Impact of Monensin Sodium and Essential Limonene Oil on the Fermentation and Chemical Composition of Total Mixed Ration Silages with Moisture Variations

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Comments for author File: Comments.pdf

Comments on the Quality of English Language

Author Response

Dear reviewer

When we conceived this experiment, we started with the premise that the presence of MON and OEL would hinder fermentation, and if this were confirmed, the production of TMR silages should proceed without these additives. However, in the first trial, the results surprised us by revealing improvements in fermentation rather than impairments.
To confirm these results, we decided to conduct a second trial using the same doses but under different moisture conditions. This approach was adopted due to the partial solubility of MON and limonene in water and to test the effect of the additives in a more intense fermentation condition (30% DM). To our surprise, the results followed the same trend as the first trial (except for ELO600), demonstrating that the additives improve fermentation.
Although we attempted to find explanations for our findings, we could not delve deeply enough. The mechanisms of action of MON, for example, have been extensively studied in ruminal bacteria, including lactobacilli that cause acidosis. However, it seems that both MON and OEL affect the bacteria present in the silage differently, although we do not have conclusive evidence. Some microbiologist colleagues informed us that, without knowing the bacterial species present in the silage, it is difficult to explain why the additives favored lactic fermentation. I agree with these researchers and am seeking partnerships to study this matter more thoroughly.
Unfortunately, we could not answer all the questions posed to us, as we lack sufficient data, but we appreciate your efforts in reviewing our manuscript. We count on your understanding.

Abstract and Introduction

1. Clarification needed:

Specify the type of essential oils used, particularly "limonene".

R: It was corrected. Thanks.

Provide the rationale for choosing the specific dosages of MON35, MON45, ELO300, and ELO600.

R: This was done in the MM. Thanks.

Clarify the transition between the first and second assays.

R: It was done in the abstract. Thanks

Detail methods for measuring fermentative losses and short chain fatty acid profiles.

R: This was done in the MM. Thanks

Include control conditions for both assays.

R: This was done in the abstract. Thanks

Provide data supporting the economic benefits of TMR ensilage.

R: It was done in the introduction. Thanks

Elaborate on findings from referenced studies ([2], [4], [5]).

R: It was done. Thanks

Expand on mechanisms of monensin and essential oils.

R: It was done. Thanks

Provide more details or references supporting higher water activity enhancing additives' action.

R: It was done. Thanks


2.  Suggestions:

Ensure consistency in unit presentation (e.g., "35 mg of monensin per kg of DM"). R: It was corrected. Thanks.

Maintain uniform reference formatting

R: Dear reviewer, in this case I think the reference in the text is correct. The journal works with continuous numbering of citations in the text, however, when we repeat the citation, the numbering remains the same. However, in the "references" section there were errors that were revised.

Add a section detailing statistical methods.

R: It was done. Thanks.

Clearly state hypotheses at the end of the introduction.

R: It was done. Thanks.

Explain how aerobic stability (AS) was measured.

R: It was done. Thanks.

Clarify why ELO600 had the highest aerobic stability given its antimicrobial properties.

R: The journal's guidelines do not allow discussions in the article abstract. Due to the numerous comments from the reviewers, the abstract exceeded the word limit. However, I believe the current version is significantly improved.




Materials and Methods

1. Clarification needed:

Specify the type of essential oil used in detail to match the abstract.

R: It was done. Thanks.

Explain the criteria used to determine the dosages of monensin and essential oils.

R: It was done. Thanks.

Provide a table detailing the exact proportions of each component in the sorghum roughage and various concentrates.

R: Dear reviewer, we are no longer able to present the composition of each of the ingredients used. We only have the composition of the TMR in our database, as shown in table 1.




2.  Suggestions:

Break down long paragraphs into bullet points or subsections for better readability, especially when detailing complex procedures.

R: It was done. Thanks.

Describe how the experimental silos were stored and monitored during the 90day period, including specific conditions apart from room temperature.

R: Dear reviewer, we no longer have measurements of the laboratory's daily temperatures over the 90 days of silo storage, only the average for the period. We know that the ambient temperature can interfere with fermentation and that it is common to observe high temperatures in tropical climates. Either way, we can guarantee that all silos have been exposed to the same environmental conditions.

