A Method for Estimating the Velocity at Which Anaerobic Metabolism Begins in Swimming Fish

Round 1

Reviewer 1 Report

I find the topic of this study of interest and important for analysing barriers to fish migration. However, the manuscript is not clear, and above all, it must be revised into English by a native speaker and undergo major edition. The objectives are poorly specified, and the methodology applied in the study is not clearly described. Results are not presented clearly, and the discussion, that is practically non-existent, needs to be improved. Therefore, in my opinion, this manuscript needs to undergo a major revision before considering it for publication.

Author Response

Comment 1：I find the topic of this study of interest and important for analyzing barriers to fish migration. However, the manuscript is not clear, and above all, it must be revised into English by a native speaker and undergo major edition. The objectives are poorly specified, and the methodology applied in the study is not clearly described. Results are not presented clearly, and the discussion, that is practically non-existent, needs to be improved. Therefore, in my opinion, this manuscript needs to undergo a major revision before considering it for publication.

Response：Thank you for your comment. We have made major revision to the manuscript: rewritten the abstract and discussion; adjusted the structure of the paper; made a lot of changes to the introduction; revised into English by a native speaker.

Reviewer 2 Report

General Comments:

This study provides an assessment of barriers, such as dams and sluices, used in waterways and their effects on fish metabolic patterns, notably swimming metabolism.  Overall the study is interesting and there is an ample amount of calculations that went into this work it appears. However, there are many aspects where critical details appear to have been overlooked and methodologies were not clearly explained. This is particularly true when it comes to the procedures used for carrying out the swim chamber respirometry studies. The authors seem as though they are presenting novel forms of analysis on these topics when in fact there is an ample amount of research that exists on this topic area which they have seemingly overlooked. The study also seems to present findings that in fact are very intuitive, yet they are presented as “major findings”, such as: oxygen consumption increases with swimming speed (yes, this has been documented for numerous species), tailbeat frequency increases as fish swim faster (yes, this is very intuitive and doesn’t warrant an entire figure or discussion).  It would be better for the authors to focus the discussion section on what the truly novel findings are and not waste time/text expounding on intuitive findings that are common amongst numerous fish species. Also, the manuscript overall needs to be edited thoroughly to resolve numerous grammar and syntax errors which are too numerous to enumerate in the following specific edits. This is an interesting study, and one that provides potentially useful information, but there are some major issues which need to be addressed prior to when/if this study would be published as noted in further comments that follow below.

Specific edits:

• The abstract is not good and needs to be significantly revised. It should be edited for English language grammar and syntax. Also, the abstract references “the calculation using the previously used formula”, yet there is no mention this previous formula. In many cases the syntax doesn’t make sense and it is unclear what the overall findings are of this study.
• Lines 36 – 37: “analyzing the migratory”. What does this mean?
• Line 49: scientific names of fish should be italicized and the word “juvenile” should not be capitalized.
• The source of the fish seems somewhat problematic, in that they were obtained from the local fish market 48 hrs before the testing procedures, and there is no indication of what the market holding conditions were for these fish. In many cases live fish markets hold fish in sub-optimal conditions, so this could definitely result in fish of poor quality being used as test subjects for this study. The authors state that the fish were then purged for 2 days prior to testing, but again it is unclear when the fish may have last eaten a meal if they were obtained from the market only 2 days earlier, purged over the course of 2 days, and then used in an experiment aimed at assessing metabolic and kinematic endpoints.
• Table 1: It is unclear whether all 31 individual fish experienced the same stepped-up swimming test, or if only some fish were used at different swimming speeds. These details need to be more clearly stated to avoid confusion, and depending on how the test was conducted the results could be interpreted rather differently.
• Lines 116 – 124: From the methods description it seems that intermittent respirometry was not used, but instead the DO and temperature were calculated at the start of the experiment and then measured every 5 minutes thereafter. Was there no flushing of the swim chamber to bring oxygen saturation of the water back to normal levels between swimming speed intervals? The methods of how the swim test was conducted need to be more clearly stated, as the current description sounds as though oxygen levels declined precipitously over the course of the experiment with no flushing.
• Figure 3 should likely appear only in “Supplementary Data” file, as such calibration figures are typically not included as primary figures in a manuscript.
• Lines 160 – 161: How did the authors determine when the fish was using aerobic or anaerobic metabolism? This needs to be defined clearly.
• In the Results section, and throughout the manuscript, swimming speeds should be represented in m/s AND in bodylengths/s (BL/s) to allow for easy comparison with other species in tested using similar methods in the literature.
• Based on Figure 5 it appears that the fish “failed” between 0.9 – 1.05 m/s swimming speed, and the oxygen consumption afterwards can likely be attributed to anaerobic metabolism. The authors do not note any evidence of a change in swimming pattern, as is typically observed when fish enter an anaerobic stage of metabolism when swimming such as the onset of burst-and-glide swimming in some species, and such evidence would help support the arguments being presented by the authors in this study.
• Line 357: The section titled “Patents” should instead be titled as “Acknowledgements” or “Author Contributions”. There are no “patents” being described, so the title does not make sense here.
• References: As noted previously, it is odd that with so much research having been conducted on the swimming of fish, swim chamber respirometry, and other aspects of fish bioenergetics that the authors have not included notable background references to support their study from such authors as: Steffensen et al., Brett and Groves (1979), Stieglitz et al. (2016, 2018), Mager et al. (2018), and many others. Many of these studies provide supporting evidence that could help explain the findings of the present research study whereas the authors only appear to include rather abstract references in their current study when in fact there is much more information out there on this topic than the authors mention in their study.

Author Response

Comment 1：The abstract is not good and needs to be significantly revised. It should be edited for English language grammar and syntax. Also, the abstract references “the calculation using the previously used formula”, yet there is no mention this previous formula. In many cases the syntax doesn’t make sense and it is unclear what the overall findings are of this study.

Response：Thank you for your comment. We have revised into English by a native speaker and rewritten the abstract as “Anaerobic metabolism begins before fish reach their critical swimming speed. Anaerobic metabolism affects the swimming ability of fish, which is not conducive to their upward tracking. The initiation of anaerobic metabolism therefore provides a better predictor of flow barriers than critical swimming speed. To estimate the anaerobic element of metabolism for swimming fish, the respiratory metabolism and swimming performance of adult crucian carp were tested in a closed tank. The swimming behavior and rate of oxygen consumption of these carp were recorded at various swimming speeds. Swimming costs were calculated via rate of oxygen consumption and hydrodynamic modeling. Then, the drag coefficient of the crucian carp during swimming was calibrated, and the velocity at which anaerobic metabolism was initiated method estimated, via the new method described herein. This study adds to our understanding of the metabolic patterns of fish at different swimming speeds.”.

