The Application of an Infrared-Based ECG Acquisitor in an Online Healthy Assessment System: The Effect of Temperature on Cardiac Function in Carp (Cyprinus carpio)

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

 This study provides insights into the application of an IR-based ECG acquisitor in an online health assessment system, specifically examining the effect of temperature on carp (Cyprinus carpio). However, several aspects could be improved or clarified:

 1 Title: The title "The Application of IR-based ECG Acquisitor in an Online Healthy Assessment System: Effect of Temperature on Carp (Cyprinus carpio)" does not accurately reflect the main objective of the study, which is solely to examine the effect of temperature on the heart function of fish.

2 Abstract:

Lines 08–11: “Global warming and widespread hydraulic construction have significantly impacted fish reproduction. Variations in river temperature affect the heart function of fish, which in turn affects their habitat distribution. Therefore, we hypothesize that the electrocardiograph (ECG) parameters of carp can indicate the stress caused by environmental factors.”

In this paragraph, several phenomena or factors (in bold) are mentioned without a clear connection to the core objectives of the study. Some of these elements are not directly related to the main purpose of the study.

Moreover, while heart function may influence the fitness or viability of fish, it does not directly determine their habitat distribution.

The abstract is 144 words, which is very short, and it lacks proper synthesis and focus.

Line 17: “This study emphasizes the importance of using real-time online fish ECG assessments to evaluate fish health status.”

This statement is too broad. Since the study is specifically focused on heart function, it cannot fully support conclusions about overall fish health status. A more accurate phrasing is recommended.

3 Introduction

- “ Today, global warming poses a serious ecological challenge for humanity [1]. The rising frequency of extreme heatwaves is associated with increased global mortality rates, primarily due to cardiovascular diseases during these events”

In fact, global warming presents a significant challenge for humanity, but it is also affecting other areas, such as aquatic organisms. There are many examples to discuss regarding the impact of global warming on these organisms."

It is better to avoid overly general introductions. The most effective approach is to focus on the specific goals and objectives of the current study.

- "Cardiac function, which can be defined as the heart's ability to supply blood to other tissues, is a critical factor in determining the health of many animals [10]. It also plays a pivotal role in oxygen delivery, regulating the aerobic metabolic capacity and physical performance of fish."

This is mentioned in lines 32-43, following the discussion on the variation in cardiac function at different temperatures. I believe this definition is not appropriately placed in this section and would suggest it be deleted.

- “Fish were among the earliest organisms used in biomonitoring, and their cardiac function is more comparable to humans than other animal models. Thus, the results of stress response evaluations in fish may reflect human stress responses [20,21].”

It is known that fish have a single circulatory system with a two-chambered heart, while humans have a double circulatory system with a four-chambered heart.

I understand the potential of this study, but that doesn’t mean I should suggest that it is relevant to humans. The value of this work lies with aquatic organisms.

Concerning references [20,21]: Bae, M.J.; Park, Y.S. Biological early warning system based on the responses of aquatic organisms to disturbances: A review. 372 Science of The Total Environment.

Vöröslakos, M.; Takeuchi, Y.; Brinyiczki, K.; Tamas, Z.; Oliva, A.; Fernández-Ruiz, A.; Kozák, G.; Kincses, Z.T.; Bela, I.; Buzsáki, 374 G.; et al. Direct effects of transcranial electric stimulation on brain circuits in rats and humans. Nature.

I have checked the references, and at no point is there any citation of an analogy between human cardiac function and fish as models.

- “Fish are especially susceptible in rivers affected by hydropower development or sensitive to climate change. Limited food availability and reduced feeding opportunities can significantly lower habitat carrying capacity and hinder fish growth, thereby influencing spawning and foraging activities [28].”

Except for “sensitive to climate change” the other factors are not related to the purpose of the study: 'The effect of temperature on the heart function of fish.'"

- “Assessing whether fish can navigate ecological engineering projects is crucial for evaluating the potential threats these projects pose to ecosystems. “

It’s a significant dilemma that calls for a multifaceted and interdisciplinary study, yet the study only addresses the effect of temperature variation.

- “"The effects of environmental factors on fish cardiac function” Which environmental factors, when the purpose is 'The evaluation of the effect of temperature on the heart function of fish'?"

- "This approach enables us to test our hypothesis and evaluate whether this online device can effectively measure fish responses to ecological engineering projects, particularly in terms of mitigating potential threats to spawning and feeding migrations."

While I recognize the potential of such a study and its implications, the methodology and findings seem speculative. Each word must be precise and focused.

- "Between ECG parameters and environmental factors” There are again reference to environmental factors, but the study only addresses the effect of temperature variation on..."

4 Materials and methods

The number of fish tested is insufficient, and their origin is questionable.

It is possible that the fish were exposed to inappropriate stress. The reference to 'from an aquarium' raises concerns: what is the exact source?

