PONE-D-21-05713

Dietary Supplementation of Alpha-lipoic Acid Mitigates the Negative Effects of Heat Stress in Poultry

PLOS ONE

Dear Dr. Mishra,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

The manuscript should be revised deeply. The main problem found in the manuscript is related to the some aspects of methodology and redaction style. The manuscript should be presented according to guidelines for authors of Plos One. Please review the referee comments and make your peer revision. Thanks for your hard work.

Please submit your revised manuscript by May 22 2021 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

• A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.
• A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.
• An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols. Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols.

We look forward to receiving your revised manuscript.

Kind regards,

Arda Yildirim, Ph.D.

Academic Editor

PLOS ONE

https://www.researchgate.net/profile/Arda_Yildirim2

Additional Editor Comments:

Firstly, thank you for your suggestion as an academic editor. This MS deals with an interesting and important topic in poultry production. Nevertheless there are still some points of concern from the reviewers, before the manuscript can be accepted for publication. Please make MS title specific because of focusing on animal material that is broiler. As there are some points unclear to me regarding the trial execution, sampling and statements in the MS, I recommend major revision.

Journal Requirements:

When submitting your revision, we need you to address these additional requirements.

1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at

https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and

https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf

2. In your Data Availability statement, you have not specified where the minimal data set underlying the results described in your manuscript can be found. PLOS defines a study's minimal data set as the underlying data used to reach the conclusions drawn in the manuscript and any additional data required to replicate the reported study findings in their entirety. All PLOS journals require that the minimal data set be made fully available. For more information about our data policy, please see http://journals.plos.org/plosone/s/data-availability.

Upon re-submitting your revised manuscript, please upload your study’s minimal underlying data set as either Supporting Information files or to a stable, public repository and include the relevant URLs, DOIs, or accession numbers within your revised cover letter. For a list of acceptable repositories, please see http://journals.plos.org/plosone/s/data-availability#loc-recommended-repositories. Any potentially identifying patient information must be fully anonymized.

Important: If there are ethical or legal restrictions to sharing your data publicly, please explain these restrictions in detail. Please see our guidelines for more information on what we consider unacceptable restrictions to publicly sharing data: http://journals.plos.org/plosone/s/data-availability#loc-unacceptable-data-access-restrictions. Note that it is not acceptable for the authors to be the sole named individuals responsible for ensuring data access.

We will update your Data Availability statement to reflect the information you provide in your cover letter.

3. Thank you for stating the following in the Acknowledgments Section of your manuscript:

[This work was supported by a Start-up grant from CTAHR University of Hawaii at Manoa, and USDA Multistate (2052R) to B.M.Apart from providing funds, these organizations were not involved in any experimental procedure and manuscript preparation.]

We note that you have provided funding information that is not currently declared in your Funding Statement. However, funding information should not appear in the Acknowledgments section or other areas of your manuscript. We will only publish funding information present in the Funding Statement section of the online submission form.

Please remove any funding-related text from the manuscript and let us know how you would like to update your Funding Statement. Currently, your Funding Statement reads as follows:

 [N/A]

Please include your amended statements within your cover letter; we will change the online submission form on your behalf.

4. We noticed you have some minor occurrence of overlapping text with the following previous publication(s), which needs to be addressed:

- https://scholarspace.manoa.hawaii.edu/handle/10125/63507

Lines 184 - 194

In your revision ensure you cite all your sources (including your own works), and quote or rephrase any duplicated text outside the methods section. Further consideration is dependent on these concerns being addressed.

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

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

Reviewer #4: Yes

**********

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

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

Reviewer #4: Yes

**********

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: Yes

Reviewer #4: Yes

**********

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

Reviewer #4: Yes

**********

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: The design and objectives of this study is interesting, and it is expected to contribute further to the knowledge base of improving broiler health by providing nutritional strategies to tackle heat-stress problem. The authors have done good work to make a comprehensive story of microbiota, immunity, heat stress parameters and performance. However, authors need to revise their work and correct some inherent concerns in the feed formulation. The other general comments and minor corrections are listed pointwise.

