Effects of Water Temperature on Growth, Hematological Measurements and Stress-Related Gene Expression of Atlantic Salmon (Salmo salar) Parr Reared in a Recirculating Aquaculture System
Round 1
Reviewer 1 Report
Comments and Suggestions for AuthorsComments on the manuscript with ID (animals-3858625-peer-review-v1): -
References: - Latin names should be written in italic.
Line 92: The authors should add the physical and chemical water properties before the start of the experiment.
Line 94: This step needs details. How did you acclimate the fish to different water temperatures? This is not simply written as you wrote.
Table 1: Add GE, NPE and ME values.
Lines 106-107: Add an appropriate reference to the dose used for inducing anesthesia. Source of MS-222 used should be added (City, Company name and country).
Line 110: Add the equations used.
Lines 112-115: Add appropriate references used for the methodologies used.
Lines 119-120 and Lines (124-127): Add codes of kits used for biochemical measurements.
Line 137: Add appropriate reference for the methodology used.
Livak, K. J., & Schmittgen, T. D. (2001). Analysis of relative gene expression data using real-time quantitative PCR and the 2− ΔΔCT method. Methods, 25(4), 402-408.
Table 2: Add primer efficiency, R2 and Pearson’s coefficient.
Comments for author File: 
Comments.pdf
Author Response
Reviewer 1
We sincerely thank the reviewer for their valuable comments and suggestions, which have helped improve the quality of our manuscript. Below, we provide a detailed response (highlighted in yellow in the manuscript) to each point raised.
Comments and Suggestions for Authors
Comment 1: References: - Latin names should be written in italic.
Response 1: In the references, the scientific name of the species has been italicized where necessary.
Comment 2: Line 92: The authors should add the physical and chemical water properties before the start of the experiment.
Response 2: We added the physical and chemical water properties during the acclimation period following the suggestion. (please see lines 107-109)
Comment 3: Line 94: This step needs details. How did you acclimate the fish to different water temperatures? This is not simply written as you wrote.
Response 3: The detailed steps to acclimate the fish to the target water temperatures have been included following the suggestion. (please see lines 110-114)
Comment 4: Table 1: Add GE, NPE and ME values.
Response 4: We have included the values of GE and NPE in Table 1 in accordance with the suggestion (please see Table 1). As we did not measure the apparent digestibility of the diet used in this study, we are unable to provide ME values at this time. We will consider measuring ADC in future studies to provide more detailed information for readers.
Comment 5: Lines 106-107: Add an appropriate reference to the dose used for inducing anesthesia.
Response: The relevant information on anesthesia for Atlantic salmon has been included following the suggestion. (please see lines 133-135)
Comment 6: Source of MS-222 used should be added (City, Company name and country).
Response 6: The source of the anesthetic has been included following the suggestion. (please see line 132)
Comment 7: Line 110: Add the equations used.
Response 7: The equations used for each of the measurements have been included following the suggestion. (please see lines 138-147)
Comment 8: Lines 112-115: Add appropriate references used for the methodologies used.
Response 8: The relevant references used for the methodologies have been provided following the suggestion. (please see lines 150-154)
Comment 9: Lines 119-120 and Lines (124-127): Add codes of kits used for biochemical measurements.
Response 9: The relevant information on the kits used for the assays have been provided following the suggestion. (please see lines 159-161; 163-164)
Comment 10: Line 137: Add appropriate reference for the methodology used. Livak, K. J., & Schmittgen, T. D. (2001). Analysis of relative gene expression data using real-time quantitative PCR and the 2− ΔΔCT method. Methods, 25(4), 402-408.
Response 10: The relevant reference has been included following the suggestion. (please see citation number 30)
Comment 11: Table 2: Add primer efficiency, R2 and Pearson’s coefficient.
