-->PONE-D-26-06317-->-->A polymorphism and expression levels of SCD and their relationships to production traits in a Japanese farmed emu population-->-->PLOS One

Dear Dr. Wada,

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Reviewers' comments:

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-->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: Partly

Reviewer #2: Yes

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

Reviewer #1: No

Reviewer #2: Yes

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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: No

Reviewer #2: No

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

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Reviewer #1: Yes

Reviewer #2: 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: Reviewer Comments

ABSTRACT:

The abstract emphasizes the study's significance, objectives, findings, and conclusions. The sentence stating that "However, the expression levels of SCD transcripts were negatively correlated with fat content (P < 0.01) and positively correlated with meat yield (P < 0.05), suggesting that increased SCD transcript levels in subcutaneous fat are associated with reduced fat content and higher meat production in emus" should be removed, as the study's focus and results do not directly support this conclusion.

Line 62-22

It should be clarified that although progeny testing can be time-consuming and expensive, it is considered highly accurate because its results reflect the combined genetic influence on quantitative traits such as meat yield, fat content, and fatty acid profile. In contrast, molecular genetic improvement is reliable only when the markers used are universal and applicable across various environments and management systems.

Line 74-79

It should be clarified that when associating genotype with production traits, the environmental conditions of emu rearing used in this study must be explicitly stated. The impact of the c.267 A>C substitution mechanism on SCD may vary across environments and management systems. Therefore, to provide a more comprehensive understanding, the research design should include testing across multiple environments and management systems.

Lines 83-91

It should be clarified that:

1.The information on rearing management, diet, and the origin of the reared emus, based on Koshiishi et al. 2022, is inaccurate because the data for this study were collected between 2015 and 2017, whereas the referenced study is from 2022. Including a brief note on the environmental conditions during data collection would improve clarity.

2.The number of datasets for male and female emus in the 266 samples should be increased.

3.A short explanation of how the melting point and adipose tissue fat composition data were obtained is recommended.

4.An explanation is needed for why 168 datasets were used for melting point analysis, whereas only 25 datasets were used for fatty acid composition.

Lines 140-143

1.The data analysis method should be explained clearly. Are the data sets for males and females analyzed separately?

2.An explanation is needed for why ANOVA and T-tests were performed using different approaches.

3.Why was an equilibrium test not conducted for the observed genotypes? Performing this test is essential to determine whether the gene allele frequencies at the emu farm are in equilibrium. This information would help discuss the results.

Line 167-175

Further clarification is needed in the Discussion section:

1.When analyzing this association, were male and female data examined separately or pooled together?

2.It is important to consider whether the findings could be affected by various factors, such as (1) the relatively small dataset; (2) allele frequencies that may not be be in equilibrium due to factors like selection, mutation, migration, or genetic drift; and (3) the large standard deviations (SD) observed in mean body weight, fat yield, and meat yield, which could result from measurement errors or individual variability within the population. These points should be discussed further.

Lines 182-183

These outcomes vary by method. The method should specify the amount of data utilized.

Lines 183-202

It should be clarified whether the relatively small data set affected the results, which showed no significant differences between genotypes in both males and females.

Lines 214-217 and 219-222

It is better to omit the term "higher or lower tendency" and instead focus on whether the difference is statistically significant, basing the discussion solely on the statistical results.

Lines 231-241

The results of this study depend on the sample size used. Therefore, the discussion section should clarify whether the processed data included male and female samples or was analyzed separately. If combined, were the data sets comparable or significantly different? The varying data sizes for males and females could affect the results.

Lines 269-274

It is best to compare only poultry research results, as comparing poultry and mammalian studies can introduce bias due to their different metabolisms and growth patterns. In the discussion, the author mainly explains the SCD gene and the factors suspected to influence it. It would be helpful to add an explanation that quantitative traits such as body weight, fatness, and fatty acid composition are influenced by multiple genes working together to express a trait in specific environments. Therefore, it’s important to discuss the roles of other genes in the SCD gene network to determine whether selecting only the SCD gene as the factor influencing body weight, fatness, and fatty acid composition in emus is appropriate or whether including other genes would strengthen the results.

Lines 320-321

The sentence "However, the expression levels of SCD transcripts were negatively correlated with fat content (P < 0.01) and positively correlated with meat yield (P < 0.05), suggests that increased SCD transcript levels in subcutaneous fat are associated with reduced fat content and higher meat production in emus" should be omitted because the research methods and design do not support this claim.

Lines 332-403

The author should focus solely on research findings related to poultry, as poultry metabolism differs from that of non-avian animals, especially mammals, with variations in growth processes and stages.

Figures

It is recommended to improve the clarity and sharpness of the images, as much of the text within the figure is hard to read.

Other remarks

1. To the best of my knowledge, this publication has not previously used emus as research subjects or examined the SCD gene.

2. The research has been approved by the research ethics committee of the Science Council of Japan and the Ethical Care and Use of Animals Committee at Tokyo University of Agriculture (approval numbers: 270049, 280002, 290096, 300126, and 2019109).

3. To my knowledge, the research was conducted according to approved protocols and did not violate publication ethics.

Reviewer #2: This article provides information on the effects of SCD polymorphism and expression levels on production traits in a Japanese farmed emu population. It is in general appropriately organized, carried out and written, however there are some points that should be corrected or clarified. Please check comments and corrections in the attached file.

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Reviewer #1: No

Reviewer #2: No

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Attachments

Attachment

Submitted filename: Reviewer comments.docx

Attachment

Submitted filename: PONE-D-26-06317_reviewer.pdf

<p>A polymorphism and expression levels of SCD and their relationships to production traits in a Japanese farmed emu population

PONE-D-26-06317R1

Dear Dr. Wada,

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,

Abbas Farmany

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

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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

**********

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

Reviewer #1: Yes

Reviewer #2: 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

**********

-->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

**********

-->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: After thoroughly reviewing the revisions by the authors of manuscript PONE-D-26-06317R1, titled "A polymorphism and expression levels of SCD and their relationships to production traits in a Japanese farmed emu population," and evaluating it against PLOS One's acceptance criteria, I present the following comments:

1. All improvement suggestions from reviewers 1 and 2 have been successfully implemented and thoroughly explained, leading to a clearer and more concise discussion.

2. Overall, the paper appears to be the author's original research, with no evidence of prior publication.

3. Following enhancements to the research methodology, the statistics and analysis are now clearer, enabling a thorough discussion of the results.

4. The conclusions have been revised to highlight the findings and remove any speculative elements unrelated to the research.

5. Overall, the paper is composed in standard English.

6. The study involved animals and followed procedures aligned with ethical standards for experimentation and research integrity.

7. The article adheres to relevant reporting guidelines and community standards regarding data availability.

After reviewing the revised manuscript, I find that this research offers valuable new insights into the under-studied field of emu research. Hence, I recommend the paper for publication in PLOS One.

Reviewer #2: Authors made the necessary amendments and the article is substantially improved. I suggest its acceptance

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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: Yes: Ronny Rachman Noor

Reviewer #2: No

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