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Nitrite Stress Induces Oxidative Stress and Leads to Muscle Quality Decreased in Wuchang Bream (Megalobrama amblycephala Yih) Juveniles

Water 2022, 14(2), 160; https://doi.org/10.3390/w14020160

by Zhenyi Hu¹

, Chenglong Qi¹, Chenzhi Lin¹ and Rong Tang^2,*

Reviewer 1: Anonymous

Reviewer 2: Anonymous

Reviewer 3: Anonymous

Reviewer 4: Anonymous

Water 2022, 14(2), 160; https://doi.org/10.3390/w14020160

Submission received: 15 November 2021 / Revised: 30 December 2021 / Accepted: 5 January 2022 / Published: 8 January 2022

(This article belongs to the Special Issue Effect of Aquatic Environment on Fish Ecology)

Round 1

Reviewer 1 Report

Well designed work. Congratulations to the working team.

Author Response

Thank you for your comments. We communicated with professional language teachers and modified the grammar and expression of the full text as much as possible. And we proofread the layout of the manuscript according to the journal's submission guidelines. The modified part has been marked in red in the manuscript.

Author Response File: Author Response.pdf

Reviewer 2 Report

The manuscript “Nitrite stress induces oxidative stress and leads to muscle quality decreased in Wuchang bream (Megalobrama amblycephala Yih) juveniles” aimed to assess the short-term effects of sodium nitrite assessing blood and oxidative stress biomarkers and muscle quality at different concentrations and endpoints in freshwater fish M. amblycephala.

The topic of the manuscript is novel for fish, especially freshwater and farmed fish, and may fit with the scope of Water. Although the study is of potential interest for the readership, I have some major comments that should be addressed before possible publication of the MS in the journal Water. Please I would suggest to revise English language and style.

_ The abstract is too descriptive. Authors should summarize the main information emerging from the data obtained.

_ L. 43. Please, add “species” after “active oxygen” in sentence “Living organisms produce a certain amount of active oxygen during normal growth”.

L 78_ the aim of this study is not fully clear and should be rephrased. The scientific approach adopted (acute toxicity test) should be clearly stated.

_ Material and methods section should be deeply improved, in order to increase clarity and scientific value of the MS. Methods should be more detailed.

1)Where was sodium nitrite purchased? What is its purity?

2) How was the cytosolic extraction performed? What sample dilution was done? How were the samples homogenized? Have they been centrifuged? Under what experimental conditions was centrifugation carried out? What are the operating principles of the kits used to measure the levels of oxidative stress biomarkers? Which instrument was used to perform the "metabolism analysis"?

3) Is there a relationship between the concentrations tested and those found in aquaculture? Authors have to specify and discuss this information.

_ Section 2.3: I suggest replacing “related indicators” with “biomarkers”.

_ Results: when biomarker responses are significantly different from control group, I suggest adding percentages to better understand the magnitude of the difference between treated and control groups.

Paragraph 4.1:

_L. 256-264. “The triggering of fish anti-stress mechanism”…“including SOD, CAT, and GPx”: please add references supporting what has been stated in this paragraph.

_ “That may mean excessive energy consumption”… “rise again to resist stress”: please add references supporting what has been stated in these sentences.

L. 268. _ … “showed a significant positive correlation with exposure concentration”: did Authors carry out the statistical correlation analysis? This is not clear from the M&M and Results. If the statistical correlation analysis has been done, please clearly state it in M&M and report the results, otherwise I suggest substituting “correlation” with a different term through the text to indicate dose-dependent biochemical responses.

L. 288_ Das [18] and other studies: please insert references related to other studies.

L. 310 Paragraph 4.2: I apologize, the relationship between the effects of temperatures and salt stress on SOD and GPx reported in previous studies and the results obtained on sodium nitrite is not clear to me. I suggest rewriting the discussion using more relevant references (e.g. effects of nitrite or nitrogen compounds on SOD, CAT and GPx in fish).

Other comments: please, use the term "decrease" with caution. When biomarker levels return to control levels after increasing, I suggest using "return to control group levels".

Moreover, use the italic for the scientific name of the species.

