Dear Dr. Sakcamduang,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

Please submit your revised manuscript by Jul 19 2025 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org . When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

• A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.
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We look forward to receiving your revised manuscript.

Kind regards,

Doa'a G. F. Al-u'datt

Academic Editor

PLOS ONE

Journal Requirements:

When submitting your revision, we need you to address these additional requirements.

1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf

2. Thank you for your submission to PLOS ONE. We note that your cover letter mentions an article with the title "Effect of Perna canaliculus oil in dogs with pulmonary hypertension", but the main manuscript's title is "Clinical Improvement in Canine Pulmonary Hypertension with Perna canaliculus Oil (PCSO-524) Add-on Therapy: Effects on Exercise Tolerance and Cough". Can you please clarify which is the correct title and check if you have submitted an incorrect cover letter or incorrect manuscript file? If one is incorrect please upload the correct file.

3. Thank you for stating the following financial disclosure:

This study was funded by the Pharmalink International Ltd. Central, Hong Kong.

Please state what role the funders took in the study. If the funders had no role, please state: "The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript."

If this statement is not correct you must amend it as needed.

Please include this amended Role of Funder statement in your cover letter; we will change the online submission form on your behalf.

4. Please remove all personal information, ensure that the data shared are in accordance with participant consent, and re-upload a fully anonymized data set.

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5. Please include captions for your Supporting Information files at the end of your manuscript, and update any in-text citations to match accordingly. Please see our Supporting Information guidelines for more information: http://journals.plos.org/plosone/s/supporting-information.

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

Reviewer #1: Partly

Reviewer #2: Yes

Reviewer #3: Partly

Reviewer #4: Yes

Reviewer #5: Yes

Reviewer #6: Partly

Reviewer #7: Yes

Reviewer #8: Partly

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2. Has the statistical analysis been performed appropriately and rigorously? -->?>

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

Reviewer #4: Yes

Reviewer #5: Yes

Reviewer #6: Yes

Reviewer #7: Yes

Reviewer #8: Yes

**********

3. Have the authors made all data underlying the findings in their manuscript fully available??>

The PLOS Data policy

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

Reviewer #4: Yes

Reviewer #5: Yes

Reviewer #6: Yes

Reviewer #7: Yes

Reviewer #8: Yes

**********

4. Is the manuscript presented in an intelligible fashion and written in standard English??>

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: No

Reviewer #4: Yes

Reviewer #5: Yes

Reviewer #6: No

Reviewer #7: Yes

Reviewer #8: Yes

**********

Reviewer #1: This is an interesting and well-designed study that evaluates the potential benefits of Perna canaliculus oil (PCSO-524) as an add-on therapy in dogs with pulmonary hypertension. The topic is relevant to clinical veterinary medicine, especially for practitioners managing canine cardiac patients.

Strengths of the manuscript:

The prospective, randomized, placebo-controlled trial design adds scientific strength and reliability to the findings.

The methods, particularly the use of standardized clinical scoring and echocardiographic assessment, are appropriately selected.

The writing is generally clear and well-organized.

Ethical approval and owner consent were obtained and clearly reported.

Suggestions for improvement:

1. Sample size: The study had a relatively small number of dogs (n=17), which limits the generalizability of the findings. Please acknowledge this limitation more clearly in the discussion.

2. Dietary influence: Since omega-3 intake from the dogs' diets may have affected the results, future studies should aim to control or record dietary intake.

3. Biomarker use: The lack of significant change in NT-proBNP concentrations deserves further exploration or explanation, especially considering its role in heart disease monitoring.

4. Clarity of figures and tables: Ensure that all supplemental tables and figures referenced (e.g., Supplemental Table 3, Figure 1) are available and labeled clearly for reviewers and readers.

5. Minor language editing: Some grammatical and typographical errors should be addressed to improve the overall clarity and professionalism of the manuscript.

Ethical and scientific integrity: There are no major concerns regarding ethical issues, dual publication, or research misconduct. The study appears to adhere to the ethical standards of animal research.

In conclusion, this study contributes valuable preliminary data on the potential use of PCSO-524 in dogs with PH. With minor revisions and clarifications, the manuscript could be suitable for publication.

