Validation of Fecal Glucocorticoid Metabolites as Non-Invasive Markers for Monitoring Stress in Common Buzzards (Buteo buteo)

Round 1

Reviewer 1 Report

This Is an interesting study providing useful methodological information for the non-invasive assessment of stress in a wild bird. There are inherent limitations in any approach to measuring stress in wild animals and this study both suffers from some of these limitations and provides hints to avoid or reduce some of them. Thus, it is likely to be of value to those who need to monitor stress in wild animals, particularly wild birds.

I find the Materials and Methods, specifically sections “2.1 Settings and examination methods” and “2.2  Biological validation”, a little jumbled. I would suggest a clearer presentation of the pre-study and main study with more distinct paragraphs for (a) treatment of the birds (from start to finish), (b) protocol for the collection of fecal samples, (c) preservation and treatment of samples, and (d) laboratory procedures,

The Discussion is a little more verbose than I would prefer but this is a matter of personal writing style so I am just hinting at it, leaving it to the authors to perhaps alter some paragraphs for a more succinct style.

Further specific points in the manuscript that require attention:

Line 57-58:         “Birds of prey play a special role as predators, since unlike prey animals they only show insufficient objectively measurable external signs of stress”

                                What is the connection between the display of signs of stress  and the role of birds of prey as predators?

Lines 62-63:       “as all pathogens and environmental contaminants in the prey animals are accumulated and reflected in them [11].”

                                Although many environmental contaminants accumulate in birds of prey, I would hesitate to say ALL of them because some contaminants may quickly leave the body by excretion and others are rapidly broken down, even though they may have time to cause severe acute toxicity (e.g. organophospates). Thus, I would prefer a less absolute wording, such as “most pathogens and …” or “usually pathogens and …”.

Lines 131-132   “[the test birds were] placed individually in quietly situated aviaries”: please elaborate briefly, mentioning any external disturbance that might have occurred, and clarify whether birds in adjacent aviaries might have interacted causing any additional stress.

Line 216              “logarithmized” might be replaced by “log-transformed”

Line 270              I do not understand what is meant by “agile and defensible”. Please use different wording. The term “unremarkable” in Lines 271 and 276 is also difficult to understand.

Line 322              I would rather not capitalize chicken/chickens since it is not a species name (as Domestic Fowl or Jungle Fowl would be). Similarly for mice and hares (Line 362).

Lines 380-381   “the weather during sampling was consistently cool and thus did not correspond to the normal temperature of 18-22 °C”: what is “normal” temperature? Please clarify.

Line 425              Correct “pool samples” to “pooled samples”

Finally, I could point out the North American rather than British/Commonwealth spelling (center, behavior, etc.) – this is up to the authors or journal to decide.

A very few points have been mentioned in Comments and Suggestions for Authors. One or two typographical errors may still exist.

Author Response

Dear reviewer,

thank you very much for your helpful comments to improve this article. Please find the detailed responses to your comments in blue letters in the attached document and the corrections in track changes in the re-submitted manuscript.

Author Response File: Author Response.docx

Reviewer 2 Report

This is an informative paper on validating fecal glucocorticoid metabolites as non-invasive markers for monitoring stress in Common Buzzards. This study provides valuable insights into monitoring wildlife stress levels, mainly in human care. The authors raise several questions (advantages of using fecal glucocorticoid metabolites as a non-invasive marker for monitoring stress in Common Buzzards?  How can the findings be applied to improve the welfare of wildlife in human care? The implications of this research for conservation efforts aimed at protecting Common Buzzards and other wildlife species?) Which they attempt to answer through the analyses of fecal sampling, a non-invasive technique.

The study employed a thorough methodology for collecting and analyzing fecal samples to measure glucocorticoid metabolites and includes detailed results of biological validation in female and male Buzzards, enhancing the clarity and transparency of the findings. The research lays the groundwork for future studies on stress in birds of prey during treatment and rehabilitation in wildlife centers, offering valuable insights into animal welfare practices.

However, the paper also suffers from some weak points that the authors must address adequately. The study involved a small sample size of six Common Buzzards, which may limit the generalization of the findings to a larger population. Further, sampling Buzzards in two rounds over four weeks may introduce variability in the results, and the acclimatization period could have influenced the stress responses observed. Also, the study focused on a relatively short sampling period of one week, which may not capture long-term variations in stress levels or account for seasonal differences. While the study did not find significant sex differences in stress responses, further exploring potential variations between male and female Buzzards could provide valuable insights. It is also possible that the limited sample size influenced these data. The paper could benefit from external validation of the findings through replication by other research groups to confirm the reliability and reproducibility of the results. The discussion section could provide a more in-depth analysis of how the research findings can be practically applied to improve the welfare of Common Buzzards and other wildlife species in human care. The study did not extensively address all possible confounding factors influencing glucocorticoid metabolite levels in fecal samples, such as diet, habitat conditions, or individual variability. Addressing these weaknesses through larger sample sizes, longer-term monitoring, external validation, and more comprehensive discussions could enhance the robustness and applicability of the research findings in future studies and conservation efforts. These drawbacks need to be mentioned and discussed.

I realize that the authors have researched and written only about captive buzzards. But there are also published studies of stress on different buzzard species from around the wild using different techniques, e.g., ptilochronology and corticosterone levels, or a combination of the two. Since the authors also recommend broader applications of the technique, I suggest the authors dedicate a paragraph or two to the different studies conducted in the wild on other Buteo spp.

Other technical remarks in the paper:

Line 59 – delete “highly”

Lines 60-61: “Only temporary keeping for health care by qualified persons is permitted” – I am not sure this claim is valid since several different entities keep raptors. For example, falconry is legal in Germany, and quite a few raptors are kept privately.

Lines 67-70 – strong language with several superlatives. Recommend to tone down the arguments.

Lines 73 to 75 – “CRH (corticotropin-releasing hormone)” Please spell out all the hormones and then give the acronym in parentheses, as you did in line 72.

Author Response

Dear reviewer,

thank you very much for your helpful comments to improve this article. Please find the detailed responses to your comments in blue letters in the attached document and the corrections in track changes in the re-submitted manuscript.

Author Response File: Author Response.docx

Round 2

Reviewer 2 Report

I have read the revised manuscript and find it much improved. The authors have corrected or justified my concerns, so it can be accepted for publication.