Review Reports - Through the Lens of Age: Using Dog Photographs to Uncover Welfare and Stress

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

It is an interesting study. Also interesting how the specialists and the volunteers score almost similar, but the authors gave a valid reason for that.

Will an accuracy of 56,7 % be sufficient? To me it seems a bit low...

264 dogs were included but 262 photo"s were viewed...where were the other 2?

Author Response

Comment 1: It is an interesting study. Also interesting how the specialists and the volunteers score almost similar, but the authors gave a valid reason for that. Will an accuracy of 56,7 % be sufficient? To me it seems a bit low...

While we acknowledge that this may seem low, it is important to note that this accuracy significantly exceeds the random chance rate of 33.3%. Studies have shown that similar visual assessments, such as in human aging (Christensen et al., 2009) and wildlife classification (Swanson et al., 2016), have comparable accuracy rates, demonstrating the validity of non-invasive visual tools.

This tool offers valuable insights into dog welfare, particularly when paired with biological markers like telomere length, providing a more comprehensive understanding of the animal’s health. Its accessible and non-invasive nature also makes it practical for use by both specialists and non-specialists. While we agree that improvements could be made, such as refining the age evaluation process, we believe this tool adds an important dimension to welfare assessment, particularly for large-scale applications where traditional methods may not be feasible.

Comment 2: 264 dogs were included but 262 photo"s were viewed...where were the other 2?

Thank you for your observation regarding the discrepancy between the number of dogs and the number of photos evaluated. Upon review, the two missing photos were excluded due to poor image quality, which did not meet our standards for the analysis, so, we uploaded 262 photos for our volunteers to analyse them. These exclusions did not affect the overall statistical analysis, as the data set used for the volunteers evaluation was 60 photos from the 262 total. In the revised manuscript, we included this clarification in the methods section to ensure transparency.

Reviewer 2 Report

Comments and Suggestions for Authors

This study investigated the potential of a new tool for dog welfare assessment that could be used by non-specialists. The new tool is based on the use of dog’s apparent age (evaluated from pictures). This methodology is considered by the authors as a non-invasive tool for assessing dog welfare.

I read carefully the manuscript. The study's topic is interesting and explores the use of evaluating dog age for animal welfare assessment. The authors hypnotized that this tool permits to simplify the animal welfare assessment. It is very interesting the correlation between the estimated age and length of telomeres

Great work is needed to improve the manuscript to be suitable for publication in this journal. Please, consider the following comments.

The main issue is related to the concept of animal welfare and the way was used in the current study.

Animal welfare is a multidimensional concept and its assessment must consider this aspect. So, for this reason is very limiting to carry out animal welfare assessment using only a tool based on the dogs’ apparent age. It is a good idea to simplify the assessment approach but this cannot be without the fact that welfare is a multifaceted and complex thing

Animal welfare can be defined in a number of different ways, but there is a growing consensus that whatever the definition, it has to include three elements: the emotional state of the animal, its biological functioning and its ability to show normal patterns of behaviour. These three elements are closely interrelated. Animal welfare comprises the state of the animal's body and mind, and the extent to which its nature (genetic traits manifest in breed and temperament) is satisfied. In addition, the welfare of nonhuman animals is a complex concept that includes the biological functioning of an animal, emotional states and opportunities to experience natural life, including the execution of natural behaviours.

For these aspects related to animal welfare suggest some references

· Manteca, X., Velarde, A., & Jones, B. (2009). Animal welfare components. In Welfare of production animals: assessment and management of risks (pp. 61-77). Wageningen Academic.

· Arndt, S. S., Goerlich, V. C., & van der Staay, F. J. (2022). A dynamic concept of animal welfare: The role of appetitive and adverse internal and external factors and the animal’s ability to adapt to them. Frontiers in Animal Science, 3, 908513.

· Englund, M. D., & Cronin, K. A. (2023). Choice, control, and animal welfare: Definitions and essential inquiries to advance animal welfare science. Frontiers in Veterinary Science, 10, 1250251.

