Bupivacaine as a euthanasia agent for African Clawed Frogs (Xenopus laevis)

Kaela Navarro; Katechan Jampachaisri; David Chu; Cholawat Pacharinsak

doi:10.1371/journal.pone.0279331

Peer Review History

Original SubmissionJune 15, 2022
1 Sep 2022 Decision Letter - Rishabh Charan Choudhary, Editor PONE-D-22-17202Bupivacaine as a euthanasia agent for African Clawed Frogs (Xenopus laevis)PLOS ONE Dear Dr. Navarro, 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 Oct 16 2022 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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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: "This work was supported by Stanford's Department of Comparative Medicine. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. The content is solely the responsibility of the authors and does not necessarily represent the official views of the Stanford Department of Comparative Medicine or any other agency of the State of California." Please include your amended statements within your cover letter; we will change the online submission form on your behalf. 6. We note that you have indicated that data from this study are available upon request. 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Please also provide contact information for a data access committee, ethics committee, or other institutional body to which data requests may be sent. b) If there are no restrictions, please upload the minimal anonymized data set necessary to replicate your study findings as either Supporting Information files or to a stable, public repository and provide us with the relevant URLs, DOIs, or accession numbers. For a list of acceptable repositories, please see http://journals.plos.org/plosone/s/data-availability#loc-recommended-repositories. We will update your Data Availability statement on your behalf to reflect the information you provide. 7. Please include your tables as part of your main manuscript and remove the individual files. Please note that supplementary tables (should remain/ be uploaded) as separate "supporting information" files [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: No Reviewer #2: No ****** 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: Dear Author, This is an interesting. However ,lease elaborate the practical utility and implication of the present and advantage over other existing models. How these dat can be used to expand knowledge in clinical or basic research files Reviewer #2: The author is willing to share raw data upon request, however states that restrictions will apply as far as posting to a public repository. It is not included with the manuscript. Manuscript review PONE-D-22-17202, entitled "Bupivacaine as a euthanasia agent for African Clawed Frogs (Xenopus laevis)". Overall Comments: This manuscript describes the immersion of African Clawed frogs in bupivacaine or TMS for humane euthanasia. This information is beneficial to the research community and describes a potential novel euthanasia agent for this species, in that bupivacaine successfully lowered the heart rate more quickly than the standard immersion agent and produced irreversible death. This give the community another, safer, agent to use. It also shows the importance of using a secondary method of euthanasia when following the AVMA Guidelines for the use of TMS in this species. The study is described well and conclusion are appropriate. Abstract, Line 31- 9 hours is stated, this should be 19. Introduction, Line 47- Please add the word “and” between endocrinology and toxicology. Materials and Methods, Line 110- Please add a sentence prior to “A celiotomy was performed…” on how it was confirmed a surgical plane of anesthesia was attained. Results, Line 139- Please add a reference to Figure 2. Discussion, Line 228-9- “brain death or irreversible” Please complete this sentence. Figure 1B. There should be an indication that frogs without a heart beat had a celiotomy performed at the 5h timepoint. ****** 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: Amit Agrawal Reviewer #2: 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.] 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.0279331.r001
Revision 1
10 Sep 2022 Author Response 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 Response: This has been revised. 2. Please include your full ethics statement in the ‘Methods’ section of your manuscript file. In your statement, please include the full name of the IRB or ethics committee who approved or waived your study, as well as whether or not you obtained informed written or verbal consent. If consent was waived for your study, please include this information in your statement as well. Response: Thank you for the comment; this was included in our manuscript and can be located in the revised version in lines 104-108. The additional requirements of anesthesia and euthanasia can be found within the methods section. 3. As part of your revision, please complete and submit a copy of the Full ARRIVE 2.0 Guidelines checklist, a document that aims to improve experimental reporting and reproducibility of animal studies for purposes of post-publication data analysis and reproducibility: https://arriveguidelines.org/sites/arrive/files/Author%20Checklist%20-%20Full.pdf (PDF). Please include your completed checklist as a Supporting Information file. Note that if your paper is accepted for publication, this checklist will be published as part of your article. Response: This checklist has been completed and uploaded as a supplemental file. 4. We note that the grant information you provided in the ‘Funding Information’ and ‘Financial Disclosure’ sections do not match. When you resubmit, please ensure that you provide the correct grant numbers for the awards you received for your study in the ‘Funding Information’ section. Response: This has been clarified in the resubmission process. 