Briefly explain the calibration process of the spectrophotometer used in section 2.2.

R: It was added in the MM. Thanks




Statistical Data Analysis

1. Clarification needed:

State the assumptions made in the statistical models used for data analysis and whether these assumptions were tested (e.g., normality, homoscedasticity).

R: The Shapiro-Wilk normality test was used to check the homogeneity of variances. We added this in MM section.

Describe how outliers were handled in the data analysis.

R: An individual observation was considered outlier when the standard deviations in relation to the mean or to the model were larger than + 3 or less than −3. We added this in MM section.




2.  Suggestions:

Specify which variables were significantly different and how these differences were interpreted when mentioning statistical significance (α ≤ 0.05).

R: It was added in the MM. Thanks.

Provide a brief explanation of the ScottKnott test and why it was chosen over other posthoc tests.

R: Since the Scott-Knott test is a clustering algorithm, it groups similar means preventing overlapping in the results, it facilitate the interpretation of results, especially in studies with many comparisons. This contrasts with the Tukey test, which performs pairwise comparisons, resulting in a matrix of significant differences that can be more complex to interpret.

Clarify what A and B represent in model (2) – are these the two moisture levels (30% and 40% DM)?

R: It was corrected. Thanks

Describe how data accuracy was ensured and if there were any calibration steps for the temperature sensors used in the data logger and temperature monitoring.

R: It was added in the MM. Thanks




Results

1. Clarification needed:

Specify the exact moisture percentages for terms "higher" and "lower" moisture used in the results for consistency.

R: It was added to the footnotes of figures and tables

Clarify if the significant differences (P < 0.05) reported are consistent across multiple repetitions.

R: This was done in MM. Thanks




2.  Suggestions:

Include footnotes in Table 2 and Table 3 to briefly explain abbreviations used (e.g., GL, EL, DMR).

R: It was added. Thanks

Interpret the implications of your findings rather than just reporting values. For instance, explain why certain treatments had higher lactic acid or NH3N values and what this means for TMR silage quality.

Thoroughly discuss the interaction effects in the second assay (Figure 1) and explain the biological significance of these interactions in detail.

R: We explained the reasons for not being able to go much deeper into the explanations at the beginning of this letter. We are open to suggestions for references that help us explain results obtained in this study.




Describe how standard errors (SEM) were calculated and the statistical methods used to ensure their accuracy.

R: We calculate the SEM by take the standard deviation (SD) and divided it by the square root of the sample size. The standard error of the mean (SEM) measures how much discrepancy is likely in a sample's mean compared with the population mean. Descriptive statistics, was used to summarized data indexes and accuracy. However, we opt to not present it, since it was not relevant for the interpretation of the results.

Provide more information on the methodology for measuring fermentation parameters like gas losses, effluent production, and DM recovery to understand how precise these measurements are.

R: It was added in the MM. Thanks

Consider environmental factors, such as temperature fluctuations during the 90day period, that could affect the fermentation process.

R: Dear reviewer, we no longer have measurements of the laboratory's daily temperatures over the 90 days of silo storage, only the average for the period. We know that the ambient temperature can interfere with fermentation and that it is common to observe high temperatures in tropical climates. Either way, we can guarantee that all silos have been exposed to the same environmental conditions.







First Assay:

Provide more context or hypothesize why no significant differences were found if stating "no difference (P > 0.05) was observed."

R: Dear reviewer, I don't understand what is being asked. Could you clarify this point?




Discuss underlying reasons for higher aerobic stability in ELO600 and MON35 treatments, referencing relevant literature.

R: Dear reviewer. Fungi are more sensitive than bacteria to essential oil, meaning larger doses of essential oil are needed to inhibit bacterial growth. In the case of this work and several others in the literature, the presence of OEL in silage ends up reducing the fungal population and improving aerobic stability. In the case of MON, I was unable to gather articles showing its effects on aerobic fungi in silage. The vast majority of studies on MON only evaluate ruminal bacterial populations. Perhaps, the mode of action of monensin on fungi is different from methanogenic bacteria.




Explain potential mechanisms by which MON and ELO reduce NH3N content in silages.

