PONE-D-21-38220Validation of the rabbit pain behaviour scale (RPBS) to assess acute postoperative pain in rabbits (Oryctolagus cuniculus)PLOS ONE

Dear Dr. Luna,

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Additional Editor Comments:

Please find the reviews of the three reviewers. Pleaser carefully carry out the suggested revisions,

King Regards,

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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: Yes

Reviewer #2: Yes

Reviewer #3: Partly

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2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: 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

Reviewer #3: 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: Yes

Reviewer #2: Yes

Reviewer #3: No

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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: I reviewed the article titled “Validation of the rabbit pain behavior Scale (RPBS) to assess acute postoperative pain in rabbits (Oryctolagus cuniculus)”.

Explanatory and descriptive factor analysis can also offer a different perspective. Or it can be recommended for further studies as a limitation.

Construct validity does not mean responsiveness. It should be corrected.

Do you think the time is appropriate for responsiveness? Provide additional evidence by providing references.

King Regards.

Reviewer #2: Summary

There are very few validated pain scales designed for use in rabbits. This study by Pinho et al. generates and validates a useful novel tool, the rabbit pain behaviour scale (RPBS) for post-operative pain. Pinho et al. demonstrate strong correlations between the RPBS and commonly used unidimensional pain scales (VAS, SDS, NS). A moderate correlation with the Rabbit Grimace Scale is established. Importantly, the authors are the first to establish a threshold at which administration of analgesics is recommended for rabbits. This study provides valuable information to the field, but I do have some concerns and suggest some revisions.

Major Comments

Lines 405-406: “The Youden index determined a score ≥ 3 as the cut-off point to distinguish rabbits in pain from those without pain.”

• Figure 4 shows that rabbits in both ovariohysterectomy groups had median RPBS scores above 5 at baseline/before surgery, which would give the false impression that these rabbits were in pain and in need of analgesics.

• The authors attribute this false positive to methodology (lines 537-546): “Motivational items (pinecones and carrots) were offered at the beginning of each recording only in rabbits undergoing orthopaedic surgery, which stimulated activity… The groups undergoing orchiectomy showed good specificity for the RPBS, even without the presence of stimulating items. This difference in behaviour compared to the OVH groups may due to sex-related behavioural differences, as males are more active than females[57].”

o The use of pinecones and carrots as enrichment prior to filming the rabbits in the Ortho group is very important and should be mentioned more explicitly in the methods section. It’s discussed very well in the discussion.

o What were the sexes of the rabbits in the Ortho group?

o Ref. 57 is not sufficient: “Sex differences in human-directed social behavior in pet rabbits”

o

Lines 485-486: The authors mention that their study was completed prior to the publication of the Bristol Rabbit Pain Scale (BRPS; Benato et al. 2021). It would be valuable to determine the correlation between the RPBS and the BRPS, although this may not be possible. A more detailed discussion of the similarities and differences between the RPBS and the BRPS is warranted.

Lines 518-525: “In this sense, the RPBS showed a high correlation with the unidimensional scales. However, as in the validation of a scale to assess acute pain in sheep [24], the correlation with the facial scale (RbtGS)[18] was only moderate. This result may be related to the lack of training of evaluators to use the RbtGS and/or the lack of refinement of the latter instrument, which did not pass through the important validation criteria such as criterion validity, intra-rater reliability, principal component analysis and internal consistency.”

• It is very concerning that the evaluators were not trained to use the Rabbit Grimace Scale, when a goal of the study was to compare outcomes of the RPBS and the RbtGS. Were the evaluators trained to use the unidimensional scales?

Line 185-187: “The videos of the 58 animals were edited by the main researcher of the study to generate 2 to 3 minute videos that proportionally represented the duration and frequency of the behaviours observed in the original videos.”

• Table 1 shows that rabbits in the ORC-Melox, ORC-Multi, OVH-Pla, and OVH-Melox were originally videotaped for 15-20 minutes. Could you please elaborate on how the videos were shortened to the final length (2-3 minutes)?

Minor Comments

Line 73-86: Please define abbreviations

Table 3 – Typo “IY” instead of “YI”

Line 255 – I suggest adding a brief discussion of the principal component analysis to the discussion section.

Table 7 – The formatting of this table is unclear

Line 604-605 – “The positive side of this bias was the demonstration of the importance of using motivational items in assessments.” Yes, this is great!

Reviewer #3: This work appears to be an extension of the work by Benato et al. ((2021) https://doi.org/10.1371/journal.pone.0252417) and adds validity to RBPS and affirms the practicality of this assessment method in clinical and research settings.

The authors have conducted a comprehensive analysis to conduct their validation. However; there are few points of concern that need to be addressed before such validation is to be accepted and recommended for use in veterinary clinics or animal research laboratories.

Methodology

Evaluation of the videos:

How did the investigator avoid subjective bias while selecting the videos for evaluation? What criteria of inclusion and exclusion were used to minimize bias? It is possible to fall into confirmatory bias and miss essential findings that could have influenced the final conclusions. Please provide detail explanation on this.

Selection and training of evaluators

As in the above comment, there may be potential bias in the process of selection and training. What did you do to avoid/minimize these biases? "All evaluators were veterinarians with residency in veterinary anesthesiology and around 4 years of experience in the area (RHP, AAJ, DSC, and MWF)". It is good that the evaluators have some relevant background; however, such evaluation is better done by professionals with diverse background. Apart from veterinarians, the assessment should be done by animal behaviorists, laboratory animal specialists and researchers with experience in handling and using rabbits. In addition, even the selection of the veterinarian evaluators should include some diversity in terms of training, location and experience (as suggestion, veterinarians from different reputable institutions and veterinary hospitals in different countries).

Conclusion

"The use of motivational items is recommended to ensure an accurate assessment when using the scale." This statement is not clear enough. Please elaborate.

Minor grammatical error corrections. There are few grammatical errors that need to be corrected. For example: Line 133: "...and the anaesthesia maintained with...." Please do through editing and proofreading.

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Reviewer #1: No

Reviewer #2: No

Reviewer #3: Yes: Erkihun Aklilu

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Validation of the rabbit pain behaviour scale (RPBS) to assess acute postoperative pain in rabbits (Oryctolagus cuniculus)

PONE-D-21-38220R1

Dear Dr. Luna,

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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Kind regards,

Fatih Özden, PhD

Academic Editor

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

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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

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3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

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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: (No Response)

Reviewer #2: Yes

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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

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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 should be accepted as it stands. I would like to thank to the authors.

King Regards

Reviewer #2: Pinho et al. have sufficiently addressed all major and minor comments in their revised manuscript and it is now well suited for publication.

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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: No