Major comments

1. What standard requirement was followed for this diet formulation? The calcium and phosphorus are extremely low.

The starter diet has high protein but comparatively low energy which is not as the right recommended ratio.

2. Were the birds weighed individually to show the standard deviation in each pen?

3. Confirm whether the gene expression calculation was based on relative standard curve or the comparative CT

method. Describe more specifically what represents the gene expression value.

4. Line 153. The citation for embedding is not properly attributed. The reviewer was not able to locate the information in

the cited article or in the reference attributed in the cited article!

5. Since the authors have stated that ALA would have improved performance via microbiota-VFA modulation, it is

expected that they provide some insight into why ALA potentially modify microbiota.

Minor comments

Line 17: add ‘is’ between ‘which’ and ‘readily’

Line 20: State clearly the adaptation and allocation period along with the replication of the treatments.

Line 48: Better say heavier breast meat. Also, it should be improved breeding and not improved chicken.

Line 50: Insert ‘the’ between ‘and’ and ‘presence’.

Line 51: High temperature is the problem of tropical condition in the context of chicken.

Line 59: Check whether it will be is or are with ALA.

Line 82: Use n for experimental unit. Either state total number of birds per treatment or just mention birds per experimental unit.

Line 91: Remove ‘with’ after ‘fed’.

Line 110: Change ‘reducing’ to ‘subtracting’.

Line 158: Is it muscularis mucosae or submucosa?

Line 200-2001: Were the reads trimmed before pairing or after pairing?

Line 227: Add significance statistics.

Line 238: Remove numerical improvement. This means there was high variation among the replicates.

Line 244-245: Check grammar.

Line 262: Change ‘remains’ to ‘remain’.

Line 273: Use correct preposition after ‘compared’. Add significance statistics.

Line 288: Remove ‘total’ before ‘concentration’.

Line 298: Change ‘have’ to ‘had’.

Line 305: Add ‘the’ before ‘heat-stressed’.

Line 378: Is decreasing crypt depth an impairment?

Figure 5: Define the box plot. State whether the whiskers are error or data range. If the whiskers are range, then why are no data points on NHS box plot whisker?

Figure 7: What is the unit of relative abundance of Lactobacillales? Is it any fractions?

Reviewer #2: Dear Authors Regarding the manuscript title Dietary Supplementation of Alpha-lipoic Acid Mitigates the Negative Effects of Heat Stress in Poultry

The scientific background of the topic was well mentioned in the introduction part. The experiment design, as well as the replicates and methods used, were very good. The results obtained were presented in tables well discussed with other author’s results. However, some observation in the present paper should be corrected and add to improve the quality of the paper.

• The title (Dietary Supplementation of Alpha-lipoic A 1 cid Mitigates the Negative Effects of Heat Stress in Poultry) it will be better if you replacing Poultry to broilers

• Table 1 Ingredients and nutrient composition of the experimental diets, need to carful checking for the following :

1- SBM , add the crude protein level 44 or 46%?

2- MEn, kcal/kg, Ca, digP , not according to the Cobb-500 requirement.

• Introduction and Discussion

Need some other references about the impact of heat stress and Growth and Lipid Metabolism Marker Genes in Broiler Chickens in poultry I recommend you read the following references:

Saki Shimamoto 1,2, Kiriko Nakamura 1, Shozo Tomonaga 3, Satoru Furukawa 4, Akira Ohtsuka and Daichi Ijiri. Effects of Cyclic High Ambient Temperature and Dietary Supplementation of Orotic Acid, a Pyrimidine Precursor, on Plasma and Muscle Metabolites in Broiler Chickens. Metabolites 2020, 10, 189; doi:10.3390/metabo10050189

Saleh, Ahmed A.; Shukry, Mustafa; Farrag, Foad; Soliman, Mohamed M.; Abdel-Moneim, Abdel-Moneim E. (2021) "Effect of Feeding Wet Feed or Wet Feed Fermented by Bacillus licheniformis on Growth Performance, Histopathology and Growth and Lipid Metabolism Marker Genes in Broiler Chickens" Animals 11, no. 1: 83.

