Factors Affecting Hair Cortisol Concentration in Domestic Dogs: A Focus on Factors Related to Dogs and Their Guardians

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

A rather interesting study, and good to see a fairly large cohort of normal dogs being used, which will add significantly to our knowledge and understanding.

 

Introduction, general point. Cortisol is a bivalent marker, and can increase in both negative arousal (stress) and positive arousal (fun). The introduction is describing the use of cortisol for potentially negative arousal only. Given that these are vet visit samples, and no behavioural markers used (just an owner/guardian estimate), it would be difficult to identify changes in cortisol as due to negative arousal.

ELISA assay in general. Were these samples done in duplicate/triplicate? If so were mean values used for analysis. What was the reproducibility?

Results, a general point. Given that you have a fairly large cohort of healthy dogs, the paper could be improved by calculating the clinical reference range for all the healthy dogs. Given the limited amount of reference range data available for cortisol, this would be a key extra impact for the paper. Similar the mean (plus SD) for each dog breed could be added to table 1, as it only has breed numbers. This would add extra information for researchers, if some breeds might have higher levels – breed variation? Up until now a lot of studies have used small cohorts and slightly different kits, etc, so great to have one large cohort under the same test ELISA system. In doing so, I think this could make a  a paper that is fairly highly cited, for this reason alone! 

References have a large number of formatting variations, missing journal information. I have listed a few examples below.

 

L44. Discusses cortisol as a measure of stress but does not mention the cut-off limit (reference range) for normal dogs, and stressed dogs.

L137. Should it be 200 rpm not 200 g/min as g is a force not a rotational/shaking rate?

L139-142. Sample dried, but no information about volumes of resuspension or buffer.

L284. Maybe, re-write the dogs/guardian’s age sentence as it can currently read as ageing together too.

L288. Maybe not effect, but more correlation as age will not directly effect/cause but can correlate with these changes .

L301. “Fair positive “ does not make sense – would “moderately positive” describe better?

L420. Missing book/Journal information.

L422. Missing Journal information.

L461. Title in CAPITOLS and missing journal information.

L464. Missing weblink to information.

L471. Missing book/journal information.

Comments on the Quality of English Language

Mostly fairly well written, understandable and clear. A couple of sentence are in need of rewording or clarification to aid understanding (see comments).

Author Response

1 Open Review

( ) I would not like to sign my review report 
(x) I would like to sign my review report 

Quality of English Language

(x) The English could be improved to more clearly express the research. 
( ) The English is fine and does not require any improvement. 

 

	Yes	Can be improved	Must be improved	Not applicable
Does the introduction provide sufficient background and include all relevant references?	( )	(x)	( )	( )
Is the research design appropriate?	( )	(x)	( )	( )
Are the methods adequately described?	( )	(x)	( )	( )
Are the results clearly presented?	(x)	( )	( )	( )
Are the conclusions supported by the results?	(x)	( )	( )	( )
Are all figures and tables clear and well-presented?	( )	(x)	( )	( )

 

Comments and Suggestions for Authors

A rather interesting study, and good to see a fairly large cohort of normal dogs being used, which will add significantly to our knowledge and understanding.

 

Introduction, general point. Cortisol is a bivalent marker, and can increase in both negative arousal (stress) and positive arousal (fun). The introduction is describing the use of cortisol for potentially negative arousal only. Given that these are vet visit samples, and no behavioural markers used (just an owner/guardian estimate), it would be difficult to identify changes in cortisol as due to negative arousal.

• 74: Yes, as correctly noted by the reviewer, this clarification concerning cortisol has been added at lines 74-76.

 

ELISA assay in general. Were these samples done in duplicate/triplicate? If so were mean values used for analysis. What was the reproducibility?

• 169: Yes, samples were analysed in duplicate and the mean was used for analysis, as now added at lines 169-170. As for reproducibility, as specified at line 171-172 tthe laboratory follows a method that was previously subjected to an internal validation process, as reported in a published study [21], where all info are reported.

 

Results, a general point. Given that you have a fairly large cohort of healthy dogs, the paper could be improved by calculating the clinical reference range for all the healthy dogs. Given the limited amount of reference range data available for cortisol, this would be a key extra impact for the paper.

• 206-208: Thank you for the suggestion. As data don’t follow normal distribution, we calculated the clinical reference range as the interval of values containing the central 95 % of a healthy population, i.e. the reference limits are the values at the 2.5th and 97.5th percentiles, respectively.

Similar the mean (plus SD) for each dog breed could be added to table 1, as it only has breed numbers. This would add extra information for researchers, if some breeds might have higher levels – breed variation? Up until now a lot of studies have used small cohorts and slightly different kits, etc, so great to have one large cohort under the same test ELISA system. In doing so, I think this could make a  a paper that is fairly highly cited, for this reason alone!

 

• 112, Table 1: Yes, Thank you for the suggestion. We had considered this as well for the groups with at least five subjects, but as we found that individual variability within the breed was very high, we had omitted it. However, as noted by the reviewer, this remains useful information to include and can now be found in table 1.

