Proteomic Analysis Reveals That Dietary Supplementation with Fish Oil Enhances Lipid Metabolism and Improves Antioxidant Capacity in the Liver of Female Scatophagus argus

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

General Comments:

The study conducted by He et al. investigated the effects of fish and soybean oils on proteomics changes in proteins associated with lipid metabolism, glucose metabolism, inflammation, antioxidant responses, and oxidative stress in female Scatophagus argus. While the study provides valuable insights into metabolic and proteomic changes, it could be repeated using male fish, fry, or growing-out fish, as the selected parameters do not directly serve ovarian development, as the authors seem to suggest. However, as a metabolic and proteomic study, it is well-conducted and could be suitable for publication after major revisions. The aim should be refocused on metabolic changes rather than directing the manuscript toward ovarian development.

Specific Comments:

1. Study Focus and Reproductive Indicators:

The study primarily focuses on the effects of different lipid sources in relation to ovarian development in female broodstock. However, the investigated parameters only consider the ovary somatic index (OSI) as a reproductive indicator, neglecting the synthesis of key substances such as vitellogenin (Vtg), which is crucial for assessing ovarian development.

2. Line 48-56: Additional references should be added to support the cited information, as this would strengthen the background and justification for the study.

3. Line 56-58: The text refers to the nutritional requirements of marine broodstock, but Scatophagus argus is a freshwater or brackish water species (tolerating up to 8 ppm salinity). This discrepancy should be clarified or corrected.

4. Materials and Methods:
   • The control group is not clearly defined. It is essential to specify which group serves as the control.
   • The number of treatments is limited. Including a mixture of fish oil and plant oil (e.g., soybean oil) as an additional treatment could provide more comprehensive insights. Furthermore, exploring other plant oils, such as corn oil, might offer a cost-effective alternative while meeting the fish's nutritional requirements.
   • The lipid profile of each oil used should be provided, as the interpretation of results heavily depends on this information.
5. Line 103: The country name should be added for clarity.

6. Vitamin C Addition:

The sentence mentioning the addition of lactic acid bacteria and vitamin C to the water is unclear. Please clarify whether vitamin C was added to the water or the diet, as this could significantly impact the interpretation of results.

7. Line 116:the number of replicates should be clarified. Is it two replicates per treatment? This information is critical for assessing the statistical robustness of the study.

8. Line 119: A separate subtitle, such as "Growth and Somatic Indices," should be added to improve the organization and readability of the manuscript.

9. Results Interpretation: The results indicate that fish fed the soybean oil diet showed higher growth and ovarian somatic index (OSI), although the differences were not statistically significant. They also exhibited lower liver and intestine somatic indices. However, these findings seem to contradict the proteomic analysis, which suggests better performance in fish fed the fish oil diet. This discrepancy should be addressed and explained in the discussion.

10. Discussion Section:

• The discussion focuses heavily on the effects of fish oil, with little to no mention of soybean oil and its impacts. A more balanced discussion, comparing the effects of both oil types, is necessary.
• The discussion is notably short and should be expanded to provide a more in-depth analysis of the results, their implications, and how they align with or differ from previous studies.

Author Response

General Comments:

The study conducted by He et al. investigated the effects of fish and soybean oils on proteomics changes in proteins associated with lipid metabolism, glucose metabolism, inflammation, antioxidant responses, and oxidative stress in female Scatophagus argus. While the study provides valuable insights into metabolic and proteomic changes, it could be repeated using male fish, fry, or growing-out fish, as the selected parameters do not directly serve ovarian development, as the authors seem to suggest. However, as a metabolic and proteomic study, it is well-conducted and could be suitable for publication after major revisions. The aim should be refocused on metabolic changes rather than directing the manuscript toward ovarian development.

Reply:The statement of the research objective has been revised and adjusted.

Specific Comments:

1.Study Focus and Reproductive Indicators:

The study primarily focuses on the effects of different lipid sources in relation to ovarian development in female broodstock. However, the investigated parameters only consider the ovary somatic index (OSI) as a reproductive indicator, neglecting the synthesis of key substances such as vitellogenin (Vtg), which is crucial for assessing ovarian development.

Reply:Thank you for your suggestions. Discussing the impact on ovarian development does indeed require evaluating these indicators. This experiment is a continuation of our previous transcriptome study. During sampling, we recorded these exogenous indicators first, but in the proteomic results, the expression of reproductive-related proteins such as VTG did not show significant differences. We have adjusted the statement of the research objective to focus on metabolic changes.

