Effects of a Nutritional Supplement (DìRelax^TM) on Anxiety in Dogs in a Randomized Control Trial Design

Round 1

Reviewer 1 Report

This paper aimed to investigate the efficacy of a nutritional supplement called DiRelaxTM on anxiety in dogs. 

This paper has some weak points in the experimental design and I think it needs some improvements.

The use of an RCT design should be identified in the title. 

It is necessary to clarify if the study is blind for dog owners and researchers involved because this is a critical bias for the results presented: you used a questionnaire addressed to owners and behavioral data from impossible task tests were analyzed by researchers of the team therefore they can be influenced in their assessment.

In general, a section of Materials and Methods should be about "experimental design" with a deep description of the trial design (e.g. parallel, etc), randomization procedures, experimental endpoints chosen (primary, secondary, tertiary), outcome measures including how and when they are assessed. Procedures to collect data need to be better described (for example, it is not clear how many days after the end of the treatment you performed the second round of the impossible task test).

The inclusion and exclusion criteria of the animal sample need improvements (e.g. have these dogs ever performed the impossible task test before? What type of anxiety we are speaking about? Which severity classification did you use to classify patients? Any other treatments used (behavioral therapy? drugs?).  Some information about the sample was missed for example breed, housing, and husbandry conditions which needed to be similar among dogs enrolled. 

How the sample size was determined is not clear and you need to specify it. Moreover, a cross-over design would be useful to improve the straightness of your data.

point 2.3 blood analysis has some mistakes (you used serum aliquots for biochemistry analysis and total blood for the hematological ones) see lines 127-133. Describe in more detail how and when you performed the blood samples.

point 2.2 the questionnaire you used needs to be added in the supplementary material (it is not the original C-BARQ but you used a non-validated questionnaire based on C-BARQ, this could be a critical point for the external validity of your results you need to evidence in the paragraph about the limitations of the study I strongly suggest you add at the end of the discussion).

2.4.3 Data Collection: add some references to justify the ethogram adopted.

Results: animals enrolled flow (a diagram is appreciated) with losses and exclusions after randomization, define dates for recruitment and follow-up, a table with demographic and clinical characteristics of the two groups.

Discussion: Not only harm but also unintended effects should be described or if you didn't have unintended effects you need to declare it. 

A paragraph about the limitations of the study is necessary. Moreover, the external validity and applicability of the trial findings need to be added.

 

 

Author Response

Please find attached the replies to your comments.

Author Response File: Author Response.docx

Reviewer 2 Report

This report details the effects of a neutraceutical on anxiety in dogs participating in an unsolvable task paradigm. Anxiety is a serious problem among pet dogs, and any effective treatments, especially those without side effects, would be welcome. However, I have some concerns about the ms as currently written, and I am also not convinced about the utility of the research beyond Italy.

Major points

My main criticism is that the methods are not adequately described. Much information is provided, but there are still many questions to answer. I will provide specific detail later in the review. I also am not convinced that the authors have interpreted their results cautiously enough. It strikes me that there are very few changes between the treatment and control group and pre-post on anxiety symptoms, but the authors interpret this to mean that the supplement has a benefit. Perhaps there is some benefit, but it seems fairly minor. If the authors have more explicit results (e.g., effect sizes, CI) to present to justify this interpretation, I would like to see it.

Finally, I did a quick Google search and found that this product is available in Italy, but I couldn't see it being available elsewhere. Is it available outside of Italy/Europe? If not, this limits the relevance outside Europe and the title and abstract should be updated to reflect this. If it is available outside Europe, this should be noted.

Specific comments

Simple summary is too technical, because 'impossible task paradigm' isn't something that the lay community would be expected to immediately understand. Suggest rewriting to clearly define with the task is, to aid comprehension by non-scientific readers.

In both simple summary and abstract, as well as intermittently throughout the report, CBARQ is used instead of C-BARQ. I believe the latter is the correct spelling. Please be consistent.