 

References have a large number of formatting variations, missing journal information. I have listed a few examples below.

• 458: Thank you for spotting this. We have now revised the list of references in order to comply with guide for authors.

 

L44. Discusses cortisol as a measure of stress but does not mention the cut-off limit (reference range) for normal dogs, and stressed dogs.

• Considering the large variation in methods, there is not a clear cut-off limit that allows to distinguish between normal and stressed dogs. However, for the reslts of the current study, we calculated a clinical reference range, as added at L.214-215.

 

L137. Should it be 200 rpm not 200 g/min as g is a force not a rotational/shaking rate?

• 159: Thank you for spotting it, “rpm” is the appropriate unit. The use of “g/min” was a translation mistake from Italian, where “g” stands for “giri”, meaning rotations.

 

L139-142. Sample dried, but no information about volumes of resuspension or buffer.

• 165: Thank you for spotting it, this information has been added at line 165.

 

L284. Maybe, re-write the dogs/guardian’s age sentence as it can currently read as ageing together too.

• 326-7: Yes, the requested correction has been made for greater clarity.

 

L288. Maybe not effect, but more correlation as age will not directly effect/cause but can correlate with these changes .

• 340-1: Yes, this correction has been added.

 

L301. “Fair positive “ does not make sense – would “moderately positive” describe better?

• 345: Yes, the term “fair” comes from a cited article [30], but indeed the term “moderate” seems to be more commonly used and clear to describe the strength of the correlation.

 

L420. Missing book/Journal information.

• 472: The missing information has been added.

L422. Missing Journal information.

• 478: The missing information has been added.

L461. Title in CAPITOLS and missing journal information.

• 536: The missing information has been added.

L464. Missing weblink to information.

• 538: The missing information has been added.

L471. Missing book/journal information.

• 546: The missing information has been added

Thank you for spotting this. We have now revised the list of references in order to comply with guide for authors.

 

Comments on the Quality of English Language

Mostly fairly well written, understandable and clear. A couple of sentence are in need of rewording or clarification to aid understanding (see comments).

Reviewer 2 Report

Comments and Suggestions for Authors

Title

The current title is too vague and does not adequately reflect the specific focus of the study. A more descriptive title is recommended, such as:
“Factors Affecting Hair Cortisol Concentration in Domestic Dogs”
or
“Influence of Biological and Clinical Variables on Hair Cortisol Levels in Companion Dogs”.

 

Abstract

• The abstract is generally clear and easy to follow; however, it lacks sufficient background information and a clear justification for the study.
• Line 22: Please define “EIA” in full at first mention, .
• Line 27: The sentence “Previous literature has shown that several factors can affect HCC in dogs, but results are often controversial” should be moved toward the beginning to provide stronger context for the research.

 

Introduction

• Line 44: Please expand on the limitations of traditional cortisol assessment methods.
• Line 52: The statement about incomplete understanding of hair follicle biology and cortisol may be misleading. The role of cortisol in hair follicle function has been well studied, and its detection in hair is supported by established physiological mechanisms. Consider revising this sentence for accuracy.

 

Materials and Methods

• Table 1: Please clarify the criteria used to categorise dog breeds in this study. Were they grouped by size, function, or based on breed classifications from an official organisation?

 

Results

• Table 3: Please include an analysis comparing cortisol levels between entire and neutered dogs, regardless of sex. This could provide valuable insight into hormonal influences on HCC.
• Table 6: A potential association between HCC and anxiety levels was observed (P = 0.087). Although not statistically significant, this trend may be biologically relevant and worth discussing, especially given the relatively small sample size.
• Line 260: Please provide more details regarding adrenal gland size. Define normal reference values, explain the criteria used to determine whether glands were classified as small or large, and consider including an ultrasound image or diagram if possible.

 

Discussion

• Line 281: Please revise this section to clearly differentiate between your findings and those reported in the literature. Specify whether the results are consistent or differ and discuss potential reasons for discrepancies.
• Line 284: Provide a physiological explanation for the influence of age on cortisol levels or adrenal function in dogs. Age-related changes in the hypothalamic-pituitary-adrenal (HPA) axis may be relevant.
• Line 329: Specify the nature of the chronic disease reported in the study. If primarily gastrointestinal (GI), this should be stated clearly, as different systems may affect cortisol regulation differently.
• Expand on the mechanisms by which chronic gastroenteritis may reduce cortisol levels. Consider including possible explanations such as malabsorption, impaired nutrient uptake, or HPA axis dysfunction.
• Clarify whether your study or previous research provides direct evidence that age influences cortisol levels in dogs. If this is a novel finding, it should be highlighted; otherwise, cite relevant supporting literature.
• Consider grouping dog breeds by size (e.g., small, medium, large) or function (e.g., working, companion) and comparing HCC among these categories to identify potential breed-related trends.

 

Author Response

2 Inizio modulo

Open Review

(x) I would not like to sign my review report 
( ) I would like to sign my review report 

Quality of English Language

( ) The English could be improved to more clearly express the research. 
(x) The English is fine and does not require any improvement. 

