A Preliminary Study on the Effects of Nitrite Exposure on Hematological Parameters, Oxidative Stress, and Immune-Related Responses in Pearl Gentian Grouper

Round 1

Reviewer 1 Report

Dear authors,

Your work presents a very interesting and important theme for the aquaculture producers: the fish's response to nitrites, a known pollutant and by-product of the degradation of ammonia and organic matter in the aquaculture production tanks.

Your work needs an extensive rewriting and reorganization of the introduction since it is very difficult to read and doesn't bring interest to the reader.

You also have several issues in the Material and Methods section, where there is no description of the experimental condition in the tanks used for the trials ( tank volume, fish density, water renewal if they are in an open, semiclosed or closed system, ...), on the description of the methodologies used for blood sampling (are the syringes heparinized, ...), and the methodology that you describe for blood serum is actually very similar to the one used for blood plasma, with raises questions about what you truly analyzed in this point.

However, the worst is that your work has a critical fault in the methodology used. Your sample number per replicate is simply insufficient to obtain any proper results. Moreover, if you are using biochemical methods to assess serum AST, ALT, alkaline phosphatase (AKP), cortisol (COR), IgM, LZM, glutathione (GSH), superoxide dismutase (SOD), and malondialdehyde (MDA) levels, the sample number should be higher than 3 per replicate. Since you had 24 fishes per tank/replicate, you should have used at least 5-10 individuals to compensate for the high variability obtained in this kind of analysis. Unfortunately, and due to this poor sampling number, your results and conclusion most probably don't reflect the experimental population response to the nitrate concentrations, jeopardizing your entire work.

Even taking into consideration that your Results and Discussion sections are well written, the low number of biological replicates per replicate and treatment leads me to suggest that this paper should be rejected.

Best regards,

Author Response

Thank you for the useful comments, which give us a big help for the later research.

Author Response File: Author Response.pdf

Reviewer 2 Report

 

This is an interesting work. The effects of nitrite exposure on healthy status on Pearl gentian grouper is evaluated by studying haematological, immune and antioxidant parameters. The graphs are appropriate and the text is clear.

  

Specific comments

Introduction

The introduction is clear and the extension appropriated.

 

Material and methods

 

Line 83 to 85. The exposure conditions could be explained including some details. For example:

-       Had the fish only 24 hours for acclimatation before the experimental condition? How were the conditions before that? In this 24 hours, it was any system for cleaning the water?

-       the capacity of the tanks where the fish was exposed could be specified.

-       Was these tanks closed systems or had any recirculation system?

-       Was the seawater obtained from the sea or it was artificial?

-       Was the nitrite levels measured before start the experiment to ensure no extra nitrite were supported?

-       Was the nitrite levels measured during the experiment?

 

Line 121-124. A two-ways ANOVA is desirable. Why Duncan post-hoc test was done? Please justify why do no use Tukey, or Bonferroni. If homogeneity of variances  (or any other parameters) was evaluated, please indicate the used test.

 

Results

The table is a little confusing and maybe the representation of the results could be improved in some way.

Some numbers in the table are confused (such as 07, 01…).

 

The quality of the pictures from MO could be improved. Sometimes the measurement and it values included in the pictures are difficult to read.

 

Any micromorphological measurement was done using image analyses for the optical microscopy images? Why? Only representative images are included. The work could improve if these measurements are done.

 

Discussion 

There is a significant decrease in cortisol levels on group control at 96 hours with respect to time 0. No discussion about that is included. If these observations are caused because the removal of the other fish in the aquaria due to sampling, are these effects evaluated for other conditions (with the different concentration of nitrite)?

Author Response

Thank you for the useful comments, which give us a big help for the later research.

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Dear authors,

First of all, thank you for your efforts to improve the manuscript. That was a huge improvement in the quality and readability of the manuscript. I only have some minor corrections/suggestions that you can check on the attached revised manuscript.

Regarding the experimental design, I understand that due to the limited number of fish per treatment you were limited to a N=3 samples for each sampling point per treatment. However I deal almost on a daily basis with this kind of biochemical analysis, and the variability due to individual metabolism and the accuracy of these tests make this N clearly insufficient, in statistical terms. It should have been 5 at the minimum. You should have taken this into account in the experimental design and increased the number of fish per treatment.

