagram of the inflection point ammonia nitrogen concentration (BPAN) of the present invention;

[0034] Figure 4 This is a schematic diagram illustrating the changes in physiological indicators of razor clams under ammonia nitrogen stress according to the present invention. Detailed Implementation

[0035] First, those skilled in the art should understand that these embodiments are merely used to explain the technical principles of the embodiments of this application and are not intended to limit the scope of protection of the embodiments of this application. Those skilled in the art can make adjustments as needed to adapt to specific application scenarios.

[0036] like Figure 1 As shown, Figure 1 This is a schematic diagram illustrating the process and structure of a method for assessing ammonia nitrogen tolerance in razor clams using infrared heartbeat measurement, according to the present invention. The present invention provides a method for assessing ammonia nitrogen tolerance in razor clams using infrared heartbeat measurement, comprising the following steps:

[0037] S1: The razor clams to be tested were raised and domesticated under the established domestication conditions to adapt them to the experimental environment;

[0038] S2: Prepare the connection of the heart rate detection device before the assessment;

[0039] S3: Take the razor clams acclimated in step S1 for heart rate assessment. After cleaning the razor clams, turn on the power and the Power Lab data acquisition host of the heart rate detection device, set the relevant parameters of the Lab Chart software, and then attach an infrared probe to the shell surface above the heart of the razor clam. Adjust the position of the infrared probe to obtain a clear and stable electrocardiogram (the waveform is "mountain" shaped with 3 peaks in one cycle).

[0040] S4: Place the razor clams with the attached sensors into a temporary holding tank filled with seawater to allow them to adapt to the detection environment;

[0041] S5: Control the seawater temperature, add ammonia nitrogen stock solution with a concentration of 1000 mg / L to the temporary holding tank for the first time, stir the seawater, and measure the ammonia nitrogen concentration of the seawater to make the ammonia nitrogen concentration in the seawater reach 90-110 mg / L;

[0042] After the heartbeat waveform of the razor clam stabilizes for 30 minutes, ammonia nitrogen stock solution is added to the temporary holding tank for the second time to make the ammonia nitrogen concentration of the seawater reach 130-160 mg / L;

[0043] Similarly, with a 30-minute interval after the heartbeat of the razor clam stabilizes following the addition of ammonia nitrogen stock solution, ammonia nitrogen stock solution was continuously added to the temporary holding tank, with each addition being 40 mg / L more than the previous addition, until the heartbeat waveform of the razor clam became disordered (“V” shape and “wave” shape, with 2 or 4 peaks in one cycle).

[0044] During the measurement process, the heart rate changes of the razor clams were recorded in real time using Lab Chart software. A scatter plot was drawn in Excel with ammonia nitrogen concentration as the X-axis and the heart rate of the razor clams as the Y-axis. Regression and curve fitting analysis were performed to obtain the corresponding curve equations. The x-coordinate value of the intersection point was calculated, which is the required inflection point ammonia nitrogen concentration BPAN value, thus completing the measurement of ammonia nitrogen tolerance of the razor clams.

[0045] As a preferred embodiment, in step S1, the acclimatization conditions include temperature, dissolved oxygen, pH, and salinity levels, with the temperature being 20-25℃, dissolved oxygen saturation above 90%, salinity between 20-22, pH at 8, and acclimatization time being 7 days.

[0046] As a preferred embodiment, in step S1, the cultivation conditions are as follows: feed Chaetoceros muscaria once every 12 hours, and promptly clean up food residue and change the water.

[0047] As a preferred embodiment, step S2 includes: connecting the other end of the infrared sensor to a signal amplifier via an RJ11 connection; connecting the signal amplifier to a data acquisition host via a BNC interface; and connecting the acquired data to a computer via a USB connection. The photocurrent generated by the heartbeat is filtered by the amplifier, and the voltage value is converted into a digital signal by the data acquisition and analysis system and input into the computer. The heart rate-related parameters are obtained using the Lab Chart Version 8.0 software.