 

	Yes	Can be improved	Must be improved	Not applicable
Does the introduction provide sufficient background and include all relevant references?	(x)	( )	( )	( )
Is the research design appropriate?	(x)	( )	( )	( )
Are the methods adequately described?	( )	(x)	( )	( )
Are the results clearly presented?	( )	(x)	( )	( )
Are the conclusions supported by the results?	(x)	( )	( )	( )
Are all figures and tables clear and well-presented?	( )	(x)	( )	( )

 

Comments and Suggestions for Authors

Title

The current title is too vague and does not adequately reflect the specific focus of the study. A more descriptive title is recommended, such as:
“Factors Affecting Hair Cortisol Concentration in Domestic Dogs”
or
“Influence of Biological and Clinical Variables on Hair Cortisol Levels in Companion Dogs”.

• Thank you for the suggestion, we have modified it in “Factors Affecting Hair Cortisol Concentration in Domestic Dogs: a focus on factors related to dogs and their guardians”

 

Abstract

• The abstract is generally clear and easy to follow; however, it lacks sufficient background information and a clear justification for the study.

• 20: The abstract has been modified taking into account the comments of both reviewers. We hope it is now more grounded and clearer.

 

• Line 22: Please define “EIA” in full at first mention.

• 25: The requested change has been made.

 

• Line 27: The sentence “Previous literature has shown that several factors can affect HCC in dogs, but results are often controversial” should be moved toward the beginning to provide stronger context for the research.

• 20: The requested change has been made, it can now be found at line 20.

 

Introduction

• Line 44: Please expand on the limitations of traditional cortisol assessment methods.

• 49: Additional limitations have been reported at lines 49-55.

 

• Line 52: The statement about incomplete understanding of hair follicle biology and cortisol may be misleading. The role of cortisol in hair follicle function has been well studied, and its detection in hair is supported by established physiological mechanisms. Consider revising this sentence for accuracy.

• 63: the sentence has been modified for greater clarity as in lines 63-67.

 

Materials and Methods

• Table 1: Please clarify the criteria used to categorise dog breeds in this study. Were they grouped by size, function, or based on breed classifications from an official organisation?

• 109: Breeds were grouped according to the number of individuals represented in our sample, as now better reported in the caption of table 1. We hope now it is clearer.

 

Results

• Table 3: Please include an analysis comparing cortisol levels between entire and neutered dogs, regardless of sex. This could provide valuable insight into hormonal influences on HCC.

• 227: We have revised as suggested, changes can be found at lines 196 and230- 233, as well as in table 3.

• Table 6: A potential association between HCC and anxiety levels was observed (P = 0.087). Although not statistically significant, this trend may be biologically relevant and worth discussing, especially given the relatively small sample size.

• 382: We agree with the reviewer, this discussion can be found at lines 382-386.

 

• Line 260: Please provide more details regarding adrenal gland size. Define normal reference values, explain the criteria used to determine whether glands were classified as small or large, and consider including an ultrasound image or diagram if possible.

L.297: We have clarified this aspect; the changes can be found at lines 122 and 297-300. We agree with the reviewer that this might represent a relevant point; however, as the number of altered adrenal glands is very limited, we believe that it is not significant to stress this point, so we did not include images. 

Discussion

• Line 281: Please revise this section to clearly differentiate between your findings and those reported in the literature. Specify whether the results are consistent or differ and discuss potential reasons for discrepancies.

• 304: We have revised as suggested, changes can be found at lines 305-388.

 

• Line 284: Provide a physiological explanation for the influence of age on cortisol levels or adrenal function in dogs. Age-related changes in the hypothalamic-pituitary-adrenal (HPA) axis may be relevant.

• 337: This aspect has been widened, changes can be found at lines 337-343.

 

• Line 329: Specify the nature of the chronic disease reported in the study. If primarily gastrointestinal (GI), this should be stated clearly, as different systems may affect cortisol regulation differently.

• 116: Thank you for the suggestion, it has been added at lines 116-118.

 

• Expand on the mechanisms by which chronic gastroenteritis may reduce cortisol levels. Consider including possible explanations such as malabsorption, impaired nutrient uptake, or HPA axis dysfunction.

• 378: Thank you for the suggestion, it has been added at lines 378-380.

 

• Clarify whether your study or previous research provides direct evidence that age influences cortisol levels in dogs. If this is a novel finding, it should be highlighted; otherwise, cite relevant supporting literature.

• 322: As reported at lines 236-239, results on the influence of age on HCC belongs to this study and some modifications have been made in the discussion at lines 322-344.

 

• Consider grouping dog breeds by size (e.g., small, medium, large) or function (e.g., working, companion) and comparing HCC among these categories to identify potential breed-related trends.

• Table 1: This is a very good suggestion, however most dogs were mongrels for which we did not collect data on size, but we’ll take it into account for future studies.

 

 

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

I am happy with this version of the manuscript. This manuscript would be useful for cortisol evaluation and animal welfare assessment.