I would suggest that you change the title of the paper to insert something like "Preliminary studies on..." because these kinds of studies can easily withstand this experimental design, that a full study will be highly criticized due to the lack of statistical soundness of the biochemical data.

Best regards,

Author Response

Dear Editors and Reviewers:

Thank you for the reviewers’ useful comments, which give us a big help for the later research. We have studied comments carefully and have made correction which we hope meet with approval. Revised portion are marked in red in the paper. The main corrections in the paper and the responds to the reviewer’s comments are as flowing: The manuscript has been revised accordingly, and the detailed corrections are listed below point by point:      

 

Reviewers' comments:

Q: Regarding the experimental design, I understand that due to the limited number of fish per treatment you were limited to a N=3 samples for each sampling point per treatment. However I deal almost on a daily basis with this kind of biochemical analysis, and the variability due to individual metabolism and the accuracy of these tests make this N clearly insufficient, in statistical terms. It should have been 5 at the minimum. You should have taken this into account in the experimental design and increased the number of fish per treatment.

Answer: Thank you for your suggestion. We have noticed this problem. Individual differences will be taken into account in the future experimental design, and the sample size will be increased to make the experimental analysis more perfect.

Q: I would suggest that you change the title of the paper to insert something like "Preliminary studies on..." because these kinds of studies can easily withstand this experimental design, that a full study will be highly criticized due to the lack of statistical soundness of the biochemical data.

Answer: According to your suggestion, we have been changed the title of the paper to " A Preliminary Study on the Effects of Nitrite Exposure on Hematological Parameters, Oxidative Stress, and Immune-related Responses in Pearl Gentian Grouper ."

The revised manuscript has been resubmitted to the journal. We are looking forward to the positive response. 

Yours sincerely,

Weiqiang Qiu and Jing Xie

Reviewer 2 Report

First of all, thank you for your efforts to improve the manuscript. That was a huge improvement in the quality and readability of the manuscript. I only have some minor corrections/suggestions.

 

-       I suggested to do micromorphological measurement using image analyses (such us Fiji or ImageJ,…). No reply about that was found.

 

-       I suggested to explain why Duncan test was done, due to its low potence depending of the number of samples… etc. and suggested Tukey, but no response was done about that.

Author Response

Dear Editors and Reviewers:

Thank you for the reviewers’ useful comments, which give us a big help for the later research. We have studied comments carefully and have made correction which we hope meet with approval. Revised portion are marked in red in the paper. The main corrections in the paper and the responds to the reviewer’s comments are as flowing: The manuscript has been revised accordingly, and the detailed corrections are listed below point by point:      

 

Reviewers' comments:

Q: I suggested to do micromorphological measurement using image analyses (such us Fiji or ImageJ,…). No reply about that was found.

Answer: Thank you for your advice. We have added relevant images and content analysis to the article (line 255-257, 264-267, 282-285, 292-295).

Q: I suggested to explain why Duncan test was done, due to its low potence depending of the number of samples… etc. and suggested Tukey, but no response was done about that.

Answer: Thank you for your suggestion. The test method in our references has been tested by Duncan and Tukey test. It is found that the result is similar. In the future experimental analysis, Tukey test will be taken into account for data analysis and appropriate analysis method will be selected. Thank you again for your suggestion (Xu, Z.; Zhang, H.; Guo, M.; Fang, D.; Mei, J.; Xie, J. Analysis of Acute Nitrite Exposure on Physiological Stress Response, Oxidative Stress, Gill Tissue Morphology and Immune Response of Large Yellow Croaker (Larimichthys crocea). Animals 2022, 12, 1791; Hosseini, H.; Esmaeili, M.; Zare, M.; Rombenso, A. Egg enrichment with n-3 fatty acids in farmed hens in sub-optimum temperature: A cold-temperament additive mix alleviates adverse effects of stress on performance and health. J. Anim. Physiol. Anim. Nutr. 2021, 1–12; Wang, Q.; Mei, J.; Cao, J.; Xie, J. Effects of Melissa officinalis L. Essential Oil in Comparison with Anaesthetics on Gill Tissue Damage, Liver Metabolism and Immune Parameters in Sea Bass (Lateolabrax maculatus) during Simulated Live Transport. Biology 2021, 11, 11.).

The revised manuscript has been resubmitted to the journal. We are looking forward to the positive response. 

Yours sincerely,

Weiqiang Qiu and Jing Xie