Ahmed A. Saleh , Mohammed S. Eltantawy , Esraa M. Gawish , Hassan H. Younis , Khairy A. Amber , Abd El-Moneim E. Abd El-Moneim & Tarek A. Ebeid (2020) Impact of Dietary Organic Mineral Supplementation on Reproductive Performance, Egg Quality Characteristics, Lipid Oxidation, Ovarian Follicular Development, and Immune Response in Laying Hens Under High Ambient Temperature. Biological Trace Element Research. 195:506–514.

Inoue H, Shimamoto S, Takahashi H, et al. Effects of astaxanthin-rich dried cell powder from Paracoccus carotinifaciens on carotenoid composition and lipid peroxidation in skeletal muscle of broiler chickens under thermo-neutral or realistic high temperature conditions. Anim Sci J. 2018;00:1–8.

Ahmed A. Saleh , Abeer A. Kirrella, Mahmoud A. O. Dawood, Tarek A. Ebeid (2019) Effect of dietary inclusion of cumin seed oil on the performance, egg quality, immune response and ovarian development in laying hens under high ambient temperature. Animal Physiology and Animal Nutrition. 103(6):1810-1817.

• Results: it will be better if you present the data for performance in table.

Reviewer #3: Heat stress is a practical problem to broiler chicks which results in over $128 million in losses for the poultry industry in the United States. The manuscript by Wasti et al examines whether using alpha lipoic acid (ALA) mitigated negative effects of heat stress in poultry chickens. The results document the beneficial effects of ALA in broiler chickens by improving gut health. The manuscript has been written in a well-orchestrated manner. However, some points have to be discussed and manuscript could be accepted only after major revisions.

General Comments:

Line 91: Remove the space error after “phases”, and also throughout the manuscript.

Line 97: Add space between “study[9]”

Line 111: No need to explain abbreviation ADG again once it already mentioned in the abstract section.

Line 238: Dietary ALA supplementation o was able to improve the FCR. What is supplementation o? Please remove it from text.

Specific Comments:

Comment 1: Figure 1,2,3,4 No word about sample size based on which the means and SEM were computed. Please add.

Comment 2: Most of the figures do not have the superscripts. Authors have to correct those figures.

Comment 3: The exact computed P-value at least for significant ones should be shown not only (P<0.05) which is a theoretical value.

Comment 4: Can authors please describe more for antioxidant defence system and its significance in poultry?

Comment 5: Please elaborate the ALA mechanism in improving growth performance of heat stress chickens in the discussion section.

Comment 6: Authors have not mentioned any dehydration-related parameters which is the most important factor to study the heat stress-related study as this causes dehydration in animals.

Comment 7: During dehydration, mainly epithelial cells hampered. Tight junction plays a major role during heat stress. The authors focused on the claudin and occludin molecules. Results showing that insignificant changes in claudin molecule but occludin is significantly higher in the treatment group. Tight junction maintains by both of these molecules. It is assuming from this result that due to heat stress, epithelial cells in the HS group individuals may experience dehydration condition which may lead to the loss of experimental animals. Is there so? If no, then how it will be justified? However, how it will be justified about the findings of villus height (higher in NHS), crypt depth (higher in NHS), and villus surface area (same in HS and ALA+HS) in relation to claudin and occludin level? Meantime, IL-4 is reduced which is an anti-inflammatory marker in the HS group.

Comment 8: Insert superscripts in Heat shock protein-related graph (Figure 2A). How it is possible the same level of HSP70 in the NHS and HS groups? It seems that the protein folding process will be the same in the control and HS group of animals. Then how HSP90 levels get reduced in the HS group?

Comment 9: Peroxiredoxin level showing different levels in the graph but it is showing the same superscripts? Why?

Comment 10: Most of the antioxidant-related parameter levels are higher in the treatment group. Meantime SOD1 and SOD2 are higher in the NHS group. How it will be justified?