R: Dear reviewer, NH3-N is an indicator of proteolysis, and for silages this is linked to Clostridium fermentation. However, only in essay 1 did MON and ELO reduce NH3-N levels. In essay 2, ELO600 had higher levels of N-NH3 and the other treatments did not differ from the control. Therefore, any statement that could link additives to reduced proteolysis would not be correct. But I'm open to suggestions about what might have happened.







Second Assay:

Discuss possible reasons for higher gas losses in MON35 and ELO600 treatments.

Elaborate on why ELO might lead to increased lactic acid production under higher moisture conditions and its impact on silage quality.

R: Dear reviewer, Only ELO300 helped with the production of lactic acid. The higher dose harmed the process. I believe this is linked to the resistance that each microorganism has to doses of ELO. I did not find research that specifically evaluated the use of Limonene on microbial populations in silage, perhaps this would help me explain better what happened.




Derive practical recommendations for farmers using TMR silages based on the interactions between additives and moisture levels.

R: It was written in the conclusion




Align findings on differences in aerobic stability between treatments and moisture levels with previous studies and discuss practical implications for silage management.

R: It was added. Thanks







Discussion

1. Clarification Needed:

Provide more details on the specific essential oils used, including their chemical composition and potential antimicrobial properties.

R: It was done, Thanks




Link results explicitly to underlying biological mechanisms. For instance, how do MON and ELO alter microbial activity?

R: It was improved, Thanks




2.  Suggestions:

Break the discussion into subsections to address key points more clearly (e.g., "Effect on Fermentation Parameters," "Impact on Nutrient Composition," "Aerobic Stability").

R: It was done in the discussion of essay 2




Add more references to support interpretations, particularly regarding mechanisms by which MON and ELO impact fermentation and microbial populations.

R: It was improved, Thanks




Elaborate on how the threshold for gas loss values below 3% indicates good fermentation quality and under what conditions lower values might not be indicative of good quality.

R: Dear reviewer, I don't understand what is being asked. Could you clarify this point?




Provide more insight into specific microbial changes due to additives leading to enhanced AS.

R: R: It was improved, Thanks




Discuss potential selectivity of different essential oils on microbial populations and how this might vary with different doses or types of oils.




Include more specific details on how NH3N and ethanol production impact overall silage quality and usability.

R:




Elaborate on how different moisture levels might influence the effectiveness of MON and ELO differently.

Discuss implications of the shift towards heterofermentative bacteria with higher ELO doses and moisture for overall nutritional quality and stability of the silage.

R: We explained the reasons for not being able to go much deeper into the explanations at the beginning of this letter. We are open to suggestions for references that help us explain results obtained in this study.




Provide more detailed information on the antimicrobial properties of limonene against specific LAB strains.

R: It was improved, Thanks




Discuss how maintaining lactic acid:acetic acid ratios might affect longterm storage and nutritional value of the silage.

Hypothesize potential long-term impacts of changes in NDF, ADF, and starch contents on animal health and performance.

R: Dear reviewer, I don't understand what is being asked. Could you clarify this point?

Discuss practical benefits for animal feed efficiency related to the observed increase in IVDMD with MON35.

R: It was improved, Thanks

Suggest specific experimental approaches or methodologies to address research gaps on the dynamics of microbial populations and the use of other ionophores and essential oils.

R: It was improved, Thanks







General Observations:

Ensure all abbreviations (e.g., DM, NH3N) are consistently defined upon first use and used uniformly throughout the text.

R: It was checked. Thanks

Be clear when discussing statistically significant results and include corresponding pvalues or reference specific results from the tables.

R: It was checked. Thanks

Doublecheck reference formatting for consistency according to the journal’s guidelines.

R: It was checked. Thanks




Authors' note: Dear evaluator, due to the numerous suggested modifications and the limited time available for revisions, we may not have fully addressed all requests. However, we are available to make further adjustments as necessary.

Reviewer 2 Report

Comments and Suggestions for Authors

It is suggested not to use abbreviations in the title. 

The value of Pi should be included in the abstract. 

In the introduction, a brief definition of essential oil should be provided to give the reader a better understanding. 

The objectives and hypotheses are clearly stated in the introduction. The reason for choosing monensin and essential oil doses should be clearly explained. 

The amount of ADF should be indicated in Table 1. 

The NFC calculation formula should be provided. 

The reason for using sorghum in the formula should be explained. 

The rationale for using the Scott-Knott test to compare averages should be clarified. 