Response 11: The values for primer efficiency, R2, and Pearson’s coefficient have been provided in the Table 2. (please see Table 2)
Reviewer 2 Report
Comments and Suggestions for AuthorsPlease write a chapter with the conclusions of this study, and I also think it would be useful to do more detailed research with citations from several current scientific studies related to the growth of Atlantic salmon (Salmo salar) in RAS (38 references for a journal like Animals are not enough), then resend it for review.
Author Response
Reviewer 2
We sincerely thank the reviewer for their valuable comments and suggestions, which have helped improve the quality of our manuscript. Below, we provide a detailed response (highlighted in green in the manuscript) to each point raised.
Comments and Suggestions for Authors
Comment 1: Please write a chapter with the conclusions of this study, and I also think it would be useful to do more detailed research with citations from several current scientific studies related to the growth of Atlantic salmon (Salmo salar) in RAS (38 references for a journal like Animals are not enough), then resend it for review.
Response 1: We have included a separate section of conclusions following the suggestion (please see lines 383-391). In addition, we have cited current studies in relation to the growth of Atlantic salmon in RAS as suggested (please see lines 81-84, 340-342, 374-381).
Reviewer 3 Report
Comments and Suggestions for Authors- Introduction
Missing recent RNA-seq / transcriptomic papers that already characterised thermal stress genes in salmon.
Does not compare the chosen 10–22 °C range with published optimal windows for parr
No mention of potential interaction between triploidy and thermal tolerance, although all-female triploids were used.
Add references to covering (i) Korean salmon temperature work, (ii) global transcriptomic evidence, (iii) triploid thermal performance, and (iv) published optimal intervals for parr. - Research design
No gradual acclimation phase described ( from 12 °C acclimation to target temperatures?).
Time frame: 60 d is short compared with commercial grow-out; smoltification not blocked or monitored.
Provide power analysis or cite pilot data; clarify acclimation protocol; discuss why 60 d is sufficient or extend discussion of limitation. - Methods – adequacy of description
No statement on randomisation of tanks or blind sampling.
Feeding “to apparent satiation” – not quantified (video, satiation curve, or biomass-based ration).
Uneaten feed collection protocol lacks detail (how long left in tank?, drying temperature?).
GPx kit manufacturer listed incomplete.
ELISA kit for cortisol – no intra-/inter-assay CV, standard curve range, or dilution factor.
RNA integrity (RIN) not reported; no evidence of genomic DNA removal.
Reference gene stability not validated.
Add randomisation scheme, feed loss methodology, complete kit details, RNA quality metrics, and reference-gene stability analysis. - Results
No correlation analysis between GPx activity and HSP gene expression.
Cortisol values are given without time-of-sampling control; diel cycle not considered.
Provide scatter or box plots; add correlation matrix; record sampling clock time. - Conclusions
“mild physiological trade-offs” at 14 °C is based only on HSP up-regulation vs 10 °C; no performance cost shown.
Does not reconcile own optimum (14 °C) with literature values of 16–19 °C for parr (Handeland et al. 2008).
No practical recommendation on acceptable upper limit for commercial RAS
Tone down claims, integrate contradicting literature, give explicit management thresholds.
Author Response
Reviewer 3
We sincerely thank the reviewer for their valuable comments and suggestions, which have helped improve the quality of our manuscript. Below, we provide a detailed response (highlighted in blue in the manuscript) to each point raised.
Comments and Suggestions for Authors
Comment 1: Introduction : Add references to covering (i) Korean salmon temperature work, (ii) global transcriptomic evidence, (iii) triploid thermal performance, and (iv) published optimal intervals for parr. Missing recent RNA-seq / transcriptomic papers that already characterised thermal stress genes in salmon. Does not compare the chosen 10–22 °C range with published optimal windows for parr. No mention of potential interaction between triploidy and thermal tolerance, although all-female triploids were used.
Response 1: In the introduction, we have added statements with relevant references, which responds to the reviewer’s comments. (please see lines 73-84, 91-94)
Comment 2: Research design : clarify acclimation protocol; discuss why 60 d is sufficient or extend discussion of limitation. No gradual acclimation phase described (from 12°C acclimation to target temperatures?). Time frame: 60 d is short compared with commercial grow-out; smoltification not blocked or monitored. Provide power analysis or cite pilot data.