· Veissier, I., & Forkman, B. (2008). The Nature of Animal Welfare Science. Annual Review of Biomedical Sciences, 10.

The introduction should be revised to emphasize animal welfare in light of the above. The tool developed by the authors could be considered as a tool that can be integrated in the assessment of dog welfare. Or maybe better it could talk about a tool that can give information on the health condition of the dog and can be integrate in a holist approach for welfare assessment. I suggest to consider this second option in order to make the study more credible and published. Moreover, it is not clear how this tool works to give information on dog welfare condition. The estimation of age is an animal-based measure (ABM) that may be added and correlated to other ABMs for the assessment of welfare.

Here some example of dog welfare assessment tool and references that can be useful for the introduction of the manuscript:

· Malkani, R., Paramasivam, S., & Wolfensohn, S. (2022). Preliminary validation of a novel tool to assess dog welfare: The Animal Welfare Assessment Grid. Frontiers in veterinary science, 9, 940017.

· Polgár, Z., Blackwell, E. J., & Rooney, N. J. (2019). Assessing the welfare of kennelled dogs—A review of animal-based measures. Applied animal behaviour science, 213, 1-13.

· Bacon, H., Walters, H., Vancia, V., Connelly, L., & Waran, N. (2019). Development of a robust canine welfare assessment protocol for use in dog (Canis familiaris) catch-neuter-return (CNR) programmes. Animals, 9(8), 564.

· Berteselli, G. V., Arena, L., Candeloro, L., Dalla Villa, P., & De Massis, F. (2019). Interobserver agreement and sensitivity to climatic conditions in sheltered dogs' welfare evaluation performed with welfare assessment protocol (Shelter Quality protocol). Journal of Veterinary Behavior, 29, 45-52.

· Berteselli, G. V., Rapagnà, C., Salini, R., Badagliacca, P., Bellucci, F., Iannino, F., & Dalla Villa, P. (2021). A Pilot Study to Develop an Assessment Tool for Dogs Undergoing Trap-Neuter-Release (TNR) in Italy. An Overview on the National Implementation of TNR Programmes. Animals, 11(11), 3107.

· Barnard, S., Pedernera, C., Velarde, A., & Dalla Villa, P. (2014). Welfare assessment protocol for shelter dogs. Istituto Zooprofilattico Sperimentale dell’Abruzzo e del Molise.

Methods

LL 74-77 specify how many photos for each dog category considered

LL 92-93 specify who performed this procedure (vet, owner, etc.) and how the samples were stored and managed, who did perform the analysis?

LL 133-140 this part is not totally clear, detail better the selection of 60 photos (from 264), how many for the dog categories. LL 138-140 this part should be moved in the results, if participants are recruited by sharing the survey. Specify if the participants were selected a priori.

LL 145-147 as said previously, please specify better how the volunteers were involved.

LL 148-155 this part is not clear. 262 or 264 photos? What is the Dog’s Life project? How this step is related with the aim of the study?

LL 172-173 Please clarify how the photos were subdivided in the dogs’ age categories.

Results

Specify the distribution of the data (normal or not-normal distribution)

LL 219 33 volunteers are the total of respondents?

LL 239 this result is related to all the participants?

LL 246 no supplementary material was available.

LL 241-242 this result is not totally clear. It is not clear the difference between specialists and not-specialists.

LL 255 this result is very interesting. Was evaluated the correlation between the type of dog (pet, lab dog, working dog, shelter dog)? It is relevant to specify this point.

Discussion

As said for introduction, also for discussion is important to integrate parts related to animal welfare, animal welfare assessment etc.

Discussion is weak and needs to be expanded

LL 282 LL 285 as said above, it could be better to talk about health condition or quality of life

I suggest to explore the possible fields in which this approach can be used, and how it works for the purpose of the study.

For example, in shelter dogs where the environmental condition can affect the health and welfare status of these animals. How this tool can be used?