Also, in some parts of the ms, the authors refer to the 'impossible task', while others refer to the 'unsolvable task'. I personally prefer the latter, but the authors should pick one and be consistent.

Introduction

L35-37 - one sentence should not be an entire para. Suggest combining this sentence with the next para.

L38 - check formatting of beginning of sentence. There should be an author's name spelled out ahead of [4].

L45 - '...about to be left alone...' is specific to separation anxiety. Consider restructuring this sentence so that it talks about general anxiety symptoms and then goes to more specific sep anx symptoms

L47 - '...anxiety-related effects...' . Effects is not the right word. Perhaps 'diagnoses' or 'disorders' would be more appropriate here.

L71-72 - awkward language. Suggest rephrasing to '...some validated behavioural tests allow measurement of stressful responses in a rigorous way'.

L71-76 - more detail is needed about the impossible task, and the results of the Passalacqua study cited. This information is critical to justify the study, so it needs to be explained clearly. How might anxiety impair the performance on the task? And what exactly is the task anyway? How does it work? Don't assume that the reader will know this, especially if practicing veterinarians are reading this for advice on how to manage anxiety using supplements. There are probably many veterinarians who are not familiar with this task, which came out of the dog cognition literature.

Methods

L97-110 - I suggest adding a table with relevant demographic info about each dog, especially their weight, since the dose is determined by mg/kg. It would even be a useful place to add the pre-post info about the blood test results to ensure that all were normal at baseline and didn't change much afterwards. At a minimum, pre-treatment blood test results should be mentioned here to confirm that there were no dogs with abnormal results, or if there were, that there wouldn't be baseline differences between the two groups.

L117 - This sentence suggests that the original C-BARQ was modified. How so? Similarly, has it been validated in Italian? Also, when were they administered? Above on L83 it says pre-post, but when exactly?

With that in mind, a flow chart of the process would be useful, given that there are many parts to this study.

L121 - how long before and after the treatment/tests?

Fig 2 - is the caption backwards? it looks like photo A is locked and photo B is unlocked. If the caption is correct, then more explanation in text will be required to explain how the box worked.

L159 - owners were asked not to feed dogs 4 hours prior, but how does that fit in with the 12 hours fasting noted in the blood test? This is why a flow chart would be helpful.

L172 - unclear phrasing in '...ensuring that the dog observed the protocols and blocked the apparatus in the unsolvable task'.

L180 - what is 'scrolling'?

L188 - was the C-BARQ analysed pre-post? or between groups? or both?

Results

Table 2- in most of the improved behaviours, apart from the last two, the wording of the item suggests that the dog is engaging in a particular behaviour in response to those scenarios, but the particular behaviour itself is not mentioned. What behaviour(s) do those items refer to? Growling?  Barking? Something else?

Table 2 - last p-value should be <0.001, not 0.000

L213 - suggest removing 'data now shown' and showing the data in the demographic table I suggested earlier.

Fig 3 L227 - does round 1 = solvable condition? Round 2 = unsolvable condition? Clarify

Discussion

At the start of the Discussion, restate the aim and the overall results, before going into the more theoretical aspects of why the study matters.

The Discussion is quite short, and it would benefit from a bit more comparison between Direlax and other available anxiety treatments. Is it more or less effective, based on existing research? What about side effects? Do more effective treatments have more side effects? What's the trade-off in those cases? Would this supplement be more appropriate for dogs with mild to moderate anxiety symptoms, while stronger pharmaceutical treatments could be reserved for severe anxiety? A para or two on this would be helpful.

L231 - suggest changing to 'Currently, they are often considered family members'. I don't think this feeling of dogs as family members is a universal phenomenon among pet owners. Most of the research into human-animal relationships uses convenience samples of people who probably love their dogs enough to complete a survey about them. We cannot assume that they represent typical dog owners.

L233 - 'in the right way' is unclear.

