-->PONE-D-26-08918-->-->Pyridoxine requirement of Pacific white shrimp (Penaeus vannamei) fed soybean meal based diet-->-->PLOS One

Dear Dr. Lee,

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Amel Mohamed El Asely

Academic Editor

PLOS One

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Reviewers' comments:

Reviewer's Responses to Questions

-->Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. -->

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Partly

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-->2. Has the statistical analysis been performed appropriately and rigorously? -->

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: No

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-->3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.-->

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: No

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-->4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.-->

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

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-->5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)-->

Reviewer #1: There are some comments and necessary points must be met before publishing for paper improvements:

-Some editing for English language is required throughout the manuscript

mention the kit's Catalog number and reference used for

non-specific immune response and antioxidant capacity and Digestive enzyme analysis , methods should be in more detail.

Reviewer #2: - Please provide references for the selected dose of tetracycline hydrochloride used in this study.

- Please verify the sequence of the forward primer for the beta-actin gene listed in the table 2.

- Authors, could you explain the difference in growth performance as presented in Table 3, specifically between P150 and P75?

- Please indicate the size of the amplicons produced by each primer set for the various genes in Table 2.

- Please clarify how non-specific amplification of genes was ruled out in this study.

Reviewer #3: The manuscript has several ambiguities that need to be addressed before consideration. These are listed below;

1. The experimental design lacks adequate justification. The rationale behind the selection of treatments, such as the use of tetracycline as a control and the inclusion levels of pyridoxine, is not clearly explained. Without a strong scientific basis for these choices, it is difficult to assess whether the design appropriately addresses the research objectives.

2. The study does not clearly define a specific hypothesis or research question. The objectives appear descriptive rather than hypothesis-driven, which weakens the work's scientific direction and limits its contribution to theory- or mechanism-based understanding.

3. There is an insufficient mechanistic explanation of the results. While improvements in growth, antioxidant activity, or immunity are reported, the manuscript does not adequately explain the biological pathways through which pyridoxine exerts these effects. This results in a largely observational study rather than a mechanistic one.

4. The statistical analysis is inadequately described and potentially insufficient. Although methods such as ANOVA and regression are mentioned, there is no clear description of the statistical model, assumptions tested, or post hoc comparisons performed. This raises concerns about the robustness and reproducibility of the results.

5. There is limited validation of results across multiple parameters. The study relies heavily on biochemical and growth indicators but lacks complementary validation (e.g., molecular markers, gene expression, or histological evidence) to support its conclusions.

6. The sample size and experimental replication are not clearly justified. Without information on statistical power or replication strategy, it is difficult to determine whether the study is adequately powered to detect meaningful differences.

7. The discussion lacks critical comparison with existing literature. Although some references are cited, the manuscript does not adequately assess whether the findings align with or conflict with previous studies, nor does it explain any discrepancies.

8. There is a possible overinterpretation of results. The conclusions extend beyond the data presented, particularly in their suggestion of broad applications of pyridoxine, without sufficient supporting evidence or acknowledgment of study limitations.

9. The study does not adequately address potential confounding factors. Variables such as environmental conditions, feed composition variability, or biological variability among experimental units are not discussed, which may influence the results.

10. Also, the manuscript lacks a clear discussion of study limitations and external validity. There is no consideration of how the findings translate to real-world conditions or other species/systems, limiting the applicability of the research.

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-->6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review?  For information about this choice, including consent withdrawal, please see our Privacy Policy.-->

Reviewer #1: No

Reviewer #2: Yes:  Khawaja Ashfaque Ahmed

Reviewer #3: No

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Pyridoxine requirement of Pacific white shrimp (Penaeus vannamei) fed soybean meal based diet

PONE-D-26-08918R1

Dear Dr. Kyeong-Jun Lee,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

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If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

Kind regards,

Amel Mohamed El Asely

Academic Editor

PLOS One

Additional Editor Comments (optional):

Reviewers' comments:

Reviewer's Responses to Questions

-->Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.-->

Reviewer #1: All comments have been addressed

Reviewer #2: All comments have been addressed

Reviewer #3: All comments have been addressed

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-->2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. -->

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

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-->3. Has the statistical analysis been performed appropriately and rigorously? -->

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

**********

-->4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.-->

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

**********

-->5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.-->

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

**********

-->6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)-->

Reviewer #1: the authors have adequately addressed the comments raised in a previous round of review and I feel that this manuscript is now acceptable for publication

the manuscript is technically sound, and the data support the conclusions

It's a nice job to revise the manuscript and address the comments.

Reviewer #2: (No Response)

Reviewer #3: Dear Author(s),

I have carefully evaluated the revised manuscript and the authors’ detailed responses to the reviewers’ comments. The authors have adequately addressed the concerns raised during the review process and have made substantial improvements to the manuscript. The revised manuscript is now scientifically sound and well-organized, and the methodology, statistical analyses, discussion, and interpretation of results have been significantly strengthened. The additional clarifications regarding experimental design, replication strategy, study limitations, and comparison with previous literature have improved the overall quality and clarity of the work. In my opinion, the manuscript is suitable for publication in its present form, and I therefore recommend acceptance. I deeply appreciate the authors’ timely responses to all the comments and suggestions raised by the reviewers.

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-->7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review?  For information about this choice, including consent withdrawal, please see our Privacy Policy.-->

Reviewer #1: No

Reviewer #2: Yes:  Khawaja Ashfaque Ahmed

Reviewer #3: No

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