Effects of Rumen Fermentation Characteristics on Stress-Related Hormones and Behavior in Sheep
Round 1
Reviewer 1 Report
Comments and Suggestions for AuthorsThis study is commendable for investigating the interplay between rumen fermentation, stress-related hormones, and behavior in sheep, which has implications for animal welfare and feed management. However, there are some aspects that could be further refined. The sample size of eight lambs may be considered relatively small, potentially limiting the generalizability of the findings. Additionally, while the study highlights correlations between variables, causality is not established, indicating a need for more in-depth research. The language used in presenting the results could be more precise; for instance, stating the specific values of the acetic acid/propionic acid ratio and sniffing behavior correlation would enhance clarity. Furthermore, it would be beneficial to explore additional factors that could contribute to the observed behaviors and hormonal responses, providing a more comprehensive understanding. In addition, the following points need further attention on the part of the authors. Firstly, in Table 1, it is recommended to avoid using abbreviations to enhance clarity for readers. Moreover, if abbreviations are used in other tables, they should be appropriately defined for better comprehension. Regarding the statistical analysis, there seems to be a discrepancy as the authors mention both two-way ANOVA and one-way ANOVA. This inconsistency needs to be addressed and clarified in order to ensure the accuracy of the results. Additionally, the adequacy of the provided data for a comprehensive paper may be a concern, and further data collection or analysis may be necessary. The reference section is notably sparse, indicating that a more extensive literature review could enhance the study's context and relevance. A more thorough exploration of relevant prior research would strengthen the study's foundation. Overall, while the study offers valuable insights, addressing these points would refine the presentation and bolster the robustness of the findings.
Author Response
Dear Reviewer,
Thank you for your insightful comments and suggestions, which have greatly helped in enhancing the quality of our manuscript. We have thoroughly revised the manuscript based on your feedback and have detailed our responses to each of your comments below. The changes made in response to your comments are highlighted in yellow and gray.
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Dear Reviewer,
Thank you for your insightful comments and suggestions, which have greatly helped in enhancing
the quality of our manuscript. We have thoroughly revised the manuscript based on your feedback
and have detailed our responses to each of your comments below. The changes made in response to
your comments are highlighted in yellow and gray.
Comment 1: “The sample size of eight lambs may be considered relatively small, potentially
limiting the generalizability of the findings.”
Response 1: We appreciate the reviewer’s point regarding our sample size. It is important to note
that while striving for robust statistical power, we also placed a high priority on the welfare of the
animals involved in our study. The invasive nature of the procedures, such as the collection of rumen
fluid via orogastric tubing and blood sampling from the jugular vein, necessitated a careful balance
between achieving meaningful statistical results and minimizing pain and distress to the animals.
We intentionally designed the experiment to keep the sample size as small as possible without
compromising the scientific validity, adhering to the principles of Replacement, Reduction, and
Refinement (the 3Rs) in animal research. We acknowledge the limitations that this ethical stance
imposes on the generalizability of our findings, and we have discussed these in the manuscript (p.
8, line 279 and 300). Future studies with larger sample sizes could potentially confirm and extend
our observations, provided that the welfare of the animals can be assured.
Comment 2: While the study highlights correlations between variables, causality is not established,
indicating a need for more in-depth research.
Response 2: Thank you for your comment regarding the distinction between correlation and
causality. Our study design, which incorporated dietary interventions, was intended to explore
causative effects. However, we understand that establishing causality in biological systems often
requires a more longitudinal approach. We have revised the discussion section to emphasize the
correlational nature of our findings and the need for further studies to investigate causality more
definitively (p. 8, line 298).
Comment 3: The language used in presenting the results could be more precise; for instance, stating
the specific values of the acetic acid/propionic acid ratio and sniffing behavior correlation would
enhance clarity.
Response 3: Thank you for your comments regarding the precision of language used to describe the
statistical findings in our manuscript. We have taken great care to ensure that our presentation of the
results is both accurate and clear. Specifically, in the results section, we have provided the exact
value of the Spearman's correlation coefficient (ρ=0.545) along with the significance level (P<0.05)
to reflect the relationship between the acetic acid/propionic acid ratio and the sniffing behavior
observed during the Open Field Test. These details are directly stated in the text and further
highlighted with an asterisk in Table 3 to emphasize their significance.
We believe this approach adequately conveys the strength and significance of the correlations to
the readers, allowing for a transparent and straightforward interpretation of the data. If there are any
additional aspects of the language precision that could be enhanced further, we welcome your
suggestions and are open to making any necessary adjustments to improve the clarity of the
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manuscript.
Comment 4: Furthermore, it would be beneficial to explore additional factors that could contribute
to the observed behaviors and hormonal responses, providing a more comprehensive understanding.
Response 4: In response to your suggestion, we have included a discussion on the potential role of
rumen microbiota in modulating stress-related behaviors and physiological responses in ruminants
(p. 8, line 304). Although our study did not directly assess the rumen microbiota, the literature from
monogastric animal models provides compelling evidence that warrants such consideration in future
ruminant research.
Comment 5: Firstly, in Table 1, it is recommended to avoid using abbreviations to enhance clarity
for readers.
