Non-invasive stress evaluation in domestic horses (Equus caballus): impact of housing conditions on sensory laterality and immunoglobulin A

The study aimed to evaluate sensory laterality and concentration of faecal immunoglobulin A (IgA) as non-invasive measures of stress in horses by comparing them with the already established measures of motor laterality and faecal glucocorticoid metabolites (FGMs). Eleven three-year-old horses were exposed to known stressful situations (change of housing, initial training) to assess the two new parameters. Sensory laterality initially shifted significantly to the left and faecal FGMs were significantly increased on the change from group to individual housing and remained high through initial training. Motor laterality shifted significantly to the left after one week of individual stabling. Faecal IgA remained unchanged throughout the experiment. We therefore suggest that sensory laterality may be helpful in assessing acute stress in horses, especially on an individual level, as it proved to be an objective behavioural parameter that is easy to observe. Comparably, motor laterality may be helpful in assessing long-lasting stress. The results indicate that stress changes sensory laterality in horses, but further research is needed on a larger sample to evaluate elevated chronic stress, as it was not clear whether the horses of the present study experienced compromised welfare, which it has been proposed may affect faecal IgA.

Page 5, line 12: The EIA is not directed against 11-oxoaetiocholanolone-17-CMO (the antibody was raised gainst that immunogen coupled to BSA). It's ok just to mention the name of the EIA (maybe include the group of FGMs picked up by this assay). Line 19-21 (also lines 34 and 35): That corrections sounds odd to me. What were intra-assay CV? If those were also large, you cannot "correct" with such a factor. By the way, I don't understand (from the wording) how it was calculated. Line 27: delete "and mixed in the freezer bag by hand". You mention this already before -so why another mixing here? Line 30 and 31: there should be a word space between a number and its dimension (1600 g; 1.2 ml; 3260 g) Page 6; 3.8. I wonder why inter-observer correlation was calculated for motor laterality (line 8), if only one one did the observations (line 10). 4. a) and onwards. What are the given increases? Taken from mean or median values? Or calculated on an individual basis and from those values the mean/median? Line 47: I was surprised to read about salivary cortisol here? At least I don't find it in Fig. 1 (and elsewhere). Page 7, line 36: Reword: …to be a good behavioural parameter for the non-invasive evaluation…. Line 41/42: The sentence is hard to understand, please reword. Line 58: The sample size is always limited, but you probably mean "low". References: Please carefully revise them. There are several refs with lacking full information (such as article number, issue or pages; e.g. 21; 29; 59,..) Ref 24: Delete "Text" Ref 29: I guess the journal is "Animals" issue/article number? Ref 53: That is unsuited here -please replace by Palme, 2019. Fig 1: As mentioned before: please modify "base" to baseline; delete the (b) -as the box is also in yellow you clearly indicate that it is the same situation (maybe rewrite to 1 d, 2 d, 7 d) Delete the "concentrations" from both y-axes (that's trivial if you give the dimension). Any explanation for the extremely high outlier in 2.? I suggest only mentioning it, but redrawing the figure with the y-axes scaled from 0 to 40! Delete the "trend" (*). It is non-significant, and just makes the figure more complicated.  Beh. 199,[229][230][231][232][233][234][235][236][237][238][239][240][241][242][243] Decision letter (RSOS-191994.R0) 02-Jan-2020 Dear Ms Marr, The editors assigned to your paper ("Non-invasive stress evaluation in domestic horses (Equus caballus): Impact of housing conditions on sensory laterality and immunoglobulin A") have now received comments from reviewers. We would like you to revise your paper in accordance with the referee and Associate Editor suggestions which can be found below (not including confidential reports to the Editor). Please note this decision does not guarantee eventual acceptance.
Please submit a copy of your revised paper before 25-Jan-2020. Please note that the revision deadline will expire at 00.00am on this date. If we do not hear from you within this time then it will be assumed that the paper has been withdrawn. In exceptional circumstances, extensions may be possible if agreed with the Editorial Office in advance. We do not allow multiple rounds of revision so we urge you to make every effort to fully address all of the comments at this stage. If deemed necessary by the Editors, your manuscript will be sent back to one or more of the original reviewers for assessment. If the original reviewers are not available, we may invite new reviewers.