Comment 2：Lines 36 – 37: “analyzing the migratory”. What does this mean?

Response：：We appreciated reviewer’s comment. The meaning is not clear and has been changed to: However, the flow velocity in the fishway is often high and could be a hydraulic barrier. It is critical to formulate a standardized approach to determining hydraulic barriers to improve the efficiency of fishway.

Comment 3：scientific names of fish should be italicized and the word “juvenile” should not be capitalized.

Response：We appreciated reviewer’s comment. We have been modified the scientific names of fish as required.

Comment 4：The source of the fish seems somewhat problematic, in that they were obtained from the local fish market 48 hrs before the testing procedures, and there is no indication of what the market holding conditions were for these fish. In many cases live fish markets hold fish in sub-optimal conditions, so this could definitely result in fish of poor quality being used as test subjects for this study. The authors state that the fish were then purged for 2 days prior to testing, but again it is unclear when the fish may have last eaten a meal if they were obtained from the market only 2 days earlier, purged over the course of 2 days, and then used in an experiment aimed at assessing metabolic and kinematic endpoints.

Response：Thank you for your comment. We apologize for not expressing clearly. All of the test fish were purchased at a time by the market staff from the fish farm and sent directly to the laboratory two days before the experiment. Have avoided the fish in sub-optimal conditions. I reorganized the language to make the source and feeding of the fish more clearly: “Fish. Crucian carp (n = 31, body mass = 260.10±7.93g, total length = 24.73 ±0.25cm, spawning period) were obtained from a fish farm in Wuhan, Hubei, China. The fish were immediately transferred to laboratory aquaria (diameter 2m, height 0.8m), where they acclimated for several days. They were fed daily and any remaining food was removed after 1 hr. Testing took place July 1 - August 10, 2019 and was carried out in Key Laboratory of Ecological Impacts of Hydraulic-Projects and Restoration of Aquatic Ecosystem of Minis-try of Water Resource, Wuhan, China., with room temperature controlled by air conditioning. The test fish were kept temporarily in the pool on site for 2d and not fed for the 24h period to being transferred, in water, to the respirometer [15]. The duration of each test was around 6 hours. Dissolved oxygen (DO) within the respirometer was maintained above 6.0mg/L [29], and the water temperature maintained at (20±1) ºC. When the DO fell below 6.0mg/L, testing was interrupted and the water was aerated [17]. Sample numbers and corresponding water velocities are shown in Table 1, below”.

Comment 5：Table 1: It is unclear whether all 31 individual fish experienced the same stepped-up swimming test, or if only some fish were used at different swimming speeds. These details need to be more clearly stated to avoid confusion, and depending on how the test was conducted the results could be interpreted rather differently.

Response：We appreciated reviewer’s comment. We add the sentence “Test fish swimming at various speed, when the speed reaches the critical swimming speed, the number of samples decreases” in Table 1 to avoid confusion.

Comment 6：Lines 116 – 124: From the methods description it seems that intermittent respirometry was not used, but instead the DO and temperature were calculated at the start of the experiment and then measured every 5 minutes thereafter. Was there no flushing of the swim chamber to bring oxygen saturation of the water back to normal levels between swimming speed intervals? The methods of how the swim test was conducted need to be more clearly stated, as the current description sounds as though oxygen levels declined precipitously over the course of the experiment with no flushing.

Response：We appreciated the reviewer’s comment. The methods of how the swim test was conducted have been more clearly stated: “At the same time, the DO (mg/L) and temperature in the respirometer were recorded every 5min for 25min. A fish was regarded as fatigued when the test fish stopped at the end of the test area, lightly patted the downstream wall for 20s and still cannot swim again [31]” and clarified as “When the DO fell below 6.0mg/L, testing was interrupted and the water was aerated [17]”.

Comment 7：Figure 3 should likely appear only in “Supplementary Data” file, as such calibration figures are typically not included as primary figures in a manuscript.

Response：We appreciated the reviewer’s comment. We have deleted the Figure 3 from manuscript and moved the Figure 3 to “Supplementary Data” file.

Comment 8：Lines 160 – 161: How did the authors determine when the fish was using aerobic or anaerobic metabolism? This needs to be defined clearly.

Response：We appreciated the reviewer’s comment. We have defined it clearly: “Studies have shown that and anaerobic metabolism starts at approximately 80% of the critical swimming speed. Therefore, we assumed that the water velocity is not greater than 0.45m/s (less than 60%U_crit), only aerobic respiration is performed in this test.”

Comment 9： In the Results section, and throughout the manuscript, swimming speeds should be represented in m/s AND in bodylengths/s (BL/s) to allow for easy comparison with other species in tested using similar methods in the literature.

Response：We appreciated the reviewer’s comment. The U_crit have been represented in m/s and in BL/s (0.85±0.032m/s,4.40±0.16 BL/s)

Comment 10：Based on Figure 5 it appears that the fish “failed” between 0.9 – 1.05 m/s swimming speed, and the oxygen consumption afterwards can likely be attributed to anaerobic metabolism. The authors do not note any evidence of a change in swimming pattern, as is typically observed when fish enter an anaerobic stage of metabolism when swimming such as the onset of burst-and-glide swimming in some species, and such evidence would help support the arguments being presented by the authors in this study.

Response：We thank reviewer’s comments. We are very sorry for note the evidence of a change in swimming pattern, in our paper, we focus on the energy model to estimate the velocity at which anaerobic starts, in the future research, we will pay more attention to the change of swimming pattern to support the arguments.

Comment 11：Line 357: The section titled “Patents” should instead be titled as “Acknowledgements” or “Author Contributions”. There are no “patents” being described, so the title does not make sense here.

Response：We appreciated the reviewer’s comment. We have deleted the title “Patents” and instead as “Author Contributions”.

Comment 12：References: As noted previously, it is odd that with so much research having been conducted on the swimming of fish, swim chamber respirometry, and other aspects of fish bioenergetics that the authors have not included notable background references to support their study from such authors as: Steffensen et al., Brett and Groves (1979), Stieglitz et al. (2016, 2018), Mager et al. (2018), and many others. Many of these studies provide supporting evidence that could help explain the findings of the present research study whereas the authors only appear to include rather abstract references in their current study when in fact there is much more information out there on this topic than the authors mention in their study.

Response：We appreciated the reviewer’s comment. We have added Steffensen and Brett’s studies to provide supporting evidence that help explain the findings. We have made major revise to introduction and discussion, many of other related studies have been referenced to provide supporting evidence to our research.