2.Line 48-56: Additional references should be added to support the cited information, as this would strengthen the background and justification for the study.

Reply:The original text has been supplemented and revised in Line 48-61.

3.Line 56-58: The text refers to the nutritional requirements of marine broodstock, but Scatophagus argus is a freshwater or brackish water species (tolerating up to 8 ppm salinity). This discrepancy should be clarified or corrected.

Reply:The original text has been supplemented and clarified in Line 61-63.

4.Materials and Methods:

The control group is not clearly defined. It is essential to specify which group serves as the control.

Reply:It has been revised in Line 131-132.

The number of treatments is limited. Including a mixture of fish oil and plant oil (e.g., soybean oil) as an additional treatment could provide more comprehensive insights. Furthermore, exploring other plant oils, such as corn oil, might offer a cost-effective alternative while meeting the fish's nutritional requirements.

Reply:Thank you for your suggestions. The study on partially replacing fish oil with plant oil is of great significance for the sustainable development of the aquaculture industry. This experiment is an extension of the transcriptome study, with the initial aim of combining transcriptome data to identify proteomic evidence supporting the conclusion that fish oil promotes ovarian development in medaka fish. Therefore, the experimental design was based on the transcriptome study, with no additional treatment groups.

The lipid profile of each oil used should be provided, as the interpretation of results heavily depends on this information.

Reply:The original text has been supplemented and revised in Table 1.

5.Line 103: The country name should be added for clarity.

Reply:The original text has been revised in Line 117.

6.Vitamin C Addition:

The sentence mentioning the addition of lactic acid bacteria and vitamin C to the water is unclear. Please clarify whether vitamin C was added to the water or the diet, as this could significantly impact the interpretation of results.

Reply:The description in the original text can be found in Line 129-130.

7.Line 116:the number of replicates should be clarified. Is it two replicates per treatment? This information is critical for assessing the statistical robustness of the study.

Reply:The original text has been supplemented and revised in Line 134-135.

8.Line 119: A separate subtitle, such as "Growth and Somatic Indices," should be added to improve the organization and readability of the manuscript.

Reply:The original text has been supplemented and revised in Line 143.

9.Results Interpretation: The results indicate that fish fed the soybean oil diet showed higher growth and ovarian somatic index (OSI), although the differences were not statistically significant. They also exhibited lower liver and intestine somatic indices. However, these findings seem to contradict the proteomic analysis, which suggests better performance in fish fed the fish oil diet. This discrepancy should be addressed and explained in the discussion.

Reply:The original text has been supplemented and revised in scetion 4.1.

10.Discussion Section:

The discussion focuses heavily on the effects of fish oil, with little to no mention of soybean oil and its impacts. A more balanced discussion, comparing the effects of both oil types, is necessary.

Reply:The discussion section of the original text has been completely revised in section 4.

The discussion is notably short and should be expanded to provide a more in-depth analysis of the results, their implications, and how they align with or differ from previous studies.

Reply:The discussion section of the original text has been completely revised in section 4 and 5.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

Proteomics analysis reveals that dietary supplementation with 2 fish oil enhances lipid metabolism and improves antioxidant 3 capacity in the liver of female Scatophagus argus

Introduction

Lines 65-76. Describes the effect of fish oil on ovarian development and functionality. Does the same occurs with vegetable oils?, Please edit the paragraph by adding supportive information.

Line 92. Add a paragraph indicating the importance of replacing “oil fish” with “vegetable oil”.  

Line 91. Please enhance this statement by adding relevant information from the transcriptomic study “Transcriptomic analyses further reveal the effects of fish oil versus soybean oil diets on liver function in S. argus”

Lines 92-98, please edit the aims of the study. Was the comparative proteomic profile between fish oil and soybean oil the main objective?, or only the fish oil diets?.

M & M

Line 108. Please add the size and weight of fish before starting the feeding trial

Line 115. Comment: In the FO and SO comparative assay, Please add a line indicating why “no” controls were included or if one of the groups can be considered a control group, please add the legend “control group” after the appropriate group. Also, add appropriate references in this section to support the study design.   

Results

Line 188. Comment: In this section the authors are describing in table 2 the key growth indicators, including CF, GSI, HSI, and VSI from 3 fishes while in the M & M section the authors are using 50 fish for FO and 50 fish for SO. Please clarify.