To revise your manuscript, log into http://mc.manuscriptcentral.com/rsos and enter your Author Centre, where you will find your manuscript title listed under "Manuscripts with Decisions." Under "Actions," click on "Create a Revision." Your manuscript number has been appended to denote a revision. Revise your manuscript and upload a new version through your Author Centre.
When submitting your revised manuscript, you must respond to the comments made by the referees and upload a file "Response to Referees" in "Section 6 -File Upload". Please use this to document how you have responded to the comments, and the adjustments you have made. In order to expedite the processing of the revised manuscript, please be as specific as possible in your response.
In addition to addressing all of the reviewers' and editor's comments please also ensure that your revised manuscript contains the following sections as appropriate before the reference list: • Ethics statement (if applicable) If your study uses humans or animals please include details of the ethical approval received, including the name of the committee that granted approval. For human studies please also detail whether informed consent was obtained. For field studies on animals please include details of all permissions, licences and/or approvals granted to carry out the fieldwork.
• Data accessibility It is a condition of publication that all supporting data are made available either as supplementary information or preferably in a suitable permanent repository. The data accessibility section should state where the article's supporting data can be accessed. This section should also include details, where possible of where to access other relevant research materials such as statistical tools, protocols, software etc can be accessed. If the data have been deposited in an external repository this section should list the database, accession number and link to the DOI for all data from the article that have been made publicly available. Data sets that have been deposited in an external repository and have a DOI should also be appropriately cited in the manuscript and included in the reference list.
If you wish to submit your supporting data or code to Dryad (http://datadryad.org/), or modify your current submission to dryad, please use the following link: http://datadryad.org/submit?journalID=RSOS&manu=RSOS-191994 • Competing interests Please declare any financial or non-financial competing interests, or state that you have no competing interests.
• Authors' contributions All submissions, other than those with a single author, must include an Authors' Contributions section which individually lists the specific contribution of each author. The list of Authors should meet all of the following criteria; 1) substantial contributions to conception and design, or acquisition of data, or analysis and interpretation of data; 2) drafting the article or revising it critically for important intellectual content; and 3) final approval of the version to be published.
All contributors who do not meet all of these criteria should be included in the acknowledgements.
We suggest the following format: AB carried out the molecular lab work, participated in data analysis, carried out sequence alignments, participated in the design of the study and drafted the manuscript; CD carried out the statistical analyses; EF collected field data; GH conceived of the study, designed the study, coordinated the study and helped draft the manuscript. All authors gave final approval for publication.
• Acknowledgements Please acknowledge anyone who contributed to the study but did not meet the authorship criteria.
• Funding statement Please list the source of funding for each author.
Once again, thank you for submitting your manuscript to Royal Society Open Science and I look forward to receiving your revision. If you have any questions at all, please do not hesitate to get in touch. The presented paper investigates sensory laterality and concentration of faecal immunoglobulin as two potential non-invasive measures of stress in horses. Both reviewers find the study interesting and well presented and they have a number of suggestions to further improve the readability of the manuscript prior to publication.