Author Response

Comment 1: This study investigated the potential of a new tool for dog welfare assessment that could be used by non-specialists. The new tool is based on the use of dog’s apparent age (evaluated from pictures). This methodology is considered by the authors as a non-invasive tool for assessing dog welfare.

Thank you for your thoughtful feedback and for recognizing the innovative potential of using apparent age as a non-invasive tool for assessing dog welfare. We acknowledge the importance of further elaborating on why this tool is considered non-invasive. By using photographs, we eliminate the need for physical contact, reducing the potential for stress or discomfort in the dogs. This approach aligns with our goal of developing a welfare assessment tool that is accessible and minimally disruptive. We would also like to emphasize that apparent age has been widely validated as a predictor of biological aging in humans (Gunn et al., 2008; Christensen et al., 2009). Our study extends this methodology to dogs, and while this is a novel application, the results suggest it can reliably estimate welfare indicators, especially when combined with biological markers like telomere length. In terms of reliability, we ensured that non-specialists were adequately supported through detailed tutorials on the Zooniverse platform. Our data showed that their performance in age prediction was comparable to that of specialists, which supports the tool’s potential for broader application. In the revised manuscript, we will expand on these points and discuss any potential limitations, such as subjective interpretation of photographs. Future improvements will include enhanced guidelines for evaluators and exploring automated image analysis to improve consistency. Thank you once again for your valuable insights, and we hope these clarifications strengthen the study.

Comment 2: I read carefully the manuscript. The study's topic is interesting and explores the use of evaluating dog age for animal welfare assessment. The authors hypnotized that this tool permits to simplify the animal welfare assessment. It is very interesting the correlation between the estimated age and length of telomeres

Thank you for your insightful feedback and for recognizing the importance of our hypothesis regarding the simplification of welfare assessments and the correlation between apparent age and telomere length. Regarding the simplification of welfare assessment, we emphasize that using apparent age as a visual tool significantly reduces the need for invasive methods like cortisol measurement, which require more time and resources. By focusing on photographs, welfare evaluations can be conducted more frequently and broadly, particularly in settings such as shelters or veterinary clinics, where non-invasive, quick assessments are highly beneficial. We appreciate your interest in the correlation between estimated age and telomere length. Our statistical analysis, using Generalized Linear Models (GLMs), confirmed that dogs with accurate age predictions had telomere lengths consistent with their chronological age, while those with prematurely graying hair showed shorter telomeres, suggesting accelerated biological aging. This finding is critical for early detection of stress-related aging, further validating apparent age as a welfare assessment tool. We also highlight that this tool is designed for non-specialists, with platforms like Zooniverse allowing public volunteers to participate in welfare assessments. This makes it accessible for a wider range of users, enabling regular welfare monitoring. We will incorporate these clarifications into the manuscript and provide more details on the statistical validation of the telomere-age correlation. Thank you once again for your valuable feedback.

Comment 3: Great work is needed to improve the manuscript to be suitable for publication in this journal. Please, consider the following comments. The main issue is related to the concept of animal welfare and the way was used in the current study. Animal welfare is a multidimensional concept and its assessment must consider this aspect. So, for this reason is very limiting to carry out animal welfare assessment using only a tool based on the dogs’ apparent age. It is a good idea to simplify the assessment approach but this cannot be without the fact that welfare is a multifaceted and complex thing. Animal welfare can be defined in a number of different ways, but there is a growing consensus that whatever the definition, it has to include three elements: the emotional state of the animal, its biological functioning and its ability to show normal patterns of behaviour. These three elements are closely interrelated. Animal welfare comprises the state of the animal's body and mind, and the extent to which its nature (genetic traits manifest in breed and temperament) is satisfied. In addition, the welfare of nonhuman animals is a complex concept that includes the biological functioning of an animal, emotional states and opportunities to experience natural life, including the execution of natural behaviours.