Author Response

1. The manuscript “Nitrite stress induces oxidative stress and leads to muscle quality decreased in Wuchang bream (Megalobrama amblycephala Yih) juveniles” aimed to assess the short-term effects of sodium nitrite assessing blood and oxidative stress biomarkers and muscle quality at different concentrations and endpoints in freshwater fish M. amblycephala.

Thank you for your affirmation of our work. Thank you very much for processing and reviewing our paper.

2. The topic of the manuscript is novel for fish, especially freshwater and farmed fish, and may fit with the scope of Water. Although the study is of potential interest for the readership, I have some major comments that should be addressed before possible publication of the MS in the journal Water. Please I would suggest to revise English language and style.

Thank you for your suggestions and related questions.

We consulted native English speakers and revised the language and style of the manuscript as much as possible.

3. The abstract is too descriptive. Authors should summarize the main information emerging from the data obtained.

Thank you for your suggestion. We have revised the abstract as follows,

L14-29: “To determine the effects of nitrite exposure on muscle quality and physiological functions in Wuchang bream (Megalobrama amblycephala). M. amblycephala juveniles were exposed to acute nitrite (0, 1, 5, 10, 20 mg/L), and the muscle and blood samples were measured at 12, 24, 48, and 96 hours. The results showed that when exposed to nitrite for 12 h, the concentration of blood glucose, cortisol, aspartate aminotransferase (AST) and alanine aminotransferase (ALT) in the 20 mg/L experimental group had the maximum value. The activity of lactate dehydrogenase (LDH) increased significantly in a dose-dependently manner, and peaked at 96 h in the 20 mg/L group. During 96 h of exposure to nitrite, the total antioxidant capacity (T-AOC) and catalase (CAT) activity in the liver of the 20 mg/L experimental group were significantly higher than those of the control group, while the concentration of muscle glycogen showed a downtrend. At 12 h and 96 h, the hardness of the four experimental groups were significantly higher than that of the control group. Our research shows that acute sodium nitrite exposure will not only cause oxidative stress and decreased muscle quality in M. amblycephala juveniles, but also will be accompanied by changes in serum biochemical index, liver antioxidant capacity, muscle physiological characteristics, and muscle physical characteristics. Preliminary speculation may be that acute nitrite exposure may cause M. amblycephala juveniles to choose to reduce muscle quality and activate antioxidant systems.”

4. L43. Please, add “species” after “active oxygen” in sentence “Living organisms produce a certain amount of active oxygen during normal growth”.

Thanks for your suggestion.

We replaced “active oxygen” with “reactive oxygen species (ROS)”.

5. L78 the aim of this study is not fully clear and should be rephrased. The scientific approach adopted (acute toxicity test) should be clearly stated.

Thanks for your suggestion.

We rephrased the aim of this study and added “We used the method of acute toxicity test” to illustrate the scientific method we adopted.

L81-87: “We used the method of acute toxicity test. M. amblycephala juveniles were exposed to sodium nitrite aqueous solution at different concentrations and at different times. Therefore, this study was aimed to comprehensive analysis the antioxidant index and muscle quality after acute exposure to sodium nitrite in M. amblycephala juveniles, and to investigate the effects of acute toxicity on serum biochemical index, liver antioxidant capacity, muscle physiological characteristics, and muscle physical characteristics.”

6. Material and methods section should be deeply improved, in order to increase clarity and scientific value of the MS. Methods should be more detailed. Where was sodium nitrite purchased? What is its purity?

Thanks for your suggestion.

The sodium nitrite was purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China), with a purity greater than or equal to 99.0%. We add “the sodium nitrite was purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China), with a purity greater than or equal to 99.0%.” in Material and methods.

7. How was the cytosolic extraction performed? What sample dilution was done? How were the samples homogenized? Have they been centrifuged? Under what experimental conditions was centrifugation carried out? What are the operating principles of the kits used to measure the levels of oxidative stress biomarkers? Which instrument was used to perform the "metabolism analysis"?