Reviewers' Comments to Author: Reviewer: 1 Comments to the Author(s) This presents a novel and useful study looking at non-invasive measures of both short-term and long-term stress in horses. This paper is well written, and only requires some small edits to enhance its readability. Page 2 L34 -insert the word "initially" before "significantly shifted to the left…" L35 -insert "and remained high through initial training" after "individual stabling." I know you are probably limited by word count, but I feel it would be useful to include the results of your IgA in the summary. L45 -change wording "…individual stabling without contact with conspecifics…" Page 3 L24 -reference 31 deals with differences in right and left hemisphere processing, but in humans, not horses. Please adjust where this reference appears. L44 -Change to read "...in free-roaming Przewalski and domestic horses.." L48 -one-sided L57 -two months (plural) Page 4 L27-29 -Maybe move the description of the box stalls to the above section 3.1 L35-36 -Can you explain more clearly what responses you expected -ie. what was your hypothesis? L47 -Please explain this in more detail as "usual practice" will differ among individuals, countries, etc. L58 -please rewrite as either "To calculate stable mean base values..." or "To calculate a stable mean base value..." Page 5 L11 -Please indicate the length of time for centrifugation. L32,33 -Please write out the acronyms EIA and ELISA. Page 6 L17 -Please explain what the "cs-tool" is. L21 -Include the test statistic and p-value for the Shapiro Wilks test. L47-49 -There is no mention of salivary cortisol analyses in the methodology. Furthermore, Fig 1 shows only values at 24h and 48h after initial training (not immediately or 2h after; nor are these time frames referred to in methodology). Also do not begin the next sentence with a numeral. Page 7 L17-18 -could it also be that training further developed the "sidedness" of particular horses? L41 -months (plural) L56-57 -However, Squibb et al. (2018) showed that behaviour did not reflect internal shifts in heart rate variability or eye temperature. Perhaps this is due to mature horses used in that study (thus trained to suppress conflict behaviour) versus the untrained horses in this study. Squibb, K. The paper submitted by Marr et al (RSOS-191994) reports a "stress" experiment (change of housing; initial training) in horses. The study aims at evaluating two new stress measures (sensory laterality and faecal IgA) by comparing them with two well established ones (FGMs and motor laterality). The latter clearly demonstrated the stressfulness of the situations, which was also found when using sensory laterality (but not faecal IgA). I think the experiment is well planed, the findings are interesting and, although preliminary, the suggested parameters (especially sensory laterality) are worth to be further investigated. Nevertheless there are several things, which to my opinion need to be revised and/or addressed adequately (see my detailed comments below) before the paper is suited for publication.

Detailed comments (ordered by line):
Page 2, line 32 (and elsewhere) I suggest adding the "s" for the plural: FGMs -in some cases the singular is needed (e.g. faecal glucocorticoid metabolite concentrations -FGM) Line 37: modify to "level, as a behavioural parameter that….(I may be mistaken, but think that observing behaviour is always non-invasive, or?) Abstract: I miss a word about unaffected IgA levels. Line 46: stress responses (plural) Line 49: better use "stressors" instead of "stress" (two times) Line 57: Minimal to non-invasive methods ,…, such as glucocorticoids in saliva or their metabolites in faeces… (what is a gentle method?) Page 3: line 0 and 1: GCs (or GC) instead of GM, because that is not only true for faeces, but a general statement. Line 39: But see Gorgasser et al (2007), where no effect was seen. I suggest to include that ref (here and/or in the discussion), which indicated that it may depend upon breed or method.
Line 41: Ref 53 is not about FGMs -I suggest to replace it with a most recent review here (Palme, 2019). Line 42: stress (no plural) or stressors. "decreased/increased" (Past Tense) Page 4: line 4: why two and three-year-old? You only mention three-year-old elsewhere? Line 16: "ad libitum" in italics 3.2. -a) and b): I suggest to combine those -there is nothing different in b) -just a few days later. It will also make the figs etc easier to comprehend. It may be helpful to point to Table S1 already here (or even include a graph in the main text illustrating the timeline of the different situations and sample collections). Line 35: "quicker" 3.3. -How fresh were collected samples? I think that's a criticial issue, as a recent study be the same research group (why not cite it?) demonstrated. Line 55 (and elsewhere): I suggest to use "baseline" (adjective) instead of base throughout the ms! Line 58: I wonder whether those 3 values were normally distributed, and think it may be better to use the median (more representative if there are outliers -and almost identical if not). Maybe good to mention that the delay time of faecal excretion is about 1 day in horses, and therefore those sampling points were chosen. Page 5, line 12: The EIA is not directed against 11-oxoaetiocholanolone-17-CMO (the antibody was raised gainst that immunogen coupled to BSA). It's ok just to mention the name of the EIA (maybe include the group of FGMs picked up by this assay). Line 19-21 (also lines 34 and 35): That corrections sounds odd to me. What were intra-assay CV? If those were also large, you cannot "correct" with such a factor. By the way, I don't understand (from the wording) how it was calculated. Line 27: delete "and mixed in the freezer bag by hand". You mention this already before -so why another mixing here? Line 30 and 31: there should be a word space between a number and its dimension (1600 g; 1.2 ml; 3260 g) Page 6; 3.8. I wonder why inter-observer correlation was calculated for motor laterality (line 8), if only one one did the observations (line 10). 