Reviewer 3 Report

This article tries to suggest a new method to determine the anaerobic component to fish swimming in the hopes of explaining performance through fish passageways and thus improve fish passageway design. While the idea is interesting, the execution of the study and especially the reporting of the results is lacking. I have identified major issues with the manuscripts detailed below.

Major Comments

1) There are already other methods to estimate anaerobic components of fish swimming, for example, estimations of EPOC - excessive post-exercise oxygen consumption. There are also measures that rely on quantification of lactic acid production and on oxygen delivery to the tissues. Thus, the claim that the authors say this is an innovative approach is incorrect.

2) The respirometry experiments are not well described and appear to not have been conducted using the standard techniques of measuring oxygen multiple times a minute and fitting a linear regression and reporting the fit of said regression. Thus, the oxygen estimates that the authors provide are likely fraught with error.

3) Fitting a linear regression to frequency of tail beat as a function of swimming speed is not a correct approach since it is clear that the relationship is asymptotic.

4) The literature on the subject is not sufficiently summarized in the introduction.

5) The manuscript needs to be edited for language and proofread by someone with both English and subject expertise to avoid the loss of information that comes from the lack of knowledge of some of the terms in English. This is particularly clear in the introduction and in the methods.

6) The purpose of the study was to study fish in passageways but all the work is very tangential to passageways. A better title and framing of the question would be appropriate, focusing on cost of transport estimates and oxygen consumption estimates. I am not sure I fully understand how the change in behavior while going through the passageway would change the metabolic requirements...

7) The discussion is not fully developed, especially in terms of the swimming kinematics.

8) There are no experimental determinations of drag, just empirical calculations based on multiple assumptions that cannot be guaranteed. There is no point in reporting this value or comparing it to other studies. An experimental determination of drag would be a much better fit for such a paper (e.g. https://www.nature.com/articles/s41598-018-30490-x)

9) There are no clear comparisons of the COT from this study to other similar studies.

10) Issues with COT and COS. Cost of transport does not include basal metabolism and it's a flaw to think that energy required for metabolic cost can be scale to the fish swimming speed and subtracted then. The energy expended to maintain the bodily functions derived from basal metabolic rate (BMR) is not going to change independently of swimming speed. Thus Figure 6B is a not correct and the rest of the paper hinges on it.

 

Author Response

Comment 1：There are already other methods to estimate anaerobic components of fish swimming, for example, estimations of EPOC - excessive post-exercise oxygen consumption. There are also measures that rely on quantification of lactic acid production and on oxygen delivery to the tissues. Thus, the claim that the authors say this is an innovative approach is incorrect.

Response：We thank reviewer’s comments. We are sorry for having used the sentence “there is currently no method to obtain the energy from anaerobic metabolism of live fish” Make you confused. We have used “The energy from anaerobic metabolism is hard to be estimated by swimming speed of live fish” instead of it to make the sentence express clearly.

Comment 2：The respirometry experiments are not well described and appear to not have been conducted using the standard techniques of measuring oxygen multiple times a minute and fitting a linear regression and reporting the fit of said regression. Thus, the oxygen estimates that the authors provide are likely fraught with error.

Response：We appreciated reviewer’s comment. The respirometry experiments are not well described. The respirometry experiments was based on Lee et al.(2002) and Cai et al.(2013). We have described respirometry experiments as: “during the test, water velocity was increased by 0.15m/s at 25-min intervals until the fish was fatigued, starting at 0.15m/s. At the same time, the DO and temperature in the respirometer were recorded every 5min for 25min (when the DO fell below 6.0mg/L, the test was interrupted and the water was aerated)”

Comment 3：Fitting a linear regression to frequency of tail beat as a function of swimming speed is not a correct approach since it is clear that the relationship is asymptotic.

Response：Thank you for your comment. We have found that the swimming speed was above U_crit did not correlate as well as the other speed. However, fitting a linear regression to frequency of tail beat as a function of swimming speed appeared in many papers (Cai et al.(2013), Tu et al.(2012), Steinhausen and Steffensen et al.(2005), Dickson et al.(2002)), we think the relationship of them was asymptotic cause by significantly reduced sample size at high velocities and we clarify it in the paper. Therefore, we have added the sentence “The deviation from the linear relationship and increased standard deviation apparent at higher velocities may be related to the significantly reduced sample sizes available at those velocities (Table 1)” in the conclusion to clarified it.

Comment 4：The literature on the subject is not sufficiently summarized in the introduction.

Response：We have made major revision to the paper and made a lot of changes to the introduction.

Comment 5：The manuscript needs to be edited for language and proofread by someone with both English and subject expertise to avoid the loss of information that comes from the lack of knowledge of some of the terms in English. This is particularly clear in the introduction and in the methods.

Response：We have made major revision to the manuscript and revised into English by a native speaker.

Comment 6：The purpose of the study was to study fish in passageways but all the work is very tangential to passageways. A better title and framing of the question would be appropriate, focusing on cost of transport estimates and oxygen consumption estimates. I am not sure I fully understand how the change in behavior while going through the passageway would change the metabolic requirements.

Response：We appreciated reviewer’s comment. The manuscript presents experiments that aim to estimate the velocity at which fish anaerobic metabolism begins, by evaluating swimming behavior, drag coefficients, and oxygen consumption and metabolism. We want to present a method for estimating the velocity at which anaerobic metabolism begin, but we fail to express it. We have made major revision to the manuscript and change the title to “A method for estimating the velocity at which anaerobic metabolism begins in swimming fish”.

Comment 7：The discussion is not fully developed, especially in terms of the swimming kinematics.

Response： We appreciated reviewer’s comment. We have rewritten the discussion especially in terms of swimming kinematics to make it fully developed.

Comment 8：There are no experimental determinations of drag, just empirical calculations based on multiple assumptions that cannot be guaranteed. There is no point in reporting this value or comparing it to other studies. An experimental determination of drag would be a much better fit for such a paper (e.g. https://www.nature.com/articles/s41598-018-30490-x)

Response：We appreciated reviewer’s comment. The paper reviewer recommends focus on the drag and drag reduction rate with no drag coefficient, our manuscript focus on the drag coefficient and drag calculate which similar to such paper (e.g. Videler et al. (1989) and Zobott et al. (2020)) and we have added the content that compare the value with their studies.

Comment 9：There are no clear comparisons of the COT from this study to other similar studies.

