Elaboration of massage technique for semen collection and examination of semen characteristics in chinchilla (Chinchilla lanigera)

Bianka Babarczi; Árpád Drobnyák; Judit Barna; Éva Váradi Kissné; Zsuzsa Szabó; Mónika Heincinger; Károly Kustos; Zsuzsanna Szőke; Barbara Végi

doi:10.1371/journal.pone.0290441

Peer Review History

Original SubmissionNovember 7, 2022
14 Feb 2023 Decision Letter - Wan-Xi Yang, Editor PONE-D-22-30686Elaboration of massage technique for semen collection and examination of semen characteristics in chinchilla (Chinchilla lanigera)PLOS ONE Dear Dr. Babarczi, 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. ACADEMIC EDITOR:All the three external reviewers have provided valuable comments. The detailed massage technique should be described, including the protocol for examination of semen characteristics in chinchilla (Chinchilla lanigera). All the questions and concerns from the three viewers should be treated seriously. A list of changes is needed for review. Please submit your revised manuscript by Mar 31 2023 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'. 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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. 3. PLOS requires an ORCID iD for the corresponding author in Editorial Manager on papers submitted after December 6th, 2016. Please ensure that you have an ORCID iD and that it is validated in Editorial Manager. To do this, go to ‘Update my Information’ (in the upper left-hand corner of the main menu), and click on the Fetch/Validate link next to the ORCID field. This will take you to the ORCID site and allow you to create a new iD or authenticate a pre-existing iD in Editorial Manager. Please see the following video for instructions on linking an ORCID iD to your Editorial Manager account: https://www.youtube.com/watch?v=_xcclfuvtxQ [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 ******** 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: Yes Reviewer #2: Yes Reviewer #3: 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 ****** 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 ****** 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 clear, concise and structured manuscript. Apart from the theriogenology studies, further investigation on the reproductive rate of chinchillas either in natural or captive could be interesting to be explored. Reviewer #2: Introduction : - lacks introduction to how the massage technique was conceived, please add a few line and cite relevant references M&M: This section should have more detailed on "technique for semen collection and examination of semen characteristics". Currently the manuscript lack depth for understanding of the technique. Please include details and figures on: 1. There was no elaboration on how the chinchilla was restrained 2. The area of anus massage and the entire penile length on a chinchilla to aid reader to understand the procedure 3. Photomicrograph of the sperm Results: Table 1 should be improved - column 4 stated as "%". What does this imply? Table 2: Row 5 - I am not sure how this data is presented? Reviewer #3: The chinchilla is an endangered species in the wild, so some research on their reproduction is very important for the species perpetuation. This manuscript developed a manual technique for semen collection which met all animal welfare requirements. In addition, the basic parameters of sperm obtained by this method was determined. This study has definite value for reproduction and animal welfare of the chinchilla. However, I have some major concerns and minor concerns for this manuscript. Major concerns: 1. In this study, the authors just analyzed the parameters of sperm obtained by the manual technique and some spermatological parameters exhibit a larger fluctuation in the different times of the year. At the same time, this manual technique works for only 66% of chinchillas. So I suggest if the authors want to show the manual technique is a better way for semen collection than the common electro-ejaculation methods, they can analyze the parameters of sperm collected by electro-ejaculation methods. And through comparison and analysis, not only the advantages of the manual technique, but also the development of this technique will show up better. 2. The authors analyzed many spermatological parameters in this study. However, what’s the relationship between the results of these parameters and the manual technique developed in the study? 3. About the manual technique, the authors did not write it clearly. I suggest it can be descripted in more detail in the methods part. 4. The discussion part is too simple. I suggest the authors performed a profound discussion on the importance of this manual technique and meaning for the reproduction of chinchillas. 5. Several brackets are italic incorrectly, such as (Chinchilla lanigera) , (Table 1) , (Fig.1)…… 6. The references of this manuscript are so old and I suggest to add some new references. ****** 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: Dr Mohd Iswadi Ismail Reviewer #2: Yes: Wan Nor Fitri Bin Wan Jaafar Reviewer #3: 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. Attachments Attachment Submitted filename: PONE-D-22-30686_reviewer.pdf https://doi.org/10.1371/journal.pone.0290441.r001