[0048] As a preferred embodiment, in step S3, the cleaning includes: cleaning the surface of the razor clam shell and wiping it dry.

[0049] As a preferred embodiment, in step S3, the relevant parameters of the Lab Chart software are set as follows: range 2-5V, low pass 1-10Hz, AC coupling, and acquisition rate 1k / s.

[0050] As a preferred embodiment, in step S4, the temporary holding time is 1-2 hours.

[0051] As a preferred embodiment, in step S5, the ammonia nitrogen concentration of the seawater after the first addition is 100±5.82 mg / L, and the ammonia nitrogen concentration of the seawater after the second addition is 140±6.13 mg / L.

[0052] As a preferred embodiment, in step S5, the seawater temperature is 20-25℃; the changes in the razor clam's heart rate include a normal heartbeat phase and an arrhythmic heartbeat phase;

[0053] During the normal heartbeat phase, the heart rate of the razor clam showed an upward trend. The linear fitting equation between the heart rate and ammonia nitrogen concentration during this phase was y = (0.05245 ± 0.01475)x + (18.8945 ± 0.6385).

[0054] During the arrhythmia phase, the heart rate of the razor clam decreased linearly with increasing exposure time. The linear regression equation between the heart rate and ammonia nitrogen concentration during this phase was y = -(0.05895 ± 0.00585)x + (42.399 ± 2.474).

[0055] As a preferred embodiment, in step S5, the ammonia nitrogen concentration BPAN value is 201.58–224.58 mg / L.

[0056] Example 1:

[0057] This embodiment describes a method for assessing the ammonia nitrogen tolerance of razor clams using infrared heartbeat testing. The specific implementation steps are as follows:

[0058] S1: Before the evaluation, the razor clams were strictly domesticated. Suitable domestication conditions were set, including temperature, dissolved oxygen, and salinity levels. The temperature was 20℃, dissolved oxygen saturation was 93%, salinity was 21, and pH was 8. The standard breeding program included feeding Chaetoceros muelleri once every 12 hours, timely cleaning of food residue, and water changes. The domestication time was 7 days.

[0059] S2: The heartbeat detection device includes an infrared sensor (CNY-70), a heartbeat monitor amplifier (AMP-03U) (Newshift@, Portugal), a Power Lab8 / 35 eight-channel high-speed research recording host (including Lab Chart Pro professional version, manufactured by ADInstruments, Australia, Shanghai) and Blu-tag (BOSTIK, Australia). The other end of the infrared sensor is connected to the signal amplifier via an RJ11 connection. The signal amplifier is then connected to the data acquisition host via a BNC interface. The acquired data is connected to the computer via USB. Figure 1 Configure the Lab Chart software to signal acquisition mode. The photocurrent generated by the heartbeat is processed by an amplifier and filtered. The voltage value is then converted into a digital signal by the data acquisition and analysis system and input into the computer. The Lab Chart Version 8.0 software is used to acquire relevant parameters of heart rate (HR).

[0060] S3: Subsequent testing was conducted on the domesticated razor clam samples. The shell length of the razor clam was 58.52±4.41mm. First, the dirt on the surface of the razor clam shell was cleaned and excess water was wiped away to facilitate the contact between the sensor and the shell surface. Then, relevant parameters were set in the Lab Chart software. The relevant parameters of the Lab Chart software were set as follows: range (2~5V), low pass (1~10Hz), AC coupling, and acquisition rate of 1k / s, which can obtain a clear and stable electrocardiogram with a heartbeat waveform.