Table 2. Legend. Add “soyabean oil” after “fish oil”

General comment for this section: It will be useful to the reader to describe the up and down regulated proteins by section: 1) fish oil. 2) Soybean oil and 3) shared proteins.

For instance, figure 2, 3 & 4 legends describe the comparative evaluation between FO and SO, but in the figure it is difficult to interpret such differences as data are plotted as a whole.

Discussion

Lines 247-250. “This study investigates the regulatory effects of fish oil and soybean oil diets on gonadal development and reproductive mechanisms in female S. argus by identifying protein-level evidence. Coupled with prior transcriptomic data from our team [14], the findings elucidate the mechanisms through which dietary fish oil promotes ovarian maturation at the protein level in broodstock”

Comment: The authors have mentioned this statement in the introduction section, but this study was focused on the comparative proteomic profile of liver from FO and SO. Is this experiment part of the transcriptomic study?, if so please clarify it in the study.  In the study design there is no evidence of a gonadal development study so far.

Section 4.1 Effects of Fish Oil on Liver Metabolism

Please, edit the whole section; the authors are describing the up and down regulated proteins associated to lipid metabolism in the liver and also in the gonads. But as mentioned above, in this study, there is no evidence of gonadal development study.

General comment: This section should be improved, the authors are performing a comparative evaluation between fish oil and soyabean oil, but no the latter is not discussed. I found the study interesting, but some edition is needed to fully address the main objective of the study. Please highlight the importance of the study and its limitation.

Conclusion: The conclusion section also needs some edition. For instance in table 2, the authors show that there is no difference in growth and index values between FO and SO.

Author Response

Reviewer2:

Comments and Suggestions for Authors
Proteomics analysis reveals that dietary supplementation with 2 fish oil enhances lipid metabolism and improves antioxidant 3 capacity in the liver of female Scatophagus argus

Introduction

Lines 65-76. Describes the effect of fish oil on ovarian development and functionality. Does the same occurs with vegetable oils?Please edit the paragraph by adding supportive information.
Reply: Plant oils mainly contain n-6 polyunsaturated fatty acids (such as linoleic acid, LA), short-chain n-3 precursors (such as alpha-linolenic acid, ALA), and fat-soluble vitamins, which can effectively enhance ovarian steroidogenesis and promote an increase in the expression of sex steroid synthase transcripts. The original paper has been revised and supplemented in Line 82-85.

Line 92. Add a paragraph indicating the importance of replacing "oil fish" with "vegetable oil".
Reply:The original paper has been revised and supplemented in Line 85-90.

Line 91. Please enhance this statement by adding relevant information from the transcriptomic study "Transcriptomic analyses further reveal the effects of fish oil versus soybean oil diets on liver function in S. argus"
Reply:The original paper has been revised and supplemented in Line 102-109.

Lines 92-98, please edit the aims of the study. Was the comparative proteomic profile between fish oil and soybean oil the main objective?, or only the fish oil diets?
Reply:The objective of the study is to focus on the effect of adding fish oil to the feed, while comparing the proteomic differences between the two groups is the method. The original paper has been revised and supplemented in Line 109-110.

M & M

Line 108. Please add the size and weight of fish before starting the feeding trial
Reply:The original paper has been revised and supplemented in Line 117-119.

Line 115. Comment: In the FO and SO comparative assay, Please add a line indicating why "no" controls were included or if one of the groups can be considered a control group, please add the legend "control group" after the appropriate group. Also, add appropriate references in this section to support the study design.
Reply:The feed must include a lipid source (oil source); without lipids, fish cannot grow properly. In this experiment, the soybean oil group serves as the control group, which has been revised and marked as the control group in the original text. The experimental design is based on relevant literature (Line 139-142).

Results

Line 188. Comment: In this section the authors are describing in table 2 the key growth indicators, including CF, GSI, HSI, and VSI from 3 fishes while in the M & M section the authors are using 50 fish for FO and 50 fish for SO. Please clarify.
Reply:A total of 50 fish were raised in both the FO group and the SO group, with 3 fish randomly selected from each group for subsequent experiments. The original text has been supplemented in Line 211-212.

Table 2. Legend. Add "soyabean oil" after "fish oil"
Reply:Done (Line 216).