4. a) and onwards. What are the given increases? Taken from mean or median values? Or calculated on an individual basis and from those values the mean/median? Line 47: I was surprised to read about salivary cortisol here? At least I don't find it in Fig. 1 (and elsewhere). Page 7, line 36: Reword: …to be a good behavioural parameter for the non-invasive evaluation…. Line 41/42: The sentence is hard to understand, please reword. Line 58: The sample size is always limited, but you probably mean "low". References: Please carefully revise them. There are several refs with lacking full information (such as article number, issue or pages; e.g. 21; 29; 59,..) Ref 24: Delete "Text" Ref 29: I guess the journal is "Animals" issue/article number? Ref 53: That is unsuited here -please replace by Palme, 2019. Fig 1: As mentioned before: please modify "base" to baseline; delete the (b) -as the box is also in yellow you clearly indicate that it is the same situation (maybe rewrite to 1 d, 2 d, 7 d) Delete the "concentrations" from both y-axes (that's trivial if you give the dimension). Any explanation for the extremely high outlier in 2.? I suggest only mentioning it, but redrawing the figure with the y-axes scaled from 0 to 40! Delete the "trend" (*). It is non-significant, and just makes the figure more complicated. Decision letter (RSOS-191994.R1) 28-Jan-2020 Dear Ms Marr, It is a pleasure to accept your manuscript entitled "Non-invasive stress evaluation in domestic horses (Equus caballus): Impact of housing conditions on sensory laterality and immunoglobulin A" in its current form for publication in Royal Society Open Science. The comments of the reviewer(s) who reviewed your manuscript are included at the foot of this letter.
Please ensure that you send to the editorial office an editable version of your accepted manuscript, and individual files for each figure and table included in your manuscript. You can send these in a zip folder if more convenient. Failure to provide these files may delay the processing of your proof. You may disregard this request if you have already provided these files to the editorial office.
You can expect to receive a proof of your article in the near future. Please contact the editorial office (openscience_proofs@royalsociety.org) and the production office (openscience@royalsociety.org) to let us know if you are likely to be away from e-mail contact --if you are going to be away, please nominate a co-author (if available) to manage the proofing process, and ensure they are copied into your email to the journal.
Due to rapid publication and an extremely tight schedule, if comments are not received, your paper may experience a delay in publication.
Please see the Royal Society Publishing guidance on how you may share your accepted author manuscript at https://royalsociety.org/journals/ethics-policies/media-embargo/.

Associate Editor's comments (Dr Claudia Wascher):
The presented paper investigates sensory laterality and concentration of faecal immunoglobulin as two potential non-invasive measures of stress in horses. Both reviewers find the study interesting and well presented and they have a number of suggestions to further improve the readability of the manuscript prior to publication.
 Thank you very much. We addressed the suggestion in a point-by-point response to the reviewers comments Reviewers' Comments to Author:

Reviewer: 1
Comments to the Author(s) This presents a novel and useful study looking at non-invasive measures of both short-term and longterm stress in horses. This paper is well written, and only requires some small edits to enhance its readability.
 Thank you very much for your valuable comments. They helped to improve the readability of our manuscript a lot.
Page 2 L34 -insert the word "initially" before "significantly shifted to the left…"  done L35 -insert "and remained high through initial training" after "individual stabling."  done I know you are probably limited by word count, but I feel it would be useful to include the results of your IgA in the summary.
 This point is well taken. You are right, the abstract is limited by word count. But we still could add 10 words and include short sentence about the IgA results L45 -change wording "…individual stabling without contact with conspecifics…"  done Page 3 L24 -reference 31 deals with differences in right and left hemisphere processing, but in humans, not horses. Please adjust where this reference appears.
 Thank you. We deleted this reference (Royet and Plailly, 2004), as it was not really suitable in this context L44 -Change to read "...in free-roaming Przewalski and domestic horses.." L48 -one-sided L57 -two months (plural)  L44, L48, L57: Thanks, done. Page 4 L27-29 -Maybe move the description of the box stalls to the above section 3.1