Thanks. Our experimental operations are strictly implemented in accordance with the kit instructions. In brief, we weigh the liver tissue, add saline, and grind at low temperature. Use a 4 ℃ centrifuge for centrifugation. Perform pre-experiment and determine the concentration of the sample according to the kit instructions. Dilute with normal saline. For metabolic analysis, we use a microplate (Infinite M200 Nanoquant, Tecan, Männedorf, Switzerland) reader to detect the value. We have complemented the experimental method.

L106-115: “Each group of nine-tailed fish was randomly selected and anesthetized with MS-222 (MCE, China). The sample is collected at 12 h, 24 h, 48 h and 96 h during the nitrite exposure. A 1 mL syringe was used to take blood from the tail artery of the experimental fish. Serum was separated by centrifugation at 3500 r/min for 30 min. The serum is stored at -80 °C. Serum is used for the detection of biochemical indicators. Use sterile surgical instruments to collect experimental fish tissues. Liquid nitrogen is used to preserve experimental tissues. The experimental tissue samples are used for anti-oxidation index detection. A fresh steak of 1 cm thickness muscle between the dorsal and caudal fin was sampled for measuring of texture parameters. 10 g muscle lateral line frozen at -80 ℃ for determination of lactic acid and glycogen.”

L123-128: “Measured on the analyzer, the kits were purchased from Wuhan Kangruijia Technology Co., Ltd (Wuhan, China). The cortisol was determined by radioimmunoassay (RIA), and the kit was purchased from Beijing Northern Institute of Biotechnology (Beijing, China). Glycogen was determined by oxidation colorimetric method, and the kit used was purchased from Nanjing Jiancheng Biochemical Corporation (Nanjing, China).”

L135-136: “The kit used was purchased from Nanjing Jiancheng Biochemical Corporation (Nan-jing, China).”

L139-140: “The detection of muscle glycogen and lactic acid were determined by colorimetric method(Infinite M200 Nanoquant, Tecan, Männedorf, Switzerland).”

8. Is there a relationship between the concentrations tested and those found in aquaculture? Authors have to specify and discuss this information.

Thank you for your suggestion.

The concentration of sodium nitrite used in our experiment is 0, 1, 5, 10, 20 mg/L. Our research group has published related articles. The 96-hour LC₅₀ of sodium nitrite to grass carp was 25.3 mg/L, and the experimental concentrations were 0, 8, 25, and 50 mg/L (Xie et al, 2019). The concentration used in the experiment using ammonia nitrogen stress of M. amblycephala was 0, 5, 10, 15, and 20 mg/L (Zhang et al, 2019). Sun has also used sodium nitrite at a concentration of 15 mg/L to expose M. amblycephala (Sun et al, 2015). We refer to the previous experimental concentration to determine the exposure concentration for this study.

Reference:

Sun, S.M.; Zhu, J.; Ge, X.P.; Zhang, C.F.; Miao, L.H.; Jiang, X.J. Cloning and Expression Analysis of a Heat Shock Protein 90 β Isoform Gene from the Gills of Wuchang Bream (Megalobrama Amblycephala Yih) Subjected to Nitrite Stress. Genet. Mol. Res. 2015, 14, 3036–3051, doi:10.4238/2015.April.10.14.

Xie, L.; Chen, S.; Yao, C.; Li, D.; Li, L.; Tang, R. Nitrite Induces Endoplasmic Reticulum Stress and Associates Apoptosis of Liver Cells in Grass Carp (Ctenopharyngodon Idella). Aquaculture 2019, 507, 275–281, doi:10.1016 /j.aquaculture. 2019.04.016.

Zhang, W.; Xia, S.; Zhu, J.; Miao, L.; Ren, M.; Lin, Y.; Ge, X.; Sun, S. Growth Performance, Physiological Response and Histology Changes of Juvenile Blunt Snout Bream, Megalobrama Amblycephala Exposed to Chronic Ammonia. Aquaculture 2019, 506, 424–436, doi:10.1016/j.aquaculture.2019.03.072.

9. Section 2.3: I suggest replacing “related indicators” with “biomarkers”.

Thanks for your suggestion.

We replaced “related indicators” with “biomarkers”. And we modified the manuscript using revision mode.