Reviewer #4: It is a well-written manuscript. The study design and data collection are sufficient. The only concern is the number of animals used in the current study is low, and the growth performance data may not be representative. There are some minor comments:

Please indicate the sample number/statistical unit in each result. For example, if you collect two samples from each replicate. The data need to be averaged within each replicate upon analysis.

Is there any reason why ALA was not fed to the birds from the beginning of the trial?

It would be great if the author could present the morality from each treatment since it may impact the performance calculation due to low birds no.

Please also describe the raising environment, such as pen size and type (batter, cage or floor pen, litter, etc. )

**********

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: No

Reviewer #3: Yes: Sahil Kalia

Reviewer #4: Yes: Chongxiao Chen

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

Attachments

Attachment

Submitted filename: Plos One review.docx

PONE-D-21-05713R1

Dietary Supplementation of Alpha-lipoic Acid Mitigates the Negative Effects of Heat Stress in Broilers

PLOS ONE

Dear Dr. Mishra,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

Please provide clear information on whether the non-standard requirement of a commercial hybrid broiler for calcium and phosphorus (available) values is met.

Please submit your revised manuscript by Aug 09 2021 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

• A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.
• A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.
• An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols. Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols.

We look forward to receiving your revised manuscript.

Kind regards,

Arda Yildirim, Ph.D.

Academic Editor

PLOS ONE

Journal Requirements:

Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.

Additional Editor Comments (if provided):

For your guidance, you can check the reviewers' comments.

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

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: (No Response)

Reviewer #2: All comments have been addressed

Reviewer #3: All comments have been addressed

**********

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

**********

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: 1. More specifically, the calcium and phosphorus values are not formulated according to the commercial birds recommendation (in this case Cobb 500). So, the authors answer is not correct. It is true that there is variation in the inclusion level of calcium and phosphorus. The authors must mention in the manuscript that they formulated a low calcium and phosphorus diet and if possible, should provide the reason for such formulation. Low Ca and P do not necessarily keep the birds safe. This is not going to affect the outcome of this study but would provide a wrong reference for other researchers that low Ca and P diet would be required to yield a standard growth performance. The authors should provide correct information. They cannot cite one reference for meeting requirement and provide reason that it is outdated, and they considered commercial recommendation, surprisingly which was even not followed. Cobb guidelines state Ca requirement to be 0.9% in the starter and 0.8% in the finisher, available phosphorus is around 0.45%. The authors have formulated the diet containing 0.75% Ca in the starter and 0.52% in the finisher, while the available phosphorus is 0.3% in the starter and 0.23% in the finisher. There is a typo: it should be available phosphorus and not dig. If the authors provide correct information in the draft and state the reason for this inclusion level then it would be ok otherwise it would be understood as a mistake in their feed formulation.

2. Also, authors should have removed 'standard curve method' from line 146 if they calculated gene expression by the comparative CT method. Figure 2 does not show any fold change (as stated in line 147) as none of the treatment has a normalized value of 1. So, it would be better to remove the fold change from the materials and methods. Besides, the authors should mention the treatment (may be the control group) that was used to calculate the ΔΔ CT.

Reviewer #2: Dear Authors Regarding the manuscript title Dietary Supplementation of Alpha-lipoic Acid Mitigates the Negative Effects of Heat Stress in Broilers

The scientific background of the topic was well mentioned in the introduction part. The experiment design, as well as the replicates and methods used, were very good. The results obtained were presented in tables well discussed with other author’s results. In addition the authors answered all inquiries and the manuscript may accept in this form.

Reviewer #3: (No Response)

**********

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

Reviewer #3: Yes: Sahil Kalia

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

Attachments

Attachment

Submitted filename: Reviewer report revision 1.docx

Dietary Supplementation of Alpha-lipoic Acid Mitigates the Negative Effects of Heat Stress in Broilers

PONE-D-21-05713R2

Dear Dr. Mishra,

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.

An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.

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,

Arda Yildirim, Ph.D.

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Thanks for reviewing and accepting all the comments and suggestions.

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

**********

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

**********

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

Reviewer #1: 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

**********

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

**********

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 comments have been addressed. Congratulations to the authors for their hard work and acceptable draft for 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