Establishment of methanogen bacterial interactions during the preweaning period of dairy cattle

Nagaraju Indugu; Kapil S. Narayan; Meagan L. Hennessy; Dipti Pitta

doi:10.1371/journal.pone.0310648

Peer Review History

Original SubmissionFebruary 23, 2024
10 Jul 2024 Decision Letter - Ibukun M. Ogunade, Editor PONE-D-24-07201Establishment of methanogen bacterial interactions during the preweaning period of dairy cattlePLOS ONE Dear Dr. Pitta, 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 Aug 24 2024 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. 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The template and more information on our requirements for LaTeX submissions can be found at http://journals.plos.org/plosone/s/latex. 4. 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 If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references.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 If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice. [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 ******** 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: Yes Reviewer #2: 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 ****** 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 ****** 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: Overall, this is a beautiful study revealing microbial dynamics in developing animals during the preweaning period of dairy cattle. This is crucial for understanding and optimizing rumen development, which significantly impacts the future productivity and health of the animals. Comments Line 84-88: Please rewrite the objective and your hypothesis to succinctly conform with the abstract. There is a disjoint in the sentence especially line 88……. which further dictates….. Sentences like this should not stand here. Line 98: Remove ad libtum from this sentence and leave as…… grain consumption was provided……… Line 99: How did you collect your fecal samples? Was the feces collected via stimulation or was picked from the floor……. It would be important to have a line of sentence describing this. Line 220: Replace accompaniment with follow up study……. Line 144: Rarefied or rarified… Typo error, this should be Rarefied. Reviewer #2: The manuscript presents a valuable contribution to understanding methanogen colonization in neonatal dairy calves and its potential impact on methane emissions. The study is methodologically sound and well-referenced, providing a comprehensive analysis of the temporal dynamics of methanogenic archaea and their interactions with bacteria. However, some information about Materials and methods are missing, likewise some improvements need to be made. Abstract: Include age and average weight of the calves at the point of the study Introduction: Add references at line 56, and 58. All acronyms should be defined. While some of the target audiences are likely familiar with technical terms, ensuring that complex terminology (e.g., "methylotrophic methanogens", "hydrogenotrophic methanogens") is clearly defined upon first use can enhance accessibility. The knowledge gap mentioned in lines 81-83 is critical but could be more explicitly stated. A clear statement of what is known versus what is not would sharpen the focus. Materials and Methods: Include the average weight of the calves at the point of the study Why wasn’t a control group (group of calves not receiving the same diet) included in the study to help provide comparative insights? Provide detailed information about the acidified milk and the grain and hay diet content (Diet Table). Why were fecal samples collected only and rumen fluid samples were not collected to give more information about the ruminal content and the microbial populations of the calves, similar to the study by Friedman et al., 2017. Details of fecal collection procedure should be included What time during the day were the fecal samples collected from each calf, was it after or before meals? Results Line 138: Were these four experimental samples removed from other analyses? Line 179 needs to be improved (Methanogenic) Line 226 shows should be show Were residuals checked for normality, and how were potential outliers handled? Discussion The transition between discussing bacterial and methanogen communities seems a bit abrupt, improving the flow would help readability Explain why acidified milk was chosen and how it might influence the microbial dynamics compared to other feeding strategies Rephrase line 232 to “Surprisingly, the correlation analysis revealed that, at any given time point, only one of the two genera was present, and they did not co-occur.” to improve clarity. Simplifying the language where possible and defining key terms would make the discussion more accessible. The use of terms like "flux" and "clusters" needs to be clearly defined in the context of microbial community dynamics to avoid ambiguity. Lines 263-266: The mention of similar interactions in mature rumen and low-methane yield phenotype cows is interesting but needs more context and explanation. How do these findings compare with other studies on methanogen-bacteria interactions? Create a sub-heading for the Conclusion ****** 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: Yes: Godstime Taiwo Reviewer #2: Yes: Modoluwamu Idowu ******** [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. https://doi.org/10.1371/journal.pone.0310648.r001
Revision 1