Response： We appreciated reviewer’s comment. We did not find the COT study of the experimental fish used. We added a comparison of the COT variation law of other fishes, and explained the reason for this law. We express it as: “the energy cost per unit distance COT reflects the energy efficiency status of the whole exercise process, the lower its value, the higher the energy efficiency. In this study, the energy cost per unit distance of crucian carp showed a decreasing trend with the in-crease of swimming speed because the contribution of standard metabolic rate to the whole oxygen consumption rate gradually decreased, which is similar to the research results of Cai et al and Steinhausen et al.”

Comment 10：Issues with COT and COS. Cost of transport does not include basal metabolism and it's a flaw to think that energy required for metabolic cost can be scale to the fish swimming speed and subtracted then. The energy expended to maintain the bodily functions derived from basal metabolic rate (BMR) is not going to change independently of swimming speed. Thus Figure 6B is a not correct and the rest of the paper hinges on it.

Response：We appreciated the reviewer’s comment. We have failed to express the energy cost relationship with swimming speed. We want to express that the energy consumption caused by swimming speed changes rather the expended to maintain the bodily functions derived from basal metabolic rate is going to change independently of swimming speed. In our test, the only variable is swimming speed, so we defined the energy consumption caused by swimming speed changes as the energy consumption at Us minus the energy consumption at still water. To make the express clearly, we deleted the parts of COS and express it as: “since the only variable in the test is swimming speed, the total mechanical power caused by the speed change can be obtained from the equation: P_s=(AMR-AMR₀)*m*13.54/3600, where AMR₀ (mg O²·h^-1·kg^-1) is the Oxygen consumed per hour per kilogram of body mass in still water.”

Reviewer 4 Report

Overview

In the manuscript Analysis of barriers in fishway based on fish metabolic patterns, the authors studied the swimming ability of the crucian carp in a closed tank. The swimming behavior and the oxygen consumption rate changes of the crucian carp were recorded under different swimming speeds, the drag coefficient during swimming was calibrated, and a method to identify the anaerobic metabolism initial velocity was proposed. Although the authors seek to relate the results to improving the efficiency of fishways, the study carried out on a respirometer, which establishes the control of variables, such as temperature, flow behavior, which does not occur in the natural environment of a fishway, because in addition to the hydraulic and environmental covariables vary with time and space, there are other covariables such as turbulence in the flow, which the apparatus cannot be resembled. In this sense, studies on respirometers do not make it possible to state what the authors highlight in the manuscript: “in order to analyze the water velocity barriers of fish upstream through fishway”. Thus, the manuscript is valid and restricted to subsidize information for the elaboration of fishway design most suitable for the species of the study: the crucian carp.

The manuscript is well written, and it has as its strong point the planning and development of the experimental design. However, the hypotheses and objectives of the study are not clearly defined, as well as the justifications and the importance for species (potamodromous), regarding the biological requirements in the use of fishways. The authors must include information with the description (scientific name, classification, geographical distribution, etc.), habitat, the biological needs that justify the use of fishway, and whether it is considered a target species.

Also, many Results data can be found in the Discussion. It is very poor and needs to be compared with other studies carried out with the use of a respirometer. Authors should substantially improve this topic to avoid compromising the final acceptance.

In addition, there are specific corrections to be made throughout the manuscript that are commented below.

Specific comments

Title

The study was carried out on a respirometer, a limited apparatus to affirm that the results obtained may refer to Analyzes of barriers in fishway, as stated in the title of the manuscript. I recommend an adjustment of the title, i.e. Optimal swim speeds by respirometer... 

Summary

The objectives of the study are not mentioned in the abstract and the methodologies used to answer each hypothesis. The authors should rewrite the Summary due to suggestions for the adjustments of the manuscript. 

Introduction

Lines 66-72: Authors must include the objectives and hypotheses of the study. Also, make it clear why the use of this species in the experiment. Is it a migratory species? If not, why did you use it? Insert information about the species: biological description (scientific name, classification, geographical distribution, etc.), habitat, reproduction. Include photo.

Line 73: The study was tested on a respirometer and not on a flume - open channel type (because it is an open conduit and provides an environment close to reality). Clarify that the respirometer (closed conduit) has limitations to represent reality.

Material and methods

Authors must include information about where the study was conducted (country, University or Institute, the name of the laboratory).

Line 101: Was PIV used to measure flow velocities? Clarify it.

Lines 103-104: Was this apparatus used to evaluate and quantify the TAIL BEAT FREQUENCY (TBF)? Describe it.

Line 117: Describe in detail how it was carried out.

Line 120: DO = Dissolved Oxygen? What is the unit? mg / L or ppm? Was oxygen saturation (%) also measured? It can be estimated from temperature, atmospheric pressure and DO. It is essential to include in the results the physical and chemical parameters of the experiment.

Line 159: Is this reference correct? Do the alpha and beta values refer specifically to the species studied, crucian carp? Clarify it.

Discussion

The discussion is not too convincing. In part the discussion repeats the results in other words rather than discussing the findings. Remove the repetitive elements of results and discuss instead critically address the finding in relation to swimming behavior and the oxygen consumption rate in crucian carp.

Some papers are essential to be considered in the manuscript (introduction and discussion), and they are listed below:

Brett, J. R. (1964). The respiratory metabolism and swimming performance of Young sockeye Salmon. Journal of the Fisheries Research Board of Canada, 21(5), 1183-1226. https://doi.org/10.1139/f64-103.

Clark, T. D., Sandblom, E., & Jutfelt, F. (2013). Aerobic scope measurements of fishes in an era of climate change: Respirometry, relevance and recommendations. Journal of Experimental Biology, 216(15), 2771-2782. https://doi.org/10.1242/jeb.084251.

Liao, J. C. 2007. A review of fish swimming mechanics and behaviour in altered flows. Phil. Trans. R. Soc. B362, 1973–1993. http://doi.org/10.1098/rstb.2007.2082

Peake, S. J., & Farrell, A. P. (2006). Fatigue is a behavioural response in respirometer-confined smallmouth bass. Journal of Fish Biology, 68(6), 1742-1755. https://doi.org/10.1111/j.0022-1112.2006.01052.x.

Rodgers GG, Tenzing P, Clark TD. Experimental methods in aquatic respirometry: the importance of mixing devices and accounting for background respiration. J Fish Biol. 2016 Jan;88(1):65-80. doi: 10.1111/jfb.12848. PMID: 26768972.