10. Results: when biomarker responses are significantly different from control group, I suggest adding percentages to better understand the magnitude of the difference between treated and control groups.

Thank you for your suggestion.

When the biomarker response was significantly different from the control group, we increased the percentage to better understand the degree of difference between the experimental group and the control group. The specific content is added to the description of the “Result”.

L184-187: “At 12 h, the serum cortisol level of the experimental groups was significantly higher than the control group, and showed a significant positive correlation with the exposure concentration. Serum cortisol in the 20 mg/L experimental group was 321% higher than that in the control group.”

L193-195: “As for changes in total cholesterol concentration, at 12 h, the total cholesterol concentration of the 20 mg/L experimental group was significantly lower than that of the control group. The value is 86% of the control group.”

L196-198: “At 96 h, the total cholesterol concentration of the 20 mg/L experimental group was significantly higher than that of the other groups. The value is 114% of the control group.”

11. Paragraph 4.1: L 256-264. “The triggering of fish anti-stress mechanism”…“including SOD, CAT, and GPx”: please add references supporting what has been stated in this paragraph.

Thanks for your suggestion.

We have added references and revised them in the manuscript.

L279-282: “The triggering of fish anti-stress mechanism is mainly through the stress of fish regulating the central hypothalamus-pituitary-renal tissue axis (HPI), releasing adrenocorticotropic hormone (ACTH), and promoting stress hormones such as catecholamines and cortisol in the fish body (Guo et al, 2020).”

L285-287: “In addition, as an oxidant, nitrite also triggers the activation of the protective effect of the fish body's antioxidant system, which triggers changes in the activity of enzymes including SOD, CAT, and GPX (Xie et al, 2019).”

Reference:

Guo, H.; Lin, W.; Wang, L.; Zhang, D.; Wu, X.; Li, L.; Li, D.; Tang, R.; Yang, L.; Qiu, Y. The Supplementation of Dietary Selenium Yeast and Green Tea‐derived Polyphenols Improves Antioxidant Capacity and Immune Response in Juvenile Wuchang Bream under Ammonia Stress. Aquac. Res. 2020, 51, 3790–3803, doi:10.1111/are.14724.

12. “That may mean excessive energy consumption”… “rise again to resist stress”: please add references supporting what has been stated in these sentences.

Thanks for your suggestion.

We have added references and revised them in the manuscript.

L301-302: “That may mean excessive energy consumption during the initial resistance to external stress and that causes a decrease in cholesterol levels (Kumaraguru et al, 1986; Cheng et al, 2012).”

L302-304: “As time goes on, the fish metabolism compensation mechanism is activated, which makes the cholesterol level recover and rise again to resist stress (Chen et al, 1993; Ciji et al, 2020).”

Reference:

Kumaraguru, A.K.; Beamish, F.W.H. Effect of Permethrin (NRDC-143) on the Bioenergetics of Rainbow Trout, Salmo Gairdneri. Aquat. Toxicol. 1986, 9, 47–58, doi:10.1016/0166-445X(86)90005-6.

Cheng, S.-Y.; Shieh, L.-W.; Chen, J.-C. Changes in Hemolymph Oxyhemocyanin, Acid–Base Balance, and Electrolytes in Marsupenaeus Japonicus under Combined Ammonia and Nitrite Stress. Aquat. Toxicol. 2013, 130–131, 132–138, doi:10.1016/j.aquatox.2012.12.015.

Chen, J.-C.; Cheng, S.-Y. Hemolymph PCO2, Hemocyanin, Protein Levels and Urea Excretions of Penaeus Monodon Exposed to Ambient Ammonia. Aquat. Toxicol. 1993, 27, 281–291, doi:10.1016/0166-445X(93)90059-A.

Ciji, A.; Akhtar, M.S. Nitrite Implications and Its Management Strategies in Aquaculture: A Review. Rev. Aquac. 2020, 12, 878–908, doi:10.1111/raq.12354.