5. Thank you for stating the following in the Acknowledgments Section of your manuscript: "We would like to thank Andrea Craig and Alberto Gaudiel for their assistance in care for the animals. The study was supported by the Stanford Department of Comparative Medicine." 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: "This work was supported by Stanford's Department of Comparative Medicine. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. The content is solely the responsibility of the authors and does not necessarily represent the official views of the Stanford Department of Comparative Medicine or any other agency of the State of California." Please include your amended statements within your cover letter; we will change the online submission form on your behalf. Response: Thank you. The funding statement in the manuscript has been removed. We have addressed the statement in our cover letter. 6. We note that you have indicated that data from this study are available upon request. PLOS only allows data to be available upon request if there are legal or ethical restrictions on sharing data publicly. For more information on unacceptable data access restrictions, please see http://journals.plos.org/plosone/s/data-availability#loc-unacceptable-data-access-restrictions. Response: We have uploaded supplementary files to provide a minimal data set. All other data are included within the manuscript. 3. Have the authors made all data underlying the findings in their manuscript fully available? The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified. Reviewer #1: No Reviewer #2: No Response: We have uploaded supplementary files to provide a minimal data set. All other necessary data are included within the manuscript. 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: Dear Author, This is interesting. However, please elaborate the practical utility and implication of the present and advantage over other existing models. How these data can be used to expand knowledge in clinical or basic research files. Response: Thank you for your comment. We have added additional information elaborating on the importance of Xenopus as an animal research model, why large group culls may be necessary, and the importance of alternative euthanasia agents in the introduction. Reviewer #2: The author is willing to share raw data upon request, however states that restrictions will apply as far as posting to a public repository. It is not included with the manuscript. Response: We have uploaded supplementary files to provide a minimal data set. All other necessary data are included within the manuscript. Reviewer 2: Abstract, Line 31- 9 hours is stated, this should be 19. Response: Thank you, this has been revised. Reviewer 2: Introduction, Line 47- Please add the word “and” between endocrinology and toxicology. Response: Thank you, this has been revised. Reviewer 2: Materials and Methods, Line 110- Please add a sentence prior to “A celiotomy was performed…” on how it was confirmed a surgical plane of anesthesia was attained. Response: This has been revised to confirm a surgical plane of anesthesia had been attained prior to celiotomy and how it was performed. Reviewer 2: Results, Line 139- Please add a reference to Figure 2. Response: This has been added. Reviewer 2: Discussion, Line 228-9- “brain death or irreversible” Please complete this sentence. Response: Thank you, this has been revised. Reviewer 2: Figure 1B. There should be an indication that frogs without a heart beat had a celiotomy performed at the 5h timepoint. Response: The figure has been updated to reflect the celiotomy at 5h and the figure text has been updated to reflect when celiotomies were performed and for which animals. Attachments Attachment Submitted filename: Response to Reviewers.docx https://doi.org/10.1371/journal.pone.0279331.r002
5 Dec 2022 Decision Letter - Michael Klymkowsky, Editor Bupivacaine as a euthanasia agent for African Clawed Frogs (Xenopus laevis) PONE-D-22-17202R1 Dear Dr. Navarro, 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, Hazel Bautista Support Staff - Lead PLOS ONE Additional Editor Comments (optional): Reviewers' comments: Reviewer's Responses to Questions Comments to the Author 1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation. Reviewer #1: All comments have been addressed Reviewer #2: All comments have been addressed ******** 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 authors have made suggested changes The practical of study has been added The data sets have been made available Reviewer #2: (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: Yes: Amanda Darbyshire ******** https://doi.org/10.1371/journal.pone.0279331.r003
Formally Accepted
12 Dec 2022 Acceptance Letter - Michael Klymkowsky, Editor PONE-D-22-17202R1 Bupivacaine as a euthanasia agent for African Clawed Frogs (Xenopus laevis) Dear Dr. Navarro: I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department. 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 plosone@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. Michael Klymkowsky Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0279331.r004

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