L260 - 'a positive trend was observed...' this was not reported in the results. If the authors want to push the idea of a 'trend' being important - something I personally disagree with but I accept that some researchers support - then they should report those results in the Results section before mentioning them in the Discussion. In this case, effect sizes and/or CIs would be helpful to interpret whether these 'trends' are actually meaningful or not.

 

 

 

 

 

 

Author Response

Please find attached the replies to your comments.

Author Response File: Author Response.docx

Reviewer 3 Report

The authors examined the effects on dog behavior and physiology of DiRelax, a nutraceutical formulated to reduce anxiety in dogs. Behavior was evaluated in two ways, completion of C-BARQ by owners and researchers scoring videotape recordings of the impossible task test. Physiological measurements included a complete blood count and blood chemistry analyses. Behavioral and physiological measures were recorded before treatment and after 30 days of treatment for dogs in the supplement group and placebo group. The authors found some behavioral differences between supplement and placebo groups (in both the C-BARQ and impossible task test) but no physiological differences. My main concerns are some lack of clarity and absence of critical information in the Materials and Methods section, as well as the failure to include the physiological data in the Results section. My specific comments are detailed below.

Lines 17-22: I suggest expanding the Simple Summary, which is very short and does not mention that physiology also was evaluated. Also, write out what C-BARQ stands for and briefly describe the questionnaire because the general public may not be familiar with it. I would also briefly describe the impossible task test.

Lines 23-31: More detail also is needed in the Abstract, specifically about the “clinical investigations” – even just mentioning complete blood count and blood chemistry would be helpful. Also include that there were no differences between supplement and placebo groups in the physiological measures – otherwise there is no information in the Abstract to back up your statement that DiRelax “can be safely administered with no adverse effects”.

Lines 35-37: It is unusual to have a single sentence paragraph – consider merging it with the next paragraph.

Line 66: Readers may be unfamiliar with the term “anamensis”, so either define the term or choose a more familiar word.

Line 77-80: While the goals of the research are clearly stated, this paragraph could be stronger if you state hypotheses tested and predictions made.

Line 82: I was a bit unclear about whether dogs were housed in facilities at University of Naples (or somewhere else?) for the full 30 days or whether owners brought them in daily for treatment with placebo or DiRelax. If housed at a facility away from home for a month or so, then some description of the housing conditions and daily care should be provided. Also, what impact could being housed away from home have on the dogs’ anxiety? This issue should be addressed in the Discussion.

Line 110: I assume that the supplement and placebo were administered once daily(?) in food (or not?), but this needs to be stated. Also, what was the composition of the placebo? More detail is needed here.

I might have missed it, but I assume that owners were unaware of whether their dog received the supplement or placebo when completing the C-BARQ at the end of the treatment? Similarly, I assume that researchers scoring the videotapes also were unaware of the group to which each dog belonged? If this information is missing, then add it if accurate.

Lines 121-122: by “after the treatment” do you mean “after 30 days of treatment”?

Line 166: The sex of an unfamiliar person has been shown to influence the behavior of dogs (e.g., Lore, R.K.; Eisenberg, F.B. Avoidance reactions of domestic dogs to unfamiliar male and female humans in a kennel setting. Appl. Anim. Behav. Sci. 1986, 15, 261–266. Wells, D.L.; Hepper, P.G. Male and female dogs respond differently to men and women. Appl. Anim. Behav. Sci. 1999, 61, 341–349 and other papers). Can you provide information on the sex of the unfamiliar individuals present at impossible task tests?

Line 180: What is the behavior listed as “scrolling”?

Lines 185-186: It is great that you checked inter-observer reliability, but it is typical to provide in the paper some measure of the values obtained (e.g., percentage of agreement, Kappa statistic, or correlation coefficient).

Lines 210-213: In order to conclude that DiRelax can be safely administered with no adverse effects on physiology and behavior, the physiology data should be included in the results section of the paper. The paper is not overly long, and the physiology values would be of interest to some readers (e.g., veterinarians).