Response 5: Thank you for your suggestion regarding the use of abbreviations in Table 1. In
accordance with your recommendation, we have revised the table to replace abbreviations with their
full terms to enhance clarity for our readers.
Comment 6: Moreover, if abbreviations are used in other tables, they should be appropriately
defined for better comprehension.
Response 6: Following your recommendation, we have now detailed the full descriptions of the
open field behaviors of table3. Additionally, we have provided explicit definitions for each of these
behavioral responses in the Methods section of our paper to ensure clear understanding for our
readers (p. 4, line 154).
Comment 7: Regarding the statistical analysis, there seems to be a discrepancy as the authors
mention both two-way ANOVA and one-way ANOVA. This inconsistency needs to be addressed
and clarified in order to ensure the accuracy of the results.
Response 7: Thank you for pointing out the inconsistency in our description of the statistical
analyses. We have carefully reviewed our manuscript and would like to confirm that all statistical
analyses were indeed conducted using two-way ANOVA. Any mention of one-way ANOVA was an
error and has now been corrected to reflect the accurate method employed (p. 4, line 170. We
appreciate your attention to detail, which has helped us improve the precision of our manuscript.
Comment 8: Additionally, the adequacy of the provided data for a comprehensive paper may be a
concern, and further data collection or analysis may be necessary.
Response 8: We appreciate your observation on the adequacy of the data presented in our manuscript.
We recognize the importance of comprehensive data to support our findings and conclusions. The
dataset reported was meticulously collected and analyzed to ensure the robustness of our study
within the ethical constraints of animal research. We have strived to present a balanced account by
discussing the limitations and context of our data collection in the manuscript. In the study, we
adhered to the principles of the 3Rs (Replacement, Reduction, and Refinement) to minimize animal
use and ensure ethical treatment. This ethical stance inevitably influences the sample size but is a
critical consideration in the field of animal research. We believe our dataset provides valuable
insights into the interplay between rumen fermentation characteristics, stress-related hormones, and
behavior in sheep, serving as a preliminary exploration of this complex field. We have now
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expanded the Discussion section to further elaborate on the potential implications of our findings
and to call for additional studies that could augment our dataset with a larger sample size or
longitudinal analyses, which would be invaluable in confirming and extending the patterns observed.
Comment 9: The reference section is notably sparse, indicating that a more extensive literature
review could enhance the study's context and relevance. A more thorough exploration of relevant
prior research would strengthen the study's foundation.
Response 9: Thank you for your constructive feedback on the reference section of our manuscript.
In response to your suggestion, we have diligently expanded our literature review to provide a more
comprehensive context for our study. We agree that a robust foundation of previous research is
essential to frame our study's contributions effectively. With the newly added citations, we have
endeavored to encapsulate a broader spectrum of related work, thereby situating our preliminary
investigation within the wider scientific discourse. We believe that these additions not only
strengthen the study’s foundation but also underscore the novelty of our research as an initial
exploration into the dietary modulation of rumen fermentation and its potential effects on stressrelated behaviors in ruminants.
Sincerely,
Author
Author Response File: 
Author Response.pdf
Reviewer 2 Report
Comments and Suggestions for AuthorsDear Authors,
The manuscript entitle ""Effects of rumen fermentation characteristics on stress-related hormones and behavior in sheep" can be improved.
Please find below my suggestions and doubts for its improvement.
With kind regards,
Reviewer
Comments for author File: Comments.pdf
Author Response
Dear Reviewer,
Thank you for your insightful comments and suggestions, which have greatly helped in enhancing the quality of our manuscript. We have thoroughly revised the manuscript based on your feedback and have detailed our responses to each of your comments below. The changes made in response to your comments are highlighted in blue and gray.
Dear Reviewer,
Thank you for your insightful comments and suggestions, which have greatly helped in enhancing
the quality of our manuscript. We have thoroughly revised the manuscript based on your feedback
and have detailed our responses to each of your comments below. The changes made in response to
your comments are highlighted in blue and gray.
Comment 1: In the study, there was a possible effect due to the habituation of the animals. Could
you please explain if the same people were involved during the experiments? Because animals seem
to be receptive to people and habits.
Response 1: Thank you for raising a valid point concerning the potential impact of human
interaction on the habituation of animals in our study. We understand the importance of controlling
for such variables to ensure the integrity of our behavioral observations. Throughout the study, from
the transition period leading up to the experiments until the final day, we implemented stringent
measures to minimize the sheep's contact with humans. The same individual was responsible for the
routine care and sampling processes to maintain consistency in human-animal interactions. For
procedures such as blood collection and rumen fluid sampling, an additional randomly selected
person assisted the primary caretaker. Importantly, during the Open Field Test (OFT), we employed
video recording to monitor the behavior of the sheep without any human presence in their line of
sight. This approach was specifically chosen to avoid any potential influence that the presence of a
person might have on the animals' behavior, ensuring that their responses were as natural and
undisturbed as possible.
Comment 2: -L39= keyword: ruminants? Could be added to know in which specie was this study
performed even if it seems obvious due to the rumen fatty volatile acids.