Reviewer #2: The specific questions and recommendations are as follows:

1:

The manuscript would benefit from a structured abstract.

2:

It is recommended to crate a section for the Ethical considerations. Under Ethical considerations

section, it is required to provide more information in the standard format.

3:

I recommend creating a new section for the study limitations.

4:

It is advised that a new section explaining any acronyms used in the article be added at the end,

following the conclusion section.

Thanks to the authors for their hard work on this Study. It’s a pleasure to review such a

contribution to the field.

Reviewer #3: Abstract : "While the abstract contains the necessary scientific content, it currently lacks the clarity and brevity expected by PLOS ONE. The sentences are overly long and dense, obscuring the main findings. I recommend reducing redundancy, improving flow, and focusing on clear communication of the objective, methods, and outcomes in under 300 words." Introduction :

The Introduction provides a reasonable overview of pulmonary hypertension (PH) in dogs, including its causes, clinical manifestations, and current treatment options such as sildenafil. It also introduces PCSO-524 and its known anti-inflammatory properties. However, the section could be improved in several ways. The transitions between ideas are somewhat abrupt, and the research gap—why adjunctive therapy is needed despite standard treatment—is not clearly articulated. The mechanistic discussion of PCSO-524's effects is overly detailed and would benefit from being more concise and clinically focused. Additionally, there are several grammatical and formatting issues (e.g., “outcomesin” in line 75) that require correction. I recommend clarifying the rationale for the study and tightening the prose to improve readability and focus.

Materials and Methods

The methodology is overall well-designed and clearly reported. The study is a prospective, randomized, placebo-controlled trial with appropriate ethical approvals and informed consent. Interventions and clinical assessments, including echocardiography, electrocardiography, and biomarker analysis, are detailed and replicable. The inclusion criteria and statistical analysis are also appropriate. Blinding is reported and appears to be well-executed. However, additional clarification on the randomization sequence generation and allocation concealment would improve transparency. Minor grammatical issues are present but do not detract from methodological rigor.

Results

The Results section presents the findings in a structured order and uses appropriate statistical methods. The within-group and between-group comparisons are clearly reported with valid p-values and consistent formatting (mean ± SD and median [range] where appropriate). Significant improvements in tricuspid regurgitation pressure gradient (TRPG) and exercise tolerance in the PCSO-524 group are appropriately highlighted. However, the section would benefit from improved language clarity and consistency in terminology (e.g., repeated use of “visit” vs. “day”). Grammatical errors and typographical mistakes (e.g., “rricuspid” instead of “tricuspid”) should be corrected. The use of vague language such as “numerically different improvement” should be revised to more standard phrasing like “a non-significant trend.” Overall, the statistical validity is strong, but presentation quality needs refinement.

Discussion and Conclusion

The Discussion section provides a broad and generally balanced interpretation of the study’s findings, particularly the observed improvements in exercise tolerance and coughing among dogs receiving PCSO-524. The authors appropriately contextualize their results within the existing literature and acknowledge key limitations, including small sample size, re-examination delays, and the absence of dietary control for omega-3 intake. These admissions strengthen the scientific integrity of the discussion.

However, the section is significantly weakened by numerous grammatical errors, awkward phrasing, and lack of narrative focus. Several paragraphs contain redundancies or imprecise statements (e.g., “may could have biased”), and the flow between ideas is inconsistent. Additionally, some claims are repeated unnecessarily or not fully supported by detailed reasoning (e.g., comparisons of TRPG reductions). References to human studies are overused in places without drawing direct relevance to the canine population. A major language edit is required to improve clarity, eliminate repetition, and present a more coherent, professional narrative.

The Conclusion, while consistent with the overall findings, is underdeveloped. It lacks clarity, contains grammatical issues, and does not adequately emphasize the statistically significant clinical improvements observed in the PCSO-524 group. Furthermore, the final sentence regarding the variability of TRPG is valid but ends the manuscript on a vague note. The conclusion should be rewritten to provide a concise, confident summary of findings and a clear recommendation for future research.