13. L 268. _ … “showed a significant positive correlation with exposure concentration”: did Authors carry out the statistical correlation analysis? This is not clear from the M&M and Results. If the statistical correlation analysis has been done, please clearly state it in M&M and report the results, otherwise I suggest substituting “correlation” with a different term through the text to indicate dose-dependent biochemical responses.

Thank you for your suggestion. We have revised the sentence.

L305-308:“In this experiment, the AST activity in the 20 mg/L experimental group was always higher than that of the control group at 12 h, 24 h, and 48 h exposure, and had the maximum value. At 96 h, there was no significant difference in AST activity between the experimental group and the control group.”

14. L 288_ Das [18] and other studies: please insert references related to other studies.

Thanks for your suggestions, we have added the following references.

[35] Das, P.C.; Ayyappan, S.; Jena, J.K.; Das, B.K. Acute Toxicity of Ammonia and Its Sub-Lethal Effects on Selected Haematological and Enzymatic Parameters of Mrigal, Cirrhinus Mrigala (Hamilton). Aquac. Res. 2004, 35, 134–143, doi:10.1111/ j.1365-2109.2004.00994.x.

[36] Bogin, E.; Marom, M.; Levi, Y. Changes in Serum, Liver and Kidneys of Cisplatin-Treated Rats; Effects of Antioxidants. Clin. Chem. Lab. Med. 1994, 32, doi:10.1515/cclm.1994.32.11.843.

15. L 310 Paragraph 4.2: I apologize, the relationship between the effects of temperatures and salt stress on SOD and GPx reported in previous studies and the results obtained on sodium nitrite is not clear to me. I suggest rewriting the discussion using more relevant references (e.g. effects of nitrite or nitrogen compounds on SOD, CAT and GPx in fish).

Thank you for your suggestion. We revised paragraph 4.2, deleted references on temperature and salinity, and added new references. The section was rewritten.

L322-338: “The function of the fish body's antioxidant system mainly depends on the changes in the activity of antioxidant enzymes. As the most important oxidase in the fish body, SOD plays an important role in the process of removing active oxygen, eliminating superoxide anion free radicals and protecting cells from temperature stress. Nitrite exposure can promote the formation of reactive oxygen species in aquatic animals, thereby inducing oxidative damage (Sun et al, 2014). The increase of SOD, CAT and GPx activity are considered to be a strategy for fish to cope with oxidative stress (Zheng et al, 2016). In this experiment, we also found that at 24 h and 48 h, SOD activity in the liver of each experimental groups increased significantly to resist oxidative stress. CAT in organisms can reduce H₂O₂ in the body and maintain the normal physiological activities of cells and the body (Parihar et al, 1997). During the period of nitrite exposure, CAT activity also increased significantly with the increase of exposure concentration to resist the damage caused by oxidative stress. In addition, there are some other antioxidant enzymes such as GPx, which can catalyze the degradation of hydrogen peroxide and hydroperoxide (Wei et al 2008) and remove lipid peroxides (Chatterjee et al, 2004). Exposure to Macrobrachium nipponense with high concentration of sodium nitrite for 24 hours will significantly reduce the GPx activity (Wang et al, 2004). However, in this study, it was not found that nitrite had a significant effect on the GPx activity in M. amblycephala juveniles.”

References

Chatterjee, N.; Pal, A.K.; Manush, S.M.; Das, T.; Mukherjee, S.C. Thermal Tolerance and Oxygen Consumption of Labeo Rohita and Cyprinus Carpio Early Fingerlings Acclimated to Three Different Temperatures. Journal of Thermal Biology 2004, 29, 265–270, doi:10.1016/j.jtherbio.2004.05.001.

Parihar, M.S.; Dubey, A.K.; Prakash, P. Responses of Superoxide Dismutase, Glutathione Peroxidase and Reduced Glutathione Antioxidant Defenses in Gills of the Freshwater Catfish (heteropneustes fossills) to Short-term Elevated Temperature. 6.

Sun, S.; Ge, X.; Zhu, J.; Xuan, F.; Jiang, X. Identification and MRNA Expression of Antioxidant Enzyme Genes Associated with the Oxidative Stress Response in the Wuchang Bream (Megalobrama Amblycephala Yih) in Response to Acute Nitrite Exposure. Comp. Biochem. Physiol. Part C Toxicol. Pharmacol. 2014, 159, 69–77, doi:10.1016/j.cbpc.2013.09.005.