Revision 1
2 May 2023 Author Response Reviewer #1: Overall, this is a clear, concise and structured manuscript. Apart from the theriogenology studies, further investigation on the reproductive rate of chinchillas either in natural or captive could be interesting to be explored. Thank you for your opinion. The topic you raise is indeed very interesting. The ultimate aim of our studies is to develop artificial insemination of chinchillas, the first step of which is to develop an effective, animal friendly sperm collection. Reviewer #2: Introduction : - lacks introduction to how the massage technique was conceived, please add a few line and cite relevant references Thank you for your comment. The digital manipulation or massage technique for sperm collection usually is commonly used in dogs, pigs and poultry among other species. In this regard, we have added references to the introduction section. Massage method for sperm collection has not yet been described in Chinchilla, we are the first to have developed it. M&M: This section should have more detailed on "technique for semen collection and examination of semen characteristics". Currently the manuscript lack depth for understanding of the technique. Please include details and figures on: 1. There was no elaboration on how the chinchilla was restrained 2. The area of anus massage and the entire penile length on a chinchilla to aid reader to understand the procedure 3. Photomicrograph of the sperm We have added a more detailed description of the massage technique in the Material and Methods, including a picture about sperm collection and spermatozoa smears. Results: Table 1 should be improved - column 4 stated as "%". What does this imply? In the fourth column, we have added the name of the column to make it easier to understand. Table 2: Row 5 - I am not sure how this data is presented? The data in row 5 present significant differences. We have added the following sentence to the title of the table, which may help you to understand it better: “There is a significant difference between letters connected by hyphens.” Reviewer #3: The chinchilla is an endangered species in the wild, so some research on their reproduction is very important for the species perpetuation. This manuscript developed a manual technique for semen collection which met all animal welfare requirements. In addition, the basic parameters of sperm obtained by this method was determined. This study has definite value for reproduction and animal welfare of the chinchilla. However, I have some major concerns and minor concerns for this manuscript. Major concerns: 1. In this study, the authors just analyzed the parameters of sperm obtained by the manual technique and some spermatological parameters exhibit a larger fluctuation in the different times of the year. At the same time, this manual technique works for only 66% of chinchillas. So I suggest if the authors want to show the manual technique is a better way for semen collection than the common electro-ejaculation methods, they can analyze the parameters of sperm collected by electro-ejaculation methods. And through comparison and analysis, not only the advantages of the manual technique, but also the development of this technique will show up better. Thank you for your comments. We put a detailed description of the developed new technique in the M&M section of the manuscript. The primary aim of our work was not to compare electroejaculation with the massage technique, but to develop a technique in a new approach and checking whether it was useable to get semen with good quality all year round. We cited electroejaculation method in the introduction section because it has been the only technique for collection semen in chinchillas so far. Since there were no data on the quality of semen from the massage technique until now, we compared them with data from the available literature. We are not trying to prove that the massage technique is much better than electroejaculation, but rather to see if this method can work without anaesthesia, expensive facilities and torturing animals. Anyway, the new technique has another advantage, namely to obtain higher amount of spermatozoa per week in total, even though only 60-70% of males respond to stimulation, since it is useable several times a week compared to the electroejaculation which can be used only once a week or every two weeks. 2. The authors analyzed many spermatological parameters in this study. However, what’s the relationship between the results of these parameters and the manual technique developed in the study? Since earlier literature data on spermatological parameters show large deviations, we could only conclude that the values obtained with the manual technique are similar and fit to the available data. Therefore, the technique can be used safely for sperm collection and insemination throughout the year, which is the basis for the development of artificial insemination of the species. 