Overall, while the interpretation of results is scientifically reasonable, a thorough rewrite of the Discussion and Conclusion sections is necessary to ensure clarity, accuracy, and proper scholarly tone.

Reviewer #4: This study is well-executed and provides valuable insights into adjunctive therapy for canine PH. Minor improvements in methodological transparency and discussion depth would enhance its impact.

Reviewer #5: no additional comments .

Reviewer #6: Dear Authors, This is a good manuscript and deserves to be published, however there are a few areas for improvement before the manuscript can be accepted.

1. Sample Size is too small. I suggest to label the title to reflect that aspect. You can call it a pilot study or something like that to justify the small sample.

2. The diet is not controlled or recorded. This could significantly influence the study outcomes as it is particularly important when evaluating a nutraceutical intervention. I suggest you acknowledge this more explicitly and, if possible, include retrospective dietary assessments.

3. Study Group 1 (PCSO-524) had significantly worse baseline coughing scores. While this may partially explain the observed improvement, it could also bias the results. I suggest that you consider conducting an ANCOVA adjusting for baseline coughing scores.

4. There is a basic need for defining and expressing the lack of objective and functional endpoints. As the study relies heavily on owner-reported outcomes, it makes it even more important to use more objective data as that will for sure strengthen the findings and improve the quality of the study. You can also discuss this limitation and how further research studies can address it.

5. Kindly elaborate on the implications of the lack of change in NT-proBNP concentrations.

6. Please elaborate the discussion on the composition and other synergistic actions of PCSO-524.

7. Ensure all abbreviations are defined at first mention in both the abstract and main text.

8. Ensure that grammatical errors are corrected before submission and that the English is up to the standards of the journal and general scientific literature.

Reviewer #7: This is a well written and interesting paper. It has great clinical application. There is so little available for this disease so it good to see some research on a natural product that will be helpful for most patients.

The statistical analysis appears to be correct and appropriate.

The data set presented is complete.

The English is appropriately written.

The references contain all the appropriate literature and does include both human and veterinary literature.

On line 273 results is spelled incorrectly.

Reviewer #8: The manuscript presents a prospective, randomized, double-blind, placebo-controlled study assessing the efficacy of Perna canaliculus oil (PCSO-524) as an add-on therapy to sildenafil in dogs with pulmonary hypertension (PH). The subject is novel and relevant, particularly as interest in nutraceuticals grows. The methodology is generally appropriate, and the manuscript is well structured. However, several issues—particularly related to small sample size, lack of dietary control, and interpretation of clinical significance—should be addressed before consideration for publication.

2. Methodology:

Design: Randomized, placebo-controlled design is appropriate.

Blinding: Adequately blinded (owners, investigators, and sponsors), which strengthens the credibility of the findings.

Sample Size: Only 17 dogs completed the study, which severely limits statistical power.

Inclusion Criteria: Well-defined, but would benefit from clearer exclusion criteria (e.g., how arthritis was ruled out).

Nutritional Confounding: No documentation of dietary intake or background EPA/DHA content, which critically affects interpretation of omega-3 related outcomes.

Suggestions for Revision:

Include a table summarizing baseline diets or clearly state the limitation due to dietary variability.

Clarify how orthopedic comorbidities were ruled out (as they may influence exercise tolerance).

Include a table summarizing baseline diets or clearly state the limitation due to dietary variability.

3. Statistical Analysis:

Suggestion: Provide confidence intervals alongside p-values to support effect estimates.

5. Interpretation and Discussion:

The discussion is thorough and references relevant literature.

However, the role of confounding variables (e.g., underlying osteoarthritis, nutrition) is under-emphasized.

The statement that PCSO-524 improved coughing and exercise tolerance should be softened to reflect the exploratory nature of this pilot study.

Suggestion:

Revise tone to reflect preliminary nature and acknowledge limitations more explicitly.

Consider recommending replication in a larger, multicenter cohort.

Final Recommendation:

Major Revision

Summary of Required Revisions:

Address Small Sample Size Limitations in the Discussion and interpret findings with appropriate caution.

Include or Discuss Diet Control and how this may have influenced omega-3 intake.