Wang, W.-N.; Wang, A.-L.; Zhang, Y.-J.; Li, Z.-H.; Wang, J.-X.; Sun, R.-Y. Effects of Nitrite on Lethal and Immune Response of Macrobrachium Nipponense. Aquaculture 2004, 232, 679–686, doi:10.1016/j.aquaculture.2003.08.018.

Wei, W.; Cuijuan, N.; Ying, G.; Li, L. The Effects of Environmental Endocrine Disruptors on Ferti-lization and Embryonic Development of The Fish liza haematocheila. 1.

Zheng, J.; Mao, Y.; Su, Y.; Wang, J. Effects of Nitrite Stress on MRNA Expression of Antioxidant Enzymes, Immune-Related Genes and Apoptosis-Related Proteins in Marsupenaeus Japonicus. Fish Shellfish Immunol. 2016, 58, 239–252, doi:10.1016/j.fsi.2016.08.058

16. Other comments: please, use the term "decrease" with caution. When biomarker levels return to control levels after increasing, I suggest using "return to control group levels". Moreover, use the italic for the scientific name of the species.

Thank you for your kind help. We re-checked the manuscript and revised the manuscript where it was used less once. And we have re-edited the full text and revised all the scientific names of the species to italics.

Author Response File: Author Response.pdf

Reviewer 3 Report

Despite the main idea of the manuscript seems good. But the mentioned problems supported that the paper needed hard work to be suitable for publication since the statistical analysis method needed to be adjusted before it could be published. As a consequence, I was unable to make any changes to the results or discussion sections. Furthermore, none of the approaches stated in the M&M section are supported by any references. there are several drawbacks found in the existing manuscript.

L 15 scientific names should be revised all over the manuscript and represented in italic form.

L 19 "and then decreased" this information need to rephrased again to be more cleared, how the same measure increased after 12 h of nitrite exposure then decreased at the same time?

L 23 "downward" strange word

L 23 and 25 the two sentences need to merged and rephrased again.

At the end of the abstract, there is no conclusion. To eliminate unclear phrases and grammatical problems, the abstract should be revised by native language speaker.

L 32 I believed the INTRODUCTION section should start with an introduction to the relevance of intensive culture systems, followed by a representation of the significant association between high stocking density and nitrite pollution in aquatic environments.

L 78 the aim of the work needs to rewritten again in a good presented manner.

The anesthetic agents employed to collect samples were not reported by the authors.

L 106 the all used methods needs adequate references (this comment should be applied into all material and methods section).

L 155 The statistical analysis procedure is inappropriate; there are two primary experimental effects (exposure duration and nitrite levels), thus Two-way ANOVA should be used. This indicate that the entire findings are incorrect and could not supported for publishing.

Author Response

1. Despite the main idea of the manuscript seems good. But the mentioned problems supported that the paper needed hard work to be suitable for publication since the statistical analysis method needed to be adjusted before it could be published. As a consequence, I was unable to make any changes to the results or discussion sections.

Thank you for your affirmation of the idea of the manuscript. We have explained and changed the statistical analysis method and experimental methods of the manuscript according to your suggestions, and hope to get your affirmation.

2. Furthermore, none of the approaches stated in the M&M section are supported by any references.

Thank you for your kind help. We added references in the M&M section.

L149: “The determination of drip loss refers to previous studies [25].”

L160-162: “Texture parameters were measured in raw flesh immediately after removal of the second transversal steak of 1 cm thickness from fish. The assay was performed following the method described by Zhang [26] with minor modifications.”

References

[25] Zhang, X.; Wang, J.; Tang, R.; He, X.; Li, L.; Takagi, Y.; Li, D. Improvement of Muscle Quality of Grass Carp (Ctenopharyngodon Idellus) With a Bio-Floating Bed in Culture Ponds. Front. Physiol. 2019, 10, 683, doi:10.3389/fphys.2019.00683.