General comment for this section: It will be useful to the reader to describe the up and down regulated proteins by section: 1) fish oil. 2) Soybean oil and 3) shared proteins.
Reply:The results section has been rewritten (section 3.2).

For instance, figure 2, 3 & 4 legends describe the comparative evaluation between FO and SO, but in the figure it is difficult to interpret such differences as data are plotted as a whole.
Reply:The results section has been rewritten (section 3.2).

Discussion

Lines 247-250. "This study investigates the regulatory effects of fish oil and soybean oil diets on gonadal development and reproductive mechanisms in female S. argus by identifying protein-level evidence. Coupled with prior transcriptomic data from our team [14], the findings elucidate the mechanisms through which dietary fish oil promotes ovarian maturation at the protein level in broodstock"
Comment: The authors have mentioned this statement in the introduction section, but this study was focused on the comparative proteomic profile of liver from FO and SO. Is this experiment part of the transcriptomic study?, if so please clarify it in the study.  In the study design there is no evidence of a gonadal development study so far.
Reply:In this study, no differentially expressed proteins related to reproductive regulation were detected. It should be noted that the liver transcriptome analysis mentioned in the text belongs to an independent experimental module, and the entire section of the original text has been revised (section 4).

Section 4.1 Effects of Fish Oil on Liver Metabolism
Please, edit the whole section; the authors are describing the up and down regulated proteins associated to lipid metabolism in the liver and also in the gonads. But as mentioned above, in this study, there is no evidence of gonadal development study.
Reply:The entire section of the original text has been revised (section 4.1).

General comment: This section should be improved, the authors are performing a comparative evaluation between fish oil and soyabean oil, but no the latter is not discussed. I found the study interesting, but some edition is needed to fully address the main objective of the study. Please highlight the importance of the study and its limitation.
Reply:The innovation lies in the application of proteomics to reveal the mechanism by which fish oil feed regulates the metabolism of Scatophagus argus liver. The limitations are as follows: 
(1)The liver transcriptome analysis experiment used different fish, so the experimental results may be influenced by factors such as different feeding conditions, genetic background, and individual differences.
(2)The experimental grouping only includes two groups, with no additional experimental groups involving gradient soybean oil replacement for fish oil. The entire section of the original text has been revised (section 4).

Conclusion: The conclusion section also needs some edition. For instance in table 2, the authors show that there is no difference in growth and index values between FO and SO.
Reply:The entire section of the original text has been revised (section 4).

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

please see in the attached document

Comments for author File: Comments.pdf

Author Response

Reviewer3:

Proteomics analysis reveals that dietary supplementation with fish oil enhances lipid metabolism and improves antioxidant capacity in the liver of female Scatophagus argus  
GENERAL conclusion on the manuscript  
This study investigated the effects of dietary fish oil supplementation on liver function in female spotted scat (Scatophagus argus) using proteomic analysis. The topic has novelty because is focusing to a new aspect of correlation in metabolic function of liver and protein expression. Compared to soybean oil, fish oil supplementation led to upregulation of proteins involved in lipid metabolism, including Commd1, Tspan8, Mb, Tmem41b, Sdf2l1, and Pex5. Additionally, proteins associated with glucose metabolism, inflammation, and antioxidant responses were elevated in the fish oil group, while proteins linked to oxidative stress were downregulated. 
These findings suggest that dietary fish oil enhances hepatic lipid metabolism and improves the liver's antioxidant capacity, potentially supporting ovarian maturation in S. argus. The results are consistent with previous transcriptomic data and provide proteomic-level evidence for the role of n3 long-chain polyunsaturated fatty acids in modulating hepatic lipid and energy metabolism, thereby influencing reproductive processes in teleost fish  
The introduction clearly defines the main idea of the study, the objectives are clearly stated. The  methods used in the studies are well formulated. The results are presented in a clear and wellformatted set of tables and graphs, which contain all the relevant information found. Overall, the  experiment is well designed, the findings discussed deeply and compared with relevant studies. The English language is perfectly adequate.  
There are some mirror comments that needs to be included into manuscript before publication.  

Specific comments  

LINE 109 Information related to feeding rate and frequency is required. Were the fish fed ad libitum?
Reply:The daily feeding rate for the Scatophagus argus in the breeding experiment was approximately 2.0-2.8%, with feeding occurring twice daily, in the morning and evening. The original text has been supplemented and revised in Line 136-138.