For these aspects related to animal welfare suggest some references

Manteca, X., Velarde, A., & Jones, B. (2009). Animal welfare components. In Welfare of production animals: assessment and management of risks(pp. 61-77). Wageningen Academic.
Arndt, S. S., Goerlich, V. C., & van der Staay, F. J. (2022). A dynamic concept of animal welfare: The role of appetitive and adverse internal and external factors and the animal’s ability to adapt to them. Frontiers in Animal Science, 3, 908513.
Englund, M. D., & Cronin, K. A. (2023). Choice, control, and animal welfare: Definitions and essential inquiries to advance animal welfare science. Frontiers in Veterinary Science, 10, 1250251.
Veissier, I., & Forkman, B. (2008). The Nature of Animal Welfare Science. Annual Review of Biomedical Sciences, 10.

Thank you for your valuable feedback regarding the concept of animal welfare. We acknowledge that animal welfare is indeed a multifaceted concept and that the current manuscript does not evaluate the full range of aspects correlated to it. In the revised version, we expanded the discussion to incorporate a more comprehensive welfare framework, which includes emotional state, biological functioning, and natural behaviors.

Actually, the apparent age tool makes part of a broader welfare assessment. In the revised manuscript (lines 386-396), we highlight its role in measuring biological aging and how it can be integrated with behavioral and physiological indicators. We also included the references you suggested, alongside additional relevant literature, to support the multidimensional approach to welfare.

We believe these changes will significantly improve the manuscript’s alignment with current welfare science and enhance its contribution to the field.

Comment 4: The introduction should be revised to emphasize animal welfare in light of the above. The tool developed by the authors could be considered as a tool that can be integrated in the assessment of dog welfare. Or maybe better it could talk about a tool that can give information on the health condition of the dog and can be integrate in a holist approach for welfare assessment. I suggest to consider this second option in order to make the study more credible and published. Moreover, it is not clear how this tool works to give information on dog welfare condition. The estimation of age is an animal-based measure (ABM) that may be added and correlated to other ABMs for the assessment of welfare.

Here some example of dog welfare assessment tool and references that can be useful for the introduction of the manuscript:

Malkani, R., Paramasivam, S., & Wolfensohn, S. (2022). Preliminary validation of a novel tool to assess dog welfare: The Animal Welfare Assessment Grid. Frontiers in veterinary science, 9, 940017.
Polgár, Z., Blackwell, E. J., & Rooney, N. J. (2019). Assessing the welfare of kennelled dogs—A review of animal-based measures. Applied animal behaviour science, 213, 1-13.
Bacon, H., Walters, H., Vancia, V., Connelly, L., & Waran, N. (2019). Development of a robust canine welfare assessment protocol for use in dog (Canis familiaris) catch-neuter-return (CNR) programmes. Animals, 9(8), 564.
Berteselli, G. V., Arena, L., Candeloro, L., Dalla Villa, P., & De Massis, F. (2019). Interobserver agreement and sensitivity to climatic conditions in sheltered dogs' welfare evaluation performed with welfare assessment protocol (Shelter Quality protocol). Journal of Veterinary Behavior, 29, 45-52.
Berteselli, G. V., Rapagnà, C., Salini, R., Badagliacca, P., Bellucci, F., Iannino, F., & Dalla Villa, P. (2021). A Pilot Study to Develop an Assessment Tool for Dogs Undergoing Trap-Neuter-Release (TNR) in Italy. An Overview on the National Implementation of TNR Programmes. Animals, 11(11), 3107.
Barnard, S., Pedernera, C., Velarde, A., & Dalla Villa, P. (2014). Welfare assessment protocol for shelter dogs. Istituto Zooprofilattico Sperimentale dell’Abruzzo e del Molise.