3 Sep 2024 Author Response Editors’ comments 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 AU: We have revised the manuscript to adhere to PLOS ONE's style requirements. This includes updates to figure citations, file naming conventions, and references to align with the guidelines provided. 2. Thank you for stating the following financial disclosure: “This research was funded by the United States Department of Agriculture National Institute of Food and Agriculture (Grant #PENV570262; https://nifa.usda.gov/us-states-and-territories/pennsylvania).” Please state what role the funders took in the study. If the funders had no role, please state: "The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript." If this statement is not correct you must amend it as needed. Please include this amended Role of Funder statement in your cover letter; we will change the online submission form on your behalf. AU: As requested, we have included the Role of Funder statement in the cover letter. 3. Please update your submission to use the PLOS LaTeX template. The template and more information on our requirements for LaTeX submissions can be found at http://journals.plos.org/plosone/s/latex. AU: We have updated our manuscript according to PLOS submission guidelines. 4. 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. AU: We have reviewed and updated references and citations 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: Overall, this is a beautiful study revealing microbial dynamics in developing animals during the preweaning period of dairy cattle. This is crucial for understanding and optimizing rumen development, which significantly impacts the future productivity and health of the animals. Thank you for your comments. We have sought to address the specific critiques below. Comments Line 84-88: Please rewrite the objective and your hypothesis to succinctly conform with the abstract. There is a disjoint in the sentence especially line 88……. which further dictates….. Sentences like this should not stand here. AU: We have revised the objective and hypothesis for clarity. The sentence has been improved as suggested (Lines 84-94). Line 98: Remove ad libtum from this sentence and leave as…… grain consumption was provided……… AU: We have removed the term "ad libitum" as suggested. Line 99: How did you collect your fecal samples? Was the feces collected via stimulation or was picked from the floor……. It would be important to have a line of sentence describing this. AU: We collected fecal samples via rectal stimulation. This information has been added to the manuscript (Lines 107-110). Line 220: Replace accompaniment with follow up study……. AU: Agreed and corrected. Line 144: Rarefied or rarified… Typo error, this should be Rarefied. AU: Thank you. We have corrected it to "Rarefied." Reviewer #2: The manuscript presents a valuable contribution to understanding methanogen colonization in neonatal dairy calves and its potential impact on methane emissions. The study is methodologically sound and well-referenced, providing a comprehensive analysis of the temporal dynamics of methanogenic archaea and their interactions with bacteria. However, some information about Materials and methods are missing, likewise some improvements need to be made. Thank you for your comments. We have sought to address the specific critiques below. Abstract: Include age and average weight of the calves at the point of the study AU: The age of the calves and average birth weight is included in the abstract as suggested (Lines 32-35). Introduction: Add references at line 56, and 58. AU: We have revised the sentences as requested and added two references: Johnson & Johnson (1995) and Wolin (1981). All acronyms should be defined. While some of the target audiences are likely familiar with technical terms, ensuring that complex terminology (e.g., "methylotrophic methanogens", "hydrogenotrophic methanogens") is clearly defined upon first use can enhance accessibility. AU: We have defined both "methylotrophic methanogens", and "hydrogenotrophic methanogens (Lines 74-77) The knowledge gap mentioned in lines 81-83 is critical but could be more explicitly stated. A clear statement of what is known versus what is not would sharpen the focus. AU: We have revised the text to clearly state the knowledge gap (Lines 84-88). Materials and Methods: Include the average weight of the calves at the point of the study AU: We included the average birth weight of the calves (lines 102-103). Why wasn’t a control group (group of calves not receiving the same diet) included in the study to help provide comparative insights? AU: The primary objective of this study was to understand the temporal patterns of methanogenic archaea, rather than to compare different diets. As such, a control group was not included. All calves followed the same dietary protocol: they received acidified milk until 8 weeks of age and were then switched to a diet of grain and hay, as per the standard feeding schedule on our dairy farm. As such there is no treatment but we compared methanogenic communities by age of calves. Provide detailed information about the acidified milk and the grain and hay diet content (Diet Table). AU: We have now included a detailed Diet Table as Supplementary Table 1 in the manuscript, which provides information on the composition of the acidified milk, grain, and hay diets. Why were fecal samples collected only and rumen fluid samples were not collected to give more information about the ruminal content and the microbial populations of the calves, similar to the study by Friedman et al., 2017. AU: Rumen is not fully developed, and calves are considered monogastric until weaning. According to Friedman et al paper, samples were collected at Wk2 and then 2 months which is not similar to our study. Our goal was to follow temporal dynamics and sampling for rumen contents at that frequency will be considered invasive sampling. Collecting rumen at birth through 4-6 weeks of age is also considered invasive and may