Additionally, the fish may have had suboptimal physical conditions.

The two-week duration of the experiment coincides with the acclimatization period for the fish to their new environment, which could lead to stress related to the installation process.

Moreover, the presence of “professional maintenance personnel” could also be a source of stress.

A more rigorous experimental design is needed, with conditions as close as possible to those in their natural environment. Furthermore, the food deprivation—'the fish were fed twice daily (7 am and 4 pm), but feeding was stopped 24 hours prior to the experiment, and no food was given during the experimental period'—adds an additional source of stress, which could accelerate the heart rate.

I disapprove of the methodology, which lacks rigor. This type of experiment is highly delicate, and I consistently stress the importance of eliminating all other stress sources for the fish to isolate the impact of temperature variation, something that was not adhered to in the presented work.

5 Discussion

- “In the context of biology, stress is defined as the non-specific response of the body to demands for change [36]. Conducting laboratory studies is fundamental for determining the relationship between stress and measurable physiological parameters.” “Carps are in the top ranks of China’s aquaculture output [37]. In the aquaculture market, carp is a commercially valuable fish. Among all freshwater fish, carp is the most extensively cultured, widely distributed, and diverse species of the Cyprinidae family. Previous studies have indicated that the optimal growth temperature for crucian carp is between 21℃ and 25℃ [38]. Therefore, in this study, the control group for the temperature experiment was set at 22℃. The water temperature for each experiment was set based on these considerations.” It should be in the introduction.

- “The responses of heart rate of fishes under different temperatures were presented in  Table 2,” It appears more as a result.

- “Electrical excitability plays a pivotal role in initiating and regulating the rhythm and contraction speed of fish hearts, making it essential for temperature-related cardiac function adjustments. Prolonged exposure to altered thermal environments triggers compensatory modifications in both ion channel expression and functionality, which can partially counterbalance the direct influence of temperature on cardiac action potentials and heart rate.” Some references should be added.

- “From our laboratory and others' research [41,42], it is evident that recording the amplitudes of various waves in fish electrocardiograms is quite challenging. This difficulty arises from the challenge of inserting two electrodes into relatively equal positions. Additionally, during the experiment, the behavior and activity of the fish can cause the electrodes to shift, which may contribute to significant experimental deviations in the ECG indicators of fish.”

I appreciate the self-critique and the acknowledgment of the experimental limitations, which is a crucial aspect of scientific research. However, I believe this discussion should be positioned just before the conclusion and future perspectives, to better suggest improvements for the conditions of future studies.

Final Decision

The study lacks rigor, such as the limited sample size (n = 27) and inappropriate experimental conditions, including the short duration of exposure (2 weeks) and the restricted temperature gradient (four temperature points: 18°C, 22°C (control group), 26°C, and 30°C). Furthermore, temperature is the sole environmental factor considered, ignoring other potential stressors that could also impact cardiac function. Although ECG parameters are measured, the study does not establish correlations with other physiological or health indicators, such as stress hormone levels, mortality, or behavioral changes. Moreover, focusing solely on Cyprinus carpio (common carp) restricts the generalizability of the results to other fish species, which may exhibit different thermal tolerance or cardiac responses.

I am unable to accept this work in its current form. Several key aspects must be addressed to ensure the study meets the required standards of scientific rigor and clarity.

 

Comments on the Quality of English Language

The English could be improved to more clearly express the research.

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

The use of the common carp (Cyprinus carpio) as a model species requires clearer justification. While the manuscript refers to C. carpio, the fish shown in Figure 1 appear to be koi carp, which casts doubt on the consistency. The authors should clarify the strain used and provide a more robust rationale for its selection, ideally based on biological or physiological relevance and not just commercial value.

In the experimental design (lines 98–114), it is unclear whether the acclimatization time was standardized for each temperature. The manuscript should state how long the fish were acclimated prior to ECG recording and whether physiological criteria were used to confirm acclimation.

The section on SOM analysis (lines 187–202) does not provide sufficient explanation. The authors should further explain the biological or physiological significance of the clusters shown in Figure 4 to help the reader interpret the results.

There also seems to be a contradiction in the results of the Pearson correlation (lines 203–213). While the text states that heart rate and wave amplitudes are negatively correlated with temperature, the coefficient given for heart rate (0.844) indicates a strong positive correlation. This needs to be clarified.

In the Discussion, some assertions, regarding the expression of ion channels (lines 239–244), are not directly supported by the data presented. It would strengthen the manuscript if the authors could link their results more clearly to these claims or cite relevant literature.

Regarding the placement of electrodes (lines 253–267), the manuscript indicates some challenges, but it is not clear how these were handled analytically. Were any records excluded or corrected? If so, a brief explanation of the statistical treatment would be helpful.

The section (lines 268–296) touches on relevant theoretical issues, but offers little in the way of practical implementation. It would be helpful if the authors could propose specific, evidence-based recommendations for temperature management in aquaculture based on their findings.