[26] Zhang, X.; Shen, Z.; Qi, T.; Xi, R.; Liang, X.; Li, L.; Tang, R.; Li, D. Slight Increases in Salinity Improve Muscle Quality of Grass Carp (Ctenopharyngodon Idellus). Fishes 2021, 6, 7, doi:10.3390/fishes6010007.

3. There are several drawbacks found in the existing manuscript.

L 15 scientific names should be revised all over the manuscript and represented in italic form.

Thank you for your suggestion. We have italicized the species name of the full text.

4. L 19 "and then decreased" this information need to rephrased again to be more cleared, how the same measure increased after 12 h of nitrite exposure then decreased at the same time?

Thank you for your suggestion. We rephrased the sentence as follow,

L17-19: “The results showed that when exposed to nitrite for 12 h, the concentration of blood glucose, cortisol, aspartate aminotransferase (AST) and alanine aminotransferase (ALT) in the 20 mg/L experimental group had the maximum value.”

5. L 23 "downward" strange word

Thank you for your suggestion. We changed “downward” to “downtrend”.

6. L 23 and 25 the two sentences need to merged and rephrased again.

Thank you for your suggestion. We revised the two sentences.

L23-24: “At 12h and 96h, the hardness of the four experimental groups was significantly higher than that of the control group.”

7. At the end of the abstract, there is no conclusion. To eliminate unclear phrases and grammatical problems, the abstract should be revised by native language speaker.

Thank you for your suggestion. We revised the abstract as follows,

L24-29: “Our research shows that acute sodium nitrite exposure will not only cause oxidative stress and decreased muscle quality in M. amblycephala juveniles, but also will be accompanied by changes in serum biochemical index, liver antioxidant capacity, muscle physiological characteristics, and muscle physical characteristics. Preliminary speculation may be that acute nitrite exposure will cause M. amblycephala juveniles to use the ability to activate the antioxidant system instead of growth.”

8. L 32 I believed the INTRODUCTION section should start with an introduction to the relevance of intensive culture systems, followed by a representation of the significant association between high stocking density and nitrite pollution in aquatic environments.

Thank you for your suggestion. We changed the third paragraph of the introduction to the first paragraph. The third paragraph mainly introduces intensive culture systems. The first and second paragraphs mainly introduce the research progress of nitrite.

9. L 78 the aim of the work needs to rewritten again in a good presented manner.

Thank you for your suggestion, we have rewritten the aim of the article.

L80-87: “In addition, there are few studies on the effects of acute exposure to sodium nitrite stress on fish muscle quality. We used the method of acute toxicity test. M. amblycephala juveniles were exposed to sodium nitrite aqueous solution at different concentrations and at different times. Therefore, this study was aimed to comprehensive analysis the antioxidant index and muscle quality after acute exposure to sodium nitrite in M. amblycephala juveniles, and to investigate the effects of acute toxicity on serum biochemical index, liver antioxidant capacity, muscle physiological characteristics, and muscle physical characteristics.”

10. The anesthetic agents employed to collect samples were not reported by the authors.

The anesthetic agent we used is MS-222, purchased from MedChemExpress (MCE, China), and we have added the introduction of the anesthetic agent in the article (L106-107).

11. L 106 the all used methods needs adequate references (this comment should be applied into all material and methods section).

Thank you for your suggestion. Our research group has been committed to studying the physiological and biochemical levels and muscle quality of fish. The research methods used in this article have been used by the research group. Muscle metabolism indexes, physiological and biochemical indexes and antioxidant indexes are all used in kits provided by Nanjing Jiancheng Biochemical Corporation (Nanjing, China). Muscle drip loss, PH value and Textural parameters are read using mathematical formulas or experimental instruments. All experimental methods are supported by relevant literature, and the specific literature is as follows,

References

Zhang, X.; Wang, J.; Tang, R.; He, X.; Li, L.; Takagi, Y.; Li, D. Improvement of Muscle Quality of Grass Carp (Ctenopharyngodon Idellus) With a Bio-Floating Bed in Culture Ponds. Front. Physiol. 2019, 10, 683, doi:10.3389/fphys.2019.00683.