Santos, Hersília de Andrade e, Viana, Edna Maria de Faria, Pompeu, Paulo Santos, & Martinez, Carlos Barreira. (2012). Optimal swim speeds by respirometer: an analysis of three neotropical species. Neotropical Ichthyology, 10(4), 805-811. https://dx.doi.org/10.1590/S1679-62252012000400013

Zhang Y, Gilbert MJH, Farrell AP. Measuring maximum oxygen uptake with an incremental swimming test and by chasing rainbow trout to exhaustion inside a respirometry chamber yields the same results. J Fish Biol. 2020 Jul;97(1):28-38. doi: 10.1111/jfb.14311. Epub 2020 Apr 6. PMI

Author Response

Comment 1：The study was carried out on a respirometer, a limited apparatus to affirm that the results obtained may refer to Analyzes of barriers in fishway, as stated in the title of the manuscript. I recommend an adjustment of the title, i.e. Optimal swim speeds by respirometer

Response：We appreciated reviewer’s comment. The manuscript aimed to estimate the velocity at which fish anaerobic metabolism begins, by evaluating swimming behavior, drag coefficients, and oxygen consumption and metabolism. We want to present a method for estimating the velocity at which anaerobic metabolism begin, but we fail to express. The title “optimal swim speeds by respirometer” is more suitable than the original. But after we made major revision to the manuscript, we think the title “A method for estimating the velocity at which anaerobic metabolism begins in swimming fish” may a better summary of the manuscript. Anyway, thank you for yours recommend.

Comment 2：The objectives of the study are not mentioned in the abstract and the methodologies used to answer each hypothesis. The authors should rewrite the Summary due to suggestions for the adjustments of the manuscript

Response：We appreciated reviewer’s comment. We rewritten the abstract as: “Anaerobic metabolism has started before fish swim at the critical swimming speed. Anaerobic metabolism will affect the swimming ability of fish, which is not conducive to the upward tracking of fish. Therefore, anaerobic metabolic is a better predictor of flow barriers than critical swimming speed. To estimate anaerobic parts of fish swimming, the respiratory metabolism and swimming performance of adult crucian carp were tested in a closed tank. The swimming behavior and the oxygen consumption rate change of the crucian carp were recorded under different swimming speeds. Swimming costs were both calculated using oxygen consumption rate and hydrodynamic model. Then, the drag coefficient of the crucian carp during swimming was calibrated, and a method to estimate the anaerobic metabolism initial velocity was proposed. This study helps to understand the metabolic patterns of fish at different swimming speeds.”

Comment 3：Lines 66-72: Authors must include the objectives and hypotheses of the study. Also, make it clear why the use of this species in the experiment. Is it a migratory species? If not, why did you use it? Insert information about the species: biological description (scientific name, classification, geographical distribution, etc.), habitat, reproduction. Include photo.

Response：We appreciated reviewer’s comment. The manuscript aimed to estimate the velocity at which fish anaerobic metabolism begins, by evaluating swimming behavior, drag coefficients, and oxygen consumption and metabolism. The method to estimate the anaerobic metabolism initial velocity was proposed and the method is also applicable to other fish. Therefore, there is no focus on describing the characteristics of the test fish.

Comment 4：Line 73: The study was tested on a respirometer and not on a flume-open channel type (because it is an open conduit and provides an environment close to reality). Clarify that the respirometer (closed conduit) has limitations to represent reality.

Response：We appreciated reviewer’s comment. We have expressed it as: “to obtain the necessary parameters of metabolism in relation to swimming speed, estimating the rate of oxygen consumption over a series of increasing velocity steps in a closed tank which can provide ample space for free swimming without sacrificing sensitivity to oxygen change by involving too large a volume of circulating water.”

Comment 5：Authors must include information about where the study was conducted (country, University or Institute, the name of the laboratory).

Response：We appreciated reviewer’s comment. We have added the sentence “the test was carried out in Key Laboratory of Ecological Impacts of Hydraulic-Projects and Restoration of Aquatic Ecosystem of Ministry of Water Resource, Wuhan, China” in the materials and methods.

Comment 6：Line 101: Was PIV used to measure flow velocities? Clarify it.

Response：We appreciated reviewer’s comment. It was used to measure water velocity and established the relationship between the adjustment frequency of the variable-speed motor x and water velocity y at the beginning. We clarified it as “. Water velocity in the respirometer was controlled by a propeller driven by a variable-speed motor; this was calibrated using a LaVision Particle Image Velocimetry system (PIV)” and “The relationship between the frequency setting of the variable-speed motor, x, and water velocity, y, was calibrated using the LaVision Particle Image Velocimetry system (PIV)”.

Comment 7：Lines 103-104: Was this apparatus used to evaluate and quantify the TAIL BEAT FREQUENCY (TBF)? Describe it.

Response：Yes, it was. We have added the sentence “Video was recorded during the test and the tail beat frequency could be obtained through frame-by-frame analysis by ProAnaly software” to describe it.

Comment 8：Line 117: Describe in detail how it was carried out

Response：We appreciated reviewer’s comment. We have described in detail how it was carried out in the “Morphometric and Statistical analysis”.

Comment 9：Line 120: DO = Dissolved Oxygen? What is the unit? mg / L or ppm? Was oxygen saturation (%) also measured? It can be estimated from temperature, atmospheric pressure and DO. It is essential to include in the results the physical and chemical parameters of the experiment.

Response：We appreciated reviewer’s comment. We have added the “Dissolved Oxygen” and the unit “mg / L” in the sentence. The oxygen saturation (%) was not measured, we focus on the energy cost and drag calculated.

Comment 10：Line 159: Is this reference correct? Do the alpha and beta values refer specifically to the species studied, crucian carp? Clarify it.

Response：We appreciated reviewer’s comment. The equation, the alpha and beta values was first proposed in Web’s(1971) paper, the reference we used was not suitable, we used Web’s(1971) paper instead of it. We have clarified alpha and beta values refer specifically to the salmons in the paper and the effect of A_s was excluded by measuring the surface area of the fish.

Comment 11：The discussion is not too convincing. In part the discussion repeats the results in other words rather than discussing the findings. Remove the repetitive elements of results and discuss instead critically address the finding in relation to swimming behavior and the oxygen consumption rate in crucian carp. Some papers are essential to be considered in the manuscript (introduction and discussion), and they are listed below

Response：We appreciated reviewer’s comment. We have made major revisions to the introduction and discussion parts, and carefully read your recommended papers, citing some of the papers related to our research.

Reviewer 5 Report

Please see attached file. 

Comments for author File: Comments.pdf

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

The comments raised in the first revision were satisfactorily answered by the authors. However, there are a few corrections, which are listed below, that authors should rectify.

 

SPECIFIC COMMENTS:

Abstract - Line 19 – The scientific name of crucian carp should be added.