[0061] S4: Place the razor clams with sensors attached in a temporary culture tank filled with seawater for 1 - 2 hours of temporary culture at a temperature of 20°C to allow them to adapt to the detection environment. After the razor clams have adapted to the environment and the heartbeat waveform has been stable for 30 minutes, add an ammonia nitrogen stock solution with a concentration of 1000 mg / L to the temporary culture tank, and gently stir the seawater using a magnetic stirrer. During this process, use a portable measuring instrument (HACH, DR3900) to measure the ammonia nitrogen concentration, so that the ammonia nitrogen concentration in the seawater reaches 100 ± 5.82 mg / L. After the razor clams' heartbeat waveforms have been stable for 30 minutes in an ammonia nitrogen water body with a concentration of 100 ± 5.82 mg / L, continue to add the ammonia nitrogen stock solution to the temporary culture tank until the ammonia nitrogen concentration in the temporary culture tank reaches 140 ± 6.13 mg / L. Similarly, with a 30 - minute interval after the razor clams' heartbeat stabilizes after adding the newly added ammonia nitrogen stock solution, continuously add the ammonia nitrogen stock solution to the temporary culture tank until the razor clams' heartbeat waveforms become disordered.

[0062] S5: During the measurement process, use Lab Chart software to record the change in the razor clams' heart rate in real - time. With the ammonia nitrogen concentration as the X - axis and the razor clams' heart rate as the Y - axis, use Excel software to perform piece - wise linear fitting on the ammonia nitrogen concentration - heart rate scatter plot to draw a scatter plot, obtain the corresponding equations and联立 them, and calculate the abscissa value of the intersection point, which is the required inflection point ammonia nitrogen concentration BPAN value.

[0063] Heartbeat regularity analysis and index development

[0064] The heartbeat of razor clams during ammonia nitrogen stress can be divided into 2 stages according to its characteristics: the normal heartbeat stage and the abnormal heartbeat stage. In the normal heartbeat stage, the heartbeat waveform of razor clams is in the shape of a "mountain", showing 3 peaks in one cycle (as shown in Figure 2 a), while in the abnormal heartbeat stage, the heartbeat waveforms of razor clams show "V" - shaped and "wave" - shaped different from the normal waveform, specifically showing 2 peaks or 4 peaks in one cycle (as shown in Figure 2 b, c, d, e). According to the change law of razor clams' heartbeat under ammonia nitrogen stress, an evaluation index for indicating the ammonia nitrogen tolerance of razor clams was developed, and the specific method is as follows:

[0065] a. In the normal heartbeat stage, the heart rate of razor clams shows an upward trend (as shown in Figure 3 ), and a linear fitting equation between the heart rate of razor clams and the ammonia nitrogen concentration in this stage is established (in this example, y=(0.05245 ± 0.01475)x+(18.8945 ± 0.6385), this equation is applicable to razor clams at a temperature of 20 - 25°C, and different temperature cultures will have an impact on it);

[0066] b. In the abnormal heartbeat stage, as the ammonia nitrogen stress concentration increases, the heartbeat waveforms of razor clams begin to show abnormalities, manifested as irregular waveforms, and their heart rates also linearly decrease with the increase in exposure time (as shown in Figure 3As shown), a linear regression equation was established between the heart rate and ammonia nitrogen concentration of razor clams at this stage (in this example, y = -(0.05895±0.00585)x + (42.399±2.474). This equation is applicable to razor clams at a temperature of 20-25℃. Different temperatures will have an impact on them).

[0067] c. Calculate the x-axis of the intersection point of the two linear equations; this is the ammonia nitrogen concentration required to cause the razor clam's heartbeat to change from normal to arrhythmic. Figure 3 In this example, the concentration is 201.58–224.58 mg / L, which is suitable for razor clams at 20–25°C (different temperatures will affect the concentration). This ammonia nitrogen concentration is defined as the ammonia nitrogen inflection point concentration BPAN.

[0068] When the culture temperature was 20℃ and other conditions met those described in Example 1 above, the linear fitting equation for the normal heartbeat phase was calculated as y = 0.0377x + 19.533, and for the arrhythmic heartbeat phase as y = -0.0531x + 39.925. The abscissa of the intersection of the two linear equations was further calculated, which represents the ammonia nitrogen concentration required to cause the razor clam's heartbeat to change from normal to arrhythmic; in this example, it was 224.58 mg / L. Figure 3 As shown in (a).

[0069] Using BPAN as an indicator of ammonia nitrogen tolerance in razor clams can assess their ammonia nitrogen tolerance.