3. About the manual technique, the authors did not write it clearly. I suggest it can be descripted in more detail in the methods part. We have added a more detailed description of the massage technique in the Material and Methods, including a picture about sperm collection and spermatozoa smears. 4. The discussion part is too simple. I suggest the authors performed a profound discussion on the importance of this manual technique and meaning for the reproduction of chinchillas. We discuss in details the results and the analysis of our study over two pages with several available references, thus do not think that the section is unsatisfactory for the professionals. We added some new statements to the section regarding the further advantage of the method. It includes all the new results, such as the fact, that only electroejaculation has been used to collect sperm until now, and only subjective estimation of motility has been used so far. Thus, we are the first to use CASA to test motility, to show first the types of sperm abnormalities in the species. Furthermore we were the first to describe a novel, effective massage technique in chinchillas. 5. Several brackets are italic incorrectly, such as (Chinchilla lanigera) , (Table 1) , (Fig.1)…… Thank you for your comment, it has been corrected in the text. 6. The references of this manuscript are so old and I suggest to add some new references. The reviewer is right that the literature used is not from today and not very much of it. However, this is because the last article on chinchilla reproduction was published in 2014. Unfortunately the researches of reproductive biology of chinchillas seems not to be a very popular topic, recently chinchillas are more commonly studied by researchers in human medical diagnostics from different aspects. If you know any recent publications, we would welcome your recommendation. Attachments Attachment Submitted filename: Response to Reviewers.docx https://doi.org/10.1371/journal.pone.0290441.r002
6 Jun 2023 Decision Letter - Wan-Xi Yang, Editor PONE-D-22-30686R1Elaboration of massage technique for semen collection and examination of semen characteristics in chinchilla (Chinchilla lanigera)PLOS ONE Dear Dr. Babarczi, 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 pay attentions to the reviewer's comments. The figures should be labled accordingly. ============================== Please submit your revised manuscript by Jul 21 2023 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, Wan-Xi Yang, 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. [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: 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 #3: Yes ****** 3. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: 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 #3: (No Response) ****** 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 #3: (No Response) ****** 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: All comments provided by the author(s) are acceptable. However, there are a few improvement need to be made; 1. Inconsistent reference list - There is inconsistent in writing the reference list. Author(s) should check the journal's format 2. The Fig 1 & Fig 2 should be labelled -A point of interest in the Fig 1 & Fig 2 should be precisely labelled 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: Yes: Mohd Iswadi Ismail Reviewer #3: 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.0290441.r003
Revision 2
13 Jul 2023 Author Response Dear Reviewer, thank you for your work, the change is you have requested, have been made. 1. Inconsistent reference list - There is inconsistent in writing the reference list. Author(s) should check the journal's format We check the format references, and we have corrected the incorrect information and the inconsistencies. 2. The Fig 1 & Fig 2 should be labelled -A point of interest in the Fig 1 & Fig 2 should be precisely labelled Based on your request we proposed the following changes to the title of the figures: Fig 1. Collection of sperm sample on a gauze pad whit massage technique base on digital manipulation Fig 2. Differentiating between live (white) and dead (pink) chinchilla spermatozoa in a stained smear using aniline blue and eosin Attachments Attachment Submitted filename: Response to Reviewers.docx https://doi.org/10.1371/journal.pone.0290441.r004
9 Aug 2023 Decision Letter - Wan-Xi Yang, Editor Elaboration of massage technique for semen collection and examination of semen characteristics in chinchilla (Chinchilla lanigera) PONE-D-22-30686R2 Dear Dr. Babarczi, 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, Wan-Xi Yang, Ph.D. Academic Editor PLOS ONE Additional Editor Comments (optional): Reviewers' comments: https://doi.org/10.1371/journal.pone.0290441.r005
Formally Accepted
22 Aug 2023 Acceptance Letter - Wan-Xi Yang, Editor PONE-D-22-30686R2 Elaboration of massage technique for semen collection and examination of semen characteristics in chinchilla (Chinchilla lanigera) Dear Dr. Babarczi: 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. Wan-Xi Yang Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0290441.r006

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