Line 260: The statement about the positive trend seen in questions other than the six that were significant is the first time this is mentioned. The positive trend should be backed up with some examples in the Results section.

A paragraph on study limitations would strengthen the Discussion section.

Minor issues:

Line 45: Change “dogs perceive” to “a dog perceives”

Line 71: Change “to measure” to “measurement of”

Line 72: Unclear what is meant by “in a strictly way”

Line 76: I would insert “on this test” after “dogs” so readers know you are describing the impossible task test.

Line 197: No cap for “Due” and change “no normal distribution” to “non normal distribution”

Line 205: Change “over” to “of the”

Line 251: Change “indicates” to “indicate”

Line 265: Change “possess” to “possesses”

Author Response

Please find attached the replies to your comments.

Author Response File: Author Response.docx

Round 2

Reviewer 1 Report

Thank you for your answers, I appreciate the revisions made to the manuscript and I think it is suitable for publication in the present form.

Author Response

Your welcome, thank you for improving the manuscript with your suggestions.

Reviewer 2 Report

This manuscript has been much improved. It is now clear exactly what happened to the dogs and when. I just have a few minor comments for further clarification.

Table 4 - are the group and time effects reported, p-values? Or something else?

Fig 3 - suggest adding in the caption that R1 = pre-treatment, and R2 = post-treatment

Discussion - Para 1 is now very long. Suggest cutting into 2 or 3 smaller paras to improve flow.

L351 - suggest changing 'fairly' to 'mild'

Author Response

REV2

This manuscript has been much improved. It is now clear exactly what happened to the dogs and when. I just have a few minor comments for further clarification.

Table 4 - are the group and time effects reported, p-values? Or something else?

Yes, the group and the time effects was evaluated but no differences between groups emerged. In the table the P value has been clarified.

Fig 3 - suggest adding in the caption that R1 = pre-treatment, and R2 = post-treatment

Done.

Discussion - Para 1 is now very long. Suggest cutting into 2 or 3 smaller paras to improve flow.

Done.

L351 - suggest changing 'fairly' to 'mild'

Done.

Reviewer 3 Report

The authors have done a nice job revising their manuscript. The manuscript is much improved, especially with respect to clarification of study methods, and inclusion of physiological data and study limitations. Although I did not suggest this change, I also appreciated the increased coverage in the first paragraph of the Discussion of pharmaceuticals and nutraceuticals used to treat anxiety in dogs. I have only a few minor comments regarding wording.

1. I would write out Randomized Control Trial in the title (the abbreviation may not be familiar to all readers).
2. Table 1: Change “Neutralization” to “Neutered”
3. Table 4 title: Do you mean “Complete blood count” rather than “Cell blood count”? Also, explain in a footnote that RMSE means root mean squared error (the abbreviation may not be familiar to all readers).
4. Informed consent statement (lines 365-367): I would change “veterinary control” to “veterinary clinical and neurological examinations and blood collection” to be more specific.

Author Response

REV3

The authors have done a nice job revising their manuscript. The manuscript is much improved, especially with respect to clarification of study methods, and inclusion of physiological data and study limitations. Although I did not suggest this change, I also appreciated the increased coverage in the first paragraph of the Discussion of pharmaceuticals and nutraceuticals used to treat anxiety in dogs. I have only a few minor comments regarding wording.

1. I would write out Randomized Control Trial in the title (the abbreviation may not be familiar to all readers).

Done.

2. Table 1: Change “Neutralization” to “Neutered”

Done.

3. Table 4 title: Do you mean “Complete blood count” rather than “Cell blood count”? Also, explain in a footnote that RMSE means root mean squared error (the abbreviation may not be familiar to all readers).

Done.

4. Informed consent statement (lines 365-367): I would change “veterinary control” to “veterinary clinical and neurological examinations and blood collection” to be more specific.

Done.