Response 2: We have added the statement regarding the acclimation protocol following the reviewer’s comment (please see lines 107–109, 110–114). We acknowledge that a feeding trial longer than 60 days would likely yield more robust results for this type of research. In future studies, we will certainly consider the reviewer’s suggestion when designing experiments. Additionally, the discussion on this issue has been included in the manuscript (please see lines 374–379). The photoperiod was set to 24 hours of light to prevent smoltification. There was a typo in the original statement (see line 115). We appreciate the suggestion to provide a power analysis or cite pilot data. Due to the specific circumstances in Korea (namely, the difficulty in obtaining sufficient parr for growth trials) only a few studies on Atlantic salmon parr have been conducted, which limits data availability for analysis. Nonetheless, we designed the experiment based on existing literature and our prior experience to ensure adequate sample sizes for reliable results. In future studies, we will consider incorporating detailed power analyses or pilot data to further validate the experimental design.
Comment 3: Methods – adequacy of description :　Add randomisation scheme, feed loss methodology, complete kit details, RNA quality metrics, and reference-gene stability analysis. No statement on randomisation of tanks or blind sampling. Feeding “to apparent satiation” – not quantified (video, satiation curve, or biomass-based ration). Uneaten feed collection protocol lacks detail (how long left in tank?, drying temperature?). GPx kit manufacturer listed incomplete. ELISA kit for cortisol – no intra-/inter-assay CV, standard curve range, or dilution factor. RNA integrity (RIN) not reported. no evidence of genomic DNA removal. Reference gene stability not validated.
Response 3: The statement regarding the randomization scheme has been provided (please see lines 117–118). An additional explanation of the feeding regime has been included (please see lines 122–125). Detailed information on cortisol analysis has been added (please see lines 172–176). Information about the GPx kit has been included (please see lines 169–170). Moreover, detailed explanations regarding RIN, genomic DNA removal, and validation of reference gene stability have been described (please see lines 180–205).
Comment 4: Results : Provide scatter or box plots; add correlation matrix; record sampling clock time. No correlation analysis between GPx activity and HSP gene expression. Cortisol values are given without time-of-sampling control; diel cycle not considered.
Response 4: We very much appreciate the suggestion to perform a correlation analysis between GPx activity and HSP gene expression. Due to our limited background understanding of the appropriate analysis for these measurements, we would be grateful for any guidance or references from the reviewer to help us perform a suitable analysis. We recognize the potential value of exploring the relationship between these variables, as it could provide valuable insights for readers.
We appreciate the reviewer's suggestion to convert the existing results into scatter or box plots. After careful consideration, we believe that the current presentation effectively conveys the data and maintains clarity within our specific context, which is also consistent with the standards commonly accepted in other related journals, to our understanding. Therefore, we prefer to retain the existing format. Nonetheless, we thank the reviewer for their valuable feedback.
We acknowledge the reviewer's comment regarding the absence of time-of-sampling control and the diel cycle consideration. To address this, all blood samples were collected between 13:00 and 14:00 under continuous light conditions (24L:0D) to minimize the impact of diel variation on cortisol levels (please see lines 167-168). We appreciate the reviewer’s insight and will consider this factor more thoroughly in future studies.
Comment 4: Conclusions : Tone down claims, integrate contradicting literature, give explicit management thresholds. “mild physiological trade-offs” at 14 °C is based only on HSP up-regulation vs 10 °C; no performance cost shown. Does not reconcile own optimum (14 °C) with literature values of 16–19 °C for parr (Handeland et al. 2008). No practical recommendation on acceptable upper limit for commercial RAS.
Response 4: We appreciate the reviewer's insightful comments. We have incorporated the comment into our conclusions (please see lines 383-391).