[0070] Example 2:

[0071] Example 2 is similar to Example 1, except that:

[0072] In step S1, the temperature is 22℃, the dissolved oxygen saturation is 90.0%, the salinity is 20, and the pH is 8.

[0073] In step S4, the first addition of mother liquor was 1000 mL, the ammonia nitrogen concentration in the seawater was 90±1.04 mg / L, and the ammonia nitrogen concentration after the second addition was 130±3.15 mg / L. The temperature was set at 22℃.

[0074] Example 3:

[0075] Example 3 is similar to Example 1, except that:

[0076] In step S1, the temperature is 25℃, the dissolved oxygen saturation is 93.0%, the salinity is 22, and the pH is 8.

[0077] In step S4, the first addition of mother liquor was 1000 mL, the ammonia nitrogen concentration in the seawater was 110±1.97 mg / L, and the ammonia nitrogen concentration after the second addition was 150±4.02 mg / L. The temperature was set at 25℃.

[0078] The method of the present invention will be described in detail below using the BPAN value of the heart rate of the razor clam in Example 1 as an indicator for evaluation, taking the rapid selection method of razor clams under ammonia nitrogen stress as an example:

[0079] I. Assessment of ammonia nitrogen tolerance time in different individuals of razor clams based on heart rate indicators:

[0080] To verify the above-mentioned method for evaluating the ammonia nitrogen tolerance of razor clams based on heartbeat, 30 one-year-old razor clams from Ninghai, Zhejiang (shell length 58.52±4.41mm, wet weight 16.04±3.08g) were selected as experimental subjects. These razor clams were placed in a temporary holding tank with an ammonia nitrogen concentration of 224.58mg / L (razor clam BPAN value) according to the above-mentioned operation method. The time T of the onset of cardiac arrhythmia in each razor clam was observed, and the heart rate (HR) at the time of cardiac arrhythmia was recorded.

[0081] Table 1 summarizes the time T (time of cardiac arrhythmia) and heart rate (HR) of razor clams during the same time period based on the heart rate index. The results show that, based on heart rate changes, the sensitivity of 30 razor clams of the same specification and batch to 224.58 mg / L ammonia nitrogen stress varied. Some razor clams were extremely sensitive to ammonia nitrogen, exhibiting cardiac arrhythmia within 15 minutes of its addition, while others showed cardiac arrhythmia only after 90 minutes. These results indicate that the BPAN index developed based on the method of this invention can effectively indicate the cardiac performance of razor clams under ammonia nitrogen stress, providing a real-time, intuitive, accurate, and quantifiable assessment of the razor clams' tolerance to ammonia nitrogen.

[0082] Table 1. Evaluation of the time to ammonia nitrogen tolerance and heart rate changes in different individuals (N=30) of razor clams based on heart rate indicators:

[0083]

[0084]

[0085] II. Assessment of physiological and biochemical changes in ammonia nitrogen tolerance in razor clams based on heart rate indicators:

[0086] This invention utilizes 391 one-year-old razor clams (shell length 58.52±4.41 mm, wet weight 16.04±3.08 g) from Ninghai County, Zhejiang Province as materials. Following the method described in this invention, the temperature and pH were strictly controlled at 20℃ and 8, respectively. After the razor clams had adapted to the temporary rearing tank for 1-2 hours, ammonia nitrogen stock solution was added to the tank to achieve an ammonia nitrogen concentration of BPAN = 224.58 mg / L (the actual ammonia nitrogen concentration in the water was 221.48±5.34 mg / L). The time of cardiac arrhythmia for each clam after BPAN ammonia nitrogen concentration stress was recorded. The cardiac arrhythmia time of each clam was then recorded and sorted. Clams with faster arrhythmia (first 10%, designated as the sensitive group) and slower arrhythmia (last 10%, designated as the tolerant group) were selected and designated as the sensitive group and the tolerant group, respectively. Next, the 76 razor clams were subjected to ammonia nitrogen stress at a BPAN concentration of 224.58 mg / L for 96 hours. Every 12 hours, 4 razor clams were selected for blood ammonia content measurement, and the number of deaths in the sensitive and tolerant groups was recorded (e.g., ...). Figure 4 (As shown). The results showed that at the same time point, there was no significant change in blood ammonia levels in either the tolerant or sensitive groups (as shown). Figure 4 As shown in Figure a), with increasing stress duration, serum ammonia levels in both the tolerant and sensitive groups showed a significant increase (P<0.05). Furthermore, 96-hour survival curves revealed (e.g.) Figure 4 As shown in b), there were significant differences between the tolerant and sensitive groups at 84h and 96h of ammonia nitrogen stress (P<0.05).