not represent true composition of rumen microbiota as rumen is still developing. Therefore, we relied on fecal sampling. Details of fecal collection procedure should be included AU: The fecal collection procedure is now described in (lines 107-110). What time during the day were the fecal samples collected from each calf, was it after or before meals? AU: We typically collect fecal samples 2h post-feeding, this information has been updated in text (Line 108). Results Line 138: Were these four experimental samples removed from other analyses? AU: Yes, we removed the four experimental samples from all microbiome analyses. Line 179 needs to be improved (Methanogenic) AU: We have replaced “Methanogenic” with “Archaea”. Line 226 shows should be show AU: We have replaced “shows” with “show”. Were residuals checked for normality, and how were potential outliers handled? AU: We used generalized estimating equations (GEE) statistical model to analyze alpha diversity and for taxonomy. In GEE, the primary focus is on the population-averaged effects rather than individual-level predictions, and as such, the normality of residuals is not a strict requirement. GEE is robust to misspecifications of the working correlation structure, and the estimates of the regression coefficients are consistent even if the residuals are not normally distributed. Therefore, we did not check residuals for normality, as it is not a prerequisite for the validity of GEE results. In our analysis, we used boxplots and interquartile range (IQR) statistics to identify potential outliers and found two animals at Week 2 that were outliers. We then ran the GEE model without these two observations. The results were consistent whether or not we included the outliers, indicating that they did not significantly impact our findings. Discussion The transition between discussing bacterial and methanogen communities seems a bit abrupt, improving the flow would help readability AU: Thank you, we have revised the sentence as suggested (Lines 268-281). Explain why acidified milk was chosen and how it might influence the microbial dynamics compared to other feeding strategies AU: We have added justification for using acidified milk (Lines 282-300). Rephrase line 232 to “Surprisingly, the correlation analysis revealed that, at any given time point, only one of the two genera was present, and they did not co-occur.” to improve clarity. AU: We have rephrased the sentence as suggested (Lines 249-264). Simplifying the language where possible and defining key terms would make the discussion more accessible. The use of terms like "flux" and "clusters" needs to be clearly defined in the context of microbial community dynamics to avoid ambiguity. AU: flux eliminated, grouping patterns added in parenthesis for clusters Lines 263-266: The mention of similar interactions in mature rumen and low-methane yield phenotype cows is interesting but needs more context and explanation. How do these findings compare with other studies on methanogen-bacteria interactions? AU: We have revised the sentences as suggested (Lines 325-333) Create a sub-heading for the Conclusion AU: A separate sub-heading has been created for the Conclusion. Attachments Attachment Submitted filename: Response to reviewers.docx https://doi.org/10.1371/journal.pone.0310648.r002
5 Sep 2024 Decision Letter - Ibukun M. Ogunade, Editor Establishment of methanogen bacterial interactions during the preweaning period of dairy cattle PONE-D-24-07201R1 Dear Dr. Pitta, 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 will be generated when your article is formally accepted. Please note, if your institution has a publishing partnership with PLOS and your article meets the relevant criteria, all or part of your publication costs will be covered. Please make sure your user information is up-to-date by logging into Editorial Manager at Editorial Manager® and clicking the ‘Update My Information' link at the top of the page. If you have any questions relating to publication charges, 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, Ibukun M. Ogunade, Ph.D. Academic Editor PLOS ONE Additional Editor Comments (optional): Reviewers' comments: https://doi.org/10.1371/journal.pone.0310648.r003
Formally Accepted
11 Sep 2024 Acceptance Letter - Ibukun M. Ogunade, Editor PONE-D-24-07201R1 PLOS ONE Dear Dr. Pitta, I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now being handed over to our production team. At this stage, our production department will prepare your paper for publication. This includes ensuring the following: * All references, tables, and figures are properly cited * All relevant supporting information is included in the manuscript submission, * There are no issues that prevent the paper from being properly typeset If revisions are needed, the production department will contact you directly to resolve them. If no revisions are needed, you will receive an email when the publication date has been set. At this time, we do not offer pre-publication proofs to authors during production of the accepted work. Please keep in mind that we are working through a large volume of accepted articles, so please give us a few weeks to review your paper and let you know the next and final steps. Lastly, if your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. 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. If we can help with anything else, please email us at customercare@plos.org. Thank you for submitting your work to PLOS ONE and supporting open access. Kind regards, PLOS ONE Editorial Office Staff on behalf of Dr. Ibukun M. Ogunade Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0310648.r004

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