We agree that animal welfare is a multifaceted concept and that our tool should be presented as providing health-related insights that can contribute to a more holistic welfare assessment. In the revised manuscript, we reframed the tool as one that complements existing welfare measures by offering non-invasive biological data related to stress and aging in lines XX-XX. While this tool provides important health information through the correlation of apparent age and telomere length, it is not intended to replace other welfare assessments but rather to enhance them. We also appreciate your suggestion to expand the literature review. We incorporated the recommended references (e.g., Malkani et al., 2022; Polgár et al., 2019) to ensure the study is situated within the broader context of established animal welfare assessment protocols. Finally, we clarified the mechanism by which the tool provides welfare insights and suggest how it could be integrated with other animal-based measures (ABMs) to build a more comprehensive welfare framework. Thank you again for your valuable input, which we believe will significantly improve the manuscript. (Lines 84-94)

Comment 5: Methods

LL 74-77 specify how many photos for each dog category considered

In the revised manuscript, we included a detailed breakdown of the number of photos used for young (0–2 years), adult (2–5 years), and senior (6+ years) dogs, it was 20 for each category. Additionally, we explained how these photos were selected to ensure consistent quality and representation within each category. In the cases there were any imbalances in the number of photos across categories, we addressed how this was accounted for in the analysis. (Currently lines 131-135)

LL 92-93 specify who performed this procedure (vet, owner, etc.) and how the samples were stored and managed, who did perform the analysis?

All the information bellow were included in the manuscript.

Personnel Involved in Sample Collection: the buccal swab samples were collected by LMLD, the dog owner, the keeper, or a veterinarian, depending on who could perform the procedure with the least discomfort to the animal. This specification will enhance the understanding of the sampling process. Lines 142-145
Sample Storage and Management: After collection, each swab was stored in a designated tube with a Dri-Capsule (Cell Projects, Kent, UK) to prevent DNA degradation. The samples were kept at room temperature until further analysis, and LMLD ensured that they were securely labeled and stored. Lines 147-149
Analytical Procedures: LMLD conducted all analyses, including DNA extraction, qPCR, and data evaluation. Lines 255-257

(Currently lines 142-147 and 248-250)

We revised the manuscript to provide a more detailed explanation of how the 60 photos were selected from the total pool of 264, including criteria such as image quality and balanced representation of dog age categories (young, adult, senior). A breakdown of how many photos were selected for each category was also included (Lines 194-197 and 203-205),

LL 145-147 as said previously, please specify better how the volunteers were involved.

Following your suggestion we provided more details about the involvement of the volunteers in lines 189–209. The volunteers participated in the study by evaluating photographs of the dogs. They received written guidelines instructing them on how to assess the apparent age of the dogs based on specific visual characteristics. This ensured consistency across all evaluations.

LL 148-155 this part is not clear. 262 or 264 photos? What is the Dog’s Life project? How this step is related with the aim of the study?

Upon review, the two missing photos were excluded due to poor image quality, which did not meet our standards for the analysis, so, we uploaded 262 photos for our volunteers to analyse them. These exclusions did not affect the overall statistical analysis, as the data set used for the volunteer’s evaluation was 60 photos from the 262 total. In the revised manuscript, we included this clarification in the methods section to ensure transparency, lines XX-XX "Dog's Life project" was the name given to my PhD project, which included various welfare assessments, such as hormonal and molecular evaluations, completed in different chapters as the study progressed. However, after considering your feedback, I agree that the mention of the "Dog’s Life project" may cause confusion. Therefore, I have removed this term from the manuscript to avoid any misunderstanding and ensure clarity regarding the aims and scope of the study.

LL 172-173 Please clarify how the photos were subdivided in the dogs’ age categories.

We classified the dogs' ages by consulting with veterinarians to gather insights on canine development and the prevalence of age-related diseases. Additionally, we utilized the guidelines established by Creevy et al. (2019), which provide a comprehensive framework for understanding canine life stages. This dual approach ensured a reliable and scientifically grounded classification system for the dogs in our study. Lines 134-138

Comment 6: Results

Specify the distribution of the data (normal or not-normal distribution)

The distribution of the data was assessed using the Shapiro-Wilk test. The results indicated that the data were normally distributed, which guided the selection of subsequent statistical analyses. This information was included in the manuscript, lines 233-235.

LL 219 33 volunteers are the total of respondents?