The results are written in accordance with the tables.

Lines 351-352: These lines should be rewritten, and the references should be revised.

Line 356: The reference should be corrected to align with the journal's style.

Line 363: Specify which microorganisms are being referred to by providing an example.

Line 415: Correct the reference formatting. This should also be done for other lines where necessary.

Line 443: Indicate which essential oils are more effective.

Rewrite the conclusion to reflect it as a conclusions section rather than a summary. Discuss what worked and didn’t work in your study, and what the implications are.

Abbreviations should be corrected.

References should be formatted according to the journal's style.

Comments on the Quality of English Language

.

Author Response

Comment 1: It is suggested not to use abbreviations in the title.

Response : It was corrected. Thanks for the observation

Comment 2: The value of Pi should be included in the abstract. 

Response : It was included. Thanks for the observation

Comment 3 In the introduction, a brief definition of essential oil should be provided to give the reader a better understanding. 

Response: It was included. Thanks for the observation

Comment 4 The objectives and hypotheses are clearly stated in the introduction. The reason for choosing monensin and essential oil doses should be clearly explained. 

Response: It was included in MM. Thanks for the observation

Comment 5 The amount of ADF should be indicated in Table 1. 

Response: It was included. Thanks for the observation

Comment 6 The NFC calculation formula should be provided. 

Response: The chemical composition was carried out using Nirs, so no formula was presented for calculating non-fibrous carbohydrates.

Comment 7 The reason for using sorghum in the formula should be explained. 

Response: Sorghum was used because it is a very common crop in the region where the experiment was carried out. An observation was made on the material and methods.

Comment 8 The rationale for using the Scott-Knott test to compare averages should be clarified.

Response: Since the Scott-Knott test groups similar means, it can facilitate the interpretation of results, especially in studies with many comparisons. This contrasts with the Tukey test, which performs pairwise comparisons, resulting in a matrix of significant differences that can be more complex to interpret.

Comment 9 The results are written in accordance with the tables.

Response: Dear reviewer, we don't understand this comment. We are unsure if there were any parameters that were described out of order or if you just said that the results were discussed in order. If there is any problem in the order of result presentation, let us know and we will modify it.

Comment 10 Lines 351-352: These lines should be rewritten, and the references should be revised.

Response: It was rewritten. Thanks for the observation

Comment 11 Line 356: The reference should be corrected to align with the journal's style.

Response: Dear reviewer, in this case I think the reference is correct. The journal works with continuous numbering of citations in the text, however, when we repeat the citation, the numbering remains the same.

Comment 12 Line 363: Specify which microorganisms are being referred to by providing an example.

Response: It was included. Thanks for the observation

Comment 13 Line 415: Correct the reference formatting. This should also be done for other lines where necessary.

Response: Dear reviewer, in this case I think the reference is correct. The journal works with continuous numbering of citations in the text, however, when we repeat the citation, the numbering remains the same.

Comment 14 Line 443: Indicate which essential oils are more effective.

Response: It was included. Thanks for the observation

Comment 15 Rewrite the conclusion to reflect it as a conclusions section rather than a summary. Discuss what worked and didn’t work in your study, and what the implications are.

Response: It was rewritten. Thanks for the observation

Comment 16 Abbreviations should be corrected.

Response: It was corrected. Thanks for the observation

Comment 17 References should be formatted according to the journal's style.

Response: References was reviewed. Thanks for the observation

Reviewer 3 Report

Comments and Suggestions for Authors

 

The application of antimicrobial additives such as monensin (MON) and limonene essential oil (ELO) in TMR (Total Mixed Ration) silages is interesting. Although the use of MON in silages is not completely new, the combination with ELO, especially at the dosages tested and in the different moisture conditions, can be considered a relatively new approach.

Suggested improvements to the text:

Lack of consistency in terms, for example:

Line 38: TMR ensilage

Line39,44: ensiled TMR

Line 45: TMR silage

Table 1; Table 3; Table 5- It is not common to use the units of dry matter as a % of fresh matter. MM-mineral matter is also not a common term, but rather crude ash

Table 3, table 5 in animal nutrition it is not common to use the term Chemical-bromatological but chemical composition.