Response 2: Thank you for your suggestion to include more specific keywords in our manuscript.
We agree that clarity is essential for readers to understand the context and focus of our study. As
recommended, we have now added 'sheep' to the list of keywords to explicitly indicate the species
used in our research.
Comment 3: L58= could you please expand a bit explaining what you mean with “strong
temperament” and how do you define it? Which characteristics are involved in an animal of this
specie to consider it with“strong temperament”.
Response 3: We appreciate your request for clarification on the term “strong temperament” as it
relates to our study. Upon reviewing our references and considering the precision of terminology,
we have updated our manuscript to replace the term "strong temperament" with "aggressive
behaviors."
Comment 4: -L75Could you please add the age average of the lambs.
Response 4: As per your suggestion, we have updated our manuscript to specify that the average
age of the lambs at the time of the study was 8 months. This information has been added to the
Materials and Methods section, Line 75.
Comment 5: Table 1: This part goes at the bottom outside part of the table. DM, dry matter; CP,
crude protein; 109 TDN, total digestible nutrient; EE, ether extract; NDF, neutral detergent fibre;
ADF, acid detergent fiber.
Response 5: Thank you for your valuable comment on the use of abbreviations in Table 1. In our
effort to enhance the readability and clarity of our manuscript, we have revised Table 1 by replacing
all abbreviations with their corresponding full terms. This change has been made to ensure that all
readers can easily understand the table content without referring to the abbreviations at the bottom
or elsewhere in the text.
Comment 6: L120=please detail the methodology of the time-resolved fluoroimmunoassay.
Response 6: Thank you for your valuable comment. We have updated our manuscript to specify that
detail of TRIFMA methodology.
Comment 7: L131= please detail the methodology or at least cite from which protocol did you
follow in order to perform this analysis.
Response 7: Thank you for your valuable comment. We have updated our manuscript to specify that
detail of HPLC methodology.
Comment 8: L143= what do you mean with this paragraph? I am assuming is the ethical approval,
should this be in here? Or placed somewhere else. It does not seem to follow up what it is written
in this part, probably at the animals and treatments sections or at the “Institutional Review Board
Statement:”
Response 8: Thank you for your valuable feedback. I apologize for any confusion caused. I have
removed the redundant sections as per your suggestion.
Comment 9: L145= 2.4. Statistical analysis should be “2.5”, numbering is repeated.
Response 9: Thank you for pointing out the numbering error in the manuscript. I have corrected it
as per your suggestion. I appreciate your attention to detail in helping improve the quality of the
manuscript.
Comment 10: L170 and L174.Figure 1 and 2 = please italicize the “P” value. For the representation
of the graphs, could you please add a small square.
Response 10: Thank you for your valuable comment. We have updated our manuscript as per your
suggestion.
Comment 11: Table 3= please remove the space between “escape attempts”.
Response 11: Thank you for your valuable comment. We have updated our manuscript as per your
suggestion.
Comment 12: L209= probably this word “instrumental” is not the adequate, could you please find
a synonym or more appropriate word.
Response 12: Thank you for your suggestion. We have replaced “instrumental” with "pivotal" as
per your recommendation.
Comment 13: L209-221= This paragraph could be shortened and be explained more precisely, since
you are speaking about habituation in the following part of the discussion.
Response: Thank you for your valuable feedback. We have revised the mentioned paragraph to make
it more succinct.
Comment 13: L211= “Open Field Test (OFT)” this OFT was already defined before, there is no
need to re do it again.
Response 13: Thank you for your valuable feedback. We have updated our manuscript as per your
suggestion.
Comment 14: L222 and L279, L285=please write instead d 2A/P” “Acetic/Propionic”.
Thank you for your valuable feedback. We have updated our manuscript as per your
recommendation.
Comment 15: L241= please cite “some studies” which citation.
Response 15: Thank you for pointing out the need for citation regarding the studies mentioned. We
would like to clarify that the reference to 'some studies' is immediately followed by an illustrative
example, 'For instance, the intravenous infusion of acetate was found to suppress the secretion of
GH in sheep,' which is duly cited [5].
Comment 16: L249=this Total Digestible Nutrients (TDN) was already defined before, please just
leave TDN.
Response16: Thank you for your valuable feedback. We have updated our manuscript as per your
recommendation.
Comment 17: L281= Even if the total VFAs in the rumen do not change (where? In your study? Or
elsewhere…because if it is in your study, it should be “did not change in our study” …
Response17: Thank you for your valuable feedback. We have updated our manuscript as per your
recommendation.
Comment 18: L289= could you please conclude which diet do you think it could help lambs to
suffer less stress. he higher concentrate or the lower one according to your investigation.
Response 18: Thank you for your constructive feedback on our conclusion. We have revised it to
more directly address the impact of different diets on stress in lambs. Despite the complexity of
dietary effects on stress response mechanisms, our observations lead us to cautiously suggest that
high-concentrate diets, even when not causing apparent ruminal disorders, may contribute to chronic
stress in livestock. We have incorporated this perspective into our conclusion, highlighting the
importance of optimizing feed composition to enhance animal welfare.
Sincerely,
Author
Author Response File: Author Response.pdf