LINE 132, 139 Please give the exact name for SDS and DTT, UA buffer
Reply:The original text has been supplemented and revised in Line 157,158 and 165.

LINE 196 The title of this section needs clarification: e.g., Comparative proteomic analysis of fish fed by using diets....
Reply:Done (Line 220).

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The author used one answer for 90% of the questions (The original text has been supplemented and revised in Line ….) this is not acceptable even the mentioned line does not match the manuscript.

As the authors confirmed the addition of vitamin C to the rearing water, I could not find any practical application of this step it is not logic or how much it cost.

The fatty acids profile was mentioned in table 1, but how it was determined was not mentioned in the M and M.

Comments on the Quality of English Language

The language needs as second check, and the general format and spacing need to be considered. 

Author Response

Comments and Suggestions for Authors

The author used one answer for 90% of the questions (The original text has been supplemented and revised in Line ….) this is not acceptable even the mentioned line does not match the manuscript.

Reply: Thank you for your suggestion. The line numbers previously referenced corresponded to the clean version of the manuscript. These have now been updated to align with the tracked-changes version that highlights all revisions. We have comprehensively elaborated on our responses and the specific modifications made in the revised text (edits in green, additions in yellow). All changes in the manuscript are marked with color-coded text and strikethroughs for clarity.

As the authors confirmed the addition of vitamin C to the rearing water, I could not find any practical application of this step it is not logic or how much it cost.

Reply: Thank you for your suggestion. Previous studies have demonstrated that waterborne vitamin C supplementation alleviates oxidative stress in zebrafish (Danio rerio) [1] and enhances immunity and antioxidant capacity in grass carp (Ctenopharyngodon idella) [2]. As you rightly noted, this intervention is not strictly indispensable. However, the current experiment serves as an extension of our prior transcriptomic research, and the experimental design—including the vitamin C administration protocol—was intentionally kept consistent with the transcriptomic study to ensure methodological continuity.

[1]Paduraru, E., Flocea, E. I., Lazado, C. C., Simionov, I. A., Nicoara, M., Ciobica, A., Faggio, C., & Jijie, R. (2021). Vitamin C Mitigates Oxidative Stress and Behavioral Impairments Induced by Deltamethrin and Lead Toxicity in Zebrafish. International journal of molecular sciences, 22(23), 12714.

[2]Iqbal, K. J., Majeed, H., Iqbal, K. J., Asghar, M., Azmat, H., Fatima, M., Khan, N., Baboo, I., Tehseen, A., Ali, W., Saeed, U., Khizar, A., Fatima, A., Nisa, S., & Davies, S. J. (2023). Administration of vitamin E and C enhances immunological and biochemical responses against toxicity of silver nanoparticles in grass carp (Ctenopharyngodon idella). PloS one, 18(4), e0284285.

The fatty acids profile was mentioned in table 1, but how it was determined was not mentioned in the M and M.

Reply: Thank you for your feedback. Comparative lipid compositions of the dietary oil formulations (with fatty acid profiles analyzed by Tongwei Company, China) are detailed in Table 1. We have supplemented this methodological description in the revised manuscript (Line 150-152).

Comments on the Quality of English Language

The language needs as second check, and the general format and spacing need to be considered.

Reply: Thank you for your suggestions. We have thoroughly reviewed and revised the entire manuscript. All modifications are marked as follows: edits in green, additions in yellow, and deletions with strikethroughs for clarity.

Reviewer 1:

General Comments:

The study conducted by He et al. investigated the effects of fish and soybean oils on proteomics changes in proteins associated with lipid metabolism, glucose metabolism, inflammation, antioxidant responses, and oxidative stress in female Scatophagus argus. While the study provides valuable insights into metabolic and proteomic changes, it could be repeated using male fish, fry, or growing-out fish, as the selected parameters do not directly serve ovarian development, as the authors seem to suggest. However, as a metabolic and proteomic study, it is well-conducted and could be suitable for publication after major revisions. The aim should be refocused on metabolic changes rather than directing the manuscript toward ovarian development.

Reply:The statement of the research objective has been revised and adjusted.

Specific Comments:

1.Study Focus and Reproductive Indicators:

The study primarily focuses on the effects of different lipid sources in relation to ovarian development in female broodstock. However, the investigated parameters only consider the ovary somatic index (OSI) as a reproductive indicator, neglecting the synthesis of key substances such as vitellogenin (Vtg), which is crucial for assessing ovarian development.