[0087] The results of the above embodiments and tests of the present invention demonstrate that the method of evaluating the ammonia nitrogen tolerance of razor clams using infrared heartbeat testing can effectively determine the tolerance of razor clams to ammonia nitrogen, thus providing a detailed and rapid experimental method for the rapid assessment of the ammonia nitrogen tolerance of razor clams, and providing technical support for the breeding of new razor clam varieties and healthy and green aquaculture in my country.

[0088] In summary, this invention provides a method for assessing the ammonia nitrogen tolerance of razor clams using infrared heart rate testing. First, the razor clams are acclimatized and then used for heart rate assessment. Relevant parameters are set in the Lab Chart software on a Power Lab data acquisition and analysis system. An infrared probe is fixed to the shell surface above the clam's heart to obtain a clear and stable electrocardiogram. The razor clams with the attached sensor are placed in a holding tank filled with seawater, and ammonia nitrogen stock solution is continuously added to the tank. Ammonia nitrogen concentration and heart rate are monitored in real time until the clam's heart rate waveform becomes disordered. A scatter plot is plotted with ammonia nitrogen concentration as the X-axis and clam heart rate as the Y-axis. Regression and curve fitting analyses are performed to obtain the corresponding curve equations. These equations are then combined, and the x-coordinate value of the intersection point is calculated, which is the required inflection point ammonia nitrogen concentration (BPAN value). Compared with traditional methods, this method has the advantages of being non-destructive, convenient, and rapid, providing technical support for the breeding of ammonia nitrogen-tolerant razor clam varieties and the establishment of resistance index systems.

[0089] In the description of this application, the references to terms such as "an embodiment," "some embodiments," "in this embodiment," "specific example," or "some examples," etc., refer to specific features, structures, materials, or characteristics described in connection with that embodiment or example, which are included in at least one embodiment or example of this application. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in a suitable manner in any one or more embodiments or examples. Moreover, without contradiction, those skilled in the art can combine and integrate the different embodiments or examples described in this specification, as well as the features of different embodiments or examples.

[0090] The above description is merely a specific embodiment of this application, but the scope of protection of this application is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in this application should be included within the scope of protection of this application. Therefore, the scope of protection of this application should be determined by the scope of the claims.