Abstract - Line 22-24 – Do you mean “Then, …, and the velocity at which anaerobic metabolism was initiated was estimated, via the new method described herein.” – Please clarify.

Line 31 – References – The formatting of the references is not consistent with the journal's instructions. Please change it accordingly throughout the manuscript.

Line 59 – The scientific name of sturgeon should be added.

Line 61 – Please add “respectively” in “… (42.75 versus 25.85J kg-1 m-1, respectively).”.

Line 80 – A comma should be added to “… closed tank, which provides ample space for free swimming, has an …”.

Line 105 – “… acclimated for several days.” – How many days? This should be specified.

Line 112 – Change to “… at 20(±1)ºC.”

Line 114 – Delete “, below”.

Table 1 – Instead of adding the sentence to the table, consider adding it to the caption, e.g., “Table 1. Number of samples at each stage. When velocity (m/s) reaches the critical swimming speed, the number of samples decreases.”.  

Line 135 – Consider change “… fish was regarded as fatigued when the test fish …” to “… fish was considered as fatigued when it …”.

Line 163-165 and Equations 5-8 – You should mention, and present, equations in order.

Line 175 – Delete “Thus,”.

Line 183 – “The error bars in figures below are represent standard errors” – Delete “are”.

Line 187 – Consider change to “The crucian carp tested under 20±1ºC water temperature conditions had a total length…”.

Line 196 – Move “(Figure 4)” to “… rate of oxygen consumption and swimming speed (Figure 4) …”.

Line 200-201 – “As water velocity increased, the rate of oxygen consumption also increased. However, the rate of increase varied over velocity intervals.” – Consider change to “As water velocity increased, the rate of oxygen consumption also increased, varying over velocity intervals.”.

Line 282 – Add “be” to “…fish can be calculated using …”.  

Line 288 – Delete “with”.

Line 303 – “The efficiency of fish in nature exceeds 90% [36].” – This was mentioned before, please delete.

Line 326 – “carp” is misspelled.

Line 359 – Change “ie” to “i.e.,”. Revise throughout the manuscript.

Author Response

Comment 1：Abstract - Line 19 – The scientific name of crucian carp should be added.

Response：Thank you for your comment. We have added the scientific name of crucian carp as “To estimate the anaerobic element of metabolism for swimming fish, the respiratory metabolism and swimming performance of adult crucian carp (Carassius auratus) were tested in a closed tank”.

Comment 2：Abstract - Line 22-24 – Do you mean “Then, …, and the velocity at which anaerobic metabolism was initiated was estimated, via the new method described herein.” – Please clarify.

Response：Thank you for your comment. We apologize for not expressing clearly.

The sentence has been revised to “Then, the drag coefficient of the crucian carp during swimming was calibrated, and the velocity at which anaerobic metabolism was initiated was estimated, via the new method described herein”.

Comment 3：Line 31 – References – The formatting of the references is not consistent with the journal's instructions. Please change it accordingly throughout the manuscript.

Response：Thank you for your comment. We have changed it accordingly throughout the manuscript.

Comment 4：Line 59 – The scientific name of sturgeon should be added.

Response：Thank you for your comment. We have added the scientific name of crucian carp as “Thiem et.al [1] did not find a difference in the rate of energy expenditure between sturgeon (Acipenser fulvescens)”.

Comment 5：Line 61 – Please add “respectively” in “… (42.75 versus 25.85J kg-1 m-1, respectively).”

Response：Thank you for your comment. We have added “respectively” in this sentence as “(42.75 versus 25.85J kg^-1 m^-1, respectively)”.

Comment 6：Line 80 – A comma should be added to “… closed tank, which provides ample space for free swimming, has an …”.

Response：Thank you for your comment. We have added a comma in this sentence as “To obtain the necessary metabolic parameters in relation to swimming speed, estimating the rate of oxygen consumption at series of increasing velocities within a closed tank, which provides ample space for free swimming has an important advantage: specifically, this avoids the reduced sensitivity to changes in oxygen levels that occur if the volume of circulating water is excessively large”.

Comment 7：Line 105 – “… acclimated for several days.” – How many days? This should be specified.

Response：Thank you for your comment. All test fish were purchased at once two days before the test and one fish was test every day. We have revised it as “The fish were immediately transferred to laboratory aquaria (diameter 2.0m, height 0.8m), where they acclimated for at least two days”.

Comment 8：Line 112 – Change to “… at 20(±1)ºC.”

Response：Thank you for your comment. We have changed it to “… at 20(±1)ºC”.

Comment 9：Line 114 – Delete “, below”.

Response：Thank you for your comment. We have delete the “, below”.

Comment 10：Table 1 – Instead of adding the sentence to the table, consider adding it to the caption, e.g., “Table 1. Number of samples at each stage. When velocity (m/s) reaches the critical swimming speed, the number of samples decreases.”.

Response：Thank you for your comment. We have revised it as “Table 1. Number of samples at each stage. When velocity (m/s) reaches the critical swimming speed, the number of samples naturally decreases”.

Comment 11：Line 135 – Consider change “… fish was regarded as fatigued when the test fish …” to “… fish was considered as fatigued when it …”.

Response：Thank you for your comment. We have revised it as “A fish was considered as fatigued when it stopped at the end of the test area, lightly patted the downstream wall for 20s and still cannot swim again”.

Comment 12：Line 163-165 and Equations 5-8 – You should mention, and present, equations in order.

Response：Thank you for your comment. We have mentioned and presented the equations in order.

Comment 13：Line 175 – Delete “Thus,”.

Response：Thank you for your comment. We have deleted it.

Comment 14：Line 183 – “The error bars in figures below are represent standard errors” – Delete “are”.

Response：Thank you for your comment. We have deleted it.

Comment 15：Line 187 – Consider change to “The crucian carp tested under 20±1ºC water temperature conditions had a total length…”.

Response：Thank you for your comment. We have revised it as “ The crucian carp tested under 20±1ºC water temperature conditions had a total length of 24.73±0.25cm, body length of 19.32±0.24cm, and body mass of 260.10±7.93g. Their U_crit was 0.85±0.032m/s (4.40±0.16 body lengths, BL/s)”.

Comment 16：Line 196 – Move “(Figure 4)” to “… rate of oxygen consumption and swimming speed (Figure 4) …”.

Response：Thank you for your comment. We have revised it as “ The relationship between rate of oxygen consumption and swimming speed (Figure 4)”.

Comment 17：Line 196 – Move “(Figure 4)” to “… rate of oxygen consumption and swimming speed (Figure 4) …”.