Reply:Thank you for your suggestions. Discussing the impact on ovarian development does indeed require evaluating these indicators. This experiment is a continuation of our previous transcriptome study. During sampling, we recorded these exogenous indicators first, but in the proteomic results, the expression of reproductive-related proteins such as VTG did not show significant differences. We have adjusted the statement of the research objective to focus on metabolic changes.

2.Line 48-56: Additional references should be added to support the cited information, as this would strengthen the background and justification for the study.

Reply:Thank you for your comment. We have supplemented the text with statements and citations based on the key terms species, lipids, and broodstock to clarify their roles in lipid utilization and reproductive physiology. These revisions are detailed in Lines 56-60 of the revised manuscript.

3.Line 56-58: The text refers to the nutritional requirements of marine broodstock, but Scatophagus argus is a freshwater or brackish water species (tolerating up to 8 ppm salinity). This discrepancy should be clarified or corrected.

Reply:Thank you for your comment. We have supplemented the text with descriptions highlighting the physiological adaptations of Scatophagus argus as a euryhaline fish species. These revisions are detailed in Lines 69-71 of the revised manuscript.

4.Materials and Methods:

The control group is not clearly defined. It is essential to specify which group serves as the control.

Reply:Thank you for your suggestion. We have explicitly emphasized in the revised manuscript (Lines 146-147) that the FO group serves as the experimental group and the SO group as the control group.

The number of treatments is limited. Including a mixture of fish oil and plant oil (e.g., soybean oil) as an additional treatment could provide more comprehensive insights. Furthermore, exploring other plant oils, such as corn oil, might offer a cost-effective alternative while meeting the fish's nutritional requirements.

Reply:Thank you for your suggestions. The study on partially replacing fish oil with plant oil is of great significance for the sustainable development of the aquaculture industry. This experiment is an extension of the transcriptome study, with the initial aim of combining transcriptome data to identify proteomic evidence supporting the conclusion that fish oil promotes ovarian development in medaka fish. Therefore, the experimental design was based on the transcriptome study, with no additional treatment groups.

The lipid profile of each oil used should be provided, as the interpretation of results heavily depends on this information.

Reply:The original text has been supplemented and revised in Table 1.

5.Line 103: The country name should be added for clarity.

Reply:The original text has been revised in Line 117.

6.Vitamin C Addition:

The sentence mentioning the addition of lactic acid bacteria and vitamin C to the water is unclear. Please clarify whether vitamin C was added to the water or the diet, as this could significantly impact the interpretation of results.

Reply:The description in the original text can be found in Line 144-145.

7.Line 116:the number of replicates should be clarified. Is it two replicates per treatment? This information is critical for assessing the statistical robustness of the study.

Reply:Thank you for your comment. We have supplemented the manuscript with the experimental design details: two replicates per group, each containing 25 fish. This information has been explicitly added in Lines 149-150 of the revised text.

8.Line 119: A separate subtitle, such as "Growth and Somatic Indices," should be added to improve the organization and readability of the manuscript.

Reply:Thank you for your suggestion. We have added the subsection as Section 2.3 in the revised manuscript (Line 159).

9.Results Interpretation: The results indicate that fish fed the soybean oil diet showed higher growth and ovarian somatic index (OSI), although the differences were not statistically significant. They also exhibited lower liver and intestine somatic indices. However, these findings seem to contradict the proteomic analysis, which suggests better performance in fish fed the fish oil diet. This discrepancy should be addressed and explained in the discussion.

Reply:Thank you for your comment. Regarding the non-significant differences in these metrics, we have supplemented the discussion in Section 4.1 by citing relevant literature and addressing potential influences such as variations in rearing conditions, genetic backgrounds, and individual differences.

10.Discussion Section:

The discussion focuses heavily on the effects of fish oil, with little to no mention of soybean oil and its impacts. A more balanced discussion, comparing the effects of both oil types, is necessary.

Reply:Thank you for your suggestion. In the Discussion section (Lines 408-415,438-442), we have supplemented descriptions highlighting that plant oils and fish oil may yield divergent outcomes across different fish species. This underscores the necessity of tailoring nutrient-specific feed formulations to species-specific requirements.