Zhang, X.; Shen, Z.; Qi, T.; Xi, R.; Liang, X.; Li, L.; Tang, R.; Li, D. Slight Increases in Salinity Improve Muscle Quality of Grass Carp (Ctenopharyngodon Idellus). Fishes 2021, 6, 7, doi:10.3390/fishes6010007.

12. L 155 The statistical analysis procedure is inappropriate; there are two primary experimental effects (exposure duration and nitrite levels), thus Two-way ANOVA should be used. This indicate that the entire findings are incorrect and could not supported for publishing.

Thank you for your suggestion.

Our research is based on exposure time or exposure concentration. And we have compared the oxidative stress level and muscle quality of different concentrations of sodium nitrite on Wuchang bream at the same time. We controlled the consistency of time variables and adopted a one-way analysis of variance. At the same time, we have compared the effect of different exposure times on the oxidative stress level and muscle quality of Wuchang bream at the same concentration level.

In order to facilitate readers to understand the full text of the experimental data, we have added the data analysis. The details are as follows,

L173-174: “All data are controlled in the same time period, and the differences between groups under different concentration conditions are analyzed.”

Author Response File: Author Response.pdf

Reviewer 4 Report

Dear Authors,

a few details marked directly in the MS needs to be considered.

Comments for author File: Comments.pdf

Author Response

1. a few details marked directly in the MS needs to be considered.

Thank you for your suggestion. We have made changes based on the details you pointed out. Please see the attachment or below.

2. L3 place the scientific title of species in italic

Thanks for your suggestion, we changed “Megalobrama amblycephala” to italic.

3. L14 dissolved in water? it is not clear

Thank you for your suggestion, the sodium nitrite we used is dissolved in water.

4. L15 italic, perform in whole MS

Thanks for your suggestion, we changed “Megalobrama amblycephala” to italic and performed in whole MS

5. L101 were the whole number of fish used? how many samples per group?

We use random sampling, and each group selects nine samples for experiment.

6. L166 Please check Fig1a for significant difference and explain superscripts

Thank you for your suggestion. We re-analyzed the experimental data. The results showed that at 96 h, the blood glucose levels of the 5 mg/L and 20 mg/L experimental groups were significantly higher than 1 mg/L and 10 mg/L experimental groups and the control group. The existing label in Figure 1a has been modified, and the different lowercase letters in the figure indicate significant differences between each other.

7. L183 According to Fig. 1f., 5, 10 & 20mg/l groups significantly differ to control? Please explain...

Thank you for your suggestion. According to Figure 1f, there was a significant difference in the LDH activity of the 5 mg/L, 10 mg/L and 20 mg/L experimental groups. We made changes in the manuscript.

8. L187 Please reformulate.

Significant difference between groups was indicated only at 12h...

Thanks for your suggestion, we have revised the section. We made changes in the manuscript.

L206-208: “At 12 h, the activity of ALT concentration in 20 mg/L exposure group was significantly higher than that of the other groups (p< 0.05), and there were no significant differences at other times (p > 0.05).”

9. L191 Please check the text of Results and compare Fig1 for significant differences

Thank you for your suggestion. We proofread the results in Figure 1. The result text of Figure 1 was checked.

10. L196 Please state the p value in the figure title. Do it in the whole MS

Thank you for your suggestion. We have added a description of the p value in the figure title. The description of the p value of the full text have been added.

11. L 202 Check the figure again and explain significance of 10mg at 24h and 1 & 10mg @ 48h exposure.

Thank you for your suggestion, we have modified it in the manuscript.

L221-227: “There was no significant difference in the SOD activity in the liver between the 10mg/L experimental group and the control group at 24h and 48h (p > 0.05). In the 24h and 48h, the SOD activity of 5mg/L and 20mg/L experimental groups were significantly higher than that of the control group (p < 0.05). At 24h, the SOD activity in the 1mg/L experimental group was significantly higher than that in the control group (p < 0.05). But at 48h, there was no significant difference between the results of the 1mg/L experimental group and the control group (p > 0.05).”

12. L 217 lowercase letter c