We had a total of 33 non-specialist volunteers and 53 specialists who assessed the photographs of the dogs. We clarified this distinction in the revised manuscript, lines 302, to ensure there is no confusion about the total number of respondents and their classifications.

LL 239 this result is related to all the participants?

Thank you for your questions regarding lines 219 and 239. To clarify, the 33 volunteers mentioned in line 219 (Currently 302) refer specifically to the non-specialist respondents, while line 239 discussed results related solely to the specialists. We recognize the need for clearer wording in the manuscript to distinguish between these groups. When we say Participants we are combining specialists and non-specialists. We revised the relevant sections to explicitly state that the paragraph in lines 331-333 pertains only to the specialists, ensuring that this distinction is clear to the readers.

The results showed in this part are related to a question asked in the survey. I suggest to add more details regarding the answers in the survey, if I understood correctly there are not only photos of dogs and questions related to the age evaluation.
We recognize the need to clarify the structure of the survey and provide more detail about the questions asked beyond the photo-based age evaluations. We added the questionnaire to the supplementary materials.

LL 246 no supplementary material was available.

We apologize for the oversight. We have now included the supplementary material, which can be accessed via the link provided in the revised manuscript. This material contains the dogs’ photographs evaluated by the volunteers that complement the main findings of our study. The supplementary material is referenced at the appropriate points in the manuscript to enhance clarity.

LL 241-242 this result is not totally clear. It is not clear the difference between specialists and not-specialists.
We acknowledge the need to clarify these categories and their associated performance in the study. In the revised manuscript, we will defined specialists as those with formal training or professional experience in dog care (e.g., veterinarians, trainers), and non-specialists as members of the general public with no specialized training. Additionally, we expanded the results section to clearly explain any differences between these two groups in their ability to estimate dog age. Where there were no significant differences, we provided an explanation for this, lines 249-252 and 326-328

LL 255 this result is very interesting. Was evaluated the correlation between the type of dog (pet, lab dog, working dog, shelter dog)? It is relevant to specify this point.

We conducted an analysis to investigate this relationship and found no significant correlation between dog type and the outcomes.

Comment 7: Discussion

As said for introduction, also for discussion is important to integrate parts related to animal welfare, animal welfare assessment etc.

We agree that these aspects are important to emphasize.

In response, we have revised the Discussion to better incorporate the relevant animal welfare concepts. Specifically, we have included a more in-depth analysis of how our findings relate to established frameworks of animal welfare, addressing the emotional, biological, and behavioral dimensions as described by Veissier & Forkman, (2008). We also expanded on the significance of comprehensive welfare assessments, considering the dynamic influences highlighted by Arndt et al. (2022) and the role of environmental factors, such as choice and control, in promoting animal well-being (Englund & Cronin, 2023). These additions ensure a more thorough discussion of how welfare assessments can be practically applied based on our study's findings.

Discussion is weak and needs to be expanded

We have made several revisions to strengthen this section:

Deeper Analysis: We have expanded the discussion of our key findings in relation to existing literature, providing a more thorough comparison with previous studies. This includes a more detailed interpretation of the results in the context of established theories and frameworks, particularly regarding dog ageing and multidimensional nature of animal welfare.
Animal Welfare and Assessment: Following your suggestion and that of other reviewers, we have integrated additional discussion on animal welfare and its assessment (referencing Arndt et al., 2022; and others), highlighting how our findings contribute to this field and the implications for welfare evaluation.
Practical Implications: We have added a section that emphasizes the practical implications of our findings, especially how they can inform future research, welfare assessment practices, and interventions aimed at improving animal well-being.
Limitations and Future Research: We have also elaborated on the limitations of our study and identified key areas for future research, which provide a more comprehensive outlook on how our work fits within the larger body of knowledge.

