-->PONE-D-25-30716-->-->A reliable in vitro rumen culture system and workflow for screening anti-methanogenic compounds-->-->PLOS ONE

Dear Dr. Sabass,

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Academic Editor

PLOS ONE

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Additional Editor Comments:

Based on the reviewers’ evaluation, the current manuscript does not fully meet our stringent requirements for publication. However, we believe it holds potential and may be accepted pending major revisions. The required corrections are substantial and must address all concerns raised by the examiners. Authors are expected to revise the manuscript thoroughly and provide a detailed response to each comment. Only after careful consideration of the revised version will a final decision be made. We encourage the authors to review the feedback closely and make the necessary improvements to enhance the quality and clarity of their submission

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

-->Comments to the Author

1. Does the manuscript report a protocol which is of utility to the research community and adds value to the published literature?-->

Reviewer #1: Yes

Reviewer #2: Yes

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-->2. Has the protocol been described in sufficient detail?

To answer this question, please click the link to protocols.io in the Materials and Methods section of the manuscript (if a link has been provided) or consult the step-by-step protocol in the Supporting Information files.

The step-by-step protocol should contain sufficient detail for another researcher to be able to reproduce all experiments and analyses.-->

Reviewer #1: Partly

Reviewer #2: Yes

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-->3. Does the protocol describe a validated method?

The manuscript must demonstrate that the protocol achieves its intended purpose: either by containing appropriate validation data, or referencing at least one original research article in which the protocol was used to generate data.-->

Reviewer #1: Yes

Reviewer #2: Yes

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-->4. If the manuscript contains new data, have the authors made this data 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: N/A

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-->5. Is the article 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 highlight any specific errors that need correcting in the box below. -->

Reviewer #1: Yes

Reviewer #2: Yes

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-->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: Overall, the manuscript is comprehensive and addresses an important issue. However, several aspects should be improved. Major revisions are suggested below to enhance the manuscript:

• The abstract is too general. Include specific results.

• Provide more quantitative data (e.g., effect sizes, statistical comparisons) to support claims about system performance.

• What is this “everyday labware”. It is vague and informal for a research article.

• There are formatting mistakes throughout the article. Like font style, alignment, and subscript. The figure numbers should be checked again.

• All the figures are not clear. Reproduce them.

• Check the reference style

• Please provide the list of abbreviations or table for abbreviations

• The discussion often lacks clarity in structure and logical flow, making it difficult to follow key conclusions.

• Discussion should include statistical comparisons or confidence intervals to assess significance across systems.

• The discussion lacks targeted reasoning.

• Enhance the interpretation of microbiome data, particularly the discrepancy between methanogen abundance and CH4 output.

• The authors should justify use of highly variable inocula.

• The final paragraph lacks a critical reflection on the system’s limitations, such as microbial drift over time, absence of host factors, or scalability to large screening efforts.

• The discussion is too short. It must be revised and explain every aspect in detail.

Reviewer #2: The authors tried to present an overview of anti-methanogenic compounds to mitigate the emission of these GHGs. However, there are several parts that need to be revised before publication.

1. The abstract of the article needs to be revised as it lacks key findings in the article.

2. The author should clarify why they chose T powder, BF, IF, 3-NOP, and rapeseed oil (NCOIL), and explain the rationale behind the concentrations of these supplements.

3. The image quality in the paper is poor. The author should revise these figures to improve readability.

4. The author should mention the material along with the method.

5. There is a lot of referencing to supplementary files (for example, S1 Fig, S2 File), but the main text lacks context.

6. There are various typo errors, such as "abbundances" (line 325). The author must improve and avoid these errors.

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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.

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Reviewer #1: Yes:  Syed Muhammad wajahat ul hasnain

Reviewer #2: No

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Attachments

Attachment

Submitted filename: PONE-D-25-30716.pdf

A reliable in vitro rumen culture system and workflow for screening anti-methanogenic compounds

PONE-D-25-30716R1

Dear Dr. Sabass,

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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Kind regards,

Bawadi Abdullah

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

recommended based on the review comments.

Reviewers' comments:

Reviewer's Responses to Questions

-->Comments to the Author

1. Does the manuscript report a protocol which is of utility to the research community and adds value to the published literature?-->

Reviewer #1: Yes

Reviewer #2: Yes

**********

-->2. Has the protocol been described in sufficient detail?

To answer this question, please click the link to protocols.io in the Materials and Methods section of the manuscript (if a link has been provided) or consult the step-by-step protocol in the Supporting Information files.

The step-by-step protocol should contain sufficient detail for another researcher to be able to reproduce all experiments and analyses.-->

Reviewer #1: Yes

Reviewer #2: Yes

**********

-->3. Does the protocol describe a validated method?

The manuscript must demonstrate that the protocol achieves its intended purpose: either by containing appropriate validation data, or referencing at least one original research article in which the protocol was used to generate data.-->

Reviewer #1: Yes

Reviewer #2: Yes

**********

-->4. If the manuscript contains new data, have the authors made this data 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: N/A

Reviewer #2: No

**********

-->5. Is the article 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 highlight any specific errors that need correcting in the box below. -->

Reviewer #1: Yes

Reviewer #2: 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 revised manuscript has addressed the earlier concerns, and the authors have made significant improvements to strengthen the overall quality of the paper. The methodology is clearly presented, and the results are now better supported with relevant discussion. I recommend the paper for acceptance in its present form without any further changes.

Reviewer #2: The manuscript has officially been accepted in its current form, and no additional changes or corrections are necessary before publication.

**********

-->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: No

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