Claims

1. A method for assessing ammonia nitrogen tolerance in razor clams using infrared heartbeat testing, characterized in that, Includes the following steps: S1: The razor clams to be tested were raised and domesticated under the established domestication conditions to adapt them to the experimental environment; S2: Prepare the connection of the heart rate detection device before the assessment; S3: Take the razor clam that has been domesticated in step S1 for heart rate assessment. After cleaning the razor clam, turn on the power and the Power Lab data acquisition host of the heartbeat detection device, set the relevant parameters of the Lab Chart software, and then attach an infrared probe to the shell surface above the heart of the razor clam. Adjust the position of the infrared probe to obtain a clear and stable electrocardiogram. S4: Place the razor clams with the attached sensors into a temporary holding tank filled with seawater to allow them to adapt to the detection environment; S5: Control the seawater temperature, add ammonia nitrogen stock solution with a concentration of 1000 mg / L to the temporary holding tank for the first time, stir the seawater, and measure the ammonia nitrogen concentration of the seawater to make the ammonia nitrogen concentration in the seawater reach 90-110 mg / L; After the heartbeat waveform of the razor clam stabilizes for 30 minutes, ammonia nitrogen stock solution is added to the temporary holding tank for the second time to make the ammonia nitrogen concentration of the seawater reach 130-160 mg / L; With a 30-minute interval after the heartbeat of the razor clam stabilizes following the addition of ammonia nitrogen stock solution, ammonia nitrogen stock solution is continuously added to the holding tank, with each addition being 40 mg / L more than the previous addition, until the heartbeat waveform of the razor clam becomes disordered. During the measurement process, the heart rate changes of the razor clams were recorded in real time using Lab Chart software. A scatter plot was drawn in Excel with ammonia nitrogen concentration as the X-axis and razor clam heart rate as the Y-axis. Regression and curve fitting analysis were performed to obtain the corresponding curve equations and solve them simultaneously. The x-coordinate value of the intersection point was calculated, which is the required inflection point ammonia nitrogen concentration BPAN value, thus completing the measurement of ammonia nitrogen tolerance of the razor clams. In step S5, the ammonia nitrogen concentration of the seawater after the first addition is 100 ± 5.82 mg / L, and the ammonia nitrogen concentration of the seawater after the second addition is 140 ± 6.13 mg / L. In step S5, the seawater temperature is 20-25℃; the heart rate changes of the razor clam include a normal heartbeat phase and an arrhythmic heartbeat phase. During the normal heartbeat phase, the heart rate of the razor clam showed an upward trend. The linear fitting equation between the heart rate and ammonia nitrogen concentration during this phase was y=(0.05245 ± 0.01475)x + (18.8945 ± 0.6385). During the arrhythmia phase, the heart rate of the razor clam decreased linearly with increasing exposure time. The linear regression equation between the heart rate and ammonia nitrogen concentration during this phase was y = -(0.05895 ± 0.00585)x + (42.399 ± 2.474).

2. The method for assessing ammonia nitrogen tolerance in razor clams using infrared heartbeat measurement according to claim 1, characterized in that, In step S1, the acclimatization conditions include temperature, dissolved oxygen, and salinity levels, with the temperature being 20-25℃, dissolved oxygen saturation above 90%, salinity between 20-22, pH at 8, and acclimatization time at 7 days.

3. The method for assessing ammonia nitrogen tolerance in razor clams using infrared heartbeat measurement according to claim 1, characterized in that, In step S1, the cultivation conditions are as follows: feed Chaetoceros muscaria once every 12 hours, and promptly clean up food residue and change the water.

4. The method for assessing ammonia nitrogen tolerance in razor clams using infrared heartbeat measurement according to claim 1, characterized in that, Step S2 includes: connecting the other end of the infrared sensor to a signal amplifier via an RJ11 connection; connecting the signal amplifier to the data acquisition host via a BNC interface; and connecting the acquired data to the computer via a USB connection. The photocurrent generated by the heartbeat is filtered by the amplifier, and the voltage value is converted into a digital signal by the data acquisition and analysis system and input into the computer. The heart rate-related parameters are obtained using the Lab Chart Version 8.0 software.

5. The method for assessing ammonia nitrogen tolerance in razor clams using infrared heartbeat measurement according to claim 1, characterized in that, In step S3, the cleaning includes cleaning the surface of the razor clam shell and wiping it dry.

6. The method for assessing ammonia nitrogen tolerance in razor clams using infrared heartbeat measurement according to claim 1, characterized in that, In step S3, the relevant parameters of the Lab Chart software are set as follows: range 2~5V, low pass 1~10Hz, AC coupling, and acquisition rate 1k / s.

7. The method for assessing ammonia nitrogen tolerance in razor clams using infrared heartbeat measurement according to claim 1, characterized in that, In step S4, the temporary holding time is 1-2 hours.

8. The method for assessing ammonia nitrogen tolerance in razor clams using infrared heartbeat measurement according to claim 7, characterized in that, In step S5, the ammonia nitrogen concentration BPAN value is 201.58~224.58 mg / L.

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