The discussion is notably short and should be expanded to provide a more in-depth analysis of the results, their implications, and how they align with or differ from previous studies.

Reply:Thank you for your suggestion. We have comprehensively revised and rephrased the Discussion section (Section 4) to include statements on plant oil substitution and species-specific feed formulation adjustments. Additionally, in the Conclusions (Section 5), we have incorporated a comparative analysis with our prior transcriptomic study, alongside a discussion of the current limitations and future research directions.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

I do not have further observations. 

Comments on the Quality of English Language

The documents need some furhter English edition

Author Response

Comments on the Quality of English Language

The documents need some furhter English edition.

Reply: Thank you for your suggestions. We have thoroughly reviewed and revised the entire manuscript. All modifications are marked as follows: edits in green, additions in yellow, and deletions with strikethroughs for clarity.

Reviewer 2:

Comments and Suggestions for Authors

Proteomics analysis reveals that dietary supplementation with 2 fish oil enhances lipid metabolism and improves antioxidant 3 capacity in the liver of female Scatophagus argus

Introduction

Lines 65-76. Describes the effect of fish oil on ovarian development and functionality. Does the same occurs with vegetable oils?Please edit the paragraph by adding supportive information.

Reply: Thank you for your valuable comment. Plant oils primarily contain n-6 polyunsaturated fatty acids (such as linoleic acid, LA), short-chain n-3 precursors (such as α-linolenic acid, ALA), and fat-soluble vitamins, which can effectively enhance ovarian steroidogenesis and increase the expression of sex steroid synthase transcripts. We have supplemented the revised manuscript (Lines 90-93) with literature describing that plant oils can also regulate reproductive functions in certain fish species. This phenomenon may be attributed to interspecific differences in the utilization of essential fatty acids, highlighting the need to tailor nutrient-specific feed formulations according to species-specific requirements.

Line 92. Add a paragraph indicating the importance of replacing "oil fish" with "vegetable oil".

Reply:Thank you for your comment. With global supplies constrained by overfishing and ecological pressures, optimizing the ratio of plant oils to fish oil in aquafeed formulations is critical for the sustainable development of aquaculture. We have revised and supplemented the manuscript (Lines 93-98) to elaborate on the advantages of plant oils and the significance of fish oil replacement.

Line 91. Please enhance this statement by adding relevant information from the transcriptomic study "Transcriptomic analyses further reveal the effects of fish oil versus soybean oil diets on liver function in S. argus"

Reply:Thank you for your comment. Fish oil supplementation enhanced hepatic lipid metabolic efficiency through coordinated regulation of lipid homeostasis genes (fasn, acox1, apob) involved in synthesis, catabolism, and transport processes. Simultaneously, it upregulated mitochondrial bioenergetic regulators (ppara, cpt1a, ucp2) governing energy transduction pathways, thereby optimizing hepatic energy allocation. Notably, fish oil demonstrated pleiotropic regulatory effects on reproductive endocrine genes (star, cyp19a1, vtg) associated with steroidogenesis and oogenesisThe original paper has been revised and supplemented in Line 110-117.

Lines 92-98, please edit the aims of the study. Was the comparative proteomic profile between fish oil and soybean oil the main objective?, or only the fish oil diets?

Reply:The objective of the study is to focus on the effect of adding fish oil to the feed, while comparing the proteomic differences between the two groups is the method. The original paper has been revised and supplemented in Line 121-123.

M & M

Line 108. Please add the size and weight of fish before starting the feeding trial

Reply:Thank you for your comment. The mean initial body weight of the experimental fish was 242.83 ± 16.10 g, with a total body length of 19.48 ± 0.36 cm. We have supplemented this baseline information in Line 132-134.

Line 115. Comment: In the FO and SO comparative assay, Please add a line indicating why "no" controls were included or if one of the groups can be considered a control group, please add the legend "control group" after the appropriate group. Also, add appropriate references in this section to support the study design.

Reply:Thank you for your feedback. The feed must include a lipid source (oil source), as fish cannot grow properly without dietary lipids. In this experiment, the soybean oil group serves as the control group, which has been explicitly labeled as such in the revised manuscript. This study is an extension of our prior transcriptomic research, aiming to identify protein-level evidence supporting the role of fish oil in promoting ovarian development in Scatophagus argus. Consequently, the experimental design aligns with the methodology of the transcriptomic study. Relevant references supporting the experimental design have been added to the revised manuscript (Lines 146,147,155-158).