Line 122-124 repeats the procedure from line 134 to line 136

Line 136-142: Although chemical analyses were not the main objective of the study, the use of standardized methods to validate the NIR analyses would have been important to ensure the accuracy and reliability of the results. Confidence in the data obtained could be strengthened by including complementary analyses using standardized conventional methods, at least on a representative fraction of the samples. This would guarantee the robustness and accuracy of the data, increasing the reliability of the study's conclusions. It should include a note on the importance of validating NIR results with conventional methods.

Line 153- 154- the ammoniacal nitrogen method should be better described

Line 164- the reference does not comply with the journal's standards

Line181- the reference does not comply with the journal's standards

Author Response

Comment 1: “The application of antimicrobial additives such as monensin (MON) and limonene essential oil (ELO) in TMR (Total Mixed Ration) silages is interesting. Although the use of MON in silages is not completely new, the combination with ELO, especially at the dosages tested and in the different moisture conditions, can be considered a relatively new approach.”

Response 1: We appreciate it. Thank you for recognizing the scientific novelty of our work.

Suggested improvements to the text:

Comment 2. “Lack of consistency in terms, for example: Line 38: TMR ensilage; Line39,44: ensiled TMR; Line 45: TMR silage. “

Response 2. It was corrected.

Comment 3. “Table 1; Table 3; Table 5- It is not common to use the units of dry matter as a % of fresh matter. MM-mineral matter is also not a common term, but rather crude ash.”

Response 3. It was corrected.

Comment 4. Table 3, table 5 in animal nutrition it is not common to use the term Chemical-bromatological but chemical composition.

Response 4. It was corrected

Comment 5. Line 122-124 repeats the procedure from line 134 to line 136.

Response 5. It was removed, thanks

Comment 6. Line 136-142: Although chemical analyses were not the main objective of the study, the use of standardized methods to validate the NIR analyses would have been important to ensure the accuracy and reliability of the results. Confidence in the data obtained could be strengthened by including complementary analyses using standardized conventional methods, at least on a representative fraction of the samples. This would guarantee the robustness and accuracy of the data, increasing the reliability of the study's conclusions. It should include a note on the importance of validating NIR results with conventional methods.

Response 6. We agree with your observation. The analyzes were carried out in a private laboratory that follows strict quality standards and, therefore, the accuracy of the results obtained by Nirs is periodically checked in relation to traditional chemical analyses. A note was made in the text regarding this specific point.

Comment 7.  Line 153- 154- the ammoniacal nitrogen method should be better described.

Response 7. It was done

Comment 8. Line 164- the reference does not comply with the journal's standards.

Response 8. It was corrected.

Comment 9. Line181- the reference does not comply with the journal's standards.

Response 9. In this case it is not a reference, it is the version/year of the statistical program.

 

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The paper can be accepted as it is. The authors have done their best to address all the queries, so it's a green flag on my end.

To authors:

Regarding the statement "no difference (P > 0.05) was observed," I expected  you to provide more context or hypotheses to explain this result? Specifically, it would be helpful to discuss potential contributing factors (e.g., sample size, data variability), details on the study design and any limitations, comparisons with existing literature, and possible explanations for the lack of significant differences. Adding these points will improve the readers' understanding of your findings.

Regarding my suggestion on the factors, To provide more clarity and improve your manuscript, please consider elaborating on the potential mechanisms by which MON (monensin) and ELO (essential oils) could influence NH3-N content, such as MON's inhibition of Gram-positive bacteria, including proteolytic Clostridia, and ELO's antimicrobial properties affecting microbial activity in silage. Additionally, discuss possible reasons for the differing results between essays, such as variations in experimental conditions, silage composition, microbial populations, or additive application rates. Comparing your findings with existing literature, particularly studies observing similar variability or specific conditions where MON and ELO were effective, would provide valuable context. Finally, suggest areas for further research to elucidate the conditions under which these additives effectively reduce NH3-N content in silages.

 

 

Comments on the Quality of English Language

The paper can be accepted as it is!

Author Response

Dear Reviewer,

We would like to express our sincere gratitude for your thorough review and valuable suggestions for our manuscript. We have carefully considered and addressed all your comments and suggestions. The modifications have been made accordingly and are highlighted in the revised manuscript.

Thank you once again for your time and effort in reviewing our work. We believe that your insights have significantly improved the quality of our manuscript.

Best regards,