Response：Thank you for your comment. We have revised it as “The relationship between rate of oxygen consumption and swimming speed (Figure 4)”.

Comment 18：Line 200-201 – “As water velocity increased, the rate of oxygen consumption also increased. However, the rate of increase varied over velocity intervals.” – Consider change to “As water velocity increased, the rate of oxygen consumption also increased, varying over velocity intervals.”.

Response：Thank you for your comment. We have revised it as “As water velocity increased, the rate of oxygen consumption also increased, varying over velocity intervals”.

Comment 19：Line 282 – Add “be” to “…fish can be calculated using …”.

Response：Thank you for your comment. We have revised it as “The drag force on a steadily moving fish can be calculated using the hydrodynamic model”.

Comment 20：Line 288 – Delete “with”.

Response：Thank you for your comment. We have revised it as “Under turbulent conditions, C_d gradually decreases as Re increases, approximately in proportion with Re^-0.2”.

Comment 21：Line 303 – “The efficiency of fish in nature exceeds 90% [36].” – This was mentioned before, please delete.

Response：Thank you for your comment. We have deleted it.

Comment 22：Line 326 – “carp” is misspelled.

Response：Thank you for your comment. We have revised it as “It was found that the drag power, P_d, of the crucian carp began to exceed their aerobic power”.

Comment 23：Line 359 – Change “ie” to “i.e.,”. Revise throughout the manuscript.

Response：Thank you for your comment. We have revised it throughout the manuscript.

Reviewer 3 Report

I appreciate the effort the authors put into addressing my comments and those of the other reviewers. The manuscript is indeed improved. There are some issues that still need to be addressed before publication.

Major comments:

Another pass through the whole manuscript by a fluent English speaker would be great to make sure all sentences are conveying the information that they were set to be.

Minor comments:

Table 1 - the added text should be either in the caption or as a note.

 

Author Response

Comment 1：Another pass through the whole manuscript by a fluent English speaker would be great to make sure all sentences are conveying the information that they were set to be.

Response：Thank you for your comment. We have invited a fluent English speaker pass through the whole manuscript.

Comment 2：Table 1 - the added text should be either in the caption or as a note.

Response：We have revised it as “Table 1. Number of samples at each stage. When velocity (m/s) reaches the critical swimming speed, the number of samples decreases”.

Reviewer 5 Report

I thank the authors for revising their manuscript according to reviewers’ comments. However, several important issues remain, which ought to be addressed prior to publication.

1. The abstract does not mention any findings of the study, nor does it present or summarise the proposed method, which should be the highlight of the paper and abstract.
2. Line 105: please be precise and state how many days.
3. Table 1: please place the sentence in the table caption. Was the sample number decreased intentionally or was it that the number of fish able to swim past the critical velocity naturally decreased?
4. A PIV system is mentioned several times in the methodology, yet no details are provided. The authors responded to my request of including details of the PIV saying that a description isn't necessary, but it is essential for completeness of their methodology. Even if omitted for brevity, the authors could refer to other publication where it is described in detail.
5. Line 191: It seems that using a polynomial fit, or a function other than linear, would better follow the trend of your data at higher velocities? The relationship between swimming speed and tail beat frequency is not necessarily always linear.
6. Statistics and sample size: I had suggested that the authors perform further statistical analysis to ensure the differing sample sizes do not skew the results, particularly when fitting equations onto the data. This has not been addressed besides acknowledging the poor fit of the equations at higher velocities, which are observed in figures 3, 4 and 5. A simple ANOVA analysis would quantify the sample size effects and inform whether other functions fit onto the data better than currently presented. Deriving a method including some poorly fitted data is unsatisfactory.
7. Figure 6: the caption refers to two blue solid lines, are they the same? Please use other colours to distinguish the data. Please also add all the figure's data series and lines in the figure legend.
8. Section 4.1: is Li et al. the only other study of crucian carp swimming performance to compare to? Also please refer as [last name] et al. not both last and first name.
9. Lines 291-295: this is confusing. Was the measured As used or not? please clarify.
10. Please revise this new text in the discussion section to remove repetitions, eg. "The efficiency of fish in nature exceeds 90% "
11. Lines 309-312: Why assume 60%Ucrit while stating that studies have shown that it is 80%Ucrit?
12. Lines 317-321: Please be specific about the what being compared. Authors response to comment 67: "... The drag coefficient is not related to surface area but the shape. ..." This is not true. Drag coefficient depends on both shape and size. Please see the classic equation you present in your manuscript (equation 4) F=0.5CdρAsUs², which is also expressed as: Cd=2F/ρAsUs². Note that Cd depends on the area, used either as a projected area or surface area.
13. Line 339: please specify that this is for the species tested at your chosen temperature, and indicate how it might change for other species and environments.
14. Line 399: please check this reference

Author Response

Comment 1：The abstract does not mention any findings of the study, nor does it present or summarise the proposed method, which should be the highlight of the paper and abstract.

Response：We appreciated reviewer’s comment. We have revised it as “anaerobic metabolism begins before fish reach their critical swimming speed. Anaerobic metabolism affects the swimming ability of fish, which is not conducive to their upward tracking. The initiation of anaerobic metabolism therefore provides a better predictor of flow barriers than critical swimming speed. To estimate the anaerobic element of metabolism for swimming fish, the respiratory metabolism and swimming performance of adult crucian carp (Carassius auratus, mass = 260.10±7.93, body length = 19.32±0.24) were tested in a closed tank at 20±1ºC. The swimming behavior and rate of oxygen consumption of these carp were recorded at various swimming speeds. Results indicate: (1) The critical swimming speed of the crucian carp was 0.85±0.032m/s (4.40±0.16 BL/s). (2) When a power function was fitted to the data, oxygen consumption, as a function of swimming speed, was determined to be AMR = 131.24 + 461.26U_s^1.27 (R² = 0.948, P < 0.001) and the power value (1.27) of U_s indicated high swimming efficiency. (3) Increased swimming speed led to increases in the tail beat frequency. (4) Swimming costs were calculated via rate of oxygen consumption and hydrodynamic modeling. Then, the drag coefficient of the crucian carp during swimming was calibrated (0.126-0.140), and the velocity at which anaerobic metabolism was initiated was estimated (0.52m/s), via the new method described herein. This study adds to our understanding of the metabolic patterns of fish at different swimming speeds.”

Comment 2：Line 105: please be precise and state how many days.

Response：Thank you for your comment. All test fish were purchased at once two days before the test and one fish was test every day. We have revised it as “The fish were immediately transferred to laboratory aquaria (diameter 2.0m, height 0.8m), where they acclimated for at least two days”.