Results

Line 188. Comment: In this section the authors are describing in table 2 the key growth indicators, including CF, GSI, HSI, and VSI from 3 fishes while in the M & M section the authors are using 50 fish for FO and 50 fish for SO. Please clarify.

Reply:A total of 50 fish were raised in both the FO group and the SO group, with 3 fish randomly selected from each group for subsequent experiments. The original text has been supplemented in Line 227-228.

Table 2. Legend. Add "soyabean oil" after "fish oil"

Reply:Done (Line 232).

General comment for this section: It will be useful to the reader to describe the up and down regulated proteins by section: 1) fish oil. 2) Soybean oil and 3) shared proteins.

Reply:Thank you for your suggestion. We have substantially revised the Results section by reorganizing the findings into three distinct categories: co-expressed proteins, soybean oil-specific proteins, and fish oil-specific proteins. These modifications, including updated figures and textual clarifications, are detailed in Section 3.2 of the revised manuscript..

For instance, figure 2, 3 & 4 legends describe the comparative evaluation between FO and SO, but in the figure it is difficult to interpret such differences as data are plotted as a whole.

Reply:Thank you for your suggestion. We have rewritten the Results section and reorganized the graphical representations (Table 2, Figures 2-8) to specifically highlight three key aspects: co-expressed proteins, soybean oil-specific proteins, and fish oil-specific proteins..

Discussion

Lines 247-250. "This study investigates the regulatory effects of fish oil and soybean oil diets on gonadal development and reproductive mechanisms in female S. argus by identifying protein-level evidence. Coupled with prior transcriptomic data from our team [14], the findings elucidate the mechanisms through which dietary fish oil promotes ovarian maturation at the protein level in broodstock"

Comment: The authors have mentioned this statement in the introduction section, but this study was focused on the comparative proteomic profile of liver from FO and SO. Is this experiment part of the transcriptomic study?, if so please clarify it in the study.  In the study design there is no evidence of a gonadal development study so far.

Reply:In this study, no differentially expressed proteins related to reproductive regulation were detected. It should be noted that the liver transcriptome analysis mentioned in the text belongs to an independent experimental module, and the entire section of the original text has been revised (section 4).

Section 4.1 Effects of Fish Oil on Liver Metabolism

Please, edit the whole section; the authors are describing the up and down regulated proteins associated to lipid metabolism in the liver and also in the gonads. But as mentioned above, in this study, there is no evidence of gonadal development study.

Reply:Thank you for your suggestion. We acknowledge the lack of differential expression evidence of reproduction-related proteins in this study. Consequently, we have removed the gonad-related content and refocused the analysis on metabolic changes. These revisions are detailed in Section 4.1 of the revised manuscript.

General comment: This section should be improved, the authors are performing a comparative evaluation between fish oil and soyabean oil, but no the latter is not discussed. I found the study interesting, but some edition is needed to fully address the main objective of the study. Please highlight the importance of the study and its limitation.

Reply:Thank you for your feedback. In the Discussion section, we have supplemented descriptions highlighting that plant oils and fish oil may yield divergent outcomes across different fish species. This underscores the necessity of tailoring nutrient-specific feed formulations to species-specific requirements.

Innovation:

The novelty of this study lies in the application of proteomics to elucidate the mechanisms by which fish oil regulates hepatic metabolism in Scatophagus argus.

Limitations:

Heterogeneity in experimental subjects: The liver transcriptome analysis utilized fish from a separate cohort, and thus the results may be influenced by variations in rearing conditions, genetic backgrounds, and individual differences.

Simplified experimental design: The study employed only two experimental groups (soybean oil vs. fish oil), omitting gradient-based replacement trials to assess incremental effects.

These revisions are comprehensively addressed in Section 4 of the revised manuscript.

Conclusion: The conclusion section also needs some edition. For instance in table 2, the authors show that there is no difference in growth and index values between FO and SO.

Reply:Thank you for your suggestion. We have supplemented the discussion regarding the lack of statistically significant differences in growth performance between the FO group and SO group. This observation aligns with prior studies demonstrating that lipid sources (e.g., plant vs. fish oil) may not universally affect growth outcomes in fish, as cited in the revised manuscript (Section 4). Relevant literature supporting this phenomenon has been incorporated to strengthen the interpretation.

Author Response File: Author Response.pdf