Saliva and plasma metabolome analysis during the five days before foaling in the mare

Lydie Nadal-Desbarats; Camille Dupuy; Frédéric Montigny; Priscila Silvana Bertevello; Cécile Douet; Amandine Gesbert; Fabrice Reigner; Ghylène Goudet

doi:10.1371/journal.pone.0344530

Peer Review History

Original SubmissionNovember 20, 2025
27 Jan 2026 Decision Letter - Juan Loor, Editor Dear Dr. Goudet, 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 Mar 13 2026 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. A 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'. 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We look forward to receiving your revised manuscript. Kind regards, Juan J Loor Academic Editor PLOS One 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. To comply with PLOS One submissions requirements, in your Methods section, please provide additional information regarding the experiments involving animals and ensure you have included details on (1) methods of sacrifice, (2) methods of anesthesia and/or analgesia, and (3) efforts to alleviate suffering. 3. We note that this submission includes NMR spectroscopy data. 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Authors should provide the acquisition data, FID files and processing parameters for each experiment, clearly labelled with the compound name and identifier, as well as a structure file for each provided dataset. See our list of recommended repositories here: https://journals.plos.org/plosone/s/recommended-repositories 4. Please expand the acronym “INRAE” (as indicated in your financial disclosure) so that it states the name of your funders in full. This information should be included in your cover letter; we will change the online submission form on your behalf. 5. Thank you for stating the following financial disclosure: “This work was supported by INRAE and Institut Français du Cheval et de l’Equitation (IFCE).” 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. 6. If the reviewer comments include a recommendation to cite specific previously published works, please review and evaluate these publications to determine whether they are relevant and should be cited. There is no requirement to cite these works unless the editor has indicated otherwise. 7. 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? Reviewer #1: Yes ******** 2. Has the statistical analysis been performed appropriately and rigorously? -->?> Reviewer #1: Yes ****** 3. Have the authors made all data underlying the findings in their manuscript fully available??> The PLOS Data policy Reviewer #1: Yes ****** 4. Is the manuscript presented in an intelligible fashion and written in standard English??> Reviewer #1: Yes ****** Reviewer #1: The study describes potential biomarkers measurable in the saliva of pregnant mares to determine parturition. This approach is particularly valuable because saliva can be collected non-invasively by non-veterinary personnel. The study is well designed, and only minor revisions are required. Abstract, line 29: Replace “level” with “concentration” and apply this change consistently throughout the manuscript whenever quantitative data are reported. Discussion, line 370: Original: “However, the lack of concomitant decrease of acetic acid levels in saliva and plasma remains questioning.” Suggested correction: “However, the lack of a concomitant decrease in acetic acid concentrations in saliva and plasma remains unclear (or unexplained).” Lines 155–156: Insert “samples” for clarity: “The 60 saliva samples were prepared by protein precipitation using cold methanol.” ****** 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 ******** [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.] To ensure your figures meet our technical requirements, please review our figure guidelines: https://journals.plos.org/plosone/s/figures You may also use PLOS’s free figure tool, NAAS, to help you prepare publication quality figures: https://journals.plos.org/plosone/s/figures#loc-tools-for-figure-preparation. NAAS will assess whether your figures meet our technical requirements by comparing each figure against our figure specifications. https://doi.org/10.1371/journal.pone.0344530.r001
Revision 1
6 Feb 2026 Author Response Journal Requirements: 1. We have modified the manuscript so that it meets PLOS ONE’s style requirements, the modifications have been highlighted. 2. Information regarding the experiments involving animals and details on methods of sacrifice, anesthesia, analgesia, and efforts to alleviate suffering have been included and highlighted in the Methods section lines 115 to 151, and in the Ethics statement section lines 452 to 464. 3. We have included the requested information about NMR spectroscopy data: 1) In the methods section (line 172 to 179 in the revised manuscript), we have already mentioned the source of the NMR instrument, the magnetic field strength, the nucleus being measured, the solvent, the temperature and the pulse sequence to supress water signal: “1H-NMR spectra were acquired at 298 K on an AVANCE III HD 600 MHz system (Bruker Biospin, Karlsruhe, Germany) equipped with a 5 mm TCI cryoprobe with z-gradient. 