LL 282 LL 285 as said above, it could be better to talk about health condition or quality of life

Thank you for your suggestion regarding the use of the terms "health condition" or "quality of life" in lines 282–285. We agree that these terms better capture the holistic aspects of animal welfare in the context of our discussion. In response, we have revised the text accordingly. Specifically, we have replaced the original phrasing with "health condition" and "quality of life" to more accurately reflect the comprehensive state of the animals’ well-being, incorporating both physical health and overall welfare. These changes are now reflected in the revised manuscript, lines XX-XX, to ensure consistency and clarity.

Comment 8: I suggest to explore the possible fields in which this approach can be used, and how it works for the purpose of the study. For example, in shelter dogs where the environmental condition can affect the health and welfare status of these animals. How this tool can be used?

We agree that this tool has wide-ranging applications, particularly in shelters where dogs are often subjected to stressful environmental conditions.

In the revised manuscript, we expanded the discussion to explore how the tool could be used in shelters, where the apparent age could serve as a non-invasive indicator of biological aging, potentially highlighting animals in need of immediate intervention. We also considered its use in other contexts, such as veterinary clinics or rescue organizations, where quick welfare assessments are beneficial.

We appreciate your input and believe these additions will strengthen the manuscript’s practical relevance.

References

Arndt, S. S., Goerlich, V. C., & van der Staay, F. J. (2022). A dynamic concept of animal welfare: The role of appetitive and adverse internal and external factors and the animal’s ability to adapt to them. Frontiers in Animal Science, 3(August), 1–21. https://doi.org/10.3389/fanim.2022.908513

Christensen, K., Thinggaard, M., McGue, M., Rexbye, H., Hjelmborg, J. V. B., Aviv, A., Gunn, D., Van Der Ouderaa, F., & Vaupel, J. W. (2009). Perceived age as clinically useful biomarker of ageing: Cohort study. BMJ (Online), 339(7735), 1433. https://doi.org/10.1136/bmj.b5262

Creevy, K. E., Grady, J., Little, S. E., Moore, G. E., Groetzinger Strickler, B., Thompson, S., & Webb, J. A. (2019). 2019 AAHA Canine Life Stage Guidelines*. Journal of the American Animal Hospital Association, 55(6), 267–290. https://doi.org/10.5326/JAAHA-MS-6999

Englund, M. D., & Cronin, K. A. (2023). Choice, control, and animal welfare: definitions and essential inquiries to advance animal welfare science. Frontiers in Veterinary Science, 10(August), 1–5. https://doi.org/10.3389/fvets.2023.1250251

Swanson, A., Kosmala, M., Lintott, C., & Packer, C. (2016). A generalized approach for producing , quantifying , and validating citizen science data from wildlife images. 30(3), 520–531. https://doi.org/10.1111/cobi.12695

Veissier, I., & Forkman, B. (2008). ARBS Annual Review of Biomedical Sciences The Nature of Animal Welfare Science 1 . Animal Welfare : Moral Issues Put in Practice. ARBS Annual Review of Biomedical Sciences, 15–26.

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

The manuscript is improved, the authors addressed all comments. In the second version of the paper some aspects should be addressed to make the article suitable for publication.

I suggest to clarify better the aim. Specify that it could be a tool which can be integrated in the welfare assessment protocol or approach. Bot in abstract and in the introduction

LL 97-102 this part seems disconnected with the previous one. This aspect about citizen science could be moved in the discussion

LL 142 specify the acronym

LL 145-150 I suppose that the samples performed by owners and vets were sent to the laboratory

LL 221 the n of volunteers involved in the project should be considered as a result

LL 249-253 Also this part should be considered as a result

LL 269Please add information about the student (vet student?)

LL 387 I suggest to rephrase “t can be integrated into…”

LL 421 I suggest to delate outcomes

LL 435 I suggest to rephrase “as a possible integrative tool for welfare assessment”

Author Response

LL 142 specify the acronym

I just noticed that I missed this one, on the file that I have attached. LMLD, it is me the first author, I conducted all the sample collections and processing along the study. If you think that more clarification is needed in the manuscript I'll gladly do it.

Kindest regards,

Luisa

Author Response File: Author Response.docx