Round 2
Reviewer 1 Report
Comments and Suggestions for AuthorsThe authors appropriately responded to the comments and questions raised by the reviewer.
Author Response
Comment : The authors appropriately responded to the comments and questions raised by the reviewer.
Response: We sincerely appreciate your feedback and are glad that our responses addressed your comments and questions satisfactorily. We remain grateful for your thorough review and valuable input.
Reviewer 2 Report
Comments and Suggestions for AuthorsThe manuscript The effects of water temperature on growth, hematological measurements, and stress-related gene expression in Atlantic salmon (Salmo salar) fry raised in a recirculating aquaculture system was investigated.
The introduction is comprehensive and well done.
Lines 105-106 provide more detail on the other features of the RAS system; it cannot only have mechanical and biological filters
Lines 107-108 specify the equipment used to measure water parameters
Line 109 specifies how many tanks were used to conduct the experiment
Line 115: specify which device you used to measure dissolved oxygen
Growth performance and sampling
Add IBW and FBW, even if they can be deduced from Wg, as this makes it easier to evaluate the growth parameters
Biochemical analysis of plasma: Specify how many specimens you analyzed
Antioxidant enzyme activity and cortisol: State how many specimens you analyzed.
The image at 201 looks strange.
The discussions are rather limited; I think more emphasis should be placed on antioxidant enzyme activity and cortisol and on gene expression. Find current studies and report on them experimentally.
Author Response
Once again, we sincerely thank the reviewer for their additional valuable comments and suggestions, which have helped improve the quality of our manuscript. Below, we provide a detailed response (highlighted in yellow in the manuscript) to each point raised.
The manuscript : The effects of water temperature on growth, hematological measurements, and stress-related gene expression in Atlantic salmon (Salmo salar) fry raised in a recirculating aquaculture system was investigated.
Comment 1 : The introduction is comprehensive and well done.
Response 1 : Thank you for your kind words. We are glad that the introduction met your expectations and provided a clear overview of our work. We appreciate your positive feedback
Comment 2 : Lines 105-106 provide more detail on the other features of the RAS system; it cannot only have mechanical and biological filters
Response 2 : We have provided more detailed information on the RAS system following the suggestion (please see lines 105-109)
Comment 3 : Lines 107-108 specify the equipment used to measure water parameters
Response 3 : The relevant information on the equipment used to measure water parameters has been included following the suggestion (please see lines 115-121)
Comment 4 : Line 109 specifies how many tanks were used to conduct the experiment
Response 4 : We have specified the tank set-up for the growth trial following the suggestion (please see lines 109-112)
Comment 5 : Line 115: specify which device you used to measure dissolved oxygen
Response 5 : We have specified the device we used to measure dissolved oxygen following the suggestion (please lines 115-116)
Comment 6 : Growth performance and sampling. Add IBW and FBW, even if they can be deduced from Wg, as this makes it easier to evaluate the growth parameters
Response 6 : We have added the initial and final weights for better understanding of the equation (please see line 156)
Comment 7 : Biochemical analysis of plasma: Specify how many specimens you analyzed
Response 7 : We have specified the number of samples for the analysis following the suggestion (please see lines 168-169)
Comment 8 : Antioxidant enzyme activity and cortisol: State how many specimens you analyzed.
Response 8 : We have specified the number of samples for the analysis following the suggestion (please see lines 180-181)
Comment 9 : The image at 201 looks strange.
Response 9 : We have increased the resolution of the image following the suggestion (please see the equation)
Comment 10 : The discussions are rather limited; I think more emphasis should be placed on antioxidant enzyme activity and cortisol and on gene expression. Find current studies and report on them experimentally.
Response 10 : We acknowledge that the previous version of the discussion section was somewhat limited. In response to the reviewer’s suggestion, we have reinforced this section by expanding the discussion on antioxidant enzyme activity, cortisol, and gene expression, incorporating insights from recent articles (please see lines 378-382, 384-390, 394-400, 402-406, 408-413)