Clarify and Justify Exclusion of Orthopedic Comorbidities or acknowledge their potential confounding effects.

Correct Minor Language Errors and Typos.

Soften Conclusions to reflect the exploratory nature of the findings.

**********

what does this mean? ). If published, this will include your full peer review and any attached files.

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Reviewer #1: Yes:  Hussein Mussa Muafa

Reviewer #2: Yes:  SEYED AMIRHOSSEIN MAZHARI

Reviewer #3: No

Reviewer #4: No

Reviewer #5: No

Reviewer #6: Yes:  Kevin Morris

Reviewer #7: No

Reviewer #8: No

**********

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/ . PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org . Please note that Supporting Information files do not need this step.

Attachments

Attachment

Submitted filename: Review 55.pdf

Attachment

Submitted filename: review report.docx

Dear Dr. Sakcamduang,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

Please submit your revised manuscript by Sep 20 2025 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org . When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

• A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.
• A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.
• An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols . Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols .

We look forward to receiving your revised manuscript.

Kind regards,

Doa'a G. F. Al-u'datt

Academic Editor

PLOS ONE

Journal Requirements:

If the reviewer comments include a recommendation to cite specific previously published works, please review and evaluate these publications to determine whether they are relevant and should be cited. There is no requirement to cite these works unless the editor has indicated otherwise. 

Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.

Additional Editor Comments:

see attached file

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

Reviewer #2: (No Response)

Reviewer #3: All comments have been addressed

Reviewer #6: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions??>

Reviewer #2: Yes

Reviewer #3: Yes

Reviewer #6: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously? -->?>

Reviewer #2: Yes

Reviewer #3: Yes

Reviewer #6: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available??>

The PLOS Data policy

Reviewer #2: Yes

Reviewer #3: Yes

Reviewer #6: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English??>

Reviewer #2: Yes

Reviewer #3: No

Reviewer #6: Yes

**********

Reviewer #2: The specific questions and recommendations are as follows:

1:

The manuscript would benefit from a structured abstract.

The revised version has the same unstructured abstract as the primary submission.

2:

It is recommended to crate a section for the Ethical considerations. Under Ethical considerations

section, it is required to provide more information in the standard format.

It is better presented in the revised version.

3:

I recommend creating a new section for the study limitations.

Has not been addressed by authors.

4:

It is advised that a new section explaining any acronyms used in the article be added at the end,

following the conclusion section.

No action by authors.

Thanks to the authors for their hard work on this Revision. It’s a pleasure to review such a

contribution to the field.

Reviewer #3: (No Response)

Reviewer #6: (No Response)

**********

what does this mean? ). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy

Reviewer #2: Yes:  SEYED AMIRHOSSEIN MAZHARI

Reviewer #3: No

Reviewer #6: Yes:  Kevin Morris

**********

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/ . PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org

Attachments

Attachment

Submitted filename: Review 55a.pdf

Clinical improvement in canine pulmonary hypertension with Perna canaliculus oil (PCSO-524) add-on therapy: effects on exercise tolerance and cough

PONE-D-25-20629R2

Dear Dr. Sakcamduang,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

An invoice will be generated when your article is formally accepted. Please note, if your institution has a publishing partnership with PLOS and your article meets the relevant criteria, all or part of your publication costs will be covered. Please make sure your user information is up-to-date by logging into Editorial Manager at Editorial Manager®  and clicking the ‘Update My Information' link at the top of the page. For questions related to billing, please contact billing support .

If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

Kind regards,

Doa'a G. F. Al-u'datt

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewer #2:

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

Reviewer #2: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions??>

Reviewer #2: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously? -->?>

Reviewer #2: I Don't Know

**********

4. Have the authors made all data underlying the findings in their manuscript fully available??>

The PLOS Data policy

Reviewer #2: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English??>

Reviewer #2: Yes

**********

Reviewer #2: Thanks to the authors for their hard work on this Revision. It’s a pleasure to review such a

contribution to the field.

**********

what does this mean? ). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy

Reviewer #2: Yes:  SEYED AMIRHOSSEIN MAZHARI

**********

Attachments

Attachment

Submitted filename: Review 55b.pdf