PONE-D-20-35024

Varying digestible isoleucine level to determine effects on performance, egg quality, serum biochemistry, and ileal protein digestibility in diets of young laying hens

PLOS ONE

Dear Dr. King,

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We look forward to receiving your revised manuscript.

Kind regards,

Mahmoud A.O. Dawood, PhD

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: 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: Yes

Reviewer #2: Yes

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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: No

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’s comments on Manuscript no PONE-D-20-35024.

The topic is very interesting and does allow knowledge transfer in the field of avian nutrition. However, the manuscript lacks certain details, which must be added. Major revision is required before it can be accepted for publication. The comments below will allow the authors to improve their manuscript:

Abstract:

Line 1 & 2: In control diet, 0.10 isoleucine was added to achieve 0.66% isoleucine levels. Mention the supplemental levels of isoleucine for all diets to achieve 0.66 to 0.84% isoleucine in complete feed.

Line 3 & 4: The authors must mention the control diet's crude protein value along with the ME values.

Line 6 &7: What is meant by weight change? Do you mean weight gain? Rephrase the term to clarify.

Line 12-14: Authors need to do a regression analysis to confirm the optimum levels of isoleucine for commercially important parameters. There may be different optimum levels for different parameters.

Introduction:

The introduction is not clear and needs to be elaborated and rewritten to justify higher isoleucine levels? What has changed, which demands higher levels?

Line 7-8: “Researchers have proposed optimum isoleucine levels to be from 550 to 660mg/hen /day”: But it is not clear how much of the supplemental isoleucine is needed in the diet to fulfil this requirement? This recommendation is for which breeds and what ages??

Line 8-9: Sentence needs revision. What do authors mean by genetic variation and age groups?

Results:

Table 1: was it for 20 to 30 weeks? Or 23 to 30 weeks?

Table 1: Units for Egg production and Feed intake is missing?

Table1: Change in bird weight? Not clear? I think authors means initial at minus final wt. If that is the case, then it should be mentioned as weight gain.

Table 1: Egg mass data is missing? Must add.

Page 6: line 2: Bodyweight change was significant for the week? Did it increase or decrease with age?

Page 6: Line 3: numerical differences mean nothing? The level of egg production was statistically lower for all treatments fed 0.75 to 0.84% supplemental isoleucine? Revise sentence to clarify what authors are trying to highlight?

Line 4: Highest numerical FCR? This is not true. Numerical differences should not be highlighted. Revise the sentence.

Table 2: Line 5-12: Needs revision: The authors mention that levels and weeks were significantly affected for external egg quality but did not explain the major differences, were they good or bad differences? The optimum level of isoleucine was different for each parameter. For example, For level as a factor, significantly higher egg weight was achieved at 0.69%, higher specific gravity was achieved at 0.84% isoleucine, and shell thickness was highest at 0.78% isoleucine.

Page 7: Internal egg quality (Supplement, Table 1):

It is not clear from the table what does propositional yolk and albumen weight means? Propositional to what? Please clarify?

For proportional yolk weigh, the optimum level of isoleucine was 0.84%. For proportional albumen weigh, the optimum level was 0.78%. The optimum level was 0.81% for yolk index, Y: A the optimum level was 0.84%. Can they all be mentioned in the text?

Regression analysis is required to determine the optimum level.

Table3: Were these levels within the expected physiological range? Not clear. Is an increase or decrease of these significant levels a good or a bad indicator? The whole section needs to be rewritten to clarify the differences between levels? Again, which level of isoleucine resulted in desired effects?

Table 3: A word is missing under quadratic (See the last row of the table?

Page 8: Discussion:

Line 11-12: Was Dong referring to egg production? Please mention.

Line 13: greater quantities? Be specific, mention what were the optimum levels for the egg production. Revise sentences.

Line 15-16: Excessive BCAAS? not clear, what is categorised as excessive levels? Revise the sentence. Which level of isoleucine can cause excessive BCAAs??

Line 16-17: Sentence needs revision: The authors need to clarify that Serotonin is an appetite suppressant. Therefore, speculation can be made that reduced feed intake at 0.75% can be associated with isoleucine level. Not clear what happened at 0.81? Please check and amend the sentence.

Page 9:

Line 3 to 4: "when less expensive feeds are used" what are less expensive feeds? Do you mean low CP feeds? Clarify the sentence.

Line 10- "Lowering isoleucine levels" What were the isoleucine levels used in Shim et al? Was the effect due to lowering of CP levels in general or was the effect specific to isoleucine levels? Not clear.

Line 13-14: what were the lower levels of isoleucine used by Dong? Add the information.

Page 10:

Line 2-3: effect of protein sources and levels of isoleucine are two different things. Clarify the sentence.

Line 3-5: Are you comparing isoleucine with Valine? Do you expect both AA's to affect the immunoglobulins in a similar way? or is it a typo? Please check and clarify.