Finally, while the conclusion is in line with the objectives of the study, it tends to overstate the practical applicability of the results. The authors should briefly point out the methodological limitations and suggest what further validation would be required before the results can be put into practice.

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

The authors indicate that they performed the Kolmogorov-Smirnov test to validate the normality of the data. However, to use an analysis of variance it is also necessary to validate homoscedasticity. Authors must add this information (which can be attached as supplementary material). On the other hand, for the study of liner regression and correlation it is necessary to ensure that the residuals follow a normal distribution and to prove that the slope and correlation coefficient are statistically significant. Authors must add this information (which can be attached as supplementary material).

Authors must indicate which multiple comparison test was used, in cases where ANOVA yielded statistically significant results.

The authors state that: “Overall, from 18°C to 26°C, the heart rate increases with rising temperature, but the increase is significantly limited when the temperature reaches 30°C.”. Therefore, it is requested which statistical test was used so that it can be stated that “the increase is significantly limited”.

Authors must present the standard-deviation as a measure of variability and not the standard-error of the mean, as it is only a measure of precision of the sample mean.

Regarding: “There is no significant difference in the average heart rate between 18°C and 22°C or between 26°C and 30°C (p > 0.05). However, there is a highly significant difference between 18°C and 26°C and between 18°C and 30°C (p < 0.01), and a significant difference between 22°C and 30°C (p < 0.05).”. How did the authors reach these conclusions? Which multiple comparisons test is used?

In the legend of figure 2c, the authors must indicate what the “*” means.

In the legend of figure 3a, the authors must indicate what the letters mean. On the other hand, one wonders why a “*” is used in figure 2 and in this case letters are used. The figures must be standardized.

Regarding: “There is a significant difference in R-wave am-171 plitude between the control group and the 26°C experimental group (p < 0.05).”. Again, how did the authors reach these conclusions? Which multiple comparisons test is used? The same question for the information presented between lines 175 and 182.

Regarding: “There is a negative correlation between rising temperature and the duration of these ECG intervals, demonstrating a shortening effect”, what is the value of the coefficient and what is the p-value? What coefficient is used?

The beginning of section 3.3 should be revised, given that you never begin a section with a table.

The p-value can never be “0.000” as indicated in table 1. “<0.001” must be entered.

The location of table 2 should be reviewed, as it is presented within the scope of the discussion of results and not within the scope of the presentation of results.

Still in table 2, what do the values ​​after the “±” sign in the 3rd column mean?

The authors refer in section “2.4. Statistical analysis” that: “The original ECG data were analyzed by correlation, regression and variance after preliminary processing.”. However, nowhere in the manuscript are the regression results presented. This information must be included and/or clarified.

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

Introduction:

- “Today, global warming poses a serious ecological challenge for humanity [1]. Ectothermic animals, such as fish, similarly encounter significant challenges from fluctuating environmental temperatures, which directly impact their cardiovascular function [2].”

There is a thematic disconnect between the two sentences: the first refers to humanity, while the second shifts focus to fish. To ensure smoother continuity, it would be more appropriate to delete "humanity" and retain only "ecological challenges."

The term "humanity" could be reintroduced later in the text, particularly in a section discussing comparative perspectives. This would allow for a more coherent exploration of potential analogies between the cardiovascular systems of fish and humans—an avenue worth investigating in future research.

- “Carps (Cyprinus carpio) are in the top ranks of China’s aquaculture output [28]. In the aquaculture market, carp is a commercially valuable fish. Among all freshwater fish, carp is the most extensively cultured, widely distributed, and diverse species of the Cyprinidae family.”

This sentence should be merged as there is a lot of repetition.

Discussion

- lines 290- 296: “Since cardiac output, defined as the blood volume pumped by the heart per unit time, is deter- 291 mined by heart rate and stroke volume, any increase in heart rate directly enhances the pumping performance of the heart. Therefore, within a certain temperature scope, the higher the temperature, the higher the heart rate. This increase in heart rate may be due to the elevated temperature leading to an increase in cardiac output to meet the higher oxygen demand.”

Please add appropriate references to this information.

 

Thank you for considering the comments and for the improvements made to the manuscript. However, it appears that some information is repeated multiple times throughout the manuscript. I would suggest condensing these sections to ensure a more focused and concise presentation of the content.

Comments on the Quality of English Language

The English could be improved to more clearly express the research.

Author Response

Please see the attachment. Thank you.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

Dear Authors

Thank you for your response. The manuscript is much improved.

I have no more additional comments.

Best regards.

 

Author Response

Thank you for your patient guidance and help, I wish you a successful work.

Reviewer 3 Report

Comments and Suggestions for Authors

The authors responded as intended.

Author Response

Thank you for your patient guidance and help, I wish you a successful work.