1H-NMR spectra were recorded with a “noesypr1d” pulse sequence with a time domain of 64 K data points, a sweep width of 12 ppm, 64 scans, an acquisition time of 4.56s and a relaxation delay of 20s. A NUS (50%) TOCSY sequence was used on the quality controls for the metabolite identifications of the biological samples. The 1H-NMR spectra were phase corrected and imported in NMRProcFlow (DOI: 10.1007/s11306-017-1178-y) for baseline correction and bucketing. Regions with a coefficient of variation across the quality control inferior to 30% were kept for the analysis.” Lines 169 to 171, the volume of solvent and the standard concentration are already mentioned: “Before NMR analysis, 210 µL of deuterated phosphate buffer (pH = 7.4; containing 3-trimethylsilylpropionic acid (TSP) at 152 µM final concentration) were added to the dried residues and transferred to 3 mm NMR tubes.” 2) The S1-Table resumes, for identified compounds in our biological samples, their chemical shifts in ppm, their multiplicities and their corresponding identifiers according to the Metabolomics Standards Initiative (MSI). 3) Full annotated representative 1H-Nuclear Magnetic Resonance spectroscopy spectra of mare saliva and plasma are in Figure 1 (A) and (B). Concerning the chemical structure, the HMDB, PubChem identifiers are mentioned in S1-Table and their SMILES identifiers related to their structure are also resumed in the S1-Table. The chemical structures were intentionally omitted from the Figure 1 to prevent visual overcrowding. The NMR data were already deposited on Zenodo Open repository. The data can be uploaded using the following link: https://doi.org/10.5281/zenodo.17589754. Line 201 to 202 of the manuscript mentioned: “The experimental data have been deposited with the DOI 10.5281/zenodo.17589754 in the Zenodo repository (https://zenodo.org).” 4. We have expanded the acronym INRAE to ‘Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement’, and we have highlighted the modifications. 5. We have stated the financial disclosure and the role of the funders and highlighted these changes lines 447 to 450: Financial disclosure This work was supported by Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE) and Institut Français du Cheval et de l’Equitation (IFCE). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. 6. The reviewer comments did not include a recommendation to cite specific previously published works. 7. We have reviewed our reference list, it is complete and correct. Response to Reviewers’ comments: 5. Review comments to the author: Abstract, line 29: Replace “level” with “concentration” throughout the manuscript We thank the reviewer to raise this question. However, the authors deliberately adopted the term “level” rather than “concentration”, because although the spectral intensities are expressed relative to a reference standard, they are not corrected for the number of protons contributing to each signal cluster and therefore cannot be interpreted as absolute concentrations. The data presented correspond to relative signal intensities; accordingly, the use of the term “level” is retained. Discussion, line 370 (373 in the revised manuscript): the sentence has been modified and highlighted. Lines 155-156 (159 in the revised manuscript): the sentence has been modified and highlighted. We have reviewed the figures so that they meet the technical requirements. Attachments Attachment Submitted filename: Response_to_Reviewers.doc https://doi.org/10.1371/journal.pone.0344530.r002
22 Feb 2026 Decision Letter - Juan Loor, Editor Saliva and plasma metabolome analysis during the five days before foaling in the mare PONE-D-25-61361R1 Dear Dr. Goudet, 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. For questions related to billing, please contact billing support . 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, Juan J Loor Academic Editor PLOS One Additional Editor Comments (optional): Reviewers' comments: https://doi.org/10.1371/journal.pone.0344530.r003
Formally Accepted
Acceptance Letter - Juan Loor, Editor PONE-D-25-61361R1 PLOS One Dear Dr. Goudet, 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 You will receive further instructions from the production team, including instructions on how to review your proof when it is ready. Please keep in mind that we are working through a large volume of accepted articles, so please give us a few days 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. You will receive an invoice from PLOS for your publication fee after your manuscript has reached the completed accept phase. If you receive an email requesting payment before acceptance or for any other service, this may be a phishing scheme. Learn how to identify phishing emails and protect your accounts at https://explore.plos.org/phishing. 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. Juan J Loor Academic Editor PLOS One https://doi.org/10.1371/journal.pone.0344530.r004

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