Comment 3：Table 1: please place the sentence in the table caption. Was the sample number decreased intentionally or was it that the number of fish able to swim past the critical velocity naturally decreased?

Response：Thank you for your comment. We have revised it as “Table 1. Number of samples at each stage. When velocity (m/s) reaches the critical swimming speed, the number of samples naturally decreases”.

Comment 4：A PIV system is mentioned several times in the methodology, yet no details are provided. The authors responded to my request of including details of the PIV saying that a description isn't necessary, but it is essential for completeness of their methodology. Even if omitted for brevity, the authors could refer to other publication where it is described in detail.

Response：We appreciated reviewer’s comment. We have referred to other publication as “the details were described in Tarrade’s paper[2]”.

Comment 5：Line 191: It seems that using a polynomial fit, or a function other than linear, would better follow the trend of your data at higher velocities? The relationship between swimming speed and tail beat frequency is not necessarily always linear.

Response：We appreciated reviewer’s comment. The relationship between swimming speed and tail beat frequency is not necessarily always linear. However, in this paper, the linear fitting is better at low velocity, and at high velocity, it may be caused by the reduction of samples, so linear fitting may better reflect the relationship.

Comment 6：Statistics and sample size: I had suggested that the authors perform further statistical analysis to ensure the differing sample sizes do not skew the results, particularly when fitting equations onto the data. This has not been addressed besides acknowledging the poor fit of the equations at higher velocities, which are observed in figures 3, 4 and 5. A simple ANOVA analysis would quantify the sample size effects and inform whether other functions fit onto the data better than currently presented. Deriving a method including some poorly fitted data is unsatisfactory.

Response：We appreciated reviewer’s comment. We have referred other publication to statistical analysis (Beamish, F., Swimming capacity[J]. Fish Physiology and Biochemistry, 1978. VII  https://doi.org/10.1007/978-1-4613-2073-9_20; Ohlberger, J., G. Staaks, F. Holker, Swimming efficiency and the influence of morphology on swimming costs in fishes[J]. Journal of Comparative Physiology B-Biochemical Systemic and Environmental Physiology, 2006. 176(1): p. 17-25.  https://doi.org/10.1007/s00360-005-0024-0; Sepulveda, C.A., J.B. Graham, D. Bernal, Aerobic metabolic rates of swimming juvenile mako sharks, Isurus oxyrinchus[J]. Marine Biology, 2007. 152(5): p. 1087-1094.  https://doi.org/10.1007/s00227-007-0757-2; Cai, L., et al., Swimming Capability and Swimming Behavior of Juvenile Acipenser schrenckii[J]. Journal of Experimental Zoology Part a-Ecological Genetics and Physiology, 2013. 319A(3): p. 149-155.  https://doi.org/10.1002/jez.1780;) and use the most relevant function to fit as much as possible.

Comment 7：Figure 6: the caption refers to two blue solid lines, are they the same? Please use other colours to distinguish the data. Please also add all the figure's data series and lines in the figure legend.

Response：We appreciated reviewer’s comment. We are sorry for mistake and have revised it as “Drag, F, and efficiency, η, of crucian carp at different swimming speeds. Drag of fish (F, mean) as function of swimming speed (Function: F = 0.0055 + 0.13Us, R² = 0.998, P < 0.001, black solid line). Trend of F relative to Us (black dotted line). Trend of η relative to U_s (blue solid line)”. The black dotted line was used to reflect the trend of the relationship of F and velocity and black solid line was used to describe the fitting relationship, they share a set of data.

Comment 8：Section 4.1: is Li et al. the only other study of crucian carp swimming performance to compare to? Also please refer as [last name] et al. not both last and first name.

Response：We appreciated reviewer’s comment. We have revised it as “The critical swimming speed of crucian carp determined by Li et al [3] was 1.19±0.024m/s (5.91±0.09 BL/s, with body length of 20.0±0.80cm). The fish used in both this and Li’s study were crucian carp of similar body length, but their source, habitat/environment and living habits were quite different.” We have only found Li’s test fish is the same as we used.

Comment 9：Lines 291-295: this is confusing. Was the measured As used or not? please clarify.

Response：We appreciated reviewer’s comment. We have revised it as “However, the incorrectness of A_s was excluded by using the measured surface area of the fish.”.

Comment 10：Please revise this new text in the discussion section to remove repetitions, eg. "The efficiency of fish in nature exceeds 90% "

Response：We appreciated reviewer’s comment. We have deleted the repetitions.

Comment 11：Lines 309-312: Why assume 60%Ucrit while stating that studies have shown that it is 80%Ucrit?

Response：We appreciated reviewer’s comment. Studies have shown that anaerobic metabolism starts at approximately 80% of the critical swimming speed, so it was reasonable to assume that only aerobic metabolism when swimming speed lower than 60%Ucrit.

Comment 12：Lines 317-321: Please be specific about the what being compared. Authors response to comment 67: "... The drag coefficient is not related to surface area but the shape. ..." This is not true. Drag coefficient depends on both shape and size. Please see the classic equation you present in your manuscript (equation 4) F=0.5CdρAsUs², which is also expressed as: Cd=2F/ρAsUs². Note that Cd depends on the area, used either as a projected area or surface area.

Response：We appreciated reviewer’s comment. We are sorry for not explain it clearly last time. The drag coefficient is not related to surface area but the shape. F=0.5CdρAsUs², which is not expressed as: Cd=2F/ρAsUs². The Cd=f (shape, Re, α), it is a dimensionless number, related to shape, Re and α (angle of attack) (Xin, Z., et al., Hydraulics[B]. 2009: China Electric Power Press. 133-137.). To specific about what being compared, we have revised it as “but smaller than the drag coefficient (0.45) for flow past a round sphere at the corresponding Reynolds number”.

Comment 13：Line 339: please specify that this is for the species tested at your chosen temperature, and indicate how it might change for other species and environments.

Response：We appreciated reviewer’s comment. We have revised it to “An approximate estimate of the carp initial velocity under 20±1ºC water temperature conditions for anaerobic metabolism of 0.52m/s was obtained, a finding consistent with the observation that excess post-exercise oxygen costs begin to be observed in the range of 60-80% U_crit [4]. This value may be different for different temperatures, species and environments.”.

Comment 14：Line 399: please check this reference.

Response：We appreciated reviewer’s comment. We have revised it to “Atique, U., WHY THERE ARE FEWER FISH UPSTREAM?[B]. 2017. 26-32.  http://doi.org/10.13140/RG.2.2.18056.93446”.