Line 8-10: The CP levels of this study is 16%. It may be that increase in CP level can affect ND titer. There are many other factors that can affect the ND titre? These other factors can be explored to describe your results. The sentence needs clarification.

Line 11: what are "protein deprived chicks"? Not clear.

Line 15-16: Line 14 to 17: needs revision: recommendation for further studies is very vague. Can they be specific?

Material and methods:

• Throughout the text, it should be made clear that isoleucine levels are total and not SID.

• Methods should also specify L- isoleucine quantity added in each diet to meet the required % isoleucine levels.

Page 11:

Line 6: Methods should also specify the type of cage system that was used in this study.

Line 12: the formula used to determine ileal CP digestibility should be provided in the manuscript.

Line 15: Hempe et al: year of publication is missing

Line 16: Victor and Carver, 1936: Reference is too old. Please replace it with any recent reference if possible.

Line 16: Haugh 1938: Reference is missing in the reference list. Again reference too old.

How internal and external egg parameters were analysed? The information must be added in the methods section.

Page 12:

Line 3: Was digesta samples freeze or oven-dried. Add the information.

Statistical analysis:

• For egg production parameters it was factorial design (L*W), how can that be analysed as one way? It has to be a factorial design?

• Data must be analysed as regression analysis to confirm optimum levels of isoleucine for different parameters.

Reviewer #2: This is the review for the manuscript PONE-D-20-35024, entitled “Varying digestible isoleucine level to determine effects on performance, egg quality, serum biochemistry, and ileal protein digestibility in diets of young laying hens” for PLOS ONE Journal.

The present manuscript presents original data, which has been collected using appropriate standard methods. However, Minor concerns still need to be addressed or clarified.

Page 3 (Line 2): Abbreviations should be clarified when first mentioned e.g., AA = Amino acids. Clarify the three limited amino acids (including tryptophan).

Page 9 (Line 12): It is not clear why shell thickness changed with isoleucine supplementation. More explanation is required

Page 10 (Line 13): No discussions for the alteration in serum glucose content.

Page 10 (Line 19): what is 490?

Page 10 (Line 20): "and the Control + 0.69". Control or basal diet?

Page 12 (Lines 2 & 3): Many citations. Use one or two.

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Reviewer #1: No

Reviewer #2: No

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Attachments

Attachment

Submitted filename: Reviewer comment PoneD 20 35024.docx

PONE-D-20-35024R1Varying digestible isoleucine level to determine effects on performance, egg quality, serum biochemistry, and ileal protein digestibility in diets of young laying hensPLOS ONE

Dear Dr. King,

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 submit your revised manuscript by Nov 25 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: https://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,

Mahmoud A.O. Dawood, PhD

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.

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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: The manuscript has been much improved. However, there are some minor corrections which are still required. Please see the comments which needs addressing:

Page 1:

Title: remove the word “digestible” as digestible levels of Isoleucine are not investigated in this study? It’s the L-Isoleucine levels that are studied in this experiment.

Page 2:

Line 16: Further studies to investigate what? Not clear.

Page 3:

Line 10: Table 1: Units are missing?

Page 6:

Line 1: Replace the word “numerically” with “statistically” because the differences were statistically different.

Page 7:

Table 1: wrong numbering: Table on page 3 is also Table 1?

Table 1. Symbol ### is similar for FCR and Weight gain: check and correct. Footnote for how FCR was calculated is missing

Page 9:

Line 12: Fermentation? After which fermentation? Clarify?

Line 17: Reference is missing

Line 19: Incomplete reference. Year is missing in reference?

Page 14:

Line 12: Were differences within an acceptable range? What is a reduction in levels indicating? Good or bad?

Line19: which viral strain?

Page 15:

Rewrite conclusion section:

Line 4 and 5: Delete the sentence. This is not your conclusion.

Line 8: Remove reference from the conclusion section. The authors can use this reference in the discussion section.

Line 11? Further studies to test what? Would you please clarify which “further” studies are required? To investigate what?

Page 15 vs 16: Material and Methods: Line 14 on page 15 says you had 10 layers per replicate, but in Line 13 on Page 16, authors say there is five birds/cage? Please check and confirm if you had 5 or 10 laying hens/replicate?

Page 17:

Line 1: Delete the line “ except for layers …..below.” Culls are not counted as Mortality.

Line 13: what was the level of celite used?

Line 20: Revise sentence. Remove the word “above” and mention that it was ileal digesta.

Reviewer #2: (No Response)

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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: Dr. Mohammed El Basuini

[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.

Varying isoleucine level to determine effects on performance, egg quality, serum biochemistry, and ileal protein digestibility in diets of young laying hens

PONE-D-20-35024R2

Dear Dr. Annie King,

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,

Mahmoud A.O. Dawood, PhD

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments: