PONE-D-23-37809Cold therapy and pain relief after hot-iron disbudding in dairy calvesPLOS ONE

Dear Dr. Lecorps,

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. See Editor Comments below

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We look forward to receiving your revised manuscript.

Kind regards,

Angel Abuelo, DVM, MRes, MSc, PhD, DABVP (Dairy), DECBHM

Academic Editor

PLOS ONE

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PONE-D-23-37809R1Cold therapy and pain relief after hot-iron disbudding in dairy calvesPLOS ONE

Dear Dr. Lecorps,

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. As per previous correspondence, there were some discrepancies between the comments of the 2 initial reviewers, and a 3rd reviewer was enlisted. Thank you for your patience in receiving an editorial decision. Collectively, there are some important concerns regarding the study design and interpretation of results. I invite you to address all reviewers' concerns by revising your manuscript accordingly.

Please submit your revised manuscript by Apr 15 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.

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,

Angel Abuelo, DVM, MRes, MSc, PhD, DABVP (Dairy), DECBHM

Academic Editor

PLOS ONE

Additional Editor Comments:

In the manuscript, please specify the type of study using adequate terminology (e.g., case-control, cross-sectional, cohort, randomized control trial, etc.)

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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: (No Response)

Reviewer #2: (No Response)

Reviewer #3: (No Response)

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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: Partly

Reviewer #2: Yes

Reviewer #3: No

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

Reviewer #1: I Don't Know

Reviewer #2: Yes

Reviewer #3: 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: Yes

Reviewer #2: Yes

Reviewer #3: 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

Reviewer #3: 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: This paper evaluates the use of "cold therapy" as a mechanism to reduce pain associated with dehorning. There are several challenges associated with the description of the study that makes it difficult to interpret the results. Furthermore, disbudding was completed very late and many different breeds were used. The practical relevance of producers using cold therapy is also a concern. Specific comments can be found below:

Line 20: Please clarify the type of analgesia provided

Line 21: It is unclear from the abstract how frequently the "cool packs" were changed or how they were placed on the calf's head and for how long. These seem like critical details to assess the practicality of this approach working.

Line 23 to 24: The calves were housed in completely separate pens when the treatments were being applied? This seems like it could introduce substantial bias as it would be impossible to control for the impact of pen or calves within the pen.

Line 39 to 41: I think it is important to note that the use of pain mitigation is low in some countries. For example, in Canada, the overwhelming majority of producers use pain control (it is a requirement), whereas in the US, most use pain control as well. This brings up another point that is worth discussing in the discussion of this paper. If few producers are using pain control, how will we convince them to use cold therapy which would seemingly be harder to manage?

Line 56 to 58: How was it determined to be beneficial? Through preference testing?

Line 85 to 86: Above, it mentions few have done this type of work making it difficult to calculate; however, here it suggests previous studies are done? Basing sample size on simply what other studies have enrolled is likely inappropriate and should be deleted. It is different to use effect sizes found in the studies to come up with a calculation.

Line 87 to 88: I assume that breeds would not be balanced between groups making it difficult to control for the likely effect that breed could have on the testing done in this study. How was that controlled for? If not controlled for, this should be noted as a limitation.

Line 88: When were calves normally weaned at this facility? Why was disbudding completed so late? Earlier disbudding would reduce inflammation and the size of the lesion. Likely important to preface the findings with the dehorning time in mind.

Line 92: Additional information is needed on the feeding program provided ahead of time.

Line 93: 190 g of milk replacer per L is very concentrated. Is this correct?

Line 94 to 95: Were groups made up to contain both treatment groups?

Line 110: How was this temperature maintained?

Line 118: Was the cold pack only applied for 20 min (or seems like 40 min per day for 6 days) and no longer? How effective is short application of cold packs in controlling inflammation? Is there any data or studies that could be cited here to provide insight into why this threshold was chosen?

Line 125: How was this milk prepared (temperature and g/L)? Was it the same milk replacer? Was time relative to last milk replacer meal controlled for? i.e., if a calf was fed milk at 8 am, was it tested at 8:30? Similarly, if a calf was fed at 8 would it be tested at 13:30? This would have a major impact on milk consumption.

Line 147 to 148: Pleas add in the brand of meloxicam and procaine used.

Line 155: This is confusing. 3.75 L were consumed per session? I assumed it went down as highlighted in Line 151 to 153. Please clarify

Line 164 to 165: Why were they excluded?

Line 171: Why are these excluded?

Line 191: How were these models evaluated to ensure model fit?

Line 233: Please provide information about the comparability of the two treatment groups including age at disbudding and breed to ensure comparable between groups.

Line 241: Pack temperature or treatment group?

Line 259: Was coloured controlled for in the model as well?

Line 300 to 301: What does "near-significant" mean? This is also not in the results as far as I can tell.

Line 314 to 317: I find this very confusing. Could a different word be used instead of trial? When were the calves disbudded and how did time relative to disbudding come into play?

Reviewer #2: This study uses approach-avoidance as well as Conditioned place preference to evaluate the effect of a cool pack on post disbudded calves. 15 calves were enrolled prior to the disbudding procedure and habituated and conditioned to reception of a cool or ambient pack while drinking milk. Calves were disbudded with local and systemic anesthesia and analgesia. The authors found that calves had a reduced latency to approach when a cool pack was offered vs. an ambient temp pack, but that calves did not display a preference to the pen where they received the cool pack.

This is a very interesting study that pairs two tests to evaluate the emotional component of pain/pain relief. The study is well written.

Comments:

L41. Is disbudding a procedure that has to be performed by a veterinarian in the UK? Is that why “workload” is cited as a reason to not provide pain mitigation? Please clarify.

L58. In that study, calves at 20d all still had necrotic buds, so is it still pain? What about sensitivity? Itchiness due to healing epithelial tissue?

L88. These calves are quite old to disbud (with most (Holstein) recommendations begin to disbud younger than 4 weeks of age). Why was the procedure done so late?

L128: Videos didn’t work for me, which was disappointing!

L134: So each calf received each pack/color combination (which stayed the same so if calf 1 had blue/cold they always got blue/cold)? Thus if that calf wanted the cold pack after disbudding, they would have to go to blue? Why not apply the cold pack to the calf post disbudding for some period of time paired to color? Couldn’t it be that cold on a “normal” calf head isn’t the same as cold on a disbudded head (soothing the inflammation, for example) and that you would have had a stronger response? Please clarify in the methods.

L343. Yes, would have been very interesting to give the calf the choice (milk meal paired with color/pack) but that would be much more difficult to habituate/train?

L346. Consider adding some discussion of calf age and how age (and bud size) might impact the calf response to this type of treatment. Would suspect that younger calves (shorter burn time) might not respond the same way? Any more information available on technique used in this study? Time of procedure (how long to burn?)?

Figure 4. Confused by the Trial 1/Trial 2/Trial 3…..what are these? There is no reference to these three trials anywhere else in the manuscript (that I can find). Please clarify.

Reviewer #3: I commend the authors for their creative approach to attempt to offer calves autonomy and additional pain relief given the long-lasting pain from disbudding. I appreciate the acknowledgement of the limitations for this study, such as the motivation for milk influencing the latency to respond in Trial 1. However, there were several conceptual areas that I found concerning or where my interpretation is that additional information would be useful.

1.) The use of an approach/aversion paradigm and conditioned place preference/aversion are not consistently described throughout the manuscript. I would benefit from some clarification and consistency.

o Ln 67 lays out the test as an approach-aversion test, while the predictions do not specify which aspect of the treatments would be aversive (ln 70-72).

o In Ede et al 2018 (10.1038/s41598-018-27669-7), the treatment was something the authors predicted that the calves would avoid (injection pain), thus naming it an approach/avoid paradigm in their work. As far as I can tell, in the current study, the prediction was that the calves would like the cold (ln 70-72), thus it becomes an approach/approach paradigm, which is more difficult to untangle in terms of what it means to the calf than what Ede et al 2018 did. Consider discussing this.

o It would be reasonable to predict that calves would find either of the packs aversive given the increased sensitivity following disbudding. This is touched on this briefly in ln 310. If you choose to continue describing the test as an approach-aversion test, I would benefit from some context for the aversive component in the introduction. In addition to references 12, 20, and 21, you may consider discussing other literature that provides evidence of sensitivity to touch following disbudding, to support the aversive component of your test (e.g.: 10.3168/jds.2021-21552; 10.3168/jds.2023-23238; 10.3168/jds.2009-2813).

o Ln 177-181 begins by describing the use of a conditioned place preference test, but ends by stating calves were tested for aversion. Ln 281-284 and Figure 5 present the preference of calves for the cold pack, but do not report any conditioned place aversion. Please clarify if conditioned place aversion was also tested or observed.

2.) The conclusions drawn are overstated and do not seem supported by the approach or data presented

o Ln 350-351: I do not see evidence presented in the current paper of the analgesic effects of cold therapy for calves. Rather, calves seek a milk reward faster with a cold pack is applied to their head compared to an ambient-temperature pack.

o Ln 26-27, 306: I do not see evidence that “suggests the cold therapy provides some form of pain relief post-disbudding” Rather, calves seek a milk reward faster with a cold pack is applied to their head compared to an ambient-temperature pack. Pain relieving characteristics were not evaluated.

o Ln 346: Further work is needed to understand possible benefits of cold therapy, rather than “confirm”.

o As the cold therapy was tied to a milk reward, it is difficult to disentangle the effects of the cold therapy from their motivation for milk. With the current methods, calves will likely approach the packs unless they are not hungry, which seems unlikely at this plane of nutrition and age of the calves. I would benefit from further discussion of the implications of this context when assessing the results.

3.) Use of the milk reward in conjunction with the compress.

o In Ln 92, it seems calves being provided their milk meals only in the arena? Was there any milk meals provided outside of the training or test sessions?

o Ln 154-155 was the remaining milk replacer fed in different location? Was this changed in any way as the reward size changed?

o Ln 187-188 given the ages, were any calves being weaned during the trial?

o The drinking speed seems rather fast (0.5L/60 sec; ln 151, 171), I would benefit from some contextualization of these speeds, how they compare to other literature, and biologically normal drinking behavior.

o What are the possible implications of initiating the suckling response to then end a training session and restart? For more context about how milk stimulates sucking, see, for example, de Passillé 2001, Sucking motivation and related problems in calves, 10.1016/s0168-1591(01)00108-3

4.) What do we know about cold therapy?

o I would benefit from additional information on the use of cold therapy.

o How long is cold therapy needed to receive the benefits of pain relief? Is it possible to provide context of timing in humans and other species?

o I appreciate that citations 29 and 30 are reviews of this topic, is there specific evidence that can be pulled to help provide additional context for this pain relief strategy?

o Is there evidence that the cold therapy actually cooled the wounds? Surface temperature of the calf’s head at the start vs after pack application?

5.) Why were the calves/trials where milk drinking persisted for longer than one minute excluded?

o What was the number of calves effected by each exclusion of data? How did those relate to treatment (e.g., Ln 165, 171)?

o The exclusion of these trials or limiting the compress contact to ~2 minutes seems to assume that the benefits from the cold are less than 60 seconds. Is there evidence to support this assumption?

6.) Additional points that I would benefit from clarification on:

o I am struggling with some of the mismatch in language used throughout the paper, particularly when discussing different metrics to assess calf welfare. For example, in ln 51, it is said that behavioral and physiological evidence provide weak evidence for animal’s emotional state, but later in the paper, there seems to be strong conclusions drawn using behavioral metrics. e.g., ln 299-300 conclusions about these responses and how they link to emotional state are made.

o There seems to be a discrepancy in the amount of discussion among results, where some considerations that are only tangential given much more space than those that are concrete and connected to results shown. For example, there a limited amount of discussion on the effects of milk allowance (ln 300-303; this is a concrete, important consideration to understand the findings), but other components that were not reported in this study, such as personality, were discussed in length (ln 333-341).

o Ln 109- 111: How was temperature maintained for the cold packs? Including this information would be helpful for others aiming to replicate your work

o Ln 146-147: Consider including details of the disbudding process, for example, the iron used and if the bud was left in or scooped out

o Ln 162-163: Could you provide an operational definition for “gazing” as part of your definition for latency to drink? How is this assessed for an animal with approximately 270 degrees in their visual field?

o Ln 199-201: Why were the data transformed through the addition of a constant to eliminate negative values? Given the interest in change from baseline, wouldn’t the negative values have meaning?

o Ln 56-58: What is the benefit of phrasing the study results in this way as opposed to contextualizing CPP, which is a component of your research?

o Ln 156-160: Why choose to conduct the CPP at the end of testing sessions when milk has been decreased rather than after session 7? Could this affect the association the calves would have made in the sessions running up to the CPP?

o Ln 87-88: Did you investigate any effects of breed?

o Ln 122-123: Was the side of the pen the color was on also altered? Or was blue always left as depicted in Figure 1?

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Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

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PONE-D-23-37809R2Cold therapy and pain relief after hot-iron disbudding in dairy calvesPLOS ONE

Dear Dr. Lecorps,

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. We appreciated the thoughtful revisions. Nevertheless, as highlighted by both reviewers, there are still issues pending that need to be addressed before the manuscript could be considered further. Please address these carefully.

Please submit your revised manuscript by Jun 20 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.

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,

Angel Abuelo, DVM, MRes, MSc, PhD, DABVP (Dairy), DECBHM

Academic Editor

PLOS ONE

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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: (No Response)

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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 #3: No

**********

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

Reviewer #1: Yes

Reviewer #3: No

**********

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: 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 #3: 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: Thanks for your responses to the comments. I understand in science, it is sometimes necessary to "push" the boundaries; however, the applicability of this is limited. This should be discussed especially when producers aren't even doing "easy" methods of pain control (i..e local + NSAID)

Line 19: Please add in the duration of the contact time

Line 95 to 97: I find this revised sentence confusing. I would delete that other studies have used 15 calves previously as it doesn't add meaning

Reviewer #3: Thanks to the authors for including the new figure, refining the use and predictions associated with the approach-aversion methodology and clarifying the conclusions about the new information generated about cold therapy. I have one outstanding concern in the R2 version.

I find the approach to excluding data (latency exceeded 30s, drinking exceeded 60s) concerning, as written. I’ve pasted the responses to Reviewers 1 and 3 about this topic below. From what I understand thus far, I am concerned about the approach taken, to exclude these data, has on the conclusions drawn.

A priori or post-hoc decision. Firstly, for the trials ended if latency was greater than 30s and drinking events were capped at 60s, I think it is important to know if this decision was made a priori or post hoc. As the authors point out, these kinds of cutoffs are common when running trial-based experiments. However, if these data were actually collected, then excluding them and imposing a 30s or 60s end was a post-hoc choice, this is worrisome to me. From my perspective, trimming a dataset to remove extreme values post hoc is not best practice.

Alternatives to exclusion. I appreciate that extreme values can affect statistical models, but exclusion should be a last resort. It is entirely possible that these responses represent an important part of the biological response of the calf. As the authors note, these represent “extremely aversive calf responses” and this was part of the question being asked. As such, exclusion is not “taking a conservative approach” as the authors describe it, at least from my perspective it is not conservative. To me, there are several options that would be preferable to the current approach of complete exclusion from a given model. Even when thresholds are set a priori, I prefer to enter a ceiling value for animals that do not meet or exceed a predetermined threshold, for example 31s for latency and 61s for drinking events, in this case (1 s beyond the cutoff). This approach allows the ceiling effect to be included while still meeting some of the assumptions of the statistical tests (e.g. normality). Another option, especially if data were collected beyond the thresholds (that is the decision about thresholds were post hoc) and actual ceilings were reached (e.g. say 80s drinking event), would be to keep all values in the dataset and explore data transformation in parametric models or use of non-parametric tests to address the model fit concerns the authors describe. Lastly, another approach I admire is to present the model outcomes with and without outliers (or extreme values in this case) included. This allows the reader to see their effect for themselves and make their own decision about how much they trust the findings.

Treatment differences. Lastly, the treatment distribution of the excluded values (or even if ceiling values are entered instead, as suggested above) should be described. I did not see a response to this aspect of my question in the authors’ response.

From the response to Reviewer #1

Line 164 to 165: Why were they excluded?

>>> Trials were ended immediately if latencies exceeded 30s. Although there is always

a cost in applying cut-off thresholds, 30s is often used in studies where calves have to

make a choice (e.g. Lecorps et al., 2018). We subsequently felt exclusion of ≥30s

latencies was the most appropriate approach. Whilst this reduced our data size slightly

and prevented us from capturing extremely aversive calf responses, including ≥30s

latencies may have had a disproportionate effect in our models. We therefore chose to

exclude these latencies from the model. To this effect, the section now reads (line 188-

192):

“If the latency for a given trial exceeded 30s, the trial was ended and the calf returned

to the lobby area, to commence the next trial, consistent with previous studies [27].

Latencies exceeding 30s were excluded from subsequent statistical analysis (21/588

trials excluded). Whilst cut-off thresholds prevent the capture of extremely aversive calf

responses, the exclusion of these latencies ensured our models were not

disproportionately influenced by extreme values.

Line 171: Why are these excluded?

We excluded calves that took greater than 60s to drink for a similar reason – given the

rate of consumptions noted previously, we considered 60s to consume the reward to

be a sufficiently long time to allow for consumption. A cut-off time was necessary to

maintain experimental timings, particularly when multiple calves per session needed to

be tested sequentially. Again, data points ≥60s drinking duration were excluded to

eliminate the effects of extreme values in our models, therefore taking a conservative

approach.

Response to Reviewer #3:

Why were the calves/trials where milk drinking persisted for longer than one minute

excluded?

o What was the number of calves effected by each exclusion of data? How did those

relate to treatment (e.g., Ln 165, 171)?

>>> Trials were ended immediately if latencies exceeded 30s. Although there is always

a cost in applying cut-off thresholds, 30s is often used in studies where calves have to

make a choice (e.g. Lecorps et al., 2018). We subsequently felt exclusion of ≥30s

latencies was the most appropriate approach. Whilst this reduced our data size slightly

and prevented us from capturing extremely aversive calf responses, including ≥30s

latencies may have had a disproportionate effect in our models, making detection of

differences between groups difficult. We therefore chose to exclude these latencies

from the model. To this effect, the section now reads (line 187-192):

“If the latency for a given trial exceeded 30s, the trial was ended and the calf returned

to the lobby area, to commence the next trial, consistent with previous studies [27].

Latencies exceeding 30s were excluded from subsequent statistical analysis (21/588

trials excluded). Whilst cut-off thresholds prevent the capture of extremely aversive calf

responses, the exclusion these latencies ensured our models were not

disproportionately influences by extreme values.”

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Reviewer #3: No

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Cold therapy and pain relief after hot-iron disbudding in dairy calves

PONE-D-23-37809R3

Dear Dr. Lecorps,

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.

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Angel Abuelo, DVM, MRes, MSc, PhD, DABVP (Dairy), DECBHM

Academic Editor

PLOS ONE

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Reviewer #1: (No Response)

Reviewer #3: All comments have been addressed

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Reviewer #1: Yes

Reviewer #3: Yes

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Reviewer #1: Thanks for your responses. I would still suggest adding in the exact timing even if there is a range into the abstract.

Reviewer #3: (No Response)

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